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.

Keywords: GRN co-option; GRN evolution; chondrocytes; head cartilage; limb cartilage.

FIGURE 1

Laser capture microdissection was used to isolate chondrocytes from the chick HH36 humerus and ceratobranchial. (A,B,J) Whole-mount Alcian blue and Alizarin red staining identified cartilage and perichondral bone in chondral bones of the chick forelimb (A,B) or hyoid (J). (C,K) Safranin O-stained section of HH36 humerus or ceratobranchial highlighted immature (IMM, red dotted outline) and mature cartilage (MAT, yellow dotted outline). (D,E,L,M) High-magnification images of IMM (D,L) and MAT (E,M) from black boxes in (C) or (K) (F–I,N–Q) Unstained sections of HH36 chick humerus or ceratobranchial before (F,N) and after (G,O) laser capture of IMM, and before (H,P) and after (I,Q) laser capture of MAT. (R,S) Trichrome-stained section of HH36 humerus or ceratobranchial showed Aniline blue staining of bone matrix in perichondral bone. Abbreviations: Bh = basihyal; c = carpal; Cb = ceratobranchial; Eb = epibranchial; h = humerus; IMM = immature chondrocytes; m = metacarpals; MAT = mature chondrocytes; p = phalanges; Pb = perichondral bone; r = radius; s = scapula; u = ulna; Uh = urohyal.

FIGURE 2

A core GRN drives chondrocyte differentiation in the limb and head. (A) Venn diagram demonstrated that limb and head chondrocytes shared almost 80% of the genes expressed above threshold (limb and head data combined before normalization and cutoff calculations). (B) Venn diagrams of IMM or MAT (separately normalized) showed that limb and head chondrocytes again shared expression of most genes. (C) Enriched biological processes in the overlap of panel A included cartilage differentiation processes, whereas limb morphogenesis and neural crest-related processes were enriched in the non-overlapping humerus and ceratobranchial portions, respectively. (D) A core cartilage gene regulatory network (GRN), compiled from the overlapping portion of panel A and published literature, included typical IMM and MAT markers highlighted in pink and yellow, respectively. Arrowheads represent positive interaction, whereas negative interactions are depicted as –|. Interactions included in this network could be direct (solid lines) or indirect (dashed lines). Novel putative cartilage genes with unknown regulatory interactions were included as single-nodes surrounded by dashed lines to the bottom right of the network, except for PTN and TNC whose regulatory interactions have been confirmed.

FIGURE 3

GCN of either limb or head chondrocyte data was organized into groups of enriched IMM or MAT genes that were negatively correlated with each other. (A,B) A GRN underlying chondrocyte differentiation was estimated from a gene co-expression network (GCN) from limb (A) or head (B) chondrocytes (separately normalized). In both limb and head GRNs, one portion contained genes upregulated in IMM (pink nodes), while another portion contained genes upregulated in MAT (yellow nodes). Typical chondrocyte differentiation genes included in the network (light blue nodes) were not differentially expressed between IMM and MAT. Representative lists of genes in each of these three categories are shown to the side of the GRNs. These candidate genes were selected because they are known to have a role during cartilage differentiation in different vertebrates, and they are listed in alphabetical order. Putative novel genes are indicated by an asterisk next to the gene name. (C,D) Isolated portions of IMM- and MAT-enriched genes from the limb (C) or head (D) GRNs. Most upregulated genes in IMM or MAT were connected by positive interactions (red lines), whereas IMM and MAT genes were mostly connected by negative interactions (blue lines).

FIGURE 4

GCN of combined limb and head chondrocyte data was organized into groups of enriched IMM or MAT genes that were negatively correlated with each other. (A) A GRN underlying chondrocyte differentiation was estimated from a GCN from limb and head chondrocytes (normalized together). One portion of the GRN contained genes upregulated in IMM (pink nodes), while another portion contained genes upregulated in MAT (yellow nodes). Typical chondrocyte differentiation genes included in the network (light blue nodes) were not differentially expressed between IMM and MAT. Representative lists of genes in each of these three categories are shown to the side of the GRNs. These candidate genes were selected because they are known to have a role during cartilage differentiation in different vertebrates, and they are listed in alphabetical order. Putative novel genes are indicated by an asterisk next to the gene name. (B) Isolated portions of IMM- and MAT-enriched genes in the GRN. Most upregulated genes in IMM or MAT were connected by positive interactions (red lines), whereas IMM and MAT genes were mostly connected by negative interactions (blue lines).

FIGURE 5

GCN of either IMM or MAT chondrocyte data was organized into groups of enriched limb or head genes that were negatively correlated with each other. (A,B) A GRN underlying IMM (A) or MAT (B) differentiation was estimated from a GCN from limb and head chondrocytes (IMM and MAT data separately normalized). In both IMM and MAT GRNs, one portion contained genes upregulated in the limb (green nodes), while another portion contained genes upregulated in the head (orange nodes). Typical chondrocyte differentiation genes included in the network (light blue nodes) were not differentially expressed between limb and head. Representative lists of genes in each of these three categories are shown to the side of the GRNs. (C,D) Isolated portions of limb- and head-enriched genes in the IMM (C) and MAT (D) GRNs. Most upregulated genes in limb or head were connected by positive interactions (red lines), whereas limb and head genes were mostly connected by negative interactions (blue lines).

FIGURE 6

Co-option of a core cartilage GRN by different embryonic populations during vertebrate evolution. The ancestral core cartilage GRN, shared in the common ancestor of vertebrates and their sister invertebrates, likely included such genes as SoxD, SoxE, and ColA expressed in cranial mesoderm or endoderm. In primitive agnathan vertebrates, cranial cartilage expanded when cranial neural crest cells co-opted a core cartilage GRN, likely building new regulatory connections between cranial neural crest genes and SoxE orthologs. Later, when paired appendages with cartilage evolved in the common ancestor of osteostracans and gnathostomes, a core cartilage GRN was additionally co-opted by lateral plate mesoderm, likely by establishing new regulatory connections between lateral plate mesoderm genes and SoxE orthologs.