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Next Generation Sequencing Quantitative Analysis of Wild Type and Shoc2 -/- sox10-positive cells in zebrafish

ABSTRACT: To determine how Shoc2-mediated signals affect expression of genes controlling specification of neural crest cells in zebrafish development, we applied RNA-sequencing. shoc2 CRISPR/Cas9 mutants that are transgenic for sox10:dsRed – marking neural crest progenitors were utilized in these experiments. mRNAs was isolated from the FACS-purified sox10:dsRed positive cells of shoc2 CRISPR/Cas9 mutant and wild type larvae. This approach allows for a robust and targeted quantitative assessment of how Shoc2-ERK1/2 signaling affects emergence of several neural crest lineages. The differences in the transcriptome of cells purified from shoc2 crispants and control embryos were determined.

ORGANISM(S): Danio rerio

PROVIDER: GSE198231 | GEO | 2023/02/27

REPOSITORIES: GEO

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Project description:It is believed that the inherent differentiation program of melanocytes during embryogenesis predisposes melanoma cells to high frequency of metastasis. Sox10, a transcription factor expressed in neural crest stem cells and a subset of progeny lineages, plays a key role in the development of melanocytes. We show that B16F10 melanoma cells transfected with siRNA specific for Sox10 display reduced migratory activity which in turn indicated that a subset of transcriptional regulatory target genes of Sox10 are likely to be involved in migration and metastasis of melanoma cells. We carried out microarray-based gene expression profiling using Sox10-specific siRNA to identify regulatory targets and found that multiple genes including melanocortin-1 receptor (Mc1R) partake in the regulation of migration. We provide evidences that a significant portion of the effect of Sox10 on migration is mediated by Mitf, a transcription factor downstream to Sox10. The involvement of Mc1R in migration was studied in detail in vivo using a murine metastasis model. Specifically, B16F10 melanoma cells treated with a specific siRNA showed reduced tendency in metastasizing to and colonizing the lung after being injected in the tail vein. These data reveal a cadre of novel regulators and mediators involved in migration and metastasis of melanoma cells that represent potential targets of therapeutic intervention. Chemically synthesized siRNA duplex (WT1-Sox10) was used to knock-down the transcription factor Sox10 in murine melanoma cell line B16F10. For the control, siRNA containing 5 nucleotide alterations (MT1-Sox10) were used. Total RNA was subsequently prepared to synthesize probes for microarray screening. A total of three pairs of replicate samples were generated each from separate transfection followed by RNA preparation. Expression values were determined and compared within each pair of WT1-Sox10 and MT1-Sox10 transfections. Genes showing over 2 fold changes in all three replicate pairs were subjected to subsequent analyses.

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Next Generation Sequencing Quantitative Analysis of Wild Type and Shoc2 -/- sox10-positive cells in zebrafish

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