During larval and early pupal development expression of rho in longitudinal vein primordia mediates the localized formation of wing veins (Sturtevant, 1995).

Boundaries in the Drosophila wing imaginal disc organize vein-specific genetic programs

Vein primordia in Drosophila form at boundaries along the A/P axis between discrete sectors of the larval wing imaginal disc. Genes involved in initiating vein development during the third larval instar are expressed either in narrow stripes corresponding to vein primordia or in broader `provein' domains consisting of cells competent to become veins. Genes specifying the alternative intervein cell fate are expressed in complementary intervein regions. The regulatory relationships between genes expressed in narrow vein primordia, in broad provein stripes and in interveins remains unknown, however. Additional evidence is presented in this paper for veins forming in narrow stripes at borders of A/P sectors. These experiments further suggest that narrow vein primordia produce secondary short-range signal(s), which activate expression of provein genes in a broad pattern in neighboring cells. Crossregulatory interactions among genes expressed in veins, proveins and interveins contribute to establishing the vein-versus-intervein pattern, and control of gene expression in vein and intervein regions must be considered on a stripe-by-stripe basis. Evidence is presented for a second set of vein-inducing boundaries lying between veins, which are referred to as paravein boundaries. It is proposed that veins develop at both vein and paravein boundaries in more primitive insects, which have up to twice the number of veins present in Drosophila. A model is presented in which different A/P boundaries organize vein-specific genetic programs to govern the development of individual veins (Biehs, 1998).

Genes involved in initiating wing vein development in third larval instar wing discs are expressed either in narrow stripes, corresponding to vein primordia, or in broader provein stripes, consisting of cells that are competent to become vein cells. For example, rhomboid (rho) and argos (aos) are expressed in narrow vein stripes, while Dl, achaete (ac), scute (sc), caupolican (caup) and araucan (ara) are expressed in broader provein domains. rho, which encodes an integral membrane protein, is expressed in all vein primordia and promotes vein formation throughout wing development by locally activating the Egfr- signaling pathway. aos encodes an Egfr antagonist, which feeds back negatively to inhibit Egfr activity. caup and the neighboring gene ara encode related homeobox genes that promote expression of vein genes such as rho and proneural genes such as ac and sc. Delta (Dl) encodes a ligand for the Notch (N) receptor, which mediates lateral inhibitory interactions among cells in vein-competent domains during pupal development. The vein pattern is also reflected by the complementary intervein expression patterns of blistered (bs), which encodes the Drosophila homolog of the Serum Response Factor (DSRF) and vein (vn), which encodes a putative Egfr ligand of the neuregulin/heregulin class. bs provides an essential general function for intervein development and is strongly downregulated in all vein primordia relative to intervein regions. vn promotes vein development and is expressed in a single strong intervein stripe running between the L3 and L4 primordia in third instar larval discs. Initiation of vein development during the third larval instar is followed by a period of vein maintenance and refinement during prepupal and pupal stages. At least three different types of cell-cell communication contribute to the refinement process: (1) lateral inhibitory signal(s) elaborated by presumptive vein cells restrict vein formation to the center of broad vein-competent domains; (2) dorsal-to-ventral signal(s) maintain vein fates in cells on the ventral surface of the wing and (3) vein continuity signal(s) promote vein formation in straight lines along the vein axis. These various signals collaborate to ensure that the dorsal and ventral components of narrow veins are strictly aligned and uninterrupted (Biehs, 1998).

To determine the precise relationships between the expression patterns of vein, provein and intervein genes, a series of double-label experiments have been performed. The primary vein marker, rho, is expressed in five sharp stripes 1-2 cells wide that are likely to correspond to the primordia for the L1-L5 longitudinal wing veins. Neuronal precursor cells for sensory organs located along the L3 vein align with the L3 stripe of rho expression in third instar wing discs. To generalize this finding to other veins, which normally are not decorated with sensory organs, the relationship was determined between the expression patterns of rho and the A101 neuronal precursor cell marker in wing primordia of Hw49c mutants, which have ectopic sensilla running along each longitudinal vein. Consistent with the premise that each stripe of rho expression in third instar wing discs corresponds to a vein primordium, ectopic neural precursors in Hw49c mutants coincide with rho-expressing cells in third instar wing discs and in early everting prepupal wings. Having confirmed that each stripe of rho expression corresponds to a longitudinal vein primordium, the relative expression patterns of various genes expressed in narrow vein stripes or broader provein stripes were determined by double-label experiments. The expression patterns of rho and Dl were compared. In mid-to-late third instar larvae, Dl is expressed in a series of four stripes 4-6 cells wide. Double-label experiments reveal that the broader stripes of Dl protein expression are centered over the narrower L1, L3, L4 and L5 rho stripes. Additionally, double-label experiments with the anti-Dl antibody and antisense RNA probes for aos, caup and ac reveal that the three stripes of aos expression coincide with the L3, L4 and L5 Dl stripes; that the three broad caup stripes straddle the narrower L1, L3 and L5 Dl stripes, and that the single dorsally restricted stripe of ac expression is coincident with the dorsal component of the L3 Dl stripe. The relationship between the expression of rho and intervein markers was determined. BS RNA and Bs protein are expressed ubiquitously in the wing pouch, but are strongly downregulated in a pattern of four stripes. The L2-L5 rho stripes are centered within the troughs of Bs downregulation, which tend to be one or two cells wider than the rho stripes (e.g. there are single rows of cells flanking rho-expressing cells not expressing either rho or high levels of Bs). These data are consistent with the previous observation that L3 sensory organ precursor cells lie within the L3 trough of Bs downregulation. Strong expression of vn is confined to the region between the L3 and L4 stripes of Dl expression. An important feature of these various double-labeling experiments is that the centers of all vein and provein stripes coincide. For example, the narrow stripes of rho expression run up the middle of the broader Dl stripes, and the yet broader domains of caup expression (7-8 cells wide) symmetrically straddle the odd-numbered Dl stripes. Also, as mentioned above, the stripes of Bs downregulation are centered over the narrower stripes of rho expression in veins. The nearly perfect nested registration of several vein, provein and intervein markers in third instar wing discs suggests that common positional cues coordinate expression of these genes in and around each vein primordium (Biehs, 1998).

Hh is produced in the posterior compartment of the wing disc and diffuses a short distance into the anterior compartment where it activates target genes such as ptc and dpp in stripes running up the center of the wing disc. The L3 and L4 vein primordia form respectively along the anterior and posterior borders of the Hh-signaling domain. In addition, a variety of evidence suggests that the anterior edge of the Hh-signaling domain defines the position of the L3 vein. To determine whether vein and intervein markers respond in concert to alterations in the level of Hh, a series of double-label experiments were performed. In GAL4-en;UAS-ptc wing discs, which have a reduced distance between the L3 and L4 vein primordia, the L3 expression patterns of Dl and rho, Dl and ac, and Dl and caup shift coordinately in a posterior direction. The relative positions of vein and intervein markers also are preserved in these discs as revealed by the concerted shift in the expression of rho and Bs. Evidence is also presented that the Hh-signaling domain in the anterior compartment normally sends a signal to adjacent posterior compartment cells to initiate L4 formation (Biehs, 1998).

Since the expression of vein, provein and intervein genes is initiated almost simultaneously in third larval instar wing discs, it is possible that crossregulatory interactions among these early acting genes, as well as continued signaling from boundaries, are important for establishing the vein pattern. To address this question, the expression of vein, provein and intervein genes was examined in early acting vein mutants, which disrupt the initiation, rather than the maintenance, of vein development. Early acting loss-of-vein mutants include the recessive mutants rhove (a cis-regulatory allele of rho that lacks detectable rho expression in vein primordia), vn1, rhove;vn1 double mutants, iroDFM2 (which behaves as an L3-specific loss-of-function allele of the iro locus and does not survive to adulthood), radius incompletus (ri) (which is a regulatory allele of the knirps/knirps-related locus) and abrupt (ab). Expression of markers in early acting extravein mutants were also examined such as the recessive net mutant and the dominant rhoSld enhancer piracy line (Biehs, 1998).

Two major conclusions can be drawn from the results of these experiments. The first of these is that crossregulatory interactions do play a significant role in establishing the initial sharp vein-versus-intervein pattern. For example, in vn1 wing discs, which lack detectable expression of the Egfr ligand vn, expression of Dl and rho is virtually eliminated in the L4 primordium. rho-mediated activation of Egfr signaling also contributes to establishing the vein pattern, since both the L3 and L4 stripes of Dl expression are severely compromised in rhove;vn1 double mutants. rho and vn also collaborate to activate ac and sc expression in the L3 primordium because expression of ac and sc in broad L3 stripes is lost in rhove;vn1 double mutants discs. The presence of isolated sc-expressing cells in rhove;vn1 discs, likely to be L3 sensory organ precursors, may explain why L3 sensilla are usually present in rhove;vn1 wings. Also supportive of the first conclusion is the fact that rho function is necessary and sufficient for initiating argos expression throughout the wing disc. The iro locus is known to play a central role in establishing the vein pattern in odd-numbered veins (Biehs, 1998).

The second major conclusion regarding crossregulatory interactions among vein, provein and intervein genes is that individual stripes of gene expression may represent independent units of regulation. This point is most obvious for the ri and ab mutants in which expression of all relevant vein, provein and intervein markers (e.g. downregulated Bs expression) is strictly dependent on ri function in L2 and on ab function in L5. The distinct behaviors of the L3, L4 and L5 Dl stripes in vn1 versus rhove;vn1 mutants described above is another example of stripe-dependent regulation of gene expression. The differential requirement for Egfr signaling to activate expression of genes in particular veins presumably reflects differing threshold requirements for Egfr signaling (Biehs, 1998).

It is proposed that vein formation is initiated at boundaries between discrete A/P sectors of the wing disc. The vein-inducing boundary for the L2 primordium is likely to be the border between spalt major-expressing and salm non-expressing (or weakly expressing) cells. The L2 primordium forms within the salm non-expressing domain of cells. The vein-inducing boundary for the L3 primordium may be the border between Hh responding cells expressing high/moderate levels of ptc and cells expressing very low levels of ptc. The L3 primordium forms within the domain of very low ptc expression. With respect to the L4 primordium, the vein-inducing boundary is likely to be the A/P compartment boundary itself. Although the L4 vein is displaced to the posterior by a few cell diameters from the A/P compartment boundary in adult flies, the L4 primordium initially abuts the A/P boundary in third instar wing discs. Currently, there is not a good candidate border known in the position of the L5 primordium. Vein-inducing boundaries might act directly to regulate gene expression in and around vein primordia, or might act through intermediate vein-organizing genes to orchestrate gene expression. Mutants lacking the function of a vein-organizing gene should lack expression of all vein markers and should not downregulate expression of intervein markers in that vein. Based on this criterion, candidate vein-organizing genes are ri for the L2 vein and ab for the L5 vein. Whether there are similar genes acting to organize gene expression in L3 and L4 remains to be determined. It is proposed that vein-inducing boundaries and/or vein-organizing genes activate expression of vein genes (e.g. rho) in narrow stripes, initiate the production of locally acting signals that activate gene expression in broader vein-competent regions centered over veins (e.g. Dl and ac/sc) and suppress expression of intervein genes (e.g. bs). These genes then engage in various vein-specific crossregulatory interactions (Biehs, 1998).

A variety of evidence indicates that biologically meaningful boundaries also run between and parallel to longitudinal vein primordia. These cryptic borders are referred to as paravein boundaries since ectopic veins (paraveins) have a strong tendency to form in these positions in a variety of extravein mutants. Four likely paravein boundaries (P2, P4, P5 and P6) can be observed in third instar wing discs. The position of the putative P4 paravein between the primordia for L3 and L4 can be revealed by a stripe of rho mis-expression in fused mutant wing discs. P4 also is marked by a short ectopic vein (between L3 and L4 in various extravein mutants); a true vein (which is found in this position in primitive insects), forms along the anterior boundary of en expression. The proposed P5 paravein boundary runs between the primordia for the L4 and L5 veins in the approximate location of the posterior border of the spalt expression domain in third instar discs. In pupal wings, it is unambiguous that P5 borders the posterior edge of salm expression. Thus, a short ectopic section of vein (P5) running between L4 and L5 in net/+ adult wings can be visualized in net/+ pupal wings as an ectopic segment of Dl expression abutting the posterior edge of the spalt domain. The positions of the P2 and P6 paraveins also are likely to be defined in third instar discs as revealed by ectopic expression of rho in net mutants. In mid-third instar wing discs, ectopic rho expression is bounded by L2 at the anterior and by L5 at the posterior. However, shortly thereafter in late third instar discs, rho expression expands anteriorly beyond L2 and posteriorly behind L5. These enlarged borders of rho mis-expression in net discs are likely to correspond to the positions of the P2 and P6 paraveins, respectively, since there are ectopic veins that run between the margin and L2 (i.e. P2) and between L5 and L6 (i.e. P6) in net adult wings. The P4 and P5 paraveins also can be marked by rows of ectopic bristles in wings of AS-C Hw49c or h1 mutants. The P4 paravein boundary appears to have been conserved during the evolution of Diptera, since a bonafide vein, which forms in this location in syrphid flies, abuts the en expression domain during pupal stages. Interestingly, other morphological features of the syrphid wing also correspond to sharp en boundaries in the pupa, suggesting that late en boundaries organize various linear features of the adult wing (Biehs, 1998).

In primitive insects, which have up to twice the number of veins as Drosophila, it is likely that vein development is initiated along boundaries corresponding to paraveins as well as veins. According to this model, veins form at all vein and paravein positions in primitive insects. Vein patterns in insects with fewer than the full complement of veins generally have been interpreted by assuming that ancestral veins are fused into a reduced number of veins (e.g. R2 and R3 being fused to generate L2 in Drosophila). Instead, it is proposed that vein formation is initiated at a subset of vein and paravein boundaries present in all insects. According to this view, the pattern of veins generated in a given insect species depends on which of these boundaries initiates vein formation. An attractive aspect of this hypothesis is that it provides a ready explanation for the curious evolutionary fact that, in nearly all major orders of insects, there are examples of species that have the primitive archetypal vein pattern. Such apparently primitive species exist in many orders where it is clear that the founding member of that group must have had fewer veins (i.e. because the great majority of species within that order share a particular subpattern of veins in addition to other more advanced characteristics). According to the vein/paravein boundary model, the archetypal vein pattern could re-emerge from an insect lineage having a simplified vein pattern by virtue of an atavistic mutation that relieves suppression of vein formation at certain paravein boundaries. Future experiments in other insect species will be required to address the origins of different venation patterns (Biehs, 1998).

Antagonism between EGFR and Wingless signalling in the larval cuticle of Drosophila

Signaling by the epidermal growth factor receptor (EGFR) plays a critical role in the segmental patterning of the ventral larval cuticle in Drosophila: by expressing either a dominant-negative EGFR molecule or Spitz, an activating ligand of EGFR, it is shown that EGFR signaling specifies the anterior denticles in each segment of the larval abdomen. rhomboid, spitz and argos are expressed in denticle rows 2 and 3, just posterior to denticle row 1 in the engrailed expression "posterior" domain of larval ectoderm. These denticles derive from a segmental zone of embryonic cells in which EGFR signaling activity is maximal (Szuts, 1997).

Both Egfr and spitz are expressed fairly uniformly throughout the embryonic epidermis. However, Spi appears to be incapable of activating Egfr unless it is processed into a secreted form; there is genetic evidence that the membrane-spanning products of the rhomboid and Star genes may be mediating this process. In young embryos (before the germ band is fully extended) rhomboid is expressed in distinct segmental stripes. These stripes remain visible until stage 16 by which time they fade away. They become circumferential, and one cell wide in the dorsal half of the embryo. In the ventral half, they are also one cell wide in the thoracic segments, a bit wider in the first abdominal segment, and at least two cells wide in all other segments. These stripes correspond roughly to the cells giving rise to denticle rows 2 and 3. argos is observed in, and adjacent to, cells expressing rhomboid, and indeed is expressed in circumferential stripes after completion of germ-band retraction. The segmental stripes of argos are fairly similar to the rhomboid stripes but a bit wider. Argos is an inhibitor of Egfr function, so Argos reduction is expected to result in overactivation of Egfr signaling. argos loss of function mutants result in an entire additional row of denticles anterior to row 1 (Szuts, 1997).

High EGFR signalling activity depend on bithorax complex gene function. In mutants lacking abdominal-A and Ultrabithorax, rhomboid expression is very weak. In these mutants, there is very little expression of Argos. These homeotic genes account for the main difference in shape between abdominal and thoracic denticle belts (Szuts, 1997).

Interaction between EGFR signaling and DE-cadherin during nervous system morphogenesis

Dynamically regulated cell adhesion plays an important role during animal morphogenesis. The formation of the visual system in Drosophila embryos has been used as a model system to investigate the function of the Drosophila classic cadherin, DE-cadherin, which is encoded by the shotgun (shg) gene. The visual system is derived from the optic placode which normally invaginates from the surface ectoderm of the embryo and gives rise to two separate structures, the larval eye (Bolwig's organ) and the optic lobe. The optic placode dissociates and undergoes apoptotic cell death in the absence of Shotgun, whereas overexpression of Shotgun results in the failure of optic placode cells to invaginate and of Bolwig's organ precursors to separate from the placode. These findings indicate that dynamically regulated levels of Shotgun are essential for normal optic placode development. Overexpression of Shotgun can disrupt Wingless signaling through titration of Armadillo out of the cytoplasm to the membrane. However, the observed defects are likely the consequence of altered Shotgun mediated adhesion rather than a result of compromising Wingless signaling, since overexpression of a Shotgun-alpha-catenin fusion protein, which lacks Armadillo binding sites, causes similar defects as Shotgun overexpression. The genetic interaction between Shotgun and the Drosophila EGF receptor homolog, Egfr, was studied. If Egfr function is eliminated, optic placode defects resemble those following Shotgun overexpression, which suggests that loss of Egfr results in an increased adhesion of optic placode cells. An interaction between Egfr and Shotgun is further supported by the finding that expression of a constitutively active Egfr enhances the phenotype of a weak shg mutation, whereas a mutation in rhomboid (rho) (an activator of the Egfr ligand Spitz) partially suppresses the shg mutant phenotype. Finally, Egfr can be co-immunoprecipitated with anti-Shotgun and anti-Armadillo antibodies from embryonic protein extracts. It is proposed that Egfr signaling plays a role in morphogenesis by modulating cell adhesion (Dumstrei, 2002).

The head ectoderm of early Drosophila embryos is subdivided into several domains that realize different morphogenetic programs. The embryonic eye field is the posterior-medial region of the procephalic neurectoderm that gives rise to the visual system, which includes the larval eye (Bolwig's organ) and adult eye, as well as the optic lobe. Around gastrulation, cells of the eye field undergo a convergent extension directed laterally. Shortly afterwards these cells form two morphologically visible placodes, one on either side of the embryo. These optic placodes sink inside and become the optic lobe primordia, epithelial double layers attached to the posterior surface of the brain. The optic placode of a stage 12-13 embryo is V-shaped, with the anterior leg of the V representing the anterior lip, which later forms the inner anlage of the optic lobe, and the posterior leg forming the posterior lip, later forming the outer anlage. As the invagination deepens and the two lips 'sink' inside the embryo, ectodermal cells that earlier surrounded the perimeter of the optic placode approach each other and eventually form a closed epidermal cover. Abundant cell death accompanies the closing of the head epidermis over the optic lobe anlage, and the subsequent separation of this anlage from the epidermis. A small number of cells that originally formed part of the posterior lip of the optic placode remain integrated in the head epidermis and form the larval eye or Bolwig's organ. As these cells move away from the optic lobe anlage their basal ends become drawn out and form axons that constitute Bolwig's nerve (Dumstrei, 2002).

Shotgun is expressed throughout the ectoderm including the eye field and its epithelial derivatives. One would expect that normal optic lobe development requires modulation of Shotgun activity to allow, for example, the segregation of the invaginating optic placodes from the surrounding ectoderm. Since cell culture studies have indicated that the mammalian EGF receptor can disrupt cadherin-based adhesion, it was of interest to see whether Drosophila Egfr is expressed in the visual system to allow for such a possibility in Drosophila as well. Egfr is expressed in a complex and dynamic pattern that closely parallels the pattern of double-phosphorylated ERK (dpERK) expression, indicating activation of the MAP kinase signaling pathway. During stage 7 both rho (an activator of Egfr signaling) and dpERK are expressed in two stripes in the head ectoderm. The expression of dpERK in these two stripes is the result of Egfr activity. The anterior stripe corresponds to part of the head midline, while the posterior stripe reaches into the eye field. Distribution of dpERK in the two stripes becomes patchy during stage 10. At the same time, the posterior stripe widens dorsally to overlap with part of the optic lobe placode. Finally, at the late extended germ band stage and during germ band retraction, dpERK becomes restricted to the optic lobe placodes and cells of the dorsal head midline. This expression pattern demonstrates that Egfr activation accompanies the determination, morphogenesis and differentiation of the embryonic visual system (Dumstrei, 2002).

On the subcellular level, Egfr is expressed diffusely on the membrane of epithelial cells and neuroblasts. Egfr overlaps with Armadillo, the Drosophila ß-catenin homolog, which is an integral component of the cadherin-catenin complex. Like Shotgun, Armadillo is concentrated strongly in the apically located zonula adherens but is also found at lower levels in the entire lateral membranes (Dumstrei, 2002).

A second type of junction, called a septate junction, develops in Drosophila epithelial cells at a slightly later stage than the zonula adherens. Septate junctions have been implicated in maintaining epithelial stability. The Coracle protein forms part of the septate junctional complex, and an antibody against Coracle serves as a sensitive marker for this junction. Applying this marker to embryos of different stages it was found that all ectodermally derived epithelia express Coracle, except for the optic lobe and the invaginations that form the stomatogastric nervous system. Accordingly, no septate junctions have been reported in previous electron microscopic investigations of these tissues. The reliance on adherens junctions alone may make the optic lobe (and stomatogastric nervous system) susceptible to changes in the stability of these junctions; such changes occur resulting from manipulations of Shotgun and Egfr (Dumstrei, 2002).

A finely adjusted level of Shotgun is required for optic placode morphogenesis, and ß-catenin, as well as EGFR signaling, is involved in this process. Reduction in Shotgun results in dissociation of the placode around the time when it normally invaginates, suggesting that the forces exerted on the epithelial sheet while folding may disrupt cell contacts. A similar phenotype was described for other epithelial invaginations, including the Malpighian tubules and stomatogastric nervous system. Abolishing Armadillo/ß-catenin function results in a similar, if somewhat weaker phenotype. If Shotgun is overexpressed, invagination is also impaired. Cells stay together in a placode-like formation (as would be expected from 'hyperadhesive' epithelial cells), but do not noticeably constrict apically. It should be noted that the interpretation of this failure of optic placode cells to constrict is complicated by the accompanying increase in cell death in surrounding head epidermal cells. This phenomenon, in addition to a direct effect of an increased amount of Shotgun in the optic placode cells, could be part of the pathology responsible for the non-invagination phenotype. By contrast, the non-disjunction of optic lobe and larval eye is likely to be a rather direct consequence of an increased amount of Shotgun expression. Interestingly, other adhesion systems, notably the Drosophila N-CAM homolog FasII, are also involved in optic lobe-larval eye separation. Thus, the down regulation of FasII by the 'anti-adhesion' molecule Beaten path is also required for normal larval eye morphogenesis (Dumstrei, 2002).

Vein-positioning in the Drosophila wing in response to Hh; new roles of Notch signaling

The Drosophila wing is a classical model for studying the generation of developmental patterns. Previous studies have suggested that vein primordia form at boundaries between discrete sectors of gene expression along the antero-posterior (A/P) axis in the larval wing imaginal disc. Observation that the vein marker rhomboid (rho) is expressed at the center of wider vein-competent domains led to the proposal that narrow vein primordia form first, and produce secondary short-range signals activating provein genes in neighboring cells. This study examined how the central L3 and L4 veins are positioned relative to the limits of expression of Collier (Col), a dose-dependent Hedgehog (Hh) target activated in the wing A/P organizer. rho expression is first activated in broad domains adjacent to Col-expressing cells and secondarily restricted to the center of these domains. This restriction, which depends upon Notch (N) signaling, sets the L3 and L4 vein primordia off the boundaries of Col expression. N activity is also required to fix the anterior limit of Col expression by locally antagonizing Hh activation, thus precisely positioning the L3 vein primordium relative to the A/P compartment boundary. Experiments using Nts mutants further indicate that these two activities of N can be temporally uncoupled. Together, these observations highlight new roles of N in topologically linking the position of veins to prepattern gene expression (Crozatier, 2003).

In the Drosophila wing, L3 vein is decorated with campaniform sensory organs (CS). Formation of the L3 vein and sensory organs is generally thought to be topologically linked through expression of rho and the proneural genes ac and sc in overlapping A/P positions in third instar wing discs. In wild-type wings, CS overlap the posterior-most row of L3 vein cells. In heterozygous N55 mutant wings, veins are wider than in wild-type, due to defective partitioning of the provein into vein and intervein cells, but selection of sensory organ precursors (SOPs) is not affected. It was observed, however, that CS still overlap the posterior-most L3 vein cells, unlike what would be predicted if widening of L3 vein due to defective vein resolution were centered over its initial coordinate position. The position of SOP relative to the A/P border is not changed in N mutant discs, indicating that in the adult, the position of L3 vein is shifted anteriorly by one or two rows of cells. In order to determine whether this shift and the defect in vein resolution are coupled, the temperature-sensitive allele Nts2 was used. When Nts2 mutants are shifted to restrictive temperature between 104 and 128 h AEL [8-32 h after puparium formation (APF)], the L3 vein is broader, indicative of abnormal vein resolution, but, unlike N55/+ wings, the CS are now centered over the broader vein. Reciprocally, when a transient temperature shift is applied at 60-80 h AEL (mid-second/mid-third larval stages), the L3 vein retains a wild-type width but, as in N55/+, its position is shifted anteriorly relative to the CS. Nts mutant analysis thus reveals a new role of N in positioning the L3 vein relative to the A/P border, temporally uncoupled from its known role in partitioning provein into vein and intervein cells at the pupal stage. This has led to a detailed investigation of the molecular mechanisms involved in positioning L3 vein (Crozatier, 2003).

Longitudinal vein primordia can be visualized in third instar larval wing discs as a series of stripes of cells expressing provein genes, alternating with domains of D-SRF expression. col activates D-SRF expression in A/P organizer cells and positions L3 vein by limiting L3 vein competence to cells expressing iro-C but not col. Therefore col transcription was examined in N55/+ third instar wing discs; it is expanded toward the anterior by one to two rows of cells. The position of the SOPs were examined, using a neuralised (neu)-lacZ reporter gene (transgenic line A101). Whereas in wild-type, one row of cells separates SOPs from the anterior limit of Col expression, SOPs are found immediately adjacent to cells expressing high levels of Col protein in N55/+ discs. Counterstaining of discs with propidium iodide (which labels all nuclei) confirms that the position of SOPs relative the A/P border (anterior limit of hh/posterior limit of Col expression) is unchanged, leading to the conclusion that reducing N activity in third instar larvae specifically results in anterior expansion of Col expression. Col expression was then examined in clones of N mutant cells generated in a heterozygous N55/+ background and spanning the A border of Col expression; it was found not to be expanded further anteriorly. col expression is established in response to Hh in a dose-dependent manner. The present data indicate that: (1) only one or two rows of cell activate col in response to Hh in the absence of Notch signaling, and (2) the same expansion on col expression results from complete absence of N or 2-fold reduction of N signaling suggesting that col expression is very dose sensitive. Thus, the expansion of col expression observed in N55/+ discs indicates that N signaling locally antagonizes Hh activation of col transcription, to precisely position the posterior limit of the L3 vein primordium. Repression of col transcription by Notch signaling has already been reported in formation of an embryonic muscle and at the wing margin but the molecular mechanisms underlying this expression remain to be determined. iro-C expression is also expanded anteriorly in late 3rd instar larval discs in N55/+ mutants, indicating that the entire L3 vein-competence domain is shifted anteriorly. The opposite, posterior shift of iro-C expression (and consequently L3 vein position) observed in col mutant discs (this correlates with the posterior shift of L3 vein observed in these mutants) is linked to the modified range of Dpp signaling resulting from lack of col activity. Similarly, it is proposed that the anterior expansion of iro-C expression in N55/+ mutant discs reflects a modified range of Dpp signaling induced by anterior extension of Col expression. Thus, in wild-type discs the cross-regulation between Hh, N and Dpp signalling allows the positioning of the L3 vein primordium in register with CS. Next, the question of the relation between the A and P boundaries of Col expression and positions of L3 and L4 veins versus proveins was addressed (Crozatier, 2003).

The current view is that vein primordia form at borders between adjacent A/P sectors of gene expression. According to this view, and based on col expression and requirement in cells along the A/P compartment boundary, the L4 and L3 vein primordia are predicted to edge the Col-expressing domain. It was observed, however, that in late 3rd instar wing discs, the col expression domain is not directly flanked by rho-expressing cells, but is separated from them by one to two rows of cells expressing neither gene and expressing Dl. This led to an examination at an earlier stage. In mid-third instar larvae, the col and rho expressing domains are immediately adjacent to one another, suggesting that the vein-centered-over-provein pattern is established secondarily as the disc continues to grow in size due to cell proliferation. Contrary to wild-type, in N55/+ mutant discs, col and rho expression domains remain juxtaposed, correlating with an increased number of rows of rho-expressing cells. These observations indicate that Notch signaling is involved in restricting rho expression and EGFR signaling to single rows of cells at the center of provein domains, probably via lateral inhibition. This process therefore operates already in mid-third instar larvae and results in a displacement of one to two cells between the positions of L3 and L4 vein primordia and the boundaries of Col expression. This displacement offers an explanation for the observation that the adult L4 vein is separated by several rows of intervein cells from the A/P compartment boundary. Although rho expression at the center of proveins in late third instar larvae likely prefigures the position of adult veins, the provein into vein and intervein resolution process can be initiated or, conversely abort later during pupal development, as shown by analysis of various mutants including Nts mutants. While consonant with the view that different A/P boundaries of prepattern gene expression in the wing primordium define the positions where provein domains are specified, the data do not support the suggestion that secondary short-range signals organize proveins around vein primordia. They rather support a sequential induction mechanism in which activation of the EGFR pathway defines vein-competent groups of cells in early 3rd instar larvae as well as promote the expression of Dl; in turn, Dl activation initiates lateral inhibitory signaling and restricts EGFR signaling to cells at the center of vein-competent domains, through a feed-back regulatory loop requiring Notch. This mechanism is consistent with the loss of the L3 and L4 stripes of Dl expression in rhove vn1 (vn, an EGFR ligand) mutants, indicating that Dl expression is dependent on EGF-R signaling. A similar EGFR-->Dl sequential induction model has recently been proposed to operate in differentiating photoreceptor cells in the developing eye of Drosophila. In conclusion, these observations highlight the importance of cross-talk between the Hh and N signaling pathways in assigning overlapping A/P positions to the L3 vein and associated sensory organs and the role of N in precisely positioning vein primordia, thus intimately linking prepattern to the vein resolution process (Crozatier, 2003).

Drosophila adult muscle precursors form a network of interconnected cells and are specified by the rhomboid-triggered EGF pathway

In Drosophila, a population of muscle-committed stem-like cells called adult muscle precursors (AMPs) keeps an undifferentiated and quiescent state during embryonic life. The embryonic AMPs are at the origin of all adult fly muscles and, as is demonstrated in this study, they express repressors of myogenic differentiation and targets of the Notch pathway known to be involved in muscle cell stemness. By targeting GFP to the AMP cell membranes, it was shown that AMPs are tightly associated with the peripheral nervous system and with a subset of differentiated muscles. They send long cellular processes running along the peripheral nerves and, by the end of embryogenesis, form a network of interconnected cells. Based on evidence from laser ablation experiments, the main role of these cellular extensions is to maintain correct spatial positioning of AMPs. To gain insights into mechanisms that lead to AMP cell specification, a gain-of-function screen was performed with a special focus on lateral AMPs expressing the homeobox gene ladybird. The data show that the rhomboid-triggered EGF signalling pathway controls both the specification and the subsequent maintenance of AMP cells. This finding is supported by the identification of EGF-secreting cells in the lateral domain and the EGF-dependent regulatory modules that drive expression of the ladybird gene in lateral AMPs. Taken together, these results reveal an unsuspected capacity of embryonic AMPs to form a cell network, and shed light on the mechanisms governing their specification and maintenance (Figeac, 2010).

In late Drosophila embryos, each abdominal hemisegment features six AMPs at stereotypical positions associated with differentiating muscle fibres. To better characterize these cells, tests were performed to see whether the Notch pathway, which is known to be required for generation of satellite cells from muscle progenitors and for keeping them ready to engage in muscle regeneration, is also active in AMPs. Analysis of a GFP reporter line, E(spl)M6-GFP, described as a read-out of the Notch pathway in Drosophila, revealed that it is co-expressed with Twist in AMPs. Also, transcripts of another Notch target, Him, specifically accumulated in AMPs. By testing several mesodermal cell markers, it was found that, in addition to Twist, two other transcription factors, Zfh1 and Cut, are expressed in all AMPs. Zfh1 expression in embryonic AMPs has been reported previously, whereas cut has been used to reveal a subset of AMPs associated with larval wing and leg imaginal discs. Despite expressing common markers, the AMPs are heterogenous and differ by the expression of muscle identity genes. For example, slouch (S59) and Pox meso are specifically expressed in ventral (V) AMPs whereas ladybird (lb) and Kruppel (Kr) display lateral (L) AMP-specific expression (Figeac, 2010).

To gain insights into AMP cell shapes and their behaviour, an E(spl) M6-GAL4 line was generated that recapitulates M6-GFP expression, and it was used it to drive a membrane-targeted GFP. It has been previously reported that AMPs are associated with the larval peripheral nervous system (PNS) and that in daughterless mutant embryos lacking all the larval sensory system, the final pattern of AMPs is deranged. This study showed that all embryonic AMPs are closely associated with both the PNS and the differentiated muscles, sitting either at the top of muscle fibres [LAMPs and dorsal (D) AMPs] or on their internal face [dorsolateral (DL) AMPs and VAMPs)]. In late embryos, the AMPs form a network of cells displaying irregular shapes and are interconnected by long cellular processes aligning PNS nerves. Connections between the AMPs initially form within the parasegments, but the AMPs very quickly send filopodia posteriorly and make contact with DLAMPs of the adjacent segment, thus interlinking all AMPs. In addition to the interconnected M6+/twi+ AMPs, a population of morphologically distinct M6+/twi- cells of unknown fate, located more internally in central and posterior regions of the abdomen, was identified (Figeac, 2010).

It has been reported that a subset of muscle progenitors divides asymmetrically and gives rise to numb-positive founder cells that undergo differentiation and to Notch-expressing AMPs. Through this pathway, six AMPs are born in each abdominal hemisegment. In contrast to founders, AMPs express the Notch target Holes in muscle (Him) and Zfh1, the Drosophila homolog of ZEB, both of which are able to counteract Mef2-driven myogenic differentiation. Interestingly, another general AMP marker, E(spl)M6, also corresponds to a Notch target, suggesting that Notch signalling could play an evolutionarily conserved role in muscle cell stemness. It operates not only in vertebrate satellite cells but also, as shown in this study, in Drosophila AMPs. Finally, it is reported that, similar to muscle progenitors, the AMPs are heterogenous and express different muscle identity genes, such as lb or slou. This strongly suggests that AMPs acquire a positional identity that makes them competent to form a given type of muscles during adult myogenesis. For example, the lateral AMPs expressing lb are at the origin of all lateral body wall muscles of the adult fly. In support of the specific positional identities of AMPs comes also the analysis of lame duck (lmd) mutant embryos known to be devoid of fusion-competent myoblasts (FCMs). In this mutant context, the number of Twi-positive and Zfh1-positive AMP-like cells is highly increased, while the number of Lbe- and Twi-positive LAMPs committed to the lateral lineage remains unchanged. Thus in the absence of lmd, some presumptive FCMs can adopt the AMP-like fate but they do not carry positional information transmitted by the identity genes such as lb (Figeac, 2010).

Based on the premise that the AMPs correspond to a novel population of transient stem cells, their shapes and behaviour were analyzed in living embryos carrying M6-GAL4 and UAS-GAP-GFP transgenes. Surprisingly it was found that shortly after their specification, the AMPs start to send cellular processes that align along the nerves of the PNS, with the result that, by the end of embryogenesis, all AMPs become linked together. Interestingly, the intersegmental connections are made via an intermediary M6+ twi- cell of unknown fate. In addition to this particular cell, which ensures the intersegmental link between AMPs, the embryos also contained other M6+ twi- non-neural cells of rounded morphology located more internally that were unconnected to the AMP cell network. The origin and identity of these cells remain unknown (Figeac, 2010).

Exploiting the possibility of following AMPs in vivo, test were performed to see how AMPs would behave if their connections were broken. Since the AMPs separated from the network by laser ablation changed shape and lost their normal positions, it is concluded that one important reason for which AMPs form a cell network is to keep precise spatial positioning. Based on the observation that AMPs send long cellular processes along the peripheral nerves, it is probable that nerves serve as a support for extending AMP cell protrusion. This possibility is supported by the abnormal pattern of AMPs observed in daughterless mutant embryos lacking the PNS and in embryos in which the PNS was affected by the Elav-GAL4 driven expression of the inducer of apoptosis, Reaper. PNS nerves might also represent a source of signals for AMPs such as Delta in order to maintain Notch activity. However, analysis of the lateral domain revealed that Delta expression was associated with the segment border muscle (SBM) precursor but not with the PNS neurons, indicating that Notch activity in lateral AMPs is regulated by Delta produced in the SBM rather than in nerves (Figeac, 2010).

Taking advantage from the restricted number of embryonic AMPs and the genetic tools available in Drosophila, a large-scale gain-of-function screen was performed to identify the genes involved in AMP specification. rho and other components of the EGF signalling pathway were found to be crucially required for both specification and maintenance of AMPs. Importantly, as reported by Krejci (2009), several components of EGF signalling are direct targets of Notch in AMPs, thus creating a link between the two signalling pathways. The high number of AMPs in EGFRCA and RAS gain-of-function contexts provides evidence that RAS signalling not only promotes muscle founder specification, but is also crucial for specifying AMPs when induced by EGF signals. Further support for a key role of the EGFR pathway is the identification of cells sending EGF to lateral AMPs and the demonstration of their role in AMP cell maintenance. It also turns out that the anti-apoptotic role of the EGFR pathway in Drosophila AMPs described in this study is conserved across evolution, since EGF signalling also promotes survival of vertebrate satellite cells (Figeac, 2010).

The evidence for a major role of the EGFR pathway in the specification and maintenance of AMPs raises important questions about EGF targets operating in these muscle-committed stem-like cells in Drosophila. lb genes have been shown to be required for specification of LAMPs, making them candidate targets of EGF signalling in the lateral region. This study shows that lb regulatory modules contain binding sites for ETS factors that act as EGFR effectors and goes on to demonstrate their crucial role in AMP enhancer activity. The proximity of the ETS binding sites and homeodomain binding sites in the AMP element suggests that an adapted spatial conformation of interacting factors is important in allowing simultaneous binding and thus maintenance of the lineage-restricted activity of this enhancer. Interestingly, the main difference between regulatory modules driving expression in differentiated muscle lineages versus regulatory modules that act in non-differentiated AMPs is the responsiveness of the latter category to extrinsic EGF signals. In opposition to this, this study found that intrinsic Mef2 inputs are sufficient to drive expression in differentiated muscle lineage. The ETS and Mef2-driven expression of these two distinct regulatory modules is positively regulated by lb, which is known to play a pivotal role in the specification of muscle lineages in the lateral domain. The specific expression of lb in a subset of AMP cells and of its ortholog Lbx1 in activated satellite cells suggests that similarities in genetic control of Drosophila and vertebrate muscle stem cells might extend beyond those discussed here (Figeac, 2010).


Activation of EGFR and ERK by rhomboid signaling regulates the consolidation and maintenance of sleep in Drosophila

Epidermal growth factor receptor (EGFR) signaling in the mammalian hypothalamus is important in the circadian regulation of activity. This study examined the role of the EGF pathway in the regulation of sleep in Drosophila. The results demonstrate that rhomboid (Rho)- and Star-mediated activation of EGFR and ERK signaling increases sleep in a dose-dependent manner, and that blockade of rhomboid (rho) expression in the nervous system decreases sleep. The requirement of rho for sleep localized to the pars intercerebralis, a part of the fly brain that is developmentally and functionally analogous to the hypothalamus in vertebrates. These results suggest that sleep and its regulation by EGFR signaling may be ancestral to insects and mammals (Foltenyi, 2007).

The findings reported here show a previously unknown role for EGFR and ERK signaling in sleep regulation and consolidation in Drosophila. In the adult fruit fly, EGFR is expressed ubiquitously throughout the nervous system, where its only known role is in the maintenance and survival of neurons. The current results demonstrate that the overexpression of EGFR pathway signaling components Rho and Star in Drosophila causes an acute, reversible and dose-dependent increase in sleep that tightly parallels an increase in phosphorylated ERK in the head. The ability of a dominant-negative EGFR to block the activation of ERK, as well as the known selectivity of Rho for these ligands, argues that the manipulation is specific to the EGFR pathway. In contrast to the increase in sleep amount after Rho overexpression, inhibiting its expression led to a significant decrease in sleep. Notably, this decrease in sleep was due to a marked shortening of the duration of sleep episodes accompanied by an elevation of sleep bout number. This observation suggests that flies have an increased need for sleep, but are unable to stay asleep, which is perhaps analogous to insomnia in humans. Therefore, it is proposed that the EGFR pathway is essential for sleep maintenance (Foltenyi, 2007).

The brain regions that appear to be involved in the influence of signaling by Rho, EGFR and ERK on sleep are the pars intercerebralis, median bundle and tritocerebrum. The cells of the pars intercerebralis contain Rho and generate EGFR ligand that activates ERK in the receiving cells in the tritocerebrum. The pars intercerebralis was identified as the region that is responsible for EGFR ligand secretion by demonstrating that inhibiting Rho in this region resulted in decreased sleep, and that the cells in that region expressed endogenous Rho. The tritocerebrum was identified though the system-wide overexpression of the EGFR ligand-processing components Rho and Star, which resulted in a localized hyperactivation of ERK. This is presumably because an ectopic presence of Rho and Star will only result in heightened EGFR signaling if the cells contain endogenous ligand precursor (Foltenyi, 2007).

Although the mushroom body is the only region of the Drosophila brain that has been reported to have an effect on sleep, no Rho expression was detected in the mushroom body, nor did inhibiting Rho with UAS-rhoDN in this structure have any effect on sleep levels. However, it is reasonable to expect that the regulation of sleep would involve multiple brain regions and pathways, and that the regulation, versus the function, of sleep could be two distinct, but linked, processes (Foltenyi, 2007).

Cells of the pars intercerebralis send out axonal projections though the median bundle and then bifurcate, innervating the tritocerebrum or running alongside the esophageal canal to innervate the endocrine gland corpora cardiaca. The results indicate that the pars intercerebralis cells innervating the corpora cardiaca are not the ones responsible for the observed decrease in sleep; Gal-4 drivers that are active in these cells did not produce a significant drop in sleep levels when expressing rho RNAi. Developmental studies have led to the postulate that the pars intercerebralis and the corpora cardiaca are the developmental equivalent of the mammalian hypothalamic-pituitary axis. The hypothalamus is a major center in the mammalian brain for the regulation of arousal, and the SCN, which is a part of the hypothalamus, has already been shown to regulate circadian activity through EGFR signaling (Foltenyi, 2007).

Vertebrate studies have only investigated EGFR signaling in the subparaventricular zone, a region located immediately adjacent to the SCN, and this region was shown not to affect total sleep levels, but does alter its timing. In addition, evidence in mammals for a role of EGF in sleep per se is equivocal. These results directly demonstrate that the disruption of EGFR ligand production affects sleep though the pars intercerebralis and not though the circadian control center of the Drosophila brain. It also suggests that the pars intercerebralis shares some functional, as well as developmental, homology with the mammalian hypothalamus through its crucial and conserved involvement in regulating sleep and its maintenance with neural hormones such as the EGFR ligands (Foltenyi, 2007).

In the fly, a single member of the EGFR family binds both the TGF-α-like family of ligands (Spitz, Gurken and Keren) and the neuregulin-like ligand Vein. In vertebrates, these ligands bind to specific ErbB family members, with ErbB-1 (EGFR) binding EGF and TGF-α, whereas ErbB-3 and ErbB-4 bind the neuregulins. In mammalian systems, ErbB-2 and ErbB-4 cofractionate, coimmunoprecipitate and colocalize in cultured rat hippocampal neurons with the postsynaptic density protein PSD-95 (also known as SAP90), and show exclusion from presynaptic terminals. Similarly, ERK colocalizes with, and directly phosphorylates, PSD-95, as is the case with the ErbB receptor-family members. In the fly, EGFR interacts with the postsynaptic density protein Discs Large (Dlg), the Drosophila homolog of PSD-95 (Foltenyi, 2007).

ERK has a role in synaptic plasticity that is conserved among Aplysia, Drosophila and mammals. A recent study shows that ERK directly phosphorylates the pore-forming α subunit of the A-type potassium channel Kv4.2, a member of the Shal-type (Shaker-like) family. This broadens the role of ERK beyond the realm of cell proliferation, differentiation, and even long-term memory consolidation, and suggests that it may also contribute to the more immediate alterations of the electrical properties of the neuronal membrane (Foltenyi, 2007).

On the basis of the current findings and the published reports on the functions of EGFR, the following cellular mechanism is proposed for sleep regulation in Drosophila. Star and Rho in the pars intercerebralis produce and secrete ligand to EGFR located at the postsynaptic membrane of neurons in the tritocerebrum, leading to the activation of ERK in these cells. The difference in staining patterns between inactive ERK clustering near synapses (data not shown) and active ERK located out in the axons indicates that the activated ERK, at least in part, translocates from the postsynaptic membrane and spreads out into the axons that fill out the tritocerebrum and other locations to which these cells project. As a result of a lack of ppERK in the cell bodies of these neurons and the reversible nature of the sleep behavior, it is unlikely that these cells are undergoing long-term synaptic structural changes associated with changes in gene expression. Instead, it is proposed that the action of ppERK occurs at the synapse or in the axon (or both), where it is possibly altering the gating of a neural receptor or channel, and thus changing the membrane properties of the cells. This modification results in an altered brain state that ultimately manifests itself in the sleep behavior of the animal. Such a model is consistent with a previously described mutation in the potassium channel shaker (Kv1.4), which has been shown to be incapable of getting much sleep (Foltenyi, 2007).

Return: see Rhomboid: Developmental Biology part 1/2

rhomboid: Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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