Top rows: The caudal fin of adult zebrafish regenerates rapidly after injury. Bottom row: Within 2 weeks the bony fin rays grow back to their initial size.
Retinoic acid signaling in the regenerating zebrafish caudal fin
The ability of adult vertebrate animals to completely regenerate organs after injury or amputation is limited to fish, salamanders and to lizards with evolved mechanisms of tail autotomy. It is an important goal to understand the molecular and cellular processes that allow adult tissue regeneration in these animals, and to understand why the ability for regeneration in humans is only very limited.
Zebrafish caudal fin regeneration requires proliferation of cells that originate in the stump tissue at the amputation surface. They form a proliferating tissue, the blastema, that gradually reconstitutes the amputated structures. Retinoic acid (RA) is a small molecule that acts as an activator of the expression of a number of target genes that are important for development, homeostasis and regeneration.
RA has a long history of interests in regeneration research for its effects on regenerating limbs in salamanders: In short, exogenously added RA respecifies the positional identity of the blastema towards more proximal cell fates. Because the resulting limb contains supernumerary regions, the effect of RA on limb regeneration has been termed "super-regeneration". Interestingly, it has long been neglected to show if RA plays a role in normal regeneration, i.e. is there a phenotype of regenerated limbs that is associated with the loss of endogenous RA?
We addressed this question and showed that controlled RA treatment does not block blastema formation, but rather enhances proliferation in the fin stump. Genetic loss-of-function experiments demonstrate that RA signaling controls proliferation during blastema formation by integrating molecular signals that both stimulate and inhibit proliferation. We also uncovered an unanticipated role for RA to maintain the fast cycling blastemal population in the adult fish by protecting them from cell death.
In current and future experiments, we are examining the effects that changes in RA signaling have on the various cell types in the fin, including the regeneration of bony fin rays. Our studies are important in showing that blastemal proliferation, the hallmark of epimorphic regeneration, is dependent on RA. This will be informative for other regeneration models, such as the zebrafish heart, in which the RA signaling pathway is activated and likely plays a similar role in controlling proliferation.
GENETIC DIFFERENCES BETWEEN FORE- AND HINDLIMB DEVELOPMENT
Fore- and hindlimbs in vertebrates are homologous structures whose development is regulated mostly by the same signaling pathways. Nevertheless, a number of key genes are differently involved in the development of both limb types and it currently is debated whether retinoic acid (RA) signaling is required at all for the formation of pelvic fins.
We are investigating whether RA is required for hindlimb development using genetic means to manipulate its signaling activity. Furthermore, we have identified mutant fish that exclusively lack pelvic fins and displays defects in the posterior body. We are working at identifying the underlying mutations using pharmacological inhibitors to some of the candidate genes, as well as rescue experiments that supply either candidate gene alone to mutants. This project will identify new genes that specifically regulates hindlimb development, with implications for understanding how positional differences in the vertebrate body axis become manifested.
Development of RA-reporter lines
We are developing reporter lines that detect changes in RA signalling in developing and regenerating cells. On the one hand, we aim to detect the presence of RA directly through constructs that respond to cellular changes in RA availability. On the other hand, we have established BAC-recombineering to produce transgenic fish that faithfully recapitulate gene expression of the RA synthetizing enzymes in zebrafish. These lines will become essential tools in the analyses of RA signaling regulation.