Chromatin states at homeoprotein loci distinguish axolotl limb segments prior to regeneration

Abstract

The salamander limb regenerates only the missing portion. Each limb segment can only form segments equivalent to- or more distal to their own identity, relying on a property termed, positional information. How positional information is encoded in limb cells has been unknown. By cell-type-specific chromatin profiling of upper arm, lower arm, and hand, we found segment-specific levels of histone H3K27me3 at limb homeoprotein gene loci but not their upstream regulators, constituting an intrinsic segment information code. During regeneration, regeneration-specific regulatory elements became active prior to the re-appearance of developmental regulatory elements. This means that, in the hand segment, the permissive chromatin state of the hand homeoprotein gene HoxA13 engages with regeneration regulatory elements, bypassing the upper limb program.

Somite-independent regeneration of the axolotl primary body axis

“…tgISceI(Mmu .Col1a2:TFPnls-T2a-ER T 2 -Cre-ER T 2 ) Etnka , tgISceI( CAGGs:LoxP-GFP-LoxP-Cherry ) Etnka and tgISceI( Caggs:LoxP-STOP-LoxP-Cherry ) Etnka transgenic lines are described previously 6,27,28 . To generate Lfng transgenic animals, mouse Lfng 29 driver element was cloned at the 5′ end of the GFPnls-T2A-ER T 2 -Cre-ER T 2 (ER-Cre-ER) cassette with flanking ISceI sites.…”

“…Collagen 1 alpha 2 (COL1A2) is an extracellular matrix (ECM) protein and in axolotl, it is expressed in dermal fibroblasts and skeleton cells (28). Staining tail sections with COL1A2 antibodies showed broad labelling of the basal lamina (that demarcates dermal fibroblasts and basal keratinocytes), skeletal and periskeletal structures, as well as fin mesenchyme.…”

Molecular basis for positional memory and its reprogrammability in limb regeneration

“…For lineage tracing, either ZRS>TFP axolotls or Hand2 lineage tracing axolotls were mated with loxP-mCherry fate mapping axolotl of genotype: tgSceI( Caggs:loxP-GFP-dead(Stop)-loxP-mCherry ) Etnka (ref 8 ) . To induce Cre/loxP recombination during development, stage 42 ZRS or Hand2 lineage tracing embryos were bathed overnight dark in 500 ml of 2 μM 4-OHT, as described in the water-based method of 46 .…”

“…2a-c) , we performed lineage tracing of embryonic Shh cells by crossing the ZRS axolotl with our previously published loxP-mCherry fate mapping axolotl ( Fig. 1c ) 8 . In this model, we expected limb cells derived from embryonic Shh lineage cells to express mCherry and cells switching on Shh after amputation to express TFP.…”

“…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 .…”