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

ARTICLE https://doi.org/10.1038/s41467-019-09403-7 OPEN Primary cilia regulate hematopoietic stem and progenitor cell specification through Notch signaling in zebrafish 1234567890():,; Zhibin Liu1,2,3, Haiqing Tu4, Yunsi Kang5, Yuanyuan Xue1,2,3, Dongyuan Ma1,2,3, Chengtian Zhao Lu Wang1,2,6 & Feng Liu 1,2,3 5, Huiyan Li4, 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. 1 State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, 100101 Beijing, China. 2 University of Chinese Academy of Sciences, 100049 Beijing, China. 3 Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, 100101 Beijing, China. 4 State Key Laboratory of Proteomics, National Center of Biomedical Analysis, 100850 Beijing, China. 5 Institute of Evolution and Marine Biodiversity, Ocean University of China, 266003 Qingdao, China. 6 State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, 300020 Tianjin, China. Correspondence and requests for materials should be addressed to L.W. (email: ) or to F.L. (email: ) NATURE COMMUNICATIONS | (2019)10:1839 | https://doi.org/10.1038/s41467-019-09403-7 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09403-7 I t has been well established that most vertebrate cells can transmit extracellular signals through a hairy-like sensory organelle, called primary cilium1. Primary cilia exist in many types of cells including endothelial cells (ECs), epithelia, fibroblasts, and others in vertebrates2. Previous studies have revealed that primary cilia have specialized functions, in shear-stress sensation3,4, chemosensation5, differentiation6, proliferation7, and maintenance of stem cells in a wide array of tissues2,8–11. Thus, defects in ciliogenesis and function usually lead to ciliopathies, such as autosomal dominant polycystic kidney disease, obesity, and others10. In the cilia system, primary cilia contain a “9 + 0” axoneme (“9” denotes nine parallel doublet microtubules and “0” denotes absence of a central pair of microtubules (MTs)). An intraflagellar transport (IFT) system, including intraflagellar transport protein88 (IFT88 or Polaris), is utilized to elongate MT axoneme. Ift88 knockdown (KD) led to a vascular impairment phenotype12. Furthermore, a calcium channel protein, Polycystin1 (PKD1), is localized in cilia, and PKD1 conventional knockout mice are embryonically lethal at E15.5 due to vascular leakages and hemorrhage13. Similarly, Polycystin2 (PKD2) KD also caused angiogenesis defects in zebrafish12. Interestingly, a recent study demonstrated that primary cilia are present in the ECs of zebrafish blood vessels12. Given that a subset of ECs in the dorsal aorta (DA) can develop into hemogenic endothelial cells (HE cells), it is tempting to speculate that the endothelial primary cilia in the DA may participate in HE specification. During hematopoietic stem and progenitor cell (HSPC) development, HE cells produce HSPCs through the endothelialto-hematopoietic transition (EHT) in vertebrates14–16. Nonetheless, our understanding of the precise regulatory mechanisms involved in HE specification is still limited17. The transcription factor runx1 is a widely used marker for HE cells at the early embryonic stage18. Deficiency of Runx1 results in impairments of EHT and definitive hematopoiesis19,20. Notch signaling is a critical regulator of runx1. In vertebrates, loss of one of Notch receptors, Notch1, causes a decrease of runx1 expression, which subsequently affects definitive hematopoiesis21–23. Furthermore, in the absence of Notch ligands, the definitive hematopoiesis is also disrupted in jagged1 null mice24 and zebrafish mindbomb mutants25. Notch signaling exerts complex regulation in HSPC development through divergent ligands and receptors26,27, as well as multiple inputs28,29. However, very little is known about the upstream factors of Notch signaling and how they initiate Notch activation. Intriguingly, it has been reported that Notch components localize in cilia and Notch signaling can be transmitted through cilia30,31. However, it remains elusive whether cilia can transduce Notch signaling in controlling definitive hematopoiesis in vertebrates. Here, we use the zebrafish as a vertebrate model and demonstrate that impairment of primary cilia formation or function leads to defects in HSPC development, especially in HE specification. Blocking primary cilia specifically in ECs causes the reduction of HE cells. Mechanistically, we uncover that Notch signaling functions downstream of endothelial primary cilia to specify HE cells properly. Altogether, our findings demonstrate that endothelial primary cilia modulate HSPC development through transducing Notch signaling. Results The dynamics of endothelial cilia during embryogenesis. To study the underlying link between cilia and hematopoiesis, primary cilia in the vascular ECs in the aorta-gonad-mesonephros (AGM) region, where the definitive hematopoiesis occurs, were characterized firstly. By visualizing a triple-transgenic line, Tg 2 (βact:Arl13b–GFP/kdrl:mCherry/runx1:en-GFP), which marks cilia, ECs and hematopoietic cells (including HSPCs), respectively, we found that primary cilia were present in ECs in the AGM region at 28 h post fertilization (hpf) (Fig. 1a). Meanwhile, runx1+kdrl+ HE cells were also ciliated (Fig. 1b), which was supported by analysis of another HE transgenic line, Tg(gfi1:GFP/ βact:Arl13b–GFP), at 28 hpf (Fig. 1b). In contrast, the cmyblabeled HSPCs in the AGM region were non-ciliated (Ac-tubulin labeled cilia) by fluorescence in situ hybridization (FISH) and Actubulin staining (Fig. 1c). Time-course analysis of a doubletransgenic line, Tg(βact:Arl13b–GFP/ kdrl:mCherry) showed that the number of primary cilia was reduced from 32 hpf and nearly absent at 52 hpf in the AGM region (Fig. 1d–f). The dynamic changes of primary cilia were co (...truncated)