Caudal Fin Regeneration in Zebrafish - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 299

22.4 STAGES OF EPIMORPHIC REGENERATION

Upon caudal fin amputation, an impressive series of regenerative stages are initiated
that result in the complete restoration of lost bone, epidermis, blood vessels, nerves,
connective tissue, and pigmentation (Fig. 22.1). This complex process, termed
epimorphic regeneration,

is completed in approximately 3 days in larval fish

(Fig. 22.2) and 2 weeks in adult fish (reviewed in Lovine, 2007). Epimorphic

regeneration is a term used to describe a regenerative process involving the formation
of a mass of undifferentiated proliferative multipotent mesenchymal cells called a
blastema (Akimenko et al., 2003). In the adult model, an initial wound healing stage,
characterized by nonproliferative lateral epithelial cell migration over the wound and
subsequent formation of the apical epidermal cap (AEC), is initiated immediately
following surgical removal of caudal fin tissue (Nechiporuk and Keating, 2002).
Second, the wound epithelium thickens and mesenchymal tissue proximal to the
amputation plane begins to disorganize. Cellular disorganization is thought to occur
as a result of growth factors that originate from the mature wound epidermis and
stimulate mesenchymal cells to dedifferentiate and proliferate as theymigrate distally
toward the area directly proximal to the AEC (Nechiporuk and Keating, 2002). In the
third stage, a series of blastemas form at the severed portion of each amputated fin ray.
Blastema formation is the main event that distinguishes regeneration from limb
development. The blastema is an accumulated mass of progenitor cells that are
thought to be pluripotent or able to produce daughter cells capable of differentiating
into a variety of cell types required to populate the regenerating tissue. Although
pluripotency is inferred from aforementioned migratory and proliferative qualities,
lineage tracing studies have not yet been completed. Therefore, it is possible that
resident stem cells or some combination of mesenchymal dedifferentiation and stem
cell proliferation gives rise to new cells in the regenerating tissue.
The immature blastema proliferates slowly with a median G2 cell cycle time of

6 h (Nechiporuk and Keating, 2002). Twenty-four hours following caudal fin amputa-
tion, blastemal cells segregate into two morphologically identical, but functionally
distinct, subpopulations. The distal blastema, located proximally to the AEC, contains
msxb positive cells, proliferates extremely slowly, and is hypothesized to specify the
regenerating boundary and direct regenerative growth (Nechiporuk and Keating, 2002).
In sharp contrast, the proximal blastema contains a rapidly proliferative mass of msxb
negative cells with a mean G2 time of

>

60min. Together, the proximal and distal
blastemas form a proliferation gradient with a 50-fold difference in proliferation across
an approximate distance of 50

<

Mor 10 cell diameters (Nechiporuk andKeating, 2002).
The next phase of regenerative outgrowth is marked by intense proliferation in the
proximal blastema. A moderately proliferative patterning zone is located immediately
proximal to the proximal blastema. The patterning zone contains newly divided cells
that migrate to appropriate locations and differentiate to populate the new tissue with
mesenchymal cells and fibroblasts. The location and functional differences between
the distal and proximal blastema and patterning zone are thought to be generated
and maintained through epithelial-mesenchymal interactions (Poss et al., 2003;
Lee et al., 2009). The final stage, regenerative termination, is not well understood.

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