DEV02011 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 - Fig. S1. Mapping and identification of the Gja1^Jrt mutation. (A) A genome scan of mice with digit fusion, using microsatellite markers that were polymorphic between the parental strains (C57BL/6J and C3H/HeJ), mapped the mutation between markers D10Mit194 and D10Mit42. C, homozygous for C3H/HeJ allele; H, heterozygous for C57BL/6J and C3H/HeJ alleles. (B) Sequencing of the Gja1 gene (Gen Bank NM_010288) revealed a G to A transition only in mice with digit fusion (Gja1^Jrt/+) compared with those without fusion (+/+).

• Supplemental Figure 2 - Fig. S2. Whole-mount Alcian Blue-Alizarin Red S staining of the calvaria of 3 day Gja1^Jrt/+ mutants revealed thin and porous bones with open foramena compared with wild-type littermates (+/+) skulls.

• Supplemental Figure 3 - Fig. S3. Localization and functional analysis of the Gja1^Jrt mutant protein. Cx43^G60S-GFP localized to both the Golgi apparatus and the cell surface when expressed in NRK, HeLa or N2A cells. NRK (B-D), HeLa (F-H) and N2A (K, L) cells were transfected with cDNA encoding human Cx43-GFP or Cx43^G60S-GFP. Similar to the endogenous expression of Cx43 in NRK cells (A, arrow), Cx43-GFP localized to cell-cell interfaces in all cell types (B,F,K, arrows), while no Cx43 was expressed in wild-type HeLa cells (E). The Cx43^G60S-GFP mutant also localized to cell-cell interfaces (C,G,L, arrows). Notably, a subpopulation of Cx43^G60S-GFP was found to co-localize with resident Golgi proteins (D, H; yellow arrows). Additionally, when HeLa cells were transfected with cDNA encoding untagged Cx43 or Cx43^G60S, both were localized to the cell surface (I,J), similar to their GFP-tagged counterparts. To highlight cell morphology, nuclei were stained with Hoechst 33342 and some cells (A-C,E-G,I,J) were immunolabeled for tubulin. Scale bar: 10 mm

• Supplemental Figure 4 - Fig. S4. Mineralization of the heart in Gja1^Jrt/+ mice. (A,B) Hematoxylin and Eosin staining of heart tissue revealed small, multifocal lesions of myocardial mineralization in 15 week Gja1Jrt/+ mice that were not seen in the littermate controls. Scale bars: 100 mm.