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Molecular and Cellular Analysis of Cartilage Regeneration
in the Lizard Anolis carolinensis
Jonathan Sankman1,2,3, Michael Ammar1,2,3, Glenn Markov1,2,3, Walter Eckalbar1,2, Kenro Kusumi1,2
1Barrett, The Honors College, and 2School of Life Sciences & 3SOLS Undergraduate Research Program,
Arizona State University, Tempe, AZ 85287
Abstract
Lizards have the ability to regrow a functional tail after it has been self-amputated. The process is unique in that it involves de novo
regeneration of a variety of different tissue types including, muscle, nerves, and hyaline cartilage. Humans are unable to repair
hyaline cartilage, and understanding the genetic control of this process in lizards would have a signi ficant impact. Soon after self-
amputation, a cluster of cells forms at the regenerating tail tip consisting of progenitor cells that may play a role in the organization
and structure of the regenerating tissues. In the green anole, Anolis carolinensis (Figure 1), this regeneration process is completed by
fifty days, and cartilaginous tissues begin to be defined by fourteen days. My analysis of the regenerating tail has shown a clear
progression of differentiation from the mesenchymal cells at the distal tip to the developing cartilaginous tube and interface with the
vertebrae at the proximal area. Using genome and transcriptome resources available for Anolis carolinensis, I have been examining
the expression of candidate genes in the regenerating tail by in situ hybridization, including the key developmental genes notch1 and
tbx6. Ongoing in situ hybridization analysis is underway for candidate genes that have been identified through a RNA-Seq next-
generation analysis of the regenerating tail.
Fig. 1. Female adult Anolis carolinensis from ASU colony (photo Karla Moeller)
Fig. 2. Cellular Analysis of Original and Regenerating Tails
A
BDC
Scale/Epidermis
Bone
Muscle
Introduction
There is no better example of de novo tissue regeneration among vertebrates than tail regeneration in lizards. Tail regeneration in
lizards is of particular interest because vital tissues that cannot be regenerated in humans are uniquely regenerated de novo in
lizards, specifically hyaline cartilage (Simpson 1970). Hyaline cartilage is an essential component of our joints. It creates a smooth
surface between bones ideal for handling movement and compression and its loss gives rise to joint dysfunction and pain. Cartilage
regeneration in mammals is difficult and is not repairable under current technologies (Steinert et al., 2008). One of the main
procedures to alleviate problems associated with hyaline cartilage loss is joint replacement surgery; however, this surgery can be
expensive, painful, and difficult to perform in areas with numerous joints such as the hands (Gunther, 2001). The loss of cartilage
from arthritis and other rheumatic conditions affects 21% of US adults (Helmick et al. 2008). Thus, new therapies that can restore
hyaline cartilage are of great interest. Lizards have shown regenerative capabilities to regrow hyaline cartilage and identifying the
genes involved is a necessary step in understanding this amazing biological process.
Since there are no comparable examples of hyaline cartilage repair in mammals, there is little research available to direct gene
expression studies. Instead, candidate genes must be selected from genetic analysis of cartilage formation and patterning in early
mammalian development. Chondrogenesis is the process of cartilage development and begins with the condensation of
undifferentiated mesenchymal cells into clusters cells that change shape, pattern, and can differentiate into cartilage cells in
response to locally secreted molecules, extracellular matrix proteins, and the expression of various specific transcriptions factors
(Motero & Hurle, 2007). Specific genes that are thought to be involved in this process include bone morphagenic proteins (BMPs)
that may play role in shaping of the condensations, type II collagen (col2a1), N-cadherin (ncad). N-cam (ncam1), and tenasci n C
(tnc), and SRY-box 9 (sox9), aggrecan (agc), nkx3.2, and fgf receptor 3 (fgfr3) (Zuscik et al., 2008). Molecular analysis through in
situ hybridization will be important for revealing the regulation of these chondrogenic genes during the cartilage repair process in the
adult Anole and relating this to early mammalian development.
Figure 2. A) Normal histology of the tail showing segmented skeletal muscle bone, and cartilage and B) two, C) four, D)
seven, E) fourteen ,and F)sixty seven day regenerating tails. Sections are fifteen microns thick and imaged at 100X.
Fig. 3. Molecular Analysis Through In Situ Hybridization
F
EMelanocyte
Chondrocytes
Ependymal Tube
Myocytes
Methods
Cellular Analysis
Frozen-sections were made of regenerating and original tails. Tails were fixed fresh and mounted in optimal cutting temperature
compound (OCT). Sectioning was performed using the CM1950UV Leica Cryostat at the Keck Bioimaging Laboratory at Arizona
State University and sections were mounted onto glass slides. Slides were used for hematoxylin and eosin staining as well and in
situ hybridization. Protocols for hemotoxylin and eosin staining have been developed and optimized for Anole frozen sections.
Cryosectioned tissues were washed in distilled water and stained with hematoxylin. Sections were then rinsed in ethanol and
stained with eosin. Sections were dehydrated in ethanol and cleared with xylene.
Molecular Analysis
Gene expression analysis was examined through in situ hybridization of frozen sections. Slides with optimal cutting temperature
(OCT) compound imbedded frozen sections were treated with proti nase K, washed, dehydrated in ethanol, and hybridized overnight
with dioxygenin labeled probe using slide chambers at 70°C. The following day, hybridizing solution was removed, followed by a
series of washes and blocking with 1% heat inactivated fetal bovine serum. Alkaline phosphate-conjugated antibody to dioxygenin
was added and the slides were incubated overnight at 4°C. A developing solution of NBT/BCIP is used to remove the phosphate
giving rise to a purple dye in areas of gene expression.
References
Gunther, K.P. (2001). Surgical Approaches for osteoarthritis. Be st Pract Res Clin Rheumatol. 15(4): 627-643.
Helmick C.G., Felson D.T., Lawrence R.C., Gabriel S., Hirsch R., Kwoh C.K., Liang M.H. , Kremers H.M., Mayes M.D., Merkel P.A., Pillemer S.R.,
Reveille J.D., Stone J.H. (2008). Estimates of the prevalence of arthritis and other rhe umatic conditions in the United States. Part I. Art hritis
Rheum. Jan;58(1):15-25.
Simpson S.B., (1970). Studies on regeneration of the lizard’s tail. American Zoologist. May;10(2): 157-165.
Steinert A., Nöth U., Tuan R. (2008). Concepts in gene therapy for cartilage repair Injury. 39(1): 97-113.
Zuscik M.J., Hilton M.J., Zhang X., Chen D., O’Keefe R.J. (2008). Regula tion of chondrogenesis and chondrocyte differentiation by stress. J
Clin Invest. Feb;118(2):429-438.
Discussion and Future Directions
Hematoxylin and eosin staining shows the progression of tail regeneration over time. The dense cluster of cells present at the
tail tip (Fig. 2, D) may consist of progenitor cells that play a role in the organizati on and structure of developing tissues. The tail
begins actively growing after seven days and muscle and cartilaginous tissues are well defined by fourteen days (Fig . 2, E).
Melanocytes are also regenerated and seen near the periphery of the tail . After fifty days (Fig. 2, F) the tail has fully
regenerated lacking vertebral bone, distinct muscle groups, and scales that are seen i n the normal tail (Fig. 2, A).
Cufflinks RNA-Seq analysis has revealed a low FPKM for notch1 in the regenerating tail which may be responsible for there
being only light stai ning. In situ hybridization will need to be performed and perfected on additi onal candidate genes that can
now be identified through a RNA-Seq next-generation analysis
Figure 3. In Situ hybridization on Anole frozen sections. Key developmental gene notch1 on A) anole embryo and B)
fourteen day regenerating tail. Sections are twenty microns thick and imaged at 40X.
Results
Cellular Analysis
Hemotoxylin and eosin staining of original and regenerating tail sections gives insight into the cellular events i nvolved in tail
regeneration (Figure 2). The original tail (A) has segmented vertebral bone and distinct muscle groups. After two days following
autotomy (B), no major changes were evident. After four days (C), a wound epithelium formed. After seven days (D), a dense
collection of cells is seen at the tail tip. A cartilaginous tube with an adjacent myocyte population can be seen after fourteen days
(E). After sixty seven days (F), skeletal muscle fibers and a functional cartilaginous tube have fully formed.
Molecular Analysis
In Situ hybridization of notch1 has been successful ly performed on Anole embryo and tail tissue (Figure 3). Dark staining was seen
in the neural tube of an anole embryo (A) and in stripes near the periphery in the regenerating tail.
Acknowledgements
This research was supported in part by funds from the Howard Hughes Medical
Institute through the Undergraduate Science Education Program and from the ASU
School of Life S ciences.
