Antagonistic cross-regulation between Wnt and Hedgehog signalling pathways controls post-embryonic retinal proliferation

Abstract

SUMMARYContinuous neurogenesis in the adult nervous system requires a delicate balance between proliferation and differentiation. Although Wnt/b-catenin and Hedgehog signalling pathways are thought to share a mitogenic function in adult neural stem/progenitor cells, it remains unclear how they interact in this process. Adult amphibians produce retinal neurons from a pool of neural stem cells localised in the ciliary marginal zone (CMZ). Surprisingly, we found that perturbations of the Wnt and Hedgehog pathways result in opposite proliferative outcomes of neural stem/progenitor cells in the CMZ. Additionally, our study revealed that Wnt and Hedgehog morphogens are produced in mutually exclusive territories of the post-embryonic retina. Using genetic and pharmacological tools, we found that the Wnt and Hedgehog pathways exhibit reciprocal inhibition. Our data suggest that Sfrp-1 and Gli3 contribute to this negative cross-regulation. Altogether, our results reveal an unexpected antagonistic interplay of Wnt and Hedgehog signals that may tightly regulate the extent of neural stem/progenitor cell proliferation in the Xenopus retina. and a transgenic male. The latter was selected beforehand as having a single transgene insertion site (as inferred by mendelian ratios in its progeny) in order to ensure homogeneous levels of GFP expression in the offspring. Construction of the LEF1-VP16 and LEF1-EnR transgenesis vectors has been described previously (Denayer et al., 2008) and transgenic X. tropicalis lines (LEF1-VP16Tg and LEF1-EnRTg) were generated as described (Sekkali et al., 2008). These constructs are fused with the dexamethasone-responsive hormone-binding domain of the human glucocorticoid receptor (GR). Expression constructs and morpholinospCS2- TCF3-VP16GR and pCS2-dnTCF3-GR (de Croze et al., 2011), pCS2-Ihh-CD2 [previously called Bhh (Locker et al., 2006)], pCS2-Smo-M2 , pCS2-cyclinA2 and pCS2-cdk2 (Decembrini et al., 2006) and pCS2-GFP (a gift from David Turner, University of Michigan, Ann Arbor, USA) were described previously. pCS2-Shh-CD2 and pCS2-Dhh-CD2 (previously called Chh) were generated by subcloning the N-terminal coding regions (devoid of the C-terminal cleavage product) of Shh and Dhh cDNAs (Ekker et al., 1995) Microinjection and in vivo DNA lipofectionCapped mRNAs encoding TCF3-VP16GR and GFP were transcribed from pCS2 plasmids after NotI digestion using the mMessage mMachine SP6 Kit (Ambion). Then, 400 pg of each mRNA was injected into two blastomeres of four-cell stage embryos. Gli3, Sfrp-1 or standard control morpholino oligonucleotides (Gene Tools) were injected into one blastomere at the one-cell stage (30 ng). Their efficacy was tested by analysing in vivo GFP fluorescence following co-injection of a chimeric GFP construct fused downstream of the morpholino-complementary sequence (supplementary material Fig. S1).Lipofection experiments were performed by cotransfecting the indicated pCS2 constructs together with pCS2-GFP at stage 18 into the presumptive region of the retina, as previously...

Involvement of the Wnt/β-catenin pathway in neurectoderm architecture in Platynereis dumerilii

“…Endo-IWR-1 acts as a stabilizer of Axin, thereby stimulating the degradation of cytoplasmic b-catenin and constitutively inhibiting the Wnt/b-catenin pathway, without altering the pool of membrane tethered b-catenin 25,27 . This molecule has been shown to reduce the amount of b-catenin and to inhibit the transcriptional response to Wnt/b-catenin signalling in developing zebrafish and Xenopus embryos and larvae 28,29 , indicating its suitability for in vivo studies. In order to confirm endo-IWR-1 phenotypes, we used PNU-74654 that prevents the interaction between b-catenin and the transcription factor TCF 26,30 .…”

Antagonism between Hedgehog and Wnt signaling pathways regulates tumorigenicity (Review)

“…In addition, treatment with IWR-1, which prevents Axin protein degradation, led to the opposite phenotype. Furthermore, morpholino-mediated GLI3 knockdown could rescue the decreased PTCH1 expression observed in BIO-treated tadpoles (83). This evidence suggests that GLI3 represents a key downstream effector of the Wnt pathway, which may account for its negative effect on Hh activity (82,93,94).…”

“…Although a number of studies have demonstrated that synergy between the two pathways promotes embryogenesis and cellular differentiation, conflicting data has also been reported that the Hh and Wnt signaling pathways are functionally antagonistic in vertebrates and invertebrates (80)(81)(82)(83)(84). The crosstalk protein SFRP1, which acts as an antagonist of Wnt signaling, was initially identified in 1998, and has subsequently been shown to be regulated by Hh in the developing spinal cord and gastric cancer cells (12,(80)(81)(82).…”

“…The crosstalk protein SFRP1, which acts as an antagonist of Wnt signaling, was initially identified in 1998, and has subsequently been shown to be regulated by Hh in the developing spinal cord and gastric cancer cells (12,(80)(81)(82). Borday et al (83) investigated the potential cross-regulation between the Wnt and Hh signaling pathways in neural stem/progenitor cells in the ciliary marginal zones using pharmacological tools. They detected Wnt activity and subsequently Hedgehog inhibition by 6-bromoindirubin-3'-oxime treatment, which worked as a selective activator of the canonical Wnt pathway.…”

“…Furthermore, Hh signaling pathway negatively regulates Wnt activity via transcriptional regulation of SFRP1, and the Wnt/β-catenin pathway downregulates Hedgehog activity through Gli3 transcriptional regulation. The reciprocal inhibition between Hh and Wnt signaling pathways regulates a delicate balance between proliferation and differentiation of neural stem cells (83).…”