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Irx3 and Irx5 in Ins2-Cre+ cells regulate hypothalamic postnatal neurogenesis and leptin response

Nature Metabolism volume 3, pages 701–713 (2021)Cite this article

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Abstract

Obesity is mainly due to excessive food intake. IRX3 and IRX5 have been suggested as determinants of obesity in connection with the intronic variants of FTO, but how these genes contribute to obesity via changes in food intake remains unclear. Here, we show that mice doubly heterozygous for Irx3 and Irx5 mutations exhibit lower food intake with enhanced hypothalamic leptin response. By lineage tracing and single-cell RNA sequencing using the Ins2-Cre system, we identify a previously unreported radial glia-like neural stem cell population with high Irx3 and Irx5 expression in early postnatal hypothalamus and demonstrate that reduced dosage of Irx3 and Irx5 promotes neurogenesis in postnatal hypothalamus leading to elevated numbers of leptin-sensing arcuate neurons. Furthermore, we find that mice with deletion of Irx3 in these cells also exhibit a similar food intake and hypothalamic phenotype. Our results illustrate that Irx3 and Irx5 play a regulatory role in hypothalamic postnatal neurogenesis and leptin response.

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Fig. 1: Irx3/5dHet mice exhibit reduced food intake and enhanced leptin response.
Fig. 2: Irx3/5dHet mice show elevated numbers of orexigenic and anorexigenic neurons in the ARC–ME.
Fig. 3: Irx3/5dHet mice exhibit elevated postnatal neurogenesis in the ARC–ME of the hypothalamus.
Fig. 4: Reduced expression of Irx3 and Irx5 increases the number of postnatally generated Ins2-Cre descendant leptin-sensing ARC neurons.
Fig. 5: scRNA-seq using Ins2-Cre system reveals Irx3- and Irx5-expressing radial glia-like NSCs in the postnatal ARC–ME.
Fig. 6: Deletion of Irx3 in the Ins2-Cre+ lineage results in improved leptin response and enhanced postnatal hypothalamic neurogenesis.

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Data availability

Drop-seq data have been deposited in the GEO under accession code GSE142243, and 10x scRNA-seq data have been deposited in the GEO under accession code GSE154969. Public datasets GSE93374 (ref. 31), GSE68177 (ref. 32), GSE100320 (ref. 42) and GSE132730 (ref. 43) were used in this study.

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Acknowledgements

We thank T. Paton, B. Thiruv, J. Zhang, B. Innes and G. Bader for assisting with the computational analysis of scRNA-seq data. The research was supported by Canadian Institutes of Health Research (MOP-136821) and the Canada Research Chairs program to C.-C.H. J.E.S. was supported by a postdoctoral fellowship award from Diabetes Canada.

Author information

Author notes

  1. Kyoung-Han Kim

    Present address: University of Ottawa Heart Institute and Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada

  2. These authors contributed equally: Joe Eun Son, Zhengchao Dou.

Authors and Affiliations

  1. Program in Developmental & Stem Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada

    Joe Eun Son, Zhengchao Dou, Kyoung-Han Kim, Siyi Wanggou, Vincent Su Bin Cha, Rong Mo, Xiaoyun Zhang, Xinyu Chen, Xi Huang & Chi-chung Hui

  2. Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada

    Zhengchao Dou, Xinyu Chen, Xi Huang & Chi-chung Hui

  3. Arthur and Sonia Labatt Brain Tumour Research Centre, The Hospital for Sick Children, Toronto, Ontario, Canada

    Siyi Wanggou & Xi Huang

  4. Department of Neurosurgery, Xiangya Hospital, Central South University, Changsha, China

    Siyi Wanggou & Xuejun Li

  5. Princess Margaret Genomics Centre, University Health Network, Toronto, Ontario, Canada

    Troy Ketela

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Contributions

J.E.S., X.L., X.H. and C.-C.H. conceived and designed the study. J.E.S., Z.D., K.-H.K., V.S.B.C. and X.C. performed the mouse experiments. J.E.S., Z.D., R.M. and X.Z. carried out the RNA and protein analyses. J.E.S., Z.D., S.W., and T.K. performed the scRNA-seq analysis. J.E.S. and C.-C.H. wrote the manuscript with input from all authors.

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Correspondence to Chi-chung Hui.

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The authors declare no competing interests.

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Peer review information Nature Metabolism thanks Seth Blackshaw and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Isabella Samuelson.

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Extended data

Extended Data Fig. 1 General phenotypes of Irx3/5dHet mice.

a-b, RNA expression analysis of (a) Irx3 and (b) Irx5 in hypothalamus, PWAT and BAT (male mice; n = 6 WT, n = 6 Irx3/5dHet). c, Representative photograph of WT vs. Irx3KO, WT vs. Irx5KO, and WT vs. Irx3/5dHet male mice at weaning (21 days of age). d, Body length (nose-to-anus distance) of WT and Irx3/5dHet mice at 14 weeks of age (male mice; n = 8/11). e, Body weight changes of WT and Irx3/5dHet female mice. The inset bar-graph shows body weight of 11 weeks old mice (n = 9/9). Data are represented as means ± SEM values. *P < 0.05; **P < 0.01 by two-tailed unpaired t test. ns: not significant.

Extended Data Fig. 2 Metabolic phenotypes of Irx3/5dHet mice.

a, Frequency distribution and b, average of adipocyte diameter in PWAT of WT and Irx3/5dHet mice (male mice; n = 6/10). c, Linear regression analysis of energy expenditure as a function of lean mass. d-e, qPCR analysis of Ucp1 mRNA expression in (d) IWAT and (e) BAT (male mice; n = 7 WT, n = 6 Irx3/5dHet). f, Representative images and quantification of the number of p-STAT3+ cells in the VMH and DMH following leptin injection (male mice; n = 3 WT-Leptin, n = 4 Irx3/5dHet-Leptin). Scale bar, 100 μm. Data are represented as means ± SEM values. *P < 0.05; **P < 0.01 by two-tailed unpaired t test. ns: not significant. g, Visualization of fluorescence-conjugated leptin (Leptin650) in the ventricular hypothalamus of wild-type and Irx3/5dHet mice. Leptin650 was found only in the blood vessels at 2 minutes post-leptin injection. Leptin650 diffused into the parenchyma of the ventricular hypothalamus at 10 minutes post-leptin injection (Top). Leptin650 uptake by tanycytes. Dashed lines indicate the ventricular tanycytic layer (Bottom). Scale bar, 100 μm. h, Representative Images of ZO-1 immunostaining in the ventricular tanycytic layer of WT and Irx3/5dHet mice. Scale bar, 50 μm.

Extended Data Fig. 3 Cellular characterization of ARC-ME from wild-type and Irx3/5dHet mice using single cell RNA sequencing (scRNA-Seq) analysis.

a, Schematic of the micro-dissected ARC-ME preparation for scRNA-Seq analysis. b, Heatmap of top marker genes for each cell cluster and annotation. c, UMAP plot of sequenced ARC-ME cells (2208 WT cells, 1407 Irx3/5dHet cells) and annotated according to known cell type markers. d, Percentage of orexigenic (Agrp+/Npy+) and anorexigenic (Pomc+/Cartpt+) neurons out of 2208 WT cells or 1407 Irx3/5dHet cells as revealed by scRNA-Seq. The number of Agrp+/Npy+ neurons is 124 for WT and 186 for Irx3/5dHet. For Pomc+/Cartpt+ neurons, the numbers are 397 and 400 for WT and Irx3/5dHet, respectively. Data represented as data-points ± 95% confidence intervals. *P < 0.05; **P < 0.01 determined using the chi-square test of independence with Bonferroni-adjusted P values. e, Gene expression analysis in micro-dissected ARC-ME (male mice; n = 14 WT, n = 16 Irx3/5dHet). Data are represented as means ± SEM values. The asterisks indicate a significant difference (*P < 0.05; **P < 0.01). P value was determined by two-tailed unpaired t test.

Extended Data Fig. 4 BrdU labelling during adult period.

a, Representative images and b, Quantification of the number of BrdU-labelled cells in the ARC (n = 5 WT, n = 4 Irx3/5dHet). 8 weeks old male mice received BrdU from for 14 days through drinking water (1 mg/mL). Data are represented as means ± SEM values. ns: not significant. P value was determined by two-tailed unpaired t test. Scale bars, 100 μm.

Extended Data Fig. 5 Identification of Ins2-Cre+ cells.

a, Schematic of the generation of Ins2-Cre;Rosa26mT/mG mice. b, Representative mT/mG images of various tissues from Ins2-Cre;Rosa26mT/mG male mice at adult stage. c, Representative fluorescence RNA in situ hybridization images of tdTomato with Npy or Cartpt in the ARC from adult Ins2-Cre;Ai14 tdTomato male mice. Arrowheads denote co-labelled cells. Scale bar, 100 μm.

Extended Data Fig. 6 scRNA-Seq analysis of Ins2-Cre;Ai14 tdTomato+ ARC-ME cells at P10.

a, Heatmap of top marker genes for each cell cluster and annotation. b, Violin plots showing orexigenic (Agrp+/Npy+) neurons and anorexigenic (Pomc+/Cartpt+) neurons in the neuronal cluster. c, Feature plots of Htra1, Mfge8, Fgf10, and Gfap. The color key indicates expression levels (red, high; gray, low). d, Proportional distribution of cell types. e, Percentage of orexigenic (Agrp+/Npy+) and anorexigenic (Pomc+/Cartpt+) neurons out of 3479 control cells or 3939 Irx3/5dHet cells as revealed by scRNA-Seq. The number of Agrp+/Npy+ neurons is 1286 in control and 2081 in Irx3/5dHet. There are 1142 and 1494 Pomc+/Cartpt+ neurons in control and Irx3/5dHet, respectively. f, Proportional distribution of radial glia-like NSCs (427-control; 430-Irx3/5dHet) identified by reference-based integration with Sox2-eGFP SVZ dataset. Data represented as datapoints ± 95% confidence intervals. *P < 0.05; **P < 0.01 determined using the chi-square test of independence with Bonferroni-adjusted P values. ns: not significant.

Extended Data Fig. 7 Analysis of the developing whole hypothalamus scRNA-Seq dataset (GSE132730).

a, UMAP plots of radial glia/progenitors, tanycytes, and immature neurons from whole hypothalamus at different developmental timepoints. b, Differential gene expression analysis shows that embryonic and postnatal radial glia/progenitors from whole hypothalamus have distinct gene expression profiles. Radial glia/progenitors from P10 hypothalamus and Ins2-Cre;Ai14 tdTom+ RG-like NSCs display highly related gene expression profiles.

Extended Data Fig. 8 Pseudotime differential trajectory analysis of RG-like NSCs, tanycytes and neurons from the Ins2-Cre;Ai14 tdTomato+ scRNA-Seq dataset.

a, Integrated diffusion map of RG-like NSCs, tanycytes and neurons from the Ins2-Cre;Ai14 tdTomato dataset reveals a single linear differentiation trajectory (Left). A line with arrow indicates pseudotime trajectory inferred by the Slingshot method. Cells are color-coded for defined pseudotime score. Expression profiles of genes for stemness (Sox2), enriched in radial glia-like NSCs (Hes5) and neurons (Tubb3) in pseudo-trajectory (Right). Cells are color-coded according to expression level. Termination with higher Sox2/Hes5 expression was chosen as the root. b, Pseudotime trajectory analysis performed using minimum-spanning tree (MST) shows a similar trajectory to the Slingshot method. c, Integrated diffusion map of RG-like NSCs, tanycytes and orexigenic (Agrp+/Npy+)/anorexigenic (Pomc+/Cartpt+) neurons from postnatal Ins2-Cre;Ai14 tdTomato+ scRNA-seq dataset reveals a single linear differentiation trajectory (Left). A line with arrow indicates pseudotime trajectory defined by Slingshot. Cells are color-coded for defined pseudotime score. Expression profiles of genes for stemness (Sox2), enriched in radial glia-like NSCs (Hes5) and orexigenic/anorexigenic neurons (Agrp and Npy for orexigenic neurons; Pomc and Cartpt for anorexigenic neurons) in pseudo-trajectory (Right). Cells are color-coded according to expression level. Termination with higher Sox2 expression was chosen as the root.

Extended Data Fig. 9 General phenotypes of Ins2-Cre;Irx3KO mice.

a, Breeding scheme for the generation of Ins2-Cre;Irx3Fl/Fl (Ins2-Cre;Irx3KO) mice. b, qPCR analysis of Irx3 and Irx5 mRNA expression in micro-dissected ARC-ME (male mice; n = 12 Cont, n = 15 Ins2-Cre;Irx3KO). c, Additional images of the fluorescence RNA in situ hybridization analysis of Irx3 expression in the ventral hypothalamus of control (n = 3) and Ins2-Cre;Irx3KO (n = 3). Arrowheads denote positive signals along the walls of the third ventricle in the control, which are absent in Ins2-Cre;Irx3KO mice. d, Body length (nose-to-anus distance) of control and Ins2-Cre;Irx3KO mice at 14 weeks of age (male mice; n = 4 Cont, n = 7 Ins2-Cre;Irx3KO). e, Linear regression analysis of energy expenditure as a function of lean mass. f, Gene expression analysis in micro-dissected ARC-ME (male mice; n = 12/15). Data are represented as means ± SEM values. The asterisks indicate a significant difference (*P < 0.05; **P < 0.01, ns: not significant). P values were determined by two-tailed unpaired t test. g, Leptin650 injection study. Leptin650 was found only in the blood vessels at 2 minutes post-leptin injection. Leptin650 diffused into the parenchyma of the ventricular hypothalamus at 10 minutes post-leptin injection (Top). Leptin650 uptake by tanycytes (Bottom). Dashed lines indicate the ventricular tanycytic layer. Scale bar, 100 μm.

Extended Data Fig. 10 Pancreatic islet phenotypes of Ins2-Cre;Irx3KO mice.

a, Representative images of pancreatic islets. b, Quantification of the islet diameter, and c, Coverage of α-cells (Glu+) or β-cells (Ins+) per islet (male mice; n = 6 Cont, n = 8 Ins2-Cre;Irx3KO). d, Plasma insulin level at postprandial and fasting conditions (male mice; n = 11/13). Pancreatic phenotypes were assessed in 3 weeks old mice. Data are represented as means ± SEM values. The asterisks indicate a significant difference (ns: not significant). P values were determined by two-tailed unpaired t test. Scale bar, 100 μm.

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Reporting Summary

Supplementary Table 1

Sequences of oligonucleotides.

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Son, J.E., Dou, Z., Kim, KH. et al. Irx3 and Irx5 in Ins2-Cre+ cells regulate hypothalamic postnatal neurogenesis and leptin response. Nat Metab 3, 701–713 (2021). https://doi.org/10.1038/s42255-021-00382-y

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