decapentaplegic

The Drosophila gene tolloid has been shown to genetically interact with dpp. The genetic interactions between tolloid and dpp suggests a model in which the Tolloid protein participates in a complex containing the DPP ligand, its protease serving to activate DPP, either directly or indirectly. Dpp's activity is modulated by Tolloid, which also has a role in the determination of dorsal cell fate (Finelli, 1994). Subsequent studies show that the target of Tolloid is in fact Short gastrulation (Sog).

Tolloid, a putative metalloprotease related to BMP-1, enhances DPP function, while SOG, an ortholog of the Xenopus organizer Chordin, inhibits DPP function. Tolloid is secreted and requires a protelytic processing step for activation. The removal of the N-terminal prodomain is not catalyzed by TLD itself, since it is removed from a putative TLD protease null mutant. Most of the TLD in embryos is in the nonprocessed form. Using epistasis tests and a Xenopus secondary axis induction assay, it has been shown that TLD negates the inhibitory effects of SOG/CHD on DPP/BMP-type ligands. Ventral overexpression in Xenopus of either a dominant negative BMP4 receptor, or a cleavage mutant of either BMP4, Noggin, Chordin, or Short gastrulation induces secondary axes in 80%-100% of injected embryos. However, when CHD or SOG mRNA are coinjected, together with an equimolar amount of TLD mRNA, secondary axis induction is blocked, suggesting that TLD is capable of inhibiting SOG or CHD function. Activated TLD is unable to inhibit secondary axis induction mediated by Noggin, the dominant negative BMP receptor, or a cleavage mutant of BMP. In transient transfection assays, TLD cleaves SOG; this cleavage is stimulated by DPP. It is proposed that formation of the embryonic DPP activity gradient involves the opposing effects of SOG inhibiting DPP, and TLD processing SOG to release DPP from the inhibitory complex (Marques, 1997).

Noggin, a protein expressed in the Spemann organizer region of the Xenopus embryo, promotes dorsal cell fate within the mesoderm and neural development within overlying ectoderm. noggin, expressed in Drosophila, promotes ventral development, specifying ventral ectoderm and CNS in the absence of all endogenous ventral-specific zygotic gene expression. Noggin blocks DPP signaling upstream of DPP receptor activation. It is proposed that, whole most or all of the DPP produced in the dorsal-most region binds to its receptors, DPP produced more laterally has an increased probability of being bound by ventrally produced Short gastrulation, and that DPP can be released from this diffusible complex by the action of a third dorsal-specific gene, perhaps tolloid (Holley, 1996).

Short gastrulation prevents DPP from suppressing neurogenesis laterally in the blastoderm embryo. It is possible to exacerbate defects in sog mutants by increasing the level of DPP. The earliest neuroectodermal marker affected in sog mutants with a double dose of dpp is rhomboid, which is normally expressed in lateral stripes 8-10 cells wide in wild-type embryos but rapidly narrows to stripes 4-6 cells across in sog mutants with elevated DPP. Similarly l'sc expression is reduced in sog mutants with elevated DPP. Surprisingly, dpp itself is induced throughout the neuroectoderm in this genetic combination. This provides the first evidence that dpp is capable of autoactivating its own expression during early embryogenesis. Ubiquitous dpp expression results in zerknüllt expression throughout the entire trunk neuroectoderm and mesoderm (Biehs, 1996).

A striking feature of the affects of DPP on neural suppression and dorsalization is that neuronal suppression is induced by a lower threshold of DPP activity than is dorsalization. Much less DPP is required to suppress expression of neuroectodermal genes than is required to activate dorsal markers. For example, brief submaximal heat induction of heat shock dpp in a wild type sog background leads to nearly maximal suppression of lethal of scute, scratch and snail expression during germ band extension, but there is no detectable ectopic expression of zerknüllt in the neuroectoderm (Biehs, 1996).

Gene dosage experiments are consistent with SOG diffusing dorsally 12 to 15 cell diameters from the lateral source of SOG mRNA to determine the limit of dorsal rhomboid expression. Thus SOG diffuses from the neuroectoderm into the presumptive mesoderm to interfere with DPP signaling. Since the effect of SOG is highly dosage dependent then it is likely that there is a gradient of SOG activity in both dorsal and ventral regions of the embryo creating a reciprocal gradient of DPP activity in the dorsal region of the embryo. In this respect, SOG displays many features of a classic morphogen (Biehs, 1996).

Screw (SCW) and DPP act together to establish distinct response boundaries within the dorsal half of the embryo, perhaps by forming heterodimers that have higher activity than homodimers of either molecule alone. Null mutations in the scw gene are phenotypically similar to moderate dpp mutants and cause dorsal cells to adopt ventral fates. scw encodes a novel TGF-beta protein and is an integral part of the signal that specifies dorsal pattern. Although scw is expressed uniformly during blastoderm stages, its effect on development appears graded and is restricted to the dorsal side of the embryo. DPP activity alone is insufficient to specify different dorsal cell fates (Arora, 1994).

Extracellular gradients of signaling molecules can specify different thresholds of gene activity in development. A gradient of Decapentaplegic (Dpp) activity subdivides the dorsal ectoderm of the Drosophila embryo into amnioserosa and dorsal epidermis. The proteins Short gastrulation (Sog) and Tolloid (Tld) are required to shape this gradient. Sog has been proposed to form an inhibitory complex with either Dpp or the related ligand Screw, and is subsequently processed by the protease Tld. Paradoxically, Sog appears to be required for amnioserosa formation, which is specified by peak Dpp signaling. Sog appears to be required for peak Dpp/Screw activity, since sog mutants lack amnioserosa. SOG transcripts are detected in two ventrolateral stripes within the presumptive neurogenic ectoderm. Several amnioserosa marker genes, including Kruppel, rhomboid and hindsight exhibit broadened patterns of expression that gradually diminish in older embryos. In contrast, the Race (Related to angiotensin converting enzyme) pattern is not transiently expanded in sog mutants; instead, by the onset of gastrulation, expression is nearly lost in central regions. Race may represent a more definitive marker for the presumptive amnioserosa than the genes used in previous studies (Ashe, 1999).

The misexpression of sog using the even-skipped stripe-2 enhancer redistributes Dpp signalling in a mutant background in which dpp is expressed throughout the embryo. Dpp activity is diminished near the Sog stripe and peak Dpp signaling is detected far from this stripe. However, a tethered form of Sog suppresses local Dpp activity without augmenting Dpp activity at a distance, indicating that diffusion of Sog may be required for enhanced Dpp activity and consequent amnioserosa formation. The long-distance stimulation of Dpp activity by Sog requires Tld, whereas Sog-mediated inhibition of Dpp does not. The heterologous Dpp inhibitor Noggin inhibits Dpp signaling but fails to augment Dpp activity. These results suggest an unusual strategy for generating a gradient threshold of growth-factor activity, whereby Sog and its protease specify peak Dpp signaling far from a localized source of Sog. Different models have been proposed to explain the requirement of Sog in generating peak Dpp activity. One invokes the diffusion of Sog-Dpp or Sog-Screw complexes away from the ventrolateral Sog stripes, thereby focusing Dpp and/or Screw at the dorsal midline. An alternative model suggests that a product resulting from the cleavage of Sog directly signals formation of the amnioserosa, possibly by augmenting the binding of Dpp or Screw to the receptors Thick veins and Saxophone (Ashe, 1999).

There are three serine-threonine tyrosine kinase receptors for dpp. Two of these, Thickveins and Saxophone, are homologous to type I TGFbeta receptors. The third, Punt, is homologous to type II TGFbeta receptors. Punt on its own is able to bind vertebrate activin but not BMP2, a vertebrate ortholog of DPP. Mutations in punt produce phenotypes similar to those exhibited by tkv, sax, and dpp mutants. Furthermore, Punt will bind BMP2 in concert with TKV or SAX, forming complexes with these receptors. It has been suggested that Punt functions as a type II receptor for DPP as part as an obligatory complex with TKV, with SAX associated in a tissue dependent manner (Letsou, 1995 and Wharton, 1995).

Axis formation in the Drosophila wing depends on the localized expression of the secreted signaling molecule Decapentaplegic (Dpp). Dpp acts directly at a distance to specify discrete spatial domains, suggesting that it functions as a morphogen. Expression levels of the Dpp receptor thick veins (tkv) are not uniform along the anterior-posterior axis of the wing imaginal disc. tkv is expressed at low levels in the center of the disc and at higher levels toward the edges of the disc. Although tkv levels are low in the center of the disc, clonal analysis has shown that tkv activity is stringently required in this region for growth and for target gene expression. Receptor levels are low where Dpp induces its targets Spalt and Omb in the wing pouch. Receptor levels increase in cells farther from the source of Dpp in the lateral regions of the disc (Lecuit, 1998).

Evidence is presented that Dpp signaling negatively regulates tkv expression and that the level of receptor influences the effective range of the Dpp gradient. High levels of tkv sensitize cells to low levels of Dpp and also appear to limit the movement of Dpp outside the wing pouch. Thus receptor levels help to shape the Dpp gradient. It was asked whether Dpp signaling regulates tkv expression by examining the effects of clones of cells expressing Dpp at lateral positions in the disc where the level of tkv is normally high. Dpp-expressing clones were marked indirectly by their ability to induce ectopic Spalt expression. tkv transcript levels are reduced where Spalt is misexpressed, suggesting that Dpp can act at a distance to repress tkv expression These results suggest that the reduced levels of tkv transcript in the center of the disc are due to downregulation by Dpp acting at a distance (Lecuit, 1998).

Are the reduced levels of the Tkv expression important for the formation of the Dpp activity gradient? To address this, an examination was made of the effects on the expression of the Dpp-target genes Spalt and Omb in clones of cells that overexpress wild-type Tkv. Tkv-expressing clones well inside the endogenous domains show little effect on either Spalt or Omb expression. Clones near the edge of the endogenous Spalt domain show increased Spalt expression and those near the edge of the Omb domain show elevated Omb expression. Tkv-expressing clones located outside but near the endogenous Spalt domain show ectopic induction of Spalt. Clones located farther from the Spalt domain do not show ectopic activation of Spalt. Together, these observations suggest that overexpressing Tkv can increase the sensitivity of cells to low levels of Dpp (Lecuit, 1998).

Tkv was overexpressed to assess the consequences of broadly elevating Tkv expression levels in the central region of the disc. Wings with elevated Tkv expression are reduced in size. The effect is stronger in the posterior compartment, with the region between veins 4 and 5 being more reduced than the region between veins 2 and 3. The region between veins 3 and 4 is relatively normal, possibly because the size of this intervein region is specified directly by Hedgehog, not by Dpp. These observations suggest that the long-range activity of Dpp in the vein 2-3 and 4-5 regions is compromised by overexpression of the Dpp receptor. Overexpressing Tkv strongly reduces the size of the Spalt domain in the Posterior (P) compartment. The effect on Spalt expression is much stronger in the P compartment than in the A compartment and the Spalt domains in both compartments appear to be less graded at their edges than in wild type. The effective range of the Dpp activity gradient appears to be limited to a few cells in the P compartment in mid- and late-third instar discs. This suggests that overexpression of receptor can limit the spread of Dpp in the P compartment. These observations suggest that high levels of the receptor might sequester ligand and limit its movement across the wing disc. The difference observe between A and P compartments when Tkv is overexpressed probably reflects the fact that cells originating in the Dpp expression domain can contribute to formation of a large part of the anterior compartment, but not to the posterior compartment. Thus cells originating in the Dpp domain could ëcarryí Dpp protein away from the source as they and their progeny are displaced by addition of new cells (the displacement process can be directly visualized by lineage tracing cells originating in the dpp-expression domain). It is concluded that artificially high levels of Thick veins outside the wing pouch appear to limit the spread of Dpp and thereby modulate the shape of the ligand gradient. In addition, the level of Tkv expression modulates the sensitivity of cells to Dpp. Thus regulation of receptor levels by Dpp modulates the shape of the Dpp gradient (Lecuit, 1998).

Shortsighted is in the DPP pathway in the eye imaginal disc. shortsighted is expressed in a hedgehog-dependent stripe in the undifferentiated cells just anterior to the morphological furrow in the eye imaginal disc. It appears to be involved in the transmission of the differentiation-inducing signal; a reduction in shortsighted function leads to a delay in differentiation and to a loss of photoreceptors in the adult. shortsighted is also required for a morphogenetic movement in the brain that reorients the second optic lobe relative to the first (Treisman, 1995).

The fact that Transforming growth factor beta at 60A mutations are dominant enhancers of a sensitized dpp pathway implicates Tgfbeta-60A in potentiating dpp signaling. This is most obvious in the visceral mesoderm of the midgut where dpp signaling is required to regulate homeotic gene expression and to maintain its own expression through a positive feedback mechanism. Although dpp signaling in the visceral mesoderm appears intact in Tgfbeta-60A mutants, a requirement for Tgfbeta-60A is revealed in tkv 6 Tgfbeta-60A double mutants. When dpp signaling is attenuated through a mutant tkv receptor, eliminating Tgfbeta-60A function reduces the signaling to below threshold level. The derepression of Sex combs reduced in the anterior midgut and the loss of expression of dpp target genes (wingless, Ultrabithorax and dpp) in the visceral mesoderm and labial in the endoderm are consistent with inadequate dpp signaling. A similar requirement for Tgfbeta-60A is observed during dorsal closure of the embryonic ectoderm (Y. Chen, 1998).

Tgfbeta-60A may form functional heterodimers with Decapentaplegic. In a signaling system with multiple interacting dimeric ligands, the interpretation of any single mutant phenotypes must consider the effect of losing both homomeric and possible heteromeric ligands. Therefore, the functions of the dpp pathway may be a composite input from Dpp homodimers, and Dpp/Scw and Dpp/Tgfbeta-60A heterodimers. Alternatively, Tgfbeta-60A homodimers may function in an additive fashion with Dpp homodimers at sites of overlapping expression. However, the loss-of-function phenotypes of dpp are as severe as the loss-of- function phenotypes of its downstream components, such as tkv or Mad, suggesting that there is very little signaling, if any at all, from Tgfbeta-60A homodimers in dpp-dependent events. Therefore, it is unlikely that Tgfbeta-60A homodimers play a significant role in dpp-dependent processes. Rather, it is thought that Dpp/Tgfbeta-60A heterodimers form at sites of overlapping expression and participate with Dpp homodimers in multiple signaling events. The broad distribution of Tgfbeta-60A proteins provides an opportunity for forming Dpp/Tgfbeta-60A heterodimers. Unlike scw null mutations, no obvious disruption of dpp signaling is observed in Tgfbeta-60A null mutants, suggesting that Dpp/Tgfbeta-60A heterodimers are not as limiting as Dpp/Scw heterodimers, but function in partially redundant manner with Dpp homodimers (Chen, 1998).

Dpp and Gbb exhibit different effective ranges in the establishment of the BMP activity gradient critical for Drosophila wing patterning

Morphogen gradients ensure the specification of different cell fates by dividing initially unpatterned cellular fields into distinct domains of gene expression. It is becoming clear that such gradients are not always simple concentration gradients of a single morphogen; however, the underlying mechanism of generating an activity gradient is poorly understood. This study indicates that the relative contributions of two BMP ligands, Gbb and Dpp, to patterning the wing imaginal disc along its A/P axis, change as a function of distance from the ligand source. Gbb acts over a long distance to establish BMP target gene boundaries and a variety of cell fates throughout the wing disc, while Dpp functions at a shorter range. On its own, Dpp is not sufficient to mediate the low-threshold responses at the end points of the activity gradient, a function that Gbb fulfills. Given that both ligands signal through the Tkv type I receptor to activate the same downstream effector, Mad, the difference in their effective ranges must reflect an inherent difference in the ligands themselves, influencing how they interact with other molecules. The existence of related ligands with different functional ranges may represent a conserved mechanism used in different species to generate robust long range activity gradients (Bangi, 2006a).

Dual function of the Drosophila Alk1/Alk2 ortholog Saxophone shapes the Bmp activity gradient in the wing imaginal disc

Wing patterning in Drosophila requires a Bmp activity gradient created by two Bmp ligands, Gbb and Dpp, and two Bmp type I receptors, Sax and Tkv. Gbb provides long-range signaling, while Dpp signals preferentially to cells near its source along the anteroposterior (AP) boundary of the wing disc. How each receptor contributes to the signaling activity of each ligand is not well understood. This study shows that while Tkv mediates signals from both Dpp and Gbb, Sax exhibits a novel function for a Bmp type I receptor: the ability to both promote and antagonize signaling. Given its high affinity for Gbb, this dual function of Sax impacts the function of Gbb in the Bmp activity gradient more profoundly than does Dpp. It is proposed that this dual function of Sax is dependent on its receptor partner. When complexed with Tkv, Sax facilitates Bmp signaling, but when alone, Sax fails to signal effectively and sequesters Gbb. Overall, this model proposes that the balance between antagonizing and promoting Bmp signaling varies across the wing pouch, modulating the level and effective range, and, thus, shaping the Bmp activity gradient. This previously unknown mechanism for modulating ligand availability and range raises important questions regarding the function of vertebrate Sax orthologs (Bangi, 2006b).

These data clarify the respective roles of Sax and Tkv in mediating Bmp signaling during wing patterning. This analysis shows that Tkv is responsible for mediating both Dpp and Gbb signals, and that Sax has a much more complex role in wing patterning than previously appreciated; Sax not only promotes signaling but also antagonizes signaling by limiting the availability of primarily the Gbb ligand. Both the antagonistic and signal promoting functions of Sax were revealed not only by gain-of-function studies but importantly, also by loss-of-function analyses. Loss of the antagonistic function of endogenous sax is evident: (1) as a broadening the pMad profile when the wing disc completely lacks sax function; and (2) as a non-autonomous increase in pMad levels in wild-type cells abutting the boundary of sax null clones. Loss of Sax-mediated signaling itself is evident: (1) in sax mutant discs as a reduction in the peak pMad levels along the AP boundary; and (2) in sax clones as a cell-autonomous reduction in pMad accumulation. Gain-of-function or overexpression studies indicate that the balance of Sax and Tkv levels in wing disc cells is crucial for proper signaling and, thus, wing patterning. Altogether, these results indicate that Sax is important in modulating Bmp signaling across the wing disc by both mediating and blocking Bmp signals, and, thus, shaping the Bmp activity gradient. How can the novel function of Sax as an antagonist be reconciled at the molecular level with the ability of Sax to promote signaling (Bangi, 2006b)?

Given that Tkv is required for all Bmp signaling in the wing disc, the simplest explanation for the fact that Sax signaling appears to depend on the presence of Tkv is that Sax can only promote signaling in a receptor complex also containing Tkv. Three different forms of Bmp receptor complexes can potentially form in wing disc cells, those composed of two type II receptor molecules and either two Tkv, two Sax or one molecule of each: Tkv-Tkv, Sax-Sax and Tkv-Sax. Overexpressing Tkv or Sax in wing disc cells enabled shifting of the balance between the relative levels of these two molecules, artificially enriching for the formation of receptor complexes homomeric for type I molecules Tkv-Tkv or Sax-Sax. Disrupting the balance of endogenous Tkv to Sax levels by overexpressing Sax immediately reveals the antagonistic function of Sax, consistent with the idea that excess Sax could be sequestering ligand in Sax-Sax receptor complexes which signal either very poorly or not at all. However, overexpression of Tkv, enriching for Tkv-Tkv complexes with high affinity for Dpp and lower affinity for Gbb, leads to increased signaling given sufficient ligand. The third receptor complex, Tkv-Sax, probably accounts for the contribution of Sax to the promotion of Bmp signaling and probably signals in vivo more efficiently than Tkv-Tkv, based on the fact that pMad levels are lower inside clones devoid of Sax than the pMad levels seen in cells at an equivalent position along the AP axis elsewhere on the disc. Loss of Tkv, by definition, eliminates signaling by both Tkv-Tkv and Tkv-Sax, leaving only Sax-Sax containing receptor complexes, which are clearly unable to elicit a pMad-mediated signal on their own. Thus, the model predicts that removing Sax function results in two opposing consequences: (1) a reduction in total Bmp signaling caused by loss of Tkv-Sax complexes, and (2) an increased availability of Bmp ligand and potential signaling caused by loss of Sax-Sax complexes. Several biochemical studies support the putative existence of functional Sax-Tkv receptor complexes. Heteromeric complexes involving different vertebrate type I receptors have been shown to contribute to a single signaling receptor complex and in Drosophila S2 cells both Sax and Tkv appear to be necessary to produce a synergistic signal (Bangi, 2006b).

It is important to note that increasing wild-type Tkv levels in the presence versus absence of excess ligand results in very different phenotypic outcomes. In contrast to Sax, increasing Tkv in the presence of excess ligand leads to a larger increase in Bmp signaling. However, at endogenous ligand levels, as Tkv levels are experimentally increased, a loss of Bmp signaling is seen that is indicative of the preference of Tkv for binding Dpp over Gbb. Clearly, both Gbb and Dpp become limiting in the presence of excess Tkv, with low level Tkv overexpression preferentially limiting Dpp-dependent signaling, while higher levels of overexpression limit both. Clearly, although overexpression of ligand and receptor together reveals a significant difference in the signaling ability of Tkv and Sax, overexpression of receptor alone in the absence of increased ligand appears to reflect only receptor ligand-binding preference (Bangi, 2006b).

Such experimental manipulations of Tkv levels can lead to the loss of Bmp signaling by limiting the range of Bmp signaling, but unlike sax, loss of endogenous tkv function never leads to an increase in Bmp signaling. Furthermore, there is no indication that Tkv is required for or involved in the antagonistic function of Sax. At endogenous levels, Sax-Sax complexes, unlike Tkv-Tkv or Tkv-Sax complexes, appear to modulate the range of Bmp signaling by sequestering ligand without any associated signaling, and, thus, Sax identifies a new previously unrecognized Bmp modulator whose signaling ability appears to depend on which receptor it partners (Bangi, 2006b).

The fact that both Dpp and Gbb are dependent on Tkv for signaling has significant implications regarding the Bmp activity gradient, given that removal of Tkv at any point along the gradient results in the loss of both Gbb and Dpp signaling, not just Dpp signaling. When both ligands are present at similar levels, the higher affinity of Dpp for Tkv means the contribution of Dpp to total Bmp signaling will be more significant than that of Gbb, and movement of Dpp across the wing disc will be affected more strongly by Tkv than that of Gbb. Thus, Gbb should and does contribute more significantly to the low points of the Bmp activity gradient, especially since competition with Dpp for binding to Tkv will also be lower in these regions (Bangi, 2006b).

These findings from receptor and ligand overexpresion experiments suggest that both the antagonistic and signal promoting functions of Sax impact Gbb signaling most significantly because of their preferential interaction. For example, although localized loss of Sax from the peripheral cells of the wing pouch leads to ectopic induction of brk, loss in more central cells does not, suggesting that the relative contribution of Sax to overall Bmp signaling is less in the central cells where Tkv must contribute more significantly given the higher level of Dpp near the AP boundary. The greater contribution of Sax to total signaling in the more peripheral cells of the wing pouch is consistent with its higher affinity for Gbb and the long-range nature of Gbb versus Dpp (Bangi, 2006b). Similarly, removal of Sax from just anterior compartment cells results in brk repression in both the anterior and posterior compartments suggesting that in the absence of Sax, anteriorly expressed Gbb can signal to the posterior-most cells of the wing pouch to effectively repress brk expression beyond its normal domain. This result indicates that endogenous Sax normally functions to not only restrict the level of Gbb signaling but also the range of Gbb. The role that Sax plays in promoting Gbb function, in particular, is detected only when sax function is completely eliminated and gbb function is also significantly compromised (Bangi, 2006b).

Given that Tkv is also required for mediating Gbb signals, of the two proposed receptor complexes that could mediate Gbb signaling (Tkv-Tkv and Tkv-Sax), which is preferentially used by Gbb in wild-type cells? It is clear that Tkv-Sax complexes are not obligatory for Gbb signaling since Gbb signaling is not abolished in sax mutants. The fact that removing Sax does not cause a gbb loss-of-function phenotype indicates that enough Gbb is made available by the loss of Sax antagonism and can signal to compensate for losing that region of total signaling that Sax normally promotes. The fact that pMad levels within a sax clone are lower then endogenous levels indicates that signaling in the clone cells containing only Tkv-Tkv is less efficient than the neighboring cells that have wild-type levels of both Sax and Tkv (Bangi, 2006b).

A synergy has been observed between co-expressed constitutively active (CA) Tkv and Sax in the early embryo and between Tkv and Sax in S2 cells in response to Dpp-Scw heterodimers, since only Dpp homodimers are able to signal efficiently in the absence of Sax. A likely, albeit minimal, contribution of Dpp-Gbb heterodimers to long-range wing patterning has been detected (Bangi, 2006a) making it is possible that Tkv-Sax complexes could respond to Dpp-Gbb heterodimers and such complexes could be particularly efficient at signaling. Given the dual function of Sax, the relative levels of Sax to Tkv are likely to be crucial for establishing a synergistic interaction. The ability of Tkv-Sax containing complexes to mediate ligand homodimers has not yet been determined in vivo and it is also not yet completely clear if the antagonism by Sax can affect heterodimers as well as homodimers. The current data indicate that the ability of Sax to promote signaling must reside with Tkv-Sax-containing complexes and the strong contribution of Gbb to the low points of the gradient with a minimal contribution by Dpp leaves open the possibility that Dpp-Gbb can signal, in addition to Gbb-Gbb, to cells far from the AP boundary (Bangi, 2006b).

Overexpression studies in the follicle cells of the Drosophila ovary produce the same results as those in the wing, indicating that the ability of Sax to block Gbb signaling is not limited to the developing wing. However, in contrast to studies in the wing disc, loss of sax from the follicle cells, as well as the embryonic midgut and neuromuscular synapse produces mutant phenotypes indicative of a loss of ligand function. It is possible that the contribution of Sax to signal promotion in these tissues may be stronger than its antagonistic function. The phenotypic outcome of sax loss of function in a particular process probably depends on the relative numbers of Sax-Sax and Sax-Tkv complexes on the cell surface and the relative binding affinity of a given Bmp ligand for these two complexes. What regulates the composition of type I receptors in a signaling complex is not yet known (Bangi, 2006b).

The ability of the Sax to block Bmp signaling may reflect its requirement to have input from another molecule to activate its kinase domain. When activated by in vitro mutagenesis, Sax and its vertebrate orthologs Alk1/Alk2 (Acvrl1 and Acvr1 - Mouse Genome Informatics) are able to phosphorylate Bmp specific R-Smads, but ligand-induced activation of Sax or Alk1/2 kinase has not been reported. Interestingly, a ligand-induced Bmp receptor complex containing Alk2 and ActRII is unable to phosphorylate Smad1. Furthermore, Alk1 has been shown to require a different type I receptor (Alk5) to activate its kinase domain. Although it has been suggest that the Alk2/ActRII complex might be unstable in vitro, it is also possible that activation of Alk2 (and of its Drosophila ortholog Sax) may depend on its partner type I receptor and/or which ligand is bound, or some other protein. Although Gbb fails to activate Sax-Sax, perhaps another Bmp ligand (i.e. Scw) can. Similarly, endoglin, related to the co-receptor betaglycan, could be important in modulating Alk1-dependent signaling given that mutations in either gene give rise to hereditary hemorrhagic telangiectasia. Sax may require a different type I receptor partner, i.e. Tkv, to activate its kinase or transduce a signal, and such a requirement may be a universal feature of the Alk1/Alk2/Sax subgroup of Bmp type I receptors (Bangi, 2006b).

The robustness of morphogen gradients may depend on negative-feedback mechanisms to buffer against environmental and genetic fluctuations. Clearly, Sax plays a crucial role in modulating the range of the Bmp activity gradient from analysis at both the level of Bmp-dependent target gene expression and the final pattern of the adult wing. The identification of the antagonistic nature of a Bmp type I receptor to modulate signaling activity by sequestering ligand without transducing a signal provides a new mechanism that contributes to the robustness of the Bmp activity gradient. It is proposed that the dual function of Sax is crucial for buffering the wing disc Bmp activity gradient against local fluctuations in ligand levels (environmental, genetic or experimentally induced). Whether this mechanism of signal modulation is evolutionarily conserved remains to be determined, but the fact that the vertebrate Sax orthologs Alk1 and Alk2 have been shown biochemically to exhibit antagonistic behaviors in vitro is interesting. Detailed analysis of these orthologs in developmental contexts will be crucial to determine whether the robustness of vertebrate Bmp activity gradients also depends on the modulation of ligand availability by specific receptors (Bangi, 2006b).

Processing of the Drosophila Sog protein creates a novel BMP inhibitory activity

Structurally unrelated neural inducers in vertebrate and invertebrate embryos have been proposed to function by binding to BMP4 or Dpp, respectively, and preventing these homologous signals from activating their receptor(s). The functions of various forms of the Drosophila Sog protein were examined using the discriminating assay of Drosophila wing development. Misexpression of Drosophila Sog, or its vertebrate counterpart Chordin, generates a very limited vein-loss phenotype. This sog misexpression phenotype is very similar to that of viable mutants of glass-bottom boat (gbb), which encodes a BMP family member. Consistent with Sog selectively interfering with Gbb signaling, Sog can block the effect of misexpressing Gbb, but not Dpp in the wing. In contrast to the limited BMP inhibitory activity of Sog, carboxy-truncated forms of Sog, referred to as Supersog, have been identified which when misexpressed cause a broad range of dpp minus mutant phenotypes (Yu, 2000).

The predicted Sog protein is 1038 amino acids in length and contains four cysteine-rich (CR) domains in the extracellular domain. The metalloprotease Tld cleaves Sog at three major sites. Supersog1 is an N-terminal fragment of Sog including CR1 plus another 114 amino acids, and contains an additional 33 amino acids derived from vector sequences at its C terminus. Supersog2, which contains the same amino acids as Supersog1 but terminates abruptly at the end of Sog sequences, also generates Supersog phenotypes, albeit slightly weaker than those observed with Supersog1. Supersog4 is an N-terminal fragment of Sog ending 80 amino acids before CR2 and includes 130 sog 3' UTR derived amino acids (Yu, 2000).

In line with its phenotypic effects, Supersog can block the effects of both misexpressing Dpp and Gbb in the wing. Vertebrate Noggin, in contrast, acts as a general inhibitor of Dpp signaling, which can interfere with the effect of overexpressing Dpp, but not Gbb. Evidence suggests that Sog processing occurs in vivo and is biologically relevant. Overexpression of intact Sog in embryos and adult wing primordia leads to the developmentally regulated processing of Sog. This in vivo processing of Sog can be duplicated in vitro by treating Sog with a combination of the metalloprotease Tolloid (Tld) plus Twisted Gastrulation (Tsg), another extracellular factor involved in Dpp signaling. In accord with this result, coexpression of intact Sog and Tsg in developing wings generates a phenotype very similar to that of Supersog. Evidence is provided that tsg functions in the embryo to generate a Supersog-like activity, since Supersog can partially rescue tsg minus mutants. Consistent with this finding, sog minus and tsg minus mutants exhibit similar dorsal patterning defects during early gastrulation. These results indicate that differential processing of Sog generates a novel BMP inhibitory activity during development and, more generally, that BMP antagonists play distinct roles in regulating the quality as well as the magnitude of BMP signaling (Yu, 2000).

The fact that pulses of Supersog1 expression delivered during the late blastoderm stage of development can partially rescue the tsg minus mutant embryos suggests that a Supersog-like activity might mediate part of tsg function in vivo. In addition, late blastoderm stage tsg minus mutant embryos display defects similar to those of sog mutants, suggesting that tsg is involved in a late function of Sog. Consistent with the view that tsg acts during early gastrulation, tsg minus mutants can not be rescued by driving expression of a tsg transgene under the control of the tld promoter, which is expressed only early during the blastoderm stage. In contrast, it is possible to rescue tsg minus mutants by driving tsg expression with promoters that continue to be expressed into early gastrulation. Several possible ways in which Supersog-like activities could contribute to this stage of development can be imagined, given that they have different ligand specificities from intact Sog and are stable to further proteolysis by Tld. Since Sog has been proposed to block the activity of Scw in embryos, it is likely that some other BMP is the preferred target of Supersog molecules. In addition, since Scw is only expressed transiently during the blastoderm stage of development, intact Sog would have no obvious target to inhibit beyond this stage. Perhaps a stable broad-spectrum BMP antagonist such as Supersog could inhibit the action of other BMPs expressed in the dorsal ectoderm during early stages of gastrulation (possibly Dpp itself) and thereby provide a form of molecular memory, which helps maintain the distinction between neural and non-neural ectoderm (Yu, 2000).

The observation that Supersog is less effective than Sog in blocking BMP signaling in the early embryo is consistent with the view that Supersog is not just a higher affinity version of Sog and suggests that Supersog is actually less effective than Sog at blocking the effect of Scw. The fact that Supersog does not inhibit Dpp itself during early blastoderm stages is likely to be the result of insufficient levels of Supersog being expressed by the heat shock vector. It is possible, however, that an endogenously produced Supersog activity (e.g. generated upon Tsg binding to Sog) has a higher affinity for Dpp than the artificially created Supersog1 construct. In any case, it is proposed that Supersog acts in the late blastoderm embryo or during early gastrulation stages rather than in the early blastoderm embryo, and that during this latter period, it is able to block the activity of a BMP (e.g. Dpp?) not recognized by Sog. It is tempting to consider a two step temporal model for the action of Sog and Supersog during embryonic dorsal-ventral patterning to account for the fact that sog mutants display a dorsal-ventral phenotype earlier than tsg minus mutants. According to one such scenario, the labile Tld-sensitive form of full-length Sog is produced from a localized source (i.e. the neuroectoderm) and diffuses dorsally to be degraded by Tld. Tld acts as a sink to create a transiently stable gradient of Sog, which creates a reciprocal gradient of Dpp activity. The Sog gradient created by this classic source/sink configuration would only be short-lived, however, since cells begin migrating when gastrulation begins. At this stage, the embryo elongates and the Dorsal gradient collapses, leading to loss of gene expression in early zygotic D/V domains. Following the establishment of the short-lived hypothetical Sog gradient, tsg expression is initiated in dorsal cells and leads to the production of stable Supersog-like molecules by switching the activity of Tld from degrading to activating Sog. Supersog-like molecules then could provide a stable record of high versus low BMP signaling domains during a subsequent step of development (Yu, 2000).

Robustness of the BMP morphogen gradient in Drosophila embryonic patterning

Developmental patterning relies on morphogen gradients, which generally involve feedback loops to buffer against perturbations caused by fluctuations in gene dosage and expression. Although many gene components involved in such feedback loops have been identified, how they work together to generate a robust pattern remains unclear. The network of extracellular proteins that patterns the dorsal region of the Drosophila embryo by establishing a graded activation of the bone morphogenic protein (BMP) pathway has been studied. The BMP activation gradient itself is robust to changes in gene dosage. Computational search for networks that support robustness shows that transport of the BMP class ligands (Scw and Dpp) into the dorsal midline by the BMP inhibitor Sog is the key event in this patterning process. The mechanism underlying robustness relies on the ability to store an excess of signaling molecules in a restricted spatial domain where Sog is largely absent. It requires extensive diffusion of the BMP-Sog complexes, coupled with restricted diffusion of the free ligands. Dpp is shown experimentally to be widely diffusible in the presence of Sog but tightly localized in its absence, thus validating a central prediction of a theoretical study (Eldar, 2002).

Graded activation of the BMP pathway subdivides the dorsal region of Drosophila embryos into several distinct domains of gene expression. This graded activation is determined by a well-characterized network of extracellular proteins, which may diffuse in the perivitelline fluid that surrounds the embryo. The patterning network is composed of two BMP class ligands (Scw and Dpp), a BMP inhibitor (Sog), a protease that cleaves Sog (Tld) and an accessory protein (Tsg), all of which are highly conserved in evolution and are used also for patterning the dorso-ventral axis of vertebrate embryos. Previous studies have suggested that patterning of the dorsal region is robust to changes in the concentrations of most of the crucial network components. For example, embryos that contain only one functional allele of scw, sog, tld or tsg are viable and do not show any apparent phenotype. Misexpression of scw or of tsg also renders the corresponding null mutants viable (Eldar, 2002).

To check whether robustness is achieved at the initial activation gradient, signaling was monitored directly by using antibodies that recognize specifically an activated, phosphorylated intermediate of the BMP pathway (pMad). Prominent graded activation in the dorsal-most eight cell rows was observed for about 1h, starting roughly 2h after fertilization at 25°C. This activation gradient was quantified in heterozygous mutants that were compromised for one of three of the crucial components of the patterning network, Scw, Sog or Tld. Whereas homozygous null mutants that completely lack the normal gene product have a deleterious effect on signaling, the heterozygotes, which should produce half the amount of the gene product, were indistinguishable from wild type. Similarly, overexpression of the Tld protein uniformly in the embryo did not alter the activation profile. The activation profile at 18°C is the same as that at 25°C. This robustness to temperature variations is marked, considering the wide array of temperature dependencies that are observed in this temperature span. By contrast, the profile of pMad is sensitive to the concentration of Dpp. The dosage sensitivity of Dpp is exceptional among morphogens and is singled out as being haploid-insufficient (Eldar, 2002).

No apparent transcriptional feedback, which might account for the robustness of dorsal patterning, has been identified so far. Robustness should thus be reflected in the design of interactions in the patterning network. To identify the mechanism underlying robustness, a general mathematical model of the dorsal patterning network was formulated. For simplicity, initial analysis was restricted to a single BMP class ligand (Scw or Dpp), a BMP inhibitor (Sog) and the protease (Tld). The general model accounted for the formation of the BMP-Sog complex, allowed for the diffusion of Sog, BMP and BMP-Sog, and allowed for the cleavage of Sog by Tld, both when Sog is free and when Sog is associated with BMP. Each reaction was characterized by a different rate constant (Eldar, 2002).

Extensive simulations were carried out to identify robust networks. At each simulation, a set of parameters (rate constants and protein concentrations) was chosen at random and the steady-state activation profile was calculated by solving three equations numerically. A set of three perturbed networks representing heterozygous situations was then generated by reducing the gene dosages of sog, tld or the BMP class ligand by a factor of two. The steady-state activation profiles defined by those networks were solved numerically and compared with the initial, nonperturbed network. A threshold was defined as a given BMP value (corresponding to the value at a third of the dorsal ectoderm in the nonperturbed network). The extent of network robustness was quantified by measuring the shift in the threshold for all three perturbed networks. Over 66,000 simulations were carried out, with each of the nine parameters allowed to vary over four orders of magnitude (Eldar, 2002).

As expected, in most cases (97.5%) the threshold position in the perturbed networks was shifted by a large extent (>50%). In most of those nonrobust cases, the BMP concentration was roughly uniform throughout the dorsal region. By contrast, Sog was distributed in a concentration gradient with its minimum in the dorsal midline, defining a reciprocal gradient of BMP activation. Thus, the key event in this nonrobust patterning mechanism is the establishment of a concentration gradient of Sog, which was governed by diffusion of Sog from its domain of expression outside the dorsal region, coupled with its cleavage by Tld inside the dorsal region. Although such a gradient has been observed, it is also compatible with other models (Eldar, 2002).

A small class of networks (198 networks, 0.3%) was identified in which a twofold reduction in the amounts of all three genes resulted in a change of less than 10% in the threshold position. Notably, in all of these robust cases, BMP was redistributed in a sharp concentration gradient that peaked in the dorsal midline. In addition, this concentration gradient decreases as a power-low distribution with an exponent n = 2, which indicates the uniqueness of the robust solution. In these cases, Sog was also distributed in a graded manner in the dorsal region. Analysis of the reaction rate constants of the robust networks showed a wide range of possibilities for most parameters. But two restrictions were apparent and defined the robust network design: (1) in the robust networks the cleavage of Sog by Tld was facilitated by the formation of the complex Sog-BMP; (2) the complex BMP-Sog was broadly diffusible, whereas free BMP was restricted (Eldar, 2002).

To identify how robustness is achieved, an idealized network was considered by assuming that free Sog is not cleaved and that free BMP does not diffuse. The steady-state activation profile defined by this network can be solved analytically; the solution reveals two aspects that are crucial for ensuring robustness. First, the BMP-Sog complex has a central role, by coupling the two processes that establish the activation gradient: BMP diffusion and Sog degradation. This coupling leads to a quantitative buffering of perturbations in gene dosage. Second, restricted diffusion of free BMP enables the system to store excess BMP in a confined spatial domain where Sog is largely absent. Changes in the concentration of BMP alter the BMP profile close to the dorsal midline but do not change its distribution in most of the dorsal region (Eldar, 2002).

The complete system, comprising Sog, Tld, Tsg, both Scw and Dpp, and their associated receptors was examined next. Two additional molecular assumptions are required to ensure the robustness of patterning. First, Sog can bind and capture the BMP class ligands even when the latter are associated with their receptors. Second, Dpp can bind Sog only when the latter is bound to Tsg. Indeed, it has been shown that, whereas Sog is sufficient for inhibiting Scw, both Tsg and Sog are required for inhibiting Dpp. This last assumption implies that Tsg functions to decouple the formation of the Scw gradient from the parallel generation of the Dpp gradient, ensuring that Scw and Dpp are transported to the dorsal midline independently by two distinct molecular entities (Eldar, 2002).

The complete model was solved numerically for different choices of rate constants. In particular, the effect of twofold changes in gene dosage was assessed. The steady-state activation profiles can be superimposed, indicating the robustness of the system. In addition, with the exception of Dpp, the expression of all other crucial network components can be altered by at least an order of magnitude before an effect on the position of a given threshold is observed. In the model, the lack of robustness to Dpp stems from its insufficient dosage. Note that the time taken to reach steady state is sensitive to these concentrations of protein. For the wide range of parameters that were used, however, the adjustment time does not exceed the patterning time. Flexible adjustment time thus facilitates the buffering of quantitative perturbations (Eldar, 2002).

This analysis has identified two principle molecular features that are essential for robust network design: first, free Sog is not cleaved efficiently -- an assumption that is supported by the in vitro finding that Sog cleavage by Tld requires BMP; second, the diffusion of free BMP is restricted. This is the central prediction of the theoretical study, namely, that Scw diffusion requires Sog, whereas Dpp diffusion requires both Sog and Tsg. Although several reports suggest that in wild-type embryos both Dpp and Scw are widely diffusible, their ability to diffuse in a sog or tsg mutant background has not been examined as yet (Eldar, 2002).

To monitor the diffusion of Scw or Dpp, the even-skipped (eve) stripe-2 enhancer (st2) was used to misexpress Dpp or Scw in a narrow stripe perpendicular to the normal BMP gradient. In transgenic embryos, dpp or scw RNA was detected in a stripe just posterior to the cephalic furrow. Initially the stripe was about 12 cells wide at early cleavage cycle 14, but refined rapidly to about 6 cells by late cycle 14. The st2-dpp and st2-scw embryos were viable, despite the high expression of these proteins as compared with their endogenous counterparts (Eldar, 2002).

The activation of the BMP pathway was monitored either by staining for pMad or by following dorsal expression of the target gene race, which requires high activation. Scw is a less potent ligand than is Dpp. This experimental setup could not be used to study Scw diffusion properties because expressing st2-scw did not alter the pattern of pMad or race expression in wild-type or sog^-/- embryos. By contrast, expression of st2-dpp led to an expansion of both markers in a region that extends far from the st2 expression domain, indicating a wide diffusion of Dpp in a wild-type background. Conversely, on expression of st2-dpp in sog^-/- or in tsg^-/- embryos, both markers were confined to a narrow stripe in the st2 domain. The width of this stripe was comparable to that of st2-dpp expression, ranging from 6 to 12 cells, indicating that Dpp does not diffuse from its domain of expression in the absence of Sog or Tsg. Taken together, these results show that both Sog and Tsg are required for Dpp diffusion, as predicted by the theoretical analysis (Eldar, 2002).

The computation ability of biochemical networks is striking when one considers that they function in a biological environment where the amounts of the network components fluctuate, the kinetics is stochastic, and sensitive interactions between different computation modules are required. Studies have examined the effect of these properties on cellular computation mechanisms, and robustness has been proposed to be a 'design principle' of biochemical networks. The applicability of this principle to morphogen gradient patterning has been shown during early development. Quantitative analysis can be used to assess rigorously the robustness of different patterning models and to exclude incompatible ones. The remaining, most plausible model points to crucial biological assumptions and serves to postulate the central feedback mechanisms. Applying the same modelling principles to other systems might identify additional 'design principles' that underlie robust patterning by morphogen gradients in development (Eldar, 2002).

Physical properties of Tld, Sog, Tsg and Dpp protein interactions are predicted to help create a sharp boundary in Bmp signals during dorsoventral patterning of the Drosophila embryo

Dorsal cell fate in Drosophila embryos is specified by an activity gradient of Decapentaplegic. Genetic and biochemical studies have revealed that the Sog, Tsg and Tld proteins modify Dpp activity at the post-transcriptional level. The predominant view is that Sog and Tsg form a strong ternary complex with Dpp that prevents it from binding to its cognate receptors in lateral regions of the embryo, while in the dorsalmost cells Tld is proposed to process Sog and thereby liberate Dpp for signaling. In this model, it is not readily apparent how Tld activity is restricted to the dorsal-most cells, since it is expressed throughout the entire dorsal domain. In this study, additional genetic and biochemical assays were developed to further probe the relationships between the Sog, Tsg, Tld and Dpp proteins. Using cell based assays, it has been found that the dynamic range over which Dpp functions for signaling is the same range in which Dpp stimulates the cleavage of Sog by Tld. In addition, the data support a role for Tsg in sensitizing the patterning mechanism to low levels of Dpp. It is proposed that the strong Dpp concentration dependence exhibited by the processing reaction, together with movement of Dpp by Sog and Tsg protein can help explain how Tld activity is confined to the dorsal-most region of the embryo through formation of a spatially dependent positive and negative reinforcement loop. Such a mechanism also explains how a sharp rather than smooth signaling boundary is formed (Shimmi, 2003).

According to the prevailing view, Sog, Tsg and Tld act to create a transport mechanism that helps promote Dpp diffusion from lateral regions of the embryos towards the dorsal side. According to this model, Sog would diffuse into the dorsal domain from its ventral lateral site of synthesis and capture Dpp, thereby preventing Dpp from binding to receptor. Net flux of Sog towards the dorsal side is envisioned to help transport Dpp and thereby increase its concentration in the dorsalmost tissue, which is destined to become the amnioserosa. Tld acts to liberate Dpp by cleaving Sog, and Dpp once released, will either be recaptured by another Sog molecule or bound to its receptors (Shimmi, 2003).

In order for the transport model to produce a Dpp concentration peak, the proper balance between binding affinities, diffusion rates and proteolytic processing is needed. Tsg has been suggested to have several activities that could influence this balance. In one model, Tsg would act to slow down the intrinsic rate of Sog cleavage by Tld. In this case, loss of Tsg is predicted to result in elevated processing of Sog. This should produce a sog loss-of-function phenotype, as is observed when molecular markers are examined. That data argues strongly against this possibility. First, it has been demonstrated that Tsg function is epistatic to Tld. If the tsg mutant phenotype is caused by excess Tld activity, then eliminating Tld should produce a tld loss-of-function phenotype. However, a tsg-like phenotype is observed where there is a general lowering and flattening of the Dpp activity gradient, as assayed by marker gene expression. In addition, biochemical studies reveal that Tsg actually enhances the ability of Tld to cleave Sog. Taken together, it is concluded that Tsg does not function during DV patterning to retard Tld proteolytic activity (Shimmi, 2003).

A second property has been attributed to Tsg: it alters the selection of Tld cleavage sites in Sog thereby producing novel Sog fragments with unique properties. In particular, a Sog fragment termed Supersog containing the first CR domain and a region of the spacer between CR1 and CR2 appears to be produced in vitro by the action of Tsg and Tld. Although the production of Supersog-like fragments are seen under the present reaction conditions described in this study, no enhancement in their production is seen upon Tsg addition. This may reflect loss of an unidentified component during purification or differences in the sensitivities of the CR1 antibodies used in the two studies. These issues are presently under examination. Whether Supersog-type molecules contribute to DV patterning in vivo is unclear. The fact that overexpression of Supersog can partially rescue tsg mutant embryos suggests that they could be important. A full resolution of the role of Supersog will need to await the results of in vivo rescue experiments employing mutants of the different Sog cleavage sites, especially those that lead to the production of Supersog-like fragments (Shimmi, 2003).

One of the primary findings in this report is that the rate of Sog cleavage is very sensitive to the level of the Dpp protein and varies substantially over a 10-fold range. Interestingly, this is the same Dpp concentration range within which low to maximal signaling occurs in S2 cell culture. Tsg sensitizes the system such that both the binding of Dpp to Sog as well as the rate of cleavage of Sog by Tld is stimulated by Tsg protein. Because in the invertebrate system, the binding of ligand to Sog is required for efficient processing of Sog, it is not surprising that the rate of Sog processing goes up in the presence of Tsg. This follows because, at a given concentration of Sog and Dpp, more complex will be formed in the presence of Tsg leading to a higher substrate concentration for the Tld protease. It is speculated that this system evolved in part to enable the embryo to produce a patterning mechanism that functions within the context of a very short developmental window. In Drosophila, the time between initial transcription of dpp during the early blastoderm stage and assignment of fate required for proper gastrulation is only about 40 minutes. In this short time-window, Dpp concentration must reach an effective signaling level. However, using a genomic Dpp-HA construct, it has been possible to visualize Dpp in the early embryo and it is present at much lower levels than in other tissues, such as the epidermis, at later stages of embryogenesis. It is proposed that under these conditions of low Dpp concentration, the presence of Tsg is required to enable Sog to bind to Dpp and to stimulate Sog cleavage in order to create a cyclic binding and release process that enables Dpp to be carried towards the dorsal midline. Furthermore, it is proposed that the intrinsic sensitivity of the cleavage reaction to the Dpp concentration is crucial for formation of a sharp signaling boundary. Thus, as the Dpp concentration drops in the lateral regions as a consequence of Dpp movement towards the dorsal side, the rate of Sog cleavage drops, allowing more Sog to enter this region and further reducing signaling in lateral regions. The movement of Dpp will simultaneously raise Dpp concentration in the dorsal region, further stimulating cleavage and clearance of Sog and thereby reinforcing Dpp signaling at the dorsal midline. This built-in positive and negative reinforcement mechanism should help establish sharp signaling boundaries by formation of steep ligand gradients, instead of the more gradual gradients that would form if Sog cleavage was not sensitive to the Dpp concentration (Shimmi, 2003).

In some vertebrate systems, DV patterning mechanisms have been conserved with respect to the molecules employed, but the polarity of axis over which they act has been inverted. Thus, in both amphibians and zebrafish, Bmp ligands specify ventral cell fates, whereas Bmp inhibitors, such as Chordin, are secreted from dorsal cells. In each of these systems, Tsg- and Tld-like proteins also contribute to axis formation, but the biochemical details of their associations appear different from those found in Drosophila. Two distinctions are most apparent and these probably have biological significance with respect to the patterning mechanism employed by these organisms. In Xenopus, the affinity of chordin for Bmps is significantly higher than Sog for Dpp; Bmps can be coimmunoprecipitated by chordin alone whereas this is not the case for the Drosophila components. In addition, once cleaved by Xolloid, at least some of the CR1 containing fragments of chordin continue to have significant affinity for the Bmp ligand preventing it from signaling (Shimmi, 2003 and references therein).

The second major difference between the Drosophila and Xenopus systems is that the Drosophila processing of Sog is dependant on prior binding of Sog to Dpp, while in Xenopus this is not the case. Rather, Chordin cleavage by Xolloid appears to be constitutive and is not enhanced by any tested ligand. Without ligand dependent cleavage, net movement of Bmps by Chordin diffusion may not readily occur nor would there be a mechanism to both positively and negatively reinforce the processing reaction. Indeed, recent studies have demonstrated that in the Drosophila embryo, Chordin does not have the ability to promote Dpp signaling at a distance, whereas Sog does. As a result, spatially enhanced Bmp concentrations and sharp signaling boundaries that result from net ligand movement by the activities of the Chordin, Xolloid and Tsg proteins may not occur in Xenopus. In fact there is no evidence in Xenopus that loss of Chordin activity actually results in a reduction in Bmp signaling in select regions of the embryo as occurs in Drosophila (Shimmi, 2003).

Despite these differences, Tsg may, nevertheless, play both positive and negative roles in modulating Bmp signaling; however, its mechanism is somewhat different. As processed fragments of Chordin still have reasonable affinity for ligand, they may need to be dislodged to allow for signaling. Tsg binding to Bmps appears to help promote this dislodgment and their ultimate degradation. In Drosophila, since Sog binds poorly to ligand in the absence of Tsg there is no need for Tsg to help promote dissociation of Sog fragments. Rather, it is its ability to help promote association of Sog with Dpp that is key to understanding its function. Tsg appears also to alter the rate of chordin proteolysis. Thus, at a high Tsg-to-chordin ratio, Chordin may be degraded and in this way Tsg might help promote signaling. It is possible that some combination of these properties is used in other vertebrates. For example, in zebrafish it has recently been shown that loss of chordin can enhance a phenotype that results from haplo-insufficiency for swirl, a gene that encodes Bmp2b. This paradoxical observation, that loss of an inhibitor exacerbates a phenotype resulting from loss of a ligand, is exactly analogous to the case of amnioserosa development in Drosophila where loss of Sog (an inhibitor) leads to less Dpp signaling in the dorsal domain. Detailed studies examining the ligand dependence of Chordin cleavage in zebrafish by minifin, the gene encoding a Tld homolog, have not been reported. It is possible therefore, that like Drosophila, this system may also employ a transport mechanism involving Tsg, Chordin and Tld that acts to boost Bmp signaling in specific tissues. It is interesting to note that the mouse homologs of Tsg, Chordin and Tld also exhibit their own distinct biochemical properties. Thus, a new Tld processing site in Chordin is induced by the presence of Tsg but this is not seen when the Xenopus components are used. Thus, it seems probable that the inherent complexity of this multi-component regulatory mechanism has provided numerous targets for evolutionary change. It is speculated that these changes account for the remarkable diversity that this mechanism exhibits with respect to the actual details by which it regulates Bmp signaling in different organisms (Shimmi, 2003).

Dally interaction with Decapentaplegic

Regulating the level of Dpp signaling is critical to its function during development. One type of molecule proposed to modulate growth factor signaling at the cell surface is an integral membrane proteoglycan. division abnormally delayed (dally), a Drosophila member of the glypican family of integral membrane proteoglycans is required for normal Dpp signaling during development, affecting cellular responses to this morphogen. Dally is required for the control of cell division in the developing visual system, the morphogenesis of the eye, wing, antenna and genitalia. Ectopic expression of dally+ can alter the patterning activity of Dpp, suggesting a role for dally+ in modulating Dpp signaling strength. Expression of the Dpp target gene, optomotor blind, is reduced in dally mutants. dally phenotypes are rescued by increasing the dosage of dpp+ and dally mutants suppress phenotypes resulting from ectopic expression of Dpp in the wing disc. Additionally, ectopic dally expression potentiates the patterning activity of ectopic DPP. These findings support a role for members of the glypican family in controlling TGF-beta/BMP activity in vivo by affecting signaling at the cell surface (Jackson, 1997).

Dpp functions as a morphogen to specify cell fate along the anteroposterior axis of the wing. Dpp is a heparin-binding protein and Dpp signal transduction is potentiated by Dally, a cell-surface heparan sulfate proteoglycan, during assembly of several adult tissues. However, the molecular mechanism by which the Dpp morphogen gradient is established and maintained is poorly understood. Evidence is shown that Dally regulates both cellular responses to Dpp and the distribution of Dpp morphogen in tissues. In the developing wing, dally expression in the wing disc is controlled by the same molecular pathways that regulate expression of thickveins, which encodes a Dpp type I receptor. Elevated levels of Dally increase the sensitivity of cells to Dpp in a cell autonomous fashion. In addition, dally affects the shape of the Dpp ligand gradient as well as its activity gradient. It is proposed that Dally serves as a co-receptor for Dpp and contributes to shaping the Dpp morphogen gradient (Fujise, 2003).

To examine the effect of dally mutations on the distribution of Dpp morphogen, Dpp-GFP was expressed in the region where it is endogenously expressed using dpp-GAL4. In wild-type discs, Dpp-GFP is detectable as intracellular punctate spots and on the surface of the receiving cells. Dpp-GFP migrates throughout the wing pouch region, forming a shallow but evident gradient. However, in dally-mutant discs, no evident gradient of Dpp distribution could be detected in the receiving cells. In general, mutant discs showed a lower level of cell surface signals, suggesting reduced stability of Dpp (Fujise, 2003).

To determine whether dally overexpression at the A/P border cells, which causes abnormal patterns of pMad, also affects Dpp ligand gradient formation, Dpp-GFP distribution was observed in discs where dally is co-expressed with Dpp-GFP using dpp-GAL4. Consistent with the pMad patterns, Dpp is restricted to the dally-overexpressing region and fails to migrate properly. This suggests that Dally binds to Dpp protein and limits its distribution. Intensity profiles of these discs show that both reduction of dally and overexpression of dally at the A/P border cells result in a shallower gradient and lower levels of Dpp in the receiving cells. Taken together, Dally regulates formation of both Dpp ligand and activity gradients. In addition, the results strongly suggest that Dally plays at least two roles in the formation of the Dpp signaling gradient: (1) it regulates the sensitivity of cells to Dpp in a cell autonomous fashion; and (2) it affects Dpp protein distribution, which is a non-autonomous effect (Fujise, 2003).

This study demonstrates that dally controls shape of both the ligand and the activity gradients of Dpp in the developing wing. How does dally contribute to the Dpp gradient formation? In vitro analyses using mammalian tissue culture cells have established that HSPGs can increase FGF signaling by stabilizing FGF/FGF receptor complexes Several lines of evidence indicate that the dosage of HSPGs is an important factor for FGF signaling. For example, sodium chlorate treatment, which inhibits the sulfation of heparan sulfate, reduces the biological response of cells to FGF; the response can be restored by an exogenous supplement of heparin. However, restoration is seen only at an optimal concentration of heparin; excess heparin competes for FGF with signaling complex, resulting in a reduction of signaling. In the Drosophila wing, ectopic expression of Dally-like, another glypican related to Dally, leads to a massive accumulation of extracellular Wg protein and compromises Wg signal transduction, suggesting that the glypicans can affect ligand stability and distribution (Fujise, 2003 and references therein).

On the basis of these studies as well as the current data, Dally would appear to have both positive and negative roles on Dpp signaling. In its positive role, Dally serves as a co-receptor for Dpp, stabilizing Dpp protein and enhancing signaling. Conversely, given that Dpp is a heparin-binding protein, Dally may bind Dpp through its heparan sulfate chains and reduce the amount of free Dpp ligands. Thus, Dally affects the Dpp gradient at two distinct steps: signal transduction (autonomous effect) and ligand distribution (non-autonomous effect). A model is proposed in which alterations in the shapes of the Dpp ligand and the activity gradients caused by dally mutations and dally overexpression are interpreted as the sum of these plus and minus effects of Dally function. In this model, Dally normally sequesters Dpp protein to some extent in A/P border cells, where dally levels are very high. Therefore, reduced levels of Dally in mutant discs may result in the release of Dpp ligand and, consequently, higher levels of signaling activity in the central region. Therefore, dally mutations may severely reduce the stability of Dpp protein as well as its signaling activity in the receiving cells. When dally is overexpressed in A/P border cells, Dpp is trapped by binding to excess Dally and fails to distribute properly (Fujise, 2003).

Although it is thought that Dally regulates the diffusion of Dpp, the results do not rule out the possibility that Dally plays a more active role in facilitating Dpp diffusion or 'carries' Dpp protein. For example, it is possible that Dally is required for the Dpp movement through the transcytosis pathway or other transport systems, such as cytonemes (Fujise, 2003).

Hox control of morphogen mobility and organ development through regulation of glypican expression

Animal bodies are composed of structures that vary in size and shape within and between species. Selector genes generate these differences by altering the expression of effector genes whose identities are largely unknown. Prime candidates for such effector genes are components of morphogen signaling pathways, which control growth and patterning during development. This study shows that in Drosophila the Hox selector gene Ultrabithorax (Ubx) modulates morphogen signaling in the haltere through transcriptional regulation of the glypican dally. Ubx, in combination with the posterior selector gene engrailed (en), represses dally expression in the posterior (P) compartment of the haltere. Compared with the serially homologous wing, where Ubx is not expressed, low levels of posterior dally in the haltere contribute to a reduced P compartment size and an overall smaller appendage size. One molecular consequence of dally repression in the posterior haltere is to reduce Dpp diffusion into and through the P compartment. These results suggest that Dpp mobility is biased towards cells with higher levels of Dally and that selector genes modulate organ development by regulating glypican levels (Crickmore, 2007).

Upon comparing Dpp signaling readouts in the wing and haltere, it was noticed that, in addition to a general narrowing of Dpp pathway activity, Dpp signaling was also asymmetric relative to its source (the AP organizer) in the haltere. Specifically, the P-Mad signal was stronger anterior to the AP organizer (roughly demarcated by the domain of peak P-Mad staining) than it was posterior to the organizer. To test if this asymmetry is due to asymmetric ligand distribution or differences in signal transduction, an extracellular staining protocol was used to examine the distribution of a Dpp::GFP fusion protein following its expression in AP organizer cells. In wing cells, Dpp::GFP was detected in a broad gradient on both sides of the AP organizer. In the haltere, the distribution of Dpp::GFP is limited in both directions owing to high tkv expression levels, but this restriction is stronger in the P direction. Dpp::GFP spread was abruptly halted a few cell diameters posterior to the haltere AP compartment boundary, contrasting with a tapering signal seen in the anterior direction. By contrast, the Gal4 driver used to express Dpp::GFP (ptc-Gal4) drove nearly symmetrical expression of a UAS-GFP transgene, demonstrating that the distribution of Dpp::GFP in the haltere is not due to asymmetric activity of the ptc-Gal4 driver. In both the wing and haltere, the pattern of extracellular Dpp::GFP was very similar to the P-Mad pattern, suggesting that Ubx does not affect Dpp signal transduction downstream of ligand binding, at least as detected with the anti-P-Mad antibody. Furthermore, in both the wing and haltere, a similar coincidence of extracellular Dpp::GFP and P-Mad patterns was observed when Dpp::GFP was expressed in clones. The correlation between the P-Mad and extracellular Dpp::GFP patterns in both the wing and haltere allows inference of extracelluar Dpp ligand distribution by visualizing P-Mad in the proceeding experiments (Crickmore, 2007).

In the wing, Dpp::GFP distribution and P-Mad staining were also asymmetric, owing to slightly higher levels of Tkv in the P compartment, which impedes diffusion. By contrast, because Tkv levels are similar on both sides of the AP boundary of the haltere, Tkv levels are unlikely to account for the Dpp signaling asymmetry in this appendage. This idea directly by providing uniform levels of UAS-tkv to both the haltere and wing. Under these conditions, P-Mad staining became symmetric in the wing, but remained asymmetric in the haltere. These results suggest that the more-restricted P-Mad staining in the P compartment of the wild-type haltere is due to a tkv-independent and haltere-specific anterior bias in the diffusion of Dpp (Crickmore, 2007).

In previous work, it was showed how the upregulation of the Dpp receptor, thickveins, in the haltere causes an overall decrease in Dpp mobility as compared with the wing, and consequently contributes to the small size of the haltere. This study shows that the HSPG dally is repressed in the P compartment of the haltere and that this regulation decreases the P:A ratio and overall size of the haltere. Posterior dally repression causes Dpp diffusion to be biased away from P cells, generating an AP asymmetry in Dpp signaling. The findings reported here therefore provide another instance wherein Ubx controls the extracellular signaling environment of the developing haltere and thereby distinguishes it from the wing (Crickmore, 2007).

The movement of most or all signaling molecules through tissues is regulated by HSPGs, including glypicans such as dally. In contrast to receptors, HSPGs control the distribution of multiple signaling molecules. Regulation of HSPG expression and activity by selector genes is therefore a potentially very powerful mechanism for shaping signaling pathway activation profiles and molding organ shapes and sizes. However, the promiscuity of HSPGs also makes it difficult to assign the morphological consequences of their expression patterns to the alteration of individual signaling pathways. Indeed, it is likely that the altered dally expression pattern in the haltere has implications for Hh, Wg and Dpp signaling, all of which control growth and patterning. This study has focused on the relationship between dally expression and Dpp signaling (Crickmore, 2007).

Dpp signaling is increased in dally⁺ clones and decreased in dally^- clones. These and other findings have suggested that Dally participates in the control of Dpp mobility. The current results add to these earlier observations by suggesting that variations in the levels of Dally between the cells of a tissue influence the direction and extent of Dpp diffusion. Specifically, it is proposed that in addition to simply being promoted by Dally, Dpp mobility is biased towards cells with higher Dally levels. This idea derives mainly from the observation that Dally can influence Dpp movement in a cell-non-autonomous manner. For example, when Dally levels are increased in the haltere P compartment, there is a shift in Dpp signaling from the A to the P compartments, as visualized by the levels of P-Mad. Similarly, knocking down Dally levels in the P compartment of the wing influences the extent and levels of P-Mad in the A compartment. If discontinuities in Dally levels can non-autonomously influence Dpp signaling across compartment borders, it follows that differences in Dally levels between cells within a compartment can also shape the Dpp signaling landscape. This might be important for wild-type wing development, where graded Dpp signaling represses dally, resulting in an inverse dally gradient that increases towards the lateral edge of the disc. It is suggested that this inverse dally gradient helps to attract Dpp to more lateral regions of the disc. Accordingly, in a dally-mutant wing disc, the Dpp gradient is less broad than in a wild-type wing disc. It is possible that other HSPGs control the mobility of signaling molecules in a similar manner (Crickmore, 2007).

Altering dally levels in either the A or P compartment changes relative compartment size, but only P compartment dally levels are relevant for total organ size. Two possible explanations are considered that link the P-specific dally repression seen in the haltere to a reduction in final organ size. Both of these scenarios (which are not mutually exclusive) focus on the role of P cells in producing Hh, which diffuses into A cells to instruct Dpp production and, consequently, controls final organ size. Importantly for both models, it was found that there is in fact less Hh detected in the P compartment of the wild-type haltere as compared with the wing. In the first model, the repression of dally reduces overall Hh production simply by reducing the size of the P compartment, which is a consequence of reduced Dpp signaling. In this scenario, fewer Hh-producing P cells result in less total Hh production from the P compartment, and therefore less Dpp produced in the A compartment. The logic of this potential mode of size regulation is interesting: a selector gene (Ubx) restricts growth factors (Wg and Dpp) from the pool of cells (the P compartment) that produces another growth factor (Hh). In the second scenario, dally repression may directly reduce the amount of Hh in the P compartment that can be transported into the A compartment. In support of this idea, Hh staining was found to be reduced in clones of cells where Dally levels are reduced through UAS-dallyRNAi (Crickmore, 2007).

Together, dally and dlp influence the mobility of all known morphogens in Drosophila. In addition to the compartmental regulation of dally, it is also noted that Dlp levels are generally lower throughout the haltere as compared with the wing. The haltere also lacks the domain of dlp repression seen at the DV boundary of the wing. Finally, it was also noticed that the expression of Notum-lacZ, an enhancer trap into a gene that encodes an HSPG-modifying enzyme, is different between the wing and haltere. The combined alteration of dally, dlp and Notum levels in the haltere is likely to have consequences for any signaling molecule that uses HSPGs for transport. When these observations are combined with those of earlier work showing that the levels of both Dpp and its receptor are regulated differently in the haltere and wing, and the observation that wg is repressed in the posterior haltere, a picture emerges in which selector genes alter the expression of multiple components of multiple signaling pathways to change morphogen signaling landscapes between tissues and thereby modify organ shapes and sizes. It is hypothesized that the summation of all signaling pathway changes may be sufficient to understand the size and shape differences between fundamentally similar epithelia such as the wing and haltere imaginal discs (Crickmore, 2007).

The range of spalt-activating Dpp signalling is reduced in endocytosis- defective Drosophila wing discs

Pattern formation along the anterior-posterior (A/P) axis of the developing Drosophila wing depends on Decapentaplegic (Dpp), a member of the conserved transforming growth factor beta (TGF beta) family of secreted proteins. Dpp is expressed in a stripe along the A/P compartment boundary of the wing imaginal disc and forms a long-range concentration gradient with morphogen-like properties that generate distinct cell fates along the A/P axis. Dpp expression and Dpp signaling have been monitored in endocytosis-mutant wing imaginal discs that develop severe pattern defects specifically along the A/P wing axis. The results show that the size of the Dpp expression domain is expanded in endocytosis-mutant wing discs. However, this expansion does not result in a concommittant expansion of the functional range of Dpp activity but rather, results in its reduction, as indicated by the reduced expression domain of the Dpp target gene spalt. The data suggest that clathrin-mediated endocytosis, a cellular process necessary for membrane recycling and vesicular trafficking, participates in Dpp action during wing development. Genetic interaction studies suggest a link between the Dpp receptors and clathrin. Impaired endocytosis does not interfere with the reception of the Dpp signal or the intracellular processing of the mediation of the signal in the responder cells, but rather affects the secretion and/or the distribution of Dpp in the developing wing cells (Gonzalez-Gaitan, 1999).

Mutations in the Drosophila alpha-adaptin gene (DAda) disrupt clathrin-mediated endocytosis prior to vesicle formation at the cell membrane. Embryos that are homozygous for a lack-of-function allele, such as DAda3, develop into normal looking larvae that die while still in their eggshells. alpha-adaptin is also expressed at high levels at the plasma membrane of developing wing imaginal disc cells during larval stages. To address a possible role for alpha-adaptin during wing development, a hypomorphic allele, D-Ada4, was generated to overcome embryonic lethality. The strongest non-lethal allelic combination, D-Ada3/D-Ada4, causes a temperature-dependent wing phenotype. At 18 degrees C, the mutant wings are normal. At 25 degrees C, wings are reduced in size and show vein pattern defects along the A/P axis. At 29 degrees C, only wing remnants with strongly enhanced pattern defects along the A/P axis are observed. Such remnants develop diagnostic dorsoventral pattern elements, such as sensilla campaniformia on the hinge and the dorsal surface of the wing blade, the dorsal and ventral hairs of the wing margin triple row, and specific dorsoventral aspect of the veins. Thus, no discernible dorsoventral wing pattern defects were found. The mutant pattern formation along the A/P axis of the endocytosis-mutant wings is affected in a manner similar to hypomorphic decapentaplegic mutants (Gonzalez-Gaitan, 1999).

It was next asked whether wing pattern defects are also observed when clathrin-mediated endocytosis is impaired by double mutant combinations as has been shown for mutants where alpha-adaptin and dynamin activities are jointly reduced. In double heterozygous mutants for clathrin heavy-chain (D-Chc) and alpha-adaptin, wings develop a temperature-dependent phenotype. At 25 degrees C and 29 degrees C, the A/P pattern defects of D-Chc/1;DAda3/1 mutant wings resemble those observed with DAda mutant wings. Furthermore, such wings developed at 18 degrees C a thickened posterior cross-vein similar to mutants of the Dpp receptor thick veins (tkv). The dpp- and tkv-like phenotypes obtained with the endocytosis-mutant combinations are consistent with the proposal that clathrin-mediated endocytosis is necessary for proper Dpp action during wing development (Gonzalez-Gaitan, 1999).

The conclusions drawn from the mutant phenotype are consistent with the finding that despite the enlarged dpp expression domain found in endocytosis impaired mutants, the range of sal-activating Dpp activity is significantly reduced to 3±4 cell diameters from the source of the signal. Recent results suggest that gradient formation and long-range signaling by secreted signaling proteins such as Dpp, Hedgehog and Wingless are modulated by regulatory feedback loops involving the receptors of these genes. Here, Dpp acts like Wingless: it negatively regulates the expression of its receptor Tkv. Since endocytosis has been shown to be a prerequisite for receptor clearance at the cell membrane, and in view of the genetic interactions between clathrin and the Dpp receptors Tkv and Put shown here, it is possible, among other explanations, that impaired endocytosis interferes with Dpp receptor levels and/or the formation of the Dpp gradient, as well as with the need to recycle receptors in order to keep signaling working effectively. Increased Tkv is likely to sequester free Dpp and thereby hinders Dpp migration, resulting in an altered shape of the Dpp gradient. This genetic link between the Dpp receptors and clathrin suggests that a process involving receptor-mediated endocytosis might participate in mediating Dpp action over distance, extending its functional range beyond some 4 cell diameters. However, the results obtained with double mutant wing discs do not distinguish between a signaling defect, a transport defect or unrelated defects such as the need to recycle receptors to maintain effective signaling (Gonzalez-Gaitan, 1999).

Mosaic analysis carried out with endocytosis-deficient wing disc cells establishes that the reception of the Dpp signal is not dependent on endocytotic events. This is clearly shown by the fact that the endocytosis-deficient cells express sal normally, whereas cell clones of comparable size lacking the Dpp receptor Tkv, which disrupts signal reception, fail to express sal. Furthermore, the results establish that the intracellular processing of Dpp signal between the activated receptors and the nuclear factor(s) required to activate the target gene sal is not dependant on clathrin-mediated endocytosis, as has been reported for Egf signaling. This leaves the possibility that impaired endocytosis affects the secretion or the propagation of the Dpp signal over distance, for example by transcytosis, or that both processes are affected at the same time. Once Dpp antibodies or functional Dpp-GFP fusions are available to visualize the Dpp gradient and the subcellular distribution of Dpp directly, these question can be addressed in the mutant combinations described here (Gonzalez-Gaitan, 1999).

Regulation of Dpp secretion

The Drosophila tumor suppressor gene lethal(2) giant larvae (lgl) encodes a cytoskeletal protein required for the change in shape and polarity acquisition of epithelial cells, and also for asymmetric division of neuroblasts. lgl also participates in the release of Decapentaplegic (Dpp), a member of the transforming growth factor ß (TGFß) family that functions in various developmental processes. During embryogenesis, lgl is required for the dpp-dependent transcriptional activation of zipper (zip), which encodes the non-muscle myosin heavy chain (NMHC), in the dorsalmost ectodermal cells -- the leading edge cells. The embryonic expression of known targets of the dpp signaling pathway, such as labial or tinman is abolished or strongly reduced in lgl mutants. lgl mutant cuticles exhibit phenotypes resembling those observed in mutated partners of the dpp signaling pathway. In addition, lgl is required downstream of dpp and upstream of its receptor Thickveins (Tkv) for the dorsoventral patterning of the ectoderm. During larval development, the expression of spalt, a dpp target, is abolished in mutant wing discs, while it is restored by a constitutively activated form of Tkv (Tkv^Q253D). Taking into account that the activation of dpp expression is unaffected in the mutant, this suggests that lgl function is not required downstream of the Dpp receptor. Finally, the function of lgl responsible for the activation of Spalt expression appears to be required only in the cells that produce Dpp, and lgl mutant somatic clones behave non autonomously. The activity of lgl is therefore positioned in the cells that produce Dpp, and not in those that respond to the Dpp signal. These results are consistent with the same role for lgl in exocytosis and secretion as that proposed for its yeast ortholog sro7/77: lgl might function in parallel or independently of its well-documented role in the control of epithelial cell polarity (Arquier, 2001).

Secretion relies on intracellular vesicular trafficking and on the polarized exocytosis machinery. Recent studies have demonstrated that Lgl function is essential for the establishment of the polarities of epithelial cells. An important issue is therefore to understand whether the role of Lgl in Dpp secretion is direct or simply a consequence of the loss of epithelial cell polarity. Analysis of the temporal requirement for Lgl function argues in favor of Lgl being necessary for the establishment of cell polarity, rather than for its maintenance. Moreover, alteration in Dpp signaling can be observed in lgl mutants in epithelial cells that are correctly polarized and this supports a direct function for Lgl in Dpp secretion (Arquier, 2001).

The epidermis is not affected in homozygous lgl4-null mutant larvae that no longer contain the maternal Lgl protein responsible for a normal embryonic development. lgl4 larvae develop a cuticle that possesses the hallmarks of a wild-type cuticle by all the criteria used, thus indicating that the apical secretion of cuticle components has not been altered. Markers for epithelial cell polarity are localized in the correct position in stage 16 embryos when Lgl is no longer detected. Likewise, lglts3 embryos in which the Lgl protein has lost its cortical location have maintained their typical epithelial cell polarity and their capacity to secrete normal cuticle components. In neuroblasts, Lgl seems to exert its action early during mitosis to recruit basal determinants to the cortex but it does not contribute to their maintenance in this latter location. The polarity of epithelial wing disc cells is preserved until the middle of the third instar larval stage, long after the maternal Lgl contribution has ceased (Arquier, 2001).

It seems reasonable to assume that there is a unique exocytosis pathway mediated by lgl to ensure both cell polarity control and secretion. Dlg and scrib might participate in this same pathway: indeed, they strongly interact genetically with lgl and share with this gene a large panel of identical mutant phenotypes. Lgl, however, does not strictly colocalize with Dlg and Scrib in either epithelial cells. In addition, the Dlg cortical localization does not require lgl function. One could therefore anticipate an lgl action, within a separate and distinct pathway, in parallel to that of dlg and scrib. Further experiments are needed to address this issue (Arquier, 2001).

In yeast, sro7/77-mediated polarized exocytosis relies on a complex regulation and interaction with the actomyosin cytoskeleton. Sro7/77 displays a strong genetic interaction with myo1 (encoding a Type II myosin homolog of NMHC) and with myo2 (encoding an unconventional Type V myosin). In addition, Myo1P can physically interact with Sro7P, in a manner resembling that prevailing between Lgl and Zipper/NMHC. These observations support the idea that Lgl serves as a functional link between the actomyosin cytoskeleton polarity and a specific polarized exocytosis pathway, although the precise function exerted by Lgl in such a process has yet to be deciphered. In yeast, as in flies, myo1 (zipper) and sro7/77 (lgl) display a negative genetic interaction. Loss-of-function alleles of lgl suppress the dorsal closure phenotype in homozygous zip mutants. Conversely, overexpression of lgl enhances the dorsal closure phenotype (Arquier, 2001).

Regulation of Dpp acylation and or trafficking by Sightless

sightless (sit) is required for the activity of Drosophila Hh in the eye and wing imaginal discs and in embryonic segmentation. sit acts in the cells that produce Hh, but does not affect hh transcription, Hh cleavage, or the accumulation of Hh protein. sit encodes a conserved transmembrane protein with homology to a family of membrane-bound acyltransferases. The Sit protein could act by acylating Hh or by promoting other modifications or trafficking events necessary for its function (Lee, 2001b).

One of the critical signals triggering photoreceptor development is Hedgehog (Hh), which is expressed at the posterior margin of the disc prior to differentiation and subsequently in the differentiating photoreceptors. Hh activates the expression of decapentaplegic (dpp) in a stripe at the front of differentiation, or morphogenetic furrow; Dpp signaling also promotes photoreceptor formation. dpp expression is lost from the morphogenetic furrow in sit mutant eye discs. Another target of Hh signaling, the proneural gene atonal, also requires sit for its expression. Despite this lack of Hh target gene expression, a hh-lacZ enhancer trap is expressed at the posterior margin of sit mutant eye discs, indicating that hh expression is established normally. This suggests that the sit phenotype could be due to a defect in Hh signaling (Lee, 2001b).

Hh signaling has been extensively studied in the wing disc, where hh is expressed in the posterior compartment and signals to cells just anterior to the compartment boundary to upregulate the expression of dpp and patched (ptc). The Hh signal is mediated by the stabilization and activation of the full-length form of the transcription factor Cubitus interruptus (Ci). This stabilization can be detected with an antibody directed against the C-terminal region of Ci, which fails to recognize the cleaved form of Ci produced in the absence of Hh signaling. sit mutant wing discs show defects consistent with a lack of Hh pathway function; ptc expression is not upregulated at the compartment boundary, and dpp expression is almost completely absent. In addition, no stabilization of full-length Ci could be detected at the compartment boundary. However, hh-lacZ is expressed at wild-type levels in sit mutant discs, indicating that hh transcription is unaffected. This implicates Sit in the Hh pathway downstream of hh transcription and upstream of Ci stabilization (Lee, 2001b).

Three Drosophila EXT genes shape morphogen gradients through synthesis of heparan sulfate proteoglycans

The signaling molecules Hedgehog (Hh), Decapentaplegic (Dpp) and Wingless (Wg) function as morphogens and organize wing patterning in Drosophila. In the screen for mutations that alter the morphogen activity, novel mutants of two Drosophila genes, sister of tout-velu (sotv) and brother of tout-velu (botv), and new alleles of toutvelu (ttv), were identified. The encoded proteins of these genes belong to an EXT family of proteins that have or are closely related to glycosyltransferase activities required for biosynthesis of heparan sulfate proteoglycans (HSPGs). Mutation in any of these genes impaired biosynthesis of HSPGs in vivo, indicating that, despite their structural similarity, they are not redundant in the HSPG biosynthesis. Protein levels and signaling activities of Hh, Dpp and Wg were reduced in the cells mutant for any of these EXT genes to a various degree, Wg signaling being the least sensitive. Moreover, all three morphogens were accumulated in the front of EXT mutant cells, suggesting that these morphogens require HSPGs to move efficiently. In contrast to previous reports that ttv is involved exclusively in Hh signaling, ttv mutations were also found to affect Dpp and Wg. These data lead to the conclusion that each of three EXT genes studied contribute to Hh, Dpp and Wg morphogen signaling. It is proposed that HSPGs facilitate the spreading of morphogens and therefore, function to generate morphogen concentration gradients (Takei, 2004).

In addition to monitoring signaling in EXT mutant cells, antibodies that recognize Hh, Dpp and Wg, and a GFP-tagged version of Dpp were used to analyze whether the levels or distribution of these morphogens had been affected. Levels of each of these proteins were significantly reduced in the mutant, both in the morphogen-expressing region and in the receiving region. For Hh, Dpp and Wg, similar results were observed in cells mutant singly for any of the EXT genes. Single mutation was not tested for the distribution of Dpp-GFP. In the morphogen-expressing region, hh expression was not downregulated, however levels of Hh protein were significantly decreased. This may indicate that Hh protein is destabilized and/or not retained efficiently on the cell surface in the absence of HSPGs. In contrast to hh, expression of the wg and dpp and levels of Wg and Dpp were decreased in the EXT clones. The decrease in dpp expression is easily accountable because Hh signaling is impaired in the absence of HSPGs. In contrast, the decrease in wg expression is not as readily explainable: cut and wg are both targets of Notch signaling, however the protein level of Cut was not altered in EXT clones. This suggests that wg is also regulated by an unknown mechanism dependent on HSPGs (Takei, 2004).

In the morphogen-receiving region, each of these proteins was significantly decreased in the clones of cells mutant for EXT genes, although a little leakage of morphogen molecules was seen even in the clones doubly mutant for ttv and botv. This suggests two possible mechanisms that do not exclude each other: in the absence of HSPGs these three morphogens are (1) destabilized and/or are not retained efficiently on the cell surface, like Hh in morphogen-expressing region, or (2) prevented from diffusing efficiently into the region consisting of EXT mutant cells. Intriguingly, close observation of the distribution of Hh strongly suggested a function for HSPGs in morphogen movement. In the wild-type discs, Hh protein synthesized in the posterior compartment appears to flow into the anterior compartment, with a moderate concentration gradient starting from the middle of the posterior compartment. However, Hh abnormally accumulates in the posterior compartment when the EXT mutant clone is in the anterior compartment along the A/P boundary. This effect is seen both in the ventral region and in the dorsal region. This suggests that Hh fails to move into the mutant cells and as a consequence accumulates in posterior cells instead. Dpp-GFP and Wg accumulation in front of the mutant clones was also apparent, however less pronounced compared with the case of Hh. Therefore it is concluded that the HSPG-dependent diffusion is the common mechanism for the movement of these three morphogens (Takei, 2004).

Abrogation of heparan sulfate synthesis in Drosophila disrupts the Wingless, Hedgehog and Decapentaplegic signaling pathways

Studies in Drosophila and vertebrate systems have demonstrated that heparan sulfate proteoglycans (HSPGs) play crucial roles in modulating growth factor signaling. Mutations have been isolated in sister of tout velu (sotv), a gene that encodes a co-polymerase that synthesizes HSPG glycosaminoglycan (GAG) chains. Phenotypic and biochemical analyses reveal that HS levels are dramatically reduced in the absence of Sotv or its partner co-polymerase Tout velu (Ttv), suggesting that both copolymerases are essential for GAG synthesis. Furthermore, mutations in sotv and ttv impair Hh, Wg and Decapentaplegic (Dpp) signaling. This contrasts with previous studies that suggested loss of ttv compromises only Hh signaling. These results may contribute to understanding the biological basis of hereditary multiple exostoses (HME), a disease associated with bone overgrowth that results from mutations in EXT1 and EXT2, the human orthologs of ttv and sotv (Bornemann, 2004).

The data provide direct evidence that HS chains are required for Dpp signaling in vivo. The core protein of the glypican Dally has been implicated in Dpp signaling based on genetic interactions and recent studies demonstrating its role in regulating the Dpp morphogen gradient in the wing. However, the contribution of HS chains to Dpp signaling has remained unclear. Dpp signaling in the wing disc is reduced in ttv or sotv mutant clones independent of effects on Hh signaling, establishing that HS GAG chains are required for optimal activity of the Dpp pathway. Although Dpp activity is clearly compromised in mutant tissue, signaling is still detectable in mutant clones located where ligand levels are the highest, such as near the AP compartment boundary. These results imply that, like Wg, Dpp can signal in the absence of HSPGs, albeit at lower efficiency (Bornemann, 2004).

Initial reports that ttv was required for Hh, but not Wg or FGF, signaling, prompted speculation that Ttv might generate a Hh-specific HSPG, and that, unlike its mammalian orthologs, Drosophila Sotv might retain significant functional activity in the absence of its partner. However, the demonstration that Hh, Wg and Dpp signaling are affected in single mutants for ttv and for sotv, together with the biochemical analysis presented in this study, suggest that the mammalian model of EXT1 and EXT2 as obligate co-polymerases and applies equally well to Drosophila. The severe and comparable reductions in HS disaccharides observed in ttv and sotv null mutant larvae lend additional support to the co-polymerase model. Moreover, the fact that the phenotype of both single and ttv, sotv double mutants is indistinguishable, strongly suggests that any residual partner activity is not biologically significant (Bornemann, 2004).

Dpp morphogen movement is independent of dynamin-mediated endocytosis but regulated by the glypican members of heparan sulfate proteoglycans

The Drosophila transforming growth factor ß homolog Dpp acts as a morphogen that forms a long-range concentration gradient to direct the anteroposterior patterning of the wing. Both planar transcytosis initiated by Dynamin-mediated endocytosis and extracellular diffusion have been proposed for Dpp movement across cells. In this work, it was found that Dpp is mainly extracellular, and its extracellular gradient coincides with its activity gradient. A blockage of endocytosis by the dynamin mutant shibire does not block Dpp movement but rather inhibits Dpp signal transduction, suggesting that endocytosis is not essential for Dpp movement but is involved in Dpp signaling. Furthermore, Dpp fails to move across cells mutant for dally and dally-like (dly), two Drosophila glypican members of heparin sulfate proteoglycan (HSPG). These results support a model in which Dpp moves along the cell surface by restricted extracellular diffusion involving the glypicans Dally and Dly (Belenkaya, 2004).

One new observation in this work is that the extracellular Dpp is broadly distributed in the wing disc. Consistent with these findings, previous biochemical analysis demonstrated that the majority of mature Dpp signaling molecules are extracellular. Importantly, the overall shape of the extracellular Dpp gradient coincides well with its activity gradient, suggesting that the extracellular Dpp gradient contributes to Dpp activity gradient in the wing disc. The observation of broadly distributed extracellular Dpp led to a re-examination of the role of Dynamin-mediated endocytosis in Dpp movement and signaling. These analyses argue that Dynamin-mediated endocytosis is not essential for Dpp movement: (1) both Dpp signaling activity and extracellular GFP-Dpp levels are not reduced in the wild-type cells behind the shi^ts1 clones that are defective in endocytosis; (2) the extracellular GFP-Dpp is also broadly distributed in endocytosis-defective wing discs homozygous for shi^ts1 at nonpermissive temperature. These data demonstrate that Dpp molecules are able to move across Dynamin-defective cells. Finally, it was found that extracellular Dpp accumulates on the cell surface of shi^ts1 mutant clones, suggesting that Dpp is able to move into shi^ts1 mutant cells and that Dynamin-mediated endocytosis is normally involved in downregulating levels of the extracellular Dpp. No accumulation of extracellular Dpp on wild-type cells was observed in front of shi^ts1 mutant clones; this would be expected if endocytosis were required for Dpp movement (Belenkaya, 2004).

While Dynamin-mediated endocytosis does not appear to be critical for Dpp movement, Dpp signaling activity is reduced cell autonomously in shi^ts1 mutant cells. This result argues that Dynamin-mediated endocytosis is an essential process for Dpp signaling. Studies in mammalian cell culture system have demonstrated the critical role of Dynamin-mediated internalization of activated TGF-β receptors in TGF-β signaling. SARA (Smad anchor for receptor activation), a FYVE finger protein enriched in early endosomes is involved in this process. Although the exact mechanism of endocytosis-mediated TGF-β signaling is still unclear, current data suggest a role of early endosomes as a signaling center for TGF-β. Consistent with this view, it has been shown that ectopic expression of the dominant-negative form of Rab5 (DRab5S43N) using engrailed-Gal4 leads to a reduction of Dpp signaling, while overexpression of Rab5 broadens the Dpp signaling. Rab5 localizes in early endosomes and is required for endosome fusion. Taken together, it is proposed that dynamin-mediated endocytosis is not directly involved in Dpp movement but is essential for Dpp signaling. Furthermore, Dynamin-mediated endocytosis can downregulate extracellular Dpp levels, thereby shaping the Dpp morphogen gradient (Belenkaya, 2004).

To investigate the role of HSPGs in Dpp morphogen gradient formation, Dpp signaling and its extracellular distribution was examined in sulfateless (sfl) and dally-dly mutant clones. dally and dly are shown to be required and partially redundant in Dpp signaling and movement in the wing disc. Two lines of evidence support the role of Dally and Dly in Dpp movement across cells: (1) Dpp signaling activity is reduced in cells behind sfl or dally-dly mutant clones; (2) extracellular Dpp levels are diminished in cells behind sfl or dally-dly mutant clones. Importantly, it was found that sfl or dally-dly mutant clones only a few cells wide can effectively block GFP-Dpp movement, suggesting that Dpp movement does not occur through 'free diffusion', by which extracellular Dpp would be expected to move across sfl or dally-dly mutant cells. Based on these observations, it is proposed that Dpp moves from cell to cell along the epithelium sheet through restricted diffusion involving Dally and Dly (Belenkaya, 2004).

If the HSPGs Dally and Dly are indeed involved in Dpp movement, observation of extracellular GFP-Dpp accumulation in front of sfl, dally-dly mutant clones would be expected. Indeed, extracellular GFP-Dpp accumulation is visible in front of sfl or dally-dly mutant clones. Consistent with this observation, Hh has been observed to accumulate abnormally in clones mutant for tout-velu (ttv) and brother of tout-velu (botv), two Drosophila EXT members involved in HS GAG chain biosynthesis. Both Wg and Dpp accumulation in front of ttv-botv clones are also observed, albeit less pronounced, compared with the case of Hh. Similarly, extracellular GFP-Dpp accumulation is relatively weak, compared with Hh accumulation. One possibility is that extracellular Dpp molecules bound by Dally and Dly in wild-type cells can still be internalized by adjacent sfl or dally-dly mutant cells through cell-cell contact, leading to a reduction of extracellular Dpp accumulation in front of sfl or dally-dly mutant cells. Consistent with this view, it was noticed that, within sfl or dally-dly mutant clones, the first row of the mutant cells immediately adjacent to wild-type cells and facing Dpp-expressing cells is still capable of transducing Dpp signaling (Belenkaya, 2004).

In addition to being required for Dpp movement, Dally and Dly are also essential for Dpp signaling in its receiving cells. Dpp signaling is reduced in sfl or dally-dly mutant cells. Reduced levels of extracellular Dpp were observed in sfl or dally-dly mutant clones. Consistent with the results in this work, clones mutant for ttv or botv as well as sister of tout-velu (sotv), members of Drosophila EXT, led to reductions in Dpp signaling and its ligand distribution when analyzed by a conventional staining protocol that revealed both extracellular and intracellular Dpp. Collectively, these data suggest that the main function of Dally and Dly in Dpp signaling is to maintain and/or concentrate the extracellular Dpp available for Dpp receptors (Belenkaya, 2004).

This study has shown that Dynamin-mediated endocytosis is not essential for Dpp movement. Dpp movement is through a cell-to-cell mechanism involving the HSPGs Dally and Dly. On the basis of these findings, it is proposed that secreted Dpp molecules in the A-P border are immediately captured by the GAG chains of Dally and Dly on the cell surface located in either the A or P compartments. The differential concentration of Dpp on the cell surface of producing cells and receiving cells drives the displacement of Dpp from one GAG chain to another toward more distant receiving cells. Alternatively, Dpp molecules bound by Dally or Dly could also move along the cell surface through a GPI linkage that is inserted in the outlet leaflet of the plasma membrane and is enriched in raft domains. In the receiving cells, Dally and Dly may present Dpp to its receptor, Tkv, that transduces Dpp signal through the Dynamin-mediated internalization process, which further downregulates extracellular Dpp levels and cell surface Tkv. Based on this model, extracellular Dpp and its receptor, Tkv, would be accumulated on the surface of Dynamin-deficient cells, and extracellular Dpp would be able to move across Dynamin-deficient cells to reach more distal cells. In sfl or dally-dly mutant clones, extracellular Dpp molecules can not be attached on the cell surface and therefore can not be transferred further to more distal cells. In this model, endocytosis is not directly involved in Dpp movement; however, through receptor-mediated internalization, Dynamin-mediated endocytosis can downregulate extracellular Dpp levels, thereby shaping the Dpp morphogen gradient. It remains to be determined how Dpp is transferred from one cell to another by the GAG chains of Dally and Dly and whether Dally and Dly play a role in preventing extracellular Dpp from degradation. Further studies are needed to determine whether other mechanisms are also involved in Dpp movement (Belenkaya, 2004).

A model for formation of the BMP activity gradient in the Drosophila embryo

The dorsoventral axis of the Drosophila embryo is patterned by a gradient of bone morphogenetic protein (BMP) ligands. In a process requiring at least three additional extracellular proteins, a broad domain of weak signaling forms and then it abruptly sharpens into a narrow dorsal midline peak. Using experimental and computational approaches, how the interactions of a multiprotein network create the unusual shape and dynamics of formation of this gradient was investigated. Starting from observations suggesting that receptor-mediated BMP degradation is an important driving force in gradient dynamics, a general model is developed that is capable of capturing both subtle aspects of gradient behavior and a level of robustness that agrees with in vivo results (Mizutani, 2005).

This study began by showing that robustness with respect to variations in the expression of single genes is not a characteristic of this system. This is an important observation, given that considerable attention has been focused lately on the robustness of morphogen-patterning systems, as well as biological signaling in general. The fact that sog^-/+ embryos eventually develop normally underscores the ability of embryos to compensate at later stages for early errors. It is not clear why marked effects of sog heterozygosity were not seen previously in previous experiments (Mizutani, 2005).

The diffusibility of BMPs in the embryo in the presence and absence of Sog was examined. By examining embryos in which Dpp is ectopically expressed, it was observed that the range of Dpp action is reduced in the absence of Sog, but still substantial, and consistent with unhindered diffusion. By observing the rate at which continuous ectopic Dpp expression gives rise to an unchanging response profile, it was also possible to infer that Dpp must undergo rapid degradation, presumably through receptor-dependent means. In these experiments, levels of expression of ectopic Dpp were not high (2.5-fold above normal when two copies of st2-dpp were present; presumably only slightly above normal when one copy was present (Mizutani, 2005).

The above observations were used to produce a simplified model of gradient formation. The goal was not necessarily to reproduce all aspects of the in vivo gradient, but rather to begin with a minimum number of elements -- and as few assumptions as possible -- and then ask which of the behaviors of the in vivo gradient could be captured. Interestingly, a great many of those behaviors emerge from a model in which a single ligand (e.g., Dpp or a Dpp/Scw heterodimer) diffuses freely, is degraded by receptors, forms a complex with Sog and Tsg, and is released from that complex when Tld cleaves Sog. These behaviors include rapid dynamics, formation of a broad domain of weak dorsal signaling that abruptly refines to a sharp midline peak, and peak narrowing or broadening when sog dosage is either increased or decreased, respectively. These behaviors depend upon the combined presence of Sog, Tsg, and Tld and are also highly sensitive to dpp dosage. Interestingly, highly localized expression of Tld and an absolute dependence of Sog cleavage on Dpp are not essential. Also not critical is the order of assembly of Dpp-Sog-Tld complexes (Mizutani, 2005).

Although the ability of the model to form a midline peak of BMP activity exemplifies the Sog/Tld-dependent 'shuttling', that mechanism does not give a complete picture of events for two reasons: (1) the abrupt onset of midline peak growth after a substantial plateau phase reflects a BMP-catalyzed chain reaction of Sog destruction that is independent of BMP transport per se; (2) calculations show that any soluble inhibitor has the ability to expand the range of action of a morphogen simply by protecting it from receptor-mediated destruction. Indeed, this effect alone could underlie some of the greater range of action of ectopically expressed Dpp in wild-type versus sog^- embryos (Mizutani, 2005).

At least one feature of the model that does not match in vivo observations, even when investigated over a wide range of parameter values, is the magnitude of the effect of sog heterozygosity on PMad peak width. The results suggest a near doubling of peak width, whereas calculations predict a more modest increase. Even accounting for the nonlinearity of immunohistochemistry and the fact that PMad may not be an instantaneous read-out of BMP receptor occupancy, the data suggest that other processes, not captured in the simple model, regulate the shape of PMad peaks. For example, it might be necessary to include the effects of a novel truncated form of Sog that promotes, rather than inhibits, BMP signaling (Mizutani, 2005).

One process that seems especially likely to shape PMad peaks is a BMP-driven, transcription-dependent feedback loop that has very recently been shown to markedly amplify high and depress low levels of BMP signaling in the Drosophila embryo. Such feedback could not only modify the shapes of PMad peaks, but also potentially explain another peculiarity of the model, which is that its peak heights and widths best fit mutant data when they are looked at up to the 30-45 min period, but not much later (i.e., not in the mathematical steady state). Since positive-feedback regulation of BMP signaling can be expected to both sharpen and maintain patterns that might otherwise have continued to evolve, it is perhaps not surprising that, at long enough times, in vivo behavior diverges from predictions of the model. Put another way, this issue serves as a reminder that, unlike BMP gradients at larval stages of Drosophila development (e.g., in the imaginal discs), the embryonic BMP gradient forms and acts so rapidly that there is little justification for assuming that steady-state calculations should reproduce in vivo observations. Indeed, it is only by considering the dynamics of gradient formation that the model presented here is able to explain the seemingly paradoxical result that decreased dorsal midline PMad staining in dpp^-/+ embryos can be rescued by lowered sog dosage, when loss of sog function, by itself, is associated with decreased dorsal midline PMad staining (Mizutani, 2005).

In summary, the results presented here indicate that known properties of the molecules required for formation of the Drosophila embryonic BMP gradient are sufficient to account for many aspects of gradient dynamics, shape, and robustness, with no need for assumptions such as lack of diffusion of free BMP, transient BMP synthesis, removal of BMP from its receptors by Sog, or attainment of a steady state. Although computational data indicate that a Sog/Tld-dependent shuttling mechanism plays a key role in shaping and timing this BMP gradient, other dynamic processes appear to participate as well (Mizutani, 2005).

Facilitated transport of a Dpp/Scw heterodimer by Sog/Tsg leads to robust patterning of the Drosophila blastoderm embryo

Patterning the dorsal surface of the Drosophila blastoderm embryo requires Decapentaplegic (Dpp) and Screw (Scw), two BMP family members. Signaling by these ligands is regulated at the extracellular level by the BMP binding proteins Sog and Tsg. Tsg and Sog play essential roles in transporting Dpp to the dorsal-most cells. Furthermore, biochemical and genetic evidence is presented that a heterodimer of Dpp and Scw, but not the Dpp homodimer, is the primary transported ligand and that the heterodimer signals synergistically through the two type I BMP receptors Tkv and Sax. It is proposed that the use of broadly distributed Dpp homodimers and spatially restricted Dpp/Scw heterodimers produces the biphasic signal that is responsible for specifying the two dorsal tissue types. Finally, it is demonstrated mathematically that heterodimer levels can be less sensitive to changes in gene dosage than homodimers, thereby providing further selective advantage for using heterodimers as morphogens (Shimmi, 2005).

The suggestion that the facilitated transport of a BMP signaling molecule might be the primary mechanism that generates pattern within the dorsal domain of the Drosophila blastoderm embryo (Holley, 1996) was a conceptual breakthrough, since it could account for the paradoxical abilities of Sog and Tsg to have both positive and negative effects on patterning. However, there was no direct evidence that either Dpp or Scw actually concentrated to the midline. In addition, it did not explain the roles of Dpp and Scw in producing the restricted high-level signaling output at the midline, as measured by p-Mad accumulation, nor did it explain how a lower level of signal was maintained in the more lateral regions to help fate the future dorsal ectoderm. Lastly, it was not apparent how the system achieves resiliency to changes in gene dosages of certain components. The experimental and computational observations described in this study have addressed these issues (Shimmi, 2005).

One of the primary findings is that Dpp and Scw form heterodimers both in tissue culture and in vivo and that these heterodimers are able to synergistically stimulate phosphorylation of Mad in cell culture. Since the Dpp/Scw heterodimers have highest affinity for Sog and Tsg, it is inferred that the heterodimer is the primary ligand transported dorsally by Sog and Tsg, resulting in high levels of p-Mad accumulation at the dorsal midline just prior to gastrulation. Consistent with this view, it was found that Dpp localization to the midline depends on Scw (Shimmi, 2005).

In addition to heterodimers being the preferred translocated species, the heterodimer model also explains the mechanism by which Scw contributes to dorsal patterning. This issue has been enigmatic since scw and its receptor, Sax, are expressed ubiquitously in the early embryo, yet signal output is limited to dorsal cells. In addition, misexpression of Scw or activated Sax produces very limited effects in most tissues, while misexpression of Dpp or activated Tkv results in very dramatic consequences. A partial resolution to this issue was suggested by the finding that coexpression of activated Sax and activated Tkv in embryos or imaginal discs produces a synergistic signal, implying that both the Sax and Tkv signals are necessary for a robust output. However, it has remained unclear whether endogenous, nonactivated receptors can produce a synergistic signal in response to ligands. As described in this study, the formation of a heterodimer between Dpp and Scw resolves these issues. In tissue culture assays, Scw homodimers produce very limited signal, while Dpp homodimers produce a moderate signal requiring only the Tkv receptor. The differential signaling ability of each homodimer explains their nonequivalence in producing patterning abnormalities when misexpressed during development. In contrast, the Dpp/Scw heterodimer is able to produce a synergistic phosphorylation of Mad that requires both the Tkv and Sax receptors; simply mixing homodimers is not sufficient. These observations demonstrate that synergistic signaling occurs at the level of receptor-mediated Mad phosphorylation and not through integration of separate signals at downstream targets. The molecular mechanism by which the Tkv and Sax receptors produce a synergistic output remains unclear (Shimmi, 2005).

Although the original role for Scw in dorsal patterning invoked formation of a heterodimer as the primary