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miR-128 regulation of jag1b and csnk1a1 in the embryonic EHT control long-term blood lineages. a,b, Quantification of erythroid (a) and lymphoid (b) progenitors by WISH against gata1a (n = 37 (WT), 37 (csnk1a1 g3′UTR), 37 (jag1b g3′UTR), 31 (WT), 30 (hsp:csnk1a1) and 28 (hsp:jag1b) embryos) and ikaros (n = 32 (WT), 34 (csnk1a1 g3′UTR), 32 (jag1b g3′UTR), 31 (WT), 31 (hsp:csnk1a1) and 30 (hsp:jag1b) embryos), respectively, at 4.5 dpf CHT or thymus. WT and hsp:csnk1a1 and hsp:jag1b were heat shocked at 24 hpf. csnk1a1 de-repression or transient gain-of-function regulate erythroid progenitor formation, while jag1b manipulations regulate lymphoid progenitors (three independent experiments; ordinary one-way ANOVA with Tukey’s multiple comparison). c, Quantification of blood cell population identified by flow cytometry of ~2-month-old WKM dissected from samples treated as in h and i. Erythrocyte fraction is increased in csnk1a1 g3′UTR and hsp:csnk1a1, while lymphoid cells fraction is increased in jag1b g3′UTR and hsp:jag1b after heat shock at 24 hpf (n = 9 (WT), 11 (csnk1a1 g3′UTR), 9 (jag1b g3′UTR), 7 (WT), 9 (hsp:csnk1a1) and 7 (hsp:jag1b) zebrafish; two-way ANOVA with multiple comparisons). d, Proposed model of nHSPC heterogeneity regulated by miR-128 regulation on Wnt and Notch signalling in vascular endothelia cells before EHT. Vascular signalling, like Wnt (purple) and Notch (yellow), limits the production of a heterogeneous pool of nHSPCs in the AGM. e, Diminishment of Wnt (light grey), like after de-repression or gain of function of csnk1a1 in the EHT, results in an increase of replicative (S), and erythroid-biased nHSPCs in the AGM and relative erythroid progenitors and mature lineages. Diminishment of Notch (light grey), like after de-repression or gain of function of jag1b in the EHT, increases G2/M nHSPCs and lymphoid-biased nHSPCs in the AGM and relative lymphoid progenitors and mature cells. All quantifications are represented with mean ± s.e.m. NS, not significant; P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001. T, thymus.
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