Morphogen gradients: new insights from DPP

Reviews

DPP gradient formation and interpretation

Morphogen gradients new insights from DPP The Drosophila TGFb family member Decapentaplegic (DPP) has been proposed to function as a morphogen to pattern cell fields in a number of developmental contexts. A series of recent reports add significantly to our knowledge of the mechanisms of DPP-gradient formation and interpretation. These reports identify additional genes and genetic circuitry necessary for this patterning system, and they highlight variations that might reflect developmental constraints within individual target cell fields.

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Steven D. Podos spodos@midway. uchicago.edu Edwin L. Ferguson elfergus@midway. uchicago.edu Department of Molecular Genetics and Cell Biology, University of Chicago, 920 East 58th Street, Chicago, IL 60637, USA. 396

n 1969, Lewis Wolpert1 put forward the model of positional information, in which cell position within a developing field is specified according to an underlying coordinate system. In the typical form of this model, a positional signaling factor (or morphogen) produced at a localized source achieves an extracellular gradient by diffusion across the target cell field (Fig. 1). Cells within the field determine their position by interpreting the concentration of the morphogen to activate specific programs of targetgene expression at discrete morphogen thresholds. The concept of positional information has served developmental biologists extremely well, and numerous extracellular signals in a variety of developmental systems have been proposed as graded positional signals (reviewed in Ref. 2). The elegance of Wolpert’s model, however, hides a host of unanswered biological questions. First, how can the wide range of morphogen concentrations present over the target cell field elicit reproducible cellular responses? Second, how is a gradient of the morphogen set up and maintained over the field? Third, how are discrete thresholds set within target cells in response to a continuously decreasing morphogen gradient? In this article, we consider several recent reports3–11 that address these questions in Drosophila for the well-established positional signal Decapentaplegic (DPP), a member of the bone morphogenetic protein (BMP) group of TGFb signaling molecules. Three main conclusions arise from these new data. First, the establishment of positional information in multiple target cell fields is not mediated solely by a graded distribution of the DPP ligand but, instead, requires synergistic signaling between two different BMP receptor– ligand pairs. Second, the developmental constraints of each target field might necessitate distinct genetic circuitry to specify positional information within the field. Third, the establishment of discrete thresholds for DPP within cell fields involves the action of a novel target gene of DPP, brinker, whose activity is repressed by dpp, and which functions to repress other dpp target genes. DPP-activity gradients effect patterning of cell fields During Drosophila development, DPP provides positional information to pattern two well-studied tissues: the wing TIG October 1999, volume 15, No. 10

imaginal disc and the embryonic ectoderm. In the wing imaginal disc, dpp is expressed in a narrow stripe of cells at the anterior–posterior (A–P) compartment boundary and is necessary both for cell proliferation and for organization of the A–P pattern across the entire imaginal disc (Fig. 2a, top). A series of experiments in 1996 (Refs 12, 13), involving ectopic activation or clonal elimination of DPP signaling, indicated that DPP can act cell nonautonomously to control transcription of the target genes spalt (sal) and optomotor-blind (omb) at successively lower thresholds across the growing disc (Fig. 2a, bottom). Although some caveats remain, most notably owing to complications arising from the cellular growth and movement that accompanies patterning in this cell field13, the favored interpretation of these results is that DPP acts as a classical long-range morphogen to pattern the wing disc. In contrast to its function in the wing disc, DPP acts at short range to provide dorsal–ventral (D–V) positional information to the embryonic ectoderm. The dpp gene is expressed by nuclei in the dorsal 40% of the embryonic circumference (Fig. 2b, top). Whereas ventral ectodermal cells that lack DPP activity differentiate as neurogenic ectoderm, different levels of DPP activity can elicit two distinct epidermal cell fates14–17: a low level of DPP activity specifies dorsal epidermis and a higher level of DPP activity can specify the extraembryonic amnioserosa (Fig. 2b, bottom). Because dpp is expressed at uniform intensity within its domain14, a DPP activity gradient must be formed by post-transcriptional modulation of DPP distribution or signaling capability18. Synergistic signaling by two BMP ligand–receptor pairs Initially, genetic and embryological experiments indicated that DPP is absolutely necessary and could, under experimental conditions, be sufficient to confer all positional values within each field of cells. However, additional complexity was suggested by findings that, in vivo, a second BMP ligand is required for the elaboration of the full range of positional values within each field. In the embryo, the screw (scw) gene is expressed around the embryonic circumference at the blastoderm stage (Fig. 2b, top), and null scw mutations cause cell-fate transformations that are similar to those caused by partial loss-of-function dpp 0168-9525/99/$ – see front matter © 1999 Elsevier Science Ltd. All rights reserved. PII: S0168-9525(99)01830-2

Reviews

DPP gradient formation and interpretation

alleles, indicating that SCW is required for maximal DPP activity19. Similarly, in the imaginal disc, the glass bottom boat (gbb, also known as 60A) gene is expressed throughout the wing pouch of the disc (Fig. 2a, top), and the patterning defects observed in hypomorphic gbb mutant wings are exacerbated by a small reduction of dpp activity, suggesting that GBB elevates DPP signaling within the disc20. Furthermore, genetic analysis of TGFb receptor function revealed that signals must be integrated downstream of multiple receptors. The heteromeric TGFb receptor complex consists of two types of serine–threonine kinases21. Upon ligand binding, the type II kinase phosphorylates the type I kinase, which then transduces the signal to downstream components22. While the type II receptor, Punt (PUT), and the type I receptor, Thick veins (TKV), are essential for all DPP signaling23–27, a second type I receptor, Saxophone (SAX), is also necessary for normal patterning in each tissue. In the embryo, SAX is required to obtain the highest levels of DPP signaling to specify amnioserosal cell fates, and in the wing disc SAX function is required to obtain normal levels of DPP signaling across the entire DPP activity gradient23–25,28. Overexpression of TKV can bypass the requirement for SAX (Ref. 25), suggesting that the two receptors could use the same intracellular signal-transduction machinery. Three reports3–5 have now clarified the interrelationships among these BMP ligands and receptors. In embryos completely lacking dpp expression, injection of increasing amounts of an mRNA encoding a mutated, constitutively activated form of the TKV receptor (TKV-A) induces the full complement of dorsal cell fates in a dose-dependent fashion3,4. Thus, signaling downstream of TKV recapitulates the embryonic response to DPP (Ref. 3). In marked contrast, injection of a constitutively activated SAX (SAX-A) mRNA has no biological effect3,4. However, when SAX-A mRNA is co-injected with low or moderate levels of TKV-A mRNA, SAX-A mRNA elevates the biological response to a given level of TKV-A mRNA (Refs 3, 4). A similar synergistic effect of ectopic TKV-A and SAX-A signaling is observed in the wing disc5. Together, these results indicate that in both tissues, SAX and TKV transmit distinct intracellular signals that must be integrated for the accurate interpretation of positional values. Ligand specificities of the TKV and SAX receptors have been established by functional criteria, primarily by determining whether dominant-negative forms of each receptor (TKV-DN and SAX-DN) can block the phenotypes caused by ectopic (or elevated) ligand activity in vivo3–5. In the embryo and the wing disc, expression of TKV-DN blocks the activity of DPP as well as the activity of SCW or GBB (depending on the tissue examined). By contrast, expression of SAX-DN blocks the activity of SCW and GBB, but has no effect on DPP activity. A separate experiment demonstrated that, in the embryo, scw function is essential for the activity of a chimeric receptor composed of the extracellular domain of SAX and the intracellular domain of TKV (Ref. 3). Moreover, SCW and GBB have full biological activity, even when expressed in cells that do not express DPP (Refs 3–5), indicating that signaling by SCW or GBB does not require the formation of heterodimers with DPP. These findings strongly suggest that SCW and GBB are necessary components of the ligand for the SAX receptor.

FIGURE 1. The ‘French flag’ model of positional information

galF hcnerF

French Flag trends in Genetics

(Top) A morphogen, produced in a restricted domain within a field of cells (left panel, black stripe), mediates the organization of the entire field into a set of discrete domains (right panel, red, white and blue stripes), which could represent either differentiation of particular cell types or the expression patterns of individual genes. (Bottom) The morphogen conveys positional information by forming an extracellular gradient (curved black line) as the result of diffusion from its source and subsequent titration or consumption within the field. Cells within the field determine their position by interpreting the morphogen concentration, resulting in their activation of specific programs of target gene expression at discrete morphogen thresholds.

Taken together, these results modify the previous paradigm of DPP action. Although experimental manipulations indicate that DPP, acting through TKV, can specify all positional values in the field in a dose-dependent fashion, the ability of DPP to specify positional values across a cell field in vivo requires synergistic signaling from a second ligand, SCW or GBB, acting through the SAX receptor (Fig. 3). While the mechanism(s) by which the SAX signal is integrated into the TKV signaling pathway are presently unknown, some predictions can be made from recent biochemical analyses of signaling downstream of the vertebrate homologs of the two receptors (Fig. 3). Although synergistic signaling between TKV and SAX is required for the elucidation of positional information in both cell fields, the biological mechanism used to specify positional information within each cell field differs. In the wing disc, diffusion of DPP from its source probably provides the necessary positional information, whereas GBB signaling provides a constant level of elevation of the DPP signal across the wing disc (Fig. 2a). By contrast, positional information in the embryonic ectoderm is specified by the spatially restricted modulation TIG October 1999, volume 15, No. 10

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FIGURE 2. Generation of positional information (a)

(b)

sal omb

dpp scw dpp gbb

AS DE NE

TKV

DPP DPP

?

?

GBB

SCW ?

SOG P

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A

?

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V

(a) In the wing imaginal disc (top), dpp is expressed in a small number of cells that border the anterior–posterior (A–P) compartment boundary in the center of the disc (black hatched area), while a gene encoding a second BMP ligand, glass bottom boat (gbb), is transcribed throughout the wing pouch (gray area). The consequent gradient of positional information results in the expression of sal in a relatively narrow band in the center of the disc (red), while omb is expressed in a broader band (white). The positional information (bottom) required to pattern the wing disc comes from synergistic signaling between DPP and GBB. Diffusion of DPP from its source provides positional information over the disc (darker gray area), while GBB probably provides a constant level of elevation of the DPP signal (lighter gray area), which is most noticeable in regions of low signaling strength away from the site of DPP production. Downregulation of TKV transcription (green line) by DPP is necessary to allow proper diffusion of DPP throughout the disc (lines ending in bars). (b) In the embryo (top), dpp is expressed at uniform levels in the dorsal (D) 40% of embryonic nuclei (black hatched area), while a gene encoding a second BMP ligand, screw (scw), is expressed uniformly around the embryonic circumference (gray area). The gradient of positional information divides the ectoderm into three tissue types: the dorsal (D) 10% of embryonic cells become extraembryonic amnioserosa (AS), dorso–lateral cells differentiate as dorsal epidermis (DE), while ventral (V) cells differentiate as neurogenic ectoderm (NE). The positional information (bottom) required to pattern the ectoderm comes from synergistic signaling between DPP and SCW. DPP is likely to signal at a constant level over the region where it is expressed (darker gray area under solid line), while much of the graded positional information within the ectoderm is likely to be specified by spatial modulation (lines ending in bars) of SCW activity (lighter gray area under solid line) by the ventrally produced inhibitor Short gastrulation (SOG, green line). A second, positive action of SOG on DPP or SCW that is required for maximal signaling dorsally is indicated by dashed arrows and lines.

of SCW and, possibly, DPP signaling by a diffusible BMP-binding protein, SOG (Fig. 2b). These differences, which are described in more detail below, might reflect the adaptation of the BMP-mediated patterning system to the particular developmental constraints of the two tissues.

Formation of a DPP gradient in the wing In the developing wing disc, DPP is expressed in a narrow stripe of cells that are adjacent to the A–P compartment boundary (Fig. 2a), and positional information is generated by the graded cell non-autonomous action of DPP away from its source, presumably by the diffusion of DPP protein (but see below for an alternative hypothesis). Although DPP diffusion across the disc cannot be observed directly, the kinetics of gradient formation have now been analysed indirectly via the expression of the DPP target genes sal and omb in response to ectopic clonal expression of different levels of DPP (Ref. 6). While clones of cells expressing DPP remain small, the secreted DPP 398

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organizes gene expression in broad domains of surrounding cells. Ectopic expression of low levels of DPP results in the differential expression of omb and sal: omb is turned on within 24 h of clone formation, while sal expression is first observed after 72 h. These results suggest that DPP receptors sense the amount of ligand continuously and, furthermore, that transcriptional output might be the result of stable ligand accumulation on the target cell surface – as has been demonstrated previously for activin signaling in cultured Xenopus animal cap cells29. By contrast, ectopic expression of high levels of DPP induces both sal and omb expression within 24 h, but both genes are expressed in the same spatial domain. It is only by 50 h after clone formation that the transcriptional boundary of omb becomes measurably wider than that of sal. These results suggest that, initially, the diffusion of DPP is restricted, resulting in a steep DPP gradient, and only after a long time period does the slope of the gradient lessen to allow differential expression of the two target genes.

Reviews

DPP gradient formation and interpretation

Two reports5,6 now show that high levels of the DPP receptor TKV are the probable cause of the slow initial phase of DPP-gradient formation and, furthermore, that DPP must downregulate the expression of its receptor to permit its own diffusion6. In wild-type discs, the TKV receptor is expressed strongly at the lateral margins of the disc but only weakly in the medial regions of the disc that are patterned by DPP signaling5,6,25. While local overexpression of wild-type TKV in a clone of cells away from the central region of the disc enhances cellular responsiveness to low levels of DPP (Ref. 6), overexpression of TKV throughout the central domain of the wing disc narrows the domains of omb and sal activation, presumably by global restriction of DPP availability at a distance from its source of production at the compartment boundary5,6. Furthermore, ectopic expression of DPP is sufficient to downregulate tkv expression within two days after clone formation6. Thus, the DPP-dependent downregulation of tkv expression is essential for proper DPP gradient formation and might be the kinetically limiting factor in DPP diffusion across the developing wing disc. These new findings might help to reconcile some of the experimental discrepancies in the original 1996 papers that reported the direct patterning activity of DPP (Refs 12, 13). In addition, the necessity for tkv downregulation might partially explain the need for GBB signaling through SAX to elaborate the full range of positional information over the developing disc. The effects of gbb mutations are most visible in the lateral regions of the disc that have low DPP signaling: in hypomorphic gbb mutants the expression pattern of omb collapses into a domain similar to that of sal (Ref. 5). In these regions of the disc, a reduced TKV receptor level, coupled with the low amount of DPP present at a distance from its source, might lessen DPP signaling below threshold levels. Thus, GBB signaling through SAX might be necessary to boost TKV signaling to elicit appropriate biological responses.

Cytonemes: does DPP diffuse? Although graded DPP activity across the wing disc is most often represented as an extracellular gradient of DPP protein, such a gradient has not been observed directly. Now, an alternative mechanism is suggested by the recent observation that lateral cells of the wing imaginal disc project long slender processes – cytonemes – that orient in a nearly parallel fashion towards the DPP-signaling center in the central region of the disc11. Whereas no specific function has yet been ascribed to cytonemes, the authors suggest a mechanism by which target cells in the lateral regions of the disc could sense DPP directly at its site of production, without the need for extracellular DPP diffusion. The graded effects of DPP signaling across the disc could then derive intracellularly from the spatial or temporal decay of the DPP signal as it is transduced from the cytoneme tip to the cell body11. Several tests might address the question of the participation of cytonemes in DPP signaling. First, numerous studies have shown that clones of DPP-expressing cells anywhere within the wing disc can organize long-range pattern in a radial fashion around the clone. Thus, for cytonemes to be the sole mechanism of non-autonomous DPP signaling within the disc, the ectopic expression of DPP must result in a reorientation or growth of cytonemes towards the DPP source, a condition that was not tested in

FIGURE 3. Synergistic TKV and SAX signaling DPP

TKV

Ligand

SCW or GBB

?

SAX

Receptor, type I subunit

? MAD

? SMADs

MED

Target genes trends in Genetics

Synergistic signaling by DPP, acting through its receptor TKV, and SCW or GBB, acting through their receptor SAX, is required to specify all positional values within each field. Whereas TKV signaling has been shown to require the SMADs MAD and MED, the signal transduction pathway downstream of SAX is currently not known. However, recent biochemical analysis of vertebrate SAX orthologs (ALK-1 and ALK-2) indicates that these receptors phosphorylate the same SMADs as do vertebrate TKV homologs (ALK-3 and ALK-6), which suggests that both SAX and TKV transduce signals through MAD (Refs 42–44), despite notable sequence differences between TKV and SAX within a structural kinase loop that contributes to SMAD substrate specificity44–46. However, in the absence of biochemical data from the Drosophila receptors, the integration between the TKV and SAX signaling pathways could formally be at any of three levels (see ‘?’): TKV receptor activation, differential effects on the SMAD complex, or at the enhancers of specific target genes.

the initial work described above11. However, DPP does not appear to cause cytoneme projection in culture; although cultured cells from the lateral region of the disc project cytonemes towards central disc cells that contain the compartment boundary (or S2 cells engineered to express FGF), cultured cells do not project cytonemes towards S2 cells engineered to express DPP (Ref. 11). A direct role for DPP in cytoneme organization in vivo might be addressed by examining the cell-autonomous effects of lesions in the DPP signal-transduction machinery on cytoneme outgrowth. As yet, long-range DPP signaling via cytonemes is not easily reconciled with the observed requirement for TKV receptor downregulation within the central region of the disc. If all disc cells use cytonemes to sense DPP at its source, then the experimental elevation of TKV receptor levels throughout the disc should effect an increase, rather than the observed decrease5,6, in DPP signaling. However, given that cytonemes appear to be a general characteristic of epidermal cells, both in arthropods and vertebrates11, much future work will be focused on understanding the role of these novel structures in growth and patterning.

Setting up the DPP gradient in the embryo In the blastoderm embryo, the gradient of DPP activity is formed rapidly – within two hours – as evidenced by region-specific activation of target genes at the onset of gastrulation. This rapidity might preclude morphogen diffusion from a localized source, particularly as the BMP receptors TKV, SAX and PUT are all provided to the embryo from the maternal genome during oogenesis and thus cannot be subject to feedback regulation, as in the TIG October 1999, volume 15, No. 10

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trends in Genetics

FIGURE 4. Transcriptional responses to the DPP gradient require BRK

BRK

DPP + GBB

SMAD BRK

SMAD BRK

SMAD BRK

omb

sal

In the schematic representation of the wing imaginal disc (top), brk transcription (green line) is repressed (line ending in bars) by BMP signaling (black line). In turn, BRK represses transcription of DPP target genes. In this context, BMP signaling refers to the integration of DPP and SCW signaling through their respective receptors. Genetic epistasis experiments indicate that full activation of lowthreshold target genes, such as omb, might require relief from BRK repression only, while full activation of higher-threshold target genes, such as sal, might require relief from BRK repression as well as direct activation by the SMAD complex. The figure (bottom) portrays one speculative mechanism for BRK and SMAD action, in which transcriptional activation by SMAD complexes (plus signs) and transcriptional repression by BRK (minus signs) are integrated directly at target gene promoters. Levels of BRK and activated SMAD complex are depicted as low (white fill), medium (gray), and high (black) within the three transcriptional domains defined by DPP and GBB signaling. Other models are equally plausible.

wing disc. Instead, the DPP and SCW ligands are expressed throughout the region where they are required for patterning, and it appears that the principal mechanism used to generate positional information is cell nonautonomous modulation of BMP-ligand signaling by a BMP-binding protein encoded by the short gastrulation (sog) gene. Initially, sog is expressed throughout the ventro-lateral region of the embryo that becomes neurogenic ectoderm. The sog gene encodes a protein with functional homology to vertebrate Chordin, which binds to and inhibits the vertebrate homolog of DPP, BMP4 (Refs 30–33). The tolloid (tld) gene, which elevates DPP activity dorsally, is expressed in the same domain as dpp and encodes a zinc metallo-protease that cleaves SOG (Refs 18, 34, 35). Thus, a simple model for the formation of a DPP activity gradient is that a ventral source of the SOG inhibitor, coupled with a dorsal sink for SOG, results in a V–D gradient of SOG that causes a reciprocal D–V gradient of DPP activity. However, two sets of experiments now show that this model is incomplete. While SOG was thought to function by inhibiting DPP directly, two papers3,4 suggest instead that SOG inhibits DPP signaling primarily by blocking SCW function (Fig. 2b). Specifically, in scw mutant embryos, injection of SOG mRNA does not block the dorsalizing activity of injected DPP mRNA, but effectively blocks the dorsalizing activity of injected SCW mRNA. Thus, spatial modulation of SCW activity by the com400

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bined action of SOG and TLD is likely to be a major component of the generation of positional information within the embryonic ectoderm. These experiments are supported by the observation that scw and tld mutant embryos have identical phenotypes, suggesting that SCW function is eliminated in the absence of TLD, possibly by sequestration in inactive complexes with SOG (Refs 18, 19). Although SOG has been primarily characterized as an inhibitor of BMP signaling, phenotypic analyses suggest that SOG also exerts a positive, cell non-autonomous function on BMP signaling that is essential for production of amnioserosa36 (Fig. 2b). This positive role of SOG has now been demonstrated directly by its misexpression from the even-skipped stripe-2 (eve s2) enhancer in a narrow stripe around the D–V circumference of the embryo10. In wild-type embryos, the dpp-dependent transcription of the Race gene marks the presumptive amnioserosa at the onset of gastrulation. Race expression is largely absent in sog mutant embryos. Ectopic sog expression from the eve s2 enhancer locally inhibits Race transcription in wildtype embryos, indicating short-range interference by SOG of DPP or SCW signaling. However, in sog mutant embryos, ectopic sog restores Race expression on the dorsal side, but only at a distance from the site of ectopic sog expression, indicating a long-range positive action of SOG upon DPP or SCW signaling. This latter activity requires extracellular SOG diffusion, because similar misexpression of a membrane-tethered mutant variant of SOG exerts a local repression, yet fails to exert a long-range activation of Race expression. In addition, the positive activity of SOG depends upon TLD function, because it is greatly compromised in embryos that are heterozygous for a tld mutation. The mechanism by which SOG mediates cell nonautonomous elevation of BMP signaling remains unknown, as does its specificity for DPP or SCW. SOG might facilitate the diffusion of a BMP ligand from the ventral regions toward the dorsal side of the embryo, whereupon TLD would cleave SOG to liberate active BMP ligand and thereby increase the dorsal BMP ligand concentration37. Alternatively, while full-length SOG inhibits BMP signaling, a TLD-mediated cleavage product of SOG might elevate BMP signaling, either directly or as an independent signal35.

Formation of discrete thresholds from a continuous gradient In Wolpert’s model1, the intracellular signal transduction machinery downstream of an extracellular morphogen must be able to activate target genes at discrete thresholds of morphogen concentration. While the DPP signaltransduction pathway has been described well in recent years, little is known about how specific transcriptional thresholds are determined within target cells. In the current paradigm of DPP signal transduction, a single class of proteins known as SMADs couples receptor activation to the control of target-gene transcription (reviewed in Ref. 21). Type I receptors, such as TKV, phosphorylate the receptor-specific SMADs, exemplified in the DPP pathway by the Mothers against dpp (Mad) gene product. The phosphorylated SMAD then associates with a general SMAD partner, represented in the DPP pathway by the Medea (Med) gene product, and the SMAD complex translocates to the nucleus where it

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DPP gradient formation and interpretation

functions together with other transcription factors to regulate target-gene expression. SMAD proteins possess direct and specific DNA-binding capacity21, and a MADbinding site is essential for the DPP-dependent transcription of the vestigial (vg) gene within a broad domain in the wing disc38. The simplicity of this pathway might allow the tight coupling of external ligand concentration to transcriptional responses, with the sensitivity of an individual target gene being dependent upon the arrangement and/ or affinities of MAD–MED-binding sites within its enhancer. Three recent reports7–9 now challenge this linear view of DPP signal transduction and, in the process, uncover an elegant mechanism for setting sharp thresholds to the DPP gradient. The papers report the characterization of a novel gene, brinker (brk), whose transcription is repressed by DPP and which functions to repress DPP target-gene transcription. The brk gene is essential for proper patterning of both the embryonic ectoderm and the wing disc, indicating that it is an integral component of the DPP system of positional information. In the wing disc, brk is expressed in the lateral-most regions of the disc adjacent to (and slightly overlapping with) cells that express omb (Fig. 4). Discs with reduced dpp expression, or clones of cells that lack either tkv or Mad activity (both of which are needed for DPP action), express brk ectopically, indicating that DPP represses brk transcription. Furthermore, in wild-type discs the central edge of the brk transcription pattern appears to be graded, suggesting that the pattern of brk transcription is a direct reflection of the DPP activity gradient. Conversely, in clones of disc cells that are mutant for the brk gene, DPP target genes are inappropriately expressed in a cell-autonomous fashion. For example, clones of brk mutant cells near the lateral edge of the disc, far from the source of DPP, express sal and omb (Refs 7–9) as well as reporters for the DPP-responsive enhancer elements in the vg and daughters against dpp (dad) genes8,9. Therefore, BRK acts as an intracellular negative regulator of DPP signaling and, as such, represents an integral component of the pathway that is necessary for proper transcriptional responses to the DPP gradient. Strikingly, the ectopic activation of sal and omb is still present in double-mutant brk Mad and brk tkv clones7,8, indicating that transcription of DPP target genes can be independent of transcriptional activation by the MAD–MED signaling complex7–9. Therefore, BRK must act differently from another DPP target gene, DAD, which also encodes a negative regulator of DPP signaling. DAD, which has limited similarity with SMADs, antagonizes DPP signaling by binding to and inhibiting the TKV receptor39,40. Although the molecular mechanism of brk function is not known, BRK is localized to the nucleus throughout its domain of expression in the imaginal discs8, suggesting that it might function directly as a transcriptional repressor. Consistent with this suggestion, all three reports note weak similarities between the sequences of BRK and known transcriptional regulators. Moreover, BRK is able to antagonize BMP signaling and induce dorsal cell fates in Xenopus embryos, suggesting that its function is conserved between arthropods and chordates9. An analysis of the effects of brk mutations on the ectopic activation of the DPP target genes sal and omb suggests possible differences in the mechanisms of tran-

scriptional control for different classes of DPP target genes. In brk single-mutant or in brk tkv double-mutant clones, omb is expressed at a level that is equivalent to that observed in its normal domain of expression. Therefore, normal omb activation can be explained as a simple relief from BRK repression without the necessity of direct input from the MAD–MED signaling complex (Fig. 4). By contrast, ectopic sal expression in brk mutant clones that lack either tkv or Mad is lower than the normal level of expression of sal in the center of the disc. Moreover, in brk clones that border the normal domain of sal expression, the ectopic sal transcription is graded, with increasing levels of sal expression towards the center of the disc, which suggests that, in these clones, the level of sal transcription is a direct output of the DPP gradient. Therefore, the normal activation of sal transcription in a sharply bounded domain requires relief from BRK repression and activation by the MAD–MED signaling complex (Fig. 4). This integration of positive and negative inputs on sal transcription might be typical of a general developmental mechanism by which a graded signal is interpreted by a field of cells to produce sharp thresholds of gene expression. One of the reports8 notes a parallel between the integration of BRK and MAD–MED inputs on sal gene expression with the integration of positive and negative transcriptional inputs to establish the sharp threshold of Krüppel expression in the syncytial blastoderm embryo in response to the hunchback gradient. A similar inference has also been drawn41 concerning the formation of a sharp threshold of snail expression in response to the gradient of nuclear Dorsal protein, although in this case the identity of the putative repressor is not known. Taken together, these data strongly suggest that for a morphogen gradient to elicit responses with sharp thresholds, it is necessary to integrate positive and negative regulators at the enhancers of target genes.

Conclusions The reports discussed here3–11 provide novel insights into the action of DPP in patterning fields of cells. They demonstrate a requirement for synergistic signaling between two ligand–receptor pairs to specify the full range of positional values within the cell fields patterned by DPP, although the synergistic signaling might play different roles in different developmental contexts. These reports also identify a novel gene, brinker, that plays an integral role in the transcriptional responses to the DPP gradient, and suggest that one function of this gene is representative of a general mechanism by which a continuous gradient of a morphogen can cause sharp thresholds in target-gene transcription. They also provide a framework for future experiments. Synergistic but dependent signaling between two different TGFb ligand–receptor pairs has not been described before, and it is currently not known whether this mechanism is widely used or is restricted to specific developmental contexts. Moreover, it will be interesting to determine whether the synergy occurs at the level of TKV receptor activation, differential effects on the SMAD complex, or at the enhancers of specific target genes (Fig. 3). Furthermore, the effects of synergistic signaling were primarily measured at the level of developmental pattern, rather than at the level of individual gene transcription, and it will be interesting to examine the interrelationships TIG October 1999, volume 15, No. 10

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between these signaling pathway(s) and the transcription of known dpp target genes, including sal, omb, brk and tkv. Lastly, a molecular dissection of the mechanisms of BRK and SMAD action should reveal the biochemical interplay between repressor and activator that is necessary for threshold formation. The results of these and other experiments will allow us to formulate a much more complete description of the interconnected pathways that

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Acknowledgements We thank V. Prince, C. Rushlow and an anonymous reviewer for helpful comments on the manuscript. Work in the authors’ laboratory was supported by a grant from the National Institutes of Health.

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Mediation of response to Dpp by brinker A recent detailed characterization1 of the effects of brk mutations on early embryonic patterning reveals that the functions of brk are similar in the wing disc and the embryo. brk is expressed in the ventro-lateral regions of the embryo in the same domain as sog. brk transcription is repressed by dpp activity, and brk, in turn, represses transcription of dpp target genes, including dpp itself. Reference 1 Jazwinska, A. et al. (1999) The role of brinker in mediating the graded response to Dpp in early Drosophila embryos. Development 126, 3323–3334

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