H~EVIEWS

D0rs0ventral pattern T h e developmental program of Drosophila melanogaster is unfolded by the sequential action of a hierarchy of genes that specify pattern elements. Maternal genes function at the top of this hierarchy to establish broad polarities along the anteroposterior and dorsoventral axes. In both systems, interactions among maternally encoded products lead to the graded expression of one or more regulatory molecules in the early embryo. While the anteroposterior axis is determined by three largely independent systems], the dorsoventral axis appears to be specified by gene products whose ultimate task is to create a nuclear gradient of dorsal tSroteifi, the ventral morphogen. Embryonic cells express distinct sets of zygotic genes and follow different developmental pathways as a result of their exposure to different levels of dorsal protein. In this review, we present a summary of the activities responsible for generating this dorsoventral polarity, with special emphasis on the role of dorsal protein.

The dorsoventral pattern Development of dorsoventral pattern of the egg chamber is initiated early in oogenesis and is established through the interaction of several maternal

formation in Drosophila: signal transduction and nuclear targeting SHUBHAGOVINDAND RUTH STEWARD The maternal determinants of dorsoventral polarity of the Drosophila embryo are derived from somatic and germ-line components of the egg chamber. During oogenesis, asymmetry seems to be established by a signal transduction process. Thisprocess is thought to provide the developing embryo with a ventral signal responsible for determining the embryonic axis. Througha set of interactions that may involve signal transduction and proteolytic cascade events, positional information is generated in the form of a graded distribution of dorsal protein in blastoderm nuclei. Different levels of dorsal protein result in asymmetric expression of zygotic genes that ultimately specify ceUfate. genes (Table 1), which are active in both the germ line and the soma. The somatic follicle cell epithelium, which surrounds the oocyte and secretes the egg shell

TABLE 1. Genes involved in dorsoventral pattern formation in Drosophila a

Dorsaltztttg

Ventraliztrtg

Maternal genes required for egg shell and embryonic polarity fs(1)Kl O (KI O) Helix-turn-helix torpedo (top) b cappuccino ( capu) gurken (grk) spire (spir) cornichon (cni)

Maternal genes required for embryonic polarity nudel (ndl) b pipe (pip) b windbeutel (wind) b gastrulation defective (gd) Serineprotease snake (snk) Serine protease easter (ea) Serine protease spi~tzle (spz ) Toll (71) Transmembrane, similar to GPlb thrombin receptor pelle (pll) tube (tub) dorsal (dl) Ventral morphogen, relfamily Zygotic genes snail (sna) twist (two

Zinc finger Helix-loop-helix

EGF receptor homolog

cactus (cact)

zerknf~llt (zen) decapentaplegic (dpp) tolloid (tld) screw (sew) shrew (srw) short gastrulation (sog) twisted gastrulation (tsg)

Homeobox TGF-~ family Similar to BMP-1

aGenes grouped according to their loss-of-function phenotype. bGenes known to function in the soma. Abbreviations: EGK epidermal growth factor; GPlb, platelet glycoprotein lb; TGF-[~, transforming growth factor-J3; BMP-1, bone morphogenetic protein-1. T1GAPRIL199t VOL. 7 ~O. 4 '610qt Ftst'~ icr ~,cien{ e P u b l i s h e r s Lid ( I ;K} 0108 - t)47() t)l S02 0(}

m

[]~IEVIEWS (chorion and vitelline membrane), shows regional differences in the shape and arrangement of cells on the dorsal and ventral sides. These differences become apparent in the pattern of the mature egg shell. Thus, when the egg is laid, it is enclosed within a multilayered egg shell and displays a well-defined anteroposterior and dorsoventral polarity. It has a fiat dorsal surface and a slightly convex ventral side. A micropyle and an operculum are present at the anterior end and two chorionic appendages are located anterodorsally. Embryonic polarity develops early in embryogenesis and is dictated by the organization of the egg chamber. The zygotic nuclei initially divide in the center of the embryo and migrate to the periphery after eight divisions, giving rise to the syncytial blastoderm. The nuclei undergo another five rounds of division and are then enclosed by membranes to form the cellular blastoderm. Dorsoventral polarity is morphologically evident by early gastrulation. A fate map shows that while the ventral-most cells of the blastoderm give rise to the mesoderm, a small group of dorsal-most cells gives rise to the extraembryonic amnioserosa. The ventrotateral neurogenic region gives rise to the ventral epidermis and the nerve cord, and the dorsolateral cells result in the dorsal epidermis 2 (Fig. 1A). The embryo develops into a larva with a rich cuticular pattern providing landmarks of polarity.

Formation of dorsoventral patterns of the egg and embryo are coupled A small group of maternal genes is known to be required for the establishment of the dorsoventral pattern of both the egg shell and the embryo. Mutations in fi(1)KlO, cappuccino and spire dorsalize both the

A

egg shell and the embryo, indicating that they are required for the formation of the ventral pattern elements3. Loss-of-function mutations in torpedo, gurken and cornichon cause ventralization of the egg shell and embryo. Thus these latter genes are required for the determination of dorsal follicular epithelium and ultimately the proper disposition of dorsal pattern elements in the embryo3,4. Analysis of germ-line mosaics has established that all these maternal genes - except torpedo - function only in the germ line3,4; torpedo was found to be soma dependent5 and to encode the Drosophila homolog of the vertebrate epidermal growth factor receptor, with an intracellular tyrosine kinase domain6,7. These observations suggest that the germ-line and somatic components in the ovary cooperate to define the dorsoventral axis of the egg chamber using signal transduction events. A model for early steps in dorsoventral pattern formation3,4 proposes asymmetric signalling between the oocyte-nurse-cell complex and the follicle cells: a signal secreted by the oocyte on the dorsal side of the egg chamber involves the activities of the gurken and cornichon genes, and is regulated by the fs(1)KlO, cappuccino and spire gene products. This signal activates the receptor (torpedo gene product) and results in the determination of the dorsoventral identities of the follicle cells. This asymmetry is then transmitted to the embryo through the vitelline membrane that is secreted by the follicle cells3,4.

Genes involved in embryonic polarity Members of another group of maternal genes - the dorsal group - and the cactus gene (Table 1) are essential for the dorsoventral polarity of the embryo,

B 4"o

I

Y

Me FIGH The fate map of the blastoderm stage embryo compared with the nuclear gradient of the dorsal protein. (A) Schematic drawing of cross-section fate map of blastoderm-stage embryo and initial expression pattern of zygotic genes. As, amnioserosa; dEE dorsal epidermis; Me, mesoderm; vNr, ventral neurogenic region. Also shown are regions of initial expression of zygotic genes in response to the nuclear gradient of dorsal protein. While twist (two and snail (sna) are expressed in the presumptive mesoderm, tolloid(tld), zerkn~llt (zen) and decapentaplegic (dpp) are expressed in the presumptive amnioserosa and dorsal ectoderm. (B) Cross-section (2 btm) through a blastoderm-stage embryo stained with anti-dorsal antibody. TTGAPRIL1991 VOL. 7 >~O.4

i ._,~i

I'~EVIEWS but not the egg shellS,9. A loss-offunction mutation in any of the dorsal group genes causes an identical 'dorsalizing' phenotype. All embryonic cells in dorsalized embryos follow a dorsal developmental pathway and therefore the embryo lacks all lateral and ventral pattern elements8,9. Conversely, loss-of-function mutations in cactus cause ventralization of the embryo: homozygous mutant females produce embryos that show a decrease or loss of dorsal epidermis, which is replaced by ventrolateral epidermis9. Since mutations i~ any,of the dorsal group genes result in the same phenotype - partial or complete dorsalization - it was proposed that the dorsal group genes participate in a common pathway. Analysis of double mutants and transplantation-rescue experiments suggested the order of their action and placed dorsal at the end of this maternal pathway9,m. Recent experiments have shown that all members of this pathway function to create a nuclear gradient of dorsal protein in the syncytial blastoderm stage embryo T M .

The ventral morphogen is dorsal protein

dorsal

vitelline membrane

i

plasma membrane

01" X

~'

(pipe, nudel, windbeutel)

~

lOllI

, • ~

/

(snake, easter, gastrulation defective) gastrulal

ventral

FIGlii A schematic representation of the dorsoventral polarity pathway leading to the formation of a nuclear gradient of dorsal protein. Early events during oogenesis in the germ line and soma generate a somatic signal on the ventral side. Dorsal group genes nudel, pi/x, and windbeutelare known to act in the soma and are thought to contribute to this signal. Germ-line factors encoded by dorsal group genes gastrulation defective, snake and easter are thought to be secreted from the embryo. All dorsal group genes acting upstream of Tollsomehow generate a ligand that activates the transmembrane Toll receptor on the ventral side. The graded activation of Toll results in the graded nuclear import of dorsal protein mediated by the activities of pelle and tube. (The model is a schematic cross-section of a blastoderm-stage embryo.)

The dorsal gene was identified on the basis of its haploinsufficient temperature-sensitive dominant phenotype. Interpretation of both recessive and dominant phenotypes led to the proposal that there must be a gradient of dorsal activity that has a highpoint at the ventral midline and decreases in the dorsal direction 13. Indeed, antibodies raised against the dorsal protein showed that it is distributed in the form of a ventral-to-dorsal nuclear gradient in the syncytial blastoderm. The regional nuclear concentration of the dorsal protein correlates with the developmental fates of the corresponding blastoderm cells 14 (Fig. 1). This is further suggested in experiments in which at least four types of radially symmetrical embryos were obtained, each of which exclusively displayed either ventral, ventrolateral, dorsolateral or dorsal pattern elementsn.R In each of these mutant embryos, the level of the nuclear dorsal protein is comparable to that of the corresponding regions in wild-type embryos. Thus, different thresholds of dorsal protein concentration can provide cells with different positional values, dividing the dorsoventral axis into broad 'domains'ILl 2. The nuclear dorsal protein gradient is formed by selective nuclear targeting: dorsal protein is uniformly distributed within the cytoplasm of unfertilized eggs and early embryos. In wild-type embryos, it becomes selectively relocalized in a ventral-to-dorsal nuclear gradient. Embryos from females that are mutant in dorsal group genes show a dorsalized phenotype and have dorsal protein only in the cytoplasm, along the entire dorsoventral axis. This observation suggests that

dorsal protein is not active in the cytoplasm and Ihal the dorsal group genes are responsible for the selective nuclear targeting of dorsal protein. Conversely, in embryos derived from cactus females, which have a ventralized phenotype, the dorsal protein gradient is shifted dorsally, suggesting that the wild-type cactus gene product is involved in retaining dorsal protein in the cytoplasm. Thus, the selective nuclear import of dorsal protein appears to be positively regulated through the dorsal group gene products, but negatively regulated by cactus lm2.

Nuclear gradient of dorsal protein: the big picture A model for the dorsoventral polarity pathway is shown in Fig. 2. Three rnembers of tllc dorsal group {pipe, nudel and windbeuteh function in tile soma ~ (D. Stein, pers. commun.). While their functions art. not yet understood, it appears that their actMties are controlled by the signal transduction events between the oocyte and the follicle cells and seem t(} be involved in information flow' from the s{}ma I)ack into the germ line on the ventral side. This ventral signal is thought to orient the embry{mk axisA+, and is processed in the embryo by the action ()f other dorsal group genes: snake, easte~ gastndation &'/'{,ctive and spatzle function upstream of Toll, while tube and pelh, function after Toll in this pathway > (K. Anderson, pets. commun.). The Toll gene product plays a key role in this pathway. Injection of wild-type cytoplasm could rescue?

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H~EVIEWS Toll- embryos in a position-dependent manner such that the site of injection defined the ventral-most region of the embryo 15. The Toll gene encodes a transmembrane protein with a large extracellular domain that has multiple copies of a leucine-rich repeat and shares homology with the Ix-chain of platelet glycoprotein lb. This domain of the glycoprotein l b is believed to act as a receptor for thrombin and von Willebrand factor in the blood-clotting cascade. It has therefore been suggested that Toll may function as a receptor 16. Antibodies directed against the Toll protein uniformly stain the plasma membrane of the syncytial blastoderm-stage embryo. These observations suggest that the dorsoventral axis of the embryo is determined through the asymmetric activation of Toll receptor by the dorsal group genesl6,17. The ligand for the Toll receptor is not known, but appears to be generated by the interactions of dorsal group gene products functioning upstream of Toll. The snake, easter and gastrulation defective genes have been cloned and their products show sequence homology to secreted serine proteases involved in complement fixation and blood clotting 18-20. It is therefore possible that these proteins are secreted from the embryo and, in concert with the somatic signal, result in the ventral activation of Toll. The asymmetric activation of the Toll protein would then indirectly result in the graded distribution of dorsal protein in the nucleus 17.

gene of the avian reticuloendotheliosis virus Rev-T. The v-rel gene causes fatal lymphoid cell tumors in chicks. It also mediates transformation of cultured spleen cells. While the functions of the v- and c-rel gene products (v-Rel/c-Rel) are not defined, there is growing evidence that the c-Rel protein is a transcription factor 24. More recently, dorsal and Rel were also found to be similar to KBF1 and the p50 subunit of NF-~:B.proteins 24~a6. The NF-KB protein was identified because of its ability to bind to the ]~B site (GGGACTITCC) in the immunoglobulin kappa light chain enhancer region 27. Initially thought to be restricted to B cells, NF-~cB is now known to be involved in the inducible expression of several genes in a variety of cell types 2s. KBF1, the transcription factor that promotes the expression of the class I major histocompatibility gene complex, has been found to be identical to the p50 subunit of NFl~:B25. The dorsal, Rel and p50/KBF1 proteins are about 50% identical over approximately 300 amino acids in the amino-terminal region. A stretch of basic amino acids that functions as a nuclear targeting signal and a possible phosphorylation site (Arg-Arg-X-Ser) are also conserved at the same relative positions in all these proteins. The carboxy-terminal ends are, however, completely divergent (Fig. 3). This suggests that while the conserved amino-terminal halves of the Rel-related proteins may possess functional similarity, the divergent carboxy-terminal domains may modulate this function in a cell-specific or species-specific manner. NF-KB is a complex of proteins consisting of at least two polypeptides, pS0 and p65 (Ref. 29). Peptide sequence data indicate that the p65 subunit also shares sequence similarity with the mouse c-Rel protein, even

The dorsal protein is similar to NF-ICBand Rel The dorsal protein shares sequence similarity with the product of the vertebrate proto-oncogene c-rel and its viral homolog v-rel (Refs 21-23), the transforming

I

-,,I

p 50

•

NLS I====11

rf~l

450 aa

( 969 aa)

P I HLS

v-rel

I

• ~==II

503 aa

P I NLS

a v i a n c-rel

,~

• ~====lJ

598 aa

P NLS I

mouse

c-rel

h u m a n c-rel

dorsal

~

q

• ~11

588 aa

P I NL$ • i=====11

619 aa

P I NLS

I

•

~

II

U

similar

II

678aa

divergent FIG~I

A schematic of the primary structure of the Ret family of proteins predicted from their cDNAsequences. The p50 subunit of NFIKBis part of a larger protein (969 amino acids). Arrow shows possible proteolytic cleavage site. P, possible phosphorylation site; NLS, nuclear localization signal. TIG APRIL1991 VOL.7 NO. 4 I~

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in the carboxy-terminal divergent region, thus including p65 in the rel cactus-dorsal family of proteins 26. complex The KBF1/p50 polypeptide is encoded by a message with an open reading frame that predicts a protein dorsal of 969 amino acids (119 kDa) and is nucleus cactus thought to be processed to a poly~,A peptide of 50 kDa (Fig. 3). p50 binds to DNA in vitro (the 119 kDa precursor does not), either as a homodimer or a heterodimer with p65 (Refs 25, 26). The DNA-binding domain shows no slmtlanty with any releasing activity known DNA-binding motifs, p65 lacks (pelle, tube ?) DNA-binding activity29. In vitro transcription experiments have shown that KBF1/p50 alone cannot activate Model for nuclear targeting of the dorsal protein based on genetic and antibody transcription. However, the p50-p65 staining data. The dorsal protein, drawn here as a homodimer (see text for complex isolated from cells is tranexplanation), is retained in the cytoplasm by the action of cactus (cactus-dorsal scriptionally competent, suggesting complex). Upon receiving a signal derived from the activities of the upstream genes that p65 may indeed be an activator through pelle and tube proteins, dorsal protein is released from cactus and of p50. The DNA-binding and dimertranslocated into the nucleus. ization domains in p50/KBF1/v-Rel have been mapped to the homologous region of the dorsoventral axis~t In these embryos, cactus is absent proteins25, 26. and dorsal protein is not retained in the cytoplasm. The nuclear targeting signal in dorsal protein is funcNuclear targeting of dorsal and NF-r,B tionally competent, but the higher levels correspondThe specific and regulated nuclear import event ing to those in the wild-type ventral and ventrolateral described for dorsal protein in the developing embryo nuclei are not attained. It appears, therefore, that in is analogous to the regulated import of the NF-~B com- addition to controlling the release of cytoplasmic dorplex in B cellsn, 12,30. The NF-gB complex is inactive in sal protein from cactus, the dorsal group genes, acting the cytoplasm of uninduced cells and is associated with through pelle and tube, also promote the graded import of dorsal protein. an inhibitor protein (II