Joints in the appendicular skeleton: Developmental mechanisms and evolutionary influences

Abstract

The joints are a diverse group of skeletal structures, and their genesis, morphogenesis, and acquisition of specialized tissues have intrigued biologists for decades. Here we review past and recent studies on important aspects of joint development, including the roles of the interzone and morphogenesis of articular cartilage. Studies have documented the requirement of interzone cells in limb joint initiation and formation of most, if not all, joint tissues. We highlight these studies and also report more detailed interzone dissection experiments in chick embryos. Articular cartilage has always received special attention owing to its complex architecture and phenotype and its importance in long-term joint function. We pay particular attention to mechanisms by which neonatal articular cartilage grows and thickens over time and eventually acquires its multi-zone structure and becomes mechanically fit in adults. These and other studies are placed in the context of evolutionary biology, specifically regarding the dramatic changes in limb joint organization during transition from aquatic to land life. We describe previous studies, and include new data, on the knee joints of aquatic axolotls that unlike those in higher vertebrates, are not cavitated, are filled with rigid fibrous tissues and resemble amphiarthroses. We show that when axolotls metamorph to life on land, their intra-knee fibrous tissue becomes sparse and seemingly more flexible and the articular cartilage becomes distinct and acquires a tidemark. In sum, there have been considerable advances toward a better understanding of limb joint development, biological responsiveness, and evolutionary influences, though much remains unclear. Future progress in these fields should also lead to creation of new developmental biology-based tools to repair and regenerate joint tissues in acute and chronic conditions.

Keywords: Articular cartilage; Joint evolution; Limb synovial joints; Morphogenesis.

Fig. 1

The interzone is essential for limb joint formation. Stage 25–26 chick embryos in ovo were subjected to microsurgical intervention to alter or remove the elbow joint interzone on the right side. The left elbow was left untouched and served as internal control. Embryos were re-incubated until day 10 (E10) at which point the elbows were dissected out and processed for histological analysis and Safranin O-fast green staining. (A) Images of E10 control elbow showing the prominent Safranin O-positive humerus (h), radius (r), and ulna (u) epiphyses flanking the developing synovial tissues and cavity. Note also the well-developed capsule (arrowhead). (B) High magnification image of boxed area in (A) showing the compact fibrous tissue (yellow brackets) covering the cartilaginous ends of the skeletal elements and an intervening and slightly less dense tissue (pink bracket). (C and D) Images of E10 elbow in which the interzone had been mechanically damaged but not removed. Area boxed in (C) is shown at higher magnification in (D). Note the considerable delay in joint development depicted by substandard separation of the opposing elements and by defective definition of the epiphyses compared to controls. Capsule appears to be unaffected (arrowhead). (E and F) Images showing absence of elbow joint and fusion of the opposing cartilaginous epiphyses after resection of the interzone. Area boxed in (E) is shown at higher magnification in (F). Note the small and relatively uniform size of chondrocytes in both intervening tissue and flanking epiphyses. (G and H) Images showing that removal of interzone and neighboring tissue led to absence of a joint and physical separation of neighboring skeletal elements. Note also the hypertrophic phenotype of chondrocytes in the opposing and truncated elements suggesting that the epiphyses were undergoing ectopic endochondral ossification typical of the diaphysis at this stage. Scale bar in (A) for C, E, and G, 250 μm; scale bar in (B) for D, F, and H, 100 μm.

Fig. 2

Tissue structure and gene expression in developing tibia articular cartilage in chick and mouse. (A and B) Images show Safranin O/fast green-stained proximal tibia epiphysis in E17 chick embryo. Area boxed in (A) is shown at higher magnification in (B). Note the presence of a prominent, thick, and fast green-positive fibrous layer (fl) facing the synovial cavity and overlaying the Safranin O-positive articular cartilage (ac). (C and D) In situ hybridization images of serial sections showing strong expression of collagen I (C, Col I) and collagen II (D, Col II) in fibrous layer and cartilage, respectively. (E and H) Images show Safranin O/fast green-stained proximal tibia epiphysis in E17.5 mouse embryo. Area boxed in (E) is shown at higher magnification in (F). Note that incipient articular cartilage (ac, yellow bracket in F) is rather narrow at this stage and is identified based on Gdf5+ cell lineage tracing (Decker et al., 2017). Its cells strongly express collagen II (H, Col II) and exhibit some residual collagen I expression (G, Col I), possibly stemming from their former mesenchymal character. (I–L) Images show Safranin O/fast green-stained proximal tibia epiphysis in neonatal P7 mouse. Area boxed in (I) is shown at higher magnification in (J). Note that the developing articular cartilage has grown in thickness (ac, yellow bracket in J), still expresses collagen II strongly (L, Col II) but no longer expresses collagen I (K, Col I). Scale bar in (A) for E and I, 350 μm; scale bar in (B) for all remaining panels, 50 μm.

Fig. 3

Timeline of vertebrate evolution through the fin-to-limb transition. Solid and dashed lines represent evolutionary timelines and currently extant species. Pictured at the bottom are enlarged cartoons of the pectoral fins/limbs of the animals indicated in the timeline and depicting the evolutionary transition from ray fin to lobe fin to Tiktaalik fin to amphibian limb. Key aspects of skeletal transitions at each stage are highlighted within these cartoons.

Fig. 4

Epiphyseal cartilage changes with adaptation to terrestrial life in spontaneously metamorphosed axolotl salamanders. Hematoxylin and eosin-stained sections of aquatic and terrestrial axolotl knee joints are depicted: femur (fe), tibia (t), and fibula (fi). (A) In 2-year-old aquatic animals, the presumptive epiphyseal cartilage (yellow bracket) is thick and continuous with the underlying growth plate (green bracket). (B and C) In 10-year-old aquatic animals, the epiphyseal cartilage is reduced in thickness (yellow brackets) but maintains morphological continuity with growth plate cartilage (green bracket). (D) In a 2-year-old metamorphosed terrestrial sibling, epiphyseal cartilage (green bracket) is much reduced in thickness compared to aquatic animal shown in (A). (E and F) By 10 years of age, a clear tidemark is visible and creates a clear histological separation from the underlying ossified growth plate (F, arrowheads). Scale bar for all panels, 100μm.

Fig. 5

Intra-joint tissue modifications during aquatic to terrestrial transition in axolotl salamanders. Images from specimens shown in Fig. 4. (A and B) The intra-joint tissue in aquatic axolotls is densely packed with fibrous cells (A) and thickness is reduced with age (B, green bracket). (C and D) By contrast, the intra-joint tissue in terrestrial axolotls displays reduced cell density, increased matrix deposition (C), and maintains thickness with age (D, green bracket). All tissue sections are stained with hematoxylin and eosin. Scale bar for panels A and C, 50 μm; scale bar for panels B and D, 150 μm.