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Homologues of the homeotic and segment polarity genes are involved in the postnatal development of the… Sabunciyan, Sarven Harutyun 2001

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HOMOLOGUES OF THE HOMEOTIC A N D SEGMENT POLARITY GENES A R E I N V O L V E D IN T H E P O S T N A T A L D E V E L O P M E N T O F T H E C A T VISUAL SYSTEM by SARVEN HARUTYUN  SABUNCIYAN  B . S c , The University of British Columbia, 1992 A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F THE REQUIREMENTS FOR T H E DEGREE OF DOCTOR OF PHILOSOPHY in T H E F A C U L T Y O F G R A D U A T E STUDIES Faculty of Medicine, Graduate Program in Neuroscience We accept this thesis as conforming to the required standard  T H E UNIVERSITY O F BRITISH C O L U M B I A August 2001 © Sarven Harutyun Sabunciyan, 2001  In presenting  this  degree at the  thesis  in  University of  freely available for reference copying  of  department publication  this or  partial fulfilment  of  British Columbia,  I agree  and study.  thesis for scholarly by  of this  his  or  her  Department The University of British Columbia Vancouver, Canada  requirements that the  I further agree  purposes  representatives.  may be It  thesis for financial gain shall not  permission.  DE-6 (2/88)  the  that  advanced  Library shall make it  by the  understood be  an  permission for extensive  granted  is  for  allowed  that without  head  of  my  copying  or  my written  ABSTRACT This thesis investigates the possible involvement o f the homologues o f the drosophila developmental genes in the postnatal development o f the cat visual system. Initially, the c D N A ' s o f homeotic genes PBX1, PBX2 and the homologues of the segment polarity genes BMP4,  BMP6, BMP type II receptor,  Wnt-1, and FrzB were partially  cloned in the cat. We report that the m R N A expression of these genes is developmentally regulated in the postnatal cat visual cortex. T o further substantiate our hypothesis that the homologues o f the drosophila developmental genes contribute to the postnatal development o f the cat visual cortex, the expression o f the beta-catenin protein was characterized in the visual system o f normally developing and deprived kittens.  The  beta-catenin protein, which is a downstream effector o f the Wnt-1 signalling pathway, is capable o f functioning both as a transcription factor and a cell adhesion molecule. Consistent with its characterized role as a transcription factor, the beta-catenin protein becomes nuclearized in L G N neurons at the end o f the period for thalamocortical plasticity. Hence, one o f the putative functions o f the beta-catenin protein in postnatal visual development is proposed to be ending thalamocortical plasticity. The role o f the beta-catenin protein in cellular adhesion is to anchor the cadherin cell adhesion molecules to the actin cytoskeleton. Interestingly, the beta-catenin/cadherin cell adhesion system in neurons is located at synapses. Fittingly, both the cadherin and the beta-catenin proteins are expressed in the neuropil of the geniculate and the visual cortex and this expression is prominent in layer I V o f the visual cortex. In addition, the temporal expression o f these proteins correlates with the critical period.  Furthermore, neuropil expression o f beta-  catenin and cadherin proteins is altered in the L G N in response to monocular deprivation. This finding suggest a role for these molecules in the competition occurring between X and Y - cell arbours. In summary, beta-catenin appears to act as a multi-functional protein and contribute to different facets o f postnatal visual development in the cat.  ii  These  findings endorse our original hypothesis that the homologues o f the drosophila developmental genes are involved in the development o f the cat visual system.  iii  TABLE OF CONTENTS Abstract Table of Contents List of Figures  .ii iv viii  Chapter! I N T R O D U C T I O N  1  1.1 G E N E R A L I N T R O D U C T I O N 1 1.2 T H E C H O I C E O F T H E C A T A S T H E A N I M A L M O D E L . 2 1.3 A N A T O M Y A N D P H Y S I O L O G Y O F T H E D E V E L O P I N G C A T V I S U A L SYSTEM 4 1.3.1 Normal development 4 1.3.2 Activity dependent nature of the developing visual system 7 1.3.3 Competitive nature of the developing visual system 9 1.3.4 Importance of concurrent visual activity 11 1.3.5 Contribution of spontaneous activity to visual development 12 1.4 C E L L U L A R A N D M O L E C U L A R M E C H A N I S M S I N V O L V E D I N V I S U A L PLASTICITY. 13 1.4.1 Hebbian mechanisms 13 1.4.2 The role of N M D A receptors in visual development 14 1.4.3 The role of metabotropic glutamate receptors in visual development. 16 1.4.4 The role of neuromodulatory neurotranmsitters i n visual development 17 1.4.5 The role of immediate early genes in visual development 18 1.4.6 The role of neurotrophins in visual development 19 1.4.7 Summary. 21 1.5 E X P E R I M E N T A L S T R A T E G I E S A I M E D A T I D E N T I F Y I N G M O L E C U L A R MECHANISMS INVOLVED IN POSTNATAL DEVELOPMENT OF THE VISUAL SYSTEM 22 1.6 D R O S O P H I L A D E V E L O P M E N T A L G E N E S 24 1.6.1 Properties of the Drosophila Developmental genes 24 1.6.2 Possible roles for segmentation genes i n visual development 26 1.7 P R O P E R T I E S O F B E T A - C A T E N I N .28 1.8 P R O P E R T I E S O F C A D H E R I N S 29 Chapter 2 H O M E O B O X A N D S E G M E N T P O L A R I T Y G E N E E X P R E S S I O N IN T H E DEVELOPING CAT VISUAL C O R T E X 31 2.0 S U M M A R Y . 2.1 I N T R O D U C T I O N 2.1.1 Possible role of homeotic genes in visual development 2.1.2 Possible role of segment polarity genes in visual development 2.1.2.1 Members of the B M P signaling pathway present in the developing visual cortex iv  31 31 32 33 34  2.1.2.2 Members of the W n t signaling pathway present i n the developing visual cortex 2.2 M E T H O D S 2.3 R E S U L T S ; 2.3.1 Technical considerations 2 3 . 2 Summary ofthe cloning strategy 2.3.3 P B X 1 2.3.4 P B X 2 2.3.5 B M P 4 2.3.6 B M P 6 2.3.7 B M P type II receptor 2.3.8 Wnt-1 2 3 . 9 Frz-B 2.3.10 Genes which could not be cloned using the R T - P C R approach 2.4 D I S C U S S I O N 2.4.1 The cloning strategy .• 2.4.2 The expression of P B X Homeotic genes i n the developing visual cortex 2.43 Segment polarity gene expression i n the developing visual cortex 2.4.4 Selection of the beta-catenin protein for further characterization in the postnatal development of the cat visual system Chapter 3  35 37 45 45 46 46 48 52 52 56 56 60 63 64 64 65 68 70  B E T A - C A T E N I N P R O T E I N E X P R E S S I O N IN T H E P O S T N A T A L D E V E L O P M E N T O F T H E CAT VISUAL SYSTEM. 72  3.0 3.1 3.2 33  SUMMARY. 72 INTRODUCTION 72 METHODS 76 RESULTS 83 33.1 Partial cloning of the cat beta-catenin c D N A 83 3.3.2 Western blot analysis of the beta-catenin protein i n the postnatally developing cat visual cortex 83 3.3.3 Western blot analysis of the beta-Catenin protein i n the postnatally developing cat L G N 87 3.3.4 Immunohistochemical localization of the beta-catenin protein in the postnatally developing cat visual cortex 89 3 3 . 5 Immunohistochemical localization of the beta-catenin protein i n the postnatally developing cat L G N 92 3 3 . 6 Nuclear beta-catenin protein levels i n the postnatally developing cat L G N . . .95 3.4 D I S C U S S I O N 95 3.4.1 Beta-Catenin protein expression in the L G N 95 3.4.2 Beta catenin protein expression i n the developing visual cortex 100 3.43 Function of the beta-catenin protein in the developing visual system 101 v  Chapter 4 C A D H E R I N P R O T E I N E X P R E S S I O N IN T H E P O S T N A T A L D E V E L O P M E N T O F T H E CAT VISUAL SYSTEM  103  4.0 4.1 4.2 4.3  SUMMARY. 103 INTRODUCTION 103 METHODS 107 RESULTS 107 4.3.1 Partial cloning of cadherin genes which are expressed i n the postnatal cat visual cortex 107 4.3.2 Selection of the pan-cadherin antibody Ill 4.3.3 Western blot analysis of cadherin protein expression i n the postnatally developing cat visual cortex 113 4.3.4 Western blot analysis of cadherin protein expression in the postnataly developing cat L G N 115 4.3.5 Immunohistochemical localization of cadherin proteins i n the postnatal visual cortex 115 4.4 D I S C U S S I O N 120  Chapter 5 A C T I V I T Y D E P E N D E N T E X P R E S S I O N O F B E T A - C A T E N I N A N D T H E C A D H E R I N PROTEINS 125 5.0 5.1 5.2 5.3  SUMMARY. 125 INTRODUCTION 125 METHODS 127 RESULTS 130 5.3.1 Effects of monocular deprivation on beta-catenin protein expression i n the postnatal cat visual cortex and the L G N 130 5.3.2 Effects of monocular deprivation on cadherin protein expression i n the postnatal cat visual cortex and the L G N 134 53.3 Effect of optic tract lesions on the expression of beta-catenin and the cadherin proteins 134 5 3 . 4 Co-immunoprecipitation of the beta-catenin and the cadherin proteins 138 5.4 D I S C U S S I O N 143 5.4.1 Activity independent regulation of the beta-catenin and the cadherin proteins in the postnatal visual cortex 144 5.4.2 Activity independent regulation of nuclear beta-catenin expression 146 5.4.3 Activity dependent changes i n beta-catenin and cadherin staining in the L G N 147  Chapter 6 S U M M A R Y A N D G E N E R A L DISCUSSION 6.1 I N T R O D U C T I O N 6.2 T H E I N V O L V E M E N T O F D E V E L O P M E N T A L G E N E S I N V I S U A L PLASTICITY vi  152 152 152  6.2.1 Evidence for the involvement of the beta-catenin protein i n postnatal visual development 155 6.2.1.1 Involvement of nuclear beta-catenin i n ending thalamocortical plasticity 155 6.2.1.2 The expression of the beta-batenin protein i n the postnataly developing cat visual cortex 158 6.2.1.3 Involvement of beta-catenin and the cadherin proteins i n X - and Y cell competition 161 6.2.1.4 Summary of the evidence implicating a role for beta-catenin in postnatal visual development 162 6.2.2 Significance of the findings presented i n this thesis 162 REFERENCES  164  vii  LIST O F FIGURES 1.1 Anatomy of the cat visual system  5  2.1 Partial c D N A sequence of the c a t P B X - 1 gene 2.2 P B X 1 gene m R N A expression i n the postnataly developing cat visual cortex 2 3 Partial c D N A sequence ofthe cat P B X - 2 gene 2.4 P B X 2 gene m R N A expression in the postnataly developing cat visual cortex 2.5 Partial c D N A sequence of the cat B M P - 4 gene 2.6 B M P 4 gene m R N A expression in the postnataly developing cat visual cortex 2.7 Partial c D N A sequence ofthe cat B M P - 6 gene 2.8 B M P 6 gene m R N A expression in the postnataly developing cat visual cortex 2.9 Partial c D N A sequence of the cat B M P type II receptor gene 2.10 Partial c D N A sequence of the cat Wnt-1 gene 2.11 Wnt-1 gene m R N A expression in the postnataly developing cat visual cortex 2.12 Partial c D N A sequence ofthe cat Frz-B gene  47 49 50 51 53 54 55 57 58 59 61 62  3.1 Post-translational regulation of beta-catenin protein expression 74 3.2 Partial c D N A sequence ofthe cat beta-catenin gene 84 3 3 Alignment of the cat and the human beta-catenin amino acid sequences 85 3.4 Beta-catenin protein expression i n the postnataly developing cat visual cortex 86 3.5 Beta-catenin protein expression i n the postnataly developing cat L G N 88 3.6 Beta-catenin antibody staining i n the visual cortex 90 3.7 Cellular location of the beta-catenin protein in the developing visual cortex 91 3.8 Expression of the beta-catenin protein in the developing cat L G N 93 3.9 Beta-catenin and SMI-32 double staining i n the L G N of older kittens 94 3.10 Beta-catenin protein expression i n the nuclei of geniculate cells during the postnatal development of the cat L G N 96 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9  Cadherin protein distribution in neurons Partial c D N A sequence of the cat cadherin-6 gene Partial c D N A sequence of the cat cadherin-8 gene Partial c D N A sequence ofthe cat cadherin-11 gene , Alignment of the cat and the human cadherin amino acid sequences Cadherin protein expression i n the postnataly developing cat visual cortex Cadherin protein expression i n the postnataly developing cat L G N Pan-cadherin antibody staining i n the visual cortex Cellular location of the cadherin proteins i n the developing visual cortex  106 108 109 110 112 114 116 118 119  5.1 Beta-catenin protein expression i n the visual cortex of normal and monocularly deprived kittens 131 5.2 Beta-catenin antibody staining in the L G N of monocularly deprived and normal kittens 132 5.3 The effect of monocular deprivation on the nuclearization of the beat-catenin protein in the L G N 133 viii  5.4 Pan-cadherin protein expression i n the visual cortex of normal and monocularly deprived kittens 5.5 Pan-cadherin antibody staining i n the L G N of monocularly deprived and normal kittens 5.6 Unilateral optic tract lesion 5.7 Neuropil expression of the beta-catenin and the cadherin proteins i n the L G N following unilateral optic tract lesions 5.8 The effect of unilateral optic tract lesions on the nuclearization of the beta-catenin protein i n the geniculate 5.9 Coimmunoprecipitation of the beta-catenin and the cadherin proteins  135 136 137 139 140 142  6.1 Summary of Drosophila developmental gene expression i n the postnatal cat visual system and their putative interactions 153 6.2 Synaptic organization mediated by the cadherin proteins 159  ix  Chapter 1 INTRODUCTION 1.1  GENERAL  INTRODUCTION  The visual areas of the brain from various species ranging from goldfish to primates have been extensively studied (Levine et al. 1993, Gaze et al. 1972, Shatz et al. 1978, Hubel et al.  1969, Kennedy et al. 1980, Van Sluyters et al. 1974, Maffei et al. 1992,  Gordon et al. 1996, Meister et al. 1991). These studies which were initially undertaken to find remedies for a variety of different visual ailments have coincidentally contributed greatly to our understanding of overall brain development and behavior. For example, the activity dependent nature of the developing visual system (Wiesel et al. 1963, Hubel et al. 1977) has emphasized the importance of environmental factors and sensory during normal brain development.  The activity dependent  synaptic  experience  rearrangements  identified in the visual system are a general characteristic of the developing brain and parallels are seen in the development of numerous brain areas (Webster et al.  1983,  Meisami et al. 1981, Westrum et al. 1986, Wooseley et al. 1976, Garraghty et al. 1992, Pons et al. 1991).  The examination of the mechanisms responsible for these synaptic  rearrangements has provided significant insight into the manner in which neural circuits are formed and refined.  Additionally, processes similar to synaptic rearrangements occurring  during the development of the visual system are postulated to be the biological basis of learning and memory (Bienenstock et al. 1982, Bear et al. 1987).  Clearly, the knowledge  obtained from these studies is not merely limited to the visual system but instead, the mechanisms involved in visual development are applicable to general brain development and function.  1  Despite the wealth of knowledge available about the physiology and the anatomy of the developing kitten visual cortex, very little is  known  regarding the molecular  mechanisms responsible for this event.  H Y P O T H E S I S : The homologues of the drosophila developmental genes are involved in the postnatal development of the kitten visual cortex.  The  developmental genes initially characterized in the drosophila are highly  conserved in all metazoa and play a prominent role in a large number of developmental events.  In order to prove our hypothesis, the expression of some of the drosophila  developmental gene homologues were examined by R T - P C R and Northern blot analysis during the critical period in kittens. T o further verify our hypothesis the properties of one these developmental genes, beta-catenin, was extensively characterized in the developing visual system.  The findings reported in this thesis suggest beta-catenin, which is the  mammalian homologue of the drosophila armadillo gene, and the cell adhesion system it gives rise to with the cadherin molecules contributes to multiple facets of visual system development in the cat.  1.2  T H E C H O I C E O F T H E C A T AS A N A N I M A L M O D E L  There are both benefits  and problems associated with using the cat as an  experimental animal model in visual neuroscience. Historically, the cat has been a popular model animal in neuroscience research. Accordingly, the physiology and the anatomy of the cat brain is well characterized. Compared to mice and rats the organization of the cat visual system closely resembles that of the primate and therefore, the cat is a more relevant animal model to study than rodents (LeVay et al. 1978, Shatz et al. 1978, Gordon et al. 1996, Fagiolini et al. 1994). Additionally, compared to the primates, the cat visual system  2  is fairly immature at birth and hence, the cat provides a much more convenient system to examine early events in visual development (LeVay et al. 1978, Shatz et al. 1978). cats are relatively inexpensive to purchase and to house compared to primates.  Also,  Finally,  since the visual areas of the cat brain develop more rapidly than the primate brain, time dependent studies are much easier to perform on these animals (Hubel et al. 1970, Hubel et al. 1969, Hubel et al. 1977, Blakemore et al. 1974, LeVay et al. 1978, Shatz et al. 1978). Unfortunately, the cat is not a suitable animal model for molecular biology. replacement or gene knockout experiments  Gene  are difficult to perform in cats because  homologous recombination occurs very rarely in cells of highly complex animals (Alberts et al. 1994, Cappechi 1989).  In addition, the cost associated with establishing transgenic  cat strains is unreasonably high. Hence, gene expression in the cat can not be manipulated by using standard molecular biological techniques.  Since the cat is not a suitable genetic  animal model, very little information is available regarding the sequence of cat genes. The above statements outline the current dilemma in visual neuroscience.  The cat possesses a  sophisticated visual system, resembling that of the primate, which is well characterized both at the neuroanatomical and neurophysiological levels. However, this system can not be manipulated at the molecular level. Therefore, when choosing an animal model to study molecular events associated with visual development, one must sacrifice either genetic modifiability or work with a simpler visual system lacking many of the attributes found in humans. This thesis has chosen the cat as its animal model because the focus of this research is to identify molecular mechanisms involved in the visual development of highly complex animals. The fact that cat c D N A sequence information is lacking is not a critical problem because many of the important neural molecules are highly conserved and these genes can be easily cloned in the cat by exploiting the extensive sequence homologies  present  amongst species. Similarly, many antibodies produced for use in other animals will cross react with cat proteins due to the high level of evolutionary conservation.  3  The difficulties  associated with modifying gene expression in complex animals is the main problem with using the cat as an experimental model.  Because of this reason, experiments aimed at  determining gene function during development can not be performed on these animals. On the other hand, cats can be subjected to visual deprivation studies which will provide pertinent information regarding the behaviour of a particular molecule during visual development.  This information can be used to form hypothesis about the function of the  molecule involved.  1.3  ANATOMY  VISUAL  1.3.1  AND PHYSIOLOGY  OF  T H E DEVELOPING  CAT  SYSTEM  Normal  Development  The eyes of the cat are located in front of the head and face forward which results in the overlap of the two visual fields required for binocular vision.  The cat visual system  exhibits "contralateral bias" because the majority of the retinal ganglion cells (about 60%) from each eye cross the midline at the optic chiasm and make connections  with the  contralateral lateral geniculate nucleus ( L G N ) (Wiesel et al. 1963) (See figure 1.1).  The  remaining retinal neurons make connections with the ipsilateral L G N . Although each L G N receives input from both eyes, the connections from each eye are kept segregated in eye specific layers.  Layer A of the L G N mainly receives input from the contralateral eye  whereas layer A l receives input from the ipsilateral eye and layer C receives connections from both eyes (Guillery 1970a). The L G N neurons connect to layer I V and to layer V I of the visual cortex.  The segregation of inputs found in the lateral geniculate nucleus is  retained in the cortex where the thalamocortical connections  from the L G N form an  alternating interdigitated series of eye specific columns referred to as "ocular dominance columns" (Hubel et al. 1969, Hubel et al. 1972, L e V a y et al. 1978). Ocular dominance  4  Figure 1.1 Anatomy ofthe Cat Visual System. The red and the blue colored lines represent the neural inputs originating from the two eyes. The inputs from the two eyes are segregated into eye specific layers in the L G N and ocular dominance columns in the visual cortex.  columns are roughly 400 - 600 microns wide and respond mainly to stimuli from only one of the eyes (Hubel et al. 1977, L e V a y et al. 1985, Lowel et al. 1987, Swindale 1988). A s determined by tracer injection experiments, ocular dominance columns are anatomically most prominent in layer IV and V I of the cortex.  However, physiological experiments  have established the presence of functional columns in layer II and HI of the cortex (Weisel et al. 1963). Physiological experiments have determined that cells located in the centers of ocular dominance columns are largely monocular and respond only to stimuli from one eye whereas cells bordering two adjacent ocular dominance columns are mainly binocular and respond equally well to stimuli from either eye (Shatz et al. 1978,  Shatz et al. 1984).  Interestingly, neither eye specific layers in the L G N (Shatz et al. 1983, Shatz et al. 1984) nor ocular dominance columns (Shatz et al. 1978) are initially present in these structures. Synapses  from either eye  are intermixed at first and they become  segregated by  simultaneous strengthening and elimination. Fortunately, ocular dominance formation in the cat visual cortex occurs postnatally and the cat provides a convenient model system for the study and manipulation of synaptic reorganization (LeVay et al. 1978,  Shatz et al.  1978). A newborn cat's visual cortex is immature to the point that migratory events associated with the formation of cortical layers are not completed (Shatz et al. 1986). The ventricular zone is present at birth and cells responsible for the formation of layers 2/3 are in the process of migrating to their proper locations in the cortical plate. B y this time, the connections from the L G N have grown into the subplate and await the completion of layer formation (Shatz et al. 1986, Friauf et al. 1990).  A role for the subplate in ocular  dominance column development is suggested by experiments which report that the ablation of this structure in early development prevents the formation of ocular dominance columns (Ghosh et al. 1990, Ghosh et al. 1992a, Ghosh et al. 1992b, Herrmann et al. 1994). Around 2 to 3 weeks of age, cortical layers become fully formed and the thalamic afferents from the L G N innervate layer IV of the cortex. Synaptic rearrangements do not occur prior  6  to the completion of cortical innervation by thalamic inputs from the subplate (Wiesel et al. 1963, Hubel et al. 1970).  Thalamocortical connections in the cortex are initially non-  segregated with regards to the two eyes.  Instead, these connections are arranged in a  retinotopic manner in the visual cortex of young kittens (LeVay et al. 1978, Tusa et al. 1978). The cues governing the formation of these initial L G N connections in the cortex are believed to be mainly chemo-attractant and chemo-repellent molecules (such as netrins and semaphorins) because these connections strictly avoid layers other than IV and V I in the cortex and thalamocortical connections are made in a retinotopic manner. Furthermore, the initial thalamocortical connections form independent of activity (Stryker et al. 1986, Reiter et al. 1986).  B y 3 to 4 weeks of age inputs from the two eyes become segregated and  ocular dominance columns are established (Levay et al. 1978).  1.3.2  Activity Dependent Nature of the Developing Visual system The most  striking aspect  of  ocular dominance  column development  is  its  dependence on normal visual activity. If visual activity to only one eye is disrupted in a young kitten (induced by eyelid suture or patching and referred to as monocular deprivation), the normal eye expands the cortical territory of its ocular dominance columns by invading the cortical area normally occupied by the occluded eye (Shatz et al. 1978, L e V a y et al. 1978, Wiesel et al. 1963, Hubel et al. 1970). This situation gives rise to a set of wide and narrow columns in the cortex which correspond to the open and deprived eye respectively.  From a cellular point of view, during monocular deprivation thalamic  neurons corresponding to the open eye expand their synaptic connections with cortical neurons at the expense of thalamocortical synapses belonging to the deprived eye (LeVay et al. 1978). Hence, in a monocularly deprived animal most cells in the cortex are responsive to stimuli from the open eye (Shatz et al. 1978).  The severity of this effect is dependent  on the length of the deprivation and the age of the animal (Hubel et al. 1970, Shatz et al. 1978). The period of time in which cats are susceptible to visual deprivation, called the "critical period", is considered to begin roughly 3 weeks after birth and end around 3 7  months of age (Wiesel et al. 1963, Hubel et al. 1970, Olson et al. 1980).  However, the 3  week to 3 month time period is only a rough estimate of overall plasticity in the cat visual cortex and the length of the critical period varies significantly between cortical layers (Daw et al. 1992). Thalamocortical connections found in layer IV lose their ability to reorganize at around 50-60 days of age (Mower et al. 1985).  The remaining layers II, III, V and VI  exhibit a gradual reduction in plasticity beginning at 6 weeks of age until the end of the critical period which is approximately at 15 months of age (Daw et al. 1992).  Thirty days  of age is designated as the height of the critical period based on single unit recording experiments performed in the cortex of monocularly deprived animals (Hubel et al. 1970, Movshon et al. 1977, Olson et al. 1975).  These experiments show that the most drastic  and rapid synaptic shifts in response to monocular deprivation occur in 30 day old kittens. Ocular dominance shifts following monocular deprivation are more prominent in the cortical hemisphere contralateral to the deprived eye because in the cat the majority of the ganglion cells cross  the optic chiasm and connect  (contralateral bias) (Hubel et al. 1963).  to the contralateral  hemisphere  After the critical period, the disruption of visual  activity does not induce significant shifts in ocular dominance (Daw et al.  1992).  However, rearing kittens in the dark can prolong the critical period and maintain plasticity in older animals (Cynader et al. 1976, Cynader et al. 1980, Cynader 1982).  The absence  of light seems to slow down the critical period but does not completely stop the age dependent loss of visual plasticity.  Although dark reared adult cats undergo ocular  dominance shifts in response to monocular deprivation, the extent of these shifts in dark reared cats is significantly less then those induced by monocular deprivation in normally developing young kittens (Cynader 1982, Mower et al. 1985, Mower 1991). In addition most of the cells exhibiting ocular dominance shifts reside in layer 2/3 of the cortex as opposed to layer 4.  Interestingly, ocular dominance columns do not form in dark reared  cats as long as they remain in the dark (Swindale 1981, Swindale 1988, Mower et al. 1985) although this issue is a point of contention (Stryker et al. 1986).  8  Additionally, the  visual system of a dark reared cat matures and loses its plasticity quite rapidly once it is exposed to light (Swindale 1988).  1.3.3  Competitive Nature of the Developing Visual System Based on the widening and shrinking of cortical ocular dominance columns in  response to monocular deprivation, the two eyes are proposed to compete for cortical space during normal development (Wiesel et al. 1965, Guillery et al. 1970b, Guillery Sherman et al. 1974, Sherman et al. 1976).  1972,  Support for this idea comes from reverse  suture studies in which one eye is initially occluded and then opened at a later date while the originally non-occluded eye is then deprived of input (Blakemore et al. 1974, Blakemore et al. 1978).  If the originally deprived eye is opened and allowed to receive normal visual  stimulation while the originally open eye is sutured shut, then the originally deprived eye begins to recover some of the cortical territory it has lost.  If this procedure is performed  early enough during the critical period, the ocular dominance columns that receive input from the initially deprived eye not only recover to their normal size but expand into the area of the originally non-occluded eye.  If this procedure is performed later on in the critical  period, the ocular dominance columns only recover to their normal width.  Reverse  suturing following the critical period does not yield a detectable change from the normal pattern. The presence of activity dependent competition for cortical space during visual system development is further verified by findings in the xenopus nervous system.  The  retinal ganglion cells in the frog completely cross the midline and innervate the contralateral tectum (which is the primary visual area in the frog) in a retinotopic manner (Gaze et al. 1972, Scalia et al. 1974). Since a tectum normally receives input from only one eye there is no competition between the two eyes for tectal space.  If a third eye is grafted onto a  tadpole then the axons originating from the retinal ganglion cells of that eye grow and innervate one of the tecta (Constantine-Paton et al. 1978).  Alternatively, one of the tecta  can be ablated, forcing axons from both eyes of the frog to grow into the non-lesioned tectum (Law et- al: 1980).  In tecta that are innervated by two eyes, the connections from  9  each eye segregate into eye specific columns which are reminiscent of the ocular dominance columns found in cats and primates (Constantine-Paton et al. 1978, Law et al. 1980). These tectal columns exhibit the same activity dependent characteristics as the ocular dominance columns (Reh et al. 1985). In addition, the blocking of N M D A receptors in the tectum of these frogs prevents the formation of eye specific columns (Cline et al. 1987, Cline et al. 1990). These findings support the notion that in the developing visual system activity dependent competition exists between two sets of connections.  Additionally, these  findings argue against intrinsic mechanisms being involved in the formation of ocular dominance columns because segregation does not occur in the absence  of activity.  Furthermore, a normal frog is unlikely to have evolved specialized molecular machinery for the formation of eye specific columns because each tectum of the frog normally receives input from only one eye. The model put forth to explain the formation of tectal columns in the three eyed frog is best summarized by the phrase "neurons that fire together wire together".  In other  words, since neurons from the same eye are more likely to fire at the same time, they will innervate the same target cells (Hebb 1949). The question that arises from this explanation is why form interdigitated columns as opposed to dividing the tectum into two continuous equal halves in which each half is driven by one of the eyes? The proposed explanation to this question is that columns are a trade-off between retinotopic organization where the innervation from the two eyes are interspersed and activity dependent synapse formation in which the innervation from the two eyes are completely segregated due to differential firing (Constantine-Paton 1981).  Conceivably, the actions of chemoattractants,  which are  responsible for the formation of the retinotopic map, in conjunction with activity dependent Hebbian mechanisms of synapse formation may give rise to the observed columnar organization.  1 0  1.3.4  Importance of Concurrent visual activity Another key aspect of visual system development in the cat is the concurrent firing of  neurons.  The importance of concurrent activity during visual development  is best  demonstrated by deficiencies observed in strabismic animals (Hubel et al. 1965, Shatz et al. 1977). Binocular cells, which are found at the borders of ocular dominance columns in the cat, arise presumably because the input from the two eyes do not have any sort of a competitive advantage over each other (Shatz et al. 1978). Since the inputs from both eyes activate the binocular cell with equal efficacy, the two inputs are able to innervate the same cortical cell. This situation can only arise if the inputs from the two eyes fire at the same time (Hebb 1949). In order for these two inputs to fire simultaneously the two eyes must be looking at the same point in the visual field.  Accordingly, if an animal is made  strabismic at an early age so that the two eyes look in different directions and do not share the same field of vision, then the cells in layer 4 of the visual cortex become almost entirely monocular (Hubel et al. 1965, V a n Sluyters et al. 1980).  In other words, since the inputs  from the two eyes rarely fire concurrently in a strabismic animal, the cortical cells become innervated only by one of the eyes.  This finding validates the notion of "cells that fire  together wire together" and stresses the necessity of normal visual activity for the proper development of the visual system. A peculiar aspect of strabismic animals is that they form much  wider  ocular dominance  columns  than normal  animals  (Lowel  1994)  and  furthermore, the borders of ocular dominance columns in strabismic animals are sharply defined (Shatz et al. 1977, Shatz et al. 1978, Hubel et al. 1965).  The number of  monocular cells can also be increased in the visual cortex by doing reverse suture experiments  (Blakemore et al. 1974).  In reverse suture experiments, one eye is initially  occluded and then opened at a later date while the originally non-occluded eye is then deprived of input.  Because the two eyes are active during different times, the inputs  become extensively segregated and most of the cortical cells become monocular.  11  1.3.5  Contribution of Spontaneous activity to visual development B y the time a primate is born, its visual cortex contains ocular dominance columns  (Hubel et al. 1977) and similarly, a newborn cat has its retinal connections segregated into eye specific layers in the L G N (Shatz 1983).  Since these animals do not receive visual  activity prenatally, how can segregated synaptic arrangements such as ocular dominance columns or eye specific L G N layers be present in newborn primates and felines?  Insight  into this question was found by studying the prenatal formation of eye specific layers in the L G N (Shatz 1983). The development of eye specific layers in the L G N is restricted to the prenatal period of life and monocular deprivation can not induce synaptic shifts in the L G N of postnatal kittens (Shatz 1983) (although, L G N neurons corresponding to the deprived eye can shrink considerably following monocular deprivation (Hickey et al. 1977)). Similar to the segregation of thalamocortical connections in the postnatal cat visual cortex, competitive interactions between the two eyes appear to give rise to the formation of eye specific L G N layers in the prenatal cat (Shatz 1990).  Since a prenatal kitten is unable to  see, normal visual stimulation does not play a role in this process.  However, injection of  T T X into eyes of prenatal kittens prevents the formation of L G N layering revealing the necessity for some sort of activity for the prenatal segregation of retinogeniculate synapses (Shatz et al. 1988, Sretavan et al. 1988).  Segregation of retino-geniculate inputs into eye  specific layers in the L G N is believed to be facilitated by spontaneous activity.  During  early development, spontaneous, random waves of activity traverse the retina (Meister et al. 1991, W o n g et al. 1993).  Cells which are physically close to each other on the retina are  likely to be included in the same wave of activity and fire together.  Since "cells that fire  together wire together", cells which are located close to each other on the retina will innervate proximal regions  in the L G N .  Because  these waves of  activity  occur  independently of each other in the two retinae, cells from the same eye are more likely to fire together. LGN.  Hence, inputs from the two eyes segregate into eye specific layers in the  Spontaneous activity is also believed to give rise to the formation of ocular 1 2  dominance columns in prenatal primates (Reiter et al.  1986,  Stryker et al.  1986).  However, the absence of ocular dominance column formation in dark reared cats is inconsistent with this notion. If spontaneous  activity is  capable of causing ocular  dominance column formation in the absence of visual activity, then how does depriving an animal of visual activity prolong the critical period and prevent the formation of ocular dominance columns?  The answer to this question is unknown but species differences  between felines and primates may explain this outcome.  1.4  CELLULAR  VISUAL  1.4.1  AND  MOLECULAR  MECHANISMS  INVOLVED  IN  PLASTICITY  Hebbian Mechanisms As outlined by Hebb (1949), synapses that are active become stronger whereas  synapses which are not used are eliminated.  The experimental paradigms of long term  potentiation (LTP) and long term depression (LTD) are proposed to be the cellular correlates of synapse formation. Long term potentiation is induced by applying a tetanic stimulus to the presynaptic cell (Andersen et al. 1977). This paradigm greatly increases the response of the postsynaptic cell to stimuli for a prolonged period of time lasting from hours to days.  Conversely during L T D , presentation of a low frequency stimulus greatly  decreases synaptic efficacy for a prolonged period of time (Lynch et al. 1977, Staton et al. 1989). L T P and L T D like processes are believed to occur during the normal development of the visual system and account for synaptic strengthening and elimination (Bienenstock et al. 1982, Bear et al. 1987, Kirkwood et al. 1994).  Specifically, L T P like events are  proposed to take place in active synapses resulting in the strengthening of these synapses whereas L T D like events are thought to be responsible for the weakening and eventual elimination of inactive synapses.  Fittingly, L T P can only be induced experimentally in the  visual cortex of young kittens and not in older animals (Komatsu et al. 1988, Kirkwood et  13  al. 1995).  Unfortunately, the existence or absence of L T P and L T D like processes in  normal visual development can not be demonstrated experimentally due to the shortcomings of  present  day electrophysiological  experimental paradigms in the  techniques.  development  of  Hence, the  the  visual  involvement  system  of  remains  these largely  speculative.  1.4.2  The role of N M D A receptors in visual development Despite the large amount of information available regarding the physiological and  anatomical development of the cat visual system, the molecular mechanisms responsible for this event have remained elusive.  A s an obvious starting point for identifying molecules  associated with visual plasticity, the role of different neurotransmitter systems in the postnatal development of the visual system has been studied. received much attention  during visual development  The N M D A receptors have  because glutamate  is the major  excitatory neurotransmitter in the visual system of all vertebrates (Heinschrnidt et al. 1987, Bear et al. 1990, Rauschecker et al. 1990).  In addition, the ability of N M D A channels to  detect concurrent activity between pre and postsynaptic cells is consistent with Hebbian models of synaptic development  (Nowak et al. 1984,  Mayer et al.  1985)  and the  importance of N M D A channels in long term potentiation is well established (Collinridge et al. 1983).  N M D A receptors are present throughout the visual system during the feline  critical period.  As would be expected of a molecule associated with visual plasticity,  N M D A receptors are more abundantly expressed in young kittens (Bode-Greuel et al. 1989, Tsumoto et al. 1987, Fox et al. 1989). Whereas the N M D A receptors are involved in eliciting visual responses in layers II/III of the cortex throughout life, they are functional in the deeper layers of the cortex (IV, V and VI) only in young kittens (Fox et al. 1989). This laminar difference in N M D A activity is believed to be partially responsible for the plastic differences amongst cortical laminae (i.e. layer II/III plastic longer than layer TV) (Daw et al. 1992). Recent studies have also shown the subunit composition of the N M D A receptors to change with age (Nase et al. 1999). The N M D A channel E P S C ' s shorten with  1 4  age in a manner paralleling the critical period (Carmignoto et al. 1992).  The expression of  the N R 2 A N M D A subunit in older animals is responsible for this change (Flint et al. 1997, Nase et al. 1999). Fittingly, N R 2 A expression can be delayed by the plasticity-prolonging paradigm of dark rearing (Nase et al. 1999).  Direct support for N M D A  receptor  involvement in ocular dominance column formation comes from three eyed frog studies in which the application of N M D A receptor antagonists cause the ocular dominance stripes to desegregate (Cline et al. 1987, Cline et al. 1990).  Similarly, the infusion of N M D A  antagonist into the kitten visual cortex interferes with plasticity (Bear et al. 1990, Bear et al. 1997). Unfortunately, the field of N M D A research is not free of controversy.  One of the  reasons for investigating N M D A receptor activity in the developing visual cortex is the putative role N M D A receptors are proposed to play in L T P and L T D . However, in kitten visual cortex slices L T P is found to rely on low threshold calcium channels and not on N M D A receptors (Komatsu et al. 1992,  Komatsu 1994).  The link between  NMDA  receptors and visual plasticity established by the use of pharmacological antagonists is also questionable.  Since pharmacologically blocking N M D A function in the cortex reduces  visual responses (Fox et al. 1989), the action of N M D A antagonists may simply block neural activity (i.e. action of these agents maybe analogous to T T X ) . receptors may not have a special role in plasticity.  Hence,  NMDA  In support of this possibility is the  finding that monocularly deprived kittens which have had their cortical N M D A receptors blocked exhibit an unexpected shift in ocular dominance towards the deprived eye (Bear et al. 1990).  The same result is obtained in monocularly deprived kittens by suppressing  general cortical activity by muscimol, a G A B A agonist (Reiter et al. 1988).  The similarity  between these two experiments suggests that the pharmacological N M D A blockers are essentially blocking neural activity to the cortex and the N M D A receptors are merely involved in eliciting visual responses. These contentious issues will hopefully be resolved by examining knockout animals lacking the expression of the N M D A N R 2 A subunit. The  1 5  retention of visual plasticity past the normal critical period in N R 2 A knockout animals would clearly establish a role for the N M D A receptors in this process.  1.4.3  The role of metabotropic glutamate receptors in visual development The metabotropic glutamate receptors have also been characterized during the  critical period. The level of phosphoinositide turnover induced in the cortex by glutamate stimulation correlates with the critical period and during the height of the critical period, animals are reported to respond more potently to glutamate (Dudek et al. 1989, Bear et al. 1991).  Since this effect is not observed with A M P A or N M D A ,  the increase in  phosphoinositide turnover is likely due to the activity of Pl-linked metabotropic glutamate receptors.  The expression of the phosphoinosityl-linked metabotropic glutamate receptor  mGluR5 in the visual cortex decreases with age (Reid et al. 1997, Daw et al. 1996).  More  importantly, following four weeks of age mGluR5 receptor's laminar distribution ceases to be ubiquitous in all layers of the cortex and becomes concentrated in layer I V . A n overall decline in immunostaining for mGluR5 is observed with age and adult-like distribution in the visual cortex is established around 3 months of age.  Clearly, the expression and the  distribution of the mGluR5 receptor closely correlate with the critical period.  However,  blockade of metabotropic glutamate receptors by the pharmacological agent M C P G fails to prevent ocular dominance plasticity (Hensch et al. 1996). Although this result refutes the involvement  of Pi-linked metabotropic glutamate  receptors  in  visual  plasticity,  the  effectiveness of M C P G at blocking PI turnover induced by metabotropic glutamate receptor activity is questionable (Huber et al. 1998).  Increase in c A M P levels by activation of  metabotropic glutamate receptors is also reported to correlate with the critical period (Reid et al. 1996).  However, since appropriate pharmacological agents are not available for  selectively blocking c A M P levels induced by metabotropic glutamate receptors, this finding has also remained largely correlative.  The conclusion derived from these studies is that  glutamate is able to induce secondary signaling pathways more effectively  during the  critical period. However, it is not clear if this excessive secondary signaling is associated  1 6  with ocular dominance column formation or if it plays a completely different role in development.  1.4.4  The  role  of  neuromodulator  neurotransmitters  in  visual  development In addition to glutamate, neuromodulator neurotransmitters are implicated to be involved in the postnatal development of the cat visual system. neurotransmitters can have either inhibitory or excitatory postsynaptic  cell  type  and  receptor  composition  Neuromodulatory  effects depending  (Krnjevic et  al.  on  1963).  the The  neuromodulatory neurotransmitters characterized in the visual cortex include acetylcholine, noradrenaline, serotonin and dopamine. A s in other sensory areas of the cortex, the visual cortex is innervated by neuromodulatory neurotransmitter producing cells from subcortical regions (Irle et al. 1984, Mizuno et al. 1969, Troiano et al. 1978, Watabe et al. 1982, Mulligan et al. 1988, Chazal et al. 1987, Tork et al. 1981).  Although the visual cortex  does not have any neurons that synthesize neuromdoulatory neurotransmitters, cortical neurons express receptors for neuromodulatory neurotransmitters and can readily respond to these neurotransmitters.  The neuromodulatory neurotransmitter receptors have  age  specific laminar distributions in the visual cortex and the expression and the distribution of these receptors correlates with the critical period (Stichel et al. 1987, Prusky et al. 1988, Prusky et al. 1990, L i u et al. 1994, A o k i et al. 1986, Jia et al. 1994, G u et al. 1990, Dyck et al. 1993a, Lewis et al. 1987).  Similar to the metabotropic glutamate receptors, the  neuromodulatory receptors are linked to c A M P or inositol phosphotidyl turnover secondary signaling pathways.  More importantly, the selective blockade of these receptors by  pharmacological agents disrupts visual plasticity and provides evidence for the involvement of neurotransmitters  such as acetylcholine  (Bear et  al.  1986,  G u et  al.  1993a),  noradrenaline (Bear et al. 1986, Kasamatsu et al. 1979, Kasamatsu et al. 1981, Kasamatsu et al. 1985a, Kasamatsu et al. 1985b, Shirokawa et al. 1987), and serotonin (Gu et al. 1995, Wang et al. 1997) in visual plasticity.  17  These neurotransmitters are believed to  control the excitability of cortical neurons by modulating the activity of N M D A receptors (Kojic et al. 1997,  Kirkwood et al. 1999).  The contribution of neuromodulatory  neurotransmitters to visual plasticity is likely to determine the threshold activation levels for the N M D A receptors.  1.4.5  The role of immediate early genes in visual development Immediate early gene (IEG) activity may also contribute to visual plasticity.  The  postnatal expression of immediate early genes in the kitten visual cortex is regulated in an age dependent manner (McCormack et al. 1992, Kaplan et al. 1995) and high levels of I E G expression can be maintained in older animals by dark rearing (Beaver et al. 1993).  More  importantly, immediate early gene expression is altered by monocular deprivation (Rosen et al.  1992).  Although immediate early gene expression  is modified by  monocular  deprivation in adult animals, visual activity is more effective at inducing immediate early gene expression in younger kittens and dark reared animals (Kaplan et al. 1996, Mower 1994).  Since the expression of these transcription factors are upregulated in the presence  of visual activity and decreased by deprivation paradigms (Mitchell et al. 1995), the immediate  early  genes  are proposed  to  be  involved in the  process  of  synaptic  rearrangements. According to this hypothesis, high immediate early gene expression levels are required to induce genetic processes which facilitate active neurons to expand their arbourization.  Alternatively, immediate early gene expression may be necessary  maintaining synapses.  for  Since in monocularly deprived animals immediate early gene  expression is high in areas of the cortex corresponding to the open eye, synapses are maintained in these areas.  Conversely, synapses are eliminated in areas of  cortex  corresponding to the closed eye because they express low levels of immediate early genes. Unfortunately, very little is known about the downstream genes that are directly regulated by the immediate early genes. Ergo, the actual molecular mechanisms activated by these genes during synaptic rearrangements are unknown.  Furthermore, the involvement of  immediate early genes in visual plasticity is based on various correlations between the  1 8  expression of these genes and the critical period. Experiments aimed at verifying the exact function of the IEG's during the critical period have not been performed due to the difficulties associated with modifying gene expression  in the cat.  Additionally, the  regulation of immediate early gene expression by visual activity following the critical period argues against a role for these proteins in visual plastcitiy.  1.4.6  The role of neurotrophins in visual development One of the best characterized family of molecules in the developing visual system is  the neurotrophins.  Neurotrophins have a diverse number of effects and are involved in  various stages of development.  The neurotrophins bind and act through specific tyrosine  kinase receptors. N G F binds to T r k A , B D N F and N T 4 - 5 bind to TrkB and N T 3 binds to T r k C (Lamballe et al. 1991, Berkemeier et al. 1991, Ip et al. 1992, Klein et al. 1991, Cordon-Cardo et al. 1991).  Initially, visual cortex neurons were proposed to release  neurotrophins in limiting amounts (in an activity dependent manner) and the  afferents  originating from the L G N were suggested to compete for these factors (Maffei et al. 1992, Harris et al. 1997).  According to this model, afferents  which successfully  neurotrophins are able to form synapses whereas afferents neurotrophins are eliminated.  receive  which do not take up  The finding that cortical infusion of N T 4 in the ferret is  capable of preventing L G N cell shrinkage associated with monocular deprivation supports this hypothesis (Riddle et al. 1995).  The case for the involvement of neurotrophins in  visual plasticity is also bolstered by the activity dependent regulation of neurotrophin expression in the visual cortex (Castren et al. 1992, Schoups et al. 1995, Torasdotter et al. 1996). However, recent findings have caused a substantial amount of confusion regarding the role of neurotrophins in visual development.  Since the ontogeny of TrkB receptor  expression is consistent with the critical period, B D N F is believed to play a prominent role in visual plasticity (Cabelli et al. 1994, Allendoerfer et al. 1994).  Infusion of B D N F or  blocking of the B D N F receptor (trkB) in normal kittens during the critical period interferes with ocular dominance  column formation and synaptic 1 9  rearrangements  induced  by  monocular deprivation (Cabelli et al. 1995, Cabelli et al. 1997, Gaulske et al. 1996). The initial interpretation of this finding was that B D N F prolongs the critical period by keeping the cortex more plastic. However, recent work on transgenic mice models overexpressing B D N F has found that an excess of B D N F actually results in a shorter critical period and the visual cortex appears to mature faster with more B D N F (Huang et al. 1999, et al. Hanover 1999).  These two paradoxical findings have challenged the validity of the original  neurotrophin hypothesis.  Additionally, cortical B D N F infusion in monocularly deprived  animals during the critical period results in an unexpected shift in ocular dominance towards the closed eye (Gaulske et al. 1996).  Furthermore, a factor which is involved in  visual plasticity should be located in layer 4 of the cortex since this is the input layer for the L G N . Unfortunately, B D N F expression in the rat visual cortex is determined to be low in layer 4 while being high in layer 2/3, 5 and 6 (Cabelli et al. 1994).  N G F infusion also  prevents ocular dominance column formation and causes a shift in ocular dominance towards the closed eye of monocularly deprived animals (Carmignoto et al.  1993,  Dommeniciet al. 1993, Dommenici et al. 1994, Gaulske et al. 1996, Maffei et al. 1992). In addition, unlike B D N F , N G F infusion is capable of inducing plasticity in the adult visual cortex (Gu et al. 1994).  However, since T r k A receptor expression in the visual  cortex is restricted to cholinergic axons emanating from the basal forebrain (Springer et al. 1987) and the level of T r k A expression in the cortex is low compared to TrkB (Allendorfer et al. 1994, Cabelli et al. 1994), the importance of N G F in the developing visual system is questionable.  The fact that relatively high levels of N G F infusion is required to cause an  effect on visual plasticity is also concerning.  Such high levels of N G F may result in  promiscuous binding of this neurotrophin with Trk receptors other than T r k A .  Another  problem with determining the role of neurotrophins in visual development is contradictory findings reported by different laboratories. For example, N G F is reported to be ineffective at preventing L G N neuron shrinkage in monocularly deprived ferrets (Riddle et al. 1995) whereas this neurotrophin appears to be capable of preventing L G N cell shrinkage in  20  monocularly deprived rats (Domenici et al. 1993). result of  species differences  or  alternatively,  Discrepancies such as these may be a methodological  issues  (such  as  the  concentration of the neurotrophin or differences in the application method) may have given rise to these paradoxical findings.  The mechanism of action of neurotrophins is also  unclear. Initially, neurotrophins were proposed to be activity induced signals involved in the strengthening and/or the maintenance of synapses (Maffei et al. 1992).  This proposed  role of neurotrophins is presumably accomplished by signaling through the Trk receptors and results in the maturation of synapses.  However, this role for the neurotrophins is  disputed by the finding that neurotrophins are able to modulate neural activity (Kang et al. 1995, Akaneya et al. 1997, Carmignoto et al. 1997).  Hence, the neurotrophins may  merely accentuate neural activity and they may not be involved in any downstream changes associated with the physical strengthening and maintenance of synapses.  1.4.7  Summary In summary, a large amount of work has been  performed identifying  physiological and anatomical characteristics of the developing visual cortex.  the  Although  several molecular factors which may play a role in visual development have been identified, the involvement of these factors in ocular dominance column formation and visual plasticity has not been definitively established.  In addition, the mechanisms  by which these  molecules contribute to visual plasticity remains unclear. For example, neurotransmitters, immediate early genes and the neurotrophins are believed to cause the activation of gene cascades via various secondary signaling pathways.  However, very little is known about  the actual identity of genes which are associated with these signaling pathways.  Clearly, a  substantial amount of work has to be performed in order to unravel the molecular mechanisms responsible for the postnatal development of the cat visual system.  2 1  1.5  EXPERIMENTAL  STRATEGIES  AIMED  AT  M O L E C U L A R M E C H A N I S M S I N V O L V E D IN V I S U A L  IDENTIFYING  PLASTICITY  Based on the current status of visual neuroscience research, it is likely that many of the proteins involved in visual plasticity have not been identified.  The identification of  genes which are directly involved in the synaptic rewiring of the visual system is currently the primary area of research interest in the visual neuroscience field. A popular approach to solving this problem is to investigate differential gene expression in the visual system following various developmental and deprivation paradigms.  For example, subtractive  hybridization has been performed to determine the gene differences between the adult visual cortex and a highly plastic kitten visual cortex (Prasad et al. 1994).  The logic behind this  experiment is that genes involved in visual plasticity should be expressed at higher levels during the critical period. Unfortunately, many genetic processes which are unrelated to visual plasticity also occur in the developing kitten brain.  Hence, the data obtained from  these experiments is difficult to interpret. A recent study has compared gene expression differences between dark reared and normal adult visual cortecies (Yang et al. 1999). experiment does not need to contend with the issue of age dependent  This  developmental  differences since both dark reared animals and normally reared animals are of the same age. Therefore, the only genetic differences between these animals should theoretically be those related to visual plasticity.  However, dark reared cats are not as plastic as young kittens  and there are also pathological conditions associated with long term confinement of kittens in darkness. Finally, genes regulated by neural activity have been identified by comparing normal visual cortecies with visual cortecies that have been silenced by T T X injections (Corriveau et al. 1998). Although the approach of studying differential gene expression is valid, it does suffer from certain serious limitations. The experimental methods available to study differential gene expression are not exhaustive and these methods will not yield all the molecular differences between two different conditions. Furthermore, the sensitivity of  22  these methods to detect differences in gene expression is an issue. is comprised of various cell types,  gene expression differences  Since the visual cortex occurring only in a  particular cell type may go undetected when gene expression is compared between two tissue samples. If other cell types in the tissue express the same gene(s) at unaltered levels, the change in gene expression exhibited by the single cell type will not be sufficient to alter the overall gene expression levels in the tissue. This problem is of concern anytime gene expression is compared between different tissues.  The standard way of overcoming this  problem is to use a homogeneous cell population in a tissue culture system.  Unfortunately,  this option in not applicable because visual development is studied at the system level and can not be recreated in a tissue culture paradigm. Nonetheless, advances in methodology may soon allow gene expression to be compared between single cell types isolated from visual cortex tissue via fluorescence activated cell sorting.  The comparison of gene  expression from single cell types in normal and manipulated animals should overcome the above mentioned problems. ( F A C S isolation of cells from tissue is routinely performed at present.  However, the amount of cells that are required for carrying out gene expression  studies exceeds the number of cells that can be generated by F A C S i n g .  Although this  experiment is potentially plausible at present using P C R based techniques, it is fraught with technical difficulties. Hence, it has not been successfully performed in the visual system.) Additionally, gene expression studies carried out on the visual cortex have focussed on the m R N A level and hence, translational and post-translational changes taking place during the critical period have not been characterized. This situation is likely to change in the recent future due to the advent of highly reproducible and easy to use two-dimensional protein systems.  In summary, although examining the differences in gene expression between  different paradigms is a valid approach to identifying the genes involved in visual plasticity, there are many technical issues to be considered when experiments.  performing these sorts of  Clearly, this approach is unlikely to identify every gene involved in visual  plasticity and it may not be the most practical approach to solving this problem.  23  A n alternate strategy to identifying the molecular events associated with visual plasticity is to apply the knowledge acquired from other developmental systems to the field of visual neuroscience. The members of a genetic cascade which was initially identified in the fruit fly drosophila melanogaster appears to be of prominent importance during development.  These genes are highly conserved amongst species and appear to be  ubiquitous to almost every type of developmental event. We hypothesized that these genes contribute to the postnatal development of the cat visual system and hence, the expression patterns of the cat homologues of the drosophila developmental genes were examined during the critical period and in various deprivation paradigms.  1.6 D R O S O P H I L A D E V E L O P M E N T A L G E N E S  1.6.1  Properties of the Drosophila Developmental genes Epigenesis in drosophila is controlled by a gene hierarchy which forms a regulatory  cascade that leads to the successive, step by step subdivision of the embryo into smaller units.  This genetic hierarchy is comprised of three general categories of transcription  factors known as the maternal genes, the segmentation genes and the homeotic genes. Homologues of these genes are present and highly conserved in all metazoa (Holland et al. 1996). The members of this gene hierarchy take part in a broad range of developmental events in various animals and are involved in the formation of a number of structures including the C N S . The necessity for homeotic and segmentation gene function in the development of the vertebrae brain is well established.  During embryonic development, the vertebrae  hindbrain is subdivided along the rostrocaudal axis into compartments  known  as  rhombomeres (Orr 1887, Vaage 1969). Rhombomeres restrict cell migration and prevent the intermixing of cells (Fraser et al. 1990). Since cells are subjected to only local cues and local cell to cell interactions, each rhombomere develops a unique phenotype (Fraser 1990). 24  The differential expression of homeotic genes amongst rhombomeres contributes to this phenotypic divergence (Wilkinson et al. 1989a, Murphy et al. 1989, Studer et al. 1996, Bell et al. 1999).  This function of the homeotic genes is analogous to their role in  drosophila development where they specify the identity of each body segment (Lewis 1978). In addition, the temporal and rostrocaudal expression of a particular homeotic gene in the both hindbrain and developing drosophila body is determined by its location on the chromosome (Wilkinson et al. 1989, Murphy et al. 1989, Lewis 1978,  Graham et al.  1989, Izpisua-Belmonte et al. 1991). A s in the hindbrain, the differential expression of the Otx and Emx homeotic gene families in the developing forebrain contributes to the anterior posterior patterning of this structure (Simeone et al. 1992, Boncinelli et al. 1993, Yoshida et al. 1997, Acampora et al. 1995, Acampora et al. 1996).  Similarly, the segment polarity  genes Wnt and Engrailed are necessary for the development of the midbrain (McMahon et al. 1990, McMahon et al. 1992, Wurst et al. 1994).  In addition to the genes mentioned  above, the activity of many other drosophila developmental genes are known to be required for the development of the C N S (Ericson et al. 1995, Wilkinson et al. 1989b, Sassai et al. 1995, Lindsell et al. 1995, De la Poma et al. 1997).  Clearly, the homologues of the  homeotic and other segmentation genes play an important role in the development of the vertebrae brain. The mammalian homologues of these drosophila developmental genes contribute to various facets of C N S development such as cell induction (Ericson et al. 1995), cell type specification (Bell et al. 1999), proliferation and differentiation (McMahon et al. McMahon et al. 1992).  1990,  However, the expression of many homeotic and segmentation  genes persists in the postnatal brain (Levine et al. 1993, Frantz et al. 1994, Zhang et al. 1998).  Although the function of these genes in postnatal developmental events has not  been thoroughly examined, homeotic and other segmentation genes are implicated to be involved in plasticity (Levine et al. 1993), apoptosis (Mehler et al. 1997, Patapoutian et al. 2000) regulation of Trk receptor expression (Zhang et.al. 1998), and synaptogenesis (Hall  25  et al. 2000). Based on the reported functions of these genes in brain development and their postnatal expression, we propose that the homologues of the drosophila developmental genes are likely to be involved in the postnatal development of the cat visual system.  1.6.2 Possible roles for segmentation genes in visual development Since the homologues  of the drosophila developmental genes are thoroughly  characterized in other developmental paradigms, speculations can be made regarding the function of these genes in the visual system. examined characteristic of the visual system.  Segregation and segmentation is a well As mentioned, retinal afferents in the L G N  segregate into eye specific layers and eye specific ocular dominance columns form in the cortex. In addition, histochemical markers such as zinc and cytochromeoxidase give rise to distinct staining patterns in the cortex (Dyck et al. 1993b). Interestingly, neural function is segregated along these lines. In the primate visual system, color and motion are subdivided into the magno- and parvo-cellular pathways which can be distinguished by their differing C O staining patterns (Livingstone et al. 1984, Dow 1974).  In the cat, which lacks color  vision, C O blobs are involved in spatial frequency (Shoham et al. 1997).  Although there  are no histochemical markers for orientation selectivity, this function is also arranged in a columnar manner where cells of the same orientation are in the same column (Rose et al. 1974). Additionally, receptors such as N M D A R 1 and serotonin (Trepel et al. 1998, Dyck et al. 1993c) are organized into columns in the cat visual cortex.  Clearly, functional and  physical compartmentalization of the visual system is evident. Since homeotic and segment polarity gene distribution is columnar (Akam 1987), a possible function of these genes may be to give rise to the observed organization of the visual system.  This notion is supported  by the finding that the drosophila serotonin receptor is expressed in columns and its expression is at least partly regulated by the pair rule gene fushi tarazu (Colas et al. 1995). Also, the transversely expressed stripe pattern of the pair rule genes in the drosophila embryo (Lawrence 1987) is reminiscent of the columnar arrangement of the interdigitated  26  ocular dominance columns. Furthermore, the transcription factor zif268 has functional domains in common with the drosophila Kruppel gene. The homologues of the drosophila developmental genes clearly have properties which are capable of giving rise to the arrangement observed in the visual system.  In  particular, the segment polarity genes have characteristics which may be relevant to visual cortex development.  These genes are usually components  of cell-to-cell  signaling  pathways (i.e. they are usually ligand, receptor or a messenger) (for a review see Ingham et al. 1991).  They are, for the most part, expressed in domains which can sometimes be  stripe-like or columnar.  As determined by experiments involving temperature sensitive  mutants in drosophila, segment polarity genes need to act during a defined window of time similar to a critical period to have an effect.  However, the most notable property of  segment polarity genes is their ability to either enhance each other's expression or to exert mutual repression on each other.  For example, in the case of mutual repression, a  reduction in the expression level of a segment polarity gene results in its domain of expression being invaded by the opposing segment polarity gene. These properties of the segment polarity genes can potentially account for the observed narrowing and widening of the ocular dominance columns occurring following monocular deprivation in young animals.  A role for segment polarity genes in visual development is supported by the  finding that the segment polarity genes wingless  and decapentepeligic are expressed in  adjoining domains in the developing drosophila visual system and they are necessary for proper retinotopic organization (Kaphingst et al. 1994).  Additionally, in the developing  chick tectum the segment polarity gene engrailed is expressed in a concentration gradient and is also required for proper retinotopic arrangement (Friedman et al. 1996).  Clearly,  these genes possess characteristics that are capable of facilitating synaptic competition and ocular dominance column formation.  In addition these genes are involved in visual  development in less complex animals.  Hence, these developmental genes, which were  27  initially characterized in the drosophila, have to be considered as ideal candidates for being involved in visual development and plasticity.  1.7  PROPERTIES OF BETA-CATENIN  Evidence for the involvement of beta-catenin, a mammalian homologue of the drosophila armadillo gene, in the postnatal development of the cat visual system is provided in this thesis. The beta-catenin protein functions both as a member of the cadherin-catenin cell adhesion system and as a transcription factor (Peifer et al. 1992, Peifer et al. 1994). The role of the beta-catenin protein during cellular adhesion is to anchor the intracellular domains of the cadherin molecules to the actin cytoskeleton.  Ordinarily, the beta-catenin  protein is rapidly degraded to prevent its accumulation in the cytosol (Aberle et al. 1997). Hence, beta-catenin protein expression is normally restricted to the periphery of the cell where it acts as a cell adhesion molecule (discussed further in the next section).  However,  beta-catenin protein expression is under tight post-translational regulation (Peifer et al. 1994). This post-translational regulation is influenced by factors such as Wnt-1 signaling, which is the homologue of the drosophila wingless gene, and the activity of the integrin linked kinase (Peifer et al. 1994, Novak et al. 1998).  The activity of these genes prevents  the rapid degradation of the beta-catenin protein and causes beta-catenin to accumulate in the cytosol (discussed in further detail in chapter 3).  The accumulated beta-catenin protein  is then translocated into the nucleus where it acts as a transcription factor (Schnieder et al. 1996).  In addition to its function as a cell adhesion molecule, we report that the beta-  catenin protein also functions both as a transcription factor in the postnatally developing cat visual system.  28  1.8  PROPERTIES O F CADHERINS  Due to the extensive interactions between the beta-catenin and the cadherin proteins, the expression of the cadherin proteins was also examined in the developing visual system of the cat. Beta-catenin and the cadherins constitute a prominent cell adhesion system in the body.  The importance of cell adhesion molecules in neuro-development and neural  connectivity is becoming apparent. Cell adhesion molecules such as N - C a m , the integrins and the cadherins are located at synapses (Persohn et al. 1989, Einheber et al. 1996, Martin et al. 1996, Uchida et al. 1996).  These molecules contribute to processes such as  synaptogenesis, target recognition, axon mobility and neuronal plasticity in the developing nervous system. Interestingly, many cell adhesion molecules are regulated by the activity of drosophila developmental genes. Cadherins are a family of calcium dependent cell adhesion molecules that have homophilic binding properties (Leckband et al. 2000).  Although heterophilic binding of  cadherins does occur, normally a particular type of cadherin molecule only forms adhesive links with cadherin molecules which are of the same type. Since they are cell adhesion molecules, the cadherins are exclusively localized to the periphery of cells.  Electron  microscopy studies have localized the beta-catenin and cadherin proteins to synapses in neuronal tissue where the beta-catenin cadherin cell adhesion system borders the active zone (Uchida et al. 1996). Since, cadherins bind in a homophilic manner, they are present both pre and postsynaptically.  The diversity in the types of cadherin and cadherin like  molecules in the central nervous system is enormous (Redies 2000).  In addition to the  classic cadherins, numerous protocadherins, which are larger versions of the cadherin molecules (Sano et al. 1993), and numerous cadherin like neural receptors,  which  essentially are modified cadherin molecules, have been discovered (Kohmura et al. 1998). The homophilic binding properties of the cadherins and the diversity of cadherin types expressed in the brain has given rise to the theory that the differential expression of these  29  proteins contributes to the precise wiring of the nervous system (Obst-Pernberg et al. 1999). The specific and restricted expression of certain cadherin types to particular neural tracts gives merit to this notion (Arndt et al. 1996). This hypothesis is also consistent with the proposed functions of the cadherins which include axon motility, target recognition and synaptogenesis (Matsunaga et al. 1988). These properties of the cadherin-catenin cell adhesion system are quite relevant to the postnatal development of the visual system. occurs during the critical period.  Clearly, pathfinding and synaptogenesis  Furthermore, the compartmental characteristics of the  visual system such as eye specific L G N layers, Cytochrome oxidase blobs and interblobs, and the magno-/parvo- tracts may arise due to differential synaptic adhesion.  In addition,  the disruption of cadherin activity is reported to prevent the induction of L T P which suggests a direct link between these molecules and neural plasticity (Tang et al. 1998). The remainder of this thesis will describe in detail our findings regarding the involvement  of  the  drosophila  developmental  development of the cat visual system.  30  gene  homologues  in  the  postnatal  Chapter 2 HOMEOTIC AND SEGMENT POLARITY GENE EXPRESSION IN T H E POSTNATALLY DEVELOPING CAT VISUAL CORTEX 2.0  SUMMARY In this chapter, the expression of various homologues of the homeotic and segment  polarity genes were examined in the postnatal cat visual cortex by R T - P C R . of the PBX1,  PBX2,  BMP4,  partially cloned in the cat.  BMP6, BMP-typell-receptor,  The c D N A ' s  Wnt-1 and FrZB genes were  Northern blot analysis revealed that the m R N A expression  pattern of the PBX1, PBX2, BMP4, and Wnt-1 genes are developmental^ regulated in the postnatal cat visual cortex. The expression and developmental regulation of these genes during the critical period gives merit to the hypothesis that the homologues  of the  drosophila developmental genes are involved in the postnatal development of the cat visual system.  2.1 INTRODUCTION The developmental genes initially identified in the drosophila appear to represent a general blueprint for development (Roelink 1996, Gellon et al. 1998, Christian 2000). These genes are highly conserved amongst species (Gellon et al. 1998, Holland et al. 1996a, Holland et al. 1996b) and are involved in the development of various structures including the nervous system (Reichert et al. 1999, Williams et al. 1998).  In fact, the  homologs of these genes play a role in almost every stage of neural development.  The  cellular decision for a neuronal or non-neuronal lineage is determined by the activity of the Notch gene (De la Pomma et al. 1997). Homologs of the homeotic and segment polarity genes are involved in cellular migration, proliferation, survival, apoptosis and target  3 1  M c M a h o n et al. 1990, M c M a h o n et al. 1992, Hall et al. 2000, Song et al. 1998, Friedman et al. 1996). The activity of homeotic genes contributes to the creation of different types of neurons (Valarche et al. 1993, Tissier-Seta et al. 1993, Pattyn et al. 2000). The expression of these genes persist into adulthood and their function is hot limited to early neural developmental events. For example, the expression of certain homeotic genes is restricted to dopamenergic neurons in the adult brain (Valarache et al. 1993) and Otx genes are distributed in a lamina specific manner in the adult cortex (Frantz et al. 1994).  Since the  members of the genetic hierarchy identified in drosophila development have such a broad role in C N S development, it is not unreasonable to assume that the homologs of these genes may play a role in the postnatal development of the visual cortex.  Accordingly, the  expression of several homeotic and segment polarity genes in the developing visual cortex were examined, and the expression of the P B X 1 , P B X 2 , B M P 4 , B M P 6 , B M P type II receptor, Wnt-1 and the Frz B genes was detected in this structure.  2.1.1 Possible Role of Homeotic Genes in Visual Development Columnar or segmental organization is a well characterized property of the cat visual cortex.  The zinc ion, cytochromeoxidase protein, serotonin receptor subtypes and  N M D A R 1 protein distribution in the cat visual cortex is columnar (Dyck et al. 1993a, Dyck et al. 1993b, Dyck et al. 1993c, Treppel et al. 1998).  In addition, ocular dominance  columns and orientation columns are present in the visual cortex (Shatz et al. 1978, Wiesel et al. 1963).  Furthermore, the cortex has a laminar arrangement with each lamina  containing distinct types of neurons (Gilbert et al. 1981).  Since one of the preeminent  molecular factors responsible for the organization and development of compartments (i.e. rhombomeres or drosophila body segments) is the homeotic genes (Lewis 1978, Akam 1987, Lawrence 1987, Murphy et al. 1989), we propose that these genes contribute to the laminar and the columnar arrangement of the cat visual cortex. The members of the homeotic gene family expressed in the postnatal visual cortex during the critical period are unknown.  A s a starting point, the expression of the  32  PBX  homeotic genes was examined in the visual cortex because the PBX genes act solely as cofactors and facilitate the function of other homeobox genes (van Dijk et al. 1995, Chang et al. 1995, Neuteboom et al. 1995).  Since PBX expression is required by several other  homeobox genes, PBX homeotic genes are more widely expressed than other homeotic genes.  Hence, they are more likely to be expressed in the visual cortex than most other  homeotic genes.  The main function of the P B X transcription factors is to facilitate  cooperative binding.  Cooperative binding refers to the process in which transcription  factors form either homodimers with themselves or heterodimers with other transcription factors (Wilson et al. 1995).  The newly formed complex alters the conformation of the  transcription factors and increases the binding affinity of these proteins for particular promoter and enhancer sequences.  Transcription factors that form complexes with co-  factors such as the PBX genes are not able to function efficiently in their absence (Wilson et al. 1995, Chang et al. 1995). Hence, the expression of the PBX genes in the developing visual cortex entails the presence of other homeobox genes in this structure. We have successfully developing visual cortex.  cloned the PBX1  The PBX  and PBX2 homeotic genes in the  homeobox gene family has evolved from the  drosophila extradenticle homeotic gene (Rauskolb et al. 1993) and has three identified members in mammals - PBX1, PBX2 and PBX3 (Kamps et al. 1990, Monica et al. 1991). These genes, which are largely identical to each other, exhibit significant divergence in their amino and carboxyl termini (Monica et al. 1991).  The expression of these genes has been  confirmed in the brain and the involvement of the PBX1  gene in olfactory system  development is reported (Redmond et al. 1996).  2.1.2 Possible role of Segment Polarity Genes in Visual Development Segment polarity genes have properties which may also be relevant to the development of the visual system.  Firstly, segment polarity genes are involved in cellular  signaling (Ingham et al. 1994, Roelink 1996, Moon et al. 1997, Nakayama et al. 2000). They are either secreted proteins used for signaling, receptors for the signaling protein or  33  downstream components of the signaling pathway.  Conceivably, the segment polarity  genes may facilitate cellular communication amongst neurons during visual development. Secondly, segment polarity genes are capable of repressing each other's expression and forming adjacent non-overlapping domains of expression (Roelink 1996, Jiang et al. 1995, Gilbert et al. 1994). It is possible that the mutually exclusive expression domains set up by the segment polarity genes may be the basis for the formation of ocular dominance columns in the postnatal visual cortex.  Appropriately, segment polarity genes are expressed in  adjacent domains and demarcate particular structures in the developing nervous system (Rubenstein et al. 1999, Joyner 1996, Mastick et al. 1996). Segment  polarity genes  give  rise  to  the  wingless  (Wnt-1  in  vertebrates),  decapentapelgic (BMP2 and BMP4 in vertebrates) and the hedgehog signaling pathways. The presence of any two of these pathways in the visual cortex would give merit to the hypothesis that segment polarity genes account for the competitive interactions occurring during the critical period. Accordingly, the expression of segment polarity genes belonging to the above mentioned signaling pathways was investigated in the postnatally developing visual cortex.  2.1.2.1 Members of the B M P Signaling Pathway Present in the Developing Visual Cortex. Bone Morphogenetic Proteins or the B M P ' s are a group of proteins that induce bone formation in mesenchymal tissue (Wozney et al. 1988).  Despite their name, the  B M P ' s function is not restricted to bone development and their expression is readily detected in various different organs (Scheld et al. 2000, McElwee et al. 2000, Cheifetz et al. 1999) including neurons (Tomizawa et al. 1995, Harland 2000).  The B M P ' s are  secreted proteins that belong to the TGF-|3 superfamily of growth factors (Massague 1990).  The B M P ' s have been found to perform a variety of functions in the developing  nervous system including neural differentiation, proliferation, and synaptogenesis (Withers  34  et al. 2000, Z h u et al. 1999, Hattori et al. 1999).  B M P ' s can also modulate neurotrophin  activity by controlling the expression of the Trk receptors (Zhang et al. 1998). The secreted protein BMP4  and its corresponding receptor BMP type II receptor  were cloned in the developing cat visual cortex.  BMP4 is the vertebrae homologue of the  drosophila decapenatpelgic gene (Padgett et al. 1993) and the presence of BMP4 along with its corresponding receptor, BMP type II receptor (Liu et al. 1995), verifies the presence of a segment polarity gene signaling pathway in the visual cortex during the critical period. The expression of BMP6  (another secreted protein belonging to the T G F - P growth factor  family) in the postnatal visual cortex was serendipitously discovered while attempting to clone B M P 7 which is a protein involved in synaptogenesis (Withers et al. 2000).  2.1.2.2 Members of the Wnt Signaling Pathway Present in the Developing Visual Cortex. The wingless gene and its effectors constitute another important segment polarity gene signaling pathway (Ramakrishna et al. 1993). drosophila wingless gene is the Wnt genes.  The vertebrae homologue of the  The Wnt genes are secreted glycoproteins  involved in cellular communication and act through the G-coupled Frizzled (Wodarz et al. 1998, Bhanot et al. 1996).  receptors  Roughly 11 different members of the Wnt  family have been identified in mammals(Dale et al. 1998).  The Wnt family can be divided  into two distinct categories based on their function (Torres et al. 1996); the Wnt-1 class and Wnt-5a class.  Interestingly, the members of the Wnt-5a class proteins antagonize the  effects of the Wnt-1 class proteins (Torres et al. 1996).  Similar to the B M P ' s the Wnt  proteins in the developing nervous system are reported to perform a variety of different functions including differentiation, migration, apoptosis, proliferation and axis formation (Patapoutian et al. 2000, McMahon et al. 1990, McMahon et al. 1992, Rubenstein et al. 1999, Smolich et al. 1994, Maloof et al. 1999, Shum et al. 1999, Wolda et al. 1993). The expression of the Wnt-1 secreted protein was investigated in the cat visual cortex because Wnt-1 is the homologue of the wingless gene and a possible inhibitor of  35  BMP4  signaling (Ramakrishna et al. 1993).  The expression of the Wnt-1 (or wingless)  signaling pathway in the visual cortex verifies the presence of a molecular mechanism which can potentially antagonize the BMP4 (decapentapelgic) signal. The expression of the Wnt-1 and BMP-4 signaling pathways during visual development gives merit to the notion that the interactions amongst segment polarity genes may be the molecular basis for the synaptic segregation occurring during the critical period.  In addition, the expression of  F r z - B , which is a secreted protein that prevents the Wnt proteins from binding to their receptor (Leyns et al. 1997), was detected.  Hence, the Frz-B gene is hypothesized to be  another candidate molecule that may be involved in synaptic segregation.  HYPOTHESIS:  The P B X homeotic genes, the Wnt-1, Frz-B signaling genes and the B M P - 4 , B M P type II receptor genes are involved in the postnatal development of the cat visual cortex.  OBJECTIVES:  1) T o identify homologues of the homeotic and the segment polarity gene families expressed in the developing cat visual cortex by performing R T - P C R . 2) T o get sequence information for the homeotic and segment polarity genes expressed in the cat and to develop specific c D N A probes for these genes in the cat. 3) T o illustrate that the expression of homeobox polarity  gene  homolouges  developmentally regulated.  36  in  the  cat  visual  and segment cortex  are  2.2 METHODS Animal Preparation A total of 17 postnatal cats were used for the experiments described in this chapter (N=3 for 15 day old kittens, N=5 for 30 day old kittens, N=4 for 120 day old kittens and N=5 for adult cats).  The animals were given an overdose of euthanol and then they were  briefly perfused with ice cold R N A s e free P B S solution. The brains were then removed, the visual cortecies were dissected, put into Falcon tubes and frozen in isoamyl alcohol. The tissue was kept frozen at - 8 0 ° C until processing.  Total R N A isolation Trizol solution was warmed to room temperature before use (storage is at 4 ° C ) . Ten to fourteen volumes of Trizol reagent was added to per unit weight of frozen brain tissue. Adding more than ten volumes of Trizol reagent was found to significantly decrease D N A contamination and slightly improve the 260/280 ratio of the purified total m R N A product.  The tissue was homogenized in trizol using a motorized homogenizer.  Efforts  were made to keep bubbles in the homogenate to a minimum. The homogenized samples were incubated for 5 minutes at room temperature to allow the complete dissociation of nucleoprotein complexes.  Zero point two volumes of chloroform was then added to the  Trizol reagent. The tubes were securely capped and shaken vigorously for 15 seconds and then incubated at room temperature for 3 minutes. The samples were then centrifuged at 12000 x g for 15 minutes at 4 ° C . After centrifugation the upper, colourless aqueous phase was carefully pipetted out and placed into a fresh tube without disturbing the interphase (which contains D N A ) or the lower, red phenol-chloroform layer containing organic phase. In order to avoid disturbing the interphase while collecting all the aqueous phase, the organic phase was back extracted by adding an equal volume of D E P C treated distilled water.  The solution was then mixed and centrifuged at 12000 x g for 5 minutes at 4 ° C .  The second extract was then pooled with the original R N A extraction. A n equal volume of  37  isopropanol was added to the R N A solution, mixed and incubated at room temperature for . 10 minutes. After the incubation, the sample was centrifuged at 12000 x g for 10 minutes at 4 ° C . The supernate was carefully removed without disturbing the R N A pellet which had formed at the bottom of the tube.  R N A pellet was washed twice by adding ice cold 75%  ethanol (the tube containing the pellet was completely filled with 75% ethanol), mixed by finger vortexing and spun at 7500 x g for 5 minutes at 4 ° C .  After the second wash, the  supernatant was carefully removed and the sample was briefly pulse centrifuged to bring down any ethanol which had not been removed. The residual ethanol was removed and the tube was pulse centrifuged once again to ensure that no ethanol had remained. Vacuum or air drying was found to severely decrease the solubility of the R N A pellet.  After all the  ethanol was removed, the R N A pellet was dissolved in D E P C treated distilled water. The total R N A was then subjected to D N A s e treatment in order to remove any residual D N A . The  D N A s e enzyme  was  removed by  phenol  chloroform extraction  followed  by  reprecipitation of the R N A in ethanol and reconstitution in D E P C treated distilled water.  mRNA  Isolation Zero point two mi's of Dynabeads Oligo (dT)25 was removed from the stock  solution and was put in a fresh 1.5ml R N A s e free Eppendorf tube.  The tube was then  placed in the Dynal M P C (the magnetic stand which collects the dynabeads) for 30 seconds and the clear solution was removed. The Dynabeads were then resuspend in 0.1ml of 2 X Binding Buffer (20mM T r i s - H C l (pH 7.5),  1.0M L i C l , 2 m M E D T A ) .  The volume of the  total R N A was adjusted to 75ug's in lOOpl's of D E P C treated water.  This solution was  mixed and heated at 6 5 ° C for 2 minutes in order to remove any secondary structures present in the R N A . During this step, the 2 X Binding buffer was removed from the dynabeads and replaced with 0.1 mi's of fresh binding buffer.  The 0.1ml solution of total  R N A was then added to the Dynabeads and the mixture was thoroughly mixed. This step was followed by a 5 minute hybridization of the Dyanbeads and the R N A on a rotating  3 8  roller at room temperature. The beads were then put on the Dynal M P C and the supernatant was removed. The beads containing the m R N A w a s then washed twice with 0.2ml's of washing buffer ( l O m M T r i s - H C l (pH 8.0), 0.15mM L i C l , I m M E D T A )  During each  wash the beads were thoroughly mixed and during the supernatant removal step, care was taken to remove all available solution.  The m R N A was then eluted off the beads by  applying 20ul's of Elution solution (2mM E D T A p H 8.0) directly to the beads and heating  at 6 5 ° C for 2 minutes.  The beads were then placed on the Dynal M P C and the remaining  m R N A solution was placed into a new R N A s e free Eppendorf tube and stored at - 8 0 ° C till use.  Reverse Transcription Reaction One microgram of poly ( A ) R N A was put into a fresh R N A s e free tube and the +  total volume of the solution was adjusted to 14uTs with DEPC-treated distilled water,  lul  of 8 5 u M oligo (dT) primer stock was added and the tubes were placed in 7 0 ° C for ten minutes in order to denature any R N A secondary structures.  The tubes were then chilled  on ice for ten minutes and the contents of the tubes were collected by brief centrifugation at room temperature. 2ul of 10X first strand buffer (0.5mM T r i s - H C l (pH 8.3), 0.75M K C 1 ,  0.03M M g C l ) , l u l of R N A s e Block Ribonuclease Inhibitor (40U/ul), l | i l of d N T P mix 2  (25mM of each d N T P ) and l u l of M M L V - R T (20U/ul) was added to the tube.  The  contents of the tubes were gently mixed, and then the tubes were briefly centrifuged. The reaction was incubated at room temperature for 10 minutes followed by a 60 minute incubation at 3 7 ° C .  After the reaction, the tubes were heated to 9 0 ° C for five minutes to  inactivate the reverse transcriptase. Five hundred microliters of distilled water was added to the reaction and this diluted R T product was used as template for the P C R reactions.  39  P C R Primer design Twenty-three base pairs long primers were designed using the primer program on the P C platform.  The human homologue of the gene of interest was used as the template for the  P C R primers.  The selected primers had a G C content of between 50% to 60% and did not  have more than 2 of the same nucleotide base adjacent to each other in their sequence.  It  was also ensured that the melting temperature of the 5 prime and the 3 prime primers did not differ more than 3 ° C .  Once candidate primers were designed, the sequence of each  primer was subjected to a Genbank searched.  In order to improve the specificity of the  P C R reaction, primers that had significant homology with animal genes other than the ones of interest were not used. Additionally, primers that had mismatches near the 3 prime end or significant mismatches with either the rat or the mouse sequence of the gene of interest were not used.  PCR Prior to P C R all plasticware (i.e. tips and tubes) and non-enzymatic solutions were U V treated in order to remove any contaminating D N A . of 4 0 m M Tricine-KOH (pH 9.2 at 2 5 ° C ) , 75p,g/ml, 0.2mM of each d N T P ,  A typical P C R reaction consisted  15mM K O A c , 3.5mM M g ( O A c ) , B S A 2  l u \ M of each primer,  transcription reaction and l p l of the 5 OX  l p l of the diluted reverse  Advantage c D N A  polymerase M i x from  Clonetech in a 50ul final volume. The cycling conditions consisted of an initial cycle at  9 4 ° C for 1 minute to remove the antibody from the Taq enzyme followed by 30-40 cycles of 9 4 ° C for 30 seconds for denaturing, 47 - 6 2 ° C for 45 seconds for annealing and 6 8 ° C for 1 minute 30 seconds for extension in a Perkin Elmer 9600 thermal cycler.  40  Three prime end labeled Oligonucleotide  Probes  T h r e e p r m i e e n d s of f i f t y m e r o g i lo n u c e lo t d ie w e r e a lb e e ld in a 2 5 ( 0 . 1 r e a c t o in  c o n t a n in i g 5ul of 5x Co r e a c t o in b u f f e r ( 0 7 .m M p o t a s s u im c a c o d y a lt e , 1 5 0 m MT r i s b a s e , 5mM CoCl, 0 5 .m M DTT, 2  p H 7 . 2 ) , 8 0 n go f t h ep r i m e r , l O u l of [a-P] d A T P and 2ul of 3 3  t e r m n ia l t r a n s f e r a s e e n z y m e . The  r e a c t o i n was  n ic u b a t e d at 3 7 ° C for  90 m n iu t e s .  U n n ic o r p o r a t e dn u c e lo t d ie sw e r er e m o v e d by N u c T r a pC o u lm n s . NucTrap Columns  N u c T r a pc o u lm n sm a n u f a c t u r e d by S t r a t a g e n ew e r eu s e d to r e m o v e u n n ic o r p o r a t e d r a d i o a c t i v en u c e lo t d ie sf r o mb o t hd o u b e l s t r a n d e d DNA be u s e d for p u r i f y i n gb o t ha lr g e DNA lx STE  (0.1M  and o l i g op r o b e s . H e n c e , t h e y ca  and s m a lo l i g op r o b e s .S e v e n t y f i v e m i c r o l i t e r s of  NaCl, 2 0 m M Tris-HCl ( p H 7 . 5 ) , O lm M E D T A ) was o la d e d o n t o the  c o u lm n and an e m p y t 10ml  s y r n ig e was a t t a c h e d . The STE was p a s s e d t h r o u g h the  c o u lm n by p u s h n ig d o w n on the s y r i n g e . A f t e r the c o u lm n was r e h y d r a t e d , the p r o b e was o la d e d o n t o the c o u lm n in a v o u lm e of 7 5 u l (STE  or w a t e r was u s e d to d i l u t e the p r o b e )  and p a s s e dt h r o u g hu s n i g a 10ml s y r i n g e . The e u la t e f r o m the c o u lm n was c o l e c t e d i n t o an E p p e n d o r ft u b e . A n o t h e r7 5 p l of lx STE was o la d e d and p a s s e d t h r o u g h the c o u lm n . A g a n i the e u la t e was c o le c t e d . S n ic e the p r o b e was r a d i o a c t i v e , a p p r o p r a it e p r e c a u t o in s s u c h as s h i e l d i n g w e r e in p a lc e d u r n i g the p u r i f i c a t i o n p r o c e d u r e . Also, the s y r n ig e was  n e v e r p u l e d b a c k w h e l i it was a t a c h e d to the c o u lm n b e c a u s e t h i s a c t o in w o u d l h a d s ir u p t e d the c o u lm n . Southern Blotting  The PCR p r o d u c t s w e r e e e lc t r o p h o r e s e d ,b l o t t e d o n t o n y o ln m e m b e r a n e s and  the  m e m b r a n e s w e r e UV fixed. T h e s e b l o t s w e r e t h e n put i n t o h y b r i d i z t i o n b u f f e r c o n t a n in i 6X SSC,  5X D e n h a r d s t ' r e a g e n t , 05 .%  SDS and O l O f g im / l s a m l o n s p e r m DNA ( w h c ih  41  was boiled for 5 minutes before being added to the mixture) and pre hybridized for at least one hour at 4 2 ° C in a shaking water bath. Then the prehybridization buffer was removed and replaced with hybridization buffer containing i million counts of the oligo probe/ml and the hybridization was allowed to proceed overnight at 4 2 ° C .  The following day, the blot  was washed twice in 2 X S S C , 0.1% S D S for five minutes at room temperature followed by a 4 2 ° C wash for 30 minutes at room temperature with the same wash buffer. temperature was then increased to 5 5 ° C  The  and the membrane was washed once with  0 . 5 X S S C , 0.1%SDS for thirty minutes followed by at least one wash with 0 . 2 X S S C , 0.1%SDS.  The membranes were then wrapped in saran wrap and exposed to X-ray film  overnight.  Cloning In order to polish the ends of the P C R product, 2.5U of Pfu D N A polymerase was added to lOiil's of the P C R product in a 0.2ml Eppendorf tube. was incubated at 7 2 ° C for 30 minutes.  The polishing reaction  After the polishing reaction the P C R product was  electrophoresed and the band of interest was gel purified. Alternatively, the polished P C R products were stored at - 2 0 ° C .  The polished P C R products were ligated into the p C R -  Script S K (+) vector by adding l u l of pCR-Script S K (+) vector (lOng/ixl), l u l of lOx  pCR-Script Direct Reaction Buffer, 0.5ul of l O m M r A T P , 5.5ul of polished P C R product,  1(0.1 of Srf I restriction enzyme (5U/pl) and l u l (4U/|il) of T4 D N A ligase. insert to vector ratio of 40:1 to 100:1 was used.  A n estimated  The tubes were mixed and incubated at  room temperature for 1 hour (an overnight incubation at 1 6 ° C following the room temperature incubation substantially improves cloning efficiency).  4 2  After the  ligation  reaction, the samples were heated at 6 5 ° C for 10 minutes to inactivate the ligase enzyme and the samples were stored on ice until the transformation reaction.  Sequencing Sequencing reactions  were performed using  the Amersham ThermoSequnase  radiolabeled terminator cycle sequencing kit. Briefly, four tubes containing 2uTs of d G T P termination master mix containing 7 . 5 u M d A T P , d C T P , d G T P , d T T P and 0.5ul's of either  [ a - P ] d d G T P , d d A T P , d d T T P or d d C T P (each at 1500Ci/mmol, 450|tCi/ml) were 33  prepared for each clone which was to be sequenced.  A mix containing the d G T P master  termination mix and the appropriate [oc- P] d d N T P was prepared and 2.5/tl's of this 33  mixture was added to the appropriate. The tubes were capped and kept on ice while the reaction mixture was made. A reaction mixture containing 2ul's of reaction buffer (260mM T r i s - H C l , pH9.5, 65 m M M g C l ) , 50-200ng of D N A , 2.5pmol of primer, and 8 units of 2  Thermo  Sequenase  polymerase  (4U/U.1  in  0.0006U/|ll  Thermoplasma  acidophilwn  inorganic pyrophospahatase; 50mM T r i s - H C l , pH8.0, I m M dithiothreitol ( D T T ) , O . l m M E D T A , 0.5% Tween-20, 0.5% Nonidet P-40, 50% glycerol), was prepared in a volume of 20ul's for each clone to be sequenced. Four point five microliters of the reaction mix was added to the tubes containing d d G T P , d d A T P , d d T T P and d d C T P termination mixes. Once the mixture was aliquoted into the appropriate tubes, the tubes were mixed, spun down and placed into a thermal cycler machine which was preheated to 9 0 ° C . cycles at 9 5 ° C for 30 sec, 6 0 ° C for 30 sec and 7 2 ° C for 2minutes were performed. the P C R reaction 4ui's  of  stop solution  (95%  formamide, 20mM E D T A ,  bromophenol blue, 0.05% xylene cyanol F F ) was added to each sample. placed in - 2 0 ° C until electrophoresis.  43  Thirty After 0.05%  Samples were  '  Labeling of Double stranded D N A The plasmid D N A was cut with appropriate enzymes and the resulting insert was electrophoresed on a 1% low temperature agarose gel and gel purified.  The amount of  purified insert D N A was estimated by running a portion of the purified insert on another gel. The D N A was labeled using the Prime-It II Random Primer Labeling Kit distributed by Stratagene.  Roughly 25ng's of D N A and lOul's of random 9mer oligonucleotide  primers (27 O D units/ml) was boiled together in a volume of 34jil's for 5 minutes.  The  tubes were then briefly centrifuged at room temperature to bring down the condensation. lOui's of 5x primer buffer provided by kit (containing 0.1 m M each of d A T P ,  d T T P ) and 5u.l's of [ a - P ] d C T P (3000Ci/mmol) was added to the tubes, 32  dGTP,  l u l o f Exo(-  )Klenow enzyme (5U/U.1) was added and the reaction was incubated at 3 7 ° C  for 30  minutes. Unincorporated nucleotides were removed by NucTrap Columns.  Northern blotting 3ixg's of m R N A  was  loaded onto  each  R N A gel.  The R N A gels  electrophoresed and then the R N A was transferred onto nylon memberanes.  were  The blot was  then prehybridized in a shaking water bath for 1 hour at 4 2 ° C in prehybridization solution. After the prehybridization, the prehybridization buffer was removed and hybridization buffer containing lmillion count per ml of probe was added to the blot.  The blot was  incubated overnight at 4 2 ° C . The following day, the blot was washed twice for 5 minutes at room temperature with 2 X S S C , 0.1% S D S solution. These washes were followed with 1 wash a t 4 2 ° C with 2 X S S C , 0.1%SDS for 30 minutes, 1 wash at 5 0 ° C with 0 . 5 X S S C , 0.1%SDS for 30 minutes, 2 washes at 5 5 ° C with 0 . 2 X S S C , 0.1%SDS.  If longer D N A  probes were used, the final washing temperature had to be increased to 6 0 ° C .  44  After the  washes, the blots were wrapped in saran wrap and exposed to X-ray film for 3 to 10 days in a light tight cassette which was stored in - 8 0 ° C . The P B X 1 , P B X 2 , B M P 4 and Wnt-1 probes were used on four different m R N A blots. After the northern blot analysis, the blots were stripped and reprobed with another marker. A blot was never reprobed with the same probe twice.  The B M P 6 northern blot  analysis was repeated twice whereas the P B X 1 , P B X 2 , W n t l and B M P 4 northern blot analysis was repeated three times (i.e. repeated on three different blots).  2.3 RESULTS 2.3.1  Technical Considerations One of the reasons the drosophila melanogaster is a popular animal model for  developmental genetic studies is its relative simplicity at the molecular level.  The vast  majority of the genes characterized in the drosophila are cloned in more complex animals where they perform similar functions (Gellon et al. 1998). This retention of function adds to the value of drosophila as a genetic animal model. However, hundreds of millions of years of evolution separate the drosophila from the mammals. During this period of time, new and more elaborate body types have arisen.  In order to achieve this complexity an  extensive amount of duplication and divergence has taken place at the genetic level (Holland et al. 1996a, Holland et al. 1996b, Williams et al. 1998). Accordingly, multiple variants or homologues of a single gene found in drosophila are present in mammals (Monica et al. 1991).  Some of the recently evolved homologues of the drosophila developmental genes  have distinct functions in mammals whereas other homologues are functionally redundant and safeguard against errors made during development (Depew et al. 1999, Bertuzzi et al. 1999).  From an experimental point of view, the presence of multiple gene variants adds  an increased level of complexity to identifying homologs of drosophila developmental genes expressed in the cat visual cortex during development.  4 5  Since the cat is not a popular genetic model, very little sequence information is available for this animal.  Accordingly, before the expression of the homolougs of the  drosophila developmental genes can be examined in the developing visual cortex, specific probes for these genes need to be obtained. Hence, attempts were made at partially cloning the c D N A ' s of various homeotic and segment polarity genes. P C R primers specific for individual genes were designed based on sequence information available from other species (mostly human).  R T - P C R was then performed on cat c D N A using low annealing  temperatures. The presence of multiple variants in mammals for drosophila developmental genes was addressed by trying to clone the mammalian homologue which most closely resembles its counterpart in drosophila both at the structural and functional levels.  2.3.2 Summary of the cloning strategy Primers were designed to human sequences of the gene which was to be cloned in the cat.  P C R was performed on cat c D N A using annealing temperatures ranging from  45°C - 62°C.  The P C R products were electrophoresed and blotted as described in the  methods section.  Since low stringency P C R yields a lot of non-specific products, gene  specific 50mer oligo probes were designed and these probes were used to screen the c D N A fragments generated by P C R via southern blot analysis.  The P C R products which were  identified by southern blot analysis were cloned and sequenced to confirm that the P C R fragment was actually from the gene of interest.  2.3.3  PBX1 PBX1  is a mammalian homologue of the extradenticle gene (Kamps et al. 1990).  Figure 2.1 shows the partial c D N A sequence of the cat PBX1  gene in the cat.  The  sequenced portions of the cat P B X 1 c D N A have 93% and 98% identity with the mouse and human P B X 1 c D N A ' s .  This P B X 1 c D N A fragment was obtained inadvertently while  attempting to partially clone the cat P B X 2 c D N A . This outcome is not surprising given the extensive homology between the different members of the P B X gene family. The P C R primers used to generate the cat P B X 1 c D N A fragment are  46  Cat  1 atcatccatcggaagttcagctccatccagatgcaacttaagcagagcacatgcgaggcc  60  Mouse:1181 a t c a t c c a c c g c a a g t t c a g c t c c a t c c a g a t g c a g c t g a a a c a g a g c a c a t g c g a g g c c  1240  Cat  :  :  llllllll  II I! 1111II1111 i ! I i 111 i I IE II II I I I I M I M M 1111III  61 g t c a t g a t c c t g c g c t c c c g g t t t c t g g a c g c g c g g c g g a a g a g a c g g a a t t t c a a c a a g  II I! 111111111 M l l l l l l l l  120  M i l l I I I 111111 I I I I I I II I I I l l l l l l I I I  Mouse:1241 g t c a t g a t c c t g c g c t c c c g g t t c c t g g a t g c g a g g c g g a a g a g a c g g a a t t t c a a c a a g  1300  : 121 c a a g c Ba la acsgtg a sa caotrcec t= g a a244 t g a abtiattst t(123), c t a t t c cExpect c a t c t c a=g c8e-63 a a 167 I d e n t i t i e s = 156/167 (93%) M i l l II M i l l II M M PMl ul sl l l/l lPl ll u sI I I I I I I I I I I S t rIaI nI d11 = Mouse:1301 c a a g c c a c a g a a a t t c t g a a t g a a t a t t t c t a t t c c c a t c t c a g c a a 1347 Cat  Cat  :  4 ccaggttggagccaacgtgcagtcacaggtggatacctttcgccatgttatcagccagac  I I I ! II i I I I I!! M M I I I I l l l l l l l l l l l l l l l  63  I I 1 1 I I I I I I I I M l M M III  Human:1206 c c a g g t t g g a g c c a a c g t g c a a t c a c a g g t g g a t a c c c t t c g c c a t g t t a t c a g c c a g a c 1265 Cat  :  64 aggaggatacagtgacggactcgcagccagtcagatgtacagtccgcagggcatcagtgc  lllllllllllllll  I! I I I I I I I I I I I I I M i l l 1 1 1 1 1 1 1 1 1 1 1 1 I I I I I I I I I I 1 1  Human:1266 aggaggatacagtgatggactcgcagccagtcagatgtacagtccgcagggcatcagtgc  Cat  123  1325  : 124 t a a t g g a g g t t g g c a g g a t g c c a c t a c c c c t t c a t c a g t g a c c t c c c c t a c a g a a g g c c c  183  1 1 I I I I I I M M I I I II1111 M M M I M M I I M M II M M M M M M M I M M Human:1326 t a a t g g a g g t t g g c a g g a t g c t a c t a c c c c t t c a t c a g t g a c c t c c c c t a c a g a a g g c c c 1385 B l a s t Score = 385 b i t s (194), Expect = e-105 I d e n t i t i e s = 206/210 (98%) Strand = Plus / Plus Cat : 184 t g g c a g t g t t c a c t c t g a t a c c t c c a a c t g 213  sequence of the cat PBX1 I I I 1Figure 1 1 1 1 12.1 I I I I Partial I I M M IcDNA I I I 1111III  Human:1386 t g g c a g t g t t c a c t c t g a t a c c t c c a a c t g 1415  47  gene.  CCATCGAACACTCGGACTATCGC  and A C C A G T T G G A G A C C T G A G A C A C G  and  these primers are specific to the human P B X 2 c D N A . Figure 2.2 is a northern blot autoradiograph showing the m R N A expression levels of the PBX1  gene in the postnatally developing cat visual cortex.  specific  45'mer  The cat P B X 1 m R N A  oligo  probe  G G A G G T C A C T G A C G A A G G G G T A G T G G C A T C C T G C C A A C C T C C A T T was designed based on the cat nucleotide sequence obtained from the above mentioned experiments. This 45'mer oligo was used as the probe in the Northern Blot autoradiograph shown in Figure 2.2. A s in humans (Kamps et al. 1990), 2 splice variants of the PBX1 gene named and PBX lb  were detected in the developing cat visual cortex.  The P B X l a  PBXla mRNA  expression in the visual cortex, which is high in 15 day old animals, is greatly reduced in 60 day and adult animals. A similar expression pattern is observed for P B X l b m R N A . Also, the P B X l b transcript is expressed at much lower levels than the P B X l a transcript.  2.3.4  PBX2 PBX2  is another mammalian homologue of the extradenticle gene (Monica et al.  1991). Figure 2.3 shows the partial c D N A sequence of the cat PBX2 c D N A has 90% identity with the human P B X 2 c D N A . generated  by  P C R using  the  primers  gene. The cat P B X 2  The cat P B X 2  cDNA  was  CCATCGAACACTCGGACTATCGC  and  A C C A G T T G G A G A C C T G A G A C A C G . These primers were designed based on sequence information available for the human P B X 2 gene. Figure 2.4 is a northern blot autoradiograph showing the m R N A expression levels of the PBX2 specific  gene in the postnatally developing cat visual cortex. The cat P B X 2 45'mer  oligo  GCAGAATAGGAATCTCCGTTGAGCCCGGGCATCCCCATAAACATGTCTCC  mRNA probe was  designed based on the cat nucleotide sequence obtained from the above mentioned cloning experiments. This 45'mer oligo was used as the probe in the Northern Blot autoradiograph shown in Figure 2.4. Similar to P B X 1 , P B X 2 m R N A expression in the visual cortex is  48  15 d a y  30 day  60 day  Adult  9.0 kb PBX la  6.0 kb 5.0 kb 4.0 kb 3.0 kb  PBX lb  2.0 kb  LOADING CONTROL  Figure 2.2:  PBX-1 Gene m R N A expression in the postnatally developing cat visual  cortex. The P B X - 1 gene expresses 2 splice variants in the developing cat visual cortex. The 7.5 kb transcript is referred to as P b x - l a and the 2.2 kb transcripts is named Pbx l b . The expression of both splice variants is downregulated in the adult. The bottom panel shows the m R N A gel preparation (stained with ethidium bromide) for this Northern blot  49  Cat  : 1  Human:783  agcaaacttgctcagatccgccacatctaccactcggagctggagaaatatgagcaggcg I I I I I I I I I II I I I I I I II I I I I I I II I I I I I I I I I I I I I I I I I I I I I I I I I I I I agcaaacttgcccagatccgtcacatataccactcggagctggagaagtatgagcaggca  Cat  : 61 t g t a a c g a g t t c a c a a c c c a c g t c a t g a a c c t g c t g a g g g a a c a g a g c c g c a c a c g g c c t I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I II I I I I I I I I I I I Human:843 t g t a a t g a g t t c a c g a c c c a t g t c a t g a a c c t g c t g a g g g a g c a g a g c c g c a c c a g g c c c  60 842 120 902  Cat  :121 g t g g c a c c c a a g g a g a t g g a g c g c a t g g t g a g c a t c a t c c a t c g g a a g t t c a g t g c c a t c 180 I I I II I I I I I I I I I I I II 11111111111111111111111 I I I I I I I I I I I I I I Human:903 g t g g c c c c c a a a g a g a t g g a a c g c a t g g t g a g c a t c a t c c a t c g a a a g t t c a g c g c c a t c 962 Cat  :181 c a g a t a c a g c t c a a g c a g a g c a c c t g t g a g g c t a t c a t g a t t c t g c g t t c c c g c t t c t t g 240 I I I I I I I I I I I I I I I I I I I I I II I I I I I I I I I I I I I I I I I I I I I I I III II Human:963 c a g a t g c a g c t g a a g c a g a g c a c c t g c g a g g c t g t g a t g a t c c t g c g c t c c c g t t t c c t g 1022  Cat  : 241 g a t g c c a g a c g a a a a c g c c g c a a c t t c a g c a a a c a g g c c a c t g a g g t c c t c a a t g a g t a t 300 I I I I I I I I I I I I I I II I I I I I I I I I I I I I I I I I II I I I I I I II I I I I I I II I I I I I I Human:1023 g a t g c c a g a c g a a a g c g c c g t a a c t t c a g c a a a c a g g c c a c t g a g g t c c t a a a t g a g t a t 1082  Cat  : 301 t t c t a c t c a c a t c t g a g t a a c c c a t a t c c t a g t g a a g a g g c t a a g g a g g a g c t t g c a a 358 I I I I I I I I II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I Human:1083 t t c t a c t c c c a c c t g a g t a a c c c a t a t c c t a g t g a g g a g g c c a a g g a g g a g c t t g c c a 1140  Figure 2.3 Partial c D N A sequence of the cat P B X 2 gene. B l a s t Score = 444 b i t s (224), Expect = e-122 I d e n t i t i e s = 324/358 (90%) Strand = Plus / Plus  50  15 d a y  30 day  60 d a y  Adult 4.0 k b  Figure 2.4 PBX-2 Gene mRNA expression in the postnatally developing cat visual cortex. The P B X - 2 gene m R N A is expressed at relatively high levels in the visual cortex of young kittens but the expression of this m R N A decreases in the adult visual cortex. The developmentally regulated expression of this transcript in the postnatal visual cortex is consistent with the notion that the P B X - 2 gene is involved in visual development. The bottom panel shows the m R N A gel preparation (stained with ethidium bromide) for this Northern blot analysis.  51  high in young kittens but the expression of this m R N A decreases in the adult.  A splice  variant is not observed for P B X 2 m R N A .  2.3.5  BMP 4 BMP  4 is the mammalian homologue of the drosophila decapentaplegic gene  (Padgett et al. 1993). Figure 2.5 shows the partial c D N A sequence of the BMP 4 gene in the cat. The sequenced portions of the cat B M P 4 c D N A have 89% and 93% identity with the human B M P 4 c D N A .  The B M P 4 c D N A was generated by P C R using the primers  G A C T T C G A G G C G A C A C T T C T G C A and G C C A C A A T C C A G T C A T T C C A G C C .  These  primers were designed based on sequence information available for the human BMP  4  gene. Figure 2.6 is a northern blot autoradiograph showing the m R N A expression levels of the BMP 4 gene in the postnatally developing cat visual cortex. The expression levels of the B M P 4 m R N A are fairly low (the autoradiograph shown in figure 2.6 was exposed for 10 days). The small smear observed in this autoradiograph suggests B M P 4 splice variants are expressed in the developing visual cortex.  This expression pattern was observed in  three different northern blots and is unlikely to be an artifact.  The possibility that the  m R N A on these northern blots were degraded can be ruled out because when these blot were stripped and reprobed with the P B X 1 probe sharp autoradiographic bands were obtained (as seen on figure 2.2). In the adult visual cortex, the higher part of the smear (or the larger splice variant) is missing whereas the lower part remains unaffected.  Hence, the  B M P 4 splice variants appear to be differentially regulated during the postnatal development of the cat visual cortex.  2.3.6  BMP 6 The partial B M P 6 c D N A sequence in the cat was obtained while trying to clone the  cat B M P 7 c D N A , which is known to be involved in synaptogenic events (Withers et al. 2000).  Figure 2.7 shows the partial c D N A sequence of this gene in the cat.  sequenced portions of the cat B M P 6 c D N A have 92% and 94% identity with the human  52  The  Cat  :  5 ctggtcttgagtatcctgagcgccccgccagtagggccaacaccgtgagaagcttccacc 64 I IIIIIIIIIIN IIIIIIIIIIII IIIII IIIIIIIIIIIIIIII IIIIIIIII I Human:388 c t g g t c t t g a g t a t c c t g a g c g c c c g g c c a g c c g g g c c a a c a c c g t g a g g a g c t t c c a c c 447 Cat  :65 a c g a a g a a c a t t t g g a g a a c a t c c c a g g g a c c a g c g a a a a c t c t g a t t t t c g t t t c c t c t 124 I I I 11 I I I I I I I I I I I I I I I I I I I I 11 II I I I I I I I I I I I I I I I I I I I I II I I I I I I Human:448 a c g a a g a a c a t c t g g a g a a c a t c c c a g g g a c c a g t g a a a a c t c t g c t t t t c g t t t c c t c t 507 Cat  :125 t t a a c c t c a g c a g c a t c c c a g a g a a c g a g g t g g t c t c t t c c g c a g a g c t t c g a c t c t t c c 184 I I I I I I I I I II I I I I I I I I I I I I I I I I I I I I I I I I II 111111111111111111 Human:508 t t a a c c t c a g c a g c a t c c c t g a g a a c g a g g t g a t c t c c t c t g c a g a g c t t c g g c t c t t c c 5 67  Cat  :185 gggagcag 192 llllllll Human:5 68 gggagcag 575 B l a s t Score = 278 b i t s (140), Expect = 7e-73 I d e n t i t i e s = 176/188 (93%) Strand = Plus / Plus The 1 4 bases i n t h e c a t sequence a r e agcc st  Cat  :  1 cttgacccgacgccagagggccaaacgcagccccaagcatcacccacagcgggcccggaa 60 I I I I I I I I I I I I I I I I II I I I I I II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I Human:1284 c t t g a c c c g a c g c c g g a g g g c c a a g c g t a g c c c t a a g c a t c a c t c a c a g c g g g c c a g g a a 1343 108 61 g a a g a a t a a g a a t c g t c g t c g c c a c t c g c t c t a c g t g g a c t t c a g c g a I I I I I I I I I I I I I II I I I I I I I I I I I I I I I I I I II I I I I I I I I Human:134 4 g a a g a a t a a g a a c t g c c g g c g c c a c t c g c t c t a t g t g g a c t t c a g c g a 1391  Cat  B l a s t Score = 127 b i t s (64), Expect I d e n t i t i e s = 97/108 (89%) Strand = Plus / Plus Figure  2.5  Partial  cDNA  sequence  53  of  the  le-27  c a t BMP 4  gene.  <u x  =s  OJ  OJ JS  O  2  c  S3  C  es 09 u «  J= —  "3 a. -3o B - o S 3 "~ £ -x 3 -c OJ (0 es es £X Z oj o > . 5. J OJ 09 es (/j -3 & B 2 B < a, es oo  if; ii i  1 •&  1*5  C  a 2 ^ |1 S3n  so .C oo * C >s es - S c e =  fi I * o • •Si d .2 T3 C > X J I)  0 a  rt  - .2 §  II 0  >  OJ  O  o  S  II  -£ 3 o 2 2§ o  1  « g < L > J3  s ii C  1 1eBS oj u  O. o o S.  . 5 e >. & P  a 5  a2 £s 09 OJ  ^  — o c D a T3 oo - ' ~ JS o B '3 c a >o Q u w 6 5 a c OJ 5 S 2  Q 8 8 S 00 es XI  U "8a fi '§ e 8 JB | ° 5 > 3 S o 11 11  t  j» 1 «  g c < M es Z  a2  s  V -D -  ,  y-  OJ  —a  a  oo  — > eS <u '—  -E  .0  -  5 "8  <u -a o-so.2 «j g |ej J  -  io l l I  Sfi  3 >M •o o 2 — o .2 OJ5 •§ -B p ^ § T3  oj  JS  a fi JS3  es  ^-  OJ  - S  Is  z oOS  Q O  —  54  o u  ii  gc  g § g. 5 o 'S OJ to O JS a S S -3 < z « n. 00 8eS2 b (11 is 1 1> 3 00 B C Q OJ OJ ' 00 oj c: r3 oj o S r* OJ o J OJ JS rt  0  B. ! H  0  B x D " D C  1i  OJ  1%  5  •<* Si . a, 2 3 . 2 P B > OJ K U T3 JS a es '09 c 5 09 * OJ •- o —j "E Q. -a oj 2 S 5 ^ B X 0909 093 -H ir a6 OJO. B -> •B w u E I E •C00 BM XOJ eS 3 z 3  Cat  :  1 caggacgtgtcgcgggtctccagcgcttcagattataacagcagtgagttgaaaacagcc 60 I I I I I I I I I I I I I I II I I I I I I I I I I I I I I I I I I II II I I I I I I I I I II I I I I I I I Human: 1353 c a g g a c g t g g c g c g g g t c t c c a g t g c t t c a g a t t a c a a c a g c a g t g a a t t g a a a a c a g c c 1412  Cat  :  Cat  :  Cat  :  61 t g c a g g a a a c a t g a g c t t t a c g t g a g c t t c c a a g a c c t g g g a t g g c a g g a c t g g a t c a t c 120 l l l l l l l l l l l l l l l l II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I Human: 1413 t g c a g g a a g c a t g a g c t g t a t g t g a g t t t c c a a g a c c t g g g a t g g c a g g a c t g g a t c a t t 1472 121 g c a c c c a a g g g c t a t g c t g c c a a t t a c t g t g a t g g a g g a t g c t c c t t c c c a c t c a a t g c a 180 I I I I I I I II I I I I I I I I II I I I I I I I I I II I I I I I I I I I I I I I I I I I I I I I I I I I M l Human: 1473 g c a c c c a a g g g c t a t g c t g c c a a t t a c t g t g a t g g a g a a t g c t c c t t c c c a c t c a a c g c a 1532 181 c a c a t g a a c g c c a c a a a c c a c g c g a t c g t g c 211 l l l l l l l l II II I I I I I I I I I I I I I I I Human: 1533 c a c a t g a a t g c a a c c a a c c a c g c g a t t g t g c 1563 B l a s t Score = 299 b i t s (151), Expect = 2e-79 I d e n t i t i e s = 196/211 (92%) Strand = Plus / Plus 1 ctgtgggtcgtgaccccacagcacaacatggggcttcagctgagcgtggtgactcgggat 60 l l l l l l l l I I I I I II I I I I I I I I I I I I I I I I I l l l l l l l l lllllllllllllll Mouse: 1153 c t g t g g g t g g t g a c a c c g c a g c a c a a c a t g g g g c t c c a g c t g a g t g t g g t g a c t c g g g a t 1212 Cat  61 g g a c t c a a c a t c a a c c c c c g a g c c g c a g g c c t g g t g g g c a g a g a c g g c c c t t a c g a c a a g 120 I I I I I I II I I I II I I I I I II II I I I I I I M I I I I I I I I I II I I I I I I I II I I M I 1272 Mouse: 1213 g g a c t c c a c g t c a a c c c c c g t g c g g c g g g c c t g g t g g g c a g a g a c g g c c c t t a c g a c a a g Cat  121 c a g c c c t t c a t g g t g g c c t t c t t c a a a g t g a g c g a g g t c c a c g t g c g c a c c a c c a g g t c a 180 I | I II II II I I I I I I I I II I I I I I I I II I I I I I I I I I I I I I I I I I I I I M II I I I I I II 127 3 c a g c c c t t c a t g g t g g c c t t c t t c a a g g t g a g c g a g g t c c a c g t g c g c a c c a c c a g g t c a 1332 Mouse: Cat  Cat  :  181 gcctccagtcggcggcggc 199 I I I I I I I I I I I I I I I II I I Mouse: 1333 g c c t c c a g t c g g c g g c g g c 1351 B l a s t Score = 307 b i t s (155), Expect = 8e-82 I d e n t i t i e s = 188/199 (94%) Strand = Plus / Plus Figure  2.7  Partial  cDNA  sequence  55  of  the  cat  BMP 6  gene.  and mouse B M P 6 c D N A ' s . the  This partial cat B M P 6 c D N A was generated by P C R using  primers  GTACGGCTGTCGAGCAGGAAGAG  TAGAGGACGGAGATGGCATTGAG  and  which are specific to the human B M P 7 gene.  During southern blot analysis, the 50mer B M P 7 probe used to detect B M P 7 c D N A fragments detected this B M P 6 c D N A fragment. Interestingly, the B M P 6 fragment is of the same size as the expected B M P 7 fragment. This outcome is not surprising given the extensive homology between different members of the B M P family. Interestingly, the cat B M P 7 c D N A could not be cloned using this P C R strategy. Figure 2.8 is a northern blot autoradiograph showing the m R N A expression levels of the B M P 6 gene in the postnatally developing cat visual cortex.  The B M P 6 gene  expresses two similar sized splice variants (more commonly known as doublets) in the visual cortex. The expression of the B M P 6 transcripts in the postnatal cat visual cortex appears to remain fairly constant throughout development.  2.3.7  B M P type II receptor Figure 2.9 shows the partial c D N A sequence of the cat BMP type II receptor gene.  This cat c D N A has 94% identity with the human B M P type II receptor c D N A . type  II  receptor  was  generated  TTCCACCTCCTGACACAACACCA  and  by  PCR  using  the  The B M P primers  CAGAATGAGCAAGACGGCAAGAG.  These primers were designed based on sequence information available for the human BMP type II receptor gene. Unfortunately, the B M P type II receptor m R N A could not be detected in the visual cortex by northern blot analysis. The failure to detect this m R N A by northern blot analysis suggests that the B M P type II receptor m R N A is expressed in very low levels in the developing visual cortex.  2.3.8 Wnt-1 Wnt-1 is a mammalian homologue of the drosophila wingless gene (Ramakrishna, et al. 1993). Figure 2.10 shows the partial c D N A sequence of the cat Wnt-1 gene. The cat  56  15 day  30 day  120 day  Adult 5.0 kb  i  4.0 kb 3.0 kb 2.5 kb 2.0 kb  LOADING CONTROL  Figure 2.8  B M P 6 Gene m R N A expression in the postnatally developing cat visual  cortex. Two similar sized splice variants (or doublets) are expressed by the B M P 6 gene (pointed out by the arrows) in the developing cat visual cortex.  The B M P 6 m R N A is  expressed throughout the critical period and in the adult visual cortex. The bottom panel shows the m R N A gel preparation (stained with ethidium bromide) for this Northern blot autoradiograph.  57  Cat  :  1 atggaacatgacaacattgtccgctttatagttggagatgagagagttactgcagatgga 60 I IIIIIIIIIIIIIIIIII IIIIIIIIIIIIIIIIIIIIIIIIIII IIIIIIIIIII I Human: 1162 a t g g a a c a t g a c a a c a t t g c c c g c t t t a t a g t t g g a g a t g a g a g a g t c a c t g c a g a t g g a 1221 Cat  :  Cat  :  61 c g c a t g g a a t a t t t g c t t g t g a t g g a g t a t . t a t c c c a a t g g a t c t c t c t g c a a g t a t t t g 120 I I I I I I I I I I I I II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I Human: 1222 c g c a t g g a a t a t t t g c t t g t g a t g g a g t a c t a t c c c a a t g g a t c t t t a t g c a a g t a t t t a 1281 121 a g t c t c c a t a c a a g t g a t t g g g t 143 I IIIIIII IIIIIIII IIII I Human: 1282 a g t c t c c a c a c a a g t g a c t g g g t 1304 Figure  2.9  Partial  cDNA  sequence o f gene .  the  cat  BMP  type  B l a s t Score = 220 b i t s (111), Expect = l e - 5 5 I d e n t i t i e s = 135/143 (94%) Strand = Plus / Plus  58  II  receptor  Cat  :  1 ggctgtcgggaaacggcgtttattttcgctatcacctccgccggggttacccattcggtg 60 I I I I II I I I I I I I I I I I I I I I I I I I I I I I I II I I I I I I I I I I I I I II I I I I I I I I I I Human: 2237 g g c t g t c g a g a a a c g g c g t t t a t c t t c g c t a t c a c c t c c g c c g g g g t c a c c c a t t c g g t g 2296 Cat  :  Cat  :  Cat  :  61 g c g c g c t c c t g c t c a g a g g g c t c c a t c g a g t c t t g c a c g t g c g a c t a t c g g c g g c g c g g c 120 I I I I I I I I I I I I I I I I I II I I I I I I I I II I I I I I I I I I I I I I I I I I I I I I I I I I Human: 2297 g c g c g c t c c t g c t c a g a a g g t t c c a t c g a a t c c t g c a c g t g t g a c t a c c g g c g g c g c g g c 2356 121 c c t g g g g g c c c c g a t t g g c a c t n n n n n n n c t g c a g c g a c a a c a t c g a c t t c g g c c g c c t c 180 II I I I I I I I I I I I I I II I I I IIIIIIIIIIIIIII IIIIIIIIIIIIII I Human: 23 57 c c c g g g g g c c c c g a c t g g c a c t g g g g g g g c t g c a g c g a c a a c a t t g a c t t c g g c c g c c t c 2416 181 ttcggcagggagtttgtggactccggggagaaggggcggg 220 I I I I I I I I I I I I I I II I I I I I I I I I I I I I I I I I I I I I I Human: 2417 ttcggccgggagttcgtggactccggggagaaggggcggg 2456 Figure  2.10  Partial  cDNA  sequence  of  the  c a t WNT  1  B l a s t Score = 325 b i t s (164), Expect = 4e-87 I d e n t i t i e s = 206/220 (93%) Strand = Plus / Plus  59  gene.  Wnt-1 c D N A has 93% identity with the human Wnt-1 c D N A . generated  by  PCR  using  the  primers  GGCCACTGTACGTGCAGAAGTTG.  The cat Wnt-1 c D N A was  CCTCTTCGGCAAGATCGTCAACC  and  These primers were designed based on sequence  information available for the human Wnt-1 gene. Figure 2.11 is a northern blot autoradiograph showing the m R N A expression levels of the Wnt-1 gene during postnatal visual cortex development. Wnt-1 m R N A expression is low at 15 days of age, significantly increased at 30 days and 120 days of age and is reduced again in the adult visual cortex. The possibility that the 15 day m R N A is degraded on this blot was ruled out by reprobing the same blot with the P B X 2 probe.  A s expected a  strong signal was obtained with the P B X 2 probe for the lane containing thel5 day old visual cortex m R N A . This finding confirms that the observed autoradiographic pattern for the Wnt-1 northern blot analysis (which was repeated 3 times) is due to differential m R N A regulation and not to m R N A degredation. This m R N A expression pattern correlates with the critical period and suggests that the Wnt-1 protein is involved in the  postnatal  development of the visual cortex.  2.3.9  Frz-B The Frz-B protein is a secreted form of the frizzled receptor (Wnt-1 binds and acts  through the frizzled receptor)(Leyns et al. 1997).  Similar to the truncated forms of the  neurotrophin Trk receptors, the secreted form of the frizzled receptor has the ligand binding domain but lacks the intracellular protein domains.  The Frz-B protein antagonizes Wnt  signaling by binding to the secreted Wnt glycoproteins and preventing them from binding to the frizzled receptors. Figure 2.12 shows the partial c D N A sequence of the F r z - B gene in the cat. The cat Frz-B c D N A has 93% identity with the Bull Wnt-1 c D N A . The cat Frz-B c D N A was generated by P C R using the primers C T G C A C C A T T G A C T T C C A G C A C G and TCGAGTTCCTGCCAGACTTCTGA.  Unfortunately, the expression of the F r z - B m R N A  could not be detected using northern blot analysis.  60  15 day  30 day  60 day  Adult 6.0 kb 5.0 kb 4.0 kb 3.0 kb 2.5 kb 2.0 kb 1.5 kb  LOADING CONTROL  Figure 2.11 WNT-1 Gene m R N A expression in the postnatally developing cat visual cortex. A t 15 days of age W N T - 1 m R N A expression is not detected i n the visual cortex. W N T - 1 m R N A expression is upregulated in the 30 day and the 120 day old kitten visual cortex. In the adult visual cortex the expression of the W N T - 1 m R N A is reduced. This developmental m R N A expression profile of the W N T - 1 gene suggests a role for this molecule during the critical period. The bottom panel shows the m R N A gel preparation (stained with ethidium bromide) for this Northern blot analysis.  61  Cat  :  1  Human: 536 Cat  :  61  Human: 596 Cat  : 121  Human: 656 Cat  : 181  Human: 716 Cat  : 241  Human: 776 Cat  g c c c a t c t g c a c c a t t g a c t t c c a g c a c g a g c c c a t c a a g c c c t g c a a g t c t g t g t g c g a 60 I I I I I II II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I g c c c a t c t g c a c c a t t g a c t t c c a g c a c g a g c c c a t c a a g c c c t g c a a g t c t g t g t g c g a 595 g c g g g c c c g g c a g g g c t g c g a g c c c a t c c t c a t c a a g t a c c g c c a c t c g t g g c c c g a g a g 120 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I II I I I I II II g c g g g c c c g g c a g g g c t g t g a g c c c a t c c t c a t c a a g t a c c g c c a c t c g t g g c c g g a a a g 655 t c t g g c c a g c g a g g a g c t g c c t g t t t a t g a c c g t g g g c t g t g c a t c t c t c c c g a g g c c a t 180 I I I I I I I I I I I I I I I I I I I II I I I I I I I I II I I I II II I I I I I I I I I I I I I I c c t g g c c t g c g a g g a g c t g c c a g t a t a t g a c c g c g g c g t g t g c a t c t c t c c g g a g g c c a t 715 cgttaccgcggacggagcggattttcctatggattctagtaatggaaactgtagagggga III II II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I cgtcactgccgacggagccgattttcctatggattccagtaatggaaactgtagaggagc  775  agccagtgaacgctgcaagtgtaagcctattagagctacacagaagacctatttccgaaa 3 00 I I I I I I I I I I I I I I I I I I I I I II I I I I I I I I I I I I I I I I I I I I I I I I I I I I II a a g c a g t g a a c g c t g c a a a t g t a a a c c a g t c a g a g c t a c a c a g a a g a c c t a t t t c c g a a a 835  : 301  Human: 836 Cat  240  caattacaactatgtcattcgggcaaaagttaaagaagtaaagaccaagagccatgatgt 360 I I I I I I I I I I I I I I II I I I I I I I I I I I I I I I I I I II I I II II I I I I I I I I I I I I I I c a a t t a c a a c t a t g t c a t t c g g g c t a a a g t t a a a g a a a t a a a g a c c a a g t g t c a t g a t g t 895  : 361  g a c t g t a g t a g t t g a g g t g a a g g a g a t t c t a a a a g c t t c t c t g g t a a a c a t t c c a a g g g a 420 I IIII IIIIII IIIIIIIIIIIIIII IIII IIIIIIIIIIIIIIIIIIIIIIIII I Human: 896- g a c t g c a g t a g t g g a g g t g a a g g a g a t t t t a a a g g c t t c t c t g g t a a a c a t t c c a a g g g a 955 : 421 g a a c g t t a a c c t t t a c a c c a g c t c t g g c t g c c t g t g t c c t c c a c t t a a t g t t a a t g a g g a 480 I II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I Human: 956 a a c t g t g a a c c t t t a t a c c a g c t c t g g c t g c c t g t g t c c t c c a c t t a a c g t t a a t g a g g a 1015  Cat  481 g t a t a t c a t c a t g g g c t a t g a a g a t g a g g a a c g c t c c a g a t t a c t g t t g g t g g a a g g t t c 540 M M II II II II I I I II II I I I I I I II II I I I I I I I I I I I I I II II I I II I I I II Human: 1016 g t a t c t c a t c a t g g g c t a c g a a g a t g a a g a g c g c t c c a g a t t a c t g t t g g t a g a a g g t t c 107 5  Cat  541 t a t t g c t g a g a a a t g g a a g g a t c g a c t t g g t a a a a a a g t t a a g c g c t g g g a t a t g a a g c t 600 II II II I I II II II I I I I II II I I I I I II II I I I I I I I I I I I I I I II I I I II I I II I I I Human: 1076 t a t t g c t g a g a a a t g g a a g g a t c g a c t t g g t a a a a a a g t t a a g c g g t g g g a t a t g a a g c t 1135  Cat  Cat  :  601 c c g c c a t c t t g g a c t c a a t a a a a g c g a t t c a a g c c a t a g t g a t t c c a c t c a g a g t 655 III I I I I I I I I I II I I I I III I I I I I II II I I I I I I II II II I I I I II I I Human: 1136 c c g t c a t c t t g g a c t g a a t a c a a g t g a t t c t a g c c a t a g t g a t t c c a c t c a g a g t 1190 Figure  2.12  Partial  cDNA  sequence  of  the  cat  FrzB  B l a s t Score = 902 b i t s (455), Expect = 0.0 I d e n t i t i e s = 605/655 (92%) Strand = Plus / Plus 62  gene.  2.3.10 Genes which could not be cloned using the R T - P C R approach Unsuccessful  efforts  were  made  at cloning  engrailed-1,  engrailed-2,  sonic  hedgehog, desert hedgehog, Indian hedgehog, chordin and noggin genes. The engrailed and hedgehog genes are extensively characterized segment polarity genes that are known to be involved in neural development (Ericson et al. 1995, Zhu et al. 1999, Friedman et al. 1996).  Chordin and noggin are secreted proteins (similar to Frz-B) that inhibit B M P  activity (Bachiller et al. 2000).  A s mentioned before, we were also unable to clone the  B M P 7 (a protein involved in synaptogenesis) c D N A in the cat. The failure to partially clone these genes does not imply that they are not expressed in the cat visual cortex during the critical period. Technical problems associated with R T P C R may have prevented us from cloning these transcripts. The presence of only a single nucleotide mismatch between the P C R primer and the c D N A template can significantly hinder the ability of the P C R primer to prime D N A elongation.  Unfortunately, the P C R  primers used in these experiments had to be designed based on sequence information available from other species and hence, nucleotide mismatches between the targeted cat c D N A templates and the P C R primers may have existed.  In addition, numerous factors  such as annealing temperature, the length of the elongation, the number of cycles, the concentration of the primers, nucleotides, template, or magnesium can effect the efficiency of a P C R reaction. Although the annealing temperature, the magnesium concentration and the number of P C R cycles were varied while attempting to clone the above mentioned c D N A ' s , it is possible that the P C R conditions used for the amplification of these transcripts may not have been optimal.  63  2.4 DISCUSSION T o briefly summarize, the m R N A ' s of the PBX1, PBX2, BMP4,  and Wnt-1 genes  are expressed in the visual cortex during the critical period in an age dependent manner. The m R N A expression of BMP type II receptor and the Frz-B genes are also detected in the developing visual cortex by R T - P C R .  The developmentally regulated expression of these  genes in the visual cortex gives merit to our original hypothesis that the homologues of the drosophila developmental genes are involved in the postnatal development of the cat visual system.  Considering the number and the diversity of developmental events homeotic and  segment polarity genes are associated with, the presence of these genes in the postnatal development of the visual cortex is not surprising. Furthermore, since the function of the homeotic and segment polarity genes are extensively characterized in other developmental models, the possible role of these genes in visual development can be speculated.  2.4.1 The cloning strategy Since many of the genes postulated to be present in the visual cortex were successfully cloned, the cloning strategy described in this thesis appears to be an efficient way of identifying homologues of the drosophila developmental genes expressed in the cat. Gene specific P C R primers were used to partially clone these developmental genes in the cat instead of degenerate P C R primers because degenerate P C R primers have a tendency to be biased for and/or against certain sequences.  Hence, it is naive to assume that all  members of a particular gene family can be detected by using degenerate primers.  More  importantly, degenerate primers need to be designed to highly conserved regions of gene families so that common sequences amongst different members of the gene family are detected and amplified. Since only short, highly conserved c D N A sequences common to various members of the same gene family can be amplified using this approach, the sequences generated by degenerate primer P C R can not be used as gene specific probes for determining the expression pattern of an individual gene (i.e. they will cross react with  64  several members of the same gene family). Eventually, gene specific P C R primers need to be designed in order to get sequence information which is unique to the gene o f interest. A s a side note, due to the extensive homology present amongst the homologues of different drosophila developmental genes, low stringency P C R utilizing primers designed to a particular gene resulted in the amplification of other genes in the same family (i.e. P B X 1 c D N A was obtained using P C R primers for P B X 2 ) .  In other words, like degenerate  primers, low stringency P C R can facilitate the cloning of multiple members of the same gene family. Furthermore, the aim of this chapter is not to characterize a single gene family but  rather, to  establish  the  expression  of  various  different  developmental gene homologues during the critical period.  types  of  drosophila  Clearly, the use of specific  primers in combination with low stringency P C R is more suited for this purpose.  2.4.2  The expression  of P B X Homeotic genes in the developing  visual  cortex The expression of the PBX homeotic genes in the developing visual cortex strongly suggests the presence of other homeobox genes in this structure because the PBX  homeotic  genes act solely as cofactors and facilitate the activity of other homeobox genes (Rauskolb et al. 1993). Transcription factors containing particular amino acid motifs form complexes with the PBX genes (Neuteboom et al. 1995, Chang et al. 1995).  Homeobox genes need  to form these complexes in order to efficiently bind certain enhancer sequences and regulate transcription. Since the PBX genes do not induce gene expression by themselves, other homeobox genes which require the PBX genes as cofactors are likely to be expressed in the developing visual cortex. The higher level of P B X 1 and P B X 2 m R N A expression in the visual cortex of young cats suggests the involvement of these genes in visual development.  T w o splice  variants of P B X 1 are found in the cat visual cortex. This finding is identical to the reported expression of the P B X 1 transcript in humans (Kamps et al. 1990) but it is different from  65  the reported expression of a single P B X 1 m R N A species in the developing olfactory bulb of the rat (Redmond et al. 1996).  Species differences between rats and cats may be  responsible for the observed splicing pattern difference.  One purpose for having splice  variants is to transport and localize transcripts to different cellular compartments.  Since  P B X 1 strictly acts as a transcription factor, the splice variants are unlikely to be transported to any cellular compartment other than the nucleus. A more likely possibility is that the P B X 1 splice variants encode proteins which have different affinities for binding different transcription factors (Monica et al. 1991).  For example, a particular homeobox gene may  only be able to form a heterodimer complex with the P B X 1 protein encoded by the smaller splice variant. It is feasible that the expression pattern of the P B X 1 m R N A splice variants in the visual cortex reflects the expression profile of the homeobox genes they interact with during development.  Homeobox genes that form transcriptional complexes with P B X 1  maybe highly expressed in the young visual cortex and the purpose for having high P B X 1 expression levels during this period maybe to provide these homeobox genes with the necessary cofactor.  Accordingly, the developmental regulation of P B X 1 and its splice  variants suggests that the homeobox genes which interact with P B X 1 are likely to be developmentally regulated in the visual cortex during the critical period. Finally, it is also possible that each splice variant is expressed by a particular cell type. P B X 2 m R N A expression pattern in the developing visual cortex is almost identical to that of P B X 1 . function of the  Since  PBX1 and PBX2 have an extensive amount of homology, the  PBX2 is speculated to be very similar to that of PBX1. The difference between  PBX1 and PBX2 genes may be limited to their ability to form complexes with different  transcription factors.  The possibility that the two PBX  genes are performing the same  function during the critical period also exists. In mammalian development redundancy of gene function is common amongst different members of a gene family.  For example,  knockout mice lacking a single member of the Dlx homeobox gene family (Dlx homeobox genes are expressed in the brain and contribute to forebrain development) do not appear to  66  be phenotypically different than normal mice (Depew et al. 1999).  Mice begin to show  noticeable phenotypic abnormalities following the knockout of at least two members of the Dlx gene family. These findings suggest that different members of the Dlx homeobox gene family are performing the same function during brain development. homology and similar m R N A expression profiles of the PBX1  Given the extensive  and PBX2  genes, this  functional redundancy may have extended into the PBX homeobox gene family. The role of P B X as a cofactor for homeobox genes containing particular amino acid motifs is well established (Neuteboom et al. 1995, Chang et al. 1995).  A s mentioned, the  presence of the PBX1 and PBX2 genes entails the expression of other homeobox genes in the developing visual cortex.  The obvious question is how are the homeobox genes  contributing to the development  of the postnatal visual cortex?  The  developmental  processes requiring segmentation have been completed well before birth, neurons have already attained their cell fates and cell division does not occur in the postnatal brain. Clearly, these well characterized functions of homeobox genes are unlikely to be taking place  during the  critical period.  However,  characteristic of the visual cortex.  compartmentalization  Columnar organization of eye  is  an  specific  inherent inputs,  orientation, neurotransmitter receptors and blob, interblob arrangement of cytochrome oxidase in the visual cortex is well documented (Hubel et al. 1963, Shatz et al. 1977, Dyck et al. 1993c, Murphy et al. 1995).  Given that the homeobox genes are involved in the  development of various different compartmental structures (i.e. rhombomeres, drosophila body segments, spinal cord), the activity of these genes may contribute to the columnar organization of the cat visual cortex.  Another function of the homeobox genes in the  central nervous system is to regulate the expression of cell adhesion molecules (Cillo et al. 1996, Wang et al. 1996, Edelman et al. 1995, Valarche et al. 1993, Lincecum et al. 1998, Packer et al. 1997, Tissier-Seta et al. 1993). B y differentially regulating the activity of cell adhesion molecules,  homeobox  genes are able to control the formation of  connections in the spinal cord (Lumsden et al. 1995, Tosney et al. 1995).  67  synaptic  It is possible  that the homeobox genes are performing a similar function in the postnatal visual cortex. Support for this hypothesis is provided by the correlation that exists between the expression of cell adhesion molecules and the critical period which will be discussed later on in this thesis (Schoop et al. 1997, Corriveau et al. 1998)  2.4.3 Segment polarity gene expression in the developing visual cortex Several members of the BMP gene family are also expressed and developmentally regulated in the visual cortex during the critical period. Splice variants which are detected for BMP6 in the visual cortex have not been observed in previous studies examining brains from other species (Tomizawa et al. 1995).  Hence, these splice variants may be novel to  the cat or alternatively, the splice variants may be novel members of the BMP family which have not been characterized.  The possible reasons for having BMP  identical to those discussed for the PBX1  splice variants is  gene (i.e. produced by different cell types,  transported to different cellular compartments or functionally different - bind different receptors).  A s mentioned, the B M P ' s are secreted proteins and the detection of the B M P  type II receptor confirms the presence of the necessary machinery for B M P signaling in the visual cortex.  The best characterized function of the B M P ' s is as signaling molecules  involved in cell to cell communication (Nakayama et al. 2000).  Signaling molecules are  believed to contribute extensively to activity dependent synaptic rearrangements occurring in the developing nervous system (Goda 1994, Fitzsimonds et al. 1998, Haydon et al. 1994, Davis et al. 1998). However, this conjecture is largely hypothetical with regards to the critical period because the identity of these signaling molecules in the developing cat visual system (with the exception of the neurotrophins) is unknown. regulated m R N A expression of the BMP4  The developmentally  signaling molecules during the critical period  suggests a role for this molecules in visual development. Out of all the transcripts examined in this chapter, Wnt-1 m R N A expression parallels the critical period most accurately. Unlike the other transcripts examined where m R N A expression decreases with age, Wnt-1 expression is low in the visual cortex of  68  kittens which are 2 weeks of age. A s discussed in the introduction, during the first 2 to 3 weeks after birth, neurons from the ventricular zone migrate into the cortical plate in order to complete the formation of the upper layers (Shatz et al. 1986). Following the completion of layer formation the thalamic connections in the subplate grow into layer I V of the cortex. Since segregation of thalamic inputs or synaptic modification does not occur before the L G N innervates the cortex, the critical period in the cat begins two to three weeks after birth. Hence, the absence of Wnt-1 expression in the visual cortex of 15 day old animals is consistent with the idea that Wnt-1 is involved in visual plasticity.  The decrease in the  expression of Wnt-1 m R N A in the adult visual cortex also correlates with the critical period. In addition to this correlation, the fact that Wnt-1 is a secreted signaling molecule capable of inhibiting the expression of the B M P 4 gene (Baker et al. 1999) makes Wnt-1 an intriguing candidate molecule in visual plasticity research. The interactions between the B M P 4 and Wnt-1 signaling pathways have the potential to account for the formation of ocular dominance columns in the developing visual cortex.  As mentioned, BMP4  is the homologue of the drosophila decapenteplegic gene  (Padgett et al. 1993) whereas Wnt-1 is the homologue of the drosophila wingless gene (Ramakrishna, et al. 1993). The DPP and the wingless genes and their homologues (i.e. BMP's and Wnt's) are capable of forming adjacent non-overlapping domains by inhibiting each others expression (Jiang et al. 1995, Baker et al. 1999, Hirsinger et al. 1994). these expression domains are in the form of interdigitated columns. domain of either one of the Wnt-1 or BMP4  Often,  If the expression  genes is downregulated via experimental  manipulation, its adjacent neighbour invades the expression domain of the downregulated gene (Penton et al. 1996, Jiang et al. 1995, Jiang et al. 1996). BMP4  These characteristics of the  and Wnt-1 genes are somewhat reminiscent of the behaviour of ocular dominance  columns following monocular deprivation. Furthermore members of the Wnt and BMP families  are implicated to be  associated  with  synaptic  modifications  and neural  communication (Withers et al. 2000, Hall et al. 2000). Moreover, both of these molecules  69  are developmentally regulated in the cat visual cortex during the critical period.  If the  release of these secreted cell signaling molecules in the developing visual cortex is regulated by neural activity, the antagonistic nature of Wnt-1 and BMP4 can theoretically give rise to ocular dominance columns and account for the anatomical and physiological  changes  associated with monocular deprivation. Additionally, the expression of the Frz-B gene in the visual cortex, which is an inhibitor of Wnt-1 activity, provides an alternative system by which ocular dominance columns can form during visual development.  The expression of  Wnt-5a type signaling genes, which antagonize Wnt-1 function (Torres et al. 1996), was not examined in this thesis but these molecules may also contribute to the formation of ocular dominance columns.  2.4.4 Selection of the Beta-Catenin Protein For Further Characterization In The Postnatal Development O f The Cat Visual System. Homologues of several homeotic and segment polarity genes were identified and shown to be developmentally regulated in the visual cortex during the critical period. Although the large body of information available about the function of these genes from other systems allows speculations to be made, the role of these genes during visual development can not be determined based solely on northern blot analysis.  The young  postnatal brain undergoes a plethora of processes which are associated with differentiation and maturation. Since these developmental processes occur at the same time as the critical period, it is difficult to assess if the homeotic and the segment polarity genes are involved in general brain development  or if they  contribute to  visual plasticity.  Further  characterization is required in order to establish a role for these genes in postnatal visual development. In order to establish the involvement of these genes in the development of the cat visual system more concretely, we decided to focus on and extensively characterize the behaviour of one of these genes in the postnatally developing cat visual cortex and lateral geniculate nucleus.  The initial strategy  for this thesis was  70  to clone  a group of  developmental genes, determine their expression profiles and then examine genes that have expression patterns paralleling the critical period.  The northern blot analysis results  reported in this chapter provide several lines of promising data and have led to some transcripts in the visual cortex (large amounts of m R N A is needed to detected these genes via northern blots) is the likely cause of this outcome.  In addition, appropriate antibodies  for the B M P 4 and W n t l proteins are not available (especially antibodies against cat proteins).  Hence, these genes could not be further characterized in the postnatally  developing visual cortex.  Accordingly, the role of these genes in visual development  remains unverified. Since the expression of the Wnt-1 m R N A correlates well with the developmental events occurring in the critical period, a reasonable approach seemed to be the investigation of downstream genes in the Wnt-1 signaling pathway. The best characterized downstream effector of Wnt-1 signaling is the beta-catenin gene, the homologue of the drosophila armadillo segment polarity gene (Moon et al. 1997). Like many other developmental genes identified in the drosophila, beta-catenin is reported to take part in a wide range of developmental events and has been intensively studied in mammals.  Because of the  attention this gene has received, molecular tools such as specific antibodies are available for the beta-catenin protein. A s reported in the next chapter, the information gathered from the examination of this protein provides further support to the hypothesis that the homologues of the drosophila developmental genes are involved in the postnatal development of the cat visual system.  7 1  Chapter 3 BETA-CATENIN PROTEIN EXPRESSION IN T H E POSTNATALLY DEVELOPING CAT VISUAL SYSTEM 3.0  SUMMARY To  further verify  our hypothesis  that the  homologues  of  the  drosophila  developmental genes are involved in the postnatal development of the cat visual system, the expression pattern of the beta-catenin protein, which is the homologue of the drosophila armadillo gene, was examined in the cortex and L G N of kittens during the critical period. Beta-catenin protein appears to be expressed at fairly constant levels in the postnatally developing cat L G N and the visual cortex as determined by western blot analysis. Immunohistochemical analysis reveals that the beta-catenin protein is expressed in the neuropil of the visual cortex and the geniculate in young animals. Although this staining pattern is  retained in the cortex  throughout life, the beta-catenin  nuclearized in L G N cells between 50-60 days of age.  protein  becomes  Based on this finding nuclear beta-  catenin protein is proposed to end the critical period for thalamocortical plasticity by altering gene expression in L G N neurons.  3.1 INTRODUCTION If the homologues of the drosophila developmental genes are involved in the postnatal development of the cat visual system then their tissue distribution and temporal expression patterns should correlate with the critical period.  Hence, we decided to  extensively characterize the protein expression of one of the homologues of the drosophila  72  developmental genes in the visual system of normally developing and deprived kittens.  In  the last chapter we reported that the temporal expression of the W n t - i m R N A in the visual cortex closely correlates with the critical period.  Since the tissue distribution of  Wnt-1  could not be determined in the cat visual cortex by standard histological techniques, the temporal expression and the tissue distribution of the beta-catenin gene was examined in the postnatally developing cat visual system.  Beta-catenin is a homologue of the drosophila  armadillo segment polarity gene and a downstream component of the Wnt-1 signaling pathway (Peifer et al. 1992, Brown et al. 1998). Unlike the wingless and decapentapelgic segment polarity genes, armadillo is not a secreted protein (Riggleman et al. 1989).  It is a protein which can act both as a  transcription factor and as a cell adhesion molecule (Peifer et al. 1992, van de Wetering et al. 1997). These functions of armadillo are conserved in the mammalian beta-catenin gene. The role of beta-catenin as a cell adhesion molecule is well established (McCrea et al. 1991, Bullions et al. 1998). Beta-catenin is found intracellularly and acts to anchor the calcium dependent cell adhesion molecules known as the cadherins to the actin cytoskeleton.  Beta-  catenin mediated attachment of cadherins to the actin cytoskeleton is required for the formation of stable adhesive links (Kitner et al. 1992, Nagafuchi et al. 1988, McCrea et al. 1991).  Interestingly, electron microscopy performed on rat brain tissue sections has  localized the beta-catenin and cadherin complexes to the synapse where they border the active zone (Uchida et al. 1996). The regulation of the beta-catenin gene is mainly at the post-translational level (See figure 3.1). Cells have one pool of beta-catenin that is attached to the cytoskeleton via the cadherins and a free pool of beta-catenin (Papkoff 1997).  Normally, cells produce a  continuous amount of beta-catenin protein which is rapidly degraded (Pai et al.  1997,  Aberle et al. 1997). The main protein responsible for beta-catenin degradation is the serine threonine kinase activity of glycogen synthase kinase 3 (GSK3) which is the mammalian homologue of the drosophila shaggy/zest white 3 gene (Aberle et al. 1997). G S K - 3 marks  73  In the absence of Wnt-1 Signaling  In the presence of Wnt-1 Signaling  Wnt-1  I  Phosphorylation  Inhibition  (GSK^)  I  BETA CATENIN,  (GSK^)  Accumulation  Ubiquitinization  Complex Formation Nuclearization  ([PROTEOSOME)  Transcription Complex  oo ° o 0o ° 0  0  Gene Regulation  Figure 3.1 Post-translational Regulation of Beta-Catenin Protein Expression.  74  beta-catenin for ubiqutinization and eventual degradation by phosophorylating it (Aberle et al. 1997).  Adenomatous polyposis coli ( A P C ) and axin also play a role in beta-catenin  degradation (Rubenfield et al. 1996, Hart et al. 1998).  Factors such as Wnt-1 signaling,  I L K (integrin linked kinase) and the activity of the dishevelled gene, which is regulated by the Notch signaling pathway, inhibit G S K - 3 activity and prevent beta-catenin degradation (Cook et al. 1996,  Yanagawa et al. 1995, Novak et al. 1998, Axelrod et al. 1996).  Inhibition of G S K - 3 leads to the increase in the free pool of beta-catenin in the cytoplasm which is then transported into the nucleus where it acts as a transcription factor (Cook et al. 1996, Novak et al. 1998). In the nucleus, beta-catenin is unable to regulate transcription by itself and instead, it functions as a cofactor for the T C F (T-cell factor)/LEF transcription factors (Clevers et al. 1997,  Huber et al. 1996, Molenaar et al. 1996, Behrens et al. 1996).  architectural  protein which  facilitates  complexes (Oosterwegel et al. 1991).  the  formation of  T C F is an  multi-protein transcriptional  The. activity of these complexes  regulate the  expression of various transcripts (Brannon et al. 1997, Riese et al. 1997) and cause cellular changes associated with beta-catenin nuclearization (Molenaar et al. 1996). Consistent with the behaviour of numerous other developmental genes, the inappropriate transcription factor activity of beta-catenin in adult tissue is involved in cancer pathogenesis (Bullions et al. 1998, Behrens 2000).  HYPOTHESIS:  The tissue distribution and the expression profile of the beta-catenin protein in the postnatally developing cat visual system correlates with the critical period.  OBJECTIVES:  1.)  T o determine  the  temporal expression  and  the  tissue  distribution of the beta-catenin protein in the postnatally developing cat visual cortex and the L G N .  75  2.)  T o determine if the beta-catenin protein is present in the  nuclei of either L G N or visual cortex  cells in the  postnatally  developing cat.  3.2 METHODS Partial cloning of beta-catenin c D N A : See methods section in chapter 2.  Preparation of Animals: For western blot analysis a total of 27 cats were sacrificed. Four animals were used for each age group with the exception of prenatal animals where N=2 and N=5 for 60 day old postnatal animals. 24 additional animals were used for immunohistochemical analysis N=4 for P(postnatal day)0, P20, P60 and adults and N=2 for P30, P45, P55,  PI20.  Animals were euthanized with an overdose of euthanyl and then perfused briefly with cold P B S buffer (pH 7.4). The brains were then removed and frozen in isoamyl alcohol ( L G N and visual cortex were dissected and pooled prior to freezing if the tissue was intended for western blot analysis). The frozen brains were stored at - 8 0 ° C until use.  Antibody: A monoclonal antibody specific against the mouse, rat and human beta-catenin proteins was purchased from Transduction Laboratories. This antibody was used in both immunohistochemistry and western blot analysis procedures.  Preparation of Protein Samples: Boiling Method: A piece of frozen brain tissue was put into suspension buffer containing 0 . 1 M N a C l , 0.01M Tris-Cl (pH 7.6), 0.001M E D T A (pH 8.0), phenylmethylsulfonyl fluoride (PMSF).  lug/ml aprotinin, lOOug/ml  The suspension buffer was cooled on ice before  use and the protease inhibitors aprotinin and P M S F were added immediately before use.  76  (Pipette tips and tubes which came into contact with P M S F were left in a N a O H solution overnight in the fume hood.) The brain tissue was teased apart using a pair of forceps.  An  equal volume of 2 X S D S gel loading buffer containing lOOmM Tris-Cl (pH 6.8), 200mM dithitothreitol, 4% S D S , 0.2% bromophenol blue was added to the sample and the solution was immediately boiled for 10 minutes.  After boiling, the sample was briefly spun down  and the high molecular weight chromosomal D N A was sheared by sonication for 1 minute at maximum speed. sample.  The purpose of this step is to decrease the viscosity of the protein  The sample was then centrifuged for 10 minutes at room temperature at 15000g  and the resultant supernatant was transferred to a new tube. western blot analysis.  This sample was used for  It was usually aliquoted in order to avoid repeated freeze thaw  cycles and placed into a - 2 0 ° C freezer for short term storage or - 8 0 ° C for long term storage.  Triple detergent Method: Ten volumes of ice cold triple detergent containing 50mM Tris-Cl (pH 150mM N a C l ,  0.02%  sodium azide, 0.1%  deoxycholate, lOOixg/ml P M S F , frozen brain tissue.  SDS,  l%Nonidet P-40,  0.5%  8.0),  sodium  litg/ml aprotinin was added to one unit per weight of  As in the last method triple detergent was cooled on ice and the  protease inhibitors aprotinin and P M S F were added immediately before use.  The brain  tissue was teased apart using a pair of forceps and the solution was incubated on ice for 30 minutes.  Every  ten minutes  solublization of the tissue.  the  solution was  pipetted several  times  to  improve  High molecular weight chromosomal D N A was sheared by  sonication for 1 minute at maximum speed. Sonication was performed on ice. The solution was then centrifuged at 15000 x g for 5 minutes and the supernatant was aliquoted in order to avoid repeated freeze thaw cycles and placed into a - 2 0 ° C freezer for short term storage or - 8 0 ° C for long term storage.  77  Preparation of Nuclear Fraction: L G N tissue was homogenized in P B S containing 2ug/ml each of Aprotinin and Leupeptin and 0.75mM P M S F until a homogeneous  solution was  obtained.  All  centrifugations were done on ice and the samples were kept on ice at all times.  The  homogenate was centrifuged and the supernatant removed.  200pl of C E R I (PIERCE)  solution was then added to the pellet containing the above listed protease inhibitors. The solution was vigorously vortexed to resuspend the cell pellet.  The mixture was then  incubated on ice for 10 minutes. 1 l u l of C E R I I solution (PIERCE) was added to the tube. The tube was vortexed at maximum setting for 5 seconds, incubated on ice for 1 minute and then vortexed again for another 5 seconds. The tubes were centrifuged at (16000 x g) for 5 minutes.  The supernatant was removed and lOOul of N E R buffer ( P I E R C E ) was  added to the insoluble pellet containing nuclei. The above listed protease inhibitors were added to the N E R buffer before use. The solution was vortexed at maximum speed for 15 seconds and incubated on ice for 40 minutes. This sample was vortexed for 15 seconds at maximum speed every 10 minutes.  After the 40 minute incubation, the tubes were  centrifuged at (16000 x g) for 10 minutes.  The supernatant containing the nuclear fraction  was then transferred to a pre-chilled tube and stored at -80°C.  Western Blot Analysis: The dry H y b o n d - E C L membrane was soaked in 100% methanol for 5 seconds and then washed in distilled water for 5 minutes. The membrane was then placed into a heat sealable plastic bag containing 5% bovine serum albumin in P B S p H 7.5 (80mM disodium hydrogen orthophosphate anhydrous, 20mM sodium dihydrogen orthophosphate, lOOmM sodium chloride) containing 0.1% Tween detergent (referred to as PBS-T) and incubated at 4 ° C overnight on a shaker table. A l l the washes and the rinses in this procedure were done  78  with P B S - T . The next day, the membrane was quickly rinsed twice, washed once at room temperature with shaking for 15 minutes and twice more for 5 minutes each at room temperature with shaking. After the washes, the membrane was incubated in a heat sealed bag with the diluted primary antibody (1:1000 dilution for cadherin, 1:2000 dilution for beta-catenin) in P B S - T for one hour at room temperature with shaking.  Unbound primary  antibody was washed off the membrane by two quick rinses, followed by a 15 minute wash at room temperature with shaking and then two more washes for 5 minutes each at room temperature with shaking.  H R P conjugated secondary antibody diluted 1:1000 in  P B S - T was then added to the membrane and the membrane was incubated in a heat sealed bag for 1 hour at room temperature with shaking.  Unbound secondary antibody was  washed off the membrane by two quick rinses, followed by a 15 minute wash once at room temperature with shaking and then four more washes for 5 minutes each at room temperature with shaking. During these washes, the detection solution (consisting of equal volumes of solution 1 and solution 2 from Amersham E C L western blotting analysis system) was prepared. The excess liquid from the washed membrane was drained onto a paper towel and the membrane was placed protein side up on a piece of Saran wrap. The remainder of the procedures described in this section were performed in the dark room. The membrane was then covered with the detection solution and incubated for exactly 1 minute.  The membrane was removed from the detection solution and wrapped in a fresh  piece of saran wrap. The membrane was put in a cassette with the protein side up and an autoradiography film (Kodak X A R ) was placed on the membrane. The exposure times for the film varied from 30 seconds to 1 minute. The E C L signal was detected by developing the film.  Immunohistochemistry: Glass slides containing 12 micron tissue sections were removed from the freezer and allowed to thaw at room temperature for at least 30 minutes.  The slides were then  fixed in an ice cold 50%methanol, 50%acetone solution for ten minutes at - 2 0 ° C .  79  The  fixation solution was made fresh each time and was cooled at - 2 0 ° C for at least one hour. After fixation, the slides were dried for an additional 30 minutes.  Wells were created by  drawing circles around each tissue section on the slide with a P A P pen.  The sections were  not allowed to dry for the remainder of the immunohistochemistry procedure. The sections were then washed three times for five minutes each with P B S buffer at room temperature while shaking.  Since fresh frozen sections contain a substantial amount of endogenous  biotin/streptavidin sites, these sites were blocked using the Z Y M E D biotin/streptavidin blocking kit.  Blocking of these sites was found to significantly decrease non-specific  background staining with the vector A B C kit which utilizes a biotin/streptavidin based amplification system.  Three drops of Reagent A , which contains streptavidin, from the  biotin/streptavidin blocking kit was put onto each individual cat brain section (per section not per slide) and the slides were incubated in a humidified chamber for 10 minutes at room temperature with shaking. Tissue sections from one day old kittens only required one drop of this solution.  After the ten minute incubation Reagent A solution was removed with a  piptteman and the slides were quickly placed into a glass container containing fresh P B S . Once all the slides had Reagent A removed, they were washed three times with shaking for five minutes each in P B S buffer.  After the final wash, excess fluid from each slide was  removed with the aid of a pipetteman and three drops of reagent B , which contains biotinm, from the biotin/streptavidin blocking kit was applied to each section and the slides were again incubated in a humidified chamber for ten minutes at room temperature with shaking. Following the incubation reagent B was removed and the slides were washed three times with shaking for five minutes each in P B S buffer.  Excess solution from the slides was  removed and 500 microliters of a 5% bovine serum albumin solution made in P B S was added to each slide and the slides were then incubated in a humidified chamber for one hour at room temperature with shaking. The purpose of this step was to block any non-specific sites. After the blocking step, the 5% B S A solution was removed and a 1%BSA solution made in P B S containing the primary antibody at a 1:100 dilution was added to the slides.  80  (The beta-catenin antibody was purchased from Transduction Laboratories).  The slides  were incubated with the primary antibody in a humidified chamber overnight at 4 ° C with shaking. Incubating for longer periods of time with the primary antibody solution (i.e. 48 or 72 hours) did not improve the final staining of the sections.  The following day, the  slides were washed three times with shaking for five minutes each in P B S buffer.  A  1%BSA solution made in P B S containing the secondary antibody at a 1:1000 dilution was added to the slides and the slides were incubated in a humidified chamber for one hour at room temperature with shaking.  While the slides were incubated with the secondary  antibody, 20 micro liters of solution B and 20 micro liters of solution C from the A B C kit were added to lOmls of a 1%BSA solution made in P B S .  The solution was mixed by  inversion and was kept undisturbed at room temperature for at least 30 minutes.  Once the  secondary antibody incubation was completed, the secondary antibody solution  was  removed and the slides were washed three times with shaking for five minutes each in P B S buffer.  The B C solution that was prepared earlier was then put onto each slide and the  slides were incubated with this solution in a humidified chamber for one hour at room temperature with shaking.  The slides were washed three times with shaking for five  minutes each in P B S buffer after the incubation. The slides were then placed in a 500ml nickel D A B solution consisting of 5g of nickel ammonium sulphate, 1.7g of imrnidazole, 50mg of D A B and 5jxl of 30% hydrogen peroxide in Tris buffer at pH7.4.  The Nickel  enhanced D A B reaction stains the slides a dark greyish blue color. Following the staining, the slides were washed three times for 5 minutes each in P B S and coversliped.  Double Labeling: The slides were fixed and processed (i.e.,  had their biotin sites blocked) as  described in the immunohistochemistry protocol. After the sections were incubated with the 5% B S A solution for 1 hour, one of the antibodies to be used in the double labeling procedure was added to the sections.  The immunohistochemistry procedure was carried  8 1  out as described. Instead of coverslipping the sections at the end of the procedure, the sections were incubated with the Z Y M E D peroxidase blocking solution for ten minutes. After the completion of the immunohistochemistry procedure, the sections were not allowed to dry. The purpose of the peroxidase blocking solution is to deactivate the activity of horseradish peroxidase enzyme remaining from the initial immunohistochemistry step. The slides were washed three times for 5 minutes each in P B S with shaking.  The slides  were then blocked for 1 hour with 5% B S A in P B S and then the second antibody to be used in the double labeling procedure was added.  The immunohistochemistry procedure  was carried out as before except for the staining step.  Instead of staining the slides in a  Nickel D A B solution, which results in a greyish-blue color, the slides were incubated with D A B alone which results in a brownish red color.  The 500 ml D A B solution contained  50mg of D A B and l u l of 30% hydrogen peroxide. After the D A B reaction, the slides were washed three times for 5 minutes each in PBS and coversliped.  Coversliping: The slides were put in distilled water for 1 minute for rehydration. The slides were then dehydrated by incubation for 1 minute in 70%, 5 minutes in 95%, and 10 minutes in 99% ethanol. The slides were then put in xylene for 20 minutes after which they were coverslipped with permount.  Nissel Staining: Buffered Neutral Red Stain was made by adding 32ml's of acetate buffer, p H 4 . 8 , (stock solution made by adding 100ml of 0.1N acetic acid to 150ml of 0.1N sodium acetate) to 800ml's of 2% aqueous neutral red solution (16g Neutral Red, 185ml 1 M acetic acid, 2.04g sodium acetate or 15ml of 1M sodium acetate solution, 200ml of distilled water, 400ml of 50% ethanol).  This solution was stored at room temperature and filtered  before each use.  82  3.3 RESULTS 3.3.1 Partial Cloning of the Cat Beta-Catenin cDNA In order to confirm the expression of the beta-catenin gene in the visual cortex during the critical period, P C R primers were designed based on the nucleotide sequence of the human beta-catenin gene. Low stringency P C R was performed as described in chapter two and the resulting bands were cloned and sequenced. Figure 3.2 shows the partial c D N A sequence of the beta-catenin gene in the cat. The cat beta-catenin c D N A has 91% identity with the human beta-catenin c D N A . generated  by  PCR  using  the  primers  The beta-catenin c D N A fragment was GGTCACCTTGCAGCTGGAATTC  and  CCTGAGC A AGTTC AC A G AGG ACC. Given that the beta-catenin gene is extensively regulated at the post-translational level (Aberle et al. 1997), the protein expression of this gene needs to be determined in the postnatally developing cat visual system.  Also, since the beta-catenin protein is capable of  entering the nucleus and acting as a transcription factor (Clevers et al. 1997), behaviour of this gene can not be assessed adequately in the visual system by simply using c D N A probes.  Therefore, finding a suitable antibody against the cat beta-catenin protein is  essential. Figure 3.3 shows the alignment of the cat and human beta-catenin amino acid sequences.  The partially cloned beta-catenin c D N A in the cat is 100% identical with the  human beta-catenin protein at the amino acid level.  This finding is not surprising  considering the human, mice and rat beta-catenin amino acid sequences are almost identical. Accordingly, beta-catenin antibodies generated for these species are likely to cross react with the cat beta-catenin protein.  3.3.2 Western Blot Analysis of the Beta-Catenin Protein in the Postnatally Developing Cat Visual Cortex The Beta-Catenin antibody was purchased from Transduction Laboratories and this antibody is reported to cross react with the human, rat and mouse beta-catenin proteins. Figure 3.4 shows the quantitative western blot analysis performed in the developing cat  83  Cat  :  1 gtgaacagggtgccattccacgactagttcagttgctggttcgtgcccatcaagataccc 60 I I II II II II II I I I I I I I I II I I I I I I I I I I I I II M I N I M I I I II I I I I I II Human:1797 g t g a g c a g g g t g c c a t t c c a c g a c t a g t t c a g t t g c t t g t t c g t g c a c a t c a g g a t a c c c 1856 Cat  : 61 a g c g c c g c a c a t c t a t g g g c g g a a c g c a g c a g c a g t t t g t g g a g g g a g t c c g t a t g g a a g 120 I II I I I I II II M i l l II II II I I I I I I I I I I I I I I I I I II I II I I II I I I Human:1857 a g c g c c g t a c g t c c a t g g g t g g g a c a c a g c a g c a a t t t g t g g a g g g g g t c c g c a t g g a a g 1916  Cat  : 121 a a a t t g t t g a a g g t t g t a c c g g a g c c c t t c a t a t c c t a g c t c g g g a t g t t c a c a a c c g a a 180 I I I I I I I I I I I I I I II I II I I I I I I II I I I I I I I I I I I I I I I I I II II I I I I II I I I I Human:1917 a a a t a g t t g a a g g t t g t a c c g g a g c c c t t c a c a t c c t a g c t c g g g a t g t t c a c a a c c g a a 1976  Cat  : 181 t c g t a a t c a g a g g a c t a a a t a c c a t t c c a t t g t t t g t g c a g c t g c t t t a t t c t c c c a t t g 240 I II I I I I I I I I II II I I I I I I II II I I I I I I I I II II I II II I I I I I I I II I I II I II Human:1977 t t g t t a t c a g a g g a c t a a a t a c c a t t c c a t t g t t t g t g c a g c t g c t t t a t t c t c c c a t t g 2036 Cat  : 241 a a a a t a t c c a a a g a g t a g c c g c a g g g g t c c t c t g t g a a c t t g c t c a g g a c a a g g a g g c t g 300 M M II II I II II I I I I I II II II I I I I I I I II II I I II I I I II II I I I I II I II I I Human:2037 a a a a c a t c c a a a g a g t a g c t g c a g g g g t c c t c t g t g a a c t t g c t c a g g a c a a g g a a g c t g 2096  Cat  : 301 c g g a a g c c a t t g a a g c g g a g g g a g c c a c g g c t c c t c t g a c a g a g c t c c t t c a c t c c a g a a 360 I I I II I l l l l l l l l I II II II I I I I I I I II II I I I I I I II I II I I I II I II I Human:2097 c a g a a g c t a t t g a a g c t g a g g g a g c c a c a g c t c c t c t g a c a g a g t t a c t t c a c t c t a g g a 2156 Cat  : 361 a c g a a g g t g t g g c a a c a t a c g c a g c t g c t g t t t t g t t c c g a a t g t c t g a g g a c a a g c c c c 420 I I II II II II II M i l l I II I I I I I II II II I I I I I I I I I I I I I II I II I I II I I I Human:2157 a t g a a g g t g t g g c g a c a t a t g c a g c t g c t g t t t t g t t c c g a a t g t c t g a g g a c a a g c c a c 2216 Cat  : 421 a g g a c t a c a a g a a g c g g c t t t c g g t g g a g c t g a c c a g t t c t c t c t t c a g a a c 472 I II I II II I II I I I II II I II I I II II I II I I I I I I I I I I I II II I Human:2217 a a g a t t a c a a g a a a c g g c t t t c a g t t g a g c t g a c c a g c t c t c t c t t c a g a a c 22 68 Figure 3.2 Partial cDNA sequence of the cat Beta-catenin gene. B l a s t Score = 634 b i t s (320), Expect = e-180 I d e n t i t i e s = 434/472 (91%) • Strand = Plus / Plus  84  Cat  : 4 73 EQGAIPRLVQLLVRAHQDTQRRTSMGGTQQQFVEGVRMEEIVEGCTGALHILARDVHNRI 2 94 EQGAIPRLVQLLVRAHQDTQRRTSMGGTQQQFVEGVRMEEIVEGCTGALHILARDVHNRI Human: 529 EQGAIPRLVQLLVRAHQDTQRRTSMGGTQQQFVEGVRMEEIVEGCTGALHILARDVHNRI 588 Cat  : 293 VIRGLNTIPLFVQLLYSPIENIQRVAAGVLCELAQDKEAAEAIEAEGATAPLTELLHSRN 114 VIRGLNTIPLFVQLLYSPIENIQRVAAGVLCELAQDKEAAEAIEAEGATAPLTELLHSRN Human: 589 VIRGLNTIPLFVQLLYSPIENIQRVAAGVLCELAQDKEAAEAIEAEGATAPLTELLHSRN 648  Cat  : 113 EGVATYAAAVLFRMSEDKPQDYKKRLSVELTSSLFRT 3 EGVATYAAAVLFRMSEDKPQDYKKRLSVELTSSLFRT Human: 64 9 EGVATYAAAVLFRMSEDKPQDYKKRLSVELTSSLFRT 685  Figure 3.3  Alignment of the cat and the human beta-catenin amino acid  B l a s t Score = S$<*£lfe>nteSJ:7 80) , Expect = 5e-84 I d e n t i t i e s = 157/157 (100%), P o s i t i v e s = 157/157 (100%)  85  P r e n a t a l 0 day  15 day  30 day 60 day 120 day A d u l t  200  kDA  120  kDA  90 kDA  50 kDA  LOADING CONTROL  Figure 3.4 Beta-Catenin protein expression in the postnatally developing cat visual  cortex. The top p a n e ls h o w s the w e s t e r nb l o ta n a y ls s i p e r f o r m e d on the d e v e o lp n i g cat v i s u a lc o r t e xu s n i g the b e t a c a t e n n i a n t b io d yp u r c h a s e df r o mT r a n s d u c t o in L a b o r a t o r i e s . T h i s a n t b io d y s p e c i f i c a ly r e c o g n z ie s as i n g l eb a n d of a r o u n d 93 kDA in v i s u a l c o r t e x p r o t e i np r e p a r a t o in so b t a n ie df r o mk i t t e n s of v a r o iu sd i f f e r e n ta g e s . S i g n i f i c a n tc h a n g e s in the e x p r e s s o in of the b e t a c a t e n n i p r o t e i n are not o b s e r v e d in the d e v e o lp n i g cat v i s u a l c o r t e x . For the o la d n ig c o n t r o l (the b o t o mp a n e l ) , the m e m b r a n eu s e d for the w e s t e r n b l o ta n a y ls s i was s t a n ie dw i t hC o o m a s e i b l u e .  86  visual cortex with the Transduction Laboratories beta-catenin antibody. The blots consist of prenatal, 0 day postnatal, 15 day postnatal, 30 day postnatal, 60 day postnatal, 120 day postnatal and adult visual cortex protein preparations. In each age a single band of roughly 93k Daltons is detected confirming the fact that the beta-catenin antibody produced by Transduction Laboratories cross reacts with the cat beta-catenin protein. A l s o , since only a single band is present in this western blot analysis, this antibody appears to specifically recognize the beta-catenin protein in the cat.  Therefore, the Transduction Laboratories  antibody is suitable for performing immunohistochemistry in the cat visual cortex to localize the distribution o f the beta-catenin protein. Since equal amounts of protein are loaded into each well (50(lg) of the blot shown in figure 3.4, these blots are quantitative and can be used to determine beta-catenin protein expression during visual cortex development. The expression pattern shown on figure 3.4 was obtained on all of the western blots. Figure 3.4 shows that the beta-catenin protein expression is fairly constant in the visual cortex during development and does not appear to be regulated in an age dependent manner. In order to verify the validity of this expression pattern, western blot analysis using the beta-catenin antibody was repeated three times on three different blots.  Since each beta-catenin antibody western blot analysis yielded a  similar result, we are confident at claiming that the pattern shown in figure 3.4 is the actual profile of beta-catenin protein expression during development. 3.3.3  Western Blot Analysis of the Beta-Catenin Protein in the Postnatally  Developing Cat L G N Figure 3.5  shows the quantitative western blot analysis performed in the  postnatally developing cat L G N with the beta-catenin antibody. The blot consists of L G N protein preparations from postnatal 0 day, 15 day, 30 day, 60 day, 120 day and adult cats. Each lane again shows a single band confirming the specificity of the beta-catenin antibody in the cat L G N . The expression of the beta-catenin protein does not appear to change with age in the postnatal L G N . A s in the cortex, the expression pattern of the beta-catenin  87  Oday  15 day  30 day  60 day  120 day  Adult  120 k D A 90 k D A  50 k D A  LOADING CONTROL  Figure 3.5  Beta-Catenin protein expression in the postnatally developing cat L G N .  The top panel shows the western blot analysis performed on the developing cat L G N using the beta-catenin antibody purchased from Transduction Laboratories. This antibody specifically recognizes a single band of around 93 k D A in L G N protein preparations obtained from kittens of various different ages.  The beta-catenin protein expression does not apper  to vary in the developing L G N . For the loading control (the bottom panel), the membrane used for the western blot analysis was stained with Coomasie blue.  88  protein in the developing L G N was confirmed by repeating the western blot analysis thrice on three different protein blots.  This expression profile of beta-catenin in the postnatal  L G N does not correspond with the critical period.  3.3.4  Immunohistochemical localization of Beta-Catenin in the Postnatally  Developing Cat Visual Cortex The Transduction Laboratory beta-catenin antibody, whose specificity in the L G N and the cortex was established by western blot analysis, was used to localize beta-catenin protein expression in developing cat visual cortex tissue. Figure 3.6 shows a 60 day old cat visual cortex tissue section subjected to immunohistochemical analysis.  Unlike the  reported expression pattern of neurotransmitter receptors, the laminar distribution of the beta-catenin protein does not change with age. The postnatal ages examined include 0 day, 20 days, 30days, 45 days, 55 days, 60 days, 120 days and adults.  Beta-catenin protein  expression is high in the upper layers of the cortex with a slight overexpression in layer 4. The layer 4 expression of the beta-catenin protein is especially prevalent in the cingulate. Beta-catenin protein expression is not restricted to the visual cortex and this protein is present in other areas of the brain. This finding consistent with the reported function of beta-catenin as a synaptic protein expressed by various types of neurons. Figure 3.7 shows the photograph of a visual cortex tissue section, which has been subjected to beta-catenin immunohistochemical analysis, taken at high magnification. This figure clearly shows that the beta-catenin protein expression is localized to the neuropil of the visual cortex. This distribution of beta-catenin is similar to the distribution of other synaptic proteins in the visual cortex such as synaptophysin. Neutral Red counter staining of the immunosection in Figure 3.7 reveals antibody staining of the beta-catenin protein is restricted to the neuropil and is absent from cell bodies of cortical neurons.  Beta-catenin  protein is not found in the nuclei of cortical neurons at any time during postnatal visual development.  This finding is surprising considering the presence of Wnt-1 m R N A  expression in the cortex during the critical period.  89  g a —•  s .s  S § 3 o  OJ  OJ  • §i  c  2 IS S O -  IE  a °  a co c *5 £s?3 _oo  f t i  z z  u  1  3  00  s  c  5  'S  d •a  1  5  c  "7 C S C X) Xi CJ  —  -  —' 12  OJ  l ! |  i  ® g  i  I I Jii •Q5 3u- S F 6 J9  — oj Xj >> 0 0 C3  —  •73 V  *  a o  o  cj Q- E—  03  £ g ob c 1  i i  *3  CO  s ii ii I  v"  !_  CJ  o «  3  3  00  93  —  a B  « ro <OJ *- > o\ B  U 'S  1 « o a  I ?I! 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S £ X Jg o «— SB JI oo c £ . re cij cn •a re oo £ Jc a. 3 E5 ° '3 a '5 D  s c  C  I  3  co  re nj  i  X  •o H B ^ u .*r :Q3  11 .£ I  cn  <u .2, g s 1 3 2"§ -1 > S u R a I $ & —' u co  —  'co U 3 3 .-3 C •S 3 a „  "re  o .2 x c c x >> 00  s s  'a  y CO  <U  3  •Si  I ts x u .E T3  ~ re "> -o ^ re « C o re B re c — 05  u >> "S u o p < a X J ^ ° -c 000 H .2 a -a 3 re  x o 3  CL  X 53  00 3  2 X„ 'E5 re  E 3 T3 c £  co *3  3.3.5  Immunohistochemical Localization of the Beta-Catenin Protein in the  Postnatally Developing Cat L G N Figure 3.8  shows immunohistochemical analysis performed in the postnatally  developing cat L G N with the beta-catenin antibody. Beta-catenin protein expression in the lateral geniculate nucleus of a 0 day old kitten is restricted to the neuropil. This pattern is similar to the beta-catenin protein distribution observed in the postnatally developing visual cortex.  The same distribution pattern is detected in the L G N of 20 day old animals.  In  kittens as old as 50 days of age, large unstained cell bodies are present in the L G N confirming the absence of the beta-catenin protein from the nuclei of geniculate cells.  In  kittens between 50 to 60 days of age, the beta-catenin protein is detected in the nuclei of L G N cells and unstained cell bodies are no longer observed. The nuclear localization of the beta-catenin protein is retained after 60 days of age and persists in the adult cat L G N . The earliest expression of beta-catenin protein in the L G N was observed in a normal 47 day old kitten.  Kittens as old as 45 days of age (n=2) lacked nuclear beta-  catenin protein in the geniculate (30 day old animals also lacked nuclear beta-catenin). Additionally, no animal exhibited an intermediate state between having nuclear beta-catenin protein (the state seen in 60 day or adult animals) or lacking nuclear beta-catenin protein (the state seen in 0 or 20 day old animals). Nuclear beta-catenin protein was either strongly expressed in the L G N or completely absent. T o confirm that the beta-catenin antibody is labeling nuclei and to determine if the beta-catenin protein is becoming nuclear in neurons, double labeling experiments were carried out.  The SMI-32 antibody, a well established neurofilament stain that specifically  labels Y-cells (Campbell et al. 1989, A n g et al. 1991, Chaudhuri et al. 1996), was used in conjunction with the beta-catenin antibody to label cat lateral geniculate tissue sections. In figure 3.9,  the brown SMI-32 labeling (the D A B stain) is clearly evident in neurons  containing the blue beta-catenin nuclei (the nickel enhanced D A B stain).  This result  demonstrates that beta-catenin nuclearization does occur in neurons. However, although  92  Oday  20 d a y  60 d a y  Adult  Figure 3.8 Expression of the beta-catenin protein in the postnataly developing cat LGN. In young postnatal animals (the top two panels) beta-catenin protein expression is restricted to the neuropil and the beta-catenin protein is not present in the nuclei of geniculate cells. Accordingly, white unstained cell bodies are observed in the L G N of young kittens (pointed to by arrows in 0 day and 20 day old animals). However, at 60 days of age nuclear beta-catenin staining is detected in the L G N (arrows) and the unstained white cell bodies are no longer observed. This staining pattern persists in the adult L G N (arrows point to nuclei). The scale bar represents 0.16 mm's.  93  Figure 3.9 Beta-catenin and SMI-32 double staining in the L G N of older kittens. The blue nickel enhanced D A B beta-catenin protein staining (arrows) is observed in the nuclei of neurons which have been labeled with the brown SMI-32 neurofilament stain (regular D A B ) .  94  the co-localization between SMI-32 and beta-catenin staining is extensive, it is not complete and there are some cells that are only labeled with the SMI-32 antibody and some cells which are strictly labeled by the beta-catenin antibody.  This finding indicates that the  nuclear localization of the beta-catenin protein is not restricted to large neurons (i.e. not specific to Y cells) and nuclearization of beta-catenin is not determined by cell size.  3.3.6  Nuclear Beta-Catenin Protein Levels  in the Postnatally  Developing  Cat L G N In order to get a more accurate measurement of the amount of beta-catenin protein present in the nuclei of L G N neurons during the critical period, nuclear protein preparations were obtained from the lateral geniculates of postnatal 0 day, 15 day, 30 day, 60 day, 120 day and adult cats. Western blot analysis performed on these nuclear L G N preparations with the beta-catenin antibody is shown in figure 3.10. Beta-catenin protein levels in nuclei of geniculate cells become elevated in kittens 60 days of age and remain high throughout adulthood.  These findings are consistent with the beta-catenin immunohistochemical  analysis described in the postnatally developing L G N (discussed further in the last section).  3.4 DISCUSSION These findings support a role for beta-catenin in the postnatal development of the visual system.  The localization of the beta-catenin protein in the nuclei of geniculate  neurons in the postnatally developing cat visual cortex correlate with the critical period. Since beta-catenin is a homologue of the armadillo segment polarity gene, the findings of this chapter solidify the hypothesis that the homologues of the drosophila developmental genes are involved in the postnatal development of the visual system.  3.4.1 Beta-Catenin Expression in the Postnatal L G N Beta-catenin is likely to perform dual functions in the postnatally developing cat L G N . In the L G N of young kittens, the expression of beta-catenin is restricted to the  95  Oday  15 day  30 day  60 day  120 day  Adult  120 k D A  Figure 3.10  Beta-Catenin protein levels in the nuclei of geniculate cells during the  postnatal development of the cat L G N . The top panel shows the western blot analysis performed on the nuclear protein preparations obtained from different aged postnatal cat L G N ' s . The beta-catenin protein is absent from the nuclear protein fraction of the L G N in cats younger than 60 days of age.  This finding is consistent with the results of the  immunohistochemical analysis described in Figure 3.8. For the loading control (the bottom panel), the membrane used for the western blot analysis was stained with Coomasie blue.  96  neuropil. This distribution pattern is consistent with the function of beta-catenin as a cell adhesion molecule. However, the beta-catenin protein levels are likely to be upregulated in a proportion of geniculate cells in postnatal kittens 60 days of age and older.  The  upregulation and the subsequent accumulation of the beta-catenin protein causes it to be transported into the nuclei of geniculate neurons where it is likely to function as a transcription factor. Interestingly, this nuclear localization of beta-catenin in L G N neurons is retained in the adult. These findings are quite surprising considering nuclear beta-catenin is usually associated with early events in development. Beta-catenin protein becomes nuclear in the L G N at the end of the critical period for thalamocortical plasticity.  Following 50-60 days of age, monocular deprivation fails to  cause ocular dominance shifts in layer 4 of the visual cortex as determined by anatomical (tracer injections) and physiological (single unit recordings) techniques (LeVay et al. 1978, LeVay et al. 1980, Mower et al. 1985, Shatz et al. 1978). Since the beta-catenin protein becomes nuclear in L G N neurons around 50 days of age, we propose nuclear beta-catenin activity to be involved in ending thalamocortical plasticity.  A s mentioned in the  introduction, beta-catenin acts as a transcription factor in the nucleus and regulates the expression of a variety of genes by forming complexes with the T C F and L E F proteins (Clevers et al. 1997, Huber et al. 1996, Molenaar et al. 1996, Behrens et al. Brannon et al. 1997, Riese et al. 1997, Molenaar et al. 1996).  1996,  Conceivably, when the  beta-catenin protein becomes nuclear in geniculate neurons, it may downregulate  the  expression of genes involved in facilitating synaptic plasticity or alternatively, beta-catenin may upregulate the expression of proteins which inhibit processes associated with synaptic rearrangements.  This model implies that synapses in layer TV of the visual cortex are  unable to undergo synaptic reorganization in kittens older than 60 days of age because thalamic processes have lost their plastic capabilities. Presumably, cortical neurons retain their plasticity past this age since beta-catenin nuclearization does not occur in the cortex. This notion is supported by the fact that plasticity is retained in the higher layers of the  97  cortex past 60 days of age and ocular dominance shifts in layer 2/3 of the cortex are detected in cats as old as 1 years of age (Daw et al. 1992).  These findings argue for a  presynaptic end to visual plasticity and stress the importance of presynaptic processes in synaptic rearrangements.  However, the possible existence of processes involved in  inhibiting plasticity specifically in layer IV of the visual cortex can not be ruled out. Nuclear beta-catenin is unlikely to be the only factor responsible for ending visual plasticity. A s mentioned, only a subset of geniculate neurons contain nuclear beta-catenin. Furthermore, nuclear beta-catenin adulthood.  is not observed  in cortical neurons  even during  Therefore, other processes associated with ending visual plasticity must be  acting on cortical cells and geniculate neurons lacking nuclear beta-catenin activity.  Beta-  catenin is viewed as a factor that causes genetic changes in a subset of L G N neurons which prevents  these neurons  from  undergoing  synaptic  rearrangements.  Additionally,  transcription factor activity of beta-catenin may only partially inhibit the plastic capabilities of neurons and other mechanisms may also be necessary  for ending thalamocortical  plasticity in geniculate neurons containing nuclear beta-catenin. T w o additional issues regarding beta-catenin nuclearization in the postnatal lateral geniculate need to be addressed.  The beta-catenin protein starts going into the nuclei of  L G N neurons in kittens that are around 50 days of age.  Often the regulatory changes  caused by transcription factors do not immediately alter the phenotypic behaviour of cells and this reason may explain why nuclear beta-catenin protein is detected in the geniculate slightly before the end of thalamocortical plasticity.  For example, if beta-catenin activity  leads to the activation of a transcription cascade, the proteins involved need to be synthesized and allowed to take effect. catenin may exhibit phenotypic lag.  More importantly, genes downregulated by beta-  Halting gene expression at the transcriptional level  does not lead to immediate cessation of gene function (Rudd et al. 1990, Thilly et al. 1978, Penman et al. 1976). Previously synthesized proteins from the inhibited gene may remain undegraded and perform their function in the absence of newly synthesized protein.  98  The  phenomenon of phenotypic lag is well documented for proteins which are highly stable and turn over slowly.  The slight disparity between the nuclearization of beta-catenin in the  L G N and the end of the period for thalamocortical plasticity can be accounted by these factors. Secondly, it is not clear why there are less nuclear beta-catenin containing cells in the L G N of adult animals.  One possibility is that nuclear beta-catenin protein causes  irreversible changes in some neurons which prevents them from being plastic whereas in other neurons beta-catenin protein may need to exert a constant level of inhibition in order to prevent plasticity. Another important implication of having nuclear beta-catenin in adult neurons involves the role beta-catenin plays in cancer pathology.  The nuclear localization of beta-  catenin in non-neuronal tissue usually occurs during early development and the presence of nuclear beta-catenin in adult cells is an aberration associated with cancer metastasis (Oyama et al. 1994, Rubinfeld et al. 1997, Korinek et al. 1997, Porifi et al. 1997).  Cancerous  adult cells containing nuclear beta-catenin lose their adhesive properties, detach from their tissue of origin and spread the disease by migrating into other organs.  In addition, beta-  catenin activity is reported to contribute to the proliferation of cancerous cells in tumors (Tetsu et al. 1999). cats.  Clearly, these events do not occur in the adult geniculate neurons of  The previous statement does not imply that beta-catenin is performing a different  function and effecting the expression of a completely different set of genes in neuronal and non-neuronal tissue.  Instead, nuclear beta-catenin may exhibit differing effects in these  tissues because certain pathways or cofactors which are present in non-neuronal cells may be absent in neurons and vice versa.  If the reported effects of the nuclear beta-catenin  protein in adult non-neuronal tissue requires the activity of certain cofactors which are lacking in neuronal cells then clearly, beta-catenin will not be able to induce the same changes in neurons. In other words, although beta-catenin may be necessary to cause the phenotypes observed during cancer, it may not be capable of carrying out this task by itself.  As a side note, examination of factors which are lacking in geniculate neurons but  99  present in cells that become cancerous with beta-catenin nuclearization may provide important information regarding the processes involved in cancer.  3.4.2  Beta catenin  expression  in the postnatally  developing  cat  visual  cortex The function of beta-catenin in the postnatally developing visual cortex appears to be as an adhesion molecule. Nuclear beta-catenin protein is not detected in the developing cat visual cortex at any age and its distribution is strictly restricted to the neuropil. A role for beta-catenin protein in cortical plasticity is suggested by the fact that its expression is slightly higher in layer IV of the visual cortex which is where thalamocortical connections reside. The known functions of the cell adhesion system beta-catenin constitutes with the cadherin proteins (such as synaptogenesis and target recognition (Huntley et al.  1999,  Riehl et al. 1996, Matsunaga et al. 1988, Obst-Pernberg et al. 1999) - discussed in detail in the next chapter) further support a role for this protein in visual plasticity. Despite the presence of Wnt-1 m R N A expression in the cortex, the beta-catenin protein does not become nuclear in cortical neurons at any age.  This finding represents the  complexity of the factors involved in Wnt-1 signaling and reflects the problems associated with determining the exact location of Wnt-1 signaling activity. In the previous chapter, the expression of F r z - B , a molecule that inhibits the function of Wnt-1 (Leyns et al. 1997), was confirmed in the developing cat visual cortex.  Several other soluble factors similar to  F r z - B , which prevent the Wnt-1 protein from binding to its receptor, are known to exist (Moon et al. 1997a, Brown et al. 1998) and are likely to be present in the visual cortex. Additionally, the Wnt-5A proteins, which are known to antagonize Wnt-1 signaling (Torres et al. 1996), may also be expressed during the critical period.  Examination of all these  molecules in the cortex is necessary in order to determine why beta-catenin does not become nuclearized in the cortex. It is important to point out that the Wnt-1 expression was detected at the m R N A level and the possibility exists that this transcript is not translated into protein.  Although the beta-catenin protein does not become localized to the nuclei of  100  cortical neurons, it is possible that Wnt-1 protein may still play a role in regulating betacatenin protein expression in the neuropil.  Alternatively, the Wnt-1 protein may be a  retrograde signal for the geniculate neurons. Support for this possibility is provided by the finding that Wnt molecules can act as retrograde messengers in the cerebellum (Hall et al. 2000).  Conceivably, the Wnt-1 molecule secreted by cortical neurons may function as  retrograde messengers and cause the nuclearization of beta-catenin in geniculate neurons thereby ending plasticity.  3.4.2 Function of beta-catenin in the developing visual system Genes regulated by nuclear beta-catenin activity in L G N neurons need to be identified in order to verify the proposed role of beta-catenin in ending the critical period for thalamocortical plasticity and to determine the mechanisms involved in this process. Theories can be formulated about the transcriptional changes caused by nuclear beta-catenin in the L G N based on studies characterizing the transcription factor function of this protein in other systems.  A s noted, beta-catenin nuclearization in adult non-neuronal cells causes  loss of adhesion allowing these cells to dislodge from their tissue of origin (Rubinfeld et al. 1997, Korinek et al. 1997, Porifi et al. 1997).  This capability of beta-catenin is attributed  to the direct inhibitory effect it exerts on cell adhesion molecules at the transcriptional level (Stewart et al. 2000, Nuruki et al. 1998). Beta-catenin is likely to control the expression of cell adhesion molecules in neurons since neurons express many of the same cell adhesion molecules as non-neuronal cells. The importance of cell adhesion molecules to the process of neuronal plasticity is beginning to emerge.  The disruption of the cadherin and the  integrin cell adhesion proteins are reported to interfere with the induction of L T P (Tang et al. 1998, Staubli et al. 1990). A correlation between cell adhesion molecule expression and visual plasticity is established by the work of Muller et al (Schoop et al. 1997) who report the adhesiveness of the visual cortex to decrease with age. Hence, we propose that nuclear beta-catenin is ending the critical period by downregulating the expression of cell adhesion molecules.  101  Since the role of beta-catenin in cell adhesion is to anchor the cadherin cell adhesion molecules to the actin cytoskeleton (Nagafuchi et al. 1988, Kitner et al. 1992, M c C r e a et al. 1991), the expression of the cadherin molecules in the postnatal visual system of the cat is investigated in the next chapter.  Interestingly, certain kinds of cadherin cell adhesion  molecules are downregulated by the transcription factor activity of beta-catenin (Stewart et al. 2000, Nuruki et al. 1998) and the ontogeny of the cadherin molecules in the developing visual cortex (reported in the next chapter) supports the hypothesis that nuclear beta-catenin activity in the L G N downregulates cell adhesion molecule expression.  102  Chapter 4 CADHERIN PROTEIN EXPRESSION IN T H E POSTNATALLY DEVELOPING CAT VISUAL SYSTEM 4.0  SUMMARY Since the role of the beta-catenin protein in cell adhesion is to anchor the cadherin  proteins to the actin cytoskeleton, the distribution of the cadherin proteins in the postnatal cat visual system was examined using a pan-cadherin antibody.  Western blot analysis  shows the expression of the cadherin proteins is downregulated in the adult cat visual cortex and the lateral geniculate nucleus.  Immunohistochemical analysis using the pan-  cadherin antibody revealed that the cadherin proteins are largely expressed in layer I V of the cortex, which is the thalamic input layer. Based on the reported properties of the cadherin cell adhesion molecules in other systems, the cadherin proteins are likely to play an organizational role in the postnatal visual system and they are likely to contribute to the proper formation of thalamocortical connections.  4.1  INTRODUCTION  Beta-catenin and the cadherin proteins constitute a prominent cell adhesion system in the body.  The role of the beta-catenin protein in this system is to anchor the cadherin  molecules to the actin cytoskeleton (McCrea et al. 1991). Since the beta-catenin protein appears to act exclusively as a cell adhesion molecule in the visual cortex and in the lateral geniculate of young kittens, the ontogeny of the cadherin molecules was investigated in the developing cat visual system.  103  The cadherin family of calcium dependent cell adhesion molecules have received a great deal of attention in neurodevelopment (Volk et al. 1984, Volk et al. 1986, Redies et al. 1996, Martinek et al. 1997, Yagi et al. 2000, Redies 2000).  The cadherin proteins are  around 130kDa in size and consist of 5 extracellular domains which are repeats of each other (Tanihara et al. 1994). The first three extracellular domain repeats contain sites for calcium binding which are necessary for stabilizing the cadherin protein and preventing its proteolysis (Nose et al. 1988, Nose et al. 1990). The extracellular domain closest to the N terminal gives rise to the cell adhesion binding sites (Nose et al. 1990).  The cadherin  molecules are typically categorized based on their binding sites since cadherin molecules bind to each other in a strictly homophilic manner (Nose et al. 1988, Miyatani et al. 1989, Shapiro et al. 1995, Leckband et al. 2000). The cadherin proteins also contain a small transmembrane domain and a highly conserved intracellular domain that binds to the catenin proteins (Nagafuchi et al. 1988, Kitner 1992, McCrea et al. 1991).  In addition, cadherin  molecules need to form homo-dimers at the conserved H A V amino acid region in order to function properly (Blaschuk et al. 1990). Numerous different types of cadherin and cadherin like molecules are expressed in the nervous system. The cadherin molecules are categorized into three general groups: the classic cadherins, protocadherins and the cadherin like neural receptors.  The classic  cadherin molecules can be subdivided further into two groups, named type I and type II, based on their amino acid sequences (Suzuki et al.  1991,  Protocadherins are larger cadherin molecules containing extracellular domains (Sano et al. 1993)  six  Tanihara et al.  1994).  or seven cadherin like  and the intracellular binding region of the  protocadherin proteins is significantly different than the classic cadherins (Sano et al. 1993). Finally, there are the cadherin like neural receptors (CNR's) which are very similar in structure to the classic cadherin cell adhesion molecules (Kohmura et al. 1998). Given the rapid rate in which new types of cadherin molecules are being discovered, additional classes of cadherin molecules are likely to be identified.  104  A s in other tissues, cadherins  are expressed in a temporally and spatially specific manner in the developing nervous system (Inoue et al. 1998, Suzuki et al. 1997, Matsunami et al. 1995, Rubenstein et al. 1999, Kimura et al. 1996, Arndt et al. 1996).  Electron microscope studies in the brain  have localized the cadherins to the synaptic terminals (Uchida et al. 1996) (figure 4.1). Involvement  of  cadherin proteins  in synaptogenesis,  neurite  extension  and  target  recognition is well established (Huntley et al. 1999, Riehl et al. 1996, Matsunaga et al. 1988, Obst-Pernberg et al. 1999).  Cadherin molecules are also expressed in specific  domains in both the adult and the developing brain (Rubenstein et al. 1999, Suzuki et al. 1997, Inoue et al. 1997). Based on these findings, the cadherin proteins are proposed to give rise to the complex and precise wiring pattern of neural connections found in the brain (Fannon et al. 1996, Martinek et al. 1997, Obst-Pernberg et al. 1999). model,  since  According to this  cadherin proteins bind in a homophilic manner, neuronal processes  expressing a particular type of cadherin molecule only make connections with other processes that express the same kind of cadherin molecule.  The large number of cadherin  protein subtypes expressed in the brain is postulated to facilitate the formation of various neural circuits.  The existence of cadherin molecules that are specifically expressed by  particular neural tracts gives merit to this hypothesis (Arndt et al. 1996, Suzuki et al. 1.997, Obst-Pernberg et al. 1999, Inoue et al. 1998, Rubenstein et al. 1999). Evidence for the involvement of cadherin proteins in activity dependent plasticity is also present.  In the developing somatosensory cortex N-cadherin expression is regulated  in an activity dependent manner (Huntley et al. 1999). In hippocampal slices, the induction of L T P is blocked by preventing cadherin dimerization (Tang et al. 1998). Neural activity also appears to stabilize and strengthen cadherin mediated synaptic adhesion (Tanaka et al. 2000).  Since events like synaptogenesis,  formation of neural circuits and activity  dependent plasticity are associated with visual development, the beta-catenin/cadherin cell adhesion system is an intriguing molecular candidate for being involved in postnatal visual system development.  105  POSTSYNAPTIC NEURON  Figure 4.1 Cadherin Protein Distribution in Neurons.  106  HYPOTHESIS:  The tissue distribution and the expression profile of the cadherin proteins in the postnatally developing cat visual system correlate with the critical period.  OBJECTIVES:  1.) T o determine temporal expression and the tissue distribution of the cadherin proteins in the postnatally developing cat visual cortex and the L G N .  4.2 METHODS  Animal preparation, Immunohistochemistry and Western blots: See Methods Section in Chapter three. 5 additional animals N = l PO, N=2 P60 and N=2 Adult had to be sacrificed for western blot analysis in order to generate more tissue. The pan-cadherin antibody was purchased from S I G M A .  4.3 RESULTS 4.3.1  Partial Cloning of Cadherin Genes Expressed in the Postnatal Cat  Visual Cortex In order to determine the different kinds of cadherin molecules expressed in the developing visual cortex, the c D N A ' s of various cat cadherin genes were partially cloned using the R T - P C R strategy described in chapter 2.  Initially, the expression of cadherin 6,  8 and 11 were examined in the developing visual cortex.  These cadherins were examined  because they demarcate neural circuits and their expression is retained in the adult brain (Suzuki et al. 1997). Figures 4.2, 4.3 and 4.4 show the partial c D N A sequences of the cat  107  Cat  :  1 ttgaccgagaaacattgctgtggcacaacattacagtaatagcaacagagatcaataatc 60 I I I II I II I I I I I I I I I I I I' I I I I I I I I I I I I I I I I I II II I I I I I I I I I I I I I I I I Human:1457 t t g a c c g a g a a a c a c t g c t a t g g c a c a a c a t t a c a g t g a t a g c a a c a g a g a t c a a t a a t c 1516 Cat  :  61 c a a a g c a a a g c a g c c g a g t c c c t c t a t a t a t t a a a g t c c t a g a t g t c a a t g a c a a t g c c c 120 I I I I I I I I I I II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I Human:1517 c a a a g c a a a g t a g t c g a g t a c c t c t a t a t a t t a a a g t t c t a g a t g t c a a t g a c a a c g c c c 1576 Cat  : 121 c g g a a t t t g c t g a a t t c t a t g a a a c c t t t g t c t g t g a a a a g g c a a a a g c a g a t c a g t t g a 180 I I I I I I I I I I I I II I I I I I I I I I I I I I I I I II I I I I I I I I I I I I I II I I II I I I I Human:1577 c a g a a t t t g c t g a g t t c t a t g a a a c t t t t g t c t g t g a a a a a g c a a a g g c a g a t c a g t t g a 1636 Cat  : 181 t t c a g a c c c t a c g a g c c a t t g a c a a a g a c g a c c c t t a t a g t g g g c a c c a a t t c t c a t t c t 240 I I I I I I I I I I I II I I I I I I I II I I I I I I I I I I I I I I I I I I I I I I II II I Human:1637 t t c a g a c c c t g c a t g c t g t t g a c a a g g a t g a c c c t t a t a g t g g a c a c c a a t t t t c g t t t t 1696 Cat  : 241 c t t t g g c c c c t g a a g c a a c c a g t g g c t c a a a c t t t a c c a t t c a a g a c a a c a a a g a t a a c a 300 I I I I I I I I I I I I I I I I I I II I I I I I I I I II I I I I I I I I I I I I I I I I I I I I I I I I I I I Human:1697 c c t t g g c c c c t g a a g c a g c c a g t g g c t c a a a c t t t a c c a t t c a a g a c a a c a a a g a c a a c a 1756 Cat  : 301 c a g c a g g a a t c t t a a c t c g g a a a a a t g g c t a t a a t a g a c a c g a g a t g a g c a c c t a t c t c t 360 I II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I Human:1757 c g g c g g g a a t c t t a a c t c g g a a a a a t g g c t a t a a t a g a c a c g a g a t g a g c a c c t a t c t c t 1816 Cat  : 361 t g c c t g t g g t c a 372 IIIIIIIIIII I Human:1817 t g c c t g t g g t c a 1828  Figure  4.2  Partial  cDNA  sequence  of  the  cat  cadherin  B l a s t Score - 507 b i t s (256), Expect = e-142 I d e n t i t i e s = 343/372 (92%) Strand = Plus / Plus  108  6  gene.  Cat  60  1  Human: 1253 g t c c t a t a a g g t t t t c c a t c g a c c g g c a c a c t g a c c t g g a g a g g c a g t t c a a c a t t a a t g 1312 Cat  120  61  Human: 1313 c a g a c g a t g g g a a g a t a a c g c t g g c a a c a c c a c t t g a c a g a g a a t t a a g t g t a t g g c a c a 1372 Cat  : 121 a c a t a a c a a t c a t t g c a a c c g a a a t t a g g a a c c a c a g t c a g a t a t c t c g a g t a c c t g t t g 180 I I I I I I I I I I I I I I I I II I I II I I I I I I I I I I I I I I I I I I I I I I M i l Human: 1373 a c a t a a c a a t c a t t g c t a c t g a a a t t a g g a a c c a c a g t c a g a t a t c a c g a g t a c c t g t t g 1432 Cat  : 181 c t a t t a a a g 189 I I I II I I II Human: 1433 c t a t t a a a g 1441 Figure 4.3  Partial cDNA sequence of the cat cadherin 8 gene.  B l a s t Score = 256 b i t s (129)., Expect = 2e-66 I d e n t i t i e s = 174/189 (92%) Strand = P l u s / P l u s  109  Cat  :  1 tattccatcgatcgtcatactgacctcgacaggtttttcactattaatccagaggatggt 60 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I II I I I I I I I II I I I I I I I I I I II I Human:1731 t a t t c c a t c g a t c g t c a c a c t g a c c t c g a c a g a t t t t t c a c t a t t a a t c c a g a g g a t g g t 1790 Cat  : 61 t t t a t t a a a a c c a c a a a a c c t t t g g a c a g a g a g g a a a c t g c c t g g c t c a a c a t a t c t g t t 120 I I I I I I I I I I I I I I II I II I I I II I I I I I I I I I II I I I I I I I I I I I I I I I I I I Human:1791 t t t a t t a a a a c t a c a a a a c c t c t g g a t a g a g a g g a a a c a g c c t g g c t c a a c a t c a c t g t c 1850 Cat  : 121 t t t g c a g c a g a a a t c c a c a a c c g g c a t c a g g a a g c c a a a g t g c c a g t g g c c a t t a g g g t c 180 I I I I II II II I I I I I I I I II I I I I I I I II I I I I I I I I I I I I I I I I I I I I II I I I II I I Human:1851 t t t g c a g c a g a a a t c c a c a a t c g g c a t c a g g a a g c c a a a g t c c c a g t g g c c a t t a g g g t c 1910 Cat  : 181 c t t g a t g t c a a c g a t a a t g c t c c c a a g t t t g c c g c c c c t t a t g a a g g c t t c a t c t g t g a g 240 I II II II I I I I I I II I I I I I II II I I II I I I I II I I I II I I I I I I I II I I I I II I I II Human:1911 c t t g a t g t c a a c g a t a a t g c t c c c a a g t t t g c t g c c c c t t a t g a a g g t t t c a t c t g t g a g 1970 Cat  : 241 a g t g a t c a g a c c a a g c c a c t t t c t a a c c a g c c a a t t g t t a c a a t t a 286 I I I II I I II II II I I I I I II II I I I I I II II I I I I I I I I I I I I I I Human:1971 a g t g a t c a g a c c a a g c c a c t t t c c a a c c a g c c a a t t g t t a c a a t t a 2016 Figure  4.4  Partial cDNA sequence of the cat cadherin 11 gene.  B l a s t Score = 440 b i t s (222), Expect = e-121 I d e n t i t i e s = .272/286 (95%) Strand = Plus / Plus  110  cadherin 6, 8 and 11 genes. The degree of identity between the human and the partially cloned cat cadherin c D N A ' s is 92% for cadherin 6 and cadherin 8, and 95% for cadherin 11.  The  cadherin  6  cDNA  was  ATGCTGCCAGGAATCCTGTCAAG  generated and  by  PCR  using  the  primers  TGACCACAGGCAAGAGATAGGTG,  cadherin 8 c D N A was generated using primers A A C C T C T G G A G C C T C C T T C T G A A and G C G T T G T C A T T G A C A T C C A G C A C , and cadherin 11 c D N A was generated using the . primers  CGATCGTCACACTGACCTCGACA  GTACAAGTCCTGCTTCTGCCGAC.  and  The amino acid sequence of these c D N A ' s and  their homology to the human amino acid sequence is shown in Figure 4.5.  A s expected,  the homology between the cat and the human cadherin genes is more extensive at the protein level. Clearly, cadherin antibodies specific to the human versions of these proteins should cross-react with the cat cadherin proteins. The presence of all three of the cadherin genes postulated to be expressed in the visual cortex led to the realization that a large number of cadherin types are likely to be expressed in this structure. The cloning and examination of each cadherin type expressed in the visual cortex is an enormous task and beyond the scope of this thesis. Additionally, it is unclear which cadherin molecule(s) is of importance in postnatal  cat  visual  development. Hence, instead of examining the expression profile of one cadherin protein, we decided to monitor the expression of several cadherin molecules  at once.  The  cumulative expression pattern of a group of cadherin molecules was determined in the developing cat visual system by using a pan-cadherin antibody capable of recognizing several different cadherin proteins.  4.3.2 Selection of the Pan-Cadherin Antibody The  pan-cadherin  antibody  produced  immunohistochemical and western blot analysis.  by  SIGMA  was  used  for  This is a well characterized antibody  which is known to cross react with several members of the classic cadherin gene family. The specificity of this antibody in other species is well established and it has been used in  1 11  CADHERIN 6 AMINO ACID SEQUENCE  Cat  : 374 DRETLLWHNITVIATEINNPKQSSRVPLYIKVLDVNDNAPEFAEFYETFVCEKAKADQLI 195 DRETLLWHNITVIATEINNPKQSSRVPLYIKVLDVNDNAPEFAEFYETFVCEKAKADQLI Human: 4 47 DRETLLWHNITVIATEINNPKQSSRVPLYIKVLDVNDNAPEFAEFYETFVCEKAKADQLI 506 Cat  : 194 QTLRAIDKDDPYSGHQFSFSLAPEATSGSNFTIQDNKDNTAGILTRKNGYNRHEMSTYLL 15 QTL A+DKDDPYSGHQFSFSLAPEA SGSNFTIQDNKDNTAGILTRKNGYNRHEMSTYLL Human: 507 QTLHAVDKDDPYSGHQFSFSLAPEAASGSNFTIQDNKDNTAGILTRKNGYNRHEMSTYLL 566 Cat  :  14 PVV 6 PVV Human: 567 PVV 569 B l a s t Score = 250 b i t s (632), Expect = 8e-67 I d e n t i t i e s = 120/123 (97%), P o s i t i v e s = 121/123 (97%) CADHERIN 8 AMINO ACID SEQUENCE  Cat  : 187 PIRFSVDRHTDLERQFNINAVVGKITQATPLDRELSVWHNITIIATEIRNHSQISRVPVA 8 PIRFS+DRHTDLERQFNINA GKIT ATPLDRELSVWHNITIIATEIRNHSQISRVPVA Human: 417 PIRFSIDRHTDLERQFNINADDGKITLATPLDRELSVWHNITIIATEIRNHSQISRVPVA 476 Cat  :  7 IK 2 IK Human: 4 77 IK 4 78 B l a s t Score = 118 b i t s (293), Expect = l e - 2 7 I d e n t i t i e s = 58/62 (93%), P o s i t i v e s = 59/62 (94%)  CADHERIN 11 AMINO ACID SEQUENCE Cat  : 285 YSIDRHTDLDRFFTINPEDGFIKTTKPLDREETAWLNISVFAAEIHNRHQEAKVPVAIRV 106 YSIDRHTDLDRFFTINPEDGFIKTTKPLDREETAWLNI+VFAAEIHNRHQEA+VPVAIRV Human: 419 YSIDRHTDLDRFFTINPEDGFIKTTKPLDREETAWLNITVFAAEIHNRHQEAQVPVAIRV 478 Cat  : 105 LDVNDNAPKFAAP 67 LDVNDNAPKFAAP Human: 479 LDVNDNAPKFAAP 491 B l a s t Score = 151 b i t s (378), Expect(2) = l e - 4 6 I d e n t i t i e s = 71/73 (97%), P o s i t i v e s = 73/73 (99%) Cat  : 70  PYEGFICESDQTKPLSNQPIVTI 2 PYEGFICESDQTKPLSNQPIVTI Human: 491 PYEGFICESDQTKPLSNQPIVTI 513 B l a s t Score = 52.7 b i t s (124), Expect(2) = l e - 4 6 I d e n t i t i e s =23/23 (100%), P o s i t i v e s = 23/23 (100%)  Figure  4.5  Alignment o f t h e c a t and t h e human amino a c i d sequences. 112  cadherin  the rat brain for immunohistochemical localization of cadherin proteins (Tang et al. 1998, Benson et al. 1998).  These experiments have shown this antibody to colocalize with  synaptophysin and other synaptic markers.  Furthermore, this antibody can block the  induction of L T P in rat hippocampal slices by interfering with cadherin function (Tang et al. 1998).  4.3.3.  Western Blot Analysis  O f Cadherin  Protein  Expression  In The  Postnatally Developing Cat Visual Cortex Figure 4.6 shows a western blot analysis performed in the postnatally developing cat visual cortex using the S I G M A pan-cadherin antibody.  This blot consists of visual  cortex protein preparations obtained from prenatal, 0 day postnatal, 15 day postnatal, 30 day postnatal, 60 day postnatal, 120 day postnatal and adult cats. In each age a single band of roughly 130kDaltons is detected confirming the fact that this antibody is able to recognize the cat cadherin proteins. Despite the use of a pan-cadherin antibody capable of recognizing multiple cadherin types, only a single band is observed in the western blot analysis shown in figure 4.6.  This outcome is not too surprising because many cadherin  proteins (especially the classic cadherin molecules) are 130 k D A in size (Tanihara et al. 1994). Since only a single band of the appropriate size is observed in this western blot, the S I G M A pan-cadherin antibody appears to specifically recognize the cadherin proteins in the cat.  Although the ability of the S I G M A pan-cadherin antibody to recognize  several  different cadherin types is well established, this antibody clearly does not recognize every possible kind of cadherin molecule expressed in the cortex.  Protocadherins which are  larger than 130 kDa and other cadherins of variant sizes are not detected by this antibody. Hence, the Sigma antibody is likely recognizing a subset of the cadherin molecules present in the visual cortex.  However, because western blot analysis using this antibody yields  only a single band, the possibility that this cadherin antibody is detecting only one cadherin protein can not be ruled out (Based on the reported expression of cadherin types in the brain and the P C R results mentioned in the cloning experiment it is unlikely that there is  113  Prenatal  Figure 4.6  Oday  15 day  30 day  60 day  120 day  Adult  Cadherin protein expression in the postnatally developing cat visual  cortex. The top panel shows the western blot analysis performed on the postnataly developing cat visual cortex using the S I G M A pan-cadherin antibody. This antibody specifically recognizes a single band of around 130 k D A in visual cortex protein preparations obtained from kittens of various different ages. The expression of these proteins is high in the young visual cortex but it declines sharply around 60 days of age. This expression pattern for the cadherin proteins is consistent with the timing of the critical period.  For the loading control (the bottom panel), the membrane used for the  western blot analysis was stained with Coomasie blue.  114  only one type of cadherin molecule expressed in the postnatal cat visual cortex).  In  addition, the pan-cadherin antibody may be biased in its ability to recognize certain types of cadherin proteins. Since equal amounts of protein was loaded into each well (100 jxg), the western blot analysis shown in figure 4.6 can be used to compare cadherin protein expression levels in the postnatally developing cat visual cortex. This expression pattern for the cadherin proteins in the developing visual cortex was confirmed by repeating the western blot analysis thrice on three different protein blots.  Clearly, cadherin protein expression in the  developing visual cortex parallels the critical period.  A high level of cadherin protein  expression is observed in the visual cortex of young kittens.  This expression decreases  around 60 days of age and cadherin protein levels remain reduced in the adult visual cortex.  4.3.4.  Western Blot Analysis of the Cadherin Protein Expression in the  Postnatally Developing Cat L G N Figure 4.7 shows western blot analysis performed in the postnatally developing cat lateral geniculate nucleus using the S I G M A pan-cadherin antibody. This blot consists of L G N protein preparations obtained from postnatal 0 day, 15 day, 30 day, 60 day, 120 day and adult cats. A s in the visual cortex, each lane in the western blot contains a single band confirming the specificity of the cadherin antibody in the cat L G N .  Also, the western blot  analysis of the cadherin proteins in the L G N was repeated three times to ensure that the observed expression pattern is not artifactual.  The expression pattern of the cadherin  proteins in the L G N is different than in the visual cortex but this expression profile is consistent with the critical period since a large decrease in cadherin protein expression occurs in the geniculate of adult cats.  4.3.5.  Immunohistochemical  Localization of  Cadherin  Proteins  in  the  Postnatal Visual Cortex The S I G M A pan-cadherin antibody characterized by western blot analysis in the last two sections was used to localize cadherin protein expression in the postnatally developing  115  Oday  15 day  30 day  60 day  120 day  Adult  133 k D A 71 k D A  42 k D A  LOADING CONTROL  Figure 4.7  Cadherin protein expression in the postnatally developing cat L G N . The  top panel shows the western blot analysis performed on the developing cat L G N using the S I G M A pan-cadherin antibody. This antibody specifically recognizes a single band of around 130 k D A in L G N protein preparations obtained from postnatal kittens of various different ages. The expression of this protein is high in the geniculate of young animals but it declines sharply in the adult L G N . For the loading control (the bottom panel), the membrane used for the western blot analysis was stained with Coomasie blue.  116  cat visual cortex. subjected  Figure 4.8 shows a 60 day old visual cortex tissue section from a cat  to irnmunohistochemical analysis  with the pan-cadherin antibody.  Since  immunohistochemical analysis was performed using a pan-cadherin antibody, the observed staining pattern likely represents the cumulative expression pattern of several different types of cadherin molecules in the visual cortex. Similar to the observed expression of the betacatenin protein discussed in the previous chapter, the laminar distribution of the cadherin protein in the developing visual cortex does not change with age. include 0 day, 20 days, 30days, 45 days, 55 days, 60 days,  Postnatal ages examined 120 days and adults.  Interestingly, this antibody strongly labels layer IV of the visual cortex, which is the thalamic input layer. Additionally, the pan-cadherin antibody staining is much more intense in visual areas of the cortex. Heavy pan-cadherin antibody staining is observed in layer rV of cortical area 17 and 18 (primary and secondary visual areas in the cat).  This staining  pattern in the visual cortex is consistent with the restricted expression patterns reported for the cadherin proteins in other brain areas.  Cadherin staining is also visible in the higher  layers of the cortex (2/3). Similar to beta-catenin and synaptophysin, cadherin staining is observed in the neuropil of the lateral geniculate and visual cortex (shown in Figure 4.9).  Neutral Red  staining clearly illustrates cadherin labeling to be outside of cell bodies. Unlike betacatenin, cadherin proteins do not have transcriptional activity and they do not translocate into the nuclei of cells.  Accordingly, cadherin protein expression is restricted to the  neuropil, as seen in Figure 4.8, throughout development in both the cortex and the L G N . This distribution of the cadherin proteins is consistent with their adhesive functions.  117  118  « 3 e *  9?  1 -s CO  ^  —  Q. O  g  § 3  U  «O .2 i "3  H •3  *  0  i l l  re  U  00 S — cn  u  3  III  C5  I z  H Z  E  GO  a.  Q O  3  .52  S 2 u  re .3  *- iS3f c a l lS 1a  •c 3 a >  1 P 5 1 8 €  S re  > ffi .5  o re  E  a  ro  SI I  8 £ fi »  s i Is § 8~  119  E  4.4 DISCUSSION The findings in this chapter support the involvement of the cadherin molecules in the postnatal development of the cat visual cortex.  Firstly, expression of these proteins in  the visual cortex closely correlates with the critical period.  Cadherin molecule expression  in the cortex decreases substantially at 60 days of age and remains low in adulthood. Secondly, cadherin proteins detected by the pan-cadherin antibody clearly label layer IV of the cortex. Layer TV is the input layer of the cortex and neurons in this layer form connections with the afferents from the geniculate.  A molecule that is expressed in  layer I V of the cortex and whose expression correlates with the critical period is likely to be involved in the reorganization of thalamocortical connections. The case for the involvement of cadherin molecules  during visual development  is further bolstered by the  characterized properties of these molecules in other areas of the brain.  well  The synaptically  localized cadherin proteins (Uchida et al. 1996) act as cell adhesion molecules and are believed to take part in such processes as synaptogenesis, target recognition, and axon migration (Arndt et al. 1996, Benson et al. 1998, Huntley et al. 1999, Riehl et al. 1996, Matsunaga et al. 1988, Obst-Pernberg et al. 1999, Redies et al. 1996, Rubenstein et al. 1999, Suzuki et al. 1997).  Unfortunately, the results of the activity dependent studies  carried out in Chapter 5 do not support the involvement of these molecules in activity dependent modifications occurring in the visual cortex. The age dependent decrease in cadherin protein expression in the developing visual system supports the proposed role of nuclear beta-catenin in downregulating the expression of cell adhesion molecules during the critical period.  The downregulation of cadherin  protein expression detected in the L G N of older kittens may be partially mediated by the transcription factor activity of the nuclear beta-catenin protein in geniculate neurons.  In  addition to its downregulation in the L G N , cadherin protein expression in the visual cortex decreases with age. L G N processes projecting into the cortex may be partially responsible for this observed decrease since cadherin molecules are located on both presynaptic and  120  postsynaptic terminals (the cadherin proteins residing in the presynaptic portion of the thalamocortical connections  are synthesized  by geniculate  neurons).  Unfortunately,  presynaptic thalamic terminals can not be separated from cortical tissue and the proteins present in these terminals had to be included in the visual cortex protein preparations used for Western Blot analysis. Since most of the cadherin protein expression is in layer IV of the visual cortex (the input layer of the L G N ) , the cadherins proteins located in thalamic terminals are likely to make a significant contribution to the overall cadherin protein levels in visual cortex protein preparations. In addition, the timing of beta-catenin nuclearization in the L G N and the decrease in cadherin expression in the visual cortex occur almost simultaneously. This finding further supports the proposed role of nuclear beta-catenin in downregulating the expression of cell adhesion molecules in L G N neurons. Additionally, the nuclearization of beta-catenin is reported to downregulate the expression of only certain types of cadherin molecules (Stewart et al. 2000, Nuruki et al. 1998).  Since the pan-  cadherin antibody used for monitoring cadherin levels may also detect cadherin proteins which are not downregulated by beta-catenin activity, the observed decrease in cadherin protein expression in the L G N and the visual cortex may not be reflective of the regulatory action imposed by nuclear beta catenin. In other words, the expression of specific types of cadherin proteins in the visual cortex and the L G N may be downregulated in a much more drastic manner during the critical period. A s an aside, the regulatory effects of the betacatenin protein in cancerous cells is not limited to the cadherins but it also encompasses other cell adhesion molecules. Hence, the expression of cell adhesion molecules other than the cadherins may be downregulated in L G N neurons by nuclear beta-catenin.  However,  beta-catenin is probably not the only factor responsible for the downregulation of cadherin protein expression in the L G N because beta-catenin nuclearization is only observed in a subset of neurons in the L G N .  The downregulation of cadherin protein expression in a  small number of cells is unlikely to cause a decrease in the overall levels of cadherin proteins in the cortex or the L G N . Regardless, there is a decrease in cell adhesion molecule  121  levels during the critical period in both the cortex and the L G N .  One of the processes  responsible for this downregulation is proposed to be the nuclear activity of beta-catenin in L G N neurons. The presence of cadherin proteins which are expressed mainly in layer IV of the visual cortex suggests these molecules to function as target recognition cues for axons originating from geniculate neurons and other cortical areas.  Since various cadherin  proteins are expressed in specific domains and demarcate specific brain areas, the cadherin proteins are believed to facilitate the proper wiring of neurons (Suzuki et al. 1997, Inoue et al. 1997, Inoue et al. 1998, Kimura et al. 1996, Matsunami et al. 1995, Redies et al. 1996, Redies  2000).  Because cadherin molecules bind in a homophilic manner (Nose et al.  1988, Nose et al. 1990, Miyatani et al. 1989, Leckband et al. 2000) the thalamocortical axons arising in the geniculate must express the same types of cadherin molecules as the cortical cells in layer IV. B y restricting the expression of a subset of cadherin molecules to layer IV and L G N neurons, axons originating from the geniculate may be guided to make connections in layer IV of the visual cortex. According to this model, since other layers of the cortex do not express cadherin proteins which are identical to those located on thalamic axons, geniculate neurons have no choice but to make connections with layer IV cortical cells. Similarly, a sub population of neurons in the cortex expressing a particular type of cadherin protein may be restricted to forming synaptic connections with neurons in the visual cortex containing the same cadherin molecules.  Hence, cadherin molecules may be  carrying out an organizational function in the visual cortex and facilitating the formation of proper connections between visual areas of the cortex and the thalamus. The involvement of N-cadherin in the organization of thalamocortical synapses  during barrel  cortex  development supports this conjecture (Huntley et al. 1999). The restricted expression domains of cadherin cell adhesion molecules provide indirect support for the involvement of homeotic genes in postnatal visual development. Cadherin protein expression patterns characterized in non-visual areas of the brain, which  122  are both lamina and brain region specific, are very reminiscent of the reported distribution pattern of the homeobox genes. A s discussed in the introduction, homeotic and segment polarity genes are expressed in specific domains and give rise to various patterns such as columns and stripes (Wilkinson et al. 1989a, Wilkinson et al. 1989b, Murphy et al. 1989, Bell et al. 1999). The similarity between cadherin molecule and homeotic gene expression is not coincidental. Homeotic and homeobox genes regulate the expression of various cell adhesion molecules (Valarache et al. 1993, Tissier-Seta et al. 1993, Edelman et al. 1995, Edelman et al. 1998, Cillo et al. 1996, Wang et al. 1996, Tosney et al. 1995) including the cadherins (Lincecum et al. 1998, Packer et al. 1997).  B y differentially regulating the  activity of cell adhesion molecules, homeotic genes are able to subdivide and cause differentiation amongst a group of similar cells.  For example,  during early brain  development, the neural tube gets divided into compartments known as rhombomeres. Each rhombomere expresses a distinct combination of homeobox genes that activates the expression of a unique set of cell adhesion molecules (Wilkinson et al. 1989a, Wilkinson et al. 1989b, Murphy et al. 1989, Bell et al. 1999).  The restriction of cell to cell interactions  and cellular migration to individual rhombomeres causes the rhombomeres to diverge from each other and leads to the formation of different nervous system structures (Fraser et al. 1990).  Similarly, the differential expression of LIM homeobox genes in the spinal cord  causes each level of the spinal cord to express a distinct set of cell adhesion molecules (Lumsden 1995, Tosney et al. 1995).  The differential expression of adhesion molecules  by spinal cord neurons enables the spinal cord to make connections  with  different  peripheral neurons. In other words, differential adhesion allows the spinal cord to become properly wired with the appropriate peripheral neurons.  In the last paragraph, this sort of  an arrangement was proposed to exist between the visual cortex and lateral geniculate afferents.  In addition, if the homeobox  genes regulate the expression  of cadherin  molecules in the developing cat visual cortex then their distribution in this structure may be similar to the distribution of the cadherin proteins.  123  The data in chapter two specifically  show the expression of various developmental genes to decrease, including the cofactor PBX family of homeobox genes, in the adult visual cortex. Hence, the expression of the PBX  homeotic genes and the cadherin molecules parallel each other during visual  development.  Concievably, the downregulation of homeotic gene expression in older  kittens may cause cell adhesion molecule expression to decrease (the data presented in this chapter and Schoop et al. 1997) in the adult visual cortex.  124  Chapter 5 ACTIVITY DEPENDENT EXPRESSION O F BETACATENIN AND T H E CADHERIN PROTEINS 5.0 SUMMARY In order to determine the effect of visual activity on beta-catenin and cadherin protein expression, immunohistochemical analysis was performed on animals subjected to various visual deprivation paradigms (monocular eyelid suture, enuclation, strabismus followed by monocular eyelid suture or enucleation and optic tract lesions).  Beta-catenin  and cadherin protein expression is not modified in the cat visual cortex by these visual deprivation paradigms. Nuclear beta-catenin protein expression also appears unaltered in the L G N of animals subjected to either monocular deprivation or optic tract lesions. However, following monocular deprivation, the beta-catenin and the cadherin proteins are expressed in higher amounts in layers of the geniculate corresponding to the deprived eye. This finding implies a role for the beta-catenin and the cadherin cell adhesion system in the competition occurring between X - and Y - cells in the postnatal L G N .  5.1 INTRODUCTION As discussed in the first chapter, synaptic rearrangements in the visual system are largely dictated by activity (Wiesel et al. 1963, Hubel et al. 1977).  Activity dependent  synaptic changes contribute not only to the development of the visual system but to the formation  of  various  other  neural structures  including  the  spinal  cord  and  the  somatosensory cortex (Webster et al. 1983, Meisami et al. 1981, Westrum et al. 1986, Wooseley et al. 1976,  Garraghty et al. 1992,  Pons et al.  1991).  The molecular  mechanisms contributing to synaptic modifications in the developing visual cortex are also  125  believed to be involved in learning, memory and synaptogenesis (Bienenstock et al. 1982, Bear et al. 1987). Essentially, activity dependent synapse formation, which is a recurring theme in neurodevelopment, is of paramount importance in the proper formation of neural circuits. Although the contribution of neural activity to synapse formation and maintenance is well established both in the visual system and elsewhere, the molecular events that facilitate these processes remain elusive. A n y molecule involved in the synaptic reorganization of the cat visual system is likely to be regulated in an activity dependent manner. with synaptic modification need to modification process.  be  present  Obviously, molecules  and functioning  associated  during the  actual  Since interfering with normal visual activity can induce synaptic  rearrangements in the postnatally developing visual system (Shatz et al. 1978, Antonini et al. 1993, Cynader et al. 1980, Cynader et al. 1982, Olson et al. 1975, Wiesel et al. 1963) the expression of molecules involved in synaptic rearrangements should be altered in response to these deprivation paradigms.  Examples of molecules regulated by visual  activity include the neurotrophins (Castren et al. 1992, Schoups et al. 1995, Torasdotter et al. 1996), immediate early genes (Kaplan et al. 1996, Mower 1994) and the cytochrome oxidase (Wong-Riley 1979) proteins.  Since molecules involved in visual plasticity are  likely to be regulated in an activity dependent manner, the expression of the beta-catenin and the cadherin proteins was examined in the visual systems of kittens subjected to various deprivation paradigms. The function and the expression of both the cadherin and beta-catenin molecules are influenced by activity in various regions of the C N S .  For example, N-cadherin is  expressed in an activity dependent manner during the formation of "barrels" in the developing somatosensory cortex (Huntley et al. 1999).  Given the similarity between  barrel cortex and visual cortex development, it is not unreasonable to assume that cadherin expression will be regulated in an activity dependent manner in the visual cortex.  Tissue  culture experiments have also shown beta-catenin protein distribution to be altered by  126  neural activity (Murase et al. 1999). Additionally, the cadherins dimerize and become more resistant to protease digestion in response  to neural activity (Tanaka et al. 2000).  Furthermore, the ability to inhibit L T P induction by preventing cadherin molecule dimerization strongly suggests the cadherins to be an integral part of activity dependent plasticity (Tang et al. 1998).  HYPOTHESIS:  Beta-catenin and the cadherin cell adhesion protein expression is regulated in an activity dependent manner in the postnatal cat visual system.  O B J E C T I V E S : 1.) T o determine i f beta-catenin or cadherin protein expression can be altered by the monocular deprivation paradigm. 2. ) T o determine if beta-catenin or cadherin protein expression can be maintained at high levels following the optic tract lesion paradigm which prolongs the critical period. 3. ) T o determine if the distribution of nuclear beta-catenin can be altered by monocular deprivation or optic tract lesion paradigms.  5.2 METHODS Animals used For monocular deprivation studies 26 kittens were used.  40 day old postnatal  kittens were monocularly deprived for 3 days N=4, for 7 days N=3, for 2 weeks N=2 or 5 weeks N=2.  Alternatively 4 kittens of the same age were monocularly enucleated for 2  weeks N=2 or 5 week N=2.  6 postnatal kittens were made strabismic around 20 days of  age and then monocularly deprived for 1 week at 40 days of age N=2, for 2 weeks N=2 or 5 weeks N=2. 2 postnatal kittens which were also strabismic were enucleated at 40 days of age and 1 was allowed to survive for 2 weeks whereas the other was allowed to survive  127  for 5 weeks. 2 animals made strabismic at postnatal day 15 and monocularly deprived for 5 days at age 55 days were also used.  1 cat made strabismic at postnatal day 15 and  monocularly deprived for 5 days at 155 days was also examined. For optic tract lesion experiments 5 postnatal kittens were used. These animals had their optic tracts lesioned around 20 days of age and allowed to survive until 90 days of age N=2 or 120 days of age N=3.  Immunoprecipitation 300-400mg of brain tissue was  placed into ice cold solubilization  solution  containing 50mM Tris-Cl (pH7.7), 0.5% NP-40, lOOmM N a C l , 2.5mM E D T A , NaF,  0 . 2 m M sodium orthovanadate, I m M sodium molybdate, 40pg/ml P M S F ,  lOmM luM  pepstatin, 0.5u.g/ml leupeptin, lu.g/ml aprotinin. Note that all the solutions and the tubes used for immunoprecipitation were cooled on ice before use and all the microcentrifugation was performed at 4 ° C . and  Additionally, the protease inhibitors P M S F , pepstatin, leupeptin  aprotinin were added from stock solutions immediately prior to use  and fresh  solubilization buffer was made each time. The brain tissue in the solubilization buffer was teased apart with forceps and the solution was allowed to sit on ice for 30 minutes.  Every  ten minutes the solution was pipetted to improve solublization of the tissue. After the thirty minutes, the solution was centrifuged at 15000 x g for 1 minute and the supernatant was transferred to a new tube (pre-cooled) on ice. Note that at times this preparation was found to be quite cloudy after only 1 spin and if the immunoprecipitation procedure was carried out using a cloudy solution, precipitates would form during the antibody incubation step. Successful immunoprecipitation could not be attained (as determined by western blot analysis) when precipitates were present during the antibody incubation step.  The  cloudiness is believed to be caused by myelin since the supernatants had a whiteish color. Spinning an additional 2 to 3 times for 1 minute at 15000 x g and aspirating the supernatant into a new tube each time resolved this problem. The protein sample was quantitated and  128  the appropriate volume of the sample containing l m g of total protein was placed into a fresh tube (300-400mg of brain tissue roughly yields 3-5mg of total protein which is enough to carry out several immunoprecipitation reactions concurrently). The total volume of the protein solution was increased to 500ul's with solubilization buffer.  60ul's of this  solution was set aside and labeled as the Pre- fraction. 8ug's of antibody was added to the  protein solution and the antibody incubation was allowed to proceed overnight at 4 ° C on a rotator.  Fresh solubilization solution was prepared the following day because protease  inhibitors do not remain active overnight. 50ul's of protein G beads were spun down in a  new eppendorf tube at 10000 x g in 4 ° C for 1 minute. 25uTs of solubilization buffer was added to the beads and the beads were spun down as before.  This wash step was repeated  3 times. After the final wash, 25ul's of solubilization buffer was added and the protein G bead slurry was added to the protein sample which had been incubating overnight.  The  antibody/protein complex was incubated with the protein G beads for 1 hour at 4 ° C on a rotator. After this incubation, the protein/antibody complex is attached to the protein G beads and not in solution. The beads were pelleted by centrifuging in 4 ° C for 1 minute at 10000 x g.  60uTs of the supernatant was set aside as the Post- fraction while the rest of  the supernatant was discarded. The pelleted protein G beads were washed 5 times with l m l of solubilization solution followed by 2 washes with a lOOmM N a C l solution to remove any proteins non-specifically bound to the beads. After the washes the beads were resuspended in 25ul's of loading buffer, boiled for 5 minutes and electrophoresed.  129  5.3 RESULTS 5.3.1  Effects  Of  Monocular  Deprivation  on  Beta-Catenin  Protein  Expression In The Postnatal Visual Cortex A n d The L G N In this set of experiments, 5-6 week old postnatal kittens had one of their eyes sutured shut or enucleated.  These kittens were allowed to survive for 3 days, 7 days, 2  weeks or 5 weeks before being sacrificed. Figure 5.1 is a picture of a visual cortex from a kitten deprived for 5 weeks stained with the beta-catenin antibody. Layer TV staining in the cortex is continuous and beta-catenin expression is not altered in response  to visual  deprivation. Hence, monocular deprivation does not appear to cause a detectable change in beta-catenin protein levels in the developing visual cortex. Changes in beta-catenin staining are also not observed in the cortex of animals deprived for shorter lengths of time. However, a clear change in the distribution of the beta-catenin protein in the L G N is observed in response to monocular deprivation.  Figure 5.2 shows beta-catenin staining  performed in the lateral geniculate of a postnatal kitten subjected to 5 weeks of monocular deprivation beginning at 5 weeks of age. Interestingly and unexpectedly, the layers of the L G N exhibiting darker beta-catenin neuropil staining correspond to the closed eye.  This  staining pattern was observed consistently in all animals which were deprived of visual activity for five weeks (normal animals monocularly deprived (n=2), strabismic animals monocularly deprived (n=2) and enucleated animals (n=2)). Beta-catenin expression in the L G N can be altered with 2 weeks of deprivation but this effect is relatively weak compared to the effect obtained with longer deprivation periods  (normal animals monocularly  deprived (n=2), strabismic animals monocularly deprived (n=2)).  Changes in beta-catenin  protein expression in the L G N are not observed in animals subjected to monocular deprivation or enucliation periods shorter than two weeks. Finally, Figure 5.3  shows nuclear beta-catenin distribution in the L G N of a  postnatal kitten monocularly deprived for 5 weeks.  Monocular deprivation or enculeation  does not appear to effect the nuclearization of the beta-catenin protein since the distribution  130  131  NORMAL  5 WEEKS MD  Figure 5.2  Beta-Catenin Antibody Staining in the L G N of Monocularly Deprived  and Normal Postnatal Kittens. The left panel is a picture of an L G N from a kitten which has been monocularly deprived for 5 weeks whereas the right panel is an L G N from a normal kitten.  The L G N shown in the monocularly deprived animal is the  geniculate which is contralateral to the deprived eye. Layer A and Layer C in this L G N , which are innervated by the deprived eye, contain stronger beta-catenin antibody staining compared to the A1 layer of the L G N which is innervated by the non-deprived, ipsilateral eye. The scale bar for this figure represents 2mm's.  132  Figure 5.3  Nuclearization of the beta-catenin protein in the postnatal cat L G N  following monocular deprivation. The picture shown in this figure is a cross section of a geniculate from a kitten which has been monocularly deprived for 5 weeks. Nuclear beta-catenin protein is found in all layers of the L G N and its widespread distribution is not distrupted by this deprivation paradigm. The deprived eye was contralateral to this LGN.  133  of nuclear beta-catenin in the L G N in normal and deprived animals is similar. Following monocular deprivation, cells containing nuclear beta-catenin are distributed through out the geniculate and they do not preferentially concentrate on either the deprived or non-deprived eye layers.  5.3.2  Effects of Monocular Deprivation on Cadherin Protein Expression In  The Postnatal Visual Cortex A n d The L G N Cadherin protein expression  was  also assessed by immunohistochemistry  postnatal kittens either monocularly deprived or enucleated at 5-6 weeks of age.  in  These  animals were sacrificed following 3 days, 7 days, 2 weeks or 5 weeks of deprivation. Similar to beta-catenin, monocular deprivation did not alter pan-cadherin antibody staining in the visual cortex of deprived animals (Figure 5.4).  However, pan-cadherin protein  expression is higher in layers of the L G N corresponding to the closed eye (figure  5.5).  The similar expression patterns of the beta-catenin and the cadherin proteins following monocular deprivation is consistent with the proposal that these molecules attach together to function as a cell adhesion system.  5.3.3  Effect of Optic Tract Lesion on the Expression of Beta-Catenin and  Cadherin Proteins B y inhibiting activity to the cortex, the optic tract lesion paradigm prolongs the critical period in the visual cortex (Singer 1982).  A n optic tract lesion is made after the  optic chiasm and unlike an optic nerve lesion where the input from only one eye is silenced, the optic tract lesion paradigm disconnects all the inputs to one L G N and its corresponding cortical hemisphere (Figure 5.6). Since optic tract lesions prolong the critical period in the geniculate and the cortex, older kittens which have had their optic tracts lesioned at an early age are expected to express plasticity related proteins at higher levels. 17 Day old postnatal kittens were subjected to unilateral optic tract lesions and allowed to survive until 120 days of age.  In this paradigm, the lesioned side of the brain retains a plastic L G N and cortex  whereas the contralateral side of the visual system essentially develops normally and loses  134  -  < s o  135  NORMAL  5 WEEKS MD  Figure 5.5  Pan-Cadherin Antibody Staining in the L G N of Monocularly Deprived  and Normal Postnatal Kittens. The left panel is a picture of an L G N from a postnatal kitten which has been monocularly deprived for 5 weeks whereas the right panel is an L G N from a normal postnatal kitten.  The L G N shown in the monocularly deprived  animal is the geniculate which is contralateral to the deprived eye. Layer A and Layer C in this L G N , which are innervated by the deprived eye, contain stronger pan-cadherin antibody staining compared to the A l layer of the L G N which is innervated by the nondeprived, ipsilateral eye. The scale bar for this figure represents 2mm's.  136  Layer IV of the Visual Cortex  Figure 5.6 Unilateral Optic Tract Lesion. The red and the blue colored lines represent the neural inputs originating from the two eyes. The black rectangle represents the location of the lesion which is after the optic chiasm. This lesion prevents visual activity from reaching the L G N and the cortex. + and - signs represent the presence or the absence of visual activity.  137  its plasticity. Hence, the unlesioned cortex and L G N can be used as a control to determine if immunohistochemical changes arise as a result of the lesions. The expression of the beta-catenin and the cadherin proteins are unaltered in cortical structures following optic tract lesions.  These findings are consistent with the reported  failure of the monocular deprivation paradigm to alter the expression of the cadherin and beta-catenin proteins in the visual cortex. However, the L G N ' s ipsilateral to the optic tract lesions exhibit higher amounts of beta-catenin and cadherin protein expression compared to the unlesioned L G N ' s (figure 5.7).  In this figure, brain tissue sections containing the  L G N ' s were digitized and psudeocolor images were produced using the N I H Image computer program.  The statistical analysis (shown in the tables) was performed by  measuring the grayscale intensity of both the lesioned and the normal L G N on each tissue section (n=10 sections). The student t-test analysis on these sections verifies the presence of a staining difference between the normal and the lesioned L G N . These findings are also consistent with the monocular deprivation studies which show activity dependent changes in the expression of beta-catenin and cadherin proteins to occur only at the thalamic level. In  addition, nuclear beta-catenin  subjected to optic tract lesions.  distribution appears  unaffected  in animals  As seen in figure 5.8 both the lesioned and the normal  geniculates appear to exhibit the same number of beta-catenin positive cells.  However, we  can not conclude that the optic tract lesion or the monocular deprivation paradigms do not modify nuclearization of beta-catenin. The level of nuclear beta-catenin in individual cells or the cells containing nuclear beta-catenin may change in response to these paradigms.  5.3.4  Co-Immunoprecipitation of  The Beta-Catenin A n d The  Cadherin  Proteins The corresponding increase in both beta-catenin and cadherin protein expression in the L G N following the deprivation paradigms described above suggests beta-catenin and the cadherin proteins to extensively interact with each other in the developing L G N . binding of the beta-catenin protein to the cadherin molecules is well established and is  138  The  c e & <D o  .2 o  8 .ta  & e  w  i.s£  -  P-value  a u n. > o a o o  2  S  in  — i  Q  0.359  Mean  d  difference  u —  z  oo Z '•B -2 o  3 I  S  -  1)  '-5 c  i  1.643  CU  v.  B tt o S -3 o o 35  cn C ce o *"* 'ce  •O O  J8 O  Number of sliides  J3  t/3  C/3  "O  u o d  C*H  OJ  E d Z  •s s o< > T3  2.002  Cr  |  cn ™  LGN  Z  of the lesioned  a o  —  C CJ  1  T3  3  a o  JCS  *J  —  2  cj co  CJ  examined  Mean Value  U  00  § I CJ  T3  cu C3 CJ  g o  CB  LGN  c co  of the normal  Mean Value  ll 2  z  . O 1) S  3"  0  I « e re s  13 •£ 8 8  •i 1 2o »C5 CJ  cu o ri a  c •e  C3  o S  IT)  re >  o V  t-value  V  -6.085  re  >  fli o oo -ca CO  —  o  C  CJ  .2 M  12  b-  2>2 CS  60  ^ s 1o  a  CJ U  00 C cu —: cs C Z  2 » * " 0 C CJ x £ : S J  o * 00 S cn -is o .2 ^° o 00 Je .S  = I O i-  "3  S  .S T3 cn O 3  cn  JS -° a.  «•) 3 3 13 ? .2 I 8>> § a B§ B S o S » J • S « 00 fa H 0 cj .zV is 00 J=  139  NORMAL LGN  LESIONED LGN  Figure 5.8  Nuclearization of the beta-catenin protein in the postnatal cat L G N  following optic tract lesions. The two L G N ' s shown in this figure are from the same 120 day old postnatal animal. The L G N shown in the top panel was allowed to develop normally whereas the L G N in the bottom panel had its optic tract lesioned at an early age. Nuclear beta-catenin protein is found in both L G N ' s and the staining pattern in the two pictures seem to be similar. Hence, unilateral optic tract lesion paradigm doesn't appear to influence the nuclearization of the beta-catenin protein.  140  required for proper cell adhesion (Kitner 1992, Nagafuchi et al. 1988, M c C r e a et al. 1991). However, cadherin molecules can interact with catenins other than beta-catenin such as plakoglobin (McCrea et al. 1991).  Since the exact type of cadherin proteins which are  being detected by the pan-cadherin antibody in the L G N are unknown, the interaction between the beta-catenin protein and the cadherin molecules detected by the pan-cadherin antibody needs to be verified. In order to demonstrate the interaction between these two molecules,  beta-catenin  and cadherin proteins were co-immunoprecipitated. In the top panel of figure 5.9, the betacatenin protein was immunoprecipitated and the proteins obtained by this procedure were electrophoresed on a gel and blotted onto a membrane.  This membrane containing the  immunoprecipitated beta-catenin protein (and presumably, all the other proteins it interacts with) was subjected to western blot analysis using the pan-cadherin antibody. The fact that cadherin proteins  are  present  in  the  immunoprecipitate  obtained  with  the  beta-  cateninantibody proves that beta-catenin and cadherin proteins detected by the pan-cadherin antibody attach to each other in the developing visual system. same figure further verifies this interaction.  The middle panel of the  During this experiment the pan-cadherin  antibody was used to immunoprecipitate the cadherin proteins and then this precipitate was screened with the beta-catenin antibody.  The positive signal observed shows that the  beta-catenin protein can be "pulled down" or co-immunoprecipitated with the cadherin proteins detected by the pan-cadherin antibody and hence, beta-catenin and the cadherin proteins are physically connected to each other in the cat visual system. For a control, the immunoprecipitation reactions were probed with antibodies for proteins that do not associate with beta-catenin or the cadherins (such as c-fos and jun). The absence of these proteins (jun was used as a control in the top panel whereas c-fos was used as a control in the middle panel) in the immunoprecipitated protein fractions shows the immunoprecipitation reactions to specifically contain proteins which interact with either beta-catenin or the cadherins. Furthermore, the bottom panel shows beta-catenin and the  141  Beta-Catenin Protein I.P./Cadherin Antibody Western Blot Analysis  LP.  c-jun CONTROL  Cadherin Protein I.P.. Beta-Catenin Antibody Western Blot Analysis Pre  LP.  Post  LP.  —  c-fos CONTROL Protein-G Negative Control Beta-Catenin LP.  Beads  Cadherin LP.  Beads  Figure 5.9 Coimmunoprecipitation of the Beta-Catenin and the Cadherin Proteins. In the top panel western blot analysis was performed on the immunoprecipitate obtained with the beta-catenin antibody using the pan-cadherin antibody. Lane a) shows the pre-fraction or the protein preparation before immunoprecipitation with the beta-catenin antibody. Lane b) is immunoprecipitation performed with 2u.g of beta-catenin antibody incubated with the protein preparation for 2 hours. The immunoprecipitation reaction shown in lane c) also contained 2u,g of beta-catenin antibody but was incubated overnight whereas the immunoprecipitation reaction in lane d) contained 8u.g of beta-catenin antibody and was incubated overnight. Lane e), f) and g) are the post-fractions (i.e. the protein preparation after the beta-catenin antibody and the proteins it attaches to are removed) corresponding to the immunoprecipitation reactions performed in lanes b), c) and d) respectively. The bottom half of the top panel shows western blot analysis performed on the same blot with the c-jun antibody (the immunoprecipitation blot was stripped and reprobed with the c-jun antibody). The c-jun protein is absent in the immunoprecipitation fraction (lanes b, c and d). The middle panel shows western blot analysis performed with the betacatenin antibody on proteins immunoprecipitated with the pan-cadherin antibody. The bottom half of the middle panel shows the reprobing of the same blot with the c-fos antibody which shows the c-fos protein not to be immunoprecipitated by the pan-cadherin antibody. The bottom panel shows a control where only beads were added to the protein preparation. Beta-catenin and the cadherin proteins are found not to be pulled down by beads alone.  142  cadherin proteins do not to simply attach to the Protein G beads.  Finally, the fact that  higher yields of co-immunoprecipitated protein could be obtained using either higher concentrations of antibody or longer incubation periods (lanes b,c and d of the top panel)illustrates periods  with  the specificity the  cadherin  of the immunoprecipitation reaction (short incubation antibody  i.e.  2  hours  repeatedly  failed  to  yield  a  immunoprecipitation product).  5.4 DISCUSSION The important finding of this chapter is that the cadherin and catenin cell adhesion molecules are upregulated in layers of the L G N corresponding to the visually deprived eye in monocularly deprived animals.  Additionally, the optic tract lesion paradigm, which  prolongs the critical period (Singer 1982), is capable of retaining high levels of beta-catenin and cadherin protein expression in the L G N .  The regulation of the beta-catenin and the  cadherin molecules by visual activity supports the involvement of these proteins in the postnatal development of the cat visual system.  Unexpectedly, changes in the expression  of these proteins in response to activity were not observed in the visual cortex. nuclear localization of beta-catenin was unaltered by visual deprivation.  Also,  Since visual  activity effects the expression of the beta-catenin and cadherin cell adhesion molecules only in the L G N , these molecules may be performing a different set of function in the geniculate as compared to the cortex.  This conjecture is not unreasonable based on the fact the  cytoarchitecture and the neuro-chemical makeup of the cortex are considerably different than the geniculate and various molecules are differentially expressed between the cortex and the L G N .  143  5.4.1  Activity Independent  Regulation of Beta-catenin  and the  Cadherin  Proteins in the Postnatal Cat Visual Cortex Whereas cadherins are clearly expressed in an activity dependent manner in the L G N , changes in the expression of these proteins in the visual cortex following monocular deprivation or optic tract lesions is not observed.  This finding is unexpected given the  expression profile of the cadherin and the beta-catenin proteins correlates well with the critical period for ocular dominance column formation. The involvement of these proteins in visual plasticity is also bolstered by the fact that the pan-cadherin antibody heavily stains the input layer of the thalamic terminals in layer IV and visual areas of the cortex. However, based on the anatomical and physiological properties of the developing visual cortex any molecular mechanism involved in synaptic rearrangements is likely to be regulated in an activity dependent manner.  Hence, the involvement of these proteins in  activity dependent synaptic modifications is somewhat  doubtful in the visual cortex.  Conceivably, the role of cadherin molecules in layer IV may be limited to facilitating the formation of proper neural circuits (discussed further in the last chapter). The absence of activity dependent regulation in the cortex is contradictory to the reported behaviour of cadherin proteins examined in other regions of the nervous systems. For example, N-cadherin expression is reported to decrease in skeletal muscles in response to neural activity (Hahn et al. 1992).  The expression of N-cadherin also decreased in  dorsal root ganglia by neural stimulation (Itoh et al. 1997). In the developing barrel cortex N-cadherin is concentrated strictly at the thalamocortical synaptic junctional complex during the initial formation of thalamocortical connections reorganizations induced by  deprivation paradigms  expression at newly forming synapses.  (Huntley et al. 1999). are accompanied  Synaptic  by N-cadherin  The cellular redistribution of the beta-catenin  protein also occurs in tissue culture neurons deprived of activity (Murase et al. 1999). Furthermore, the activity dependent regulation of these proteins in the geniculate is reported in this chapter. Since these findings contradict the reported activity independent regulation  144  of beta-catenin and the cadherin proteins in the visual cortex, the possibility that technical problems are responsible for this outcome needs to be considered.  A possible technical  problem may have arisen from the use of a pan cadherin antibody.  As noted in the  previous chapter, the differential expression of cadherin molecules allows the formation of various neural circuits. The pan-cadherin antibody heavily labels layer IV and the visual areas of the cortex.  Hence, these cadherin proteins are likely to contribute to the  organization of the visual cortex and facilitate appropriate connections to form in layer I V . However, the connections that are made onto layer IV are not strictly from the lateral geniculate nucleus. Layer I V receives extensive input from layer V I , the claustrum (LeVay et al. 1981) and neuromodulatory neurons from various subcortical areas (Irle et al. 1984, Mizuno et al. 1969, Troiano et al. 1978, Watabe et al. 1982, Mulligan et al. 1988, Chazal et al. 1987, Tork et al. 1981).  In order for these connections to form appropriately,  cadherin types other than those necessary for thalamocortical connections expressed in layer TV of the visual cortex.  need to be  Since some of the non-thalamic connections in  layer IV form in an activity independent manner (Perkel et al. 1984), layer IV is likely to contain cadherin molecules that are not regulated by activity. cadherin antibody dependence.  does  not  recognize  cadherin molecules  Unfortunately, the panbased  The pan-cadherin staining in the visual cortex  on  their  following  activity  monocular  deprivation represents the cumulative protein distribution pattern of both activity dependent and independent cadherin genes.  Hence, the presence of cadherin molecules regulated in  an activity independent manner in layer IV of the cortex may have prevented the staining pattern of the pan-cadherin antibody from changing following monocular deprivation.  In  other words, there may be cadherin molecules in layer IV of the visual cortex whose expression is regulated by visual activity. The expression of individual types of cadherin molecules needs to be examined in order to determine if activity dependent members of this cell adhesion family are expressed in the visual cortex.  In addition, since beta-catenin  interacts with various different cadherin proteins, its expression reflects the cumulative  145  expression pattern of the classical cadherins.  Therefore, the constancy of beta-catenin  expression in layer I V of the cortex following monocular deprivation may be attributable to the presence of cadherin molecules regulated in an activity independent manner.  5.4.2 Activity Independent Regulation of Nuclear Beta-Catenin Expression Another important finding of this chapter is that beta-catenin nuclearization appears to occur in an activity independent manner.  Neurons containing nuclear beta-catenin are  present in animals subjected to optic tract lesions and the distribution of neurons containing nuclear beta-catenin do not correspond to specific layers of the L G N in monocularly deprived animals. Beta-catenin nuclearization is not proposed to be involved in causing synaptic modifications because nuclear beta-catenin in the L G N is not observed until around 7 weeks of age but monocular deprivation before this period causes significant shifts in ocular dominance.  Instead, beta-catenin is believed to cause changes at the  transcriptional level that inhibits the neuron from undergoing synaptic reorganization. Therefore,  the  absence  of  nuclear beta-catenin  redistribution following  deprivation is not inconsistent with the suggested function for this protein.  monocular In addition,  paradigms prolonging the critical period act on higher layers of the cortex and these paradigms are ineffective at prolonging the critical period for thalamocortical neurons (Mower et al. 1985).  Hence, the findings of the optic tract lesion experiments can not be  deemed as contradicting the proposed role of beta-catenin thalamocortical plasticity.  nuclearization in ending  Furthermore, the presence of age related factors involved in  ending the critical period is well established. Experimental paradigms can only prolong the critical period and are unable to maintain a high level of visual plasticity in older cats (Cynader et al. 1980, 1982).  A n adult dark reared cat is not as plastic as a thirty day old  kitten and accordingly, the synaptic reorganization resulting from monocular deprivation is not nearly as drastic in these animals. Hence, beta-catenin nuclearization may also be an age  related, activity independent  molecular mechanism  thalamocortical plasticity.  146  contributing  to  the  end  of  Although changes in beta-catenin nuclearization in response to visual deprivation are not observed in this chapter, the possibility that visual activity effects nuclearization of beta-catenin can not be disregraded.  Feasibly, the level of beta-catenin protein in the  nucleus may be altered in individual cells by monocular deprivation or optic tract lesions. Alternatively, cells containing nuclear beta-catenin  may be different  in monocularly  deprived animals.  5.4.3 Activity Dependent Changes in Beta-Catenin and Cadherin staining in the L G N Beta-catenin and cadherin protein expression is altered in the L G N of kittens following monocular deprivation. Also, the expression level of these proteins is greater in the geniculate of older cats subjected to optic tract lesions.  This activity  dependent  regulation of beta-catenin and the cadherin proteins supports the involvement of these proteins in the postnatal development of the cat visual system.  We are tempted to devise a  presynaptic molecular model for ocular dominance column formation based on the activity dependent regulation of cadherin and beta-catenin  protein expression  Although this conjecture is supported by various observations (i.e.  in the L G N .  layer TV staining,  expression correlating with the critical period), this hypothesis can not be justified because changes in cadherin or beta-catenin staining in layer IV thalamocortical connections is not observed in response to monocular deprivation. Therefore, the function of these molecules in the L G N is likely to be associated with postnatal developmental events occurring in this structure rather than the cortex. Unlike the visual cortex, eye specific layers form in the cat geniculate prior to birth (Shatz 1983, Shatz et al. 1984).  Furthermore, reorganization of  eye specific layers in the L G N does not take place postnatally and unlike the ocular dominance columns of the cortex, eye specific layers of the L G N in kittens do not expand and contract postnatally in response to activity dependent paradigms such as monocular deprivation (Shatz et al. 1988, Garraghty et al. 1988).  However, cellular competition  between different neuronal classes does exist in the postnatal kitten L G N and this cellular  147  competition is affected by visual activity (Guillery et al. 1970b, Garrey, et al. 1977, Sur et al. 1984, Sur 1988). Increases in the neuropil staining of beta-catenin and pan-cadherin antibody is observed in the A and C lamina of the L G N following monocular deprivation.  The A  lamina is innervated by the X cells and the Y cell from the contralateral eye (Guillery 1970a, Guillery et al. 1970b).  The A l lamina is innervated by the X and Y cells from the  ipsilateral eye. The C lamina is innervated by Y cells from the contralateral eye and W cells of the ipsilateral eye (Guillery 1970a, Guillery et al. 1970b). X cells are not found in layer C of the L G N . The retinogeniculocortical pathways constituted by X and Y cells give rise to morphologically and functionally different neural streams.  The X pathway in the cat is  somewhat analogous to the parvocellular pathway (the cat lacks extensive color vision) and is involved in detecting spatial frequency (Shoham et al. 1997).  Similarly, the Y pathway  in the cat is involved in motion processing and is equivalent to the magnocellular pathway in the monkey (Frishman et al. 1983). A s the magno- and the parvo- names imply, the X cells are smaller in size as compared to Y cells.  The development of these two cell types  also differs in the postnatal lateral geniculate nucleus. Compared to the adult L G N the X-cell terminal fields are wider in the A and the A l layers of the kitten L G N whereas the Y-cell terminal fields in the same layers of the kitten geniculate are much narrower than in the adult L G N (Sur et al. 1984). The contraction of X-cell terminals and the expansion of Y-cell terminals in the L G N occurs gradually with age (Sur 1988). The adult terminal field patterns for X - and Y-cells in the L G N is achieved in kittens around 12 weeks of age (Sur 1988). Interestingly, the development of Y-cells is much more susceptible to the effects of monocular deprivation (Friedlander et al. 1982, Garraghty et al. 1986a). In a monocularly deprived animal, the Y-cells fail to expand their arbors in A layers of the L G N (i.e. A or A l but not C) corresponding to the deprived eye (Garraghty et al. 1986a, Garraghty et al. 1986b).  In these deprived A layers, the age  related shrinkage of X-cell terminals does not occur and the X-cells retain large terminal  148  fields (Garraghty et al. 1986a, Garraghty et al. 1986b). of visual activity stunts the growth of Y-cell arbors.  These data imply that the absence  The growth of the Y-cell arbors is  hypothesized to be accompanied by competitive pruning of X-cell connections and hence, giving rise to the observed shrinkage of X-cell arbors with age.  However, the observed  competition between X and Y cells is not simply based on activity dependent competition because Y cells grow normally and expand their arbors in the C laminae even in the absence of visual activity.  The expansion of Y cell arbors occurs in C laminae not only in the  absence of monocular deprivation but also during binocular deprivation where there is absolutely no visual input in this lamina.  The ability of Y cells to mature normally in  lamina C is attributed to the absence of X cell arbors in this layer of the L G N .  The lack of  activity in layer C is inconsequential to Y cells because they do not have to compete with X cells in this layer. Hence, both cellular and activity dependent competition appears to be involved in determining the final terminal fields of X - and Y-cells in the postnatally developing L G N . The activity dependent regulation of beta-catenin and the cadherin molecules in the geniculate suggests the involvement of these molecules in the development of X and Y cell connections in the postnatal L G N .  The higher expression levels of the beta-catenin and the  cadherin proteins corresponding to the deprived A layers of the L G N may favour the retention of X cell inputs as opposed to the formation of Y cell connections.  The  overexpression of beta-catenin and the cadherins may either stabilize the X-cell connections or alternatively, these cell adhesion molecules may inhibit Y-cell arbours from expanding. The higher expression levels of these molecules in layer C of the L G N is irrelevant since X cells are not present in this laminae.  Changes in the cytoskeleton associated with the  development of X - and Y - cell arbours are reported and support the proposed role for betacatenin and the cadherins in the competition between X and Y cells (Bickford et al. 1998). SMI-32 protein is a neurofilament protein that is  selectively expressed  by Y-cells  (Chaudhuri et al. 1986). Electron microscope studies have revealed the presence of this  149  protein in synapses(Bickford et al.  1998).  Following monocular deprivation, the  expression of SMI-32 decreases in the neuropil of A layers corresponding to the deprived eye (Bickford et al. 1998). Neuropil staining of SMI-32 is retained in the C laminae since Y cells expand their arbors in this layer lacking X cells. Compared to SMI-32, beta-catenin and cadherin expression have the opposite response to visual deprivation. These findings support the notion that overexpression of beta-catenin and the cadherin proteins favours the retention of X-cell connections.  (The beta-catenin and the cadherin protein expression in  the L G N can not be cell type specific like SMI-32 (i.e. these proteins can not be expressed by X-cells alone) because these proteins are expressed in layer C of the L G N lacking X cells and containing Y cells.) It is unclear if higher levels of beta-catenin and cadherin expression in the L G N following optic tract lesions are associated with retention of plasticity. Firstly, optic tract lesions can only maintain high levels of beta-catenin and cadherin protein expression in the L G N of older animals and not in the cortex (although the reasons for this finding may be identical to the problems associated with the use of the pan cadherin antibody discussed earlier).  The distinct responses of these two visual structures to the same deprivation  paradigm are hard to explain in terms of ocular dominance plasticity.  Secondly, paradigms  extending the critical period for ocular dominance formation fail to prolong the period during which X and Y cell competition occurs.  Possibly, the higher expression levels of  these proteins following optic tract lesions in the L G N may simply reflect the upregulation of beta-catenin and the cadherins in the absence of activity. The regulatory effect of activity on these molecules may be specific to the L G N and may not transpire in cortical structures. This possibility is in agreement with the results of the monocular deprivation studies. The experiments discussed in this section support a role for beta-catenin/cadherin cell adhesion system in the postnatal development of X - and Y - cell terminals in the postnatal L G N .  The alteration in beta-catenin and cadherin protein expression following  monocular deprivation suggests these molecules to be involved in the cellular and activity  150  dependent competition taking place between X - and Y - cell arbours. Since beta-catenin is the homolgue of the armadillo segment polarity gene, this result supports the hypothesis that segment polarity genes are involved in the synaptic organization of the postnatal cat visual system (discussed in chapter 2). Clearly, these findings bolster the argument that the homologues of the drosophila developmental genes are involved in the development of the postnatal cat visual system.  151  Chapter 6 SUMMARY AND GENERAL DISCUSSION 6.1  INTRODUCTION  The  am i of t h i s t h e s s i is to e s t a b l i s h the h y p o t h e s s i t h a t the h o m o o lg u e s of  the  d r o s o p h a l i d e v e o lp m e n t a lg e n e s are n iv o v le d in the p o s t n a t a ld e v e o lp m e n t of the cat v i s u a l  s y s t e m . In o r d e r to a c h e iv et h i s goal, s e v e r a l h o m e o c t i and s e g m e n tp o l a r i t y g e n e s w e p a r t i a ly c o ln e d in the cat and t h e i rd e v e o lp m e n t a lr e g u a lt o in d u r n i g the critical p e r o i d was  d e m o n s t r a t e d by N o r t h e r nb l o ta n a l y s i s . To p r o v d ie f u r t h e re v d ie n c e for t h i s h y p o t h e s s i the e x p r e s s o i n of the b e t a c a t e n n i p r o t e i n , the m a m m a a iln h o m o o lg u e of the armadillo s e g m e n tp o l a r i t y g e n e , was  c h a r a c t e r z ie d in the v i s u a l c o r t e x and the LGN  of n o r m a y l  d e v e o lp n i g and v i s u a ly d e p r v ie dk i t t e n s . The f i n d i n g s of t h i st h e s s i i n d i c a t eb e t a c a t e n n i to be a m u l t i f u n c t i o n a lp r o t e i nw h c ih is l i k e l y to c o n t r b iu t e to v a r o iu s a s p e c t s of p o s t n a t a l v i s u a l d e v e o lp m e n t in the cat b r a i n . The e v d ie n c ep r o v d ie d for the n iv o v le m e n t of b e t a c a t e n n i in v i s u a ld e v e o lp m e n ts t r o n g y l e n d o r s e s our o r i g i n a lh y p o t h e s s i t h a t the m a m m a a iln g e n e s o r i g i n a t i n g f r o m the d r o s o p h a l i d e v e o lp m e n t a l g e n e s c o n t r b iu t e to the p o s t n a t a l d e v e o lp m e n t of the cat v i s u a ls y s t e m .  6.2  T H E INVOLVEMENT OF DEVELOPMENTAL GENES IN  VISUAL  PLASTICITY  V a r o iu sc l a s s i c d e v e o lp m e n t a l g e n e s are e x p r e s s e d in the v i s u a l s y s t e md u r n i g the critical p e r i o d . F g iu r e 6.1 b r i e f l y s u m m a z r ie s the f i n d i n g s of t h i s t h e s s i and i lu s t r a t e s the p u t a t v ie f u n c t o in s of t h e s e g e n e s . The  PBX1 and PBX2 h o m e o c t i g e n e s , w h c ih are  k n o w nc o f a c t o r s of o t h e rh o m e o b o xg e n e s , are e x p r e s s e dd u r n i g the critical p e r o i d (van  152  153  Dijk et al. 1995, Chang et al. 1995, Neuteboom et al. 1995). Hence, additional homeobox genes that form transcriptional complexes with these PBX genes are quite likely to be expressed in the developing visual cortex. Also, the expression of the siamois homeobox gene is likely to be present in the L G N of kittens over the age of 60 days because the expression of this protein is upregulated by the nuclear activity of beta-catenin (Brannon et al. 1997). The possible relevance of these genes to the postnatal development of the visual system arises from the reported involvement of homeotic genes in segmentation and compartmentalization (Lewis 1978, Akam 1987, Lawrence 1987, Murphy et al. 1989). W e believe that homeotic genes are likely to contribute to the organization of the visual cortex and these genes may be involved in the formation of structures such as ocular dominance columns, cytochrome-oxidase blobs, and interblobs. In addition, the bone morphogenic and wnt signaling pathways, which have originated from the decapentaplegic (Padgett et al. 1993) and wingless (Ramakrishna et al. 1993) segment polarity genes, are expressed in the developing cat visual cortex.  The  importance of cellular communication and signaling in the development of the visual system is well established. Although the B M P and Wnt signaling pathways were not extensively characterized in the visual cortex, their presence and age dependent regulation is indicative of a role for these molecules in visual development. Since the Wnt and the B M P pathways can act in a competitive and antagonizing manner towards each other, the action of these genes may potentially be the molecular basis for the synaptic organization occurring in the developing visual cortex. The presence and the developmentally regulated expression of the segment polarity and homeotic genes in the visual cortex gives merit to our initial hypothesis.  However, the  strongest evidence for the involvement of the homologues of the drosophila developmental genes in visual development arises from the characterization of the beta-catenin gene, the homologue of the armadillo gene (Peifer et al. 1992).  (For detailed discussion of the  subjects discussed below please refer to the appropriate chapter in the thesis)  154  6.2.1  Evidence for the involvement of the beta-catenin protein in postnatal  visual  development The data obtained in this thesis indicate that the beta-catenin protein is performing  two distinct functions in the postnataly developing cat visual system. Since the beta-catenin protein is present in the nuclei of L G N neurons and the neuropil of the cortex and the geniculate, this protein is likely to act both as a transcription factor and as a cell adhesion molecule in the developing visual system. Because the transcription factor activity of betacatenin is quite distinct from its adhesive function, there is no reason to assume that these two separate functions of the beta-catenin molecule contribute to the same aspect of visual development. Hence, in addition to having multiple functions the beta-catenin protein may be contributing to different facets of postnatal development in the cat visual system.  6.2.1.1  Involvement  of  Nuclear  beta-catenin  in ending  thalamocortical  plasticity Beta-catenin is translocated into the nuclei of L G N neurons at the end of the period for thalamocortical plasticity. The simultaneous occurrence of these two events implies a role for beta-catenin in ending thalamocortical plasticity. B y acting as a transcription factor and altering the expression of various genes, beta-catenin is proposed to inhibit plasticity in L G N neurons. Since nuclear beta-catenin is only observed in geniculate neurons and not in cortical neurons, neurons in the L G N are postulated to lose their plasticity before cortical neurons. This possibility is supported by the fact that thalamocortical plasticity is lost prior to cortical plasticity (LeVay et al. 1980, Mower et al. 1985).  Higher layers of the cortex  (such as layer 2/3) retain plasticity until 1 years of age whereas thalamocortical connections of layer TV do not undergo any further synaptic rearrangements following roughly 50-60 days of age (Mower et al. 1985, Daw et al. 1992). The absence of plasticity in layer TV of the cortex following 50-60 days of age can be explained by the inability of the geniculate neurons to modify their synaptic connections.  The importance or the involvement of  cortical factors in determining ocular dominance plasticity is not being disputed by these  155  findings.  The nuclear activity of beta-catenin is merely being presented as a presynaptic  mechanism that contributes to ending thalamocortical plasticity. Nuclear beta-catenin distribution in the L G N does not appear to be modified by activity dependent paradigms such as monocular deprivation or optic tract lesions. Betacatenin nuclearization is not proposed to be involved in causing synaptic modifications because nuclear beta-catenin in the L G N is not observed until around 7 weeks of age but monocular deprivation before this period causes significant shifts in ocular dominance. Hence, the results of the monocular deprivation experiments do not contradict the proposed role of nuclear beta-catenin as a plasticity-inhibiting factor. In addition, the failure of the optic tract lesion paradigm to prevent or to delay beta-catenin nuclearization in the L G N does not contradict the proposed function of nuclear beta-catenin because plasticityprolonging paradigms are ineffective at prolonging thalamocortical plasticity (Mower et al. 1985).  A s determined by single unit recordings, ocular dominance shifts in dark reared  kittens (subjected to monocular deprivation) occur largely in cortical layers other than layer IV (Cynader et al. 1980, Cynader 1982).  Accordingly, anatomical changes in ocular  dominance columns can not be induced in these animals by monocular deprivation (Mower et al. 1985).  These findings indicate the critical period for thalamocortical plasticity to be  largely age dependent.  Age related events involved in ending visual plasticity for non-  thalamocortical connections also exist, since older cats subjected to plasticity-prolonging procedures are less plastic than normally reared young kittens (Cynader et al.  1980,  Cynader 1982). In summary, our proposal of an age related end to plasticity in a subset of LGN  neurons brought about by the transcriptional factor activity of beta-catenin  is  consistent with the reported characteristics of the developing visual system. Concievably, beta-catenin may inhibit the plastic capabilities of geniculate neurons by acting as a transcription factor and downregulating the expression of cell adhesion molecules.  The involvement of cell adhesion molecules in neuronal plasticity is well  established and a role for these molecules in visual plasticity is supported.  156  Nuclear beta-  catenin activity in non-neuronal adult tissue is associated with cancer and cancer metastasis (Tetsu et al. 1999, Porfiri et al. 1997, Bullions et al. 1998).  Nuclear beta-catenin activity  in cancerous cells downregulates the expression of cell adhesion molecules.  Accordingly,  these cancerous cells lose their adhesive properties and detach from their tissue of origin (Oyama et al. 1994).  Although the nuclearization of beta-catenin in the L G N does not  cause cancer, it may cause an overall decrease  in synaptic cell adhesion  expression (in addition to the one observed for the cadherins).  molecule  This decrease in cell  adhesion molecule expression is proposed to be responsible for the cessation of plasticity in the geniculate.  In support of this hypothesis is the findings of Schoop et. al. 1997 who  report the adhesiveness of the visual cortex to decrease in an age related manner. Coincidentally, this decrease in cortical adhesiveness parallels the critical period.  In  addition, the expression of M H C like cell adhesion molecules in the developing visual cortex and the L G N correlate with the critical period and the expression of these cell adhesion molecules is regulated by visual activity (Corriveau et al. 1998). Also, cell adhesion molecules are required in processes such as synaptogenesis and target recognition (Edelman et al. 1998, Uryu et al. 1999, Lilienbaum et al. 1995, Matsunaga et al. 1988). Finally, the downregulation of cadherin protein expression in the visual system of older kittens (reported in this thesis) supports the involvement of cell adhesion molecules in visual plasticity. If high levels of cell adhesion molecules are required to facilitate synaptic rearrangements (i.e. cell adhesion molecules are expressed more abundantly in younger animals), then the downregulation of these proteins by nuclear beta-catenin activity may cause thalamocortical connections to lose their plasticity. The possibility that beta-catenin ends thalamocortical plasticity by alternate means can not be ruled out considering the activity of nuclear beta-catenin is likely to influence the expression of numerous genes. However, based on the data currently available regarding the function of this protein, the above proposal appears to be the most logical.  157  6.2.1.2  The Expression  of  the  Beta-Catenin  protein  in the  postnataly  developing cat visual cortex The expression of the beta-catenin protein in the postnatal visual cortex is restricted to the neuropil. This distribution of the beta-catenin protein is consistent with its role as a cell adhesion molecule (McCrea et al. 1991).  Since the role of the beta-catenin protein in  cell adhesion is to anchor the cadherin molecules to the actin cytoskeleton, the expression of the cadherin proteins was also examined in the developing visual cortex. The role of the beta-catenin/cadherin cell adhesion system in the developing visual cortex appears to facilitate the formation and wiring of neural circuits. The expression of both the beta-catenin and the cadherin proteins is most prominent in layer I V of the cortex. In addition, the pan-cadherin antibody strongly labels the visual areas of the cortex.  A  well-characterized property of the cadherins is that they specifically label certain corticalregions and contribute to the wiring of neural circuits (Rubenstein et al. 1999, Suzuki et al. 1997, Inoue et al. 1997, Huntley et al. 1999, Riehl et al. 1996, Matsunaga et al. 1988, Obst-Pernberg et al. 1999).  The model by which cadherin molecules are believed to give  rise to specific neural circuits is summarized in figure 6.2.  In this figure there are three  hypothetical neurons that express A-cadherin, B-cadherin or A - and B - cadherin.  Due to  the homophilic binding properties of cadherin molecules, cells expressing A-cadherin can only make connections with other cells expressing A - cadherin. The same situation is true for cells expressing B-cadherin. However, cells expressing A - and B - cadherin can make connections with both. We propose that the cadherin proteins expressed in layer TV of the visual cortex may be providing a target for axons originating from various different areas of the brain. Axons originating from brain areas that make connections with layer IV such as the thalamus, clastrum and layer V I are likely to express at least some of the same cadherin molecules as layer IV. Based on the model presented in figure 6.2, these axons will be able to make connections with layer IV neurons if they express the same types of cadherins. Each neural pathway connecting to layer I V (for example the callastrum and  158  159  thalamic connections) is likely to express different cadherin molecules so that each type of input can form synapses with appropriate neurons. Although a correlation between cadherin protein expression and the critical period exists in the visual cortex, these proteins appear to be expressed in an activity independent manner in this structure. Feasibly, these proteins may contribute to the age related decrease in plasticity in the visual cortex and may not be involved in activity dependent synaptic modifications.  However,  activity  dependent  changes  in cortical cadherin protein  expression may have been missed due to the use of a pan-cadherin antibody. The fact that some connections in layer IV are made in an activity independent manner supports this notion (Perkel et al. 1984).  Activity dependent regulation of cadherin and beta-catenin  expression has been discovered in various other neural structures (Huntley et al. 1999, Murase et al. 1999, Tanaka et al. 2000, Tang et al. 1998).  The activity dependent changes  reported in the expression of the beta-catenin and the cadherin proteins in the L G N verifies the ability of visual activity to regulate the expression of these molecules.  Hence, the  examination of individual cadherin protein types is likely to identify specific species of cadherin molecules which are regulated by neural activity in the developing visual cortex. The discovery of such molecules would confirm our belief that the beta-catenin/cadherin cell adhesion system is involved in synaptic modification events taking place during the formation of ocular dominance columns. A s an aside, these data are advocating further examination of cell adhesion molecule expression in the developing visual cortex.  It seems intuitive that cell adhesion molecules,  which are the glue that form and hold synapses together, would be involved in the wiring of neural circuits.  A n obvious target for research is identifying and characterizing the  cadherin molecules that are specific to various neural streams.  Based on previous research  (Suzuki et al. 1997, Arndt et al. 1996, Kimura et al. 1996), cell adhesion molecules that are specific to both anatomical and functional neural pathways in the visual system are likely to exist.  Characterizing the expression patterns of these molecules will be an arduous task  160  given the number of different cadherin, protocadherin and cadherin like  molecules  expressed in the brain. In addition, the integrins constitute another cell adhesion system which may be involved in visual development.  Certain members of the integrin family are  localized to the synapse and they contribute to neural development and neural plasticity (Einhebner et al. 1996, Lilenbaum et al. 1995, Martin et al. 1996, Staubli et al. 1990). Efforts should also be made towards better understanding the role of C A M ' s , which do not require calcium for their function, in visual development.  6.2.1.3  Involvement of the Beta-catenin and the Cadherin proteins in X -  and Y - cell competition  Beta-catenin and cadherin protein expression in the geniculate decreases with age. Following monocular deprivation, beta-catenin and cadherin protein levels increase in layers of the L G N which receive input from the deprived eye.  Since changes in the  expression of these proteins are not detected in the cortex in response to activity, activity dependent regulation of the beta-catenin and the cadherin proteins in the geniculate is unlikely to be involved in the development of thalamocortical connections.  The only well  established activity dependent event occurring in the postnatal L G N is the development of X and Y cell connections (Guillery et al. 1970b, Garrey, et al. 1977, Sur et al. 1984, Sur 1988).  Hence, the beta-catenin and cadherin proteins expressed  in the postnataly  developing L G N are likely to contribute to this event. The altered levels of cadherin and beta-catenin protein expression in the neuropil of the L G N in response to monocular deprivation is proposed to provide the X-cell arbours with a competitive advantage and prevent the Y-cells from taking over X-cell terminals.  This proposed role of the beta-  catenin/cadherin cell adhesion system is consistent with the reported function of other cytoskeletal proteins, such as SMI-32, during the postnatal development of X - and Y-cell terminal fields in the geniculate (Bickford et al. 1998). The evidence for the involvement of  161  the beta-catenin protein in X - and Y - cell competition supports the hypothesis that segment polarity genes may be involved in synaptic organization occuring during the critical period.  6.2.1.4  Summary of the evidence  implicating a role for the beta-catenin  protein in postnatal visual development The data discussed in this section provide several lines of evidence supporting the involvement of beta-catenin in the postnatal development of the cat visual system.  First,  the beta-catenin protein along with the cadherin proteins is expressed in layer rV of the visual cortex and is proposed to play an organizational role.  Second, the beta-catenin  protein becomes nuclear in a subset of L G N neurons at the end of the critical period for thalamocortical plasticity. Third, the expression of beta-catenin is regulated in an activity dependent manner in the developing L G N and it may be involved in the competition taking place between X - and Y - cells for synaptic terminals. Taken together, these data strongly endorse the involvement of beta-catenin in visual development and ergo, these data bolster the hypothesis that the mammalian counterparts of the drosophila developmental genes are involved in the postnatal development of the cat visual system.  6.2.2  Significance of the findings presented in this thesis W e have established the expression of the homeotic and segment polarity genes in  the visual cortex during the critical period and provided evidence for their involvement in visual development.  The segment polarity and homeotic genes comprise two large gene  families. Unfortunately, the expression of only a few of these genes could be investigated in this thesis. experiments  Hence, the results of this thesis can be used as a basis for designing to  investigate  the  function  of  other  homologues  of  the  drosophila  developmental genes in visual development. For example, based on the results presented in this thesis, it is not unreasonable to assume that the Notch signaling pathway or the pair rule genes are playing a role in the postnatal development of the cat visual system.  In  addition, hypothesis regarding the function of the developmental genes identified in Chapter 2 can be formed and the expression of these genes can be further characterized in  162  the visual system. Also, examining the activity of genes that are known to interact with the homeobox and segment polarity genes identified in chapter 2 (or beta-catenin) can expand upon the data presented in this thesis. 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