Project name: Microarray based gene expression comparison between wild type and yap-1(ys38) C. elegans embyos

Description: Although multiple determinants for establishing polarity in membranes of epithelial cells have been identified, the mechanism for maintaining the apicobasal polarity is not fully understood. Here, we show that the conserved Hippo kinase pathway plays a role in the maintenance of apicobasal polarity in the developing intestine of Caenorhabditis elegans. We screened suppressors of the mutation in wts-1, the gene encoding the LATS kinase homolog, whose deficiency leads to the disturbance of the apicobasal polarity of the intestinal cells and the eventual death of organisms. Multiple alleles of yap-1 and egl-44, each encoding the worm homologs of YAP/Yki and TEAD/Sd, respectively, were identified as suppressors. WTS-1 directly bound to YAP-1 and inhibited its nuclear accumulation in intestinal cells. We also found that NFM-1, the worm homolog of NF2/Merlin, functioned in the same genetic pathway as WTS-1 to regulate YAP-1 for maintaining cellular polarity. The transcriptome analysis using microarray identified several target candidates of the YAP-1-EGL-44 complex including TAT-2, encoding a putative P-type ATPase. In summary, we have delineated the conserved Hippo pathway in worms consisting of NFM-1-WTS-1-YAP-1-EGL-44 and prove that the proper regulation of YAP-1 by upstream NFM-1 and WTS-1 is esssential for the maintenance of apicobasal membrane identities of the growing intestineOverall design: To identify target genes of the Hippo pathway for cellular polarity maintenance, we compared the expression levels of all the genes of yap-1(ys38) mutants to those of wild type worms using a microarray experiment. To collect embryos of N2 and yap-1(ys38), gravid adults from 10 plates of 10cm NGM were bleached. Total RNA was extracted from 3 biological replicates by the freezing-thawing method. The isolated RNA was qualified and used as templates for cDNA synthesis in the Genome Research Facility of the Seoul National University using an Affymetrix chip (Seolin bio, Seoul, Korea) containing 22,625 gene probes targeting 22,150 genes from C. elegans. The chip data were analyzed using the Microarray Suite version 5.0 (MAS 5.0) with global scaling performed as the normalization method using Genplex ver.3.0 (istech.inc). The results of one sample of N2 were significantly different from those of the other two replicates, therefore were excluded from the analysis. Significant genes were obtained by calculating fold-change and Welch’s T-test (P-value ≤ 0.05) using Volcano Plot and filtering A (absent) by detection call.

Data type: Transcriptome or Gene expression

Sample scope: Multiisolate

Relevance: ModelOrganism

Last updated: 2019-07-01