Project description:The goal of this study is to investigate the molecular mechanism of lhx1 on regulation of pronephros formation during the early embryonic development. In the vertebrate embryo the kidney is derived from the intermediate mesoderm. The LIM-class homeobox transcription factor lhx1 is expressed early in the intermediate mesoderm and is one of the first genes to be expressed in the nephric mesenchyme. The animal cap cells can be induced by treatment of activin and retinoic acid to differentiate into pronephros tissue. In this study we investigated the role of Lhx1 in differentiation of pronephros by depleting lhx1 in the organ culture system. We generated the gene expression profile of early pronephros tissue, and demonstrated that expression of genes from all the kidney domains is affected by the absence of lhx1. Taken together our results highlight an essential role for Lhx1 in pronephros formation. lhx1 is involved in driving specification of intermediate mesoderm into nephrogenic mesenchyme. Lhx1 is initially expressed throughout the entire intermediate mesoderm. To determine the role of lhx1 pronephros formation, we performed a microarray analysis using an explant culture system. Xenopus tissue explants can be surgically isolated and cultured under specific conditions to be driven towards many distinct tissue types. Formation of pronephric cell fates is induced by culturing isolated explants in the presence of Activin and RA (AcRA). Treatment of dissected explants of stage 9 blastulae embryos with 10ng/ml Activin and 1x10-4 M retinoic acid can induce differentiation of the pluripotent ectoderm into pan-kidney tissue. For this experiment, both blastomeres of 2-cell embryos were injected with a total of 800pg lhx1 DEED-AS. Explants were dissected and treated with AcRA and expression of pax8 at stage 15 (based on timing of paired control whole embryos) was analyzed. We observed a lack of induction of pax8 expression in lhx1-depleted explants under AcRA treatment conditions in which expression of this gene is normally induced. Based on this observation, microarray analysis was carried out to identify genes whose expression is affected by the absence of lhx1. Explants of injected embryos with 800pg of lhx1 DEED-AS were dissected, treated with pronephric tissue inductive conditions (AcRA) and harvested after 24 hours incubation at 14C (Fig. S5B). The sibling control embryos reached stage 12.5. Explants from uninjected embryos +AcRA and -AcRA as well as explants from DEED injected embryos -AcRA were also harvested. Approximately 12 caps were pooled for each RNA preparation and the analysis was performed using triplicates.

Project description:Studies on the early embryonic development of Xenopus laevis contributed much to the understanding of vertebrate patterning. Gastrula stages are of particular interest because establishment of the axis and germ layer formation take place during these stages. While many genes belonging to several signaling pathways including FGF, Wnt and TGF-beta, have been implicated in patterning the gastrula embryo, the hierarchical interactions between these factors are incompletely known. To study this question, we took advantage of microarray technology to create a regional gene expression profile for the Xenopus gastrula. Stage 10 Xenopus embryos were dissected into four portions. The dorsal marginal zone including the blastopore and some ectoderm and dorsal yolk plug, composed mostly of endomesoderm; the ventral marginal zone, also containing a portion of the yolk plug; the animal cap, dissected just above the floor of the blastocoel; and the vegetal region, composed of the central part of the yolk plug. To avoid possible cross contamination that might blur the microrarray data, thin junctional regions between the explants were removed. The dissected explants were homogenized in Stat 60 (TEL TEST), RNA was precipitated by isopropanol, treated with DNase I, and purified using the RNeasy kit (Qiagen). Biotinylated probe was prepared from 100 ng total RNA using the OVATION RNA amplification system (Nugen Technologies, Inc). The probes were hybridized to Affymetrix Xenopus Chips containing features that represent about 15,000 genes according to the manufacture’s instructions. Hybridized arrays were further processed by the GeneChip Fluidics system (Affymetrix), and signals were detected by the GeneChip Scanner (Affymetrix). Gene expression profiles were analyzed by the GCOS software (Affymetrix). The analysis showed that 100 transcripts were enriched in the dorsal explant (dorsal vs. ventral, signal log2 ratio>1.5), including the known dorsal markers Chordin, gsc, Admp; 90 transcripts were enriched in the ventral explant (ventral vs. dorsal, signal log2 ratio>1.5) including Sizzled, bambi, PV.1; 449 transcripts were enriched in the vegetal explant (vegetal vs. dorsal, vegetal vs. ventral, vegetal vs. animal cap, all signal log2 ratio>1.5), including Mixer, Sox17; 70 transcripts were enriched in the animal cap (animal cap vs. vegetal, signal log2 ratio>1.5; animal cap vs. dorsal, signal log2 ratio>1; animal cap vs. ventral, signal log2 ratio>1) including Epidermal type I cytokeratin and forkhead-2. RT-PCR was used to check the enrichment of some of the unknown genes; the enrichment of 8 of 9 ventral genes, and 9 of 12 dorsal genes was confirmed in these experiments. Keywords: Xenopus, gastrula, explant, gene expression, embryonic, gene regulation

Project description:Studies on the early embryonic development of Xenopus laevis contributed much to the understanding of vertebrate patterning. Gastrula stages are of particular interest because establishment of the axis and germ layer formation take place during these stages. While many genes belonging to several signaling pathways including FGF, Wnt and TGF-beta, have been implicated in patterning the gastrula embryo, the hierarchical interactions between these factors are incompletely known. To study this question, we took advantage of microarray technology to create a regional gene expression profile for the Xenopus gastrula. Stage 10 Xenopus embryos were dissected into four portions. The dorsal marginal zone including the blastopore and some ectoderm and dorsal yolk plug, composed mostly of endomesoderm; the ventral marginal zone, also containing a portion of the yolk plug; the animal cap, dissected just above the floor of the blastocoel; and the vegetal region, composed of the central part of the yolk plug. To avoid possible cross contamination that might blur the microrarray data, thin junctional regions between the explants were removed. The dissected explants were homogenized in Stat 60 (TEL TEST), RNA was precipitated by isopropanol, treated with DNase I, and purified using the RNeasy kit (Qiagen). Biotinylated probe was prepared from 100 ng total RNA using the OVATION RNA amplification system (Nugen Technologies, Inc). The probes were hybridized to Affymetrix Xenopus Chips containing features that represent about 15,000 genes according to the manufactureâs instructions. Hybridized arrays were further processed by the GeneChip Fluidics system (Affymetrix), and signals were detected by the GeneChip Scanner (Affymetrix). Gene expression profiles were analyzed by the GCOS software (Affymetrix). The analysis showed that 100 transcripts were enriched in the dorsal explant (dorsal vs. ventral, signal log2 ratio>1.5), including the known dorsal markers Chordin, gsc, Admp; 90 transcripts were enriched in the ventral explant (ventral vs. dorsal, signal log2 ratio>1.5) including Sizzled, bambi, PV.1; 449 transcripts were enriched in the vegetal explant (vegetal vs. dorsal, vegetal vs. ventral, vegetal vs. animal cap, all signal log2 ratio>1.5), including Mixer, Sox17ï¡.; 70 transcripts were enriched in the animal cap (animal cap vs. vegetal, signal log2 ratio>1.5; animal cap vs. dorsal, signal log2 ratio>1; animal cap vs. ventral, signal log2 ratio>1) including Epidermal type I cytokeratin and forkhead-2. RT-PCR was used to check the enrichment of some of the unknown genes; the enrichment of 8 of 9 ventral genes, and 9 of 12 dorsal genes was confirmed in these experiments. Experiment Overall Design: Ceate a regional gene expression profile in Xenopus gastrula, and predict gene expression pattern by comparing gene expression in different explant.

Project description:The Ptf1a gene has essential functions during several stages of pancreas development. It is expressed in the naM-CM-/ve endoderm and required pancreas cell fate specification; it is also required later in the differentiation and maintenance of acinar cells. To identify the regulatory genetic program downstream of Ptf1a required for early pancreatic fate acquisition, we used microarrays to perform a comprehensive gene expression analysis of Ptf1a overexpressing endodermal tissue at NF32 and NF36. The results revealed an up-regulation on 1142 probe sets over 2-fold. Additional analyses, by in situ hybridizations, identified 9 genes that were endodermally expressed after the onset of endogenous Ptf1a; STXBP1, putative transmembrane protein TA-2, C25H, IGFBP1, IRF1, HALPN3, Hey1, sestrin 1, syndecan-4. These results provide insight into the regulatory network activated by Ptf1a during early pancreas development. In order to identify downstream targets of Ptf1a, two microarrays were performed at different time-points. The two microarrays compared control pancreatic tissue (GFP) and pancreatic tissue over-expressing Ptf1a (Ptf1a+GFP). The first microarray was performed at NF32, hence renamed MA32, 8 hours after the initial expression of endogenous Ptf1a. A second microarray was performed at NF36 (MA36), 8 hours after the first one. Ptf1a+gfp mRNA or gfp mRNA alone was injected into the two dorso-vegetal blastomeres of eight-cell embryos, targeting the anterior endoderm, and 40/48 hours later the anterior endoderm was dissected out. Approximated 15 endoderm explants were pooled for each RNA preparation, and both control and experimental samples were collected from the same batch of embryos; this was done in triplicate at NF32 and in quadruplicate at NF36 (Fig. 1A). For the NF32 microarray, we used the Affymetrix 3M-bM-^@M-^Y Xenopus laevis Genome 2.0 GeneChip, whereas for the NF36 microarray we used the Affymetrix 3M-bM-^@M-^Y Xenopus laevis Genome 1.0 GeneChip; the NF36 microarray was performed prior to the release of the 2.0 GeneChip.

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Bhh signaling in pronephric development

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