Axial Skeletal Formation in Regenerating Lizard Tail

Lizard tail regeneration, particularly the cartilage regeneration, is a compelling phenomenon to be studied as it has been widely known that the regenerative capacity of cartilage after injury/trauma is very limited (Tuan et al., 2013). Urodeles are capable to regenerate tissues similar to the originals (Lozito & Tuan, 2015) while the regenerated tail of lizard is considered as an imperfect replica due to the several anatomical differences (Fisher et al., 2012). One stand out structure of those imperfections is that the axial skeleton of regenerated tail is lack of bony structures, but instead, it is composed of cartilage (Alibardi & Meyer-Rochow, 1989). The caudal vertebrae of the original tail will be replaced by a continuous, unsegmented cartilage tube surrounding the growing ependymal canal. This cartilage tube only exists in the regenerated tail of lizard and is maintained throughout the life of the organism. An experiment conducted by Lozito & Tuan (2015) showed that only the proximal region of cartilage tube undergo ossification which disproves the previous belief that cartilage tube resist ossification. Although cartilage tube is only partially ossified, Alibardi (2010) mentioned that calcification also occurs in the inner and outer edges of the cartilage tube. It is still unclear where the new cartilaginous cells originate from but there are possibilities that these cells derive from the multiplication of cells from the original vertebrae or by metaplasia from other connective cells (Alibardi, 2015a). Preliminary studies conducted by Alibardi (2014) showed the presence of putative progenitor/stem cells in the tail stump. Understanding how lizards are able to produce and grow cartilage tube in regenerated tail might be helpful in engineering cartilage tissue for osteoarthritis treatment or even replicating this ability in human years to come (Lozito & Tuan, 2016).

Skeletal system in lizard is continuously growing which might be related to the existence of stem cells in the growing centers of various bones (Pritchard & Ruzicka, 1950). Stem cells located in the intervertebral cartilages, epiphyses and perichondrium/periosteum are needed for the growth of vertebrae and long bones, and also for the capability to recover after injury or trauma (Alibardi, 2015b). An experiment performed by Alibardi (2010) indicated that those cells are present in the intervertebral and intraosseus tissues of lizard caudal vertebrae. 

Cartilage is usually involved in bone development process, making the resistance of cartilage tube to ossify is even more fascinating. Endochondral ossification is one of the two currently known processes of bone formation, in which the bone is formed from cartilage, for example during vertebrate limb development and urodele regeneration (Karp et al., 2000; Kronenberg, 2006). This process is initiated by the condensation of mesenchymal cells, followed by cell differentiation into chondrocytes. These cells actively proliferate and begin to deposit a small amount of extracellular matrix rich in type-II collagen (Col2) and sulphated glycosaminoglycans (GAGs) (DeLise et al., 2000; Tsang et al., 2014; Lozito & Tuan, 2015). Various maturity states of chondrocytes yield the formation of growth plate where the most mature cells stop proliferating and undergo hypertrophy, indicated by morphological changes such as significant increase in cell volume and specific gene expressions (Sun & Beier, 2014). A specialized matrix consisting of collagen type X (Col10) is produced by hypertrophic chondrocytes which express alkaline phosphatase to begin calcification of matrix (Adams et al., 2007; Anderson et al., 2004; van der Eerden et al., 2003). Bone morphogenetic protein 6 (BMP6) and vascular endothelial growth factor (VEGF) are secreted by the hypertrophic chondrocytes, the latter promotes the growth of blood vessels from the surrounding tissues (Zelzer et al., 2001). Mesenchymal cells and pre-osteoblasts enter the cartilage template through the invading capillaries, replacing the apoptotic hypertrophic chondrocytes (Dirckx et al., 2013). Mesenchymal cells differentiate into osteoblasts and start to produce bone matrix, replacing the cartilage matrix cleared by cathepsin-K-positive osteoclasts. Endochondral ossification ceases when the growth plate closes and the cartilage has been replaced by bone (Lozito & Tuan, 2015). Hutchins et al. (2014) ran RNA-Seq analysis of Anolis carolinensis regenerated tail (25 dpa), and the results showed that the proximal part of the regenerated tail expresses genes involved in lizard chondrogenesis. The chondrogenesis can be categorized into cartilage condensation, chondrocyte differentiation and cartilage development. Based on this analysis, the genes required for cartilage condensation are Acan, MGP, and Col2a1; chondrocyte differentiation: Col9a1, Acan, Col2a1, and Col11a2; cartilage development: BMP3, Col9a1, Lect1, Pax7, Acan, MGP, Col2a1, and Col11a2.