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Temperature-Dependent Vitamin D Signaling Regulates Developmental Trajectory Associated with Diapause in an Annual Killifish

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Temperature-Dependent Vitamin D Signaling Regulates Developmental Trajectory Associated with Diapause in an Annual Killifish Temperature-dependent vitamin D signaling regulates developmental trajectory associated with diapause in an annual killifish Amie L. T. Romneya,b, Erin M. Davisa, Meranda M. Coronaa, Josiah T. Wagnerc, and Jason E. Podrabskya,1 aDepartment of Biology, Portland State University, Portland, OR 97207; bSchool of Veterinary Medicine, Anatomy, Physiology, and Cell Biology, University of California, Davis, CA 95616; and cKnight Cancer Early Detection Advanced Research Center, Oregon Health and Science University, Portland, OR 97201 Edited by David M. Kingsley, Stanford University, Stanford, CA, and approved October 10, 2018 (received for review March 16, 2018) The mechanisms that integrate environmental signals into devel- Exposure to increased temperature (30 °C compared with 20 °C to opmental programs remain largely uncharacterized. Nuclear recep- 25 °C) and light promotes the escape trajectory (6, 7). Biochemi- tors (NRs) are ligand-regulated transcription factors that orchestrate cally, the trajectories diverge early in development, before the the expression of complex phenotypes. The vitamin D receptor formation of the embryonic axis, as evidenced by reduced insulin- α (VDR)isanNRactivatedby1 ,25-dihydroxyvitamin D3 [1,25(OH)2D3], like growth factor (IGF) expression in diapause-bound embryos (8). a hormone derived from 7-dehydrocholesterol (7-DHC). VDR A critical window of development where increased temperature signaling is best known for regulating calcium homeostasis in causes irreversible commitment to the escape trajectory occurs mammals, but recent evidence suggests a diversity of uncharac- during early somitogenesis in embryos possessing 10 to 20 pairs of terized roles. In response to incubation temperature, embryos of somites. Importantly, these temperatures are within the bounds the annual killifish Austrofundulus limnaeus can develop along experienced by embryos under field conditions. Thus, embryos of two alternative trajectories: active development and diapause. annual killifishes offer an unprecedented opportunity to study ge- These trajectories diverge early in development, from a biochemical, nome−environment interactions during vertebrate development in morphological, and physiological perspective. We manipulated an ecologically and evolutionarily relevant context (1, 9, 10). BIOLOGY incubation temperature to induce the two trajectories and profiled Here we report that vitamin D3 synthesis and signaling promotes DEVELOPMENTAL changes in gene expression using RNA sequencing and weighted active development in fish embryos, and inhibition of vitamin D gene coexpression network analysis. We report that transcripts in- 3 synthesis leads to developmental arrest. In this paper we describe volved in 1,25(OH) D synthesis and signaling are expressed in a 2 3 vitamin D signaling as a pathway that regulates developmental trajectory-specific manner. Furthermore, exposure of embryos to 3 trajectory and metabolic dormancy, and allows for the integration of vitamin D3 analogs and Δ4-dafachronic acid directs continuous de- velopment under diapause-inducing conditions. Conversely, block- environmental information into the developmental program. ing synthesis of 1,25(OH)2D3 induces diapause in A. limnaeus and a Results diapause-like state in zebrafish, suggesting vitamin D signaling is Key Gene Regulatory Networks Are Unique to Each Developmental critical for normal vertebrate development. These data support vitamin D signaling as a molecular pathway that can regulate de- Trajectory. General results for the RNA sequencing (RNAseq) velopmental trajectory and metabolic dormancy in a vertebrate. analysis are provided in SI Appendix, Fig. S1 and Table S1 and Interestingly, the VDR is homologous to the daf-12 and ecdysone NRs that regulate dormancy in Caenorhabditis elegans and Dro- Significance sophila. We suggest that 7-DHC−derived hormones and their as- sociated NRs represent a conserved pathway for the integration of Here we describe a molecular pathway that regulates dormancy environmental information into developmental programs associ- in a vertebrate and highlights a mechanism that integrates en- ated with life history transitions in animals. vironmental cues into a developmental program that has clear ecological and evolutionary significance. Further, we provide nuclear receptors | life history | dormancy | phenotypic plasticity compelling evidence that vitamin D signaling is critical for normal vertebrate development and can induce a diapause-like arrest in here is a growing appreciation for the importance of envi- embryos of zebrafish. The vitamin D receptor is homologous to Tronmental exposures during development in the determination nuclear receptors (NRs) that regulate dormancy in Caenorhabditis of a wide range of complex phenotypes beyond classic examples elegans and Drosophila. This conservation of function suggests a of phenotypic plasticity (e.g., fetal programming in mammalian conserved role for hormones derived from 7-DHC and their asso- embryos). Despite their profound influence on form and func- ciated NRs in the control of metabolic dormancy and major life tion, the mechanisms by which phenotypes are modified by en- history transitions in animals. vironmental factors are largely undefined and unexplored. The annual killifish Austrofundulus limnaeus survives in ephem- Author contributions: A.L.T.R., E.M.D., J.T.W., and J.E.P. designed research; A.L.T.R., E.M.D., M.M.C., J.T.W., and J.E.P. performed research; A.L.T.R., E.M.D., M.M.C., J.T.W., eral ponds by producing stress-tolerant embryos that survive for and J.E.P. analyzed data; and A.L.T.R. and J.E.P. wrote the paper. months encased in drying mud. Embryos survive by entering into The authors declare no conflict of interest. three distinct stages of developmental arrest termed diapause I, II, This article is a PNAS Direct Submission. and III (1–3). Diapause is a state of profound metabolic depression Published under the PNAS license. and extreme stress tolerance; diapause II embryos can survive for Data deposition: The original data sets are available via the National Center for Biotech- months without access to oxygen or liquid water (2, 4). Entrance nology Information’s (NCBI) Sequence Read Archive, https://www.ncbi.nlm.nih.gov/sra into diapause II (diapause from here forward) is the result of an (accession nos. SRX1032655, SRX1032657, SRX1032658, SRX1032664–SRX1032702;BioProject alternative developmental trajectory that is physiologically, mor- ID PRJNA272154). phologically, and biochemically unique compared with “escape” 1To whom correspondence should be addressed. Email: [email protected]. embryos that develop continuously until hatching or entrance into This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. diapause III (5, 6). Developmental phenotype can be influenced 1073/pnas.1804590115/-/DCSupplemental. by maternal provisioning and by the embryonic environment. www.pnas.org/cgi/doi/10.1073/pnas.1804590115 PNAS Latest Articles | 1of6 Downloaded by guest on September 26, 2021 Fig. 1. WGCNA of RNAseq data in embryos of ABC A. limnaeus developing along alternative developmental trajectories. To identify trajectory-specific gene expres- sion patterns, embryos were reared 20 °C or 30 °C to induce the diapause or escape trajectories, re- spectively. Embryos (n = 3 groups of 20 embryos) were sampled at six morphological stages that bracket the temperature-sensitive window where develop- mental trajectory is determined: DC, dispersed cell stage; NK, neural keel stage; 6S, 6-somite embryo; 10S, 10-somite embryo; 16S, 16-somite embryo; 20S, 20- somite embryo; 24S, 24-somite embryo. (A)Heat map of module eigengene (based on weighted aver- age of module gene expression) correlations to each stage of development along the diapause trajectory (positive is red, and negative is white). (Upper) Modules − are significantly associated (r > 0.50; P value < 10 2) with a developmental stage. (Lower) Modules are not significantly associated. Modules with asterisks are not preserved between diapause and escape networks (Zsummary statistic < 10). (B) Heat maps of expression level reported as fragments per kilobase of transcript per million mapped reads (FPKM) for the light yellow module that contains a daf-36−like cholesterol desa- turase (LOC106533739) and IL-1β as hub genes. (C) Cytoscape (version 3.5.1) visualization of the light yellow module, with yellow circles representing the genes and the connecting lines representing similarity strengths (thickness) (44), based on topological overlap determined by WGCNA. Datasets S1 and S2.Weightedgenecoexpressionnetworkanalysis phenotype, and the key importance of daf-12 signaling for reg- (WGCNA) was used to construct 21 gene coexpression modules ulating dauer dormancy in C. elegans, we decided to focus further from the more than 16,000 transcripts expressed in embryos de- on this pathway. veloping along the diapause trajectory (11) (SI Appendix,Fig.S2). Network topologies of 12 modules were preserved in the escape trajectory and likely represent conserved transcriptional pathways critical to normal development (11, 12). We focused on the AB remaining nine modules that represent diapause- and escape- specific networks. Based on a measure of intramodular connec- tivity (kME), we identified 3,034 intramodular “hub” genes − (kME > 0.90, P < 10 8; Dataset S3) in the diapause trajectory as a means to discover key regulators of each module (11). Notably, within 24 h of temperature transfer [dispersed cell
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