INTACT-olotl: A simplified system for rapid cell type-specific profiling during axolotl limb regeneration
- 1Wake Forest University, Wake Forest Institute of Regenerative Medicine
Abstract
Regenerating tissues like the axolotl (Ambystoma mexicanum) limb draw from many cell types, but isolating specific cell populations remains a bottleneck. Standard approaches rely on enzymatic dissociation and fluorescence-activated cell sorting (FACS), which are slow, costly, and require specialized instrumentation. We adapted INTACT (Isolation of Nuclei Tagged in specific Cell Types) to the axolotl, generating a transgenic line (INTACT-olotl) expressing Sun1-sfGFP in limb connective tissue, the population that carries limb positional information, dedifferentiates following injury, and forms the majority of the regenerative blastema. Combining an optimized nuclei isolation protocol with anti-GFP nanobody capture enriched labeled nuclei from ~34.6% of input to 80-89% purity. Relative to dissociation and FACS, this recovers roughly 30-fold more target nuclei in less than half the time and without a cytometer. Bead-bound nuclei are directly applied to combinatorial-barcoding snRNA-seq, eliminating fluorescence-activated nuclei sorting (FANS). Profiling INTACT+ connective tissue during TGF{beta} inhibition revealed a loss of extracellular matrix remodeling with upregulated pro-fibrotic and inflammatory gene programs, resembling an unresolved wound environment. These changes reverse and regeneration resumes upon drug washout, indicating that functional TGF{beta} signaling is required for the transition from wound resolution to blastema formation.
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BibTeX
@misc{barrett2026intactolotl,
title = {{INTACT-olotl: A simplified system for rapid cell type-specific profiling during axolotl limb regeneration}},
author = {J. E. Barrett and R. Ailuri and Z. Amos and T. Curtis and S. Rock and J. D. Currie},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.18.752769},
url = {https://www.biorxiv.org/content/10.64898/2026.09.18.752769v1},
note = {CC BY-ND},
}
RIS
TY - UNPB
TI - INTACT-olotl: A simplified system for rapid cell type-specific profiling during axolotl limb regeneration
AU - J. E. Barrett
AU - R. Ailuri
AU - Z. Amos
AU - T. Curtis
AU - S. Rock
AU - J. D. Currie
PY - 2026
DA - 2026/09/18
PB - bioRxiv
DO - 10.64898/2026.09.18.752769
UR - https://www.biorxiv.org/content/10.64898/2026.09.18.752769v1
AB - Regenerating tissues like the axolotl (Ambystoma mexicanum) limb draw from many cell types, but isolating specific cell populations remains a bottleneck. Standard approaches rely on enzymatic dissociation and fluorescence-activated cell sorting (FACS), which are slow, costly, and require specialized instrumentation. We adapted INTACT (Isolation of Nuclei Tagged in specific Cell Types) to the axolotl, generating a transgenic line (INTACT-olotl) expressing Sun1-sfGFP in limb connective tissue, the population that carries limb positional information, dedifferentiates following injury, and forms the majority of the regenerative blastema. Combining an optimized nuclei isolation protocol with anti-GFP nanobody capture enriched labeled nuclei from ~34.6% of input to 80-89% purity. Relative to dissociation and FACS, this recovers roughly 30-fold more target nuclei in less than half the time and without a cytometer. Bead-bound nuclei are directly applied to combinatorial-barcoding snRNA-seq, eliminating fluorescence-activated nuclei sorting (FANS). Profiling INTACT+ connective tissue during TGF{beta} inhibition revealed a loss of extracellular matrix remodeling with upregulated pro-fibrotic and inflammatory gene programs, resembling an unresolved wound environment. These changes reverse and regeneration resumes upon drug washout, indicating that functional TGF{beta} signaling is required for the transition from wound resolution to blastema formation.
ER -