Nlrc3 signaling is indispensable for hematopoietic stem cell emergence via Notch signaling in vertebrates

Abstract

Hematopoietic stem and progenitor cells generate all the lineages of blood cells throughout the lifespan of vertebrates. The emergence of hematopoietic stem and progenitor cells is finely tuned by a variety of signaling pathways. Previous studies have revealed the roles of pattern-recognition receptors such as Toll-like receptors and RIG-I-like receptors in hematopoiesis. In this study, we find that Nlrc3, a nucleotide-binding domain leucine-rich repeat containing family gene, is highly expressed in hematopoietic differentiation stages in vivo and vitro and is required in hematopoiesis in zebrafish. Mechanistically, nlrc3 activates the Notch pathway and the downstream gene of Notch hey1. Furthermore, NF-kB signaling acts upstream of nlrc3 to enhance its transcriptional activity. Finally, we find that Nlrc3 signaling is conserved in the regulation of murine embryonic hematopoiesis. Taken together, our findings uncover an indispensable role of Nlrc3 signaling in hematopoietic stem and progenitor cell emergence and provide insights into inflammation-related hematopoietic ontogeny and the in vitro expansion of hematopoietic stem and progenitor cells.

Fig. 1. NLRC3 is highly expressed during vertebrates HSPC development.

a Schematic paradigm of zebrafish caudal hematopoietic tissue (CHT) tissue for single-cell RNA-seq (scRNA-seq) profiling. Bubble plot of scRNA-Seq data demonstrating the expression of nucleotide-binding domain leucine-rich repeat (NLR) family members, including Nlrc3 and related inflammatory genes in the clusters of endothelial cells (ECs) and Hematopoietic stem and progenitor cells (HSPCs). b Schematic paradigm of mouse embryonic Aorta Gonad Mesonephros (AGM) for scRNA-seq profiling. Bubble plot of scRNA-Seq data demonstrating the expression of NLR family members, including Nlrc3 and related inflammatory genes in the clusters of venous endothelial cells (VECs), arterial endothelial cells (AECs), pre-hemogenic endothelial cells (pre-HECs), hemogenic endothelial cells (HECs), HSPCs, fetal liver (FL)-derived HSPCs. c Schematic paradigm of human embryonic AGM for scRNA-seq profiling. Bubble plot of scRNA-Seq data demonstrating the expression of NLR family members, including NLRC3 and inflammatory genes in the clusters of venous endothelium (VE), arterial endothelium (AE), hemogenic endothelium (HE), HSPCs and Non-HSCs. Illustrations created with BioRender.com. Source data are provided as a Source Data file.

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.

Fig. 3. Nlrc3 signaling is imperative for HSPC differentiation in zebrafish.

a Expression of the lymphoid marker rag1 in nlrc3 morphants and mutants and the thymic epithelial cell marker foxn1 in the thymus of control embryos, nlrc3 morphants, and mutants at 96 hpf (black arrowheads). b Snapshot of lck⁺ cells in Tg (lck:EGFP) embryos in the thymus of control embryos and nlrc3 morphants at 5 dpf (white arrowheads). c Expression of nlrc3 and the lymphoid genes ikaros, rag1, lck, and il7r in control embryos and nlrc3 morphants at 96 hpf by qPCR. *P = 0.0229, 0.0197, ***P = 0.0009. d Expression of the erythroid marker gata1a in control embryos and nlrc3 morphants by WISH at 96 hpf (black arrowheads). e Expression of nlrc3 and the erythroid gene gata1a in control embryos and nlrc3 morphants at 96 hpf by qPCR. **P = 0.0011. f Expression of the myeloid marker mpx in control and nlrc3 morphants by WISH at 120 hpf (black arrowheads). g Expression of nlrc3 and the myeloid gene ikaros, l-plastin, pu.1, lyz, and csf1a in control embryos and nlrc3 mutants at 96 hpf by qPCR. *P = 0.0178, 0.0197, **P = 0.0049, 0.0011, ***P = 0.0007, 0.0003. h Expression of nlrc3 and the HSPC genes runx1 and cmyb in control, nlrc3 morphants, and nlrc3 morphants with overexpression of nlrc3 mRNA at 30 hpf by qPCR. *P = 0.0286, 0.0272, 0.0158, **P = 0.0040, 0.0047, 0.0068. i Expression of the HSPC marker runx1 in control embryos, nlrc3 morphants, and nlrc3 morphants with overexpression of nlrc3 mRNA at 48 hpf by WISH (black arrowheads). j Expression of the lymphoid marker rag1 in control embryos, nlrc3 morphants, and nlrc3 morphants with overexpression of nlrc3 mRNA at 96 hpf by WISH (black arrowheads). 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 two-tailed, unpaired Student’s t-test in (c, e, g). Two-way ANOVA with Tukey’s post hoc test in (h). n = 3 biological replicates. Scale bars, 100 μm in (a, b, d, f, i, j). Illustrations created with BioRender.com. Source data are provided as a Source Data file.

Fig. 4. RNA-seq demonstrates the regulatory mechanism underlying nlrc3 in HSPC emergence.

a Schematic representation of RNA-seq analysis in this study. b Principal-component analysis (PCA) of six sequenced samples shown by the first three principal components (PC1–PC2). Control MO 1, blue; Control MO 2, light blue; Control MO 3, violet; nlrc3 MO 1, yellow; nlrc3 MO 2, green; nlrc3 MO 3, red; c Heatmap showing differential expression of hematologic-associated genes. The mark * indicates that there is a significant difference between the control group and morphant group with fold change >2 or fold change <0.5 and p-value < 0.05. (4) FDR < 0.05. d Representative statistically enriched biological process (BP), molecular function (MF) and cellular component (CC) associated genes of the upregulated and downregulated genes in nlrc3 morphant group compared with the control group. e Representative KEGG enrichment scatter plots of the signaling pathways in the nlrc3 morphant group compared with the control group. The plots were drawn based on the R version 4.1.3 and ggplot version 3.3.3. The statistical tests were one-sided and adjustments were made for multiple comparisons. f Expression of Notch genes in control embryos and nlrc3 morphants at 26 hpf by RNA-seq. Error bars, mean ± s.d., ns = 0.0554, 0.0645, 0.1207, *P = 0.0224, **P = 0.0065, 0.0029, 0.0066, ***P = 0.0006, 0.0004, ****P < 0.0001, the exact p-values mentioned above are listed from left to right, by using two-tailed, unpaired Student’s t-test. n = 3 biological replicates. g Enrichment plot of the hematopoietic cell lineages between differentially regulated genes in the nlrc3 morphant group compared with the control group by GSEA (Gene Set Enrichment Analysis). P = 0.0465. h Enrichment plot of the Notch signaling pathway between differentially regulated genes in the nlrc3 morphant group compared with the control group by GSEA. P = 0.0003. GSEA of nlrc3 morphant versus control group transcriptional profiles using software GSEA (v4.1.0) and MSigDB, the statistical tests were one-sided and adjustments were made for multiple comparisons. Black bars indicate the individual genes, enrichment is in green. Normalized enrichment score = NES in (g, h). Source data are provided as a Source Data file.

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.

Fig. 6. Nlrc3 is downstream of the NF-kB signaling pathway for regulating HSPC emergence.

a Expression of NF-kB genes in control embryos and nlrc3 morphants at 26 hpf by RNA-seq. ns = 0.2176, 0.4498, 0.6039,0.1870, *P = 0.0224, 0.0311, **P = 0.0022, 0.0018, 0.0083, ***P = 0.0002, ****P < 0.0001. b Expression of nlrc3, runx1, and the NF-kB target gene il1b in control embryos, nlrc3 mutants, nlrc3 mutants with ikbaa Mo, and treatment group with ikbaa Mo at 30 hpf by qPCR. **P = 0.0041, 0.0053, ***P = 0.0001, ****P < 0.0001. c, d Expression of the HSPC marker runx1 in control embryos, ikbaa morphants, nlrc3 mutants, and nlrc3 mutants with ikbaa morphants at 28 hpf by WISH (black arrowheads) with quantification (d) ns = 0.3163, 0.9187, ****P < 0.0001, n = 12, 12, 14, 18 embryos. e, f Expression of the HSPC marker cmyb in control embryos, nlrc3 mutants, nlrc3 mutants with overexpression of nlrc3 mRNA, treatment group with NF-kB inhibitor JSH-23, and treatment group with NF-kB inhibitor JSH-23 and overexpression of nlrc3 mRNA at 48 hpf by WISH (black arrowheads) with quantification (f) ****P < 0.0001, n = 12, 21, 20, 11, 13 embryos. g, h 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 NF-kB inhibitor JSH-23, and treatment group with NF-kB inhibitor JSH-23 and overexpression of nlrc3 mRNA with quantification (h). *P = 0.0387, ****P < 0.0001, n = 17, 19, 16 embryos. i Expression of HSPC genes and NF-kB genes in control embryos, treatment group with NF-kB inhibitor JSH-23, and treatment group with NF-kB inhibitor JSH-23 with overexpression of nlrc3 mRNA at 30 hpf by qPCR. ns = 0.8444, 0.9785, *P = 0.0179, ***P = 0.0005, ****P < 0.0001. 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 two-tailed, unpaired Student’s t-test in (a), one-way ANOVA with –Sidak test in (d, f, h), two-way ANOVA with Tukey’s post hoc test in (b, i), n = 3 biological replicates in (a, b, i). For the box plots in (d, f, h), 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 (c, e, g). Source data are provided as a Source Data file.

Fig. 7. Nlrc3 signaling is conserved in the regulation of vertebrate hematopoiesis.

a Schematic representation of AGM-derived hematopoietic endothelial (HE) cells. b Representative view of the hematopoietic cells (round) generated from 500 HE cells after co-culture with OP9 stromal cells for 4 days. c, d Flow cytometry results showing that after 4 days of OP9 co-culture, the number of c-Kit⁺CD45⁺ cells in Nlrc3 embryos decreased with quantification (d). ***P = 0.0001, n = 7, 6 embryos. e Schematic representation of fetal liver-derived cells performed in this study and flow cytometry results showing that the number of LT-HSC (Lin⁻ c-Kit⁺ Sca-1⁺ CD150⁺ CD48⁻) cells in Nlrc3 embryos decreased. f Percentage of LT-HSCs in 14.5 embryonic fetal liver of control embryos and Nlrc3 embryos. ***P = 0.0006, n = 5 embryos. g Quantification of LSK in 14.5 embryonic fetal liver of control embryos and Nlrc3 embryos. ****P < 0.0001, n = 6 embryos. h CFU-C assay of fetal liver showing that the number of CFU-mix, CFU-granulocyte-macrophage (CFU-GM), and CFU-erythroid (CFU-E) was decreased upon knock-down of Nlrc3. *P = 0.0114, 0.0132, ***P = 0.0007, n = 3 biological replicates. i Schematic representation of bone marrow transplantation experiments performed in this study. j A total of 5 × 10⁵ fetal liver-derived cells sorted from WT or Nlrc3^−/− mice were transplanted with 5 × 10⁵ rescue cells into recipient mice. Peripheral blood (PB) analysis for the percentage of engrafted donor cells at the indicated number of weeks post-transplantation. *P = 0.0473, **P = 0.0038, n = 4 biological replicates. k At 16 weeks post-transplantation, the PB of recipient mice transplanted with control or Nlrc3^−/− was analyzed for the percentage of donor-derived B, T, and myeloid cells. **P = 0.0075, n = 3 biological replicates. l Flow cytometry results show the proportion of donor with control or Nlrc3^−/− in the BM of recipient mice at 16 weeks post-transplantation n = 5 biological replicates. m Quantification of the proportion of the donor-derived cell in BM. **P = 0.0055, n = 4 biological replicates. n Expression of Nlrc3, Runx1, Il1b, and Notch-related genes in WT, Nlrc3^−/− whole FL at E14.5 by qPCR. ns = 0.8168, *P = 0.0137, 0.0205, 0.0107, **P = 0.0078, 0.0039, 0.0063, ****P < 0.0001. Error bars, mean ± s.d., the exact p-values mentioned above are listed from left to right by using two-tailed, unpaired Student’s t-test in (d, f, g, j, k, m, n). Illustrations created with BioRender.com. Source data are provided as a Source Data file.