br Results br Discussion This work represents a number

Results
Discussion This work represents a number of important advances for the study of axolotl regeneration. First, we have shown for several genomic loci that TALENs and CRISPRs can induce targeted deletions in a large proportion of cells from injected animals. In all cases, CRISPRs yielded higher viability of injected animals and higher penetrance of the phenotype, reflecting deletion of both protoporphyrin ix within often a large majority of the cells. The Tyr and Sox2 experiments show strong cellular phenotypes associated with homozygous gene deletion. In addition, for the GFP-transgene, we have shown germline transmission of the deletion after TALEN-mediated gene deletion. We therefore conclude that CRISPRs will be an important mode of generating gene deletions and other genome modifications in the axolotl for regeneration studies. Our work examining CRISPR-mediated Sox2 deletion showed mild phenotypes during development but a strong inhibition of spinal cord regeneration after tail amputation. The lack of a developmental phenotype due to SOX2 knockdown was corroborated by the injection of anti-Sox2 morpholinos that knocked down SOX2 immunostaining in the embryo but yielded the same frequency of normally developing embryos as control-injected cohorts. It is interesting that no knockdown experiments for Sox2 have been reported in amphibian and zebrafish embryos, raising the possibility that in these vertebrates, Sox2 is either not solely responsible for maintenance of pluripotent cells or that maternal stores allow survival through early stages. Indeed, when we examined Sox3, its distribution overlapped with Sox2 expression in the embryo, suggesting it can compensate for lack of Sox2, especially since Sox3 has been shown to be upstream of Sox2 in Xenopus embryos (Rogers et al., 2009). During regeneration, axolotl Sox3 was downregulated in the spinal cord, which would be consistent with an enhanced sensitivity of the regenerate to Sox2 knockdown. Our analysis of tail regeneration in the Sox2-CRISPR animals showed that despite maintaining a number of radial glial cell markers, Sox2-deleted spinal cord shows defective proliferation of GFAP-positive cells and therefore, reduced outgrowth of the spinal cord at 6 and 10 days postamputation. In other work, we have defined the cell-cycle characteristics of SOX2+PCNA+-positive cells before and after regeneration and found that in the uninjured state, resident cells cycle slowly, on average once every 14 days. Upon tail amputation, the cell cycle accelerates to a 4-day cycle (A. Rodrigo-Albors, personal communication). Given that the SOX2−TUJ1−NEUN− cells at the amputation plane maintained expression of PCNA and normal levels of EdU incorporation before regeneration, we interpret this result to mean that the SOX2+ cells that would normally accelerate their cell cycle are, in the absence of SOX2, unable to accelerate their cell cycle in response to injury cues. The role of SOX2 in promoting proliferation of neural stem/progenitor cells has previously been documented in the brain and in CNS tumors (Favaro et al., 2009, 2014), and our work would support a role for SOX2 in injury mediated rapid cell cycles required to expand the pool of neural stem cells to reconstitute the missing spinal cord.
Experimental Procedures For further details, see the Supplemental Experimental Procedures. All other methods were performed according to standard protocols.
Author Contributions
Acknowledgments
Introduction Embryonic stem cells (ESCs) are originally derived from the inner cell mass. They are pluripotent cells that can self-renew and differentiate into multiple cell types upon appropriate stimuli. Mouse ESCs (mESCs) possess a distinctive global chromatin structure that is characterized by hyperdynamic plasticity and bivalent domains marked by both active H3K4me3 (trimethylation of histone 3 lysine 4) and repressive H3K27me3 (trimethylation of histone 3 lysine 4) in the promoter regions of lineage-specific genes (Bernstein et al., 2006; Meshorer et al., 2006). In human ESCs (hESCs), H3K4me1 (monomethylation at lysine 4 of histone 3), H3K27ac (acetylation at lysine 27 of histone 3), and p300 marked chromatin loci were recently identified as active enhancers that drive gene expression (Rada-Iglesias et al., 2011). Conversely, a lack of H3K27ac but enrichment with H3K27me3 is linked to repressed but “poised” elements for genes active in early development (Rada-Iglesias et al., 2011). The switch from self-renewal to differentiation requires the participation of multiple epigenetic factors, including chromatin regulators, noncoding RNAs, and histone modifiers (Surani et al., 2007; Tay et al., 2008).