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Changes in nasal morphology during primary palate formation in the C57BL/6J mouse embryo as revealed… Nagy, Denis 1992

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CHANGES IN NASAL MORPHOLOGY DURING PRIMARY PALATE FORMATIONIN THE C57BL/6J MOUSE EMBRYO AS REVEALED BYTRANSMISSION ELECTRON MICROSCOPY AND HISTOCHEMISTRYByDENIS NAGYB.Sc. The University of British Columbia, 1987A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIES(Department of Clinical Dental Sciences)We accept this thesis as conforming to the required standardTHE UNIVERSITY OF BRITISH COLUMBIAMarch 1992© Denis Nagy, 1992In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.Department of Clinical Dental SciencesThe University of British ColumbiaVancouver, CanadaDateDE-6 (2/88)ABSTRACTPrimary palate formation in the C57BU6J mouse embryo involvesthree facial prominences. These are the medial nasal prominence (MNP), lateralnasal prominence (LNP), and the maxillary prominence (MxP). The MNP andLNP come together and fuse forming a nasal fin which is a transient structure thatpersists for a time and then regresses. Mesenchymal cells invade the nasal finand form a mesenchymal bridge.In this study, I look at the morphological changes in nasal fin epithelium andits basal lamina as the fin regresses. I also describe the spatial and temporaldistribution of filamentous actin (F-actin) at the time of primary palate formation.Morphological changes in the developing nasal cavity are illustrated byperforming 3-Dimensional reconstructions from serial sections. This information isincorporated into a model describing cellular events that take place duringprimary palate formation.C57BU6J female mice were time mated with detection of a vaginal plugbeing called Day 0 of pregnancy. Females were sacrificed at times ranging fromDay 10 to 13, and embryos were removed from the uteri and placed in salinebuffer. The embryos were staged developmentally according to tail somite (T.S.)number and then prepared for either fluorescence or transmission electronmicroscopy. A total of 25 embryos were collected ranging from 7 to 27 T.S.Specimens used in the F-actin study were fixed and then snap frozen in liquidnitrogen in preparation for cryostat sectioning. Sections were stained with achemical probe for F-actin, NBD-phallacidin, which is a fluorescent phallotoxinthat stains specifically only actin of the filamentous type.There appear to be different zones or domains of epithelium within the nasalcavity, nasal fin, and facial prominences as the primary palate is forming withrespect to F-actin staining. This reflects different states of epithelial differentiationin these various regions. F-actin appears to be distributed uniformly all aroundthe nasal cavity with higher concentrations in apical epithelium facing the lumen.Also at regions where the nasal cavity bends or evaginates, as detected by the 3-D reconstructions, F-actin content in epithelium at these locations increasesdramatically. The facial prominence epithelium stains with reduced intensityespecially in regions of presumptive fusion. Nasal fin epithelium of the fused LNPand MNP stains weakly with a disorganized appearance compared to epitheliumin other areas.In the TEM study I found that prior to nasal fin formation the basal lamina ofthe MNP and LNP starts to break down before the prominences come intocontact. Once the nasal fin forms, the zone of basal lamina destruction becomeslarger. At the point of basal lamina destruction mesenchymal cell processes aswell as epithelial cell processes touch and penetrate the lamina. Once themesenchymal bridge has formed, new basal lamina beneath the base of thenasal cavity and oral cavity epithelia is formed.Comparisons were made of changes in the basal lamina during primarypalate formation and in other developmental systems such as Mullerian ductregression, thyroid formation and secondary palate formation. Similarities anddifferences between these systems and the primary palate were observed. Alsothe actin distribution in the developing nasal cavity was compared to that seen inthe salivary gland.The primary palate may be another example of a system where an epithelialcomponent is transformed to a mesenchymal component with timing of basallamina changes being important.ivTABLE OF CONTENTSPAGEAbstract 	 iiList of Tables 	 viiList of Figures 	 viiiAcknowledgments	xiii1. INTRODUCTION 11.1 General Introduction1.2 Primary Palate Formation in the Mouse. 21.3 Cellular Events Associated with Upper Lip and Nasal 4Cavity Formation.1.3 Epithelial-Mesenchymal Interactions in Development. 7The basement membrane.1.5 Some Examples of Epithelial to Mesenchymal Transformationin Development.141.6 Actin in Non Muscle Cells 171.7 Thesis Project 232. MATERIALS AND METHODS 242.1 Mouse Collection Procedures. 242.2 Tissue Preparation for Staining With Hematoxylinand Eosin.242.3 NBD-Phallacidin as a Probe for F-Actin. 252.4 NBD-Phallacidin Protocol for F-actin Staining. 26v2.5 Transmission Electron Microscope Protocol for Basal Lamina Study. 272.6 3-Dimensional Reconstruction of the Nasal Cavity. 283. RESULTS 313.1 Nasal Fin Formation and Regression. 313.2 F-Actin Distribution at Specific Tail Somite Number. 313.3 Basal Lamina Changes During Primary Palate Formation- 41Transmission Electron Microscopy Results.4. Discussion 1074.1 Possible Role of F-Actin in Regulating Nasal Morphologyin light of the 3-D Reconstructions.1074.2 Timing of Basal Lamina Disappearance During Primary 109Palate formation4.3 Description of Hays "Fixed Cortex" Model Applied to 112Epithelial-Mesenchymal Transformation.4.4 Fixed Cortex Model compared to the Primary Palate Model 1155. Conclusions 1176. Bibliography 119viLIST OF TABLESPAGETABLE 1	30v i iLIST OF FIGURESFIGURE	PAGE1 	 a) A LM frontal section showing the MNP and LNP of an	 5011 T.S. embryo stained with H&E taken anteriorly.b) A 12 T.S. embryoc) A 14 T.S. embryod) A 16 T.S. embryoe) An 18 T.S. embryo2 	 a) A LM frontal section showing the MNP and LNP of an 	 5211 T.S embryo stained with H&E taken anteriorly.b) A 12 T.S. embryoc) A 14 T.S. embryod) A 16 T.S. embryoe) An 18 T.S embryo3 	 a) A LM frontal section showing the MNP and LNP of a 	 5413 T.S. embryo stained with H&E.b) A LM section stained with NBD-ph.c) A LM section at the dorsal aspect of the nasalcavity stained with NBD-ph.d) A LM section at the medial aspect of the nasal cavitystained with NBD-ph.e) A LM section of the nasal fin stained with NBD-ph.4 	 a) A LM frontal section showing the MNP and LNP of a 13 T.S 	 56embryo stained with H&E.viiib) A LM section showing the MNP LNP and MxP stainedwith NBD-ph.c) A LM section of the medial side of the nasal cavitystained with NBD-ph.d) A LM section of the nasal fin stained with NBD-ph.5 	 a) A LM frontal section showing the MNP and LNP of a 13 T.S. 	 58embryo stained with H&E.b) A LM section showing the MNP and LNP stained withNBD-ph.c) A LM section of the nasal cavity stained with NBD-ph.d) A LM section of the nasal fin stained with NBD-ph.6 	 a) A LM frontal section showing the MNP and LNP of a 	 6015 T.S. embryo stained with H&E.b) A LM section showing the MNP and LNP stained withNBD-ph.c) A LM section of the LNP stained with NBD-ph.d) A LM section of the MNP stained with NBD-ph.7 	 a) A LM frontal section of a 15 T.S. embryo stained with NBD-ph. 	 62b) A LM section of the medial side of the nasal cavity stainedwith NBD-ph.c) A LM section of the top of the nasal cavity stained withNBD-ph.8 	 A LM section of the base of the nasal cavity of the specimen in 	 64Fig. 7 (15 T.S) stained with NBD-ph.9 	 a) A LM frontal section of a 16 T.S. embryo stained with NBD-ph. 	 66b) A LM section of the nasal cavity stained with NBD-ph.10 a) A LM frontal section of a 17 T.S. embryo stained with NBD-ph. 	 68ixb) A LM section of the top of the nasal cavity stained withNBD-ph.11 A LM section of the 17 T.S. embryo in Fig. 10 showing the base 	 70of the nasal cavity and the mesenchymal bridge stained withNBD-ph.12 a) A LM frontal section of an 18 T.S. embryo stained with NBD-ph. 	 72b) A LM section of the top of the nasal cavity stained withNBD-ph.13 A LM section of the 18 T.S. embryo in Fig. 12 showing a 	 74mesenchymal bridge stained with NBD-ph.14 a) A LM frontal section of a 19 T.S. embryo stained with NBD-ph. 	 76b) A LM section of the top of the nasal cavity stained withNBD-ph.15 A LM section of the 19 T.S. embryo in Fig. 14 showing a nasal 	 78fin stained with NBD-ph.16 a) A LM frontal section of a 27 T.S. embryo stained with NBD-ph. 	 80b) A section of the left nasal cavity at its midpoint stained withNBD-ph.c) A section of the mesenchymal bridge stained with NBD-ph.d) A section of the nasal cavity near its base stained withNBD-ph.17 a) A TEM of a 7 T.S. embryo showing the basal layer of 	 82epithelial cells of the MNP.b) MNP epithelia located further ventrally.c) MNP epithelia a few cells further ventrally.18 a) A TEM of a 10 T.S. embryo showing the basal layer of 	 84epithelial cells of the MNP.xb) Epithelial cells at the same level but on the LNP.c) A region of the MNP in a more ventral position.19 a) A TEM of an 11 T.S. embryo showing MNP epithelia. 	 86b) Epithelial cells at the same level as (a) but on the LNP.c) Epithelial cells of the LNP located further ventrally.20 a) A TEM of an 11 T.S. embryo showing MNP epithelia. 	 88b) Epithelial cells at the same level as (a) but on the LNP.c) A necrotic epithelial cell on the MNP side of the nasal fin.21 a) A TEM of a 12 T.S. embryo showing nasal fin epithelium that 	 90appears necrotic.b) Epithelial cells located further ventrally to (a).22 a) A TEM of a 13 T.S. embryo showing MNP basal epithelia. 	 92b) MNP epithelium located ventrally from (a).c) LNP epithelium at a corresponding position to (a).d) LNP epithelium in a ventral position to (c).23 a) A TEM of a 15 T.S. embryo showing LNP epithelia. 	 94b) LNP epithelia at higher magnification showing a region ofcontact between a mesenchymal cell process and the basallamina.24 a) A TEM of a 19 T.S. embryo showing a nasal fin. 	 96b) An epithelial cell on the LNP side of the nasal fin in (a).25 a) A TEM of a 19 T.S. embryo showing the tip of a nasal fin. 	 98b) The nasal fin in (a) at higher magnification showing basallamina destruction.x i26 a) A TEM of the 19 T.S. embryo shown in Figs. 25 and 26 showing 	 100mesenchymal cell processes contacting the basal lamina on theMNP side.b) A more ventral location to (a) showing cell process contactwith the basal lamina and possibly the epithelial cell.27 a) A TEM of a 21 T.S. embryo showing nasal fin epithelium on the 	 102LNP side.b) Epithelium ventral to (a).c) Epithelium ventral to (b).d) Epithelium ventral to (c).28 a) A TEM of a 27 T.S. embryo showing epithelium at the base 	 104of the nasal cavity where a mesenchymal bridge has formed.b) An epithelial cell lateral to (a) showing an intact basal lamina.29 a) A computer generated 3-D reconstruction of the nasal cavity 	 106of an 11 T.S. embryo. -nasal cavity epithelium, -LNPepithelium, -MNP epithelium.b) A 12 T.S. nasal cavity.c) A 14 T.S. nasal cavity.d) A 16 T.S. nasal cavity.e) An 18 T.S nasal cavity.x i iAcknowledgmentsI wish to thank Dr. Virginia M. Diewert for giving me the opportunity to workin her lab and for her advice throughout my thesis project. Also, I would like tothank Mrs. Barb Tait for her technical assistance preparing tissue for lightmicroscopy and Mr. Andre Wong for his help sectioning material for electronmicroscopy. Dr. W. Vogl provided much helpful advice during the datainterpretation as did other committee members Dr. M. Harris, and Dr. V. J. Uitto.This work was supported by the Medical Research Council of Canada grantnumber MT 4543.1. INTRODUCTION1.1 General IntroductionThe human primary palate forms during stage 17 (O'Rahilly and Muller,1987) at about 41 days postfertilization (Moore,1989). The mouse model hasbeen used in studies involving primary palate formation to investigate bothnormal and abnormal development of facial structures (Reed, 1933; Trasler,1968;Gaare and Langman,1977a,b). It has been shown that different strains of micehave different susceptibilities toward developing certain facial clefts (Trasler,1968). Specifically, NJ mice develop lateral cleft lips and C57BL mice developmedial clefts. The C57BL strain does not develop lateral cleft lips so it is aconvenient strain in which to study normal primary palate formation. Failure of theprimary palate to form may result in the facial abnormality known as cleft lip(Warbrick, 1960). In humans, cleft lip occurs with an incidence of 1 in 600 births inCaucasian populations, and as high as 1 in 363 in North American Indianpopulations (Lowry and Renwick, 1969). It is thought that embryonic face shapemay be one factor contributing to cleft formation (Fraser and Pashayan, 1970;Trasler, 1968). It is also thought that a genetic component (Juriloff,1986) as wellas a number of environmental factors (Bornstein et al., 1969; Davidson et al.,1969) may contribute to producing this facial abnormality. The exact mechanismbehind cleft lip formation is still not known. It is hoped that by understanding howthe upper lip develops normally one could then better speculate what goes wrongduring abnormal development.11.2 Primary Palate Formation in the MouseA portion of the migrating neural crest population comes to rest in an areathat will soon become the embryonic face. In fact, these neural crest cellsconstitute almost the entire facial mesenchymal cell population above thedeveloping oral cavity (Johnston and Sulik,1980). The mid-face develops first asthickenings, called the nasal placodes, of ectoderm on either side of the face. Themesenchyme at the sides of the placodes then condenses and proliferates as dothe epithelial cells of the placodes themselves (Pourtois, 1972). This results in anapparent sinking inward of the placode and a rising of the medial and lateral rimsto form a primitive nasal pit or groove. This olfactory pit becomes deeper as thelateral and medial nasal prominences (LNP and MNP) grow. The nasal pit isexposed to the maxillary prominence (MxP) at its floor. The maxillary prominenceis formed by the proximal half of the mandibular arch that then grows forward atits tip to eventually meet up with the LNP and MNP (Johnston and Sulik,1980).Johnston speaks of the maxillary prominence as being "overwhelmed" by theMNP and LNP. These prominences are extremely well developed in the mouseprobably due to the mouse having a highly evolved sense of smell. The actualformation of the primary palate in the mouse starts at the bottom of the nasal pit, inthe isthmus which is the region between the MNP and LNP. The MNP and theLNP converge superior to the isthmus with the medial surface of the LNP meetingthe lateral surface of the MNP. Fusion of the LNP and MNP occurs around the2opening of the nasal pit as well as inside the pit. Fusion then proceeds in anantero-inferior direction. These two epithelial surfaces form a plate called theepithelial plate of Hochstetter or the nasal fin (Patten, 1968). The nasal fin is atransient structure that persists during the developmental period corresponding to11 and 12 tail somites in the C57 mouse. There is question as to what actuallyhappens to the epithelial cells of the nasal fin. Lejour (1969) states that thesecells undergo necrosis while others feel that the epithelial cells may migrate awayand become a part of the surrounding mesenchymal cell population(Patten,1968). It is even speculated that the epithelial cells may transform intomesenchymal cells such as is proposed to occur during secondary palateformation (Fitchett and Hay, 1989). Then at about 13 T.S., the nasal fin starts toregress (Gaare and Langman, 1977b) and the epithelial seam is disrupted.Mesenchymal cells from the medial and lateral side break through the nasal finand form what is called a mesenchymal bridge (MB).Little is known, at the ultrastructural level, about the mechanism of nasal finregression. It is not known whether contact is necessary to initiate nasal finbreakdown or whether it depends on a pre-existing genetic program inherent tothe cells. Like all epithelial cells, the cells of the nasal fin have an underlyingbasement membrane. It also is not known what role the basement membranemay play in nasal fin regression; that is, whether or not it must disappear firstbefore the mesenchymal bridge can form, or does it merely play a passive role.Once formed, the mesenchymal bridge grows and enlarges becoming morerobust and it is here that the primary palate is considered to have formed. Failure3or incomplete fusion of the MNP and LNP and subsequent insufficient MBformation may result in a cleft lip. As the face and especially the brain grows anddevelops the resulting forces generated may tear the facial prominences apart.There are a number of theories, some of which will be discussed later, as to howa cleft lip may form.1.3	 Cellular Events Associated With Upper Lip and Nasal Cavity Formation.Fusion between the epithelial linings of the MNP and LNP transforms thenasal pit into a primitive nasal cavity and forms an epithelial seam called thenasal fin. Only certain regions of the epithelium come into contact however, andremaining regions become part of the nasal and oral cavities.Gaare and Langman (1980) investigated the process of epithelial fusionand studied DNA synthesis at stages prior to contact of the MNP and LNP. Theyfound that the level of DNA synthesis in presumptive fusing epithelia and non-fusing epithelia decreased as the facial prominences came into contact. Theyconcluded that since both types of epithelia, and not just the pre-fusion epithelia,showed decreased DNA synthesis, it was unlikely that the reduced level of DNAsynthetic activity was an indication that the nasal fin was undergoing cell death.They felt that cells of the nasal fin were probably being incorporated intoneighboring epithelial linings of the growing nasal and oral cavities. They hadobserved epithelial cell death in some cells of the nasal fin in an earlier study(Gaare and Langman, 1977a) and said that direct contact was not necessary to4trigger cell death. Using an acid phosphatase procedure that tests for lysosomalactivity they found that adjacent epithelial cells transform to macrophages whoselysosomes digest the degenerating epithelial cells.Gaare and Langman (1977b), also studied the epithelial cell surfaces ofthe approaching LNP and MNP using ruthenium red which is a positive dye thatbinds to negatively charged glycoproteins comprising proteoglycan moietieswithin the outer surface of the plasma membrane. They found that prior to contact,the pre-fusing superficial epithelia extend cell projections that are coated withglycoproteins, which they believed were necessary to mediate initial adhesionbetween the MNP and LNP.The distribution of surface coat material has also been studied using lectins(Burk et al., 1979). Concanavalin A was used to bind surface coat material on pre-fusing and fused facial prominences. It was found that there was increasedsynthesis of surface coat material as the MNP and LNP were fusing and still afurther increase once fusion had occurred. They concluded that this glycoproteinsurface coat may aid adhesion and fusion of the facial prominences.In a scanning electron microscope study (Yee and Abbott, 1978) looking atprimary palate formation in the chick embryo, it was observed that prior to fusion,epithelium from the MNP, LNP and MxP started to exhibit long, slender filamentsthat extended towards the point of fusion. They believe that these "prefusionfilaments" may function in alignment or adhesion of the facial prominences.More recent studies have demonstrated that within the population ofprefusion epithelium in the MNP and LNP a unique cell type may exist (Kosaka et5al., 1985; Kosaka and Eto, 1986). These cells have been called superficial cellsand they are the ones that are thought to bridge the initial gap between the MNPand LNP. They possess well developed junctional complexes with associatedintermediate filaments and microfilaments subjacent to the junctions. Thesesuperficial cells are located at the fusion site at a region of transition betweensquamous surface ectoderm of the primitive oral cavity and the pseudo-stratifiedcolumnar epithelium in the nasal pit. Kosaka states that superficial cellsresemble the neural crest cells that make initial contact during neural tubeclosure. These cells like neural crest cells make initial contact first betweenlamellapodia, then between interdigitating cell projections. Fusing epitheliashowed some cell death but so did epithelial cells at much earlier stages and atother locations such as at the nasal placode and early nasal pit. Kosakaproposes that epithelial cell death during primary palate formation is not onlyrelated to the fusion process between LNP and MNP but also to morphogeneticevents in the growing face such as invagination of the nasal placode and growthof the prominences.Recently, investigators have developed in vitro models to study the processof fusion between LNP and MNP (Gibson et al., 1989; Forbes and Steffek, 1989;Forbes et al., 1989). Epithelial cells in organ culture were found to extendfilopodia across the fusion site that attach to the opposite facial prominenceforming extensive bridges. These processes are thought to help during earlyfusion. This same group went on to compare the fusion process in a strain of micesusceptible to cleft lip. They used A/WySn mice, a strain with a 20-25% cleft lip6frequency (Kalter,1979.) and found that the epithelial cells of the MNP and LNP inthese mice had reduced epithelial bridging and fewer cell projections at thepoints of fusion compared to a non-cleft lip strain. This reduction in epithelialsurface activity was proposed to account for the observed differences in facialclefting in the two strains.1.4 Epithelial-Mesenchymal Interactions in Development: The basementmembrane.During embryogenesis there is constant interaction between adjacenttissues while a particular organ system is forming. These tissues are separatedby substance known as the extracellular matrix (ECM). Grobstein in 1954proposed that the ECM mediated these tissue interactions and was responsiblefor changes in tissue morphology as various organs developed.In the early vertebrate embryo there exists a basic dichotomy of tissueorganization. Cells can be grouped into one of two classes. The first is epitheliawhich consists of cells closely attached and linked by tight junctions, adherensjunctions, and desmosomes (Bernfield et al., 1984). They form a sheet orcovering that sits on top of an ECM. The cells facing the lumen usually have aspecialized surface containing microvilli and a distinct glycoprotein surface coat.The basal surface of an epithelial cell faces a specialized type of ECM producedby the epithelial cell called the basement membrane.The other class of cell is called mesenchyme and they migrate through ECM.7Mesenchymal cells can form clusters of cells grouped together but they are notalways attached to their neighbors. The early embryo is made up mostly ofepithelia. Mesenchymal cells are derived from these epithelia from sheets ofcells that break away from the original cell population and migrate to newlocations within the developing embryo (Noden, 1984; Moore, 1989). Primarymesenchyme forms from the primitive streak. Most of the mesenchyme of thedeveloping face comes from the neural crest. This is a cell population that breaksaway from neural ectoderm cells of the closing neural tube. As a few cells changeshape in an epithelial sheet the form of the sheet will change possiblyinvaginating forming a tube or evaginating forming a bud. Cell shape changesstarting with just a few cells can become morphologic changes at the tissue level.Epithelial cells that fold into buds such as the lung, liver, prostate and the kidneycollecting tubules will go on to branch (Farquar et al, 1981). The budding andbranching is due to specific epithelial-mesenchymal interactions that occur atprecise times over short distances while an organ is developing. The interactionthat takes place effects both tissues involved (Gurdon, 1987).It is a common belief that the ECM is capable of controlling cell behaviorsince the ECM is closely associated with the cell surface. Molecules that link thecells to the ECM are closely associated with the cell membrane. They are integralglycoproteins that span the cell membrane and are thought to be able to interactwith the cells cytoskeletal elements (Uitto and Larjava,1991). These moleculescan be in the form of protein receptors or proteoglycan receptors. An example isthe recently discovered heparan sulfate proteoglycan named syndecan. This8molecule can bind a number of ECM components such as different collagens,fibronectin and tenascin, a molecule thought to be important in development(Halter, et al., 1989). Syndecan is able to bind to cytoskeletal actin microfilamentsand is thought to possibly be involved in cell-matrix and cell-cell interactions. It isthought that the arrangement of the cytoskeleton and the regulation of cell shapedepends a great deal on the surrounding ECM. Cell shape changes on a multi-cell level could lead to morphological shape changes at the tissue level(Bernfield, M. 1984). Different interacting tissues can reciprocally modify theothers ECM which may act as a signal for a group of cells to undergo a specificevent such as mitosis or protein synthesis.Epithelial cells and mesenchymal cells have different ECMs. The matrix isdifferent in composition and in how it associates with adjacent cells. Bothepithelial cells and mesenchymal cells are capable of making their own ECM.Epithelial cells synthesize a specialized type of ECM called a basal lamina andmesenchymal cells embed themselves in an interstitial matrix. The interstitialmatrix of embryonic tissues is a highly hydrophilic gel, rich in interstitial fluid andthe glycosaminoglycan hyaluronic acid. Fibronectin is often present as arecollagen types I, Ill, and V. Chondroitin and heparan sulfate are also present.These molecules and others, are thought possibly to be involved in certaininduction mechanisms that take place during morphogenesis. The basal laminais specific for epithelial cells and is often called the basement membrane orbasement lamina. Prior to the use of the electron microscope in biology the termbasement membrane referred to the PAS-positive layer around epithelia and9muscle fibers visible with the light microscope. With the electron microscope itbecame apparent that the basement membrane was made up of componentparts. The term basal lamina is an electron microscopic term that refers to themoderately electron dense layer found around epithelia, nerve, muscle andadipose tissue (Farquhar et al., 1981). In most locations the basal lamina consistsof a lamina densa that is 20-50 nm thick, a lamina lucida which is less electrondense and is 1 Onm thick. The basal lamina consists of functional elements thatregulate cell activities, and structural elements that impart a physical stability tothe tissue. In every basal lamina there are certain components that may differ inrelative amounts but are always present. These are: type IV collagen laminin,heparan sulfate proteoglycan, nidogen and bullous pemphigoid antigen(Charonis and Tsilibary, 1990; Kleinman et al., 1987). It is thought that thesemolecules in different proportions may impart to each basal lamina a certainspecificity compared to others (Dziadek and Timpl,1985; Wan et al., 1984). Allbasement membrane molecules are able to interact with each other. Theseassociations, and others, with surrounding tissue and ECM may play a role inpromoting growth and differentiation during development (Ferguson, 1984).Recently, investigators looked at the spatial and temporal distribution ofbasement membrane components during maxillary process formation in the chickembryo (Xu, et al., 1990). They mapped the spatial and temporal distribution oflaminin and type IV collagen in the face and found that regions that wereundergoing rapid growth and expansion, such as the lateral surface of themaxillary process, showed less intense staining for type IV collagen. Regions that10were not undergoing rapid growth such as the roof of the stomodeum, stainedwith high intensity for type IV collagen. Laminin staining was uniformly high inboth locations. They proposed that type IV collagen may confer structural stabilityto slow growing regions.Epithelial-mesenchymal interactions are thought to be involved in thedevelopment of many organ systems in the body. Tooth, thyroid, lung, bone,kidney, mammary gland, salivary gland, gut, and pancreas are all thought to formas a result of interactions between epithelial and mesenchymal components(Mina and Kollar,1987; Slavkin et al., 1984; Van Exan and Hal1,1983;Aufderheide and Ekblom, 1988). By performing tissue recombinations betweendifferent tissue types and even between different species it has been found thatthe mesenchymal component instructs the epithelium to follow a specificdifferentiation pathway. For example, Slavkin et al. (1984) used dentalmesenchyme to induce the expression of enamel proteins in dental epithelium.He also stimulated undifferentiated lung mesenchyme to induce buds of epitheliato differentiate into alveolar type II cells. Hall in 1982 performed recombinationsbetween mandibular arch epithelium and mesenchyme and inducedchondrogenesis and osteogenesis in mandibular mesenchyme. He observed thatdifferent regions of mandibular epithelium induced a greater inductive responsein the mesenchyme. He and others (Mina and Kollar, 1982; Van Exan and Hall,1984; Lesot; et al., 1986) have hypothesized that inductive signals passingbetween the epithelia and mesenchyme may reside in the basal lamina. Directcell-cell contact is not thought to be necessary for induction to occur although11some investigators believe cell contact may be important (Saber, 1989). Thisstudy performed recombinations between maxillary prominence epithelium andmesenchyme in the developing face. They performed homotypic (maxillaryepithelium and maxillary mesenchyme), heterotypic (limb epithelium andmaxillary mesenchyme), and heterochronic (epithelium and mesenchyme fromdifferent stages of development) recombinations and found that the mesenchymewould only remain viable if it was placed in contact with the epithelium beingtested.The morphogenesis of the submandibular salivary gland has often beencited as a model system for tissue interactions. The gland develops initially as asheet of endodermally derived epithelial cells that starts to bud into mesenchymalcells that are derived from the neural crest (Bernfield et al., 1984). The budelongates and forms a stalk so the structure now looks like a lollipop. Thennotches start to form at the distal end of the bulb that then deepen and form clefts.This branching process repeats itself over and over. Eventually the epitheliumbecomes the secretory apparatus of the gland and the mesenchyme gives rise tothe vascular supply and the supportive stroma. If the mesenchyme is removedfrom this system, branching ceases and the epithelial cells flatten and eventuallydie. Thus the mesenchyme is thought to be responsible for epithelial cellproliferation and epithelial integrity. Cell proliferation occurs most rapidly at thedistal ends of the lobules. In order for notches and new clefts to form at thelobules distal ends the epithelial cells must first change shape. This is thought tobe an actin mediated event. Basally arranged actin microfilaments contract giving12the epithelial cells a wedge shaped appearance (Spooner, 1973). These cells atthe cleft have a much lower rate of mitosis than do the epithelial cells at thegrowing tip. It was found by histochemistry that the clefts contain abundantglycosaminoglycan and collagens type I and IV that are thought to aidstabilization. The basal lamina is thick in the clefts but it is thinned anddiscontinuous at the growing distal tips. There are direct contacts betweenepithelial and mesenchymal cells at the growing tips that penetrate the basallamina (Cutler and Chaudry, 1973) and these may be locations for cell-cellcommunication of some sort. The basal lamina is thought to help) maintain thelobular epithelial morphology of the gland. Banerjee et al. (1977) and Bernfield(1981) propose that the basal lamina is influenced by the mesenchyme tobecome discontinuous or become stabilized at different places at different timesas the salivary gland develops. Presumably the mesenchyme turns on and offsynthesis of basal lamina components by the epithelium.Recently, the branching phenomenon of the developing salivary gland wasstudied in organ-culture to determine what effect the mesenchyme had onepithelial ability to branch (Takahashi and Nogawa, 1991). Epithelium wasseparated from mesenchyme by a membrane filter. Normal branchingphenomenon was only observed when the epithelium was exposed to Matrigel, areconstituted matrix of basement membrane components. They used amembrane filter with a pore size small enough to prevent direct cell-cell contactand still observed normal branching if the Matrigel was present. It was concludedthat some sort of diffusible factor produced by the mesenchyme was necessary to13induce epithelial branching.1.4 Some Examples of Epithelial to Mesenchymal Transformations inDevelopment Associated With Basal Lamina Destruction.In a number of developmental systems where an epithelial component isknown to disappear and be replaced by a mesenchymal component there is ashared phenomenon that takes place. This is that the epithelial basal lamina mustfirst break down before the epithelial cells are removed. The exact fate of theepithelial cells is as yet controversial and it still is not completely clear whether ornot they 1) die as a result of a programmed cell death phenomenon, 2) migrateaway into the mesenchymal cell population, 3) transform into mesenchymal cells,or 4) become incorporated into nasal and oral epithelium. In some systems it isthought that epithelial cells might become phagocytic like macrophages anddegrade neighboring epithelial tissue. The developing organ systems to whichthis phenomenon might apply are: thyroid, lens, Mullerian duct and secondarypalate formation.Greenburg and Hay in 1988, studied thyroid follicular epithelium in vitro andfound that isolated thyroid follicles that had their basal laminas enzymaticallyremoved could transform into mesenchyme-like cells when grown on type Icollagen gels. The epithelial cells switch from making type IV collagen to type Icharacteristic of mesenchymal cells. They also develop mesenchymal cellpolarity, become elongate and develop pseudopodia and filopodia. In addition14they cease to make thyroglobulin. The newly formed mesenchymal cells elongatefrom the basal surface,detach from the follicle and invade the surroundingcollagen matrix gel as single bipolar cells. Their cytoplasm becomes filamentouslike mesenchymal cells and the intermediate filament production switches fromkeratin tonofilaments associated with desmosomes to vimentin characteristic ofmesenchymal cells. There is no coexpression of keratin and vimentin. It isthought that a vimentin cytoskeleton is required for migration through a collagenmatrix. It has been concluded that transformation from epithelium to mesenchymeis carefully controlled and only occurs in appropriate areas at predictable times.Greenburg and Hay (1986) reported that anterior lens epithelium, whensuspended in type I collagen matrices, gives rise to freely migratingmesenchymal-like cells. This transformation was also inhibited if the epithelialcells were grown on basal lamina substrate.The regression of the Mullerian duct is another example of an epithelial tomesenchymal transformation where the basal lamina first disappears. TheMullerian duct is a structure that persists only if the phenotype is female. In themale this structure regresses. There is degeneration and phagocytosis ofepithelial and mesenchymal cells, removal of the ductal basal lamina, and a lossof the distinction between the epithelium and the mesenchyme (Donahoe et al.1984). This study reports that basal lamina integrity is lost prior to duct regressionand this occurred only in areas where mesenchymal cell processes touch thebasal lamina. Epithelial cell processes also extend into and through the basallamina and come into contact with mesenchymal cells and their processes.15Disappearance of the basal lamina occurs only if the duct is destined to regress.They do report limited cell death in the regressing duct but they believe cellmigration of epithelial cells into the mesenchymal cell compartment is a moreimportant event. The study does not however report whether there was a switchfrom keratin to vimentin intermediate filament production in the transformingepithelial cells. Heparan sulfate proteoglycan synthesis did stop at the time oftransformation and fibronectin lysis increased dramatically around the regressingduct. There is also increased hyaluronic acid production at the site where theepithelial cells are thought to be transforming which is thought to aid in motility ofboth epithelial and mesenchymal cells. The epithelial cells that apparentlytransform still express glucosamine residues even though they are nowmesenchymal cells. Synthesis of laminin and type IV collagen also diminishes asthe ductal basal lamina is degraded.A recent example where there is evidence of an epithelial to mesenchymaltransformation is during secondary palate formation (Fitchett and Hay, 1989).They investigated the fusion of palatine shelves in rat. These shelves rotate andapproach each other in a medial direction and eventually fuse. The palatineshelves are lined with an epithelium and it is the medial edge epithelium (MEE)that comes into contact. The outer cells of the MEE initially slough off beforecontact is made. The two shelves then adhere and the epithelial seam thins anddisappears leaving only islands of epithelial cells. Then these islands alsodisappear leaving mesenchymal cell confluence. This study reports thatprogrammed cell death is probably not a major mechanism behind the epithelial16seams disappearance. There is cell death but only in the outermost peridermalcells of the MEE. Again the epithelial cells that are presumptive mesenchymalcells extend pseudopodia and filopodia before they migrate into themesenchymal cell compartment. The cells of the MEE seam also lose a keratintype intermediate filament complement and gain a vimentin type cytoskeleton.The basal lamina is continuous prior to fusion and is contacted by numerousmesenchymal cell processes. The basal lamina then thins at the time contact ismade and soon becomes discontinuous and patchy and eventually disappears.Hemidesmosomes are infrequently seen beneath the epithelial seam attachingthe epithelia to the basal lamina. In another study looking at secondary palateformation, Ferguson (1988) reports that prior to contact of approaching palatalshelves, the medial edge epithelium still possess an intact basal lamina. He usedtype IV collagen as a marker. The epithelial cells stain intensely for desmoplakin,a protein associated with desmosomes, until fusion occurs. After fusiondesmoplakin staining is reduced as the epithelial cells lose their desmosomalattachments to their neighbors. The basal lamina becomes discontinuous andfragmented after fusion occurs and TEM shows mesenchymal cell processesextending through it.1.5 Actin in Non Muscle CellsDuring development there are many shape changes that occur in theforming tissues. To understand the basis for morphogenesis in embryonic tissues17it is necessary to know where cells divide, migrate, die, or change shape. Anumber of investigators have proposed that components of the cytoskeletoncould be involved in the coordinated shape changes often seen in developmentalsystems (Priess and Hirsh, 1986; Madreperla and Adler, 1989). These studieshave attributed cell and tissue shape changes to the actin microfilament networkand microtubules within the cytoplasm. A few of these examples will bediscussed.Actin is a major constituent protein of non muscle cells (Pollard, 1981). It isfound in the periphery of most cells organized as filaments. It forms much of theso called "cytoplasmic matrix" and is a major component of the microfilamentsystem. Globular monomeric forms of the actin molecule exist called G-actin.Filamentous forms also exist called F-actin. Many actin binding proteins regulatethe size of actin filaments and the relationships of one filament to another(Pollard, 1981). Actin filaments can form either networks or bundles. Filamentbundles can be of random arrangement and these usually have contractileproperties, or they may be unipolar and tightly crosslinked with these havingmore structural properties (Vogl, 1989; Emerman and Vogl, 1986). Contractilerings located in the cleavage furrow of dividing cells, the zonula adherensjunctions of most epithelial cells and fibroblast stress fibers are an example ofcontractile bundles. Actin filaments have been shown to be involved in numerousmotility type events such as endocytosis, secretion, phagocytosis, celltranslocation and intracellular vesicular movement. They also have morestructural roles as in the cores of microvilli and stereocilia (Vogl, 1989). Many of18actin's functions involve an interaction between actin and the cell membrane. It isby this interaction that actin may provide the cell with a mechanism of: 1)establishing specific domains within a region of membrane, 2) internalizingregions of a membrane, 3) moving a membraneous organelle to a differentlocation within the cell, 4) generating changes in the shape of the cell, and 5)moving the cell (Vog1,1989). It is the last two mechanisms I am looking at duringprimary palate formation and changes in nasal morphology.A good example illustrating the possible role of actin in morphogenesis ispresented in a study by Preiss and Hirsh (1986). They investigated the growthpattern observed in the Caeenorhabditis elegans embryo which undergoes rapidelongation of its body structure in the anterior-posterior axis. As the embryoelongates there is almost no division or migration of cells. Instead the cellsthroughout the embryo appear to change shape in a simultaneous andcoordinated manner. It is thought that actin microfilaments constrict andmicrotubules distribute such that the embryo as a whole decreases incircumference and elongates. The cytoskeletal organization of certain cells withinthe embryo determines the embryo's shape during elongation while anextracellular cuticle maintains the body shape after elongation.Retinal photoreceptors have also been studied to determine the role ofmicrotubules and microfilaments in developing and maintaining the polarizedshape of these cells (Madreperla and Adler, 1989). This study detectedimmunocytochemically longitudinally oriented actin microfilaments andmicrotubules in these photoreceptors and they postulated that within the retinal19photoreceptor cell there exists continuously active, oppositely directed,microtubule- and actin-dependent forces. These forces depending on how theybalance, may be a determining factor in forming and keeping the shape andpolarity of photoreceptor cells.Other studies have taken a developing organ system and examined it in vitrounder certain experimental conditions in order to test the idea of contractileproteins such as actin and myosin being involved in cell and tissue shapechanges. Hilfer et al. (1977) examined thyroid placodes in the presence of acontractile medium consisting of Triton X-100 and ATP. Normally thyroidplacodes take approximately 7 hours to evaginate in ovo, but in the presence ofcontractile medium, evagination was observed in minutes. They found that thecells involved in the shape changes within the placode were located at theperiphery and the sharp bends that formed in contractile medium could not beexplained by a simple pinching of cell apices at the point of folding. Instead theyproposed that several forces may be acting at the site of evagination. The samecontractile medium was used by Smuts (1981) in the study of nasal pit formationin the mouse. Previously it was thought that mesenchymal proliferation centerswere responsible for the rise in the MNP and LNP on either side of the nasalplacode (Lejour,1969). Then it was thought that both epithelium andmesenchyme actively participate in the forming of the facial prominences (Portois,1972). Minkoff and Kuntz (1977) found that the centers of mesenchyme did notundergo an increase in cell proliferation throughout the time of nasal placodeinvagination.20The morphological appearance of the forming nasal pits and facialprominences resembles the evagination of lens and thyroid (Hilfer et al., 1977).Both of these organs undergo early morphological movements in the absence ofa contribution by mesenchymal cells. Development of a nasal pit normally takesabout 8 hours from the placodal stage to an invaginated nasal pit. But in thepresence of Triton X and ATP, this invagination occurred in 5 minutes (Hilfer, etal. 1977). The contraction medium stimulated the production of a smallindentation in a newly formed placode region and a deep, pitlike invagination in afully pseudostratified placodal epithelium. The heightened prominences werestrictly epithelium and the mesenchyme had not changed position or appearance.They proposed that the rapid time for this precocious invagination does not allowthe mesenchyme to add cells by either mitosis or cell migration to support theepithelium.There has been much research done on the arrangement actin filamentstake on in a number of different cell types. Most types of epithelial cells exhibitactin associated junctional complexes called zonula adherens junctions. Thesejunctions have contractile bundles of actin filaments extending circumferentiallyalong the cytoplasmic surface of the junctional membrane (Drenckhahn andFranz, 1986). Alpha actinin and vinculin are actin associated proteins that arelocated on the cytoplasmic side of the membrane. These proteins are thought toanchor actin filaments to the plasma membrane. Microfilaments can associatewith the membrane laterally, where the filaments lie parallel to the cytoplasmicsurface or end on, where the microfilaments seem to terminate at the surface21(Rogalski and Singer,1985). Cleavage furrows of dividing cells and stress fibersare examples of lateral associations while end on associations include theplaque like focal adhesion sites formed by fibroblasts to their substrata or witheach other. Actin filament bundles have been shown to be present in specializedadherens junctions of Sertoli cells and are thought to play a functional role duringspermatogenesis (Vogl and Soucy, 1985). F-actin is also found in myoepithelialcells of the mammary gland where these bundles are thought to control cell sizeand shape during lactation (Emerman and Vog1,1986). During the developmentof the chicken eye, retinal pigmented epithelial cells extend apical projectionscontaining abundant F-actin. These projections contain actin arranged inparacrystalline bundles that are thought to be more structural than contractile innature (Owaribe and Eguchi, 1985). Cultured epithelial cells show differentdistributions of F-actin as they make a transition from a stationary to a motile state(Takeuchi, 1987). In a stationary state the cells are polygonal and F-actin isdeposited along cell borders. The cells then become hemispherical and actindistributes along the inner cell surface. Then as the cells become motile theyextend lamellae that contains an amorphous mass of F-actin at its distal end.Tucker et al. (1985) have studied neural crest (NC) migration and hypothesizedthat since cultured NC cells have actin microfilaments distributed at the cell cortexand not as localized dense focal contacts, the NC cells move through embryonicECM making only weak adhesions with their substratum. They postulate thatother embryonic cell types may generate stronger forces on the delicate ECM thatwould restrict their migration and thus set up morphogenetic events. Tomasek22and Hay (1984) cultured avian embryonic corneal fibroblasts in ECM and foundthat locomotory behavior was accompanied by adhesion of the cells to collagenfibrils in the ECM. The fibroblasts may then move by an interaction between themyosin rich cytosol and the F-actin rich cell cortex. By injecting phallotoxins intolive cultured 3T3 fibroblasts, Wang (1987) showed that some actin filamentsundergo continuous movement and reorganization in living cells.1.6 Thesis ProjectIn this study I will describe the spatial and temporal distribution of F-actin inepithelium of the developing nasal cavity, nasal fin, and the facial prominencesduring the time period corresponding to primary palate formation. I will go on todescribe changes in the basal lamina during nasal fin formation and regressionusing transmission electron microscopy. In the discussion, I will compare myobservations to literature on other developmental systems.232. MATERIALS AND METHODS2.1 MOUSE COLLECTION PROCEDURESC57BU6J mice were used for this study. Females were mated with malesbetween 5 p.m. and 8 a.m., at which time the females were checked for thepresence of a vaginal plug. Detection of a plug was considered to be day 0 ofpregnancy. 12 midnight was considered as the time of conception. The mice werefed Mouse Chow and water ad libitum. The animal quarters were illuminated for12 hours each day from 6 am to 6 pm. Pregnant females were sacrificed atspecific times on the 10th, 11th, 12th, and 13th days of pregnancy. Ages ofembryos were assumed, for example, to be 10 Days 8h (10/8) at 8 a.m. on the11th day. Embryos were dissected from the uterus and placed in phosphate-buffered saline (PBS) at 4 °C. Before fixation the embryos were staged under abinocular dissecting microscope according to the number of somites from thecaudal edge of the hind limb to the end of the tail (tail somites: T.S.). Aftercounting the number of T.S. the embryos were prepared for either staining withNBD-phallacidin or observation using transmission electron microscopy. For thisstudy 14 embryos were used for the F-actin experiments and 15 embryos wereused for the TEM study with mice ranging from 7 T.S. to 27 T.S.2.2 Tissue Preparation for Staining with Hematoxylin and Eosin (H/E)Embryos were dissected from the uterus and placed in Bouins fixative. Aftera tail somite count was made, the heads were removed and placed in anAutotechnicon Model 2A for tissue processing. The tissue was dehydrated in agraded ethanol series: 1x75%, 2x95%, and 3x100%, and then cleared in24Chloroform. Each step in the procedure was for 20 minutes. The tissues wereembedded in Paraplast Plus Tissue Embedding Medium containing dimethylsulphoxide for rapid tissue infiltration. The melting point of the wax was 56-57 °C.The heads were then placed in a mould and orientated such that frontal sectionscould be taken of the faces. Sections were cut at a thickness of 7 micrometers ona microtome, mounted on glass slides, and dried in a 50 °C oven.For staining with H/E sections were first dewaxed in xylene and thenrehydrated in ethanol: 100%, 95%, 70% and washed in water. They were thenstained in hematoxylin for 3 minutes and rinsed in tap water. This was followed bya dip in saturated lithium carbonate and the sections were washed again inwater. Slides were then stained in eosin for 1 minute and then dehydratedthrough 3 baths of absolute alcohol and then cleared in xylene. The sectionswere then mounted in Entellan and photographed.2.3 NBD-phallacidin as a Probe for Filamentous ActinPhallacidin is one of the phallotoxins isolated from the American variety ofthe deadly Amanita phalloides mushroom. This compound has a low molecularweight (847 daltons) and thus is able to readily penetrate tissue. Phallacidin is abicyclic peptide with a free carboxyl group to which the fluorophoreNitrobenzoxadiazole (NBD) is attached. Phallotoxins bind to both large and smallF-actin but are unable to bind monomeric G-actin. Phalloidin is anotherphallotoxin with molecular weight 789 daltons. This molecule has a higher affinityfor F-actin than does phallacidin and in this study it was used as a control. NBD isa small fluorescent molecule of 165 dalton molecular weight that is excited by25visible light and produces a yellow fluorescence when coupled to primaryamines. The phallotoxins are highly specific (stain F-actin at nanomolarconcentrations), are water soluble, and remain stable for a number of days.These characteristics make them very convenient probes for labeling andidentifying F-actin in tissue sections.2.4 NBD-phallacidin Protocol for F-actin StudyC57BU6J mouse embryos were dissected from the uterus at differentchronological ages and placed in 0.2 M phosphate-buffered saline (PBS). Theembryos were then staged developmentally by counting the number of tailsomites (T.S.). After T.S. counting the heads were removed. Fixation wasperformed in 3.7% paraformaldehyde for 30 minutes. A cryoprotection step wasused to prevent tissue damage due to ice crystal formation upon freezing. Thiswas performed using 40% sucrose for 12 hours. The specimens were then snap-frozen in hexane cooled to -70 °C. in liquid nitrogen. Embryos were embedded inOCT embedding media and oriented such that true frontal sections of the headscould be taken. The heads were sectioned on a cryostat at 8 to 10 micrometersthickness. Sections were placed on poly-lysine coated slides to prevent themfrom floating off the slide at later steps in the procedure. They were then post-fixed in acetone for 5 minutes and then allowed to air dry for 30 minutes. Thetissue was rehydrated in PBS for 10 minutes and then incubated in one of thefollowing treatment solutions: 1) PBS (a control for autofluorescence of thetissue); 2) PBS+ 1.65 x 10- 6 M NBD-phallacidin (fluorescent probe for filamentous26actin); 3) PBS+ 1.65 x 10-6 M NBD-phallacidin + 1.04 x 10-4 M phalloidin(competitive specificity control); and 4) PBS + 1.04 x 10-4 M phalloidin (control forphalloidin treatment). Samples were incubated in the above solutions for 30minutes and then washed 3 times in 100 microliters of PBS. This was followed bydehydration in a graded series of alcohols; 70%,95%, 2x100%, and then intoxylene. Sections were then mounted in Flo-Texx. Samples were viewed under aZeiss D-7082 Oberkochen standard ultraviolet microscope and photographedusing Fuji P-1600 film at 800 ASA.2.5 Transmission Electron Microscopy Protocol for Basal Lamina Study.C57BL/6J mouse embryos were dissected from the uterus and placed in0.1 M PBS for somite counting. The primary fixation was in 2.5% glutaraldehyde at4 °C for 1 hour. This was followed by 3 washes, 5 minutes each in 0.1 M PBS.After a 1 minute rinse in distilled water the tissue was fixed again this time in 1%osmium tetroxide for 1 hour. Osmium tetroxide was made as a 4% stock solutionwhich was diluted to 2% in distilled water and then to 1°/0 in 0.2 M PBS. Thissecondary fixation was followed by two 5 minute washes in 0.1 M PBS and a 1minute rinse in distilled water. Dehydration was performed in a graded series ofethanols starting with 30% at 4 °C for 10 minutes, 50% at 4 °C for 10 minutes andthen 2% uranyl acetate at 4 °C for 30 minutes. Next the tissue was placed in 70%at 4 °C for 10 minutes, then in 90% at room temperature for 15 minutes followedby two 15 minute steps in 100% alcohol. Substitution was carried out in 1 part27propylene oxide: 1 part 100% ethanol first for 15 minutes and then in straightpropylene oxide for 15 minutes. Embedding was first in 1 part epon: 1 partpropylene oxide for 1 hour and then 1 part propylene oxide: 3 parts epon leftovernight. Next the tissue was placed in straight epon plus catalyst for 1 hour. Thesamples were then placed in an incubator at 37 °C for 24 hours and then 60 °Cfor 48 hours. The blocks were trimmed and thin sections were taken using adiamond knife. Silver/grey sections were floated and collected oncarbon/colloidin coated 200 mesh Copper grids, counterstained with 2% uranylacetate for 30 minutes and with lead citrate for 5 minutes. Five to ten sectionswere analyzed at each of the stages collected. The samples were then observedon a Phillips 300 transmission electron microscope and photographed usingKodak 4489 electron microscopic film. The film was developed for 2 minutes withKodak D19 full strength, rinsed in tap water and fixed for 4 minutes in Kodak rapidfixer.2.6 3-Dimensional Reconstruction of the Nasal CavityFive C57 mouse embryos (11,12,14,16, and 18 T.S.) were serially sectionedafter being embedded in paraffin. The sections were taken at 7 micrometersthickness. A series of sections was chosen that illustrated the nasal cavity shapechange anteriorly to posteriorly. Every second section (14 micrometers apart) wasphotographed using Fuji 100 film and made into 35 mm slides. These slides werethen viewed through a Caramate slide projector and the faces were traced on28acetate transparencies. The three dimensional reconstruction program wasimplemented on a Hewlett-Packard 1000 Series E, minicomputer. It consists oftwo programs, one for data acquisition and the other for data display. Dataacquisition is through the program BCDIG and data display is through BC VIEW.Each tissue section is plotted as a contour outlining the section. Contours arethen plotted sequentially and observed at 40 degrees to the left and 0 degrees onthe vertical plane.29TABLE 1 	 SAMPLE SIZESFor the F-actin study 12 embryos were used:C57BL/6J 	 13 T.S.13 T.S.13 T.S.II	15 T.S.15 T.S.16 T.S.17 T.S.II	18 T.S.IV	18-20 T.S." 11d 11hII	19 T.S.27 T.S.For the basal lamina study 13 embryos were collected:C57BL/6J 	 7 T.S.II	10 T.S.Il	10 T.S.II	11 T.S.II	11 T.S.„ 	 11 T.S.12 T.S.II	13 T.S.14 T.S.II	15 T.S.19 T.S.II	21 T.S.II	27 T.S.3. RESULTS3.1 Nasal Fin Formation and RegressionFigures 1 and 2 show a series of C57 mouse embryos from 11 to 18 T.Ssectioned in the frontal plane and stained with Hematoxylin and Eosin. These areintended to illustrate the various stages of primary palate formation. The LNP andMNP can be seen to approach each other and then fuse forming an epithelialseam known as the nasal fin (Fig. 1d). One can observe different stages ofprimary palate formation in a single embryo depending on the level of section.The nasal fin persists anteriorly before being replaced by a mesenchymal bridge.That is why Figure 1 e shows MNP and LNP still prior to fusion and Figure 2eshows the same specimen in a more posterior region where a mesenchymalbridge has already formed.3.2 F-actin Distribution at specific tail somite number13 T.SVarious stages of prominence fusion were observed in the 13 T.S. embryosexamined. Figure 3 shows LNP and MNP just prior to contact. Epithelium of thefuture nasal cavity is already stratified and possibly pseudo-stratifiedcharacteristic of fully differentiated olfactory epithelia. The roof of the nasal cavityis characterized by having epithelium that is several layers thick. This continuestowards the nasal cavity base where there is a gradual transition to an epithelialcell layer of only a few cells. The epithelia that lay in the presumptive fusion area31of the two facial prominences varied in thickness but generally consists of fewercell layers than nasal cavity epithelium.In all embryos examined at 13 T.S., F-actin was found to concentrate inepithelia toward the apex of nasal cavity epithelia and it then gradually becamemore diffuse toward basal epithelia (Figs. 3c,d and 5c,d). This apical-basaldifferential was observed around the nasal cavity until the nasal cavity basewhere epithelium that lay in the pre-fusion region of the facial prominencesseemed to lose its apical actin (Fig. 5c). In the 13 T.S. embryo examined thatalready had its MNP and LNP fused, the apical actin was re-established inregions facing the closed nasal cavity (Fig. 4d). Within the nasal fin howeverapical epithelial cells from the MNP and LNP that were now in contact had losttheir apical actin (Fig. 4d). This point of transition can be seen in Figure 3e wherethe apical epithelial cells of the nasal fin appear to be disorganized and only spotdensities of actin remain. These may be zonulae adherens junctions.Mesenchymal cells stained around the periphery of each cell and within cellprocesses (Fig. 6c). Mesenchymal cells take on a stellate appearance and areseparated from their neighbours by extracellular matrix. At the junction separatingepithelium from mesenchyme in nasal cavity epithelium there were observedconcentrations of F-actin at the most basal aspect of the epithelium just above thebasement membrane(Fig. 3d,e). This appeared to be arranged parallel to thebasement membrane although it was difficult to discern the type of filamentarrangement.3215 T.S.At 15 T.S. one of the embryos examined was sectioned more anteriorly thanthe other . Figure 6 shows a frontal section through the face where the MNP andLNP have not yet fused. The facial prominences are further apart in Figure 6abecause this section was taken in a more anterior position relative to the sectionsstained with NBD-phallacidin. Prominent blood vessels can be seen in the MNPand the LNP. The epithelium of the nasal cavity is multi-layered and appears tobe of the pseudostratified type. Again, looking at Figure 6b, it can be seen thatstaining for F-actin is most intense in epithelia toward the top of the nasal cavity.This intensity diminishes toward the base of the nasal cavity and toward the facialprominences. Along the LNP it can be seen that there is a transition zone roughlyat the point where nasal epithelium starts to become oral epithelium (Fig. 6c).There is a marked decrease in the amount of F-actin present at this region. TheMNP does not show a transition region as seen in the LNP epithelium. Actinstaining was more intense in MNP epithelia. In apical epithelium certain cellsshowed spot densities of actin that were not seen in basal epithelium (Fig. 6d).There were however basally distributed actin filaments in epithelium just abovethe basement membrane that seemed to run parallel to the membrane. In themore posterior section (Fig. 7), the nasal fin has already broken down and hasbeen replaced with a mesenchymal bridge. The thickening of epithelium seenmedial to the nasal cavity is the naso-vomer organ which is a specializedepithelium designed for olfaction. This structure is quite pronounced in rodentsbut it is a vestigial organ in humans. Actin staining is again quite pronounced in33the most apical epithelium of the nasal cavity which can be more than 10 cellsthick in some locations. In epithelium heading basally, actin appears to distributearound the cell periphery in the cortex of these epithelial cells. Intensity varies butagain it appears to diminish in the basal direction. There seems to be increasedactin content in apical epithelial cells located in regions of the nasal cavity wherethere is a bend and the epithelium changes its orientation. This is especiallypronounced at the top of the nasal cavity but is also seen medially (Fig. 7b and c).Figure 8 is the same specimen as Figure 7 and it shows the nasal cavity moreventrally toward its base. It can be seen how the medial and lateral sides of thecavity appear to stain differently for actin when actually the epithelium has justbeen sectioned along a different plane. The more lateral aspect shows epithelialcells cut en face with the plane of section going through a layer of cells such thatit just catches a sheet of circumferential bundles of actin filaments coursingaround individual epithelial cells. Actin staining is quite intense in this region aswell as through the epithelial layers heading basally. Toward the more lateralaspect, the actin staining pattern more closely resembles nasal cavity epitheliumencountered previously with higher actin content in the apical epithelia comparedto the basal epithelia. It is interesting that even though there is a bend in theepithelium at the base of the nasal cavity one does not observe the increase inactin content of apical epithelium in this region as seen at the top of the cavity.This epithelium instead appears to have very little actin. Perhaps this epitheliumis somehow different than other regions of nasal epithelium in that it is at adifferent level of differentiation. One does see the occasional spot density of F-34actin that may be due to the presence of adherens junctions (Fig. 8). The mostbasal layer of epithelial cells that contacts the underlying mesenchyme seems tohave re-established its characteristic pattern of actin filament arrangement wherethe filaments appear to align parallel to the basement membrane. The nasal finhas disappeared at this point and has been replaced by a mesenchymal bridge.This would imply that the organization of F-actin in epithelium of this region wouldhave been temporarily disrupted while the mesenchymal bridge was forming andthat it would then have to re-assemble once the nasal fin epithelium haddisappeared. Mesenchymal cells also stain for actin, some more intensely thanothers. Mesenchymal cell processes containing F-actin can be seen to extendand come in contact with the epithelial cell layers (Fig. 6c and d). Theseprocesses also contact other mesenchymal cells. Mesenchymal cells beneath thenasal cavity comprising the mesenchymal bridge in Figure 8 stain quite intenselyfor F-actin.16 T.SThe embryo examined at 16 T.S. shows a frontal section through the face ofan embryo where the nasal fin has broken down and been replaced with amesenchymal bridge. The MNP and LNP can be seen in Figure 9 and a portion ofthe maxillary prominence is also included. This nasal cavity provides a goodexample of the F-actin distribution seen at areas where there is a bend in thenasal cavity. At the top one observes high concentrations of actin in apicalepithelia that radiates out in all directions proceeding basally. There is an abrupt35transition at the junction between epithelium and mesenchyme. Actin filamentstake on an arrangement parallel to the basement membrane but it is difficult to tellwhether the epithelial cells or the mesenchymal cells contain the actin arrangedlike this (Fig. 9). As one proceeds toward the base of the nasal cavity, the highconcentration of actin in apical regions of the epithelium continues and appearsto be relatively equal on both medial and lateral sides. This high actin densitydiminishes however about midway down the cavity. The apical epithelia on thelateral side shows this most apparently. The epithelia toward the more medialside continues to exhibit high concentrations of actin. The nasal cavity bulgesmedially and the epithelium increases the number of layers in this region. Wherethe epithelium bends and reorients to progress laterally there is a highconcentration of actin at the apex of epithelia at the bend that seems to radiateout in finger-like projections towards basal epithelia. These fingers extend to onlyabout halfway through the stratified epithelia. Then, as was the case at 15 T.S.,there is a marked decrease in the amount of actin seen in apical regions ofepithelia located at the base of the nasal cavity.1 7.T.S.At 17 T.S. the tissue has been torn beneath the brain cavity; however thenasal cavity remained completely intact. Figure 10 shows a face again in thefrontal plane where the MNP, LNP, and MxP are clearly visible. One can also seea thickening of epithelium on the medial side of the nasal cavity that is the naso-vomer organ. The heart can also be seen although it is damaged in this36preparation. At higher power, the nasal cavity can be seen to stain abundantlywith F-actin. At this later stage of development it can be seen that the shape of thenasal cavity has become more complex. The epithelium takes on numerous foldsand evaginations as development proceeds. The top of the nasal cavity containsthe characteristic high concentration of apical actin in epithelium closest to thecavity lumen. The finger-like projections of F-actin only extend to about one thirdthe thickness of the nasal epithelium and they disappear. Progressing laterallythere is a gradual bend in the nasal cavity that has a high content of actin in itsepithelium. The medial side at this same level contains only a thin but intenseband of actin at the most apical aspect of the epithelium next to the cavity lumen.These may actually be adherens junctions connecting epithelial cells together.The medial and lateral layers of epithelium at the base of the nasal cavity in thisspecimen approximate and continue along side one another. It appears thatthese epithelial layers are still a component of the nasal fin which has just startedto break down (see Fig. 11). The actin staining appears more intense in thisregion of contact because there are now two layers of apical epitheliumcontributing to the fluorescence. On the lateral side of the face at the base of thenasal cavity lumen,the epithelium becomes quite disorganized. It is difficult to seewhere the epithelium ends and the mesenchyme begins in this region. Possiblythe epithelium is undergoing some morphogenetic shape changes. Theepithelium here seems to thin and not every epithelial cell stains for actin with thesame intensity. At the extreme base of the nasal cavity, where the mesenchymalbridge is just one or two cells wide, the actin staining is quite faint and perhaps37the epithelium is still reorganizing and new actin filaments that will comprise thebasal epithelium have not yet been synthesized. Across the mesenchymal bridgeone can see future oral epithelium. Part of the nasal fin still persists in thisepithelium as a bud or thickening (Fig.11). There appears to be a concentration ofactin filaments in the basal layers of this oral epithelium similar to that seen innasal epithelium previously. Mesenchymal cells toward the medial side betweenthe nasal cavity and the naso-vomer organ stain quite intensely for F-actin(Fig.11) as do the mesenchymal cells on the medial side of the mesenchymalbridge. On the lateral side of the mesenchymal bridge the mesenchymal cells stillstain for actin but do so with less intensity.18 T.S.This specimen (Fig. 12) although at a higher level of development than theprevious 17 T.S. embryo shows almost the same stage of nasal fin regressionand mesenchymal bridge formation. This embryo at 18 T.S. has a mesenchymalbridge that is slightly larger and more robust than the 17 T.S. specimen. All threefacial prominences can be identified easily and it is interesting to note that theMxP has appeared prominently in specimens where the mesenchymal bridgehas started to form. In this example there is differential distribution of actinstaining in different regions of the nasal cavity. At the top, there is thecharacteristic high concentration of F-actin where there is a bend in theepithelium. The more apical epithelium stains more intensely than does the basalepithelium again. The finger-like projections of F-actin radiate out in the basal38direction. Midway down the nasal cavity on the lateral side there appears to beabundant spot densities of actin possibly due to adherens junctions. On themedial side of the nasal cavity the plane of section has exposed epithelial cellscut en face as seen previously in Figure 10. It is notable that even when there isonly a small bend or directional change in the epithelium lining the nasal cavityas the epithelium evaginates, that one still observes an increase in actin stainingintensity (Fig. 12 and 13). The epithelium beneath the cells cut en face showsactin filaments arranged differently than that observed previously. Towards thebase of the nasal cavity, the apical regions of epithelium continue to show strongfluorescence. In Figure 12 on the lateral side, the epithelial-mesenchymaljunction is very prominent. One can see numerous focal densities of F-actin in thebasal epithelium that demarcates the transition to mesenchyme. Themesenchymal bridge in Figure 13 appears to be three or four cells wide at thispoint. The basal epithelium of the nasal epithelium appears disorganized and theepithelial-mesenchymal junction is not apparent. These cells may be undergoingrearrangement and the final organization of F-actin has not yet been established.The same could be said about the oral epithelium at the base of themesenchymal bridge. Its epithelial cells also appear to be somewhatdisorganized (Fig.13).19 T.S.Figure 14a illustrates that the nasal fin persists for a certain time before itregresses. The section was taken in the anterior of the face where the nasal fin3 9epithelium was still present. In any one embryo it may be possible to observe allstages of primary palate formation. In this case we are still observing the nasal fincomprising epithelium from the MNP and LNP. The nasal cavity shows prominentactin staining in apical epithelium which becomes more diffuse basally. Towardsthe base of the nasal cavity the apical actin concentrations continue but to alesser extent (Fig.15). The epithelial-mesenchymal junction is quite apparentaround the base of the nasal cavity. It does however seem to disappear on bothlateral and medial sides of the nasal fin. The basal actin then reappears at theoral side of the nasal fin (Fig. 15). This may indicate a different type of epitheliummay be present along different regions of the nasal fin. In these regions that maybe just about to disperse and break up there are no spot densities of F-actin andpossibly therefore no adherens junctions. This region of no actin filamentassembly in basal epithelium corresponds to the area of initial mesenchymalbridge formation seen in Figures 11 and 13.27 T.S.The primary palate in this embryo (Fig. 16) has formed completely at thisstage of development. The mesenchymal bridge is quite robust and there is nonasal fin present. The shape of the nasal cavity has changed to that of a moredifferentiated state. Figure 16a shows how at points where the nasal cavity bendsone still finds increased apical actin concentration. Nasal cavity epithelium stillshows an increased actin distribution in apical epithelium with a networkappearance of intermediate epithelial layers and then a prominent parallel40arrangement at the epithelial-mesenchymal junction (Fig. 16a,b,c).Actin is generally more uniformly distributed all around the nasal cavity;however there are higher concentrations at bends. Actin at the apex of epitheliumis more uniform around the nasal cavity as well.3.3 Basal Lamina Changes During Primary Palate Formation- TEMResults.7 T.S.At 7 T.S. the LNP and MNP have not yet fused and what is shown in Figure17 is a high magnification view of the epithelium comprising the MNP. The mostbasal layer of epithelial cells is shown and a quite intact basal lamina can easilybe distinguished. The epithelial cells themselves contain abundantpolyribosomes as well as numerous cisternae of rough endoplasmic reticulum(RER). The epithelial cells can be seen to extend cell processes toward the basallamina, but at no point do these processes extend through it. Stacks of the Golgiapparatus can also frequently be seen in the epithelial cells. These cells wouldseem to be highly metabolically active due to the large number of mitochondriapresent (Fig. 17b). Also apparent are a number of endocytotic vesicles that may infact be clathrin coated pits located just above the basal lamina (Fig. 17b,c).Mesenchymal cells that underlie the epithelial cell layer also contain abundantclusters of free polyribosomes, mitochondria, but relatively less RER. Themesenchymal cells appear attached to neighbouring cells through junctionalcomplexes (Fig. 17b,c). Endocytotic vesicles can also be seen in the occasional41mesenchymal cell as well (Fig. 17b).10 T.S.The MNP and LNP have not yet made contact at this stage of development.Basal epithelial cells that are in the presumptive fusion area of the MNP and LNPhave a basal lamina that is still intact (Fig. 18a,b). the epithelial cells look activeand contain many free polyribosomes and mitochondria. In the LNP basalepithelia there are what appear to be endocytotic vesicles within the cellcytoplasm as well as fused to the plasma membrane on the side of the cellopposite to the basal lamina (Fig. 18b). At this point the epithelial cells and themesenchymal cells do not extend cell processes into the basal lamina, but on theMNP further down in the direction of the oral cavity one does see cell processesfrom both epithelial and mesenchymal cells (Fig. 18c). In Fig 18c, a cell processfrom the epithelial cell seems to extend into the basal lamina and even breakthrough it. The opposing mesenchymal cell also extends a cell process thatmakes a direct cell-cell contact with the epithelial cell. There may even be ajunctional complex forming between these cells. One can also observe debrispossibly basal lamina remnants and other extracellular matrix material.11 T.S.At this stage the MNP and LNP have fused and formed a nasal fin. Theepithelial cells on the side of the MNP have become fragmented and their basallamina has become patchy and discontinuous (Fig. 19a). It appears as though the42epithelial cell processes have penetrated the basal lamina. The epithelial cellsthemselves appear alive with no signs of necrosis at this level of the nasal fin.Endocytotic vesicles also appear in some of the epithelial cells. Mesenchymalcells also extend cell processes that approach the basal lamina. On the side ofthe LNP the basal lamina is still intact and no damage is apparent. The epithelialcells have started to extend small cell processes into the basal lamina but thesehave not yet broken through it (Fig. 19b). Mesenchymal cells also extend cellprocesses but they do not come in close contact with the basal lamina. The nucleiof the epithelial and mesenchymal cells in Figure 19b is very euchromaticindicative of a high level of cell activity. Mitochondria are also quite abundant inboth these cell types.Figure 20 shows an embryo at the same number of tail somites where anasal fin has formed and there does seem to be some indicators of necrosis insome of the epithelial cells comprising the nasal fin. The basal lamina isfragmented in some areas although the epithelial cells do not possess anelaborate cell process network yet. The basal lamina appears patchy on both theLNP and MNP sides of the nasal fin (Fig. 20a,b). Figure 20c shows an epithelialcell within the nasal fin that is about six cells thick at this point that looks necrotic.The cell appears vesiculated and the nucleus is condensed. Multivesicularbodies are also present.4312 T.S.The embryo shown at 12 T.S. has a nasal fin that has not yet been brokendown and there is an increase in the amount of necrotic activity in these epithelialcells compared to that seen in the 11 T.S. example. Figure 21 a, and b shows anumber of epithelial cells comprising the nasal fin on the MNP side. One canobserve that some of the cells have condensed nuclei, tertiary lysosomes andelectron dense multivesicular bodies. The cells appear to be fragmented andthere are also large intercellular spaces some of which could be lumen of bloodvessels. Figure 21 b is a portion of the nasal fin below 21 a in a more ventralposition. The mesenchymal cells show no signs of necrosis. These cells havelarge euchromatic nuclei many free polyribosomes and abundant mitochondria.13 T.S.The nasal fin epithelia shown in Figure 22 illustrates that although theepithelia should be starting to regress by 13 T.S. certain regions exist completelyintact. Nasal fin breakdown does not occur simultaneously in all epithelial cellscomprising the fin. Depending on where the section was taken one could observedifferent stages of primary palate formation. The nasal fin may be regressing inthis embryonic face in a more posterior position, but this section was takenanteriorly. The epithelial cells of the MNP appear quite active with manypolyribosomes in the cell cytoplasm and abundant mitochondria and RER. Incontrast to the cells shown in Fig. 21, this portion of the nasal fin shows no areasof necrosis. Some of the epithelial cells of the fin more toward the oral cavity have44started to extend cell processes into the intact basal lamina but these cellextensions have not penetrated through it yet (Fig.22b). On the lateral side theseepithelial cells lie flat against their basal lamina still and no cell processes areevident. The mesenchymal cells at this stage are very fragmented and their cellprocesses extend and approach the basal lamina but do not penetrate it. Themesenchymal cells also contain abundant mitochondria indicative of their beinghighly active metabolically.15 T.S.The nasal fin shown in Figure 23 is one that is likely to regress quite soon.The lateral side of the fin is illustrated. Epithelial cells appear fragmented andhave developed numerous cell processes that come in close contact with but donot pass through the basal lamina. The basal lamina is still quite intact althoughportions of it may have started to breakdown (Fig. 23b). The mesenchymal cellprocesses appear to contain microfilaments as shown in Fig. 23a. Theseprocesses may extend and touch the basal lamina and possibly even theepithelial cell on the other side. Figure 23b shows a higher magnification view ofa mesenchymal cell process in contact with the epithelial basal lamina. There isan electron dense region just at the point of contact possibly the site of a cell-matrix interaction of some kind.4519 T.S.By 19 T.S. the nasal fin has broken down and mesenchymal cells haveinvaded the region. The fin may still persist in anterior locations however. Figure24 and 25 show the same nasal fin just at a location where it is breaking down.The fin is perhaps six cells thick and is composed of epithelial cells from bothmedial and lateral facial prominences. The surface cells in contact with epitheliaof the opposing prominence are somewhat squamous in appearance whereasthe more basal cells are more cuboidal in shape (Fig. 24a). The cells appearquite healthy and no necrotic activity is seen. Figure 24b shows a highmagnification view of an epithelial cell on the lateral side of the nasal finillustrating an intact basal lamina with one possible endocytotic vesicle. Figure 25illustrates how the basal lamina appears just at the top of the nasal fin that hasregressed and is exposed to mesenchymal cells that are beginning to form amesenchymal bridge. The lamina is quite fragmented and patchy at the tip of thenasal fin (Fig. 25b). It is unclear whether it is still being degraded at this point orwhether it is being resynthesized. It is also difficult to tell whether the epithelialcell at the uppermost tip of the nasal fin is necrotic or whether it is preparing tomigrate and become a constituent cell of the surrounding mesenchymal cellpopulation. Figure 25a illustrates how at the tip of the regressing nasal fin thecells are very fragmented and it is not clear whether these cells are epithelial ormesenchymal in nature. Figure 26 shows the same nasal fin as Figs. 24 and 25but this figure shows epithelial cells of the MNP side of the nasal fin more in theventral direction toward the future oral cavity. One can see mesenchymal cell46processes making direct contacts with the basal lamina and possibly theepithelial cells as well (Fig. 26a,b). Figure 26c shows a mesenchymal cell that isabout to divide. Its chromatin is condensed and it remains attached toneighbouring mesenchymal cells via tight junctions.21 T.S.Figure 27 illustrates an anterior region of the face where a nasal fin stilltemporarily persists but is most likely about to regress. A section of the basal layerof epithelial cells on the lateral side of the fin is shown. The epithelial cell in Fig.27a has extended a cell process through the basal lamina. The lamina isfragmented and discontinuous in regions where epithelial cell processes arelocated. The epithelial cells show no necrotic activity and appear to be quitemetabolically active. The cell processes of both the epithelial and mesenchymalcells contain microfilaments that can not be seen in other parts of the cells.27 T.S.By 27 T.S. the mesenchymal bridge has formed and the primary palate isconsidered to be completely formed. Figure 28 shows two basal epithelial cellsthat comprise the base of the nasal cavity and the top of the mesenchymal bridge.This surface is no longer denuded and it appears as though the basal laminaunderlying the epithelial cells in the region has been repaired and laid downagain. Figure 28a shows an area where basal lamina material appears to bethicker than normal and may be a site where resynthesis is occurring. The47epithelial cells here now will go on to differentiate and become components of thenasal cavity epithelia. It is interesting that the mesenchymal cell processes arestill present in the resynthesizing stages of primary palate formation and onecould speculate that they may be involved in cell-cell communication or signallingbetween epithelia and mesenchyme.3-Dimensional ReconstructionsFigure 29 shows a series of developing nasal cavities during the time ofprimary palate formation. The reconstructions make it easier to interpret the 2-Dsectioned material and the changing nasal morphology can be better visualized.Early in primary palate formation, at 11 and 12 T.S., the nasal cavity has a simpleshape resembling a tube. The nasal fin is about to form as the MNP and LNPapproach and fuse. Later, its shape becomes more complex as the region growsand expands (Figure 29c-e). The epithelium bends at a number of locations and ifone compares this to Figures 9 and10, the actin staining in apical regions ofepithelium at these bends can be seen.48FIGURE 1. a) An 11 T.S. C57BL/6J mouse embryo frontal section takenanteriorly. These specimens were embedded in paraffin and stained withhematoxylin and eosin. Facial prominences not yet fused. MNP, medial nasalprominence; LNP, lateral nasal prominence. b) 12 T.S. Prominences not yetfused. c) 14 T.S. Prefusion stage. d) 16 T.S. Nasal fin has formed (NF). e) 18 T.S.Prefusion stage. NC, nasal cavity; OC, future oral cavity.49p r. 1 ANT• N./.MNPro : 	 1 • 	 1. 	 •.449	 444-$1,0kcle--"' -At;	 AA/W. •- 4 '':/r% 	 • 	 " 050FIGURE 2. The same embryos as Figure 1 sectioned in a more posterior position.a) 11 T.S. A nasal fin has formed (NF). MNP, medial nasal prominence; LNP,lateral nasal prominence; Mxp, maxillary prominence; NC, nasal cavity. b)12 T.S.Nasal fin has formed. NV, nasovomer organ. c) 14 T.S. Nasal fin has regressedand a mesenchymal bridge has formed (MB). d) 16 T.S. A nasal fin still persists.e)18 T.S. A mesenchymal bridge has formed and will now continue to enlarge asthe primary palate.51NC; Vi•frivou74141r1X4ttosli• ._.....•• 	 t.--1 • e	 a .• 	f-rmipIr., -- -. 44 I. j‘a - ,,s,44ft •IAv• •	 4 •••4 .	 p • es:.•r 	 rw'<47 '	 •6447)1Vi.X.Pa 	 . 	 ,4•   • .1-II':• -14, 1411-*" s••111;„?.bra deg• 6_ -.e. -	 iigi is.Vi vi' .....' • ' .	 • •.9. , ..„.... •• • 	 „1".. , - / 0151 17, Oliiii% 	 • 	 . i• v- 	 •• 	 44• 4.. 	 ■ys , 4,.... .... 	 .........4 ..... •  ...... • • -b.... s,ttel, 	 lit • • • ;FIG 2	POST52FIGURE 3. A 13 T.S. face. a) Prefusion stage stained with hematoxylin and eosin(H&E) showing MNP and LNP just prior to contact. b) Same specimen stainedwith NBD-phallacidin. c) Top of the nasal cavity. Note the intense fluorescence ofthe apical epithelial cells. E, epithelium; M, mesenchyme. d) Wall of the nasalcavity on the medial side. e) Nasal fin region. Note the spot densities of F-actin(arrows).5354FIGURE 4. a) 13 T.S. embryo showing a nasal fin stained with H&E. b) Sectionstained with NBD-phallacidin. MxP, maxillary prominence. c) Nasal cavity towardthe lateral side. Note the mesenchymal cell processes (arrows) extending toneighbouring cells and how the level of fluorescence varies. d) A nasal fin. Notehow the apical epithelial cells of the nasal fin do not fluoresce.55F I G 4- .4k1 7,7*7*;;‘14102Ct4,•sww.	 4Ve fa- 6.•41,IMP"-Ap•VZOS.•$. • 4:_osgAst.-1■94 ■1/ 'tau, 	 w-11Pr.P...49.41.r1 .4n.' 	 al*.ot .,;•_•13. 	 •"i•■■%ffdr.j..elto.%WAX.4191,epjl *#%.• •(41_0...t 0zot /ti.)X Fiitkrf56FIGURE 5. a)13 T.S. section stained with H&E showing MNP and LNP just priorto fusion. b) Section stained with NBD-Phallacidin. c) View of the nasal cavity justapproaching the nasal fin. Note the disappearance of fluorescent activity in apicalepithelial cells (arrow). d) Nasal fin. Note the minimal fluorescent activity in nasalfin epithelia.57FIG5	i;f -Art- - - 	 7l- -I- ''.0:37412v1:4-•14.ie7....v '411bak I 	 • 6,.. 	 4.:. -i.	 .7 	 • •,INFAN40. 0.-"•4•:?, 441.	s 	"? 4_. !M. 04VOInk_ ,141..ft':".0_,47471., Ripit=i;;Ot **::4'-ip.— ■ 'It .• vvie•-•,...,... A.r-,I . 	OP'''	 ■	6 	 . 7U ?VIA .	:-.0 — PP"Pteli. 	 ,,.,. •.-z.r•"=;%* aPtd!II CN..I A. dk„,.. 46„ 	 i'■.nart .1:7144E.`a•V.A*..1'. 	 I. W.' zeJ .a.":v• 	 4?"' i ISA* qet 	 IA... 'I-rP.;.„, ,...: 	 • nz mhz. 1"'pre	lo .....- 4.,..z.... 	 .VI-t.•,4re• • "'-dr .V ..of 4 	 r2 *4 idb:e. ■ at., .$ \%4N ,. --Or4e*Ykk.4. t.t_sgSa 17);t4A • --.: •*- 4 	 ,ll'ellit4AreNIra Al 1 40,.rj grx.'4, 	 iga 	 1 154., q; 444 4 :;.• 1P;aetteer• Voli Weer • ' 	• 	 4%1 . . .., 	 tt,• 1 4, . k ,* 	 Ii 	 a a 	 1":* 	 *It ge? ,i. 	 v •.1.58FIGURE 6. a) A 15 T.S. frontal section during the pre-fusion stage stained withH&E. b) A section further posteriorly stained with NBD-phallacidin. c) Closer viewof the LNP. Note the transition zone where epithelium stops fluorescing (arrow).d) The MNP with a blood vessel.5960FIGURE 7. a) A 15 T.S. embryo stained with NBD-phallacidin. NV, nasovomerorgan; H, heart. b) Nasal cavity on the medial side. Note the intense stainingapically (arrow). c) Top of the nasal cavity. Note the bend in the epithelium andthe intense fluorescence in this region (arrows).61FIG 76 2FIGURE 8. Base of the nasal cavity. M, medial, L, lateral. Note the medial nasalepithelium cut en face . Spot densities of F-actin (arrows).636 4FIGURE 9. a) A 16 T.S. embryo stained with NBD-phallacidin. Note the increasedlevel of fluorescence at points in the nasal cavity where there is a bend orevagination. b) Higher power view of the nasal cavity. Observe the differentialdistribution of F-actin at different locations around the cavity. Note the finger-likeextensions of actin at the evagination points (arrows). Note also the absence ofapical actin staining at the base of the nasal cavity. Dotted line, the epithelialmesenchymal junction.65FIG 96 6FIGURE 10. a) A 17 T.S. embryo stained with NBD-phallacidin. b) Top of thenasal cavity. Note the intense actin staining in mesenchymal cells on the medialside between the naso-vomer organ. Also see the increased fluorescent activityat bends in the nasal cavity.67FIG 106 8FIGURE 11. Same 17 T.S. embryo as Figure 10 but at the base of the nasalcavity. Note the disorganized appearance of these epithelial cells at the base.Note also the mesenchymal bridge that is one cell wide (large arrow). OE, oralepithelium.6970FIGURE 12. a) An 18 T.S. embryo stained with NBD-Phallacidin. Note themesenchymal bridge. b) The top of the nasal cavity. Note the epithelial cells onthe medial side of the cavity have been sectioned en face. See how even at smallbends or evaginations (E) one still sees an increase in the amount of F-actin.Observe the epithelial-mesenchymal junction (dotted line).7 17 2FIGURE 13. The base of the nasal cavity of the same 18 T.S. embryo shown inthe previous figure. See also the mesenchymal bridge (MB) here perhaps threecells wide. BV, blood vessel.737 4FIGURE 14. a) A 19 T.S. embryo stained with NBD-phallacidin b) Top of thenasal cavity. M, medial side. Note the differential actin distribution in the nasalcavity epithelia.757 6FIGURE 15. A close up view of the nasal fin of the previous 19 T.S. embryo. Notethe spot densities of F-actin in epithelium at the base of the nasal cavity. Alsonotice how the actin fluorescence at the epithelial-mesenchymal junctiondisappears at areas where the mesenchymal bridge is about to form (betweenthe arrows).777 8FIGURE 16. a) A 27 T.S. embryo stained with NBD-phallacidin. Note the site ofpossible ingrowth of olfactory nerves (N). The primary palate has now formedcompletely. b) This is a region of the nasal cavity on the left side of the face. Notethe differential actin distribution in apical versus basal epithelium. c) This is asection of the left nasal cavity at its base. This is also the top of the mesenchymalbridge (MB). d) A section of the left nasal cavity indicating the slightly reducedlevel of fluorescence in this region of apical epithelium.79FIGURE 168 0FIGURE 17. a) A transmission electron micrograph (TEM) of a 7 T.S embryoshowing the basal layer of epithelial cells of the MNP. At this stage ofdevelopment the LNP and MNP have not yet fused. Note the epithelial cellprocess extending into the basal lamina (arrows). b) A view of these basalepithelia in a more ventral position. Note the endocytotic vesicle (V). c) A viewfurther ventrally showing how the basal lamina is continuous at this stage ofdevelopment. (Bar=1 micrometer)811,„fl•• •<• AI.0..1! • e31..?,71r,:t*r -2•rit .0 	 ?..	• 	-4‘,0	 ' 	 • '4 -"it'7•4:",* , - . 	 . 	 .st,"1,•' ,.. ‘if,4 	 • 	 ,vV t 	 for 	•F IG 17 A•*7:4!)."' 	t 	•et 	•  14v 4'.	44- t:- 	• • C ' 	-;454.14k % -,i.-^ -0 ..-2,...--_,........j. - 	* . a4:, „ vy14' 	 ''' ' #. oo dr str, ' ,, o.„7,,4` ' .*:e r'4O'C'''$."- b.')■r■ -' q .'s, 1,1":Cr.; ■44:,:i 	,,:.,:,:.1,'''.., 	 d• 	 r 	 • 	 -3.' ' VII" •atfte, • ' 	 - 	 .... s—at A`i • -ci • , 	 ,•:•ir .4Na;:,•''.. 	 "	,,v\ 	 CI'.*to 	1  0.. •-) ,;•• r 	 %del,- ••• 	 td1 44.• ..:	t	 •82FIGURE 18. a) A TEM of a 10 T.S. embryo where the facial prominences havenot yet fused. The basal lamina is continuous and intact. M-medial nasalprominence. b) This micrograph was taken at about the same level as (a) but onthe lateral side. L, lateral nasal prominence. Note the endocytotic vesicles. c) Aregion on the MNP in a more ventral position showing an epithelial cell processextending into the basal lamina. (Bar=1 micrometer). Arrows show the basallamina.83CDLL8 4FIGURE 19. a) A TEM of an 11 T.S. embryo where the LNP and MNP haveformed a nasal fin. The micrograph was taken on the MNP showing regions ofbasal lamina destruction. Note the epithelial cell processes penetrating the basallamina and the debris of degraded extracellular matrix remaining. b) A view froma similar level on the side of the LNP showing an intact and still continuous basallamina. c) A necrotic epithelial cell within the nasal fin. (Bar=1 micrometer).858 6FIGURE 20. a) A TEM of an 11 T.S. embryo that has formed a nasal fin. Themicrograph was taken on the MNP side. Note that the basal lamina is continuousbut patchy in regions. b) This is a micrograph of the basal epithelia at about thesame level as (a) but on the LNP side. See how the basal lamina is patchy andstarting to fragment. c) This is an epithelial cell on the MNP side that appears tobe necrotic. (Bar=1 micrometer)878 8FIGURE 21. a) A TEM of a 12 T.S. embryo where a nasal fin has formed and hasnot yet broken down. Note the necrotic activity in some of the epithelial cells. b)This micrograph is of a more ventral position showing necrotic epithelial cellscontaining tertiary lysosomes and multivesicular bodies. (Bar=1 micrometer)89FIG 2112TS4 - 	 • 	 •c1/4••A.90FIGURE 22. a) A TEM of a 13 T.S. embryo where the MNP and LNP have formeda nasal fin. This is a view from the MNP showing a continuous and intact basallamina. b) This is a more ventral position on the MNP showing a continuous basallamina and epithelial cell processes extending into but not through it. c) Shownhere is a corresponding position on the LNP side of the nasal fin. Basal lamina isintact and continuous. This continues ventrally in d. (Bar=1 micrometer)912I-Q-J9 2FIGURE 23. a) A TEM of a 15 T.S. embryo where a nasal fin has formed and islikely to soon regress. Shown is a view from the LNP. Note the fragmentedepithelial cells and the mesenchymal cell process. b) A higher magnification viewof the point of mesenchymal cell contact with the basal lamina. Note the electrondense region. Also see the fragmented basal lamina material (arrow). (Bar=1micrometer)9315 TS'$411g‘SI., 	 4; 	 • 41.ati ","fs- 	 ,-• " $- .p••4).112'A',..14.0re 	,• e:"F;1/4...Nof. Y.' 4• -46•r /;„,'140.."1*t*".:1, 4lg... v.,.• r -.*.).% •94FIGURE 24. a) A TEM of a 19 T.S. embryo sectioned anteriorly where a nasal finstill persists. Above this in a dorsal direction the nasal fin has regressed. Observethat the fin consists of about six cell layers. b) This is an epithelial cell on the LNPside of the nasal fin showing an intact basal lamina and possibly an endocytoticvesicle (V). (Bar=1 micrometer)959 619TFIGURE 25. a) A TEM showing the tip of a nasal fin in the same 19 T.S. embryoas Figure 25 and (b) is the same view at a higher magnification. Notice thefragmented appearance of the cells close to the tip of the nasal fin as well as thebasal lamina debris (arrows). A, artifact (Bar= 1 micrometer)9719TS 9 8 FIGURE 26. a) This is a TEM of the residual nasal fin as seen from the MNP ofthe 19 T.S. embryo shown in Figs. 25 and 26. The mesenchymal cell processesmake direct contact with the epithelial basal lamina and possibly with theepithelial cell itself. Possibly the site of a cell-cell interaction. b) Mesenchymal cellprocesses contact the basal lamina in a more ventral location as well. c) Amicrograph showing a mesenchymal cell about to divide. This cell is located onthe LNP side of the nasal fin near the tip. (Bar=1 micrometer)99100FIGURE 27. a) A TEM of a 21 T.S. embryo where a nasal fin still persists sincethis section was taken anteriorly. These micrographs are from the LNP. Anepithelial cell process has penetrated the basal lamina which is patchy anddiscontinuous. The epithelial and mesenchymal cell processes appear to containmicrofilaments (MF). Progressing ventrally along the nasal fin (b,c, and d) onecan see patchy basal lamina and epithelial cell processes extending into it. Onealso sees a number of endocytotic vesicles (V). (bar=1 micrometer)101• 7• •A ** 	 ;P.4 V4 ••';3•I•7:.1, 	 4*•<,	 1 •0.zE 	 _.14t. 	 Nte;'!. .41t/e•N	 ;;.%•• s....1" r.;: 	 4.64,71„ torco•"•*4 "'""44.4•If. 	 .• 	 •• •. 	 .EM.4‘44i— —044/4	 4?„ ; '''''!" 	 s* ` „ ,	 , <11. or Jii, 4• • ' . 	 •,.. 	 A ''''' - . a. •-•'Ivi ,i ,o• , 	 :A-- : ,,. - ' ..*1. 	 ,,ly . 	 -.. 	 ',.1 .. ,.. . ,	 . 	 ., 	 ..4."-•'A.%. ' 4A '. A ‘	 A... ' ...."01r. ..;:rt4'' AV., ,	 A , . t.41, '1.4 . 4 ', ;4	 9.• "*"4 47 1. 	 : . e7..t ' ,, .....'. 1 ' ii, 4 	 ', 	 •..4% . i'..4.11 '...C't 'S."' 	'".. • it''''.• '4;4.. < ''<t; ''* . •V '4*-0,4 	 t;... .; -0,334,,, , . ..•,.--• 	 .. -' '11. :).e, . -"k	 • % .4 4`'11. •- „tsti• " `„....: ,i...„..4...:,64‘.;,.1:t.i , ..7..:):;;.;r..•t)tiL,,. Xf.t.e4 ,',; , 	 ,' • ' 	 . 	 --,-.:- t, 'f ...	 .4't! „ . 	 .. 	 I, 	 1- Y•fr. ''''40 4*. ''''' i•-:4); :, A '::".-• l' :.: ..	 - u I.'. 	 , L''''4- . .	 .....ii.- 'A:0;7 - % ,-i". ' . 'f's■"'''-:'..'-,..:„ .."1...,,,, ,.‘-••,:ik,	-,- •1k.,, :-....e.),...'.....,...,..,... 04 7'",,,' ;,. 	 6 	 •f<, „„::.,„,...... ,. „.• • a	 .	 ,,,, 	 „ 	 ,*;Ittp.ooler ac.• .	 4,.. ...It't• ?AI•_ 	 ■;•,,„, !'•102FIGURE 28. a) A TEM of a 27 T.S. embryo where the primary palate hascompletely formed. This is a view from the most dorsal aspect of themesenchymal bridge at the base of the nasal cavity. Observe the newlysynthesized basal lamina (arrows) in a). b) An epithelial cell just lateral to the onein a). Note that the basal lamina is completely intact. E, epithelial cell, MB,mesenchymal bridge. (Bar=1 micrometer)103104FIGURE 29. A series of three dimensional reconstructions of the developingnasal cavity during the time of primary palate formation. Nasal epithelium, medialnasal prominence epithelium, and lateral nasal prominence epithelium can beseen. a) An 11 T.S. embryo. b) 12 T.S. c) 14 T.S. d) 16 T.S. e) 18 T.S.105FIG 291 064. DISCUSSION4.1 Possible Role of F-actin in Regulating Nasal Morphology in Light of the 3-Dimensional ReconstructionsThe nasal cavity was shown to undergo dramatic changes in shapeanteriorly to posteriorly. This shape change became more pronounced as thenasal cavity continued to differentiate and develop. At 11 and 12 T.S., the cavityresembled a hollow tube that formed a ridge on its most dorsal aspect and anevagination towards its medial aspect . The cavity by 14 T.S. started to bend in itsmid region and by 16 T.S. this bending became more pronounced .After visualizing the shape of the nasal cavity from the 3-D reconstructions, itwas easier to analyze the actin staining pattern from 2-D sections. Actindistribution was uniform all the way around the nasal cavity, however stainingintensity was increased at points where there was a bend. Particularly, the apicallayers of epithelium were more intense than basal layers. The epithelium liningthe primitive nasal cavity showed a constant pattern of actin distribution at allstages of development. Occasionally, spot densities of actin staining were seen inepithelium facing the lumen that could represent actin associated adherensjunctions. Possibly, actin in these regions could provide structural support to thenasal cavity. This was similar to thyroid evagination studied by Hilfer et al. (1977)where the apical epithelial cells contained more microfilaments in their cytoplasmthan did basal epithelial cells. My results in the nasal cavity contradicted the actin107arrangement found in salivary gland morphogenesis (Spooner,1973) where thebasal cells constricted due to F-actin contraction causing the cells to assume awedge shape. The system in the salivary gland is an invagination rather than anevagination as in the nasal cavity and could explain the basal and apicaldifferences in staining.I observed different patterns of actin distribution in the facial prominences atdifferent stages of primary palate formation. Prior to fusion, the actin staining seenin the epithelium that lined the prominences was similar to the nasal cavity justdescribed with apical regions staining more intensely. As the MNP and LNPapproached each other about to fuse, the epithelium of the presumptive fusionsite of each process showed a marked decrease in staining intensity. Once theprominences had fused forming a nasal fin, this epithelium showed more diffuseconcentrations of actin. Also, in epithelium comprising the nasal fin, spot densitieswere no longer observed. Possibly, these cells were in the process of losing theirattachments to neighbouring cells and were preparing to migrate. They may havelost junctional contact.The differences in actin distribution observed in the nasal cavity versus thefacial processes may have reflected epithelium that was at a different level ofdifferentiation. Nasal epithelium may have represented a different epithelialdomain than facial prominence epithelium. Croucher and Tickle (1989) studiednasal placode epithelium in chick embryos and found that different cell surfacemolecules and extracellular matrix molecules existed in different regions of theolfactory epithelia. They found that olfactory epithelium expressed different cell108surface molecules than respiratory epithelium of the nasal passages. Myobservations suggest that different epithelial domains in mouse nasal epitheliumshow differential actin staining.4.2 Timing of Basal Lamina Disappearance During Primary Palate FormationDuring primary palate formation there were a series of observablemorphological changes that took place within the environment of the LNP andMNP epithelium. This epithelium was always in close proximity to its basal laminawhich in turn was closely apposed to mesenchymal cells underneath. It would beexpected that epithelial and mesenchymal components interact such that theevents of nasal fin formation and regression, as well as mesenchymal bridgeformation, took place in a carefully timed and coordinated manner. With theelectron microscope, these interactions could only be visualized on amorphological and descriptive level, but there were observable changes in theshape of the epithelial cells and mesenchymal cells that could be described.Specifically, prior to fusion of the facial prominences, the epithelial cells wereseen to rest against their basal lamina with the point of contact being quite flat.Mesenchymal cells had an elongated appearance and portions of these cellsextended outwards as cellular projections. These projections were observed tobe closely associated or in close proximity to the basal lamina. At alldevelopmental stages looked at from 7 to 27 T.S. mesenchymal cell processeswere observed approaching the basal lamina and often in contact with it.109Epithelial cells also extended their own cell processes toward their basal laminaas the MNP and LNP came together. As early as 10 T.S. which was still in theprefusion stage, epithelial cell processes extended into and through the basallamina. There appeared to be basal lamina fragmentation of the basal laminaoccurring in regions where there were mesenchymal cell processes, but in otherregions the basal lamina was still completely intact. This observation of basallamina destruction before MNP and LNP epithelial cells make contact wasdifferent from the situation described by some authors during secondary palateformation (Fitchett and Hay, 1989; Ferguson 1988). There, the basal lamina wasintact until the palatal shelves had fused. Then it became patchy, fragmented, anddisappeared. In the primary palate these events occurred earlier prior to fusion.Once the nasal fin had formed there was rapid basal lamina destruction asepithelial and mesenchymal cell processes penetrated through it. Thisdestruction was localized to specific regions of the nasal fin however. Oftentoward the nasal cavity the basal lamina remained intact and sometimesdestruction was only seen on the side of one facial prominence. One of theembryos at 11 T.S. had a nasal fin where on the MNP side, the basal lamina wasbreaking down while on the LNP side it remained completely intact but generallythis process appeared to be simultaneous on both MNP and LNP sides. Thebasal lamina became patchy and discontinuous beneath presumptive fusionepithelium only. Other portions of the facial prominences had an intact basallamina during primary palate formation. At sites where the basal lamina wasfragmenting one could often see vesicles in epithelial cells that may have been110endocytotic possibly taking in components of the lamina aiding in its dissolution.Regions of necrosis were observed in the nasal fins of a few embryos butthis was rare. Cells contained tertiary lysosomes and multivesicular bodies butsurrounding epithelial cells remained metabolically active and vital.During primary palate formation there were no residual epithelial cell restsas seen in secondary palate as the epithelial seam starts to thin (Fitchett and Hay,1989). In primary palate once the nasal fin started to regress the epithelial cellsquickly disappeared and mesenchymal cells invaded the area. The epithelialrests were still bound by basal lamina in secondary palate formation but noanalogous structures were seen in this study. Once the basal laminadisappeared, it did not leave any remnants behind. At the denuded edge of aregressing nasal fin, the epithelium adjacent to the mesenchymal bridge haddeposits of basal lamina material beneath it. This material appeared quite thickon the MNP side and it was impossible to tell whether this represented degradedbasal lamina or lamina that was being resynthesized as the mesenchymal bridgewas enlarging. Presumably there would be some epithelial "shuffling" andrearrangement taking place as the tissue was taking on a new level oforganization.In a number of the nasal fins studied, there were numerous points of contactbetween the cell processes of mesenchymal cells and the epithelial basallamina. A 15 T.S. nasal fin showed a mesenchymal cell process in contact withthe basal lamina but not through to the epithelial cell. There appeared to be anelectron dense layer at the point where the mesenchymal cell touched the basal111lamina. This could be some sort of communication point. Sometimesmesenchymal cells contacted the basal lamina and the epithelial cell underneath.This would imply a direct cell-cell contact as is thought to occur in facialprominence epithelium and mesenchyme where mesenchymal cell contact wasnecessary to maintain maxillary epithelium viability (Saber et al., 1989). Theepithelial and mesenchymal cell processes observed in this study often appearedto contain microfilaments. Possibly these filaments served to maintain thestructural integrity of the process.The primary palate is considered to have formed once the nasal fin hasdisappeared and the mesenchymal bridge has enlarged. The timing involved innasal fin formation and regression is likely important. The basal lamina mustbreak down before the epithelial surfaces make contact. I observed that once thenasal fin was formed, the areas of basal lamina destruction were already quiteextensive, and the epithelial cells became displaced presumably allowingmesenchymal cells to move in.4.3 Description of Hays "Fixed Cortex" Model applied to Epithelial toMesenchymal Transformation.Recently a new model has been published that attempts to describe amechanism by which epithelial cells that transform to mesenchyme can moveaway from the epithelial cell population and become a part of the surroundingmesenchymal cell population (Hay,1989; Bilozur and Hay, 1989). This theorywould explain how epithelial cells in a number of developmental systems that are112thought to involve epithelial to mesenchymal transformation are able to leavetheir original epithelial arrangement. Systems like thyroid, lens, Mullerian ductregression and secondary palate development are all potential candidates wherean epithelial to mesenchymal transformation is thought to occur. They are alsopossible systems that this model could serve to explain.The fixed cortex theory was originally applied to fibroblasts and it stated thatthe fibroblast cell surface consisting of an actin rich cortex and plasmalemma isleft behind as the myosin and intermediate filament rich endoplasm slidescontinuously forward along the actin cortex into a new front end of the cell.Essentially, the front end of a moving fibroblast becomes the rear end and is leftbehind as a membrane bound actin cortex attached to the ECM. This theory isapplied by Hay to explain the movement out of epithelium of newly formingmesenchymal cells during epithelial to mesenchymal transformation in theembryo. She puts forward the idea that myosin in the endoplasm is attached tomicrotubules and intermediate filaments such that a force is produced that coulddrive the whole inner part of the cell into the F-actin rich front end of the cell. Theintermediate filaments of the epithelial cells have switched from a keratin typecharacteristic of epithelial cells to vimentin which characterizes mesenchymalcells. Vimentin-actin interactions with ECM may be a major factor in the ability of acell to become elongated and form an actin rich new front end. Bilozur and Hay(1989) propose that a master gene is turned on during epithelial-mesenchymaltransformations that turns on a mesenchymal cell phenotype. The epithelial cellcould now produce collagen type I, fibronectin, and the cytoskeletal system of a113mesenchymal cell. As the epithelial cell becomes a newly forming mesenchymalcell it extends actin rich filopodia into the ECM. The leading end of the cell isbeing rebuilt with new actin, new membrane, and new ECM receptors. Thetrailing end of the cell that still has epithelial cell characteristics is left behind atsites of the zonula adherens junctions and cell adhesion proteins. The epitheliumleaves the old fixed surface behind and moves the endoplasm containing thenucleus out of this shell.1144.4 Applying the Fixed Cortex Model to the Primary Palate ModelIt is interesting to speculate whether an epithelial to mesenchymaltransformation takes place during primary palate formation as the nasal finepithelium regresses. If such a system were operating would it follow theprinciples of the fixed cortex theory? This would imply that the epithelial cells ofthe nasal fin that were destined to regress must lose their basal lamina first beforethey can transform into a mesenchymal phenotype. That would mean that nasalfin regression was similar to thyroid placode evagination, Mullerian ductregression, and secondary palate formation which are all systems where thismodel could be applied. Once the basal lamina had disappeared, only certainepithelial cells, that no longer had their basal lamina intact, could then migrateinto the surrounding mesenchymal cell population. As the mesenchymal bridgeincreased in size, more and more epithelial cells would be transforming intomesenchyme. The TEM observations in this study do support the idea that onlycertain regions of the nasal fin break down at any one point in time; these regionsbeing places where mesenchymal cell processes contacted the basal lamina.Also, regions of epithelium at various stages of differentiation existed that staineddifferentially for actin. Around the nasal cavity would be one type of epithelium.This was a pseudostratified primitive olfactory epithelium that contained abundantF-actin in its apical epithelial layers facing the lumen. The epithelium of the MNPand LNP that was in the region of presumptive fusion was another epithelial115domain. This epithelium would go on to fuse and form the nasal fin. It did notexhibit apical actin staining. Only the occasional spot or plaque of F-actin,possibly zonula adherens junctions existed within nasal fin epithelium . Thiswould correspond to the part of the epithelial cell left behind as the mesenchymalcomponent was migrating away in the Hay model. There appeared to be moreadherens junctions in nasal epithelium compared to nasal fin epithelium. Thiscould be due to the nasal fin being transitory and it would not be practical to havethese epithelial cells as firmly attached to each other as in olfactory epithelium.The other epithelial domain was the future oral epithelium where there was notintense actin staining in apical epithelium like that seen in nasal epithelium.My results were consistent with the Hay model in that primary palateformation could be a system where an epithelial to mesenchymal transformationtakes place. There is a transitory epithelial cell population that eventuallydisappears and is replaced with mesenchymal cells. Also the basal laminabreaks down and can no longer act as a barrier.Further studies need to be done to look at nasal fin regression in order tounderstand whether a transformation to mesenchyme takes place in thisepithelium. Immunohistochemistry would be useful to detect whether there was aswitch to a vimentin type intermediate filament production in cells of the nasal fin,or were they still producing keratin.The basal lamina phenomenon should be studied using immunological tagsto basement membrane components. This should be done at the light andelectron microscopic levels to better understand its breakdown as the epithelium116of the prominences fuse. My results indicated that the basal lamina breaks downprior to fusion of the MNP and LNP, but it would be interesting to know whether allof its component molecules disappeared simultaneously or sequentially duringprimary palate formation.5. CONCLUSIONS-Differential staining for F-actin was observed in epithelium of the nasal cavity,facial processes, and nasal fin.-In nasal cavity epithelium staining was uniform with higher concentrations ofactin in apical regions of epithelium towards the lumen and at locations wherethere was bending.-In the LNP and MNP epithelium, staining was uniform with occasional spotdensities of actin seen mostly in epithelium that was not in the presumptive fusionzone.-After fusion, LNP and MNP epithelium formed the nasal fin, and actin stainingwas greatly reduced and disorganized. Spot densities were less frequent in nasalfin epithelium.-Results of this study were consistent with the Hay Fixed Cortex Theory.117-The basal lamina regresses prior to contact of the LNP and MNP but only inregions of presumptive fusion. 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