Spatially-resolved Fzd1- and Fzd2-governed fibroblast signaling axes differentially gate regenerative versus fibrotic dermal repair

Central Hypothesis Two genetically and spatially segregated dermal fibroblast lineages—defined by stable Fzd1 or Fzd2 receptor expression—establish parallel Wnt-dependent signaling circuits that (i) compete for control of the wound bed, (ii) differentially activate β-catenin–ECM versus non-canonical JNK/STAT3 programs, and (iii) can be independently re-tuned to convert fibrotic healing into scar-free regeneration. Specific Aims & Experimental Strategy 1. Lineage authentication and spatial mapping. • Mouse strains: Fzd1CreERT2;R26Confetti, Fzd2CreERT2;R26Confetti, and double heterozygotes. 4-OHT (75 mg/kg) administered P56 ×2 prior to wounding to capture fibroblasts in homeostasis. n ≥ 6 mice/sex/genotype. • Single-cell RNA-seq (10x Genomics, 8,000 cells/mouse) at D0, D3, D7, D14 post-injury (6-mm full-thickness punch and 1-cm linear incision). Stable, non-overlapping clusters predicted by lineage-specific barcodes and transcriptomic inertia. • Light-sheet 3-D imaging of cleared skin (SHANEL) with machine-learning heat-mapping (Imaris + Cellpose) to quantify Fzd1+ vs Fzd2+ cell densities in 50-µm³ voxels across wound depth and length. 2. Mechanistic coupling of receptor usage to downstream programs. • Conditional β-catenin knockout (Fzd1CreERT2;Ctnnb1fl/fl) and Dvl2/JNK inhibitor administration (SP600125, 30 mg/kg) in Fzd2CreERT2 mice. • Axin2-luciferase reporter activity, TOPFlash assays in FACS-isolated fibroblasts, phospho-JNK and pSTAT3 multiplex IF. • Proteomic secretome profiling (TMT-LC-MS/MS) to identify paracrine cues influencing endothelial and epidermal progenitors; CRISPRi knockdown of top candidates (e.g., CCN2, VEGFA) in co-culture spheroids. 3. Functional re-balancing of healing trajectories. • Biomaterial patch (electrospun PLGA) delivering α11β1-targeted lipid nanoparticles carrying Fzd1 siRNA; release half-life ≈ 48 h. Control: scrambled siRNA. • Endpoints (n ≥ 10 wounds/condition): tensile strength, collagen I:III ratio (SHG microscopy), myofibroblast longevity (αSMA + Ki67), vascular density (IB4), re-epithelialization rate (K14 staining), and biomechanical pliability (indentation elastography) at D21 and D56. • Combined Fzd1/Fzd2 blockade to test redundancy; expectation: impaired closure and nullification of regenerative benefit, revealing non-overlapping but complementary roles. 4. Translational validation. • Human: CRISPR-engineered FZD1- or FZD2-GFP knock-in neonatal dermal fibroblasts seeded into organotypic skin equivalents; micro-wounding followed by live imaging and RNA-seq. • Large-animal (porcine) 2-cm dorsal wounds treated with Fzd1 siRNA patch; histology and optical coherence tomography at 8 weeks. Predicted Impact & Risk-Weighted Innovation • Demonstrates that scar formation is not an inevitable default but a negotiable outcome dictated by a competitive Fzd1/Fzd2 fibroblast rheostat. • Introduces the concept of intra-wound “territorial” fibroblast patterning as a therapeutic entry point, moving the field beyond global anti-fibrotic strategies. • Provides a modular biomaterial-gene therapy system that could be rapidly adapted for human trials. • Risk: redundancy with other Frizzled receptors may blunt effects; mitigated by multiplex CRISPR screens and combinatorial inhibitor testing. Key Deliverables & Milestones (Year 1-2) • Fate-map atlas of Fzd1+ vs Fzd2+ fibroblasts across time and space. • Causal proof of β-catenin vs JNK/STAT3 branch specificity for fibrosis vs angiogenesis. • Pre-clinical efficacy data showing ≥50 % reduction in scar thickness without delayed closure in mice and ≥30 % in pigs. If validated, this work will redefine fibroblast heterogeneity in wound biology and open a precision-wound-engineering paradigm wherein specific fibroblast lineages are dialed up or down to achieve functional, aesthetically superior regeneration.