Browsing by Subject "Axolotl"

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    Comparative analysis of the molecular pathways involved in spinal cor injury in axolotls vs. mammaals
    (2015-04) Diaz Quiroz, Juan Felipe
    Spinal cord injuries (SCI) in mammals are major causes of physical disabilities. In contrast the Mexican salamander (axolotl) possesses amazing regenerative capabilities and can regenerate a fully functional spinal cord after injury. We have attempted to identify the molecular determinants underlying this evolutionary divergence by undertaking a detailed comparative analysis of a regenerative model, the axolotl spinal cord, and a corresponding non-regenerative system, rat spinal cord after injury. This approach identified a small number of highly conserved microRNAs that are differentially regulated in axolotl versus. Detailed in vivo studies of one of these microRNAs, miR-125b that is highly expressed in axolotl but low in rat has identified it as a key regulator of the regenerative response in axolotl. We also found that upregulation of miR-125b after injury improves SC repair in rats. In addition, we have identified SEMA4D as a target gene regulated by this microRNA after SCI in both axolotl and rat. We also studied how this miRNA is regulated in the axolotl to promote regeneration after SCI. We found that in the absence of injury, actin cytoskeleton de-polymerization induced by Cytochalasin D (Cyto D) induced a decrease in miR-125b expression similar to the decrease induced by injury in axolotl. Analyses of the regulatory region of the miR-125b axolotl gene revealed predicted binding sites for c-Fos. When we performed SCI in axolotls, we observed an increase in the expression of c-Fos 1 day after injury. As expected, we found that in the absence of injury, treatment with Cyto D induced similar changes in c-Fos expression similar to injury. Lastly, we proposed the development of a 3D in vitro co-culture model system to study at the cellular and molecular level how miR-125b SCI regulates the response to injury in rats and whether miR-125b expression is regulated by biomechanical activation of the Rho-c-Fos pathway. The proposed model will allow better imaging, better spatial and temporal control over modulating microRNA and gene expression in different cells at different time points. Our overall data suggest that dynamic changes in the actin cytoskeleton after injury induces changes in miR-125b expression, possibly through activation of cFos in the RhoA pathway, to create a permissive environment for regeneration in axolotls.
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    A comparative approach to deciphering the molecular mechanisms of scar-free wound healing
    (2017-02) Erickson, Jami
    Although mammals form scars upon skin wound healing, the Mexican “Axolotl” salamander has the extraordinary ability to heal wounds scar-free. While axolotl skin histologically resembles mammalian skin, molecular details that prevent scar formation during axolotl wound healing are largely unknown. To address this knowledge gap, we performed transcriptional profiling during axolotl cutaneous wound healing. We analyzed genes that displayed differential gene expression during axolotl wound healing compared to previously published human gene expression profiling data. We found that Sal-like 4 (Sall4) expression was increased early during axolotl skin regeneration, but did not increase in humans until later time points. We hypothesize that early increase in expression of SALL4 after injury is required for scar-free wound healing. To test this hypothesis, we depleted SALL4 in vivo during wound healing. We found that when SALL4 is depleted, we see excessive Collagen I and XII deposition that occurs earlier and is not fully remodeled, resulting in a scar-like phenotype. To determine how SALL4 expression is regulated during wound healing, we sought to identify which microRNAs post-transcriptionally regulate SALL4. We found that miR-219 is able to regulate expression of axolotl SALL4 during wound healing. Further, when we ectopically increase miR-219 levels during axolotl wound healing, we find early excessive collagen deposition, mirroring the SALL4 depletion phenotype. Additionally, we found that miR-103, not miR-219, is able to regulate human SALL4. Thus, revealing one mechanism that could explain the different SALL4 expression profiles seen in axolotls vs. humans. Lastly, we describe how to use a Dual-Fluorescent green fluorescent protein (GFP)-Reporter/ monomeric red fluorescent protein (mRFP)- Sensor (DFRS) plasmid to quantitate the dynamics of specific miRNAs over time. This system allows researchers to obtain relative quantifications for microRNA levels during biological processes over time. This will allow researchers explore the expression dynamics of any microRNA over time in vivo.
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    Molecular Mechanisms Regulating the Pro-Regenerative Glial Cell Response to Spinal Cord Injury in Axolotl
    (2018-11) Sabin, Keith
    Axolotl salamanders have the remarkable ability to functionally regenerate after spinal cord injury. In response to injury, glial cells adjacent to the lesion undergo a pro-regenerative response, proliferate and migrate to reconnect the spinal cord and create a permissive environment for axon regeneration. This is in stark contrast to the mammalian response to spinal cord injury. Damaged astrocytes undergo reactive gliosis and contribute to a glial and fibrotic scar by secreting axon growth inhibitory molecules like chondroitin sulfate proteoglycans and collagens. This ultimately results in failed axon regeneration and a loss of sensory and motor function below the lesion. Why the pro-regenerative glial cell response in axolotl is so different from mammalian astrocytes and the identities of pro-regenerative downstream molecular pathways were not well known. To this end, we identified a dynamic change in glial cell membrane potential that was necessary for the pro-regenerative glial cell response to injury. Disruption of glial cell depolarization by either genetic or pharmacologic approaches inhibited the pro-regenerative glial cell response to injury and blocked spinal cord regeneration. Transcriptional profiling and biochemical approaches identified the ERK/c-Fos signaling pathway as key effector molecules downstream of glial cell depolarization. Investigations into the identity of the c-Fos binding partner revealed that JunB, not the canonical c-Jun, is the c-Fos binding partner in axolotl glial cells. While reactive astrocytes in mammals express AP-1cFos/cJun which functions to promote reactive gliosis, glial scar formation, and inhibit spinal cord regeneration, AP-1cFos/JunB represses expression of reactive gliosis associated genes. Therefore, we hypothesized that differential composition of AP-1 could regulate the different cellular responses to injury. Consistent with our hypothesis, the ectopic overexpression of AP-1cFos/cJun in axolotl glial cells leads to defects in axon regeneration, similar to mammals. To determine how glial cells repress c-Jun expression, we identified a miR-200a binding site in the 3’ untranslated region of axolotl c-Jun transcript. Using in vivo and in vitro approaches, we showed that axolotl c-Jun is a direct target of miR-200a. Additionally, inhibition of miR-200a leads to axon regeneration defects reminiscent of the AP-1cFos/cJun overexpression phenotype. Finally, transcriptomic profiling of miR-200a inhibitor-electroporated spinal cords revealed differential expression of a subset of genes involved with reactive gliosis, the glial scar, extracellular matrix remodeling, inflammation, migration, and axon guidance compared to control spinal cords. Collectively these results reveal that miR-200a inhibits signaling networks involved with reactive gliosis, the glial scar, and other processes necessary for spinal cord regeneration. Further examination of the RNA sequencing data revealed that miR-200a inhibition led to the expression of the mesoderm transcription factor Brachyury and down-regulation of a host of neural genes, including Sox2. This expression profile is reminiscent of a more developmentally primitive spinal cord progenitor population called neuromesodermal progenitors. This suggests that miR-200a inhibition led to the loss of the neural identity and acquisition of a more neuromesodermal progenitor-like state. Subsequent analysis revealed miR-200a inhibition indirectly promotes Brachyury expression, specifically in axolotl glial cells, perhaps via modulation of FGF and Wnt signaling molecules. Whether modulation of Brachyury expression is sufficient to induce glial cells to fully dedifferentiate into NMPs and contribute to mesoderm-derived tissues (muscle/cartilage) during regeneration is not clear. In summary, my thesis research identified an injury-induced change in glial cell membrane potential that was necessary for the pro-regenerative glial cell response to spinal cord injury. The injury-induced change in glial cell membrane potential is up-stream of ERK signaling and c-Fos expression. In damaged mammalian astrocytes, c-Fos heterodimerizes with c-Jun to promote reactive gliosis and glial scar formation. However, a majority of axolotl glial cells do not express c-Jun and instead c-Fos heterodimerizes with JunB to form AP-1cFos/JunB. AP-1cFos/JunB functions to inhibit reactive gliosis, glial scar formation, and promote the pro-regenerative glial cell response. Ectopic overexpression of AP-1cFos/cJun in axolotl glial cells inhibits spinal cord regeneration. Axolotl glial cells express the microRNA miR-200a, which functions to repress c-Jun expression. Inhibition of miR-200a blocks spinal cord regeneration, leading to differential expression of genes involved with reactive gliosis and glial scar formation. Finally, miR-200a may play an additional role in stabilizing the neural identity of neural progenitor cells during axolotl spinal cord regeneration. Inhibition of miR-200a could result in dedifferentiation of glial cells to a more neuromesodermal progenitor-like identity, perhaps by modulating Wnt and FGF signaling.