1 BLOS2 negatively regulates Notch signaling during neural and hematopoietic stem and 2 progenitor cell development 3 4 Wenwen Zhou 1, 6 #, Qiuping He 2, 6 #, Chunxia Zhang 2, 6, Xin He 1, Zongbin Cui 3, 5 Feng Liu 2, 6 *, and Wei Li 1, 4, 5 * 6 7 1 State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and 8 Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China; 9 2 State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of 10 Sciences, Beijing 100101, China 11 3 State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, 12 Chinese Academy of Sciences, Wuhan 430072, China 13 4 Center for Medical Genetics, Beijing Children’s Hospital, Capital Medical University; 14 Beijing Pediatric Research Institute; MOE Key Laboratory of Major Diseases in Children, 15 Beijing 100045, China; 16 5 Center of Alzheimer's Disease, Beijing Institute for Brain Disorders, Beijing 100053, China; 17 6 University of Chinese Academy of Sciences, Beijing 100039, China. 18 19 # Co-first authors. 20 * Correspondence should be addressed to: 21 Wei Li, Ph.D., Beijing Children’s Hospital, Capital Medical University, 56 Nan-Li-Shi Road, 1 22 Xicheng District, Beijing 100045, China. Tel: +86-10-5961-6628; Fax: +86-5971-8699; 23 e-mail: [email protected]; 24 Feng Liu, Ph.D., State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese 25 Academy of Sciences, Beijing 100101, China. Tel.: +86 (10) 64807307; Fax: +86 (10) 26 64807313; Email: [email protected]. 27 28 Running Title: BLOS2 negatively regulates Notch signaling 29 30 Key Words: BLOS2; Notch; stem and progenitor cell development; neurogenesis; 31 hematopoiesis; endo-lysosomal trafficking 32 33 The authors declare that no competing interests exist. 34 35 36 37 38 39 40 41 42 43 2 44 Abstract 45 46 Notch signaling plays a crucial role in the control of proliferation and differentiation of stem 47 and progenitor cells during embryogenesis or organogenesis, but its regulation is incompletely 48 understood. BLOS2, encoded by the Bloc1s2 gene, is a shared subunit of two lysosomal 49 trafficking complexes, biogenesis of lysosome-related organelles complex-1 (BLOC-1) and 50 BLOC-1 related complex. Bloc1s2-/- mice were embryonic lethal and exhibited defects in 51 cortical development and hematopoiesis. Loss of BLOS2 resulted in elevated Notch signaling, 52 which consequently increased the proliferation of neural progenitor cells and inhibited 53 neuronal differentiation in cortices. Likewise, ablation of bloc1s2 in zebrafish or mice led to 54 increased hematopoietic stem and progenitor cell production in the aorta-gonad-mesonephros 55 region. BLOS2 physically interacted with Notch1 in endo-lysosomal trafficking of Notch1. 56 Our findings suggest that BLOS2 is a novel negative player in regulating Notch signaling 57 through lysosomal trafficking by controlling multiple stem and progenitor cell homeostasis in 58 vertebrates. 59 60 Impact Statement 61 62 The conserved mechanism of Notch down-regulation by endo-lysosomal trafficking plays 63 important roles in the proliferation and differentiation of stem and progenitor cells during 64 embryogenesis or organogenesis in vertebrates. 65 3 66 Introduction 67 68 Notch signaling is a highly conserved cell-to-cell signaling pathway and its function in cell 69 fate determination makes it essential for embryogenesis and organogenesis, including 70 neurogenesis (Traiffort and Ferent, 2015), vasculogenesis (Krebs et al., 2000) and somite 71 segregation (Wright et al., 2011). Cortical neurogenesis requires the exquisite coordination of 72 neural progenitor cell (NPC) proliferation and differentiation to generate the complex and 73 functional cerebral cortex, which is precisely regulated in temporal and spatial patterns (Gal 74 et al., 2006; Guillemot, 2005). Notch signaling plays a crucial role in the control of 75 proliferation of NPCs and neuronal differentiation during corticogenesis (Fortini, 2009; 76 Kopan and Ilagan, 2009; Pierfelice et al., 2011). Notch signals activate expression of basic 77 helix loop helix (bHLH) Hes transcription factors that maintain NPCs undifferentiated by 78 down-regulating proneural gene expression (Bertrand et al., 2002; Kageyama et al., 2007; 79 Ohtsuka et al., 2001; Ross et al., 2003). The neurogenic transition occurs through stable 80 down-regulation of Notch target genes, leading to up-regulation of proneural genes and 81 neuronal differentiation (Kawaguchi et al., 2008; Shimojo et al., 2008). Hyperactivation of 82 Notch signaling, such as due to loss of Numb and Numblike, results in neural progenitor 83 hyperproliferation and impaired neuronal differentiation in the mouse brain (Hoeck et al., 84 2010; Li et al., 2003a; Rodriguez et al., 2012). 85 Moreover, Notch signaling pathway has emerged to be a key regulator in definitive 86 hematopoiesis. It regulates artery/vein specification (Lawson et al., 2002), as well as 87 hemogenic endothelium (HE) or hematopoietic stem and progenitor cell (HSPC) specification 4 88 through downstream factors cell-autonomously (Guiu et al., 2014; Jang et al., 2015) or 89 non-cell-autonomously (Clements et al., 2011; Kobayashi et al., 2014). Therefore, tight 90 spatio-temporal regulation of Notch activity for its location, strength and duration is essential 91 for the homeostasis and differentiation of NPCs or HSPCs. Current studies have been more 92 focused on the activation of Notch in animals. However, the precise control of Notch turnoff 93 is largely unknown but extremely important in the orchestration of the Notch signals in 94 development. 95 Notch receptor trafficking has been regarded as an important element in the regulation of 96 Notch signaling (Brou, 2009; Fortini and Bilder, 2009). The endocytic trafficking of Notch 97 receptor results in either transportation to lysosomes for degradation via multi-vesicular 98 bodies (MVBs) and late endosomes or recycling back to the plasma membrane for ligand 99 binding and activation (Le Borgne, 2006). When endocytic trafficking of Notch receptor 100 destined for lysosomal degradation is disrupted, Notch receptor accumulates in endosomes 101 undergoing proteolytic cleavage and ectopic activation in a ligand-independent manner 102 (Furthauer and Gonzalez-Gaitan, 2009; Moberg et al., 2005; Thompson et al., 2005; Vaccari 103 and Bilder, 2005; Vaccari et al., 2008). In Drosophila, Deltex interacts with another E3 104 ubiquitin ligase, Su(Dx), to activate ligand-independent Notch proteolysis and signaling 105 (Cornell et al., 1999). The HOPS and AP-3 complex are required for the Deltex-regulated 106 activation of Notch in the endosomal trafficking pathway (Wilkin, et al., 2008). Beyond flies, 107 several mammalian proteins have been identified to regulate Notch lysosomal degradation 108 through the vacuolar H(+) ATPase (Faronato et al., 2015; Kobia et al., 2014; Lange at al., 109 2011; Sethi et al., 2010). However, additional regulators involved in Notch endocytic 5 110 trafficking remain to be elucidated. 111 BLOS2 (encoded by the Bloc1s2 gene) is a subunit of biogenesis of lysosome-related 112 organelles complex-1 (BLOC-1), which has been reported to function in endo-lysosomal 113 trafficking and biogenesis of lysosome-related organelles (LRO) (John Peter et al., 2013; 114 Setty et al., 2007; Starcevic and Dell'Angelica, 2004; Wei and Li, 2013). A recent report 115 reveals that BLOS2 is also a subunit of BLOC-1 related complex (BORC), which regulates 116 the positioning of lysosomes (Pu et al., 2015). In addition, BLOS2 is likely associated with 117 the centrosome to function in regulating transcription (Sun et al., 2008). Thus, BLOS2 might 118 be a multi-functional protein and involved in regulating several cellular processes. 119 Several subunits of BLOC-1, such as dysbindin, snapin and BLOS1, mediate transport of 120 membrane receptors including dopamine receptor 2 (D2R), NMDA receptor subtype 2A 121 (NR2A), and epidermal growth factor receptor (EGFR) from endosomes to lysosomes for 122 degradation (Cai et al., 2010; Ji et al., 2009; Marley and von Zastrow, 2010; Tang et al., 2009; 123 Zhang et al., 2014a). Mice that lack BLOS1 or snapin are embryonic lethal, suggesting that 124 these two subunits of BLOC-1 might play pivotal roles in embryonic development (Tian et al., 125 2005; Zhang et al., 2014a). BLOS2, together with BLOS1 and snapin, are shared subunits of 126 BLOC-1 and BORC (Langemeyer and Ungermann, 2015; Pu et al., 2015). Whether Bloc1s2 127 knockout mice are embryonic lethal has not been reported. In addition, how BLOS2 functions 128 in endo-lysosomal trafficking has not been clearly defined. We herein describe that BLOS2 is 129 a novel negative regulator of Notch signaling mediated by lysosomal trafficking, which is 130 critical for NPC or HSPC development in vertebrates including zebrafish and mouse. 131 6 132 Results 133 134 BLOS2 is required for embryonic cortical morphogenesis and neurogenesis 135 To study the function of BLOS2 in vivo, we generated Bloc1s2 knockout mice by replacing 136 exons 1-4 of the Bloc1s2 gene with the phosphoglycerate kinase-Neo (PGK-Neo) cassette 137 (Figure 1-figure supplement 1A). By genotyping and immunoblotting, we confirmed the 138 replacement of exons 1-4 and null BLOS2 protein in multiple tissues of Bloc1s2-/- neonate 139 mice, while BLOS2 was highly expressed in brain, spleen and intestine in wild-type (WT) 140 neonate mice (Figure 1-figure supplement 1B and C). 141 Genotyping of 1-week-old progeny