89epidermis, (2) endothelial cells contribute only to the vasculature of the regenerate,
(3) osteoblasts precursors of the limb give rise only to regenerated bone, and (4) as
a negative result, transplanted hematopoietic stem cells do not contribute to the
regenerate. These fi nding are consistent with the conclusion that cells involved in
the regeneration of the urodele limb are lineage-restricted [ 29 ], so it is tempting to
draw the general conclusion that the regeneration process does not involve the re-
programming of cell types [ 18 , 27 ]. Yet, it is important to point out that a number of
cell types have yet to be carefully tested and there is considerable evidence that
support the participation of multipotent progenitor cells in other injury repair mod-
els [ 30 ]. The question of whether progenitor cells arise from a population of stem
cells versus the de-differentiation of mature cells has yet to be addressed in this
mammalian regenerative response.
The second critical question to address involves how cells are recruited to formthe blastema at the amputation wound site. During regeneration, cells migrate from
different regions of the amputation wound to form the centrally located blastema
[ 31 , 32 ]. Previous studies have implicated Stromal Cell Derived Factor-1 (SDF-
- / CXCR4 signaling in the cellular recruitment to sites of BMP2 -induced ectopic
bone formation, bone repair, and zebrafi sh fi n regeneration [ 33 – 35 ]. A comprehen-
sive study investigating the role of SDF- 1 / CXCR4 signaling in blastema formation
and digit regeneration was carried out [ 36 ] and a summary of this study follows.
Immunohistochemical and in situ hybridization studies focused on the regenerating
digit identifi ed cells expressing SDF- 1 in the blastema, wound epidermis, and bone
marrow vasculature, and other cells expressing CXCR4 in the wound epidermis and
within the blastema (Fig. 5.4a–d ). Immunostaining for Phospho-CXCR4, used to
identify the SDF-1 mediated activation of CXCR4, showed positive signal localized
to the wound epidermis and the vasculature proximal to the blastema (Fig. 5.4e ). In
line with this, CD31+ endothelial cells within the blastema tested immunopositive
for SDF- 1 , suggesting endothelial cells may function in cellular recruitment during
regeneration (Fig. 5.4f ). Primary cultures of blastema cells express CXCR4 and
display a dose-dependent response to SDF1 in transwell migration assays that is
inhibited by AMD3100, a known antagonist for CXCR4 signaling. To test the
in vivo role of SDF-1/CXCR4 signaling in neonatal digit regeneration, systemic
injections of AMD3100 resulted in a signifi cant attenuation in regenerated bone
length compared to vehicle control treated digits (Fig. 5.4g ). Importantly, no differ-
ence was found in bone length of the developing P3 digit after AMD3100 systemic
treatment, providing evidence that SDF- 1 / CXCR4 signaling is specifi c to the regen-
eration response. The BMP2 -induced regeneration response was used to provide a
gain of function test for a role of SDF1 recruitment in regeneration. First, introduc-
ing labeled blastema cells in conjunction with a BMP2 bead resulted in a chemotactic
response with labeled cells aggregating around the BMP2 source. Transwell migra-
tion assays demonstrated that BMP2 itself does not infl uence blastema cell migra-
tion indicating that the in vivo response was indirect. BMP2 treatment of P2
amputations was found to induce both SDF- 1 and CXCR4 expression and SDF-1
expression co-localized with endothelial cells in the amputation wound (Fig. 5.4h–
m ). A BMP2/SDF-1 link was confi rmed in vitro by studies showing that BMP2
5 Digit Regeneration in Mammals