Project description:Purpose: The goal of this study was to evaluate changes in the transcriptome profile during periocular neural crest differentiation into corneal endothelium and keratocytes. Methods: RNA profiles of chick embryonic day (E3) periocular neural crest, E5 corneal endothelium, and E7 corneal endothelium plus keratocytes were generated in triplicate by deep sequencing using Illumina HS4000. Bowtie2 and HISAT were used to map clean reads to reference gene and genome, respectively. An average mapping ratio of 76.26% to the reference gene and 93.10% to the genome were generated with Galgal5 reference assembly.Transcripts were normalized and presented as Fragments Per Kilobase Million (FPKM). Differentially expressed genes between pNCvsEn and pNCvsKEn were examined. Expression of some candidate genes was validated by in situ hybridization. Results: 790 transcripts were enriched between pNC and En, and 865 transcripts were enriched between pNC and KEn. Enriched transcripts correspond with KEGG pathways involved in cell proliferation, synthesis of extracellular matrix, focal adhesion, metabolism, and cancer. The RNA-Seq data serves as platform for further analyses of the molecular networks involved in NCC differentiation into corneal cells, and provides insights into genes involved in corneal dysgenesis and adult diseases.
Project description:Cells were isolated from mouse embryonic neural crest stem cells at culture day 2 (NCSC), from day 7 in vitro differentiated progeny (NCP) and day 2 epidermal neural crest stem cells from bulge explants of adult whisker follicles (EPI-NCSC). Keywords: LongSAGE embryonic neural crest stem cells at culture day 2 (NCSC), from day 7 in vitro differentiated progeny (NCP) and day 2 epidermal neural crest stem cells from bulge explants of adult whisker follicles (EPI-NCSC).
Project description:Cells were isolated from mouse embryonic neural crest stem cells at culture day 2 (NCSC), from day 7 in vitro differentiated progeny (NCP) and day 2 epidermal neural crest stem cells from bulge explants of adult whisker follicles (EPI-NCSC). Keywords: LongSAGE
Project description:Ectoderm-derived neural crest is a transient structure arising during early embryogenesis in vertebrates. Neural crest consists of four derivatives based on their anterior- to posterior location along the body axis; cranial, vagal, trunk and sacral, respectively. We recently showed that trunk neural crest-specific gene MOXD1 functions as a tumor suppressor in trunk neural crest-derived childhood cancer form neuroblastoma and is essential for proper development of healthy adrenal glands. However, the role of MOXD1 during early embryogenesis is not known. Here, we conditionally knocked out MOXD1 in trunk neural crest cells before they become lineage-committed, using a CRISPR/Cas9 approach in chick embryos. Assessment of embryo growth showed that knockout of MOXD1 delayed development with knockout embryos being smaller. RNA sequencing of trunk-derived neural crest cells from control and knockout embryos showed enrichment of genes connected to gland development, copper ion metabolism and neuroblastoma progression. In conclusion, MOXD1 is important during early and prolonged embryonic development with effects on gland formation, possibly mediated via its role in copper metabolism.
Project description:We observed impaired neural crest cell differentiation from human embryonic stem cells that harbor nonsense mutations in the Polycomb gene ASXL1. To investigate the underlying molecular mechanisms, we harvested neural crest differentiation cultures from wildtype and heterozygous ASXL1 mutant lines at day 7 of a neural crest differentiation protocol and performed total RNA sequencing using the Illumina HiSeq2500 system. Analyses of global transcriptomes revealed profound changes between wildtype and mutant cultures, and we identified downregulation of the neural crest transcription factor ZIC1.
Project description:Neural crest cells exemplify cellular diversification from a multipotent progenitor population. However, the full sequence of molecular choices governing the emergence of neural crest heterogeneity from the ectoderm remains elusive. Gene regulatory networks govern these steps of embryonic development and cell specification towards definitive neural crest. Here, we combine ultra-dense single cell transcriptomes with machine-learning strategies and experimental validation to provide a comprehensive gene regulatory network driving vertebrate neural crest fate diversification, from induction to early migration stages. Transcription factor connectome and bifurcation analyses demonstrate emergence of early neural crest fates at the neural plate stage, alongside an unbiased multipotent neural crest lineage persisting until after epithelial-mesenchymal transition. We also define a new and transient neural border zone state, preceding choice between neural crest and placodes during gastrulation. Theis combination of experimental tests, with Machine Learning broadly applicable to single cell transcriptomics, deciphers the circuits driving cranial and vagal neural crest formation and provides a general model for investigating vertebrate GRNs in development, evolution and disease.
Project description:Neural crest cells exemplify cellular diversification from a multipotent progenitor population. However, the full sequence of molecular choices governing the emergence of neural crest heterogeneity from the ectoderm remains elusive. Gene regulatory networks govern these steps of embryonic development and cell specification towards definitive neural crest. Here, we combine ultra-dense single cell transcriptomes with machine-learning strategies and experimental validation to provide a comprehensive gene regulatory network driving vertebrate neural crest fate diversification, from induction to early migration stages. Transcription factor connectome and bifurcation analyses demonstrate emergence of early neural crest fates at the neural plate stage, alongside an unbiased multipotent neural crest lineage persisting until after epithelial-mesenchymal transition. We also define a new and transient neural border zone state, preceding choice between neural crest and placodes during gastrulation. Theis combination of experimental tests, with Machine Learning broadly applicable to single cell transcriptomics, deciphers the circuits driving cranial and vagal neural crest formation and provides a general model for investigating vertebrate GRNs in development, evolution and disease.
Project description:Neural crest cells exemplify cellular diversification from a multipotent progenitor population. However, the full sequence of molecular choices governing the emergence of neural crest heterogeneity from the ectoderm remains elusive. Gene regulatory networks govern these steps of embryonic development and cell specification towards definitive neural crest. Here, we combine ultra-dense single cell transcriptomes with machine-learning strategies and experimental validation to provide a comprehensive gene regulatory network driving vertebrate neural crest fate diversification, from induction to early migration stages. Transcription factor connectome and bifurcation analyses demonstrate emergence of early neural crest fates at the neural plate stage, alongside an unbiased multipotent neural crest lineage persisting until after epithelial-mesenchymal transition. We also define a new and transient neural border zone state, preceding choice between neural crest and placodes during gastrulation. Theis combination of experimental tests, with Machine Learning broadly applicable to single cell transcriptomics, deciphers the circuits driving cranial and vagal neural crest formation and provides a general model for investigating vertebrate GRNs in development, evolution and disease.
Project description:We observed impaired neural crest cell differentiation from human embryonic stem cells that harbor nonsense mutations in the Polycomb gene ASXL1. To investigate the underlying molecular mechanisms, we harvested neural crest differentiation cultures from wildtype and homozygous ASXL1 mutant lines at day 7 of a neural crest differentiation protocol and performed total RNA sequencing using the Illumina NextSeq system. Analyses of global transcriptomes revealed profound changes between wildtype and mutant cultures, and we identified downregulation of several genes of the neural crest regulatory network, most prominently of the transcription factor ZIC1.