tretinoin and 5-bromo-4-chloro-3-indolyl-beta-galactoside

Retinoic acid (RA), an active vitamin A derivative, is essential for mammalian spermatogenesis. Genetic studies have revealed that oxidation of vitamin A to retinal by retinol dehydrogenase 10 (RDH10) is critical for embryonic RA biosynthesis. However, physiological roles of RDH10 in postnatal RA synthesis remain unclear, given that Rdh10 loss-of-function mutations lead to early embryonic lethality. We conducted in vivo genetic studies of Rdh10 in postnatal mouse testes and found that an RDH10 deficiency in Sertoli cells, but not in germ cells, results in a mild germ cell depletion phenotype. A deficiency of RDH10 in both Sertoli and germ cells in juvenile mice results in a blockage of spermatogonial differentiation, similar to that seen in vitamin A-deficient animals. This defect in spermatogenesis arises from a complete deficiency in juvenile testicular RA synthesis and can be rescued by retinoid administration. Thus, in juvenile mice, the primary, but not exclusive, source of RA in the testes is Sertoli cells. In contrast, adult Rdh10-deficient mice exhibit phenotypically normal spermatogenesis, indicating that during development a change occurs in either the cellular source of RA or the retinaldehyde dehydrogenase involved in RA synthesis.

Topics: Alcohol Oxidoreductases; Animals; DNA Primers; Galactosides; Histological Techniques; Immunohistochemistry; In Situ Nick-End Labeling; Indoles; Male; Mice; Mice, Knockout; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Sertoli Cells; Spermatogenesis; Testis; Tretinoin; Vitamin A

The progress of molecular genetics has enabled us to identify the genes responsible for congenital heart malformations. However, recent studies suggest that congenital heart diseases are induced not only by mutations in certain genes, but also by abnormal maternal factors. A high concentration of maternal retinoic acid (RA), the active derivative of vitamin A, is well known as a teratogenic agent that can cause developmental defects. Our previous studies have shown that the maternal administration of RA to mice within a narrow developmental window induces outflow tract (OFT) septum defects, a condition that closely resembles human transposition of the great arteries (TGA), although the responsible factors and pathogenic mechanisms of the TGA induced by RA remain unknown. We herein demonstrate that the expression of Tbx2 in the OFT myocardium is responsive to RA, and its downregulation is associated with abnormal OFT development. We found that RA could directly downregulate the Tbx2 expression through a functional retinoic acid response element (RARE) in the Tbx2 promoter region, which is also required for the initiation of Tbx2 transcription during OFT development. Tgfb2 expression was also downregulated in the RA-treated OFT region and was upregulated by Tbx2 in a culture system. Moreover, defective epithelial-mesenchymal transition caused by the excess RA was rescued by the addition of Tgfβ2 in an organ culture system. These data suggest that RA signaling participates in the Tbx2 transcriptional mechanism during OFT development and that the Tbx2-Tgfβ2 cascade is one of the key pathways involved in inducing the TGA phenotype.

Topics: Animals; Chromatin Immunoprecipitation; DNA Primers; Endocardial Cushion Defects; Female; Galactosides; Gene Expression Regulation, Developmental; Immunohistochemistry; In Situ Hybridization; Indoles; Luciferases; Maternal-Fetal Exchange; Mice; Microarray Analysis; Myocardium; Pregnancy; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; T-Box Domain Proteins; Transforming Growth Factor beta2; Tretinoin

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.

Topics: Alcohol Oxidoreductases; Animals; Body Patterning; Branchial Region; Galactosides; Gene Expression Regulation, Developmental; Gene Targeting; Histological Techniques; In Situ Hybridization; Indoles; Mice; Mice, Knockout; Models, Biological; Retinaldehyde; Rhombencephalon; Signal Transduction; Tretinoin; Vitamin A

A central issue during embryonic development is to define how different signals cooperate in generating unique cell types. To address this issue, we focused on the function and the regulation of the proneural gene Neurogenin2 (Neurog2) during early mouse spinal neurogenesis. We showed that Neurog2 is first expressed in cells within the neural plate anterior to the node from the 5 somite-stage. The analysis of Neurog2 mutants established a role for this gene in triggering neural differentiation during spinal cord elongation. We identified a 798 base pair enhancer element (Neurog2-798) upstream of the Neurog2 coding sequence that directs the early caudal expression of Neurog2. Embryo culture experiments showed that Retinoic Acid (RA), Sonic hedgehog (Shh) and Fibroblast Growth Factor signals act in concert on this enhancer to control the spatial and temporal induction of Neurog2. We further demonstrated by transgenesis that two RA response elements and a Gli binding site within the Neurog2-798 element are absolutely required for its activity, strongly suggesting that the regulation of Neurog2 early expression by RA and Shh signals is direct. Our data thus support a model where signal integration at the level of a single enhancer constitutes a key mechanism to control the onset of neurogenesis.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Electrophoretic Mobility Shift Assay; Fibroblast Growth Factors; Galactosides; Gene Expression Regulation, Developmental; Hedgehog Proteins; Immunohistochemistry; In Situ Hybridization; Indoles; Mice; Mice, Transgenic; Nerve Tissue Proteins; Oligonucleotides; Signal Transduction; Spinal Cord; Tretinoin

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.

Topics: Aldehyde Oxidoreductases; Animals; Blood Vessels; Body Patterning; Brain; Extremities; Galactosides; Homeostasis; Immunohistochemistry; In Situ Hybridization; Indoles; Mice; Mice, Knockout; Microscopy, Electron, Scanning; Mutation; NADPH-Ferrihemoprotein Reductase; Tretinoin

Exogenous retinoic acid (RA) induces marked effects on limb patterning, but the precise role of endogenous RA in this process has remained unknown. We have studied the role of RA in mouse limb development by focusing on CYP26B1, a cytochrome P450 enzyme that inactivates RA. Cyp26b1 was shown to be expressed in the distal region of the developing limb bud, and mice that lack CYP26B1 exhibited severe limb malformation (meromelia). The lack of CYP26B1 resulted in spreading of the RA signal toward the distal end of the developing limb and induced proximodistal patterning defects characterized by expansion of proximal identity and restriction of distal identity. CYP26B1 deficiency also induced pronounced apoptosis in the developing limb and delayed chondrocyte maturation. Wild-type embryos exposed to excess RA phenocopied the limb defects of Cyp26b1(-/-) mice. These observations suggest that RA acts as a morphogen to determine proximodistal identity, and that CYP26B1 prevents apoptosis and promotes chondrocyte maturation, in the developing limb.

Topics: Abnormalities, Drug-Induced; Aging; Aldehyde Oxidoreductases; Animals; Animals, Newborn; Body Patterning; Bromodeoxyuridine; Carcinoma; Cell Death; Cell Differentiation; Cell Line, Tumor; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Embryo, Mammalian; Embryonic Induction; Extremities; Female; Fibroblast Growth Factor 8; Fibroblast Growth Factors; Galactosides; Gene Expression Regulation, Developmental; High Mobility Group Proteins; Homeodomain Proteins; In Situ Hybridization; In Situ Nick-End Labeling; Indoles; Limb Buds; Male; Mesoderm; Mice; Mice, Knockout; Models, Biological; Pregnancy; Retinoic Acid 4-Hydroxylase; SOX9 Transcription Factor; Time Factors; Trans-Activators; Transcription Factors; Transfection; Tretinoin

The germ cell nuclear factor (GCNF, NR6A1) is a nuclear orphan receptor that functions as a transcriptional repressor and is transiently expressed in mammalian carcinoma cells during retinoic acid (RA) induced neuronal differentiation. During Xenopus laevis development, the spatiotemporal expression pattern of embryonic GCNF (xEmGCNF) suggests a role in anteroposterior specification of the neuroectoderm. Here, we show that RA treatment of Xenopus embryos enhances xEmGCNF expression. Moreover, we present evidence for the relevance of this finding in the context of primary neurogenesis and hindbrain development. During early development of the central nervous system, RA signals promote posterior transformation of the neuroectoderm and increase the number of cells undergoing primary neurogenesis. Our loss-of-function analyses using a xEmGCNF-specific morpholino antisense oligonucleotide indicate that xEmGCNF is required for the effect of RA on primary neurogenesis. This may be caused by transcriptional regulation of the gene encoding the RA-degrading enzyme CYP26, since this gene is derepressed after depletion of xEmGCNF and an antimorph of xEmGCNF directly activates transcription of CYP26, also in absence of protein synthesis. The effect of xEmGCNF knockdown on hindbrain patterning is similar to conditions of reduced RA signaling, which may be caused by a reduction of RAR gamma expression specifically in the presumptive hindbrain.

Topics: Animals; Blotting, Western; Brain; Cycloheximide; Cytochrome P-450 Enzyme System; DNA-Binding Proteins; Galactosides; Gene Expression Regulation, Developmental; In Situ Hybridization; Indoles; Neurons; Nuclear Receptor Subfamily 6, Group A, Member 1; Protein Biosynthesis; Protein Synthesis Inhibitors; Receptors, Cytoplasmic and Nuclear; Retinoic Acid 4-Hydroxylase; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Time Factors; Tretinoin; Xenopus

The molecular mechanism underlying the 3' to 5' polarity of induction of mouse Hox genes is still elusive. While relief from a cluster-encompassing repression was shown to lead to all Hoxd genes being expressed like the 3'most of them, Hoxd1 (Kondo and Duboule, 1999), the molecular basis of initial activation of this 3'most gene, is not understood yet. We show that, already before primitive streak formation, prior to initial expression of the first Hox gene, a dramatic transcriptional stimulation of the 3'most genes, Hoxb1 and Hoxb2, is observed upon a short pulse of exogenous retinoic acid (RA), whereas it is not in the case for more 5', cluster-internal, RA-responsive Hoxb genes. In contrast, the RA-responding Hoxb1lacZ transgene that faithfully mimics the endogenous gene (Marshall et al., 1994) did not exhibit the sensitivity of Hoxb1 to precocious activation. We conclude that polarity in initial activation of Hoxb genes reflects a greater availability of 3'Hox genes for transcription, suggesting a pre-existing (susceptibility to) opening of the chromatin structure at the 3' extremity of the cluster. We discuss the data in the context of prevailing models involving differential chromatin opening in the directionality of clustered Hox gene transcription, and regarding the importance of the cluster context for correct timing of initial Hox gene expression.Interestingly, Cdx1 manifested the same early transcriptional availability as Hoxb1.

Topics: Animals; Chromatin; Galactosides; Gene Expression Regulation, Developmental; Homeodomain Proteins; In Situ Hybridization; Indoles; Lac Operon; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; RNA, Messenger; Time Factors; Transcription Factors; Transcription, Genetic; Transgenes; Tretinoin

Eph receptors have been implicated in cell-to-cell interaction during embryogenesis. We generated EphA2 mutant mice using a gene trap method. Homozygous mutant mice developed short and kinky tails. In situ hybridization using a Brachyury probe found the notochord to be abnormally bifurcated at the caudal end between 11.5 and 12.5 days post coitum. EphA2 was expressed at the tip of the tail notochord, while one of its ligands, ephrinA1, was at the tail bud in normal mice. In contrast, EphA2-deficient notochordal cells were spread broadly into the tail bud. These observations suggest that EphA2 and its ligands are involved in the positioning of the tail notochord through repulsive signals between cells expressing these molecules on the surface.

Topics: Animals; Base Sequence; Blotting, Northern; Blotting, Southern; Cell Communication; DNA, Complementary; Ephrin-A1; Female; Fetal Proteins; Galactosides; Genetic Vectors; Hedgehog Proteins; Homozygote; In Situ Hybridization; Indoles; Ligands; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Mice, Transgenic; Models, Genetic; Molecular Sequence Data; Notochord; Phenotype; Protein Binding; Protein Biosynthesis; Proteins; Receptor Protein-Tyrosine Kinases; Receptor, EphA2; Signal Transduction; T-Box Domain Proteins; Tail; Time Factors; Trans-Activators; Tretinoin