Cartilage is a specialized tissue that provides mechanical support and contributes to joint function. The research of cartilage development and its disorders is crucial for understanding the etiology of various diseases, including osteoarthritis and chondrodysplasia [22]. Particularly, exploring the specific signaling molecules or regulatory factors for chondrogenic could provide important reference for the mesenchymal stem cells (MSC)-based therapy for cartilage regeneration [23]. For the past few years, single-cell transcriptomic analysis has emerged as a powerful tool for identifying key regulators and markers of cartilage development [8, 24]. Our investigation into the dynamic processes of cartilage development and differentiation during critical embryonic stages in mice has provided a deeper understanding of the morphological changes, genetic underpinnings, and the identification of pivotal genetic regulators. Through the integration of hematoxylin-eosin staining, scRNA sequencing, RT-qPCR, and immunohistochemistry, we have uncovered significant gene expression differences that drive the development of cartilage, reflecting both the intrinsic developmental program and its biomechanical adaptation needs. These findings provide an important basis for further exploring the regulatory networks of cartilage development, which will hopefully serve to promote the research of cartilage regeneration and the prevention of osteoarthritis or other diseases.
From E13.5 to E18.5, the condylar cartilage and tibial growth plate cartilage of embryonic mouse experienced rapid development, meanwhile, exhibited certain differences in the stratification, morphology and arrangement of chondrocytes[8]. The chondrocytes were derived from the mesenchymal cells. Interestingly, the differentiation of mesenchymal cells in tibial growth plate cartilage were later than that in condylar cartilage, at E13.5, the condylar cartilage of embryonic mouse was a cluster of mesenchymal cells, while the tibial growth plate cartilage of some embryos had not started to develop. This phenomenon suggested that the development of condylar cartilage may be regulated by the specific growth factors, previous research have indicated that the growth factor TGF-β and the transcription factor Sox9 both played key roles in the development, formation and repair of cartilage[25, 26], thus the differences in the expression of these regulatory factors during early development may induce earlier differentiation of mesenchymal cells in condylar cartilage. However, the earlier development of condylar cartilage may also be influenced by other regulatory factors that newly identified by our research, which will be discussed in detail below.
Mesenchymal stem cells originate from the mesoderm and ectoderm in the early stage of development, they are mature stem cells with self-replicating ability and multidirection differentiation potential [27]. Under specific induction conditions, mesenchymal stem cells could differentiate into adipose cells, cartilage cells, bone cells or other tissue cells [27]. Therefore, mesenchymal stem cell therapy is considered as a viable strategy for the treatment of cartilage injury[28, 29], however, further investigation highlights the critical importance of precise regulation during the maturation and hypertrophy of chondrocytes. Chondrocyte hypertrophy is a pivotal phase in cartilage development; however, in therapeutic contexts aiming for cartilage repair, it is crucial to induce chondrogenesis without proceeding to unwanted bone formation[30–32]. Our research provided a valuable reference for solving this problem, utilizing scRNA-seq to identify genes that are highly expressed specifically during the chondrogenesis stage but do not show significant changes in expression during the subsequent hypertrophy phase. These genes, being differentially expressed at crucial stages of cartilage formation, suggest that their regulation could potentially allow for the growth of cartilage tissue while absent further hypertrophy and endochondral bone formation. This involves the application of specific biological factors at defined times, targeting these genes to guide MSCs towards chondrocyte differentiation and maturation without crossing the threshold into hypertrophy. Such insights could lead to more effective strategies in regenerative medicine, particularly for cartilage repair, by harnessing the potential of MSCs for precise tissue engineering.
By removing the DEGs in chondrocyte clusters that also showed significantly increased expression during bone development, we further identified 5 genes Bgn, Ucma, Fmod, Msmp and 1500015O10Rik as the specific markers for cartilage development. Previous studies have confirmed that genes Bgn, Ucma, and Fmod could be involved in the regulation of cartilage development [16–18], however, no studies have deeply explored the roles of these genes in cartilage repair and cartilage regeneration, and the related functional mouse models were rarely studied. Interestingly, Bgn and Fmod, as members of the SLRPs family, play critical roles in the pathogenesis of OA[33, 34]. These proteins interact with collagens to modulate fibril formation and bind to various cell surface receptors, growth factors, and other ECM components, thereby influencing cellular functions crucial for maintaining joint integrity. The disruption of these interactions, as observed in SLRP-deficient mouse models, leads to altered collagen networks, changes in chondrocyte proliferation and differentiation, and modifications in ECM turnover, which contribute to the development and progression of OA. Additionally, SLRPs are implicated in mechanisms beyond the cartilage itself, such as influencing subchondral bone structure and muscle weakness, further complicating the OA pathogenic landscape. Therefore, Bgn and Fmod, through their extensive involvement in regulating ECM structure and cellular signaling within the joint, underscore the complexity of OA pathogenesis, highlighting the interplay between genetic, molecular, and biomechanical factors in the disease's onset and progression. This comparative analysis has allowed us to identify potential markers specific to cartilage development, opening new avenues for understanding the complexity of cartilage formation and the distinct molecular pathways that govern it. The validation of these candidate regulatory factors through RT-qPCR and immunohistochemistry has confirmed their roles in cartilage development, reinforcing their potential as markers for cartilage development and as targets for therapeutic intervention.
Otherwise, among these genes, we found several novel regulators, including Msmp and 1500015O10Rik, which were previously unknown to be involved in the cartilage development. Specifically, gene Msmp encodes a member of the beta-microseminoprotein family, the encoded protein may play a role in prostate cancer tumorigenesis [35]. The gene 1500015O10Rik is synonymous with gene Ecrg4, previous research have suggested that gene Ecrg4 is a marker of articular chondrocyte differentiation and cartilage destruction [36]. And these genes were enriched in the pathways of “protein digestion and absorption”, “ECM − receptor interaction” and “focal adhesion”, which mainly affected the development of extracellular matrix. As an important part of the articular cartilage, extracellular matrix could maintain the living environment of chondrocytes and exchange signals with the outside of the chondrocytes. The homeostasis of extracellular matrix maintains the normal function of chondrocytes, and the loss of extracellular matrix could lead to diseases such as osteoarthritis and deformation of joint structure [37]. Thus these genes whose expression increased significantly during cartilage development could also serve as the key regulators for extracellular matrix remodeling, and these newly identified regulatory factors are whorthy of further research.
Our study has identified key genes and pathways implicated in the developmental process of cartilage formation in embryonic mice, offering insights that could potentially guide future regenerative therapies. However, we acknowledge several limitations inherent to our experimental design and methodology that merit consideration. Firstly, our investigation employs a single species focus, relying exclusively on embryonic mouse models. While mice are widely used in biomedical research due to their genetic congruence with humans and their expedited developmental timelines, it is imperative to note that findings derived from mouse models may not invariably mirror human biological processes due to interspecies differences. This limitation underscores the necessity for cautious interpretation of our results within the broader context of human biology. Additionally, our analysis is constrained by a developmental stage limitation, focusing on cartilage development at two specific embryonic stages (E13.5 and E18.5). Although these stages are pivotal for understanding cartilage formation in mice, our study may overlook critical stages or transitions that could provide deeper insights into the regulatory mechanisms of differentiation. Future studies should consider a broader range of developmental stages to capture the full spectrum of cartilage development. Our research also confronts a limitation in the number of identified regulatory factors. Although we have successfully pinpointed five genes as specific markers for cartilage development, the intricate nature of cartilage formation suggests the involvement of a more extensive array of genes and regulatory factors. The genes identified herein serve as an important foundation, yet they represent only a segment of the vast regulatory network governing cartilage development. Concerning experimental validation, we employed hematoxylin-eosin staining, immunohistochemistry, and RT-qPCR. While these techniques are instrumental for our analysis, they come with limitations regarding quantification accuracy, sensitivity, and the potential omission of less abundant or transiently expressed regulatory factors. Exploring additional validation methods could enhance the robustness of our findings. In terms of functional analysis, our study incorporates functional enrichment analysis to elucidate the roles of differentially expressed genes. However, we did not conduct direct functional assays to validate the specific contributions of these genes to cartilage development and regeneration. Such empirical validation is crucial for confirming the biological significance of the identified genes and should be addressed in future work. Our investigation also highlights a gap in elucidating regulatory mechanisms, identifying key genes and pathways without delving into the precise regulatory dynamics. A comprehensive understanding of how these genes interact within the cellular environment to guide cartilage development is essential and warrants further study. Lastly, regarding the translation to therapeutics, our findings lay a foundational basis for informing future regenerative therapies. Nevertheless, translating this knowledge into viable treatments for human cartilage-related ailments demands extensive follow-up research, particularly focusing on safety, dosage, delivery mechanisms, and long-term efficacy.