vein

DEVELOPMENTAL BIOLOGY (part 2/2)

Larva and Pupa

vein is expressed in the wing, haltere, leg and eye-antenna discs in a complex and developmentally dynamic pattern. In the second and early-third instars, vein is expressed in the proximal region of the wing disc, which gives rise to the notum. In the mid-third instar, vein is expressed intensely in the notum and transcription begins in the presumptive wing anlage. During the late-third instar, the expression in the wing disc resolves into a number of domains. vein is expressed in a swath across the region which gives rise to the lateral notum, as well as along the posterior edge of the disc, in regions that give rise to hinge structures, the mesopleura, and in an incomplete stripe across the wing pouch straddling the AP boundary. The position of the wing-pouch stripe was determined relative to argos expression, which marks the presumptive veins. The vein stripe lies in the intervein region between presumptive longitudinal veins L3 and L4 extending closer to L4 so that there is approximately a one-cell overlap between argos expression in L4 and vein expression. The position of vein was also determined relative to cubitus interruptus, which is expressed in the anterior compartment. A partial overlap with cells expressing ci shows that vn expression straddles the AP compartment boundary and includes both anterior and posterior cells (Simcox, 1996).

After puparium formation (APF) weak expression is seen in other intervein regions and expression is found in the L3 sensilla, the anterior wing margin, the posterior wing margin and in cells likely to include the campaniform sensilla. By 30 hours, APF expression around the wing margin and in the sensilla of L3 ceases, and vein is expressed exclusively in all intervein regions. Within each intervein region, vein is expressed more intensely in the cells bordering the preveins. Cells not expressing vein form a narrow strip (about two cells wide). Pupal wings stained for both vein and the vein cell markers rhomboid and argos show no gap between the rho/argos expressing cells and the vein expressing cells (Simcox, 1996).

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 Hw^49c 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 Hw^49c 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 rho^ve (a cis-regulatory allele of rho that lacks detectable rho expression in vein primordia), vn¹, rho^ve;vn¹ double mutants, iro^DFM2 (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 rho^Sld 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 rho^ve;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 rho^ve;vn¹ double mutants discs. The presence of isolated sc-expressing cells in rho^ve;vn¹ discs, likely to be L3 sensory organ precursors, may explain why L3 sensilla are usually present in rho^ve;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 rho^ve;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).

The subdivision of the Drosophila wing imaginal disc into dorsoventral (DV) compartments and limb-body wall (wing-notum) primordia depends on Epidermal growth factor receptor (Egfr) signaling, which heritably activates apterous (ap) in D compartment cells and maintains Iroquois Complex (Iro-C) gene expression in prospective notum cells. The source, identity and mode of action of the Egfr ligand(s) that specify these subdivisions has been examined. Of the three known ligands for the Drosophila Egfr, only Vein (Vn), but not Spitz or Gurken, is required for wing disc development. Vn activity is required specifically in the dorsoproximal region of the wing disc for ap and Iro-C gene expression. However, ectopic expression of Vn in other locations does not reorganize ap or Iro-C gene expression. Hence, Vn appears to play a permissive rather than an instructive role in organizing the DV and wing-notum segregations, implying the existance of other localized factors that control where Vn-Egfr signaling is effective. After ap is heritably activated, the level of Egfr activity declines in D compartment cells as they proliferate and move ventrally, away from the source of the instructive ligand. Evidence is presented that this reduction is necessary for D and V compartment cells to interact along the compartment boundary to induce signals, like Wingless (Wg), which organize the subsequent growth and differentiation of the wing primordium (Zecca, 2002).

Thus Vn is required for the generation of an instructive Egfr ligand in the dorsal region of the disc, and this result poses the question of what this ligand is. One possibility is that Vn itself is the ligand, but that it must be converted from an inert (or weakly active) precursor to an active (or more potent) form. Accordingly, the instructive cue would be a dorsally localized processing or activating factor that executes this conversion, serving a role analogous to that of Rho in activating Spi. This processing activity is unlikely to be Rho itself, because Rho does not appear to be required for either the DV or wing-notum segregations. In principle, a Vn-converting activity could be provided by one of several additional Rho-like proteins that have recently been identified. However, Vn differs from Spi in that nascent Vn is likely to be a secreted protein, whereas nascent Spi is a transmembrane protein from which the EGF-containing extracellular domain is cleaved and released to generate the active Egfr ligand. Hence, Vn and Spi might be activated by distinct molecular mechanisms, with only Spi and structurally related Spi-like ligands requiring the action of Rho family proteins. Curiously, substitution of the single EGF repeat in Vn by that in Spi creates a potent, Rho-independent ligand for the Egfr. Hence, Vn might normally be converted to an active form by a mechanism that modifies the structure or conformation of its EGF repeat (Zecca, 2002).

An alternative possibility is that the instructive Egfr signal is a presently unidentified Egfr ligand or co-factor that acts in conjunction with Vn (e.g. by forming a heterodimer with Vn or by potentiating Egfr activity by some other mechanism). Because indiscriminate expression of Vn does not ectopically activate either ap or the Iro-C genes, it would be expected that such a ligand or co-factor would be locally expressed in the dorsal region of the disc. At least one additional spi-like gene has recently been identified in the Drosophila genome, providing a possible candidate for a Vn co-ligand. It is noted that a single vn mutant disc was obtained in which a clone of laterally situated Vn-expressing cells appears to act non-autonomously to activate ap expression in a nearby, more dorsal patch of cells. Although only one such exceptional disc was observed, it raises the possibility that Vn secreted by one cell can act jointly with a factor expressed by other cells to generate the instructive Egfr ligand. Such non-autonomy is consistent with a mechanism in which Vn functions in conjunction with another ligand, or in which Vn is converted from a weak to potent Egfr ligand after it is secreted (Zecca, 2002).

A third possibility is that nascent Vn does not require processing to become an effective Egfr ligand, but instead that its ability to activate the Egfr is restricted to dorsoproximal regions of the wing disc by repressors generated in more ventrally situated cells. Because activated Spi induces both ap and Iro-C gene expression in the ventral region of the disc, such repressors might be selective for Vn itself, rather than being general inhibitors of Egfr activity. Alternatively, activated Spi might be a more potent activator of Egfr activity than Vn, allowing it to drive Egfr signaling in the presence of repressors that render Vn ineffective. Wg could, in principle, be such a repressor, as it is active ventrally and represses Vn-dependent Egfr signaling in early wing discs. However, during later development, Wg is expressed in a broad stripe in the presumptive notum that overlaps the domain of Iro-C gene expression in the prospective lateral notum. Hence, at least at this stage, Wg does not appear to block the Vn-dependent activation of the Egfr that maintains Iro-C gene expression (Zecca, 2002).

All cells within the wing imaginal disc require a minimum level of Egfr/Ras activity to sustain a normal rate of proliferation. It is not known whether this activity reflects the basal activity of the Egfr/Ras transduction pathway, or the response of the receptor to a specific ligand. However, it is clear that this low level of Egfr/Ras activity does not require Vn dependent Egfr signaling, since it has been shown that ectopic expression of Ap in vn mutant discs can rescue growth and differentiation of the wing primordium. This result demonstrates that the absence of wing development in vn mutant discs is an indirect consequence of the failure to establish an ap^ON-ap^OFF interface (Zecca, 2002).

During normal development, the ap and Iro-C genes are initially activated in overlapping dorsoproximal domains in response to Egfr signaling, and hence, at this early stage, it appears that most or all D compartment cells are exposed to relatively high levels of Egfr/Ras signaling. Thereafter, as the wing disc grows, ventrally situated D compartment cells inherit the 'on' state of ap expression, even as they populate areas of the disc progressively farther from the domain of high Egfr/Ras signaling and sustained Iro-C expression. It is suggested that the progressive reduction of Egfr/Ras activity in these ventrally situated D cells enables them to interact with neighboring V compartment cells to induce Wg and Vg expression and stimulate growth of the wing primordium. By contrast, early induced clones of Ras^V12-expressing cells autonomously express ap and experience persistent high levels of Ras activation, as indicated by sustained expression of the Iro-C genes. As a consequence, the ectopic DV boundary cannot shift outside of the domain of high Egfr/Ras signaling. Cells flanking this ectopic DV boundary fail to engage in the reciprocal induction of Wg and Vg expression or to stimulate growth. Hence, the ap^ON-ap^OFF interface may normally have to shift to a region of relatively low Egfr activity for the DV boundary to acquire wing organizer activity (Zecca, 2002).

Early induced clones that express Egfr^lambda, the constitutively active form of the Egfr, can induce the formation of ectopic D compartments that retain organizer activity. However, the level of constitutive Egfr/Ras activity in such Egfr^lambda-expressing clones appears to be significantly lower than in clones of Ras^V12-expressing cells. Consistent with this, it is found that ectopic expression of Egfr^lambda considerably reduces but does not completely eliminate vg expression. Hence, it is inferred that the levels of Ras activation in Egfr^lambda-expressing cells are not sufficiently high to prevent productive interactions between D and V compartment cells, thus allowing the ectopic DV compartment boundary to acquire organizer activity (Zecca, 2002).

How might Egfr signaling regulate the capacity of the DV compartment boundary to function as an organizer? One possibility is that high levels of Egfr/Ras activity block the ability of cells to transduce Notch signals. During normal development, D and V cells engage in a positive auto-feedback loop of Delta/Notch and Serrate/Notch signaling that drives the reciprocal induction of Wg and Vg expression on both sides of the DV compartment boundary. Hence, if high levels of Egfr/Ras activity block Notch signal transduction, then persistent high levels of Ras activity on even one side of the DV boundary would suffice to disrupt the feedback loop and block the reciprocal induction of Wg and other 'boundary' genes. Accordingly, the DV boundary might have to be located in a region of low Egfr activity in order to allow reciprocal Notch signaling to induce the expression of these, and perhaps other, organizer genes (Zecca, 2002).

Another possibility is that the ap^ON-ap^OFF interface may only be able to function as an organizer when cells on both sides are of prospective wing type. Prior to the initial activation of ap and the Iro-C genes, the nascent wing disc appears to be subdivided into mutually antagonistic domains of Egfr and Wg signaling that at least transiently define the incipient notum and wing primordia. Because ap and the Iro-C genes are initially activated in response to a common source of Egfr signaling, most or all D cells at this stage may be notum type. It is only later, when ventrally situated D cells move out of range of Vn-dependent Egfr signaling and switch to being wing type, that inductive interactions occur across the DV boundary to create a new and stable source of Wg signaling. It is suggested that cells on both sides of the DV boundary may have to be of wing type for the boundary to have organizer activity. One possible reason for why this might be the case is that vg, the selector-like gene that defines the wing state, is itself an integral component of the reciprocal signaling mechanism that allows D and V cells to induce the expression of DV boundary genes. High levels of Egfr/Ras signaling actively maintain Iro-C gene expression (and hence the notum state) and block vg expression. Hence, the DV boundary may normally have to shift ventrally, into a domain of low Egfr/Ras signaling and high Wg signaling that defines the incipient wing state, to allow the positive feedback loop of inductive signaling to initiate across the DV compartment boundary. Once this loop is established, it would provide a stable source of Wg and other signals generated along the DV boundary that govern the subsequent growth and differentiation of the wing blade (Zecca, 2002).

Programmed cell death and context dependent activation of the EGF pathway (via Vein) regulate gliogenesis in the Drosophila olfactory system

In the Drosophila antenna, sensory lineages selected by the basic helix-loop-helix transcription factor Atonal are gliogenic while those specified by the related protein Amos are not. What are the mechanisms that cause the two lineages to act differentially? Ectopic expression of the Baculovirus inhibitor of apoptosis protein (p35) rescues glial cells from the Amos-derived lineages, suggesting that precursors are removed by programmed cell death. In the wildtype, glial precursors express the extracellular-signal regulated kinase (phosphoERK) transiently, and antagonism of Epidermal growth factor pathway signaling compromises their development. It is suggested that all sensory lineages on the antenna are competent to produce glia but only those specified by Atonal respond to EGF signaling and survive. These results underscore the importance of developmental context of cell lineages in their responses to non-autonomous signaling in the choice between survival and death (Sen, 2004).

Several lines of investigation have ascertained that the first cells to divide in the sensory lineages are the secondary progenitors: PIIa, PIIb and PIIc. The numbers of sensory cells undergoing division at different times in the developing antenna were estimated by staining mitotic nuclei with antibodies against phosphorylated histone. A peak of cell division was observed between 16 and 24 h after puparium formation (APF). It has been considered that only in those sensory lineages specified by Ato, PIIb produces a glial cell and a tertiary progenitor, PIIIb, which in turn divides to form the sheath cell and a neuron. In Amos dependent lineages, PIIb is believed to directly give rise to a neuron and a sheath cell. The difference between the two lineages could be entirely dependent on the nature of the proneural genes activated; Amos, for example, could direct a non-gliogenic lineage. Alternatively, the two proneural genes could specify similar division patterns but the glial cell precursor in Amos-lineages could be removed by PCD, resulting in non-gliogenic lineages (Sen, 2004).

To test the latter possibility, cell death profiles were examined in developing pupal antennae using the terminal transferase assay (TUNEL) and attempts were made to correlate the timing of PCD with cell division profiles discussed above. The appearance of TUNEL-positive cells peaked between 22 and 24 h APF consistent with the occurrence of PCD immediately after division of secondary progenitors (Sen, 2004).

TUNEL reactions were performed on 22-24 h APF antennae from lz-Gal4; UAS-lacZnls and ato-Gal4; UAS-lacZnls animals. Double labeling with antibodies against ß-galactosidase marked sensory cells arising from the Lz and Ato lineages. Lz::lacZ overlaps the regions of the antennal disc where amos expression occurs and labels all the basiconic and trichoid sensilla in the mature (36 h APF) antenna. Hence for the purpose of this study, all cells in which lz-Gal4 expresses will be considered to belong to the Amos-dependent lineages. ato-Gal4 drives reporter activity in proneural domains of the disc and subsequently in all cells of the coeloconic sense organs (Sen, 2004).

Most of the apoptotic nuclei observed during olfactory sense organ development co-localized with Lz::LacZ suggesting that death occurred mainly within the 'Amos-dependent' sensory clusters. Only very few TUNEL-positive cells were detected in regions where ato-lacZ expressed and these did not co-localize with the reporter expression. If PCD is the mechanism used to remove glial precursors from Amos lineages, then their rescue would be expected to result in additional peripheral glia in the antenna (Sen, 2004).

The GAL4/UAS system was used to target ectopic expression of baculovirus inhibitor of apoptosis protein (p35) to different cell types within the developing antennal disc. distalless⁹⁸¹-Gal4 (henceforth called dll-Gal4), which drives expression in all cells of the antennal disc, resulted in the formation of >300 glial cells as compared to ~100 in the wildtype. Other sensory cells--neurons, sheath, socket and shaft cells--within sense organs were unaffected. Ectopic expression of p35 specifically in Ato lineages (ato::p35) did not alter glial number. This means that the `additional' glial cells rescued in dll::p35 must arise from lineages other than Ato. Mis-expression of p35 in Amos-dependent lineages using lz-Gal4, on the other hand, resulted in a significant increase in glial number. While other explanations are possible, it is believed that the somewhat lower number of glia obtained in lz::p35 as compared to dll::p35 could be accounted for by the strength of the P(Gal4) driver (Sen, 2004) (Sen, 2004).

In order to identify the cell within the Amos lineage that is fated to die, the cellular events during development of sense organs were re-examined. At approximately 12-14 h APF, most sensory cells are associated in clusters of secondary progenitors. Two cells in each cluster -- PIIb and PIIc -- express the homeodomain protein Prospero (Pros). pros-Gal4;UAS-GFP recapitulates Pros expression at this stage and marks PIIb and PIIc and their progeny in all olfactory lineages. In the wildtype, a Repo-positive cell was associated with only a few of the total sensory clusters, these were all located within the coeloconic domain of the antenna. Targeted expression of p35 using pros-Gal4 increased glial number indicating that cells which are the progeny of either PIIb or PIIc could be rescued from apoptosis. In the pros-Gal UAS-2XEGFP/UAS-p35 genotype, a glial cell was associated with most clusters at 18 h APF rather than in Ato lineages alone (Sen, 2004).

In order to directly visualize the cell undergoing apoptosis, 22-24 h APF antenna from the neu^A101 strain were stained with antibodies against ß-galactosidase to mark the sensory cells and with TUNEL. Sensory clusters located in basiconic and trichoid domains of the pupal antenna each had a single associated TUNEL positive cell. Since TUNEL reactivity data does not reflect the initiation of the death program, developing antennae were also stained at different time points with an antibody that recognized the activated caspase -- Drice. At 20 h APF, a single Drice-positive cell was found within each sensory cluster within the basiconic and trichoid domains of the pupal antenna. This cell also expressed low levels of Pros suggesting that it could arise from either PIIb or PIIc. This means that the PIIb/c in Amos lineages, like that in Ato, divides to give rise to a PIIIb and its sibling. The sibling in the former lineage was not previously detected because it expresses only low levels of Pros and soon dies. Since this cell is capable of expressing the glial-identity gene repo when rescued from death, it is denoted as a glial precursor (Sen, 2004).

How is apoptosis of a specific cell within the lineage regulated? In Drosophila three genes [reaper (rpr), grim and head involution defective (hid)] which all map under the Df(3L)H99 are necessary for the initiation of the death program. Heterozygotes of Df(3L)H99 show a small but significant increase in glial number over that of normal controls. hid-lacZ was used to follow expression during antennal development; reporter activity occurs at low levels ubiquitously including in glial cells. Levels of reporter expression indicate somewhat higher hid transcription in glia rescued by p35 mis-expression. The presence of Hid in the 'normal' glial precursors suggests a mechanism dependent on possible trophic factors to keep cells alive. In several other systems signaling, mainly through the EGFR pathway, results in an antagonism of Hid action and transcription. The sustained levels of hid transcription in the rescued glia, is not unexpected since inhibitors of apoptosis act by antagonizing a downstream event of caspase activation, rather than on Hid itself (Sen, 2004).

The peripheral olfactory glia ensheath the axonal fascicles as they project towards the brain. Antennae from animals deficient for ato (ato¹/Df(3R)p¹³), lack a large fraction of glia; the ~30 which remain, appear to arise elsewhere and migrate into the third segment of the antenna somewhat later during development (Sen, 2004).

Is death the default fate for all glial precursors and are there signals that keep Ato-glia alive? Several studies have provided compelling evidence for the role of receptor tyrosine kinase signaling in glial survival. In order to test this in the olfactory glia, 14-16 h APF pupal antennae were stained with antibodies against Pros and the phosphorylated form of ERK (pERK). The PIIb and PIIc cells are recognized by expression of Pros, which becomes asymmetrically localized in PIIb during mitosis. At this stage sensory cells do not express pERK. pERK was detected in the daughter of the first secondary progenitor to divide (probably PIIb). Staining with anti-Repo antibodies shows that this cell is glial. Clusters showing pERK expressing cells were observed only in regions of the pupal antenna from where the coeloconic sensilla originate and not in the Amos-lineage sensory clusters. pERK expression decays rapidly as Repo rises; only occasional cells can be detected that show immunoreactivity to both (Sen, 2004).

The presence of pERK in nascent glial precursors is evidence for receptor tyrosine kinase pathway signaling in these cells. Since EGFR activation is a well recognized signal for glial survival, the role of this pathway in glial cell development was tested. In order to demonstrate a role for EGF signaling during glial formation in the antenna, a hs-Gal4 strain and carefully timed heat-pulses were used to drive transient expression of various antagonists of the pathway. Ectopic expression of a dominant negative form of human Ras (DN-RasN17) between 14 and 15 h APF, severely reduced the numbers of antennal glia. Similarly, expression of the inhibitory ligand Argos or a dominant negative construct of Drosophila EGFR (DN-EGFR) compromises glial development. Since abrogation of signaling could, in principle, affect other developmental events, antenna from 36 h APF pupae of relevant genotypes were stained with support cell and neuronal markers to ascertain that these treatments did not affect sense organ development generally (Sen, 2004) (Sen, 2004).

Interference with EGFR activity affects glial number, suggesting that signaling is required for glial development and/or survival. Cells in the Amos-lineage also produce glial precursors, which do not express pERK. This implies that Amos-lineage glial precursors fail to experience EGF signaling and are fated to die. This hypothesis could, in principle, be tested by constitutively activating the pathway in Amos lineages. These experiments were not possible to carry out since activation of EGFR at the time when Amos-dependent secondary progenitors were undergoing division resulted in pupal lethality. The spatial and temporal expression of Vein-lacZ and Sprouty-lacZ (Sty-LacZ) supports a role in development of glia and requires further genetic analysis. Vein-LacZ was first detected at 18 h APF and expression was elevated at 20 h APF in a domain of the pupal antenna occupied mainly by coeloconic sensilla. The fact that this is the region of the antenna from where most glia originate, is interesting in the light of data from other systems that demonstrate that Vein acts as gliotrophin (Sen, 2004).

The spatial and temporal pattern of Vein and Sty expression is consistent with a role for these ligands in glial development. The region where Vein is expressed shows high pERK activity suggesting activation of the EFGR pathway in the coeloconic (Ato) domains of the developing antenna. Sty, however, is present in the basiconic and trichoid (Amos) domains of the antenna. At 10-14 h, when the secondary progenitors have not yet divided, Sty expressing cells lie adjacent to the PIIb and PIIc; these cells are labeled by Pros. This localization is consistent with a role for Sty in antagonizing EGF activity in the secondary progenitors (Sen, 2004) (Sen, 2004).

This work provides one more instance where PCD plays a crucial role in the selection of a specific population of cell types during development although the mechanisms employed still need to be elucidated. How does the development context of lineages of cells within a single epidermal field together with non-autonomous cues result in distinct consequences? The Drosophila antenna is a valuable system to address these issues because of the diversity of morphologically and molecularly distinct cell types located in a highly stereotyped architecture and the wealth of reagents available for study (Sen, 2004).

Notch signalling coordinates tissue growth and wing fate specification in Drosophila; Tissue size modulates the activity range of the signalling molecules Wingless and Vein

During the development of a given organ, tissue growth and fate specification are simultaneously controlled by the activity of a discrete number of signalling molecules. These two processes are extraordinarily coordinated in the Drosophila wing primordium, which extensively proliferates during larval development to give rise to the dorsal thoracic body wall and the adult wing. The developmental decision between wing and body wall is defined by the opposing activities of two secreted signalling molecules, Wingless and the EGF receptor ligand Vein. Notch signalling is involved in the determination of a variety of cell fates, including growth and cell survival. Evidence is presented that growth of the wing primordium mediated by the activity of Notch is required for wing fate specification. The data indicate that tissue size modulates the activity range of the signalling molecules Wingless and Vein. These results highlight a crucial role of Notch in linking proliferation and fate specification in the developing wing primordium (Rafel, 2008).

The expression of Wg in the most ventral part of the wing disc specifies the wing field at the same time as restricting Vn expression to the most dorsal part. Vn is required to block the responsiveness of body wall cells to Wg. Thus, the relative concentration of the diffusible proteins Wg and Vn experienced by disc cells directs their wing versus body wall fate. It is interesting to note that the expression of these two molecules is established long before the wing field is induced in the presumptive wing primordium. Wg expression starts long before wing field specification takes place, as revealed by the later induction of Nub expression and the reduction in the expression of the body wall cell marker Tsh. It is therefore proposed that tissue growth modulates the cellular response to these signalling molecules and controls, in time, wing fate specification. In the early wing primordium, Vn might reach every wing cell, thereby blocking responsiveness to Wg and repressing wing fate specification. Growth induced by Notch activity might pull the sources of Wg and Vn apart and, thus, most ventral cells might not sense sufficient Vn levels, so Wg would be able to induce wing fate. Interestingly, the overexpression of Wg or overactivation of its signalling pathway is able to bypass the requirement of growth in this process, indicating that the cells sense the relative levels of Wg and Vn. Once the wing field has been specified, Wg starts to be expressed along the presumptive wing margin, where it exerts a fundamental function in the maintenance of the Notch-dependent organizing center along the DV boundary. Note that the organizing activity of Notch at the DV boundary takes place long after the early function of Notch revealed in this work, which is involved in promoting growth and facilitating wing fate specification. As revealed by the expression of the Notch target E(spl)m-β, it is not until late in the second instar that the expression of Notch is restricted to the DV boundary. During the process of wing fate specification that takes place during second instar, it is uniformly expressed in the whole wing disc. These results imply that growth also facilitates the reiterative use of signalling molecules, such as Wg and Notch, to exert different functions during the development of a multicellular organ like the wing primordium (Rafel, 2008).

At the same time that wing and body wall fate specification takes place in the wing primordium, Vn is involved in the induction of apterous expression in the dorsal region. Consistent with the model proposed above, the activity of Vn, as monitored by the expression of apterous, was modulated by tissue growth. In the absence of Notch activity, even though Vn expression is not affected, Vn appears to reach every wing cell, as apterous expression was expanded ventrally. Increased levels of Wg expression or growth promoted by CycE appear to re-establish the dorsally restricted range of activity of Vn, as apterous expansion was blocked under these circumstances (Rafel, 2008).

Growth promoted by Notch has also been shown to be directly involved in the specification of the eye within the Drosophila eye-antenna primordium, a process that also depends upon the opposing activities of two secreted signalling molecules, in this case Dpp and Wg. Thus, Notch coordinates in a very elegant manner both eye and wing primordia tissue growth and eye/wing specification, by modulating the response of the cells to the activities of signalling molecules. These results indicate that the same mechanism might be commonly used in animal development to coordinate tissue growth and fate specification (Rafel, 2008).

The evolution of wings was crucial in the process of adaptation, allowing insects to escape predators or colonize new niches. The loss and recovery of wings has occurred during the course of evolution. This would suggest that wing developmental pathways are conserved in wingless insects and are being re-used. According to the current results, it is speculated that adaptive changes in animal size could modulate the cellular response to signalling molecules such as Wg, thereby helping to drive some of these extraordinary reversible transitions (Rafel, 2008).

EGFR, Wingless and JAK/STAT signaling cooperatively maintain Drosophila intestinal stem cells

Tissue-specific adult stem cells are commonly associated with local niche for their maintenance and function. In the adult Drosophila midgut, the surrounding visceral muscle maintains intestinal stem cells (ISCs) by stimulating Wingless (Wg) and JAK/STAT pathway activities, whereas cytokine production in mature enterocytes also induces ISC division and epithelial regeneration, especially in response to stress. This study shows that EGFR/Ras/ERK signaling is another important participant in promoting ISC maintenance and division in healthy intestine. The EGFR ligand Vein is specifically expressed in muscle cells and is important for ISC maintenance and proliferation. Two additional EGFR ligands, Spitz and Keren, function redundantly as possible autocrine signals to promote ISC maintenance and proliferation. Notably, over-activated EGFR signaling could partially replace Wg or JAK/STAT signaling for ISC maintenance and division, and vice versa. Moreover, although disrupting any single one of the three signaling pathways shows mild and progressive ISC loss over time, simultaneous disruption of them all leads to rapid and complete ISC elimination. Taken together, these data suggest that Drosophila midgut ISCs are maintained cooperatively by multiple signaling pathway activities and reinforce the notion that visceral muscle is a critical component of the ISC niche (Xu, 2011).

Adult stem cells commonly interact with special microenvironment for their maintenance and function. Many adult stem cells, best represented by germline stem cells in Drosophila and C. elegans, require one primary maintenance signal from the niche while additional signals may contribute to niche integrity. ISCs in the Drosophila midgut do not seem to fit into this model. Instead, they require cooperative interactions of three major signaling pathways, including EGFR, Wg and JAK/STAT signaling, for long-term maintenance. Importantly, Wg or JAK/STAT signaling over-activation is able to compensate for ISC maintenance and proliferation defects caused by EGFR signaling disruption, and vice versa. Therefore, ISCs could be governed by a robust mechanism, signaling pathways could compensate with each other to safeguard ISC maintenance. The mechanisms of the molecular interactions among these pathways in ISC maintenance remains to be investigated. In mammals, ISCs in the small intestine are primarily controlled by Wnt signaling pathways, and there are other ISC specific markers not controlled by Wnt signaling. In addition, mammalian ISCs in vitro strictly depend on both EGFR and Wnt signals, indicating that EGFR and Wnt signaling may also cooperatively control mammalian ISC fate. It is suggested that combinatory signaling control of stem cell maintenance could be a general mechanism for ISCs throughout evolution (Xu, 2011).

The involvement of EGFR signaling in Drosophila ISC regulation may bring out important implications to understanding of intestinal diseases, in which multiple signaling events could be involved. For example, in addition to Wnt signaling mutation, gain-of-function K-Ras mutations are frequently associated with colorectal cancers in humans. Moreover, activation of Wnt signaling caused by the loss of adenomatous polyposis coli (APC) in humans initiates intestinal adenoma, but its progression to carcinoma may require additional mutations. Interestingly, albeit controversial, Ras signaling activation is suggested to be essential for nuclear β-catenin localization, and for promoting adenoma to carcinoma transition. In the Drosophila midgut, loss of APC1/2 genes also leads to intestinal hyperplasia because of ISC overproliferation. Given that EGFR signaling is generally activated in ISCs, it would be interesting to determine the requirements of EGFR signaling activation in APC-loss-induced intestinal hyperplasia in Drosophila, which might provide insights into disease mechanisms in mammals and humans (Xu, 2011).

Previous studies suggest that intestinal VM structures the microenvironment for ISCs by producing Wg and Upd maintenance signals. This study identified Vn, an EGFR ligand, as another important ISC maintenance signal produced from the muscular niche. Therefore, ISCs are maintained by multiple signals produced from the muscular niche. In addition, Spi and Krn, two additional EGFR ligands, were identified that function redundantly as possible autocrine signals to regulate ISCs. These observations are consistent with a previous observation that paracrine and autocrine EGFR signaling regulates the proliferation of AMPs during larval stages, suggesting that this mechanism is continuously utilized to regulate adult ISCs for their maintenance and proliferation. The only difference is that the proliferation of AMP cells is unaffected when without autocrine Spi and Krn, due to redundant Vn signal from the VM, whereas autocrine Spi/Krn and paracrine Vn signals are all essential in adult intestine for normal ISC maintenance and proliferation. It was found that Vn and secreted form of Spi have similar roles in promoting ISC maintenance and activation, but additional regulatory or functional relationships among these ligands require further investigation, as the necessity of multiple EGFR ligands is still not completely understood. It is known that secreted/activated Spi and Krn are diffusible signals, but clonal analysis data show that Spi and Krn can display autonomous phenotypes. This observation indicates that these two ligands could behave as very short range signals in the intestinal epithelium, or they could diffuse over long distance but the effective levels of EGFR activation could only be achieved in cells where the ligands are produced. Interestingly, palmitoylation of Spi is shown to be important for restricting Spi diffusion in order to increase its local concentration required for its biological function. Whether such modification occurs in intestine is unknown, but it is speculated that Vn, Spi and Krn, along with the possibly modified forms, may have different EGFR activation levels or kinetics, and only with them together effective activation threshold could be reached and sustained in ISCs to control ISC behavior. Therefore, a working model is proposed that ISCs may require both paracrine and autocrine mechanisms in order to achieve appropriate EGFR signaling activation for ISC maintenance and proliferation.

Mechanisms of JAK/STAT signaling activation is rather complex. In addition to Upd expression from the VM, its expression could also be detected in epithelial cells with great variability in different reports, possibly due to variable culture conditions. Upon injury or pathogenic bacterial infection, damaged ECs and pre-ECs are able to produce extra cytokine signals, including Upd, Upd2 and Upd3, to activate JAK/STAT pathway in ISCs to promote ISC division and tissue regeneration. Several very recent studies suggest that EGFR signaling also mediates intestinal regeneration under those stress conditions in addition to its requirement for normal ISC proliferation. Therefore, in addition to basal paracrine and autocrine signaling mechanisms that maintain intestinal homeostasis under normal conditions, feedback regulations could be employed or enhanced under stress conditions to accelerate ISC division and epithelial regeneration (Xu, 2011).

Evidence so far has indicated a central role of N signaling in controlling ISC self-renewal. N is necessary and sufficient for ISC differentiation. In addition, the downstream transcriptional repressor Hairless is also necessary and sufficient for ISC self-renewal by preventing transcription of N targeting genes in ISCs. Therefore, N inhibition could be a central mechanism for ISC fate maintenance in Drosophila. High Dl expression in ISCs may lead to N inhibition, though how Dl expression is maintained in ISCs at the transcriptional level is not clear yet. Hyperactivation of EGFR, Wg or JAK/STAT signaling is able to induce extra Dl⁺ cells, suggesting that these three pathways might cooperatively promote Dl expression in ISCs. It is also possible that these pathways regulate Dl expression indirectly. As Dl-N could have an intrinsically regulatory loop for maintaining Dl expression and suppressing N activation, these pathways could indirectly regulate Dl expression by targeting any component within the regulatory loop. Identifying their respective target genes by these signaling pathways in ISCs would be an important starting point to address this question (Xu, 2011).

Adult

EGF signaling regulates the proliferation of intestinal stem cells in Drosophila

Precise control of somatic stem cell proliferation is crucial to ensure maintenance of tissue homeostasis in high-turnover tissues. In Drosophila, intestinal stem cells (ISCs) are essential for homeostatic turnover of the intestinal epithelium and ensure epithelial regeneration after tissue damage. To accommodate these functions, ISC proliferation is regulated dynamically by various growth factors and stress signaling pathways. How these signals are integrated is poorly understood. This study shows that EGF receptor signaling is required to maintain the proliferative capacity of ISCs. The EGF ligand Vein is expressed in the muscle surrounding the intestinal epithelium, providing a permissive signal for ISC proliferation. The AP-1 transcription factor FOS serves as a convergence point for this signal and for the Jun N-terminal kinase (JNK) pathway, which promotes ISC proliferation in response to stress. These results support the notion that the visceral muscle serves as a functional 'niche' for ISCs, and identify FOS as a central integrator of a niche-derived permissive signal with stress-induced instructive signals, adjusting ISC proliferation to environmental conditions (Biteau, 2011).

These findings establish a crucial role for EGF signaling in the regulation of ISC proliferation, and thus support the notion that the visceral muscle surrounding the intestinal epithelium has the characteristics of a functional niche. vein expression in the muscle maintains the competence of ISCs to enter rapid proliferation in responses to stress and JNK signaling, and is thus expected to regulate epithelial homeostasis. Interestingly, it was found that both the EGFR-mediated permissive signal and the JNK-derived inductive signal are relayed by FOS, establishing an integrated molecular mechanism for the control of ISC proliferation (Biteau, 2011).

Many stem cell populations are regulated by their microenvironments, and larval ISC progenitors are regulated by a transient niche (Mathur, 2010). However, ISCs in adult flies apparently lack such a closely associated cell population within the intestinal epithelium. By contrast, control of ISC maintenance by muscle-derived Wingless suggested this tissue as a potential functional niche for adult ISCs. The current results support and extend this idea by identifying a second growth factor derived from the visceral muscle that controls ISC proliferation. In its regulation of stem cell function through Wingless and Vein, and in the close association of ISCs and muscle cells, the muscle thus shares characteristics of stem cell niches in other systems, yet it also differs from these in important ways. In mammals, as well as in the Drosophila and C. elegans gonads, the niche of most stem cell populations maintains stem cell quiescence and prevents differentiation. The EGF signal originating from the muscle, however, maintains the capacity of ISCs to divide, allowing these cells to respond to stimulating signals while not affecting ISC differentiation. Interestingly, EGFR signaling has not been described so far as crucial for interactions between the niche and stem cell populations in other systems, and these findings raise the possibility that this signaling pathway might also regulate the function of other stem cell populations in both invertebrates and vertebrates (Biteau, 2011).

Whereas knocking down the expression of vein in the muscle partially affects the ability of ISCs to proliferate under normal conditions and in response to stress, the inhibition of EGFR completely abolishes stem cell division. This might reflect the inefficiency of the vein^RNAi constructs used in this study, but might also suggest a contribution of other EGFR ligands to the regulation of ISC function. Accordingly, a genome-wide analysis of the transcriptional response of the adult intestine to bacterial infection suggests that expression of vein, as well as of two other genes encoding EGFR ligands, Keren and spitz, is increased after immune challenge. However, the potential role for these additional EGF-like ligands in regulating ISC function remains to be investigated and the cells expressing spitz and Keren in the adult intestine have yet to be identified (Biteau, 2011).

ISC function is regulated by systemic insulin-like peptides expressed by neurosecretory cells in the brain, muscle-derived vein and wingless, local unpaired cytokine expressed by ECs, and cell-intrinsic signals. These multiple signals are integrated in ISCs to adapt their proliferation rate and differentiation program to environmental and physiological challenges. To fully understand stem cell regulation in this high-turnover tissue, the molecular structure of this signaling network has to be unraveled. The findings of this study introduce the transcription factor FOS as a crucial regulator of ISC proliferation that integrates mitogenic and stress signals, and indicate that JNK and ERK regulate FOS activity directly by phosphorylation on distinct residues, controlling ISC proliferation in a combinatorial fashion. This signal-specific mode of FOS regulation by ERK and JNK in Drosophila had previously been described in the context of morphogenetic movements (in which FOS is regulated by JNK) and of eye and wing growth during development (in which it is regulated by ERK and JNK) (Biteau, 2011).

How FOS promotes ISC proliferation remains unclear. In developing imaginal discs, inhibition of FOS causes an accumulation of cells in the G2/M phase of the cell cycle, probably owing to a loss of Cyclin B expression, an essential regulator of the G2/M transition. Interestingly, in ISCs, expression of Fos^RNAi not only inhibits stress-induced accumulation of pH3⁺ cells, but also represses BrdU incorporation, indicating that FOS regulates entry into S phase. In these cells, FOS might thus regulate the transcription of essential S phase components. Further studies will be required to identify such ISC-specific FOS target genes (Biteau, 2011).

The maintenance of stem cells in a primed state, ready to respond to inductive mitogenic stress signals, is likely to be crucial for high-turnover tissues like the intestinal epithelium, which require rapid activation of stem cell division for an efficient regenerative response to tissue damage. At the same time, this enhanced mitogenic potential of ISCs might contribute to the loss of tissue homeostasis in the aging gut, and contribute to cancer formation in mammalian intestinal epithelia. Interestingly, a conserved role of AP-1 transcription factors and JNK signaling in the regulation of intestinal stem cell proliferation and intestinal cancer is emerging in mice. JNK activation is sufficient to induce cell proliferation in the intestinal crypt and increases tumor incidence and tumor growth in an inflammation-induced colon cancer model. These effects of JNK signaling are mediated by the FOS binding partner JUN, as shown by the requirement for JNK-mediated phosphorylation of JUN for APC^min/+-induced tumorigenesis. Strikingly, ISC-specific activation of WNT signaling, by mutating APC or expressing an active form of ß-catenin or wingless itself, is sufficient to induce the formation of tumor-like stem cell clusters in the fly intestine. A potential interaction of WNT signaling with JNK and JUN or FOS in ISCs remains to be tested in Drosophila. Interestingly, increased FOS activity has also recently been shown to be sufficient to promote hematopoietic stem cell self-renewal in mice, further illustrating the conserved function of FOS in the regulation of stem cell function (Deneault, 2009). AP-1 transcription factors are thus emerging as conserved essential regulators of stem cell function and the current findings provide an important starting point for further studies characterizing stem cell-specific signaling networks that integrate mitogenic, survival and stress signals to control stem cell maintenance, quiescence and proliferation, and thus influence the balance between regeneration and tumor suppression in high turnover tissues (Biteau, 2011).

Regional control of Drosophila gut stem cell proliferation: EGF establishes GSSC proliferative set point & controls emergence from quiescence

Adult stem cells vary widely in their rates of proliferation. Some stem cells are constitutively active, while others divide only in response to injury. The mechanism controlling this differential proliferative set point is not well understood. The anterior-posterior (A/P) axis of the adult Drosophila midgut has a segmental organization, displaying physiological compartmentalization and region-specific epithelia. These distinct midgut regions are maintained by defined stem cell populations with unique division schedules, providing an excellent experimental model with which to investigate this question. This study has focused on the quiescent gastric stem cells (GSSCs) of the acidic copper cell region (CCR), which exhibit the greatest period of latency between divisions of all characterized gut stem cells, to define the molecular basis of differential stem cell activity. Molecular genetic analysis demonstrates that the mitogenic EGF signaling pathway is a limiting factor controlling GSSC proliferation. Under baseline conditions, when GSSCs are largely quiescent, the lowest levels of EGF ligands in the midgut are found in the CCR. However, acute epithelial injury by enteric pathogens leads to an increase in EGF ligand expression, specifically Spitz and Vein, in the CCR and rapid expansion of the GSSC lineage. Thus, the unique proliferative set points for gut stem cells residing in physiologically distinct compartments are governed by regional control of niche signals along the A/P axis (Strand, 2013).

The CCR epithelium is the exclusive site of large acid-secreting copper cells responsible for generating a low pH compartment in the midgut. Gastric stem cells in the CCR are normally quiescent but are robustly stimulated to replenish the unique differentiated cells of the gastric epithelium in response to injury by enteric pathogens or heat stress. This study resolvse outstanding issues related to the GSSC lineage, demonstrating the presence of tripotent GSSC lineages in the CCR. In addition, wa central role is demonstrated for the conserved EGF signaling pathway in controlling the emergence of gastric stem cells from quiescence. Taken together, two key differences between GSSCs and intestinal stem cells (ISCs) are now evident: the unique region specific cell lineages that they support (copper, interstitial, enteroendocrine vs. enterocyte and enteroendocrine) and their activity levels (quiescent vs. active). Thus, maintenance of physiologically and functionally distinct compartments of the adult midgut depends upon the activity of distinct stem cell lineages (Strand, 2013).

What is the nature of the unique molecular program that governs the observed differences in GSSC and ISC proliferative behavior? This study indicates that regional differences in gut stem cell proliferation are controlled by regional differences in EGF ligand availability. First, reporters of EGF pathway activity are normally very low in the CCR under baseline conditions, when GSSCs are quiescent. However, damage to the gastric epithelium by enteric infection increases local EGF ligand expression and Erk phosphorylation. This EGF activation directly correlates with an observed increase in proliferating GSSCs. Second, ectopic activation of the EGF pathway is sufficient to cell-autonomously promote GSSC proliferation in the absence of environmental challenge. Finally, functional EGF signaling is required for GSSC proliferation following enteric infection and for GSSC lineage expansion. Importantly, these studies of GSSCs in the CCR are similar to previous studies demonstrating that EGF signals are an essential part of the core niche program controlling the ISC lineage. Thus, regional control of EGF ligands, and perhaps other regulators of EGF pathway activity, are essential in generating gastrointestinal stem cell niches with distinct proliferative set points (Strand, 2013).

In this light, it is worth noting that over-expression of epidermal growth factors and their receptors are associated with human gastric cancer, the second leading cause of cancer-related deaths worldwide. In addition, Ménétrier’s disease is a hyperproliferative disorder of the stomach caused by over-expression of the EGF ligand TGF-α. Over production of TGF-α and increased EGF signaling is associated with an expansion of surface mucous cells and a reduction in parietal and chief cells. Gastric stem cells are the proposed cell-of-origin in Ménétrier’s disease, but this has not been directly tested due to a lack of gastric stem cell specific markers in the murine system. Advances in understanding how EGF ligand availability controls activity of the acid-secreting gastric stem cell lineage in Drosophila raises the possibility that hyperplastic conditions associated with the human stomach might arise when ectopic EGF ligands draw resident stem cells out of their quiescent state (Strand, 2013).

EGF signaling appears to be only one aspect of the region specific program controlling gastric stem cells in the adult copper cell region. Previous studies have shown that a Delta-lacZ enhancer trap line was not present in GSSCs under baseline conditions. In the course of this study, it was observed that Pseudomonas entomophila challenge also leads to an increase in Delta ligand expression in dividing cells, suggesting a role for Delta/Notch signaling in the GSSC lineage. In addition, elegant studies of GSSCs under baseline conditions have recently shown that the secreted BMP/Dpp signaling pathway is both necessary and sufficient to specify copper cells in the adult midgut and acts via the labial transcription factor. Interestingly, while the highest levels of Dpp pathway reporters are detected in the CCR, manipulation of the BMP/Dpp pathway did not affect GSSC proliferation. Thus, the GSSC lineage is influenced by secreted niche factors, which independently control both GSSC proliferation and cell fate specification (Strand, 2013).

In conclusion, understanding GI regionality and homeostatic diversity along the A/P axis is important for several reasons. It is now possible to gain insight into how the modification of a core GI niche program, which adapts each stem cell to its compartment specific physiology, leads to difference in lineage output. Second, disruption of regional identity along the GI tract is associated with a class of precancerous conditions called metaplasias, in which one region of the GI tract takes on the attributes of another. Finally, both the establishment and maintenance of tumorigenic lineages exhibit marked preferences along the A/P axis of the gut. The striking similarities between vertebrate and invertebrate GI biology, suggest that delving deeper into the mechanisms underlying Drosophila midgut regionalization will continue to provide important insights into these fundamental biological problems (Strand, 2013).

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