INTRODUCTION

Dorsoventral (DV) patterning of the Drosophila embryodepends on processes acting during both oogenesis andembryogenesis. DV asymmetry is first apparent duringmidoogenesis when the oocyte nucleus moves to the anterior-dorsal side (see Spradling, 1993), leading to the asymmetricallocalization of gurken (grk) mRNA and hence of the TGFα-like Grk protein in close proximity to the oocyte nucleus(Neuman-Silberberg and Schüpbach, 1993; Roth andSchüpbach, 1994). Grk plays an instructive role in signalingoverlying somatic follicle cells to assume a dorsal fate (Rothand Schüpbach, 1994) since, in grk− mutants, ventral regionsof the eggshell are expanded at the expense of dorsal domains.This initial DV patterning of the follicle cells leads tospecification of eggshell polarity and generation of spatialinformation, which is ultimately conveyed to the embryo (seeMorisato and Anderson, 1995; Ray and Schüpbach, 1996 forreviews).

Other maternal genes have been identified that are requiredfor establishment of the embryonic DV axis (Anderson andNüsslein-Volhard, 1984; Schüpbach and Wieschaus, 1989). Ofthese 12 maternal effect “dorsal group” loci, loss-of-functionalleles of 11 genes lead to dorsalization of the embryo, whileloss-of-function mutations in one (cactus) generates embryoswith a ventralized phenotype (Roth et al., 1991). The dorsalgroup genes form a linear genetic cascade, which regulatessignaling through the Toll (Tl) receptor pathway (Hashimoto

et al., 1991; Stein et al., 1991; Stein and Nüsslein-Volhard,1992). Upstream of Tl, the genes pipe (pip), nudel (ndl)windbeutel (wind), gastrulation defective (gd), snake (snk),easter (ea) and spätzle (spz)constitute an extracellularproteolytic cascade required for processing of the putative Tlligand Spätzle (Stein and Nüsslein-Volhard, 1992; Morisatoand Anderson, 1994; Schneider et al., 1994). Three dorsalgroup genes, wind, pip andndl, are required in somatic cellsrather than the oocyte, consistent with models in which spatialinformation from the follicle cells is conveyed to the embryoto establish polarity along the DV axis (Nilson and Schüpbach,1998). These genes are expressed transiently in the follicularepithelium during midoogenesis in response to grk-mediatedEGF-R signaling. grk is also thought to activate a secondpathway for restricting ventral fates which refines the DVpattern established by dorsal group genes (Ray and Schüpbach,1996).

Downstream of the Tl receptor, the cytoplasmic domain ofwhich is related to Interleukin Receptors, products of thematernal genes tube, pelle, cactus and dorsal transduce thesignal within the embryo (Anderson et al., 1985; Roth et al.,1991; Hecht and Anderson, 1993; Shelton and Wasserman,1993). These genes encode cytoplasmic components of asignaling cascade, which culminates in translocation of Dorsal,a member of the NFκB family of transcription factors(Steward, 1987), into nuclei of blastoderm stage embryos. Akey regulator of Dorsal nuclear translocation is Cactus, whichis an IkB-related cytoplasmic factor that binds to Dorsal

3631Development 127, 3631-3644 (2000)Printed in Great Britain © The Company of Biologists Limited 2000DEV8734

The short gastrulation (sog)and decapentaplegic (dpp) genesfunction antagonistically in the early Drosophila zygote topattern the dorsoventral (DV) axis of the embryo. Thisinterplay between sogand dppdetermines the extent of theneuroectoderm and subdivides the dorsal ectoderm intotwo territories. Here, we present evidence that sogand dppalso play opposing roles during oogenesis in patterning theDV axis of the embryo. We show that maternally producedDpp increases levels of the IκB-related protein Cactusand reduces the magnitude of the nuclear concentrationgradient of the NFκB-related Dorsal protein, and that Soglimits this effect. We present evidence suggesting that Dpp

signaling increases Cactus levels by reducing a signal-independent component of Cactus degradation. Epistasisexperiments reveal that sogand dppact downstream of, orin parallel to, the Toll receptor to reduce translocation ofDorsal protein into the nucleus. These results broaden therole previously defined for sog and dpp in establishingthe embryonic DV axis and reveal a novel form ofcrossregulation between the NFκB and TGFβ signalingpathways in pattern formation.

Key words: Oogenesis, sog, dpp, Dorsoventral patterning, dorsal,Drosophila

SUMMARY

sog and dpp exert opposing maternal functions to modify Toll signaling and

pattern the dorsoventral axis of the Drosophila embryo

H. Araujo and E. Bier*

Department of Biology and Center for Molecular Genetics, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA92093-0349, USA*Author for correspondence (e-mail: bier@biomail.ucsd.edu)

Accepted 31 May; published on WWW 20 July 2000

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(Whalen and Steward, 1993). In response to Tl signaling,Cactus is degraded and Dorsal becomes free to enter thenucleus. In addition to Tl-mediated degradation of Cactus,there is a signal-independent mechanism for degrading Cactus,which may be mediated by a Casein-KinaseII-like activity (Liuet al., 1996).

The final result of graded activation of Tl signaling bySpätzle is the formation of a nuclear gradient of Dorsal, withmaximum concentrations in ventral nuclei of the embryo,lower levels laterally and none in the dorsal region (Roth et al.,1989; Rushlow et al., 1989; Steward, 1989). Different levels ofnuclear Dorsal specify the three primary domains of geneexpression in a threshold-dependent fashion. High levels ofDorsal ventrally initiate expression of genes (e.g. snail) in thepresumptive mesoderm, while lower levels of Dorsal laterallyactivate expression of genes such asrhomboid, vein, ventralnervous system defective(vnd), short gastrulation(sog) andbrinker in the neuroectoderm (Kosman et al., 1991; Ray et al.,1991; Ip et al., 1992b; François et al., 1994; Mellerick andNirenberg, 1995; Jazwinska et al., 1999a,b; Minami et al,1999). The absence of Dorsal in the dorsal region of theembryo permits expression of genes such as decapentaplegic(dpp), which are otherwise repressed by Dorsal (Ray et al.,1991). This pattern of DV gene expression is subsequentlyrefined and maintained by the action of zygotically active genesexpressed in the various DV domains. dpp, which encodes aprotein orthologous to vertebrate BMP2 and BMP4 (Padgett etal., 1987), suppresses lateral neural fates and functions in adose-dependent fashion to promote dorsal cell fates in thedorsal domain of the embryo (Irish and Gelbart, 1987; Ray etal., 1991; Arora and Nüsslein-Volhard, 1992; Ferguson andAnderson, 1992; Wharton et al., 1993). In the lateral region,Dpp is antagonized by the products of the sog (Ferguson andAnderson, 1992; Biehs et al., 1996) andbrinker (brk) genes(Jazwinska et al., 1999a,b; Minami et al., 1999) permittingcells to assume default neuroectodermal fates (Bier, 1997;Bray, 1999).

Here, we report on a maternal role for sog and dpp inpatterning the DV axis of the embryo. We show that theinterplay between maternal sog and dpp in DV patterninginfluences the formation of the nuclear Dorsal gradient. Wefind that elevating the gene dose of dpp or reducing the doseof sog maternally compromises Tl signaling in a sensitizedgenetic background. Similarly, eliminating sog function fromfollicle cells during oogenesis results in a reduction in thewidth of the ventral mesoderm. Epistasis experiments indicatethat Dpp signaling acts downstream of, or in parallel to, the Tlreceptor. Our results suggest that the mechanism by which Dppsignaling reduces nuclear translocation of Dorsal is to inhibita signal-independent form of Cactus degradation. We showthat this maternal activation of the Dpp pathway leads to acrossregulatory modulation of the Tl signaling pathway,providing the first evidence for interaction between NFκB- andTGFβ-regulated pathways in Drosophila.

MATERIALS AND METHODS

Fly stocksCanton S or w1118strains were used as wild type. The sogU2 and sogP1

alleles are described in François et al. (1994). The CyODpdpp

chromosome carrying a genomic insert of the dppcoding region andsequences required for expression during early embryogenesis waskindly supplied by Dr W. Gelbart (Harvard University). The 8xhsdppstock was kindly provided by Dr Ronald Blackman (Chemgenics,Boston) and construction of the 8xhssog is described in Yu et al.(1996). Dp (2;1)G146, dpp(+), referred to as Dp(dpp), contains aduplication of the dpplocus on the first chromosome and was obtainedfrom Bloomington Stock Center. sog6/Dp(sog)Y contains aduplication of sogon the Y chromosome (reported in Wickline andLindsley, 1997). Dorsal alleles used were dlI5 and dl1. Stocks forproducing sog− follicle cell clones were obtained from Dr J. Duffy(Harvard Medical School, Boston). All other mutants, balancers andchromosomal markers used in this study (Lindsley and Zimm, 1992)were obtained either from the Bloomington Indiana Stock Center orthe Umea Stock Center.

Production of follicle cell clonesWe generated sog− follicle cell clones using the e22-GAL4 driver toexpress Flp in the presumptive follicular stem cells (Duffy et al.,1998). sogU2 FRT 18E/FM7c mothers were crossed to GFP 18E; [e22-GAL4][UAS-Flp]/CyO males. Female progeny of the genotype sogU2

FRT 18E/GFP 18E; [e22-GAL4][UAS-Flp]/+ were then crossed to w−

males at 29°C. The pattern of GFP expression in egg chambersshowed that approximately 30% of egg chambers had follicle cellclones. In more than 10% of egg chambers, all follicle cells coveringthe oocyte failed to express GFP, and were therefore homozygous sog− mutant. Embryos were collected and processed for in situhybridization or aged to 36 hours for analysis of the cuticles. Notethat 25% of the progeny from such a cross were sog−, which resultedin embryos having a characteristic sog− cuticle phenotype (Zusman etal., 1988). To analyze the effect of sog− follicle cell clones withouteliminating zygotic sog, sog(U2) FRT 18E/GFP FRT 18E; [e22-GAL4][UAS-Flp]/+ mothers were crossed to +/Dp(sog) Y males,which contain a duplication of sog on the Y chromosome. In thiscrossing scheme, no sog− embryos are generated and the phenotypesobserved can be attributed to the presence of sog− follicle cell clones.

In situ hybridization and antibody stainingDouble label in situ hybridization in embryos was performed withdigoxigenin- and biotin-labeled RNA probes as described previously(O’Neill et al., 1994). Ovaries were dissected from 3- to 5-day-oldfemales, and in situ hybridization was performed with antisense RNAprobes (Morimoto et al., 1996). Antibody staining in embryos wasperformed as described in Sturtevant et al. (1993). The monoclonalanti-Dorsal antibody was kindly provided by Dr R. Steward and wasused at a 1:20 dilution.

Embryo sectioningFollowing histochemical labeling procedures, stained embryos wereprepared for sectioning as described in Hemavathy et al. (1997). 4 µmtransverse sections were cut on a Sorvall Microtome, mounted inPermount and viewed under DIC optics.

Eggshell and cuticle preparationsFor cuticle preparations, mutant embryos were collected on grapeplates after having been aged for 36 hours at 25°C, dechorionated in50% sodium hypochlorite and washed three times in 0.7% NaCl,0.04% Triton X-100. After washing, embryos were incubated for 2hours at 65°C in a 1:4 glycerol:acetic acid mix. Cuticles and eggshellswere mounted under a coverslip in a drop of Hoyer’s medium(Nüsslein-Volhard and Wieschaus, 1980) and the slides were returnedto 65°C for 24 hours. Cuticles were analyzed and photographed underdark-field illumination.

Heat-shock experiments3- to 5-day-old females were heat shocked at 38°C for the timesindicated in the legends and then immediately transferred with

H. Araujo and E. Bier

3633Maternal Dpp opposes Tl signaling

appropriate males to agar grape plates for embryo collection. Embryoswere collected during specific time windows after heat shock and werethen either processed immediately for eggshell or protein lysatepreparations, or were aged for cuticle preparations. For analysis ofgrk, pipe and pnt expression in ovaries, females were heat shockedfor 30 minutes (8xhssog) or 15 minutes (8xhsdpp). After 1 hourof recovery, ovaries were dissected and processed for in situhybridization.

Immunoblot analysisFor analysis of Dorsal and Cactus proteins, total protein extracts wereprepared by homogenizing 0- to 1-hour-old dechorionated embryos inelectrophoresis sample buffer (50 mM Tris 6.8, 100 mM DTT, 2%SDS, 0.1% Bromophenol blue; 10% glycerol). Extracts wereseparated by SDS-PAGE on 8% gels and electroblotted onto PVDFmembranes (BIORAD). The dilutions for primary antibodies were:monoclonal anti-Dorsal 1:20, polyclonal anti-Cactus 1:1000(provided by Dr S. Wasserman, UCSD), and monoclonal anti-Tubulin1:500 (DM1α, Sigma). After probing immunoblots with anti-Dorsalor anti-Cactus, membranes were stripped of antibodies in 200 mMglycine buffer pH 2.0 and reprobed with anti-Tubulin. Immunecomplexes were visualized using horseradish-peroxidase-conjugatedsecondary antibody and chemiluminescent enhancer system (Pierce).For quantitation of immunoblots bands from autoradiograms ofseveral exposures were quantitated using the Histogram function ofPhotoshop, by determining the intensity of black in negative images.

RNA synthesis and injectionsThe cact∆PESTand cactnSA4constructs have been described previously(Reach et al., 1996). RNA synthesis was performed usingMEGAscript in vitro transcription kit (Ambion). RNA concentrationswere defined by ultraviolet absorption and RNA was deposited in theposterior end of embryos as described (Liu et al., 1997). Cuticles wereprepared as described above.

RESULTS

sog and dpp function antagonistically duringoogenesis to pattern the dorsal-ventral axis of theembryoIn previous experiments, we observed a dosage-sensitiveinteraction between zygotically expressed sog and maternallyproduced dorsalin regulating neuroectodermal gene expression(Biehs et al., 1996). In analyzing this phenotype further, wediscovered that, in addition to this zygotic effect, sog alsointeracts maternally with dl to alter gene expression along theentire DV axis of the embryo. dl is sensitive to gene dosage, asmothers heterozygous for a dl null allele produce embryos thatare weakly dorsalized (Rushlow et al., 1989b; Steward, 1989).Lowering the dose of dl reduces the width of the mesodermfrom 18 cells to 12-14 cells (Fig. 1B; Table 1, row 2). Areduction in the dose of maternal sog enhances this dl−/+phenotype dramatically. Mothers double heterozygous for nullalleles of sog and dl (referred to as sog/+; dl/+ mothershereafter) produce embryos in which the width of the mesodermis greatly reduced and expression of neuroectodermal genessuch as vnd and sog invades the ventralmost region of theembryo (Fig. 1C,D). As this phenotype is observed at similarfrequencies (40%) in embryos with or without functionalzygotic copies of sog (Table 1, rows 3,4), and cannot begenerated by reducing the level of sog solely in the zygote(Table 1, row 5), the influence of sogin determining the size ofthe mesoderm is strictly maternal. Consistent with the knownrole of sog in antagonizing Dpp signaling, increasing thematernal dose of dpp in females heterozygous for dl results inthe same ventral shift in zygotic expression of vnd and sog (Fig.

Table 1. Expression of neuroectodermal markers in the ventral presumptive mesodermPenetrance of Width range of specified region (number of cells)ventral vnd

invasion at 70% Ventral sna Ventral sna Lateral sogegg length expression at expression at expression at

Genotype (as in Fig. 1D) 70% egg length 50% egg length 50% egg length

1 Wild type 0 8-12 12-18 16-182 dl−/+ mothers <3% 8-10 (**) 8-14 16-183 sog−/+; dl−/+ mothers (sog−/+ and +/+ embryos) 42% (§) 0-10 0-14 16-184 sog−/+; dl−/+ mothers (sog− embryos) 39% (*) 0-10 0-14 ND5 dl−/+ mothers × sog−/Dp(sog)Y <3% 8-10(**) 10-14 16-186 Dp(dpp)/+;dl−/+ mothers (includes maternal comp.) 64% 0-10 0-14 16-187 dl− +/+ Dp(dpp) mothers (Dp only zygotic component) <3% 8-10(**) 8-14 16-188 dl−/+ mothers × 8xhsdpp(4xhs embryos) <3% 8-10(**) 8-14 12-142

9 sog−/Dp(dpp); dl−/+ mothers (includes maternal comp.) 88% 0-10 0-14 16-1810 sog−/+ mothers (sog−/+ and +/+ embryos) 0 8-14 10-18 16-1811 sog−/+ mothers × 8xhsdpp(4xhs embryos) 0 8-12 10-18 12-142

12 sog−/+; Dp(dpp)/+ mothers (Dp only zygotic comp.) 0 8-12 10-18 16-1813 sog−/ Dp(dpp) mothers (includes maternal comp.) 0 8-12 10-18 16-1814 dl− +/+ twi− or dl−/+ mothers × twi−/+1 ND 0-10 8-14 16-1815 sog−/+; twi−/+ mothers 0 8-12 10-18 16-1816 twi−/+ embryos 0 8-12 12-18 16-18

1Mellerick and Nirenberg, 1995; 2Biehs et al, 1996. *not significantly different from sog+embryos derived from the same mothers. **range excluding the small number (<3%) of embryos that display complete invasion of neuroectodermal gene expression in embryos derived from dl−/+

mothers.§14% of these embryos display the extreme phenotype depicted in Fig. 1C, while embryos from dl/+ mothers never displayed the extreme phenotype. The primary alleles used in this Table were sogU2 and dlI5. 14% of embryos from sog/+; dl/+ mothers display the extreme phenotype in which the mesoderm is

completely invaded by neuroectodermal gene expression, in contrast to embryos from dl/+ mothers, which never display the phenotype. Other alleles tested forthe maternal interaction of sogand dorsal were: psog(a hypomorphic allele), dl1 (Lindsley, 1992), Df(dl) deficiency uncovering dorsal, and cactE10, a cactusallele whose product constitutively binds to Dorsal inhibiting its translocation to the nucleus. Double heterozygous combinations using these alleles behavedsimilarly to those depicted on the Table (% of invasion of vndexpression as follows: sogU2; Df(dl) 23%, sogU2; cactE10 70%, sogU2; dl1 61% and psog; dlI5 30%).More than 100 embryos were used for each condition in order to determine the penetrance of phenotypes described. N.D.=not determined.

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1E; Table 1, row 6). In contrast, increasing the dose of dppzygotically had no effect on embryos laid by dl/+ mothers(Table 1; rows 7,8), even when large amounts of dpp areprovided through heat shock (Fig. 1F; Table 1 rows 8, 11).Induction of dpp expression by heat shock, however, reducesthe width of lateral gene expression as described previously(Biehs et al, 1996; Table 1, rows 8, 11). Combined reduction insogand elevation in dpp levels in dl/+ mothers increases thefrequency of affected embryos but not the average severity ofthe phenotype (Table 1, row 9). Ventral expression of otherneuroectodermal markers such as lethal of scute, rhomboidandvein is also observed during the cellular blastoderm stage ofembryos derived from sog/+;dl/+ or Dp(dpp)/+;dl/+ mothers(data not shown). The maternal interaction between dl andsog is distinct from the interaction previously described with

zygotic sog, as indicated by the fact that the first is independentof the dose of zygotic sog, while the latter is only observed inthe absence of zygotic sog(Biehs et al., 1996).

Another phenotype observed in embryos derived fromsog/+;dl/+ or Dp(dpp)/+;dl/+ mothers is ventral extension of thecephalic furrow (arrow in Fig. 1J), similar to that observed intwi/+ embryos derived from dl/+ mothers (Ip et al., 1992a;Mellerick and Nirenberg, 1995; Table 1 rows 14,15). In addition,the domain of ventral cells expressing mesodermal genes suchas sna (Fig. 1G,H) and twi (not shown) is greatly reduced.Expression of sna is largely complementary to that ofneuroectodermal genes such as sog in embryos derived fromsog/+;dl/+ or Dp(dpp)/+;dl/+ mothers, although there issome degree of overlap (Fig. 1H, insert). This overlap inneuroectodermal and mesodermal gene expression is abnormal

H. Araujo and E. Bier

Fig. 1. Maternal sogand dppcontribute to patterning the DV axis of the embryo. (A-F) RNA in situ hybridization showing expression of sog(brown) and vnd(blue) in blastoderm stage embryos. (A) In wild-type embryos, vndand sogexpression is excluded from ventralmost regions.(B) In embryos derived from dl/+ mothers, the ventral mesodermal territory is narrowed. (C,D) In embryos derived from sog/+;dl /+ mothers,expression of sogand vnd sometimes invades ventral regions completely (C), but in most cases is observed only anteriorly where the cephalicfurrow will form (D). (E) Embryos laid by Dp(2;1)dpp/+;dl/+ mothers. (F) Embryos derived from dl−/+ mothers in which ectopic expression ofdpp is induced during embryogenesis (by heat shocking 4xhsdpp embryos), do not exhibit ventral expansion of neuroectodermal geneexpression. However, lateral expression of vnd is narrowed in such embryos. (G,H) Expression of sog (brown) and snail (blue) in wild-typeembryos (G) and embryos derived from sog/+;dl/+ mothers. The domain of ventral sna expression is narrowed and is frequently eliminated at70% egg length in embryos derived from sog/+;dl/+ mothers (arrow) (H). sogexpression invades the ventral mesoderm where snaexpression isexcluded. There is also some degree of overlap between sna and sog expression domains (insert). (I,J) Expression of sog (brown) and sim (blue)in wild-type (I) and embryos derived from sog/+;dl/+ mothers (J). The mesectoderm forms along the new neuroectodermal-mesodermal border,although there are some interruptions in simstaining (insert). The cephalic furrow extends abnormally around the entire circumference of theembryo (arrows). (K,L) Expression of sog (brown) and dpp (blue) in wild-type (K) and embryos derived from sog/+;dl/+ mothers (L), showingthat dpp and sogexpression domains abut in both cases. (M-P) sog− follicle cell clones were generated using the FLP-FRT method (seeMethods). (M) A wild-type embryonic cuticle. (N) Non-viable embryos had cuticles with partial or total absence of denticles. (O) Expression ofsog(brown) and sna(blue) in an embryo derived from mothers containing sog− follicle cell clones. Ventral expression of mesodermal genes isreduced in regions where lateral expression of sogexpands. Note that this embryo is zygotically sog+ (e.g. it has a lateral stripe of sogexpression). (P) Expression of sna (brown) and lateral rho (blue). In wild-type embryos, the mesodermal and neuroectodermal domains do notoverlap (top panel) while, in embryos derived from mothers containing follicle cell clones (bottom panel), several rows of cells at themesoderm/neuroectoderm border express both genes. Embryos are shown from a ventral perspective with anterior to the left and posterior to theright. The embryo in Fig. 1C is shown from a ventrolateral perspective to show lateral expression of sogand vnd.

3635Maternal Dpp opposes Tl signaling

since these expression domains are mutually exclusive in wild-type embryos. The mesectoderm, which forms at the borderbetween the neuroectodermal and mesodermal territories, is alsodisplaced ventrally in embryos derived from sog/+;dl/+ mothersas indicated by the expression of the single minded(sim) gene(Fig. 1I,J), which often is interrupted by gaps (Fig. 1J, insert).Since the width of the mesodermal domain is reduced while thatof the neuroectoderm remains constant in embryos derived fromsog/+;dl/+ or Dp(dpp)/+;dl/+ mothers, the dorsal region shouldbe expanded in these embryos (Table I, rows 3,4, 6). Consistentwith this expectation, the dorsal domain of dpp-expressing cellsstill abuts that of sog in lateral cells, which is shifted ventrally(Fig. 1K,L). These data reveal that sogand dppplay both zygoticand maternal roles, to influence embryonic DV patterning. sogis expressed zygotically in lateral cells where it functions toprevent dpp from autoactivating in the lateral neuroectoderm,and contributes to creating graded Dpp activity in the dorsalregion of the embryo. In contrast to these localized requirementsfor zygotic sogand dpp, the maternal activities of these genesspecify the extent and position of all DV domains.

Maternal sog activity is required in follicle cells andnot in the germlineSince lowering the dose of sogin dl/+ mothers alters the patternof zygotic gene expression along the embryonic DV axis, weexamined the consequences of eliminating sog function ingermline versus somatic cells of the egg chamber. We generatedsog− null germline clones using the Flp-DFS method (Chou andPerrimon 1992) and found no eggshell or cuticle phenotypes (notshown), consistent with the lack of detectable sogexpression inthe germline (see below). Similarly, dpp is not required in thegermline (Irish and Gelbart, 1987). In order to study the role ofDpp signaling in follicle cells, we ideally would generate clonesof dpp− follicle cells. Unfortunately, it is not possible to analyzethis later function of dppin follicle cells by clonal analysis, sincedpp is required earlier in oogenesis (Spradling, 1993). It ispossible, however, to generate loss-of-function sog clones infollicle cells using e22-GAL4 to drive expression of UAS-Flp inall follicle stem cells, which results in a high frequency of largefollicle cell clones in control experiments (see Methods). In theseexperiments, we crossed e22-GAL4; UAS-Flp females to males

Fig. 2. Maternal Dpp acts downstream of or in parallel to the Tl receptor. (A) Mothers carrying one copy of the dominant Tl3 allele produceembryos with strongly ventralized cuticles. (B) Heat-shock induction of 4xhsdpp mothers carrying the dominant Tl3 allele (Tl3/+ ;4xhsdpp,heatshocked 0-6 hours before egg laying) results in embryos that have weakly ventralized cuticles, as revealed by narrower denticle belts thatencircle the embryo. (C) Homozygous gd3 mothers produce embryos with a dorsalized phenotype. (D) Cuticles of embryos derived from gd7

grkHK;Tl3/+ have a more ventral character than those from gd7/gd7;Tl3/+, which resembles that of embryos from Tl3/+ mothers (compare with Aand E). (E) Cuticles of embryos derived from gd7/gd7;Tl3/+ mothers have a lateralized and apolar phenotype (L1). (F) A duplication of dpp ingd7Dp(dpp)/gd7;Tl3/+ mothers modifies the lateralized pattern, resulting in phenotypes with a uniform more dorsal character (L2). (G,H) Highmagnification view of the denticle patterns shown in E and F. (G) Cuticles generated by gd7/gd7;Tl3/+ mothers. (H) Cuticles generated bygd7Dp(dpp)/gd7;Tl3/+ mothers. Denticles are finer, less heavily pigmented and sometimes absent. (I,J) Embryos generated by gd2/gd2;Tl3/+mothers maintain some residual polarity as shown by the expression pattern of sog(blue) and Twist protein (brown). (I) Providing a duplicationof dppzygotically has no effect on the gd2/gd2;Tl3/+ phenotype. (J) Increasing the dose of Dpp maternally, however, as in embryos laid bygd2Dp(dpp)/gd2;Tl3/+ mothers, results in embryos in which sogis expressed along the entire DV axis and Twist expression is eliminatedventrally.

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containing a duplication of sogon the Y chromosome so that thegenotype of all embryos was sog+ and any embryonic phenotypeobserved could be attributed solely to the absence of maternalsog. Non-viable embryos collected from mothers containingsog− follicle cell clones exhibited a reduction of ventral denticlesin parts or along all the AP axis of the embryo (Fig. 1N). In themost extreme cases, all ventral denticles were absent (data notshown). Females producing sog− follicle clones also generatedembryos with altered patterns of zygotic gene expression alongthe DV axis, as exemplified by the altered expression of sna inembryos that were zygotically sog+ (Fig. 1O, note zygotic lateralsog expression). In addition, ventrolateral expression of rhoexpanded and overlapped the ventral domain of sna expressionby 2-3 cells in some of these embryos (Fig. 1P). The phenotypesassociated with sog− follicle cell clones are similar to but moresevere than those observed in embryos derived from sog/+;dl/+mothers, and can be accounted for as varying shifts of dorsal cellfates towards more ventral fates and a blurring between ventraland lateral domains of gene expression.

The dorsalizing maternal activity of Dpp functionsdownstream of or in parallel to the Tl receptorTo determine where sogand dpp function in the maternal DVpatterning hierarchy relative to other genes, we asked whethermaternal Dpp signaling acts upstream or downstream of the Tlreceptor. We addressed the epistatic relationship between dppand Tl, by examining the effect of altering the dose of dpp onthe ventralized (V1) embryonic phenotype caused by thedominant Tl3 allele also known as Tl9Q (Anderson et al., 1985).In embryos derived from Tl3/+ mothers containing a duplicationof dpp(Dp(dpp)/+; Tl3/+), we observed that a percentage (13%)exhibited weakly ventralized instead of strongly ventralizedcuticle phenotypes in which the denticle belts were narrowerand no longer encircled the entire circumference of the embryo(Table 2). We observed a more penetrant modification of thedominant ventralizing Tl3 phenotype when the maternal dose ofdpp was increased by heat shocking flies carrying a hs-dppconstruct, which resulted in 50-80% of embryos having weaklyventralized cuticles (Fig. 2A,B).

Modification of the Tl3/+ phenotype suggests that sogand

dpp interact with the maternal dorsal group pathway. However,since one copy of wild-type Tl in Tl3/+ mothers remainsresponsive to normal maternal signaling by Spätzle, it was notpossible to conclude from these experiments whether sogand dpp function upstream or downstream of Tl. In order toaddress this question, we examined embryos derived frommothers containing one copy of the Tl3 allele that alsowere homozygous for a loss-of-function mutant allele ofgastrulation defective(gd7). These mothers are thought to beunable to generate any functional Spätzle signal and thereforeTl signaling in embryos derived from these mothers is believedto result solely from the constitutively active Tl3 receptor(Anderson et al., 1985). Embryos collected from double mutantgd7/gd7;Tl3/+ mothers have lateralized cuticles as defined bytheir early gastrulation pattern and presence of thin bands ofdenticle belts encircling the embryo (referred to as the L1phenotype, Anderson et al., 1985) (Fig. 2E). A significantfraction of embryos laid by gd7/gd7;Tl3/+ females carrying aduplication of dpp(65%), however, have a strongly lateralizedphenotype associated with a more dorsal character (referred toas the L2 phenotype). This L2 phenotype is characterized byfewer, finer or absent denticles and a lateralized gastrulationpattern where the germband does not extend dorsally and nopolarity is visible (Fig. 2F-H; Table 2). Since dpp is able tomodify a phenotype generated by the constitutively active Tlreceptor alone, we conclude that maternal dpp patterning actsin parallel to, or downstream of, gdand most likely Tl, to affectDV patterning of the embryo.

We also examined the effect of increasing the dose ofmaternal Dpp on the phenotype of embryos collected fromgd2/gd2;Tl3/+ mothers, which have very low levels of Spätzlesignaling and, as a consequence, some residual DV polarity(Fig. 2I,J) (gd2 is a strong, but not null, gdallele). This residualDV polarity is revealed by the presence of bothneuroectodermal and mesodermal gene expression domains(same as Fig. 2I). Consistent with the results described above,indicating that increased Dpp activity reduces Tl signaling, anextra copy of dpp shifts cell fates dorsally and eliminates allDV polarity in embryos from gd2/gd2;Tl3/+ mothers (Fig. 2J).In contrast to the effect of providing dpp maternally, adding

H. Araujo and E. Bier

Table 2. Maternal dppaffects DV patterning by reducing signaling through the Tl receptorWeakly

Dorsalized Lateralized Lateralized ventralized Ventralized Maternal genotype (%) (%) L2 (%) L1 (%) (%) V1

Tl3/+ 0 0 0 0 100Dp(dpp)/+; Tl3/+ 0 0 0 13 87sogU2/+; Tl3/+ 0 0 0 3 97gd/gd 100 0 0 0 0gdDp(dpp)/gd 100 0 0 0 0gd7/gd7; Tl3/+ 0 0 100 0 0gd7Dp(dpp)/gd7; Tl3/+ 0 66 34 0 0gd2/gd2; Tl3/+ 0 0 97* 1.5 1.5gd2Dp(dpp)/gd2; Tl3/+ 0 53 43 4 0gd2/gd2;Tl3/+ × Dp(dpp)/Y 0 0 98.5 0.7 0.7

The polarity of the embryos follows the classification of Anderson et al. (1985). Ventralized embryos have denticle belts in a disorganized fashion over theembryo and present an asymmetry in the DV axis, both in the cuticular and gastrulation patterns. The ‘weakly ventralized’ class of embryos has denticle beltswhich are expanded dorsally. The denticle belts in these embryos are also narrower than in strongly ventralized embryos. Lateralized embryos are more elongatedthan ventralized and show no observable polarity in the cuticular pattern. Their gastrulation pattern includes failure to extend the germ band and absence offormation of the mesoderm in the ventral furrow. L2 cuticles (e.g. Fig. 3F,H) have a more dorsal character than L1, characterized by finer denticles (e.g. Fig.3E,G). When the gd allele is not specified the results are valid for both gd2 and gd7. The presence of the dppduplication also increased greatly the percentage ofunfertilized embryos (gd/gd; Tl/+=4% versus gd Dp/gd; Tl/+=54%).

*This class is not purely lateralized since some residual polarity is still observed (see Fig. 3).

3637Maternal Dpp opposes Tl signaling

extra copies of dppzygotically had no effect on the DV polarityof embryos laid by gd2/gd2;Tl3/+ mothers (Fig. 2I; Table 2).

Sog and Dpp modulate nuclear accumulation ofDorsalThe ventral shift in the fate map of embryos derived fromsog/+;dl/+ or Dp(dpp)/+;dl/+ mothers suggests that the Dorsalgradient is reduced in magnitude in these embryos. Such adecrease could result from a reduction in total levels of Dorsal,an alteration in the shape of the Dorsal gradient or changesin the ability of Dorsal to activate/repress expression ofdownstream target genes. In order to distinguish among thesevarious possibilities, we compared the distribution of Dorsalprotein in transverse sections of blastoderm embryos collectedfrom dl−/+ mothers with that of embryos derived fromsog/+;dl/+ or Dp(dpp)/+;dl/+ mothers (Fig. 3A-C). When thedose of maternal dl is decreased by 50%, the width of thedomain of cells with high or moderate levels of Dorsal stainingis narrowed from approximately 24-26 cells in wild-typeembryos to 20-22 cells (Fig. 3A,B). In embryos derived fromsog/+;dl/+ or Dp(dpp)/+;dl/+ mothers, the extent of strong ormoderate Dorsal staining is reduced further to 14-16 cells (Fig.3C – see figure legend for further details). These data indicatethat the ventral shift in gene expression presented in Fig. 1results from a reduction in the width of the Dorsal gradient inthese embryos.

To distinguish whether the contraction of the Dorsal gradientresulted from a decrease in the total amount of Dorsal proteinthat is synthesized and imported into the oocyte or fromreduced nuclear transport of Dorsal into ventralmost nuclei, weassayed the total levels of Dorsal protein in early (e.g. 0-1 hour)embryos by quantitative immunoblotting. No significantchange in the amount of Dorsal was apparent in earlyblastoderm embryos derived from sog/+;dl/+ orDp(dpp)/+;dl/+ mothers beyond that attributed to the 50%reduction of dl gene dose indl−/+ mothers (Fig. 3D andlegend). We conclude that the maternal sog and dpp do notmodify the translation or stability of Dorsal, but rather affectthe nuclear accumulation of Dorsal in ventral and lateralembryonic nuclei.

Dorsal protein is prevented from translocating to the nucleusby binding to Cactus in the cytoplasm (Whalen and Steward,1993; Belvin et al., 1995). We investigated whether thereduction in the Dorsal gradient observed in embryos derivedfrom mothers with elevated levels of Dpp signaling might beassociated with altering the amount of Cactus protein (Fig. 3E).We found that the amount of Cactus is increased in embryosderived from sog Dp(dpp)/+;dl/+ mothers compared to that ofembryos derived fromdl−/+ mothers. This result is consistentwith maternal Dpp signaling inhibiting degradation of Cactus,or increasing Cactus synthesis, which would reduce nucleartranslocation of Dorsal.

Maternal sog modifies signal-independent CactusdegradationAs mentioned above, we observed elevated levels of Cactusprotein in embryos collected from sog Dp(dpp)/+;dl/+ mothers.This increase in Cactus could mediate at least part of the effectof Dpp signaling on nuclear translocation of Dorsal sinceproteolysis of Cactus protein is an essential regulated step tofree Dorsal for nuclear translocation. Two modes of Cactus

degradation have been described, one dependent on Tl signaling,and the other, referred to as signal-independent degradation,which is regulated by a Casein KinaseII-like activity (Sheltonand Wasserman, 1993; Liu et al., 1997). Signal-independentCactus degradation seems essential to maintain the correctbalance between Cactus and Dorsal since any Cactus protein notbound to Dorsal is degraded (Whalen and Steward, 1993). RNAinjection experiments have shown that amino-terminal serineresidues of Cactus are necessary for signal responsiveness. Bymutating these residues to alanine (cactnSA4), signal-dependentdegradation can be prevented. In contrast, removing a carboxyl-terminal PEST domain (cact∆PEST) inhibits signal-independentdegradation (Reach et al., 1996; Liu et al., 1997).

To determine whether either of the two known Cactusdegradation pathways is likely to be the target of maternalSog/Dpp signaling, we compared the phenotypes of embryoscollected from heat-shocked 8xhssog or wild-type controlmothers, which were injected with either cactnSA4or cact∆PEST

RNA (Table 3). The rationale of this experiment is thatoverexpression of sog during oogenesis produces embryos withventralized cuticles, while injecting either degradation resistantCactus construct dorsalizes embryos. We reasoned that itshould be possible to determine whether signal-dependent or -independent degradation of Cactus was reduced by inducingsog expression maternally and scoring the effect on theresulting cuticles. In these experiments (Table 3), we observedthat maternal induction of sogexpression significantly reducedthe percentage of embryos fully dorsalized by injection ofcactnSA4, which is resistant to signal-dependent Cactusdegradation (e.g. from 44% to 15%). In contrast, maternalinduction of sog expression did not affect the fraction ofstrongly dorsalized embryos resulting from injection ofcact∆PEST, which is refractory to signal-independent Cactusdegradation (e.g. 49% versus 46%). Presumably the stronglydorsalized cuticles resulting from injection of dominantnegative Cactus RNAs represent embryos in which one or theother pathways of Cactus degradation has been nearlycompletely blocked. Thus, the ability of maternally expressedsogto reduce the effect of cactnSA4RNA injection most likelyresults from reducing the effect of Dpp signaling on Cactussignal-independent degradation (i.e. the remaining pathwaythat can still degrade Cactus-nSA4). Although this assumptionmay not fully apply to all injected embryos given that a rangein cuticle phenotypes was observed, the fact that maternal

Table 3. Increasing maternal sogreduces signal-independent Cactus degradation

Number Wild type or Strongly scored weakly dorsalized dorsalized

Wild type, ∆ PEST 168 51% (n=85) 49% (83)8xhs sog, ∆ PEST 82 54% (44) 46% (38)Wild type, SA4 50 56% (28) 44% (22)8xhs sog, SA4 61 85% (52) 15% (9)

Wild-type embryos and embryos derived from 8xhssogmothers (15minutes heat shock performed 18-24 hours before injection) were injectedwith cact∆PESTor cactnSA4RNA at a concentration of 1 µg/ml. For unhatchedlarvae, cuticles were obtained and examined around the site of microinjection.Embryos of the strongly dorsalized class exhibited a severe reduction orcomplete absence of denticles, and often did not have Filzkörper. Wild-typecuticles and cuticles displaying only a slight reduction in the ventral denticleband width were grouped in the same class.

3638

overexpression of sog only reduced the fraction of fullydorsalized embryos derived from cactnSA4-injected embryosstrongly suggests that maternal Dpp signaling functionsprimarily by reducing signal-independent Cactus degradationand that Sog can block this effect of Dpp.

A dorsalizing grk -dependent activity functions inparallel to Tl signalingRoth and Schüpbach (1994) have previously providedcompelling evidence for the existence of a second patterningsystem regulated by EGF-R signaling, whichrefines the DV pattern established by Toll.The existence of this pathway can be inferredfrom the observation that embryos derivedfrom mothers carrying strong mutations incomponents of the EGF-R pathway do nothave a single expanded domain of Tl activity,as is observed with dominant ventralizingalleles of the dorsal group genes, but ratherform two separated foci of strong Tl signaling(Roth and Schüpbach, 1994). To determinewhether there is an activity of grk exertedon Tl signaling in the absence of Gd/Tlactivation, we examined the phenotype ofembryos derived from gd;grk;Tl3/+ triplemutant mothers. These mutant embryos differfrom those derived from gd;Tl3 double mutantmothers in that they also lack activity ofputative grk-dependent functions that mightact in parallel to the Gd/Tl pathway. Weobserved that cuticles of embryos collectedfrom gd;grk;Tl3 mothers have a more ventralcharacter than those derived from gd;Tl3

mothers (Fig. 3D,E). This observation isconsistent with grk activating a pathwayfunctioning in parallel to Gd/Tl, whichnormally exerts a dorsalizing influence. TheDpp pathway is a candidate for this secondgrk-dependent DV patterning activity asincreasing the level of dpp has the oppositeeffect to eliminating grk function.

sog and dpp are expressed in closeproximity during stage 10 ofoogenesisAs sogand dpp are expressed in adjacent orcomplementary domains in the early embryo(François et al., 1994) and in pupal wings (Yuet al., 1996), we examined whether thesegenes would be expressed in similarcorrelated patterns during oogenesis. Wefound that sog and dpp RNA transcripts areindeed expressed in close proximity duringcertain stages of oogenesis. dpp expressionis first detected during stages 8 to 10 ofoogenesis in stretch cells and in a subsetof follicle cells at the anterior end of theoocyte, and subsequently in the centripetallymigrating follicle cells (Twombly et al.,1996). Similarly, sog expression (Fig. 4) isfirst observed at stage 9 in a broad domain of

anterior follicle cells and then refines to a narrow stripe ofanterior follicle cells overlying the oocyte between stages 9 and10A in a pattern that is reminiscent of dpp expression at thisstage. Based on its position at the extreme anterior end of theoocyte, the stripe of sog-staining cells is likely to overlap withdpp-expressing cells or lie immediately adjacent to them. Theanterior stripe of sogexpression then collapses to a semicircleof ventral anterior follicle cells during stage 10B (Fig. 4C). sogexpression is not detected in germline cells at any stage ofoogenesis.

H. Araujo and E. Bier

Fig. 3. Reducing thelevels of maternal sogand dppmodifies thenuclear Dl gradient.Distribution of Dorsalprotein in cell nuclei intransverse sections of(A) wild-type embryos,(B) embryos derivedfrom dl/+ mothers, or

(C) embryos derived from Dp(dpp)/+;dl/+ mothers. Thedomain of ventral nuclei that exhibit strong and moderatelevels of staining is indicated by arrows, which correspondapproximately to 24-26 cells (wt), 20-22 cells (dl/+mothers), and 14-16 cells (Dp(dpp)/+;dl/+). We alsodetermined the number of cells with strong nuclear Dorsalstaining that was clearly higher than the levels ofcytoplasmic staining, which respectively were: 18 cells(wt), 14-16 cells (dl/+ mothers), and 10-12 cells(Dp(dpp)/+;dl/+). Analysis of several sections confirmedthat differences in the number of strongly stained nuclei

was not due to differences in the intensity of the stain. (D) The total amount of Dorsalprotein produced was assayed by immunoblot analysis. Total protein extracts of 0- to 1-hour-old embryos generated from wild-type (wt) (lane 1), dl/+ (lane 2 and 3),Dp(dpp)/+;dl/+ (lane 4) and sog/Dp(dpp);dl/+(lane 5) mothers were run on 8% SDS-PAGE and analyzed for the total content of Dorsal protein (upper panels).Approximately 5 µg were loaded per lane. The same blot was subsequently probed withanti-Tubulin (lower panels) to assure that equivalent amounts of protein were loaded.Embryos generated from dl/+ mothers produced roughly half the amount of Dorsalprotein (compare lanes 1 and 2). This amount is not reduced further by lowering thedose of maternal sogor by increasing the number of copies of maternal dpp (lanes3,4,5). The immunoblot data is representative of 5 different experiments. These datawere quantitated by densitometry for each experiment (see Materials and Methods) andthe ratio of the anti-Dorsal/Tubulin signal was determined for embryos derived frommothers of the genotype sog/Dp(dpp);dl−/+ as well as from the control dl/+ mothers. Theratio of the experimental to control values (e.g. the anti-Dorsal/Tubulin ratios) wascalculated for each experiment and then averaged, yielding a value of 1.05, which isconsistent with no significant difference between the total levels of Dorsal protein inembryos derived from sog/Dp(dpp);dl−/+ or dl/+ mothers. (E) The total amount ofCactus protein produced was assayed by immunoblot analysis. The exposure shown wasin the linear range of the film. Total protein extracts of 0-1h old embryos generated fromdl/+ (lane 1) and sog/Dp(dpp);dl/+(lane 2) mothers were run on 8% SDS-PAGE asabove and assayed for the total content of Cactus protein. The same blot wassubsequently probed with anti-Tubulin (lower panels) to assure that equivalent amountsof protein were loaded for each sample.

3639Maternal Dpp opposes Tl signaling

sog and dpp exert opposing effects whenmisexpressed during oogenesisAs complement to the loss-of-function studies presentedabove, we mis-expressed sog or dpp during specificdevelopmental stages in order to gain insight into when thesegenes might be influencing DV patterning of the embryo. Weobserved that heat induction of sogexpression during a broadperiod of oogenesis leads to ventralization of the embryoniccuticle as revealed by expansion of ventral denticle bands,while similar overexpression of maternal dpp leads todorsalization and narrow or absent denticle bands (Fig. 5E-H).The embryonic effects of overexpressing sog or dpp wereobserved with heat shocks supplied from midoogenesis (asearly as 30 hours before egg laying) to shortly before egglaying (0-6 hours before egg laying). It is difficult to determinethe phenocritical period for the effect of sogand dppon cuticlepatterning with great precision from these experiments,however, since overexpression of these genes results in a highpercentage of unfertilized eggs during much of oogenesis.

sog and dpp also play a separate maternal role inpatterning the eggshellThe misexpression studies described above reveal that sog anddpp also play a role in eggshell patterning. Decreased Dppsignaling in anterior follicle cells or misexpression of dppresult in defects in patterning the AP axis of the eggshell(Twombly et al., 1996; Fig. 5C), whereas overexpression of sogduring the same period (stage 10) leads to ventralization ofeggshell structures (Fig. 5A,B), rather than to AP patterningdefects. Loss-of-function studies are consistent with thesefindings since in 30% of the eggs derived from egg chambersin which sog− follicle cell clones were induced the oppositephenotype was observed (e.g. the dorsal appendages are

thinner and spaced further apart, Fig. 5D). The reciprocalphenotypes resulting from loss of sog function versus sogmisexpression suggest that sog normally limits peak Dppsignaling to the dorsal anterior midline of follicle cells in theegg chamber. The phenocritical periods for the eggshellphenotypes generated by sogand dpp misexpression coincide(24-30 hours before egg laying; Fig. 5J), consistent with thesetwo genes interacting during this stage of oogenesis.

The eggshell and embryonic phenotypes resulting from sogmisexpression or sog− follicle cell clones are consistent withsog playing a common role in eggshell and embryo DVpatterning. However, a variety of evidence indicates that theactions of sogand dpp in eggshell and embryonic patterningare distinct. For example, targeted overexpression of sogor dppin follicle cells during stage 10 of oogenesis by means of theGAL4/UAS system alters embryonic DV patterning in afashion similar to that achieved by heat-shock induction (Fig.5G,H). In contrast to its effect on embryonic patterning,however, misexpression of sog with two different GAL4drivers (55B-GAL4 or CY2-GAL4), does not generate anyeggshell defects. On the other hand, a high percentage (Fig. 5I,82%) of all collected embryos hatch with no cuticular defectsduring periods when more than half of the eggs generated fromheat-shocked 8xhssogmothers have eggshell defects (Fig. 5J,24-30 hours, 59%). These experimental manipulationsuncouple the effects of sog and dpp and reveal that these genesexert independent functions to pattern the eggshell versus theembryo.

Sog and Dpp do not affect expression of maternalgenes involved in DV patterning of the embryo.We analyzed the maternal functions of sog and dpp withregard to embryonic patterning in greater detail by

Fig. 4. Expression of sogand dppduringoogenesis. RNA in situ hybridizationshowing sogexpression (A-E) or dppexpression (F) in egg chambers. sogisfirst expressed by stage 9 in a broaddomain of migrating follicle cells (A),and is further refined during stage 10A(B) to a stripe of ventral anterior folliclecells (C). A transient expression of sogina small group of follicle cells directlyabove the oocyte nucleus can also be seenat stage 10B (D, arrow). At the samestage (10B), dpp is expressed in theanterior follicle cells encircling the wholeegg chamber (F). At stage 12 and 13 (E),sog staining is observed in nurse-cell-associated follicle cells (NFC).Overexpression of sogor dppby heatshocking flies containing 8xhssog(H,K)or 8xhsdpp(I,L) modifies the expressionof pointed(G,H,I), but not pipe (J-L) infollicle cells. In control experiments,ectopic expression of sogand dppproduced by heat shock resulted inwidespread expression of sogor dpp(notshown). Dorsal is up and anterior to theleft in all panels.

3640

misexpressing these genes during oogenesis and examiningexpression of various DV marker genes. The distribution andlevels of EGF-R ligand Grk, which regulates both embryonicand eggshell patterning (see Nilson and Schüpbach, 1999)were not altered by overexpression of either sogor dpp (notshown). Similarly, overexpression of sogor dpphad no effecton the localized expression of pipe in ventral follicle cells(Fig. 4J-L). Since it has been suggested that pipe restrictsactivation of upstream elements in the Gd/Tl pathway to theventral portion of the embryo (Sen et al., 1998), these data areconsistent with the maternal roles of sog and dpp onembryogenesis acting later downstream of the Tl receptor. Incontrast, misexpression of either sog or dpp altered theexpression of genes required for patterning the eggshell suchas pointed(pnt), which encodes an ETS domain transcriptionfactor. pnt is expressed in the dorsal anteriormost follicle cellsand plays a role in positioning the dorsal appendages of theeggshell, but has no known maternal role in embryonicDV patterning (Morimoto et al., 1996) (Fig. 4G-I).Overexpression of sog inhibits ventral expression of pnt in

anterior follicle cells, while dpp nearly abolishes pntexpression. Since overexpression of pnt inhibits appendageformation (Deng and Bownes, 1997), it is possible that sogand dpp contribute to restricting pnt expression to theprimordia of the dorsal appendages. The observation thatoverexpression of sog or dpp during midoogenesis altersexpression of genes involved in eggshell patterning, but doesnot affect the expression of key maternal genes required forembryonic development, supports the view that sogand dppplay separate roles in eggshell and embryonic patterning.

DISCUSSION

sog antagonizes dpp during oogenesis to regulateembryonic DV patterningIn this study, we show that dpp functions maternally tosuppress nuclear translocation of Dorsal triggered by the Tlsignaling pathway in the early Drosophilaembryo. We showthat the maternal effect of Dpp signaling is opposed by a

H. Araujo and E. Bier

Fig. 5.Phenotypes ofmaternal overexpressionof sogand dpp. (A) Awild-type eggshell. Thedorsal appendages (da)form at a fixed anglerelative to each other.Bars indicate the APextent of the operculum,extending from themicropile (mp) to thebase of the dorsal

appendages. (B) Eggshells derived from 8xhssogmothers (embryo collection 24-30 hours after a 30 minute heat shock during oogenesis) havedorsal appendages shifted closer together or fused dorsally. (C) Eggshells derived from 8xhsdppmothers have defects in anterior structuressuch as enlargement of anterior eggshell structures (e.g. the operculum) and abnormal or absent dorsal appendages. Eggs were collected 24-30hours after 15 minute maternal heat shock. (D) An eggshell derived from mothers containing sog− follicle cell clones. The dorsal appendagesare thinner and the distance between their bases is increased. (E) A wild-type embryonic cuticle with organized denticles belts on the ventralside of the embryo. (F) Embryos from heat-shocked 8xhssogmothers (embryos collected 0-6 hours after a 30 minute heat shock) haveventralized cuticles, with denticle belts encircling the embryo and no dorsal structures (e.g. Filzkörper). Embryos collected from heat-shocked8xhsdppmothers (embryos collected 0-6 hours after a 15 minute heat shock) have dorsalized cuticles (not shown). (G) Driving expression of1xUASdppwith the GAL4 55B line, which driveslacZ expression in the anterior follicle cells, results in a dorsalized cuticle.(H) Overexpression of sogwith the CY2 GAL4 line, which drives expression in all follicle cells surrounding the oocyte, leads to expansion ofventral denticle bands (compare with bar in E) and sometimes to gaps in the denticle belts (arrows). (I,J) A plot of the effects of sogor dppoverexpression on embryonic viability (I) or on eggshell morphogenesis (J). Mothers containing 8xhssogor 8xhsdppwere heat shocked for 15or 5 minutes, respectively, at 38°C. Eggs were collected for 6 hour periods at the times indicated after heat shock. The percentage of viabilityand eggshell defects were determined for the same experimental sample. Curves represent characteristic profiles observed in three differentexperiments.

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3641Maternal Dpp opposes Tl signaling

strictly maternal function of sog. In accord with its expressionin an anterior ring of follicle cells, clonal analysis indicatesthat sog function is required in follicle cells but not in theoocyte.

The maternal function of sog in embryonic patterning isdistinct from the previously reported zygotic functions of sogin protecting the neuroectoderm from invasion by Dppsignaling and in patterning the dorsal region of the embryo.Thus, maternal Sog and Dpp proteins do not simply augmenttheir normal effects during zygotic patterning. For example, thezygotic genotype of embryos (e.g. sog+ versus sog−) has noeffect on the phenotype of embryos derived from sog−/+;dl/+mothers. Furthermore, whereas overexpression of maternal soginduces strong ventralization of the cuticle, the most extremephenotype resulting from overexpression of zygotic sog orinjection of high concentrations of sogmRNA into blastodermembryos is only a slight expansion of the ventral denticle belts(Holley et al., 1995; Biehs et al., 1996). Similarly, the maternalphenotypes associated with reduction or elimination of sogfunction result in ventral shifts of gene expression patternsalong the entire DV axis, whereas mutants lacking the zygoticfunction of sog have localized defects in gene expressionpatterns confined to the neuroectodermal and dorsal regions ofthe embryo, but do not display shifts in the relative position ofthese gene expression domains.

In addition to their maternal role in patterning the embryo,sog and dpp also influence eggshell patterning duringoogenesis. The maternal activities of sogand dpp in patterningthe eggshell versus the embryo can be uncoupled, however, byseveral experimental manipulations. Thus, sog and dpp mostlikely function in two independent capacities to pattern theeggshell versus the embryo. As the focus of this paper is on

embryonic patterning, we will not discuss the roles of soganddpp in eggshell patterning further in this report.

Maternal Dpp signaling intersects the Gd/Tl pathwaydownstream of the Tl receptorA variety of data indicates that maternal Dpp signaling actsdownstream of, or in parallel to, the Tl receptor. Most critically,increasing the gene dose of dpp in mothers carrying only anactivated signal-independent form of the Tl receptor has adorsalizing effect. This result indicates that Dpp signaling isunlikely to affect the production or distribution of the Spätzlesignal, but rather functions during early embryogenesis whenthe Tl receptor is activated by a ventral source of Spätzle. Otherdata are consistent with Dpp signaling affecting Tl signalingin the early embryo instead of during midoogenesis when sogand dpp are expressed. For example, it is possible to alterembryonic patterning by misexpressing dppor sogduring lateoogenesis, when communication between follicle cells and theoocyte is no longer possible. In addition, misexpression of dppor sog during midoogenesis has no effect on grk or pipeexpression, two genes expressed in follicle cells that play keyroles in patterning the DV axis of the embryo.

Dpp signaling increases Cactus levels bysuppressing signal-independent Cactus degradationThe observation that elevated Dpp signaling increases Cactuslevels is consistent with the view that Dpp signaling intersectsthe Gd/Tl pathway downstream of the Tl receptor to reducenuclear translocation of Dorsal. RNA injection experimentssuggest that the increase in Cactus results from Dpp signalingreducing the efficiency of a signal-independent form of Cactusdegradation. This conclusion is based on the observation that

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Fig. 6. Model for maternal sog and dppfunction during early embryogenesis. Inthis model, Sog and Dpp are producedduring oogenesis and are delivered tothe perivitelline space, but do notfunction until early embryogenesis. Theactivation of Dpp receptors in theembryonic plasma membrane sends asignal that blocks signal-independentdegradation of Cactus, leading toelevated Cactus levels, which binds toDorsal and inhibits its nucleartranslocation. Sog, which is present inthe perivitelline space, opposes Dppactivity leading to elevated Dorsalnuclear translocation.

3642

the dorsalized phenotype resulting from injection of a mutantform of Cactus that is resistant to signal-dependent degradationcan be partially reversed by reducing Dpp signaling (e.g. viaectopic sogexpression). In contrast, the dorsalized phenotyperesulting from injection of a mutant form of Cactus that isrefractory to signal-independent Cactus degradation is notaltered by reducing Dpp signaling. This result can most easilybe understood if the effect of Dpp signaling were to reduce theefficiency of signal-independent Cactus degradation. LoweringDpp signaling would increase signal-independent Cactusdegradation and therefore would have little affect on a mutantform (e.g. ∆PEST) that is resistant to this pathway. In contrast,the total levels of a mutant form of Cactus that is sensitive tosignal-independent degradation (e.g. snA4) would be expectedto decrease if this latter pathway became more active. Theseresults suggest that the mechanism by which Dpp signalingdecreases nuclear Dorsal levels is to increase the level ofCactus protein, which in turn leads to a higher proportion ofDorsal being sequestered in an inactive Cactus/Dorsal complex(Fig. 6).

Crossregulatory interactions between the Tl andDpp pathwaysIn vertebrates, crossregulation between the interleukin andTGFβ pathways has been described, particularly in theregulation of the inflammatory response (reviewed in Letterioand Roberts, 1998). For example, TGFβ signalingcounterbalances the effect of proinflamatory cytokines bylimiting the production of IFNδ and increasing the expressionof IL-1 Receptor Antagonist (Fargeas et al., 1992). In contrastto the numerous effects of TGFβ on regulating interleukin geneexpression, little has been reported regarding crossregulationbetween the actual signal transducing elements of the TGFβand interleukin pathways. The crossregulation between Tl andDpp signaling described in this study provides the first modelgenetic system for examining the mechanistic basis of theintersection between these pathways and for studyingsuppression of the inflammatory response by TGFß signaling.

A model for maternal Sog and Dpp functionIn aggregate, the results described above support models inwhich Sog and Dpp proteins are produced by the follicle cellsand then are delivered to the embryo (Fig. 6). These proteinscould be deposited in the vitelline membrane or in the oocyteplasma membrane, or might be sequestered in the perivitellinespace and remain there protected until early embryogenesis.The fact that sogand dppare expressed in follicle cells of stage10 egg chambers, around the time that follicle cells aresecreting major structural proteins of the vitelline envelope, isconsistent with their products being delivered to the vitellinemembrane or perivitelline space (Fargnoli and Waring, 1982;St Johnston and Nüsslein-Volhard, 1992). Since sogand dppare secreted proteins, they could be exported like componentsof the vitelline membrane to the extracellular compartmentbetween the follicle cells and the oocyte. After stage 13, thevitelline membrane is thought to be an impermeant barrierseparating the oocyte from follicle cells making it unlikely thatsogand dpp products are transferred after this time. A similarmodel has been proposed to explain the functions of the dorsalgroup gene nudel(Hong and Hashimoto, 1995; LeMosy et al.,1998) and of the maternal terminal system gene torsolike(tsl)

(Savant-Bhonsale and Montell, 1993). Both of these genes areexpressed during midoogenesis, long before their activity isrequired during early embryogenesis. According to this model,the Sog and Dpp proteins would remain in the perivitellinespace until early embryogenesis, when the Tl pathway isactivated by Spätzle. In the early embryo, maternal Dpp woulddecrease the level of Tl-mediated nuclear translocation ofDorsal by decreasing Cactus signal-independent degradationthrough a pathway acting in parallel to Tl. Presumably, sogantagonizes the action of dpp, resulting in maximal nuclearDorsal translocation.

Consistent with the view that Sog and Dpp proteins are madeearly (e.g. midoogenesis), but act later in the early embryo,induction of sogexpression during midoogenesis by use of aheat-shock sogconstruct increases levels of a Sog fragment inthe early embryo detected by a specific anti-Sog antibody (notshown). Thus, Sog protein produced during midoogenesis canbe stably stored for a protracted period until the onset ofembryogenesis. In contrast to Sog protein, sog mRNA does notperdure at detectable levels in early pre-blastoderm embryos inthese experiments (not shown). The fragment of Sog generatedin these experiments is the same size (60 kDa) as one that mayhave activity during pupal development (Yu et al., 2000).

The role of maternal sog and dpp in establishing theDorsal gradientThere are several unanswered questions regarding howmaternal Dpp signaling contributes to embryonic DVpatterning. An important remaining question is how maternalDpp signaling contributes to defining discrete zones of geneexpression along the DV axis? Two leading possibilities, whichare not necessarily mutually exclusive are: (1) sog and dppfunction to determine the relative proportions and positions ofthe different primary DV domains, and (2) Dpp signaling isnecessary to sharpen borders between embryonic DVterritories. There is good evidence in support of the firstpossibility, since the extents of DV expression domains can bealtered by increasing maternal Dpp activity. As mentionedabove, maternally produced Dpp results in a ventral shift of allDV domains, presumably by lowering the amount of nuclearDorsal in cells along the entire DV axis. Our results alsosupport a role for maternal sog and dppin refining the normallysharp borders between different territories, since altering thematernal dose of sogor dppgenerates overlapping expressionof mesodermal and neuroectodermal genes.

Another question is by what mechanism does maternal sogoppose Dpp in patterning the embryo? Perhaps sogis necessaryto inhibit Dpp signaling through a specific receptor subtypesuch as the Sax receptor (Neul and Ferguson, 1998; Nguyen etal., 1998) or to restrict Dpp signaling to a specific type of Dppreceptor (e.g. mediated only by Tkv). Alternatively, sogcouldbe involved in antagonizing another BMP molecule in additionto Dpp, which also functions in embryonic DV patterning.

In summary, the results presented in this study indicate thatmaternal components of Dpp signaling modify elements thatconverge with signaling downstream of the Tl receptor byregulating Cactus levels and nuclear translocation of Dorsal.Our analysis suggests that maternal sog and dpp function todefine the relative proportions of embryonic DV domains andmay play a role in creating sharp borders between thesedomains. Further experiments will be necessary to determine

H. Araujo and E. Bier

3643Maternal Dpp opposes Tl signaling

the mechanism by which maternal sog and dpp function andhow interactions between the Tl and Dpp pathways collaborateto pattern the DV axis of the Drosophilaembryo.

We thank Steve Wasserman, Par Taub, Trudi Schüpbach andHannele Ruohola-Baker for valuable discussions and helpfulcomments on the manuscript, Steve Wasserman for providing thecactnSA4and cact∆PESTconstructs, Ruth Steward for the anti-Twist andanti-Dorsal antibodies, David Stein for the pipecDNA, Joseph Duffyfor fly stocks to make follicle cell clones, Brian Biehs for help within situ hybridizations and assembling the final figures, and Dan Angfor assistance with the figures. This work was supported by NIH grantno. NS29870 to E. B. and by CNPq-Brasil and Fogarty Fellowshipsto H. A.

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