Determination of Wing Cell Fate by the Escargot and Snail Genes in Drosophila

Development 122, 1059-1067 (1996) 1059 Printed in Great Britain © The Company of Biologists Limited 1996 DEV5054

Determination of wing cell fate by the escargot and snail genes in Drosophila

Naoyuki Fuse1,2,*, Susumu Hirose2 and Shigeo Hayashi1,4,† 1Genetic Stock Research Center, 2Department of Developmental Genetics, National Institute of Genetics, Mishima, Shizuoka-ken 411, Japan *Present address: Department of Molecular Biology and Genetics, Johns Hopkins University, School of Medicine, Baltimore, MD 21205, USA †Author for correspondence (e-mail: [email protected])

SUMMARY

Insect appendages such as the wing and the leg are formed expression induced from the hsp70 promoter rescued the in response to inductive signals in the embryonic field. In escargot snail double mutant phenotype with the effects Drosophila, cells receiving such signals initiate developmen- confined to the prospective wing cells. Similar DNA binding tal programs which allow them to become imaginal discs. specificities of Escargot and Snail suggest that they control Subsequently, these discs autonomously organize patterns the same set of genes required for wing development. We specific for each appendage. We here report that two related thus propose the following scenario for early wing disc transcription factors, Escargot and Snail that are expressed development. Prospective wing cells respond to the in the embryonic wing disc, function as intrinsic determi- induction by turning on escargot and snail transcription, nants of the wing cell fate. In escargot or snail mutant and become competent for regulation by Escargot and embryos, wing-specific expression of Snail, Vestigial and β- Snail. Such cells initiate auto- and crossregulatory circuits galactosidase regulated by escargot enhancer were found as of escargot and snail. The sustained Escargot and Snail well as in wild-type embryos. However, in escargot snail expression then activates vestigial and other target genes double mutant embryos, wing development proceeded until that are essential for wing development. This maintains the stage 13, but the marker expression was not maintained in commitment to the wing cell fate and induces wing-specific later stages, and the invagination of the primordium was cell shape change. absent. From such analyses, it was concluded that Escargot and Snail expression in the wing disc are maintained by Key words: wing development, escargot, snail, cell fate their auto- and crossactivation. Ubiquitous escargot or snail maintenance, autoregulation, crossregulation, Drosophila

INTRODUCTION signals that allocate the leg primordium within the ectodermal field established by the activities of segment polarity genes. In During development, groups of cells assume specific fates support of this idea, Simcox et al. (1989) used the embryo in according to positional information. Two mechanisms, vivo culture technique to show that wg is essential for the extrinsic induction and intrinsic determination, are required for formation of imaginal discs. Using a temperature sensitive wg these processes. Cells receiving inductive signals begin to allele, Cohen et al. (1993) demonstrated that the Dll expression express intrinsic determinants, acquire a specific cell fate, and in the leg primordium requires wg activity at about 5 hours of differentiate to form specific patterns autonomously. One development, roughly corresponding to the time when the rows example is imaginal discs of Drosophila (Cohen, 1993). In of wg and dpp expression overlap. dpp has been shown to exert embryos, each imaginal primordium is allocated to a specific a strong organizing activity in the establishment of the position in the ectoderm and invaginates to form a sac-like embryonic dorsoventral axis and in patterning imaginal discs imaginal disc. Subsequently, each performs a series of (Ferguson and Anderson, 1992; Zecca et al., 1995), but its role autonomous events to organize adult external structures. in imaginal disc induction needs to be further studied. The disc The first sign of imaginal disc induction is the expression of development continues after the overlap between Dll the homeobox gene Distal-less (Dll) in the prospective leg expression and the intersection of the wg and dpp rows are lost imaginal disc (Cohen, 1990). In stage 11 embryos, the leg (Cohen et al., 1993), suggesting that the induced cells must primordia, visualized by the Dll RNA expression, appears in activate an intrinsic determinant to irreversibly commit them clusters of cells that overlap the intersection between the to imaginal cell fate. Ventral leg and dorsal wing primordia dorsoventral row of cells expressing the segment polarity gene appear to originate from a common imaginal primordium. The wingless (wg) and the anterior-posterior row of decapenta- cell lineage tracing study has shown that in stage 12, the wing plegic- (dpp) expressing cells (Cohen et al., 1993). Since Wg disc cells expressing vestigial (vg) segregate and move dorsally and Dpp are secreted signaling molecules (Padget et al., 1987; away from Dll-expressing cells (Cohen et al., 1993). Rijsewijk et al., 1987), they were proposed to be the inductive Although the intersection between wg- and dpp-expressing 1060 N. Fuse, S. Hirose and S. Hayashi rows exist in all the trunk segments, wings and legs form only studied the function of two closely linked genes, escargot in the thorax. This was shown to be due to the negative regula- (snail) and snail (sna). esg and sna encode transcriptional reg- tion by homeotic genes. In the abdomen, genes in the bithorax ulators with similar C2H2 type zinc finger domains (76% amino complex repress leg and wing formation (Bate and Martinez acid identity; Boulay et al., 1987; Whiteley et al., 1992). esg Arias, 1991; Simcox et al., 1991; Vachon et al., 1992; Carroll et is expressed in most imaginal primordia found in the embryo al., 1995) and in the first thoracic segment, wing formation was repressed by the Sex comb reduced gene in the Antennapedia complex (Carroll et al., 1995). In embryos mutant for Antenna- pedia, which is responsible for the identity of parasegment 4 and 5, formation of the leg and wing primordium was detectable (Mann, 1994; Carroll et al., 1995), suggesting that these appendages are formed as a default in the ‘ground state’ of segmental identity (Lewis, 1978). We must therefore seek a putative intrinsic determinant of imaginal disc formation outside the homeotic gene complex. The nuclear proteins, Dll and Vg are the earliest known markers for the leg and wing imaginal discs, and are required for pattern formation along the PD axis in the adult (Cohen and Jürgens, 1989; Cohen, 1990; Williams et al., 1991). However, their involvement in imaginal disc formation is not clear since imaginal discs are formed in the absence of Dll or vg (Williams et al., 1991; Cohen et al., 1993). To identify an intrinsic determinant of the imaginal disc, we

Fig. 2. Esg and Sna are expressed in the wing primordium. Wild type embryos double-stained with anti-Esg (green) and anti-Sna (red) antibodies. All embryos are oriented as dorsal up and anterior to the left. (A) In a stage 5 embryo, Sna is expressed in the ventral region, the Fig. 1. Esg and Sna have similar DNA binding specificities. prospective mesoderm. Esg is expressed in the dorsal region. There is (A,B) DNA binding specificities of Esg (A) and Sna (B). no overlap, demonstrating the specificity of each antibody. (B) In a Recombinant GST-Esg and GST-Sna fusion proteins bind to 32P- stage 13 embryo, Esg and Sna begin to be expressed in wing (w) and labeled DNA containing the E2 box. Addition of unlabeled DNA haltere (h) primordia which appear yellow (arrowheads). (C) In a stage containing wild-type (closed circle) or mutant E2 box sequences 15 embryo, wing (w), haltere (h) and genital (g) discs are stained (others) competed with this binding. Amounts of probe DNA bound yellow, indicating colocalization of Esg and Sna proteins. to the proteins expressed as percentages relative to the control (D) Enlarged view of a wing and a haltere disc which expressed both without specific competitor (Y-axis) were plotted against the ratio of Esg and Sna. An anterior spiracle (a) expressed only Esg. (E) Ventral competitor to probe (X-axis). The competition profiles of Esg (A) part of the same embryo as shown in D. From left to right, a pair of the and Sna (B) are very similar. (C) Oligonucleotide sequences used as first thoracic leg disc and a second and a third leg disc (slightly out of competitors. Central parts of the 24 mer double strand focus) are seen. The majority of leg disc cells express only Esg. A oligonucleotides are shown. Sequences corresponding to the Esg small subset of leg disc cells expressing both Esg and Sna are binding consensus (Fuse et al., 1994) are underlined. indicated by arrowheads. Bar, 125 µm for A-C and 45 µm for D and E. Overlapping activity of zinc finger proteins 1061

Fig. 3. esg and sna are required for Sna expression in the wing disc. All embryos were stained with anti-Sna antibody. (A) Stage 15 control embryo (sna1/CyO). (B) esgG66B embryo. Wing (arrowhead, w), haltere (h) and genital (g) discs express Sna as in the control (A). (C) Ventral view of a sna1 embryo. sna1 mutation causes the malformation of the whole body, but the wing, haltere and genital discs express the truncated Sna protein. (D) Ventral view of an esgG66B sna1 embryo. Sna expression in the wing, haltere and genital discs is abolished. (E) Ventral view of a heat treated esgG66B sna1 HSesg embryo. Sna expression is restored in the wing and haltere discs (arrowhead). (F,G) High magnification views of the wing disc in stage 15-16 embryos. (F) Control embryo (esgG66B sna1/CyO). Sna-expressing cells invaginate to form a sac-like wing disc. (G) An example of heat shock treated esgG66B sna1 HSesg embryo. Such embryos showed a wide range of wing disc phenotype from no invagination at all to nearly complete invagination shown here. Bar: 125 µm (A-E), 12.5 µm (F,G). and in the larva (Whiteley et al., 1992; Hartenstein and Jan, 1992; Kassis, 1994). sna1 has a small deletion within the coding 1992; Hayashi et al., 1993; Younossi-Hartenstein et al., 1993) region and is genetically null (Grau et al., 1984; Boulay et al., 1987). and has been shown to be required for the maintenance of The esg sna double mutant chromosome was made by recombination G66B 1 G66B 1 R diploidy of some imaginal cells (Hayashi et al., 1993; Fuse et between esg and sna . y w; esg /sna FRT40 neo females al., 1994; Hayashi, 1996). sna is initially expressed and were crossed with y w; Gla/CyO males. Recombinants were selected as w+ and neoR progenies, and then tested for complementation with required in the prospective mesoderm (Simpson, 1983; Grau sna and esg alleles. For analyses of embryonic phenotype, we used et al., 1984; Alberga et al., 1991). In later stages, sna is esgG66B FRT40/CyO actin-lacZ, sna1 FRT40/CyO and esgG66B sna1 expressed in wing, haltere and genital discs at the same stage FRT40/CyO stocks. esg mutant embryos were identified by the lack when esg is expressed in these discs (Alberga et al., 1991). In of staining with anti-Esg antibody. sna mutant and esg sna double this work, we studied the function of esg and sna in the early mutant embryos were identified by the sna phenotype. Other stocks stage of wing disc development and report that esg and sna act are described by Lindsley and Zimm (1992). as intrinsic determinants of the wing cell fate.

MATERIALS AND METHODS Fly stocks esgG66B has a complete deletion of the esg coding region with P[engrailed (en)-lacZ w+] inserted into the esg locus (Whiteley et al.,

Fig. 4. Vg expression in mutant embryos. All embryos were stained with anti-Vg antibody. (A) Stage 15 esgG66B embryo. Vg was expressed normally in wing and haltere discs (arrowheads). Vg was also expressed in muscle, CNS and sense organs (arrow). The sense organ expression was found in both thoracic and abdominal segments and in the same dorsoventral level as the wing disc. An identical expression pattern was observed in control (esgG66B/+) embryos (data not shown). (B) sna1 embryo. Vg expression was abolished in embryonic muscle, but remained in wing and haltere discs (arrowheads) and sense organs (arrow). (C) esgG66B sna1 embryo. Vg expression was abolished in wing and haltere discs, but remained in sense organs (arrow). (D-F) Rescue of the double mutant phenotype. (D) Heat shock treated esgG66B sna1 embryo. Vg expression in wing and haltere discs was absent. (E) Heat shock treated esgG66B sna1 HSesg embryo. Vg expression was restored in wing and haltere discs (arrowheads). (F) Heat shock treated esgG66B sna1 HSsna embryo. Vg expression was restored as in E. 2 and 3 indicate the second and third thoracic segments. Bar, 125 µm. 1062 N. Fuse, S. Hirose and S. Hayashi

DNA binding experiments produced and used for gel mobility shift assays (Ip et al., 1992; Glutathione S-transferase (GST)-Esg and GST-Sna fusion proteins Fuse et al., 1994). Both proteins bound to 32P-labeled DNA were prepared from E. coli as described by Ip et al. (1992); Fuse et containing the E2 box (Fig. 1C closed circle). Addition of al. (1994). The GST-Sna protein was purified from the inclusion body. excess DNA containing the E2 box or its derivatives competed The prepared proteins were >50% pure as judged by a Coomasie with this binding. Competition profiles of the five competitor Brilliant Blue staining after SDS-polyacrylamide gel electrophoresis. DNAs (Fig. 1C) were indistinguishable for the two proteins Gel mobility shift assay was performed as described by Fuse et al. 32 (Fig. 1A,B), indicating that Esg and Sna have similar DNA (1994). The double strand oligomers, which were used as a P- binding specificities. Next, expression patterns of Esg and Sna labeled probe and unlabeled competitors, were 5′-GCGGCC-N - 10 were compared in embryos by double-label immunostaining TTTG-3′ with a 5′-TCGA-3′ overhang on each strand. Sequences for (Fig. 2; Alberga et al., 1991; Whiteley et al., 1992). In early N10 are indicated in Fig. 1C. In a 10 µl reaction mixture, the probe DNA (Fig. 1C closed circle, final 1 fmol/µl) and GST-Esg or GST- embryos, Esg expression starts in the dorsal side of the embryo Sna (approximately 10 pg/µl) were incubated with or without various whereas Sna is expressed in prospective mesoderm on the concentration of competitors (10, 50 and 100 fmol/µl). The probe ventral side (Fig. 2A). In stage 13, the wing and haltere bound to the protein was separated from the free probe by elec- primordia that have segregated out from the leg primordia trophoresis and quantitated by Fuji BAS2000. GST alone did not bind begin to express Esg and Sna (Fig. 2B). Expression of the two the probe DNA (data not shown). proteins in the wing and haltere primordia continues in stage 15 embryos (Fig. 2C) when they invaginate to form imaginal Immunohistochemistry discs (Fig. 3F, data not shown) and Esg expression in most of Immunostaining was performed as described by Hayashi et al. (1993). the other tissues disappears. In these discs, a strict cell to cell For double-label immunostaining, Oregon R embryos were incubated with rat anti-Esg (Fuse et al., 1994) and rabbit anti-Sna (a gift from correspondence of Esg and Sna expression was observed (Fig. Rolf Reuter) antibodies. Subsequently, the embryos were incubated 2D). In leg discs that express Esg, only a subset of the cells with biotin-conjugated goat anti-rat IgG antibody (Jackson lab) and coexpressed Sna (Fig. 2E). then with FITC-conjugated streptavidin (Vector lab) and Cy3-conjugated anti-rabbit IgG antibody (Chemicon). The embryos were Phenotypes of the esg sna double mutant embryo observed with a Zeiss Axioplan microscope. For analyses of mutant The expression of the two transcription factors with similar phenotypes, embryos were fixed and incubated with rabbit anti-Sna DNA binding specificities in these imaginal discs raised the antibody, biotin-conjugated goat anti-rabbit IgG antibody (Jackson possibility that Esg and Sna cooperate to play important roles lab) and ABC complex (Vector lab), and then developed in in the early stage of wing and haltere development. To test this diaminobenzidine. Alternatively, embryos were incubated with rabbit possibility, we compared the phenotypes of esg sna double anti-Vg antibody (Williams et al., 1991), biotin-conjugated goat anti- mutant embryos with those of esg and sna single mutant rabbit IgG antibody (Jackson lab) and FITC-conjugated streptavidin embryos. We describe below the situation in the wing disc, but (Vector lab). Stained embryos were examined using a confocal microscope (LSM410, Carl Zeiss). For a simultaneous detection of β-galac- exactly the same observations were made for the haltere disc. G66B 1 tosidase (β-gal) and D-α-catenin, embryos were incubated with rabbit We used two null mutations, esg and sna for this study 1 anti-β-gal (Cappel) and rat anti-D-α-catenin monoclonal antibody (Materials and Methods). Since sna has a small deletion (Oda et al., 1993), with biotin-conjugated goat anti-rat IgG antibody, within the coding region (Grau et al., 1984; Boulay et al., and finally, with a mixture of Cy3-conjugated anti-rabbit IgG and 1987), a truncated Sna protein, which does not function, is FITC-conjugated streptavidin. detectable in sna1 mutant embryos by anti-Sna antibody (Figs 3C, 6E). We used Sna expression as a marker for the wing disc. Rescue experiments by heat inducible constructs In esg mutant embryos, the wing disc expressed Sna normally To make the HSsna construct, sna ORF flanked by XbaI and EcoRI (Fig. 3B). The sna1 mutation results in the malformation of the sites was amplified by RT-PCR and inserted into the pCaSpeR-hs whole body due to the requirement of sna for mesoderm devel- vector (Thummel and Pirrota, 1991). Transformants were established by the germ line P-transformation method (Rubin and Spradling, opment. But the truncated Sna protein was expressed in invagi- 1982). HSesg, HS∆zf (Fuse et al., 1994) or HSsna was introduced into nated wing discs (Fig. 3C). The number of Sna-expressing cells the esg sna double mutant chromosome by recombination. Recombi- in the imaginal discs appears to be lower than that in the control nants were checked for heat shock induced ubiquitous Esg or Sna and esg mutant embryos (see also Fig. 6E). It is not clear expression (data not shown). To rescue the double mutant phenotype, whether this is caused by the sna mutation itself or by the 10- to 11.5-hour old embryos were given four or five heat shocks at secondary effect of body malformation. Such Sna expression 37¡C for 20 minutes every 90 minutes. After the last heat shock, the was abolished in esg sna double mutant embryos (Fig. 3D). embryos were incubated at 25¡C for 70 minutes, and were stained esgG66B sna1 placed in trans to the deficiency Df(2L)A48 with anti-Vg or anti-Sna antibody. (Ashburner et al., 1982) results in the same phenotype as esgG66B sna1 homozygotes (data not shown), indicating that no other recessive mutation contributes to the phenotypes. In the RESULTS embryos which have only one dose of the wild-type esg or sna gene (esgG66B sna1/sna1 or esgG66B sna1/esgG66B, respectively), Similarity in DNA binding specificities and the Sna marker was expressed in wing discs (data not shown). expression patterns of Esg and Sna These results indicate that both esg and sna are required for esg and sna encode transcriptional regulators with similar zinc proper Sna expression in wing discs and that the loss of one of finger domains (Boulay et al., 1987; Whiteley et al., 1992). the genes can be compensated by one copy of the other gene. Using recombinant proteins produced in E. coli, we compared To confirm the double mutant phenotype, we also examined DNA binding specificities of Esg and Sna. GST fusion proteins the expression of two additional markers for wing disc. The containing the zinc finger domain of each protein were second wing disc marker, Vg was expressed in the wing disc Overlapping activity of zinc finger proteins 1063 in esg or sna mutant embryos (Fig. 4A,B) as well as in the (Fig. 3E). Such cells partially invaginated to form wing discs control embryos (data not shown). However, in esg sna double (Fig. 3G). Among 76 heat shocked esgG66B sna1 HSesg mutants, the Vg expression was lost (Fig. 4C). esgG66B used in embryos stained with anti-Sna, 39 (51%) had restored Sna this study has a complete deletion of the esg coding region with expression in at least one wing or haltere primordium. In 24 of P[en-lacZ] inserted into the esg locus (Whiteley et al., 1992; them (32%), the Sna-positive cells were found to be invagi- Kassis, 1994). Since en-lacZ is regulated by endogenous esg nated. As a control, esgG66B sna1 HS∆zf (zinc finger domain enhancer, in esgG66B embryos, β-gal was expressed in the wing deleted esg derivative; Fuse et al., 1994) embryos were disc as well as in leg discs, anterior spiracle, tracheal pits and similarly treated. None of them (n=58) expressed Sna in the most of the other tissues which normally express esg (Whiteley wing primordium (data not shown). These results demonstrate et al., 1992; Fig. 5B). Therefore, we would like to designate that esg and sna have overlapping functions that are essential the P[en-lacZ] inserted into the esg locus as esg-lacZ, and use for wing disc formation. the expression of esg-lacZ as a third marker for the wing disc. In both esgG66B and esgG66B sna1/sna1 embryos, esg-lacZ were Failure of wing fate maintenance in the double expressed in the invaginated wing disc as well as in the control mutant embryo (Fig. 5A-C). Similarly to the above observations, such The response to ubiquitous Esg and Sna expression were esg-lacZ expression was undetectable in esgG66B sna1 double confined to cells in a small region where the wing disc mutant embryos (Fig. 5D). Thus, the expression of the three normally forms. This suggests that such cells have already wing disc markers were abolished in esg sna double mutant acquired a potential to respond to Esg and Sna. We therefore embryos, while they were detected in both the single mutants, examined the mutant embryos when Sna expression was first indicating that overlapping activities of esg and sna are detectable in the wing primordium. In stage 13 embryos, Sna required for proper wing disc development. expression was detectable in the wing primordium within the In wild-type embryos, after the onset of esg and sna ectoderm in the control, sna and esg mutant embryos (Fig. expression, the wing primordial cells constrict their apical 6A,B, data not shown). The level of Sna expression varied surface, and then invaginate basally to form the disc structure from cell to cell. The Sna expression continued and became (Bate and Martinez Arias, 1991; Fig. 3F). To determine stronger and more uniform during stage 15 (Fig. 6D,E, data not whether the wing primordium invaginates normally in esg sna shown). However, in esg sna double mutants, the initial Sna double mutant embryo, we examined embryos double-labeled expression was detected in stage 13 (Fig. 6C), but was not with anti-D-α-catenin and anti-β-gal antibodies. β-gal detectable in stage 15 when the wing primordium normally expressed from the esg-lacZ was distributed uniformly within would have completed invagination (Fig. 6F). To determine the cell body, and D-α-catenin was localized in the adherence whether the loss of Sna expression is due to the death of the junction along the apical circumference of the cells (Oda et al., wing primordium, we examined cell death by the in situ nick 1993; Fig. 5A). Apical constrictions of wing primordium cells translation method (Hay et al., 1994). In the double mutant were detected as condensed yellow staining (Fig. 5 arrowhead), embryo, no sign of cell death was detectable in the prospec- and the invaginated cell bodies were seen in esgG66B and tive wing disc, although massive cell death in the endoderm esgG66B sna1/sna1 mutant embryos (Fig. 5B,C) as well as in the due to the sna mutation was clearly visible (data not shown). control embryos (Fig. 5A,E). However, such apical constric- These results suggest that in the double mutant, the wing pri- tion did not occur in esgG66B sna1 double mutant embryos (Fig. mordium begins initial development until stage 13, but fails to 5D,F). These results indicate that the wing disc is absent in the maintain the fate in later stages. double mutant, and that esg and sna are required for the wing- specific cell shape change. Requirement for esg and sna in the genital disc In contrast to the situation in the wing disc, leg discs formed Esg and Sna are also coexpressed in the genital disc (Fig. 2C). normally in esg sna double mutant embryo as well as in both Sna expression in the genital disc was lost in the esg sna double single mutants (Fig. 5G,H; data not shown). This result mutant as in the case of the wing disc (Fig. 3D) and was indicates that the genetic interaction between esg and sna restored by repeated induction of HSesg (data not shown). observed in the wing disc does not occur in the leg disc where These results suggest that esg and sna are required for the expression of the two genes overlaps only in a few cells (Fig. proper development of the genital disc as well. However, at 2E). present, we do not know which step of genital disc development is regulated by the two genes. Rescue of the double mutant phenotypes by ubiquitous Esg and Sna expression To further verify the role of esg and sna in wing development, we examined whether Esg or Sna supplied from transgenes can DISCUSSION rescue the esg sna double mutant phenotype. We introduced hsp70-esg (HSesg; Fuse et al., 1994) and hsp70-sna (HSsna) Overlapping function of two zinc finger proteins into the double mutant chromosome. The ubiquitous Esg or We have shown that esg and sna are required for early wing Sna expression induced by heat shock restored Vg expression development. When both of the genes were absent, the wing in the wing primordium of the double mutant (Fig. 4E,F). Such imaginal disc failed to develop. One copy of either of the genes rescues were dependent on heat shock treatment (data not was sufficient for the wing primordium to express the marker shown), and the heat shock alone without the transgenes did genes and to invaginate. The ubiquitous expression of either of not have such an effect (Fig. 4D). The ubiquitous expression the genes was able to restore the marker gene expression and of Esg also restored the Sna expression in the wing primordium cell invagination in the wing primordium. Thus, the functions 1064 N. Fuse, S. Hirose and S. Hayashi

Fig. 5. esg-lacZ expression and apical constriction of wing primordium in mutant embryos. All embryo were stained with anti-βgal (red) and anti- D-α-catenin (green) antibodies. Horizontal optical sections of esgG66B sna1/CyO (A), esgG66B (B), esgG66B sna1/sna1 (C) and esgG66B sna1 (D) embryos. Wing (w) and haltere (h) discs identifiable by esg-lacZ expression and apical accumulation of D-α-catenin (yellow condensed staining, arrowhead) are found next to tracheal histoblasts (t). Wing and haltere discs are visible in A-C, but not in D. Anterior spiracle (a) is visible in D. Apical accumulation of D-α-catenin in wing and haltere discs is also visible next to a tracheal pit (tp) in the lateral view of esgG66B sna1/CyO embryo (E; inset shows apical constriction of wing primordium at higher magnification) but not in esgG66B sna1 embryo (F). In contrast, leg imaginal discs (l) are visible in a parasagittal optical section of an esgG66B embryo (G) as well as in a esgG66B sna1 (H) embryo. Bar, 20 µm. of Esg and Sna in wing disc development are interchangeable. Similar DNA binding specificities of the two proteins suggests that they control the same set of genes required for wing development. Overlapping or ‘redundant’ activity of related basic helix- loop-helix transcription factors during neurogenesis and myogenesis have been reported (reviewed by Jan and Jan, 1993). Given the result of this study, other aspects of development are also likely to be regulated by overlapping activity of multiple transcription factors belonging to the same family. Therefore results from genetic experiment in which a single gene is disrupted should be interpreted with caution.

Fate of wing primordium in the esg sna double mutant embryo We have shown that in the esg sna double mutant, Sna expression initiated normally. We also observed that Vg was transiently expressed in wing primordium of the double mutant (our unpublished observation). Although we were unable to detect such transient expression of esg-lacZ in the double mutant, probably due to the low level of the lacZ expression, it is very likely that the initial stage of wing development Fig. 6. esg and sna maintain marker expression of wing disc. All proceeded normally in the absence of esg and sna until stage embryos were stained with anti-Sna antibody. (A, D) Control embryo 13. However, the following three points demonstrate that wing (sna1/CyO). (B, E) sna1 embryo. (C, F) esgG66B sna1 embryo. development is discontinued in later stages. First, the (A-C) Embryos in stage 13. (D-G) Stage 15 embryo. At stage 13, expression of the three marker genes (sna, vg and esg-lacZ) Sna began to be expressed in wing and haltere primordia (arrowheads) in the control (A) and sna mutants (B) and in esg sna was abolished. Second, the apical constriction of the pri- double mutants (C). Sna expression continued and Sna accumulated mordium cells was not observed. Third, the cell death of the in wing and haltere discs in the stage 15 control (D) and sna mutant primordium did not occur. These observations support the idea (E) embryos. However, in esg sna double mutant embryos (F), such that the absence of esg and sna allows wing primordium to expression was lost (arrowheads). T2 and T3 indicate the second and transform into larval epidermis. Larval epidermal cells have a third thoracic segments. Bar: 50 µm. Overlapping activity of zinc finger proteins 1065 characteristic microvilli structure over their apical surface and less likely. An alternative, more simple explanation is that Esg secrete cuticle in late stage embryos (Martinez Arias, 1993). and Sna function both as an activator and as a repressor, We did not examine these properties of the larval epidermis in depending upon the target promoter context. For example, a wing primordia of double mutant embryos because we were cellular coactivator protein may mediate transcriptional acti- unable to positively identify the primordium after it lost the vation by an otherwise silent DNA binding protein. Another marker gene expression. possibility is that Esg and Sna interfere with a transcriptional repressor, thereby allowing transcriptional activation by an Auto- and crossregulation of esg and sna activator binding at a different site. A case of a single protein Our study revealed the interesting regulatory mechanisms of that functions both as an activator and as a repressor of tran- Esg and Sna expression in the wing primordium. The Sna scription has been reported for the C2H2 type zinc finger tran- expression seen in the esg mutants was not seen in the esg sna scription factor Krüppel (Kr). In tissue culture cells, a low con- double mutants (Fig. 3B,D), indicating that Sna expression is centration of Kr activates a target promoter containing a Kr activated by sna itself. Similarly, Sna expression seen in the binding site and at high concentration, Kr repress the same sna mutants was not seen when the esg activity was addition- promoter (Sauer and Jäckel, 1991). Since our arguments are ally removed (Fig. 3C,D), indicating that esg activates Sna based solely on genetic data, any interaction involving Esg and expression. The activation of Sna by esg and sna itself are Sna could be indirect. To resolve this issue, a molecular likely to account for the accumulation of Sna in stage 15 analysis of interaction between Esg, Sna and their target embryos (Fig. 6D). Furthermore, it is likely that the expression promoters is necessary. Analysis of the esg or sna enhancer of Esg is also regulated by similar mechanisms. The esg-lacZ responsible for expression in the imaginal discs is a key to expression seen in the esg mutant embryo was not seen in the understanding the function of Esg and Sna at a molecular level. esg sna double mutant (Fig. 5B,D), indicating that sna activates the esg enhancer. Similarly, the esg-lacZ expression A two step model for wing disc formation seen in esgG66B sna1/sna1 embryos was not seen when one We propose that the wing disc formation can be separated into copy of wild-type esg was additionally removed (Fig. 5C,D), two steps. The first step is determination by an extrinsic signal indicating that the esg enhancer is activated by esg itself. Since which induces vg, esg and sna transcription (Fig. 7A). Such an the esg-lacZ expression appears to reflect endogenous esg inducer could be the combined activity of Dpp and Wg or a expression, these results suggest that Esg expression in the transient intrinsic gene activity induced by an external signal. wing disc is regulated by esg itself and sna. Taken together, Since the transcriptions are dependent on an external signal our observations suggest that Esg and Sna expression in wing source, the determination is not fixed in this stage. In the discs is maintained by their auto- and crossactivation. Sna second step, esg and sna initiate an intrinsic program of auto- expression in prospective mesoderm is also regulated by sna and crossactivations to stabilize their own expression (Fig. 7B). itself in the early embryo (Ray et al., 1991). Autoactivation It should be noted that the ubiquitous esg or sna expression was found for several genes such as fushi-tarazu (Hiromi and restored wing disc formation only in the wing primordium of Gehring, 1987), even-skipped (Frasch et al., 1988), Ultra- the double mutant. This point highlights the second role of the bithorax (Bienz and Tremml, 1988) and en (DiNardo et al., putative inductive signal in establishing the competence of the 1988), and was implicated for stable, sometimes heritable wing primordium to respond to esg and sna. Thus, only those maintenance of gene activity. In the case of esg and sna, the cells that transiently express esg and sna, and have acquired crossactivation by each other in addition to the autoactivation the competence can turn on the auto- and crossregulatory is likely to give an additional advantage to maintain their stable circuits of Esg and Sna, and enables further wing development. expression. We speculate that this two-fold restriction is important for Transcriptional control by Esg and Sna The finding that Esg and Sna activate gene expression is in Step 1 Step 2 sharp contrast to the previous findings that Sna functions as a Inductive cue direct repressor of rhomboid transcription in the embryo (Ip et al., 1992; Gray et al., 1994), and Esg acts as a repressor of transcription in tissue culture cells (Fuse et al., 1994). One expla- esg sna vg esg + Competence nation may be that the function of Esg and Sna is to repress sna vg & others genes that determine larval character, thereby allowing expression of wing disc-specific genes. In such cases, the roles Fig. 7. The two step model for the wing fate determination. Step 1: of Esg and Sna in promoting wing disc development are by stage 13, induction by extrinsic signals define two properties of indirect and permissive. However, in the heat shock rescue the wing primordium. Induced cells turn on vg, esg and sna experiment, the level of Esg or Sna expression that was suffi- transcription (left, blue state) and become competent to respond to cient to rescue the esg sna double mutant phenotype did not Esg and Sna (right, red state). In this stage, the cell’s properties have adverse effects on the development of larval tissue (Figs depends upon external signals and the cells are not fully committed. 3 and 4). Therefore, if Esg and Sna function by repressing Step 2: after stage 13, cells that have the two properties (yellow) initiate auto- and crossregulatory circuits to stabilize Esg and Sna larval cell fate, such a function must be restricted to the cells expression and the cells are irreversibly committed to wing fate. At that were induced to differentiate as wing imaginal disc. Thus, the same time, Esg and Sna regulate vg and other genes essential for the putative wing inductive signal has two effects. One wing development to promote invagination and pattern formation. promotes wing differentiation and the other represses larval Since this model is derived from genetic data, any interaction development. We consider this explanation complicated and indicated in this figure could be indirect. 1066 N. Fuse, S. Hirose and S. Hayashi defining a sharp border between imaginal and larval cells. Esg small chromosome region of Drosophila melanogaster containing the and Sna then control subordinate genes essential for wing structural gene for alcohol dehydrogenase. IV: Scutoid, an antimorphic development, such as vg (Fig. 7B). Since the wing cell fate was mutation. Genetics 102, 401-420. Bate, M. and Martinez Arias, A. (1991). The embryonic origin of imaginal not maintained in the esg sna double mutant, the auto- and discs in Drosophila. Development 112, 755-761. crossactivations by esg and sna are likely to be responsible for Bienz, M. and Tremml, G. (1988). 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Biol. 198, 157-169. and abdominal histoblasts in the embryo (Whiteley et al., 1992; DiNardo, S., Sher, E., Heemskerk-Jorgens, J., Kassis, J. and O’Farrell, P. (1988). Two-tiered regulation of spatially patterned engrailed gene Hartenstein and Jan., 1992; Hayashi et al., 1993; Younossi- expression during Drosophila embryogenesis. Nature 332, 604-609. Hartenstein et al., 1993). The role of esg in the development Ferguson, E. L. and Anderson, K. V. (1992). decapentaplegic acts as a of these cells during embryogenesis is not clear because these morphogen to organize dorso-ventral pattern in the Drosophila. Cell 71, 451- cells were apparent in strong esg mutant embryos (Hayashi et 461. Frasch, M., Warrior, R., Tugwood, J. and Levine, M. (1988). Molecular al., 1993; Fig. 5G). Given the result of this study, it is con- analysis of even-skipped mutants in Drosophila development. 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