BLOS2 Negatively Regulates Notch Signaling During Neural and Hematopoietic Stem And

1 BLOS2 negatively regulates Notch signaling during neural and hematopoietic stem and

2 progenitor cell development

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 *

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.

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].

28 Running Title: BLOS2 negatively regulates Notch signaling

30 Key Words: BLOS2; Notch; stem and progenitor cell development; neurogenesis;

31 hematopoiesis; endo-lysosomal trafficking

33 The authors declare that no competing interests exist.

44 Abstract

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.

60 Impact Statement

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.

66 Introduction

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

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

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

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 derived from crossing Bloc1s2+/- mice revealed the

142 absence of Bloc1s2-/- animals, implying Bloc1s2-/- mice were mostly embryonic lethal. Very

143 few homozygous mutant pups were born alive, and these died within a few hours of birth.

144 This feature is similar to Bloc1s1-/- and snapin knockout mice (Tian et al., 2005; Zhang et al.,

145 2014a) (Supplementary Table 1), suggesting that the shared three subunits of BLOC-1 and

146 BORC are essential for embryonic development. Bloc1s2-/- embryos or neonates manifested a

147 variety of developmental defects, including impaired hematopoiesis (E12.5) (Figure 1A), loss

148 of eye pigmentation (E14.5) (Figure 1B), craniofacial malformation (P0) (Figure 1C), and

149 smaller brain size (E14.5) (Figure 1D).

150 We next focused on the phenotypes in brain development and hematopoiesis.

151 Histological analyses revealed severe morphological defects in the developing cerebral cortex

152 of Bloc1s2-/- embryos. Nissl staining of brains prepared from E14.5 Bloc1s2-/- embryos showed

153 a thinner cerebral cortex as well as enlarged lateral ventricles (Figure 1E and F). Further

154 examination of the cortical structure under higher magnification revealed that the Bloc1s2-/-

155 cortical plate (CP) and intermediate zone (IZ) were remarkably thinner than WT, while the

156 thickness of the ventricular zone (VZ) was comparable between WT and mutant (Figure 1G

157 and H). Due to the significant reduction in cortical wall thickness, we next investigated the

158 effect of loss of BLOS2 on early cortical neurogenesis by immunostaining with deep

159 layer-specific markers Ctip2 (layer V and VI) and Tbr1 (layer VI) (Lakomá et al., 2011). At

160 E12.5 and E14.5, Ctip2+ early-born layer V and VI neurons and Tbr1+ layer VI neurons were

161 both significantly reduced in number in the Bloc1s2-/- cortex compared to the control (Figure

162 1I to K), suggesting that BLOS2 ablation impairs early cortical neurogenesis. Taken together,

163 these observations suggest that loss of BLOS2 disrupts proper cortical neurogenesis and

164 morphogenesis.

165

166 BLOS2 regulates proliferation and differentiation of NPCs

167 To further investigate the pathogenesis of the thinner cortex of Bloc1s2-/- mice and the severe

168 impairment of cortical neurogenesis in the Bloc1s2-/- cortex, we analyzed apoptosis,

169 differentiation, and proliferation during early cortical development. We performed the

170 terminal deoxynucleotidyltransferase-mediated dUTP end-labeling (TUNEL) assay to assess

171 cell death in WT and Bloc1s2-/- mice (Li et al., 2003). No significant change in TUNEL

172 signals was observed in Bloc1s2-/- mice at E14.5, suggesting that the decrease in cortical

173 neurons of Bloc1s2-/- mice does not arise from apoptotic cell death (Figure 2A and B). To

174 determine whether Bloc1s2-/- mice display neuronal differentiation defects, we examined

175 neurogenesis by the bromodeoxyuridine (BrdU)/Ki67 cell-cycle exit assay (Siegenthaler et al.,

176 2009). BrdU was injected into pregnant dams at E13.5, and embryos were collected 18 hours

177 later and processed for BrdU/Ki67 immunostaining. Cells remaining in cycle are BrdU+

178 Ki67+, whereas cells exited from cell cycle are BrdU+ only. At E14.5, less BrdU+ Ki67- cells

179 left the cell cycle in Bloc1s2-/- mice (Figure 2C and D), accompanied by a significant

180 reduction of Tuj1+ early differentiated neurons compared to WT mice (Li et al., 2003) (Figure

181 2E and F), suggesting impaired early neuronal differentiation of NPCs in the Bloc1s2-/- cortex.

182 We next explored whether proliferation of NPCs is altered in the Bloc1s2-/- cortex. We

183 counted the number of Pax6+ radial glia/apical progenitors in Bloc1s2-/- and WT animals.

184 Pax6 immunostaining on coronal sections from E14.5 brains revealed a 52% increase in

185 Pax6+ cells in the mutant when compared with the WT, suggesting that the functional loss of

186 BLOS2 results in an increase in the proliferation of Pax6+ radial glia cells (Figure 2G and H).

187 Moreover, we administered a 2-hour BrdU pulse to pregnant dams at E14.5 to label all cells in

188 S phase and measured the ratio of cycling cells that were in S phase in WT and Bloc1s2-/-

189 animals (Siegenthaler et al., 2009). Double labeling of BrdU and Ki67 showed an increased

190 labelling index (BrdU+ Ki67+/Ki67+) for cortical progenitors in Bloc1s2-/- mice, suggesting

191 increased proliferation of NPCs in the mutant (Figure 2I and J). Furthermore, neural precursor

192 proliferation was also assessed using the mitotic marker phosphohistone-3 (PH3) (Rash et al.,

193 2011). When compared with WT, Bloc1s2-/- cortex showed an increased density of PH3+ cells

194 in the VZ at both E13.5 and E14.5 (Figure 2K and L). Taken together, NPCs in the Bloc1s2-/-

195 cortex were arrested in a proliferative state, inhibiting normal neurogenesis. Our data

196 demonstrate that BLOS2 is important for the transition from proliferation to differentiation of

197 NPCs.

198

199 BLOS2 ablation leads to elevated Notch signaling in neural progenitor cells

200 We then investigated the potential signaling pathways involving BLOS2 in the transition from

201 proliferation to differentiation of NPCs in the cortex. We tested the Notch pathway for the

202 following reasons. Notch signaling plays central roles in the coordination of proliferation of

203 NPCs and neuronal differentiation during neurogenesis (Fortini, 2009; Kopan and Ilagan,

204 2009). Moreover, overexpression of transcription factors Hes1 and Hes5, which mediate

205 Notch signaling, results in neural progenitor hyperproliferation and impaired neuronal

206 differentiation in the brain (Ohtsuka et al., 2001). Similarly, loss of Numb and Numblike in

207 dorsal forebrain leads to the hyperactivation of Notch signaling, increases neural progenitor

208 proliferation, delays cell cycle exit and inhibits neuronal differentiation, mimicking the

209 Bloc1s2-/- phenotypes in NPCs (Li et al., 2003a).

210 To determine whether BLOS2 functions in the control of proliferation and differentiation

211 of NPCs through regulation of Notch signaling, we first examined the production of cleaved

212 Notch (or NICD) using anti-Notch1 V1744, an antibody specific for NICD1. Immunoblotting

213 analyses of cortex extracts derived from Bloc1s2-/- mutants and WT littermates showed that

214 the Bloc1s2-/- cortices contained higher levels of NICD1 than WT animals, indicative of

215 elevated Notch signaling. Likewise, we found an increase in the level of Notch1

216 transmembrane subunit (NTM) in the mutant cortex (Figure 3A and B). Immunohistochemical

217 analyses revealed that Notch1 was mainly detected in the ventricular and subventricular areas.

218 In E14.5 Bloc1s2-/- animals, Notch1 intensity was increased compared to the controls (Figure

219 3C and D). The elevated level of Notch1 (NTM) in Bloc1s2-/- animals potentially explained

220 their higher level of NICD1. To exclude the possibility of higher expression of Notch ligands

221 in mutant mice, Delta-like 1(Dll1) and Jagged1 (Jag1), two Notch ligands reported to be

222 expressed in neural progenitors, were examined and there was no difference between WT and

223 Bloc1s2-/- cortices (Figure 3-figure supplement 1A and B). In addition, we did not find

224 apparent changes in Itch and Dtx2 in Bloc1s2-/- cells, two mammalian homologs of Su(Dx)

225 and Deltex in flies (Figure 3-figure supplement 1C), suggesting that the upregulated Notch1

226 activity is independent of the Deltex-Su(Dx) E3-ubiquitin pathway (Cornell et al., 1999).

227 Next we assessed the expression levels of Notch target genes Hes1, Hes5 and Fabp7.

228 Quantitative reverse transcriptase PCR (qRT-PCR) analyses showed that the levels of Hes1,

229 Hes5, Fabp7 mRNA were increased in the Bloc1s2-/- cortex at E14.5 (Figure 3E-G). The

230 overall protein levels of Hes1, Hes5 and Fabp7 were likewise increased in the Bloc1s2-/-

231 cortex lysates, indicative of elevated Notch signaling during neurogenesis in mutant cortices

232 (Figure 3H and I). We confirmed the effect of BLOS2 on Notch signaling in E14.5 mouse

233 embryonic fibroblasts (MEFs). Hes1, Hes5 and Fabp7 were down-regulated in

234 BLOS2-overexpressed MEFs (Figure 3-figure supplement 1D-F) in which Notch1 was

235 reduced and less colocalized with late endosome marker LBPA (Figure 3-figure supplement

236 2A to D). Furthermore, we observed increased colocalization of Notch1 with LBPA, but

237 decreased colocalization of Notch1 with LAMP1 (a lysosome marker) in cultured NPCs

238 (Figure 3-figure supplement 2E to H).

239 In contrast, the levels of Notch1 (NTM), NICD1 and Hes5 were significantly increased in

240 Bloc1s2-/- MEFs. In addition, increased levels of Notch1 (NTM) and Notch signaling in

241 Bloc1s2-/- MEFs were mostly rescued by overexpression of full-length FLAG-BLOS2 (Figure

242 3-figure supplement 1G and H). Furthermore, the elevated Hes1 in Bloc1s2-/- MEFs could be

243 rescued by the treatment of a γ-secretase inhibitor, DAPT (Figure 3-figure supplement 3A).

244 To further confirm the effects of Notch elevation on NPC, we found that the average

245 diameter of neurospheres derived from E14.5 NPCs of Bloc1s2-/- mice was significantly

246 increased which could be rescued by the treatment of DAPT (Figure 3-figure supplement 3B).

247 Likewise, neuronal differentiation of NPCs from Bloc1s2-/- mice was greatly inhibited which

248 could be recovered by the treatment of DAPT (Figure 3-figure supplement 3C-E). These

249 collective data suggest that loss of BLOS2 results in up-regulation of Notch signaling in

250 neural progenitors, which inhibits cortical neurogenesis in Bloc1s2-/- mice.

251

252 Loss of BLOS2 in mouse results in increased frequency and number of HSPCs, and

253 elevated Notch signaling in the AGM region

254 To explore whether embryonic hematopoiesis is impaired as shown in Figure 1A, we analyzed

255 the property of HSPCs located in the aorta-gonad-mesonephros (AGM) region of Bloc1s2-/-

256 knockout mice. We confirmed that BLOS2 was null in the AGM of Bloc1s2-/- mice (Figure

257 4A). Cell sorting analysis showed that Bloc1s2 mRNA was expressed in endothelial cells and

258 in HSPCs and non-hematopoietic cells in the AGM, suggesting its ubiquitous expression in

259 hematopoietic lineages and surrounding cells (Figure 4B). Strikingly, immunofluorescence

260 analysis showed that Runx1 (a HSPC marker) expression was increased in the AGM and even

261 expanded into the dorsal region of the dorsal aorta (Figure 4C), suggesting that more HSPCs

262 were produced in Bloc1s2-/- mice. The cell proportion of c-Kit+CD34+ HSPCs from E11 AGM

263 in Bloc1s2-/- embryos was higher than that in Bloc1s2+/+ embryos (Figure 4D), further

264 confirming this notion. The colony-forming unit-cell (CFU-C) assay showed decreased

265 colony formation ability (CFU-Mix) with relatively unchanged CFU-E and drastically

266 decreased CFU-GM from AGM tissues in Bloc1s2-/- mice (Figure 4E), suggesting that the

267 differentiation of HSPCs is greatly inhibited. To test whether the increased HSPC production

268 was due to increased cell proliferation, we performed Ki67 staining assay and the results

269 showed more Ki67 positive cells observed in the AGM of Bloc1s2-/- mice (Figure 4F).

270 Together, these data suggest that BLOS2 deficiency leads to increased HSPC production in

271 mouse.

272 Since increased Notch signaling was observed in Bloc1s2-/- brains, we wondered whether

273 this also holds true in the AGM region. As expected, the protein level of both Notch1 and

274 NICD in the AGM region was increased in Bloc1s2-/- mice (Figure 4G). FACS analysis with

275 purified CD31+ cells further confirmed this result (Figure 4H). We found increased

276 colocalization of Notch1 with LBPA and decreased colocalization of Notch1 with LAMP1in

277 the AGM region (Figure 4-figure supplement 1A to D). Next, we examined the expression of

278 several Notch target genes. qRT-PCR analysis showed that the expression of Hey1 and Hey2

279 was significantly upregulated, suggesting that Notch signaling is enhanced (Figure 4I).

280

281 BLOS2 regulates HSPC development through inhibition of Notch activity in zebrafish

282 To determine whether BLOS2 is evolutionarily conserved in HSPC development in

283 vertebrates, we first examined the expression of bloc1s2 in zebrafish. We found that bloc1s2

284 was expressed in neurons and vessels (Figure 5-figure supplement 1A), especially in

285 endothelial cells (Figure 5-figure supplement 1B), which indicates a potential role of bloc1s2

286 in zebrafish hematopoiesis. To study its function in zebrafish, bloc1s2 mutants were generated

287 by CRISPR/Cas9 systems. The gRNA was designed to target 3 of the 4 bloc1s2 transcripts

288 (Figure 5-figure supplement 2A), and the expression of bloc1s2 was significantly decreased,

289 but not abolished due to the expression of transcript bloc1s2-201 in the bloc1s2 mutant fish

290 which we designated as a hypomorphic mutant, bloc1s2 hypo (Figure 5A to C; Figure 5-figure

291 supplement 2B to D). To explore its function in zebrafish hematopoiesis, we examined the

292 expression of HSPC markers in the AGM region of bloc1s2 mutants. Strikingly, we found that

293 the protein level of Runx1 was increased in bloc1s2 mutants (Figure 5D). In addition, the

294 mRNA expression of runx1 and cmyb (another HSPC marker) was also increased in the AGM

295 region (Figure 5E and F). To further confirm this HSPC phenotype, bloc1s2 homozygous

296 mutants were outcrossed with Tg(flk1:mcherry;cmyb:GFP) transgenic line and finally we

297 obtained the bloc1s2 homozygotes in Tg(flk1:mcherry;cmyb:GFP) background. Confocal

298 imaging showed an increased number of hemogenic endothelium (HE) and emerging HSPCs

299 in the bloc1s2 mutants (Figure 5G). Moreover, the increased expression of HSPC markers

300 (runx1 and cmyb) in bloc1s2 mutants were rescued by overexpression of bloc1s2 itself

301 (Figure 5-figure supplement 1C and D), indicating the specific function of bloc1s2 in HSPC

302 development.

303 The emerging HSPCs are derived from specified HE through a process named

304 endothelial-to-hematopoietic transition (EHT). Thus, the increased HSPCs may be a result of

305 high proliferation ratio of HE or emerging HSPCs. To test this hypothesis, BrdU and PH3

306 assays were performed. As expected, the BrdU signals around the dorsal aorta were

307 significantly increased (Figure 5-figure supplement 1E). To investigate whether the increased

308 HSPCs in bloc1s2 mutants can differentiate into blood cells normally, the expression of

309 lymphoid (rag1), erythroid (gata1) and myeloid (pu1) markers was examined. We found that

310 lymphoid differentiation was increased, while erythroid and myeloid differentiation was

311 impaired (Figure 5H). Taken together, these data suggest that ablation of BLOS2 promotes

312 HSPC generation, and also facilitates lymphoid differentiation at the expense of erythroid and

313 myeloid differentiation.

314 We next explored whether BLOS2 regulates HSPC generation through Notch signaling

315 pathway in a similar manner to NPC production. We firstly determined the expression of

316 Notch targets in bloc1s2 mutants. It showed that hey2 and ephrinB2a expression was

317 increased (Figure 5-figure supplement 3A and B). Notch1 protein level was also increased in

318 bloc1s2 mutants (Figure 5-figure supplement 3C). Moreover, we obtained the bloc1s2

319 homozygotes in Notch reporter line in Tg(fli1a:EGFP;tp1:dsRed) background. We found that

320 the tp1+ signals in the dorsal aorta were increased in the bloc1s2 mutants (Figure 5-figure

321 supplement 3D). To further confirm whether the increase of Notch activity mediated the

322 increase of HSPC generation in bloc1s2 mutants, a γ-secretase inhibitor DBZ was used to

323 perform the rescue experiments. As expected, the increased expression of Notch targets hey2

324 and ephrinB2a, HSPC markers runx1 and cmyb in bloc1s2 mutants was blocked by the DBZ

325 treatment (Figure 5-figure supplement 3E and F). Consistent with the zebrafish data, DBZ

326 treatment inhibited the expansion of c-Kit+CD34+ HSPCs from E11 AGM in Bloc1s2-/- mouse

327 embryos (Figure 5-figure supplement 3G). Taken together, these results suggest that elevated

328 Notch signaling leads to the increase of HSPC generation in zebrafish and mice, and that the

329 conservation of BLOS2 regulated Notch signaling is an important axis during HSPC

330 development in vertebrates.

331

332 BLOS2 is required for lysosomal degradation of Notch1

333 To further investigate the underlying mechanism utilized by BLOS2 in regulating the protein

334 level of Notch1 (NTM), we first investigated the expression of Notch1 in the WT and Bloc1s2-/-

335 cortices. qRT-PCR analysis showed that there was no significant change in Notch1 mRNA

336 level between WT and Bloc1s2-/- cortices at E14.5 (Figure 6A), suggesting that the increase of

337 Notch1 (NTM) in the mutant cortex does not arise from the increased transcription of Notch1.

338 Intracellular Notch1 (NTM) is targeted either to the proteosomal or to the

339 endosomal/lysosomal pathway for protein degradation (Andersen et al., 2012). To determine

340 whether proteasomal or lysosomal degradation of Notch1 is impaired in Bloc1s2-/- MEFs, we

341 determined the response of Notch1 (NTM) to lysosome or proteasome inhibition. Upon

342 treatment of WT MEFs with leupeptin (an inhibitor of lysosomal proteolysis) or MG132 (a

343 proteasome inhibitor), we found that Notch1 (NTM) degradation was suppressed either by

344 leupeptin or MG132, indicating that a portion of Notch1 (NTM) is degraded in lysosomes and

345 part of Notch1 (NTM) is degraded by the proteasome (Figure 6B). In Bloc1s2-/- MEFs, we

346 observed no effect of leupeptin treatment on Notch1 (NTM) levels, suggesting that increased

347 Notch1 (NTM) shown in Bloc1s2-/- MEFs probably is due to reduced Notch1 (NTM) degradation

348 by lysosomes (Figure 6C and D). However, Notch1 (NTM) degradation was suppressed by

349 MG132 in Bloc1s2-/- MEFs similar to the effect in WT MEFs, implicating that the proteasomal

350 pathway of Notch1 (NTM) degradation is not affected by loss of BLOS2 (Figure 6C and D).

351 These results suggest that BLOS2 is likely required for lysosomal degradation of Notch1

352 (NTM).

353 In order to find out whether the inhibition of Notch activity in zebrafish bloc1s2-/- also

354 resulted from impaired lysosomal degradation, we used lysosomal inhibitor chloroquine to

355 treat the bloc1s2 mRNA overexpressed embryos. It showed that the decreased Notch1

356 expression in bloc1s2 overexpressed embryos was restored by the chloroquine treatment, but

357 not by treatment with proteasomal inhibitor MG132 (Figure 6E and F), supporting that

358 BLOS2 inhibits Notch signaling pathway mainly through lysosome degradation. Altogether,

359 these results demonstrated that BLOS2 regulates NPC or HSPC development through

360 inhibition of Notch activity in a lysosome-dependent manner.

361

362 BLOS2 interacts with NICD during lysosomal trafficking of Notch1

363 The fact that BLOS2 is a shared subunit of BLOC-1 and BORC, which has been reported to

364 function in endo-lysosomal trafficking, implies the possible impairment of Notch1 in

365 endo-lysosomal trafficking with loss of BLOS2. We first performed a 5-20% (w/v) OptiPrep

366 gradient separation of the homogenates of WT and Bloc1s2-/- brains (Zhang et al., 2014b).

367 Using organellar markers, EEA1 (early endosomes), CD63 (MVBs and late endosomes),

368 LAMP1 (lysosomes), and GM130 (Golgi), we observed that the localization of Notch1 (NTM)

369 exhibited a marked increase in the EEA1 and CD63-labeled fractions in Bloc1s2-/- brains

370 compared to WT, indicative of accumulation of Notch1 (NTM) in endosomes with loss of

371 BLOS2 (Figure 7A and B). We further examined Notch1 receptor localization by co-labeling

372 cells with Notch1 and markers of endosomes and lysosomes (EEA1, LBPA and LAMP1)

373 (Delevoye et al., 2009; Tomas et al., 2015). Compared to WT cells, we observed slightly

374 increased endogenous Notch1 localization in EEA1-labeled early endosomes (Figure 7C and

375 F) and significantly increased localization in LBPA-labeled MVBs and late endosomes in

376 Bloc1s2-/- MEFs (Figure 7D and G). Using structured illumination microscopy (SIM), we

377 confirmed the accumulation of Notch1 receptor in LBPA-positive vesicles in Bloc1s2-/- MEFs

378 (Figure 7-figure supplement 1A). However, we did not observe apparent accumulation of

379 Notch1 in Bloc1s2-/- MEFs labeled with internalized TfA568 after treatment for 30 min

380 representing recycling endosomes (Figure 7-figure supplement 1B) (Delevoye et al., 2016). In

381 contrast, loss of BLOS2 resulted in a significantly decreased Notch1 localization in

382 LAMP1-labeled lysosomes (Figure 7E and H). These results suggested that the

383 endo-lysosomal trafficking of Notch1 receptor is altered in Bloc1s2-/- MEFs, and that the

384 accumulated Notch1 in the endosomes promotes its signaling.

385 Finally, we evaluated whether BLOS2 physically interacts with Notch1 to mediate its

386 lysosomal targeting. In our co-immunoprecipitation (Co-IP) assays, we found that BLOS2

387 bound to the Notch1 intracellular domain (NICD1) in HEK293T cells expressing

388 3×Flag-NICD1 and Myc-BLOS2 (Figure 8A). By endogenous IP, BLOS2 was precipitated by

389 anti-Notch1 with cells dissected from mouse E11 AGM tissues (Figure 8B), supporting that

390 BLOS2 and Notch1 indeed interact with each other. We also found that the fusion protein

391 Flag-BLOS2 signal colocalized with the signal of 6×Myc-Notch1 in HeLa cells (Figure 8C),

392 which partially co-stained with LysoTracker (Figure 8-figure supplement 2A). These results

393 suggest that BLOS2 mediates the endo-lysosomal trafficking of Notch1 probably via its

394 interaction with NICD1 region.

395 BLOS2 is a shared subunit of BLOC-1 and BORC (Langemeyer and Ungermann, 2015;

396 Pu et al., 2015). It is puzzling whether BLOC-1 functions as a universal mechanism in

397 mediating the endo-lysosomal trafficking of different signaling receptors or different BLOC-1

398 subunits play specific roles in mediating different signaling receptors. We first investigated

399 whether Notch1 levels were altered in mutants with loss of other BLOC-1 or BORC subunits.

400 We chose mouse mutants of Pldn pa (Huang et al., 1999) or Dtnbp1 sdy (Li et al., 2003b),

401 Kxd1-/- (Yang et al., 2012), and Bloc1s1-/- (Zhang et al., 2014a) to represent BLOC-1 only,

402 BORC only, and shared BLOC-1 and BORC, respectively. We observed the destabilization of

403 two BLOC-1 subunits (BLOS1 and dysbindin) and drastic reduction of KXD1 in Bloc1s2-/-

404 MEFs, suggesting its tight associations with BLOC-1 and BORC subunits (Figure 8-figure

405 supplement 1A and B). However, we did not observe apparent changes of Notch1 levels in

406 MEFs isolated from the Pldn pa, Kxd1-/-, or Bloc1s1-/- mice (Figure 8D and E). Both BLOS1

407 and BLOS2 are shared subunits of BORC and BLOC-1, and both of these two mutants are

408 embryonic lethal. However, we did not observe apparent change of Notch1 level in the

409 Bloc1s1-/- embryonic brain (Figure 8F and G).

410 Next we examined whether other endocytosed receptors were also altered in Bloc1s1-/-

411 mutant cells. We did not find apparent changes of EGFR levels in either Bloc1s1-/- MEFs

412 (Figure 8-figure supplement 2B and C) or Bloc1s1-/- cortices (Figure 8-figure supplement 2D

413 and E). Furthermore, the lysosomal number and function in Bloc1s1-/- MEFs were apparently

414 normal (Figure 8-figure supplement 2F and G; Figure 8-figure supplement 3). These results

415 suggest that the mechanism utilized by BLOS2 specifically regulates Notch1, but is not a

416 general mechanism for lysosomal degradation of other endocytosed receptors, such as EGFR.

417 Taken together, these results suggest that BLOS2 may function as a specific regulator of

418 Notch signaling, which is likely independent of BLOC-1 or BORC.

419

420 Discussion

421

422 In this study, we have described that a novel player, BLOS2, is involved in endo-lysosomal

423 trafficking of Notch1 by acting as a negative regulator of Notch signaling. In WT cells,

424 Notch1 receptor is endocytosed to early endosomes, where the receptor is either trafficked

425 back to the membrane by recycling endosomes or is directed to lysosomal degradation

426 through MVBs and late endosomes. In mutant cells, loss of BLOS2 resulted in impaired

427 endo-lysosomal trafficking of Notch1, which consequently caused the accumulation of a large

428 fraction of Notch1 in the MVBs and late endosomes. The hyperactivation of Notch signaling

429 led to the defective embryonic corticogenesis and hematopoiesis in the Bloc1s2-/- mutants. We

430 also observed interaction between BLOS2 and Notch1, suggesting that BLOS2 most likely

431 mediates endo-lysosomal trafficking of Notch1 (a model in Figure 8-figure supplement 1C).

432 Lysosomal degradation of Notch receptors prevents ectopic Notch activation by turning off

433 the signals. Our observation that BLOS2 functions as a negative regulator of Notch signaling

434 provides new mechanistic insights into the fine-tuning of Notch signaling by endo-lysosomal

435 trafficking.

436 The control of proliferation and differentiation of NPCs must be precisely regulated by

437 extrinsic and intracellular signals, so that cortical neurons are organized into speciﬁc layers.

438 At developmental stages, it is extraordinarily sensitive to the dosage of Notch signaling. We

439 here showed that loss of BLOS2 increased the number of NPCs, delayed cell cycle exit,

440 inhibited neuronal differentiation and consequently led to morphological defects of the cortex.

441 The phenocopy of cortical developmental defects in Bloc1s2-/- mutant to Emx1IREScre-mediated

442 numb and numblike double mutants led us to investigate whether BLOS2 is implicated in the

443 negative regulation of Notch signaling (Li et al., 2003a). Indeed, our results showed that loss

444 of BLOS2 resulted in the up-regulation of Notch signaling in embryonic cortex, which led to

445 the developmental arrest of NPCs from proliferation to differentiation. Our findings reveal

446 that BLOS2 is a new player involved in the control of cortical progenitor self-renewal and

447 generation of cortical neurons.

448 Notch signaling is remarkably pleiotropic and could be important for the development of

449 almost all tissues in metazoans (Louvi and Artavanis-Tsakonas, 2006). We have observed

450 increased HSPC production in both mouse and zebrafish embryos upon the up-regulation of

451 Notch signaling due to the loss-of-function of BLOS2. This renders that more profound

452 effects could be explained under this mechanistic controlling of Notch signaling. For example,

453 previous studies have shown that Notch signaling plays important roles in the development of

454 the neural crest and the retinal pigment epithelium (RPE) (Bao and Cepko, 1997; Lee et al.,

455 2005). Abnormal eye pigmentation and craniofacial malformation in Bloc1s2-/- mice, as we

456 have described in Figure 1, could be likely attributable to the disruption of Notch signaling

457 during neural crest development. However, loss of eye pigment could also result from the

458 disrupted melanosomal biogenesis as BLOS2 may play a role in BLOC-1 in melanosomal

459 protein trafficking. The detailed mechanisms for these defects await further investigation.

460 Nonetheless, Bloc1s2-/- mutant provides a new tool to study the developmental regulation of

461 Notch signaling in embryogenesis and organogenesis.

462 The cellular mechanisms underlying the control of the strength of Notch signaling are

463 important for biological processes (Hori et al., 2012). Recent studies have shown that

464 endo-lysosomal trafficking of the Notch receptor plays important roles in the regulation of

465 Notch signaling (Fortini, 2009; Yamamoto et al., 2010). Endocytic trafficking has been

466 considered to play a negative role in signaling pathways by transporting of Notch receptor to

467 lysosomes for degradation. Also it is important for the activation of Notch signaling, which

468 may occur not only on the cell membrane but also in endocytosed vesicles or endosomes

469 (Hori et al., 2011; Sorkin and von Zastrow, 2009). Accumulation of Notch receptor in

470 endosomes, caused by mutations in genes involved in endo-lysosomal trafficking, leads to

471 ectopic activation of Notch signaling (Fortini and Bilder, 2009; Furthauer and

472 Gonzalez-Gaitan, 2009). Several proteins involved in endo-lysosomal trafficking, such as

473 endosomal sorting complex required for transport (ESCRT) complexes, lethal (2) giant discs

474 (Lgd), the homotypic fusion and vacuole protein sorting (HOPS) complex, and the AP-3

475 complex, have been shown to regulate Notch signaling in flies (Jaekel and Klein, 2006;

476 Moberg et al., 2005; Thompson et al., 2005; Vaccari and Bilder, 2005; Vaccari et al., 2008;

477 Vaccari et al., 2009; Wilkin et al., 2008). To our knowledge, this is the first report of

478 developmental defects of these endo-lysosomal trafficking complexes in vertebrates

479 attributable to upregulated Notch signaling. Interestingly, loss of Lgd function leads to

480 ectopic upregulation of Notch signaling and Lgd specifically acts on Notch endo-lysosomal

481 trafficking (Jaekel and Klein, 2006). It is speculative that BLOS2 may play a similar role of

482 Lgd by functioning as an adaptor of ESCRTs in vertebrates. BLOS2 is a shared subunit of

483 both BLOC-1 and BORC (Pu et al., 2015). Although BLOC-1 and BORC have been

484 implicated in endo-lysosomal trafficking (Cai et al., 2010; Ji et al., 2009; Pu et al., 2015;

485 Zhang et al., 2014a), our results have shown that BLOS2 regulates Notch signaling likely

486 independently of BLOC-1 or BORC. Loss of BLOS2 leads to the accumulation of Notch in

487 the endosomes. In comparison, when other BLOC-1 subunit, BLOS1 or snapin, is deficient,

488 EGFR or D2R is accumulated in the plasma membrane where EGF or D2 signaling is

489 upregulated in a ligand-depend manner (Supplementary Table 1) (Cai et al., 2010; Ji et al.,

490 2009; Zhang et al., 2014a). The interesting point here is that the mechanism utilized by

491 BLOS2 specifically regulates Notch1, but is not a general mechanism for lysosomal

492 degradation of other endocytosed receptors, such as EGFR. There could be several

493 hypotheses. Firstly, different interacting partners of BLOC-1 subunits are operative to expand

494 their involvement in different pathways. Our recent results have shown that different subunits

495 of BLOC-1, such as dysbindin, muted, and BLOS1, may have unique functions in lysosomal

496 trafficking (e.g. endo-lysosomal trafficking, LRO biogenesis, and autophagy) mediated by

497 specific interacting partners (Hao et al., 2015; Wang et al., 2014; Yuan et al., 2015; Zhang et

498 al., 2014a). In addition, BLOS2 may have a function in the nucleus (Sun et al., 2008).

499 Secondly, the residual activity of the destabilized subunit outside of BLOC-1 may function in

500 some way that is different from a null mutation itself, suggesting that phenotypes are

501 differentially sensitive to genetic dosages of loss-of-function BLOC-1 alleles (Mullin et al.,

502 2015).

503 HSPCs are capable of self-renewal and differentiation into all lineages of blood

504 including erythroid, myeloid and lymphoid lineages. HSPCs are now widely used in

505 transplantation to cure multiple diseases. Limitation of the donors to provide sufficient

506 HSPCs makes it urgent to produce HSPCs ex vivo or in vitro. Our results revealed the

507 inhibitory role of BLOS2 in HSPC production. Manipulations of BLOS2 expression in

508 HSPCs may be practical in use. Down-regulation of BLOS2 by genome-editing of HSPCs

509 might facilitate its production, which is highly demanded in clinical applications. In contrast,

510 overexpression of BLOS2 promoted HSPC differentiation, which may improve the

511 differentiation efficiency. Interestingly, the differentiation activity of excessive HSPCs was

512 abnormal as they generated more T cells at the expense of erythroid and myeloid

513 differentiation. This phenotype is similar to the T-cell acute lymphoblastic leukemia (T-ALL),

514 which is also caused by the hyperactivation of Notch signaling pathway (Tzoneva and

515 Ferrando, 2012). The mimicking T-ALL phenotypes in BLOS2 knockouts have provided

516 potential zebrafish or mouse models of T-ALL for clinical investigation. In addition to this

517 point, modulation of BLOS2 or the lysosomal degradation pathway may serve as a potential

518 therapeutic target in controlling T-ALL progression in patients.

519

520 Materials and Methods

521

522 Animals and genotyping

523 Bloc1s2 knockout mice (Bloc1s2-/-) were generated by gene targeting in 129/J-derived

524 embryonic stem cells and bred in C57BL/6J at the animal facility of the Institute of Genetics

525 and Developmental Biology (IGDB), Chinese Academy of Sciences. Other mouse mutants

526 (Pldn pa, Dtnbp1 sdy, Kxd1-KO, Bloc1s1-KO) used in this study have been described (Huang et

527 al., 1999; Li et al., 2003b; Yang et al., 2012; Zhang et al., 2014a). All animal procedures were

528 approved by the Institutional Animal Care and Use Committee of IGDB (KYD2006-002).

529 WT and mutant Bloc1s2-/- alleles were identified using the following primers: 5’-TGG GCT

530 TAC AGA TGT TGT ATC AC-3’, 5’-GCC AAG ATG CAG ACT CAG TAG CAG-3’ and

531 5’-TGG GCT TAC AGA TGT TGT ATC AC-3’.

532 Zebrafish strains including Tubingen, cmyb:GFP (North et al., 2007), kdrl:mCherry

533 (Bertrand et al., 2010), fli1a:EGFP (Lawson and Weinstein, 2002), tp1:dsRED (Parsons et al.,

534 2009) and bloc1s2-/- mutants were raised and kept at 28.5 °C in the standard circulating water

535 system. The embryos were obtained by natural spawning. This study was approved by the

536 Ethical Review Committee in the Institute of Zoology, Chinese Academy of Sciences, China.

537

538 Immunohistochemistry and immunofluorescence staining

539 Embryonic brains were dissected and fixed by immersion with 4% PFA and cryoprotected

540 with 30% sucrose at 4°C. Tissues were embedded in Tissue-Tek OCT (Sakura Finetek) and

541 sectioned on a Cryostat (Leica CM1900). For immunohistochemistry, coronal sections of 40

542 μm thickness were incubated with blocking solution containing 0.01 M phosphate-buffered

543 saline (PBS) supplemented with 5% goat serum, 0.3% Triton X-100 and 3% bovine serum

544 albumin (BSA). Primary antibodies were diluted in blocking solution and incubated overnight

545 at 4°C. After washing, sections were incubated with fluorochrome-conjugated secondary

546 antibody for 2 h at room temperature. Finally, the sections were incubated with DAPI and

547 mounted. For BrdU/Ki67 immunostaining, denaturation of DNA is required. Sections were

548 incubated in 2M HCl for 30 min at 37°C, followed by neutralization in 0.1 M sodium

549 tetraborate buffer (pH 9.0) for 10 min at room temperature. For Nissl staining, serial frozen

550 sections were stained with 0.1% cresyl violet and then dehydrated with graded alcohols.

551 For immunofluorescence staining, cells were fixed in 4% paraformaldehyde (PFA) for 30

552 min and washed three times in PBS. Cells were blocked for 1.5 hours in blocking solution

553 (PBS containing 3% BSA and 0.1% Triton X-100). Cells were then incubated at 4°C

554 overnight with primary antibodies diluted in blocking solution, washed and incubated with

555 secondary antibodies for 2 h at room temperature. After washing, cells were incubated with

556 DAPI and mounted. Primary antibodies used were as follows: rat anti-Ctip2 (1:500, Abcam,

557 ab18465), rabbit anti-Tbr1 (1:200, Abcam, ab31940), rat anti-BrdU (1:100, AbCam, ab6326),

558 rabbit anti-Ki67 (1:500, AbCam, ab15580), mouse anti-Tuj1 (1:1000, Abcam, ab7751), rabbit

559 anti-Pax6 (1:500, Covance Research Products, PRB-278P), rat anti-PH3 (1:1000, Abcam,

560 ab10543), rabbit anti-Notch1 (1:400, Cell Signaling Technology, 3608), mouse anti-EEA1

561 (1:100, BD Biosciences, 610457), mouse anti-LBPA (1:200, Echelon, Z-PLBPA), mouse

562 anti-LAMP1 (1:500, BD Biosciences, 553792).

563 The mouse embryos were processed for cryosectioning (10 μM) and then did

564 immunofluorescence assay as described previously. Briefly, Bloc1s2+/+ and Bloc1s2-/- embryos

565 at E11 were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) for 12 hours

566 at 4 °C. The sections were blocked in 5% BSA containing 0.3% Triton X-100 for 1 hour, and

567 then incubated with Runx1 (Abcam, ab92336) and CD31 (Abcam) antibody overnight at 4 °C

568 and Alexa 488 and 561 labeled secondary antibody (Invitrogen) for 2 hours. After that, the

569 sections were counterstained with DAPI and all fluorescence images were acquired by Nikon

570 confocal A1 (Nikon, Japan). BrdU and PH3 assays in zebrafish were performed as described

571 previously (Lu et al., 2015; Wang et al., 2013).

572

573 Immunoblotting

574 Tissues of Bloc1s2-/- mice and their WT littermates were dissected and homogenized in RIPA

575 buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% DOC, 0.1% SDS, 50 mM Tris, pH 8.0) and

576 the protein content was determined with Protein Assay (Bio-Rad). The dissected trunk regions

577 of zebrafish embryos were homogenized with cell lysis buffer with proteinase inhibitors.

578 Lysates were separated in SDS-PAGE gels and immunoblotted with the indicated antibodies.

579 Rabbit BLOS2 polyclonal antibodies was generated against a His-tagged fusion protein

580 corresponding to the mouse full-length Bloc1s2 gene. Other antibodies used in this study were

581 as follows: rabbit anti-Notch1 (1:2000, Cell Signaling Technology, 3608), rabbit anti-Fabp7

582 (1:5000, Millipore, ABN14), rat anti-Hes1 (1:1000, MBL, D134-3), rabbit anti-Hes5 (1:1000,

583 Millipore, AB5708), mouse anti-EEA1 (1:1000, BD Biosciences, 610457), mouse anti-CD63

584 (1:1000, Millipore, NG1944051), mouse anti-GM130 (1:1000, BD Biosciences, 610822),

585 mouse anti-Flag (1:5000, Sigma, F3165), rabbit anti-Myc (1:5000, Sigma, C3956), mouse

586 anti-β-actin (1:10000, Sigma, A5441), anti-Runx1 (1:200, AnaSpec, 55593), anti-Notch1

587 (1:200, Abcam, ab65297), anti-EGFR (1:5000, Fitzgerald, 20R-ER004), anti-Cathepsin D

588 (1:1000, SantaCruz, sc-6486). Blots from three independent experiments were quantified

589 using ImageJ (NIH) and represented by relative intensity versus that of control. β-actin was

590 used as an internal control to normalize band intensity.

591

592 Quantitative real-time polymerase chain reaction (qRT-PCR) analysis

593 Total RNA was extracted isolated from WT and Bloc1s2-/- dissected cortices at E14.5 with

594 RNeasy (Qiagen) and used to generate first-strand cDNA with the Superscript III cDNA

595 synthesis system (Invitrogen) following the manufacturer’s instructions. qRT-PCR analysis

596 was performed using SYBR PrimeScript Ready Mix (Takara) in an ABI 7900 sequence

597 detection system (Applied Biosystems). GAPDH expression was used for normalization.

598 Total RNAs are from the dissected trunk region of zebrafish embryos. The PCR primers are

599 listed in Supplementary file 1-Supplementary Table 2.

600

601 Cell culture, transfection and co-immunoprecipitation assay

602 WT and Bloc1s2-/- MEFs were isolated from each of the E14.5 embryos. MEFs, HEK293T or

603 HeLa cells were all cultured in DMEM (Thermo Scientific) supplemented with 10% FBS

604 (Hyclone Laboratories, Logan, UT) and 1% penicillin/streptomycin at 37°C, 5% CO2.

605 Ultrastructures of MEFs were examined by transmission electronic microscopy by following

606 the procedures described before (Zhang et al., 2014a).

607 WT and Bloc1s2-/- neurospheres derived from E14.5 NPCs were cultured as described

608 (Louis and Reynolds, 2005). Neurospheres were cultured in DMEM/F12 medium with 1%

609 B27 supplement (Gibco), bFGF (20 ng/ ml, R&D), EGF (20 ng/ml, R&D) and 1%

610 penicillin/streptomycin at 37°C, 5% CO2. For immunofluorescence staining, mouse NPCs

611 were cultured in dishes pre-coated with poly-D-lysine and in the DMEM/F12 medium with

612 1% B27 supplement (Gibco), bFGF, EGF and 1% penicillin/streptomycin.

613 Plasmids were transfected into HEK293T and HeLa cells with Lipo2000 (Invitrogen)

614 and into MEFs with MACSfectin Reagent (Miltenyi Biotec) according to the manufacturer’s

615 protocol. The coding sequence of BLOS2 was amplified by PCR from mouse brain cDNA,

616 and cloned into the pCMV-tag2B vector (with Flag-tag) and pCMV-tag3B vector (with

617 Myc-tag). The pCS2 Notch1 Full Length-6MT (plasmid 41728) and 3 × Flag-NICD1

618 (plasmid 20183) were purchased from Addgene. Co-immunoprecipitation (Co-IP) assay was

619 performed as described (Zhang et al., 2014). Transfected HEK-293T cells were harvested and

620 lysed in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 and

621 protease inhibitors. The lysate was incubated overnight with monoclonal anti-FLAG M2

622 antibody (Sigma) at 4°C. After washing, the samples were eluted with elution buffer (5 μg/μL

623 3 × FLAG peptide) and immunoblotted with anti-Myc or anti-Flag antibody.

624 The procedure of endogenous co-IP was performed as reported previously (Zhang et al.,

625 2015). Briefly, E11 mouse AGM regions were dissected and lysed in RIPA buffer and

626 incubated with Notch1 (Abcam, ab128076, 1:100) antibody for 4 h at 4 °C. Protein A+G

627 agarose (Santa, SC-2003) was added next to the mixture which was incubated for another 3-4

628 h. Beads were collected for western blotting using Notch1 and rabbit BLOS2 polyclonal

629 antibodies.

630

631 BrdU labeling, cell cycle analysis and TUNEL assay

632 Embryonic brains were collected 2 hours after BrdU (Sigma) injection (50 mg/kg body

633 weight) into pregnant dams at E13.5. BrdU/Ki67 immunostaining was performed as described

634 above. We calculated proliferation index as (BrdU+ Ki67+)/Ki67+ where Ki67 marks all

635 cycling cells. To assess cell cycle exit, we injected BrdU intraperitoneally to pregnant dams

636 18 hours prior to embryo harvest at E13.5-E14.5, and the cell-cycle exit index was calculated

637 as (BrdU+ Ki67–)/BrdU+ (Siegenthaler et al., 2009).

638 To analyze the cell cycle status of HSPCs were stained with antibodies against

639 c-Kit-APC and CD34-PE (eBioscience) followed by fixation and permeabilization with

640 Foxp3/Transcription Factor Staining Buffer Set (eBiosciences) according to the

641 manufacturer’s protocol. After that, the cells were incubated with Ki67-FITC (eBioscience)

642 and stained with 7-amino-actinomycin D (7AAD) (eBioscience). The cells were analyzed

643 with the MoFlo XDP (Beckman Coulter).

644 The TUNEL assay was performed with the In Situ Cell Death Detection Kit, POD

645 (Roche) according to the manufacturer’s protocol.

646

647 Flow cytometry

648 Single cells obtained from E11 AGMs were dissociated by collagenase. Then antibody

649 staining was performed for 30 mins at 4 °C using antibodies specific to c-Kit-APC, CD31-PE,

650 CD41-FITC, CD45-APC, CD34-PE and TER119-PE-CY7 (eBioscience). The cells were

651 analyzed with the MoFlo XDP (Beckman Coulter).

652

653 Colony-forming unit-cell assay

654 Colony-forming unit-cell (CFU-C) assay was performed as following: Each well plated with

655 600 c-Kit+CD34+ HSPCs from E11 Bloc1s2+/+ and Bloc1s2-/- embryos was carefully harvested

656 to ultra-low attachment 24-well plates (Costar) and cultured in CFU-C media. The cells were

657 incubated at 37 °C in 5% CO2 with 100% humidity for 7-10 days, and the number of each

658 type of colonies was counted according to morphology. The experiment was repeated in

659 triplicate.

660

661 mRNA microinjection and whole mount in situ hybridization

662 For mRNA synthesis, zebrafish bloc1s2 full-length CDS were cloned into the pCS2+ vector.

663 Capped mRNA was synthesized using the mMessage mMachine SP6 kit (Ambion) and

664 purified by RNA purification kit (Tiangen, China). bloc1s2 mRNA were injected into 1

665 cell-stage wildtype and bloc1s2-/- embryos at the yolk/blastomere boundary.

666 Whole mount in situ hybridization for embryos was performed using ZF-A4 in situ

667 hybridization machine (Zfand, China) as described previously (Wang et al., 2011), with

668 probes including bloc1s2, runx1, cmyb, rag1, gata1, pu1, hey2 and ephrinB2a.

669

670 Pharmacological treatments

671 To block lysosomal or proteasomal degradation, MEFs were treated with the proteasomal

672 inhibitors MG132 (10 µM) or the lysosomal inhibitors leupeptin (100 µM) for four hours.

673 Cells were lysed in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100

674 and protease inhibitors. The cell lysates were then subjected to SDS-PAGE and

675 immunoblotted with the indicative antibodies.

676 To block Notch signaling, WT and Bloc1s2-/- MEFs were incubated with a γ-secretase

677 inhibitor DAPT (25 μM, Sigma, D5942) for five hours, and Hes1 expression was detected by

678 qRT-PCR analysis. WT and Bloc1s2-/- neurospheres were treated with DAPT (1 μM) for 72 h

679 (Hoeck et al., 2010).

680 WT and bloc1s2-/- zebrafish embryos at 18 hpf were treated with a γ-secretase inhibitor

681 DBZ (4μM, Sigma, SML0649), MG132 (10μM, Sigma, C2211) or chloroquine (50μM,

682 Sigma, C6628) respectively.

683 One embryo equivalent (ee) cells in AGM regions were treated with DMSO or DBZ (4

684 μM; Sigma, SML0649). The cells were incubated at 37 °C in 5% CO2 with 100% humidity

685 for 4 days. Semi-adherent cells were then carefully harvested and the antibody staining was

686 performed as described previously, using antibodies specific to c-Kit-APC and CD45-PE

687 (eBioscience).

688

689 High-resolution imaging

690 Photomicrographs of Nissl-stained sections were obtained on a Leica SCN400 Brightfield

691 scanner and confocal images were acquired with an ECLIPSE Ti-C2 confocal microscope

692 equipped with NIS-Elements argon software (Nikon, Japan). We also performed structured

693 illumination microscopy (SIM) on an OMX V4 system (DeltaVision OMX microscope, GE

694 Healthcare). Images were further analyzed with Image-Pro Plus and ImageJ software. For

695 morphological analysis, equivalent sections of Bloc1s2-/- and WT littermate brains were

696 chosen on the basis of common morphological landmarks. Total thickness of the cerebral

697 cortex, and thickness of the CP, IZ, and VZ were measured in Nissl-stained coronal sections

698 of forebrains with Image-Pro Plus software. To calculate LV and brain volumes, the area from

699 each serial section was estimated and volumes were estimated by summing the areas across

700 sections and multiplying by slice thickness. For in vivo cell counting, numbers of

701 marker-positive cells were counted and normalized to 100 µm of apical surface, defining a

702 radial unit in more than four serial sections from at least three different animals per group. For

703 Notch1 immunostaining, mean fluorescence values were measured in the VZ area and

704 fluorescence background was subtracted in at least four serial sections from more than three

705 different animals per group. For colocalization analysis in MEFs in vitro, at least 100 cells

706 were counted in at least five different fields from at least three independent experiments.

707 Mander’s overlap coefficient K1 was calculated with NIS-Elements argon software. The

708 zebrafish samples were prepared as previously reported (Renaud et al., 2011) and images

709 were captured by Nikon A1 confocal microscopy (Nikon, Japan).

710

711 OptiPrep gradient assay

712 The fractionation assay was performed using OptiPrep gradient method as described (Zhang

713 et al., 2014b). The dissected WT and Bloc1s2-/- brains at E14.5 were immediately

714 homogenized with HB lysis buffer (250 mM sucrose, 20 mM Tris-HCl, pH 7.4, 1 mM EDTA).

715 An equal volume of 20% and 5% Optiprep (Axis-Shield, Norway) were placed into the tube

716 in sequence and the tissue lysate were placed onto the top of the gradient. The sample was

717 centrifuged at 28,000 rpm (TLS-55, Beckman, USA) for 14 hours at 4°C. Eleven fractions

718 were collected from the top and equal aliquots from each fraction were analyzed for

719 immunoblotting.

720

721 Statistical analysis

722 Statistical analysws were performed with Student’s t test or analysis of variance, two-tailed

723 using SPSS 11.5. qRT-PCR results were normalized as described (Park et al., 2015). Sample

724 sizes are indicated in the figure legends. All data are presented as mean ± s.e.m. We choose P

725 = 0.05 as a cutoff for statistical significance. In the figures, we use the star signs to represent

726 different ranges of P values, * P < 0.05, ** P < 0.01, *** P < 0.001.

727

728 Acknowledgements

729

730 We are very thankful to Dr. Richard T. Swank who provided the Pldn pa and Dtnbp1 sdy

731 mutants for this study and proofread this manuscript. We thank Dr. Xiang Gao’s lab for

732 generating the Bloc1s2+/- mice. We are thankful to Mr. Feng Zhang for his technical support.

733 We thank Dr. Hua Han and Dr. Zhiheng Xu for discussions and suggestions.

734

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990

991 Figure Legends

992

993 Figure 1. BLOS2 deficiency impaired cortical development.

994 (A-D) Gross examination of whole mount WT (Bloc1s2+/+) and Bloc1s2-/- embryos at E12.5,

995 E14.5, and newborns (P0). Note that impaired hematopoiesis (A), loss of eye pigmentation

996 (B), the craniofacial malformation (C), and smaller brain size (D) of Bloc1s2-/- mice are

997 indicated by the arrows compared with WT littermates at E12.5, E14.5, and newborns. (E)

998 Coronal sections of E14.5 brains with Nissl staining of WT and Bloc1s2-/- embryos. The black

999 boxes indicate the areas at higher magnification shown in (G). The enlarged lateral ventricles

1000 (LV) are shown. (F) Volumetric analysis of the LV in WT and Bloc1s2-/- brains at E14.5. The

1001 values for LV volume in Bloc1s2-/- mice are indicated as percentages of the corresponding WT

1002 values. The ratio of the volume of LV to brain is also indicated as percentages of the

1003 corresponding WT values. ***, P < 0.001 (Student’s t-test); n = 6 mice. (G) The cortical plate

1004 (CP), intermediate zone (IZ), and ventricular zone (VZ) are labeled and compared. In

1005 Bloc1s2-/- brain, the thinner CP and IZ are shown, but VZ is almost normal. (H) Quantification

1006 of the thickness of the neocortex, CP, IZ and VZ in WT and Bloc1s2-/- brains at E14.5. 212.85

1007 ± 10.39 (neocortex), 44.48 ± 3.01 (CP), 72.12 ± 6.46 (IZ), 94.66 ± 3.78 (VZ) for WT; 162.61

1008 ± 1.99 (neocortex), 26.12 ± 1.27 (CP), 48.00 ± 1.26 (IZ), 87.17 ± 2.95 (VZ) for Bloc1s2-/- . **,

1009 P < 0.01 (IZ); ***, P < 0.001 (neocortex and CP); n.s., not significant, P = 0.137 (VZ); n = 6 46

1010 mice. (I) Immunohistochemistry (Ctip2 and Tbr1) on sections of WT and Bloc1s2-/- brains at

1011 E12.5 and E14.5. The numbers of Ctip2+ and Tbr1+ cells were decreased in the Bloc1s2-/-

1012 brain at E12.5 and E14.5. (J and K) Quantification of neuronal number at E12.5 and E14.5.

1013 24.00 ± 0.62 (Ctip2+) , 22.08 ± 0.33 (Tbr1+) for WT at E12.5, 12.22 ± 0.17 (Ctip2+), 12.00 ±

1014 0.68 (Tbr1+) for Bloc1s2-/- at E12.5; 48.40 ± 1.73 (Ctip2+), 27.78 ± 1.66 (Tbr1+) for WT at

1015 E14.5, 30.1 ± 1.41 (Ctip2+), 14.55 ± 0.66 (Tbr1+) for Bloc1s2-/- at E14.5 . ***, P < 0.001; n =

1016 9. All graphs are mean ± s.e.m. Scale bars: 1 mm in (D); 100 µm in (E); 50 µm in (I).

1017

1018 Figure 2. Neural progenitor proliferation and neurogenesis were altered in the Bloc1s2-/-

1019 cortex.

1020 (A) TUNEL assay on sections from WT and Bloc1s2-/- embryos at E14.5. No significant

1021 difference in the number of apoptotic cells (arrows) in the Bloc1s2-/- neocortex was detected

1022 as compared with WT littermates. (B) Quantification of TUNEL+ cells in the WT and

1023 Bloc1s2-/- cortices. Results represent the average number of TUNEL-positive cells per

1024 imaging field at ×20 magnification within the cortex. 1.33 ± 0.40 for WT, 1.53 ± 0.54 for

1025 Bloc1s2-/-. P = 0.19, n.s., not significant (Student’s t-test); n = 6 mice. (C) Representative

1026 staining from BrdU/Ki67 cell-cycle exit assay in the WT and Bloc1s2-/- cortices. In the

1027 cell-cycle exit assay at E13.5 to E14.5, a BrdU pulse was followed 18 hours later by double

1028 labeling of BrdU and Ki67. BrdU+/Ki67– cells (arrows) were considered to be cells exited

1029 from the cell cycle. Pictures showed that the number of cells exited from the cell cycle was

1030 decreased in mutant cortex compared with WT. (D) Quantification of the proportion of cells

1031 leaving the cycle [BrdU+/Ki67– over total BrdU+] in WT and Bloc1s2-/- cortices. 52.61 ± 3.48

1032 for WT, 33.29 ± 2.95 for Bloc1s2-/-. **, P < 0.01; n = 9 mice. (E) Immunohistochemistry

1033 staining of Tuj1 on sections of WT and Bloc1s2-/- brains at E14.5. The number of Tuj1+ cells

1034 was decreased in the Bloc1s2-/- brain. (F) Quantification of the thickness of the Tuj1+

1035 neuronal layer at E14.5. 189.70 ± 6.54 for WT, 126.84 ± 10.88 for Bloc1s2-/-. ***, P < 0.001;

1036 n = 9 mice. (G) Immunohistochemistry staining of Pax6 on sections of WT and Bloc1s2-/-

1037 brains at E14.5. The number of Pax6+ cells was increased in the Bloc1s2-/- brain. (H)

1038 Quantification of the number of Pax6+ cells at E14.5. 74.36 ± 3.88 for WT, 113.29 ± 4.95 for

1039 Bloc1s2-/-. ***, P < 0.001; n = 9 mice. (I) Staining of brain sections for BrdU and Ki67

1040 following a 2-hour BrdU pulse at E14.5. Pictures showed an increase in the proportion of

1041 cycling cells in S-phase in the Bloc1s2-/- cortex compared with WT. (J) Quantification of the

1042 BrdU labeling index in WT and Bloc1s2-/- cortices. BrdU labeling index is the percentage of

1043 Ki67+ cells that incorporated BrdU. 57.69 ± 1.52 for WT, 70.79 ± 3.27 for Bloc1s2-/-; **, P <

1044 0.01; n = 9 mice. (K) Immunohistochemistry staining of PH3 on sections of WT and

1045 Bloc1s2-/- brains at E13.5 and E14.5. Pictures showed an increase in PH3+ cells in the VZ in

1046 the Bloc1s2-/- cortex at E13.5 and E14.5. (L) Quantification of the number of PH3+ cells in the

1047 VZ at E13.5 and E14.5. 13.31 ± 1.43 for WT at E13.5, 23.49 ± 1.90 for Bloc1s2-/- at E13.5;

1048 4.51 ± 0.25 for WT at E14.5, 7.21 ± 0.66 for Bloc1s2-/- at E14.5. ***, P < 0.001; n = 9 mice

1049 per group. All graphs are mean ± s.e.m. Scale bars: 50 μm in A, C, E, G, I and K.

1050

1051 Figure 3. Loss of BLOS2 led to increased Notch signaling in mouse brain.

1052 (A) Immunoblotting analysis of NICD1 and Notch1 (NTM) in cortical extracts derived from

1053 WT and Bloc1s2-/- mice at E14.5 (Figure 3A-source data 1 and 2). It showed that the levels of

1054 NICD1 and Notch1 (NTM) were significantly increased in the Bloc1s2-/- cortex lysates. (B)

1055 Quantification of NICD1 and Notch1 (NTM) relative to WT cortex. Data are presented as fold

1056 change normalized to the mean of WT ± s.e.m. ***, P < 0.001 (Student’s t-test); n = 9 mice.

1057 (C) Immunohistochemistry staining of Notch1 on sections of WT and Bloc1s2-/- brains at

1058 E14.5. Insets are high-magnification images of the boxed regions. Increased Notch1

1059 expression was observed in Bloc1s2-/- cortices at E14.5. Scale bar: 50 μm. (D) Quantification

1060 of signal intensity of Notch1. **, P < 0.01; n = 9 mice. (E-G) qRT-PCR analysis for Hes1,

1061 Hes5 and Fabp7 of the WT and Bloc1s2-/- dissected cortices at E14.5. The expression of Hes1,

1062 Hes5 and Fabp7 were significantly higher in the Bloc1s2-/- cortex. Data are presented as fold

1063 change normalized to the mean of WT. *, P < 0.05; ***, P < 0.001; n = 9 mice. (H)

1064 Immunoblot analysis of Fabp7, Hes1 and Hes5 in cortical extracts derived from WT and

1065 Bloc1s2-/- mice at E14.5 (Figure 3H-source data 1 and 2). (I) Quantification of Fabp7, Hes1

1066 and Hes5 relative to WT cortex. Data are presented as fold change normalized to the mean of

1067 WT. ***, P < 0.001; n = 9 mice.

1068

1069 Figure 4. Loss of BLOS2 drives HSPCs from quiescence to rapid proliferation and results

1070 in increased frequency and number of HSPCs and elevated Notch signaling.

1071 (A) Immunoblotting of BLOS2 in the E11.5 AGM region of Bloc1s2+/+ and Bloc1s2-/- embryos

1072 (Figure 4A-source data 1). The cells in the AGM region were immunoblotted with

1073 anti-BLOS2 and anti-β-actin. (B) qRT-PCR results showed that Bloc1s2 was enriched in

1074 CD31+CD41-CD45-TER119- endothelial cells. Each bar represents the mean ± s.e.m of three

1075 independent samples. *, P < 0.05; **, P < 0.01 (Student’s t-test). (C) The

1076 immunofluorescence of Runx1 and CD31 expression in the E11 AGM region of Bloc1s2+/+ and

1077 Bloc1s2-/- embryos. Quantification of CD31+ Runx1+ cell numbers in the DA (dorsal aorta). *,

1078 P < 0.05 (n = 3 mice). (D) The cell proportion of c-Kit+CD34+ HSPC from E11 AGM in

1079 Bloc1s2+/+ and Bloc1s2-/- embryos. *, P < 0.05 (n = 3 mice). (E) Each well plated with 600

1080 c-Kit+CD34+ HSPCs from E11 Bloc1s2+/+ and Bloc1s2-/- embryos was used for CFU-C assay.

1081 Seven days later, the amount of colonies was quantified. Each bar represents the mean ± s.e.m

1082 of three independent samples. CFU-MIX: 15.67 ± 2.51 for WT, 8.33 ± 0.58 for Bloc1s2-/- (*,

1083 P < 0.05); CFU-E: 4.33 ± 0.71 for WT, 4.67 ± 0.65 for Bloc1s2-/- (P = 0.423); CFU-GM:

1084 10.33 ± 1.63 for WT, 3.33 ± 0.545 for Bloc1s2-/- (*, P < 0.05). (F) Bloc1s2 deficient

1085 c-Kit+CD34+ HSPCs have increased proliferation, indicated as Ki67+ 7AAD+

1086 (7-amino-actinomycin D). (G) Immunoblotting of Notch signaling in the AGM region. The

1087 cells in the AGM region were immunoblotted with anti-Notch1, anti-NICD and anti-β-actin

1088 (Figure 4G-source data 1). The right panel is the quantitative analysis of the western blotting

1089 results. Each bar represents the mean ± s.e.m of three independent samples. *, P < 0.05, **, P

1090 < 0.01. (H) Fluorescence-activated cell sorting (FACS) analysis of Notch1 expression on

1091 CD31+CD41-CD45-TER119- endothelial cells in Bloc1s2+/+ or Bloc1s2-/- embryos. (I) qPCR

1092 analysis of hey1 and hey2 expression in CD31+CD41-CD45-TER119- endothelial cells from

1093 Bloc1s2+/+ or Bloc1s2-/- embryos. Each bar represents the mean ± s.e.m of three independent

1094 samples. *, P < 0.05, **, P < 0.01.

1095

1096 Figure 5. bloc1s2 deficiency promotes HSPC production in zebrafish.

1097 (A-C) The whole mount in situ hybridization (WISH) (A), qRT-PCR (B) and immunoblotting

1098 (C) results (Figure 5C-source data 1) showed that the expression of bloc1s2 at RNA and

1099 protein level in bloc1s2 mutants was decreased but not abolished compared to control

1100 embryos at 24 hpf. Each bar represents the mean ± s.e.m of three independent experiments. **,

1101 P <0.01, ***, P <0.001 (Student’s t-test). (D) The Runx1 protein level was increased in

1102 bloc1s2 mutants (Figure 5D-source data 1). The right panel is the quantitative analysis of the

1103 western blotting results. Each bar represents the mean ± s.e.m of three independent

1104 experiments. ***, P < 0.001. (E) The WISH results showed the expression of runx1 at 24 hpf

1105 and 36 hpf, and cmyb expression at 36 hpf were increased in bloc1s2 mutants. Black

1106 arrowheads mark expression of runx1 and cmyb in the AGM region. (F) qRT-PCR results

1107 from the dissected trunk region showed that expression of cmyb at 36 hpf was significantly

1108 increased in bloc1s2 mutants. Each bar represents the mean ± s.e.m of three independent

1109 experiments. *, P < 0.05. (G) We generated the bloc1s2 mutants in Tg

1110 (flk1:mCherry;cmyb:GFP) background. The upper panels showed that the number of

1111 flk1+cmyb+ cells in outcrossed embryos was increased. White arrowheads mark flk1+cmyb+

1112 cells in the AGM region at 36 hpf. The lower panel is the quantitative analysis of the western

1113 blotting results. Each bar represents the mean ± s.e.m of 7 embryos. **, P < 0.01. (H) rag1

1114 expression was increased in the thymus region, but gata1 and pu1 expression was decreased

1115 in the CHT region in bloc1s2 mutants at 4.5 dpf. Black dashed circles mark rag1 expression

1116 in thymus region. Black arrowheads mark expression of gata1 and pu1 in the CHT region.

1117

1118 Figure 6. Lysosomal degradation of Notch1 was impaired in mouse and zebrafish

1119 Bloc1s2-/- cells.

1120 (A) qRT-PCR analysis for Notch1 of the WT and Bloc1s2-/- dissected cortices at E14.5. No

1121 change in Notch1 mRNA levels between WT and Bloc1s2-/- cortices was observed. Data are

1122 presented as fold change normalized to the mean of WT. n.s., not significant, P = 0.94

1123 (Student’ s t-test; n = 9 mice). (B) WT and Bloc1s2-/- MEFs incubated with or without

1124 leupeptin (100 µM) or MG132 (10 µM) for 4 h at 37°C and immunoblotted for Notch1 (NTM)

1125 (Figure 6B-source data 1). (C and D) Quantification of Notch1 (NTM) relative to that in

1126 untreated WT MEFs. **, P < 0.01; ***, P < 0.001; n.s., not significant, P = 0.876 (n = 6). All

1127 graphs are mean ± s.e.m. (E) Control and zebrafish bloc1s2 mRNA overexpressed embryos

1128 were treated with 50 μM chloroquine from 18 hpf to 36 hpf. The Immunoblotting results

1129 (Figure 6E-source data 1) showed that the decreased Notch1 protein level in bloc1s2

1130 overexpressed embryos was recovered by chloroquine treatment, while not changed by

1131 MG132 treatment. (F) Quantitative analysis of the immunoblotting results. Each bar

1132 represents the mean ± s.e.m of three independent experiments. *, P < 0.05; ***, P < 0.001.

1133

1134 Figure 7. The endolysosomal trafficking of Notch1 was altered in Bloc1s2-/- MEFs.

1135 (A) OptiPrep gradient fractionation of WT and Bloc1s2-/- brains at E14.5 probed for Notch1

1136 (NTM), BLOS2, EEA1 (endosome marker), CD63 (MVBs and late endosomes marker),

1137 LAMP1 (lysosome marker), GM130 (Golgi marker) (Figure 7A-source data 1, 2 and 3).

1138 Notch (NTM) distribution exhibited a marked increase in the EEA1 and CD63-labeled

1139 fractions in Bloc1s2-/- brain tissues. (B) Quantification of the Notch1 (NTM) in (A) displayed as

1140 the proportion in each fraction relative to the total fractions. (C to E) WT and Bloc1s2-/- MEFs

1141 immunostained with antibodies against endogenous Notch1 (green) and EEA1/LBPA/LAMP1

1142 (red). It revealed increased endogenous Notch1 localization in EEA1 and LBPA-positive 52

1143 vesicles, and decreased Notch1 localization in LAMP1-labeled vesicles in Bloc1s2-/- MEFs.

1144 Scale bars: 5 μm. (F to H) Quantification of Mander’s overlap coefficient K1 representing

1145 co-localizations of Notch1 with EEA1 and LBPA and LAMP1, respectively. *, P < 0.05, ***,

1146 P < 0.001 (Student’s t-test; n = 100 cells). All graphs are mean ± s.e.m.

1147

1148 Figure 8. BLOS2 physically interacts with NICD1 and functions independent of BLOC-1

1149 and BORC.

1150 (A) Immunoblotting analysis of Co-IP performed in HEK293T cells. Flag-NICD1 co-eluted

1151 with Myc-BLOS2, but Flag-alone did not (Figure 8A-source data 1 and 2). (B) Endogenous

1152 co-immunoprecipitation of Notch1 with BLOS2 in AGM region (Figure 8B-source data 1). IP,

1153 immunoprecipitation. (C) Partial colocalization of BLOS2 and Notch1 in HeLa cells

1154 transfected with the Flag-BLOS2 and 6×Myc-Notch1 constructs. Scale bar: 5 μm. (D)

1155 Immunoblotting analysis of Notch1 (NTM) in WT, Pldnpa, Kxd1-KO, and Bloc1s1-KO MEFs

1156 (Figure 8D-source data 1). No changes in Notch1 (NTM) levels in Pldnpa, Kxd1-KO, and

1157 Bloc1s1-KO MEFs were observed. (E) Quantification of levels of Notch1 (NTM) relative to

1158 WT MEFs. Data are presented as fold change normalized to the mean of WT. P = 0.457

1159 (Pldnpa), 0.901 (Kxd1-KO), 0.178 (Bloc1s1-KO) (Student’s t-test; n = 9). (F) Immunoblotting

1160 analysis of Notch1 (NTM) in brain extracts derived from WT and Bloc1s1-/- mice at E10.5

1161 (Figure 8F-source data 1). No change in Notch1 (NTM) levels in the Bloc1s1-/- cortex lysates

1162 was observed. (G) Quantification of Notch1 (NTM) relative to WT cortex. Data are presented

1163 as fold change normalized to the mean of WT. P = 0.264 (Student’s t-test; n = 9).

1164

1165 Figure Supplements

1166

1167 Figure 1-figure supplement 1. Generation of Bloc1s2 knockout mice.

1168 (A) Schematic diagram of knockout strategy. Exons 1-4 of Bloc1s2 gene are replaced by

1169 PGK-Neo cassette. (B) Genomic PCR using primers shown in (A). The WT allele gives rise

1170 to a 645-bp product by primers A and B, while a 485-bp product amplified by primers A and

1171 C indicates the null allele. (C) Immunoblotting analysis of BLOS2 protein in multiple tissues

1172 isolated from WT and Bloc1s2-/- neonate mice (Figure 1-figure supplement 1C-source data 1).

1173 BLOS2 protein is not detectable in various tissues of the Bloc1s2-/- mice. β-actin is served as a

1174 loading control. In this figure, +/+, +/- and -/- indicate genotypes of WT, heterozygote and

1175 homozygote, respectively.

1176

1177 Figure 3-figure supplement 1. Unchanged Notch ligands in cortices and elevated Notch

1178 levels in MEFs.

1179 (A) qRT-PCR analysis for Dll1 of the WT and Bloc1s2-/- dissected cortices at E14.5. No

1180 change in Dll1 mRNA levels between WT and Bloc1s2-/- cortex was observed. Data are

1181 presented as fold change normalized to the mean of WT. P = 0.766, n.s., not significant

1182 (Student’s t-test; n = 9). (B) Immunoblotting analysis of Jag1 in cortical extracts derived from

1183 WT and Bloc1s2-/- mice at E14.5 (Figure 3-figure supplement 1B-source data 1). No change

1184 in Jag1 levels between WT and Bloc1s2-/- cortices was observed. Quantification of Jag1

1185 relative to WT cortex. Data are presented as fold change normalized to the mean of WT. P =

1186 0.986, n.s., not significant (n = 9). (C) qRT-PCR analysis for Itch and Dtx2 expression in WT

1187 and Bloc1s2-/- MEFs. No changes in Itch and Dtx2 levels in WT and Bloc1s2-/- MEFs were

1188 observed. Data are presented as fold change normalized to the mean of WT. P = 0.776 (Itch),

1189 P = 0.071 (Dtx2), n.s., not significant (n = 3 mice). (D-F) qRT-PCR analyses of Hes1, Hes5

1190 and Fabp7 in BLOS2-overexpressed MEFs. The expression of Hes1, Hes5 and Fabp7 was

1191 significantly decreased in full-length FLAG-BLOS2 overexpressed WT MEFs. Data are

1192 presented as fold change normalized to the mean of WT. *, P < 0.05; **, P < 0.01 (n = 9). (G)

1193 Immunoblot analysis of Notch1 (NTM), NICD1 and Hes5 in WT and Bloc1s2-/- MEFs (Figure

1194 3-figure supplement 1G-source data 1 and 2). The levels of Notch1 (NTM), NICD1 and Hes5

1195 were all significantly increased in Bloc1s2-/- MEFs. Increased levels of Notch1 (NTM) and

1196 Notch signaling in Bloc1s2-/- MEFs were mostly rescued by overexpression of full-length

1197 FLAG-BLOS2. (H) Quantification of levels of Notch1 (NTM), NICD1 and Hes5 relative to

1198 WT MEFs. Data are presented as fold change normalized to the mean of WT. ***, P < 0.001

1199 (n = 9).

1200

1201 Figure 3-figure supplement 2. Endosomal Notch1 levels are reduced in BLOS2

1202 overexpressed MEFs but increased in BLOS2-KO NPCs.

1203 (A) Immunoblotting analysis of Notch1 (NTM) in WT and overexpressed of full-length

1204 Flag-BLOS2 MEFs (Figure 3-figure supplement 2A-source data 1). It showed that the levels

1205 of Notch1 (NTM) were significantly decreased by overexpression of full-length Flag-BLOS2.

1206 (B) Quantification of Notch1 (NTM) relative to WT. Data are presented as fold changes

1207 normalized to the mean of WT ± s.e.m. *, P < 0.05 (Student’s t-test; n = 3 ). (C) WT + FLAG

1208 and WT + FLAG-BLOS2 MEFs immunostained with antibodies against endogenous Notch1

1209 (red) and LBPA (green). It revealed decreased endogenous Notch1 localization in

1210 LBPA-positive vesicles in overexpressed full-length FLAG-BLOS2 MEFs. Scale bar: 10 μm.

1211 (D) Quantification of Mander’s overlap coefficient K1 representing co-localizations of

1212 Notch1 with LBPA in (C). *, P < 0.05 (n = 100 cells). All graphs are mean ± s.e.m. (E, F)

1213 Immunostaining of NPCs derived from E14.5 WT and Bloc1s2-/- neurospheres with antibodies

1214 against endogenous Notch1 (red) and LBPA/LAMP1 (green). It revealed increased

1215 endogenous Notch1 localization in LBPA-positive vesicles, and decreased Notch1

1216 localization in LAMP1-labeled vesicles in Bloc1s2-/- NPCs. Scale bars : 50 μm. (G, H)

1217 Quantification of Mander’s overlap coefficient K1 representing co-localizations of Notch1

1218 with LBPA and LAMP1, respectively. *, P < 0.05, **, P < 0.01 (n = 100 cells). All graphs are

1219 mean ± s.e.m.

1220

1221 Figure 3-figure supplement 3. Rescue assays by the treatment of a γ-secretase inhibitor

1222 DAPT.

1223 (A) qRT-PCR analysis for Hes1 of WT and Bloc1s2-/- MEFs incubated with DAPT or DMSO

1224 (CTRL). Data are presented as fold change normalized to the mean of WT. ***, P < 0.001

1225 (Student’s t-test; n = 9). (B) Representative images of neurospheres derived from E14.5 NPCs

1226 (WT-Ctrl, KO-Ctrl and KO-DAPT) are shown. Quantification of neurosphere diameters (μm):

1227 58.49 ± 2.34 for WT-Ctrl, 110.15 ± 3.84 for KO-Ctrl, 60.56 ± 2.53 for KO-DAPT. ***, P <

1228 0.001 (n = 3 mice). (C) Immunostaining of neurospheres derived from WT-Ctrl, KO-Ctrl and

1229 KO-DAPT NPCs with antibodies against Nestin (NPC marker) or Tuj1 (neuron marker). (D

1230 and E) Quantification of the percentage (%) of Nestin-positive (proliferation) and

1231 Tuj1-positive (differentiation) cells in cultured neurospheres. 41.28 ± 1.31 (Nestin+), 33.66 ±

1232 2.85 (Tuj1+) for WT-CtrlL; 81.56 ± 2.49 (Nestin+), 11.68 ± 1.06 (Tuj1+) for KO-Ctrl; 36.98 ±

1233 1.15 (Nestin+), 27.09 ± 1.13 (Tuj1+) for KO-DAPT. ***, P < 0.001 (Student’s t-test; n = 3

1234 mice). All graphs are mean ± s.e.m. Scale bars: 100 μm in (B); 50 μm in (C).

1235

1236 Figure 4-figure supplement 1. Endosomal Notch1 levels are increased in BLOS2-KO

1237 AGM cells.

1238 (A, B) Immunostaining of HSPCs derived from E11.5 WT and BLOS2-KO AGM region with

1239 antibodies against endogenous Notch1 (red) and LBPA/LAMP1 (green). It revealed increased

1240 endogenous Notch1 localization in LBPA-positive vesicles, and decreased Notch1

1241 localization in LAMP1-labeled vesicles in Bloc1s2-/- cells. Scale bars: 50 μm. (C, D)

1242 Quantification of Mander’s overlap coefficient K1 representing co-localizations of Notch1

1243 with LBPA and LAMP1, respectively. **, P < 0.01, ***, P < 0.001 (Student’s t-test; n = 100

1244 cells). All graphs are mean ± s.e.m.

1245

1246 Figure 5-figure supplement 1. BLOS2 regulation of HSPC development in zebrafish is

1247 gene-specific.

1248 (A) WISH results showed expression of bloc1s2 at 24 hpf and 36 hpf. Black arrowheads mark

1249 expression of bloc1s2 in the AGM region and CHT region. (B) Double fluorescence in situ

1250 hybridization showed that the expression of bloc1s2 co-localized with endothelial cell marker

1251 fli1a. (C) Control and bloc1s2 mutant embryos were injected with bloc1s2 full-length mRNAs.

1252 WISH results showed that the increased expression of runx1 in bloc1s2 mutants at 36 hpf was

1253 rescued by the overexpression of bloc1s2. (D) qRT-PCR results from the dissected trunk

1254 region showed that the increased expression of runx1 and cmyb in bloc1s2 mutants at 24 hpf

1255 was rescued by the overexpression of bloc1s2. Each bar represents the mean ± s.e.m of three

1256 independent experiments. *, P < 0.05, **, P < 0.01, ***, P < 0.001 (Student’s t-test). (E) The

1257 left panel showed the BrdU assay in the frozen sections of control and bloc1s2 mutants at 36

1258 hpf. The white dashed lines label the outline of the dorsal aorta and cardinal vein. The right

1259 panel is the quantification of BrdU signals around the vessels per section. Each bar represents

1260 the mean ± s.e.m of 13 embryos. *, P < 0.05.

1261

1262 Figure 5-figure supplement 2. Genome editing of zebrafish bloc1s2 by Cas9 technology.

1263 (A) Schematic diagrams show zebrafish bloc1s2 gene, four potential transcripts from

1264 ENSEMBL, gRNA sequence and its target sites. Sanger sequencing of PCR amplicons from

1265 one fish line indicated that Cas9-mediated mutation at the target site resulted in a CCGT

1266 insertion. Homozygous mutants used in this study were all from this fish line and the CCGT

1267 insertion resulted in a reading frame shift and a truncated 8 aa peptide for three of bloc1s2

1268 transcripts (001, 002 and 003), but no alteration on the transcript-201. (B) A comparison of

1269 the encoded proteins of four bloc1s2 transcripts. Note that the transcript-201 differs from -001

1270 or -003 by missing N-terminal 10 aa. (C) The calculated molecular weights (MWs) of four

1271 bloc1s2 isoforms. Note that the encoded protein of transcript-201 is slightly smaller than the

1272 -001isoform. (D) BLOS2 was decreased in the AGM region of both bloc1s2+/- and bloc1s2-/-

1273 zebrafish (Figure 5-figure supplement 2D-source data 1). β-actin was a loading control. Note that

1274 the band shown in bloc1s2-/- mutant is likely the isoform-201, therefore designated as bloc1s2 hypo

1275 elsewhere.

1276

1277 Figure 5-figure supplement 3. Increased Notch activity mediates the HSPC phenotype in

1278 zebrafish or mouse bloc1s2 mutants.

1279 (A) WISH results showed that the expression of Notch targets hey2 and ephrinB2a was

1280 increased in the dorsal aorta of bloc1s2 mutants. (B) qRT-PCR results from the dissected

1281 trunk region showed that expression of hey2 and ephrinB2a was significantly increased in

1282 bloc1s2 mutants. Each bar represents the mean ± s.e.m of three independent experiments. **P

1283 < 0.01 (Student’s t-test). (C) Western Blot results showed that the protein level of Notch1 was

1284 increased in bloc1s2 mutants (Figure 5-figure supplement 3C-source data 1). The right panel

1285 is the quantitative analysis of the western blotting results. Each bar represents the mean ±

1286 s.e.m of three independent experiments. ***, P < 0.001. (D) We generated bloc1s2 mutants in

1287 Tg (fli1a:EGFP;tp1:dsRed) background. The left panels showed that the number of fli1a+tp1+

1288 cells in bloc1s2 mutant embryos was increased. White arrowheads mark the fli1a+tp1+ cells in

1289 the AGM region at 36 hpf. The right panel is the quantification. Each bar represents the mean

1290 ± s.e.m of 6 embryos. **, P < 0.01. (E) Control and bloc1s2 mutants were treated with 4 μM

1291 DMSO or DBZ from 18 hpf to 36 hpf. qRT-PCR results from the dissected trunk region

1292 showed that the increased expression of hey2, ephrinB2a, runx1 and cmyb in bloc1s2 mutants

1293 at 36 hpf was rescued by the DBZ treatment. Each bar represents the mean ± s.e.m of three

1294 independent experiments. *, P < 0.05, **, P < 0.01, ***, P < 0.001. (F) WISH results showed

1295 that the increased expression of runx1 and cmyb in bloc1s2 mutants at 36 hpf was rescued by

1296 the DBZ treatment. Black arrowheads mark expression of runx1 and cmyb in the AGM region.

1297 (G) DBZ inhibited expansion of c-Kit+CD34+ HSPCs from E11 AGM in Bloc1s2-/- mouse

1298 embryos.

1299

1300

1301 Figure 7-figure supplement 1. Subcellular localization of Notch1 in late endosomes and

1302 recycling endosomes.

1303 (A) Double labeling of Notch1 (green) and LBPA (red) in WT and Bloc1s2-/- MEFs imaged by

1304 structured illumination microscopy (SIM). Arrow points out the accumulation of Notch1

1305 receptor in LBPA-positive vesicles. Scale bar: 5 μm. (B) WT and Bloc1s2-/- MEFs that had

1306 internalized TfA568 for 30 min were immunostained with antibodies against endogenous

1307 Notch1. No apparent change in the localization of Notch1 in TfA568-positive vesicles was

1308 observed.

1309

1310 Figure 8-figure supplement 1. Destabilization of other BLOC-1 or BORC subunits in

1311 Bloc1s2-/- MEFs and schematic model of lysosomal degradation of Notch1 mediated by

1312 BLOS2.

1313 (A) Immunoblot analysis of dysbindin, BLOS1, and KXD1 in WT and Bloc1s2-/- MEFs

1314 (Figure 8-figure supplement 1A-source data 1 and 2). The levels of dysbindin and BLOS1

1315 were destabilized and KXD1 was significantly reduced in Bloc1s2-/- MEFs. (B) Quantification

1316 of levels of dysbindin, BLOS1, and KXD1 relative to WT MEFs. Data are presented as fold

1317 change normalized to the mean of WT. ***, P < 0.001 (Student’s t-test; n = 9). (C) A

1318 proposed model. In WT cells, BLOS2 is required for the lysosomal degradation of Notch1.

1319 The Notch1 receptor is endocytosed to early endosomes, where the receptor is either

1320 trafficked back to the membrane by recycling endosomes or it is directed to lysosomal

1321 degradation through MVBs and late endosomes. However, in the mutant cells, loss of BLOS2

1322 results in impaired endo-lysosomal trafficking of Notch1. Consequently, a large fraction of

1323 Notch1 accumulates in the MVBs and late endosomes, where active NICD1 (red) is produced.

1324 This enhanced activation of Notch signaling likely causes the defective embryonic

1325 corticogenesis in the mutant mice. PM, plasma membrane; EE, early endosome; MVB,

1326 multivesicular bodies.

1327

1328 Figure 8-figure supplement 2. Unaltered lysosomal functions and EGFR steady-state

1329 levels in Bloc1s2-/- MEFs or tissues.

1330 (A) Partial colocalization of BLOS2, Notch1 and LysoTracker in HeLa cells transfected with

1331 the Flag-BLOS2 and 6×Myc-Notch1 constructs. Scale bar: 10 μm. (B) Immunoblotting

1332 analysis of EGFR (Figure 8-figure supplement 2B-source data 1). No changes in EGFR levels

1333 in WT and Bloc1s2-/- MEFs were observed, while in control group, significantly increased

1334 EGFR were seen by leupeptin (100 µM) treatment. (C) Quantification of EGFR relative to

1335 that in untreated WT MEFs. *, P < 0.05 (H2O vs. Leupeptin); n.s., not significant, P = 0.933

1336 (WT vs. KO), P = 0.131 (WT vs. WT+Flag-BLOS2) (Student’s t-test; n = 3). All graphs are

1337 mean ± s.e.m. (D) Immunoblotting analysis of EGFR in cortical extracts from WT and

1338 Bloc1s2-/- mice at E14.5 (Figure 8-figure supplement 2D-source data 1). (E) Quantification of

1339 EGFR relative to WT cortex. Data are presented as fold change normalized to the mean of

1340 WT ± s.e.m. P = 0.235, n.s., not significant (n = 9). (F) Immunoblotting analysis of cathepsin

1341 D in WT and Bloc1s2-/- MEFs (Figure 8-figure supplement 2F-source data 1). There were no

1342 apparent changes of immature and mature forms of cathepsin D protein levels between WT

1343 and Bloc1s2-/- MEFs. (G) Ratio of mature versus immature cathepsin D in WT and Bloc1s2-/-

1344 MEFs by quantifying the related bands on the western blots in A. P = 0.766, n.s., not

1345 significant (n = 3).

1346

1347 Figure 8-figure supplement 3. Ultrastructures of endo-lysosomal organelles.

1348 (A) Representative EM pictures of WT and BLOS2-KO MEFs. Scale bars, 0.5 μm. (B)

1349 Representative enlarged pictures of endo-lysosomal organelles. The definition of these

1350 organelles is following the description in a previous report (Zhang et al., 2014). MVB:

1351 multivesicular body; LE: late endosome; EL: endolysosome; LYS: lysosome. (C)

1352 Quantitative data for the comparison of the number of the endo-lysosomal organelles per cell

1353 between WT and BLOS2-KO MEFs. n.s., not significant (MVB: P = 0.668; LE: P = 0.143;

1354 LYS: P = 0.780); **, P < 0.01 (Student’s t-test); n = 7 cells.

1355

1356 Supplementary Table 1. A comparison of phenotypes for BLOS2-KO, BLOS1-KO and

1357 Snapin-KO mouse embryos or MEFs.

1358

1359 Supplementary Table 2. The PCR primers used in quantitative PCR assay.

(Figure 1)

A B C D Bloc1s2+/+ Bloc1s2-/- Bloc1s2+/+ Bloc1s2-/- Bloc1s2+/+ Bloc1s2-/- Bloc1s2+/+ Bloc1s2-/-

E12.5 E14.5 P0 E14.5

E F G H E14.5 Bloc1s2+/+ E14.5 Bloc1s2-/- E14.5 Bloc1s2+/+ E14.5 Bloc1s2-/- 300 250 WT KO WT KO *** *** 200 *** 200 CP 150 100 n.s. 100 IZ 50 **

Relative volume (%) volume Relative *** VZ

0 (um) thickness Layer 0 LV LV / whole brain Neocortex CP IZ VZ

I Ctip2 Tbr1 +/+ -/- +/+ -/- J K

60 E14.5 30 E12.5 WT KO WT KO

E12.5 50 25 40 20 *** 30 Ctip2 Tbr1 15 *** *** 20 +/+ -/- +/+ -/- 10 *** 5 10 0

E14.5 0 Number of Cells/100 um Radial Unit

Number of Cells/100 um Radial Unit Ctip2 Tbr1 Ctip2 Tbr1 (Figure 2) A B C D TUNEL Nuclei 3 E13.5 E14.5

80 +/+ -/- 70 2.5 (%) +/+ -/- n.s. + 60 2 50 /BrdU 1.5 + 40 ** Ki67

/ svz

cells/field 30 BrdU + ‐ E14.5 1 20 BrdU vz 0.5 Ki67 10 TUNEL 0 0 Bloc1s2+/+ Bloc1s2-/- Bloc1s2+/+ Bloc1s2-/- E F G H 140 Tuj1 Pax6 250 120 *** Cells/

+/+ -/- +/+ -/- + 200 100 80 150 *** 60 100 E14.5 40 E14.5

50 Unit um Radial 100 20 Number of Pax6 Number 0 0 +/+ -/- Bloc1s2+/+ Bloc1s2-/- Bloc1s2 Bloc1s2 Thickness of Tuj1 layer (um) layer Tuj1 of Thickness PH3 +/+ -/- I J BrdU labeling index K L E13.5 80 ** 30 WT KO +/+ -/-

70 E13.5 25 *** (%) (%) VZ + 60 VZ 50 20 /Ki67 +

Ki67 +/+ -/-

/ 40 15

Ki67 30

+ 10 Cells/100 um VZ Cells/100 *** BrdU

20 + 5 BrdU 10 E14.5 0 VZ PH3 0 Bloc1s2+/+ Bloc1s2-/- VZ E13.5 E14.5

(Figure 4) (Figure 5) (Figure 6) 2 Notch1 A B

1.5 WT MEF KO MEF WT MEF KO MEF n.s. CTRL Leu CTRL Leu CTRL MG CTRL MG 1 Notch1 (NTM) 0.5

β‐actin Relative Gene Expression Gene Relative 0 Bloc1s2+/+ Bloc1s2-/-

C D 4 n.s. Ctrl ** 3.5 Leupeptin 6 *** Ctrl MG132 3 ** 5 ** ) level )

2.5 level ) ** TM 4 2 TM 3 1.5 1 2 Notch1 (N Notch1

0.5 (N Notch1 1 0 0 WT MEF KO MEF WT MEF KO MEF EF 5% 20% A B Fraction 1 2 3 4 5 6 7 8 9 10 11 )

(Figure 7) ‐ WT Notch1 (NTM) TM 0.3 ‐ KO Notch1 (NTM) WT KO 0.2 ‐ WT EEA1 0.1 ‐ WT CD63

‐ WT LAMP1 0 Percent of total Notch1 (N 1234567891011 ‐ WT GM130

C Notch1 EEA1 Merge D Notch1 LBPA Merge

WT WT Notch1 EEA1 Merge Notch1 LBPA Merge

Bloc1s2 KO Bloc1s2 KO E Notch1 LAMP1 Merge

F G H Notch1 and Notch1 and Notch1 and LBPA LAMP1 1 EEA1 1.5 WT * 1.00 Notch1 Merge LAMP1 1 *** *** 0.5 0.50 0.5 coefficient K1 coefficient coefficient K1 coefficient coefficient K1 coefficient Mander’s overlap Mander’s overlap

0 0 Mander’s overlap 0.00 Bloc1s2 KO Bloc1s2+/+ Bloc1s2-/- Bloc1s2+/+ Bloc1s2-/- Bloc1s2+/+ Bloc1s2-/- (Figure 8) A 3xFlag-alone + - 3xFlag-NICD1 - + Myc-BLOS2 + + B Myc-BLOS2 IP: Flag BLOS2 Flag-NICD1

Myc-BLOS2 Notch1 Input Flag-NICD1

Flag-BLOS2 6xMyc-Notch1 Merge HeLa

D E 1.5 F G 1.5 E10.5 brain MEF WT Bloc1s1-/-

C57BL/6J Pldn pa Kxd1-/- Bloc1s1-/- 1 Notch1 1 TM WT) to ) (relative

) (relative to WT) to ) (relative (N ) TM

Notch1 TM (NTM) BLOS1 0.5 0.5 β-actin

β-actin

0 0 Notch1 Normalized (N WT Bloc1s1 KO Normalized Notch1 (N C57BL/6J Pldnpa Kxd1-/- Bloc1s1-/-