Deficiency of the SMOC2 matricellular protein impairs bone healing and produces age-dependent bone loss.

Secreted extracellular matrix components which regulate craniofacial development could be reactivated and play roles in adult wound healing. We report a patient with a loss-of-function of the secreted matricellular protein SMOC2 (SPARC related modular calcium binding 2) presenting severe oligodontia, microdontia, tooth root deficiencies, alveolar bone hypoplasia, and a range of skeletal malformations. Turning to a mouse model, Smoc2-GFP reporter expression indicates SMOC2 dynamically marks a range of dental and bone progenitors. While germline Smoc2 homozygous mutants are viable, tooth number anomalies, reduced tooth size, altered enamel prism patterning, and spontaneous age-induced periodontal bone and root loss are observed in this mouse model. Whole-genome RNA-sequencing analysis of embryonic day (E) 14.5 cap stage molars revealed reductions in early expressed enamel matrix components (Odontogenic ameloblast-associated protein) and dentin dysplasia targets (Dentin matrix acidic phosphoprotein 1). We tested if like other matricellular proteins SMOC2 was required for regenerative repair. We found that the Smoc2-GFP reporter was reactivated in adjacent periodontal tissues 4 days after tooth avulsion injury. Following maxillary tooth injury, Smoc2-/- mutants had increased osteoclast activity and bone resorption surrounding the extracted molar. Interestingly, a 10-day treatment with the cyclooxygenase 2 (COX2) inhibitor ibuprofen (30 mg/kg body weight) blocked tooth injury-induced bone loss in Smoc2-/- mutants, reducing matrix metalloprotease (Mmp)9. Collectively, our results indicate that endogenous SMOC2 blocks injury-induced jaw bone osteonecrosis and offsets age-induced periodontal decay.

The role of nuclear receptors in the differentiation of oligodendrocyte precursor cells derived from fetal and adult neural stem cells.

Oligodendrocyte precursor cells (OPCs) differentiation from multipotent neural stem cells (NSCs) into mature oligodendrocytes is driven by thyroid hormone and mediated by thyroid hormone receptors (TRs). We show that several nuclear receptors display strong changes in expression levels between fetal and adult NSCs, with an overexpression of TRß and a lower expression of RXRγ in adult. Such changes may determine the reduced capacity of adult OPCs to differentiate as supported by reduced yield of maturation and compromised mRNA expression of key genes. RXRγ may be the determinant of these differences, on the evidence of reduced number of mature oligodendrocytes and increased number of proliferating OPCs in RXRγ-/- cultures. Such data also points to RXRγ as an important regulator of the cell cycle exit, as proved by the dysregulation of T3-induced cell cycle exit-related genes. Our data highlight the biological differences between fetal and adult OPCs and demonstrate the essential role of RXRγ in the T3-mediated OPCs maturation process.

Meningeal retinoic acid contributes to neocortical lamination and radial migration during mouse brain development.

Retinoic acid (RA) is a diffusible molecule involved in early forebrain patterning. Its later production in the meninges by the retinaldehyde dehydrogenase RALDH2 coincides with the time of cortical neuron generation. A function of RA in this process has not been adressed directly as Raldh2-/- mouse mutants are embryonic lethal. Here, we used a conditional genetic strategy to inactivate Raldh2 just prior to onset of its expression in the developing meninges. This inactivation does not affect the formation of the cortical progenitor populations, their rate of division, or timing of differentiation. However, migration of late-born cortical neurons is delayed, with neurons stalling in the intermediate zone and exhibiting an abnormal multipolar morphology. This suggests that RA controls the multipolar-to-bipolar transition that occurs in the intermediate zone and allows neurons to start locomotion in the cortical plate. Our work also shows a role for RA in cortical lamination, as deep layers are expanded and a subset of layer IV neurons are not formed in the Raldh2-ablated mutants. These data demonstrate that meninges are a source of extrinsic signals important for cortical development.

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.

Involvement of retinol dehydrogenase 10 in embryonic patterning and rescue of its loss of function by maternal retinaldehyde treatment.

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 (ß-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.

Early mouse caudal development relies on crosstalk between retinoic acid, Shh and Fgf signalling pathways.

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.

Rescue of cytochrome P450 oxidoreductase (Por) mouse mutants reveals functions in vasculogenesis, brain and limb patterning linked to retinoic acid homeostasis.

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.

Retinaldehyde dehydrogenase 2 and Hoxc8 are required in the murine brachial spinal cord for the specification of Lim1+ motoneurons and the correct distribution of Islet1+ motoneurons.

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.

The regional pattern of retinoic acid synthesis by RALDH2 is essential for the development of posterior pharyngeal arches and the enteric nervous system.

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.

Embryonic retinoic acid synthesis is required for forelimb growth and anteroposterior patterning in the mouse.

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).

Genetic evidence that oxidative derivatives of retinoic acid are not involved in retinoid signaling during mouse development.

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.