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:A balance between cell survival and apoptosis is essential for animal development. Although proper development involves multiple interactions between germ layers, little is known about the intercellular and intertissue signaling pathways that promote cell survival in neighboring or distant germ layers . We show that serum- and glucocorticoid-inducible kinase 1 (SGK1) promoted ectodermal cell survival during early Xenopus embryogenesis through a non-cell-autonomous mechanism. Dorsal depletion of SGK1 in Xenopus embryos resulted in shortened axes and reduced head structures with defective eyes, and ventral depletion led to defective tail morphologies. Although the gene encoding SGK1 was mainly expressed in the endoderm and dorsal mesoderm, knockdown of SGK1 caused excessive apoptosis in the ectoderm. SGK1-depleted ectodermal explants showed little or no apoptosis, suggesting non-cell-autonomous effects of SGK1 on ectodermal cells. Microarray analysis revealed that SGK1 knockdown increased the expression of genes encoding FADD and caspase-10, components of the death-inducing signaling complex (DISC). Inhibition of DISC function suppressed excessive apoptosis in SGK1-knockdown embryos. SGK1 acted through the transcription factor nuclear factor kappaB to stimulate production of bone morphogenetic protein 7 (BMP7), and overexpression of BMP7 in SGK1-knockdown embryos reduced the abundance of DISC components. We show that phosphoinositide 3-kinase (PI3K) functioned upstream of SGK1, thus revealing an endodermal and mesodermal pathway from PI3K to SGK1 to NF-kappaB that produces BMP7, which provides a survival signal to the ectoderm by decreasing DISC function. To identify genes whose expression levels are regulated by SGK1 in Xenopus development, we performed genome-wide analysis by using Affymetrix GeneChip oligonucleotide microarrays. We performed two independent experiments. For each microarray experiment, we radially injected control-morpholino or xSGK1-morpholinos into 4-cell embryos, cultured the embryos until stage 12, extracted total RNAs using Trizol (Invitrogen) and purified them on RNeasy columns (Qiagen). Synthesis of cDNA, in vitro transcription and biotin labeling of cRNA, and hybridization to the Xenopus laevis genome array (Affymetrix) were performed according to the Affymetrix protocol (Two-Cycle Target Labeling Assays). Hybridized arrays were scanned using an Affymetrix GeneChip Scanner. Scanned chip images were analyzed with GeneChip Operating Software (GCOS) v. 1.4.

Project description:Sox9 acts together with Sox5 or Sox6 as a master regulator for chondrocyte differentiation; however, how these transcription factors functionally interact and collaborate to regulate chondrogenesis remains unclear. Here we show that the protein kinase MLTK plays an essential role in the onset of chondrogenesis through triggering the induction of Sox6 by Sox9. Knockdown of MLTK in Xenopus embryos results in drastic loss of craniofacial cartilages without defects in neural crest formation. We also find that Sox6 is specifically induced during craniofacial chondrogenesis and this induction is inhibited by MLTK knockdown. Remarkably, Sox6-knockdown embryos display essentially the same phenotype as the MLTK-knockdown embryos; the drastic loss of cartilages and the marked down-regulation of genes involved in chondrogenesis. Microarray analysis reveals a remarkable similarity between Sox6-depleted and MLTK-depleted embryos in their gene expression pattern. Moreover, we find that ectopic expression of MLTK can induce Sox6 expression in a Sox9-dependent manner. These results identify a novel, key regulator for chondrogenesis. We used microarrays to describe the genome-wide gene expression profiles of xMLTK-depleted and xSox6-depleted embryos. CoMO, xMLTK-MO, or xSox6-MO was injected into the animal pole of all blastomeres (10 ng/cell) at the four-cell stage. The heads of embryos were cut out and harvested at 72 hours after fertilization (corresponding to about stage 41). Total RNA was extracted using TRIsol reagent, treated with DNase (TURBO DNase, Ambion), and then purified using RNeasy Mini Kit (QIAGEN) according to the manufacturerM-bM-^@M-^Ys instruction. The quality of total RNA was assessed using the Agilent 2100 BioAnalyzer. Reverse transcription to synthesize first-strand cDNA, second-strand cDNA synthesis, in vitro transcription to synthesize Biotin-modified aRNA, aRNA purification and fragmentation of the labeled aRNA were performed using the GeneChip 3M-bM-^@M-^Y IVT Express Kit and hybridization to the Affymetrix GeneChip Xenopus laevis Genome 2.0 Array was performed using the GeneChip Hybridization Wash and Stain Kit according to the manufacturerM-bM-^@M-^Ys instruction. Hybridized arrays were scanned using an Affymetrix GeneChip Scanner. Scanned Chip images were analyzed with GeneChip operating Software v.1.4 (GCOS) and GeneSpring GX 11.0.2. (Agilent technologies).

Project description:We identified a novel paralog of the antioxidant response element transcription factor, Nrf2 in zebrafish. To determine whether the paralogs Nrf2a and Nrf2b regulate different sets of genes, we performed loss-of-function experiments followed by microarray-based gene expression profiling, performed on embryos in which expression of Nrf2a, Nrf2b, or both paralogs was knocked down. Embryos at the 1-4 cell stage were injected with 3-5 nl of a control morpholino (MO) or gene-specific MO to knockdown Nrf2a, Nrf2b, or a Nrf2a+b combination. At 48 hpf, triplicate groups of 5 embryos were exposed to 2 M-BM-5M of tert butyl hydroquinone or 2% DSMO for 4 hours. At 52 hpf, embryos were fixed in RNA Later and stored at -80M-BM-0C. RNA was extracted, bioanalyzed, labeled with Cy3 and hybridized to the Agilent V3 4x44K zebrafish microarray (cat. #G2519F-026437) at the Genome Technology Core of the Whitehead Institute (Cambridge, MA). Raw array data obtained from the Whitehead Institute were extracted using Agilent's feature extraction software using background detrending (spatial and multiplicative).Prior to normalization, Cy3 values below 5 were set to 5. The data were then normalized using the non-linear scaling method based on rank invariant probes. After normalization but before statistical analyses, probes not significantly above background in all microarrays were removed (3147 probes in all; based on AgilentM-bM-^@M-^Ys 2.6 standard deviation method). None of the probes were saturated for Cy3 signal on any microarray, so no further filtering was applied. There were a total of 40,456 probes for statistical analyses. Statistical tests were performed using MeV v4. Data were log transformed and median centered for each probe. An ANOVA was run for morpholino injection, with p-value based on 1000 permutations of the data and alpha of 0.01. The probes found significant in the ANOVA were subsequently examined using Rank Product (RP) analysis to identify probes up- and down-regulated by the MO injection For each RP test, a two-class unpaired RP analysis was performed using 100 permutations of the data with a false discovery rate (FDR) M-bM-^IM-$10%.

Project description:Analysis of epithelial explants injected with the intracellular domain of Notch (ICD) to block the formation of multi-ciliate and proton secreting cells or with dominant negative human Mastermind (HMM) to induce the formation of ectopic multi-ciliate and proton secreting cells. Results show which genes are up or down-regulated when HMM is compared to ICD. Specialized epithelial cells in the amphibian skin play important roles in ion transport, but how they arise developmentally is largely unknown. Here we show that proton-secreting cells (PSCs) differentiate in the X. laevis larval skin soon after gastrulation, based on the expression of “kidney” specific form of the H+v-ATPase that localizes to the plasma membrane, orthologs of the Cl-/HCO3- antiporters ae1 and pendrin, and two isoforms of carbonic anhydrase. Like PSCs in other species, we show that the expression of these genes is likely to be driven by an ortholog of foxi1, which is also sufficient to promote the formation of PSC precursors. Strikingly, the PSCs form in the skin as two distinct subtypes that resemble the alpha- and beta-intercalated cells of the kidney. The alpha-subtype expresses ae1 and localizes H+v-ATPases to the apical plasma membrane, whereas the beta-subtype expresses pendrin and localizes the H+v-ATPase cytosolically or basolaterally. These two subtypes are specified during early differentiation, and can be distinguished based on the expression of ubp1, a transcription factor in the grainyhead family. Finally, we show that ectopic expression ubp1 is sufficient to promote the differentiation of beta-subtypes while repressing alpha-subtypes. These results have implications for how PSCs are specified in vertebrates and become functionally heterogeneous. 2-cell stage Xenopus embryos were injected with synthetic mRNA encoding ICD or HMM. At stage 9/10 the presumptive ectoderm was cut off the embryo and cultured on a fibronectin coated coverslip. At stage 22 RNA was harvested from explants and used as the starting material for arrays.

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Analysis of miRNA abundance in early gastrula stage lin28 knockdown Xenopus embryos

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