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The homeotic and segmentation genes interact differently with the polycomb-group genes in Drosophila… McKeon, Joanie 1989

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T H E H O M E O T I C A N D S E G M E N T A T I O N G E N E S I N T E R A C T D I F F E R E N T L Y W I T H T H E POLYCOMB-GROVP G E N E S IN DROSOPHILA MELANOGASTER by J O A N I E M C K E O N B . S c , University of Western Ontario, 1985 A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R O F S C I E N C E in T H E F A C U L T Y O F G R A D U A T E S T U D I E S ( Z O O L O G Y ) We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A September 1989 (c) Joanie McKeon, 1989 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of - Z QO \ o The University of British Columbia Vancouver, Canada DE-6 (2/88) Abstract Members of the Polycomb (Pc)-group of genes have similar phenotypes, including posterior transformation of embryonic and adult segments, sensitivity to changes in dosage of the bithorax and Antennapedia complexes, and enhancement of Polycomb phenotypes in transheterozygotes. However, genetic and molecular evidence suggests that not all members of the Pc-group have equivalent functions. I examined the protein distribution of the three homeotic genes Ultrabithorax, Antennapedia, and Sex combs reduced, and the segmentation gene engrailed to directly assay Pc-group function. The timing, severity, tissue-specificity, and pattern of gene expression differed in each mutant. These results support the hypothesis that different members of the Pc-group have different functions. M y results indicate that it is likely that there is at least one pathway in which Polycomb and extra sex combs are at the head, and that some Pc-group genes function in subpathways. I have also shown that some members of the Pc-group function in regulating the segmentation as well as the homeotic genes and therefore have a more general role in development. i i T A B L E O F C O N T E N T S P A G E A B S T R A C T ii T A B L E O F C O N T E N T S iii L I S T O F T A B L E S v L I S T O F F I G U R E S vi A C K N O W L E D G E M E N T v i i i A B B R E V I A T I O N S vii G E N E R A L I N T R O D U C T I O N 1 C H A P T E R 1: Interaction between Ubx and the 10 Pc-group genes I N T R O D U C T I O N 10 M A T E R I A L S A N D M E T H O D S 12 R E S U L T S 18 D I S C U S S I O N 34 C H A P T E R 2: Interaction between Antp and Scr and 42 the Pc-group genes I N T R O D U C T I O N 42 M A T E R I A L S A N D M E T H O D S 44 R E S U L T S 45 D I S C U S S I O N 61 C H A P T E R 3: Interaction between en and the 68 Pc-group genes I N T R O D U C T I O N 68 M A T E R I A L S A N D M E T H O D S 69 R E S U L T S 70 ii i D I S C U S S I O N G E N E R A L D I S C U S S I O N R E F E R E N C E S L I S T O F T A B L E S T A B L E 1. Comparison of Pc-group genes. T A B L E 2. Stages of Drosophila embryogenesis. T A B L E 3. Ubx protein distribution in Pc-group embryos. T A B L E 4. Antp protein distribution in Pc-group embryos. T A B L E 5. Scr protein distribution in Pc-group embryos. L I S T O F F I G U R E S P A G E F I G U R E 1. Ubx protein expression in stage 11 embryos. 20 F I G U R E 2. Ubx protein expression in stage 13 embryos. 22 F I G U R E 3. Ubx protein expression in stage 14 embryos. 24 F I G U R E 4. Ubx protein expression in stage 16 embryos, 26 lateral view. F I G U R E 5. Ubx protein expression in stage 16 embryos, 28 ventral view. F I G U R E 6. Antp protein expression in stage 11 and 46 stage 12 embryos. F I G U R E 7. Antp protein expression in stage 14 embryos. 48 F I G U R E 8. Antp protein expression in stage 16 embryos. 50 F I G U R E 9. Scr protein expression in stage 12 embryos. 55 F I G U R E 10. Scr protein expression in stage 16 embryos. 57 F I G U R E 11. en protein expression in stage 11 and 72 stage 12 embryos. F I G U R E 12. ftz protein expression in stage 5 embryos. 74 F I G U R E 13. en protein expression in stage 16 embryos, 76 ventral view. F I G U R E 14. en protein expression in stage 16 embryos, 78 lateral view. v i ABBREVIATIONS Abbreviations used in this text are those accepted as standard by the Proceedings of the National Academy of Sciences (USA), pp. vi-vii (1987). v i i ; A C K N O W L E D G E M E N T I would like to thank my supervisor, Dr . Hugh Brock, for his encouragement, excellent professional advice, and the gift of this project. Thanks also to my fellow zoology geneticists, especially Sally Freeman, A m y Hedrick, Ian Whitehead, and Marco DeCamil l is for invaluable discussions, encouragement, and support. Especially, I would like to thank my husband, Barry, for his fortitude, encouragement, and unequivocal support. v i i i G E N E R A L I N T R O D U C T I O N This thesis examines the relationship between the members of the Polycomb (Pc)-group genes in Drosophila melanogaster to understand their interaction with the segmentation and homeotic genes. The Pc-group genes are required for correct determination of segment number and identity, but their role is poorly understood. Drosophila is an excellent organism in which to study early development because genetic and molecular analysis can be combined. The embryo has been fate-mapped (Lohs-Schardin et al., 1979), and early development has been described (Turner and Mahowald 1976; 1977; 1979; Campos-Ortega and Harteinstein, 1985). The genetic components of early development are being dissected, and a number of genes have been identified which regulate determination and pattern formation. For more detailed descriptions I refer the reader to recent reviews (Doe and Scott, 1988; Ingham, 1988; A k a m , 1987; Duncan, 1987; Nusslein-Volhard et al., 1987; Scott and Carrol l , 1987). First I will clarify a point of nomenclature. Recently it has been proposed that gene regulation in the embryo occurs not through segments but through parasegments (PS), each composed of the posterior compartment of one segment through the anterior compartment of the next segment (Martinez-Arias and Lawrence, 1985). Therefore I will use the term parasegments to describe many of the observations below. Genetic control of early Drosophila development The earliest event in Drosophila development is establishment of the primary axes, which are under the control of the maternal effect genes (Nusslen-Volhard, 1979). Approximately 11 genes have been identified that interact to set up the dorsal-ventral axis. 1 Establishing the anterior-posterior axis is more complex and three groups of genes each using three different processes are involved in coordinating three different regions of the embryo (see review Nusslein-Volhard et al, 1987). The resulting distribution of these maternal gene products acts to regulate the expression of genes that act further downstream. These are the zygotically-expressed segmentation genes, that are required to subdivide the body into segments (Nusslein-Volhard and Wiechaus, 1980), and homeotic genes that specify segment identity (Lewis, 1978). Many genetic screens have been performed to isolate zygotic genes that affect embryonic and larval segmentation patterning, and as a result three classes of segmentation genes have been identified (Nusslein-Volhard and Weichaus, 1980; Nusslein-Volhard et al., 1984; Wiechaus et al., 1984; Jurgens et al., 1984). The gap genes hunchback (hb), Kruppel (Kr), knirps (kni), and giant (gt) cause the absence of contiguous segments along the body axis as homozygous mutants. The pair-rule genes affect alternate body segments; and 8 pair-rule genes have been identified: even-skipped (eve), hairy (h), paired (prd), fushi tarazu (ftz), runt (run), odd-skipped (odd), odd-paired (opa), and sloppy-paired (sip). Absence of the pair-rule genes cause missing or defective alternate segments. The third class of segmentation genes are the segment polarity genes, which disrupt parts of single segments, and include engrailed (en), armadillo (arm), hedgehog (hh), gooseberry (gsb), patched (ptc), and wingless (wg). Some of these genes (eg. hb) also show maternal effects. In a recent screen for genes that affect both maternal and zygotic activity, many more genes involved in embryonic patterning have been discovered (Perrimon et al., 1989). Several segmentation genes have been isolated by molecular techniques. The spatial expression patterns of these genes have been determined by in situ hybridization, or by 2 probing embryos with antibodies. These patterns generally correspond to the regions that are visibly affeeted by mutations in the segmentation gene. For example, pair rule genes are expressed in stripes in alternate segments, and the segment polarity genes are expressed in stripes in every segment (reviewed in A k a m , 1987). The time of expression of each gene shows that the gap genes are the earliest to act, followed by the pair-rule genes, and that the segment polarity genes are the last to be expressed. The segmentation genes act hierarchically to subdivide the body into smaller and smaller units (Meinhardt, 1986). It has been shown that the gap genes are regulated by the maternal genes (Gaul and Jackie, 1987; Tautz , 1988) and by other gap genes (Jackie et al, 1986; Mohler et al, 1989). The pair rule genes act after the gap genes and their pattern of expression is altered in maternal and gap mutant embryos (Ingham et al., 1985; Carrol l and Scott, 1986; Carroll et al, 1986b; Ingham et al, 1986; Frasch and Levine, 1987; Howard, 1988; Ingham and Gergen, 1988). The pair rule genes also exhibit cross regulation and can be placed into a hierarchy, such that some of the genes (eg run and h) act before other pair rule genes (eg eve and ftz) and the correct expression of the later are dependent on the former (Howard and Ingham, 1986; Carrol l and Scott, 1986; Hard ing et al., 1986; Mart inez-Arias and White, 1988; Ingham and Gergen, 1988). Final ly, the segment polarity genes respond to the transient prepattern set up by the pair-rule genes (Howard and Ingham, 1986; DiNardo and O'Farrel l , 1987; Ingham et al., 1988). Molecular analysis of the segmentation genes suggests how some of this regulation might occur. A t least one anterior-posterior gene, bicoid, and four of the segmentation genes, ftz, eve, prd, and en contain a homeobox (Frigerio et al., 1986; McGinnis et al., 1984; Scott and Weiner 1984; MacDonald et al, 1986; Harding et al, 1985; Bopp et al, 1986; Poole et al, 1985). Homeoboxes were originally described in homeotic genes and are homologous to part 3 of the yeast mating type protein sequences (Shepherd et al., 1984; Laughon and Scott, 1984), and probably serve as transcription factors. Th is idea is supported by the fact that en protein has been shown to bind to specific D N A sequences (Desplan et al., 1985; 1988), ftz protein specifically activates transcription of certain promoters in vitro (Winslow et al., 1989), and eve has been shown to bind to en upstream regulatory sequences (Hoey and Levine, 1988). Similarly, two of the gap genes, hb and Kr, are homologous to the Xenopus transcription factor TFI I IA (Tautz et al., 1987; Rosenberg et al., 1986). kni also has sequences related to the 'zinc finger' of hb and Kr, but in addition, kni encodes the D N A -binding motif characteristic of ligand-dependent nuclear receptor molecules (Nauber et al., 1988). Gap gene response elements of the eve promoter have been isolated (Harding et al., 1989; Goto et al., 1989). Therefore it is likely that some of these genes directly regulate genes below them in the hierarchy. The identity of individual segments depends on the correct expression of the homeotic genes. The homeotic genes are arranged in two complexes, the bithorax complex (BX-C) which specifies the posterior thoracic and abdominal segments of the fly (Lewis 1978), and the Antennepedia complex (ANT-C) which controls the anterior thoracic and head segments (Kaufmann et al., 1980). In the absence of both complexes, the embryo develops as a series of identical thoracic segments. The B X - C consists of three genes: Ultrabithorax (Ubx) which is needed in the thoracic segments, abdominal A (abd-A) which is required in parasegments 7 to 10, and Abdominal B (Abd-B) which is required in parasegments 11 to 14 (Sanchez-Herrero et al., 1985). Although there appear to be only three protein-coding genes in the B X - C , each of these genes is controlled by regulatory regions that are segment or compartment specific (Bender et al., 1983; Karch et al., 1985). The A N T - C consists of a number of genes including the homeotic loci Antennapedia (Antp) which is required in the 4 thorax, Sex combs reduced (Scr) required in PS2, and Deformed (Dfd), proboscipedia (pb), and labial (lab) required in the head segments (Mahaffey et al., 1989). Molecular analysis has shown that the homeotic genes also contain homeoboxes (Regulski et al, 1985). Th is suggests that the products of the homeotic genes may act as transcriptional activators or repressors of other genes that are in turn responsible for making segmental structures. In situ hybridization of A N T - C and B X - C genes to tissue sections show the regions of maximal expression correspond to the regions most affected in mutant embryos (Scott and Carrol l , 1987 and A k a m 1987 for reviews). These studies indicate that the pattern of expression of the homeotic genes is intricate. Peifer et al (1987) suggest the mosaic pattern of homeotic genes in each parasegment determines the identity of that segment. The regulatory processes that restrict the expression of A N T - C and B X - C genes are complex. The correct spatial expression of the homeotic genes appears to depend on both maternal and early acting zygotic segmentation genes (Carroll et al, 1986a; Ingham and Mart inez-Arias, 1986; Ingham et al, 1988; White and Lehmann, 1986; Harding and Levine, 1988; Irish et al, 1989); a hierarchy of cross-regulatory interactions (Harding et al, 1985; Hafen et al, 1984; Struhl , 1982; Struhl and White, 1985; Riley et al, 1987); and by two other groups of genes. The trithorax group appears to act as positive regulators of homeotic gene expression (Kennison and T a m k u n , 1988), and the Polycomb-group which appears to act as negative regulators. The Pc-group genes are the subject of this thesis. Polycomb-group genes The Pc-group has about thirty loci with similar phenotypes (Jurgens, 1985). The Pc-group genes are thought to be negative regulators of the A N T - C and B X - C . It has been 5 suggested that the Pc-group genes work through a complex regulatory network and therefore generally have the same function in development (Jurgens, 1985). However, more recent studies suggest the Pc-group may have a more general function in determination, and may not be as closely related as previously thought. The purpose of this thesis is to examine the similarities and differences of the Pc-group, to better understand their role in early development. The Pc-group genes include Polycomb (Pc) (Lewis, 1978; Duncan and Lewis, 1982), extra sex combs (esc) (Struhl, 1981), Polycomblike (Pel) (Duncan, 1982), super sex combs (sxc) (Ingham, 1984), Additional sex combs (Asx), Posterior sex combs (Psc), Sex comb on midleg (Scm) (Jurgens, 1985), Sex comb extra (See) (Breen and Duncan, 1986), polyhomeotic (ph) (Dura et al, 1985), polycombeotic (pco) (Shearn, Hersperger and Hersperger, 1978), and pleiohomeotic (pho) (previously refered to as lethal(4)29) (Breen and Duncan, 1986). Support for grouping the Pc-group comes from their similar phenotypes and the enhanced phenotypes of double mutant combinations. The Pc-group genes all cause homeotic transformations similar to those described for gain of function mutations in the A N T - C and the B X - C . These transformations are not directly due to lack of the Pc-group gene products, but rather appear to be due to the inoUscriminate activation of the homeotic genes of the A N T - C and B X - C (Struhl, 1981; Duncan and Lewis, 1982; Duncan, 1982; Ingham, 1984; Dura et al., 1985). Thus the phenotype of a P c - B X - C - embryo is the same as a B X - C - embryo. The phenotypes of Pc-group mutants are affected by changing doses of the B X - C or A N T - C . Some of the Pc-group genes were isolated as dominant enhancers of Pc (Duncan, 1982; Dura et al, 1985; Breen and Duncan, 1986; Kennison and T a m k u n , 1988). The homeotic transformations seen in any single Pc-group mutant are strongly enhanced in double mutants for the genes tested (Jurgens, 1985), strongly supporting the idea that the Pc-group 6 genes form a group of functionally related genes. However, genetic studies also show differences between the Pc-group genes in terms of their maternal and zygotic contributions, their importance in imaginal development, and their interactions with the B X - C . These are summarized in Table 1. The Pc-group genes differ dramatically in terms of whether the maternal contribution is important for normal development. In the Pc-group genes tested, if the maternal contribution is removed in addition to the zygotic contribution, the phenotype exhibited by the embryos is stronger than the zygotic phenotype, and embryos show strong posteriorly directed transformations (Breen and Duncan, 1986; Ingham 1984; D u r a et al., 1988). However, relative to each other there are differences in the strengths of the phenotypes and ph and pho show the strongest effects and die prior to cuticle formation (Dura et al., 1988; Breen and Duncan, 1986). The embryonic phenotypes of the Pc-group also vary with regard to the strengths of the homeotic transformations observed. Pc and esc show extreme transformations of many or all body segments to PS14, whereas the other members of the Pc-group show less extensive transformations. The imaginal requirement of some of the Pc-group genes has been examined by studying the behavior of epidermal clones generated by somatic crossing-over in the imaginal discs (Duncan and Lewis 1982; Duncan, 1982; Struhl and Brower, 1982; Ingham 1984; Dura et al., 1988). Pc, Pel, ph, pco, and sxc are all required for imaginal development, but esc, Asx, and Psc are not. Final ly, while all the Pc-group genes examined are affected by the changes in number of doses of the B X - C they are affected in different ways. P c , esc, Pel, and sxc are enhanced by extra copies of the B X - C , whereas ph, Asx, and Psc are suppressed. Therefore, despite the fact the Pc-group have certain phenotypes in common, in detail they are different from each other. Recently, Zink and Paro (1989) presented molecular evidence that the Pc-group may 7 T a b l e 1. G e n e t i c c o m p a r i s o n o f t h e P c - g r o u p l o c i Locus Maternal Effect Embryonic Phenotype Clonal Analysis Change if B X - C dose increased Pc + +++ positive enhanced esc ++ ++++ wild-type enhanced Pel + +/- positive enhanced Scm + + n.d. n.d. See + ++ n.d. n.d. Asx + + wild-type suppressed sxc ++ + positive enhanced pho +++ positive n.d. ph +++ ++ cell lethal suppressed Psc n.d. + wild-type suppressed pco n.d. + positive n.d. +++ = strong phenotype relative to other members of the Pc-group ++ = mi ld phenotype relative to other members of the Pc-group + = weak phenotype relative to other members of the Pc-group n.d.= not done 8 function as a regulatory network. They raised antibodies against the Pc protein and showed using immunostaining techniques that Pc binds to sixty discrete sites on the polytene chromosome. Encouragingly these sites include the A N T - C and the B X - C . Pc also bound to the sites of Scm, Asx, Psc, sxc, ph, and pco, indicating that these genes are regulated by Pc. However, no binding was found at esc, Pel, See, or pho. Therefore it is likely that some of the Pc-group genes may be involved in a regulatory network with Pc at the head. There is increasing evidence to suggest that Pc-group genes may also have more general functions in development. Embryos arising from mothers with homozygous sxc (Ingham, 1984) Pel, or pho (Breen and Duncan, 1986) germline clones have segmentation defects. Asx shows even-numbered segment defects in homozygous embryos, and show odd-numbered tergite defects in heterozygous adults (Brock, unpublished). These results suggest Pc-group genes may be required for correct expression of the segmentation genes. Psc acts as an enhancer of transvection (Wu, personal communication) suggesting that Psc may have some role in chromosome pairing or attachment to the nuclear membranes, ph is required for growth of the ventral epiderm and axonal outgrowth (Dura et al., 1987; Smouse et al., 1988). Final ly, pco was originally isolated due to its small disc phenotype (Shearn, Hersperger and Hersperger, 1978). This thesis examines the distribution of the Ubx, Antp, Scr, and en gene products in embryos mutant for the Pc-group genes to see if Pc-group genes behave similary or differently. 9 C H A P T E R 1: I N T E R A C T I O N B E T W E E N UBXAND T H E P C - G R O U P G E N E S INTRODUCTION The number of genes required to regulate the expression of Ubx is large. The complex structure of the Ubx gene and the precise localization of its products provide indications of the levels of control required for its correct expression. The primary transcript of the Ubx gene is alternatively spliced to yield at least five different transcripts (O'Connor et al., 1988; Kornfield et al., 1989). Ubx products are expressed in specific cell nuclei in the epidermis, the C N S , the somatic musculature, the visceral mesoderm, and the imaginal discs of developing flies (Akam, 1983; White and Wilcox, 1984; Beachy et al., 1985; Bienz et al., 1988). It is possible that the different members of the Pc-group genes may function to regulate correct expression of the different protein products of Ubx, or they may have difTering functions in specific tissues. Alternatively the differing temporal requirements of the Pc-group suggests they may have different roles in regulating Ubx in embryos and adults. In situ hybridization studies have shown that the initial distribution of Ubx transcripts is unaffected in esc nul l embryos, but later in development Ubx transcripts accumulate inappropriately (Struhl and A k a m , 1985). Simi lar results have been obtained in Pc (Weeden et al., 1986) and ph (Dura and Ingham, 1988; Smouse et al., 1988). Therefore, it appears the Pc-group genes are not required to initiate segment-specific expression of the Ubx, but instead it appears the Pc-group genes are required to ensure the stability of segment-specific gene expression. If the Pc-group genes are involve in cell memory it is possible to imagine a number of regulatory components required, or different requirements necessary at clifferent times in development. 10 Final ly, it is also possible that some members of the Pc-group have no specific role in regulation of Ubx and other homeotic genes, but upset general processes which impinge on their correct regulation. Th is idea is supported by the fact that general disruptions such as heat-shock induce phenocopies of dominant mutants of the B X - C (Santamaria, 1979; Dura and Santamaria, 1983). Th is chapter examines the spatial distribution of the Ubx gene product in embryos mutant for Pc-group genes, to determine if these genes function similarly. 11 MATERIALS AND METHODS F l y culture Flies were raised on standard cornmeal, sucrose, agar media supplemented with baker's yeast at 25<>C. Tegosept was added as a mold inhibitor. Canton S flies were used to generate wild-type embryos for comparison with the Pc-group embryos. Where possible null alleles of the gene of interest were used to allow comparison of the results. For the work described below I have chosen to analyze the Pc3 allele. Based on dosage studies, Duncan and Lewis (1982) show that Pc3 is not a nul l allele of the Pc locus, but is weakly antimorphic. However the Pc3 allele exhibits a stronger homeotic transformation phenotype than Pc null alleles, and has been the allele most often used in previous studies. esc2 is an apparent nul l allele which arose spontaneously on the CyO balancer. esc5 is an apparent null allele induced with E M S . (Struhl, 1981) Pclw6 is a point mutation that is associated with stronger adult homeotic transformations than the Pell and Pcl% alleles described by Duncan (1982). Unl ike these two alleles, PclwG embryos show homeotic transformations (Sato et al., 1984). Duncan (1982) suggests that Pctt and Pc/2 are amorphic or strongly hypomorphic because they are indistinguishable from two deficiencies which uncover the locus in complementation tests. Therefore, Pclw6 also is amorphic or strongly hypomorphic. Only one allele of See has been recovered. Because a deficiency for the locus of See has not yet been identified, the nature of the Seel allele is unknown (Breen and Duncan, 12 1986). ScmPl is probably a null allele because similar embryonic phenotypes are observed in homozygous or hemizygous animals (Breen and Duncan, 1986). A nul l allele for Asx has not been identified. Therefore for this study I used Df(2R)trix. Df(2R)trix is a small deficiency that uncovers the Asx locus (Breen and Duncan, 1986). sxc3 is described by Ingham (1984) and is most likely close to the amorphic state of the locus. pho(A) was used in this study. It is an amorphic allele of 1(4)29 isolated by Rob Denell . In this study I examined two alleles of the ph locus. Two mutagenic events are required to generate nul l alleles of the ph locus. ph&03 was isolated after a second round of mutagenesis of the hypomorphic allele ph^09 (Dura et al, 1987) and is amorphic. Df(l)JA52 is a deficiency that uncovers the ph locus. The same mutant phenotype is seen in ph&03 and Df(l)JA52 embryos (Dura et al, 1987). The Psc* allele was recovered as a revertant of the lethal interaction between Su(z)2l and Psc and is probably a null allele for Psc (Wu, personal communication). pcol902 i 8 an EMS- induced nul l allele (Shearn, personal communication). E m b r y o collection Balanced stocks were crossed inter se to allow collection of heterozygous progeny. To obtain esc embryos it is necessary to abolish both the maternal and zygotic contributions. Therefore esc^/Gyl females were crossed to esc^/Gla males, and esc%/esc5 virgins were collected. These females were then back-crossed to esc^/Gla males and their embryos were 13 examined. These embryos are referred to as esc'. The ph stock was maintained as ph5Q3/Yw+/FM7c. ph^03/FM7c virgins were crossed to ph503/Yw+ males and their embryos were examined. To collect embryos, flies were transferred to laving bottles and embryos were collected on 1.5% agar laying trays coated with egg laying medium (5% acetic acid, 10% ethanol, and 5% baker's yeast). The flies were allowed to lay from zero to nine hours or overnight. Embryos were staged under the microscope according to Campos-Ortega and Hartenstein (1985). Mutant embryos were distinguished from wild-type by coincidence between the expected frequency of mutants (0.25) and the aberrant expression patterns of the proteins studied. For esc, two classes of embryos are expected each with a frequency of 0.5, because fathers were heterozygous. A t least 30 and up to 100 embryos at each stage were looked at for each allele examined. The expected number of embryos with aberrant expression patterns were counted and fell within the expected value for the test, x^ =3.84 a=0.05, except where noted below. Embryonic stages A brief summary of the developmental stages of the Drosophila embryo indicating the readily visible distinguishing characteristics of each stage is presented in Table 2. For a more detailed account I refer the reader to Campos-Ortega and Hartenstein (1985). Antibodies Embryos were stained with monoclonal antibodies against Ubx which were generously supplied by Danny Brower. The Ubx monoclonal antibody is F P 3.38 and 14 T a b l e 2. S tages o f E m b r y o n i c D e v e l o p m e n t o f Drosophila melanogaster Stage Hours post Distinguishing fertilization characteristics I to 4 0 to 2:10 first 13 cleavage divisions, ending in syncytial blastoderm. 5 2:10 to 2:50 cellular blastoderm 6 and 7 2:50 to 3:10 gastrulation 8 3:10 to 3:40 rapid germ band extension 9 and 10 3:40 to 5:20 slow germ band extension II 5:20 to 7:20 no major morphogenetic change; growth stage 12 7:20 to 9:20 germ band shortening; segmentation prominent; C N S separates itself completely from epidermis 13 9:20 to 10:20 begins with the completion of germ germ band shortening and ends with the beginning of head involution; no dorsal closure; hindgut opens into the anus; C N S well differentiated 14 10:20 to 11:20 beginning of head involution; dorsal closure initiated; hindgut grows and opens in the anus 15 11:20 to 13:00 dorsal closure completed; dorsal segmentation obvious; middle midgut constriction forms; contraction of C N S begins 16 13:00 to 16:00 all three midgut constrictions form; proventriculus and gastric caeca appear; head involution completed; C N S continues to shorten 15 probably recognizes all Ubx products (White and Wilcox, 1984). The antibody was used at a dilution of 1:1000. Antibody staining Embryos were collected on a nylon mesh by washing laying trays with H2O, and then rinsed three times in embryo wash (0.4% N a C l and 0.03% Triton X-100). The chorion was removed by incubating the embryos in 50% bleach for three to five minutes. Embryos were rinsed three times in embryo wash, and transferred with a paintbrush into an Eppendorf tube containing 700 ul of fixative (0.1 M P I P E S p H 6.9, 2 m M E G T A , 1 m M MgSC>4, and 4% formaldehyde), and 700 ul heptane. The tubes were shaken vigorously for 30 seconds and then rotated on a rotary shaker for twenty minutes. After fixation, the lower aqueous phase and any embryos at the bottom were removed with a Pasteur pipet and 700 ul of 90% methanol and 10% E G T A was added to the tubes. The tubes were vortexed vigorously to remove the vitelline membranes. Devitellinized embryos settled to the bottom of the tube. The top organic layer and the membranes left at the interface were pipetted off. The embryos were washed three times for two minutes each in 1 ml P B T (IX P B S , 0.2% serum albumin, 0.1% TritonX-100), and then washed six times with rotation in P B T for thirty minutes each. After the last wash the P B T was removed, and 200 ul of the appropriate dilution of the monoclonal antibody was added. The embryos were incubated overnight at 40C on the rotator. The primary antibody was removed and the embryos were washed three times for two minutes each in P B T at room temperature, and then six times for 30 minutes each on the rotator. 200 ul of a 1:5000 dilution of peroxidase conjugated Goat anti-mouse IgG (H + L) (Jackson Immunoresearch Laboratories) was added and the embryos were incubated 16 overnight at 4^C. The secondary antibody was removed and the embryos were again washed three times for two minutes each in P B T at room temperature and six times for 30 minutes each with rotation. The embryos were then rinsed two times in T B S ( lOmM Tr is p H 7.5, 150 m M N a C l , and 1 m M E D T A ) . The T B S was removed and 200 ul of 1 mg/ml maminobenzidine in T B S was added. 2 ul of 3% hydrogen peroxide was added as a catalyst and the staining reaction proceeded for two to ten minutes or unti l staining was observed under the microscope. The reaction was stopped by washing two times with T B S . The embryos were taken through a series of ethanol washes (30%, 50%, 70%, 90%, and three times 95%) for five minutes each. The ethanol was removed and the embryos were cleared in Gary's Magic Mountant (2 vol Canada Balsam :1 volume methyl salicylate), placed on slides and covered with coverslips. The embryos were observed on a Zeiss Axiophot microscope using differential interference contrast optics. Photos were taken on Fuji slide film A S A 100. A b b r e v i a t i o n s For sake of brevity the following abbreviations are used: Ubx, Antp, Scr, en, ftz, and Dfd refers to the gene; and U B X , A N T P , S C R , E N , F T Z and D F D refers to the protein product of their respective genes. 17 Results The wild-type pattern of Ubx protein expression has been previously described and is complex both spatially and temporally (White and Wilcox 1984, 1985a, 1985b; Beachy et al., 1985; Carrol l et al, 1988; Bienz and Tremml , 1988). For comparison with the mutant patterns, I will describe and illustrate the wild-type U B X pattern. U B X is first expressed at the extended germ band stage in the epidermis in embryonic cell nuclei from parasegment (PS) 5 to PS 13 (Figure 1A). PS6 shows the highest level of staining, PS7 to PS 12 staining decreases in intensity posteriorly. There is a lower level of U B X expression in PS5 and PS13. Intensity of staining is graded and is greater in the posterior part of each parasegment and decreases anteriorly. The pattern of staining remains the same in the epidermis after germ band shortening. Staining of nuclei of cells in the C N S and visceral mesoderm becomes visible at stage 13 (Figure 2A). Figure 3A shows that by stage 15, cell nuclei from the C N S become more heavily labelled than epidermal cell nuclei. White and Wilcox (1984) report that PS 13 staining is detectable in the C N S in wild type embyros but more weakly than PS12. I did not detect PS13 staining in the C N S of wild-type embryos. In the visceral mesoderm, a belt of U B X expression is seen starting at stage 13. In stage 14 and 15 embryos this belt extends around the midgut, is approximately one metamere wide and is in register with PS7 in the epidermis (Figure 3A). Bienz and Tremml (1988) have determined that U B X expression in the visceral mesoderm is in PS7. A t stage 16 the second midgut constriction coincides with the posterior l imit of U B X expression in the visceral mesoderm and U B X is never seen in the first midgut constriction (Figure 4A). In all the Pc-group mutants, except Psc and pco, the initial distribution of U B X remains indistinguishable from the wild-type distribution. In esc- embryos from esc- mothers an effect on Ubx protein pattern is first seen at stage 11 (Figure 2B). Nine percent of 18 embryos stained strongly for U B X in PS5 and PS4. A t the end of germ band shortening, low levels of U B X accumulate throughout the germ band. The protein expression parallels the pattern of m R N A expression in esc- embryos (Struhl and A k a m , 1985). A s development proceeds the protein becomes expressed more anteriorly, and by stage 13 there is low level expression of U B X in the C N S from PS 13 to the developing brain (Figure 2B). This pattern of expression remains unti l the cuticle hardens and it is no longer possible to monitor expression (Figure 3B and 4B). The pattern of cells labelled in each parasegment is very different from wild-type (compare Figure 5A to Figure 5B). There is a barely detectable level of background labelling and stronger specific labelling in a few cells in each parasegment. Struhl and White (1985) examined Ubx protein expression in esc- embryos using fluorescent staining and determined that every parasegment has the same staining pattern as P S 13. Figures 3B and 4B shows that the intensity of staining of U B X in the visceral mesoderm is decreased and there also appears to be a low level of ectopic expression into the more anterior gut regions. Correspondingly, Struhl and A k a m (1985) reported Ubx m R N A transcripts throughout the visceral mesoderm of esc embryos. A t stage 16 in wild-type embryos the proventriculus and three constrictions of the midgut appear. In esc embryos these constrictions never fully form and the proventriculus does not develop properly (compare Figure 4 A and Figure 4B). The epidermis also appears to have low level of expression that grows weaker with age. In esc/+ embryos from esc mothers, there is ectopic expression of U B X into PS3 in the C N S and also into the anterior midgut (not shown). Pc& embryos develop normally up to stage 13 when ectopic expression of U B X is seen in the C N S . The protein extends more anteriorly with age in the C N S and there is a even level of expression in each parasegment (Figure 2C and 3C). By stage 16 the protein is expressed from PS14 to the brain with almost equal intensity in each parasegment (Figure 19 Figure 1. Ubx expression i n germ band extended Pc-group embryos. Photographs of anti-Ubx antibody staining i n whole-mount wild-type (A), esc (B), sxc- (C), pho- (D), ph- (E), Psc- (F), and pco- (G) embryos at germ band extension. La tera l view w i t h anterior to the left and dorsal up. The arrowhead marks parasegment 6. 20 2.1 Figure 2. Ubx expression in stage 13 Pc-group embryos. Photographs of anti-Ubx antibody staining in whole-mount wild-type (A, J), esc- (B), Pc- (C), Pel- (D), Scm- (E), Asx- (F, L) , sxc- (G), pho- (H), Psc- (I) and ph- embryos at stage 13. (A -1) Lateral view with the plane of focus on the C N S . (J, K, L) Lateral view with plane of focus on the epidermis. Anterior is to the left and doral is up. The arrowhead marks parasegment 6. 22 23 Figure 3. Ubx expression in stage 14-15 Pc-group embryos. Photographs of anti-Ubx antibody staining in whole-mount wild-type (A), esc- (B), P c (C), Pel- (D), Scm- (E), See- (F), Asx- (G), sxc- (¥l),pho- (l),ph- (J),pco- (K) embryos at stage 14 or 15. Lateral view with plane of focus on the C N S . Anterior is to the left and dorsal is up. The arrowhead marks parasegment 6. 24 25 Figure 4. Ubx expression in stage 16 Pc-group embryos, lateral view. Photographs of anti-Ubx antibody staining in whole-mount wild-type (A), esc (B), Pc (C), Pel- (D), Scm- (E), See- (F), Asx- (G), sxc (H), pho- (I), ph- (J), Psc (K), and pco- (L) embryos at stage 16. Lateral view with plane of focus on C N S and proventriculus. Anterior is to the left and dorsal is up. The arrowhead marks parasegment 6. 26 27 Figure 5. Ubx expression in stage 16 Pc-group embryos, ventral view. Photographs of anti-Ubx antibody staining in whole-mount wild-type (A), esc (B), Pc (C), Pel- (D), Scm- (E), See- (F), Asx- (G), sxc (R),pho- (I),ph- (J), Psc (K), and pco- (L) embryos at stage 16. Ventral view with plane of focus on the C N S . Anterior is to the left and dorsal is up. The arrowhead marks parasegment 6. 28 4C). Figure 5C shows there is still heterogeneity within each parasegment and the repeated pattern appears to be that of wild-type PS 12 staining. Anterior to PS6 this pattern appears to be altered slightly. Wi th increasing age there is a decrease in protein level in the posterior parasegments, whereas anteriorly, the levels increase. Figures 3C and 4C show ectopic expression of U B X in the entire anterior midgut. A s with esc, the normal constrictions that one sees in wild-type animals do not form and this is clearly seen in stage 16 embryos where the proventriculus forms but the gut remains a fairly straight tube (compare Figure 4 A and 4C). In Pel; Scm; See; and Asx- embryos there also is ectopic expression of U B X in the C N S but not as far anterior as esc or Pc3, and it is possible to distinguish PS6 because of its elevated expression relative to the other parasegments. The first detection of ectopic expression occurs later in Pel-, Scm-, See-, and Asx- embryos than in esc or Pc3 embryos. However there are differences between their U B X expression patterns in the C N S and midgut. None of these mutants have any upset in ectodermal expression of U B X . In Pel- embryos the gut staining becomes ectopic before any defects in the C N S are detectable. Figure 2D shows an early stage 13 embryo with patches of U B X staining in the very anterior portion of the midgut where Scr protein staining is normally seen. Late in stage 13 there U B X is expressed into PS4 in the C N S . Figure 3D shows that by stage 15 the labelling has reached P S l . The brain has a few weak patches labelled by stage 16, and at this stage U B X is expressed along almost the entire l ining of the midgut (Figure 4D). In the midgut, some constrictions form but they are different, and not as extensive as in wild-type. The pattern of expression of U B X in the C N S of Pel- embryos is abnormal (Figure 5D). It differs from esc or Pc? embryos because PS6 stains more strongly relative to the other parasegments (compare Figures 4B and 4C with 4D and Figures 5B and 5C with 5D). In 30 PS7 to PS12 there is a specific subset of neurons staining but this pattern is different than wild-type (compare Figure 5D with 5A). In PS5, PS4 and PS3 most of the cells are expressing U B X with an intensity that is slightly lower than PS6. Some Pel- embryos have abnormal morphology (data not shown). In Scm- embryos the earliest ectopic U B X expression is seen at the stage 13-14 boundary. Label l ing expands anteriorly with age and Figure 3 E shows a stage 14 Scm-embryo. Figure 4 E shows that by stage 16 U B X has reached into PS2, but it never extends into the brain. After stage 16 the level of expression increases anterior to PS6 and decreases posteriorly. Unl ike esc- and Pc3 embryos, PS6 stains more intensely than PS7-12 (compare Figure 4B and 4C with 4 E and Figures 5B and 5C with 5E). PS7-12 appear wild-type (compare Figure 5 E and 5A). The pattern of labelling in PS3, PS4 and PS5 is similar to each other and it appears most of the cells are expressing U B X (Figure 5E). There is a mi ld defect in midgut staining and at all stages after stage 13 two patches of U B X are expressed in the very anterior midgut (Figures 2 E , 3 E and 4E). Howeyer, gut morphology is normal, although its development is slower than wild-type. In See- embryos weak ectopic expression is not seen until stage 14 (Figure 3F). Figure 4 F shows that labelling never extends into the brain. The U B X labelling in See-embryos is similar to that of Scm- embryos (compare Figures 4 F and 4E). It is possible to distinguish PS6 from PS7 to 12, which are wild-type, and ectopic expression is seen in PS3, PS4 and PS5. However, the pattern of cell labelling in PS3, PS4, and PS5 differs in Scm-and See- embryos (compare Figure 5 E and 5F). In See- embryos not all cells in PS3, PS4, and PS5 stain. Figure 5F shows that the midline neuroblasts stain strongly and there is weak or no label moving laterally. Visceral mesoderm staining is normal. In Asx- embryos there is a mild C N S defect and strong ectopic expression in the 31 midgut. Expression of U B X into PS4 occurs at stage 13 and is detectable in the presumptive neurogenic region and transiently in the epidermis (Figure 2 F and 2L). Staining is very weak and only appears in a portion of the cells of this parasegment. The intensity of labelling of the midline cells in PS5 increases. There are also increased levels of labelling in PS13 in the which is clearly seen from stage 13 onwards (Figures 2F, 4G, 5G), whereas in wild-type the staining is undetectable. In the midgut U B X is expressed in parasegments anterior to PS7 and appears initially at stage 13 (Figure 2F). By stage 16 almost the entire anterior midgut is labelled (Figure 4G). sxc and pho- embryos do not have ectopic U B X expression. However there is a noticeable change in the intensity of U B X staining of the parasegments relative to one another. In sxc mutants the intensity of labelling of PS6 equals the intensity of PS7 and PS8. This is first detectable at stage 10 (Figure 1C, compare to Figure 1A) in the ectoderm and continues to be seen in the C N S of older embryos (Figures 2G, 3 H , 4 H , 5H). In pho-embryos the intensity of PS6 decreases to equal PS7_which equals PS8 and PS9, which are greater in intensity than 10, 11 and 12. In some cases labelling in PS6 remains slightly stronger. Th is is first detected at stage 11 in the epidermis (Figure ID) and is seen at all stages in the C N S (Figures 4 H and 41). the overall intensity of staining is reduced in sxc and pho- embryos relative to wild-type (compare Figures 5 H and 51 to 5A). ph-, Psc and pco- embryos exhibit abnormal morphology, ph- embryos develop normally unti l 12 hours, after which there is extensive cell death in the ventral cuticle and there are problems with head involution (Dura et al. , 1987). I examined two ph alleles, a deficiency for the locus Df(l)JA52, and a nul l allele ph503. Similar results were seen for both mutants. A t stage 10-11 there is ectopic expression of U B X in the ectoderm into PS3 and PS4 and the intensity of staining in PS5 increases (Figure IE). A t stage 13 ectopic 32 epidermal labelling continues to be detectable (Figure 2K). In later stages there is no detectable U B X expression in any tissue including the C N S , ectoderm or midgut (Figures 3J , 4 J and 5J). Th is is in contrast to the results obtained by Dura and Ingham (1988) who detect U B X in head segments after germ band retraction. The development and defects of Psc- embryos are variable. Psc- embryos show abnormal development by stage 10 and U B X staining is blotchy at this time (Figure IF). A s development proceeds it becomes increasingly difficult to stage these embryos as the head, gut, and tail appear to have different developmental rates. U B X staining remains blotchy with increasing age but appears to obey the anterior (PS5) and posterior (PS 13) boundaries of wild-type expression (Figures 21 and 4K). U B X expression is a good marker to demonstrate the abnormal development of the C N S in Psc- embryo and the defects seen are quite variable. The C N S is broader and may be twisted, and often there may be holes (Figure 4 K and 5K). Figure 4 K also shows that sometimes not enough neuromeres are stained, and this may reflect segmentation problems. Midgut staining appears to be wild-type, but often the gut is distorted and morphology is abnormal (Figure 4K). pco- embryos start developing abnormally at the start of gastrulation (Figure 1G). The germ band does not extend properly and other early invaginations are atypical. By the time U B X staining originates the expression is blotchy and it is difficult to stage the embryo as the morphology is grossly upset (Figure 3K). It is possible to differentiate the nervous tissue and there is labelHng present here. Figure 4 L shows head involution is completely aberrant, and there is no staining in the anterior part of the embryo. It is not possible to distinguish gut tissue staining. 33 DISCUSSION' This chapter examines how the different members of the Pc-group interact with the Ubx gene product to determine whether these genes act similarly or differently. Table 3 provides a summary of the results. A l l the members of the Pc-group affect the expression of U B X , but do so in different ways. There are differences in intensity of staining, in ectopic expression, in t iming of the effects, and in the tissue specificity. In some members of the group the effects on U B X seem to be a by-product of general problems due to abnormal development. Some of the effects overlap between members of the Pc-group. Based on these similarities and differences it will be argued below that the members of this group can be divided into subgroups based on their interactions with Ubx. However, Pc (Haynie, 1983), esc (Struhl, 1981), Pel, Asx, See, Scm, pho (Breen and Duncan, 1986), and ph (Dura et al., 1988) have maternal effects. Therefore, if the maternal contribution of Pc-group product is high, or perdures late into embryogenesis, then the studies described above may underestimate the requirement for a given Pc-group gene. This can be seen for example, comparing esc- embryos derived from heterozygous mothers and homozygous mothers, where there are only weak effects in U B X distribution in the former, but very strong effects in the latter. Nevertheless, the results described here suggest that different Pc-group genes have different functions in development. Pc and esc have the strongest effects on Ubx of the Pc-group, shown by the extent and uniformity of ectopic U B X expression. Th is is consistent with the genetic evidence that shows that Pc and esc are negative regulators of the B X - C and that the absence of Pc and esc products leads to indiscriminate expression of all B X - C products. Th is hypothesis is supported genetically by the transformation of most segments toward the eighth abdominal segment (Lewis, 1978; Duncan and Lewis, 1982; Struhl , 1981,1983), and molecularly as 34 Table 3. Ubx protein expression in Pc-group embryos Defect Nervous System Pc ectopic in brain, PS2-4,13 uniform intensity=PSl2 esc ectopic in brain, PS2-4, 13 uniform intensity=PS13 Pel ectopic weak in brain, PS 1-4 Scm ectopic in P S 1-4 See ectopic in PS 1-4 Asx ectopic in PS4/PS13 sxc intensity differences PS6=7=8 pho intensity (lifferences PS6=7 ph suppressed Psc blotchy, abnormal morphology pco blotchy, abnormal morphology Stage First Observed 13 11 13-14 13-14 14 14/13 10 11 11-12 8 8 Gut ectopic suppressed ectopic patches in Scr domain ectopic + suppressed + 35 abd-A and Abd-B have been shown to be ectopically expressed in both Pc (Weeden et al., 1986) and esc (Struhl and White, 1985) embryos. The results cited also show that these genes are not involved in the initiation of Ubx gene expression, but are involved in the maintenance of the determined state (Struhl and A k a m , 1985) because these genes do not determine where Ubx is to be expressed but do ensure that Ubx remains off in those parasegments in which it is not initially turned on. Nevertheless, my results also show that Pc and esc affect U B X differently. esc and Pc embryos differ in t iming of effects on U B X and the pattern of expression of U B X within each parasegment. In esc embryos, U B X is expressed ectopically at stage 11 in both the epidermis and the presumptive neurogenic regions, whereas in Pc$ embryos U B X does not become ectopically expressed unti l stage 13 and was not detected in the epidermis. Th is agrees with the genetic information which indicates that esc is required earlier than Pc (Struhl and Brower, 1982). The stage 16 pattern of expression varies between these two genes. Both have low levels of expression throughoutthe C N S from PS 14 to the brain, but in esc embryos the pattern of U B X in every parasegment is that of PS 13 whereas in Pc3 embryos the pattern in each parasegment resembles wild-type PS 12. Struhl and White (1985) have shown that the pattern of Ubx gene products in the abdomen depends on the additive, yet independent, activities of the abd-A and Abd-B gene functions. They suggest that the repeated pattern of U B X expression in esc embryos is because all the B X - C genes are mdiscriminately expressed. One way to account for the difference between esc and Pc on Ubx would be if esc and Pc had different effects on abd-A or Abd-B, or possibly another yet unidentified gene. This may be likely considering the number of Pc binding sites on polytene chromosomes (Zink and Paro, 1989). Alternatively the difference may depend on the maternal contribution of the Pc product. It is not possible to conclude which alternative is 36 correct, but determining the exact pattern of expression of abd-A and Abd-B gene products in the C N S of Pc- and esc- embryos will help resolve these choices. The similarities and differences between esc and Pc can also be seen in the morphology and U B X staining of the midgut, esc- embryos seem to have a more extreme effect because the proventriculus does not form properly, and the U B X staining is very weak. In both Pc- and esc- embryos, the level of staining in the gut is decreased relative to wild-type, suggesting that abd-A and Abd-B are turned on more anteriorly in the gut also, since the abd-B gene represses Ubx protein expression in the gut (Bienz and Tremml , 1988). A s in the C N S , the level of repression of U B X in the midgut of esc- embryos is greater than in Pc-embryos, indicating that esc and Pc have different effects on abd-A and Abd-B. These differences may also be a result of the maternal effect of esc. Genetic studies have also indicted Pc and esc have independent roles. Struhl (1983) examined the dependence of the esc- phenotype on the number of copies of the Pc+ gene and found that in both esc+ and esc- animals, the effects of altering the dosage of the Pc+ gene on the expression of the B X - C genes appears to be the same. He concludes that esc and Pc have additive and independent effects on the expression of the B X - C genes. This is supported by the f inding that Pc protein does not bind to esc in polytene chromosomes (Zink and Paro, 1989) and that the transheterozygote esc/+\Pc3/+ do not enhance the effects of either single mutant (Campbell and Brock, unpublished). This is not surprising based on the different temporal requirements of the two genes (Struhl, 1981; Struhl and Brower, 1982). Therefore either Pc and esc act at different times on the same process, or affect different processes. Scm, See, Pel, and Asx share a number of features that suggest they are likely to be required for a subset of Pc function. They share ectopic expression and apparent lack of effect on abd-A and Abd-B, plus later action than Pc or esc. None of these genes affect U B X 37 expression as strongly as Pc or esc since U B X does not extend as far into the head, and PS6 retains its high intensity relative to the other parasegments. These genes probably are not affecting abd-A or Abd-B in the C N S or the selector genes in the head. The effect of Scm, See, Pel, and Asx on U B X is always after an effect has been detected in Pc- embryos. Therefore these genes could act later in a pathway regulated by Pc. Alternatively, they could act independently on part of the process(es) affected by Pc. Looking at the effect of Pc on the expression of these genes will help to answer this question. Jurgens (1985) and Breen and Duncan (1986) determined that mutations in Pel, Scm, and See cause very similar embryonic phenotypes. In these mutants the abdominal segments are affected and the thoracic segments never are. Th is seems odd in light of the results presented here because U B X expression in the abdominal C N S appears to be wild-type, whereas it is upset in the thoracic segments. What about the epidermal transformations? A t the stage at which ectopic expression was seen in the C N S of most of the embryos studied, the epidermal staining grows weaker than C N S staining. If at this stage the more posterior B X - C genes are overexpressed in the epidermis it may not be possible to score the change in expression of U B X and this was missed. It will be important to examine the expression of the abd-A and Abd-B genes in all of the Pc-group mutant embryos to see which affect these genes in both the epidermis and the C N S . It is not possible to determine whether See, Scm, Pel, and Asx actually function together in development, or if they are involved in different processes that result in similar phenotypes. Many of the genetic studies support the idea that the genes are a natural group. See, Scm, and Pel enhance the expressivity and penetrance of adult transformations of each other when they are transheterozygotes (Campbell and Brock, unpublished). In homozygous double mutant embryos for combinations of Pel, Scm, and Asx, the embryonic 38 transformations are enhanced and show strong posteriorly directed transformations resembling amorphic Pc (Jurgens, 1985). However some Pel (Duncan, 1982) and A s * (Brock, unpublished) have more restricted homeotic phenotypes. Asx also shows stronger head defects (Jurgens, 1985) and it, along with Pel, is implicated in regulation of the segmentation genes (Brock, unpublished; see chapter three; Ingham, 1984). Molecular evidence also argues both ways. Pc binds to the Scm and Asx locations on polytene chromosome (Zink and Paro, 1989), but there is no indication whether this leads to positive or negative regulation. Th is suggests that Scm and Asx are regulated by Pc and may be involved in subfunctions. Specifically Scm may have a stronger effect in the C N S , and Asx may have a stronger effect in the gut. Nevertheless, Pc does not bind to See or Pel. This suggests that Pc does not regulate Pel or See directly. The data do not rule out the possibility that some unidentified gene intervenes between Pc and See or Pel. It seems likely that Pel and See will affect processes at similar levels in the hierarchy of processes regulated by Pc as Asx or Scm, even if Pel and See act independently of Asx and Scm. In sxc and pho- embryos, only intensity differences were detected in U B X staining, without ectopic expression. Th is is very different from the effects seen with Scm, See, Pel, and Asx that affect ectopicness, but not intensity of U B X expression. Since homeotic transformations are only weak, or not observed in homozygous sxc- or pho- embryos (Ingham, 1984; Breen and Duncan, 1986), the weak upset of U B X may be insufficient to cause a cuticle phenotype. sxc and pho must affect Ubx regulation differently than the genes described above. Ei ther they upset abd-A and Abd-B regulation only in PS12-6, or they specifically regulate Ubx expression in PS6. Both sxc and pho have strong maternal effects, which may mask the extent of U B X upset. sxc and pho have other similarities, which are differentiate them from the rest of the 39 Pc-group. Both sxc' and pho' animals die as pharate adults (Ingham, 1984; Breen and Duncan, 1986), whereas for all the other Pc-group mutants die as embryos or first instar larva. Th is suggests that sxc and pho may have stronger effects on homeotic gene expression in the imaginal discs. Ingham (1985) showed that U B X is misexpressed in the wing disc and second leg disc and in the C N S in sxc- third instar larvae. It has yet to be seen if the same is true for pho, and this should be tested. ph is unique in the Pc-group as it differs in nearly every respect from the rest, ph is the only member of the group that completely suppresses the expression of U B X in the C N S . ph is involved in other processes besides regulation of the homeotic genes. Embryos lacking the ph product undergo extensive cell death in the ventral cuticle (Dura, et al., 1987) and have problems with axonal outgrowth (Smouse et al., 1988). The maternal effect seen with ph embryos is the strongest of all the group and ph- embryos from homozygous mothers die before the cuticle is laid down (Dura et al., 1988). The expression of U B X is also interesting since U B X is ectopic in the epidermis and then at a later stage completely suppressed in the C N S , showing that this gene has different effects in two different tissues, ph is likely to have a general function in development. Psc and pco- embryos also have abnormal morphology that appears separable from their homeotic phenotypes. Most striking is the generality and early onset of the morphological upsets and the early stages at which they affect U B X expression. While they do not seem to affect the early boundaries of U B X expression, they seem to affect the control of U B X as the expression is blotchy across the parasegments. It does not seem likely that these genes are related to ph because U B X expression is not suppressed, and in all three the general appearance of the embryos are different. Psc and pco have a more general affect on development than simply regulating homeotic gene expression. 40 Psc has a general function in development because it enhances the transvection phenotype of zeste (Wu, personal communication). However, genetic studies indicate that Psc strongly enhances the homeotic transformations observed in Asx, Pel, Scm (Jurgens, 1985), and Pc (Campbell and Brock, unpublished), and Pc binds to the Psc site on polytene chromosomes (Zink and Paro, 1989). Therefore, either Psc has a specific role in regulation of Ubx or it has a general role in gene regulation that is necessary for correct deployment of these genes. The latter may be true as Psc is related to Su(Z)2 (Adler, personal communication) which suppresses the zeste phenotype. This study was not able to examine the effects of maternal effects on these genes. However studying zygotic regulation of U B X expression has allowed me to show there are clear differences between members of the Pc-group. Pc and esc appear to be the genes at the head of a regulatory process or processes required for regulation of the homeotic genes. Scm, See, Pel, and Asx act later in this process, and are probably subordinate to Pc. sxc and pho appear to act differently than the genes described above. These data strongly suggest that ph, Psc, and pco have more general functions in development that impinge on the process regulated by Pc. 41 C H A P T E R 2 : I N T E R A C T I O N B E T W E E N ANTP A N D SCR A N D T H E P C - G R O U P G E N E S INTRODUCTION The A N T - C is a complex of genes required for proper development of the head and thorax. The Pc-group phenotypes resemble dominant gain of function mutations in the A N T -C complex, especially those of Antp and Scr, supporting the hypothesis that Pc-group genes interact with genes in the A N T - C . Th is suggestion is supported by the observation that like the B X - C , changing the dose of the A N T - C complex enhances or suppresses the phenotype of Pc-group mutations. Wedeen et al. (1986) have shown that Antp is completely derepressed in the C N S of Per embryos, and Smouse et al. (1988) showed that Antp is repressed in the C N S of ph- embryos. However, much less attention has been paid to the interaction of the Pc-group genes with the A N T - C compared to their interaction with the B X - C . Examining the distribution of A N T P and S C R in Pc-group mutants will show if the Pc-group regulates expression of the A N T - C . It will also show i f regulation of A N T P and S C R is similar in each Pc-group mutation. Final ly, examination of A N T P and S C R distribution should confirm the suggestions made in Chapter 1 about the specificity and t iming of Pc-group function. The extra sex comb phenotype of adults is thought to result from the ectopic expression of Scr i n meso and metathoracic segments (Struhl, 1982). Glicksman and Brower (1988) have shown that the distribution of S C R in imaginal discs confirms this hypothesis, since S C R is found in the meso and metathoracic leg disc as well as the prothoracic disc. However, the distribution of S C R in embryos has not been examined. Therefore, the studies in this chapter will show if Pc-group regulation of homeotic genes is the same in embryos and imaginal discs. 42 A s discussed above, for Abd-B, abd-A, and Ubx, the more posteriorly expressed genes regulate the expression of more anteriorly expressed genes (Struhl and White, 1985; Hafen et al., 1984; Harding et al., 1985; Carrol l et al, 1986; Riley et al, 1987). However, the nature of these cross-regulatory interactions has been less well-studied in the thorax, and appears to be more complex than in the abdomen (Riley et al, 1987). If mutations in Pc-group genes do cause ectopic expression of genes in the B X - C and A N T - C , it may be possible to examine the cross-regulatory interactions among the homeotic genes. 43 MATERIALS AND METHODS A l l stocks and methods are the same as described in chapter one. Embryos were stained with monoclonal antibodies against Scr which were generously supplied by Marcie Gl icksman (Glicksman and Brower, 1988), and Antp which was a gift of Matt Scott (Carroll et al., 1986a). The Scr antibody is derived from ascites fluid and used at a dilution of 1:100. The Antp antibody is from ascites fluid and used at a dilution of 1:1000. 44 RESULTS Antp p r o t e i n e x p r e s s i o n The wild-type distribution of Antp protein has been described by Wirz et al. (1986) and Carrol l et al. (1986a). A N T P is first detected at the extended germ band stage in ectoderm in the presumptive thoracic region in the anterior ventrolateral regions of the embryo from labial to first abdominal segment. Staining is predominantly nuclear. As the germ band shortens, the expression of A N T P persists in ventrolateral cells of PS4 and 5, but diminishes in PS6 (Figure 6A). The level of ectodermal staining decreases as the germ band shortens and only a small subset of cells are labelled by 16 hours. Expression of the protein is visible in the C N S early in neurogenesis at stage 11 (Figure 6A). Staining is visible in the ganglia from PS4 to PS13. A t this stage the levels of protein in thoracic and abdominal cells are comparable. By the end of stage 12, the thoracic levels of A N T P are higher than in the abdomen, and PS4 and PS5 label strongly (Figure 6E). Ear ly there appears to be a transient accumulation of A N T P into PS3 and PS2, which later retracts and Figure 8A shows that the anterior boundary of expression becomes PS4. Wirz et al. (1986) report the expression of A N T P into the head of wild-type embryos, but I do not detect it. A N T P expression in the visceral mesoderm is detectable late in stage 13 and probably corresponds to PS6 (Figure 7A). Th is labelling continues throughout gut development and marks the first midgut constriction, which is obvious at stage 16 (Figure 8A). In the Pc-group embryos the initial distribution of the Antp protein is indistinguishable from that of wild-type and there are no noticeable upsets in ectodermal staining. In esc- embryos from esc- mothers the pattern of A N T P expression diverges from wild-type at stage 11 (Figure 6B, compare to Figure 6A). There is ectopic A N T P expression 45 Figure 6. Antp expression in Pc-group embryos at germ extension and germ band retraction. Photographs of anti-Antp antibody staining in whole-mount wild-type (A, E) , esc (B, F) , Pc (C, G), ph- (D, H), Pel- (I), See- (J), Asx- (K), and Psc- embryos. (A - D) Germ band extended embryos. (E - L) Embryos undergoing germ band shortening. Lateral view with plane of focus on C N S . Anterior is to the left and dorsal is up. 46 47 Figure 7. Antp expression in stage 14 Pc-group embryos. Photographs of anti-Antp antibody staining in whole-mount wild-type (A), esc (B), Pc (C), Pel- (D), Scm- (E), See- (F), Asx- (G), sxc (H), ph- (I), Psc- (J), and pco- (K) embryos at stage 14. Lateral view with plane of focus on C N S . Anterior is to the left and dorsal is up. 48 Figure 8. Antp expression in stage 16 Pc-group embryos. Photographs of anti-Antp antibody staining in whole-mount wild-type (A), esc (B), Pc (C), Pel- (D), Scm- (E), Sec (F), Asx- (G), ph- (H), Psc (I), and pco- (K) embryos. Lateral view with plane of focus on C N S . Anterior is to the left and dorsal is up. 50 51 in PS2 and PS3 in the presumptive neurogenic region. With increasing age A N T P extends more anteriorly and by stage 12 has reached PS1. Also with increasing age the intensity of PS4 and PS5 quickly diminishes and by stage 13 there is low level ectopic expression throughout the C N S including the brain and PS 14 (Figure 7B). There is no detectable expression of A N T P in the visceral mesoderm (Figures 7B and 8B). In esc/+ embryos from esc mothers, there is mild ectopic expression of Antp protein in the C N S starting at stage 13 which never extends past PS2 (not shown). Again the gut is suppressed. In Pc3 embryos there is ectopic expression of A N T P in the C N S starting at late stage 11 (Figure 6C). Wi th increasing age A N T P is expressed ectopically more anteriorly (Figure 6G) and by stage 15 there is an even level of labelling extending from the brain to PS 14 (Figure 7C). A t stage 16 there seems to be a decrease in labelling posteriorly, and the intensity of staining in the brain becomes stronger (Figure 8C). There is no gut staining. In See; Scm-, and Pel- embryos there is ectopic expression of A N T P in the C N S . However, this .aberrant expression is first detected, at later stages than in Pc3 or esc embryos. In Pel- embryos ectopic expression is first detected at late stage 12, early stage 13 (Figure 61, compare to 6 F and 6G). By stage 14 there is strong expression into the brain (Figure 7D). The protein is suppressed in the midgut. See- embryos develop ectopic expression late in stage 12 to early stage 13 at which point it is just detectable (Figure 6J) compared with esc and Pc3 embryos which have strong ectopic expression at this time (Figure 6 F and 6G). Anterior labelling slowly builds so that at late stage 13 there is labelling into PS3 and by stage 15 there is weak spotty labelling into the brain (Figure 7E). Bra in labelling never reaches the level of Pc, esc, or Pel (compare Figure 8F to 8B, 8C and 8D). The midgut is normal. The pattern of A N T P in Scm- embryos is similar to the staining pattern of See- embryos, but mi ld ectopic expression is first detectable at stage 13. Figure 7 E 52 shows that by stage 15 A N T P staining weakly reaches the brain. The visceral mesoderm stains normally. In Asx- embryos ectopic expression of A N T P in PS3 is first detected early during early in stage 13. The expression never extends further anteriorly and persists unti l it is no longer possible to monitor expression (Figure 7 G and 8G). There is suppression of A N T P in the gut of Asx- embryos (Figure 7 G and 8G). There is a slight but detectable difference in sxc- embryos which is first seen at stage 11. There is a barely noticeable new expression of A N T P into PS3 and this persists unti l stage 15 (Figure 7H). However, this was not detectable in any of the embryos at stage 16. In pho- embryos there is no upset in pattern in the stages studied. Both ph503 and Df(l)JA52 embryos showed a similar pattern of A N T P labelling, different then that described for ph505 by Smouse et al. (1988). In ph- embryos there is ectopic expression starting at stage 11 in the presumptive neurogenic region (Figure 6D), and by stage 12 it has reached the brain (Figure 6H). Figure 8 H shows that by stage 16 A N T P is strongly expressed in the brain. There is some midgut staining but it is difficult to determine i f it is similar to the wild-type pattern, as the gut morphology is upset (Figure 8H). Psc- embryos show a variable phenotype and also variable expression of A N T P . Figure 6 L shows early ectodermal expression is blotchy but appears to obey the wild type boundary. The morphology of the embryos is abnormal and variable and the same is true for the A N T P staining. The nervous system is abnormal and holes are identified in the staining pattern. Sometimes there are not enough segments expressing the A N T P . Occasionally the staining respects the wild-type boundary, but at other times there is ectopic expression into what appears to be PS2 (Figure 7 K and Figure 81). Gut staining is variable, 53 and ranges from near wild-type to being completely suppressed. In pco- embryos, A N T P expression is blotchy and generally appears in al l tissues except for in the head region (Figure 7J). Figure 8 K shows that the morphology of the C N S is abnormal and A N T P staining extends into the brain. Scr p r o t e i n e x p r e s s i o n The localization of the Scr protein in wild-type embryos has been well documented (Mahaffey and Kaufman, 1987; Riley et al, 1987; Carroll et al, 1988; LeMotte et al, 1989), and will be briefly described here. S C R is expressed in the nucleus of cells of PS2 starting early in embryonic development (3 hours). The strength of the signal increases with time until a very strong signal is seen at germband retraction. Dur ing extension of the germ band staining continues to be seen in PS2 and labelling appears in PS3, but is restricted to lateral positions. A s development proceeds there is a continual shift of S C R expression from the parasegmental to the segmental register, happening earlier in the lateral positions than the ventral positions. Dur ing germ band retraction the labial and first thoracic segments are labelled. Initial visceral mesoderm staining and C N S staining also is also seen at this time, but become obvious at stage 13. In the C N S , expression is in a single neuromere, the suboesophageal ganglion. Therefore in the epidermis S C R is expressed in two segments, while in the C N S only one segment is labelled. S C R is expressed in the anteriormost midgut in two patches. LeMotte et al. (1989) report two patches of staining at the posteriormost midgut, as well, but these patches were not seen in this study. In esc- embryos there is low level ectopic expression of S C R in the epidermis which extends posteriorly in the embryo throughout the abdominal segments but only dorso-laterally (Figure 9A). The level of protein expression in this region appears to decrease with 54 Figure 9. Scr expression in late stage 12 Pc-group embryos. Photographs of anti-Scr antibody staining in whole-mount esc- (A), Pc- (B), and Asx- (C) embryos. (A) Dorsal view. Anterior is to the left. (B) Lateral view with plane of focus on epidermis. Anterior is to the left and dorsal is up. 55 56 Figure 10. Scr expression in stage 16 Pc-group embryos. Photographs of anti-Scr antibody staining in whole-mount wild-type (A), esc- (B), Pc- (C), PcV (D), Scm- (E), Asx- (F), ph- (G, H), and Psc- (I,J) embryos at stage 16. Ventral view with plane of focus on the C N S . Anterior is to the left. 57 58 age, and is not detectable in later staged embryos. However specific cells in the P N S do remain labelled in PS2 to PS7 (not shown). Figure 10B shows a stage 16 esc embryo stained for S C R , and compared to wild-type (Figure 10A) large differences in staining can be seen. The labial lobes stain but do not fuse. S C R is a good marker to show head involution problems. There is complete suppression of S C R in the C N S and visceral mesoderm. In the epidermis of Pc3 at stage 12, S C R staining extends posteriorly to PS4 and PS5 in a small patch located laterally (Figure 9B). This staining disappears with increasing age. Riley et al. (1987) looked at a nul l Pc allele {PcRl) and found the same ectopic expression into PS4 and PS5 but also observed staining laterally in the abdominal segments. A t stage 16 there is a large difference in S C R compare with wild-type (compare Figure IOC and 10A). Head involution appears to be fairly normal and the labial lobes fuse and migrate forward. Final ly, there is complete suppression of the S C R in the C N S and visceral mesoderm. See, Pel, and Asx all have less dramatic effects on S C R expression. In Seel embryos there is no ectopic expression of the protein, but the number or intensity of cells staining is decreased relative to wild-type (compare Figure 10E to 10A). The same phenotype is seen in Pel- embryos, but there is an intermediate level of staining between that of See- and wild-type (compare Figure 10D, 10E and 10A). In Asx- embryos there is mild ectopic expression of S C R into PS3 and 4 in the lateral region (like Pc3) at stage 12 (Figure 9C), which disappears with increasing age. The C N S staining is wild-type but labelling at stages 14 to 16 indicates abnormal head development. The head is rotated with in relation to the body. There is complete suppression of S C R in the visceral mesoderm (Figure 10F). In ph- embryos, both ph503 and Df(l)JA52, beginning at stage 12, low levels of protein label the maxillary segment (not shown). Later, head involution does not take place 59 and there are three patches of staining corresponding to the clypeolabrum, the maxillary, and labial regions, and the procephalic lobe stains weakly (Figures 10G and 10H). There is complete suppression of S C R in the nuclei of the C N S and visceral mesoderm. These results are similar to those obtained by D u r a and Ingham (1988) for a third nul l allele of ph (ph&05). In Df(l)JA52 embryos there is ectopic expression of S C R in the posterior end of the embryo and it appears to stain sensory organs (compare 10H and 10G). The defects in Psc embryos are variable. In some embryos S C R staining shows the occurrence of abnormal head involution (Figure 101). In embryos where head involution does take place, the staining in the C N S is weakly suppressed relative to wild-type (compare Figure 10J and 10A). Midgut staining is also variable and ranges from wild-type staining to complete suppression. In Scm-, sxc, and pho- embryos there is no change in S C R compared with wild-type. 60 DISCUSSION A s with U B X , there are differences in expression of A N T P and S C R between members of the Pc-group. These are summarized in Table 4 and Table 5. The Pc-group behaves similarly with respect to A N T P as U B X and this supports the same division of the Pc-group as in chapter one. Generally the first detection of ectopic A N T P expression in esc; Pc; Pel; See; Scm-, and Asx- embryos occurs earlier than that of U B X , but follows the same general pattern as U B X . In esc; Pc-, and ph- embryos the effects in the C N S are stronger than Pel-, See; Scm-, or Asx- judging by time of expression, extension into the brain and decrease in intensity of staining of PS4 and PS5 relative to the other parasegments. In the gut the suppression of A N T P is associated with the ectopic expression of U B X , except for ph-embryos where both are suppressed, sxc- and pho- embryos show little or no effects on A N T P expression. Psc and pco- embryos again show early, general problems of development and are unlikely to be members of the Pc-group. The ectopic expression of A N T P in ph&~03 and Df(l)JA52 was surprising, as a previous study reported the suppression of A N T P in the C N S in a third nul l allele of ph (ph505) (Smouse et al., 1988). This difference may reflect subfunctions of the ph gene, ph is a complex locus which requires two mutagenic events to obtain a nul l allele (Dura et al., 1988) and is known to be involved in different developmental processes (Dura et al., 1988; Smouse et al., 1988). Df(l)JA52 deletes most of the gene but part of proximal portion remains unaffected. It is possible that this region of the gene is affected in the ph505 allele, but not in ph503. Examination of A N T P in other ph mutants and the molecular characterization of the locus will tell more about ph function. Probably the most surprising result is the fact that S C R is not ectopically expressed in the C N S of the Pc-group and that it is completely suppressed in the C N S of esc, Pc3, and 61 T a b l e 4 . A n t p p r o t e i n e x p r e s s i o n i n P c - g r o u p e m b r y o s Nervous System Gut Defect Pc ectopic to brain esc ectopic to brain Pel ectopic to brain Scm ectopic in PS 2-4 weak into brain See Ectopic in PS 2-4 weak into brain Asx ectopic into PS3 sxc pho ph Psc pco mild ectopic into PS2 ectopic into brain abnormal morphology blotchy expression ectopic into PS2 abnormal morphology blotchy expression ectopic into brain Stage First Observed 11 11-12 12 late 13 early 13 13 12 11 10 10 suppressed suppressed suppressed suppressed variable; weakly suppressed 62 T a b l e 5 . Scr p r o t e i n d i s t r i b u t i o n i n P c - g r o u p e m b r y o s esc Pc Pel Scm See Asx sxc pho ph Psc pco Epidermis ectopic to PS 14 mild ectopic t o P S 4 mi ld ectopic t o P S 4 + blotchy; abnormal morphology blotchy; abnormal morphology Nervous System suppressed suppressed very weakly suppressed weakly suppressed suppressed blotchy blotchy Gut suppressed suppressed suppressed suppressed weakly suppressed 63 ph' embryos. It has been speculated that the extra sex comb phenotype found in adults is the result of misexpression of the Scr gene product into PS3 and 4 (Struhl, 1983; Duncan, 1982; Ingham, 1985). The results in this study indicate that Pc-group genes affect Scr expression differently in imaginal and embryonic tissue, or possibly, differently in epidermal versus nervous tissue. These results suggest that the regulation of Scr by the Pc-group genes is complex. This has been examined in esc embryos. In esc larva, from heterozygous mothers, S C R is expressed at increased levels or in new locations in more posterior leg discs (Glicksman and Brower, 1988). In Pc, esc-, and Asx- embryos there is low level ectopic expression posteriorly directed in the epidermis, but it usually limited to the lateral and dorsal positions. For these genes it is possible that the effect on S C R is restricted to the epidermal tissue. Another possibility is that the homeotic transformation of second and third thoracic segments does not require the ectopic expression of S C R at all, but may instead be due to altered expression of A N T P , or a combination of both. Certain alleles of Antp are also capable of causing transformations of the second and third thoracic legs to the first thoracic leg (Hazelrigg and Kaufman, 1983) and genetic data suggest Antp and Scr may interact in controlling at least adult structures (Hazelrigg and Kaufmann, 1983; Kaufmann and Abbott, 1984). Riley et al. (1987) looked at the expression of S C R in Mscl embryos. Mscl is an allele of Scr that causes transformations of the first thoracic to second thoracic segments, as well as transformations of the second and third thoracic segments to the first thoracic segment. They do not see ectopic expression of S C R in embryos and suggest that levels of A N T P may also be important. Experiments will have to be done to determine whether there are combinatorial interactions to direct the development of the prothoracic segment, or whether ectopic S C R expression simply does not occur in embryos and the adult homeotic 64 transformations are due to S C R being expressed ectopically in imaginal discs. That S C R is suppressed in the embryonic C N S of esc- embryos is also surprising because Gl icksman and Brower (1988) have shown that the larval C N S expression of S C R is largely or completely unchanged by esc mutations from heterozygous mothers. Taken together with the results presented here this indicates that the maternally supplied esc is sufficient to maintain the wild-type expression of Scr in the C N S throughout larval development. Th is supports the idea of Struhl and A k a m (1985) that esc may make the off-state of homeotic gene expression stable for the rest of development. Alternatively it may serve as a temporary mechanism for keeping the appropriate genes off unti l an independent and more permanent system of regulation, perhaps Pc, is set up. The experiments presented here allow examination of the effect of extended ectopic A N T P expression on S C R . It is surprising that there is no effect (as judged by Scm, Pel) because data to date has indicated that the more posteriorly acting homeotic genes negatively regulate the more anteriorly acting ones (Struhl and White, 1986; Hafen et al., 1984; Harding et al., 1985; Carrol l et al, 1986; Riley et al., 1987). However, the regulation of Scr by Antp is limited because the changes in S C R expression in the epidermis of Antp-embryos are subtle and Riley et al. (1987) suggest that other genes must be involved in restricting Scr expression in the thorax. Gibson and Gehring (1988) used an Antp gene under the regulation of a heat-shock promoter to indiscriminately express the Antp gene at germ band extension, and found no changes in the expression of S C R . They suggest that the failure to detect a change in S C R expression may reflect a requirement for continuous production of high levels of Antp protein, or could imply that other factors apart from Antp itself are required to repress Scr in regions where Scr is normally expressed. Since the experiments presented here permit extended ectopic expression of A N T P on Scr and yet 65 show no change in S C R expression, this supports the hypothesis that other factors are involved in Scr regulation. Ubx has been implicated in regulating Scr (Struhl, 1982). This study also indicates that the other factors involved in S C R suppression may be the abd-A and Abd-B genes. S C R is completely suppressed in the two Pc-group mutants where it is known that the abdominal genes are overexpressed in more anterior regions (Weeden et al, 1986; Struhl and White, 1985) and weak or no effects are seen in the rest of the group where the abdominal genes are probably not expressed (see chapter one). It can be predicted then that S C R will be ectopically expressed more posteriorly in Antp-BX-C-embryos than in Antp-Ubx- which will be stronger than in Antp- embryos. Alternatively, if there is a combinatorial effect, as suggested above, the levels of expression of a regulating gene may be important and not simply the on/off state of the genes involved. Another candidate for the regulation of Scr is the Dfd gene. D F D is normally expressed just anterior to S C R in the S I and S2 ganglia in the C N S (Mahaffey et al, 1989). In Pc- embryos, D F D is located in subpopulations of neurons within the brain, all three subesophageal ganglia, and in each ventral ganglion as posterior as PS 13a (Weeden et al, 1986). Therefore, it may be the ectopic expression of D F D which leads to suppression of S C R . It is less likely that Dfd is the contributing factor since in the epidermis of Dfd-embryos S C R expression remains wild-type, indicating that Dfd does not affect Scr (Riley et al, 1987). However, the C N S was not looked at, and the interactions may be different here. Final ly it is possible that esc, Pc, and ph may be acting directly on the Scr gene. This chapter supports the idea that the Pc-group is not functionally equivalent. It also sheds some light on the interactions involved between the homeotic genes, especially those acting in the the first thoracic segment, and indicates the regulation may be more complex than previously thought. It will be necessary to test these interactions by looking at 66 the distribution of Scr and Dfd gene products in different homeotic mutants or double and triple mutants. Understanding these interactions will make the analysis of Pc-group function easier to interpret. 67 C H A P T E R 3: I N T E R A C T I O N B E T W E E N EN A N D T H E P C - G R O U P G E N E S INTRODUCTION Previous experiments suggest that the Pc-group genes might have more general effects than the regulation of homeotic gene expression. Embryos produced from mosaic females with mutant germlines homozygous for Pel, pho, (Breen and Duncan, 1986), and sxc (Ingham, 1984) show segmentation defects. Asx adults have dominant segment defects confined to odd-numbered tergites, and homozygous Asx- embryos have even-numbered segment defects (Nicholls, Slade, Campbell , and Brock, unpublished). Final ly, increasing the dose of esc+ is lethal with one dose of eve+ (Slade and Brock, unpublished). Therefore, it seemed reasonable to examine the effect of Pc-group mutations on the distribution of en. en is sensitive to the earlier acting gap and pair-rule genes (Howard and Ingham, 1986; DiNardo and O'Farrel l , 1987; Ingham et al., 1988; Mart inez-Arias and White, 1988), and is therefore a good indicator of any segmentation upset. Many of the segmentation genes are also expressed in the developing nervous system (Baker, 1987; Knipple et al., 1985; Bopp et al., 1986; Tautz et al., 1987; MacDonald et al., 1986; Patel et al., 1989). Many show a different periodicity or pattern in the C N S than they do at the blastoderm stage and these differences may reflect differences in the regulation (Doe et al., 1988a). The segmentation genes also probably have different functions in the C N S (Doe et al, 1988a; 1988b). If Pc-group genes regulate segmentation gene expression, then different effects might be seen in the epidermis and the C N S . Th is chapter examines these possibilities. 68 MATERIAL AND METHODS A l l stocks and procedures are described in Chapter 1. The antibody used was raised against the inverted (inv) gene product which is homologous to the en, gene and the antibody cross-reacts with each protein. Both gene products have the same spatial distribution (Coleman et al., 1987) and I will refer to the staining as that of the en gene product. The function of inv is unknown and may be a pseudogene. The antibody was generously supplied by Cory Goodman. The ftz antibody was supplied by Henry Krause and is polyclonal antibody raised against the ftz protein in rabbits (Krause and Gehring, 1988). The secondary antibody used with this primary was peroxidase conjugated IgG (H+L) Goat anti-rabbit (Jackson Immunoresearch Laboratories). 69 RESULTS En Antibody staining The wild-type distribution of en protein has been described previously (DiNardo et al., 1985). The protein is expressed in the posterior compartments of all segments in the epidermis and in a subset of C N S neurons. By early germ band extension E N is equivalent in intensity in all segments and is expressed in 15 stripes of a single cell width with 3 nonlabelled cells intervening. These 15 stripes correspond to the three head, three thoracic and nine abdominal primordia (Figure 11A). A s germ band extension occurs the labelling increases in width from one to two to three cells. After the germ band has extended, E N begins to accumulate in discrete regions of the head and this expression is patchy, not in stripes. In stage 12-16 embryos, E N is expressed in about 18 C N S nuclei per hemisegment in a pattern that is claimed to be segmentally repeated (reported in Doe et al., 1988a). However, the pattern of E N expression has not been fully described. In my hands, more cells stain with en antibody in anterior segments than in posterior ones. Because individual cells were not followed through development, it is not possible to identify individual cells. The pattern seen in any given segment depends upon the plane of focus. Nevertheless, I describe here the pattern seen in PS6 of wild-type embryos for comparison with the mutant patterns, because differences in the pattern are readily identified. A s seen in Figure 13A, there is a cluster of 3-5 midline neuroblast (MNB) in each segment, that are flanked by 4-5 lateral neuroblasts (LNB) (Patel et al., 1989). Further lateral there are two prominent neuroblasts that lie on the edge bordering the epidermis, that I will call edge neuroblasts (ENB). Final ly , there are about 6 small cells that lie just anterior to the M N B , L N B , and E B cells that I will 70 refer to as anterior neuroblasts (ANB). In a lateral view, with the plane of focus being on the lumen of the proventriculus, every segment has 7 stained cells. The pattern varies from anterior to posterior, but their is always a clear gap between the stained cells, and the stained cells occupy more than half the dorsal/ventral extent of the C N S (see Figure 14A). For most of the Pc-group mutants there is no deviation of early E N expression relative to wild-type. In pco- embryos, the abnormal E N pattern is most likely a reflection of abnormal gastrulation patterns and lack of germ band extension. Figure 11B shows a pco-embryo at early stage 8 and the abnormal gastrulation pattern can be seen. There is lack of germ band extension and the invaginations form aberrantly. However E N spacing is normal and 15 stripes can be distinguished. The distribution of ftz protein in blastoderm stage embryos was examined and no difference was detectable between pco- and wild-type embryos. Later staged embryos show blotchy expression of E N (Figure 11H). Psc- embryos have upset E N pattern and this is seen as soon as the 15 stripes are distinguishable. The spacing is irregular and many of the stripes are broken or crooked (Figure 11C). Some embryos do not have the full complement of stripes. Because of this the distribution of F T Z in blastoderm stage embryos was examined. The pattern of ftz protein is dramatically upset, but variable, in Psc- embryos (compare Figures 12A, 12B and 12C). The first and seventh stripes are normal, but the internal stripes are irregular or missing. Stripes number 3-4 are affected most often, and then stripes 2 to 5. Later E N expression continues to be abnormal (Figure 111) and is blotchy in the C N S . In Asx- embryos the distribution of E N is upset in 9.5% of the embryos scored. This is lower than the expected frequency (25%) and probably reflects the low penetrance of this phenotype. The normal number of E N stripes are present but the spacing is irregular and Figure 11D shows that stripes 1 and 2, stripes 5 and 6, stripes 11 and 12, and stripes 13 and 71 Figure 11. en expression in Pc-group embryos at germ band extension and germ band retraction. Photographs of anti-e/i antibody staining in whole-mount wild-type (A, E , G), pco- (B, H), Psc- (C, I), Asx- (D), and ph- (F, J) embryos. Anterior is to the left and dorsal is up. (A - D) Lateral views of embryos at germ band extension with plane of focus through the middle of the embryo. (E, F) Lateral views of embryos at germ band extension with plane of focus on the epidermis. (G - J) Lateral views of embryos at germ band shortening with plane of focus on the epidermis. 72 73 Figure 12. ftz expression in Psc embryos at stage 5. Photographs of anti-ftz antibody staining in whole-mount wild-type (A) and Psc- embryos (B, C). (A, C) Lateral view with anterior to the left and dorsal up. (B) ventral view with anterior to the left. 74 75 Figure 13. en expression in stage 16 Pc-group embryos, ventral view. Photographs of anti-en antibody staining in whole-mount wild-type (A), esc- (B), Pc- (C), Pcl-(D), Scm- (E), See- (F), and ph- (G, H) stage 16 embryos. Ventral view with plane of focus on the C N S . Anterior is to the left. 76 77 Figure 14. en expression in stage 16 Pc-group embryos, lateral view. Photographs of anti-en antibody staining in whole-mount wild-type (A), esc- (B), Pc- (C), Pel-(D), Scm- (E), and See- (F) stage 16 embryos. Lateral view with plane of focus on the C N S . Anterior is to the left. 78 79 14 are closer to each other, resulting in larger gaps between other stripes. The pattern of E N expression in the C N S appears to be normal although there is the occasional embryo with abnormal spacing. In ph- embryos the initial distribution of E N is normal but starts to deviate at the beginning of stage 12. Figure 11F shows E N starts to be expressed more anteriorly within each parasegment relative to wild-type (Figure H E ) . By the end of stage 12 there is ectopic expression of E N in the ectoderm throughout most of the embryo, and specifically there is a second stripe of staining in the anterior of each segment (Figure 11J). The labial and maxillary segments are strongly stained. E N is suppressed in the C N S of ph- embryos. The pattern is completely suppressed in Df(l)JA52 embryos, but there are some cells which remain weakly labelled in ph503 embryos (compare Figure 13G to 13H). In Pc-, esc-, See-, Scm-, and Pel- embryos the epidermal pattern of E N expression is wild-type. Later, differences in E N staining in the C N S become apparent. These differences will be described for ventral and lateral views of the C N S of stage 16 embryos. In esc- embryos, the number of cells stained is about half that of wild-type. Figure 13B shows a ventral view of a stage 16 esc- embryo. On average, 3 M N B s , 3 L N B s , 1 E N B , and 3 A N B cells stain per hemisegment. The staining of the M N B cells is more intense relative to the other cells. The reduction in the number of A N B s makes the staining pattern seem more confined to the posterior region of each segment. In a lateral view, only six cells stain in each segment (Figure 14B). The C N S is thinner dorso-ventrally than in wild-type embryos (compare Figure 14B and 14A). Th is abnormal pattern is seen in PS 1-14. Fewer cells are detected with en antibody in each hemisegment of Pc- embryos. A s seen in Figure 13C, 2 M N B cells, 3 L N B cells, 2 E N B cells, and 3-4 A N B cells in the midline stain. The spacing between the M N B and L N B cells is greater and more irregular. In 80 lateral view, 9 cells stain in each segment (Figure 14C). Seven of the cells resemble the wild-type pattern, but in additon two more cells in the anterior part of the segment are also detected. Differences in E N staining in the C N S in other mutants are more subtle, but detectable. Figure 13F shows that in See- embryos, staining of the M N B and E N B cells appears normal. There are fewer L N B cells than in wild-type embryos. The main difference is that the staining intensity of the A N B cells is increased relative to the M N B and L N B cells. These cell lie more anterior and medial than in wild-type embryos. Laterally, 7 cells are detectable per segment, but their distribution is different because they are more clumped, occupy less area, and there are fewer cells ventrally (Figure 14F). These differences are seen in all 14 parasegments. The staining pattern of E N in Scm- embryos resembles that of See- embryos except that the midline A N B s do not appear to be so heavily stained, and there are fewer A N B cells laterally (compare Figure 13E and 13F). A s seen in Figure 13E the staining pattern seems to be more abnormal in segments 1-6 than in 7-14. The E N staining viewed laterally also resembles that found in See- embryos, but the differences in anterior versus posterior segments are more marked (compare Figures 14E and 14F). P S l - 6 look less like the wild-type pattern than do PS 1-14 (compare Figure 14E and 14A). Most Pel- embryos appeared to have normal E N staining in the C N S . Occasionally, abnormal staining was found. A s shown in Figure 13D, these embryos have fewer L N B cells staining, and there is a nearly complete absence of A N B staining. Laterally, the stained cells appear to be more clumped than in wild-type embryos (Figure 14D). In sxc- and pho-embryos, the pattern of engrailed staining was wild-type. 81 DISCUSSION The results presented here support the idea that some of the Pc-group have a wider function in that they are involved in regulation of the segmentation genes. The Asx results support this idea since the segmentation defects observed by Brock et al. (unpublished) are cuticular and early en expression regulates the segmentation of the epidermis. The penetrance of upset E N was low and agrees with the low penetrance (10%) of even-numbered segmentation defects seen in Asx- embryos (Slade and Brock, unpublished), and this may reflect a maternal requirement for the Asx gene product. However Breen and Duncan (1986) do not report segmentation defects in Asx- embryos derived from mothers with homozygous mutant germlines. ph also upset the epidermal pattern of E N , but segmentation defects were not detected in ph- embryos since ph- embryos die lacking ventral cuticle which has the markers needed to see segment defects. sxc-, pho-, and Pel- embryos showed no changes in E N expression. However it is likely that changes in E N expression will be observed in sxc-, pho-, or Pel- mutant embryos derived from a homozygous maternal germline because these embryos exhibit segmentation defects (Ingham, 1984; Breen and Duncan, 1986). Therefore it will be necessary to look at E N expression in embryos derived from sxc, pho-, and Pel- mosaic mothers to determine if these mutants upset en regulation. The epidermal expression of E N was not upset in esc-, Pc-, Sec, and Scm- embryos. This supports the idea that these genes have a specific function, namely the regulation of the homeotic genes. Alternatively, segmentation defects may only be seen i f the maternal contribution is eliminated. However, segmentation defects have not been reported in embryos derived from homozygous mutant mothers. The expression of E N is upset in Psc- and pco- embryos, but these embryos have 82 morphological defects as well. Therefore the effects seen on E N may be the result of the pleiotropic effects of the Psc and pco genes, pco was isolated in a screen for small disc phenotypes and therefore the wild-type product may be required for growth (Shearn, personal communication). It is possible that both the homeotic and segmentation phenotypes of pco are indirect effects of pco on growth. Th is is likely because morphological upsets in pco- embryos are observed before en protein is expressed, and the earlier acting pair-rule gene ftz is not affected. Psc also has been implicated in more general functions in development. Psc acts as an enhancer of transvection and may have a role in chromosomal pairing. Th is may explain the varied phenotypes of Psc. A s shown in this thesis Psc is involved in regulation of the homeotic and segmentation genes, but also has abnormal morphology. Adler et al. (personal communication) also describe ventral to dorsal transformations associated with the Psc phenotype. Recently, Smouse and Perrimon (personal communication) have shown that Psc is required for normal axonal outgrouwth. Therefore whatever the function of Psc may be, it interferes with many processes required in development. A n unexpected result was obtained in that the pattern of E N expression is altered in the C N S of esc-, Pc-, Scm-, See- and sometimes Pel- embryos. Th is is the first demonstration that many Pc-group genes may have different functions at different times in development. It is not possible to determine whether this is a direct or indirect effect of mutations in the Pc-group gene. It is entirely possible that en regulation is different in the C N S , and the differences seen in the Pc-group embryos may be a reflection of the upset in the homeotic genes. The expression of E N in the C N S of Scm- embryos is peculiar because the anterior segments seem to be more affected than the posterior segments. Therefore either Scm has a 83 regional effect on en expression, or disturbs the expression of another gene which has a regional effect on E N expression. Bustur ia and Morata (1988) noticed that P c - B X - O clones in the wing transgressed the anterior-posterior boundary, and therefore tested the expression of en in P c - B X - O cell clones in the wing and abdominal tissue of adult flies. They found that en is derepressed in imaginal P c - B X - O cell. Th is is only seen in clones in the thoracic region but not in the abdominal region. Th is suggests that Pc regulates en in imaginal cells. The derepression of en seen in the Pc- imaginal discs is in contrast to the mild effects seen in the Pc- C N S . However, as discussed in chapter 2, Scr regulation varies between imaginal discs and the C N S , so it is entirely possible that en is also under different regulation in these two tissues. 84 G E N E R A L D I S C U S S I O N The Pc-group genes have an important function in the regulation of early development but the exact nature of this function is unclear. M y results show that some members of the Pc-group function in regulating the segmentation genes as well as the homeotic genes. This is in agreement with the suggestion that different members of the Pc-group have a more general role in development (Dura et al., 1985). A more general role is also confirmed by the upset morphology and the distribution of Ubx, Antp, Scr, and en proteins in ph, Psc, and pco embryos. Although maternal effects were not looked at, my results also suggest that esc, Pc, Pel, Scm, See, Asx, sxc, and pho have specific, but different roles in regulating the genes of the A N T - C and B X - C . This may help to explain the differences in the mutant phenotypes observed in Pc-group embryos and adults, esc and Pc have the strongest embryonic phenotypes and also the strongest effect on the distribution of U B X , A N T P , and S C R . However, differences in temporal requirements and patterns of protein distributions also suggest they have independent roles. For Pel, Scm, See, and Asx both their mutant phenotype and affects on the distribution of U B X , A N T P , and S C R are less severe than P c or esc. sxc and pho have different temporal requirements than the rest of the group. Therefore, t iming and specificity make it unlikely that one multimeric complex is responsible for the Pc-group phenotype. It is more likely that there is at least one pathway in which Pc and esc are at the head, and that some Pc-group genes function in subpathways. It is also likely that some members of the Pc-group have more pleiotropic functions needed for proper expression of other genes in the Pc-group. Some of the differences in the expression of Ubx, Antp, and Scr in Pc-group embryos 85 suggest that some affect all the genes in the B X - C and A N T - C , and others do not. Therefore it is necessary that the distribution of abd-A, Abd-B, and Dfd gene products be examined in all members of the Pc-group. It will be necessary to determine the nature of the gene products of the Pc-group. Most of the Pc-group genes have been, or are in the process of being, cloned. It will be interesting to see if, like Pc, the other members of the Pc-group bind D N A . 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