A shocking connection: calcineurin interacts with bioelectric signalling to modulate allometric growth and final size of the regenerating zebrafish fin, without permanently changing positional identity
Achieving and maintaining body proportions during development and regeneration is critical for the correct performance of organisms. Allometric growth (as opposed to isometric) is characterized by differential growth of one part of the body relative to the rest, and it happens during certain stages of development (e.g. limb outgrowth) and during epimorphic regeneration, where only the regenerate grows. A fascinating feature of regeneration is that regardless of the amount of tissue lost, recovery of the original size takes roughly the same amount of time (i.e. proximally amputated limbs grow faster than distally amputated ones), suggesting that growth rate of the regenerate is guided by the positional information stored in the remaining structures. Intriguingly, bioelectricity seems involved in the control of zebrafish fin size during regeneration, as a gain-of-function mutation (alf)  in the potassium channel-encoding kcnk5b leads to fin overgrowth (although this gene is dispensable to achieve normal fin size), while a loss-of-function mutation in the gap junction-encoding connexin43 (sof)  leads to shorter than normal fins. Of importance, these changes in size tend to be caused by elongation or shortening of the equally spaced bony segments that form the ray of the fins (see Figure), rather than by changing he number of segments. Interestingly, it was recently shown that treating the regenerates with FK506 (a calcineurin inhibitor) increases the growth rate and the final size of the regenerated fin, apparently by promoting expansion of the proximal domain . It remains unclear to what extent patterning and size control mechanisms are coupled, and what the underlying mechanisms might be.
Daane et al. explore the relationship between patterning and growth control mechanisms during fin regeneration by combining size-affecting mutations with FK506 treatment. They make four electrifying discoveries:
The effect of calcineurin inhibition on fin size and growth rate is independent of the intrinsic “size memory”. Indeed, FK506-treated WT and sof regenerates grow to similarly sized, longer-than-normal fins, while treated alf fins do not show any increase ingrowth rate or size as compared to untreated alf
The growth effects of FK506 are suppressed by loss-of-function mutations in kcnk5b. They also find that FK506 does not induce expression of kcnk5b, suggesting that the drug affects activity of the channel.
FK506 treatment leads to decreased conductance in cells expressing zebrafish kcnk5b, suggesting interaction between calcineurin and the potassium channel. Moreover, when a critical residue of the putative calcineurin binding site in KCNK5B is mutated, conductance is as low as with WT channels treated with FK506, and no longer affected by FK506 treatment. Aptly, CRISPR-generated truncation of KCNK5B before the calcineurin binding site leads to overgrowth of the targeted somatic clones.
When the authors resected fins that had overgrown due to previous treatment with FK506, but that were no longer treated, the new size reached was never longer than the original pre-treatment size. The authors conclude that FK506 does not respecify positional identity, but rather enhances fin growth rate.
What I like about this preprint
This study shows for the first time that bioelectric and calcineurin signalling interact during fin regeneration, revealing a key node in the interdependence between growth rate and positional information. Their finding that this interaction is epistatic to the mechanisms that instruct overall size has important implications for regenerative medicine.
1) Kujawski et al. proposed that calcineurin inhibition re-specifies positional information because they observed upregulation of retinoic acid (RA) machinery (a marker of proximal blastemas) and distal displacement of the ray branching points . If FK506 just increases growth rate, as Daane et al. propose, could it be that RA, which is required for blastema formation and proliferation  is the growth-promoting signal? What is the status of RA signalling in alf mutants?
2) If the time required for regeneration does not change between FK506-treated and untreated animals, the prediction of the authors’ model would be that the bony segments of all regions of the fin distal to the amputation plane are elongated (Figure 1c), as opposed to only the proximal ones if positional information is re-specified (Figure 1b). Is that the case? Interestingly, the authors show here and in  that in alf mutants most bony segments are elongated.
3) Both increased and decreased conductance due to mutations of kcnk5b have the same growth-enhancing effect. The authors speculate that both changes in conductance may result in similar regulation of calcineurin activity, but this has to be proven experimentally.
4) Although pectoral fins present the advantage of being paired, which the use of the unmanipulated one as internal control, they are problematic, as they do not regenerate as efficiently in males as in females . Did the authors study just females?
The diversity of forms in Nature is frequently derived from variation in the proportions of organs and tissues. The mechanisms underlying the ability of organs and tissues to attain proper proportions are generally unknown. Through systematic genetic screens we have identified potassium channel signaling as being important for specifying fin size in the zebrafish. Getting a handle on how changes in conductance and/or membrane polarity could regulate such patterning across a structure was difficult, in part, as connections between tissue-level changes in polarity and that of classical signaling pathways active in development have not been easily joined. Work from Christopher Antos’ group showed that inhibition of the activity of calcineurin modulates size of fins in zebrafish. They postulated that changes in calcineurin affected proximal fate determination leading to perdurance of that fate and larger fins. What is surprising to us is that we were able to show that bioelectric signaling through the potassium channel Kcnk5b is required for the growth caused by calcineurin inhibition. Additionally, we show that these factors specify fin growth in a process that is not driven by inherent changes in positional identity. We suggest that this signaling node acts as a molecular rheostat, able to integrate bioelectric and calcium-mediated signaling systems to dial relative growth rates guiding ultimate fin size.
Answer to specific comments
Quite interesting follow up. We did try RA treatment, similar to the axolotl work of Maden. However, unfortunately RA causes arrest of the blastemal and even in release there is unfortunate pathology that makes analysis of pattern complicated.
You are right. All segments are longer, as described in the original paper about the alf mutants (Perathoner 2014).
Oddly enough, we never see male suppression of regeneration. This maybe a strain specific thing, and the extent of this is unclear. This fact led us to also back up the study using caudal fin – which exhibits the same effect. Thus, we have not separated sex.
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