Musashi and Plasticity of Xenopus and Axolotl Spinal Cord Ependymal Cells.

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.

The short toes mutation of the axolotl.

The axolotl mutant strain, short toes (s/s), can regenerate spinal cord and tail, but not limbs. This makes s/s potentially very useful for limb regeneration studies. This mutant merits a new examination that integrates the original description of the mutant, existing experimental studies, new data and current thinking about stem cells and regeneration. There are still major gaps in information about this mutant; the gene(s) causing the defects has not yet been discovered, and even the histological description is incomplete, especially regarding muscle abnormalities. In the short toes limb, MyHC (myosin heavy chain)-1, MyHC-2b and pax7 are down-regulated. In particular, all three MyHC genes and pax7 are highly expressed in the normal limb, but almost lost in the s/s limb. MyHC genes are one of the main components of skeletal muscle, and Pax7 is the skeletal muscle satellite cell marker. Histological experiments confirm that severe s/s has lost most skeletal muscle and myosin. These results suggest that skeletal muscle, which includes satellite cells, could play an important role in axolotl limb regeneration.

Expression patterns of chick Musashi-1 in the developing nervous system.

Vertebrate homologues of musashi have recently been referred to as neural stem cell markers because of their expression patterns and RNA-binding interactions. In the context of the notch signaling pathway, Musashi-1 (Msi-1) is a regulator of neural cell generation, cooperating with notch to maintain mitosis. In an effort to identify definitive stem cell markers of the neural retina, a portion of the Msi-1 cDNA was cloned, and the expression of Msi-1 in the chick eye was analyzed. Using an Msi-1-specific antibody and RNA probe, we show that expression of Msi-1 in the early neural tube is consistent with neural stem identity. In the neural retina, expression starts shortly before embryonic day 3 (E3) and continues up to and including E18. A BrdU incorporation assay shows Msi-1 to be found in both proliferating and differentiating cells of E5 neural retina. At E8 (when proliferation is complete in the fundus of the retina) and E18 (mature retina) Msi-1 expression was found in the ciliary marginal zone (CMZ) as well as in a subpopulation of differentiated cells, including photoreceptors and ganglion cells.

Spinal cord regeneration: intrinsic properties and emerging mechanisms.

Injured spinal cord regenerates in adult fish and urodele amphibians, young tadpoles of anuran amphibians, lizard tails, embryonic birds and mammals, and in adults of at least some strains of mice. The extent of this regeneration is described with respect to axonal regrowth, neurogenesis, glial responses, and maintenance of an 'embryonic' environment. The regeneration process in amphibian spinal cord demonstrates that gap replacement and caudal regeneration share some properties with developing spinal cord. This review considers the extent to which intrinsically regenerating spinal cord demonstrates neural stem cell behavior and to what extent anterior-posterior and dorsal-ventral patterning might be involved.