Abstract:Retinoic acid signaling is required at several steps during the development of the spinal cord, from the specification of generic properties to the final acquisition of neuronal subtype identities, including its role in trunk neural crest development. These functions are associated with the production of retinoic acid in specific tissues and are highly dependent on context. Here, we review the defects associated with retinoic acid signaling manipulations, mostly in chick and mouse models, trying to separate the different processes where retinoic acid signaling is involved and to highlight common features, such as its ability to promote transitions along the neuronal differentiation cascade.Keywords: retinoic acid; spinal cord; neural tube; axis elongation; neural crest; motor neuron; dorso-ventral pattern; rostro-caudal pattern; neurogenesis
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Most cases of congenital spinal deformities were sporadic and without strong evidence of heritability. The etiology of congenital spinal deformities is still elusive and assumed to be multi-factorial. The current study seeks to elucidate the effect of maternal vitamin A deficiency and the production of congenital spinal deformities in the offsping. Thirty two female rats were randomized into two groups: control group, which was fed a normal diet; vitamin A deficient group, which were given vitamin A-deficient diet from at least 2 weeks before mating till delivery. Three random neonatal rats from each group were killed the next day of parturition. Female rats were fed an AIN-93G diet sufficient in vitamin A to feed the rest of neonates for two weeks until euthanasia. Serum levels of vitamin A were assessed in the adult and filial rats. Anteroposterior (AP) spine radiographs were obtained at week 2 after delivery to evaluate the presence of the skeletal abnormalities especially of spinal deformities. Liver and vertebral body expression of retinaldehyde dehydrogenase (RALDHs) and RARs mRNA was assessed by reverse transcription-real time PCR. VAD neonates displayed many skeletal malformations in the cervical, thoracic, the pelvic and sacral and limbs regions. The incidence of congenital scoliosis was 13.79% (8/58) in the filial rats of vitamin A deficiency group and 0% in the control group. Furthermore, vitamin A deficiency negatively regulate the liver and verterbral body mRNA levels of RALDH1, RALDH2, RALDH3, RAR-α, RAR-β and RAR-γ. Vitamin A deficiency in pregnancy may induce congenital spinal deformities in the postnatal rats. The decreases of RALDHs and RARs mRNA expression induced by vitamin A deprivation suggest that vertebral birth defects may be caused by a defect in RA signaling pathway during somitogenesis.
Congenital spinal deformities are not uncommon with an incidence of approximately 1 per 1,000 live births [1]. Vertebral anomalies may arise from defects in the development of the axial skeleton and are often associated with intraspinal abnormalities (e.g. myelopathy and paraplegia) and other organ defects (e.g. congenital heart disease and kidney defect) [2], [3]. The exact causes of these conditions have not yet been identified. The etiology is thought to be multifactorial, involving both the environmental and genetic factors. Chemical exposure, vitamin B6, and certain drugs have been implicated in the disturbance of vertebral formation [4].
There has been much research work focusing on the relationship between the skeletal malformation and VAD in embryonic developments. VAD rats exhibit hypoplastic cranial bones, defects of the thyroid, cricoid and tracheal cartilages as well as agenesis of rhe neural arch of cervical vertebrae 1 and ectopic bone in the dorsal regions of C1, malformation of the sternal and pelvic regions [24]. However, the relationship between the congenital spinal deformities (congential scolisosis) and VAD remains poorly understood. The aim of the present study is to examine if severe VAD in pregnant rats could increase the incidence of congenital spinal deformities in filial rats. The effects of VAD on the mRNA expression levels of several components of RA signaling, including RALDH1, RALDH2, RALDH3, RAR-α, RAR-β and RAR-γ, were also investigated.
The remaining 58 neonates in the VAD group and 56 neonates in the control group were physically examined for spinal deformities. No gross spinal deformities were noticed in all neonatal rats. However, when examined by radiography at post-gestational age of 7 weeks (day 61 after the vaginal plug was documented in the mother), 13.79% (8/58) of the neonatal rats in the VAD group were found to have congenital spinal anomalies in the thoracic regions of the spine (e.g. fused ribs, hemivertebrate, fused vertebrate), many of which were present in multiple regions in the same rats (Fig. 1B). This is strikingly similar to the human clinical situation in which congenital spinal deformities are not noted on gross examination but subsequently discovered on incidental radiographic examination or with the gradual development of spinal deformities as the child grows. The radiographic appearances of the murine spinal deformities are similar to those humans. None in the control group developed any radiographically detectable spinal deformities. Representative radiographs of a normal and a deformed spine from each group are shown in Fig. 1.
How might VAD be acting to interrupt signalling? RA is the active form of vitamin A and plays a crucial role in stimulating nuclear receptor signaling during development. RA synthesis is a two-step process. The first step, oxidation of retinol to retinaldehyde, is catalyzed by several members of the alcohol dehydrogenase family (Adh1, Ad3,and Adh4); the second step, oxidation of retinaldehyde to RA, is catalyzed by three members of the aldehyde dehydrogenase family (RALDH1, RALDH2, RALDH3) and is irreversible [35]. RA synthesis is controlled both spatially and temporally and three RALDHs identified as catalysts for the second step are expressed in dynamic, nonoverlapping spatiotemporal patterns, indicating that this step is tissue- and time-restricted [37]. In vitro studies have shown that RA serves as a ligand for two families of nuclear receptors, namely, RARs and retinoid X receptor (RXRs), both of which bind DNA as heterodimers and directly regulate gene expression [17], [38]. Previous studies have demonstrated that RA is required for embryogenesis and functions through RARs, whereas RXRs are undetectable in mouse embryos [37]. In these studies, RA was shown to be essential for the development of several organs, including the hindbrain, spinal cord, heart, eye, skeleton, forelimb buds, lung, pancreas and genitourinary tract. Moreover, recent studies showed that loss of RA signaling results in left-right asymmetry of somites in mouse, chick, or zebrafish embryos, in which one side has fewer somites than the other [12], [13]. Administration of RA maternally to RA-deficient mouse embryos also restores normal axial turning and normal spinal column development [37]. These findings suggest that RA signaling is implicated in somitogenesis as well as development of other organs and VAD induced reduction RA signaling may be a common cause of congenital spinal deformities and other organ defects.
Another important finding of our study was that VAD caused a decrease in the mRNA levels of RALDH1, RALDH2, RALDH3, RAR-α, RAR-β and RAR-γ in the liver and RALDH1, RALDH2, RALDH3, RAR-αand RAR-β in the vertebral body. Precious studies have demonstrated that RA was synthesized during vertebrate evolution by these three RALDHs (RALDH1, RALDH2 and RALDH3). RA also mediates its effects on embryogenesis exclusively through RARs but not RXRs [19]. Robinson et al [39]using a transcriptomic approach, they compared RA-exposed and nonexposed rat embryos to identify overlapping and nonoverlapping effects of RA on RNA expression, and their finding had indicated that 845 genes were identified to be significantly time-dependent altered by RA in parallel with morphpological and RA induced upregulation of expression of three enzymes, CYP26A1, CYP26B1, and DHRS3, which are known involved in the breakdown of RA. On the other hand, many reports have demonstracted that VAD suppressed RALDHs, RARs and RXRs mRNA expression in many organs of VAD rats [40], [41]. Moreover, the expressions of RAR-α and RAR-β are also severely downregulated in the VAD embryo in the avian [41], [42]. Our results also have observed that VAD suppressed RALDHs and RARs mRNA expression in the liver and vertebral body of VAD neonatal rats at the day of birth. Therefore, our findings suggest that maternal VAD during pregnancy induced congenital spinal deformities in offspring might also be associated with decrease in RA signalling caused by VAD.
The findings of the present study may have implications for understanding the etiology and mechanism of human congenital spinal deformities. Given that VAD is still a serious and prevalent public health problem in the world especially among pregnant women and children and dietary vitamin A is only available from limited sources, including vegetables in the form of β-carotene or meat as retinyl esters, VAD may be an important cause of human congenital spinal deformities [43]. Three signaling pathways have been proposed to regulate the segmentation clock, namely the Notch, Wnt, and Fgf pathways. Thus far, mutations in the Notch ligand DLL3, LFNG,MESP2, Notch ligand JAGGED1 and polymorphisms in the Tbx6 gene have been identified to be associated with congenital scoliosis in humans [44]. Strikingly, most of these genes are implicated in the modulation of segmentation clock. Our study shows that RA signaling is implicated in the pathogenesis of human congenital spinal deformities. The role of RA signaling in the regulation of segmentation clock, however, awaits further investigation. Moreover, not all rats in the VAD group develop congenital spinal deformities, suggesting that defects in RA signaling may only confer partial susceptibility. 2ff7e9595c
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