Calcium signaling in vertebrate embryonic patterning andmorphogenesis

Diane C. Slusarski, Ph.D. andDepartment of Biological Sciences, University of Iowa, Iowa City, IA 52242, Phone: 319.335.3229,FAX: 319.335.1069, Email: diane-slusarski@uiowa.edu

Francisco Pelegri, Ph.D.Laboratory of Genetics, University of Wisconsin – Madison, Madison, WI 53706, Phone:608.265.9286, FAX: 608.262.2976, Email: fjpelegri@wisc.edu

AbstractSignaling pathways that rely on the controlled release and/or accumulation of calcium ions areimportant in a variety of developmental events in the vertebrate embryo, affecting cell fatespecification and morphogenesis. One such major developmentally important pathway is the Wnt/calcium signaling pathway, which, through its antagonism of Wnt/β-catenin signaling, appears toregulate the formation of the early embryonic organizer. In addition, the Wnt/calcium pathway sharescomponents with another non-canonical Wnt pathway involved in planar cell polarity, suggestingthat these two pathways form a loose network involved in polarized cell migratory movements thatfashion the vertebrate body plan. Furthermore, left-right axis determination, neural induction andsomite formation also display dynamic calcium release, which may be critical in these patterningevents. Finally, we summarize recent evidence that propose a role for calcium signaling in stem cellbiology and human developmental disorders.

A great variety of developmental processes, from fertilization to organ formation and function,are dependent on the dynamic release of calcium (Ca2+) ions. This review will focus on therole of Ca2+-mediated signals in patterning events in animal embryos, such as cell fatespecification and morphogenesis. The reader is referred to reviews that address the role ofCa2+ signaling in other biological processes, such as egg activation and fertilization (Santellaet al., 2004), cellular cleavage (Webb and Miller, 2003; Baluska et al., 2006), neuronaldevelopment (Archer et al., 1998) and cell death (Berridge et al., 1998; Chinopoulos andAdam-Vizi, 2006). We will first describe current models of Ca2+-mediated cellular signaling,such as the organelles and proteins important for Ca2+ dynamics and their interpretation byCa2+-sensitive factors. Later, we summarize current knowledge on the role of Ca2+ signalingin cell fate decisions in the vertebrate embryo, from the cellular blastoderm throughorganogenesis and the stem cell niche. Finally, we present current known associations betweenCa2+ signaling pathways and human developmental disorders.

An overview of calcium signaling pathwaysCa2+ ions are not metabolized by the cell. Instead, Ca2+ acts as a second messenger in the cellby forming ionic gradients within or outside the cell. Such gradients originate through Ca2+

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Published in final edited form as:Dev Biol. 2007 July 1; 307(1): 1–13. doi:10.1016/j.ydbio.2007.04.043.

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mobilization across membranes, either the plasma membrane or the membrane of intracellularCa2+-storing organelles (Figure 1). The resulting Ca2+ increases are regulated by the location,extent and duration of the ion channel opening, and when interpreted by Ca2+-sensitivemediators result in local or global signaling events that implement cellular responses.

In non-excitable (non-neuronal) cells, the majority of intracellular Ca2+ release occurs throughinositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ channels present in the endoplasmic reticulum(ER) membrane. Other channels, present in other cellular organelles, can also contribute tointracellular Ca2+ release, such as the ryanodine receptors (RyR) in the ER, NAADP-triggeredreceptors in lysosome-like organelles and ion exchange channels in mitochondria (reviewedin Berridge et al., 2003). There is extensive feedback between Ca2+ release circuits. Forexample, Ca2+ released from the ER can bind back to receptors (IP3 receptors (IP3Rs) andRyRs) and stimulate Ca2+-induced Ca2+ release influencing neighboring receptors andpotentially triggering a regenerative Ca2+ wave (Berridge, 1997; Berridge et al., 2003;Roderick et al., 2003a). In addition, continued stimulation and/or depletion of ER storesactivates a store operated Ca2+ entry (SOC) influx pathway located at the plasma membrane(Parekh and Putney, 2005).

A number of studies have implicated a signal transduction pathway dependent on thephosphatidylinositol (PI) cycle leading to Ca2+ release from intracellular organelles in earlydevelopmental cell decisions. This is corroborated by studies that demonstrate broadexpression of IP3R subtypes beginning at early developmental stages (Kume et al., 1993; Kumeet al., 1997b; Rosemblit et al., 1999). In comparison, the RyR is thought to have a major rolein striated muscle function and its expression only occurs as organogenesis proceeds,particularly in skeletal and cardiac muscle. The PI cycle is activated in response to manyhormones and growth factors that bind to cell surface receptors. Two predominant receptorclasses are the G protein-coupled receptor class (GPCR) and the receptor tyrosine kinase (RTK)class. Extracellular ligand stimulation of these receptors activates a PI-specific phospholipaseC (PLC) (Figure 1). GPCRs generally activate PLC-β while RTKs generally stimulate PLC-γ. Activated PLC converts membrane bound phosphatidylinositol (4,5) bisphosphate (PIP2)into IP3 and lipophilic diacylglycerol (DAG). IP3 subsequently binds to receptors locatedprincipally on the endoplasmic reticulum (ER) and activates the IP3R, triggering the rapidrelease of Ca2+ into the cytosol of the cell. At the same time, DAG produced by PIP2 hydrolysiscan act as an additional second messenger to further activate pathway downstream targets suchas Protein Kinase C (PKC; see below).

Effectors and interpretation of calcium signalsRelative to cytosolic Ca2+ levels, cellular stimulation has been shown to induce a transientincrease or oscillations of Ca2+ (Bootman et al., 2001), and in some systems these tworesponses may occur simultaneously (Gerbino et al., 2005). Much of the newly releasedcytosolic Ca2+ is quickly bound by Ca2+ binding proteins (Falcke, 2003). Some of theseproteins act as Ca2+ buffers while other proteins become activated components of signaltransduction pathways. For example, calmodulin, a member of the EF-hand protein family thatrepresents the most abundant family of eukaryotic Ca2+ binding proteins (Haiech et al.,2004), is activated by cooperative binding of Ca2+ ions and subsequently activates proteinkinases, phosphatases, ion transporters and cytoskeletal proteins. One particularly notable classis the Ca2+/calmodulin-dependent kinase (CaMK) family (Hoeflich and Ikura, 2002; see Table1 for a summary of Ca2+ signaling regulators described in this review).

Another major target of activated calmodulin is the protein phosphatase calcineurin, whichactivates the nuclear factor of activated T cells (NFAT). Calcineurin phosphorylates NFATproteins, promoting their nuclear localization and assembly with partner proteins to form

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transcription complexes. Rephosphorylation by an unknown priming kinase and glycogensynthase kinase-3 (GSK-3) leads to NFAT export from the nucleus (Beals et al., 1997; Graefet al., 1999), ending their cycle of activation (reviewed in Schulz and Yutzey, 2004). Anotherset of molecular targets of PI cycle activation is constituted by the protein kinase C (PKC)isozymes, which are activated by both DAG (produced by PIP2 hydrolysis) and freeintracellular Ca2+ (Sakai et al., 1997; Oancea and Meyer, 1998; Shirai et al., 1998; Violin etal., 2003). In addition to triggering specific cellular inductive responses, intracellular Ca2+

concentrations can affect the general state of the cell, for example the levels of protein synthesisand folding (Roderick et al., 2003b) and the decision to undergo apoptosis (Berridge et al.,1998). A review of other known Ca2+-sensitive factors can be found in Ikura et al., (2002).

A particularly important emerging concept is the idea that ubiquitous Ca2+ can trigger variousspecific cellular responses by virtue of differences in the amplitude, frequency and duration ofintracellular Ca2+ oscillations. Such oscillations can be derived from changes in upstream stepswithin the PI cycle, such as G-protein activity (Luo et al., 2001; Rey et al., 2005), PLC activity(Thore et al., 2004; Nomikos et al., 2005), and IP3 levels (Hirose et al., 1999; McCarron etal., 2004). Oscillatory small molecules such as IP3 may be transmitted to other cells via gapjunctions (Lin et al., 2004), a phenomenon that may be of significance in the regulation of axisinduction in the zebrafish blastula (see below). Feedback from activated Ca2+ binding proteinsadds another layer of complexity to the dynamics of Ca2+ release and removal. For example,IP3 R activity integrates signals from small molecules and proteins, including PKC and Ca2+/Calmodulin dependent protein kinase II (CaMKII; Nadif Kasri et al., 2004; Patterson et al.,2004).

Many Ca2+-binding proteins sense the frequency of intracellular Ca2+ increases. In the case ofCaMKII, such ability has been shown to depend on the synergism between Ca2+/calmodulinbound to each of the multimeric CaMKII subunits and the activity of the kinase domain (DeKoninck and Schulman, 1998; Dupont and Goldbeter, 1998). Of interest, the frequency-dependent response to Ca2+ oscillations can be modulated by the use of alternative CaMKIIsplice variants (Bayer et al., 2002), suggesting that gene regulation may further modify thecellular response to variations in intracellular Ca2+. The transcriptional regulatory activity ofNFAT has also been shown to be exquisitely sensitive to the frequency of IP3 and Ca2+

oscillations, presumably via changes in calcineurin activity (Dolmetsch et al., 1997; Dolmetschet al., 1998; Li et al., 1998). Other studies have shown that Ca2+ oscillation frequencies mediateCa2+-dependent activation of Ras family effector G-proteins and the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) cascade (Walker et al.,2004; Kupzig et al., 2005).

Calcium transients and axis inductionAxis induction in vertebrates has been shown to be dependent on the activity of the Wntsignaling network (Figure 2) (reviewed in Pelegri, 2003;Weaver and Kimelman, 2004; see alsoTao et al., 2005). Activation of the so-called canonical Wnt pathway results in the inhibitionof a complex; composed of GSK-3, Axin/Conductin, the adenomatous polyposis tumorsuppressor protein (APC) and Diversin, which normally targets the β-catenin protein fordegradation via ubiquitination and the proteasome complex (Figure 2A) (reviewed in Polakis,2000). Inactivation of the β-catenin-degradation complex by Wnt signaling results in thestabilization and nuclear accumulation of β-catenin protein, thus this pathway has been termedthe Wnt/β-catenin pathway. Nuclear β-catenin in turn interacts with members of the LEF/TCFtranscription factor family to promote the activation of downstream target genes involved inaxis specification.

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However, other Wnt pathways, either in parallel or part of a complex signaling network, appearto interact with the Wnt/β-catenin pathway in the early specification of the embryonic axis. InXenopus and zebrafish, one of these pathways involves the PI cycle and Ca2+ release (Figure2B). Classical studies linking PI cycle activity to body plan determination reported the abilityof lithium, an inhibitor of inositol turnover (Berridge et al., 1989), to induce dorsal cell fatesin Xenopus (Kao et al., 1986; Kao and Elinson, 1989; Kao and Elinson, 1998), and similareffects were obtained in the zebrafish embryo (Stachel et al., 1993; Aanstad and Whitaker,1999). Lithium-induced expansion of dorsal structures in the embryo can be rescued bysupplying an intermediate of the PI cycle (myo-inositol; Busa and Gimlich, 1989), indicatingthat indeed the PI cycle is a primary target with regards to the effects of this agent on dorsalcell fate specification. Moreover, the effects of lithium were most pronounced when exposureoccurred on the ventral side of the embryo, suggesting that in the embryo PI cycle activity isnormally high on the ventral side and low in the dorsal side. Subsequent findings indicated thatanother endogenous target of lithium is the β-catenin degradation complex component GSK-3,which when inhibited promotes dorsal axis induction (Klein and Melton, 1996; Stambolic etal., 1996). Exogenous myo-inositol can also suppress the effects of GSK-3 inhibition(Hedgepeth et al., 1997), further supporting the notion that PI cycle activity and Wnt/β-cateninsignaling act in parallel to regulate axis induction. It remains to be determined whether lithiumaffects additional targets involved in axis induction.

Several pieces of evidence in zebrafish and Xenopus further support a requirement for PI cycleactivity in dorsoventral patterning. Xenopus embryos injected with antibodies that disruptIP3R function displayed expanded dorsal structures with the loss of ventral structures (Kumeet al., 1997a). A similar dorsalization effect can be observed in the zebrafish after injection ofIP3R blocking antibodies as well as treatments with other PI-cycle inhibitors (Westfall et al.,2003b). Together, these studies suggested that high levels of PI cycle activity promote ventralcell fates, possibly by counteracting the axis-inducing Wnt/β-catenin signaling pathway.

The findings of an involvement for PI cycle activity in axis induction agree well with theobserved spontaneous increase in IP3 levels in the Xenopus embryo at the blastula stage (Busaand Gimlich, 1989; Maslanski et al., 1992). Moreover, beginning at the 32-cell stage, thezebrafish embryo exhibits rapid aperiodic Ca2+ release that persists until the midblastulatransition stage (Reinhard et al., 1995; Slusarski et al., 1997a; Slusarski et al., 1997b),consistent with the idea that the increased IP3 levels may trigger Ca2+ release during thesestages. This idea has been corroborated by drug inhibition studies that indicate that theseCa2+ transients depend on PLC activity and IP3-dependent Ca2+ release from the ER (Slusarskiet al., 1997a; Slusarski et al., 1997b).

Inhibition of G-protein signaling suppresses Ca2+ release in zebrafish (Slusarski et al.,1997a; Ahumada et al., 2002), indicating that the Ca2+ release pathway occurs downstream ofa G-protein coupled receptor (as opposed to a G-protein-independent pathway of PLCactivation such as that triggered by Fibroblast Growth Factor – see below). Of interest areCa2+ transients in the zebrafish blastula that originate in external cellular layers, the envelopinglayer (EVL) and yolk syncitial layer (YSL) (Reinhard et al., 1995; Slusarski et al., 1997b).Although the EVL and YSL are extraembryonic (Kimmel et al., 1995), it has been proposedthat signaling from these layers becomes transmitted into the blastula cells that will form theembryo proper. There is accumulating evidence that this does occur between the YSL and theoverlying deep cells (Mizuno et al., 1996; Ober and Schulte-Merker, 1999; Rodaway et al.,1999), and has been proposed to occur between the EVL and the cells below (Westfall et al.,2003a; Westfall et al., 2003b; Lyman-Gingerich et al., 2005). The mechanism of theintercellular transmission of this Ca2+ remains unknown, although it is possibly mediated bygap junctions present in zebrafish blastula cells (Bozhkova and Voronov, 1997), which have

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been shown to be involved in the transmission of Ca2+-releasing small molecules such as IP3(Clair et al., 2001).

In vertebrate embryos, while over-expression of a subset of Wnts induces hyperdorsalizationand ectopic axes by virtue of Wnt/β-catenin signaling activity (Moon et al., 1993b; Du et al.,1995; Kelly et al., 1995; Dale, 1998; Moon and Kimelman, 1998), a second Wnt class(including Wnt-5A, -4, and -11) appears to act independently of β-catenin function (Kühl etal., 2000b). Emerging evidence suggests that the ability of Wnt ligands to activate differentsignaling pathways, β-catenin-dependent (or canonical) and β-catenin-independent (or non-canonical) appears to be dependent on timing of expression and receptor context. In thezebrafish embryo, Wnt-5 overexpression results in an increase in the frequency of intracellularCa2+ release in a manner that is dependent on G-protein activity and the PI cycle (Slusarski etal., 1997a; Slusarski et al., 1997b), thus linking this Wnt family activity to IP3-dependentCa2+ release and defining the Wnt/Ca2+ signaling pathway. Various studies have shown thatthere are common components, between the Wnt/Ca2+ and another non-canonical Wntpathway, the planar cell polarity (Wnt/PCP) pathway, involved in the polarization of cells inDrosophila and vertebrate species (reviewed in Wallingford et al., 2002; Strutt, 2003). Thesecommon components suggest that non-canonical Wnt signaling activity can be viewed as acomplex network with cellular outputs identified by Ca2+ modulation and polarized cellmovement (Mlodzik, 2002).

The link between non-canonical Wnt pathway activation and axis induction was initiallysuggested by the apparent antagonism of certain pairs of Wnt ligands when expressed inXenopus and zebrafish embryos (Moon et al., 1993b; Slusarski et al., 1997b). Expression ofligands that activate Wnt/β-catenin signaling in these embryos, such as Wnt-8, results in ectopicaxis induction. However, coexpression of these Wnt ligands with others which, when expressedon their own do not promote Wnt/β-catenin activation, such as Wnt-5A, suppresses this axis-induction effect. Stimulating Ca2+ release, for example via activation of the Serotonin receptor,also antagonizes Xwnt-8 induced expansion of the dorsal domains (Slusarski et al., 1997b),suggesting that Wnt-5 antagonism of Wnt/β-catenin is mediated by Ca2+ release. On the otherhand, pharmacological or genetic reduction of the Wnt/Ca2+ pathway in zebrafish embryosgenerates ectopic accumulation of nuclear β-catenin and activation of β-catenin transcriptionaltargets (Westfall et al., 2003a; Westfall et al., 2003b), and G-protein inhibition is able todorsalize Xenopus embryos (Kume et al., 2000). These observations are consistent with a modelin which IP3-dependent Ca2+ release, promoted by Wnt/Ca2+ signaling activity, negativelyregulates the Wnt/β-catenin signaling pathway and therefore axis induction (Figure 2).

Further support of this idea comes from the analysis of a mutation in the zebrafish maternalgene hecate, where an increase in Ca2+ release frequency is associated with a strong inhibitionof dorsal axis induction (Lyman-Gingerich et al., 2005). Pharmacological inhibition studiesindicated that the ectopic Ca2+ release observed in hecate embryos depends on Wnt/Ca2+

pathway components, and interference with Ca2+ dynamics was shown to rescue the defectsin dorsal cell fate specification observed in these mutants. Importantly, the level of Wnt/β-catenin activity does not affect the frequency of endogenous Ca2+ transients (Westfall et al.,2003a; Lyman-Gingerich et al., 2005), in agreement with a causal relationship between Ca2+

release and the inhibition of dorsal axis induction.

In the zebrafish, Wnt-5 has been shown to correspond to the genetically defined gene pipetail(ppt; Rauch et al., 1997), a gene which when mutated results in zygotic defects in the extensionof the axis during somitogenesis (Hammerschmidt et al., 1996; Kilian et al., 2003). Thepossibility that Wnt-5/Ppt itself is the endogenous activator of Wnt/Ca2+ signaling in thezebrafish embryo was determined by testing for maternal-effects caused by germ linehomozygosity for Wnt-5/ppt. Zebrafish embryos lacking maternal Wnt-5/ppt function exhibit

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a reduction in the frequency of Ca2+ transients and nuclear β-catenin stabilization, as well asinduce dorsalized phenotypes, which become more prevalent if they are additionally mutantfor zygotic Wnt-5/ppt (Westfall et al., 2003a). Thus, the gain- and loss-of-function effects ofWnt-5 suggest that this factor is an early endogenous signal involved in Wnt/Ca2+ activationand the regulation of dorsal axis induction.

Several Ca2+-sensitive factors have been implicated as potential downstream mediators of Wnt/Ca2+ antagonism of Wnt/β-catenin signaling. In Xenopus, CaMKII is activated by Wnt andFrizzled (Fz) receptors to promote ventral cell fates (Kühl et al., 2000a). In the zebrafishembryo, expression of constitutively active CaMKII can similarly lead to axis induction defects(Westfall and Slusarski, unpublished observations). Moreover, CaMKII activation rescues thezygotic Wnt-5/ppt phenotype, showing that CaMKII activity occurs downstream of Wnt/Ca2+ pathway activation, at least during the gastrulation stages. Other studies in Xenopus haveshown that Wnt-5A induces nuclear translocation of the calcineurin target transcription factorNFAT (Saneyoshi et al., 2002). The same studies also show that the expression of activatedNFAT ventralizes Xenopus embryos and antagonizes Wnt/β-catenin activity while, conversely,expression of dominant negative NFAT induces ectopic axis formation and expression of dorsaltarget genes. Additionally, the Drosophila segment polarity gene naked cuticle (nkd) has beenshown to antagonize Wnt/β-catenin activity in a manner dependent on its EF-hand Ca2+-binding motif (Zeng et al., 2000; Rousset et al., 2001; Wharton et al., 2001; Li et al., 2005).Thus, multiple Ca2+-sensitive factors may be likely candidates to regulate Wnt/Ca2+ signalingand axis induction, although some of these studies have the caveat that the observed effects onaxis induction depend on the expression of activated or dominant negative proteins. Forexample, maternally provided Wnt-11 has been shown to be the endogenous signal involvedin Wnt/β-catenin activation and axis induction in Xenopus (Tao et al., 2005), and Wnt-5A,when coexpressed with the appropriate Frizzled receptor, can also induce Wnt/β-cateninsignaling (Mikels and Nusse, 2006). Yet genetic loss of Wnt-11 function in the zebrafishsupport a clear role in cell movement and no indication of a role in axis formation (Heisenberget al., 2000). Loss of function studies using genetic mutations or functional knock-downapproaches should be helpful in discerning the identities of the endogenous factors involvedin this process.

The precise nature of the regulation of the Wnt/β-catenin pathway by Ca2+-sensitive mediatorsis also not fully understood. In the zebrafish blastula embryo, this regulation may occurupstream or at the level of β-catenin accumulation, as suggested by the reduction of nuclearβ-catenin in hecate mutant embryos (Lyman-Gingerich et al., 2005), and the ectopicaccumulation of nuclear β-catenin in embryos where Ca2+ release is inhibited (Westfall etal., 2003b). In Xenopus, calcineurin/NFAT activity appears to regulate Wnt/β-catenin signalingby modulating the activity of the GSK-3-dependent β-catenin-degradation complex (Saneyoshiet al., 2002), suggesting a possible mechanism for this regulation. However, there is alsoprecedent for other modes of GSK-3-independent regulation of β-catenin stability, as in thevertebrate limb, where Wnt-5A promotes the degradation of β-catenin in a manner dependentinstead on the Siah-APC-Ebi E3 ubiquitin ligase complex (Topol et al., 2003). The proteasecalpain has also been shown to mediate the Ca2+-dependent degradation of β-cateninindependently of the GSK-3-containing β-catenin degradation complex (Li and Iyengar,2002). Similarly, activated PKC can promote β-catenin degradation through a GSK-3-independent mechanism (Gwak et al., 2006). Furthermore, it remains a possibility that Wnt/Ca2+ may also regulate dorsal induction in a manner independent of β-catenin itself, as hasbeen proposed in various cellular systems where CaMKII acts through a mitogen activatedprotein kinase (MAPK) pathway to directly regulate the activity of Tcf family transcriptionfactors (Ishitani et al., 1999; Meneghini et al., 1999; Rocheleau et al., 1999; Ishitani et al.,2003a; Ishitani et al., 2003b).

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The emerging picture is made additionally complex by the possibility that Ca2+-sensitivetargets may not only affect Wnt/β-catenin activity, but may also feed back to modify the activityof Wnt/Ca2+ signaling. For example, increased DAG and Ca2+ levels caused by Wnt/Ca2+

pathway activation trigger the recruitment of PKC to the plasma membrane in early vertebrateembryos (Berridge, 1993; Sheldahl et al., 1999; Sheldahl et al., 2003) and this activated kinaseboth regulates common Wnt pathway components such as Dishevelled (Dsh; Kinoshita et al.,2003) and provides negative feedback on Ca2+ oscillations (Codazzi et al., 2001; Halet et al.,2004).

While the role of Wnt/Ca2+ in axis induction is becoming increasingly substantiated in thevertebrate embryo, less certain is the significance of Ca2+ release mediated by other signalingpathways such as Fibroblast Growth Factor (FGF). As with other members of the RTK family,ligand stimulation of FGF receptors activates PLC-γ (Mohammadi et al., 1991), hydrolyzesPIP2, into IP3 and DAG, and leads to the subsequent release of Ca2+ from IP3-sensitiveintracellular stores (Figure 1). In Xenopus, activation of FGF signaling induces mesoderm inthe blastula embryo (Kimelman and Kirschner, 1987; Slack et al, 1987; Kimelman et al.,1988) as well as Ca2+ efflux in oocytes (Muslin et al., 1994). However, althoughphosphorylation of PLC-γ by the FGF receptor has been shown to be developmentallyassociated with mesoderm induction in Xenopus (Ryan and Gillespie, 1994; Ryan et al.,1998), a mutation in the FGF receptor that renders it unable to either activate PLC-γ or triggerCa2+ release does not interfere with its mesoderm-inducing ability (Muslin et al., 1994). Thus,PLC-γ activation, and presumably FGF-induced Ca2+ release, does not appear to be necessaryfor mesoderm induction.

Studies in the zebrafish system have shown an additional role for FGF, which is dorsallyexpressed during gastrulation, in the establishment of dorsoventral patterning (reviewed inThisse and Thisse, 2005). This later role appears to occur independently of the early Wnt/β-catenin pathway involved in axis induction, and instead occurs by the repression of the ventralinducing BMP factors in dorsal regions. Of interest, Palma et al. have shown a role for Ca2+

signaling in determining dorsal cell fates during gastrulation (Palma et al., 2001), and notventral cell fates as suggested by the Ca2+ -dependent inhibition of axis induction normallyobserved in the blastula embryo (Westfall et al., 2003a; Westfall et al., 2003b; Lyman-Gingerich et al., 2005). Further studies are needed to determine whether FGF-mediated Ca2+

signaling has a role in the promotion of dorsal fates in the gastrulating embryo.

Global waves and morphogenesis during vertebrate gastrulationDuring gastrulation, vertebrate embryos undergo a variety of morphogenetic movementsinstrumental for the development of the body plan (reviewed in Keller, 2002; Wallingford etal., 2002), including the dorsally-directed migration that results in axis thickening (dorsalconvergence) and the lateral intercalation of axial cells that results in its elongation (axisextension). Recent studies suggest that Ca2+ release may be involved in the orchestration ofsuch morphogenetic movements involving cell polarization. Waves of Ca2+ mobilization,associated with waves of tissue contraction, can be observed in dorsal explants of gastrulatingXenopus embryos (Wallingford et al., 2001). Similarly, intercellular Ca2+ waves have beenobserved at the margin of gastrulating zebrafish embryos (Gilland et al., 1999). A causalrelationship between Ca2+ waves and morphogenesis is supported by the finding that, inXenopus embryos, pharmacological inhibition of such waves results in convergent extensiondefects without affecting cell fate (Wallingford et al., 2001). Similarly, zebrafish embryoszygotically mutant for Wnt-5/ppt, which exhibit a reduction in Ca2+ transient frequency(Westfall et al., 2003a), exhibit defects in axis extension (Hammerschmidt et al., 1996; Kilianet al., 2003). As mentioned above, expression of activated CaMKII can rescue the convergenceextension defect characteristic of Wnt-5/ppt mutants (Westfall et al., 2003a), indicating that

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CaMKII may mediate the effects of Ca2+ in this process. These data suggest the possibilitythat these Ca2+ waves coordinate convergent extension (C–E) during vertebrate gastrulation.

Convergent extension in the vertebrate embryo, a result of the polarization of migrating cells,is considered analogous to the PCP pathway involved in the polarization of epithelial cells inthe Drosophila cuticle (Solnica-Krezel, 2005). Wnt genes that result in the activation ofCa2+ release in the blastula embryo, such as Wnt-5 (Slusarski et al., 1997b; Westfall et al.,2003b), can also alter morphogenetic movements later during gastrulation (Moon et al.,1993a; Ungar et al., 1995). Recent studies indicate that that Wnt/Ca2+ and Wnt/PCP pathwaysshare common components and may even be part of a loosely connected network (Sheldahl etal., 2003). The observations that interference with either Ca2+ release or Wnt/PCP signalingresults in convergence extension defects suggests that this non-canonical Wnt signalingnetwork is involved in convergence extension (Figure 2B).

Indeed, in addition to Wnt-5/ppt, mutations in other genes involved in non-canonical Wntsignaling result in cell movement defects in zebrafish. This is the case for Wnt-11/silberblick(Heisenberg et al., 2000), the Wnt receptor Frizzled-2 (Oishi et al., 2006), the putativetransmembrane protein Strabismus/trilobite (Jessen et al., 2002; Park and Moon, 2002), andthe intracellular protein Prickle (Veeman et al., 2003). In addition, expression of Prickle(Veeman et al., 2003), Frizzled-2 (Slusarski et al., 1997a), Strabismus (DCS unpublished),Wnt-4, -5 and -11 (Westfall et al., 2003a) all stimulate Ca2+ release in zebrafish. Likewise, themutant form of Dsh that retains the ability to signal through the PCP pathway but not the Wnt/β-catenin pathway is also able to activate the Wnt/Ca2+ cascade in Xenopus and zebrafish(Sheldahl et al., 2003). On the other hand, pharmacological reagents that suppress Fz2-inducedCa2+ release in zebrafish lead to altered gastrulation movements (Slusarski et al., 1997a;Ahumada et al., 2002). Similarly, a requirement for G-protein signaling in gastrulation wasrecently demonstrated by antisense morpholino oligonucleotide knockdown of Gα12 andGα13 and the use of dominant negative constructs (Lin et al., 2005). These observations areconsistent with the possibility that Wnt/Ca2+ signaling, possibly dependent upon G-proteinactivity, is important for cell polarization involved in vertebrate morphogenesis.

Oishi et al., (2006) report that the knock down of the putative phosphorylation-dependentcytoskeletal regulatory molecule, duboraya (dub), synergizes with a Frizzled-2 knock down,to produce embryos with shorter anteroposterior axes and undulating notochords, a phenotypeconsistent with convergence extension defects. These studies also show that phosphorylationof dub, known to be essential for its function, is influenced by the expression of proteins thatstimulate Ca2+ release in zebrafish embryos (Liu et al., 1999; Ahumada et al., 2002; Sheldahlet al., 2003). Thus, it is possible that Wnt/Ca2+ signaling results in the activation of dub viaphosphorylation, although further study is required to confirm this hypothesis.

Ca2+ as a second messenger regulating cellular movements has been demonstrated in manycell types and most likely has a multi-fold role in coordinating epiboly and gastrulationmovements in the embryo. Drawing a parallel between neural outgrowth and gastrulation,transient Ca2+ release has been proposed to influence neuronal outgrowth by regulating cellularsecretion and organization of the cytoskeleton (reviewed in Spitzer, 2006). Thus, secretion ofdiffusible molecules, such as the Wnts, and the generation of new cell contacts could enableinductive interactions among cells. In addition, cellular microdomains, (including receptors,their associated proteins and Ca2+ pumps) have been described in polarized epithelial cells(reviewed in Kiselyov et al., 2006). The polarized distribution of Fz and other core PCPcomponents could lead to differential Ca2+ dynamics across a cell, or sheet of cells, andinfluence cell adhesion and motility. Further insight into downstream targets could also bedrawn from the growing tips of plants, which integrate small GTPases, PI cycle, Ca2+ andprotein kinases to mediate actin cytoskeletal reorganization and membrane trafficking

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(reviewed in Cole and Fowler, 2006). Investigation of Ca2+ release dynamics in zebrafishepiboly and convergence extension mutants may further correlate intracellular Ca2+ withcoordinated or polarized cell movements.

Calcium, cilia and left-right patterningEvidence from several vertebrate model systems suggests that the positioning of the internalorgans across the Left-Right (L-R) axis, presaged by the asymmetric expression of a group ofgenes (Levin, 2005), is modulated by Ca2+ signaling. In mice, the symmetry-breaking eventin left-right polarity is thought to arise from a directional flow generated by the rotation ofmonocilia in the embryonic node (Nonaka et al., 1998; Okada et al., 1999). Similar monociliaare observed in the chick node and the zebrafish Kupffer’s vesicle (KV), where they areproposed to serve a similar function as in the mouse node. In these analogous structures, ciliabeat in the same direction, creating a leftward nodal flow. In the mouse, this flow has beenproposed to stimulate mechanosensory cilia to trigger an elevation in intracellular Ca2+ levelsin cells along the left edge of the node (McGrath et al., 2003). Intracellular Ca2+ increases witha left-sided bias near the zebrafish KV have also been detected (Sarmah et al., 2005). Elevatedintracellular Ca2+ is thought to act as a second messenger, via an unknown mechanism, toultimately induce left-sided gene expression. This model is further supported by theobservation that the asymmetry in node Ca2+ levels is lost in mouse embryos homozygous formutations in the Polycystic kidney disease gene (Pkd-2), a Ca2+-permeable ion channel, andthat these mutants exhibit laterality defects (McGrath et al., 2003).

In chick embryos, it is not known if there is a similar asymmetry of intracellular Ca2+ asobserved in the mouse node and zebrafish KV. However in chick, it appears thatextracellular Ca2+ levels may be higher transiently on the left side. This asymmetry wasabolished after treatment with ompremazole, an inhibitor of H+/K+ ATPase, which also causedL-R defects specifically the reversal of heart looping. These results led the authors to proposethat differential H+/K+ ATPase activity sets up a spatial gradient of extracellular Ca2+, whichis subsequently transduced to activate asymmetric gene expression on the left side (Raya etal., 2004). Thus, evidence of a role for Ca2+ in L-R patterning is very tantalizing, but manyquestions and issues remain to be addressed; such as the Ca2+ sources, the Ca2+-dependentresponders and the precise role of extracellular versus intracellular Ca2+ in the induction andmaintenance of laterality signals.

Recently, PCP components have been linked with cilia function and laterality. It has long beenknown that PCP-mediated cell polarization is required for the proper placement of cilia inDrosophila wing cells. However, only very recent studies suggest a similar function for PCPsignaling in vertebrate cells. Indeed, Frizzled-2 knockdown, in addition to C-E defects, resultsin a reduction in cilia length and number within the zebrafish KV (Oishi et al., 2006). The sameauthors report a similar defect caused by functional knock down of the cytoskeletal regulatorduboraya. Although the precise role of the Ca2+ releasing factor Fz2 and its proposed targetduboraya in PCP signaling (see above) and cilliogenesis needs to be better substantiated, thesefindings suggest an association of Wnt/PCP and Ca2+-releasing genes with cilia generation,maintenance and/or function.

Calcium signaling and organogenesisOther aspects of organogenesis impacted by Ca2+ release involve the induction of the neuralprecursor cells, which will give rise to the Peripheral and Central Nervous Systems. The roleof Ca2+ in neural induction has been extensively described in a recent review (Webb et al.,2005) and we describe here only some basic findings. Periodic Ca2+ fluxes are observed inanterior dorsal ectoderm during stages of presumptive neural patterning in Xenopus, wherethey increase in amplitude at a time coincident with neural induction (Leclerc et al., 2000).

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Similarly, zebrafish embryos also exhibit intercellular Ca2+ waves in the prospective dorsalregion (Créton et al., 1998; Gilland et al., 1999). Ca2+ release from L-type Ca2+ channelspresent in the plasma membrane is required to induce neural specific genes in Xenopus (Leclercet al., 1999; Leclerc et al., 2000; Leclerc et al., 2003) and the newt Pleurodeles waltl (Moreauet al., 1994). However, manipulations that inhibit Ca2+ release and neural induction also altergastrulation movements (Leclerc et al., 2000; Palma et al., 2001; Wallingford and Harland,2001), making it difficult to use pharmacological agents to separate the effects of Ca2+ signalingon gastrulation and neural patterning.

Neural induction involves interaction between Bone Morphogenetic Proteins (BMPs) and theirantagonists, such as chordin and noggin (De Robertis and Kuroda, 2004). In Pleurodelesexplants, noggin application triggers an increase in Ca2+ release (Leclerc et al., 1999). Whetherthis Ca2+ transient occurs by the direct activation of Ca2+ release by noggin or via other noggin-modulated pathways, such as BMP signaling, has yet to be determined, as well as whetherthese events occur in the context of the whole animal.

In addition to neural induction, Ca2+ signaling has been implicated in the formation of thesomites, which will give rise to muscle, cartilage and bones. Somites are derived from paraxialmesoderm, where Ca2+ release activity has been reported during the segmentation period(Créton et al., 1998; Webb and Miller, 2000). Ca2+ release activity has also been reported inisolated Xenopus myocytes (Ferrari and Spitzer, 1999) and in mature somites in whole zebrafishembryos (Ashworth, 2004). Ca2+ release inhibition alters myotome patterning (Ferrari andSpitzer, 1999). In addition, elimination of calcineurin activity in Xenopus embryos abolishedsomite formation and led to additional later organogenesis defects in the heart, kidney and gutlooping (Yoshida et al., 2004). Recent work has linked bilateral somite formation to L-Rasymmetry signals (Kawakami et al., 2005; Vermot et al., 2005; Vermot and Pourquie,2005). It has yet to be determined whether this coupling of L-R and somite formation processesis directly linked to Ca2+ fluxes.

There is significant evidence suggesting a role for the calcineurin/NFAT pathway in thedevelopment of the cardiovascular and skeletal muscle systems, which has been presented inextensive recent reviews (Hogan et al., 2003; Wilkins and Molkentin, 2004). Future studiesshould aim at clarifying the regulatory pathways involved in Ca2+ release and modulationinvolved in these processes.

Calcium and the stem cell nicheSeveral studies are beginning to show a role for Ca2+ signaling in stem cell development.Human bone marrow-derived mesenchymal stem cells (hMSDs), show Ca2+ oscillations thatare dependent on both Ca2+ release from IP3Rs in the ER as well as Ca2+ entry and extrusionvia plasma membrane ion pumps and Na+- Ca2+ exchangers (Kawano et al., 2002; Kawanoet al., 2003). Further studies found that the Ca2+ oscillations depend on an autocrine/paracrinesignaling pathway, where secreted ATP stimulates P2Y1 receptors to activate PLC-β toproduce IP3 (Kawano et al., 2006). These same studies showed that the translocation of thedownstream transcription factor NFAT is dependent on the ATP-induced Ca2+ oscillations,and that these oscillations and NFAT nuclear translocation disappeared as hMSCsdifferentiated into adipocytes. Conversely, increases in intracellular Ca2+ result in theinhibition of differentiation of human adipocytes (Ntambi and Takova, 1996). These studiessuggest a link between intracellular Ca2+ oscillations and the maintenance of undifferentiatedhMSCs.

Another interesting report has shown a role for extracellular Ca2+, present in the endostealsurface of the bone marrow and sensed by the seven transmembrane–spanning Ca2+-sensingreceptor (CaR), in the migration and homing of mammalian haematopoietic stem cells (HSCs;

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Adams et al., 2005). In this case, however, Ca2+ signaling does not appear to influence theability of HSCs to proliferate or differentiate. As stem cells corresponding to other cell typesare studied, it will be interesting to determine how common the involvement of Ca2+ signalingis in stem cell specification, homing and maintenance.

Human developmental disorders involving calcium-sensitive factorsDefects in the regulation of Ca2+-sensitive factors may underlie a variety of developmentalhuman syndromes. Two genes within the critical region responsible for Down’s syndrome,DSCR1 and the nuclear serine/threonine kinase DYRK1A, act synergistically to prevent thenuclear translocation of the calcineurin target NFAT (Arron et al., 2006). This and previousstudies have shown that calcineurin- and NFAT-deficient mice, as well as Dscr1- and Dyrk1a-overexpressing mice, show phenotypes similar to those of human Down’s syndrome, includingneurological, skeletal, cardiovascular and immunological defects (Arron et al., 2006). Theauthors propose that a 1.5 fold-increase in dosage of the DSCR1 and DYRK1A genesdestabilizes a regulatory circuit leading to reduced NFAT activity and Down syndromefeatures. A potential for disrupted Ca2+ regulation of the calcineurin/NFAT pathway resultingin Down’s syndrome is further supported by the conservation across species of pathwaysregulating NFAT nuclear localization, namely activation by intracellular Ca2+ increase andcalcineurin, and inhibition by DYRK kinases (Gwack et al., 2006). However, further analysiswill be required to determine the precise role of Ca2+ signaling in Down’s syndrome.

Recent studies have implicated a role of Ca2+ signaling misregulation in another humandevelopmental disorder, Noonan syndrome, which is associated with facial dysmorphia,disproportionate short stature, increased risk of leukemia and congenital heart defects (Noonan,1968; Allanson, 1987). This syndrome is thought to be caused by mutations in a src homology2-containing protein tyrosine phosphatase (SHP-2/PTPN11), which cause its constitutiveactivation (Tartaglia, 2001; Tartaglia, 2003; Araki, 2004). Gain-of-function mutants of SHP-2/PTPN11 enhanced FGF-2-mediated Ca2+ oscillations in fibroblasts, as well as spontaneousCa2+ oscillations in cardiomyocytes (Uhlén et al., 2006). Together with the known role of thecalcineurin/NFAT pathway in cardiac morphogenesis (Hogan et al., 2003; Schulz and Yutzey,2004; Wilkins and Molkentin, 2004), these data suggest that at least some aspects of Noonansyndrome may be caused by increased frequency of Ca2+ oscillations and overactivation ofcalcineurin/NFAT signaling.

ConclusionOne of the most intriguing questions in biology is how ubiquitous signals can be used to conveyspecific information. Ca2+ signaling constitutes an excellent example of this challenge, sinceit is important for basic cellular processes, from cell division to cell death, and also appears toregulate a variety of specific events involved in patterning and morphogenesis. An importantpart of the solution to this problem appears to be that information can be encoded throughvariations in amplitude, length and frequency of Ca2+ oscillations. Our understanding of themechanisms that regulate these oscillations, and the processes involved in translating theireffects into cellular responses, is still in its infancy. Other important avenues of research willaddress how such basic information branches into coordinated pathways involving both cellfate specification and morphogenesis. The exciting recent findings that suggest thatmisregulation of Ca2+ signaling pathways is involved in a number of human developmentaldisorders, imparts signinficant urgency to the quest toward their understanding, as it may resultin therapies to treat these genetic disorders.

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Fig. 1.Schematic diagram of cellular Ca2+ sources in non-excitable cells. (1) Stimulation of the cellswith agonists and growth factors leads to the activation of GPCR and RTK. (2) This leads toactivation of PLC isoforms, which catalyze the hydrolysis of PIP2 giving rise to IP3 and DAG.(3) IP3 binds to its receptor (IP3R) on the ER and triggers Ca2+ release from the store. (4) Oneaspect of CICR involves Ca2+ binding to the high affinity Ca2+-activation sites on IP3R andRyR inducing the channels to open. (5) Intracellular Ca2+ is rapidly bound by Ca2+ bindingproteins, which leads to their activation. (6) DAG is another second messenger which activatesPKC among other targets. (7) Clearance of cytoplasmic Ca2+, shown by dashed lines, occursby Ca2+ extrusion via plasmalemmal pumps and Na+/Ca2+ exchange as well as by uptake intointracellular stores (8). GPCR, G protein-coupled receptor; RTK, receptor protein tyrosinekinase; PLC, phosphoinositidespecific phospholipase C; PIP2, membranephosphatidylinositol-4,5-biphosphate; PLC, Protein Kinase C; DAG, diacylglycerol; IP3,inositol-1,4,5-trisphosphate; IP3R, IP3-receptor; ER, endoplasmic reticulum; RYR, ryanodinereceptor; CaMK II, Ca2+calmodulin – dependent kinase II; CICR, Ca2+ induced Ca2+ release;NFAT, nuclear factor of activated T-cells.

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Fig. 2.Schematic diagram of the Wnt signaling network. Highlighted are key components identifiedin the (A) Wnt/β-catenin and the (B) Wnt/Ca2+ signaling pathways. When the so-called“canonical” or Wnt/β-catenin path is inactive, a degradation complex, including Axin, GSK-3,and APC, phosphorylates β-catenin inducing its rapid destruction by the proteasome. Once theFrizzled(Fz)/LRP co-receptor complex is bound by Wnt, Fz interacts with Dsh, which modifiesthe destruction complex and leads to β-catenin stabilization. Nuclear β-catenin interacts withLEF/TCF to promote the transcription of Wnt target genes. The so-called “non-canonical”Wnts are thus named as they appear to act independently of β-catenin. Wnt binding to Fz leadsto activation of Dsh, an increase in intracellular Ca2+ and activation of PKC. Increasedintracellular Ca2+ can then lead to a secondary activation of PKC as well as to activation ofCaMKII and NFAT. Increased intracellular Ca2+ and activated calcium sensors have beenshown to antagonize β-catenin, noted as red bars. The PCP pathway also signals through Fzand Dsh which then signals through small GTPases (Rho) and C-Jun N-terminal kinase (JNK)to modulate cytoskeletal elements. The PCP pathway utilizes core components, shown are stbmand pk. Fz, Dsh and pk are all capable of activating Ca2+ release. Fz, Frizzled; LRP, low densitylipoprotein receptor; APC, adenomatous polyposis coli; GSK-3, glycogen synthase kinase 3;Dsh, Dishevelled; TCF, T cell factor; LEF, lymphoid enhancer factor; PKC, protein kinase C;PLC, phospholipase C; JNK, c-jun NH2-terminal kinase; stbm, Strabismus; pk, Prickle.

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Table 1Regulators of calcium signaling with an inferred developmental role

Factor Type Role Process affected References

Wnt-5/Ppt extracellular ligand activates Ca2+ transients axis induction /convergence extension

Slusarski, et al., 1997a,b;Westfall et al., 2003

hecate unknown regulates Ca2+ transientfrequency

axis induction Lyman-Gingerich et al., 2005

CaMKII EF-hand Ca2+–binding kinase regulates target protein factors axis induction /convergence extension

Kühl et al., 2000

calcineurin Ca2+-dependent phosphatase promotes NFAT nucleartranslocation

axis induction / organformation

Saneyoshi et al., 2002; Yoshidaet al., 2004

NFAT transcription factor regulates target gene expression axis induction / stem cellmaintenance / organformation

Saneyoshi et al., 2002; Kawanoet al., 2006; Shulz and Yutzey,2004; Wilkins and Molkentin,2004

Pkd-2 Ca2+-permeable ion channel required for Ca2+-asymmetry inthe node

left-right asymmetry McGrath et al., 2003

CaR seven-transmembrane Ca2+-sensing receptor required for import ofextracelllar Ca2+

stem cell homing Adams et al., 2005

DYRK1A nuclear serine/threonine kinase prevents nuclear translocationof NFAT

defects associated withDown’s syndrome

Arron et al., 2006

SHP-2/PTPN11 src homology tyrosine phosphatase activates Ca2+ transients defects associated withNoonan syndrome

Uhlén et al., 2006

Dev Biol. Author manuscript; available in PMC 2009 August 19.