Tig1 regulates proximo-distal identity during salamander limb regeneration

Abstract

Salamander limb regeneration is an accurate process which gives rise exclusively to the missing structures, irrespective of the amputation level. This suggests that cells in the stump have an awareness of their spatial location, a property termed positional identity. Little is known about how positional identity is encoded, in salamanders or other biological systems. Through single-cell RNAseq analysis, we identified Tig1/Rarres1 as a potential determinant of proximal identity. Tig1 encodes a conserved cell surface molecule, is regulated by retinoic acid and exhibits a graded expression along the proximo-distal axis of the limb. Its overexpression leads to regeneration defects in the distal elements and elicits proximal displacement of blastema cells, while its neutralisation blocks proximo-distal cell surface interactions. Critically, Tig1 reprogrammes distal cells to a proximal identity, upregulating Prod1 and inhibiting Hoxa13 and distal transcriptional networks. Thus, Tig1 is a central cell surface determinant of proximal identity in the salamander limb.

Sequencing and chromosome-scale assembly of the giantPleurodeles waltlgenome

“…4a, see also Suppl. Data 6 for a general pipeline for microsynteny analysis), recently implicated in the regulation of proximo-distal identity during salamander limb regeneration (Oliveira et al, 2022). Despite clear variations in gene size, we uncovered a remarkable conservation of loci organisation, with the presence of Tig1 neighbours Schip1 and Mfsd1 at one end, and of Gfm1, Mlf1 and Shox2 at the other end of the Tig1 locus, a structure that is recapitulated across representative vertebrate genomes (Fig.…”

“…C) Tig1 overexpression affects expression of nearby genes in A. mexicanum. Bars represent ratios of gene expression in Tig1 or Tig1 P155A overexpressing cells versus control cells (analysis based on dataset from (Oliveira et al, 2022)). D) Heatmap depicting differences in gene expression of Tig1 and its genomic neighbours during P. waltl limb regeneration.…”

Molecular basis for positional memory and its reprogrammability in limb regeneration

“…For the RA pathway, the aldehyde dehydrogenase enzyme Aldh1a3 was enriched anteriorly whereas its responder gene, encoding for the retinoic acid receptor responder 1 ( Rarres1 , also known as Tig1 ), was enriched posteriorly. Tig1/Rarres1 was recently implicated in proximal-distal positional identity through misexpression assays and effects on genes known to be involved in proximal-distal patterning 7 . However, the loss of function phenotype of Tig1/Rarres1 in vivo is not known, nor is it known if this gene can reprogramme positional memory for subsequent regeneration.…”

“…Among the salamander limb cells, connective tissue cells are known to encode positional information 4,5 , and any positional discontinuities in the limb are likely recognised at both gene expression and chromatin levels. For example, proximal and distal connective tissue cells express different levels of the cell surface molecules Prod1 and Tig1 and harbour differential epigenetic marking on Hox genes and other patterning gene loci [6][7][8] . Similarly, dorsal, but not ventral, connective tissue cells transcribe the transcription factor Lmx1b 9,10 .…”

Senescent cells enhance newt limb regeneration by promoting muscle dedifferentiation

“…Axolotls (A. mexicanum) were obtained from Neil Hardy Aquatica (Croydon, UK) and from the axolotl facility at TUD-CRTD Center for Regenerative Therapies Dresden (Dresden, Germany). Animals were maintained in individual aquaria at ~18-20 °C, as previously described (Oliveira et al, 2022). Axolotls of the leucistic (d/d) strain were used in all experiments.…”