Jose F. de Celis

Roles of the Notch gene in Drosophila wing morphogenesis

The Notch gene encodes a transmembrane protein that functions as a receptor of intercellular signals in many developmental processes of Drosophila. We study here the Notch function in wing morphogenesis and vein patterning in genetic mosaics of both Notch null and Notch gain-of-function alleles. Cell proliferation and differentiation properties of mutant Notch cells define three different Notch requirements in the wing: in imaginal disc cell proliferation, in restriction of vein differentiation and in margin formation. The study of Notch mosaics in different mutant backgrounds reveals that Notch activity during epidermal cell proliferation and wing vein differentiation is exerted by its regulation of a common group of genes involved in the specification and restriction of vein competent regions.

Two-step process for photoreceptor formation in Drosophila.

The formation of photoreceptor cells (PRCs) in Drosophila serves as a paradigm for understanding neuronal determination and differentiation. During larval stages, a precise series of sequential inductive processes leads to the recruitment of eight distinct PRCs (R1–R8). But, final photoreceptor differentiation, including rhabdomere morphogenesis and opsin expression, is completed four days later, during pupal development. It is thought that photoreceptor cell fate is irreversibly established during larval development, when each photoreceptor expresses a particular set of transcriptional regulators and sends its projection to different layers of the optic lobes. Here, we show that the spalt (sal) gene complex encodes two transcription factors that are required late in pupation for photoreceptor differentiation. In the absence of the sal complex, rhabdomere morphology and expression of opsin genes in the inner PRCs R7 and R8 are changed to become identical to those of outer R1–R6 PRCs. However, these cells maintain their normal projections to the medulla part of the optic lobe, and not to the lamina where outer PRCs project. These data indicate that photoreceptor differentiation occurs as a two-step process. First, during larval development, the photoreceptor neurons become committed and send their axonal projections to their targets in the brain. Second, terminal differentiation is executed during pupal development and the photoreceptors adopt their final cellular properties.

Regulation and function of Spalt proteins during animal development

The genes of the spalt (sal) family play fundamental roles during animal development. The two members of this family in Drosophila, spalt (sal) and spalt-related (salr) encode Zn-finger transcription factors that link the Decapentaplegic (Dpp)/BMP signalling pathway to the patterning of the wing. They are regulated by the Dpp pathway in the wing disc, and they were shown to mediate some of the morphogenetic activities of the Dpp/BMP4 secreted ligand. The sal genes were initially found by virtue of mutations that produce homeotic transformations in the head and tail of the Drosophila embryo. Since then, a number of other requirements have been associated to these genes in Drosophila, including morphogenesis of the respiratory system, cell fate specification of sensory organs and the differentiation of several photoreceptor cells, among others. Vertebrate sal orthologues (spalt-like/sall) have also important developmental roles during neural development and organogenesis, and at least two human sall genes are linked to the genetic diseases Townes Brocks Syndrome (TBS; SALL1 ) and Okihiro Syndrome (OS; SALL4 ). In this review, we will summarize the main characteristics of the sall genes and proteins, pointing out to the similarities in their developmental roles during Drosophila and vertebrate development.

Drosophila laminins act as key regulators of basement membrane assembly and morphogenesis

Laminins are heterotrimeric molecules found in all basement membranes. In mammals, they have been involved in diverse developmental processes, from gastrulation to tissue maintenance. The Drosophila genome encodes two laminin α chains, one β and one Γ, which form two distinct laminin trimers. So far, only mutations affecting one or other trimer have been analysed. In order to study embryonic development in the complete absence of laminins, we mutated the gene encoding the sole laminin β chain in Drosophila, LanB1, so that no trimers can be made. We show that LanB1 mutant embryos develop until the end of embryogenesis. Electron microscopy analysis of mutant embryos reveals that the basement membranes are absent and the remaining extracellular material appears disorganised and diffuse. Accordingly, abnormal accumulation of major basement membrane components, such as Collagen IV and Perlecan, is observed in mutant tissues. In addition, we show that elimination of LanB1 prevents the normal morphogenesis of most organs and tissues, including the gut, trachea, muscles and nervous system. In spite of the above structural roles for laminins, our results unravel novel functions in cell adhesion, migration and rearrangement. We propose that while an early function of laminins in gastrulation is not conserved in Drosophila and mammals, their function in basement membrane assembly and organogenesis seems to be maintained throughout evolution.

Conserved cross-interactions in Drosophila and Xenopus between Ras/MAPK signaling and the dual-specificity phosphatase MKP3

The extracellular signal‐regulated kinase (ERK) is a key transducer of the epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR) signaling pathways, and its function is required in multiple processes during animal development. The activity of ERK depends on the phosphorylation state of conserved threonine and tyrosine residues, and this state is regulated by different kinases and phosphatases. A family of phosphatases with specificity toward both threonine and tyrosine residues in ERK (dual‐specificity phosphatases) play a conserved role in its dephosphorylation and consequent inactivation. Here, we characterize the function of the dual‐specificity phosphatase MKP3 in Drosophila EGFR and Xenopus FGFR signaling. The function of MKP3 is required during Drosophila wing vein formation and Xenopus anteroposterior neural patterning. We find that the expression of the MKP3 gene is localized in places of high EGFR and FGFR signaling. Furthermore, this restricted expression depends on ERK function both in Drosophila and Xenopus, suggesting that MKP3 constitutes a conserved negative feedback loop on the activity of the Ras/ERK signaling pathway. Developmental Dynamics 232:695–708, 2005.

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.

The Complex Tale of the achaete–scute Complex: A Paradigmatic Case in the Analysis of Gene Organization and Function During Development

The achaete–scute gene complex (AS-C) contains four genes encoding transcription factors of the bHLH family, achaete, scute, lethal of scute, and asense located in 40 kb of DNA containing multiple cis-regulatory position-specific enhancers. These genes play a key role in the commitment of epidermal cells toward a neural fate, promoting the formation of both sensory organs in the peripheral nervous system (bristles) of the adult and of neuroblasts in the central nervous system of the embryo. The analysis of the AS-C initially focused on the variations in positional specificity of effects of achaete (ac) and scute (sc) alleles on macrochaete bristle pattern in the Drosophila adult epidermis, and from there it evolved as a key entry point into understanding the molecular bases of pattern formation and cell commitment. In this perspective, we describe how the study of the AS-C has contributed to the understanding of eukaryotic gene organization and the dissection of the developmental mechanisms underlying pattern formation.

A Gain-of-Function Screen Identifying Genes Required for Growth and Pattern Formation of the Drosophila melanogaster Wing

The Drosophila melanogaster wing is a model system for analyzing the genetic control of organ size, shape, and pattern formation. The formation of the wing involves a variety of processes, such as cell growth, proliferation, pattern formation, and differentiation. These developmental processes are under genetic control, and many genes participating in specific aspects of wing development have already being characterized. In this work, we aim to identify novel genes regulating wing growth and patterning. To this end, we have carried out a gain-of-function screen generating novel P-UAS (upstream activating sequences) insertions allowing forced gene expression. We produced 3340 novel P-UAS insertions and isolated 300 that cause a variety of wing phenotypes in combination with a Gal4 driver expressed exclusively in the central domain of the presumptive wing blade. The mapping of these P-UAS insertion sites allowed us to identify the gene that causes the gain-of-function phenotypes. We show that a fraction of these phenotypes are related to the induction of cell death in the domain of ectopic gene expression. Finally, we present a preliminary characterization of a gene identified in the screen, the function of which is required for the development of the L5 longitudinal vein.

A Gain-of-Function Screen Identifying Genes Required for Vein Formation in the Drosophila melanogaster Wing

The formation of the Drosophila wing involves developmental processes such as cell proliferation, pattern formation, and cell differentiation that are common to all multicellular organisms. The genes controlling these cellular behaviors are conserved throughout the animal kingdom, and the genetic analysis of wing development has been instrumental in their identification and functional characterization. The wing is a postembryonic structure, and most loss-of-function mutations are lethal in homozygous flies before metamorphosis. In this manner, loss-of-function genetic screens aiming to identify genes affecting wing formation have not been systematically utilized. As an alternative, a number of genetic searches have utilized the phenotypic consequences of gene gain-of-expression, as a method more efficient to search for genes required during imaginal development. Here we present the results of a gain-of-function screen designed to identify genes involved in the formation of the wing veins. We generated 13,000 P-GS insertions of a P element containing UAS sequences (P-GS) and combined them with a Gal4 driver expressed mainly in the developing pupal veins. We selected 500 P-GSs that, in combination with the Gal4 driver, result in modifications of the veins, changes in the morphology of the wing, or defects in the differentiation of the trichomes. The P-element insertion sites were mapped to the genomic sequence, identifying 373 gene candidates to participate in wing morphogenesis and vein formation.

Role of the Drosophila Non-Visual ß-Arrestin Kurtz in Hedgehog Signalling

The non-visual ß-arrestins are cytosolic proteins highly conserved across species that participate in a variety of signalling events, including plasma membrane receptor degradation, recycling, and signalling, and that can also act as scaffolding for kinases such as MAPK and Akt/PI3K. In Drosophila melanogaster, there is only a single non-visual ß-arrestin, encoded by kurtz, whose function is essential for neuronal activity. We have addressed the participation of Kurtz in signalling during the development of the imaginal discs, epithelial tissues requiring the activity of the Hedgehog, Wingless, EGFR, Notch, Insulin, and TGFβ pathways. Surprisingly, we found that the complete elimination of kurtz by genetic techniques has no major consequences in imaginal cells. In contrast, the over-expression of Kurtz in the wing disc causes a phenotype identical to the loss of Hedgehog signalling and prevents the expression of Hedgehog targets in the corresponding wing discs. The mechanism by which Kurtz antagonises Hedgehog signalling is to promote Smoothened internalization and degradation in a clathrin- and proteosomal-dependent manner. Intriguingly, the effects of Kurtz on Smoothened are independent of Gprk2 activity and of the activation state of the receptor. Our results suggest fundamental differences in the molecular mechanisms regulating receptor turnover and signalling in vertebrates and invertebrates, and they could provide important insights into divergent evolution of Hedgehog signalling in these organisms.