The lack of gene-knockout technologies in many animal models of regeneration can be a problem to study gene function during this process. Using RNAi or morpholinos to produce knockdowns can somehow compensate these limitations. Recently, new methods of gene editing have been developed, including TALENs (transcriptional activator-like effector nucleases) and CRISPR (clustered regularly interspaced short palindromic repeat). Now, a recent paper from the laboratory of Elly Tanaka reports for the first time on the use of these novel technologies to study the effects of knocking out a Sox2 homologue during axolotl regeneration (http://www.ncbi.nlm.nih.gov/pubmed/25241743).
As a first step to study gene deletion driven by TALENs and CRISPRs the authors tried to knock out a genomically inserted GFP-transgene in their strain of germline-transgenic GFP-axolotls. They injected the TALEN mRNAs or CRISPR RNAs into freshly laid embryos. Both methods turned out to be successful although CRISPRs appeared more efficient. Next, they used both methods to knockout an endogenous gene, a tyrosinase homologue. This gene is not essential for development and gives a pigmentation defect easily detectable. Again here, both methods worked although CRISPR-mediated knockouts were more penetrant and efficient.
Then, they moved to study the effects of knocking out Sox2, an important gene required for neural stem/progenitor cells maintenance and expansion in other animals, during both axolotl development and regeneration. Thus, they injected several different CRISPR RNAs into fertilized eggs at the single-cell stage and analysed them at 13 days post injection. Of 487 eggs injected with a particular Sox2-gRNA, 403 survived and grew up to normal sizes. Of those, 274 showed a curved body and many had excess blood in the olfactory bulb are. Also, they displayed a severe reduction of Sox2-positive cells in this olfactory bulb. Remarkably, and in contrast to mice in which Sox2 knockout is lethal, axolotls can apparently survive without this gene. To further corroborate these results, the authors knocked-down Sox2 using morpholinos and obtained a similar viability as for the CRISPR-mediated knockouts.
When analysing in more details the defects at the cellular level in the Sox2 knockout animals the authors found that although the expression of Sox2 in the cells lining the spinal cord lumen was lacking, those animals had an apparently normal organization of the spinal cord with normal NEU+ neurons, as well as normal expression of other neural stem cell markers such as GFAP and ZO-1 and the proliferation markers PCNA and phosphohistone H3. Then, the authors analysed the regenerative capabilities of these animals knockout for Sox2. To do that, they amputated the tail of those Sox2-CRISPR animals that showed a curved-body phenotype (and that were those that had a higher penetrance of deletions). Remarkably, Sox2 knockout axolotls showed reduced or lack of spinal cord in the regenerated tail. At day 6 of regeneration this reduction in the length of the regenerated spinal cord was not correlated with a shorter regenerated overall tail. By day 10, however, the Sox2 knockout animals also displayed a mild reduction of the overall length of the regenerated tail.
After a series of experiments with different markers the authors concluded that the deletion of Sox2 in neural stem cells resulted in a defect in the proliferative expansion of neural stem cells specifically after tail amputation. Compared to normal regenerating controls, it seems that in Sox2 knockout animals neural stem cells were not able to accelerate their cell cycle after amputation and a higher percentage of them appeared to remain in G1 or G2/M. Thus, the lack of Sox2 hampered proliferation and expansion of the neural stem/progenitor cell pool.
Finally, and with the aim of better understanding the different effects seen in embryogenesis and regeneration after knocking out Sox2, the authors analysed the expression of Sox3 because of the relationship between both genes in other models. Interestingly, here they found that during axolotl embryogenesis Sox3 showed indistinguishable expression patterns compared to Sox2, which would argue that Sox3 could compensate the lack of Sox2 during embryogenesis after Sox2-CRISPR knockout. On the contrary, during regeneration Sox3 was downregulated in the regenerating spinal cord, suggesting that the lack of Sox2 in those Sox2 knockout axolotls could not be compensated by the expression of Sox3.
In summary, this study shows that TALENs and CRISPRs can induce specific gene deletions in axolotls and be used to study regeneration in these animals at the genetic level. Remarkably, whereas Sox2 knockouts were viable and were developed with only mild morphological defects, these same animals failed to regenerate the spinal cord, revealing a regeneration-specific need for this gene in axolotls.