Anterior Repression of Hunchback 1575

Development 127, 1573-1582 (2000) 1573 Printed in Great Britain © The Company of Biologists Limited 2000 DEV2518

Persistence of Hunchback in the terminal region of the Drosophila blastoderm embryo impairs anterior development

Florence Janody1, Joachim Reischl2 and Nathalie Dostatni1,* 1LGPD, IBDM, Parc Scientiﬁque de Luminy, Case 907, 13288 Marseille Cedex 09, France 2Lehrstuhl für Genetik, Universität Bayreuth, Universitätsstr. 30, 95447 Bayreuth, Germany *Author for correspondence (e-mail: [email protected])

Accepted 3 February; published on WWW 21 March 2000

SUMMARY

Anterior terminal development is controlled by several at early stages of a labral (cap’n’collar) and two foregut zygotic genes that are positively regulated at the anterior (wingless and hedgehog) determinants that are positively pole of Drosophila blastoderm embryos by the anterior controlled by bicoid and torso. These results uncovered an (bicoid) and the terminal (torso) maternal determinants. antagonism between hunchback and bicoid at the anterior Most Bicoid target genes, however, are ﬁrst expressed at pole, whereas the two genes are known to act in concert for syncitial blastoderm as anterior caps, which retract from most anterior segmented development. They suggest that the anterior pole upon activation of Torso. To better the repression of hunchback by torso is required to prevent understand the interaction between Bicoid and Torso, a this antagonism and to promote anterior terminal derivative of the Gal4/UAS system was used to selectively development, depending mostly on bicoid activity. express the best characterised Bicoid target gene, hunchback, at the anterior pole when its expression should be repressed by Torso. Persistence of hunchback at the pole Key words: Torso signal transduction cascade, Bicoid morphogen, mimics most of the torso phenotype and leads to repression hunchback, Drosophila

INTRODUCTION (Strecker et al., 1986) whereas hkb mutant embryos develop all patterned elements of the head (Weigel et al., 1990). Since head Determination of the antero-posterior axis of the Drosophila defects of embryos derived from torso females are more extensive embryo requires the activity of the anterior, the posterior and the than those of tll mutants, the existence of at least one additional terminal systems (St Johnston and Nüsslein-Volhard, 1992). The zygotic gene mediating terminal information at the anterior has activity of the morphogen Bicoid (Bcd) gradient determines been postulated (Cohen and Jürgens, 1991; Sprenger and anterior development by controlling target gene expression in Nüsslein-Volhard, 1993). Several zygotic mutants lack labral distinct anterior domains, while the activation of the Torso structures that are formed in hkb, tll or double mutant embryos receptor tyrosine kinase (RTK) specifies patterns at the terminalia (Sprenger and Nüsslein-Volhard, 1993). Among them, of the embryo: the acron at the anterior and the telson at the cap’n’collar (cnc) is expressed at blastoderm stage in the labral posterior. The acron is defined as the cuticular region, which is territory under the positive control of torso and bicoid (Mohler lacking in the maternal terminal group mutants (Schupbach and et al., 1995). This control does not involve any of the zygotic Wieschaus, 1986a); it includes the labrum, parts of the brain and genes tested so far (Mohler, 1993), suggesting that cnc could the optic lobes, and large portions of the cephalopharyngeal directly mediate part of the terminal system activity at the skeleton encompassing most of the non-segmental acron (Jürgens anterior. et al., 1986). These structures arise from domains of the embryo In addition to participating in head structure formation, the that lie anteriormost before head involution occurs (Jürgens et al., terminal system plays a key role in the formation of the gut 1986). Terminal activity is mediated by activation of the Torso (Schupbach and Wieschaus, 1986b). The anlagen of this RTK at both ends of the embryo, by a ligand locally activated by complex structure are localised at the polar regions of the the polar follicle cells. This process leads to activation of the ras blastoderm embryo (for a review see Skaer, 1993). About 3 signal transduction cascade (Perrimon and Desplan, 1994), which hours after egg deposition, cells at the anterior/ventral region allows zygotic derepression of tailless (tll) and huckebein (hkb), invaginate to form the stomodeum that gives rise to the foregut, possibly by inactivation the Groucho maternal corepressor which will differentiate in the pharynx, oesophagus and part of (Paroush et al., 1997). In the posterior region, hkb and tll are the proventriculus. The stomatogastric nervous system (SNS) sufficient to mediate all the maternal terminal function (Weigel originates from the anlage of the foregut and differentiates a et al., 1990). At the anterior, tll is required for the formation of network of peripheral ganglia (Hartenstein, 1997). At the most of the brain and parts of the cephalopharyngeal skeleton posterior pole the combined activity of tll and hkb is sufficient 1574 F. Janody, J. Reischl and N. Dostatni to relay the terminal information in inducing hindgut and anterior pole although double mutant analysis, with hb zygotic posterior midgut formation (Casanova, 1990; Strecker et al., mutant embryos derived from torso mutant females, indicates 1986; Weigel et al., 1990). At the anterior, formation of the that it is not sufficient. Our analysis uncovers an antagonism foregut might be dependent on hkb and tll function (Reuter and between hb and bcd at the tip of the embryo that is critical for Leptin, 1994; Strecker et al., 1986), but it probably also terminal development. It suggests that a major role of torso requires a positive input from the terminal system independent at the anterior might be to prevent anterior segmental of tll and hkb (Weigel et al., 1990). The two segment polarity development, through repression of hb, at the expense of genes, wingless (wg) and hedgehog (hh), are required for anterior terminal development, which relies mostly on Bcd foregut formation (Pankratz and Hoch, 1995) and their specific activity. expression in foregut territories at blastoderm stages is probably important (Mohler et al., 1995). As observed for early MATERIALS AND METHODS anterior expression of cnc, anteriormost expression of wg and hh in the blastoderm is under the positive control of torso and Plasmids bcd (Mohler, 1995), suggesting that these two genes might also The transcription unit pnos-Gal4-GCN4-Bcd3′UTR was constructed be critical mediators of terminal activity at the anterior pole. by successive cloning steps, inserted into the KpnI and NotI restriction In the absence of bcd the acron, developing at the anterior, sites of the plasmid pCasPer4, which was then used to transform is replaced by a telson developing normally at the posterior. Drosophila germlines. Briefly the pnos sequence consists of an approximately 1000 bp fragment, the nanos promoter and the nanos This indicates that while the terminal system is sufficient to ′ establish a telson, the combined activities of both terminal and 5 untranslated region (UTR) (Gavis and Lehmann, 1992). The sequence coding for the first 94 amino acids of the Gal4 DNA binding anterior determinants are required to establish the acron protein was obtained from pCG-Gal4(1-94) (Tanaka et al., 1994). It (Frohnhöfer and Nüsslein-Volhard, 1986) and suggests an contains an XbaI restriction site at its 3′ end and was cloned such that interaction between the anterior and the terminal systems. the Gal4 ATG replaces the Nanos ATG at an NdeI restriction site. The Transcription of Bcd target genes such as hunchback (hb) and sequence coding for three copies of the GCN4 activation domain orthodenticle (otd) is first activated, during syncitial (Ronchi et al., 1993) was amplified by PCR, cloned in-frame blastoderm, as an anterior cap, and subsequently repressed, at downstream of Gal4 (1-94) sequence in the XbaI restriction site and, the anterior pole, during cellularisation in response to Torso using compatible BamHI and BglII restriction sites, downstream. This activity (Driever and Nüsslein-Volhard, 1989; Finkelstein and sequence was followed by the EcoRV-XbaI restriction fragment ′ Perrimon, 1990; Ronchi et al., 1993). Bcd transcriptional containing the Bcd 3 UTR (Berleth et al., 1988). activity itself is directly repressed at the pole by activation of Drosophila stocks and transgenic lines the Torso pathway leading to the anterior repression of Bcd Mutant alleles used were torPM (Klingler et al., 1988), hkbAtllG targets (Bellaïche et al., 1996). The inhibition of Bcd activity (Ronchi et al., 1993), hb14F21 (Lehmann and Nüsslein-Volhard, 1987) by Torso requires the maternal terminal genes D-Raf and D- and bcdE1 (Frohnhöfer and Nüsslein-Volhard, 1986). All transgenic sor (MEK), and is independent of tll and hkb (Ronchi et al., stocks contained four copies of the corresponding P-element insertion. 1993; Janody et al., 2000), indicating that this process is an Gal4-GCN4 transgenic females were crossed with y−w− males alternative output of the terminal system at the anterior. The carrying, or not, the appropriate responder transgenes. Embryos were Bcd protein is phosphorylated on MAP kinase sites in the collected at 25¡C. Injections for transgenic lines were performed as embryo (Janody et al., 2000) and some of these described by Bellaïche et al. (1996). phosphorylations are dependent on torso (Ronchi et al., 1993). Whole-mount in situ hybridisation and However, site-directed mutagenesis associated with immunocytochemistry transgenesis indicates that phosphorylation of Bcd is not Digoxigenin-labelled RNA probes and in situ hybridisation were done involved in the downregulation of its activity by Torso (Janody as in Bellaïche et al. (1996) from pKS derivatives containing the et al., 2000). coding sequences of the different genes studied. The anti-digoxigenin To understand the role of Torso-induced repression of Bcd antibody coupled to alkaline phosphatase (Boehringer) was pre- targets, hunchback (hb) and orthodenticle (otd) were expressed absorbed and used at 1/2000 dilution. Immunocytochemistry was at the anterior pole at the time when they are normally performed as in Macdonald and Struhl (1986) using the anti-Otd repressed by Torso (during cellularisation). This was antibody (Wieschaus et al., 1992) at 1/2000 dilution and the anti-Hb performed using a derivative of the Gal4/UAS system with a antibody (Kosman et al., 1998) at 1/1000 dilution. Embryos were driver allowing the expression of a Gal4-GCN4 fusion protein mounted in 80% glycerol and photographed using Nomarski optics. as a maternal anterior gradient. Our results show that Feeding assay and larval gut dissection expression of otd, using this system, induces defects in For the feeding assay, larvae were allowed to grow on grape agar structures that originate from the posterior region of the head plates and yeast dyed with Neutral Red (Fluka). 4 hours feeding was and that are unlikely to be due to the persistence of otd at the allowed for visualising gut motility. The larval guts were dissected in pole. In contrast, the Gal4-GCN4 driver used with a UAS-hb Ringer’s medium and immediately mounted in 80% glycerol. transgene prevents the development of most labral structures, foregut derivatives and the SNS. It leads to the absence of expression of wg oesophageal, hh foregut and cnc labral RESULTS domains without disturbing early expression of hkb and tll. These experiments show that the persistence of hb during Targeted expression of hb and otd at the anterior cellularisation impairs terminal development. They strongly pole during cellularisation argue that Torso-induced anterior repression of hb is necessary To analyse the role of Torso-induced anterior repression of Bcd to mediate a large part of the terminal system activity at the targets, hb and otd expression was maintained at the anterior Anterior repression of hunchback 1575

Table 1. Lethalities induced by hb and otd expression under the control of the Gal4-GCN4 driver

Number of Number of Number of NomenclatureMaternal genotype Paternal genotype dead embryos dead larvae adults wt*Gal4-GCN4 wt (y −− w ) 61084 Hb+ Gal4-GCN4 UAS-hb 80 20 0 Otd+ Gal4-GCN4 UAS-otd 60 32 8 bcd−/+ : wt*Gal4-GCN4 : bcd−/+ wt (y −− w ) 51283 bcd−/+ : Hb+ Gal4-GCN4 : bcd−/+ UAS-hb 99 1 0 Designation of embryos with specific maternal and paternal genotypes are indicated. 100 embryos were scored for lethality in each case. pole of the blastoderm embryo when it is normally repressed by apparent. A less severe phenotype was observed in 16% of Hb+ Torso. For this purpose, a derivative of the Gal4 system (Brand embryos, which showed mostly truncations of a part of the dorsal and Perrimon, 1993) was developed. The driver consisted of a bridge, distortions of the pharyngeal wall and defects of the fusion protein (Gal4-GCN4) between the Gal4 DNA binding labrum derivatives, which were either absent, just truncated or domain and three copies of the yeast GCN4 activation domain, displaced (Fig. 2D). Most remaining Hb+ embryos (52%) which was expressed as a maternal anterior gradient under the exhibited head involution defects that might be secondary. Thus, control of the nanos promoter and the Bcd 3′UTR localisation almost half of the Hb+ embryos exhibited defects in acronal signal (Macdonald and Struhl, 1988). As shown in Fig. 1A, a structures (including the labrum), which are controlled by the UAS-lacZ reporter transgene was efficiently expressed by the terminal system. maternal Gal4-GCN4 driver as an anterior cap during the 48% of the Otd+ embryos exhibited a weak distortion of the process of cellularisation and it was not repressed by Torso at pharyngeal wall and defects in mouth hooks, mostly lacking the pole. When the Gal4-GCN4 driver was used to activate the internal part (Fig. 2E), whereas most remaining embryos UAS-hb and UAS-otd target transgenes this allowed the (51%) exhibited head involution defects, as observed with Hb+ persistence of either hb (compare Fig. 1F with H) or otd embryos. Thus, except for the weak phenotype of the (compare Fig. 1G with K) expression during cellularisation. pharyngeal wall, which might be a labral derivative (Mohler et The persistence of hb expression at the pole did not affect otd al., 1995), acronal derivatives, including the labrum, appeared endogenous expression (Fig. 1I) and, conversely, the persistence normal in Otd+ embryos and most defects involved structures of otd expression at the pole did not affect hb endogenous that originate from the maxillary segment located in the expression (Fig. 1J). In addition, both Hb and Otd proteins posterior part of the head. were expressed at physiological levels at the anterior pole of mid-cellularising embryos (compare Fig. 1B,C with D,E, Expression of a labral determinant, cap’n’collar, is respectively). The Gal4-GCN4/UAS-hb system only appeared altered in Hb+ embryos to overcome repression of hb at the pole (compare Fig. 1F with To characterise these head phenotypes, the expression pattern of H), indicating that the use of the Gal4-GCN4 driver might be genes involved in head development was analysed in Hb+ and Otd+ suitable to analyse the role of Torso-induced anterior repression embryos. Expression of the two target genes of the Torso pathway, of hb. In contrast, the Gal4-GCN4/UAS-otd system not only tll and hkb, was not affected in these embryos (data not shown). overcame otd anterior repression at the pole but also overcame The expression of cap’n’collar (cnc), which is involved in the ventral repression of otd and induced a change in the posterior formation of most labral structures (Mohler et al., 1995), is border of otd expression, which became more diffuse (compare normally activated at the beginning of cellularisation as an anterior Fig. 1G with K). Thus, the use of the Gal4-GCN4 driver with cap and a more posterior stripe (Mohler et al., 1991; Fig. 3A), and the UAS-otd transgene might induce phenotypes that will not be this expression is maintained throughout embryonic development caused by the lack of anterior repression of this gene. (Mohler et al., 1991). cnc anterior cap of expression never appeared in embryos from torso females (Fig. 3B) but cnc normally Labrum formation does not occur in Hb+ embryos expressed in the tll−/hkb− double mutant embryo (Fig. 3C), To understand the developmental consequences of hb and otd indicating that it is dependent on Torso activity (Mohler, 1993) but repression at the pole, we analysed the lethality and defects of that the two target genes of the Torso pathway are dispensable for embryos carrying either the UAS-hb or the UAS-otd transgenes its expression. The early anterior cap of cnc expression was normal and derived from mothers carrying the Gal4-GCN4 driver. For in Hb+ and Otd+ embryos (not shown), but its maintenance during simplification, these embryos are hereafter named Hb+ and Otd+. germ band extension was not observed in Hb+ embryos although As shown in Table 1, the maternal Gal4-GCN4 transgene by itself it was normal in Otd+ embryos (compare Fig. 3D with E,F). Since (wt*) exhibited very low lethality. In contrast, Hb+ and Otd+ cnc is involved in the development of labral structures (Mohler et embryos exhibited 80% and 60% embryonic lethality, al., 1995), the absence of the labral expression domain of cnc at respectively (Table 1). 30% of the Hb+ embryos exhibited a stage 10 in Hb+ embryos provides a molecular explanation for part strong head phenotype mimicking the torso loss-of-function of the head defects observed with Hb+ embryos. However, since phenotype (compare Fig. 2B and C): the median tooth and the hb expression is modified much earlier in Hb+ embryos, it is not epistomal sclerite were absent, the pharyngeal arms of the mouth clear whether the lack of maintenance of the cnc labral expression skeleton, including the dorsal bridge, the dorsal arm and the domain at later stages is a direct or an indirect consequence of hb ventral arm, were extremely reduced and the oesophagus was not persistence in these embryos. 1576 F. Janody, J. Reischl and N. Dostatni

Fig. 1. The Gal4-GCN4 driver. In situ hybridisation with lacZ (A), hb (F,H,J) and otd (G,I,K) probes and immunocytochemistry with anti-Hb (B,D) and anti-Otd (C,E) antibodies on wild-type embryos (B,C,F,G) and embryos from females carrying the Gal4-GCN4 driver crossed with males carrying the UAS-lacZ (A), UAS-hb (D,H,I) and UAS-otd (E,J,K) transgenes. Early expression of the cnc anterior cap is under the positive food motility in the gut of escaping larvae. The red coloured yeast control of bcd and torso, and independent of known zygotic gap fed to wild-type larvae stained their gut throughout its length and genes (Mohler, 1993). Since the lethality of Hb+ embryos was rapidly accumulated in the midgut (Fig. 4A). In contrast, all Hb+ increased when the maternal copy number of bcd was reduced to and most Otd+ larvae had feeding problems since the food never one (Table 1), the expression of cnc was also analysed at reached the midgut and accumulated in the foregut (Fig. 4B-C). blastoderm in wild type (wt), Hb+ and Otd+ embryos derived from bcd−/+ females. The reduction of bcd copy number from two to one did not signiﬁcantly modify cnc early expression in otherwise wild-type embryos: the anterior cap and the posterior stripe were still clearly present and they were only slightly reduced and shifted towards the anterior (compare Fig. 3A and G). In contrast, the anterior cap of cnc expression was drastically reduced in Hb+ embryos derived from bcd−/+ females (Fig. 3H) when compared to its expression in Otd+ (Fig. 3I) or wild-type (Fig. 3G) embryos derived from bcd−/+ females. These observations suggest that early expression of cnc is activated by Bcd and repressed by Hb when the latter is expressed at the pole during cellularisation. In wild- type and Hb+ embryos, the posterior stripe of cnc expression remained unchanged both during germ band extension (compare Fig. 3D and E) and at blastoderm stage (not shown). In contrast, it was often reduced in Otd+ embryos during germ band extension (Fig. 3F) and at blastoderm stage (not shown) and it was absent in Otd+ embryos derived from bcd−/+ females (Fig. 3I). This stripe of cnc expression prevents maxillary development in favour of mandibular development (Mohler et al., 1995) and its reduction in Otd+ embryos is probably related to the mouth hooks defects observed.

Gut formation is perturbed in Hb+ and Otd+ embryos Fig. 2. Head phenotypes induced by hb and otd expression under the control of the + Gal4-GCN4 driver. Nomarski photographs of head structures at the end of The few larvae emerging from Hb embryos never embryogenesis of wild-type embryos (A), embryos from torso females (B), embryos + became adults and only a few Otd embryos from females carrying the Gal4-GCN4 driver crossed with males carrying the UAS-hb survived larval stages to become adults (Table 1). (Hb+; C,D) or the UAS-otd (Otd+; E) transgenes. Head structures are indicated in A: To understand the lethality at larval stages, a feeding mouth hook (mh), median tooth (mt), epistomal sclerite (es), dorsal bridge (db), assay was used that allowed the visualisation of dorsal arm (da), posterior pharyngeal wall (ppw), eosophagus (eo), ventral plate (vp). Anterior repression of hunchback 1577

Fig. 3. Labral expression of cnc is modiﬁed in Hb+ embryos. In situ hybridisation with a cnc probe. Blastoderm embryos were wild type (A), from torso females (tor−; B), double mutant tll−/hkb− (C) or from bcd−/+ females carrying the Gal4-GCN4 driver, crossed with y−w− (bcd−/+: wt*; G), with UAS-hb (bcd−/+: Hb+; H) or with UAS-otd (bcd−/+: Otd+; I) males. Stage-10 embryos were from females carrying the Gal4- GCN4 driver crossed with y−w− (wt*; D), with UAS-hb (Hb+; E) or with UAS-otd (Otd+; F) males. tll−/hkb− double mutant embryos were identiﬁed using a simultaneous staining with a fushi tarazu (ftz) probe (C). Arrowheads indicate missing or reduced labelling (B,E,F,H,I). Lr, the labral expression domain of cnc (D).

The morphology of dissected foreguts of Hb+ larvae indicated reached the proventriculus, which exhibited an abnormal accumulation of the coloured yeast in the pharynx and the morphology. In most Otd+ larvae, the coloured yeast was oesophagus, which was drastically shortened (compare Fig. engorged at the caudal end of the oesophagus and associated with 4D,E); the gastric caeca were slightly altered and the food never defects of the proventriculus and the gastric caeca (Fig. 4F).

Fig. 4. Larval phenotypes induced by hb and otd expression under the control of the Gal4-GCN4 driver. Larvae were fed with Neutral Red-stained yeast and photographed alive (A-C) or used for foregut dissections (D-F). Larvae were from females carrying the Gal4- GCN4 driver crossed with y− w− (wt*; A,D), with UAS-hb (Hb+; B,E) or with UAS-otd (Otd+; C,F) males. Arrowheads indicate the accumulated food in the foregut (B,C), delineate the eosophagus (D,E) and indicate the abnormal gastric caeca (F). The eosophagus (eo), the proventriculus (pv) and the gastric caeca (gc) are indicated D. 1578 F. Janody, J. Reischl and N. Dostatni

These phenotypes, for both Hb+ and Otd+ larvae, are reminiscent These observations indicate that formation of the SNS is of the ‘cardiac arrest’ phenotype observed with alleles of the altered in Hb+ and Otd+ embryos. The absence of SNS wingless (wg), hedgehog (hh) and defective-proventriculus (dve) development in Hb+ embryos is a likely consequence of the genes (Fuss and Hoch, 1998; Nakagoshi et al., 1998; Pankratz absence of eosophageal wg expression at earlier stages and Hoch, 1995). (Gonzalez-Gaitan and Jäckle, 1995). Finally, except for the SNS precursors, fkh expression was normal in the head of Hb+ Early anterior expression of wg and hh is altered in and Otd+ embryos at this stage (compare Fig. 6D with E,F). Hb+ embryos Since the gut phenotypes observed in Hb+ and Otd+ larvae Repression of hb is not sufficient to mediate Torso- were very similar to the wg and hh gut phenotypes (Pankratz induced expression of cnc, wg and hh and Hoch, 1995), expression of these genes was analysed. wg Our results indicate that early anterior expression of a labral expression in wild-type embryos initiates during cellularisation determinant, cnc, and of two foregut determinants, wg and hh, with three expression domains: the oesophageal domain (ES), is repressed when zygotic expression of hb is allowed to persist which appears as a spot at 95-100% egg length (EL), the at the anterior pole of the Drosophila blastoderm embryo. cephalic domain, which is located dorsally (75-85% EL), and Expression of cnc, wg and hh is under the positive regulation a posterior stripe (Mohler, 1995; Fig. 5A). The wg esophageal of bcd and torso but no zygotic gene has yet been implicated (ES) domain is absent in embryos from torso females (Mohler, in this control (Mohler, 1993, 1995). This suggests that the Hb 1995; Fig. 5D). It was usually absent or poorly expressed in Hb+ embryos (Fig. 5B) and appeared normal in tll−/hkb− double mutant (Fig. 5E) and Otd+ (Fig. 5C) embryos. Similar results were observed for early zygotic expression of hh, which is initiated at late blastoderm stage, in two broad anterior domains: the foregut (FG) and the cephalic domains (Mohler, 1995; Fig. 5F). The FG domain of hh expression was not initiated in Hb+ embryos (Fig. 5G) nor in embryos from torso females (Fig. 5I; Mohler, 1995), while it was normally expressed in Otd+ embryos (Fig. 5H) and in tll−/hkb− double mutant embryos (Fig. 5J). By the end of stage 9, wg expression in the ES domain and hh expression in the FG domain are maintained by the fork head (fkh) gene expression (Mohler et al., 1995) and they follow the formation of the stomodeum. In Hb+ embryos and in embryos from torso females, expression of wg ES domain and hh FG domain was not reinitiated nor was it maintained (not shown). In contrast, both genes were normally expressed at this stage in tll−/hkb− double mutant and Otd+ embryos. These observations indicate that early anterior expression of wg and hh is dependent on the activation of the Torso cascade at the pole but does not require tll and hkb. They also indicate that the absence of hb repression prevents initiation and subsequent maintenance of expression of wg ES and hh FG domains. They suggest that Torso-induced anterior repression of hb is necessary for the correct expression of wg and hh in the ES and FG domains, respectively. To determine whether defects of foregut innervation could also be involved in the gut phenotypes of Hb+ and Otd+ larvae, the formation of the stomatogastric nervous system (SNS) of these larvae was investigated. During embryonic stage 10, SNS development becomes morphologically distinct by the appearance of three evenly spaced dorsal invaginations composed of small groups of cells that can be visualised by fkh and Krüppel (Kr) expression Fig. 5. The eosophageal domain of wingless (wg) expression and the foregut domain of hedgehog (hh) expression are absent in Hb+ embryos. In situ (Gonzalez-Gaitan and Jäckle, 1995; Fig. 6A,D). fkh hybridisation with wg (A-E) and hh (F-J) probes on blastoderm embryos from and Kr expression at the position of the SNS anlage females carrying the Gal4-GCN4 driver crossed with y−w−(wt*; A,F), with UAS- was never observed in Hb+ embryos (Fig. 6B,E) and hb (Hb+; B,G) or with UAS-otd (Otd+; C,H) males, from torso females (tor−; the number of SNS precursor invaginations was D,I). tll−/hkb− double mutant embryos were identiﬁed with a double staining for drastically reduced in most Otd+ embryos (Fig. 6C,F). ftz (tll−/hkb−; E,J). Arrowheads indicate missing expression domains. Anterior repression of hunchback 1579

Fig. 6. The expression of Kr and fkh in the anlage of the SNS is absent in Hb+ embryos and reduced in Otd+ embryos. In situ hybridisation with Kr (A-C) and fkh (D-F) probes on stage-10 embryos from females carrying the Gal4-GCN4 driver crossed with y−w− (wt*; A,D), with UAS-hb (Hb+; B,E) or with UAS-otd (Otd+; C,F) males. Numbers (1, 2 and 3) indicate the three distinct SNS anlage invaginations (A,C,D,F). The arrowheads indicate the missing invaginations (B,C,E,F). protein is able to repress the three genes cnc, wg and hh, and to the persistence of a given target gene at the pole or to its that torso-induced anterior repression of hb is necessary for expression in more posterior regions. their positive control by torso. To determine whether the Since the anterior domain of hb endogenous expression is positive control of cnc, wg and hh by torso could be the result very broad and strong, expression of hb from the Gal4-GCN4 of a double negative control involving hb, expression of these driver only induces persistence of hb at the pole and genes was analysed in hb zygotic mutant embryos derived from overexpression of hb in its own expression domain. Several torso females. If the lack of early anterior expression of cnc, reasons suggest that the latter has no effect on development: wg and hh was solely due to the absence of repression of hb ﬁrst, no phenotype affecting structures of the segmented part of at the pole, expression of these genes should be recovered in the head is observed with Hb+ embryos; second, the overall hb− embryos derived from torso females. As shown in Fig. 7, expression of cnc, wg and hh in Hb+ embryos is not shifted early anterior expression of cnc (A), wg (B) and hh (C) was towards the posterior as would be expected for an increased not recovered in hb− embryos derived from torso females morphogenetic activity of the Hb protein. In contrast, the whereas it was normal in hb− embryos (D-F). This indicates defects observed in Hb+ embryos involve the labrum and the that, although necessary, the anterior repression of hb is not foregut derivatives, which originate from the anteriormost sufficient to mediate Torso positive control on cnc, wg and hh domain of the blastoderm embryo where hb is normally early anterior expression.

DISCUSSION

Terminal mis-expression of hb and otd at the anterior The anterior domain of Torso activity encompasses the anteriormost expression domains of cnc, wg and hh (Fig. 8A) and overlaps the domain where Bcd targets are repressed during cellularisation. The Gal4-GCN4 driver is able to activate reporter gene expression in this domain but also in a more posterior region (Fig. 1), and the phenotypes observed with this system could be due either

Fig. 7. Repression of hb by Torso is not sufficient for cnc, wg and hh early anterior expression. In situ hybridisation on blastoderm hb− zygotic embryos with cnc, wg and hh probes. hb− embryos were identiﬁed through their ftz expression pattern detected with a simultaneous staining using a ftz probe. Embryos were from torso (A-C) and wild-type (D-F) females. 1580 F. Janody, J. Reischl and N. Dostatni

A) C)

Torso Labrum Non-segmental Bcd (mt, es, acron ppw, db) (db, da, vp) ? ? Hb Foregut (ph, eo, pv)

cnc wg and hh Anterior midgut (labrum) (foregut)

END OF B) BLASTODERM STAGE 9 STAGE 10 EMBRYOGENESIS LARVAL STAGE

Initiation of No maintenance of No expression Absence of food wg ES domain wg ES domain of kr and fkh progression in the foregut strongly reduced in the SNS anlage

Reduced size of eosophagus

Persistence of hb Initiation of No maintenance of defects in proventriculus expression at the anterior hh FG domain hh FG domain formation pole during cellularisation strongly reduced

Reduced initiation No expression of of cnc anterior cap cnc labral domain absence of labrum

Fig. 8. The role of hb repression at the anterior pole of the Drosophila embryo. (A) Summary of fate map (Jürgens et al., 1986) and cnc, wg and hh expression data (Mohler, 1993, 1995) in the most anterior region of the blastoderm embryo. The labrum territory will give rise to the median tooth (mt), the epistomal sclerite (es), the dorsal bridge (db) and the posterior pharyngeal wall (ppw). The territory of the non-segmental acron will give rise to the dorsal bridge (db), the dorsal arm (da) and the ventral plate (vp). The foregut territory will give rise to the pharynx (ph), the eosophagus (eo) and part of the proventriculus (pv). The anterior expression domain of cnc is in yellow and of hh and wg in blue. (B) The persistence of hb induced several distinct expression defects and phenotypes. Defects framed in yellow are observed in embryos from torso females (Mohler et al., 1995; Skaer, 1993). The foregut does not form in embryos from torso females. (C) Model for the positive control of cnc, wg and hh by torso and bcd at the anterior pole. Hb is a positive target of Bcd (black arrow). If not repressed by Torso at the pole, Hb would repress the expression of both the labral determinant cnc and the two determinants of foregut formation wg and hh (interrupted lines). repressed by Torso (Fig. 8A). The expression of cnc, wg and hh The persistence of hb at the anterior pole impairs in this domain is required for the formation of these structures foregut and labrum formation and is perturbed in Hb+ embryos. Finally, since early Fate-map studies have defined territories at the surface of the blastoderm expression of cnc, wg and hh is affected in Hb+ blastoderm embryo that give rise to specific developmental fates embryos, these expression defects are almost certainly a direct (Jürgens et al., 1986): cells that will form the stomodeum extend consequence of hb persistence at the pole during cellularisation. from 100% to around 95% egg length (EL); the labrum anlage In contrast, the defects of Otd+ embryos are probably not due extends dorsally from 98% EL to 85% EL; the non-segmental to otd persistence at the anterior poles for two reasons: first, the acron extends more towards the posterior (Fig. 8A). The Hb+ mouth hooks that are affected originate from the posterior embryos exhibited a large set of phenotypes involving structures segmented part of the head; second, the defects in these structures that originate from these territories. This diversity, probably due are correlated with a reduction/failure of expression of the cnc to variability in the strength of Gal4-GCN4, allowed the posterior stripe. The phenotype of larvae derived from Otd+ identification of distinct developmental steps that exhibit different embryos suggests defects in foregut and/or anterior midgut sensitivity to the expression of the Hb protein. The weaker formation. Since early expression of wg, hh and fkh in foregut phenotype of Hb+ embryos was a normal embryonic territories is normal, early foregut differentiation is probably development and emergence of larvae dying of defects in foregut normal in these embryos. In contrast, the development of the derivatives. Consistently, wg eosophageal and hh foregut domains gastric caeca is largely perturbed, suggesting that anterior midgut appeared absent or strongly reduced in most Hb+ embryos (Fig. formation is probably altered. Further analysis, however, is 8B). This indicates that early expression of wg and hh in foregut required to determine whether this defect is due to the persistence territories is highly sensitive to the persistence of hb at the pole. of otd at the pole or to the absence of otd ventral repression. An intermediate phenotype exhibited, in addition to foregut Anterior repression of hunchback 1581 disruption, the absence of labral derivatives, which can be al., 1996; Ronchi et al., 1993), appears as an anterior cap at correlated with a reduction of cnc labral expression (Fig. 8B). syncycial blastoderm and it is repressed from the pole by Torso Although cnc labral expression is clearly absent in most Hb+ during cellularisation. In contrast, the first anterior expression embryos during germ band elongation, the sensitivity of cnc cnc, wg and hh is only detectable at the pole at the beginning labral expression at the blastoderm stage is only revealed in a of cellularisation and it is not repressed by Torso. The sensitised system when the bcd copy number is reduced. This molecular mechanism leading to this atypical regulation of cnc, indicates that, whereas wg eosophageal and hh foregut expression wg and hh by Bcd remains to be understood (Fig. 8C). domains are easily disrupted by the persistence of hb at the pole, cnc expression is less sensitive to hb expression. This could hb antagonises bcd at the pole reflect differences in the sensitivity of wg, hh and cnc promoters Blastoderm expression of cnc, wg and hh is under positive to Hb. Alternatively, we cannot exclude the possibility that the control of both torso and bcd and it is repressed by hb when the Gal4-GCN4 protein gradient allows strong expression of the Hb expression of the latter is allowed to persist during protein at the pole and weaker expression in more distal regions, cellularisation. These observations indicate that the Hb and Bcd such as those where cnc labral expression domain lie at proteins have opposite effects on the regulation of cnc, wg and blastoderm stage (Fig. 8A). The stronger phenotype was very hh expression at the anterior pole and the subsequent formation similar to the torso loss-of-function phenotype: it showed, in of the labrum and the foregut derivatives. This is further addition to labrum disruption, defects in the dorsal arms, which supported by two observations: first, the lethality of Hb+ originate from slightly more posterior regions of the head embryos was increased when Bcd dosage was reduced; second, (Jürgens et al., 1986). The formation of these structures is under the anterior cap of cnc expression was drastically reduced in terminal control and the close similarity observed between this Hb+ embryos expressing only one copy of the bcd gene whereas strong phenotype and the phenotype of embryos from torso the reduction of bcd gene dosage in otherwise wild-type females (Fig. 2B,C) strongly argues that these phenotypes are embryos had almost no effect. This suggest an antagonism linked. However, we have not been able to identify a zygotic gene between hb and bcd at the anterior pole. A major role of torso whose expression would be specifically modified at blastoderm at the anterior would be to repress hb to prevent this antagonism, stages and which would be responsible for defects in dorsal arms which impairs anterior terminal development (Fig. 8C). formation. Several lines of evidence strongly support the notion that the Torso-induced anterior repression of hb is Hb protein is acting both as a repressor and activator of necessary for terminal development transcription. It contains six zinc fingers and binds DNA specifically (Stanojevic et al., 1989; Treisman and Desplan, The results of the present study led us to propose that Torso- 1989). It interacts directly with dMi-2, a protein possibly induced repression of hb is necessary for anterior terminal involved in histone deacetylation, and genetic studies suggest development and mediates a large part of the terminal activity at that this interaction might be critical for Polycomb-mediated the anterior. The analysis of cnc, wg and hh expression in hb silencing of HOX genes (Kehle et al., 1998). In contrast, both mutant embryos derived from torso females also indicates that maternal and zygotic components of hb are involved in most repression of hb by torso is not sufficient to allow expression of aspects of anterior and central segmental development these genes under bcd control. Since the maternal component of (Simpson-Brose et al., 1994; Struhl et al., 1992). hb regulates hb remained expressed in hb zygotic mutant embryos from torso positively the expression of most Bcd targets at the anterior and females, it might still contribute to the repression of cnc, wg and this regulation is mediated through Hb binding sites (Simpson- hh. Alternatively, the Torso cascade might also act on cnc, wg Brose et al., 1994; Small et al., 1991). It has been proposed that and hh early anterior expression, independently of hb, through hb is required for bcd to execute all of its function (Simpson- another transcription factor (Fig. 8C). This factor may be induced Brose et al., 1994). Our analysis indicates that bcd activity, in by the Torso pathway to activate cnc, wg and hh transcription or, inducing anteriormost expression of cnc, wg and hh and the it may be another Bcd target repressed at the pole by Torso. This subsequent formation of labrum and foregut derivatives, is putative factor would then act, like Hb, by repressing cnc, wg and antagonised by the persistence of the Hb protein at the pole in hh expression. Since Torso is known to repress the expression of the cellularising embryo. It suggests that hb might be several Bcd targets, several candidates could fulfil this function. dispensable for bcd function in patterning the anterior terminalia. otd is a direct target of Bcd (Gao and Finkelstein, 1998) but cnc, This is further supported by the observation that in the absence wg and hh were normally expressed in Otd+ embryos, suggesting of all hb function there are still a labrum and foregut structures that maintenance of otd expression at the anterior pole does not (E. Wimmer and C. Desplan, personal communication). repress these genes. The tll gene, which encodes a transcription factor and behaves as a classical Bcd target, is a possible candidate. We are indebted to Ernst Wimmer and Claude Desplan for their Since the repression of cnc, wg and hh in Hb+ embryos constant support during the process of this work and to Claude occurs as soon as expression of hb is allowed to persist, it is Desplan for allowing us to start this work in his laboratory. We would likely that the negative regulation of cnc, wg and hh by Hb is like to thank Ernst Wimmer and Thomas Lecuit for the UAS-hb/UAS- otd and the UAS-lacZ transgenic stocks, respectively; Winshipp Herr, direct. cnc, wg and hh are also positively regulated by Bcd but Liz Gavis and Gary Struhl for constructs; Jim Mohler, Laurence their pattern of expression at blastoderm stages indicates that Röder, Corinne Angelats, Alfrun Eckner and Steve Kerridge for this regulation is atypical. The expression of direct zygotic cDNAs reagents to make probes; Bob Finkelstein, David Kosman and targets of Bcd such as hb (Driever et al., 1989), otd (Finkelstein John Reinitz for antibodies. We are grateful to Claude Desplan, Yacine and Perrimon, 1990; Gao and Finkelstein, 1998) or a synthetic Graba and Bernard Jacq for critical reading of the manuscript. F.J. was Bcd reporter containing only Bcd binding sites (Bellaïche et supported by MRT and ARC fellowships, J.R. by the Boehringer 1582 F. Janody, J. Reischl and N. Dostatni

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