Robert P. Ray

Dystroglycan and perlecan provide a basal cue required for epithelial polarity during energetic stress.

Summary Dystroglycan localizes to the basal domain of epithelial cells and has been reported to play a role in apical-basal polarity. Here, we show that Dystroglycan null mutant follicle cells have normal apical-basal polarity, but lose the planar polarity of their basal actin stress fibers, a phenotype it shares with Dystrophin mutants. However, unlike Dystrophin mutants, mutants in Dystroglycan or in its extracellular matrix ligand Perlecan lose polarity under energetic stress. The maintenance of epithelial polarity under energetic stress requires the activation of Myosin II by the cellular energy sensor AMPK. Starved Dystroglycan or Perlecan null cells activate AMPK normally, but do not activate Myosin II. Thus, Perlecan signaling through Dystroglycan may determine where Myosin II can be activated by AMPK, thereby providing the basal polarity cue for the low-energy epithelial polarity pathway. Since Dystroglycan is often downregulated in tumors, loss of this pathway may play a role in cancer progression.

Crossveinless-c is a RhoGAP required for actin reorganisation during morphogenesis

Members of the Rho family of small GTPases are required for many of the morphogenetic processes required to shape the animal body. The activity of this family is regulated in part by a class of proteins known as RhoGTPase Activating Proteins (RhoGAPs) that catalyse the conversion of RhoGTPases to their inactive state. In our search for genes that regulate Drosophila morphogenesis, we have isolated several lethal alleles of crossveinless-c (cv-c). Molecular characterisation reveals that cv-c encodes the RhoGAP protein RhoGAP88C. During embryonic development, cv-c is expressed in tissues undergoing morphogenetic movements; phenotypic analysis of the mutants reveals defects in the morphogenesis of these tissues. Genetic interactions between cv-c and RhoGTPase mutants indicate that Rho1, Rac1 and Rac2 are substrates for Cv-c, and suggest that the substrate specificity might be regulated in a tissue-dependent manner. In the absence of cv-c activity, tubulogenesis in the renal or Malpighian tubules fails and they collapse into a cyst-like sack. Further analysis of the role of cv-c in the Malpighian tubules demonstrates that its activity is required to regulate the reorganisation of the actin cytoskeleton during the process of convergent extension. In addition, overexpression of cv-c in the developing tubules gives rise to actin-associated membrane extensions. Thus, Cv-c function is required in tissues actively undergoing morphogenesis, and we propose that its role is to regulate RhoGTPase activity to promote the coordinated organisation of the actin cytoskeleton, possibly by stabilising plasma membrane/actin cytoskeleton interactions.

Twisted Perspective: New Insights into Extracellular Modulation of BMP Signaling during Development

TGF-␤-like ligands, the Bone Morphogenetic Proteins (BMPs), is the direct modulation of ligand activity by extracellular factors (reviewed in De Robertis and Sasai, 1996; Figure 1). In amphibians, opposing activities of BMP4 and its antagonist Chordin (Chd) are responsible for subdivision of the embryonic dorsal–ventral axis. A Brown University similar role for BMP4 and Chd orthologs has been impli-Providence, Rhode Island 02912 cated in dorsal–ventral patterning of the zebrafish embryo (Mullins, 1998). Thus, in the vertebrates, BMP activity specifies ventral fates in the early embryo, and is During the past several decades, considerable research antagonized by localized expression of Chd orthologs efforts have elucidated the central components of many dorsally. In Drosophila, the same relationship exists be-signal transduction pathways in animal systems. More tween the BMP homologs decapentaplegic (dpp) and recently attention has shifted to the importance of mod-screw (scw) and the Chd ortholog short gastrulation ulating intercellular signaling processes during develop-(sog), but the axis is inverted: dpp and scw specify dorsal ment, as it is clear that fine-tuned regulation of signaling fates in the embryo and are antagonized by sog, which systems in vivo results in subtle distinctions at the cellu-is expressed ventrally. In both vertebrates and inverte-lar level. Extracellular modulators have been a particular brates, the activity of Chd orthologs is antagonized by a focus of investigation, including proteins that affect re-family of metalloproteases including Drosophila Tolloid ceptor stability, ligand function, or ligand availability. In (Tld), Xenopus Xolloid (Xol), Human BMP-1, and their these latter types of regulation, proteins such as proteo-orthologs in other vertebrates. Biochemical studies on glycans, heparan sulfate modifying enzymes, and prote-the Xenopus, Drosophila, and mouse proteins have ases have been recognized as key players, as have demonstrated that Tld/Xol/BMP1 cleavage inactivates a diverse class of diffusible modulators that influence Sog/Chd and promotes BMP signaling (Marqué s et al., ligand activity by inhibiting or facilitating its function (see references contained within reviews by Capdevila Xol proteins function as BMP agonists. modulator that influences the availability of BMP ligands, on the Drosophila gene twisted gastrulation (tsg) and a family of the TGF-␤ superfamily of signaling molecules. its vertebrate orthologs reveal another level of complex-It appears that for TGF-␤ signaling in general a major ity in the extracellular modulation of BMP signaling dur-means of signal regulation is the modulation of ligand ing development. tsg was first identified in Drosophila as availability and as yet this system provides …

Patterned Anchorage to the Apical Extracellular Matrix Defines Tissue Shape in the Developing Appendages of Drosophila.

Summary How tissues acquire their characteristic shape is a fundamental unresolved question in biology. While genes have been characterized that control local mechanical forces to elongate epithelial tissues, genes controlling global forces in epithelia have yet to be identified. Here, we describe a genetic pathway that shapes appendages in Drosophila by defining the pattern of global tensile forces in the tissue. In the appendages, shape arises from tension generated by cell constriction and localized anchorage of the epithelium to the cuticle via the apical extracellular-matrix protein Dumpy (Dp). Altering Dp expression in the developing wing results in predictable changes in wing shape that can be simulated by a computational model that incorporates only tissue contraction and localized anchorage. Three other wing shape genes, narrow, tapered, and lanceolate, encode components of a pathway that modulates Dp distribution in the wing to refine the global force pattern and thus wing shape.

Rapid evolution of a novel signalling mechanism by concerted duplication and divergence of a BMP ligand and its extracellular modulators

Gene duplication and divergence is widely considered to be a fundamental mechanism for generating evolutionary novelties. The Bone Morphogenetic Proteins (BMPs) are a diverse family of signalling molecules found in all metazoan genomes that have evolved by duplication and divergence from a small number of ancestral types. In the fruit fly Drosophila, there are three BMPs: Decapentaplegic (Dpp) and Glass bottom boat (Gbb), which are the orthologues of vertebrate BMP2/4 and BMP5/6/7/8, respectively, and Screw (Scw), which, at the sequence level, is equally divergent from Dpp and Gbb. It has recently been shown that Scw has arisen from a duplication of Gbb in the lineage leading to higher Diptera. We show that since this duplication event, Gbb has maintained the ancestral BMP5/6/7/8 functionality while Scw has rapidly diverged. The evolution of Scw was accompanied by duplication and divergence of a suite of extracellular regulators that continue to diverge together in the higher Diptera. In addition, Scw has become restricted in its receptor specificity: Gbb proteins can signal through the Type I receptors Thick veins (Tkv) and Saxophone (Sax), while Scw signals through Sax. Thus, in a relatively short span of evolutionary time, the duplication event that gave rise to Scw produced not only a novel ligand but also a novel signalling mode that is functionally distinct from the ancestral Gbb mode. Our results demonstrate the plasticity of the BMP pathway not only in evolving new family members and new functions but also new signalling modes by redeploying key regulators in the pathway.

Different Requirements for Proteolytic Processing of Bone Morphogenetic Protein 5/6/7/8 Ligands in Drosophila melanogaster

Background: Bone morphogenetic proteins require proteolytic processing to generate the mature ligand. Results: The BMPs Gbb and Scw have three cleavage sites, and BMP7 has one, which are differentially required to produce the functional ligand. Conclusion: Gbb, Scw, and BMP7 have distinct processing requirements despite being closely related. Significance: Rapid evolution of cleavage sites is a general mechanism for fine-tuning BMP ligand activity to function. Bone morphogenetic proteins (BMPs) are synthesized as proproteins that undergo proteolytic processing by furin/subtilisin proprotein convertases to release the active ligand. Here we study processing of BMP5/6/7/8 proteins, including the Drosophila orthologs Glass Bottom Boat (Gbb) and Screw (Scw) and human BMP7. Gbb and Scw have three functional furin/subtilisin proprotein convertase cleavage sites; two between the prodomain and ligand domain, which we call the Main and Shadow sites, and one within the prodomain, which we call the Pro site. In Gbb each site can be cleaved independently, although efficient cleavage at the Shadow site requires cleavage at the Main site, and remarkably, none of the sites is essential for Gbb function. Rather, Gbb must be processed at either the Pro or Main site to produce a functional ligand. Like Gbb, the Pro and Main sites in Scw can be cleaved independently, but cleavage at the Shadow site is dependent on cleavage at the Main site. However, both Pro and Main sites are essential for Scw function. Thus, Gbb and Scw have different processing requirements. The BMP7 ligand rescues gbb mutants in Drosophila, but full-length BMP7 cannot, showing that functional differences in the prodomain limit the BMP7 activity in flies. Furthermore, unlike Gbb, cleavage-resistant BMP7, although non-functional in rescue assays, activates the downstream signaling cascade and thus retains some functionality. Our data show that cleavage requirements evolve rapidly, supporting the notion that changes in post-translational processing are used to create functional diversity between BMPs within and between species.

Apical and Basal Matrix Remodeling Control Epithelial Morphogenesis

Summary Epithelial tissues can elongate in two dimensions by polarized cell intercalation, oriented cell division, or cell shape change, owing to local or global actomyosin contractile forces acting in the plane of the tissue. In addition, epithelia can undergo morphogenetic change in three dimensions. We show that elongation of the wings and legs of Drosophila involves a columnar-to-cuboidal cell shape change that reduces cell height and expands cell width. Remodeling of the apical extracellular matrix by the Stubble protease and basal matrix by MMP1/2 proteases induces wing and leg elongation. Matrix remodeling does not occur in the haltere, a limb that fails to elongate. Limb elongation is made anisotropic by planar polarized Myosin-II, which drives convergent extension along the proximal-distal axis. Subsequently, Myosin-II relocalizes to lateral membranes to accelerate columnar-to-cuboidal transition and isotropic tissue expansion. Thus, matrix remodeling induces dynamic changes in actomyosin contractility to drive epithelial morphogenesis in three dimensions.

13-P113 Mutation of Furin cleavage sites in Drosophila BMPs

Gli repressor formation via Protein kinase A (PKA) activation. In the current study, we generated a chondrocyte-specific knockout mouse strain of Suppressor of Fused (Sufu) and demonstrated that loss of Sufu leads to elevated expression of Hh target genes, Gli1 and Patched 1 (Ptch1), in the growth plate. Histological analysis illustrated an increase of proliferating cells and a reduction of the hypertrophic zone in Sufu-deficient growth plate mimicking that seen in Ptch1 knockout mice. These observations indicate that Sufu plays a negative role in Hh signaling during chondrocyte development. Furthermore, explant culture studies revealed that Sufu is a key regulator in controlling the effect of PTHrP on chondrocyte differentiation. In vertebrates, primary cilia are required for proper control of Gli activities in Hh signal transduction. Intriguingly, using micromass culture, we showed that PTHrP regulates Ptch1 expression through a cilia-independent mechanism during chondrocyte differentiation. We are currently investigating the molecular action of Sufu in PTHrP-dependent chondrocyte differentiation and the role of PTHrP in cilia-independent Hh signaling. The results of these studies will be presented.