Project description:The integrity of the mammalian epidermis is essential for organism survival, and it depends on a balance of proliferation and differentiation in the resident stem cell population. The kinase Ripk4 and the transcription factor Irf6 are mutated in severe developmental syndromes in humans, and mice lacking these genes display epidermal hyperproliferation and soft tissue fusions, resulting in neonatal lethality. However, the mechanism by which these genes control epidermal differentiation in vivo is unknown. By generating various mouse knock-out and knock-in strains we demonstrate that in vivo the role of Ripk4 in development is dependent on its kinase activity, Ripk4 and Irf6 function cell autonomously in the epidermis,Ripk4 and Irf6 lie on a linear pathway and phosphorylation of Irf6 on Serine413 and Serine424 is essential to prime it for activation. This priming then allows Ripk4 to phosphorylate Irf6 on Serine90, which ensures Irf6 activation. We then use RNA-seq, ChIP-seq and ATAC-seq analysis to define the global transcriptional targets of Irf6 in epidermal differentiation. Collectively, our results explain how Ripk4 activates Irf6, and how this pathway ensures epidermal differentiation and a functional barrier. This is crucial for understanding the etiology of developmental syndromes that are characterized by orofacial, skin and genital abnormalities.
Project description:The integrity of the mammalian epidermis is essential for organism survival, and it depends on a balance of proliferation and differentiation in the resident stem cell population. The kinase Ripk4 and the transcription factor Irf6 are mutated in severe developmental syndromes in humans, and mice lacking these genes display epidermal hyperproliferation and soft tissue fusions, resulting in neonatal lethality. However, the mechanism by which these genes control epidermal differentiation in vivo is unknown. By generating various mouse knock-out and knock-in strains we demonstrate that in vivo the role of Ripk4 in development is dependent on its kinase activity, Ripk4 and Irf6 function cell autonomously in the epidermis,Ripk4 and Irf6 lie on a linear pathway and phosphorylation of Irf6 on Serine413 and Serine424 is essential to prime it for activation. This priming then allows Ripk4 to phosphorylate Irf6 on Serine90, which ensures Irf6 activation. We then use RNA-seq, ChIP-seq and ATAC-seq analysis to define the global transcriptional targets of Irf6 in epidermal differentiation. Collectively, our results explain how Ripk4 activates Irf6, and how this pathway ensures epidermal differentiation and a functional barrier. This is crucial for understanding the etiology of developmental syndromes that are characterized by orofacial, skin and genital abnormalities.
Project description:Transcription factor paralogs may share a common role (e.g. Hox) in staged or overlapping expression in specific tissues. In other examples, members have distinct roles in a range of embryologic, differentiation or response pathways (e.g. Tbx, Pax). For the Interferon Regulatory Factor (IRF) family of transcription factors, mice deficient in Irf1, Irf2, Irf3, Irf4, Irf5, Irf7, Irf8 or Irf9, have defects in the immune response but display no embryologic abnormalities. Mice deficient for Irf6 have not been reported, but in humans, mutations in IRF6 cause two Mendelian orofacial clefting syndrome, and genetic variation in IRF6 confers risk for isolated cleft lip and palate. Mice deficient for Irf6 have abnormal skin, limb and craniofacial development. Histological and gene expression analyses indicate that the primary defect is in keratinocyte differentiation and proliferation. This study describes a novel role for an IRF family member in epidermal development. Keywords: Comparison of tissue from two genotypes
Project description:We report transcriptional profiles of 4hpf zebrafish embryos from either wild-type or a CRISPR-targeted irf6 loss-of-function mutant. Irf6 is a transcription factor implicated in syndromic and nonsyndromic forms of cleft palate. To define critical genes involved in palate development, we harvested RNA from irf6-/- embryos before an embryonic lethal stage in zebrafish and carried out RNA-sequencing and subsequent analysis. We identify top differentially expressed genes that include genes involved in periderm development, genes expressed in developmental processes (such as Fgfs and Wnt-pathway genes), and genes involved in orofacial clefting. We identify a crucial gene esrp1 that we chose for further validation and downstream analysis.
Project description:We conducted RNA-Seq (using Direct Ligation of Adapters to first strand cDNA) in neurons from E16.5 mouse embryonic cortices from WT and SMCX KO mice and harvested after 10 days in vitro culture.
Project description:We conducted RNA-Seq (using Direct Ligation of Adapters to first strand cDNA) in neurons from E16.5 mouse embryonic cortices from WT and SMCX KO mice and harvested after 10 days in vitro culture. We sequenced RNA samples after 10 days in vitro cultures in biological and technical duplicates. 4 RNA samples from WT and SMCX KO neurons. We also sequenced RNA samples from same neurons after stimulation with KCl for 60 mins. So a total of 8 RNA-Seq samples.
Project description:We conducted ChIP-Seq of H3K4 methylation in neurons isolated from E16.5 mouse embryonic cortices from WT and SMCX KO mice and harvested after 10 days in vitro culture.