Dental and extra-oral clinical features in 41 patients with WNT10A gene mutations: A multicentric genotype-phenotype study.

WNT10A gene encodes a canonical wingless pathway signaling molecule involved in cell fate specification as well as morphogenetic patterning of the developing ectoderm, nervous system, skeleton, and tooth. In patients, WNT10A mutations are responsible for ectodermal-derived pathologies including isolated hypo-oligodontia, tricho-odonto-onycho-dermal dysplasia and Schöpf-Schulz-Passarge syndrome (SSPS). Here we describe the dental, ectodermal, and extra-ectodermal phenotypic features of a cohort of 41 patients from 32 unrelated families. Correlations with WNT10A molecular status (heterozygous carrier, compound heterozygous, homozygous) and patient's phenotypes were performed. Mild to severe oligodontia was observed in all patients bearing biallelic WNT10A mutations. However, patients with compound heterozygous mutations presented no significant difference in phenotypes compared with homozygous individuals. Anomalies in tooth morphology were frequently observed with heterozygous patients displaying hypodontia. No signs of SSPS, especially eyelids cysts, were detected in our cohort. Interestingly, extra-ectodermal signs consisted of skeletal, neurological and vascular anomalies, the latter suggesting a wider phenotypic spectrum associated with WNT10A mutations. Indeed, the Wnt pathway plays a crucial role in skeletal development, lipid metabolism, and neurogenesis, potentially explaining patient's clinical manifestations.

Specific expression of the retinoic acid-synthesizing enzyme RALDH2 during mouse inner ear development.

Retinoid binding proteins and nuclear receptors are expressed in the developing mouse inner ear. Here, we report that the retinaldehyde dehydrogenase 2 (Raldh2) gene, whose product is involved in the enzymatic generation of retinoic acid (RA), exhibits a restricted expression pattern during mouse inner ear ontogenesis. The Raldh2 gene is first expressed at embryonic day (E) 10.5 in a V-shaped medio-dorsal region of the otocyst outer epithelium, which evolves as two separate domains upon otocyst morphogenesis. At E14.5, Raldh2 is expressed in two areas of the utricle epithelium and specific regions of the saccule and cochlear mesenchyme. Later, Raldh2 transcripts are restricted to two cochlear areas, the stria vascularis and Reissner membrane. Raldh2 mesenchymal expression did not correlate with migrating neural crest-derived melanoblasts. These restricted expression domains may correspond to specific sites of RA synthesis during inner ear morphogenesis.

Embryonic retinoic acid synthesis is essential for heart morphogenesis in the mouse.

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.

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.

Retinoic acid synthesis and hindbrain patterning in the mouse embryo.

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.

Expression of the transcriptional intermediary factor TIF1alpha during mouse development and in the reproductive organs.

Nuclear receptors are important regulators of development and reproduction whose action can be modulated by transcriptional intermediary factors (TIFs). In situ hybridization was used to investigate the expression pattern of the putative nuclear receptor mediator TIF1alpha during mouse embryogenesis and adult life. TIF1alpha is ubiquitously expressed until midgestation. At 12.5 gestational days, TIF1alpha is preferentially expressed in the developing central and peripheral nervous system. Differential expression persists until perinatal stages, with high expression in the brain, nasal epithelium and within proliferating regions of the kidney and teeth. In the adult, TIF1alpha expression is predominant in both the male and female gonads. Immunogold electron microscopy revealed that TIF1alpha protein is most abundant in the nuclei of male germ cells at various stages of their maturation.

Embryonic retinoic acid synthesis is essential for early mouse post-implantation development.

A number of studies have suggested that the active derivative of vitamin A, retinoic acid (RA), may be important for early development of mammalian embryos. Severe vitamin A deprivation in rodents results in maternal infertility, precluding a thorough investigation of the role of RA during embryogenesis. Here we show that production of RA by the retinaldehyde dehydrogenase-2 (Raldh2) enzyme is required for mouse embryo survival and early morphogenesis. Raldh2 is an NAD-dependent aldehyde dehydrogenase with high substrate specificity for retinaldehyde. Its pattern of expression during mouse development has suggested that it may be responsible for embryonic RA synthesis. We generated a targeted disruption of the mouse Raldh2 gene and found that Raldh2-/- embryos, which die at midgestation without undergoing axial rotation (body turning), exhibit shortening along the anterioposterior axis and do not form limb buds. Their heart consists of a single, medial, dilated cavity. Their frontonasal region is truncated and their otocysts are severely reduced. These defects result from a block in embryonic RA synthesis, as shown by the lack of activity of RA-responsive transgenes, the altered expression of an RA-target homeobox gene and the near full rescue of the mutant phenotype by maternal RA administration. Our data establish that RA synthesized by the post-implantation mammalian embryo is an essential developmental hormone whose lack leads to early embryo death.

Expression of T:G mismatch-specific thymidine-DNA glycosylase and DNA methyl transferase genes during development and tumorigenesis.

In situ hybridization was used to characterize the expression pattern of the T:G mismatch-specific thymidine-DNA glycosylase (TDG) gene, encoding a DNA repair enzyme which corrects G:T mismatches that result from the hydrolytic deamination of 5-methyl cytosines. TDG transcripts were uniformly and ubiquitously expressed from 7.5-13.5 days post-coitum, but were then markedly enriched in specific tissues of the developing fetus. At 14.5 gestational days, TDG was strongly expressed in the developing nervous system, thymus, lung, liver, kidney and intestine. At later stages, high levels of expression were detected in the thymus, brain, nasal epithelium and within proliferating regions of the intestine, skin, kidney, teeth and bone. This pattern of expression strongly correlated with those of the methyl transferase (MTase) gene, coding for the enzyme which specifically methylates CpG dinucleotides, and the p53 tumour suppressor gene. However, TDG and MTase were differentially expressed during maturation of the male and female germline. We also report that tumors occuring in mice which overexpress MMTV-v-Ha-ras or MMTV-c-myc transgenes or mice heterozygous for p53 gene disruption, all show elevated TDG and MTase expression specific to the transformed tissue.

MDM2 expression during mouse embryogenesis and the requirement of p53.

We compared mouse embryonic expression of the MDM2 proto-oncogene, p21WAF1/CIP1 and their transcriptional regulator, p53. MDM2 expression is ubiquitous from 7.5 to 11.5 days post coitum (dpc) and more restricted from 12.5 dpc, with the highest levels in the testes and neural tube. From 14.5 to 18.5 dpc, the nasal respiratory epithelium expresses high levels of MDM2 RNA and protein and p21WAF1/CIP1 RNA, in both wild type and p53 null embryos. MDM2 expression during development is tissue-specific and, like p21WAF1/CIP1, is independent of p53. MDM2 may have a developmental role after 6.5 dpc, when MDM2 null mice die (Jones, S.N., Roe, A.E., Donehower, L.A., Bradley, A., 1995. Rescue of embryonic lethality in Mdm2-deficient mice by absence of p53. Nature 378, 206-208; Montes de Oca Luna, R., Wagner, D.S., Lozano, G., 1995. Rescue of early embryonic lethality in mdm2-deficient mice by deletion of p53. Nature 378, 203-206).

Restricted expression and retinoic acid-induced downregulation of the retinaldehyde dehydrogenase type 2 (RALDH-2) gene during mouse development.

Retinaldehyde dehydrogenase type 2 (RALDH-2) was identified as a major retinoic acid generating enzyme in the early embryo. Here we report the expression domains of the RALDH-2 gene during mouse embryogenesis, which are likely to indicate regions of endogenous retinoic acid (RA) synthesis. During early gastrulation, RALDH-2 is expressed in the mesoderm adjacent to the node and primitive streak. At the headfold stage, mesodermal expression is restricted to posterior regions up to the base of the headfolds. Later, RALDH-2 is transiently expressed in the undifferentiated somites and the optic vesicles, and more persistently along the lateral walls of the intraembryonic coelom and around the hindgut diverticulum. The RALDH-2 expression domains in differentiating limbs, which include presumptive interdigital regions, coincide with, but slightly precede, those of the RA-inducible RAR beta gene. The RALDH-2 gene is also expressed in specific regions of the developing head, including the tooth buds, inner ear, meninges and pituitary gland, and in several viscera. Administration of a teratogenic dose of RA at embryonic day 8.5 results in downregulation of RALDH-2 transcript levels in caudal regions of the embryo, and may reflect a mechanism of negative feedback regulation of RA synthesis.

A potent far-upstream enhancer in the mouse pro alpha 2(I) collagen gene regulates expression of reporter genes in transgenic mice.

We have identified three DNase I-hypersensitive sites in chromatin between 15 and 17 kb upstream of the mouse pro alpha 2 (I) collagen gene. These sites were detected in cells that produce type I collagen but not in cells that do not express these genes. A construction containing the sequences from -17 kb to +54 bp of the mouse pro alpha 2 (I) collagen gene, cloned upstream of either the Escherichia coli beta-galactosidase or the firefly luciferase reporter gene, showed strong enhancer activity in transgenic mice when compared with the levels seen previously in animals harboring shorter promoter fragments. Especially high levels of expression of the reporter gene were seen in dermis, fascia, and the fibrous layers of many internal organs. High levels of expression could also be detected in some osteoblastic cells. When various fragments of the 5' flanking sequences were cloned upstream of the 350-bp proximal pro alpha 2(I) collagen promoter linked to the lacZ gene, the cis-acting elements responsible for enhancement were localized in the region between -13.5 and -19.5 kb, the same region that contains the three DNase I-hypersensitive sites. Moreover, the DNA segment from -13.5 to -19.5 kb was also able to drive the cell-specific expression of a 220-bp mouse pro alpha 1(I) collagen promoter, which is silent in transgenic mice. Hence, our data suggest that a far-upstream enhancer element plays a role in regulating high levels of expression of the mouse pro alpha 2(I) collagen gene.

Morphological and molecular characterization of retinoic acid-induced limb duplications in mice.

This study reports a morphological, skeletal, and molecular characterization of the supernumerary limbs induced by systemic administration of all-trans retinoic acid to egg-cylinder stage mouse embryos. As initially described by Rutledge et al. (Proc. Natl. Acad. Sci. USA 91, 5436, 1994), we have found that oral administration of all-trans retinoic 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 of Sonic hedgehog and Hoxd-13 was 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.

Coordinate patterns of expression of type I and III collagens during mouse development.

The extracellular proteins types I and III collagen are abundantly expressed during development. Here, the patterns of the pro alpha 1(I), pro alpha 2(I), and pro alpha 1(III) collagen mRNAs are systematically examined from 7.5 to 17.5 days of development (E7.5 to E17.5) in the mouse using in situ hybridization with specific riboprobes. Coordinated expression of pro alpha 1(I) and pro alpha 2(I) collagen mRNA was found throughout development in all regions examined. Widespread type I collagen expression starting at E8.5 occurred in embryonic mesoderm, sclerotomes, dermatomes, and in the forming connective tissues. After E14.5, regions of ossification showed highest levels of type I collagen expression. Pro alpha 1(III) collagen expression was specific to and coordinated with patterns of type I collagen expression in many fibroblast-containing tissues. No expression of type III collagen occurred in osteoblasts. This comprehensive study of the transcripts of abundantly expressed structural proteins should provide a basis for comparison of other key extracellular matrix molecules and serve as a reference for studies on the patterns of activities of various promoter/enhancer-reporter gene constructions of type I and III collagen genes in transgenic mice.

Developmental expression of a type II collagen/beta-galactosidase fusion gene in transgenic mice.

The correct temporal and spatial expression of the type II collagen gene is believed to be important for normal development and growth of the skeleton and the eye, i.e., tissues where the protein product is predominantly found. To study transcriptional activation of type II collagen gene in skeletal and nonskeletal tissues we produced transgenic mice carrying murine proalpha1(II) collagen/beta-galactosidase fusion gene constructs. The expression of the fusion gene was found to depend on the presence of intron 1 deleted failed to reveal any beta-galactosidase activity confirming the important role of regulatory sequences within intron 1 of the gene. High-level expression of the functional construct was clearly confined to cartilaginous tissues but transient low-level expression was also observed in extraskeletal locations, such as the developing brain and the notochord. The results demonstrate that the regulatory elements in the proalpha1(II) collagen/beta-galactosidase fusion gene construct confer both temporal and spatial specificity indistinguishable from that of the endogenous proalpha1(II) collagen gene as determined by the presence of the corresponding mRNA by in situ hybridization. Furthermore the beta-galactosidase activity correlated well with the progression of chondrogenesis as seen by staining of whole mouse embryos with Alizarin red S and Alcian blue in the hybrid mouse strain used for microinjections. The transgenic mouse line produced should prove useful for studies on various aspects of chondrogenesis. Furthermore, the data shows that the regulatory elements present in the construct are sufficient for targetting the expression of other genes in cartilage.

Osteoblast-specific expression of the alpha 2(I) collagen promoter in transgenic mice: correlation with the distribution of TGF-beta 1.

To begin to assess the transcriptional mechanisms that regulate type I collagen gene expression in differentiating osteoblasts, we have sought to determine the minimal promoter sequences that confer osteoblast-specific expression to the alpha 2(I) collagen gene during murine development. Transgenic mice were generated harboring DNA constructs in which the -2000, -500, and -350 to +54 regions located upstream of the start of transcription were linked to the Escherichia coli beta-galactosidase reporter gene (LacZ). Histochemical staining using X-gal indicated that the -2000 lacZ transgene was strongly expressed in newly differentiated and fully functional osteoblasts at intramembranous and endochondral sites of ossification. The promoter was also active in osteocytes in regions of bone remodeling within alveolar bone. The temporal and spatial activity of this region of the promoter closely resembled the developmental patterns of expression of the endogenous alpha 2(I) collagen gene as determined by in situ hybridization. The cis-acting elements within the 500 and 350 bp segments of the alpha 2(I) collagen promoter also drove reporter gene expression in forming osteoblasts, although levels of transgene expression were not as marked as that seen with the 2000 bp promoter. Furthermore, the synthesis and secretion of TGF-beta 1 in osteogenic zones coincided with areas where the alpha 2(I) collagen promoter constructs were transcriptionally active. Since a nuclear factor 1 binding site present at -300 has been shown to mediate the effects of TGF-beta 1 on the alpha 2(I) collagen promoter, these data support a role for TGF-beta 1 in the control of this gene during development.

Minimal DNA sequences that control the cell lineage-specific expression of the pro alpha 2(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 -2,000 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 bulbus arteriosus, valves of the developing heart, sclerotomes, meninges, limb buds, connective tissue fascia between muscle fibers, osteoblasts in newly formed bones, fibroblasts in tendons, periosteum, dermis, and peritoneal membranes. The pattern of beta-galactosidase activity was similar and included within the extracellular immunohistochemical localization pattern of transforming growth factor-beta 1 (TGF-beta 1). The -315(-)-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' to a very short alpha 2(I) collagen promoter (-40(-)+54) showed preferential activity in tail and skin of 4-wk-old transgenic mice. Except for low expression of the transgene in bone, this pattern mimics the expression of the endogenous pro alpha 2(I) collagen gene. We propose the hypothesis that the tissue-specific expression of the pro alpha 2(I) collagen gene during embryogenesis is controlled by both TGF-beta 1 and cell-specific transcription factors; one of these could interact directly or indirectly with either the -315(-)-284 or the -40(-)+54 segment.

Beta-globin enhancers target expression of a heterologous gene to erythroid tissues of transgenic mice.

To examine the role of human beta-globin enhancers in tissue-specific and developmental regulation, a hybrid beta-globin-simian virus 40 gene was analyzed in transgenic mice. A beta-globin DNA fragment containing two previously defined enhancers stimulated transcription from the simian virus 40 promoter in a tissue- and stage-specific pattern similar to that of the normal beta-globin gene. These results help to define the functions of beta-globin regulatory elements and suggest an approach for targeted expression of heterologous genes in erythroid cells in vivo.

Plasma delta-9-tetrahydrocannabinol in pregnant sheep and fetus after inhalation of smoke from a marijuana cigarette.

Seven pregnant ewes were prepared with open-ended tracheal T tubes and with catheters in maternal femoral artery and in central circulation of fetus. Several days postoperatively, at 129-132 days gestation, ewes inhaled smoke from one marijuana cigarette containing 3.19% delta-9-tetrahydrocannabinol (delta-9-THC). Smoke was produced continuously in a hand-held chamber and delivered to the protruding arm of the tracheal tube. Samples of maternal and fetal blood were taken during the 8-10 minute smoking period and at intervals up to 24 hours. Delta-9-THC was detected in maternal plasma at 3 minutes and peaked at 10 minutes. Fetal plasma delta-9-THC reached detectable levels in 5 animals by 10 minutes. Maximum mean level was reached at 90 minutes and remained nearly constant until the fourth hour. Fetal delta-9-THC levels remained lower than maternal levels at all times. The terminal half-life of delta-9-THC in fetal and maternal plasma exceeded 10 hours.