Connexin expression and gap junction communication compartments in the developing mouse limb

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Abstract
Fundamental to the understanding of mouse limb morphogenesis and pattern formation is the need to elucidate the spatial and temporal distribution of gap junction proteins (connexins, Cx) and cell-cell communication compartments. To this end, we used immunofluorescence and confocal microscopy together with 3-dimensional reconstruction software to map the distribution of Cx43 and Cx32 in 11–14.5 days postcoitum (dpc) mouse limbs. Cx43 was strictly localized to the apical ectodermal ridge (AER) and non-ridge ectoderm throughout all stages of mouse limb development studied. Cx32, on the other hand, was abundant in the mesenchyme with only low levels of expression in the 11–13.5 dpc ectoderm. However, at 14–14.5 dpc there was a clear increase in Cx32 expression in the ectoderm. Double labeling for connexins and confocal microscopy revealed Cx43 and Cx32 in the same optical section of the basal cells of the ectoderm but in separate plaques. Lucifer yellow dye injections showed that the cells of the AER were in direct communication with the nonridge ectoderm but dye was never observed to spread to the mesenchyme. Cells of the mesenchyme were coupled to each other but to a much lesser extent than cells of the ectoderm. Finally, although there was an increase in Cx32 expression in the ectoderm at 14–14.5 dpc, this was not correlated with any detectable change in communication compartments. Thus, the lack of dye transfer between the ectoderm and underlying mesenchyme from the peak of AER height through its decline suggests that bulk transfer of morphogens between these two layers is not necessary for mouse limb development.