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Canonical Wnt signalling is known to be involved in the regulation of differentiation and proliferation in the context of endodermal organogenesis. Wnt mediated beta-catenin activation is understood to be modulated by secreted Frizzled-related proteins, such as XsFRP5, which is dynamically expressed in the prospective liver/ventral pancreatic precursor cells during late neurula stages, becoming liver specific at tailbud stages and shifting to the posteriorstomach/anterior duodenum territory during tadpole stages of Xenopus embryogenesis. These expression characteristics prompted us to analyse the function of XsFRP5 in the context of endodermal organogenesis. We demonstrate that XsFRP5 can form a complex with and inhibit a multitude of different Wnt ligands, including both canonical and non-canonical ones. Knockdown of XsFRP5 results in transient pancreatic hypoplasia as well as in an enlargement of the stomach. In VegT-injected animal cap explants, XsFRP5 can induce expression of exocrine but not endocrine pancreatic marker genes. Both, its expression characteristics as well as its interactions with XsFRP5, define Wnt2b as a putative target for XsFRP5 in vivo. Knockdown of Wnt2b results in a hypoplastic stomach as well as in hypoplasia of the pancreas. On the basis of these findings we propose that XsFRP5 exerts an early regulatory function in the specification of the ventralpancreas, as well as a late function in controlling stomach size via inhibition of Wnt signalling.
Fig. 3. Knockdown of XsFRP5 expression by morpholino injection leads to a hypoplastic ventralpancreas and stomach enlargement. (A–U) Endodermal marker gene expression analysis by whole-mount in situ hybridisation. 5 ng mmMO1 or a combination of 5 ng MO1 and 2 ng MO2 were 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 32 (A–F), stage 37/38 (G–L) and stage 40 embryos (M–U). The red bars indicate the width of the Sox2 expression domain (P–R). The red arrowheads mark the ventral pancreatic anlage (C, F, I, L, O). (V) Vibratome sections of stage 37/38 control and MO1 + MO2-injected embryos stained for Ptf1a/p48. The blue arrows indicate the dorsal pancreatic anlage and the red arrows mark the ventral anlagen. (W) The size of the ventral pancreatic anlagen was quantified in control, mmMO- and MO1 + MO2-injected embryos stained for Ptf1a/p48 or XPDIp. The average pixel number in the controls was set to 100%. Significance was calculated using Student's T Test and is marked with asterisks. (X) Vibratome sections of stage 32 and stage 37/38 embryos, as well as stage 40 gut explants were quantified for proliferative cells using pH3-labelling. 10 consecutive sections of 3 embryos/explants were scored for pH3+ cells in the anteriorendoderm 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 is marked with asterisks.
Fig. 5. Comparative expression analysis of XsFRP5 and different Xwnts. DIG-UTP labelled antisense probes against XsFRP5 (A–C), Xwnt2b (D–F), Xwnt5a (G–I) and Xwnt11R (J–L) were used for whole-mount in situ hybridisation of stage 35 embryos (A, D, G, J), stage 40 embryos (B. E, H, K) and stage 40 gut tube and heart-explants (C, F, I, L). Corresponding sections are shown in A′–K′. he = heart, li = liver, lu = lung, st = stomach/duodenum, oe = oesophagus, and pr = pronephros.
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 anteriorendoderm 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.
