miR-184 has multiple roles in Drosophila female germline and early zygotic development

The regulation of gene expression during early development is very complex. In nonplacental organisms, the mother initiates and controls much of this process by placing mRNA transcripts in well-defined concentrations and locations within the developing egg. In many instances, these maternal 'determinants' serve as morphogens -- their absolute and relative concentrations are therefore crucial and under elaborate regulation, which includes mechanisms for transporting and localizing transcripts and tight control of their translation. During the midblastula transition, many of the maternal messages are destroyed, and zygotic expression takes over to mediate embryonic pattern formation and subsequent development. Mechanistic understanding of this early posttranscriptional regulation of maternally provided transcripts is still fragmentary, partly due to the difficulties in studying RNA-protein interactions and their lack of sequence specificity (Iovino, 2009).

Genomically encoded microRNAs (miRNAs) represent a new layer of posttranscriptional gene regulation that might play an important role in this context. miRNAs bind to specific sequences within the 3'UTRs of mRNAs, leading to degradation of the targeted mRNA or inhibition of protein synthesis. The nature and extent of their role in biological processes are still being debated, but both studies in which miRNA function is abolished wholesale by disrupting their biogenesis and analyses of individual miRNA genes reveal a strong requirement in the control of stem cell fate and in early embryonic development, with higher fishes providing an apparent exception (Iovino, 2009).

In Drosophila, the role of miRNAs in regulating stem cell behavior in the ovaries has been investigated by mosaic analysis of mutants that abrogate miRNA biogenesis. Presumably due to the perdurance of mature miRNAs, mutant clones show age-dependent phenotypes: after 12 days, the number of developing egg chambers is significantly depleted due to reduced division of germline stem cells; longer-term studies show a gradual loss of both germline and somatic stem cells; in both cases, the underlying causes are unclear. Forty-three miRNAs are expressed in the Drosophila germline, but none of their functions have been described (Iovino, 2009).

This study reports the genomic knockout of the highly conserved miRNA mir-184, which is expressed in the female germline and has assumed control over multiple steps in oogenesis and early embryogenesis in Drosophila. A range of phenotypes of varying penetrance was observed, several of the responsible targets were identified, and their protein levels are were shown to be tuned by miR-184 in vivo. These results support the notion that an individual miRNA can exert phenotypically relevant control over multiple biological processes, and provide insight into the molecular mechanisms of miRNA-mediated regulation in female germline development (Iovino, 2009).

miR-184 was originally identified by expression cloning from the small RNA fraction of Drosophila embryos, but is conserved from insects to humans. Northern analysis shows expression of miR-184 throughout the life cycle, with a relatively weak maternal contribution but strong subsequent zygotic expression; notably, strong expression was found in ovaries. RNA in situ hybridization using the primary transcript as probe shows strong expression in a highly dynamic pattern throughout embryogenesis. miR-184 is also one of the few miRNAs that are expressed in Schneider (S2) cells in significant copy number (Iovino, 2009).

mir-184 is a single copy gene and lies isolated within a 50 kb region on the right arm of the second chromosome. The genomic region is rich in extant P element insertions, including several FRT site-containing elements (PBac{WH}, P-element{XP}; Exelixis Collection), which were used to generate an FLP-induced deletion of 22 kb between the elements PBac{WH}f05119 and P{XP}d08710. To be able to carry out rescue and misexpression experiments, a UAS-mir-184 strain was generated, that contains 1.5 kb of genomic sequence surrounding the mir-184 gene (1 kb upstream, 0.5 kb downstream) (Iovino, 2009).

Δmir-184 zygotic mutant flies eclose at a normal Mendelian ratio and appear morphologically normal, indicating that loss of zygotic expression has no detectable effect on adult viability and no obvious effect on development and overall morphology, which is surprising given the strong and complex expression of the mir-184 transcript throughout embryogenesis. Among adults homozygous for Δmir-184, male fertility is normal; however, females lay far fewer eggs than in wild-type, and the eggs and embryos that are produced show severe abnormalities. Strikingly, the defects become progressively worse over time: young (2- to 3-day-old) Δmir-184 females lay 5-10 eggs per day, which represents <10% of wild-type production. Approximately 70% of the eggs have normal (external) morphology and are fertilized; however, most of these embryos (85%) show severe defects in anteroposterior patterning, and many also show severe defects during cellularization; only about 1% of all progeny develop to adulthood. As the females age, egg production declines further and the number of eggs with an abnormal external morphology increases. Eggs from 3- to 4-day-old females are typically smaller than wild-type, and many show defects in dorsoventral patterning of the egg shell, as judged by the position and length of the dorsal appendages. Δmir-184 females that are 5 days or older lay almost no eggs. Thus, progressive failure of egg production is the prevalent phenotype in the Δmir-184 mutant and supersedes all others within a week. However, its incomplete or delayed penetrance makes it possible to observe a range of distinct other defects as well, indicating that miR-184 function is required for multiple successive steps of oogenesis and early embryogenesis (Iovino, 2009).

The observed phenotypes point to a requirement for miR-184 in either the female germline itself or in the somatic cells of the ovary. RNA in situ hybridization in ovaries is often difficult, and it was not possible to obtain consistent interpretable results when attempting to detect the miR-184 primary transcript. To determine where the requirement lies, UAS-mir-184 transgene was expressed in different cell populations of the ovary using established Gal4 drivers, and under which conditions the sterility phenotype can be rescued was examined, mindful of the possibility that ectopic or even overexpression might lead to phenotypic defects by itself. nos-Gal4VP16 drives expression in the germline cells, C587-Gal4 in most somatic cells of the ovary excluding the cap cells, and GR1-Gal4 drives expression in the follicle cells that envelope the oocyte and produce the egg shell. Expression of mir-184 in the germline (nos-Gal4VP16) strongly rescues the sterility of Δmir-184 females: egg production approaches wild-type levels, and almost all eggs and embryos appear morphologically normal. This indicates that miR-184 is required in the germline, which is consistent with the fact that expression of mature miR-184 is detected in northerns of freshly laid eggs/embryos, that is, prior to the onset of zygotic transcription. Notably, substantial rescue of egg production, although not egg morphology, was observed by simply introducing UAS-mir-184 into the Δmir-184 background. This suggests that, due to the inclusion of 1 kb upstream sequence, the UAS-mir-184 transgene on its own drives moderate expression in the germline. Northern analysis of ovaries from Δmir-184 females that carry the UAS-mir-184 transgene indeed reveals weak expression of mature miR-184, at about 10% of the level observed in wild-type, indicating that the 1 kb upstream sequence included in the UAS construct contains at least part of a germline promoter. Expression of mir-184 in the somatic cell populations of the ovary leads to different results: driving expression using C587-Gal4 has no effect beyond that of UAS-mir-184 alone, whereas driving expression in the follicle cells (GR1-Gal4) leads to severe sterility, suggesting that ectopic or overexpression of mir-184 in follicle cells is in itself detrimental to oogenesis (Iovino, 2009).

miR-184, strongly expressed in the germline and deposited in the egg, regulates several distinct steps during oogenesis and early embryogenesis, including stem cell differentiation and axis formation of both egg chamber and embryo. The underlying molecular mechanism were characterized by identifying three relevant miR-184 targets. Female germline development has long been known to be a carefully regulated process in which the spatiotemporal pattern and activity level of key factors is kept in check by multiple levels of control. The current results show that miR-184 provides a crucial additional layer of regulation. Interestingly, miR-184 does not target the key developmental regulators and morphogens themselves but components involved in their regulation, namely a signal transduction receptor, a transport factor, and a general transcription factor (Iovino, 2009).

Developmentally, the first process miR-184 regulates is the interaction between somatic niche and germline stem cells. Previous genetic analysis of this process has focused on the role of TKV in mediating the DPP signal in stem cell maintenance and cystoblast differentiation. It has now been demonstrated that miR-184-mediated translational repression of SAX protein levels, potentially combined with indirect effects on TKV protein distribution, are a crucial mechanism in dampening DPP signal reception and thus promoting cystoblast differentiation. The substantial rescue of egg production that was observed when halving the gene dose of sax suggests that the lack of cystoblast differentiation (and the subsequent loss of germline stem cells) is responsible for the reduction and ultimate loss of fertility in Δmir-184 mutants (Iovino, 2009).

miR-184's role in establishing egg chamber polarity is more complex. miRNAs have frequently been viewed as performing a clean-up task - suppressing translation of residual transcript after developmental decisions have been made. The misregulation of K10 in Δmir-184 mutants argues that precocious translation, even within the proper cell (oocyte), may also be deleterious. However, the mechanistic connection between the early overproduction and the later depletion of K10 protein is currently not understood. Because actively translated transcripts are generally considered to be more protected against degradation, a partial loss of K10 transcript seems unlikely. Given that K10 mRNA is bound by translational regulators (Bicaudal D and Egalitarian) and K10 protein interacts with other proteins (Squid), it is possible that these factors themselves are limiting and titrated away by the precocious translation and strong accumulation of K10 protein, but the possibility that other miR-184 targets not yet implicated in dorsoventral patterning of the egg are also involved cannot be excluded (Iovino, 2009).

Finally, in early embryonic development, miR-184 tunes the potent transcriptional repressor TTK69, thereby ensuring the proper timing of pair rule gene expression and anterior-posterior patterning. Several additional phenotypes are readily visible in the mutant that indicate miR-184's involvement in processes known to be tightly regulated, such as the transition into the vitellogenic state, which is stringently controlled by several hormone systems, but also in processes where this is unexpected, such as cortical nuclear migration in the syncytial blastoderm. Detection of the entire range of distinct phenotypes in the Δmir-184 mutant was only possible due to their partial penetrance; however, eventually the requirement for GSC differentiation becomes absolute and, thus, within a week, the loss of egg production supersedes all other phenotypes (Iovino, 2009).

The phenotypes observed in the Δmir-184 mutant partially overlap with those seen in mutants in which miRNA biogenesis is disrupted. However, these experiments are difficult to compare: biogenesis mutants presumably affect all 43 miRNAs normally expressed in the germline, causing additional phenotypes that are likely to epistatically mask effects visible in Δmir-184; in addition, these studies have to be conducted under mosaic conditions, where perdurance of mature miRNAs may add another layer of complication. The polarity and vitellogenesis defects but not the germarium overgrowth that were found in the Δmir-184 mutant have been reported for dcr-1 germline clones. Conversely, dcr-1 germline clones show cell-autonomous cell-cycle defects that were not observed in this study, and GSC maintenance defects cannot be observed in the Δmir-184 mutant, due to its rapid tumorous growth and subsequent regression phenotype (Iovino, 2009).

This study also sheds light on important mechanistic aspects of miRNA function. Most of the defects in the Δmir-184 mutant can be rescued by germline-specific expression of mir-184, indicating that the miRNA is coexpressed with its targets in the same cell and tunes their expression. Loss of mir-184 function leads to increases in protein level in the 2- to 5-fold range, with the mutant showing increased variability in protein level compared to wild-type, concordant with the observed incomplete penetrance and variability in phenotype. The findings support the idea that miRNAs regulate a large number of different targets in vivo. Depending on the stoichiometry and affinity between miRNA and mRNA as well as the critical level of the cognate protein, some of this regulation, although quantifiable at the expression level, may be phenotypically silent. However, the fact that several distinct and molecularly attributable defects are observed in the Δmir-184 mutant clearly indicates that the loss of proper tuning of protein levels frequently becomes phenotypically visible. This is consistent with the longstanding knowledge that many biological processes are sensitive to changes in the activity level of their key components (Iovino, 2009).

Both genetic and molecular analyses demonstrate the key role of the maternal component of miR-184. miR-184 is strongly expressed in the ovaries and later in a highly dynamic pattern throughout embryogenesis, but a pronounced difference was observed in phenotypic impact: loss of the zygotic component has no discernable effect on adult morphology and viability, yet loss from the female germline results in severe morphologic defects in oogenesis and embryonic development. Notably, much of this germline requirement can be rescued by much lower levels of miR-184 than are expressed in wild-type. Moreover, the maternal contribution of miR-184 persists stably through the first 3 hr of development and is then slowly degraded with a half-life of ~ 3 hr. This long perdurance is common to many maternally provided transcripts and typically results in rescue into larval stages and beyond. Thus, it is quite possible that also in the case of miR-184, the persisting maternal contribution rescues whatever zygotic function the miRNA may have, implying that the high level and complex pattern of its embryonic expression might be (partially) redundant (Iovino, 2009).

The remarkable functionality carried by low concentrations of the miRNA highlights the need for complete removal of the maternal contributions of miRNAs when undertaking functional studies. Surprisingly, this consideration has frequently been neglected in current genetic analyses of Drosophila miRNAs, despite the fact that many of those under investigation have weak (similar to miR-184) or even strong maternal contributions (e.g., miR-6 and miR-286). This disregard of maternal contribution and of functional redundancy between family members may be partially responsible for the unusual situation that for Drosophila miRNAs, primarily postembryonic and more subtle phenotypes have been reported, whereas for most vertebrate miRNAs, severe, even embryonic, phenotypes are observed (Iovino, 2009).

Another intriguing finding of this study is that while miR-184 itself is highly conserved, two of the three miR-184 target sites identified are only partially conserved across the Drosophilids, suggesting that the acquisition of molecular targets and thus of regulatory function is in evolutionary flux. The fact that poorly conserved sites and even sites with mismatch in the 5' seed region can confer significant and phenotypically relevant repression, draws into question, from a developmental biologist's perspective, the rationale for filtering computational target site predictions based on evolutionary conservation and of applying overly stringent seed matching rules. The results suggest that considering other features of target candidates, such as site accessibility, can provide an important complement to purely sequence-based approaches (Iovino, 2009).

Protein Interactions

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).

The Drosophila type II receptor, Wishful thinking, binds BMP and myoglianin to activate multiple TGFβfamily signaling pathways

Wishful thinking (Wit) is a Drosophila transforming growth factor-β (TGFβ) superfamily type II receptor most related to the mammalian bone morphogenetic protein (BMP) type II receptor, BMPRII. To better understand its function, a biochemical approach was undertaken to establish the ligand binding repertoire and downstream signaling pathway. It was observed that BMP4 and BMP7, bound to receptor complexes comprised of Wit and the type I receptor Thickveins and Saxophone to activate a BMP-like signaling pathway. Further it was demonstrated that both Myoglianin and its most closely related mammalian ligand, Myostatin, interacted with a Wit and Baboon (Babo) type II-type I receptor complex to activate TGFβ/activin-like signaling pathways. These results thereby demonstrate that Wit binds multiple ligands to activate both BMP and TGFβ-like signaling pathways. Given that Myoglianin is expressed in muscle and glial-derived cells, these results also suggest that Wit may mediate Myoglianin-dependent signals in the nervous system (Lee-Hoeflich, 2005).

To provide insight into the molecular mechanisms of Wit that contribute to the biological functions of Wit, this study has characterized the Wit interacting ligands, their compatible type I receptor partners, and their downstream signaling pathways. The binding of BMP7, the mammalian ligand most related to Gbb, to Wit is in agreement with and gives biochemical evidence for results obtained from genetic analysis indicating that Wit mediates Gbb-activated BMP signaling in collaboration with the type I receptors, Tkv and Sax. The demonstration of the binding of BMP4, a functional ortholog of Dpp, to the receptor complex also suggests the possibility of Wit mediating Dpp signals. Dpp is not expressed in muscle or motoneurons but Wit is widely expressed in the central nervous system from embryonic stages suggesting that this putative Dpp signaling might regulate early developmental processes other than NMJ formation. The data showing that a receptor complex comprised of Wit and Tkv can activate MAD phosphorylation is also in agreement with the observation of impaired phosphorylation of MAD in Wit deficient flies and provides further support for a role of Wit in mediating BMP signaling (Lee-Hoeflich, 2005).

While the expression of dActivin in the developing nervous system and its proposed function in neuronal remodeling downstream of Wit or Punt have led to a suggestion that dActivin might induce Wit-mediated activin signaling, this study observed that mammalian activin, which is most closely related to dActivin, does not bind to Wit. One possible explanation for this discrepancy is that Wit might bind ligands other than dActivin and that this indirectly compensates for the lack of Punt-dActivin interaction. An attempt to produce dActivin in mammalian cells using a heterologous system was unsuccessful, thus the possibility cannot be eliminated that mammalian and Drosophila activins have different binding specificities. Generation of dActivin null flies or cell clones and testing for functional equivalence in rescue experiments should help resolve this issue (Lee-Hoeflich, 2005).

Alternatively, it is speculated that Wit might mediate activin signaling via Myoglianin since myostatin, the mammalian ligand most closely related to Myoglianin, activates a TGFβ/activin-like pathway. Accordingly, it was found that both myoglianin and myostatin bind to the Wit and Babo receptor complex. Furthermore, it was observed that coexpression of Wit and Babo induces dSmad2 phosphorylation and mediates myostatin-induced transcriptional activation of a TGFβ/activin-responsive reporter. In agreement to these observations, ectopic expression of Wit induces dSmad2 phosphorylation in insect S2 cells. Retrograde signaling between target-derived factors and the presynaptic terminal is crucial for NMJ development. Since Drosophila Myoglianin is abundantly expressed in muscle at late developmental stages and since Wit-mediated retrograde signaling had been identified previously, it is postulated that Myoglianin might activate a novel retrograde Wit signaling pathway. Interestingly, myostatin inhibits the BMP7 signaling response by competitive binding to type II receptor, ActRIIB, thus the binding of Myoglianin to Wit might also affect Gbb-mediated signaling and thus contribute to NMJ formation. Generation of flies harboring myoglianin loss-of-function mutations will shed light on these issues. These observations underscore the diverse mechanisms controlling Wit signaling and add impetus to further experiments in the context of the Wit receptor (Lee-Hoeflich, 2005).

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

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.