Table of contents

Targets of DPP in the eye

decapentaplegic mediates the effects of hedgehog in tissue patterning by regulating the expression of tissue-specific genes. In the eye disc, the transcription factors eyeless, eyes absent, sine oculis and dachshund participate with these signaling molecules in a complex regulatory network that results in the initiation of eye development. Analysis of functional relationships in the early eye disc indicates that hh and dpp play no role in regulating ey, but are required for eya, so and dac expression. Ey is expressed throughout the eye portion of the wild-type eye disc during early larval stages, prior to MF initiation. Eya and Dac are expressed throughout the posterior half of the eye imaginal disc, with stronger expression at the posterior margin. Ey is expressed normally in homozygous Mad1-2 clones that touch the posterior margin and in clones that are positioned internally in the disc, indicating that Dpp signaling is not required for Ey expression prior to MF initiation. In contrast, neither Eya nor Dac is expressed in homozygous Mad1-2 clones that touch the margin of the eye disc. In addition, Eya and Dac are not expressed, or are expressed weakly, in internal clones that lie well anterior of the posterior margin. However, strong Eya and Dac expression is observed in internal clones that lie within a few cell diameters of the posterior margin. Like Eya and Dac protein, SO mRNA is expressed in the posterior region of the eye disc prior to MF initiation. Mad1-2 posterior margin clones fail to express so. These results suggest that dpp function is required to induce or maintain Eya, SO and Dac expression, but not Ey expression, at the posterior margin prior to MF initiation. This function is consistent with the pattern of DPP mRNA expression along the posterior and lateral margins at this stage of eye disc development. Whereas dpp is not necessary for Eya and Dac expression in internal, posterior regions of the early eye disc, it does play a role in regulating Eya and Dac expression in internal, anterior regions of the disc. Although DPP mRNA expression does not extend to the very center of the eye disc, it is expressed in a significant proportion of the interior of the disc. The possibility that dpp may regulate gene expression in more central regions may be attributed to the fact that it encodes a diffusible molecule (Curtiss, 2000).

Restoring expression of eya in loss-of-function dpp mutant backgrounds is sufficient to induce so and dac expression and to rescue eye development. Thus, once expressed, eya can carry out its functions in the absence of dpp. These experiments indicate that dpp functions downstream of or in parallel with ey, but upstream of eya, so and dac. Additional control is provided by a feedback loop that maintains expression of eya and so and includes dpp. The fact that exogenous overexpression of ey, eya, so and dac interferes with wild-type eye development demonstrates the importance of such a complicated mechanism for maintaining proper levels of these factors during early eye development. Whereas initiation of eye development fails in either Hh or Dpp signaling mutants, the subsequent progression of the morphogenetic furrow is only slowed down. However, clones that are simultaneously mutant for Hh and Dpp signaling components completely block furrow progression and eye differentiation, suggesting that Hh and Dpp serve partially redundant functions in this process. Interestingly, furrow-associated expression of eya, so and dac is not affected by double mutant tissue, suggesting that some other factor(s) regulates their expression during furrow progression (Curtiss, 2000).

The lack of eya, so and dac expression in Mad1-2 clones that lie at the margins of the eye disc prior to MF initiation reflects a role for dpp in controlling early eye gene expression at these stages of eye development. Evidence from several studies suggests that ey acts together with dpp at or near the top of the hierarchy: (1) ey expression is not regulated by dpp; (2) ey and dpp are both required for eya, so and dac expression prior to MF initiation; (3) ey is not capable of rescuing dppblk eye development or of inducing ectopic eyes in regions of imaginal discs in which dpp is not already expressed. These observations suggest that ey functions upstream of or in parallel with dpp. The possibility that ey is responsible for dpp expression, leading indirectly to eya, so and dac expression, is unlikely. Since ey cannot induce ectopic eyes without a source of dpp, it probably cannot induce dpp expression, at least not in the absence of factors that are specific to the eye disc. Moreover, Ey protein binds to the regulatory region of so, suggesting it is directly involved in so regulation. Thus, it is likely that ey and dpp cooperate to induce expression of the other early eye genes (Curtiss, 2000).

Such cooperation could achieve two ends. (1) ey is expressed throughout the eye disc and from embryonic stages of development through MF initiation. However, induction of eya, so and dac expression and MF initiation occurs approximately 48 hours later, around the time of the transition between second and third instars. Moreover, eya, so and dac are not expressed throughout the eye disc as ey is, but have stronger levels of expression around the margins than in other regions. The initiation of dpp expression at the posterior margin at approximately the same time suggests that it could be the spatiotemporal signal that sets the MF in motion. (2) dpp induces expression of tissue-specific genes as part of its role in patterning many diverse structures in Drosophila. An interaction with ey could be essential to ensuring that in the eye imaginal disc dpp initiates factors that are appropriate to eye development, such as eya, so and dac (Curtiss, 2000).

Loss of DPP signaling in the Drosophila eye can lead to ectopic expression of wingless, suggesting that Mothers against dpp negatively regulates wingless transcription. Mutant Mad clones are found to express wingless in eye imaginal discs. Similarly, bifurcations of antennae are associated with mutant Mad clone. Such clones express wg and are overgrown when located in the dorsal region of the antennal disc. Thus the antagonistic effect of DPP signaling in wg expression is also observed in other discs and might be a general mechanism during Drosophila imaginal disc development (Wiersdorff, 1996).

During eye development in Drosophila, cell cycle progression is coordinated with differentiation. Prior to differentiation, cells arrest in G1 phase anterior to and within the morphogenetic furrow. Decapentaplegic is required to establish this G1 arrest, since Dpp-unresponsive cells located in the anterior half of the morphogenetic furrow show ectopic S phases and ectopic expression of the cell cycle regulators Cyclins A, E and B. Conversely, ubiquitous over-expression of Dpp in the eye imaginal disc transiently inhibits S phase without affecting Cyclin E or Cyclin A abundance. This Dpp-mediated inhibition of S phase occurs independent of the Cyclin A inhibitor Roughex and of the expression of Dacapo, a Cyclin E-Cdk2 inhibitor. Furthermore, Dpp-signaling genes interact genetically with a hypomorphic cyclin E allele. Taken together these results suggest that Dpp acts to induce G1 arrest in the anterior part of the morphogenetic furrow by a novel inhibitory mechanism. In addition, these results provide evidence for a Dpp-independent mechanism that acts in the posterior part of the morphogenetic furrow to maintain G1 arrest (Horsfield, 1998).

It has been suggested that Dpp may lead to G1 arrest within the morphogenetic furrow (MF) by regulating the G2 to M phase transition in cells anterior to the MF (Penton, 1997). This study showed that tkv, sax or shn mutant clones, which cannot respond to Dpp, fail to arrest in G1 and continue to express the G2 marker Cyclin B within the MF. This implies that these cells are delayed in G2 and it was proposed that Dpp has a role in inducing mitosis anterior to the MF (Penton, 1997). However, this conclusion contrasts with the established role of mammalian TGF-beta in G1 arrest, which acts by inducing G1 cyclin-Cdk inhibitors. Furthermore, the expression of other cell cycle markers in these Dpp-unresponsive clones was not examined, so it could not be concluded whether this apparent G2 block was the only cell cycle defect caused by the absence of Dpp signaling. In the present study, G1/S phase markers were examined as well as G2-M phase markers in Dpp-unresponsive clones and the effect of ectopic over-expression of Dpp on S phases was determined in eye discs. Contrary to previous conclusions (Penton, 1997), the current results provide evidence that Dpp induces G1 arrest, rather than promoting the G2 to M phase progression, in the MF during eye development (Horsfield, 1998).

Dpp has a proliferative role in first and second instar eye discs, and in wing and leg discs. How can the proliferative function of Dpp be reconciled with its role in G1 arrest in the third instar eye disc? One possibility is that Dpp acts through cell cycle regulators that are expressed or activated only when the eye disc begins to differentiate. This would permit Dpp to act as a negative regulator of the cell cycle in a tissue- or temporal-specific manner. This tissue-specific cell cycle regulation may also be present in antennal discs, since ubiquitous over-expression of Dpp also inhibits S phases in antennal discs, but not in other imaginal discs or brain lobes. Tissue-specificity is also exhibited by mammalian TGF-beta, which causes G1 arrest in epithelial cells but induces proliferation in other tissues (Horsfield, 1998 and references).

Taken together these results suggest that Dpp triggers G1 arrest in the MF, but it is not clear how this arrest takes place. Dpp-mediated G1 arrest occurs downstream of Cyclin E or Cyclin A protein accumulation. Given its similarity to TGF-beta, it is possible that Dpp induces G1 arrest by leading to the inhibition of Cyclin E-Cdk2 activity by the induction of a p27 Cdk inhibitor. However, it is unlikely that Dpp-mediated G1 arrest involves the Drosophila p21/p27 homolog, Dacapo, since Dacapo expression is only detected in the posterior region of the MF and in differentiating cells. Ectopic Dpp can inhibit S phases in cells where Dacapo expression is undectable. Furthermore, the Cyclin A inhibitor Roughex is not involved in Dpp-mediated G1 arrest. This study proposes a model for G1 arrest and differentiation during eye development. It is proposed that Dpp acts by inducing a novel inhibitor that abrogates Cyclin E-Cdk2 function, leading to G1 arrest within the MF. Although dpp is expressed within the MF, Dpp could diffuse more anteriorly where it may act upon G1 cells approaching the MF in the asynchronously dividing region. This mechanism alone would result in a higher proportion of cells at later stages of the cell cycle, perhaps accounting for the higher number of mitoses observed immediately anterior to the MF. Since Dpp-unresponsive cells eventually arrest in G1 in the posterior part of the MF, it appears that a Dpp-independent mechanism operates in this region to maintain cells in G1. This mechanism may involve the Cyclin E-Cdk2 inhibitor, Dacapo, which is expressed in differentiating cells in the posterior part of the MF or another unidentified G1-S inhibitor. The factor that induces expression of this proposed G1-S inhibitor is also unknown. The Cyclin A inhibitor Rux, also acts throughout the MF to prevent the inappropriate activation of Cyclin A-Cdk1. It is significant that there appear to be at least three G1-arrest mechanisms operating within the MF. Synchronization of cells in G1 may be a prerequisite for cells to correctly receive and respond to neural differentiation signals in a coordinated manner. The importance of this G1 arrest is stressed by the aberrant retinal patterning that occurs when G1 arrest is disrupted. In conclusion, evidence has been provided that dpp plays an important role in mediating G1 arrest in the anterior part of the MF. In addition, it appears that a Dpp-independent mechanism acts in the posterior part of the MF to maintain cells in G1. Understanding how these controls are integrated to direct eye development may prove to be the key to uncovering the link between tissue patterning and cell cycle regulation (Horsfield, 1998 and references).

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

Retinal cell fate determination in Drosophila is controlled by an interactive network of retinal determination (RD) genes, including eyeless, eyes absent, sine oculis and dachshund. The role of decapentaplegic in this pathway was investigated. During eye development, while eyeless transcription does not depend on dpp activity, the expression of eyes absent, sine oculis and dachshund are greatly reduced in a dpp mutant background. dpp signaling acts synergistically with, and at multiple levels within, the retinal determination network to induce eyes absent, sine oculis and dachshund expression and ectopic eye formation. These results suggest a mechanism by which a general patterning signal such as Decapentaplegic cooperates reiteratively with tissue-specific factors to determine distinct cell fates during development (Chen, 1999).

During ectopic photoreceptor determination there is a tight correlation between the location of ectopic eyes and the endogenous pattern of dpp expression. In particular, the dpp-GAL4 driver is the most efficient means of retinal induction by any of the RD genes: ubiquitous eyeless (ey) expression induces downstream genes only in the vicinity of the anteroposterior (AP) compartment boundary of discs where dpp is normally expressed. These results suggested that dpp signaling may be essential for the RD genes to specify retinal cell fates. dpp is normally expressed along the AP boundary of the larval wing disc. The GAL4 line 30A drives gene expression in a ring that surrounds the wing pouch, which will become the wing blade in the adult. The 30A ring pattern corresponds to tissue that will form the hinge of the adult wing and overlaps endogenous dpp at only two spots. When ey is misexpressed using 30A-GAL4, ectopic eye formation is induced only at two positions: dorsal and ventral to the pouch at the AP boundary. One explanation for this phenomenon is that dpp activity is essential for ey to induce ectopic eye development. Coexpression of dpp and ey is sufficient to expand the domain of ectopic retinal development induced by ey alone. To test whether dpp and ey act synergistically to induce RD genes, mRNA levels of ey, eya, so and dac were measured in a dpp loss-of-function background. ey is normally expressed throughout the entire eye disc prior to MF initiation and anterior to the furrow during MF progression. In dpp mutants, the eye-antennal disc is much smaller than in wild-type due to a proliferation defect, and MF initiation and photoreceptor development does not occur. Nevertheless, EY mRNA is still detectable in dpp mutant eye discs throughout second and third instar larval development. In contrast, although eya is still expressed in the ocellar region, almost no EYA, SO or DAC mRNA is detected in dpp mutant eye discs prepared from second or third instar larvae. These data indicate that dpp is not essential for ey expression but is required upstream of eya, so and dac in the eye disc (Chen, 1999).

If eya and dac are the primary downstream targets of dpp during eye development, then it should be possible to bypass the requirement for dpp and induce ectopic eye formation by overexpressing ey with eya or dac. While targeted expression of either eya or dac alone driven by 30A-GAL4 is unable to induce photoreceptor development, strong synergistic induction of ectopic eye formation is observed when ey is coexpressed with either dac or eya. Although there is clear synergy between ey and dac or eya, ectopic photoreceptor induction in both imaginal discs and adults is still limited to the vicinity of the AP boundary and the source of dpp signaling. Moreover, photoreceptor differentiation is still restricted to the vicinity of the AP boundary when ey, dac, eya and so are simultaneously induced by 30A-GAL4, indicating that dpp and ey must regulate other essential targets in this process (Chen, 1999).

It is possible that dpp signaling might cooperate directly and exclusively with ey. Alternatively, dpp could interact at multiple levels within this pathway. To distinguish these two models, a test was performed to see whether dpp functions synergistically with eya and so to regulate the expression of dac. No ectopic dac expression is induced by so alone: targeted expression of eya induces ectopic dac expression only at a single ventral spot on the AP boundary of the wing disc when driven by 30A-GAL4. Consistent with the idea that the Eya and So proteins function cooperatively as a complex, strong synergistic induction of dac is observed when eya and so are coexpressed. However, dac expression is still restricted mainly to places where endogenous dpp is present. In contrast, when dpp is coexpressed with eya, strong dac expression is induced all along the ventral-posterior pouch margin Moreover, ectopic Dac is detected around the entire circumference of the wing pouch as a result of dpp, eya and so coexpression. Since coexpression of dpp, eya and so is sufficient to induce dac expression in places where dpp and ey cannot, it is concluded that dpp interacts with the network at multiple levels to control the expression of retinal determination genes. Consistent with this interpretation, no induction of ey transcription could be detected in response to misexpression of dpp, eya and so with 30A-GAL4 (Chen, 1999).

Thus dpp signaling is reiteratively used to regulate gene expression within the retinal cell fate determination pathway in Drosophila. Specifically, dpp signaling enables ey to induce strong eya, so and dac expression in the posterior, but not anterior, wing disc compartment. In contrast, dpp functions synergistically with eya and so to activate the expression of dac in both compartments. This activation of dac expression by dpp, eya and so is unlikely to result from feedback induction of ey for two reasons: (1) targeted expression of ey and dpp is unable to induce dac in the anterior wing disc compartment, and (2) ectopic ey transcription is not detected in response to misexpression of dpp, eya and so driven by 30A-GAL4 in the wing disc. Thus, these data suggest that dpp signaling interacts with the retinal determination pathway at (at least) two levels to regulate RD gene expression. Interestingly, while targeted expression of dpp, eya and so with 30A-GAL4 is unable to induce ey expression or ectopic photoreceptor development in the wing disc, coexpression of eya and so using dpp-GAL4 is sufficient to induce ey expression and photoreceptor development in the antennal disc. These differences most likely reflect the unique transcriptional environments present in the specific portions of each imaginal disc tested in these assays (Chen, 1999).

The Drosophila eye disc is a sac of single layer epithelium with two opposing sides: the peripodial membrane (PM) and the disc proper (DP). Retinal morphogenesis is organized by Notch signaling at the dorsoventral (DV) boundary in the DP. Functions of the PM in coordinating growth and patterning of the DP are unknown. The secreted proteins Hedgehog, Wingless, and Decapentaplegic are expressed in the PM. From there they control DP expression of the Notch ligands Delta and Serrate. Peripodial clones expressing Hedgehog induce Serrate in the DP while loss of peripodial Hedgehog disrupts disc growth. Furthermore, PM cells extend cellular processes to the DP. Therefore, peripodial signaling is critical for eye pattern formation and may be mediated by peripodial processes (Cho, 2000).

Restricted localization of Hh-, Wg-, and Dpp-LacZ+ expressing cells along the DV axis in L1 discs suggests that these signals might act upstream of N. To test this idea, Hh activity was removed using a temperature-sensitive allele; Wg was ectopically expressed using hs-wg, or Dpp activity was removed by using a heteroallelic combination of the two dpp alleles, and then the expression patterns of the N ligands Dl and Ser were visualized in the eye discs. In L2 wild-type discs, Dl is preferentially expressed in the dorsal domain, while Ser is enriched along the DV midline of the DP. Both Dl and Ser are also present in the PM at a low level. In hhts2 mutants shifted to restrictive temperature during the early L1 stage, both Dl and Ser are uniformly expressed in dorsal and ventral domains. Ubiquitous Wg overexpression causes variable defects in Dl pattern such as significant reduction in the dorsal domain except near the margin or mislocalization to the ventral domain. Wg overexpression also causes mislocalization of Ser to the dorsal DP. dppe12/dppd14 mutant discs showed similar disruption of the DV-specific Dl and Ser pattern, indicating the necessity of Hh, Wg, and Dpp in DV patterning (Cho, 2000).

The complex interplay of Hh, Wg, and Dpp signaling has been studied for initiation and progression of the morphogenetic furrow. This study has examined much earlier stages of eye development to determine whether these same molecules organize DV patterning prior to retinal differentiation; it has been demonstrated that: (1) Hh, Wg, and Dpp display distinct DV expression patterns in the PM in early discs; (2) their signals are essential for domain-specific expression of Dl and Ser in the DP, and (3) signaling from the PM to DP is important for patterning in the DP. These findings provide a novel view of how eye discs are patterned, a model suggesting Hh, Wg, and Dpp signal from the PM to the DP by means of cellular processes (Cho, 2000).

Soon after the embryo hatches, wg- and dpp-LacZ+ cells appear in the dorsal and ventral domains of the disc, respectively. This suggests that the eye disc is already subdivided into dorsal and ventral fates. Consistent with this data, analyses of genetic mosaics have indicated that the eye disc consists of dorsal and ventral compartments of different cell lineages and of different cell affinities. Subsequent to the initial appearance of wg- and dpp-LacZ+ cells, these two types of cells are juxtaposed in the DV midline of PM and seem to be mutually exclusive in later stages. Such an antagonistic interaction between Wg and Dpp is a common theme that has emerged from studies in limb disc patterning and may play a crucial role in defining domains in the PM (Cho, 2000).

In addition to DV subdivisions, the dorsal domain of L1 discs appears to be further divided into anterior-posterior subdomains. This is based on the expression of Wg in the anterior but not in the posterior dorsal domain, while Dpp is expressed in the opposite pattern. The anterior and posterior subdomains may correspond to the anlage for the head and the dorsal eye, respectively. It has been shown that Wg expressed in the vertex and gena primordia is important for head capsule formation, while Dpp is antagonistic to this process. Interestingly, many new types of wg-LacZ+ PM cells appear during the L2 stage and occupy either DV midline or anterior dorsal domain. Perhaps some of these wg-LacZ+ PM cells may play important roles in specifying head fate of the anterior dorsal domain (Cho, 2000).

The PM is an important source of inductive signals to control cell fates within the DP. According to the presented model, Hh acts differentially to localize Wg- and Dpp-expressing cells to the dorsal and ventral domains of the PM, respectively, in the L1 disc. Establishment of DV domains in the PM governs subsequent signaling from the PM to the DP for controlling the DV specificity and the level of Dl/Ser expression. This idea is supported by observations that ectopic Hh expression in the PM cells can induce Ser expression in the DP, consistent with spatiotemporal correlation of Hh and Ser expression pattern in the L1 and L2 discs (Cho, 2000).

Although this study has focused on signaling from the PM to the DP, the signaling may be bidirectional. It is conceivable that the extension of peripodial processes may depend on specific signaling cues provided from the DP. Such bidirectional signaling may be essential to coordinate DV boundary formation and disc growth in both layers. Whether the signaling molecules that are transferred from the PM cells are Hh/Wg/Dpp themselves and/or other molecules remains unanswered. Interestingly, Patched (Ptc), the receptor for Hh that is known to be upregulated transcriptionally by Hh signaling, is expressed in the DP but is more abundant in the PM. This suggests that Hh signaling may occur laterally and vertically, within the PM layer as well as between the two layers (Cho, 2000).

It has been shown that ectopic Hh+ clones generated anterior to the furrow induce ectopic furrow and retinal differentiation. Evidence presented here suggests that hh- or ectopic Hh+ clones in the PM and margin but not the DP can induce pattern changes in the DP. These data are also consistent with the observation that retinal differentiation is abolished in hh- clones induced in the disc margin but not in the middle of the eye field (Cho, 2000).

Another important question raised by the prospect of interepithelial signaling is how the signals are transmitted from the PM cells to the DP. One possibility is that signaling molecules are secreted from PM cells directly to the underlying region of the DP. Alternatively, these molecules may be transported to the DP via peripodial trans-lumenal extensions that contact specific target cells or reach in the vicinity of target areas in the DP. Hh signaling may be mediated by peripodial processes, although the former possibility cannot be excluded. Ser expression in the DP induced by ectopic peripodial Hh signaling often extends beyond the region directly underneath the Hh+ PM cells. Hh may diffuse from the processes to reach other nearby DP cells. Alternatively, PM cells may extend longer processes than what can be detected in the fixed tissues. It is also possible that the inductive event occurs earlier when the two cell populations are in closer contact and subsequently become displaced relative to one another as the epithelium grows (Cho, 2000).

Recent studies have shown that disc cells send out long and thin cytoplasmic extensions, named cytonemes. Cytonemes are actin-based extensions that grow from the apical surface of the DP cells toward the signaling center, the anterior-posterior boundary of the wing disc. Some of the peripodial extensions described in this study also show cytoneme-like long and thin processes, although it is not known whether the processes are also actin-based. The peripodial processes observed can be readily seen in fixed tissues, unlike cytonemes that cannot be detected in fixed discs. Furthermore, cytonemes extend from the DP cells and grow on the apical surface of the DP, while peripodial processes extend from the apical surface of PM cells across the disc lumen to the DP. In addition, the observation of different shapes of processes suggests that peripodial processes exist in multiple types (Cho, 2000).

Inductive signaling between two cell layers is an important mechanism of morphogenesis in vertebrate development. For instance, BMP4 signaling between optic vesicle and surface ectoderm is important for lens induction in vertebrates. Wnt signaling between the ectoderm and the mesoderm is also crucial for proper dorsoventral limb patterning. First shown to occur during Drosophila leg disc regeneration and now in the eye, peripodial signaling to the DP may be analogous to such inductive signaling in vertebrates. This study illustrates a novel mechanism of interepithelial signaling between PM and DP layers and its importance in eye disc patterning. Significantly, ablation or genetic disruption of the PM also affects development of the DP, providing additional evidence for peripodial signaling. Precise localization of receptors and downstream components for Hh, Wg, and Dpp in early eye discs will help in understanding how these signals are transmitted between the PM and the DP (Cho, 2000).

In Drosophila, the development of the compound eye depends on the movement of a morphogenetic furrow (MF) from the posterior (P) to the anterior (A) of the eye imaginal disc. Several subdomains along the A-P axis of the eye disc have been described that express distinct combinations of transcription factors. One subdomain, anterior to the MF, expresses two homeobox genes, eyeless (ey) and homothorax (hth), and the zinc-finger gene teashirt (tsh). Evidence suggests that this combination of transcription factors may function as a complex and that their combination plays at least two roles in eye development: it blocks the expression of later-acting transcription factors in the eye development cascade, and it promotes cell proliferation. A key step in the transition from an immature proliferative state to a committed state in eye development is the repression of hth by the BMP-4 homolog Dpp (Bessa, 2002).

Anterior to the MF, at least three cell types can be distinguished by the patterns of Hth, Ey, and Tsh expression. The most anterior domain in the eye field, which is next to the antennal portion of the eye-antennal imaginal disc, expresses Hth, but not Tsh or Ey. In a slightly more posterior domain, all three of these factors are coexpressed (region II). In a more posterior domain, Tsh and Ey, but not Hth, are coexpressed. This domain, which also expresses hairy, is equivalent to the pre-proneural (PPN) domain. The MF, marked by the expression of Dpp, is immediately posterior to the PPN domain, and therefore abuts Tsh + Ey-expressing cells (Bessa, 2002).

Domain II is the only region of the eye-antennal imaginal disc that strongly expresses all three of these transcription factors. Posterior to the MF, Hth, but not Tsh or Ey, is expressed in cells committed to become pigment cells. Hth and Ey, but not Tsh, are coexpressed in a narrow row of margin cells that frame the eye field and separate the main epithelium of the eye disc from the peripodial membrane. Finally, Hth is also strongly expressed in peripodial cells, whereas Ey and Tsh are weakly expressed in a subset of these cells (Bessa, 2002).

The patterns of Tsh, Ey, and Hth expression in the anterior of the eye disc suggest that hth is repressed by a signal coming from the MF. A good candidate for this signal is Dpp because it can act at long range ahead of the furrow. This idea was tested by eliminating the activity of the Dpp pathway by generating clones of cells mutant for Dpp's downstream transcription factor, mothers against Dpp (mad). mad- clones de-repress hth, consistent with the idea that Dpp represses hth. De-repression of hth was observed in mad- clones that touched the posterior margin of the eye disc as well as in clones within the PPN domain. However, mad- clones close to the MF only partially de-repressed hth, suggesting that other signals present in the MF and acting at short range can also repress hth. One such signal may be Hh, which is sufficient for furrow propagation in the absence of Dpp signaling (Bessa, 2002).

The de-repression of hth in mad- clones suggests that Dpp, expressed from the MF, acts at long range to repress hth. In contrast, tsh and ey are expressed in cells adjacent to the MF, suggesting that these genes are not as sensitive to repression by Dpp. To test this directly, the Dpp pathway was activated in clones of cells by expressing an activated form of the Dpp receptor, Thick veins (Tkv*). Expression of Tkv* completely represses hth, but fails to repress ey. tsh was also not repressed in most Tkv* clones. However, tsh expression was reduced in some clones, suggesting that high levels of Dpp activity may be able to repress tsh. The complete repression of hth, but not ey or tsh, by Tkv* is consistent with the idea that Dpp represses hth, but not ey or tsh, as the MF moves anteriorly. Because ey and tsh are also repressed as the MF moves, there must be another signal coming from the furrow that acts at short range to repress these genes. This signal could be Dl, Hh, or a third, as-yet unidentified, signal (Bessa, 2002).

The complementary patterns of Hth versus So, Eya, and Dac at the transition between domain II and the PPN domain suggested that these factors may also be playing a role in hth repression. To test this idea, clones of cells mutant for eya were examined. eya- clones de-repress hth. Part of this de-repression is probably due to the fact that dpp expression requires eya. However, the de-repression of hth is observed in all eya- cells, even in cells that are next to wild-type, dpp-expressing cells. Thus, Dpp expressed in wild-type neighboring cells is not able to repress hth in adjacent eya- cells. These data suggest that eya is required for Dpp to repress hth in the PPN domain. hth was also de-repressed in dac- clones, suggesting that dac also plays a role in hth repression (Bessa, 2002).

The definition of the PPN domain stems from the observation that the induction of neural cell fates in the eye disc requires at least two signals downstream of Hh. The first signal is Dpp, which creates a zone of cells ahead of the MF, termed the PPN domain, which is competent to receive a second, proneural-inducing signal. Cells in the PPN domain express high levels of hairy. Only cells that receive the Dpp signal are able to respond to the second, shorter-acting signal. This second signal is Dl, which is expressed by cells in and behind the furrow and is required for the down-regulation of hairy. In addition to Dl, neural induction, in particular the initiation of ato expression, may also require another signal that is transduced by the ser/thr kinase raf (Bessa, 2002).

hth has been linked to the PPN domain in three ways: (1) in wild-type eye discs, hth expression abuts hairy expression; (2) Hth represses hairy -- these data suggest that hth defines the anterior limit of hairy expression (3) Dpp is a repressor of hth. Together, these results suggest that the anterior limit of the PPN domain is defined by hth expression, and that, as the MF moves anteriorly, hth is repressed by Dpp, allowing the PPN domain and hairy expression to shift anteriorly. In these experiments, only some anterior hth- clones de-repressed hairy. This result is interpreted as suggesting that hairy expression is both activated by Dpp and repressed by hth. Consequently, hth- cells that do not receive enough Dpp would still be unable to express hairy (Bessa, 2002).

In the absence of Dpp signaling, the MF is still able to progress across the eye disc because other signals, such as Hh, are sufficient for furrow progression. This is consistent with the inference that other short-range signals present in the MF can also repress hth. However, the furrow moves more slowly when it confronts cells that cannot respond to Dpp. The slower progression of the furrow could, in part, be because these cells express Hth. Interestingly, Dpp is not expressed in the MF during retinal morphogenesis in the beetle Tribolium or the grasshopper Schistocerca. The use of dpp in eye development may have been necessary in faster-growing insects like Drosophila to increase the speed of eye morphogenesis. Cells at the lateral and posterior edges of the Drosophila eye disc and in the far anterior of the disc continue to express hth and contribute to non-eye portions of the adult head. Moreover, eya- eye disc cells continue to express hth and contribute to non-eye regions of the head. Taken together, these observations suggest that changing the potency of Dpp's ability to repress hth could be used as a way to both modulate the pace of eye development and to control the ratio of eye-to-head tissue (Bessa, 2002).

Regulation of the retinal determination gene dachshund in the embryonic head and developing eye

Drosophila eye development is controlled by a conserved network of retinal determination (RD) genes. The RD genes encode nuclear proteins that form complexes and function in concert with extracellular signal-regulated transcription factors. Identification of the genomic regulatory elements that govern the eye-specific expression of the RD genes will allow a better understanding of how spatial and temporal control of gene expression occurs during early eye development. Conserved non-coding sequences (CNCSs) between five Drosophilids were compared along the ~40 kb genomic locus of the RD gene dachshund (dac). This analysis uncovers two separate eye enhancers, in intron eight and the 3' non-coding regions of the dac locus, defined by clusters of highly conserved sequences. Loss- and gain-of-function analyses suggest that the 3' eye enhancer is synergistically activated by a combination of eya, so and dpp signaling, and only indirectly activated by ey, whereas the 5' eye enhancer is primarily regulated by ey, acting in concert with eya and so. Disrupting conserved So-binding sites in the 3' eye enhancer prevents reporter expression in vivo. These results suggest that the two eye enhancers act redundantly and in concert with each other to integrate distinct upstream inputs and direct the eye-specific expression of dac (Anderson, 2006).

The smallest fragment in the 3' dac eye enhancer that can respond to dpp, eya and so is 3EE194 bp, which is centered around two CNCS blocks of ~40 bp and 20 bp. These two CNCS blocks are also common to all active fragments of the 3' eye enhancer. These two evolutionarily conserved stretches were scanned for known, genetically upstream transcription factor binding sites. The 40 bp conserved stretch contains two putative consensus So-binding sites, S1-5'-CGATAT and S2-5'-CGATAC, compared with the consensus 5'-(C/T)GATA(C/T) described previously. Each of these putative So-binding sites in 3EE were mutated individually and in combination to test their requirement for normal enhancer activity in vivo. Mutation of individual So-binding sites causes a severe reduction, but not complete elimination, of enhancer activity in vivo. However, simultaneous mutation of both So binding sites completely abolishes enhancer activity in vivo. These results, coupled with loss-and gain-of-function analyses with dpp, eya and so, suggest that So binds to the 3' eye enhancer directly and nucleates a protein complex that includes Eya to regulate 3EE. However, despite much effort using a wide variety of binding conditions, it was not possible to demonstrate specific, direct binding of So protein to oligos that contain these So-binding sites. The 5' eye enhancer, which has four CNCS blocks, were scanned for potential upstream transcription factor binding sites and no strong candidate binding sites were found within the CNCS blocks (Anderson, 2006).

Loss- and gain-of-function analyses with the two eye enhancers suggest that each enhancer is regulated by a distinct set of protein complexes. The 5' eye enhancer is activated by a combination of ey, eya and so, but is not activated by Dpp signaling. 5EE is activated by ectopic ey expression even in eya and so mutants, suggesting that it is regulated exclusively by ey. However, somewhat paradoxically, expression of 5EE, the intron 8 enhancer, is lost in eya and so mutants even though ectopic expression of a combination of dpp, eya and so does not activate this enhancer. Furthermore, driving high levels of ey in so1 mutant eye discs restores 5EE-lacZ expression. Coupled together, these results suggest that 5EE is primarily regulated by ey but that the regulation of 5EE by ey also requires eya and so (Anderson, 2006).

By contrast, the 3' dac eye enhancer is regulated by a combination of eya, so and dpp signaling, but is not directly dependent on ey. 3EE-GFP expression is lost in eya2 and so1 mutant eye discs, and in posterior margin mad1-2 mutant clones. Furthermore, ey cannot bypass the requirement for eya and so to activate 3EE. Conversely, 3EE is strongly induced by co-expression of eya and so. Moreover, dpp signaling via the tkv receptor can synergize with eya and so to induce 3EE in ectopic expression assays. Furthermore, neither Mad nor Medea, the intracellular transducers of Dpp signaling, is sufficient to bypass the requirement for activation of the Dpp receptor Tkv in these assays. Thus, it is concluded that events downstream of Dpp-Tkv signaling, such as the phosphorylation of Mad, are essential for the synergistic activation of the 3' dac eye enhancer by eya and so. Taken together, these results suggest that there are distinct requirements for the activation of the 5' and 3' dac eye enhancers. However, the exact nature of the protein complexes that regulate 5EE and 3EE remain to be determined (Anderson, 2006).

Morphogenetic furrow (MF) initiation is completely blocked in posterior margin dac3-null mutant clones. However, dac3 clones that do not include any part of the posterior margin develop and do not prevent MF progression, but do cause defects in ommatidial cell number and organization. This dichotomy in dac function is reflected in the two eye enhancers characterized in this study. Analysis of dac7 homozygotes demonstrates that the 3' eye enhancer is dispensable for MF initiation and progression. It is proposed that in dac7 mutants, the intact 5EE enhancer is sufficiently activated by ey to drive high enough levels of dac expression to initiate and complete retinal morphogenesis. However, dac7 mutants have readily observable defects in ommatidial organization. Thus, it is further proposed that this lack of normal patterning in dac7 mutants is most likely due to the loss of 3EE, which normally acts in concert with 5EE after MF initiation, to integrate patterning inputs from extracellular signaling molecules such as Dpp with tissue-specific upstream regulators such as ey, eya and so. However, it is not known if the 3' eye enhancer is sufficient to initiate dac expression in the absence of the 5' eye enhancer (Anderson, 2006).

Based on the results, a two-step model is proposed for the regulation of dac expression in the eye. First, the initiation of dac expression in the eye disc is dependent on Ey binding to 5EE. However, Ey is fully functional only when So and Eya are present. It is possible that Ey recruits So and Eya to 5EE, but a model is favored in which Ey bound to 5EE cooperates with an So/Eya complex bound to 3EE to initiate dac expression in the eye. After initiation of the MF, dac expression is maintained by an Eya and So complex bound to 3EE. In addition, 3EE can integrate patterning information received via dpp signaling, thereby allowing the precise spatial and temporal expression of dac in the eye. This two part retinal enhancer ensures that dac expression is initiated only after ey activates eya and so expression. Thus, the dac eye enhancers provide a unique model with which the sequential activation of RD proteins allows the progressive formation of specialized protein complexes that can activate retinal specific genes (Anderson, 2006).

The redundancy in dac enhancer activity also explains the inability to isolate eye-specific alleles of dac, despite multiple genetic screens. The modular nature of the two enhancers and their potential ability to act independently or in concert suggest that both enhancers must be disrupted to block high levels of transcription of dac. Thus, two independent hits in the same generation, a phenomenon that occurs infrequently in genetic screens, would be required to obtain an eye-specific allele in dac (Anderson, 2006).

Despite much investigation, very few direct targets of RD proteins, especially for Eya and So, have been identified. One study suggests that So can bind to and regulate an eye-specific enhancer of the lz gene. However, lz is not expressed early during eye development and is required only for differentiation of individual cell types. The results suggest that regulation of dac expression occurs via the interaction of two independent eye enhancers that are likely to be bound by Ey, Eya and So, and respond to dpp signaling. This analysis of the 3' eye enhancer suggests that two putative conserved So-binding sites are essential for 3EE activity in vivo. Mutation of individual So-binding sites dramatically reduces, but does not completely eliminate, reporter expression in the eye. Mutating both predicted So-binding sites completely blocks enhancer activity in vivo. Thus, it is concluded that So binds to 3EE via these conserved binding sites. However, it has not been possible to demonstrate a direct specific interaction of either So alone or a combination of Eya and So with oligos that contain these putative So-binding sites in vitro. It is possible that other unidentified proteins are required for stabilizing the Eya and So complex. Furthermore, the 194 bp fragment that responds to ectopic expression of dpp, eya, and so contains no conserved or predicted Mad-binding sites. This raises the intriguing possibility that dpp signaling activates other genes, which then directly act with eya and so to regulate the 3' eye enhancer. Alternatively, a large complex that includes Eya, So and the intracellular transducers of dpp signaling, such as Mad and Medea, may be responsible for activation of 3EE. Similarly, the results suggest that the 5' eye enhancer is regulated primarily by ey. However, it is unclear whether Ey directly binds 5EE. Furthermore, Ey is fully functional only in the presence of Eya and So. Thus, Ey either independently recruits Eya and So into a 5' complex or is activated by virtue of its proximity to the So/Eya complex bound to the 3' enhancer or both (Anderson, 2006).

The exact order and dynamics of protein complex assembly at 5EE and 3EE requires further investigation. However, the two dac eye enhancers are extremely useful tools with which to investigate fundamental issues about the mechanism of RD protein action. One significant issue concerns the mechanism of Eya function during eye development. Eya consists of two major conserved domains, an N-terminal domain that has phosphatase activity in vitro and a C-terminal domain that can function as a transactivator in cell culture assays. So contains a conserved Six domain and a DNA binding homeodomain. However, it is unclear if Eya provides phosphatase activity, transactivator function, or both, in this complex. Characterization of the components of the protein complexes that regulates dac expression may uncover the targets of Eya phosphatase activity during eye development. Thus, the isolation of two eye enhancers with distinct regulation provides very useful tools with which to study protein complex formation and function during Drosophila retinal specification and determination (Anderson, 2006).

Hedgehog and Dpp signaling induce cadherin Cad86C expression in the morphogenetic furrow during Drosophila eye development

During Drosophila eye development, cell differentiation is preceded by the formation of a morphogenetic furrow, which progresses across the epithelium from posterior to anterior. Cells within the morphogenetic furrow are apically constricted and shortened along their apical-basal axis. However, how these cell shape changes and, thus, the progression of the morphogenetic furrow are controlled is not well understood. This study shows that cells simultaneously lacking Hedgehog and Dpp signal transduction fail to shorten and do not enter the morphogenetic furrow. Moreover, a gene, cadherin Cad86C, has been identified that is highly expressed in cells of the leading flank of the morphogenetic furrow. Ectopic activation of either the Hedgehog or Dpp signal transduction pathway results in elevated Cad86C expression. Conversely, simultaneous loss of both Hedgehog and Dpp signal transduction leads to decreased Cad86C expression. Finally, ectopic expression of the extracellular region and transmembrane domain of Cad86C in either eye-antennal imaginal discs or wing imaginal discs results in apical constriction and shortening of cells. It is concluded that Hedgehog and Dpp signaling promote the shortening of cells within the morphogenetic furrow. Induction of Cad86C expression might be one mechanism through which Hedgehog and Dpp promote these cell shape changes (Schlichting, 2008).

The progression of the morphogenetic furrow provides an example of a developmentally regulated cell shape change. This paper studied the signaling pathways that regulate this cell shape change and has identified a transcriptional target of these pathways. The Hedgehog and Dpp signaling pathways both promote the shape change of cells that normally occurs in the morphogenetic furrow. Moreover, Cad86C, which is expressed in cells of the morphogenetic furrow was identified and evidence is provided that expression of this gene is regulated by both Hedgehog and Dpp signaling. Finally, Cad86C possesses, among known cadherins, an unique activity to organize elongated epithelial folds. The data suggest that Cad86C is a transcriptional target gene of Hedgehog and Dpp in the morphogenetic furrow. Furthermore, the data are consistent with the notion that Cad86C might be one effector that acts downstream of Hedgehog and Dpp signaling to help execute the cell shape changes associated with the progression of the morphogenetic furrow (Schlichting, 2008).

The conclusion that Cad86C expression in the morphogenetic furrow is regulated by Hedgehog and Dpp signal transduction is derived from the analysis of loss-of-function mutants in these signaling pathways and from the ectopic activation of these signaling pathways through expression of activated components. The level of Cad86C protein is highly reduced in smo3 tkva12 clones straddling the normal position of the morphogenetic furrow, whereas Cad86C protein is still detectable in smo3 or tkva12 single mutant clones. Conversely, expression of a constitutively active form of the Hedgehog-regulated transcription factor Ci, CiPKA4, or a constitutively active form of the Dpp receptor Thickveins, TkvQ253D, resulted in increased levels of Cad86C protein. Two observations indicate that Hedgehog and Dpp signal transduction regulate the expression of Cad86C mainly at a transcriptional level. First, the abundance of Cad86C RNA is highly increased in the morphogenetic furrow of wild-type eye imaginal discs and, second, Cad86C RNA is highly reduced when hedgehog activity (and Dpp expression) is impaired in hhts2 mutant eye imaginal discs. In the first intron of Cad86C, a cluster of three putative Ci binding sites have been identified based on their sequence similarity to the Gli/Ci consensus binding sequence. This provides a first indication that Cad86C might be a direct transcriptional target of the Hedgehog signaling pathway. In Cad86C71C mutants, in which these putative Ci binding sites are deleted, Cad86C RNA appears to be normally expressed in the morphogenetic furrow, demonstrating that these sites are not essential for Cad86C expression in the morphogenetic furrow. This is consistent with the finding that the Hedgehog signal transduction pathway is not essential for Cad86C expression in cells of the morphogenetic furrow and, that in its absence, the Dpp signaling pathway can promote expression of Cad86C. Cad86C expression, in addition, might be controlled also at a posttranscriptional level, since Cad86C RNA, but not Cad86C protein, is detected in some cells posterior to the morphogenetic furrow (Schlichting, 2008).

In contrast to other known cadherins, Cad86C possesses an unique activity to organize elongated folds in epithelia. This activity appears to be mediated by the cadherin repeats, since expression of a deletion variant of Cad86C, Cad86C-EXTRA-HA, in which the intracellular region is missing, still induces epithelial folds. Since cadherin repeats mediate the binding between cadherin molecules, it is speculated that expression of Cad86C-HA promotes epithelial folding through its interaction with a cadherin. No evidence is found that Cad86C interacts homophilically in cells of the morphogenetic furrow. Cad86C might, therefore, interact with a different kind of cadherin to promote epithelial folding. Candidates for Cad86C-interacting cadherins include the non-classical cadherins Cad74A, Cad88C, and Cad96Cb, which, during embryonic spiracle development, are expressed in sub-sets of cells adjacent to Cad86C (Lovegrove, 2006). Among these three cadherins, it was found that Cad88C is expressed in a complementary pattern to Cad86C. However, adult flies homozygous mutant for Cad86C and Cad88C had an apparently normal eye size, indicating that Cad88C is, if at all, not an essential interacting partner for Cad86C during morphogenetic furrow progression (Schlichting, 2008).

Cad86C-HA induces epithelial folding non-cell-autonomously, indicating that an imbalance in the expression level of Cad86C between neighboring cells might result in cell shortening. It is noted, however, that Cad86C184A mutant clones in the wing imaginal disc and eye imaginal disc are not associated with epithelial folds, perhaps because the absolute difference in Cad86C expression between mutant cells and neighboring control cells is only modest (Schlichting, 2008).

Similar to the expression of Cad86C-HA, expression of an activated form of the regulatory light chain of non-muscle Myosin II has recently been shown to promote epithelial folding in the eye imaginal disc (Corrigall, 2007; Escudero, 2007). The finding that Cad86C-EXTRA-HA promotes epithelial folding indicates that Cad86C does not directly interact with non-muscle Myosin II to bring about cell shape changes. Future studies will need to examine the relationship between Cad86C and non-muscle Myosin II (Schlichting, 2008).

This study found that both the Hedgehog and Dpp signaling pathways operate to promote the cell shape changes that normally occur in the morphogenetic furrow. It is tempting to speculate that Cad86C acts downstream of Hedgehog and Dpp signal transduction to promote the progression of the morphogenetic furrow. This speculation is mainly based on three observations. First, Cad86C protein is present at high levels in cells of the leading flank of the morphogenetic furrow, the cells that undergo apical constriction and shortening first. Second, Cad86C expression in the eye imaginal disc is regulated by Hedgehog and Dpp signal transduction, the two signal transduction pathways that promote the progression of the morphogenetic furrow. And third, ectopic expression of Cad86C-HA in clones of cells results in apical cell constriction and cell shortening, cell shape changes typically associated with the progression of the morphogenetic furrow. However, since attempsts to detect a genetic requirement for Cad86C in morphogenetic furrow progression failed, it remains possible that Cad86C may play a role during eye development that is unrelated to morphogenetic furrow progression (Schlichting, 2008).

The morphogenetic furrow moves at a speed of one ommatidial cluster in approximately two hours. Based on these results, the following model is proposed of how the morphogenetic furrow progresses. Cells leaving the morphogenetic furrow start to differentiate and express Hedgehog. Hedgehog signals anteriorly to induce the expression of dpp in cells of the morphogenetic furrow. In response to Hedgehog and Dpp signaling, several target genes, including Cad86C, are induced in cells of the leading flank of the morphogenetic furrow. The resulting proteins promote the apical constriction and shortening of the leading edge cells, a process recently shown to require non-muscle Myosin II. The apical constriction and shortening of the leading edge cells then moves the leading flank of the furrow anteriorly. As cells proceed through the center of the morphogenetic furrow, Hedgehog signal transduction is switched off and target gene expression ceases. Downregulation of Cad86C expression in the center of the morphogenetic furrow appears to be important, since sustained expression of Cad86C-HA prevents cells from elongating. The cessation of target gene expression, therefore, might allow cells to extend to their normal length and shape and, thus, to leave the morphogenetic furrow. These cells will then start to differentiate and express Hedgehog (Schlichting, 2008).

Cad86C possesses an unique activity to induce elongated folds in epithelia. The identification of Cad86C interacting proteins will be important to elucidate the mechanisms by which Cad86C promotes epithelial fold formation. Identification of Cad86C interacting proteins, as well as the identification of additional Hedgehog and Dpp target genes, promises to shed further light on the cell biological mechanisms underlying morphogenetic furrow progression (Schlichting, 2008).

Bowl functions downstream of Dpp in the antennal disc

In Drosophila, antennae and legs are serially homologous appendages, and yet they develop into organs of very different structure and function. This implies that different genetic mechanisms operate onto a common developmental ground state to produce antennae and legs. Still few such mechanisms have been uncovered. During leg development, bowl, a member of the odd-skipped gene family, has been shown to participate in the formation of the leg segmental joints. This study reports that, in the antennal disc, bowl has a dramatically different role: bowl is expressed in the ventral antennal disc to prevent inappropriate expression of wg early during development. The removal of bowl function leads to the activation of wg in the dpp-expressing domain. This ectopic expression of wg, together with dpp, results in a new proximo–distal axis that promotes non-autonomous antennal duplications. The role of bowl in suppressing a supernumerary PD axis is maintained even when the antennal disc is homeotically transformed into a leg-like appendage. Therefore, bowl is part of a genetic program that suppresses the formation of supernumerary appendages specifically in the fly’s head (Brás-Pereira, 2008).

In Drosophila, antennae, mouthparts, legs and genitalia are considered to be serially homologous ventral appendages. This means that despite their very different structure and function, they are thought to develop from a common developmental ground state. It is the segment-specific selector gene expression that, acting upon this ground state, defines their specific morphologies. Of these ventral appendages, the development of the leg is best understood. The leg primordium is set aside as a cluster of epidermal cells, composed of a distal population, that expresses Distal-less (Dll) and a proximal one, expressing homothorax (hth), teashirt and escargot. This early genetic subdivision corresponds to the proximo–distal (PD) telopodite–coxopodite subdivision of the insect appendages. hedgehog (hh), expressed by posterior cells within the leg primordium, triggers the expression of the decapentaplegic (dpp) and wingless (wg) signaling molecules in anterior cells which, through mutual repression, become expressed in a dorsal and a ventral wedge, respectively. wg and dpp expressions only coincide at the center of the leg disc and it is this confluence of maximal signaling that defines the distal tip of the future leg and triggers growth. The larval development of the leg primordium –called leg imaginal disc – progresses by the successive definition of intermediate domains of gene expression that specify the segments of the leg (coxa, trocanter, femur, tibia and tarsus) are defined. During late larval life, leg development becomes wg/dpp-independent, and the distal disc tip becomes a source of EGFR signaling, which is responsible of the further segmentation of the tarsus into the five tarsomeres and the terminal claw. Growth and segmentation of the leg also depends on Notch signaling. Activation of Notch by its ligands Delta (Dl) and Serrate (Ser) is necessary for the disc to grow, and the overlapped expression of Dl and Ser in concentric rings defines the position of the joints of the leg segments as the cells immediately distal to these rings (Brás-Pereira, 2008 and references therein).

The odd-skipped family of genes, odd-skipped (odd), drumstick (drm) and sister of odd and bowl (sob) are among the Notch targets in legs. These genes are expressed in concentric rings at the prospective leg joints, just distal to the Dl/Ser ring domains. A fourth member of the family, brother of odd with entrails limited (bowl), has a more widespread expression pattern. Genetic data indicate that bowl is required for the segmentation of the leg, and that the localized co-expression of the other family members allows (probably in a redundant fashion) the activation of bowl at the prospective joints. Further molecular and genetic experiments show that, at least during embryogenesis, the product of the gene lines blocks bowl function by directly binding to Bowl and preventing its nuclear accumulation. Drm and likely Odd are able to competitively displace Lines from Bowl, thus allowing Bowl to become nuclear and functional (Brás-Pereira, 2008 and references therein).

The distinct antennal development is promoted by the distal maintenance of hth expression in the antennal disc, resulting in the co-expression of hth and Dll. This co-expression selects the antennal fate. Compared to the leg, the antenna is a much shorter appendage, with four segments (antennal (a) segments 1–3, plus a distal arista), and functions in olfaction, through the specialization of its a3 segment. The antennal disc does not develop as an independent disc, like the leg one, but forms part of the eye–antennal disc complex. This disc comprises cells derived from several embryonic head segments and the unsegmented acron. All the cells of the eye–antennal disc complex express the Pax6 genes eyeless (ey) and twin-of-eyeless during first larval stage (L1), but during L2, only the posterior two-thirds of the complex express Pax6 genes, while the anterior third expresses cut (ct). The L2 ct and Pax6 domains correspond to the antennal and eye discs, respectively. The smaller size and fewer segments of the adult antenna when compared to the leg correlate with a different expression of the Dl and Ser ligands in antennal and leg discs. Accordingly, the antennal disc has only two odd-expressing rings, instead of the six present in leg discs. The different control of growth and segmentation in the antenna indicates that there must be mechanisms operating differently in antennal and leg discs (Brás-Pereira, 2008 and references therein).

The fact that bowl has been placed downstream of Notch signal in the elaboration of distal leg patterning prompted a test of whether bowl had any function during antennal development, and if it did, whether it was similar to its role during leg segmentation. The results indicate that, during antennal disc development, bowl has a dramatically different role: bowl is expressed at early stages in the ventral antennal disc, where it prevents inappropriate expression of wg. If bowl is removed, the activation of wg results in non-autonomous antennal duplications. bowl is still required to prevent PD axis duplication in homeotically transformed antennal discs, which indicates that there are genetic differences between head and thorax discs that are selector gene independent (Brás-Pereira, 2008). .

During the development of the antennal disc, bowl has two phases of expression: an early expression in the ventral disc, required to maintain wg repressed, and a later one in concentric rings. Both phases have antennal-specific properties. The early bowl expression and function is unique to the antenna. Its expression in rings associated to prospective joints, which recapitulates the ring expression in leg discs, does not seem required for joint formation in the antenna, contrary to what has been described in the legs. In addition, bowl is still required to repress a ventral supernumerary PD axis even if the antenna has been homeotically transformed into a leg-like appendage by overexpression of the leg selector Antp. All these results indicate that the development of the head structures deriving from the antennal disc depends not only on the activity of selector genes, but also on a cephalic-specific genetic program. Supporting this claim, it was found that the expression of eyg, an antennal-specific marker, is maintained in homeotically transformed antennal discs (Brás-Pereira, 2008).

These cephalic vs. thoracic differences might reflect the very different developmental histories of antennal and leg discs. While each leg disc primordium is formed from cells derived from just two adjacent parasegments (or one embryonic segment), the antennal disc is part of a composite disc, the eye–antennal disc, which forms by the fusion of imaginal primordia derived from several embryonic head segments [the labial, antennal, intercalary, mandibular and maxillary segments plus the unsegmented acron. The coalescence of all these primordia in a single imaginal disc might have required the repression of some domains of gene expression carried along by precursor cells. In this sense bowl might have been recruited to block wg expression in the ventral cells of the antennal disc during the early stages of its development. The lack of bowl at this stage would release wg expression which, in turn and with dpp, would trigger the development of a new appendage (Brás-Pereira, 2008).

The repressive function of bowl might extend to other parts of the eye–antennal eye disc. bowl minus clones in the ventral region of the stem that connects the antennal and eye disc lobes develop autonomously into eye tissue. In contrast to the antennal suppressing function, bowl is required autonomously to repress eye development. This autonomy indicates that either the signals normally operating to spread retinal differentiation in the normal eye are not produced in these ectopic retinal patches, or that the wild type tissue is refractory to these signals. At present, no choice can be made between these two hypotheses. It was noticed, however, that the overexpression of the bowl inhibitor Lines driven by the dpp-GAL4 driver leads to two phenotypic outcomes: antennal duplication or ectopic ventral eyelet. Interestingly, only in one case out of more than 20 discs analyzed these two phenotypes co-occurred. This suggests that the cells in the sensitive region adopt collectively only one two fates, antenna or eye, and that deciding upon one excludes the other. Since wg normally acts by limiting the eye field, eye fate might be blocked in those ventral bowl minusclones derepressing wg. In addition, it is noted that this ct, ey-expressing region is particularly prone to develop into eye upon genetic perturbations. For example, it is this region that is preferentially transformed into eye when hth function is removed or when tsh is ectopically expressed. Perhaps, the unique fact that this region co-expresses antennal and eye determinants makes its fate more ambiguous. In the absence of bowl, hth might tilt the equilibrium towards head capsule or antennal development, while the opposite fate – eye – would be adopted in the presence of tsh and ey. It will be interesting to determine whether functional relationships between bowl and these factors exist to determine specific fates within the eye disc (Brás-Pereira, 2008).

Mechanistically, bowl function seems to lie downstream of hh and dpp. In bowl minus cells associated with an antennal duplication, hh is still expressed and the Hh-coreceptor patched is normally up-regulated in anterior cells abutting the hh-expressing domain, which indicates correct hh-signaling. Accordingly, wg derepression in bowl minus cells occurs closest to the P cells, as expected for a hh target gene. In the embryo, bowl has also been placed downstream of hh during the process of epidermal differentiation (Brás-Pereira, 2008).

In the antenna, as in the leg disc, the dpp and wg signaling pathways repress each other to establish two opposing wedges of dpp and wg expression. In bowl minus clones, though, dpp expression, monitored by a lacZ-expressing reporter, is not turned off, despite the induction of wg expression ventrally. Although this might be due to the perdurance of the LacZ product, bowl minus cells accumulate normal levels of phosphorylated-Mad. This indicates that bowl-mutant cells transduce the dpp signal. Therefore, these results suggest that bowl is required for the mutual repression of wg and dpp in the ventral portion of the antennal disc. Nevertheless, bowl is not sufficient to repress wg in the antenna. Simple explanations for this fact have been ruled out, such as low levels of the induced Bowl protein, or its retention in the cytoplasm. This insufficiency is not due to the inhibition by Lines, because even in the presence of Drm, which prevents Lines from binding to Bowl, this latter is still unable to repress wg. Although further work is required to identify which other factor or factors collaborate with bowl during ventral antennal disc development, the simplest explanation would be that Bowl acts in concert with a factor induced by dpp. This is because bowl cannot block the ectopic wg expression in ventral antennal cells devoid of dpp signal. Nevertheless, when bowl expression is forced in the leg disc using the ptc-GAL4 driver, wg is repressed by bowl cell-autonomously in the most distal region of the disc, but not in the more proximal domain. This result strengthens the idea that bowl acts as a wg repressor. Such repression takes place in the distal part of its domain, closest to the dpp source, which also supports the claim that bowl requires the dpp signaling to repress wg (Brás-Pereira, 2008).

This study has shown that bowl is expressed in the ventral antennal disc, the realm of the dpp pathway, and that dpp signaling can activate bowl transcription in this disc. These results suggest that high levels of dpp induce bowl which, in turn, is required to prevent inappropriate expression of wg in the antennal disc together with the dpp pathway. Two are the likely sources of Dpp: the wedge of dpp that can be visualized using the dpp-disc enhancer reporters in the antenna, and a ventral disc expression that is controlled by a separate enhancer. This enhancer drives dpp expression in the prospective ventral head region, close to the region where bowl is transcribed in early discs (Brás-Pereira, 2008).

bowl and the related genes odd and drm show a late pattern of expression in rings, similar to the one deployed in leg discs. But contrary to their requirement for leg segmentation, bowl seems to be dispensable for antennal segmentation. A similar situation has been described for the gene dachshund (dac). dac is expressed in the medial segment of both leg and antennal discs, but while loss of dac in the leg leads to the loss of intermediate adult leg structures, the antenna develops normally. These results might reflect the fact that, although antennal and leg discs have specific developmental programs, the mechanisms for generating the PD axis are shared by both appendages. This mechanism would call a similar battery of genes, even if only a subset of them is effectively used for the development of each appendage. In fact, ectopic activation of the Notch pathway by overexpression of the ligand Delta induces ectopic expression of drm in the antenna. This indicates that, similarly to what happens in the leg discs, ring expression of Odd-family genes in the antenna might also be under Notch control. In this sense, in the antenna the segmentation function might have been taken over by other(s) member(s) of the Odd family, expressed as well in the future joints (Brás-Pereira, 2008).

In summary, the results show that the zinc-finger encoding gene bowl is part of a cephalic-specific program that represses appendage formation in the ventral eye–antennal disc. Here, bowl is required to repress wg, downstream of dpp, to prevent the generation of supernumerary antennae. These extra appendages might arise from some silenced primordium in the proximal part of the antenna, which would be normally fated to become part of the head capsule. In addition, bowl also silences the development into eye of another cell population of the prospective head that presents mixed expression of antenna and eye selector genes. The repressive action of bowl that is described here might have been essential for the coalescence of cells deriving from several different embryonic cephalic segments into a single imaginal disc, as well as for the formation of the head structures of adult cyclorraphan flies, such as Drosophila (Brás-Pereira, 2008).

Increased avidity for Dpp/BMP2 maintains the proliferation of progenitors-like cells in the Drosophila eye

During organ development, the progenitor state is transient, and depends on specific combinations of transcription factors and extracellular signals. Not surprisingly, abnormal maintenance of progenitor transcription factors may lead to tissue overgrowth, and the concurrence of signals from the local environment is often critical to trigger this overgrowth. Therefore, identifying specific combinations of transcription factors/signals promoting (or opposing) proliferation in progenitors is essential to understand normal development and disease. This study used the Drosophila eye as a model where the transcription factors hth and tsh are transiently expressed in eye progenitors causing the expansion of the progenitor pool. However, if their co-expression is maintained experimentally, cell proliferation continues and differentiation is halted. Hth+Tsh-induced tissue overgrowth was shown to require the BMP2 Dpp and the abnormal hyperactivation of its pathway. Rather than using autocrine Dpp expression, Hth+Tsh cells increase their avidity for Dpp, produced locally, by upregulating extracellular matrix components. During normal development, Dpp represses hth and tsh ensuring that the progenitor state is transient. However, cells in which Hth+Tsh expression is forcibly maintained use Dpp to enhance their proliferation (Neto, 2016).

Abnormal maintenance of transcription factors that promote an undifferentiated, proliferative state is often an initiating event in tumors. However, abnormal growth is dependent on specific non-autonomous signals provided by the microenvironment. This study used an experimental system that results in continuous growth to identify these signals and the mechanism of action. In this system, the GAL4-driven maintenance during eye development of hth and tsh, two transcription factors normally transiently co-expressed in eye progenitors, cause cells to increase their avidity for Dpp. This, in turn, leads to a hyper-activation of the pathway, which is necessary to maintain the proliferative/undifferentiated phenotype. The increased avidity for Dpp was shown to be mediated, at least partly, through increased expression of the proteoglycans components encoded by dally and dlp, functionally modified by slf (Neto, 2016).

Progenitor cells, forced to maintain Hth and Tsh (hth+tsh progenitor-like cells) trap Dpp produced at local sources, which then causes an increased in intracellular signaling. The mechanism responsible of this trapping seems to be the increase of extracellular matrix (ECM) components. First, a cell-autonomous increase was found in dally transcription and Dlp membrane levels, the two glypican moieties of heparane sulphate proteoglycans. Second, the RNAi-mediated attenuation of sfl function, a gene encoding an enzyme required for the biosynthesis of these proteoglycans, is required for the overgrowth/eye-suppression phenotype induced by hth+tsh maintenance. A third line of support comes from examination of the effects of hth+tsh or hth+tsh+slf RNAi on the pMad profiles. Considering that the Dpp production remains unaltered, hth+tsh tissue shows an increase in both pMad signal amplitude and range, which is consistent with the increase in proteoglycans simultaneously augmenting Dpp diffusion and stability. On the contrary, reducing proteoglycan biosynthesis in hth+tsh+slf RNAi cells results in the retraction of the pMad signaling range back towards control values, which again is expected if Dpp's diffusion depends on proteoglycans (Neto, 2016).

By forcing the expression of hth and tsh in eye precursors, these cells are exposed to signaling levels higher than they would normally encounter. This is because during normal eye development Dpp, produced at the furrow, represses first hth and then, closer to the furrow, also tsh, so that the cells approaching the furrow and receiving the highest Dpp levels no longer co-express hth and tsh. The loss of hth marks the transition between proliferation/undifferentiation and cell quiescence/commitment. This transition coincides with a transient proliferative wave (the so-called 'first mitotic wave') that precedes entry into G1. This transition zone corresponds to a region where low, but not null, levels of Hth and pMad signals overlap. If the interaction between hth+tsh and the Dpp pathway described in this study were to hold also in the zone of hth/Dpp signal overlap during normal eye development (remember that hth-positive cells co-express normally tsh too), one prediction would be that the mitotic wave would be lost if either hth or dpp-signaling were removed. Indeed this has been shown to be the case: RNAi-mediated attenuation of hth or abrogation of Dpp signaling result in the loss of the first mitotic wave. However, it is not thought that the mechanisms driving Dpp-mediated proliferation of optix> hth+tsh cells are necessarily the same as those operating normally in hth+tsh-expressing progenitors during eye development, because of the following experiment. Discs were generated expressing in their dorsal domain an RNAi targeting Hth's partner, the Pbx gene extradenticle (exd). In the absence of Exd, Hth is degraded. Therefore, a depletion of Exd causes an effective loss of Hth. Knowing that in optix>hth+tsh the stability and diffusion of Dpp were increased, the prediction would be that the loss of hth (in exd-depleted cells) should cause a decrease in both the stability and diffusion of Dpp. However, when the dorsal ('exd-') with the ventral ('exd+') pMad profiles of D>exdRNAi discs was quantified, it was found that both the stability and diffusion of Dpp increased by the loss of hth. This result suggests that during normal eye development hth (perhaps together with tsh) influences Dpp signaling, but the mechanisms described in this study as triggered by forced hth+tsh expression are likely different (Neto, 2016).

The upregulation of dally and dlp by hth+tsh is likely the consequence of the transcriptional activity of Hth+Tsh in partnership with the YAP/TAZ homologue, Yki, as previous work showed that loss of the protocadherin genes fat (ft) and dachsous (ds) , which causes the activation of Yki, results in an upregulation of dally and dlp in the wing primordium. In fact, previous studies have found, in imaginal tissues, binding of Yki and Hth to nearby sites on the dlp locus, suggesting that some of this regulation might be direct. All these data make Yki a necessary component of the molecular machinery responsible for the increased avidity of hth+tsh cells for Dpp. However, in the eye primordium, the overexpression of Yki induces a different phenotype than hth+tsh. More importantly, in the eye primordium, yki+ clones do not cause the autonomous upregulation of pMad signal that hth+tsh clones do. Therefore, a specific stoichiometry among Hth, Tsh and Yki is likely necessary to induce the Dpp signaling-dependent properties of hth+tsh cells, at least in the developing eye. Alternatively, Hth and Tsh may activate Yki-independent targets that would be required for the full expression of the phenotype. Recently, another study has found that Yki and the Dpp pathway synergize in stimulating tissue overgrowth, both in eye and wing primordia, through the physical association between Yki and Mad. The current results suggest that hth+tsh progenitor-like cells establish a positive feedback, in which the growth promoting activity of the Hth:Tsh:Yki complex would be enhanced by increasing levels of pMad activated by Dpp. This feedback would be region-specific, as it depends on sources of Dpp that are localized within the eye primordium. Further work is needed to investigate the molecular mechanisms behind this feedback. Finally, it has been shown recently that tissue growth promoted by the PI3K/PTEN and TSC/TOR nutrient-sensing pathways also requires Dally, which, in turn, increases the avidity of the growing tissue for Dpp. Therefore, increasing the avidity for Dpp by augmenting proteoglycan levels may be a common strategy of tissues to sustain their growth (Neto, 2016).

Table of contents

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

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