Fig. 4. Functional Npn1 is required for the Sema3A- and Sema3C-mediated SMG branching morphogenesis in mice. (A) Anti-Npn1 antibody dose-dependently abolished SMG cleft formation promoted by Sema3A or Sema3C in the SMG co-cultures. Complete inhibition could be achieved in the presence of 5 µg/ml neutralizing antibody. Representative explants are shown and the summary of six independent experiments is shown in the bar graph. Paired t-test: ^*, P<0.05; ^**, P<0.01. (B) Npn1-AP fusion proteins, but not AP proteins, blocked Sema3A-mediated branching activity in the SMG co-cultures. Note that Npn1-AP fusion protein alone could block the endogenous branching activities (Mock) (upper panels). By contrast, application of Npn2-Fc fusion proteins in the co-cultures had no effects on the SMG branching activity (lower panels). Representative explants were shown and the summary of five independent experiments is shown in the bar graph. Paired t-test: ^*, P<0.05; ^**, P<0.01. (C) Sema3A-AP bound the epithelial buds in the SMG cultured ex vivo for 24 hours. As a control, when the Sema3A-AP conditioned medium was depleted of the AP-fusion proteins by pre-incubation with Npn1-transfected COS cells, the binding activity on the epithelial buds was greatly reduced. (D) Sema3A mRNA was detected in the SMG epithelial buds cultured ex vivo (a) and in E15.5 embryonic SMG (b). The expression was distributed as a gradient with the highest level at the front end of the terminal bud. Immunofluorescence staining of E-cadherin highlighted the epithelial buds in a. Area within dashed line, epithelial bud. (E) Semi-quantitative RT-PCR analysis confirmed that Sema3A transcript was mainly in the SMG epithelium. Scale bars: 100 µm. C, mock-transfected COS cells; Epi, epithelium; IF, immunofluorescence; ISH, RNA in situ hybridization; M, mesenchyme.