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Fig. 6. Knockdown of Xwnt2b expression causes foregut hypoplasia. (A) Specificity control for the Xwnt2b-Splice Morpholino (SpliMO). 20 ng SpliMO were injected animally into both blastomeres at the two cell stage and embryos/animal cap explants were analysed for the Xwnt2b transcript by RT-PCR at different developmental stages. M = Fastruler™ DNA ladder Low Range (Fermentas). (B–I) Marker gene expression analysis after Xwnt2b knockdown by whole-mount in situ hybridisation. A total amount of 20 ng SpliMO was injected into the vegetal pole of all four blastomeres at the four-cell stage. LacZ activity was used as a lineage tracer. Whole-mount in situ hybridisation was performed with stage 37/38 (B, C) and stage 40 embryos (D–I). The red arrowheads mark the hypoplastic pancreas (E) and stomach/duodenum (I) as detected by staining with XPDIp and fibrinogen probes, respectively. The red bars indicate the width of the Sox2 expression domain (F, G). (J) Vibratome sections of control and SpliMO-injected embryos, as well as of gut explants were analysed for proliferative cells using pH3-labelling. 10 consecutive sections of 3 embryos/explants were scored for pH3+ cells in the anterior endoderm at stages 32 and 37/38, and in the stomach/pancreas/liver area of stage 40 gut explants. The mean value of pH3+ cells in the controls was set to 100%. Significance was calculated using Student's T Test and marked with asterisks. (K) Quantification of the size of the Sox2-expression domain in control, MO1 + MO2-, Xwnt2b-SpliMO- and double injected gut explants as well as Xwnt2b-MO and Xwnt2b-SpliMO2 injected gut explants. The average pixel number in the controls was set to 100%. Significance was calculated using Student's T Test and marked with asterisks. |
