decapentaplegic

DEVELOPMENTAL BIOLOGY part 1/3

Embryonic

Early dpp expression pattern in the ectoderm is dynamic, consisting of three phases. Phase I, in which dpp is expressed in a broad dorsal domain, depends on elements in the dpp second intron that interact with the Dorsal transcription factor to repress transcription ventrally. In contrast, in phases II and III, dpp is expressed first in broad longitudinal stripes (phase II) and subsequently in narrow longitudinal stripes (phase III) (Schwyter, 1995). dpp is also expressed in the visceral mesodermal midgut, where it regulates formation of caeca. DPP has a major role in compartment formation between visceral segments and is expressed there during the process of segmentation (Manek, 1994).

dpp expression in the gut, at least some of which is presumably endodermal, includes the presumptive pharynx, a portion of the presumptive exophagus, the primordia of the gastric caeca, the parasegment 7 region of the midgut, and a portion of the presumptive hindgut. As these expression patterns are generated from promoter fragments, the finding of DPP expression in the endoderm should remain controvertial until confirmed (Jackson, 1994).

The Drosophila visceral mesoderm (VM) is a favorite system for studying the regulation of target genes by Hox proteins. The VM is formed by cells from only the anterior subdivision of each mesodermal parasegment (PS). The VM itself acquires modular anterior-posterior subdivisions similar to those found in the ectoderm. Mesodemal cells located just under the engrailed-expressing cells in the posterior ectodermal compartment have been called the mesodermal "P domain." The dorsal-most cells of the mesodermal P domain in each PS express the homeobox gene bagpipe (bap); they detach from the mesodermal fold and move inward toward the center of the embryo. These bap-expressing cells form the VM progenitor groups. The VM subdivisions, and the metameric expression of Connectin, form in response to ectodermal production of secreted signals encoded by the segment polarity genes hedgehog and wingless and are independent of Hox gene activity. A cascade of induction from ectoderm to mesoderm to endoderm thus subdivides the gut tissues along the A-P axis. Induction of VM subdivisions may converge with Hox-mediated information to refine spatial patterning in the VM. Con patches align with ectodermal engrailed stripes, so the VM subdivisions correspond to PS 2-12 boundaries in the VM. The PS boundaries demarcated by Connectin in the VM can be used to map expression domains of Hox genes and their targets with high resolution. The resultant map suggests a model for the origins of VM-specific Hox expression in which Hox domains clonally inherited from blastoderm ancestors are modified by diffusible signals acting on VM-specific enhancers (Bilder, 1998b).

Since Con expression marks the imprint of ectodermal PS boundaries on the VM, Con patches can be used to precisely map the domains of Hox gene transcription in relation to Con patches. teashirt is expressed in two domains. The anterior midgut domain extends from visceral mesoderm segment (VS) 4 to mid-VS 6, where it shares a posterior boundary with Antennapedia; the central midgut domain extends several cells to either side of the VS 8 boundary. dpp is also expressed in two domains: at the gastric caeca, it is found in the A domain of VS2 and the P domain of VS 3, while in the central midgut it extends from the A domain of VS 6 to terminate just anterior to the VS 8 boundary. wg is expressed just anterior to the VS 8 boundary, with some cells after stage 12 lying in VS 8. pnt is expressed throughout VS 8, although expression is not seen until early stage 13. At stage 13, the two domains of odd paired (opa) expression extend from the P domain of VS 4 to the VS 6 boundary and from VS 9 through VS 11 (Bilder, 1998b).

The formation of the tracheal network in Drosophila is driven by stereotyped migration of cells from the tracheal pits. No cell divisions take place during tracheal migration and the number of cells in each branch is fixed. This work examines the basis for the determination of tracheal branch fates, prior to the onset of migration. The EGF receptor pathway is activated by localized processing of the ligand Spitz in the tracheal placodes and is responsible for the capacity to form the dorsal trunk and visceral branch. Prominent double phosphorylation of Erk (Rolled) is detected in the tracheal placodes at stage 10-11. This pattern is Efgr-dependent and is abolished in rhomboid mutants. The double phosphorylated Erk domain is broader than the region of rhomboid expression. Since Rhomboid is known to regulate Spitz processing, this pattern probably reflects the diffusion of the secreted form of Spitz originating within the rhomboid-expressing cells, in the central part of the placode. In mutants for Egfr, tracheal pits appear normal, although certain tracheal branches fail to develop: specifically, the dorsal trunk and visceral branch are missing or incomplete. spalt mutants show specific defects in the migration of dorsal trunk cells, pointing to an important role for spalt in subdivision of tracheal fates. The Dpp pathway is induced in the tracheal pit by local presentation of Dpp from the adjacent dorsal and ventral ectodermal cells. This pathway patterns the dorsal and lateral branches. Elimination of both Dpp and Egfr pathways blocks migration of all tracheal branches. Antagonistic interactions between the two pathways are demonstrated. The opposing activities of two pathways may refine the final determination of tracheal branch fates. Egfr-dependent activation of Erk (Rolled) in the tracheal placode precedes the activation of the same pathway by Breathless. Only after Egfr induction is diminished, does a new double phosphorylated Erk pattern appear, induced by Breathless. It is proposed that two opposing gradients of Dpp and Spitz are operating within the placode. the cells in the center of the tracheal placode encounter high concentrations of secreted Spitz, and low or negligable levels of Dpp. Conversely, the cells located at the dorsal and ventral domains of the placode encounter high concentrations of Dpp and low levels of secreted Spitz. Therefore, induction by the Egfr and Dpp pathways creates three subsets of cells: dorsal, central and ventral (Wappner, 1997).

Wing and leg precursors of Drosophila are recruited from a common pool of ectodermal cells expressing the homeobox gene Dll. Induction by Dpp promotes this cell fate decision toward the wing and proximal leg. The receptor tyrosine kinase Egfr antagonizes the wing-promoting function of Dpp and allows recruitment of leg precursor cells from uncommitted ectodermal cells. By monitoring the spatial distribution of cells responding to Dpp and Egfr, it has been shown that nuclear transduction of the two signals peaks at different positions along the dorsoventral axis when the fates of wing and leg discs are specified and that the balance of the two signals assessed within the nucleus determines the number of cells recruited to the wing. Differential activation of the two signals and the cross talk between them critically affect this cell fate choice (Kubota, 2000).

In a screen for genes expressed in the embryonic limb primordia, rhomboid was found to be transiently expressed in the central part of Dll-expressing limb primordia in stage 11 embryos. rho transcription is the rate-limiting step of the activation of an EGFR ligand Spitz. As expected from the role of rho as a stimulator of Egfr, a transient expression of an activated, phosphorylated form of MAPK (dpMAPK) is detected in the nucleus of limb primordial cells surrounding the rho-expressing cells. The dpMAPK expression starts after the initiation of Dll transcription and diminishes before the separation of the wing and leg disc primordium. The dpMAPK expression is undetectable in null mutants of rho or Egfr. The peak of dpMAPK expression is located ventrally to the cells expressing dpp. The results suggest that rho-mediated stimulation of Egfr and MAPK occurs at the time of cell fate specification of wing and leg discs (Kubota, 2000).

The spatial distribution of cells responding to Dpp and its relationship to Egfr signals was studied. To this end, an antibody specific to phosphorylated C-terminal sequence of Mad was produced. The phosphorylated sequence corresponds to the site at which the type I BMP receptor phosphorylates SMad1. The antibody detects an antigen distributed in a pattern similar to, but broader than, that of DPP mRNA. This immunoreactivity is dependent on Dpp signaling, as it is absent in stage 11 mutants of thick veins encoding type I Dpp receptor and in dpp mutants. This indicates that other extant TGFbeta-related signaling molecules present in Drosophila embryos do not substitute for Dpp to induce this immunoreactivity. Conversely, ectopic expression of Dpp results in high accumulation of this immunoreactivity. These results suggest that the antibody detects a Dpp-specific signaling event, most likely the phosphorylation and nuclear transport of Mad. Hereafter, the immunoreactivity detected by this antibody is called pSSVS (Kubota, 2000).

pSSVS is found mainly localized in the nucleus and distributed in regions a few cells wider in diameter than those of dpp-expressing cells. These properties are consistent with the previous findings that Mad transduces the Dpp signal to the nucleus. Double labeling of pSSVS and DLL mRNA shows that pSSVS expression is higher in the dorsal region of Dll-expressing cells. Combined with the double-labeling results of dpMAPK and Dll or dpp, it is concluded that cells responding to Dpp and Egfr overlap, but the peak of the responses are shifted. Such differential distribution of the two signals results in an arrangement of cells responding to a different strength of Dpp and Egfr along the dorsoventral axis (Kubota, 2000).

To study the role of Egfr at the stage of wing and leg cell fate determination, specific marker gene expression was examined in Egfr signaling mutants. DLL mRNA is expressed in the entire limb primordium at stage 11 and becomes restricted to distal leg cells at stage 15. Esg protein expression was used to detect both wing and proximal leg cells. In rho mutants, the size of limb primordia at stage 11 is the same as the control, but the later development of leg discs is abnormal. The number of leg disc cells expressing Dll and/or Esg at stage 15 is reduced, and these cells no longer show the circular arrangement typical of leg disc precursors. Amorphic mutation of Egfr cause a ventral expansion of limb primordia as a result of a loss of the early function of Egfr, but the expression of leg markers is severely reduced at stage 15. A similar phenotype is observed in mutants lacking both maternal and paternal copies of Dsor1, which encodes a MAP kinase kinase. In all cases described above, Esg-expressing cells at the dorsal part of leg discs are most frequently lost, suggesting that the development of dorsoproximal leg cells is most sensitive to the loss of Egfr activity. In contrast, wing and leg disc development is normal in vein mutants, suggesting the putative ligand of Egfr encoded by this gene is dispensable. These results suggest that MAPK activation induced by Rho and Egfr is essential for normal leg development (Kubota, 2000).

The temporal requirement for Egfr was studied by the temperature-sensitive allele Egfr^f1. When the temperature is increased to the restrictive temperature at 5 hours after egg laying (AEL) prior to the induction of the limb primordium, the expression of Dll is expanded to the ventral midline, as was also observed with the strong Egfr mutants. When the temperature is increased at 6 hour AEL, the initial Dll expression is not altered, but the leg disc development is severely affected. Only mild defect is found in leg discs when the temperature is increased at 7 hours AEL, suggesting that Egfr must function between 6 and 7 hours AEL to correctly specify the leg cell fate. This is the time when the transient activation of MAPK is observed. Furthermore, whether Egfr is required autonomously in limb primordial cells was examined by expressing a dominant-negative form of Egfr using Dll-Gal4. Expression of this driver starts in the limb primordium at stage 11 and persists in a subset of wing discs and in entire leg discs at stage 15 because of the persistence of Gal4 activity. Imaginal disc-specific inhibition of Egfr interfers with leg disc development, while leaving the wing disc intact. These results demonstrated that a transient activation of Egfr in stage 11 limb primordia is essential for the leg disc development (Kubota, 2000).

In contrast to the severe defects in leg discs, none of the mutations in Egfr signaling interfer with wing disc formation. In these mutants, wing primordia consistently express Esg and another wing disc marker Vestigial, and invaginate to form discs. However, an increase in the number of wing disc cells has been noted in Egfr signaling mutants. This effect was analyzed in rho mutants; unlike Egfr mutants, in rho mutants the number of limb primordial cells at stage 11 is the same as the control. The number of Esg-expressing wing disc cells in rho mutants is increased compared to the control, while the number of the proximal leg disc cells is severely reduced. It is concluded that Egfr signaling is required to limit wing disc cell differentiation in limb primordial cells that are not yet fully committed. It is inferred that a subset of prospective leg cells that do not receive a sufficient amount of Egfr signaling fail to differentiate as proximal leg and instead adopt a wing fate (Kubota, 2000).

The increase in the number of wing disc cells in rho mutants resembles the overexpression phenotype of Dpp and raises a possibility that Egfr might prevent wing disc development by negatively regulating Dpp signaling. Such a cross talk could occur at several levels including the following: (1) regulation of dpp transcription, (2) signal transduction from Dpp receptors to the nucleus, and (3) transcriptional regulation of downstream target genes. The analyses excluded the first two possibilities for two reasons. (1) The expression pattern of DPP mRNA is unaffected by the mutation of rho. A previous report showing an expansion of dpp expression in Egfr mutants probably reflects the global patterning role of Egfr in the earlier stage. (2) pSSVS expression around limb primordia does not change in rho mutants. Conversely, the expression pattern of dpMAPK is not changed by a null mutation of tkv. These results suggest that the differential distribution of cells responding to Dpp and Egfr is set up independently of each other's activity (Kubota, 2000).

Dpp and Egfr were found to antagonize each other after signal transduction into the nucleus. Hyperactivation of Egfr by an ectopic expression of an Egfr ligand Spitz causes a great accumulation of dpMAPK. As expected from the negative effect of Egfr on the wing development, this treatment completely eliminates wing disc formation and, in addition, causes a malformation of the leg disc. Since it was found that cells migrating out of the leg primordium express dpMAPK, it is unlikely that the failure in wing disc formation is due to the prevention of cell migration or to cell death. It has been suggested that hyperactivation of Egfr prevents limb primordial cells from adopting the wing cell fate. It is likely that those cells adopt the epidermal fate instead. Overexpression of Dpp causes an accumulation of pSSVS and an increase in the number of wing disc cells. Coexpression of Dpp with Spi partially restores the development of both wing and leg discs, suggesting that wing disc development overcomes the negative effect of Egfr if provided with a sufficient amount of Dpp. The restored wing primordia migrate with high levels of pSSVS and dpMAPK, further supporting the notion that Dpp and Egfr signals are transduced independently of one another (Kubota, 2000).

dad is an immediate transcriptional target gene of Dpp, the expression of which closely parallels that of pSSVS expression in embryos and is inducible by Dpp. dad expression is not affected in Egfr or rho mutants. Furthermore, elevated dad expression induced by Dpp is not affected by sSpi, suggesting that at least one of the immediate transcriptional responses to Dpp is unaffected by elevated Egfr signaling (Kubota, 2000).

The antagonism between Dpp and Egfr during wing disc development raises a question: what is the default state of the wing and leg primordia in the absence of the two signals? Double mutant phenotypes of Dpp and Egfr signaling were examined. tkv mutants lack wing discs and their leg discs are malformed. This phenotype reflects a disc cell autonomous requirement for Dpp signaling, because the phenotype is reproduced by the disc-specific inhibition of Dpp signaling by dad, which inhibits Mad. The phenotype of either tkv;rho or tkv;Egfr double mutants is a simple addition of each mutation, in which wing discs are lost completely and leg discs are severely reduced. Since Dll-expressing limb primordial cells are present in tkv;Egfr double mutants in stage 11, it has been concluded that these cells fail to differentiate as wing discs and their ability to differentiate as leg discs is also compromised. A few Esg-positive cells remain at the position of the leg, and it is speculated that this reflects the presence of a second leg-inducing signal. These results suggest that Dpp is absolutely required for wing disc development irrespective of the activity of Egfr (Kubota, 2000).

Egfr affects the choice of wing vs. leg developmental options differently; it promotes leg development while it inhibits wing development. These two activities of Egfr are the earliest of known events of leg specification, and occur prior to the establishment of proximodistal axis in the leg. In the absence of late functions of Dpp and Egfr, limb primordia are specified but fail to differentiate into wing disc and most of leg disc. Thus it is proposed that early limb primordium at stage 11 consists of cells not yet fully committed to either wing or leg disc fate, and the cells are exposed to different amounts of Dpp and Egfr signaling according to their dorsoventral location. Dpp recruits the cells to the wing disc fate. Egfr antagonizes the cellular response to the wing-inducing function of Dpp and allows the development of wing discs only in the dorsal region. Thus the dorsoventral difference in Dpp and Egfr signaling in the limb primordium provides key information to the separation and differentiation of the wing and leg discs. In contrast to the opposing roles of Dpp and Egfr on wing disc development, leg discs requires both signals. The effect of the loss of Egfr activity on leg disc development is not compensated for by a simultaneous loss of Dpp signaling, indicating that Egfr has an additional activity to promote leg development separately from its role to antagonize Dpp. Because dorsal and ventral limb primordial cells respond to Egfr differently, it is speculated that at least one additional dorsoventral factor influences leg disc formation at stage 11. This idea is consistent with the fact that residual proximal leg cells can still be induced in the almost complete absence of Egfr and Dpp activity. One candidate for the factor is Wg, which is expressed in the limb primordium (Kubota, 2000).

The nuclear transduction of the Dpp signal, as visualized by the distribution of pSSVS and expression of dad, is unaffected by Egfr. The results suggest that the antagonistic effect of Egfr on Dpp signaling occurs after transduction into the nucleus. Therefore, the mechanism of SMad inhibition by direct phosphorylation by MAP kinase does not play a major role in this case (Kubota, 2000).

The finding that Egfr is activated in the limb primordium and prevents wing disc formation suggests that Egfr is a key factor in the diversification of the wing and leg fate. It is proposed that the differential activation of Dpp and Egfr, and the dorsal cell migration brings a subset of limb primordial cells out of the range of Egfr signaling, and thereby allows Dpp to induce wing development. It follows that dorsally migrating cells acquire the wing cell identity only after the separation from leg-promoting signals. Consistent with this idea, expression of wing-specific markers Vg and Sna, start only after the separation of the two primordia. Mechanisms that promote the dorsal cell migration remain to be identified. Given that the basic genetic components for the induction of the wing and leg have been identified in the model organism Drosophila, it can now be asked how the genetic mechanism of wing and leg specification has evolved by comparing the expression and function of these genes in limb primordial cells of primitive insects (Kubota, 2000).

Stepwise formation of a SMAD activity gradient during dorsal-ventral patterning of the Drosophila embryo

Genetic evidence suggests that the Drosophila ectoderm is patterned by a spatial gradient of bone morphogenetic protein (BMP). Patterns have been compared of two related cellular responses - signal-dependent phosphorylation of the BMP-regulated R-SMAD, MAD, and signal-dependent changes in levels and sub-cellular distribution of the co-SMAD Medea. Nuclear accumulation of Medea requires a BMP signal during blastoderm and gastrula stages. During this period, nuclear co-SMAD responses occur in three distinct patterns. At the end of blastoderm, a broad dorsal domain of weak SMAD response is detected. During early gastrulation, this domain narrows to a thin stripe of strong SMAD response at the dorsal midline. SMAD response levels continue to rise in the dorsal midline region during gastrulation, and flanking plateaus of weak responses are detected in dorsolateral cells. Thus, the thresholds for gene expression responses are implicit in the levels of SMAD responses during gastrulation. Both BMP ligands, DPP and Screw, are required for nuclear co-SMAD responses during these stages. The BMP antagonist Short gastrulation (Sog) is required to elevate peak responses at the dorsal midline as well as to depress responses in dorsolateral cells. The midline SMAD response gradient can form in embryos with reduced dpp gene dosage, but the peak level is reduced. These data support a model in which weak BMP activity during blastoderm defines the boundary between ventral neurogenic ectoderm and dorsal ectoderm. Subsequently, BMP activity creates a step gradient of SMAD responses that patterns the amnioserosa and dorsomedial ectoderm (Sutherland, 2003).

These in vivo studies validate the molecular model for signal-dependent nuclear accumulation of Medea. Nuclear accumulation of Medea requires both competence to oligomerize and MAD. Nuclear accumulation is signal dependent, requiring both BMP ligands, Dpp and Scw. Conversely, all cells accumulated nuclear Medea in the presence of constitutively active Tkv receptor. At these stages, any independent contribution from activin-like signals is below the detection limit (Sutherland, 2003).

Furthermore, levels of Medea determine the strength of BMP responses at these stages. Medea overexpression leads to expansion of the dorsal-most fate, with increased numbers of amnioserosa cells. Signal-dependence for nuclear accumulation is retained. Decreased Medea exacerbates loss of amnioserosa from reduced Dpp levels (Sutherland, 2003).

The intensity of Medea staining was surprisingly sensitive to signal activity. However, steady-state levels of Medea are unaffected by the level of BMP activity. The antibodies appear highly sensitive to a Medea conformation that is prevalent in the nucleus, most probably an active SMAD complex. This sensitivity makes nuclear Medea an excellent assay to distinguish spatial patterns of endogenous BMP activity (Sutherland, 2003).

In wild-type embryos, two transitions in the distribution of BMP activity are evident. Many cellular blastoderm embryos lack detectable levels of nuclear Medea, but a few have low levels of nuclear Medea in a broad dorsal domain, with little gradation. From the proportion of cellular blastoderm embryos with this pattern, the duration of nuclear Medea appears to be brief. These data parallel reports of broad, weak PMad staining during mid-cellularization, except that nuclear Medea is detected later and in a broader pattern. The time lag between the earliest reported detection of PMad and detection of nuclear Medea probably stems from a combination of technical differences and the time necessary for nuclear accumulation. In sum, initial BMP activity is weak and distributed broadly in dorsal regions. Low BMP activity at this phase is required to maintain the early phase of zen expression (Sutherland, 2003).

Onset of gastrulation is associated with a dramatic change in the domain of nuclear Medea, which narrows to a tight midline stripe of cells while staining levels intensify. PMad shows a similar transition to a narrower domain, but earlier. Thus, lateral SMAD responses became undetectable just as a steep activity gradient forms along the dorsal midline (Sutherland, 2003).

A third response pattern arises during mid-gastrulation: dorsolateral domains of cells exhibit low levels of nuclear Medea. Response levels remain high in the dorsal-most cells, even as they move laterally during gastrulation. Levels fall off rapidly over a few cells on either side, with a sharp transition to flanking plateaus of weak responses. The subcellular distribution of Medea is unchanging in ventral and ventrolateral cells. The full BMP response domain does not extend as far ventrally as it does during blastoderm, even though many dorsal cells move laterally during germband extension. Thus, the lateral-most cells with responses at blastoderm have decreased responses during gastrulation (Sutherland, 2003).

In sum, the dorsal midline stripe of SMAD responses corresponds to a steep BMP activity gradient, with thresholds that correlate with patterning markers. The edges of the Medea peak response correlates precisely with the position of dorsal cephalic markers during stage 8, the cycle 14 mitotic domains 1, 3 and 5. The second phase of zen expression occurs in cells with peak PMad responses at the end of stage 5. Flanking cells with lower PMad levels correlate with the broader expression domain for the BMP target genes tailup and u-shaped. The full Medea response domain correlates approximately with the expression domain for u-shaped and extends into the presumptive dorsomedial ectoderm. The sharp transitions in SMAD response levels predict expression boundaries for BMP-responsive genes (Sutherland, 2003).

Similarly, in the wing primordium, a BMP gradient creates sharp transitions in PMad levels, which match gene expression boundaries. However, BMP activity is modulated by different mechanisms in this tissue. dpp is expressed in a narrow stripe at the center, and ligand spreads to nearby cells over a period of hours. In contrast, the early embryonic BMP activity gradient forms rapidly, and is narrower than the expression domains for dpp and scw. Extracellular binding proteins form the embryonic BMP activity gradient (Sutherland, 2003).

The final width of the midline peak response is sensitive to gene dosage for both dpp and sog. It is broader when dpp dosage is increased, and narrower with only one copy of dpp. Similarly, the width of the stripe is broader, but more variable, when sog levels are reduced. The response domain is broadest in sog null embryos; however, the level of response is significantly reduced. This is distinct from the effect of increased dpp dosage, in which the response domain is broader, but normal SMAD response levels are achieved or exceeded (Sutherland, 2003).

The role of Sog as both a short-range inhibitor and a long-range potentiator of dorsal patterning has led to a proposal that Sog transports BMP ligands from lateral regions to the dorsal midline. Biochemical analyses suggest mechanisms for Sog-BMP binding and release. Computational analysis have defined conditions under which transport could occur with these mechanisms. The transition from weak, broad SMAD responses to narrow, strong responses is consistent with concentration of BMP activity at the dorsal midline, and the loss of this transition with loss of Sog is consistent with a Sog-dependent transport model. However, there are significant differences between the current results and the assumptions used to develop the computational model. These include the presence of a midline SMAD response in dpp–/+ embryos and the sensitivity to reduced sog dosage. It will be important to refine future computational models to fit the complete set of BMP response data (Sutherland, 2003).

Both BMP ligands, Dpp and Scw, are required to form the dorsal-midline gradient. However, scw mutant embryos retain a small amount of dorsal ectoderm, with concomitant expansion of ventral ectoderm. Surprisingly, the weak dorsolateral Medea response is lost in scw embryos. It is concluded that the full Medea response domain encompasses the cell fates that are lost in scw mutants, amnioserosa and dorsomedial ectoderm. It appears that dorsal cells can acquire a dorsolateral fate without gastrula BMP activity (Sutherland, 2003).

Mutants with expanded ventral ectoderm show reduced SMAD responses during the first phase of BMP activity. PMad was not detected in blastoderm tld embryos. Homozygotes for moderate dpp alleles have lower PMad levels during blastoderm. Conversely, sog embryos have a slightly expanded PMad response during blastoderm, and a slight expansion of dorsal ectoderm. Thus, BMP activity during blastoderm positions the boundary between dorsal and ventral ectoderm (Sutherland, 2003).

Mutations that shift the boundary between amnioserosa and dorsal ectoderm show altered SMAD responses in the third phase of BMP activity, the dorsal-midline gradient. dpp-/+ embryos have variable reductions in midline SMAD responses and in the number of amnioserosa cells. Strikingly, sog null embryos have little amnioserosa and a strong reduction in SMAD response levels during gastrulation. Thus, SMAD response levels during gastrulation are critical for amnioserosa specification (Sutherland, 2003).

Taken together, these data suggest a multi-step model for DV patterning of the embryonic ectoderm, incorporating aspects of the two previous models. In a previous gradient model, ectodermal fates are subdivided simultaneously by a continuous BMP gradient involving Dpp and Scw. In the successive cell-fate decision model, amnioserosa is specified by dorsal-midline Dpp+Scw activity, and the dorsal ectoderm by Dpp alone at stage 9 (Sutherland, 2003).

Instead, it is proposed that the blastoderm phase of weak BMP activity establishes a dorsal ectoderm domain. Mutations that shift the boundary between dorsal and ventral ectoderm also have altered SMAD responses at this stage. It is at this stage that SMADs compete with Brinker to regulate the first phase of zen expression. Furthermore, this early signal maintains BMP activity, for the late-blastoderm domain of dpp expression is set by competition between BMPs, Sog and Brinker. BMP activity subsequently maintains the dorsal boundary for brinker expression. Thus, BMP activity at blastoderm defines a dorsal domain where dpp is expressed and brinker is not (Sutherland, 2003).

After cellularization is complete, a step gradient of BMP activity subdivides the dorsal region into amnioserosa, dorsomedial ectoderm and dorsolateral ectoderm. Peak activity levels determine the amount of amnioserosa. Flanking shoulders of weak activity specify the dorsomedial ectoderm. It is proposed that the dorsolateral ectoderm experiences a transient BMP response during late blastoderm, but little or no response during gastrulation. In sum, the dorsal-midline gradient of BMP activity specifies at least three cell fates (Sutherland, 2003).

BMP activity in the dorsal ectoderm does not end with germband extension. During stage 9, PMad is detected throughout the dorsal ectoderm and amnioserosa, and might finalize determination of dorsal ectoderm fates. Dpp expression within the dorsal ectoderm contributes to combinatoral regulation of gene expression patterns in subsets of dorsal ectodermal cells. However, the ventral boundary of dpp expression in the stage 9 dorsal ectoderm must be defined by earlier events (Sutherland, 2003).

The step gradient of SMAD responses is maintained during the morphogenetic movements of gastrulation and germband extension. The peak response is maintained only in cells that initially reside at the dorsal midline, even though ventral ectoderm moves to a dorsal position during stages 7 and 8. The BMP activity gradient is thought to form by diffusion in the perivitelline fluid; however, dorsal cells 'remember' their BMP exposure as they move laterally. It is probable that the ligand distribution is established prior to the time that peak SMAD responses are detected, and activity persists through cell biological mechanisms. For example, ligand may bind to the extracellular matrix, so that it remains associated with dorsal cells. Alternatively, receptor-ligand complexes may continue to signal following endocytosis. Understanding the intracellular modulation of BMP responses will be important to understand how extracellular morphogen gradients are translated into a stable pattern of cell fates (Sutherland, 2003).

Establishment of the leading edge during embryonic development

The leading edge (LE) is a single row of cells in the Drosophila embryonic epidermis that marks the boundary between two fields of cells: the amnioserosa and the dorsal ectoderm. LE cells play a crucial role in the morphogenetic process of dorsal closure and eventually form the dorsal midline of the embryo. Mutations that block LE differentiation result in a failure of dorsal closure and embryonic lethality. How LE cells are specified remains unclear. To explore whether LE cells are specified in response to early dorsoventral patterning information or whether they arise secondarily, the extent of amnioserosa and dorsal ectoderm was altered genetically, and LE cell fate was assayed. No expansion of LE fate is observed in dorsalized or ventralized mutants. Furthermore, the LE fate arises as a single row of cells, wherever amnioserosa tissue and dorsal epidermis are physically juxtaposed. Taken together these data indicate that LE formation is a secondary consequence of early zygotic dorsal patterning signals. In particular, proper LE specification requires the function of genes such as u-shaped and hindsight, which are direct transcriptional targets of the early Decapentaplegic/Screw patterning gradient, to establish a competency zone from which LE arises. It is proposed that subsequent inductive signaling between amnioserosa and dorsal ectoderm restricts the formation of LE to a single row of cells (Stronach, 2001).

Using mutations that influence DV patterning, it is possible to alter the size and distribution of BMP target gene expression patterns, which indicate the extent of amnioserosa and dorsal ectodermal cell fates. If LE fate was specified directly by a particular threshold level of BMP signal, then one would expect LE fate to be perturbed in concert with amnioserosa and dorsal ectoderm fates in DV mutants. Mutations in genes such as dorsal, Toll, brinker and short gastrulation alter the size of BMP target gene expression domains; however, these mutants failed to alter specification of LE fate. Among these genotypes, brk and sog specifically modulate the shape of the BMP signaling gradient in a region where LE fate might arise, yet LE formation in these mutants is fundamentally normal. Furthermore, in dorsalized embryos, LE cells were observed regularly at the boundary between amnioserosa and dorsal ectoderm even when the morphology of these tissues was severely disrupted. Islands of amnioserosa cells within a field of ectoderm were consistently surrounded with a single row of LE cells, independent of the number of amnioserosa cells constituting the island. The converse situation also occurred; again, a single row of LE cells formed at the boundary between the ectoderm and amnioserosa (Stronach, 2001).

DV mutants were also analyzed to determine whether a decrease in BMP signaling activity converts amnioserosa to LE as predicted by a gradient patterning model. A range of ventralizing mutations (cactus, sog, screw, dpp) displaying progressive loss of amnioserosa tissue did not give rise to embryos with an expanded domain of LE cells. In fact, LE cells were not detected in the absence of amnioserosa. No situation was found in which an altered BMP gradient was associated with expanded LE fate, thus the prediction of a direct gradient response model does not explain LE fate specification (Stronach, 2001).

Notably, DV mutant embryos that perturb the BMP gradient, also perturb the expression domains of target genes, including u-shaped and hindsight, without altering LE specification. However, loss of ush and hnt function results in specific and distinct perturbations in LE formation. Thus, the interpretation that LE fate specification is not a direct early response to the BMP gradient is favored, but rather is a secondary consequence of the specification of dorsal fates through the action of BMP target genes like ush and hnt (Stronach, 2001).

Taken together, these results raise the possibility that amnioserosa may be required for LE formation. To address the function of amnioserosa for LE specification, puc enhancer expression was examined in several mutants of the U-shaped class, including u-shaped (ush) and hindsight (hnt). Incidentally, the dorsal expression domains of these genes are directly regulated by DV patterning signals. In these mutant embryos, the amnioserosa tissue is fated normally and begins to differentiate up to stage 11, but then degenerates prematurely. In both ush and hnt mutants, programmed cell death takes place over the course of a few hours, with elimination of amnioserosa cells by stage 13 -- the time when dorsal closure would normally commence. Unexpectedly, different patterns of expression were observed with the puc enhancer in the two mutants. In ush embryos, ß-gal-positive cells were not detected. In contrast, hnt mutant embryos displayed Puc-positive LE cells at the edge of the dorsal ectoderm, albeit with less uniform expression than normally observed. To confirm these observations, the accumulation of dpp mRNA in the LE was examined. Similar to puc enhancer expression, differential expression of dpp was observed in ush versus hnt mutant embryos. ush mutant embryos show a consistent and significant reduction in LE dpp expression, although residual dpp transcripts are seen. dpp expression appears relatively normal in hnt mutant embryos (Stronach, 2001).

In addition to the differential expression of two LE markers in the U-shaped mutants, ectopic expression of LE markers is observed only in hnt mutant embryos. ß-gal-positive cells were observed in the region of the amnioserosa in hnt mutants as early as stage 11, raising the possibility that this could be an example of expanded LE cell fates. These cells adopt only partial LE cell fate, for the following reasons. These cells do not express the LE marker Fasciclin III, but do express two other LE molecules, albeit aberrantly. puc, for example, is expressed precociously in these cells, preceding Fasciclin III expression in the ectoderm, and dpp is rarely but reproducibly expressed. Additionally, cells in this region express amnioserosa fate markers such as race, through stage 11. Thus, based on the possibility that these cells may co-express LE and amnioserosa markers during stage 11, their identity cannot be unequivocally determined. These results may indicate that these cells are of mixed fate. The presence of ectopic LE-like cells in hnt mutant embryos, coupled with the severe reduction of LE fate markers in ush mutants, suggest that the distinction between amnioserosa and LE is a secondary consequence of Hnt and Ush functions, not a direct result of specific BMP signaling thresholds (Stronach, 2001).

If LE cells are specified as a secondary consequence of DV patterning gradients, then what additional mechanisms are at work to define LE as a single row of cells? The data are consistent with several mechanisms. One possibility is that specification of the LE involves the combinatorial action of nested sets of transcriptional regulators, including Hnt dorsally and Ush in a broader domain. Accordingly, loss of Hnt function is predicted to result in a failure to differentiate amnioserosa, coupled with dorsal expansion of more lateral fates, such as the LE. Consistent with this model, hnt mutant embryos display Puc-positive cells with partial LE character in the region of the dying amnioserosa during stage 11. These results suggest that Hnt may be necessary to distinguish amnioserosa from LE fate at the time of extended germ band stage. This timing is late, relative to the timing of the early BMP threshold response, further supporting the notion that LE specification is a secondary consequence of initial BMP signaling (Stronach, 2001).

Ush may play a role in differentiation of more lateral fates adjacent to the amnioserosa and the Hnt expression domain. Indeed, Ush function is essential for LE development because LE does not form in ush mutant embryos. Based on these results, it is imagined that Ush could define a competency zone from which LE cells arise, or Ush could participate in generating or modulating a signal(s) for communication between the differentiating amnioserosa and dorsal ectoderm. Ush is related to mammalian zinc-finger protein family, Friend of GATA (FOG), which has been shown to participate as a cofactor with GATA transcription factors. Together, these protein complexes regulate cell fate determination multiple times during both mammalian and Drosophila development. Interestingly, FOG2, a mammalian homolog of Ush, appears to be required during an inductive signaling event between two distinct tissues in the mouse heart, suggesting that inductive processes in development may commonly use the function of Ush family members. It has not been determined whether the function of Ush in LE cell specification is localized to the amnioserosa, the dorsal ectoderm, or both. Experiments to replace Ush function in a tissue-specific manner should address this issue (Stronach, 2001).

Although transcriptional targets of BMP signaling, such as ush and hnt, among others, define at least three specific threshold responses, the size difference between the nested expression domains of these markers still fails to account for a cell fate defined by a single row of cells. An additional mechanism to explain the spatially restricted stripe of LE cells is through an inductive signaling event. From the analysis of dorsalized mutants, it is observed that LE forms as a result of the juxtaposition of amnioserosa tissue with dorsal ectoderm, which may provide spatially limited activation of the JNK pathway. Thus, restricted expression of JNK target genes, such as puc and dpp may be a direct result of a signal that specifies LE (Stronach, 2001).

Communication between the amnioserosa and the dorsal ectoderm during embryogenesis has been suggested in two cases recently: (1) Hnt expression in the amnioserosa is required nonautonomously for proper cell rearrangements in the dorsal ectoderm, associated with retraction of the embryonic germband; (2) the raw gene product appears to be expressed in the amnioserosa, though it influences the activity of the JNK pathway in the ectoderm during dorsal closure. As amnioserosa and ectoderm develop, they may acquire different cell affinities, which cause them to sort into separate domains or islands (in the case of dorsalized embryos), displaying smooth borders at their interface. A difference in cell adhesion at the boundary may be sufficient to generate signaling for LE specification similar to inductive mechanisms at work at the compartmental boundaries of larval imaginal discs. The challenge now will be to identify molecules that may participate in an inductive signal (Stronach, 2001).

These results suggest that a multistep process determines the LE as a single row of cells. LE does not form directly in response to discrete intermediate levels of BMP signaling activity, but forms secondarily by the action of transcriptional regulators that are themselves BMP target genes. Among these targets, Hnt and Ush define a LE competency zone that is expanded in hnt mutants and eliminated in ush mutants. It is proposed that from within the competency zone, LE fate is further refined to a single row by an unknown inductive signal generated by the physical juxtaposition of amnioserosa with dorsal ectoderm. This signal activates the JNK pathway that regulates localized expression of dpp and puc (Stronach, 2001).

Shark (SH2 domain ankyrin repeat kinase) is a Drosophila nonreceptor tyrosine kinase that contains from amino to carboxyl terminus, a Src homology 2 (SH2) domain (N-SH2), five ankyrin repeats, a second SH2 domain (C-SH2), a proline-rich and basic region, and a tyrosine kinase domain. Analysis of the phenotypes associated with a shark loss-of-function mutation demonstrate that Shark activity is essential for the migration of the dorsolateral epidermis of the embryo during dorsal closure (DC). Shark kinase functions in DC upstream of Dpp expression by leading edge (LE) cells (Fernandez, 2000).

Because no obvious genetic interactions were obtained between Shark and mutations of the JNK pathway, tests were performed to see whether constitutive activation of the JNK or the Dpp pathway could rescue the shark¹ DC phenotype. When shark¹ GLCs were generated in the background of flies carrying a shark¹ chromosome with an inserted transposon expressing an activated form of c-Jun (hs-SEjunAsp), shark¹ DC defects were completely rescued, in some cases, as determined by the decreased penetrance of embryonic lethality (~10% lower than the fully penetrant 50% observed without the expression of hs-SEjunAsp) and by the complete or partial enclosure observed in unhatched embryos. These results are consistent with the action of Shark upstream of bsk (JNK) in the JNK pathway in LE cells (Fernandez, 2000).

Graded maternal short gastrulation protein contributes to embryonic dorsal-ventral patterning by delayed induction

Establishment of the dorsal–ventral (DV) axis of the Drosophila embryo depends on ventral activation of the maternal Toll pathway, which creates a gradient of the NFkappaB/c-rel-related transcription factor Dorsal. Signaling through the maternal BMP pathway also alters the dorsal gradient, probably by regulating degradation of the IkB homologue Cactus. The BMP4 homologue decapentaplegic (dpp) and the BMP antagonist short gastrulation (sog) are expressed by follicle cells during mid-oogenesis, but it is unknown how they affect embryonic patterning following fertilization. This study provides evidence that maternal Sog and Dpp proteins are secreted into the perivitelline space where they remain until early embryogenesis to modulate Cactus degradation, enabling their dual function in patterning the eggshell and embryo. Metalloproteases encoded by tolloid (tld) and tolkin (tok), which cleave Sog, are expressed by follicle cells and are required to generate DV asymmetry in the Dpp signal. Expression of tld and tok is ventrally restricted by the TGF-α ligand encoded by gurken, suggesting that signaling via the EGF receptor pathway may regulate embryonic patterning through two independent mechanisms: by restricting the expression of pipe and thereby activation of Toll signaling and by spatially regulating BMP activity (Carneiro, 2006).

This study has shown that sog, dpp, and tld act during oogenesis to promote the formation of dorsal anterior structures of the eggshell and to establish the embryonic DV axis. According to a proposed model, Sog is produced in follicle cells and is processed into different forms depending on DV location and stored in the perivitelline space. These forms of Sog then persist until early stages of embryogenesis at which time they act by a delayed induction mechanism to alter signaling mediated by maternally derived Dpp. It is proposed that an asymmetric distribution of Sog peptides is produced through the action of the ventrally localized Tld and Tok metalloproteases. Different forms of Sog act locally to inhibit Dpp signaling ventrally (e.g., N-Sog) or diffuse over considerable distances to concentrate Dpp dorsally (e.g., full-length Sog or C-Sog). According to this model, a dorsal-to-ventral gradient of Dpp activity is formed in the perivitelline space that counteracts and sharpens the inverse gradient of nuclear dorsal (Carneiro, 2006).

An important finding in this study is that Sog protein produced by follicle cells is secreted into the perivitelline space where it persists until the end of oogenesis and early embryogenesis, prior to initiation of zygotic sog expression. One way maternal Sog fragments might influence DV patterning in the embryo is to modify zygotic Dpp signaling. However, maternal Dpp signaling is involved in establishing the relative positions of the ventral mesoderm versus the lateral neuroectodermal territories, while zygotic Dpp activity determines the relative positions of dorsal and lateral domains. These distinct phenotypes suggest that maternal Sog acts by modulating the maternal rather than the zygotic component of Dpp signaling (Carneiro, 2006).

This analysis also suggests that the Dpp synthesized by follicle cells is secreted into the perivitelline space and stored there until advanced stages of oogenesis. These maternally synthesized Sog and Dpp proteins may act on the embryo following fertilization when signaling through the Toll pathway is initiated. Several lines of evidence support this hypothesis. (1) Through epistatic analysis, it was shown that maternal Dpp does not act upstream of the Toll receptor. Therefore, genes expressed in the follicle cell epithelium that regulate DV patterning exclusively via the Toll pathway should not be targets of maternal Dpp signaling. Alternatively, undescribed non-Toll mediators of DV patterning could potentially be targets of maternal Dpp in the follicular epithelium. (2) Blocking Tkv receptor function or reducing maternal Dpp activity (by 8xhssog, in follicle cells has no effect on the pattern of pip expression. It has been shown that maternal dpp does not alter grk expression. Thus, no evidence was found that the embryonic effects here described in this study are due to alterations in patterning of the follicular epithelium. (3) Maternal dpp signaling increases the levels of Cactus protein in the embryo by a mechanism that is independent of Toll. Finally, inhibition of Tkv with tkvDN expressed with an early maternal driver alters the embryonic expression domains of ventral and lateral genes such as vnd and snail, which are targets of dorsal activation but not of zygotic BMP signaling. tkvDN expression also alters expression of DV genes in lateralized embryos, which lack dorsal ectoderm and early zygotic dpp expression. In aggregate, these various data support the view that maternal dpp and sog act by delayed induction on the embryo itself. The possibility cannot be ruled out, however, that the embryonic DV phenotypes described in this study result from the combined effects of direct and indirect maternal dpp signaling with the predominant effect being direct (Carneiro, 2006).

Delayed inductive activities have been proposed for a variety of proteins synthesized during oogenesis. For example, activation of the terminal system relies on delayed inductive activity of the secreted product of the torsolike gene (tsl), which is expressed by follicle cells at the two poles of the oocyte and associates with the vitelline membrane. ndl has a dual action on chorion integrity and embryonic patterning. The embryonic patterning function of ndl is thought to be mediated by Nudel protein that is secreted into the perivitelline space where it associates with the embryonic plasma membrane and initiates a proteolytic cascade. It is proposed that Sog and Dpp secreted by follicle cells also serve two roles. First, they contribute to patterning the follicle cell epithelium and chorion, and secondly, they are transferred to and stored in the perivitelline space where it is proposed that they function after fertilization to modify Toll patterning in the embryo (Carneiro, 2006).

During embryogenesis, Sog protein diffuses dorsally from the neuroectoderm and may carry Dpp dorsally in a complex with Tld, Tsg, and Scw, resulting in the generation of peak Dpp activity in the dorsal midline. The spatial distribution of maternal Sog, Dpp, Tld and Tok during oogenesis could also create asymmetric BMP activity. Since tld and tok are expressed only in ventral follicle cells, a ventral-to-dorsal gradient of Sog fragments is likely to be produced. Because cleavage of Sog by Drosophila Tld and Tok is dependent on the amount of Dpp, cleavage of Sog by Tld and Tok should be increased near the source of Dpp, generating an oblique gradient of Sog fragments in the egg chamber. The existence of such a gradient is supported by the greater staining seen in anterior ventral cells with the anti-Sog 8A antiserum during stage 10B. However, greater asymmetry may exist as a result of differential distribution of an array of Sog fragments throughout the egg chamber. Unfortunately, visualization of such asymmetry would be hard to achieve due to limitations in the ability to recognize several fragments by existing Sog antisera (Carneiro, 2006).

The analysis of marked sog− and tld− follicle cell clones suggests that the mobility of Sog fragments in the extracellular compartment may contribute to creating a maternal Dpp activity gradient. Such clones resulted in different Sog staining patterns in the perivitelline space adjacent to the clones depending on where they were located along the DV axis. The staining pattern observed with the 8A antibody suggests that ventrally generated N-Sog cleavage products may be less diffusible than intact Sog or than C-Sog and remain restricted to their site of production. In contrast, full-length Sog and C-Sog fragments appear to diffuse more readily (Carneiro, 2006).

Diffusion of Dpp may also contribute to patterning the eggshell. The expression of dpp in anterior follicle cells is consistent with its role in the formation of dorsal anterior chorionic structures. An anterior-to-posterior gradient of Dpp activity in dorsal regions of the egg chamber is suggested by the Dpp-dependent activation of the A359 enhancer trap and graded repression of bunched along the AP axis. In addition, BR-C expression is lost in mad− clones away from the source of Dpp. sog is likely to contribute to establishing this BMP gradient since ventral sog−clones act non-cell-autonomously to decrease the size of the operculum. Since ventral tld− clones also alter the extent and angle of the operculum, Tld may process Sog to generate a fragment that diffuses and carries Dpp to a dorsal anterior location, concentrating and thus enhancing Dpp activity. Further evidence that a fragment with such activity exists derives from the observation that overexpression of a C-terminal Sog fragment generates chorionic phenotypes that strongly resemble dpp overexpression (Carneiro, 2006).

A dorsally produced form of Sog also appears to participate in patterning the eggshell since sog− clones located dorsally result in fusion of dorsal appendages along the dorsal midline. DV positioning of the dorsal appendages depends on several factors, most critically on EGFR signaling. In contrast, mild overexpression of dpp generates fusion of the dorsal appendages. Considering the well-established role of Sog in modulating Dpp activity, the fused appendage phenotype generated by dorsal sog− clones most likely reflects the loss of Dpp antagonism exerted by Sog (Carneiro, 2006).

In addition to the activities described above, N-Sog fragments which remain ventrally restricted could exert Supersog-like activity, antagonizing BMPs while acquiring resistance to further cleavage and degradation by Tld. This ventrally restricted activity most likely patterns the embryo but does not affect dorsal positioning of eggshell structures, which depends on the combined activity of Dpp/BMPR signaling and dorsally generated Grk/EGFR signals (Carneiro, 2006).

The assortment of Sog fragments in egg chambers is very similar to that in the embryo. Full-length and processed forms of Sog generated by Tld during oogenesis might remain asymmetrically distributed during embryogenesis and exert distinct activities. This hypothesis is in agreement with the effect of tld− and sog− follicle cell clones on the embryo. In the majority of cases, tld− follicle cell clones result in ventralized cuticles, indicating that Tld generates some activity that synergizes with Dpp. Reciprocally, the great majority of sog− follicle cell clones result in dorsalized cuticles and embryos, indicating that Sog primarily acts by antagonizing Dpp. Since only ventral sog− clones generate cuticle defects, ventrally produced Sog presumably generates a ventralizing activity that blocks Dpp locally. In contrast, since in a minority of cases ventral shifts are observed in embryonic gene expression domains resulting from sog− clones, as well as a minority of dorsalized cuticles from tld− clones, there may also be a form of Sog that can enhance Dpp signaling. This positive BMP promoting activity could be generated ventrally, as suggested above in the case of chorion patterning (Carneiro, 2006).

A model depicting the proposed effects of different Sog forms on formation of the chorion and embryonic patterning is presented. According to this model, ventrally restricted Tld cleaves Sog near the Dpp source in ventral anterior follicle cells generating N-Sog and C-Sog. It is suggested that N-Sog fragments remain restricted near ventral anterior cells to antagonize Dpp, while C-Sog fragments diffuse dorsally concentrating Dpp in dorsal anterior cells that direct formation of the operculum. This asymmetric production of Sog molecules would generate a dorsal-to-ventral gradient of Dpp, with the highest levels dorsally near the anterior Dpp source. Although direct visualization of the predicted resulting Dpp gradient in the embryo is hard to achieve with the tools available, it is proposed that such a similarly oriented gradient persists until early embryogenesis based on the asymmetric pattern of Dpp-GFP distribution during late oogenesis and the observed alterations in embryonic gene expression domains resulting from modifications in maternal Dpp signaling (Carneiro, 2006).

The slope of the Dl nuclear gradient ultimately defines the extent of the mesoderm (Mes), neuroectoderm (NE), and dorsal ectoderm (DE). A uniform increase or decrease in nuclear Dl along the DV axis can only alter the extent of the Mes and DE and positioning of the NE, while a change in the slope of the gradient will modify the extent of NE territories such as the vnd expression domain. Under all conditions that Dpp signaling was altered, modifications were observed in the width of the vnd domain. This suggests that graded maternal Dpp signaling helps determine the slope of the dorsal gradient. Earlier studies suggested that Dpp inhibits Cactus degradation and as a consequence decreases Dl translocation into the nucleus. Increased Dpp signaling should result in more Dl retained in the cytoplasm, with consequent narrowing of the mesoderm and ventral shift in lateral and dorsal expression domains. Conversely, inhibition of Dpp signaling would result in increased levels of Dl becoming available for nuclear translocation. Considering the proposal that maternal Dpp is highest dorsally, and that Cactus may also act to prevent Dl diffusion along the DV axis, decreasing Dpp should lower Cactus levels in dorsal–lateral regions of the embryo and result in the redistribution of free Dl from ventral to lateral regions. As a consequence of this redistribution of Dl, there would be a slight decrease in Dl levels ventrally and an increase laterally that would have the net effect of flattening the gradient. Such a mechanism would require a certain degree of mobility of dorsal dimers in the syncytial blastoderm. In future studies, it will be interesting to determine the relative mobilities of Dl/Cactus complexes in the cytoplasm (Carneiro, 2006).

Maternal BMP signaling may also increase the robustness of dorsal patterning. The prevailing view of DV patterning is that signaling through the Toll pathway is sufficient to generate threshold-dependent activation of several dorsal target genes along the entire DV axis. Activation of Toll triggered by the ON/OFF pip expression pattern must be transformed into a ventrally centered gradient of Toll signaling. Several mechanisms may contribute to generate this gradient, based on autoregulatory feedback mechanisms. Although the Toll system may be internally robust, regulatory inputs from other signaling pathways could also contribute further to its stability, such as suggested for the wntD pathway and for maternal Dpp. While a significant body of evidence supports the standard view that establishment of the dorsal gradient through the Toll pathway is central to DV axis specification, the maternal Dpp pathway may constitute an important secondary mechanism that sharpens and ensures robustness and stability of the dorsal gradient in response to a rapidly changing embryonic environment (Carneiro, 2006).

The initiating event in maternal DV patterning is localized activation of the Grk/EGFR pathway in dorsal cells. Grk functions by restricting the expression of both pip and tld/tok, providing two potentially independent means for spatially regulating the activity of Toll and Dpp. This dual action of the Grk/EGFR pathway is consistent with analysis in which it was found that embryonic cuticles from gd−; grk−; Tl[3] mothers displayed a phenotype distinct from those collected from gd−; Tl[3] mothers. While cuticles from both genotypes had denticle belts surrounding the entire circumference of the embryo, cuticles from gd−; grk−; Tl[3] mothers were more elongated than those from gd−; Tl[3] mothers and exhibited a more ventral character. This suggests that grk provides an additional signal for asymmetry downstream or in parallel to gd. It is suggested that the hypothetical system proposed acts downstream of grk/EGFR and in parallel to Toll may be the Dpp pathway (Carneiro, 2006).

Dpp and the abdomen

The adult abdominal epidermis develops during the pupal stage from groups of cells called histoblast nests, which differ from imaginal discs in two important respects: (1) abdominal histoblasts do not invaginate during embryogenesis, but remain part of the larval epidermis and secrete larval cuticle, and (2) they do not proliferate during the larval stages. After pupariation, histoblasts multiply rapidly and migrate to replace the polyploid larval epidermal cells (LEC). As the individual nests grow and merge, LEC are destroyed only upon contact with histoblasts, so that the continuity of the pupal epidermis is maintained at all times. The replacement of LEC by histoblasts is completed by 40-42 hours after puparium formation (APF). The epidermis of each abdominal segment is produced by three bilateral pairs of histoblast nests: the anterior dorsal nests produce the tergite; the posterior dorsal nests form the flexible intertergal cuticle, and the ventral nests produce the sternite and pleura. In addition, a spiracular nest produces a small patch of epidermis around each spiracle. Dorsally, each segment is composed of a sclerotized, pigmented tergite and flexible, unpigmented intertergal cuticle that is normally folded underneath the tergite. All cells in the sternites, pleura and tergites secrete 3-4 trichomes per cell. The wide-based, curved trichomes secreted by pleural cells are distinct from the thinner, straighter sternal and tergal trichomes. In addition, sternites and tergites, but not the pleura, contain arrays of bristles. Dorsoventral patterning is also present within tergites, since the dark pigment band at the posterior edge of each tergite is wider medially than laterally. Some segments deviate from the typical pattern. For example, the first abdominal segment (A1) lacks a sternite. Also, in the male, A7 lacks both a sternite and a tergite, A6 lacks bristles on its sternite, and A5 and A6 have uniformly darkly pigmented tergites (Kopp, 1999 and references).

The adult abdominal epidermis is formed during the first 40-42 hours of pupal development. At pupariation, the abdominal epidermis is composed predominantly of the polyploid LEC, which are easily distinguishable from the much smaller, diploid histoblasts. At this stage, the anterior dorsal histoblast nest (aDHN) contains 13-19 cells; the posterior dorsal nest (pDHN) contains 5-8 cells, and the ventral nest (VHN) contains 9-13 cells. Histoblasts begin to proliferate and migrate to supplant the LEC soon after pupariation. At 18-20 hours APF, the aDHN and pDHN merge to form a single dorsal histoblast nest (DHN). The DHN merges with the VHN and the spiracular anlage between 20 and 28 hours APF. The spiracle, located at the lateral midline, marks the boundary between ventral and dorsal histoblasts, and eventually the boundary between the pleura and the tergite. The fusion of histoblast nests of consecutive segments begins at 28 hours APF and proceeds until 40-42 hours APF, when the formation of a continuous adult epidermis is completed by the fusion of contralateral nests at the dorsal and ventral midlines. Morphological differentiation of the epidermis into sternite, tergite and pleural territories becomes evident shortly thereafter. These regions can be distinguished at 45 hours APF by differences in the shape and arrangement of cells and by the pattern of developing adult muscles (Kopp, 1999).

The origin of the adult wg and dpp patterns can be traced to the early pupal stage. At the time of fusion of the aDHN and pDHN (18 hours APF), wg and dpp expression domains encompass both adult and larval cells, and are limited to the posterior region of the anterior compartment. Within this zone, the patterns of wg and dpp are largely complementary along the DV axis. wg is expressed in a dorsal-posterior sector of the aDHN and the adjacent dorso-lateral LEC, as well as in a ventral sector of the VHN and the adjacent ventro-lateral LEC. wg is not expressed in the dorsal part of the VHN, in the ventral part of the aDHN or in the lateral LEC between the two nests. wg expression is also weak or absent in the LEC near the dorsal and ventral midlines. dpp is expressed in a dorsal sector of the VHN and in a few cells at the dorsal margin of the aDHN. dpp expression is also seen in the lateral LEC between the VHN and aDHN, and in the dorsal LEC between contralateral dorsal nests. The pupal expression of wg evolves from the pattern present in the larva, where wg is expressed in a circumferential stripe along the AP compartment boundary. The early pupal pattern develops by gradual elimination of expression at the ventral, dorsal and lateral midlines. dpp is not expressed in the epidermis of third instar larvae. However, the expression of dpp in the pupa is reminiscent of the embryo, where it is expressed in mid-dorsal and ventro-lateral stripes. Thus, the pupal expression of dpp may reflect some memory of this embryonic pattern (Kopp, 1999 and references).

The patterns of wg and dpp expression established by 18 hours APF are maintained during the subsequent growth of the histoblast nests. At the time of fusion of the VHN and DHN (24-28 hours APF), wg expression is seen in sectors in the ventral third of the VHN and in the dorsal half of the DHN. dpp is expressed in a stripe in the dorsal two-thirds of the VHN and in a group of 30-40 cells at the dorsal DHN margin. dpp expression also extends transiently into the ventral DHN margin; this expression lasts for only a few hours, and encompasses about 15 cells at its peak (Kopp, 1999).

The complementary expression patterns of wg and dpp are retained in the newly formed adult epidermis at 40-42 hours APF. dpp is expressed in a transverse stripe in the presumptive pleura and in a wedge-shaped stripe along the dorsal midline of the tergite. The limits of pleural expression of dpp coincide precisely with the sternite-pleura and tergite-pleura boundaries. wg is expressed in the sternite and in the medial tergite, but is excluded from the dorsal midline. Neither gene is expressed in a large lateral region of the tergite. The expression of wg and dpp remains restricted to the posterior region of the anterior compartment, with sharply defined posterior and graded anterior boundaries. Double labelling with Engrailed shows that the posterior limit of dpp expression coincides with the compartment boundary. Based on morphological landmarks and on the pattern of lacZ expression in wg-lacZ/dpp-lacZ pupae, the same appears to be true for wg (Kopp, 1999).

The division into dorsal tergite, ventral sternite and ventro-lateral pleural cuticle is largely specified during the pupal stage by Wingless, Decapentaplegic and Egf receptor signaling. Expression of wg and dpp is activated at the posterior edge of the anterior compartment by Hedgehog signaling. Within this region, wg and dpp are expressed in domains that are mutually exclusive along the dorso-ventral axis: wg is expressed in the sternite and medio-lateral tergite, whereas dpp expression is confined to the pleura and the dorsal midline. Neither gene is expressed in the lateral tergite. Tergite and sternite cell fates are specified by Wg signaling. Egfr acts synergistically with Wg to promote tergite and sternite identities, and Wg and Egfr activities are opposed by Dpp signaling, which promotes pleural identity. Wg and Dpp interact antagonistically at two levels:(1) their expression is confined to complementary domains by mutual transcriptional repression and (2) Wg and Dpp compete directly with one another by exerting opposite effects on cell fate. Egfr signaling does not affect the expression of wg or dpp, indicating that it interacts with Wg and Dpp at the level of cell fate determination. Within the tergite, the requirements for Wg and Egfr function are roughly complementary: Wg is required mainly in the medial region, whereas Egfr is most important laterally. Dpp signaling at the dorsal midline controls dorso-ventral patterning within the tergite by promoting pigmentation in the medial region (Kopp, 1999).

The major conclusion of this report is that much of the dorso-ventral (DV) patterning of the adult abdomen is determined by antagonistic interactions between Dpp, which specifies pleural cell fate, and Wg and Egfr signaling, which together specify tergite and sternite fates. Expression of wg and dpp is activated at the posterior edge of the anterior compartment by Hh signaling. Within this zone, wg and dpp are expressed in complementary patterns along the DV axis: wg is expressed in the presumptive sternite and in the medio-lateral region of the tergite, whereas dpp is expressed in the presumptive pleura and at the dorsal midline of the tergite. Although the pattern of Egfr activation in the abdomen has not been determined, Egfr signaling is most important in the lateral tergite, a region where neither wg nor dpp are expressed (Kopp, 1999).

Dpp signaling and the dorsolateral peripheral nervous system

Transforming growth factor ß signaling mediated by Decapentaplegic and Screw is known to be involved in defining the border of the ventral neurogenic region in the fruitfly. A second phase of Decapentaplegic signaling occurs in a broad dorsal ectodermal region. The dorsolateral peripheral nervous system forms within the region where this second phase of signaling occurs. Decapentaplegic activity is required for development of many of the dorsal and lateral peripheral nervous system neurons. Double mutant analysis of the Decapentaplegic signaling mediator Schnurri and the inhibitor Brinker indicates that formation of these neurons requires Decapentaplegic signaling, and their absence in the mutant is mediated by a counteracting repression by Brinker. Interestingly, the ventral peripheral neurons that form outside the Decapentaplegic signaling domain depend on Brinker to develop. The role of Decapentaplegic signaling on dorsal and lateral peripheral neurons is strikingly similar to the known role of Transforming growth factor ß signaling in specifying dorsal cell fates of the lateral (later dorsal) nervous system in chordates (Halocythia, zebrafish, Xenopus, chicken and mouse). It points to an evolutionarily conserved mechanism specifying dorsal cell fates in the nervous system of both protostomes and deuterostomes (Rusten, 2002).

The second phase of Dpp signaling, covering most if not all the dorsal ectoderm, starts at stage 9 and lasts until stage 10/11 [3.40 to 5.20 hours after egg laying (AEL)]. Initially proneural clusters (PNCs) and later sensory organ precursors (SOPs), singled out within each PNCs, can be visualized by the expression of the proneural genes achaete (ac, 4.20-7.20 hours AEL), atonal (ato, 5-6.30 hours AEL) and amos (amo, 5.20-6 hours AEL). Thus, the second wave of Dpp signaling precedes and overlaps with the development of the PNCs and SOPs. The domain of Dpp signaling was examined using an enhancer trap lacZ line inserted in the gene daughters against dpp (dad), a target of Dpp. Double immunofluorescence staining shows that dorsally located Ac and Ato positive PNCs and SOPs originate inside the dad-lacZ positive region, suggesting that they have received, or still receive, Dpp signaling. However, a subset of PNCs and SOPs are ventral to the dad-lacZ domain. As the PNS neuronal precursors differentiate close to the position where they originate, it can be concluded that a part of the dorsal PNS forms within an active Dpp signaling region (Rusten, 2002).

The embryonic abdominal (A) PNS of Drosophila consists of three bilateral clusters of neurons (ventral, lateral and dorsal) per segment, which can be most especially appreciated in the serially homologous segments A1-A7. In order to investigate whether the second phase of Dpp signaling is necessary for patterning the PNS, mutant alleles for a gene involved in the Dpp signaling pathway, schnurri (shn), were examined. This gene encodes a zinc-finger transcription factor that is necessary for the transcription of some Dpp target genes and binds directly to the main Dpp mediator Mothers against Dpp (Mad). Unlike the zygotic mutants of dpp, scw, tolloid (tld) or mad, shn mutants have no effect on the initial dpp/scw governed dorsoventral patterning of the blastoderm. They express normally the early Dpp target genes, such as pannier (pnr, stage 7), dpp itself in the dorsal ectoderm (stage 9) and Krüppel (Kr) (which is a marker for the amnioserosa), showing that the dorsal ectoderm is correctly specified. By contrast, several Dpp target genes that are expressed following the second phase of Dpp signaling are affected in shn zygotic mutants: at stage 11, the expression of genes responsive to Dpp signaling, such as dad, pnr, spalt or dpp itself is reduced or lost. Thus, any failures in PNS formation, which are observed in shn mutant embryos, must originate from the second rather than the first phase of Dpp signaling and are likely to be mediated by Shn. PNS malformations were sought in strong shn zygotic mutant embryos using the ubiquitous PNS neuronal marker 22C10. Homozygous shn¹ and shn^k00401 fail to undergo dorsal closure and show severe defects of PNS development. A strong reduction in number of neurons is observed, especially in the dorsal and lateral PNS clusters, although it is difficult to determine exactly which neurons are affected because of the dorsal closure failure. Therefore, another allele, shn^k04412, which does undergo dorsal closure, was also examined. In these embryos, position and identity of PNS neurons could be more clearly assigned. In homozygosity, as well as in transheterozygosity over shn¹, this mutant shows a reduction in the number of dorsal and lateral neurons, similar to the other mutants analyzed. These results are consistent with a role for Shn-mediated Dpp signaling in the formation of the dorsal and lateral PNS (Rusten, 2002).

A different way to interfere with the second phase of Dpp signaling is to express specific inhibitors once the initial dorsoventral patterning is accomplished. Brk is a nuclear protein that negatively regulates Dpp-induced genes and is expressed ventrally in a complementary pattern to Dpp in the embryo. Sog is a secreted protein that can bind to Dpp and inhibit it from signaling, and Supersog (Ssog) is a hyperactive inhibitory fragment of Sog. In order to avoid interference with the first wave of Dpp signaling (stage 5 to 7, 2.10-3.10 hours AEL), brk and ssog were misexpressed from stage 8 (3.10 hours AEL) to interfere with the second phase (stage 9 to 10/11, 3.40-5.20 hours AEL). UAS-brk expression in segments T2-A3, which is driven by the Kr-Gal4 driver, and ubiquitous expression of ssog in the entire embryo, produced using a HS-ssog construct, leads to reduced number of neurons in the dorsal and lateral PNS. The effects are less severe for Ssog misexpression than for UAS-brk misexpression and notable for shn mutations. Approximately 20% of the embryos expressing ubiquitous ssog do not undergo dorsal closure, similar to the phenotype observed when strong alleles of shn are analyzed. The HS-ssog produces a manifest decrease in phosphorylated-Mad (p-Mad) in the dorsal region. This indicates a reduction in Dpp signaling responsible for the phenotype. The residual p-Mad staining observed in some embryos might be the reason why Ssog misexpression leads to less severe effects than UAS-brk misexpression or shn mutations (Rusten, 2002).

In all these mutant backgrounds the dorsal and lateral PNS clusters show a severe reduction in the number of neurons. No major differences are found depending on the neuronal type: the percentage of external sensory organ neurons lost is similar to the loss of neurons in the chordotonal organs. The penetrance of this effect, as measured in the differentiated PNS clusters, varies among abdominal segments. The average reduction in neuronal number ranges from 25% (HS-ssog) to 41% (shn^k00401) in the dorsal cluster and 8% (HS-ssog) to 52% (shn^k00401) in the lateral cluster. By contrast, the ventral cluster is less affected because it shows 2% (HS-ssog) to 18% reduction (shn^k00401). The lateral pentascolopodial organ shows migration defects in these embryos, but the other sensory organs are located in their expected relative positions (Rusten, 2002).

The reduced number of neurons observed in the dorsal and lateral PNS when Dpp signaling is impeded could result from lack of proneural gene expression, which is known to be necessary for PNC and SOP formation. The expression of ato and ac was analyzed to examine the specification of progenitor cell subclasses in mutant backgrounds defective for Dpp signaling. The development of the serially homologous abdominal segments A1 to A7 is similar and very synchronous. Thus, in the wild type, whenever a specific number of PNCs and SOPs appear in one abdominal segment, a similar pattern is observed in the other abdominal segments as well. This is not true for shn^k04412 mutants and for embryos expressing ubiquitous ssog, where the numbers of Ac and Ato positive SOPs and PNCs vary among the abdominal segments. This is consistent with the variably penetrant phenotypes observed in differentiated PNS among abdominal segments. In embryos expressing Kr-Gal4;UAS-brk, loss of Ato- and Ac-positive PNCs and SOPs was observed specifically in the abdominal segments A1-A3 where brk was misexpressed, when compared with abdominal segments A4-A7 that served as an internal reference. The reduced numbers of Ato- and Ac-positive neuronal progenitors appear to result from failure of PNC formation rather than an increase in cell death ratio: apoptosis does not appear to increase in segments expressing brk compared with the other abdominal segments. Taken together, these results suggest that reduction in the number of neurons is produced by failure in proneural gene expression (Rusten, 2002).

Hedgehog and Decapentaplegic instruct polarized growth of cell extensions in the Drosophila trachea

The trachea is a respiratory organ consisting of a network of tubular epithelia that delivers outside air directly to target organs. The tracheal primordium forms six primary branches that migrate towards specific target tissues expressing Branchless (Bnl), a Drosophila homolog of FGF. Bnl activates Breathless (Btl), an FGF receptor, at the tip of primary branches and cell process formation. The dorsal branch (DB) migrates toward the dorsal midline, where it fuses with another DB from the contralateral side. Two specialized cells are present at each DB tip. Fusion cells lead the migration and form anastomoses of tracheal tubules, whereas terminal cells extend a long cell process called a terminal branch. The terminal branch is also present in other branches such as visceral and ganglionic branches, and in all cases spreads over the surface of target tissues and serves as an interface for gas exchange by extending unicellular processes containing a dead-ended lumen, a structure known as the tracheole. Terminal branching in postembryonic stages is regulated by Bnl, which is induced as a hypoxic response. The regulation of terminal branch migration in the CNS uses the same molecules involved in axon guidance. Current knowledge is lacking, however, on the regulation of directed terminal branch growth over the epidermis, as well as the mechanism by which it is positioned over the epidermis to maximize oxygen transfer. The guidance roles of the morphogens Hedgehog and Decapentaplegic during directed outgrowth of cytoplasmic extensions in the Drosophila embryonic trachea was investigated. A subset of tracheal terminal cells adheres to the internal surface of the epidermis and elongates cytoplasmic processes called terminal branches. Hedgehog promotes terminal branch spreading and its extension over the posterior compartment of the epidermis. Decapentaplegic, which is expressed at the onset of terminal branching, restricts dorsal extension of the terminal branch and ensures its monopolar growth. Orthogonal expression of Hedgehog and Decapentaplegic in the epidermis instructs monopolar extension of the terminal branch along the posterior compartment, thereby matching the pattern of airway growth with that of the epidermis (Kato, 2004).

Terminal branches extend numerous cell processes that rapidly and repeatedly extend and retract in many directions. Although cell processes that extend anteriorly and dorsally are unstable, a subset of cell processes that extend ventrally along the posterior (P) segmental compartment becomes selectively stabilized. The behavior of terminal cells in the anterior compartment is strikingly different from that in the P compartment. In the anterior compartment, the number and size of the cell processes are much smaller, suggesting that the P compartment constitutes the preferred substrate for terminal cell spreading (Kato, 2004).

Hh is important for promoting terminal cell spreading. Hh is secreted from the P compartment and forms symmetrical gradients of cellular responses in both the anterior and posterior directions within the epidermis. It is proposed that Hh stimulates the adhesion of terminal cells to the epidermis by activating Ci. Because Hh signaling is submaximal in terminal cells, terminal branch filopodia that extend randomly would be preferentially stabilized near the source of Hh. Thus, terminal cell bodies are placed at the point of highest Hh concentration and terminal branches are stabilized at the apex of the Hh concentration gradient. Because terminal cell growth continues while the level of Hh signaling remains below maximum within terminal cells, terminal branches would be expected to extend along the P compartment (Kato, 2004).

The mechanism(s) of terminal branch guidance by Hh through regulation of Ci-dependent transcription differs from those that guide the behavior of the growth cone, which is primarily regulated at the level of cytoskeletal motility. The latter mechanism has the advantage of maintaining a small cell-surface area receiving guidance cues to minimize the chances of making aberrant connections during synapse formation. Terminal cells, however, use the entire basal cell surface to receive a guidance signal and to stabilize their association with the epidermis. This mechanism of terminal branching by cell spreading meets the physiological requirement that the terminal branch serves as an interface for gas exchange (Kato, 2004).

Hh signaling has been implicated in another cell adhesion-related process, namely cell sorting behavior at the AP compartmental boundary in the wing imaginal disc. Because this behavior is regulated transcriptionally by Ci, there may be a common downstream target of Ci that acts in both wing disc cells and terminal cells (Kato, 2004).

Terminal branch extension is limited to the AP compartmental border, suggesting that there is an additional mechanism that shifts the terminal branch to the anterior side of the P compartment. Bnl was expressed as short stripes in the dorsal epidermis (DE) at the time of terminal branching. It has been reported that mesodermal cells also contribute to correct patterning of dorsal branch (DB). It will be interesting to address guidance functions of those components on terminal branch outgrowth (Kato, 2004).

Dpp is expressed at the dorsal edge of the DE during dorsal closure. This corresponds to the time when terminal branch outgrowth in the DB starts, suggesting that Dpp affects the initial stage of terminal branch outgrowth. Prolonged activation of Dpp signaling in terminal cells by expression of Dpp prevents its elongation. These observations suggest that in normal development Dpp prevents dorsally directed terminal branch extension at the onset of terminal branching, thereby shunting terminal branch extension towards the ventral direction. It is proposed that Dpp converts the initial bipolar shape of a terminal branch into one that is monopolar. Once terminal branch extension is initiated, its direction may be maintained by localized Bnl expression at the AP compartment border. Whether this inhibitory effect of Dpp is mediated by direct signaling to cytoskeletons at the cell periphery or mediated by a nuclear transduction of the signal, remains to be determined (Kato, 2004).

Terminal cells undergo an enormous increase in cell volume and surface area during terminal branching, which continues throughout embryonic and post-embryonic stages. FGF signaling promotes this process in two ways, first by activating the target gene SRF, which is required for terminal branch growth, and second by stimulating rapid filopodial movement. These two FGF signaling effects seem to be independent of Hh and Dpp. The FGF ligand Bnl is expressed in epidermal cells beneath terminal cells during terminal branching. Because this expression is limited to a relatively small region, it is considered unlikely that FGF signaling is sufficient to provide vectorial information for terminal branching. It is suggested that FGF-driven growth and the motility of terminal branches are restricted to the P compartment by Hh signaling, wherein they are further limited by Dpp to establish a monopolar growth pattern (Kato, 2004).

Hh functions as both a cell fate determinant and a guidance molecule for terminal branch extension. But how are these two distinct Hh functions coordinated? Inactivation of Hh after initiation of terminal branching via a temperature shift of hh^ts2 mutant embryos causes a loss of terminal cells, suggesting that maintenance of tracheal cell fate also depends on a late Hh function. Thus, the striped expression of Hh in the epidermis is used simultaneously for epidermal patterning, tracheal cell fate determination and terminal branch guidance, exemplifying a simple strategy to coordinate the patterning of complex organs having multiple tissue types (Kato, 2004).

Proximodistal subdivision of Drosophila legs and wings: the elbow-no ocelli gene complex serves as a mediator of the function of the Wg and Dpp signaling systems

Appendages are thought to have arisen during evolution as outgrowths from the body wall of primitive bilateria. In Drosophila, subsets of body wall cells are set aside as appendage precursors through the action of secreted signaling proteins that direct localized expression of transcription factors. The Drosophila homeodomain protein Distal-less is expressed in the leg primordia and required for formation of legs, but not wings. The homeodomain protein Nubbin is expressed in the wing primordia and required for formation of wings, but not legs. Given that insect legs and wings have a common developmental and evolutionary origin, attempts were made to identify genes that underlie the specification of all appendage primordia. Evidence is presented that the zinc-finger proteins encoded by the elbow and no ocelli genes act in leg and wing primordia to repress body wall-specifying genes and thereby direct appendage formation (Weihe, 2004).

Evidence suggests that the el and noc genes serve as mediators of the function of the Wg and Dpp signaling systems in specification of the appendage field within the imaginal discs. El and Noc are induced by Wg and Dpp and are required to repress the proximally expressed proteins Hth and Tsh. Previous work had identified Dll as a gene required for appendage formation in leg and antenna, and nub as a gene required for wing. This report identifies El and Noc as a pair of zinc-finger proteins that function in both ventral and dorsal appendages. However, there are interesting differences in the way that they do so, when examined in detail (Weihe, 2004).

Dll expression is required for the formation of all leg and antenna elements in the ventral (leg) discs, and until this work Dll was the earliest known marker for the distal region leg disc. Previous work has shown that repression of Hth and Tsh by Dpp and Wg was not required for expression of Dll in the leg, nor could Dll repress Hth and Tsh. Thus an essential mediator of the effects of Wg and Dpp was missing. The current results present evidence that El and Noc serve this function, since their removal leads to ectopic expression of Hth and Tsh. Removal of El and Noc does not cause loss of Dll expression, so it is concluded that Wg and Dpp act independently to induce El and Noc expression and Dll to define the distal region of the leg disc (Weihe, 2004).

The situation differs slightly in the wing. Repression of Tsh is the earliest marker for specification of the distal wing region, preceding the onset of Hth repression or of Nub induction. Loss of Tsh and Hth are required to allow Nub expression. Ectopic expression of Hth and Tsh and loss of Nub is observed in clones lacking El and Noc activity. Thus in the wing, expression of the distal marker Nub cannot be demonstrated to be independent of El and Noc (because ectopic Hth can repress Nub, but not Dll). The vestigial gene is also important for wing development and has been proposed to be a wing specifying gene. However, Vestigial is expressed all along the DV boundary of the wing, both in the wing primordium and in the body wall. This led to the suggestion that while Vestigial is essential for wing development, its expression cannot be taken as a molecular marker for wing identity per se, particularly at early stages. For this reason analysis of the relationship between El, Noc and Vestigial was not performed in this study (Weihe, 2004).

Is the repression of trunk genes needed to specify appendage, as opposed to body wall, in wing and leg discs? In the wing disc the answer appears to be yes; repression of 'trunk genes' like hth is necessary to make the remaining part of the disc competent to form the appendage. However, in the leg the situation is more complex. Coexpression of Dll and Hth does not disrupt proximal-distal axis formation, but leads to homeotic transformation of leg tissue into antennal tissue. Hth is not repressed and limited to proximal areas in the antenna. However, loss of el and noc activities in the leg disc leads to loss of distal leg tissue without any evident transformation into antennal tissue. Thus, El and Noc may regulate the expression of other 'trunk genes', whose restricted expression is required to make the remaining leg and antenna disc competent to form the appendage (Weihe, 2004).

The regional requirements for El and Noc highlight another interesting difference between leg and wing disc development. el noc double mutant cells are excluded from contributing to the tarsal region of the leg but not from contributing to the femur and tibia. Lineage tracing has shown a considerable net flux of cells from the proximal (Tsh-expressing domain) into femur and tibia. While there is no boundary of lineage restriction separating these domains, cells must be able to change from expressing the proximal marker Hth to expressing the distal marker Dll in order to move from one territory to the other. The wing in contrast does not appear to normally exhibit this large net flux of cells from proximal to distal and the el noc double mutant cells are excluded from contributing to the entire wing region. Clonal analysis has suggested that el noc double mutant cells attempt to sort out toward proximal territory, or if that fails, they can be lost from the disc, apparently by sorting out perpendicular to the epithelium. These observations suggest that El and Noc activity may contribute to the production of proximal-distal differences in cell affinities and thereby may help to maintain segregation of these cell populations during development (Weihe, 2004).

Embryonic development of the Drosophila corpus cardiacum

The development of the Drosophila neuroendocrine gland, the corpus cardiacum (CC) was investigated, along with the role of regulatory genes and signaling pathways in CC morphogenesis. CC progenitors segregate from the blastoderm as part of the anterior lip of the ventral furrow. Among the early genetic determinants expressed and required in this domain are the genes giant (gt) and sine oculis (so). During the extended germ band stage, CC progenitor cells form a paired cluster of 6–8 cells sandwiched in between the inner surface of the protocerebrum and the foregut. While flanking the protocerebrum, CC progenitors are in direct contact with the neural precursors that give rise to the pars intercerebralis, the part of the brain whose neurons later innervate the CC. At this stage, the CC progenitors turn on the homeobox gene glass (gl), which is essential for the differentiation of the CC. During germ band retraction, CC progenitors increase in number and migrate posteriorly, passing underneath the brain commissure and attaching themselves to the primordia of the corpora allata (CA). During dorsal closure, the CC and CA move around the anterior aorta to become the ring gland (see Image). Signaling pathways that shape the determination and morphogenesis of the CC are decapentaplegic (dpp) and its antagonist short gastrulation (sog), as well as hedgehog (hh) and heartless (htl; a Drosophila FGFR homolog). Sog is expressed in the midventral domain from where CC progenitors originate, and these cells are completely absent in sog mutants. Dpp and hh are expressed in the anterior visceral head mesoderm and the foregut, respectively; both of these tissues flank the CC. Loss of hh and dpp results in defects in CC proliferation and migration. Htl appears in the somatic mesoderm of the head and trunk. Although mutations of htl do not cause direct effects on the early development of the CC, the later formation of the ring gland is highly abnormal due to the absence of the aorta in these mutants. Defects in the CC are also caused by mutations that severely reduce the protocerebrum, including tailless (tll), suggesting that additional signaling events exist between brain and CC progenitors. The parallels between neuroendocrine development in Drosophila and vertebrates are discussed (De Velasco, 2004).

In the larva, the ring gland forms a large and conspicuous structure located anterior to the brain and connected to the brain by a pair of tracheal branches and the paired nerve of the corpus cardiacum (NCC). Three different glands, the corpus allatum (CA; dorsally), prothoracic gland (laterally), and corpus cardiacum (CC; ventrally) form part of the ring gland. By far, most of its volume is taken up by the prothoracic gland whose cells, the source of ecdysone, grow in size and number as larval development progresses, whereas the cells of the CC remain small and do not appear to proliferate. Both the CC and CA, as well as axons innervating the ring gland, are FasII positive from the late embryonic stage onward. Labeling of the CC is stronger and starts earlier (stage 11) than that of the CA (stage 15), which makes it easy to distinguish between the two structures in the embryo. Another convenient marker of the CC is adipokinetic hormone (AKH), which is expressed exclusively in the CC from late embryonic stages onward (De Velasco, 2004).

The ring gland of the mature embryo is situated posterior to the brain hemispheres. The CC and CA occupy their positions ventral and dorsal to the aorta, respectively. The prothoracic gland cannot yet be recognized as a separate entity, possibly due to the fact that its precursors are small and few in number. Cells of the CC number around eight on each side and are arranged in a U-shape around the floor of the aorta. All cells are spindle shaped and send short processes ventromedially where they meet and form a bundle attached to the ventral wall of the aorta (subaortic processes) (De Velasco, 2004).

Several signaling pathways, notably Shh, BMP, and BMP antagonists, Wnt and FGF, specify the fate map of the head in vertebrates and also control later morphogenetic events shaping head structures. The same signaling pathways are active at multiple stages in Drosophila head development, and the pattern of activity and requirement of these pathways in regard to CC development was therefore investigated (De Velasco, 2004). .

The first signal acting zygotically in the Drosophila head is the BMP homolog Dpp, which forms a dorsoventral gradient across the blastoderm. The homolog of the BMP antagonist Chordin, short gastrulation (Sog), is expressed in the ventral blastoderm, overlapping with the ventral furrow. Loss of sog results in the absence of the CC, while the SNS is still present, which reflects ventral origin of the CC. Sog seems to be the only signal, of those tested, required for CC determination, since mutation of all other pathways does not eliminate the CC but merely effects its size, shape, or location (De Velasco, 2004).

Following its early widespread dorsal expression, Dpp becomes more confined during gastrulation to a narrow mid-dorsal stripe and an anterior cap that corresponds to parts of the anlagen of the esophagus and epipharynx. From this domain segregates the most anterior population of head mesoderm cells that give rise to the visceral muscle of the esophagus and which maintain Dpp expression. The visceral mesoderm of the esophagus flanks both CC and SNS. Loss of Dpp causes absence of the SNS; the CC is still present and expresses AKH but does not migrate posteriorly (De Velasco, 2004).

Activity of the MAPK signaling pathway is widespread in the Drosophila head from gastrulation onward. Beside a wide anterior and posterior domain traversing the lateral and dorsal domain of the head ectoderm, the primordia of the foregut, including the SNS, and head mesoderm show a dynamic MAPK activity. At least two RTKs, EGFR and FGFR/heartless, drive the MAPK pathway in the embryonic head. EGFR is responsible for activation in the ectoderm and foregut. Loss of EGFR causes widespread cell death in the head and the absence of the SNS. The CC is still present, although reduced in size. Activation of MAPK by Heartless (Htl) occurs in a narrow anterior domain of head mesoderm that gives rise to the dorsal pharyngeal muscles. The foregut, SNS, and CC develop rather normally in htl mutants. However, the CC shows variable defects in shape and location, which are most likely due to the absence of the aorta and CA, both of which are derivatives of the dorsal mesoderm, which is defective in htl loss of function and to which the CC is normally attached (De Velasco, 2004).

Dpp signalling orchestrates dorsal closure by regulating cell shape changes both in the amnioserosa and in the epidermis

During the final stages of embryogenesis, the Drosophila embryo exhibits a dorsal hole covered by a simple epithelium of large cells termed the amnioserosa (AS). Dorsal closure is the process whereby this hole is closed through the coordination of cellular activities within both the AS and the epidermis. Genetic analysis has shown that signalling through Jun N-terminal Kinase (JNK) and Decapentaplegic (Dpp), a Drosophila member of the BMP/TGF-β family of secreted factors, controls these activities. JNK activates the expression of dpp in the dorsal-most epidermal cells, and subsequently Dpp acts as a secreted signal to control the elongation of lateral epidermis. This analysis shows that Dpp function not only affects the epidermal cells, but also the AS. Embryos defective in Dpp signalling display defects in AS cell shape changes, specifically in the reduction of their apical surface areas, leading to defective AS contraction. These data also demonstrate that Dpp regulates adhesion between epidermis and AS, and mediates expression of the transcription factor U-shaped in a gradient across both the AS and the epidermis. In summary, this study shows that Dpp plays a crucial role in coordinating the activity of the AS and its interactions with the LE cells during dorsal closure (Fernández, 2007).

Several studies have implicated Dpp signalling in the process of Drosophila dorsal closure. The expression of dpp in the LE cells and the observation that a failure of Dpp signalling leads to defects in the dorso-lateral epidermal cells led to the suggestion that Dpp acts as a secreted factor regulating epidermal morphogenesis during dorsal closure. During dorsal closure zygotic mutant embryos lacking Dpp receptor activity (tkv) display specific defects in the epidermal cells that can be rescued in a tissue autonomous manner. The LE cells fail to elongate properly in a coordinated manner and display aberrant morphologies. Moreover, the LE cells do not organise microtubule bundles in the dorsoventral axis as in the wild-type. The more lateral epidermal cells begin to elongate but eventually this movement fails. Together these defects in Dpp signalling mutants prevent the zipping of the epidermal fronts at both ends, confirming and extending previous suggestions of a role for Dpp signalling in the epidermal cells (Fernández, 2007).

In addition to the defects in the cytoskeleton of the epidermal cells, this study shows that tkv mutant embryos display severe defects in the behaviour of the AS cells that can be rescued by activation of Dpp signalling in a tissue autonomous manner. The importance of the AS for germ band retraction and dorsal closure has been demonstrated by laser ablation experiments and by cell ablation using tissue specific drivers to express toxins in the AS. However, the signals that control the AS movements still remain elusive. The current results show that one of these signals is likely to be Dpp coming for the LE cells. In this regard, it is notable that during vertebrate wound healing, a process similar to dorsal closure, TGFβ elicits a paracrine response in both the epidermis cells adjacent to the LE as well as in the mesenchymal cells underlying the wound (Fernández, 2007).

In addition to tissue autonomous defects in the AS and the epidermis, it was observed that the adherens junctions between these two tissues are defective in tkv mutants as reflected in low levels of Armadillo staining. Consistent with this, PTyr levels, which are largely localised to the adherens junctions, are downregulated in Dpp signalling mutants at the late stages of dorsal closure. Eventually this interface breaks up and the tissues separate from each other resulting in a dorsal hole. It is difficult to clearly distinguish if the defects in the adherens junctional markers are a cause or a consequence of the detachments that we observe between the AS and the epidermis in tkv mutant embryos. However, it is clear that Dpp signalling is involved in maintaining the AS-LE interface integrity. Very recent work (Wada, 2008) has confirmed this possibility showing that Dpp can regulate integrin activity at the interface between the AS and the LE (Fernández, 2007).

Together these results identify several discrete requirements for Dpp signalling during dorsal closure and suggest that Dpp is acting as an orchestrator of morphogenetic movements to ensure that the elongation of the epidermis and the contraction of the AS occur in a coordinated manner. How Dpp signalling controls these morphogenetic processes is not clear? Two studies have shown that Dpp is involved in ensuring the correct architecture of epithelial cells in the wing disc. In this epithelium, tkv mutant cells lose their normal columnar organisation, round up and are extruded from the tissue. Additionally, these cells display abnormal microtubule polarity similar to that of the LE cells of tkv mutants. It is possible that Dpp signalling has an effect on microtubule organisation by activating the localised expression of regulatory proteins, but these targets still remain to be identified (Fernández, 2007).

Previous studies have shown that Dpp signalling acts downstream of JNK signalling during dorsal closure. This conclusion is mainly based on two observations: the expression of dpp in the LE cells during dorsal closure is absent in JNK mutants and ectopic activation of Dpp signalling can rescue the defects of JNK mutants. However, the phenotype of the epidermal cells is different in JNK and Dpp signalling mutants, and more significantly it was shown that the contraction of the AS is not compromised in the absence of JNK signalling. This suggests that Dpp signalling is acting independently of JNK signalling in the AS, and that it is only its later function within the LE that is dependent on JNK activation. This hypothesis is consistent with the observation that only the LE expression of dpp is absent in JNK mutants (Fernández, 2007).

The requirement for Dpp signalling in the AS correlates with earlier patterns of dpp expression, in particular with the broad dorsal epidermal expression in the extended germ band, which is not strongly disrupted in JNK mutants. Cytoskeletal rearrangements of the AS cells are required for germ band retraction and tkv mutants show variable but clear defects in this process. It is possible that early Dpp signalling acts on the AS to regulate germ band retraction and that this early activity also initiates cellular activities that are required later for the contraction of the AS during dorsal closure (Fernández, 2007).

The data suggest a model in which Dpp mediates temporally and spatially separable functions during the process of dorsal closure. The results show that dpp expression at the extended germ band stage is required to regulate the behaviour of the AS, in a JNK independent manner. Subsequently, during dorsal closure stages, JNK drives the expression of dpp at the LE cells, which is necessary for epidermal morphogenesis and appears to contribute to the adhesive integrity of the interface between the LE cells and AS. During the early phase of closure the main force that drives the process seems to be provided by the AS which pulls the epidermal cells in tow. While the epidermis elongates dorsally the AS cells actively constrict their apical surface. In a tkv mutant where the AS cells are not actively constricting the process can be overcome by overexpressing a constitutively active form of Tkv in the epidermis. Also, if the epidermis is maintained mutant and the AS cells express the constitutively active form of the receptor, the dorsal gap is again closed. In both cases closure is achieved at a slower rate and the final pattern is imperfect compared to the wild-type situation. These results suggest that, closure is accomplished by the cooperative and active contribution of both tissues regulated by Dpp signalling. The fact that tkv mutants can be rescued when an activated form of Tkv is expressed either in the AS cells or in the ectodermal cells also suggests that Dpp signalling may not be required in a completely cell autonomous manner. Dpp could act through a relay mechanism in the nearby tissue to induce a diffusible signalling factor required for dorsal closure. However, this possibility seems unlikely as such putative factor and the corresponding signalling cascade have not been identified. An alternative explanation is based on the regulation of adhesion at the LE/AS interface by Dpp; activation of the Dpp pathway on either side of the interface may be sufficient to strengthen the adhesion and rescue the tkv mutant phenotype, at least partially. In any case, Dpp seems to be acting as a coordinator of dorsal closure to ensure that the cell shape changes in the epidermis and in the AS result in the desired final pattern (Fernández, 2007).

Larval and pupal stages (part 1/2)

Dpp and the Eye-antennal Disc

The eye-antennal imaginal discs of Drosophila melanogaster form the head capsule, the eyes and the antenna of the adult fly. Unlike the limb primordia, each eye-antennal disc gives rise to morphologically and functionally distinct structures. As a result, these discs provide an excellent model system for determining how the fates of primordia are specified during development. An investigation has been carried out of how the adjacent primordia of the compound eye and dorsal head vertex are specified. Subdivision of the eye-antennal disc is not based on compartmentalization: this is in contrast to the basis for subdivision in the wing and leg discs. Therefore, selector gene-mediated division of the disc into compartments, mediated by engrailed and invected, as in the wing disc for example, is not likely to be the basis for regionalization within the antennal primordium. Instead, in this region, the genes wingless and orthodenticle are expressed throughout the entire second instar eye-antennal disc, conferring a default fate of dorsal vertex cuticle. Mutations that decrease dpp expression in the eye primordia lead to the formation of severely reduced eyes. Similarly, the loss of otd or wg function in the vertex primordia causes the elimination of dorsal head structures (Royet, 1997).

Transplantation experiments show that the eye primordium occupies most of the posterior half of the eye-antennal disc (the so-called 'eye disc'). The head vertex forms from the dorsomedial region of the disc, while the antenna develops from the anterior half of the disc (the so-called 'antennal disc'). During the early third instar stage (70-80 hours after egg laying), dpp is expressed in a horseshoe-shaped domain along the ventral, posterior and dorsal periphery of the eye disc. Dorsal dpp expression does not extend as far anteriorly as ventral expression, but instead ends at the vertex primordium. At this stage, otd expression covers the vertex primordium and extends along the edge of the antennal disc. The posterior boundary of otd expression in the vertex anlage coincides, approximately, with the anterior boundary of the dpp domain. At the same stage of disc development, wg is expressed in two regions of the eye disc. One region corresponds to the future gena (the lateral part of the head capsule bounded above by the eye) and the other to the head vertex (Royet, 1997).

dpp expression prevents dorsal head development in the eye primordium. Flies homozygous for the dppd-blk allele that reduces dpp activity in the eye primordium greatly reduces the compound eye giving rise to an eye with only a few residual ommatidia. In these mutants the eyes are largely replaced by frons cuticle, which normally appears only on the dorsal areas of the head. This ectopic frons lies between the orbital cuticle and the remaining ommatidia, and to the anterior, between the shingle cuticle and the ommatidia. In other eye loss mutants, such as sine oculis or eyes absent, the eyes are completely lost but are not replaced by ectopic frons. This suggests that dorsal head cuticle does not result simply from loss of the eyes, but is caused instead by loss of dpp function. Clones of Mothers against dpp, coding for a protein involved in transmission of the Dpp signal, likewise transform ommatidia into frons (Royet, 1997).

Activation of decapentaplegic expression in the posterior eye disc eliminates wg and otd expression, thereby permitting eye differentiation. In dppd-blk mutants, the otd domain expands toward the anlagen of the shingle cuticle and the compound eyes, consistent with the location of ectopic frons cuticle on dppd-blk mutant heads. wg expression also expands in these mutant discs. Ectopic activation of the wingless pathway (the result of the generation of clones mutant for shaggy/zeste-white 3) in the eye primordium induces otd expression and vertex formation. Loss of shaggy function results in constitutively activated wg signaling and ectopic otd expression. This suggests that otd expression in the vertex primordium is normally activated or maintained by wingless. Early activation of dpp depends on hedgehog expression in the eye anlage prior to morphogenetic furrow formation. Loss of hh activity during the second instar larval stage eliminates dpp expression along the posterior and lateral margins of the eye disc and in the antennal primordium. This loss of dpp expression is associated with a dramatic expansion of the otd expression domain. wg expression also expands into the eye primordium (Royet, 1997).

Unlike the limb discs, which derive from single trunk segments, each eye-antennal disc arises from multiple embryonic head segments. Divisions between segment primordia within the disc could contribute to certain aspects of regional specification. It is proposed that wg and otd expression in the eye-antennal discs are inherited from the embryo, where the two genes are expressed in segments from which these discs are derived. The almost ubiquitous expression of these two genes serves to program the early disc for a vertex fate. Later, hh expression in the posterior region of the future eye disc induces dpp expression along the margins of the eye primordium. dpp represses wg, permitting the formation of the eye primordium (Royet, 1997).

Development of the Drosophila retina occurs asynchronously. The leading edge of differentiation, its front marked by the morphogenetic furrow, progresses across the eye disc epithelium over a 2 day period. The mechanism by which this front advances suggest that developing retinal cells behind the furrow drive the progression of morphogenesis utilizing the products of the hedgehog and decapentaplegic genes. Analysis of hh and dpp genetic mosaics indicates that the products of these genes act as diffusible signals in this process. Expression of dpp in the morphogenetic furrow is closely correlated with the progression of the furrow under a variety of conditions. HH, synthesized by differentiating cells, induces the expression of dpp, which appears to be a primary mediator of furrow movement (Heberlein, 1993).

Pattern formation in the eye imaginal disc is initiated at the posterior edge and moves in a wave toward the anterior; the front of this wave is called the morphogenetic furrow (MF). DPP is required for proliferation and initiation of pattern formation at the posterior edge of the eye disc. It has also been suggested that DPP is the principal mediator of Hedgehog function in driving progression of the MF across the disc. This paper shows that ectopic DPP expression is sufficient to induce a duplicated eye disc with normal shape, MF progression, neuronal cluster formation and direction of axon outgrowth. Induction of ectopic eye development occurs preferentially along the anterior margin of the eye disc. Ectopic DPP expressing clones, situated away from the margins, induce neither proliferation nor patterning. The DPP signaling pathway is shown to be under tight transcriptional and post-transcriptional control within different spatial domains in the developing eye disc. DPP expressing clones located in the middle of the eye disc (i.e. in a region competent to induce dpp and morphogenetic furrows in response to ectopic HH) neither induce ectopic morphogenetic furrows nor endogeneous dpp. DPP positively controls its own expression, as evidenced by the absence of DPP expression in Mad mutant clones. Also, DPP suppresses wingless transcription. In contrast to the wing disc, DPP does not appear to be the principal mediator of Hedgehog function in the eye. Whereas eye tissue away from the margins can respond to HH, it is not competent to respond to ectopic DPP. DPP, WG and HH control proliferation and patterning in essentially all imaginal discs studied. However, their relationship to one another differs from tissue to tissue. In contrast to the developing wing, in the eye disc DPP does not fall strictly under the control of HH, nor is it the principal mediator of HH function. The antagonistic relationship between DPP and WG in the eye disc is similar to that in the leg, however it differs from that in the wing disc (Pignoni, 1997).

Decapentaplegic is expressed at the disc's posterior margin prior to initiation. Under the control of hh, it is expressed in the furrow, during MF progression. While dpp has been implicated in eye disc growth and morphogenesis, its precise role in retinal differentiation has not been determined. To address the role of dpp in initiation and progression of retinal differentiation, the consequences of reduced and increased dpp function have been analyzed during eye development. dpp is not only required for normal MF initiation, but is sufficient to induce ectopic initiation of differentiation. Inappropriate initiation is normally inhibited by wingless. Loss of dpp function is accompanied by expansion of wg expression, while increased dpp function leads to loss of wg transcription. In addition, dpp is required to maintain, and sufficient to induce, its own expression along the disc's margins. It is thought that dpp autoregulation and dpp-mediated inhibition of wg expression are required for the coordinated regulation of furrow initiation and progression. In the later stages of retinal differentiation, reduction of dpp function leads to an arrest in MF progression (Chanut, 1997a).

The progression of retinal morphogenesis in the Drosophila eye is controlled to a large extent by Hedgehog (HH), a signaling protein emanating from differentiating photoreceptor cells. Adjacent, more anterior cells in the morphogenetic furrow respond to HH by expressing dpp, suggesting that the relationship between HH and DPP might be similar to that in the limb imaginal discs where DPP mediates the organizing activity of HH. This study contradicts that suggestion. Analysis of somatic clones of cells lacking the DPP receptors Punt or Tkv reveals that DPP plays only a minor role in furrow progression and no critical role in subsequent ommatidial development. Within tkv and punt clones traversing the furrow at the time of dissection, neuronal differentiation, as shown by ELAV staining, is somewhat retarded, especially in the middle of large clones. The function of DPP in this context must be nonessential or redundant as the furrow is only slightly slowed, but not stopped. Normal ommatidial development occurs in the complete absence of DPP. In contrast, HH-independent dpp expression around the posterior and lateral margins of the first and second instar eye discs is important for the growth of the eye disc and for initiation of the morphogenetic furrow at these margins. Tkv and Punt are absolutely required for cell proliferation in the early developing eye imaginal disc. tkv clones are severly restricted in their ability to grow, implying a strong requirement for the DPP signal for cell proliferation in the early eye disc. There is a posterior requirement for punt function in eye development, which suggests a role for DPP signaling in the initiation of the furrow at the posterior margin Adult eyes containing predominantly punt mutant tissue are regularly observed, but such eyes always have some wild-type tissue at the posterior margin. Both punt and tkv clones cause local overproliferation and block neural differentiation. The tissue in these marginal clones must die, as loss of head cuticle and eye structures is observed in eyes containing mutant clones (Burke, 1996).

The posteriorly expressed signaling molecules Hedgehog and Decapentaplegic drive photoreceptor differentiation in the Drosophila eye disc, while at the anterior lateral margins Wingless expression blocks ectopic differentiation. Mutations in axin prevent photoreceptor differentiation and leads to tissue overgrowth; both these effects are due to ectopic activation of the Wingless pathway. In addition, ectopic Wingless signaling causes posterior cells to take on an anterior identity, reorienting the direction of morphogenetic furrow progression in neighboring wild-type cells. Signaling by Dpp and Hh normally blocks the posterior expression of anterior markers such as Eyeless. Wingless signaling is not required to maintain anterior Eyeless expression and in combination with Dpp signaling can promote Ey downregulation, suggesting that additional molecules contribute to anterior identity. Along the dorsoventral axis of the eye disc, Wingless signaling is sufficient to promote dorsal expression of the Iroquois gene mirror, even in the absence of the upstream factor pannier. However, Wingless signaling does not lead to ventral mirror expression, implying the existence of ventral repressors (Lee, 2001).

Loss of axin function at the posterior margin results in outgrowths from the disc, over-riding the normal control of organ size. axin mutant clones also form smooth borders with surrounding cells, suggesting that their ability to adhere to