(G-267) Phagocytes and their Role in Lizard Tail Regeneration

Subtrack: Articular Cartilage, Meniscus and Joints

Osteoarthritis is a crippling disease that affects millions of people annually. The disease involves the degradation of articular cartilage in certain joints. Humans are unable to regenerate cartilage, and the current treatments for these cartilage pathologies are not sufficient for long-term management. Conversely, lizards are able to regenerate large amounts of cartilage when they regrow amputated tails. Studying how lizards regenerate their tails provides a unique opportunity to develop new treatments for osteoarthritis.

Lizards are the closest evolutionary relatives to humans that can regenerate a significant portion of their body—their tails—through blastema-based chondrogenesis. Regenerated lizard tails are boneless with tail skeletons consisting of single cartilage tubes surrounding ependyma-derived regenerated spinal cords [1]. To date, no in-depth analyses of the cells responsible for lizard tail regeneration exist, and the biological mechanisms responsible for lizard tail regeneration are not fully understood. We have conducted a myriad of experiments to better understand the cellular aspects of lizard tail regeneration. We identified a certain phagocyte of pericytic (non-hematopoietic) origin dubbed “septoclasts”. Unlike other phagocytes such as macrophages and osteoclasts, septoclasts originate from pericytes and mesenchymal stromal cells rather than monocytes. All phagocyte types play important roles in lizard tail regeneration, but septoclasts exhibit a more involved role in chondrogenesis as demonstrated by our data. This project aims to decipher the role that phagocytes, specifically septoclasts, play in lizard tail regeneration. Understanding these cells marks the first step toward developing lizard-cell-based therapies for orthopedic pathologies.

We conducted single cell RNA sequencing on tail samples of Anolis carolinensis (AC) to identify different cell populations in regenerating lizard tails. To validate the phagocyte clusters present in the sequencing data, we conducted fluorescence in situ hybridization (FISH) of regenerating lizard tails to produce a spatiotemporal map displaying the location of the phagocytes at different stages of tail regeneration. AC original tails were amputated at day 0, and regenerated tails were collected every 7 days post-amputation (DPA) up until 21 DPA. Amputated regenerated tail samples were then sectioned in a Leica CM1860 cryotome. Using FISH, the sections were stained with three phagocyte markers displayed in the sequencing data—CTSK, CTSB, and Col4a1—and Sulf1, a known chondrogenesis marker. Stained sections were imaged using a Keyence BZ-X800 fluorescent microscope. The different phagocyte populations were quantified through counting. This procedure was repeated for 7, 14, and 21 DPA. By combining each of the imaged stains and quantification data, we created a map showing the locative distribution of different phagocytes at various times in the regeneration process.

To study the necessity of phagocytes for regeneration, the FISH procedure above was repeated with lizards treated with clodronate liposomes, a known phagocyte depletion agent. ACs were injected with clodronate or control PBS liposomes 72 and 48 hours pre-amputation. Regenerated tails were collected 14 DPA using the FISH procedure described above.

The RNA sequencing data revealed three distinct phagocyte populations expressing different combinations of phagocyte markers (Figure 1). Macrophages are CTSB+, Col4a1-, and CTSK-; osteoclasts are CTSK+, Col4a1+, and CTSK-; septoclasts are CTSB-, Col4a1+, and CTSK+. The spatiotemporal map created in response to the sequencing data illuminated multiple features about phagocytes in regenerating lizard tails, especially septoclasts. Septoclasts are most numerous between 7-21 DPA (Figure 2d) and mostly cluster at the distal tip of the regenerating tail (Figure 2c, 2c’’). Comparing the expression of Sulf1 with the phagocytes’ locations demonstrated which cells induced fibroblastic chondrogenesis. We previously proved that Sulf1 is an important gene in upregulating chondrogenesis in fibroblasts [2]. The expression of Sulf1 in the distal tip (Figures 2b, 2c) and the presence of septoclasts in the same area (Figures 2b’’, 2c’’) suggest that they may help incite fibroblastic chondrogenesis. We hypothesize that specific biomolecules secreted by septoclasts recruit fibroblasts for chondrogenesis, resulting in the upregulation of Sulf1. Macrophages and osteoclasts remained near the amputation plane and lacked significant Sulf1 co-expression, suggesting that they have less chondrogenic potential than septoclasts in the regenerating blastema.

Additional experiments confirmed septoclasts’ role in fibroblastic chondrogenesis. We have previously shown that clodronate liposome treatment depletes phagocyte populations in regenerated tails [3]. Compared to control regenerated tails treated with PBS, the clodronate liposome treated tails failed to form a blastema (Figure 3). In the clodronate liposome treated lizards, no phagocytes were detected in the regenerating tail. In addition, there was no expression of Sulf1 in the clodronate liposome treated lizards. Overall, this data suggests that phagocytes, including septoclasts, are necessary for proper blastema formation and thus, proper tail regeneration.

Future experiments will further study these cells. Currently, we are using flow cytometry to isolate these cells and analyze them through RNA sequencing, ATAC sequencing, and bisulfite sequencing. These genetic analyses will reveal more nuanced differences between the phagocytes. With further study of these cells, we can work to induce similar cartilage regeneration in mouse models. If successful, our cartilage regeneration research can be utilized to treat orthopedic diseases like osteoarthritis, ameliorating patients’ experiences.

[1] Lozito, T. P. & Tuan, R. S. Lizard tail skeletal regeneration combines aspects of fracture healing and blastema-based regeneration. Development 143, 2946–2957 (2016).

[2] Vonk, A. C. et al. Lizard Blastema Organoid Model Recapitulates Regenerated Tail Chondrogenesis. Journal of Developmental Biology 10, 12 (2022).

[3] Londono, R. et al. Single cell sequencing analysis of lizard phagocytic cell populations and their role in tail regeneration. Journal of Immunology and Regenerative Medicine 8, 100029 (2020).