The Role of the dpp-Group Genes in Dorsoventral Patterning of the Drosophila Embryo

THE ROLE OF THE dpp-GROUP GENES IN DORSOVENTRAL PATTERNING OF THE DROSOPHlLA EMBRYO

Christine Rushlow and Siegfried Roth

Introduction . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . , . . . . . , . . . . . . . . . . . . . . . . . . . . . . B. dl as a Transcriptional Repressor . . . . . . . . , . . . . . V. The dpp-Group Genes . . . . . . . . . . . . . . . . . . . . . . A. dpp is a Signaling Molecule . . . . . . . . . . . . . . . . B. dpp is a Component of a Graded Patterning Process . . . C. dpp is a Morphogen . . . . . . . . . . . . . . . . . . . . D. tolloid Encodes a Potential Protease . . . . . . . . . . . . E. screw Encodes Another Member of the TGFP Superfamily F. Additional Genes Required to Enhance dpp Activity . . .

I.

11. The Dorsoventral Pattern . . . . . . 111. The doixrl Morphogen Gradicnt . . IV. Targets of the cll Gradient . . . . . . A. dl as a Transcriptional Activator

Advances in Developmental Binlogy Volume 4, pages 27-82. Copyright 0 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-969-9

27

. , . . . . . . . . . . . . . . . . . . . . . . . .

28 29 34 37 38 40 42 43 45 48 49 51 53

28

CHRISTINE RUSHLOW and SIEGFRIED ROTH

G. short gastrulation Inhibits dpp Activity . . . . . . . . . . . . 54 13. twisted gustrulation and zen Act Downstream or in Parallel to dpp . . . . . . . . . . . . . . . . . . . . . . 58 VI. Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 VII. Formation and Function of the dpp Gradient: Summary and Prospectives . . . . . . . . . . . . . . . . . . . . . 65 A. The dpp Gradient is Dependent on, but not Completely Determined by, the d/ Gradient . . . . . . . . . . 65 B. The dl and dpp Morphogen Gradients Establish Different Parts of the DV Pattern . . . . . . . . . . 67 VIII. A Comparison with Vertebrates . . . . . . . . . . . . . . . . . . . 70 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . 73 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

1.

INTRODUCTION

Morphogens are regulatory molecules distributed in monotonic gradients. They specify cell fate in a concentration-dependent manner so that cells acquire different fates based on the amount of morphogen to which they are exposed. The existence of morphogens had long been suggested, but only recently have genetic and molecular techniques allowed the visualization of morphogen gradients and the study of their formation and function. The most detailed picture of the role of morphogen gradients in development emerged from work carried out on Drosophilu embryogenesis. Genetic saturation screens have led to the identification of the vast majority of the genes requircd for patterning of the early embryo (Jurgens et al., 1984; Nusslein-Volhard et al., 1984; Wieschaus et al., 1984; Schupbach and Wieschaus, 1986, 1989). The comprehensiveness of this information allowed a detailed mechanistic analysis of the principles that govern the fonnation of the body axes during early embryogenesis. This analysis revealed the importance of morphogen gradients for both the large-scale organization and the pattern refinement along the two body axes. The following general principles seem to apply: ( I ) Pattern fonnation along the anteroposterior (AP) and dorsoventral (DV) body axes occurs largely independent from each other (reviewed in St. Johnston and Niisslein-Volhard, 1992). Mutations affecting one axis normally do not perturb the pattern along the other axis. Therefore, each axis can be studied separately. (2) Maternal and zygotic contributions have to be distinguished. Under the control of maternally supplied products, morphogen gradients are generated with long-range effects on

The dpp-group GeIJeS

29

polarity and pattern of the body axes. In the case of the anterior and DV systems, the gradients consist of transcription factors which regulate the expression of zygotic genes in a concentration-dependent manner (reviewed in St. Johnston and Nusslein-Volhard, 1992). This process converts the information contained in the maternal morphogen gradient into a more refined pattern of gene expression domains. ( 3 ) In some instances, further pattern refinement again employs a morphogen gradient (reviewed in Anderson et al., 1992). The purpose of this chapter is to investigate such a transition from a maternally generated morphogen gradient to a zygotic gradient which controls further pattern refinement. In Drosoplzila, the DV pattern depends on a single, maternally generated gradient, the nuclear concentration gradient of the dorsal ( d l ) gene product (reviewed in Anderson et al., 1992). This gradient has peak levels ventrally and i t directly organizes the pattern on the ventral side ofthe embryo. However, the patterning of the dorsal half of the embryo, though also dependent on the dl protein gradient, occurs more indirectly via the formation of the dpp morphogen gradient with the highest activity levels i n the dorsalmost regions (Ferguson and Anderson, 1992a; Wharton et al., 1993). In the following, we first describe the differences between patterning on the ventral and the dorsal sides of the Drosophila embryo, then the genes involved in the formation and interpretation of the dpp activity gradient.

If. THE DORSOVENTRAL PATTERN The dorsoventral (DV) pattern of the Drosophila embryo can be subdivided into four major regions, the anlagen of which can be traced back to the cellular blastoderm stage (schcniatized in Figure 1). The developmental fate of these regions can be distinguished by virtue of region-specific morphogenetic movements during gastrulation (Figure 1b), by the stereotypic pattern of mitotic divisions during cell cycle 14 (Figure la), and by the various differentiated structures they produce (Figure lc; Campos-Ortega and Hartenstein, 1985; Wieschaus and NussleinVolhard, 1986; Foe, 1989). The ventralmost part of the pattern gives rise to the mesoderm. It is derived from an 18 cell-wide strip of ventrally located cells of the blastoderm embryo (mitotic domain 10, 610) which invaginates in a morphogenetic movement called ventral furrow formation. The mesoderm gives rise to somatic and visceral musculature, fat body, and gonads.

A

A

/

.

-

dorsal ectoderm

- - .- - - - - - - -

-w

ventral ectoderm

\jP mesectoderm

6

C

dorsal folds

dorsal hairs

\ I

pole cells

Fk

-.cephalic furrow

ventral furrow

ventral denticle bands

Figure 7. Fate map of the D V axis. Embryos are oriented so that anterior is to the left and dorsal is up. (A) Lateral (left) and transverse (right) views of the cellular blastoderm embryo. The main subdivisions of the DV axis (left) and the mitotic domains (6)at the beginning of gastrulation (right) are indicated. The dorsalmost region gives rise to the amnioserosa (domain A). Dorsolateral cells give rise to dorsal ectoderm including dorsal epidermis, the peripheral nervous system, and trachea. The dorsolateral regions comprises mitotic domains 19 and 1 1 . Ventrolateral cells give rise to the ventral ectoderm including the central nervous system and ventral epidermis, and comprises domains N and M. The ventralmost region will become mesoderm (domain 10). Abutting the mesoderm, the rnesectoderm (domain 14) forms as a single-cell-wide stripe that becomes the ventral midline after ventral furrow formation. (B) An embryo undergoing gastrulation reveals region-specific morphogenetic movements. The cephalic furrow appears laterally. The ventral furrow forms as a longitudinal cleft along the ventral midline between 20% and 80% egg length. At the posterior end a plate forms which carries the pole cells towards the dorsal side of the embryo when the germ band starts to extend. As the pole cells shift dorsally, the anterior and posterior dorsal folds begin to form. (C) Cuticle preparation of a first instar larva shows the characteristic array of ventral denticle bands (or belts) and the finer hairs of the dorsal epidermis. Also visible are the head skeleton and the Filzkiirper (Fk) which derives from a defined dorsolateral position within the dorsal ectoderni. 30

The dpp-group Genes

31

The ventrolateral region is called neuroectoderm or ventral neurogenic region or ventral ectodenn. It gives rise to both ventral epidermis and central nervous system (CNS), and is derived from an approximately 20 cell-wide strip of cells on either side of the mesoderm. The pattern of mitotic divisions reveals an early subdivision of this region. Abutting the mesoderm, the mesectoderni (domain 14) forms as a single-cell-wide strip that becomes the ventral midline after ventral furrow formation, and generates the specialized glial cells and neurons of the midline (Crews el al., 1988). Lateral to the mesectoderm mitotic domains M and N can be distinguished which contain CNS and ventral epidermis precursors. The embryonic cuticle produced from these regions contains characteristic heavily pigmented ventral denticles (Figure I c). The dorsolateral region (approximately 16 cells wide at cellular blastoderm) gives rise to dorsal epidermis, the peripheral nervous system, and the tracheal system. The cephalic fold which forms during early gastrulation is a morphogenetic marker for both the dorsolateral and ventrolateral regions (Figure 1 b). The dorsolateral region harbors two mitotic domains: domains 1 1 and 19. The latter is a single cell-wide strip which demarcates the region dorsally. The cuticle produced by the dorsal epidermis is characterized by a lawn of fine hairs (Figure lc). In the terminal regions of the embryo, distinct cuticular structures are derived from specific positions within the dorsolateral region. For example, the labrum (the median tooth) and the spiracle hairs (a structure protecting the tracheal openings posteriorly) are derived from a more dorsal position than the antenna1 sense organs and the Filzkoi-per (a tracheal specialization shown in Figure 1c). The dorsalmost region is a five cell-wide domain which gives rise to the amnioserosa, an extraembryonic tissue. As the germband begins to elongate along the dorsal side of the embryo, two dorsal folds (the anterior and posterior transverse folds) appear (Figure 1b). The cells in this region do not divide after cellularization (domain A). They undergo a change in shape from the blastoderin cuboidal to a flat squamous shape, and their nuclei become polyploid and enlarged. The differentiated amnioserosa cells become compressed and displaced laterally during germband elongation. During germband shortening they spread out and provide a covering over the dorsal side. Later, as the embryo undergoes dorsal closure, the amnioserosa is internalized and undergoes apoptosis (Abrams et al., 1993). The morphological subdivisions of the DV pattern are partially reflected in the requirement for specific zygotic gene activities (summarized

Table 7. Dorsoventral Genes’ ~ _ _ _ ~

Embryonic dorsoventral tissues affected

Gene w

h\

MS MS

snail (sria)”* twist (iwi)’?* rhomboid ( r h ~ )also ~ ’ called ~ veinlet (ve) star ( S p 6 spitz pi)^'^ pointed @rif)5,6 single-minded ( ~ i m ) ’ , ~

‘

decapentaplegic (dpp) tolloid (tld) screw ( s ~ w ) ~ ” ~ shrew (srw)I4 short gastrulation (sog)I8

MES, VE MES, VE MES, VE MES, VE MES, VE AS, DE, VE AS, DE AS, DE AS AS, VE

Expression

-

_ _ _ _ _ _ ~ ~ ~ _ ___ Tvpe ofprotein function

zinc finger3 transcription factor basic helix-loop-helix (bHLH) transcription factor4 transmembrane protein7 transmembrane protein’ TGFn-like protein’ ETS-like protein, transcription factor” bHLH transcription factor’ I BMP-214. TGFP-like secreted signaling r n o l e c ~ l e ’ ~ BMP-1, metalloprotea~e’~ TGFP-like secreted signaling molecule1’ not known potential secreted signaling rnole~ule’~

twisted gastrulation (tsg)” zerkniillt (zen)’* Mothers against dpp (Mad)’4 Medea (Med)24 thick veins (tl~l,)~*-~’ saxophone (sax)”

AS AS

z)lg

AS, DE AS, ? AS, DE, VE AS

mat. zyg mat, zyg mat, zyg mat. zyg

w!z

potential connective tissue growth factor (C-lFG)-likeprotein2’ homeodomain transcription factorz3 nor:el protein25

not known 3 M P type I receptor2G3o BMP type I 32

: Key: MS: mesoderm; MES: Inesectudmn; VE: ventral epidennis; DE: dorsal epidcnnis; A: amnioserosa; mat: rriaternal; ~ y g zygotic

’& w

I . Siiiipson (1983).’2.Nusslein-Vblhard et al. (1984). 3. Boulay c t al. (19S7). 4. Thisse ct al. (1988). 5 Mayer and Nus~lciI1-Vnlhard(,1988). 6 . K i l n and Crews (1993). 7. Bier el al. (1990). 8. Kolodkin et al. (1994). 9. Rutlcdgc ct a!. (1992). 10. Kl6rnbt (,1993). 11, Namhu al. (1991) 12. Irish :ind Gclbari (1987). 1 3 . Padgettctal. (1987). 14. Jurgcns e t a ) .(1984). IS. Shimell etal. (1991). 16. Aroraand Niisslein-Volhard (1992). 17. A w n et al (1994). 18. Zusman et al. (1988). 19. FranGois et al. (1994). 2,O. Zusman and Wieschaus (1985). 21. Mason et al. (1994) 22. Wakimoto et a1 (1984). 23. Rushlow et al. (1987a). 21. Raftery et al. (1995). 25. Scketsky et al. (1995). 26. Lindsley and Zimm (1992). 27. Schupbach and Wieschaus (1989).28. Terracol and Lengyel (1994). 29, Nellen etal. (1994). 30. Penton et al. (1994). 3!. Bmmmef et al. (1994). 32. Xie et al. (1994).

Notes: *Included are the mesoderm-forming genes, the spi-group genes, the dpp-genes. and genes that interact with dpp.

34

CHRISTINE RUSHLOW And SIEGFRIED ROTH

in Table 1 ;reviewed in Rushlow and Arora, 1990; Ferguson and Anderson, 1991). The establishment of the mesodermal anlagen requires the two genes, twist ( M i ) and snail (sna; Simpson, 1983). In the absence of twi orstza, the ventral furrow does not form and mesoderm differentiation does not occur. The dorsal and dorsolateral regions ofthe embryo require the activities of seven genes: decapentnplegic (dpp),tolloid (tld),screw (scw),shrew (srw),twisted gastrulation (tsg),short gastrulation (sog), and zerkizullt (zen; Jurgens et al., 1984; Nusslein-Volhard et al., 1984; Wieschaus et al., 1984; Wakimoto et al., 1984). Since the phenotypes caused by mutations in these genes are similar to those produced by dpp alleles of different strength (Wharton et al., 1993), and since, as will be discussed later, they either act to modulate dpp activity, or they function downstream of dpp (Ferguson and Anderson, 1992a), we call these genes collectively, the dpp group (Arora and Niisslein-Volhard, 1992). The genes described so far are either required in the mesoderm or in the dorsal half of the embryo. No zygotic mutations have been isolated which delete the entire ventrolateral region. However, the spitz-group of genes which includes rhomboid (rho; Bier et al., 1990) and singleminded (sim;Crews et al., 1988), for example, are involved in the specification of the mesectoderm and subregions of the ventral epidermis (Mayer and Nusslein-Volhard, 1988; Kim and Crews, 1993). Interestingly, not all cells along the DV axis are committed simultaneously to execute a specific differentiation program. While cells in the ventral half of the embryo are already committed at early gastrulation, the cells of the dorsal half retain a considerable regulative capacity for several hours after gastrulation (Technau and Campos-Ortega, 1987). This might be a reflection of the fact that the pattern i n both halves of the embryo are established in different ways. While the pattern in the ventral half of the embryo is directly dependent on the dl morphogen gradient, the dorsal half of the embryo is organized by a dpp activity gradient (Anderson et al., 1992).

111. THE dorsal MORPHOGEN GRADIENT While the AP axis of the Drosophila embryo is patterned by the independent action of three groups of maternal genes, the DV axis is organized by a single group of maternal genes which functions to establish a single morphogen gradient (reviewed by St. Johnston and NussleinVolhard, 1992; Anderson et al., 1992). These genes are the 11 dorsal-

The dpp-group Gene5

35

group genes and cacfzi.9(cacf).In the absence of the activity of any one of the dorsal-group genes, the resulting mutant embryos lack all DV polarity. They differentiate dorsal epidermis at all positions of the embryonic circumference and entirely lack lateral and ventral structures. This and the analysis of partial loss-of-function alleles demonstrates that the highest requirement for dorsal-group gene activity is at the ventral side and that lower amounts of activity correspond to ventrolateral and dorsolateral positions (Nusslein-Volhard, 1979; Anderson et al., 1985a, 1985b). The dorsal-group genes mediate a signal transduction process (reviewed by Anderson et al., 1992). Spatial information present at the ventral side of the inner egg shell, the vitelline membrane, initiates the formation of a proteolytic cascade which leads to the localized production of an extracellular ligand molecule (Stein et al., 1991). This ligand protein which is released is a proteolytic fragment of the spiitzle (sp) into the fluid-filled space surrounding the embryo, the perivitelline space (Stein and Nusslein-Volhard. 1992; Morisato and Anderson, 1994; Schneider et al., 1994). The proteolytic spz fragment binds to the transmembrane protein Toll (77) which is uniformly present in the plasma membrane surrounding the embryo and functions as a receptor molecule (Hashimoto et ai., 1988; Schneider et al., I99 1 ). Thereby, the TI receptor becomes activated only at the ventral side of the embryo. Activated TI receptor initiates an intracellular signaling cascade which stimulates the releaseofdlfromthe inhibitorcact(R0thet al., 1991; Geisleretal., 1992; Kidd, 1992) and results in the transport of the dl protein from the cytoplasm to the nucleus. Although the quantity of dl protein remains uniform throughout the embryo, a gradient in the subcellular localization is established such that dl protein is mostly nuclear in ventral regions, mostly cytoplasmic in dorsal regions, and partitioned between the nucleus and cytoplasm in lateral regions (Roth et al., 1989; Rushlow et al., 1989; Steward, 1989). A high magnification view of the gradient is shown in Figure 2. It is in the ventrolateral region where the gradient appears steepest. The shape of the nuclear concentration gradient of dl protein is deteimined, at lcast in part, by a diffusion gradient ofthe active spz fragment in the perivitelline space (Roth, 1993; Morisato and Anderson, 1994; Schneider et al., 1994). Nuclear dl protein acts as a morphogen to specify pattern and position along the DV axis (Roth et al., 1989; Jiang and Levine, 1993). Since all the morphogentic effects of the dl protein depend on its nuclear concentration we use in the following the tern1 “dl morphogen” instead of

D

/"

I tld

n sim

[Ill rho

twi sna

scw

Figure 2. The dorsal morphogen gradient regulates D V zygotic gene expression. A high magnification view of the dl gradient is shown on the left. It was derived from a region of one side of a cellular blastoderm embryo (top) that was labeled with anti-dl and rhodamine-conjugated secondary antibodies, Thus, staining appears white. Notice that dlstaining in the ventral region i s predominantly nuclear, while that in the dorsal region is mostly cytoplasmic. In the ventrolateral region staining i s partitioned between the cytoplasm and nuclei; close inspection of this region will reveal the steepest part of the nuclear gradient. The subdivisions of the DV axis are indicated with horizontal lines (MS, mesoderm; MES, mesectoderm; VE, ventral ectoderm; DE, dorsal ectoderm; AS, amnioserosa).The limits, but not the relative levels, of zygotic D V gene expression are summarized as dark rectangles to the right of the dl gradient (see text for references). Expression patterns that refine during gastrulation are summarized as lighter colored rectangles. dl protein differentially activates the expression of twit m a , rho, and possibly sog; dl represses the expression of dpp, tld, Zen, and possibly tsg. 36

The dpp-group Genes

37

“nuclear dl protein.” A correlation between pattern elements and dl morphogen concentrations can be established through the analysis of various maternal-effect mutations which change both the dl moi-phogen distribution and the DV pattern. Especially informative are mutations which cause a uniform distribution of the dl morphogen. They give rise to embryos which are radially symmetric, that is, they differentiate the same cell type(s) at all DV positions (Anderson et al., 1985b). Such apolar embryos can be generated with: ( I ) a unifoimly high dl moiphogen concentration which will differentiate only mesoderm, (2) an intermediate dl concentration differentiating only neuroectoderm, ( 3 ) low dl concentrations differentiating only certain dorsolateral structures, but no dorsal epidermis, or (4) undetectable amounts of dl differentiating dorsal epidermis and amnioserosa (Roth et al., 1989). Thus, the generation of at least these four differentiation states is not dependent on the interaction between distinct parts ofthe DV pattern. Rather, they are determined by specific thresholds of dl morphogen concentrations. Interestingly, these four DV differentiation states do not completely match the subdivisions of the DV pattern based on morphological criteria. The absence of the n’l morphogen elicits cell fates which are derived from two different regions of the wildtype embryo, the dorsolaterally-derived dorsal epidermis and the dorsally-derived amnioserosa (Konrad et al., 1988). In these embiyos, amnioserosa cells are found randomly dispersed among dorsal epidermal cells around the entire embryonic circumference. This observation demonstrates that the pattern elements of the dorsal side ofthe embryo are not entirely determined by distinct dorsal morphogen concentrations. However, the polarization of the dl region which brings the pattern elements into a specific array so that the amnioserosa occupies the dorsalmost position flanked by dorsal epidermis on both sides, is still dependent on the establishment of the d/ gradient on the ventral side. The four differentiation states identified by genetic and morphological means correspond to three dl protein thresholds. However the molecular analysis of dl target genes (discussed later) indicates that additional thresholds exist within the mesoderm and within the neuroectoderm.

IV. TARGETS OF THE dl GRADIENT The dl protein belongs to the rel/NFKB-family of transcription factors (Steward, 1987; reviewed in Rushlow and Warrior, 1992). It regulates the transcription of several DV zygotic genes, that is, genes required to

38

CHRISTINE KUSHLOW and SIEGFRIED ROTH

specify different parts of the DV pattern (reviewed in Ip and Levine, 1992). At a given DV position the transcriptional activity o f a specitic target gene depends on the quantity o f dl protein present in the nuclei at this position, and on the quantity and quality of df binding sites located in its regulatory sequences. Since the d I protein can act as both a transcriptional activator or repressor, its graded nuclear distribution elicits a variety of different target gene expression patterns (schematized in Figure 2). For example, dl protein activates the expression of mi,sna, and rho, and it represses the expression o f d p p , tld, and zen (reviewed in Ip and Levine, 1992). The dl binding sites in the promoter regions ofthesc target genes have been identified, and mutations that disrupt dl binding in vitro have been shown to affect the expression of corresponding promoterlacZ fusion genes in vivo (Ip et al., 199I , 1992a, 1992b; Jiang et al., 1991; Pan et al., 1991;Thisse et al., 1991 ; Rushlow and Warrior, 1992; Huang et al., 1993; Kirov et al., 1993). These studies have revealed why the pattern on the ventral side of the embryo is directly determined by the shape of the dorsal morphogen gradient while the pattern on the dorsal side emerges as a secondary consequence of the gradient. We first give a brief description of the patterning on the ventral side to highlight the differences between ventral and dorsal patterning. A. dl as a Transcriptional Activator

Several dl protein thresholds can be distinguished on the ventral side of the embryo which correspond to specific domains ofgenc expression. i and sna genes which are responsible for mesoderm differentiaThe m tion, are activated by high levels o f dl in a ventral region which IS approximately 18 cells wide (Jiang and Levine, 1993). Both encode putative transcription factors, mi IS a bHLH protein (Thisse et al., 19SS), and sna is a zinc finger protein (Boulay et a]., I987j, and thus, it is likely that twi and sna regulate the expression o f downstrcam target genes. I t appears that m a preferentially acts as a repressor o f gencs involved in the specification of veiitrolateral fates (mesectoderm, neuroectodermj while twi is responsible for the activation of genes required for mesodermal differentiation (Kosinan et al., 1991; Leptin, 1991). Within the mesodermal region, dl has peak levels only in the ventralmost 12-14 cells. Studies by Jiang and Levine (1993) demonstrated that low affinity dl-binding sites present in one part of the twi promoter are

The dpp-group Genes

39

only sufficient for twi expression in the ventralmost 12-14 cells where there are peak levels of dl protein. Expression in the entire mesodermal region requires a combination of low affinity dl-binding sites with bHLH protein-binding sites. The ubiquitously expressed (maternally derived) bHLH proteins, daughterless and T4, a member of the achaete-scute (AC-S) complex, have been shown to be involved in mesoderm specification (Gonzalez-Crespo and Levine, 1993). Furthermore, mi,being itself a bHLH protein, seems to be autoregulative and probably binds to its own promoter. The lateral borders ofsiza and twi expression differ markedly (Kosman et ai., 1991). twi expression diminishes at its borders much like the dl gradient though with a steeper slope. sna however, shows a high level of expression throughout the presumptive mesoderm and diminishes abruptly at the mesoderm-neuroectoderm boundary. A study of the sna promoter has shown that it contains both dl and m i binding sites (Ip et al., 1992a). It seems that dland twi are both activators ofsna transcription and that they function multiplicatively to ensure strong, uniform expression of sna throughout the ventral domain. The sharp sna border may be formed by multiplying the shallow dl gradient with the steeper m i gradient. rho is initially expressed in ventrolateral stripes which encompass the ventral half of the presumptive neuroectoderm (Bier et al., 1990). The ventral limit of the rho stripe abuts the siza border precisely (see Figure 2). The analysis of the rho promoter shows that it contains a small element which harbors both dl and sna binding sites (Ip et al., 1992b). sna binding leads to repression of rho transcription. Although dl is capable of activating rho in the ventral region, sna acts to keep rho transcription off. rho expression extends considerably further toward the lateral side than twi. Thus, it is activated by lower levels of dl protein than those required for twi expression. This response to low levels of dl protein requires not only high affinity dl binding sites, but in addition, as in the case of the mesoderm, bHLH protein binding sites. Thus, like the mesoderm, the specification of the mesectoderm and parts of the neuroectoderm occurs via a cooperation of ubiquitously present bHLH proteins and spatially restricted nuclear dl protein. It is conceivable that similar mechanisms will govern the expression of sog whose transcripts also accumulate in a ventrolateral stripe that extends even further to the dorsal side than that of rho (see Figure 2;

40

CHRISTINE RUSHLOW and SIEGFRIED ROTH

FranCois et al., 1994). In this case, promoter elements might exist which respond to even lower dl protein concentrations. In summary, the patterning of the ventral side of the embryo employs two principles: (1) dl protein acts, in part, in concert with uniformly distributed bHLH proteins as a concentration-dependent transcriptional activator, and (2) some of the dl target genes are themselves transcriptional regulators that enhance or repress the transcriptional activation by dl protein. Thus, the pattern on the ventral side of the embryo appears to be a direct consequence of a small number of gene regulatory interactions. B. dl as a Transcriptional Repressor

As mentioned above, dl also acts as a transcriptional repressor of dpp, tld, and Zen whose activities are required to specify dorsal and dorsolatera1 pattern elements (reviewed in Ip and Levine, 1992). In the wildtype embryo, these genes are initially expressed in a dorsal-on, ventral-off pattern (Figures 2 and 3; Doyle et al., 1986; St. Johnston and Gelbart, 1987; Shimell et al., 1991). In embryos from dl mutant females, their expression domains extend along the entire DV axis (Rushlow et al., 1987; Ray et al., 1991; Shimell eta]., 1991). The uniform expression of dpp, tld, and zen in these mutant embryos corresponds to the apolar, dorsalized phenotype described above. The repression of dpp, tld, and Zen occurs in the ventral and ventrolateral regions. Thus, very low dl protein concentrations can mediate repression. In fact, it has been shown that the strong dl binding sites in the zen promoter have at least a five-fold higher affinity for dl protein than those of the tud promoter (Jiang et al., 1991; Thisse et al., 1991). How can dl protein act as a transcriptional activator and as a transcriptional repressor in the same nucleus, for example, in a ventral position? Regulatory elements in the dpp, tld, and Zen genes that mediate ventral repression have been identified and are called ventral repression elements (VRE; Doyle et al., 1989; Huang et al., 1993; Jackson and Hoffmann, 1994; Kirov et a]., 1994). They were shown to contain dl binding sites, and mutations in these sites abolish ventral repression. However, the dl binding sites themselves are not sufficient to explain repression. dl binding sites alone, present in an artificial promoter, induce transcriptional activation (Jiang et a]., 1992). Thus, dl protein mediates activation through these binding sites when they are taken out of context

The dpp-group Genes

41

of the Zen promoter. Therefore, sequences other than dl binding sites in the VRE must be important for repression. Kirov et al. (1 993) and Jiang et al. (1993) showed that pyrimidine-rich sequences that lie adjacent to the dl binding sites in the zen VRE are required for repression. When these sequences are mutated, but the dl binding sites left intact, dldependent repression is abolished. Moreover, the mutated VRE now confers dl-dependent activation. Thus, by mutating the pyrimidine (T)rich sequences, the silencer element is converted to an enhancer element, and dl mediates activation rather than repression. It was proposed that another molecule, a co-repressor, binds next to dl and together they mediate repression. Recently, Lehming et al. (1994) described a candidate for the corepressor. Using a yeast system to isolate Drosophilu cDNAs encoding inhibitors of dl protein activity, they found an HMG (high mobility group) protein that converts dl, as well as its vertebrate counterpart, NF-KB,from an activator to a repressor. It is not yet known ifthis protein, called DSPl (Drosophila switch protein), binds to the VRE and/or interacts with the dl protein to confer repression. HMG proteins were first isolated as components of chromatin and it is possible that formation of a protein-DNA complex could interfere with the assembly of a stable initiation complex at the transcription start site. In contrast to the genes activated by dl, the genes so far known to be repressed by dl display similar expression domains (Doyle et al., 1986; St. Johnston and Gelbart, 1987; Shimell et al., 1991). dpp, tld, and zen are confined to a domain comprising about 40% of the embryonic circumference, and although there is some gradation of their expression patterns at the dorsolateral border, presumably due to the dl protein gradient, the borders are approximately the same (Figure 3). Maternaleffect mutations which lead to partial dorsalizations of the DV pattern affect the expression domains of all three genes in a similar manner (Figure 3). It seems, therefore, that the patterning of the dorsal side of the embryo is not initiated by a mechanism of differential repression analogous to the differential activation important for ventral patterning. However, as mentioned above, the dl gradient still has some organizing influence on the patterning of the dorsal side. The remainder of this review considers how members of the dpp group-genes establish a zygotic DV morphogen gradient, presumably of dpp activity, that organizes the pattern in the dorsal half of the embryo.

42

CHRISTINE RUSHLOW and SIEGFRIED ROTH

tld

dPP

zen

D1

Figure 3. dl represses tid, dpp, and Zen. wildtype (wt; top) and D1 (bottom) stage 5 embryos were hybridized with RNA probes synthesized from cDNA templates of tld (left), dpp (center), or Zen (right), embedded, and sectioned. In wildtype dlseems to repress tld, dpp, and zen in a similar manner. To enhance potential differences between the expression domains females were used of tld, dpp, and Zen, embryos derived from Tr632/T15BRE for in situ hybridizations. These embryos show a strong expansion of dorsal fates (D1 phenotype according to Anderson et al., 1985), and have therefore enlarged expressiondomains of tld, dpp, and Zen. The tlddomain seems to be slightly larger than the dppdomain, and the dppdomain seems to be slightly larger than that of zen. But even this strong dorsalization does not reveal a qualitative difference in the expression of these three genes, such that, for example, one of them would show a complete derepression (uniform expression).

V. THE dppGROUP GENES

As mentioned above seven zygotic genes, the dpp group, are required for the specification of pattern in the dorsal region of the embryo (Arora and Nusslein-Volhard, 1992; Ray, 1993). Loss of hnction mutants of six genes-dpp, tld, sew, srw, tsg, and zen-cause a ventralized phenotype in which a deletion of dorsal parts of the pattern is compensated by an expansion of lateral and/or ventrolateral regions (summarized in Figure 4). Mutations in the seventh gene, sog, also cause a partial loss of dorsal fates, but in addition, the extent of the ventral ectoderm is reduced. All

The dpp-group Genes

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WI

VB

M

Figure 4. Fate map changes in the dpp-group mutants. Adapted from Ray, 1993. Circles represent cross sections through the middle of cellular blastoderm embryos. Each embryo is labeled wildtype (wt) or mutant for each of the dpp-group genes. Regions along the DV axis are differentially shaded and represent mitotic domains (top, left; Foe, 1989). The fate map shifts were determined from the analysis of mitotic domains and regionspecific markers (Arora et al., 1992, Ray, 1993; Mason et al., 1994). The dorsalmost region, domain A, i s affected in all dpp-group mutants. Successive dorsal pattern elements are deleted (domains 19, 11) and ventral elements expanded as the mutant phenotype becomes more ventralized; dpp null embryos (bottom, right) lack all dorsal structures.

the members of the dpp group are early-acting zygotic genes so that their effect is already visible shortly after cellularization when the first morphogenetic movements occur. Mutants of dpp-group genes lead to abnormal gastrulation in which the laterally derived cephalic fold is displaced to a dorsal position and germband extension does not occur normally. The following sections are centered around the dpp gene and how other genes affect or respond to its activity. Many of the genes have been characterized at the molecular level, and in some cases vertebrate counterparts have been identified (summarized in Table 1). A. dpp is a Signaling Molecule

The decapentaplegic (dpp) gene is necessary for several different morphogenetic processes during Drosophilu development (Spencer et

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al., 1982). Besides its role in DV patterning (Irish and Gelbart, 1987), it is also involved in midgut morphogenesis (Panganiban et al., 1990), eye development (Heberlein et al., 1993; Kaphingst and Kunes, 1994), and in the establishment of the proximodistal axis of adult appendages (Campbell et al., 1993; Basler and Struhl, 1994). In fact, the name dpp derives from its requirement for the normal development of all 15 major imaginal discs. For the purpose of this chapter we focus only on the role of dpp in DV patterning of the early embryo. The dpp gene product is homologous to members of the transforming growth factor-p (TGFP) superfamily of secreted growth and differentiation factors (Padgett et al., 1987; reviewed in Lyons et al., 1992). Thus, dpp does not instruct cell fate based on direct transcriptional regulation of downstream genes as is the case for the dl morphogen. Rather, dpp acts at the level of intercellular communication. In accordance with these molecular findings, genetic mosaic experiments have shown that dpp can act nonautonomously in imaginal discs (Posakony et al., 1990). Furthermore, characterization of dpp activity and protein localization in the embryonic gut has shown that the dpp protein can undergo limited diffusion (Panganiban et al., 1990). Thus, dpp has properties of an extracellular signaling molecule. More recently, cell surface receptors have been identified that appear to mediate the dpp signal (Table 1 and see below; Childs et al., 1993; Brummel et al., 1994; Nellen et al., 1994; Penton et al., 1994; Xie et al., 1994). The members of the TGFP-superfamily are synthesized as large precursor molecules which are cleaved to release a mature carboxyterminal segment of 110-140 amino acids (reviewed in Massague et al., 1994). The active forms are disulfide-linked homodimers or heterodimers of this carboxy-terminal segment. All members show sequence similarity to the prototype, TGF P l , particularly in the active domain where the most conserved feature is the spacing of seven cysteine residues. The superfamily, which currently includes at least 24 members, has several subgroups. The main groups are the TGFPs, the decapentaplegic-Vg-related (DVR) proteins (including the bone morphogenetic proteins, or BMPs), and the activins (reviewed in Kingsley, 1994a). dpp is most related to the vertebrate bone morphogenetic proteins BMP-2 and BMP-4 (about 75% identical in the mature signaling portion of the molecule). In fact BMP-4 is capable of rescuing a dpp mutant embryo (Padgett et al., 1993) and conversely, dpp protein when implanted in rats engenders the same response of ectopic bone formation as shown by all other BMPs (Sampath et al., 1993). This demonstrates

The dpp-group Genes

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the great degree of conservation between the vertebrate and fly molecules.

B. dpp is a Component of a Graded Patterning Process The characteristic feature of the dpp-group genes is that mutations in any of these genes cause a loss of dorsal pattern elements (reviewed in Ferguson and Anderson, 1991 ). For most of the dpp-group genes multiple alleles exist which can be ordered in an allelic series. The phenotypic effects caused by these alleles have been analyzed on the basis of changes in the cuticle pattern, the patterns of mitotic domains or the expression of molecular markers (Arora and Niisslein-Volhard, 1992; Ray, 1993; Arora et al., 1994; Franqois et al., 1994; Mason et al., 1994). The most complete phenotypic analysis was derived from the large number of embryonic lethal dpp alleles (Wharton et al., 1993). Partial loss-of-function alleles of dpp were ordered in a series in which the weak alleles affect only the dorsalmost structure, the amnioserosa. These weak alleles could be tightly ordered among themselves to show progressive deletion of the amnioserosa by counting the number of amnioserosa cells. Stronger alleles delete, in addition to the amnioserosa, dorsolateral structures. The progressive loss of these structures is compensated by an expansion and a dorsal shift in position of more ventral pattern elements such as the ventral denticle bands (see Figure 5 ) . Finally, the complete lack of dpp causes not only a loss of all dorsal and dorsolateral structures, but also the loss of dorsal parts of the ventral epidermis. The remaining ventral epidermis replaces the deleted structures so that the mutant embryos consist only of ventral epidermis and mesoderm (Figures 4 and 5).

The correlation between allelic strength and the progressive loss of dorsal pattern elements reveals a graded requirement for dpp activity such that high levels of dpp activity specify the amnioserosa while progressively lower levels specify dorsal epidermis and some parts of the ventral epidermis. Furthermore, the phenotypic series comprises a continuum of fate shifts suggesting that the requirement for dpp activity is continuous, and that dpp is part of a system which generates a gradient of positional information. Allelic series exhibiting a continuum of ventralized phenotypes have also been described for other dpp-group genes, such as tld and scw (Ferguson and Anderson, 1992a; Ray, 1993; Arora et al., 1994). The phenotypes are similar if not identical to those caused by dpp alleles of

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CHRISTINE RUSHLOW and SIEGFRIED ROTH

The dpp-group Genes

47

corresponding strength, indicating that these genes are part of a system that generates positional information in a graded manner. However, even the strongest alleles of any of the other dpp-group genes do not cause phenotypes as severe as dpp null alleles (Figures 4 and 5). tld and sew are required for the production of the amnioserosa and the dorsalmost parts of the dorsal epidermis. Zen, tsg, and SYW, and to some degree sog, are only required for the differentiation of the amnioserosa (Figures 4 and 5). Double mutants between dpp null alleles and alleles of any of the other dpp-group genes do not lead to phenotypes stronger than the dpp null phenotype (Arora and Niisslein-Volhard, 1992). Thus, none of the other dpp-group genes seem to contribute to the DV pattern in a dppindependent way. Another feature which distinguishes dpp from the other dpp-group genes is its dosage-sensitivity (Spencer et al., 1982). dpp is haplo-lethal. Embryos heterozygous for a deficiency of the dpp region are weakly ventralized (Irish and Gelbart, 1987). This suggests that the level of dpp gene product is critical for proper development. Furthermore, increasing the wildtype gene dosage of dpp can shift cell fates along the DV axis, so that more amnioserosa is present with increased dpp copy number (Wharton et al., 1993). A normal diploid embryo has an average of 130 amnioserosa cells; increasing the dpp copy number to four, increases the number to 325.1100. A transformation of epidermal cells into amnioserosa cells can also be observed if the dpp copy number is changed in the background of apolar dorsalized embryos (dl mutant embryos). As

Figure 5. The larval cuticle produced by mutations of dpp-group genes. Dark-field micrographs of wildtype (wt) and mutant embryos. Embryos are oriented so that anterior is up and ventral is front. A. The cuticular phenotype of a wt embryo demonstrating the normal array of ventral denticle bands and the Filzkorper (Fk). B. t/dB4 mutant embryo showing a partially ventralized embryo. The ventral denticle bands are expanded at the expense of the dorsal epidermis and amnioserosa. C. zenw36mutant embryo showing a weakly ventralized embryo. The Filzkorper are abnormal and internalized, the head is not involuted, and the anterior portion of the gut is externalized. D. dppH1n46 mutant embryo showing a strongly ventralized phenotype. Denticle bands encircle the entire DV circumference at the causes a expense of dorsal epidermis and amnioserosa. E. dpph1n-r27/dpph1n-r92 less severe phenotype resemblingthat of strong tldalleles (compare E and B). F n-r4/dpph In-r2 7 causes a weak ventralization, similar to that of zen mutations (compare F and C).

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mentioned earlier these embryos differentiate both dorsal epidermal cells and amnioserosa cells if they have a wildtype dosage of dpp. They differentiate exclusively dorsal epidermis if they have only one copy of dpp, and exclusively amnioserosa (in the segmented body region) if they have four copies. This demonstrates that at least the distinction between amnioserosa and dorsal epidermis depends critically on the level of dpp activity. The experiments mentioned so far reveal that dpp is a crucial component of a system which generates an activity gradient with peak levels in the dorsalmost region. C. dpp is a Morphogen

Ferguson and Anderson ( I 992b) showed that the injection ofdpp RNA into blastoderm embryos can rescue the dpp mutant phenotype. They also demonstrated that dpp RNA injections into wildtype embryos can cause a dorsalization of the pattern. A dorsalization can even be achieved if apolar embryos are used as recipients which normally differentiate only ventral epidermis. These apolar embryos do not express some of the dpp-group genes (see below) indicating that dpp can become active even in the absence of other dpp-group genes. In these experiments, the degree of dorsalization depends on the amount of injected RNA. A steep dose response relationship was observed: two to fourfold increases in the concentration of injected RNA elicit progressively more dorsal cell fates. High levels of dpp drive amnioserosa development, intermediate dpp levels drive dorsal epidermal development, and low levels of dpp permit development of ventral ectoderm. Moreover, local dpp activity can also direct a surprising degree of patterning within the entire ectoderm. Local injections of dpp RNA into apolar embryos polarize the gastrulation movements and produce the array of DV structures typical for the wildtype pattern, including dorsal hairs, lateral denticles, and trapezoidal denticle bands. Thus, in these experiments dpp can define both embryonic polarity and organize detailed pattern. The fact that dpp can function at least partially independently from other dpp-group genes, and that local RNA injections induce DV patterning in a dose-dependent manner strongly suggests that dpp itself acts as a morphogen. More support for this idea comes from further genetic experiments. The similarity of the ventralized phenotypes caused by mutations in dpp and other members of the dpp-group seems to preclude the unambiguous hierarchic ordering of these genes. However, a relationship among the

The dpp-group Genes

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dpp-group genes could be established on the basis of gene dosage studies (Ferguson and Anderson, 1992a). For example, doubling the dpp gene dosage completely suppresses the phenotypes of weak tld mutants and partially suppresses tld null mutants. This result is significant because it demonstrates that tld function is not absolutely required for dpp activity. In contrast doubling the tld gene dosage does not change the dpp null phenotype. Because an increase in the dosage of dpp can partially bypass the requirement for tld, but an increase of tld gene dosage does not suppress the dpp mutant phenotype, it appears that tld acts to elevate the activity of dpp. Similar results were found for srw and scw, indicating that they are also upstream of dpp and function to increase dpp activity (Ferguson and Anderson, 1992a; Arora et al., 1994). In contrast, thezen and tsg phenotypes cannot be suppressed by additional dpp copies which places them downstream or in parallel to dpp (Ferguson and Anderson, 1992a). Interestingly, increasing the dpp gene dosage in a sog mutant background leads to a new phenotype (Ferguson and Anderson, 1992a). As mentioned above, among the dpp-group genes, sog is unusual since sog mutants show a reduction of dorsalmost fates and ventrolateral fates at the same time (see discussion below). If the dpp gene dosage is increased in a sog mutant background, the ventrolateral region (the neuroectoderm) is even further reduced while the region producing dorsal epidermis is expanded. This indicates that sog might normally inhibit dpp activity in ventral regions. In summary, these genetic and injection experiments strongly suggest that an activated form of dpp forms a morphogen gradient in the dorsal half of the embryo. This gradient is responsible not only for determining cell fate and spatial pattern in dorsal regions, but it also has an organizing influence on the neuroectoderm (Ferguson and Anderson, 1992a). The other dpp-group genes are either upstream of dpp and modulate its activity in a positive (tld, SCW, and s w ) or negative (sog) way, or they are potential targets of dpp function (zen, tsg). Since all but one of the dpp-group genes have been cloned, and more recently, dpp receptors have been identified, a molecular picture emerges of how the dpp activity gradient is established. D. tolloid Encodes a Potential Protease

The genetic data which suggest that tld acts to enhance dpp activity find support from the molecular analysis of the tld gene (Shimell et al.,

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CHRISTINE RUSHLOW and SIEGFRIED ROTH

1991). tld transcripts accumulate like dpp transcripts in the dorsal 4050% of the blastoderm embryo (Figure 3). Thus, tld and dpp products

colocalize in the dorsal half of the embryo. tld encodes a protein of an approximate molecular weight of I16 kD which possesses an N-terminal signal sequence. It is therefore, as dpp, most likely a secreted protein. The tld protein has three sequence motifs: an N-terminal region with similarity to the astacin family of metalloproteases, two EGF-like repeats, and five copies of the CUB repeat which was first found in the human complement proteins C 1r and C 1s. However, most significantly, the overall structure of the tld protein is similar to the human bone morphogenetic protein 1 (BMP-I) (Wozney et al., 1988). tldand BMP-1 are 4 1% identical. The BMPs were originally isolated as components of a protein complex that can induce ectopic bone morphogenesis in rats (Wang et al., 1988; Wozney et a]., 1988). Besides BMP-1, all other so far identified BMPs are TGFP family members (Wozney et al., 1988; Celeste et al., 1990), and as mentioned above two of these, BMP-2 and BMP-4, are 75% identical in their active carboxy terminus to the dpp protein. Since the mammalian homologues of tld and dpp (BMP-1 and BMP-2/4) are present in a complex, it is likely that tld and dpp proteins also physically associate. The biochemical function of the tld/BMP- 1 protease is currently unknown. It is not clear whether BMP- 1 is the elusive maturation enzyme responsible for cleaving the pro-forms of TGFP-type molecules. Most TGFP-type molecules are cleaved at a site which suggests that a subtilisin-like activity is responsible for the processing event (Ozkaynak et a]., 1992; Barr, 1991). It could be that additional processing sites exist which modify the activity of TGFP-type molecules. Alternatively, the tldiBMP- 1 protease could function in a later step such as the liberation of the mature peptide from the latent complex. Due to the difficulty to obtain biochemical data on BMP-I in mammalian systems, the genetic and molecular analysis of the tld locus b m m e s especially important. The tld alleles show a complex complementation behavior (Ferguson and Anderson, 1992b). Some of the alleles are antimorphic. Embryos heterozygous for weak dpp alleles and antimorphic tld alleles die with a weakly ventralized phenotype. In contrast, embryos carrying a deficiency of the tld locus and the same dpp allele are hlly viable. If tld acts as a protease, then the antimorphic alleles could encode products that are still able to form a complex with dpp, although they are proteolytically inactive and consequently render the complex inactive. Childs and O’Connor (1993) have sequenced the antimorphic

The dpp-group Genes

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tld alleles and find, in fact, that the molecular lesions cluster in the protease domain. While the early expression pattern of tld RNA closely resembles that of dpp RNA, the post cellularization pattern of tld transcripts diverges from that of dpp (Shimell et al., 1991). Maybe, tld has additional dpp-independent functions and conversely, some of the later dpp f h c tions may not require tld. This is especially clear for dpp’s function in imaginal disc development during late larval and pupal stages, since the temperature sensitive period for tld function does not extend past the cellular blastoderm stage (Ferguson and Anderson, 1992a). Interestingly, two other genes have been discovered in Drosophila which are homologous to tld. They have been named tolloid-related-] (tlr-I) and tolloidrelated-2 (tlr-2; Nguyen et al., 1994). tlr-1 is located immediately proximal to the tld gene and is 62% identical to tld. It is required during larval and pupal stages of development and thus may augment some of the later dpp functions.

E. screw Encodes Another Member of the TGFV Superfamily

Like tld, scw enhances dpp function, but is not absolutely required for dpp activity (Arora et al., 1994). This is evident from the fact that the scw mutant phenotype can be rescued by injection of dpp RNA. Thus, scw does not act downstream of dpp, and the dpp signal transduction pathway is functional in scw mutant embryos. scw encodes a novel BMP-like member of the TGFP superfamily (Arora et al., 1994). It shares 40% identity with dpp in the carboxy-terminal active region. The greatest sequence conservation between scw and a vertebrate family member is seen with BMP-6 (49%). This is considerably less than the conservation between dpp and BMP-2 and BMP-4, suggesting that scw is not an ortholog of a known vertebrate member of the TGFP superfamily. Unlike tld which is expressed only on the dorsal side of blastoderm embryos, the scw gene is ubiquitously expressed during early stages of embryogenesis (Arora et al., 1994). Moderate levels of scw transcripts are first detected in a stage 4 embryo toward the end of nuclear cycle 10. The level of scw RNAs rapidly increases during cycles 11-1 2 until the cellular blastoderm stage (stage 5 ) when they decline very rapidly to below detection. Although ubiquitous, Arora et al. demonstrated that scw expression on the dorsal side is suficient for normal development by testing a tld-promoter-scw fusion gene in a scw mutant background. The

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lack of scw transcripts in ventral regions had no developmental consequences. This suggests that scw activity is restricted to dorsal regions. It is possible that the activity of scw is restricted by translation of scw message only in dorsal cells or by postranslational modification of scw protein in a limited region of the embryo. For example, the dorsally localized tld protein could interact with either scw or dpp, or with both proteins. scw and tld have identical loss-of-function phenotypes. Thus, scw activation, though not dpp activation, could be entirely meditated by tld. Given the structural similarity of scw and dpp, it is possible that scw functions as a signaling molecule by forming heterodimers with dpp (Arora et al., 1994). Protein-protein interactions between dpp and scw are consistent with the observation of genetic interactions. Specific gain-of-function scw alleles have been identified that fail to complement a recessive, partial loss-of-function dpp allele (Raftery et al., 1995). Embryos carrying a single copy of both mutations die with a partially ventralized phenotype. In contrast, embryos carrying a deficiency for the scw locus and the same allele of dpp are completely viable, indicating that the defective product encoded by the gain-of-function scw allele can block the activity of the remaining functional copy of dpp. Thus, these alleles behave like the tld alleles described above as antimorphs. The antimorphic scw proteins may be incapable of signal transduction, but still sequester active dpp molecules and thus reduce the effective levels of dpp in the embryo. On the basis of the putative physical association of dpp and scw proteins, Arora et al. (1994) propose a model which explains why scw is only required to specify amnioserosa and parts of the dorsal epidermis while dpp has a broader requirement. They suggest that the activity gradient that specifies dorsal pattern may be composed of both scwldpp heterodimers and dpp homodimers. scw and dpp could act combinatorily such that scwldpp heterodimers elicit a stronger response than dpp homodimers. scw homodimers would be inactive since they should be formed in a dpp null background where scw appears not to be active. The assumption that,dpp homodimers elicit a response explains why the scw mutant phenotype is less severe than that of dpp. The stronger signaling response elicited by heterodimers may result from a higher affinity of the heterodimer for a common set of receptors. Besides scw, one other TGFP-like molecule has been discovered in Drosophila and named by its chromosomal localization, 60A (Wharton et al., 1991). dpp and 60A are 55% identical to each other. While dpp is

The dpp-group Genes

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more closely related to BMP-2 and -4 (75% identity), 60A seems to be related to BMP-5, -6, and -7 (70% identity). Like dpp and scw, 60A is expressed during embryogenesis, however, its pattern of expression is broader than that of dpp (Wharton et al., 1991). The function of 60A is not known since mutations in the locus have not yet been reported. A first hint for a role in development comes from ectopic expression studies. Although both dpp and 60A can induce ectopic bone formation in rats, they elicit different responses if they are ectopically expressed during Drosophila development (Staehling-Hampton et al., 1994). While ectopic expression of dpp induces the formation of dorsal epidermis in ventrolateral positions, ectopic 60A expression has no detectable effects on embryogenesis. Thus, 60A seems not to be involved in DV patterning, nor can its ectopic expression interfere with the function of endogenous dPP. F. Additional Genes Required to Enhance dpp Activity One of the dpp-group genes, shrew (sw) has not yet been cloned. In contrast to tld and SCW, it is required only for the highest levels of dpp activity, since null mutations of s w affect only the amnioserosa (Figure 4;Ferguson and Anderson, 1992a). An increase in the dose of dpp can bypass the requirement for the sw gene. Thus, like tld and scw, snv acts upstream of dpp to increase dpp activity. Genetic screens have been performed to isolate other posi tive-acting components of the dpp signaling pathway (Ferguson and Anderson, 1992a). The assumption was made that such components might be rate-limiting, and should, therefore, enhance the dpp mutant phenotype. Interestingly, in a screen for further zygotic mutations, only alleles of the already known dpp-group genes, tld and scw, were isolated. This reinforces the notion that tld and scw are pivotal elements of the dpp activity gradient. Screens were also performed looking for maternal enhancers of the dpp phenotype, which led to the discovery of two new loci, Mothers against dpp (Mud) and Medea (Med; Raftery et al., 1995). Mutations in these genes cause pupal lethality and exhibit phenotypes which resemble those caused by mutations affecting late dpp functions (gut defects, imaginal disc defects). This indicates that Mud and Medencode rate-limiting components integral to dpp pathways throughout development. Although in the maternal screens multiple alleles of Mud and Med have been isolated, saturation was not achieved since two other maternal genes

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have been found which enhance the dpp phenotype. These are thick veins ( t h ) and saxophone (sax), both encoding potential dpp receptors (see below). It has not been determined so far whether Mad and Med act upstream or downstream of dpp. MAD has been cloned and characterized and does not contain informative sequence motifs (Sekelsky et al., 1995). However, given that the MAD protein lacks a secretion signal sequence or transmembrane domain, it seems likely that MAD acts within the cells that produce dpp or within the cells that receive the dpp signal. G. Short Gastrulation Inhibits dpp Activity

Among the dpp-group genes, sog is unusual since mutations cause amnioserosa defects and, at the same time, reduce the extent of the ventral epidermis (Figure 4; Ferguson and Anderson, 1992a). Mutations in other dpp-group genes cause either an expansion of the ventral epidermis or do not affect the ventral epidermis. Surprisingly, the dorsally-located defects caused by sog mutations are due to a requirement of sog activity in ventral regions of the embryo as defined by genetic mosaic studies (Zusman et al., 1988). A better understanding of the sog phenotype resulted from a manipulation of the dpp gene dosage in a sog mutant background. Increasing the dpp gene dosage in embryos with normal sog function affects the amount of amnioserosa, but leaves ventral fates largely unchanged (Wharton et al., 1993). However, sog mutant embryos with three copies of dpp already have a dramatically reduced ventral epidermis (Ferguson and Anderson, 1992a), and with four copies of dpp, they virtually lack all ventral epidermis (shown in Figure 6d). In these embryos the dorsal epidermis is expanded at the expense of the ventral epidermis. This suggests that sog acts to inhibit dpp activity in the ventrolateral region where the genetic mosaic experiments previously located its requirement. An inhibitory action of sog on dpp also explains why sog mutations, unlike any other dpp-group mutation, can rescue the dpp hap1o:insufficiency to a significant extent (Frangois et al., 1994). Although the defects on the dorsal side of sog mutant embryos result ultimately in a loss of dorsalmost cell fates (amnioserosa), initially, the dorsalmost region seems to be expanded (Rushlow and Levine, 1990; Ray et al., 1991; Frangois et al., 1994). This can be demonstrated by changes in the dorsal rho expression domain. Besides the ventrolateral expression domains of rho described above, rho is also expressed in a

The dpp-group Genes

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dorsal domain which is L T l O cells wide in wildtype embryos. In sog mutant embryos, the dorsal rho domain is 20 cells wide indicating an expansion of dorsalmost fates (Bier et al., 1990). However, normal pattern refinement of dpp and zen (see below) does not occur in this expanded region (Rushlow and Levine, 1990; Ray et al., 1991). Thus, sog seems to be required for the normal subdivision of the dorsal region which is a prerequisite for the establishment of the amnioserosa. Interestingly, while the ventral defects of sog are enhanced by extra copies of dpp, the dorsal defects are suppressed (Ferguson and Anderson, 1992a). Thus, in contrast to sog’s inhibitory action on dpp at the ventral side, sog seems to enhance dpp activity at the dorsal side. These observations suggest that sog is responsible for both the dorsal maximum and the lateral extent of the dpp activity gradient. Thus, sog may redistribute dpp activity to keep it high in dorsal regions and low in ventrolateral regions (Ferguson and Anderson, 1991). Recently the sog gene has been cloned (Franqois et al., 1994). As predicted from genetic mosaic data, sog transcripts accumulate in a broad ventrolateral stripe, 14-1 6 cells wide, during cycle 14. The sog stripe is broader than the ventrolateral rho stripe; it extends one to two cells beyond rho ventrally, and 4-6 cells beyond rho dorsally (see Figure 2). The dorsalmost cells expressing sog abut the ventralmost cells expressing dpp, although the boundary between the sog and dpp expression domains is not absolute, as a few cells express low levels of both transcripts. Expression of sog is progressively lost dorsally during the late blastoderm stage and during early gastrulation. By germband extension sog transcripts are confined to the mesectoderm. sog encodes a protein with a single long hydrophobic domain beginning 56 amino acids from the amino terminus (Franqois et al., 1994). This domain could either serve as a transmembrane domain or as an internal signal sequence. Thus, it is not clear if sog remains membranebound or whether it is secreted. The putative extracellular portion of the sog protein contains four repeats of a novel motif defined by the spacing of 10 cysteine residues (CR 1-CR4). These repeats are distantly related to domains present in thrombospondin, procollagen, van Willebrand factor, laminins and genes of the CEF-1O/CTGF/PlG-M2 family. Some of these cysteine-rich repeats present in sog are separated by dibasic pairs of amino acids which could serve as cleavage sites for tyrosine-like serine proteases. Thus, they might be released as diffusible peptides following proteolytic processing. This would explain the non-autonomous genetic behavior of sog mutations. The peptides could diffuse

CHRISTINE RUSHLOW and SIEGFRIED ROTH

56

4 x dpp'

A

-

sog 4 x dpp'

The dpp-group Genes

57

toward the dorsal side of the embryo and possibly interact with dpp directly. Support for this model comes from studies on procollagen; a region of the procollagen domain in type IV collagen and thrombospondin has been shown to bind TGFP (Paralkaret al., 1991; Murphy-Ullrich et al., 1992). Moreover, the procollagen domain is biologically active as a soluble factor. To explain the sog mutant phenotype one may assume that sog binds dpp and thereby limits its diffusion toward the ventral side. Preventing dpp diffusion may also be necessary to maintain the highest dpp levels dorsally. Alternatively, .Tog may interact with other dpp-group genes, with the dpp receptors or activate its own receptors. Interestingly, although sog does not encode a transcription factor, sog exerts an influence on dpp transcription during the late cellular blastoderm stage (Figure 6; Roth, unpublished results). The dpp expression domain is slightly expanded to comprise 50% of the egg circumference in sog mutant embryos rather than 40% as in wildtype (Roth, data not shown). If in asog mutant background, the dpp gene dosage is increased, an additional expansion of the dpp expression domain occurs (Figures 6b and 6c) which is not observed if the dpp dosage is increased in a wildtype background (Figure 6a). sog mutant embryos with four copies of dpp show high levels of dpp transcripts along the entire embryonic circumference except in the presumptive mesodermal region. This corresponds to the complete loss of the ventral epidermis (Figure 6d). Taking into account the molecular data, sog’s influence on dpp transcription must be indirect. If sog has its own receptor, a consequence of sog signaling could be the transcriptional repression of dpp. However, it is also possible that dpp activates its own transcription and that sog figure 6. sog inhibits dpp in ventral regions. Embryos with four copies of the dpp gene that were otherwise wt (A) or mutant for sog (B-D) were hybridized with a dpp antisense R N A probe (A-C) or aged for cuticle preparations (D).Lateral views (A,B,D) or ventral views (C) are shown. The dpp expression pattern is slightly expanded (to 50%) in an embryo with four copies of dpp (A), compared to an embryo with the normal two copies (40%; see Figure 3). However, in the absence of sog, this pattern expands greatly to encompass 80% of the circumference, excluding only the presumptive mesodermal (B,C). Thus, the ventrolateral region of the embryo now expresses dpp. This results in a fate change of ventral ectoderm to dorsal ectoderm, and the embryo is entirely covered with dorsal epidermis (D). Thus sog acts to inhibit dpp transcription in ventrolateral regions.

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inhibits this auto-regulation of dpp, for example by restricting the diffusion of activated dpp protein. Recently, a Xenopus gene, chordin, was described which seems to be an ortholog of sog (Sasai et al., 1994). sog and chordin have the same overall protein organization. The four cysteine repeats are present in the same relative positions in the two proteins. In addition, the cysteine repeats are the regions with highest similarity between the proteins (e.g., 47% identity for the first repeat; FranGois and Bier, 1995). Interestingly, chordin seems to play a similar role in the DV patterning of Xenopus as sog does in Drosophila. This observation yields a new perspective for a comparison of DV patterning in insects and vertebrates (see below). H. fwisfedgasfrubfion and zen Act Downstream or in Parallel to dpp

Extra copies of the dpp gene are unable to suppress tsg or zen mutations, suggesting that they act downstream or in parallel to dpp, rather than upstream like tld, scw, and S M (Ferguson and Anderson, 1992a). There are several differences in the effects that tsg and zen mutations have on dorsal cell fate determination when compared with mutations in the other dpp-group genes. They affect cell fate of the dorsal midline cells (presumptive amnioserosa and dorsalmost regions of the head) but not of the dorsal ectoderm (Figure 4; Wakimoto et al., 1984; Zusman and Wieschaus, 1985). Using several criteria to determine the fate of cells in mutant embryos (cuticular phenotypes, mitotic domains, and specific cell marker expression patterns), there appears to be a shift in fate of dorsal midline cells to dorsolateral fates (Arora and NiissleinVolhard, 1992; Mason et al., 1994). There does not appear to be an accompanying expansion of the more ventrolateral regions, as is the case in tld, scw, and dpp mutants (see Figure 5 ) . Thus, it seems that zen and tsg are dorsal midline specific genes which act in concert with, or in response to, the highest levels of dpp activity. The tsg gene has recently been cloned and characterized (Mason et al., 1994). Initially tsg transcripts are expressed in the dorsal half of the embryo (see Figure 2), but not uniformly along the AP axis. tsg transcripts appear in two domains along the AP axis, a broad domain in the middle of the embryo that looks like a saddle over the dorsal midline, and an anterior cap. This pattern quickly refines during cellularization; transcripts disappear from the dorsal midline from both domains to give a bilaterally symmetrical pattern which refines into a series of four

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diffuse stripes along the AP axis. Transcripts fade during the rapid phase of germband elongation. Curiously, the expression pattern at the end of cellularization does not include all regions of the fate map that are affected by tsg mutations. Some of the dorsal midline cells whose behavior is affected in tsg mutants are more than I0 cells removed from the tsg-expressing domains after cellularization. It is possible that the early low level tsg gene products present at the beginning of cellularization are functional. Alternatively, the protein product may act as a signal to surrounding cells or be part of a signal transduction process. The lack of cell autonomy in mosaic studies (Zusman and Wieschaus, 1985) supports the latter view. The predicted tsg protein appears to be secreted since a potential signal sequence is present at the amino-terminus (Mason et al., 1994). A homology search identified human connective tissue growth factor (CTGF; Bradham et al., 1991), and other related proteins, as sharing a limited region of similarity with tsg in the amino half of the protein. The distinguishing features of this family of proteins is an overall structure exhibiting cysteine-rich amino and carboxy-terminal domains flanking a cysteine-free nonconserved central core. The carboxy-terminal region of one of the cysteine-rich domains in the CTGF family of proteins is related to the cysteine-rich repeats in sog. Two facts about CTGF make the homology with tsg interesting. First, CTGF is a powerful chemoattractant (Bradham et al., 1991). The cytoskeletal rearrangements induced by CTGF during chemoattraction may be similar to those that occur to dorsal cells during amnioserosa differentiation. Second, CTGF is active in mammalian cells that are becoming committed to bone formation, as are the BMP proteins (Mason et al., 1994). Thus dpp, tld, SCW, and tsg act in concert as do BMP-I, BMP2/4, CTGF, and CYR61, a relative of CTGF, in their respective developmental processes. As mentioned before, zen is initially expressed in the dorsal half of the embryo (Doyle et al., 1986; see Figure 2). The factors responsible for its activation (as well as that of dpp and tld) are unknown, but assumed to be general transcriptional activators. Zen is not expressed in ventral regions due to ventral repression by dl proteins (Rushlow et al., 1987). During cellularization, zen RNA and protein refine into a stripe along the dorsal midline about 5-6 cells wide. The length of the stripe extends from the optic lobe region to the presumptive posterior midgut. It is not clear if Zen protein functions early in cellularization or only after refinement into the dorsal stripe. zen is the only dpp-group gene

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that encodes a transcription factor. The Zen gene is located in the Antennapedia complex on the third chromosome, and like the other genes in the complex, it contains a homeobox (Doyle et af., 1986; Rushlow et al., 1987). The zen protein is a sequence specific DNA binding protein and was demonstrated to function as a transcriptional activator in transient cotransfection assays (Hoey and Levine, 1988; Han et al., 1989). Thus, Zen was speculated to be a direct regulator of downstream genes involved in the differentiation of the amnioserosa. Zen expression is affected by all other dpp-group genes (Rushlow and Levine, 1990; Ray et af., 1991 ). dpp activity is necessary for maintaining Zen transcription in the dorsal half of the embryo. In dpp, tld, and scw mutants, zen transcripts fade from dorsal regions early in cell cycle 14 and protein is never detected. Experiments where dpp transcripts were injected into young embryos showed that high levels of dpp are sufficient to induce zen transcription (Ferguson and Anderson, 1992b). tsg and sog are necessary for zen refinement into the dorsal stripe (Rushlow and Levine, 1990; Ray et al., 1991). In tsg mutants, zen RNA remains broad and fades earlier than normal. In sog mutants, the zen pattern also remains broad but does not fade as early; Zen protein levels do not, however, reach maximal levels as they do in the wildtype dorsal stripe. Thus, tsg and sog have a similar effect on the refinement of zen RNAs. If the dpp activity gradient is responsible for inducing high levels of Zen in the dorsalmost region at the late blastoderm stage, and this high level of zen is required for those cells to undergo differentiation into amnioserosa, then high levels of zen should be sufficient for amnioserosa formation. Ectopic expression studies support this idea. Expression of Zen driven by the hsp70 promoter in early gastrulating embryos resulted in an expansion of the amnioserosa (Figure 7; Rushlow, unpublished results). Figures 7a-7c show embryos stained with anti-Kriippel (Kr) antibodies which provides a marker for differentiated amnioserosa cells (Hoch et al., 1990; Jacob et al., 199'1). The amnioserosa of a wildtype embryo contains about 130 Kr-staining cells (Figure 7a; Ray et al., 1991). When Zen is expressed in all regions of a gastrulating embryo, the number of Kr-staining cells increases to 2-3 times the wildtype number (Figures 7b and 7c), reminiscent of the situation where extra copies of dpp are present in an otherwise normal embryo (Wharton et al., 1993). Note that even in the more extreme case (Figure 7c), the amnioserosa does not expand into

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cuticle

anti-Kr wt A

hszen

B

C Figure 7. Ectopic expression of zen causes an expansion of the amnioserosa. Embryos undergoinggerm band extension (stage 7) were stained with anti-Kr and peroxidase-conjugated secondary antibodies (A-C), or aged for cuticle preparations (D-F). Heat shock experiments were performed on transformant embryos carrying an hsp70 promoter-zencDNA fusion gene: 3-4 h old embryos were heat shocked at 38°C for 45 minutes. Note the expansion of the amnioserosa in heat-shocked embryos (B and C; C has more amnioserosa than B) when compared to wt (A). Heat-shocked embryos display defects in dorsal closure and germband retraction which might be caused by the enlarged amnioserosa (E,F). Some embryos have reduced denticle belts (F).

the entire ventrolateral region, that is, ventral denticles are still present (Figure 70. This could be a reflection of the timing of Zen ectopic expression; gastrulation may be too late to change the fate of ventral cells, but not of dorsal cells. Heat shock induction performed earlier than gastrulation had detrimental effects on the embryos. Alternatively, perhaps another factor (tsg?) must also be ectopically expressed in ventral regions in order to induce proper amnioserosa development.

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VI. RECEPTORS In order to understand how different concentrations ofdpp elicit different cellular responses, the downstream components of the dpp signaling pathway must be analyzed. A first step in this direction was the molecular cloning of dpp receptors which became possible after receptors for members of the TGFP superfamily had been identified in vertebrates (Childs et al., 1993; Brummel et al., 1994; Nellen et al.. 1994; Penton et al., 1994; Wrana et al., 1994b; Xie et al., 1994). Biochemical studies using mammalian tissue culture cells indicated that members of the TGFP superfamily bind to receptors consisting of multiple components (reviewed in Kingsley, 1994a). Originally three molecular size groups, called receptor type I (53 kD), type I1 (70-85 kD), and type 111 (200-400 kD) were identified. Receptor type 111 turned out to be a membrane proteoglycan that binds the TGFP isoforms and facilitates TGFP binding to the receptor proper. Receptor types I and 11 proved to be the components actually involved in signal transduction. They both encode transmembrane serinehhreonine kinases which are only distantly related to each other (40% amino acid identity). In order to constitute a hnctional receptor, both type I and type I1 receptors have to be present. Different sets oftype I and 11 receptors exist for the different subgroups of the TGFP superfamily. For example, type I and I1 activin receptors are distinct from the corresponding pairs of receptors which bind the TGFPs or the BMPs. Recently a mechanism of activation of the TGFP receptor has been described which shows that type I and I1 receptors fulfill different functions during signal transduction (reviewed in Wrana et a]., 1994a). The type I1 transmembrane serine/threonine kinase shows a constitutive autophosphorylation and is able to bind ligand in the absence of type I receptor. However, no signal transduction occurs if the receptor type I is not present. Only ligand bound by receptor type I1 can be recognized by r w p t o r type I. As a result, a stable ternary complex forms between the ligand and the two receptors. This complex formation enables receptor type I1 to phosphorylate receptor type I. Phosphorylated receptor type I mediates the signal to intracellular targets. The knowledge of the mammalian receptor sequences led to the identification of related DrosophiZu genes via homology cloning. First, the two Drosophila receptors with the closest similarity to vertebrate activin type I and I1 receptors were identified (Childs et al., 1993; Wrana et al., 1994b). These Drosophila activin receptor homologs are expressed

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maternally, during embryonic and imaginal disc development. If they are co-expressed in tissue culture cells they bind mammalian activin efficiently. Due to the lack of mutations in the corresponding Drosophilu genes, the developmental function of these receptors has not been elucidated. We currently do not know the corresponding Drosophila ligand(s). When the Drosophila activin receptor type I was co-expressed with a BMP-2 type I1 receptor (C. elegans daf-4) it was unable to bind BMP-2 (Wrana et al., I994b). This and the close relationship of BMP-2 to dpp and 60A suggests that the Drosophila activin receptor type I does not mediate the biological effects of dpp or 60A. Homology screening led to the identification of two further type I receptors which corresponded to the Drosophila genes sax and tkv (Brummel et al., 1994; Nellen et al., 1994; Penton et al., 1994; Xie et al., 1994). A comparative sequence analysis revealed that they do not appear to be orthologous to any currently known type I receptor, nor are they closely related to each other or the Drosophila activin receptor type I. Both receptors, if co-expressed with a BMP-2 type I1 receptor (C. elegans daf-4) bind BMP-2 with high affinity (Brummel et al., 1994; Penton et al., 1994). For tkv, the binding of dpp was also demonstrated (Penton et al., 1994). Thus, both may function as dpp receptors in vivo. Both the sax and tkv receptors are maternally expressed and uniformly present in the early embryo (Affolter et al., 1994; Brummel et al, 1994; Penton et a]., 1994). In later stages of development only sax shows ubiquitous expression. tkv, in contrast, displays a complex and dynamic pattern of expression. During cellularization, tkv transcripts accumulate at the dorsal side of the embryo. Shortly before gastrulation transcripts start to accumulate ventraily in the region which gives rise to the mesoderm. Later, the invaginated mesodermal cells continue to express high levels oftranscripts and, in addition, specific regions in the neuroectoderm, the tracheal placodes and visceral mesoderm accumulate tkv transcripts. The phenotypes caused by mutations in tkv and sax genetically support the assumption that both receptors are involved in dpp signaling (Affolter et al., 1994; Brummel et al., 1994; Nellenet al., 1994; Pentonet al., 1994; Terracol and Lengyel, 1994; Xie et al., 1994). Loss of function alleles of tkv are embryonic lethal and cause cuticular defects. They had been first identified in a screen for mutations affecting the larval cuticle pattern (the gene had been called sluter [slu];Niisslein-Volhard et al., 1984). However, this cuticular phenotype results from a late requirement of tkv during dorsal closure while its earlier requirement during DV patterning

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is masked by maternally supplied tkv transcripts (Affolter et al., 1994). Using germ-line mosaics, embryos could be produced which lacked both maternal and zygotic tkv gene products (Nellen et al., 1994). These embryos show a severe ventralization which is indistinguishable from the ventralized phenotype caused by a complete loss of dpp function. Severely ventralized embryos are also produced by females which are transheterozygous for certain strong tkv alleles. Using tkv alleles of different strength the same range of phenotypes can be produced which is displayed by the allelic series of hypomorphic dpp alleles (Terracol and Lengyel, 1994). In contrast to tkv, sax mutations do not appear to be zygotically lethal (Nellen et al., 1994). sax was discovered in a screen for maternal-effect mutations (Schupbach and Wieschaus, 1989). The maternal phenotype of the strongest known sax alleles is a weak ventralization (loss of amnioserosa) which resembles the weakest embryonic phenotypes caused by partial loss of dpp function. Thus, although sax is present uniformly in the early embryo, it appears to be required only to interpret peak levels of the dpp gradient. Genetic interactions between dpp and saxltkv further support the proposed molecular interactions. For example, sax and tkv alleles that encode full-length proteins with point mutations in their kinase domain enhance the phenotype of weak dpp mutations in a dominant negative manner. It seems that these mutant proteins are able to interact with and sequester limiting amounts of dpp protein on the outside of the cells without being able to activate the kinase domain within the cell (Nellen et al., 1994). In addition to defects in DV pattern formation of the early embryo, both tkv and sax mutations display defects in later patterning events (Nellen et al., 1994; Brummel et al., 1994; Penton et al., 1994). Like dpp, tkv and sax are required for normal midgut morphogenesis and for patterning in the imaginal discs. Genetic interactions between dpp and saxltkv have also been reported for these late functions. Thus, tkv and sax might be generic dpp receptors which are required throughout development for the multiple aspects of dpp function. The identification of dpp and activin receptors in Drosophila together with the possibility to perform large-scale genetic screens (Raftery et al., 1995) makes it conceivable that work with Drosophila will help to elucidate the intracellular signaling pathway of TGFj3-like molecules. This pathway has remained largely elusive in studies with mammalian cells. In mammalian tissue culture the expression of several genes is known to be modulated as a consequence of TGFP signaling (reviewed

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in Attisano et al., 1994). TGFP can control transcription both positively as in the case of collagen and TGFP itself, as well as negatively in the case of transin/stromlysin and c-myc. The growth inhibitory action of TGFP on human keratinocytes might involve a downregulation of cyclin E expression. The nature of these transcriptional responses appear to be quite complex and although both positive and negative TGFP-response elements have been identified, the chain of events between the reception ofthe signal and changes in transcriptional activity remains to be defined. Only recently, a protein was characterized (FKBP- 12) that seems to specifically interact with the type I TGFP receptor (Wang et al., 1994).

VII. FORMATION AND FUNCTION OF THE dpp GRADIENT SUMMARY AND PROSPECTIVES

A. The dpp Gradient is Dependent on, but not Completely Determined by, the dl Gradient

The dpp activity gradient is related to the dl gradient via two distinct levels of negative interactions. First, the dpp gradient is confined to the dorsal side of the embryo by the dl-dependent transcriptional repression of dpp itself and of at least one of its activators, tld (St. Johnston and Gelbart, 1987; Shimell et al., 1991). However, the repression of transcription of dpp and other dpp-group genes is not sufficient to explain the formation of the dpp activity gradient. Although there might be slight differences in the expression domains of dpp and tld (Figure 3), these differences appear to be too small to confer the necessary spatial information for the patterning of the dorsal half of the embryo. A second mechanism has to be postulated by which the dl gradient influences the dpp activity gradient. Since there are no detectable differences in the nuclear dl protein concentrations in the dorsal half of the embryonic circumference, it is likely that such a mechanism is initiated in the ventrolateral region and spreads from there towards the dorsal side (Ferguson and Anderson, 1992a, 1992b; Wharton et a]., 1993). df might activate genes in the ventrolateral region whose products diffuse into the dpp expression domain and antagonize dpp activity. The antagonistic influence on dpp might be achieved in several distinct ways. 1. It could occur at the transcriptional level. However, the dpp transcription seems to be uniform in its expression domain (Figure 3).

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2. dpp translation could be controlled such that a dpp protein gradient forms with a high point at the dorsal side. 3. The processing and modification of dpp could be controlled either by proteins which interact with dpp itself or with activators of dpp (tld,scw, or snu). 4. The antagonistic mechanism might even act downstream of dpp. A signaling protein (sog) emanating from the ventrolateral side might bind dpp directly or have its own receptors on the dorsal side. The activation of these receptors could lead to a down-regulation of the dpp signaling pathway. If the latter possibility would apply, a dpp activity gradient would form without an actual gradient of (activated) dpp protein. Although this possibility cannot be excluded, the injection of dpp RNA into Drosophila embryos (Ferguson and Anderson, 1992b) and studies with activin in vertebrates (Green and Smith, 1990; Green et al., 1992; Gurdon et al., 1994) suggest that physical gradients of TGFP-like molecules can exist, and that they can exert morphogenetic functions. Genetic and molecular studies reveal that sog is a candidate for a gene which is activated by dl in the ventrolateral region and which antagonizes dpp activity in a non-autonomous way (Zusman et al., 1988; Ferguson and Anderson, 1992a; FranGois et al., 1994). It remains to be shown whether sog protein is in fact secreted and diffuses into the dpp expression domain. As mentioned above, although sog is not a transcription factor, it represses dpp transcription in the ventrolateral region (Figure 6). This observation might indicate that dpp is auto-regulative so that dpp signaling in the embryo induces dpp transcription. sog could interfere with such an auto-regulative mechanism thereby precluding the spreading of dpp transcription toward the ventral side. Although the dpp activity gradient forms in response to the dl morphogen gradient, its shape seems not to be completely determined by the shape of the maternal gradient. For example, the five cell-wide domain giving rise to the amnioserosa is not determined by a specific dl morphogen threshold. Instead it depends on a specific activity level of dpp (Ferguson and Anderson, 1992b; Wharton et a\., 1993). How can the dpp activity gradient on the one hand depend on the dl gradient, and on the other hand contain more spatial information than the maternal gradient does? Mechanisms involved in pattern-sharpening might include positive and negative autoregulation (Meinhardt, 1982). The analysis of dpp null alleles which initially produce normal amounts of dpp RNA reveals

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that dpp is required to maintain its own transcription (Ray et al., 199 1). In addition, the spreading of dpp transcription in sog mutant embryos can be explained by an auto-inductive mechanism. Thus, these two observations are in agreement with a positive autoregulation of dpp. Furthermore, the analysis of the refinement of dpp expression also indicates negative feedback loops (Ray et al., 1991). dpp expression is down-regulated in the presumptive amnioserosa region where its initial requirements are highest. This down-regulation depends on genes which themselves are activated by dpp. For example, in a Zen mutant embryo, dpp refinement does not occur. A more systematic analysis of these regulatory interactions might reveal that a combination of positive and negative autoregulation is required to generate refined spatial information. B. The dl and dpp Morphogen Gradients Establish Different Parts of the D V Pattern

The DV pattern of the Drosophilu embryo seems to be specified by at least two gradients whose functions are already required before gastrulation, the maternal dl gradient which acts ventrally and the zygotic dpp gradient which organizes the dorsal pattern. Ventrally, the activation of twi and SNU is crucial for both the establishment of the mesoderm and the mesectoderm. Dorsally, the dpp activity gradient establishes the domains of amnioserosa and dorsal epidermis. It seems that the ventrolateral region, the neurogenic ectodenn, is the default state of the DV pattern which forms in the absence of either mesoderm or dorsal epidermis specification. This can be inferred form the phenotypic analysis of zygotic DV mutants. Both mesodermal genes and dpp seem to cause a repression ofneuroectodermal cell fates. Thus, in asna twi double mutant the lack of the mesoderm is compensated by a shift of the neuroectoderm toward the ventral side (Leptin and Grunewald, 1990; Kosman et al., 1991; Leptin, 1991; Raoetal., 1991), whileinadppmutant theneuroectoderm expands toward the dorsal side (Irish and Gelbart, 1987; Wharton et al., 1993). It also seems that at least parts of the neuroectoderm do not require dl-dependent gene activation. As described above, the complete lack of dl function leads to embryos which develop only dorsal cell fates since dpp is uniformly expressed along the DV axis. If, in such a dl mutant background, dpp activity is removed, embryos result which develop ventral epidermis along their entire circumference (Irish and Gelbart, 1987; Wharton et al., 1993). Thus, the dorsalized phenotype of

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dl mutant embryos is due to the repression of neuroectodermal development by dpp, and neuroectoderm can form even in the absence of nuclear dl protein. Therefore, the neuroectoderm is the ground state from which the dl and dpp gradients define ventral and dorsal pattern elements, respectively. Although both dpp and the mesodermal genes repress neuroectoderm development, they also exert an organizing and inductive influence on some parts of the neuroectoderm. Mesoderm specification leads to the establishment of the mesectoderm which induces ventral parts of the ventral epidermis (Kim and Crews, 1993). In sna twi double mutants both mesoderm and mesectoderm are completely lacking and although the mesoderm is replaced by the ventral epidermis, the amount of ventral epidermis seems to be reduced (Arora and Niisslein-Volhard, 1992). Similarly, dpp is not only repressing ventral ectodermal fates, but it seems to be required, as discussed above, for the formation and organization of dorsal parts of the ventral ectoderm (Wharton et al., 1993). Since an organizing influence on the neuroectoderm emanates from both the ventral and the dorsal sides, it is interesting to study embryos which at the same time lack the mesoderm and the dorsal ectoderm. Such embryos result if dpp, sna, and twi gene activities are simultaneously missing (Figure 8; Roth, unpublished results). The triple mutant embryos show an expansion of the neuroectoderm along the entire DV axis (Figure 8a). In this respect they resemble embryos derived from a dl, dpp double mutant (Irish and Gelbart, 1987). However, in contrast to those, they have polarity during gastrulation. The triple mutant embryos (Figure 8c) exhibit a polarized germband extension. Since these embryos have normal dl gradients, they show localized gene expression before gastrulation. For example, rho is expressed in a broad domain which is continuous on the ventral side since sna-dependent repression of rho does not occur (Figure 8b). However, in later stages rho transcripts completely disappear from the ventral side (Figure 8c), since later rho is expressed in the mesectoderm which is not established in the absence of mesoderm formation (Nambu et al., 1990). It is not known whether the early expression of rho has any function, or whether rho.plays any role in the absence of mesectoderm formation. Similarly, several dpp-group genes (sog, zen, tld) might be locally transcribed in the triple mutant, since their early transcription pattern depends on dl. But analogous to the case of rho, it is not known whether they have any function if dpp is not present. Thus, it is not clear where the residual polarity in the dpp sna twi triple mutant embryo originates. dl might play

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dpp' sna- twi'

Figure 8. dpp sna twi triple mutant embryos lack mesoderm, mesectoderm; and dorsal ectoderm. A. Cuticular phenotype of the triple mutant, dPPHin46,* sna"'; Df(2R)twF''. Ventral denticles encircle the embryo. B and C. Stage 5 (B) and 6 (C) embryos were hybridized with rho probes. Mesoderm formation does not occur, rho expression diminishes in the ventral region and, is completely absent ventrally in embryos slightly older than (C). Despite this, the triple mutant embryos show a polarized germ band extension.

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a not yet identified role in the patterning of the neuroectoderm, some spi-group genes (e.g., rho) might have early functions independent from mesectoderm specification, and finally, not all functions of the dpp-group genes might be mediated by dpp. Whatever the final answer will be, the residual polarity in the triple mutant indicates that a simple model which invokes only mesoderdmesectoderm specification ventrally and dorsal ectoderdamnioserosa specification dorsally is not sufficient to explain all aspects ofpatterning along the embryonic DV axis. A further analysis of the triple mutant promises new insights in the function of either the spi-group or the dpp-group genes.

VIII. A COMPARISON WITH VERTEBRATES Most of the zygotic DV genes in Drosophila have homologs in vertebrates. For example, twi and sna homologs exist in vertebrates which are expressed in the mesoderm and could play a role in mesoderm specification (Hopwood et al., 1989; Sargent and Bennett, 1990; Wolf et al., 1991; Smith et a!., 1992; Hammerschmidt and Nusslein-Volhard, 1993). The close relationship of dpp and BMP2/4 has been confirmed even with functional assays (Padgett et a]., 1993; Sampath et a]., 1993), and the finding that tld is homologous to BMPl suggests that details of the regulative mechanisms which activate TGFP-like molecules are conserved between insects and vertebrates (Shimell et al., 1991). Originally it seemed that the dpp-group genes and the BMPs were involved in different developmental processes, DV patterning and bone formation, respectively (reviewed in Kingsley, 1994b). Other TGFP-like molecules, only distantly related to the BMPs and dpp, have been implicated in DV patterning in vertebrates. More specifically, they have been shown to be involved in mesoderm induction (reviewed in Kessler and Melton, 1994). In Xenopus, during cleavage stages, cells of the vegetal pole, which will later form the endoderm, induce the overlaying marginal zone cells to form mesoderm. The two best-studied mesoderm-inducing TGFP-like molecules are Vgl and activin. Vgl. is a maternally expressed TGFP-like molecule whose mRNA is localized to the vegetal pole of Xenopus oocytes and cleavage stage embryos (Weeks and Melton, 1987). Although the Vgl precursor protein is abundantly expressed, the cleaved mature form has yet to be detected, suggesting that the processing of Vgl is tightly regulated (Dale et al., 1993; Thomsen and Melton, 1993). A stringent regulation of Vgl processing is also suggested by the observation that Vgl RNA injections into

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Xenopus embryos have little or no effects. In comparison, although the full activation of dpp is a highly regulated process during normal development (see above), injection of dpp RNAs into Drosophila embryos always caused a dose-dependent dorsalization (Ferguson and Anderson, 1992b). The phenotypic effects of Vgl could only be investigated using hybrid Vgl molecules consisting of an N-terminal proregion from a BMP and the C-terminal region from Vgl. Such hybrid molecules appeared to be processed after injection, so that mature Vgl was released. These injection studies showed that mature Vgl is a potent inducer of dorsal mesoderm (Dale et al., 1993; Thomsen and Melton, 1993). Dose-response studies have been performed for another mesoderminducing TGFP-like molecule, activin, which showed that different activin concentrations elicit different developmental fates (Green and Smith, 1990). Recently, it was shown using combinations of Xenopus vegetal and animal cap tissues that activin can form a morphogentic gradient (Gurdon et al., 1994). The selection of genes expressed by a cell was found to be determined by its distance from the activin source, indicating that activin regulates gene expression in a concentrationdependent manner. Therefore, the existence of an activin concentration gradient was proposed which comprises at least 10 cell diameters (300um in Xenopus animal pole tissue) and requires a few hours for its formation. This gradient can form by passive diffusion since activin can bypass cells that do not themselves respond to the signal nor synthesize protein. These experiments strongly suggest that morphogen gradients of TGFP-like molecules can form in embryonic tissues of a vertebrate. The same principles should apply for Drosophila tissues. There are no apparent physiochemical reasons to exclude the idea that diffusion can contribute to the formation of a gradient of activated dpp. A closer comparison between DV patterning in insects and vertebrates is implied by the recent discovery that BMP-4 is also a mesoderm indwer, and that it is required for the DV patterning of the marginal zone (Dale et al., 1992; Jones et al., 1992; Fainsod et al., 1994). BMP-4 is both maternally and zygotically expressed in Xenopus. In mesoderm induction assays, BMP-4 induces only ventral mesodermal tissues, in contrast to Vgl and activin which can also induce dorsal mesoderm. In experimental situations, BMP-4 expression modifies the induction by activin, resulting in suppression of dorsal mesoderm formation. Thus, it appears that the ventralizing BMP-4 signal is able to override dorsalizing signals. Studies with a truncated BMP-4 receptor show that in the absence of

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BMP-4 signaling, dorsal mesoderm forms even in ventral regions of the embryo (Graff et al., 1994; Maeno et al., 1994). This suggests that ventral is not the ground state for all mesoderm as had been inferred, for example, from the completely ventralized phenotype of UV-treated embryos. Interestingly, Xenopus embryos express, in addition to the close dpp homolog, BMP-4, also the sog homolog, chordin (Franqois et al., 1994; Sasai et al., 1994; Franqois and Bier, 1995). While BMP-4 is expressed in the ventral marginal zone, chordin expression is detected in the dorsal marginal zone. Thus, BMP-4 and chordin are expressed in different and potentially abutting domains. The relationship of their expression domains might be similar to those of dpp and sog in Drosophila. Injection of chordin RNA into Xenopus embryos demonstrates that chordin is a potent dorsalizing factor (Sasai et al., 1994). chordin 5 biological effects seem to be opposite to BMP-4 and thus, chordin might antagonize BMP-4 function, like sog antagonizes dpp function. Corresponding molecules involved in DV patterning in both insects and vertebrates seem to fulfill opposing functions. BMP-4 is ventralizing in vertebrates while dpp is dorsalizing in insects; chordin is dorsalizing in vertebrates while sog is ventralizing in insects. These findings have led to the revival of an old discussion. In 1822, Geoffroy St. Hilaire proposed that the arthropod body plan is like the vertebrate body plan turned upside down (Niibler-Jung and Arendt, 1994). According to this idea the longitudinal nerve cords of insects and vertebrates derive from the same centralized nervous system in their common ancestor. Because the nerve cord is located ventrally in insects and dorsally in vertebrates, the ventral side of insects would then correspond to the dorsal side of vertebrates. To explain how a common ancestor can give rise to both vertebrates and insects (arthropods), an inversion of the DV axis during early chordate evolution was suggested (Arendt and Niibler-Jung, 1994). This inversion must result from a change in the gastrulation behavior. The vertebrate mode of gastrulation can be conceptually derived from that efpolychaete annelids in a way which implies an inversion of the DV axis. However, this inversiun hypothesis is challenged by the existence of a group of hemichordates (enteropneusta) which possess both a ventral and a dorsal nerve cord (Peterson, 1995). According to an alternative, and less radical proposal, vertebrates are derived from ciliated larvae resembling those of modern echinoderms (Lacalli, 1995). This idea accounts for a partial inversion of the vertebrate DV axis relative to that of insects. A solution to these interesting evolutionary questions requires that molecular studies are carried out not only in

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Drosophilu and vertebrates, but that t h e y are also extended to primitive invertebrates. However, even w i t h o u t a knowIedge of t h e precise evolutionary relationship, it is already a p p a r e n t that the molecules and mechanisms discovered in Drosophila not only have counterparts i n vertebrates, but that they govern similar developmental processes (for other examples see Scott, 1994).

ACKNOWLEDGMENTS We would like to thank several of our colleagues for valuable and stimulating discussions that enabled us to write this manuscript: Laurel Raftery, Rob Ray, Manfred Frasch, Chip Ferguson, Ethan Bier, Kristi Wharton, Kavita Arora, Mike O’Connor, Liz Mason, Larry Marsh, and Rick Padgett. Manfred Frasch first observed the dpp expression changes in sog mutant embryos with multiple copies of dpp. We thank Rob Ray for help in generating the dpp,sna,twi triple mutant, and Ethan Bier for the hsCasper plasmid. We are grateful to Trudi Schiipbach and Paul Wassarman for their patience. We acknowledge Rob Ray and Kavita Arora for providing parts of Figures 1,4, and 5 . We thank Andreas Bergmann, Stefan Schulte-Merker, and Mary Mullins for critical reading of the manuscript.

REFERENCES Abrams, J.M., White, K., Fessler, L.I., & Steller, H. (1993). Programmed cell death during Drosophilu embryogenesis. Development I1 7.29-43. Affolter, M., Nellen, D., Nussbaumer, U., & Basler, K. (1994). Multiple requirements for the receptor serindthreonine kinase thick veins reveal novel functions of TGFP homologs during Drosophila embryogenesis. Development f20,3 105-3 I 17. Anderson, K.V., Jurgens, G., & Nusslein-Volhard. C. ( 1985a). Establishment of dorsalventral polarity in the Drosophilu embryo: Genetic studies on the role of the Toll gene product. Cell 42,779-789. Anderson, K.V., Bokla, L., & Nusslein-Volhard, C. (1985b). Establishment of dorsalventral polarity in the Drosophila embryo: The induction ofpolarity by the Toll gene product. Cell 42, 79 1-798. . Anderson, K.V., Schneider, D.S., Morisato, D., Jin, Y., & Ferguson, E.L. (1992). Extiacellular morphogens in Drosophila embryonic dorsal-ventral patterning. Cold Spring Harbor Symp. Quant. Biol. 57,40%417. Arendt, D., & Nubler-Jung, K. (1994). Inversion of the dorsoventral axis. Nature 371, 26. Arora, K., & Nusslein-Volhard, C. (1992). Altered mitotic domains reveal fate map changes in Drosophila embryos mutant for zygotic dorsoventral patterning genes. Development 114, 1003-1024.

74

CHRISTINE RUSHLOW and SIEGFRIED ROTH

Arora, K., Levine, M., & O’Connor, M.B. ( 1994). The screw geneencodes a ubiquitously expressed ineinber of the TGF-P family required for specification of dorsal cell fates in the Drosophila embryo. Genes & Dev. 8,2588-2601. Attisano, L., Wrana, J.L., Lopez-Casillas, F., & Massague, J. (1994). TGF-P receptors and actions. Biochem. Biophys. Acta 1222,7140. Barr, P.J. ( 199 I). Mammalian subtilisins: The long-sought dibasic processing endoproteases. Cell 66, 1-3. Basler, K., & Struhl., G. ( 1994). Compartment boundaries and the control of Drosophila limb pattern by hedgehog protein. Nature 368,208-2 14. Bier, E., Jan, L.Y., & Jan, Y.N. (1990). rhomboid, a gene required for dorsoventral axis establishment and peripheral nervous system development in Drosophila melanogaster. Genes & Dev. 4 , 190-203. Boulay, J.-L., Dennefeld, C., & Alberga, A. (1987). The Drosophila developmental gene snail encodes a protein with nucleic acid binding fingers. Nature 330, 395-398. Bradham, D.M., Igarashi, A., Potter, R.L., & Grotendorst, G. (1991). Connective tissue growth factor: A cysteine-rich secreted by human vascular endothelial cells is related to the SRC-induced immediate early gene product CEF-10. J. Cell Biol. 114, 1285-1294. Brummel, T.J., Twombly, V., Marques, G., Wrana, J.L., Newfeld, S.J., Attisano, L., Massague, J., O’Connor, M.B., & Gelbart, W.M. (1994). Characterization and relationship of Dpp receptors encoded by the saxophone and thick veins genes in Drosophila. Cell 7&25 1-261. Campbell, G., Weaver, T., & Tomlinson, A. (1993). Axis specification in the developing Drosophila appendage: The role of wingless,decapentaplegic and the homeobox gene aristaless. Cell 74, 1 1 13-1 123. Campos-Ortega, J.A., & Hartenstein, V. ( 1985). The Embryonic Development OfDrosophila melunogaster. Springer Verlag, Berlin. Childs, S.R., & O’Connor, M.B. (1993). Two domains of the tolloid protein contribute to its unusual genetic interaction with decapentaplegic. Dev. Biol. 162,209-220. Childs, S.R., Wrana, J.L., Arora, K., Attisano, L., O’Connor, M.B., & Massague, J. (1993). Identification of a Drosophila activin receptor. Proc. Natl. Acad. Sci. USA 90,9475-9479. Crews, S.T.,Thomas, J.B.,&Goodman, C.S. (1988). The Drosophilasiiigle-mindedgene encodes a nuclear protein with sequence similarity to theper gene product. Cell 52, 143-15 1. Dale, L., Howes, G., Price, B.M.J., & Smith, J.C. (1992). Bone morphogenetic protein - . Q: ‘A ventralizing factor in Xenopus development. Development I f 5,573-585. Dale, L., Matthews, G., & Colman, A:( 1993). Secretion and mesoderm-inducing activity of the TGF-beta-related domain of Xenopus Vgl . EMBO J. 12,447 1-4480. Doyle! H.J., Harding, K., Hoey, T., & Levine, M. (1986). Transcripts encoded by a hoineobox gene are restricted to dorsal tissues of Diasophila embryos. Nature 323, 76-79. Doyle, H.J., Kraut, R., & Levine, M. (1 989). Spatial regulation of zerkniillt: A dorsalventral patterning gene in Drosophila. Genes & Dev. 3, 15 18-1533. Fainsod, A., Steinbeisser, H., & De Robertis, E.M. (1994). On the function of BMP-4 in patterning the marginal zone of theXenopus embryo. EMBO J. 13,5015-5025.

The dpp-group Genes

75

Ferguson, E.L., & Anderson, K.V. (1991 1. Dorsal-ventral pattern formation in thc Drosophilu embryo: The role of zygotically active genes. Current Topics Dev. Biol. 25, 1 7 4 3 . Ferguson, E.L., & Anderson, K.V. (1992a). Localized enhancement and repression of the activity of the TGF-P family member, decapentaplegic, is necessary for dorsal-ventral pattern formation in the Drosophila embryo. Development 114, 583-597. Ferguson, E.L., & Anderson, K.V. (1992b). Decupeiztuplegic acts as a morphogen to organize dorsal-ventral pattern in the Drosophilu embryo. Cell 71,451461. Foe, V. ( 1989). Mitotic domains rcveal early commitment of cells in Drosophila embryos. Development 107, 1-22. Franqois, V., & Bier, E. ( 1 995). Xenopus chordin and Drosoplzilu short gastrulation genes encode homologous proteins functioning in dorsal-ventral axis formation. Cell 80, 19-20. Franqois, V., Solloway, M., O’Neill. J.W., Emery, J., & Bier, E. (1994). Dorsal-ventral patterning of the Di-mophila embryo depends on a putative negative growth factor encoded by the slzor-t gastrulution gene. Genes & Dev. 8,2602-261 6. Geisler, R., Bergmann, A., Hiromi, Y., & Nusslein-Volhard, C. (1992). cactus, a gene involved in dorsoventral pattern formation of Drosophilu, is related to the I kappa B gene family of vertebrates. Cell 71,6 13-62 I . Gonzalez-Crespo, S., & Levine, M. ( 1993). interactions between dorm/ and helix-loophelix proteins initiate the differentiation of the embryonic mesoderm and neuroectoderm in Drosophilu. Genes & Dev. 7, 1703-1 7 13. Graff, J.M., Thies, R.S., Song, J.J., Celeste, A.J., & Melton, D.A. (1994). Studies with a Xenopus BMP receptor suggest that ventral mesoderm-inducing signals override dorsal signals in vivo. Cell 79, 169-1 79. Green, J.B.A., & Smith, J.C. (1990). Graded changes in dose of a Xenopus activin A homologue elicit stepwise transitions in embryonic cell fate. Nature 347,391-394. Green, J.B.A., New, H.V., & Smith, J.C. (1992). Responses of embryonicXenopus cells to activin and FGF are separated by multiple dose thresholds and correspond to distinct axes of the mesoderm. Cell 71, 731-739. Gurdon. J.B., Harger, P., Mitchell, A,, & Lemaire, P. (1994). Activin signalling and response to a morphogen gradient. Nature 371,487492. Green, J.B:A., New, H.V., & Smith, J.C. (1992). Responses of embryonicXenopu.7 cells to activin and FGF are separated by multiple dose thresholds and correspond to distinct axes of the mesoderm. Cell 7 1 , 73 1-739. Hammerschmidt, M., & Nusslein-Volhard, C. (1 993). The expression of a zebrafish gene homologous to Drosophilu snail suggests a conserved function in invertebrate and vertebrate gastrulation. Development 119, 1107-1 11 8. Han, K.,.Levine, M . S . , & Manley, J.L. (1989). Synergistic activation and repression of transcription by Dvosophilu homeobox proteins. Cell 56, 573-583. Hashimoto, C., Hudson, K.L., & Anderson, K.V. (1988). The Toll gene of Drosophila, required for dorsal-ventral embryonic polarity, appears to encode a transmembrane protein. Cell 52,269-279. Heberlein, U., Wolff, T.. & Rubin, G.M. (1993). The TGF beta homolog dpp and the segment polarity gene hedgehog are required for propagation of a morphogenetic wave in the Drosophilu retina. Cell 75,913-926.

76

CHRISTINE RUSHLOW and SIEGFRIED ROTH

Hoch, M., Schroder, C., Seifert, E., & Jackle, H. (1990). Cis-acting control elements for Kruppel expression in the Di-osophilu embryo. EMBO J. 9,2587-2595. Hoey, T., & Levine, M. (1988). Divergent homeobox proteins recognize similar DNA sequences in Di-osophilu. Nature 332, 858-86 I . Hopwood, N.D., Pluck, A., & Gurdon, J.B. (1989). A Xenopus mRNA related to Drosophilu rwist is expressed in response to induction in the mesoderm and the neural crest. Cell 59, 893-903. Huang, J.D., Schwyter, D.H., Shirokawa, J.M., & Courey, A.J. (1993). The interplay between multiple enhancer and silencer elements defines the pattern of decapentuplegic expression. Genes Dev. 7,696704. Ip, Y.T., Kraut, R., Levine, M., & Rushlow, C.A. (1991). The dorsal morphogen is a sequence-specific DNA-binding protein that interacts with a long-range repression element in Drosophila. Cell 64,439-446. Ip, Y.T., & Levine, M. ( 1992). The role of the dorsul morphogen gradient in Drosophilu embryogenesis. Sem. Dev. Biol. 3, 15-23. Ip, Y.T., Park, R.E., Kosman, D., Bier, E., & Levine, M. (1992a). The dorsal gradient morphogen regulates stripes of rhomboid expression in the presumptive neuroectoderm of the Drosophilu embryo. Genes & Dev. 6. 1728-1739. Ip, Y.T., Park, R.E., Kosman, D., Yazdanbakhsh, K., & Levine, M. (1992b). Dorsal-twist interactions establish snail expression in the presumptive mesoderm of the Drosophila embryo. Genes & Dev. 6, 1518-1530. Irish, V.F., & Gelbart, W.M. (I 987). The decupentaplegic gene is required for dorsal-ventral patterning of the Drosophilu embryo. Genes & Dev. I , 868479. Jackson, P.D., & Hoffmann, EM. (1994). Embryonic expression patterns of the Dtvsophila decupentaplegic gene: Separate regulatory elements control blastoderm expression and lateral ectodermal expression. Dev. Dyn. 199,2844. Jacob, Y., Sather, S., Martin, J.R., & 0110,R. (1991). Analysis ofKruppel control elements reveals that localized expression results from the interaction of multiple subelements. Proc. Natl. Acad. Sci. USAR8,5912-5916. Jiang, J., & Levine, M. ( 1 993). Binding affinities and cooperative interactions with bHLH activators delimit threshold responses to the dorsal gradient morphogen. Cell 72, 741-752. Jiang, J., Kosman, D., Ip, Y.T., & Levine, M. (1991). The dorsal morphogen gradient regulates the mesoderm determinant twist in early Drosophilu embryos. Genes & Dev. 5, 1881-1891. Jiang, J., Rushlow, C.A., Zhou, Q., Small, S., & Levine, M. (1992). Individual dorsal binding sites mediate activation and repression in the Drosophilu embryo. EMBO J. 11,3147-3154. Jiang, J., Cai, H., Zhou, Q., & Levine, M. (1993). Conversion of a dorsal-dependent silencer into an enhancer: Evidence for dorsal corepressors. EMBO J. 12, 3201. 3209. Johes, C.M., Lyons, K.M., Lapan, P.M., Wright, C.V.E., & Hogan, B.J.M. ( 1992). DVR-4 (bone morphogenetic protein-4) as a postero-ventralizing factor in Xenopus mesoderm induction. Development 115,639447. Jurgens, G., Wieschaus, E., & Nusslein-Volhard, C. (1984). Mutations affecting the pattern of the larval cuticle in Drosophilu melunoguster.11: Zygotic loci on the third chromosome. Roux’s Archives of Developmental Biology 193,283-295.

The dpp-group Genes

77

Kaphingst, K., & Kunes, S. (1994). Pattern formation in the visual centers of the Drosophila brain: wingless acts via decapentaglegic to specify the dorsoventral axis. Cell 78,437-448. Kessler, D.S., & Melton, D.A. ( I 994). Vertebrate embryonic induction: Mesodermal and neural patterning. Science 266, 59-04, Kidd, S . (1992). Characterization of the Drosophila cactus locus and analysis of interactions between cactus and dorsal proteins. Cell 71, 623435. Kim, S.H., & Crews, S.T. (1993). Influence of Drosophila ventral epidermal development by the CNS midline cells and spitz class genes. Development 118, 893-90 !. Kingsley, D.M. (1994a). The TGF-beta superfamily: New members, new receptors, and new genetic tests of function in different organisms. Genes & Dev. 8, 133-146. Kingsley, D.M. (1994b). What do BMPs do in mammals? Clues from the mouse short ear mutation. Trends Genet. 10, 1 6 2 1 . Kirov, N., Zhelnin, L., Shah, J., & Rushlow, C. (1993). Conversion o f a silencer into an enhancer: Evidence for a co-repressor in dorsal-mediated repression in Drosophila. EMBO J. 12,3193-3199. Kirov, N., Chiids, S., O’Connor, M., & Rushlow, C. (1994). The Duosophila dorsal morphogen represses the tolloid gene by interacting with a silencer element. Mol. Cell. Biol. 14,713-722. Konrad, K.D., Goralski, T.J.. & Mahowald, A.P. (1988). Developmental genetics of the gastrulation defective locus in Drosophila melanogaster. Developmental Biology 127, 133-142. Kosman, D., Ip, Y.T., Levine. M., & Arora, K. (1 99 I). Establishment of the mesodermneuroectoderm boundary in the Drosophila embryo. Science 254, I 1 8-1 22. Lacalli, T.C. (1995). Dorsoventral axis inversion. Nature 373, 110-1 1 1 . Lehming, N., Thanos, D., Brickman, J.M., Ma, J., Maniatis, T., & Ptashne. M. (1994). An HMG-like protein that can switch a transcriptional activator to a repressor. Nature 371, 175-179. Leptin, M. (199 I). twist and srzail as positive and negative regulators during Dmvophila mesoderm development. Genes & Dev. 5, 1.568-1576. Leptin, M., & Grunewald, B. (1990). Cell shape changes during gastrulation in Drosophi/a. Development 110, 73-84. Lyons, K.M., Jones, C.M., & Hogan, B.L. (1992). The TGF-beta-related DVR gene family in mammalian development. Ciba Found. Symp. 16.5, 219-230; 230-234. Maeno, M., Ong, C.R., Suzuki, A,, Ueno, N., & Kung, H.-F. ( 1994). A truncated bone morphogenetic protein 4 receptor alters the fate of ventral mesoderm to dorsal mesoderm: Roles of animal pole tissue in the development of the ventral mesoderm. Proc. Natl. Acad. Sci. USA 91, 10260-10264. Mason, E.D., Konrad, K.D., Webb, C.D., & Marsh, L.J. (1994). Dorsal midline fate in Dro%Yophilaembryos requires twisted gastrulation, a gene encoding a secreted protein related to human connective tissue growth factor. Genes & Dev. 8, 1489IiOl. Massague, J., Attisano, L., & Wrana, J.L. (1994). The TGF-P family and its composite receptors. Trends Cell Biol. 4, 172-178. Mayer, U., & Nusslein-Volhard, C. (1988). A group of genes required for pattern formation in the ventral ectoderm of the Dimophila embryo. Genes and Development 2, 1 4 9 6 15 11.

78

CHRISTINE RUSHLOW and SIEGFRIED ROTH

Meinhardt, H. (1982). Models of Biological Pattern Formation.Academic Press, London. Morisato, D., & Anderson, K.V. (1994). The spatzle gene encodes a component of the extracellular signaling pathway establishing the dorsal-ventral pattern of the Drosophila embryo. Cell 76,677488. Murphy-Ullrich, J.E., Schultz-Cherry, S., & Hook, M. (1992). Transforming growth factor$ complexes with thrombospondin. Mol. Biol. Cell. 3. 181-188. Nambu, J.R., Frank, R.G., Hu, S., & Crews, S.T. (1990). The single-minded gene of Drosophila is required for the expression of genes important for the development of CNS midline cells. Cell 63, 63-75. Nellen, D., Affolter, M., & Basler, K. (1994). Receptor serinekhreonine kinases implicated in the control of Dro.sophila body pattern by decapentaplegic. Cell 78, 225-237. Nguyen, T., Jamal, J., Shimell, M.J., Arora, K., & O’Connor, M.B. (1994). Characterization of tolloid related-I: A BMP- I -like product that is required during larval and pupal states of Drosophila development. Dev. Biol. 166, 569-586. Nubler, K., & Arendt, D. (1994). Is ventral in insects dorsal in vertebrates. Roux’s Arch. Dev. Biol. 203,357-366. Nusslein-Volhard, C. (1979). Maternal effect mutations that alter the spatial coordinates ofthe embryo ofDrosophilamelanogaster. In: Determinants ofspatial Organization (Subtelny, S . , & Koenigsberg, I.R., Eds.). Academic Press, New York, pp. 185-2 11. Nusslein-Volhard, C., Wieschaus, E., & Kluding, J. (1984). Mutations affecting the pattern of the larval cuticle in Drosophila nielanogaster I: Zygotic loci on the second chromosome. Roux’s Archives of Developmental Biology 193.267-282. Ozkaynak, E., Rueger, D.C., Drier, E.A., Corbett, C., Ridge, R.J., Sampath, T.K., & Oppermann, H. (1990). Op-l cDNA encodes an osteogenic protein, in the TGF-P family. EMBO J. 9,2085-2093. Padgett, R.W., Johnson, R.D.S., & Gelbart, W.M. (1987). A transcript from aDrosophila pattern gene predicts a protein homologous to the transforming growth factor-p family. Nature 325, 81-84. Padgett, R.W., Wozney, J.M., & Gelbart, W.M. (1993). Human BMP sequences can confer normal dorsal-ventral patterning in the Drosophila embryo. Proc. Natl. Acad. Sci. USA 942905-2909. Pan, D., Huang, J-D., & Courey, A.J. (1991). Functional analysisofthe Drosopfiila twist promoter reveals a dorsal-binding ventral activator region. Genes & Dev. 5 , 18921901. Panganiban, G.E., Rashka, K.E., Neitzel, M.D., & Hoffmann, EM. (1990). Biochemical characterization of the Drosophila DPP protein, a member of the transforming growth factor p family of growth factors. Molecular and Cellular Biology 10, 2669-2677. Paralkar, V.M., Hamkonds, R.G., & Reddi, A.H. (1991). Transforming growth factor beta type 1 binds to collagen IV of basement membrane matrix: Implications for development. Dev. Biol. 143, 303-308. Penton, A,, Chen, Y., Staehling-Hampton, K., Wrana, J.L., Attisano, L., Szidonya, J., Cassill, J.A., Massague, J., & Hoffmann, F.M. (1994). Identification of two bone rnorphogenetic protein Type I receptors in Drosophila and evidence that Brk25D is a decapentaplegic receptor. Cell 78,239-250. Peterson, K.J. (1995). Dorsoventral axis inversion. Nature 373, I 11-1 12.

The dpp-group Genes

79

Posakony, L.G., Raftery, L.A., & Gelbart, W.M. (1990). Wing formation in Drosophilu rnelunoguster requires decupei~ruplegicgene function along the anterior-posterior compartment boundary. Mech. Dev. 33,6%82. Raftery, L.A., Twombly, V., Wharton, K., & Gelbart, W.M. (1995). Genetics screens to identify elements of the decapentuplegic signaling pathway in Drosophila. Genetics 139,241-254. Rao, Y., Vaessin, H., Jan, L.Y., & Jan, Y.-N. (1991). Neuroectoderm in Drosophilu embryos is dependent on the mesoderm for positioning but not for formation. Genes and Development 5, 1577-1588. Ray, R.B. (1993). Dorsal-ventral patterning in the Drosophila embryo. Ph.D. thesis, Harvard University. Ray, R.P., Arora, K., Niisslein-Volhard, C., & Gelbart, W.M. (1991). The control of cell fate along the dorsal-ventral axis of the Drosophila embryo. Development 113, 35-54. Roth, S. ( 1 993). Mechanisms of dorsal-ventral axis determination in Drosophila embryos revealed by cytoplasmic transplantations. Development I 1 7, 1385-1396. Roth, S., Stein, D., & Niisslein-Volhard. C. (1989). Agradient ofnuclear localization of the Dorsal protein determines dorsoventral pattern in the Drosophila embryo. Cell 59, I 1 89-1 202. Roth, S., Hiromi, Y., Godt, D., & Nusslein-Volhard, C. (1991). Cactus. a maternal gene required for proper formation of the dorsoventral morphogen gradient in Drosophih embryos. Development 112, 37 1-388. Rushlow, C.A., & Arora, K. (1990). Dorsal-ventral polarity and pattern formation in Drosophila. Pattern formation in Drosophilu. Sem. Cell Biol. I , 173-184. Rushlow, C., & Levine, M. (1990). Role of the zer.kiziillt gene in dorsal-ventral pattern formation in Drosophila. Adv. Genet. 27. 277-307. Rushlow, C., & Warrior, R. ( 1 992). The re1 family of proteins. BioEssays 14, 8%95. Rushlow, C.A., Frasch, M., Doyle, H., & Levine, M. (1987). Maternal regulation of zerkniillt: A homeobox gene controlling differentiation of dorsal tissues in Dvosophilu. Nature 330,583-586. Rushlow, C.A.. Han, K., Manley, J.L., & Levine, M. (1989). The graded distribution of the dorsuf morphogen is initiated by selective nuclear transport in Dmsophilu. Cell 59, 1165-1177. Rutledge, B.J., Zhang, K., Bier, E., Jan, Y.N., & Perrimon, N. (1992). The Drosophila spitz gene encodes a putative EGF-like growth factor involved in dorsal-ventral axis formation and neurogenesis. Genes & Dev. 6, 1503-1 5 17. Sampath, T.K., Rashka, K.E., Doctor, J.S., Tucker, R.F., & Hoffman, F.M. (1993). Dtvsophila transforming growth .factor j3 superfamily proteins induce endochondral bone formation in mammals. Proc. Natl. Acad. Sci. USA 90,60044008. Sargent,'M.G., & Bennett, M.F. (1990). Identification in Xenopus of a structural homoIogue of the Drosophila gene snail. Development 109,967-973. Sasai, Y., Lu, B., Steinbeisser, H., Geissert, D., Gont, L.K., & De Robertis, E.M. (1994). Xenopus chordin: A novel dorsalizing factor activated by organizer-specific homeobox genes. Cell 79,779-790. Schneider, D.S., Hudson, K.L., Lin, T.Y., & Anderson, K.V. (1991). Dominant and recessive mutations define functional domains of Toft, a transmembrane protein

80

CHRISTINE RUSHLOW and SIEGFRIED ROTH

required for dorsal-ventral polarity in the Drosophila embryo. Genes & Dev. 5 , 797-807. Schneider, D.S., Jin, Y., Morisato, D., & Anderson, K.V. (1994). Aprocessed form ofthe Spatzle protein defines dorsal-ventral polarity in the Drosophila embryo. Development 120, 1243-1250. Schupbach, T., & Wieschaus, E. (1989). Female sterile mutations on the second chromosome of Drosophifa melanogaster I. Maternal effect mutations. Genetics 121, 101-1 17. Schupbach, T., & Wieschaus, E. ( I 986). Maternal-effect mutations altering the anteriorposterior pattern of the Drosophila embryo. Roux’s Archives of Developmental Biology 195, 302-317. Scott, M.P. (1994). Intimations of a creature. Cell 79, 11 21-1 124. Sekelsky, J.J., Newfeld, S.J., Raftery, L.A., Chartoff, E.H., & Gelbart, W.M. (1995). Genetic characterization and cloning of Mothers against dpp, a gene required for decapentaplegic function in Drosophila melanogaster. Genetics 139, 1347-1 358. Shimell, M.J., Ferguson, E.L., Childs, S.R., & O’Connor, M.B. (1991). The Drosophila dorsal-ventral patterning gene tolloid is related to human bone morphogenetic protein I.Cell 67,469-48 1. Simpson, P. (1983). Maternal-zygotic gene interactions during formation of the dorsoventrai pattern in Drosophila embryos. Genetics 105, 6 15-632. Smith, D.E., Franco del Amo, F., & Gridley, T. (1992). Isolation of Sna, a mouse gene homologous to the Dvosophila genes snail and escargot: Its expression pattern suggests multiple roles during postimplantation development. Development 116, 1033-1039. Spencer, F.A., Hoffmann, F.M., & Gelbart, W.M. (1982). Decapentaplegic: A gene complex affecting morphogenesis in Drosophila melanogaster. Cell 28,45 1-46 1. St. Johnston, R.D., & Gelbart, W.M. (1987). Decapentaplegic transcripts are localized along the dorsal-ventral axis of the Drosophila embryo. EMBO J. 6,2785-2791. St. Johnston, R.D., & Nusslein-Volhard, C. ( 1 992). The origin of pattern andpolarity in the Drosophila embryo. Cell 68,201-2 19. Staehling-Hampton, K., Jackson, P.D., Clark, M.J., Brand, A.H., & Hoffmann, F.M. ( 1994). Specificity ofbone morphogenetic protein-related factors: Cell fate and gene expression changes in Drosophila embryos induced by decapentaplegic but not 6OA. Cell Growth & Diff. 5,585-593. Stein, D., & Nusslein-Volhard, C. ( 1992). Multiple extracellular activities in Drosophila egg perivitelline fluid are required for establishment of embryonic dorsal-ventral polarity. Cell 68,429-440. Stein, D., Roth, S., Vogelsang, E., & Nusslein-Volhard, C. (1991). The polarity of the dorsoventral axis in the Drosophila embryo is defined by an extracellular signal. Cell 65, 725-735. Steward, R. (1987). dorsal, an embryonic polarity gene in Drosophila is homologous to the vertebrate proto-oncogene, c-re/.Science 238,692694. Steward, R. (1989). Relocalization of the dorsal protein from the cytoplasm to the nucleus correlates with its function. Cell 59, 117%-1188. Technau, G.M., & Campos-Ortega, J.A. (1987). Cell autonomy of expression of neurogenic genes ofDrosophilu melanogaster. Proc. Natl. Acad. Sci. USA84,4500-4504.

The dpp-group Genes

81

Terracol, R., & Lengyel. J.A. ( 1994). The thick veiris gene of Drosophila is required for dorsoventral polarity of the embryo. Genetics 138, 165-178. Thisse, B., Stoetzel, C., Gorostiza-Thisse, C., & Perrin-Schmitt, F. (1988). Sequence of the twist gene and nuclear localization of its protein in endomesodermal cells of early Drosophila embryos. EMBO J. 7,2 175-2 183. Thisse, C., Perrin-Schmitt, F., Stoetzel, C., & Thisse, B. (1991). Sequence-specific transactivation of the Di*o.sophilatwist gene by the dorsal gene product. Cell 65, 1191-1201. Thomsen, G.H., & Melton, D.A. (1993). Processed Vgl protein is an axial mesoderm inducer in Xenopus. Cell 74,433441. Wakimoto, B.T., Turner, F.R., & Kaufinan, T.C. (1984). Defects in embryogenesis in mutants associated with the Antennapedia gene complex of Drosophila rnelanogaster. Dev. Biol. 102, 147-1 72. Wang, E.A., Rosen, V., Cordes, P., Hewick, R.M., Kriz, M.J., Luxenberg, D.P., Sibley, B.S., & Wozney, J.M. (1988). Purification and characterization of other distinct bone-inducing factors. Proc. Natl. Acad. Sci. USA 85, 9484-9488. Wang, T., Donahoe, P.K., & Zervos, A S . (1994). Specific interaction oftype 1 receptors of the TGF-P family with the iinmunophilin FKBP- 12. Science 265,674-676. Weeks, D.L., & Melton, D.A. (1987). A maternal mRNA localized to the vegetal hemisphere in Xenopirs eggs codes for a growth factor related to TGF-P. Cell 51, 86 1-867. Wharton, K.A., Thomsen, G.H., & Gelbart, W.M. ( I99 I). Drosophila 60A gene, a new TGF-P family member is closely related to human bone morphogenetic proteins. Proc. Natl. Acad. Sci. USA 88,9214-9218. Wharton, K.A., Ray, R.P., & Gelbart, W.M. (1993). An activity gradient of decapentaplegic is necessary for the specification of dorsal pattern elements in the Drosophila embryo. Development 117, 807-422. Wieschaus, E., & Nusslein-Volhard, C. ( 1986). Looking at embryos. In: Dro.sopliila:A Practical Approach (Roberts, D:B.. Ed.). IRL Press, Washington DC, pp. 199-227. Wieschaus, E., Nusslein-Volhard, C., & Jurgens, G. (1984). Mutations affecting the pattern of the larval cuticle in Drosophila melanogaster: 11. Zygotic loci on the X-chromosome and fourth chromosome. Wilhelm Roux’s Arch. Dev. Biol. 193, 296-307. Wolf, C., Thisse, C., Stoetzel, C., Thisse, B., Gerlinger, P., & Perrin-Schmitt, F. (1991). The M-twist ofMus is expressed in subsets of mesodermal cells and is closely related to the Xenopus X-twi and the Drosophila twist genes. Dev. Biol. 143,363-373. Wozney, J.M., Rosen, V., Celeste, A.J., Mitsock, L.M., Whitters, M.J., Kriz, R.W., Hewick, R.M., & Wang, E.A. ( 1988).Novel regulators ofbone formation: Molecular clones and activities. Science 242, 1528-1 534. Wrana, J.L., Attisano, L., Wieser, R., Ventura, F., & Massague, J. (1994a). Mechanisms of activation of the TGF-P receptor. Nature 370,341-347. Wrana, J.L., Tran, H., Attisano, L., Arora, K., Childs, S.R., Massague, J., & O’Connor, M.B. (1 994b). Two distinct transmembrane serinehhreonine kinases from Drosophila melanogaster form an activin receptor complex. Mol. Cell. Biol. 14,944-950. Xie, T., Finelli, A.L., & Padgett, R.W. (1994). The Drosophila saxophone gene: Aserine threonine kinase receptor ofthe TGF-beta superfamily. Science 263, 1756-1 759.

82

C H R I S T I N E R U S H L O W and SIEGFRIED R O T H

Zusman, S.B., & Weischaus, E. (1985). Requirement for zygotic gene activity during gastrulation in Drosophila melanogaster. Dev. Biol. 111,359-371. Zusman, S.B., Sweeton, D., & Wieschaus, E.F. (1988). Short gastrulation, a mutation causing delays in stage specific cell shape changes duringgastrulation in Drosophila melanogaster. Dev. Biol. 129,417427.