Project description:Recently a new neonatal diabetes syndrome, Mitchell-Riley syndrome, was discovered. To identify the genetic cause of the syndrome homozygosity mapping was used, several chromosomal regions were linked to Mitchell-Riley syndrome. In situ hybridization of genes from one such region using model organism Xenopus laevis identified RFX6 as a potential candidate gene; mutant forms of RFX6 were subsequently found in Mitchell-Riley patients. Analysis of the expression pattern of RFX6 in Xenopus development shows it is expressed broadly in the endoderm early in development, and later RFX6 becomes restricted to the endocrine cells of the gut and pancreas. Morpholino knockdown of RFX6 in Xenopus caused a loss of pancreas marker gene expression. Injection of exogenous wild type RFX6 rescued the morpholino phenotype in Xenopus tadpoles. Attempts to rescue the loss-of-function phenotype using mutant forms of RFX6 found in Mitchell-Riley patients were unsuccessful suggesting the changes lead to loss-of-function and could be the cause of Mitchell-Riley syndrome. Microarray analysis of gene expression in knockdown tissue suggested a downregulation in marker genes for lung, stomach and heart, ambiguous results for the liver, and an upregulation in kidney marker gene expression. RT-PCR and in situ hybridization confirms a loss of lung, stomach and heart gene expression, no change in liver marker hex and an upregulation in kidney marker KcnJ1. The fact that the morpholino phenotype affects multiple organs suggests that RFX6 has a broad role early in endoderm development. Xenopus laevis embryos were injected with morpholinos, either mismatch control (MM) or RFX6 start site (MO1), at the 8-cell stage. The foreguts of the resulting tadpoles were dissected at 3 different stages of development, NF30, NF40 and NF44.
Project description:To adapt to its changing dietary environment, the digestive tract is extensively remodeled from the embryo to the adult during vertebrate development. Xenopus laevis metamorphosis is an excellent model system for studying mammalian gastrointestinal development and is used to determine the genes and signaling programs essential for intestinal development and maturation. The metamorphosing intestine can be divided into four distinct developmental time points and these were analyzed with X. laevis microarrays. Due to the high level of conservation in developmental signaling programs and homology to mammalian genes, annotations and bioinformatics analysis were based on human orthologs. Clustering of the expression patterns revealed co-expressed genes involved in essential cell processes such as apoptosis and proliferation. The two largest clusters of genes have expression peaks and troughs at the climax of metamorphosis respectively. Novel conserved gene ontology categories regulated during this period include transcriptional activity, signal transduction, and metabolic processes. Interestingly, the induced genes associated with metamorphic climax correlated with the gene expression peaks observed around birth in the mouse intestine. Thus both mouse and amphibian, share similarities at the molecular levels for intestinal maturation and remodeling, which appears to be under the influence of increasing levels of circulating thyroid hormone. Moreover, our genome-wide analysis of the intestine during development identified larval/embryo- and adult-specific genes. Detailed analysis revealed 17 larval specific genes that may represent molecular markers for human colonic cancers, while many adult specific genes are associated with dietary enzymes. This global developmental expression study provides the first detailed molecular description of intestinal remodeling and maturation during postembryonic development, which should help improve our understanding of intestinal organogenesis and human diseases. This study significantly contributes towards our understanding of the dynamics of molecular regulation during development and tissue renewal, which is important for future basic and clinical research and for medicinal applications. Time series experiment through natural metamorphosis of intestine in Xenopus laevis. Biological replicates: 3 replicates for each stage of major change during amphibian metamorphosis (except in the climax where 2 bioogical replicates were used). Universal reference design was used instead of dye-swap design for 2-color hybridizations.
Project description:RNA-seq technology was used to identify differentially localized transcripts from Xenopus laevis and Xenopus tropicalis stage VI oocytes. Besides the discovery of a group of novel animally enriched RNAs, this study revealed a surprisingly low conservation of vegetal RNA localization between the two frog species. mRNA profiles of Xenopus laevis and Xenopus tropicalis animal and vegetal oocyte halves were generated by RNA-seq technology. For Xenopus laevis, animal and vegetal oocyte RNA preparations from two different females were generated in duplicates. For Xenopus tropicalis, animal and vegetal oocyte RNA preparations from two different females were analyzed.
Project description:Xenopus laevis embryos were injected with mRNA for EFTFs at 2-cell stage. Animal caps collected at stage 9, cultured to the equivalent of stage 15 and RNA extracted. Four biological replicates of the EFTF-injected and GFP-injected (control) caps were used to profile transcript expression patterns using Affymetrix Xenopus Laevis GeneChip microarrays. Experiment Overall Design: Xenopus laevis embryos were injected with mRNA for EFTFs at 2-cell stage. Animal caps collected at stage 9, cultured to the equivalent of stage 15 and RNA extracted. Four biological replicates of the EFTF-injected and GFP-injected (control) caps were used to profile transcript expression patterns using Affymetrix Xenopus Laevis GeneChip microarrays.