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Early downstream targets of Notch signaling which contribute to multiciliate cell fate

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) or a DNA binding mutant of Mastermind (DBM) to induce the formation of ectopic multi-ciliate and proton secreting cells. Results show which genes are up or down-regulated when DBM/HMM are compared to ICD. Epithelial cells projecting hundreds of motile cilia are prominent in the respiratory system, brain ependyma and female reproductive tract where they generate a vigorous fluid flow along luminal surfaces. Despite their importance for organ function, how multiciliate cells arise developmentally in diverse epithelia is poorly understood. Notch signaling has been shown in a variety of tissues, including the Xenopus epithelium, to regulate the formation of multiciliate cell precursors. In order to identify early downstream targets of Notch signaling which contribute to multiciliate cell fate we blocked or activated Notch signaling in explants of Xenopus epithelium and looked at changes in gene expression to identify candidates that regulate multiciliate cell fate. Here we identify a novel coiled-coil protein, called multicilin (MCI), whose expression is Notch-regulated and restricted to epithelia where multiciliate cells forms in Xenopus embryos. Inhibiting MCI activity blocks the formation of multiciliate cell precursors while ectopic MCI activity is sufficient to induce multiciliate cell differentiation within epithelial progenitors.

ORGANISM(S): Xenopus laevis

PROVIDER: GSE32331 | GEO | 2012/03/01

SECONDARY ACCESSION(S): PRJNA147263

REPOSITORIES: GEO

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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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Early downstream targets of Notch signaling which contribute to multiciliate cell fate

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