Cellular and molecular mechanisms of synovial joint and articular cartilage formation

Abstract

Synovial joints and articular cartilage play crucial roles in the skeletal function, but relatively little is actually known about their embryonic development. Here we first focused on the interzone, a thin mesenchymal cell layer forming at future joint sites that is widely thought to be critical for joint and articular cartilage development. To determine interzone cell origin and fate, we microinjected the vital fluorescent dye DiI at several peri-joint sites in chick limbs and monitored the behavior and fate of labeled cells over time. Peri-joint mesenchymal cells located immediately adjacent to incipient joints migrated, became part of the interzone, and were eventually found in epiphyseal articular layer and joint capsule. Interzone cells isolated and reared in vitro expressed typical phenotypic markers, including GDF-5, Wnt-14, and CD-44, and differentiated into chondrocytes over time. To determine the molecular mechanisms of articular chondrocyte formation, we carried out additional studies on the ets transcription factor family member ERG and its alternatively spliced variant C-1-1 that we previously found to be expressed in developing avian articular chondrocytes. We cloned the human counterpart of avian C-1-1 (ERGp55Delta81) and conditionally expressed it in transgenic mice under cartilage-specific Col2 gene promotor-enhancer control. The entire transgenic mouse limb chondrocyte population exhibited an immature articular-like phenotype and a virtual lack of growth plate formation and chondrocyte maturation compared to wild-type littermate. Together, our studies reveal that peri-joint mesenchymal cells take part in interzone and articular layer formation, interzone cells can differentiate into chondrocytes, and acquisition of a permanent articular chondrocyte phenotype is aided and perhaps dictated by ets transcription factor ERG.

FIGURE 1

Schematic representation of the major steps of synovial joint formation. Mesenchymal pre-chondrogenic condensations appearing in the early limb bud exhibit no over signs of joint formation and are thus uninterrupted (A). Unknown up-stream mechanisms possibly involving Hox genes determine the exact location for joint initiation, and the first overt morphological sign of joint formation is a further gathering and condensation of cells (B). Soon after, the interzone becomes readily recognizable as a thicker and quite compact structure oriented perpendicularly to long axis of the long bone anlagen and composed of mesenchymal cells connected by gap junctions (C). The interzone and its adjacent epiphyseal cartilaginous tissue initiate a morphogenetic process that will eventually mold the joint into its required and distinct three-dimensional configuration (D). Eventually, all the distinct components of a mature joint form and include articular cartilage and capsule (E).

FIGURE 2

Fluorescent images of chick embryo digit joints. DiI was microinjected in the immediate vicinity of incipient metatarsal-phalangeal joint in Day 6.5 chick embryos in ovo (A, arrow) or at a distance of about 0.3 mm (B, arrow). Eggs were re-incubated and examined 48 h later. Limbs were sectioned and examined under fluorescence microscopy. Note in (A) that labeled peri-joint cells appear to have migrated into the developing joint from the point of injection (arrow) to locations along the joint (arrowheads). Note in (B) that labeled cells have migrated from the injection point (arrow) as well, but not into the joint.

FIGURE 3

Microscopic and RT-PCR analyses of interzone cells in culture. Interzones were microsurgically isolated from Day 6.5 chick embryo digits, dissociated into single cell suspensions, and grown in monolayer. (A), phase microscopic image of day 2 cells exhibiting a fibroblastic and flat morphology; (B), phase microscopic image of day 7 cells exhibiting a round and refractile morphology; (C) RT-PCR analysis of genes expressed by freshly-isolated interzone cells, namely Wnt-14 (lane 1), GDF-5 (lane 2), Gli-3 (lane 3), CD-44 (lane 4) and collagen IIA (lane 5); and (D) RT-PCR analysis showing that the day 7 cultures express high levels of collagen IX and aggrecan genes (lanes 2 and 4) compared to lower levels in day 2 cultures (lanes 1 and 3).

FIGURE 4

Schematic representation of the major human ERG variants. Indicated are the major domains, including the N-terminal region, the alternatively-spliced 81 and 72 bp exons in the middle region, the est DNA-binding domain (ETS), and the C-terminal region.

FIGURE 5

Anatomical, histological and in situ hybridization analyses of developing skeleton. Day E18.5 wild-type (WT) mouse embryos and transgenic embryos expressing human ERGp55Δ81 in cartilage (TG) were processed for skeletal anatomical analysis (A and B), histology (C and D) and in situ hybridization analysis of collagen X gene expression (E and F). Note in WT that the skeleton and long bone growth plate have normal appearances (A and C) and normal strong expression of maturation marker collagen X (E). In contrast, the transgenic skeleton is much smaller (B), the growth plate is completely disorganized and composed of uniform chondrocytes displaying an immature small-sized appearance (D), and there is poor expression of collagen X (F).