Integrin Signaling Regulates Spindle Orientation in Drosophila to Preserve the Follicular-Epithelium Monolayer

SummaryEpithelia act as important physiological barriers and as structural components of tissues and organs. In the Drosophila ovary, follicle cells envelop the germline cysts to form a monolayer epithelium. During division, the orientation of the mitotic spindle in follicle cells is such that both daughter cells remain within the same plane, and the simple structure of the follicular epithelium is thus preserved. Here we show that integrins, heterodimeric transmembrane receptors that connect the extracellular matrix to the cell's cytoskeleton [1, 2], are required for maintaining the ovarian monolayer epithelium in Drosophila. Mosaic egg chambers containing integrin mutant follicle cells develop stratified epithelia at both poles. This stratification is due neither to abnormal cell proliferation nor to defects in the apical-basal polarity of the mutant cells. Instead, integrin function is required for the correct orientation of the mitotic apparatus both in mutant cells and in their immediately adjacent wild-type neighbors. We further demonstrate that integrin-mediated signaling, rather than adhesion, is sufficient for maintaining the integrity of the follicular epithelium. The above data show that integrins are necessary for preserving the simple organization of a specialized epithelium and link integrin-mediated signaling to the correct orientation of the mitotic spindle in this epithelial cell type

Generation and characterization of mecp2 mutant.

Motivation: Mecp2 gene encodes the Methyl CpG binding protein (MeCP2). This protein is known for being an important regulator of gene expression that interacts with methylated and unmethylated genomic DNA regions, and enhances or silences transcriptional processes. Different mutations of the MeCP2 protein in humans can lead to a variety of symptoms in Rett syndrome (RTT), a rare disease which causes abnormalities during female brain development as well as acute mental and physical disability. These misfunctions are caused by mutations in one or two of the domains that can be found in the MeCP2 protein: the methyl binding domain (MBD) and the transcriptional repression domain (TRD). The aim of this study is the generation of a knock-out (KO) mutant of the mecp2 gene in zebrafish by employing the CRISPR/Cas9 technique. Once we obtain the homozygous mutant, we will elucidate the significance of this mutation by deep RNA sequencing (RNA-Seq), focusing on the differences between transcripts present in wild type (WT) and homozygous mutant fish. These results will give us an indication of which genes are potentially regulated by the MeCP2 protein, which could render zebrafish as a useful model system aimed at understanding RTT etiology in vivo.Methods: To create the mecp2 KO, we designed sgRNA guides targeting the mecp2 exon 2, using the CRISPRscan software. We then tested the sgRNAs in F0 by microinjection of single-cell stage zebra-fish embryos. The embryos were allowed to develop to 24-48 hours post fertilization (hpf), after which they were genotyped. Amplification of regions of interest by PCR followed by electrophoresis in agarose gel, showed us that the sgRNAs worked, as we could see a band different to that of the WT (471bp). After the raising of the F0, we genotyped the mutant individually to identify founders. The founders of interest were named as follows: (i) mecp2 26⚦, with a deletion of 181bp, (ii) mecp2 27, ⚦with deletion of 15bp and (iii) mecp2 13,⚦ with a deletion of 8bp. These founders were out-crossed with WT zebra-fish to stabilize the mutation (F1), and the offspring was genotyped and raised. We then genotyped the F1 generation by extracting the DNA from fin tissue (fin-clip); it is expected that 25% of this generation should be heterozygous. Once we have sequenced the heterozygous candidates for the mecp2 gene, we will in-cross a male and a female with the same mutation, in order to have a homozygous F2. Finally, we will genotype the F2 individually at 72h, extracting the DNA and RNA at the same time, using the qiagen DNAeasy blood and tissue kit for DNA and Trizol for RNA. The RNA of the confirmed homozygous mutants will be sent for RNA-Seq analysis.Results and conclusions: We have generated heterozygous mecp2 mutants, that we have to genotype, from the founders mentioned above (F1). The deletions sequenced cause the generation of a stop codon, so the MeCP2 protein should not be expressed in homozygous fish. The next step will be generating the homozygous generation in order to study the phenotype and perform the RNA-Seq at 72h of embryo development. Once we obtain the results of the RNA-seq analysis, we will be able to explain which are the genes whose expression is modulated by MeCP2 and that are potentially implicated in RTT

Generation and characterization of mecp2 mutant.

Motivation: Mecp2 gene encodes the Methyl CpG binding protein (MeCP2). This protein is known for being an importantregulator of gene expression that interacts with methylated and unmethylated genomic DNA regions, and enhances orsilences transcriptional processes. Different mutations of the MeCP2 protein in humans can lead to a variety of symptoms inRett syndrome (RTT), a rare disease which causes abnormalities during female brain development as well as acute mentaland physical disability. These misfunctions are caused by mutations in one or two of the domains that can be found in theMeCP2 protein: the methyl binding domain (MBD) and the transcriptional repression domain (TRD). The aim of this study isthe generation of a knock-out (KO) mutant of the mecp2 gene in zebrafish by employing the CRISPR/Cas9 technique. Oncewe obtain the homozygous mutant, we will elucidate the significance of this mutation by deep RNA sequencing (RNA-Seq),focusing on the differences between transcripts present in wild type (WT) and homozygous mutant fish. These results will giveus an indication of which genes are potentially regulated by the MeCP2 protein, which could render zebrafish as a usefulmodel system aimed at understanding RTT etiology in vivo.Methods: To create the mecp2 KO, we designed sgRNA guides targeting the mecp2 exon 2, using the CRISPRscan software.We then tested the sgRNAs in F0 by microinjection of single-cell stage zebra-fish embryos. The embryos were allowed todevelop to 24-48 hours post fertilization (hpf), after which they were genotyped. Amplification of regions of interest by PCRfollowed by electrophoresis in agarose gel, showed us that the sgRNAs worked, as we could see a band different to that of theWT (471bp). After the raising of the F0, we genotyped the mutant individually to identify founders. The founders of interestwere named as follows: (i) mecp2 26⚦, with a deletion of 181bp, (ii) mecp2 27 ,⚦ with deletion of 15bp and (iii) mecp2 13 ,⚦with a deletion of 8bp. These founders were out-crossed with WT zebra-fish to stabilize the mutation (F1), and the offspringwas genotyped and raised. We then genotyped the F1 generation by extracting the DNA from fin tissue (fin-clip); it is expectedthat 25% of this generation should be heterozygous. Once we have sequenced the heterozygous candidates for the mecp2gene, we will in-cross a male and a female with the same mutation, in order to have a homozygous F2. Finally, we willgenotype the F2 individually at 72h, extracting the DNA and RNA at the same time, using the qiagen DNAeasy blood andtissue kit for DNA and Trizol for RNA. The RNA of the confirmed homozygous mutants will be sent for RNA-Seq analysis.Results and conclusions: We have generated heterozygous mecp2 mutants, that we have to genotype, from the foundersmentioned above (F1). The deletions sequenced cause the generation of a stop codon, so the MeCP2 protein should not beexpressed in homozygous fish. The next step will be generating the homozygous generation in order to study the phenotypeand perform the RNA-Seq at 72h of embryo development. Once we obtain the results of the RNA-seq analysis, we will be ableto explain which are the genes whose expression is modulated by MeCP2 and that are potentially implicated in RT

Integrins contribute to the establishment and maintenance of cell polarity in the follicular epithelium of the Drosophila ovary

8 páginas, 5 figuras.The generation of epithelial cell polarity is a key process during development. Although the induction and orientation of cell polarity by cell-cell and cell-extracellular matrix (ECM) interactions is well established, the molecular mechanisms by which signals from the ECM control cell polarity in developing epithelial tissues remain poorly understood. Here, we have used the follicular epithelium of the Drosophila ovary to investigate the role that integrins, the main cell-ECM receptors, play in the establishment of apicobasal polarity. Mature follicle cells have an apical side facing the germ line and a basal side in contact with a basement membrane. Our results show that integrins - presumably via interactions with the basement membrane - play a reinforcing role in follicle cell polarization, as they are required to establish and/or maintain follicle cell membrane asymmetry only when contact with the germ line is prevented. We suggest that the primary cue for polarization of the follicular epithelium is contact with the germline cells. In addition, while interfering with apical and lateral polarization cues leads to apoptosis, we show here that inhibition of contact with the basement membrane mediated by integrins does not affect cell survival. Finally, we provide evidence to suggest that integrins are required to orientate epithelial polarity in vivo.We thank the Bloomington Stock Centre for fly stocks. Research in our laboratories is funded by the Spanish Ministerio de Ciencia y Tecnología(MCYT) (BFU2006-10934 to A.G-R; BFU2004-02840/BMC to M.D.M-B),by the EMBO Young Investigator Programme and by the Junta deAndalucía (CVI-280 and PO6-CVI-01592). L. C-R was supported by afellowship funded by the EMBO Young Investigator Programme and A. F-M was supported by a FPI studentship from the MCYT and by an I3P-CSIC contract. The institutional support from the Junta de Andalucía tothe CABD is acknowledged.Peer reviewe

The Ste20 kinase misshapen is essential for the invasive behaviour of ovarian epithelial cells in Drosophila

7 páginas, 5 figuras.Stationary-to-migratory transitions of epithelial cells have a key role in development and tumour progression. Border cell migration is a powerful system in which to investigate this transition in living organisms. Here, we identify the Ste20-like kinase misshapen (msn) as a novel regulator of border-cell migration in Drosophila. Expression of msn in border cells is independent of the transcription factor slow border cells and of inputs from all pathways that are known to control border-cell migration. The msn gene functions to modulate the levels and/or distribution of Drosophila E-cadherin to promote the invasive migratory behaviour of border cells.Funding from the Spanish Ministerio de Ciencia e Innovación (MICINN), the EMBO Young Investigator Programme and the Junta de Andalucía is acknowledged.Peer reviewe

Optic cup morphogenesis: a model to study the cellular and molecular bases of basal constriction

Resumen del trabajo presentado a ZING Conferences: Genes, Epigenetics and Evolution in Eye Development and Disease, celebrada en Cambridge (UK) del 28 de octubre al 1 de septiembre de 2014.Shaping the vertebrate eye into a hemispherical organ requires the inward folding of the polarized retinal neuroepithelium. Optic cup morphogenesis is however an atypical model for epithelial morphogenesis. In contrast to well-known apical constrictions described in Drosophila epithelia, it involves the contraction of the tissue towards its basal surface. Understanding which cellular and molecular aspects of basal constriction are specific and which are shared with apical constrictions is a long-term objective in the laboratory. In particular we focus on developmental regulators that, operating directly on basic cell properties, such as cell adhesion, cell shape or cell contractility, control eye morphogenesis. One of these developmental effectors, the transmembrane regulator of polarized integrin endocytosis Opo, plays an essential role in optic cup formation and hence it has been a main research topic in our group. Through a combination of transcriptomic, genetic, and cell biological approaches, we are currently identifying novel components of the molecular machinery involved in optic cup folding. Here we explore the mechanisms coupling the activation of the retinal gene regulatory network to the constriction-driven folding of the retinal epithelium. We show that which vsx2, operating through the effector gene opo, acts as a central transcriptional node that coordinates neural retina patterning and optic cup invagination in zebrafish.Peer Reviewe

A role for the chaperone Hsp70 in the regulation of border cell migration in the Drosophila ovary

11 páginas, 6 figuras.Unravelling the molecular mechanisms that govern cell migration is of great importance towards understanding both normal embryogenesis and physiological and pathological processes occurring in the adult. Migration of border cells (BCs) during Drosophila oogenesis provides a simple and attractive model in which to address this problem. Here, we show that the molecular chaperone Hsp70 is required for BC migration. Thus, BCs lacking all Hsp70 genes present in the fly genome fail to reorganize their actin cytoskeleton, resulting in migration defects. Similar defects are found when the Hsp70 co-chaperone DnaJ-1, the Drosophila homolog of the human Hsp40, is overexpressed specifically in BCs. In addition, we provide biochemical and genetic evidence for an interaction between DnaJ-1 and PDGF/VEGF receptor (PVR), which is also required for actin-mediated BC migration. Furthermore, our results showing that PVR also interacts genetically with Hsp70 suggest that a mechanism by which the DnaJ-1/Hsp70 chaperone complex regulates BC migration is by modulating PVR function.Research in our laboratories is funded by the Spanish Ministerio de Ciencia y Tecnología (MCYT) (BMC2003-01512 and BFU2006-10934 to A.G.-R; BFU2004-02840/BMC and BFU2007-64715/BMC to M.D.M.-B), by the EMBO Young Investigator Programme and by the Junta de Andalucía (CVI-280 and P06-CVI-01592). L.C. was supported by a fellowship funded by the EMBO Young Investigator Programme and A.F.-M. was supported by a FPI studentship from the MCYT and by an I3P-CSIC contract. C.M.L. was supported by an EMBO long-term fellowship. The institutional support from the Junta de Andalucía to the CABD is acknowledged.Peer reviewe

Numb/Numbl-Opo Antagonism Controls Retinal Epithelium Morphogenesis by Regulating Integrin Endocytosis

Polarized trafficking of adhesion receptors plays a pivotal role in controlling cellular behavior during morphogenesis. Particularly, clathrin-dependent endocytosis of integrins has long been acknowledged as essential for cell migration. However, little is known about the contribution of integrin trafficking to epithelial tissue morphogenesis. Here we show how the transmembrane protein Opo, previously described for its essential role during optic cup folding, plays a fundamental role in this process. Through interaction with the PTB domain of the clathrin adaptors Numb and Numbl via an integrin-like NPxF motif, Opo antagonizes Numb/Numbl function and acts as a negative regulator of integrin endocytosis in vivo. Accordingly, numb/numbl gain-of-function experiments in teleost embryos mimic the retinal malformations observed in opo mutants. We propose that developmental regulator Opo enables polarized integrin localization by modulating Numb/Numbl, thus directing the basal constriction that shapes the vertebrate retina epithelium.The programs “Intramural/CSIC” 200920I212, Juan de la Cierva, and FCT (Portugal) supported O.B., M.D.-M., and I.G.-R., respectively. This work was supported by grants BFU2008-04362 and BFU2011-22916.Peer reviewe

Numb/Numbl-Opo antagonism controls retinal epithelium morphogenesis by regulating integrin endocytosis

Polarized trafficking of adhesion receptors plays a pivotal role in controlling cellular behavior during morphogenesis. Particularly, clathrin-dependent endocytosis of integrins has long been acknowledged as essential for cell migration. However, little is known about the contribution of integrin trafficking to epithelial tissue morphogenesis. Here we show how the transmembrane protein Opo, previously described for its essential role during optic cup folding, plays a fundamental role in this process. Through interaction with the PTB domain of the clathrin adaptors Numb and Numbl via an integrin-like NPxF motif, Opo antagonizes Numb/Numbl function and acts as a negative regulator of integrin endocytosis in vivo. Accordingly, numb/numbl gain-of-function experiments in teleost embryos mimic the retinal malformations observed in opo mutants. We propose that developmental regulator Opo enables polarized integrin localization by modulating Numb/Numbl, thus directing the basal constriction that shapes the vertebrate retina epithelium.info:eu-repo/semantics/publishedVersio