decapentaplegic

Effects of Mutation or Deletion (part 2/3)

Dpp and neurogenesis

DPP surpresses neurogenesis in dorsal cells of embryos. To assess the role of dpp in regulating neurogenesis, the effect of this signaling pathway on neurogenesis in dorsal cells was examined. The dorsal cuticle of embryos lacking dpp activity appears to be ventralized. However, this inference is tenuous as the number of differentiated neurons is reduced, not expanded in late dpp mutant embryos. In early gastrulating mutants, on the other hand, dorsal expression is observed of neuroectodermal markers such as thick veins and lethal of scute. Similarly, neuroblasts visualized with markers such as scratch and snail subsequently form ectopically in the dorsal region of dpp mutants. Consistent with dpp acting early to suppress initiation of neurogenesis, ectopic expression of the proneural gene lethal of scute is first detectable in dorsal cells of late blastoderm stage dpp mutants. Paradoxically, the increased number of neuroblasts in dpp mutants does not generate a hypertrophied differentiated nervous system. Thus, dpp mutants may lack a late positive role for dpp in neuronal maturation or may hyperactivate pathways functioning to inhibit subsequent steps in neurogenesis (Biehs, 1996).

Dpp and terminalia

This study reports the expression pattern of Dll in the genital disc, the requirement of Dll activity for the development of the terminalia and the activation of Dll by the combined action of the morphogenetic signals Wingless (Wg) and Decapentaplegic (Dpp). In Drosophila, the terminalia comprise the entire set of internal and external genitalia (with the exception of the gonads), and includes the hindgut and the anal structures. They arise from a single imaginal disc of ventral origin which is of complex organization and shows bilateral symmetry. The genital disc shows extreme sexual dimorphism. Early in development, the anlage of the genital disc of both sexes consists of three primordia: the female genital primordium (FGP); the male genital primordium (MGP), and the anal primordium (AP). In both sexes, only two of the three primordia develop: the corresponding genital primordium and the anal primordium. These in turn develop, according to the genetic sex, into female or male analia. The undeveloped genital primordium is the repressed primordium (either RFP or RMP, for the respective female and male genital primordia) (Gorfinkiel, 1999).

During the development of the two components of the anal primordium -- the hindgut and the analia -- only the latter is dependent on Dll and hedgehog (hh) function. The hindgut is defined by the expression of the homeobox gene even-skipped. The lack of Dll function in the anal primordia transforms the anal tissue into hindgut by the extension of the eve domain. Meanwhile targeted ectopic Dll represses eve expression and hindgut formation. The Dll requirement for the development of both anal plates in males and only for the dorsal anal plate in females, provides further evidence for the previously held idea that the analia arise from two primordia. In addition, evaluation was made of the requirement for the optomotor-blind (omb) gene which, as in the leg and antenna, is located downstream of Dpp. These results suggest that the terminalia show similar behavior as the leg disc or the antennal part of the eye-antennal disc, consistent with both the proposed ventral origin of the genital disc and the evolutive consideration of the terminalia as an ancestral appendage (Gorfinkiel, 1999).

The requirement for the Hh signal in Dll activation might be mediated by Wg and Dpp signals. This occurs in other ventral discs. Dll expression arises at the juxtaposition of Wg and Dpp expressing cells as revealed by double staining for Dll and Dpp, and Dll and Wg. In both genital and anal primordia, Dll expressing cells overlap those that express wg and dpp. It has been previously reported that the ectopic expression of both Wg and Dpp produces several phenotypic alterations in both female and male terminalia. Similar types of transformations are also induced by the lack of function of either patched (ptc) or Protein kinase A (Pka). In these mutants, the Hh pathway is constitutively active giving rise to the derepression of Wg and Dpp. The lack of Pka function in the genital disc induces ectopic Dll. This Dll induction requires both Wg and Dpp signals in the same cells since Dll is not activated in Pka2;dpp2 and in Pka2;wg2 double mutant clones, as occurs in other discs of ventral origin (Gorfinkiel, 1999).

In the male repressed primordium (RMP) of the female genital disc, wg is expressed but not dpp. Consequently, Dll is not expressed because Dll is only activated in cells that express both dpp and wg. Ectopic Dpp expression in the wg expression domain driven by the MS248-GAL4 line induces Dll 'de novo' in the RMP, which shows an increase in size. However, these changes do not allow the development of adult structures from this primordium since there is no activation of the male specifc cyto-differentiation genes because the genetic sex has not changed. Dll is not activated in the repressed female primordium (RFP) of the male genital disc despite the fact that, in this primordium, both wg and dpp are normally expressed. This activation does not occur even if the levels of Dpp are increased. These results suggest that specific genes expressed in the RFP can exert a negative control of Dll expression (Gorfinkiel, 1999).

In order to find other genes involved in the development of the terminal structures, the expression pattern and the functional requirement for optomotor-blind (omb) were examined. This gene encodes a protein with a DNA-binding domain (T domain) and behaves as a downstream gene of the Hh pathway in other imaginal discs. In the genital disc, Omb is detected in the dpp expression domains, abutting the wg expressing cells. This behaviour of omb expression is similar to that found in the leg and antennal discs. In the genital disc, omb is also regulated by the Hh signaling pathway since Pka2 clones also ectopically express omb. The phenotypes produced due to omb lack of function using the allele omb282 were examined; homozygous females for this allele could not be obtained but some male pharates were analyz

ed. In males, the dorsal bristles of the claspers and the hypandrium bristles are absent. Also, the hypandrium is devoid of hairs and the hypandrium fragma is reduced. Surprisingly, the anal plates are mostly somewhat enlarged in the ventral region and reduced in the dorsal areas. The structures affected in omb2 are duplicated when omb is overexpressed in the dpp domain using the dpp-GAL4/UAS-omb combination. In males, the dorsal bristles of the clasper and the hypandrium bristles are duplicated. These phenotypes are similar to the ones obtained as a result of ectopic Dpp (Gorfinkiel, 1999).

The hindgut of the Drosophila embryo is subdivided into three major domains, the small intestine, large intestine, and rectum, each of which is characterized by specific gene expression. The expression of wingless, hedgehog, decapentaplegic, and engrailed corresponds to the generation or growth of particular domains of the hindgut. wg, expressed in the prospective anal pads, is necessary for activation of hh in the adjacent prospective rectum. hh is expressed in the prospective rectum, which forms anterior to the anal pads, and is necessary for the expression of dpp at the posterior end of the adjacent large intestine. wg and hh are also necessary for the development of their own expression domains, anal pads, and rectum, respectively. dpp, in turn, causes the growth of the large intestine, promoting DNA replication. en defines the dorsal domain of the large intestine, repressing dpp in this domain. A one-cell-wide domain, which delineates the anterior and posterior borders of the large intestine and its internal border between the dorsal and ventral domains, is induced by the activity of en. A model is proposed for the gene regulatory pathways leading to the subdivision of the hindgut into domains (Takashima, 2001).

The term 'tissue compartments' can be used to indicate the domains of the gut. In this report, the term 'domain' is used in order to avoid confusion with the term 'developmental compartment', which has been defined by clonal analysis of the wing disc. To clarify the use of anatomical descriptions, the organization of the hindgut domains, as revealed by specific gene expression patterns is described. The most anterior domain of the hindgut, which is just posterior to the midgut, is the small intestine. The small intestine is followed by the large intestine, then the rectum. The large intestine is further subdivided into a ventral and a dorsal domain. A one-cell-wide domain, which was designated as h4, forms at the anterior and posterior borders of the large intestine, as well as at the border between the dorsal and ventral domains of the large intestine. The cells in these regions are designated collectively 'border cells'. Until the end of stage 12, the hindgut tube is situated on the midline of the body, and is left-right symmetric. During early stage 13, the hindgut rotates to the left, resulting in the original dorsal and ventral domains coming to face the left and right side of the body, respectively. The orifice of the rectum (the anal slit) is surrounded by the anal pads, the development of which is tightly linked to that of the hindgut (Takashima, 2001).

wg, hh, and dpp are expressed in the hindgut of the Drosophila embryo. The expression patterns of these genes have been re-examined in detail to define their exact spatial relationship. wg is expressed throughout the proctodeum at stage 9, then soon becomes restricted to two separate regions: (1) the primordium of the anal pads, which surrounds the posterior opening of the hindgut, and (2) a narrow ring anterior to the small intestine. The expression in these two domains persists throughout embryogenesis (Takashima, 2001).

dpp is first expressed at early stage 11 as a narrow ring anterior to the prospective rectum. After early stage 12, a weak expression appears in the ventral domain of the large intestine, which partly overlaps the former dpp-positive domain. en, initially expressed throughout the hindgut primordium at stage 9, is soon restricted to the dorsal domain of the large intestine. The en-positive dorsal domain and the dpp-positive ventral domain do not overlap when examined by double staining for En protein and DPP mRNA. The expression of en continues throughout embryogenesis and larval stages (Takashima, 2001).

The border cells differentiate at the anterior and posterior border of the large intestine and at the border between the dorsal and ventral domains of the large intestine. The border cells are first detected at stage 12 by lacZ expression of some enhancer-trap strains, and after stage 14, the cells are distinguished by marked expression of Crb and dead ringer. By double staining for En and beta-galactosidase protein of border cell-specific enhancer trap lines, the border cells are found to abut the En-positive domain and to express no En protein, suggesting that dpp-positive cells abutting the en-positive domain differentiate into border cells. It is noteworthy that the spatial organization of en, hh, wg, and dpp domains is quite different from that of the segmented epidermis or the imaginal discs, suggesting that a different patterning mechanism is working in the hindgut (Takashima, 2001).

In wg mutants, dpp expression in the large intestine is completely abolished. The ectopic expression of wg slightly upregulates the initial dpp expression in the large intestine at stage 11, but has repressed dpp throughout the hindgut by late stage 12. The relationship between wg activity in the anal pads and dpp expression in the large intestine seems to be indirect and complicated (Takashima, 2001).

en expression in the dorsal domain of the large intestine, in contrast to that of dpp and hh, is not affected in wg embryos. These results suggest that the defects of the hindgut in wg mutants are partly mediated by failure of hh expression in the future rectum. It should be noted that the defects of the large intestine in the wg mutant are more drastic than those in either hh or dpp mutants. There may exist some pathway of wg action that is not mediated by hh and dpp (Takashima, 2001).

hh is expressed in the prospective rectum and small intestine after stage 11. The hindgut of the hh mutant embryo is shorter than that of wild-type (i.e. about 70% that of wild-type at stage 16. After stage 12 in wild-type embryos, the prospective rectum is recognized by a slightly enlarged lumen at the posterior end of the proctodeum. In hh embryos, the rectum is initially almost normal in size at early stage 12, but after early stage 13 it begins to degenerate, and becomes scarcely recognizable at stage 16. Consequently, the posterior border of en expression in the dorsal domain is more proximate to the orifice. The small intestine is also reduced in hh mutant embryos, but this defect is not so drastic when compared with that of the rectum. Growth of the large intestine, which occurs in wild-type embryos after stage 12, is suppressed in hh mutants, resulting in a short hindgut. In hh embryos, dpp expression in the region just anterior to the prospective rectum becomes very weak, but dpp expression in the ventral domain of the large intestine is not affected or, if anything, appears to be enhanced. A dpp mutation, in contrast, does not affect hh expression in the future rectum. These results indicate that hh expression in the prospective rectum is necessary for the development of the rectum itself, and also for sustaining the normal dpp level in the posterior end of the large intestine. Inductive effects of hh on dpp expression in the large intestine has been demonstrated by the ectopic expression of hh. Ectopic expression of hh in the posterior half of the large intestine by mating the UAS-hh strain with the hairy-GAL4 strain, which expresses GAL4 in the posterior half of the hindgut including most of the large intestine, results in markedly expanded dpp expression in the posterior portion of the large intestine, including both the ventral and dorsal regions (Takashima, 2001).

dpp is expressed in two overlapping regions of the large intestine; these regions appear to be regulated independently. dpp expression at the posterior end of the large intestine depends on hh activity in the adjacent rectum, whereas the weak expression of dpp in the ventral domain of the large intestine is not affected in the hh mutant. In the dorsal domain of the large intestine, where dpp is not expressed except in the posterior-most portion, en is expressed throughout development. Double staining for En protein and dpp mRNA reveal that the en-domain and the dpp-domain do not overlap. To analyze the regulatory relationship between dpp and en, dpp expression was examined in an en mutant, in which en and its paralog invected (inv) are deficient. Expression of dpp expands to the dorsal domain of the large intestine in the en mutant, but overall morphology of the hindgut is almost normal except for a slight overgrowth. Repression of dpp by en is also demonstrated by ectopic expression of en. When en is expressed throughout the hindgut with the GAL4-UAS system, dpp expression in the hindgut becomes very weak except in the posterior-most portion of the large intestine, where the hh signal from the adjacent rectum activates dpp expression (Takashima, 2001).

It should be noted that wg and hh mutations result in a short hindgut, and these mutations are associated with the reduction of dpp expression in the large intestine. It is very likely that suppression of the growth of the large intestine correlates with the decrease in dpp expression. The effect of dpp mutation on the development of the hindgut was therefore examined (Takashima, 2001).

The hindgut of the dpp mutant embryo is of almost normal length based on observation of its overall morphology. However, by in situ hybridization with a byn probe, which detects the whole hindgut and anal pads, strong homozygous dpp mutant embryos show a significantly shorter hindgut. In these embryos, the anal pads and posterior abdomen are abnormally internalized, forming a tube-like structure continuous to the hindgut orifice. dpp mutation does not affect hh expression in the small intestine or rectum, and these parts develop almost normally. The short hindgut observed in dpp mutants could be a consequence of the failure of normal growth of the large intestine. Consistent with this idea, when dpp is ectopically expressed throughout the hindgut by the GAL4-UAS system, excessive growth of the hindgut is induced. Excessive growth is observed only when the patched-GAL4, in which GAL4 strongly expressed throughout hindgut in stages 9-11, is used as a driver (Takashima, 2001).

The growth of the hindgut after stage 12 is not associated with cell division but with endoreplication. DNA synthesis in the hindgut cells of dpp mutant and wild-type embryos was compared by incorporation of BrdU. In wild-type embryos, all epithelial cells of the large intestine incorporate BrdU. It is noteworthy that only the large intestine shows DNA replication in the hindgut. In dpp embryos, BrdU incorporation is completely abolished (Takashima, 2001).

Dpp and wing morphogenesis

In the Drosophila wing imaginal disc, the Hedgehog (Hh) signal molecule induces the expression of decapentaplegic in a band of cells abutting the anteroposterior (A/P) compartment border. It has been proposed that Dpp organizes the patterning of the entire wing disc. This proposal was tested by studying the response to distinct levels of ectopic expression of Hh and Dpp, using the sensory organ precursors (SOPs) of the wing and notum and the presumptive wing veins as positional markers. Mis-expression of Dpp does not mimic all the effects of mis-expression of Hh. Dpp specifies the position of most SOPs in the notum and of some of them in the wing. Close to the A/P compartment border, however, SOPs are specified by Hh rather than by Dpp alone. Late signaling by Hh, after setting up dpp expression, is responsible for the formation of vein 3 and the scutellar region, and also for the determination of the distance between veins 3 and 4. One of the genes that mediates the Hh signal is the zinc-finger protein Cubitus interruptus (Ci). Ectopic Ci has an effect on SOP distribution similar to that observed by the induction of Hh expression. These results indicate that Hh has a Dpp-independent morphogenetic effect in the region of the wing disc near the A/P border (Mullor, 1997).

Multiple BMPs are required for growth and patterning of the Drosophila wing. The Drosophila BMP gene, Tgfbeta-60A, exhibits a requirement in wing morphogenesis distinct from that shown previously for dpp. Tgfbeta-60A mutants exhibit a loss of pattern elements in the wing, particularly those derived from cells in the posterior compartment, consistent with the Tgfbeta-60A mRNA and protein expression pattern. Individuals homozygous for null alleles of the Tgfbeta-60A gene, exhibit embryonic defects in gut morphogenesis and result in early larval lethality. The structures in the wing that are affected most dramatically by mutations in Tgfbeta-60A, the PCV and L5, are those that are least sensitive to the reduction or absence of dpp. The ACV and longitudinal veins L2 and L4, lying on either side of the A/P boundary, have been shown to be most sensitive to the loss of Dpp signaling, consistent with the proposal that Dpp organizes wing pattern via a morphogen gradient emanating from the A/P boundary. (It has been proposed that longitudinal vein L3 is fated by a different mechanism and as a result is not sensitive to the level of Dpp signaling). Since mutations in the Tgfbeta-60A locus indicate that Tgfbeta-60A is essential for establishing cell identity in the wing, especially within the posterior compartment, the possibility was investigated that Tgfbeta-60A and Dpp signal together to provide positional information for the entire wing. No dominant genetic interactions are observed between alleles of Tgfbeta-60A and dpp, therefore, recombinant chromosomes were constructed using alleles of dpp that result in an overall lowering of dpp expression in the imaginal discs. Individuals heterozygous for such hypomorphic dpp alleles are phenotypically wild type. As homozygotes or in various heteroallelic combinations, these dpp alleles can be lethal or can generate adults with appendage defects ranging from minor loss of vein material to truncations of the entire appendage. An individual heterozygous for a hypomorphic dpp allele and homozygous or transheterozygous for Tgfbeta-60A alleles exhibits wing phenotypes qualitatively different from those observed in the Tgfbeta-60A mutant alone. Tgfbeta-60A mutants heterozygous for a dpp hypomorphic allele exhibit a significant loss of L4 (>60% lack more than half of L4) and a more frequent loss of the ACV. Tgfbeta-60A mutants heterozygous for a more severe dpp allele, not only show a greater loss of L4, ACV and L2 vein material (>90% lack more than half of L4 and 100% lack the entire ACV including a thinning of L2), but also a loss of intervein tissue, especially between L2/L3 and L4/L5, as exhibited by a reduction in the overall size of the wing. In addition to defects in wing patterning, abnormalities were noted in the proximal/distal organization of the legs, a process in which dpp is known to play a central role. The common defects exhibited by the Tgfbeta-60A mutants combined with dpp hypomorphic alleles are truncations and/or apparent fusions of the distal most tarsal segments of the male prothoracic leg. These data indicate that heteroallelic combinations of Tgfbeta-60A and dpp result in phenotypes more pronounced and distinct from phenotypes observed as a result of mutations in either gene alone. These new phenotypes are not simply additive. Individual pattern elements are affected differently as a result of these heteroallelic combinations, for example, a greater loss of L4 is seen while the loss of L5 is reduced. These results suggest that the requirement for both dpp and Tgfbeta-60A in earlier stages of larval development, presumably at times of high cell proliferation, is not met in such complex genotypes. Yet the suppression of the ACV defect indicates that Tgfbeta-60A does not solely act to modulate levels of Dpp signaling; rather, as the ratio of Tgfbeta-60A to dpp changes across the wing imaginal disc, the specification of a pattern element is affected. Different relative levels of Tgfbeta-60A to Dpp signaling would result in different positional information. The readout may be either synergistic or antagonistic, depending on the particular positional point within the developing wing (Khalsa, 1998).

Tgfbeta-60A alleles have been shown to genetically interact with mutations in BMP type I receptor genes, tkv and sax. The Dpp signal is mediated by two different BMP type I receptors, Tkv and Sax, during wing morphogenesis as well as during other stages of development The possibility of a genetic interaction between alleles of Tgfbeta-60A and alleles of tkv or sax was investigated to address the relative importance of these receptors in mediating the signals resulting from the actions of Tgfbeta-60A and Dpp. Recombinants were constructed between gbb-60A 4 or gbb-60A 1 and several alleles of tkv and sax. The addition into a Tgfbeta-60A mutant background of a chromosomal deficiency that removes the tkv locus, results in a severe mutant wing phenotype with a dramatic loss of both the PCV and ACV and most of L4 and L5. In addition, distal gaps are present in L2 and L3. A less extreme phenotype is seen with tkv6, a hypomorphic allele that retains significant receptor function. The observed interaction between tkv and Tgfbeta-60A cannot be explained solely as a secondary consequence of lowering Dpp signaling readout by the mutation of a receptor that mediates Dpp signaling. These data suggest that Tkv is able to mediate Tgfbeta-60A signaling and that it may do so in different ways at different times during development. The effect of reducing the Tgfbeta-60A copy number was investigated in flies compromised for functional Tkv receptor. Reducing Tgfbeta-60A in a tkv mutant background produces a further thickening of wing veins. This result suggests that Tgfbeta-60A may play a role in vein differentiation itself and/or in the tkv/dpp feedback loop important in defining the boundaries of the vein. Genetic combinations used to investigate the potential interaction between Tgfbeta-60A and sax alleles indicate a reduction in viability for Tgfbeta-60A mutant genotypes containing a single copy of a sax null allele. This reduction in viability suggests that lowering both Tgfbeta-60A and sax compromises development. The wing phenotype of the few viable adults recovered is similar to a very severe Tgfbeta-60A mutant wing phenotype, with a substantial loss of L5, complete loss of the PCV and ACV and loss of half of L4. Clearly the levels of Tgfbeta-60A signaling and Sax function are dependent on one another (Khalsa, 1998).

Based on genetic analysis and expression studies, it has been concluded that Tgfbeta-60A must signal primarily as a homodimer to provide patterning information in the wing imaginal disc. Tgfbeta-60A and dpp genetically interact and specific aspects of this interaction are synergistic while others are antagonistic. It is proposed that the positional information received by a cell at a particular location within the wing imaginal disc depends on the balance of Dpp to Tgfbeta-60A signaling. Furthermore, the critical ratio of Tgfbeta-60A to Dpp signaling appears to be mediated by both Tkv and Sax type I receptors (Khalsa, 1998).

Formation of the longitudinal veins (LVs) of the Drosophila wing involves the interplay among Dpp, Egf and Notch pathways. Formation of crossveins (CVs: see Derivatives of the wing disc) present a paradoxical problem. As shown both morphologically and using molecular markers, the definitive CVs are not formed until long after the initial specification of the LVs. The CVs therefore must form within territory that has already been specified as intervein. The CVs must also interconnect with existing LVs at a time when the Delta expressed by the LVs is thought to inhibit vein formation in adjacent cells. Mechanisms must exist that override both intervein specification and the lateral inhibition of veins, allowing the formation of continuous, interconnected vein tissue. BMP-like signaling plays a special role in the formation of the CVs from within intervein territory. BMP-like signals also help maintain the connections between the LVs and the margin of the wing. crossveinless 2 (cv-2) is a critical factor in these processes, as it is expressed more highly in the CVs and the ends of the LVs and is required for the high levels of BMP-like signaling observed in these regions (Conley, 2000). The cv-2 mutation was first identified by Benedetto Nicoletti in 1962 (FlyBase: Cv-2 site). The structure of the Cv-2 protein strongly suggests that these effects are direct, and that Cv-2 is a novel player in the BMP-like signaling pathway (Conley, 2000).

Both Dpp and Gbb vein signals are mediated largely by the type I receptor Thickveins, rather than the alternate type I receptor Saxophone. Cells lacking Tkv do not form veins, but removal of Sax does not reliably remove veins. However, not all veins are equally sensitive to reductions in Dpp and Gbb signaling. The hypomorphic gbb⁴ mutation shows complete loss of the cross veins (CVs), but only slight loss of the ends of the LVs. Sog encodes a Chordin-like molecule that inhibits BMP-like signaling; both Sog and Chordin are thought to bind to and sequester ligands, preventing the activation of receptors. Overexpressing Sog in the wing specifically blocks formation of the CVs and the ends of the LVs. The secreted Tolloid proteases, similar to vertebrate BMP1s, can increase BMP signaling by cleaving and inactivating Chordin or Sog. Loss of tolkin (also known as tolloid-related) blocks formation of the CVs and the tips of the LVs. Overexpressing a dominant negative form of Sax again induces a similar phenotype (Conley, 2000 and references therein).

Such phenotypes are very reminiscent of the crossveinless class of mutations in Drosophila (reviewed in Garcia-Bellido, 1992). Strong reductions in crossveinless 2 (cv-2) function have been shown to remove the posterior CV (PCV), the anterior CV (ACV), and the ends of the LVs. However, despite the possibility that the crossveinless genes encode novel players in BMP-like signaling, none have been characterized and the sensitivity of CVs to BMP-like signaling has not been explained. Evidence is presented that cv-2 encodes a novel member of the BMP-like signaling pathway, expressed in and required for high levels of BMP-like signaling in the developing cross veins. The Cv-2 protein contains five cysteine-rich domains similar to those known to bind BMP-like ligands, strongly suggesting that Cv-2 directly modulates Dpp or Gbb activity (Conley, 2000 and references therein).

dpp and gbb mutations both disrupt CV formation. Weak cv-2 alleles are strengthened by dpp and gbb loss-of-function mutations. cv-2^225-3/cv-2³⁵¹¹ flies never lack the entire PCV, but 50% of gbb 4 cv-2^225-3/cv-2 3511 flies lack the entire PCV. Similarly, cv-2³⁵¹¹/Df(2R)Pu-D17 only rarely disrupt the ACV, but dpp^d6 cv-2³⁵¹¹/Df(2R)Pu-D17 commonly does. However, cv-2 cannot dominantly enhance earlier dpp-dependent patterning in the wings: dpp^d5 Df(2R)Pu-D17 /dpp^hr4 wings look no worse than dpp^d5/dpp^hr4 wings. To provide a more direct link between cv-2 and Dpp and Gbb signaling, Mad activation was examined in mutant pupal wings. In cv-2¹ adults, the PCV is more reliably disrupted than the ACV; the anti-p-Mad staining normally found near the PCV in 19, 22, 26 and 36 hours after pupariation wings is lost or disrupted in cv-2¹ homozygotes, as is the reduction of anti-DSRF in the PCV. In adults of the stronger allelic combination cv-2¹/Df(2R)Pu-D17, the ACV is also often lost along with the ends of some of the LVs. Interestingly, no disruption of the ACV or LV anti-p-Mad staining cv-2¹/Df(2R)Pu-D17 pupal wings is detected at 21 or 25 hours after pupariation; only at 36 hours after pupariation is staining lost from the ACV. This indicates that cv-2 is required not only to initiate Mad activity in the PCV, but also to maintain that activity in the ACV (Conley, 2000).

The Drosophila BMP5/6/7/8 homolog, glass bottom boat (gbb), has been shown to be involved in proliferation and vein patterning in the wing disc. To better understand the roles for gbb in wing development, as well as its relationship with decapentaplegic, clonal analysis was used to define the functional foci of gbb during wing development. gbb has both local and long-range functions in the disc that coincide both spatially and functionally with the established functions of dpp, suggesting that both BMPs contribute to the same processes during wing development. Indeed, comparison of the mutant phenotypes of dpp and gbb hypomorphs and null clones shows that both BMPs act locally along the longitudinal and cross veins to affect the process of vein promotion during pupal development, and long-range from a single focus along the A/P compartment boundary to affect the processes of disc proliferation and vein specification during larval development. Moreover, duplications of dpp are able to rescue many of the phenotypes associated with gbb mutants and clones, indicating that the functions of gbb are at least partially redundant with those of dpp. While this relationship is similar to that described for dpp and the BMP screw (scw) in the embryo, the mechanisms underlying both local and long-range functions of gbb and dpp in the wing are different. For the local foci, gbb function is confined to the regions of the veins that require the highest levels of dpp signaling, suggesting that gbb acts to augment dpp signaling in the same way as scw is proposed to do in the embryo. However, unlike scw-dependent signals in the embryo, these gbb signals are not transduced by the Type I receptor saxophone (sax), thus, the cooperativity between gbb and dpp is not achieved by signaling through distinct receptor complexes. For the long-range focus along the A/P compartment boundary, gbb function does not appear to affect the high point of the dpp gradient, but, rather, appears to be required for low points, which is the reciprocal of the relationship between dpp and scw in the embryo. Moreover, these functions of gbb also do not require the Type I receptor sax. Given these results, it is concluded that the relationships between gbb and dpp in the wing disc represent novel paradigms for how multiple BMP ligands signal during development, and that signaling by multiple BMPs involves a variety of different inter-ligand relationships that depend on the developmental context in which they act (Ray, 2001).

gbb has two distinct types of functions: local and long range. The local foci are confined to the posterior compartment, and affect the promotion of the posterior cross-vein (PCV) and the distal tips of the longitudinal veins L4 and L5. The long-range focus lies in the anterior compartment comprising a broad band of cells along the A/P compartment boundary and affects disc proliferation and the specification of L5. These gbb foci are coincident with the foci for dpp in the disc, and many of the phenotypes associated with the gbb clones are rescued by additional copies of the dpp locus. Thus, gbb and dpp contribute to the same functions in the disc and gbb functions are to some extent redundant with those of dpp. Comparison of the foci and phenotypes of gbb and dpp mutants and clones indicates that the relationship between gbb and dpp is different for different functions. For promotion of distal tips of L4 and L5, gbb function is restricted to those areas that require the highest levels of dpp signaling, and since these phenotypes can be rescued with additional copies of dpp, it is concluded that gbb is required to augment the levels of dpp signaling. For promotion of the PCV, the case is not so clear. Both gbb and dpp are required for PCV promotion. However, since dpp duplications do not rescue this phenotype, it is possible that gbb and dpp act independently or that the contribution of gbb to this process is sufficiently great that it cannot be compensated for by the additional doses of dpp. The requirement for gbb in the specification of L5 is not consistent with an augmentation of dpp signaling, since gbb mutants and clones do not affect structures specified by the high point of the dpp gradient. Rather, gbb clones affect structures far from the source along the A/P compartment boundary, suggesting that gbb signaling contributes to the low levels of BMP signaling at the extremes of the gradient (Ray, 2001).

Mutant phenotypes are observed only in gbb clones when the mutant tissue encompasses the entirety of the focus on both the dorsal and ventral surfaces of the wing. For example, clones that occupy the dorsal-anterior quadrant of the wing exhibit no defects in the patterning or size of the wing, while clones that occupy both the dorsal-anterior and ventral-anterior quadrants affect both these aspects of wing development. One explanation for this phenomenon is that Gbb exhibits long-range non-autonomy in the disc, and, in fact, there is some evidence for this, since it has been found that small patches of wild-type cells along the A/P compartment boundary in the context of a large mutant clone are able to rescue loss of L5 completely in the posterior compartment. However, gbb clearly does not act in a broadly non-autonomous fashion in all of its functions: gbb clones that cover the PCV or distal L5 exhibit vein defects that respect the clone boundaries, indicating that the presumptive vein cells within the clone cannot be rescued by the wild-type Gbb present in the adjacent cells. For these functions, the 'rescue' observed in single-sided clones implies pattern regulation occurring between the two wing surfaces. Indeed, it has long been asserted that there are signaling events between the dorsal and ventral surfaces of the wing (as have been shown for several genes) whereby loss of veins on one surface can be compensated for by the wild-type pattern in the opposing surface. The requirement for dorsal-ventral overlap observed with gbb mutant clones is indicative of such a signaling mechanism, and given these results, as well as those from previous studies that have shown a requirement for dorsal-ventral overlap in clones of dpp and sog, it is plausible that the BMPs themselves might be responsible for mediating these signaling processes (Ray, 2001).

Perhaps the most striking result from clonal analysis is that the requirements for gbb in the wing disc are localized even though the gene is widely expressed. This result implies that Gbb activity is in some way restricted post-transcriptionally. Two models seem the most likely to account for this effect. First, since it has been shown that all gbb foci are coincident with sites of dpp expression in the disc, it is possible that Gbb and Dpp form heterodimers, and that Gbb is only active in this form. Heterodimer formation has been documented for a number of different TGFß superfamily members, and in some cases heterodimers and homodimers have been shown to have distinct properties. For example, heterodimers of BMP2 or BMP4 and BMP7 are much more potent in the induction of ventral mesoderm and bone induction than their respective homodimers. Activins and Inhibins illustrate a different relationship: the homodimeric Activins having the opposite biological effects of the heteromeric Inhibins (Ray, 2001).

An alternative model is that the restriction of gbb function in the disc is achieved through local activation of Gbb homodimers, which may be achieved by specific agonists expressed within the foci or antagonists expressed everywhere else. Possible agonists include the Drosophila BMP-1 homologs tolloid and tolkin, or Drosophila homologs of the subtilisin-like proprotein convertases or furins, that are thought to be involved in the cleavage of BMP pro-proteins into the active ligand. In addition, the recently characterized secreted protein crossveinless 2 (cv-2) may act as an agonist of BMP signaling specifically in the presumptive crossveins. The antagonist sog is a likely candidate for restricting BMP activity during pupal development (i.e. for vein promotion functions) as it has been shown to be expressed in all intervein cells at this time. Moreover, there is some evidence that sog function in the wing may specifically antagonize gbb, and thus may very well account for the restriction of gbb function to the presumptive veins (Ray, 2001).

Clonal analysis has identified four processes that require gbb during wing development, disc proliferation, specification of the L5 vein territory, promotion of the PCV and promotion of the longitudinal veins L4 and L5. Based on the criteria of comparisons of gbb clone phenotypes with dpp and sax phenotypes, the ability for the gbb mutant phenotypes to be rescued by additional copies of dpp, and the spatial requirements for gbb during wing development, it is clear that each of these functions employs a different relationship between dpp and gbb, and each of these relationships is distinct from that which has been established for dpp and scw in the embryo (Ray, 2001).

During animal development, epithelial cell fates are specified according to spatial position by extracellular signaling pathways. Among these, the transforming growth factor beta/bone morphogenetic protein (TGF-beta/BMP) pathways are evolutionarily conserved and play crucial roles in the development and homeostasis of a wide range of multicellular tissues. This study shows that in the developing Drosophila wing imaginal epithelium, cell clones deprived of the BMP-like ligand Decapentaplegic (DPP) do not die as previously thought but rather extrude from the cell layer as viable cysts exhibiting marked abnormalities in cell shape and cytoskeletal organization. It is proposed that in addition to assigning cell fates, a crucial developmental function of DPP/BMP signaling is the position-specific control of epithelial architecture (Gibson, 2005).

Non-lethal stress treatments (X-radiation or heat shock) administered to Drosophila imaginal discs induce massive apoptosis, which may eliminate more that 50% of the cells. Yet the discs are able to recover to form final structures of normal size and pattern. Thus, the surviving cells have to undergo additional proliferation to compensate for the cell loss. The finding that apoptotic cells ectopically express dpp and wg suggested that ectopic Dpp/Wg signalling might be responsible for compensatory proliferation. This hypothesis was tested by analysing the response to irradiation-induced apoptosis of disc compartments that are mutant for dpp, for wg, or for both. There is compensatory proliferation in these compartments, indicating that the ectopic Dpp/Wg signalling generated by apoptotic cells is not involved. However, this ectopic Dpp/Wg signalling is responsible for the hyperplastic overgrowths that appear when apoptotic ('undead') cells are kept alive with the caspase inhibitor P35. The ectopic Dpp/Wg signalling and the overgrowths caused by undead cells are due to a non-apoptotic function of the JNK pathway. It is proposed that the compensatory growth is simply a homeostatic response of wing compartments, which resume growth after massive cellular loss until they reach the final correct size. The ectopic Dpp/Wg signalling associated with apoptosis is inconsequential in compartments with normal apoptotic cells, which die soon after the stress event. In compartments containing undead cells, the adventitious Dpp/Wg signalling results in hyperplastic overgrowths (Pérez-Garijo, 2009).

The involvement of Dpp/Wg signalling in compensatory proliferation was suggested by the finding that dpp and wg are expressed in apoptotic cells. This, together with the observation of increased proliferation in the vicinity of the apoptotic cells, led to the model that compensatory proliferation is caused by the mitogenic activity of the ectopic Dpp and Wg signals emitted by apoptotic cells (Pérez-Garijo, 2009).

In irradiated discs, the ectopic Dpp/Wg signalling generated by the apoptotic cells is superimposed on the normal Dpp/Wg signalling. The latter is essential for the normal growth of the wing compartments; in dpp^d12 homozygous discs the wings are reduced to a rudiment, and is shown in this study the lack of wg activity results in smaller compartments. These experiments have tested the role of the ectopic Dpp/Wg signalling in size restoration of irradiated discs, that is, the contribution of the apoptotic cells to the process (Pérez-Garijo, 2009).

The ability of P compartments to compensate growth in conditions in which apoptotic cells can produce neither the Dpp nor the Wg signal, or are defective in both signals, was analyzed. The results indicate that the model of compensatory proliferation mentioned above is incorrect. The elimination of ectopic dpp and wg functions in wing discs subjected to massive apoptosis does not impede the restoration of normal size and pattern; in other words, there is compensatory growth without contribution of the Dpp and Wg signals emitted by the apoptotic cells (Pérez-Garijo, 2009).

Having studied compensatory growth only in P compartments, it is just conceivable that Dpp and Wg originated by apoptotic cells in the A compartment might diffuse to the P compartment where they could induce the additional growth necessary to compensate size. This is very unlikely for two reasons. (1) The undead apoptotic cells induce additional proliferation only in their own vicinity. Thus, it is hard to imagine that Dpp/Wg of anterior origin could have an affect on proliferation extending to the entire posterior compartment. Moreover, in the current experiments the cells are not protected by P35; they are not undead cells but regular apoptotic cells that die soon after initiating apoptosis. Therefore the proliferation stimulus they provide would be very short lived. (2) If the Dpp and Wg of apoptotic origin were able to travel a long way across compartment borders, it would be expected that the overgrowths produced by undead cells were not restricted to compartments. For example, in irradiated hh>p35 discs or in en>hid + p35 (and other similar genotypes), in which undead cells belong to the P compartment, the A compartment should also overgrow, stimulated by the Dpp and Wg of posterior origin. In all cases reported, the effect is essentially restricted to the posterior compartment (Pérez-Garijo, 2009).

Thus, although the dpp and wg genes are activated in apoptotic cells, their function appears to be inconsequential. So what is the mechanism responsible for the compensatory growth? One possibility is the existence of some other hitherto undetected signal with mitogenic properties. Although this possibility cannot be ruled out, it appears unlikely because Dpp and Wg are the major growth signals identified in the wing disc after many years of studies. The Dpp pathway has been shown to play a major role in inducing growth in the wing disc; in absence of Dpp activity wing growth is much reduced and an excess of Dpp activity causes additional growth. Moreover, in the experiments in which apoptotic cells are protected with P35, it was found that the absence of Dpp and Wg prevents the appearance of overgrowths, strongly suggesting that these signals are responsible for the additional growth associated with apoptotic cells (Pérez-Garijo, 2009).

It is believed that compensatory growth does not require any special mechanism involving the participation of apoptotic cells. It is the normal process that regulates compartment size that is responsible for restoring normal size after massive apoptosis. It has been shown recently that A and P compartments are autonomous units of size control in the wing disc, i.e., A and P compartments grow autonomously until they reach the final correct size. It has also been shown that the size control mechanism is highly homeostatic. It can adjust to changes in cell size and number, and to differential cell division rates -- alterations in any of these parameters do not produce changes in the final compartment size. As stated above, only the overproduction of Dpp results in breakdown of the size control mechanism (Pérez-Garijo, 2009).

It thought that the compensatory growth after the loss of cells because of irradiation (or any other stress event) is another example of the versatility of the size control mechanism. It is proposed that the massive cell death caused by the irradiation would be equivalent to making the compartment smaller. The irradiated compartment would then restore the correct size simply by performing some additional division. It would be, in effect, an overall regeneration process of the entire blastema, which would be achieved by lengthening the proliferation period, an idea that is supported by observations such that damage to growing discs results in a prolonged growth period. Even a loss of 50% of the cells can be restored if all of the surviving cells divided once. In the wing disc, the length of the division period is about 8-12 hours and therefore only a short delay may be sufficient to allow time for recovery. Thus, irradiated discs would, after some delay caused by the stress, resume growth and the normal control mechanism would stop growth once compartments have reached the final size (Pérez-Garijo, 2009).

Although the ectopic Dpp and Wg signals do not have a role in compensatory proliferation, they are required for the appearance of overgrowths caused by undead cells. A key difference between undead cells and normal apoptotic cells is that the former persistently express Dpp and Wg (probably as a result of JNK activity). In irradiated posterior compartments that comprise undead and non-apoptotic cells, such as, for example, in irradiated hh>p35 discs, the undead cells keep producing the Dpp and Wg signals from shortly after the irradiation and until the end of the proliferation period of the disc. Thus, the non-apoptotic cells receive a continuous supply of the Dpp and Wg mitogens from the undead ones. The result is an overgrowth, which is also associated with abnormal cell differentiation. Both additional growth and abnormal differentiation would be expected in these circumstances, as Dpp and Wg are growth inducers as well as morphogens determining cell pattern and differentiation (Pérez-Garijo, 2009).

The overall conclusion from the above is that the ectopic Dpp and Wg signals generated by apoptotic cells are irrelevant for compensatory proliferation, but are prime factors in the generation of hyperplastic overgrowths caused by undead cells. The question then is why are dpp and wg activated in normal apoptotic cells. It is thought that their activity is a collateral effect of the activation of the JNK pathway after an apoptotic stimulus: γ-irradiation induces JNK activity in the wing disc and radiation-induced apoptosis depends on JNK activity. As expected, in these experiments X-irradiation also induced JNK activity (Pérez-Garijo, 2009).

The function of the JNK pathway appears to be required for the ectopic expression of wg and dpp in apoptotic cells. In experiments in which cell death is blocked with P35 after apoptosis induction, the JNK pathway becomes continuously activated in undead cells and appears to be associated with ectopic wg expression. It is therefore possible that the ectopic activation of dpp and wg in the apoptotic cells could be a consequence of JNK function, rather than a consequence of the apoptotic program. The results strongly support this view: direct activation of JNK via the UAS-hep^act construct in dronc mutant discs, in which apoptosis is much reduced, induces wg and dpp expression. Furthermore, these mutant discs show hyperplastic overgrowths in the spalt domain, where JNK is active (Pérez-Garijo, 2009).

It has been shown that JNK activity induces several cellular functions: the initiation of the apoptotic program, and also other non-apoptotic functions, such as the capacity for cell migration and the ability to induce dpp. It is probable that normal apoptotic cells acquire these other JNK-dependent properties, but that they die very quickly and so these other functions have minimal effects. This is different in undead cells because the JNK activity becomes persistent and, therefore, they can manifest some or all of the JNK non-apoptotic functions: these cells can move and invade neighbouring compartments, and express dpp and wg continuously. It is thought that it is the persistent manifestation of these two non-apoptotic JNK-mediated properties, dpp/wg activation and the induction of cell migration that causes the hyperplastic overgrowth (Pérez-Garijo, 2009).

The implication of the Dpp and Wg signals in hyperplastic overgrowths in Drosophila might have some general significance as their vertebrate homologues, BMP/TGFβ and Wnt, are known to be involved in the generation of tumours in mammals. Moreover, inappropriate function of the JNK pathway is also connected with tumour formation in vertebrates. It is speculated that situations similar to those described in this study might also occur in mammalian cells in which caspase activity is blocked, by virus infections or other causes. This could result in continuous activation of the JNK pathway and, subsequently, of BMP/TGFβ and Wnt, and could eventually produce a tumour (Pérez-Garijo, 2009).

Decapentaplegic and growth control in the developing Drosophila wing

As a central model for morphogen action during animal development, the bone morphogenetic protein 2/4 (BMP2/4)-like ligand Decapentaplegic is proposed to form a long-range signalling gradient that directs both growth and pattern formation during Drosophila wing disc development. While the patterning role of Dpp secreted from a stripe of cells along the anterior-posterior compartmental boundary is well established, the mechanism by which a Dpp gradient directs uniform cell proliferation remains controversial and poorly understood. To determine the precise spatiotemporal requirements for Dpp during wing disc development, this study used CRISPR-Cas9-mediated genome editing to generate a flippase recognition target (FRT)-dependent conditional null allele. By genetically removing Dpp from its endogenous stripe domain, the requirement was confirmed of Dpp for the activation of a downstream phospho-Mothers against dpp gradient and the regulation of the patterning targets spalt, optomotor blind and brinker. Surprisingly, however, third-instar wing blade primordia devoid of compartmental dpp expression maintain relatively normal rates of cell proliferation and exhibit only mild defects in growth. These results indicate that during the latter half of larval development, the Dpp morphogen gradient emanating from the anterior-posterior compartment boundary is not directly required for wing disc growth (Akiyama, 2015).

Dpp spreading is required for medial but not for lateral wing disc growth

Drosophila Decapentaplegic has served as a paradigm to study morphogen-dependent growth control. However, the role of a Dpp gradient in tissue growth remains highly controversial. Two fundamentally different models have been proposed: the 'temporal rule' model suggests that all cells of the wing imaginal disc divide upon a 50% increase in Dpp signalling, whereas the 'growth equalization model' suggests that Dpp is only essential for proliferation control of the central cells. To discriminate between these two models, morphotrap, a membrane-tethered anti-green fluorescent protein (GFP) nanobody was generated and used, that enables immobilization of enhanced (e)GFP::Dpp on the cell surface, thereby abolishing Dpp gradient formation. In the absence of Dpp spreading, wing disc patterning is lost; however, lateral cells still divide at normal rates. These data are consistent with the growth equalization model, but do not fit a global temporal rule model in the wing imaginal disc (Harmansa, 2015).

Read-out of dynamic morphogen gradients on growing domains

Quantitative data from the Drosophila wing imaginal disc reveals that the amplitude of the Decapentaplegic (Dpp) morphogen gradient increases continuously. It is an open question how cells can determine their relative position within a domain based on a continuously increasing gradient. This study shows that pre-steady state diffusion-based dispersal of morphogens results in a zone within the growing domain where the concentration remains constant over the patterning period. The position of the zone that is predicted based on quantitative data for the Dpp morphogen corresponds to where the Dpp-dependent gene expression boundaries of spalt (sal) and daughters against dpp (dad) emerge. The model also suggests that genes that are scaling and are expressed at lateral positions are either under the control of a different read-out mechanism or under the control of a different morphogen. The patterning mechanism explains the extraordinary robustness that is observed for variations in Dpp production, and offers an explanation for the dual role of Dpp in controlling patterning and growth. Pre-steady-state dynamics are pervasive in morphogen-controlled systems, thus making this a probable general mechanism for the scaled read-out of morphogen gradients in growing developmental systems (Fried, 2015).

Dpp and dorsal closure

One of the fundamental events in insect metamorphosis is the replacement of larval tissues by imaginal tissues. Shortly after pupariation the imaginal discs evaginate to assume their positions at the surface of the prepupal animal. This is a very precise process that is only beginning to be understood. In Drosophila, during embryonic dorsal closure, the epithelial cells push the amnioserosa cells, which contract and eventually invaginate in the body cavity. In contrast, during pupariation the imaginal cells crawl over the passive larval tissue following a very accurate temporal and spatial pattern. Spreading is driven by filopodia and actin bridges that, protruding from the leading edge, mediate the stretching of the imaginal epithelia. Although interfering with JNK (Jun N-terminal kinase) and dpp produces similar phenotypic effects suppressing closure, their effects at the cellular level are different. The loss of JNK activity alters the adhesion properties of larval cells and leads to the detachment of the imaginal and larval tissues. The absence of dpp signaling affects the actin cytoskeleton, blocks the emission of filopodia, and promotes the collapse of the leading edge of the imaginal tissues. Interestingly, these effects are very similar to those observed after interfering with JNK and dpp signaling during embryonic dorsal closure (Martin-Blanco, 2000).

Hypomorphic mutations in dpp cause a thoracic cleft phenotype reminiscent of that observed in hep¹. Further, this failure in thorax closure also is observed in several mutant combinations of the dpp receptors thick veins (tkv) and punt, and in the dpp signal transducer medea. dpp in imaginal discs is expressed in a very complex and dynamic pattern, and it accumulates in the stalk cells that will generate the imaginal leading edge. Later, during closure, dpp is found in the anterior part of the dorsal midline, in a fraction of the cells expressing puc. Interestingly, dpp expression is not affected in a hep^r75-null background, in contrast to the embryo, where the expression of dpp in the most dorsal epidermis is controlled by JNK signaling (Martin-Blanco, 2000).

To study the cellular activities depending on dpp during thorax closure, dpp signaling was interfered with by expressing a dominant negative form of the receptor Tkv (Tkv^DN). When Tkv^DN is expressed in the pannier domain, it leads to a strong cleft phenotype, where both heminota remain extremely contracted and isolated. In this condition, an intervening naked cuticle is found joining the heminota. A more restricted ectopic expression is generated with the MZ980-Gal4 line. In this combination, which probably represents a partial reduction in dpp activity, the thorax cleft is attenuated and cuticular polarity defects are observed at the midline. Occasional individuals with stronger phenotypes (similar to those obtained with Pnr-Gal4) are recovered, suggesting that leading-edge cells are determinant for the spreading of the imaginal tissue (Martin-Blanco, 2000).

In contrast to JNK activity-deficient animals, interfering with dpp signaling does not appear to affect the integrity of the larval epidermis. The lack of spreading of the imaginal sheets in the absence of Tkv activity appears to be due to extreme compacting of the actin cytoskeleton at the leading edge. No filopodia appear to be generated from the imaginal epithelium, and imaginal cells progressively are pulled together, causing bunching of the epidermis. This phenotype is reminiscent of the cellular defects of embryos mutant for tkv. In these embryos, during embryonic dorsal closure, epidermal cells appear to elongate correctly, but they become misdirected, generating a phenotype of epidermal bunching. Taken together, these data indicate that dpp signaling during thorax closure is involved in the maintenance of cell polarity and the control of the actin cytoskeleton (Martin-Blanco, 2000).

Imaginal disc epithelia have the general characteristics of other epithelia in Drosophila and in other organisms. The discs have a basal surface lined with a fibrous basal lamina and an apical surface at which cells are connected at their ends by a series of specialized junctions, including zonula adherens, gap, and septate junctions. Before eversion, the squamous cells of the peripodial epithelium are folded and adhere to the basal lamina. Just before eversion takes place, the cells detach from the basal lamina; the epithelial cells then columnarize and the accompanying contraction forces the discs to evert through the peripodial stalks. Stalk widening and disc eversion appear to result from microfilament contraction, which leads to dramatic changes in cell shape, rather than from changes in membrane adhesiveness (Martin-Blanco, 2000).

There are some differences between dorsal closure and imaginal spreading. During embryonic closure, the amnioserosa and the epidermal cells keep their relative positions constant, and despite occasional delaminations, amnioserosa cells remain in place until the very end of the process. They detach from the overlying epidermis only upon closure completion, become dispersed into the body cavity, and undergo apoptosis. By contrast, during disc spreading, imaginal cells crawl over the larval epidermis. In this process, larval cells are left below and behind and eventually delaminate from the edges (Martin-Blanco, 2000).

Spreading and fusion of epidermal cells could be directed by different mechanisms. In adult vertebrates, cells at the edge of cutaneous wounds extend lamellipodia and drag themselves forward. In contrast, in vertebrate embryos, wound-edge cells remain blunt-faced and the force to draw the wound edges together seems to be provided by a purse-string-like contraction of a thick cable of actin at the leading edge. This mechanism also applies to some developmental processes, such as Xenopus gastrulation and the late stages of C. elegans ventral enclosure. In Drosophila, purse-string contraction appears to be the mechanical force leading embryonic dorsal closure. Actin and non-muscle myosin accumulate at the leading edge of the epithelium; mutations in hep or in zipper (the gene coding for non-muscle myosin) that abolish actin and non-muscle myosin accumulation yield dorsal-open phenotypes (Martin-Blanco, 2000).

These data suggest that the spreading of the imaginal epithelium is active and led by cells at the boundary, although a contribution of the rest of the cells of the epithelia may be possible. Forward locomotion of imaginal cells probably will involve contraction of intracellular actomyosin filaments. Thick filopodia connecting cells to the contralateral heminota are also observed. These multibranched filopodia, which protrude out of leading-edge cells, expand over the larval surface and eventually form actin bridges. Upon contact, they seem to exert a mechanical force, pulling the imaginal tissues together.

The mechanical role of thick filopodia involved in imaginal spreading is conserved in other developmental processes such as gastrulation in the sea urchin embryo and the epiboly of the C. elegans hypodermis. In sea urchin, primary and secondary mesenchyme cells extend filopodia as they move, making contacts with the ectoderm. During ventral enclosure in the nematode, leading cells display actin-rich filopodia; treatment with cytochalasin D immediately halts the process (Martin-Blanco, 2000).

One important characteristic of epithelial fusion in a developmental context is the precise recognition of the contralateral parts. During embryonic dorsal closure in Drosophila, a perfect match links the anterior and posterior compartments of each segment across the midline. During pupariation, the spreading of the imaginal tissues results in the alignment of notal landmarks along the anterior-posterior axis. When rare mismatches occur, anterior cells never match to posterior ones; rather, they meet anterior cells of distinct contralateral segments. This accurate identification suggests that different positional values must be present in different cells at the leading edge and, importantly, a mechanism should exist that allows perception of these differences at a distance (Martin-Blanco, 2000).

Thorax closure starts at the anterior end of the wing disc, proceeds through the most posterior region, and, finally, fills the gap. This regulated cadence also has been observed in an independent study. Timing also is regulated during embryonic dorsal closure. In this process, spreading and fusion proceed from both ends of the embryo, showing segmental periodicity (Martin-Blanco, 2000).

Contact guidance is a mechanism that directs migration or spreading; in discs, contact guidance could be mediated by filopodial tracts making appropriate informative contacts at the contralateral discs. This situation would be reminiscent of sea urchin gastrulation, where thin filopodia are involved in cell-cell signaling. This function also has been suggested for cytonemes, actin-rich, thin filopodia present on Drosophila imaginal discs (Martin-Blanco, 2000).

The loss of dpp signal during embryonic dorsal closure causes the pulling together of the leading edges of segments into bunches. This phenotype appears to be due to defects in the leading-edge cytoskeleton, resulting in a misregulated contraction. Similar defects are seen after interfering with dpp signaling during imaginal disc spreading. Bunches develop at the leading edge, resulting in the excessive contraction of the epithelium. The role of dpp in imaginal and embryonic fusion appears to be conserved in related developmental processes. During palate fusion in vertebrates, the transforming growth factor beta3, a homolog of Dpp, is expressed in the cells that will form the palate suture. Mutations in transforming growth factor beta3 cause palate clefts in homozygous null mice. In these mice, in contrast to wild-type animals, filopodia-like structures are not present on the surface of the medial-edge epithelial cells. Thus, dpp appears to be involved in the regulation of cytoskeleton dynamics along the leading edge of epithelia, although how its activity is implemented is not known (Martin-Blanco, 2000).

Dpp and leg morphogenesis

Continued: Decapentaplegic Effects of Mutation part 3/3

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