Expression of connexin 43 (Cx43) is critical for normal hematopoiesis by Encarnacion Montecino-Rodriguez, Hyosuk Leathers, and Kenneth Dorshkind Blood Volume 96(3):917-924 August 1, 2000 ©2000 by American Society of Hematology

Representative blot showing identification of Cx43+/+, Cx43+/−, and Cx43−/−mice by PCR.Liver DNA from mice was amplified using neomycin and Cx43 primers as described in “Materials and methods.”. Representative blot showing identification of Cx43+/+, Cx43+/−, and Cx43−/−mice by PCR.Liver DNA from mice was amplified using neomycin and Cx43 primers as described in “Materials and methods.” Encarnacion Montecino-Rodriguez et al. Blood 2000;96:917-924 ©2000 by American Society of Hematology

Thymus cellularity.(A) Late gestation Cx43+/+ (n = 4), CX43+/− (n = 12), and Cx43−/− (n = 6) mice. Thymus cellularity.(A) Late gestation Cx43+/+ (n = 4), CX43+/− (n = 12), and Cx43−/− (n = 6) mice. (B) Neonatal Cx43+/+ (n = 6), Cx43+/− (n = 5), and Cx43−/− (n = 3) mice. (C) Young adult Cx43+/+ (n = 4) and Cx43+/− (n = 4) mice. The level of statistical significance between different groups of mice is indicated. Encarnacion Montecino-Rodriguez et al. Blood 2000;96:917-924 ©2000 by American Society of Hematology

Frequency of CD4−CD8−, CD4+CD8+, CD4+, and CD8+thymocyte subpopulations.(A) Late gestation Cx43+/+ (n = 4), Cx43+/− (n = 12), and Cx43−/− (n = 6) embryos. Frequency of CD4−CD8−, CD4+CD8+, CD4+, and CD8+thymocyte subpopulations.(A) Late gestation Cx43+/+ (n = 4), Cx43+/− (n = 12), and Cx43−/− (n = 6) embryos. (B) Neonatal Cx43+/+ (n = 6), Cx43+/− (n = 5), and Cx43−/− (n = 3) mice. (C) Young adult Cx43+/+ (n = 4) and Cx43+/− (n = 4) mice. The level of statistical significance for each thymocyte subpopulation between different groups of mice is indicated. Encarnacion Montecino-Rodriguez et al. Blood 2000;96:917-924 ©2000 by American Society of Hematology

Cellularity in the bone marrow, thymus, and spleen Cellularity in the bone marrow, thymus, and spleen.(A) Bone marrow (BM) from 2 femurs and 2 tibiae); (B) thymus (THY); and (C) spleen (SPL) from Cx43+/+ and Cx43−/− mice 9 days after treatment with 5-FU or saline. Cellularity in the bone marrow, thymus, and spleen.(A) Bone marrow (BM) from 2 femurs and 2 tibiae); (B) thymus (THY); and (C) spleen (SPL) from Cx43+/+ and Cx43−/− mice 9 days after treatment with 5-FU or saline. Three to 4 mice of each genotype received the indicated treatment, and animals were processed individually. The ratio of recovery calculated as described in Materials and methods is indicated in parenthesis. The level of statistical significance between the different experimental groups is indicated. The results shown are from 1 of 2 identical experiments. Mice were 5 weeks of age when treated with 5-FU. Encarnacion Montecino-Rodriguez et al. Blood 2000;96:917-924 ©2000 by American Society of Hematology

Phenotypes.The phenotypes are shown for CD45R+ (A), TER-119+(B), and CD11b+ (C) cells in the bone marrow of Cx43+/+ and Cx43+/− mice 9 days after 5-FU or saline treatment. Phenotypes.The phenotypes are shown for CD45R+ (A), TER-119+(B), and CD11b+ (C) cells in the bone marrow of Cx43+/+ and Cx43+/− mice 9 days after 5-FU or saline treatment. Mice are the same as those described in the legend to Figure 4. The level of statistical significance between the different experimental groups is indicated. Encarnacion Montecino-Rodriguez et al. Blood 2000;96:917-924 ©2000 by American Society of Hematology

Thymus cellularity in Cx43+/+ or Cx43+/− mice after bone marrow transplantation.(A) The transplantation protocol. Thymus cellularity in Cx43+/+ or Cx43+/− mice after bone marrow transplantation.(A) The transplantation protocol. (B) Thymus celllularity in Cx43+/+ or Cx43+/− recipients of Cx43+/+ or Cx43+/− bone marrow cells. (C) PCR analysis of testes, bone marrow (BM), and thymus (THY) from Cx43+/+ or Cx43+/− mice grafted with either Cx43+/+ or Cx43+/− bone marrow cells as indicated. Testis DNA was used to confirm the identity of the recipient mouse. Note that the genetic composition of the thymus of Cx43+/− mice that received Cx43+/+ bone marrow is not completely donor derived. Only 2 of 3 to 4 mice from each group are shown. The asterisk indicates values significantly different at P < .05. Encarnacion Montecino-Rodriguez et al. Blood 2000;96:917-924 ©2000 by American Society of Hematology

B lymphopoiesis in long-term bone marrow cultures from Cx43+/− and Cx43+/+ mice.(A) After establishment of confluent adherent layers from Cx43+/+ or Cx43+/− bone marrow cells, cultures were recharged from the same pool of Cx43+/+ or Cx43+/− bone marrow, res... B lymphopoiesis in long-term bone marrow cultures from Cx43+/− and Cx43+/+ mice.(A) After establishment of confluent adherent layers from Cx43+/+ or Cx43+/− bone marrow cells, cultures were recharged from the same pool of Cx43+/+ or Cx43+/− bone marrow, respectively, and maintained under myeloid long-term culture conditions for 3 weeks before transfer to B lymphoid culture conditions. Cells were harvested from cultures at weekly intervals thereafter and the frequency of CD45R+sIgM− and CD45R+sIgM+ cells determined. Groups different at ** = P < .0005; * = P < .005. There were no significant differences in cellularity between the cultures established with Cx43+/+ and Cx43+/− bone marrow (data not shown). Data are based on individual analysis of 8 cultures per experimental group. (B) Expression of Cx43 mRNA in sorted Lin−, Sca-1+, c-kit+ stem cells, sorted CD43+CD45R+ pro-B cells, and heterogeneous bone marrow stroma depleted of hematopoietic cells. RT-PCR was performed as described in “Materials and methods.” Encarnacion Montecino-Rodriguez et al. Blood 2000;96:917-924 ©2000 by American Society of Hematology