Organs, limbs, fins and tails are made of multiple tissues whose growth is controlled by specific signals and genetic programmes. All these different cell populations must work together during development or regeneration to form a complete structure that is the right size in relation to the rest of the body. Growing evidence suggests that this synchronicity might be down to electric signals, which are created by movements of charged particles in and out of cells.

In particular, previous work has identified two factors that control the development of fins in fish: the Kcnk5b potassium-leak channel, which allows positive ions to cross the cell membrane; and an enzyme called calcineurin, which can modify the activity of proteins. Kcnk5b and calcineurin seem to play similar roles in the proportional growth of the fins in relation to the body, but exactly how was unknown.

To investigate this question, Yi et al. used genetically modified zebrafish to show how the Kcnk5b channel could control genes responsible for appendage growth. However, their tests on different cell types revealed that potassium movement through the Kcnk5b channel leads to different sets of developmental genes being turned on, depending on the tissue type of the cell. This could explain how one type of signal (in this case, movement of ions) can coordinate the growth of a wide range of tissues that use different combinations of developmental genes to form. Kcnk5b therefore appears to coordinate the regulation of the various combinations of genes needed for different fin tissues to develop, so that every component grows in a proportional, synchronized manner. Yi et al. also showed that calcineurin can modify the Kcnk5b channel to control its activity. In turn, this affects the movement of potassium ions across the membrane, changing electrical activity and, as a consequence, the proportional growth of the fin.

Further work should explore how Kcnk5b and calcineurin link to other signals that regulate the size of fins and limbs. Ultimately, a finer understanding of the molecules controlling the growth of body parts will be useful in fields such as regenerative medicine or stem cell biology, which attempt to build organs for clinical therapies.

Tissue scaling involves the coordinated control of developmental programs, since anatomical structures consist of different tissues that form in a coordinated manner and grow proportionally with each other and with the body. While there are several developmental signals known to regulate cell proliferation and tissue formation, mechanisms that concomitantly activate several developmental signals to synchronize the growth of multi-tissue appendages and organs in a manner that is coordinated with body proportions remain poorly defined.

There is growing evidence that several biological phenomena involved in tissue generation and growth are influenced by electrophysiological changes in ‘non-excitable’ cells (Sundelacruz et al., 2009). Several cell behaviors are affected by the addition of electric currents (McCaig et al., 2005): cell migration, cell proliferation, cell differentiation, gene transcription and consequently tissue formation are all altered by the application of an exogenous current (Baer and Colello, 2016; Bartel et al., 1989; Blackiston et al., 2009; Borgens et al., 1977; Geremia et al., 2007; Sundelacruz et al., 2009; Yasuda, 1974; Zhao et al., 2002). The culmination of these findings have led to the hypothesis that bioelectrical fields exist that have higher order organizational non-cell-autonomous properties in the development of anatomical structures (for review, see Levin, 2014; Messerli and Graham, 2011].

As a regulator of membrane potential, K⁺ conductance is an important component of the electrophysiological properties of cells. Evidence that illustrates the importance of K⁺ conductance in tissue formation comes from studies in which disruption of inward rectifying K⁺ channels of the Kir2 family can cause cranial facial defects, abnormal number of digits and reduced digit size (Andersen et al., 1971; Canún et al., 1999; Sansone et al., 1997; Tawil et al., 1994; Yoon et al., 2006a; Yoon et al., 2006b; Zaritsky et al., 2000). A striking finding concerning the coordinated control of cell behavior is the formation of eye structures by overexpressing different ion channels that alter membrane potential in early Xenopus embryos (Pai et al., 2012): overexpression and activation of a glycine-gated chloride channel in cells that form the eye interferes with eye formation, while overexpression of a dominant-negative K⁺-ATP channel simulates ectopic eye formation even in unexpected locations on the body (Pai et al., 2012). These findings illustrate that changes in the membrane potential of cells can have significant impacts on the development of anatomical structures. However, how electrophysiological information associates with the multiple necessary signals that control formation and/or growth of multi-tissue structures remains unclear.

The development of body structures not only involves forming tissues, it also involves coordinating the growth of each contributing tissue cell. To form organs that correctly scale with the body, each tissue grows either isometrically (grows with the same rate as the body) or allometrically (disproportionally grows in relation to the growth of the body). The zebrafish mutants another long fin (alf), long fin (lof), and schleier (schl) display continued allometric growth of each appendage from the juvenile stage into the adult stage (Lanni et al., 2019; Perathoner et al., 2014; Stewart et al., 2020). The dominant allometric growth phenotype of alf is due to mutations in the transmembrane pore region of kcnk5b (Perathoner et al., 2014), encoding a two-pore K⁺-leak channel that regulates membrane potential by outward flow of K⁺ from the cell (Goldstein et al., 2001). The dominant phenotype of lof is linked to elevated expression of a voltage-gated potassium channel Kcnh2a (Stewart et al., 2020). The phenotype of schl is due to dominant-negative mutations in kcc4 (Lanni et al., 2019), encoding a K⁺-Cl^- cotransporter that regulates intracellular K⁺ levels in a chloride-dependent manner (Marcoux et al., 2017). alf, lof, and schl demonstrate the importance of K⁺ conductance and ultimately of electrophysiological signals for the correct body-to-appendage proportions. Despite the connection between K⁺ conductance and the proportional growth of the fins, it remains unclear how K⁺-mediated signal translates into coordinated growth of the fish appendage and how any K⁺ channel is regulated to scale tissue.

We show that activity of the single two-pore K⁺-leak channel Kcnk5b is sufficient to induce the activation of at least two components, Shh and Lef1, of two important morphogen pathways, not only in the adult fin but also in the larva. Our data also indicates that this induction is cell autonomous, arguing that increases in membrane potential caused by Kcnk5b regulate growth through intracellular regulation of these developmental pathways. Overexpression of kcnk5b or one of two other two-pore kcnk channels in different mammalian cell lines showed variable activation of genes belonging to different developmental signal transduction mechanisms, supporting the conclusions that the different developmental mechanisms needed to scale fish appendages can be regulated by the same electrophysiological change induced by potassium leak and that the combinatorial activation of the different developmental mechanisms by Kcnk5b is cell-type dependent. Lastly, we provide evidence for how post-translational modification of Kcnk5b at Serine345 by calcineurin regulates its electrophysiological activity and consequently the scaling of zebrafish fins. Thus, we describe an endogenous cell-autonomous mechanism through which electrophysiological signals can induce and coordinate specific morphogen and growth factor signals to mediate the scaling of an anatomical structure.

Anatomical structures consist of a combination of different tissue types that develop and grow in a coordinated manner. Recent discoveries show that K⁺ channels regulate the scaling of fish appendages, but it is still unclear how this electrophysiological signal controls several diverse developmental phenomena within this anatomical structure to achieve coordinated developmental growth. Our results reveal that this in vivo electrical signal has a hierarchical effect on specific developmental genes in the fish fins and larva.

Two-pore K⁺-leak channels such as Kcnk5b allow K⁺ to cross the membrane to establish an electrochemical equilibrium, this activity directly affects the membrane potential of the cell (Goldstein et al., 2001). Normally, the concentration of K⁺ is higher on the cytoplasmic side of the plasma membrane due to continual active transport of K⁺ into the cell by the ATP-dependent Na⁺/K⁺ pumps (Shattock et al., 2015). As a leak channel, opening of Kcnk5b causes a flow of K⁺ out of the cell, which hyperpolarizes the membrane potential (Goldstein et al., 2001). The previous findings that mutations that increase Kcnk5b channel activity are associated with maintaining allometric growth (Perathoner et al., 2014) argue that such changes in membrane potential promote disproportional growth. Our current findings further these previous findings by showing that conditional induction of Kcnk channel activity is sufficient to induce morphogen pathways (Figures 1, 2 and 4) in different in vivo and in vitro contexts, demonstrating transcriptional control of particular developmental mechanisms by different two-pore K⁺-leak channels.

In addition to K⁺-leak channels, cells regulate intracellular K⁺ through different channels and exchangers. Inward rectifying K⁺ channels allow K⁺ to enter the cell along the ion’s electrochemical gradient. Exchangers will exchange K⁺ with different substrates (e.g. Na⁺) to facilitate the entry or removal of K⁺ based on the concentration gradient of K⁺ and the exchanged substrate. Previous findings show the importance of the inward rectifying K⁺ channel Kir2 for cranial-facial and digit defects in humans (Andersen et al., 1971; Canún et al., 1999; Sansone et al., 1997; Tawil et al., 1994; Yoon et al., 2006a; Yoon et al., 2006b). Knockout of the mouse Kir2 channels results in similar head and digit defects (Zaritsky et al., 2000), and dominant-negative inhibition of the Drosophila Kir2 leads to wing appendage defects that are analogous to the human and mouse appendage defects (Dahal et al., 2012). While the mammalian phenotypes remain unexplained, the defects in the Drosophila wings have been linked to reduced Dpp (BMP) signaling (Dahal et al., 2012), suggesting that intracellular K⁺ homeostasis is important for BMP signaling.

Removal of an ATP-sensitive K⁺ channel in the early Xenopus embryo disrupts eye formation, while ectopic expression of this channel will produce ectopic eyes in the head and in locations that were not considered to be competent for producing eyes (Pai et al., 2012). The ability to ectopically generate eyes was linked to electrophysiological hyperpolarization of the cells and the activation of Pax6-eyeless gene (Pai et al., 2012), a master regulator for eye development (Chow et al., 1999; Halder et al., 1995). In planaria, shortly after wounding, membrane depolarization acts as an early anterior signal that is sufficient (even when induced on the posterior side) to promote the consequent formation of all the anterior structures of the planarian head by inducing notum expression, which inhibits β-catenin-dependent Wnt signal transduction (Durant et al., 2019). Furthermore, it was recently revealed that in the Drosophila wing discs, depolarization events promote membrane localization of the transmembrane receptor smoothened to promote Shh signaling and that membrane potential values are patterned within the wing disc (Emmons-Bell and Hariharan, 2021). These discoveries show that electrophysiological changes are important signals in the formation and growth of anatomical structures.

A recent finding showed that calcineurin inhibition or increased activity of the voltage-gated Kcnh2 channel (lof mutant) promotes regenerative outgrowth from the proximal side of a mid-fin excavation, a site that normally does not mount a regenerative outgrowth response (Cao et al., 2021). Our findings help explain how calcineurin-regulated and electrophysiological changes from potassium channels can lead to broader tissue organizing phenomena by showing the inductive effect that increasing K⁺ conductance (and calcineurin’s regulation of K⁺ conductance) can have on a broad number of developmental pathways, which is important for coordinating the organized formation of tissues and organs. Furthermore, we posit that the effect of the activity of the Kcnk5b channel is broader than the traditional mechanisms of growth factor/morphogen signaling pathways, because it is not confined to specific signal transduction mechanisms; rather, it has variable broad effects, such as activation of several developmental signals in the adult fin (Figure 1), the larva (Figure 2) and different mammalian cell lines (Figure 4). While we observed that Kcnk5b is sufficient to promote lef1-mediated transcription (Figures 1–3), we did not observe direct activation of axin2, a down-stream target gene of β-catenin activity (Figure 1—figure supplement 1C). We posit that Kcnk5b activity does not directly activate canonical Wnt, but primes cells for increased β-catenin activity upon the reception of a Wnt signal through up-regulation of Lef1. Ultimately, continued Kcnk5b activity must lead to increased β-catenin-dependent Wnt signaling, since all evidence indicates that Wnt signaling is required for allometric fin outgrowth (Kawakami et al., 2006; Stoick-Cooper et al., 2007), and allometric fin growth is the phenotype that we get by kcnk5b or kcnk9 overexpression in amputated and unamputated fins (Figure 6E–L). We propose that the competence to activate different developmental pathways by electrophysiological changes is because the responding cells are either primed to activate them or the pathways are already active. It will be important to find out how this electrophysiological signal coordinates the activity of these developmental signals. In this regard, only few factors are known that regulate shh and lef1 transcription. Thus, our finding that an electrophysiological mechanism is involved not only provides a new understanding of how electrophysiology acts as an inductive signal, it also may lead to the discovery of molecular mechanisms that control the expression of these mediators of important morphogen signals.

The scaling activity of Kcnk5b includes all the tissues of the entire appendages of the fish (Perathoner et al., 2014). Previous findings implicate broader intercellular electrophysiological gradients as a mechanism for tissue growth (Adams and Levin, 2013). Electrophysiological measurements of animal tissues show that electric fields are generated and are important in vivo (Borges et al., 1979; Jenkins et al., 1996; McGinnis and Vanable, 1986), which suggests the existence of in vivo bioelectric information that regulates physiological phenomena. However, from our transplantation experiments, we observe that Kcnk5b’s effect is cell autonomous (Figure 3). Consequently, the question arises about how the activity of this K⁺-leak channel relates to a broad, coordinated phenotype of scaling the several tissues of the fin. An answer is that the autonomous transcriptional programs include morphogens. What is unclear is whether a limited number of cells in the fin control the growth and organizing information so that Kcnk5b only needs to act on a limited number of cell types, or whether Kcnk5b regulates proportional growth at multiple levels and that the cell autonomous transcriptional response that we observe is one outcome of a combination of intracellular and intercellular responses induced by Kcnk5b.

Changes in membrane potential from alterations in K⁺ conductance are also associated with the progression through the cell cycle (Blackiston et al., 2009; Urrego et al., 2014), because K⁺ channel activity increases at specific cell cycle phases (Urrego et al., 2014), and inhibition of K⁺ channel activity leads to cell cycle arrest in many different tissue cell types (Blackiston et al., 2009). It is possible that this phenomenon explains part of Kcnk5b’s ability to promote allometric growth. We do not yet know whether other phenomena linked to the activity of mammalian Kcnk5 [influence cell tonicity (Niemeyer et al., 2001), metabolic acidosis and alkalinization (Warth et al., 2004), CO₂/O₂ chemosensing in retrotrapezoid nucleus neurons (Flores et al., 2011) and apoptosis in lymphocytes and neurons (Göb et al., 2015; Nam et al., 2011)] are involved in appendage scaling.

We previously showed that calcineurin inhibition shifts isometric growth to allometric growth (Kujawski et al., 2014). Subsequently, Daane et al. showed that this effect is reversible in that removal of calcineurin inhibitors restores isometric growth. The authors also suggest an alternative mechanism for calcineurin regulation of Kcnk5b in its C-terminus (Daane et al., 2018); however, their model posits that active calcineurin promotes the activity of Kcnk5b, which does not yet explain how calcineurin inhibition and increased Kcnk5b activity each promote allometric growth (Kujawski et al., 2014; Perathoner et al., 2014). In either case, these data implicate calcineurin as a molecular switch governing isometric versus allometric growth control. Our findings provide a mechanism for how this switch acts to scale the fish appendages by directly regulating the activity of Kcnk5b through the dephosphorylation and phosphorylation of a specific serine (Figure 7). The ability to mimic or block calcineurin regulation of this K⁺-leak channel (Figure 5), whose activity levels directly translate into the extent of allometric growth (Figure 6), defines how calcineurin inhibition expands clonal populations during fin regeneration (Tornini et al., 2016). However, as we observed from both calcineurin inhibition (Kujawski et al., 2014) and from conditionally inducing Kcnk5b activity (Figure 6), the induced allometric growth of the entire fin is more than expanding clonal populations, since the outcome is not tumorigenesis. Instead, the growth is coordinated among all the tissues (Figure 6G–I,K,L; Kujawski et al., 2014; Perathoner et al., 2014), and our finding that Kcnk5b activates several developmental pathways (Figures 1, 2 and 4) argues that calcineurin activity acting on Kcnk5b regulates more than cell cycle progression.

Figure 7

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An important next step is to learn how the calcineurin-Kcnk5b circuit is integrated into the broader mechanisms that scale the appendages. Calcineurin is a Ca²⁺-dependent enzyme which suggests that intracellular Ca²⁺ is involved in scaling information. Ca²⁺ is a broad second messenger that can activate several downstream Ca²⁺-dependent enzymes, so broad changes in its subcellular levels likely have multiple effects. It remains unclear whether there is a specific intracellular distribution pattern that leads to calcineurin-mediated control of scaling. It is also possible that Ca²⁺-mediated activation of calcineurin—and consequent restoration of isometric growth—is so dominant that other Ca²⁺-mediated activities have little effect.

Two mechanisms that regulate proportional growth of organs are vitamin D and Hippo signaling. Increasing vitamin D signaling enhance the growth of the entire body, including the fins (Han et al., 2019). We propose that vitamin D is a systemic body signal that ultimately leads to the increase in Kcnk5b signaling. It is also possible that this hormone acts independently of Kcnk5b. In Drosophila, the Hippo pathway regulates brain size and size of the imaginal discs (Poon et al., 2016; Rogulja et al., 2008). Mice overexpressing a nuclear version of the Hippo-signaling component Yap1 in the adult liver develop significantly enlarged livers (Camargo et al., 2007; Dong et al., 2007). The Hippo signal transduction pathway consists of several core components that can be regulated by different factors at plasma membrane and within the cell (Yu and Guan, 2013), so there are several possible nodes of interaction between of Kcnk5b and Hippo cascade. It is also possible the Hippo-mediated transcription regulates kcnk5b expression or channel activity.

Connexin43 also regulates proportional growth of the fins, since mutations that reduce the intercellular connectivity of connexin43 produce adult fins that are half the size as the fins of wild-type siblings (Hoptak-Solga et al., 2007; Iovine et al., 2005). The connective nature of these intercellular junction proteins indicate that direct communication between intracellular compartments of tissue cells is an important component of the scaling mechanism of the fins. Our observation that Kcnk5b cell-autonomously activates the 7XTCF-Xla.sam:mCherry reporter (Figure 3) indicates that it is not due to intracellular transfer of K⁺. It is still unclear whether the disruption of intracellular trafficking of other ions (such as Na⁺ or Ca²⁺) or of other factors is responsible for the connexin43’s effect on scaling.

Kcnk5b’s ability to activate the Lef1-dependent reporter cell autonomously in different tissue types supports the conclusion that K⁺ conductance has the potential to regulate developmental transcription in a broad range of tissues (Figure 3B–E). The observation that neuronal cells in the brain and myocytes in the trunk muscle respond similarly to non-excitable cells elsewhere in the body suggests that even cells that harbor action potentials use K⁺ conductance to regulate gene expression. However, when we either stably expressed or transient transfected Kcnk5b in different cell lines, we did not observe consistent activation of Lef1 or Shh (Figure 4). We posit that the competencies of the cultured cell lines are different from the developmental competencies of the in vivo cells. Also, the mammalian cell lines are cancer cells, so they may already have an increased transcriptional base line for the selected developmental genes that the two-pore potassium-leak channels can only partially affect.

While we observed similarities between Kcnk5b and Kcnk9 or Kcnk10 in HEK cells, in Hela and N2A cells, we observed only partially similar profiles for the selected genes (while Kcnk9 and Kcnk10 were similar) (Figure 4). We postulate that the observed differences may come from one or both of the following explanations. First, different responses from channels of the same ion-type are due to different levels of membrane potential changes, which results in different levels of gene transcription. Second, these channels have different intracellular sequences, which may determine other unknown intermolecular interactions that have different signal transduction properties. In any case, we did observe that transgenic overexpression of Kcnk9 produces a similar allometric growth of the caudal fin (Figure 6K,L). It remains to be explored whether intermolecular interactions between the channels and another protein contribute to the scaling of other organs as well as how other electrophysiological mechanisms that control membrane potential have the same growth effect.

In conclusion, we show how a specific electrophysiological mechanism activates important morphogen pathways to scale tissues in different in vivo contexts. We propose the observed diversity in morphogen and growth factor expression to Kcnk5b activity explains why the increased activity of Kcnk5b produces the diverse transcriptional response in the different tissues associated with the observed coordinated outgrowth of the entire fin. Also, we show how changes in phosphorylation of S345 in the cytoplasmic C-terminus can be regulated by calcineurin to directly control electrophysiological activity of the channel to scale the fin. Thus, we offer an in vivo paradigm in which membrane potential acts as potent regulator of coordinated developmental signaling, and we show how the two-pore K⁺-leak channel Kcnk5b is involved in the initial activation of specific developmental mechanisms that lead to the scaling of the fish fin appendages.

All data generated are included in the manscript and files.

1. 1. Adams DS
   2. Levin M

   (2013) Endogenous voltage gradients as mediators of cell-cell communication: strategies for investigating bioelectrical signals during pattern formation

   Cell and Tissue Research 352:95–122.

   https://doi.org/10.1007/s00441-012-1329-4

   • PubMed
   • Google Scholar
2. 1. Andersen ED
   2. Krasilnikoff PA
   3. Overvad H

   (1971) Intermittent muscular weakness, Extrasystoles, and multiple developmental anomalies

   Acta Paediatrica 60:559–564.

   https://doi.org/10.1111/j.1651-2227.1971.tb06990.x

   • Google Scholar
3. 1. Baer ML
   2. Colello RJ

   (2016) Endogenous bioelectric fields: a putative regulator of wound repair and regeneration in the central nervous system

   Neural Regeneration Research 11:861–864.

   https://doi.org/10.4103/1673-5374.184446

   • PubMed
   • Google Scholar
4. 1. Balciunas D
   2. Wangensteen KJ
   3. Wilber A
   4. Bell J
   5. Geurts A
   6. Sivasubbu S
   7. Wang X
   8. Hackett PB
   9. Largaespada DA
   10. McIvor RS
   11. Ekker SC

   (2006) Harnessing a high cargo-capacity transposon for genetic applications in vertebrates

   PLOS Genetics 2:e169.

   https://doi.org/10.1371/journal.pgen.0020169

   • PubMed
   • Google Scholar
5. 1. Bartel DP
   2. Sheng M
   3. Lau LF
   4. Greenberg ME

   (1989) Growth factors and membrane depolarization activate distinct programs of early response gene expression: dissociation of fos and jun induction

   Genes & Development 3:304–313.

   https://doi.org/10.1101/gad.3.3.304

   • PubMed
   • Google Scholar
6. 1. Blackiston DJ
   2. McLaughlin KA
   3. Levin M

   (2009) Bioelectric controls of cell proliferation: ion channels, membrane voltage and the cell cycle

   Cell Cycle 8:3527–3536.

   https://doi.org/10.4161/cc.8.21.9888

   • PubMed
   • Google Scholar
7. 1. Borgens RB
   2. Vanable JW
   3. Jaffe LF

   (1977) Bioelectricity and regeneration. I. initiation of frog limb regeneration by minute currents

   Journal of Experimental Zoology 200:403–416.

   https://doi.org/10.1002/jez.1402000310

   • Google Scholar
8. 1. Borges RB
   2. Vanable JW
   3. Jaffe LF

   (1979) Reduction of sodium dependent stump currents disturbs urodele limb regeneration

   Journal of Experimental Zoology 208:377–386.

   https://doi.org/10.1002/jez.1402090304

   • Google Scholar
9. Book

   1. Brand M
   2. Granato M
   3. Nüsslein-Volhard C

   (2002) Keeping and raising zebrafish

   In: Nüsslein-Volhard C, Dahm R, editors. Zebrafish: A Practical Approach. Oxford University Press. pp. 7–38.

   https://doi.org/10.1086/382400

   • Google Scholar
10. 1. Camargo FD
    2. Gokhale S
    3. Johnnidis JB
    4. Fu D
    5. Bell GW
    6. Jaenisch R
    7. Brummelkamp TR

    (2007) YAP1 increases organ size and expands undifferentiated progenitor cells

    Current Biology 17:2054–2060.

    https://doi.org/10.1016/j.cub.2007.10.039

    • PubMed
    • Google Scholar
11. 1. Canún S
    2. Pérez N
    3. Beirana LG

    (1999) Andersen syndrome autosomal dominant in three generations

    American Journal of Medical Genetics 85:147–156.

    https://doi.org/10.1002/(SICI)1096-8628(19990716)85:2<147::AID-AJMG9>3.0.CO;2-0

    • PubMed
    • Google Scholar
12. 1. Cao Z
    2. Meng Y
    3. Gong F
    4. Xu Z
    5. Liu F
    6. Fang M
    7. Zou L
    8. Liao X
    9. Wang X
    10. Luo L
    11. Li X
    12. Lu H

    (2021) Calcineurin controls proximodistal blastema polarity in zebrafish fin regeneration

    PNAS 118:e2009539118.

    https://doi.org/10.1073/pnas.2009539118

    • PubMed
    • Google Scholar
13. 1. Chow RL
    2. Altmann CR
    3. Lang RA
    4. Hemmati-Brivanlou A

    (1999)

    Pax6 induces ectopic eyes in a vertebrate

    Development 126:4213–4222.

    • PubMed
    • Google Scholar
14. 1. Daane JM
    2. Lanni J
    3. Rothenberg I
    4. Seebohm G
    5. Higdon CW
    6. Johnson SL
    7. Harris MP

    (2018) Bioelectric-calcineurin signaling module regulates allometric growth and size of the zebrafish fin

    Scientific Reports 8:10391.

    https://doi.org/10.1038/s41598-018-28450-6

    • PubMed
    • Google Scholar
15. 1. Dahal GR
    2. Rawson J
    3. Gassaway B
    4. Kwok B
    5. Tong Y
    6. Ptácek LJ
    7. Bates E

    (2012) An inwardly rectifying K+ channel is required for patterning

    Development 139:3653–3664.

    https://doi.org/10.1242/dev.078592

    • PubMed
    • Google Scholar
16. 1. Dong J
    2. Feldmann G
    3. Huang J
    4. Wu S
    5. Zhang N
    6. Comerford SA
    7. Gayyed MF
    8. Anders RA
    9. Maitra A
    10. Pan D

    (2007) Elucidation of a universal size-control mechanism in Drosophila and mammals

    Cell 130:1120–1133.

    https://doi.org/10.1016/j.cell.2007.07.019

    • PubMed
    • Google Scholar
17. 1. Durant F
    2. Bischof J
    3. Fields C
    4. Morokuma J
    5. LaPalme J
    6. Hoi A
    7. Levin M

    (2019) The Role of Early Bioelectric Signals in the Regeneration of Planarian Anterior/Posterior Polarity

    Biophysical Journal 116:948–961.

    https://doi.org/10.1016/j.bpj.2019.01.029

    • Google Scholar
18. 1. Emmons-Bell M
    2. Hariharan IK

    (2021) Membrane potential regulates hedgehog signaling in the Drosophila wing imaginal disc

    EMBO Reports 22:e51861.

    https://doi.org/10.15252/embr.202051861

    • PubMed
    • Google Scholar
19. 1. Flores CA
    2. Cid LP
    3. Niemeyer MI
    4. Sepúlveda FV

    (2011) B lymphocytes taken to task: a role for a background conductance K2P K⁺ channel in B cells. focus on "Expression of TASK-2 and its upregulation by B cell receptor stimulation in WEHI-231 mouse immature B cells"

    American Journal of Physiology. Cell Physiology 300:C976–C978.

    https://doi.org/10.1152/ajpcell.00050.2011

    • PubMed
    • Google Scholar
20. 1. Geremia NM
    2. Gordon T
    3. Brushart TM
    4. Al-Majed AA
    5. Verge VMK

    (2007) Electrical stimulation promotes sensory neuron regeneration and growth-associated gene expression

    Experimental Neurology 205:347–359.

    https://doi.org/10.1016/j.expneurol.2007.01.040

    • Google Scholar
21. 1. Göb E
    2. Bittner S
    3. Bobak N
    4. Kraft P
    5. Göbel K
    6. Langhauser F
    7. Homola GA
    8. Brede M
    9. Budde T
    10. Meuth SG
    11. Kleinschnitz C

    (2015) The two-pore domain potassium channel KCNK5 deteriorates outcome in ischemic neurodegeneration

    Pflügers Archiv - European Journal of Physiology 467:973–987.

    https://doi.org/10.1007/s00424-014-1626-8

    • Google Scholar
22. 1. Goldstein SA
    2. Bockenhauer D
    3. O'Kelly I
    4. Zilberberg N

    (2001) Potassium leak channels and the KCNK family of two-P-domain subunits

    Nature Reviews Neuroscience 2:175–184.

    https://doi.org/10.1038/35058574

    • PubMed
    • Google Scholar
23. 1. Grigoriu S
    2. Bond R
    3. Cossio P
    4. Chen JA
    5. Ly N
    6. Hummer G
    7. Page R
    8. Cyert MS
    9. Peti W

    (2013) The molecular mechanism of substrate engagement and immunosuppressant inhibition of calcineurin

    PLOS Biology 11:e1001492.

    https://doi.org/10.1371/journal.pbio.1001492

    • PubMed
    • Google Scholar
24. 1. Halder G
    2. Callaerts P
    3. Gehring WJ

    (1995) Induction of ectopic eyes by targeted expression of the eyeless gene in Drosophila

    Science 267:1788–1792.

    https://doi.org/10.1126/science.7892602

    • PubMed
    • Google Scholar
25. 1. Han Y
    2. Chen A
    3. Umansky KB
    4. Oonk KA
    5. Choi WY
    6. Dickson AL
    7. Ou J
    8. Cigliola V
    9. Yifa O
    10. Cao J
    11. Tornini VA
    12. Cox BD
    13. Tzahor E
    14. Poss KD

    (2019) Vitamin D stimulates cardiomyocyte proliferation and controls organ size and regeneration in zebrafish

    Developmental Cell 48:853–863.

    https://doi.org/10.1016/j.devcel.2019.01.001

    • PubMed
    • Google Scholar
26. 1. Hoptak-Solga AD
    2. Klein KA
    3. DeRosa AM
    4. White TW
    5. Iovine MK

    (2007) Zebrafish short fin mutations in connexin43 lead to aberrant gap junctional intercellular communication

    FEBS Letters 581:3297–3302.

    https://doi.org/10.1016/j.febslet.2007.06.030

    • PubMed
    • Google Scholar
27. 1. Iovine MK
    2. Higgins EP
    3. Hindes A
    4. Coblitz B
    5. Johnson SL

    (2005) Mutations in connexin43 (GJA1) perturb bone growth in zebrafish fins

    Developmental Biology 278:208–219.

    https://doi.org/10.1016/j.ydbio.2004.11.005

    • PubMed
    • Google Scholar
28. 1. Jenkins LS
    2. Duerstock BS
    3. Borgens RB

    (1996) Reduction of the current of injury leaving the amputation inhibits limb regeneration in the red spotted newt

    Developmental Biology 178:251–262.

    https://doi.org/10.1006/dbio.1996.0216

    • PubMed
    • Google Scholar
29. 1. Kawakami Y
    2. Rodriguez Esteban C
    3. Raya M
    4. Kawakami H
    5. Martí M
    6. Dubova I
    7. Izpisúa Belmonte JC

    (2006) Wnt/beta-catenin signaling regulates vertebrate limb regeneration

    Genes & Development 20:3232–3237.

    https://doi.org/10.1101/gad.1475106

    • PubMed
    • Google Scholar
30. 1. Kujawski S
    2. Lin W
    3. Kitte F
    4. Börmel M
    5. Fuchs S
    6. Arulmozhivarman G
    7. Vogt S
    8. Theil D
    9. Zhang Y
    10. Antos CL

    (2014) Calcineurin regulates coordinated outgrowth of zebrafish regenerating fins developmental

    Cell 28:573–587.

    https://doi.org/10.1016/j.devcel.2014.01.019

    • Google Scholar
31. 1. Lanni JS
    2. Peal D
    3. Ekstrom L
    4. Chen H
    5. Stanclift C
    6. Bowen ME
    7. Mercado A
    8. Gamba G
    9. Kahle KT
    10. Harris MP

    (2019) Integrated K+ channel and K+cl- cotransporter functions are required for the coordination of size and proportion during development

    Developmental Biology 456:164–178.

    https://doi.org/10.1016/j.ydbio.2019.08.016

    • PubMed
    • Google Scholar
32. 1. Levin M

    (2014) Molecular bioelectricity: how endogenous voltage potentials control cell behavior and instruct pattern regulation in vivo

    Molecular Biology of the Cell 25:3835–3850.

    https://doi.org/10.1091/mbc.e13-12-0708

    • PubMed
    • Google Scholar
33. 1. Marcoux AA
    2. Garneau AP
    3. Frenette-Cotton R
    4. Slimani S
    5. Mac-Way F
    6. Isenring P

    (2017) Molecular features and physiological roles of K + -Cl − cotransporter 4 (KCC4)

    Biochimica Et Biophysica Acta (BBA) - General Subjects 1861:3154–3166.

    https://doi.org/10.1016/j.bbagen.2017.09.007

    • Google Scholar
34. 1. McCaig CD
    2. Rajnicek AM
    3. Song B
    4. Zhao M

    (2005) Controlling cell behavior electrically: current views and future potential

    Physiological Reviews 85:943–978.

    https://doi.org/10.1152/physrev.00020.2004

    • PubMed
    • Google Scholar
35. 1. McGinnis ME
    2. Vanable JW

    (1986) Electrical fields in Notophthalmus viridescens limb stumps

    Developmental Biology 116:184–193.

    https://doi.org/10.1016/0012-1606(86)90055-2

    • Google Scholar
36. 1. McLaughlin KA
    2. Levin M

    (2018) Bioelectric signaling in regeneration: mechanisms of ionic controls of growth and form

    Developmental Biology 433:177–189.

    https://doi.org/10.1016/j.ydbio.2017.08.032

    • PubMed
    • Google Scholar
37. 1. Messerli MA
    2. Graham DM

    (2011) Extracellular electrical fields direct wound healing and regeneration

    The Biological Bulletin 221:79–92.

    https://doi.org/10.1086/BBLv221n1p79

    • PubMed
    • Google Scholar
38. 1. Moro E
    2. Ozhan-Kizil G
    3. Mongera A
    4. Beis D
    5. Wierzbicki C
    6. Young RM
    7. Bournele D
    8. Domenichini A
    9. Valdivia LE
    10. Lum L
    11. Chen C
    12. Amatruda JF
    13. Tiso N
    14. Weidinger G
    15. Argenton F

    (2012) In vivo wnt signaling tracing through a transgenic biosensor fish reveals novel activity domains

    Developmental Biology 366:327–340.

    https://doi.org/10.1016/j.ydbio.2012.03.023

    • PubMed
    • Google Scholar
39. 1. Morokuma J
    2. Blackiston D
    3. Adams DS
    4. Seebohm G
    5. Trimmer B
    6. Levin M

    (2008) Modulation of potassium channel function confers a hyperproliferative invasive phenotype on embryonic stem cells

    PNAS 105:16608–16613.

    https://doi.org/10.1073/pnas.0808328105

    • PubMed
    • Google Scholar
40. 1. Nam JH
    2. Shin DH
    3. Zheng H
    4. Lee DS
    5. Park SJ
    6. Park KS
    7. Kim SJ

    (2011) Expression of TASK-2 and its upregulation by B cell receptor stimulation in WEHI-231 mouse immature B cells

    American Journal of Physiology-Cell Physiology 300:C1013–C1022.

    https://doi.org/10.1152/ajpcell.00475.2010

    • PubMed
    • Google Scholar
41. 1. Niemeyer MI
    2. Cid LP
    3. Barros LF
    4. Sepúlveda FV
    5. Niemeyer M
    6. Pena-Münzenmayer G

    (2001) Modulation of the Two-pore domain Acid-sensitive K+ channel TASK-2 (KCNK5) by changes in cell volume

    Journal of Biological Chemistry 276:43166–43174.

    https://doi.org/10.1074/jbc.M107192200

    • Google Scholar
42. 1. Pai VP
    2. Aw S
    3. Shomrat T
    4. Lemire JM
    5. Levin M

    (2012) Transmembrane voltage potential controls embryonic eye patterning in xenopus laevis

    Development 139:313–323.

    https://doi.org/10.1242/dev.073759

    • PubMed
    • Google Scholar
43. 1. Pai VP
    2. Lemire JM
    3. Chen Y
    4. Lin G
    5. Levin M

    (2015) Local and long-range endogenous resting potential gradients antagonistically regulate apoptosis and proliferation in the embryonic CNS

    The International Journal of Developmental Biology 59:327–340.

    https://doi.org/10.1387/ijdb.150197ml

    • PubMed
    • Google Scholar
44. 1. Perathoner S
    2. Daane JM
    3. Henrion U
    4. Seebohm G
    5. Higdon CW
    6. Johnson SL
    7. Nüsslein-Volhard C
    8. Harris MP

    (2014) Bioelectric Signaling Regulates Size in Zebrafish Fins

    PLOS Genetics 10:e1004080.

    https://doi.org/10.1371/journal.pgen.1004080

    • Google Scholar
45. 1. Poon CLC
    2. Mitchell KA
    3. Kondo S
    4. Cheng LY
    5. Harvey KF

    (2016) The Hippo Pathway Regulates Neuroblasts and Brain Size in Drosophila melanogaster

    Current Biology 26:1034–1042.

    https://doi.org/10.1016/j.cub.2016.02.009

    • Google Scholar
46. 1. Rogulja D
    2. Rauskolb C
    3. Irvine KD

    (2008) Morphogen Control of Wing Growth through the Fat Signaling Pathway

    Developmental Cell 15:309–321.

    https://doi.org/10.1016/j.devcel.2008.06.003

    • Google Scholar
47. 1. Sansone V
    2. Griggs RC
    3. Meola G
    4. Ptácek LJ
    5. Barohn R
    6. Iannaccone S
    7. Bryan W
    8. Baker N
    9. Janas SJ
    10. Scott W
    11. Ririe D
    12. Tawil R

    (1997) Andersen's syndrome: A distinct periodic paralysis

    Annals of Neurology 42:305–312.

    https://doi.org/10.1002/ana.410420306

    • Google Scholar
48. 1. Shattock MJ
    2. Ottolia M
    3. Bers DM
    4. Blaustein MP
    5. Boguslavskyi A
    6. Bossuyt J
    7. Bridge JHB
    8. Chen-Izu Y
    9. Clancy CE
    10. Edwards A

    (2015) Na+/Ca2+ exchange and na+/K+-ATPase in the heart

    Journal of Physiology 593:1361–1382.

    https://doi.org/10.1113/jphysiol.2014.282319

    • Google Scholar
49. 1. Shen Y
    2. Wu S-Y
    3. Rancic V
    4. Aggarwal A
    5. Qian Y
    6. Miyashita S-I
    7. Ballanyi K
    8. Campbell RE
    9. Dong M

    (2019) Genetically encoded fluorescent indicators for imaging intracellular potassium ion concentration

    Communications Biology 2:0269-2.

    https://doi.org/10.1038/s42003-018-0269-2

    • Google Scholar
50. Preprint

    1. Stewart S
    2. Bleu HKL
    3. Yette GA
    4. Henner AL
    5. Braunstein JA
    6. Stankunas K

    (2020) Longfin causes cis-ectopic expression of the kcnh2a ether-a-go-go-ectopic expression of the kcnh2a ether-a-go-go K+ channel to autonomously prolong fin outgrowth.

    bioRxiv.

    https://doi.org/10.1101/790329

    • Google Scholar
51. 1. Stoick-Cooper CL
    2. Weidinger G
    3. Riehle KJ
    4. Hubbert C
    5. Major MB
    6. Fausto N
    7. Moon RT

    (2007) Distinct wnt signaling pathways have opposing roles in appendage regeneration

    Development 134:479–489.

    https://doi.org/10.1242/dev.001123

    • PubMed
    • Google Scholar
52. 1. Sundelacruz S
    2. Levin M
    3. Kaplan DL

    (2009) Role of membrane potential in the regulation of cell proliferation and differentiation

    Stem Cell Reviews and Reports 5:231–246.

    https://doi.org/10.1007/s12015-009-9080-2

    • PubMed
    • Google Scholar
53. 1. Tawil R
    2. Ptacek LJ
    3. Pavlakis SG
    4. DeVivo DC
    5. Penn AS
    6. Ozdemir C
    7. Griggs RC

    (1994) Andersen's syndrome: potassium-sensitive periodic paralysis, ventricular ectopy, and dysmorphic features

    Annals of Neurology 35:326–330.

    https://doi.org/10.1002/ana.410350313

    • PubMed
    • Google Scholar
54. 1. Tornini VA
    2. Puliafito A
    3. Slota LA
    4. Thompson JD
    5. Nachtrab G
    6. Kaushik AL
    7. Kapsimali M
    8. Primo L
    9. Di Talia S
    10. Poss KD

    (2016) Live monitoring of blastemal cell contributions during appendage regeneration

    Current Biology 26:2981–2991.

    https://doi.org/10.1016/j.cub.2016.08.072

    • PubMed
    • Google Scholar
55. 1. Urrego D
    2. Tomczak AP
    3. Zahed F
    4. Stühmer W
    5. Pardo LA

    (2014) Potassium channels in cell cycle and cell proliferation

    Philosophical Transactions of the Royal Society B: Biological Sciences 369:20130094.

    https://doi.org/10.1098/rstb.2013.0094

    • Google Scholar
56. 1. Warth R
    2. Barrière H
    3. Meneton P
    4. Bloch M
    5. Thomas J
    6. Tauc M
    7. Heitzmann D
    8. Romeo E
    9. Verrey F
    10. Mengual R
    11. Guy N
    12. Bendahhou S
    13. Lesage F
    14. Poujeol P
    15. Barhanin J

    (2004) Proximal renal tubular acidosis in TASK2 K+ channel-deficient mice reveals a mechanism for stabilizing bicarbonate transport

    PNAS 101:8215–8220.

    https://doi.org/10.1073/pnas.0400081101

    • PubMed
    • Google Scholar
57. 1. Yasuda I

    (1974) Mechanical and electrical callus Clinical Studies

    Annals New York Academy of Sciences 238:457–465.

    https://doi.org/10.1111/j.1749-6632.1974.tb26812.x

    • Google Scholar
58. 1. Yoon G
    2. Quitania L
    3. Kramer JH
    4. Fu YH
    5. Miller BL
    6. Ptácek LJ

    (2006a) Andersen-Tawil syndrome: definition of a neurocognitive phenotype

    Neurology 66:1703–1710.

    https://doi.org/10.1212/01.wnl.0000218214.64942.64

    • PubMed
    • Google Scholar
59. 1. Yoon G
    2. Oberoi S
    3. Tristani-Firouzi M
    4. Etheridge SP
    5. Quitania L
    6. Kramer JH
    7. Miller BL
    8. Fu YH
    9. Ptáček LJ

    (2006b) Andersen-Tawil syndrome: prospective cohort analysis and expansion of the phenotype

    American Journal of Medical Genetics Part A 140A:312–321.

    https://doi.org/10.1002/ajmg.a.31092

    • Google Scholar
60. 1. Yu FX
    2. Guan KL

    (2013) The hippo pathway: regulators and regulations

    Genes & Development 27:355–371.

    https://doi.org/10.1101/gad.210773.112

    • PubMed
    • Google Scholar
61. 1. Zaritsky JJ
    2. Eckman DM
    3. Wellman GC
    4. Nelson MT
    5. Schwarz TL

    (2000) Targeted disruption of Kir2.1 and Kir2.2 genes reveals the essential role of the inwardly rectifying K(+) current in K(+)-mediated vasodilation

    Circulation Research 87:160–166.

    https://doi.org/10.1161/01.res.87.2.160

    • PubMed
    • Google Scholar
62. 1. Zhao M
    2. Pu J
    3. Forrester JV
    4. McCaig CD

    (2002) Membrane lipids, EGF receptors, and intracellular signals colocalize and are polarized in epithelial cells moving directionally in a physiological electric field

    The FASEB Journal 16:857–859.

    https://doi.org/10.1096/fj.01-0811fje

    • PubMed
    • Google Scholar

Acceptance summary:

The manuscript investigates the relationship between calcineurin and Kcnk5b in regulation of allometric versus isometric fin growth in zebrafish. The manuscript also presents interesting results supporting a cell-autonomous effect of Kcnk5b, rather than non-autonomous bioelectric action, proposed for channels by several earlier publications in the field. Towards understanding the molecular mechanism via which Kcnk5b activity regulates fin growth the authors provide evidence of changes in Wnt and Shh signaling and/or expression, providing a model for how Kcnk5b could exert its effects. Finally, Ser to Alanine and Ser to Glutamic acid mutations, – nice transgenic and cell culture work, support a model that regulation of Ser435 phosphorylation by Calcineurin regulates fin growth. Therefore, this work advances understanding of how potassium channels regulate allometric fin growth, which would be of interest to the scientific community.

Decision letter after peer review:

Thank you for submitting your article "An endogenous scaling mechanism that controls a K⁺-leak channel to regulate morphogen and growth factor transcription" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Lilianna Solnica-Krezel as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Marianne Bronner as the Senior Editor.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv (if it is not already there) along with this decision letter and a formal designation that the manuscript is "in revision at eLife". Please let us know if you would like to pursue this option. (If your work is more suitable for medRxiv, you will need to post the preprint yourself, as the mechanisms for us to do so are still in development.)

Summary:

Mechanisms regulating proportional growth during development and regeneration are poorly understood. The manuscript investigates the relationship between calcineurin and Kcnk5b in regulation of allometric versus isometric fin growth in zebrafish. The manuscript also presents interesting results supporting a cell-autonomous effect of Kcnk5b, rather than non-autonomous bioelectric action, currently prevailing model. Towards understanding the molecular mechanism via which Kcnk5b activity regulates fin growth the authors provide evidence of changes in Wnt and Shh signaling and/or expression, providing a model for how Kcnk5b could exert its effects. Finally, Ser to Alanine and Ser to Glutamic acid mutations in transgenic zebrafish and cell culture, support a model that regulation of Ser435 phosphorylation by Calcineurin regulates fin growth. This work advances understanding of how potassium channels regulate allometric fin growth.

Essential revisions:

The manuscript reports large amount of data, which is in general well presented. However, several conclusions require further experimental support, toning down and/or clarification. These and other concerns need to be addressed before the manuscript becomes suitable for publication.

1. In the manuscript, the control for the heat-shock Tg[hsp70:kcnk5b-GFP] transgenic animals is the transgenic line not subjected to heat shock. Since heat-shock is known to induce significant transcriptional changes, the authors need to repeat all experiments comparing heat-shocked wild-types with heat-shocked transgenics. This is particularly important since some of the results are unexpected (msx being up after 6, but not 12 hours, aldh1 strongly up after 6 but down after 12 hours).

2. The gene expression changes presented in Figure 1 are impressive. However, interpreting the changes in lef1 as evidence for alteration of Wnt signaling, and changes of mxb as alterations of BMP signaling, requires further support. While lef1 is a good direct Wnt target in many tissues, it can be regulated in the fin in a Wnt independent manner (Wehner et al. Cell Reports 2014). Moreover, it is not clear that msxb is only regulated by BMP signaling and thus could serve as readout for pathway activity. Similar to the larval experiments, authors should use a Wnt reporter to support their findings that Wnt signaling is regulated. For Hedgehog signaling, patched is often used as readout, and authors should also assay for its expression.

3. The authors conclude increased nuclear localization of b-catenin in Tg[hsp70:kcnk5b-GFP] upon HS, without altered overall b-catenin fluorescence intensity. However, in the presented panels anti-β catenin intensity appears higher in the HS transgenic tissue. Figure 1H: instead of showing single channels for the overview, single channels should be shown for the higher mag, so readers can assess themselves whether there is nuclear β-catenin. Also, please clarify the thickness of sections and whether we are looking at a regular IFF or confocal sections?

4. The mosaic experiments are interesting, but their description and documentation should be improved. Moreover, the conclusions are not fully supported by the data.

– Line 193-194 The authors state "Moreover, in all tissues, the ectopic mCherry expression was always limited to the Kcnk5b-positive cells (Figure 3B-D). However, the graph in Figure 3D, show many more mCherry positive cells than GFP positive cells? Isn't this consistent with the non-autonomous model? 10)

– Second, why are the numbers of mCherry+ cells much higher in 3 of the counted tissues in the graph in D than of the GFP+ cells? If this included endogenous mCherry expression, including endogenous expression domains is meaningless for the purpose of showing whether induction is cell-autonomous. Authors must concentrate on domains where there is no endogenous mCherry "background".

– Third, it needs to be considered that Wnt ligands induced by transgene expression might not spread far considering their hydrophobic nature. Thus, proving that there is no mCherry induction outside the GFP domain must be done at very high cellular resolution and not in those low-resolution images the authors provide.

– Figure 3A cartoon needs to be revised as the text states that "we raised mosaic embryos as larva (should be larvae), but the figure shows analyses of mosaic blastulae. When exactly at the larval stage heat shock has been applied? On line 185 the authors talk about "chimeric embryos" – please clarify. Also, please provide the number of experimental "chimeric animals (embryo or larvae)".

5. It's interesting that authors can detect increased growth of the fin fold relative to the body, although the channel and the presumed downstream signals are induced everywhere. So what about overall larval growth? Does this also increase?

6. Based on the results presented in Figure 4, the authors conclude that "Transfection of HEK cells with these two other channels resulted in the same transcriptional profile as Kcnk5b. However, similar changes were observed for SHH and PEA3, but not for LEF1, ALDH1a2 or MSX1, contrasting the results for kcn5b in HEK293T in Figure 4Bb. This undermines the authors' conclusion that "that the transcriptional response to Kcnk5b is a response to the electrophysiological changes associated with intracellular K⁺ 221 leak." To further test this, such comparative experiments should be performed also in HeLa and N2 cells, in which Kcn5b channel induces distinct transcriptional responses.

7. Authors assay for gene expression in stably transfected cells. Continuous expression of the channel might have many effects on the physiology / proliferation of the cells, which preclude meaningful conclusions from transcriptional changes. Experiments should also be performed with inducible expression systems.

8. The experiments testing the specificity of Calcineurin regulation of Kcnk5b are compelling as are the experiments testing the role of S345 and its phosphorylation. However, Ser345 phosphorylation can be only inferred from these experiments. To make this conclusion, a more direct evidence of S345 would be required. Therefore, it is an overstatement "we observed a linear relationship between the proportional length of the unamputated lobes and the phosphorylation status of the channels", this should be revised as "presumptive phosphorylation".

9. According to Figure 6B the difference between the proportional length of the unamputated lobes for wild-type Kcnk5b channel and the phosphomimetic S345E form is not significant. This could be due to relatively small number of experimental animals; however, the authors need to acknowledge this. It is also possible that in vivo effect of this mutation are not as strong as suggested by electrophysiology. The most elegant way to test effects of these mutations would be to edit them into the endogenous kcnk5b locus, although this would require significant amount of time and is not essential.

10. The authors conclude "kcnk5bS345E mutant was resistant to calcineurin-mediated inhibition (Figure 6D)". However, it appears that Calcineurin addition enhanced the mutant channel activity? Are these differences significant?

11. The number of experimental animals, and the number of experiments performed should be clearly indicated in figures or figure legends.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for submitting your article "An endogenous scaling mechanism that controls a K⁺-leak channel to regulate morphogen and growth factor transcription" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Marianne Bronner as the Senior Editor.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

We would like to draw your attention to changes in our policy on revisions we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, when editors judge that a submitted work as a whole belongs in eLife but that some conclusions require a modest amount of additional new data, as they do with your paper, we are asking that the manuscript be revised to either limit claims to those supported by data in hand, or to explicitly state that the relevant conclusions require additional supporting data.

Our expectation is that the authors will eventually carry out the additional experiments and report on how they affect the relevant conclusions either in a preprint on bioRxiv or medRxiv, or if appropriate, as a Research Advance in eLife, either of which would be linked to the original paper.

Essential revisions:

There was consensus among the reviewers that the revised manuscript was significantly improved. However, some conclusions in the Abstract and/or the main manuscript require tempering down and/or clarification. The following concerns need to be addressed before the manuscript becomes suitable for publication.

While the authors have now added the appropriate controls at least for some experiments, they do not adjust their conclusions based on the new data. While an upregulation is observed of lef1 and shh after 6 hours, albeit much less dramatically as when compared to non-heat shocked fish (about 1.5 to 2 fold with the appropriate new controls, compared to 30 to 40 fold in the old data), the effect after 12 hours has disappeared. In fact, some of the results like downregulation of lef1 after 12 hours have disappeared as well. However, the authors still state "The elevated expression of shh and lef1 continued 12 hours after the single pulse (Figure 1Ba Figure 1—figure supplement Figure 1B)". This is clearly not what the Figure 1—figure supplement Figure 1B.

The new results representing properly-controlled experiments should be moved to the main figures. These new results need to be appropriately incorporated into the authors conclusions.

Appropriate controls using heat-shocked WT without the Tg[hsp70:kcnk5b-GFP] transgene, should be also performed for other experiments, e.g. much of figure 2.

In their response letter, the authors agree with the reviewers that "lef1 activation may be independent" of Wnt signaling and alone cannot be used as a reporter for Wnt signaling. The authors changed some of the conclusions in the Results. However, the abstract still states "We show the activation of Kcnk5b is sufficient to activate several development programs, the most responsive of which are shh and lef1". "Developmental programs" is an overstatement and this needs to be rephrased. Similarly, the Abstract states "Kcnk5b can induce the expression of these developmental programs in cultured mammalian cell lines". The phrase "developmental programs" should be removed as it implies a gene or signaling cascade(s) for which there is insufficient evidence.

In response to the request to clarify whether data points in qPCR graphs are biological replicates, the authors claim they added that information, but it is very unclear. E.g in the legend to Figure 1 they write "…each experiment represents an N of 3 or more, with each N having 2 or more replicates". What does that mean? One is concerned that "replicates" are technical replicates and the impressively large sample sizes used for the qPCR experiments throughout the paper include technical replicates. In the response letter the authors state that sufficient number of biological replicates have been performed. This needs to be clearly presented in the manuscript.

Moreover, the authors agree with the reviewers that posttranslational modification of serine 345 in Kcnk5b is "presumptive" Yet, the Abstract states "We also demonstrate how post translational modification of serine 345 in Kcnk5b by calcineurin regulates channel activity and controls these developmental programs to scale the fin." The authors discuss their efforts to generate appropriate transgenic or genome edited lines to more directly test this presumptive serine modification. These are challenging experiments. However, without such experiments, one cannot make these definitive conclusions in the Abstract (or elsewhere in the manuscript).

Essential revisions:

The manuscript reports large amount of data, which is in general well presented. However, several conclusions require further experimental support, toning down and/or clarification. These and other concerns need to be addressed before the manuscript becomes suitable for publication.

1. In the manuscript, the control for the heat-shock Tg[hsp70:kcnk5b-GFP] transgenic animals is the transgenic line not subjected to heat shock. Since heat-shock is known to induce significant transcriptional changes, the authors need to repeat all experiments comparing heat-shocked wild-types with heat-shocked transgenics. This is particularly important since some of the results are unexpected (msx being up after 6, but not 12 hours, aldh1 strongly up after 6 but down after 12 hours).

We understand the concern, and we added new experiments that compare wild-type (AB background genetic strain) heat-shocked fish with Tg[hsp70:kcnk5b-GFP] additional heat-shocked fish. These data are included as Figure 1—figure supplement Figure 1, and they show the same gene activation trend.

We posit the differences between the 6 and 12 hour time points as evidence of a close relationship between Kcnk5b activity and the transcriptional activity of these genes. From our qRT-PCR experiments, we see almost all of the heat-shock-induced expression of the channel reduced to near background levels by 12 hours, which correlates with the mentioned reduction in the gene expression between 6 to 12 hours.

2. The gene expression changes presented in Figure 1 are impressive. However, interpreting the changes in lef1 as evidence for alteration of Wnt signaling, and changes of mxb as alterations of BMP signaling, requires further support. While lef1 is a good direct Wnt target in many tissues, it can be regulated in the fin in a Wnt independent manner (Wehner et al. Cell Reports 2014). Moreover, it is not clear that msxb is only regulated by BMP signaling and thus could serve as readout for pathway activity. Similar to the larval experiments, authors should use a Wnt reporter to support their findings that Wnt signaling is regulated. For Hedgehog signaling, patched is often used as readout, and authors should also assay for its expression.

As requested, we assess patched1, patched2 and bmp2 and observed up-regulation (Figure 1—figure supplement Figure 1H). We kept the reference of msxb to BMP signaling, because the increased bmp transcription that we did see although was slight, it was statistically significant. To address the reviewer’s point about β-catenin regulated transcription, we also examined the expression of other β-catenin-activated genes: axin2, cyclin D and myc, but we did not see upregulation (data placed in Suppl. Figure 1I). Thus, as the reviewer suggested might be the case, lef1 activation may be independent. These results altered our interpretation, and consequently, we have modified our text to focus on lef1 transcription and less on direct β-catenin-dependent Wnt activation. We still do see more β-catenin nuclear localization from the counted confocal images of the immunohistochemistry sections, and ultimately one can expect β-catenin-dependent Wnt to be activated indirectly, since the phenotype involves patterned fin growth even for the unamputated fins. We provide the following hypotheses to these observations in the Discussion section:

“While we observed that Kcnk5b is sufficient to promote lef1-mediated transcription (Figures 1-3), we did not observe direct activation of down-stream target genes of β-catenin activity (Figure 1—figure supplement Figure 1I). We posit that Kcnk5b activity does not directly activate canonical Wnt, but primes cells for increased β-catenin activity upon the reception of a Wnt signal through up-regulation of Lef1. Ultimately, continued Kcnk5b activity must lead to increased β-catenin-dependent Wnt signaling, since evidence indicates that it is required for fin outgrowth (Kawakami et al., 2006; Stoick-Cooper et al., 2007), which is the phenotype we get by Kcnk5b activation in amputated and unamputated fin (Figure E-I).”

3. The authors conclude increased nuclear localization of b-catenin in Tg[hsp70:kcnk5b-GFP] upon HS, without altered overall β-catenin fluorescence intensity. However, in the presented panels anti-β catenin intensity appears higher in the HS transgenic tissue. Figure 1H: instead of showing single channels for the overview, single channels should be shown for the higher mag, so readers can assess themselves whether there is nuclear β-catenin. Also, please clarify the thickness of sections and whether we are looking at a regular IFF or confocal sections?

We understand the reviewer’s concern. The sections are of 10 µm thick and the images are taken by confocal microscopy. Each confocal image as shown in Figure 1H is 0.45 μm. We now include this information in the figure legend: “Confocal planes (0.45 μm) of immunohistochemistry stained 10 μm sections of…” (lines 569-570). We decided to use the sections with the highest intensities and clearest histology for each group for the panels, but this clearly raised a discrepancy in difference in expression. We have gone through all of our stainings and now provide panels that are representative of our general observations and of our quantitative analyses. In addition, we added confocal serial sections as supplemental data (Figure 1—figure supplement Figure 2E-J) and high-light a few nuclei to show what we mean by nuclear β-catenin nuclear staining. We also provided the higher magnification image panels. Each point in the graph represents a combined total of β-catenin nuclear distribution for one confocal section. We sectioned > 6 fins (3 different experiments) for each group, so our quantitative analyses should reliably reflect what is occurring in fin.

4. The mosaic experiments are interesting, but their description and documentation should be improved. Moreover, the conclusions are not fully supported by the data.

– Line 193-194 The authors state "Moreover, in all tissues, the ectopic mCherry expression was always limited to the Kcnk5b-positive cells (Figure 3B-D). However, the graph in Figure 3D, show many more mCherry positive cells than GFP positive cells? Isn't this consistent with the non-autonomous model?

– Second, why are the numbers of mCherry+ cells much higher in 3 of the counted tissues in the graph in D than of the GFP+ cells? If this included endogenous mCherry expression, including endogenous expression domains is meaningless for the purpose of showing whether induction is cell-autonomous. Authors must concentrate on domains where there is no endogenous mCherry "background".

To clarify the point about ectopic versus normal expression of the mCherry reporter, the graphed mCherry positive cells lacking GFP are endogenous cells that normally expressed the reporter during embryonic/larval development. We included these cells to represent the cells normally expressing β-catenin-Wnt signaling, because we were concerned that if we didn’t, then an alternative argument could be made that there should be some cells that endogenously express the reporter, and there might be concern that the recipient fish were not transgenic for the reporter. However, we do see the review’s point. To address both issues better, we removed the measurements of the endogenous cells and reconfirmed the mCherry genotypes by qPCR on the fish parents and recipient fish harboring the donor cells. All the animals are homozygous for the mCherry reporter.

We are very certain about our conclusion that all ectopic mCherry expression is limited to only the cells expressing Kcnk5b-GFP. Even muscle, which does already express the reporter, shows that the muscle cells expressing Kcnk5b-GFP have elevated cell-autonomous expression of the Lef1-regulated mCherry compare to their neighbors lacking it (Figure 3Bj-l; 3Cr-t). We have provided more evidence from more transplantations as well as higher magnifications (Figure 3Da-c; Figure 3—figure supplement Figure 1Ia-Li) and three-dimensional composites (different axes rotations) from confocal z-stacks (Figure 3Dd-f; Figure 3—figure supplement Figure 1Gh,i-Jh,i) as additional evidence.

– Third, it needs to be considered that Wnt ligands induced by transgene expression might not spread far considering their hydrophobic nature. Thus, proving that there is no mCherry induction outside the GFP domain must be done at very high cellular resolution and not in those low-resolution images the authors provide.

We have provided higher resolution images of individual cells from several different examples and three-dimensional composites from z-stacks (Figure 3D; Figure 3—figure supplement Figure 1Ia-Li) to show that only cells harboring Kcnk5b-GFP induced mCherry expression.

– Figure 3A cartoon needs to be revised as the text states that "we raised mosaic embryos as larva (should be larvae), but the figure shows analyses of mosaic blastulae. When exactly at the larval stage heat shock has been applied? On line 185 the authors talk about "chimeric embryos" – please clarify. Also, please provide the number of experimental "chimeric animals (embryo or larvae)".

We have revised the figure cartoon to show make clear when the heat shocks were applied and that the cells were assessed in 48- and 72-hour old fish. By chimeric animals, we mean the recipient fish embryos that harbored the incorporated the transplanted donor cells. We specified this more clearly in the text. “we observed ectopic activation of 7XTCF-Xla.sam:mCherry reporter only in donor cells of chimeric 48 hpf and 72 hpf fish (recipient 7XTCF-Xla.sam:mCherry fish harboring transplanted cells from Tg[hsp70:kcnk5b-GFP;7XTCF-Xla.sam:mCherry] embryos).”

5. It's interesting that authors can detect increased growth of the fin fold relative to the body, although the channel and the presumed downstream signals are induced everywhere. So what about overall larval growth? Does this also increase?

To address this point, we provide body (nose-to-base of somatic musculature) measurements with our dorsal-to-ventral measurements. Unexpectedly, we observe that the body lengths are reduced by the kcnk5b expression. Despite this, the average lengths of the anterior-to-posterior finfolds are increased. We include these data in Figure 3—figure supplement Figure 1G and 2H. We speculate that the reduced body size stems from negative effects on different body systems such as the central nervous system and circulatory system. We had to modify the heat-shock method to perform a daily heat-shock protocol, since continued heat shock after the heart formation lead to significant mortality.

6. Based on the results presented in Figure 4, the authors conclude that "Transfection of HEK cells with these two other channels resulted in the same transcriptional profile as Kcnk5b. However, similar changes were observed for SHH and PEA3, but not for LEF1, ALDH1a2 or MSX1, contrasting the results for kcn5b in HEK293T in Figure 4Bb. This undermines the authors' conclusion that "that the transcriptional response to Kcnk5b is a response to the electrophysiological changes associated with intracellular K⁺ 221 leak." To further test this, such comparative experiments should be performed also in HeLa and N2 cells, in which Kcn5b channel induces distinct transcriptional responses.

We understand this point, and we modified our conclusions about the effects of Kcnk5b.

“However, when we either stably expressed or transient transfected Kcnk5b in different cell lines, we did not observe consistent activation of Lef1 or Shh (Figure 4). […] It remains to be explored whether intermolecular interactions between the channels and another protein contribute to the scaling of other organs as well as how other electrophysiological mechanisms that control membrane potential have the same growth effect”

We also did the requested transfection experiments for Kcnk9 and Kcnk10 in Hela and N2A cells, and we observed several profile differences between how Kcnk5b affects transcription and how Kcnk9 and 10 affect transcription from expression analyses. These data support the reviewer’s point. Consequently, we have modified the text in the Results section:

“When we tested whether Kcnk9 and Kcnk10 produce similar transcription profiles as Kcnk5b in HELA and N2A cells, we observed similar profiles between Kcnk9 and Kcnk10 (Figure 4H-L), but differences between their outcomes and the transcriptional outcome of Kcnk5b. […] We propose that the variability in genes that are transcribed may explain why the solitary change in the activity of this channel in all cells of the fin leads to the variable transcriptional responses that are needed to promote coordinated growth of a multi-tissue anatomical structure.”

And in the Discussion section:

“While we observed similarities between Kcnk5b and Kcnk9 or Kcnk10 in HEK cells, in Hela and N2A cells, we observed only partially similar profiles for the selected genes (while Kcnk9 and Kcnk10 were similar) (Figure 4). We postulate that the observed differences may come from one or more of the following explanations. First, different responses from channels of the same ion-type are due to different levels of membrane potential changes, which results in different levels of gene transcription. Second, these channels have different intracellular sequences, which may determine other unknown intermolecular interactions that have different signal transduction properties. In any case, we did observe that transgenic overexpression of Kcnk9 produces a similar allometric growth of the caudal fin (Figure 6K,L)”

7. Authors assay for gene expression in stably transfected cells. Continuous expression of the channel might have many effects on the physiology / proliferation of the cells, which preclude meaningful conclusions from transcriptional changes. Experiments should also be performed with inducible expression systems.

We added transient transfection experiments for Kcnk5b and they show the same responses, albeit, the expression levels are not as high. We included this data in Figure 4—figure supplement Figure 2A,B and referenced it in the text (seelines 227-228).

8. The experiments testing the specificity of Calcineurin regulation of Kcnk5b are compelling as are the experiments testing the role of S345 and its phosphorylation. However, Ser345 phosphorylation can be only inferred from these experiments. To make this conclusion, a more direct evidence of S345 would be required. Therefore, it is an overstatement "we observed a linear relationship between the proportional length of the unamputated lobes and the phosphorylation status of the channels", this should be revised as "presumptive phosphorylation".

We understand the reviewer’s point and revised the text to state “presumptive phosphorylation”.

9. According to Figure 6B the difference between the proportional length of the unamputated lobes for wild-type Kcnk5b channel and the phosphomimetic S345E form is not significant. This could be due to relatively small number of experimental animals; however, the authors need to acknowledge this. It is also possible that in vivo effect of this mutation are not as strong as suggested by electrophysiology. The most elegant way to test effects of these mutations would be to edit them into the endogenous kcnk5b locus, although this would require significant amount of time and is not essential.

We agree with this point, and we have increased the numbers to see whether the difference becomes statistically significant. By increasing the numbers, we do reach a statistical significance (Figure 6I). We posit that the lack of growth difference in the unamputated fin is due to the relatively low growth rate of uninjured fins.

We have tried to mutate the endogenous locus a few times using different CRISPR-based methods, but we as of yet have not been able to mutate the proline (serine not possible because the codon does not contain the required specific nucleotides for base switching to alanine/glutamic acid) or to induce recombination with a homologous sequences that contain the desired mutations. While we agree that this is experiment would be a good test, because of continued inability to target the endogenous site, we are thankful that this is not required.

10. The authors conclude "kcnk5bS345E mutant was resistant to calcineurin-mediated inhibition (Figure 6D)". However, it appears that Calcineurin addition enhanced the mutant channel activity? Are these differences significant?

The observed trends are not significant. We incorporated the results of the significance tests to show that apparent differences between any two groups are not significant.

11. The number of experimental animals, and the number of experiments performed should be clearly indicated in figures or figure legends.

We have rechecked each figure legend and placed this information in the legend. We have also provided the raw data values for each experiments in the accompanying files that eLife links to the figures.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Essential revisions:

There was consensus among the reviewers that the revised manuscript was significantly improved. However, some conclusions in the Abstract and/or the main manuscript require tempering down and/or clarification. The following concerns need to be addressed before the manuscript becomes suitable for publication.

While the authors have now added the appropriate controls at least for some experiments, they do not adjust their conclusions based on the new data. While an upregulation is observed of lef1 and shh after 6 hours, albeit much less dramatically as when compared to non-heat shocked fish (about 1.5 to 2 fold with the appropriate new controls, compared to 30 to 40 fold in the old data), the effect after 12 hours has disappeared. In fact, some of the results like downregulation of lef1 after 12 hours have disappeared as well. However, the authors still state "The elevated expression of shh and lef1 continued 12 hours after the single pulse (Figure 1Ba Figure 1—figure supplement Figure 1B)". This is clearly not what the Figure 1—figure supplement Figure 1B.

The new results representing properly-controlled experiments should be moved to the main figures. These new results need to be appropriately incorporated into the authors conclusions.

We understand the concerns and apologize for the misunderstanding. Since previously kept the original results in the main figure 1, so we maintained the original text. We have now replaced these figure panels with the previous supplementary panels as requested and have revised the text accordingly (lines 129-149). We have also replaced the mRNA in situ experiments for shh and lef1 (Figure 1E and F) with experiments that contain the heat-shock wild-type controls to make clear that heat shock alone does not induce the expression of these genes. The expression patterns are the same as the previous data (which showed that transgenic fish that were not heat shocked do not induce the expression of these genes).

Appropriate controls using heat-shocked WT without the Tg[hsp70:kcnk5b-GFP] transgene, should be also performed for other experiments, e.g. much of figure 2.

We added the requested controls to those figure panels that lacked them and joined them in one figure panel (Figure 2A). We have also provided new in situ results for shh expression the fish pectoral fin bud (Figure 2B) to further support the qRT-PCR results (now with the additional heat shock controls). The other figure panels already included non-transgenic heat-shocked fish.

In their response letter, the authors agree with the reviewers that "lef1 activation may be independent" of Wnt signaling and alone cannot be used as a reporter for Wnt signaling. The authors changed some of the conclusions in the Results. However, the abstract still states "We show the activation of Kcnk5b is sufficient to activate several development programs, the most responsive of which are shh and lef1". "Developmental programs" is an overstatement and this needs to be rephrased. Similarly, the Abstract states "Kcnk5b can induce the expression of these developmental programs in cultured mammalian cell lines". The phrase "developmental programs" should be removed as it implies a gene or signaling cascade(s) for which there is insufficient evidence.

We used the “developmental program” term, not only because of the activation of the genes important for development, but that ultimately the all developmental programs required for fin growth (which the formation/development of new tissues) must be activated to induce the allometric growth phenotype that we observe in Figure 6. However, we understand the concerns, so we made the requested changes and hope that our current conclusions address the issue.

In response to the request to clarify whether data points in qPCR graphs are biological replicates, the authors claim they added that information, but it is very unclear. E.g in the legend to Figure 1 they write "…each experiment represents an N of 3 or more, with each N having 2 or more replicates". What does that mean? One is concerned that "replicates" are technical replicates and the impressively large sample sizes used for the qPCR experiments throughout the paper include technical replicates. In the response letter the authors state that sufficient number of biological replicates have been performed. This needs to be clearly presented in the manuscript.

We apologize for the ambiguity. We detailed the representation of our data.

Moreover, the authors agree with the reviewers that posttranslational modification of serine 345 in Kcnk5b is "presumptive" Yet, the Abstract states "We also demonstrate how post translational modification of serine 345 in Kcnk5b by calcineurin regulates channel activity and controls these developmental programs to scale the fin." The authors discuss their efforts to generate appropriate transgenic or genome edited lines to more directly test this presumptive serine modification. These are challenging experiments. However, without such experiments, one cannot make these definitive conclusions in the Abstract (or elsewhere in the manuscript).

We apologize for missing this change. We have revised accordingly.

Author details

1. Chao Yi

   1. School of Life Sciences and Technology, ShanghaiTech University, Shanghai, China
   2. CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China

   Contribution

   Data curation, Formal analysis, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

2. Tim WGM Spitters

   School of Life Sciences and Technology, ShanghaiTech University, Shanghai, China

   Contribution

   Data curation, Investigation, Writing - review and editing

   Contributed equally with

   Ezz Al-Din Ahmed Al-Far

   Competing interests

   No competing interests declared

3. Ezz Al-Din Ahmed Al-Far

   Institut für Pharmakologie und Toxikologie, Technische Universität Dresden, Dresden, Germany

   Contribution

   Formal analysis, Investigation, Writing - review and editing

   Contributed equally with

   Tim WGM Spitters

   Competing interests

   No competing interests declared

4. Sen Wang

   1. School of Life Sciences and Technology, ShanghaiTech University, Shanghai, China
   2. CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China

   Contribution

   Investigation

   Competing interests

   No competing interests declared

5. TianLong Xiong

   1. School of Life Sciences and Technology, ShanghaiTech University, Shanghai, China
   2. CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China

   Contribution

   Investigation

   Competing interests

   No competing interests declared

6. Simian Cai

   School of Life Sciences and Technology, ShanghaiTech University, Shanghai, China

   Contribution

   Investigation

   Competing interests

   No competing interests declared

7. Xin Yan

   School of Life Sciences and Technology, ShanghaiTech University, Shanghai, China

   Contribution

   Investigation

   Competing interests

   No competing interests declared

8. Kaomei Guan

   Institut für Pharmakologie und Toxikologie, Technische Universität Dresden, Dresden, Germany

   Contribution

   Resources, Writing - review and editing

   Competing interests

   No competing interests declared

9. Michael Wagner

   1. Institut für Pharmakologie und Toxikologie, Technische Universität Dresden, Dresden, Germany
   2. Klinik für Innere Medizin und Kardiologie, Herzzentrum Dresden, Technische Universität Dresden, Dresden, Germany

   Contribution

   Formal analysis, Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

10. Ali El-Armouche

    Institut für Pharmakologie und Toxikologie, Technische Universität Dresden, Dresden, Germany

    Contribution

    Resources, Writing - review and editing

    Competing interests

    No competing interests declared

11. Christopher L Antos

    1. School of Life Sciences and Technology, ShanghaiTech University, Shanghai, China
    2. Institut für Pharmakologie und Toxikologie, Technische Universität Dresden, Dresden, Germany

    Contribution

    Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing - original draft, Project administration, Writing - review and editing

    For correspondence

    clantos@shanghaitech.edu.cn

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-8881-8568

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