Fig. 2: Nlrc3 signaling is indispensable for HSPC production in zebrafish.

a, b Confocal imaging showing the number of hemogenic endothelium and emerging HSPCs in runx1⁺kdrl⁺ cells from Tg (runx1:EGFP/kdrl:mCherry) embryos at 28 hpf in the AGM (white arrowheads) in control embryos and nlrc3 morphants with quantification (b). ****P < 0.0001, n = 11, 10 embryos. c, d Confocal imaging showing the number of cmyb⁺kdrl⁺ cells in Tg (cmyb:EGFP/kdrl:mCherry) embryos at 48 hpf in the AGM (white arrowheads) in control embryos and nlrc3 morphants with quantification (d). ****P < 0.0001, n = 10, 9 embryos. e, f Confocal imaging showing the number of HSPCs in Tg (CD41:GFP) embryos at 72 hpf in the CHT (white arrowheads) in control embryos and nlrc3 morphants with quantification (f). ****P < 0.0001, n = 9, 13 embryos. g, h Expression of the HSPC marker runx1 in nlrc3 morphants and mutants in the AGM region at 28 hpf by whole mount in situ hybridization (WISH) (black arrowheads) with quantification (h) ****P < 0.0001, n = 18, 32, 21 embryos. i, j Expression of the HSPC marker cmyb in nlrc3 morphants and mutants in the region of AGM and CHT at 36 hpf by WISH (black arrowheads) with quantification (j) ****P < 0.0001, n = 25, 37, 18 embryos. k Expression of nlrc3 and the HSPC genes runx1, cmyb in control embryos and nlrc3 morphants at 28 hpf by qPCR. *P = 0.0462, ***P = 0.0006, 0.0010, n = 3 biological replicates. Error bars, mean ± s.d., ****P < 0.0001, by using two-tailed, unpaired Student’s t-test in (b, d, f, k), one-way ANOVA – Sidak test in (h, j). For the box plots in (b, d, f, h, j), box limits extend from the 25th to 75th percentile, while the middle line represents the median. Whiskers extend to the largest value no further than 1.5 times the inter-quartile range (IQR) from each box hinge. Scale bars, 100 μm in (a, c, e, g, i). Illustrations created with BioRender.com. Source data are provided as a Source Data file.