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

The WNT5A-mediated Wnt/Planar Cell Polarity (Wnt/PCP) pathway plays a key role in vertebrate development, particularly in limb morphogenesis. Robinow Syndrome (RS) is a rare genetic disorder characterized primarily by craniofacial malformations and limb shortening that is linked to mutations in multiple Wnt/PCP genes. The pathogenic WNT5A point mutation, Cys83Ser (C83S), is one of the most-studied RS-associated variants to date. It has been described as a loss-of-function, hypomorphic, or dominant-negative variant based on overexpression studies in vitro and in vivo. However, a mammalian model that mimics the C83S condition in human RS patients has not yet been established, and methods to distinguish between Wnt/PCP loss-of-function and gain-of-function in vivo phenotypes remain limited. In this study, we present a novel image-based method, local misalignment score (LMS), for in situ visualization and quantification of cell alignment within long bones during late embryonic development, providing a reliable and specific in vivo readout of aberrant Wnt/PCP-associated phenotypes. Using this method to assess chondrocyte orientation across mouse limb regions, we found that the heterozygous germline Wnt5a-C83S point mutation in mice induces profound chondrocyte orientation defects. This phenotype is distinct from the disrupted chondrocyte orientation with spatially patterned severity observed in homozygous Wnt5a conditional knockout (Wnt5a-cKO) limbs, which represent a Wnt5a loss-of-function model, and from those in Wnt5a-LSL knock-in limbs, where ectopic Wnt5a expression disrupts the endogenous gradient rather than producing a true Wnt5a gain-of-function effect. We further performed a comprehensive in vitro analysis of Wnt5a-C83S in C3H10T1/2 cells using a luciferase-based KIF26B reporter system along with other established Wnt signaling readouts. The results show that the C83S mutation does not exert dominant-negative effects on Wnt/PCP signaling, consistent with our in vivo findings. In summary, our work provides new insights into the putative gain-of-function or neomorphic nature of the RS-related WNT5A mutation and its impact on WNT5A gradient-dependent limb development. We highlight the reliability of LMS as an in vivo morphological measure of chondrocyte orientation that reveals defects linked to aberrant Wnt/PCP activity. When combined with other spatially resolved readouts, LMS enables location-based evaluation of pathogenic mechanisms.