Previous studies showed that conotruncal heart malformations can arise with the increase or decrease in α1 connexin function in neural crest cells. To elucidate the possible basis for the quantitative requirement for α1 connexin gap junctions in cardiac development, a neural crest outgrowth culture system was used to examine migration of neural crest cells derived from CMV43 transgenic embryos overexpressing α1 connexins, and from α1 connexin knockout (KO) mice and FC transgenic mice expressing a dominant-negative α1 connexin fusion protein. These studies showed that the migration rate of cardiac neural crest was increased in the CMV43 embryos, but decreased in the FC transgenic and α1 connexin KO embryos. Migration changes occurred in step with connexin gene or transgene dosage in the homozygous vs. hemizygous α1 connexin KO and CMV43 embryos, respectively. Dye coupling analysis in neural crest cells in the outgrowth cultures and also in the living embryos showed an elevation of gap junction communication in the CMV43 transgenic mice, while a reduction was observed in the FC transgenic and α1 connexin KO mice. Further analysis using oleamide to downregulate gap junction communication in nontransgenic outgrowth cultures showed that this independent method of reducing gap junction communication in cardiac crest cells also resulted in a reduction in the rate of crest migration. To determine the possible relevance of these findings to neural crest migration in vivo, a lacZ transgene was used to visualize the distribution of cardiac neural crest cells in the outflow tract. These studies showed more lacZ-positive cells in the outflow septum in the CMV43 transgenic mice, while a reduction was observed in the α1 connexin KO mice. Surprisingly, this was accompanied by cell proliferation changes, not in the cardiac neural crest cells, but in the myocardium— an elevation in the CMV43 mice vs. a reduction in the α1 connexin KO mice. The latter observation suggests that cardiac neural crest cells may have a role in modulating growth and development of non–neural crest– derived tissues. Overall, these findings suggest that gap junction communication mediated by α1 connexins plays an important role in cardiac neural crest migration. Furthermore, they indicate that cardiac neural crest perturbation is the likely underlying cause for heart defects in mice with the gain or loss of α1 connexin function.

Connexin 43 (Cx43α1) gap junction has been shown to have an essential role in mediating functional coupling of neural crest cells and in modulating neural crest cell migration. Here, we showed that N-cadherin and wnt1 are required for efficient dye coupling but not for the expression of Cx43α1 gap junctions in neural crest cells. Cell motility was found to be altered in the N-cadherin–deficient neural crest cells, but the alterations were different from that elicited by Cx43α1 deficiency. In contrast, wnt1-deficient neural crest cells showed no discernible change in cell motility. These observations suggest that dye coupling may not be a good measure of gap junction communication relevant to motility. Alternatively, Cx43α1 may serve a novel function in motility. We observed that p120 catenin (p120ctn), an Armadillo protein known to modulate cell motility, is colocalized not only with N-cadherin but also with Cx43α1. Moreover, the subcellular distribution of p120ctn was altered with N-cadherin or Cx43α1 deficiency. Based on these findings, we propose a model in which Cx43α1 and N-cadherin may modulate neural crest cell motility by engaging in a dynamic cross-talk with the cell's locomotory apparatus through p120ctn signaling.

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Summary The cardiac trabeculae are sheet-like structures extending from the myocardium that function to increase surface area. A lack of trabeculation causes embryonic lethality due to compromised cardiac function. To understand the cellular and molecular mechanisms of trabecular formation, we genetically labeled individual cardiomyocytes prior to trabeculation via the brainbow multicolor system, and traced and analyzed the labeled cells during trabeculation by whole-embryo clearing and imaging. The clones derived from labeled single cells displayed four different geometric patterns that are derived from different patterns of oriented cell division (OCD) and migration. Of the four types of clones, the inner, transmural, and mixed clones contributed to trabecular cardiomyocytes. Further studies showed that perpendicular OCD is an extrinsic asymmetric cell division that putatively contributes to trabecular regional specification. Furthermore, N-Cadherin deletion in labeled clones disrupted the clonal patterns. In summary, our data demonstrate that OCD contributes to trabecular morphogenesis and specification.

Although gap junctions are not known to be important in mediating cell‐cell interactions amongst migratory cells, our studies showed that the connexin 43 (Cx43) gap junction gene is widely expressed in mouse neural crest cell lineages. Using in situ hybridization analysis, Cx43 expression was detected in presumptive neural crest cells emerging from the neural folds of the early postimplantation embryo. Neural crest expression of the Cx43 gap junction gene was also indicated by the analysis of transgenic mice containing a lacZ reporter construct driven by the Cx43 promoter. In neural tube explant cultures of these transgenic mice, lacZ expression was observed in the emerging neural crest outgrowths. Whole mount X‐gal staining of these transgenic embryos at various stages of development showed lacZ expression in neural crest cells distributed along the entire craniocaudal axis, with expression found in both cranial and trunk neural crest cells contributing to a wide range of embryonic tissues. This included presumptive cardiac neural crest cells localized in the heart. In light of the widespread expression of Cx43 in neural crest cell lineages, dye injection studies were carried out to determine if neural crest cells are functionally coupled via gap junctions. Such studies revealed extensive dye coupling among presumptive neural crest cells, thus demonstrating that these migratory cells are indeed gap junctional communication competent. In total, these observations suggest that gap junctions may play a role in mouse neural crest development. This possibility is particularly intriguing given the recent finding that the Cx43 knockout mice die of defects associated with the outflow tract [Reaume et al., 1995], a region of the heart in which neural crest cells are required for normal development. Dev. Genet. 20: 119–132, 1997. © 1997 Wiley‐Liss, Inc.

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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The incidence of KD in children has increased over time, and the development of CAL decreased in the past 5 years in Shanghai. Earlier treatment with IVIG (<5 days) was associated with reduced CAL among patients with KD.