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The present thesis is a contribution to unravel the molecular mechanisms that underlie urodele regeneration. Urodele amphibians (newts and salamanders) are among the few vertebrates with the remarkable ability to regenerate lost body appendages, like the limbs and the tail. Urodele tail and limb regeneration occurs via blastemal epimorphic regeneration. A blastema is a mound of progenitor cells that accumulates at the amputation plane and eventually gives rise to the missing structures. It is known today that dedifferentiating muscle fibers at the amputation plane contribute to the blastema cell pool, but how this process occurs on the cellular and molecular level is hardly understood, which is in part due to the lack of molecular methods to test gene function in urodeles. Furthermore, little is known about how coordinated growth and patterning occurs during urodele regeneration, and if the patterning mechanisms in regeneration are related to the ones in development. The goal of this study was to better understand these processes on the molecular level. To address these questions, I first established several methods in our model systems, which are the mexican salamander Ambystoma mexicanum (axolotl) and a cell line derived from the newt Notophthalmus viridescens. In order to monitor gene expression on a cellular level during regeneration, I worked out a good in situ hybridization protocol on axolotl tissue cryosections. To be able to test gene function, I established electroporation conditions to both overexpress genes in the cultured newt cells and to deliver morpholinos into axolotl cells in vivo and newt cells in culture. I demonstrate here that morpholinos are an effective tool to downregulate protein expression in urodele cells in vivo and in culture. Testing the role of two candidate genes in muscle fiber dedifferentiation, the homeobox containing transcription factor Msx1 and Rad, a GTP-binding protein of a new Ras-related protein family, revealed that neither seems to play a major role in muscle dedifferentiation, both in culture and in vivo. In addition to testing gene function I have examined the muscle dedifferentiation process in more detail. I show here that dedifferentiating muscle fiber nuclei undergo morphological changes that are likely due to chromatin remodeling events. I also demonstrate that the axolotl spinal cord expresses embryonic dorsoventral (d/v) patterning markers of the neural tube. The transcription factors Msx1, Pax7 and Pax6 are expressed in their respective d/v domains in both the differentiated and the regenerating axolotl spinal cord. Furthermore, the secreted signaling molecule sonic hedgehog (Shh) is expressed in the floor plate in both the differentiated and the regenerating cord. Using a chemical inhibitor (cyclopamine) and an activator of the hedgehog pathway, I discovered that hedgehog signaling is required for overall tail regeneration. Blocking hedgehog signaling does not only result in d/v patterning defects of the regenerating spinal cord, but it also strongly reduces blastema cell proliferation. In addition, I identified cartilage and putative muscle progenitor cells in the blastema, marked by the expression of the transcription factors Sox9 and Pax7, respectively. Both progenitor populations are reduced in the blastema in the absence of hedgehog signaling. The continuous expression of marker genes for embryonic progenitor cell domains in the mature axolotl may be related to their ability to regenerate.

The establishment of transgenesisi in axolotls is crucial for studying development and regeneration, as it would allow for long-term fate tracing as well as gene expression analysis, therefore we were interested in both obtaining animals expresing the transgene with little mosaicism in F0 generation and transgenesis. We demonstrate here that plasmid injection into one cell stage axolotl embryo generates transgenic animals that display germline transmission of a transgene. However, the efficiency of simple plasmid injection is very low, expression of the transgene is mosaic and seems to be promoter dependant. We have tested several methods of transgenesis developed in other systems. First we used Adeno-Associated Viral Terminal Repeats inserted into the injected construct to enhance the expression level of the transgene and reduce mosaicism. However, in the axolotl system we do not observe the enhancement of expression. Moreover, the expression appeared to be transient and disappeared after two months. Further, we tested the effect of the inclusion of ISceI meganuclease in the injections, succesful transgenesis method in the medaka system. It resulted in a higher percentage of F0 animals displaying strong , stable expression throughout the body. This represents the first demonstration in the axolotl of germline transmission of the transgene. Using this technique we have generated a germline transgenic anima expressing GFP ubiquitously in all tissue examined. We have used this anima to study cell fate in the dirsal fin during development. We have discovered a contribution of somite cells to dorsal fin mesenchyme in the axolotl, which was previously assumed to derive solely from neural crest. We have also studied the role of blood during tail regeneration by transplanting the ventral blood-forming region from GFP+ embryos into unlabeled host. During tail regeneration, we do not observe GFP+ cells contributing to muscle or nerve, suggesting that during tail regeneration blood stem cells do not undergo significant plasticity. We are interested in characterization of pluripotency of blastema cells. Previously, it has been shown that neural progenitor cells form the spinal cord can transdifferentiate to muscle and other tissue types in the regenerating tail. To test if blastema cells have the potency of differentiating into a neural tissue , we transplanted GFP+ 4day blastema into an injured spinal cord. Our result shows that blastema cells don't seem to contribute to the regenerating spinal cord.

[Truncated abstract] Developmentally, the rodent retinocollicular projection is often thought of as a homogenous projection of retinal ganglion cell (RGC) axons, however the extensive period of RGC neurogenesis and sequential arrival of their axons into central targets such as the superior colliulus (SC) suggests otherwise. RGC axons are already present in the developing SC at embryonic (E) day 16.5-17. RGCs born on E15 have innervated the SC by birth, whereas axons derived from RGCs that are born last (E19) do not grow into the SC until postnatal (P) days 4-6 (Dallimore et al., 2002). These observations may go someway to explaining why, after SC lesions in rats at P2, there is greater growth distal to the lesion site compared to lesions made at P6 (Tan and Harvey, 1997b). It may be that the post lesion growth is simply de novo growth of axons from late-born RGCs rather than regeneration of pre-existing, injured axons. Early and late cohorts of growing RGC axons presumably encounter different developmental terrains as they grow from retina to central targets, possibly resulting in differences in developmental milestones and growth potentials. There may also be differences in guidance cues, further suggesting that gene expression in early vs late born RGCs may differ. To examine differences between early (E15) and late (E19) born RGCs during development, the time-course and extent of programmed RGC death in normal rat pups, and RGC death following the removal of target-derived trophic factors, was assessed. ... On the other hand, LCM captured GCL analysed for gene expression at P0 and P7 revealed decreases in AKT, Math5, Notch1, c-jun, DCC, Arginase-1 mRNA levels and a considerable decrease in GAP-43 expression. It is not surprising to see differences in gene expression between whole eye and the more specific GCL samples, as the cells in all layers of the retina have very different functions and different developmental profiles. It is important to note decreases in mRNA expression in the GCL for a number of the genes analysed at P0 and P7, reflecting cessation of RGC death and completion of axonal growth into central visual targets. I also examined at the protein level expression of DCC, Arginase1, c-Jun and Bcl-2 at birth (P0) in BrdU labeled RGCs born on E15 or E19. When comparing the percentage of double labelled cells compared to the total number of cells expressing each protein, Bcl-2, c-Jun and Arg1 were expressed more in E15 RGCs (22.90%, 72.71%, and 16.44% respectively in E15 RGCs, compared with 0.52%, 13.17% and 3.59% in E19 RGCs). In contrast, DCC was expressed more at birth in E19 RGCs (18.05% in E19 RGCs compared with 9.23% in E15 RGCs). This shows there is clearly a difference in the expression of proteins in the two cohorts of RGCs, which is consistent with PCR data and with their growth state as their axons encounter the changes in the newborn brain. The overall findings of this research suggest that seemingly homogenous populations of neurons are quite different in their developmental profile and in their response to injury. This work may provide new ways of determining better strategies for CNS repair and the most effective way of targeting cells for regeneration and survival.

The aim of the thesis is to create methodological papers that will serve to teach or practice massage. There is outlined the history and the beginning of the word massage. There are deseribed the masseur touches, their technique and indications. The suitability and unsuitability of massage, effects, effects on the human body. It lists and uses of funds and equipment for sports massage. Based on the literature and captured images are developed methodology sheets for your convenience and are accompanied by photographs. The leaves have been tested in practice, a masseur in the fitness center.

Newts have the incredible ability to regenerate many different organs and tissues as adults, including the limbs. Limb regeneration occurs via the dedifferentiation of stump tissue and the formation of a blastema, which provides the majority of cells for the regenerate. Despite all that we have learned about dedifferentiation and blastema formation, the cellular and molecular mechanisms underlying these processes are still poorly understood. We used representational difference analysis (RDA) to identify genes involved in the early dedifferentiation process in newt forelimb regeneration. Our analysis identified approximately 410 unique genes that were differentially regulated during this process. Microarray analysis was used to determine the expression profile of these genes throughout limb and tail regeneration. We used quantitative PCR (qPCR) to validate the expression of a subset of these genes [β-catenin, wntless, dapper, thymosin-β 4 (Tβ4), and thymosin-β 10/15 (Tβ10/15)] in regenerating limb and tail tissue, as well as in differentiating newt myoblasts. We also verified the expression of these genes in the regenerating newt limb using immunohistochemistry (IHC) and in situ hybridization (ISH). Finally, we performed a functional analysis on β-catenin, wntless, dapper, and Tβ4 by overexpressing these genes in mouse myoblasts to examine their effects on differentiation and potential roles in dedifferentiation. Quantitative PCR verified the expression of β-catenin, wntless, dapper, and Tβ4 during limb regeneration and IHC/ISH localized the β-catenin and Tβ4 proteins to the blastema during regeneration. Tβ10/15 was shown by qPCR to be expressed in the tail during regeneration. Overexpression of newt β-catenin, wntless, dapper, and Tβ4 in mouse myoblasts showed that each of these genes has an inhibitory effect on the differentiation of myoblasts into myotubes and, therefore, may play a role in promoting or maintaining the dedifferentiated state. Our work has identified a large number of genes with potential roles in regulating the dedifferentiation process during newt forelimb regeneration. We have also laid a framework from which much more work can be done by drawing on the genes we have identified and the microarray data, which indicate ideal follow-up candidates. Our analysis of specific genes has also increased our understanding of the molecular events occurring during the dedifferentiation process in the regenerating newt limb.

The present thesis is a contribution to unravel the molecular mechanisms that underlie urodele regeneration. Urodele amphibians (newts and salamanders) are among the few vertebrates with the remarkable ability to regenerate lost body appendages, like the limbs and the tail. Urodele tail and limb regeneration occurs via blastemal epimorphic regeneration. A blastema is a mound of progenitor cells that accumulates at the amputation plane and eventually gives rise to the missing structures. It is known today that dedifferentiating muscle fibers at the amputation plane contribute to the blastema cell pool, but how this process occurs on the cellular and molecular level is hardly understood, which is in part due to the lack of molecular methods to test gene function in urodeles. Furthermore, little is known about how coordinated growth and patterning occurs during urodele regeneration, and if the patterning mechanisms in regeneration are related to the ones in development. The goal of this study was to better understand these processes on the molecular level. To address these questions, I first established several methods in our model systems, which are the mexican salamander Ambystoma mexicanum (axolotl) and a cell line derived from the newt Notophthalmus viridescens. In order to monitor gene expression on a cellular level during regeneration, I worked out a good in situ hybridization protocol on axolotl tissue cryosections. To be able to test gene function, I established electroporation conditions to both overexpress genes in the cultured newt cells and to deliver morpholinos into axolotl cells in vivo and newt cells in culture. I demonstrate here that morpholinos are an effective tool to downregulate protein expression in urodele cells in vivo and in culture. Testing the role of two candidate genes in muscle fiber dedifferentiation, the homeobox containing transcription factor Msx1 and Rad, a GTP-binding protein of a new Ras-related protein family, revealed that neither seems to play a major role in muscle dedifferentiation, both in culture and in vivo. In addition to testing gene function I have examined the muscle dedifferentiation process in more detail. I show here that dedifferentiating muscle fiber nuclei undergo morphological changes that are likely due to chromatin remodeling events. I also demonstrate that the axolotl spinal cord expresses embryonic dorsoventral (d/v) patterning markers of the neural tube. The transcription factors Msx1, Pax7 and Pax6 are expressed in their respective d/v domains in both the differentiated and the regenerating axolotl spinal cord. Furthermore, the secreted signaling molecule sonic hedgehog (Shh) is expressed in the floor plate in both the differentiated and the regenerating cord. Using a chemical inhibitor (cyclopamine) and an activator of the hedgehog pathway, I discovered that hedgehog signaling is required for overall tail regeneration. Blocking hedgehog signaling does not only result in d/v patterning defects of the regenerating spinal cord, but it also strongly reduces blastema cell proliferation. In addition, I identified cartilage and putative muscle progenitor cells in the blastema, marked by the expression of the transcription factors Sox9 and Pax7, respectively. Both progenitor populations are reduced in the blastema in the absence of hedgehog signaling. The continuous expression of marker genes for embryonic progenitor cell domains in the mature axolotl may be related to their ability to regenerate.

The establishment of transgenesisi in axolotls is crucial for studying development and regeneration, as it would allow for long-term fate tracing as well as gene expression analysis, therefore we were interested in both obtaining animals expresing the transgene with little mosaicism in F0 generation and transgenesis. We demonstrate here that plasmid injection into one cell stage axolotl embryo generates transgenic animals that display germline transmission of a transgene. However, the efficiency of simple plasmid injection is very low, expression of the transgene is mosaic and seems to be promoter dependant. We have tested several methods of transgenesis developed in other systems. First we used Adeno-Associated Viral Terminal Repeats inserted into the injected construct to enhance the expression level of the transgene and reduce mosaicism. However, in the axolotl system we do not observe the enhancement of expression. Moreover, the expression appeared to be transient and disappeared after two months. Further, we tested the effect of the inclusion of ISceI meganuclease in the injections, succesful transgenesis method in the medaka system. It resulted in a higher percentage of F0 animals displaying strong , stable expression throughout the body. This represents the first demonstration in the axolotl of germline transmission of the transgene. Using this technique we have generated a germline transgenic anima expressing GFP ubiquitously in all tissue examined. We have used this anima to study cell fate in the dirsal fin during development. We have discovered a contribution of somite cells to dorsal fin mesenchyme in the axolotl, which was previously assumed to derive solely from neural crest. We have also studied the role of blood during tail regeneration by transplanting the ventral blood-forming region from GFP+ embryos into unlabeled host. During tail regeneration, we do not observe GFP+ cells contributing to muscle or nerve, suggesting that during tail regeneration blood stem cells do not undergo significant plasticity. We are interested in characterization of pluripotency of blastema cells. Previously, it has been shown that neural progenitor cells form the spinal cord can transdifferentiate to muscle and other tissue types in the regenerating tail. To test if blastema cells have the potency of differentiating into a neural tissue , we transplanted GFP+ 4day blastema into an injured spinal cord. Our result shows that blastema cells don't seem to contribute to the regenerating spinal cord.

Indiana University-Purdue University Indianapolis (IUPUI) / Retinoids play an important role in tissue patterning during development as well as in epithelial formation and health. In the mammalian central nervous system, the meninges are a source of retinoids for brain tissue. Retinoid production has been described in juvenile Axolotl ependymal cells. Retinoid effects may possess a significant role in the regeneration-permissive interaction of the meninges and ependyma of the Axolotl spinal cord after penetrating injury. During spinal cord regeneration in urodele amphibians, the pattern of retinoid production changes as the meninges interact with the injury-reactive ependymal cells reconstructing the injured spinal cord. In order to determine which components of the retinoid metabolism and intracellular signaling pathway act in Urodele spinal cord regeneration, we employed antibody/horseradish peroxidase staining of both intact and regenerating Axolotl spinal cord tissues obtained from adult animals as well as cell culture techniques to determine expression of three retinoid pathway components: Cellular Retinoic Acid Binding Protein II (CRABP 2), Cellular Retinol Binding Protein I (CRBP 1), and Retinaldehyde Dehydrogenase II (RALDH 2). Current results demonstrate the following in the intact cord: 1) CRBP 1 is expressed in the pia and dura mater meningeal layers, in gray matter neurons (including their axonal processes), and the ependymal cell radial processes that produce the glia limitans, 2) CRABP 2 is expressed in the arachnoid and/or dura mater meningeal layers surrounding the spinal cord, and 3) RALDH 2 is expressed in the meninges as well as cytoplasm of grey matter neurons and some ependymal/sub-ependymal cells. In the regenerating cord, CRBP 1 is expressed in ependymal cells that are undergoing epithelial-to-mesenchymal transition (EMT), as is CRABP 2. RALDH 2 staining is very strong in the reactive meninges; in addition, expression is also upregulated in the cytoplasmic and perinuclear regions of reactive grey matter neurons, including motor neurons and in the apical region of ependymal. Preliminary studies culturing reactive meninges and ependymal cells together suggested that the meninges could drive re-epithelialization of the reactive ependymal cells. Experiments to characterize this interaction show an unusual proliferation pattern: Proliferating Cell Nuclear Antigen (PCNA) labeling is present in intact and regenerating cord ependymal cells. However, in culture, the presence of meninges results in no proliferation proximal to the explant, but extensive proliferation in leading cell outgrowth; also, the cultured meninges is positive for RALDH2. In summary, the intact adult cord shows meningeal production of RA, which is upregulated following injury; in addition, during this time, RA production is upregulated in the adult ependymal cells as well. In culture, the reactive meninges appears to modulate the behavior of reactive ependymal cells.

Bare-root, container, and root production method (RPM™) seedlings of two oak species (Nuttall (Quercus texana Buckley), cherrybark (Q. pagoda Ell.)) were planted on lands damaged by Hurricane Katrina in southern Mississippi to compare the height growth, groundline diameter growth and survival of the different planting stocks. Tree shelters were applied to half of the bare-root seedlings to determine their effect on the height and groundline diameter growth and survival of the seedlings. RPM seedlings exhibited significantly greater height and groundline diameter growth than bare-root or container seedlings after one growing season. Bare-root seedlings exhibited significantly greater height and groundline diameter growth than container seedlings. Tree shelters significantly increased height growth of bare-root seedlings; however, sheltered bare-root seedlings exhibited significantly less groundline diameter growth than non-sheltered seedlings. Cherrybark oak exhibited greater height growth than Nuttall oak, while Nuttall oak exhibited greater groundline diameter growth than cherrybark across all planting stocks.