saxophone is expressed uniformly in early development but soon disappears. Expression reappears with time, and by stage 9 it is found uniformly. Expression disappears again by stage 16 (Brummel, 1994).

The BMP pathway patterns the dorsal region of the Drosophila embryo. Using an antibody recognizing phosphorylated Mad (pMad), signaling was followed directly. In wild-type embryos, a biphasic activation pattern is observed. At the cellular blastoderm stage, high pMad levels are detected only in the dorsal-most cell rows that give rise to amnioserosa. This accumulation of pMad requires the ligand Screw (Scw), the Short gastrulation (Sog) protein, and cleavage of their complex by Tolloid (Tld). When the inhibitory activity of Sog is removed, Mad phosphorylation is expanded. In spite of the uniform expression of Scw, pMad expansion is restricted to the dorsal domain of the embryo where Dpp is expressed. This demonstrates that Mad phosphorylation requires simultaneous activation by Scw and Dpp. Indeed, the early pMad pattern is abolished when either the Scw receptor Saxophone (Sax), the Dpp receptor Thickveins (Tkv), or Dpp are removed. After germ band extension, a uniform accumulation of pMad is observed in the entire dorsal domain of the embryo, with a sharp border at the junction with the neuroectoderm. From this stage onward, activation by Scw is no longer required, and Dpp suffices to induce high levels of pMad. In these subsequent phases pMad accumulates normally in the presence of ectopic Sog, in contrast to the early phase, indicating that Sog is only capable of blocking activation by Scw and not by Dpp (Dorfman, 2001).

Normally Sog may form a graded distribution in the dorsal region, which is essential for patterning. When the Sog/Scw complex is cleaved by Tld, Scw is released and can bind either Sog or Sax. The data suggest that in regions closer to the neuroectoderm, the levels of Sog are high and titrate the free ligand. In the dorsal-most region however, where Sog levels are low, the released Scw has a greater probability of binding and activating the Sax receptor, rather than being trapped again by Sog. Thus, the graded distribution of Sog is critical for generating the reciprocal distribution of Scw, and the ensuing activation profile (Dorfman, 2001).

Activation of Tkv by Dpp is essential for the appearance of the early pMad pattern, corresponding to the future amnioserosa cells. At this stage, distinct cell fates are also induced in the dorsolateral cells, as reflected by expression of pnr and repression of msh expression. It is assumed that low levels of activation that may be induced by Dpp alone, but not detected by pMad antibodies, are responsible for these fates. Elimination of Dpp or Tkv leads to complete absence of early, as well as late, pMad patterns. Thus, Scw is not sufficient for the early activation phase, and the presence of Dpp is crucial. Cooperativity between Scw and Dpp occurs at the level of receptor activation. One possibility is that the observed pMad levels reflect only an additive effect of Scw and Dpp signaling. Indeed, the number of dpp copies has a profound effect on signaling levels and the shape of the early pMad distribution. Alternatively, it is possible that there is a synergistic interaction between Scw and Dpp signaling. In this case, the requirement of both ligands for the production of the early pMad pattern may indicate that synergy occurs at the level of receptor activation. Phosphorylation of Mad may require the formation of heterotetrameric receptors, containing both Sax/Put and Tkv/Put pairs. Cross linking experiments of the vertebrate receptors support this model (Dorfman, 2001).

Scw is required for generating the pMad pattern only in the early phase. All subsequent patterns rely only on Dpp. This feature may be explained differently by each of the above two models. If Scw and Dpp are required additively in the early phase, higher levels of Dpp may suffice to induce the pMad pattern at later stages. The autoregulatory effects of Dpp on its transcription may account for the elevation in Dpp levels. Alternatively, if Scw and Dpp signaling is synergistic, why is such a synergism necessary only in the early phase? In the early embryo, a maternal transcript encoding an inhibitor of BMP signaling may be translated, to block signaling by Sax/Put or Tkv/Put dimers. Such inhibitor(s) may be displaced only in ligand-bound heterotetrameric receptor complexes. The maternal transcripts of the inhibitor(s) may diminish by stage 9, to allow pMad production by activation of Tkv/Put alone (Dorfman, 2001).

TGF-beta comprise a superfamily of secreted proteins with diverse functions in patterning and cell division control. TGF-beta signaling has been implicated in synapse assembly and plasticity in both vertebrate and invertebrate systems. wishful thinking, a Drosophila gene that encodes a protein related to BMP type II receptors, has been shown to be required for the normal function and development of the neuromuscular junction (NMJ). These findings suggest that a TGF-beta-related ligand activates a signaling cascade involving type I and II receptors and the Smad family of transcription factors to orchestrate the assembly of the NMJ. This study demonstrates that the TGF-beta type I receptor Saxophone and the downstream transcription factor Mothers against dpp (Mad) are essential for the normal structural and functional development of the Drosophila NMJ, a synapse that displays activity-dependent plasticity (Rawson, 2003).

Neurophysiological defects and neuronal gene deregulation in Drosophila mir-124 mutants

miR-124 is conserved in sequence and neuronal expression across the animal kingdom and is predicted to have hundreds of mRNA targets. Diverse defects in neural development and function were reported from miR-124 antisense studies in vertebrates, but a nematode knockout of mir-124 surprisingly lacked detectable phenotypes. To provide genetic insight from Drosophila, its single mir-124 locus was deleted, and it was found to be dispensable for gross aspects of neural specification and differentiation. In contrast, a variety of mutant phenotypes were detected that were rescuable by a mir-124 genomic transgene, including short lifespan, increased dendrite variation, impaired larval locomotion, and aberrant synaptic release at the NMJ. These phenotypes reflect extensive requirements of miR-124 even under optimal culture conditions. Comparison of the transcriptomes of cells from wild-type and mir-124 mutant animals, purified on the basis of mir-124 promoter activity, revealed broad upregulation of direct miR-124 targets. However, in contrast to the proposed mutual exclusion model for miR-124 function, its functional targets were relatively highly expressed in miR-124-expressing cells and were not enriched in genes annotated with epidermal expression. A notable aspect of the direct miR-124 network was coordinate targeting of five positive components in the retrograde BMP signaling pathway, whose activation in neurons increases synaptic release at the NMJ, similar to mir-124 mutants. Derepression of the direct miR-124 target network also had many secondary effects, including over-activity of other post-transcriptional repressors and a net incomplete transition from a neuroblast to a neuronal gene expression signature. Altogether, these studies demonstrate complex consequences of miR-124 loss on neural gene expression and neurophysiology (Sun, 2012).

microRNAs (miRNAs) are ~22 nucleotide (nt) regulatory RNAs that function primarily as post-transcriptional repressors. In animals, miRNAs have propensity to target mRNAs via 6-7 nt motifs complementary to their 5' ends, termed 'seed' regions. This limited pairing requirement has allowed most miRNAs to capture large target networks. Analysis of multigenome alignments indicates that typical human miRNAs have hundreds of conserved targets, and that a majority of protein-coding genes are under miRNA control. The extraordinary breadth of animal miRNA:target networks has been extensively validated by transcriptome and proteome studies (Sun, 2012).

miR-124 is strictly conserved in both primary sequence and spatial expression pattern, being restricted to the nervous system of diverse metazoans, including flies, nematodes Aplysia, and all vertebrates studied. Such conservation implies substantial functions of miR-124 in controlling neural gene expression. miR-124 has been a popular model for genomewide investigations of miRNA targeting principles. For example, studies of miR-124 yielded the first demonstration of the downregulation of hundreds of direct targets detected by transcriptome analysis, and that this activity was driven by the miRNA seed region. In addition, miR-124 provided one of the first illustrations of spatially anticorrelated expression of a miRNA and its targets and direct identification of Ago-bound target sites (Sun, 2012).

Functional studies have connected vertebrate miR-124 to various aspects of neural specification or differentiation. Studies in chick ascribed miR-124 as a proneural factor that inhibits the anti-neural phosphatase SCP1. However, no substantial effect of miR-124 on chick neurogenesis was found in a parallel study, although miR-124 was observed to repress neural progenitor genes such as laminin gamma1 and integrin beta1. In the embryonic mammalian brain, miR-124 was reported to direct neural differentiation by targeting polypyrimidine tract binding protein 1 (PTBP1), a global repressor of alternative splicing in non-neural cells. In the adult mammalian brain, miR-124 promoted neural differentiation of the immediate progenitors, the transit-amplifying cells (TAs). Here, miR-124 was shown to directly target the transcription factor Sox9, which maintains TAs and is downregulated during neural differentiation. Other mammalian studies bolster the concept that miR-124 promotes neurogenesis or neural differentiation. One mechanism involves direct repression by miR-124 of Baf53a, a neural progenitor-specific chromatin regulator that must be exchanged for a neural-specific homolog to consolidate neural fate. However, complicating the picture is the recent report that Xenopus miR-124 represses neurogenesis by directly targeting the proneural bHLH factor NeuroD1 (Sun, 2012 and references therein).

All vertebrate miR-124 loss-of-function studies have relied on antisense strategies and have yet to be validated by bona fide mutant alleles. However, as the three vertebrate mir-124 loci are co-expressed in the nervous system, analysis of the null situation will require a triple knockout. So far, a mir-124 knockout has only been described in C. elegans, which harbors a single copy of this gene. Like most other miRNA mutants in this species, the loss of miR-124 did not cause obvious developmental, physiological or behavioral phenotypes. Nevertheless, comparison of gene expression in mir-124-expressing cells from wildtype and mir-124 mutant animals revealed strong enrichment in miR-124 target sites amongst upregulated transcripts, revealing the impact of miR-124 on neuronal gene expression (Clark, 2010). The broad, but phenotypically-tolerated, misregulation of miR-124 targets in this species is potentially consistent with the 'fine-tuning' model for miRNA regulation (Sun, 2012).

This study analyzed a knockout of the sole mir-124 gene in Drosophila. Although this mutant is viable and exhibits grossly normal patterning, numerous phenotypes were documented, including short lifespan, increased variation in the number of dendritic branches of sensory neurons, decreased locomotion and aberrant synaptic release at CNS motoneuron synapses. All of these phenotypes were rescued by a single copy of a 19 kilobase (kb) genomic transgene encompassing the mir-124 locus. A transcriptional reporter of mir-124 was generated that recapitulated the CNS expression of endogenous pri-mir-124, and this was used to purify mir-124-expressing cells from stage-matched wild-type and mir-124-mutant embryos. Transcriptome analysis revealed strong enrichment of direct miR-124 targets amongst genes upregulated in mir-124-mutant cells. The miR-124 target network included coordinate repression of multiple components in the retrograde BMP signaling pathway, whose activity controls synaptic release. Loss of miR-124 further correlated with increased activity of other neural miRNAs and the neural translational regulator Pumilio, and had the net effect of impairing transition from the neuroblast to neuronal gene expression signature. Altogether, it was demonstrated that endogenous miR-124 has substantial impact on CNS gene expression, which underlie its requirement for organismal behavior and physiology (Sun, 2012).

These studies of Drosophila mir-124 demonstrate that its loss is compatible with grossly normal neural development and differentiation, despite broad changes in gene expression and global upregulation of direct miR-124 targets. Nevertheless, many clear defects are detected in these mutants, including short lifespan of adult males, defective larval locomotion, and aberrant synaptic transmission. The latter phenotype is perhaps reminiscent of reports that inhibition of Aplysia miR-124 similarly results in an increase in evoked EPSP amplitude. These phenotypes were confirmed phenotypes to be due to miR-124 loss, as shown by their rescue by a mir-124 genomic transgene. Importantly, these phenotypes were obvious even under optimal culture conditions, demonstrating palpable requirements for this miRNA in the intact animal. It remains to be seen if synaptic overactivity in the mir-124 mutant can be directly linked to the behavioral defects observed at the organismal level. The electrophysiological defects in mir-124 mutants phenocopy activation of BMP signaling at the synapse, and miR-124 directly targets multiple components of this pathway. Still, it remains possible that the many other gene expression changes in mir-124 mutant neurons contribute to its loss of function phenotype. The detailed in vivo transcriptome-wide analysis of endogenous miR-124 targets sets the stage for future studies of how individual targets might affect different settings of miR-124 function (Sun, 2012).

Only a handful of other miRNA mutants are lethal or exhibit overt morphological defects, suggesting that many miRNAs serve as robustness factors. For example, a Drosophila mir-7 mutant exhibits minor cell specification defects, but these are enhanced by heat shock. In addition, the introduction of many C. elegans 'benign' miRNA mutants into genetically sensitized backgrounds uncovers a high frequency of phenotypes. Interestingly, miR-124 is not required for normal dendrite formation per se, but its absence caused a broader distribution of dendrite numbers on ddaD and ddaE neurons, i.e. a 'robustness' defect. It is speculated that environmental or genetic stress may reveal additional requirements for miR-124 in development and differentiation of the nervous system (Sun, 2012).

In light of the broad roles ascribed to endogenous miR-124 in neurogenesis, neural differentiation, and neural physiology (Gao, 2010), all from antisense strategies, the extensive negative data from the current Drosophila mir-124 knockout are equally compelling. While the relevant neural subpopulation may not have been examined, these studies indicate that miR-124 is not required for gross aspects of neurogenesis and differentiation in the embryonic and larval nervous system. Similarly, C. elegans deleted for mir-124, which is expressed mostly in ciliated sensory neurons, do not reveal obvious defects in neural development (Clark, 2010). Given that these invertebrate orthologs of miR-124 are identical in sequence to their vertebrate counterparts, and are highly and specifically expressed in their respective nervous systems, there is not strong reason a priori to suspect that miR-124 should not have comparable requirements amongst different animals. The analysis of vertebrate mir-124 knockouts is therefore highly anticipated (Sun, 2012).

The Drosophila system has been critical for elucidating fundamental features of miRNA target recognition in animals, and for studying specific miRNA-target interactions that mediate phenotype. However, it has been little-used to analyze the effects of miRNA-mediated gene regulation in the animal at the transcriptome-wide level. Perhaps the clearest example is the broad upregulation of maternal transcripts in early embryos lacking the mir-309 cluster. However, most miRNAs are tissue or cell-specific, and while it is much simpler to profile transcripts from whole flies, the inclusion of irrelevant cells can mask the action of the miRNA. For example, only 4/200 transcripts upregulated in mir-8 mutant pupae appeared to be direct conserved targets (Sun, 2012).

By purifying cognate miRNA-expressing cells from wild-type and miRNA-mutant backgrounds, this study succeeded in assessing transcriptome-wide effects of genetic removal of miR-124 with precision. The data provide a new perspective on the utilization of 'anti-targeting' in Drosophila. Previously, miR-124 was selected as a particularly compelling case in which its Drosophila targets were depleted for in situ terms related to nervous system development, and enriched for terms related to epidermal development. Since these tissues derive from a common developmental progenitor, the neuroectoderm, this led to a model in which miR-124 may solidify the neural fate by widespread suppression of epidermal genes that should be absent from neurons. This bioinformatic correlation has not been confirmed using an independently-derived set of miRNA targets (Sun, 2012).

Nevertheless, two observations suggest that the feature of mutual exclusion in the Drosophila miR-124 network is of subtle consequence. First, derepressed target genes were not enriched for epidermally-expressed genes. This is consistent with the view that on the transcriptome-wide level, the exclusion of epidermal genes from miR-124-expressing cells is primarily enforced by transcriptional mechanisms. Second, miR-124 targets were preferentially amongst the higher-expressed transcripts in miR-124+ cells, even in wild-type. Moreover, as well-conserved targets were expressed at overall higher absolute levels than poorly-conserved targets in miR-124+ cells, it is concluded that a dominant feature of the miR-124 target network has selected for substantial co-expression of the miRNA and its targets, perhaps to fine-tune their levels. This viewpoint is consistent with analyses of miR-124 targets in human, indicating a unifying theme for this particular miRNA across animals (Sun, 2012).

Early manifestations of the miRNA world emerged from pervasive control of the C. elegans heterochronic pathway and the D. melanogaster Notch pathway by miRNAs, and a few similar situations have been documented, i.e. direct targeting throughout the branched amino acid catabolism pathway by miR-277 or repression of multiple components of fatty acid metabolism by miR-33. Nevertheless, it is rare for such dedicated target networks to be seen amongst the miRNA oeuvre. Amongst the broad network of miR-124 targets, coordinate targeting of multiple components of the retrograde BMP signaling pathway is striking, including all three receptors (Sax/Tkv/Wit), the downstream transcription factor (Mad) and its cofactor (Medea). It was recently shown that misexpression of activated Sax and Tkv receptors in motoneurons increases evoked excitatory junctional potentials without affecting spontaneous activity, very similar to that of mir-124 mutants. This study extends this finding by analysis of activated Tkv alone. Therefore, deregulation of BMP signaling may contribute to the electrophysiological defects observed in mir-124 mutants (Sun, 2012).

Still, a 'one size fits all' description of miR-124 activity is not appropriate, since a number of functional miR-124 targets were observed whose predominant activities are in epidermal or other non-neural derivatives. Thus, the large miR-124 network accommodates a range of target properties. Derepression of a sufficient number of such non-neural transcripts may contribute collectively to the incomplete capacity of mir-124 mutant cells to transition from a neuroblast to neuronal gene expression signature (Sun, 2012).

One may speculate that dysfunction of miRNAs, which have large networks of targets, may trigger global changes in other modes of gene regulation. For example, overexpression of individual miRNAs or siRNAs can de-repress endogenous regulation via non-cognate miRNAs, possibly reflecting a titration mechanism. In addition to a global effect on neuroblast-to-neural transition, it was observed that genes downregulated upon in vivo loss of miR-124 were enriched for seeds of K box miRNAs and miR-10-5p. This is potentially consistent with a model in which absence of this abundant miRNA frees up AGO1 complexes to accept other neural miRNAs, yielding their overactivity. Another plausible mechanism might be that miR-124 represses a transcriptional repressor of these other miRNAs (Sun, 2012).

Pumilio binding sites were strongly associated with downregulated transcripts in mir-124 mutants. Pumilio is well-characterized as a neural RNA binding protein and translational regulator, and affects synaptic function and dendrite morphogenesis, which was also observed to be miR-124-regulated settings. Predictions of conserved miRNA binding sites (e.g., TargetScan or mirSVR) did not identify miR-124 target sites in the annotated pumilio 3' UTR or CDS; however modENCODE data revealed that pumilio transcription extends ~2 kb downstream of its annotated 3' end. The regulatory potential of such long pumilio 3' UTR isoforms remains to be studied. Other possibilities are that miR-124 regulates a transcriptional regulator of pumilio, or that Pumilio activity is altered in mir-124 mutants. Future studies should address the cross-talk of post-transcriptional regulation in neurons mediated by miR-124, neuronal miRNAs and Pumilio (Sun, 2012).

Haemocytes control stem cell activity in the Drosophila intestine

Coordination of stem cell activity with inflammatory responses is critical for regeneration and homeostasis of barrier epithelia. The temporal sequence of cell interactions during injury-induced regeneration is only beginning to be understood. This study shows that intestinal stem cells (ISCs) are regulated by macrophage-like haemocytes during the early phase of regenerative responses of the Drosophila intestinal epithelium. On tissue damage, haemocytes were recruited to the intestine and secreted the BMP homologue DPP, inducing ISC proliferation by activating the type I receptor Saxophone and the Smad homologue SMOX. Activated ISCs then switched their response to DPP by inducing expression of Thickveins, a second type I receptor that had previously been shown to re-establish ISC quiescence by activating MAD. The interaction between haemocytes and ISCs promoted infection resistance, but also contributed to the development of intestinal dysplasia in ageing flies. The study proposes that similar interactions influence pathologies such as inflammatory bowel disease and colorectal cancer in humans (Ayyaz, 2015).

Coordination of stem cell activity with inflammatory responses is critical for regeneration and homeostasis of barrier epithelia. The temporal sequence of cell interactions during injury-induced regeneration is only beginning to be understood. This study shows that intestinal stem cells (ISCs) are regulated by macrophage-like haemocytes during the early phase of regenerative responses of the Drosophila intestinal epithelium. On tissue damage, haemocytes are recruited to the intestine and secrete the BMP homologue DPP, inducing ISC proliferation by activating the type I receptor Saxophone and the Smad homologue SMOX. Activated ISCs then switch their response to DPP by inducing expression of Thickveins, a second type I receptor that has previously been shown to re-establish ISC quiescence by activating MAD. The interaction between haemocytes and ISCs promotes infection resistance, but also contributes to the development of intestinal dysplasia in ageing flies. It is proposed that similar interactions influence pathologies such as inflammatory bowel disease and colorectal cancer in humans (Ayyaz, 2015).

The results extend the current model for the control of epithelial regeneration in the wake of acute infections in the Drosophila intestine. It is proposed that the control of ISC proliferation by haemocyte-derived DPP integrates with the previously described regulation of ISC proliferation by local signals from the epithelium and the visceral muscle, allowing precise temporal control of ISC proliferation in response to tissue damage, inflammation and infection (Ayyaz, 2015).

The association of haemocytes with the intestine is extensive, and can be dynamically increased on infection or damage. In this respect, the current observations parallel the invasion of subepithelial layers of the vertebrate intestine by blood cells that induce proliferative responses of crypt stem cells during infection. A role for macrophages and myeloid cells in promoting tissue repair and regeneration has been described in adult salamanders and in mammals, where TGFβ ligands secreted by these immune cells can inhibit ISC proliferation, but can also contribute to tumour progression. The results provide a conceptual framework for immune cell/stem cell interactions in these contexts (Ayyaz, 2015).

The observation that DPP/SAX/SMOX signalling is required for UPD-induced proliferation of ISCs suggests that SAX/SMOX signalling cooperates with JAK/STAT and EGFR signalling in the induction of ISC proliferation. Accordingly, while constitutive activation of EGFR/RAS or JAK/STAT signalling in ISCs is sufficient to promote ISC proliferation cell autonomously, this study found that this partially depends on Smox. Even in these gain-of-function conditions, ISC proliferation can thus be fully induced only in the presence of basal SMOX activity. As short-term overexpression of DPP in haemocytes does not induce ISC proliferation, it is further proposed that DPP/SAX/SMOX signalling can activate ISCs only when JAK/STAT and/or EGFR signalling are activated in parallel. However, long-term overexpression of DPP in haemocytes results in increased ISC proliferation, suggesting that chronic activation of immune cells disrupts normal signalling mechanisms and results in ISC activation even in the absence of tissue damage (Ayyaz, 2015).

BMP TGFβ signalling pathways are critical for metazoan growth and development and have been well characterized in flies. Multiple ligands, receptors and transcription factors with highly context-dependent interactions and function have been described. This complexity is reflected by the sometimes conflicting studies exploring DPP/TKV/SAX signalling in the adult intestine. These studies consistently highlight two important aspects of BMP signalling in the adult Drosophila gut: ISCs can undergo opposite proliferative responses to BMP signals; and there are various sources of DPP that differentially influence ISC function in specific conditions. By characterizing the temporal regulation of BMP signalling activity in ISCs, the results resolve some of these conflicts: it is proposed that early in the regenerative response, haemocyte-derived DPP triggers ISC proliferation by activating SAX/SMOX signalling, and ISC quiescence is re-established by muscle-derived DPP as soon as TKV becomes expressed. Of note, some of the conflicting conclusions described in the literature may have originated from problems with the genetic tools used in some studies. This study have used two independent RNAi lines (BL25782 and BL33618) that effectively decrease dpp mRNA levels in haemocytes when expressed using HmlΔ::Gal4 (Ayyaz, 2015).

The close association of haemocytes with the type IV collagen Viking suggests that the stimulation of ISC proliferation by haemocyte-derived DPP may also be controlled at the level of ligand availability, as suggested previously for DPP from other sources. The regulation of SAX/SMOX signalling by DPP observed in this study is surprising, but consistent with earlier reports showing that SAX can respond to DPP in certain contexts. Biochemical studies have suggested that heterotetrameric complexes between the type II receptor PUNT and the type I receptors SAX and TKV can bind DPP, and complexes with TKV/TKV homodimers preferentially bind DPP, and complexes with SAX/SAX homodimers preferentially bind GBB. In the absence of TKV, SAX has been proposed to sequester GBB, shaping the GBB activity gradient, but to fail to signal effectively. Expression of GBB in the midgut epithelium has recently been described, and ligand heterodimers between GBB and DPP are well established. Consistent with earlier reports, this study found that GBB knockdown in ECs significantly reduces ISC proliferation in response to infection. Complex interactions between haemocyte-derived DPP, epithelial GBB, and ISC-expressed SAX, PUNT and TKV thus probably shape the response of ISCs to damage, and will be an interesting area of further study (Ayyaz, 2015).

Similar complexities exist in the regulation of transcription factors by SAX and TKV. Canonically, SMOX is regulated by Activin ligands (Activin, Dawdle, Myoglianin and maybe more), and the type I receptor Baboon. This study has tested the role of Activin and Dawdle in ISC regulation, and, in contrast to DPP, this study could not detect a requirement for these factors in the induction of ISC proliferation after Ecc15 infection. Furthermore, the data establish a requirement for haemocyte-derived DPP as well as for SAX expression in ISCs in the nuclear translocation of SMOX after a challenge. This study thus indicates that in this context, SAX responds to DPP and regulates SMOX. Regulation of SMOX by SAX has been described before, yet SAX is also known to promote MAD phosphorylation, but only in the presence of TKV. Consistent with such observations, this study has detected MAD phosphorylation in ISCs only in the late recovery phase on bacterial infection, when TKV is simultaneously induced in ISCs. During this recovery phase, ISCs maintain high SAX expression, but SMOX nuclear localization is not detected anymore, suggesting that SAX cannot activate SMOX in the presence of TKV, and might actually divert signals towards MAD instead. The data also suggest that Medea (the Drosophila SMAD4 homologue) is not required for SMOX activity. Although surprising, this observation is consistent with recent reports that SMAD proteins in mammals can translocate into the nucleus and activate target genes in a SMAD4-independent manner. The specific signalling readouts in ISCs when these cells are exposed to various BMP ligands and are expressing different combinations of receptors are thus likely to be complex (Ayyaz, 2015).

The current findings demonstrate that the control of ISC proliferation by haemocyte-derived DPP is critical for tolerance against enteropathogens, but contributes to ageing-associated epithelial dysfunction, highlighting the importance of tightly controlled interactions between blood cells and stem cells in this tissue. Nevertheless, where haemocytes themselves are required for normal lifespan, loss of haemocyte-derived DPP does not impact lifespan. One interpretation of this finding is that beneficial (improved gut homeostasis) and deleterious (for example, reduced immune competence of the gut epithelium) consequences of reduced haemocyte-derived DPP cancel each other out over the lifespan of the animal. It will be interesting to test this hypothesis in future studies. Ageing is associated with systemic inflammation, and a role for immune cells in promoting inflammation in ageing vertebrates has been proposed. In humans, recruitment of immune cells to the gut is required for proper stem cell proliferation in response to luminal microbes, and prolonged inflammatory bowel disease further contributes to cancer development. It is thus anticipated that conserved macrophage/stem cell interactions influence the aetiology and progression of such diseases. The data confirm a role for haemocytes in age-related intestinal dysplasia in the fly intestine, and provide mechanistic insight into the causes for this deregulation. It can be anticipated that similar interactions between macrophages and intestinal stem cells may contribute to the development of IBDs, intestinal cancers, and general loss of homeostasis in the ageing human intestine (Ayyaz, 2015).

Effects of Mutation or Deletion

In a dpp null mutant, all dorsal cell fates are missing and the embryos are completely ventralized. In contrast, embryos mutant for scw are partially ventralized and lack amnioserosa but differentiate a reduced dorsal ectoderm. The relative severity of the dpp and scw mutant phenotypes does not correlate with their expression patterns, since scw is transcribed uniformly at the syncitial blastoderm stage and dpp expression is restricted to the dorsal side of the embryo. One explanation for the different efficacies of the two ligands could be that they differ in abundance or have different affinities for their receptors. Alternatively, the ligands could evoke qualitatively different responses, perhaps by acting through different receptors. To distinguish between these alternatives, the ability of SCW mRNA to restore dorsal pattern in dpp null embryos was assayed. If the difference in the scw and dpp mutant phenotypes simply reflects their effective concentrations, excess Scw protein should compensate for the loss of dpp function. Injected Scw protein fails to restore amnioserosa in embryos that lack dpp function. This suggests that Scw and Dpp act in qualitatively distinct ways. While it had been postulated that dimerization between Scw and Dpp potentiates Dpp signaling by the formation of a potent Scw/Dpp dimer, this has been shown not to be the case. Expression of Scw in ventral cells in which Dpp is absent, rescues a scw mutant phenotype. Because Scw/Dpp dimers are likely to form intracellularly, these results strongly argue that formation of Scw/Dpp heterodimers is not a prerequisite for the biological activity of Scw in the embryo (Nguyen, 1998).

To understand the basis for the differential response of the embryo to Scw and Dpp signaling, the interaction of the ligands with the two type I receptors Sax and Tkv was examined. Using dominant-negative forms of the type I receptors Sax and Tkv, it is demonstrated that Sax mediates the Scw signal, while Tkv is required for both Dpp and Scw activity. While Dpp/Tkv signaling is obligatorily required, Scw/Sax activity is necessary but not sufficient for dorsal patterning. Tkv function is required for the response to both ligands, while the ability of Sax-DN to interfere specifically with Scw and not Dpp signaling strongly argues that Sax preferentially mediates the response to Scw. Sax and Tkv act synergistically, suggesting a mechanism for integration of the Scw and Dpp signals. Further, it is shown that the extracellular protein Sog can antagonize Scw, thus limiting its ability to augment Dpp signaling in a graded manner (Nguyen, 1998).

Genetic and phenotypic studies have established that sog and dpp exert opposing influences on dorsal patterning, leading to the suggestion that Sog functions as an antagonist of Dpp activity. Levels of Sog that do not affect Dpp signaling can block the ability of Scw to promote dorsal cell fates. The ability of Sog to specifically interfere with Scw does not conflict with previous studies showing a genetic antagonism of dpp activity by sog. Since Scw augments Dpp signaling, the inhibition of Scw activity by Sog is equivalent to antagonism of Dpp. In fact, results from earlier studies support the assertion that Sog preferentially targets Scw activity in the embryo. Thus, it is proposed that one way by which Sog mediates its negative effect on dorsal patterning is by antagonizing Scw function (Nguyen, 1998).

These data are also inconsistent with a central role for sog in modulating Dpp activity in late development. Ectopic expression of Sog in the wing disc using a variety of GAL4 drivers causes no significant phenotypic defects. This is quite striking given the prominent role of Dpp in organizing pattern along the anterior-posterior axis in the wing disc. It is worth noting that the loss of posterior crossveins caused by expression of Sog is similar to the defect caused by Sax-DN, rather than Tkv-DN. An explanation for the failure of Sog to target Dpp could be that Dpp is bound to extracellular matrix components or forms a high-affinity complex with its receptor. Alternatively, the observation that Xenopus Noggin can severely ventralize Drosophila embryos raises the possibility that a Noggin-like factor may be the functionally relevant Dpp antagonist (Nguyen, 1998).

If Sog primarily blocks Scw activity during embryogenesis, the role of Tolloid may be to potentiate Scw signaling by releasing it from an inhibitory complex. Scw can promote Tld-dependent cleavage of Sog. This may explain why the loss of tld function results in a partially ventralized phenotype similar to that of scw- mutants, rather than the complete ventralization typical of dpp null embryos. The observation that embryos lacking both scw and tld function do not display a more severe phenotype is also compatible with this view (Nguyen, 1998).

Mutations that abolish sax result in phenotypes similar to those with a partial loss of DPP; mutations that abolish tkv activity show phenotypes similar to those with a complete loss of DPP function. (Nellen, 1994).

dpp embryos and embryos from sax mothers exhibit similar mutant phenotypes during early gastrulation. These two loci exhibit genetic interactions, suggesting they are utilized in the same pathway (Xie, 1994).

hindsight expression in the amnioserosa is regulated by the dorsoventral pathway. Dorsal Hnt protein expression is reduced in genetically ventralized mutant embryos such as those produced by saxophone or cactus females. Reciprocally, dorsal Hnt expression expands ventrally in dorsalized embryos. Anterior midgut expression of Hnt is also affected by the dorsoventral pathway (Yip, 1997).

decapentaplegic is required for patterning of anterior eggshell structures, reflecting expression of dpp in anterior somatic follicle cells. Mutations in sax result in a block in oogenesis associated with egg chamber degeneration and a failure to transfer nurse cell contents to the oocyte. This suggests that the dpp signal is transmitted from the soma to the germline during oogenesis (Twombly, 1996).

The imaginal disk expression of the TGF-ß superfamily member DPP in a narrow stripe of cells along the anterior-posterior compartment boundary is essential for proper growth and patterning of the Drosophila appendages. DPP receptor function was examined to understand how this localized DPP expression produces its global effects on appendage development. Clones of saxophone or thick veins (tkv) mutant cells, defective in one of the two type I receptors for DPP, show shifts in cell fate along the anterior-posterior axis. In the adult wing, clones that are homozygous for a null allele of sax or a hypomorphic allele of tkv show shifts to more anterior fates when the clone is in the anterior compartment and to more posterior fates when the clone is in the posterior compartment. The effect of these clones on the expression pattern of the downstream gene spalt-major also correlates with these specific shifts in cell fate. The shift in cell fate is explained by assuming that the cells in mutant clones act as though they see a lower than normal DPP concentration. Thus cell fate along the A/P axis is directly related to the perceived DPP level. It is concluded that cell fate is directly related to the distance of cells from the source of DPP at the A/P axis and that DPP is responsible for patterning of the entire wing blade in direct response to the long-range DPP signal. The similar effects of sax null and tkv hypomorphic clones indicate that the primary difference in the function of these two receptors during wing patterning is that TKV transmits more of the DPP signal than does SAX. These results are consistent with a model in which a gradient of DPP reaches all cells in the developing wing blade to direct anterior-posterior pattern. While current evidence suggests that TKV is absolutely required for DPP signaling, there appears to be no such absolute requirement for SAX. Thus DPP receptor complexes that lack a TKV subunit cannot transmit a sufficient level of DPP signal to trigger a biological response in the receiving cell. In contrast, receptor complexes lacking SAX subunits are still capable of significant signal reception and downstream signaling (Singer, 1997).

In order to directly address the role of Medea in the Dpp pathway, the ability of Medea mutants to suppress ectopic signaling from Dpp receptors was tested. A constitutively activated Dpp Type I receptor, Saxophone (Sax*), was generated by the substitution of a single amino acid (Q263D) near the GS box of the intracellular domain. The activated receptor is expressed in a spatially controlled manner using the GAL4-UAS system. Under engrailedGAL4(enGAL4) control, UAS-Sax* produces a phenotype in the wing, characterized by posterior defects, such as overgrowth and ectopic venation. Removal of a single copy of a gene that is required for Sax signaling, for example Mad, suppresses this phenotype. This same suppression is observed when a single copy of Medea is removed from enGal4,UAS-Sax* transgenic flies. Since two type I receptors have been identified for Dpp, the ability of Medea to suppress signaling from the other activated receptor, Thick veins (Tkv*) was tested. Interestingly, Medea does not show the same ability to suppress a Tkv* phenotype, typified by ectopic vein material and severe blistering. In fact, a subset of Medea alleles showed very low levels of suppression, and to a much lesser extent than Mad. One explanation for the differential ability of Medea mutants to suppress the Sax* and Tkv* phenotypes is that the two activated receptors achieve different levels of signaling. In addition, Medea may not be a limiting component in Tkv* signaling. Therefore, the removal of one copy of Medea may be insufficient to affect the high levels of Tkv* signaling, but may be enough to influence the weaker Sax* signal (Das, 1998).

spinster (spin), which encodes a multipass transmembrane protein, has been identified in a genetic screen for genes that control synapse development. spin mutant synapses reveal a 200% increase in bouton number and a deficit in presynaptic release. spin is expressed in both nerve and muscle and is required both pre- and postsynaptically for normal synaptic growth. Spin has been localized to a late endosomal compartment and evidence is presented for altered endosomal/lysosomal function in spin mutants. Evidence is presented that synaptic overgrowth in spin is caused by enhanced/misregulated TGF-ß signaling. TGF-ß receptor mutants show dose-dependent suppression of synaptic overgrowth in spin. Furthermore, mutations in Dad, an inhibitory Smad, cause synapse overgrowth. A model is presented for synaptic growth control with implications for the etiology of lysosomal storage and neurodegenerative disease (Sweeney, 2002).

To determine whether synaptic overgrowth in spin is caused by enhanced TGF-ß signaling, it was asked whether TGF-ß signaling is necessary for synaptic overgrowth in spin. The type II receptor mutation wishful thinking causes a severe decrease in bouton number at the NMJ. Type I TGF-ß receptors are known to function in concert with type II receptors, and the type I receptors tkv and sax participate in synaptic growth regulation in this system. Third instar larva mutant for sax or tkv have smaller neuromuscular synapses. This study confirms that there is a significant decrease in bouton number in wit, and that there is a similar decrease in bouton number in both tkv and sax. These receptors are shown to function in the larval motoneurons by demonstrating that pMAD staining in the cell bodies of larval motoneurons requires wit or sax. In this experiment, the larval CNS was costained with pMAD and anti-evenskipped, which labels a subset of motoneurons (Sweeney, 2002).

A genetic analysis of the TGF-ß receptor mutations wit, tkv, and sax in combination with spin demonstrates that TGF-ß signaling is necessary for synaptic overgrowth in spin. Heterozygous mutations in tkv, sax, and wit do not alter synaptic bouton numbers at the NMJ. Heterozygous mutations in tkv, sax, and wit suppress synaptic overgrowth when placed in the spin mutant background. Bouton numbers are significantly reduced in each case where a single copy of a receptor is mutated in combination with spin. Bouton numbers were quantified in each of the double mutant combinations of tkv, sax, or wit with spin. In each case, when both copies of a receptor were removed, synaptic overgrowth was suppressed in the spin mutant background further than when only a single copy of a receptor was mutated. These data demonstrate that TGF-ß receptor mutations suppress synaptic overgrowth in spin in a dose-dependent manner. Furthermore, since bouton numbers return to wild-type, or below wild-type levels, it demonstrates that TGF-ß signaling is necessary for synaptic overgrowth in spin. Taken together with the increase in bouton numbers seen in dad, these data support the conclusion that enhanced or misregulated TGF-ß signaling is a major determinant of synaptic overgrowth in spin. It is hypothesized that altered endosomal function due to loss of Spin causes enhanced TGF-ß signaling and subsequent synaptic overgrowth. Future experiments will be necessary to determine whether enhanced signaling is due to increased receptor number at the plasma membrane, or an inability to stop signaling within the late endosomal system (Sweeney, 2002).

Germline stem cell number in the ovary is regulated by mechanisms that control Dpp signaling: Mutations in sax increase the number of GSCs

The available experimental data support the hypothesis that the cap cells (CpCs) at the anterior tip of the germarium form an environmental niche for germline stem cells (GSCs) of the Drosophila ovary. Each GSC undergoes an asymmetric self-renewal division that gives rise to both a GSC, which remains associated with the CpCs, and a more posterior located cystoblast (CB). The CB upregulates expression of the novel gene, bag of marbles (bam), which is necessary for germline differentiation. Decapentaplegic (Dpp), a BMP2/4 homolog, has been postulated to act as a highly localized niche signal that maintains a GSC fate solely by repressing bam transcription. The role of Dpp in GSC maintenance has been examined in more detail. In contrast to the above model, it is found that an enhancer trap inserted near the Dpp target gene, Daughters against Dpp (Dad), is expressed in additional somatic cells within the germarium, suggesting that Dpp protein may be distributed throughout the anterior germarium. However, Dad-lacZ expression within the germline is present only in GSCs and to a lower level in CBs, suggesting there are mechanisms that actively restrict Dpp signaling in germ cells. One function of Bam is to block Dpp signaling downstream of Dpp receptor activation, thus establishing the existence of a negative feedback loop between the action of the two genes. Moreover, in females doubly mutant for bam and the ubiquitin protein ligase Smurf, the number of germ cells responsive to Dpp is greatly increased relative to the number observed in either single mutant. These data indicate that there are multiple, genetically redundant mechanisms that act within the germline to downregulate Dpp signaling in the Cb and its descendants, and raise the possibility that a Cb and its descendants must become refractory to Dpp signaling in order for germline differentiation to occur (Casanueva, 2004).

The prevalent model for Dpp action within the ovary is that it is a local niche signal whose activity is permissive for GSC maintenance. In this model, only GSCs within the niche are exposed to Dpp protein and removal of the CB from the niche lessens or eliminates exposure to the ligand. Moreover, the only postulated function of Dpp is to repress the transcription of bam within the GSCs. The data presented in this paper reveal additional aspects of Dpp function in GSC maintenance. The results strongly suggest that Dpp ligand is not restricted to the niche but rather is present throughout the anterior germarium. Data is presented that the observed specificity of Dpp signaling to the GSCs and CBs is due to functionally redundant mechanisms that operate in the germline to actively downregulate Dpp signaling during GSC differentiation. One of these mechanisms is Bam itself, thus establishing a negative feedback loop between the actions of the two genes. These findings indicate GSC differentiation is correlated with downregulation of Dpp signaling, raising the possibility that Dpp signaling plays an active role in GSC maintenance, and that GSC differentiation requires both the presence of Bam and the absence of Dpp signaling (Casanueva, 2004).

If GSCs and CBs are exposed to equivalent amounts of Dpp protein, as is suggested by both the transcription pattern of the Dpp gene and the expression of Dad-lacZ in the CpCs of the niche and the ISCs posterior to the niche, then it is likely that the observed reduction in Dad-lacZ expression between the GSC and the CB results from intracellular modulation of the strength of the Dpp signal. One hallmark of the GSC is its invariant plane of division. It is proposed that the differential Dpp signaling between the GSC and CB sign results from an intracellular modulation of Dpp signal strength between the two daughter cells, either by the asymmetric segregation of one or more cellular components that modulate Dpp signaling, or by loss of a contact-based niche signal that elevates Dpp signaling preferentially within the GSCs. Removal of the CB cell from the niche thus results in partial downregulation of Dpp signaling. A lower level of Dpp signaling in the CB cell results in the transcription of Bam, which plays multiple roles in CB differentiation, one of which is to cause the daughters of the CB cell to become refractory to further Dpp signaling. Thus, sequential regulatory mechanisms cooperate to ensure an irreversible change in the fate of the GSC cell within two generations (Casanueva, 2004).

Loss-of-function mutations in Smurf and gain-of-function mutations in sax increase the number of GSCs, suggesting these genes may perturb the proposed intracellular modulation of Dpp signaling that occurs between the GSC and CB. However, these data are not sufficient to determine whether this proposed modulatory pathway acts through direct regulation of the functions of one or both of these gene products, or whether the proposed pathway acts in parallel to these genes. In the embryo, loss of Smurf activity results in a ligand-dependent elevation of Dpp signaling that has greater, but not indefinite, perdurance (Podos, 2001), suggesting that Dpp signaling in Smurf mutants, and by inference sax mutants, is still responsive both to the amount of ligand and to the presence of other negative regulatory mechanisms. In the ovary, the Dad-lacZ-expressing germ cells in the Smurf and sax mutants fill the region of the anterior germarium that roughly corresponds to the spatial extent of Dad-lacZ expression in the somatic cells of region 1 and 2A of a wild-type germarium, suggesting that potentially all germ cells in region 1 and 2A of the Smurf and sax germaria are equally and fully responsive to the Dpp ligand. It is proposed that GSCs in the Smurf and sax germaria ultimately undergo normal differentiation because in the more posterior regions of the germaria the amount of Dpp ligand may be reduced to a level that allows bam transcription, which further reduces Dpp signaling and causes cyst differentiation (Casanueva, 2004).

The reduction in Dpp signaling between the GSC and the CB releases Bam from Dpp-dependent transcriptional repression, and one, but not the only, function of Bam is to downregulate Dpp signaling downstream of receptor activation prior to overt GSC differentiation. This is the first molecular action ascribed to Bam, and these data could provide an entry point to elucidate the biochemical basis of the function of Bam in CB differentiation. Further work will be necessary to determine whether the action of Bam on the Dpp pathway is direct or indirect, whether Bam action results in the reduction or complete elimination of Dpp signaling in the developing cysts, and which step in the intracellular Dpp signal transduction pathway or expression of Dpp target genes is affected by Bam action. However, it is possible that initial insights into Bam function can be made by comparing the thresholds for Dpp signaling readouts in the developing wing disc of the larva to the data obtained in the germarium. In the wing disc, Dpp diffuses from a limited source to form a gradient throughout the disc that displays different thresholds for multiple signaling readouts. Specifically, Dad-lacZ is transcribed in response to high and intermediate levels of Dpp, but does not respond to the lowest levels of ligand. An antibody exists that recognizes the active phosphorylated form of Mad, pMad. In the wing disc, high level staining with the pMad antibody is present in only a subset of cells that express high levels of Dad-lacZ, suggesting that in this tissue the pMad antibody is less sensitive to Dpp signaling than is Dad-lacZ expression. Intriguingly, in the ovariole pMad staining is visible in the GSCs, CBs and the developing cysts. Because Dad-lacZ expression was never observed in the developing cysts, these results could suggest that the relative sensitivities of these two reagents are reversed within the germline. Alternatively, if the reagents have the same relative sensitivities in the two tissues, the data suggest that Bam could act, probably at a post-transcriptional level, to downregulate Dpp signaling downstream of Mad activation (Casanueva, 2004).

The pattern of Dad-lacZ expression observed in the Smurf; bam and sax; bam double mutant ovarioles is qualitatively different from that observed in any of the single mutant ovarioles. Although Dad-lacZ expression is observed only at the anterior tip of the germarium of each single mutant, many, but not all, of the double mutant ovarioles contain germ cells throughout the ovariole that express high levels of Dad-lacZ. From these data, it is concluded that two redundant pathways downregulate Dpp signaling in the germline, and that in the single mutants, the action of the remaining active pathway is sufficient to constrain Dpp responsiveness to the anterior tip of the germarium. However, not all doubly mutant ovarioles display a spatial expansion of Dpp signaling, and this variability can even be observed in ovarioles from a single female. It is proposed that the observed variability results because the Smurf and sax mutations have modulatory effects on Dpp signaling that are both dependent on the presence of ligand and are sensitive to additional mechanisms that downregulate Dpp signaling. In both the Smurf; bam and sax; bam ovarioles, the germ cells that express Dad-lacZ are observed throughout the ovariole, but are more likely to be near somatic cells. It is possible that the variability in Dad-lacZ expression occurs because of a non-uniform distribution of the Dpp ligand. Nevertheless, there is not a consistent correlation between the domains of Dad-lacZ expression in the somatic and germ cells, suggesting that there may be additional germline intrinsic factors that affect Dpp signaling (Casanueva, 2004).


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

date revised: 15 July 2015

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