A calcineurin-mediated scaling mechanism that controls a K⁺-leak channel to regulate morphogen and growth factor transcription

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.

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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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