Effects of Mutation or Deletion (part 3/3)

Dpp and leg morphogenesis

Genetically mosaic flies were constructed which lack a functional dpp or wg gene in portions of their leg epidermis, and the leg cuticle was examined for defects. Although dpp has been shown to be transcribed both ventally and dorsally, virtually the only dpp-null clones that affect leg anatomy are those which reside dorsally. Conversely, wg-null clones only cause leg defects when they reside ventrally. Both findings are consistent with models of leg development in which the future tip of the leg is specified by an interaction between dpp and wg at the center of the leg disc. Null clones can cause mirror-image cuticular duplications, confined to individual leg segemnts. Double-ventral, mirror-image patterns are observed with dpp-null clones, and double-dorsal patterns with wg null clones. Clones that are doubly mutant (null for both dpp and wg) manifest reduced frequencies for both types of duplications. Duplications can include cells from surrounding non-mutant territory. Such nonautonomy implies that both genes are involved in positional signaling, not merely in the maintenance of cellular identities. However, neither gene product appears to function as a morphogen for the entire leg disc, since the effects of each gene's null clones are restricted to a discrete part of the circumference. Interestingly, the circumferential domains where dpp and wg are needed are complementary to one another (Held, 1996).

In the Drosophila leg disc, wingless and decapentaplegic are expressed in a ventral-anterior and a dorsal-anterior stripe of cells, respectively. This pattern of expression is essential for proper limb development. While the Hedgehog (Hh) pathway regulates dpp and wg expression in the anterior-posterior (A/P) axis, mechanisms specifying their expression in the dorsal-ventral (D/V) axis are not well understood. Evidence is presented that supernumerary limbs (slimb) mutant clones in the disc deregulate wg and dpp expression in the D/V axis. This suggests for the first time that their expression in the D/V axis is actively regulated during imaginal disc development. Furthermore, slimb is unique in that it also deregulates wg and dpp in the A/P axis. The misexpression phenotypes of slimb- clones indicate that the regulation of wg and dpp expression is coordinated in both axes, and that slimb plays an essential role in integrating A/P and D/V signals for proper patterning during development. Genetic analysis further reveals that slimb intersects the A/P pathway upstream of smoothened (smo) (Theodosiou, 1998).

slimb was identified in a mutant screen. To identify recessive overproliferation mutations in genes that are lethal in homozygous mutant animals, genetic screens were performed in mosaic flies containing homozygous mutant patches in otherwise wild-type backgrounds. Two classes of recessive overproliferation mutations have been identified. Mutations of the first group cause mutant cells to undergo extensive proliferation and form unpatterned, tumorous outgrowths in mosaic adults. Mutations of the second group induce both patterned and irregular outgrowths. slimb affects developmental signals that regulate cell proliferation and pattern organization. The slimb transcript encodes a Cdc4-related protein containing F-box and WD-40 motifs. Jiang (1998) has independently reported the identification of this gene. Using a Drosophila slimb cDNA, a human homolog (H-slimb) has been isolated. The fly and human proteins share 78% amino acid identity throughout, suggesting that slimb is functionally conserved (Theodosiou, 1998).

slimb-induced outgrowths are reminiscent of the phenotypes caused by misexpression of dpp and wg. dpp and wg expression were examined in slimb mosaic leg discs using wg-lacZ and dpp-lacZ reporter genes. slimb clones ectopically express both wg and dpp in a cell-autonomous fashion. slimb mutant clones deregulate wg and dpp in both D/V and A/P axes. Ectopic wg expression is observed in both ventral and dorsal regions. Similar results are also observed for dpp. In slimb mutant clones situated within or near the endogenous dpp expression zone, dpp is expressed in the mutant cells but down-regulated in adjacent wild-type cells. Previously it had been shown that Wg and Dpp signaling mutually antagonize each other's expression, which prevents expression of the two molecules in the same cells. Ectopic expression of both wg and dpp in slimb- clones in the dorsal-anterior of the leg disc indicates a disruption of this mutual antagonism. To test whether ectopic wg and dpp expression are responsible for the outgrowth phenotype in slimb mosaic animals, flies were generated carrying clones of cells mutant for both slimb and wg, or slimb and dpp. In comparison to slimb mutant clones, double mutant clones do not cause any significant outgrowths. Therefore, Wg and Dpp are two primary effector molecules responsible for the induction of outgrowths in slimb mosaic animals. These results are consistent with previous observations that wg and dpp are both required for defining the proximodistal outgrowth center (Theodosiou, 1998).

The slimb phenotype differs from those of all previously known genes, in that it is the first gene found to deregulate both wg and dpp expression in the D/V axis. Disrupting components of the Hh signaling pathway deregulate wg and dpp only along the A/P axis. Thus, the control of wg and dpp expression in the D/V axis is not disrupted by disruption of the Hh pathway. The mechanism restricting wg and dpp in the D/V axis is not known. The mutant phenotype of slimb- clones in discs provides the first evidence that wg and dpp expression in the D/V axis is actively regulated during imaginal disc development, and is not solely defined during embryonic development. Since the Hh pathway regulates wg and dpp expression in the A/P axis, these results suggest that a pathway different from Hh may operate in imaginal discs to restrict their expression in the D/V axis. This pathway cannot be either the Wg or Dpp signaling pathway since inactivation of Wg or Dpp signaling is known to affect either dpp or wg expression, but not both. The slimb phenotypes described here were not observed in the previous study which used weak slimb alleles and revealed only A/P defects (Jiang, 1998). Jiang proposed that Slimb protein normally targets Ci and Arm for processing or degradation by the ubiquitin/proteasome pathway, and that Hh and Wg regulate gene expression, at least in part, by inducing changes in Ci and Arm, which protect both Ci and Arm from Slimb-mediated proteolysis. The phenotypic differences probably reflect the fact that a null allele was used in the current study instead of hypermorphic alleles. In addition to D/V defects, slimb mutant clones also deregulate wg and dpp expression in the A/P axis. slimb is the first identified gene that regulates both wg and dpp expression in the A/P as well as D/V axes (Theodosiou, 1998).

To further explore how slimb regulation and function correlates with A/P signaling, double mutant analysis was carried out with slimb mutants and with mutants of hh and smo. No reduction of outgrowths was observed in slimb-, hh- double mutant clones. Furthermore, slimb mutant clones have no effect on hh expression. This indicates that slimb acts downstream or independent of Hh signaling. In contrast, slimb-, smo- double mutant clones almost completely suppress slimb induced outgrowths. Consistent with the adult phenotype, discs carrying slimb- , smo- clones fail to ectopically express either dpp or wg. These data suggest that slimb intersects the A/P signal upstream of smo. Jiang (1998) suggested that slimb acts downstream of smo. This difference may be explained by the use of different alleles for smo and slimb. The Slimb product contains WD-40 repeats believed to act as a scaffold for the binding of multiple proteins. It is possible that this structure may allow for proteins such as Smo and components of a D/V pathway to converge. The Slimb-related protein Cdc4 from Saccharomyces cerevisiae along with Cdc53, and Cdc34 are part of the ubiquitin proteolysis machinery. The current data that Slimb acts upstream of Smo, together with its sequence homology with Cdc4, suggests that Slimb could be involved in the regulation of Smo protein degradation (Theodosiou, 1998).

The origins of the Drosophila leg revealed by the cis-regulatory architecture of the Distalless gene

Limb development requires the elaboration of a proximodistal (PD) axis, which forms orthogonally to previously defined dorsoventral (DV) and anteroposterior (AP) axes. In arthropods, the PD axis of the adult leg is subdivided into two broad domains, a proximal coxopodite and a distal telopodite. This study shows that the progressive subdivision of the PD axis into these two domains occurs during embryogenesis and is reflected in the cis-regulatory architecture of the Distalless (Dll) gene. Dll protein in the thorax was first detected during embryonic stage 11, and continues to be visualized in this region until the end of embryogenesis. Early Dll expression, governed by the Dll304 enhancer, is in cells that can give rise to both domains of the leg as well as to the entire dorsal (wing) appendage. A few hours after Dll304 is activated, the activity of this enhancer fades, and two later-acting enhancers assume control over Dll expression. The LT enhancer is expressed in cells that will give rise to the entire telopodite, and only the telopodite. By contrast, cells that activate the DKO ("Distalless Keilin Organ") enhancer will give rise to a leg-associated larval sensory structure known as the Keilin's organ (KO). Cells that activate neither LT nor DKO, but had activated Dll304, will give rise to the coxopodite. In addition, the trans-acting signals controlling the LT and DKO enhancers are described; surprisingly, the coxopodite progenitors begin to proliferate ~24 hours earlier than the telopodite progenitors. Together, these findings provide a complete and high-resolution fate map of the Drosophila appendage primordia, linking the primary domains to specific cis-regulatory elements in Dll (McKay, 2009).

To determine how each of the cell fates in the limb primordia is specified, genetic experiments were carried out to identify the regulators of the LT and DKO enhancers. Consistent with LT's dependency on wg and dpp for leg disc expression, LT is activated in the embryo in cells that receive both inputs, as monitored by anti-Wg and anti-PMad staining. To determine whether wg is required for LT activity, a temperature-sensitive allele of wg was used to allow earlier Dll activation. Switching the embryos to the restrictive temperature at stage 11 resulted in the absence of LT activity, despite the presence of Dll protein (probably derived from Dll304 activity. In addition, ectopic activation of the wg pathway [using an activated form of armadillo (arm*)] resulted in more LT-lacZ-expressing cells (McKay, 2009).

Like wg, the dpp pathway is necessary for LT-lacZ expression in leg discs. Paradoxically, dpp signaling represses Dll in the embryo because dpp mutants show an expansion in Dll304-lacZ expression. By contrast, LT-lacZ is not expressed in dpp null embryos. LT-lacZ, but not Dll protein, was also repressed by two dpp pathway repressors, Dad and brk. Conversely, stimulation of the dpp pathway [using an activated form of the Dpp receptor (TkvQD)] resulted in ectopic activation of LT ventrally (McKay, 2009).

Taken together, these data demonstrate that LT is activated by Wg and Dpp in the embryonic limb primordia, just as it (and Dll) is in the leg disc. Similarly, DKO activity also requires Wg and Dpp input (McKay, 2009).

Although LT is activated by wg and dpp in the leg primordia, these signals are also present in each abdominal segment. Consequently, there must be additional factors that restrict LT activity to the thorax. One possibility is that LT is repressed by the abdominal Hox factors, such as Dll304. Alternatively, LT might be regulated by Dll, itself. In Dll null embryos LT-lacZ was initially expressed in a stripe of cells instead of a ring, but then expression decayed. Ectopic expression of Dll resulted in weak ectopic expression of LT-lacZ in the thorax and abdomen. These data suggest that LT activity is restricted to the thorax in part because of the earlier restriction of Dll304 activity to the thorax (McKay, 2009).

The related zinc-finger transcription factors encoded by buttonhead (btd) and Sp1 are also expressed in the limb primordia and are also required for ventral appendage specification. In strong btd hypomorphs, the activity of LT was still detected but the number of cells expressing LT-lacZ was decreased and its pattern was disrupted. LT-lacZ expression was completely eliminated in animals bearing a large deficiency that removes both btd and Sp1. By contrast, Dll304 was activated normally in these animals (data not shown). Importantly, LT-lacZ expression was rescued by expressing btd in these deficiency embryos. By contrast, expressing Dll, tkvQD, or arm* did not rescue LT expression in these deficiency embryos. Ectopic expression of btd resulted in weak ectopic activation of LT-lacZ in cells of the thorax and abdomen. Strikingly, the simultaneous expression of Dll and btd resulted in robust ectopic expression of LT-lacZ in abdominal segments in the equivalent ventrolateral position as the thoracic limb primordia. btd and Dll were not sufficient to activate LT in wg null embryos (data not shown). These data indicate that the thoracic-specific expression of the LT enhancer is controlled by the combined activities of btd and/or Sp1, Dll and the wg and dpp pathways (McKay, 2009).

Although the data suggest that LT is activated by a combination of Wg, Dpp, Btd and Dll, these activators are also present in the precursors of the KO, which activate DKO instead of LT. Because the KO is a sensory structure, the role of members of the achaete-scute complex (ASC) that are expressed in these cells was tested. In embryos hemizygous for a deficiency that removes the achaete-scute complex, LT-lacZ expression was expanded at the expense of the Ct-expressing cells. Consistently, ectopic expression of the ASC gene asense (ase) repressed LT and increased the number of Ct-expressing cells. These data suggest that there is a mutual antagonism between the progenitors of the telopodite and those of the KO. It was also found that DKO-lacZ expression in the leg primordia was lost in Dll or btd null embryos, consistent with the loss of KOs in these mutants. DKO activity was also lost from the limb primordia in embryos deficient for the ASC. These results indicate that DKO is activated by the same genes that promote LT expression but, in addition, requires proneural input from the ASC (McKay, 2009).

One of the most interesting findings from this work is that the temporal control of Dll expression in the limb primordia by three cis-regulatory elements is linked to cell-type specification. The earliest acting element, Dll304, is active throughout the appendage primordia. At the time Dll304 is active, the cells are multipotent and can give rise to any part of the dorsal or ventral appendages, or KO. A few hours later, Dll304 activity fades, and two alternative cis-regulatory elements become active. Together, these two elements allow for the uninterrupted and uniform expression of Dll within the appendage primordia. However, their activation correlates with a higher degree of refinement in cell fate potential: LT, active in only the outer ring of the appendage primordia, is only expressed in the progenitors of the telopodite. By contrast, DKO, active in the cells within the LT ring, is only expressed in the progenitors of the KO. Thus, although the pattern of Dll protein appears unchanged, the control over Dll expression has shifted from singular control by Dll304 to dual control by LT and DKO. Moreover, not only is there a molecular handoff from Dll304 to LT and DKO, the two later enhancers both require the earlier expression of Dll. Thus, the logic of ventral primordia refinement depends on a cascade of Dll regulatory elements in which the later ones depend on the activity of an earlier one (McKay, 2009).

The high-resolution view of the embryonic limb primordia provided in this study allows clarification of some contradictions that currently exist in the literature. Initial expression of Dll in the thorax overlaps entirely with Hth-nExd (referring to nuclear Extradenticle). Subsequently, hth expression is lost from most, but not all, of the Dll-expressing cells of the leg primordia. The first reports describing these changes failed to recognize the persistent overlap between Dll and Hth-nExd in some cells. As a result, and partly because of the analogy with the third instar leg disc, the predominant view of this fate map became that the Dll-positive, Hth-nExd-negative cells of the embryonic primordia gave rise to the telopodite, while the surrounding Hth-positive cells gave rise to the coxopodite. The expression pattern of esg, a gene required for the maintenance of diploidy, was also misinterpreted as being a marker exclusively of proximal leg fates. Counter to these earlier studies, the current experiments unambiguously show that the Dll-positive, Hth-nExd-negative cells in the center of the primordia give rise to the KO, the ring of Dll-positive, Esg-positive, Hth-nExd-positive cells gives rise to the telopodite, and the remaining Esg-positive, Dll-negative cells give rise to the coxopodite (McKay, 2009).

The spurious expression of DKO-lacZ in Dll-non-expressing cells outside the leg primorida complicates the interpretation of several experiments. Attempts to refine DKO activity by changing the size of the cloned fragment proved unsuccessful. Nevertheless, the evidence supports the idea that DKO-positive, Dll-positive cells of the leg primordia give rise to the Keilin's organ, and not the adult appendage (McKay, 2009).

The progenitors of the coxopodite begin to proliferate at approximately 48 hours of development, consistent with previous measurements of leg imaginal disc growth, whereas the progenitors of the telopodite do not resume proliferating for an additional 12 to 24 hours. According to estimates of the cell cycle time in leg discs, this difference in the onset of proliferation results in one to two additional cell divisions in the coxopodite, consistent with images of late second instar leg discs presented in this study. Why might the telopodite and coxopodite begin proliferation at different times? One possibility is that the cells of the coxopodite give rise to the peripodial epithelium that covers the leg imaginal disc, and therefore require additional cell divisions relative to the telopodite. It is also possible that the telopodite is delayed because the neurons of the Keilin's organ serve a pathfinding role for larval-born neurons that innervate the adult limb. Perhaps this pathfinding function requires that the KO and telopodite remain associated with each other through the second instar. Consistently, the leg is the only imaginal disc that has not invaginated as a sac-like structure in newly hatched first instar larvae (McKay, 2009).

A possible explanation for the delay in the onset of telopodite proliferation is the persistent co-expression of hth and Dll in these cells; hth (and tsh) expression is turned off in these cells at about the same time they begin to proliferate. Consistent with this idea, maintaining the expression of hth throughout the primordia blocks the proliferation of the telopodite. Also noteworthy is the finding that the genes no ocelli and elbow have been shown to mediate the ability of Wg and Dpp to repress coxopodite fates. Together with the current findings, it is possible that the activation of these two genes in the LT-expressing progenitors is the trigger that turns off hth and tsh in these cells (McKay, 2009).

The experiments suggest that once LT is activated, and under normal growth conditions, there is a lineage restriction between the telopodite and coxopodite. By contrast, previous lineage-tracing experiments using tsh-Gal4 concluded that the progeny of proximal cells could adopt more distal leg fates. However, tsh is still expressed in the telopodite progenitors far into the second instar, providing an explanation for these results. In contrast to this early restriction, there is no evidence for a later lineage restriction within the telopodite. For example, the progeny of a Dll-positive cell can lose Dll expression and contribute to the dac-only domain (McKay, 2009).

Interestingly, the lineage restriction between coxopodite and telopodite is not defined by the presence or absence of Hth-nExd or Tsh because both progenitor populations express hth and tsh after their fates have been specified. By contrast, when these two domains are specified, the telopodite expresses Dll, while the coxopodite does not, suggesting that Dll may be important for the lineage restriction. However, later in development, some cells in the telopodite lose Dll expression and express dac, but continue to respect the coxopodite-telopodite boundary. Thus, either Dll expression in the telopodite is somehow remembered or the telopodite-coxopodite boundary can be maintained by dac, which is expressed in place of Dll immediately adjacent to the telopodite-coxopodite boundary. Also noteworthy is the finding that clones originating in the coxopodite can contribute to the trochanter, the segment inbetween the proximal and distal components of the adult leg that expresses both Dll and hth in third instar imaginal discs. However, the progeny of such clones do not contribute to fates more distal than the trochanter. Likewise, a clone originating in the telopodite can also contribute to the trochanter, but will not grow more proximally into the coxa. Thus, the lineage restriction uncovered here seems to be determined by distinct combinations of transcription factors expressed in the coxopodite and telopodite progenitors at stage 14. The progeny of cells that express Dll, tsh and hth can populate the telopodite or trochanter, whereas the progeny of cells that express tsh and hth, but not Dll, can populate the coxopodite or trochanter. In light of Minute-positive results, however, the lineage restriction between coxopodite and telopodite does not satisfy the classical definition of a compartment boundary. A similar non-compartment lineage restriction has also been documented along the PD axis of the developing Drosophila wing (McKay, 2009).

Dpp and eye morphogenesis

Decapentaplegic (Dpp) regulates many aspects of imaginal disc growth and patterning in Drosophila. This study analysed the phenotype of an eye-specific dpp allele, dppblk, which causes a reduction in the size of the retina due to a loss of ventral ommatidia. Prior to the onset of differentiation, dppblk eye discs are normal regarding size, shape, and ability to express dorsal and ventral markers. However, expression of a dpp-lacZ reporter is reduced at the ventral margin. Additional dorsoventral asymmetry appears during retinal differentiation: the morphogenetic furrow (MF) initiates normally at the posterior tip of the disc, but fails to propagate into the ventral epithelium (see Progression of the morphogenetic furrow across the eye disc). This defect can be rescued by increasing dpp expression along the ventral margin by local removal of patched function. It is proposed that the primary defect in dppblk is an inability to activate dpp expression properly at the ventral margin. It is unknown whether this inability is due to insufficient expression of Dpp in dppblk to support autoregulation, or due to a specific loss of enhancer sequences required for dpp autoregulation in dppblk. The deficiency in dppblk has two consequences: it prevents initiation from the ventral margin, and it renders the ventral epithelium unresponsive to differentiation signals emanating from the MF. Rotation polarization is maintained in dppblk discs, suggesting that dppblk has no effect on neural cell polarity (Chanut, 1997b).

Neuronal differentiation in the Drosophila retinal primordium (the eye imaginal disc) begins at the posterior tip of the disc and progresses anteriorly as a wave. The morphogenetic furrow (MF) marks the boundary between undifferentiated anterior cells and differentiating posterior cells. Anterior progression of differentiation is driven by Hedgehog, synthesized by cells located posterior to the MF. hedgehog , which is expressed prior to the start of differentiation along the disc's posterior margin, also plays a crucial role in the initiation of differentiation. Using a temperature-sensitive allele it has been shown that hh is normally required at the posterior margin to maintain the expression of both decapentaplegic (dpp) and the proneural gene atonal. In addition, ectopic differentiation driven by ectopic dpp expression or loss of wingless function requires hh. Consistent with this is the observation that ectopic dpp induces the expression of hh along the anterior margin even in the absence of differentiation. Taken together, these data reveal a novel positive regulatory loop between dpp and hh that is essential for the initiation of differentiation in the eye disc (Borod, 1998).

The DPP requirement for cell fate specification and cell cycle synchronization in the developing Drosophila eye was examined by determining whether cells defective for thickveins, saxophone or schnurri show abnormalities in cell division or differentiation. Clones mutant for a null allele of tkv that are anterior or posterior to the morphogenetic furrow have amounts of Cyclin B that are indistinguishable from those in surrounding cells. In contrast, tkv clones that span the MF maintain cyclin B expression in the anterior part of the furrow, even though the surrounding cells arrested in G1 have no detectable Cyclin B. Maintenance of cyclin B expression is thought to indicate a failure of cell cycle progression, as Cyclin B levels decline in M phase. Mitotic figures are not observed in clones in the anterior half of the MF. The phenotype observed in the clones is similar to defects caused by mutations in division abnormally delayed (dally), which is required for G2-M progression ahead of the furrow. Mutations in dally and dpp display genetic interactions in development of the eye, antenna, and genitalia, which suggests that dally augments Dpp function. The behavior of Dpp-receptor mutant clones supports a role for Dpp in controlling progression through G2-M as a means of synchronizing the divisions that accompany differentiation of the eye disc. Cell fate, however, is unaffected by receptor mutation, as revealed by the expression of atonal, a proneural gene required for retinal precursor cell 8 (R8) determination. Because atonal expression is maintained in tkv clones, hh must not act through dpp to induce its expression, and thus dpp mediates a subset of hh functions in the MF (Penton, 1997).

During Drosophila eye development, Hedgehog (Hh) protein secreted by maturing photoreceptors directs a wave of differentiation that sweeps anteriorly across the retinal primordium. The crest of this wave is marked by the morphogenetic furrow, a visible indentation that demarcates the boundary between developing photoreceptors located posteriorly and undifferentiated cells located anteriorly. Evidence is presented that Hh controls progression of the furrow by inducing the expression of two downstream signals. The first signal, Decapentaplegic (Dpp), acts at long range on undifferentiated cells anterior to the furrow, causing them to enter a 'pre-proneural' state marked by upregulated expression of the transcription factor Hairy. Acquisition of the pre-proneural state appears essential for all prospective retinal cells to enter the proneural pathway and differentiate as photoreceptors. The second signal, presently unknown, acts at short range and is transduced via activation of the Serine-Threonine kinase Raf. Activation of Raf is both necessary and sufficient to cause pre-proneural cells to become proneural, a transition marked by downregulation of Hairy and upregulation of the proneural activator, Atonal (Ato), which initiates differentiation of the R8 photoreceptor. The R8 photoreceptor then organizes the recruitment of the remaining photoreceptors (R1-R7) through additional rounds of Raf activation in neighboring pre-proneural cells. Dpp signaling is not essential for establishing either the pre-proneural or proneural states, or for progression of the furrow. Instead, Dpp signaling appears to increase the rate of furrow progression by accelerating the transition to the pre-proneural state. In the abnormal situation in which Dpp signaling is blocked, Hh signaling can induce undifferentiated cells to become pre-proneural but does so less efficiently than Dpp, resulting in a retarded rate of furrow progression and the formation of a rudimentary eye (Greenwood, 1999).

One candidate for a secondary signal, which acts downstream of Hh in the developing retina, is the TGF-beta homolog Dpp. Dpp is induced by Hh just anterior to the morphogenetic furrow. Moreover, experiments in other discs have established that Dpp can act at long range from its source to mediate the organizing activity of Hh on more anteriorly situated tissue. However, previous studies have shown that Dpp signaling is not essential for either photoreceptor differentiation or propagation of the furrow once photoreceptor differentiation initiates at the posterior edge of the eye primordium. These findings challenge the notion that Dpp mediates the organizing activity of Hh in front of the furrow. To test whether Dpp has such an organizing role, two kinds of experiments were performed. In the first, Dpp or activated Thickveins (Tkv), a type I TGFbeta receptor required for all known Dpp activities, was ectopically expressed anterior to the furrow. In the second, Dpp expression or Tkv activity was blocked. The results of these experiments indicate that Dpp signaling is both necessary and sufficient to upregulate Hairy expression anterior to the furrow and to maintain the normal rate of furrow progression, but that it is neither necessary nor sufficient to activate Ato expression and initiate photoreceptor differentiation in more posterior cells (Greenwood, 1999).

The dppblk mutation is associated with a deletion of cis-acting regulatory sequences that are essential for Hh-dependent transcription of dpp in the eye. As a result, Dpp signaling in the eye disc is abolished or severely reduced anterior to the furrow, and the resulting eye is greatly reduced in size in both the dorsal-ventral and antero-posterior axis. Hairy expression in wild-type and dppblk disks were compared, using the upregulation of Cubitus interruptis (Ci), a protein that is stabilized in response to Hh signaling, as a marker of the position at which the furrow should normally form. In wild-type eye discs, Ci accumulates to peak levels in a dorso-ventral stripe of cells just posterior to the stripe of peak Hairy expression, consistent with the finding that Hairy expression is repressed in response to Hh signaling within the furrow, but is activated by Dpp signaling anterior to the furrow. In contrast, the stripe of maximal Hairy expression is displaced posteriorly in dppblk discs relative to the stripe of maximal Ci expression. Moreover, the furrow appears to have moved only a small distance from the posterior edge of the presumptive eye primordium, even in eye discs from mature third instar larvae, consistent with the 'small eye' phenotype observed in the adult. These results indicate that Dpp signaling is normally required to activate high level Hairy expression in a stripe positioned just anterior to the furrow. They also indicate that Dpp signaling is necessary to sustain the normal rate of furrow progression. Finally, they suggest that Dpp signaling influences the response of cells to peak levels of Hh signal transduction: Hairy expression is downregulated in these cells in wild-type discs, but not in dppblk discs (Greenwood, 1999).

This analysis indicates that Hh orchestrates Hairy and Ato expression as well as furrow progression in a manner that depends on Dpp signaling ahead of the furrow. Yet Dpp signaling is not sufficient to activate Ato or to initiate photoreceptor differentiation. Conversely, cells devoid of Smo, and hence unable to receive Hh, can still be induced by neighboring wild-type cells to express Ato and make photoreceptors. These findings argue for an additional signal involved in mediating the organizing activity of Hh on photoreceptor differentiation and furrow progression (Greenwood, 1999).

Neuron-glia interactions are crucial for the establishment of normal connectivity in the nervous system during development, but the molecular signals involved in these interactions are largely unknown. Differentiating photoreceptors in the developing Drosophila eye influence the proliferative and migratory behavior of the subretinal glia through the diffusible factors Decapentaplegic (Dpp) and Hedgehog (Hh). Subretinal glial precursors originate in the optic stalk and migrate into the eye imaginal disc concomitant with the onset of differentiation of photoreceptors. Their presence in the eye disc, in turn, is necessary for guiding photoreceptor axons from the eye disc into the optic stalk. Proliferation and migration of the glia are separable processes, and Dpp promotes both the proliferation and motility of the glia, whereas Hh appears to promote only their motility; neither specifies the direction of migration. Evidence is presented that Dpp and Hh act on the glia in parallel and through the regulation of transcription. Ectopic migration of subretinal glia can result in the ectopic projection of photoreceptor axons. This study suggests a novel function for Hh in regulating migratory behavior and provides further evidence for a complex mutual dependence between glial and neuronal cells during development (Rangarajan, 2001).

In the developing Drosophila visual system, the eye disc is connected to the optic lobe by the optic stalk. Photoreceptor cells are generated in the eye disc in a posterior-to-anterior progression; the wave of differentiation is marked at its front by an indentation of the disc epithelium called the morphogenetic furrow. The photoreceptors extend axons into the basal layer of the eye disc, where they turn and proceed posteriorly to exit the disc through the optic stalk. The subretinal glia (also known as retinal basal glia) originate from precursor cells in the optic stalk; the glial precursors in the stalk already express the glial-specific marker Repo. The glia begin to migrate into the eye disc with the onset of the differentiation of photoreceptors, but do not require the presence of axons in the stalk to find their way into the eye disc. Once in the eye disc, the glia migrate anteriorly on the basal surface of the eye epithelium; the anterior border of their migration lies typically 2-4 rows of ommatidia posterior to the morphogenetic furrow, which is where axons sent out by the differentiating photoreceptors begin to turn posteriorly. Thus, glia are present only in the axonal portion of the eye disc. The glial cells associate closely with the photoreceptor axons and develop extensive processes that surround the axons and fill the intervening space. The development of the subretinal glia thus involves proliferation, migration, and terminal differentiation events, and it is closely tied, both spatially and temporally, to the development of the photoreceptors (Rangarajan, 2001).

The coordination of neuronal and glial development is crucial for the establishment of functional circuitry in the visual system. Normal photoreceptor axon projections depend on the proper positioning of the subretinal glia: Glia have to be present in the eye disc near the entrance to the optic stalk for photoreceptor axons to find their way into the stalk; if glia are induced to mismigrate to ectopic positions in the anterior, undifferentiated portion of the eye disc, axons frequently follow. To prevent anterior misprojection of axons it is therefore imperative that the migration of the subretinal glia be restricted to the differentiating portion of the eye disc. In addition, the number of glia in the eye disc has to be roughly proportional to the number of ommatidia to ensure proper wrapping and insulation of the photoreceptor axons. This means that the glial population has to expand quickly in response to the growing number of ommatidia progressively forming in the eye disc. This matching of cell numbers requires the appropriate modulation of glial proliferation and migration (Rangarajan, 2001).

Neuron-glia interactions have also been shown to play a role in oligodendrocyte and astrocyte development in the vertebrate visual system. Retinal ganglion cell axons signal to the glia in various ways to promote their proliferation and survival, thereby ensuring precise matching of the two cell populations. However, there has been no evidence to date for neuronal regulation of glial migration other than through haptotaxis (Rangarajan, 2001).

Dpp loss of function experiments show a substantial reduction in the number or percentage of mutant glia in the eye disc, indicating that a disruption of Dpp signaling impairs the accumulation of subretinal glia in the eye disc. This phenotype could be the result of an effect of Dpp on the proliferation, the survival, or the migration of the glia. To determine which aspect of glial development is affected, a gain of function approach was taken. Dpp overactivity leads to a pronounced increase in the number of glial cells in both eye disc and optic stalk, and to ectopic migration of the glia beyond their normal anterior boundary. The increase in cell number, coupled with the fact that there is no appreciable cell death in wild-type glia at this stage of eye development, suggests that Dpp stimulates glial cell proliferation (Rangarajan, 2001).

Subretinal glia migrate from the optic stalk into eye discs only when photoreceptors have begun to differentiate in the eye disc, but they do not require axons as a substrate for their migration. They also migrate towards ectopic islands of differentiating photoreceptors, again without axonal substrate. These findings suggest that differentiating photoreceptors regulate the migration of the subretinal glia over a distance, either by secreting diffusible molecules or by preferentially stabilizing far-reaching glial filopodia. The present study demonstrates that the diffusible factors Dpp and Hh are among the signals the differentiating photoreceptors send to promote subretinal glial motility. Gain-of-function experiments show that overactivity of Dpp as well as of Hh signaling leads subretinal glial cells to overshoot their normal anterior boundary, either in a relatively narrow stream or in a broad front. In the case of Dpp, this ectopic migration of the glia is accompanied by severe overproliferation, and therefore the possibility that the changes in migratory behavior are merely a secondary consequence of the increase in cell number had to be considered. However, severe overproliferation of glia in otherwise wild type eye discs does not lead to ectopic migration. This is true not only when overproliferation is induced by acceleration of the cell cycle, but also when it is caused by Ras overactivity, which presumably mimics the effects of a wide range of growth factors. Similarly, overproliferation of glia in the optic stalk of eya mutants, which lack photoreceptors, does not induce migration into the eye disc. Conversely, ectopic migration of glia is observed in eye discs with Hh overactivity, i.e., in discs in which the number of glial cells is largely normal. These results demonstrate that overproliferation in itself is not sufficient to induce abnormal migration of glial cells and that proliferation and migration are in fact independently regulated processes (Rangarajan, 2001).

The ectopic migrations of glial cells that was observed are not preferentially directed towards the sources of Dpp or Hh. Moreover, similar migratory phenotypes can be induced by expression of constitutively active components of the Dpp and Hh pathways within the glial cells. Such cell-autonomous activation of the pathway precludes the use of information about ligand distribution as a positional cue. Both findings therefore argue that Dpp and Hh exert their effect on the glia not by providing positional information, but by stimulating motility. This motogenic effect may be due to an enhanced ability to migrate or to an impaired ability to respond to an inhibitory signal. In any case, both Dpp and Hh appear to act by regulating transcription within the glial cells, through their canonical signal transduction machinery (Rangarajan, 2001).

One of the best understood motogens is scatter factor (SF), which disperses cohesive colonies of epithelial cells by stimulating random motility. However, it can also act as a mitogen (hepatocyte growth factor), morphogen, trophic factor, or chemoattractant depending on the cellular context; in all cases, its effects are mediated by the receptor tyrosine kinase c-Met. The actual scattering induced by SF occurs only 4-6 h after addition of SF and is inhibited by cycloheximide, suggesting that regulation of gene expression is involved. Thus, Dpp and Hh resemble SF both in terms of phenotypic effect and with regard to their mechanism of action (Rangarajan, 2001).

The function of Hh as a motogen is novel. Although the molecule is being studied in many contexts, a role in cell motility has not been attributed to it to date. Dpp and its vertebrate counterparts, in contrast, have been implicated in the control of migratory processes during development. Dpp plays a role in tracheal cell migration along the dorso-ventral body axis and in the dorsal migration of the ectoderm during dorsal closure of the Drosophila embryo. During wound healing, the migration of various cell types, including astrocytes, depends on TGFß (Rangarajan, 2001).

Why are not more clear-cut effects on the migratory behavior of the subretinal glia observed in loss of function experiments? Mosaic data confirm at least for Dpp that the molecule is required for the normal accumulation of glia in the eye disc. But because of the limitation of the mosaic technique it is difficult to distinguish between effects on proliferation and on motility and to discern specific migratory effects: the failure of ombGAL4 to drive recombination to completion prior to entry of the glia into the eye disc means that at least some glia arrive in the eye disc and migrate anteriorly as heterozygotes, i.e., before undergoing recombination to become homozygous mutant. Another major reason for the lack of a more pronounced loss of function effect is most likely that the Dpp and Hh signal transduction pathways are (partially) functionally redundant with other pathways in regulating glial cell development (Rangarajan, 2001).

Ras signaling, possibly triggered by diffusible RTK ligands, is very likely to play a role in regulating both the proliferative and migratory behavior of the subretinal glia. Generally, Dpp and Hh must be part of a more complex network of signals regulating glial development in the eye disc. For example, it is not yet known how the sharp anterior boundary of glial migration behind the morphogenetic furrow is established. Possible mechanisms include an attractive substrate or signal in the posterior, or an inhibitory signal in the anterior. Since in wild type the distribution of both Dpp and Hh extends further to the anterior than the glial cells migrate, this mechanism also has to be able to counteract the motogenic effects of Dpp and Hh (Rangarajan, 2001).

How do Dpp and Hh exert their effects on subretinal glial migration? Since Hh and Dpp are known to regulate each other's expression in other tissues, it would be conceivable that they act in sequence. However, neither dpp nor hh is expressed in the subretinal glial cells, which rules out the possibility that they mutually regulate each other’s transcription and implies that Dpp and Hh both act in a paracrine fashion. It therefore seems likely that Dpp and Hh signaling converge to control the transcription of genes required for cell motility (or possibly for reading the stop signal). These targets could include genes coding for cytoskeletal components, proteins regulating cell adhesion, or enzymes to degrade the extracellular matrix. The idea that such genes could be transcriptionally controlled by Dpp or Hh is supported by the recent finding that in the Drosophila wing compartment, cell-sorting at the anterior/posterior boundary is under the opposing transcriptional control of Hh and En, suggesting that these molecules regulate the expression of a single cell adhesion molecule. The identification of transcriptional targets of Dpp and Hh signaling will provide important insights into the mechanism by which these signals regulate glial cell motility (Rangarajan, 2001).

Neural determination in the Drosophila eye occurs progressively. A diffusible signal, Dpp, causes undetermined cells first to adopt a 'pre-proneural' state in which they are primed to start differentiating. A second signal is required to trigger the activation of the transcription factor Atonal, which causes the cells to initiate overt photoreceptor neurone differentiation. Both Dpp and the second signal are dependent on Hedgehog (Hh) signaling. Previous work has shown that the Notch signaling pathway also has a proneural role in the eye (as well as a later, opposite function when it restricts the number of cells becoming photoreceptors -- a process of lateral inhibition). It is not clear how the early proneural role of Notch integrates with the other signaling pathways involved. Evidence suggests that Notch activation by its ligand Delta is the second Hh-dependent signal required for neural determination. Notch activity normally only triggers Atonal expression in cells that have adopted the pre-proneural state induced by Dpp. Notch drives the transition from pre-proneural to proneural by downregulating two repressors of Atonal: Hairy and Extramacrochaetae (Baonza, 2001).

Loss of Notch signaling leads to a loss of neural differentiation. Cells within clones of a null allele of Notch fail to upregulate Atonal expression from its initial low, uniform level. This implies that Notch signaling is required for the initiation of neural development but not for the first low level expression of Atonal. To examine in detail the role of Notch signaling in promoting neural differentiation, clones of cells expressing the Notch ligand Delta were made and their ability to induce neural differentiation was examined. In the wing disc, similar ectopic expression of Delta in clones induces the activation of Notch signaling within the clone as well as non-autonomously in cells surrounding it (Baonza, 2001).

Clones were generated using the Gal4/UAS system combined with the Flip-out technique and third instar larval eye discs were labelled with different markers to assess neural development. The phenotype of Delta-expressing clones depends on their position with respect to the morphogenetic furrow. Clones in the anterior part of the disc have no effect unless they are within 12-15 cell diameters of the furrow. Within this zone close to the furrow, Delta induces the ectopic expression of Atonal, both autonomously within the clone and non-autonomously, in cells surrounding the clone. In some of these clones there are also cells ectopically expressing the neural antigen Elav. This indicates that once Atonal expression is activated, the full neural program is initiated. Thus, the primary proneural function of Notch signaling is the activation of Atonal (Baonza, 2001).

Consistent with the neural-promoting properties of Delta, clones that span the furrow from posterior to anterior cause the anterior displacement of Atonal and Elav expression. This displacement implies that the furrow accelerates as it moves through the clone. In the region of these clones that lies posterior to the furrow, the domain of Atonal expression is expanded and the Atonal-expressing cells are disorganized and more numerous. In this region repression of neural differentiation, visualized with the expression of Elav, is also observed. This later phenotype reflects the function of Notch signaling pathway in preventing neural differentiation posterior to the morphogenetic furrow (Baonza, 2001).

Similar clones were also produced expressing the alternative Notch ligand, Serrate, and unlike Delta-expressing cells, these clones cause no neural induction ahead of the furrow. Conversely, when posterior to the furrow, Ser-expressing clones behave like those expressing Delta and prevent neural differentiation. This implies that anterior to the furrow, the two Notch ligands are not equivalent in their ability to activate the receptor. The reason for this has not been explored, but it is noted that the Notch glycosyltransferase Fringe, which makes Notch resistant to Serrate, is strongly expressed anterior to the furrow. The inability of Serrate to induce proneural Notch signaling is consistent with previous reports, which show that loss of Serrate caused no effects on eye development (Baonza, 2001).

These results imply that there is a zone of about 12-15 cell diameters ahead of the morphogenetic furrow, where the activation of Notch signaling by Delta, but not by Serrate, is sufficient to trigger neural fate (Baonza, 2001).

The simplest explanation of these results is that some signal or signals emanating from the cells posterior to, or within, the morphogenetic furrow are necessary for the specification of a neural competence zone ahead of the furrow. Within this zone, cells can respond to Delta-induced Notch activation by upregulating Atonal expression. A candidate for such a signal is the secreted protein Dpp. Dpp is expressed within the furrow in response to Hh signaling and has been proposed to define a 'pre-proneural' state in a zone anterior to the furrow. In order to analyse whether the function of Dpp is sufficient to generate the condition necessary for the neural activation by Notch signaling, clones that simultaneously express ectopic dpp and Delta were produced (Baonza, 2001).

Clones of cells that express dpp alone only induce neural differentiation along the margin of the eye discs; internal clones have no effect on neural induction. By contrast, clones that co-express Dl and dpp trigger neural differentiation everywhere ahead of the furrow. In all the clones studied, ectopic expression of Atonal and Elav was observed. The induction of neural differentiation occurs in all the cells surrounding the clone and not, as in Delta-expressing clones, only in the cells within the competence zone. In most of the clones analyzed, Atonal expression was found to be associated with an ectopic morphogenetic furrow induced by the clones. Thus, it is possible to observe clones with ectopic Atonal expression several cells away from the border of the clone and with Atonal expression restricted to isolated cells within the clone, reproducing the pattern of Atonal expression of the endogenous furrow. One interpretation of this result is that once Atonal is activated within and in the cells surrounding the clone, the normal cascade of ommatidial development is triggered, inducing an ectopic furrow that begins to move away from the clone (Baonza, 2001).

These observations lead to the conclusion that the expression of dpp is sufficient to enable all cells anterior to the furrow to activate neural differentiation in response to Notch. It is postulated that during normal development, Dpp primes the cells to become competent to differentiate neurally in response to Notch signaling, at a range of 12-15 cells anterior to the furrow (Baonza, 2001).

Loss of Dpp signaling during eye development causes furrow progression to slow down but not to stop: partial redundancy allows Hh signaling to induce neural differentiation in cells in which the Dpp signaling is blocked. Furthermore, clones of ectopic expression of Hh always induce neural differentiation and an ectopic furrow, even beyond the zone of Dpp-influenced cells, indicating that Hh is sufficient to trigger neural differentiation. The current model is that Dpp is important for furrow progression to occur efficiently and at a normal rate, but that it is not essential for neural differentiation to occur. This study shows that Dpp signaling has an important role in promoting the proneural function of Notch signaling by generating the 'pre-proneural' state ahead of the furrow. This does not, however, rule out the possibility that Hh signaling could also produce a similar effect. If the function of Dpp signaling can be rescued by Hh signaling, then it would be expected that the effects of ectopic activation of Notch signaling would be identical in a background where Dpp signaling is blocked (because in this case, Hh would replace Dpp function) (Baonza, 2001).

An examination was made of the effect of the ectopic expression of Delta when Dpp signaling is blocked, by inducing clones that co-express Delta and the negative Dpp signal regulator brinker. The use of brinker expression was evaluated as a way of inhibiting Dpp function in the eye by examining the phenotype of clones of brinker-expressing cells. brinker-expressing clones indeed mimic mad null clones in their ability to prevent the initiation of the morphogenetic furrow when they occur at the posterior margin of the disc (Baonza, 2001).

Double clones of brinker- and Delta-expressing cells only activate Atonal expression when they lie within four to five cells of the morphogenetic furrow. In addition, the position of the endogenous morphogenetic furrow is only slightly altered compared with control clones expressing Dl alone. Thus, the proneural action of ectopic Notch signaling anterior to the morphogenetic furrow is substantially reduced in cells in which Dpp signaling is inhibited. These results suggest that despite some partially rescuing short-range signal near the furrow (which is presumed to be Hh), Dpp signaling is required for the longer range ability of cells to initiate neural differentiation in response to Notch activation (Baonza, 2001).

The fact that the ectopic expression of Dpp does not reproduce the effects cause by the overexpression of Hh, indicates that additional Hh-dependent signals are needed to promote neural differentiation. The results suggest that Notch signaling could be one of these. According to this model blocking Notch and Dpp signaling would be sufficient to prevent neural differentiation, since it would block both Hh-induced intermediate signals. To analyze this possibility, double mutant clones of the strong Delta allele Dlrev10 and the medea allele med8 were induced. Medea is the Drosophila homologue of the mammalian MAD-related protein Smad4, and is required for transduction of the Dpp signal. Clones of med8 along the posterior eye margin cause similar phenotypes to mad minus clones, preventing the initiation of the morphogenetic furrow. Internal clones of med8 can reduce the expression of Atonal, especially the initial uniform expression. Occasionally (1/17), the expression of Atonal is totally removed in part of the clone. These phenotypes are similar to those described when Dpp signaling is blocked in mutant clones of the Dpp receptor thick vein (tkv). One phenotype of med8 clones could be found not accounted for by phenotypes caused by loss of other members of the pathway: in some clones (6/11) posterior to the morphogenetic furrow, Atonal is ectopically expressed, always in isolated cells. The basis for this phenotype is not understood, but it does not affect the region anterior to the furrow, which is under consideration here (Baonza, 2001).

Double Dlrev10;med8 mutant clones show a combination of the phenotypes observed in independent mutant clones of Delta and med. Thus, all internal clones analysed show Delta-like reduction of Atonal expression. In some of these clones there are regions where Atonal expression is totally lost, a phenotype observed in med clones. Also as in medea clones, posterior Dlrev10;Med8 clones are found that express Atonal ectopically. However, in this case, the Atonal expression is in clusters of cells, reflecting the fact that lateral inhibition is blocked in the absence of Delta (Baonza, 2001).

These results indicate that the initial expression of Atonal can be induced in the absence of Notch and Dpp signaling, implying that Hh signaling can, directly or via yet another intermediate, overcome the loss of function of both pathways (Baonza, 2001).

The progression of the morphogenetic furrow correlates with the modulated expression of the negative regulators of Atonal expression, Emc and Hairy. Hairy is expressed in a broad stripe anterior to the furrow and rapidly switched off in the furrow. Emc protein is present in all cells but the highest levels are present in a dorsoventral stripe of cells anterior to the domain of Hairy expression, whereas the lowest levels are observed in the furrow. Thus, the increase of Atonal expression in the proneural groups within the furrow is associated with the downregulation of both Emc and Hairy. Whether this downregulation of Emc and Hairy is mediated by Notch was tested by analyzing the expression of Emc and Hairy when Notch signaling is blocked and when it is ectopically activated). It is concluded from these results that Delta/Notch signaling promotes Atonal activation and neural differentiation by downregulating the repressors Hairy and Emc (Baonza, 2001).

Thus a model is proposed specifically to integrate proneural Notch signaling into the concept of a progression of cell states, from undetermined to pre-proneural to proneural. Hh in the cells posterior to the morphogenetic furrow activates the expression of Dpp in the furrow. The data support the idea that as Dpp acts at a longer range than Hh, this relays a signal to a zone extending about 15 cells anterior to the furrow, priming these cells for differentiation. This makes cells competent to receive a later signal that upregulates Atonal expression, thereby initiating overt neural differentiation. This second signal is also dependent on Hh, but operates only much closer to the furrow: the evidence implies that it consists of Delta activating Notch signaling. The initial 'pre-proneural' state is molecularly defined by the accumulation of the repressors of atonal transcription Hairy and Emc, as well as by the positive regulator of Atonal, the HLH transcription factor Daughterless. Therefore, although Atonal and Daughterless are both expressed in this pre-proneural zone, neural differentiation is not initiated, as Hairy and Emc ensure that Atonal activity remains below a threshold. The Hh-dependent activation of Delta/Notch signaling triggers the transition from this pre-proneural state to the proneural state by downregulating both Hairy and Emc. This negative regulation of the Atonal repressors is sufficient to allow the accumulation of active Atonal in the proneural groups to a level where R8 determination is initiated (Baonza, 2001).

Novel function of the class I bHLH protein Daughterless in the negative regulation of proneural gene expression in the Drosophila eye

Two types of basic helix-loop-helix (bHLH) family transcription factor have functions in neurogenesis. Class II bHLH proteins are expressed in tissue-specific patterns, whereas class I proteins are broadly expressed as general cofactors for class II proteins. The Drosophila class I factor Daughterless (Da) is upregulated by Hedgehog (Hh) and Decapentaplegic (Dpp) signalling during retinal neurogenesis. The data suggest that Da is accumulated in the cells surrounding the neuronal precursor cells to repress the proneural gene atonal (ato), thereby generating a single R8 neuron from each proneural cluster. Upregulation of Da depends on Notch signalling, and, in turn, induces the expression of the Enhancer-of-split proteins for the repression of ato. It is proposed that the dual functions of Da--as a proneural and as an anti-proneural factor--are crucial for initial neural patterning in the eye (Lim, 2008).

Da is upregulated in the furrow region. Surprisingly, however, it was found that there are two distinct patterns of Da upregulation. The first pattern is a broad, low-level upregulation in the furrow (hereafter referred to as basal level). The second pattern is a stronger expression of Da (hereafter referred to as high level) selectively in the non-neural cells surrounding the Ato-positive R8 cells between proneural clusters. Tests were perfomed to see whether this previously unrecognized pattern of expression of Da is specific by examining eye discs containing da loss-of-function (LOF) clones. Both the basal and high-level expressions of Da in the furrow were lost in the LOF clones of da3, a null allele, showing the specificity of the pattern of Da expression (Lim, 2008).

The basal level of Da upregulation overlaps with the domain of Ato expression near the furrow, where they function together to regulate neurogenesis. As the furrow progression and expression of Ato are controlled by Hh and Dpp signalling, it was reasoned that regulation of Da expression in the furrow might be linked to these signalling pathways (Lim, 2008).

To test whether Hh signalling is required for the expression of Da, Da expression was examined in hh1 mutant eye discs in which the production of Hh ceases after the mid-third instar stage, resulting in reduced expression of Ato and arrest of furrow progression. The expression of Da was downregulated in hh1 mutant eye discs. LOF clones of smoothened (smo), a crucial component for Hh signal transduction, were generated. Da expression was significantly reduced in smo mutant clones spanning the furrow, suggesting that Hh signalling is required for the expression of Da. However, the expression of Da was not completely eliminated in hh1 mutant eye discs or in smo LOF clones. As Dpp signalling is partly required for the expression of Ato, whether Dpp signalling is also necessary for the expression of Da was tested by analysing LOF clones of mad (mothers against dpp), an essential factor for Dpp signalling transduction. Da expression showed little reduction in mad mutant clones, indicating that Dpp signalling by itself is not essential for Da expression. By contrast, the expression of Da was almost completely abolished in LOF clones of smo and mad double-mutant cells in the furrow region. Thus, the Hh and Dpp signalling pathways are crucial but partly redundant for the expression of Da. It was also found that loss of function of Ato reduced the level of Da expression in the furrow. Therefore, several factors, including Ato, coordinate the accumulation of Da in the furrow (Lim, 2008).

To test whether the upregulation of Da in the furrow has a function in neurogenesis, da3 LOF clones were generated and the effects of da mutation on the expression of Ato and neuronal differentiation were examined. Loss of da resulted in ectopic expansion of Ato expression in the mutant clone, suggesting that Da is crucial for repressing the expression of Ato (Lim, 2008).

Despite ectopic expression of Ato, most of the cells in da LOF mutant clones could not differentiate into photoreceptor cells, as indicated by the lack of neuronal markers such as Senseless (R8 marker) and Elav (pan-neural marker). Hence, the expression of ectopic Ato is insufficient to induce retinal differentiation in the absence of Da. However, local differentiation was occasionally detected near the posterior end of some clones. This might be due to the perdurance of Da in LOF clones, although other possibilities, such as partial non-autonomy or partial independence of photoreceptor differentiation from Da in the posterior region of the eye disc, cannot be excluded (Lim, 2008).

To support the idea that a high level of Da expression is required for the repression of Ato, a temperature-sensitive allele of da (dats) was examined that causes conditional partial loss of function of Da at the restrictive temperature. In dats mutant eye discs, Ato was expressed in several cells rather than a single R8 cell per proneural cluster. In addition, the effects of conditional expression of Da was tested by temperature shifts of heat-shock (hs)-da flies. Ato was repressed by the overexpression of Da after a longer heat shock but not after a shorter heat shock. These observations support the idea that enriched Da expression in the cells surrounding each R8 cell is required for generating a single R8 cell by the inhibition of Ato expression (Lim, 2008).

The expanded expression of Ato in da mutant clones might, in part, be due to the failure of da mutant cells to induce lateral inhibition of Ato expression. It is also possible that Da might be involved in the cell-autonomous repression of Ato expression. To test this possibility, Da was overexpressed in the dorsoventral margin of the eye disc using the optomotor blind (omb)-Gal4 driver. The overexpression of Da downregulated Ato expression in the expression domain of omb. Furthermore, the overexpression of Da in the antenna disc using the dpp-Gal4 driver resulted in Ato repression in the expression domain of dpp. Taken together, these data from LOF and overexpression analyses suggest that the high-level expression of Da is necessary and sufficient for the cell-autonomous repression of Ato during the selection of R8 (Lim, 2008).

Both Da and Notch (N) are essential for the selection of R8 by repressing Ato expression in non-R8 precursors within proneural clusters. Hence, Da might be involved in N-dependent lateral inhibition. Furthermore, the overexpression of ASC proneural factors, together with Da, can synergize with Suppressor of hairless and N to activate the expression of Enhancer-of-split (E(spl)) in cultured cells. Since E(spl) is expressed complementary to the expression of Ato in the same cells expressing a high level of Da, whether Da alone could regulate the expression of E(spl) was tested in vivo. The expression of E(spl) proteins was reduced in da3 mutant cells, showing that Da is required for the expression of E(spl) in vivo. Furthermore, the overexpression of Da with dpp-Gal4 could induce the expression of ectopic E(spl) in the dpp domain of the antenna disc. These results indicate that a high level of Da expression is necessary and sufficient for the activation of E(spl) expression (Lim, 2008).

Since E(spl) is the main mediator of N signalling, Ato repression by a high level of Da might be dependent on the expression of E(spl). To test this possibility, the MARCM method was used to generate E(spl) LOF clones in which the expression of Da is induced by tubulin (tub)-Gal4. Da overexpression in E(spl) LOF clones did not show a significant repression of Ato. Similarly, overexpression of E(spl)mδ in da LOF clones did not show noticeable repression of Ato. These data suggest that both Da and E(spl) are required for positive feedback regulation and for repression of Ato during lateral inhibition. However, it is also possible that other bHLH family genes of the E(spl) complex loci might be required, or that the overexpression of E(spl) or Da by tub-Gal4 in MARCM assays might not be strong enough to repress the expression of ato. By contrast, Da expression by dpp-Gal4 induces the expression of E(spl), even in the proximal sector of the antenna disc where Ato is not expressed. amos, the proneural gene for olfactory sensilla, is not expressed in the antenna disc at this time. Thus, a high level of Da can induce E(spl) in the absence of Ato, although Da might act with other class II proteins to promote the expression of E(spl) (Lim, 2008).

Since N signalling is activated in the same cells surrounding R8 founder neurons, whether Da expression is affected was examined by removing the function of N using a temperature-sensitive allele, Nts. The loss of function of N at the restrictive temperature resulted in several Ato-positive cells per proneural cluster. Furthermore, the transient loss of N activity abolished the high-level of Da expression between the proneural clusters but did not eliminate the basal level of Da expression in the same cells. This suggests that N signalling is essential for the high-level upregulation of Da expression. Since the expression of da is regulated by Hh and Dpp signalling, as well as Ato, it is possible that the regulation of Da by Hh and Dpp might be mediated by Ato-dependent N signalling in the non-R8 precursor cells (Lim, 2008).

To investigate further the role of N signalling in the expression of Da, whether E(spl) proteins mediate the function of N in inducing a high level of Da expression was examined. Loss of E(spl) caused ectopic expression of Ato in E(spl) mutant clones because of the lack of N-mediated lateral inhibition. Interestingly, the high level of Da expression was suppressed, but the basal level of Da expression was still detected in E(spl) mutant clones, as seen in Nts mutant eye discs. Thus, E(spl) is required for the high level but not for the basal level of Da expression. In contrast to da3 LOF mutant cells that fail to differentiate in spite of ectopic Ato expression, E(spl) LOF mutant cells not only expressed ectopic Ato but also differentiated into ectopic photoreceptors. Thus, the basal level of Da expression remaining in E(spl) LOF clones is sufficient for the formation of a functional complex with Ato to induce neural differentiation (Lim, 2008).

On the basis of the above observations, a model is proposed in which Da has dual functions as a proneural and as an anti-proneural factor depending on the expression level during early retinal neurogenesis . The anti-proneural function of Da proposed in this model provides an explanation for the abnormal upregulation of Ato in da mutant cells in the furrow, although the LOF experiments are also consistent with the pre-existing view that Da promotes the function of Ato. In Ato-positive neural precursors, low levels of Da expression are sufficient to form heterodimers with Ato to function as a proneural factor. In neighbouring cells, the N-E(spl) pathway further upregulates the expression of Da, which, in turn, induces more expression of E(spl). This putative feedback regulation might provide a mechanism for more effective lateral inhibition of Ato expression for the selection of R8. Interestingly, Da can form a homodimer and bind to DNA in vitro. Thus, in Ato-negative cells surrounding the R8 precursors, a high level of Da expression might enforce the formation of Da homodimers and/or heterodimers with other unknown bHLH proteins to repress the expression of ato. It would be interesting to see whether mammalian type I bHLH proteins such as E proteins might also be specifically regulated to have distinct developmental functions as seen in the case of Da (Lim, 2008).

DPP signaling controls development of the lamina glia required for retinal axon targeting in the visual system of Drosophila

The Drosophila visual system consists of the compound eyes and the optic ganglia in the brain. Among the eight photoreceptor (R) neurons, axons from the R1-R6 neurons stop between two layers of glial cells in the lamina, the most superficial ganglion in the optic lobe. Although it has been suggested that the lamina glia serve as intermediate targets of R axons, little is known about the mechanisms by which these cells develop. DPP signaling has been shown to play a key role in this process. dpp is expressed at the margin of the lamina target region, where glial precursors reside. The generation of clones mutant for Medea, the DPP signal transducer, or inhibition of DPP signaling in this region results in defects in R neuron projection patterns and in the lamina morphology; these defects are caused by defects in the differentiation of the lamina glial cells. glial cells missing is expressed shortly after glia precursors start to differentiate and migrate. Its expression depends on DPP; gcm is reduced or absent in dpp mutants or Medea clones, and ectopic activation of DPP signaling induces ectopic expression of gcm and Repo. In addition, R axon projections and lamina glia development are impaired by the expression of a dominant-negative form of gcm, suggesting that gcm indeed controls the differentiation of lamina glial cells. These results suggest that DPP signaling mediates the maturation of the lamina glia required for the correct R axon projection pattern by controlling the expression of gcm (Yoshida, 2005).

dpp is expressed in the dorsal and ventral margins of the posterior region of the optic lobe, adjacent to the cells expressing wg, which induces dpp expression. Glial cells in the lamina target region arise from these regions and migrate into the lamina target region as they contact R axons. Axons from R1-R6 neurons stop between two rows of glial cell layers, the epithelial and marginal layers, and form the lamina plexus. The third row of glial cells, the medulla glia, is located just beneath the marginal glia. The homeodomain protein Repo is expressed in these glial cells (Yoshida, 2005).

The expression pattern of dpp-lacZ, an enhancer-trap allele of dpp, was compared with the expression pattern of Repo. At a stage prior to glia differentiation and migration, expression of the dpp reporter is detected in the dorsal and ventral margins of the lamina target region. dpp continues to be expressed at the margins of the lamina target region throughout the third larval instar (Yoshida, 2005).

wg at the posterior-most domain induces the expression of dpp and omb. Some wg-expressing cells extend projections towards the lamina target region. These cells extend scaffold axons along which the lamina glia migrate. Thus, it was possible that the wg signal is involved in the migration and/or differentiation of lamina glia. However, partial elimination of Wg activity with a wgts allele does not cause a specific defect in glia migration. Therefore, wg may play a role in organizing domains in the visual cortex by activating/repressing various genes, rather than contributing to the generation of specific cell types (Yoshida, 2005).

Medea is required for lamina glia development. Medea encodes a co-SMAD and mediates a range of DPP/BMP/TGFß signaling events. In addition to dpp, four related genes -- glass bottom boat (gbb), screw, activin and activin2 -- have been identified in Drosophila. GBB signals through TKV/Saxophone (SAX) and Wishful Thinking (WIT) type I and type II receptors, respectively. Activin uses Baboon as a type I receptor, and Punt and WIT as type II receptors. Brains mutant for gbb and wit were examined, but no defects in lamina glia development were observed. It is concluded that it is highly likely that dpp is the ligand responsible for lamina glia development. However, the possibility that one or more of the DPP-related ligands acts redundantly in this process cannot be excluded (Yoshida, 2005).

In the embryo, gcm initiates the specification of glial cells from neural cells of various lineages. gcm expression is strictly controlled to ensure the correct separation of glial versus neuronal cell fate. Analysis of the cis-regulatory elements of gcm suggests that gcm expression depends on multiple regulatory elements to allow the control of lineage-specific transcription and autoregulation. The analysis carried out in this study suggests that a different situation exists in the optic lobe; gcm is expressed in the glia and the lamina neuronal cells, and is required for the differentiation of these cell types. In addition, differentiation is controlled differently in the lamina and in the glia. In the lamina, gcm expression seems to be controlled by hh, and in the glia, by dpp. These results suggest that gcm is controlled and functioning in a different manner in the optic lobe. Uncovering the mechanisms of the control and function of gcm would probably prove an intriguing focus for future research (Yoshida, 2005).

DPP and its vertebrate homolog BMP play crucial roles in many aspects of development by controlling patterning, cell growth and differentiation. This analysis reveals a role for DPP signaling in lamina glia differentiation in the Drosophila visual system. DPP has also been reported to function in several aspects of visual center development; for instance, DPP signaling has been shown to be involved in the proliferation and migration of the subretinal glia in eye disc development, which plays an important role in the R axon navigation. In addition, defects have been reported in the medulla neuropile in dpp mutant animals, suggesting a role for dpp in neuronal fate specification. Furthermore, tkv is expressed in lamina precursor cells just ahead of the lamina furrow, where these cells meet R axons and start to differentiate. Although this possibility is one of the things that prompted an examination of the role of DPP signaling in lamina development, no defects were uncovered when Mad or Medea clones were generated in the OPC or the lamina. Moreover, dpp appears to be expressed in the inner proliferation center (IPC), which will form the lobula, in addition to its expression in the dorsal and ventral marginal domains. Thus, dpp may be required for some aspects of lobula development. Unfortunately, this cannot be easily addressed at this moment because of a lack of appropriate markers. Further study of the requirements for dpp in the lamina, the medulla, the lobula and other cell types could lead to a more comprehensive understanding of how DPP signaling controls differentiation and other events during development of the visual system (Yoshida, 2005).

Dynamic decapentaplegic signaling regulates patterning and adhesion in the Drosophila pupal retina; Rst activity opposes DE-cadherin-mediated cell adhesion

The correct organization of cells within an epithelium is essential for proper tissue and organ morphogenesis. The role of Decapentaplegic/Bone morphogenetic protein (Dpp/BMP) signaling in cellular morphogenesis during epithelial development is poorly understood. In this paper, the developing Drosophila pupal retina -- looking specifically at the reorganization of glial-like support cells that lie between the retinal ommatidia -- was used to better understand the role of Dpp signaling during epithelial patterning. The results indicate that Dpp pathway activity is tightly regulated across time in the pupal retina and that epithelial cells in this tissue require Dpp signaling to achieve their correct shape and position within the ommatidial hexagon. These results point to the Dpp pathway as a third component and functional link between two adhesion systems, Hibris-Roughest and DE-cadherin. A balanced interplay between these three systems is essential for epithelial patterning during morphogenesis of the pupal retina. Importantly, a similar functional connection has been identified between Dpp activity and DE-cadherin and Rho1 during cell fate determination in the wing, suggesting a broader link between Dpp function and junctional integrity during epithelial development (Cordero, 2007).

Loss of Dpp pathway activity results in a loss of epithelial integrity, but the function of Dpp signaling during maturation of developing epithelia is not fully understood. This study shows that reducing the activity of components of the Dpp pathway leads to abnormal Interommatidial precursor cells (IPC) shape and organization within the ommatidial hexagonal pattern. This activity is linked to fine regulation of apical junction components and is required to maintain stable cell-cell contacts during cell movements within the epithelium. The expression of Dpp in primary pigment cells and the segregation of its receptors to the neighboring IPCs suggest a model in which Dpp acts in the primaries to organize local IPCs through the dynamic control of apical junctions. This view is supported by the dynamic changes in p-Mad activity in the neighboring IPCs, which is highest during the stage (20-26 hours APF) when IPCs rearrangements are maximal (Cordero, 2007).

The role of Dpp in cellular morphogenesis during epithelial development is poorly understood. Therefore, advantage was taken of the unique stereotyped pattern of the pupal retina to study cell behavior as morphogenesis progresses, focusing on events at the single-cell level. In situ visualization experiments suggest that IPCs with reduced Tkv activity are incapable of maintaining their cell-cell contacts and are subject to aberrant changes in their cell shape. Further emphasizing the link with cellular adhesion, this function of Dpp signaling involves DE-cadherin and Rho1, which are essential regulators of cell adhesion and cell shape (Cordero, 2007).

Several lines of evidence are provided indicating that Rst is a negative regulator of Dpp signaling. Previous work has demonstrated that Rst directs IPC movements through selective cell adhesion: IPCs seek to maximize their Rst-mediated contacts with primaries while decreasing contacts with their neighbors. Additionally, reducing Rst activity leads to a failure of initial cell movement. Consistent with these results, Rst activity opposes DE-cadherin-mediated cell adhesion. One model to account for these observations is that cells require a balance between cell movement provided by Hibris-Rst and the stability of cell-cell contacts provided by Dpp signaling. Live visualization supports the view that reducing Dpp activity leaves cells with an imbalance, as IPCs move toward their proper positions but fail to stabilize cell-cell contacts or lock stably into their final positions. Furthermore, downregulation of Dpp signaling leads to unstable DE-cadherin IPC-IPC junctions. Conversely, loss of rst results in loss of cell movements, which can be compensated by either reducing cell adhesion or Dpp signaling activity, again supporting the importance of maintaining a balance between the Hbs-Rst and the Dpp-DE-cadherin systems. Perhaps Dpp (and, by extension, BMP) activity is utilized in the adult for similar functions -- for example, as a 'proof-reading' mechanism to remove aberrant cells from an epithelium (Cordero, 2007).

The results in the wing raise the interesting possibility that regulation of DE-cadherin and Rho1-dependent cell shape and cell adhesion might be a characteristic of Dpp pathway activity common to other biological systems. Similar to the pupal retina, epithelial cells in the wing disc with reduced Dpp signaling displayed abnormal morphologies and were unable to maintain their positions. In the case of the wing, these defects were manifested as viable cysts of mutant cells that were basally excluded from the epithelium. The mechanisms involved in such cell behaviors remain unknown. The results suggest that the role of Dpp signaling during wing patterning also involves DE-cadherin and Rho1. The experiments do not distinguish whether the defects in wing cell fates are a direct or a secondary effect of altered cell adhesion, although altering DE-cadherin activity by itself was not sufficient to cause such defects. Cell adhesion and cell fate have been related previously: for example, Rho-dependent cell shape changes can influence fate decisions in stem cells. Despite the commonalities observed, tissue-specific factors are likely to regulate Dpp-dependent epithelial patterning: for example, Rst does not appear to have a role in wing development, and no changes in retinal Tubulin distribution reported has been reported for the wing (Cordero, 2007).

Dpp is the closest ortholog of vertebrate BMP2/4, and it appears to be active during cellular morphogenesis in a number of contexts including the developing vertebrate eye. Interestingly, and similar to observations for IPCs, fiber cells in the developing vertebrate lens show high levels of p-SMAD activity during the period of cell elongation. Loss of the Type I receptor ALK3 (also known as BMPR1A) or expression of the inhibitor noggin led to abnormal morphogenesis of these fiber cells including mispositioning and failure to elongate; requirements for E-cadherin (also known as cadherin 1) and RHOA function have not been explored (Cordero, 2007).

Finally, Rst does regulate developmental processes other than IPC patterning. For example, Rst is expressed in retinal axons and is required for correct targeting of those axons into the larval brain lobes. Interestingly, Dpp signaling also has a role in this process. Genetic interactions between rst3 and members of the Dpp pathway in the arrangement of these descending axons, raising the intriguing possibility that the two systems act together in axon targeting as well (Cordero, 2007).

These results provide evidence to support a model in which the Dpp pathway acts as an intermediary between the Rst and DE-cadherin adhesion systems. A balanced interplay between these three systems is essential to regulate epithelial cell movements, cell shape and cell-cell contacts during morphogenesis of the pupal retina. Several questions emerge from this study. For example, the data suggest that Rst acts on Dpp signaling by regulating surface-associated Tkv. Immunoprecipitation experiments failed to identify a physical interaction between Rst and Tkv, suggesting intermediate steps remain to be identified. Also, the transcription factor Mad is required to regulate IPC patterning, but the transcriptional targets that link Dpp signaling to DE-cadherin and Rho1 are unknown. A better understanding of the links between these three pathways should help shed light on the mechanisms that regulate the fine cellular events required during patterning of developing epithelia (Cordero, 2007).

Dpp and oogenesis

decapentaplegic is required for patterning of anterior eggshell structures, reflecting expression of dpp in anterior somatic follicle cells. Mutations in sax results in a block in oogenesis associated with egg chamber degeneration and a failure to transfer nurse cell contents to the oocyte. This suggests that the dpp signal is transmitted form the soma to the germline during oogenesis (Twombly, 1996).

The Drosophila eggshell, which has a pair of chorionic appendages (dorsal appendages) located asymmetrically along both the anterior/posterior and dorsal/ventral axes, provides a good model to study signal instructed morphogenesis. Broad-Complex, a gene encoding zinc-finger transcription factors, is essential for the morphogenesis of dorsal appendages and is expressed in a bilaterally symmetrical pattern in the lateral-dorsal-anterior follicle cells during late oogenesis. This pattern of expression is induced and specified along the dorsoventral axis by an epidermal growth factor receptor signaling pathway, which in the oocyte includes Gurken, a localized transforming growth factor alpha-like molecule. In the surrounding somatic follicle cells, Torpedo, the Drosophila EGF receptor homolog that functions as the target of Gurken specifies BR-C expression. The precisely localized expression of BR-C along the AP axis requires a separate signaling pathway, initiated by a transforming growth factor-beta homolog, Decapentaplegic, in nearby follicle cells. The fact that BR-C expression is missing in the anterior most columnar follicle cells suggests that there is probably another repressor produced in these cells. If Dpp signaling acts as a repressor of BR-C expression in oogenesis, a decrease in Dpp levels should result in the expression of BR-C in the anterior most collumnar cells -- cells where it is not found in wild-type eggshells. This was confirmed using a temperature sensitive dpp mutant. When dpp is ectopically expressed in most of the follicle cells, BR-C expression is found over the middle part of the oocyte at a more posterior postion than in wild-type egg chambers, resulting in a more posterior localization of the dorsal appendages. These two signaling pathways (Gurken functioning from the oocyte and Dpp functioning from the follicle cells) co-ordinately specify patches of follicle cells to express the Broad-Complex in a unique position with respect to both DV and AP axes respectively, and which, in turn direct the differentiation of the dorsal appendages in the correct position on the eggshell (Deng, 1997).

Stem cells are thought to occupy special local environments, or niches, established by neighboring cells that give them the capability for self-renewal. Each ovariole in the Drosophila ovary contains two germline stem cells surrounded by a group of differentiated somatic cells that express hedgehog and wingless. The BMP2/4 homolog decapentaplegic (dpp) is specifically required to maintain female germline stem cells and promote their division. Overexpression of dpp blocks germline stem cell differentiation. Overexpressing dpp for 3 days after eclosion produces tumorous germaria. Large germline cells filling germarial regions 1 and 2a contain spectrosomes but showed no evidence of cyst formation. In regions 2b and 3, 16-cell cysts are observed that probably derive from differentiated cystoblasts, or cysts that had formed before the first heat shock. This phenotype is very similar to that of bag of marbles (bam) and benign gonial cell neoplasm (bgcn) mutants. These results suggest that ectopic Dpp inhibits cystoblast differentiation but does not block cyst formation and maturation (Xie, 1998).

Mutations in dpp or its receptor (saxophone) accelerate stem cell loss and retard stem cell division. Mutant germline stem cell clones were constructed to show that the dpp signal is directly received by germline stem cells. punt, thickveins, mad, Medea and Dad are all shown to be required cell-autonomously for germline stem cell maintenance; punt, tkv, mad, and Med are shown to be required cell-autonomously to stimulate germline stem cell division. During aging, the number and activity of stem cells is thought to be reduced. The level of dpp signaling is shown to control the life span and division rate of germline stem cells. Reduced dpp signaling causes premature stem cell loss. Perhaps more surprising is the observation that putative increases in signaling, caused by removal of Dad activity from stem cells, causes them to be maintained longer. This finding suggests that dpp signaling not only is necessary, but may sometimes be rate limiting for stem cell maintenance. This is the first example where stem cell life span has been extended in an intact organism. Thus, dpp signaling helps define a niche that controls germline stem cell proliferation (Xie, 1998).

The "niche" hypothesis postulates that stem cells reside in optimal microenvironments or niches. When a stem cell divides, only one daughter can remain in the niche, while the other becomes committed to differentiate. A stem cell within the niche would have a high probability of self-renewal but a low probability of entry into the differentiation pathway. This model is consistent with the observations that stem cells require the addition of growth factors for proliferation and differentiation in many in vitro culture systems. The molecular nature of the microenvironment within a niche has yet to be defined in any system; however, the Drosophila germarium appears to contain such a niche. In the anterior, the stem cells abut terminal filament and cap cells, which both express hh, while only the latter express wg and armadillo. Stem cell daughters lie more to the posterior and probably directly contact inner germarial sheath cells, which express hh and patched. This asymmetry in structure and signals may allow germline stem cells to receive different levels of signals from their daughters. The existence of the germline stem cell niche is also consistent with stem cell proliferation when dpp is overexpressed. Under these conditions, the size of the niche may be substantially enlarged. Conversely, reduction of dpp function may weaken the ability of the niche to maintain germline stem cells, leading to accelerated losses. These results suggest that dpp is an essential niche signal. However, dpp likely interacts with other signals from surrounding somatic cells to make a functional niche for germline stem cells. Nonetheless, the identification of dpp as a key niche signal should greatly facilitate efforts to culture Drosophila germline stem cells in vitro (Xie, 1998 and references).

Technical limitations have so far prevented the identification of the source of the dpp signal that is received by germline stem cells. Ideally, analysis of clones of a null dpp allele would reveal which cells signal the germ line. However, the somatic cells adjacent to the stem cells cease division early in ovary development, making the induction of specific small clones difficult. The pattern of dpp expression in the germarium should also provide some insight into the origins of the signal. However, the only available dpp-lacZ fusion line and whole mount in situ experiments have failed to detect expression in the germarium, although follicle cell expression in late stage egg chambers is observed (Xie, 1998).

Germ line stem cell differentiation in Drosophila requires gap junctions and proceeds via an intermediate state.

The zero population growth (zpg) locus of Drosophila encodes a germline-specific gap junction protein, Innexin 4, that is required for survival of differentiating early germ cells during gametogenesis in both sexes. Zpg is required during oogenesis for the survival of the germ line stem cell daughter as it moves away from the niche and begins to differentiate. Germ-line stem cells (GSCs) lacking Zpg can divide, but the daughter cell destined to differentiate dies. These results suggest that zpg may be necessary for the differentiation process itself, as well as for survival of differentiated germ cells, and that zpg probably acts in parallel to bam and bgcn. The differentiation of the GSC to a cystoblast is gradual, and it is suggested many of the germ cells in 'stem cell tumors' caused either by strong mutations in bam or by overexpression of Dpp may be at an intermediate state between GSCs and cystoblasts. These findings suggest that germ line stem cells differentiate upon losing contact with their niche, that gap junction mediated cell-cell interactions are required for germ cell differentiation, and that in Drosophila germ line stem cell differentiation to a cystoblast is gradual. (Gilboa, 2003).

To investigate further a possible role for Zpg-mediated intercellular communication in the development of germ cell tumors, the genetic interaction between dpp and zpg was analyzed. It has been proposed that an increased Dpp signal induces over-proliferation of GSC-like cells. It was therefore reasoned that an increased Dpp signal could induce zpg cells to over-proliferate. To test this, flies carrying several insertions of a heat-shock dpp transgene (hs-dpp) were crossed into a zpg background. Flies were heat shocked, and then dissected and stained to reveal the germ line and spectrosomes. Control animals heterozygous for zpg, carrying a subset of the hs-dpp transgenes, had more single germ cells with spherical spectrosomes than did wild type. Homozygous zpg animals, which carried at least the same number of hs-dpp transgenes as the control, did not show an increase in germ cell number. To test whether zpg cells could respond to a Dpp signal, ovaries of zpg animals were stained with antibodies against phosphorylated Mad (p-Mad). Mad is a component of the Dpp signal transduction pathway and is phosphorylated upon activation of the pathway. In wild type, p-Mad staining can be detected in GSCs, cystoblasts and dividing cysts in region 1 of the germarium. The highest level of staining is observed in early germ cells; staining then gradually declines towards the posterior. p-Mad is also detected in the single cells that accumulate following Dpp overexpression. Similarly, p-Mad staining is detected in zpg germ cells, suggesting that the mutant cells are able to receive the Dpp signal. There may be several explanations for the failure of zpg cells to proliferate or survive in response to a Dpp signal. (1) The Dpp pathway could be blocked downstream of Mad in zpg cells. (2) zpg cells may not be able to survive when unattached to the tip of the ovary. (3) Proliferating cells in hs-dpp flies, that move away from the niche, are in a more differentiated state than the cells in the niche, and therefore die in a zpg background (Gilboa, 2003).

bam tumor cells and germ cells proliferating after Dpp overexpression (hs-dpp tumor cells) are considered to be GSCs because of their round spectrosomes and lack of BamC staining. Yet, these cells do not accumulate in a zpg background. One possible explanation for this observation is that bam and hs-dpp cells, as they move away from the niche, are at an intermediate state (pre-cystoblast) between a stem cell and a cystoblast, and that cystoblast development and survival requires Zpg. To determine whether an intermediate state between GSCs and cystoblasts exists in wild type, ovarioles were triple-labeled with anti-Vasa, 1B1 monoclonal antibody and anti-BamC, to mark the germ line, spectrosomes and cystoblasts, respectively. BamC antibody stains cysts of 4 or 8 cells strongly. Two-cell cysts had notably weaker staining. Only rarely were cystoblasts, i.e., single cells, stained with anti-BamC. In many ovarioles, single cells with a spherical spectrosome were observed that were removed from the stem cell position yet did not stain for the cystoblast marker BamC. The number of cells were counted that carried a spherical spectrosome and did not stain with anti-BamC. These cells would comprise the GSC population plus the presumptive intermediate population. Of 100 ovarioles scored, most had between 3 and 5 single cells that did not stain with anti-BamC. The average number of these cells was 3.9. This is a greater number than the average number of GSCs that populate an ovariole (between 2 and 3), as determined by cell-lineage analysis and electron microscopy. These data support the hypothesis that an intermediate state between a stem cell and a cystoblast exists in wild type (Gilboa, 2003).

An intermediate cell population between slowly dividing stem cells and differentiating cells, described as transit amplifying cells, is a common feature in stem cell systems, including those giving rise to blood cells, skin and the gut epithelium. These are the products of stem cell division and have limited potential to divide prior to differentiation. In principle, the existence of a transit amplifying cell population in the Drosophila germ line could be suggested if the GSC division rate is too low to account for the number of cysts/egg chambers produced (including dying ones) in a set period of time. Acquiring this information, especially in region 1 of the germarium has proved to be difficult. Therefore the products of possible division of cells were directly examined at the transition state. If a pre-cystoblast cell amplifies, it should give rise to more than one egg chamber. By contrast, a cystoblast divides incompletely, giving rise to a two-cell cyst and, eventually, to one egg chamber. To distinguish between 'intermediate-state' divisions and cystoblast divisions, mosaic analysis was conducted in wild-type germaria using the FLP-FRT marker system, in which cells that are produced by mitotic recombination are marked as twin-spots by the copy number of the gene (lacZ), encoding the marker ß-Galactosidase (0 or 2 copies). Recombination in GSCs would result in a marked GSC, a string of marked cysts arising from its subsequent division, and one cyst that is the twin-spot of the original recombination event. A recombination event in the cystoblast would not be observed under the experimental conditions because marked cells within a cyst share the diffusable ß-Gal. It was reasoned that, if a cell at the transition state divides, twin-spot cysts would be observed even in germaria where GSCs are not marked. Such a situation could also arise if a marked GSC left the niche and differentiated. However, GSC loss is unlikely to affect this analysis, since the half-life of wild-type GSCs is 4.6 weeks, whereas the females were dissected 2-3 days after induction of Flipase. Of 177 ovarioles scored, 104 contained no marked GSCs and no marked cysts (~60%). 73 ovarioles contained marked cysts and a marked GSC (~40%). Ovarioles were observed that contained only marked cysts and marked GSCs. These results led to the conclusion that cells at the transition state in wild type do not divide at an appreciable rate but proceed to differentiate to a cystoblast. Thus, they do not constitute the equivalent of a transient amplifying population. By contrast, tumor cells in bam mutants, or when Dpp is overexpressed, continue to divide, since their differentiation is blocked (Gilboa, 2003).

Thus, zpg ovarioles contain single germ cells at the anterior tip. Most of these cells do not reach the cystoblast stage. Since zpg cells are not arrested at a particular stage of their cell cycle, and can divide, it is concluded that GSCs that lack zpg divide to give another stem cell and a daughter cell that dies at early stages of differentiation. Accordingly, overexpression of bam, which is necessary and sufficient to promote germ cell differentiation, kills zpg cells (Gilboa, 2003).

zpg encodes innexin 4, one of eight innexins in Drosophila (Phelan, 2001; Stebbings, 2002; Tazuke, 2002). Innexins are the functional homologs of the vertebrate connexins, or gap junction proteins (Phelan, 2001). In mammalian oogenesis, gap junctions have been implicated in cell-cell signaling. Early luteinization of granulosa cells is observed either when the oocyte is physically removed from immature wild-type follicles, or in mice lacking connexin 37 (Simon, 1997), suggesting a gap junction-mediated signaling mechanism between the oocyte and granulosa cells (Gilboa, 2003).

Zpg is required for the survival of the germ line stem cell daughter as it moves away from the niche and begins to differentiate. From its expression pattern, and the specificity of its function, it is clear that Zpg acts in a germ line autonomous way. However, it is not yet known whether Zpg facilitates communication between germ cells, or between germ line and soma. The germarium is a compact structure where early germ cells contact each other, the somatic cap cells contact GSCs, and inner-sheath cells contact GSCs and their daughters. Further study is needed to clarify which cells communicate with germ cells through Zpg-gap junctions. Likewise, the nature of the requirement for zpg in GSC differentiation is still uncertain. Gap junctions may be used to supply the GSC daughter cell with nutrients, or with a survival factor required for its subsequent growth. Alternatively, gap junctions may be used to deposit a factor that is required for the differentiation process itself, rather than for survival, while an accessory mechanism eliminates cells that begin differentiating without that factor. The major argument in favor of a role for Zpg in differentiation at the stem cell stage comes from the phenotypic analysis of zpg, pum double mutants in which, unlike in pum single mutants, GSCs do not differentiate. Although Pum-mediated repression is removed in the double mutants, GSCs cannot differentiate as they may lack a differentiation signal provided by Zpg. It is harder to imagine how nutritional deficiency per se could block differentiation of the double-mutant cells because single-mutant zpg cells do begin to differentiate (and then die) (Gilboa, 2003).

Recent studies suggest that the niche promotes stem cell fate through Dpp signaling. This may be achieved through repression of bam. GSCs are also tightly tethered to the niche via adherens junctions. Other, as-yet unidentified mechanisms may be used by the niche to protect GSCs (Gilboa, 2003).

Once germ cells leave the niche they activate the differentiation pathway. It is proposed that differentiation of GCSs to cystoblasts is not direct but proceeds via an intermediate state. Most wild-type cells, which by their position within the germarium were judged to be cystoblasts, are not stained with BamC antibody. This finding concurs with Ohlstein (1997), who observed cytoplasmic Bam just before the cystoblasts divide to form a two-cell cyst and proposed the existence of an intermediate/pre-cystoblast state between GSCs and cystoblasts. In other stem cell systems, the intermediate cell population has a biological function, namely increasing the progeny of a single stem cell division. The results indicate that in Drosophila females, cells at the intermediate state do not constitute a transit-amplifying cell population. However, the 'pre-cystoblast state' may have a different biological significance. A vacant niche can be filled by a neighboring GSC that divides 'sideways' instead of along the anteroposterior axis. An alternative for filling a vacant GSC position might be for a cell at the intermediate state to reoccupy the niche (Gilboa, 2003).

The suggested model, adding a transitory state between the stem cell and the cystoblast, raises an interesting question. Is the differentiation of a GSC to a cystoblast a continuous process or a discrete one? It is notable that none of the markers that are currently used is specific to the stem cell, the cystoblast or the intermediate. Bam is present (in its fusomal form) already in the stem cell, and BamC gradually accumulates in the cystoblast. Pumilio protein and phosphorylated Mad are also detected from GSCs to cystoblasts and early cysts. In other stem cell systems, including mammalian hematopoiesis, many intermediate cell types are known, and can be isolated by specific marker combinations. Due to the relative lack of markers, the isolation of these 'cell types' from Drosophila ovaries is currently impossible. The overlap of expression patterns of the proteins that are known to have a role in stem cell maintenance and differentiation may indicate that differentiation is gradual, and possibly reversible (Gilboa, 2003).

It has been suggested that in bam or bgcn mutant ovaries, or when Dpp is overexpressed, the germaria are filled with GSC tumor cells. The findings of an intermediate cell population in wild type raises the possibility that GSC tumor cells share some properties with precystoblasts. Both these populations are single, do not stain for BamC, but exist outside of the niche. Some support to the analogy between pre-cystoblasts and 'GSC tumors' is evident in the fact that the latter do not survive past the niche in a zpg background. Thus, the zpg double mutants allow two cell populations in 'GSC tumors' to be distinguish - cells that are inside or outside of the niche. It is suggest that when Dpp is overexpressed, or in bam/bgcn mutants, cells outside the niche cannot fully activate the differentiation program and are at an intermediate state between a GSC and a cystoblast. These cells die in a zpg background, whereas the tumor cells in contact with the niche survive. The results thus suggest that beyond Zpg gap junctions and Dpp signaling, there must be additional signaling between GSCs and the niche, which helps maintain GSCs. Additional markers are needed to determine unequivocally whether bam tumors are similar to Dpp tumors, and whether they share properties with wild-type precystoblasts (Gilboa, 2003).

Differentiation of the stem cell daughter requires gap junctions. It is assumed that zpg is acting in parallel to pum, dpp, bam and bgcn because the double mutants showed incomplete epistasis of zpg over each of these genes. Although a role for gap junction proteins has been established in mammalian oogenesis (Ackert, 2001; Carabatsos, 2000; Juneja, 1999; Simon, 1997), this is thought to be the first instance where a gap junction protein is shown to control stem cell differentiation. What passes through these gap junctions, and which cells are connected to GSCs through Zpg channels, is still unknown. One intriguing option is that Zpg forms part of the channels that connect GSCs to the surrounding somatic niche cells. If so, that would suggest that the niche in the Drosophila germarium is necessary, not only for stem cell maintenance, but also for stem cell differentiation (Gilboa, 2003).

TSC1/2 tumour suppressor complex maintains Drosophila germline stem cells by preventing differentiation

Tuberous sclerosis complex human disease gene products TSC1 and TSC2 form a functional complex that negatively regulates target of rapamycin (TOR), an evolutionarily conserved kinase that plays a central role in cell growth and metabolism. This study describes a novel role of TSC1/2 in controlling stem cell maintenance. In the Drosophila ovary, disruption of either the Tsc1 or Tsc2 gene in germline stem cells (GSCs) leads to precocious GSC differentiation and loss. The GSC loss can be rescued by treatment with TORC1 inhibitor rapamycin, or by eliminating S6K, a TORC1 downstream effecter, suggesting that precocious differentiation of Tsc1/2 mutant GSC is due to hyperactivation of TORC1. One well-studied mechanism for GSC maintenance is that BMP signals from the niche directly repress the expression of a differentiation-promoting gene bag of marbles (bam) in GSCs. In Tsc1/2 mutant GSCs, BMP signalling activity is downregulated, but bam expression is still repressed. Moreover, Tsc1 bam double mutant GSCs could differentiate into early cystocytes, suggesting that TSC1/2 controls GSC differentiation via both BMP-Bam-dependent and -independent pathways. Taken together, these results suggest that TSC prevents precocious GSC differentiation by inhibiting TORC1 activity and subsequently differentiation-promoting programs. As TSC1/2-TORC1 signalling is highly conserved from Drosophila to mammals, it could have a similar role in controlling stem cell behaviour in mammals, including humans (Sun 2010).

TSC1/2 is known to regulate cell growth via inhibition on TORC1. This study demonstrates that it also functions by inhibiting the activity of TORC1 to maintain GSCs. Treatment with rapamycin, a TORC1-specific inhibitor, can completely rescue GSC loss in Tsc1 mutants. In addition, eliminating S6K, which functions downstream of TORC1 in regulating protein translation, could also completely rescue GSC loss in Tsc2 mutants. Interestingly, the daughters of Tor mutant GSCs can differentiate into germline cyst properly, indicating that TOR is normally not required for differentiation, but its hyperactivation in Tsc1/2 mutants drives precocious GSC differentiation. The simplest explanation of the delayed cystoblast differentiation in rapamycin-treated females might be a non-specific effect of drug treatment. However, it is also possible that TORC1 inhibition by rapamycin might cause repression of some, but not all, aspects of TOR function, which leads to uncoordinated development and/or differentiation of cystoblasts in response to GSC division. Consistently, accumulated cystoblasts where also observed when overexpressing both Tsc1 and Tsc2 in the germline. Together with the observation that TSC1/2-TORC1 signaling controls cell growth of germline cysts, this study suggests that TSC1/2-TORC1 may serve as a signaling integration point that orchestrates germline division, differentiation and development in order to control egg production in response to the local micro-environment and the system environment of the animals (Sun 2010).

In the Drosophila ovary, BMP signaling from the niche directly suppresses bam expression in GSCs to prevent differentiation. This signaling is crucial for GSC maintenance. As revealed by pMad expression, BMP signaling activity is significantly downregulated in Tsc1 mutant GSCs. This study also demonstrated that downregulation of pMad in Tsc1 mutant GSCs is mediated by TORC1 hyperactivation, as rapamycin treatment is able to restore the downregulated pMad level. However, TOR is not required for proper BMP signaling activity because pMad expression is not altered in rapamycin-treated germaria. Therefore, only TORC1 hyperactivation could inhibit BMP signaling in GSCs through unknown mechanisms, and this inhibitory effect occurs specifically in GSCs, as BMP signaling activity is not altered in Tsc1 mutant imaginal disc cells (Sun 2010).

Logically, bam expression could be derepressed in Tsc1 mutant GSCs as a consequence of BMP pathway downregulation. Surprisingly, no significant upregulation of bam-GFP expression could be detected in mutant GSCs, although in other GSCs that were compromised by BMP signaling, such as tkv mutant and mad mutant GSCs, bam transcription is significantly upregulated. Nevertheless, there might still be residual BMP signaling activities in Tsc1/2 mutant GSCs that are sufficient to suppress bam expression. Consistent with this notion is the observation that bam-GFP is not obviously upregulated in aged GSCs, even if BMP signaling activity has been significantly reduced. Together with the observation that bam mutation could not rescue the differentiation of Tsc1 mutant germ cells, it is suggested that the compromised BMP signaling activity may not be primarily responsible for Tsc1/2 mutant GSC loss. It is not clear why the effect of TSC1/2 on BMP signaling occurs specifically in GSCs. Possibly, Tsc1/2 mutant GSCs, once induced, have already primed for differentiation through a Bam-independent mechanism, which may trigger a positive feedback signal to inhibit BMP signaling activity, in order to facilitate differentiation (Sun 2010).

This study also reveals a BMP-Bam-independent mechanism that probably underlies the major role of TSC1/2-TORC1 signaling in GSC maintenance. The phenotype of Tsc1 bam double mutant germ cells differs from the bam alone mutant germ cells, as the double mutant GSCs can still become lost from the niche over time and undergo further differentiation into early cystocytes. Interestingly, the phenotype of Tsc1/2 mutant GSCs is similar to that of pelota (pelo) mutants. Pelo encodes a translational release factor-like protein and may regulate GSC maintenance at the translational level. In pelo mutant GSCs, there is also a downregulation of BMP signaling but no obvious upregulation of bam expression, and bam pelo double mutant germ cells are able to undergo similar limited differentiation into cystocytes, suggesting that TSC1/2 and Pelo might function in the same or parallel pathway to control GSC differentiation. It is proposed that similar to Pelo, TSC1/2 might function in a parallel pathway with the BMP-Bam pathway to control GSC differentiation, possibly by regulating the translation of differentiation-related mRNAs (Sun 2010).

Pum and Nos, which are known to function together to repress translation of the target mRNAs in embryos, are also essential for GSC maintenance. Recent genetic and biochemical studies suggest that Bam/Bgcn may directly inhibit the function of Pum/Nos to allow cystoblast differentiation. However, BMP signaling activation is able to prevent differentiation of nos mutant primordial germ cells, indicating that Pum/Nos could also function in parallel with the BMP-Bam pathway to control germ cell differentiation. In the future, it would be important to determine the functional relationships between the TSC1/2-TORC1 pathway, Pelo and Pum/Nos in regulating GSCs, and whether these factors, together with the microRNA pathway, target similar mRNAs to control GSC differentiation (Sun 2010).

This study has identified a novel role of TSC1/2 in controlling GSC maintenance and differentiation in the Drosophila ovary. Increasing evidence also suggests similar roles for TSC1/2-TOR signaling in regulating adult stem cell differentiation in mammals. For example, TSC1/2-mTOR signaling is also required for maintaining the quiescence of haematopoietic stem cells (HSCs), as Tsc1 deletion drives HSCs from quiescence to rapid cycling, which compromises HSC self-renewal. Thus, TSC1/2-TOR signaling could have an evolutionarily conserved role in regulating stem cell maintenance and differentiation from Drosophila to mammals (Sun 2010).

Determination of EGFR signaling output by opposing gradients of BMP and JAK/STAT activity

A relatively small number of signaling pathways drive a wide range of developmental decisions, but how this versatility in signaling outcome is generated is not clear. In the Drosophila follicular epithelium, localized epidermal growth factor receptor (EGFR) activation induces distinct cell fates depending on its location. Posterior follicle cells respond to EGFR activity by expressing the T-box transcription factors Midline and H15, while anterior cells respond by expressing the homeodomain transcription factor Mirror. This study shows that the choice between these alternative outputs of EGFR signaling is regulated by antiparallel gradients of JAK/STAT and BMP pathway activity and that mutual repression between Midline/H15 and Mirror generates a bistable switch that toggles between alternative EGFR signaling outcomes. JAK/STAT and BMP pathway input is integrated through their joint and opposing regulation of both sides of this switch. By converting this positional information into a binary decision between EGFR signaling outcomes, this regulatory network ultimately allows the same ligand-receptor pair to establish both the anterior-posterior (AP) and dorsal-ventral (DV) axes of the issue (Fregoso Lomas, 2016).

This study shows that the choice between two alternative Grk/EGFR signaling outcomes in the follicular epithelium depends on positional input provided by Upd and Dpp. At the posterior, the presence of Upd allows Grk to induce Mid/H15 while, at the anterior, Grk together with Dpp positively regulates Mirr. In this context, Upd and Dpp serve to define the response to Grk/EGFR signaling, since they are not sufficient to induce Mid/H15 and Mirr, respectively, in the absence of Grk (Fregoso Lomas, 2016).

Mutual repression is demonstrated between Mid/H15 and Mirr that is proposed to generate a double-negative feedback circuit that toggles the system between anterior and posterior outcomes. Moreover, in addition to their mutual regulation, analysis of double-mutant clones reveals that Upd and Dpp each regulate both Mid/H15 and Mirr and, thus, each provides input to both sides of this circuit. Upd is required for the expression of Mid and H15 even in the absence of a functional mirr gene, demonstrating that Upd is required for Mid/H15 expression independent of its ability to repress Mirr. Similarly, Dpp signaling can repress Mid independently of its positive effect on Mirr. The choice of Grk/EGFR signaling outcome in this context thus depends not only on mutual repression between these alternate targets but also on their opposing regulation by Upd and Dpp (Fregoso Lomas, 2016).

It is proposed that these elements define a bistable network that controls the choice between two alternative outcomes of Grk/EGFR signaling. These outcomes are irreversible -- e.g., posterior EGFR signaling in later stages cannot induce Mirr unless Mid and H15 are absent - and mutually exclusive, and the factors described in this study include two key elements found in bistable networks: feedback and non-linearity. The feedback in this case is provided by the reciprocal repression between Mirr and Mid/H15, generating a double-negative feedback loop that reinforces the choice of signaling outcome. In addition, bistability requires non-linearity in the response of the circuit to its upstream regulators, which makes the switch more sensitive to graded inputs. It is proposed that, in the follicular epithelium, this is achieved by the joint opposing regulation of the feedback circuit by both Dpp and Upd; each activates one side of the switch while repressing the other, biasing the outcome in the same direction (Fregoso Lomas, 2016).

These alternative responses to Grk are separated in time, as the source of Grk moves from posterior to anterior during the course of development. An important element that determines the choice between them is the early pattern of Mirr expression. In early stages of oogenesis, Mirr is Grk independent and is restricted to the main body follicle cells, due to its repression in the terminal regions by Upd. These main-body follicle cells correspond to the future anterior region of the columnar epithelium, and it is proposed that this early expression of Mirr predisposes them to express Mirr instead of Mid/H15 when Grk adopts its final dorsal anterior localization. Such a role for the early phase of Mirr expression is also consistent with the DV asymmetry of the Mid expression domain; as Grk moves anteriorly, leaving the range of posterior Upd and entering this domain of early Mirr expression and Dpp pathway activity, only the peak dorsal levels of Grk are capable of inducing Mid (Fregoso Lomas, 2016).

These observations also provide an example of how antiparallel signaling gradients can be integrated during epithelial patterning. Tissue patterning by opposing morphogen gradients is observed in developmental contexts as diverse as the Drosophila blastoderm and vertebrate neural tube, where they engage downstream transcriptional networks whose dynamic properties generate reproducible gene expression boundaries. Mutual repression between downstream transcription factors helps define the position and boundaries of cell fate domains, but how the opposing gradients are integrated is not well understood. This study shows that, in the follicular epithelium, the opposing Upd and Dpp gradients are integrated at the level of the Mirr-Mid/H15 feedback circuit. This integration occurs not only at the level of the mutual repression between Mid/H15 and Mirr but also by the ability of each gradient to regulate both sides of this circuit (Fregoso Lomas, 2016).

Together, these elements define the framework of a regulatory network that integrates localized positional information to regulate a binary choice of EGFR signaling outcome. The results allow construction of a model that both accounts for how an individual cell responds to Grk/EGFR signaling and explains how these spatial inputs are integrated across the epithelium to generate a defined pattern of Mid/H15 and Mirr expression, ultimately defining the pattern of the eggshell. Mirr is required for the generation of the high- and low-Broad domains, and Mid/H15 expression is required to define the posterior limit of these domains. The ability of Dpp and Upd to influence the outcome of EGFR signaling allows a single signaling input, namely localized secretion of Grk by the oocyte, to generate multiple distinct outputs that are localized in space and time, thus establishing both the AP and DV polarity of the epithelium and generating a complex and reproducible pattern of cell fates (Fregoso Lomas, 2016).

DPP-mediated TGFβ signaling regulates juvenile hormone biosynthesis by activating the expression of juvenile hormone acid methyltransferase

Juvenile hormone (JH) biosynthesis in the corpus allatum (CA) is regulated by neuropeptides and neurotransmitters produced in the brain. However, little is known about how these neural signals induce changes in JH biosynthesis. This study reports a novel function of TGFβ signaling in transferring brain signals into transcriptional changes of JH acid methyltransferase (jhamt), a key regulatory enzyme of JH biosynthesis. A Drosophila genetic screen identified that Tkv and Mad are required for JH-mediated suppression of broad (br) expression in young larvae. Further investigation demonstrated that TGFβ signaling stimulates JH biosynthesis by upregulating jhamt expression. Moreover, dpp hypomorphic mutants also induces precocious br expression. The pupal lethality of these dpp mutants is partially rescued by an exogenous JH agonist. Finally, dpp is specifically expressed in the CA cells of ring glands, and its expression profile in the CA correlates with that of jhamt and matched JH levels in the hemolymph. Reduced dpp expression was detected in larvae mutant for Nmdar1, a CA-expressed glutamate receptor. Taken together, it is concluded that the neurotransmitter glutamate promotes dpp expression in the CA, which stimulates JH biosynthesis through Tkv and Mad by upregulating jhamt transcription at the early larval stages to prevent premature metamorphosis (Huang, 2011).

The functions of the TGFβ superfamily and other morphogens in regulating insect metamorphosis are rarely reported. In two independent genetic screens, it was discovered that Drosophila TGFβ signaling controls two different aspects of insect metamorphosis. In a previous study, it was found that Baboon (Babo) and dSmad2-mediated TGFβ signaling regulates larval neuron remodeling, which is part of the insect central nervous system metamorphosis induced by 20E during the pupal stage. Further investigation revealed that Babo/dSmad2-mediated TGFβ signaling controls larval neuron remodeling through regulating the expression of EcR-B1, a specific isoform of the 20E receptor (Huang, 2011).

This paper reports several findings. First, br is precociously expressed in 2nd instar tkv and Mad mutant larvae. Second, the precocious br expression phenotype in tkv and Mad mutant larvae can be suppressed by exogenous JH agonist (JHA). Third, Tkv and Mad repressed br expression in a non-cell-autonomous manner. Fourth, the presence of Mad in the CA is sufficient to repress br expression in the fat body (FB). Fifth, jhamt mRNA levels and JHAMT activity were significantly reduced in the Mad-deficient larvae. These results demonstrate that Tkv- and Mad-mediated signaling is required in the CA to activate jhamt expression and thus JH biosynthesis, which in turn controls insect metamorphosis (Huang, 2011).

The Drosophila genome encodes two TGFβ type II receptors, Punt (Put) and Wishful thinking (Wit). The genetic screen failed to identify a role for either of these receptors in the regulation of JH biosynthesis. Put and Wit are most probably functionally redundant in this biological event, as in the case of TGFβ-mediated mushroom body neuron remodeling (Huang, 2011).

Dpp is a key morphogen that controls dorsal/ventral polarity, segmental compartment determination and imaginal disc patterning. Dpp function usually depends on its gradient distribution. In an attempt to identify the ligand for Tkv/Mad-mediated TGFβ signaling in the CA, a novel, gradient-independent role for Dpp was discovered that controls JH biosynthesis. Dpp is the ligand of Tkv, which regulates jhamt transcription. Loss of Dpp, even RNAi reduction of Dpp in the CA specifically, causes precocious br expression at the early larval stages, which phenocopies tkv and Mad mutants. Phenotypes of dpp, including precocious br expression and lethality, are at least partially rescued by JHA treatment or ectopic jhamt expression in the CA. Notably, dpp-lacZ is strictly expressed in the CA cells, but not in the other two types of endocrine cells in the ring gland: the prothoracic gland and corpus cardiacum cells. The developmental expression profile of dpp in the CA is always consistent with that of jhamt. Finally, dpp expression in the CA may be directly controlled by neurotransmitter signals in the brain, which is supported by reduced dpp and jhamt transcription levels in the Nmdar1 mutant wandering larvae (Huang, 2011).

Several lines of evidence suggest that Met is a crucial regulator at or near the top of a JH signaling hierarchy, possibly acting as a JH receptor. However, null Met mutants of Drosophila are completely viable, which is unexpected if Met is a JH receptor. A recent investigation indicated that another Drosophila bHLH-PAS protein, Germ cell-expressed (Gce), which has more than 50% homology to Met, may function redundantly to Met in transducing JH signaling (Baumann, 2010). Because Met is on the X chromosome in the fly genome, it was not covered by the genetic screen. The br protein in the FBs of a Met null allele, Met27, was tested at the 2nd instar larval stage, and precocious br expression was observed. Importantly, this precocious br expression phenotype could not be suppressed by exogenous JHA. This result not only supports the previous reports regarding the function of Met in transducing JH signaling but also suggests that the precocious br expression is a more sensitive indicator for the reduced JH activity in Drosophila compared with precocious metamorphosis, lethality and other phenotypes (Huang, 2011).

Kr-h1 was reported to act downstream of Met in mediating JH action. Studies in both Drosophila and Tribolium reveal that, at the pupal stages, exogenous JHA induces Kr-h1 expression, which in turn upregulates br expression. The genetic screen successfully identified that Kr-h1 is cell-autonomously required for the suppression of br expression at young larval stages. Precocious br expression occurred in the FBs of Kr-h1 mutants and was not suppressed by JHA treatment. Therefore, these studies further suggest that Kr-h1 functions as a JH signaling component in mediating insect metamorphosis. However, the finding shows that, at the larval stages of Drosophila, the JH-induced Kr-h1 suppresses, rather than stimulates, br expression. This result is consistent with the facts that Kr-h1 functions to prevent Tribolium metamorphosis and Br is a crucial factor in promoting pupa formation (Huang, 2011).

In summary, this study has found a novel function of Dpp, Tkv and Mad-mediated TGFβ signaling in controlling insect metamorphosis. As summarized in a model, the brain sends neurotransmitters, such as glutamate, to the CA through neuronal axons. Glutamate interacts with its receptor (NMDAR) on the surface of CA cells to induce dpp expression. Dpp protein produced and secreted by CA cells forms a complex with TGFβ type I receptor (Tkv) and type II receptor on the membrane of CA cells, followed by phosphorylation and activation of Tkv. Activated Tkv in turn phosphorylates Mad, which is imported into the nucleus together with co-Smad and stimulates jhamt expression. JHAMT in CA cells transforms JH acid into JH, which is released into hemolymph. The presence of JH in young larvae prevents premature metamorphosis through Met/Gce and Kr-h1 by suppressing the expression of br, a crucial gene in initiating insect metamorphosis (Huang, 2011).

Population genetics and evolution of Dpp

The sequence level organization of a significant portion of the dpp locus in Drosophila melanogaster has been analyzed and interspecific comparisons with D. simulans, D. pseudoobscura and D. virilis have been used to explore the molecular evolution of the gene. Interspecific analysis identifies significant selective constraint on both the nucleotide and amino acid sequences. As expected, interspecific comparison of protein coding sequences shows that the C-terminal ligand region is highly conserved. However, the central portion of the protein is also conserved, while the N-terminal third, the proregion, is quite variable. The central portion (domain 2) is conserved to the level seen in the ligand region, suggesting an important role for this portion of the protein in DPP function. For the related protein, TBF-ß1, there is biochemical evidence that the proregion is essential for secretion and dimerization. The pattern of amino acid conservation seen for DPP suggests that this function resides only in domain 2 of the proregion. Comparison of noncoding regions reveals significant stretches of nucleotide identity in the 3' untranslated portion of exon 3 and in the intron between exons 2 and 3. An examination of cDNA sequences representing five classes of dpp transcripts indicates that these transcripts encode the same polypeptide (Newfeld, 1997a).

A study of polymorphism and species divergence of the dpp gene of Drosophila has been made. Eighteen lines from a population of D. melanogaster were sequenced for 5200 bp of the Hin region of the gene, coding for the dpp polypeptide. A comparison was made with sequence from D. simulans. Ninety-six silent polymorphisms and three amino acid replacement polymorphisms were found. The overall silent polymorphism (0.0247) is low, but haplotype diversity (0.0066 for effectively silent sites and 0.0054 for all sites) is in the range found for enzyme loci. Amino acid variation is absent in the N-terminal signal peptide, the C-terminal TGF-beta peptide and in the N-terminal half of the pro-protein region. At the nucleotide level there is strong conservation in the middle half of the large-intron and in the 3' untranslated sequence of the last exon. The 3' untranslated conservation, which is perfect for 110 bp among all the divergent species, is unexplained. There is strong positive linkage disequilibrium among polymorphic sites, with stretches of apparent gene conversion among originally divergent sequences. The population apparently is a migration mixture of divergent clades (Richter, 1997).

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decapentaplegic: Biological Overview | Evolutionary Homologs | Transcriptional regulation | Targets of activity | Protein Interactions | Post-transcriptional Regulation | Developmental Biology | References

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