After amputation of an appendage in certain salamanders and fish, new cartilage or bone structures of correct size and pattern emerge from a mound of proliferative tissue called the blastema (Brockes and Kumar, 2005
). A major objective in the field has been to define the cellular source(s) of regenerated skeletal elements. This includes identifying cell types within the appendage stump that normally give rise to regenerated cartilage or bone after amputation, as well as identifying cells that have the developmental capacity to create skeleton under additional conditions (Poss, 2010
; Tanaka and Reddien, 2011
). Proposed sources are the differentiated chondrocytes and osteoblasts themselves, or non-skeletal cells that undergo new differentiation or trans-differentiation events after amputation.
Grafting experiments in amphibians performed over the past century have attempted to resolve this issue. Surgical transplantation of dissected cartilage or bone indicated that skeletal tissues wholly or predominantly contribute like tissue, suggesting that lineage is restricted throughout blastema formation and patterning (Namenwirth, 1974
; Steen, 1968
; Steen, 1970
). Yet, other experiments, including the transplantation of dye-labeled muscle cells to limb blastemas or non-skeletal tissue to irradiated limbs, indicated that additional cell types may act as progenitors for bone or cartilage (Lo et al., 1993
; Morrison et al., 2006
). A recent study of axolotl limb regeneration examined the contributions of tissues grafted from transgenic animals constitutively expressing a fluorescent reporter protein. These experiments generated the prevailing model for axolotl limb cartilage regeneration, which is that cartilage cells predominantly contribute like tissue, while one or more cell populations within the dermis also has the potential to form cartilage (Kragl et al., 2009
Tissue grafts can be ineffective at resolving certain key questions of tissue origin, such as: 1) how host tissue naturally participates in regeneration; 2) the extent to which specific cell types contribute during regeneration; and 3) whether cells in the stump undergo developmental changes like de-differentiation in the process of creating new structures. Very recently, three studies examined similar questions during fin regeneration in zebrafish by genetic lineage-tracing of specific cell types. Adult zebrafish fins contain several cylinder-shaped, segmented bony fin rays that are lined by osteoblasts and encase fibroblasts, blood vessels, nerves, and pigment cells. By inducible fate-mapping of cells expressing the intermediate osteoblast marker osterix
, Knopf and colleagues found that existing osteoblasts undergo partial de-differentiation, as defined by reduced expression of osteoblast markers, after which they proliferate and contribute solely to regenerated bone structures (Knopf et al., 2011
). Tu and Johnson assessed the mosaicism of transgenes injected into embryos during rapid cell division, and found that transgenic clones containing labeled osteoblasts within regenerated fins do not possess other cell types (Tu and Johnson, 2011
). Sousa and colleagues used live imaging of labeled osteocalcin
-expressing cells to indicate contribution of differentiated osteoblasts to the regenerate (Sousa et al., 2011
). Together, these studies supported a common conception that osteoblasts in the regenerate derive predominantly or wholly from the de-differentiation, proliferation, and migration of lineage-restricted stump osteoblasts.
Here, by creating a system to facilitate inducible ablation of resident osteoblasts in adult fins, we examined the extent to which zebrafish fin regeneration is dependent on these cells. We found by lineage-tracing of existing osteoblasts that they are restricted to contributing like cells during regeneration, in agreement with recent published work. Unexpectedly however, ablation of ostensibly all osteoblasts prior to amputation did not slow down the rate of zebrafish fin regeneration. Instead, new osteoblasts arose from cells that differentiated de novo after amputation, a result confirmed by genetic fate-mapping. Our findings indicate that stump osteoblasts are a dispensable source for regenerating appendage bone, and provide important new context for understanding mechanisms of robust skeletal regeneration.