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

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.