Myelination is a cellular adaptation allowing rapid conduction along axons. We have investigated peripheral axons of the zebrafish maxillary barbel (ZMB), an optically clear sensory appendage. Each barbel carries taste buds, solitary chemosensory cells, and epithelial nerve endings, all of which regenerate after amputation (LeClair and Topczewski [2010] PLoS One 5:e8737). The ZMB contains axons from the facial nerve; however, myelination within the barbel itself has not been established. Transcripts of myelin basic protein (mbp) are expressed in normal and regenerating adult barbels, indicating activity in both maintenance and repair. Myelin was confirmed in situ by using toluidine blue, an anti-MBP antibody, and transmission electron microscopy (TEM). The adult ZMB contains ~180 small-diameter axons (<2 μm), approximately 60% of which are myelinated. Developmental myelination was observed via whole-mount immunohistochemistry 4-6 weeks postfertilization, showing myelin sheaths lagging behind growing axons. Early-regenerating axons (10 days postsurgery), having no or few myelin layers, were disorganized within a fibroblast-rich collagenous scar. Twenty-eight days postsurgery, barbel axons had grown out several millimeters and were organized with compact myelin sheaths. Fiber types and axon areas were similar between normal and regenerated tissue; within 4 weeks, regenerating axons restored ~85% of normal myelin thickness. Regenerating barbels express multiple promyelinating transcription factors (sox10, oct6 = pou3f1; krox20a/b = egr2a/b) typical of Schwann cells. These observations extend our understanding of the zebrafish peripheral nervous system within a little-studied sensory appendage. The accessible ZMB provides a novel context for studying axon regeneration, Schwann cell migration, and remyelination in a model vertebrate.
Xenopus laevis tadpoles have the capacity to regenerate their spinal cord after an injury; however, the mechanisms involved in this process are unknown. We hypothesize that neural progenitors are necessary for spinal cord regeneration. In order to evaluate that, we have studied Sox2 function, a neural progenitor marker, during spinal cord regeneration. We found that Sox2 expression was upregulated after tail amputation and spinal cord transection, two different models of spinal cord injury. Additionally, we observed Sox2+ cells undergoing proliferation and LRC increase in response to tail amputation. The activation of neural progenitors suggests a role for ependymal regrowth and neurogenesis in X. laevis CNS regeneration. We also analyzed the role of neural progenitors during spinal cord regeneration by the overexpression of a dominant negative form of Sox2 after amputation. Experimental reduction of Sox2 activity diminished proliferation of spinal cord resident cells and impairs tail regeneration. Furthermore, Sox2 expression levels are correlated with functional recovery after transection at different stages of metamorphosis. We concluded that Sox2+ cells are necessary for spinal cord and tail regeneration and lead to a model whereby spinal cord damage activates tissue specific progenitor proliferation.Program/Abstract #382 miRNAS and regulation of retinoid signaling in the regenerating adult newt spinal cord Appendage regeneration is a complex process unique to only a few species of adult vertebrates, including the Urodele amphibians. One current focus involves the identification of molecules and signaling pathways that control the formation of the tail blastema, as well as the outgrowth and patterning of the regenerating spinal cord. Recent efforts in our lab have centered on defining the upstream and downstream regulators of retinoid signaling that result in the production of a functional caudal spinal cord after tail amputation. We have previously demonstrated that inhibition of retinoid signaling through the RARβ2 receptor using a specific antagonist, LE135, inhibits the outgrowth of the ependymal tube during the first 6 days after tail amputation. We now demonstrate that miR133a is significantly down regulated in the ependymal tube during this same period. Our most recent results with microRNA-based profiling have identified at least 18 highly conserved miRNAs that display significant changes in expression in tail regenerates treated with LE-135 compared to DMSO treated control regenerates. An analysis of the expression patterns and putative functions of these potential regulators of retinoid signaling are underway.Amphibian Axolotl (Ambystoma mexicanum) can regenerate its limbs after amputation. During the early stage of regeneration, a G2 maintaining protein, ecotropic viral integration site 5 (EVI5), has dramatically increased, which suggests the delay of cells entering mitosis. Aurora B Kinase (ABK) assists to degrade EVI5, which moves cells from G2 to mitosis. Unfortunately, sequence information of E...
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