Limb Mesoderm and Head Ectomesenchyme Both Express a Core Transcriptional Program During Chondrocyte Differentiation

Abstract

To explain how cartilage appeared in different parts of the vertebrate body at discrete times during evolution, we hypothesize that different embryonic populations co-opted expression of a core gene regulatory network (GRN) driving chondrocyte differentiation. To test this hypothesis, laser-capture microdissection coupled with RNA-seq was used to reveal chondrocyte transcriptomes in the developing chick humerus and ceratobranchial, which are mesoderm- and neural crest-derived, respectively. During endochondral ossification, two general types of chondrocytes differentiate. Immature chondrocytes (IMM) represent the early stages of cartilage differentiation, while mature chondrocytes (MAT) undergo additional stages of differentiation, including hypertrophy and stimulating matrix mineralization and degradation. Venn diagram analyses generally revealed a high degree of conservation between chondrocyte transcriptomes of the limb and head, including SOX9, COL2A1, and ACAN expression. Typical maturation genes, such as COL10A1, IBSP, and SPP1, were upregulated in MAT compared to IMM in both limb and head chondrocytes. Gene co-expression network (GCN) analyses of limb and head chondrocyte transcriptomes estimated the core GRN governing cartilage differentiation. Two discrete portions of the GCN contained genes that were differentially expressed in limb or head chondrocytes, but these genes were enriched for biological processes related to limb/forelimb morphogenesis or neural crest-dependent processes, respectively, perhaps simply reflecting the embryonic origin of the cells. A core GRN driving cartilage differentiation in limb and head was revealed that included typical chondrocyte differentiation and maturation markers, as well as putative novel “chondrocyte” genes. Conservation of a core transcriptional program during chondrocyte differentiation in both the limb and head suggest that the same core GRN was co-opted when cartilage appeared in different regions of the skeleton during vertebrate evolution.

Proteoglycan inhibition of canonical BMP-dependent cartilage maturation delays endochondral ossification

“…To evaluate whether BMP signalling was active in developing cartilage of zebrafish, laser capture microdissection of cranial cartilage was performed, avoiding perichondral cells, followed by RNA sequencing ( Gomez-Picos et al, 2022 ; Nguyen et al, 2023 ). Several members of the BMP signalling pathway were expressed in cranial cartilage of 6 days post-fertilization (dpf) wild-type zebrafish larvae ( Table S1 ).…”

Distinct Gene Regulatory Dynamics Drive Skeletogenic Cell Fate Convergence During Vertebrate Embryogenesis

“…Across our three anatomical sampling sites, we focused on ‘broad’ clusters that likely contained cells transitioning from a mesenchymal precursor state towards a skeletogenic fate (Fig.1D-F, color coded), and re-clustered them at finer resolutions. Within these ‘fine’ clusters, we again used expression profiles of known marker genes, as well as two previously identified early chondrogenic gene co-expression modules, ‘IMM’ 20 and ‘RED’ 21 , to assess their respective skeletogenic differentiation. In each embryonic lineage, we identified a ‘fine’ cluster enriched for an early skeletogenic, i.e.…”

“…Indeed, skeletogenic cells of the three lineages investigated here all start out as embryonic epithelia and undergo an epithelial-to-mesenchymal transition (EMT), before migrating to their respective anatomical locations in the periphery 37-39 . There, they form mesenchymal condensations and appear to initiate a core skeletogenic program which – at a global scale – shows similarity in its overall gene regulatory state 20,40,41 (Fig.1H). We also noted a shared increase in ribosomal protein transcription, potentially to meet the translational needs for increased extracellular matrix protein production and secretion 42 (Fig.S4A).…”

“…A second round of clustering was conducted on select mesenchyme populations, with resolutions of 0.4 (somite, limb) and 0.5 (nasal) (=‘fine clustering’). Cell type assignments of clusters were based on visual inspection of known marker gene expression patterns, and the activity of the two previously identified early chondrogenic gene expression modules ‘IMM’ 20 and ‘RED’ 21 using the Seurat function ‘AddModuleScore’.…”

“…Indeed, skeletogenic cells of the three lineages investigated here all start out as embryonic epithelia and undergo an epithelial-tomesenchymal transition (EMT), before migrating to their respective anatomical locations in the periphery [37][38][39] . There, they form mesenchymal condensations and appear to initiate a core skeletogenic program which -at a global scale -shows similarity in its overall gene regulatory state 20,40,41 (Fig. 1H).…”