Nlrc3 signaling is required for hematopoietic stem cell emergence by activating Notch signaling in vertebrates
Purpose: Since Nlrc3 signaling is imperative for HSPCs production in zebrafish, to further determine the regulatory mechanism by which nlrc3 signaling regulates HSPCs. Bulk RNA sequencing analysis is performed to dissect the function and molecular mechanism between the control groups and the nlrc3 morphants groups. Methods: The GFP+ cells in Tg(fli1a:eGFP) zebrafish embryos at 28 hpf were sorted, which is the stage that EHT occurs in the VDA and the site of hemogenic endothelial cells onset. The mRNA profiles of these cells in wild-type (WT) and Nlrc3 knockdown were deep sequencing, in triplicate, using Illumina GAIIx. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT–PCR validation was performed using TaqMan and SYBR Green assays. Results: Using an optimized data analysis workflow, we mapped about 35 million sequence reads per sample and identified 4153 transcripts showing differential expression between the of WT and  Nlrc3 morphants, with a fold change =1.5 and p value <0.05. Based on the results of RNA seq, some signaling pathways essential for  the the Nlrc3 regulates the onset of HSPCs in vertebrate are dissected. Including Notch, WNT, NF-kB. Conclusions: Hemogenic endothelial cells mRNA profiles of WT groups and Nlrc3 knockdown groups were deep sequenced and anlysis. Based on the results of RNA seq, we performed  experiments to validate the up and downstream pathway involved in the the Nlrc3 regulates the onset of HSCPs in vertebrate. Overall design: mRNA profiling by high throughput sequencing to dissect the function and molecular mechanism by which nlrc3 signaling regulates the HSPCs in vertebrate.