thickveins

DEVELOPMENTAL BIOLOGY

Embryonic

tkv expression is uniform in early embryos, but by stage 5, expression is more apparent on the dorsal side. By late stage 5,TKV is detected in seven stripes on the ventral side and in a discountinuous pattern on the dorsal side. By stage 9 [Images], expression is restricted to the mesoderm. By stage 16, TKV is restricted to the gastric caeca primordia, first and fourth midgut chambers, hindgut and the head region (Penton, 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).

A presynaptic endosomal trafficking pathway controls synaptic growth signaling

Structural remodeling of synapses in response to growth signals leads to long-lasting alterations in neuronal function in many systems. Synaptic growth factor receptors alter their signaling properties during transit through the endocytic pathway, but the mechanisms controlling cargo traffic between endocytic compartments remain unclear. Nwk (Nervous Wreck) is a presynaptic F-BAR/SH3 protein that regulates synaptic growth signaling in Drosophila. This study shows that Nwk acts through a physical interaction with sorting nexin 16 (SNX16). SNX16 promotes synaptic growth signaling by activated bone morphogenic protein receptors, and live imaging in neurons reveals that SNX16-positive early endosomes undergo transient interactions with Nwk-containing recycling endosomes. An alternative signal termination pathway was identified in the absence of Snx16 that is controlled by endosomal sorting complex required for transport (ESCRT)-mediated internalization of receptors into the endosomal lumen. These results define a presynaptic trafficking pathway mediated by SNX16, NWK, and the ESCRT complex that functions to control synaptic growth signaling at the interface between endosomal compartments (Rodal, 2011).

Endocytic membrane traffic regulates signaling by synaptic growth factor receptors and may be used as a point of control in tuning receptor traffic in response to neuronal activity. The neuronal F-BAR/SH3 protein Nwk is involved in endocytic membrane traffic that attenuates synaptic growth signaling, but the mechanism by which it acts has been unknown. This study has identified a physical interaction between Nwk and the early endosomal protein SNX16 that is critical for down-regulating the synaptic growth-promoting activity of SNX16. This interaction may bring together the actin-polymerizing activity of the first SH3 domain of Nwk with the potential lipid-binding/tubulating activities of the F-BAR domain of Nwk and the PX domain of SNX16, driving tubule-based membrane flux from early to recycling endosomes (Rodal, 2011).

Sorting nexins form a large family of proteins that share a common phosphoinositide-binding PX domain and are involved in diverse aspects of membrane traffic (Cullen, 2008). The best-characterized members of this family are the sorting nexin-BAR family, including Snx1 (tied to endosome-to-Golgi traffic) and Snx9 (tied to the internalization step of endocytosis), which each contains both a lipid-tubulating BAR domain and a PX domain. Crystal structures and functional studies of Snx9 have shown that its BAR and PX domains form a single lipid-binding and -deforming module with combined specificities that neither domain exhibits alone. As such, SNX16-Nwk interactions may form an analogous F-BAR/PX module via an intermolecular rather than intramolecular interaction. It has not been possible to purify sufficient amounts of SNX16 to directly test its effects on lipid binding by Nwk; therefore, further analysis of their interaction will require an approach to isolate SNX16 in vitro. Interestingly, the SNX16-Nwk interaction depends on a region of Nwk surrounding its second SH3 domain, raising the possibility that this region of Nwk may exert intramolecular effects on the amino-terminal F-BAR domain, as has previously been shown for other F-BAR proteins (Rao, 2010). Furthermore, Nwk binds to the coiled-coil region of SNX16, which is involved in SNX16 dimerization, suggesting that Nwk may act on SNX16 by affecting its dimerization state (Rodal, 2011).

Mammalian SNX16 has been implicated in the trafficking of the EGF receptor from early to late endosomes, which may mediate down-regulation of receptor signaling. However, the mechanism by which SNX16 promotes this trafficking step is not understood and no loss-of-function studies on SNX16 have been reported. A mutant of mammalian SNX16 that lacks 60 aa corresponding to the Drosophila SNX16 Nwk-binding site blocks trafficking of SNX16 and EGF to late endosomes, leading to increased EGF signaling. Interestingly, the F-BAR protein FBP17 has been reported to interact with SNX2. These results raise the possibility that BAR family-sorting nexin interactions may be broadly used to control membrane traffic in cells (Rodal, 2011).

Drosophila SNX16 localizes to an early endosomal compartment at the NMJ defined by the small GTPases Rab4 and Rab5. This compartment accumulates signaling receptors, such as the BMP receptors Tkv and Wit, and signaling is amplified when receptors are stalled in this compartment, suggesting that the SNX16 compartment is an active site of signaling. By identifying specific Snx16 mutants that disrupt interactions with Nwk, it was possible to separate the requirements for receptor down-regulation at early endosomes and show that activated receptors specifically require SNX16-Nwk-mediated traffic to recycle to the plasma membrane (Rodal, 2011).

Previous studies have shown that Nwk colocalizes with the recycling endosome marker Rab11 in fixed tissue and cooperates with Cdc42, which is thought to function at the recycling endosome, to activate WASp/Arp2/3-mediated actin polymerization. Together with the result that rab11 mutants exhibit synaptic overgrowth similar to nwk mutants (Khodosh, 2006), it is concluded that Nwk functions at recycling endosomes, which are downstream of early endosomes. This study shows that Nwk tagged with fluorescent proteins localizes to novel punctate structures in heterologous cells and nerve terminals. These puncta had not been observed using anti-Nwk antibodies against either endogenous or overexpressed Nwk in fixed tissue, as they are poorly preserved upon fixation. In living synapses, Rab11 colocalizes with Nwk to mobile puncta, SNX16-containing early endosomes interact transiently with Nwk puncta, and inhibition of the GTPase dynamin blocks separation of these compartments. Dynamin inhibition has previously been shown to disrupt endosomal function in the fly NMJ, it cannot be certain that the collapse of SNX16-Nwk compartments under these conditions is caused by the specific interactions of these proteins with dynamin. However, because SNX16-Nwk interactions lead to the down-regulation of synaptic growth signals, the data are consistent with a model in which the exchange of receptors from the SNX16 endosome to the Nwk/Rab11 endosome leads to the attenuation of receptor signaling. Binding of Nwk to the cytoplasmic tail of the BMP receptor Tkv may mediate this event (O'Connor-Giles, 2008), but further experiments will be required to directly examine cargo transfer in the future. Because there are significant cytoplasmic pools of both Nwk and SNX16, the contribution of these soluble proteins to membrane traffic in the nerve terminal or the possibility that overexpression of Nwk and Snx16 to visualize live trafficking events does not faithfully recapitulate the behaviors of endogenous proteins cannot be excluded. However, the transient interaction of Nwk and SNX16 puncta correlates well with genetic results showing that nwk attenuates a synaptic growth-promoting activity of Snx16 at early endosomes (Rodal, 2011).

Because Nwk- and SNX16-labeled compartments transiently interact in nerve terminals, temporal control of the interaction between their lipid-binding domains may provide a mechanism to acutely drive membrane tubulation in a regulated fashion. Furthermore, the association of SNX16 with endosomes depends on phosphoinositides, as the phosphatidylinositol 3-kinase inhibitor wortmannin disrupts localization of SNX16 in cultured cells, indicating that regulation of phospholipid composition may also contribute to the membrane-deforming activities of SNX16 and Nwk (Rodal, 2011).

This study found that Snx16 loss-of-function mutants suppress synaptic overgrowth resulting from loss of Nwk-mediated traffic as well as from activation of Wg and BMP signaling pathways. These data suggest that SNX16 plays an active role in promoting synaptic growth at the endosome, aside from its function in signal attenuation through Nwk. Snx16 was found to be required to restrict synaptic growth when MVB formation is hampered in hrs mutants, suggesting that receptor entry into the endosomal lumen is an alternative signal attenuation pathway. Furthermore, it was found that overexpression of a mutant Snx16 that cannot bind Nwk promotes the accumulation of endosomal structures at the NMJ and drives excess synaptic growth, suggesting that Snx16 may play a role in MVB maturation and acts at the branch point between endosomal sorting pathways. Defining the mechanism by which SNX16 promotes synaptic growth will require further structure-function analysis of the active domains of the protein aside from its Nwk-binding coiled coil (Rodal, 2011).

Traffic through endocytic compartments has proven to be a critical point of regulation in the nervous system. Synaptic vesicle endocytosis is controlled by synaptic activity through calcium-dependent dephosphorylation of endocytic proteins, and postsynaptic trafficking of AMPA receptors through the recycling endosome is increased in response to activity via the calcium-dependent motor myosin V. Rab5-positive compartments at the Drosophila NMJ have been previously characterized for their role in the synaptic vesicle cycle, and it will be interesting to determine how receptor-mediated endocytosis and synaptic vesicle endocytosis are coordinately or separately regulated in response to activity. Synaptic growth at the Drosophila NMJ is positively regulated by calcium influx through voltage-gated calcium channels, and endosome number increases in response to activity. It is tempting to speculate that calcium influx acts through conserved mechanisms for modulating membrane dynamics to delay the attenuation of receptor signaling by SNX16-Nwk-mediated traffic, leading to increased synapse growth in response to activity. A key future goal will be to determine specific points of activity-dependent regulation of membrane traffic in presynaptic endosomes (Rodal, 2011).

Larval

During vein differentiation dpp is expressed in the pupal veins under the control of genes that establish vein territories in the imaginal disc. Both dpp and thick veins are differentially expressed in vein territories during pupal development. dpp and tkv regulate one another by a feedback mechanism in which Tkv activity represses dpp expression. Dpp, acting through its receptor Thick veins, activates vein differentiation and restricts expression of both veinlet and the Notch-ligand Delta to the developing veins. Ectopic dpp expression or Tkv activation in the wing disc result in the differentiation of ectopic veins. Outside of vein territories, the repression of dpp by the widely expressed Tkv could participate in restricting dpp expression to the veins. It is possible that the observed down-regulation of tkv expression in vein cells participates in generating the levels of Tkv activation necessary to activate vein differentiation, but insufficient to repress dpp expression. The expression of dpp and tkv in vein territories depends (either directly or indirectly) on EGF-receptor activity, because the transcription of these genes is not activated when Egf-R is reduced (as in veinlet and vein mutant wings). Once Dpp is established in the veins, local activation of Tkv in these cells is required both for the maintenance of veinlet and Delta expression and for the veins to differentiate. In dpp mutants, the vein thickening observed in Notch mutants is elimated. Conversely, Notch gain-of-function alleles that lead to the truncation of veins results in very pronounced vein loss in combination with both dpp and tkv mutants. In dpp mutants, Delta and E(spl)mß, which normally takes place in vein territories, is lost. In summary, genetic combinations between mutations that increase or reduce Notch, veinlet and dpp activities suggest that the maintenance of the vein differentiation state during pupal development involves cross-regulatory interactions between these pathways (de Celis, 1997).

Formation of the longitudinal veins (LVs) of the Drosophila wing involves the interplay among Dpp, Egf and Notch pathways. Formation of crossveins (CVs: see Derivatives of the wing disc) present a paradoxical problem. As shown both morphologically and using molecular markers, the definitive CVs are not formed until long after the initial specification of the LVs. The CVs therefore must form within territory that has already been specified as intervein. The CVs must also interconnect with existing LVs at a time when the Delta expressed by the LVs is thought to inhibit vein formation in adjacent cells. Mechanisms must exist that override both intervein specification and the lateral inhibition of veins, allowing the formation of continuous, interconnected vein tissue. BMP-like signaling plays a special role in the formation of the CVs from within intervein territory. BMP-like signals also help maintain the connections between the LVs and the margin of the wing. crossveinless 2 (cv-2) is a critical factor in these processes, as it is expressed more highly in the CVs and the ends of the LVs and is required for the high levels of BMP-like signaling observed in these regions (Conley, 2000). The cv-2 mutation was first identified by Benedetto Nicoletti in 1962 (FlyBase: Cv-2 site). The structure of the Cv-2 protein strongly suggests that these effects are direct, and that Cv-2 is a novel player in the BMP-like signaling pathway (Conley, 2000).

Both Dpp and Gbb vein signals are mediated largely by the type I receptor Thickveins, rather than the alternate type I receptor Saxophone. Cells lacking Tkv do not form veins, but removal of Sax does not reliably remove veins. However, not all veins are equally sensitive to reductions in Dpp and Gbb signaling. The hypomorphic gbb⁴ mutation shows complete loss of the cross veins (CVs), but only slight loss of the ends of the LVs. Sog encodes a Chordin-like molecule that inhibits BMP-like signaling; both Sog and Chordin are thought to bind to and sequester ligands, preventing the activation of receptors. Overexpressing Sog in the wing specifically blocks formation of the CVs and the ends of the LVs. The secreted Tolloid proteases, similar to vertebrate BMP1s, can increase BMP signaling by cleaving and inactivating Chordin or Sog. Loss of tolkin (also known as tolloid-related) blocks formation of the CVs and the tips of the LVs. Overexpressing a dominant negative form of Sax again induces a similar phenotype (Conley, 2000 and references therein).

Such phenotypes are very reminiscent of the crossveinless class of mutations in Drosophila (reviewed in Garcia-Bellido, 1992). Strong reductions in crossveinless 2 (cv-2) function have been shown to remove the posterior CV (PCV), the anterior CV (ACV), and the ends of the LVs. However, despite the possibility that the crossveinless genes encode novel players in BMP-like signaling, none have been characterized and the sensitivity of CVs to BMP-like signaling has not been explained. Evidence is presented that cv-2 encodes a novel member of the BMP-like signaling pathway, expressed in and required for high levels of BMP-like signaling in the developing cross veins. The Cv-2 protein contains five cysteine-rich domains similar to those known to bind BMP-like ligands, strongly suggesting that Cv-2 directly modulates Dpp or Gbb activity (Conley, 2000 and references therein).

dpp and gbb mutations both disrupt CV formation. Weak cv-2 alleles are strengthened by dpp and gbb loss-of-function mutations. cv-2^225-3/cv-2³⁵¹¹ flies never lack the entire PCV, but 50% of gbb 4 cv-2^225-3/cv-2 3511 flies lack the entire PCV. Similarly, cv-2³⁵¹¹/Df(2R)Pu-D17 only rarely disrupt the ACV, but dpp^d6 cv-2³⁵¹¹/Df(2R)Pu-D17 commonly does. However, cv-2 cannot dominantly enhance earlier dpp-dependent patterning in the wings: dpp^d5 Df(2R)Pu-D17 /dpp^hr4 wings look no worse than dpp^d5/dpp^hr4 wings. To provide a more direct link between cv-2 and Dpp and Gbb signaling, Mad activation was examined in mutant pupal wings. In cv-2¹ adults, the PCV is more reliably disrupted than the ACV; the anti-p-Mad staining normally found near the PCV in 19, 22, 26 and 36 hours after pupariation wings is lost or disrupted in cv-2¹ homozygotes, as is the reduction of anti-DSRF in the PCV. In adults of the stronger allelic combination cv-2¹/Df(2R)Pu-D17, the ACV is also often lost along with the ends of some of the LVs. Interestingly, no disruption of the ACV or LV anti-p-Mad staining cv-2¹/Df(2R)Pu-D17 pupal wings is detected at 21 or 25 hours after pupariation; only at 36 hours after pupariation is staining lost from the ACV. This indicates that cv-2 is required not only to initiate Mad activity in the PCV, but also to maintain that activity in the ACV (Conley, 2000).

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).

Bone morphogenetic protein- and mating-dependent secretory cell growth and migration in the Drosophila accessory gland

The paired male accessory glands of Drosophila melanogaster enhance sperm function, stimulate egg production, and reduce female receptivity to other males by releasing a complex mixture of glycoproteins from a secretory epithelium into seminal fluid. A small subpopulation of about 40 specialized secretory cells, called secondary cells, resides at the distal tip of each gland. These cells grow via mechanisms promoted by mating. If aging males mate repeatedly, a subset of these cells delaminates from and migrates along the apical surface of the glandular epithelium toward the proximal end of the gland. Remarkably, these secretory cells can transfer to females with sperm during mating. The frequency of this event increases with age, so that more than 50% of triple-mated, 18-d-old males transfer secondary cells to females. Bone morphogenetic protein signaling specifically in secondary cells is needed to drive all of these processes and is required for the accessory gland to produce its normal effects on female postmating behavior in multiply mated males. It is concluded that secondary cells are secretory cells with unusual migratory properties that can allow them to be transferred to females, and that these properties are a consequence of signaling that is required for secondary cells to maintain their normal reproductive functions as males age and mate (Leiblich, 2012).

The secondary cells of the male fly accessory gland selectively grow during aging in adults, a process enhanced by repeated mating. These cells exhibit a range of behaviors, induced by mating, that are atypical of secretory cells in glands, including active delamination and migration. Although migrating cells were initially observed in less than 5% of repeatedly mated males, introducing a delay between two previous matings and dissecting the resulting 18-d-old males revealed migrating cells in all animals, suggesting that this process is common in aged, mated animals (Leiblich, 2012).

The growth, delamination and migratory activities of secondary cells all require cell-autonomous BMP signaling. One or more of these BMP-regulated processes modulates long-term, postmating behavior in females, particularly when males are repeatedly mated over short periods of time, requiring rapid replenishment of luminal content in the accessory gland. Although the numbers of vacuoles in secondary cells with high levels of BMP signaling seem more variable than controls, vacuole number in Dad-expressing secondary cells appears relatively normal, suggesting that reduced BMP signaling does not simply block the general secretory machinery. However, reduced signaling presumably affects the synthesis or function of one or more secondary cell products, leading either to direct effects in mated females or to indirect effects through modulation of main cell function or products in males (Leiblich, 2012).

Unexpectedly, some secondary cells are transferred to females after multiple matings, particularly in aged flies, raising the possibility that these delaminating cells continue to function together with sperm even outside the male. Transfer is not essential for these cells to mediate their BMP-regulated effects in females, because not all mated females receive these cells. However, it is possible that transfer could contribute to changes in accessory gland function as the glandular epithelium undergoes BMP-dependent structural alterations during aging and mating. A recent study from Minami (2012) indicates that secondary cells are required for normal male fecundity and effects on female postmating behaviors. The current work now clearly demonstrates that BMP-mediated events in secondary cells are involved in maintaining these latter functions specifically during adulthood (Leiblich, 2012).

The data highlight some surprising parallels between the accessory gland and the prostate, in addition to those previously reported. Like the prostate, the structure of the accessory gland epithelium changes significantly with age. Furthermore, BMP signaling is implicated in normal prostate development and in the progression of prostate cancer. Importantly, prostate cells have been identified in human semen and the phenotype of these cells may be altered in prostate cancer. Although many of these cells are likely to have sloughed off from the epithelium, the current study raises the possibility that some actively delaminate into seminal fluid (Leiblich, 2012).

The secondary cells of the accessory gland require BMP signaling to regulate the synthesis or function of one or more important components of the seminal fluid as flies age and mate. However, this signaling simultaneously drives cell loss and changes in the morphology and function of the epithelium, which appears to lack regenerative capacity in flies. The prostate gland of most human males over 50 y of age is hyperplastic, and it is tempting to speculate that this reflects a regenerative response to similar events in this organ. A more detailed analysis of secondary cell biology should help to further elucidate the processes that underlie functional changes in the accessory gland epithelium and test whether these are shared by male reproductive glands in other organisms (Leiblich, 2012).

Smad-independent BMP signaling in somatic cells limits the size of the germline stem cell pool

In developing organisms, proper tuning of the number of stem cells within a niche is critical for the maintenance of adult tissues; however, the involved mechanisms remain largely unclear. This study demonstrated that Thickveins (Tkv), a type I bone morphogenetic protein (BMP) receptor, acts in the Drosophila developing ovarian soma through a Smad-independent pathway to shape the distribution of BMP signal within the niche, impacting germline stem cell (GSC) recruitment and maintenance. Somatic Tkv promotes Egfr signaling to silence transcription of Dally, which localizes BMP signals on the cell surface. In parallel, Tkv promotes Hh signaling, which promotes escort cell cellular protrusions and upregulates expression of the Drosophila BMP homolog, Dpp, forming a positive feedback loop that enhances Tkv signaling and strengthens the niche boundary. These results reveal a role for non-canonical BMP signaling in the soma during GSC establishment and generally illustrate how complex, cell-specific BMP signaling mediates niche-stem cell interactions (Tseng, 2018).

Self-restrained regulation of stem cell niche activity by niche components in the Drosophila testis

Most, if not all, stem cells reside in a defined microenvironment, called the niche. Short-ranged niche signal must be tightly controlled to be active only inside the niche to maintain the proper balance of stem cell self-renewal verse differentiation. However, how niche components restrict localized niche signal activation remains largely unknown. This study finds that Thickveins (Tkv, a type I receptor of the Dpp signaling pathway) in cyst stem cells (CySCs) of the testis niche prevents Dpp signaling activation outside of the niche. Tkv functions as Dpp trap/sink to spatially restrain Dpp signaling inside the niche. This self-restrained regulation of niche activity by Tkv in CySCs is independent of the canonical Dpp signaling pathway. These data demonstrate the critical roles of niche components (CySCs) in the self-restrained regulation of niche activity, which could be shed light on niche activity regulation in general (Xu, 2018).

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