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The neural crest plays a crucial part in cardiac development. Cells of the cardiac subpopulation of cranial neural crest migrate from the hindbrain into the outflow tract of the heart where they contribute to the septum that divides the pulmonary and aortic channels. In Splotch mutant mice, which lack a functional Pax3 gene, migration of cardiac neural crest is deficient and aorticopulmonary septation does not occur. Downstream genes through which Pax3 regulates cardiac neural crest development are unknown. Here, using a combination of genetic and molecular approaches, we show that the deficiency of cardiac neural crest development in the Splotch mutant is caused by upregulation of Msx2, a homeobox gene with a well-documented role as a regulator of BMP signaling. We provide evidence, moreover, that Pax3 represses Msx2 expression via a direct effect on a conserved Pax3 binding site in the Msx2 promoter. These results establish Msx2 as an effector of Pax3 in cardiac neural crest development.

Neural crest cells are multipotential stem cells that contribute extensively to vertebrate development and give rise to various cell and tissue types. Determination of the fate of mammalian neural crest has been inhibited by the lack of appropriate markers. Here, we make use of a two-component genetic system for indelibly marking the progeny of the cranial neural crest during tooth and mandible development. In the first mouse line, Cre recombinase is expressed under the control of the Wnt1 promoter as a transgene. Significantly, Wnt1 transgene expression is limited to the migrating neural crest cells that are derived from the dorsal CNS. The second mouse line, the ROSA26 conditional reporter (R26R), serves as a substrate for the Cre-mediated recombination. Using this two-component genetic system, we have systematically followed the migration and differentiation of the cranial neural crest (CNC) cells from E9.5 to 6 weeks after birth. Our results demonstrate, for the first time, that CNC cells contribute to the formation of condensed dental mesenchyme, dental papilla, odontoblasts, dentine matrix, pulp, cementum, periodontal ligaments, chondrocytes in Meckel’s cartilage, mandible, the articulating disc of temporomandibular joint and branchial arch nerve ganglia. More importantly, there is a dynamic distribution of CNC- and non-CNC-derived cells during tooth and mandibular morphogenesis. These results are a first step towards a comprehensive understanding of neural crest cell migration and differentiation during mammalian craniofacial development. Furthermore, this transgenic model also provides a new tool for cell lineage analysis and genetic manipulation of neural-crest-derived components in normal and abnormal embryogenesis.

A subpopulation of neural crest termed the cardiac neural crest is required in avian embryos to initiate reorganization of the outflow tract of the developing cardiovascular system. In mammalian embryos, it has not been previously experimentally possible to study the long-term fate of this population, although there is strong inference that a similar population exists and is perturbed in a number of genetic and teratogenic contexts. We have employed a two-component genetic system based on Cre/lox recombination to label indelibly the entire mouse neural crest population at the time of its formation, and to detect it at any time thereafter. Labeled cells are detected throughout gestation and in postnatal stages in major tissues that are known or predicted to be derived from neural crest. Labeling is highly specific and highly efficient. In the region of the heart, neural-crest-derived cells surround the pharyngeal arch arteries from the time of their formation and undergo an altered distribution coincident with the reorganization of these vessels. Labeled cells populate the aorticopulmonary septum and conotruncal cushions prior to and during overt septation of the outflow tract, and surround the thymus and thyroid as these organs form. Neural-crest-derived mesenchymal cells are abundantly distributed in midgestation (E9.5-12.5), and adult derivatives of the third, fourth and sixth pharyngeal arch arteries retain a substantial contribution of labeled cells. However, the population of neural-crest-derived cells that infiltrates the conotruncus and which surrounds the noncardiac pharyngeal organs is either overgrown or selectively eliminated as development proceeds, resulting for these tissues in a modest to marginal contribution in late fetal and postnatal life.

Germline mutation in mice of the retinoic acid receptor gene RXRα results in a proliferative failure of cardiomyocytes, which leads to an underdeveloped ventricular chamber and midgestation lethality. Mutation of the cell cycle regulator N-myc gene also leads to an apparently identical phenotype. In this study, we demonstrate by chimera analysis that the cardiomyocyte phenotype in RXRα−/−embryos is a non-cell-autonomous phenotype. In chimeric embryos made with embryonic stem cells lacking RXRα, cardiomyocytes deficient in RXRα develop normally and contribute to the ventricular chamber wall in a normal manner. Because the ventricular hypoplastic phenotype reemerges in highly chimeric embryos, we conclude that RXRα functions in a non-myocyte lineage of the heart to induce cardiomyocyte proliferation and accumulation, in a manner that is quantitatively sensitive. We further show that RXRα is not epistatic to N-myc, and that RXRα and N-myc regulate convergent obligate pathways of cardiomyocyte maturation.

Embryonic exposure to the vitamin A metabolite retinoic acid (RA) causes malformations in numerous developing tissues, including the limbs, which serves as a model system of retinoic acid action. RA treatment of wild-type mouse embryos results in digit truncations and long bone reductions. These effects are mediated by products of the retinoic acid and retinoid X receptor genes (RARs and RXRs), members of the nuclear receptor family of liganddependent transcription factors. Mouse embryos homozygous for a mutation in the RXRα gene appear normal in limb development, although such embryos are phenotypically affected in other tissues. We now describe resistance to limb malformations normally induced by teratogenic RA exposure in the RXRα−/− background. RA treatments that cause limb defects in 100% of wild-type embryos fail to elicit malformations in RXRα homozygotes, implicating RXRα as a component in the teratogenic process in the limbs. Heterozygous embryos are intermediate in sensitivity to RA, suggesting the importance of RXRα gene dosage in limb teratogenesis. Expression of the RA-inducible gene RARβ2 was equivalent between wild-type and homozygous embryos after RA treatment. RA treatment also did not distinguish between wild-type and RXRα−/− embryos in the spatial expression of sonic hedgehog (Shh) and hoxd-12, two other genes implicated in limb development. However, the quantitative level of hoxd-12 expression was elevated in RXRα−/− embryos. These observations indicate that transcriptional processes which are inappropriately regulated in the mouse limb by exogenous RA require RXRα for their execution, and that specific teratogenic processes, as well as specific normal developmental processes under vitamin A control, occur through individual members of the RXR and RAR families.

RXRα null mutant mice display ocular and cardiac malformations, liver developmental delay, and die from cardiac failure around embryonic day (E) 14.5 pc. To dissect the molecular basis of the RXRα-associated cardiomyopathy, we performed subtractive hybridization and systematically characterized putative downstream target genes that were selectively lacking in the mutant embryos, both at early (E10.5) and late (E13.5) stages of mouse embryonic development. Approximately 50% of the subtracted clones (61/115) encoded proteins involved in intermediary metabolism and electron transport, suggesting an energy deficiency in the RXRα−/− embryos. In particular, clone G1, which encodes subunit 14.5b of the NADH-ubiquinone dehydrogenase complex, displayed a dose-dependent expression in the wild-type, heterozygous and RXRα mutant mice. This gene was also downregulated in a retinoid-deficient rat embryo model. ATP content and medium Acyl-CoA dehydrogenase mRNA were lower in RXRα mutant hearts compared to wild-type mice. Ultrastructural studies showed that the density of mitochondria per myocyte was higher in the RXRα mutant compared to wild-type littermates. We propose a model whereby defects in intermediary metabolism may be a causative factor of the RXRα−/− phenotype and resembles an embryonic form of dilated cardiomyopathy.