[show abstract][hide abstract] ABSTRACT: Retinoic acid (RA), an active vitamin A metabolite, is a key signaling molecule in vertebrate embryos. Morphogenetic RA gradients are thought to be set up by tissue-specific actions of retinaldehyde dehydrogenases (RALDHs) and catabolizing enzymes. According to the species, two enzymatic pathways (β-carotene cleavage and retinol oxidation) generate retinaldehyde, the substrate of RALDHs. Placental species depend on maternal retinol transferred to the embryo. The retinol-to-retinaldehyde conversion was thought to be achieved by several redundant enzymes; however, a random mutagenesis screen identified retinol dehydrogenase 10 [Rdh10(Trex) allele; Sandell LL, et al. (2007) Genes Dev 21:1113-1124] as responsible for a homozygous lethal phenotype with features of RA deficiency. We report here the production and characterization of unique murine Rdh10 loss-of-function alleles generated by gene targeting. We show that although Rdh10(-/-) mutants die at an earlier stage than Rdh10(Trex) mutants, their molecular patterning defects do not reflect a complete state of RA deficiency. Furthermore, we were able to correct most developmental abnormalities by administering retinaldehyde to pregnant mothers, thereby obtaining viable Rdh10(-/-) mutants. This demonstrates the rescue of an embryonic lethal phenotype by simple maternal administration of the missing retinoid compound. These results underscore the importance of maternal retinoids in preventing congenital birth defects, and lead to a revised model of the importance of RDH10 and RALDHs in controlling embryonic RA distribution.
Proceedings of the National Academy of Sciences 09/2011; 108(40):16687-92. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: The progressive generation of embryonic trunk structures relies on the proper patterning of the caudal epiblast, which involves the integration of several signalling pathways. We have investigated the function of retinoic acid (RA) signalling during this process. We show that, in addition to posterior mesendoderm, primitive streak and node cells transiently express the RA-synthesizing enzyme Raldh2 prior to the headfold stage. RA-responsive cells (detected by the RA-activated RARE-lacZ transgene) are additionally found in the epiblast layer. Analysis of RA-deficient Raldh2(-/-) mutants reveals early caudal patterning defects, with an expansion of primitive streak and mesodermal markers at the expense of markers of the prospective neuroepithelium. As a result, many genes involved in neurogenesis and/or patterning of the embryonic spinal cord are affected in their expression. We demonstrate that RA signalling is required at late gastrulation stages for mesodermal and neural progenitors to respond to the Shh signal. Whole-embryo culture experiments indicate that the proper response of cells to Shh requires two RA-dependent mechanisms: (1) a balanced antagonism between Fgf and RA signals, and (2) a RA-mediated repression of Gli2 expression. Thus, an interplay between RA, Fgf and Shh signalling is likely to be an important mechanism underpinning the tight regulation of caudal embryonic development.
Development 03/2009; 136(4):665-76. · 6.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cytochrome P450 oxidoreductase (POR) acts as an electron donor for all cytochrome P450 enzymes. Knockout mouse Por(-/-) mutants, which are early embryonic (E9.5) lethal, have been found to have overall elevated retinoic acid (RA) levels, leading to the idea that POR early developmental function is mainly linked to the activity of the CYP26 RA-metabolizing enzymes (Otto et al., Mol. Cell. Biol. 23, 6103-6116). By crossing Por mutants with a RA-reporter lacZ transgene, we show that Por(-/-) embryos exhibit both elevated and ectopic RA signaling activity e.g. in cephalic and caudal tissues. Two strategies were used to functionally demonstrate that decreasing retinoid levels can reverse Por(-/-) phenotypic defects, (i) by culturing Por(-/-) embryos in defined serum-free medium, and (ii) by generating compound mutants defective in RA synthesis due to haploinsufficiency of the retinaldehyde dehydrogenase 2 (Raldh2) gene. Both approaches clearly improved the Por(-/-) early phenotype, the latter allowing mutants to be recovered up until E13.5. Abnormal brain patterning, with posteriorization of hindbrain cell fates and defective mid- and forebrain development and vascular defects were rescued in E9.5 Por(-/-) embryos. E13.5 Por(-/-); Raldh2(+/-) embryos exhibited abdominal/caudal and limb defects that strikingly phenocopy those of Cyp26a1(-/-) and Cyp26b1(-/-) mutants, respectively. Por(-/-); Raldh2(+/-) limb buds were truncated and proximalized and the anterior-posterior patterning system was not established. Thus, POR function is indispensable for the proper regulation of RA levels and tissue distribution not only during early embryonic development but also in later morphogenesis and molecular patterning of the brain, abdominal/caudal region and limbs.
[show abstract][hide abstract] ABSTRACT: Retinoic acid (RA) activity plays sequential roles during the development of the ventral spinal cord. Here, we have investigated the functions of local RA synthesis in the process of motoneuron specification and early differentiation using a conditional knockout strategy that ablates the function of the retinaldehyde dehydrogenase 2 (Raldh2) synthesizing enzyme essentially in brachial motoneurons, and later in mesenchymal cells at the base of the forelimb. Mutant (Raldh2L-/-) embryos display an early embryonic loss of a subset of Lim1+ brachial motoneurons, a mispositioning of Islet1+ neurons and inappropriate axonal projections of one of the nerves innervating extensor limb muscles, which lead to an adult forepaw neuromuscular defect. The molecular basis of the Raldh2L-/- phenotype relies in part on the deregulation of Hoxc8, which in turn regulates the RA receptor RARbeta. We further show that Hoxc8 mutant mice, which exhibit a similar congenital forepaw defect, display at embryonic stages molecular defects that phenocopy the Raldh2L-/- motoneuron abnormalities. Thus, interdependent RA signaling and Hox gene functions are required for the specification of brachial motoneurons in the mouse.
Development 05/2005; 132(7):1611-21. · 6.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: Targeted inactivation of the mouse retinaldehyde dehydrogenase 2 (RALDH2/ALDH1a2), the enzyme responsible for early embryonic retinoic acid synthesis, is embryonic lethal because of defects in early heart morphogenesis. Transient maternal RA supplementation from E7.5 to (at least) E8.5 rescues most of these defects, but the supplemented Raldh2(-/-) mutants die prenatally, from a lack of septation of the heart outflow tract (Niederreither, K., Vermot, J., Messaddeq, N., Schuhbaur, B., Chambon, P. and Dollé, P. (2001). Development 128, 1019-1031). We have investigated the developmental basis for this defect, and found that the RA-supplemented Raldh2(-/-) embryos exhibit impaired development of their posterior (3rd-6th) branchial arch region. While the development of the first and second arches and their derivatives, as well as the formation of the first branchial pouch, appear to proceed normally, more posterior pharyngeal pouches fail to form and the pharyngeal endoderm develops a rudimentary, pouch-like structure. All derivatives of the posterior branchial arches are affected. These include the aortic arches, pouch-derived organs (thymus, parathyroid gland) and post-otic neural crest cells, which fail to establish segmental migratory pathways and are misrouted caudally. Patterning and axonal outgrowth of the posterior (9th-12th) cranial nerves is also altered. Vagal crest deficiency in Raldh2(-/-) mutants leads to agenesis of the enteric ganglia, a condition reminiscent of human Hirschprung's disease. In addition, we provide evidence that: (i) wildtype Raldh2 expression is restricted to the posteriormost pharyngeal mesoderm; (ii) endogenous RA response occurs in both the pharyngeal endoderm and mesoderm, and extends more rostrally than Raldh2 expression up to the 2nd arch; (iii) RA target genes (Hoxa1, Hoxb1) are downregulated in both the pharyngeal endoderm and mesoderm of mutant embryos. Thus, RALDH2 plays a crucial role in producing RA required for pharyngeal development, and RA is one of the diffusible mesodermal signals that pattern the pharyngeal endoderm.
Development 07/2003; 130(11):2525-34. · 6.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: Numerous studies, often performed on avian embryos, have implicated retinoic acid (RA) in the control of limb bud growth and patterning. Here we have investigated whether the lack of endogenous RA synthesis affects limb morphogenesis in mutant mouse embryos deficient for the retinaldehyde dehydrogenase 2 (Raldh2/Aldh1a2). These mutants, which have no detectable embryonic RA except in the developing retina, die at E9.5-E10 without any evidence of limb bud formation, but maternal RA supplementation through oral gavage from E7.5 can extend their survival. Such survivors exhibit highly reduced forelimb rudiments, but apparently normal hindlimbs. By providing RA within maternal food, we found both a stage- and dose-dependency for rescue of forelimb growth and patterning. Following RA supplementation from E7.5 to 8.5, mutant forelimbs are markedly hypoplastic and lack anteroposterior (AP) patterning, with a single medial cartilage and 1-2 digit rudiments. RA provided until E9.5 significantly rescues forelimb growth, but cannot restore normal AP patterning. Increasing the RA dose rescues the hypodactyly, but leads to lack of asymmetry of the digit pattern, with abnormally long first digit or symmetrical polydactyly. Mutant forelimb buds are characterized by lack of expression or abnormal distal distribution of Sonic hedgehog (Shh) transcripts, sometimes with highest expression anteriorly. Downregulation or ectopic anterior expression of Fgf4 is also seen. As a result, genes such as Bmp2 or Hoxd genes are expressed symmetrically along the AP axis of the forelimb buds, and/or later, of the autopod. We suggest that RA signaling cooperates with a posteriorly restricted factor such as dHand, to generate a functional zone of polarizing activity (ZPA).
Development 09/2002; 129(15):3563-74. · 6.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: Retinoic acid, the active derivative of vitamin A (retinol), is a hormonal signaling molecule that acts in developing and adult tissues. The Cyp26a1 (cytochrome p450, 26) protein metabolizes retinoic acid into more polar hydroxylated and oxidized derivatives. Whether some of these derivatives are biologically active metabolites has been debated. Cyp26a1(-/-) mouse fetuses have lethal morphogenetic phenotypes mimicking those generated by excess retinoic acid administration, indicating that human CYP26A1 may be essential in controlling retinoic acid levels during development. This hypothesis suggests that the Cyp26a1(-/-) phenotype could be rescued under conditions in which embryonic retinoic acid levels are decreased. We show that Cyp26a1(-/-) mice are phenotypically rescued by heterozygous disruption of Aldh1a2 (also known as Raldh2), which encodes a retinaldehyde dehydrogenase responsible for the synthesis of retinoic acid during early embryonic development. Aldh1a2 haploinsufficiency prevents the appearance of spina bifida and rescues the development of posterior structures (sacral/caudal vertebrae, hindgut, urogenital tract), while partly preventing cervical vertebral transformations and hindbrain pattern alterations in Cyp26a1(-/-) mice. Thus, some of these double-mutant mice can reach adulthood. This study is the first report of a mutation acting as a dominant suppressor of a lethal morphogenetic mutation in mammals. We provide genetic evidence that ALDH1A2 and CYP26A1 activities concurrently establish local embryonic retinoic acid levels that must be finely tuned to allow posterior organ development and to prevent spina bifida.
[show abstract][hide abstract] ABSTRACT: Retinoic acid (RA), the active derivative of vitamin A, has been implicated in various steps of cardiovascular development, but its contribution to early heart morphogenesis has not been clearly established in a mammalian system. To block endogenous RA synthesis, we have disrupted the gene encoding RALDH2, the first retinaldehyde dehydrogenase whose expression has been detected during early mouse post-implantation development. We describe here the heart abnormalities of the RA-deficient Raldh2 mutants that die in utero at gestational day 10.5. The embryonic heart tube forms properly, but fails to undergo rightward looping and, instead, forms a medial distended cavity. Expression of early heart determination factors is not altered in mutants, and the defect in heart looping does not appear to involve the Nodal/Lefty/Pitx2 pathway. Histological and molecular analysis reveal distinct anteroposterior components in the mutant heart tube, although posterior chamber (atria and sinus venosus) development is severely impaired. Instead of forming trabeculae, the developing ventricular myocardium consists of a thick layer of loosely attached cells. Ultrastructural analysis shows that most of the ventricular wall consists of prematurely differentiated cardiomyocytes, whereas undifferentiated cells remain clustered rostrally. We conclude that embryonic RA synthesis is required for realization of heart looping, development of posterior chambers and proper differentiation of ventricular cardiomyocytes. Nevertheless, the precise location of this synthesis may not be crucial, as these defects can mostly be rescued by systemic (maternal) RA administration. However, cardiac neural crest cells cannot be properly rescued in Raldh2(-/- )embryos, leading to outflow tract septation defects.
Development 05/2001; 128(7):1019-31. · 6.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: Targeted disruption of the murine retinaldehyde dehydrogenase 2 (Raldh2) gene precludes embryonic retinoic acid (RA) synthesis, leading to midgestational lethality (Niederreither, K., Subbarayan, V., Dolle, P. and Chambon, P. (1999). Nature Genet. 21, 444-448). We describe here the effects of this RA deficiency on the development of the hindbrain and associated neural crest. Morphological segmentation is impaired throughout the hindbrain of Raldh2-/- embryos, but its caudal portion becomes preferentially reduced in size during development. Specification of the midbrain region and of the rostralmost rhombomeres is apparently normal in the absence of RA synthesis. In contrast, marked alterations are seen throughout the caudal hindbrain of mutant embryos. Instead of being expressed in two alternate rhombomeres (r3 and r5), Krox20 is expressed in a single broad domain, correlating with an abnormal expansion of the r2-r3 marker Meis2. Instead of forming a defined r4, Hoxb1- and Wnt8A-expressing cells are scattered throughout the caudal hindbrain, whereas r5/r8 markers such as kreisler or group 3/4 Hox genes are undetectable or markedly downregulated. Lack of alternate Eph receptor gene expression could explain the failure to establish rhombomere boundaries. Increased apoptosis and altered migratory pathways of the posterior rhombencephalic neural crest cells are associated with impaired branchial arch morphogenesis in mutant embryos. We conclude that RA produced by the embryo is required to generate posterior cell fates in the developing mouse hindbrain, its absence leading to an abnormal r3 (and, to a lesser extent, r4) identity of the caudal hindbrain cells.
Development 02/2000; 127(1):75-85. · 6.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: Pluripotent mouse P19 embryonal carcinoma (EC) cells have been extensively used as a developmental model system because they can differentiate in the presence of retinoic acid (RA) into derivatives of all three germ layers depending on RA dosage and culture conditions. The expression of several genes has been shown to be induced in RA-treated P19 EC cells and, interestingly, some of these genes may play important roles during mouse embryogenesis. In view of the increasing evidence that RA is a crucial signaling molecule during vertebrate development, we have initiated a study aimed at the systematic isolation of genes whose expression is induced in P19 cells at various times after exposure to RA. We describe here an efficient differential subtractive hybridization cloning strategy which was used to identify additional RA-responsive genes in P19 cells. Fifty different cDNA fragments corresponding to RA-induced genes were isolated. Ten cDNAs represent known genes, 4 of which have already been described as RA-inducible, while the remaining 40 correspond to novel genes. Many of these cDNA sequences represent low-abundance mRNAs. Kinetic analysis of mRNA accumulation following RA treatment allowed us to characterize four classes of RA-responsive genes. We also report the sequence and expression pattern in mouse embryos and adult tissues of one of these novel RA-inducible genes, Stra1, and show that it corresponds to the mouse ligand for the Cek5 receptor protein-tyrosine kinase.
[show abstract][hide abstract] ABSTRACT: Vertebrate Hoxd genes are sequentially activated during the morphogenesis and pattern formation of the limb. Using the approach of gene disruption via homologous recombination in embryonic stem cells, we have assessed the function of the last gene of the complex, Hoxd-13. Mutant mice displayed skeletal alterations along all body axes suggesting the existence of a general multiaxial patterning system. In limbs, abnormalities such as a reduction in the length of some bony elements, loss of phalanges, bone fusions, and the presence of an extra element were observed. We propose that the mutation induces local heterochrony, as illustrated by an important retardation in limb morphogenesis. The relevance of these observations to our understanding of the development and evolution of the tetrapod limb is discussed.
[show abstract][hide abstract] ABSTRACT: An rp lambda 5 cDNA encoding a ribosomal protein (r-protein) and a pseudogenic form of the corresponding gene (rp lambda 7) have been cloned from mouse. This cDNA codes for a highly basic protein of 160 amino acids (aa) with a deduced M(r) of 17,601, and most likely represents the species homolog of a recently cloned rat cDNA, which has been proposed to encode a homolog of the yeast r-protein, YL43. The entire rp lambda 5 gene encompasses less than 1.5 kb of genomic DNA and apparently is composed of only two exons, as deduced from sequence comparison with its very similar pseudogenic variant, rp lambda 7. Southern analysis, using the rp lambda 5 cDNA as a probe, indicates the existence of a great number of highly related sequences in the mouse genome. The mRNA for rp lambda 5 is approximately 800 nucleotides (nt) long and is found to be ubiquitously expressed at high levels in embryonic and adult mouse tissues, as shown by Northern and in situ analyses. Retinoic acid (RA) seems to have a moderate down-regulatory effect on this mRNA in differentiating P19 embryonal carcinoma cells. Several degenerate/nondegenerate RA-response element (RARE) motifs are found within 560 bp upstream from the degenerate start codon in rp lambda 7. However, it is unknown whether this RA effect is exerted at the transcriptional and/or posttranscriptional levels.