Browsing by Subject "Limb Regeneration"

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    Amphibian Limb Regeneration and Cell Cycle Regulation
    (Office of the Vice Chancellor for Research, 2013-04-05) Ziliak, Tyler
    Partial or full regeneration of limbs is an exclusive capability owned by a few amphibians, and this occurrence is often a potent factor to the survival of a species or its ability to thrive in an environment. A restricted ability, a greater understanding would have a profound impact and clear applications in the treating of human diseases, ailments, and injuries. Further grasping the mechanisms behind natural regeneration and its occurrence at different stages and under different stimuli may allow for mimicry in the influence of tissues for human benefit. In an antecedent study, it was observed that ecotropic viral integrative factor 5 (EVI5) was perpetuating itself at a greater rate in sample tissues of the regenerationcompetent axolotl salamander (Ambystoma mexicanum) as opposed to control tissue where this increase was null. EVI5 arrests the cells after Synthesis phase and G2 phase before the cells enter into Mitosis until prospective cells dedifferentiate and there is a an established blastema-the instrument by which regeneration continues. This study wished to show what proteins are expressed in the blastema tissue at these stages and to ascertain these previous findings. Samples of the axolotl were taken at 1, 4, and 7 days post-amputation where the amputation was made either midway or two-thirds towards the distal end of the tibia-fibula pairing. Samples were embedded and cut into sections with a cryostat at approximately -20 degrees Celsius. Immunofluorescent staining was utilized with EVI5 as primary antibody for the target site and anti-goat as the second antibody; in addition, H&E (hematoxylin and eosin) staining was employed to more definitively identify the nuclear structures of the cut and stained sections. Slides were observed under microscope to decipher protein expression and compare results. Positive identification of EVI5 reinforces its importance in delaying Mitosis so that a blastema can form and regeneration can occur.
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    Matrix Metalloproteinases Expression during Limb Regeneration
    (Office of the Vice Chancellor for Research, 2010-04-09) SANTOSH, N.; MAHMOUDI, B. S.; WINDSOR, L. J.; STOCUM, D. L.; SONG, F.
    Axolotl (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.
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    Regulation of EVI5, VEGF and P53bp2 during Amphibian Limb Regeneration
    (Office of the Vice Chancellor for Research, 2013-04-05) Elkhatib, Wiaam
    Understanding limb regeneration on a molecular level could lead to new methods of healing for humans, therefore revolutionizing current medical treatments. The axolotl salamander possesses capabilities of limb regeneration that are lost in the Xenopus laevis froglet. The hypothesized reason is that elevated levels of EVI5 (ecotropic viral integration site 5) binding protein allow the axolotl to regenerate by delaying the mitosis of dedifferentiated cells until they have established a blastema. VEGF (vascular endothelial growth factor) and P53bp2 (tumor protein 53 binding protein 2) genes also take part in this process by stimulating blood vessel formation and regulating apoptosis and cell growth in regenerated tissue. The objective of this study is to clone EVI5, VEGF, and P53BP2 cDNA that can be used to detect their mRNA transcripts during limb regeneration in the axolotl and Xenopus laevis. To accomplish this, RNA is extracted from axolotl and Xenopus laevis limb tissue using an RNeasy kit. Total RNA concentration is then measured spectrophotometrically. RT-PCR (reverse transcription polymerase chain reaction) is used to clone the cDNAs, which are identified by Agarose gel electrophoresis and later sequenced for verification. It took half a year to get high enough RNA concentrations from both species’ tissues and then clone the three genes. The EVI5 band size was determined to be about 200bps, VEGF about 370bps, and P53bp2 about 500bps using the Agarose gel electrophoresis, signifying successful gene cloning. The long-term goal is to determine the role these genes play in limb regeneration with the aim of applying that knowledge to new medical treatments.