Fig. 3

Wls is specifically expressed and produced in HbChP. a Gene expression analysis by real-time qPCR of Wntless (Wls) expression in TelChP and HbChP at E12.5 and E17.5. Wls gene expression was normalized against expression level of β-actin in each condition. Graph shows n = 3 biologically independent samples; error bars represent mean ± s.d.; P-values (two-tailed Student’s t-test with unequal variance) for differences between analysed ChPs: * P < 0.05. TelChP vs HbChP: E12.5 P = 0.0261; E17.5 P = 0.0237. Biological replicates are indicated in the graph. b In situ hybridization analysis of HbChP coronal sections confirms Wls expression levels difference between E13.5 TelChP and HbChP, n = 6. Wls and Wnt5a expression in TelChP region is restricted mostly to CH (asterisk) and not to TelChP epithelium. Scale bar: 50 μm. Inset images show higher magnification view of fluorescent signal corresponding to Wls and Wnt5a transcripts in corresponding TelChP and HbChP epithelium. Scale bar: 10 μm. c Western blot analysis of WLS levels in tissue lysates of TelChP and HbChP isolated at E14.5 and E17.5, n = 3. β-actin serves as a loading control. d Representative images from immunofluorescent analysis of TelChP and HbChP at E14.5 displaying presence of WLS only in the HbChP, n = 4. WLS is absent from the TelChP, but present in CH (asterisk). Scale bar: 100 μm. e Detailed view of Wnt5a and Wls transcripts distribution highlights correlation between levels of Wls and Wnt5a in individual HbChP epithelial cells at E13.5 (arrowhead - Wnt5a^high/Wls^high; empty arrowhead - Wnt5a^low/Wls^low), n = 6. Scale bar: 5 μm. f Magnified image of immunofluorescent analysis of HbChP epithelium at E14.5 shows high degree of correlation between WNT5A and WLS levels observed within HbChP epithelial cells (arrowheads), n = 4. Scale bar: 5 μm. Source data are provided as a Source Data file