Project description:Transcription factors, which regulate the spatiotemporal patterns of gene expression during organogenesis, often regulate multiple aspects of tissue morphogenesis, including cell-type specification, cell proliferation, cell death, cell polarity, cell shape, cell arrangement and cell migration. In this work, we describe a distinct role for Ribbon (Rib) in controlling cell shape changes during elongation of the Drosophila salivary gland (SG). Notably, the morphogenetic changes in rib mutants occurred without effects on general SG cell attributes such as specification, proliferation and apoptosis. Moreover, the abnormal cell/organ shape in rib mutants occurred without compromising epithelial-specific morphological attributes such as apicobasal polarity and junctional integrity. To identify the genes regulated by Rib that control cell and organ shape, we performed ChIP-seq analysis in embryos driving rib expression specifically in the SGs. To learn if the Rib binding sites identified in the ChIP-seq analysis were linked to changes in gene expression through transcriptional activation, repression, or both, we performed microarray analysis comparing RNA samples from age-matched wild-type and rib null embryos. From the superposed ChIP-seq and microarray gene expression data, we identified 60 genomic sites of bound Rib most likely to regulate SG-specific gene expression. We confirmed several of the identified Rib targets by qRT-pCR and/or in situ hybridization. Our results indicate that Rib regulates cell shape change in the Drosophila salivary gland via a diverse array of targets through both transcriptional activation and repression. Furthermore, our results suggest that a critical component of the SG morphogenetic gene network involving Rib is its autoregulation.
Project description:Transcription factors, which regulate the spatiotemporal patterns of gene expression during organogenesis, often regulate multiple aspects of tissue morphogenesis, including cell-type specification, cell proliferation, cell death, cell polarity, cell shape, cell arrangement and cell migration. In this work, we describe a distinct role for Ribbon (Rib) in controlling cell shape changes during elongation of the Drosophila salivary gland (SG). Notably, the morphogenetic changes in rib mutants occurred without effects on general SG cell attributes such as specification, proliferation and apoptosis. Moreover, the abnormal cell/organ shape in rib mutants occurred without compromising epithelial-specific morphological attributes such as apicobasal polarity and junctional integrity. To identify the genes regulated by Rib that control cell and organ shape, we performed ChIP-seq analysis in embryos driving rib expression specifically in the SGs. To learn if the Rib binding sites identified in the ChIP-seq analysis were linked to changes in gene expression through transcriptional activation, repression, or both, we performed microarray analysis comparing RNA samples from age-matched wild-type and rib null embryos. From the superposed ChIP-seq and microarray gene expression data, we identified 60 genomic sites of bound Rib most likely to regulate SG-specific gene expression. We confirmed several of the identified Rib targets by qRT-pCR and/or in situ hybridization. Our results indicate that Rib regulates cell shape change in the Drosophila salivary gland via a diverse array of targets through both transcriptional activation and repression. Furthermore, our results suggest that a critical component of the SG morphogenetic gene network involving Rib is its autoregulation. Three independent collections of stage 11 â 16 rib1/ribP7 embryos and three of wild-type embryos were used for hybridization to Drosophila Genome 2.0 Chips. Scanned intensity values were normalized using RMA (Partek software) and statistical analysis analyses were performed using the Spotfire software package (TIBCO). Target genes were identified as those that were upregulated/downregulated (1.5-fold change cutoff, P < 0.05) in rib1/ribP7 embryos when compared with Oregon R controls.
Project description:Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This methods paper describes how to isolate mid-to-late stage follicles (Stage 10B-14) and utilize them to provide new insights into the molecular and morphologic events occurring during tight windows of developmental time. Isolated follicles can be used for a variety of experimental techniques, including in vitro development assays, live imaging, mRNA expression analysis and western blot analysis of proteins. Follicles at Stage 10B (S10B) or later will complete development in culture; this allows one to combine genetic or pharmacologic perturbations with in vitro development to define the effects of such manipulations on the processes occurring during specific periods of development. Additionally, because these follicles develop in culture, they are ideally suited for live imaging studies, which often reveal new mechanisms that mediate morphological events. Isolated follicles can also be used for molecular analyses. For example, changes in gene expression that result from genetic perturbations can be defined for specific developmental windows. Additionally, protein level, stability, and/or posttranslational modification state during a particular stage of follicle development can be examined through western blot analyses. Thus, stage-specific isolation of Drosophila follicles provides a rich source of information into widely conserved processes of development and morphogenesis.
Project description:To determine the genes directly and indirectly under the control of the Grainy head transcription factor during late stages of Drosophila embryogenesis.
Project description:Transcription factors, which regulate the spatiotemporal patterns of gene expression during organogenesis, often regulate multiple aspects of tissue morphogenesis, including cell-type specification, cell proliferation, cell death, cell polarity, cell shape, cell arrangement and cell migration. In this work, we describe a distinct role for Ribbon (Rib) in controlling cell shape changes during elongation of the Drosophila salivary gland (SG). Notably, the morphogenetic changes in rib mutants occurred without effects on general SG cell attributes such as specification, proliferation and apoptosis. Moreover, the abnormal cell/organ shape in rib mutants occurred without compromising epithelial-specific morphological attributes such as apicobasal polarity and junctional integrity. To identify the genes regulated by Rib that control cell and organ shape, we performed ChIP-seq analysis in embryos driving rib expression specifically in the SGs. To learn if the Rib binding sites identified in the ChIP-seq analysis were linked to changes in gene expression through transcriptional activation, repression, or both, we performed microarray analysis comparing RNA samples from age-matched wild-type and rib null embryos. From the superposed ChIP-seq and microarray gene expression data, we identified 60 genomic sites of bound Rib most likely to regulate SG-specific gene expression. We confirmed several of the identified Rib targets by qRT-pCR and/or in situ hybridization. Our results indicate that Rib regulates cell shape change in the Drosophila salivary gland via a diverse array of targets through both transcriptional activation and repression. Furthermore, our results suggest that a critical component of the SG morphogenetic gene network involving Rib is its autoregulation.
Project description:The somatic muscles of Drosophila develop in a complex pattern that is repeated in each embryonic hemi-segment. During early development, progenitor cells fuse to form a syncytial muscle, which further differentiates via expression of muscle-specific factors that induce specific responses to external signals to regulate late-stage processes such as migration and attachment. Initial communication between somatic muscles and the epidermal tendon cells is critical for both of these processes. However, later establishment of attachments between longitudinal muscles at the segmental borders is largely independent of the muscle-epidermal attachment signals, and relatively little is known about how this event is regulated. Using a combination of null mutations and a truncated version of Sd that binds Vg but not DNA, we show that Vestigial (Vg) is required in ventral longitudinal muscles to induce formation of stable intermuscular attachments. In several muscles, this activity may be independent of Sd. Furthermore, the cell-specific differentiation events induced by Vg in two cells fated to form attachments are coordinated by Drosophila epidermal growth factor signaling. Thus, Vg is a key factor to induce specific changes in ventral longitudinal muscles 1-4 identity and is required for these cells to be competent to form stable intermuscular attachments with each other.
Project description:BackgroundThe common house spider Parasteatoda tepidariorum represents an emerging new model organism of arthropod evolutionary and developmental (EvoDevo) studies. Recent technical advances have resulted in the first single-cell sequencing (SCS) data on this species allowing deeper insights to be gained into its early development, but mid-to-late stage embryos were not included in these pioneering studies.ResultsTherefore, we performed SCS on mid-to-late stage embryos of Parasteatoda and characterized resulting cell clusters by means of in-silico analysis (comparison of key markers of each cluster with previously published information on these genes). In-silico prediction of the nature of each cluster was then tested/verified by means of additional in-situ hybridization experiments with additional markers of each cluster.ConclusionsOur data show that SCS data reliably group cells with similar genetic fingerprints into more or less distinct clusters, and thus allows identification of developing cell types on a broader level, such as the distinction of ectodermal, mesodermal and endodermal cell lineages, as well as the identification of distinct developing tissues such as subtypes of nervous tissue cells, the developing heart, or the ventral sulcus (VS). In comparison with recent other SCS studies on the same species, our data represent later developmental stages, and thus provide insights into different stages of developing cell types and tissues such as differentiating neurons and the VS that are only present at these later stages.
Project description:Notch and Delta proteins generate Notch signaling that specifies cell fates during animal development. There is an intriguing phenomenon in Drosophila embryogenesis that has not received much attention and whose significance to embryogenesis is unknown. Notch and Delta mRNAs expressed in early-stage embryos are shorter than their counterparts in mid-stage embryos. We show here that the difference in sizes is due to mRNA 3' processing at alternate polyadenylation sites. While the early-stage Notch mRNA has a lower protein-producing potential than the mid-stage Notch mRNA, the early-stage Delta mRNA has a higher protein-producing potential than the mid-stage Delta mRNA. Our data can explain the complementary patterns of Notch and Delta protein levels in early- and mid-stage embryos. Our data also raise the possibility that the manner and regulation of Notch signaling change in the course of embryogenesis and that this change is effected by 3' UTR and mRNA 3' processing factors.
Project description:To determine the genes directly and indirectly under the control of the Grainy head transcription factor during late stages of Drosophila embryogenesis. Total RNA from pre-larval (late stage 16 and early stage 17) grhIM homozygous (+; cn, grhIM, bw, sp; +) and wild-type (y; cn, bw, sp; +) embryos were compared.
Project description:The ETS transcriptional repressor Yan functions as part of a developmental switch that in response to receptor tyrosine kinase signaling, transitions from a high-Yan to a low-Yan state to direct specification of a variety of cell fates. To date very few direct target genes have been identified, nor is it clear how their expression is buffered against developmental noise to prevent inappropriate oscillations between states. Following investigation of its genome-wide chromatin occupancy profile, we noticed a striking signature at developmentally important genes whereby Yan associates with chromatin in regions of high-peak density that span multiple kilobases which partially relies upon SAM-domain mediated self-association. We speculate that the high-density Yan occupancy signature may reveal a novel mechanism that buffers the expression of critical developmental regulators against intrinsic and environmental noise. The supplementary bed file contains Yan binding regions.