Abstract
The differentiated state of spinal cord ependymal cells in regeneration-competent amphibians varies between a constitutively active state in what is essentially a developing organism, the tadpole of the frog Xenopus laevis, and a quiescent, activatable state in a slowly growing adult salamander Ambystoma mexicanum, the Axolotl. Ependymal cells are epithelial in intact spinal cord of all vertebrates. After transection, body region ependymal epithelium in both Xenopus and the Axolotl disorganizes for regenerative outgrowth (gap replacement). Injury-reactive ependymal cells serve as a stem/progenitor cell population in regeneration and reconstruct the central canal. Expression patterns of mRNA and protein for the stem/progenitor cell-maintenance Notch signaling pathway mRNA-binding protein Musashi (msi) change with life stage and regeneration competence. Msi-1 is missing (immunohistochemistry), or at very low levels (polymerase chain reaction, PCR), in both intact regeneration-competent adult Axolotl cord and intact non-regeneration-competent Xenopus tadpole (Nieuwkoop and Faber stage 62+, NF 62+). The critical correlation for successful regeneration is msi-1 expression/upregulation after injury in the ependymal outgrowth and stump-region ependymal cells. msi-1 and msi-2 isoforms were cloned for the Axolotl as well as previously unknown isoforms of Xenopus msi-2. Intact Xenopus spinal cord ependymal cells show a loss of msi-1 expression between regeneration-competent (NF 50–53) and non-regenerating stages (NF 62+) and in post-metamorphosis froglets, while msi-2 displays a lower molecular weight isoform in non-regenerating cord. In the Axolotl, embryos and juveniles maintain Msi-1 expression in the intact cord. In the adult Axolotl, Msi-1 is absent, but upregulates after injury. Msi-2 levels are more variable among Axolotl life stages: rising between late tailbud embryos and juveniles and decreasing in adult cord. Cultures of regeneration-competent Xenopus tadpole cord and injury-responsive adult Axolotl cord ependymal cells showed an identical growth factor response. Epidermal growth factor (EGF) maintains mesenchymal outgrowth in vitro, the cells are proliferative and maintain msi-1 expression. Non-regeneration competent Xenopus ependymal cells, NF 62+, failed to attach or grow well in EGF+ medium. Ependymal Msi-1 expression in vivo and in vitro is a strong indicator of regeneration competence in the amphibian spinal cord.
Highlights
In all vertebrates, the ependymal cells that line the central canal of the spinal cord play essential roles in normal spinal cord structure and physiology
In anuran amphibians, such as Xenopus, regenerative capacity in the central nervous system (CNS) is one of many processes affected by progress toward and through metamorphosis (Shi, 2000)
Three washes were performed with Tris–HCl buffered saline containing 0.05% Tween-20 (TBST) and sections were incubated with 1% blocking reagent (DIG Nucleic Acid Detection Kit: Roche) in TBST
Summary
The ependymal cells (ependymoglia) that line the central canal of the spinal cord play essential roles in normal spinal cord structure and physiology (rev. Oksche and Ueck, 1976; Reichenbach and Wolberg, 2013; Jiménez et al, 2014; Pannese, 2015; Moore, 2016). The extent to which ependymal epithelium disorganizes during regeneration is species and location specific (Clarke and Ferretti, 1998; Chernoff et al, 2003; Gargioli and Slack, 2004; Zukor et al, 2011) In anuran amphibians, such as Xenopus, regenerative capacity in the central nervous system (CNS) is one of many processes affected by progress toward and through metamorphosis (Shi, 2000). Additional ependymal roles in cord regeneration include the interaction of reactive ependymal cells with the reactive meningeal connective tissue to prevent fibrotic/meningeal scar formation, including production of matrix metalloproteinases to remove extracellular matrix material (Chernoff et al, 2000; Zukor et al, 2011) Not all of these regenerative processes have been documented at every stage of the urodele life cycle. Epidermal growth factor (EGF) is known to maintain injury-reactive Axolotl ependymal cells in a mesenchymal state in vitro, and EGF-containing defined medium is shown here to support mesenchymal growth from Xenopus ependymal cells, as well (Chernoff et al, 1990; O’Hara and Chernoff, 1994)
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