Sonsoles Campuzano

DaPKC-dependent phosphorylation of Crumbs is required for epithelial cell polarity in Drosophila

Both in Drosophila and vertebrate epithelial cells, the establishment of apicobasal polarity requires the apically localized, membrane-associated Par-3–Par-6–aPKC protein complex. In Drosophila, this complex colocalizes with the Crumbs–Stardust (Sdt)–Pals1-associated TJ protein (Patj) complex. Genetic and molecular analyses suggest a functional relationship between them. We show, by overexpression of a kinase-dead Drosophila atypical PKC (DaPKC), the requirement for the kinase activity of DaPKC to maintain the position of apical determinants and to restrict the localization of basolateral ones. We demonstrate a novel physical interaction between the apical complexes, via direct binding of DaPKC to both Crb and Patj, and identify Crumbs as a phosphorylation target of DaPKC. This phosphorylation of Crumbs is functionally significant. Thus, a nonphosphorylatable Crumbs protein behaves in vivo as a dominant negative. Moreover, the phenotypic effect of overexpressing wild-type Crumbs is suppressed by reducing DaPKC activity. These results provide a mechanistic framework for the functional interaction between the Par-3–Par-6–aPKC and Crumbs–Sdt–Patj complexes based in the posttranslational modification of Crb by DaPKC.

Molecular analysis of the asense gene, a member of the achaete-scute complex of Drosophila melanogaster, and its novel role in optic lobe development.

The achaete‐scute complex (AS‐C) comprises five genetic regions: achaete, scute (sc) alpha, lethal of sc, sc beta and sc gamma. Each region promotes the determination and positional specification of different, but partially overlapping, subsets of neural elements of Drosophila. In this work, we report a molecular characterization of the sc gamma region. It comprises 22 kb of DNA and contains two transcription units, only one of which, named asense (ase), seems involved in neurogenesis. ase encodes a protein that shares with other three AS‐C proteins a domain containing a helix‐‐loop‐‐helix motif characteristic of a group of DNA‐binding proteins. In the embryo, ase is expressed in neural precursor cells, a pattern consistent with the known requirement of sc gamma for the development of the larval nervous system. In late third‐instar larvae, the gene is expressed in developing structures of the central nervous system (CNS), namely the anlagen of the optic lobes and in many cells, including neuroblasts, of the central brain and ventral ganglia. Its removal leads to anatomical defects in the adult optic lobes. This is the first demonstration of a role for the AS‐C in the development of the adult CNS.

The achaete-scute complex is expressed in neurogenic regions of Drosophila embryos

The achaete‐scute gene complex (AS‐C) of Drosophila melanogaster is involved in the development of the embryonic central nervous system and the cuticular sensory organs of both larva and adult. We have determined the embryonic spatial distributions of three transcripts encoded in the achaete, scute α and lethal of scute regions of the complex. The RNAs are present mainly between the blastoderm/early gastrula and the stage of germ band shortening. The patterns of expression are complex and evolve rapidly, affecting most or all of the known neurogenic regions. Gene expression precedes and is concomitant with the histological appearance of precursors of neural cells. These results support a role for the AS‐C in determination and early differentiation of embryonic neural cells.

Half a century of neural prepatterning: the story of a few bristles and many genes

In 1954, Curt Stern proposed the concept of the neural prepattern, meaning the underlying positional information in an undifferentiated epithelium that determined where neural differentiation could take place. Subsequent work gave a molecular basis to this concept, which was equated to a combination of transcription factors deployed in partially overlapping spatial domains that regulated proneural genes and, thereby, neural differentiation. Here, we review the work that, in the past few years, has identified many prepattern genes and has disclosed that their function is not limited to the regulation of proneural genes.

GH3, a novel proapoptotic domain in Drosophila Grim, promotes a mitochondrial death pathway.

Grim encodes a protein required for programmed cell death in Drosophila. The Grim N‐terminus induces apoptosis by disrupting IAP blockage of caspases; however, N‐terminally‐deleted Grim retains pro apoptotic activity. We describe GH3, a 15 amino acid internal Grim domain absolutely required for its proapoptotic activity and sufficient to induce cell death when fused to heterologous carrier proteins. A GH3 homology region is present in the Drosophila proapoptotic proteins Reaper and Sickle. The GH3 domain and the homologous regions in Reaper and Sickle are predicted to be structured as amphipathic α‐helixes. During apoptosis induction, Grim colocalizes with mitochondria and cytochrome c in a GH3‐dependent but N‐terminal‐ and caspase activity‐independent manner. When Grim is overexpressed in vivo, both the N‐terminal and the GH3 domains are equally necessary, and cooperate for apoptosis induction. The N‐terminal and GH3 Grim domains thus activate independent apoptotic pathways that synergize to induce programmed cell death efficiently.

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.

DNA map of mutations at the scute locus of Drosophila melanogaster

The achaete‐scute gene complex (AS‐C) of Drosophila melanogaster is involved in the differentiation of innervated elements in the adult (chaetes) and in the embryo (central nervous system). Genetically, the AS‐C is subdivided into four regions: achaete, scute α, lethal of scute, and scute β. Using a previously cloned fragment of scute DNA, we have now cloned 62 kb of wild‐type DNA from the scute region. No repetitive sequences have been detected in this stretch of DNA. Of 16 scute mutants with chromosomal rearrangements studied (inversions, deletions, and translocations), nine, included genetically in scute β, have breakpoints in the cloned region. The remaining rearrangements, which genetically correspond to scute α, map outside and to the left of the cloned region. Of nine scute ‘point mutants’ studied, eight have large DNA alterations within the cloned region. These alterations include insertions (five) and deletions (three). The DNA alterations found in both ‘point mutants’ and rearrangements are interspersed and scattered over 40 kb. The relationship between the sites of the DNA alterations and the mutant phenotypes are discussed.

An ancient genomic regulatory block conserved across bilaterians and its dismantling in tetrapods by retrogene replacement

Developmental genes are regulated by complex, distantly located cis-regulatory modules (CRMs), often forming genomic regulatory blocks (GRBs) that are conserved among vertebrates and among insects. We have investigated GRBs associated with Iroquois homeobox genes in 39 metazoans. Despite 600 million years of independent evolution, Iroquois genes are linked to ankyrin-repeat-containing Sowah genes in nearly all studied bilaterians. We show that Iroquois-specific CRMs populate the Sowah locus, suggesting that regulatory constraints underlie the maintenance of the Iroquois-Sowah syntenic block. Surprisingly, tetrapod Sowah orthologs are intronless and not associated with Iroquois; however, teleost and elephant shark data demonstrate that this is a derived feature, and that many Iroquois-CRMs were ancestrally located within Sowah introns. Retroposition, gene, and genome duplication have allowed selective elimination of Sowah exons from the Iroquois regulatory landscape while keeping associated CRMs, resulting in large associated gene deserts. These results highlight the importance of CRMs in imposing constraints to genome architecture, even across large phylogenetic distances, and of gene duplication-mediated genetic redundancy to disentangle these constraints, increasing genomic plasticity.

Mirror represses pipe expression in follicle cells to initiate dorsoventral axis formation in Drosophila

Dorsoventral (DV) axis formation in Drosophila begins with selective activation of EGFR, a receptor tyrosine kinase (RTK), in dorsal-anterior (DA) ovarian follicle cells. A critical event regulated by EGFR signaling is the repression of the sulfotransferase-encoding gene pipe in dorsal follicle cells, but how this occurs remains unclear. Here we show that Mirror (Mirr), a homeodomain transcription factor induced by EGFR signaling in DA follicle cells, directly represses pipe expression by binding to a conserved element in the pipe regulatory region. In addition, we find that the HMG-box protein Capicua (Cic) supports pipe expression in ventral follicle cells by repressing Mirr in this region. Interestingly, this role of Cic resembles its function in regulating anteroposterior (AP) body patterning, where Cic supports gap gene expression in central regions of the embryo by repressing Tailless, a repressor induced by RTK signaling at the embryonic poles. Thus, related RTK-Cic repressor circuits regulate the early stages of Drosophila DV and AP body axis formation.

Antagonistic and cooperative actions of the EGFR and Dpp pathways on the iroquois genes regulate Drosophila mesothorax specification and patterning.

In Drosophila, restricted expression of the Iroquois complex (Iro-C) genes in the proximal region of the wing imaginal disc contributes to its territorial subdivision, specifying first the development of the notum versus the wing hinge, and subsequently, that of the lateral versus medial notum. Iro-C expression is under the control of the EGFR and Dpp signalling pathways. To analyze how both pathways cooperate in the regulation of Iro-C, we isolated several wing disc-specific cis-regulatory elements of the complex. One of these (IroRE2) integrates competing inputs of the EGFR and Dpp pathways, mediated by the transcription factors Pointed (downstream of EGFR pathway) and Pannier/U-shaped and Mothers against Dpp (Mad), in the case of Dpp. By contrast, a second element (IroRE1) mediates activation by both the EGFR and Dpp pathways, thus promoting expression of Iro-C in a region of elevated levels of Dpp signalling, the prospective lateral notum near the anterior-posterior compartment boundary. These results help define the molecular mechanisms of the interplay between the EGFR and Dpp pathways in the specification and patterning of the notum.