Wnt- and RA-signaling in pancreas development

We identified the Wnt-receptor Fzd4 and its secreted variant Fzd4s as direct RA-targets with a possible role in pancreas development. Therefore, we asked about the role of Wnt-signaling in pancreas development. Previous studies proposed that Wnt-signaling needs to be repressed for foregut maintenance and therefore to allow a proper pancreas development. In Xenopus, McLin and colleagues found several foregut markers repressed upon Wnt8 over-expression (McLin etal., 2007).

Furthermore, Li and colleagues identified the secreted Wnt-inhibitor sfrp5 to be expressed in the early foregut epithelium of Xenopus embryos. A downregulation of functional sfrp5 leads to smaller foregut domains and in contrast the ectopic sfrp5 expression results in an expanded foregut domain at the expense of the hindgut (Li et al., 2008). However, a more recent study found evidences for the requirement of

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low Wnt-signaling activity for foregut maintenance. It was shown that the depletion of Wnt-receptor Fzd7 in the foregut results in pancreas agenesis in Xenopus and that a low expression level of this Wnt-receptor is essential for foregut maintenance (Zhang et al., 2013a). Moreover, sfrps that were initially thought to be exclusively negative Wnt-signaling modulators emerged as biphasic regulators in a concentration dependent manner (Mii and Taira, 2009). These findings suggest a regulatory mechanism that ensures an appropriate Wnt-signaling activity in the foregut.

Our finding of Wnt-receptors Fzd4 and Fzd4s involved in pancreas development is further supported by transcriptome analysis of hepatic and pancreatic progenitors in mouse. The transcriptome of bi-potential hepato-pancreatic progenitors was compared to the transcriptome of developed dorsal and ventral pancreatic buds and the liver bud (Rodríguez-Seguel et al., 2013). They found intracellular Wnt-signaling transducers like Disheveled to be expressed equally in all samples whereas Wnt-ligands, receptors and co-receptors were strongly downregulated in liver progenitors. Among these differentially expressed Wnt-components, Fzd4 and its ligand Wnt5a were found. It was demonstrated that endodermal explants from Xenopus embryos treated with soluble Wnt5a exhibit an enhanced expression of Pdx1 and Ptf1a. Furthermore, liver cells treated with Wnt5a strongly induce Pdx1 expression (Rodríguez-Seguel et al., 2013). They suggest that non-canonical Wnt-signaling is a potential promotor of pancreatic fate. We found only Fzd4/Fzd4s expression regulated by RA and not Wnt5a expression. However, we observed an effect of treatment on Wnt-signaling in our explant system. Two hours after RA-addition the activity of both, canonical and non-canonical Wnt-signaling reporter was decreased. Thereby, the non-canonical Wnt-reporter was slightly stronger affected.

The described effect of RA-treatment on canonical Wnt-signaling is consistent with several other studies. Zhang and colleagues identified Ndrg1a as RA-target and demonstrated that the inhibitory function of Ndrg1 on canonical Wnt-signaling is required for foregut development (Zhang et al., 2013b). However, Ndrg1 was not differentially expressed upon RA-treatment in our system. One explanation for this could be the late time point of Ndrg1 induction by RA that was observed in stage 16 embryos earliest, but we searched for RA-targets that were induced within two hours after RA-addition. Another study using mouse ESCs also found the negative regulatory effect of RA on canonical Wnt-signaling (Osei-sarfo and Gudas, 2014). In addition, they found non-canonical Wnt-signaling activated by RA. This activatory effect of RA on non-canonical Wnt-signaling was also described by Harada and colleagues (Harada et al., 2007). There, RA-inducible G-protein-coupled receptors

107 were found to bind receptors and thereby activating non-canonical Wnt-signaling. These findings seem to be contradictory to our observed negative regulation of non-canonical Wnt-reporter activity by RA-treatment. However, the term “non-canonical Wnt-signaling” comprises two different pathways. The planar cell polarity (PCP) pathway that involves Rho GTPase and JNK and on the other hand the calcium pathway that involves calcium-sensitive kinases and PKC (reviewed in Nusse, 2012). Our non-canonical Wnt-reporter system is based on an Atf2-response element that is activated by the PCP-pathway (Ohkawara and Niehrs, 2010). In contrast, both studies used a reporter that contains a binding site for the transcription factor NFAT. This transcription factor is activated by the Wnt/calcium pathway (Dejmek et al., 2006). Thus, the effect of RA on the NFAT-reporter needs to be tested in our system. It is possible that RA-signaling has a biphasic activity on different non-canonical Wnt-signaling pathways.

We further examined the effect of Fzd4-knockdown on Wnt-signaling reporter activity in the explant system. We found canonical Wnt-reporter activity only slightly and not significantly increased upon Fzd4-downregulation. In contrast, non-canonical Atf2-reporter activity was significantly increased. This finding complies with the observed decrease in non-canonical Wnt-reporter activity upon RA addition.

Hence, these data suggest that the negative regulatory effect of RA on non-canonical Wnt-signaling is mediated by Fzd4 and/or Fzd4s. However, Fzd4 as well as Fzd4s were shown to positively regulate non-canonical Wnt/PCP-signaling (Descamps et al., 2012; Gorny et al., 2013). Therefore, the effect of Fzd4-downregulation on non-canonical Wnt-signaling needs to be further investigated.

Moreover, it remains to be tested whether the downregulation of Wnt-signaling by RA is mediated by Fzd4 and if this Fzd4-function is required for pancreas specification.

A connection of Hnf1b and Wnt-signaling was shown in zebrafish. Lancman and colleagues demonstrated that Hnf1b and Wnt2b synergistically function in the specification of hepato-pancreatic progenitors (Lancman et al., 2013). Thus, it is necessary to examine if a combined activity of Hnf1b and Fzd4 and/or Fzd4s is sufficient to substitute for RA in pancreas specification.

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