Canonical Wnt signaling and the regulation of divergent mesenchymal Fgf8 expression in axolotl limb development and regeneration

Abstract

The expression of Fibroblast growth factors (Fgf) ligands in a specialized epithelial compartment, the Apical Ectodermal Ridge (AER), is a conserved feature of limb development across vertebrate species. In vertebrates, Fgf 4, 8, 9, and 17 are all expressed in the AER. An exception to this paradigm is the salamander (axolotl) developing and regenerating limb, where key Fgf ligands are expressed in the mesenchyme. The mesenchymal expression of Amex.Fgf8 in axolotl has been suggested to be critical for regeneration. To date, there is little knowledge regarding what controls Amex.Fgf8 expression in the axolotl limb mesenchyme. A large body of mouse and chick studies have defined a set of transcription factors and canonical Wnt signaling as the main regulators of epidermal Fgf8 expression in these organisms. In this study, we address the hypothesis that alterations to one or more of these components during evolution has resulted in mesenchymal Amex.Fgf8 expression in the axolotl. To sensitively quantify gene expression with spatial precision, we combined optical clearing of whole-mount axolotl limb tissue with single molecule fluorescen in situ hybridization and a semi-automated quantification pipeline. Several candidate upstream components were found expressed in the axolotl ectoderm, indicating that they are not direct regulators of Amex.Fgf8 expression. We found that Amex.Wnt3a is expressed in axolotl limb epidermis, similarly to chicken and mouse. However, unlike in amniotes, Wnt target genes are activated preferentially in limb mesenchyme rather than in epidermis. Inhibition and activation of Wnt signaling results in downregulation and upregulation of mesenchymal Amex.Fgf8 expression respectively. These results implicate a shift in tissue responsiveness to canonical Wnt signaling from epidermis to mesenchyme as one step contributing to the unique mesenchymal Amex.Fgf8 expression seen in the axolotl.

Multi-species atlas resolves an axolotl limb development and regeneration paradox

“…Then, we surveyed the established AER markers to determine the transcriptional similarities of these axolotl cells with the AER in other species ( Figures 1E and S5 ). The large majority of axolotl cells in this cluster co-expressed many of the AER markers 1 , such as Wnt5a and Msx2 , whilst some others (e.g., Fgf8 ) were absent (Figures 1E and S5) , in alignment with recent reports 4, 7 . A further transcriptome-wide comparison found not only a high similarity of AER-like axolotl cells to the AER cells in other species, but also that this population was distinct from non-AER basal ectodermal cells (Figures 1F and S6) .…”

“…Axolotl AER cells had comparable signaling potential to the AER in other species (Figures 1G and S7) . Meanwhile, individual pathway examination revealed the axolotl AER differs in paralog and co-factor expressions, particularly in the FGF and WNT pathways (Figure S8) , complementing and extending previous observations 4, 7, 21 . We could not detect noticeable differences in transcriptional levels associated with DELTAs, BMPs, or TGFbs (Figure S8) .…”

“…The copyright holder for this preprint this version posted March 1, 2023. ; https://doi.org/10.1101/2023.03.01.530572 doi: bioRxiv preprint with recent reports 4,7 . A further transcriptome-wide comparison found not only a high similarity of AER-like axolotl cells to the AER cells in other species, but also that this population was distinct from non-AER basal ectodermal cells (Figures 1F and S6).…”

“…The AER supplies critical signaling ligands and its spatial organization contribute to the morphogen gradients to form a correctly patterned limb 2 . Interestingly, the leading limb regeneration model organism, the axolotl 3 , was suggested not to have an AER, as they lack a morphological ridge structure and some of the molecular AER markers [4][5][6][7] . Despite these findings, as .…”

Thyroid hormone receptor knockout prevents the loss of Xenopus tail regeneration capacity at metamorphic climax

“…It would be interesting to investigate whether RXR-RAR heterodimers play a role in tail regeneration in TRDKO. In addition, Wnt and FGF pathways are well-known signaling pathways implicated in tail and limb regeneration [ 26 , 72 , 73 ]. We found that wnt3a , wnt5a , fgf10 and fgf8 were expressed at much higher levels in the patterning and outgrowth period in the TRDKO tail compared to wild type tail after amputation at stage 61.…”