Work

Local Misalignment Scoring Reveals Spatially Uniform Chondrocyte Disorganization in a Wnt5a-C83S Knock-in Model of Robinow Syndrome

Year: 2025

Type: preprint

Abstract: WNT5A-driven Wnt/Planar Cell Polarity (Wnt/PCP) signaling is essential for vertebrate limb morphogenesis, and pathogenic WNT5A variants cause autosomal dominant Robinow Syndrome (RS). The recurrent Cys83Ser (C83S) substitution is among the best-studied RS-associated alleles and has been variably described as hypomorphic, loss-of-function, or dominant-negative based largely on overexpression in non-mammalian systems. However, a physiologically relevant mammalian model and robust in vivo readouts that distinguish loss- from gain-of-function Wnt/PCP perturbations have been lacking. Here, we introduce a quantitative image-based metric, the local misalignment score (LMS), which visualizes and measures cell orientation in situ across long bones during late embryonic development. LMS captures local coherence of chondrocyte alignment independently of embryo age, sectioning depth, or canonical Wnt activity, and selectively recognizes Wnt/PCP defects in Wnt5a and Wntless conditional knockouts, but not in Lrp5/6-deficient limbs. Using LMS, we show that a CRISPR/Cas9-engineered Wnt5a-C83S mouse exhibits profound and uniform chondrocyte orientation defects and limb shortening that are mechanistically distinct from the spatially patterned phenotypes in Wnt5a loss-of-functon(Wnt5a-cKO) and ectopic-expression (Wnt5a-LSL) models, and occur without SOX9 downregulation or severe distal digit defects. Complementary in vitro analyses using a KIF26B-NanoLuc Wnt/PCP reporter and biochemical readouts demonstrate that WNT5A-C83S has markedly reduced signaling activity but does not inhibit wild-type WNT5A in autocrine, paracrine, or juxtracrine contexts, arguing against a dominant-negative mechanism. Together, these data support a model in which WNT5A-C83S behaves as a hypomorphic, non–dominant-negative variant that perturbs Wnt/PCP gradient-dependent limb development through altered, rather than simply reduced, signaling output. More broadly, LMS provides a scalable morphological readout for spatially resolved interrogation of Wnt/PCP-associated defects in complex tissues.

Source: Research Square

DOI: 10.21203/rs.3.rs-8283811/v1