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Evolution of a novel adrenal cell type that promotes parental care

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Abstract

Cell types with specialized functions fundamentally regulate animal behaviour, and yet the genetic mechanisms that underlie the emergence of novel cell types and their consequences for behaviour are not well understood1. Here we show that the monogamous oldfield mouse (Peromyscus polionotus) has recently evolved a novel cell type in the adrenal gland that expresses the enzyme AKR1C18, which converts progesterone into 20α-hydroxyprogesterone. We then demonstrate that 20α-hydroxyprogesterone is more abundant in oldfield mice, where it induces monogamous-typical parental behaviours, than in the closely related promiscuous deer mice (Peromyscus maniculatus). Using quantitative trait locus mapping in a cross between these species, we ultimately find interspecific genetic variation that drives expression of the nuclear protein GADD45A and the glycoprotein tenascin N, which contribute to the emergence and function of this cell type in oldfield mice. Our results provide an example by which the recent evolution of a new cell type in a gland outside the brain contributes to the evolution of social behaviour.

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Fig. 1: Oldfield mice have larger adrenal glands that recently evolved the ability to produce more 20α-OHP than deer mice.
Fig. 2: 20α-OHP and its metabolite allo-diol increase parenting behaviours.
Fig. 3: Molecular and cellular characterization of adrenal glands reveals the zona inaudita in oldfield mice.
Fig. 4: Quantitative genetic analysis of zona inaudita identifies prominent roles for Gadd45a and Tnn.

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Data availability

Sequence data are available at NCBI Sequence Read Archive under BioProject ID PRJNA1094591. Source data are provided with this paper.

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Acknowledgements

This work was supported by the following grants: Searle Scholarship, Klingenstein-Simons Fellowship in Neuroscience, Sloan Foundation Fellowship, and National Institutes of Health (NIH) R00HD084732, R21HD106241 and R35GM143051 to A.B.; the Simons Society of Fellows Junior Fellowship 855220 to J.R.M.; Canadian Institutes of Health Research Project Grant 426405 to K.K.S.; Tishman Fellowship to S.L.; and SFARI Bridge to Independence award and Rose F. Kennedy Intellectual and Developmental Disabilities Research Center pilot grant to S.R. Imaging was performed with support from the Zuckerman Institute’s Cellular Imaging platform, which received grant S10OD023587-01 from the NIH. Columbia University’s Shared Research Computing Facility, where computations were performed, was supported by NIH grant G20RR030893-01 and NYSTAR contract C090171. L. Hammond and H. Ibarra provided training and advice on microscope imaging, C. Zhang shared reagents, L. Remedio shared reagents and advice on tissue clearing, H. Pan and M. Hiller helped run TOGA to find orthologues, and H. Hoesktra provided beach mice and cloudland mice adrenals. M. Tosches, R. Axel, S. Siegelbaum and D. Bambah-Mukku and his students provided comments on the manuscript. C. Everett created the diagram in Fig. 4f. Schematics in Fig. 2a,i and Extended Data Fig. 3 were created using BioRender.com.

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  1. These authors contributed equally: Natalie Niepoth, Jennifer R. Merritt

Authors and Affiliations

  1. Zuckerman Mind Brain Behavior Institute, Columbia University, New York, NY, USA

    Natalie Niepoth, Jennifer R. Merritt, Michelle Uminski, Emily Lei, Victoria S. Esquibies, Ina B. Bando, Kimberly Hernandez, Christoph Gebhardt & Andres Bendesky

  2. Department of Ecology, Evolution and Environmental Biology, Columbia University, New York, NY, USA

    Natalie Niepoth, Jennifer R. Merritt, Michelle Uminski, Emily Lei, Victoria S. Esquibies, Ina B. Bando, Kimberly Hernandez, Christoph Gebhardt & Andres Bendesky

  3. Department of Chemistry and Biochemistry, Manhattan College, New York, NY, USA

    Sarah A. Wacker

  4. Department of Neuroscience, Albert Einstein College of Medicine, New York, NY, USA

    Stefano Lutzu & Stephanie Rudolph

  5. Department of Psychiatry and Behavioral Sciences, Albert Einstein College of Medicine, New York, NY, USA

    Stefano Lutzu & Stephanie Rudolph

  6. Department of Psychology, University of British Columbia, Vancouver, British Columbia, Canada

    Asmita Poudel & Kiran K. Soma

  7. Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, British Columbia, Canada

    Asmita Poudel & Kiran K. Soma

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  1. Natalie Niepoth
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Contributions

N.N. and A.B. conceived and designed the study. N.N. conducted all transcriptomic and genetic experiments and analyses, with contribution from M.U. and J.R.M. J.R.M. performed behaviour assays with contributions from V.S.E., I.B.B., K.H., M.U. and N.N. N.N. and M.U. performed adrenal histology. N.N. and E.L. characterized adrenal mass and volume. K.H. developed the pair-bonding assay. C.G. and J.R.M. built behaviour testing apparatus. S.A.W. performed AKR1C18 biochemistry experiments. K.K.S. and A.P. developed LC–MS/MS steroid quantification methods and quantified steroid levels. S.R. and S.L. conducted electrophysiology experiments and analysis. N.N., J.R.M. and A.B. wrote the paper with input from all authors.

Corresponding author

Correspondence to Andres Bendesky.

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Extended data figures and tables

Extended Data Fig. 1 Anatomical, molecular, and biochemical characterization of deer- and oldfield adrenal glands.

a, Adrenal weight at postnatal day 0.5. Lines at median. b, Adult adrenal weight by age. P-values by generalized linear model. Band is 95% confidence interval. c, Adrenal medulla volume. Lines at median. P-values by generalized linear model. d, Representative adrenal section from the zona fasciculata (zF) of the deer- and oldfield mouse adrenal cortex. Cyp11b1 (green) labeled by in situ hybridization and counterstained with DAPI (red). Five independent biological replicates per species yielded similar results. Scale bar, 20 µm. e, Scatterplot of gene expression by species and sex. f, Boxplots of the expression of steroidogenic enzymes in the corticosterone- and 20α-OHP synthesis pathways in the adrenal. Boxplot hinges are 25th and 75th quartiles, whiskers are 1.5× interquartile range, line at median. g, Total steroid levels in the adrenal gland of deer and oldfield mice. Lines at median. P-values by generalized linear model. DOC, deoxycorticosterone. h, Expression of Akr1c18 in the ovaries and testes of oldfield and deer mice. Lines at median. P-values by two-sided t-test. i, Akr1c18 (red) labeled by in situ hybridization in the ovary of a deer mouse and counterstained with DAPI (blue). Three independent biological replicates per species yielded similar results. Scale bar, 0.5 mm. j, Circulating 20α-OHP levels in the blood of oldfield mice after adrenalectomy. Lines at median. P-values by generalized linear model.

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Extended Data Fig. 2 Effects of 20α-OHP on alloparental and parental care.

Male and female care for pups as measured by proportion of time spent huddling pups, proportion of time spent grooming pups, fraction of pups retrieved to the nest, and nest quality score (from 0 to 4) in a, unmated deer mice, b, unmated oldfield mice, and c, deer mouse fathers. Boxplot hinges are 25th and 75th quartiles, whiskers are 1.5× interquartile range, line at median. P-values by generalized linear model for proportion of time huddling, proportion of time grooming the pup, proportion of pups retrieved, and nest quality. P-values for proportion of animals that retrieved at least one pup by Fisher’s exact test. d, Proportion of pup attacks by species, treatment, and reproductive experience.

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Extended Data Fig. 3 Partner preference is not affected by 20α-OHP.

Top: Schematic of the experimental design of the partner preference test. Bottom: Observations from the same breeding pairs are connected by lines (10 min partner preference test). P-values by generalized linear model (effect of trial type).

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Extended Data Fig. 4 20α-OHP is converted to allo-diol in the brain of deer- and oldfield mice.

a, Concentration of allo-diol, allopregnanolone and progesterone in deer- and oldfield mouse cerebellum and hypothalamus after incubation with 20α-OHP, as measured by LC-MS/MS. P-values by two-sided t-test.

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Extended Data Fig. 5 Effects of allo-diol on alloparental and parental care, and on δGABAAR.

Male and female care for pups as measured by proportion of time spent huddling pups, proportion of time spent grooming pups, fraction of pups retrieved to the nest, and nest quality score in a, unmated deer mice, b, unmated oldfield mice, and c, oldfield parents. Boxplot hinges are 25th and 75th quartiles, whiskers are 1.5× interquartile range, line at median. P-values by generalized linear model for proportion of time huddling, proportion of time grooming the pup, proportion of pups retrieved, and nest quality. P-values for proportion of animals that retrieved at least one pup by Fisher’s exact test. d, Proportion of pup attacks by species, treatment, and reproductive experience. e, Baseline tonic GABA receptor currents with leak current subtracted, after gabazine application. f, Input resistance (Ri) under different pharmacological conditions. No change in Ri in the presence of allo-diol and 20α-OHP suggests no effect on GABA receptor currents. A decrease in Ri in the presence of THIP is consistent with an increase in GABA receptor currents. This effect is diminished when THIP is co-applied with allo-diol but not with 20α-OHP. Ri increases in gabazine when all GABA receptors are blocked. P-values by two-sided one-sample t-test (μ = 0) and two-sample two-sided t-tests. Bars denote the mean ± SEM.

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Extended Data Fig. 6 UMAP visualization of the house-, deer-, and oldfield mouse adrenal.

a, UMAP of integrated analysis of house mouse, deer mouse, and oldfield mouse adrenal nuclei. b, UMAP showing Akr1c18 expression in adrenal cells, marking the X zone in house mice and the zona inaudita in oldfield mice. c, UMAP of integrated analysis of deer mouse and oldfield mouse nuclei, then split by sex. d, Volcano plot of differential adrenal gene expression between deer- and oldfield mouse (purple: higher in deer mouse, green: higher in oldfield mouse, grey: n.s.). Akr1c18 and extracellular matrix gene markers of the zona inaudita (from Fig. 3g) are highlighted. False Discovery Rate=0.05. A medulla, adrenergic medulla; NA medulla, noradrenergic medulla.

Extended Data Fig. 7 Expression of top marker genes of the adrenal zones of deer mice and oldfield mice.

Violin plots denoting the top two markers of each adrenal cell type.

Extended Data Fig. 8 Dot plots of marker genes of the zona inaudita of oldfield mice and the X zone of house mice.

Expression of transcription factors (TFs), extracellular matrix (ECM) genes, and other genes upregulated in the zona inaudita or X zone. Note that in adults, the X zone is only present in unmated females.

Extended Data Fig. 9 Cis-regulation of Gadd45a contributes to transcription factor module expression.

a, Expression of Akr1c18 in the adrenal of female and male deer × oldfield F2 hybrids. b, Dot plot of Gadd45a, Tnn, and Akr1c18 expression in adrenal cortex cell types. c, Gadd45a expression by genotype at the Gadd45a locus in F2-hybrid males. Boxplot hinges are 25th and 75th quartiles, whiskers are 1.5× interquartile range, line at median. P-value by ANOVA. Logarithm of the odds (LOD) across the genome of d, Hif1a and e, Runx2 expression. f, LOD across the genome for the TF module and the TF module excluding Gadd45a. g, LOD of the TF module and the TF module controlling for Gadd45a expression. Dashed lines denote genome-wide threshold of significance (α = 0.05).

Extended Data Fig. 10 Cis-regulation of Tnn contributes to extracellular matrix module expression.

a, Expression of Tnn in deer and oldfield mice. P-value by two-sided t-test. b, Allele-specific expression of Tnn in deer × oldfield F1 hybrids. P-value by paired two-sided t-test. c, Correlation between Akr1c18 expression and Tnn expression across development of oldfield mice. P-value by bivariate correlation, R denotes Pearson’s correlation coefficient. d, Logarithm of the odds (LOD) across the genome of Cdkn2a, Podnl1, Serpine1, and Timp1 expression. e, LOD of the ECM module and the ECM module without Tnn. f, LOD of the ECM module and the ECM module controlling for Tnn expression. g, LOD of Akr1c18 expression and Akr1c18 expression controlling for Tnn expression. Dashed lines denote genome-wide threshold of significance (α = 0.05). h, Tnn expression as a function of genotype at the Tnn locus in F2-hybrid males. Akr1c18 expression as a function of genotype at the Tnn locus (i) or the Akr1c18 locus (j) in F2-hybrid males. h,i,j Boxplot hinges are 25th and 75th quartiles, whiskers are 1.5× interquartile range, line at median. P-values by ANOVA.

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Supplementary information

Supplementary Information

Supplementary Methods, Supplementary Table 3, Supplementary Notes 1 and 2, Supplementary References and Supplementary Figs. 1–3.

Reporting Summary

Supplementary Table 1

Adrenal cell cluster markers

Supplementary Table 2

One-to-one orthologues

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Niepoth, N., Merritt, J.R., Uminski, M. et al. Evolution of a novel adrenal cell type that promotes parental care. Nature (2024). https://doi.org/10.1038/s41586-024-07423-y

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