Chapter 7

Organ and Appendage Regeneration

in the Axolotl

Johanna E. Farkas, Piril Erler, Polina D. Freitas, Alexandra E. Sweeney,

and James R. Monaghan

Abstract Regeneration is a remarkable feat of biology. It requires an organ sys-

tem – most often consisting of many different cell types – to stop its specialized
function and step back in ontological time. Regeneration overcomes the general
understanding that development is a one-way street. We now know that there is
great variability in regeneration capacity across phylogeny, likely because animals
need to have some mechanism in place to survive injuries or diseases that it will
encounter. Some animals, including humans, meet this need by closing the wound
as quickly as possible and making due with the deficit. Other animals, such as the
axolotl described here, instead regenerate the damaged or missing tissue.
Ambystoma mexicanum, commonly known as the axolotl, is uncommon among
vertebrates because of its superior regenerative abilities. References of their unique
abilities cross from the scientific to the main stream media, but they are also useful
animals for understanding the mechanisms that regulate regeneration. As axolotls
are tetrapods that can breed all year-round and accept grafts between adults, they
possess some major advantages that make them uniquely suited to be animal models
of regeneration. In terms of appendage regeneration, there is currently a lack of
animal models for complex regeneration. The two dominant models are zebrafish,
which regenerate caudal fins throughout life, and Xenopus frogs, which can regener-
ate limbs and tails early in development. Although these systems have provided
important insights into vertebrate regeneration, the axolotl limb is particularly well
suited to study adult appendage regeneration. Axolotls are capable of regenerating
complete adult limbs that are morphologically similar to human limbs using endo-
chondral ossification. In contrast, the zebrafish dermal caudal fin skeleton has no

J.E. Farkas • P. Erler • P.D. Freitas • J.R. Monaghan (*)

Department of Biology, Northeastern University,
360 Huntington Avenue, Boston, MA 02115-5000, USA
e-mail: [Link]@[Link]
A.E. Sweeney
Department of Biology, Northeastern University,
360 Huntington Avenue, Boston, MA 02115-5000, USA
School of Life Sciences, Queen’s Medical Centre, Nottingham University,
Nottingham NG7 2UH, UK

© Springer International Publishing Switzerland 2016 223

G. Steinhoff (ed.), Regenerative Medicine - from Protocol to Patient,
DOI 10.1007/978-3-319-27583-3_7
224 J.E. Farkas et al.

mammalian counterpart, and the fin skeleton regenerates by direct ossification from
mature osteocytes (Sousa et al. Development, 138(18):3897–3905, 2011; Knopf
et al. Dev Cell 20(5):713–724, 2011). Xenopus regenerates early in development,
but its ability to regenerate patterned skeletal structures is absent in adulthood.
Therefore, the axolotl is among the best vertebrate models for adult joint
regeneration.
Although axolotls have been studied for almost 200 years, only recently have
technological advances helped revive the axolotl into a model organism in modern
regeneration biology (Voss et al. Cold Spring Harbor Protoc 2009(8), 2009). The
axolotl is becoming a chosen model for regenerative biology because it can regener-
ate more completely than any other vertebrate in the majority of organs studied.
Modern genomic tools are available including microarray analysis (Monaghan et al.
J Neurochem 101(1):27–40, 2007; BMC Biol 7, 2009; Biol Open. 2012; Campbell
et al. Dev Dyn: An Off Publ Am Assoc Anat 240(7):1826–1840, 2011), RNAseq
(Monaghan et al. BMC Biol 7, 2009; Stewart et al. PLoS Comput Biol 9(3):e1002936,
2013; Knapp et al. PLoS One 8(5):e61352, 2013), a genomic map (Smith et al.
Genetics 171(3):1161–1171, 2005a), genomic sequence data (Smith et al. BMC
Genomics 10:19, 2009), and bioinformatic databases (Smith et al. BMC Genomics
6:181, 2005b). Functional testing of genes is also available through the generation
of transgenics (Sobkow et al. Dev Biol 290(2):386–397, 2006; Monaghan and
Maden, Dev Biol 368(1):63–75, 2012a; Khattak et al. Nat Protoc 9(3):529–540,
2014; Whited et al. Proc Natl Acad Sci U S A 109(34):13662–13667, 2012), knock-
down of genes by morpholinos (Schnapp et al. Development 132(14):3243–3253,
2005; Zhu et al. Dev Biol 370(1):42–51, 2012), over-expression of genes by electro-
poration (Mercader et al. Development 132(18):4131–4142, 2005) and viruses
(Whited et al. Development 140(5):1137–1146, 2013; Khattak et al. BMC Dev Biol
13:17, 2013), and cell tracking by tissue grafting between GFP and white axolotls
(Nacu et al. Cold Spring Harbor Protoc 2009(8), 2009). With this array of modern
tools, married with the qualities that have made the axolotl a subject of research for
hundreds of years, the axolotl system has become a powerful model to dissect the
mechanisms that regulate development and regeneration.
Here, we will highlight what is known about the axolotl’s regenerative abilities
and discuss the mechanisms that regulate regeneration of each organ system. It is
generally assumed that the axolotl has the ability to regenerate most if not all of its
tissues, but a survey of tissue regeneration has yet to be performed in this animal
model. We will focus upon the regenerative capacity of the axolotl, but it is neces-
sary to include examples of regeneration in the newt, Xenopus laevis, and zebrafish
because in some aspect these species have been studied in more detail than in the
axolotl model.

Keywords Axolotl • Ambystoma mexicanum • Cardiac regeneration • Nervous sys-

tem • Limb regeneration • Spinal cord • Wound healing • Immune system •
Extracellular matrix
7 Organ and Appendage Regeneration in the Axolotl 225

7.1 Axolotl Limb Regeneration

Though the axolotl possesses many extraordinary regenerative capabilities, its abil-
ity to fully regenerate amputated limbs is among its most striking and well-studied.
Urodele salamanders are the only vertebrates capable of regenerating limbs through-
out adulthood, and thus this process has been the subject of scientific study and
fascination for more than two centuries (Spallanzani 1769). Though many of the
molecular mechanisms underlying limb regeneration remain poorly-understood,
past studies have nevertheless elucidated the major steps and some underlying
mechanisms of the process.
Axolotl limb regeneration occurs in a series of stages that are morphologically
and transcriptionally distinct (Voss et al. 2015). Amputation of the limb induces
rapid vasoconstriction, which serves to minimize blood loss from the injury. Clotting
is very rapid, as is epidermal migration and closure of the wound site. So long as
excess bone is trimmed and kept from protruding from the wound site, wound clo-
sure will occur within 24 h post-amputation. Over the next several days, the wound
epithelium thickens and forms a structure called the apical epithelial cap (AEC),
which intimately contacts the mesenchyme in the absence of the dermis. This AEC
is a crucial component of limb regeneration- if it is prevented from forming or
replaced with fully-thickened epidermis, regeneration does not take place (Tassava
and Garling 1979; Loyd and Tassava 1980). It is believed that the AEC secretes a
host of factors, including metalloproteases, which are necessary for breaking down
the extracellular matrix (ECM) of the underlying tissue, thus permitting and guiding
cell migration and accumulation in the mesenchyme (Thornton 1960; Yang and
Bryant 1994; Yang et al. 1999; Lévesque et al. 2007). Once a critical mass of dedif-
ferentiated cells accumulates below the wound epithelium, the next stage of regen-
eration is initiated and a proliferative mass called the blastema is formed. Although
the overall rate of limb regeneration largely depends on the size of the animal, the
switch from wound healing and cell accumulation to blastema formation generally
occurs at around 10 days post-amputation (DPA). Once the blastema has formed,
cell cycling and proliferation increases dramatically (Loyd and Tassava 1980) as
blastemal cells divide rapidly and promote the outward growth of the regenerating
structure.

7.2 Characteristics of the Blastema

Though it appears to be a homogeneous population of near-identical cells, the blas-

tema is in fact comprised of local (within 1–2 mm of the wound site (Butler 1935;
Butler and O’Brien 1942)) dedifferentiated cells arising from several different tis-
sue types, including fibroblasts, Schwann cells, and satellite cells (Monaghan and
Maden 2012b). This last cell type represents a crucial distinction between axolotls,
which are members of the family Ambystomatidae, and newts, which are members
226 J.E. Farkas et al.

of the family Salamandridae. Though newts and axolotls bear superficial similari-
ties and are both studied for their regenerative capabilities, the families in fact
diverged 145 million years ago (Zhang and Wake 2009) and certain differences have
been noted in their regenerative mechanisms. Of note is the fact that while satellite
cells dedifferentiate and contribute to the blastema in the axolotl, in newts it appears
that myocytes themselves dedifferentiate (Sandoval-Guzman et al. 2014). Caution
is thus advised when applying findings from the newt to the axolotl, and vice versa.
However, one feature that remains constant across both examples of limb regenera-
tion is the fact that all blastemal cells are lineage-restricted: that is, once regenera-
tion is nearly complete, they re-differentiate back into their tissues of origin. Thus,
satellite cells eventually differentiate into myocytes while Schwann cells will only
become Schwann cells. The lone exception is that of fibroblasts, which can differ-
entiate into either fibroblasts or chondrocytes (Kragl et al. 2009). Dedifferentiated
fibroblasts make up a disproportionately large percentage of the regenerating blas-
tema (Muneoka et al. 1986), and their plasticity allows the limb to fully regenerate
even if all skeletal elements are removed prior to amputation (Thornton 1938; Foret
1970). Although the mechanisms behind this cellular “memory” remain unknown,
one can safely qualify the blastema as a collection of generally lineage-restricted,
dedifferentiated cells arising from multiple tissue types.

7.3 Nerve Dependence and Molecular Mechanisms of Limb

Regeneration

One curious characteristic of blastemal formation and growth is the fact that it is
nerve-dependent. If the limb is denervated prior to or shortly after amputation via
simple transection of the brachial nerves, the wound heals over without incident but
formation of the blastema does not occur and regeneration does not go forward.
This phenomenon has been the source of scientific curiosity and study since it was
first discovered in the early 1800s (Todd 1823), and nerve dependence is in fact
found in numerous examples of regeneration and wound healing across phylogeny.
From starfish arm regeneration to mammalian ear punch healing, an intact nerve
source appears to be imperative for proper regenerative growth (Kumar and Brockes
2012). Though the molecular mechanisms underlying this nerve dependence remain
poorly-understood in the axolotl, a host of studies spanning the past half-century
have done much to elucidate the roles of these nerves during limb regeneration.
Nerves heavily invade the wound epithelium within several days after amputa-
tion, and they appear to be critical for the maintenance of the AEC, as early dener-
vation eventually results in the collapse of the AEC and the impairment of blastema
formation. Nerves further appear to be necessary for the maintenance of blastemal
proliferation. Denervation once the blastema has formed starkly reduces the prolif-
eration of blastemal cells (Tassava et al. 1974; Maden 1978; Goldhamer and Tassava
7 Organ and Appendage Regeneration in the Axolotl 227

1987), though it does not appear to affect cell differentiation or limb patterning.
Thus, denervation well after blastema formation (at around 15 DPA) actually
induces the formation of a miniature, fully-patterned limb (Schotté and Butler 1944;
Singer and Craven 1948; Powell 1969). Axolotl peripheral nerves are themselves
capable of regeneration and will in fact rapidly regrow after denervation.
Consequently, studies involving limb denervation must be careful to re-denervate
every 7–10 days and cannot last longer than approximately 20 days, after which re-
denervation is effectively impossible and regeneration of the limb goes forward.
This characteristic represents another divergence between newts and axolotls, as
amputated newt limbs do not recover from brachial nerve transection and will not
regenerate even up to 72 days post-denervation (Liversage and McLaughlin 1983).
A series of elegant experiments performed by Marcus Singer in the 1950s char-
acterized the nature of nerve dependence in the regenerating axolotl limb. Singer
found that a certain number of nerve fibers is necessary for regeneration, although
this amount varies depending on the site of amputation- if the number of nerves
present falls below a specific threshold, regeneration does not take place. Singer
also found that the critical function of the nerves does not involve action potentials
or neurotransmitter release, nor does it require sensory or motor neuron innervation
in particular. Instead, nerve support of the wound epithelium and blastema appears
to be trophic in nature (Singer 1952, 1964). These experiments suggest that nerves-
and the dorsal root ganglia, implantations of which are capable of rescuing regen-
eration in denervated limbs (Kamrin and Singer 1959; Tomlinson and Tassava 1987;
Goldhamer et al. 1992)- release factors which are critical for inducing blastema
formation and maintaining blastemal proliferation. The precise identities of these
factors remain largely unknown, although evidence has been gathered in support of
many- including neuregulin-1 (Wang et al. 2000), transferrin (Kiffmeyer et al. 1991;
Mescher et al. 1997), fibroblast growth factors (Satoh et al. 2011), and anterior gra-
dient protein (Kumar et al. 2007). Identification of these critical nerve-derived fac-
tors thus constitutes a major topic of regenerative science moving forward.
The early invasion of macrophages into the wound site is also critical for limb
regeneration. Total macrophage ablation prevents regeneration and induces aber-
rant fibrotic deposition in the wound site (Godwin et al. 2013). Limb regeneration
like all forms of axolotl regeneration is a totally scar-free process that occurs with
minimal collagen deposition (Seifert et al. 2012; Levesque et al. 2010). Instead of
collagen, a dynamic network of fibronectin provides loose structure for the regen-
erating blastema (Maden and Keeble 1987; Rao et al. 2009). It is therefore possible
that macrophages, which peak in number at approximately 4 DPA (Godwin et al.
2013), are necessary for maintaining a permissive regenerating environment early
after injury. Further studies have also demonstrated that macrophages play a role in
clearing senescent cells from the wound site and blastema during regeneration
(Yun et al. 2015). It is thus believed that macrophages have multiple crucial func-
tions in axolotl limb regeneration, and the study of these functions remains an
ongoing process.
228 J.E. Farkas et al.

7.4 Limb Patterning and Mammalian Appendage

Regeneration

One extraordinary characteristic of axolotl limb regeneration is the fact that perfect
regeneration occurs regardless of the site of amputation. Thus, amputation at the
shoulder will result in regeneration of the entire limb, while amputation distal to the
elbow joint will regenerate only distal tissues. Considerable research has therefore
been devoted to elucidating the molecular underpinnings of axolotl limb patterning.
Studies have found that fibroblast-derived blastemal cells demonstrate a “memory”
of their initial position (Kragl et al. 2009), and this position is in some way expressed
on the cell surface, as proximal cells cultured in vitro reliably engulf distal cells
(Nardi and Stocum 1984). These cells are thus capable of detecting discrepancies in
the proximal/distal and dorsal/ventral axes and subsequently intercalating any miss-
ing structures, though the stability of this positional memory varies and can be
reprogrammed in early and distal blastemal cells if they come into contact with
more stable proximal cells (McCusker and Gardiner 2013). A host of studies have
demonstrated that retinoic acid (RA) plays crucial roles during limb patterning.
Application of exogenous RA early after amputation induces its expression in
(Monaghan and Maden 2012a) and proximalizes (Niazi et al. 1985; Keeble and
Maden 1989) fibroblast-derived blastemal cells, completely erasing distal positional
memory and suggesting that positional cell memory is in some way maintained via
a proximodistal retinoic acid gradient throughout the limb (Scadding and Maden
1994). Meanwhile, the salamander-specific cell surface protein Prod1, which is
upregulated in the blastema in response to RA (da Silva et al. 2002), may mediate
cell adhesion differences. Prod1 ablation impairs proximal cell engulfment (da
Silva et al. 2002) and overexpression of the protein proximalizes distal blastemal
cells (Echeverri and Tanaka 2005). The molecular mechanisms of limb patterning
remain under investigation, and as salamander limb patterning is itself a topic vast
enough to fill an entire chapter, numerous reviews have covered the process in
greater detail (Stocum and Cameron 2011; McCusker and Gardiner 2014; Mariani
2010; McCusker et al. 2015).
The axolotl’s extraordinary ability to fully regenerate amputated limbs sets it
apart from mammals and virtually all other vertebrates outside the urodeles, but
some regenerative mechanisms are conserved across phylogeny. Mammalian
appendage regeneration is very limited in scope: mice can regenerate only the tips of
their digits (Borgens 1982) while human children are capable of regenerating ampu-
tated fingertips so long as the wound is not immediately sealed after injury
(Illingworth 1974). Like axolotl limb regeneration, mammalian digit tip regeneration
relies on a heterogeneous mix of lineage-restricted progenitor cells (Lehoczky et al.
2011) and is disrupted if there is no intact nerve source, although nerve dependency
in this case appears to affect tissue patterning more than cell proliferation (Rinkevich
et al. 2014). However, nerve dependency during mouse ear hole punch regeneration
appears to align closely with salamander nerve dependency, as denervation of the ear
prevents blastema formation and induces necrosis (Buckley et al. 2012). This over-
7 Organ and Appendage Regeneration in the Axolotl 229

lap further underlined by the conserved presence of msx1, a homeobox-containing

gene that is highly upregulated during both mammalian digit tip (Reginelli et al.
1995; Allan et al. 2006) and axolotl limb regeneration (Koshiba et al. 1998). Axolotl
limb regeneration thus shares some essential similarities with mammalian digit tip
regeneration, and consequently it offers an enticing model for the study and advance-
ment of regenerative medicine in mammals.

7.5 Nervous System Regeneration

Axolotls have the striking ability to regenerate their central nervous system after
multiple injury paradigms including tail amputation, spinal cord transection, spinal
cord crush, and brain injury ((Chernoff et al. 2003; Chernoff 1996) for reviews).
Considering the variability of CNS morphology and injury types, the axolotl’s abil-
ity to regenerate after so many different injuries is remarkable. Reasons for the
axolotl’s regenerative capabilities are unknown, but traits including ongoing wide-
spread adult neurogenesis, a lack of a glial scarring after injury, and the overall
retention of embryonic characteristics likely contribute. However, axolotls are not
unique in their regenerative ability as teleost fish, other salamanders, and lizards can
regenerate CNS axons in the tail, while embryonic birds and mammals can regener-
ate CNS tissue to some extent ((Chernoff et al. 2003; Tanaka and Ferretti 2009) for
reviews).

7.6 Brain Regeneration

Brain regeneration has been demonstrated mainly in the telencephalon (Kirsche

and Kirsche 1964a, b; Richter 1968). The axolotl brain looks similar in structure to
the embryonic mouse brain. Neural stem cells with radial processes send processes
out to the pial surface throughout the animal’s life. These neural progenitor cells
proliferate throughout the uninjured adult axolotl nervous system, with the highest
level of proliferation occurring in the telencephalon (Maden et al. 2013; Richter
and Kranz 1981), and differentiate into NeuN+ neurons. Injury induces extensive
proliferation of neural progenitor cells in ventricular zones, which then migrate and
differentiate into several different cell types such as mature and immature neurons,
astrocytes, and radial glial cells (Maden et al. 2013). Ongoing neurogenesis in
regions capable of regeneration may provide an essential pool of active progenitor
cells required to mount a regenerative response. Interestingly, these proliferation
trends are not unique to axolotls. The human brain has active adult stem cell niches
in the subventricular zone and subgranular zone of the hippocampus (Eriksson
et al. 1998; Spalding et al. 2005). In addition, injury in adult rats induces extensive
proliferation capable of ameliorating the damage produced by stroke induction,
230 J.E. Farkas et al.

though the environment surrounding the lesion normally prevents successful heal-
ing (Arvidsson et al. 2002). It is intriguing to think that the availability or induction
of neural progenitor cell proliferation could enhance regenerative ability in
mammals.

7.7 Spinal Cord Regeneration

Regeneration of the spinal cord can occur anywhere along the rostro-caudal axis of
the animal after spinal cord crush, transection, or tail amputation. Tail amputation is
the simplest and most repeatable injury model and does not have detrimental effects
such as paralysis. Therefore, it has become the most widely adopted model for CNS
injury. Tail amputation is followed by regression of the spinal cord by ~0.5 mm
from the amputation plane accompanied by a rapid immune response with an influx
of leukocytes to the wound site. Over the first few days, cell death of neurons near
the amputation plane is apparent along with degeneration of white matter (Monaghan
et al. 2007; Zhang et al. 2003). By 3 days after amputation, neural progenitor cells
have migrated to generate a terminal bulb at the caudal end of the spinal cord, which
is surrounded by blastema cells. By approximately 7 days after injury, cell prolifera-
tion of neural progenitor cells and axon regeneration of spared neurons ensues in a
caudal direction. The migration and proliferation of the neural progenitor cells lin-
ing the central canal is a conserved mechanism for all animals that retain the ability
to regenerate their spinal cord (Gaete et al. 2012; McHedlishvili et al. 2007). The
cells about 500 um proximal to the tip of the damaged spinal cord give rise to all of
the central and peripheral nervous system components of the regenerated tail
(McHedlishvili et al. 2007, 2012). Most neurons in the regenerated spinal cord arise
from new neurogenesis, although a subset of neurons in the new cord arise from
translocation of differentiated neurons located proximal to the injury site (Zhang
et al. 2003). The importance of the neural progenitor population for regeneration
was clearly demonstrated using CRISPR/Cas 9-mediated genomic ablation of the
important neural stem cell gene Sox2, which showed that axolotls with nearly 100
% Sox2 deletion had defective spinal cord regeneration (Fei et al. 2014). This find-
ing is extremely important in our search for the differences between molecular
mechanisms of mammals and axolotls, as even conditional Sox2 knockouts cause
developmental problems in mice (Ferri et al. 2013). Future knockout studies are
likely to elucidate the genetic networks that are essential for axolotl CNS
regeneration.
Spinal cord regeneration after tail amputation is considerably different than spi-
nal cord transection or crush because it occurs within a regenerating tail blastema.
Furthermore, tail amputation necessitates the regeneration of multiple tissues types
beyond the neural tissue, grows only in a single direction, and is nerve dependent
(Holtzer 1956). Both injury models rely upon the proliferation of existing stem
neural stem cells of the spinal cord, which are analogous to the mammalian radial
glia seen only during development (Tanaka and Ferretti 2009). Axolotl spinal cord
transection may thus provide the most beneficial insights into possible therapies for
mammalian CNS injury. After transection, the terminal bulb is generated by 5 days
7 Organ and Appendage Regeneration in the Axolotl 231

post injury and rejoins the proximal to distal stumps by 15 days post injury. Axon
regeneration begins between 7 and 15 days and axons cross the lesion site by 21
days (Hui et al. 2013). Axon tracing studies after spinal cord transection have dem-
onstrated that the regenerated portion of the spinal cord is thinner than the uninjured
portion at 6–12 weeks post-transection, but eventually – sometimes up to 23 months
after transection – all of the axonal connections are reestablished (Clarke et al.
1988; Davis et al. 1990).
The axolotl’s ability to regenerate its spinal cord may be explained by a combina-
tion of pro-regenerative events that overcome inhibitory signals. For example, in
contrast to mammals, axolotls do not form a glial scar after spinal cord lesion.
Regeneration of the CNS in mammals is impaired by the formation of a glial scar
comprising of astrocytes and inhibitory proteoglycans (Fawcett and Asher 1999).
This barrier is thought to be initially beneficial because it prevents the spread of
damage throughout the system. However, it becomes detrimental when axons begin
sprouting across the injury site and are stopped by the glial scar. Similar glial cell
migration after injury exists in salamanders, but these processes establish a permis-
sive environment that promotes CNS regeneration (O’Hara and Chernoff 1994;
Zukor et al. 2011). A brief spike in apoptosis might enable lower vertebrates to
eliminate injured cells and prevent the spread of tissue damage (Sirbulescu and
Zupanc 2009). Furthermore, expression of matrix metalloproteinases, which chew
up extracellular debris, may limit the deposition of scar tissue and provide neces-
sary remodeling of the extracellular matrix (Chernoff et al. 2000). Mammals also
express myelin proteins such as Nogo-A, Oligodendrocyte myelin glycoprotein,
and myelin-associated glycoprotein whose release after injury may inhibit axon
regeneration (Yiu and He 2006). Regenerative species possess each of these mole-
cules so it is not the presence or absence of these molecules in lower vertebrates that
imparts regenerative ability (Hui et al. 2013; Shypitsyna et al. 2011), although their
presence does not interfere with healing and regeneration. Lastly, upregulation of
axon growth-inhibiting molecules such as Semaphorin 4D also limits regeneration
in the mammalian CNS (Moreau-Fauvarque et al. 2003). Interestingly, comparisons
of microRNA expression between regenerating axolotls and spinal-lesioned rats
showed that miR-125b is downregulated in axolotls during regeneration. Over-
expression or inhibition of miR-125b inhibits proper axolotl spinal cord regenera-
tion and increasing miR-125b levels in rats decreased Semaphorin 4D, which
improved functional recovery after spinal cord lesion (Macdonald-Obermann and
Pike 2014). Altogether, several factors may contribute to the axolotl’s ability to
regenerate. Functional studies that test the role of each of these factors should elu-
cidate the mechanisms that enable CNS regeneration.

7.8 Scarless Wound Healing

Axolotl skin facilitates cutaneous gas exchanges and protects the body from exter-
nal damage. The axolotl can regenerate wounded epidermis, dermis, and dermal
organs without forming scar tissue, and this ability is present throughout its
232 J.E. Farkas et al.

lifespan. This is in contrast to mammals, which repair skin wounds via accumula-
tion of fibroblasts in the area and replacing the original architecture of the skin with
a dense collagenous matrix, ultimately resulting in loss of elasticity, pigmentation,
and sensation (Martin 1997; Seifert and Maden 2014). This is an imperfect solution
that does not repair the integrity of the tissue, but instead acts to keep pathogens
from entering the body. The scar-free wound repair process in the axolotl provides
us with the means to understand what may lie beyond these limits. Deciphering the
molecular mechanisms that are responsible for axolotl skin regeneration after injury
will not only grant us aesthetically pleasing healing but may also allow for complete
functional recovery beyond fibrotic scar formation.
Both mammalian and axolotl skin is composed of an epidermis and dermis which
are separated by the basal lamina/membrane. In both classes, a hypodermis is also
present beneath the dermal layer adjacent to the muscle and connective tissue.
Mammalian wound repair of the skin starts with bleeding and platelet accumulation
and is followed by fibrin clot formation. Next, cytokine-directed migration of
immune cells to the wound bed causes inflammation. Once the immune cells clear
the debris of dead or damaged cells, new tissue formation starts with the prolifera-
tion and migration of keratinocytes from the edges of the wound to re-epithelize
underneath the scab. Mammalian re-epithelialization takes up to 10 days (Gurtner
et al. 2008). Fibroblasts deposit mainly collagen as they remodel the new ECM and
scar tissue forms as a result. In contrast, in the axolotl there is minimal bleeding and
no scab formation upon comparable injury. Immune cell migration and inflamma-
tion is minimal. This is followed by re-epithelialization that is 5–10 times faster in
comparison to mammalian tissues (Ferris et al. 2010). Re-epithelialization occurs in
a vortex motion (Tanner et al. 2009). Once the remodeling of the extracellular
matrix is complete, there is minimal collagen deposition and the wound is repaired
with a total lack of scar formation.
Full thickness excisional flank wounds are induced by removing the epidermis,
basement membrane and the dermis of the skin. Upon this excisional skin injury, the
axolotl completes scar-free wound healing in 80 days (Levesque et al. 2010; Seifert
et al. 2012). The axolotl is the only adult tetrapod that can heal full thickness exci-
sional flank wounds scar-free (Seifert et al. 2012; Levesque et al. 2010), while only
the fetal stage of other animals shows a similar regenerative capacity (Seifert and
Maden 2014; Gurtner et al. 2008; Adzick and Longaker 1992). Anurans have some
ability to regenerate dermal tissues, but also retain features of a mammalian-like
fibrotic scar (Bertolotti et al. 2013; Yannas et al. 1996). The aquatic axolotl has a
mucogenic epidermis whereas the terrestrial axolotl has a keratinized epidermis. Of
interest for translational work, the keratinized epithelium of the terrestrial axolotl
resembles mammalian skin composition (Seifert et al. 2012; Page et al. 2009).
Surprisingly, scarless wound healing also takes place in the adult terrestrial axolotl
(Seifert et al. 2012), albeit at a slower rate. Overall, it is important to note that in
comparison to mammalian wound healing in both the aquatic and the terrestrial
axolotl, hemostasis and re-epithelialization occur faster, inflammation is lower, and
ECM deposition is delayed, granting scarless wound healing (Seifert et al. 2012).
Given the compositional similarities, the metamorphic/terrestrial axolotl has great
7 Organ and Appendage Regeneration in the Axolotl 233

potential to aid in our understanding of the molecular and cellular events that enable
the physiological outcome of scar-free wound healing.

7.9 Extracellular Matrix Formation and Matrix

Metalloproteases

Fibronectin, collagen, and tenascin-C are ECM components which are synthesized
by fibroblasts. The ECM composition determines the outcome of how a wound will
close. Thus, the expression levels and timely deposition of these components are
tremendously influential in promoting scarless wound healing. During mammalian
wound healing, there is a transient deposition of fibronectin, which is later replaced
by collagen and localized tenascin-C deposition at the edges of the wound bed.
Contrastingly, in the axolotl, tenascin-C is abundant across the wound bed and
maintains high levels throughout the dermal regeneration. Additionally, collagen
deposition in the axolotl follows a pattern similar to that of mammalian wound
repair. However, alongside the continuously elevated levels of tenascin-C, fibronec-
tin levels are much lower in the wound bed resulting in a different ECM composi-
tion that is capable of scar-free healing (Seifert et al. 2012).
Upon early deposition of these ECM components to the wound bed, the mam-
malian wound is repaired mainly by fibrotic tissue. In contrast, there is a delay of
ECM deposition in the axolotl. This phenomenon is attributed to the matrix metal-
loproteases (MMPs) that are abundant during the healing process. These proteases
inhibit new ECM formation by degrading ECM components (Seifert et al. 2012). In
the axolotl, MMP1, MMP19, MMP28, MMP9 and MMP3/10a & b are highly
expressed before and during the re-epithelialization process and aid in keratinocyte
migration from the edges of the wound bed. While MMP1, MMP19 and MMP28
expression is returned to baseline levels following the completion of re-
epithelialization, MMP9 and MMP3/10a & b maintain high levels of expression.
MMP2 expression is also elevated after re-epithelialization is complete. When
treated with broad-spectrum MMP inhibitors, the axolotl excision wounds are
unable to re-epithelialize (Ferris et al. 2010). Thus, in order for a successful re-
epithelialization, keratinocyte migration is greatly dependent on MMP activity.
MMP9, MMP3/10a & b and MMP2 are conserved proteases capable of degrading
fibronectin and are also present in the mammalian tissue. Besides fibronectin degra-
dation, high levels of MMP3/10a & b and MMP2 in the axolotl degrade collagen
type I and II, and the persisting activity of these three MMPs may be considered
important mechanisms of the scarless wound healing process. Fetal mammalian tis-
sue, which is capable of scarless healing, also presents higher levels of these three
MMPs when compared to the adult mammalian tissue during wound healing, fur-
ther supporting the importance of MMP activity (Namazi et al. 2011; Li et al. 2006).
Furthermore, ECM composition and substrate stiffness play an important role in
the rate of re-epithelialization. When skin explants from terrestrial axolotls were
234 J.E. Farkas et al.

cultured on collagen beds wound closure was completed. However, wound closure
was unsuccessful when explants were challenged with ECM components of fibro-
nectin, laminin, or tenascin (Huang et al. 2015). These findings once again support
the importance of MMP function in degradation of ECM components during scar-
less wound healing and the avoidance of fibrotic scar formation. In addition, the
substrate stiffness on which the explants were cultured also had a reverse effect on
the plasticity of the epithelial cells, as it slowed the rate of re-epithelialization
(Huang et al. 2015).

7.10 Molecular Mechanisms of Scarless Wound Healing

The molecular mechanisms of scarless excisional wound healing are not yet fully
understood. In mammalian wound repair, myofibroblasts that express α-SMA trig-
ger inflammatory, angiogenic, and fibrotic factors and also orchestrate fibrotic tissue
formation (Wynn 2008; Bellayr et al. 2010). Given this specificity, α-SMA is used
as a fibrotic marker in mammalian tissues. Therefore, α-SMA expression was inves-
tigated in the axolotl to determine myofibroblast involvement in the wound healing
process. α-SMA expression was not detected at the site of post excisional skin
injury (Levesque et al. 2010). Only minor α-SMA activity was observed in the base-
ment membrane in later stages of wound healing. These findings overall indicate a
lack of myofibroblast activity and a subsequent lack of fibrotic tissue formation in
the axolotl, which results in scarless wound healing.
It is also known that TGF-β1 signaling triggers the production of α-SMA express-
ing cells in mammals and is crucial for wound healing (Desmouliere et al. 2005).
Increased TGF-β1 secretion from macrophages initially stimulates ECM compo-
nents but later leads to scar formation in mammals (Bellayr et al. 2010). Contrasting
with adult mammals, fetal models exhibit lower TGF-β1 signaling and thus exhibit
scarless wound healing (Zgheib et al. 2014). A recent study showed that axolotl
TGF-β1 expression was elevated 1 h post injury in the surrounding epidermis of an
excisional wound but ceased 4 days post-injury. Specifically, TGF-β1 was initially
expressed by the migrating epidermis, and later only by the epidermis and the new
ECM (Levesque et al. 2010). Similar expression patterns of TGF-β1 have also been
seen in fetal mammalian models (Martin et al. 1993). Thus, we can conclude that
TGF-β1 signaling results in fibrosis in adult mammals but does not affect the scar-
less wound healing of the axolotl due to its transient pattern of expression. Further
investigation of the regulators of this pathway may be crucial in understanding scar-
less wound healing.
In addition, the molecular limits of scarless wound healing in the axolotl were
established when excisional wounds were challenged with the drug bleomyosin and
scar formation was subsequently observed (Levesque et al. 2010). The resulting
fibrotic tissue was composed of accumulated fibronectin that thickened the epider-
mis. The regeneration of the basement membrane was also impaired upon bleomyo-
sin treatment. Even though it was established that the axolotl possesses the necessary
7 Organ and Appendage Regeneration in the Axolotl 235

mechanisms to retain fibrosis in the presence of a chemical inhibitor, the collagen

deposition was minimal, whereas collagen plays a major role in mammalian scar
tissue. In addition, there was no increase in α-SMA expressing cells, even though
fibrotic tissue was present (Levesque et al. 2010). To further understand regenera-
tive factors, a study isolated AmbLOXe from limb blastemas. AmbLOXe is highly
similar to the mammalian LOX gene and is a lypoxigenase that mediates the inflam-
matory responses important for wound healing. Transfecting human cells (human
osteosarcoma and keratinocyte cell lines) with AmbLOXe lead to increased cellular
migration (Menger et al. 2011). These findings suggest that utilizing regenerative
factors from the axolotl may also enhance scar-free wound healing in mammalian
systems.
The adult axolotl is an outstanding model to study scar-free wound repair. The
axolotl not only provides us with a convenient translational model, but it also uti-
lizes existing conserved mechanisms for regeneration, therefore providing relevant
insight into scarless wound healing. The metamorphic/terrestrial axolotl is espe-
cially promising given its skin composition and its resemblance to mammalian tis-
sue. Overall, scarless wound healing is affected by many factors. Rapid
re-epithelialization, high activity of matrix metalloproteases which delay new ECM
formation, high levels of tenascin-C and a lack of collagen deposition which aid in
remodeling new ECM, lack of α-SMA expressing myofibroblasts, transient TGF-β1
signaling, and low inflammatory responses are all contributing factors to scarless
wound healing in the axolotl. Further studies will reveal how conserved mecha-
nisms are regulated differently in the adult axolotl compared to mammals, with the
ultimate goal of orchestrating molecular and cellular mechanisms of wound healing
in order to generate the complete recovery of healthy, non-fibrotic skin in
mammals.

7.11 Cardiac Regeneration

Regeneration of the heart myocardium is observed to some extent in most verte-

brates including zebrafish (Poss et al. 2002), mice (Porrello et al. 2011), frogs
(Rumyantsev 1966), and newts (Oberpriller and Oberpriller 1974). It is commonly
believed that lower vertebrates can recover function throughout life while mice can
only regenerate up to 7 days after birth (Porrello et al. 2011). Although only three
studies have experimentally demonstrated myocardial regeneration in the axolotl,
the evidence supports that axolotls can regenerate adult myocardial tissue.
Regeneration of the cardiac tissue after 10–15 % partial ventricular amputation is
demonstrated in all three of these studies (Flink 2002; Vargas-Gonzalez et al. 2005;
Cano-Martinez et al. 2010). In the axolotl, the completeness of regeneration has not
been assessed yet. In newts, it is known that cardiac regeneration occurs without any
presence of a scar (Witman et al. 2011). Continuous BrdU labeling from 14 to 21
days post injury in axolotls showed that the majority of BrdU+ cells were found
within 75–125 μm of the injury site. In addition, co-staining with cardiomyocyte-
specific antibodies showed that 74.3 % of epicardial cells and 12.8 % of
236 J.E. Farkas et al.

cardiomyocytes were BrdU+. Some BrdU+ cells were found as far as 750 μm away
from the injury site suggesting a widespread proliferative response after cardiac
injury (Flink 2002). Most functional recovery occurs within 30–90 days post injury,
which is preceded by cardiomyocyte proliferation (Cano-Martinez et al. 2010).
Co-labeling of proliferative markers with cardiomyocyte markers suggest that car-
diomyocyte dedifferentiation drives the regenerative process (Flink 2002; Vargas-
Gonzalez et al. 2005; Cano-Martinez et al. 2010), which is supported by the fact that
adult ventricular cardiomyocytes in newts can readily proliferate in vitro (Mercer
et al. 2013; Nag et al. 1979; Tate et al. 1989). Furthermore, genes involved in the
embryonic cardiogenic programming including Hand2, Nkx.2, Gata4, Islet1, and
Gata5 are all upregulated during newt cardiac regeneration with a significant pro-
portion of Gata4 and Islet1 expression co-localizing with cardiomyocyte markers
supporting the likelihood of cardiomyocyte dedifferentiation (Witman et al. 2011).
Based on the evidence, it is likely that axolotls regenerate both by a local injury
response (epimorphic) and a widespread organ-wide (compensatory) mechanism. A
similar mechanism of cardiomyocyte proliferation was observed in endogenous car-
diomyocytes during zebrafish regeneration after ventricular cryoinjury (Sallin et al.
2015). Moreover, cellular lineage tracing in zebrafish have shown that dedifferentia-
tion of resident cardiomyocytes generate the new myocardium in zebrafish (Kikuchi
et al. 2010; Jopling et al. 2010; Zhang et al. 2013), which also seems to be the case
in the regenerating neonatal mouse (Porrello et al. 2011, 2013; Mahmoud et al.
2013). Although a more comprehensive analysis of heart regeneration is required in
amphibians to elucidate their mechanism of regeneration, it is intriguing to think
that all animals with the capability to regenerate heart myocardium do so using the
same basic mechanisms.

7.12 Early Wound Healing and the Role of the Immune

System

Regeneration across all tissue types is a complex process dependent on inflamma-

tion, tissue remodeling, and tissue formation. Blood and its circulation plays a major
role in orchestrating these events by closing off the initial wound and carrying nec-
essary factors to the wound site. Here, we will cover what is known about the axo-
lotl’s clotting factors and the immune system’s role in regeneration, with a particular
emphasis on the role of leukocytes for this process.

7.13 Clotting Factors

An early step in regeneration is the formation of a clot mediated by cleavage of

plasma fibrinogen by thrombin. The clot not only closes off the wound to prevent
infection, but may also provide activating factors required for regeneration. Axolotl
blood clots within several minutes after amputation, and the wound epidermis
7 Organ and Appendage Regeneration in the Axolotl 237

surrounds the clot within 12–24 h depending upon the size of the wound (Sobkow
et al. 2006). It is possible that clotting factors released early after injury may be an
inductive signal. Indeed, a thrombin-generated ligand is known to induce newt myo-
tubes to re-enter the cell cycle in culture (Tanaka et al. 1997), supporting the hypoth-
esis that thrombin is required for regeneration. In the regenerating newt lens,
thrombin activity is present and required for regeneration (Imokawa and Brockes
2003). Thrombin seems to be associated with regenerating tissues, but it is not
known whether it is sufficient to induce a regenerative response. Furthermore, the
thrombin-mediated ligand or its downstream targets have not been identified, mak-
ing it difficult to know what role it plays during regeneration. Regardless, clotting is
associated with the early injury response and therefore is a prime candidate for
inducing a regenerative response.

7.14 Inflammation and the Immune System

The axolotl immune system is comprised of an innate immune system and a rudi-
mentary adaptive immune system. Axolotls are deemed relatively immunodeficient
due to the fact that they only produce two immunoglobulin classes (IgM and IgY) –
neither of which are anamnestic – and overall humoral and cytotoxic responses are
slow or non-existent (Tournefier et al. 1988; Chen and Robert 2011; Godwin and
Rosenthal 2014). IgM is produced by lymphocytes within the spleen around 7
weeks post-fertilization, though IgY is not detected until the axolotl reaches 7
months old (Fellah et al. 1989). It has been surmised that inflammation and immu-
nomodulation may be implicated in regenerative ability because there is an inverse
relationship between the maturation of the immune system and capacity to regener-
ate (Mescher and Neff 2005; Harty et al. 2003; King et al. 2012). Inflammation is an
initial response to wounding. Upon injury to mammalian tissue, cytokines direct
immune cells to the area of the assault, thus creating inflammation and inducing
scar formation (Wynn 2008). Contrastingly, the larval axolotl lacks neutrophils and
macrophages that migrate to the wound bed, and the adult axolotl has a low number
of neutrophils found in the wound bed (Levesque et al. 2010; Seifert et al. 2012;
Ziegels 1971). This reduced inflammatory response correlates with scar-free wound
healing of the axolotl (Seifert et al. 2012), which is reminiscent of the scar-free
wound healing capabilities of fetal mammals (Namazi et al. 2011). This is sup-
ported by studies which have shown that after 24 weeks of gestation, onset of a high
inflammatory response is consistent with low regenerative capacity and scar forma-
tion in mammals (Yates et al. 2012; Adzick and Lorenz 1994; Xue and Jackson
2015). Despite this clear inverse correlation, the molecular mechanisms behind how
the immune cells affect regeneration is poorly understood.
Macrophages specifically seem to be important in the regeneration process.
Their roles are unclear, but they likely contribute through the phagocytosis of debris
following initial injury, breakdown of extracellular matrix (ECM) and promotion of
its reconstruction, release of pro- and subsequently anti-inflammatory cytokines,
238 J.E. Farkas et al.

and mobilization of stem cells. Depletion of macrophages in the axolotl limb imme-
diately after injury prevents blastema formation, and depletion of baseline-level
macrophages 15 days post injury will prolong total regeneration time (Godwin et al.
2013). In mouse bone marrow studies, macrophage depletion caused hematopoietic
stem cells to egress from the niche due to a loss of paracrine homeostasis factors
such as Cxcl12 (Chow et al. 2011). In zebrafish, depletion of macrophages also
leads to defective caudal fin regeneration (Li et al. 2012). Macrophage depletion has
also been implicated in prevention of heart regeneration in the adult axolotl, though
if they are required for merely debris clearance, cytokine signaling, or a greater
paracrine role is still not clear (Pinto et al. 2014). Overall, the ability of macro-
phages to alter their environment physically and through molecular signaling is
clearly essential to tissue regeneration, though the extent of these mechanisms are
not yet fully understood. While the immune system is implicated in many aspects of
regeneration, the entire blood lineage itself shows a high level of regenerative capac-
ity in the axolotl. Ablation of the liver or spleen by targeted irradiation of the adult
axolotl will lead to anemia and eventually death (Lopez et al. 2014). Furthermore,
the liver and spleen may provide different niches as the HSPC populations of the
spleen are 1000-fold enriched in lymphoblastic populations compared to the periph-
ery of the liver (Lopez and Scott 2015).

7.15 Conclusion

Based upon the examples in this review, axolotls utilize multiple mechanisms to
coordinate a regenerative response, but it is clear that with time the animal will fully
regenerate its limb, tail, spinal cord, and heart. In all injuries models studied, injury
initiates a local infiltration of leukocytes, which is associated with migration of cells
nearby the injury. Migration of cells is followed with a sustained proliferation of
progenitor cells that arise either from dedifferentiation or recruitment of local adult
stem cells, most commonly located near the injury site. Differentiation of progeni-
tor cells is a recapitulation of development of each organ, coupled with enhanced
growth until the injured tissue is replaced. The molecular processes that initiate a
regenerative response or regulate when an organ stops growing are critical questions
that need to be elucidated to in order to understand the regenerative process.
The axolotl’s regenerative abilities are not limited to its brain, spinal cord, nerve,
tail, heart, limb and skin regeneration. The regenerative ability of other organs are
highlighted in Table 7.1. Considering the broad regenerative ability that has been
described here, it is likely that most if not all organs and tissues can regenerate at
some point in their lifetime. For example, it was previously thought that axolotls
could not regenerate their lens. This idea was recently overturned, showing that for
a duration of 2 weeks after hatching/Stage 44, axolotls possess the ability to regen-
erate the lens from the ventral or the dorsal iris or simultaneously from both
(Suetsugu-Maki et al. 2012). Overall, given its wide variety of organ and appendage
regenerating capabilities, the axolotl is a compelling model for the study of adult
7 Organ and Appendage Regeneration in the Axolotl 239

Table 7.1 Table includes original citations that describe the regenerative ability of each animal
group
Tissue Species All citations
Retina Ambystoma– intermediate Keefe (1973), Stone (1950)
Newts – yes
Brain Ambystoma– yes Kirsche and Kirsche (1964b), Parish et al. (2007),
Newts – yes and Minelli and Del Grande (1974)
Spinal cord Ambystoma– yes McHedlishvili et al. (2012, Butler and Ward (1965,
Newts – yes 1967), Egar and Singer (1972), and Piatt (1955)
Lens Ambystoma– up to 2 Eguchi (1963), Suetsugu-Maki et al. (2012), and
weeks post hatching Collucci (1891)
Newts – yes
Jaw & Newts – yes Ghosh et al. (1994), Goss and Stagg (1958)
teeth
Heart Ambystoma – yes Oberpriller and Oberpriller (1974), Flink (2002),
Newts – yes Vargas-Gonzalez et al. (2005), and Cano-Martinez
et al. (2010)
Lungs Unknown
Liver Ambystoma – yes Williams (1961), Heberlein (1930)
Newts – yes
Spleen Newts – yes Garavini (1977)
Gall Unknown
bladder
Pancreas Newts – intermediate Vethamany-Globus and Liversage (1973)
Stomach Unknown
Intestine Newts – yes O’Steen and Walker (1962), O’Steen (1958), and
Grubb (1975)
Kidneys Newts – no Scadding and Liversage (1974)
Testis Newts – yes Flament et al. (2009), Uchida and Hanaoka (1949),
and Bois and Beaumont (1927)
Ovaries Unknown
Lateral line Ambystoma – yes Jones and Corwin (1993), Jorgensen and Flock
(1976), Stone (1933,1937), and Jørgensen and Flock
(1976)
Tail Ambystoma – yes Holtzer (1956), Iten and Bryant (1976)
Newts – yes
Limb Ambystoma – yes Schotté and Butler (1944); Iten and Bryant (1973),
Newts – yes and Chalkley (1954)
Skin Ambystoma – yes Seifert et al. (2012), Levesque et al. (2010), and
Ziegels (1971)
Note that the references are not comprehensive. “Newt” includes references to Cynops pyrrhogas-
ter, Triturus cristatus, Pleurodeles waltl, and Notophalamus viridescens. “Ambystoma” includes
references to Ambystoma maculatum or Ambystoma mexicanum (axolotl). Missing values indicate
the organ has not been studied in all animals
240 J.E. Farkas et al.

tissue regeneration. A broad survey of organ regeneration should provide much

insight into the common and divergent mechanisms that regulate regeneration.

References

Adzick NS, Longaker MT (1992) Scarless fetal healing. Therapeutic implications. Ann Surg
215(1):3–7
Adzick NS, Lorenz HP (1994) Cells, matrix, growth factors, and the surgeon. The biology of scar-
less fetal wound repair. Ann Surg 220(1):10–18
Allan CH et al (2006) Tissue response and Msx1 expression after human fetal digit tip amputation
in vitro. Wound Repair Regen 14(4):398–404
Arvidsson A et al (2002) Neuronal replacement from endogenous precursors in the adult brain
after stroke. Nat Med 8(9):963–970
Bellayr IH, Walters TJ, Li Y (2010) Scarless wound healing. J Am Coll Certif Wound Spec
2(2):40–43
Bertolotti E, Malagoli D, Franchini A (2013) Skin wound healing in different aged Xenopus laevis.
J Morphol 274(8):956–964
Bois AD, Beaumont JD (1927) Intersexualité phénotypique dans la gonade mâle du triton. C R Soc
Biol 97:1323–1324
Borgens RB (1982) Mice regrow the tips of their foretoes. Science 217(4561):747–750
Buckley G et al (2012) Denervation affects regenerative responses in MRL/MpJ and repair in
C57BL/6 ear wounds. J Anat 220(1):3–12
Butler EG (1935) Studies on limb regeneration in X-rayed amblystoma larvae. Anat Rec
62(3):295–307
Butler EG, O’Brien JP (1942) Effects of localized x-radiation on regeneration of the urodele limb.
Anat Rec 84(4):407–413
Butler EG, Ward MB (1965) Reconstitution of the spinal cord following ablation in urodele larvae.
J Exp Zool 160(1):47–65
Butler EG, Ward MB (1967) Reconstitution of the spinal cord after ablation in adult Triturus. Dev
Biol 15(5):464–486
Campbell LJ et al (2011) Gene expression profile of the regeneration epithelium during axolotl
limb regeneration. Dev Dyn: An Off Publ Am Assoc Anat 240(7):1826–1840
Cano-Martinez A et al (2010) Functional and structural regeneration in the axolotl heart
(Ambystoma mexicanum) after partial ventricular amputation. Arch Cardiol Mex
80(2):79–86
Chalkley DT (1954) A quantitative histological analysis of forelimb regeneration in triturus viride-
scens. J Morphol 94(1):21–70
Chen G, Robert J (2011) Antiviral immunity in amphibians. Viruses 3(11):2065–2086
Chernoff EA (1996) Spinal cord regeneration: a phenomenon unique to urodeles? Int J Dev Biol
40(4):823–831
Chernoff EA et al (2000) Matrix metalloproteinase production in regenerating axolotl spinal cord.
Wound Repair Regen 8(4):282–291
Chernoff EA et al (2003) Urodele spinal cord regeneration and related processes. Dev Dyn
226(2):295–307
Chow A, Brown BD, Merad M (2011) Studying the mononuclear phagocyte system in the molecu-
lar age. Nat Rev Immunol 11(11):788–798
Clarke JD, Alexander R, Holder N (1988) Regeneration of descending axons in the spinal cord of
the axolotl. Neurosci Lett 89(1):1–6
Collucci V (1891) Sulla rigenerazione parziale deell’occhio nei tritoni: Isogenesi esvilluppo-
Studio seprimentale. Mem R Accad Sci lst Bologna Ser 5(1):593–629
da Silva SM, Gates PB, Brockes JP (2002) The newt ortholog of CD59 is implicated in proxi-
modistal identity during amphibian limb regeneration. Dev Cell 3(4):547–555
7 Organ and Appendage Regeneration in the Axolotl 241

Davis BM et al (1990) Time course of salamander spinal cord regeneration and recovery of swim-
ming: HRP retrograde pathway tracing and kinematic analysis. Exp Neurol 108(3):198–213
Desmouliere A, Chaponnier C, Gabbiani G (2005) Tissue repair, contraction, and the myofibro-
blast. Wound Repair Regen 13(1):7–12
Echeverri K, Tanaka EM (2005) Proximodistal patterning during limb regeneration. Dev Biol
279(2):391–401
Egar M, Singer M (1972) The role of ependyma in spinal cord regeneration in the urodele, Triturus.
Exp Neurol 37(2):422–430
Eguchi G (1963) Electron microscopic studies on lens regeneration. Embryologia 8(1):45–62
Eriksson PS et al (1998) Neurogenesis in the adult human hippocampus. Nat Med
4(11):1313–1317
Fawcett JW, Asher RA (1999) The glial scar and central nervous system repair. Brain Res Bull
49(6):377–391
Fei JF et al (2014) CRISPR-mediated genomic deletion of Sox2 in the axolotl shows a requirement
in spinal cord neural stem cell amplification during tail regeneration. Stem Cell Rep
3(3):444–459
Fellah JS et al (1989) Ontogeny of immunoglobulin expression in the Mexican axolotl.
Development 107(2):253–263
Ferri A et al (2013) Sox2 is required for embryonic development of the ventral telencephalon
through the activation of the ventral determinants Nkx2.1 and Shh. Development
140(6):1250–1261
Ferris DR et al (2010) Ex vivo generation of a functional and regenerative wound epithelium from
axolotl (Ambystoma mexicanum) skin. Dev Growth Differ 52(8):715–724
Flament SP et al (2009) Lifelong testicular differentiation in Pleurodeles waltl (Amphibia,
Caudata). Reprod Biol Endocrinol 7:21
Flink IL (2002) Cell cycle reentry of ventricular and atrial cardiomyocytes and cells within the
epicardium following amputation of the ventricular apex in the axolotl, Amblystoma mexica-
num: confocal microscopic immunofluorescent image analysis of bromodeoxyuridine-labeled
nuclei. Anat Embryol (Berl) 205(3):235–244
Foret JE (1970) Regeneration of larval urodele limbs containing homoplastic transplants. J Exp
Zool 175(3):297–321
Gaete M et al (2012) Spinal cord regeneration in Xenopus tadpoles proceeds through activation of
Sox2-positive cells. Neural Dev 7:13
Garavini C (1977) Regeneration of the spleen in Triturus cristatus. Arch Ital Anat Embriol
82(4):319–325
Ghosh S, Thorogood P, Ferretti P (1994) Regenerative capability of upper and lower jaws in the
newt. Int J Dev Biol 38(3):479–490
Godwin JW, Rosenthal N (2014) Scar-free wound healing and regeneration in amphibians: immu-
nological influences on regenerative success. Differentiation 87(1–2):66–75
Godwin JW, Pinto AR, Rosenthal NA (2013) Macrophages are required for adult salamander limb
regeneration. Proc Natl Acad Sci U S A 110(23):9415–9420
Goldhamer DJ, Tassava RA (1987) An analysis of proliferative activity in innervated and dener-
vated forelimb regenerates of the newt. Notophthalmus Viridescens Dev 100(4):619–628
Goldhamer DJ, Tomlinson BL, Tassava RA (1992) Ganglia implantation as a means of supplying
neurotrophic stimulation to the newt regeneration blastema: cell-cycle effects in innervated and
denervated limbs. J Exp Zool 262(1):71–80
Goss RJ, Stagg MW (1958) Regeneration of lower jaws in adult newts. J Morphol
102(2):289–309
Grubb RB (1975) An autoradiographic study of the origin of intestinal blastemal cells in the newt,
Notophthalmus viridescens. Dev Biol 47(1):185–195
Gurtner GC et al (2008) Wound repair and regeneration. Nature 453(7193):314–321
Harty M et al (2003) Regeneration or scarring: an immunologic perspective. Dev Dyn
226(2):268–279
Heberlein JML (1930) Uber regeneration innerer organe beim axolotl. G. Fischer, Thüringische
Landesuniversität Jena
242 J.E. Farkas et al.

Holtzer SW (1956) The inductive activity of the spinal cord in urodele tail regeneration. J Morphol
99(1):1–39
Huang TY et al (2015) Cooperative regulation of substrate stiffness and extracellular matrix pro-
teins in skin wound healing of axolotls. Biomed Res Int 2015:712546
Hui SP et al (2013) Expression pattern of Nogo-A, MAG, and NgR in regenerating urodele spinal
cord. Dev Dyn 242(7):847–860
Illingworth CM (1974) Trapped fingers and amputated finger tips in children. J Pediatr Surg
9(6):853–858
Imokawa Y, Brockes JP (2003) Selective activation of thrombin is a critical determinant for verte-
brate lens regeneration. Curr Biol 13(10):877–881
Iten L, Bryant S (1973) Forelimb regeneration from different levels of amputation in the newt,
Notophthalmus viridescens: length, rate, and stages. Wilhelm Roux’ Archiv für
Entwicklungsmechanik der Organismen 173(4):263–282
Iten LE, Bryant SV (1976) Stages of tail regeneration in the adult newt, Notophthalmus virides-
cens. J Exp Zool 196(3):283–292
Jones JE, Corwin JT (1993) Replacement of lateral line sensory organs during tail regeneration in
salamanders: identification of progenitor cells and analysis of leukocyte activity. J Neurosci
13(3):1022–1034
Jopling C et al (2010) Zebrafish heart regeneration occurs by cardiomyocyte dedifferentiation and
proliferation. Nature 464(7288):606–609
Jorgensen JM, Flock A (1976) Non-innervated sense organs of the lateral line: development in the
regenerating tail of the salamander Ambystoma mexicanum. J Neurocytol 5(1):33–41
Jørgensen JM, Flock Å (1976) Non-innervated sense organs of the lateral line: development in the
regenerating tail of the salamanderAmbystoma mexicanum. J Neurocytol 5(1):33–41
Kamrin AA, Singer M (1959) The growth influence of spinal ganglia implanted into the denervated
forelimb regenerate of the newt. Triturus J Morphol 104:415–439
Keeble S, Maden M (1989) The relationship among retinoid structure, affinity for retinoic acid-
binding protein, and ability to respecify pattern in the regenerating axolotl limb. Dev Biol
132(1):26–34
Keefe JR (1973) An analysis of urodelian retinal regeneration: I. Studies of the cellular source of
retinal regeneration in Notophthalmus viridescens utilizing 3H-thymidine and colchicine.
J Exp Zool 184(2):185–206
Khattak S et al (2013) Foamy virus for efficient gene transfer in regeneration studies. BMC Dev
Biol 13:17
Khattak S et al (2014) Optimized axolotl (Ambystoma mexicanum) husbandry, breeding, meta-
morphosis, transgenesis and tamoxifen-mediated recombination. Nat Protoc 9(3):529–540
Kiffmeyer WR, Tomusk EV, Mescher AL (1991) Axonal transport and release of transferrin in
nerves of regenerating amphibian limbs. Dev Biol 147(2):392–402
Kikuchi K et al (2010) Primary contribution to zebrafish heart regeneration by gata4+ cardiomyo-
cytes. Nature 464(7288):601–605
King MW, Neff AW, Mescher AL (2012) The developing Xenopus limb as a model for studies on
the balance between inflammation and regeneration. Anat Rec (Hoboken)
295(10):1552–1561
Kirsche K, Kirsche W (1964a) Compensatory hyperplasia and regeneration in the telencephalon of
Ambystoma mexicanum after resection of a hemisphere. Z Mikrosk Anat Forsch 71:505–521
Kirsche K, Kirsche W (1964b) Regenerative processes in the telencephalon of Ambystoma mexi-
canum. J Hirnforsch 7:421–436
Knapp D et al (2013) Comparative transcriptional profiling of the axolotl limb identifies a tripartite
regeneration-specific gene program. PLoS One 8(5):e61352
Knopf F et al (2011) Bone regenerates via dedifferentiation of osteoblasts in the zebrafish fin. Dev
Cell 20(5):713–724
Koshiba K et al (1998) Expression of Msx genes in regenerating and developing limbs of axolotl.
J Exp Zool 282(6):703–714
7 Organ and Appendage Regeneration in the Axolotl 243

Kragl M et al (2009) Cells keep a memory of their tissue origin during axolotl limb regeneration.
Nature 460(7251):60–65
Kumar A, Brockes JP (2012) Nerve dependence in tissue, organ, and appendage regeneration.
Trends Neurosci 35(11):691–699
Kumar A et al (2007) Molecular basis for the nerve dependence of limb regeneration in an adult
vertebrate. Science (New York, N.Y.) 318(5851):772–777
Lehoczky JA, Robert B, Tabin CJ (2011) Mouse digit tip regeneration is mediated by fate-restricted
progenitor cells. Proc Natl Acad Sci U S A 108(51):20609–20614
Lévesque M et al (2007) Transforming growth factor: β signaling is essential for limb regeneration
in Axolotls. PLoS One 2(11):e1227
Levesque M, Villiard E, Roy S (2010) Skin wound healing in axolotls: a scarless process. J Exp
Zool B Mol Dev Evol 314(8):684–697
Li WJ et al (2006) Expression of matrix metalloproteinases and their inhibitors in fetal skin and
their biological significance. Zhongguo Wei Zhong Bing Ji Jiu Yi Xue 18(5):303–306
Li L et al (2012) Live imaging reveals differing roles of macrophages and neutrophils during
zebrafish tail fin regeneration. J Biol Chem 287(30):25353–25360
Liversage RA, McLaughlin DS (1983) Effects of delayed amputation on denervated forelimbs of
adult newt. J Embryol Exp Morphol 75:1–10
Lopez D, Scott EW (2015) Generation of axolotl hematopoietic chimeras. J Biol Methods 2:10
Lopez D et al (2014) Mapping hematopoiesis in a fully regenerative vertebrate: the axolotl. Blood
124(8):1232–1241
Loyd RM, Tassava RA (1980) DNA synthesis and mitosis in adult newt limbs following amputa-
tion and insertion into the body cavity. J Exp Zool 214(1):61–69
Macdonald-Obermann JL, Pike LJ (2014) Different epidermal growth factor (EGF) receptor
ligands show distinct kinetics and biased or partial agonism for homodimer and heterodimer
formation. J Biol Chem 289(38):26178–26188
Maden M (1978) Neurotrophic control of the cell cycle during amphibian limb regeneration.
J Embryol Exp Morphol 48:169–175
Maden M, Keeble S (1987) The role of cartilage and fibronectin during respecification of pattern
induced in the regenerating amphibian limb by retinoic acid. Differentiation 36(3):175–184
Maden M, Manwell LA, Ormerod BK (2013) Proliferation zones in the axolotl brain and regenera-
tion of the telencephalon. Neural Dev 8(1):1
Mahmoud AI et al (2013) Meis1 regulates postnatal cardiomyocyte cell cycle arrest. Nature
497(7448):249–253
Mariani FV (2010) Proximal to distal patterning during limb development and regeneration: a
review of converging disciplines. Regen Med 5(3):451–462
Martin P (1997) Wound healing – aiming for perfect skin regeneration. Science 276(5309):75–81
Martin P et al (1993) Rapid induction and clearance of TGF beta 1 is an early response to wound-
ing in the mouse embryo. Dev Genet 14(3):225–238
McCusker CD, Gardiner DM (2013) Positional information is reprogrammed in blastema cells of
the regenerating limb of the axolotl (Ambystoma mexicanum). PLoS One 8(9):e77064
McCusker CD, Gardiner DM (2014) Understanding positional cues in salamander limb regenera-
tion: implications for optimizing cell-based regenerative therapies. Dis Model Mech
7(6):593–599
McCusker C, Bryant SV, Gardiner DM (2015) The axolotl limb blastema: cellular and molecular
mechanisms driving blastema formation and limb regeneration in tetrapods. Regeneration
2(2):54–71
McHedlishvili L et al (2007) A clonal analysis of neural progenitors during axolotl spinal cord
regeneration reveals evidence for both spatially restricted and multipotent progenitors.
Development (Cambridge, England) 134(11):2083–2093
McHedlishvili L et al (2012) Reconstitution of the central and peripheral nervous system during
salamander tail regeneration. Proc Natl Acad Sci U S A 109(34):E2258–E2266
244 J.E. Farkas et al.

Menger B et al (2011) AmbLOXe – an epidermal lipoxygenase of the Mexican axolotl in the con-
text of amphibian regeneration and its impact on human wound closure in vitro. Ann Surg
253(2):410–418
Mercader N, Tanaka EM, Torres M (2005) Proximodistal identity during vertebrate limb regenera-
tion is regulated by Meis homeodomain proteins. Development (Cambridge, England)
132(18):4131–4142
Mercer SE, Odelberg SJ, Simon HG (2013) A dynamic spatiotemporal extracellular matrix facili-
tates epicardial-mediated vertebrate heart regeneration. Dev Biol 382(2):457–469
Mescher AL, Neff AW (2005) Regenerative capacity and the developing immune system. Adv
Biochem Eng Biotechnol 93:39–66
Mescher AL et al (1997) Transferrin is necessary and sufficient for the neural effect on growth in
amphibian limb regeneration blastemas. Develop Growth Differ 39(6):677–684
Minelli G, Del Grande P (1974) Localization and quantitative analysis of the elements leading to
the regeneration of the optic tectum in the adult Triturus cristatus carnifex. Z Mikrosk Anat
Forsch 88(2):209–224
Monaghan JR, Maden M (2012a) Visualization of retinoic acid signaling in transgenic axolotls
during limb development and regeneration. Dev Biol 368(1):63–75
Monaghan JR, Maden M (2012b) Cellular plasticity during vertebrate appendage regeneration.
Curr Top Microbiol Immunol 367:53–74
Monaghan JR et al (2007) Early gene expression during natural spinal cord regeneration in the
salamander Ambystoma mexicanum. J Neurochem 101(1):27–40
Monaghan JR et al (2009) Microarray and cDNA sequence analysis of transcription during nerve-
dependent limb regeneration. BMC Biol 7
Monaghan JR et al (2012) Gene expression patterns specific to the regenerating limb of the
Mexican axolotl. Biol Open 1(10):937–948
Moreau-Fauvarque C et al (2003) The transmembrane semaphorin Sema4D/CD100, an inhibitor
of axonal growth, is expressed on oligodendrocytes and upregulated after CNS lesion.
J Neurosci 23(27):9229–9239
Muneoka K, Fox WF, Bryant SV (1986) Cellular contribution from dermis and cartilage to the
regenerating limb blastema in axolotls. Dev Biol 116(1):256–260
Nacu E et al (2009) Axolotl (Ambystoma mexicanum) embryonic transplantation methods. Cold
Spring Harbor Protoc 2009(8):pdb-prot5265
Nag AC, Healy CJ, Cheng M (1979) DNA synthesis and mitosis in adult amphibian cardiac muscle
cells in vitro. Science 205(4412):1281–1282
Namazi MR, Fallahzadeh MK, Schwartz RA (2011) Strategies for prevention of scars: what can
we learn from fetal skin? Int J Dermatol 50(1):85–93
Nardi JB, Stocum DL (1984) Surface properties of regenerating limb cells: evidence for gradation
along the proximodistal axis. Differentiation 25(1–3):27–31
Niazi IA, Pescitelli MJ, Stocum DL (1985) Stage-dependent effects of retinoic acid on regenerat-
ing urodele limbs Wilhelm. Rouxs Arch Dev Biol 194(6):355–363
O’Hara CM, Chernoff EA (1994) Growth factor modulation of injury-reactive ependymal cell
proliferation and migration. Tissue Cell 26(4):599–611
O’Steen WK (1958) Regeneration of the intestine in adult urodeles. J Morphol 103(3):435–477
O’Steen WK, Walker BE (1962) Radioautographic studies of regeneration in the common newt
III. Regeneration and repair of the intestine. Anat Rec 142(2):179–187
Oberpriller JO, Oberpriller JC (1974) Response of the adult newt ventricle to injury. J Exp Zool
187(2):249–253
Page RB et al (2009) A model of transcriptional and morphological changes during thyroid
hormone-induced metamorphosis of the axolotl. Gen Comp Endocrinol 162(2):219–232
Parish CL et al (2007) Midbrain dopaminergic neurogenesis and behavioural recovery in a sala-
mander lesion-induced regeneration model. Development 134(15):2881–2887
Piatt J (1955) Regeneration of the spinal cord in the salamander. J Exp Zool 129(1):177–207
7 Organ and Appendage Regeneration in the Axolotl 245

Pinto AR, Godwin JW, Rosenthal NA (2014) Macrophages in cardiac homeostasis, injury
responses and progenitor cell mobilisation. Stem Cell Res 13(3 Pt B):705–714
Porrello ER et al (2011) Transient regenerative potential of the neonatal mouse heart. Science
331(6020):1078–1080
Porrello ER et al (2013) Regulation of neonatal and adult mammalian heart regeneration by the
miR-15 family. Proc Natl Acad Sci U S A 110(1):187–192
Poss KD, Wilson LG, Keating MT (2002) Heart regeneration in zebrafish. Science
298(5601):2188–2190
Powell JA (1969) Analysis of histogenesis and regenerative ability of denervated forelimb regener-
ates of Triturus viridescens. J Exp Zool 170(2):125–147
Rao N et al (2009) Proteomic analysis of blastema formation in regenerating axolotl limbs. BMC
Biol 7:83
Reginelli AD et al (1995) Digit tip regeneration correlates with regions of Msx1 (Hox 7) expres-
sion in fetal and newborn mice. Development 121(4):1065–1076
Richter W (1968) Regenerative processes following removal of the caudal sector of the telencepha-
lon including the telencephalo-diencephalic border region in Ambystoma mexicanum (In
German). J Hirnforsch 10(6):515–534
Richter W, Kranz D (1981) Autoradiographic investigations on postnatal proliferative activity of
the telencephalic and diencephalic matrix-zones in the axolotl (Ambystoma mexicanum), with
special references to the olfactory organ (author’s transl). Z Mikrosk Anat Forsch
95(6):883–904
Rinkevich Y et al (2014) Clonal analysis reveals nerve-dependent and independent roles on mam-
malian hind limb tissue maintenance and regeneration. Proc Natl Acad Sci U S A
111(27):9846–9851
Rumyantsev PP (1966) Autoradiographic study on the synthesis of DNA, RNA, and proteins in
normal cardiac muscle cells and those changed by experimental injury. Folia Histochem
Cytochem (Krakow) 4(4):397–424
Sallin P et al (2015) A dual epimorphic and compensatory mode of heart regeneration in zebrafish.
Dev Biol 399(1):27–40
Sandoval-Guzman T et al (2014) Fundamental differences in dedifferentiation and stem cell
recruitment during skeletal muscle regeneration in two salamander species. Cell Stem Cell
14(2):174–187
Satoh A et al (2011) Blastema induction in aneurogenic state and Prrx-1 regulation by MMPs and
FGFs in Ambystoma mexicanum limb regeneration. Dev Biol 355(2):263–274
Scadding SR, Liversage RA (1974) Studies on the response of the adult newt kidney to partial
nephrectomy. Am J Anat 140(3):349–367
Scadding SR, Maden M (1994) Retinoic acid gradients during limb regeneration. Dev Biol
162(2):608–617
Schnapp E et al (2005) Hedgehog signaling controls dorsoventral patterning, blastema cell prolif-
eration and cartilage induction during axolotl tail regeneration. Development (Cambridge,
England) 132(14):3243–3253
Schotté OE, Butler EG (1944) Phases in regeneration of the urodele limb and their dependence
upon the nervous system. J Exp Zool 97(2):95–121
Seifert AW, Maden M (2014) New insights into vertebrate skin regeneration. Int Rev Cell Mol Biol
310:129–169
Seifert AW et al (2012) Skin regeneration in adult axolotls: a blueprint for scar-free healing in
vertebrates. PloS One 7(4):e32875
Shypitsyna A et al (2011) Origin of Nogo-A by domain shuffling in an early jawed vertebrate. Mol
Biol Evol 28(4):1363–1370
Singer M (1952) The influence of the nerve in regeneration of the amphibian extremity. Q Rev Biol
27(2):169–200
Singer M (1964) The trophic quality of the neuron: some theoretical considerations. Prog Brain
Res 13:228–232
246 J.E. Farkas et al.

Singer M, Craven L (1948) The growth and morphogenesis of the regenerating forelimb of adult
Triturus following denervation at various stages of development. J Exp Zool 108(2):279–308
Sirbulescu RF, Zupanc GK (2009) Dynamics of caspase-3-mediated apoptosis during spinal cord
regeneration in the teleost fish, Apteronotus leptorhynchus. Brain Res 1304:14–25
Smith JJ et al (2005a) A comprehensive expressed sequence tag linkage map for tiger salamander
and Mexican axolotl: enabling gene mapping and comparative genomics in ambystoma.
Genetics 171(3):1161–1171
Smith JJ et al (2005b) Sal-site: integrating new and existing ambystomatid salamander research
and informational resources. BMC Genomics 6:181
Smith JJ et al (2009) Genic regions of a large salamander genome contain long introns and novel
genes. BMC Genomics 10:19
Sobkow L et al (2006) A germline GFP transgenic axolotl and its use to track cell fate: dual origin
of the fin mesenchyme during development and the fate of blood cells during regeneration. Dev
Biol 290(2):386–397
Sousa S et al (2011) Differentiated skeletal cells contribute to blastema formation during zebrafish
fin regeneration. Development (Cambridge, England) 138(18):3897–3905
Spalding KL et al (2005) Retrospective birth dating of cells in humans. Cell 122(1):133–143
Spallanzani L (1769) An essay on animal reproductions (Translated from the Italian, 1768, by
M. Maty).T. Becket, London
Stewart R et al (2013) Comparative RNA-seq analysis in the unsequenced axolotl: the oncogene
burst highlights early gene expression in the blastema. PLoS Comput Biol 9(3):e1002936
Stocum DL, Cameron JA (2011) Looking proximally and distally: 100 years of limb regeneration
and beyond. Dev Dyn: An Off Publ Am Assoc Anat 240(5):943–968
Stone LS (1933) The development of lateral-line sense organs in amphibians observed in living
and vital-stained preparations. J Comp Neurol 57(3):507–540
Stone LS (1937) Further experimental studies of the development of lateral-line sense organs in
amphibians observed in living preparations. J Comp Neurol 68(1):83–115
Stone LS (1950) Neural retina degeneration followed by regeneration from surviving retinal pig-
ment cells in grafted adult salamander eyes. Anat Rec 106(1):89–109
Suetsugu-Maki R et al (2012) Lens regeneration in axolotl: new evidence of developmental plas-
ticity. BMC Biol 10:103
Tanaka EM, Ferretti P (2009) Considering the evolution of regeneration in the central nervous
system. Nat Rev Neurosci 10(10):713–723
Tanaka EM et al (1997) Newt myotubes reenter the cell cycle by phosphorylation of the retinoblas-
toma protein. J Cell Biol 136(1):155–165
Tanner K et al (2009) Coherent movement of cell layers during wound healing by image correla-
tion spectroscopy. Biophys J 97(7):2098–2106
Tassava RA, Garling DJ (1979) Regenerative responses in larval axolotl limbs with skin grafts over
the amputation surface. J Exp Zool 208(1):97–110
Tassava RA, Bennett LL, Zitnik GD (1974) DNA synthesis without mitosis in amputated dener-
vated forelimbs of larval axolotls. J Exp Zool 190(1):111–116
Tate JM, Oberpriller JO, Oberpriller JC (1989) Analysis of DNA synthesis in cell cultures of the
adult newt cardiac myocyte. Tissue Cell 21(3):335–342
Thornton CS (1938) The histogenesis of the regenerating fore limb of larval Amblystoma after
exarticulation of the humerus. J Morphol 62(2):219–241
Thornton CS (1960) Influence of an eccentric epidermal cap on limb regeneration in Amblystoma
larvae. Dev Biol 2:551–569
Todd TJ (1823) On the process of reproduction of the members of the aquatic salamander. Q J Sci
Lit Arts 16:84–96
Tomlinson BL, Tassava RA (1987) Dorsal root ganglia grafts stimulate regeneration of denervated
urodele forelimbs: timing of graft implantation with respect to denervation. Development
99(2):173–186
7 Organ and Appendage Regeneration in the Axolotl 247

Tournefier A, Fellah S, Charlemagne J (1988) Monoclonal antibodies to axolotl immunoglobulins

specific for different heavy chains isotypes expressed by independent lymphocyte subpopula-
tions. Immunol Lett 18(2):145–148
Uchida T, Hanaoka K (1949) The occurence of oviform cells by hormonal injection in the regener-
ated testes of a newt. Cytologia 15:109–130
Vargas-Gonzalez A et al (2005) Myocardial regeneration in Ambystoma mexicanum after surgical
injury. Arch Cardiol Mex 75(Suppl 3):S3-21-9
Vethamany-Globus S, Liversage RA (1973) Effects of insulin insufficiency on forelimb and tail
regeneration in adult Diemictylus viridescens. J Embryol Exp Morphol 30(2):427–447
Voss SR, Epperlein HH, Tanaka EM (2009) Ambystoma mexicanum, the axolotl: a versatile
amphibian model for regeneration, development, and evolution studies. Cold Spring Harbor
Protoc 2009(8):pdb.emo128
Voss SR et al (2015) Gene expression during the first 28 days of axolotl limb regeneration I:
experimental design and global analysis of gene expression. Regeneration 2(3):120–136
Wang L, Marchionni MA, Tassava RA (2000) Cloning and neuronal expression of a type III newt
neuregulin and rescue of denervated, nerve-dependent newt limb blastemas by rhGGF2.
J Neurobiol 43(2):150–158
Whited JL, Lehoczky JA, Tabin CJ (2012) Inducible genetic system for the axolotl. Proc Natl Acad
Sci U S A 109(34):13662–13667
Whited JL et al (2013) Pseudotyped retroviruses for infecting axolotl in vivo and in vitro.
Development 140(5):1137–1146
Williams DD (1961) Liver Regeneration in the Newt. Triturus viridescens. Physiol Zool
34(3):256–259
Witman N et al (2011) Recapitulation of developmental cardiogenesis governs the morphological
and functional regeneration of adult newt hearts following injury. Dev Biol 354(1):67–76
Wynn TA (2008) Cellular and molecular mechanisms of fibrosis. J Pathol 214(2):199–210
Xue M, Jackson CJ (2015) Extracellular matrix reorganization during wound healing and its
impact on abnormal scarring. Adv Wound Care (New Rochelle) 4(3):119–136
Yang EV, Bryant SV (1994) Developmental regulation of a matrix metalloproteinase during regen-
eration of axolotl appendages. Dev Biol 166(2):696–703
Yang EV et al (1999) Expression of Mmp-9 and related matrix metalloproteinase genes during
axolotl limb regeneration. Dev Dyn 216(1):2–9
Yannas IV, Colt J, Wai YC (1996) Wound contraction and scar synthesis during development of the
amphibian Rana catesbeiana. Wound Repair Regen 4(1):29–39
Yates CC, Hebda P, Wells A (2012) Skin wound healing and scarring: fetal wounds and regenera-
tive restitution. Birth Defects Res C Embryo Today 96(4):325–333
Yiu G, He Z (2006) Glial inhibition of CNS axon regeneration. Nat Rev Neurosci 7(8):617–627
Yun MH, Davaapil H, Brockes JP (2015) Recurrent turnover of senescent cells during regeneration
of a complex structure. Elife 4:e05505
Zgheib C, Xu J, Liechty KW (2014) Targeting inflammatory cytokines and extracellular matrix
composition to promote wound regeneration. Adv Wound Care (New Rochelle) 3(4):344–355
Zhang P, Wake DB (2009) Higher-level salamander relationships and divergence dates inferred
from complete mitochondrial genomes. Mol Phylogenet Evol 53(2):492–508
Zhang F, Ferretti P, Clarke JD (2003) Recruitment of postmitotic neurons into the regenerating
spinal cord of urodeles. Dev Dyn 226(2):341–348
Zhang R et al (2013) In vivo cardiac reprogramming contributes to zebrafish heart regeneration.
Nature 498(7455):497–501
Zhu W et al (2012) Activation of germline-specific genes is required for limb regeneration in the
Mexican axolotl. Dev Biol 370(1):42–51
Ziegels J (1971) The melanocytes of the axolotl. Their modifications during skin regeneration.
Arch Biol (Liege) 82(3):407–428
Zukor KA, Kent DT, Odelberg SJ (2011) Meningeal cells and glia establish a permissive environ-
ment for axon regeneration after spinal cord injury in newts. Neural Dev 6(1):1