290
Xenopus
order of urodeles and anurans (e.g. Xenopus laevis), have
been among the favorite models of choice to study regeneration since the beginnings of experimental biology (Freitas et
al. 2019). Lorenzo Spallanzani demonstrated that the tail of
salamanders, including its spinal cord, grows back after tail
amputation (Spallanzani 1768). A century later, George H.
Lewes demonstrated that anurans also were able to regenerate the spinal cord (Lewes 1859), followed by the important fnding that the regenerative capability decreased with
tadpole development (Hooker 1925), which was later corroborated with modern approaches (Sims 1962; Filoni et al.
1984 ).
During the second half of the 20th century, taking advantage of the progress of microscopic techniques, a more
detailed description of spinal cord regeneration process was
attained in salamanders, axolotl, and frogs, mainly Xenopus.
One set of observations demonstrated that, in contrast to
mammals, axonal outgrowth is an important component of
spinal cord reconstitution. Axonal growth from severed neurons rostral and caudal to the injury site was demonstrated
in salamanders (Piatt 1955), although it can take as long as
23 months to recover the number of axons observed in the
uninjured animals (Clarke et al. 1988; Davis et al. 1989).
Similarly, in X. laevis tadpoles, axons growing through the
injury site was demonstrated (Sims 1962; Filoni et al. 1984).
Studies in Rana catesbiana and X. laevis tadpoles showed
descending axonal projections that regenerate and cross the
injury site, but regeneration of ascending (sensory) axons
was not observed at any stage (Forehand and Farel 1982;
Beattie et al. 1990). The absence of regeneration of ascending axons was also reported in newts ( Zukor et al. 2011).
In line with the absence of functional recovery, the axonal
regenerative capacity in X. laevis was no longer observed in
animals transected after tail resorption (Beattie et al. 1990).
A second group of responses triggered by SCI, revealed
by descriptive studies in salamanders, is the activation of
ependymoglial cell proliferation. Ependymoglial cells are
probably the equivalent to mammalian radial glial cells and
neural stem cells (NSCs) and are found lining the central
canal of the spinal cord of salamanders (Freitas et al. 2019).
These proliferative ependymoglial cells (Stefanelli 1951)
lead to neurogenesis, which seems to be a necessary process
for the reconstruction of the nervous tissue (Butler and Ward
1965; Benraiss et al. 1999). In addition, the basal processes
of these cells build an ependymoglial tube or bridge in the
injury site, providing a pathway and permissive substrate for
axonal regeneration (Egar and Singer 1972). Similarly, after
spinal cord transection in X. laevis, axons grow through the
ablation gap in association with a bridge of ependymal processes (Michel and Reier 1979; Filoni et al. 1984). These
observations show that the environment is permissive for
axon growth, probably due to the absence of a glial scar, in
salamanders and in X. laevis R-stages.
The advances from the last century established a very
detailed description of the response to SCI in urodeles and
anurans but failed to provide a mechanistic explanation of
the process. In summary, they described the presence of
active axon regeneration, proliferation of putative NSPCs
that give rise to new neurons, and presence of a regenerative-permissive environment, a response that is lost with
metamorphosis in the case of X. laevis.
20.3. PRESENT STATUS OF THE FIELD
20.3.1. X. LAEVIS: A MODEL ORGANISM TO
STUDY SPINAL CORD REGENERATION
As indicated previously, one of the great advantages of
X. laevis as a model organism to study spinal cord regeneration is the possibility to perform experiments in R- and
NR-stages. This comparison allows the identif cation of the
cellular, molecular, and genetic mechanisms involved in
regeneration and those that are responsible for the loss of
the regenerative capacities (Gaete et al. 2012; Lee-Liu et al.
2014; Muñoz et al. 2015; Lee-Liu et al. 2018). In addition,
the NR-stages provide an experimental paradigm to test new
therapies such as genetic manipulation and pharmacological treatments that could enhance the regenerative capacities
(Phipps et al. 2020).
Many methodological developments make X. laevis an
amenable system to perform functional studies (Harland and
Grainger 2011), including the generation of transgenic lines
(Amaya and Kroll 1999); mutagenesis using CRISPR and
TALEN systems (Nakajima and Yaoita 2015; Nakayama et
al. 2020); injection and electroporation of DNA, mRNA, or
morpholinos (Blum et al. 2015; Gomez et al. 2003; Bestman
et al. 2006; Eide et al. 2000); and intracoelomic injection
of drugs (Edwards-Faret et al. 2017). Additionally, standardized protocols for optimized husbandry have been
established, allowing the acquisition of a large number of
regenerative tadpoles in three weeks and non-regenerative
froglets in two months, making them easily accessible for
the isolation of material and to have enough animals for statistically robust experimental procedures (Edwards-Faret et
al. 2017).
The study of spinal cord regeneration in frogs can be
approached using two main types of injury methods: tail
amputation and spinal cord transection or resection. Tail
amputation removes the entire tail, including tissues such
as muscle, notochord, and spinal cord, followed by their
regeneration (Beck et al. 2003). The fnding that there is a
refractory period (NF stage 46–48) during which tail regeneration does not occur, and instead it only heals, has been
very useful to understand tail, including spinal cord, regeneration (Slack et al. 2008). Using this injury method, it has
been demonstrated that cellular dedifferentiation does not
occur; instead, the spinal cord and notochord are regenerated from their corresponding pre-existing tissue in the tail
stump, indicating that cell-lineage restriction is maintained
during this process (Gargioli and Slack 2004). Similarly, the
regenerated muscle originates from skeletal muscle satellite cells (Gargioli and Slack 2004), and melanophores arise
from melanophore precursors present in the tail (Lin et al.
2007). In addition, tail amputation has been a fertile ground
Xenopus
order of urodeles and anurans (e.g. Xenopus laevis), have
been among the favorite models of choice to study regeneration since the beginnings of experimental biology (Freitas et
al. 2019). Lorenzo Spallanzani demonstrated that the tail of
salamanders, including its spinal cord, grows back after tail
amputation (Spallanzani 1768). A century later, George H.
Lewes demonstrated that anurans also were able to regenerate the spinal cord (Lewes 1859), followed by the important fnding that the regenerative capability decreased with
tadpole development (Hooker 1925), which was later corroborated with modern approaches (Sims 1962; Filoni et al.
1984 ).
During the second half of the 20th century, taking advantage of the progress of microscopic techniques, a more
detailed description of spinal cord regeneration process was
attained in salamanders, axolotl, and frogs, mainly Xenopus.
One set of observations demonstrated that, in contrast to
mammals, axonal outgrowth is an important component of
spinal cord reconstitution. Axonal growth from severed neurons rostral and caudal to the injury site was demonstrated
in salamanders (Piatt 1955), although it can take as long as
23 months to recover the number of axons observed in the
uninjured animals (Clarke et al. 1988; Davis et al. 1989).
Similarly, in X. laevis tadpoles, axons growing through the
injury site was demonstrated (Sims 1962; Filoni et al. 1984).
Studies in Rana catesbiana and X. laevis tadpoles showed
descending axonal projections that regenerate and cross the
injury site, but regeneration of ascending (sensory) axons
was not observed at any stage (Forehand and Farel 1982;
Beattie et al. 1990). The absence of regeneration of ascending axons was also reported in newts ( Zukor et al. 2011).
In line with the absence of functional recovery, the axonal
regenerative capacity in X. laevis was no longer observed in
animals transected after tail resorption (Beattie et al. 1990).
A second group of responses triggered by SCI, revealed
by descriptive studies in salamanders, is the activation of
ependymoglial cell proliferation. Ependymoglial cells are
probably the equivalent to mammalian radial glial cells and
neural stem cells (NSCs) and are found lining the central
canal of the spinal cord of salamanders (Freitas et al. 2019).
These proliferative ependymoglial cells (Stefanelli 1951)
lead to neurogenesis, which seems to be a necessary process
for the reconstruction of the nervous tissue (Butler and Ward
1965; Benraiss et al. 1999). In addition, the basal processes
of these cells build an ependymoglial tube or bridge in the
injury site, providing a pathway and permissive substrate for
axonal regeneration (Egar and Singer 1972). Similarly, after
spinal cord transection in X. laevis, axons grow through the
ablation gap in association with a bridge of ependymal processes (Michel and Reier 1979; Filoni et al. 1984). These
observations show that the environment is permissive for
axon growth, probably due to the absence of a glial scar, in
salamanders and in X. laevis R-stages.
The advances from the last century established a very
detailed description of the response to SCI in urodeles and
anurans but failed to provide a mechanistic explanation of
the process. In summary, they described the presence of
active axon regeneration, proliferation of putative NSPCs
that give rise to new neurons, and presence of a regenerative-permissive environment, a response that is lost with
metamorphosis in the case of X. laevis.
20.3. PRESENT STATUS OF THE FIELD
20.3.1. X. LAEVIS: A MODEL ORGANISM TO
STUDY SPINAL CORD REGENERATION
As indicated previously, one of the great advantages of
X. laevis as a model organism to study spinal cord regeneration is the possibility to perform experiments in R- and
NR-stages. This comparison allows the identif cation of the
cellular, molecular, and genetic mechanisms involved in
regeneration and those that are responsible for the loss of
the regenerative capacities (Gaete et al. 2012; Lee-Liu et al.
2014; Muñoz et al. 2015; Lee-Liu et al. 2018). In addition,
the NR-stages provide an experimental paradigm to test new
therapies such as genetic manipulation and pharmacological treatments that could enhance the regenerative capacities
(Phipps et al. 2020).
Many methodological developments make X. laevis an
amenable system to perform functional studies (Harland and
Grainger 2011), including the generation of transgenic lines
(Amaya and Kroll 1999); mutagenesis using CRISPR and
TALEN systems (Nakajima and Yaoita 2015; Nakayama et
al. 2020); injection and electroporation of DNA, mRNA, or
morpholinos (Blum et al. 2015; Gomez et al. 2003; Bestman
et al. 2006; Eide et al. 2000); and intracoelomic injection
of drugs (Edwards-Faret et al. 2017). Additionally, standardized protocols for optimized husbandry have been
established, allowing the acquisition of a large number of
regenerative tadpoles in three weeks and non-regenerative
froglets in two months, making them easily accessible for
the isolation of material and to have enough animals for statistically robust experimental procedures (Edwards-Faret et
al. 2017).
The study of spinal cord regeneration in frogs can be
approached using two main types of injury methods: tail
amputation and spinal cord transection or resection. Tail
amputation removes the entire tail, including tissues such
as muscle, notochord, and spinal cord, followed by their
regeneration (Beck et al. 2003). The fnding that there is a
refractory period (NF stage 46–48) during which tail regeneration does not occur, and instead it only heals, has been
very useful to understand tail, including spinal cord, regeneration (Slack et al. 2008). Using this injury method, it has
been demonstrated that cellular dedifferentiation does not
occur; instead, the spinal cord and notochord are regenerated from their corresponding pre-existing tissue in the tail
stump, indicating that cell-lineage restriction is maintained
during this process (Gargioli and Slack 2004). Similarly, the
regenerated muscle originates from skeletal muscle satellite cells (Gargioli and Slack 2004), and melanophores arise
from melanophore precursors present in the tail (Lin et al.
2007). In addition, tail amputation has been a fertile ground