Hypercholesterolemia, the driving force of atherosclerosis, accelerates the expansion and mobilization of hematopoietic stem and progenitor cells (HSPCs). The molecular determinants connecting hypercholesterolemia with hematopoiesis are unclear. Here we report that a somite-derived pro-hematopoietic cue, AIBP, orchestrates HSPC emergence from the hemogenic endothelium, a type of specialized endothelium manifesting hematopoietic potential. Mechanistically, AIBP-mediated cholesterol efflux activates endothelial Srebp2, the master transcription factor for cholesterol biosynthesis, which in turn transactivates Notch and promotes HSPC emergence. Srebp2 inhibition impairs hypercholesterolemia-induced HSPC expansion. Srebp2 activation and Notch upregulation are associated with HSPC expansion in hypercholesterolemic human subjects. Genome-wide ChIP-seq, RNA-seq, and ATAC-seq indicate that Srebp2 trans-regulates Notch pathway genes required for hematopoiesis. Our studies outline an AIBP-regulated Srebp2-dependent paradigm for HSPC emergence in development and HPSC expansion in atherosclerotic cardiovascular disease.

Since its discovery, Chikungunya fever caused by a virus (CHIKV) has ravaged most of Africa and Southeast Asia. Despite there being more than a million reported cases in India alone and the seriousness of the disease in the chronic phase, a clear understanding of the disease pathogenesis and host response remains elusive. Here, we use microarray technology and quantitative PCR method to establish the complete miRNA, snoRNA and mRNA signature of host response upon CHIKV infection in human cell line infection model, HEK293T. The results were further validated in human primary cells (dermal fibroblasts). miRNA expression profiling revealed regulation of 152 miRNAs post CHIKV infection. An interesting overlap in miRNA signature was seen majorly with HCV, HPV and HIV1 virus. The microarray data further validated by qRT-PCR revealed induction of miR-744, miR-638, miR-503 and others among the top upregulated miRNAs. Notably, we found induction of snoRNAs belonging to C/D cluster including close paralogs of U3, U44, U76 and U78 snoRNAs. Genes were found to be differentially expressed along 3 major pathways; TGF-β, endocytosis and the cell cycle pathways. qRT-PCR data confirmed strong induction of TGF-β (SMAD6, JUN, SKIL) and endocytosis pathway (CXCR4, HSPA8, ADRB1) genes while downregulation of cell cycle genes (CDC27 and CDC23). Interestingly, use of TGF-β inhibitor, SB-431542, increased CHIKV mediated cell death. Overall, this study aims at providing the first complete transcriptome signature of host response upon CHIKV infection to aid identification of possible biomarkers and therapeutic targets.

BackgroundOligodontia is a severe form of tooth agenesis characterized by the absence of six or more permanent teeth. Oligodontia has complex etiology and variations in numerous genes have been suggested as causal for the condition.MethodsWe applied whole‐exome sequencing (WES) to identify the cause of oligodontia in a 9‐year‐old girl missing 11 permanent teeth. Protein modeling and functional analysis in zebrafish were also performed to understand the impact of identified variants on the phenotype.ResultsWe identified a novel compound heterozygous missense mutation in WNT10A (c.637G>A:p.Gly213Ser and c.1070C>T:p.Thr357Ile) as the likely cause of autosomal recessive oligodontia in the child. Affected residues are located in conserved regions and variants are predicted to be highly deleterious for potentially destabilizing the protein fold and inhibiting normal protein function. Functional studies in zebrafish embryos showed that wnt10a is expressed in the craniofacies at critical time points for tooth development, and that perturbations of wnt10a expression impaired normal tooth development and arrested tooth development at 5 days postfertilization (dpf). Furthermore, mRNA expression levels of additional tooth development genes were directly correlated with wnt10a expression; expression of msx1, dlx2b, eda, and axin2 was decreased upon wnt10a knockdown, and increased upon wnt10a overexpression.ConclusionsOur results reveal a novel compound heterozygous variant in WNT10A as pathogenic for oligodontia, and demonstrate that perturbations of wnt10a expression in zebrafish may directly and/or indirectly affect tooth development recapitulating the agenesis phenotype observed in humans.

Summary The CAP superfamily member, CRISPLD2, has previously been shown to be associated with nonsyndromic cleft lip and palate (NSCLP) in human populations and to be essential for normal craniofacial development in the zebrafish. Additionally, in rodent models, CRISPLD2 has been shown to play a role in normal lung and kidney development. However, the specific role of CRISPLD2 during these developmental processes has yet to be determined. In this study, it was demonstrated that Crispld2 protein localizes to the orofacial region of the zebrafish embryo and knockdown of crispld2 resulted in abnormal migration of neural crest cells (NCCs) during both early and late time points. An increase in cell death after crispld2 knockdown as well as an increase in apoptotic marker genes was also shown. This data suggests that Crispld2 modulates the migration, differentiation, and/or survival of NCCs during early craniofacial development. These results indicate an important role for Crispld2 in NCC migration during craniofacial development and suggests involvement of Crispld2 in cell viability during formation of the orofacies. genesis 53:660–667, 2015.

Application of CRISPR-Cas9 technology in diverse organisms has resulted in an explosion of genome modification efforts. To expand the toolbox of applications, we have created an E. coli Exonuclease I (sbcB) - Cas9 fusion that has altered enzymatic activity in zebrafish embryos. This Cas9 variant has increased mutation efficiency and favors longer deletions relative to wild type Cas9. We anticipate that this variant will allow for more efficient screening for F0 phenotypes and mutation of a larger spectrum of genomic targets including deletion of regulatory regions and creating loss of function mutations in transcription units with poor sequence conservation such as lncRNAs where larger deletions may be required for loss of function.