{"id":448656,"date":"2024-01-02T19:00:00","date_gmt":"2024-01-03T00:00:00","guid":{"rendered":"https:\/\/platohealth.ai\/nlrc3-signaling-is-indispensable-for-hematopoietic-stem-cell-emergence-via-notch-signaling-in-vertebrates-nature-communications\/"},"modified":"2024-01-03T22:58:01","modified_gmt":"2024-01-04T03:58:01","slug":"nlrc3-signaling-is-indispensable-for-hematopoietic-stem-cell-emergence-via-notch-signaling-in-vertebrates-nature-communications","status":"publish","type":"post","link":"https:\/\/platohealth.ai\/nlrc3-signaling-is-indispensable-for-hematopoietic-stem-cell-emergence-via-notch-signaling-in-vertebrates-nature-communications\/","title":{"rendered":"Nlrc3 signaling is indispensable for hematopoietic stem cell emergence via Notch signaling in vertebrates – Nature Communications","gt_translate_keys":[{"key":"rendered","format":"text"}]},"content":{"rendered":"
Previous studies in vertebrate embryos uncovered the role of inflammatory signaling in HSPC emergence. To explore the potential pathways involved in hematopoietic ontogeny and differentiation in this stage, we screened the dynamic expression of NLR family genes and other genes related to inflammation in the published scRNA-seq data in vertebrates\u2019 embryos during HSPC development. In a data profiling caudal hematopoietic tissue (CHT) of zebrafish at 3.5 and 4.5 days postfertilization (dpf)27<\/a><\/sup>, Compared to many other NLR family paralogs, including nlrp1<\/i>, nlrp3<\/i>, nlrc3l<\/i>, nlrc5<\/i>, nlrc6<\/i>, nlrx1<\/i> and nlrp16<\/i>, the expression of nlrc3<\/i> is relatively higher in the ECs and HSPCs subpopulations (Fig. 1a<\/a>). To further investigate the expression of nlrc3<\/i> in mammalian models, we analyzed published single-cell transcriptomic data profiling mouse embryo from 9.5 days post coitus (dpc) to 11.5 dpc28<\/a><\/sup>. Comparison of Aorta Gonad Mesonephros (AGM)\u2013derived HSPCs and fetal liver (FL)-derived HSPCs with venous endothelial cells (VECs), arterial endothelial cells (AECs), pre-hemogenic endothelial cells (pre-HECs) and hemogenic endothelial cells (HECs), the expression of Nlrc3<\/i> increases with hematopoietic maturation, especially when comparing HECs with HSPCs, which is the stage that hemogenic endothelial to hematopoietic transition (EHT) occurs (Fig. 1b<\/a>). And the expression of Nlrc3<\/i> is not only highly expressed among the NLR family genes but also like that of Tlr4<\/i>, which has been reported to be involved in the regulation of HSPC development1<\/a><\/sup>. Furthermore, in the latest single-cell transcriptome map of human hematopoietic tissues from the first trimester29<\/a><\/sup>, the expression trend of NLRC3<\/i> was also validated to be enriched in venous endothelium (VE), arterial endothelium (AE), hemogenic endothelium (HE), HSCs and HPCs which are defined as Non-HSCs (Fig. 1c<\/a>), especially the differentiation stage from HE to HSCs.<\/p>\n a<\/b> 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<\/i> and related inflammatory genes in the clusters of endothelial cells (ECs) and Hematopoietic stem and progenitor cells (HSPCs). b<\/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<\/i> 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<\/b> 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<\/i> 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.<\/p>\n<\/div>\n<\/div>\n Although rather in the primitive stage, the hematopoietic differentiation system using embryonic stem cells (ESCs) is a convenient research model that simulates and observes the in vivo embryonic hematopoiesis, in which cells gradually differentiate into the mesoderm, HE, and HSPCs stages, and are accompanied by specific different molecular markers at different stages31<\/a>,32<\/a><\/sup>. To further verify the expression of Nlrc3<\/i> in vitro, we compared FL- and bone marrow (BM)-derived HSPCs with HSC-like cells derived from mouse ESCs (Supplementary Fig. 1a<\/a>)30<\/a><\/sup>. Differential gene expression analysis using RNA-seq revealed that among the NLR family genes, Nlrc3<\/i> expression was highly enriched with the expression of genes that have been reported to regulate the emergence of HSCs, including Tlr4<\/i>1<\/a><\/sup> and IFN-\u03b3<\/i>16<\/a><\/sup> (Supplementary Fig. 1b<\/a>).<\/p>\n We further validated our results by employing the in vitro HSC-like cell differentiation system using human embryonic stem cells (hESCs) (Supplementary Fig. 1c<\/a>). We examined the dynamic change of NLRC3<\/i> during the 12-day hematopoietic differentiation process by qPCR and found that compared with day 0, the expression of NLRC3<\/i> gradually increased along with the hematopoietic differentiation from hESCs (Supplementary Fig. 1d<\/a>). Moreover, we isolated cells from different stages (hESCs at Day 0, CD309+<\/sup> mesoderm cells at Day 3, CD31+<\/sup>CD34+<\/sup> HE cells at Day 6, CD43+<\/sup> HSPCs cells at Day 9, and CD45+<\/sup> HSPCs at Day 12). Our qPCR results showed that, compared with undifferentiated hESCs, the expression of NLRC3<\/i> in HE was significantly increased by approximately 300-fold and was even higher in CD43+<\/sup> and CD45+<\/sup> HSPCs (Supplementary Fig. 1e<\/a>). TLR4<\/i> and NLPR3<\/i>, which have been reported to be regulators of HSPCs emergence and differentiation, showed a similar trend to NLRC3<\/i> (Supplementary Fig. 1f, g<\/a>). Overall, these results suggest that NLRC3<\/i> was highly expressed during embryonic HSPC development in vivo and vitro and might play an important role in HSPC emergence in vertebrates.<\/p>\n To investigate whether nlrc3<\/i> signaling is required for in vivo HSPC emergence, we synthesized probes from the full-length mRNA of nlrc3<\/i> and utilized them to observe the in-situ expression of nlrc3<\/i> in zebrafish at different developmental stages. The WISH experiment demonstrated that nlrc3<\/i> was expressed from the 1-cell stage and, importantly, showed specific expression in the AGM region of zebrafish at 24\u201328hpf, which coincides with the onset of HSPC generation. There was also a high expression of nlrc3<\/i> at 72hpf in the CHT region, which follows a similar spatiotemporal expression pattern as HSPC generation, indicating that nlrc3<\/i> may be involved in the development of HSPCs (Supplementary Fig. 2a<\/a>). By co-staining with arterial endothelial-specific markers dlc<\/i> and efnb2<\/i> (Supplementary Fig. 2b, c<\/a>), hematopoietic stem\/progenitor cell-specific markers runx1<\/i> and cmyb<\/i> (Supplementary Fig. 2d, e<\/a>), we found that co-staining nlrc3<\/i> with these probes can effectively enhance the expression of HE and HSPCs. These results suggest that nlrc3<\/i> may be involved in the regulation of this process in zebrafish.<\/p>\n In order to validate this hypothesis, loss-of-function experiments were performed using zebrafish with a targeted Morpholino (MO) (Supplementary Fig. 3a<\/a>). We observed the floor of the DA region in Tg (runx1<\/i>:EGFP\/ kdrl<\/i>:mCherry) double-transgenic embryos at 28 hpf by confocal microscopy, runx1<\/i> is a conserved HSPC marker and kdrl<\/i> is a vascular endothelial-specific marker, the runx1<\/i>+<\/sup>kdrl<\/i>+<\/sup> cells signifies HSPCs occurs through the hemogenic endothelial (HE) to hematopoietic transition (EHT) process is this time point, and the number of runx1<\/i>+<\/sup>kdrl<\/i>+<\/sup> HSPCs of morphants was significantly lower than that in control embryos (Fig. 2a, b<\/a>). By using the Tg(cmyb<\/i>:EGFP\/kdrl<\/i>:mCherry) double-transgenic line at 48 hpf, cmyb<\/i> is another conserved HSPC marker, we found that the number of cmyb<\/i>+<\/sup>kdrl<\/i>+<\/sup> HSPCs of morphants within the ventral wall of the dorsal aorta (VDA) markedly decreased in nlrc3<\/i> morphants (Fig. 2c, d<\/a>). Tg (CD41<\/i>: GFP), which is a well-established transgenic line with HSPC expansion in the CHT, also had reduced expression in morphants at 72 hpf (Fig. 2e, f<\/a>). Furthermore, we performed whole-mount in situ hybridization (WISH) to measure the expression of runx1<\/i> and cmyb<\/i>, which are nascent HSPC markers at early developmental stages, and found that the expression of runx1<\/i> and cmyb<\/i> was significantly reduced at 28 hpf and at 36 hpf, respectively, in the aortic floor of nlrc3<\/i> morphants compared with their wild-type siblings (Fig. 2g, j<\/a>). To further demonstrate the role of nlrc3<\/i> in HSPC emergence, we generated nlrc3<\/i> mutant by the CRISPR\/Cas9, 29\u2009bp base is knocked out in homozygotes (Supplementary Fig. 2b, c<\/a>). The expression of nlrc3<\/i> was abrogated in mutants at the site of HSPC occurrence at 24\u201336 hpf (Supplementary Fig. 3d, e<\/a>). Consistent with previous results, the expression of runx1<\/i> and cmyb<\/i> was decreased in mutants by WISH at 28 hpf and 36 hpf, respectively (Fig. 2g\u2013j<\/a>). With this mutant, we also validated the downregulation of hematopoietic markers and nlrc3<\/i> at 28 hpf by qPCR (Fig. 2k<\/a>). The similar phenotype between morphants and mutants allows for the experimental design to be tailored based on specific needs. To obtain more comprehensive evidence, we examined gata2b<\/i>, a critical early hematopoietic marker12<\/a><\/sup>. Through qPCR, we found a downregulation of gata2b<\/i> in the mutant at 28 hpf. Therefore, we synthesized the full-length mRNA of gata2b<\/i> and overexpressed it. The reduction in runx1<\/i> at 28hpf and cmyb<\/i> at 36 hpf due to nlrc3<\/i> deficiency was partially restored by the rescue of gata2b<\/i> (Supplementary Fig. 3f, g<\/a>). This result was also confirmed by qPCR analysis (Supplementary Fig. 3h<\/a>). These results indicated that nlrc3<\/i> was required for HSPC emergence and expansion.<\/p>\n a<\/b>, b<\/b> Confocal imaging showing the number of hemogenic endothelium and emerging HSPCs in runx1<\/i>+<\/sup>kdrl<\/i>+<\/sup> cells from Tg (runx1<\/i>:EGFP\/kdrl<\/i>:mCherry) embryos at 28 hpf in the AGM (white arrowheads) in control embryos and nlrc3<\/i> morphants with quantification (b<\/b>). ****P<\/i>\u2009<\u20090.0001, n<\/i>\u2009=\u200911, 10 embryos. c<\/b>, d<\/b> Confocal imaging showing the number of cmyb<\/i>+<\/sup>kdrl<\/i>+<\/sup> cells in Tg (cmyb<\/i>:EGFP\/kdrl<\/i>:mCherry) embryos at 48 hpf in the AGM (white arrowheads) in control embryos and nlrc3<\/i> morphants with quantification (d<\/b>). ****P<\/i>\u2009<\u20090.0001, n<\/i>\u2009=\u200910, 9 embryos. e<\/b>, f<\/b> Confocal imaging showing the number of HSPCs in Tg (CD41<\/i>:GFP) embryos at 72 hpf in the CHT (white arrowheads) in control embryos and nlrc3<\/i> morphants with quantification (f<\/b>). ****P<\/i>\u2009<\u20090.0001, n<\/i>\u2009=\u20099, 13 embryos. g<\/b>, h<\/b> Expression of the HSPC marker runx1<\/i> in nlrc3<\/i> morphants and mutants in the AGM region at 28 hpf by whole mount in situ hybridization (WISH) (black arrowheads) with quantification (h<\/b>) ****P<\/i>\u2009<\u20090.0001, n<\/i>\u2009=\u200918, 32, 21 embryos. i<\/b>, j<\/b> Expression of the HSPC marker cmyb<\/i> in nlrc3<\/i> morphants and mutants in the region of AGM and CHT at 36 hpf by WISH (black arrowheads) with quantification (j<\/b>) ****P<\/i>\u2009<\u20090.0001, n<\/i>\u2009=\u200925, 37, 18 embryos. k<\/b> Expression of nlrc3<\/i> and the HSPC genes runx1<\/i>, cmyb<\/i> in control embryos and nlrc3<\/i> morphants at 28 hpf by qPCR. *P<\/i>\u2009=\u20090.0462, ***P<\/i>\u2009=\u20090.0006, 0.0010, n<\/i>\u2009=\u20093 biological replicates. Error bars, mean \u00b1 s.d., ****P<\/i>\u2009<\u20090.0001, by using two-tailed, unpaired Student\u2019s t<\/i>-test in (b<\/b>, d<\/b>, f<\/b>, k<\/b>), one-way ANOVA \u2013 Sidak test in (h<\/b>, j<\/b>). For the box plots in (b<\/b>, d<\/b>, f<\/b>, h<\/b>, j<\/b>), 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 \u03bcm in (a<\/b>, c<\/b>, e<\/b>, g<\/b>, i<\/b>). Illustrations created with BioRender.com. Source data are provided as a Source Data file.<\/p>\n<\/div>\n<\/div>\n To further evaluate the effect of nlrc3<\/i> on hematopoietic differentiation, we examined the expression of rag1<\/i>, which specifically indicates the differentiation capability of HSPCs for T cell lineages. WISH results showed that rag1<\/i> expression was markedly reduced in both morphants and mutants (Fig. 3a<\/a>). A similar phenotype was observed in Tg (lck<\/i>:EGFP) transgenic morphants at 120 hpf (Fig. 3b<\/a>), while lymphoid genes, including ikaros<\/i>, rag1<\/i>, lck<\/i>, and il7r<\/i>, were downregulated in mutants, as assessed by qPCR (Fig. 3c<\/a>). However, the expression of the thymic epithelial cell marker foxn1<\/i> was normal (Fig. 3a<\/a>), suggesting that T-cell defects in these deficient embryos resulted from early HSPC defects. The expression of gata1a<\/i>, an erythrocyte-specific marker, was decreased in the posterior blood island (PBI) and trunk regions of morphants at 36 hpf and 96 hpf, respectively (Supplementary Fig. 4a<\/a>, Fig. 3d<\/a>), which was further verified by qPCR analysis in mutants (Fig. 3e<\/a>). We also examined the phenotypes of Tg (lcr<\/i>:EGFP) and Tg (gata1a<\/i>:DsRed) transgenic morphants at 60 hpf (Supplementary Fig. 4b, c<\/a>). These two transgenic lines are capable of tracking erythrocytes in blood vessels, and the results strongly suggest that nlrc3<\/i> signaling is essential for definitive erythropoiesis. Myeloid lineage differentiation was also impaired in mutants, as assayed by mpx<\/i> at 96 hpf and l-plastin<\/i> at 120 hpf by WISH (Fig. 3f<\/a>, Supplementary Fig. 4f<\/a>). Similar phenotypes were assessed in Tg (mpx<\/i>:EGFP) and Tg (lyz<\/i>:DsRed) transgenic morphants as the neutrophil marker at 60 hpf and 72 hpf, respectively, in the CHT region (Supplementary Fig. 4d, e<\/a>), and the expression of myeloid-lineage genes in mutants at 96 hpf was found to be decreased by qPCR (Fig. 3g<\/a>). These results indicated that HSPC differentiation was impaired when nlrc3<\/i> signaling was disturbed.<\/p>\n<\/a><\/div>\n
\n Nlrc3<\/i> signaling is indispensable for HSPC production in zebrafish<\/h3>\n
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