Fig. 2

LIX1 is required for mesenchymal progenitor proliferation and smooth muscle cell determination in the developing stomach. a LIX1 whole-mount in situ hybridization of E4.5 gastrointestinal tracts. Scale bars, 1 mm. White arrowheads show the down-regulation of LIX1 expression in ShLIX1-expressing stomachs. b SM22 whole-mount in situ hybridization of gastrointestinal tracts. Black bars locate the change in the SM22-expression domain. c RT-qPCR analysis of relative mRNA expression. Data were normalized to GAPDH expression. Normalized expression levels were converted to fold changes. Values are presented as the mean ± standard error of the mean (SEM) of n = 12 controls vs. n = 10 ShLIX1-expressing stomachs. *P < 0.05; **P < 0.01 by one-tailed (LIX1, αSMA and SM22) or two-tailed (BARX1, MYOCD and SRF) Mann–Whitney tests. d Serial transverse sections of stomachs analysed either by in situ hybridization using the retroviral Envelop (ENV) riboprobe (scale bars, 500 μm) or by immunofluorescence with anti-αSMA antibodies. Nuclei were visualized with Hoechst. Black arrowheads in the ENV panels indicate the area that is imaged at high power in the αSMA panels. e Serial transverse sections of stomachs analysed either by in situ hybridization using the ENV riboprobe (scale bars, 500 μm) or by immunofluorescence using anti-PH3 antibodies. Nuclei are visualized with Hoechst. Black arrowheads in the ENV panels indicate the area imaged at high power in the PΗ3 panels. Graph represents the quantification of PH3-positive cells. Normalized expression levels were converted to fold changes. Values are presented as the mean ± SEM of n = 12 control vs. n = 10 ShLIX1-expressing stomachs. ***P < 0.001 by two-tailed Mann–Whitney test. f RT-qPCR analysis of relative mRNA expression in control and ShLIX1-expressing stomachs. Data were normalized to GAPDH expression. Normalized expression levels were converted to fold changes. Values are presented as the mean ± SEM of n = 12 controls vs. n = 10 ShLIX1-expressing stomachs. *P < 0.05; **P < 0.01 by two-tailed Mann–Whitney tests