Gap junction-mediated cell-cell communication modulates mouse neural crest migration

Abstract

Previous studies showed that conotruncal heart malformations can arise with the increase or decrease in alpha1 connexin function in neural crest cells. To elucidate the possible basis for the quantitative requirement for alpha1 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 alpha1 connexins, and from alpha1 connexin knockout (KO) mice and FC transgenic mice expressing a dominant-negative alpha1 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 alpha1 connexin KO embryos. Migration changes occurred in step with connexin gene or transgene dosage in the homozygous vs. hemizygous alpha1 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 alpha1 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 alpha1 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 alpha1 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 alpha1 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 alpha1 connexin function.

Figure 1

Neural crest migration from neural tube explant cultures. Neural crest cells rapidly emerge from the explanted neural tube fragment and migrate out as a monolayer of cells surrounding the central dense mass of neuroepithelial tissue. Note the increase in the area of the neural crest outgrowth from 24 h (A) to 48 h (B) of culture.

Figure 2

Analysis of cell proliferation in neural crest outgrowth cultures. To quantitate cell proliferation in the neural crest outgrowth, BrdU incorporation was examined. Proliferating cells can be readily distinguished as those that are darkly stained after immunodetection with an anti-BrdU antibody (A). Such cultures were subsequently stained with hematoxylin for counting total cell number in the outgrowth (B). Note that A and B are pictures of the same outgrowth culture.

Figure 3

Increased α₁ connexin gap junction plaques in the CMV43 crest cells. Immunostaining with a α₁ connexin antibody revealed increased α₁ connexin expression in the homozygous CMV43 neural crest outgrowth (B) as compared with neural crest cells from nontransgenic embryos (A). The punctate pattern of immunostaining is consistent with the localization α₁ connexins in gap junction plaques. In the control nontransgenic neural crest outgrowths, very small punctate immunostaining can be observed, although this is hard to record photographically.

Figure 4

Neural crest cells in fetal hearts of CMV43 and α₁ connexin KO mice. The distribution of cardiac neural crest cells in E14.5 fetal hearts were examined using a lacZ reporter transgene driven by the α₁ connexin promoter (Lo et al., 1997). Shown are the front and side views of X-gal–stained hearts from wild-type, CMV43, and α₁ connexin KO mice. (A and E) Wild-type mouse heart. The white asterisk in A denotes presumptive neural crest cells in the conus, perhaps comprising cells in the closing seam of the aorticopulmonary septum. (B and F) Homozygous CMV43 heart. The black arrow in F denotes mild bulging of the conotruncus. Note a greater abundance of lacZ staining in the conus (B, white asterisk), suggesting an increased abundance of neural crest cells in the outflow septum. (C and G) Homozygous α₁ connexin KO heart with severe conotruncal malformation. Note the very low level of lacZ staining in the conus (white asterisk), suggesting that fewer neural crest cells are present in the outflow septum. This heart exhibits a more severe phenotype consisting of very prominent thinning of the conotruncal myocardium (region denoted by black arrows). The white arrow in G denotes acute bend in aorta. (D and H) Homozygous α₁ connexin KO heart with mild conotruncal malformation. This heart shows less prominent thinning of the conotruncal myocardium (region denoted by black arrows) as compared with the heart in (C and H). Interestingly, the level of lacZ staining in the conus is close to normal (D, white asterisk). The white arrow in H denotes an acute bend in the aorta. p, pulmonary trunk; a, aorta; rv, right ventricle.

Figure 5

Presumptive neural crest cells in the outflow septum of E14.5 fetal hearts. Histological analysis of the X-gal–stained E14.5 fetal hearts expressing the lacZ reporter gene used to track the distribution of cardiac neural crest cells shows the presence of presumptive neural crest cells in the wall of the aorta (Ao) and also in the outflow septum (O). Note that compared with the nontransgenic heart (A), there is an increased abundance of lacZ-expressing cells in the outflow septum and the wall of the aorta of the CMV43 heart (C), while lacZ-expressing cells in these same regions appeared to be decreased in the KO heart (B). Section in A was stained with hematoxylin-eosin, while sections in B and C were immunostained with PCNA antibody (B) and myosin heavy chain antibody (C). PI, pulmonary infundibulum.

Figure 6

Cell proliferation changes in the ventricular myocardium. Sections of the E14.5 fetal heart were examined for cell proliferation by immunostaining with an antibody to the PCNA. Compared with the wild-type nontransgenic heart (WT), there was a marked increase in PCNA immunostaining in the ventricular myocardium of the homozygous transgenic heart (CMV43). This is indicative of increased myocyte cell proliferation with the overexpression of α₁ connexin gap junctions in neural crest cells. In contrast, in the homozygous α₁ connexin KO mouse heart (Cx43 −/−), there was a reduction in cells exhibiting PCNA expression, thus indicating a paucity of myocyte cell proliferation with the deletion of α₁ connexin function.