Mechanisms Underlying Influence of Bioelectricity in Development

To execute the intricate process of development, cells coordinate across tissues and organs to determine where each cell divides and differentiates. This coordination requires complex communication between cells. Growing evidence suggests that bioelectrical signals controlled via ion channels contribute to cell communication during development. Ion channels collectively regulate the transmembrane potential of cells, and their function plays a conserved role in the development of organisms from flies to humans. Spontaneous calcium oscillations can be found in nearly every cell type and tissue, and disruption of these oscillations leads to defects in development. However, the mechanism by which bioelectricity regulates development is still unclear. Ion channels play essential roles in the processes of cell death, proliferation, migration, and in each of the major canonical developmental signaling pathways. Previous reviews focus on evidence for one potential mechanism by which bioelectricity affects morphogenesis, but there is evidence that supports multiple different mechanisms which are not mutually exclusive. Evidence supports bioelectricity contributing to development through multiple different mechanisms. Here, we review evidence for the importance of bioelectricity in morphogenesis and provide a comprehensive review of the evidence for several potential mechanisms by which ion channels may act in developmental processes.

Keywords: bioelectricity, ion channels, signaling, signaling pathways, prolifieration, apoptosis

The process by which a single fertilized egg develops into a multicellular organism is a remarkable feat of biology. For most plants and animals, the fertilized egg must undergo dozens of repeated divisions with various lineages of cells proliferating, migrating, and differentiating at exactly the correct times and places within 3D space to form the specialized tissues and organs of the adult organism. This process requires a vast amount of information to be transmitted and processed for the organism to form correctly, and yet this process occurs in every multicellular species.

Development is robust, with organisms and tissues able to withstand damage or induced errors during development and still ultimately develop correctly. This remarkable ability to develop correctly after perturbation can be seen in the development of twins. In the early stages of mammalian development embryos can be completely split into two, and each half can go on to produce a fully developed organism. This splitting can even occur spontaneously as late as 14 days post-fertilization in human embryos and result in the correct development of a set of twins (Hall, 2003). Thus, the process of development is not simply an unfolding of a single developmental pathway encoded rigidly within genetics. Developing organisms can also correct early damage. The imaginal discs in developing Drosophila can regenerate after damage or ablation during early development and go on to form functional adult appendages (Smith-Bolton et al., 2009). Severe morphological abnormalities can be induced in Xenopus during early craniofacial development and yet go on to later self-correct (Vandenberg et al., 2012; Pinet et al., 2019). This amazing ability of cells and tissues to respond to environmental changes and develop needed structures can additionally be seen in regenerating organisms. Planarians, zebrafish, Xenopus, and axolotls can regenerate entire damaged or amputated organs and limbs (Reddien and Alvarado, 2004; Roy and Gatien, 2008; Gemberling et al., 2013; Phipps et al., 2020). An extreme example can be seen in Hydra vulgaris (freshwater polyps) which are able to completely reaggregate and regenerate from dispersed cells in suspension (Gierer et al., 1972).

This extraordinary ability of tissues and cells to develop correctly even when facing environmental perturbations raises one of the fundamental questions of developmental biology: how do cells communicate and coordinate in a tissue-wide manner to guide development? How does each cell know when and where to proliferate, migrate, and differentiate even in the face of perturbation?

Much of this tissue wide coordination is attributed to the morphogen signaling pathways. These morphogens, including members of the bone morphogenetic protein (BMP) pathway, Wnt pathway, Hedgehog pathway, are secreted proteins that form a concentration gradient across tissues, giving cells positional information based on the concentrations of the various morphogens. According to the morphogen concentration hypothesis, the precise concentration of each of these morphogens activates various genetic pathways that tell each cell what type of cell to differentiate into and where to differentiate (Rogers and Schier, 2011). While morphogen signaling and other canonical signaling pathways (such as Notch signaling) help explain how cells can communicate with each other across space, there is still much that is not understood about how cells precisely coordinate the spatial distribution as well as timing of cellular processes required for development. How exactly morphogen gradients are regulated is a growing question in the field, as the passive diffusion model does not adequately explain gradient formation. Recently, there has been growing evidence that in addition to the classic molecular developmental signaling pathways that cells use to coordinate development, cells use electrical signaling via ion channels to communicate (Harris, 2021; Levin, 2021). This field of research, known as developmental bioelectricity, is growing rapidly. Here, we review the evidence that ion channels are important for development in humans and other organisms, the potential mechanisms by which ion channels may be regulating development, and the next steps and barriers within the field of developmental bioelectricity.

Ion channels sit within the cell membrane or organelle membranes of the cell and help regulate the levels of calcium, sodium, potassium, chloride, and other charged molecules within the cytoplasm and the other compartments in the cell. The difference in ion concentrations within the cell and in the extracellular space creates a transmembrane potential (Vmem). All cells have a resting membrane potential, but the exact value of Vmem varies by cell type. In general, differentiated cells are hyperpolarized relative to stem cells (Sundelacruz et al., 2009). Rapid changes in Vmem are essential for the functioning of excitable cells such as neurons and myocytes as these changes induce the release of neurotransmitters or contraction of muscle cells. In excitable cells these rapid changes are called action potentials. Action potentials occur when an influx of positively charged sodium depolarizes the cell, activating voltage-gated calcium channels that mediate calcium entrance. The influx of calcium activates calcium-sensitive proteins that then mediate neurotransmitter containing vesicle fusion. Eventually, potassium channels open to allow an efflux of positively charged potassium to repolarize the membrane.

While non-excitable cells do not exhibit the same rapid changes in Vmem that exitable cells do, there is growing evidence that there are Vmem patterns and slow changes in Vmem across developing cells that influence development (Levin, 2014; Harris, 2021; Levin, 2021). Regions of relatively depolarized and hyperpolarized cells have been found within multiple different developing organisms from flies to mammals, suggesting that Vmem patterns play a conserved role in development (Figure 1). In the Drosophila larval wing disc, a stripe of cells along the anterior posterior (A/P) boundary is depolarized relative to the other cells and this Vmem pattern is important for wing development (Emmons-Bell and Hariharan, 2021). In developing mouse and chick limb buds, regions of the limb undergoing chondrogenesis shift from a relatively hyperpolarized state to a relatively depolarized state over time as chondrogenesis occurs (Atsuta et al., 2019). Disruption of this depolarized state hinders chondrogenesis (Atsuta et al., 2019). A similar patterning in Vmem can be found in developing Xenopus embryos, where dynamic regions of hyperpolarized and depolarized cells in the ectoderm change throughout development (Vandenberg et al., 2011). Clusters of hyperpolarized cells in developing Xenopus mark the developing eyes, and perturbation of this pattern disrupts eye formation (Pai et al., 2012). In developing mouse and chick limb buds, regions of the limb undergoing chondrogenesis shift from a relatively hyperpolarized state to a relatively depolarized state over time as chondrogenesis occurs (Atsuta et al., 2019). These studies suggest that Vmem may play a role in development and a wide number of studies confirm that disrupting ion channels—which collectively control the Vmem—leads to developmental defects.

Ion channel mediated electrical signaling is essential for the proper development of many organisms ranging from planarians to humans. In humans, a set of syndromes known as channelopathies are associated with mutations in ion channel genes. Most of these channelopathies lead to defects in the functioning of the heart or the brain, consistent with the known important roles of ion channels in those organs. However, many of these channelopathies are also associated with morphological defects, suggesting that ion channels play a role in the development of organs and tissues that is not limited to those tissues traditionally associated with ion channel function such as the brain. For example, Andersen-Tawil Syndrome, a channelopathy associated with a mutation in the inwardly rectifying potassium channel Kir2.1, leads to multiple morphological and craniofacial defects including short stature, low-set ears, small-lower jaw, cleft palate, clinodactyly, and syndactyly (Tawil et al., 1994; Plaster et al., 2001; Perez-Riera et al., 2020). Disruption of the homologous channel in Xenopus, Drosophila melanogaster, and mice also leads to developmental defects within those organisms, suggesting a conserved role for Kir2.1 in development (Dahal et al., 2012; Adams et al., 2016; Dahal et al., 2017; Belus et al., 2018). Timothy Syndrome, caused by gain-of-function mutations in the calcium channel Cav1.2, is associated with multiple developmental defects including fusion of the digits of the hands or feet (syndactyly) and craniofacial defects in humans (Splawski et al., 2004; Splawski et al., 2005). Mouse and zebrafish models of Timothy Syndrome recapitulate the craniofacial defects, suggesting a conserved role for Cav1.2 in development (Ramachandran et al., 2013).

Other human syndromes that are associated with ion channel mutations and that lead to morphological defects include Temple-Baraitser Syndrome, associated with mutations in the voltage-gated potassium channel KCNH1, Birk-Barel Syndrome, associated with mutations in the two-pore domain potassium channel KCNK9, Keppen-Lubinsky Syndrome, associated with mutations in the inwardly rectifying potassium channel KCNJ6, and CLIFAHDD Syndrome, associated with mutations in the sodium leak channel NALCN (Barel et al., 2008; Chong et al., 2015; Masotti et al., 2015; Simons et al., 2015). Each of these syndromes lead to various craniofacial and digital defects (Barel et al., 2008; Chong et al., 2015; Masotti et al., 2015; Simons et al., 2015).

Outside of syndromic channelopathies there is additional evidence that disruption of ion channel function can lead to developmental defects within specific tissues and organs. For example, cystic fibrosis is caused by disruption of the epithelial chloride channel cystic fibrosis transmembrane regulator (CFTR) (Davies et al., 2007). Disruption of CFTR leads to a reduced ability of the lungs to clear bacteria and the production of viscous mucus that disrupts proper lung functioning (Davies et al., 2007). Recent evidence, however, also indicates that CFTR is required for proper development of the lungs (Larson and Cohen, 2005; Amaral et al., 2020). CFTR is expressed at very high levels during fetal lung development, and patients with cystic fibrosis present with abnormal lung development as early as 17–19 weeks gestation (Gosden and Gosden, 1984; Larson and Cohen, 2005). In CFTR knockout mice, transient expression of a normal copy of CFTR in utero rescues lethality and some of the lung and intestinal phenotypes of cystic fibrosis even when this CFTR is no longer expressed after birth, suggesting that CFTR is particularly important during development (Larson et al., 2000a). Overexpression of CFTR in both mice and primates leads to increases in proliferation and differentiation of fetal lung secretory cells, suggesting that CFTR contributes to timing of proliferation and differentiation within the lung during development (Larson et al., 2000a; Larson et al., 2000b). The identification of multiple human syndromes and developmental defects associated with ion channel disruption is just one of the many lines of evidence suggesting that ion channels are essential for development.

Disruption of ion channel function has been associated with developmental defects within many non-human organisms, suggesting that ion channels play a conserved role in morphogenesis. Dozens of ion channel mutations impacting ion channels of nearly every category have been linked to developmental defects in worms, flies, frogs, fish, and mice (Pai et al., 2012; George et al., 2019; Srivastava et al., 2021). Ion channels are important for regulating both the size and patterning of various tissues. In zebrafish, mutations in the potassium channel gene kcnk5b lead to enlarged fins while mutations in the gap junction gene connexin43 lead to shorter fins, suggesting that ion channel signaling is important for regulating the growth and proportional size of the fins (Iovine et al., 2005; Perathoner et al., 2014; Daane et al., 2018). Mutations in many different calcium, potassium, sodium, and chloride channels in Caenorhabditis elegans have been associated with changes in body length or girth, suggesting that ion channels also regulate body size in C. elegans (Srivastava et al., 2021). In D. melanogaster a screen of wing development identified 44 ion channels important for regulating both wing size and vein patterning, suggesting bioelectrical signaling is important for the overall development of the wing (Dahal et al., 2012; George et al., 2019). Loss of Kir2.1 in mice, the inwardly rectifying potassium channel associated with Anderson-Tawil Syndrome in humans, causes craniofacial and digital defects, suggesting that Kir2.1 is important for patterning of those structures (Dahal et al., 2012; Belus et al., 2018). Injection of a dominant-negative form of the inwardly rectifying potassium channel Kir2.1 in frogs also leads to abnormal craniofacial development (Adams et al., 2016).

These craniofacial defects are recapitulated by expression and activation of a light-activated cation channel or a light activated hydrogen pump, suggesting that the role of ion channel function in craniofacial development is not limited to Kir2.1 (Adams et al., 2016). The myriad of ion channels that contribute to morphogenesis in organisms ranging from worms to humans supports the hypothesis that bioelectricity plays an important role in guiding development.

Interestingly, ion channels are important for the establishment of anterior-posterior polarity and tissue identity. For example, trunk fragments of planarians (planarians with both tail and head amputated) usually regenerate both the head and the tail at the proper ends. However, brief treatment of trunk fragments with 8-OH, a gap junction inhibitor, can lead to the regeneration of two heads, creating double-headed animals (Durant et al., 2017). This change in the body axis plan appears to be permanent, with double-headed flatworms continuing to generate two heads after subsequent amputations even when all 8-OH has been removed (Durant et al., 2017). Treating planarian trunk fragments with ionophores to alter the resting membrane potential and depolarize the cells also results in the regeneration of double-headed organisms, suggesting that it is the depolarization of cells that regulates the development of the body axis (Durant et al., 2019). In Xenopus embryos, injection of mRNA encoding a dominant-negative form of the potassium channel Kir6.1 was sufficient to induce the formation of ectopic eyes, suggesting bioelectricity can regulate tissue identity as well (Pai et al., 2012). Together, these data strongly support the importance of bioelectrical signaling in development, including regulation of the body axis, regulation of body and body part size, and regulation of patterning.

Ion channels that conduct sodium, potassium, calcium, and chloride can influence the levels of cytoplasmic calcium. For example, voltage-gated calcium channels open in response to a depolarized membrane potential. Calcium release from the endoplasmic reticulum is regulated in part by ion channels that conduct other ions. Interestingly, spontaneous calcium oscillations in developing tissues exist in diverse organisms. Excitable cells such as neurons, muscle cells, or pancreatic beta cells communicate and perform their functions through rapid changes in intracellular concentrations of ions to generate action potentials. While most other cells do not propagate action potentials in the same way, many different cells and tissues propagate calcium transients and waves. Calcium waves that propagate spontaneously or in response to stimuli have been found in mesenchymal stem cells (Kawano et al., 2002), chondrocytes (Kono et al., 2006), osteoblasts (Godin et al., 2007), keratinocytes (Tsutsumi et al., 2009), endothelial cells (Uhrenholt et al., 2007; Yokota et al., 2015; Justet et al., 2019), and epithelial cells (Nathanson, 1994; Evans and Sanderson, 1999; Nihei et al., 2003). These calcium oscillations are due to rapid changes in cytosolic calcium. Calcium is stored in the endoplasmic reticulum and the mitochondria. Calcium channels and gap junctions located in the cell membrane as well as channels in the endoplasmic reticulum have been found to play a key role in the propagation of spontaneous calcium waves (Uhlén and Fritz, 2010). Calcium levels in the cytosol rise when calcium is brought across the cell membrane by gap junctions or activated-voltage gated calcium channels, or when the ER stores of calcium are released into the cytoplasm (Uhlén and Fritz, 2010).

This increase in cytosolic calcium is then brought back down either by movement of the calcium through gap junctions into other cells, by being pumped back into the ER through the ATPase SERCA, being pumped out of the cell, or by being taken up by mitochondria (Uhlén and Fritz, 2010). The change in calcium levels between the cytosol, the ER, and mitochondria lead to the propagation of the calcium oscillations that are observed in cells (Uhlén and Fritz, 2010). The function of these dynamic changes in calcium is not well understood. Recently, however, these calcium waves and transients have been found within wide number of developing tissues, and disruption of the calcium oscillations disrupt morphogenesis, suggesting that calcium dynamics may help coordinate development.

Disruption of these dynamic changes in calcium in some tissues can lead to abnormal development. In D. melanogaster, the larval developing wing epithelium propagates calcium waves both in response to wounding and spontaneously in vivo (Narciso et al., 2015; Restrepo and Basler, 2016; Balaji et al., 2017; Brodskiy et al., 2019). Disruption of these calcium waves either pharmacologically or through mutations impacting ion channels required for these calcium waves is associated with disruption in proper Drosophila wing development (Brodskiy et al., 2019). Blue pansy butterflies also spontaneously propagate calcium waves and transients during pupal wing development and disruption of these oscillations leads to malformed scale-development and eye spot formation in the wing (Ohno and Otaki, 2015). In these blue pansy butterflies, many of the calcium oscillations appear to originate from the future eye spot of the developing wing, suggesting that the calcium oscillations may instruct development of this structure in the wing (Ohno and Otaki, 2015). Calcium oscillations in developing tissues are not limited to invertebrates (Slusarski and Pelegri, 2007). Calcium oscillations have been found in budding chick feather buds and inhibition of these calcium oscillations disrupts cell migration and feather bud formation (Li et al., 2018). Calcium oscillations occur in cultured primary mouse embryonic palate cells (Isner and Bates, 2021) and they have also been imaged during early embryonic development in zebrafish (Webb and Miller, 2006), Xenopus (Wallingford et al., 2001), and mouse embryos (reviewed in Stewart and Davis, 2019). The existence of these calcium oscillations within many different organisms and tissues during development paired with the evidence that blocking them leads to developmental defects suggests that calcium oscillations are important for development.

The growing understanding of the role of spontaneous calcium oscillations and bioelectrical signaling in non-neuronal cells is reminiscent of what is known about the evolutionary development of synapses and neural connections. Research into the potential origins of neuronal synapses suggests that many synaptic proteins likely originated in non-neuronal cells before being co-opted by neurons (Ovsepian and Vesselkin, 2014; Ovsepian, 2017; Ovsepian et al., 2020). The potential use of calcium oscillations and bioelectrical signaling in the development of non-neuronal tissues may support the hypothesis that this rudimentary bioelectrical signaling may have evolved over time to help develop the fine-tuned chemical synapse in neural connections (Ovsepian and Vesselkin, 2014; Ovsepian, 2017; Ovsepian et al., 2020).

While it is becoming increasingly evident that ion channels are essential for proper development, the mechanism by which they act is still unclear. How do cells within tissues and organs use bioelectricity during development?

Recent studies suggest that it is the differences in the Vmem across cells that is important for development, rather than specific ion channels or ions. For example, in Xenopus laevis disruption of the homolog of Kir2.1, the channel associated with Anderson-Tawil Syndrome in humans, leads to craniofacial defects (Adams et al., 2016). These defects were recapitulated by optogenetic activation of a non-specific cation channel or optogenetic activation of a hydrogen pump, both expected to cause similar changes in the Vmem as Kir2.1 disruption (Adams et al., 2016). In contrast, disruption of a sodium-hydrogen exchanger that was expected to be electroneutral and cause no change in Vmem led to no disruption of craniofacial development (Adams et al., 2016). In Xenopus embryos the development of ectopic eyes can be induced by the injection of ion channel expression constructs that depolarize regions of the embryo (Pai et al., 2012). This induction of ectopic eyes is not limited to a single ion channel construct, however, and a variety of different constructs lead to the same ectopic eye formation (Pai et al., 2012). These results suggests that it is the overall Vmem which is collectively controlled by ion channels—rather than the specific identity of the channels or ions—that is important for morphological development (Adams et al., 2016).

While it is clear that Vmem is important for development, less is known about the downstream molecular mechanism by which bioelectricity regulates morphogenesis. There are several potential mechanisms by which bioelectricity may play a role in development. Calcium is required for various cellular processes that feed directly into development. What lessons can we learn from excitable cells, like neurons and pancreatic beta cells? In these cells, sodium, potassium, and chloride channels determine Vmem. Several calcium channels open or close in response to a particular Vmem. Thus, channels that do not conduct calcium contribute to intracellular calcium concentration. Could Vmem play a central role as a regulator of calcium, which mediates proliferation, apoptosis, cell cycle control, cell polarity, cell migration, and even molecular signaling? Evidence for each of these potential mechanisms is described below.

Ion channels play an important role in cell death pathways including both apoptosis and necrosis (Figure 2, and reviewed in Lang et al., 2006; Bortner and Cidlowski, 2014). Potassium leaves the cell during early apoptosis leading to a depletion of intracellular potassium (Bortner et al., 1997; Yu, 2003). This loss of potassium is important for the apoptotic pathway and inhibition of potassium efflux can prevent apoptosis (Bortner et al., 1997; Yu, 2003). Potassium is one of the most abundant ions within the cell and physiological concentrations of potassium have an inhibitory effect on caspase and nuclease activity (Hughes et al., 1997; Dallaporta et al., 1998). A number of potassium channels have been identified as playing an important role in mediating apoptosis. These include outward delayed rectifier (IK) channels, voltage-gated potassium channels, the inward rectifier Kir1.1, and multiple calcium activated potassium channels (reviewed in Yu, 2003). Drops in potassium levels lead to shrinkage of the cell due to changes in osmolarity, and this shrinkage may contribute to apoptosis (Yu, 2003). While lowering potassium concentration by itself is not sufficient to induce apoptosis, potassium depletion facilitates apoptosis and may be a universal part of the apoptotic pathway (Hughes et al., 1997; Dallaporta et al., 1998; Yu, 2003).

Calcium acts as a key signaling molecule in apoptosis. Calcium is stored at high concentrations in the ER and the mitochondria, and at a lower concentration in the cytosol. An imbalance of these calcium stores can lead to cell death (Figure 2A, reviewed in Orrenius et al., 2003; Rizzuto et al., 2003; Zhivotovsky and Orrenius, 2011). Calcium was first associated with cell death when it was found that cells killed by withholding oxygen or treating with a cytotoxic drug had a dramatic rise in calcium content (Chien et al., 1977; Schanne et al., 1980). Cytosolic calcium increases during apoptosis (Martikainen et al., 1991; Kruman et al., 1998), and overactivation or disruption of calcium channels increases cell death. Disruption or overactivation of the ER calcium regulating channels inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs), ryanodine receptors (RyRs), and SERCA all increase cell death (Ruiz et al., 2010; Kiviluoto et al., 2012; Sehgal et al., 2017).

There are multiple pathways by which an imbalance in calcium levels can induce cell death (Figures 2A,B). One pathway is via the induction of prolonged ER stress (Figure 2A). The ER is an essential organelle required for protein folding and processing (Adams et al., 2019). Inside the ER, a wide variety of chaperones help process and fold new proteins, and many of these chaperones require calcium to function correctly (Adams et al., 2019). If levels of calcium in the ER drop, the ability of chaperones to efficiently fold proteins is reduced, and misfolded or unfolded proteins accumulate, a situation known as ER stress (Adams et al., 2019). ER stress initially induces the unfolded protein response (UPR) pathway (Adams et al., 2019). Long term chronic ER stress, however, leads to the expression or activation of C/EBP-homologous protein (CHOP), c-Jun N-terminal kinase (JNK), caspases, and other pro-apoptotic proteins, to induce apoptosis (Nakagawa et al., 2000; Hiramatsu et al., 2015).

High intracellular calcium can induce cell death through mitochondria (Figure 2B). Mitochondria take up calcium from the cytoplasm. Sharp rises in cytoplasmic calcium can overload calcium in the mitochondria which induces the opening of the permeability transition pore (PTP), a complex in the inner mitochondrial membrane (Williams et al., 2013; Bonora and Pinton, 2014). Extreme PTP activation causes swelling and rupture of the mitochondria resulting in necrosis, while milder activation of PTP can lead to leakage of cytochrome C and the induction of apoptosis (Bonora and Pinton, 2014).

Changes in sodium and chloride flux have also been reported during apoptosis (Figure 2C). Sodium levels increase within the cell during apoptosis, and activation of voltage-gated sodium channels (VGNCs) via the VGNC activator veratridine can induce apoptosis in neurons (Dargent et al., 1996; Koike et al., 2000; Arrebola et al., 2005). Chloride flux is important for apoptosis and blocking chloride channels can block apoptosis, perhaps because this ion regulates cell volume (Okada et al., 2004; Okada et al., 2006).

Ion channels play an important role in cell death pathways, and mediation of apoptosis is one mechanism by which bioelectricity influences development.

Ion channels play help regulate cell proliferation. The transmembrane potential of cells changes over the course of the cell cycle (Sachs et al., 1974; Wonderlin et al., 1995). It was observed as early as the 1970s that depolarization could induce mitosis of neuronal precursors (Stillwell et al., 1973; Cone and Cone, 1976). It is now known that calcium, potassium, sodium, and chloride all play roles in regulating the cell cycle (Blackiston et al., 2009).

Calcium plays an essential role in regulating the cell cycle at nearly every transition step (Humeau et al., 2018) (Figure 3). The cell cycle is controlled by cyclin-dependent protein kinases (CDKs) that activate upon binding to a cyclin. Expression of each of the cyclins regulates the CDK complexes and guides entry into the next phase of the cell cycle. Calcium feeds into the cell cycle primarily by regulating calmodulin (CaM) and calcineurin (CaN) (Humeau et al., 2018). CaM is a protein that is activated upon binding of calcium. Ca2+/CaM can directly regulate CDKs and cyclins or act through activation of calcineurin, a phosphatase that activates upon Ca2+/CaM binding (Kahl and Means, 2003). Together Ca2+/CaM and Ca2+/CaM activated-calcineurin regulate many of the CDKs and cyclins (Kahl and Means, 2003). For example, calcium acts through CaM or CaN activation to regulate the levels CDK1, CDK2, cyclin A, cyclin D, and cyclin E within various cell types (Colomer et al., 1994; Tomono et al., 1998; Kahl and Means, 2004; Humeau et al., 2018) (Figure 3).

In addition to acting through CaM and its downstream pathways, calcium oscillations are important for regulating cell cycle phase transitions. Calcium oscillations have been found to be important for the G1/S phase transition. The store-operated calcium entry (SOCE) pathway, a pathway in which the calcium channels Stim and Orai work to bring calcium into the cell upon ER calcium depletion, was found to be upregulated during G1/S phase transition and downregulated prior to the G2/M phase transition (Chen et al., 2016) (Figure 3). Blocking SOCE can lead to G1 arrest (Short et al., 1993; Chen et al., 2016). Treating cells with calcium blockers can also lead to inhibition of the metaphase-anaphase transition, suggesting that calcium helps regulate the mitotic spindle checkpoint in addition to the G1/S phase transition (Xu et al., 2003). Together these data suggest that calcium is an important second messenger for regulating both cell death and cell life pathways.