A reevaluation of X-irradiation-induced phocomelia and proximodistal limb patterning.

Phocomelia is a devastating, rare congenital limb malformation in which the long bones are shorter than normal, with the upper portion of the limb being most severely affected. In extreme cases, the hands or fingers are attached directly to the shoulder and the most proximal elements (those closest to the shoulder) are entirely missing. This disorder, previously known in both autosomal recessive and sporadic forms, showed a marked increase in incidence in the early 1960s due to the tragic toxicological effects of the drug thalidomide, which had been prescribed as a mild sedative. This human birth defect is mimicked in developing chick limb buds exposed to X-irradiation. Both X-irradiation and thalidomide-induced phocomelia have been interpreted as patterning defects in the context of the progress zone model, which states that a cell's proximodistal identity is determined by the length of time spent in a distal limb region termed the 'progress zone'. Indeed, studies of X-irradiation-induced phocomelia have served as one of the two major experimental lines of evidence supporting the validity of the progress zone model. Here, using a combination of molecular analysis and lineage tracing in chick, we show that X-irradiation-induced phocomelia is fundamentally not a patterning defect, but rather results from a time-dependent loss of skeletal progenitors. Because skeletal condensation proceeds from the shoulder to fingers (in a proximal to distal direction), the proximal elements are differentially affected in limb buds exposed to radiation at early stages. This conclusion changes the framework for considering the effect of thalidomide and other forms of phocomelia, suggesting the possibility that the aetiology lies not in a defect in the patterning process, but rather in progenitor cell survival and differentiation. Moreover, molecular evidence that proximodistal patterning is unaffected after X-irradiation does not support the predictions of the progress zone model.

Ionizing radiation-induced gene modulations, cytokine content changes and telomere shortening in mouse fetuses exhibiting forelimb defects.

Several lines of evidence have linked limb teratogenesis to radiation-induced apoptosis and to the p53 status in murine fetuses. In previous reports, we studied the occurrence of various malformations after intrauterine irradiation and showed that these malformations were modulated by p53-deficiency as well as by the developmental stage at which embryos were irradiated. In this new study, we focused onto one particular phenotype namely forelimb defects to further unravel the cellular and molecular mechanisms underlying this malformation. We measured various parameters expected to be directly or indirectly influenced by irradiation damage. The mouse fetuses were irradiated at day 12 p.c. (post conception) and examined for forelimb defects on gestational days 15, 16, 17 and 19 of development. The release of inflammatory cytokines was determined in the amniotic fluid on day 16 p.c. and the mean telomere lengths assessed at days 12, 13 and 19 p.c. Differential gene expression within the forelimb bud tissues was determined using Real Time quantitative PCR (RTqPCR) 24 h following irradiation. Apoptosis was investigated in the normal and malformed fetuses using the TUNEL assay and RTqPCR. First, we found that irradiated fetuses with forelimb defects displayed excessive apoptosis in the predigital regions. Besides, overexpression of the pro-apoptotic Bax gene indicates a mitochondrial-mediated cell death. Secondly, our results showed overexpression of MKK3 and MKK7 (members of the stress-activated MAP kinase family) within the malformed fetuses. The latter could be involved in radiation-induced apoptosis through activation of the p38 and JNK pathways. Thirdly, we found that irradiated fetuses exhibiting forelimb defects showed a marked telomere shortening. Interestingly, telomere shortening was observed as the malformations became apparent. Fourthly, we measured cytokine levels in the amniotic fluid and detected a considerable inflammatory reaction among the irradiated fetuses as evidenced by the increase in pro-inflammatory cytokine levels. Altogether, our data suggest that transcriptional modulations of apoptotic, inflammation, stress, and DNA damage players are early events in radiation-induced forelimb defects. These changes resulted in harsh developmental conditions as indicated by a marked increase in cytokine levels in the amniotic fluid and telomere shortening, two features concomitant with the onset of the forelimb defect phenotype in our study.

Infrared radiation influence on molt and regeneration of Neohelice granulata Dana, 1851 (Grapsidae, Sesarminae).

This paper analyzes the influence of infrared radiation (IR) on regeneration, after autotomy of limb buds of Neohelice granulata and consequently the time molt. Eyestalks were ablated to synchronize the start of molt. Afterward, animals were autotomized of five pereopods and divided into control and irradiated groups. The irradiated group was treated for 30 min daily until molt. Limb buds from five animals of days 4, 16 and 20 were collected and histological sections were made from them. These sections were photographed and chitin and epithelium content measured. Another group was made, and after 15 days limb buds were extracted to analyze mitochondrial enzymatic activity from complex I and II. The irradiated group showed a significant reduction in molt time (19.38+/-1.22 days) compared with the control group (32.69+/-1.57 days) and also a significant increase in mitochondrial complex I (388.9+/-27.94%) and II (175.63+/-7.66%) in the irradiated group when compared with the control group (100+/-17.90; 100+/-7.82, respectively). However, these effects were not accompanied by histological alterations in relation to chitin and epithelium. This way, it was possible to demonstrate that IR increases complex I and II activity, reduces the time molt and consequently increases the appendage regeneration rate.

Organ culture studies on the development of mouse embryo limb buds under EMF influence.

PURPOSE: To investigate the effects of an electromagnetic field (EMF) on limb bud development in vitro, an organ culture system was applied. MATERIALS AND METHODS: Three test groups of amputated mouse limb buds included the experimental (E) group which received EMF (50 Hz/13.1 mT, for 2 h), a sham (Sh) group exposed to no EMF treatment and the control (C) group. The limb buds of E and Sh groups (n = 20 per group) were amputated from mouse embryos on day 11.5 of development and cultured in minimum essential medium Eagle (MEM Eagle), supplemented with 15% human embryo cord serum, for 2 days, while those of group C (n = 20) were removed on day 13.5 of development. All samples were fixed in Bouin's fluid, embedded in paraffin, serially sectioned (5 microm thick) and stained with Hematoxylin and Eosin. Limb bud measurements were performed using a scaled graticule. RESULTS: Morphological and histological examinations showed significant changes in the experimental limb bud group as compared with the sham and control groups. The growth rate in both fore and hindlimb buds in proximal-distal (P-D) and anterior-posterior (A-P) axes were significantly increased. Chondrocyte counts and mitotic figures of mesenchymal and red blood cells were significantly increased as compared with those of sham and control groups. There was also a significant reduction of mesenchymal cell counts, while no significant difference was observed in the degenerated cell counts among the three groups. CONCLUSIONS: These findings suggest that EMF, under the conditions applied, has progressive effects on the limb bud development and that both proliferation and differentiation can be stimulated in vitro.

Adaptive response in embryogenesis: I. Dose and timing of radiation for reduction of prenatal death and congenital malformation during the late period of organogenesis.

An adaptive response was demonstrated during embryogenesis in mice. Whole-body irradiation at a dose of 0-50 cGy was given to condition pregnant ICR mice on day 9 to day 11 of gestation. Then their whole bodies were exposed to a challenging dose of 5 Gy on the next day. The numbers of living fetuses, prenatal deaths and living fetuses with external gross malformations were determined on day 19. A conditioning dose of 30 cGy on day 11 significantly increased the rate of living fetuses and reduced the incidence of congenital malformations induced by a 5-Gy dose on day 12. This indicates the existence of a critical dose and timing for administering a conditioning dose for radioadaptation during the late period of organogenesis in mice. The possible mechanisms involved are discussed.

Radiation-induced apoptosis and limb teratogenesis in embryonic mice.

In utero irradiation of the fetus during the period of organogenesis induces a dramatic increase in malformation. However, the mechanisms underlying the teratogenesis remain to be elucidated. In the present study, the correlation between radiation-induced apoptosis and limb malformation was examined in mice. The mice were exposed to X rays in utero on day 11 of gestation during the period of organogenesis of limb buds. A marked increase in the number of apoptotic cells in the predigital regions in the forelimb buds was detected 4 h after irradiation. The preinterdigital regions of the forelimb buds did not show such an increase at the same time. Aphlangy and ectrodactyly were the main types of anomalies observed on day 19 in the limbs of the fetuses irradiated with 5 Gy. The increases in prenatal death and teratogenesis in limb digits in living fetuses were dependent on dose. The possible mechanisms involved are discussed.

Prenatal radiation-induced limb defects mediated by Trp53-dependent apoptosis in mice.

We reported previously that in utero radiation-induced apoptosis in the predigital regions of embryonic limb buds was responsible for digital defects in mice. To investigate the possible involvement of the Trp53 gene, the present study was conducted using embryonic C57BL/6J mice with different Trp53 status. Susceptibility to radiation-induced apoptosis in the predigital regions and digital defects depended on both Trp53 status and the radiation dose; i.e., Trp53 wild-type (Trp53(+/+)) mice appeared to be the most sensitive, Trp53 heterozygous (Trp53(+/-)) mice were intermediate, and Trp53 knockout (Trp53(-/-)) mice were the most resistant. These results indicate that induction of apoptosis and digital defects by prenatal irradiation in the later period of organogenesis are mediated by the Trp53 gene. These findings suggest that the wild-type Trp53 gene may be an intrinsic genetic susceptibility factor that is responsible for certain congenital defects induced by prenatal irradiation.

Adaptive response in embryogenesis: IV. Protective and detrimental bystander effects induced by X radiation in cultured limb bud cells of fetal mice.

The radioadaptive response and the bystander effect represent important phenomena in radiobiology that have an impact on novel biological response mechanisms and risk estimates. Micromass cultures of limb bud cells provide an in vitro cellular maturation system in which the progression of cell proliferation and differentiation parallels that in vivo. This paper presents for the first time evidence for the correlation and interaction in a micromass culture system between the radioadaptive response and the bystander effect. A radioadaptive response was induced in limb bud cells of embryonic day 11 ICR mice. Conditioning irradiation of the embryonic day 11 cells with 0.3 Gy resulted in a significant protective effect against the occurrence of apoptosis, inhibition of cell proliferation, and differentiation induced by a challenging dose of 5 Gy given the next day. Both protective and detrimental bystander effects were observed; namely, irradiating 50% of the embryonic day 11 cells with 0.3 Gy led to a successful induction of the protective effect, and irradiating 70% of the embryonic day 12 cells with 5 Gy produced a detrimental effect comparable to that seen when all the cells were irradiated. Further, the bystander effect was markedly decreased by pretreatment of the cells with an inhibitor to block the gap junction-mediated intercellular communication. These results indicate that the bystander effect plays an important role in both the induction of a protective effect by the conditioning dose and the detrimental effect of the challenge irradiation. Gap junction-mediated intercellular communication was suggested to be involved in the induction of the bystander effect.

Stimulation of axon growth from the spinal cord by a regenerating limb blastema in newts.

The effects of limb blastemas of Pleurodeles waltl on axon growth from fragments of spinal cord were studied in vitro. Cultured in a defined medium, spinal cord fragments regenerated sparse, short axons. The culture of spinal fragments in the presence of blastemas greatly enhanced the length, number and survival of axons. Testing separately each of the two components of the blastema showed that only the mesenchyme exerts a neurotropic effect on the spinal fragments. Other tissues such as muscle or skin had a limited neurotrophic effect. Additionally, the neurotrophic activity of blastemas seems to be dependent of its proliferation status. Compared with blastemas of regenerating limbs from young animals, irradiated blastemas (devoid of mitotic activity) and blastemas of regenerating limbs from old animals or differentiated blastemas (both characterized by a low mitotic activity), exhibited a weaker neurotrophic influence. The blastema neurotrophic factor is not an attachment molecule but a soluble one and cannot be nerve growth factor (NGF) or fibroblast growth factor (FGF). It has a relatively low molecular weight (less than 15 kDa) and its protein nature was ascertained by its sensitivity to heating and proteases. As the production of this mesenchyme-derived neurotrophic factor depends upon mesenchymal cell proliferation of the blastema, we suggest that there is loop of positive regulation between spinal nerves and blastema. Blastema tissues may stimulate nerve regeneration allowing the stimulation of proliferation of blastema cells by regenerating nerve fibers. Alternatively, blastema cells may produce a neurotrophic factor whose secretion might be dependent on cell proliferation.

Effects of UVC irradiation on cultured mouse embryonic limb bud cells.

Effects of UVC irradiation (UVC) at a dose range from 0 to 30 J/m(2) were investigated on the cultured embryonic limb bud cells (LBC), isolated from fore- and hindlimbs of day 11 mouse embryos. Although dose-dependent inhibition was found for both cellular proliferation and chondrogenesis, the chondrogenic proteoglycan (PG) synthesis was more sensitive to UVC than cellular proliferation when compared at ID(50), the inhibitory dose that reduced assessment value by 50% of the control. No significant difference in induction and repair kinetics of UV-induced cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts ((6-4)PPs) were found between LBC and NIH3T3 mouse cell line. The fluorescent light (FL) treatment of LBC pre or post UVC irradiation did not affect repair kinetics of CPDs and (6-4)PPs, cellular proliferation, formation of PG-producing nodule and the PG synthesis.

Gene silencing of Tead3 abrogates radiation-induced adaptive response in cultured mouse limb bud cells.

There is a crucial need to better understand the effects of low-doses of ionizing radiation in fetal models. Radiation-induced adaptive response (AR) was described in mouse embryos pre-exposed in utero to low-doses of X-rays, which exhibited lower apoptotic levels in the limb bud. We previously described AR-specific gene modulations in the mouse embryo. In this study, we evaluated the role of three candidate genes in the apoptotic AR in a micromass culture of limb bud cells: Csf1, Cacna1a and Tead3. Gene silencing of these three genes abrogated AR. Knowing that TEAD3 protein levels are significantly higher in adapted cells and that YAP/TAZ/TEAD are involved in the control of cell proliferation and apoptosis, we suggest that modulation of Tead3 could play a role in the induction of AR in our model, seen as a reduction of radiation-induced apoptosis and a stimulation of proliferation and differentiation in limb bud cells.

Anteroposterior axis formation in Xenopus limb bud recombinants: a model of pattern formation during limb regeneration.

We previously showed that recombinant limb buds with dissociated and reaggregated mesenchyme develop more than 30 digits in Xenopus laevis, which exhibits different capacities for limb regeneration at different developmental stages (Yokoyama et al. [1998] Dev Biol 196:1-10). Cell-cell contact among anterior- and posterior-derived mesenchymal cells is required for anteroposterior (AP) axis formation of recombinant limbs in an intercalary manner. However, whether one-way induction from posterior cells to anterior cells as proposed by the polarizing zone model or interactions between anterior and posterior cells evoke the AP axis formation in recombinant limbs remains unclear. In this study, we found, by a combination of X-ray irradiation and a recombinant limb technique, that not one-way induction but interactions between anterior and posterior cells accompanied by cell contribution are indispensable for AP axis formation in recombinant limbs. Shh was expressed in posterior-derived not anterior-derived cells. We propose that the recombinant limb is an excellent model for examining the axis formation mechanism in regenerating limbs because, as in recombinant limbs, cell-cell contact among cells derived from different positions of an amputation plane occurs in the blastema of regenerating limbs.