Unknown,Transcriptomics,Genomics,Proteomics

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Analysis of Notch signaling on Xenopus epidermis

ABSTRACT: 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.

ORGANISM(S): Xenopus laevis

SUBMITTER: Ian Quigley

PROVIDER: E-GEOD-23844 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Cutaneous lupus erythematosus (CLE) is an autoimmune disease that localizes to the skin and is known to contain elevated glycosaminoglycans (GAGs) on Hale’s stain of skin biopsy specimens. Recently, different GAG species have been shown to have distinct effects on the recruitment and activation of immune cells and stimulation of cytokine production (Taylor and Gallo, FASEB, 2006; 20: 9-22). Thus, we speculate that the elevated GAGs observed in CLE play a role in the local inflammatory process that produces skin lesions in these patients. In order to further investigate a molecular basis for the elevated expression of these GAGs in CLE skin lesions, we would like to determine the gene expression profiles of GAG synthesis, degradation, and modifier genes in lesional and non-lesional skin samples from CLE patients and compare to those from healthy controls. A microarray approach will give us a broader understanding of the genetic regulation of the expression of various GAG species in CLE skin. We will then be able to target future quantitative gene expression experiments by real-time RT-PCR to the genes that are shown to be involved in CLE. In order to accomplish our goal, we would like to examine the GAG gene expression profiles of DLE, TLE, and SCLE subtypes due to the differences in CS and HA staining that we found among these subtypes. Since HA and CS are elevated in DLE and HA in TLE, but not in SCLE, the SCLE samples will also serve as an internal control. We would like to examine both lesional and non-lesional skin biopsies to determine if CLE skin prior to developing a lesion is different at the genetic level from healthy control skin and how it changes once a lesion does develop. We will separate the dermis from the epidermis of the skin biopsies and extract RNA just from the dermis to enrich for dermal fibroblast RNA. We aim to submit four patient biopsies per subtype as well as four samples from healthy control skin for comparison. This number is necessary in order to account for the biologic variability among different patients. We would submit more samples per subtype but are limited by availability of patients in clinic. Thus, we will have a total of 28 samples to submit for microarray. This study design will allow us to analyze the GAG gene expression profiles among different CLE subtypes and enable us to identify which GAc

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Analysis of Notch signaling on Xenopus epidermis

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