# Cloning and Expression of Col10a1 Gene and Its Response to Wnt/TGF-β Signaling Inhibitors in the Chinese Three-Keeled Pond Turtle (Mauremys reevesii)

## TL;DR

This study explores how the Col10a1 gene contributes to carapace development in Chinese three-keeled pond turtles and how it is regulated by Wnt and TGF-β signaling pathways.

## Contribution

The study provides novel insights into Col10a1's role in turtle carapace development and its regulation by Wnt/β-catenin and TGF-β/Smad pathways.

## Key findings

- Col10a1 expression increases during embryonic carapace ossification and collagen deposition.
- Wnt/β-catenin pathway inhibition downregulates Col10a1, while TGF-β/Smad inhibition upregulates it.
- Col10a1 shows widespread expression in adult tissues, with high levels in brain, kidneys, and liver.

## Abstract

The formation of Mauremys reevesii carapaces requires precisely regulated collagen deposition and ossification. The Col10a1 gene may play a key role in this process, but its specific function remains unclear. In this study, we aim to investigate the role of Col10a1 in M. reevesii carapace formation through molecular cloning, expression profiling, and functional characterization. We successfully cloned the Col10a1 gene and characterized its molecular properties using bioinformatics tools. Furthermore, we sought through spatiotemporal expression analysis at key embryonic developmental stages and in multiple adult tissues, combined with in vitro experiments using M. reevesii carapace-derived cells, to investigate the potential regulatory mechanisms of the Col10a1. Our findings provide important insights into the molecular mechanisms underlying M. reevesii carapace development and lay a foundation for further investigations into reptilian carapace formation mechanisms.

The formation of the Chinese three-keeled pond turtle (Mauremys reevesii) carapace is a complex biological event involving developmental processes such as collagen deposition and ossification. As a key regulator of collagen deposition and ossification, Col10a1 may play a crucial role in carapace development, though its specific mechanism remains unclear. To investigate the role of Col10a1 during carapace development and its regulatory mechanisms, we cloned its cDNA sequence and performed bioinformatic analysis. This revealed that Col10a1 encodes a stable, hydrophilic, and basic protein, with phylogenetic analysis showing closest evolutionary relationships to other reptiles and the greatest divergence from fish. Further RT-qPCR analysis examined Col10a1 expression patterns in M. reevesii embryos at stages 14, 18, and 22, as well as in various tissues of adult males and females. Results indicated that during embryonic development, Col10a1 expression levels progressively increased alongside the progression of carapace ossification and collagen deposition, suggesting its involvement in regulating this process. In adult tissues, Col10a1 exhibited widespread expression, with particularly high levels in the brain, kidneys, and liver, suggesting potential specialized functions in these organs. Finally, in vitro experiments demonstrated that inhibition of the Wnt/β-catenin pathway with salinomycin sodium salt downregulated the expression of both its target genes (Sp5, Myc, Ccnd1) and Col10a1. In contrast, inhibition of the TGF-β/Smad pathway with oxymatrine suppressed its target genes (Serpine1, Cdkn1a) but concomitantly upregulated Col10a1. These results suggest that Col10a1 expression may be positively regulated by the Wnt/β-catenin pathway and negatively regulated by the TGF-β/Smad pathway. Our findings provide novel insights into the molecular mechanisms governing carapace development and collagen deposition in M. reevesii, laying a crucial foundation for further investigations into the regulatory networks involving Col10a1 during carapace formation.