Matrix Metalloproteinases Expression during Limb Regeneration

poster abstractAxolotl (regeneration-competent) is one of the unique vertebrates which can regenerate missing organs such as limbs, jaws, spinal cord, and tail anytime during their life cycle. There also exists a recessive mutant of axolotl which has a phenotype called short toes (s/s, regenerationdeficient). The s/s mutant can regenerate its tail and spinal cord but cannot maintain the growth of the limb blastema, which results in the failure of limb regeneration. Remodeling of extracellular matrix (ECM) during early blastema formation, also known as histolysis, leads to the release of stem cells and activation of various growth factors. Therefore, histolysis is considered to be a crucial step in regenerating the exact replica of missing limbs in axolotls. Matrix metalloproteinases (MMPs) are zinc dependent endopeptidase that have been suggested to play roles in histolysis. However, it still remains unclear if histolysis is different in limb regeneration between regeneration competent and deficient animals. In this study, we analyzed the expression patterns of MMPs and the tissue inhibitors of the MMPs (TIMPs) in axolotl and s/s utilizing MMP arrays (RayBiotech, Inc., Norcross, GA), zymography and western blots. The cut-off limbs of the axolotls and s/s were used as controls. The animals were allowed to regenerate and the blastema was collected at three stages: epidermis closure (EC), dedifferentiation (DD), and early bud (EB). The total proteins were extracted from all the samples. 20 μg of protein was used to perform MMP arrays according to manufacturer’s protocol. They detected MMP-1, -2, -3, -8, -9, -10, and -13, as well as TIMP-1, -2 and -4 in the controls, EC, DD and EB samples from axolotl and s/s. Gelatin zymograghy with 20 μg of protein confirmed that MMP-2 and -9 were expressed at all the same time points in the axolotl and s/s samples. The expression patterns of MMP-9 were similar in the axolotl and s/s until the DD stage. While later in the EB stage, the axolotl showed a decrease in MMP-9 expression and s/s showed increased expression. Western blots were performed with 40 μg of protein using MMP-2 and -9 antibodies, and confirmed the zymography results. These results suggested that the expression patterns of the MMPs, especially MMP-9, are different in regeneration competent and deficient animals. One of the keys for a healthy blastema formation, which can multiply and later repattern into the missing limb, might be the release of the critical amount of MMP at the right time. This study was supported by an IUSD start-up grant to F. Song and a grant from W. M. Keck Foundation to D. L. Stocum

Solution Structure and Phylogenetics of Prod1, a Member of the Three-Finger Protein Superfamily Implicated in Salamander Limb Regeneration

Prod1 is a cell-surface molecule of the three-finger protein (TFP) superfamily involved in the specification of newt limb PD identity. The TFP superfamily is a highly diverse group of metazoan proteins that includes snake venom toxins, mammalian transmembrane receptors and miscellaneous signaling molecules..The available data suggest that Prod1, and thereby its role in encoding PD identity, is restricted to salamanders. The lack of comparable limb-regenerative capability in other adult vertebrates could be correlated with the absence of the Prod1 gene

The Homeobox Transcription Factor Barx2 Regulates Plasticity of Young Primary Myofibers

Adult mammalian muscle retains incredible plasticity. Muscle growth and repair involves the activation of undifferentiated myogenic precursors called satellite cells. In some circumstances, it has been proposed that existing myofibers may also cleave and produce a pool of proliferative cells that can re-differentiate into new fibers. Such myofiber dedifferentiation has been observed in the salamander blastema where it may occur in parallel with satellite cell activation. Moreover, ectopic expression of the homeodomain transcription factor Msx1 in differentiated C2C12 myotubes has been shown to induce their dedifferentiation. While it remains unclear whether dedifferentiation and redifferentiaton occurs endogenously in mammalian muscle, there is considerable interest in induced dedifferentiation as a possible regenerative tool.We previously showed that the homeobox protein Barx2 promotes myoblast differentiation. Here we report that ectopic expression of Barx2 in young immature myotubes derived from cell lines and primary mouse myoblasts, caused cleavage of the syncytium and downregulation of differentiation markers. Microinjection of Barx2 cDNA into immature myotubes derived from primary cells led to cleavage and formation of mononucleated cells that were able to proliferate. However, injection of Barx2 cDNA into mature myotubes did not cause cleavage. Barx2 expression in C2C12 myotubes increased the expression of cyclin D1, which may promote cell cycle re-entry. We also observed differential muscle gene regulation by Barx2 at early and late stages of muscle differentiation which may be due to differential recruitment of transcriptional activator or repressor complexes to muscle specific genes by Barx2.We show that Barx2 regulates plasticity of immature myofibers and might act as a molecular switch controlling cell differentiation and proliferation

Proteomes and Signalling Pathways of Antler Stem Cells

As the only known example of complete organ regeneration in mammals, deer antler in the growing or velvet phase is of major interest in developmental biology. This regeneration event initiates from self-renewing antler stem cells that exhibit pluripotency. At present, it remains unclear how the activation and quiescence of antler stem cells are regulated. Therefore, in the present study proteins that were differentially expressed between the antler stem cells and somatic cells (facial periosteum) were identified by a gel-based proteomic technique, and analysed using Ingenuity Pathway Analysis software. Several molecular pathways (PI3K/Akt, ERK/MAPK, p38 MAPK, etc.) were found to be activated during proliferation. Also expressed were the transcription factors POU5F1, SOX2, NANOG and MYC, which are key markers of embryonic stem cells. Expression of these proteins was confirmed in both cultured cells and fresh tissues by Western blot analysis. Therefore, the molecular pathways and transcription factors identified in the current study are common to embryonic and adult stem cells. However, expression of embryonic stem cell transcription factors would suggest that antler stem cells are, potentially, an intermediary stem cell type between embryonic and the more specialized tissue-specific stem cells like those residing in muscle, fat or from a hematopoietic origin. The retention of this embryonic, pluripotent lineage may be of fundamental importance for the subsequent regenerative capacity of antlers