Interactive Fly, Drosophila

Body structures of Drosophila develop through transient developmental units, termed parasegments, with boundaries lying between the adjacent expression domains of wingless and engrailed. Parasegments are transformed into the morphologically distinct segments that remain fixed. Segment borders are established adjacent and posterior to each engrailed domain. They are marked by single rows of stripe expressing cells that develop into epidermal muscle attachment sites. The positioning of these cells is achieved through repression of Hedgehog signal transduction by Wingless signaling at the parasegment boundary. The nuclear mediators of the two signaling pathways, Cubitus interruptus and Pangolin, function as activator and symmetry-breaking repressor of stripe expression, respectively (Piepenburg, 2000).

A cis-acting element of stripe (sr) has been identified that specifically directs gene expression in segment border cells during embryogenesis. This element was used to illuminate the molecular mechanism underlying segment border selection. The results show that Hedgehog (Hh) signaling can activate gene expression in two rows of cells, one on each side of the engrailed (en) expression domain. However, anterior Hh signaling causes the maintainance of wingless expression anterior to the PS boundary. Wg in turn antagonizes Hh-dependent gene expression and thereby prevents the formation of segment border cells anterior to the en domain. Hh and Wg activities relevant for the selection of segment border cells are mediated by functional binding sites of their nuclear mediators, Cubitus interruptus (Ci) and Pangolin (Pan), respectively within the sr cis-acing element. The data suggest that the segment border is established in response to the asymmetry of Wg signaling at the PS boundary (Piepenburg, 2000).

stripe (sr) is expressed in all precursors of the epidermal muscle attachment sites, including those marking the segment border in the Drosophila larvae. To obtain an early molecular marker for the segment border corresponding to the row of cells posteriorly adjacent to the en expression domain, a 1.9 kb enhancer element of the sr gene (sr1.9) was isolated that is both necessary and sufficient to direct transgene-dependent lacZ expression in segment border precursor cells in a dorsal and lateral position of the embryo. Expression of the reporter gene is activated in parallel with sr, which is first expressed during late stage 10. At this time, the initial equal distribution of Wg has already become asymmetric, meaning that the protein spreads anteriorly over a range of maximally five cells but is restricted to only one row of cells directly adjoining the posterior margin of the expression domain. sr acts as a transcription factor required for setting up the cell fate of the muscle attachment sites which mark the segment border of the fly. Thus, sr1.9-dependent reporter gene expression can be employed to study the transregulatory requirement for positioning the segment border cells within the PS (Piepenburg, 2000).

Subfragments of the sr1.9 element lacking sr239 failed to activate discernible gene expression, whereas sr239 directs sr1.9-like gene expression in the row of cells posterior to the en domain. Moreover, sr239-dependent gene activation could be achieved upon ectopic CiZn/C (the active form of Ci) expression within the en domain. Thus, the Ci binding site-containing sr239 element is both necessary and sufficient to activate gene expression in segment border precursor cells, and it is sufficient to mediate gene activation in response to dominant active Ci. It was next asked whether the two Ci binding sites within the sr239 element are necessary to mediate Hh-dependent gene activation. For this experiment, sr239 variants lacking either one or both Ci binding sites were generated. sr239 variants lacking only one functional Ci binding site can mediate gene expression in the correct spatial pattern, but the level of expression is strongly reduced. In contrast, lack of both Ci binding sites abolished Hh/Ci-dependent gene activation completely. In summary, these findings establish that Ci activates gene expression in segment border cells. They confirm, by direct means, that Hh signaling acts not only anteriorly and across the PS boundary to maintain Wg activity, but functions in a symmetric fashion and thereby determines the position of the segment border within the PS (Piepenburg, 2000).

Since Hh signaling appears to be symmetric, it was of interest to know why the sr1.9 element fails to mediate gene activation in the row of cells anterior to the en domain. An explanation for this phenomenon would be that signaling by Wg causes region-specific repression, preventing gene activation by Hh-dependent Ci. To test this proposal, Wg was ectopically expressed in the ptc domain and the change of sr1.9-driven gene expression was examined in such embryos. Furthermore, the sr1.9-mediated gene activation was analyzed in embryos mutant for lines (lin), sloppy paired (slp), and naked (nkd). Each of these mutant embryos express en, but the wg pattern is altered (Piepenburg, 2000 and references therein).

sr1.9-mediated gene expression was abolished in response to ectopic Wg activity. The same effect was observed in nkd mutant embryos where Wg is expressed at each side of an enlarged en domain. Conversely, in slp and lin mutants where Wg activity is not maintained, sr1.9-mediated gene expression is found in two rows of cells, one on each side of the en domain. Furthermore, sr1.9-mediated gene expression was also observed in cells anterior to the region of en expression in embryos in which late Wg activity was abolished due to a temperature-sensitive wg mutation. This establishes that the repression of Hh action in the cells anterior to the wild-type en domain is dependent on Wg activity (Piepenburg, 2000).

To explore how Wg exerts its repressing function, whether the Drosophila TCF/Lef1 homolog Pangolin (Pan), the nuclear mediator of Wg activity, can directly interact with the sr239 element was examined. Pan in vitro binding sites were found next to and partially overlapping the Ci binding sites. Deletion of one Pan binding site that leaves the Ci binding sites intact (sr239DeltaPan), resulted in gene activation anterior to the en domain. In contrast to sr239-mediated gene expression that can be suppressed by ptc-Gal4-driven Wg activation, sr239?Pan-mediated gene expression is not abolished in response to ectopic Wg activity (Piepenburg, 2000).

It is known that Pan can associate with corepressors such as dCBP or Groucho. Upon reception of the Wg signal, Pan is switched into an activator of transcription by association with Armadillo, a coactivator of Wg target genes. The findings in this study suggest an alternative mechanism since the Pan binding sites of the sr1.9 and sr239 elements mediate Pan-dependent repression in cells with high Wg activity. This repression is necessary and sufficient to antagonize Ci-dependent transcriptional activation. Pan could thereby exert this function by competing sterically for Ci binding, by short-range quenching of Ci-mediated gene activation, or by active repression. Each way, Pan would allow for the formation of only one row of segment border cells within each PS by repressing the Hh-dependent sr activation in the wg domain (Piepenburg, 2000).

Transcriptional Regulation

In the Drosophila embryo, the correct association of muscle cells with their specific ectodermally derived tendon cells, also known as epidermal muscle attachment or EMA cells, is achieved through reciprocal interactions between these two distinct cell types. Vein, a neuregulin-like factor secreted by the approaching myotube, activates the EGF-receptor signaling pathway within the tendon cells to initiate tendon cell differentiation. kakapo is expressed in the tendons and is essential for muscle-dependent tendon cell differentiation. Kakapo is a large intracellular protein and contains structural domains also found in cytoskeletal-related vertebrate proteins (including plakin, dystrophin, and Gas2 family members). kakapo mutant embryos exhibit abnormal muscle-dependent tendon cell differentiation. The expression of delilah, stripe, and beta1 tubulin is induced in the epidermal attachment cells as a result of the EGF-receptor pathway activation by the neuregulin-like growth factor, Vein (Yarnitzky, 1997). Vein is secreted by mesodermal cells underlying the EMA cells. Vein protein localization is restricted to the muscle-tendon junctional site in wild-type embryos. However, in kak mutant embryos, Vein protein is not localized and appears rather diffuse. This altered pattern of Vein may explain the multiple number of cells expressing delilah and stripe: since Vein is not strictly localized at a given muscle-tendon junction site, it apparently weakly activates the EGF-receptor pathway in neighboring cells as well. It is presumed that the only cells that can respond to the ectopic Vein protein are the competent population of EMA cells, defined by the early expression of stripe. These cells express stripe during early developmental stages in a muscle-independent manner and normally lose their stripe expression by stage 16 of embryonic development. When these competent EMA cells receive the muscle-derived Vein signal, the expression of stripe and delilah is reactivated. It appears that only this population of cells is capable of responding to Vein, since the pattern of the ectopic Stripe- or Delilah-expressing cells in the kak mutant embryos resembles that of the early population of Stripe-expressing cells. The reduced levels of beta1 tubulin mRNA in the mutant tendon cells may also result from the abnormal pattern of Vein localization, since lower levels of Vein may not be sufficient to induce maximal beta1 tubulin expression. It therefore appears that the primary defect in kak mutant embryos stems from the lack of Vein accumulation at the muscle-tendon junctional site (Strumpf, 1998).

Changes in the extracellular matrix (ECM) govern the differentiation of many cell types during embryogenesis. Integrins are cell matrix receptors that play a major role in cell-ECM adhesion and in transmitting signals from the ECM inside the cell to regulate gene expression. In this paper, it is shown that the PS integrins are required at the muscle attachment sites of the Drosophila embryo to regulate tendon cell differentiation. The analysis of the requirements of the individual alpha subunits, alphaPS1 and alphaPS2, demonstrates that both PS1 and PS2 integrins are involved in this process. In the absence of PS integrin function, the expression of tendon cell-specific genes such as stripe and beta1 tubulin is not maintained. In addition, embryos lacking the PS integrins also exhibit reduced levels of activated MAPK. This reduction is probably due to a downregulation of the epidermal growth factor receptor (Egfr) pathway, since an activated form of the Egfr can rescue the phenotype of embryos mutant for the PS integrins. Furthermore, the levels of the Egfr ligand Vein at the muscle attachment sites are reduced in PS mutant embryos. Altogether, these results lead to a model in which integrin-mediated adhesion plays a role in regulating tendon cell differentiation by modulating the activity of the Egfr pathway at the level of its ligand Vein (Martin-Bermudo, 2000).

Two physiologically distinct types of muscles, the direct and indirect flight muscles, develop from myoblasts associated with the Drosophila wing disc. The direct flight muscles (DFMs) are specified by the expression of Apterous, a Lim homeodomain protein, in groups of myoblasts. This suggests a mechanism of cell-fate specification by labelling groups of fusion competent myoblasts, in contrast to mechanisms in the embryo, where muscle cell fate is specified by single founder myoblasts. In addition, Apterous is expressed in the developing adult epidermal muscle attachment sites. Here, it functions to regulate the expression of stripe, a gene that is an important element of early patterning of muscle fibers, from the epidermis. These results, which may have broad implications, suggest novel mechanisms of muscle patterning in the adult, in contrast to embryonic myogenesis (Ghazi, 2000).

In examining the adult expression pattern of embryonic muscle founder markers, expression of an ap reporter was observed in a pattern that suggested specific roles in myogenesis. The DFMs, located dorsolaterally in each hemisegment of the adult mesothorax, show reporter gene activity. No staining was seen in the indirect flight muscles (IFMs), which constitute the bulk of the muscles of the dorsal mesothorax. Amongst DFMs, the most conveniently identifiable ones are muscles 49-58. Muscles 49 and 51-55, show staining for the ap lacZ reporter gene in different planes of foci (Ghazi, 2000).

Epidermal attachment sites for muscles in the adult fly are identifiable by anatomical examination and by expression of stripe (sr). sr marks all muscle attachment cells, both in the embryo and the adult. The pattern of sr expression during pupal development has been studied and the attachment sites for the IFMs identified. ap lacZ adult expression is also seen in the thoracic epidermis in the regions where muscles attach (Ghazi, 2000).

On the third instar wing imaginal disc the presumptive notum shows low ap lacZ and Ap protein expression distinct from the high levels seen in the presumptive dorsal wing. Although the presumptive notum is a dorsal structure, ap does not seem to have a selector function to define 'dorsalness' in the mesothoracic trunk as it does in the dorsal wing blade. ap expression in the pupal epidermis changes temporally beginning with an early expression in broad regions of the dorsal notal epidermis and a subsequent localization to restricted domains, including the attachment sites of the DLMs. At 18 hours after puparium formation (APF), ap lacZ expression is seen in regions that included the developing anterior attachment sites of the DLMs. This expression eventually narrows down to the anterior attachment sites and very closely abuts the posterior attachment sites of the DLMs. This can be observed by simultaneous labelling of developing pupae for ap and sr. By 36 hours APF, when a complete set of DLM fibers is in place, ap co-localization with individual muscle-attached sr-expressing tendon cells is clearly seen. This attachment site expression continues in the adult. The same pattern is seen on labelling with Ap-specific antibodies (Ghazi, 2000).

Expression of ap in epidermal attachment sites of muscles and the phenotypes noticed in ap mutants suggests a regulatory role for the gene in the development of muscle attachment sites. stripe (sr), the earliest known marker for epidermal attachment sites, was chosen as a potential target for regulation by ap in mediating its epidermal function, and its expression was examined in wing discs of ap⁴ homozygotes. sr is crucial for differentiation of epidermal cells into muscle-attached tendon cells. sr is expressed in discrete domains in the wing disc, which will form the attachment sites of adult thoracic muscles. sr expression in the disc commences very late in third larval instar and is consistently seen as pupation is initiated. Hence the 0 hours APF white prepupal stage was chosen for examination of sr expression in ap mutants. sr expression is either completely lost or drastically reduced in ap⁴ wing discs. Animals that reach pupal stages show severe reduction in sr levels. These results are further strengthened by the observation that ap and sr interact with each other genetically to affect IFM development. The ap⁴ mutation is completely recessive, as is a sr recessive lethal. In a transheterozygous combination, the two alleles show defective IFMs in a significant population of animals. Further, an enhancer trap insertion at the sr locus that shows a very mild recessive phenotype, enhances the IFM defects of ap⁴ and such animals also show a dark midnotal stripe that is characteristic of strong, viable sr alleles. This suggests that ap functions in IFM patterning by influencing attachment site development by the regulation of sr (Ghazi, 2000).

How repeating striped patterns arise across cellular fields is unclear. To address this the repeating pattern of Stripe (Sr) expression across the parasegment (PS) was examined in Drosophila. This pattern is generated in two steps. Initially, the ligands Hedgehog (Hh) and Wingless (Wg) subdivide the PS into smaller territories. Next, the ligands Hh, Spitz (Spi), and Wg each emanate from a specific territory and induce Sr expression in an adjacent territory. The width of Sr expression is determined by signaling strength. Finally, an enhancer trap in the sr gene detects the response to Spi and Wg, but not to Hh, implying the existence of separable control elements in the sr gene. Thus, a distinct inductive event is used to initiate each element of the repeating striped pattern (Hatini, 2001).

The repeating pattern of Stripe (Sr) expression across the parasegment (PS) is generated by inductive inputs from three spatially localized ligand sources. The ligands, Hh, Spi, and Wg, emitted by En, Ve, and Wg territories, respectively, control Sr expression in cells adjacent to each ligand source. There are three notable features to this regulation: (1) each ligand-producing territory induces Sr expression in the adjacent territory; (2) the induction is asymmetric, either anterior or posterior to each source; (3) the induction is initiated at the high level of signaling achieved near the source, limiting expression of Sr to a narrow row of cells. Because these same ligands act more broadly in cuticle cell fate specification, these results also suggest that the ligands and signaling territories operate in a fundamentally distinct way in order to construct a repeating striped pattern. These observations reveal a strategy used to generate a repeating striped pattern across a cellular field that may be used generally (Hatini, 2001).

Each Sr row is initiated adjacent to a different ligand source. The induction of Sr was limited to a narrow row of cells at each position. Manipulating either the ligand level, or the sensitivity of cells to a specific signaling pathway, leads to a broadened territory of Sr induction. Thus, local activity gradients of Hh, Spi, and Wg are each generated, and a threshold for activation of Sr is only surpassed in cells adjacent to each source. The gradient landscape of Spi and Wg is sculpted using the inducible antagonists Argos and Naked, respectively. Although how the activity landscape for Hh is sculpted was not specifically addressed, the Hh pathway also makes use of an inducible antagonist. It is likely that Hh spread is limited by binding to the Hh receptor Ptc, which is upregulated by Hh input (Hatini, 2001).

To generate the repeating striped tendon pattern, the Sr gene must be able to respond to each of three different ligands. To account for this, it is expected that the Sr promoter is modular, and each Sr row is induced via a separable, cis-acting response element. An enhancer trap P-insertion in the sr gene (sr⁰³⁹⁹⁹) provides evidence for this since it detects the response to Spi and Wg, but not to Hh, implying that the P-insertion separates response elements in the sr gene. A separable Sr promoter element controlling Hh-dependent expression has been identified. Although this element operates only in dorsal and lateral epidermis, and not ventrally where Sr is expressed in repeating striped pattern, this observation strongly suggests that the control elements will be modular. Furthermore, in this dorsal/lateral element, the presence of functional, consensus Cubitus interruptus (Ci) DNA binding sites suggests direct regulation of Sr by the Hh signaling pathway. The obvious analogy is to the modularity of regulatory regions of certain pair-rule genes, which are able to integrate non-periodic information in order to generate periodicity. Note that the induction of the sr gene is limited to cells bordering each ligand source, even though each of the signals can act across several cell diameters. It is predicted that a given Sr response-element is configured to sense and respond only to a particularly high threshold level of each ligand (Hatini, 2001).

The ligands controlling Sr expression emanate from each of three territories across the PS. These territories are established by the primary organizing signals, Wg and Hh. In the earliest step, cross regulation between Wg and En/Hh-expressing cells stabilizes each ligand's expression and consolidates these two territories. In addition, through negative regulation, both Wg and Hh limit the expression of Ser to a central territory within the PS. Finally, signals from the En/Hh territory induce Ve expression in two cell rows just posterior to the En/Hh territory. The exact width of the Ve-expressing territory is adjusted as local input from the Ser territory induces a third Ve-expressing cell row. Thus, Hh and Wg act indirectly by defining and limiting each other's expression territory, as well as that of downstream ligands. All of these ligands then organize the repeating pattern. Three ligands induce Sr expression at specific positions across the PS, while the role of Ser reveals a particularly interesting spatial cue. Although the second row of Sr is induced in the anterior-most row of Ser-expressing cells, Ser expression is not necessary for this. Rather, Ser dictates the spacing between Sr row 1 and 2, because it defines the breadth of the Ve territory and thereby the position of the first non-Ve cell that can induce Sr in response to Spi-Egfr signaling (Hatini, 2001).

Sr expression is induced asymmetrically relative to each ligand source. For instance, Hh induces Sr posterior to the En territory, but not in the En territory or anterior to it. Wg imparts asymmetry to Hh/En signaling, and thereby prevents Ve expression anterior to the En/Hh territory. In exactly the same way, via antagonism of Hh signaling, Wg appears to block Sr expression anterior to the En/Hh territory, because the removal of Wg function allows Sr expression anterior to the En/Hh territory. The Wg signaling pathway imposes asymmetry to the dorsal/lateral Sr regulatory element via consensus Pangolin DNA binding sites. This principle is likely to extend to the ventral control of Sr for the generation of one element of the repeating striped pattern (Hatini, 2001).

One reason why Hh signaling cannot induce Sr expression in the En cells is that En represses expression of the Hh signal transducer, Ci. Nevertheless, it is still necessary to explain why signals from both the Ve and Wg territories cannot induce Sr in the En cells, even though each signal definitely acts on these cells to specify cuticle fate. A clue comes from the observation that when the En territory is not maintained Sr is induced symmetrically relative to the Wg or to the Ve sources. Thus, it is proposed that the En protein prevents Sr induction by Wg or Egfr inputs by repressing Sr expression. This is supported by the observation that activating Wg signaling at high levels in En cells still does not lead to Sr expression. To explain why Sr is not induced by Spi in the Ve territory, nor by Wg in the Wg territory, it is inferred that there is a specific block to autocrine signaling in each territory. Interestingly, this block is specific to Sr induction, and not to other outcomes of signaling, such as cuticle fate specification. It suggests a lack of an activator essential to induce Sr expression or expression of a repressor that blocks such a response in the Ve and Wg territories (Hatini, 2001).

The same ligands establish strikingly distinct patterns across the same cellular field. While the cuticle pattern comprises a diversity of cell types, the Sr expression pattern reflects the near-periodic specification of the same cell type. These distinct outcomes arise because the same ligands act in a fundamentally different manner in these two processes. As an example, Wg specifies smooth cuticle in a broad region anterior to the Wg territory, in the Wg territory, and posterior to the Wg territory (in anterior En/Hh cells). However, as is shown in this study, Wg induces Sr only anterior to the Wg territory in a narrow region, and not in the Wg territory or posterior to it. Also, Spi, through Egfr function, induces denticles over a broad region, both in the Ve territory and anterior to it in a subset of En/Hh cells. However, Egfr function induces Sr only in a narrow region posterior to the Ve territory. Thus, despite the broad effects of Wg and Egfr on cuticle pattern, the effect of Wg and Egfr in building the repeating striped pattern is constrained to a narrow region of cells. As a final example of the distinction between control of denticle pattern and control of repeating striped pattern, in the same row of cells, cuticle fate is specified by Spi while tendon fate is specified by Hh input. The unique effects of these ligands on Sr expression are crucial for the establishment of the repeating striped pattern. Thus, the information encoded in the signaling territories is decoded in different ways to achieve both repeating pattern and cell-type diversity across the same field (Hatini, 2001).

The generation of near-periodic Sr pattern across the PS is conceptually similar to the two-step process that is used in establishing the periodic body plan through pair-rule gene expression in syncitial embryos. Initially, primary pattern organizing centers are established at the boundaries of a field of naive nuclei or cells. In the first step, these centers establish patterned expression of secondary organizing genes across the field, subdividing the field into distinct gene expression territories. In the second step, the information encoded in these territories is used to initiate a repeating striped gene expression pattern. In the embryo, Bicoid together with Hunchback and Nanos organize expression of the gap genes. Territories of gap gene expression are then used to establish the periodic pattern of primary pair-rule gene expression. In the PS, Wg and Hh organize overall parasegmental pattern by first defining each other's territory and then the territories of secondary regulatory genes, Ser and Ve. The signaling territories are then used to establish near-periodic expression of Sr. Note that although the conceptual similarity is striking, the mechanisms generating these two repeating patterns are distinct. The pair-rule gene expression pattern is established in a unique, syncitial system by diffusion of transcriptional regulators in a common cytoplasm, whereas Sr expression is established across an epithelial monolayer by communication between cells via inter-cellular signaling systems. In addition, while a balance between diffusible activators and repressors determines pair-rule gene expression at any point along the syncitial embryo, the unique properties of the signaling territories across the PS determine Sr expression. In particular, the juxtaposition of pairs of territories, one that sends a signal with one that can initiate Sr expression in response to the signal, is utilized to initiate Sr expression adjacent to the boundaries between these territories (Hatini, 2001).

A near-periodic striped pattern of veins is produced in the developing wing disc. Emerging evidence suggests that the two-step strategy may also apply to this system, and that unique properties of putative signaling territories are used to initiate wing veins adjacent to at least two territories across the wing blade. In the first step, combined action of Hh and Dpp establishes different territories of downstream regulatory genes across the A/P axis of the future wing blade. While Hh establishes a territory of Ptc expression adjacent to the compartment boundary, Dpp acts more broadly across the wing and establishes two nested territories of Spalt and Optomotor-blind expression. In the second step, veins are induced adjacent to at least two territories, suggesting that an unknown ligand emanates from one territory and induces the vein in the adjacent territory. The possibility that the two-step process described here for the PS is used across the wing blade suggests that this may be a general strategy for creating repeating striped patterns across other cellular fields (Hatini, 2001).

In Drosophila, muscles attach to epidermal tendon cells are specified by the gene stripe (sr). Flight muscle attachment sites are prefigured on the wing imaginal disc by sr expression in discrete domains. The mechanisms underlying the specification of these domains of sr expression have been examined. The concerted activities of the wingless (wg), decapentaplegic (dpp) and Notch (N) signaling pathways, and the prepattern genes pannier (pnr) and u-shaped (ush) establish domains of sr expression. N is required for initiation of sr expression. pnr is a positive regulator of sr, and is inhibited by ush in this function. The Wg signal differentially influences the formation of different sr domains. These results identify the multiple regulatory elements involved in the positioning of Drosophila flight muscle attachment sites (Ghazi, 2003).

Much of the presumptive notum is in the anterior compartment in which there are four domains of sr expression. One of these is in the medial region (a) and gives rise to the anterior tendon cells, to which dorsal longitudinal muscles (DLMs) attach. The remaining three are in the lateral region (b, c and d) and provide dorsal attachments for dorsoventral muscles (DVMs). In the posterior compartment, sr is expressed in a narrow band that eventually forms the posterior insertion site for DLMs. The positioning of different sr domains on the wing imaginal disc have been examined with respect to prepattern and pattern forming genes expressed in this region (Ghazi, 2003).

The Wg gradient in the presumptive notum controls sr transcription differentially and keeps different sr domains distinct. The actual regulation of sr by wg appears to be very complex. Lateral domain c appears more sensitive to perturbations in wg signaling as compared to b. This is interesting since all the cells of domain b receive uniform levels of Wg whereas cells at the posterior border of c, and those bordering the posterior sr domain receive high Wg. One possibility is that the latter cells block progress of the Wg gradient and thus determine responses of cells further away. This may be brought about by targeting Wg to lysosomes and degrading it, as in the embryo. Another possibility, not exclusive of the first, could be that the domain and levels of wg transcription determine the range and gradient of Wg. The precise definition of the domain of wg transcription could be by a mechanism similar to that used in the wing margin. While the data suggest that the posterior and lateral-most domain do not receive Wg and may lie outside its purview, the formal possibility still exists that wg affects these domains in some other unknown way (Ghazi, 2003).

wg controls sr expression by in segment border cells of the Drosophila embryo. Wg signaling restricts sr activation to a single row of cells. In the presumptive notum on the wing disc, hh expression is restricted to a very narrow region, which forms the posterior compartment. Its effects in the disc are mediated by dpp, which serves multiple functions. Dpp is required for induction of wg expression, as it positively regulates pnr, which in turn activates wg. However, once wg is induced, Dpp tightly restricts its domain. This antagonism is required for correct positioning of the DC bristles. This antagonism also defines domains of sr. It is unclear if dpp directly regulates sr, or its effect is by control of other genes. The similarity between sr phenotypes observed on expansion of wg expression, and in dpp mutants, is suggestive of its effects being mediated by wg only, but it is also possible that it influences sr expression directly (Ghazi, 2003).

Pnr, a GATA-binding protein normally functions as a transcriptional activator and is antagonized by Ush in its function. Loss of function pnr mutants show no sr expression in the domain covered by pnr. This, along with sr expansion in mutants of ush, would suggest that pnr activates sr in the notum, and is inhibited by ush. However, there is also loss of sr expression in pnr `gain of function' mutants. The reason for this is not completely clear. One possibility is that since the mutation causes an increase in wg activity in the region this may cause a down-regulation of sr. This is supported by a similar effect seen on misexpression of activated armadillo in the pnr domain. Results with both pnr and ush have been taken into account to suggest that pnr positively regulates sr and is antagonized by ush (Ghazi, 2003).

Most sr expression commences in regions of low ush. Phenotypes of ush mutants, and ush misexpression experiments, also indicate that the gene inhibits sr, in keeping with the simplistic scenario that ush antagonizes pnr-mediated activation of sr. However, the medial sr domain is partially covered by ush proximally. Further, pnr is known to be required for positive induction of ush in the embryonic epidermis and in some loss of function allelic combinations of pnr, such as pnr^VX6/pnr^VX1, there is reduced ush expression on the disc. So how is sr initiated in the region where Pnr and Ush are co-expressed? The answer to this is not known but probably lies in levels of Ush and Pnr at that position. In loss of function ush mutants ectopic dorsocentral bristles form but post vertical (PV) bristles are missing, suggesting that the Pnr-Ush complex acts as a repressor of the DC enhancer, but as activator of the enhancer of PV bristles. Such observations have indicated complex, context dependent interactions between Pnr and Ush in determining cell fate and could explain the regulation of sr expression in the medial notal region (Ghazi, 2003).

These results indicate that each sr domain is regulated by a combination of prepattern genes and signaling molecules. But, a precise description of the 'combinatorial code' for regulation of each sr domain is beyond the scope of this work and can be achieved by generation of domain specific markers for sr. Based on expression pattern data, and existing literature, it is suggested that high levels of Pnr, low (or absence of) Ush and moderate levels of Wg determine the initial induction of domain a. The distinction between medial (a) and lateral (b-d) domains is established by the presence of very high levels of Wg (the cells where the Wg gradient originates). Lateral expression domains are probably induced in domains controlled by the lateral prepattern gene iro. The differences between different lateral domains arise as a result of expression of different genes in the region. For instance, the lateral-most domain d appears to be regulated by ush and does not encounter Wg at all. Whereas, all cells of b receive uniformly moderate levels of Wg, only cells at the borders of c receive high Wg levels, and these differences result in the distinct identities of the two domains. Dpp, either through its effects on these regulatory genes and/or through direct effects on sr influences the process (Ghazi, 2003).

The role of N as a potential regulator of sr was examined, since it is known to influence multiple events in wing disc morphogenesis from proliferation to bristle patterning. Using a temperature sensitive allele (N^ts), the protein function was inactivated by growing animals at non-permissive temperatures during the third larval instar. sr expression was examined at 0 h APF. Loss of sr expression is observed in these animals. In hemizygous males, this effect is most severe and sr expression is completely abolished. Females, with one normal copy of N, showed faint sr expression. This suggested that N may be required for initiation of sr expression. A dominant negative form of N (N^dn), was expressed in the pnr domain; abolition of sr expression was found. This was observed most clearly in the anterior medial domain covered by pnr. The lateral domains showed some reduction in sr as well. In a gain of function experiment, a constitutively active form of N (N^intra) was expressed in the same region and results in an increase in sr-lacZ. The N ligand Ser is known to regulate sr expression in the embryonic segment border cells. Mis-expression of Ser in the presumptive notum region resulted in loss of sr expression. Together, these results show that the initiation of sr expression relies on N, which is antagonized by Ser in this activity (Ghazi, 2003).

Prepatterning the Drosophila notum: the three genes of the iroquois complex play intrinsically distinct roles -- Regulation of stripe expression

The Drosophila thorax exhibits 11 pairs of large sensory organs (macrochaetes) identified by their unique position. Remarkably precise, this pattern provides an excellent model system to study the genetic basis of pattern formation. In imaginal wing discs, the achaete-scute proneural genes are expressed in clusters of cells that prefigure the positions of each macrochaete. The activities of prepatterning genes provide positional cues controlling this expression pattern. The three homeobox genes clustered in the iroquois complex (araucan, caupolican and mirror) are such prepattern genes. mirror is generally characterized as performing functions predominantly different from the other iroquois genes. Conversely, araucan and caupolican are described in previous studies as performing redundant functions in most if not all processes in which they are involved. The question of the specific role of each iroquois gene in the prepattern of the notum was addressed, and it was clearly demonstrated that they are intrinsically different in their contribution to this process: caupolican and mirror, but not araucan, are required for the neural patterning of the lateral notum. However, when caupolican and/or mirror expression is reduced, araucan loss of function has an effect on thoracic bristles development. Moreover, the overexpression of araucan is able to rescue caupolican loss of function. It is concluded that, although retaining some common functionalities, the Drosophila iroquois genes are in the process of diversification. In addition, caupolican and mirror are required for stripe expression and, therefore, to specify the muscular attachment sites prepattern. Thus, caupolican and mirror may act as common prepattern genes for all structures in the lateral notum (Ikmi, 2008).

In earlier works, the iro-C genes functions were mainly assessed from studies of mutations affecting solely mirr, of deficiencies deleting the whole complex or of rearrangements susceptible to affect several genes, and on misexpression experiments. In order to unravel the respective roles of ara, caup and mirr and to analyze their possible functional redundancy in the neural prepatterning of the notum, this study has combined the analysis of LoF mutations of these genes with a functional replacement approach (Ikmi, 2008).

mirr appears to be required for the formation of four out of the seven lateral macrochaetes (PS, pSA, aPA and pPA): their loss is elicited by LoF alleles of mirr and by a dominant allele (mirr^G8. The proneural clusters as well as the SOPs corresponding to the macrochaetes unaffected by mirr mutations may reside outside (aNP) or inside (pNP) the mirr domain of expression. Therefore the requirement or dispensability of mirr for the patterning of the bristles in the lateral notum appear to depend partly but not only on its domain of expression. All the phenotypes observed in this study were elicited by mild perturbations of the mirr function, compatible with viability of adults. Therefore, a role of mirr in the patterning of the other lateral macrochaetes cannot be completely excluded (Ikmi, 2008).

In the prospective lateral notum, ara and caup appear to have mostly identical domains of expression, which, at least, enclose all the SOPs for lateral macrochaetes. These authors have suggested that there is a functional redundancy of these two genes on the grounds of their similar protein-coding sequences and patterns of expression, and this is generally admitted. However no evidence of this has been reported to date. Notably no LoF mutations affecting only one of these two genes have been described previously. This study characterized mutations abolishing (or drastically reducing) caup expression without an appreciable effect on ara and mirr (e.g. iro1 or caup^G3) and null (or strong hypomorph) mutations of ara that do not affect significantly caup or mirr (e.g. TSI or ara^G4) (Ikmi, 2008).

Six transposable element (TE) insertions were characterized in caup, and it was shown that they behave like an allelic serie of caup hypomorphic mutations: flies homozygous for these insertions are viable and display phenotypes ranging from wt or the loss of only a few macrochaetes to the loss or a strong frequency decrease of all the seven lateral macrochaetes (strong Iroquois phenotype). These phenotypes are aggravated when these TE insertions are heterozygous over an iro-C deficiency and even more over the iro1 rearrangement, which has a breakpoint in the first caup intron. Heterozygous iro1/Df-BSI L3 larvae display no caup expression (or an extremely low level) although ara and mirr levels are similar to the control conditions. Similarly, the strongest P allele of caup (caup^G3) causes a drastic reduction or an absence of caup expression without affecting notably ara and mirr, neither in expression level nor in expression domain in the notum. Therefore, it can be assumed that caup is required for the patterning of all lateral macrochaetes and, presumably, for the direct or indirect activation of sc expression in the lateral notum (Ikmi, 2008).

Besides inactivating caup expression, the P[Gal4] insertions in the caup gene reproduce its expression pattern. By combining such a Gal4 driver with an UAS-caup transgene, it was shown that caup overexpression in its normal domain of expression is able to rescue the caup LoF phenotype to an almost wt situation. The frequency of occurrence of all the seven lateral macrochaetes is either normal or at least elevated as compared to the mutant situation. Taken together, loss and gain of function approaches demonstrate that caup is required for the patterning of all the lateral macrochaetes of the notum (Ikmi, 2008).

The same approach was applied to study the loss of function of ara, and the effects were analyzed of five insertions and an inversion (TSI) in the ara gene. In ara^G4/TSI and TSI/Df-BSI L3 larvae, ara expression is absent or strongly reduced while caup and mirr expression are not significantly affected as compared to the control situations. These larvae die as pupae, showing the requirement of ara for viability. The very rare adult escapers do not present any bristle defects. The same is true for the four other ara hypomorphic mutants studied. In summary, the sole LoF of ara is never associated with a loss of macrochaetes. Consequently, conversely to caup, ara is dispensable for the patterning of all the lateral bristles of the notum (Ikmi, 2008).

In conclusion, the Iro proteins are different for their contribution to the neural patterning of the lateral notum. Only Caup and Mirr are required for the patterning of the lateral macrochaetes and therefore to control the activation of expression of ac and sc in the corresponding proneural clusters (Ikmi, 2008).

When it is strongly overexpressed in the caup domain of expression, Ara, although not required, is sufficient to rescue the lack of lateral macrochaetes phenotype elicited by caup LoF. Therefore, unexpectedly considering the LoF results, when overexpressed Ara can carry out the function of Caup in the neural patterning of the notum. This property may play a biological role, as can be seen in the exacerbation of the phenotypic defects elicited by ara LoF in sensitized conditions (reduction of caup and/or mirr function). Although to a much less extent, Mirr is also able to perform in part the Caup function, as seen from the limited rescue of PS, aNP and aSA macrochaetae by mirr overexpression in a caup LoF background. This functional difference between Ara and Mirr correlates well to their sequence divergence with Caup. A possible explanation for the difference observed between the LoF of ara and caup is that Ara, in physiological conditions, has a lower activity than Caup to promote proneural genes expression. The increase of the amount of the Ara protein to non-physiological levels probably increases the expression of proneural genes sufficiently to compensate for the loss of Caup activity. This is in good agreement with the findings that Drosophila Iro proteins bind in vitro the same sequence but differ in their strength of binding to this site. Consequently, owing essentially to the proteins intrinsic properties, the Iro transcription factors do not regulate in vivo the same target genes (Ikmi, 2008).

It has been suggested that a common network of prepattern genes regulates all structures of the notum. Indeed, in the prospective medial notum, pnr is required for both ac-sc and sr expression, which respectively specify the development of bristles and tendon precursors. Preliminary observations indicate that pnr also regulates pigment patterns in medial notum (Ikmi, 2008).

In the wing discs of larvae mutant for caup or for mirr, two (b and d) out of the three domains of sr expression located in the prospective lateral notum are missing. Furthermore, a mirr mutant partially affects sr expression in the two remaining domains (a and c). In addition, adult flies lacking either caup or mirr display falling wings, suggesting an abnormal attachment of indirect flight muscles. Therefore, caup and mirr activate sr expression and in consequence participate to the specification of lateral flight muscle attachment sites. It had been shown that Iro proteins can form homodimers and heterodimers that bind in vitro the same palindromic site called Iro Binding Site (IBS; Bilioni, 2005). Thus, it is possible that the activity of a Caup-Mirr heterodimer controls the regulation of sr expression in regions (b) and (d). The sr expression pattern is normal in the wing discs of larvae lacking the ara function. Hence ara is not required for the expression of sr in the prospective notum. Nonetheless, a reduction of ara function, as seen in hypomorphic mutants or escapers to the lethality, leads to heldout wings. This phenotype is often observed in flies carrying mutations that affect direct flight muscles. It is thus possible that ara regulates the specification of direct flight muscle attachment sites. In summary, here again, the iro-C genes products are not functionally equivalent in their contribution to the muscular attachment sites prepattern (Ikmi, 2008).

Although there are no strong evidences that iro-C regulates pigment patterns, preliminary reports suggest that this is the case. From all these data, the iro-C genes caup and mirr appear as common prepattern genes for the specification of all the structures in the lateral notum, similarly to pnr in the medial notum (Ikmi, 2008).

In addition, pnr and iro-C domains partially overlap each other at the virtual border between the medial and the lateral notum. The DC macrochaetes and the 'a' sr domain can be affected in caup and mirr mutants. These structures are dependant on the pnr function. Therefore, both pnr and the iro-C appear to prepattern a region of the notum, at the intersection of their expression domains, which could correspond to the medial-lateral band of wg expression overlapping these domains (Ikmi, 2008).

mirr LoF leads to embryonic lethality. Although the targets of ara are yet to be identified, ara LoF causes pupal lethality, whereas flies lacking caup function are viable and exhibit developmental defects. Thus, ara, caup and mirr play distinct biological functions during development. These different roles can be attributed in part to differences in expression pattern. For instance, mirr is the first iro-C gene that is detected during embryogenesis and it is the only iro-C gene that is expressed and plays a role in oogenesis. However, this diversity in expression domains cannot account for all the observed functional differences. The overexpression of these three proteins in the same domains (here with the same caup-Gal4 driver) have different consequences: while overexpression of caup and ara to more than 10 times the normal level is compatible with viability, the overexpression of mirr to the same level is lethal. When expressed at levels compatible with viability, mirr is unable to rescue the caup LoF while ara is. Thus, the differences in the iro-C genes roles cannot be only attributed to differences in their patterns of expression but rather should also be due to differences in their coding sequences (Ikmi, 2008).

In mammals, there are two clusters of three iroquois genes (IrxA and IrxB). Expression patterns, LoF and misexpression studies reveal a similar situation where the Irx genes may have redundant or non-redundant roles, depending on the gene and/or on the developmental process (Ikmi, 2008).

The roles of the Drosophila iro-C genes have been documented in numerous other developmental processes, for instance: eye dorsal-ventral patterning, wing veins patterning, wing-body wall boundary, dorsal-ventral axis formation in oogenesis. This study has put together the tools and conditions allowing to address the question of the specific roles and/or of the functional redundancy of the iro-C gene in these other developmental processes (Ikmi, 2008).

The long-term fates for duplicated genes include inactivation, maintenance and diversification. Maintenance of duplicated developmental genes can be the result of selective pressure exerted through dosage requirements and/or contribution to the genetic and developmental robustness necessary to reproducibly elaborate correct patterning of diverse territories. Alternatively, the subfunctionalization model proposes that, after a duplication of genes, each copy may sustain deleterious mutations in different structural and/or regulatory elements. Eventually, the ancestral functions are partitioned between the copies that are both retained (Ikmi, 2008).

An interesting example is the ac-sc complex. Similarly to ara and caup, ac and sc arose from the most recent duplication event in this complex. It has been shown that ac, but not sc, is dispensable for the development of the sensory bristles. Two hypotheses have been proposed for the reason why evolution has retained ac: first, an as yet undiscovered function; second (the favored hypothesis), a contribution to genetic robustness. However the situations of the ac-sc and iro complexes are not identical: no phenotypic consequence of the ac LoF in has been found in an otherwise wt background while this study observed ara LoF cause lethality. The reasons why the three Drosophila iro-C genes are maintained appear thus different and may proceed from the two (non-exclusive) hypotheses mentioned above. mirror has clearly evolved to perform different functions from ara and caup. This can be seen in expression pattern as well as in LoF phenotypes and in the functional properties of the protein. This study has provided evidences for the functional divergence between ara and caup: first, the LoF of ara is lethal while the LoF of caup is not; second, caup is required for the neural and the muscular prepatterning of the prospective notum, while ara is dispensable. Additionally, subtle differences exist in their expression pattern in the L3 wing disc. Other differences have yet to be characterized more precisely at other stages and in other tissues and their role investigated. The three iro-C genes appear in the process of diversification and subfunctionalization, both in their expression domains and in the functions of their encoded proteins. In addition, the partial ability of ara to perform caup functions, at least in the neural patterning of the notum, may contribute to buffer this patterning against intrinsic and extrinsic perturbations. It could thus be retained by a selective pressure at work on fly populations surviving in the wild (Ikmi, 2008).

Targets of Activity

The number of epidermal cells expressing the muscle attahment markers groovin, delilah and ß1-tubulin is greatly reduced and their normally stereotyped expression patterns are strongly disturbed in sr mutants (Frommer, 1996).

Thrombospondin, acting downstream of stripe, mediates in the formation of the myotendinous junction

Organogenesis of the somatic musculature in Drosophila is directed by the precise adhesion between migrating myotubes and their corresponding ectodermally derived tendon cells. Whereas the PS integrins mediate the adhesion between these two cell types, their extracellular matrix (ECM) ligands have been only partially characterized. This study shows that the ECM protein Thrombospondin (Tsp), produced by tendon cells, is essential for the formation of the integrin-mediated myotendinous junction. Tsp expression is induced by the tendon-specific transcription factor Stripe, and accumulates at the myotendinous junction following the association between the muscle and the tendon cell. In tsp mutant embryos, migrating somatic muscles fail to attach to tendon cells and often form hemiadherens junctions with their neighboring muscle cells, resulting in nonfunctional somatic musculature. Talin accumulation at the cytoplasmic faces of the muscles and tendons is greatly reduced, implicating Tsp as a potential integrin ligand. Consistently, purified Tsp C-terminal domain polypeptide mediates spreading of PS2 integrin-expressing S2 cells in a KGD- and PS2-integrin-dependent manner. A model is proposed in which the myotendinous junction is formed by the specific association of Tsp with multiple muscle-specific PS2 integrin receptors and a subsequent consolidation of the junction by enhanced tendon-specific production of Tsp secreted into the junctional space (Subramanian, 2007).

Tsp was initially recovered in a microarray screen for genes that are downstream of Stripe by comparing the gene expression profile of embryos overexpressing Stripe in the ectoderm with that of wild-type embryos. Further analysis showed that in stripe mutant embryos Tsp protein is still detected, possibly because of earlier Stripe-independent transcriptional input. Importantly, overexpression of Stripe using the engrailed-gal4 driver leads to a significant induction of Tsp expression, confirming the microarray results and the ability of Stripe to induce Tsp expression. Because Stripe expression is greatly upregulated following muscle-tendon interaction, it is assumed that Stripe-dependent Tsp induction is linked to muscle-tendon interaction. It is concluded that Tsp distribution is dynamic and correlates with the biogenesis of adherens junction formation (Subramanian, 2007).

Drosophila tsp is expressed in all ectodermal tendon precursor cells, strongly enriched in those positioned at the segment border of the embryo. Furthermore, tsp is expressed in all differentiated tendon cells after muscle contact. Therefore, tsp is expressed in all cells that have previously been identified by the expression of stripe. stripe encodes an EGR-type Zn-finger transcription factor that is required for tendon cell differentiation. Like stripe, the initial expression of tsp is controlled by Hedgehog signaling at the segment borders and requires stripe activity only during the later stages when the tendon cells are already differentiated. These results suggest that the genes stripe and tsp are activated in parallel by Hh-dependent Ci activity, and that stripe activity maintains the expression of tsp during the later stages when Ci activity has ceased (Chanana, 2007).

Post-transcriptional Regulation

Differential RNA metabolism regulates a wide array of developmental processes. A mechanism is described that controls the transition from premature Drosophila tendon precursors into mature muscle-bound tendon cells. This mechanism is based on the opposing activities of two isoforms of the RNA binding protein How. While the isoform How(L) is a negative regulator of Stripe, the key modulator of tendon cell differentiation, How(S) isoform elevates Stripe levels, thereby releasing the differentiation arrest induced by How(L). The opposing activities of the How isoforms are manifested by differential rates of mRNA degradation of the target Stripe mRNA. This mechanism is conserved, as the mammalian RNA binding Quaking proteins may similarly affect the levels of Krox20, a regulator of Schwann cell maturation (Nabel-Rosen, 2002).

RNA binding proteins of the Signal Transduction and Activation of RNA (STAR) family may regulate gene expression at various levels, e.g., at the level of nuclear export of the target mRNA, at the level of mRNA stability, and at the translation level. The How proteins appear to exert their activity through their effect on mRNA stability. How(L) appears to induce rapid degradation of the target RNA, an activity that is tightly coupled to its nuclear retention and depends on the presence of the nuclear retention signal that is conserved in QKI-5. It has been suggested that How(L) may prevent nuclear export of its target mRNA. Indeed, in embryos overexpressing How(L), the mRNA of gfp-sr3'UTR is occasionally detected in the nucleus. Presently, it is not possible to determine whether the primary effect of How(L) is retention of the target mRNA in the nucleus followed by degradation of the target mRNA or vice versa. How(S) increases the stability of the same target RNA. The fact that How(S) is present both in the nucleus and in the cytoplasm raises the possibility that the association of How(S) with its target RNA during and following its nuclear export leads to mRNA stabilization. A number of RNA binding proteins possess both nuclear and cytoplasmic functions, e.g., proteins that affect both mRNA export and mRNA stability. Similarly, How proteins may affect both nuclear-cytoplasm shuttling and mRNA stability. The differential association of each of the How proteins with distinct protein partners presumably leads to their opposing effects on mRNA stability. A possible mechanism for the counteraction effect of How(S) may arise from its association with How(L), eliminating the repression by How(L). Indeed, How(S) and How(L) are coprecipitated from Schneider cells following their cotransfection together with the gfp-sr3'UTR (Nabel-Rosen, 2002).

A recent report suggests that a sequence (TGE) in the 3'UTR of C. elegans tra-2, essential for Gld-1 binding, mediates deadenylation and poly(A)-dependent translation repression. Poly(A) deadenylation may also lead to mRNA degradation. Since a sequence motif that is partially related to TGE is also present in the stripe and krox20 3'UTR, degradation of the target mRNA by How(L) may be based on a similar mechanism. Recently, the two cytoplasmic hnRNPs, K and E1, have been shown to inhibit translation of lipoxigenase mRNA by preventing its attachment to the 60S ribosomal unit (Ostareck, 2001). The possibility cannot be excluded that How(S), in addition to its positive effect on mRNA stability, may also facilitate translation efficacy (Nabel-Rosen, 2002).

The results suggest that the relative amount of How(L) and How(S) during different stages of embryonic or adult development regulate the switch between the premature and mature state of differentiation of tendon cells. In early stages of embryonic development, How(L) prevails, Stripe expression is downregulated, and differentiation is arrested. In later stages of embryonic development, How(S) is upregulated, overriding How(L) repression and facilitating Stripe expression. The difference in Stripe mRNA levels may be further enhanced by Stripe transcriptional autoregulation. What could be the mechanism that regulates the relative levels of How proteins during tendon cell maturation? Northern analysis suggests that the total levels of How(L) mRNA are low throughout embryonic development, relative to those of How(S) mRNA. At the protein level, the proportion of the two proteins is inverted; How(S) protein levels are low and increase only during late stages of embryonic development. This suggests that How(S) is posttranscriptionally regulated. Indeed, transgenic flies carrying How(S) with its unique 3'UTR exhibit almost undetectable levels of How(S) protein following induction by the gal4 driver. When this 3'UTR is deleted, the expression levels of How(S) are significantly higher. The expression of How(S) appears to be elevated by Vein-mediated activation of the ->F receptor pathway in tendon cells following muscle-tendon association. The molecular link between ->F receptor activation and the upregulation of How(S) has yet to be elucidated. A recent report suggesting that ERK phosphorylation of hnRNP-K drives cytoplasmic accumulation of hnRNP-K may be of relevance if, similarly to hnRNP-K, How(S) undergoes ERK-dependent phosphorylation (Nabel-Rosen, 2002).

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).

DEVELOPMENTAL BIOLOGY

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

date revised: 25 August 2007

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