whereas peritoneal cavity B-2 B cells and even more B-1 B cells showed reduced expression. Compared to the B cell compartment, calponin-3-GFP expression in T cells was in general weaker and restricted to the early developmental subsets. Fig 4. High expression of calponin-3 throughout B cell development. A. Staining pattern and gating strategies for cells isolated from the bone marrow, the spleen, lymph nodes and the peritoneal cavity. Numbers indicate the respective populations as analyzed in B. Expression of calponin-3-GFP in different B cell subsets derived from a Cnn3 ki f/f mouse or a +/+ littermate. Cells were isolated, stained and analyzed as indicated in A. Bar graphs indicate the ratio of the GFP MFI of ki f/f cells versus that of +/+ cells. Data represent 4 independent experiments. For statistical analysis, normalized GFP MFI values of control and ki f/f littermates were compared by a paired t-test. LN, lymph node; PC, peritoneal cavity. doi:10.1371/journal.pone.0128385.g004 9 / 16 Calponin-3 in B Lymphocyte Development Calponin-3 is dispensable for early B cell development Together, our in vitro results and the in vivo expression pattern suggested a putative role of calponin-3 in early B cell development. To test this, we crossed our Cnn3 ki f/f mice with a CMV-Cre transgenic strain to excise the floxed mini gene, thereby generating a null STA 4783 site allele. However, homozygeous calponin-3 knockout mice displayed an extensive growth of neuronal tissue during early embryonic development, were born with a severe exencephaly and died immediately postnatal. To enable investigation of calponin-3 in the B cells, we hence restricted deletion of the Cnn3 gene to the B cell compartment by crossing our mice with the mb1-cre mouse strain. Percentages of non-B cells, pro-/ 
pre-B cells, immature and recirculating mature B cells in the bone marrow of these mice were comparable to that of controls. Moreover, when stimulated with an antibody that crosslinks the pre-BCR, overall induced tyrosine phosphorylation as measured by western blotting was identical for both genotypes. Furthermore, the kinetic of induced calcium flux in Cnn3 ko d/d versus littermate control cells was comparable. Reflecting this normal functional capacity and distribution of cells in the bone marrow, we were furthermore unable to find any statistically significant differences in the percentages and the signaling capacity of splenic B cells as measured by induced calcium flux. This suggests that calponin-3, albeit highly expressed, is either not functionally involved in early B cell development or that its loss can be compensated, e.g. by calponin 2. To rule this out, we crossed our Cnn3 ki f/f mb1-cre mice with a floxed Cnn2 strain PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19711918 previously described. However, the B cell-specific Fig 5. Deletion of calponin-3 results in exencephaly. A. Targeted Cnn3 locus before and after Cre-mediated deletion, illustrated as in Fig 2A. B. Reflected light images of Cnn3 ko d/d embryos their +/+ littermates as a control. C. Southern blot analysis of a control mouse, a heterozygous Cnn3 and a homozygous Cnn3 knockout mouse. BamHI-digested genomic DNA from the respective mice was separated by gel electrophoresis, blotted and hybridized with the 3′ external probe. Arrows indicate the positions of fragments corresponding to the wild-type as well PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19710274 as the deleted allele. D. Fetal brain tissue of a homozygous Cnn3 knockout and a control littermate was lysed, subjected to SDS-PAGE and western blotting and
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