Biology subjects

Currie, J. D.

Publications and source records attributed to Currie, J. D..

4 recordsLinked to original sources

bioRxiv2026

INTACT-olotl: A simplified system for rapid cell type-specific profiling during axolotl limb regeneration

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.

developmental biology↗

bioRxiv2025

Enhancer-directed gene delivery for digit regeneration based on conserved epidermal factors

Limb loss remains a significant clinical challenge, but regenerative medicine approaches such as gene therapy offer a promising strategy to trigger endogenous regeneration programs. Optimal vector configurations and molecular targets for appendicular skeletal repair are not well defined. Here, we leveraged insights from species with a high endogenous capacity for appendage regeneration to design an enhancer-directed gene delivery platform that functions during mouse digit regeneration, a well characterized model for partial limb regeneration in mammals. Single-cell RNA sequencing of zebrafish caudal fin regeneration, combined with expression data in regenerating salamander limbs and mouse digit tips, implicated the SP family of transcription factors as conserved, epidermally-expressed mediators of appendage regrowth. Null mutants of Sp8 demonstrated impaired limb regeneration in salamanders, while conditional knockout of Sp6 and/or Sp8 in the mouse basal epidermis resulted in defective bony digit tip regeneration, involving an IL-17 mediated osteoclastogenic program. Spatiotemporally focused expression of FGF8, a known target of SP factors, using a zebrafish-derived tissue regeneration enhancer element via adeno-associated viral vectors, could partially rescue digit tip regeneration in SP knockout mice and accelerate digit regeneration in wildtype mice. Our results demonstrate a contextual gene therapy approach to address limb loss based on genes like SP transcription factors conserved across multiple contexts of appendage regeneration. Significance StatementInstructing regeneration of complex structures in mammals remains an unsolved problem. Gene therapy offers a compelling approach to foster endogenous regeneration by delivering therapeutic gene products to specific cells post injury. We identified a conserved regeneration-linked epidermal transcriptional program in mouse digit regeneration centered on the SP6 and SP8 transcription factors, involving inflammatory responses from osteoclasts. We engineered AAVs harboring a zebrafish tissue regeneration enhancer to direct FGF8 expression in the epidermis after amputation. This enhancer directed delivery partially rescued impaired digit regeneration in Sp6 and Sp8 conditional knockout mice and accelerated regrowth in wildtype digits. Our work links developmental signaling to adult regeneration and establishes a modular, injury site specific gene therapy framework that enables new interventions for limb healing.

genetics↗

bioRxiv2022

Osteoclast-mediated resorption primes the skeleton for successful integration during axolotl limb regeneration

Early events during axolotl limb regeneration include an immune response and the formation of a wound epithelium. These events are linked to a clearance of damaged tissue prior to blastema formation and regeneration of the missing structures. Here, we report the resorption of calcified skeletal tissue as an active, cell-driven and highly regulated event. This process, carried out by osteoclasts, is essential for a successful integration of the newly formed skeleton. Indeed, the extent of resorption is directly correlated with the integration efficiency. Moreover, we identified the wound epithelium as a major regulator of skeletal resorption, likely creating a zone of influence in which signals involved in recruitment/differentiation of osteoclasts are released. Finally, we reported a correlation between resorption and blastema formation, particularly, a coordination of resorption with cartilage condensation. In sum, our results identify resorption as a major event upon amputation, playing a critical role in the overall process of skeletal regeneration.

developmental biology↗

bioRxiv2019

The Prrx1 limb enhancer marks an adult population of injury-responsive, multipotent dermal fibroblasts

The heterogeneity of adult tissues has been posited to contribute toward the loss of regenerative potential in mammals. Here we characterize an adult population of dermal fibroblasts that maintain expression of a Prrx1 enhancer which originally marked mesenchymal limb progenitors. Prrx1 enhancer-positive cells (Prrx1enh+) make up a small subset of adult dermal cells (~0.2%) and reside mainly within specific dermal perivascular and hair follicle niches. Upon injury, however, Prrx1enh+ cells readily migrate into the wound bed and amplify on average 16-fold beyond their uninjured numbers. Additionally, Prrx1enh+ cells emigrate out of their dermal niches following wounding and contribute to subcutaneous tissue. Prrx1enh+ cells are uniquely injury-responsive and do not contribute to tissue homeostasis or enriched by neonatal-like Wnt signaling. Prrx1enh+ cells represent a potent regenerative cell population that, despite being a meager minority in adult skin, demonstrate the potential to tip the balance of mammalian wound healing toward scar-free healing.

developmental biology↗