decapentaplegic


PROTEIN INTERACTIONS

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 ‘carry’ 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 (TkvQ253D). 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 shits1 clones that are defective in endocytosis; (2) the extracellular GFP-Dpp is also broadly distributed in endocytosis-defective wing discs homozygous for shits1 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 shits1 mutant clones, suggesting that Dpp is able to move into shits1 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 shits1 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 shits1 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, 2005a).

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, 2005a).

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, 2005a).

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, 2005a).

Although the original role for Scw in dorsal patterning invoked formation of a heterodimer as the primary signaling species, this model fell into disfavor because ventral injection of scw mRNA or ventral expression of scw from the twist promoter can partially rescue amnioserosa formation. Since disulfide-linked heterodimer formation of TGF-β type ligands is known to occur in the Golgi during the secretion process, ventral expression of Scw without Dpp should preclude formation of heterodimers, and, therefore, any rescuing activity should be brought about by homodimers. Although some rescue was observed in these experiments, it is important to note that even multiple copies of ventrally expressed Scw do not lead to viability. In contrast, a single copy of Scw expressed in the dorsal domain using the tld promoter gives complete viability and fertility. In addition, these experiments assume that there is no internalization within the dorsal domain of Scw homodimers followed by isomerization with Dpp and resecretion. This possibility is mechanistically very similar to models in which Dpp is proposed to undergo transcytosis. Therefore, while ventral overexpression of Scw homodimers may have some ability to compensate for loss of Scw dorsally, normal patterning is most efficiently achieved when Scw is expressed in a domain in which heterodimers can form (Shimmi, 2005a).

BMP-directed patterning of dorsal blastoderm cells ultimately results in the specification of two tissues, amnioserosa and dorsal ectoderm. In general, these tissues derive from cells receiving high and low BMP signal, respectively. Whether there are additional cell fate subdivisions specified within the steep signaling transition zone is not clear, although cells can discriminate subtle signaling differences as evidenced by the slightly wider expression pattern of the BMP target genes rho and usp compared to zen and race. Although both Dpp and Scw are required to establish the high point of signaling necessary to specify amnioserosa, only Dpp is needed to specify dorsal ectoderm. This is consistent with observations that the Dpp/Scw heterodimer will be preferentially concentrated at the midline because of its high affinity for Sog and Tsg. In contrast, Dpp and Scw homodimers will be more broadly distributed because of their lower affinities for Sog and Tsg. Although the different species cannot be directly distinguished in vivo, analysis of downstream target genes in a scw mutant embryo revealed that there is sufficient BMP activity to activate pnr transcription, but its pattern is very wide, consistent with the observed broad distribution of Dpp homodimers. In the wild-type case, Dpp and Scw homodimers, together with a small number of heterodimers that escape from Sog and Tsg, may contribute to signaling in the lateral ectoderm, since the pnr signal is stronger in wild-type than in scw mutants. These homodimers also likely signal in a repressive manner to prevent ectopic transcription of neurogenic genes within the dorsal domain. Thus, patterning of dorsal tissue appears to take advantage of the differing properties of homo- and hetero-dimers to establish a biphasic signaling state. Specifically, selective transport of the heterodimer and synergistic receptor signaling produce a restricted high point and amnioserosa cell fate, while Dpp and Scw homodimers generate a broad low level of signal that help fate the future dorsal ectoderm and restrict neurogenic activity to more lateral regions. It is likely that the full specification of dorsal ectoderm does not occur until a second round of dpp transcription takes place after germ band extension. It is also likely that additional components help reinforce the formation of the biphasic state, since recent genetic data indicate that tight localization of Dpp to the midline requires an initial phase of low-level Dpp signal reception (E.L. Ferguson, personal communication). The suggestion is that this initial low-level Dpp signal induces expression of an additional component that participates in the localization process. The identity of this component remains elusive (Shimmi, 2005a).

Lastly, it is noted that employment of heterodimers in early embryonic patterning may be a common theme. In zebrafish, both BMP2b and BMP7 are required for dorsal-ventral patterning, and loss-of-function mutations in each gene exhibit identical severely dorsalized phenotypes. Since this phenotype is not enhanced in double mutants and overexpression of these two gene products reveals synergy in the ventralization of wild-type embryos, it has been suggested that BMP2a/BMP7 heterodimers are the primary molecules that specify ventral cell fates in this organism. These observations further highlight the overall similarity in the molecular components used to pattern the early zebrafish and Drosophila (Shimmi, 2005a).

Use of the Dpp/Scw heterodimer provides the patterning system with an effective buffer at a very early step in dorsal cell fate specification. Buffering for reductions of Scw or Dpp is predominantly determined by the relative monomer production rates, and if Scw is in slight excess with respect to Dpp, reductions in the levels of Scw will have little effect on the output Dpp/Scw heterodimer, regardless of the specific choices of parameters (Shimmi, 2005a).

Patterning is also resilient to reductions of Sog and Tsg. Sog and Tsg have synergistic BMP binding activity and the concentration of Sog/Tsg in the PV space is governed by the interaction of reaction and diffusion. The Sog/Tsg ratio can be computed as described for Dpp/Scw to determine the compensation in this subsystem, and the results are different from those for Dpp/Scw. Now there are two distinct solution regions, one for small β (β is the ratio of the wild-type production rates for monomer Dpp to monomer Scw) where many choices of parameters provide significant compensation for reductions of gene dosage, and one for large β where there is virtually no compensation. Because the behavior for large β and small β is very different, this analysis can explain the compensation for reductions in either Sog or Tsg but not both. This suggests that other mechanisms must be involved to explain the experimentally observed resilience in both sog and tsg heterozygous embryos. These could include the following: (1) the spatial separation of Sog and Tsg expression, (2) downstream kinetic mechanisms that compensate after Sog/Tsg formation, or (3) both. Both may contribute, but the following focuses on the possible effects of compensation in downstream kinetic interactions (Shimmi, 2005a).

After Sog/Tsg formation, the next step downstream is the binding of the inhibitor Sog/Tsg to Dpp/Scw. Experimentally, it is observed that Tolloid cleavage of Sog is greatly enhanced when bound to Dpp/Scw and is enhanced in the presence of Tsg. In addition, a previous mathematical model of BMP patterning suggested that cleavage of Sog (only when bound in the complex Sog/BMP) is a requirement for the system to exhibit resilience to changes in gene dose of sog, tsg, or scw. These data support the idea that Dpp/Scw transported from the broad dorsal region must be released from the Sog/Tsg/Dpp/Scw complex. Interestingly, the local dynamics of Sog/Tsg + Dpp/Scw complex formation are completely analogous to the local dynamics for Sog + Tsg complex formation. This suggests that, if the level of Dpp/Scw or Sog/Tsg is decreased from the original wt levels, the output complex Sog/Tsg/Dpp/Scw would be less affected. Taken together, the Sog/Tsg and Sog/Tsg/Dpp/Scw steps lead to a cascade in which the compensation in the first step is enhanced in the second step. In effect, the output from one complex formation stage becomes the input substrate for the next stage. Of course, the level of buffering achieved depends on the system parameters. The output suggests that patterning would be most compensated for reduction of Scw, followed by Tsg, then Sog, and lastly Dpp. Of course, other downstream steps may also contribute to compensation (Shimmi, 2005a).

In reality, patterning involves diffusive transport as well, but the analysis shows how a cascade of stages can produce compensation in the kinetic steps. When the full BMP patterning model that incorporates transport is compared to a previous model mediated by homodimers and monomers, there are approximately 100 times more 'robust' hits when scw+/−, sog+/−, tsg+/−, and tld+/− cases are considered. In principle, the binding cascade analysis extends to other systems and can be used to explore other changes of input, including overexpression of a protein (Shimmi, 2005a).

The crossveinless gene encodes a new member of the Twisted gastrulation family of BMP-binding proteins which, with Short gastrulation, promotes BMP signaling in the crossveins of the Drosophila wing

In the early Drosophila embryo, Bone morphogenetic protein (BMP) activity is positively and negatively regulated by the BMP-binding proteins Short gastrulation (Sog) and Twisted gastrulation (Tsg). A similar mechanism operates during crossvein formation, utilizing Sog and a new member of the tsg gene family, encoded by the crossveinless (cv) locus. The initial specification of crossvein fate in the Drosophila wing requires signaling mediated by Dpp and Gbb, two members of the BMP family. cv is required for the promotion of BMP signaling in the crossveins. Large sog clones disrupt posterior crossvein formation, suggesting that Sog and Cv act together in this context. sog and cv can have both positive and negative effects on BMP signaling in the wing. Moreover, Cv is functionally equivalent to Tsg, since Tsg and Cv can substitute for each other's activity. It is also confirmed that Tsg and Cv have similar biochemical activities: Sog/Cv complex binds a Dpp/Gbb heterodimer with high affinity. Taken together, these studies suggest that Sog and Cv promote BMP signaling by transporting a BMP heterodimer from the longitudinal veins into the crossvein regions (Shimmi, 2005b).

One interesting aspect of BMP signaling in many developmental contexts is that its activity can be regulated at the extracellular level by a number of secreted factors. In Drosophila, these include the products of the short gastrulation (sog), twisted gastrulation (tsg), and tolloid (tld) genes. All three genes were identified as modulators of BMP signaling in the early embryo, and their developmental functions have been well characterized. At the blastoderm stage, BMP signals provided by the dorsally expressed Decapentaplegic (Dpp), and by the generally expressed Screw (Scw), a second ligand that forms a heterodimer with Dpp (Shimmi, 2005a), instruct cells to adopt either amnioserosa or dorsal ectoderm fate. Proper subdivision into these two cell types requires the action of Sog, Tsg, and Tld. Sog and Tsg are BMP-binding proteins that make a high-affinity complex with the Dpp/Scw heterodimer. This complex reduces BMP signaling in the dorsal-lateral regions by blocking the ability of the heterodimer to bind to receptors. Thus, a major role of the Sog/Tsg complex is to antagonize signaling, and similar activity has been found for the vertebrate homologs Chordin and Tsg (Shimmi, 2005b and references therein).

However, the Sog/Tsg complex also stimulates BMP signaling in the dorsal-most cells of Drosophila embryo; it is thought to do so by protecting the ligand from degradation and enabling it to diffuse over long distances. Since sog is expressed in ventral-lateral cells adjacent to the dorsal cells that express dpp, scw, and tsg, the net flux of Sog towards the dorsal side provides a driving force that concentrates BMP heterodimer in the dorsal-most region of the embryo. The ligand is then released for signaling by Tld, an extracellular metalloprotease that cleaves Sog in a BMP-dependent manner. Concentration of the heterodimer to the dorsal-most cells by this facilitated transport mechanism provides a high level signal that specifies amnioserosa cell fate, while dorsal-lateral cells receive less BMP signal and become dorsal ectoderm (Shimmi, 2005b and references therein).

The ability of Tsg to stimulate BMP signaling is apparently not limited to the early Drosophila embryo. Vertebrate Tsg can stimulate BMP signaling in some circumstances, and is required to stimulate high levels of BMP signaling during axis formation in the zebrafish embryo. However, in these cases, Tsg may act, not via a transport mechanism, but by antagonizing Chordin's ability to inhibit BMP signaling. Tsg increases the rate at which Chordin and Sog are cleaved and thus inactivated by Tolloid-like protease. Nonetheless, zebrafish Chordin can also apparently stimulate BMP signaling in some circumstances (Shimmi, 2005b and references therein).

This paper reports another context in which both Sog and a novel Tsg family member stimulate BMP signaling at the developing crossveins in the Drosophila pupal wing. The Drosophila wing has proven to be an attractive model system for elucidating molecular mechanisms that regulate growth and patterning. A major attribute of this system is the stereotypical array of veins that develop along the wing surfaces. These thickenings of the ectodermal cuticle serve both structural support roles for flight and act as channels for the supply of nutrients to the wing cells. For the geneticist, they provide a key set of morphological landmarks for identification of genes that affect the patterning process. Analysis of many classical mutations that alter vein cell fate and patterning have revealed the fundamental roles played by three highly conserved growth factor signaling pathways. For the five longitudinal veins (L1-L5) that form along the proximal-distal axis, a key initiating event is the localized expression of Epidermal growth factor (EGF) signaling components in the vein primordial cells during late imaginal disc development. In response to EGF receptor signaling, Delta is expressed along the veins and induces Notch to inhibit vein formation in neighboring cells. Subsequently, during pupal stages, EGF receptor signaling induces expression of dpp within the developing longitudinal veins. Expression of dpp in the longitudinal veins is required for maintenance of EGF receptor signaling and final vein differentiation, especially at the distal tips (Shimmi, 2005b and references therein).

In addition to the five longitudinal veins, two other shorter veins form perpendicular to the longitudinal veins; these are the anterior crossvein (ACV), which forms between L3 and L4, and the posterior crossvein (PCV), which forms between L4 and L5. Unlike the longitudinal veins, the crossveins do not rely on early EGF signaling for their initial specification. Instead, their formation is initiated during pupal stage by localized BMP signaling, which requires Dpp. However, in the case of the PCV, Dpp is not initially produced in the crossvein, but instead diffuses into the PCV region from the longitudinal veins. Dpp does not act alone during this process since mutations in glass bottom boat (gbb), a member of the BMP5/6/7 subfamily, also eliminate the PCV. Gbb is widely expressed during pupal wing development, but an analysis of gbb mutant clones has suggested that the active BMP component for PCV specification might be a heterodimer of Dpp and Gbb formed in the longitudinal veins, since the PCV is lost only when the clone includes cells of the longitudinal veins where Dpp is produced (Shimmi, 2005b and references therein).

Like the embryo, modulation of BMP signaling in the PCV also appears to involve several additional secreted proteins. For example, mutations in tolloid-related (tlr; also known as tolkin) and crossveinless 2 (cv-2) eliminate PCV formation by preventing BMP signals in the primordial PCV cells. The tlr gene encodes a Tolloid-like metalloprotease that is able to cleave Sog, while cv-2 encodes a protein containing 5 cysteine-rich (CR) domains, similar to the BMP-binding modules found in Sog (Shimmi, 2005b).

The similarity of these proteins to those involved in patterning the early Drosophila embryo suggests that correct specification of the PCV likely involves establishing a spatially regulated distribution of BMP ligand(s) through the activity of extracellular modulatory factors. This report provides additional evidence supporting this hypothesis by cloning the crossveinless (cv) gene and analyzing its function. cv encodes a new member of the tsg gene family. Like mutations in cv-2 and tlr, loss of cv prevents accumulation of phosphorylated Mad (pMad), the active form of the major transcription effector of BMP signaling, in the crossvein cells. In addition, ectopic expression studies were used to show that Cv and Tsg have functionally related activities, since each can substitute for the other in vivo, and ectopic expression of Cv and Sog phenocopies co-expression of Tsg and Sog. These observations led to a re-examination of the role of Sog during wing vein development. Previous clonal analyses suggested that Sog acts as a dedicated antagonist of BMP signaling and helps maintain longitudinal vein integrity. However, this study shows that Sog is in fact required for BMP signaling in the PCV, since large sog clones inhibit PCV formation. These data suggest that, as in the early embryo, Sog plays a dual role in promoting and inhibiting BMP signaling. In light of these results, the biochemical properties of Cv were examined, and it was found that, like Tsg, it can form a high-affinity oligomeric complex with Sog and BMP heterodimers (Shimmi, 2005b).

Taken together, these results suggest that similar mechanisms govern PCV development and early embryonic development. In the embryo, a Dpp/Scw heterodimer specifies amnioserosa fate following ligand transport from lateral to dorsal-most regions through the action of Sog and Tsg. Processing of the complex by Tld then enables signaling in a restricted spatial domain. It is speculated that formation of the PCV likely requires selective transport of a Dpp/Gbb heterodimer from the longitudinal veins to the PCV competent zone through the action of Sog and the Tsg-like protein Cv. As in the embryo, the Tolloid-related enzyme may release the ligand through processing of the Sog/Cv/BMP complex to generate a spatially restricted pattern of signaling in the PCV. This example illustrates how, in different developmental contexts, related molecules and common mechanistic processes can achieve new patterning outcomes (Shimmi, 2005b).

The formation of Drosophila wing veins is a very sensitive system for examining the activity of BMP signaling within the context of a developmental patterning process. Two distinct aspects of the BMP signaling process in veins have been recognized. (1) BMP signals are produced by the developing longitudinal veins where they act locally to help maintain the vein fate earlier specified by EGF signaling. (2) BMP signals produced in the longitudinal veins act at longer range to initiate BMP signaling in the crossveins. This study shows evidence that this long-range signaling requires the activity of both Sog and a Tsg-like molecule encoded by the cv gene. Thus, within the context of the crossveins, Cv and Sog play positive roles in BMP signaling. Based on analogy to the embryonic patterning system, these results suggest that Cv and Sog aid in the transport of BMP ligands from producing cells to receiving cells in the posterior crossvein competent zone (Shimmi, 2005b).

Since Tsg and Cv showed a similar domain structure, attempts were made to determine if they were functionally equivalent by expressing one in place of the other during either embryonic or pupal development. These experiments showed that these two products are, to some extent, genetically interchangeable. However, Tsg and Cv may have been optimized for a particular developmental function that likely represents interactions with a particular ligand, i.e., Dpp/Scw heterodimers in the case of Tsg and Dpp/Gbb heterodimers in the case of Cv. A recent phylogenetic comparison of the Cv and Tsg proteins from different insect species suggests that these two proteins fall into distinct families, one Cv-like and one Tsg-like). In addition, under conditions of overexpression, cv and tsg exhibit enhanced genetic interactions with different BMP ligands. For instance, cv interacts better with gbb than with dpp. While no difference was seen in the ability of Sog and Tsg versus Sog and Cv to bind to Dpp/Gbb heterodimers, these data are qualitative. Thus, it is possible that these two protein complexes could have different affinities for different ligands that are optimal for their particular developmental function (Shimmi, 2005b).

A similar observation has recently been made for Tld and Tlr proteins. These two metalloproteases show very similar overall structure and both cleave Sog in the same positions, but with different kinetics and site preferences. In this case, the two proteins cannot substitute for the other and it has been proposed that this represents optimization of catalytic activity for a fast (Tld in the embryo) or slow (Tlr in pupal wing vein) developmental function (Shimmi, 2005b).

In the early embryo, Tsg and Sog function together to help redistribute BMP signals from their broad initial distribution profiles throughout the dorsal half of the embryo into a narrow stripe of cells centered on the dorsal midline. In this model, Tsg and Sog play both positive and negative roles. The positive role comes via transport and the resulting increase in BMP concentration at the dorsal midline. The negative role comes from blocking access of the ligand to receptors in the lateral regions during BMP transport (Shimmi, 2005b).

The process of PCV formation appears remarkably similar, at least in terms of the BMP signaling components employed. Both Sog and the Tsg-like molecule Cv are required for BMP signaling, as indicated by the accumulation of pMad, in the developing crossveins. Thus, both Cv and Sog play positive roles in augmenting BMP signaling during crossvein formation. This positive role may also come from facilitating transport of BMPs, since co-expression of Cv and Sog in the posterior compartment resulted in ectopic vein formation and BMP signaling gains in the anterior compartment. Similar, although less penetrant, effects on anterior venation have been observed when Sog alone is misexpressed (Shimmi, 2005b).

In addition, Cv and Sog may also inhibit BMP signaling around the longitudinal veins. A sog mutant clonal analysis has demonstrated a requirement for Sog in keeping longitudinal veins straight and narrow. In the absence of Sog, the veins meandered. A similar effect on the longitudinal veins is seen when cv is lost, with an expansion in pMad accumulation around the longitudinal veins. Thus, Cv and Sog may function together to restrict the range of Dpp signaling along the longitudinal veins (Shimmi, 2005b).

Two other similarities between embryonic patterning and PCV formation are worth noting. In the embryo, the Tld metalloprotease is required to release ligand from the inhibitor complex of Sog and Tsg. Likewise, the Tolloid-related protease Tlr is required for PCV formation. Tlr is expressed in the pupal wings, when it is required for crossvein pMad, and has recently been shown to process Sog at the same three sites as does Tld (Serpe, 2005). Thus, it seems likely that Tlr is needed to release a BMP ligand for signaling in the PCV competent zone (Shimmi, 2005b).

There may also be strong similarities between the embryo and crossvein patterning in their use of ligands. In the embryo, both Dpp and Scw are needed to specify the amnioserosa, while for PCV specification, both Dpp and Gbb are required. Sog and Tsg show the highest affinity for the heterodimer of Dpp/Scw in the embryo, suggesting that this is the primary transported ligand. Similarly, the ligand with highest affinity for Cv and Sog is a heterodimer of Dpp and Gbb. Interestingly, gbb mutant clonal analysis has shown that the PCV is lost only when a gbb clone encompasses adjacent longitudinal vein material. Since the longitudinal veins serve as the source of Dpp during PCV specification, these observations are consistent with the notion that a heterodimer of Dpp and Gbb is the primary ligand that specifies PCV formation (Shimmi, 2005b).

Although similarities between embryonic dorsoventral patterning and PCV formation are striking, there are clear differences. The most notable is the geometry of the system. Why is the long-range signaling from the longitudinal veins limited to the crossvein regions? Examination of the expression patterns of several components may provide some clues. Tkv expression is reduced in the crossveins, and since binding to receptor is a major impediment to diffusion in wing discs, this might enhance net flux of ligand into the area of reduced Tkv expression. However, down-regulation of tkv in the PCV actually depends on high levels of BMP signal (Ralston, 2005). Therefore, it is not likely that reduced Tkv expression provides a channel for ligand flow; rather, it may reinforce a flux direction that is initiated by other means (Shimmi, 2005b).

In this regard, it is notable that sog expression is also reduced in the crossvein regions and this is independent of BMP signaling (Ralston, 2005). As in the embryo, Sog flux from areas of high expression, i.e., intervein regions in the wing, into areas of low expression, the crossvein zones, might provide the proper positional information. Consistent with this view is the observation that uniform expression of Sog eliminates the initial stages of crossvein development. However, there are inconsistencies in this simple model. While misexpression of Sog can lead to loss of the crossvein, normally positioned crossveins appear when Sog misexpression is coupled with ubiquitous expression of Cv-2 (Ralston, 2005), suggesting that crossvein positioning can be independent of the sog expression pattern. Similarly, loss of sog from clones does not induce ectopic crossveins (this study) (Shimmi, 2005b).

Another possibility is that the cleavage of Sog is spatially regulated. In the embryo, Tld is expressed in the dorsal domain, and since its ability to process ligand is dependent on the Dpp concentration, the processing rate will be highest at the dorsal midline. However, in the pupal wing, tlr is not expressed at higher levels in the crossvein zones; instead, it is high in the entire intervein. Therefore, it is not clear how the ligand would be released from a complex of Cv and Sog specifically in the crossveins. Moreover, uniform expression of tlr causes only mild expansion of the crossveins (Shimmi, 2005b).

Perhaps the key to understanding the differences between the embryonic patterning process and that of the crossveins will be determining the mechanism of action of other gene products that are required for crossvein formation. These include cv-c, cv-d, detached, and the cv-2 gene products. Among these genes, only the cv-2 product has been identified; it is a large secreted factor that contains CR domains, similar to those found in Sog, and is expressed in the developing crossveins. The major distinction between Sog and Cv-2 is that Cv-2 contains a Von Willebrand type D domain found on many blood-clotting proteins that is not present in Sog. In Chordin, the CR modules are responsible for BMP binding and vertebrate Cv-2 homologs have also been shown to bind BMPs. Depending on the assay used, vertebrate Cv-2 homologs can either inhibit or promote signaling. Cv-2 does not seem to be required in the early embryo, yet it is essential for crossvein formation. Drosophila Cv-2, like its vertebrate counterparts, can also bind BMPs and, although it is a secreted protein, Cv-2 can associate with the cell surface. One possibility is that it captures BMPs, perhaps from a Sog/Cv complex, and keeps them close to the cell surface and in this way promotes BMP signaling by keeping the local BMP concentration high. It may also play a more direct role as a coreceptor (Shimmi, 2005b).

Nonetheless, while cv-2 is expressed in the crossveins, and is required for BMP signaling there, ubiquitous expression of cv-2 does not disrupt the positioning of the crossveins, even when coupled with ubiquitous expression of sog. Thus, other genes must act in conjunction with or upstream of these BMP modulators to help establish the crossvein competent zone. It is interesting to note in this regard that mutations in CDC42 induce ectopic crossveins, suggesting that it might be involved in the process that selects the site of crossvein formation (Shimmi, 2005b).

Normally, tsg is expressed only in the early blastoderm embryo. However, sog is expressed at several other developmental stages. In fact, this was one of the motivations to look for additional Tsg homologs, so that it might be determined if Sog always utilizes a Tsg-like partner or whether in some developmental processes it might act alone. One late embryonic process in which Sog has been implicated is to regulate tracheal morphogenesis. As in vein formation, tracheal patterning requires input from the EGFR and Dpp pathways. However, in this case, each pathway is antagonistic to the other. Normally, sog is expressed as a dorsal stripe abutting the tracheal pits, and in sog mutant embryos, hyper-activation of Dpp leads to a loss of dorsal trunk and a reduction in visceral branches. It was therefore interesting that cv is also expressed in and around the tracheal pits, but tsg is not expressed at this stage. However, no alteration was observed in tracheal development in cv mutants. Indeed, these embryos appear fully viable and the resulting adults are fertile. These results suggest that Cv has no other essential role in development. The pattern of cv expression around the tracheal pits may reflect a prior evolutionary involvement in tracheal development that is now provided by Sog alone or perhaps by Sog in conjunction with some other unknown BMP modulatory factor (Shimmi, 2005b).

Drosophila Follistatin exhibits unique structural modifications and interacts with several TGF-beta family members

Follistatin (FS) is one of several secreted proteins that modulate the activity of TGF-β family members during development. The structural and functional analysis of Drosophila Follistatin (dFS) reveals important differences between dFS and its vertebrate orthologues: it is larger, more positively charged, and proteolytically processed. dFS primarily inhibits signaling of Drosophila Activin (dACT) but can also inhibit other ligands like Decapentaplegic (DPP). In contrast, the presence of dFS enhances signaling of the Activin-like protein Dawdle (DAW), indicating that dFS exhibits a dual function in promoting and inhibiting signaling of TGF-β ligands. In addition, FS proteins may also function in facilitating ligand diffusion. Mutants of daw are rescued in significant numbers by expression of vertebrate FS proteins. Since two PiggyBac insertions in dfs are not lethal, it appears that the function of dFS is non-essential or functionally redundant (Bickel, 2008).

Polypeptide cytokines of the transforming growth factor β (TGF-β) family control a wide range of developmental and physiological functions in higher eukaryotes. This diverse group of signaling molecules provides positional information required for axis formation and tissue specification, controls various processes such as tissue growth, cell death, and pathfinding of axons in the nervous system, and prevents differentiation of embryonic stem cells. Many components of this pathway have been linked to tumor formation in humans. The highest degree of sequence conservation between various family members is found within the C-terminal domains, which are released as dimers by proteolytic processing. Similarities in sequence and biological activities allow these factors to be divided into at least two distinct subgroups: Bone Morphogenetic Proteins (BMPs) and Activins/Inhibins/TGF-βs. The latter group exhibits an additional intramolecular disulfide bond at the N-terminus after processing. In Drosophila, there are four Activins/TGF-βs, Drosophila Activin (dACT), Dawdle (DAW, also known as Activin-like protein at 23, ALP23, and Anti-Activin, AACT), Myoglianin (MYO), and Maverick (MAV), and three BMP-type ligands, Decapentaplegic (DPP), Screw (SCW), and Glass Bottom Boat (GBB). Each ligand dimer forms a complex with two type II and two type I receptor serine/threonine kinases that phosphorylate SMAD transcription factors. BMP-type ligands signal primarily through the type I receptors thick veins (TKV) and Saxophone (SAX) and activate Mothers against DPP (MAD). Activins/TGF-β-type ligands are believed to signal through the type I receptor Baboon (BABO), which in turn activates primarily dSMAD2 but to a minor extent also MAD (Bickel, 2008)

TGF-β signaling is regulated by various extracellular proteins. Antagonists like Follistatin (FS), Noggin, Chordin/Short Gastrulation, and DAN/Cerberus bind ligands and prevent interactions with receptors and signaling. In some species, they exhibit overlapping and redundant functions. Recently, it was shown that the simultaneous depletion of FS, Noggin, and Chordin in Xenopus tropicalis results in transformation of ventral into dorsal tissue during embryogenesis (Bickel, 2008).

Follistatin was first identified as an inhibitor of Activin in vertebrates. Subsequent studies showed that it also binds other ligands with lower affinities including BMP 2, 4, 6, 7, and Myostatin. Knockout mice of fs die shortly after birth. They are smaller and exhibit defects in skeletal and muscle development. Recently, the crystal structure of the human FS-Activin complex was resolved. It provides valuable insight into the function of the different FS domains and a basis to explain the mechanism of ligand inhibition (Bickel, 2008 and references therein)

This study has analyzed the function of Drosophila Follistatin (dFS). Like vertebrate FS proteins, dFS is subdivided into a N-terminal domain (N) and three FS domains (FS1-3). However, dFS is substantially larger than its vertebrate homologues due to a large basic insertion into FS1. Interestingly, dFS is proteolytically processed, and small processed forms of dFS are able to bind to ligands like dACT. This result suggests a possible different inhibitory mechanism: ligands bound to processed dFS can bind to type II receptors but cannot recruit type I receptors. Consequently, processed dFS might not only sequester ligands but also prevent unbound ligands from interacting with receptor complexes. Among the seven Drosophila TGF-β ligands, dFS primarily inhibits dACT but can also inhibit signaling of other ligands like DPP. In contrast, dFS can augment signaling of the TGF-β member DAW. These results suggest that dFS might exhibit dual functions in facilitating and inhibiting TGF-β signaling. Analysis of two PiggyBac insertions in dFS reveals that they affect dfs expression. Since homozygous animals of these lines are viable and phenotypically wild type, it is assumed that the function of dFS is non-essential or functionally redundant. Taken together, this study reveals interesting differences between the mechanisms of modulating TGF-β signaling by dFS and its vertebrate orthologues (Bickel, 2008)

The primary structure of dFS shows both similarities and differences compared to its vertebrate orthologues. dFS is divided into a N-terminal domain (N) and three FS domains (FS1-3) that can be further subdivided into EGF-like and Kazal protease inhibitor-like domains. Compared to its vertebrate orthologues, dFS is substantially larger due to an insertion of 260 amino acids. The insertion contains 55 positively charged amino acids (pI > 10) and is located after the heparin binding site in FS1. In contrast to Drosophila, vertebrates produce a second, long isoform of FS by alternative splicing (FS-315). This form contains a C-terminal extension with many negatively charged amino acids that reduce adhesion to sulfates of proteoglycans at the cell surface. FS-315 is the major mammalian circulating form (Bickel, 2008)

To analyze the function of dFS, dfs cDNA was cloned into expression vectors for tissue culture cells and transgenic flies. From comparison to vertebrate orthologues, the start site was assumed to be at nucleotide 577. At this position, the SignalP server website predicts a signal sequence (MALLIGLLLLNFRLTAA-GTCW) that is cleaved equivalently to the vertebrate FS proteins. However, expression of a construct that contains this predicted signal sequence but lacks the first 279 nucleotides does not result in a translated protein in culture cells and growth inhibition in transgenic flies. In contrast, the complete cDNA is translated in tissue culture cells and inhibits growth in vivo. There are three potential start codons upstream of the predicted signal peptide sequence. Based on the consensus for Drosophila start sites, the most likely start codon is at nucleotide 181-183 (CAACA-ATG). It is noted that there is a full-length dfs cDNA that is 671 nucleotides longer, GH04473 (NM_144119). This cDNA extends the coding sequence by 62 additional amino acids. Although conserved between different Drosophila species, the absence of this additional sequence in the shorter cDNA does not prevent translation and secretion of a growth inhibitory protein. Interestingly, the full-length cDNA does not encode an N-terminal signal peptide sequence either. Instead, several protein analysis programs predict three potential transmembrane domains within the 211 amino acid leader. Thus, in contrast to vertebrate FS proteins, the secretion of dFS might involve an initial membrane-anchoring step (Bickel, 2008)

There are several differences in the structure of dFS and its vertebrate orthologues. One unique characteristic of dFS is the unusual signal trailer that contains three potential transmembrane domains. The results suggest that cleavage likely occurs after the third transmembrane domain at the equivalent position of vertebrate FS proteins. No evidence was found that this feature affects secretion or alters the function of dFS (Bickel, 2008)

A major difference between dFS and its vertebrate orthologues is the large basic insertion. This modification is likely consequential and may alter the activity of dFS. Based on protein folding prediction and the structure of the human FS-Activin complex, the insertion is not well structured and is located after the heparin binding site within the EGF sub-domain of FS1. Since this area projects away from Activin, the positively charged amino acids likely increase the affinity of dFS to heparin sulfate proteoglycans on the cell surface and do not contribute to Activin binding. It is conceivable that this feature leads to a reduction of diffusion and an increase in local concentration of dFS. In addition, it may also reduce diffusion of ligands and enhance the stability of ligand-receptor complexes. Vertebrates have adopted an opposite strategy. They generate a long form of FS by alternative splicing, which contains a negatively charged C-terminal tail that reduces interactions with heparin sulfate. The long form is the major endocrine form in humans. Since it does not efficiently interact with the cell surface, it is not clear whether this form primarily inhibits Activin signaling or actually enhances Activin circulation by preventing unwanted interactions with neighboring cells (Bickel, 2008)

The FS-Activin complex shows that the N domain binds to the wrist region of Activin blocking interaction with the type I receptor. The FS2 domain binds to the type II receptor binding site of Activin. In humans, two FS proteins can encircle an Activin dimer preventing interactions with both types of receptors. It was found experimentally that Activin bound to FS still can interact with the type II receptor. This result is explained if only one molecule of FS is bound to an Activin dimer leaving the second Activin monomer free to bind to receptors. Unlike vertebrate FS proteins, this study found that dFS is proteolytically processed at the C-terminus into two major forms. Based on the migration of epitope tagged forms of dFS on Western blots, the small form contains at least the N and FS1 domains and lacks FS3 and probably part of FS2. Since the small form is immunoprecipitated by dACT, it appears that it can bind to dACT. What is the possible role of this small processed form? The following model suggests that the small form could potentially be a stronger inhibitor: if two small processed dFS proteins without FS2 bind to dACT monomers, they prevent interactions with type I but not type II receptors. A dFS-dACT-PUT complex is inactive, since dACT cannot recruit the type I receptor BABO. In this scenario, the small dFS forms would not only inactivate bound dACT but also reduce interactions of free dACT with type II receptors. If type II receptors were limiting, the small dFS forms would be able to reduce dACT signaling more efficiently than the full-length protein. This hypothesis can be tested by expressing transgenes that encode the small form of dFS. To perform these experiments, the structures of the small and large forms of dFS are currently being determined (Bickel, 2008)

In most experiments, dFS was seen to function as an inhibitor of TGF-β signaling. As a major exception, dFS enhances rather than inhibits DAW signaling in tissue culture assays. Interestingly, unlike other ligands, DAW does not increase but reduces the viability of animals that over-express dFS. This observation suggests that over-expression of dFS may not only be lethal due to reducing the activities of various TGF-β family members but also due to increased signaling of ligands like DAW. If DAW and dFS physically interact, the question arises how dFS can increase ligand signaling. In the early embryo, it has been shown that Short Gastrulation (SOG), the Drosophila orthologues of the vertebrate Chordin protein, not only inhibits signaling of DPP but also facilitates diffusion, which is necessary for the formation of the peak levels of the DPP gradient. The formation of the DPP gradient depends on the equilibrium between bound and unbound DPP by SOG. Similarly, it is conceivable that ligand binding by the positively charged dFS can reduce ligand diffusion. If the concentration or the affinity of dFS for a ligand is high, the ligand is not released or bound again, and signaling is inhibited. Alternatively, if the concentration of dFS or its affinity for a ligand is low, dFS binding can locally increase ligand concentration, while subsequent ligand release will enhance signal transduction. Consistent with such a model, in tissue culture assays dFS rather increases than decreases the level of MAD activation by DPP, while presumably higher levels of dFS inhibit DPP signaling in the wing. Such a dual function could probably be at work during dorsal-ventral axis formation. While dact is not expressed in the early embryo, dfs is present in a dorsal stripe. This pattern corresponds with the highest levels of DPP signaling. Expression of dfs is rather late in the formation of the DPP gradient and is potentially regulated by DPP. It is conceivable that initial low levels of dFS could, like SOG, function to increase the local concentration of DPP and contribute to the formation and maintenance of the dorsal peak activity of DPP signaling. In contrast, higher levels of dFS protein that accumulate by the end of the dorsal-ventral axis formation process may inhibit DPP and contribute to terminating the dorsal DPP signal. Recently, a computational model that described ligand distribution and signaling in the presence of a cell surface BMP-binding protein was developed that supports this idea. A dual function of FS proteins in facilitating and inhibiting TGF-β signaling is also supported by the finding that fs mutants in mice exhibit overlapping phenotypes with activin knock out animals. Consequently, FS was originally described as enhancer of Activin signaling. Finally, facilitating diffusion and redistribution of dACT and potentially other ligands is probably the best mechanism to explain why dFS and vertebrate FS proteins from frogs and humans can partially compensate for the lack of DAW. It is speculated that the vertebrate FS proteins exhibit lower affinities for Drosophila ligands like dACT and diffuse better since they lack the basic insertion of dFS. Most likely, these differences could explain why expression of the vertebrate but not dFS can rescue small but significant numbers of daw mutant animals. Taken together, it is possible that distinct affinities for dFS account for the different interactions seen with various ligands. Further studies are necessary to investigate such a possible dual function of dfs in early embryos and during development (Bickel, 2008).

Two PiggyBac insertions affect dfs transcription. Interestingly, both lines are homozygous viable and do not show any obvious pattern defects. These results suggest that the function of dFS not essential for viability. In mice, a mutation in fs results in various defects with lethal consequences. In contrast, FS, Chordin, and Noggin exhibit overlapping functions in X. tropicalis. It is necessary to reduce all three inhibitors to transform ventral into dorsal tissue during embryogenesis of this species. Although there is no Noggin-like protein in Drosophila, it is possible that dFS also shares overlapping functions with SOG. Expression of sog overlaps with dfs in many tissues, and it is possible that SOG can substitute for dFS in its absence (Bickel, 2008)

The analysis of dfs RNA levels in homozygous f00897 flies shows that they are substantially reduced. Thus, this insertion can be regarded as a hypomorphic allele. It is conceivable that the amount of protein synthesized in this mutant is sufficient for normal development. However, further reduction of dFS by combining f00897 with the deficiency Df(2R)Exel7135 that entirely removes dfs does not affect viability either. In contrast to f00897, the PiggyBac insertion in e03941 clearly disrupts transcription of a full-length mRNA. Based on the location of the insertion, the truncated mRNA likely encodes an altered protein that still contains the N, FS1, and FS2 domains. It lacks the entire FS3 domain and likely contains additional C-terminal amino acids due to altered or absence of splicing. Since the small form is still able to bind dACT, a partially functional dFS protein could still be present in e03941 flies, if proper processing would occur. However, it is unlikely that such an altered protein is processed correctly into the small form of dFS. Since no homozygous lethal lines were obtained in the FRT-mediated deletion screen, it appears that dfs is not lethal. Taken together, the lack of any obvious phenotypes in homozygous f00897 and e03941 lines suggests that dFS is not essential for normal development or is redundant like in some vertebrate species (Bickel, 2008)

Dally regulates Dpp morphogen gradient formation by stabilizing Dpp on the cell surface

Decapentaplegic, a Drosophila homologue of bone morphogenetic proteins, acts as a morphogen to regulate patterning along the anterior-posterior axis of the developing wing. Previous studies showed that Dally, a heparan sulfate proteoglycan, regulates both the distribution of Dpp morphogen and cellular responses to Dpp. However, the molecular mechanism by which Dally affects the Dpp morphogen gradient remains to be elucidated. This study characterized activity, stability, and gradient formation of a truncated form of Dpp (DppΔN), which lacks a short domain at the N-terminus essential for its interaction with Dally. DppΔN shows the same signaling activity and protein stability as wild-type Dpp in vitro but has a shorter half-life in vivo, suggesting that Dally stabilizes Dpp in the extracellular matrix. Furthermore, genetic interaction experiments revealed that Dally antagonizes the effect of Thickveins (Tkv; a Dpp type I receptor) on Dpp signaling. Given that Tkv can downregulate Dpp signaling by receptor-mediated endocytosis of Dpp, the ability of dally to antagonize tkv suggests that Dally inhibits this process. Based on these observations, a model is proposed in which Dally regulates Dpp distribution and signaling by disrupting receptor-mediated internalization and degradation of the Dpp-receptor complex (Akiyama, 2008)

The Dpp pathway is regulated by multiple cell surface and extracellular factors. In the developing wing, Dally is one of the key molecules that modulate Dpp signaling. It affects the shape of the Dpp ligand gradient (protein distribution) as well as its activity gradient (spatial patterns of signaling activity). Dally and Dpp expressed in S2 tissue culture cells are coimmunoprecipitated, suggesting that Dally forms a complex with Dpp. It was also observed that Dally colocalizes with Dpp and Tkv in cells. In addition, Dally enhances Dpp signaling in a cell autonomous fashion. These findings suggest that Dally acts as a Dpp co-receptor at least in some developmental contexts. Interestingly, however, in embryos and in imaginal disc cells close to Dpp-expressing cells, Dpp can mediate signaling without Dally, indicating that HS is not absolutely required for all BMP-dependent processes in vivo (Akiyama, 2008)

DppΔN, which does not bind to heparin, fails to interact with Dally. The easiest interpretation of this result is that wild-type Dpp interacts with Dally via its HS chains. However, a recent study using Surface Plasmon Resonance showed that binding of BMP4 to Dally is not fully inhibited by excess HS. Also, a mutant form of Dally, which does not undergo HS modification, is able to significantly rescue dally mutant phenotypes. These findings suggest that the BMP-glypican interaction is not entirely dependent on the HS chains. One possible explanation for the failure of DppΔN to bind to Dally is that Dpp normally binds to Dally through both the HS chains and its protein core, and DppΔN has reduced affinities for both sites (Akiyama, 2008)

Although DppΔN lacks the ability to interact with Dally, it shows normal in vitro protein stability and signaling activity. Therefore, this truncated form of Dpp provided a powerful system to gain insight into the functions of Dally in distribution and signaling of the Dpp morphogen: this molecule was used to elucidate the consequences of lacking the ability to bind HSPGs. In the wing disc, DppΔN cannot form a normal gradient: only a low level of DppΔN was detected in the Dpp-receiving cells. Notably, this pattern of DppΔN resembles the Dpp ligand and activity gradients observed in dally mutant wing discs. A series of in vitro and in vivo Dpp stability assays suggested that DppΔN forms a shallow gradient because it is remarkably unstable in the matrix and that the stability of Dpp depends on its interaction with Dally (Akiyama, 2008)

Genetic experiments revealed that Tkv and Dally have opposite effects on Dpp gradient formation during wing development. Dally and Tkv share some common properties as components of the Dpp signaling complex: they both autonomously enhance Dpp signaling and limit migration of Dpp by binding to Dpp protein. Nevertheless, tkv and dally mutually suppress one another’s pMad gradient phenotypes. Consistent with the genetic interactions observed in dally and tkv mutants, the pMad phenotype produced by overexpression of tkv was significantly restored by coexpression of dally. These observations indicate that dally antagonizes tkv in Dpp signaling. Since it has been proposed that Tkv promotes Dpp degradation by receptor-mediated endocytosis, dally may stabilize Dpp by inhibiting this process (Akiyama, 2008)

Altogether, these studies suggest that Dally serves as a co-receptor for Dpp and regulates its signaling as well as gradient formation by disrupting the degradation of the Dpp-receptor complex. In this model, the Dpp signaling complex with Dally co-receptor would remain longer on the cell surface or in the early endosomes to mediate signaling for a prolonged period of time. In contrast, in the absence of Dally, the complex would be relatively quickly degraded. This possible role of Dally can account for the shrinkage of the Dpp gradient in dally mutant wing discs. However, the possibility cannot be excluded that DppΔN is lost from the cell surface by lack of retention and further diffuses away (Akiyama, 2008)

A previous kinetic analysis of FGF degradation in cultured mammalian vascular smooth muscle cells also showed that HSPG co-receptors can enhance FGF signaling by stabilizing FGF. In these cells, the intracellular processing of FGF-2 occurred in stages: low molecular weight (LMW) intermediate fragments accumulated at the first step. Blocking HS synthesis by treatment of cells with sodium chlorate substantially reduced the half-life of these LMW intermediates, indicating that HSPGs inhibit a certain step of the intracellular degradation of FGF-2. HSPGs have also been implicated in the endocytosis and degradation of Wg. Wg protein is endocytosed from both apical and basal surfaces of the wing disc and degraded by cells to downregulate the levels of Wg protein in the extracellular space. It has been proposed that Dally-like (Dlp), the second Drosophila glypican, regulates the Wg gradient by stimulating the translocation of Wg protein from both the apical and basal membranes to the lateral side, a less active region of endocytosis, thereby inhibiting degradation of Wg protein (Akiyama, 2008)

Interestingly, DppΔN behaved differently in vivo from the previously reported mutant Xenopus BMP4 lacking the heparin-binding site. Although the action range of BMP4 is restricted to the ventral side during Xenopus embryogenesis, the truncated BMP4 migrated further in the embryo. In addition, heparitinase treatment of embryos also resulted in long-range diffusion of BMP4. These findings led to the conclusion that HSPGs trap BMP4 in the extracellular matrix to restrict its distribution in the Xenopus embryo. This activity of HSPGs seems to be opposite to that of Dally in the Dpp receiving cells of the Drosophila wing, where the major role of Dally is to stabilize Dpp protein. In general, ligands that fail to be retained on the cell surface can have any of the following fates: they may (1) migrate further and act as a ligand somewhere else, (2) be degraded by extracellular proteases, or (3) be internalized by endocytosis and degraded intracellularly. Theoretically, a mixture of all these phenomena can happen at the same time in a given tissue. However, which of these predominates may depend on cellular and extracellular environmental conditions such as concentrations of proteases in the matrix and the rate of endocytosis. Therefore, in the absence of HS, whether a major fraction of ligands is degraded or migrates further can be tissue-dependent, and the effects of HSPGs on BMP gradients will vary depending on the developmental context: a mutant BMP4 molecule moves further in a frog embryo, but DppΔN is degraded in Drosophila wing (Akiyama, 2008)

Although the results presented in this study support a role for HSPGs in Dpp stability, they do not rule out the possible involvement of HSPGs in migration of Dpp protein from cell to cell. The gradient of DppΔN is significantly narrower than that of wild-type Dpp, raising the argument that HS-binding plays a role also in normal Dpp migration in a tissue. Further studies will be required to determine whether or not HSPGs affect morphogen movement. This study also provides new insight into functional aspects of Dpp processing. Mature forms of Dpp generated by differential cleavages are likely to show different affinities for proteoglycans in the matrix. Therefore, they may have different half-lives and/or spatial distribution patterns in vivo. The biological significance of the occurrence of differently processed forms remains to be elucidated (Akiyama, 2008)

Type IV collagens regulate BMP signalling in Drosophila

Dorsal-ventral patterning in vertebrate and invertebrate embryos is mediated by a conserved system of secreted proteins that establishes a bone morphogenetic protein (BMP) gradient. Although the Drosophila embryonic Decapentaplegic (Dpp) gradient has served as a model to understand how morphogen gradients are established, no role for the extracellular matrix has been previously described. This study shows that type IV collagen extracellular matrix proteins bind Dpp and regulate its signalling in both the Drosophila embryo and ovary. Evidence is provided that the interaction between Dpp and type IV collagen augments Dpp signalling in the embryo by promoting gradient formation, yet it restricts the signalling range in the ovary through sequestration of the Dpp ligand. Together, these results identify a critical function of type IV collagens in modulating Dpp in the extracellular space during Drosophila development. On the basis of findings that human type IV collagen binds BMP4, it is predicted that this role of type IV collagens will be conserved (Wang, 2008).

There are two type IV collagen proteins in Drosophila, Viking (Vkg) and Dcg1 (also known as Cg25C). The ability was tested of secreted biologically active, epitope-tagged Dpp purified from the media of transfected Drosophila S2 tissue culture cells to bind to the amino- and carboxy-terminal non-collagenous domains of Vkg and Dcg1. Dpp-haemagglutinin (HA) binds to the C-terminal but not the N-terminal domains of both Vkg and Dcg1, whereas denatured Dpp-HA protein does not. Dpp/Scw heterodimers also bind to the Vkg and Dcg1 C-terminal domains. Surface plasmon resonance analyses show that the binding between Dpp and glutathione S-transferase (GST)-VkgC or GST-DcgC is saturable and has dissociation constants (Kd) of 0.75 and 0.65 microM, respectively (Wang, 2008).

Deletion analysis of VkgC identified a region required for Dpp interaction which, when aligned with the equivalent region of Dcg1, shows a short conserved sequence. Deletion of five of these amino acids from the Vkg C-terminal domain severely attenuates the interaction between Vkg and both Dpp and Dpp/Scw ligands. As these binding studies used GST fusion proteins purified from bacteria, the results were confirmed using GST-VkgC and GST-VkgCDelta proteins secreted into the media of transfected S2 cells. In addition, further mutational analysis was performed of the Vkg sequences required for Dpp interaction. The amino acids in Vkg that are necessary for Dpp interaction are present in a sequence that is conserved in mosquito, worm, mouse and human type IV collagens. Alignment of all the known type IV collagen sequences from these species identifies a consensus Y/FI/VSRCXVCE, which may function as a conserved BMP-binding module. In support of this, saturable binding has been shown between human full-length triple-helical type IV collagen and BMP4 with a Kd of 92 nM (Wang, 2008).

The data demonstrate that the interaction between Dpp and collagen IV is an essential aspect of correct signalling in the Drosophila germarium and early embryo. In wild-type germaria, it is suggested that Dpp secreted from the niche binds to Vkg, which restricts Dpp signalling range from the source. In mutant germaria with reduced Vkg protein, less Dpp will be bound by Vkg, resulting in an increased Dpp signalling range which downregulates bam transcription in more cells, thereby increasing GSC number (Wang, 2008).

In the embryo, a model is favored whereby binding of Dpp/Scw to type IV collagens facilitates assembly of the Dpp/Scw-Sog-Tsg complex. Tolloid (Tld) cleavage of this complex releases Dpp/Scw, which can rebind type IV collagens. In the presence of Sog, the inhibitory complex will be reassembled, whereas in the absence of Sog, type IV collagens will promote Dpp/Scw-receptor interactions. This latter function may require the unusual apical distribution of Vkg protein in the embryo, since Dpp seems to predominantly interact with its receptor apically in the embryo. In the dorsal ectoderm, initial Dpp signalling enhances subsequent Dpp signalling by the activation of an as yet unidentified target gene in a positive feedback loop. By promoting Dpp-receptor interactions at the dorsal midline leading to target gene activation, type IV collagens will facilitate the amplification of signalling by positive feedback (Wang, 2008).

The model explains the phenotype of embryos from vkg/+ females; the reduced amount of type IV collagens would impair assembly of the Dpp/Scw-Sog-Tsg complex and initial gradient formation. Disruption of the early gradient, in combination with reduced receptor interactions in type IV collagen mutant embryos, will reduce target gene expression and positive feedback, further decreasing subsequent signalling. As a result, the peak Dpp target genes are lost and intermediate thresholds are thinner (Wang, 2008).

In addition to the role of type IV collagens in regulating Dpp signalling in the early embryo that is described here, integrins (other principal constituents of basal lamina) are required for apposition of the amnioserosa and yolk sac to mediate proper germ band retraction and dorsal closure during later embryonic development. Therefore, basal lamina components have repeated roles in dorsal-ventral patterning of the fly embryo. Different types of extracellular matrix proteins also modulate BMP signalling at other development stages, for example, heparan sulphate proteoglycans regulate Dpp movement in the Drosophila wing. In vertebrates, type IV collagens are not only transcriptional targets of BMP signalling, but they also bind BMP4 and have been suggested to potentiate signalling in tissue culture cells. It is proposed that the conserved sequence that were identified will function as a BMP-binding module, and that type IV collagens will affect BMP signalling during vertebrate development (Wang, 2008).

Hemocyte-secreted type IV collagen enhances BMP signaling to guide renal tubule morphogenesis in Drosophila

Details of the mechanisms that determine the shape and positioning of organs in the body cavity remain largely obscure. This study shows that stereotypic positioning of outgrowing Drosophila renal tubules depends on signaling in a subset of tubule cells and results from enhanced sensitivity to guidance signals by targeted matrix deposition. VEGF/PDGF ligands from the tubules attract hemocytes, which secrete components of the basement membrane to ensheath them. Collagen IV sensitizes tubule cells to localized BMP guidance cues. Signaling results in pathway activation in a subset of tubule cells that lead outgrowth through the body cavity. Failure of hemocyte migration, loss of collagen IV, or abrogation of BMP signaling results in tubule misrouting and defective organ shape and positioning. Such regulated interplay between cell-cell and cell-matrix interactions is likely to have wide relevance in organogenesis and congenital disease (Bunt, 2010).

As the renal tubules extend through the body cavity, two processes occur; they elongate through cell rearrangements and they make precise, guided movements with respect to other tissues. A major source of the motive force required for tubule extension is the convergent-extension movements of the tubule cells themselves. As the tubules are continuous with the hindgut and thus have a fixed point proximally, these movements result in a distal-directed extensive force. This study shows that in addition the normal morphogenesis of the anterior tubules depends on tissue guidance involving the coordinated activity of the PDGF/VEGF and BMP signaling pathways. Abrogation of either pathway has no effect on convergent-extension movements in the tubules but leads to failure of their normal pathfinding through the body cavity (Bunt, 2010).

PVF ligands (Pvf1-3) expressed by the tubules attract migrating hemocytes to form short-term associations with them, during which hemocytes secrete components of the BM. The presence of collagen IV in the matrix ensheathing anterior tubule cells primes their response to local sources of the BMP pathway ligand, Dpp. Thus, interference with hemocyte secretion of collagen IV, whether by preventing hemocyte migration, by preventing their attraction to the tubules, or by abrogating hemocyte expression and/or processing of collagen IV, results in failure of BMP pathway activation in tubule cells and consequent misrouting of the anterior tubules. The tissue interactions that govern the guided outgrowth of the anterior tubules are summarized in Tissue Interactions Underlie Anterior Tubule Morphogenesis (Bunt, 2010).

As the tubules elongate, a distinct but dynamic subset of cells in the kink region responds sequentially to Dpp guidance cues from dorsal epidermal cells, the midgut, and, more anteriorly, gastric cecal visceral mesoderm and leads forward extension. Activation of the pathway targets, pMad and Dad, in these leading cells ensures that as the tubules project through the body cavity they take a stereotypical route. Loss of Dpp expression in the midgut or repression of BMP signaling in the tubules leads to stalling of their forward movement. Misexpression of Dpp is sufficient to cause tubule misrouting, in which the kink regions project toward the ectopic source. In accordance with these findings defective tubule morphogenesis has been described in embryos lacking the BMP receptors Thick veins (type 1) or Punt (type II), as well as in embryos mutant for schnurri, which encodes a pathway transcriptional regulator shown to be active during embryogenesis (Bunt, 2010 and references therein).

Strikingly only cells in the kink show pathway activation. The current evidence suggests that leading kink cells respond directionally to local gradients of Dpp and that they receive the highest level of ligand, which would account for the restricted domain of activation. However, as the kink region extends beyond the Dpp source, more posterior cells experience high levels of signal but show no pathway activation, indicating that other factors must differentiate between the leading and trailing cells. Segregation into leading and following populations is a common feature of collective cell migration and tubule branching and extension during organogenesis. Leading cells in outgrowing Drosophila trachea, migrating border cells and mammalian ureteric bud formation show distinct patterns of gene expression, respond differentially to external signals, and may repress pathway activation in their neighbors. Thus, tubule kink cells could themselves restrict the domain of pathway response (Bunt, 2010)

As well as their roles in determining cell fate, survival, and growth in Drosophila, TGF-β superfamily signals regulate tissue morphogenesis and have been shown to influence the invasive behavior of metastatic tumors. This study shows, through loss- and gain-of-function analysis, that Dpp also acts as a chemoattractant during organogenesis to determine the path of renal tubule extension though the body cavity. TGF-β superfamily signaling can induce epithelial-to-mesenchymal (EMT) transition through the expression of Snail- and ZEB-family members, which act to repress cell adhesion and polarity, leading to increased motility and, in the case of cancers, to single-cell metastatic activity. Such changes in kink cells could explain their role in pathfinding. However, recent evidence suggests that collective cell migration of epithelial tissues can occur without full EMT and kink cells remain polarized, ensheathed in ECM during tubule elongation (Bunt, 2010).

Ninov (2010) has shown that pathway activation through pMAD leads to increased actin dynamics and E-cadherin turnover in outgrowing histoblasts, resulting in reduced cell adhesion and enhanced cell motility through filopodial/lamellipodial extensions. The current results reveal similar lamellipodial extensions in kink cells, in line with Vasilyev (2009), who demonstrated directional basal lamellipodia in cells of the extending pronephric tubules of zebrafish. It is possible that the production of lamellipodia and tubule navigation also depends on Mad-independent effects on cytoskeletal regulators such as cdc42 (Bunt, 2010).

The current analysis reveals that deposition of ECM is a prerequisite for BMP signaling in tubule guidance. TGF-β/BMP signaling can be modified both by soluble ECM components such as HSPGs and also by architectural, fibrillar elements. The current evidence indicates that for normal tubule outgrowth collagen IV is the crucial component of the BM; it is deposited before tubule elongation (cf. perlecan deposited after elongation), is uniquely contributed by the hemocytes (the tubules express laminins as well as the hemocytes), and the effects of collagen IV loss of function mimic the failure of hemocyte migration to the tubules (whether in collagen IV mutants or in embryos lacking the function of lysyl hydroxylase or dSparc, factors that are required for normal collagen IV processing and deposition) (Bunt, 2010).

Collagen IV sharpens the dorsoventral gradient of BMP signaling in early Drosophila embryos through enhanced ligand-mediated activation (Wang, 2008), which depends on a conserved BMP-binding domain in the C-terminal region of collagen IV. Wang (2008) propose a two-step process in which the binding of Dpp/Screw ligand hetereodimers to collagen IV facilitates the formation of a complex between Dpp/Scw dimers, Sog, and Tsg. Tolloid cleavage of the complex releases ligand dimers, which become active on rebinding to collagen IV dorsally where Sog is absent. This study now shows that basement membrane collagen IV also acts during organogenesis to facilitate BMP signaling in a specialized region of tubule cells. Whereas the mechanism of activation could be as outlined by Wang (2008), early requirements for Dpp signaling in tubule development (Hatton-Ellis, 2007) complicate further analysis (Bunt, 2010).

Although the forward extension of the anterior tubules is important for their morphogenesis, it is likely that other factors regulate their navigation through the body cavity. The kink region dips ventrally and the distal tips extend dorsally late in embryogenesis so that specialized cells at the distal tip contact dorsal structures. Further, morphogenesis of the posterior tubules is unaffected by the repression of BMP signaling; they migrate posteriorly, crossing the hindgut and adopt their normal position in the body cavity, with their tip cells contacting hindgut visceral nerves. It is probable that the coordination of multiple inputs controls the morphogenetic movements of all four tubules (Bunt, 2010).

This study has highlighted the importance of multiple tissue interactions in the outgrowth of Drosophila renal tubules, between the tubules and hemocytes, and, as a consequence of this interaction, with guidepost tissues such as the midgut visceral mesoderm. Similar interactions occur during the specification and recruitment of renal tubule cells, in the branching of the ureteric bud and in the formation of the glomerulus. In vertebrate nephrogenesis kidney medullary and cortical tubules extend, taking up stereotypical positions with respect to blood vessels, with which they later interact to maintain tissue homeostasis. TGF-β superfamily signaling plays multiple roles early in vertebrate kidney development so that analysis of signaling during renal tubule morphogenesis requires conditional alleles or specialized reagents. Such studies reveal requirements for TGF-β superfamily signaling in the morphogenesis of the pronephric tubules and duct in Xenopus, and for the maintenance and morphogenesis of mammalian nephrogenic mesenchyme. VEGF is expressed in early renal mesenchyme and ureteric bud and later in glomeruli, where it is essential for glomerular capillary growth. It will be exciting to discover whether a combination of VEGF/PDGF ligands in renal tissues and spatially restricted TGF-beta superfamily guidance cues underpins the coordinated morphogenesis of these spatially linked renal/blood systems, as has now been shown ro occur in Drosophila (Bunt, 2010).

The BMP-binding protein Crossveinless 2 is a short-range, concentration-dependent, biphasic modulator of BMP signaling in Drosophila

In Drosophila, the secreted BMP-binding protein Short gastrulation (Sog) inhibits signaling by sequestering BMPs from receptors, but enhances signaling by transporting BMPs through tissues. Crossveinless 2 (Cv-2) is also a secreted BMP-binding protein that enhances or inhibits BMP signaling. Unlike Sog, however, Cv-2 does not promote signaling by transporting BMPs. Rather, Cv-2 binds cell surfaces and heparan sulfate proteoglygans and acts over a short range. Cv-2 binds the type I BMP receptor Thickveins (Tkv), and this study shows that the exchange of BMPs between Cv-2 and receptor can produce the observed biphasic response to Cv-2 concentration, where low levels promote and high levels inhibit signaling. Importantly, the concentration or type of BMP present can determine whether Cv-2 promotes or inhibits signaling. Cv-2 expression is controlled by BMP signaling, and these combined properties enable Cv-2 to exquisitely tune BMP signaling (Serpe, 2008).

Cv-2 modulates BMP signaling in the Drosophila wing by a mechanism distinct from that of Sog. BMP signaling in the early stages of PCV development depends, in large part, on BMPs being produced in the adjacent longitudinal veins, and endogenous Sog acts over a long range to promote signaling in this context, likely by transporting BMPs from the longitudinal veins into the PCV region. Both Sog and Cv-2 are biphasic, as low levels promote and high levels inhibit BMP signaling. However, Cv-2 acts over a short range within the PCV, precluding a direct role in the long-range transport of ligands from the longitudinal veins. The short-range action of Cv-2 is likely to involve binding to cell surface proteins such as Dally, and strongly suggests that Cv-2 acts on cells receiving the BMP signal. Moreover, Cv-2 can stimulate signaling in vitro, where the transport or stability of BMPs in the medium is unlikely to be an issue (Serpe, 2008).

Consistent with a role in reception, it was found that Cv-2 binds not only BMPs, but also the type I BMP receptor Tkv and vertebrate BMPR-IA and -IB. It is therefore proposed that the binding between Cv-2 and receptor facilitates transfer and signaling of BMPs via formation of a transient, nonsignaling complex containing Cv-2, type I receptor, and BMPs. At moderate levels, Cv-2 moves ligand from the extracellular space onto receptors via this complex, while at higher levels Cv-2 antagonizes signaling by sequestering ligand in the complex. The inability of this complex to signal is consistent with studies suggesting that Cv-2 binds to the BMP “knuckle” epitope used to bind type II BMP receptors (Serpe, 2008).

Computational analyses also predict that the relative affinities of different BMPs for Cv-2 or receptors will influence the effect of Cv-2 upon signaling. Although the vertebrate counterparts of BMP ligands appear to have similar affinities for Cv-2, they have different affinities for their receptors, and the model predicts that this alone can alter the activity of Cv-2. Indeed, in cell culture assays Cv-2 only antagonizes Dpp signaling, but has biphasic effects on Gbb signaling. This could explain why a vertebrate member of the Cv-2/Kielin-like family, mouse KCP, stimulates BMP-2 signaling but inhibits TGF-β and Activin signaling in vitro. Likewise, in the early Drosophila embryo, where a different set of BMP ligands act, it was found that loss of endogenous cv-2 actually expands BMP signaling, opposite to the effects of Cv-2 loss in the PCV. Thus, Cv-2 activity is highly context dependent (Serpe, 2008).

Fundamental to the proposed model is the formation of a transient complex containing Cv-2, BMP, and the receptor. Tripartite complexes have been demonstrated to form between follistatin, type I receptor, and BMP ligands, and this study found that Cv-2 and the extracellular portion of BMPR-IB simultaneously coimmunoprecipitate with Dpp. Similarly, the vertebrate type I receptor can coprecipitate both BMP and mouse KCP. Although the tripartite intermediate was not directly demonstrated, this might reflect the transient nature of this complex due to very rapid on-off kinetics. In fact, modeling predicts the intermediate is a low-affinity, transient complex (Serpe, 2008).

It is important to recognize that Cv-2 does not act as an obligate coreceptor in the described model. Rather, Cv-2 is modulatory, consistent with the fact that Cv-2 does not participate in BMP signaling in many contexts. In fact, the model requires that the tripartite complex does not signal, and it is only after Cv-2 is displaced that the type I receptor is free to signal. This is in contrast to the activity of coreceptors like Cripto, which is required for binding of the TGF-β family member Nodal to type I receptors and formation of signaling complexes with type II receptors. While Cripto can antagonize signaling, this involves non-Nodal ligands. In contrast, Cv-2 can promote or antagonize the signaling mediated by a single type of ligand such as Gbb (Serpe, 2008).

The functional, structural, and regulatory aspects of Drosophila Cv-2 show remarkable conservation with its vertebrate homologs in terms of HSPG binding, cleavage, and feedback by BMP signaling. Despite these similarities, a different mechanism was recently proposed to explain the ability of zebrafish Cv-2 to either promote or inhibit signaling; the cleavage of Cv-2 was proposed to convert Cv-2 from an antagonist to an agonist (Rentzsch, 2006). In support of this model was the observation that an uncleavable form of Cv-2 was more potent at dorsalizing zebrafish embryos (indicating a loss of BMP signaling) than was the full-length cleavable form, and that an N-terminal fragment lacking the vWFD domain ventralized embryos (indicating a gain in BMP signaling). Processing did not dramatically alter the KD of zebrafish Cv-2 for BMP binding, but apparently blocked its ability to bind HSPGs. Thus, the authors proposed that uncleaved Cv-2 binds HSPGs to sequester BMPs, while cleaved Cv-2 promoted signaling in a tissue-specific manner by an unknown mechanism (Serpe, 2008).

Little support was found for this model in Drosophila. Blocking cleavage did not create a strictly inhibitory molecule, since both wild-type and uncleavable Drosophila Cv-2 acted in a biphasic fashion. Moreover, both cleaved and uncleaved forms of Drosophila Cv-2 bound Dally and cell surfaces. Also no evidence was found of differential cleavage among cell types or developmental stages. Evidence from other secreted proteins suggests that GD-PH cleavages like that in Cv-2 occur via an autocatalytic process triggered by the low pH found within the late secretory compartments. Indeed, evidence was found of constitutive, pH-dependent Cv-2 cleavage in vitro, suggestive of an unpatterned, autocatalytic process in vivo (Serpe, 2008).

Nonetheless, conservation of the cleavage site among species suggests that cleavage plays an important role, and it was found that cleavage of Drosophila Cv-2 lowers its affinity for BMPs in vitro. However, similar manipulations of zebrafish Cv-2 did not greatly affect its KD for BMP. These may represent true species-specific differences, or they may result from differences in the binding assays used: the immobilization of proteins in the Biacore analyses of zebrafish Cv-2, or the presence of additional factors in the conditioned S2 cell medium present in coimmunoprecipitation assays. Since Drosophila Cv-2 can rescue the knockdown of zebrafish Cv-2, any species-specific differences are likely quantitative, rather than qualitative (Serpe, 2008).

In zebrafish, Chordin largely antagonizes BMP signaling, and thus Cv-2 and Chordin have essentially opposite effects on BMP signaling. However, loss of Cv-2 ameliorates only a subset of the gain-of-signaling phenotypes caused by loss of Chordin. Thus, Cv-2 has been proposed to promote signaling by two distinct mechanisms, one that depends on Chordin and one that is independent of Chordin. The current model can explain the Chordin-independent effect of Cv-2 and suggests that the Chordin-dependent effect may result from competition between Chordin and Cv-2 for BMPs. Since Cv-2 can block binding between BMPs and Chordin, the presence of Cv-2 will impact the amount of Chordin-bound BMP. In the absence of Chordin, the amount of free BMPs is likely to be higher, and the effect of Cv-2 in promoting signaling would not be as prominent (Serpe, 2008).

The situation is different in the Drosophila wing, where both Sog and Cv-2 promote signaling in the developing PCV. A model has emerged in which Sog and Cv (Tsg2) facilitate transport of BMPs into the PCV competent zone, where processing by Tlr leads to release of BMPs, and capture by Cv-2 for presentation to receptors. Thus, Sog and Cv-2 act coordinately, through independent mechanisms, to promote BMP signaling during PCV specification. Intriguingly, binding between Cv-2 and Sog have been detected in vitro, and this may provide a direct connection between the two systems by facilitating the exchange of BMPs from Sog to Cv-2 and thus onto the receptor (Serpe, 2008).

The data presented in this study indicate that Cv-2 can have remarkably versatile effects on signaling depending on the particular context in which it acts, providing an explanation for the contradictory effects observed for members of Cv-2/Kielin family in different developmental contexts. In addition, it was demonstrated that coupling the extracellular effects with positive feedback on the production of Cv-2 itself can lead to bistable signaling wherein a very sharp transition can be generated between cells that receive high versus low levels of signal. This positive feedback thus provides a mechanism for positionally refining signaling. However, the ability of Cv-2 to promote signaling apparently does not rely solely on spatial patterns of Cv-2, Sog, and Cv expression: Cv-2 promotes signaling in cell culture, and the PCV is formed in wings in which Cv-2, Sog, and Cv are overexpressed throughout the posterior compartment. The current model of Cv-2 function shows how a cell surface ligand-binding molecule can act locally to either promote or inhibit signaling. It is noted that this model may be applicable to other molecules such as the HSPGs that have been proposed to both activate and inhibit signaling (Serpe, 2008).

Enzymatic regulation of pattern: BMP4 binds CUB domains of Tolloids and inhibits proteinase activity

In Xenopus embryos, a dorsal-ventral patterning gradient is generated by diffusing Chordin/bone morphogenetic protein (BMP) complexes cleaved by BMP1/Tolloid metalloproteinases in the ventral side. A new BMP1/Tolloid assay was developed using a fluorogenic Chordin peptide substrate, and an unexpected negative feedback loop for BMP4 was identified, in which BMP4 inhibits Tolloid enzyme activity noncompetitively. BMP4 binds directly to the CUB (Complement 1r/s, Uegf [a sea urchin embryonic protein] and BMP1) domains of BMP1 and Drosophila Tolloid with high affinity. Binding to CUB domains inhibits BMP4 signaling. These findings provide a molecular explanation for a long-standing genetical puzzle in which antimorphic Drosophila tolloid mutant alleles displayed anti-BMP effects. The extensive Drosophila genetics available supports the relevance of the interaction described here at endogenous physiological levels. Many extracellular proteins contain CUB domains; the binding of CUB domains to BMP4 suggests a possible general function in binding transforming growth factor-beta (TGF-beta) superfamily members. Mathematical modeling indicates that feedback inhibition by BMP ligands acts on the ventral side, while on the dorsal side the main regulator of BMP1/Tolloid enzymatic activity is the binding to its substrate, Chordin (Lee, 2009).

The differentiation of cell types along the vertebrate D-V axis is regulated by an extracellular network of BMPs and their regulators, such as Chordin, BMP1/Tolloid, Tsg, and Crossveinless-2, in animals as diverse as Xenopus, Drosophila, zebrafish, amphioxus, hemichordates, and spiders. In addition, in the vertebrates, additional extracellular BMP antagonists such as Noggin and Follistatin cooperate with the anti-BMP activity of Chordin. The complexity of this biochemical pathway raises the question of why so many components and regulatory interactions are required to establish a simple gradient of BMP signaling through the transcription factors Smad1/5/8. One reason is that a stable gradient must be robustly maintained through many hours of development (from blastula until the end of gastrulation) at a time during which the three embryonic germ layers are undergoing massive morphogenetic movements. In addition, the frog embryo must have the ability to adapt to changes in temperature in its environment (Lee, 2009)

The patterning system must be resilient, given the self-regulating nature of development. When Xenopus embryos are cut in half, they will attempt to regenerate an embryo as perfect as possible, producing in some cases identical twins. This implies that cells in the dorsal and ventral poles of the early embryo communicate with each other, forming a self-regulating embryonic field. At a molecular level, these cell-cell communications can be explained by a pathway in which dorsal BMPs (ADMP and BMP2) and their antagonist, Chordin, are repressed at the transcriptional level by BMP signaling, while on the ventral side, BMP4/7 and CV2 are activated by the same signal, providing a self-regulating system. The key controlling element in this D-V conversation is provided by BMP1/Tolloid enzymes that degrade Chordin/BMP complexes releasing active BMP that are regulated by the Sizzled/Ogon-secreted competitive inhibitor. In this study, a novel regulatory node in the D-V patterning pathway, in which BMP4 serves as a feedback inhibitor of the BMP1 and Tolloid-related enzymes, was introduced (Lee, 2009).

A synthetic Chordin octapeptide spanning the C-terminal cleavage site that fluoresces when cleaved by Tolloids provided a quantitative enzymatic assay. This new assay was essential to the work, because both Chordin and Sog become better substrates for Tolloids when bound to BMP. It is therefore not possible to conduct a biochemical study on the digestion of full-length Chordin/Sog plus or minus BMP, because BMP affects both the substrate and the enzyme. The conformational change in the Chordin/Sog substrate would have precluded the discovery of the inhibition of enzyme activity by BMP4 (Lee, 2009).

Inhibition of BMP1/tolloids by BMP4 was specific, because it was not observed with other proteins such as Activin A, Tsg, Follistatin, and Noggin. The kinetics followed those of a Michaelis-Menten noncompetitive inhibition. This meant that BMP4 affected the activity of the enzyme by binding to a site distinct from the catalytic center. BMP4 was found to bind directly to CUB domains with high affinity. The Ki or inhibition constant (concentration at which half of the enzyme is bound to the inhibitor) for BMP1 was in the 40 nM range, and in the 14-20 nM range when measured by direct binding. This is within physiological levels, since the Km (Michaelis constant or affinity of the enzyme for its substrate) of BMP/Tolloids for Chordin substrate was between 17 and 25 nM, and of 96 nM for its BMP1/PCP activity (Lee, 2009).

The ventral center of the Xenopus gastrula expresses a chordin-like protein called CV-2 that strongly binds Chordin/BMP complexes transported from more dorsal regions of the embryo and facilitates BMP signaling through its cognate receptors after cleavage of Chordin by BMP1/tolloids. This suggests that in vivo free BMP is locally concentrated at sites of high CV2 and chordinase activity; it is in these regions that the negative feedback loop should be most effective. Not only will the BMP levels be highest, but also the Chordin levels will be lowest. The affinities of the interaction between BMP4/Tsg/Chordin and Tolloid may also be enhanced by the recently described Olfactomedin-related adaptor protein Ont-1, which brings together Chordin and tolloids (Lee, 2009).

The importance of the interaction between Tolloid and BMPs for developmental patterning in vivo is suggested by Drosophila genetics. A very large allelic series of tolloid mutants has been obtained that display a graded series of patterning defects along the D-V axis in Drosophila. This suggests that Tolloid provides a rate-limiting step during patterning. Therefore, any decrease in its activity caused by binding of BMPs would be expected to regulate the signaling gradient. The antimorphic tolloid mutations, which are proteolytically inactive but display anti-BMP effects, demonstrate that endogenous Tolloid enzyme is expressed at high enough levels to function antagonistically toward Dpp in vivo. Thus, at least in Drosophila, the interactions between Tolloid and BMPs discovered in this study function at physiological concentrations of D-V pathway components (Lee, 2009).

There previously had been isolated reports showing that DN-BMP1/tolloids dorsalized Xenopus ventral mesoderm, which should lack Chordin. One possible interpretation for these results was the presence of a Chordin counterpart, such as CV2, expressed in the high-BMP regions of the embryo. However, it was later found that CV2 is resistant to degradation by tolloids/BMP1. Instead, it was found that the anti-BMP effect of DN-tolloids, which can take place in Chordin-depleted embryos, are due to the sequestration of BMP ligands through direct binding to CUB domains (Lee, 2009).

It was initially hoped that the second site mutations described in Drosophila Tolloid CUB domains would point to amino acid residues critical for Tolloid binding of BMP4. Instead, all second site mutations affected Tolloid/BMP1 protein secretion. These second site antimorphic revertants behave essentially as null mutations of tolloid because they are not secreted. It is likely that the original antimorphic mutants displayed anti-Dpp effects because they bound BMPs in the Drosophila embryo (Lee, 2009).

CUB domains are also required for enzymatic activity. In the case of BMP1/PCP, it has been shown that the procollagen substrate is not efficiently recognized when CUB2 of BMP1 is deleted. However, the protease domain plus CUB1 is sufficient for BMP1 chordinase activity. In the case of Drosophila Tolloid, CUB4 and CUB5 are required to cleave Sog, and for Xolloid, CUB1 and CUB2 are required for recognition and cleavage of Chordin. Thus, CUB domains in Tolloid/BMP1 have specific functions in substrate recognition. CUB domains are also required for secretion, in addition to serving as inhibitory BMP-binding sites. As an interaction between the BMP1 prodomain and BMP4 has also been reported, it should be noted that the prodomain was lacking in all the CUB domain constructs used in the present study (Lee, 2009).

CUB domains are present in many secreted or transmembrane proteins, but their biochemical function remains unknown. The human genome contains 56 different loci encoding CUB domain-containing proteins. The finding that the CUB domains of BMP1 and Tolloid bind BMP4 suggests the exciting possibility that CUB domains may serve as binding modules for TGF-β superfamily ligands in other extracellular proteins as well. In the future it will be interesting to investigate, for example, the binding properties of the CUB domains found in Complement components C1r and C1s, which function in the opsonization of antigens. Another interesting protein is CUB domain-binding protein 1 (CDCP-1), a transmembrane receptor with three CUB domains that activates the Src tyrosine protein kinase and promotes metastases in human cancers; TGF-β also promotes metastases. Other CUB domain-containing proteins include membrane frizzled-related protein (MFRP), in which mutations in CUB domains cause nanophthalmos; procollagen C-peptidase enhancer (PCPE), known as a potent enhancer of BMP1/PCP activity in procollagen processing; the WNT transmembrane coreceptor Kremen; and many other extracellular or transmembrane proteins (Lee, 2009).

The effects of enzymatic inhibition -- in this case, noncompetitive inhibition by BMP4 -- were integrated into a reaction-diffusion model to understand its effect on the BMP morphogen gradient of the early Xenopus embryo. This mathematical modeling predicted that Tld activity will be inhibited in ventral regions in which BMPs are present in high concentrations. An unexpected finding was that Chordin itself is a major regulator of BMP/Tolloid activity. At high concentrations, such as in the dorsal side of the frog gastrula and likely in the fly ventral blastoderm, Chd/Sog complexed with Tld is predicted to decrease the availability of free (active) BMP/Tolloid. This will inhibit degradation of Chordin-BMP complexes, preventing local BMP release and signaling, enabling the complex to diffuse further (Lee, 2009).

These observations suggest that the Tolloid inhibition by BMP also takes place in fruit flies, which provide a system much more amenable to the visualization of gradients, and for which sophisticated mathematical modeling already exists. In the future, it will be interesting to investigate whether CUB domains generally serve as BMP or TGF-β superfamily-binding modules. This approach has been productive in the case of the CR/vWFc domains of Chordin, which function as BMP-binding modules in many proteins (Lee, 2009).

The present study suggests that the antimorphic revertant mutations, were based on direct Dpp-Tolloid associations and were indicators of a crucial step in the formation or maintenance of the self-adjusting D-V morphogen gradient. The findings in Drosophila and Xenopus also suggest that this extracellular negative feedback regulation was already present in the patterning system of Urbilateria, the last common ancestor of all bilateral animals that lived more than 535 million years ago. Finally, the direct binding of BMP4 to BMP1 explains why highly purified bone-inducing protein preparations contained BMP1/Tolloid in addition to BMP2-7. It may be worthwhile to explore the value of BMP1 or its CUB domains as a delivery system for BMPs in therapeutic interventions, such as the repair of bone fractures (Lee, 2009).

Shaping BMP Morphogen Gradients through Enzyme-Substrate Interactions

Bone morphogenetic proteins (BMPs) regulate dorsal/ventral (D/V) patterning across the animal kingdom; however, the biochemical properties of certain pathway components can vary according to species-specific developmental requirements. For example, Tolloid (Tld)-like metalloproteases cleave vertebrate BMP-binding proteins called Chordins constitutively, while the Drosophila Chordin ortholog, Short gastrulation (Sog), is only cleaved efficiently when bound to BMPs. This study identified Sog characteristics responsible for making its cleavage dependent on BMP binding. 'Chordin-like' variants that are processed independently of BMPs changed the steep BMP gradient found in Drosophila embryos to a shallower profile, analogous to that observed in some vertebrate embryos. This change ultimately affected cell fate allocation and tissue size and resulted in increased variability of patterning. Thus, the acquisition of BMP-dependent Sog processing during evolution appears to facilitate long-range ligand diffusion and formation of a robust morphogen gradient, enabling the bistable BMP signaling outputs required for early Drosophila patterning (Peluso, 2011).

To identify and characterize the Tld processing sites in Sog, the Sog cleavage fragments were purified and sequenced using tagged proteins generated in S2 insect cell cultures. The intermediate Sog cleavage fragments were captured using suboptimal amounts of enzyme and Dpp, the obligatory cosubstrate. The three major processing sites in Sog are in close proximity to the Cys-rich BMP binding modules. The positions of processing sites 1 and 3 correspond to the two major processing sites in Chordin. Sequencing of the N-termini revealed a conserved Asp residue at position P10, a hallmark of the astacin family of proteases that includes Tld, a conserved aliphatic residue (V) in position P3, and no significant homology with other Tld/BMP-1 substrates (Peluso, 2011).

Replacement of all four residues at processing site 1 (V183ALD) with Ala rendered Sog virtually uncleavable at this site in vitro. Full-length mutant Sog was still degraded over time, likely due to processing at the remaining unmodified sites, but the speed of its degradation was reduced. Additional Ala replacements at site 2 (V728PGD) further slowed down the Sog destruction. When only the conserved D (position P10) was replaced with E at processing site 1 were similar effects observed: undetectable cleavage at site 1 and a slower overall degradation of the mutant Sog. In this system, the precise cleavage kinetics at individual sites could not be observed, but the overall Sog destruction in various uncleavable Sog mutants was clearly slowed down, likely because of blocked/reduced cleavage at the modified site(s). Also any mutations at site 3 induced constitutive cleavage at this site, thus site 3 was kept intact for these studies (Peluso, 2011).

To test if these mutations (Sog-u) could render Sog uncleavable in vivo, Sog gain-of-function phenotypes were examined. Overexpression of Sog in the wing imaginal disc produces very mild phenotypes of venation defects. Sog together with Tsg produces a more potent BMP inhibitor; their combined overexpression inhibits BMP signaling and results in smaller wings with altered patterns of venation. Co-overexpression of Sog and Tsg with Tolloid-related (Tlr) is able to reverse the small wing phenotype and restore normal patterning in the case of wildtype Sog, but not in the case of Sog-u. In fact, overexpression of Sog-u by itself produced a significant loss of posterior crossvein, a structure that requires peak BMP signaling, suggesting that Sog-u is a better BMP inhibitor than the wild-type Sog. Moreover, the loss of posterior crossvein tissue was exacerbated when Tlr was coexpressed with Sog-u, indicating further reduction in the BMP activity. This is likely due to Tlr degrading endogenous Sog but not Sog-u. Thus, Sog-u appears resistant to cleavage and degradation in vivo and may act as a dominant negative by prolonged binding of the BMP ligands (Peluso, 2011).

Unlike in vertebrates, Drosophila Tld and Tlr process Sog only when bound to a BMP-type ligand. The binding of Sog to Tld requires several Tld protein interaction motifs besides the protease domain. Nevertheless, the requirement for the obligatory cosubstrate for Sog processing is thought to indicate a BMP-induced conformational modification that allows the Sog-BMP complex, but not Sog alone to fit into the catalytic pocket of the enzyme. In contrast, Chordin, which exhibits BMP-independent processing, should bind and fit into Tld's catalytic pocket without the need for a BMP-induced conformational change. Indeed, in spite of limited conservation between Sog and Chordin (40% similarity, 22% identity), it was found that Drosophila Tld can cleave the vertebrate Chordin in a BMP-independent manner (Peluso, 2011).

To focus on the enzyme-substrate interactions for Sog and Chordin, the Tld catalytic domain was modeled using the crystal structures available for related enzymes, the crayfish Astacin (the founder member of this zinc metalloprotease family), and the human Tld catalytic domain. As previously described, the catalytic pocket of Tld enzymes appears very tight in the proximity of the catalytic Zn, where scissile bonds align, and has a relatively wide cavity that accommodates residues P3 and P2 in the substrate. Bulky, hydrophobic residues in the substrate, such as in Chordin, might facilitate enzyme-substrate binding. Indeed, when the effect of changing processing site 1 was examined in Sog to either Chordin site 1 (Sog-1Ch1) or Chordin site 2 (Sog-1Ch2) indications were found of Tld cleavage at these sites in the absence of Dpp, although this cleavage was extremely weak. In addition, an aromatic residue in position P3 in the substrate could potentially stack against the aromatic ring, a key position near the active site, to further lower the substrate-enzyme binding energy and facilitate substrate binding (Peluso, 2011).

These predictions were tested and found that indeed changing several residues at the processing site could alter the cosubstrate requirements. For example, Sog processing at mutated site 1 (V183ALDV to FYGDP) occurred independently of the cosubstrate (Peluso, 2011).

This processing was enhanced when Dpp was added to the reaction partly because cleavage at unmodified sites 2 and 3 could not happen in the absence of the cosubstrate. Addition of Tsg similarly enhanced the processing of wild-type and mutant Sog. Nonetheless, when Dpp was in limiting amounts, the mutant Sog was processed more efficiently than the wild-type protein. Edman degradation confirmed that processing occurred at the expected G185-D covalent bond in the mutated site, and that mutagenesis did not create any promiscuous cleavage. Similar changes at processing site 2, separately or with site 1, further enhanced the speed of Sog degradation. The strongest effect was seen for a Sog variant in which both sites 1 and 2 were rendered BMP-independent for processing, designated Sog-i for 'independent of BMP for cleavage.' At the sequence level Sog-i is very different from Chordin, but it resembles Chordin in how it is processed by Tld: Sog-i exhibits significant BMP-independent processing by Tld, which is enhanced in the presence of BMP ligands. To emphasize these similarities Sog-i is referred as 'Chordinlike' Sog (Peluso, 2011).

Tests were performed to see if these changes impact Sog's ability to bind BMPs and/or inhibit their signaling. Purified Sog and Sog-i were found to be indistinguishable in their binding to Dpp homodimers and Dpp/Scw heterodimers in co-ip experiments. Also, in both cases, addition of Tsg equally increased Sog binding to the BMPs. The inhibitory activities of Sog and Sog-i on BMP signaling were compared in a cell-based assay; in the presence of Tsg, Sog inhibits Dpp-induced signaling in a concentration dependent manner. Equivalent amounts of Sog-i and Tsg produced a similar inhibitory response (Peluso, 2011).

While the BMP binding properties of Sog-i appeared to be largely unaffected, it was predicted that this 'Chordin-like' Sog would resemble Chordin when introduced into fly embryos, and be less efficient in promoting long-range BMP signaling. To model this process, a previously published spatiotemporal patterning model was modified by adding the BMP-independent processing of Sog. Briefly, the rate for Tld-mediated processing of Sog increases when the rate of BMP-dependent or BMP-independent cleavage increases. An increase in the rate of Tld processing will modify the Sog protein levels and the shapes of the Sog and Sog/Tsg distributions in the model. This results in a simultaneous reduction in the inhibition of Dpp signaling laterally and a reduction in the Dpp accumulation near the dorsal midline. To quantify the effect of BMP-independent cleavage of Sog-i on the net transport of BMP molecules toward the dorsal midline, the net diffusive flux of BMP ligand in the embryo was calculated by summing the contributions of free BMP and Sog-bound BMP. The flux provides the magnitude and direction of transport driven by the gradient of concentration. The Sog-i simulation clearly indicated a lower net transport toward the midline than the simulation with Sog-WT. Also investigated in this model was whether increased Sog-i expression could improve the transport toward the midline and whether the reduction in transport is solely the result of a reduction in Sog levels. Increasing the production of Sog-i increases the total amount of Sog-i in the system; however, transport of Dpp/Scw toward the midline is still reduced even with significantly increased levels of Sog-i greater than in Sog-WT embryos with normal patterning (Peluso, 2011).

To test the biological effect of these Sog variants on the BMP morphogen gradient profile, transgenic fly lines were constructed that allowed for normal spatial and temporal expression of Sog proteins at endogenous levels. The neural-ectoderm expression of tagged and nontagged Sog proteins, Sog-WT, Sog-WT-HA, and Sog-i-HA, in all of the transgenic lines obtained, overlapped the sog mRNA endogenous pattern. The relative Sog levels in these transgenic lines were quantified by immunofluorescence using anti-HA antibodies, anti-Sog antibodies, or both. It was found that indeed these transgenic lines have similar levels of Sog protein. In addition, all of the transgenic lines expressing Sog-WT (either HA-tagged or not-tagged) rescued the sogYL26 mutants and trans-heterozygous combinations (sog-/-) to viable, and fertile adults (Peluso, 2011).

The profile of the BMP morphogen gradient was examined in stage 5 embryos by following the accumulation of activated/ phosphorylated Mad (P-Mad), the effector of the BMP signaling pathway. In the absence of Sog, the facilitated diffusion of BMP ligands does not occur and Dpp remains uniformly distributed over the dorsal domain. No gradient of BMP activity is generated, thus the P-Mad levels are low and constant over the entire dorsal domain of sog-/- mutant embryos. In contrast, stage 5 wild-type embryos have a sharp, step gradient of BMP signaling, in which P-Mad levels are high in the dorsal most cells and rapidly drop off to undetectable levels in more lateral regions. The P-Mad positive domain is wider and slightly reduced in intensity in heterozygous (sog+/-) embryos. Among the sog alleles tested, the sogYL26/+ heterozygous embryos showed the widest P-Mad profile. The HA-tagged or untagged sog-WT transgenes were equally effective in restoring the sharp P-Mad profile in sog-/- embryos when in two copies, suggesting that the tag did not alter Sog activity. In contrast, addition of two sog-i copies to any sog-/- background produced a wide P-Mad positive domain with reduced signal intensities. In the latter embryos the boundaries of the P-Mad positive domains were more diffuse, with reduced slopes evident in the cross-section profile. Analogous studies of race expression, downstream of BMP signaling in the presumptive amnioserosa, indicated a similar effect. This suggests that Sog-i is indeed less efficient in supporting an adequate Dpp/Scw-Sog/Tsg flux toward the midline and consequently the formation of the steep BMP distribution profile (Peluso, 2011).

To quantify the differences in BMP signaling profiles between wild-type, sog+/-, and sog-/- embryos with 2x sog-i or 2x sog-WT transgenes, the P-Mad fluorescent staining of each embryo was decomposed into the product of an amplitude multiplied by the P-Mad distribution 'shape'. In brief, for each embryo, a region of interest was selected that encompasses a 4-cell-wide band centered at 33% embryo length. Each embryo was then processed through a Savitzky-Golay filter that removed noise while preserving the shape of the distribution and allowed for reliable calculation of the slope of the P-Mad profile (Peluso, 2011).

Shape was quantified by measuring the spatial-derivative of P-Mad in the cross-section. Starting on the left of a P-Mad cross-section plot, the derivative will be positive and change in magnitude at each position along the D/V axis directly proportional to the slope of P-Mad. As the slope decreases near the dorsal midline, the value of the derivative is approximately zero and then negative for the right side of the distribution where P-Mad is decreasing. The local average P-Mad slope for the population of 2x sog-WT embryos at each spatial location was virtually indistinguishable from the WT P-Mad slope. In contrast, the population average P-Mad slope for 2x sog-i embryos was noticeably shallower than WT and 2x sog-WT embryos with a lower magnitude of the spatial derivative near the midline and a higher magnitude in the lateral dorsal ectoderm. Moreover, differences in the overall intensity of P-Mad staining and/or in embryo-to-embryo variability do not account for this observation; even when the scaling for each population was chosen so the population means would have the same peak P-Mad levels, or when the absolute value of the local P-Mad slope was used for each individual in each population to calculate the population distributions of slopes, the differences remain clear: the replacement of sog-WT with sog-i leads to broader, shallower P-Mad profiles (Peluso, 2011).

The difference between the P-Mad profiles in WT and 2x sog-i was not equivalent to a decrease in the total amount of Sog protein in the system. Distributions of P-Mad slopes in both sog+/- and 2x sog-i differed from WT embryos, but the perturbations were not equivalent. For sog+/-, Sog protein levels were reduced approximately 50%, and the position of the peak slope shifted laterally away from the dorsal midline; however, the magnitude of the slope was still significantly greater than the magnitude of the slope for 2x sog-i embryos, though slightly less than WT. This means that the P-Mad profile in sog+/- is wider, but the steepness of the BMP activity gradient is similar to the steepness of the WT gradient. In contrast, the slope of the P-Mad profile in 2x sog-i embryos was significantly lower than WT embryos near where their peaks overlap and significantly greater than WT in the lateral dorsal ectoderm (Peluso, 2011).

Prior to gastrulation high levels of BMP signaling at the dorsal midline in early Drosophila embryos specify amnioserosa, an extraembryonic tissue required for gastrulation. The sharp and narrow BMP signaling domain in WT embryos induced formation of an amnioserosa field of approximately 200 cells in stage 13 embryos. The sog+/- heterozygous embryos have a wider BMP signaling domain that produced a larger amnioserosa field, about 50% bigger than that of the WT embryos. The spatial extent of the BMP signaling field above a certain threshold but below wild-type peak values appears to determine how many amnioserosa cells will be specified and consequently the size of the ensuing tissue (Peluso, 2011).

When sog-WT transgenes replaced the endogenous sog, the amnioserosa cell numbers were rescued to wild-type levels. However, addition of two sog-i copies to any sog-/- background tested produced statistically significant increases in the amnioserosa fields. The biological consequences of replacing Sog with Sog-i could not be explained simply by quantity differences between the sog-WT and sog-i transgenes. First, shallow BMP gradient profiles, broader target gene expression domains, along with increased cell allocation/ amnioserosa fields were observed using multiple independent sog-i transgenic lines with expression levels comparable with those of the sog-WT lines. Second, additional copies of sog-WT and sog-i transgenes did not significantly impact the BMP gradient profiles or amnioserosa fields. The P-Mad positive domains were more intense in either 4x groups, but in the sog-/-; 4x sog-i embryos the signaling domain remained wide, the boundaries diffuse and the slopes of the cross-section profiles reduced; also, the sog-/-; 4x sog-i embryos had significant embryo-to-embryo variability (Peluso, 2011).

To further search for alternative explanations for the Sog-i effects, Sog-WT and Sog-i versions of the 3D embryonic model were optimized against the 4x population data and it was asked whether the experimental observations could be captured by changes in Sog affinity to Dpp or changes in the processing rate of Sog by Tld independent of Dpp. It was found that the model with an enhanced processing rate achieved a greater fit: a modest increase in the processing of Sog by Tld without Dpp (increase BMP independent processing from about 8% to 19% of Tld processing rate in presence of Dpp), resulted in signaling profiles that matched the experimental data very well (Peluso, 2011).

In the 2x sog-WT simulations, the net reaction rate was negative in the lateral portions of the dorsal region and reached maximum near the dorsal midline. In the simulations for the 2x sog-i, the peak rate of Dpp release occurred laterally halfway between the neural-ectoderm and dorsal region. In contrast, no models obtained by decreasing the binding between Sog and Dpp (10x or more) could capture the experimentally observed loss of sharp boundaries. Thus, the shift in the net rate of cleavage, in conjunction with less effective net flux, produced less accumulation of Dpp near the dorsal midline in simulations of sog-i embryos (Peluso, 2011).

In conclusion, it was found that several residues at the Tld processing site make Sog dependent on a (BMP) cosubstrate for processing. Mutating these residues reduced the transport range of Sog-BMP complexes in vivo and altered the shape of the BMP signaling profiles and consequent cell fate allocation. Interestingly, BMP-dependent Chordin cleavage was also a requirement in mathematical modeling for scale invariance of Xenopus embryos. Here the cosubstrate requirement ensured transport of both ADMP and the BMP ligands and the reestablishment of a well-proportioned DV axis. How might shuttling of ligands persist in the absence of BMP-dependent cleavage of Chordin? An intriguing possibility is that Sizzled-mediated repression of Xolloid spatially restricts Chordin processing providing a nonuniform Chordin sink. In mathematical models of embryo patterning, lowering the processing rate of Tld results in signaling distributions that are sharper and result in a greater net transport of BMP ligands away from the Sog/Chordin source (Peluso, 2011).

An interesting and unexpected outcome of the comparison between sog-WT and sog-i embryos suggests that BMP-dependent Sog processing reduces embryo-to-embryo variability in P-Mad levels. Both sog-WT and sog-i embryos show sensitivity of P-Mad to gene dosage. However, when the coefficient of variation (standard deviation/width) within each genotype was calculated, it was found that embryos with one or two sog-WT copies showed less variability in signaling width than their 2x sog-i counterparts. The variability is greater at nearly all threshold positions, and the variability within this population increased dramatically at higher threshold levels. This suggests that BMP-dependent Sog destruction may reduce embryo-to-embryo variability between individuals in a population of the same genotype to provide robust patterning of the dorsal structures (Peluso, 2011).

Altogether, these results indicate that a 'Chordin-like' Sog is less able to reliably support patterning of the early Drosophila embryo. By modifying the Sog-Tld substrate-enzyme interaction with just a few residue changes, it appears that a new developmental function for Sog evolved that ensured reliable shuttling of BMPs and robust patterning. Further refinement of this shuttling mechanism, such as its speed or its directionality, expanded the repertoire of cell fate specification by BMP morphogen gradients and was likely exploited for diversified patterning during natural evolution (Peluso, 2011).

Formation of the long range Dpp morphogen gradient

Dpp acts as a secreted morphogen in the Drosophila wing disc, and spreads through the target tissue in order to form a long range concentration gradient. Despite extensive studies, the mechanism by which the Dpp gradient is formed remains controversial. Two opposing mechanisms have been proposed: receptor-mediated transcytosis (RMT) and restricted extracellular diffusion (RED). In these scenarios the receptor for Dpp plays different roles. In the RMT model it is essential for endocytosis, re-secretion, and thus transport of Dpp, whereas in the RED model it merely modulates Dpp distribution by binding it at the cell surface for internalization and subsequent degradation. This study analyzed the effect of receptor mutant clones on the Dpp profile in quantitative mathematical models representing transport by either RMT or RED. Novel genetic tools were then used, experimentally monitoring the actual Dpp gradient in wing discs containing receptor gain-of-function and loss-of-function clones. Gain-of-function clones reveal that Dpp binds in vivo strongly to the type I receptor Thick veins, but not to the type II receptor Punt. Importantly, results with the loss-of-function clones then refute the RMT model for Dpp gradient formation, while supporting the RED model in which the majority of Dpp is not bound to Thick veins. Together these results show that receptor-mediated transcytosis cannot account for Dpp gradient formation, and support restricted extracellular diffusion as the main mechanism for Dpp dispersal. The properties of this mechanism, in which only a minority of Dpp is receptor-bound, may facilitate long-range distribution (Schwank, 2011).

One outcome of the modeling was the prediction that RMT and RED mechanisms could be discriminated by analyzing Dpp levels behind receptor mutant clones. While in the transcytosis model these levels should be significantly decreased, they would be almost unaltered in the diffusion model. This difference stems from the uptake of Dpp by its receptors, which is an essential feature for morphogen transport by RMT, but not by RED. The experimental results revealed that neither GFP:Dpp levels nor Dpp signaling activity is reduced behind receptor mutant clones, excluding a significant role for receptor-mediated transcytosis in Dpp gradient formation. Important support for this conclusion was provided by situations where 'islands' of wild-type cells received Dpp signal despite being surrounded by mutant tissue, ruling out the possibility that Dpp reaches the distal side of receptor mutant clones by being transported around the clones. When analyzing the GFP:Dpp distribution in mosaic tissues, it was also found that the Dpp levels are not significantly reduced within receptor mutant clones. While this outcome further argues against the RMT model, it is consistent with the 'external-unbound limit case scenario,' representing RED with the majority of Dpp not being bound to Tkv. Indeed, in the GOF experiments the ratio of unbound Dpp could be narrowed down to approximately 60%-80% (Schwank, 2011).

If transcytosis is modeled in a receptor-independent manner, the effects on Dpp distribution by receptor mutant clones do not differ significantly from those in the restricted extracellular diffusion scenario. Thus, receptor-independent transcytosis, for example via fluid phase uptake, remains a possible mechanism for Dpp gradient formation. Several other studies, however, support the restricted extracellular diffusion model. Based on theoretical grounds, it has been proposed that diffusive mechanisms for Dpp gradient formation are more likely than non-diffusive ones. Moreover, experimental studies on heparan sulfate proteoglycans (HSPGs), in particular glypicans, demonstrated the necessity of an intact ECM for morphogen movement. In the Drosophila wing disc, clones mutant for the glypicans Dally and Dally-like (Dlp) disrupted the formation of the Dpp gradient. Dally was also shown to bind Dpp, to stabilize it on the cell surface, and to influence its mobility (Schwank, 2011).

However, although the evidence that glypicans assist extracellular diffusion of Dpp seems compelling, alternative or additional functions of glypicans in Dpp distribution cannot be excluded. For example, a recent study suggests that apically localized Dlp binds to the Wingless (Wg) morphogen in the Wg producing region, undergoes internalization, and thereby redistributes Wg to the basolateral compartment where Wg spreads to form a long-range gradient. It is possible that recycling of glypicans is also involved in Dpp relocalization and that this process is important for Dpp movement. Consistent with such a notion, another study reported that dynamin-dependent endocytosis is necessary for Dpp movement. Blocking such a ubiquitous cellular machinery, however, not only inhibits the recycling of receptors and glypicans, but may also change the composition and distribution of glypicans in the ECM, which in turn might impede extracellular diffusion. Given that the phenotypes of receptor clones fully conform to the simplest model of Dpp movement along the ECM (restricted extracellular diffusion), the view is favored that the main function of glypicans for Dpp gradient formation is to facilitate Dpp diffusion along the ECM (Schwank, 2011).

The observation that receptor mutant clones do not have a major effect on the Dpp gradient contradicts previous observations in which ablation of tkv in small lateral clones leads to an accumulation of Dpp at the side of the clone facing the source, arguing for a block of Dpp movement within such clones. The different results could be explained by the presence of brk in the previous study. The ectopic up-regulation of brk in tkv mutant clones, which in most cases leads to clone elimination, most likely also causes drastic changes in the transcriptional program in 'escaper' cells. Thus the sharp increase in GFP:Dpp levels at the proximal edge inside tkv mutant clones (facing the Dpp source) could be accounted for by increased levels of Dpp binding proteins, a theory which is supported by the fact that Dpp accumulation was strictly clone-autonomous and not in cells ahead of the clones. In the current experimental setup, such secondary effects were avoided by simultaneously removing tkv together with brk. As the negative control (Mad brk clones) shows, the signaling state of these cells (Dpp signaling off, no Brk) does not significantly alter the Dpp profile (Schwank, 2011).

Transport along cytonemes is another proposed model for the dispersal of Dpp. In its simplest implementation, this model assumes that imaginal disc cells form filopodial extensions towards the Dpp producing region and that Dpp is shuttled along these extensions by binding to Tkv. In this scenario, Tkv GOF clones would not only lead to an increase of receptors inside the clones, but also along the cytonemes, and thus affect the Dpp profile also ahead of the clones. This, however, was not observed in the current experiments, and the restricted extracellular diffusion model is favored over the cytoneme model for Dpp gradient formation (Schwank, 2011).

During development morphogens function as short-range or long-range signals in order to specify cell fates within a tissue. For example, during wing disc development the range of Hh signaling is relatively short compared to that of Dpp, with a functional range of approximately 10 cells versus 40 cells, respectively. It is likely that properties of the transport system are important determinants of the range of a morphogen. In the restricted diffusion model, morphogen spreading is impeded by ECM proteins and cell surface receptors, which efficiently trap their ligand at the cell surface and direct it to degradation. Thus one mechanism to control the range of a morphogen gradient is regulating the receptor levels. Indeed, the Hh as well as the Dpp system appear to make use of this strategy to regulate their range. The Hh signal limits its range by upregulating the expression of its binding receptor Patched (Ptc), while the Dpp signal broadens its range by downregulating the expression of its receptor Tkv. The effects of Tkv LOF and GOF clones on the Dpp profile suggest that the majority of Dpp is not bound to the receptor Tkv. It is tempting to speculate that the Dpp-Tkv binding properties represent an additional property of the Dpp signaling system that facilitates the formation of a long-range gradient, by assuring that the majority of Dpp remains in a free and unbound state. Just like lower receptor levels, a lower binding constant would contribute to the spread of Dpp, due to reduced immobilization and degradation of Dpp. It remains to be seen if the ratio of bound to unbound ligand differs for long- versus short-range morphogens and if this ratio represents a general means to regulate the range of morphogen gradients (Schwank, 2011).

Regulation of Dpp activity by tissue-specific cleavage of an upstream site within the prodomain

BMP4 is synthesized as an inactive precursor that is cleaved at two sites during maturation: initially at a site (S1) adjacent to the ligand domain, and then at an upstream site (S2) within the prodomain. Cleavage at the second site regulates the stability of mature BMP4 and this in turn influences its signaling intensity and range of action. The Drosophila ortholog of BMP4, Dpp, functions as a long- or short-range signaling molecule in the wing disc or embryonic midgut, respectively but mechanisms that differentially regulate its bioactivity in these tissues have not been explored. The current studies demonstrate, by dpp mutant rescue, that cleavage at the S2 site of proDpp is required for development of the wing and leg imaginal discs, whereas cleavage at the S1 site is sufficient to rescue Dpp function in the midgut. Both the S1 and S2 sites of proDpp are cleaved in the wing disc, and S2-cleavage is essential to generate sufficient ligand to exceed the threshold for pMAD activation at both short- and long-range in most cells. By contrast, proDpp is cleaved at the S1 site alone in the embryonic mesoderm and this generates sufficient ligand to activate physiological target genes in neighboring cells. These studies provide the first biochemical and genetic evidence that selective cleavage of the S2 site of proDPP provides a tissue-specific mechanism for regulating Dpp activity, and that differential cleavage can contribute to, but is not an absolute determinant of signaling range (Sopory, 2010).

Endogenous Dpp functions as a morphogen, to instruct cells of their fate in a concentration-dependent fashion. Many studies have described the critical role that extracellular factors play in shaping the Dpp activity gradient by facilitating or inhibiting Dpp movement and/or by modulating the ability of signal receiving cells to perceive a given concentration of ligand. The Dpp receptor Thick veins, for example, sensitizes cells to low levels of Dpp but also limits Dpp movement when present at high levels. Similarly, the secreted Dpp binding proteins Short gastrulation and Twisted gastrulation inhibit Dpp from binding to Thick veins, thereby blocking local signaling but facilitating long-range diffusion. The current studies demonstrate that proteolytic activation of the precursor in signal sending cells also contributes to differences in Dpp bioactivity in various tissues. Specifically, in the wing disc, Dpp is cleaved at both the S1 and the S2 sites and the latter cleavage is essential to generate steady state levels of ligand that surpass the threshold for induction of pMAD at both short- and long-range, over much of the disc. By contrast, in the embryonic gut Dpp is cleaved only at the S1 site, and this generates sufficient ligand to activate physiological target genes in neighboring cells. Future studies will be required to determine whether cleavage at the S1 site alone is necessary to prevent aberrant spread of the ligand beyond immediately adjacent cells in the gut (Sopory, 2010).

It is possible that the ability of proDppS2KK(in which the furin consensus motif at the S2 site had been disabled) to rescue labial expression in the gut, and its inability to rescue growth and patterning in the wing reflect differences in the level of expression of precursor proteins due to the use of different drivers in the two tissues, rather than differences in use of the S2 site. This is unlikely, however, since precursor protein is always present in excess of cleavage products, while BMP4/Dpp signaling activity is determined by the rate of precursor folding, dimerization and cleavage, and by the rate of ligand turnover. In the current studies, when proDppS2KK was expressed in the wing using the MS1096-Gal4 driver, precursor protein was present at high levels and yet very low steady state levels of mature Dpp were detected. Furthermore, induction of pMAD was not observed in these wing discs, even when incubated at higher temperature to maximize GAL4 activity. Thus, in the wing disc but not in the gut Dpp activity is tightly regulated by cleavage of the S2 site (Sopory, 2010).

Several models have been proposed for how Dpp moves across cells in the wing, all of which invoke endocytic trafficking to generate or shape the gradient. One model suggests that Dpp is actively transported by endocytosis, intracellular transport and exocytosis while other models postulate that Dpp moves by extracellular diffusion and/or by extracellular transport facilitated by binding proteins such as Short gastrulation and Crossveinless. More recent studies argue that Dpp is passed from cell to cell after binding to the HSPGs, Dally and Dally-like (Dly). These studies reveal that endocytosis is required for signal transduction and for lysosomal degradation of Dpp, which shapes the activity gradient. This latter finding provides a feasible explanation for why ligand generated by cleavage at the S1 site alone is unable to signal even at short range over most of the wing disc. Specifically, it is proposed that the ligand is preferentially targeted for degradation, and thus does not accumulate to high enough steady state levels to surpass those needed for signal activation (Sopory, 2010).

Previous studies have shown that the prodomain/ligand complex generated by cleavage of BMP4 at the S1 site alone is targeted to the lysosome, but it is not known whether this occurs directly from the biosynthetic pathway in signal sending cells, or following secretion and receptor-mediated endocytosis in signal receiving cells. It has been shown that deletion of the heparin binding motifs on BMP4 partially stabilizes the ligand/prodomain complex generated by cleavage at the S1 site alone, suggesting that degradation may occur following endocytosis in a process that is facilitated by binding to cell surface HSPGs, such as Dally. If Dpp is regulated in the same way, this might contribute to the ability of DppS2KK to active pMAD when expressed in cells on the periphery of the wing disc, since levels of Dally are at their lowest, and levels of Thick veins at their highest in these cells, thereby minimizing lysosomal targeting while maximizing signal activation. Although recent studies suggest that Dally disrupts, rather than enhances internalization of native Dpp in the wing, it may function differently in the context of a ligand/prodomain complex generated by cleavage at the S1 site alone. This complex might also be endocytosed after binding to crossveinless2/BMPER, which has been shown to direct receptor-mediated internalization and lysosomal targeting of BMP4. Alternatively, Dpp generated by cleavage of the precursor at the S1 site alone may be targeted directly for degradation within the biosynthetic pathway, prior to secretion, consistent with its inability to activate pMAD cell autonomously in cells located in central regions of the disc. Analysis of Dpp maturation within or adjacent to clones of cells that are defective for components of the lysosomal trafficking machinery, or for HSPGs may shed light on this question and on the mechanisms by which the prodomain directs intracellular trafficking (Sopory, 2010).

A recent independent analysis of Dpp maturation agrees with the finding that cleavage at the S2 site is essential to generate sufficient mature Dpp to support wing development. In that study, the distribution of mature Dpp was imaged directly, by extracellular staining of wing discs, and ligand cleaved from an S2-mutant precursor was not detected outside of cells. This is consistent with biochemical analysis showing that steady state levels of ligand generated from this precursor are very low. It was concluded that cleavage of the S2 site occurs first, and is an obligate requirement for cleavage of downstream sites such that mature ligand cannot be generated from proDppS2KK. This remains a feasible explanation, since it is not possible to definitively identify the order of cleavage of Dpp in vivo. However, current data showing that the S1 site can be cleaved independently of the S2 site in Drosophila cell lines and embryos, and that cleavage of the S2 site is dispensable for Dpp function in the embryonic midgut support an alternate interpretation. Specifically, it is proposed that failure to cleave the S2 site in the wing disc leads to rapid degradation of mature Dpp generated by cleavage at the S1 site, consistent with biochemical analysis of BMP4 processing and degradation in vertebrate embryos. Since it was not possible to visualize the small ligand fragment, which is generated by cleavage N-terminal to the myc-epitope tag in the construct analyzed in this study, the possibility that mutation of the S2 site affects processing at this third site, leading to differences in signaling efficacy, cannot be ruled out. This seems unlikely, however, since cleavage of the third site was shown to be dispensable for Dpp function, at least in the wing (Sopory, 2010).

Although a model in which cleavage at the S2 site is regulated in a tissue-specific manner is currently favored and this in turn determines the rate of degradation of the mature ligand in the wing versus the gut, it is possible that both sites are cleaved in the wing and the gut, and that these tissues instead differ in their ability to traffic or degrade S1-cleaved DPP. For example, in the wing cleavage products generated by S1-cleavage may be recognized by a trafficking receptor that targets them to the lysosome, whereas in the gut, this receptor may be absent. This model offers an alternate explanation for the observation that steady state levels of S1-only cleaved prodomain generated from either the wild type or S2KK-mutant precursor protein are very low in the wing, but relatively high in the gut. Specifically, this may reflect differential stability of the S1-cleavage products in the wing versus the gut, rather than differential use of the S2 cleavage site in the wild type precursor. However, the data showing that steady state levels of S2-cleaved prodomain generated from the wild type precursor are barely detectable in the mesoderm, but abundant in the wing cannot be accounted for by the differential stability model, and are more consistent with selective, tissue-specific cleavage of the S2 site. Conversely, the finding that equivalent levels of cleavage products are generated and secreted from cultured Drosophila S2 cells regardless of whether the S2 site is cleaved supports the differential stability model, and suggests that S2 cells are unable to traffic or degrade the S1-cleaved prodomain/ligand complex. An analogous study in the same S2 cell line, however, reported the opposite result: that significantly lower steady state levels of mature Dpp are detected in the media of cells expressing proDppS2KK relative to those expressing DppWT. A likely explanation for these contradictory results is use, in the current study, of the actin-GAL4 driver to express proDpp in S2 cells, leading to supraphysiological levels of cleavage products that saturate the lysosomal trafficking machinery. In support of this proposal, steady state levels of cleavage products generated by wild type proBMP4 are significantly higher than those synthesized from S2-cleavage mutant variants in Xenopus embryos, oocytes, or mammalian cultured cells expressing low amounts of each precursor protein, but these differences disappear as the dosage is increased above a certain threshold (Sopory, 2010).

Although the current results show that cleavage of the S2 site of proDpp correlates with long-range signaling in vivo, S2 cleavage is not an absolute determinant of signaling range, but is instead one of many factors that influence the distance over which Dpp signals. For example, spatial and temporal expression of dpp is tightly controlled at the transcriptional level, and cells differ in their ability to respond to Dpp based on the level of receptors and other cofactors present at the cell surface. Varying any one of these parameters perturbs the Dpp activity gradient. Importantly, the current studies show that cleavage of the S2 site is required for long- and short-range signaling in cells that are normally responsive to this ligand in the wing, but not for short-range signaling to cells that normally respond to endogenous Dpp in the gut. Thus, differential use of the S2 site can influence signaling range when assayed in the context of cells that normally respond to endogenous Dpp. As with all regulatory mechanisms, however, it is possible to bypass this control if the precursor is ectopically expressed at high levels outside of the endogenous Dpp signaling domain (Sopory, 2010).

The current studies provide the first biochemical evidence that the S2 site of Dpp/BMP4 is cleaved in a tissue-specific fashion, and future studies will be required to determine how this is regulated. It has recently been shown that two members of the PC family, furin and PC6 function redundantly to cleave the S1 and the S2 sites of proBMP4, whereas a third PC, possibly PC7, functions to selectively cleave the S1 site, possibly in a developmentally regulated fashion in vertebrate embryos. These studies raise the possibility that tissue-specific cleavage of proBMP4 is regulated by differential expression of a site-specific protease. The results of RNAi knockdown studies in S2 cells show that DFur1 and DFur2 function redundantly to cleave both the S1 and the S2 sites of Dpp, although DFur1 may preferentially cleave the S2 site. The latter result raises the possibility that the S2 site is cleaved in all tissues that express DFur1, and not in tissues that express only DFur2. This simple hypothesis is not supported by the observations that DFur2 also contributes to S2 site cleavage and that DFur1 is expressed in the embryonic mesoderm, where the S2 site is not cleaved. Because PC activity is tightly regulated post-translationally, RNA expression does not necessarily indicate that functional protein is present and thus further in vivo analysis will be required to stringently test this hypothesis (Sopory, 2010).

BMP4 and Dpp play highly conserved roles in patterning the limbs, and during specification and differentiation of the endoderm layer. The current studies suggest that Dpp/BMP4 maturation and activity may be regulated differently in these tissues in vertebrates relative to invertebrates. Specifically, the current studies show that S2 cleavage is essential for Dpp function in the wing disc, but not in the gut whereas analysis of mice carrying a point mutation that prevents cleavage of the S2 site of BMP4 demonstrates an opposite requirement for S2 cleavage in analogous tissues in mammals. At present, nothing is known about which sites are cleaved in proBmp4 in the limb versus the gut in mammals. Further studies will be required to determine whether and how tissue-specific use of the S2 site differs between vertebrates and flies (Sopory, 2010).

magu is required for germline stem cell self-renewal through BMP signaling in the Drosophila testis

Understanding how stem cells are maintained in their microenvironment (the niche) is vital for their application in regenerative medicine. Studies of Drosophila male germline stem cells (GSCs) have served as a paradigm in niche-stem cell biology. It is known that the BMP and JAK-STAT pathways are necessary for the maintenance of GSCs in the testis. However, recent work strongly suggests that BMP signaling is the primary pathway leading to GSC self-renewal (Leatherman, 2010). This study shows that magu controls GSC maintenance by modulating the BMP pathway. magu, a putative matricellular protein, a secreted protein that could regulate cell-matrix interactions, is specifically expressed from hub cells, and accumulates at the testis tip. Testes from magu mutants exhibit a reduced number of GSCs, yet maintain a normal population of somatic stem cells and hub cells. Additionally, BMP pathway activity is reduced, whereas JAK-STAT activation is retained in mutant testes. Finally, GSC loss caused by the magu mutation is suppressed by overactivating the BMP pathway in the germline (Zheng, 2011).

This study shows that magu plays an important role in GSC maintenance. Strong evidence is provided that it does so by modulating BMP activation in germ cells. magu encodes a secreted protein of the SPARC/BM-40/osteonectin family, recently shown to ensure the proper activity gradient for the BMP morphogen, Dpp, across the developing wing epithelium (Vuilleumier, 2010). The role characterized for magu in the testis niche exhibits some similarities as well as differences to that proposed for the wing (Zheng, 2011).

It has been shown that the BMP pathway is activated and required in GSCs, whereas the JAK-STAT pathway is activated and required in both GSCs and CySCs. The data shows that magu is required for maintenance of GSCs, but not CySCs, and that BMP activation was impaired in germ cells adjacent to the hub in magu mutants. It was also found that forcing activation of the BMP pathway in germ cells substantively rescues the magu phenotype. Thus, it is concluded that the primary role of magu in the testis niche is to modulate BMP signaling and thereby maintain GSCs (Zheng, 2011).

Superficially, these results suggest that magu works in a manner similar to that described in the wing epithelium, where magu facilitates the transport of BMP ligands to establish the proper signaling gradient. However, there are several differences comparing the wing with the testis niche (Zheng, 2011).

The most obvious is that to control wing patterning, BMP signaling is graded and must be effective over a long range. Thus, Dpp is expressed from a stripe of cells in the center of the wing disk, while the region where BMP activation is modulated by magu is located far laterally, many tens of cells away from the ligand source (Vuilleumier, 2010). In striking contrast to this situation, BMP ligands are produced in hub cells and CySCs of testes, which are directly adjacent to GSCs, where pathway activation is required. In the testis, there is no documented graded requirement, and, if anything, it is likely that pathway activation must be restricted to cells near the niche to ensure that few cells take on stem cell character. Therefore, while magu is thought to assist the movement of Dpp over a long range in the wing (Vuilleumier, 2010), there is no need for long-range transport for GSC maintenance in the testis. This distinction between the two systems suggests that key mechanistic differences remain to be uncovered for how magu affects BMP signaling (Zheng, 2011).

One way that magu supports robust signaling far from the BMP ligand source in the wing is that magu gene expression is engaged by a feedback circuit in order to be used as a positive modulator of signaling. Thus, magu expression is repressed in areas of relatively high signaling, and that repression is relieved in regions of low signaling. Its action in the low signaling region is to promote signaling even though these areas are far from the ligand source (Vuilleumier, 2010). In fact, expressing magu ectopically in the area of high signaling serves to dampen signaling there, while enhances signal at a distance, presumably by promoting movement or stabilization of the ligand. In the testis niche, there is some evidence for feedback regulation, as a reporter construct mutated for pMad/Medea/Schnurri complex binding sites (Vuilleumier, 2010) is expressed more robustly, and in more hub cells. However, in contrast to the wing, there is no evidence that this negative feedback regulation is necessary in the testis niche, as overexpression of magu had no discernable effect on GSC numbers (data not shown) (Zheng, 2011).

One other potential difference between the wing and testis niche is that the BMP ligands acted on by magu might differ in the two systems. Vuilleumier haS addressed the function of magu with respect to Dpp, the principal BMP ligand used globally for wing patterning. However, the major BMP ligand for male GSC maintenance appears to be Gbb. This difference could have consequences for the mechanism by which magu influences BMP signaling comparing the two systems. For example, although Dpp does not interact directly with magu (Vuilleumier, 2010), the potential remains that magu might bind to Gbb for GSC maintenance (Zheng, 2011).

In this regard, it is worth noting that gbb is expressed throughout the wing, and that compromising gbb function does generate a wing vein phenotype similar to magu mutants. Thus, in the wing, even though the focus has been on Dpp, perhaps there is an effect also on Gbb transport and/or signaling. Thus, further investigation of the modulation of BMP signaling by magu in both the wing and testis niche should be revealing (Zheng, 2011).

The fact that overexpressing a constitutively active form of BMP type I receptor in the germline can rescue the GSC phenotype suggests that magu acts upstream of receptor binding. This is in agreement with its proposed role in the wing and also preliminary analysis in zebrafish (Vuilleumier, 2010). There are a number of membrane-associated and secreted factors that magu might influence to modulate BMP signaling (Zheng, 2011).

In the wing, magu interacts directly with Dally, a HSPG (heparan sulfate proteoglycan) (Vuilleumier, 2010). Interestingly, Dally and its homologue Dally-like (Dlp) are also important for male GSC maintenance. While no genetic interactions has been found between magu and dally, dlp or several other genes needed for HSPG biosynthesis, some preliminary data indicate that overexpressing dlp in the germ cells can increase the fraction of testes retaining GSCs among magu mutants. Dlp has been shown to be enriched among hub cells, this study has had no success in reproducing this suggestive distribution. Therefore, further experiments are needed to test for interactions between magu and Dlp or other HSPGs in GSC maintenance (Zheng, 2011).

Given that magu is secreted from hub cells, its localization could have suggested a more specific hypothesis for its action in the testis niche. However, magu protein localization among cells of the niche appears complex. An antibody raised against an N-terminal portion of magu exhibits punctate signal restricted among hub cells, and at the hub-GSC interface, but this serum was effective only sporadically. A second serum directed against a C-terminal peptide (Vuilleumier, 2010) robustly exhibits the same punctate pattern among hub cells, but also reveals a slightly extended distribution among stem cells and their daughters near the hub. Additionally, this serum revealed strong punctate signal likely among the extracellular matrix (ECM) near the hub. It is not possible at this time to distinguish whether the pool of magu associated with ECM or the more generally distributed pool is active for GSC maintenance (Zheng, 2011).

However, considering the close proximity of hub cells to GSCs, it is simplest to envision that magu acts along the hub cell-germline stem cell interface, where the interaction of BMP ligands and receptors occurs. It is possible that magu facilitates interactions between BMPs and their receptors via formation of ternary ligand/magu/receptor complexes. This model has been shown for Crossveinless 2 (Cv2), an extracellular BMP modulator engaged for crossvein patterning in the wing. Cv2 can also bind to Dally, and the Cv2-HSPG interaction is likely important for normal BMP signaling in crossvein patterning. magu and its vertebrate orthologues SMOC1/2 have two Thyroglobulin type-1 repeats. It has been shown that proteins with such repeats can inhibit extracellular proteases. Thus, although Cv2 appears to have no effect on the function of Tolkin, the protease promoting BMP signaling in crossvein patterning, it is reasonable to speculate that magu may function as a protease inhibitor to protect BMP ligands from being degraded by extracellular proteases (Zheng, 2011).

Alternatively, the enrichment observed among the ECM is interesting. Among the family of proteins to which magu belongs, SPARC interacts with type IV Collagen, a component of basement membranes, and SMOC1/2 are associated with basement membranes. Interestingly, Viking (Vkg), the type IV collagen in Drosophila, is involved in the female GSC maintenance, potentially by sequestration of Dpp, thereby restricting BMP signaling in the germarium. It would be interesting to investigate whether Vkg also plays a similar role in the testis, and interacts with magu to maintain a normal number of GSCs (Zheng, 2011).

The niche-dependent feedback loop generates a BMP activity gradient to determine the germline stem cell fate.

Stem cells interact with surrounding stromal cells (or niche) via signaling pathways to precisely balance stem cell self-renewal and differentiation. However, little is known about how niche signals are transduced dynamically and differentially to stem cells and their intermediate progeny and how the fate switch of stem cell to differentiating cell is initiated. The Drosophila ovarian germline stem cells (GSCs) have provided a heuristic model for studying the stem cell and niche interaction. Previous studies demonstrated that the niche-dependent BMP signaling is essential for GSC self-renewal via silencing bam transcription in GSCs. The Fused (Fu)/Smurf complex has been shown to degrade the BMP type I receptor Tkv allowing for bam expression in differentiating cystoblasts (CBs). However, how the Fu is differentially regulated in GSCs and CBs remains unclear. This study reports that a niche-dependent feedback loop involving Tkv and Fu produces a steep gradient of BMP activity and determines GSC fate. Importantly, it was shown that Fu and graded BMP activity dynamically develop within an intermediate cell, the precursor of CBs, during GSC-to-CB transition. Mathematic modeling reveals a bistable behavior of the feedback-loop system in controlling the bam transcriptional on/off switch and determining GSC fate (Xia, 2012).

In the feedback loop model to show how the GSC fate is regulated. In the model, the external BMP signal cues stimulate phosphorylation of Tkv protein, the activated Tkv then promotes the synthesis rate of phosphorylated Mad (pMad), and pMad promotes the degradation of Fu protein and represses the transcription of bam. Meanwhile, degradation of the activated Tkv is also controlled by Fu. To assess the dynamic properties of this feedback loop, it was assumed that the transcriptions of genes tkv, mad, and fu are sufficient and that the degradation rate of pMad and the synthesis rate of Fu protein are constants. The network diagram of the feedback loop clearly points out two characteristics of the model: first, the microenvironment-derived BMP ligands serve as a key external signal, the strengths of which are differentially sensed by GSCs, pre-CBs, and CBs, thereby regulating the dynamic expression of the activated Tkv, pMad, and Fu during the asymmetric division of GSCs. Second, although the transcription of the bam gene is regulated negatively by Tkv/pMad, the expressions (and/or regulations) of the activated Tkv, pMad, and Fu are independently of the status of the Bam protein (Xia, 2012).

The dynamic analysis reveals the bistable behavior (i.e., switch behavior) of the system and how the system dynamics respond to the strength of external BMP ligand activity. Specifically, the strong external BMP ligand activity (in GSCs) will lead to a low expression level of Fu as well as high expression levels of the activated Tkv and pMad. Conversely, the weak external BMP ligand activity (in CBs) will lead to a high level of Fu expression (and low levels of the activated Tkv and pMad expression). However, for the transitional stage with intermediate BMP signaling (in pre-CBs), both high and low levels of Fu and pMad expression exist. These theoretical predictions not only exactly match the experimental data, but they also bring an insightful physical interpretation for why the niche dependence of BMP signaling determines the fate of stem cells by precisely balancing of stem cell renewal and differentiation. The current model permits the proposal of a comprehensive description of the action of niche signaling that governs the decision between stem cells and differentiating cells (Xia, 2012).

Crossveinless d is a vitellogenin-like lipoprotein that binds BMPs and HSPGs, and is required for normal BMP signaling in the Drosophila wing.

The sensitivity of the posterior crossvein in the pupal wing of Drosophila to reductions in the levels and range of BMP signaling has been used to isolate and characterize novel regulators of this pathway. This study shows that crossveinless d (cv-d) mutations, which disrupt BMP signaling during the development of the posterior crossvein, mutate a lipoprotein that is similar to the vitellogenins that comprise the major constituents of yolk in animal embryos. Cv-d is made in the liver-like fat body and other tissues, and can diffuse into the pupal wing via the hemolymph. Cv-d binds to the BMPs Dpp and Gbb through its Vg domain, and to heparan sulfate proteoglycans, which are well-known for their role in BMP movement and accumulation in the wing. Cv-d acts over a long range in vivo, and does not have BMP co-receptor-like activity in vitro. It is suggested that, instead, it affects the range of BMP movement in the pupal wing, probably as part of a lipid-BMP-lipoprotein complex, similar to the role proposed for the apolipophorin lipid transport proteins in Hedgehog and Wnt movement (Chen, 2012).

The evidence indicates that Cv-d, a member of the Cvd/160MEP family of Vtg-like lipoproteins, acts over a long range to promote BMP signaling in the developing PCV of the Drosophila wing, probably after having been delivered to the wing via the hemolymph. Cv-d binds both BMPs and HSPGs, and Cv-d activity in vivo requires the presence of its BMP-binding Vg domain. As Cv-d does not promote signaling in vitro, in vivo it more likely acts by increasing the movement of Dpp and Gbb from the LVs or their accumulation in the PCV region. This is consistent with the initial BMP signaling defects found near the center of cv-d mutant posterior cross veins (PCVs). The later appearance of defects near the LVs may be caused by the LV-specific expression of vein-inhibiting signals such as Delta and Argos. cv-d1 and cv-d13 also reduce the number of adult jump muscle fibers, the development of which is sensitive to changes in BMP signaling and loss of Crossveinless-Twisted gastrulation 2 (Cv-Tsg2). Thus, Cv-d probably regulates BMP signaling in at least two different contexts (Chen, 2012).

The activity of Cv-d in BMP signaling has interesting parallels to the signaling activities of the Drosophila Apolipophorins, which have a similar domain structure to Cv-d/160MEP proteins. Apolipophorins shuttle lipids from the fat body and digestive tract to other tissues via the hemolymph, but their role is not limited to lipid delivery (Chen, 2012).

Apolipophorins can increase the range over which signaling proteins move in the wing disc, an effect thought to be mediated by diffusion of an extracellular complex that contains lipids, Apolipophorins and lipid-linked signaling proteins such as Hedgehog, Wnts and HSPGs. Although BMPs are not lipid-linked proteins, Cv-d activity also requires both BMP and lipid binding motifs, consistent with BMP movement via a lipid-lipoprotein complex. The loss of Apolipophorins can also affect signaling by loss of lipid delivery, and thus lipid-dependent intracellular signaling, such as the lipid-mediated signal transduction triggered when Hedgehog binds to its receptor Patched. However, the evidence does not support a similar role for Cv-d-mediated lipid delivery in BMP signaling. Lipids have not been linked to the transduction of canonical BMP signaling, and reducing lipid delivery to wings through lipid starvation or reduction in Drosophila Apolipophorin expression greatly shrinks the size of the wing but does not alter venation or BMP signaling. Cv-d complexes also carry less lipid than apolipophorins. Lipid content in apolipophorin complexes is 30- 60%, but in complexes containing the Apis Cv-d homolog VHDL, it is only 10%. Like VHDL complexes, Cv-d complexes are higher density (lower lipid content) than Apolipophorin complexes (Chen, 2012).

The LDL-like receptors that mediate lipoprotein uptake do not appear to play a role in PCV development. Loss of the LRP1, LPR1&2, Megalin or the Vtg receptor Yolkless produces viable adults with normal crossveins. The only remaining LDL receptors in Drosophila are CG8909/MEGF7, which has been detected in neuronal tissue but not in wing imaginal discs, and the LRP5/6 homolog Arrow, which is required for Wingless/Wnt signaling during wing disc patterning but has no known role in BMP signaling. Thus, it is likely that the effect of Cv-d on BMP signaling is mediated by BMP binding, rather than via lipid delivery alone (Chen, 2012).

Although Vtgs are best known as the major components of yolk, there is a growing awareness that Vtg-like lipoproteins can have functions outside the yolk, such as immune protection and spermegg recognition in vertebrates, and clotting and melanization in arthropod hemolymph. Cv-d demonstrates a new and important function in BMP signaling. Is this function shared by other Vtg family proteins? Intriguingly, Xenopus Lipovitellin 1, a Vg domain-containing fragment of VtgA2, was isolated as a BMP4-binding protein, and purified VtgA2 bound to purified BMP4 and ActivinA (but not to TGF-β1) in surface plasmon resonance assays. Although the requirement for Vtg in the nutrition of early vertebrate embryos makes it difficult to interpret tests of Vtg function, the evolution of two different Vtg-family proteins in insects, the yolk Vtg and Cvd/ 160MEP families, may provide a 'natural experiment' that has allowed separation of the yolk and non-yolk roles of this protein family, demonstrating for the first time the importance of Vtg-BMP interaction (Chen, 2012).

Multistep molecular mechanism for bone morphogenetic protein extracellular transport in the Drosophila embryo

In the Drosophila embryo, formation of a bone morphogenetic protein (BMP) morphogen gradient requires transport of a heterodimer of the BMPs Decapentaplegic (Dpp) and Screw (Scw) in a protein shuttling complex. Although the core components of the shuttling complex--Short Gastrulation (Sog) and Twisted Gastrulation (Tsg)--have been identified, key aspects of this shuttling system remain mechanistically unresolved. Recently, it was discovered that the extracellular matrix protein collagen IV is important for BMP gradient formation. This study formulates a molecular mechanism of BMP shuttling that is catalyzed by collagen IV. Dpp is shown to be the only BMP ligand in Drosophila that binds collagen IV. A collagen IV binding-deficient Dpp mutant signals at longer range in vivo, indicating that collagen IV functions to immobilize free Dpp in the embryo. In vivo evidence is provided that collagen IV functions as a scaffold to promote shuttling complex assembly in a multistep process. After binding of Dpp/Scw and Sog to collagen IV, protein interactions are remodeled, generating an intermediate complex in which Dpp/Scw-Sog is poised for release by Tsg through specific disruption of a collagen IV-Sog interaction. Because all components are evolutionarily conserved, it is proposed that regulation of BMP shuttling and immobilization through extracellular matrix interactions is widely used, both during development and in tissue homeostasis, to achieve a precise extracellular BMP distribution (Sawala, 2012).

There is ample experimental and theoretical support for the notion that BMP gradient formation in the early embryo involves the concentration of the most potent signaling species, the Dpp/Scw heterodimer, at the dorsal midline in a process involving Sog and Tsg. This study presents in vivo evidence for a role of collagen IV in two key aspects of this shuttling model, which have remained mechanistically unresolved. First, collagen IV functions to immobilize free Dpp, explaining why Sog and Tsg are needed for Dpp movement. Second, collagen IV acts as a scaffold for assembly of the Dpp/Scw-Sog-Tsg shuttling complex. The advantage to BMP gradient formation of assembling the shuttling complex on collagen IV has been suggested by analysis of organism-scale mathematical models. These models reveal that the in vitro binding affinity between BMPs and Sog is too low to account for the rate of shuttling complex formation required in vivo. However, by acting as a scaffold, collagen IV would increase complex formation by locally concentrating Dpp/Scw and Sog. Models with a 10–20% reduction in diffusion rates for Dpp/Scw and Sog and an increased apparent affinity of Dpp/Scw for Sog, show the best fit to in vivo data (Sawala, 2012).

The molecular model of shuttling complex assembly occurs in three steps. The first step involves independent binding of Dpp/Scw and Sog to collagen IV. The ability of Dpp-Δa to signal long range in sog embryos, where wild-type Dpp is trapped in its expression stripe, provides in vivo evidence that the Dpp-collagen IV interaction restricts movement of free Dpp ligands. The result also demonstrates that Sog and Tsg promote long-range movement of Dpp because they release Dpp from collagen IV, and not simply because they prevent Dpp–receptor interactions. Restriction of Dpp diffusion by collagen IV may stabilize the gradient by preventing ventral movement of Dpp/Scw after release from Sog/Tsg and promoting Dpp/Scw–receptor interactions at the dorsal midline. It will be interesting, ultimately, to directly visualize Dpp and Dpp-Δa directly in sog and tsg mutant embryos. Although current methods allow detection of high levels of receptor-bound Dpp, there are technical limitations associated with specifically detecting the pools of Dpp that would be informative here, i.e., Dpp/Scw heterodimer within the shuttling complex or Dpp-Δa/Scw diffusing between cells. The data show that Scw is unable to bind the NC1 domain of collagen IV. This lack of collagen IV-dependent immobilization can explain why Scw, unlike Dpp, is capable of long-range signaling in the absence of Sog (Sawala, 2012).

Step 2 of shuttling complex assembly involves remodeling of the protein interactions to generate a poised intermediate. Specifically, step 2 is driven by Scw-mediated disruption of the Sog CR4–collagen IV interaction, so that Dpp/Scw is transferred from collagen IV to the Sog CR3-CR4 domains. Scw displacement of the Sog CR4 domain from collagen IV provides molecular insight as to why Scw is needed for Dpp transport. In addition to the binding preference of Sog and Tsg for the Dpp/Scw heterodimer, only Scw has a high affinity for the Sog CR4 domain. Therefore, Dpp/Scw can be released from collagen IV into the shuttling complex, whereas the Dpp homodimer remains trapped on collagen IV (Sawala, 2012).

In the final step of the model, Tsg mobilizes the shuttling complex by disrupting the Sog CR1–collagen IV interaction. It has been noted that tsg mutants display a more severe reduction in BMP signaling than sog and sog tsg double mutants. This observation has been attributed to a potential Sog-independent pro-BMP activity of Tsg at the level of receptor binding. A second contributing factor is suggested by the model, where Sog and Tsg act at distinct steps to allow formation of the shuttling complex. In tsg mutants, Dpp/Scw is loaded onto Sog by collagen IV, but remains locked in this inhibitory poised complex, so that the only BMPs capable of signaling are Dpp and Scw homodimers, which are less potent than the Dpp/Scw heterodimer. By contrast, in sog or sog tsg mutants, Dpp/Scw is not shuttled dorsally but is still capable of signaling locally, adding to signaling by Dpp and Scw homodimers. The weaker level of Dpp/Scw signaling in tsg mutants also provides support for the proposed order of steps 2 and 3 in the assembly process, because this order gives rise to the inhibitory intermediate of Dpp/Scw-Sog. Previously it was shown that an N-terminal fragment of Sog, called Supersog, which contains the CR1 domain and a portion of the stem, can partially rescue the loss of peak Dpp/Scw signaling in tsg embryos. The model suggests that this property of Supersog comes from the ability of its CR1 domain to compete with full-length Sog for binding to collagen IV, thereby releasing Sog-Dpp/Scw, similar to the role of Tsg in shuttling complex assembly. It is noted that the CR1–collagen IV interaction appears weaker than that of CR4–collagen IV, which may facilitate release of Dpp/Scw by Tsg or Supersog-like fragments. After Tsg-mediated release from collagen IV, the mobile shuttling complex can diffuse randomly. Upon Tolloid cleavage of Sog, the liberated Dpp/Scw heterodimer rebinds collagen IV, which either promotes receptor binding or a further round of shuttling complex assembly, depending on the local concentration of Sog (Sawala, 2012).

In addition to collagen IV, the basic region in Dpp/BMP2/4 also binds to heparan sulfate proteoglycans (HSPGs), which can either restrict or enhance BMP long-range movement. Indeed, this study found that an HSPG-binding mutant, Dpp-ΔN, also binds only weakly to collagen IV, suggesting that the collagen IV- and HSPG-binding sites on Dpp overlap. It will be interesting to test how HSPGs and collagen IV interact to regulate BMP activity in tissues where they are coexpressed, such as the early vertebrate embryo. In the early Drosophila embryo, the absence of glycosaminoglycan chains, which largely mediate binding of HSPG to Dpp, make it possible to specifically focus on the Dpp–collagen IV interaction (Sawala, 2012).

A shuttling-based mechanism of BMP transport is also used in a number of other developmental contexts, including the early vertebrate embryo, specification of the vertebral field in mice, and establishment of the posterior cross-vein territory in the Drosophila wing disk. Restriction of BMP movement may also be important in other contexts, including several where collagen IV was already shown to regulate a short-range Dpp signal, such as the ovarian stem cell niche and the tip of malpighian tubules. The basic collagen IV binding motif is highly conserved among the Dpp/BMP2/4 subfamily and is also found in some other BMPs, including BMP3, consistent with reports that BMP3 and BMP4 can bind collagen IV. Overall, these findings support the idea that the collagen IV–BMP interaction is a conserved aspect of extracellular BMP regulation and suggest that the function of collagen IV in both long-range BMP shuttling and local restriction of BMP movement will impact on a number of other contexts in both flies and vertebrates (Sawala, 2012).

Cytoneme-mediated delivery of hedgehog regulates the expression of bone morphogenetic proteins to maintain germline stem cells in Drosophila

Stem cells reside in specialised microenvironments, or niches, which often contain support cells that control stem cell maintenance and proliferation. Hedgehog (Hh) proteins mediate homeostasis in several adult niches, but a detailed understanding of Hh signalling in stem cell regulation is lacking. Studying the Drosophila female germline stem cell (GSC) niche, this study shows that Hh acts as a critical juxtacrine signal to maintain the normal GSC population of the ovary. Hh production in cap cells, a type of niche support cells, is regulated by the Engrailed transcription factor. Hh is then secreted to a second, adjacent population of niche cells, the escort cells, where it activates transcription of the GSC essential factors Decapentaplegic (Dpp) and Glass bottom boat (Gbb). In wild-type niches, Hh protein decorates short filopodia that originate in the support cap cells and that are functionally relevant, as they are required to transduce the Hh pathway in the escort cells and to maintain a normal population of GSCs. These filopodia, reminiscent of wing disc cytonemes, grow several fold in length if Hh signalling is impaired within the niche. Because these long cytonemes project directionally towards the signalling-deficient region, cap cells sense and react to the strength of Hh pathway transduction in the niche. Thus, the GSC niche responds to insufficient Hh signalling by increasing the range of Hh spreading. Although the signal(s) perceived by the cap cells and the receptor(s) involved are still unknown, these results emphasize the integration of signals necessary to maintain a functional niche and the plasticity of cellular niches to respond to challenging physiological conditions (Rojas-Ríos, 2012).

The study of the mechanisms behind Hh signalling in the Drosophila ovary has allowed the identification of Hh-coated cytonemes in a cellular stem cell niche, emphasizing the idea that cytonemes mediate spreading of the activating signal from the producing cells. Recently, it has been reported that the Hh protein localises to long, basal cellular extensions in the wing disk (Callejo, 2011). In addition, filopodial extensions in the wing, eye, and tracheal system of Drosophila have been shown to segregate signalling receptors on their surface, thus restricting the activation of signalling pathways in receiving cells (Roy, 2011). Hence, cytonemes, as conduits for signalling proteins, may be extended by receiving cells (and so are involved in uptake) or may be extended by producing cells (and so are involved in delivery and release) (Rojas-Ríos, 2012).

Interfering with actin polymerisation in adult niches leads to a significant reduction in the number of cap cells (CpCs) growing Hh cytonemes, concomitant with precocious stem cell differentiation, demonstrating that these actin-rich structures are required to prevent stem cell loss and thus are functionally relevant. Importantly, because this study disturbed actin dynamics in post-mitotic CpCs that still produce wild-type levels of Hh protein and express CpC markers (but fail to activate the Hh pathway in ECs), the observed effects on stem cell maintenance are most likely specific to Hh delivery from CpCs to their target ECs via short cytonemes. This interpretation is further reinforced by the observation that CpCs can sense decreased Hh levels and/or a dysfunction in the transduction of the Hh pathway in the niche and respond to it by growing Hh-rich membrane bridges up to 6-fold longer than in controls. In this regard, it is interesting to note that the two lipid modifications found in mature Hh protein act as membrane anchors and give secreted Hh a high affinity for membranes and signalling capacities. In fact, it has been recently described that a lipid-unmodified form of Hh unable to signal does not decorate filopodia-like structures in the wing imaginal disc epithelium, confirming the link between Hh transport along cytonemes and Hh signalling (Callejo, 2011). Thus, cytonemes may ensure specific targeting of the Hh ligand to the receiving germline cells in a context of intense signalling between niche cells and the GSCs. Interestingly, in both en- and smo- mosaic niches, the long processes projected towards the signalling-deficient area of the niche, which showed that competent CpCs sense the strength of Hh signalling activity in the microenvironment. While the nature of the signal perceived by the CpCs or the receptor(s) involved in the process are unknown, it is postulated that Hh-decorated filopodial extensions represent the cellular synapsis required for signal transmission that is established between the Hh-producing cells (the CpCs) and the Hh-receiving cells (the ECs). In this scenario (and because Ptc, the Hh receptor, is a target of the pathway) the membranes of mutant ECs, in which the transduction of the pathway is compromised, contain lower Ptc levels. Thus, longer and perhaps more stable projections ought to be produced to allow proper signalling. In addition, the larger the number of en mutant cells (and hence the stronger the deficit in Hh ligand concentration or target gene regulation), the longer the cellular projections decorated with Hh, which indicates that the niche response is graded depending on the degree of signalling shortage (Rojas-Ríos, 2012).

Do the longer cytonemes found in mosaic germaria represent structures created de novo, or do they simply reflect a pre-existing meshwork of thin intercellular bridges that can regulate the amount of Hh protein in transit across them? Because an anti-Hh antibody was utilised to detect the cytonemes and all attempts to identify other markers for these structures have failed, it is not possible to discriminate between these two possibilities. In any case, since no increased was detected in Hh levels in wild-type CpCs that contained cytonemes relative to those that did not, it is clear that long filopodia do not arise solely by augmenting Hh production in the CpCs. Rather, if long cytonemes are not synthesised in response to a Hh signalling shortage and if they already existed in the niche, they ought to restrict Hh spreading independently of significant Hh production. Furthermore, because the strength of Hh signalling in the niche determines the distance of Hh spreading, either cytoneme growth or Hh transport (or both) are regulated by the ability of the CpCs to sense the Hh signalling output (Rojas-Ríos, 2012).

The demonstration that a challenged GSC niche can respond to insufficient signalling by the cytoneme-mediated delivery of the stem cell survival factor Hh over long distances has wider implications. Niche cells have been shown to send cellular processes to their supporting stem cells in several other scenarios: the Drosophila ECs of the ovary and the lymph gland, the ovarian niche of earwigs, and the germline mitotic region in the hermaphrodite Caenorhabditis elegans. Similarly, wing and eye disc cells project cytonemes to the signalling centre of the disc. However, definitive proof that the thin filopodia described in the lymph gland, the earwig ovary, or imaginal discs deliver signals from the producing to the effector cells is lacking. The current findings strongly suggest that cytonemes have a role in transmitting niche signals over distance, a feature that may underlie the characteristic response of more complex stem cell niches to challenging physiological conditions. Careful analysis of the architecture of sophisticated niches, such as the bone marrow trabecular zone for mouse haematopoietic stem cells, will be needed to further test this hypothesis and to determine whether it represents a conserved mechanism for stem cell niche signalling (Rojas-Ríos, 2012).

Nanotubes mediate niche-stem-cell signalling in the Drosophila testis

Stem cell niches provide resident stem cells with signals that specify their identity. Niche signals act over a short range such that only stem cells but not their differentiating progeny receive the self-renewing signals. However, the cellular mechanisms that limit niche signalling to stem cells remain poorly understood. This study shows that the Drosophila male germline stem cells form previously unrecognized structures, microtubule-based nanotubes, which extend into the hub, a major niche component. Microtubule-based nanotubes are observed specifically within germline stem cell populations, and require intraflagellar transport proteins for their formation. The bone morphogenetic protein (BMP) receptor Tkv localizes to microtubule-based nanotubes. Perturbation of microtubule-based nanotubes compromises activation of Dpp signalling within germline stem cells, leading to germline stem cell loss. Moreover, Dpp ligand and Tkv receptor interaction is necessary and sufficient for microtubule-based nanotube formation. The study proposes that microtubule-based nanotubes provide a novel mechanism for selective receptor-ligand interaction, contributing to the short-range nature of niche-stem-cell signalling (Inaba, 2015).

The Drosophila testis represents an excellent model system to study niche-stem-cell interactions because of its well-defined anatomy: eight to ten germline stem cells (GSCs) are attached to a cluster of somatic hub cells, which serve as a major component of the stem cell niche. The hub secretes at least two ligands: the cytokine-like ligand Unpaired (Upd), and a BMP ligand Decapentaplegic (Dpp), both of which regulate GSC maintenance. GSCs typically divide asymmetrically, so that one daughter of the stem cell division remains attached to the hub and retains stem cell identity, while the other daughter, called a gonialblast, is displaced away from the hub and initiates differentiatio. Given the close proximity of GSCs and gonialblasts, the ligands (Upd and Dpp) must act over a short range so that signalling is only active in stem cells, but not in differentiating germ cells. The basis for this sharp boundary of pathway activation remains poorly understood (Inaba, 2015).

Using green fluorescent protein (GFP)-α1-tubulin84B expressed in germ cells (nos-gal4>UAS-GFP-αtub), this study found that GSCs form protrusions, referred to as microtubule-based (MT)-nanotubes hereafter, that extend into the hub. MT-nanotubes are sensitive to fixation similar to other thin protrusions reported so far, such as tunnelling nanotubes, explaining why they have escaped detection in previous studies. MT-nanotubes appear to be specific to GSCs: 6.67 MT-nanotubes were observed per testis in the GSC population (or 0.82 per cell). The average thickness and length of MT-nanotubes are 0.43 ± 0.29 µm (at the base of MT-nanotube) and 3.32 ± 1.6 µm, respectively. These GSC MT-nanotubes are uniformly oriented towards the hub area. By contrast, differentiating germ cells showed only 0.44 MT-nanotubes per testis (or <0.002 per cell), without any particular orientation when present. MT-nanotubes were sensitive to colcemid, the microtubule-depolymerizing drug, but not to the actin polymerization inhibitor cytochalasin B, suggesting that MT-nanotubes are microtubule-based structures. MT-nanotubes were not observed in mitotic GSCs, and GSCs form new MT-nanotubes as they exit from mitosis. By contrast, MT-nanotubes in interphase GSCs were stably maintained for up to 1 h of time-lapse live imaging. Although cell-cycle-dependent formation of MT-nanotube resembles that of primary cilia, MT-nanotubes are distinct structures, in that they lack acetylated microtubules and are sensitive to fixation. Furthermore, a considerable fraction of GSCs form multiple MT-nanotubes per cell (54% of GSCs with MT-nanotubes), and MT-nanotubes are not always associated with the centrosome/basal body, as is the case for the primary cilia (Inaba, 2015).

To examine the geometric relationship between MT-nanotubes and hub cells further, MT-nanotubes were imaged in combination with various cell membrane markers, followed by three-dimensional rendering. Although the MT-nanotubes are best visualized in unfixed testes that express GFP-αTub in germ cells, adding a low concentration (1 μM) of taxol to the fixative preserves MT-nanotubes, allowing immunofluorescence staining. First, Armadillo (Arm, β-catenin) staining, which marks adherens junctions formed at hub cell/hub cell as well as hub cell/GSC boundaries, revealed that adherens junctions do not form on the surface of MT-nanotubes. Using FM4-64 styryl dye, it was found that the MT-nanotubes are ensheathed by membrane lipids. Furthermore, myristoylation/palmitoylation site GFP (myrGFP), a membrane marker, expressed in either the germline or hub cells illuminated MT-nanotubes, suggesting that the surface membrane of a MT-nanotube is juxtaposed to hub-cell plasma membrane (Inaba, 2015).

Genes were examined that regulate primary cilia and cytonemes for their possible involvement in MT-nanotube formation. RNA interference (RNAi)-mediated knockdown of oseg2 (IFT172), osm6 (IFT52) and che-13 (IFT57), components of the intraflagellar transport (IFT)-B complex that are required for primary cilium anterograde transport and assembly, significantly reduced the length and the frequency of MT-nanotubes. Knockdown of Dlic, a dynein intermediate chain required for retrograde transport in primary cilia<, also reduced the MT-nanotube length and frequency. Knockdown of klp10A, a Drosophila homologue of mammalian kif24 (a MT-depolymerizing kinesin of the kinesin-13 family, which suppresses precocious cilia formation), resulted in abnormally thick/bulged MT-nanotubes. No significant changes were observed in MT-nanotube morphology upon knockdown of IFT-A retrograde transport genes, such as oseg1 and oseg3 (Inaba, 2015).

Endogenous Klp10A localized to MT-nanotubes both in wild-type testes and in GFP-αTub-expressing testes. GFP-Oseg2 (IFT-B), GFP-Oseg1, GFP-Oseg3 (IFT-A) and Dlic also localized to the MT-nanotubes when expressed in germ cells. The localization of IFT-A components to MT-nanotubes, without detectable morphological abnormality upon mutation/knockdown, is reminiscent of the observation that most of the genes for IFT-A are not required for primary cilia assembly. Expression of a dominant negative form of Dia (DiaDN) or a temperature-sensitive form of Shi (Shits) in germ cells (nos-gal4>UAS-diaDN or UAS-shits), which perturb cytoneme formation, did not influence the morphology or frequency of MT-nanotubes in GSCs. Taken together, these results show that primary cilia proteins localize to MT-nanotubes and regulate their formation (Inaba, 2015).

In search of the possible involvement of MT-nanotubes in hub-GSC signalling, it was found that the Dpp receptor, Thickveins (Tkv), expressed in germ cells (nos-gal4>tkv-GFP) was observed within the hub region, in contrast to GFP alone, which remained within the germ cells. A GFP protein trap of Tkv (in which GFP tags Tkv at the endogenous locus) also showed the same localization pattern as Tkv-GFP expressed by nos-gal4. By inducing GSC clones that co-express Tkv-mCherry and GFP-αTub, it was found that Tkv-mCherry localizes along the MT-nanotubes as puncta. Furthermore, using live observation, Tkv-mCherry puncta were observed to move along the MT-nanotubes marked with GFP-αTub, suggesting that Tkv is transported towards the hub along the MT-nanotubes. It should be noted that, in the course of this study, it was noticed that mCherry itself localized to the hub when expressed in germ cells, similar to Tkv-GFP and Tkv-mCherry. Importantly, the receptor for Upd, Domeless (Dome), predominantly stayed in the cell body of GSCs, demonstrating the specificity/selectivity of MT-nanotubes in trafficking specific components of the niche signalling pathways. A reporter of ligand-bound Tkv, TIPF localized to the hub region together with Tkv-mCherry, in addition to its reported localization at the hub-GSC interface. Furthermore, Dpp-GFP expressed by hub cells co-localized with Tkv-mCherry expressed in germline. These results suggest that ligand (Dpp)-receptor (Tkv) engagement and activation occurs at the interface of the MT-nanotube surface and the hub cell plasma membrane. Knockdown of IFT-B components (oseg2RNAi, che-13RNAi or osm6RNAi), which reduces MT-nanotube formation, resulted in reduction of the number of Tkv-GFP puncta in the hub area, concomitant with increased membrane localization of Tkv-GFP. A similar trend was observed upon treatment of the testes with colcemid, suggesting that MT-nanotubes are required for trafficking of Tkv into the hub area. By contrast, knockdown of Klp10A, which causes thickening of MT-nanotubes, led to an increase in the number of Tkv-GFP puncta in the hub area. Taken together, these data suggest that Tkv is trafficked into the hub via MT-nanotubes, where it interacts with Dpp secreted from the hub (Inaba, 2015).

Knockdown of klp10A (klp10ARNAi) led to elevated phosphorylated Mad (pMad) levels, a readout of Dpp pathway activation, in GSCs. By contrast, RNAi-mediated knockdown of oseg2, osm6 and che-13 (IFT-B components), which causes shortening of MT-nanotubes, reduced the levels of pMad in GSCs. Dad-LacZ, another readout of Dpp signalling activation, exhibited clear upregulation upon knockdown of klp10A. GSC clones of che-13RNAi, osm6RNAi or oseg2452 were lost rapidly compared with control clones, consistent with the idea that MT-nanotubes help to promote Dpp signal transduction. Knockdown of oseg2, che-13 and osm6 did not visibly affect cytoplasmic microtubules, suggesting that GSC maintenance defects upon knockdown of these genes are probably mediated by their role in MT-nanotube formation. Global RNAi knockdown of these genes in all GSCs using nos-gal4 did not cause a significant decrease in GSC numbers , indicating that compromised Dpp signalling due to MT-nanotube reduction leads to a competitive disadvantage in regards to GSC maintenance only when surrounded by wild-type GSCs (Inaba, 2015).

When klp10ARNAi GSC clones were induced, pMad levels specifically increased in those GSC clones, indicating that Klp10A acts cell-autonomously in GSCs to influence Dpp signal transduction. Importantly, klp10ARNAi spermatogonia did not show a significant elevation in pMad level compared with control spermatogonia, demonstrating that the role of Klp10A in regulation of Dpp pathway is specific to GSCs. pMad levels did not change in spermatogonia upon manipulation of MT-nanotube formation. GSC clones of klp10ARNAi or klp10A null mutant (klp10A24) did not dominate in the niche, despite upregulation of pMad, possibly because of its known role in mitosis. Importantly, these conditions did not significantly change STAT92E levels, which reflect Upd-JAK-STAT signalling in GSCs, revealing the selective requirement of MT-nanotubes in Dpp signalling. Together, these results demonstrate that MT-nanotubes specifically promote Dpp signalling and their role in enhancing the Dpp pathway is GSC specific (Inaba, 2015).

Since cytonemes are induced/stabilized by the signalling molecules themselves, the possible involvement of Dpp in MT-nanotube formation was explored First, it was found that a temperature-sensitive dpp mutant (dpphr56/dpphr4) exhibited a dramatic decrease in the frequency of MT-nanotubes (0.067 MT-nanotubes per GSC) and the remaining MT-nanotubes were significantly thinner. Knockdown of tkv (tkvRNAi) in GSCs also resulted in reduced length and frequency of MT-nanotubes. Conversely, overexpression of Tkv (tkvOE) in germ cells led to significantly longer MT-nanotubes. Interestingly, expression of a dominant negative Tkv (tkvDN), which has intact ligand-binding domain but lacks its intracellular GS domain and kinase domain, resulted in thickening of MT-nanotubes, rather than reducing the thickness/length. This indicates that ligand-receptor interaction, but not downstream signalling events, is sufficient to induce MT-nanotube formation. Strikingly, upon ectopic expression of Dpp in somatic cyst cells (tj-lexA>dpp), spermatogonia/spermatocytes were observed to have numerous MT-nanotubes, suggesting that Dpp is necessary and sufficient to induce or stabilize MT-nanotubes in the neighbouring germ cells. In turn, MT-nanotubes may promote selective ligand-receptor interaction between hub and GSCs, leading to spatially confined self-renewal (Inaba, 2015).

This study shows that previously unrecognized structures, MT-nanotubes, extend into the hub to mediate Dpp signalling. It is proposed that MT-nanotubes form a specialized cell surface area, where productive ligand-receptor interaction occurs. In this manner, only GSCs can access the source of highest ligand concentration in the niche via MT-nanotubes, whereas gonialblasts do not experience the threshold of signal transduction necessary for self-renewal, contributing to the short-range nature of niche signalling. In summary, the results reported here illuminate a novel mechanism by which the niche specifies stem cell identity in a highly selective manner (Inaba, 2015).

Adaptive protein divergence of BMP ligands takes place under developmental and evolutionary constraints

The bone morphogenetic protein (BMP) signaling network, comprising evolutionary conserved BMP2/4/Decapentaplegic (Dpp) and Chordin/Short gastrulation (Sog), is widely utilized for dorsal-ventral (DV) patterning during animal development. A similar network is required for posterior crossvein (PCV) formation in the Drosophila pupal wing. Although both transcriptional and post-transcriptional regulation of co-factors in the network appears to give rise to tissue-specific and species-specific properties, their mechanisms are incompletely understood. In Drosophila, BMP5-8 type ligands, Screw (Scw) and /aGlass bottom boat (Gbb), form heterodimers with Dpp for DV patterning and PCV development, respectively. Sequence analysis indicates that the Scw ligand contains two N-glycosylation motifs; one being highly conserved between BMP2/4 and BMP5-8 type ligands, and the other being Scw ligand-specific. The data reveal that N-glycosylation of the Scw ligand boosts BMP signaling both in cell culture and in the embryo. In contrast, N-glycosylation modifications of Gbb or Scw ligands reduce the consistency of PCV development. These results suggest that tolerance for structural changes of BMP5-8 type ligands is dependent on developmental constraints. Furthermore, gain and loss of N-glycosylation motifs in conserved signaling molecules under evolutionary constraints appear to constitute flexible modules to adapt to developmental processes (Tauscher, 2016).

This study provides insights into how evolutionary and developmental pressures shape molecules after their divergence from a common ancestor. A conserved N-glycosylation motif exists, which is specific for BMP-type ligands throughout various animal species. In addition, it was observed that the BMP5-8-type ligand Scw contains a unique N-glycosylation motif that helps to maintain a peak level of BMP signal in the embryo. In contrast, N-glycosylation modifications of BMP-type ligands reduce the consistency in PCV development. These observations provide insights into how evolutionarily conserved signaling molecules adapt to developmental processes (Tauscher, 2016).

The significance of N-glycosylation of the TGF-β-type ligands has been studied previously. For example, N-glycosylation of the BMP2 prodomain affects the folding and secretion of ligands, and non-glycosylated BMP2 and BMP6 produced in bacterial cells appear to be less active than the glycosylated ligands. Addition of an N-glycosylation motif in Nodal changes the stability of ligands, resulting in an increased signaling range. These facts suggest that N-glycosylation of ligands may play significant roles in vivo. However, these roles have been largely unexplored because of a lack of in vivo model systems. By employing both in vivo studies and cell-based experiments, this study has investigated how N-glycosylation modifications of the BMP-type ligands impact developmental processes. The in vivo rescue experiments revealed that these motifs are crucial for fly viability and are required to achieve peak level BMP signaling. Loss of the Scw-specific motif leads to a reduced impact on BMP signaling in the embryo compared with the effect of the conserved motif but also to less signaling capacity when compared to ScwWT, resulting in lower viability of g.scwN1Q rescued flies. On the other hand, integration of the Scw-specific N-glycosylation motif into its paralog Gbb (Scw-Gbb chimera) is not sufficient to provide functionality in the early embryo. This suggests that the critical changes responsible for the differing specificity of the Gbb and Scw ligands that developed after gene duplication may be differences in the primary sequences other than N-glycosylation motifs (Tauscher, 2016).

As reported in the case of Nodal, adding N-glycosylation sites to ligands may change protein stability/secretion and therefore may affect in vivo phenotypes. In the case of Scw, it is presumed that acquisition of the unique N-glycosylation motif has no drastic effect on protein stability/secretion, but instead directly affects the signaling outcome. First, equal amounts of differentially glycosylated ligands show different signaling intensities in the cell-based assay. Second, expression of differentially glycosylated ligands showed different signaling intensities in the embryo when they are expressed in identical genetic backgrounds. Third, the total protein levels in both cell lysates and supernatants for ScwWT, ScwN1Q and ScwN2Q are equivalent when they are expressed in S2 cells. Thus, these results suggest that changing the number and positions of N-glycosylation motifs may impact signaling intensities both in vivo and in vitro without significantly changing protein stability/secretion. In contrast, non-glycosylated Scw ligand (ScwN1_N2Q) appears to be less efficiently secreted. These facts suggest that at least one N-glycosylation site of Scw is crucial for maintaining protein stability/secretion, but their number or position may not be essential for secretion (Tauscher, 2016).

Interestingly, N-glycosylation of the ligands did not provide any advantage for PCV formation. Instead, the Scw ligand lacking both N-glycosylation motifs (ScwN1_N2Q) most efficiently restored the PCV-less phenotypes in gbb mutant wings. It is hypothesized that N-glycosylation of BMP ligands does not always benefit extracellular trafficking of ligands. Highly glycosylated ligands may interact with enriched extracellular matrix (ECM) at the basal side of wing epithelia and reduce the ligand mobility regulated by the BMP network. Alternatively, differential expression of key molecules may explain different phenotypes between embryogenesis and crossvein development. It has been previously reported that the heparan sulfate proteoglycan (HSPG) Dally impacts BMP signaling in various contexts. Dally plays a role in Dpp gradient formation in the wing imaginal disc by stabilizing Dpp and it increases the signaling of Gbb and Dpp in Drosophila S2 cells. In addition, lack of Dally and Dally-like protein (Dlp) affects PCV formation in the wing. Interestingly, HSPGs are absent within the first 3 hours of embryogenesis, which is the only time frame of scw expression. Based on these facts, it appears that Scw and HSPGs are mutually exclusive. This may partly explain why non-glycosylated Scw is functional for PCV development but not for embryonic DV patterning. Furthermore, the ScwN1_N2Q:Dpp heterodimer is likely to be a primary ligand responsible for BMP signaling in the PCV region. Since Dpp carries the conserved N-glycosylation motif, the ScwN1_N2Q:Dpp heterodimer contains one N-glycosylation site, although ScwN1_N2Q lacks N-glycosylation site. The N-glycosylation site of Dpp may help facilitate ScwN1_N2Q:Dpp heterodimer secretion (Tauscher, 2016).

Why is a unique N-glycosylation site acquired in the Scw ligand? scw is exclusively expressed in the early embryo, which is in contrast to the usually recurrent activity of signaling molecules at different stages of development. The favored model is that random mutations create differential N-glycosylation motifs in otherwise functionally redundant and conserved ligands. These novel motifs lead to structural changes that confer either advantages or disadvantages, depending on the developmental context. Since a positive feedback mechanism is crucial for DV patterning in Drosophila, acquisition of the unique N-glycosylation site could bring an advantage to Scw signaling. In contrast, in a wide range of species including humans, BMP2/4- and BMP5-8-type ligands are repeatedly utilized for development at different stages and in different positions. Therefore, to provide robustness and reproducibility in various contexts, vertebrate BMP2/4 and BMP5-8 contain only one N-glycosylation site to impose developmental constraints: stronger signaling than a non-glycosylated ligand, and less impeded extracellular trafficking than additionally glycosylated ligands. Consistently, Gbb has been shown to function at various developmental stages (Tauscher, 2016).

Although various co-factors of the BMP network have been identified among species, it remains to be addressed how they adapted to different developmental stages and different species. The scw allele was originally identified as a DV patterning defect and was determined to encode a BMP5-8-type protein. It was then proposed that scw originates from gene duplication of gbb in the branch leading to higher Diptera, a highly diverged branch in the arthropod lineage. Hence, gbb and scw provide an outstanding opportunity to investigate evolutionary divergence of protein structures. In Drosophila, gbb and scw are expressed in distinct patterns, but both function as co-factors of the BMP network. A recent study indicates both Gbb and Scw are utilized for DV patterning in the scuttle fly. gbb expression was also described in the early embryo of the lower Dipteran Clogmia albipunctata, in which the scw gene was not found. These facts indicate a possibility that Gbb acts as a co-factor of the BMP network for DV patterning in most arthropod species and that Scw evolved specifically for DV patterning in higher Diptera after duplication of the scw-like gene gbb. Further studies are needed to elucidate how Gbb lost the capacity to transduce signals in the Drosophila blastoderm embryo (Tauscher, 2016).

In summary, these data reveal that two BMP5-8-type ligands, Scw and Gbb, which function as co-factors of the BMP network, provide a unique model to investigate how orthologous proteins evolve under developmental and evolutionary constraints. Further studies in this context will help elucidate how evolutionarily conserved molecules generate diversified structures in the animal kingdom (Tauscher, 2016).

Basement membrane manipulation in Drosophila wing discs affects Dpp retention but not growth mechanoregulation

Basement membranes (BMs) are extracellular matrix polymers basally underlying epithelia, where they regulate cell signaling and tissue mechanics. Constriction by the BM shapes Drosophila wing discs, a well-characterized model of tissue growth. Recently, the hypothesis that mechanical factors govern wing growth has received much attention, but it has not been definitively tested. This study manipulated BM composition to cause dramatic changes in tissue tension. Increased tissue compression was found when perlecan was knocked down did not affect adult wing size. BM elimination, decreasing compression, reduced wing size but did not visibly affect Hippo signaling, widely postulated to mediate growth mechanoregulation. BM elimination, in contrast, attenuated signaling by bone morphogenetic protein/transforming growth factor beta ligand Dpp, which was not efficiently retained within the tissue and escaped to the body cavity. These results challenge mechanoregulation of wing growth, while uncovering a function of BMs in preserving a growth-promoting tissue environment (Ma, 2017).

Basement membranes (BMs) are laminar polymers of extracellular matrix proteins which underlie epithelia and surround organs in all animals. The main components of BMs are collagen IV, nidogen, laminin, and perlecan, all conserved from insects to humans. Despite long-known conservation, ubiquity in animal tissues, and extensive biochemical knowledge, understanding of the developmental roles of BMs is comparatively poor. Nonetheless, significant progress has been made in recent years with the help of model organisms, such as Drosophila melanogaster and Caenorhabditis elegans, thanks to limited genetic redundancy of BM components in these systems. In this way, it has been shown in the fruit fly Drosophila that collagen IV is required for full Dpp activity in dorsal cells of the embryo and for the response to Dpp of renal tubules. In addition, BMs are now known to play an essential role in mechanically shaping tissues: in the absence of a BM, tissues such as the egg follicleand the larval imaginal discs uffer profound deformations (Ma, 2017).

Drosophila adult wings develop from the pouch region of the wing imaginal disc, a widely studied model for tissue growth regulation. The wing pouch of the third instar larva (L3 stage) is a highly columnar monolayered epithelium where each cell attaches to the BM. Recently, the hypothesis that mechanical factors contribute to the regulation of wing growth has gathered considerable momentum. The observations that cell compression is higher at the center of the pouch and that compression increases during larval development have led to several models postulating a negative effect of compression on growth. This negative effect of compression on growth is invoked to solve the apparent paradox that combined concentration of growth promoters Dpp and Wingless (Wg) is higher at the center of the pouch, yet the distribution of cell proliferation is roughly homogeneous throughout the disc. In this context, the Hippo signaling pathway, known to respond to cell contact, cell crowding, and cytoskeletal tension has been postulated as a mediator of mechanical inputs into wing growth. However, the difficulty of experimentally changing tissue constriction in an internally developing organ has precluded definitive testing of this hypothesis (Ma, 2017).

To investigate the developmental role of the BM and explore the influence of mechanical factors on wing growth, this study subjected wing discs to different BM manipulations changing tissue constriction in order to assess their effect on disc development and adult wing size. The results show a lack of effect of mechanical constriction on Hippo signaling and wing growth. In contrast, BM was foudn to contribute to tissue growth by enhancing tissular retention of Dpp (Ma, 2017).

The results of the experiments changing tissue constriction through BM manipulation are difficult to reconcile with a physiological role of cell compression in regulation of normal wing growth, a central tenet of wing growth mechanoregulation models. Increase in compression when perlecan was knocked down, and decreased compression when the BM was degraded, both failed to produce the predicted effects: smaller and larger wings, respectively. In contrast to the results in the larval wing, tissue size regulation by cell crowding and apoptosis has been shown to occur in the notum during metamorphosis. Since both the wing and the notum derive from the same imaginal disc, it follows that mechanical effects on size must be highly dependent on the specific developmental context (Ma, 2017).

The failure to observe changes in Hippo activity after dramatic changes in tissue shape also challenges the role of Hippo signaling in regulating wing growth in response to compression. Nonetheless, several manipulations of cytoskeletal components clearly influence Hippo signaling in the wing, affecting growth. Because the actin-rich zonula adherens is the physical locus where Hippo signaling complexes assemble, Hippo signaling may act as a critical sensor of cell polarity or cell contact. According to the current results, however, it does not act in the wing as a tension-growth feedback regulator slowing growth in response to cell crowding (Ma, 2017).

Discs made of larger, fewer cells have long been known to give rise to normally sized adult wings, indicating that some parameter different from cell numbers contributes to defining final wing size, for instance some physical dimension of the tissue such as planar area or tissue volume. BM manipulations dramatically changed apical area and height of individual cells and of the tissue as a whole, but they may not have changed cell size, as suggested by the fact that cell density in the adult wing did not change. These findings, therefore, would be consistent with a model in which tissue mass or volume contributes to determination of final wing size. Normally sized discs and adult wings made of larger, fewer cells, in addition, offer a further argument against mechanical regulation of wing growth, as these larger cells would display very different physical properties in terms of their apical areas and the tensions supported by their membranes and cytoskeletons (Ma, 2017).

Even though no mechanical effects on Hippo signaling or wing growth were detected following profound tissue deformations, it cannot be completely rule out that BM manipulations caused secondary effects that negated putative effects of mechanical signals. Such is the case, it is arguee, of the discs flattened by BM elimination. These discs gave rise to smaller adult wings, an effect that further experiments indicate is a result of the specific requirement of the BM in Dpp signaling. Nonetheless, this study also failed to detect changes in cell proliferation or adult wing size when discs were flattened in vivo through direct application of force. Importantly, a contribution of the directionality of compression is also a possibility that cannot be rule out, as cells in the periphery of both act > troli and rn > Mmp2 discs change their apical discs change their apical area, but maintain the tendency of the wild-type to align their major axis tangentially to the center of the disc. Therefore, if the vector of the compression rather than its magnitude is readable by a cell or its neighbors, the results cannot rule out a role for this in regulating wing growth. This pattern of cell orientation has been attributed to a slightly higher proliferation rate in the center of the wing pouch, a fact overlooked in the past and possibly responsible in the first place for the higher cell compression in the center of the wing. BM modifications, therefore, would not affect this intrinsically different proliferation rate in the central and peripheral wing regions. The results, finally, do not rule out the possibility that more extreme mechanical inputs could impact wing growth, for instance in wound healing or damage-stimulated growth (Ma, 2017).

Despite the lack of influence on Hippo signaling in the BM manipulations, the data show that the BM itself is required to preserve a growth-promoting environment by hindering diffusion of Dpp out of the disc. Collagen IV, the main component of BMs, physically interacts with Dpp through the C-terminal NC1 domains of both collagen IV chains. The effects of collagen IV loss on Dpp signaling in the wing, the dorsal blastoderm and germarium, and renal tubules are all consistent with a role of collagen IV in Dpp concentration. Elimination of the BM, however, did not seem to affect signaling by the other diffusible ligands Wg and Hh, which are, unlike Dpp, quite hydrophobic and may not require a mechanism preventing their escape from the tissue. The role of the BM in maintaining the concentration of extracellular ligands, therefore, may not be general, but ligand specific or specific to Dpp (Ma, 2017).

A role has been attributed to Dpp signaling in modulating cell height in the wing epithelium. Even though the current experiments eliminating the BM caused both a Dpp deficit and decreased cell height, it is unlikely that the effects on cell height in this experiment are caused by the Dpp deficit. First, the effects of collagenase treatment on disc morphology are immediate, which is difficult to explain as a deficit in Dpp signaling, specially a transcriptionally mediated effect. Second, discs in which the BM was simultaneously degraded and Dpp signaling was activated were still flattened, supporting the idea that effects on tissue shape elicited by BM degradation are not due to a Dpp deficit (Ma, 2017).

Since Dpp does not seem to accumulate basally in the wing disc, it is hypothesized that transient binding of Dpp allows the wing BM to act as a semipermeable barrier hindering Dpp diffusion, although not completely preventing it. This is a function that other BMs are long known to serve in the vertebrate kidney or the blood-brain barrier. Indeed, the results showing homogeneously high levels of Dpp signaling in the disc when Dpp was expressed in the fat body demonstrate an ability of Dpp to cross the BM. This result has also implications for understanding of Dpp signaling in the wing, as it shows that Dpp presentation by apical cytonemes is not absolutely required for signaling. A function of the BM in limiting basal escape of Dpp is, in addition, highly consistent with recent findings showing that a Dpp.GFP fusion could be immobilized at the BM, with effects on patterning and growth similar to the ones observed when the BM was eliminated. The findings support a critical role for basolaterally diffusing Dpp against a competing hypothesis stating that the functional Dpp gradient forms apically. It must be noted, however, that the role of the medial Dpp stripe in regulating growth has been called into question during the third larval instar, when a non-stripe source in the anterior compartment would serve this growth-promoting function instead. Because BM elimination reduces not just medial spalt and pMad, but also growth, it follows that the BM is required to maintain the concentration of Dpp from both sources: the medial stripe and the unknown anterior non-stripe source (Ma, 2017).

Given the conservation of BM components and Dpp, BM degradation and epithelial-to-mesenchymal transitions may enhance BMP/TGF-β signaling across tissue layers in development. The results also suggest a way in which tumoral BM degradation could contribute to tissue signaling misregulation in cancer by allowing escape of these diffusible signals. Finally, the visualization of an apico-basal gradient of Dpp in this highly columnar epithelium calls for the inclusion of the apico-basal dimension in future quantitative studies of Dpp gradient formation (Ma, 2017).


decapentaplegic: Biological Overview | Evolutionary Homologs | Transcriptional regulation | Targets of activity | Developmental Biology | Effect of mutation | References

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