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 nonridge 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. 0 1993 Wiley-Liss, Inc.
CHD1 is a conserved chromatin remodeling factor that localizes to active genes and functions in nucleosome assembly and positioning as well as histone turnover. Mouse CHD1 is required for the maintenance of stem cell pluripotency while human CHD1 may function as a tumor suppressor. To investigate the action of CHD1 on higher order chromatin structure in differentiated cells, we examined the consequences of loss of CHD1 and over-expression of CHD1 on polytene chromosomes from salivary glands of third instar Drosophila melanogaster larvae. We observed that chromosome structure is sensitive to the amount of this remodeler. Loss of CHD1 resulted in alterations of chromosome structure and an increase in the heterochromatin protein HP1a, while over-expression of CHD1 disrupted higher order chromatin structure and caused a decrease in levels of HP1a. Over-expression of an ATPase inactive form of CHD1 did not result in severe chromosomal defects, suggesting that the ATPase activity is required for this in vivo phenotype. Interestingly, changes in CHD1 protein levels did not correlate with changes in the levels of the euchromatin mark H3K4me3 or elongating RNA Polymerase II. Thus, while CHD1 is localized to transcriptionally active regions of the genome, it can function to alter the levels of HP1a, perhaps through changes in methylation of H3K9.
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