Interactive Fly, Drosophila

Dorsal-ventral patterning within the embryonic ectoderm of Drosophila requires two type I TGFbeta receptors, Tkv and Sax, as well as two TGFbeta ligands, Dpp and Screw. In embryos lacking dpp signaling, increasing the level of Tkv activity promotes progressively more dorsal cell types, while activation of Sax alone has no phenotypic consequences. However, Sax activity synergizes with Tkv activity to promote dorsal development. To determine the interrelationship between the signaling pathways downstream of the Tkv and Sax receptors, an assay was carried out of the phenotypic consequences of activating each signaling pathway separately in embryos that lack dpp expression. Increasing levels of activation of Tkv signaling recapitulate embryonic dorsal-ventral pattern, as measured by the dosage-dependent production of dorsal epidermal and amnioserosal cell fates. In contrast, activation of the Sax signaling pathway alone does not promote formation of any dorsal structures. However, the activated Sax receptor synergizes with the activated Tkv receptor in production of both dorsal epidermis and amnioserosal cell fates. From these data it is concluded that, while the functions of both receptors are necessary for in vivo patterning, elevation of Tkv signaling can bypass the requirement for Sax signaling. Furthermore, the data indicate that Sax signaling is dependent on Tkv signaling for phenotypic consequences and that Sax signaling elevates the biological response to a given level of Tkv signaling (Neul, 1998).

Functional experiments suggest the two receptors have different ligands: Dpp acts through Tkv, and Scw acts through Sax. Furthermore, Sog, a negative regulator of this patterning process, preferentially blocks Scw activity. To establish functional interactions between the Scw ligand and the Sax receptor, use was made of the ability of scw mutant embryos to produce amnioserosa in response to injection of either DPP or SCW mRNAs. Injection of mRNA encoding a dominant-negative Sax receptor is able to block the biological activity of injected SCW mRNA but is unable to block the activity of injected DPP mRNA. These findings were extended by showing that scw function is required for the ability of a chimeric receptor containing the extracellular domain of Sax fused to the intracellular domain of Tkv to rescue a tkv mutant. Taken together, these results strongly suggest that Scw is an obligate component of the Sax ligand. Furthermore, because ventral expression of scw in cells that do not express dpp is sufficient to rescue a scw mutant, Scw-DPP heterodimers appear not to be essential for the generation of wild-type pattern, raising the possibility that Scw homodimers are the in vivo ligand for the Sax receptor (Neul, 1998).

Injection of SOG mRNA blocks the biological response of scw mutants to injection of SCW mRNA, but not to injection of DPP mRNA. These results strongly suggest that Sog, which has been genetically characterized as a negative regulator of Dpp activity, functions primarily to modulate Scw activity over the dorsal-ventral axis. These data thus suggest that an activity gradient of dpp results from the differential spatial modulation of Scw activity by Sog. This could happen by either of two mechanisms. One possibility is that the existence of a local ventral source for Sog and the presence of a 'sink' for Sog in the dorsal regions of the embryo (the cleavage of Sog by Tld) could result in a ventral-to-dorsal gradient of Sog. The binding of Sog to Scw could thereby result in the formation of a reciprocal dorsal-to-ventral gradient of scw activity. A second model for the action of Sog posits that Sog facilitates the directional diffusion of the Scw ligand from the lateral to the dorsal regions of the embryo. Specifically, Sog binding to Scw shields the ligand from binding to its ubiquitously localized receptors and thereby allows the Scw-Sog complex to diffuse in the perivitelline space. Dorsally located Tolloid then cleaves Sog, releasing the Scw ligand from the inhibitor. The action of Sog would thus lead to increased dorsal localization of Scw and increased activity of the Sax pathway, ultimately resulting in formation of amnioserosa. This facilitated diffusion model implies that one function of Sog is to elevate Dpp/Scw signaling dorsally. This model would directly explain the reduction in amnioserosa observed in sog mutants and would account for the cell nonautonomous function of Scw, revealed by ventral injections of SCW mRNA. Moreover, this model could also provide an explanation for a puzzling aspect of the phenotype of embryos that lack the nuclear gradient of dorsal gene product. Such dorsalized embryos have a pattern of zygotic gene expression around the embryonic circumference that is similar to that of the most dorsal cells in the wild-type embryo. However, only a small number of cells in dorsalized embryos differentiate as amnioserosa; the great majority of cells in these embryos differentiate as dorsal ectoderm. An increase in dpp gene dosage in dorsalized embryos is sufficient to increase the number of amnioserosal cells. Thus, it appears that despite the pattern of gene expression in dorsalized embryos, the level of dpp/scw signaling is not sufficient to fate amnioserosa. Dorsalized embryos do not express sog; thus, the lack of 'facilitated diffusion' of the Scw ligand mediated by Sog could be the cause of this phenotype (Neul, 1998 and references).

It is proposed that the original function of Dpp might have been to mediate dose-independent cell fate decisions. The ability of Dpp to function in a dose-dependent manner was acquired evolutionarily by the recruitment of a second signaling system whose output could modulate Tkv activity, but whose biological function was dependent on Dpp. The genetic compartmentalization inherent within this circuitry would have ensured the increased evolutionary capacity of such a patterning system. Specifically, genetic alterations in components of the modulatory signaling pathway could lead to significant phenotypic variability without disruption of the original cell fate choice mediated by Dpp. Thus, this genetic circuitry could have been a component in the generation of diverse body plans (Neul, 1998). Drosophila punt gene encodes a type II TGF-beta receptor able to bind activin on its own, but not BMP2, a vertebrate ortholog of DPP. Mutations in punt produce phenotypes similar to those exhibited by thick veins, sax, and dpp mutants. Furthermore, Punt will bind BMP2 in concert with TKV or SAX, forming complexes with these receptors. Punt functions as a type II TGF-beta receptor for DPP. It has been proposed that BMP signaling in vertebrates may also involve the sharing of such type II receptors by diverse ligands (Letsou, 1995). BMP-2, a human homolog of DPP, binds to SAX when tested in human cells transduced with a sax cDNA expression vector (Brummel, 1994).

The immunophilin FKBP12 binds to the cytoplasmic domain of TGFß type I receptors and is released upon a ligand-induced, type II receptor mediated phosphorylation of the type I receptor. Blocking FKBP12/type I receptor interaction with immunophilin FK506 nonfunctional derivatives enhances the ligand activity, indicating that FKBP12 binding is inhibitory to the signaling pathways of the TGFß family ligands. Overexpression of FKBP12 specifically inhibits pathways activated by TGFß, and point mutations in FKBP12 abolish its inhibitory activity. FKBP12 functions as an immunosuppressive in vertebrates by binding macrolides FK506 and rapamycin and recruiting and thereby inactivating calcineurin and the serine kinase FRAP, respectively, resulting in the blockage of the signaling pathways mediated by calcineurin or FRAP. Since calcineurin is a serine/threonine phosphatase while type I receptors are serine/threonine kinases, and phosphorylation of the type I receptor as well as its downstream substrates is essential for signaling via the type I receptor, one plausible mechanism is proposed for FKBP12 action whereby calcineurin could inhibit type I signaling activity by dephosphorylating type I receptor or its bound substrates. A novel cDNA that is 66% identical to mouse FKBP12 was isolated as the predominant interactor for Drosophila type I receptor Thick veins. FKBP12 interacts with Saxophone as well (Wang, 1996).

Medea is involved in transmitting signals from Saxophone into the nucleus. It is proposed that Medea 17 and especially Medea 15 are compromised in the dosage-sensitive specification of amnioserosa, but that both mutant proteins retain a separable function required for the specification of dorsolateral cell fates in the embryo. What could the two separately mutable activities of Medea represent? One possibility is that each activity represents a differential capacity to transduce a signal downstream of each of the two type I Dpp receptors, Tkv and Sax. Embryos that lack both maternal and zygotic tkv activity differentiate no dorsal structures, similar to the complete loss of Medea. In contrast, although the phenotypes of embryos completely lacking sax activity have not been reported because of a requirement for sax during oogenesis, existing mutations in sax result only in the loss of amnioserosa, similar to the phenotype caused by the Med 15 mutation. These parallels suggest that Med 15 and Med 17 mutants may be defective in the response to signals downstream of the Sax receptor, while still transducing signals from the Tkv receptor. In light of this proposal, it is noted that both Med 15 and Med 17 have amino acid substitutions in loop 3, an element of the Smad4 crystal structure that is implicated in productive heteromeric interactions with activated receptor-specific Smad proteins. The mutant Medea proteins might therefore have a diminished capacity to form particular heteromeric complexes with Mad in response to signaling by one receptor but not another. Alternatively, the mutant Medea proteins could have selective disruptions in interactions with other components of the signaling system, such as factors that may collaborate with Mad and Medea to regulate expression of specific target genes. Full evaluation of this proposal awaits biochemical characterization of signaling downstream of the Tkv and Sax receptors in vivo (Hudson, 1998).

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, 2006).

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, 2006)?

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, 2006).

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, 2006).

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, 2006).

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, 2006).

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, 2006). 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, 2006).

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, 2006).

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, 2006).

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, 2006).

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, 2006).

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, 2006).

saxophone: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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