Sol Sotillos

Compartmentalisation of Rho regulators directs cell invagination during tissue morphogenesis

During development, small RhoGTPases control the precise cell shape changes and movements that underlie morphogenesis. Their activity must be tightly regulated in time and space, but little is known about how Rho regulators (RhoGEFs and RhoGAPs) perform this function in the embryo. Taking advantage of a new probe that allows the visualisation of small RhoGTPase activity in Drosophila, we present evidence that Rho1 is apically activated and essential for epithelial cell invagination, a common morphogenetic movement during embryogenesis. In the posterior spiracles of the fly embryo, this asymmetric activation is achieved by at least two mechanisms: the apical enrichment of Rho1; and the opposing distribution of Rho activators and inhibitors to distinct compartments of the cell membrane. At least two Rho1 activators, RhoGEF2 and RhoGEF64C are localised apically, whereas the Rho inhibitor RhoGAP Cv-c localises at the basolateral membrane. Furthermore, the mRNA of RhoGEF64C is also apically enriched, depending on signals present within its open reading frame, suggesting that apical transport of RhoGEF mRNA followed by local translation is a mechanism to spatially restrict Rho1 activity during epithelial cell invagination.

Regulated Crb accumulation controls apical constriction and invagination in Drosophila tracheal cells.

Many epithelial tissues undergo extensive remodelling during morphogenesis. How their epithelial features, such as apicobasal polarity or adhesion, are maintained and remodelled and how adhesion and polarity proteins contribute to morphogenesis are two important questions in development. Here, we approach these issues by investigating the role of the apical determinant protein Crumbs (Crb) during the morphogenesis of the embryonic Drosophila tracheal system. Crb accumulates differentially throughout tracheal development and is required for different tracheal events. The earliest requirement for Crb is for tracheal invagination, which is preceded by an enhanced accumulation of Crb in the invagination domain. There, Crb, acting in parallel with the epidermal growth factor receptor (Egfr) pathway, is required for tracheal cell apical constriction and for organising an actomyosin complex, which we propose is mediated by Crb recruitment of moesin (Moe). The ability of a Crb isoform unable to rescue polarity in crb mutants to otherwise rescue their invagination phenotype, and the converse inability of a FERM-binding domain mutant Crb to rescue faulty invagination, support our hypothesis that it is the absence of Crb-dependent Moe enrichment, and not the polarity defect, that mainly underlies the crb invagination phenotype. This hypothesis is supported by the phenotype of lethal giant larvae (lgl); crb double mutants. These results unveil a link between Crb and the organisation of the actin cytoskeleton during morphogenesis.

Interactions between the Notch, EGFR, and decapentaplegic signaling pathways regulate vein differentiation during Drosophila pupal wing development.

The formation of longitudinal veins in the Drosophila wing involves cell interactions mediated by the conserved signaling pathways Decapentaplegic (Dpp), Notch, and epidermal growth factor receptor (EGFR). Interactions between Notch and EGFR taking place in the wing disc divide each vein into a central domain, where EGFR is active, and two boundary domains where Notch is active. The expression of decapentaplegic (dpp) is activated in the veins during pupal development, and we have generated Gal4 drivers using the regulatory region that drives dpp expression at this stage. By using these drivers, we studied the relationships between the Notch, EGFR, and Dpp signaling pathways that occur during pupal development. Our results indicate that the interactions between EGFR and Notch initiated in the imaginal disc are maintained throughout pupal development and contribute to determine the places where dpp is expressed. Once dpp expression is initiated, Dpp and EGFR activities in the provein maintain each other and, in cooperation, determine vein cell differentiation. Developmental Dynamics 232:738–752, 2005.

Genetic control of morphogenesis - Hox induced organogenesis of the posterior spiracles

The posterior spiracle has become one of the best systems to study how Hox genes control morphogenesis. Interaction of Abdominal-B (ABD-B) with dorso ventral and intrasegmental positional information leads to the local activation of ABD-B primary targets in the dorsal region of the eighth abdominal segment (A8). Primary targets pattern the spiracle subdividing it into two broad areas: external stigmatophore vs. internal spiracular chamber precursor cells. Primary targets then activate secondary targets and modulate the expression of signalling molecules in the spiracle primordium creating unique spiracle positional values. This genetic cascade activates the realisator genes that modulate the cell behaviours causing invagination, elongation and cell rearrangements responsible for spiracle morphogenesis. The spiracle realisators that have been identified to date correspond to cell adhesion proteins, cytoskeleton regulators and cell polarity molecules. Interestingly, these realisators localise to different apico-basal locations in the cell (RhoGEF apical, Crumbs subapical, E-cadherin in the adherens junction, RhoGAP basolateral). Therefore, the Hox anterior-posterior code is converted in the cell into apico-basal information required to implement the posterior spiracle morphogenetic program. We believe this may be a common characteristic for Hox induced organogenesis.

JAK-STAT pathway in Drosophila morphogenesis: From organ selector to cell behavior regulator

One of the main contributions of Drosophila to the JAK-STAT field is the study of morphogenesis. JAK-STAT signaling controls the formation of many different structures through surprisingly different morphogenetic behaviors that include induction of cell rearrangements, invagination, folding of tissues, modulation of cell shape, and migration. This variability may be explained by the many transcription factors and signaling molecules STAT regulates at early stages of development. But is STAT just acting as an upstream inducer of morphogenesis or does it have a more direct role in controlling cell behaviors? Here we review what is known about how the canonical phosphorylation of STAT contributes to shaping the embryonic and imaginal structures.

Src kinases mediate the interaction of the apical determinant Bazooka/PAR3 with STAT92E and increase signalling efficiency in Drosophila ectodermal cells

Intercellular communication depends on the correct organization of the signal transduction complexes. In many signalling pathways, the mechanisms controlling the overall cell polarity also localize components of these pathways to different domains of the plasma membrane. In the Drosophila ectoderm, the JAK/STAT pathway components are highly polarized with apical localization of the receptor, the associated kinase and the STAT92E protein itself. The apical localization of STAT92E is independent of the receptor complex and is due to its direct association with the apical determining protein Bazooka (Baz). Here, we find that Baz-STAT92E interaction depends on the presence of the Drosophila Src kinases. In the absence of Src, STAT92E cannot bind to Baz in cells or in whole embryos, and this correlates with an impairment of JAK/STAT signalling function. We believe that the requirement of Src proteins for STAT92E apical localization is mediated through Baz, as we can co-precipitate Src with Baz but not with STAT92E. This is the first time that a functional link between cell polarity, the JAK/STAT signalling pathway and the Src kinases has been established in a whole organism.

Forces shaping a Hox morphogenetic gene network

The Abdominal-B selector protein induces organogenesis of the posterior spiracles by coordinating an organ-specific gene network. The complexity of this network begs the questions of how it originated and what selective pressures drove its formation. Given that the network likely formed in a piecemeal fashion, with elements recruited sequentially, we studied the consequences of expressing individual effectors of this network in naive epithelial cells. We found that, with exception of the Crossveinless-c (Cv-c) Rho GTPase-activating protein, most effectors exert little morphogenetic effect by themselves. In contrast, Cv-c expression causes cell motility and down-regulates epithelial polarity and cell adhesion proteins. These effects differ in cells endogenously expressing Cv-c, which have acquired compensatory mechanisms. In spiracle cells, the down-regulation of polarity and E-cadherin expression caused by Cv-c–induced Rho1 inactivation are compensated for by the simultaneous spiracle up-regulation of guanine nucleotide exchange factor (GEF) proteins, cell polarity, and adhesion molecules. Other epithelial cells that have coopted Cv-c to their morphogenetic gene networks are also resistant to Cv-c’s deleterious effects. We propose that cooption of a novel morphogenetic regulator to a selector cascade causes cellular instability, resulting in strong selective pressure that leads that same cascade to recruit molecules that compensate it. This experimental-based hypothesis proposes how the frequently observed complex organogenetic gene networks are put together.

JAK/STAT Signalling: STAT Cannot Play with Ken and Barbie

Transcriptional responses to the activation of a signalling pathway are cell-specific. New data show that the sequence-specific transcriptional repressors of the KEN/BCL-6 family play an important role in the selection of STAT targets in vertebrates and invertebrates, indicating that all STAT proteins may share this ancestral mechanism.

Functional analysis of the Drosophila RhoGAP Cv-c protein and its equivalence to the human DLC3 and DLC1 proteins

RhoGAP proteins control the precise regulation of the ubiquitous small RhoGTPases. The Drosophila Crossveinless-c (Cv-c) RhoGAP is homologous to the human tumour suppressor proteins Deleted in Liver Cancer 1–3 (DLC1-3) sharing an identical arrangement of SAM, GAP and START protein domains. Here we analyse in Drosophila the requirement of each Cv-c domain to its function and cellular localization. We show that the basolateral membrane association of Cv-c is key for its epithelial function and find that the GAP domain targeted to the membrane can perform its RhoGAP activity independently of the rest of the protein, implying the SAM and START domains perform regulatory roles. We propose the SAM domain has a repressor effect over the GAP domain that is counteracted by the START domain, while the basolateral localization is mediated by a central, non-conserved Cv-c region. We find that DLC3 and Cv-c expression in the Drosophila ectoderm cause identical effects. In contrast, DLC1 is inactive but becomes functional if the central non-conserved DLC1 domain is substituted for that of Cv-c. Thus, these RhoGAP proteins are functionally equivalent, opening up the use of Drosophila as an in vivo model to analyse pharmacologically and genetically the human DLC proteins.