Transcriptional Regulation

Functional intertwining of Dpp and EGFR signaling during Drosophila endoderm induction

Decapentaplegic has a prime function during endoderm induction in Drosophila. Dpp is secreted from the outer cell layer of the embryonic midgut (the visceral mesoderm), where Dpp's main source of expression in parasegment ps7 depends directly on the homeotic gene Ultrabithorax (Ubx). In the same cell layer, Dpp stimulates expression of another extracellular signal, Wingless (Wg), in a neighboring parasegment (ps8), which in turn feeds back to ps7 to stimulate Ubx expression. Thus, Dpp is part of a "parautocrine" feedback loop of Ubx (i.e., an autocrine feedback loop based partly on paracrine action) that sustains its own expression through Dpp and Wg. Dpp also spreads to the inner layer of the embryonic midgut, the endoderm, where it synergizes with Wg to induce expression of the homeotic gene labial (lab). To achieve this, Dpp locally elevates the endodermal expression levels of Drosophila D-Fos with which it cooperates to induce lab. Differentiation of various cell types in the larval gut depends on these inductive effects of Dpp and Wg. A cAMP response element (CRE) from the Ubx midgut enhancer has been shown to be necessary and to some extent sufficient to mediate the Dpp response in the embryonic midgut (Eresh, 1997).

CREs are known to be signal-responsive elements, not only for cAMP signaling as described initially but also for other signals including ones acting through Ras. This prompted an investigation of whether any other signal may play a part in the Dpp response. This led to the discovery that the Drosophila epidermal growth factor receptor (Egfr) has a critical function during endoderm induction. Acting as a secondary signal with a permissive role in this same process is Vein, a neuregulin-like ligand that stimulates the epidermal growth factor receptor and Ras signaling. Dpp and Wg up-regulate vein expression in the midgut mesoderm in two regions overlapping the Dpp sources. This up-regulation depends on dpp and wg. Vein is thus a secondary signal of Dpp and Wg, and it stimulates homeotic gene expression in both cell layers of the midgut (Szuts, 1998).

EGFR expression is thought to be fairly ubiquitous in the embryo. However, vein transcripts are found in a highly restricted pattern, primarily in the embryonic mesoderm. In the midgut too, vein expression is spatially regulated, as follows: vein transcripts in the midgut are restricted to the visceral mesoderm. Initially, during stage 13, low levels of vein expression are seen at intervals throughout the midgut mesoderm. However, soon after the formation of the midgut epithelium, vein transcripts start to accumulate locally, and two main domains of prominent vein expression develop, one in the anterior and one in the middle midgut. Anteriorly, vein expression spans approximately ps2-ps4 and is strongest around the ps3/ps4 junction, that is, posterior to the gastric caeca. In the middle midgut, there is a fairly wide band of low vein expression spanning approximately ps6-ps10, with strongly up-regulated expression levels throughout ps7 (and trailing into anterior ps8). Posterior ps7 becomes the most prominent site of vein expression in the midgut. Finally, a narrow band with low levels of vein transcripts is seen at the posterior end of the midgut. The two main expression domains of vein overlap the two domains of Dpp expression in the visceral mesoderm (in ps3 and ps7), but each of them is considerably wider than the corresponding dpp domain. vein expression in the visceral mesoderm is severely diminished in dpps4 mutants. The prominent band of vein expression in ps7 is no longer seen, and expression in ps4 is reduced too. Instead, the strongest expression of vein in these mutants is seen at a novel location, at the ps5/ps6 junction around the incipient first midgut constriction (this ps5/ps6 expression is higher than in the wild type, and can be used to identify young dpp mutant embryos). It is concluded that dpp is required for the localized up-regulation of vein expression in the midgut (Szuts, 1998).

vein expression is also strongly diminished in wg mutants. vein expression can still be seen at moderate levels in the ps4 region, but vein expression is barely visible elsewhere in the midgut of these mutants. In particular, there are only traces of vein expression in the ps7/ps8 region, and expression at both midgut ends is almost undetectable. Clearly, wg plays an essential role as well in up-regulating vein expression. dpp and wg are sufficient to position the two domains of vein up-regulation. High mesodermal Wg causes very strong vein expression in ps2-ps7, significantly stronger than that caused in this region by mesodermal Dpp expression alone. This indicates that wg cooperates with dpp in positioning vein up-regulation. It is shown that neither Dpp for Egfr signaling is particularly effective in the absence of the other. Thus these two pathways are functionally interdependent and that they synergize with each other, revealing functional intertwining (Szuts, 1998).

The mutant analysis suggests strongly that Vein is the main, if not the only, ligand that stimulates Egfr in the embryonic midgut. This contrasts with other tissues, mainly of ectodermal origin, in which Spitz is the main Egfr ligand. Interestingly, Vein also has a major role during an inductive process between muscle and epidermis: Vein is secreted from muscle cells and triggers differentiation of the receiving epidermal cells into tendon cells. These functions of Vein during inductive processes between different cell layers suggest that the molecular properties of Vein are particularly suited to such processes that require the signal to cross basal membranes. Similarly, the extensive mesodermal expression of Vein may mean that this signal protein is particularly well-adapted to its production in this cell layer. Note that Vein is similar to mammalian neuregulins that appear to function in developmental contexts that involve communication between different cell layers (Szutz, 1998 and references).

The transcriptional response elements for the Dpp signal in midgut enhancers from homeotic target genes are bipartite, comprising CRE sites as well as binding sites for the Dpp signal-transducing protein Mad. Of these sites, the CRE seems to function primarily in the response to Ras, the secondary signal of Dpp. It is also shown that the Dpp response element in the labial enhancer comprises CREs and Mad binding sites. The results with the labial enhancer confirm the conclusions derived from the Ubx enhancer, namely that the response element to Dpp signaling is bipartite and contains Mad binding sites as well as CREs. The latter are critical in both cell layers for the signal response, whereas the former seem less criticial in the endoderm than in the visceral mesoderm. Perhaps this reflects the fact that lab is the ultimate target gene of the endoderm induction and that its enhancer clearly integrates a number of distinct positional inputs, some of which may be partially redundant (Szuts, 1998).

Why should there be this secondary signal whose role is entirely permissive, namely to assist the primary signal in implementing its tasks? Two kinds of answers are proposed. The first one is based on the observation that lack of Vein/Egfr signaling in the midgut appears to make cells sick and perhaps causes them to die. Therefore, Vein/Egfr signaling may serve as a "survival signal." Intriguingly, cell survival in embryos lacking vein or Egfr function appear to be affected preferentially near the two Dpp sources (where vein expression is up-regulated). Perhaps high levels of Dpp signaling can cause cell death; if so, vein signaling may be up-regulated to counteract a putative local deleterious effect of Dpp. A precedent for such a scenario may be found in the developing chick limb bud where the cell death-inducing properties of BMP (a TGF-beta-like signal) seem to be antagonized locally by a signal triggering the Ras pathway. However, although antagonistic effects between Egfr- and TGF-beta-type signaling have been observed, the evidence provided here suggests strongly that Vein/Egfr and Dpp both act positively in the embryonic midgut of Drosophila. Furthermore, they synergize with each other in the transcriptional stimulation of target genes. This observed synergy parallels cooperation between Ras and TGF-beta signaling during epithelial tumor progression. It is therefore thought unlikely that Vein functions in the midgut entirely as a survival signal near Dpp sources (Szuts, 1998).

The second kind of answer builds on the observations that indicate functional interdependence and synergy between the two signaling pathways in stimulating transcription of target genes. This could be beneficial for developmental systems in two ways: (1) if cells need to be costimulated by cooperating primary and secondary signals, this would serve to sharpen their signal response. This putative sharpening effect may be a contributory factor in sharp responses to signaling thresholds such as those observed in the Xenopus embryo.(2) The need for costimulation would safeguard against fortuitous and random stimulation of cells by any one signal, thus improving the reliability of their signal response. And although a requirement for the secondary signal is observed throughout the functional realm of the primary signal, it is envisaged that the role of the secondary signal is particularly critical in remote cells where the distribution of the primary signal becomes shallow, imprecise, and unreliable. Therefore, the secondary signal may provide primarily "remote stimulation." Whatever the case, it seems very likely that the use of a functionally coupled primary-secondary signal system results in a refinement and stabilization of positional information and in a degree of precision of this information that could not be conferred by one signal alone. Functional intertwining of a secondary and a primary signal may represent a mechanistic solution of how morphogens such as Dpp and activins work. Perhaps, signaling pathways do not function on their own in eliciting multiple different cellular responses, as envisaged by the purest version of the morphogen concept (Szuts, 1998).

Vein expression is induced by the EGF receptor pathway to provide a positive feedback loop in patterning the Drosophila embryonic ventral ectoderm

Although there is a single EGF receptor in Drosophila, multiple ligands activate it. This work explores the role of two ligands, Spitz and Vein, in the embryonic ventral ectoderm. Spitz is a potent ligand, whereas Vein is an intrinsically weak activating ligand. Prior to gastrulation, vein is expressed in the future neuroectoderm. This early phase of expression appears to cooperate with Spitz in the induction of medial neuroblast cell fates. Following gastrulation at stage 9, vein expression in the neuroectoderm becomes confined to three to four cell rows on either side of the midline. This pattern gradually restricts, such that by stage 11 only one cell row on each side of the midline expresses vein. Secreted Spitz, emanating from the midline, triggers expression of vein in the ventral-most rows of cells, by inducing expression of the ETS domain transcription factor Pointed P1. In the absence of Vein, lateral cell fates are not induced when Spitz levels are compromised. The positive feedback loop of Vein generates a robust mechanism for patterning the ventral ectoderm (Golembo, 1999).

Expression of vein is observed in positions adjacent to the ventral midline, where activation of the Egfr pathway by secreted Spitz emanating from the midline is maximal. This raised the possibility that vein expression may be triggered by Spitz. In rhomboid mutant embryos in which active Spitz molecules are not produced, no expression of vein is observed at stage 11, demonstrating that Rho/Spitz are indeed necessary for vein expression in the ventral ectoderm. To test if Egfr activation is sufficient to induce vein expression in the embryo, the pathway was ectopically activated at two different stages. Rho was ectopically expressed at stage 9 in the central domain of the embryo by Kruppel-Gal4, and this gives rise to ectopic vein expression in the same region. At stage 11 secreted Spitz was expressed in the engrailed domains, resulting in the induction of vein expression in the same pattern. These experiments demonstrate that high levels of Egfr activation are sufficient for inducing vein expression (Golembo, 1999).

Induction of gene expression by Egfr in the ventral-most rows of cells occurs for the argos, orthodenticle, and tartan genes. Induction is obtained through inactivation of Yan, an ETS domain transcriptional repressor, and induction of Pointed P1, an ETS domain transcriptional activator. To test if induction of vein by Egfr is also mediated by Pointed P1, vein expression was examined in pointed mutant embryos. Traces of expression are observed in the midline in stage 10 embryos only, whereas no expression is displayed by the ventral-most and lateral ectodermal cell rows at stage 11, thus showing that vein induction is defective in pointed mutants. Elimination of Pointed activity can also be obtained in the following manner: an activated form of Yan, in which the inactivating MAP kinase phosphorylation sites have been mutated, blocks the activity of Pointed by competing for the same DNA-binding sites. Indeed, when activated, Yan is ectopically expressed in the Kruppel domain the endogenous expression of vein is abolished in that region. To examine if Pointed P1 is sufficient for induction, vein expression was examined in embryos in which Pointed P1 was expressed in the Kruppel domain. Indeed, expression of vein in the same domain is observed. These results demonstrate that under conditions of ectopic expression, Pointed P1 is necessary and sufficient for vein expression (Golembo, 1999).

Two different nested cell fates are induced by the Egfr pathway in the ventral ectoderm, depending on the distance of the cells from the midline. The cell rows closest to the midline assume the ventral-most fate, as reflected by the expression of target genes such as otd and argos. Intermediate levels of Egfr activation induce lateral cell fates, reflected by the expression of FasIII, which is observed in five rows of cells on each side of the midline. Both ventral-most and lateral markers are eliminated in mutants for Egfr, as well as in mutants that abolish Spitz or its processing (spitz, rho, or Star). Conversely, ectopic secreted Spitz or Rho is capable of expanding expression of both lateral and ventral-most fates. To examine the role of Vein in the ventral ectoderm, vein null mutants were examined for the expression of marker genes. No defects in the expression of otd or FasIII were observed. These results indicate that at this level of resolution, the function of Vein is redundant. This is also consistent with normal levels of activated MAP kinase that are observed at stage 9 in vein mutant embryos. A role for Vein is revealed under conditions in which the level of Spitz is compromised. Flies that are heterozygous for a null allele of Spitz are viable. Normal patterning of the ventral ectoderm takes place in heterozygous spitz embryos, as monitored by the expression pattern of FasIII. Embryos were generated that are homozygous for the vein null allele and carry only one functional copy of spitz. In these embryos, patterning of the ventral-most cells is normal, as reflected by the expression of otd or FasIII. However, more lateral cells fail to express FasIII. These results indicate that when Spitz levels are compromised in heterozygous embryos, the cell row closest to the midline undergoes normal patterning. However, the levels of Spitz may be too low to pattern the more lateral cells. Under these conditions, induction of Vein appears to be critical to facilitate Egfr activation in these cells. Upon ectopic expression of Vein, the ventral-most markers are not induced at all or only intermittently, thus reflecting the reduced inherent activity of Vein. To test directly the biological activity of Vein in a controlled setting, it is necessary to eliminate endogenous signaling by Spitz, as well as the presence of Argos. This can be achieved in embryos that are mutated for rhomboid, and thus exhibit no expression of the ventral-most markers (such as argos) or lateral markers. Ectopic expression of Vein in rho mutant embryos is capable of restoring FasIII expression, but did not induce otd expression. Thus, Vein is capable of inducing the lateral cell fates in the absence of secreted Spitz (Golembo, 1999).

Two distinct mechanisms have been proposed for patterning by morphogens. In one case, a gradient of a single morphogen gives rise to distinct cell fates caused by the induction of different levels of signaling in the same pathway. The complementary scenario involves a relay mechanism: an initial induction by the primary morphogen induces the production of a relay factor triggering another signaling pathway, to pattern the more distant cells. This work describes a combination of the two models. Only the EGF receptor cascade patterns the ventral ectoderm. However, the primary signal, Spitz, induces a relay mechanism by triggering expression of Vein, another ligand of Egfr. Again, it is important to emphasize that although the restricted spatial distribution of secreted Spitz is critical for correct patterning, Vein and Argos distribution may be more uniform. Argos reduces the overall level of EGF receptor signaling, whereas Vein provides a lower level of activation, capable of inducing only the lateral cell fates. vein expression is also induced by the EGF-receptor pathway in follicle cells within the dorsal-anterior corner of the egg chamber. It thus appears that Vein may provide a positive feedback loop in several tissues that are patterned by EGF receptor activity (Golembo, 1999 and references).

Hedgehog activates the EGF receptor pathway during Drosophila head development

Another facet of the activity of Vein that should be considered is its sensitivity to Argos inhibition. If the lateral cells have not encountered sufficient levels of Spitz prior to the induction of Argos, their capacity for activation in the presence of Argos is severely compromised, and relies on the continued availability of secreted Spitz from the midline. The induction of Vein in the ventral-most cells, which takes place in parallel to the induction of Argos, may help to overcome this problem. Vein is capable of activating Egfr in the presence of Argos: in embryos heterozygous for spitz, the activity of Vein induces the lateral fates. This takes place in a situation in which argos is expressed in the ventral-most cells. Vein itself is not capable of inducing expression of argos. In conclusion, this work has revealed a powerful regulatory network, orchestrated by the sequential utilization of Spitz and Vein, two ligands of the EGF receptor with different properties. Induction of Vein takes place once the ventral-most cell fates already have been determined by high Spitz levels. Vein expression prolongs the time window of activation of the Egfr pathway to ensure that the lateral cell fates will be specified correctly. Vein is suited to this task, since it is a less potent ligand than Spitz, capable of inducing only the lateral cell fates. Vein can activate Egfr even in the presence of Argos, thus balancing the parallel negative-feedback loop of Argos (Golembo, 1999).

The Hedgehog (Hh) and Epidermal growth factor receptor (Egfr) signaling pathways play critical roles in pattern formation and cell proliferation in invertebrates and vertebrates. In this study, a direct link between these two pathways is demonstrated in Drosophila. Hh and Egfr signaling are each required for the formation of a specific region of the head of the adult fruitfly. hh and vein (vn), which encodes a ligand of the Drosophila Egfr, are expressed in adjacent domains within the imaginal primordium of this region. Using loss- and gain-of-function approaches, it has been demonstrated that Hh activates vn expression. Hh activation of vn is mediated through the gene cubitus interruptus (ci) and this activation requires the C-terminal region of the Ci protein. wingless (wg) represses vn expression, thereby limiting the domain of Egfr signaling (Amin, 1999).

The dorsal head capsule, which lies between the compound eyes, contains three morphologically distinct domains. The medial domain includes the ocelli and their associated bristles, which lie on the triangular ocellar cuticle. The mediolateral region contains the frons cuticle, which consists of a series of closely spaced parallel ridges. The lateral region is occupied by the orbital cuticle, which contains a stereotypical pattern of bristles. The head capsule forms primarily from the two eye-antennal imaginal discs. Each half of the dorsal head derives from a primordium in the disc immediately adjacent to the anlage of the compound eye. During the pupal stage, the two discs fuse at what will form the midline of the dorsal head capsule (Amin, 1999).

hh is expressed within the medial domain of the dorsal head capsule. Specifically, it is expressed in the interocellar cuticle, which contains the small interocellar bristles. Using a vn-lacZ strain, vn is found to be expressed in the dorsal head capsule. vn expression lies primarily within the mediolateral frons cuticle, near but not immediately adjacent to the region of hh transcription. The regions of hh and vn expression in the dorsal head primordium of the eye-antennal disc are compared. Consistent with its expression on the adult head capsule, hh is expressed in the region of this primordium that lies between the precursor cells of the ocelli. vn is expressed in the wing and haltere discs, but its expression in the eye-antennal disc has not been described. Using both the vn-lacZ strain and in situ hybridization with a vn probe, vn is also found to be expressed in the dorsal head primordium. As on the adult head, vn expression lies near that of hh. Double-labeling shows that the domains of hh and vn expression are immediately adjacent to each other. In situ hybridization reveals that vn is also expressed at low levels in the morphogenetic furrow. Eliminating Hh function during head development results in the deletion of the entire medial domain, including the interocellar cuticle and bristles, and the ocelli and their associated bristles. This region is replaced by frons cuticle, which is normally confined to the mediolateral region of the head capsule. Ectopic hh expression generates ectopic medial structures at more lateral positions. Hh is therefore necessary for the specification of the medial domain and sufficient to direct more lateral regions of the dorsal head towards a medial fate (Amin, 1999).

Particular combinations of Egfr alleles cause a reduction in the size of the ocelli and the loss of the two ocellar bristles, which flank the medial ocellus. Since vn is expressed within the dorsal head primordium, the effects of eliminating either vn expression or Egfr-mediated signaling on head development were determined. Examination of vn mutant clones shows that Vn is required for the development of some, but not all, of the Hh-dependent medial head structures. The ocelli and ocellar bristles are deleted and the postvertical bristles, which lie near the lateral ocelli, are also lost. However, most of the interocellar cuticle is retained, indicating that the vn dorsal head phenotype is less global than that caused by loss of Hh function. Since vn encodes a ligand for Egfr, the effects of eliminating Egfr-mediated signaling on head development were examined. To do so, the GAL4/UAS system was used to express a dominant negative form of the Egfr (DN-DER) across the entire dorsal head primordium. DN-DER expression eliminates the same structures deleted in vn clones, suggesting that vn is primarily responsible for activating Egfr signaling in this region. As was the case for Vn, the interocellar cuticle is retained in the absence of Egfr signaling (Amin, 1999).

Since the hh mutant phenotype is more extensive than either the vn or Egfr phenotypes, a test was made to determine whether Hh acts upstream of the Egfr pathway. Using a temperature-sensitive hh allele (hhts2), Hh function was eliminated during the third instar larval stage. Loss of Hh eliminates or strongly reduces vn expression in both the dorsal head primordium and the morphogenetic furrow. To determine whether Hh can induce vn expression outside the dorsal head primordium, ectopic hh expression was induced using the Flp recombinase technique. Hh is found to be capable of activating vn in other regions of the eye disc. A disc-specific enhancer from the dpp gene was used to induce ectopic hh expression using the GAL4/UAS system. This enhancer drives reporter gene expression at the posterior and lateral margins of the third instar eye disc as well as in a portion of the antennal anlage. Ectopic hh expression induced by this enhancer severely disrupts eye-antennal disc morphology. It also induces a band of ectopic vn expression anterior to the region of hh transcription. Combined with the previous results, these experiments demonstrate that Hh is necessary for vn expression in the dorsal head primordium, and is sufficient to induce ectopic vn expression in other regions of the disc (Amin, 1999).

To test whether Hh is also required to activate Egfr-mediated signaling, a monoclonal antibody that specifically recognizes the active, dually phosphorylated form of mitogen-activated protein kinase (dp-ERK) was used. dp-ERK is expressed at high levels in the morphogenetic furrow, and at lower levels in ommatidia posterior to the furrow. When the anti-dp-ERK signal is allowed to develop for longer periods, weaker expression appears in cells within the dorsal head primordium. Eliminating Hh function reduces or eliminates dp-ERK expression both in these cells and in the furrow. Hh mediated induction of the Egfr pathway has been shown to be medated by Cubitus interruptus. Expression of an N-terminal fragment of Ci with repressor activity reduces or eliminates vn expression in the dorsal head analage. On the contrary, expression of the Ci155 activator increases the intensity and extent of vn expression and causes the ocelli to increase in size and fuse (Amin, 1999).

wingless is broadly expressed throughout the early eye-antennal disc, where it confers a default state of head cuticle. Later, wg expression becomes restricted to the primordia of the orbital cuticle and ptilinium, and to a portion of the antennal anlage. Just as hh expression is medially adjacent to that of vn on the adult head capsule, wg expression abuts vn in the frons both laterally and anteriorly. Loss of Wg signaling causes the deletion of both the frons and orbital cuticles. To determine whether Wg participates in vn regulation, a temperature-sensitive allele was used to eliminate Wg function during second instar development. In contrast to Hh, Wg negatively regulates vn. Loss of Wg activity during this time window expands the domain of vn expression in the dorsal head primordium and induces ectopic vn expression in other regions of the eye-antennal disc (Amin, 1999).

Connecting Hh, Dpp and EGF signalling in patterning of the Drosophila wing; the pivotal role of collier/knot in the AP organiser

Hedgehog (Hh) signaling from posterior (P) to anterior (A) cells is the primary determinant of AP polarity in the limb field in insects and vertebrates. Hh acts in part by inducing expression of Decapentaplegic (Dpp), but how Hh and Dpp together pattern the central region of the Drosophila wing remains largely unknown. The role played by Collier (Col), a dose-dependent Hh target activated in cells along the AP boundary (the AP organizer in the imaginal wing disc) has been examined. col mutant wings are smaller than wild type and lack L4 vein, in addition to missing the L3-L4 intervein and mis-positioning of the anterior L3 vein. These phenotypes are linked to col requirement for the local upregulation of both emc and N, two genes involved in the control of cell proliferation, the EGFR ligand Vein and the intervein determination gene blistered. Attenuation of Dpp signaling in the AP organizer is also col dependent and, in conjunction with Vein upregulation, required for formation of L4 vein. A model recapitulating the molecular interplay between the Hh, Dpp and EGF signaling pathways in the wing AP organizer is presented (Crozatier, 2002).

Col regulates vn transcription in the AP organizer. This expression of vn is required for formation of L4 vein. The loss of this vein in col1 mutants could not, however, be rescued by expressing high level of Vn in the AP organizer, suggesting that a second signal dependent upon Col is also required. The specific loss of L4 vein is observed in conditions of reduced levels of Dpp signaling caused by ubiquitous expression of either Tkv or TkvDN. Together with this, the col1 wing phenotype and Col requirement for downregulation of Dpp signaling in the AP organizer suggests a role for Dpp signaling in formation of L4 vein. Indeed, expressing a dominant-negative form of Tkv (TkvDN) in the AP organizer results in L4 loss. At first sight, it may appear contradictory that either upregulation (in col mutants) or downregulation (by expressing TkvDN) of Dpp signaling in the AP organizer leads to the preferential loss of posterior L4 vein. In both cases, however, there is increased sequestering of Dpp in the AP organizer, which limits its range of diffusion and signaling in posterior cells. Therefore attenuation of Dpp signaling in the AP organizer and increased signaling in posterior flanking cells appears to be required in addition to Vn activity for formation of L4 vein. By modulating Dpp signaling and vn transcription in cells receiving high doses of Hh, Col thus links Hh short-range activity to both positioning of the anterior L3 vein and formation of the posterior L4 vein (Crozatier, 2002).

Negative regulation of Egfr/Ras pathway by Ultrabithorax during haltere development in Drosophila

In Drosophila, wings and halteres are the dorsal appendages of the second and third thoracic segments, respectively. In the third thoracic segment, homeotic selector gene Ultrabithorax (Ubx) suppresses wing development to mediate haltere development. Halteres lack stout sensory bristles of the wing margin and veins that reticulate the wing blade. Furthermore, wing and haltere epithelia differ in the size, shape, spacing and number of cuticular hairs. The differential development of wing and haltere, thus, constitutes a good genetic system to study cell fate determination. Down-regulation of Egfr/Ras pathway is critical for haltere fate specification: over-expression of positive components of this pathway causes significant haltere-to-wing transformations. RNA in situ, immunohistochemistry, and epistasis genetic experiments suggest that Ubx negatively regulates the expression of the ligand vein as well as the receptor Egf-r to down-regulate the signaling pathway. Electromobility shift assays further suggest that Egf-r is a potential direct target of Ubx. These results and other recent findings suggest that homeotic genes may regulate cell fate determination by directly regulating few steps at the top of the hierarchy of selected signal transduction pathways (Pallavi, 2006).

To identify potential targets of Ubx and thereby mechanism of its function, a gain-of-function genetics strategy was employed. Ubx-GAL4 driver, is expressed in the entire anterior compartment of the haltere imaginal disc. Ubx-GAL4 is also a null allele of Ubx and exhibits characteristic dominant phenotype; the presence of wing-type sensory bristles in the capitellum of the haltere. This GAL4 driver provides a fortuitous sensitive background to carry out large-scale screens for identifying the suppressors and enhancers of Ubx function, which otherwise may be less efficient in a wild type background. Indeed, over-expression of Vestigial (Vg), a pro-wing gene and a target of Ubx function, in the developing haltere results in very high degree of haltere-to-wing homeotic transformations, A candidate gene screen was employed to identify downstream targets of Ubx, in which various genes known to be involved in wing development were ectopically expressed in the developing haltere using the Ubx-GAL4 driver. Criterion for defining haltere-to-wing transformation in this study was the presence of wing-type sensory bristles, although increase in haltere size and enhanced pigmentation was frequently observed. For comparison between different genotypes, the degree of transformation was estimated by counting the number of sensory bristles on the haltere capitellum. UAS stocks for a large number of such wing patterning genes were crossed to Ubx-GAL4 driver and were scored for enhancement and suppression of the dominant phenotype of heterozygous Ubx. Progeny for many of the crosses resulted in early embryonic or early larval lethality, reflecting the fact that the GAL4 driver expresses at early stages during embryonic development. Nevertheless, strong haltere-to-wing transformations were observed upon over-expression/mis-expression of most of the positive components of the Egfr/Ras pathway. For example, the bristle number in the capitellum of the Ubx-GAL4 haltere increased when positive components such as Vn or Egf-r were over-expressed and over-expression of negative components such as Aos completely suppressed the heterozygous Ubx phenotype (Pallavi, 2006).

A significant finding of this study is the down-regulation of Egfr/Ras pathway in haltere discs by Ubx. Earlier reports suggest that a short-range signal originating from the D/V boundary activates Egfr/Ras pathway in a zone of cells on the edges of the D/V boundary and that this activation is essential for vg transcription (Nagaraj, 1999). Egfr/Ras pathway has also been implicated in the developmental events along the A/P axis: in wing vein specification. Consistent with the down-regulation of both A/P and D/V signaling events in haltere discs, expression of most of the Egfr/Ras pathway components is repressed in the entire haltere pouch. Observations on the strengths of haltere-to-wing transformations (at the margin bristle level) in different genetic backgrounds establish the specificity of genetic interactions between Egfr/Ras pathway and Ubx during haltere development (Pallavi, 2006).

The abovementioned results on the down-regulation of Egfr/Ras pathway in haltere discs by Ubx are consistent with the previously reported genetic screen for the modifiers of homeotic genes, which indicates that Ras1 activity modulates functions of the homeotic loci Sex combs reduced (Scr) and Ubx. For example, haploinsufficient (haltere-to-wing) phenotype of Ubx109/+ is significantly enhanced in Gap1Ubx109/Gap1 adults (Gap1 is a negative regulator of the Egfr/Ras pathway). This effect of Gap1 is reversed in Gap1Ubx109/Gap1Rase1b individuals due to the antagonistic roles of Gap1 and Ras1 in the Egfr/Ras pathway (Pallavi, 2006).

All the components of the Egfr/Ras pathway tested so far are differentially expressed between wing and haltere discs. This suggests the utility of developing wings and halteres as assay systems to identify novel components of Egfr/Ras pathway. Indeed, enhancer-trap screens and microarray analyses to identify genes that are differentially expressed between wing and haltere discs have resulted in the identification of CG32062 (Drosophila homologue of human ataxin-2 binding protein) and Mapmodulin (Drosophila homologue of human Inhibitor-1 of protein phosphatase-2A) as potential modulators of Egfr/Ras pathway (Pallavi, 2006).

The activation of dpERK1/ERK2 and aos in the haltere pouch by the ectopic expression of vn or Egf-r suggests that Ubx regulates Egfr/Ras pathway at ligand as well as receptor levels. Clonal analysis of Ubx function (loss of Ubx in haltere discs and gain in wing discs) demonstrates that Ubx controls vn expression in a cell-autonomous manner. Inability of ectopic Hh to activate vn expression in haltere discs suggests that Ubx functions downstream of Hh to repress vn expression. Putative Ubx-binding sites are present in the cis-regulatory regions of both vn and Egf-r, further suggesting their direct regulation by Ubx. Indeed, electromobility shift experiments suggest that, at least, Egf-r is probably a direct target of Ubx function. Thus, it is likely that Ubx independently down-regulates both vn and Egf-r. Ubx-mediated down-regulation of vn and Egf-r appears to be critical; over-expression of normal or the activated form of Egf-r induced stronger phenotypes when expressed in aos heterozygous background than in Ubx heterozygous background. However, Ubx may exert some influence on the pathway downstream of the receptor, since the strength of the phenotypes induced by the over-expression of Vn or Egf-r was stronger in Ubx heterozygous background. At dpERK1/ERK2 level too, the activation was stronger when Egf-r was over-expressed in Ubx heterozygous genetic background than in the wild type background (Pallavi, 2006).

It is interesting to note that Ubx regulates Egfr/Ras pathway at the level of the receptor itself. Although Egfr/Ras pathway is auto-regulated at several levels including the transcription of Egf-r itself, external factors regulating Egfr/Ras pathway during various developmental events mostly act at the level of the ligand/s or downstream effectors such as MAPK or transcription factors Yan, Pointed, etc. This study has found that the receptor itself is a direct target of Ubx indicating a novel mode of regulation of this pathway (Pallavi, 2006).

Specification of the larval oenocyte has been shown to be dependent on the regulation of just one principal target Rho by the homeotic gene abdominal A. Similarly, Hox proteins AbdA and AbdB specify the lineage of the embryonic NB6-4 neuroblast in abdominal segments by down-regulating CycE. Differential expression of CycE is both required and sufficient to generate segmental differences in NB6-4 lineage. This study reports that down-regulation of Vn and Egf-r is critical for Ubx-mediated suppression of wing margin bristles in the haltere. These results suggest that one common mechanism by which homeotic genes may regulate cell fate determination is by directly regulating few steps at the top of the hierarchy of selected signal transduction pathways. In contrast, Wingless and Decapentaplegic signaling pathways, which regulate more complex traits such as wing growth and shape, are regulated by Ubx at multiple levels in the hierarchy of those pathways (Pallavi, 2006 and references therein).

Although absence of veins in the haltere could be attributed to down-regulation of Egfr/Ras pathway, activation of sensory bristle development in Ubx+/Ubx+ halteres over-expressing positive components of Egfr/Ras pathway suggests a role for this pathway in cell fate specification in the wing margin. So far, no direct role for Egfr/Ras pathway has been assigned in the specification of sensory bristles of the wing margin, although it is known to specify macrochaete of the notum. Indeed, preliminary investigations suggest that Wg pathway induces EGFR/Ras pathway expression in cells immediately adjacent to the D/V boundary, and the latter pathway is required and sufficient to specify sensory organs of the wing margin (Pallavi, 2006).

The bristle development in the transformed halteres appears to be organized in two parallel rows when various components of Egfr/Ras pathway are over-expressed in Ubx heterozygous background, while the bristles are positioned in a disorganized way when phenotypes are induced in wild type background. This could be due to partial de-repression of D/V signaling in Ubx heterozygous background, which may allow appropriate positioning of the zone of margin bristle development (Pallavi, 2006).

Moleskin is essential for the formation of the myotendinous junction in Drosophila

It is the precise connectivity between skeletal muscles and their corresponding tendon cells to form a functional myotendinous junction (MTJ) that allows for the force generation required for muscle contraction and organismal movement. The Drosophila MTJ is composed of secreted extracellular matrix (ECM) proteins deposited between integrin-mediated hemi-adherens junctions on the surface of muscle and tendon cells. This paper identifies a novel, cytoplasmic role for the canonical nuclear import protein Moleskin (Msk) in Drosophila embryonic somatic muscle attachment. Msk protein is enriched at muscle attachment sites in late embryogenesis and msk mutant embryos exhibit a failure in muscle-tendon cell attachment. Although the muscle-tendon attachment sites are reduced in size, components of the integrin complexes and ECM proteins are properly localized in msk mutant embryos. However, msk mutants fail to localize phosphorylated focal adhesion kinase (pFAK) to the sites of muscle-tendon cell junctions. In addition, the tendon cell specific proteins Stripe (Sr) and activated mitogen-activated protein kinase (MAPK) are reduced in msk mutant embryos. Rescue experiments demonstrate that Msk is required in the muscle cell, but not in the tendon cells. Moreover, muscle attachment defects due to loss of Msk are rescued by an activated form of MAPK or the secreted epidermal growth factor receptor (Egfr) ligand Vein. Taken together, these findings provide strong evidence that Msk signals non-autonomously through the Vein-Egfr signaling pathway for late tendon cell late differentiation and/or maintenance (Liu, 2011).

In Drosophila, the formation of a stable myotendinous junction is essential to withstand the force of muscle contraction required for larval hatching. Proper formation of the MTJ requires proper integrin heterodimer formation at the junctions between both muscle cells and tendon cells for a permanent linkage to the ECM proteins deposited between these two cell types. The precise mechanism by which the muscle cells signal to the tendon cells to form and maintain the semi-adherens junctions that comprise the stable MTJ is still being elucidated. This study shows that the canonical nuclear import protein Msk is essential for Drosophila somatic muscle attachment. Moreover, a model is provided explaining how Msk may function in non-cell autonomously from the muscle to the tendon cell for proper MTJ maintenance. Though vein mRNA is produced in the myotubes, Vein protein is secreted and is restricted to the junctions at muscle-tendon attachment sites. Msk signals through the secreted Egfr ligand Vein to mediate cross-talk between the muscle and tendon cells. The binding of Vein to the tendon-expressed Egfr activates a signaling cascade through activated MAPK. Activated MAPK translocates to the nucleus and with SrA, activates downstream genes to induce terminal differentiation in the tendon cells. An inability of the tendon cells to maintain activated MAPK and Sr activity would affect the amounts of target proteins required to maintain stable muscle-tendon adhesion. For example, a decrease in Tsp deposition into the ECM would result in smaller attachment sites and an inability to maintain a tight integrin-ECM association (Liu, 2011).

Msk plays a general role in myogenesis as defects in msk mutant embryos were observed in all hemisegments and affected all muscle groups. The variable penetrance, which was classified as either major (Class I) or moderate (Class II) muscle detachment phenotypes, present in msk mutant embryos is likely due to the presence of maternal msk transcript. Attempts to further knockdown Msk levels by removal of maternal load resulted in non-viable egg chambers, consistent with a requirement for Msk function in cell viability. It is possible that a further decrease, but not complete loss in Msk function, could result in earlier defects in myogenesis, since muscle patterning defects, predominantly characterized by missing muscles, were observed in a subset of msk mutant embryos (Liu, 2011).

After cell fate determination is established in somatic muscle development, myoblast fusion and myotube migration begin to proceed simultaneously in stage 13 embryos until the final muscle pattern is completed. As the migrating myotubes approach their target tendon cells, the αPS2ΒPS integrin heterodimer begins to accumulate at the leading edge of the muscle. This integrin complex is required for at least two separate events: (1) to serve as a transmembrane link between the internal actin cytoskeleton and the ECM components Tsp and Tig; and (2) for the proper localization and/or accumulation of Vein. The accumulation of Vein at the sites of muscle-tendon interactions is necessary for activation of the Egfr pathway and subsequent late tendon cell differentiation. In these mature, muscle-linked tendon cells, SrB expression is positively regulated. SrB also turns on the downstream transcriptional target Tsp, resulting in more Tsp secretion and subsequent strengthening of the MTJ through integrin binding (Liu, 2011).

The results suggest that Msk affects the later stages of tendon cell maturation and MTJ formation and/or maintenance. (1) No obvious defects were observed in myoblast fusion or the guidance of muscles to their correct target tendon cell. (2) Msk protein expression, visualized by both antibody immunolocalization and a fluorescently-tagged Msk fusion protein, demonstrates that enrichment of Msk protein at the future muscle-tendon attachment sites occurs after stage 15. The appearance of Msk protein localization corresponds to the timing of MTJ junction formation, but it does not rule out the possibility that Msk has a role in earlier myogenic events. (3) The muscle detachment phenotypes observed in msk mutant embryos are consistent with the myospheroid phenotype observed for other genes, including myospheroid (βPS int), inflated (αPS2 int), and rhea (Talin), which are well-characterized for their role in embryonic muscle attachment. Finally, in all msk mutants examined, regardless of the severity of the muscle detachment phenotypes, MTJ formation occurred in the correct location. Although the muscle attachment sites were smaller in msk mutants than in WT embryos, they were initially formed correctly, but not capable of reaching their mature size. These data taken together indicate that the muscle-specific αPS2βPS integrin complex initially forms an attachment to the ECM proteins Tig and Tsp. However, as mature tendon cell induction is compromised in msk mutant embryos, whereby the tendon cells are not able to produce and secrete proper levels of Tsp protein. Thus, the size of the mature MTJ is reduced and results in a decreased affinity at the muscle-tendon junctions (Liu, 2011).

Sr is a key factor in tendon cell differentiation in the embryo and fly thorax. In the embryo, SrB is essential for early tendon cell induction and SrA is activated for later tendon cell maturation. Furthermore, ectopic expression of Sr can act as a guidance cue for migrating myotubes as ectopic expression of Sr in epidermal cells, the salivary glands, or the CNS results in muscle patterning defects where muscles take the incorrect route and/or become attached to ectopic cells expressing Sr. Even though the data shows that Msk is required for nuclear Sr in the tendon cells, ectopic expression of Msk is not sufficient for tendon cell induction based upon three lines of experimentation. First, ectopic Msk expression in either the muscle or epidermis resulted in aberrant muscle attachment, but not misguided myotubes. All muscles were found to be in the correct position, regardless of Msk expression in domains outside of the normal hemisegments. Second, ectopic Msk expression was not sufficient to induce either early or elevated Sr levels. Third, expression of Msk in the salivary glands or CNS did not result in misguided muscles toward these locations (Liu, 2011).

The tissue-specific rescue experiments show that Msk is required in the muscle cell for proper muscle-tendon attachment to occur. Thus, two mechanisms that are not mutually exclusive are proposed by which Msk may be functioning in the muscle cells to exert its effect on tendon cell maturation. First, Msk may be required directly and/or indirectly for the localization and/or accumulation of secreted Vein. As antibodies against Vein were not available for testing this possibility, it was shown that reintroducing Vein in msk mutants could rescue muscle attachment defects. Second, Msk may act via an integrin-dependent mechanism to modulate adhesion, which is explained in detail below (Liu, 2011).

From these studies, Msk localizes to the ends of muscles at the sites of muscle attachment. It is proposed that this localization of Msk recruits other proteins to the sites of muscle attachment to sequester proteins near the cell periphery and/or to modulate integrin affinity at the muscle attachment site. First, the absence of pFAK localization in msk mutants strongly suggests that Msk is essential for pFAK localization to the muscle-tendon attachment site. Surprisingly, mutations in FAK do not result in embryonic muscle attachment defects. However, pFAK localization is also lost in integrin mutants, suggesting that pFAK is involved in undefined events in myogenesis. If Msk serves as a scaffold protein to localize pFAK and/or other molecules to the sites of muscle-tendon cell attachment, these proteins may play an accessory role in integrin-mediated adhesion. One idea is that a Msk-pFAK complex may serve to limit the signaling function of integrins so its adhesive role predominates in MTJ formation. It is well-established that integrins play both adhesive and signaling roles in cell migration and development. Clustering of the cytoplasmic tails of βPS-integrins initiates a downstream signaling pathway that regulates gene expression in the Drosophila midgut, but is not sufficient to induce tendon cell differentiation in formation of the MTJ. This suggests that integrin-mediated adhesion is required to assemble ECM components and influence the ability of Vein to activate the Egfr pathway. While loss of FAK activity does not result in somatic muscle defects, overexpression of FAK does. Muscles that have detached from the epidermis as a result of FAK overexpression still retain βPS2 integrins at the muscle ends. As observed in mammalian systems, this raises the possibility that pFAK may play a role in integrin complex disassembly. Excess pFAK may either displace proteins that bind to the cytoplasmic domain of integrins or excessively phosphorylate proteins resulting in integrin complex turnover and a decrease in stable adhesion. Alternatively, pFAK may exhibit redundancy with another protein at the attachment sites. There is precedence for this in the fly as pFAK functions redundantly with the tyrosine kinase Src downstream of integrins in the larval neuromuscular junctions (NMJs) to restrict NMJ growth. Furthermore, in FAK mutants, phosphotyrosine signal is still observed at the sites of muscle attachment, supporting the idea that another tyrosine kinase is functional. A viable candidate may be Src42A, as it is also expressed at the sites of muscle attachment in the embryo (Liu, 2011).

The canonical role for Msk is to import proteins, such as activated MAPK into the nucleus. However, not activated MAPK or nuclear Msk were detected in the nuclei of muscle cells. As this study has shown, strong Msk immunolocalization is detected at the MTJ in stage 16 embryos, but not in the nuclei of developing muscles in stages 13-15. It cannot be ruled out that Msk is present at low levels in the muscle, and was not detectable. It is true that the YFP-Msk fusion protein was detected in the nucleus, but this may be due to the over-expression of the protein. While it is possible that nuclear Msk and MAPK in muscles are required for some aspects of myogenesis, this requires further analysis (Liu, 2011).

Future experiments will determine the precise mechanism by which Msk influences Vein secretion, localization, and/or accumulation at muscle-tendon cell attachment sites. Is it mediated through integrins? Is phosphorylation of Msk essential for activity? Msk is tyrosine phosphorylated in response to insulin and PS integrins, although the kinase remains unknown. FAK may be an example of a kinase that can phosphorylate Msk at the muscle attachment sites (Liu, 2011).

In mammalian studies, the canonical role for Importin-7 is in nuclear import. Other roles for cytoplasmic Importin-7 have not been examined. Thus, it will be interesting to uncover new roles for Importin-7, specifically in vertebrate muscle development (Liu, 2011).

Dpp-induced Egfr signaling triggers postembryonic wing development in Drosophila

The acquisition of flight contributed to the success of insects and winged forms are present in most orders. Key to understanding the origin of wings will be knowledge of the earliest postembryonic events promoting wing outgrowth. The Drosophila melanogaster wing is intensely studied as a model appendage, and yet little is known about the beginning of wing outgrowth. Vein (Vn) is a neuregulin-like ligand for the EGF receptor (Egfr), which is necessary for global development of the early Drosophila wing disc. vn is not expressed in the embryonic wing primordium and thus has to be induced de novo in the nascent larval wing disc. Decapentaplegic (Dpp), a Bone Morphogenetic Protein (BMP) family member, provides the instructive signal for initiating vn expression. The signaling involves paracrine communication between two epithelia in the early disc. Once initiated, vn expression is amplified and maintained by autocrine signaling mediated by the E-twenty six (ETS)-factor PointedP2 (PntP2). This interplay of paracrine and autocrine signaling underlies the spatial and temporal pattern of induction of Vn/Egfr target genes and explains both body wall development and wing outgrowth. It is possible this gene regulatory network governing expression of an EGF ligand is conserved and reflects a common origin of insect wings (Paul, 2012).

Deciphering gene regulatory networks (GRNs) is critical for understanding the causation of development, and a large network that explains endoderm development in the sea urchin has been elaborated . The Drosophila early wing disc provides a tractable system for GRN analysis because it is a relatively small field of cells and many genes involved in the process are known. This study has developed knowledge of the GRN involving Egfr signaling activated by its ligand Vn. It is a key circuit because Vn/Egfr signaling induces the ap and iro-C genes, which are required for development of the major territories of wing and body wall beginning in the first instar. The spatial and temporal control of vn expression involves two major inputs: initiation by paracrine Dpp signaling and maintenance by a positive Vn/Egfr autocrine feedback loop. These are direct, positive, early acting inputs into the vn promoter. The Dpp signal emanates from the peripodial epithelium and is unidirectional, activating vn only in the disc proper. This directionality is important because ectopic activation of Egfr in the peripodial epithelium antagonizes its development and transforms the cells into columnar cells characteristic of the disc proper, which develop into body wall structures. It is not known why Dpp signaling is apparently only active in cells across the lumen from the expression domain in the first instar. It is possible some factor required for Dpp signaling is differentially expressed or Dpp signaling is mediated by cellular processes as has been proposed for the early eye disc. There are also negative inputs that operate slightly later in development and function to limit the spatial extent of vn expression. Productive Vn/Egfr signaling is confined to the future body wall because pntP2 expression focuses proximally as development proceeds. There is also an early acting negative input from Wg signaling that has been defined genetically. wg expression begins in distal cells in the first instar wing disc, which is consistent with a role in repressing vn expression in cells that will become the future wing (Paul, 2012).

The direct feedback loop involving vn expression is an example of a regulatory circuit governing a 'community effect,' a term coined by Gurdon to describe the change in transcription that occurs when cells are isolated from their neighbors. This phenomenon suggested that proximity to other cells serves as a mechanism to sustain expression of particular genes important for a collective cell fate. The term has been used to describe a developmental event that is the result of a ligand inducing its own expression in a neighboring cell. In this way, the signal is propagated through a field of cells, which, as a 'community,' then express a similar repertoire of target genes. Examples of this type of subcircuit operating in pattern formation have been described that involve TGF-β, FGF, and Wnt ligands. This study reports a case in which an EGF ligand is implicated. Adding an additional ligand class to the list supports the idea that the strategy is widespread in development. In a normal developmental context, a secreted EGF ligand, such as Vn, can be produced as part of a positive feedback loop provided there are tight controls that limit the operation of the loop both spatially and temporally. Clearly, if there were no limits, a runaway situation would result and too many cells would become part of the 'community,' or the state of activity would be perpetuated beyond a certain developmental window. Indeed this seems to be the case in some disease contexts involving EGF ligands, including neuregulin (the vertebrate equivalent to Vn), where autocrine loops sustain the continued growth of cancer cells (Paul, 2012).

It is widely thought that flight contributed significantly to the success of insects and consequently the evolution of the wing is of great interest. There are two major theories; the first holds that wings derived from a proximal branch of the leg and the second that wings derived from a paranotal lobe extending from the dorsal body wall. In a combination of these ideas, it has been proposed that the wing may have a leg origin but that the ability to form a flat wing-like structure depends upon proximity to the dorsal-lateral boundary in the side body (the paranotal lobe in wingless forms). Dorsal appendages such as the tracheal gill or stylus are like the Drosophila wing in that they express wg and vg and may represent evolutionary precursors to the wing. Unlike the wing, however, they do not form close to a region where ap is also expressed, which may be essential for an outgrowth to form a flat structure like the wing. Vn/Egfr signaling is upstream of ap and therefore a prerequisite for wing formation. This study trace the circuit regulating ap expression back earlier to the induction of vn in a transient stripe triggered by Dpp signaling. This initiates broad Vn/Egfr/PntP2 signaling that extends distally and ap is induced throughout the dorsal compartment, where, together with wg and vg, it acts to produce the wing. Continued Egfr activation, however, blocks wing development, and hence the domain of active signaling is shortly thereafter restricted to proximal cells by the absence of PntP2 in distal cells. In proximal cells where PntP2 persists, a feedback loop is established and body wall development is promoted. The changing spatial domain of vn (from a stripe to a proximal wedge) establishes a prepattern that is permissive for both wing and body wall development. It will be interesting to determine if Egfr signaling underlies body wall formation in other species and if a similar transient spatial extension of this activity correlates with winged morphs (Paul, 2012).

Local control of intestinal stem cell homeostasis by enteroendocrine cells in the adult Drosophila midgut

Enteroendocrine cells populate gastrointestinal tissues and are known to translate local cues into systemic responses through the release of hormones into the bloodstream. This study reports a novel function of enteroendocrine cells acting as local regulators of intestinal stem cell (ISC) proliferation through modulation of the mesenchymal stem cell niche in the Drosophila midgut. This paracrine signaling acts to constrain ISC proliferation within the epithelial compartment. Mechanistically, midgut enteroendocrine cells secrete the neuroendocrine hormone Bursicon, which acts (beyond its known roles in development) as a paracrine factor on the visceral muscle (VM). Bursicon binding to its receptor, DLGR2 (Rickets), the ortholog of mammalian leucine-rich repeat-containing G protein-coupled receptors (LGR4-6), represses the production of the VM-derived EGF-like growth factor Vein through activation of cAMP. This study has therefore identified a novel paradigm in the regulation of ISC quiescence involving the conserved ligand/receptor Bursicon/DLGR2 and a previously unrecognized tissue-intrinsic role of enteroendocrine cells (Scopelliti, 2014).

Bursicon, also known as the tanning hormone, has been studied for decades due its essential role as the last hormone in the cascade of Ecdysis. In all invertebrate metazoa, this endocrine cascade is fundamental to coordinate molting events during animal lifetime, and in holometabolous insects, such as Drosophila, it control metamorphosis. Fly gene expression data suggest that the endocrine hormones and their cognate receptors involved in key stages of development may have other roles during adult animal life. However, these functional roles are largely unknown. This study is the first to demonstrate a role of Bursicon beyond development (Scopelliti, 2014).

A model is suggested in which Bursicon from enteroendocrine cells in the posterior midgut acts through DLGR2 to increase the production of cAMP within the VM, a mesenchymal ISC niche. This signaling limits the production of niche-derived, EGF-like Vein, leading to ISC quiescence. Burs protein expression was detected via immunolabeling in approximately 50% of the enteroendocrine cells of the posterior midgut, which appeared in stochastic spatial distribution within the most posterior segment of the adult midgut. Given that the percentage of enteroendocrine cells expressing Burs remained constant, it is likely that Burs expression might label a subtype of enteroendocrine cells within the midgut (Scopelliti, 2014).

The evidence indicates that burs mRNA levels are upregulated in the midgut during the phase of relative ISC quiescence in mature animals under homeostatic conditions. Conversely, during the phase of growth of the young immature gut or the dysplastic phase of the aging gut (both characterized by relative high rates of ISC proliferation) burs levels were relatively low, and burs overexpression was sufficient to suppress ISC proliferation. Therefore, the results provide the first demonstration of a tissue-intrinsic role of enteroendocrine cells, which drives homeostatic stem cell quiescence in the adult Drosophila midgut. Future studies should further characterize the upstream mechanisms controlling Burs production in the midgut, which might be linked to the yet undefined events involved in the regulation of overall tissue size and proliferation (Scopelliti, 2014).

Enteroendocrine cells are well known for their ability to mediate interorgan communication via hormone secretion into the bloodstream. The results demonstrate a novel, local role for enteroendocrine cells as paracrine regulators of stem cell proliferation. Such a mechanism could be phylogenetically conserved and take place in the mammalian intestine and other tissues of the gastrointestinal tract. This may therefore represent an unappreciated but yet important function of these cells beyond their conventional endocrine role (Scopelliti, 2014).

Mammalian LGRs are thought to drive ISC proliferation acting as receptors for the Wnt agonists R-spondins, which are unrelated to Bursicon and absent in the fly genome. Accordingly, no changes were detected in either Wg levels or signaling in burs or rk mutant midguts (Scopelliti, 2014).

Unexpectedly for a Wnt agonists and positive regulators of ISC proliferation, recent studies suggest that LGRs can act as tumor suppressors in colorectal cancer. Moreover, mammalian LGRs have also been shown to be activated by alternative ligands and promote cAMP signaling after the binding of yet unknown ligand(s). Therefore, it is likely that an unidentified functional homolog of Bursicon may act as an additional LGR ligand in mammals, driving ISC quiescence by regulating mitogenic signals from the surrounding niche as described here. Remarkably, DLGR2 shows closer sequence homology to the still poorly characterized LGR4, which (consistent with the Drosophila data) is expressed by the murine intestinal smooth-muscle layers and can signal via cAMP production. Consistent with the model, a recent study correlates loss-of-function mutations in LGR4 with multiple types of human epithelial carcinomas. Therefore, the results uncovered a novel biological role for LGRs, which is likely to impact mammalian stem cell research by providing a mechanistic framework for the so far correlative mammalian evidence toward a potential role of LGRs as tumor suppressor genes (Scopelliti, 2014).

Altogether, these results demonstrate a novel paradigm in the regulation of intestinal homeostasis involving the conserved ligand/receptor Bursicon/DLGR2 and a previously unrecognized tissue-intrinsic role of enteroendocrine cells, which may provide insights into other stem cell based systems (Scopelliti, 2014).

vein: Biological Overview | Evolutionary Homologs | Protein Interactions | Developmental Biology | Effects of Mutation | References

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