Local BMP receptor activation at adherens junctions in the Drosophila germline stem cell niche

According to the stem cell niche synapse hypothesis postulated for the mammalian haematopoietic system, spatial specificity of niche signals is maximized by subcellularly restricting signalling to cadherin-based adherens junctions between individual niche and stem cells. However, such a synapse has never been observed directly, in part, because tools to detect active growth factor receptors with subcellular resolution were not available. This study describes a novel fluorescence-based reporter that directly visualizes bone morphogenetic protein (BMP) receptor activation and shows that in the Drosophila testis a BMP niche signal is transmitted preferentially at adherens junctions between hub and germline stem cells, resembling the proposed synapse organization. Ligand secretion involves the exocyst complex and the Rap activator Gef26, both of which are also required for Cadherin trafficking towards adherens junctions. It is therefore proposed that local generation of the BMP signal is achieved through shared use of the Cadherin transport machinery (Michel, 2011).

In keeping with the stem cell niche synapse hypothesis, a BMP niche signal in the Drosophila testis is transduced at subcellularly confined sites associated with adherens junctions between hub cells and GSCs. Although BMP ligands are also produced by the somatic CySCs, BMP receptor activation is not detected at the GSC surfaces facing the CySCs. There are several nonexclusive explanations that may contribute to this observation. Either, niche signalling is indeed dominated by the homodimeric Dpp or heterodimeric Dpp/Gbb ligands that are produced preferentially by the hub cells. In support of this idea, Dpp but not Gbb can fully suppress bam transcription upon ectopic expression, and is, at least in the wing, thought to have higher signalling activity. Alternatively, signalling from the CySCs may occur diffusely over the entire GSC surface and thus become diluted below the detection threshold of the reporter. Finally, based on the expression profile of the BMP ligands signalling from the CySCs is presumably dominated by Gbb and may therefore preferentially act through the alternative type I BMP receptor Saxophone, thus avoiding detection by a Tkv-specific reporter (Michel, 2011).

However, without artificial Jak/Stat pathway over-activation in the somatic cells of the testis, the CySC-derived BMP signal is by itself not sufficient to maintain GSC fate. Consequently, GSC detachment form the hub induces Bam derepression6 indicating a loss of BMP pathway activation. The junction-associated BMP signal from the hub to the germline, described in this study, is therefore essential for GSC maintenance (Michel, 2011).

In addition, this study shows that trafficking of both Dpp and DE-Cadherin in the hub cells involves the exocyst complex and the Rab11-positive recycling compartment. It is proposed that the local release of the junctional BMP signal is achieved through this shared use of intracellular machinery. Admittedly, RNAi-mediated inactivation of the exocyst complex is bound to have pleiotropic effects, and it cannot be excluded that the secretion of Upd or other growth factors may not also be affected. Can the observed loss of GSC stemness following exocyst knockdown therefore be directly attributed to a loss of BMP signalling from the hub? This is believed to be the case, because loss of Jak/Stat signalling in the germline would primarily be expected to affect adhesion of the GSCs to the hub. Although this loss of contact secondarily causes Bam derepression, Bam expression was also observed in GSCs still adhering to the hub. As Bam expression indicates a loss of BMP signalling also in the testis, this is attributed directly to the loss of the junction-associated BMP signal that is directly detected using a reporter that detects BMP receptor activation (Michel, 2011).

Future studies are required to address what directs Dpp secretion within the hub cells towards the adherens junctions with the overlying GSCs rather than towards those facing the adjacent hub cells. In addition, how the BMP ligands are confined after secretion to prevent lateral diffusion away from the site of release can now be studied. It is likely that for the latter proteoglycans has an essential role (Michel, 2011).

Finally, it was shown that the exocyst is also required for generation of the Dpp signal in the wing disc, where it forms a long-range morphogen gradient rather than a contact-dependent niche signal. It will be interesting to test whether this reflects a specific requirement of planar transcytosis, with the junctions forming a two dimensional network of signalling synapses. Alternatively, as suggested by zebrafish experiments, subcellularly restricted signal transduction at intercellular junctions may be a more general mechanism operating also in systems where BMP ligands spread through extracellular diffusion (Michel, 2011).

Disruption of axonal transport perturbs Bone Morphogenetic Protein (BMP) - signaling and contributes to synaptic abnormalities in rwo neurodegenerative diseases

Formation of new synapses or maintenance of existing synapses requires the delivery of synaptic components from the soma to the nerve termini via axonal transport. One pathway that is important in synapse formation, maintenance and function of the Drosophila neuromuscular junction (NMJ) is the bone morphogenetic protein (BMP)-signaling pathway. This study shows that perturbations in axonal transport directly disrupt BMP signaling, as measured by its downstream signal, phospho Mad (p-Mad). Components of the BMP pathway genetically interact with both kinesin-1 and dynein motor proteins. Thick vein (TKV) vesicle motility is also perturbed by reductions in kinesin-1 or dynein motors. Interestingly, dynein mutations severely disrupted p-Mad signaling while kinesin-1 mutants showed a mild reduction in p-Mad signal intensity. Similar to mutants in components of the BMP pathway, both kinesin-1 and dynein motor protein mutants also show synaptic morphological defects. Strikingly TKV motility and p-Mad signaling are disrupted in larvae expressing two human disease proteins; expansions of glutamine repeats (polyQ77) and human amyloid precursor protein (APP; see Drosophila Appl) with a familial Alzheimer's disease (AD) mutation (APPswe). Consistent with axonal transport defects, larvae expressing these disease proteins show accumulations of synaptic proteins along axons and synaptic abnormalities. Taken together these results suggest that similar to the NGF-TrkA signaling endosome, a BMP signaling endosome that directly interacts with molecular motors likely exists. Thus problems in axonal transport occurs early, perturbs BMP signaling, and likely contributes to the synaptic abnormalities observed in these two diseases (Kang, 2014 - Open access: 25127478).

Effects of Mutation

Genes that interact with thick veins

A screen was carried out for dominant enhancer mutations magnifying the effects of a hypomorphic allele of thick veins, a type I receptor for dpp. tkv 6 is a mutation in a splice acceptor site that results in aberrant in-frame splicing, deleting two extracellular amino acids of the receptor. When expressed in COS1 cells, the mutant receptor fails to bind BMP-2 homodimers. However, tkv 6 behaves genetically as a hypomorph. In contrast to the embryonic lethal tkv null alleles, tkv 6 is homozygous viable: the only visible phenotype is the thickened wing veins. All other imaginal-disc-derived structures of tkv 6 homozygotes appear normal. Interestingly, tkv 6/Df(2L)tkv2 flies are phenotypically identical to tkv 6 homozygotes. To test if tkv 6 is a suitable genetic background for a modifier screen, the effects of lowering the activity of other known dpp pathway components were examined. Heterozygous mutations in shn or punt enhance the tkv 6 homozygous phenotype. In the tkv 6 background, shn IB is a dominant enhancer of the venation pattern in the wing and the proximal/distal patterning of the leg. In the wing, longitudinal vein 2 fails to reach the wing margin. In the leg, distal elements such as claws and distal tarsal segments are deleted. Such phenotypes are reminiscent of hypomorphic dpp phenotypes. punt 135 also enhances the tkv 6 phenotypes. Based on these observations, it was reasoned that the dpp signaling output through the mutant receptor tkv 6 is near the threshold for proper patterning of the imaginal discs. The tkv 6 mutation is therefore an appropriate genetic background for identifying new components essential for mediating dpp signaling. Enhancers of tkv 6 are phenotypically similar to dpp mutants. The enhancers are recessive lethal in a wild-type background. tkv 6 homozygotes that are heterozygous for the enhancer mutations have defects in imaginal disc development. During pupal development, the dorsal proximal region of the two wing imaginal discs fuse to form the adult notum. A heterozygous mutation, mapping to Tgfbeta-60A, causes deletions of distal and dorsal structures and occasional duplication of ventrolateral structures such as sex combs on male prothoracic legs. These phenotypes are indistinguishable from those of dpp disk alleles, suggesting that this enhancer acts in the dpp signal transduction pathway (Y. Chen, 1998).

Meiotic mapping and complementation tests have established seven complementation groups for the enhancers. New alleles of tkv, Mad, Medea and punt have been recovered. Of the five Mad alleles, three have point mutations in the coding region. Missense mutations were found in both new punt alleles. The tkv D17 and Med D5 allelism is based on genetic non-complementation. A tkv transgene rescues the lethality of tkv D17 homozygotes, supporting the view that D17 is a tkv allele. Genetic and molecular characterizations of the D4 complementation group reveal that it corresponds to the Tgfbeta-60A gene. Tgfbeta-60A encodes a BMP-7 homolog, isolated on the basis of its sequence homology. Its function has been unknown due to the lack of mutations in Tgfbeta-60A. Three alleles of Tgfbeta-60A were confirmed by sequencing the mutant alleles. 60A D8 and 60A D20 are nonsense mutations in the prodomain due to single nucleotide substitutions. 60A D4 has one nucleotide deletion, causing a frame-shift premature stop in the prodomain (Y. Chen, 1998).

p38 mitogen-activated protein kinase (p38) has been extensively studied as a stress-responsive kinase, but its role in development remains unknown. D-p38's inhibit antimicrobial peptide production in cultured cells. D-p38's are also activated by stress-inducing and inflammatory stimuli, such as UV irradiation, high osmolarity, heat, serum starvation, H202 and LPS. Drosophila has two p38 genes: D-p38a, which maps to 95E4-to-95F1, and D-p38b , which maps to the 34D region. To elucidate the developmental function of the Drosophila p38's, various genetic and pharmacological manipulations were used to interfere with their functions: expression of a dominant-negative form of D-p38b; expression of antisense D-p38b RNA; reduction of the D-p38 gene dosage, and treatment with the p38 inhibitor SB203580. Expression of a dominant-negative D-p38b in the wing imaginal disc causes a decapentaplegic-like phenotype and enhances the phenotype of a dpp mutant. Inhibition of D-p38b function also causes the suppression of the wing phenotype induced by constitutively active Tkv (TkvCA). Mosaic analysis reveals that D-p38b regulates the Tkv-dependent transcription of the optomotor-blind (omb) gene in non-Dpp-producing cells, indicating that the site of D-p38b action is downstream of Tkv. Furthermore, forced expression of TkvCA induces an increase in the phosphorylated active form(s) of D-p38(s). These results demonstrate that p38, in addition to its role as a transducer of emergency stress signaling, may function to modulate Dpp signaling. It is hypothesized that a D-p38b cascade exists that can be involved in Dpp signaling and that includes D-MKK3 and a homolog of TGF-beta-activated kinase 1 (TAK1). While Mad is directly phosphorylated by Tkv, the activation of D-p38 by TkvCA may be indirect. It is also possible that one or more transcription factors functioning in the Dpp response requires prior phosphorylation mediated by the D-p38 cascade, a process that is regulated independent of Tkv (Adachi-Yamada, 1999).

The Drosophila tumor suppressor gene lethal(2) giant larvae (lgl) participates in the release of Decapentaplegic (Dpp), a member of the transforming growth factor ß (TGFß) family that functions in various developmental processes. lgl is required downstream of dpp and upstream of its receptor Thickveins (Tkv) for the dorsoventral patterning of the ectoderm. During larval development, the expression of spalt, a dpp target, is abolished in mutant wing discs, while it is restored by a constitutively activated form of Tkv (TkvQ253D). Taking into account that the activation of dpp expression is unaffected in the mutant, this suggests that lgl function is not required downstream of the Dpp receptor. The function of lgl responsible for the activation of Spalt expression appears to be required only in the cells that produce Dpp, and lgl mutant somatic clones behave non autonomously. The activity of lgl is therefore positioned in the cells that produce Dpp, and not in those that respond to the Dpp signal. These results are consistent with the same role for lgl in exocytosis and secretion as that proposed for its yeast ortholog sro7/77: lgl might function in parallel or independently of its well-documented role in the control of epithelial cell polarity (Arquier, 2001).

spinster (spin), which encodes a multipass transmembrane protein, has been identified in a genetic screen for genes that control synapse development. spin mutant synapses reveal a 200% increase in bouton number and a deficit in presynaptic release. spin is expressed in both nerve and muscle and is required both pre- and postsynaptically for normal synaptic growth. Spin has been localized to a late endosomal compartment and evidence is presented for altered endosomal/lysosomal function in spin mutants. Evidence is presented that synaptic overgrowth in spin is caused by enhanced/misregulated TGF-ß signaling. TGF-ß receptor mutants show dose-dependent suppression of synaptic overgrowth in spin. Furthermore, mutations in Dad, an inhibitory Smad, cause synapse overgrowth. A model is presented for synaptic growth control with implications for the etiology of lysosomal storage and neurodegenerative disease (Sweeney, 2002).

To determine whether synaptic overgrowth in spin is caused by enhanced TGF-ß signaling, it was asked whether TGF-ß signaling is necessary for synaptic overgrowth in spin. The type II receptor mutation wishful thinking causes a severe decrease in bouton number at the NMJ. Type I TGF-ß receptors are known to function in concert with type II receptors, and the type I receptors tkv and sax participate in synaptic growth regulation in this system. Third instar larva mutant for sax or tkv have smaller neuromuscular synapses. This study confirms that there is a significant decrease in bouton number in wit, and that there is a similar decrease in bouton number in both tkv and sax. These receptors are shown to function in the larval motoneurons by demonstrating that pMAD staining in the cell bodies of larval motoneurons requires wit or sax. In this experiment, the larval CNS was costained with pMAD and anti-evenskipped, which labels a subset of motoneurons (Sweeney, 2002).

A genetic analysis of the TGF-ß receptor mutations wit, tkv, and sax in combination with spin demonstrates that TGF-ß signaling is necessary for synaptic overgrowth in spin. Heterozygous mutations in tkv, sax, and wit do not alter synaptic bouton numbers at the NMJ. Heterozygous mutations in tkv, sax, and wit suppress synaptic overgrowth when placed in the spin mutant background. Bouton numbers are significantly reduced in each case where a single copy of a receptor is mutated in combination with spin. Bouton numbers were quantified in each of the double mutant combinations of tkv, sax, or wit with spin. In each case, when both copies of a receptor were removed, synaptic overgrowth was suppressed in the spin mutant background further than when only a single copy of a receptor was mutated. These data demonstrate that TGF-ß receptor mutations suppress synaptic overgrowth in spin in a dose-dependent manner. Furthermore, since bouton numbers return to wild-type, or below wild-type levels, it demonstrates that TGF-ß signaling is necessary for synaptic overgrowth in spin. Taken together with the increase in bouton numbers seen in dad, these data support the conclusion that enhanced or misregulated TGF-ß signaling is a major determinant of synaptic overgrowth in spin. It is hypothesized that altered endosomal function due to loss of Spin causes enhanced TGF-ß signaling and subsequent synaptic overgrowth. Future experiments will be necessary to determine whether enhanced signaling is due to increased receptor number at the plasma membrane, or an inability to stop signaling within the late endosomal system (Sweeney, 2002).

Overactivation of receptor tyrosine kinases (RTKs) has been linked to tumorigenesis. To understand how a hyperactivated RTK functions differently from wild-type RTK, a genome-wide systematic survey was conducted for genes that are required for signaling by a gain-of-function mutant Drosophila RTK Torso (Tor). Chromosomal deficiencies were screened for suppression of a gain-of-function mutation tor (torGOF); this screen led to the identification of 26 genomic regions that, when in half dosage, suppress the defects caused by torGOF. Testing of candidate genes in these regions revealed many genes known to be involved in Tor signaling (such as those encoding the Ras-MAPK cassette, adaptor and structural molecules of RTK signaling, and downstream target genes of Tor), confirming the specificity of this genetic screen. Importantly, this screen also identified components of the TGFß (Dpp) and JAK/STAT pathways as being required for TorGOF signaling. Specifically, it was found that reducing the dosage of thickveins (tkv), Mothers against dpp (Mad), or STAT92E (aka marelle), respectively, suppress torGOF phenotypes. Furthermore, it has been demonstrated that in torGOF embryos, dpp is ectopically expressed and thus may contribute to the patterning defects. These results demonstrate an essential requirement of noncanonical signaling pathways for a persistently activated RTK to cause pathological defects in an organism (Lia, 2003).

Traditional screens aiming at identifying genes regulating development have relied on mutagenesis. A new gene has been identified involved in bristle development, identified through the use of natural variation and selection. Drosophila melanogaster bears a pattern of 11 macrochaetes per heminotum. From a population initially sampled in Marrakech, a strain was selected for an increased number of thoracic macrochaetes. Using recombination and single nucleotide polymorphisms, the factor responsible was mapped to a single locus on the third chromosome, poils au dos (French for 'hairy back'), that encodes a zinc-finger-ZAD protein. The original, as well as new, presumed null alleles of poils au dos are associated with ectopic achaete-scute expression that results in the additional bristles. This suggests a possible role for Poils au dos as a repressor of achaete and scute. Ectopic expression appears to be independent of the activity of known cis-regulatory enhancer sequences at the achaete–scute complex that mediate activation at specific sites on the notum. The target sequences for Poils au dos activity were mapped to a 14 kb region around scute. In addition, pad has been shown to interact synergistically with the repressor hairy and with Dpp signaling in posterior and anterior regions of the notum, respectively (Gibert, 2005).

Mutations in very few genes have been shown to induce ectopic bristles in the anterior region of the notum. Some ectopic bristles can be induced in this region by reduction in Dpp signaling late in development. A genetic interaction between pad and Dpp signaling was tested using mutations in the receptors punt (put) and thickveins (tkv). A strong genetic interaction was observed between pad1 and putP1. Trans-heterozygous putP1/pad1 flies have ectopic DC bristles whereas each of the single heterozygotes displays a wild-type pattern. Flies homozygous for the hypomorphic mutation tkv1 occasionally have ectopic bristles anterior to the aDC at 18°C. The phenotype is strongly enhanced in the anterior region of the notum of double mutant tkv1; pad1 flies grown at 25°C. In particular, many more ectopic bristles are visible around the prescutal suture than in pad1 alone (Gibert, 2005).

Dally regulates Dpp morphogen gradient formation by stabilizing Dpp on the cell surface: Dally antagonizes the effect of Thickveins on Dpp signaling

Decapentaplegic, a Drosophila homologue of bone morphogenetic proteins, acts as a morphogen to regulate patterning along the anterior-posterior axis of the developing wing. Previous studies showed that Dally, a heparan sulfate proteoglycan, regulates both the distribution of Dpp morphogen and cellular responses to Dpp. However, the molecular mechanism by which Dally affects the Dpp morphogen gradient remains to be elucidated. This study characterized activity, stability, and gradient formation of a truncated form of Dpp (DppΔN), which lacks a short domain at the N-terminus essential for its interaction with Dally. DppΔN shows the same signaling activity and protein stability as wild-type Dpp in vitro but has a shorter half-life in vivo, suggesting that Dally stabilizes Dpp in the extracellular matrix. Furthermore, genetic interaction experiments revealed that Dally antagonizes the effect of Thickveins (Tkv; a Dpp type I receptor) on Dpp signaling. Given that Tkv can downregulate Dpp signaling by receptor-mediated endocytosis of Dpp, the ability of dally to antagonize tkv suggests that Dally inhibits this process. Based on these observations, a model is proposed in which Dally regulates Dpp distribution and signaling by disrupting receptor-mediated internalization and degradation of the Dpp-receptor complex (Akiyama, 2008)

The Dpp pathway is regulated by multiple cell surface and extracellular factors. In the developing wing, Dally is one of the key molecules that modulate Dpp signaling. It affects the shape of the Dpp ligand gradient (protein distribution) as well as its activity gradient (spatial patterns of signaling activity). Dally and Dpp expressed in S2 tissue culture cells are coimmunoprecipitated, suggesting that Dally forms a complex with Dpp. It was also observed that Dally colocalizes with Dpp and Tkv in cells. In addition, Dally enhances Dpp signaling in a cell autonomous fashion. These findings suggest that Dally acts as a Dpp co-receptor at least in some developmental contexts. Interestingly, however, in embryos and in imaginal disc cells close to Dpp-expressing cells, Dpp can mediate signaling without Dally, indicating that HS is not absolutely required for all BMP-dependent processes in vivo (Akiyama, 2008)

DppΔN, which does not bind to heparin, fails to interact with Dally. The easiest interpretation of this result is that wild-type Dpp interacts with Dally via its HS chains. However, a recent study using Surface Plasmon Resonance showed that binding of BMP4 to Dally is not fully inhibited by excess HS. Also, a mutant form of Dally, which does not undergo HS modification, is able to significantly rescue dally mutant phenotypes. These findings suggest that the BMP-glypican interaction is not entirely dependent on the HS chains. One possible explanation for the failure of DppΔN to bind to Dally is that Dpp normally binds to Dally through both the HS chains and its protein core, and DppΔN has reduced affinities for both sites (Akiyama, 2008)

Although DppΔN lacks the ability to interact with Dally, it shows normal in vitro protein stability and signaling activity. Therefore, this truncated form of Dpp provided a powerful system to gain insight into the functions of Dally in distribution and signaling of the Dpp morphogen: this molecule was used to elucidate the consequences of lacking the ability to bind HSPGs. In the wing disc, DppΔN cannot form a normal gradient: only a low level of DppΔN was detected in the Dpp-receiving cells. Notably, this pattern of DppΔN resembles the Dpp ligand and activity gradients observed in dally mutant wing discs. A series of in vitro and in vivo Dpp stability assays suggested that DppΔN forms a shallow gradient because it is remarkably unstable in the matrix and that the stability of Dpp depends on its interaction with Dally (Akiyama, 2008)

Genetic experiments revealed that Tkv and Dally have opposite effects on Dpp gradient formation during wing development. Dally and Tkv share some common properties as components of the Dpp signaling complex: they both autonomously enhance Dpp signaling and limit migration of Dpp by binding to Dpp protein. Nevertheless, tkv and dally mutually suppress one another’s pMad gradient phenotypes. Consistent with the genetic interactions observed in dally and tkv mutants, the pMad phenotype produced by overexpression of tkv was significantly restored by coexpression of dally. These observations indicate that dally antagonizes tkv in Dpp signaling. Since it has been proposed that Tkv promotes Dpp degradation by receptor-mediated endocytosis, dally may stabilize Dpp by inhibiting this process (Akiyama, 2008)

Altogether, these studies suggest that Dally serves as a co-receptor for Dpp and regulates its signaling as well as gradient formation by disrupting the degradation of the Dpp-receptor complex. In this model, the Dpp signaling complex with Dally co-receptor would remain longer on the cell surface or in the early endosomes to mediate signaling for a prolonged period of time. In contrast, in the absence of Dally, the complex would be relatively quickly degraded. This possible role of Dally can account for the shrinkage of the Dpp gradient in dally mutant wing discs. However, the possibility cannot be excluded that DppΔN is lost from the cell surface by lack of retention and further diffuses away (Akiyama, 2008)

A previous kinetic analysis of FGF degradation in cultured mammalian vascular smooth muscle cells also showed that HSPG co-receptors can enhance FGF signaling by stabilizing FGF. In these cells, the intracellular processing of FGF-2 occurred in stages: low molecular weight (LMW) intermediate fragments accumulated at the first step. Blocking HS synthesis by treatment of cells with sodium chlorate substantially reduced the half-life of these LMW intermediates, indicating that HSPGs inhibit a certain step of the intracellular degradation of FGF-2. HSPGs have also been implicated in the endocytosis and degradation of Wg. Wg protein is endocytosed from both apical and basal surfaces of the wing disc and degraded by cells to downregulate the levels of Wg protein in the extracellular space. It has been proposed that Dally-like (Dlp), the second Drosophila glypican, regulates the Wg gradient by stimulating the translocation of Wg protein from both the apical and basal membranes to the lateral side, a less active region of endocytosis, thereby inhibiting degradation of Wg protein (Akiyama, 2008)

Interestingly, DppΔN behaved differently in vivo from the previously reported mutant Xenopus BMP4 lacking the heparin-binding site. Although the action range of BMP4 is restricted to the ventral side during Xenopus embryogenesis, the truncated BMP4 migrated further in the embryo. In addition, heparitinase treatment of embryos also resulted in long-range diffusion of BMP4. These findings led to the conclusion that HSPGs trap BMP4 in the extracellular matrix to restrict its distribution in the Xenopus embryo. This activity of HSPGs seems to be opposite to that of Dally in the Dpp receiving cells of the Drosophila wing, where the major role of Dally is to stabilize Dpp protein. In general, ligands that fail to be retained on the cell surface can have any of the following fates: they may (1) migrate further and act as a ligand somewhere else, (2) be degraded by extracellular proteases, or (3) be internalized by endocytosis and degraded intracellularly. Theoretically, a mixture of all these phenomena can happen at the same time in a given tissue. However, which of these predominates may depend on cellular and extracellular environmental conditions such as concentrations of proteases in the matrix and the rate of endocytosis. Therefore, in the absence of HS, whether a major fraction of ligands is degraded or migrates further can be tissue-dependent, and the effects of HSPGs on BMP gradients will vary depending on the developmental context: a mutant BMP4 molecule moves further in a frog embryo, but DppΔN is degraded in Drosophila wing (Akiyama, 2008)

Although the results presented in this study support a role for HSPGs in Dpp stability, they do not rule out the possible involvement of HSPGs in migration of Dpp protein from cell to cell. The gradient of DppΔN is significantly narrower than that of wild-type Dpp, raising the argument that HS-binding plays a role also in normal Dpp migration in a tissue. Further studies will be required to determine whether or not HSPGs affect morphogen movement. This study also provides new insight into functional aspects of Dpp processing. Mature forms of Dpp generated by differential cleavages are likely to show different affinities for proteoglycans in the matrix. Therefore, they may have different half-lives and/or spatial distribution patterns in vivo. The biological significance of the occurrence of differently processed forms remains to be elucidated (Akiyama, 2008)

Thick veins in the amnioserosa

In a dpp null mutant, all dorsal cell fates are missing and the embryos are completely ventralized. In contrast, embryos mutant for scw are partially ventralized and lack amnioserosa but differentiate a reduced dorsal ectoderm. The relative severity of the dpp and scw mutant phenotypes does not correlate with their expression patterns, since scw is transcribed uniformly at the syncitial blastoderm stage and dpp expression is restricted to the dorsal side of the embryo. One explanation for the different efficacies of the two ligands could be that they differ in abundance or have different affinities for their receptors. Alternatively, the ligands could evoke qualitatively different responses, perhaps by acting through different receptors. To distinguish between these alternatives, the ability of SCW mRNA to restore dorsal pattern in dpp null embryos was assayed. If the difference in the scw and dpp mutant phenotypes simply reflects their effective concentrations, excess Scw protein should compensate for the loss of dpp function. Injected Scw protein fails to restore amnioserosa in embryos that lack dpp function. This suggests that Scw and Dpp act in qualitatively distinct ways. While it had been postulated that dimerization between Scw and Dpp potentiates Dpp signaling by the formation of a potent Scw/Dpp dimer, this has been shown not to be the case. Expression of Scw in ventral cells in which Dpp is absent, rescues a scw mutant phenotype. Because Scw/Dpp dimers are likely to form intracellularly, these results strongly argue that formation of Scw/Dpp heterodimers is not a prerequisite for the biological activity of Scw in the embryo (Nguyen, 1998).

To understand the basis for the differential response of the embryo to Scw and Dpp signaling, the interaction of the ligands with the two type I receptors Sax and Tkv was examined. Using dominant-negative forms of the type I receptors Sax and Tkv, it is demonstrated that Sax mediates the Scw signal, while Tkv is required for both Dpp and Scw activity. While Dpp/Tkv signaling is obligatorily required, Scw/Sax activity is necessary but not sufficient for dorsal patterning. Tkv function is required for the response to both ligands, while the ability of Sax-DN to interfere specifically with Scw and not Dpp signaling strongly argues that Sax preferentially mediates the response to Scw. Sax and Tkv act synergistically, suggesting a mechanism for integration of the Scw and Dpp signals. Further, it is shown that the extracellular protein Sog can antagonize Scw, thus limiting its ability to augment Dpp signaling in a graded manner (Nguyen, 1998).

Genetic and phenotypic studies have established that sog and dpp exert opposing influences on dorsal patterning, leading to the suggestion that Sog functions as an antagonist of Dpp activity. Levels of Sog that do not affect Dpp signaling can block the ability of Scw to promote dorsal cell fates. The ability of Sog to specifically interfere with Scw does not conflict with previous studies showing a genetic antagonism of dpp activity by sog. Since Scw augments Dpp signaling, the inhibition of Scw activity by Sog is equivalent to antagonism of Dpp. In fact, results from earlier studies support the assertion that Sog preferentially targets Scw activity in the embryo. Thus, it is proposed that one way by which Sog mediates its negative effect on dorsal patterning is by antagonizing Scw function (Nguyen, 1998).

These data are also inconsistent with a central role for sog in modulating Dpp activity in late development. Ectopic expression of Sog in the wing disc using a variety of GAL4 drivers causes no significant phenotypic defects. This is quite striking given the prominent role of Dpp in organizing pattern along the anterior-posterior axis in the wing disc. It is worth noting that the loss of posterior crossveins caused by expression of Sog is similar to the defect caused by Sax-DN, rather than Tkv-DN. An explanation for the failure of Sog to target Dpp could be that Dpp is bound to extracellular matrix components or forms a high-affinity complex with its receptor. Alternatively, the observation that Xenopus Noggin can severely ventralize Drosophila embryos raises the possibility that a Noggin-like factor may be the functionally relevant Dpp antagonist (Nguyen, 1998).

If Sog primarily blocks Scw activity during embryogenesis, the role of Tolloid may be to potentiate Scw signaling by releasing it from an inhibitory complex. Scw can promote Tld-dependent cleavage of Sog. This may explain why the loss of tld function results in a partially ventralized phenotype similar to that of scw- mutants, rather than the complete ventralization typical of dpp null embryos. The observation that embryos lacking both scw and tld function do not display a more severe phenotype is also compatible with this view (Nguyen, 1998).

Medea is involved in transmitting signals from Thick veins into the nucleus. It is proposed that Medea 17 and especially Medea 15 are compromised in the dosage-sensitive specification of amnioserosa, but that both mutant proteins retain a separable function required for the specification of dorsolateral cell fates in the embryo. What could the two separately mutable activities of Medea represent? One possibility is that each activity represents a differential capacity to transduce a signal downstream of each of the two type I Dpp receptors, Tkv and Sax. Embryos that lack both maternal and zygotic tkv activity differentiate no dorsal structures, similar to the complete loss of Medea. In contrast, although the phenotypes of embryos completely lacking sax activity have not been reported because of a requirement for sax during oogenesis, existing mutations in sax result only in the loss of amnioserosa, similar to the phenotype caused by the Med 15 mutation. These parallels suggest that Med 15 and Med 17 mutants may be defective in the response to signals downstream of the Sax receptor, while still transducing signals from the Tkv receptor. In light of this proposal, it is noted that both Med 15 and Med 17 have amino acid substitutions in loop 3, an element of the Smad4 crystal structure that is implicated in productive heteromeric interactions with activated receptor-specific Smad proteins. The mutant Medea proteins might therefore have a diminished capacity to form particular heteromeric complexes with Mad in response to signaling by one receptor but not another. Alternatively, the mutant Medea proteins could have selective disruptions in interactions with other components of the signaling system, such as factors that may collaborate with Mad and Medea to regulate expression of specific target genes. Full evaluation of this proposal awaits biochemical characterization of signaling downstream of the Tkv and Sax receptors in vivo (Hudson, 1998).

Previous studies have established Dpp’s role as a morphogen in patterning the embryonic ectoderm. dpp signaling is also required for dorsal closure of the embryonic ectoderm. How, if at all, does the level of Tgfbeta-60A affect the phenotype of the embryonic ectoderm? The cuticle phenotypes of single and double mutants were compared. Since tkv 6 homozygotes are viable and Tgfbeta-60A mutants show no obvious defects until late in development, the cuticular patterns of these mutants are essentially normal. However, tkv 6 Tgfbeta-60A homozygote embryos die and exhibit head defects and an excessive ventral curvature. Although the double mutant cuticles bear some resemblance to hypomorphic dpp mutants, they do not exhibit an obvious expansion of the ventral denticle belts. However, the double mutant phenotype suggests that when dpp signaling is compromised in the embryonic ectoderm, removing Tgfbeta-60A activity further attenuates dpp signaling. The relatively mild phenotype of the double mutant embryo might reflect partial rescue by the maternal contribution of wild-type Tkv receptors. Indeed, a quarter of the embryos produced by mothers homozygous for tkv 6 and heterozygous for Tgfbeta-60A exhibit a dorsal open phenotype similar to that of zygotic tkv null embryos. Therefore, in the absence of maternally provided wild-type Tkv, tkv 6 Tgfbeta-60A double mutant embryos exhibit a phenotype indicative of defective dpp signaling during the process of dorsal closure (Y, Chen, 1998).

A test of a constitutively active form of Jra (DJun) determined it could rescue the dorsal open phenotype in misshapen msn mutant embryos. Previous studies have shown that activated Djun rescues the bsk phenotype, indicating that one of the main functions of JNK is to phosphorylate and activate Jun. A constitutively active form of Jra was made by replacing the JNK phosphorylation sites with acidic residues. To test whether this activated Djun rescues the dorsal open phenotype in msn mutant embryos, it was expressed under the control of the hsp70 heat shock promoter in the msn mutant background. Expression of activated Djun rescues the dorsal open phenotype in most of the msn mutant embryos; heat shock decreased the number of embryos with a dorsal open phenotype from about 50%. In addition, expression of an activated form of tkv, tkvQ253D, also rescues the dorsal open phenotype in msn mutant embryos. GAL4 driven by the ectoderm-specific promoter at 69B was used to direct the expression of UAS-tkvQ253D in msn mutant embryos. This expression of activated tkv partially rescues the dorsal open phenotype caused by msn; it also has a dorsalizing effect on the ventral ectoderm of the embryos related to the earlier function of dpp in establishing the dorsoventral axis, which served to mark embryos expressing activated tkv. Thus, these findings provide genetic evidence that msn functions upstream of the JNK MAP kinase module in leading edge cells (Su, 1998).

Mutations that abolish sax or tkv activity cause phenotypes similar to partial or complete loss of activity of the TGF beta homolog Decapentaplegic (Nellen, 1994). These animals lack an embryonic hypoderm and have a hole in the dorsal surface of the cuticle.

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

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

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

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

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

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

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

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

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

Cytoskeletal dynamics and supracellular organisation of cell shape fluctuations during dorsal closure

Fluctuations in the shape of amnioserosa (AS) cells during Drosophila dorsal closure (DC) provide an ideal system with which to understand contractile epithelia, both in terms of the cellular mechanisms and how tissue behaviour emerges from the activity of individual cells. Using quantitative image analysis this study shows that apical shape fluctuations are driven by the medial cytoskeleton, with periodic foci of contractile myosin and actin travelling across cell apices. Shape changes were mostly anisotropic and neighbouring cells were often, but transiently, organised into strings with parallel deformations. During the early stages of DC, shape fluctuations with long cycle lengths produced no net tissue contraction. Cycle lengths shortened with the onset of net tissue contraction, followed by a damping of fluctuation amplitude. Eventually, fluctuations became undetectable as AS cells contracted rapidly. These transitions are accompanied by an increase in apical myosin, both at cell-cell junctions and medially, the latter ultimately forming a coherent, but still dynamic, sheet across cells. Mutants with increased myosin activity or actin polymerisation exhibited precocious cell contraction through changes in the subcellular localisation of myosin. thick veins mutant embryos, which exhibited defects in the actin cable at the leading edge, showed similar timings of fluctuation damping to the wild type, suggesting that damping is an autonomous property of the AS. These results suggest that cell shape fluctuations are a property of cells with low and increasing levels of apical myosin, and that medial and junctional myosin populations combine to contract AS cell apices and drive DC (Blanchard, 2010).

The process of DC relies on the coordination of the elongation of epidermal cells and contraction of the AS. Throughout the early stages of this process the AS cells exhibit fluctuations of their apical area. This fluctuating behaviour is driven by transient actin-myosin accumulations at the apical cortex of cells, involving the assembly, contraction and disassembly of large-scale actin and myosin structures. The formation and disassembly of foci could result from the self-organising properties of myosin and actin and the presence of specific regulators, which are known to spontaneously form active networks, asters and rings in vitro. Tension generated by the contraction of neighbouring cells, the sudden loss of tension due to detachment of actin from the cell membrane, spontaneous catastrophic collapse, and the stretching of the apical membrane resulting in the influx of ions such as calcium, could all contribute to the timing and location of fluctuations (Blanchard, 2010).

Differences in the absolute and relative strengths of actin-myosin structures in distinct subcellular populations provide an explanation for the patterns that were observed in wild-type and mutant embryos. The observations show that throughout DC the amounts of myosin increase in two subcellular populations: in a cortical ring and in a medial apical network. The amount of myosin at cell-cell junctions determines the shape of cell membranes: low levels lead to wiggly membranes and increasing levels of junctional myosin lead to the straightening of the membranes. The amount of actin-myosin in the medial population determines the fluctuating behaviour of cells: low levels lead to low-frequency fluctuations and no tissue contraction, as seen in the early phase of DC; intermediate levels lead to high-frequency fluctuations and slow tissue contraction, as seen in the slow phase of DC; and high levels lead to a coherent, but dynamic, sheet of actin-myosin across cells that was strongly contractile, as seen in the wild-type fast phase (Blanchard, 2010).

Increasing the amounts of both junctional and medial myosin in ASGal4/UAS-ctMLCK embryos led to premature apical contraction, precocious straight cell membranes and isotropic cell shapes, which are a signature of high cortical tension. By contrast, in ASGal4/UAS-DiaCA embryos, expressing an activated form of Diaphanous, low junctional myosin levels throughout DC gave rise to wiggly apical membranes. However, these cells showed precocious apical contraction and no discernible apical shape fluctuations. An increase in myosin levels was observed at a sub-apical position, which suggests that in these cells contraction is not only apical but spans the sub-apical and lateral axis. This sub-apical and lateral contraction of AS cells could be an impediment to apical shape fluctuations, as apical contraction must be accommodated by basal or lateral expansion (Blanchard, 2010).

Overactivating myosin led to apical blebbing in AS cells. Blebbing results from an increase in the ratio of cortical tension to cortex-membrane adhesion and is associated with transient actin-myosin accumulations similar to those observed in this study. No evidence was found for blebs in AS cells in wild-type embryos. It is suggested that dynamic actin-myosin structures are the more general property of cells, which in combination with high cortical tension or weak cortex-membrane adhesion can lead to blebs (Blanchard, 2010).

Previous results have shown that neighbours oscillate mainly in anti-phase (Solon, 2009). The current results reveal a more subtle picture, with anti-phase correlation predominantly in one orientation and in-phase correlation perpendicular to this. This combination results in interesting patterns, with rows or patches of cells that become synchronised for short periods of time and represent an emergent property of the system. It is expected that some sort of multicellular pattern is inevitable because of the requirement to maintain a coherent epithelium while cells fluctuate. However, the patterns also suggest that cell fluctuations can become entrained locally and for short periods. Analysis of the dynamics of cell shape suggests that cell contraction is the active process, but that fluctuating behaviour of a cell can be influenced and the timing of active contraction altered by the forces generated by immediate neighbours and more distant cells. Thus, the organisation of apical contractions and expansions at the multicellular scale arises from the feedback in both directions between intrinsic cell behaviour and mechanical context (Blanchard, 2010).

This study was undertaken to understand how changes in actin-myosin behaviour at a subcellular scale resulted in the patterned contraction of the AS. There is a gradual increase in the rate of contraction of individual cells that strongly accelerates with the onset of zippering behaviour. These changes were correlated primarily with a shortening of fluctuation cycle length. The current results suggest that these changes result from an increase in both apical medial and junctional myosin levels. The overall increase in myosin levels and the formation of a continuous actin-myosin network could provide the molecular basis for the transition of the AS to a more solid tissue (Blanchard, 2010).

What causes the increase in apical myosin levels? One possibility is that it is induced by a chemical or mechanical signal from the epidermis. A radial gradient of fluctuations (Solon, 2009) and of the rate of contraction of AS cells (Gorfinkiel, 2009) suggests that the epidermis is providing some information for the patterned contraction of the AS. However, the analysis of tkv mutants shows that even when the mechanical properties of dorsal-most epidermal cells have been altered and Dpp signalling is compromised, AS cells change their fluctuation behaviour in a similar pattern to the wild type and finally contract. This reveals that several processes that are individually redundant ensure DC. DC could result from an AS-autonomous programme of increasing medial and junctional myosin (and/or actin), through changes in the dynamics of actin and myosin activity, of intracellular trafficking or of cell adhesion. Alternatively, apical myosin might increase as a result of the fluctuating behaviour of AS cells and the build-up of tension due to neighbour contractions. Whatever the mechanism, it is likely that an increase in myosin activity involves activation of the Rho GTPase, which has a central role both in integrating mechanical and structural cues and in regulating myosin-based tension (Blanchard, 2010).

AS cells fluctuate at low frequencies for a long period during early DC without any tissue contraction. High-frequency fluctuations drive moderate cell and tissue contraction during the slow phase, before they disappear in the transition to rapid tissue contraction. This raises the question of whether fluctuations have a function, as it is at least theoretically possible that contraction could be achieved in a smooth manner. One can speculate that cell fluctuations would be a way to maintain a basic level of cell activity that could be turned easily into morphogenetically relevant behaviours. Cell fluctuations could, more simply, be an epiphenomenon of the self-organised dynamics of actin and myosin. Alternatively, pulsatile contraction might ensure that differences in apical tension are equilibrated between neighbouring cells, ensuring coordination in the contraction of cells across the tissue. This analysis of neighbour relations suggests that fluctuations allow for a certain degree of coordination between cells. A combination of empirical investigation and modelling will be crucial to understand the importance of fluctuations per se during morphogenesis (Blanchard, 2010).

Thick veins in the midgut

Mothers against dpp (Mad) is the prototype of a family of genes required for signaling by TGF-beta related ligands. In Drosophila, Mad is specifically required in cells responding to Decapentaplegic (Dpp) signals. The role of Mad in Dpp-mediated signaling was examined by utilizing tkvQ199D, an activated form of the Dpp type I receptor serine-threonine kinase thick veins (tkv). In the midgut, dpp is expressed in the visceral mesoderm of parasegments 3 and 7. In response to Dpp signals, cells expressing dpp in parasegment 3 repress the expression of the homeotic gene Sex combs reduced. Dpp signals are also required to maintain dpp expression in parasegment 3 through an autocrine feedback loop. However, cells in parasegment 4 do not appear to be affected by Dpp signals; Scr is expressed while dpp is not. In ps7, the homeotic gene Ultrabithorax initiates dpp expression. Subsequently, Dpp functions in an autocrine manner to maintain Ubx and thus dpp expression. In ps7, Dpp also signals between germ layers to the underlying endoderm. Within the midgut endoderm, which does not express dpp, expression of the homeotic gene labial is dependent on Dpp signals (Newfeld, 1997).

In the embryonic midgut, tkvQ199D mimics Dpp-mediated inductive interactions. There is an anterior expansion of labial midgut endoderm expression in response to ubiquitously expressed tkvQ199D. In early stage tkvQ199D embryos, dpp expressin is expanded to include ps4, ps5 and ps6. In late stage tkvQ199D embryos, the expanded domain of expression is maintained at very high levels, while in late stage Mad mull tkvQ199D embryos, this is not observed. Analysis of Scr expression in Mad null embryos, combined with tkvQ199D, reveals an anterior expansion of Scr, showing that Mad and dpp are required for repressing Scr. Mad function is epistatic to tkvQ199D in the repression of Scr. Thus homozygous Mad mutations block signaling by tkvQ199D and appropriate responses to signaling by tkvQ199D are restored by expression of MAD protein in Dpp-target cells (Newfeld, 1997).

Thick veins in trachea

Dpp controls tracheal cell migration along the dorsoventral body axis of the Drosophila embryo. The requirement for Dpp is revealed by two manipulations: (1) the overexpression of Dpp using a heat-shock promoter and (2) use of mutations in the Dpp receptors thickveins and punt. The failure of tracheal cells to receive the DPP signal from adjacent dorsal and ventral cells results in the absence of dorsal and ventral migrations. Ectopic Dpp signaling can reprogram cells in the center of the placode to adopt a dorsoventral migration behavior. The effects observed in response to ectopic Dpp signaling are also observed upon the tracheal-specific expression of a constitutive active Dpp type I receptor (TKV[Q253D]). The alterations in migration behavior are similar for constitutively active receptor and for Dpp ectopic expression, indicating that the Dpp signal is received and transmitted in tracheal cells to control their migration behavior. Whereas, lack of Dpp signaling results in a failure of tracheal cells to migrate along the dorsoventral axis without significantly affecting anterior migrations, ubiquitous Dpp signaling suppresses anterior migrations without interfering with dorsoventral migration (Vincent, 1997).

Dpp signaling determines localized gene expression patterns in the developing tracheal placode, and is also required for the dorsal expression of the recently identified Branchless (Bnl) guidance molecule, the ligand of the Breathless (Btl) receptor. spalt (sal) is strongly expressed in dorsal trunk cells in stage 14 embryos and is necessary for the directed anterior migration of these cells. sal is expressed in the dorsal trunk in punt and tkv mutant embryos, indicating that Dpp does not regulate sal expression. However, embryos in which the Dpp signaling pathway has been activated in all tracheal cells at the placode stage fail to accumulate Sal. This lack of Sal expression correlates with the absence of the dorsal trunk upon ectopic Dpp signaling. In contrast to sal the gene knirps is activated in the developing tracheal system in all the branches (dorsal branch, ganglionic branch, and lateral trunk) that are thought to be under the control of Dpp. kni expression is lost in tkv mutants; (kni expression only persists in the visceral branches of tkv mutants). kni expression is turned on in all tracheal cells after constitutive Dpp signaling. The requirement for Dpp for the correct expression of the ligand branchless (bnl) was revealed using tkv and punt mutants. The dorsal-most patches of bnl expression which prefigure the formation of the dorsal branches, are severely reduced in punt mutant embryos and absent in tkv mutants. Thus, Dpp plays a dual role during tracheal cell migration. It is required to control the dorsal expression of the Bnl ligand. In addition, the Dpp signal recruits groups of dorsal and ventral tracheal cells and programs them to migrate in dorsal and ventral directions (Vincent, 1997).

Decapentaplegic (Dpp) signaling determines the number of cells that migrate dorsally to form the dorsal primary branch during tracheal development. Dpp is expressed in dorsolateral epidermal clusters located near the tips of the outgrowing dorsal branches. The Dpp receptor, Thick veins, is expressed in all tracheal cells during embryogenesis and is required for in dorsal branch outgrowth ectopic activation of fusion markers in cells of the dorsal branch. Dpp signaling is required for the differentiation of one of three different cell types in the dorsal branches, the fusion cell. In Mad mutant embryos or in embryos expressing dominant negative constructs of the two type I Dpp receptors in the trachea the number of cells expressing fusion cell-specific marker genes is reduced and fusion of the dorsal branches is defective. Ectopic expression of Dpp or the activated form of the Dpp receptor Tkv in all tracheal cells induces ectopic fusions of the tracheal lumen and ectopic expression of fusion gene markers in all tracheal branches. Delta is among the fusion marker genes that are activated in the trachea in response to ectopic Dpp signaling. In conditional Notch loss of function mutants, additional tracheal cells adopt the fusion cell fate. Ectopic expression of an activated form of the Notch receptor in fusion cells results in suppression of fusion cell markers and disruption of the branch fusion. The number of cells that express the fusion cell markers in response to ectopic Dpp signaling is increased in Notch ts1 mutants, suggesting that the two signaling pathways have opposing effects in the selection of the fusion cells in the dorsal branches (Steneberg, 1999).

The Drosophila tracheal system is a model for the study of the mechanisms that guide cell migration. The general conclusion from many studies is that migration of tracheal cells relies on directional cues provided by nearby cells. However, very little is known about which paths are followed by the migrating tracheal cells and what kind of interactions they establish to move in the appropriate direction. An analysis has been carried out of how tracheal cells migrate relative to their surroundings and which tissues participate in tracheal cell migration. Cells in different branches are found exploit different strategies for their migration; while some migrate through preexisting grooves, others make their way through homogeneous cell populations. Alternative migratory pathways of tracheal cells are associated with distinct subsets of mesodermal cells and a model is proposed for the allocation of groups of tracheal cells to different branches. These results show how adjacent tissues influence morphogenesis of the tracheal system and offer a model for understanding how organ formation is determined by its genetic program and by the surrounding topological constraints (Franch-Marro, 2000).

Tracheal cells are first specified as clusters of ectodermal cells at the embryonic surface. Since tracheal cells invaginate and form the tracheal pits they occupy the grooves between the muscle precursors of adjacent metameres. The formation of this groove is independent of tracheal invagination because it also forms between metameres that do not have tracheal placodes and it also develops in trh mutant embryos, which do not undergo tracheal invagination. A subset of the tracheal cells moves anteriorly, whereas another subset moves posteriorly until they reach the cells from the adjacent placodes. These cells will form the dorsal trunk, the most prominent tracheal branch that spans the embryo longitudinally. Those cells migrate across the adjacent precursors of somatic muscles and separate the precursors of the most dorsal muscles from the precursors of more ventral muscles. Other cells, those from the dorsal side of the tracheal pit, move dorsally along the longitudinal groove to form the dorsal branches that will end up fusing with the dorsal branches coming from the contralateral hemisegments. In the ventral side, the tracheal cells follow two different paths along the two clusters of lateral muscle precursors at each side of the groove. Anterior ventral cells will form the anterior lateral trunk while the posterior ventral cells will form the posterior lateral trunk. Finally, another group of cells from a midposition in the tracheal pit will migrate inward and will form the visceral branch (Franch-Marro, 2000).

To elucidate how the cells of the dorsal trunk migrate across the precursors of somatic muscles it was first determined whether they recognized some kind of preexisting gap between the most dorsal and the remaining muscle precursors. Thus, an examination was carried out to see whether the gap between these muscle precursors is also established in the absence of migration of the cells of the dorsal trunk or, alternatively, is a consequence of the migration of these tracheal cells. First, the situation of muscle precursors was analyzed in breathless (btl) mutant embryos in which there is no tracheal migration. Second, embryos were studied in which a constitutive form of the Tkv receptor was induced specifically in the tracheal cells; in these embryos, the cells that would normally migrate anteriorly or posteriorly to form the tracheal trunk are instead forced to migrate in the dorsoventral axis. In both cases, no topological segregation between the most dorsal and the remaining precursor cells was observed, indicating that migration of the tracheal cells separates the two subsets of muscle precursors cells at a stage at which the somatic muscle precursors have been specified. Thus, the cells of the dorsal trunk appear to open up their way through a contiguous population of cells (Franch-Marro, 2000).

In the developing tracheal system of Drosophila, six major branches arise by guided cell migration from a sac-like structure. The chemoattractant Branchless/FGF (Bnl) appears to guide cell migration and is essential for the formation of all tracheal branches, while Decapentaplegic signaling is strictly required for the formation of a subset of branches, the dorsal and ventral branches. Using in vivo confocal video microscopy, it has been found that the two signaling systems affect different cellular functions required for branching morphogenesis. Bnl/FGF signaling affects the formation of dynamic filopodia, possibly controlling cytoskeletal activity and motility as such, and Dpp controls cellular functions allowing branch morphogenesis and outgrowth (Ribeiro, 2002).

The formation of tracheal branches via directed cell migration requires input from other signaling systems in addition to Bnl/FGF. Activation of the Dpp signal transduction cascade is essential in dorsal and ventral tracheal cells prior to migration for the subsequent formation of dorsal and ventral (ganglionic and lateral trunk anterior and posterior) branches. In the absence of the Dpp receptors Thick veins (Tkv) or Punt (Put), dorsal branches completely fail to develop and ventral branches are strongly affected. Dpp induces the expression of the genes kni and knrl in the ventral and dorsal cells of the placode; in the absence of these two nuclear proteins, dorsal branches are absent and ventral branches are strongly abnormal (Ribeiro, 2002).

Knowing that Bnl/FGF acts as a chemoattractant for tracheal cells, and having shown above that Bnl/FGF signaling induces filopodial activity, one must wonder why cells need input from the Dpp signaling cascade for a directed movement to the Bnl/FGF source. Is the Dpp response a prerequisite for the subsequent induction of filopodia by Bnl/FGF? Or do dorsal branch cells respond to Bnl/FGF with the formation of filopodia even in the absence of Dpp signaling input, yet fail to migrate properly? In order to find out how these different signaling systems interact in vivo, the cytoskeletal activity of tracheal cells was examined in the absence of Dpp signaling, with particular emphasis on dorsal branches. However, both tkv and put mutants lack dorsal expression of bnl; therefore, they not only lack the Dpp signaling input but also the Bnl/FGF signaling input. In line with the absence of dorsal bnl expression, cellular extensions were not observed in dorsal tracheal cells in put mutants when analyzed in vivo using the GFP-actin fusion protein (Ribeiro, 2002).

To circumvent the problem of the absence of dorsal bnl expression in mutants defective in Dpp signaling, use was made of the inhibitory SMAD protein encoded by the Drosophila Daughters against dpp (Dad) gene. Specific inhibition of Dpp signaling in tracheal cells via trachea-specific ectopic expression of Dad led to the absence of dorsal branches, despite the presence of bnl expression on the dorsal side of the embryo. Consistent with the absence of dorsal branches upon ectopic expression of Dad, kni expression was not detectable in dorsal tracheal cells. Loss of dorsal branches was also readily visible in the later larval stages; no dorsal branches were observed in third instar larvae upon the expression of Dad in the tracheal system during embryogenesis. In embryos and in larvae expressing Dad, stump-like dorsal outgrowths were occasionally observed at positions where dorsal branches form in wild-type animals. It is argued that these stumps are misrouted dorsal trunk outgrowths; such outgrowths are never observed in tkv or put mutants, presumably due to the lack of bnl expression dorsal to the invaginating placode. It is concluded from these experiments that ectopic expression of Dad mimics the tkv and put mutant phenotypes with regard to the lack of dorsal branch formation, and that dorsal branches fail to form through guided cell migration in this particular Dpp loss-of-function situation despite the presence of dorsal bnl expression (Ribeiro, 2002).

To investigate the possible cell shape changes or cytoskeletal rearrangements in dorsal tracheal cells in the absence of Dpp signaling in vivo, confocal imaging was performed of living embryos expressing both a Dad and a GFP-tagged actin transgene in the developing trachea. Confirming the observations made in fixed embryos and in third instar larvae, the phenotype observed in late embryonic stages (stages 15 and 16) in vivo is the complete absence of dorsal branches. However, analysis of a time-lapse study of three-dimensional reconstructions, in which tracheal GFP-actin dynamics were recorded in an interval of 5 min for 135 min, revealed a strikingly different picture. Unlike put mutants, embryos in which Dpp signaling is inhibited specifically in tracheal cells by ectopic expression of Dad clearly show dorsal outgrowths and filopodial activities in positions where dorsal branches normally form. These outgrowths look bud-like and showed dynamic filopodial extensions, but never refine to single-cell diameter, tubular dorsal branches. Although tracheal cells migrated dorsally, they never migrated over a large distance, and in most cases all the cells forming these buds eventually reintegrated into the main dorsal trunk, leading to a general absence of dorsal branches (Ribeiro, 2002).

These results demonstrate that in the absence of Dpp signaling, tracheal cells close to the dorsal bnl-expressing ectodermal cells are able to form actin-containing filopodial extensions and initiate dorsal migration. However, the lack of Dpp signaling, which results in the lack of expression of the kni/knrl target genes, leads to failure to form a dorsal branch, and the short, bud-like dorsal outgrowths eventually reintegrate into the main dorsal trunk. Consistent with this interpretation, cells forming the initial dorsal outgrowth in Dad-expressing embryos in rare cases generated a dorsal trunk-sized lumen. These dorsally directed stumps of dorsal trunk were also visible in third instar larvae. Such dorsal trunk-like buds are also seen in mutants that lack Dpp-induced kni/knrl in the tracheal system, indicating that dorsal migration also takes place in these mutants. These buds are never observed in put mutants, presumably due to the lack of dorsal expression of the chemoattractant Bnl/FGF (Ribeiro, 2002).

Thick veins in the Salivary Gland

Salivary gland formation in the Drosophila embryo is linked to the expression of the homeotic gene Sex combs reduced (Scr). When Scr function is missing, salivary glands do not form, and when Scr is expressed everywhere, salivary glands form in new places. However, not every cell that expresses Scr is recruited to a salivary gland fate. Along the anterior-posterior axis, the posteriorly expressed proteins encoded by the teashirt (tsh) and Abdominal-B (Abd-B) genes block Scr activation of salivary gland genes. Along the dorsal-ventral axis, the secreted signaling molecule encoded by decapentaplegic prevents activation of salivary gland genes by Scr in dorsal regions of parasegment 2. Five downstream components in the Dpp signaling cascade required to block salivary gland gene activation have been identified: two known receptors (the type I receptor encoded by the thick veins gene and the type II receptor encoded by the punt gene); two of the four known Drosophila members of the Smad family of proteins which transduce signals from the receptors to the nucleus [Mothers against dpp (Mad) and Medea (Med)], and a large zinc-finger transcription factor encoded by the schnurri (shn) gene. The expression patterns of d-CrebA and Trachealess were examined in embryos missing zygotic function of schnurri. In embryos homozygous for shn, a dorsal expansion of salivary gland protein expression is observed. The presence of amnioserosa, an extreme dorsal cell type, suggests that embryos lacking zygotic shn function are not ventralized, as are embryos missing maternal and zygotic function of tkv, pt, Mad, or Med or missing zygotic function of dpp. These results reveal how anterior-posterior and dorsal-ventral patterning information is integrated at the level of organ-specific gene expression (Henderson, 1999).

Thick veins in the Malpighian tubules

During Drosophila embryogenesis the Malpighian tubules evaginate from the hindgut anlage and in a series of morphogenetic events form two pairs of long narrow tubes, each pair emptying into the hindgut through a single ureter. Some of the genes that are involved in specifying the cell type of the tubules have been described. Mutations of previously described genes were surveyed and ten were identified that are required for morphogenesis of the Malpighian tubules. Of those ten, four block tubule development at early stages; four block later stages of development, and two, rib and raw, alter the shape of the tubules without arresting specific morphogenetic events. Three of the genes, sna, twi, and trh, are known to encode transcription factors and are therefore likely to be part of the network of genes that dictate the Malpighian tubule pattern of gene expression (Jack, 1999).

Mutations of punt interfere with rearrangement without apparently reducing the number of cells in the tubules. The tubules of punt embryos are more than two cells wide in various locations along their length. punt encodes the type II receptor for Dpp. The punt type II receptor and the type I receptor encoded by tkv typically act together to transduce the Dpp signal, and a transcription factor encoded by shn is a downstream effector of Dpp signaling. Mutations of tkv or shn normally have phenotypes nearly identical to put. However, thickened Malpighian tubules have not been observed in either tkv or shn mutants. Although the difference in phenotype could be due to residual activity in the tkv and shn 1 alleles examined, these results suggest that other proteins may be involved in transmitting the signal through Punt to cause tubule elongation (Jack, 1999).

Thick veins and mesoderm

The final overall shape of the salivary gland and its position within the developing embryo arise as a consequence of both its intrinsic properties and its interactions with surrounding tissues. This study focuses on the role of directed cell migration in shaping and positioning the Drosophila salivary gland. The salivary gland turns and migrates along the visceral mesoderm (VM) to become properly oriented with respect to the overall embryo. Salivary gland posterior migration requires the activities of genes that position the visceral mesoderm precursors, such as heartless, thickveins, and tinman, but does not require a differentiated visceral mesoderm. A role for integrin function in salivary gland migration is demonstrated. Although the mutations affecting salivary gland motility and directional migration cause defects in the final positioning of the salivary gland, most do not affect the length or diameter of the salivary gland tube. These findings suggest that salivary tube dimensions may be an intrinsic property of salivary gland cells (Bradley, 2003).

These studies suggest that the VM is required for salivary gland migration and predicts that other mutations affecting VM formation would cause similar salivary gland defects. During development, mesodermal precursor cells that migrate dorsally are exposed to the dorsally localized Decapentaplegic (DPP) signaling molecule, which is required to specify dorsal mesoderm derivatives, including cardiac and VM precursors. In embryos lacking thickveins (tkv), which encodes a DPP receptor, breaks of variable width and position in the VM are observed, similar to the breaks observed in the htl VM. Correspondingly, salivary gland migration defects were observed in tkv mutants similar to those seen in htl mutants. Such migration defects were not observed, however, in embryos expressing a negative regulator of the DPP pathway, Daughters against dpp. Together, these results suggest that DPP signaling, like FGFR1, is not required in the salivary gland cells for their normal migration, but rather that DPP signaling is required for VM formation, which in turn is required for the directed migration of the salivary gland (Bradley, 2003).

In htl, tkv, and tinman, the residual fragments of VM express Fas3, have a VM-like structure, and are able to direct salivary gland migration if present along its migratory path. Thus, the residual structures appeared to be differentiated VM with wild-type properties. To determine whether salivary gland migration requires a differentiated VM, embryos with mutations in the VM-specific gene biniou (bin) were examined. In bin mutant embryos, VM precursors segregate from dorsal mesoderm and move internally where they coalesce into the typical VM band; however, all tested VM-specific genes, including Fas3, fail to be expressed in bin mutants. Thus, an intact structure formed from VM precursors is present in bin mutants, but the VM precursor cells fail to express markers indicative of differentiation from a general mesodermal cell into a VM-specific cell. The salivary glands in bin mutants had no defects in turning or posterior migration, suggesting that guidance of salivary gland posterior migration by the VM requires neither the terminal differentiation of the precursors nor the function of any VM gene whose expression is bin-dependent (Bradley, 2003).

The VM forms a contiguous structure that may physically block salivary cells from further dorsal movement, thereby causing the cells to move posteriorly, in the path of least resistance. Alternatively or additionally, there may be a bin-independent factor (or factors) that guides salivary gland migration in a more instructive way, perhaps via a secreted signal or a transmembrane guidance molecule. If the mesodermal cue were informational, a signaling pathway functioning within salivary gland cells would have to be involved. A screen of several candidate pathways revealed that mutations disrupting the FGFR1-, FGFR2-, EGF-, DPP-, JNK-, or Wg-signaling pathway did not have phenotypes consistent with a role in the salivary cells for their migration. Thus, focus was placed on molecules known to have a more direct role in migration, specifically the integrin family of cell adhesion molecules, which are heterodimers of two transmembrane proteins, an alpha and a ß subunit. In Drosophila, each of the five identified alpha subunits (alphaPS1-5) is thought to dimerize with the ßPS subunit encoded by the myospheroid (mys) gene. The alpha subunit of alphaPS2ßPS (PS2) integrin is expressed in all mesodermal cells beginning at a very early stage, suggesting that PS2 integrin is likely to be present in the VM precursor cells prior to bin-dependent differentiation. Indeed, alphaPS2 RNA expression was observed in the mesoderm of binR22 homozygotes. In embryos mutant for inflated (if), the gene encoding the alphaPS2 subunit, migration of two tissues along the VM is affected, the endoderm and the tracheal visceral branch. Thus, the PS2 integrin is required to make the VM a suitable substrate for the migration of at least two distinct cell populations (Bradley, 2003).

Whether PS2 integrin is required for salivary gland migration was examined by staining if mutant embryos for several salivary gland proteins. In if homozygotes, salivary cells appear to invaginate normally. The first group of salivary cells to be internalized reaches the approximate level of the wild-type turning point but fails to migrate. During subsequent stages, the remaining if salivary cells continue to internalize, but the distal tip remains at the approximate VM turning point, and the tube is often slightly bent. By late stages, if salivary tubes are frequently folded in half with the distal tips oriented anteriorly. The apparent lack of salivary gland migration in if mutants is distinct from the mismigration phenotypes in htl, hbr, tkv, and tin mutants (Bradley, 2003).

Thick veins and determination of wing vs. leg cell fates

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

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

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

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

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

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

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

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

The role of Dpp signaling in maintaining the Drosophila wing anteroposterior compartment boundary as revealed by tkv mutation

The subdivision of the developing Drosophila wing into anterior (A) and posterior (P) compartments is important for its development. The activities of the selector genes engrailed and invected in posterior cells and the transduction of the Hedgehog signal in anterior cells are required for maintaining the A/P boundary. Based on a previous study, it has been proposed that the signaling molecule Decapentaplegic (Dpp) is also important for this function by signaling from anterior to posterior cells. However, it has not been known whether and in which cells Dpp signal transduction is required for maintaining the A/P boundary. The role of the Dpp signal transduction pathway and the epistatic relationship of Dpp and Hedgehog signaling in maintaining the A/P boundary has been analyzed by clonal analysis. A transcriptional response to Dpp involving the T-box protein Optomotor-blind is required to maintain the A/P boundary. Further, Dpp signal transduction is required in anterior cells, but not in posterior cells, indicating that anterior to posterior signaling by Dpp is not important for maintaining the A/P boundary. Finally, evidence is provided that Dpp signaling acts downstream of or in parallel with Hedgehog signaling to maintain the A/P boundary. It is proposed that Dpp signaling is required for anterior cells to interpret the Hedgehog signal in order to specify segregation properties important for maintaining the A/P boundary (Shen, 2005).

For many years, it was thought that En and Inv regulated the segregation of A and P cells by specifying a P-type cell segregation in a cell-autonomous fashion. Recent work has challenged this view by showing that a unidirectional Hh-mediated signal from P to A cells is required to specify the A-type segregation behavior of A cells and that the role of En and Inv is mainly to control Hh signaling. Based on the findings that A cells signal back to P cells via Dpp and that wings from flies hypomorphic for dpp have a distorted A/P boundary, it has been proposed that A to P signaling by Dpp might also be important to maintain the A/P boundary. However, whether Dpp signal transduction is required for the maintenance of the A/P boundary and in which cells the Dpp signal is required remained unknown. By analyzing clones mutant for tkv, mad, and omb, several independent lines of evidence are provided that Dpp signal transduction is required to maintain the A/P boundary and that it is only required in A cells, but not in P cells. Thus, the results do not support the hypothesis that A to P signaling by Dpp is required to maintain the A/P boundary. Instead, the results suggest that Dpp signaling within Dpp-producing A cells is required to maintain the A/P boundary (Shen, 2005).

Through analysis of mutant clones located at the A/P boundary lacking the activity of the type I Dpp receptor Tkv, evidence is provided that the reception of the Dpp signal in A cells is required to maintain the A/P boundary. When generated in the P compartment, a few tkvbsk clones displace the A/P boundary to a small extent: this is attributed to the unusual round shape of these clones. However, the majority of P tkvbsk clones do not displace the A/P boundary, suggesting that the reception of the Dpp signal is not required in P cells to maintain the A/P boundary. In contrast, mutant clones generated in the A compartment at the A/P boundary displace the position of the A/P boundary toward P, indicating that the reception of the Dpp signal is required in A cells to maintain the A/P boundary (Shen, 2005).

How does the reception of the Dpp signal control cell segregation at the A/P boundary? Although the molecular basis is unknown, a cell's segregation behavior presumably depends on its cytoskeletal or surface properties (cell affinity). Members of the TGFβ superfamily have been observed in other systems to be able to activate regulators of the actin cytoskeleton independently of Mad/Smad transcription factors, raising the possibility that Dpp reception could control cell segregation by directly altering structural components of the responding cells. Alternatively, Dpp could control the segregation of cells by regulating the transcription of one or several target genes. To distinguish between these possibilities, the role of downstream components of the Dpp signal transduction pathway were analyzed. Three independent lines of evidence is provided that a transcriptional response to the Dpp signal is required to maintain the A/P boundary. (1) The segregation behaviors of madbsk and tkvbsk clones are indistinguishable. Like tkvbsk clones, A madbsk clones displace the A/P boundary toward P, indicating a role for the transcription factor Mad in A cells to maintain the A/P boundary. (2) madbrk clones respect the A/P boundary, indicating that repression of brk transcription by Mad is important for normal A/P cell segregation. (3) A omb clones displace the A/P boundary toward P. The frequency and extent of the boundary displacement of A omb, tkvbsk, and madbsk clones is comparable, suggesting that the Dpp target gene omb is the main mediator of this aspect of the Dpp signal. In contrast to omb clones, most A clones mutant for the Dpp target gene sal do not displace the A/P boundary, indicating that sal does not play an important role in maintaining the A/P boundary. Together, these data suggest that the transduction of the Dpp signal controlling the maintenance of the A/P boundary bifurcates at the level of the Dpp target genes (Shen, 2005).

Cells of tkvbsk, madbsk, and omb clones displacing the A/P boundary do not appear to intermingle well with P cells. In fact, within the entire wing disc pouch, these mutant clones have a round shape and smooth borders, suggesting that these mutant cells in general do not intermingle freely with wild-type cells. Similar clone shapes have been reported upon mutation or misexpression of several genes, including mutants in the Dpp target gene sal and misexpression of a constitutively active form of Tkv. The round shapes and smooth borders of clones have been attributed to differences in the affinity of clone cells for their neighbors, suggesting that Tkv, Mad, and the Dpp target genes omb and sal may affect some aspects of wing pouch cell affinity. Therefore, the inability of A tkvbsk, madbsk, and omb clones displacing the A/P boundary to intermingle well with P cells is attributed to this particular role (Shen, 2005).

Taken together, this analysis indicates two roles for Dpp signal transduction: (1) it provides some aspects of the cell affinity of both A and P wing pouch cells; (2) it is required in A cells to specify an A cell affinity important for maintaining the A/P boundary. These two roles of Dpp signal transduction could either be related or distinct. The finding that the Dpp target gene sal is required for the first role, but not the second, provides a first indication that these two roles are implemented by partially distinct molecular mechanisms (Shen, 2005).

How might Omb regulate the segregation behavior of cells at the A/P boundary? Recent work has shown that Omb has at least two roles during the patterning of the Drosophila wing. First, Omb is required for the expression of two Dpp target genes sal and vestigial (vg) (del Alamo Rodriguez, 2004). Since sal mutant clones do respect the A/P boundary, the role of Omb in maintaining the A/P boundary cannot depend on sal induction. Since Vg is required for wing cell proliferation, its role in maintaining the A/P boundary cannot be tested. Second, Omb is involved in shaping the expression pattern of tkv along the A/P axis of the wing disc (del Alamo Rodriguez, 2004). The expression of tkv is reduced in Dpp-producing A cells along the A/P boundary. This reduction of tkv expression is mediated by the transcription factor Master of thickveins (Mtv, also known as Brakeless and Scribbler, which is expressed in these cells in response to the Hh signal. Since both tkv and mtv are upregulated in omb mutant clones, it has been proposed that Omb is required for Mtv to repress tkv (del Alamo Rodriguez, 2004). However, reduction of tkv transcription in A cells does not seem to be important for the segregation of cells at the A/P boundary, because A clones either mutant for mtv, in which tkv levels are increased, or overexpressing tkv, respect the A/P boundary. Thus, neither the role of Omb in repressing tkv nor in activating sal transcription appears to be important for Omb's function in maintaining the A/P boundary. Therefore, other target genes of Omb must exist that mediate Omb's function in maintaining the A/P boundary (Shen, 2005).

Anterior cells at the A/P boundary have been shown to require Hh signal transduction to segregate from P cells. Evidence is provided that A cells in addition need to transduce the Dpp signal for normal segregation. What is the epistatic relationship between Hh and Dpp signaling? The activity of the Hh transduction pathway is not affected in either tkvbsk or madbsk clones as monitored by the expression of the Hh target gene ptc, indicating that Hh signal transduction does not require Dpp signal transduction components for its activity. However, the Dpp target gene omb appears to be important for A cells to interpret the Hh signal because the ability of Ci to specify A-type segregation properties depends, in part, on the activity of Omb. Thus, Dpp signaling acts either downstream of or in parallel with Hh signaling in maintaining the A/P boundary (Shen, 2005).

Previously, three transcription factors, a transcriptional activator form of Ci (hereafter referred to as Ci[act]), En, and Inv, have been shown to be required for the segregation of A and P cells. Evidence exists for the involvement of a fourth transcription factor, the T-box protein Omb. Omb is further shown to act downstream of or in parallel with Ci. How could these four transcription factors regulate the segregation of A and P cells? In a simple model, Ci[act], En, Inv, and Omb could regulate the segregation of A and P cells by controlling the transcription of the same set of target genes that may encode cell affinity molecules or regulate the activity of cell affinity molecules. Omb is activated in both A and P cells in a broad domain centered around the A/P boundary by Dpp signaling where Omb may upregulate the expression of this putative target gene(s). The activity of Ci[act] is restricted to Hh-responding A cells along the A/P boundary. In these A cells, the target gene(s) would be further induced. En and Inv expressions are mainly confined to P cells in which they are known to act as repressors of transcription. Thus, En and Inv would repress the putative target gene(s) in P cells. The abrupt difference in the expression of putative target gene(s) would contribute to the segregation of A and P cells. Anterior clones (but not P clones) of cells lacking Omb would displace the A/P boundary because normally the putative target gene would be highly expressed in A cells, but not in P cells, where it would be repressed by En and Inv. Omb may therefore provide a basal affinity to cells in the center of the wing disc that is modified by Ci[act], En, and Inv to create a sharp difference of this affinity in cells on both sides of the A/P boundary. In an alternative model, Omb, Ci[act], En, and Inv would regulate distinct sets of genes. To distinguish among these models, it will be necessary to identify the Ci[act], En, Inv, and Omb target genes mediating cell segregation (Shen, 2005).

The precise position and shape of the Dpp organizer along the A side of the A/P boundary are important for normal growth and patterning of the wing. It has been proposed that the segregation of cells at the A/P boundary contributes to maintain this precise position and shape of the Dpp organizer in the growing wing disc epithelium. It is intriguing to notice that the Dpp-organizing activity itself plays a role in the segregation of A and P cells, suggesting that the Dpp-organizing activity contributes to maintain its own position. It will be interesting to investigate whether other organizers associated with compartment boundaries have similar functions (Shen, 2005).

Dependence of Drosophila wing imaginal disc cytonemes on Decapentaplegic

The anterior/posterior (A/P) and dorsal/ventral (D/V) compartment borders that subdivide the wing imaginal discs of Drosophila third instar larvae are each associated with a developmental organizer. Decapentaplegic, a member of the transforming growth factor-ß superfamily, embodies the activity of the A/P organizer. It is produced at the A/P organizer and distributes in a gradient of decreasing concentration to regulate target genes, functioning non-autonomously to regulate growth and patterning of both the anterior and posterior compartments. Wingless is produced at the D/V organizer and embodies its activity. The mechanisms that distribute Dpp and Wg are not known, but proposed mechanisms include extracellular diffusion, successive transfers between neighbouring cells, vesicle-mediated movement, and direct transfer via cytonemes. Cytonemes are actin-based filopodial extensions that have been found to orient towards the A/P organizer from outlying cells. This study shows that in the wing disc, cytonemes orient toward both the A/P and D/V organizers, and that their presence and orientation correlates with Dpp signalling. The Dpp receptor, Thickveins (Tkv), is present in punctae that move along cytonemes. These observations are consistent with a role for cytonemes in signal transduction (Hsiung, 2005).

Cytonemes appear as fluorescent strands emanating from the apical surface of disc cells that express green fluorescent protein (GFP). Using standard epifluorescence microscopy, cytonemes are visible only if neighbouring cells have low background fluorescence, only in unfixed discs, and only if they extend in a single optical plane. The contour of the apical surface of the notum primordium is rather flat, and cytonemes can be imaged in this region in discs that are suspended in liquid. However, the wing pouch primordium is convex, and cytonemes can be imaged only in discs that have been slightly flattened. The fragile nature of disc cells requires that physical and osmotic insults be minimized, and the methods developed to image wing cytonemes avoid rupture, delamination and other responses to injury (Hsiung, 2005).

Small clones (averaging 10-15 cells) expressing CD8-GFP are visible in a speckled pattern. High-magnification views of clones in similar discs reveals cytonemes extending outwards from some, but not all clones. On the basis of the presence or absence of cytonemes and on the orientation of cytonemes, three regions of the disc can be distinguished. In the wing blade primordium, approximately 20% of the clones extended cytonemes oriented toward either the A/P or D/V compartment borders. More than 95% of these clones had cytonemes oriented toward one of the two borders, and <5% had cytonemes oriented toward both. It has not been possible to establish whether all cells extend cytonemes, whether cells can extend more than one cytoneme, or whether a single cell can extend cytonemes toward both axes. A/P cytonemes as long as 80.2 microm have been recorded; the average length in these preparations is 20.8 microm. D/V cytonemes were shorter, averaging 8.8 microm (Hsiung, 2005).

Clones in the notum primordium radiate cytonemes in all directions, without a consistent bias toward either the A/P or D/V axes of the disc. Cytonemes associated with notum clones averaged 7.4 microm in length, almost 65% shorter than A/P cytonemes in the wing primordium. Unlike cells in the wing and notum primordia, cells in the hinge/pleural primordium do not extend cytonemes. These hinge/pleural cells were examined by expressing GFP in clones and by using an enhancer trap expressed in the hinge region, but no cell extensions were observed in either case (Hsiung, 2005).

Although Dpp is essential for cell survival in the notum, there is no indication that the A/P compartment border in the notum has an associated organizing centre, and it is ambiguous whether Dpp functions as a morphogen in the notum region. Dpp is apparently not required for either growth or cell survival in the hinge/pleural region. Thus, directional cytonemes are present in the wing primordium (where Dpp functions as a morphogen); cytonemes are present and 'omni-directional' in the notum (where Dpp may function only to support cell proliferation), and cytonemes are absent in the hinge/pleural region, where Dpp function appears not to be required. These three distinct cell types -- cells with A/P- or D/V-oriented cytonemes, cells with cytonemes lacking a directional bias, or cells without cytonemes -- could each be imaged in a restricted and defined region of the same disc (Hsiung, 2005).

Whether the shape and distribution of cytonemes in wing discs correlates with the presence of Dpp was tested. When Dpp levels were reduced at the A/P organizer using temperature-sensitive mutants of either dpp (dppts) or hedgehog (hhts) (dpp expression depends upon Hh signalling), cytonemes in the wing primordium were affected. At the permissive temperature (18°C) in mutant discs, or at either the permissive or non-permissive (29°C) temperatures in normal discs, cytonemes emanating from cells at the lateral flanks of discs oriented toward the A/P organizer. After incubation at 29°C, however, cytonemes in hhts and dppts mutant discs are more numerous (> twofold), and are not uniformly oriented toward the A/P organizer region. In these mutant discs, curved and bent cytonemes, and cytonemes crossing over each other were observed. Cytonemes with such shapes are never observed under normal conditions, or if mutant larvae are returned to the permissive temperature after a period of incubation at 29°C (Hsiung, 2005).

To test whether Dpp is sufficient for cytoneme induction, cytonemes were imaged in discs in which Dpp was expressed ubiquitously. In control discs, cells project cytonemes toward A/P and D/V axes only, but in discs with heat-shock-induced Dpp (hs-dpp) >50% of the clones projected cytonemes outwards in all directions. These cytonemes were significantly shorter than those in untreated discs, averaging about 10.6 microm in length. Even more striking were the cells in the hinge domain, which normally do not extend cytonemes. Under conditions of ubiquitous Dpp expression, cells in the hinge domain extend cytonemes in apparently random orientations. The ability to image cytonemes is limited to preparations in which discs have been extracted from larvae and the cytonemes are static, but their varied appearance under the conditions tested illustrates that they are dynamic in vivo. Although a model is favored in which they extend from cells in random directions but become stabilized when functional contacts are made with signalling cells, the possibility that their directionality is directly influenced by extracellular cues cannot be excluded (Hsiung, 2005).

The distribution of the Dpp receptor Tkv was monitored, as a Tkv-GFP fusion protein. When expressed in the lateral flanks of wing discs, most of the fluorescence is localized to the plasma membrane of expressing cells. However, bright, motile punctae were also present in more central regions, as far as 30 microm from the edge of the expression domain. These punctae are motile, moving in both anterograde and retrograde directions, and some images clearly revealed their association with cytonemes. Trafficking of these punctae was approximately 5-7 microm s-1, a rate consistent with measured rates of vesicular movement on actin filaments. The resolution of these studies could not establish whether these filaments were inside or on cytonemes (Hsiung, 2005).

The distribution of Tkv-GFP punctae around clones was plotted in discs with normal expression of Dpp or with ubiquitous Dpp expression. In normal discs, Tkv-GFP punctae are polarized in the direction of the A/P border. In contrast, Tkv-GFP punctae in heat-shocked hs-dpp discs are more numerous and project in various directions all around the circumference of the clones. Since these patterns of Tkv-GFP punctae could be imaged in unflattened discs, their distribution was compared in both flattened and unflattened discs. No differences were detected between the two conditions with respect to either the total number of punctae, or to the distance from or position relative to the clones. This confirms that the slight flattening used to image cytonemes does not generate or substantially alter these structures (Hsiung, 2005).

Previous work has demonstrated that cytonemes bind phalloidin (a specific F-actin-binding protein) and can be labelled with an actin-GFP fusion protein, suggesting that cytonemes are actin-based. To test whether cytonemes and the movement of Tkv-GFP punctae is actin-dependent, discs containing clones of Tkv-GFP-expressing cells were treated with cytochalasin D, an actin-binding drug. The number of Tkv-GFP punctae at a distance from the cell bodies was dramatically reduced in treated discs. Bright punctae were observed on the surface of cells expressing Tkv-GFP, and they appeared to move along the surface of the cells even in the presence of drug. In contrast, the bright punctate fluorescence distant from GFP-expressing cells was not motile. These observations suggest that cytonemes can function as vehicles for active, actin-based transport of receptors (Hsiung, 2005).

To better document the structure of cytonemes, optical sections of cytoneme-producing clones were reconstructed to render their three-dimensional structure. In such images, cytonemes labelled with CD8-GFP as well as cytonemes containing Tkv-GFP punctae, were observed that extend from the apical surface of the disc columnar epithelial cells. In contrast, expression of a human guanine nucleotide exchange factor (GEF) protein, Vav-GFP, which has been shown to localize to filopodia in vertebrate cells, labels basal filopodia when expressed in wing disc cells. This preferential placement of proteins into different types of filopodial extensions indicates that apical and basal extensions are structurally distinct: it suggests that these cell extensions may be functionally distinct, and it implies the existence of a mechanism for sorting proteins to specific types of extensions (Hsiung, 2005).

Dpp is synthesized and secreted by a narrow stripe of 5-7 cells adjacent to the A/P compartment border in the wing primordium, and it distributes in a gradient of decreasing concentration that extends across the wing pouch. A concentration gradient does not imply a mechanism for distribution; it is conceivable that cytonemes sense and respond to Dpp but do not ferry it. However, on the basis of the results presented in this study, cytoneme-based transport is considered to be an attractive possibility. As this work shows, the Dpp receptor Tkv is present in cytonemes, and the presence, orientation and shape of cytonemes in wing discs correlates with what is known about the different roles that Dpp has in the wing, notum and hinge primordia. Moreover, cytonemes change in response to conditions of Dpp gain-of-function and loss-of-function. These correlations are consistent with the idea that Dpp moves from its source in an oriented manner imposed by the directionality of these cellular extensions. Several recent studies reported have cellular extensions in Drosophila cells that correlate with signalling by Branchless (a Drosophila FGF), Notch and Scabrous, extensions in spider cells that correlate with signalling by Dpp, and extensions in mammalian cells that correlate with signalling by epidermal growth factor. The widespread occurrence of cytonemes and cytoneme-like filopodia suggests that their role in long-distance signalling might be a general one, one that might permit selective signalling in ways that enable cells to regulate both release and uptake of signals (Hsiung, 2005).

Dpp signaling promotes the cuboidal-to-columnar shape transition of Drosophila wing disc epithelia by regulating Rho1

Morphogenesis is largely driven by changes in the shape of individual cells. However, how cell shape is regulated in developing animals is not well understood. This study shows that the onset of TGFbeta/Dpp signaling activity correlates with the transition from cuboidal to columnar cell shape in developing Drosophila melanogaster wing disc epithelia. Dpp signaling is necessary for maintaining this elongated columnar cell shape and overactivation of the Dpp signaling pathway results in precocious cell elongation. Moreover, evidence is provided that Dpp signaling controls the subcellular distribution of the activities of the small GTPase Rho1 and the regulatory light chain of non-muscle myosin II (MRLC). Alteration of Rho1 or MRLC activity has a profound effect on apical-basal cell length. Finally, it was demonstrated that a decrease in Rho1 or MRLC activity rescues the shortening of cells with compromised Dpp signaling. These results identify a cell-autonomous role for Dpp signaling in promoting and maintaining the elongated columnar shape of wing disc cells and suggest that Dpp signaling acts by regulating Rho1 and MRLC (Widmann, 2009).

Cell extrusion was observed when Dpp signaling was locally reduced in tkva12 bsk- clones, but not when it was reduced throughout the dorsal compartment by expression of Dad. This indicates that cell extrusion is a consequence of the sharp boundary of Dpp signaling at the clone border. One of the first morphological consequences of the loss of Dpp signaling in tkva12 bsk- clones was the apical constriction of mutant cells and surrounding control cells. Apical constriction correlated with increased staining intensities of F-actin and P-MRLC, a marker for active non-muscle myosin II, at the apicolateral side of tkva12 bsk- and neighboring wild-type cells. The formation of a similar actin-myosin ring has been previously demonstrated during the extrusion of apoptotic cells, and it has been proposed that contraction of this ring squeezes cells out of the epithelium. It is currently unclear whether these increased staining intensities reflect an increase in the total amount of F-actin and P-MRLC in tkva12 bsk- mutant clones, or whether they are instead merely a consequence of the apical constriction of cells. Nevertheless, these findings are consistent with the view that contraction of an actin-myosin ring might contribute to the extrusion of tkva12 bsk- cells. Apical cell constriction was paralleled with cell shortening along the apical-basal axis. Based on the observation that reduction in Dpp signaling throughout the wing disc pouch resulted in apical-basal cell shortening, but not in apical cell constriction, it is speculated that cell shortening, and thus the formation of an inappropriate cell shape, might be an initial event leading to the extrusion of tkva12 bsk- cells. If so, cell extrusion might not represent a specific response to eliminate slow-growing or apoptotic cells, but rather represents a general response to inappropriate cell function or morphology. In the wild type, cell extrusion might be instrumental in maximizing tissue fitness by removing cells with inappropriate function or morphology (Widmann, 2009).

The basal membrane of tkva12 bsk- cells and neighboring control cells, identified by PSβ-integrin labeling, became apposed. Since this led to a reduction in the lateral contact between mutant and neighboring control cells, this apposition might help to dislodge tkva12 bsk- cells from the remaining epithelium, and thereby, might aid the extrusion process. It is also noted that extruded tkva12 bsk- cells displayed features reminiscent of epithelial-to-mesenchymal transition (EMT). In particular, a strong decrease in E-cadherin, a hallmark of EMT and actin-rich processes were observed in extruded tkva12 bsk- cells. Interestingly, a role for Dpp/BMPs in preventing EMT has also been identified in vertebrates. Mouse BMP7, which is related to Dpp, for example, is required for counteracting EMT associated with renal fibrosis. Decreased E-cadherin levels have also recently been reported following the extrusion of cells deficient for C-terminal Src kinase from Drosophila epithelia, indicating that this might be a more common consequence of cell extrusion (Widmann, 2009).

Reduced apical-basal cell length was observed when Dpp signaling was severely reduced, either in clones or throughout the wing disc pouch; however, apical cell constriction, fold formation and cell extrusion were only detected by clonal reduction of Dpp signaling. Instead, cells were apically widened and did not extrude when Dpp signaling was reduced throughout the dorsal compartment. These experiments therefore allowed the effects of sharp boundaries of Dpp signaling at clone borders to be separated from cell-autonomous functions of Dpp signaling. They demonstrate that the cell-autonomous function of Dpp signaling is not to prevent apical cell constriction, folding and cell extrusion, but rather to maintain proper columnar cell shape. Moreover, three further observations suggest that Dpp signaling has an instructive role that drives cell elongation. (1) In the wild type, an increase in Dpp signal transduction activity correlated with apical-basal cell elongation in second instar larval discs. (2) In wing discs of late third instar larvae, Dpp signal transduction activity correlated with apical-basal cell length along the anteroposterior axis. (3) Activation of Dpp signaling, by expressing the constitutively active Dpp receptor TkvQ-D, resulted in precocious cell elongation and apical cell narrowing during early larval development. These findings indicate that Dpp signaling is an important trigger for the cuboidal-to-columnar transition in cell shape that occurs during mid-larval development (Widmann, 2009).

How does Dpp signaling promote the apical-basal elongation of wing disc cells? Compartmentalization of Rho1 activity has been recognized as being important for shaping cells and tissues. In the wild-type wing disc, Rho1 protein is enriched and the activity of the Rho1 sensor is increased at the apicolateral side, and more moderately at the basal side, of elongated cells. By contrast, Rho1 activity is more uniform in cuboidal cells, and overexpression of RhoGEF2, which leads to uniform distribution of this protein and presumably also uniform Rho1 activity, resulted in a cuboidal cell shape. Rho1, when present at the apicolateral side of cells, might therefore have a function in stabilizing or promoting cell elongation. Since the apicolateral increase in Rho1 sensor activity correlated with an increase of P-MRLC at a similar location, this function of Rho1 might be mediated by myosin II. The observation that a decrease in the bulk of Rho1 activity, either through expression of Rho1N19 or rho1dsRNA, resulted in cell elongation rather than in cell shortening, further suggests that the compartmentalization of Rho1 activity is important for shaping wing disc cells. Future studies will need to examine the morphogenetic consequences of locally modulating the activity of Rho1 (Widmann, 2009).

The results provide strong evidence for a functional link between Dpp signaling and Rho1-myosin II. Shortening of cells with compromised Dpp signaling could be rescued by a decrease in Rho1 or MRLC activity. In particular, the expression of MbsN300, an activated form of a subunit of myosin light chain phosphatase, which in wild-type wing discs did not significantly alter cell length, did rescue the shortening of Dpp-compromised cells. This indicates a specific interaction between Dpp signaling and Mbs-myosin II. The data further suggest that Dpp signaling controls apical-basal cell length by compartmentalizing Rho1 protein abundance and/or activity. (1) In late third instar wing discs, apicolateral enrichment of Rho1 protein and Rho1 sensor activity directly correlated with the local level of Dpp signal transduction activity. (2) Rho1 protein abundance and Rho1 sensor activity were decreased at the apicolateral side of cells when Dpp signal transduction was compromised by expression of Dad. (3) Rho1 protein and Rho1 sensor activity were increased at the apicolateral side and also at the basal side of cells when Dpp signal transduction was activated during early development by expression of TkvQ-D (Widmann, 2009).

Local activation of Rho1 and myosin II can lead to contraction of actin-myosin filaments, which can increase the cortical tension that is important for the shaping of cells during various developmental processes. By compartmentalizing Rho1 activity, Dpp signaling might promote both apical-basal cell elongation and apical cell narrowing. An increase in tension at the apicolateral cell cortex might promote apical cell narrowing. At the same time, a relative decrease in cortical tension laterally, compared with that on the apicolateral side, might allow cells to elongate through intrinsic cytoskeletal forces and/or extrinsic forces imposed by the growth of the epithelium. In this model, Dpp signaling directs the cuboidal-to-columnar shape transition of wing disc cells by increasing the Rho1 and myosin II activities at the apicolateral side of cells. The local increase of Rho1 and myosin II activities might then shift the balance of tension between the apicolateral cell cortex and the lateral cell cortex towards an increased tension at the apicolateral cell cortex (Widmann, 2009).

The results identify a Dpp-Brk-Rho1-myosin II pathway controlling cell shape in the wing disc epithelium. The elimination of Brk function in mad- mutant cells allowed these cells to maintain a normal columnar cell shape, indicating that Dpp controls epithelial morphogenesis through repression of Brk. Since Brk acts as a transcriptional repressor, the link between Brk and Rho1 is most probably established through an unknown Brk-repressible gene. The identification of genes transcriptionally repressed by Brk will thus be important for determination of how Dpp signaling controls Rho1 and thereby, epithelial cell shape. The finding that Dpp signaling has a cell-autonomous morphogenetic function indicates that Dpp signaling provides a connection between cell-fate specification, cell growth and the control of morphogenesis. It, thereby, might help to facilitate the coordination of these processes during wing disc development (Widmann, 2009).

Given the evolutionary conserved functions of Rho and myosin II, it is anticipated that the mechanisms regulating columnar cell shape, which are describe in this study for the wing disc, will also operate in a wide range of other epithelia. Moreover, the role of TGFβ/Dpp signaling in patterned morphogenesis appears to be conserved in vertebrates, raising the possibility that Rho and myosin II are common mediators of TGFβ/Dpp signaling (Widmann, 2009).

Thick veins in spermatogenesis

The continuous and steady supply of transient cell types such as skin, blood and gut depends crucially on the controlled proliferation of stem cells and their transit amplifying progeny. Although it is thought that signaling to and from support cells might play a key role in these processes, few signals that might mediate this interaction have been identified. During spermatogenesis in Drosophila, the asymmetric division of each germ line stem cell results in its self-renewal and the production of a committed progenitor that undergoes four mitotic divisions before differentiating while remaining in intimate contact with somatic support cells. TGF-ß signaling pathway components punt and schnurri have been shown to be required in the somatic support cells to restrict germ cell proliferation. This study showns, by contrast, that the maintenance and proliferation of germ line stem cells and their progeny depends upon their ability to transduce the activity of a somatically expressed TGF-ß ligand, the BMP5/8 ortholog Glass Bottom Boat. TGF-ß signaling represses the expression of the Bam protein, which is both necessary and sufficient for germ cell differentiation, thereby maintaining germ line stem cells and spermatogonia in their proliferative state (Shivdasani, 2003).

In order to test the requirement for TGF-β signaling in the germ line, the behavior of marked germ line clones lacking the activity of various TGF-β signaling pathway components was investigated. Germ line stem cells mutant for tkv or put (a type II TGF-β receptor) and spermatocytes lacking the activity of tkv, put, or mad (a transcription factor required for the regulation of TGF-β target genes) were generated but did not persist to the same extent as wild-type clones, as evidenced by assessing the ratio of the number of testes containing at least one germ line clone to the number of testes containing wild-type control clones. Sporadically (approximately 4% of cases), cysts containing eight, rather than 16, spermatocytes were observed, implying that the fourth spermatogonial division had not been complete. Such a scenario might have arisen due to the transient persistence of Tkv, Mad, or Put protein after the respective wild-type allele was lost. Together, these clonal analysis data suggest that TGF-β signaling is required for both germ line stem cell maintenance and spermatogonial proliferation. No requirement was found for schnurri (shn), the product of which is frequently required in Dpp signaling, in the germ line for these processes (Shivdasani, 2003).

Thick veins in the wing

Continued: Effects of mutation part 2/2

thickveins: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | References

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