Fig. 5: Nlrc3 regulates HSPC production via activating Notch signaling.

a, b Confocal imaging showing Tg (tp1:EGFP; kdrl:mCherry) embryos at 30 hpf in the floor of the DA (white arrowheads) in control embryos, nlrc3 morphants, and nlrc3 morphants with overexpression of nlrc3 mRNA with quantification (b). *P = 0.0394, 0.0221, n = 8, 7, 7 embryos. c, d Confocal imaging showing the number of CD41⁺ cells at 72 hpf in the CHT (white arrowheads) of Tg (CD41:GFP) embryos in the control embryos, treatment group with Notch inhibitor DAPT, and treatment group with Notch inhibitor DAPT with overexpression of nlrc3 mRNA with quantification (d). ns = 0.6905, ****P < 0.0001, n = 17, 12, 11 embryos. e Expression of nlrc3, runx1, and NOTCH family genes in the control embryos, treatment group with the Notch inhibitor DAPT, and treatment group with the Notch inhibitor DAPT with overexpression of nlrc3 mRNA at 30 hpf by qPCR. ns > 0.9999, = 0.2217, 0.4466, *P = 0.0149, 0.0498, **P = 0.0047, 0.0049, ***P = 0.0004, 0.0002, ****P < 0.0001. f hsp70:Gal4; UAS:NICD-myc embryos injected with control MO and nlrc3 MO, WISH for runx1 was performed at 28 hpf. g, h Confocal imaging showing the number of runx1⁺kdrl⁺ cells in Tg (runx1:EGFP/kdrl:mCherry) embryos at 32 hpf in the AGM (white arrowheads) in control embryos, nlrc3 morphants, and nlrc3 morphants with overexpression of hey1 mRNA with quantification (h). **P = 0.0021, ***P = 0.0003, n = 11, 10, 10 embryos. i, j Confocal imaging showing the number of hematopoietic cells in Tg (CD41:GFP) embryos at 72 hpf in the CHT (white arrowheads) in control embryos, nlrc3 morphants, and nlrc3 morphants with overexpression of hey1 mRNA with quantification (j). *P = 0.0380, ****P < 0.0001, n = 18, 17, 16 embryos. k Expression of the HSPC marker runx1 in control embryos, nlrc3 mutants, treatment group with Notch inhibitor DAPT and treatment group with Notch inhibitor DAPT with overexpression of hey1 mRNA at 30 hpf by WISH. Error bars, mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, the exact p-values mentioned above are listed from left to right, by using one-way ANOVA with –Sidak test in (b, d, h, j), two-way ANOVA with Tukey’s post hoc test in (e), n = 3 biological replicates in (e). For the box plots in (b, d, 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 IQR from each box hinge. Scale bars, 100 μm in (a, c, f, g, i, k). Source data are provided as a Source Data file.