Tissue- and stage-specific control of homeotic and segmentation gene expression in Drosophila embryos by the polyhomeotic gene

Development 103, 733-741 (1988) Printed in Great Britain The Company of Biologists Limited 1988 733 Tissue- and stage-specific control of homeotic and segmentation gene expression in Drosophila embryos by the polyhomeotic gene JEAN-MAURICE DURA 1 ' 2 and PHILIP INGHAM 1 1 ICRF, Developmental Biology Unit, Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK 2 Centre de Genetique Moleculaire/CNRS 91190 Gtfsur Yvette, France Summary The distributions of the products of the homeotic genes Sex combs reduced (Scr) and Ultrabithorax (Ubx) and of the segmentation genes, fushi tarazu (ftz), even skipped (eve) and engrailed (en) have been monitored in polyhomeotic (ph) mutant embryos. None of the genes monitored show abnormal expression at the blastoderm stage in the absence of zygotic ph expression. Both Scr and Ubx are ectopically expressed in the epidermis of ph embryos, confirming the earlier proposal, based on genetic analysis, that/?/i + acts as a negative regulator of Antennapedia (ANT-C) and bithorax (BX-C) complex genes. At the shortened germ band stage, en is also ectopically expressed, mainly in the anterior region of each segment. In contrast to these effects in the epidermis, the expression of en, Ubx, Scr and ftz is largely or completely suppressed in the central nervous system, whereas eve becomes ectopically expressed in most neurones. Key words: antibody staining, Drosophila, homeotic genes, spatial regulation, nervous system. Introduction The development of the Drosophila embryo depends upon the precise spatial and temporal regulation of expression of a number of genes controlling the specification of cell fate. Many of these genes are first transcribed in relatively simple patterns at the blastoderm stage, but subsequently exhibit increasingly complex expression patterns as embryogenesis proceeds (reviewed by Akam, 1987). One approach to analysing the mechanisms underlying this complex level of control is to isolate mutations that alter the regulatory parameters of particular genes. Such an approach has led to the identification of a large group of mutations, the common phenotype of which is suggestive of an alteration in the regulation of the bithorax and Antennapedia homeotic gene complexes (for a review see Ingham, 1985a). A subset of these genes has been called the Polycomb (Pc) group (Jurgens, 1985) after Pc, the first member of the group to be described (Lewis, 1978). Pc group genes act as negative regulators of homeotic genes (Lewis, 1978; Struhl, 1981; Duncan, 1982; Ingham, 1984; Dura et al. 1985, 1987; Jurgens, 1985). In at least two cases, this repressing activity has been shown to be exerted only after the initial patterns of homeotic gene expression have been established (Struhl & Akam, 1985; Wedeen et al. 1986). This maintenance function also depends upon genes within the ANT-C and BX-C themselves (Hafen et al. 1984a; Harding et al. 1985; Struhl & White, 1985). Thus the Pc group genes can be considered to mediate the interactions between homeotic genes which serve to elaborate the simple patterns of gene expression established at the blastoderm stage. polyhomeotic (ph) is a complex genetic locus (Dura et al. 1987), which, by most criteria, belongs to the Pc group of genes. Viable alleles of ph show various homeotic transformations of imaginal structures which are best understood as resulting from the inappropriate expression of genes within the ANT-C and BX-C (Dura et al. 1985). Similarly some embryonic lethal alleles display transformations of the ventral cuticle similar to those caused by other Pc group mutations (Dura et al. 1987). In contrast to the latter, however, the amorphic condition of ph results in massive cell death during germ-band shortening (Dura et al. 1987). Although this necrosis is widespread it is restricted to the ventral epidermis, the

734 J. -M. Dura and P. Ingham ventral nerve cord and dorsal epidermis being unaffected. In this study, we have tested directly the proposed role of ph + in regulating the expression of the BX-C and ANT-C by analysing the distribution of the Ubx and Scr proteins in ph embryos. In an effort to discover the basis of the pleiotropic effects of ph, we have also analysed the expression of the homeoboxcontaining segmentation genes ftz, eve and en in ph embryos. We find that ph + is required for the regulation of expression of all these genes in the developing germ band. Strikingly, the nature of this requirement differs between epidermal and neural cells, implying tissue-specific functions for the ph gene product. Materials and methods Fly strains and culture conditions For the analysis of wild-type patterns of gene expression Oregon R embryos were used. Mutant embryos bearing a null allele of poly ho meo tic ph were produced by the strain y ph 505 //FM7c (Dura et al. 1987). Flies and embryos were maintained at 25 C on standard culture medium. Antibody staining Embryos were stained with antibodies against ftz (H. Krause, personal communication), eve (Frasch etal. 1987), Scr (Glicksman & Brower, 1988), Ubx (White & Wilcox, 1984) and en (DiNardo et al. 1985). The staining procedure has been previously described (White & Lehmann, 1986 as modified by Ingham & Martinez-Arias, 1986). Mutant embryos were distinguished from wild type by coincidence between the expected frequency (0-25) and aberrant expression patterns of the genes studied. Results Homeobox gene expression at the blastoderm stage does not require zygotic ph expression We analysed the distribution of ftz and eve proteins and of Ubx transcripts in blastoderm embryos derived from heterozygous ph flies. In no case did we observe deviations from the normal wild-type patterns of expression (data not shown). Thus we infer that zygotic expression of ph + is not required for the establishment of the normal blastoderm prepattern of gene expression. The effect of simultaneous absence of zygotic and maternally derived ph product has not been tested. Regulation of homeotic gene expression by ph in the germ band Expression of the Sex combs reduced (Scr) gene was monitored with a monoclonal antibody raised against the Scr gene product generously supplied by Marcie Glicksman (Glicksman & Brower, 1988). In wildtype, Scr expression can first be detected in the early extended germ band before formation of the gnathal buds. At this stage, it is restricted to parasegment 2. Subsequently Scr protein is detectable in the posterior part of the maxillary, all the labial and the anterior part of the first thoracic segment (Mahaffey & Kaufman, 1987; Riley etal. 1987; see Fig. 1A). This pattern of labelling becomes more intense once germ band shortening is completed (Fig. 1C). As head involution takes place, cell nuclei from the part of the ventral nerve cord that corresponds to the labial and Tl segments are heavily stained by Scr antibody (Fig. IE). The pattern of Scr expression in ph embryos departs from the wild type at the beginning of germ band shortening. A low level of Scr protein can be detected in maxillary and sometimes in T2 segments (Fig. IB). In fully shortened ph embryos, staining appeared in the dorsal part of the clypeolabrum and of the procephalic lobe. Nearly all nuclei of the maxillary segment expressed Scr protein at the same level as those of the labial segment. The labial and Tl segments were labelled to a lower level than wild type. Later, labelling in Tl completely disappears. Labelling in the other thoracic and abdominal segments is not above background (Fig. ID). At the stage when head involution would normally take place during wild-type development, ph embryos exhibit three regions of heavily stained nuclei which correspond to the procephalic, the maxillary and the labial regions. In these embryos, nuclei from the ventral nerve cord completely lack any detectable Scr expression (Fig. IF). The pattern of Ubx protein distribution in wildtype embryos revealed with the monoclonal antibody FP 3.38 has previously been described (see White & Lehmann, 1986). Briefly, at the extended germ band stage, Ubx protein is expressed in embryonic cell nuclei from PS 5 to PS 13 although to a lower level in PS 5 and PS 13 than in the others (Fig. 2A). After germ band retraction has been completed, abdominal segments 1 to 7 show a high level of Ubx protein expression. Ubx protein is expressed to a much lower level or not at all in the posterior part of the dorsal region of these segments. Two one-cell-nucleus-wide bands are detected in the anterior and posterior parts of T3, along with a single one-nucleus-wide band in the posterior part of T2 (Fig. 2C). When head involution is completed cell nuclei from the ventral nerve cord become more heavily labelled than epidermal cell nuclei (Fig. 2E). In ph embryos, Ubx protein appears ectopically throughout PS 0 to PS 5 towards the end of the extended germ band stage. This expression is not homogeneous but rather is confined to specific nuclei within each parasegment, some of

Homeobox-gene expression in ph embryos 735 which show a high level of Ubx expression (Fig. 2B). The pattern of expression appears consistent between different embryos. After germ band retraction is completed, ph embryos show ectopic Ubx protein expression in all the head and thoracic segments (Fig. 2D). The levels of accumulation in the first abdominal segments are lower than in wild type but still show the characteristic anteroposterior gra- dation. At later stages, cell nuclei of the ventral nerve cord stain less intensely than those of epidermal cells, a situation which is the reverse of that of wild type (Fig. 2F). A rabbit antiserum directed against protein products of the engrailed locus was used to detect en expression (DiNardo etal. 1985). Wild-type embryos stained with this antiserum show a characteristic Dh 1A L T1 T2 B L T1 ' T2 M Fig. 1. Scr protein expression in 7- to 11-h-old ph + or ph embryos. A, C and E show wild-type embryos. B,D and F show ph embryos of similar ages. Except otherwise stated, embryos are anterior to the left and dorsal upwards. (A,B) Late extended germ band stage embryos. In ph + embryos, Scr protein expression is restricted to the posterior compartment of the maxillary, all the labial and the anterior part of the first thoracic segment. In ph embryos, staining appears in the anterior part of the maxillary segment and in the second thoracic segment. (C,D) Fully retracted germ band stage embryos. In ph + embryos, the pattern of labelling is similar but more intense to the one in panel A. In ph embryos, ectopic staining appears in the dorsal part of the clypeolabrum (arrowhead, here the staining is out of focus), in the procephalic lobe and in the maxillary segment. The labial segment is less stained than in wild type. Labelling in the thoracic and abdominal segments seems not above background. (E,F) Ventral view of embryos from early head involution stage. The ventral nervous system of Tl and the labial segment is clearly stained in wild type. No expression of Scr protein is detected in the central nervous system of ph embryos. Abbreviations: CNS, central nervous system; L, labial segment; P, procephalic lobe; M, maxillary segment; 77 and 72, first and second thoracic segments.

736 J.-M. Dura and P. Ingham ph O 5 \6 7 I d 8 \T1\ IN c NS Fig. 2. Ubx protein expression in 7- to ll-h-oldp/i + or ph embryos. A,C,E show wild-type embryos; B,D, and F show ph embryos of similar ages. (A,B) Extended germ band stage embryos. Ubx expression is low in parasegment 5 and high in parasegments 6, 7 and 8 in wild-type embryos (the other parasegments are located dorsally and therefore not visible on this photograph). In ph embryos, parasegments 6, 7 and 8 show wild-type expression but specific nuclei anterior to parasegment 6 present ectopical staining. Panels (C,D) Retracted germ gand stage embryos. In ph + embryos, the pattern of Ubx protein expression is restricted to: a single one-nucleus-wide stripe in the posterior compartment of T2, two one-nucleus-wide stripes in the anterior and posterior parts of T3 and all of abdominal segments 1-7. In ph embryos, ectopic expression of Ubx protein is detected in thoracic and head segments, along with a decrease of expression in the abdominal segments. (E,F) Early head involution stage embryos. At this stage, the central nervous system of ph embryos is less intensely stained than the epidermis, contrary to the situation in ph + embryos. labelling pattern. At the extended germ band stage, 15 stripes two to three nuclei wide are visible. These 15 stripes correspond to the three head (mandibular, maxillary and labial), three thoracic and nine abdominal primordia. After full germ band retraction, en expression is detected in the posterior part of each segment (Fig. 3A,D,F). In ph embryos, a novel pattern of en expression becomes apparent at the beginning of germ band retraction. Most of the nuclei in each segmental unit appear stained although the most intensive staining is found in posterior nuclei. At this stage, nuclei from the most anterior region of each segment did not seem to express en protein. In fully shortened ph embryos, almost all nuclei of the maxillary and labial segments were intensely stained (Fig. 3G). However, unlabelled patches were sometimes found in the middle of labelled regions. In the thoracic and abdominal segments of these embryos,

Homeobox-gene expression in ph embryos 737 two stripes of nuclei, one anterior and one posterior, were stained with the en antibody (Fig. 3C). The middle region of the segments was stained to a somewhat lower level but still above background. In some embryos staining is present in the ventral part of the head (Fig. 3B). As head involution begins in wildtype embryos, nuclei in the ventral nerve cord are clearly stained by en antiserum (Fig. 3D). In ph embryos from the same developmental stage, en expression was undetectable in the nerve cord (Fig. 3E). ph regulates pair-rule gene expression in the nervous system To determine whether the reduction or absence of en, Scr and Ubx expression in the ventral nerve cord reflects a general non-specific requirement forp/i + in this tissue, we monitored the expression of the pair- 3A J\ B M Fig. 3. en protein expression in 9- to 11-h-old ph + or ph embryos. (A,D,F) Wild-type embryos; (B,C,E,G) ph embryos of similar ages. Except where otherwise stated, embryos are anterior to the left and dorsal upwards. Retracted germ band stage embryos. In wild-type embryos, (A) en protein expression is restricted to the posterior compartment of each segment. In ph embryos, ectopic expression of en protein is detected in the anterior compartment of each segment: (B) en expression is present in the ventral part of the head as well as in the middle region of each segment. (C) Ectopic en expression is restricted to a one-nucleus-wide stripe in each anterior compartment. This embryo is dorsal downwards. D shows a ventral view of a ph + embryo when head involution begins. The central nervous system is clearly stained. In ph embryos (E), there is complete suppression of en expression in the CNS. F shows a lateral view of the head of a ph + embryo. The posterior compartments of the labial and the maxillary segment are stained by the en antibody. In ph embryos (G), both the anterior and posterior compartments express en protein. Abbreviations: CNS, central nervous system; L, labial segment; M, maxillary segment.

738 J.-M. Dura and P. Ingham 4A B D Fig. 4. Expression of //?z in the developing nervous system of a wild-type (A) and ph (B) shortened germ band stage embryo. At a similar stage eve is expressed in a smaller number of neurones in each segment (C). In the absence of ph +, eve becomes expressed throughout the ventral nerve cord (D). The organization of the nervous system is revealed by staining with the monoclonal antibody 22C10 (Zipursky et al. 1984). The segmentally organized array of longitudinal and commissural axon fascicles typical of a 12 h wild-type embryo (E) is clearly disrupted in a ph embryo at the same stage (F). rule genes ftz and eve, both of which show highly specific expression patterns in the nervous system (Carroll & Scott, 1986; Macdonald et al. 1986; Frasch et al. 1987). Neuronal expression of ftz protein is almost completely eliminated in ph embryos (compare Fig. 4A and B). In contrast, eve protein is expressed in virtually all the cells of the ventral nerve cord of ph embryos (compare Fig. 4C and D). These altered patterns of gene expression are accompanied by the aberrant organization of the ventral nerve cord (Fig. 4E and F). Discussion Previous analysis of the patterns of homeotic gene expression have identified two distinct stages in their regulation. The initial patterns require the functions of various segmentation genes (Ingham et al. 1986; Ingham & Martinez-Arias, 1986; White & Lehman, 1986; Harding & Levine, 1988) whereas their maintenance depends upon at least two members of the Pc group genes, namely esc (Struhl & Akam, 1985) and Pc (Wedeen et al. 1986), and on the homeotic genes themselves (Hafen etal. 1984a,b; Harding etal. 1985; Struhl & White, 1985). Although ph shares some of the characteristics of the Pc group genes, it differs in its unusual genetic and molecular organization and its role in the ventral epidermis development (Dura et al. 1987). The results of our analysis demonstrate that ph is involved not only in the maintenance of expression of the ANT-C and BX-C but also in the regulation of other homeobox-containing genes. Ectopic expression of Scr and Ubx proteins in the epidermis of ph embryos supports the role originally inferred for ph +, namely repression of the ANT-C and BX-C. The novel pattern of Scr protein distri-

Homeobox-gene expression in ph embryos 739 bution may indicate that the maxillary segment is homeotically transformed into a labial segment in ph embryos. Part of the procephalic lobe and of the clypeolabrum also show ectopic expression of Scr protein. The level of Scr labelling in the labial segment seems reduced compared to wild type. Adult males hemizygous for viable hypomorphic alleles of ph often bear extra sex combs on the second and third legs (Dura et al. 1985) which should correspond to an inappropriate expression of the Scr gene product, normally required only in the first legs (Wakimoto & Kaufman, 1981; Struhl, 1982). However, Scr protein is not expressed at a high level within the thoracic segments of ph embryos. One explanation could be that the lack of ph + product derepresses other homeotic genes in these segments (this is at least true for Ubx) which in turn will diminish Scr expression. In the adults, the lack of ph + product is not complete, since only hypomorphic alleles are viable. In this situation, the balance between ectopic Ubx and Scr expression may be such that the Scr-induced state is favoured and becomes stable. This effect is a common feature of the adult phenotypes of all the Pc group mutations. Curiously, there is a dorsal-ventral difference; in the dorsal discs, the cells follow a more posterior level of development, consistent with high expression of Ubx, not Scr. The ectopic pattern of Ubx protein expression within the thoracic segments of ph embryos is indeed in good accordance with the adult phenotype of hypomorphic ph alleles which show a partial transformation of wing to haltere (Dura et al. 1985). Not all cell nuclei respond equally to the lack of ph + zygotic product since reproducible heterogeneity in Ubx labelling was observed in ph embryos before complete germ band retraction. When the germ band is fully retracted, ph embryos exhibit a lower level of Ubx protein expression within abdominal segments than in wild type. This may indicate that other homeotic genes within BX-C, namely abda and AbdB, are also inappropriately expressed in these segments and thus decrease the expression of Ubx. The zygotic requirement for the ph + product is similar to the maternal requirement for esc + (Struhl & Akam, 1985). In both cases, the absence of product has no effect on the initial pattern of Ubx transcription. It seems likely that all the Pc group gene products are required only after the initiation of homeotic gene transcription is completed (see Ingham, 1985a), mediating the interactions that result in the complex patterns of expression observed in late embryonic stages. That this function is not limited to the regulation of the ANT-C and BX-C genes is shown by our analysis of the expression of other homeobox-containing genes. In ph embryos both the posterior and anterior part of each segment express en protein. This finding was unexpected since there is no evidence of a transformation of anterior to posterior structures in ph mutants which might ensue from such ectopic expression. However, we note that mutations of trx, a gene which formally appears equivalent to a positive regulator of the BX-C, cause an apparent en" transformation in the wing (Ingham, 19856). Heterogeneous Ubx expression in abdominal segments of the wild-type embryo is controlled by en gene product (Martinez- Arias & White, 1988). Interestingly such heterogeneity is still apparent in ph embryos despite the ectopic expression of en, implying that expression of en is not in itself sufficient to repress Ubx. At the shortened-germ-band stage, ph embryos exhibit high level ectopic expression of en, Ubx and Scr in the epidermal cells, yet the normal expression of all three genes in the central nervous system is virtually completely suppressed. Such a loss of expression would be expected to presage cell death. However, although there is considerable necrosis in the ventral epidermis shortly after this stage, no significant cell death has been observed in the ventral nerve cord of ph embryos (unpublished observations). Moreover we find that, whereas ftz expression is similarly suppressed at this stage, there is a massive overexpression of eve. In contrast to the highly specific pattern of eve expression typical of wild type (Macdonald et al. 1986; Frasch et al. 1987; Doe et al. 1988), virtually every neurone expresses eve in the nerve cord of ph embryos. Concomitant with these altered patterns of expression we observe highly abnormal patterns of fasciculation. The regulatory relationships between the homeotic and segmentation genes in the nervous system are at present poorly understood, but it is known that they differ from those which obtain at the blastoderm (Doe et al. 1988). Thus, for instance, whereas the pattern of eve expression in the blastoderm is independent of ftz function, absence of ftz activity from specific neurones results in the elimination of eve expression (Doe et al. 1988). The multiple effects of ph on gene expression in the nervous system may thus be in part indirect. For instance, it is possible that the ectopic expression of eve is itself responsible for the inhibition of expression of ftz, en, Ubx and Scr. Whatever the mechanism, it seems likely that the altered fasciculation seen in ph embryos is a consequence of these altered patterns of gene expression. The functional complexity of ph, apparent from its pleiotropic phenotype, has been confirmed here by our analysis of gene expression in mutant embryos. We find that ph exerts a stage- and tissue-specific control over the expression of a number of homeobox-containing genes. Such specificity of a trans-

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