Primary cilia regulate hematopoietic stem and progenitor cell specification through Notch signaling in zebrafish

Hematopoietic stem and progenitor cells (HSPCs) are capable of producing all mature blood lineages, as well as maintaining the self-renewal ability throughout life. The hairy-like organelle, cilium, is present in most types of vertebrate cells, and plays important roles in various biological processes. However, it is unclear whether and how cilia regulate HSPC development in vertebrates. Here, we show that cilia-specific genes, involved in primary cilia formation and function, are required for HSPC development, especially in hemogenic endothelium (HE) specification in zebrafish embryos. Blocking primary cilia formation or function by genetic or chemical manipulations impairs HSPC development. Mechanistically, we uncover that primary cilia in endothelial cells transduce Notch signal to the earliest HE for proper HSPC specification during embryogenesis. Altogether, our findings reveal a pivotal role of endothelial primary cilia in HSPC development, and may shed lights into in vitro directed differentiation of HSPCs.

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Policy information about availability of computer code Data collection For RNA-seq data, the sequencing reads were undertaken with BGISEQ-500 in Beijing Genomic Institution. For images captured by a Nikon A1 confocal laser microscope, we used NIS-Elements AR4.13.00 64-bit. For collecting the western blotting data, we used Imagequant LAS 4000 . For collecting the whole mount in situ data, we used NIS-Elements F . For collecting the transmission electron microscope, we used system software in JEM1400 electron microscope. For collecting the live imaging of zebrafish embryos, we used NIS-Elements F. For the fluorescence data collection in human cells, we used Zeiss LSM 880 or DeltaVision Image Restoration Microscope.

Data analysis
For RNA-seq data analysis, reads were aligned to the zv11 genome assembly using HISAT (Hierarchical Indexing for Spliced Alignment of Transcripts) (http://www.nature.com/articles/nmeth.3317) and differentially expressed genes were identified with software. For image analysis, we used Volocity 6.0, Image J, Imaris 9.0, NIS-Elements AR4.13.00 64-bit and photoshop CS6. Statistical analysis were down by GraphPad Prism 6.01 software.
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Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability The RNA-seq data presented in the paper was deposited at NCBI under accession number SRP140462 [https://www.ncbi.nlm.nih.gov/sra/?term=SRP140462]. All the

October 2018
other relevant data supported this study are provided as a Source Data file. All the relevant data supported this study from the corresponding author are available upon request.

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