Ionising Radiation Induces Promoter DNA Hypomethylation and Perturbs Transcriptional Activity of Genes Involved in Morphogenesis during Gastrulation in Zebrafish.

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 (< 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.

Bone morphogenetic protein 1 regulates dorsal-ventral patterning in early Xenopus embryos by degrading chordin, a BMP4 antagonist.

Bone morphogenetic protein 1 (BMP1) is a metalloprotease that ventralises dorsal mesoderm when overexpressed in early Xenopus embryos. Here we show that Xenopus BMP1 blocks the dorsalising activity of chordin, but not noggin or DeltaxBMPR, when coexpressed in the ventral marginal zone and degrades chordin protein in vitro. We also show that a dominant-negative mutation for XBMP1 (dnBMP1) dorsalises ventral mesoderm in vivo, and blocks degradation of chordin by both XBMP1 and Xolloid, a closely related Xenopus metalloprotease, in vitro. dnBMP1 does not dorsalise ventral mesoderm in UV-irradiated embryos, demonstrating that this activity is dependent upon a functional organiser--the natural source of chordin in Xenopus gastrulae. Our results suggest that XBMP1 may regulate the availability of chordin during vertebrate embryogenesis.

Difference in sensitivity of inner cell mass and trophectoderm to X-irradiation in mouse blastocysts.

Pregnant B6C3F1 mice were exposed to a single whole body X-irradiation on day 4 (73-74 hr postconception) of gestation. In experiment 1, they were sacrificed at 2, 4, 6, or 9 hr after a dose of 2 Gy, and their embryos were removed and examined with light and electron microscopy. In experiment 2, dose-response effects of irradiation on the embryos were examined 4 hr after doses of 0-4 Gy. In experiment 3, DNA fragmentation (a marker of apoptosis) was observed by 3'-OH nick-end labeling technique. In inner cell mass (ICM) and trophectoderm (TE) of blastocysts exposed to 2 Gy, cells with cytoplasmic degeneration, or dead cells phagocytosed by their neighboring cells, were found. Although morphological features of these dying cells did not reveal typical characteristics of apoptosis such as nuclear condensation and membrane blebbing, DNA fragmentation was detected by nick-end labeling technique. The degenerated cytoplasm consisted of aggregating ribosomes. Degenerated cells began to increase from 2 hr after irradiation and reached maximal at 4 hr in both ICM and TE. The incidences of degenerated cells in ICM were higher than those in TE at any time point. These findings provide evidence that cell death observed in blastocysts after X-irradiation is apoptotic and sensitivity of the two groups of cells (ICM and TE) to X-rays is different.

Formation of distal structures from stumps of chick wing buds at stages 24-25 following the grafting of quail tissue from X-irradiated distal limb buds.

To examine the regulatory activity of the proximal region of chick limb buds, distal halves of chick wing buds at stages 24-25 were removed, and the distal tips of X-irradiated stage 21 quail limb buds with an apical ectodermal ridge (AER) were grafted onto the chick stumps. The host stumps formed only humerus after removal of the donor distal tips or with tip mesoderm alone. However, when distal tips with an AER were grafted onto the stumps, one or two cartilage elements of host origin were formed at the distal end of the humerus. To examine whether the stump cells have changed to cells of a more distal, progress zone (PZ) cells, the chick stumps with quail tips were immunostained with antibody against AV-1, which reacts with the anterior PZ region of chick limb buds and is specific for an antigen that is expressed under the control of the AER. Within two days of grafting, cells positive for the antigen reappeared in the stump tissues. These results suggest that the some of the stump cells may be converted to PZ cells and their positional values may change to those of more distal structures under the influence of the AER.

MRI effects on craniofacial size and crown-rump length in C57BL/6J mice in 1.5T fields.

Previous studies have shown magnetic resonance imaging to be potentially teratogenic for eye development. An investigation was undertaken to ascertain the effects of 1.5 Tesla magnetic resonance imaging fields on crown-rump length and craniofacial perimeter, two less sensitive teratologic end points of the C57BL/6J mouse. A sham control and two experimental groups of dams (N = 12, 11, 12) placed in different magnet locations were exposed to magnetic resonance imaging fields under clinically realistic conditions. A T-2 weighted spin-echo technique of 36-minutes duration was used on each dam. Crown-rump length was chosen because it is a standard measure of embryotoxicity. Craniofacial perimeter was evaluated because of its relationship to the anterior neural plate, the same region in the developing embryo that gives rise to the eyes. Magnetic resonance imaging fields were found to produce crown-rump and craniofacial perimeter measures smaller than control animals (p < 0.05) when exposed at the isocenter (p = 0.038 and 0.008 respectively). Groups exposed to magnetic resonance imaging fields at the magnet entrance showed p values of 0.004 and 0.053 respectively. The results confirmed magnetic resonance imaging teratogenicity in the C57BL/6J mouse and demonstrates the need for further experimental investigation to ascertain the clinical safety of magnetic resonance imaging for humans.

Cellular interactions involved in the determination of the early C. elegans embryo.

Classical work implied that early nematode embryogenesis is completely mosaic. This view was lately challenged by the demonstration that in C. elegans an early interaction has to occur to induce the production of muscle from a blastomere. Here, early embryonic blastomeres were inactivated by laser microsurgery. The cell lineages of irradiated embryos were compared to those of intact embryos. It is shown that one blastomere, MS, is required for the specification of mesodermal pharyngeal fates and another blastomere, P2, for the specification of hypodermal fates from the descendants of the AB blastomere, whereas the proper specification of the nervous system requires the presence of both. The irradiation of a third blastomere shows that interactions also occur within the ectoderm. I propose that the body plan of the C. elegans embryo may be established by two primary signals followed by secondary interactions. The suggested mechanisms are reminiscent of those involved in amphibian development.

Cutaneous nerves of the embryonic chick wing do not develop in regions denuded of ectoderm.

Peripheral nerves travel to their targets along precise routes, and it is likely that different cues provide guidance at different stages of the journey. In a developing chick limb, the cutaneous nerve fibres follow at first deep mixed nerve trunks, in company with motor axons; they branch from these trunks at predictable points and approach the skin; they then ramify profusely to form a plexus at a precisely defined depth beneath the ectoderm, at exactly the same level as the blood vascular plexus. To analyse the role of signals from the target patch of skin in regulating cutaneous nerve development, we have ablated patches of dorsal wing ectoderm using short-wave ultraviolet irradiation at E4 (embryonic day 4), approximately one day before nerves grow into the limb bud. The irradiated patches remain denuded of ectoderm for more than a week, by which time the cutaneous nerve plexus on the contralateral control side is well developed and can be revealed by whole-mount silver staining. Where the ectoderm has been ablated, no cutaneous nerve plexus forms, and the nerve branches that normally would have diverged from the neighbouring mixed nerve trunk to innervate the missing patch of skin are absent - ab initio, apparently. The routes of the mixed nerve trunks are not affected. Partial ablation of the territory of a cutaneous nerve branch often leads to loss of the whole nerve branch; the intact skin territory thus left vacant is invaded by ramifications from the remaining cutaneous branches, as expected if the normal extent of a cutaneous nerve's territory is regulated by competition. Where there is an ectodermal lesion, cutaneous innervation stops precisely at its boundary, even though the vascular plexus extends for some distance beyond this margin, beneath the denuded surface. The data suggest that the embryonic skin is required firstly to trigger divergence of cutaneous nerve branches from the mixed nerve trunks, and secondly, once the nerve fibres have reached the skin, to supply a trophic cue (probably NGF) encouraging growth of a plexus; at the same time, the embryonic skin generates a signal inhibiting nerves from approaching closer than about 70 microns to the surface.

Normal development of the skeleton in chick limb buds devoid of dorsal ectoderm.

It has been suggested that the ectoderm on the dorsal and ventral faces of the limb bud plays a part in controlling the pattern of cartilage differentiation. To test this, the dorsal wing bud ectoderm in the chick embryo was destroyed by irradiation with ultraviolet light at stage 17-19, at the very beginning of limb bud development, but the apical ectodermal ridge was spared. The irradiated ectoderm disappeared within 24 hr (by stage 23-24) and did not regenerate thereafter; thus the dorsal surface of the limb bud was kept denuded throughout most of the period of skeletal pattern formation. By 6 or 7 days after the irradiation (stage 35), when the rudiments of all the adult skeletal elements are normally present in recognizable form, the irradiated wings could be placed into two categories, those that were approximately normal in shape and those that had curled dorsally. All of these limbs were reduced in size, to varying degrees, when compared to their controls and lacked dorsal soft tissues. The limbs that were normal in shape, however, even though sometimes denuded over practically the whole extent of their dorsal surface, almost always had a complete and normally proportioned cartilage pattern, suggesting that ectoderm (other than the apical ectodermal ridge) does not exert any direct control over the development of the limb cartilage pattern. However, many of those limbs that had curled as a result of the irradiation did have major pattern deformities, suggesting that the topology of cartilage differentiation does depend on the shape of the limb bud.

Further characterization of the effects of ultraviolet irradiation of the amphibian egg.

UV-irradiation of the vegetal hemisphere of amphibian eggs leads to developmental abnormalities in neural morphogenesis. The possibility that the egg's transient sensitivity to irradiation could be due to pigmentation changes was examined in albino eggs. The tissue specificity of the effects of irradiation was analyzed by exchanging the ectoderm between irradiated and control embryos.