held out wings

Transcriptional Regulation

The early expression of How protein is apparently under the control of twist. A direct role for twist in the mesodermal expression of how is further suggested by the existence of four putative Twist binding-sites in a 80 base pair intronic fragment. Also consistent with this hypothesis is the fact that How is expressed in the precursor cells of adult muscles that re-express twist. Early expression is not modified in either snail or tinman mutants. The expression is not affected in a mef2 mutant in which late differentiation stages of muscular cells is altered (Zaffran, 1997).

In order to test if how is transcriptionally regulated by ecdysone (see Ecdysone receptor), third larval instar tissues were isolated before the increase in ecdysone titer. They were cultured for 8 hours, and a physiologically high titer of ecdysone was added to cultures for varying periods of time. RNA was extracted from the tissues, electorphoresed, transferred to a membrane, and hybridized with a radiolabelled how probe. how is induced by ecdysone in cultured organs (Baehrecke, 1997).

Muscle-dependent maturation of tendon cells is induced by post-transcriptional regulation of stripeA

Terminal differentiation of single cells selected from a group of equivalent precursors may be random, or may be regulated by external signals. In the Drosophila embryo, maturation of a single tendon cell from a field of competent precursors is triggered by muscle-dependent signaling. The transcription factor Stripe induces both the precursor cell phenotype, as well as the terminal differentiation of muscle-bound tendons. The mechanism by which Stripe activates these distinct differentiation programs remained unclear. This study demonstrates that each differentiation state is associated with a distinct Stripe isoform and that the Stripe isoforms direct different transcriptional outputs. Importantly, the transition to the mature differentiation state is triggered post-transcriptionally by enhanced production of the stripeA splice variant, which is typical of the tendon mature state. This elevation is mediated by the RNA-binding protein How(S), with levels sensitive to muscle-dependent signals. In how mutant embryos the expression of StripeA is significantly reduced, while overexpression of How(S) enhances StripeA protein as well as mRNA levels in embryos. Analysis of the expression of a stripeA minigene in S-2 cells suggests that this elevation may be due to enhanced splicing of stripeA. Consistently, stripeA mRNA is specifically reduced in embryos mutant for the splicing factor Crooked neck (Crn), which physically interacts with How(S). Thus, a mechanism is generated by which tendon cell terminal differentiation is maintained and reinforced by the approaching muscle (Volohonsky, 2007).

This study demonstrates the involvement of post-transcriptional control in a cell-differentiation process that must be coupled to muscle-tendon interaction. Terminal differentiation of tendons involves a major reorganization of the microtubule and actin networks. Such processes are presumably not compatible with embryonic morphogenetic movements such as germ band retraction. Thus, it is essential to spatially and temporally restrict differentiation to single muscle-bound tendon cells. Indeed, the results show that premature overexpression of StripeA in the entire ectoderm leads to severe defects in germ band retraction (Volohonsky, 2007).

Stripe mediates both the determination of precursor cells as well as their maturation and ability to undergo specific temporal and spatial regulation. The current findings suggest both negative and positive feedback loops, based on post-transcriptional regulation of stripe splice variants that on one hand maintain non-bound tendon cells at the precursor state, and on the other hand enable irreversible differentiation of muscle-bound tendons (Volohonsky, 2007).

Whereas some tissue differentiation processes (e.g. tracheal development) initiate upon the expression of a key transcription factor, which autoregulates its own expression, thus leading to a unidirectional differentiation route, other cells (e.g. cells in the proneural region) go through an intermediate stage of a field of competent precursors, in which only additional local interactions lead to irreversible differentiation. Maturation of tendon cells follows the latter path, although the selection mechanism is based on regulation at the post-transcription level (Volohonsky, 2007).

The following model is used to explain the transition between the two phases of tendon cell development: the initial expression of stripeB is induced by segment polarity-dependent signals. StripeB defines a set of tendon precursor cells. StripeB then reinforces its own expression and in addition induces How(L) expression, which in turn suppresses stripeB mRNA levels, thus keeping StripeB levels constant throughout embryonic development. This is supported by experiments that show that StripeB overexpression leads to elevation in How(L) and in StripeB itself. Following myotube extension and adhesion to a tendon precursor cell, How(S) levels are elevated in the muscle-adherent tendon cells, presumably due to EGFR activation. How(S) associates with the splicing factor Crn and the complex shuttles into the nucleus, where it binds to stripeA intronic sequences and elevates its mRNA levels, by enhancing its splicing and maintaining the stability of the spliced mRNA. The resulting muscle-bound tendon cell expresses high StripeA levels, which further drive the expression of genes required for terminal tendon differentiation (e.g. shot, how), as inferred from StripeA overexpression experiments. This regulatory mechanism couples muscle binding and tendon cell maturation, while preventing differentiation of additional, non-bound, precursors (Volohonsky, 2007).

RNA-binding proteins can function as adaptor units promoting the assembly of large protein complexes that control the various aspects of RNA metabolism. How, together with Quaking and GLD-1, belongs to the Star family of RNA-binding proteins, the members of which often regulate more than one facet of RNA metabolism. For example, GLD-1 has been suggested to regulate mRNA stability as well as translation of some of its targets. Similarly Quaking controls mRNA stability as well as RNA splicing, and possibly also mRNA nuclear export and localization. It appears that How proteins also exhibit a wide range of activities on RNA metabolism. While the effect of How(L) and How(S) on stripe mRNA stability has been demonstrated previously, this study suggests that How(S) has an additional activity in regulating the splicing of stripeA. Consistent with this study, How has been identified in a dsRNA-based screen for alternative splicing regulators, as a protein required for specific splicing of exons within two out of five tested genes, paralytic (exons A/I), and Dscam (exon 4), in S-2 cells. Previous studies suggested that the ability of How proteins to stabilize stripe mRNA is mediated by the 3' UTR of stripe. However, the splicing of stripeA appears to be regulated by its specific intronic sequences (Volohonsky, 2007).

By contrast to How(L), which is localized specifically in the nucleus, How(S) is distributed both in the nucleus and the cytoplasm. However, when How(S) is retained in the nucleus by the addition of an NLS sequence, it loses its effect on the mRNA levels of its target. What could be the molecular explanation for the involvement of How(S) in splicing? It is suggested that How(S) binds to a cytoplasmic splicing factor and recruits it to the nucleus, where it is targeted to bind stripeA-specific intronic sequences. This may enhance the splicing of stripeA-specific exons. A candidate splicing factor is Crn. Crn is a general, well-conserved splicing factor that is expressed by a wide range of cell types and is distributed both in the nucleus and the cytoplasm. In a parallel study, it was demonstrated that crn and how mutants exhibit closely related phenotypes, affecting glial cell maturation. Importantly, both Crn and How(S) proteins [but not How(S)-NLS] coprecipitate from S-2 cell extracts, indicating that both proteins are associated in a common protein complex in the cytoplasm (Edenfeld, 2006). In addition, when Crn is myristoylated and transfected into S-2 cells together with How(S), How(S) is relocated to the membrane (Edenfeld, 2006). Furthermore, in crn mutants StripeA, but not StripeB, levels are reduced, and this is reflected in the reduction of Shot levels (Volohonsky, 2007).

These results support a model in which How(S) interacts with Crn in the cell cytoplasm, shuttles into the nucleus and facilitates stripeA splicing, and possibly mRNA stability, leading to StripeA protein elevation. A similar mechanism may operate in the Quaking-dependent facilitation of myelin-associated glycoprotein splicing (Volohonsky, 2007).

In summary, a molecular mechanism has been described that is based on post-transcriptional control, by which cell differentiation is induced and maintained by local interactions with neighboring cells (Volohonsky, 2007).

Dissection of the target specificity of the RNA-binding protein HOW reveals dpp mRNA as a novel HOW target

Regulation of RNA metabolism plays a major role in controlling gene expression during developmental processes. The Drosophila RNA-binding protein Held out wing (HOW), regulates an array of developmental processes in embryonic and adult growth. The primary sequence and secondary structural requirements for the HOW response element (HRE) has been characterized; this site (ACUAA) is necessary and sufficient for HOW binding. Based on this analysis, the Drosophila TGFß homolog, dpp, was identified as a novel direct target for HOW negative regulation in the wing imaginal disc. The binding of the repressor isoform HOW(L) to the dpp 3' untranslated region (UTR) leads to a reduction of GFP-dpp3'UTR reporter levels in S-2 cells, in an HRE site-dependent manner. Moreover, co-expression of HOW(L) in the wing imaginal disc with a dpp-GFP fusion construct led to a reduction in DPP-GFP levels in a dpp-3'UTR-dependent manner. Conversely, a reduction of the endogenous levels of HOW by targeted expression of HOW-specific double-stranded RNA led to a corresponding elevation in dpp mRNA level in the wing imaginal disc. Thus, by characterizing the RNA sequences that bind HOW, a novel aspect has been demonstrated of regulation, at the mRNA level, of Drosophila DPP (Israeli, 2007).

It has been shown that HOW binds directly to the 3'UTR of stripe. To characterize the HOW-binding sites further, the stripe 3'UTR 1.2 kb sequence was truncated into smaller fragments, which were individually transcribed in vitro and labeled with biotin. These fragments were tested for HOW binding by adding in vitro-translated HOW tagged with hemagglutinin (HA) to the biotin-labeled RNA followed by precipitation of the RNA complexes using avidin-conjugated magnetic beads. The presence of HOW on the beads was then tested by western blot analysis using anti-HA antibodies. As a control for non-specific binding, a mutant HOW variant (HOW^m) was used, that carries a mis-sense mutation in the KH domain exchanging arginine at position 185 to cysteine, mimicking the severe loss-of-function how^e44 allele. HOW^m does not exhibit RNA-binding activity. This analysis allowed selection of two HOW-binding fragments (a and b) in which the sequence ACUAA, which was similar, but not identical, to the GLD-1 hexanucleotide-binding site in tra-2, was identified. In fragment a, there are three repeats of this sequence, and fragment b contains one such sequence (Israeli, 2007).

It is concluded that the sequence ACUAA represents the primary HRE. Importantly, one of the HRE sequences (at position 766) is conserved in the 3'UTR of stripe in Drosophila pseudoobscura. Moreover, three repeats of the pentamer AAUAA (which also binds HOW, but to a lesser extent) were identied that are conserved between the two Drosophila species. Thus the HOW-binding site NA(C>A)UAA closely resembles that of STAR proteins from other species, although it is not identical. The binding of HOW was studied in the context of the entire stripe 3'UTR, and it was demonstrated that deletion of these four sites indeed abrogates the responsiveness of the stripe 3'UTR to HOW (Israeli, 2007).

Because a pentanucleotide sequence would be relatively abundant within the 3'UTRs of many RNAs, it was suspected that additional restrictions might exist in addition to the primary sequence ACUAA. Analysis of the distinct HOW-binding sites in the stripe 3'UTR using the Mfold program showed that high-affinity binding for HOW occurs when the binding site (ACUAA) is included within a single-stranded loop. However, secondary-structure predictions of large RNA fragments (larger than 30-40 nucleotides) using the Mfold program resulted in numerous alternatives. To test whether a loop secondary structure is essential for the binding of HOW, HRE-containing loops of distinct sizes were constructed, fused to the 3' end of the stripe 3'UTR fragment (1-225), which does not bind HOW. It was found that single-stranded loops that are larger than 12 nucleotides and contain a single HRE site exhibit significant binding, whereas loops smaller than 12 nucleotides did not exhibit specific binding to HOW. Presumably, these loops are too small to allow this binding (Israeli, 2007).

Structural studies helped identify a novel HOW target, namely dpp mRNA, in the wing imaginal disc. Normally, the repressor isoform of HOW, HOW(L), reduces dpp mRNA levels in the wing imaginal disc and in the pupal wing, leading to reduced DPP protein levels during the establishment of the anteroposterior axis, and later during wing vein formation. Presumably, in the absence of HOW(L), higher DPP levels at the source would alter the overall shape of the DPP gradient, thus expanding the Spalt expression domain. The phenotype of ectopic veins obtained by continuous expression of HOW(L) dsRNA in the pupal wings supports an additional role for HOW(L) in repressing dpp mRNA at later stages of wing development (Israeli, 2007).

The sensitivity of the embryo to DPP levels has been demonstrated by the DPP haplo-insufficient phenotype. This sensitivity is also exhibited in the wing imaginal disc by the observation that endogenous dpp can be replaced by UAS-GFP-dpp driven by dpp-gal4 only at low temperatures (16°C or 19°C), at which the Gal4 protein is significantly less active. Because the responsiveness of the cells to DPP levels is highly sensitive, it is necessary to tightly regulate the levels of DPP protein; for example, by constitutive reduction of its mRNA levels in DPP-secreting cells by the HOW(L) protein (Israeli, 2007).

Cell divisions in the Drosophila embryonic mesoderm are repressed via posttranscriptional regulation of string/cdc25 by HOW

Cell-cycle progression is tightly regulated during embryonic development. In the Drosophila early embryo, the levels of String/Cdc25 define the precise timing and sites of cell divisions. However, cell-cycle progression is arrested in the mesoderm of gastrulating embryos despite a positive transcriptional string/cdc25 activation provided by the mesoderm-specific action of Twist. Whereas String/Cdc25 is negatively regulated by Tribbles in the mesoderm at these embryonic stages, the factor(s) controlling string/cdc25 mRNA levels has yet to be elucidated. This study shows that the repressor isoform of the Drosophila RNA binding protein Held Out Wing [HOW(L)] is required to inhibit mesodermal cell division during gastrulation. Embryos mutant for how exhibit an excess of cell divisions, leading to delayed mesoderm invagination. The levels of the mitotic activator string/cdc25 mRNA in these embryos were significantly elevated. Protein-RNA precipitation experiments show that HOW(L) binds string/cdc25 mRNA. Overexpression of HOW(L) in Schneider cells reduces specifically the steady-state mRNA levels of a gfp reporter fused to string/cdc25 untranslated region (3'UTR). These results suggest that in wild-type embryos, string/cdc25 mRNA levels are downregulated by the repressor isoform HOW(L), which binds directly to string/cdc25 mRNA and regulates its degradation. Thus, this study proposes a novel posttranscriptional mechanism controlling cell-cycle progression in the Drosophila embryo (Nabel-Rosen, 2005; full text of article).

String/Cdc25 is a limiting factor that controls cell-cycle progression in early embryonic stages after cellularization. Both string/cdc25 mRNA and String/Cdc25 protein are extremely unstable (T_1/2 < 15 min). The instability of the mRNA and protein allows for a sensitive response of String/Cdc25 levels to transcriptional regulation by various transcription factors operating in pattern formation in the embryo. It has been reported that, in addition to the time of initiation of string/cdc25 transcription, accumulation of string/cdc25 mRNA is slower in mitotic domain 10 (MD10) than in MD2. This is consistent with lower mRNA levels detected in MD10 in relation to MD2 in wild-type embryos. This study provides a molecular basis for string/cdc25 mRNA instability. In situ hybridization with string antisense probe as well as RT-PCR experiments demonstrated that, in how mutant embryos, string/cdc25 is upregulated. Moreover, protein-RNA binding experiments showed a direct binding between HOW and string RNA, and in Schneider cells a gfp-string3'UTR reporter mRNA is specifically degraded in the presence of HOW(L). Collectively, these experiments are consistent with HOW(L) being the major factor responsible for string instability in the early embryo (Nabel-Rosen, 2005).

A consensus RNA binding site, (U>A/C/G)ACUAA, has been recently described for the binding of the STAR protein Gld-1. The same sequence has also been characterized in as being a consensus RNA binding site for HOW. Importantly, this sequence, GACUAA, is present in the string 3'UTR. These results are consistent with the idea that HOW binds the relaxed consensus sequence described for Gld-1, which is also present in string 3′UTR. Interestingly, this sequence appears also in the C. elegans cdc25/string homolog, suggesting that Gld-1 (similarly to HOW in Drosophila) may control cdc25/string in C. elegans (Nabel-Rosen, 2005).

The arrest of cell-cycle progression in the invaginating mesoderm must be transient because immediately after the invagination process the cells undergo a round of cell division. Thus, String/Cdc25 protein levels must be downregulated to a narrow time window to enable mesoderm invagination. This time frame may be achieved in the following manner: Twist, a regulator of mesoderm fate, activates the transcription of string/cdc25 and HOW. Maternal HOW, as well as zygotic HOW shown previously to be downstream of Twist, compromises string/cdc25 mRNA levels, and Tribbles (which requires Twist and Snail to perform its activity) reduces String/Cdc25 protein levels at this stage. Thus, in parallel to string/cdc25 transcriptional activation, Twist provides a double safe mechanism that silences string at the mRNA via HOW activity and String protein via Tribbles activity in MD10. The activity of both HOW and Tribbles should enable the eventual accumulation of String/Cdc25 protein at the end of the invagination process to allow cell-cycle progression at this stage. Therefore, both HOW's and Tribbles's inhibitory effect may not be highly efficient. This, together with the constitutive transcriptional activation of string by Twist, may lead to the eventual accumulation of string mRNA and protein levels, overcoming the negative control imposed by HOW and Tribbles. Alternatively, String accumulation may be caused by a more direct inhibition of both HOW and Tribbles activities, possibly by signaling pathways that operate in the mesoderm after its invagination (Nabel-Rosen, 2005).

Maternal HOW appears to reduce string mRNA levels in the lateral ectoderm in stage-5 and -6 embryos in addition to MD10. It is therefore possible that the extra cell divisions detected in these regions may have an additional indirect effect on mesoderm invagination (Nabel-Rosen, 2005).

It is instructive to ask whether HOW regulates additional processes during mesoderm development. Although zygotic how mutants do not exhibit mesoderm defects until late developmental stages, the how germline clone embryos do show significant mesoderm aberrations. The entire somatic muscle pattern of these embryos is severely disrupted, presumably owing to accumulation of defects. At this stage, it is impossible to distinguish between primary and secondary effects induced by the complete lack of HOW. The muscle defects detected in how germline clone embryos suggest that HOW has a broader function in the mesoderm and that it may regulate the levels of an array of essential genes necessary for appropriate mesoderm development. The identification of such genes should elucidate the full regulatory range of HOW activity (Nabel-Rosen, 2005).

Finally, regulation at the level of mRNA metabolism by STAR family proteins has been shown to occur in several developmental systems, for example, C. elegans gld-1 and mammalian quaking. These proteins exhibit a wide range of activities, affecting RNA splicing, mRNA nuclear export, mRNA stability, and possibly others. The advantage of such regulation is the ability to respond rapidly to external signals by controlling the mRNA levels of an array of target genes. The synchronization between muscle-cell differentiation and cell-cycle progression may be based on the activities of both HOW and Tribbles, but the molecular link between both processes has yet to be elucidated (Nabel-Rosen, 2005).

Post-transcriptional repression of the Drosophila midkine and pleiotrophin homolog miple by HOW is essential for correct mesoderm spreading

The even spreading of mesoderm cells in the Drosophila embryo is essential for its proper patterning by ectodermally derived signals. In how germline clone embryos, defects in mesoderm spreading lead to a partial loss of dorsal mesoderm derivatives. HOW is an RNA-binding protein that is thought to regulate diverse mRNA targets. To identify direct HOW targets, a series of selection methods were implemented on mRNAs whose levels were elevated in how germline clone embryos during the stage of mesoderm spreading. Four mRNAs were found to be specifically elevated in the mesoderm of how germline clone embryos and to exhibit specific binding to HOW via their 3' UTRs. Importantly, overexpression of three of these genes phenocopied the mesoderm-spreading phenotype of how germline clone embryos. Further analysis showed that overexpressing one of these genes, miple, a Drosophila midkine and pleiotrophin heparin-binding growth factor, in the mesoderm leads to abnormal scattered MAPK activation, a phenotype that might explain the abnormal mesoderm spreading. In addition, the number of EVE-positive cells, which are responsive to receptor tyrosine kinase (RTK) signaling, was increased following Miple overexpression in the mesoderm and appeared to be dependent on Heartless function. In summary, these analysis suggests that HOW downregulates the levels of a number of mRNA species in the mesoderm in order to enable proper mesoderm spreading during early embryogenesis (Toledano-Katchalski, 2007).

Previous analysis of how germline clones suggested that HOW is essential for correct mesoderm spreading over the ectoderm (Nabel-Rosen, 2005), a process that is required for the spatial patterning of the mesoderm layer by ectoderm-derived signals. This phenotype could not result from the earlier effect of extra cell division during gastrulation detected in the how germline clone embryos, because mesoderm invagination and gastrulation were eventually completed in these embryos. Furthermore, mesoderm spreading was unaffected in tribbles (trbl) mutant embryos, which exhibit a similar defect of extra cell divisions during gastrulation that leads to delayed and unsynchronized mesoderm invagination (Toledano-Katchalski, 2007).

Regulation of mesoderm-specific mRNA levels by HOW might contribute to the spatial and temporal control of gene expression during mesoderm spreading. The genome analysis was designed to identify mRNAs whose levels might be directly controlled by the repressor isoform HOW(L) in the mesoderm. Such targets should be normally repressed to enable even spreading of the mesoderm. Three out of the four HOW targets identified in this screen, namely falten, CG31638 and LAP (CG2520) are contributed maternally, and therefore HOW(L)-dependent repression in the mesoderm might be essential for reducing their levels in this tissue to enable proper mesoderm spreading. This scenario is supported by the defective mesoderm spreading induced by overexpression of falten and CG31638. Miple does not appear to be maternally contributed according to expression data and in situ analysis. It is not clear, however, which transcription factor is responsible for miple induction. Because miple mRNA was detected in mesoderm derivatives at stage 11, it might be induced by mesoderm-specific transcription factors such as Twist, MEF2 and/or Tinman, which are expressed in the mesoderm during spreading. In that case, to abrogate the effects of Miple, it would be necessary to block miple expression during mesoderm spreading. These data suggest that this is the role of HOW(L), because in its absence, miple mRNA is significantly elevated in the mesoderm. Thus, HOW(L) in the mesoderm of gastrulating embryos is necessary to reduce maternal mRNA expression and, in addition, to reduce the levels of gene products whose expression is not compatible with early mesoderm development, but might be required shortly after the process of mesoderm spreading has been completed. Thus, HOW(L) is essential to enable temporal morphogenetic processes in the mesoderm during its spreading over the ectoderm (Toledano-Katchalski, 2007).

Miple was further analyzed because its vertebrate homologs, midkine and pleiotropin, are involved in cell migration and are associated with receptor tyrosine kinase (RTK) signaling (Stoica, 2002). Therefore, its downregulation by HOW(L) might contribute to the restricted dorsal activation of the HTL-dependent signaling during mesoderm spreading. Moreover, the putative heparin-binding motif of Miple could affect the affinity of the HTL ligands to the HTL receptor, thereby modulating the strength of HTL-dependent signaling (Toledano-Katchalski, 2007).

Indeed, the findings suggest that downregulation of Miple levels in the mesoderm is essential for correct mesoderm spreading, because Miple overexpression leads to impaired mesoderm spreading. The disordered pattern of MAPK phosphorylation (detected by anti-dpERK antibody) observed following Miple overexpression might be the primary cause for the mesoderm spreading defect. In wild-type embryos, MAPK activation is detected only at the most dorsal cells of the spreading mesodermal cells. The mechanism by which this spatial MAPK activation is achieved is not clear. It has been suggested that MAPK activation takes place only in cells that directly contact the ectoderm. In that case, Miple might trigger prolonged mesoderm-ectoderm cell contacts and this could delay mesoderm spreading. Indirect evidence, especially the observation that overexpression of an activated form of HTL does not lead to an ectopic dpERK signal in the entire mesoderm, led to the suggestion that a constitutive inhibitory input of MAPK activation is present in mesoderm cells (Wilson, 2005). This inhibitory activity was suggested to be overcome only in cells that form close contact with the ectoderm. It is unlikely that the role of Miple is to counteract this inhibitory signal, because overexpression of Miple has an effect not only on MAPK activation in early mesoderm spreading but also on the late HTL-dependent signaling in the dorsal EVE-positive cells, in which this inhibitory signal has not been implicated. Therefore the possibility is favored that Miple enhances HTL signaling, and that this enhancement is reflected by MAPK activation in both early and later stages of mesoderm development (Toledano-Katchalski, 2007).

The elevation of the dpERK signal detected following overexpression of Miple might be mediated by HTL activation, because no other RTK has been shown to be expressed in the mesoderm at the stage of gastrulation. Although the increased number of EVE-expressing cells detected in the dorsal mesoderm clusters following overexpression of Miple is eliminated in embryos lacking active HTL receptor, the possibility cannot be excluded that the lack of EVE-positive cells in the dorsal mesoderm might stem from the failure of the htl mutant mesoderm cells to reach the most dorsal locations (Toledano-Katchalski, 2007).

In vertebrates, midkine and pleiotrophin have been identified by phage display as potential high-affinity ligands for the human receptor tyrosine kinase ALK (Stoica, 2002). Although the possible role of Miple in ALK-dependent signaling cannot be excluded, ALK is not expressed in the early stages of mesoderm spreading, and does not overlap with the EVE-expressing clusters; thus, it is unlikely to affect the increased number of EVE-expressing cells. Receptor tyrosine phosphatase-zeta has been implicated as a putative pleiotrophin receptor (Milev, 1998). If a similar receptor exists in Drosophila, it might respond to Miple overexpression by altering MAPK levels (Toledano-Katchalski, 2007).

It is possible that the heparin-binding domain of Miple enhances the activity of the HTL ligands. In vertebrates, heparin-containing proteins act as co-ligands to FGFs by inducing their dimerization. Miple is a heparin-binding protein, because it binds specifically to a heparin column. The contribution of heparan sulfate proteoglycans to proper mesoderm spreading in Drosophila had been demonstrated by the requirement of two enzymes, Sugarless and Sulfateless, for this process. Moreover, a genetic interaction between mutations in each of these enzymes and the two FGF receptors HTL and Breathless (BTL) was demonstrated. Overexpression of Miple during mesoderm spreading might, on the one hand, compete with endogenous heparan sulfate proteoglycan for Thisbe and Pyramus binding, and thus could inhibit their ability to activate the HTL-dependent signaling. In contrast, Miple might also activate the HTL pathway by replacing the endogenous heparan sulfate proteoglycan that is normally involved in activation of the FGF8-like ligands. These dual activities might interfere with the normal dorsal-restricted MAPK activation in the mesoderm (Toledano-Katchalski, 2007).

In wild-type embryos, miple is downregulated by HOW(L) in the mesoderm; however, its mRNA expression is detected at later developmental stages, including in the ventral midline and in the brain (Englund, 2006). In midline glial cells, a second FGF receptor, Breathless, has been implicated in the promotion of cell migration at stages 12-13 of embryonic development. At this stage, Miple might contribute to the spatial and temporal control of Breathless activation. Such a scenario must be tested directly in miple mutant embryos (Toledano-Katchalski, 2007).

Although the mesoderm spreading phenotype of how germline clone embryos is not fully penetrant and is detected in only a few segments, the contribution of HOW activity is crucial because of the secondary effect that non-homogenous mesoderm spreading exhibits on the development of the heart and dorsal somatic mesoderm. HOW(L) appears to function in the mesoderm as a safety mechanism to prevent mis-expression of either maternally contributed genes or genes whose early transcriptional activation in the mesoderm might interfere with the normal development of the mesoderm. An example of similar repressive activity of HOW(L) is its activity in the reduction of string levels (Nabel-Rosen, 2005) in the gastrulating embryo to prevent premature cell division during mesoderm invagination (Toledano-Katchalski, 2007).

In summary, this study reveals the crucial function of the STAR family member HOW(L) in enabling proper mesoderm development via the repression of specific mRNAs provided either maternally, or expressed prematurely in a specific tissue. HOW(L) and its vertebrate homolog, QKI5, are expressed in wide range of tissues during early developmental stages, and might function in these tissues in a similar fashion to enable proper embryonic and tissue development (Toledano-Katchalski, 2007).

Targets of Activity

The selective sensitivity of cells to programmed cell death (PCD) depends on the positive and negative death-inducing signals that converge into the apoptotic pathway. In Drosophila, the midline glial (MG) cells undergo selective death during development. This study shows that the long isoform of the RNA-binding protein Held Out Wing (HOW(L)) is essential for enhancing the sensitivity of the MG cells to PCD. In how mutant embryos, the number of MG cells was elevated. This phenotype could be rescued by midline expression of the HOW(L) repressor isoform. In how mutant embryos, the levels of the caspase inhibitor of apoptosis, Diap1 were elevated, in parallel to reduction in the levels of activated caspase. Similarly, reducing the levels of HOW in S2 cells led to elevation of Diap1, whereas over expression of HOW(L) promoted reduction of Diap1 protein as well as mRNA levels. Importantly, deletion of the two HOW binding sites from diap1 3'UTR abrogated HOW-dependent repression of Diap1, suggesting that HOW represses diap1 by binding to its 3'UTR. These results suggest that HOW(L) enhances the sensitivity of MG cells to apoptotic signals by reducing the levels of diap1 in these cells in, demonstrating a novel mode of regulation of PCD at the mRNA level (Reuveny, 2009).

The sensitivity of cells to apoptotic signals depends on the balance between the pro-apoptic and anti-apoptotic signals expressed within a cell in a given developmental context. MG cells represent a unique system in which to study apoptosis because only a small subset of the cells (2-3 out of 6) are doomed to die, and the death must be executed in a relative short period of time (around 3 h), during the migration of the AMG pair towards the next segment. HOW functions to enhance the sensitivity of these cells to the pro apoptotic signals by reducing the levels of the anti-apoptotic protein, Diap1 (Reuveny, 2009).

Regulation through pro apoptotic signals, e.g. the activities of Reaper, Grim and Hid (RGH) proteins, or the anti-apoptotic signal, by influencing the activity of Diap1, enables cells to respond to a wide array of signaling pathways. The convergence of these signals in a single cell determines not only whether the cell will undergo PCD, but also the timing during development at which this process will occur. In case of MG cells, the timing is critical, as the cells die prior to their arrival to the commissure, and thus do not receive the survival signal through MAPK activation (Reuveny, 2009).

RGH proteins were shown to affect the levels of Diap1 at several regulatory stages. Reaper and Hid affect Diap1 levels via ubiquitination and proteosomal degradation. In addition, Morgue has been shown to promote Diap1 degradation whereas Reaper was shown to also inhibit translation of diap1. Despite the expression of Hid and Reaper in MG cells, only partial PCD is induced in these cells, possibly due to high levels of Diap1 (Reuveny, 2009).

Previous data has suggested that HOW(L) mediates developmental processes in other tissues (e.g., mesoderm, tendon cells), by a temporal reduction of the levels of key regulatory proteins. For example, in gastrulating embryos, HOW(L) reduces the mRNA levels of string/cdc25 to arrest cell division during mesoderm invagination, and at a later stage, HOW(L) reduces the levels of miple1 to allow mesoderm spreading. Similarly, this study shows that HOW contributes to the timing of MG cell apoptosis by reducing the levels of Diap1, thereby sensitizing these cells to pro-apoptotic signals. An effect of HOW on cell division through regulation of String is not favored by this study, since HOW is detected in the midline cells only at stage 12-13 at which the MG cells do not divide anymore (Reuveny, 2009).

Several lines of evidence support the idea that HOW(L) might affect MG cell apoptosis through its repression of Diap1 levels. First, it was shown by antibody staining that Diap1 levels are elevated in how mutants. Second, reducing HOW levels by introducing HOW-specific dsRNA or elevating HOW(L) levels in S2 cells leads to corresponding opposing effects on Diap1 protein expression, elevation of Diap1 when HOW is reduced and reduction of Diap1 when HOW(L) is elevated. The diap1 3' UTR contains two binding sites for HOW, and is capable of binding to HOW(L) in vitro. Nevertheless, a corresponding elevation of diap1 mRNA could not be detected in the S2 cells in which HOW was knocked down by dsRNA, possibly due to a continuous positive transcriptional input of diap1 in these cells. However, a reduction of diap1 mRNA and protein levels was induced, following over expression of HOW(L) in S2 cells and in embryos. Also, whether the splicing pattern of diap1 was altered in S2 cells depleted of HOW was examined, since HOW was demonstrated to mediate alternative splicing in other tissues. To this end, an RT-PCR was performed with primers specific for each of the three diap1 splice variants; however, no change in the pattern of diap1 splicing was observed (Reuveny, 2009).

Thus, HOW(L) might affect Diap1 protein levels by repressing both its mRNA levels as well as its translation. Alternatively it could affect Diap1 indirectly by influencing the levels of an upstream regulator of Diap1. The results support a direct effect of HOW through its association with the HOW-binding sites in diap1 3' UTR, since deletion of these sites abrogated the reduction of Diap1 detected in the presence of HOW(L) (Reuveny, 2009).

Gld-1 the C. elegans orthologue of HOW affects both translation and stability of its target mRNAs, apparently by affecting the length of the polyA tail of the target mRNA. HOW(L) might act in a similar fashion on diap1 mRNA (Reuveny, 2009).

Whereas HOW(L) does not induce apoptosis in other tissues, where it is highly expressed (e.g. mesoderm, tendon cells etc.), it was shown to have pro apoptotic effects in MG cells and in the adult fly eye. It is suspected that in these tissues, a delicate balance between the levels of the pro apoptotic and anti apoptotic proteins is maintained, so that the cells become highly sensitive to Diap1 levels, and thus are responsive to reduced or elevated levels of HOW(L) (Reuveny, 2009).

Interestingly, one isoform of the mammalian orthologue of HOW, Quaking7 (QKI-7), has been shown to induce apoptosis of fibroblasts and primary rat oligodendrocytes. The molecular mechanism of QKI-7-induced apoptotic activity has yet to be elucidated, but the unique C' terminal tail of QKI-7 appears to be necessary for this apoptotic activity. In contrast, C. elegans GLD-1 was shown to repress the levels of cep1 an orthologue of mammalian P53. In that system, GLD-1 exhibits an anti apoptotic effect. It appears therefore, that STAR proteins are not dedicated to a defined direction of apoptotic regulation. Rather, their basic activity is to elevate or reduce the levels of critical components in the process to enable the execution of PCD or to allow other developmental process to occur (Reuveny, 2009).

Previous studies demonstrated that how transcription is induced in response to the activation of the Ecdyson (Ecd) pathway however, the biological significance of this induction was not clear. During larval stages, a high titer of Ecd acts through the ecdyson receptor EcR/Ultraspiracle nuclear receptor heterodimer to signal puparium formation and destruction of several larval tissues including the midgut and salivary glands. The Ecd pathway triggers a transcriptional cascade that culminates in rpr and hid induction to initiate tissue destruction. Interestingly, the Ecd pathway induces parallel repression of diap1 via the activity of the CREB binding protein, CBP. CBP is both necessary and sufficient to down-regulate Diap1, providing the cells with the competence to die. Whereas the contribution of CBP to MG cell apoptosis has yet to be elucidated, it is possible that in this system, in parallel to the induction of CBP transcription, the Ecd pathway triggers HOW(L) transcription to enhance Diap1 destruction, possibly due to a need to induce rapid death of the MG cells (Reuveny, 2009).

To address whether HOW(L) is sufficient to rescue the excess in MG cells in disembodied (dib) mutant embryos, defective in ecdysone biosynthetic, HOW(L) was overexpressed in dib mutant embryos that carried the MG-specific enhancer trap, AA142, using the sim-gal driver. Embryos over expressing HOW(L) in dib mutant embryos still maintained a high number of MG cells, suggesting that HOW(L) is not sufficient to rescue the dib mutant phenotype. Importantly, the levels of Diap1 in MG cells in these embryos were significantly reduced (Reuveny, 2009).

The inability of HOW(L) to reduce the number of MG cells following its over expression in the dib mutant embryos might be explained by the involvement of the Ecd pathway not only in PCD but also in repression of MG cell division in an earlier developmental stage. Also, since the Ecdysone pathway positively regulates Hid, it is possible that the MG cells did not contain enough pro-apoptotic signals to induce PCD, and therefore it is not surprising that HOW(L) did not provide rescue of the MG cell number (Reuveny, 2009).

In summary, this study have identified the KH-domain RNA-binding protein, HOW, as a novel regulator of PCD in MG cells, likely acting as a regulator of Diap1 translation and/or stability. It is proposed that HOW provides the MG cells with enhanced sensitivity to the pro apoptotic effects of Hid and Reaper, triggering the rapid apoptosis of MG cells during their migration (Reuveny, 2009).

The regulation of glial-specific splicing of Neurexin IV requires HOW and Cdk12 activity

The differentiation of the blood-brain barrier (BBB) is an essential process in the development of a complex nervous system and depends on alternative splicing. In the fly BBB, glial cells establish intensive septate junctions that require the cell-adhesion molecule Neurexin IV. Alternative splicing generates two different Neurexin IV isoforms: Neurexin IV^exon3, which is found in cells that form septate junctions, and Neurexin IV^exon4, which is found in neurons that form no septate junctions. This study shows that the formation of the BBB depends on the RNA-binding protein HOW (Held out wings), which triggers glial specific splicing of Neurexin IV^exon3. Using a set of splice reporters, it was shown that one HOW-binding site is needed to include one of the two mutually exclusive exons 3 and 4, whereas binding at the three further motifs is needed to exclude exon 4. The differential splicing is controlled by nuclear access of HOW and can be induced in neurons following expression of nuclear HOW. Using a novel in vivo two-color splicing detector, a screened was carried out for genes required for full HOW activity. This approach identified Cyclin-dependent kinase 12 (Cdk12) and the splicesosomal component Prp40 as major determinants in regulating HOW-dependent splicing of Neurexin IV. Thus, in addition to the control of nuclear localization of HOW, the phosphorylation of the C-terminal domain of the RNA polymerase II by Cdk12 provides an elegant mechanism in regulating timed splicing of newly synthesized mRNA molecules (Rodrigues, 2012).

Cdk12 protein is a nuclear localized kinase that phosphorylates the C-terminal domain (CTD) of the RNA polymerase II during transcriptional elongation (Bartkowiak, 2010). The phosphorylated CTD is bound by Prp40, a subunit of the U1 snRNP. Prp40, in turn, has been shown to interact with the HOW-binding protein Crn/Clf1. Thus, Cdk12 is in a position to facilitate splicing of pre-mRNAs that have bound the HOW protein (Rodrigues, 2012).

Differential splicing is a key element in generating the amazing complexity of higher nervous systems. Through relatively few regulatory elements, a single gene can generate several different isoforms with potential distinct cellular functions. In Drosophila, differential splicing is required for the correct glial development. This study has dissected the role of the STAR-family member HOW in controlling such a differential splicing event at the Nrx-IV locus, which is pivotal for the generation of the BBB (Rodrigues, 2012).

Nrx-IV exons 3 and 4 are spliced in a mutually exclusive manner. They share DNA sequence identity of 60% and encode related Discoidin domains, which provide distinct adhesive properties. Within glial cells, expression of Nrx-IV^exon3 predominates and participates in the formation of septate junctions. Interestingly, the binding partner of Nrx-IV at the Drosophila septate junctions, Neuroglian, or the Caspr-binding partner at the septate-like junctions in vertebrates, Neurofascin, are also subject to cell-type specific, differential splicing (Rodrigues, 2012).

Differential splicing appears to be of more general relevance during the formation of septate junctions. The fly homologue of the membrane-skeleton protein 4.1, Coracle, binds to Nrx-IV and mediates the linkage of the septate junctions to the cytoskeleton. Differential splicing of coracle generates at least four different splice variants that encode four distinct proteins. RT-PCR experiments indicate that the Coracle-PB isoform is generated in a HOW-dependent manner (Rodrigues, 2012).

STAR proteins, like HOW, bind sequence motifs in the pre-mRNA of their targets. Following site-specific mutation of all HOW response elements (HREs), it was shown that HRE1 may be needed for general exon definition. The mutation of this sequence motif leads to increased exon skipping of both exon 3 and exon 4, suggesting a crucial role for HRE1 in general splicing, possibly affecting the branch point of this intron. The HRE2, HRE3 and HRE4 elements influence mutually exclusive splicing. Upon mutation of these motifs, both exons are left in the mRNA more frequently, which suggests their function in exon selection. Such an effect was not observed in neurons. Thus, these HREs seem to play a role in exon selection (Rodrigues, 2012).

The HOW isoforms share an identical KH RNA-binding domain. HOW(S) predominantly localizes to the cytoplasm and HOW(L) is found mostly in the nucleus of glial cells. This study showed that nuclear HOW is sufficient for the induction of glial-specific splicing in neurons. Interestingly, both HOW isoforms can partially rescue the how mutant phenotype. HOW(S) appears to have higher rescuing abilities. As both transgenes are inserted in the same chromosomal landing site, resulting in identical expression levels, it is assumed that HOW(S) must be efficiently transported into the nucleus to promote Nrx-IV splicing. Because, following overexpression of the HOW(S), most of the protein stays in the cytoplasm, the shuttle mechanism(s) directing HOW(S) into the nucleus must be very tightly regulated. Possibly, HOW(S) has better rescuing abilities as HOW(S), but not HOW(L), can facilitate the nuclear import of the splice factor Crn (Rodrigues, 2012).

STAR family proteins are phosphorylated on several residues. In the past, it has been established that the HOW homolog Sam68 is phosphorylated by MAPK the regulation of which is controlled by Raf. Indeed, expression of a dominant-negative Raf protein in glia shifted the splicing pattern towards the neuronal form, suggesting a role for receptor tyrosine kinase signaling for glial differentiation as it has been demonstrated at several other instances (Rodrigues, 2012).

In addition, it is noted that silencing of Cdk12 resulted in a shift of the splicing pattern towards the neuronal form. Cdk12 is a broadly expressed serine/threonine kinase that also contains stretches of arginine- and serine-rich sequences (SR domains) known to be present in RNA-processing proteins, which regulate splicing, nuclear export and stability of the mRNA. Drosophila Cdk12 is associated with the C-terminal domain (CTD) of the RNA polymerase II (RNAPII) and phosphorylates Ser2 (Bartkowiak, 2010). The CTD of RNAPII acts as an assembly platform that controls transcription and pre-mRNA processing. Phosphorylated CTD in turn is recognized by Prp40, which belongs to the U1 snRNP. Moreover, a direct interaction between PrP40 and Crocked neck like factor 1 (Clf1), which binds HOW, has been demonstrated. Thus, phosphorylation of CTD by Cdk12 (Bartkowiak, 2010) recruits the assembly of the spliceosome at specific pre-mRNA targets defined by binding of HOW. In line with this model, it is noted that silencing of Prp40 also alters Nrx-IV splicing (Rodrigues, 2012).

In Drosophila, Cdk12 associates with Cyclin K (Bartkowiak, 2010), which is required for its catalytic activity. The activity of cyclins can be regulated by RTK signaling and thus might present a link that connects the Raf/MAPK pathway with a direct control of splicing activity. Additionally, CTD phosphorylation could be linked to MAPK activity in former studies. Cdk12 is expressed in the nucleus of almost all cells. To further decipher the role of Cdk12 during splicing, a loss-of-function allele was used. Homozygous mutant animals are lethal at the beginning of larval development. However, these mutants show no splicing defects, most probably owing to strong maternal contributions (Rodrigues, 2012).

The formation of the BBB implies the maturation of septate junctions only in fully differentiated subperineurial glial cells. Thus, the timing of splicing of pre-mRNAs encoding septate junction proteins is crucial and most likely regulated by two independent signaling cascades. It is proposed that the mRNA-binding protein HOW integrates these signaling events and is key in determining cellular differentiation (Rodrigues, 2012).

Protein Interactions

The splicing factor crooked neck associates with the RNA-binding protein HOW to control glial cell maturation in Drosophila

In both vertebrates and invertebrates, glial cells wrap axonal processes to ensure electrical conductance. Crooked neck (Crn), the Drosophila homolog of the yeast Clf1p splicing factor, directs peripheral glial cell maturation. crooked neck is expressed and required in glial cells to control migration and axonal wrapping. Within the cytoplasm, Crn interacts with the RNA-binding protein HOW and then translocates to the nucleus where the Crn/HOW complex controls glial differentiation by facilitating splicing of specific target genes. By using a GFP-exon trap approach, some of the in vivo target genes were identified that encode proteins localized in autocellular septate junctions. Thus, glial cell differentiation is controlled by a cytoplasmic assembly of splicing components, which upon translocation to the nucleus promote the splicing of genes involved in the assembly of cellular junctions (Edenfeld, 2006).

Most glial cells of the Drosophila PNS are born in the CNS and migrate toward their final destination. Subsequently, the cell body follows to then initiate wrapping of the axons. crn and how are both required for the initiation of axonal wrapping. The proteins encoded by crn and how are involved in the regulation of splicing of components of the septate junctions that are required for glial cell differentiation. Crn and HOW(S) interact in the cytosol to control their nuclear import, providing a simple mechanism to couple glial and neuronal cell differentiation (Edenfeld, 2006).

crn encodes an unusual TPR-containing protein whose function is essential for embryonic development. The Crn protein is found in the cytosol and in nuclear “speckles” (Raisin-Tani, 2002). Previous genetic and biochemical evidence has already suggested that Crn and its homologs participate in the assembly and the control of the splicing machinery. A mutation of the yeast crooked neck ortholog results in the accumulation of unspliced pre-mRNAs and, furthermore, Crn-like proteins are needed for pre-mRNA splicing in vitro. Crn is found in two functional complexes with and without snRNA and via its N-terminal TRPs helps to assemble the intact spliceosome (Wang, 2003). Within the spliceosome, the Crn homolog assists in the initial spliceosome assembly and also binds the phospho-CTD of the RNA polymerase II (Gasch, 2005). In Drosophila crn mutants, changes were observed in the splicing pattern suggesting that Crn modulates splicing preferences during alternative splicing (Edenfeld, 2006).

Alternative splicing employs differential use of 5' or 3' splice sites and has evolved as an efficient way to achieve a functional diversification and regulation of gene products. The basic splicing mechanism first requires the correct choice of 5' and 3' splice junctions and subsequently the assembly of the spliceosome. While Crn can facilitate spliceosome assembly, it does not directly participate in the selection of specific splice junctions, since the Crn protein is not able to bind to RNA. However, in Drosophila, Crn does regulate alternative splicing of few specific target genes, implying the existence of interaction partners that direct the Crn protein to these target RNAs (Burnette, 1999; Park, 2004; Edenfeld, 2006 and references therein).

This study has identified the HOW(S) protein as such an interaction partner that is likely able to recruit Crn to specific splicing targets. The HOW proteins contain an hnRNP K homology (KH) motif and exhibit specific RNA-binding activities. The KH motif is found in the GSG domain (GRP33, Sam68, GLD) shared by the Signal Transduction and Activation of RNA (STAR) family of proteins. The how locus is genetically complex: it encodes two antagonizing splice variants, HOW(S) and HOW(L), the functions of which were thoroughly analyzed in tendon cells. This study shows that HOW(L) is involved in the instability of stripe mRNAs, whereas HOW(S) is involved in mRNA stability as well as in the control of stripe A splicing (Volohonsky, 2007). Since HOW(S) associates with Crn, a direct influence on splicing can be anticipated (Edenfeld, 2006).

The proposed cytoplasmic-nuclear shuttling of a Crn/HOW(S) complex furthermore allows the linking of extracellular signals to a direct control of splicing. Concerning glial cell differentiation, this suggests that wrapping of axonal fascicles is not only dependent on a transcriptional control. It is rather likely that neuronal signals help to efficiently couple glial and neuronal differentiation by directly influencing the splicing pattern. The nature of such a signal is still elusive, however. As described for the HOW-related protein Sam68, phosphorylation may be important to control the interaction of Crn and HOW in the cytoplasm and thus the transport of the complex into the nucleus. In this respect, it is interesting to note that the development of oenocytes, which is impaired in both crn and how mutants, requires EGF-receptor signaling and that neuronal EGF-receptor signaling has been shown to regulate glial expression of neuroglian (Edenfeld, 2006).

In agreement with such a model is the finding that the how mutant phenotype resembles the phenotype caused by the loss of crn. Furthermore, Crn is able to bind only cytosolic HOW(S). If HOW(L) expression is forced to the cytosol, it can also bind to Crn, confirming that the interaction of Crn and HOW occurs in the cytosol. The assembly of the Crn/HOW complex is crucial to precisely regulate the nuclear concentration of these splice factors, which in turn is relevant for alternative splicing. Within the nucleus, HOW(S) binds to a consensus sequence with a length of only five nucleotides, making the quest for specific target genes difficult. To nevertheless get insight into this important functional aspect, a collection of strains was utilized in which endogenous genes were tagged by the insertion of a GFP-exon. In this study two components of the septate junctions were identified that form important autocellular junctions needed to stabilize glial cell morphology as candidate targets for Crn. Glial septate junctions are morphologically established by the end of embryogenesis. In line with the notion that neurexinIV is a target gene of crn, no septate junctions were detected in crn mutant glial cells. Furthermore, dye-penetration experiments show that the blood-brain barrier, which crucially depends on the presence of septate junctions, is not established in crn mutants (Edenfeld, 2006).

In addition to regulating splicing, Crn and HOW proteins may also have additional functions. For example, members of the hnRNP-A/B family of RNA-binding proteins are able to regulate alternative splicing of the Drosophila P element transposase and the Ubx gene. However, the function of the hnRNP-A/B family member Hrp48 is not restricted to the control of RNA splicing since it is also involved in the control of oskar mRNA localization in the Drosophila oocyte. Such a dual specificity of the Hrp48 RNA-binding protein in regulating RNA splicing and RNA transport has also been suggested for the Crn-binding partner HOW. In addition, it was recently demonstrated that the yeast Crn homolog also affects DNA replication, and first phenotypic analyses of the Drosophila crn mutant led to the proposal that Crn might play a role in regulation of cell divisions (K. Zhang, 1991). However, no abnormal cell number was observed for the peripheral glial cells, suggesting that at least during glial development crn has no function during the cell cycle (Edenfeld, 2006).

The data lead to a model underlying glial cell differentiation that may not only be applicable for Drosophila. The vertebrate homolog of how is the quaking gene, which is also required for glial differentiation. quaking viable mutants initially develop normally but then show tremors due to severe myelination defects. The mutant phenotype is caused by a deletion in the promoter region of the quaking gene that encodes several alternatively spliced mRNAs. The quaking viable deletion abrogates the expression of QKI-6 and QKI-7 in myelinating cells of the brain. The complete loss of quaking transcripts results in early lethality. quaking and how mutants not only share a defect in axonal wrapping. Moreover, the corresponding gene products appear to have different functions in the nucleus and the cytosol. Whereas QKI-5 is strictly nuclear, QKI-6 and QKI-7 are able to shuttle between the cytosol and the nucleus as it has been observed for the HOW(S) protein. The position of putative QKI binding sites close to tissue-regulated exons was found to be conserved in mice and is similar to what was found for the putative HOW binding sites in neurexinIV. Thus, although invertebrates and vertebrates have long been thought to follow very different routes toward glial differentiation, the underlying molecular control of glial wrapping may be conserved (Edenfeld, 2006).

Phosphorylation of the Drosophila melanogaster RNA-binding protein HOW by MAPK/ERK enhances its dimerization and activity

Drosophila melanogaster Held Out Wings (HOW) is a conserved RNA-binding protein (RBP) belonging to the STAR family, whose closest mammalian ortholog Quaking (QKI) has been implicated in embryonic development and nervous system myelination. The HOW RBP modulates a variety of developmental processes by controlling mRNA levels and the splicing profile of multiple key regulatory genes; however, mechanisms regulating its activity in tissues have yet to be elucidated. This study links receptor tyrosine kinase (RTK) signaling to the regulation of QKI subfamily of STAR proteins, by showing that HOW undergoes phosphorylation by MAPK/ERK. Importantly, that this modification facilitates HOW dimerization and potentiates its ability to bind RNA and regulate its levels. Employing an antibody that specifically recognizes phosphorylated HOW, this study shows that HOW is phosphorylated in embryonic muscles and heart cardioblasts in vivo, thus documenting for the first time Serine/Threonine (Ser/Thr) phosphorylation of a STAR protein in the context of an intact organism. The sallimus/D-titin (sls) gene was identified as a novel muscle target of HOW-mediated negative regulation and further show that this regulation is phosphorylation-dependent, underscoring the physiological relevance of this modification. Importantly, it was demonstrated that HOW Thr phosphorylation is reduced following muscle-specific knock down of Drosophila MAPK rolled and that, correspondingly, Sls is elevated in these muscles, similarly to the HOW RNAi effect. Taken together, these results provide a coherent mechanism of differential HOW activation; MAPK/ERK-dependent phosphorylation of HOW promotes the formation of HOW dimers and thus enhances its activity in controlling mRNA levels of key muscle-specific genes. Hence, these findings bridge between MAPK/ERK signaling and RNA regulation in developing muscles (Nir, 2012).

DEVELOPMENTAL BIOLOGY

Embryonic

In spite of the strong maternal expression of the 3.6 kb transcript, no protein is detected before the onset of gastrulation, suggesting that the maternally supplied mRNA is either not translated or that the translated protein is highly unstable (Zaffran, 1997).

how mRNA is first detected in mesodermal precursors on the ventral side of the embryo at the onset of gastrulation. how transcription continues to be detected in presumptive mesoderm cells at the germ band extension stage. As germ band retraction occurs, how transcription is detected in the cells destined to form somatic and visceral muscle cells. Expression is also detected in the pharyngeal muscles at the anterior end of the embryo. how transcription is detected in cardiac precursors and muscle attachment cells of the epidermis as dorsal closure occurs. Late during embyogenesis, how transcription is restricted to the heart and muscle attachment sites of the epidermis (Baehrecke, 1997).

When the different mesodermal lineages segregate, the expression of How becomes restricted to the myogenic lineage, including the cardioblasts and probably all the myoblasts. Antibodies directed against the protein demonstrate that How is localized to the nucleus (Zaffran, 1997).

Genetic control of cell morphogenesis during Drosophila melanogaster cardiac tube formation

Tubulogenesis is an essential component of organ development, yet the underlying cellular mechanisms are poorly understood. This study analyzed the formation of the Drosophila cardiac lumen that arises from the migration and subsequent coalescence of bilateral rows of cardioblasts. This study of cell behavior using three-dimensional and time-lapse imaging and the distribution of cell polarity markers reveals a new mechanism of tubulogenesis in which repulsion of prepatterned luminal domains with basal membrane properties and cell shape remodeling constitute the main driving forces. Furthermore, a genetic pathway is identified in which roundabout, slit, held out wings, and dystroglycan control cardiac lumen formation by establishing nonadherent luminal membranes and regulating cell shape changes. From these data a model is proposed for Drosophila cardiac lumen formation, which differs, both at a cellular and molecular level, from current models of epithelial tubulogenesis. It is suggested that this new example of tube formation may be helpful in studying vertebrate heart tube formation and primary vasculogenesis (Medioni, 2008).

The analysis provided here establishes the cellular basis of lumen formation of the Drosophila cardiac tube. The lumen of the tube is formed from the migration of two bilateral rows of polarized cardioblasts (CBs), which join at the dorsal midline. One main result of this study is the characterization of two types of cell membrane domains directly involved in lumen formation, the luminal domains (L domains) and adherent domains (J domains). Adherens junctions that are responsible for sealing the tube originate from the J domain, whereas the membrane walls of the lumen originate from the L domain (Medioni, 2008).

Remarkably, the L domain displays characteristics of basal membranes, revealed by expression of molecular markers normally associated with a basal membrane. Furthermore, specification of the L and J domains takes place very early in the tubulogenesis process, significantly before coalescence of the bilateral rows of CBs at the dorsal midline. Finally, during CB migration, membrane domains undergo remodeling, concomitant with profound cell shape changes. These two cellular processes appear to be closely connected and are probably regulated by the cellular environment of the CBs composed by the overlying dorsal ectoderm and the amnioserosa cells. These interactions will be investigated in a future work (Medioni, 2008).

The mechanism of Drosophila cardiac lumen formation reported in this study is thus notably different from the previously described mechanisms of epithelial tubulogenesis. In epithelial tubulogenesis, after receiving a polarization signal that sets apicobasal polarity, the cells or group of cells establish a basal surface and generate vesicles carrying apical membrane proteins. The vesicles are targeted to the prospective apical region, where they fuse with the existing membrane or with each other to form a lumen. Finally, continued vesicle fusion and apical secretion expand the lumen (Medioni, 2008).

In contrast, constriction of the leading edge domain during cardioblast (CB) migration, precise control of cell shape changes, and delimitation of specific membrane domains appear to be the driving forces of Drosophila cardiac lumen formation. Cells forming the dorsal vessel have the features of migrating cells. In contrast to epithelial tubulogenesis, which involves apical membrane domains, the apex of polarized CBs constricts, forms adherens junctions, and consequently does not constitute the L domain. Instead, the luminal membrane domain possesses basal membrane characteristics, as is also the case in endothelial cells. Moreover, the size of the cardiac lumen is determined by the isotropic growth of CBs, and not, as in other models, by anisotropic extension of the L domain involving apical membrane vesicles.

Finally, the genetic control of the process involves gene products of slit, robo, how, and dg, which are not known regulators of lumen formation in epithelial tubes (Medioni, 2008).

This study leads to the identification of a genetic pathway, including slit, robo, how, and dg, controlling membrane domain specification and dynamics during cardiac lumen formation. Within this pathway, Slit appears to play a central role and a previously unrecognized function in cell morphogenesis (Medioni, 2008).

Several studies have shown that Slit-Robo function is essential for cardiac tube formation by controlling the proper migration, cohesion, and alignment of the two rows of CBs. The results reported in this study show that Slit is also involved in the correct specification of the L domain and its distinct features with respect to the adjacent J domains. Activation of Slit-Robo signaling determines the respective size of these two types of domains (Medioni, 2008).

The data suggest that activation of this pathway inhibits the formation of adherens junctions. This possibility is supported by recent findings in chick retina cells, where activation of the Slit-Robo pathway leads to the inactivation of β-catenin (Arm in Drosphila), resulting in the dissociation of N-cadherin from the junctional complex and preventing the formation of adherens junctions. Consistent with these observations, DE-Cad (Shg) is expressed in the J domains of CBs and is required for cardiac tube morphogenesis. Moreover, slit and shg show genetic interaction in cardiac tube morphogenesis. In the absence of slit function, the size of the L domain is strongly reduced, suggesting that Slit-Robo signaling prevents the formation of Arm/DE-Cad-mediated adherens junctions in the L domain (Medioni, 2008). How encodes an RNA-binding protein involved in mRNA metabolism, and given its exclusive nuclear localization at this stage of development, How may regulate slit splicing. In the absence of the How protein, the gene splicing could be affected, producing a Slit protein unable to correctly localize at the L domain. This hypothesis is consistent with the fact that expression of wild-type Slit in CBs can suppress the effect of how18 mutation on Slit localization and lumen formation. How has also recently been shown to regulate the splicing of neuronal membrane proteins such as neurexin. Moreover, How is expressed in the midline glia with Slit and Dg, suggesting that interaction among these three genes is part of a general mechanism by which junctions and lumen formation are controlled (Medioni, 2008).

A model is preposed for the genetic control of lumen formation in the cardiac tube. According to this model, How could directly regulate Slit by controlling its splicing and targeting the luminal compartment. Consequently, Slit binds to Robo activating the signaling pathway, which in turn inhibits Arm/DE-Cad-mediated adherens junction formation in the luminal compartment, leading then to the specification of distinct J and L domains. Parallel to this, activation of Slit-Robo signaling modulates the actin cytoskeleton and triggers CB cell shape remodeling required for lumen formation and growth. As How is able to act on many targets, it could also directly control the actin cytoskeleton by targeting an actin-binding molecule. Concerning Dg, it was shown that dg and slit genetically interact; however, overexpression of Slit does not rescue the lumen phenotype observed in dg mutants, contrasting with how mutations. Thus, it is proposes that Dg could regulate Slit localization at the L domain by its function in the specification and differentiation of the L domain, and therefore acts parallel to slit for lumen formation, behaving, for example, as a coreceptor of Robo. In addition, Dg could control actin cytoskeleton dynamics via its interaction with Dystrophin (Medioni, 2008).

The data clearly show that cardiac tube formation in Drosophila differs substantially from all other described mechanisms of tubulogenesis. Is this mechanism of tubulogenesis unique or is it shared with other organs and/or other organisms? Primary vasculogenesis in vertebrates leads to the formation of large median vessels, the dorsal aorta and the cardinal vein. These vessels arise from migrating mesenchymal cells of the lateral mesoderm, termed angioblasts, that are organized in bilateral groups of cells. Angioblasts migrate toward the midline as a cohort of cells, coalesce, and form a lumen. At this stage, as in flies, cells around the lumen show a crescentlike shape and an extracellular matrix is deposited at the internal face of luminal membranes. Similar cellular events are also observed during the formation of the primitive cardiac tube in vertebrates, suggesting that a common mechanism of tubulogenesis might exist for all tubes that arise from the coalescence of migrating bilateral mesenchymal cells (Medioni, 2008).

The Drosophila cardiac tube, or dorsal vessel, shares many similarities with the cardiovascular system of vertebrates. A significant fraction of genes expressed in the Drosophila cardiac tube are also annotated to be expressed in vertebrate blood vessels, suggesting that vasculogenesis and dorsal vessel morphogenesis might share common genetic regulators (Medioni, 2008).

Finally, components of the genetic pathway controlling cardiac lumen formation that are described in this study have potentially similar functions in vertebrates. It has been shown that numerous proteins involved in axon guidance are expressed in vertebrate blood vessels. In particular, the Slit-Robo signaling pathway has been involved in promoting tumor vascularization, hSlit2 being expressed in tumor cells and hRobo1 in endothelial cells. Moreover, mSlit3 has been implicated in mammalian cardiogenesis, and Quaking, the mouse homologue of How, is required for vasculogenesis and expressed in the developing heart (Medioni, 2008).

In conclusion, analysis of CB morphogenesis during development of the Drosophila cardiovascular system provides evidence for a new model of biological tube formation. It is proposed that this mechanism might also be used for the formation of the large median vessels and primitive heart tube in vertebrates (Medioni, 2008).

Larval

how transcription increases at metamorphosis. Similar to the pattern of expression during embryogenesis, how transcription is detected in muscle and muscle attachment cells during the onset of metamorphosis. how is detected in adult muscle cell precursors that are attached to the wing imaginal disc (Baehrecke, 1997).

Effects of Mutation or Deletion

Animals lacking how function die late during embryogenesis, possessing defects that seem to occur sometime between myoblast fusion and muscle cell attachment. A lethal mutant dies with the most posterior region of the cuticle arrested above the dorsal surface, presumably cause by failure to complete germ band retraction. howe44 mutants have normal differentiated and fused myotubules, but with variable expressivity the myotubes of some embryos appear to migrate but are disorganized and don't possess the proper pattern of attachment while the myotubules of other embryos show no signs of migration and actually appear to have fewer myoblasts. Partial loss-of-function results in lethality during the metamorphosis from a larva to an adult. Most howr17 homozygotes die during metamorphosis with their heads stuck inside their thoraxes. A small number of howr17 homozygotes escape as adults that do not fold their wings properly and, thus, possess the phenotype for which this gene is named. These flies also have blisters on their wings (Baehrecke, 1997).

Hypomorphic mutant alleles of how produce adult animals that have lost the ability to fly; they keep their wings on the horizontal, at a 90∞ angle to the body axis (Zaffran, 1997).

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date revised: 10 February 2013


  

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