The ability to regenerate injured tissues is a fascinating phenomenon. Some organisms, such as the hydra or the axolotl, possess extensive regenerative capacity. Others, including our own species, Homo sapiens, have much more limited regenerative capacity. Is this inability to regenerate hardwired into our biology, or can our cells be “tricked” into carrying out a regenerative program?
The first step is to understand the genetic programs governing regeneration in organisms which do it really well. The model system we study – the zebrafish – has an innate ability to regenerate many different tissues, including the heart. Major cardiac injury, inflicted either by surgical removal of the tip of the ventricle or application of a cryoprobe, is repaired within one to three months, with full physiological recovery.
We use zebrafish because they combine vertebrate biology (over 80% of human genes have orthologs in zebrafish!) with many advantageous features of invertebrates, such as external development of optically transparent embryos, small size, and relatively simple and inexpensive maintenance.
The genetic program of regeneration is thought to overlap the genetic program of development. Indeed, all major developmental signaling pathways, from Wnt to BMP and Retinoic Acid, are required for regeneration. Herein lies a major challenge: it is impossible to study regeneration in an organism which fails to reach adulthood due to developmental defects. To test if a given gene is required for regeneration, we use conditional mutagenesis to inactivate it in adult animals.
Over the years, we have been perfecting transposon-based gene trap vectors to ensure that they produce complete loss of function, or null, phenotypes when they integrate into genes. We engineered our gene traps to contain loxP sites for reversion using Cre recombinase (Balciuniene et al., 2013, and references therein). The next step was to equip a gene trap with modified loxP and FRT sites for fully conditional mutagenesis. We performed a pilot gene trap screen, among other recovering an integration into the Tbx5a gene, among others. Mutations in the human ortholog Tbx5 cause the Holt-Oram syndrome. Remarkably, zebrafish with impaired Tbx5a function display phenotypes remarkably similar to those seen in Holt-Oram patients. By conditionally inactivating tbx5a in adult cardiomyocytes, we have shown that Tbx5a is also required for cardiac regeneration in zebrafish (Grajevskaja et al., 2018). We are currently performing epitope tagging of Tbx5a (see below) in order to identify which critical downstream genes this transcription factor regulates to facilitate regeneration.
Emergence of highly efficient CRISPR/Cas9 mutagenesis tools prompted us to investigate if they could be adapted for precise modification of target loci. We found that CRISPR/Cas9 can be readily used to integrate epitope-coding sequences into zebrafish genes. Rather unexpectedly, we found that the C- and N-termini of the transcription factors we are interested in are quite highly evolutionarily conserved. We therefore decided to insert epitope-coding sequences into the “middle” of the gene, using somewhat lower levels of evolutionary conservation as a guide (Burg et al., 2016). We are currently epitope tagging several additional transcription factors.
Relatively high efficiency of oligonucleotide-mediated repair of double strand breaks induced by CRISPR/Cas9 has prompted us to adapt this method for conditional mutagenesis in zebrafish (Burg et al., 2018). We sequentially integrated two loxP sites into the tbx20 gene to engineer a conditional mutant, and converted a previously published fleer gene trap mutant (Balciuniene at al., 2013) into a conditional mutant by integrating a second loxP site. We are currently testing if tbx20 and/or fleer are required for regeneration, and completing the engineering of conditional aldh1a2 and tcf21 mutants.