Connexin 43 (Cx43) knockout mice and transgenic mice (CMV43) overexpressing the Cx43 gap junction gene exhibit heart defects involving the conotruncus and right ventricle. Based on the heart phenotype and Cx43 gene and transgene expression pattern, we previously proposed that the heart defects may reflect a role for gap junctions in the modulation of cardiac neural crest development. To further elucidate the mechanism by which these heart defects may arise, fetal heart structure and function in these transgenic and knockout mice were examined by magnetic resonance microscopy and Doppler echocardiography. Magnetic resonance microscopy of E14.5 fetuses revealed an enlargement of the right ventricular chamber in the heterozygous Cx43 knockout and CMV43 transgenic mice. This was accompanied by thinning of the chamber wall. In the homozygous Cx43 knockout mouse, heart malformation was also restricted to the right ventricle. This was generally characterized by two pouches at the base of the pulmonary outflow tract, but occasionally hearts with a single pouch were found. Magnetic resonance microscopy showed in some of the CMV43 and Cx43 knockout mice an attenuation of the ductus arteriosus, a phenotype which may be indicative of outflow tract obstruction. This was confirmed by the in utero Doppler echocardiography, which showed increased outflow velocity in E12.5 to 14.5 CMV43 and Cx43 knockout fetuses. In some of these fetuses, Doppler analysis also revealed arrhythmia and absence of isovolemic contraction time. Further examination of these hearts by histology and immunohistochemistry showed abnormal myocardial development in the conotruncus. Particularly interesting was the presence of abundant subendocardial fibrous tissue expressing smooth muscle actin. In the developing heart, such mesenchyme in the outflow tract is usually considered neural crest-derived tissue. Together, these results confirm the importance of Cx43 gene dosage in conotruncal heart development and suggest that this likely involves a role for Cx43 gap junctions in cardiac crest development. In future studies, these transgenic mice may serve as valuable animal models for further studying the role of gap junctions and cardiac crest cells in conotruncal heart development.

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Patterning along the left/right axes helps establish the orientation of visceral organ asymmetries, a process which is of fundamental importance to the viability of an organism. A linkage between left/right and axial patterning is indicated by the finding that a number of genes involved in left/right patterning also play a role in anteroposterior and dorsoventral patterning. We have recovered a spontaneous mouse mutation causing left/right patterning defects together with defects in anteroposterior and dorsoventral patterning. This mutation is recessive lethal and was named no turning (nt) because the mutant embryos fail to undergo embryonic turning. nt embryos exhibit cranial neural tube closure defects and malformed somites and are caudally truncated. Development of the heart arrests at the looped heart tube stage, with cardiovascular defects indicated by ballooning of the pericardial sac and the pooling of blood in various regions of the embryo. Interestingly, in nt embryos, the direction of heart looping was randomized. Nodal and lefty, two genes that are normally expressed only in the left lateral plate mesoderm, show expression in the right and left lateral plate mesoderm. Lefty, which is normally also expressed in the floorplate, is not found in the prospective floor plate of nt embryos. This suggests the possibility of notochordal defects. This was confirmed by histological analysis and the examination of sonic hedgehog, Brachyury, and HNF-3 beta gene expression. These studies showed that the notochord is present in the early nt embryo, but degenerates as development progresses. Overall, these findings support the hypothesis that the notochord plays an active role in left/right patterning. Our results suggest that nt may participate in this process by modulating the notochordal expression of HNF-3 beta.

Transgenic mice were generated containing a cytomegaloviral promoter driven construct (CMV43) expressing the gap junction polylpeptide connexin 43. RNA and protein analysis confirmed that the transgene was being expressed. In situ hybridization analysis of embryo sections revealed that transgene expression was targeted to the dorsal neural tube and in subpopulations of neural crest cells. This expression pattern was identical to that seen in transgenic mice harboring other constructs driven by the cytomegaloviral promoter (Kothary, R., Barton, S. C., Franz, T., Norris, M. L., Hettle, S. and Surani, M. A. H. (1991) Mech. Develop. 35, 25–31; Koedood, M., Fitchel, A., Meier, P. and Mitchell, P. (1995) J. Virol. 69, 2194–2207), and corresponded to a subset of the endogenous Cx43 expression domains. Significantly, dye injection studies showed that transgene expression resulted in an increase in gap junctional communication. Though viable and fertile, these transgenic mice exhibited reduced postnatal viability. Examination of embryos at various stages of development revealed developmental perturbations consisting of cranial neural tube defects (NTD) and heart malformations. Interestingly, breeding of the CMV43 transgene into the Cx43 knockout mice extended postnatal viability of mice homozygote for the Cx43 knockout allele, indicating that the CMV43 trangsene may partially complement the Cx43 deletion. Both the Cx43 knockout and the CMV43 transgenic mice exhibit heart defects associated with malformations in the conotruncus, a region of the heart in which neural crest derivatives are known to have important roles during development. Together with our results indicating neural-crest-specific expression of the transgene in our CMV-based constructs, these observations strongly suggest a role for Cx43-mediated gap junctional communication in neural crest development. Furthermore, these observations indicate that the precise level of Cx43 function may be of critical importance in downstream events involving these migratory cell populations. As such, the CMV43 mouse may represent a powerful new model system for examining the role of extracardiac cell populations in cardiac morphogenesis and other developmental processes.

We have examined fetal thymic development in the trisomy 16