Morphological and Molecular Characterization of Retinoic Acid-Induced Limb Duplications in Mice

AbstractThis study reports a morphological, skeletal, and molecular characterization of the supernumerary limbs induced by systemic administration of all-transretinoic acid to egg-cylinder stage mouse embryos. As initially described by Rutledgeet al.(Proc. Natl. Acad. Sci. USA91, 5436, 1994), we have found that oral administration of all-transretinoic acid (70 mg/kg body weight) at 5.5 days postcoitum induced the formation of supernumerary limbs. Most often, these arose as a pair of extra buds located caudally and ventrally to the normal (orthotopic) hindlimb buds without duplication of the lower body axis. The resulting one or two supernumerary hindlimbs were connected to an imperfectly mirror-image-duplicated pelvic girdle. Variable truncations of the stylopodium and zeugopodium skeleton, as well as abnormal splitting of the distal skeleton, were frequently observed. The apical ectodermal ridge of the extra limb buds expressed expected growth factor genes. However, an ectopic anterior expression ofSonic hedgehogandHoxd-13was seen in the supernumerary buds, suggesting that these buds would incorporate potential polarizing cells of the hindlimb or genital field and generate an ectopic polarizing zone. This is consistent with the reverse orientation of most supernumerary limbs at later stages. Some of the buds did not express limb-specific markers and were thus expected to degenerate or form nonlimb structures, as observed in an adult specimen. Less frequently, extra limb buds with normal polarity were associated to a duplicated lower body axis. Retinoic acid also generated a novel type of duplication in which “twin” hindlimbs with two parallel apical ectodermal ridges and zones of polarizing activity arose on one side of the embryo

Hox genes define distinct progenitor sub-domains within the second heart field

AbstractMuch of the heart, including the atria, right ventricle and outflow tract (OFT) is derived from a progenitor cell population termed the second heart field (SHF) that contributes progressively to the embryonic heart during cardiac looping. Several studies have revealed anterior-posterior patterning of the SHF, since the anterior region (anterior heart field) contributes to right ventricular and OFT myocardium whereas the posterior region gives rise to the atria. We have previously shown that Retinoic Acid (RA) signal participates to this patterning. We now show that Hoxb1, Hoxa1, and Hoxa3, as downstream RA targets, are expressed in distinct sub-domains within the SHF. Our genetic lineage tracing analysis revealed that Hoxb1, Hoxa1 and Hoxa3-expressing cardiac progenitor cells contribute to both atria and the inferior wall of the OFT, which subsequently gives rise to myocardium at the base of pulmonary trunk. By contrast to Hoxb1Cre, the contribution of Hoxa1-enhIII-Cre and Hoxa3Cre-labeled cells is restricted to the distal regions of the OFT suggesting that proximo-distal patterning of the OFT is related to SHF sub-domains characterized by combinatorial Hox genes expression. Manipulation of RA signaling pathways showed that RA is required for the correct deployment of Hox-expressing SHF cells. This report provides new insights into the regulatory gene network in SHF cells contributing to the atria and sub-pulmonary myocardium

Retinoic Acid Controls the Bilateral Symmetry of Somite Formation in the Mouse Embryo

A striking characteristic of vertebrate embryos is their bilaterally symmetric body plan, which is particularly obvious at the level of the somites and their derivatives such as the vertebral column. Segmentation of the presomitic mesoderm must therefore be tightly coordinated along the left and right embryonic sides. We show that mutant mice defective for retinoic acid synthesis exhibit delayed somite formation on the right side. Asymmetric somite formation correlates with a left-right desynchronization of the segmentation clock oscillations. These data implicate retinoic acid as an endogenous signal that maintains the bilateral synchrony of mesoderm segmentation, and therefore controls bilateral symmetry, in vertebrate embryos

Retinoic acid deficiency alters second heart field formation

Retinoic acid (RA), the active derivative of vitamin A, has been implicated in various steps of cardiovascular development. The retinaldehyde dehydrogenase 2 (RALDH2) enzyme catalyzes the second oxidative step in RA biosynthesis and its loss of function creates a severe embryonic RA deficiency. Raldh2(−/−) knockout embryos fail to undergo heart looping and have impaired atrial and sinus venosus development. To understand the mechanism(s) producing these changes, we examined the contribution of the second heart field (SHF) to pharyngeal mesoderm, atria, and outflow tract in Raldh2(−/−) embryos. RA deficiency alters SHF gene expression in two ways. First, Raldh2(−/−) embryos exhibited a posterior expansion of anterior markers of the SHF, including Tbx1, Fgf8, and the Mlc1v-nlacZ-24/Fgf10 reporter transgene as well as of Islet1. This occurred at early somite stages, when cardiac defects became irreversible in an avian vitamin A-deficiency model, indicating that endogenous RA is required to restrict the SHF posteriorly. Explant studies showed that this expanded progenitor population cannot differentiate properly. Second, RA up-regulated cardiac Bmp expression levels at the looping stage. The contribution of the SHF to both inflow and outflow poles was perturbed under RA deficiency, creating a disorganization of the heart tube. We also investigated genetic cross-talk between Nkx2.5 and RA signaling by generating double mutant mice. Strikingly, Nkx2.5 deficiency was able to rescue molecular defects in the posterior region of the Raldh2(−/−) mutant heart, in a gene dosage-dependent manner

Retinoic Acid Excess Impairs Amelogenesis Inducing Enamel Defects.

Abnormalities of enamel matrix proteins deposition, mineralization, or degradation during tooth development are responsible for a spectrum of either genetic diseases termed Amelogenesis imperfecta or acquired enamel defects. To assess if environmental/nutritional factors can exacerbate enamel defects, we investigated the role of the active form of vitamin A, retinoic acid (RA). Robust expression of RA-degrading enzymes Cyp26b1 and Cyp26c1 in developing murine teeth suggested RA excess would reduce tooth hard tissue mineralization, adversely affecting enamel. We employed a protocol where RA was supplied to pregnant mice as a food supplement, at a concentration estimated to result in moderate elevations in serum RA levels. This supplementation led to severe enamel defects in adult mice born from pregnant dams, with most severe alterations observed for treatments from embryonic day (E)12.5 to E16.5. We identified the enamel matrix proteins enamelin (Enam), ameloblastin (Ambn), and odontogenic ameloblast-associated protein (Odam) as target genes affected by excess RA, exhibiting mRNA reductions of over 20-fold in lower incisors at E16.5. RA treatments also affected bone formation, reducing mineralization. Accordingly, craniofacial ossification was drastically reduced after 2 days of treatment (E14.5). Massive RNA-sequencing (RNA-seq) was performed on E14.5 and E16.5 lower incisors. Reductions in Runx2 (a key transcriptional regulator of bone and enamel differentiation) and its targets were observed at E14.5 in RA-exposed embryos. RNA-seq analysis further indicated that bone growth factors, extracellular matrix, and calcium homeostasis were perturbed. Genes mutated in human AI (ENAM, AMBN, AMELX, AMTN, KLK4) were reduced in expression at E16.5. Our observations support a model in which elevated RA signaling at fetal stages affects dental cell lineages. Thereafter enamel protein production is impaired, leading to permanent enamel alterations.journal article20162017 01 06importe

Enamel and dental anomalies in latent-transforming growth factor beta-binding protein 3 mutant mice.

Latent-transforming growth factor beta-binding protein 3 (LTBP-3) is important for craniofacial morphogenesis and hard tissue mineralization, as it is essential for activation of transforming growth factor-β (TGF-β). To investigate the role of LTBP-3 in tooth formation we performed micro-computed tomography (micro-CT), histology, and scanning electron microscopy analyses of adult Ltbp3-/- mice. The Ltbp3-/- mutants presented with unique craniofacial malformations and reductions in enamel formation that began at the matrix formation stage. Organization of maturation-stage ameloblasts was severely disrupted. The lateral side of the incisor was affected most. Reduced enamel mineralization, modification of the enamel prism pattern, and enamel nodules were observed throughout the incisors, as revealed by scanning electron microscopy. Molar roots had internal irregular bulbous-like formations. The cementum thickness was reduced, and microscopic dentinal tubules showed minor nanostructural changes. Thus, LTBP-3 is required for ameloblast differentiation and for the formation of decussating enamel prisms, to prevent enamel nodule formation, and for proper root morphogenesis. Also, and consistent with the role of TGF-β signaling during mineralization, almost all craniofacial bone components were affected in Ltbp3-/- mice, especially those involving the upper jaw and snout. This mouse model demonstrates phenotypic overlap with Verloes Bourguignon syndrome, also caused by mutation of LTBP3, which is hallmarked by craniofacial anomalies and amelogenesis imperfecta phenotypes.journal article2017 Febimporte

Retinoic Acid-Dependent Signaling Pathways and Lineage Events in the Developing Mouse Spinal Cord

Studies in avian models have demonstrated an involvement of retinoid signaling in early neural tube patterning. The roles of this signaling pathway at later stages of spinal cord development are only partly characterized. Here we use Raldh2-null mouse mutants rescued from early embryonic lethality to study the consequences of lack of endogenous retinoic acid (RA) in the differentiating spinal cord. Mid-gestation RA deficiency produces prominent structural and molecular deficiencies in dorsal regions of the spinal cord. While targets of Wnt signaling in the dorsal neuronal lineage are unaltered, reductions in Fibroblast Growth Factor (FGF) and Notch signaling are clearly observed. We further provide evidence that endogenous RA is capable of driving stem cell differentiation. Raldh2 deficiency results in a decreased number of spinal cord derived neurospheres, which exhibit a reduced differentiation potential. Raldh2-null neurospheres have a decreased number of cells expressing the neuronal marker β-III-tubulin, while the nestin-positive cell population is increased. Hence, in vivo retinoid deficiency impaired neural stem cell growth. We propose that RA has separable functions in the developing spinal cord to (i) maintain high levels of FGF and Notch signaling and (ii) drive stem cell differentiation, thus restricting both the numbers and the pluripotent character of neural stem cells

Minimal DNA sequences that control the cell lineage-specific expression of the proalpha2(I) collagen promoter in transgenic mice

The pattern of expression of the pro$\alpha$2(I) collagen gene is highly tissue-specific in adult mice and shows its strongest expression in bones, tendons, and skin. Transgenic mice were generated harboring promoter fragments of the mouse pro$\alpha$2(I) collagen gene linked to the Escherichia coli $\beta$-galactosidase or firefly luciferase genes to examine the activity of these promoters during development. A region of the mouse pro$\alpha$2(I) collagen promoter between $-$2000 and +54 exhibited a pattern of $\beta$-galactosidase activity during embryonic development that corresponded to the expression pattern of the endogenous pro$\alpha$2(I) collagen gene as determined by in situ hybridization. A similar pattern of activity was also observed with much smaller promoter fragments containing either 500 or 350 bp of upstream sequence relative to the start of transcription. Embryonic regions expressing high levels of $\beta$-galactosidase activity included the valves of the developing heart, sclerotomes, meninges, limb buds, connective tissue fascia between muscle fibers, osteoblasts, tendon, periosteum, dermis, and peritoneal membranes. The pattern of $\beta$-galactosidase activity was similar to the extracellular immunohistochemical localization of transforming growth factor-$\beta$1 (TGF-$\beta$1). The $-$315 to $-$284 region of the pro$\alpha$2(I) collagen promoter was previously shown to mediate the stimulatory effects of TGF-$\beta$1 on the pro$\alpha$2(I) collagen promoter in DNA transfection experiments with cultured fibroblasts. A construct containing this sequence tandemly repeated 5\sp\prime to both a very short $\alpha$2(I) collagen promoter ($-$40 to +54) and a heterologous minimal promoter showed preferential activity in tail and skin of 4-week old transgenic mice. The pattern of expression mimics that of the $-$350 to +54 pro$\alpha$2(I) collagen promoter linked to a luciferase reporter gene in transgenic mice

Expression of enzymes synthesizing (aldehyde dehydrogenase 1 and reinaldehyde dehydrogenase 2) and metabolizaing (Cyp26) retinoic acid in the mouse female reproductive system

Vitamin A is required for female reproduction. Rodent uterine cells are able to synthesize retinoic acid (RA), the active vitamin A derivative, and express RA receptors. Here, we report that two RA-synthesizing enzymes [aldehyde dehydrogenase 1 (Aldh1) and retinaldehyde dehydrogenase 2 (Raldh2)] and a cytochrome P450 (Cyp26) that metabolizes vitamin A and RA into more polar metabolites exhibit dynamic expression patterns in the mouse uterus, both during the ovarian cycle and during early pregnancy. Aldh1 expression is up-regulated during diestrus and proestrus in the uterine glands, whereas Raldh2 is highly induced in the endometrial stroma in metestrus. Cyp26 expression, which is not detectable during the normal ovarian cycle, is strongly induced in the uterine luminal epithelium, 24 h after human CG hormonal administration. Raldh2 stromal expression also strongly responds to gonadotropin (PMSG and human CG) induction. Furthermore, Raldh2 expression can be hormonally induced in stromal cells of the vagina and cervix. All three enzymes exhibit differential expression profiles during early pregnancy. Aldh1 glandular expression is sharply induced at 2.5 gestational days, whereas Raldh2 stromal expression increases more steadily until the implantation phase. Cyp26 epithelial expression is strongly induced between 3.5-4.5 gestational days, i.e. when the developing blastocysts colonize the uterine lumen. These data suggest a need for precise regulation of RA synthesis and/or metabolism, in both cycling and pregnant uterus