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Embryonic development is particularly vulnerable to stress and DNA damage, as mutations can accumulate through cell proliferation in a wide number of cells and organs. However, the biological effects of chronic exposure to ionising radiation (IR) at low and moderate dose rates (&lt, 6 mGy/h) remain largely controversial, raising concerns for environmental protection. The present study focuses on the molecular effects of IR (0.005 to 50 mGy/h) on zebrafish embryos at the gastrula stage (6 hpf), at both the transcriptomics and epigenetics levels. Our results show that exposure to IR modifies the expression of genes involved in mitochondrial activity from 0.5 to 50 mGy/h. In addition, important developmental pathways, namely, the Notch, retinoic acid, BMP and Wnt signalling pathways, were altered at 5 and 50 mGy/h. Transcriptional changes of genes involved in the morphogenesis of the ectoderm and mesoderm were detected at all dose rates, but were prominent from 0.5 to 50 mGy/h. At the epigenetic level, exposure to IR induced a hypomethylation of DNA in the promoter of genes that colocalised with both H3K27me3 and H3Kme4 histone marks and correlated with changes in transcriptional activity. Finally, pathway enrichment analysis demonstrated that the DNA methylation changes occurred in the promoter of important developmental genes, including morphogenesis of the ectoderm and mesoderm. Together, these results show that the transcriptional program regulating morphogenesis in gastrulating embryos was modified at dose rates greater than or equal to 0.5 mGy/h, which might predict potential neurogenesis and somitogenesis defects observed at similar dose rates later in development.

International audience; contamination of the environment after the chernobyl and fukushima Daiichi nuclear power plant (npp) disasters led to the exposure of a large number of humans and wild animals to radioactive substances. However, the sub-lethal consequences induced by these absorbed radiological doses remain understudied and the long-term biological impacts largely unknown. We assessed the biological effects of chronic exposure to ionizing radiation (IR) on embryonic development by exposing zebrafish embryo from fertilization and up to 120 hours post-fertilization (hpf) at dose rates of 0.5 mGy/h, 5 mGy/h and 50 mGy/h, thereby encompassing the field of low dose rates defined at 6 mGy/h. Chronic exposure to IR altered larval behaviour in a light-dark locomotor test and affected cardiac activity at a dose rate as low as 0.5 mGy/h. The multi-omics analysis of transcriptome, proteome and transcription factor binding sites in the promoters of the deregulated genes, collectively points towards perturbations of neurogenesis, muscle development, and retinoic acid (RA) signaling after chronic exposure to iR. Whole-mount RnA in situ hybridization confirmed the impaired expression of the transcription factors her4.4 in the central nervous system and myogenin in the developing muscles of exposed embryos. At the organ level, the assessment of muscle histology by transmission electron microscopy (TEM) demonstrated myofibers disruption and altered neuromuscular junctions in exposed larvae at 5 mGy/h and 50 mGy/h. The integration of these multi-level data demonstrates that chronic exposure to low dose rates of iR has an impact on neuronal and muscle progenitor cells, that could lead to motility defects in free swimming larvae at 120 hpf. The mechanistic understanding of these effects allows us to propose a model where deregulation of RA signaling by chronic exposure to IR has pleiotropic effects on neurogenesis and muscle development.

a Tg(−505/−310 (unc45b)gata2:gfp) embryos were injected with hsp90a morpholinos (hsp90a-mo) or with hsp90a-mo together with a mix of CRISPR RNA directed against hsf1 and cas9 mRNA (hsp90a-mo + guide hsf1). We note a decrease of GFP in the hsf1/hsp90a double knock-down compared with the hsp90a single morphant. b–b’ Injection of the plasmid encoding a Hsf1–mOrange fusion protein does not activate the unc45b promoter. Expression of TFP reporter in hsf1-morange injected embryos (+hsf1:morange) (a, b) is as high as in uninjected tg(−1.8unc45b:tfp) embryos (cont) (a). c–h’ Heat shock triggers unc45b up-regulation. −505/−310(unc45b)gata2:gfp injected embryos (c, d, c’, d’), Tg(−505/−310 (unc45b)gata2:gfp) (e, f, e’, f’) and tg(−1.8unc45b:tfp) (g, h, g’, h’) transgenic embryos were raised at 28 °C until 48 hpf and heat shock at 37 °C for 12 hours (d, d’, f, f’, h, h’). Embryos shown in (c, c’, e, e, g, g’) were kept at 28 °C. The heat shock embryos show up-regulation of GFP in skeletal and cardiac muscles, demonstrating that the regulatory sequences of unc45b react to heat shock. (PDF 1668 kb)

Summary of the transgenes used to map the regulatory elements mediating the misfolded myosin response A-B: tg(-1.8unc45b:tfp) (“1”) and its deletion derivatives were expressed transiently (trs) or as transgenesstably integrated into the genome (stb). By serial deletion analysis 645 bp upstream of the ATG of unc45bwas identified to still drive basal muscle expression and respond to unc45b deficiency (B). Furthertruncation of the unc45b sequences to the region from -369 bp to +390 bp abolished the response.C-E: Refined analysis of the unc45b regulatory sequences contained in tg(-645/+533unc45b:tfp). Theunc45b deletion fragments were cloned in front of the gata2 promoter (blue bars). Transgenes containingthe 195 bp fragment (“16”, D) harboring sequences from -505 to -310 drove GFP expression in skeletalmuscle (D, cont) and reacted to the accumulation of unfolded myosin (D, +unc45b-mo). Deletion constructs12-20 retained basal muscle expression and responded to misfolded myosin. In contrast, constructs 21-24give no GFP expression (see E).Further mutations of the 195 bp unc45b fragment separates the regulatory sequences responsible for basalmuscle expression and the response to misfolded myosin. Construct 28 drove basal GFP expression inskeletal muscles but did not respond when co-injected with the unc45b-mo (G). Construct 30 (55 bp)reacted similarly (H). These constructs retained a binding site of the Mef2 transcription factor. Constructs 31-37 lack the muscle basal expression but still respond to misfolded myosin (I). Thus, the region from -445 to -425 is important to mediate the response. This region contains a homology to the heat shock responseelement (HSE, J). The HSE was mutated by introduction of 4 point mutations (asterisks J, F, I) in constructs“26”, “38”, “41” and “42”. Mutation of the HSE leads to loss of the misfolded myosin response. Abbreviations:ME: muscle expression. The numbers above constructs indicate position relative to the ATG of unc45b. (PDF 2497 kb)

a, b Heatmap of Euclidian distance of RNASeq experiments. Pairwise comparison of conditions is displayed as a matrix of Euclidean distances with red colors indicating similarities (short distance) and yellow dissimilarities (high distance) between conditions. a RNA was prepared from either wild-type siblings (C1 and C2) or unc45b mutants (U) at 24, 48 and 72 hpf. Biological repeats are indicated by the numbers (1–3). C control, U unc45 −/− . b Clustering of Euclidean distances of the unc45b, hsp90a and ache mutants and their respective wild-type siblings at 72 hpf. c–e Fold-change expression of selected genes between wild-type and unc45b mutant embryos at 24, 48 and 72 hpf as detected in the RNASeq data. The expression of unc45b (c), hsp90a (d) and smyd1b (e) mRNA steadily increases from 24–72 hpf. f, g Log2 fold change of genes found upregulated at 72 hpf in unc45b and hsp90a mutants but not in ache mutants (f), or upregulated in the three mutants (g). Note that the level of upregulation in ache mutant is really low compared with the unc45b and hsp90a mutants. h–j MA plots displaying log10 (mean expression) between control and unc45b −/− mutants against log2 (fold change) at three developmental stages obtained with DESeq: 24 hpf (h), 48 hpf (i), 72 hpf (j). Genes up- or down-regulated with FDR 1.5 are shown in red. Numbers indicate the total number of detected genes with FDR 1.5. The error bars indicate "standard deviation". (PDF 3965 kb)

Chart representing the mean TFP or GFP intensity of transgenic embryos. a Mean pixel intensity of tg(−1.8unc45b:tfp) embryos compared with tg(−1.8unc45b:tfp) injected with either unc45b-mo or hsp90a-mo, showing twice as much TFP fluorescence in the morphant compared with wild-type embryos. b Mean pixel intensity of tg(−1.8unc45b:tfp) embryos compared with tg(−1.8unc45b:tfp) unc45b mutant showing twice as much TFP fluorescence in the mutant compared with the wild-type embryos. c Mean pixel intensity of tg(−505/−310(unc45b)gata2:gfp) embryos compared with tg(−505/−310(unc45b)gata2:gfp) injected with hsp90a-mo. The GFP intensity in the morphant is increased six times compared with wild type. d Mean pixel intensity of tg(−505/−310(unc45b)gata2:gfp) embryos compared to tg(−505/−310(unc45b)gata2:gfp) injected with unc45b-mo or unc45b-mo and hsf1-mo. The hsf1-mo abolished the increased GFP expression obtained with unc45b-mo alone. e Mean pixel intensity of tg(−505/−310(unc45b)gata2:gfp) embryos compared with tg(−505/−310(unc45b)gata2:gfp) injected with unc45b-mo or unc45b-mo and hsf2-mo. The hsf2-mo does not reduce the increased GFP expression obtained with unc45b-mo alone. f Mean pixel intensity of tg(−505/−310(unc45b)gata2:gfp) embryos compared with tg(−505/−310(unc45b)gata2:gfp) injected with unc45b-mo or unc45b-mo and hsf1cont-mo. The hsf1cont-mo does not reduce the increased GFP expression obtained with unc45b-mo alone. For each measurement at least three embryos were examined. For each chart the measured embryos are progeny of homozygous transgenics crossed with wild-type adult fish, and were exposed to the same light intensity. The error bars indicate "standard deviation". (PDF 498 kb)

a, b Sequence comparison of the unc45b region mediating the misfolded myosin response with homologous regions in the unc45b genes of other fish species. a Scheme of the 195-bp zebrafish unc45b fragment (−505 to −310 relative to the ATG) containing the HSE at position −446/−422 and the Mef2 binding site at position −335/−313. The recognition sequence is depicted below each site. b Comparison of the zebrafish unc45b sequence from −505 to −310 with regions in the unc45b genes of four other fish species revealed the presence of a conserved HSE element (black ovals). c–e Injection of −505/−405(unc45b)gata2:gfp plasmid into wild-type embryos (cont) (c) do not generate any GFP expression, whereas co-injection with unc45b-mo activates the regulatory sequence (c) (unc45b-mo). Examination of the GFP fibrils revealed classic striations (d, e, z) (Z-line). f injection of −505/−405(unc45b)gata2:gfp plasmid into hel mutant (hel−/−) or wild-type siblings (cont) do not lead to GFP0 expression. g, h Injection of −505/−310(unc45b)gata2:gfp plasmid into wild type (cont) (g, h), unc45b morphant (unc45b-mo) (g) or hel mutants (hel−/−) (h) show a GFP upregulation in unc45b morphant but not in hel mutants. (PDF 3322 kb)

The cornea is a central component of the camera eye of vertebrates and even slight corneal disturbances severely affect vision. The transcription factor PAX6 is required for normal eye development, namely the proper separation of the lens from the developing cornea and the formation of the iris and anterior chamber. Human PAX6 mutations are associated with severe ocular disorders such as aniridia, Peters anomaly and chronic limbal stem cell insufficiency. To develop the zebrafish as a model for corneal disease, we first performed transcriptome and in situ expression analysis to identify marker genes to characterise the cornea in normal and pathological conditions. We show that, at 7 days post fertilisation (dpf), the zebrafish cornea expresses the majority of marker genes (67/84 tested genes) found also expressed in the cornea of juvenile and adult stages. We also characterised homozygous pax6b mutants. Mutant embryos have a thick cornea, iris hypoplasia, a shallow anterior chamber and a small lens. Ultrastructure analysis revealed a disrupted corneal endothelium. pax6b mutants show loss of corneal epithelial gene expression including regulatory genes (sox3, tfap2a, foxc1a and pitx2). In contrast, several genes (pitx2, ctnnb2, dcn and fabp7a) were ectopically expressed in the malformed corneal endothelium. Lack of pax6b function leads to severe disturbance of the corneal gene regulatory programme.