Karen Staehling-Hampton, Allen S. Laughon and F. Michael Hoffmann*. McArdle Laboratory for ...... expression of zen and dpp (Roth et al., 1989; Rushlow et al.,. 1989; Steward .... Heberlein, U., Wolff, T. and Rubin, G. M. (1993). The TGFβ ...
Development 121, 3393-3403 (1995) Printed in Great Britain © The Company of Biologists Limited 1995
3393
A Drosophila protein related to the human zinc finger transcription factor PRDII/MBPI/HIV-EP1 is required for dpp signaling Karen Staehling-Hampton, Allen S. Laughon and F. Michael Hoffmann* McArdle Laboratory for Cancer Research and the Laboratory of Genetics, University of Wisconsin Medical School, 1400 University Avenue, Madison, WI 53706, USA *Author for correspondence
SUMMARY Little is known about the signal transduction pathways by which cells respond to mammalian TGF-βs or to decapentaplegic (dpp), a Drosophila TGF-β-related factor. Here we describe the genetic and molecular characterization of Drosophila schnurri (shn), a putative transcription factor implicated in dpp signaling. The shn protein has eight zinc fingers and is related to a human transcription factor, PRDII/MBPI/HIV-EP1, that binds to nuclear factor-κBbinding sites and activates transcription from the HIV long terminal repeat (LTR). shn mRNA is expressed in a dynamic pattern in the embryo that includes most of the known target tissues of dpp, including the dorsal blastoderm, the mesodermal germlayer and parasegments 4 and 7 of the midgut. Mutations in shn affect several develop-
mental processes regulated by dpp including induction of visceral mesoderm cell fate, dorsal/ventral patterning of the lateral ectoderm and wing vein formation. Absence of shn function blocks the expanded expression of the homeodomain protein bagpipe in the embryonic mesoderm caused by ectopic dpp expression, illustrating a requirement for shn function downstream of dpp action. We conclude that shn function is critical for cells to respond properly to dpp and propose that shn protein is the first identified downstream component of the signal transduction pathway used by dpp and its receptors.
INTRODUCTION
endoderm (Immerglück et al., 1990; Panganiban et al., 1990; Reuter et al., 1990; Bienz, 1994; Staehling-Hampton and Hoffmann, 1994). During the larval stages, dpp expression is activated by the product of the hedgehog gene in specific sets of cells in the imaginal disks (Basler and Struhl, 1994). dpp is required for the proliferation, differentiation and proximaldistal patterning of the imaginal disks and the migration of the morphogenetic furrow across the eye imaginal disk (Heberlein et al., 1993; Masucci et al., 1990; Bryant, 1988). Cells respond to dpp or other TGF-β-related factors through the activation of transmembrane serine/threonine kinase receptors (Massagué et al., 1994; Miyazono et al., 1994). In Drosophila, four transmembrane serine/threonine kinases that are similar to mammalian TGF-β receptors have been identified (Childs et al., 1993; Penton et al., 1994; Nellen et al., 1994; Brummel et al., 1994; Xie et al., 1994; Letsou et al., 1995; Ruberte et al., 1995). Three of these, thick veins (tkv), saxophone (sax) and Atr I, have sequence characteristics of type I receptors. One of the Drosophila receptors, punt, is similar in sequence to other type II receptors. Molecular evidence indicates that the tkv, sax and punt receptors can bind dpp or its closest mammalian relative BMP2 (Penton et al., 1994; Brummel et al., 1994; Letsou et al., 1995). Atr I and punt also bind to mammalian activin (Childs et al., 1993; Wrana et al., 1994). Analysis of phenotypes caused by mutations in tkv, sax and punt has implicated tkv and punt in virtually all dpp
The transforming growth factor beta (TGF-β) superfamily consists of more than 24 secreted factors that play key roles in cellular differentiation and proliferation (Kingsley, 1994). The high degree of evolutionary conservation of this family of proteins is indicated by the identification of three members in Drosophila, decapentaplegic (dpp), 60A and screw (Padgett et al., 1987; Doctor et al., 1992; Wharton et al., 1991; Arora et al., 1994). Genetic analysis has implicated these factors in a variety of developmental processes. For example, embryos that lack dpp are completely ventralized and fail to form dorsally derived structures (Ferguson and Anderson, 1992; Irish and Gelbart, 1987). Injection of increasing amounts of dpp mRNA causes progressive increase in dorsal cell fates, suggesting that an activity gradient of dpp instructs different dorsal/ventral ectodermal cell fates along the gradient (Ferguson and Anderson, 1992). Recently, it has been reported that dorsal ectodermal expression of dpp is also critical for the induction of specific cell fates in the underlying mesodermal cell layer that gives rise to the visceral mesoderm (Frasch, 1995; StaehlingHampton et al., 1994). Later in embryogenesis, dpp is involved in maintaining patterns of homeotic gene expression in the midgut visceral mesoderm and in the induction of the homeotic gene labial across germ layers in the developing midgut
Key words: Drosophila, transforming growth factor-beta, decapentaplegic, mesoderm development
3394 K. Staehling-Hampton, A. S. Laughon and F. M. Hoffmann
Fig. 1. Genetic enhancement of dpp and tkv phenotypes by an allele of shn. Wings from adult flies are shown. shnIB/dpps5 flies have a terminal gap at the end of the fourth longitudinal vein (B, arrow) This phenotype is rescued by addition of a dpp transgene (A). A slight thickening of the wing veins occurs in flies heterozygous for Df(2L)tkv2 and tkv1, an adult viable allele (C). Introducing one copy of shnIB into the tkv1/(Df)tkv background greatly enhances the mutant phenotype (D). The L2, L3, L4 and L5 wing veins are severely thickened and do not extend to the tip of the wing blade (D).
responses and sax in a subset of dpp responses. Mutations in Atr I have not been reported. By analogy to the mechanism of ligand activation of the mammalian TGF-β receptors, it is believed that ligand-induced oligomerization of the Drosophila type II and type I receptors results in the phosphorylation and activation of type I receptors by type II receptors (Wrana et al., 1992). However, it is not currently known what proteins signal downstream of the activated type I receptors. In this report, we describe the molecular and genetic characterization of schnurri (shn). shn is a putative zinc finger transcription factor related to human PRDII/MBP1/HIV-EP1 (Fan and Maniatis, 1990; Baldwin et al., 1990; Maekawa et al., 1989). Analysis of shn mRNA expression and mutant phenotypes indicates that it is expressed in tissues that respond to dpp signaling and that its expression is required for these responses to dpp. shn mutations cause a zygotic embryonic phenotype similar to that caused by mutations in the dpp receptors tkv and punt, absence of dorsal hypoderm resulting in a dorsal hole in the cuticle. shn mutant alleles interact genetically during wing formation with dpp alleles and with alleles of tkv. Furthermore, dpp-induced mesoderm gene expression is blocked in shn mutants. We propose that the signal transduction pathway(s) which are initiated when dpp activates its receptors require shn function to effect dpp-induced cell fate changes. MATERIALS AND METHODS Drosophila stocks shnIB, tkv1, Df(2L)tkv2 and dpps5 are described in Lindsley and Zimm (1992). twist-GAL4 and UAS-dpp flies are described in StaehlingHampton et al. (1994) and Staehling-Hampton and Hoffmann (1994). P. element stock P{ry[+], P\T:lacZ:Hsp70 Km[r] ori=PZ} I(2)04738, cn/CyO; ry[506] was obtained from the Indiana stock center (Bloomington, IN).
Table 1. Interaction of dorsal-open genes with dpp and tkv alleles
schnurri basket punt anterior open lethal(2)1A109
Heterozygous phenotype with dpps5
Heterozygous phenotype with Df(2L)tkv2
Reduced expression of labial
91 76 53 100
41 40
Yes No Yes No No
Numbers represent the percentage of heterozygous progeny with a wing vein phenotype. Alleles of other dorsal-open genes, yurt, kayak, pannier and canoe, were tested but did not show an interaction. None of the dorsal-open mutations showed a strong interaction with alleles of screw or 60A, the other two TGF-β-related factors in Drosophila or with alleles of saxophone and punt, two other Drosophila TGF-β receptors (data not shown).
Molecular analysis of shn Genomic DNA from adult flies with the P. element insertion was prepared according to Ashburner (1989) except that a different lysis buffer (0.1 M Tris-HCL pH 9.0; 0.1 M EDTA; 1% SDS) was used. Plasmid rescue was done using the procedure of Karpen and Spradling, Carnegie Institute (personal communication). The rescued DNA was transformed into maximum efficiency DH5α cells (GIBCO BRL). A 4 kb HindIII fragment of plasmid-rescued genomic DNA was labeled with digoxigenin and used as a probe for embryo and polytene chromosome in situ hybridizations. The 4 kb genomic fragment hybridized to the shn genomic region (47E-48B) and detected a dorsal specific message in blastoderm embryos. The same genomic fragment was then used as a probe to isolate cDNAs from a 4-8 hour embryonic cDNA library (gift of Nick Brown). The shn cDNAs were sequenced by standard dideoxy chain termination protocols (Sequenase, US Biochemicals). The longest cDNA recovered was 6.5 kb and was truncated at the 5′ end. To obtain more 5′ sequence, RACE PCR was done using the Marathon amplification cDNA kit (Clontech) according to the manufacturer’s protocol. The RACE products ranged in size from 3.5 to 2.0 kb and were identical
dpp signaling requires schnurri 3395 except for length. With the RACE sequence, the total length of the cDNA was 9 kb. Sequence alignments were done using the Bestfit program developed by the Genetics Computer Group (Madison, WI).
dpp pathway, we examined the genetic interactions between nine different dorsal-open mutations and alleles of dpp or tkv (Table 1). We also examined the dorsal-open mutant embryos for changes in the expression of the homeotic gene labial, a target of the dpp signaling pathway in the embryonic midgut (Staehling-Hampton and Hoffmann, 1994; Panganiban et al., 1990; Immerglück et al., 1990; Reuter et al., 1990).
In situ localization and antibody staining The anti-fasIII monoclonal was a gift of M. Bate, University of Cambridge and was originally described in Patel et al. (1987). snail antibody made in rabbits was a gift of Sean Carroll, University of Wisconsin, and was originally 1 MENATTTTAKHYKTAGKQYKTNKVNNTRRATAAAAAAATTTVTTAATPTKKRTYRETATA described in Alberga et al. (1991). fasIII (1:100) and snail (1:500) antibody stainings were done 61 TTVTQRSTNKANIAAAIALAAATEATASASASATATATDATLTASKAAATAAATTDAASG according to the methods of Michael Bate, Uni121 NSNSSSKPSTSTRDKLGEVPLPTVDSNHIISNNNNNNNNNNTSNNNNNNHHSDNSISENN versity of Cambridge (personal communication); 181 YEFKSRDKASLSDSKMTLQQMAAVSSNQEPVAPNVANTMSNSSIINSQQTTVNATPATDE Horse radish peroxidase (HRP) detection was done 241 APLGDRSNISRYLHKKFKRLASTTEVDSWSATNGGGALQASGDAVRTTSLSSNSSLSPPP using the Vectastain Elite ABC kit developed by 301 TTPLANGHHLMTQQQSHQQVQQQQQQQTPPPSVAIHEFSANFVNSNHVNAIGHEESIISN Vector laboratories, Inc. anti-shn3 antibody stainings were done 361 SGYKAAGRTRTPLANSNSNTNSTSNSNSNHAANATLSPASFAQHQQLTQPTTVSPGIPGG according to the methods of Carroll and Scott 421 AAAPNPACEKSGRYVCQYCNLICAKPSVLEKHIRAHTNERPYPCDTCGIAFKTKSNLYKH (1985) with the following modifications. The shn 481 CRSRSHAARARGLEVPADADDGLSDQDAELSNSSSELPSRAGSPYEEPINSPTPSPSTLS primary antibody was preabsorbed overnight at 541 AAKSAYIQQPPLPTYMQQLPLGSPAAGTLPPTTADNHHSATAQHRQSIDYKPYKPKFHNA 4°C in a large volume of wild-type embryos in PBS 601 SMYSCSSKELQQQQQQLQIQQQQQQHQLAQQKLSIQLPLVQQPSLAHPTLSPSTQMKMKH + 0.1% Triton X-100 and 0.2% BSA. To block background staining from endogenous peroxi661 HINSHQIQLQLQQQQSLLAQQSLLAAMPPGGVYYLGQPSYYNQDTAAAIHQHALAIQQFQ dases, the embryos were treated for 10 minutes in 721 IHLAQQQQQQQQQHPPLVKATPPMQQPPSLPPQQLVRANSQISSVATAPATPTPAANLST 3% H2O2 after fixation. The shn3 antibody was 781 FASSSGGKQVNVAKVQEHISKLISQNEAIVENKEILLQKKYPKQLSRSRSFNNANSNNAS used at a concentration of 1 µg/ml. 841 QHGSNASALHANNSHNNTNAQMPERETKVNLAQAIFQKQQHQLQQQQQQQITQQQQQQLE In situ hybridization to mRNA in embryos for RNA utilized digoxigenin-labeled cDNA or RNA 901 QQNYYTYIQQQQECQQQQLEPPNGVVKRNAYKPGVVMTTPVKQQQQLPPPPSPLPMQTMQ probes (made using the Genius kit (Boehringer961 YRQDPATPVTKIEQPTTAVPVMPLNLSAKPKPTLVTVPVSSLSTSSLAPTPTTSTNPTSS Mannheim)) according to the methods of Tautz and 1021 SKTAPPPAQVNNSIIKNLLLNARGLAVPIGEGDDAVYSCPICASEFRSADDLKLHNSTYC Pfeiffle (1989). 1081 QDASSSAPMSPASSPFRSNSISLSLPELKSHMANSKNPLSLAKLAWSQLKTKRSSLVLSR
Production of shn3 antibodies A 39×103 Mr C-terminal segment containing the last three shn zinc fingers was expressed and purified as a histidine-tagged fusion. A DNA fragment extending from ScaI at 6737 to BstBI at 8300 was inserted into pRSETA (Invitrogen) at the PvuII and BstBI sites. The resulting 43×103 Mr fusion protein was extracted from inclusion bodies in 6 M guanidine/20 mM NaPO4, pH 7.8 and affinity purified on a Probond Ni2+ column (Invitrogen) by adsorption, washing in 8 M urea/500 mM NaCl/20 mM NaPO4, pH 7.8, and elution in wash buffer adjusted to pH 4. Eluted protein was dialysed against PBS (5 mM NaPO4, pH 7.5/137 mM NaCl) plus 10 µM ZnSO4 and used to immunize rabbits.
1141 LSAAQTPARTSTVTAPTVTASAPAPAVATVTAPSSAPAPQIEALRFVDAPLPSPGPLLGK 1201 TPLVDYAQQSTPRKAQDSVVITKMHEDRQFVIEAQPAKRIKTSDLVVASSSQQPTSFNFS 1261 FNNQNSSNSVPELQSSKEERLRRFTSSGGSMIPISECPDLDNSPKMIRTPLLSGGSFQDV 1321 SVKVNNETGSSSKERKLMALVSGSGLLGVSSGPQHFQFPPINSITAFNPLTLPPMSGGDK 1381 TTPVTPIPHVPGMPGPGSLTPQMPLLPPPPQQLQLPIPSSRGRSPNRKQPSPLLLGGGSG 1441 ELKALSPFGGVQNVPSEFSRQPPTPAQRQALQWNSKETPKKAPFNFLRMADNVKTTEPEV 1501 RHFNLENVISGKQQELPLTPLHVDTPNGNAPEETSPVASASASAKSKFLRPTSLPLKPGT 1561 FTPKRHHGITPTANTLPLISPETPRPSKSCVQLYLNGHAYTYLGLKCSTKMFYCTVNCPQ 1621 PSYVAGMHKLSMYSVWQVCEENQPHPLGFKLKQVMALYDSRQRMLGNGSSTAMAGSGKLS 1681 YNLVASQQIVSSPSTSSTSSAFYQGPLKTPPTVTIAALSEANVAAKANEEAQAKKLETSP 1741 SGQPLVGGYESHEDYTYIRGRGRGRYVCSECGIRCKKPSMLKKHIRTHTDVRPFTCSHCN 1801 FSFKTKGNLTKHMQSKTHFKKCIELGINPGPMPPDSEFLDVDMDFDQQSSTSAGGRTSSM 1861 AGESDSDDYSDNESESSDTDESKSRQKEHEAARGLLSLSMTPPIPQSVSPYPQLQDTPLP 1921 AASPANSIGSSGSQPKRLVCSFTSPKPPFDYQKQEQYYSNPEESKPKRSVANEESAPMDL 1981 TKPRGSILLISPSPVSVPAHDLPKSQAQQMHDVIFGTSGNESGFMKTLISVSDKVRISAE
RESULTS Genetic analysis and cloning of schnurri Animals mutant for the dpp receptors, tkv or punt, lack embryonic dorsal hypoderm and have a hole in the dorsal surface of the cuticle (Penton et al., 1994; Nellen et al., 1994; Brummel et al., 1994). Other mutations that have a ‘dorsal-open’ phenotype have been isolated in genetic screens for embryonic lethal mutations in Drosophila (Jürgens et al., 1984). To investigate whether some of these genes encode products that function on the
2041 MEEQAKHEAEGEDVQLQTYIKEHALHQAKIKQSQFSRSYLINTLYTAASPVMSSSSTLFT 2101 ANSRPVMSVNEVPSIEVHEVKTPEAIESPRSAPEQAPVILAQIAQEENEEPNIAEPHNAN 2161 LPAAVQQPDVNEFTGVLGNPTAPPTSSVTATSVSTTTAAPVAPSSTANSTQPAQRTVIVG 2221 EDGFKSSTPTSKIGDLQHVSYGRGVPPAPIAGDARPTCTMCSKTFQRQHQLTLHMNIHYM 2281 ERKFKCEPCSISFRTQGHLQKHERSEAHKNKVMMTSTFGVPTTSNPRPFECTDCKIAFRI 2341 HGHLAKHLRSKTHVQKLECLQKLPFGTYAEIERAGISLTEIDTSDCENSLISLKLLAQKL 2401 QEKDPGKLSSYTTPSGMMQLAQDAAGPVSQDSASEDGFSAAIASAIASLDNDSAGNTPKR 2461 ASSMSEDETVANGLNHSLKRRLPGSFSSTGEESDNPPESSGEKRARSGQELPLPVAVPVA 2521 ASAAASN
Fig. 2. Complete protein sequence of schnurri. Boxes highlight the first (amino acids 436-486), second (amino acids 1768-1818) and third (amino acids 2258-2353) sets of Zn fingers. The single C-C-X13-H-C-type Zn finger motif is underlined twice. The domain rich in acidic amino acids is underlined with a thin line and 9 S/TPXK/R repeats are underlined in bold.
3396 K. Staehling-Hampton, A. S. Laughon and F. M. Hoffmann One of the dorsal-open mutations, schnurri (shn), interacted genetically with dpp alleles and with tkv alleles and showed reduced midgut expression of labial. 91% of shnIB/dpps5 heterozygotes had mutant wings with a shortened L4 wing vein (Fig. 1B). The shortened wing vein phenotype was rescued by addition of a dpp transgene into the background (Fig. 1A) and was indistinguishable from wild type. Flies with one copy of an adult viable tkv allele, tkv1, over a deficiency of tkv, Df(2L)tkv2, have wing veins that are slightly thickened at the tip (Fig. 1C; Penton et al., 1994). Introducing one copy of shnIB into a tkv1/Df(2L)tkv2 background greatly enhanced the thickened vein phenotype and led to terminal gaps in veins L2, L3, L4 and L5 (Fig. 1D). Some of the tkv1/Df(2L)tkv2, shnIB flies were also missing terminal leg claws (data not shown), a phenotype associated with reduced dpp expression in the imaginal disks (Spencer et al., 1982). The expression of the homeotic gene labial was reduced in the midgut endoderm of shnIB homozygotes (data not shown), indicating that expression of a specific dpp target gene was affected in shn mutants. A P element insertion line was obtained that failed to complement the embryonic dorsal-open phenotype of shnIB (data not shown). The P element insertion allele also showed heterozygous genetic interactions with dpps5 (data not shown). A 4 kb piece of genomic DNA flanking the transposon insertion was recovered by plasmid rescue of the P element and used to identify shn cDNAs (see Materials and Methods). The shn cDNA has an open reading frame of 7584 base pairs with approximately 0.2 kb of 5′ and 1.2 kb of 3′ untranslated sequence and is predicted to encode a 272×103 Mr (2528 amino acid) protein (Fig. 2). The predicted shn protein sequence includes 8 regions with sequence similarity to C2-H2-type zinc fingers (Klug and Rhodes, 1987) (Fig. 2). The Zn fingers are clustered in two pairs and one triplet. There is also a region similar in sequence to acidic transcriptional activation domains (Fig. 2). Between the first and second predicted Zn finger pairs, there is a
Fig. 3. Pattern of schnurri mRNA expression in wild-type, dpp null and shn mutant embryos. shn antisense probes (B) but not sense probes (A) detect a maternal message in early embryos. shn mRNAs are expressed dorsally at the blastoderm stage (C). As embryogenesis proceeds, shn can be detected in the invaginated germ band (D, arrowhead) and is expressed in the mesoderm during germ band extension (E, solid arrow). shn mRNA is undetectable in embryos homozygous for the P element insertion (E, open arrow). In germ band retracted embryos, shn is expressed in the endoderm (F, arrows). After the posterior and anterior midgut fuse shn is expressed at higher levels in two domains in the midgut (G, arrowheads). This expression is transient and is not detected after the midgut constrictions form (data not shown). In dpp null embryos, shn is expressed in the mesoderm (H, arrows) and in dorsal ectoderm (H, arrowheads). The genotype of the dpp null embryos is dpp61/dpp61. dpp null embryos were identified by their severe ventralized morphology.
dpp signaling requires schnurri 3397 region, C-C-X13-H-C, that may also form a single Zn finger and seven repeats of the amino acid sequence S/TPXK/R (Fig. 2). S/TPXK/R motifs are putative DNA-binding domains and may be regulated by phosphorylation (Hill et al., 1990; Suzuki, 1989; Churchill and Suzuki, 1989). schnurri mRNAs are expressed in the dorsal blastoderm and in the mesoderm The expression pattern of the shn message in embryos is similar to the pattern of mRNA for the dpp receptor tkv (Penton et al., 1994; Brummel et al., 1994). Like tkv, the shn message is maternal and was expressed faintly but uniformly in early embryos (Fig. 3B). In contrast to the antisense probe (Fig. 3B), a sense probe made to shn did not detect a message in early embryos (Fig. 3A). During the blastoderm stage, shn mRNA was restricted to the dorsal half of the embryo similar to the pattern of dpp (St Johnston and Gelbart, 1987) and tkv mRNAs (Fig. 3C). As embryogenesis proceeded, shn was detected in the presumptive mesoderm of the invaginating ventral furrow (Fig. 3D, arrowhead) and was expressed in the mesoderm throughout germ band extension (Fig. 3E, solid arrow). tkv mRNA is also detected in the mesoderm throughout gastrulation (Penton et al., 1994; Brummel et al., 1994). In germ band retracted embryos, shn was expressed in the endoderm (Fig. 3F, arrows). After fusion of the posterior and anterior midgut, shn was detected in two domains corresponding to parasegment 4 and 7 (Fig. 3G, arrowheads). The shn midgut domains are transient and are only detected prior to the formation of the midgut constrictions. In dpp null embryos shn mRNA is detected in the mesoderm (Fig. 3H, arrows) and dorsal ectoderm (Fig. 3H, arrowheads), showing that dpp is not required for shn expression. The specificity of the probe for shn mRNA was confirmed by the absence of detectable expression in shn mutant embryos (Fig. 3E, open arrow). Expression of shn protein in wild-type and shn mutant embryos To demonstrate that the cDNA we cloned is shn, wild-type and shn mutant embryos were stained with antibodies made to the third zinc finger set of the putative shn cDNA. In wild-type germ band retracted embryos, shn protein was detected in the endoderm (Fig. 4A, arrowheads) and in the foregut (Fig. 4A, arrow). In embryos homozygous for the shn P element insertion no protein was detected in either the foregut or the endoderm (Fig. 4B). This was consistent with the absence of shn mRNA in shn P element homozygotes (Fig. 3E). In embryos homozygous for the chemically induced shnIB allele, no staining for shn protein was observed (Fig. 4C). Therefore, two different mutant alleles cause the absence of detectable shn product. This evidence, together with the isolation of the DNA using the shn P element insertion and the localization of the cloned DNA on polytene chromosomes to the shn region, all support the conclusion that the cloned cDNA is shn. A more complete description of the shn protein expression pattern will be presented in a future manuscript. Expression of dpp, bagpipe, fasIII and snail in shn mutants The dorsally restricted expression pattern of shn suggested that shn might be required for the dorsal blastoderm expression of either dpp or tkv. To test this possibility, we examined the
Fig. 4. Expression of shn protein in wild-type and shn mutant embryos. In wild-type germ band retracted embryos (A), shn protein is expressed in the endoderm (A, arrowheads) and in the foregut (A, arrow). In embryos homozygous for the shn P element insertion (B), no protein is detected in either the foregut (B, arrow) or the endoderm (B, arrowheads). In embryos homozygous for the shnIB allele (C), shn protein is undetectable in the foregut (C, arrow) and endoderm (C, arrowheads). Approximately 25% of the embryos (n=40) produced from a cross of shn P/CyO males and females or shnIB/CyO males and females lacked endodermal and foregut expression.
expression of dpp or tkv mRNA in a population of embryos from a cross of shnIB/CyO males and females. 25% of the embryos would have homozygous shn mutations. 90% of cellular blastoderm embryos (n=132) showed normal dorsal expression of dpp and 96% (n=67) showed normal dorsal expression of tkv. The small percentage of embryos not staining is likely do to problems with fixation or probe permeability. We conclude that the absence of zygotic shn product does not alter early expression patterns of dpp or tkv. The later shn mRNA expression pattern suggested that shn might be involved in mesodermal gene expression and in the
3398 K. Staehling-Hampton, A. S. Laughon and F. M. Hoffmann
Fig. 5. Abnormal expression of dpp, bagpipe, fasIII and snail in schnurri mutant embryos. dpp mRNA was detected in discrete domains along the developing gut tube in the pharynx (p), esophagus (e), gastric caeca (gc) parasegment 7 of the midgut (mg) and hindgut (hg) in wild-type germ bandretracted embryos (A). In shnIB homozygotes (B), dpp was not detected in the gastric caeca (gc) or the midgut (mg), but could be seen in the pharynx (p), esophagus (e) and the hindgut (hg). dpp mRNA in the ectoderm (marked by arrowheads) also was reduced in shnIB mutants (B) compared to wild-type embryos (A). In wild-type embryos (C), bagpipe (bap) mRNA was detected in patches in the dorsal mesoderm at stage 10. In embryos homozygous for the shn P element insertion (D) bap expression in the mesoderm was greatly reduced. The same embryos were photographed at a midline focal plane to show that the wild-type (E) and the shn mutant (F) are at approximately the same stage in development. Expression of fasciclin III (fasIII) protein in the visceral mesoderm of wild-type (G) and shnIB mutants (H) at stage 12. Notice how the domain of fasIII expression is continuous and uniform in wild-type embryos compared with the discontinuous and globular expression in shnIB embryos. In germ band retracted embryos, snail protein was detected in the imaginal disk precursors of wild-type embryos (I, arrowheads) but not in shnIB homozygotes (J, arrowheads). In B and J, shn mutant embryos were identified by their dorsal open morphology. For D and F, the total number of embryos at stage 10/11 were counted (n=92) and approximately 25% had reduced levels of bagpipe expression.
dpp signaling requires schnurri 3399 regulation of midgut gene expression. In the visceral mesoderm of the midgut, dpp regulates its own expression in the gastric caeca primordia (parasegment 4) and parasegment 7 of the visceral mesoderm (Staehling-Hampton and Hoffmann, 1994; Hursh et al., 1993). Expression of dpp in the gastric caeca primordia and parasegment 7 visceral mesoderm was absent in shn mutants but dpp expression in other regions of the alimentary canal (pharynx, esophagus and hindgut) was unaffected (Fig. 5A,B). Note, however, that the intensity of the narrow lateral stripe of ectodermal dpp expression also was reduced in shn mutants during germ band retraction (Fig. 5A,B arrowheads). The morphology of the midgut in shn mutants is abnormal and the gut constrictions do not form. Round midguts without constrictions are characteristic of mutants that fail to differentiate visceral mesoderm such as bagpipe (bap) and tinman (Bodmer, 1993; Azpiazu and Frasch, 1993). To determine if visceral mesoderm differentiation is abnormal in shn mutants, we examined the expression of two visceral mesoderm markers bap (Azpiazu and Frasch, 1993) and fasicilin III (fasIII) (Bate, 1993; Patel et al., 1987). Visceral mesoderm bap mRNA expression was reduced in shn mutants (Fig. 5C-F) and the uniform expression pattern of fasIII protein in the visceral mesoderm was broken up into discrete pieces (Fig. 5G,H). The imaginal disk primordia form in the lateral ectoderm at sites approximating the intersection of dpp expression and expression of wingless, the wnt1 growth factor homologue in Drosophila (Cohen et al., 1993). In shn mutant embryos, the dorsal/ventral patterning of the cuticle is largely normal with the exception of the most dorsal cuticular structures. However, when shnIB mutant embryos were stained with antibodies to snail (Alberga et al., 1991), a marker of the wing and haltere primordia, no expression in the imaginal disk precursors was seen (Fig. 5I,J). The absence of snail expression and the reduction of the lateral stripe of dpp expression (Fig. 5B) in this region of the embryo indicates a defect in the cell fates at this lateral position along the dorsal/ventral axis in shn mutant embryos. The effects of shn mutations on the mesoderm and lateral ectoderm implicate it in dpp functions. Although the mRNA expression pattern was most consistent with shn being involved in responding to the dpp signal rather than regulation of dpp expression, we tested the effects of shn mutations on dpp responses caused by ectopic expression of dpp to confirm its involvement in response to or ‘downstream of’ dpp. In wildtype embryos bap is expressed in dorsal/lateral patches on both sides of the ventral midline (Fig. 6A). When dpp is expressed ectopically throughout the mesoderm by expressing UAS-dpp in the twist-GAL4 pattern (see Brand and Perrimon (1993) for a description of the GAL4/UAS system), the expression of bap spreads and extends across the midline (Fig. 6B and StaehlingHampton et al., 1994). In a shn mutant background, however, the expansion of bap by ectopic dpp was blocked (Fig. 6C) suggesting that shn functions downstream of dpp in mesodermal induction. DISCUSSION The function of shn in dpp signaling In Drosophila, the identification of gene products acting on signaling pathways has occurred by analysis of genes with
Fig. 6. Inhibition of ectopic dpp responses in schnurri mutant embryos. Ventral view of bap expression in wild-type embryos (A) and in twist-GAL4;UAS-dpp embryos (B). The domain of bap expression is expanded and extends across the ventral midline in twist-GAL4;UAS-dpp embryos (B). Introducing one copy of shnIB into the twist-GAL4;UAS-dpp background completely blocks the expansion of bap expression by ectopic dpp (C). Late stage 10 embryos are shown. 100% of the twist-GAL4;UAS-dpp embryos displayed ectopic bap expression. Approximately one quarter of the embryos from a cross of twist-GAL4; shnIB/CyO females and shnIB/CyO UAS-dpp males did not have ectopic bap expression.
similar null phenotypes, e.g., the cascade of maternal effect genes responsible for dorsal-ventral axis formation (Chasan and Anderson, 1994), or by second-site modifier screens, e.g. the signal transduction pathway downstream of the sevenless receptor tyrosine kinase (Simon, 1994). To identify other gene products on the dpp signal transduction pathway, we examined genes whose mutant phenotypes were similar to the dorsalopen cuticle caused by mutations in the dpp receptor tkv. Mutations in one of these genes, shn, interacted with alleles of dpp and alleles of tkv. The expression of dpp target genes was reduced in shn mutants suggesting that shn is required for the expression of some dpp target genes. Mutations in other genes causing a dorsal-open phenotype did not exhibit additional interactions with dpp mutations. Several of these other genes have also recently been cloned including pannier (Ramain et al., 1993; Winick et al., 1993) and canoe (Miyamoto et al., 1995). pannier encodes a GATA transcription factor that regulates achaete and scute expression and functions during neural precursor development (Ramain et al., 1993). pannier mRNA is expressed dorsally in blastoderm embryos and its expression is absent in dpp mutant embryos (Winick et al., 1993). This is in contrast to shn mRNA expression in the early
3400 K. Staehling-Hampton, A. S. Laughon and F. M. Hoffmann embryo which is unaffected in dpp null mutant embryos (Fig. haltere disk primordia is not detectable. The defects in dorsal 3H). Thus, pannier may be directly or indirectly a target of dpp hypoderm and the lateral ectoderm correspond to two regions signaling whereas shn protein is more likely to participate in of late dpp expression, the narrow stripe at the dorsal edge of the dpp signaling cascade. The product of the canoe gene is a the ectoderm and the narrow stripe in the lateral ectoderm (St novel protein with a GLGF/DHR motif that is thought to Johnston and Gelbart, 1988; Jackson and Hoffmann, 1994). function in the same developmental pathways as Notch and The function of dpp expression in these two stripes is not scabrous (Miyamoto et al., 1995). Thus, we expect that interknown as mutations that specifically remove these aspects of ference with dpp signaling is only one of several causes for the the dpp expression pattern have not been recovered. Further dorsal-open phenotype. analysis of the shn mutant phenotype may provide new insights At late germ band extension, when the mesoderm differeninto the functions of the mid-embryonic lateral stripe of ectotiates into subtypes of mesoderm, shn and tkv are expressed in dermal dpp expression. the mesoderm, whereas dpp expression at this stage is confined shn is related to human PRDII/MBPI/HIV-EP1 to the overlying ectoderm. This expression pattern suggested that shn might function in tissues responding to dpp rather than Computer database searches revealed that shn is related to in tissues producing dpp. Expression of dpp in the apposing PRDII/MBPI/HIV-EP1, a mammalian transcription factor (Fan ectoderm is both necessary and sufficient to induce the expression of visceral mesoderm-specific genes such as bap (Frasch, 1995; Staehling-Hampton et al., 1994). In shn mutants, bap expression is A. reduced and visceral mesoderm development is abnormal as revealed by fasIII protein expression. In order to test whether shn might be exerting its effect through the regulation of dpp expression, we schnurri expressed the dpp cDNA under the control of an C 2528 AA artificial promoter responsive to the yeast GAL4 upstream activation sequence (UAS). The 67% expansion of bap expression by ectopic dpp was 78% blocked in shn mutants supporting the model that PRDII/MBP1/ shn acts downstream of dpp during mesoderm HIV-EP1 induction. It will be important to determine whether C shn protein binds to target sites in bap and activates N 2717 AA bap transcription directly. dpp and labial expression are reduced in the midgut of shn mutants, indicating that shn =500AA expression in the midgut (Fig. 3G) may be necessary for the induction of labial in the B endoderm by dpp in the visceral mesoderm and/or for the autoregulation of dpp in the visceral mesoderm. However, the general defectiveness of a. the midgut revealed by fas III staining suggests that 429 EKSGRYVCQYCNLICAKPSVLEKHIRAHTNERPYPCDTCGIAFKTKSNLY 478 the absence of normal patterns of dpp and labial :| |:|:|:||| ||||||| ||||:|| |||||| ||| :|||||||| expression in the midgut should be interpreted with 401 KKQGKYICEYCNRACAKPSVLLKHIRSHTGERPYPCVTCGFSFKTKSNLY 450 caution as they may be an indirect consequence of 479 KHCRSRSHAAR 489 the incomplete differentiation of the midgut. To || :| :| : determine if shn functions in the induction of labial 451 KHKKSHAHTIK 461 or in the autoregulation of dpp, mutations that remove or overexpress shn specifically at later b. embryonic stages, e. g., the germ band retracted 1747 GGYESHEDYTYIRGRGRGRYVCSECGIRCKKPSMLKKHIRTHTDVRPFTC 1796 stage, are needed. If a role for shn can be established |||:|:|:| ||||||||:|:| ||||||||||||||||||||||||: | in the visceral mesoderm responses to dpp, it will 2068 GGYKSNEEYVYIRGRGRGKYICEECGIRCKKPSMLKKHIRTHTDVRPYHC 2117 be of interest to determine whether the dpp-respon1797 SHCNFSFKTKGNLTKHMQSKTHFKKCIELGINPG 1830 sive regulatory regions identified in Ultrabithorax ::|||||||||||||||:|| | |||::|||: | (Ubx) (Thüringer et al., 1993), labial (Tremml and 2118 TYCNFSFKTKGNLTKHMKSKAHSKKCVDLGISVG 2152 Bienz, 1992) and dpp (Hursch et al., 1993) have Fig. 7. Comparison of schnurri and PRDII/MBP1/HIV-EP1 protein domains. Line binding sites for shn protein. drawing of shn and PRDII/MBP1/HIV-EP1 showing the conserved functional The requirement for shn in the embryonic domains (A). Groups of ovals represent the sets of Zn fingers. A single oval ectoderm is revealed by the defects in dorsal represents the C-C-X13-H-C-type zinc finger motif and the black circle represents hypoderm as well as the defects in the lateral the region rich in acidic residues. The percent of amino acid identity between the ectoderm. The narrow lateral stripe of dpp first and second zinc finger pairs of shn and PRDII/MBP1/HIV-EP1 are shown. expression in the lateral ectoderm is greatly dimin- (B) Alignment of the first (a) and second (b) Zn finger regions of shn (top) with ished and the expression of snail in the wing and those of PRDII/MBP1/HIV-EP1 (bottom).
.
dpp signaling requires schnurri 3401 and Maniatis, 1990; Baldwin et al., 1990; Maekawa et al., 1989). PRDII/MBPI/HIV-EP1 is one member of a family of related zinc finger transcription factors that bind to nuclear factor κB (NF-κB)-related sites (Fan and Maniatis, 1990; Baldwin et al., 1990; Maekawa et al., 1989). PRDII/MBPI/HIV-EP1 related proteins bind to sites in the major histocompatability complex class I genes (Van ’T Veer et al., 1992; Baldwin et al., 1990; Mitchelmore et al., 1990; Rustgi et al., 1990), the Kappa immunoglobulin genes (Baldwin et al., 1990; Rustgi et al., 1990), human interferonβ gene (Fan and Maniatis, 1990), β2-microglobulin gene (Baldwin et al., 1990), rat angiotensinogen gene (Ron et al., 1991), the αA-crystalin gene (Nakamura et al., 1990), α1-antitrypsin gene (Mitchelmore et al., 1990), the recombination signal sequences (Rss) of immunoglobulin and T-cell receptor genes (Wu et al., 1993) and the HIV long terminal repeat (LTR) (Nomura et al., 1991; Maekawa et al., 1989; Seeler et al., 1994). PRDII/MBPI/HIV-EP1 binding to NF-κB sites in the HIV-1 LTR can activate HIV-1 gene expression (Seeler et al., 1994), however, no other functional role for PRDII/MBPI/HIV-EP1 related factors has been demonstrated. PRDII/MBPI/HIV-EP1 mRNA is induced by viral infection and by growth factors (Fan and Maniatis, 1990; Baldwin et al., 1990) suggesting that the proteins have the potential to be regulated by growth factor or viral induced signaling. Like shn, PRDII/MBPI/HIV-EP1 has pairs of widely spaced Zn fingers (Fig. 7A). The shn and PRDII/MBPI/HIV-EP1 Zn fingers are remarkably conserved with amino acid identities of 67% and 78% (Fig. 7A,B). PRDII/MBPI/HIV-EP1 also has a single C-C-X13-H-C zinc finger motif and a region rich in acidic amino acids. However, unlike shn, PRDII/MBPI/HIVEP1 does not have a third triplet of Zn fingers and has only two S/TPXK/R repeats. Other PRDII/MBPI/HIV-EP1-related factors, Rc and MBP2, have 5 and 11 S/TPXK/R repeats respectably (Wu et al., 1993; Van ’T Veer et al., 1992). We conclude that shn and PRDII/MBPI/HIV-EP1 are members of the same gene family but that there may be a closer mammalian homologue of shn. Since the first two pairs of Zn fingers in shn and PRDII/MBPI/HIV-EP1 are highly conserved it is possible that shn, like PRDII/MBPI/HIV-EP1, binds to NF-κB-related sites. Two Drosophila proteins that bind to NF-κB-related sites are dorsal and the dorsal-related protein Dif. dorsal protein is present in the nucleus of ventral blastoderm cells where it is critical for activating expression of twist and repressing expression of zen and dpp (Roth et al., 1989; Rushlow et al., 1989; Steward, 1989). Dif functions in the Drosophila immune response (Ip et al., 1993). The analysis of shn mutant phenotypes has not yet addressed whether there are any functional interactions between shn and dorsal or shn and Dif that might indicate competition of the proteins for binding sites at target genes. The expression pattern of shn mRNA, the effects of shn mutations on dpp-induced responses and the sequence similarities of shn to mammalian zinc-finger transcription factors suggest that shn is a critical factor on a signaling pathway activated by dpp. Genetic screens for second-site modifiers of dpp mutant phenotypes have recovered mutations in other genes whose products may be important for dpp signaling (Raftery et al., 1995). The first of these genes to be molecularly cloned, mad, encodes a novel protein whose sequence has
unfortunately not provided any immediate clues to its molecular role in dpp signal transduction (Sekelsky et al., 1995). Further genetic dissection of the dpp signaling pathway will be facilitated by strategies that sensitize the pathway at different points to recover second-site modifiers. The identification of shn as a component on the dpp signaling pathway therefore provides new opportunities for sensitizing the pathway in an effort to identify additional gene products involved. The similarity of shn to known DNA-binding proteins also suggests that shn will be useful in the identification of additional target genes regulated by dpp signaling. This might be accomplished for example by procedures used recently to identify new target genes of Ultrabithorax (Mastick et al., 1995). Identification of target genes will be particularly useful in further understanding the roles of dpp in the imaginal disks since very little is known about the molecular mechanisms for dpp’s action in distal patterning of legs and wings and migration of the morphogenetic furrow in the eye. Finally, the evolutionary conservation of the TGF-β-related ligands and their receptors raises the promise that other components on the signaling pathway will also be well conserved and that a mammalian homologue of shn will be involved in some of the responses to TGF-β-related factors in mammalian cells. We thank Drs Michael Bate and Sean Carroll for antibodies, the Indiana Stock Center for fly stocks and Patricia Rower-Nutter for assistance with antibody stainings and DNA sequencing. We thank Dr Rahul Warrior for discussions and Drs Marcus Affolter and Konrad Basler for communication of unpublished results. This work was supported by grants from the NIH: RR06610 to F. M. H., Cancer Center Core support CA07175 and Predoctoral Training Grant CA09135 support for K. S-H; F. M. H. is the recipient of a Faculty Research Award from the American Cancer Society.
REFERENCES Arora, K., Levine, M. S. and O’Connor, M. B. (1994). The screw gene encodes a ubiquitously expressed member of the TGF-β family required for specification of dorsal cell fates in the Drosophila embryo. Genes Dev. 8, 2588-2601. Alberga, A., Boulay, J.-L., Kempe, E., Dennefeld C. and Haenlin, M. (1991). The snail gene required for mesoderm formation in Drosophila is expressed dynamically in derivatives of all three germ layers. Development 111, 983-992. Ashburner, M. (1989). Drosophila, A Laboratory Manual. Cold Spring Harbor Laboratory Press. Azpiazu, N. and Frasch, M. (1993). tinman and bagpipe: two homeo box genes that determine cell fates in the dorsal mesoderm of Drosophila. Genes Dev. 7, 1325-1340. Baldwin, A. S. Jr., LeClair, K. P., Singh, H. and Sharp, P. A. (1990). A large protein containing zinc finger domains binds to related sequence elements in the enhancers of the class I major histocompatability complex and kappa immunoglobulin genes. Mol. Cell. Biol. 10, 1406-1414. Basler, K. and Struhl, G. (1994). Hedgehog, a product of posterior compartment cells in Drosophila, organizes anterior compartment pattern. Nature 368, 208-214. Bate, M. (1993). The mesoderm and its derivatives. In The Development of Drosophila Melanogaster. (eds. M. Bate and A. Martinez-Arias), pp. 10131090. Cold Spring Harbor Laboratory Press. Bienz, M. (1994). Homeotic genes and positional signaling in Drosophila viscera. Trends in Genet. 10, 22-26. Bodmer, R. (1993). The gene tinman is required for specification of the heart and visceral muscles in Drosophila. Development 118, 719-729. Brand, A. H. and Perrimon, N. (1993). Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401-415.
3402 K. Staehling-Hampton, A. S. Laughon and F. M. Hoffmann Brummel, T. J., Twombly, V., Marqués, G., Wrana, J. L., Newfeld, S. J., Attisano, L., Massagué, J., O’Connor, M. B. and Gelbart, W. M. (1994). Characterization and relationship of dpp receptors encoded by the saxophone and thick veins genes in Drosophila. Cell 78, 251-261. Bryant, P. J. (1988). Localized cell death caused by mutations in a Drosophila gene coding for a transforming growth factor-β homolog. Dev. Biol. 128, 386-395. Carroll, S. B. and Scott, M. P. (1985). Localization of the fushi tarazu protein during Drosophila embryogenesis. Cell 43, 47-57. Chasan, R. and Anderson, K. V. (1994). Maternal control of dorsal-ventral polarity and pattern in the embryo. In The Development of Drosophila melanogaster. (eds. M. Bate and A. Martinez-Arias) pp. 387-424. Cold Spring Harbor Laboratory Press. Childs, S. R., Wrana, J. L., Arora, K., Attisano, L., O’Connor, M. B. and Massagué, J. (1993). Identification of a Drosophila activin receptor. Proc. Natl. Acad. Sci. USA 90, 9475-9479. Churchill, M. E. A. and Sukuki, M. (1989). ‘SPKK’ motifs prefer to bind DNA at A/T-rich sites. EMBO J. 8, 4189-4195. Cohen, B., Simcox, A. A. and Cohen, S. M. (1993). Allocation of the imaginal primordia in the Drosophila embryo. Development 117, 597-608. Doctor, J. S., Jackson, P. D., Rashka, K. E., Visalli, M. and Hoffmann, F. M. (1992). Sequence, biochemical characterization, and developmental expression of a new member of the TGF-β superfamily in Drosophila melanogaster. Dev. Biol. 151, 491-505. Fan, C.-M. and Maniatis, T. (1990). A DNA-binding protein containing two widely separated zinc finger motifs that recognize the same DNA sequence. Genes Dev. 4, 29-42. Ferguson, E. L. and Anderson, K. V. (1992). decapentaplegic acts as a morphogen to organize dorsal-ventral pattern in the Drosophila embryo. Cell 71, 451-461. Frasch, M. (1995). Induction of visceral and cardiac mesoderm by ectodermal Dpp in the early Drosophila embryo. Nature 374, 464-467. Heberlein, U., Wolff, T. and Rubin, G. M. (1993). The TGFβ homolog dpp and the segment polarity gene hedgehog are required for propagation of a morphogenetic wave in the Drosophila retina. Cell 75, 913-926. Hill, C. S., Packman, L. C. and Thomas, J. O. (1990). Phosphorylation at clustered -Ser-Pro-X-Lys/Arg- motifs in sperm specific histones H1 and H2B. EMBO J. 9, 805-813. Hursh, D.A., Padgett, R.W., and Gelbart, W.M. (1993). Cross regulation of decapentaplegic and Utrabithorax transcription in the embryonic visceral mesoderm of Drosophila. Development 117, 1211-1222. Immerglück, K., Lawrence, P. A. and Bienz, M. (1990). Induction across germ layers in Drosophila mediated by a genetic cascade. Cell 62, 261-268. Ip, Y. T., Reach, M., Engstrom, Y., Kadalayil, L., Cai, H., GonzálezCrespo, S., Tatei, K. and Levine, M. (1993). Dif, a dorsal-related gene that mediates an immune response in Drosophila. Cell 75, 753-763. Irish, V. I. and Gelbart, W. M. (1987). The decapentaplegic gene is required for dorsal-ventral patterning of the Drosophila embryo. Genes Dev. 1, 868879. Jackson, P. D. and Hoffmann, F. M. (1994). Embryonic expression patterns of the Drosophila decapentaplegic gene: Separate regulatory elements control blastoderm expression and lateral ectodermal expression. Dev. Dynamics 199, 28-44. Jürgens, G. E., Wieschaus, E., Nüsslein-Volhard, C. and Kluding, H. W. (1984). Mutations affecting the pattern of the larval cuticle in Drosophila melanogaster. Roux’s Arch. Dev. Biol. 193, 283-295. Kingsley, D. M. (1994). The TGF-β superfamily: new members, new receptors, and new genetic tests of function in different organisms. Genes Dev. 8, 133-146. Klug, A. and Rhodes, D. (1987). ‘Zinc fingers’: A novel protein motif for nucleic acid recognition. Trends Biochem. Sci. 12, 464-469. Letsou, A., Arora, K., Wrana, J. L., Simin, K., Twombly, V., Jamal, J., Staehling-Hampton, K., Hoffmann, F. M., Gelbart, W. M., Massagué, J. and O’Connor, M. B. (1995). Drosophila dpp signaling is mediated by the punt gene product: a dual ligand type II receptor of the TGF-β receptor family. Cell 80, 899-908. Lindsley, D. L. and Zimm, G. G. (1992). The Genome of Drosophila melanogaster. San Diego, CA: Academic Press, Inc. Maekawa, T., Sakura, H., Sudo, T. and Ishii, S. (1989). Putative metal structure of the human immunodeficiency virus type 1 enhancer binding protein HIV-EP1 J. Biol. Chem. 264, 14591-14593. Massagué, J., Attisano, L. and Wrana, J. L. (1994). The TGF-β family and its composite receptors. Trends Cell Biol. 4, 172-178. Mastick, G.S., McKay, R., Oligino, T., Donovan, K. and López, A.J. (1995).
Identification of target genes regulated by homeotic proteins in Drosophila melanogaster through genetic selection of Ultrabithorax protein-binding sites in yeast. Genetics 139, 349-363. Masucci, J. D., Miltenberger, R. J. and Hoffmann, F. M. (1990). Patternspecific expression of the Drosophila decapentaplegic gene in imaginal disks is regulated by 3’ cis-regulatory elements Genes Dev. 4, 2011-2023. Mitchelmore, C., Traboni, C. and Cortese, R. (1990). Isolation of two cDNAs encoding zinc finger proteins which bind to the α1-antitrypsin promoter and to the major histocompatibility complex class I enhancer. Nucleic Acids Research 19, 141-147. Miyamoto, H., Nihonmatsu, I., Kondo, S., Ueda, R., Togashi, S., Hirata, K., Ikegami, Y. and Yamamoto, D. (1995). canoe encodes a novel protein containing a GLGF/DHR motif and functions with Notch and scabrous in common developmental pathways in Drosophila. Genes Dev. 9, 612-625. Miyazono, K., Ten Dijke, P., Ichijo, H. and Heldin, C. H. (1994). Receptors for transforming growth factor-β. Adv. Immunology 55, 181-220. Nakamura, T., Donovan, D. M., Hamada, K., Sax, C. M., Norman, B., Flanagan, J. R., Ozato, K., Westphal H. and Piatigorsky, J. (1990). Regulation of the mouse αA-crystallin gene: Isolation of a cDNA encoding a protein that binds to a cis sequence motif shared with the major histocompatibility complex class I gene and other genes. Mol. Cell. Biol. 10, 3700-3708. Nellen, D., Affolter, M. and Basler, K. (1994). Receptor serine/threonine kinases implicated in the control of Drosophila body pattern by decapentaplegic. Cell 78, 225-237. Nomura, N., Zhao, M.-J., Nagase, T., Maekawa, T., Ishizaki, R., Tabata, S. and Ishii, S. (1991). HIV-EP2, a new member of the gene family encoding the human immunodeficiency virus type I enhancer-binding protein. J. Biol. Chem. 266, 8590-8594. Padgett, R. W., St Johnston, R. D. and Gelbart, W. M. (1987). A transcript from a Drosophila pattern gene predicts a protein homologous to the transforming growth factor-β family. Nature 325, 81-84. Panganiban, G. E. F., Reuter, R., Scott, M. P. and Hoffmann, F. M. (1990). A Drosophila growth factor homolog, decapentaplegic, regulates homeotic gene expression within and across germ layers during midgut morphogenesis. Development 110, 1041-1050. Patel, N. H., Snow, P. M. and Goodman, C. S. (1987). Characterization and cloning of fasciclin III: A glycoprotein expressed on a subset of neurons and axon pathways in Drosophila. Cell 48, 975-988. Penton, A., Chen, Y., Staehling-Hampton, K., Wrana, J. L., Attisano, L., Szidonya, J., Cassill, J. A., Massagué, J. and Hoffmann, F. M. (1994). Identification of two bone morphogenetic protein type I receptors in Drosophila and evidence that Brk25D is a decapentaplegic receptor. Cell 78, 239-250. Raftery, L. A., Twombly, V., Wharton, K. and Gelbart, W. M. (1995). Genetic screens to identify elements of the decapentaplegic signaling pathway in Drosophila. Genetics 139, 241-254. Ramain, P., Heitzler, P., Haenlin, M. and Simpson, P. (1993). pannier, a negative regulator of achaete and scute in Drosophila, encodes a zinc finger protein with homology to the vertebrate transcription factor GATA-1. Development 119, 1277-1291. Reuter, R., Panganiban, G. E. F., Hoffmann, F. M. and Scott, M. P. (1990). Homeotic genes regulate the spatial expression of putative growth factors in the visceral mesoderm of Drosophila embryos. Development 110, 10311040. Ron, D., Brasier, A. R. and J. F. Habener, J. F. (1991). Angiotensinogen gene-inducible enhancer-binding protein 1, a member of a new family of large nuclear proteins that recognize nuclear factor κB-binding sites through a zinc finger motif Mol. Cell. Biol. 11, 2887-2895. Roth, S., Stein, D. and Nusslein-Volhard, C. (1989). A gradient of nuclear localization of the dorsal protein determines dorsoventral pattern in the Drosophila embryo. Cell 59, 1189-1202. Ruberte, E., Marty, T., Nellen, D., Affolter, M. and Basler, K. (1995). An absolute requirement for both the type II and type I receptors, punt and thickveins for dpp signaling in vivo. Cell 80, 889-897. Rushlow, C. A., Han, K., Manley, J. L. and Levine, M. (1989). The graded distribution of the dorsal morphogen is initiated by selective nuclear transport in Drosophila. Cell 59, 1165-1177. Rustgi, A. K., Van ‘T Veer, L. J. and Bernards, R. (1990). Two genes encode factors with NF-κB- and H2TF1-like DNA-binding properties. Proc. Natl. Acad. Sci. USA 87, 8707-8710. Seeler, J.-S., Muchardt, C., Suessle, A. and Gaynor, R. B. (1994). Transcription factor PRDII-BF1 activates human Immunodeficiency virus type 1 gene expression. J. Virology 68, 1002-1009.
dpp signaling requires schnurri 3403 Sekelsky, J. J., Newfeld, S. J., Raftery, L. A., Chartoff, E. H. and Gelbart, W. M. (1995). Genetic characterization and cloning of Mothers against dpp, a gene required for decapentaplegic function in Drosophila melanogaster. Genetics 139, 1347-1358. Simon, M. A. (1994). Signal transduction during the development of the Drosophila R7 photoreceptor. Dev. Biol. 166, 431-442. Spencer, F.A., Hoffmann, F.M., and Gelbart, W.M. (1982) Decapentaplegic: A gene complex affecting morphogenesis in Drosophila melanogaster. Cell 28, 451-461. St Johnston, R. D. and Gelbart, W. M. (1987). decapentaplegic transcripts are localized along the dorsal-ventral axis of the Drosophila embryo. EMBO J. 6, 2785-2791. Staehling-Hampton, K., Hoffmann, F. M., Baylies, M. K., Rushton, E. and Bate M. (1994). dpp induces mesodermal gene expression in Drosophila. Nature 372, 783-786. Staehling-Hampton, K. and Hoffmann, F. M. (1994). Ectopic decapentaplegic in the Drosophila midgut alters the expression of five homeotic genes, dpp and wingless, causing morphological defects. Dev. Biol. 164, 502-512. Steward, R. (1989). Relocalization of the dorsal protein from the cytoplasm to the nucleus correlates with its function. Cell 59, 1179-1188. Suzuki, M. (1989). SPXX, a frequent sequence motif in gene regulatory proteins. J. Mol. Biol. 207, 61-84. Tautz, D. and Pfeiffle, C. (1989). A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila reveals translational control of the segmentation gene hunchback. Chromosoma (Berlin) 98, 81-85. Thüringer, F., Cohen, S.M., and Bienz, M. (1993). Dissection of an indirect autoregulatory response of a homeotic Drosophila gene. EMBO J. 12, 24192430. Tremml, G. and Bienz, M. (1992). Induction of labial expression in the Drosophila endoderm: response elements for dpp signaling and for autoregulation. Development 116, 447-456.
Van ’T Veer, L. J., Lutz, P. M., Isselbacher, K. J. and Bernards, R. (1992). Structure and expression of major histocompatibility complex-binding protein 2, a 275-kDa zinc finger protein that binds to an enhancer of major histocompatability complex class I genes. Proc. Natl. Acad. Sci. USA 89, 8971-8975. Wharton, K. A., Thomsen, G. H. and Gelbart, W. M. (1991). Drosophila 60A gene, a new TGF-β family member is closely related to human bone morphogenetic proteins. Proc. Natl. Acad. Sci. USA 88, 9214-9218. Winick, J., Abel, T., Leonard, M. W., Michelson, A. M., Chardon-Loriaux, I., Holmgren, R. A., Maniatis, T. and Engel, J. D. (1993). A GATA family transcription factor is expressed along the embryonic dorsoventral axis in Drosophila melanogaster. Development 119, 1055-1065. Wrana, J. L., Attisano, L., Cárcamo, J., Zentella, A., Doody, J., Laiho, M., Wang, X.-F. and Massagué, J. (1992). TGFβ signals through a heteromeric protein kinase receptor complex. Cell 71, 1003-1014. Wrana, J. L., Tran, H., Attisano, L., Arora, K., Childs, S. R., Massagué, J. and O’Connor, M. B. (1994). Two distinct transmembrane serine/threonine kinases from Drosophila melanogaster form an activin receptor complex. Mol. Cell Biol. 14, 944-950. Wu, L.-C., Mak, C.-H., Dear, N., Boehm, T., Foroni, L. and Rabbitts, T. H. (1993). Molecular cloning of a zinc finger protein which binds to the heptamer of the signal sequence for V(D)J recombination. Nucleic Acids Res. 21, 5067-5073. Xie, T., Finelli, A. L. and Padgett, R. W. (1994). The Drosophila saxophone gene: A serine-threonine kinase receptor of the TGF-β superfamily. Science 263, 1756-1759. (Accepted 14 July 1995)
Note added in proof The Genbank accession number for the schnurri sequence is U31368
