The switch in question involves R-spondins, a family of four secreted glycoproteins that act as potent amplifiers of canonical WNT signaling. RSPOs are best known for their role in stem cell biology, where they bind LGR4/5/6 receptors and the E3 ubiquitin ligases ZNRF3 and RNF43, thereby stabilizing WNT receptors at the plasma membrane and preventing their internalization. What has remained murky is whether RSPO-mediated signaling plays any role in the muscle stromal compartment. Using single-cell RNA sequencing of whole hindlimb muscle from C57BL/6J mice, the researchers found that Rspo gene expression was concentrated almost exclusively in the FAP population, marked by Pdgfra, Thy1 and Ly6a, with Rspo3 showing the highest expression among all four family members. Droplet digital PCR on freshly isolated FAPs confirmed that Rspo3 was the most abundant, with Rspo2 running a close second, while muscle stem cells expressed both genes only weakly.

That expression pattern raised an obvious question: if FAPs are making their own R-spondins, what are those proteins doing to FAP fate? The answer, it turns out, is that they are restraining the cells from becoming fat. When the researchers cultured FAPs in adipogenic induction medium supplemented with recombinant RSPO2 or RSPO3 alone, lipid accumulation, measured by Oil Red O staining at days 7 and 10 of differentiation, was barely affected. But when either RSPO was combined with WNT3A, the canonical WNT ligand, the anti-adipogenic effect was dramatically enhanced. Western blotting revealed that the combination treatment strongly suppressed PPARγ and C/EBPα, the two master transcription factors that drive adipogenesis, as well as FABP4, a mature adipocyte marker, during the early phase of differentiation.

To test whether endogenous R-spondins perform a similar function, the team used pools of four siRNAs each to knock down Rspo2, Rspo3, or both genes simultaneously in cultured FAPs. Individual knockdowns produced only modest increases in lipid accumulation, but the dual knockdown, which achieved over 60 percent reduction of both transcripts, markedly enhanced adipogenic differentiation, with elevated expression of PPARγ, C/EBPα and FABP4 detectable as early as day 3. The redundancy between the two ligands, the authors note, is consistent with overlapping activities described among RSPO family members in other tissues, and suggests that basal RSPO signaling helps maintain FAPs in a non-adipogenic state under normal conditions, likely through an autocrine mechanism in which the progenitors regulate their own fate.

Mechanistically, the researchers traced the anti-adipogenic effect to β-catenin, the central transcriptional co-activator of canonical WNT signaling. When they knocked down Ctnnb1, the gene encoding β-catenin, using siRNA, the inhibitory effect of RSPO3 and WNT3A on lipid accumulation and adipogenic marker expression was partially reversed. Conversely, when they used FAPs derived from mice carrying a β-catenin gain-of-function allele, in which Cre-mediated excision of exon 3 removes the phosphorylation sites that normally target β-catenin for degradation, adipogenic differentiation was strongly suppressed. Activation was confirmed by roughly 60-fold and 30-fold increases in Axin2, a known downstream WNT target, at days 3 and 10 of differentiation respectively. Together, these experiments establish canonical WNT/β-catenin signaling as the central mediator of RSPO function in FAPs.

The in vivo validation came from a glycerol-induced muscle injury model, in which 50 percent glycerol injected into the tibialis anterior muscle promotes ectopic adipogenesis. When the researchers delivered recombinant RSPO3 and WNT3A proteins directly into the injured muscle over two consecutive days, FABP4 immunofluorescence intensity was significantly reduced compared to vehicle controls, with the highest-intensity FABP4-positive cells, the top 10 percent, particularly depleted. PLIN1 immunofluorescence showed a similar trend toward lower adipocyte density, and RT-qPCR confirmed decreased expression of adipogenic genes in the treated muscle. A retrospective power analysis based on mouse-level corrected total cell fluorescence values calculated a statistical power of approximately 99.97 percent with four mice per group, underscoring the robustness of the effect.

Transcriptomic profiling of the RSPO3/WNT3A-treated muscles provided a systems-level view of what the treatment was doing. RNA sequencing identified 1,122 upregulated and 1,270 downregulated genes at a fold change threshold of 1.5 and an FDR cutoff of 0.05. Pathway enrichment analysis revealed that downregulated genes were strongly enriched in adipogenesis, PPAR signaling, and oxidative phosphorylation, consistent with the metabolic reprogramming that accompanies adipocyte differentiation. Transcription factor enrichment using the TRRUST database identified SREBF1, PPARG and CEBPA as central nodes, and ten downstream adipogenic targets, including Adipoq, Retn, Fabp4 and Slc2a4, were coordinately suppressed. Interestingly, MyoD and MEF2C, two major myogenic regulators, were also identified among the affected transcription factors, suggesting that RSPO3/WNT3A may influence myogenesis during muscle regeneration as well.

On the upregulated side, the picture was more complicated. Genes enriched in focal adhesion, cell adhesion, and PI3K-Akt-mTOR signaling pathways were elevated, with several extracellular matrix genes, including Col1a1, Col1a2, Col5a1 and Fn1, showing increased expression, potentially regulated by SP1, STAT3 and SMAD3. Preliminary results showed that both RSPO2 and RSPO3, in combination with WNT3A, increased COL1A1 and CTGF protein expression in cultured FAPs, raising the possibility that suppression of adipogenesis is accompanied by a reciprocal shift toward ECM production and fibrogenic processes. Notably, despite the expected activation of canonical WNT signaling, pathway enrichment analyses did not prominently identify WNT-related terms, which the authors attribute to the transient nature of WNT target gene activation by negative feedback and to the timing of muscle harvest after RSPO/WNT administration.

The in vivo loss-of-function experiments, however, left the endogenous role of R-spondins somewhat inconclusive. The researchers used conditional Rspo3 knockout mice, achieving over 90 percent ablation efficiency, combined with electroporation of Rspo2 siRNA into the injured tibialis anterior muscle, which achieved roughly 30 to 50 percent knockdown. Across four groups, control, Rspo2 knockdown, Rspo3 conditional knockout, and combined knockdown/knockout, no significant differences in adipogenesis were observed by FABP4 corrected total cell fluorescence or adipogenic marker gene expression. The authors suggest that the suboptimal Rspo2 knockdown, compensatory mechanisms from other RSPO family members, or the complexity of the in vivo niche, where multiple signaling pathways converge to regulate FAP fate, may explain the discrepancy with the in vitro results. A genetic Rspo2/Rspo3 double knockout model, they conclude, will be necessary to fully resolve the contribution of endogenous RSPO signaling in vivo.

Nevertheless, the study identifies RSPO2 and RSPO3 as previously unrecognized suppressors of FAP adipogenesis acting through canonical WNT/β-catenin signaling, and suggests a potential therapeutic strategy to modulate FAP behavior, preserving their native functions in muscle homeostasis and regeneration while limiting pathological adipogenesis. The implications extend to conditions characterized by pathological intramuscular fat accumulation, including Duchenne muscular dystrophy, aging-related sarcopenia, and metabolic disorders, where FAP-derived fat infiltration degrades muscle function. The authors caution, however, that further investigation into the balance between adipogenic and fibrogenic outcomes will be essential before such findings can be translated into clinical applications, given the preliminary evidence that RSPO/WNT signaling may also promote a fibrogenic phenotype in FAPs. Whether delivering RSPO proteins, or small molecules that mimic their WNT-potentiating activity, can safely tip the fate of muscle-resident progenitors away from fat and toward repair remains an open question, but the molecular switch they have uncovered now offers a concrete target for intervention.

Subject of Research: R-spondins suppress adipogenic differentiation in fibro-adipogenic progenitors via autocrine WNT/β-catenin signaling

Article Title: R-spondins suppress adipogenic differentiation in fibro-adipogenic progenitors via autocrine WNT/β-catenin signaling

Article References: Fransisca, J., Oh, Y., Sarmito, B., Alymkulova, N., Hwang, Y., & Yoon, J. K. (2026). R-spondins suppress adipogenic differentiation in fibro-adipogenic progenitors via autocrine WNT/β-catenin signaling. Frontiers in Cell and Developmental Biology, 14, Article 1942626. https://doi.org/10.3389/fcell.2026.1942626

Image Credits: AI Generated

DOI: 10.3389/fcell.2026.1942626

Keywords: R-spondins, suppress, adipogenic, differentiation, fibro-adipogenic, progenitors, autocrine, catenin, signaling, scientific research