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1 An element for development: Calcium signaling in mammalian reproduction and development 2 3 Teneale A. Stewarta,b and Felicity M. Davisa,b,* 4 5 6 7 aMater Research Institute-The University of Queensland, Faculty of Medicine, The University of 8 Queensland, Woolloongabba, QLD, Australia 9 bTranslational Research Institute, Woolloongabba, QLD, Australia 10 *Corresponding author at: Mater Research Institute-The University of Queensland, Faculty of Medicine, 11 The University of Queensland, Woolloongabba, QLD, Australia. Email address: [email protected] (F. 12 Davis) 13 14 15 16 17 18 19 Keywords: Calcium channels, calcium signaling, signal transduction, reproduction, fertilization, 20 embryogenesis, development, stem cells. 21 22 Abstract 23 Life begins with calcium. It is the language that a sperm cell uses to respond to instructions from the female 24 reproductive tract to alter its swimming pattern and gain the force required to penetrate the outer layers of the 25 . The first heartbeat transpires from spontaneous calcium oscillations in embryonic cardiomyocytes. 26 The dynamic balance of calcium between auditory hair cells and the fluid they bathe in enables us to hear our 27 first sound, and our interpretation and response to this sound requires rapid calcium flux through neuronal 28 voltage-sensitive calcium channels. Calcium signaling can decode and integrate informational cues from both 29 the chemical and mechanical cellular microenvironment to drive the form and function of many mammalian 30 organ-systems. Here, we highlight roles for the intracellular calcium signal in the reproductive- and 31 developmental- biology of mammals. A greater appreciation of the signaling pathways that initiate and support 32 life has wide-ranging significance for the fields of reproductive science, neonatology and regenerative 33 medicine. Furthermore, as developmental programs are often reactivated in cancer, an improved 34 understanding of the signaling pathways that underpin mammalian development has important implications 35 for cancer research. 36 1

37 1.0 Introduction 38 Mammalian reproduction and development depend on coordinated programs of cell division, 39 specification, migration and death. These events are organized in space, time and scale by short- and long- 40 range chemical and mechanical signals [1]. As a second messenger, the intracellular calcium signal is capable 41 of decoding and integrating inputs from both the chemical and physical extracellular environment [2]. Calcium 42 signaling also controls cell division, differentiation, migration and death in a range of human and rodent cell 43 types [3]. Thus, calcium-mediated signal transduction is thought to underpin many of the fundamental 44 processes involved in the development and propagation of mammals. 45 The ability of calcium to translate diverse extracellular signals into specific cellular responses is 46 attributed to the ‘calcium signaling toolkit’—that is, the spatiotemporal expression of more than 100 calcium 47 channels, pumps, exchangers, sensors and buffers [2]. A message received, for example, through ligand- 48 activation of certain receptor tyrosine kinases (including the epidermal growth factor receptor and fibroblast 49 growth factor receptor) produces an increase in intracellular calcium via a phospholipase C (PLC)-inositol 50 trisphosphate (InsP3) pathway, which results in endoplasmic reticulum (ER) calcium release through InsP3 51 receptors (InsP3Rs) [4,5]. The nature of the response can depend on various factors, including: 1) the intensity 52 and directionality of the instigating signal [6]; 2) the ER morphology, abundance and subcellular localization 53 [7–9]; 3) the isoform-specific expression [10], tetramer composition [11], post-translational modification [12], 54 stability [13] and mobility state of InsP3Rs [14]; and 4) signal amplification and propagation through calcium- 55 induced-calcium-release events [15]. A second wave of signaling then ensues as ER calcium store depletion 56 is detected by specific ER calcium sensors, STIM1 and STIM2, with additional diversity encoded by alternate 57 splicing [16]. These bind to and activate entry of calcium through store-operated channels, of which there are 58 three known isoforms with differing properties: ORAI1, ORAI2 and ORAI3 (mammalian specific) [4], with 59 further signal complexity arising, for example, through mitochondrial calcium buffering upon opening of these 60 plasmalemmal channels [17]. This is only one example of the complexity and diversity in the calcium 61 response. Touch, stretch, shear stress, G- coupled receptor (GPCR) and purinergic receptor activation 62 are other examples of outside-in signaling that may be decoded through increases in intracellular calcium, 63 with possible integration of coincident signaling events [2]. Thus, whilst the calcium ion per se is only a single 64 biochemical messenger, it is one that can exquisitely vary its properties in space, time and amplitude, through 65 a calcium signaling toolkit that is highly amenable to pharmacological modulation [18]. It is essential that we 66 understand how this integrated communication-control system specifies key aspects of reproduction and 67 development in mammals. 68 In this review, we discuss established and proposed roles for intracellular calcium signaling in 69 mammalian reproduction and development (Fig. 1). We begin by examining roles for the calcium signal in 70 the production of functionally mature haploid gametes, their fusion at fertilization and the successful transition 71 to an early . We review calcium-mediated control of and morphogenesis in the 72 developing embryo and provide a snapshot of some of the calcium channels, transporters and binding 2

73 whose genetic deficiency produces embryonic lethality in rodent models (Table 1), suggestive of an obligatory 74 role in . Finally, we conclude by discussing roles for the calcium signal in the 75 regulation of adult stem/progenitor cells during postnatal development, and highlight recent methodological 76 advances that can be used to improve our understanding of calcium signal-dependent developmental 77 programs. The intracellular language encoded by calcium undoubtedly underpins key events in mammalian 78 reproduction and development; roles will continue to be unveiled as the field evolves and seminal discoveries 79 in lower vertebrate systems begin to be translated to the mammalian arena. 80 81 2.0 Calcium signaling in mammalian reproduction. 82 2.1 Sperm production and maturation. The production of sperm in the seminiferous tubules of the 83 testis is a complex and expeditious process, occurring over 34.5 days in mice [19] and involving the 84 coordinated expression of more than 2,300 [20]. Spermatogonial stem cells reside in the basal 85 compartment of the seminiferous tubules. These cells are capable of self-renewal and progressively give rise 86 to committed spermatogonia, spermatocytes and spermatids [21]. Elongated spermatids, which are 87 morphologically complete although functionally immature, are eventually released into the lumen of the 88 seminiferous tubules, where they undergo further maturation in the epididymis as they transit toward the 89 caudal region for storage prior to ejaculation [21]. More than 350 genes are implicated in or 90 sub-fertility in rodent models [22], including the store-operated calcium entry (SOCE) channel Orai1 [23], 91 the P2x1 purinergic receptor [24], cationic channel of sperm (CatSper1-4, d, z) [25] and the plasma membrane 92 calcium ATPase (Pmca4) [26,27] (Fig. 1). 93 Male mice with a null mutation in the encoding ORAI1 are infertile [4,23]. No viable sperm can 94 be recovered from the cauda epididymis of sexually-mature Orai1-null mice and histological analyses of 95 seminiferous tubules reveal marked defects in the integrity and number of round and elongated spermatids. 96 Defects in sperm production are progressive, with seminiferous tubules from mature adult mice exhibiting 97 atrophy, dilation and in some cases evidence of germ cell depletion [23]. Further studies using somatic- and 98 germ cell- specific knockout models are required to determine the precise role(s) of ORAI1 channels in sperm 99 production and maturation. Indeed, it is currently unclear whether the observed sterility of Orai1-null male 100 mice is attributable to a direct and essential role for this channel in spermatogenesis (e.g., mitotic stem cell 101 divisions, or spermiogenesis) or whether it has an indirect role in sperm production and maturation 102 via the supporting and interstitial cells in the testis (e.g., Sertoli or Leydig cells). Defects may also be related 103 to the productive trafficking of sperm through the male reproductive tract [28], the extent of fluid reabsorption 104 in the efferent ductules [29], or a multiplicity of factors [21]. 105 The final passage of sperm through the male reproductive tract during ejaculation is dependent on 106 contraction of the smooth muscle cells that surround the vas deferens [24,28,30]. Contractile force is 107 diminished in mice that are deficient for the gene encoding the calcium-permeable ligand-gated 108 P2X1, and P2x1-null male mice are correspondingly sub-fertile [24]. Similarly, male mice with a targeted 3

109 deletion of the gene encoding the α1A-adrenoceptor as well as α1A/α1B/α1D-adrenoceptor triple knockout mice 110 are infertile, with abnormal vas deferens contractility [30]. Moreover, defects in smooth muscle contractility

111 and fertility are enhanced in male mice lacking both P2X1 receptors and α1A-adrenoceptors, despite normal

112 testicular morphology, sperm quality and motility [28]. As α1-adrenoceptors signal through a pathway that 113 involves phospholipase C (PLC) and inositol 1,4,5-trisphosphate (InsP3), compound infertility in this model 114 may be due to impaired calcium-contraction coupling in smooth muscle cells of the vas deferens. The male 115 sterility observed in the Orai1-null model [23] may also be in-part attributable to reduced SOCE downstream

116 of α1-adrenoceptor activation, producing defects in calcium-contraction coupling. 117 Normal, healthy sperm achieve functional competence in the female reproductive tract following 118 ejaculation. This process, known as capacitation, involves elevated intracellular pH and calcium, and a 119 transformation in the pattern of flagellar beating to a more powerful, asymmetric motion, which facilitates 120 penetration of the cumulus and zona pellucida of mammalian (reviewed in Ref [31]). Calcium- 121 permeable CATSPER channels are localized to the flagella of sperm and are essential for hyperactivated 122 motility. Male mice lacking genes encoding the α-subunits (CATSPER1-4) [25,32,33] or the auxiliary 123 subunits CATSPERδ [34] and CATSPERζ [35] have defects in sperm motility producing infertility or sub- 124 fertility in these models. In humans, infertility has been observed in men with mutations or deletions in the 125 genes encoding CATPSER1 [36] and CATPSER2 [37,38]. In the absence of significant amounts of ER [31], 126 increases in intracellular calcium levels via CATSPER channels are largely returned to basal levels via the 127 action of plasmalemmal calcium efflux pumps [26,27]. Pmca4 is the most highly expressed PMCA isoform 128 in mouse testes [26] and Pmca4-null male mice are infertile and have defects in hyperactivated motility and 129 evidence of flagellar calcium overload [26,27]. The calcium signal is thus exquisitely controlled throughout 130 the male reproductive tract and in male gametes to ensure sperm production, maturation and survival, which 131 is essential for mammalian procreation. As plasma membrane channels represent attractive targets for 132 therapeutic manipulation, the exploitation of one or more of these pathways may lead to novel, non-steroidal 133 therapeutics for male contraption. 134 2.2 Egg-to-embryo transition. Sperm-egg fusion triggers a series of repetitive calcium transients in the 135 oocyte, which are responsible for initiating embryogenesis under normal physiological conditions [39,40]. 136 These oscillations are triggered by a sperm-specific isozyme of phospholipase C (PLCζ), which is expressed 137 on the perinuclear theca adjacent to the acrosome [41]. Sperm-derived PLCζ cleaves phosphatidylinositol 4,5-

138 bisphosphate (PIP2) present in the oocyte, to generate InsP3 and instigate calcium release from ER calcium 139 stores. The importance of PLCζ-mediated calcium signaling for optimal oocyte development, however, has 140 remained the subject of investigation and debate [40,42,43]. Recently, studies performing in vitro fertilization 141 or intracytoplasmic sperm injection using sperm derived from Plcz1-null mice, revealed an absence of the 142 calcium oscillations that are typical of mammalian oocyte activation [42]. Nevertheless, a small number of 143 eggs fertilized by Plcz1-null sperm in this study were able to develop to the stage [42], and male

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144 Plcz1-null mice were shown to be merely sub-fertile, not infertile [42,43] (in contrast to humans with 145 mutations in the gene encoding PLCζ [44]). In a separate study using in vitro fertilization in rodent models, 146 Plcz1-null sperm were capable of eliciting a calcium response, albeit highly irregular in nature [40,43]. The 147 mechanisms underpinning PLCζ-independent calcium transients, their role in mice and men and the fidelity 148 of derived using PLCζ-independent mechanisms are yet to be fully understood. 149 PLCζ-InsP3-mediated calcium oscillations in the oocyte rely on calcium entry across the plasma 150 membrane [45]. The classical pathway for calcium influx downstream of InsP3-mediated ER calcium store 151 depletion is store-operated entry via STIM-ORAI [4]. However, whilst pharmacological inhibition of store- 152 operated channels successfully blocks thapsigargin-mediated calcium entry in freshly ovulated eggs, these 153 inhibitors fail to prevent calcium oscillations and egg activation. These data indicate that a STIM-ORAI- 154 independent calcium entry pathway is likely to be responsible for egg activation [45]. Indeed, in a decisive in 155 vivo study using Orai1-null female mice, it was demonstrated that SOCE via ORAI1 is dispensable for murine 156 egg activation and fertility [46]. This work was later confirmed in calcium imaging experiments using oocytes 157 derived from Orai1-null mice as well as those derived from Stim1, Stim2 and Stim1/Stim2 conditional 158 knockout mice [47]. The calcium- and magnesium- permeable transient receptor melastatin 7 (TRPM7) 159 channel and the Cav3.2 voltage-activated channel, encoded by Cacna1h, have recently been demonstrated to 160 be largely responsible for the calcium entry that supports calcium oscillations in mammalian oocytes [48]. It 161 should be noted, however, that similar to in vivo matings using Plcz1-null male mice, female mice lacking 162 both Trpm7 and Cacna1h in oocytes are merely sub-fertile in in vivo mating studies [48], suggestive of 163 alternate pathways for egg activation signaling. 164 2.3 The placenta. Transfer of calcium from the maternal to the fetal circulation is required for prenatal 165 calcium-dependent signal transduction (discussed in Section 3.0) and bone mineralization [49]. It has been 166 estimated that the human placenta transfers approximately 30 g of calcium across the layer, and 167 that the majority of this transfer takes place during the third trimester [50]. Thus, similar to secretory cells in 168 the mammary gland [46], placental trophoblast cells are tasked with transporting large amounts of calcium 169 across their cytosol to support offspring development, whilst avoiding calcium-mediated cytotoxicity [49]. A 170 number of calcium channels and transporters are expressed in the placenta and endothelial cells of the placenta 171 [49,51], including the genes encoding PMCA1-4 [49,52], sodium-calcium exchanger -1 and -2 (NCX1 and 2) 172 [49,52,53], TRPV6 [49,54] and PIEZO1 [55], where they may regulate placental perfusion, transport and 173 ultimately embryonic viability [53,55,56]. Further studies are required to establish the precise calcium 174 signaling toolkit of the placenta and extraembryonic structures; to characterize the specific roles of these 175 channels and transporters in placental calcium transfer, signaling and vascularization; and to definitively 176 determine how their aberrant expression or dysfunction may lead to gestational complications. 177 178 3.0

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179 The calcium signal is an important participant in the evolution of the developing embryo from a 180 fertilized oocyte, enabling a multitude of biologically distinct and complex processes, such as cell fate 181 determination and organogenesis (reviewed in [1,2]). Such a feat is made possible by the selective expression 182 and engagement of specific components of the calcium signaling toolkit, at precise points in space and time 183 [2]. Based on studies in other vertebrate model systems—primarily Xenopus, zebrafish and medaka fish— 184 there is strong evidence supporting a role for the calcium signal throughout the embryonic development of 185 vertebrate animals [1]. Early embryonic development in mammals involves complex and dynamic cellular 186 interactions and rearrangements required for the determination of the mammalian body plan. This includes 187 the derivation of the three germ layers (, and ), from which the somatic tissues 188 of the developing embryo are derived (reviewed in [57,58]). Whilst our understanding of the signal 189 transduction pathways that drive mechanisms of multicellularity in mammals lags behind that of lower 190 vertebrates, accumulating evidence supports both redundant and essential roles for calcium signaling in 191 mammalian embryonic development and organogenesis (Fig. 1 and Table 1) [26,59–65]. In the following 192 section we limit the discussion to studies that utilize whole embryos and explanted embryonic tissue to 193 investigate three key stages of mammalian embryogenesis: left-right patterning, cardiac development and 194 kidney organogenesis. These three embryonic events, whilst fundamentally distinct in time, space and nature, 195 each appear to be in-part regulated by the versatile calcium signal. 196 3.1 Left-right patterning and asymmetric morphogenesis. Asymmetric morphogenesis, characterized 197 by distinct left-right asymmetry, or ‘patterning’, of the internal organs and vasculature across the mediolateral 198 plane, is fundamental to normal mammalian development [66–69]. In the mouse embryo, left-right patterning 199 is initiated around E7.5 by a cilia-driven asymmetric fluid flow (‘nodal flow’ [70]) originating from within 200 the ventral node, a transient midline structure present during early somitogenesis [67,69,71,72]. Nodal flow 201 sensing by cells peripheral to the ventral node activates downstream signaling activities, including the Nodal 202 signaling pathway, culminating in left-right patterning of the and situs-specific 203 morphogenesis (reviewed in [66,69,71,72]). The mechanisms underlying detection of nodal flow, of which 204 mechanosensing and/or chemosensing are two prevailing models, and its translation to localized signaling 205 events that are asymmetrically transferred to the lateral plate mesoderm are not well understood, though 206 evidence supports a role for calcium signaling [73,74] (for comprehensive reviews refer to [72,75]). Early 207 reports of a role for calcium signaling in sensing nodal flow suggested an essential role for the calcium 208 permeable channel polycystin-2 (PKD2) protein in normal mouse embryonic left-right patterning [76]. The 209 whole body Pkd2 mutant phenotype is embryonic lethal (Table 1), with animals exhibiting altered Nodal 210 expression and laterality defects [76], in addition to cardiovascular and renal defects [60,76]. Subsequent 211 studies confirmed PKD2 localization to primary cilia of the embryonic node [73,77], where together with 212 -like 1 (PKD1L1) protein [78,79], it is proposed to facilitate flow sensing in nodal crown cells 213 [73,77,78]. While increased calcium levels have been detected in crown cells engineered to express the 214 genetically-encoded calcium indicator (GECI) GCaMP2 in response to nodal flow, this is not dependent on 6

215 PKD2 expression [77]. However, it has been suggested that the frequency of calcium signals is reduced in 216 embryonic mouse nodes lacking Pkd2, relative to wildtype embryos [80], and PKD2-dependent calcium 217 increases have been reported in perinodal cells, implicating a role in the transfer of the calcium signal to the 218 lateral plate mesoderm [73]. Despite reports that polycystin family members, including PKD2, function as 219 mechanosensing complexes in other biological contexts [81], recent studies employing transgenic mice 220 engineered to express a GECI with a reference fluorescent protein for ratioing in primary cilia, demonstrated 221 that deflection of nodal cilia does not initiate localized calcium signals [82]. These findings bring into question 222 previous hypotheses linking mechanical activation of primary cilia at the node and PKD2-mediated calcium 223 increases [73,77], suggesting alternative mechanisms underlie the altered calcium signaling and embryonic 224 laterality defects associated with genetic deletion of PKD2 [82]. Of interest, are observations that 225 pharmacological modulators of components of the SOCE pathway can alter nodal calcium signals [80], as 226 well as the perinodal expression of a transcriptional enhancer (ANE) [77], which is implicated in the Nodal 227 signaling pathway during mouse embryonic left-right patterning. Node calcium signals were absent in mouse 228 embryos pretreated with the sarco/endoplasmic reticulum calcium ATPase (SERCA) inhibitor, thapsigargin 229 [80], which depletes ER calcium stores and activates SOCE. Moreover, thapsigargin, micromolar 230 concentrations of gadolinium (Gd3+) [83], and 2-ABP [84] each disrupted left-right asymmetric ANE activity 231 in the embryonic mouse node [77]. Many questions remain regarding the role of specific calcium signaling 232 pathways (and their functional compensation) in the establishment of mammalian left-right asymmetry, 233 including members of the SOCE family of proteins such as ORAI1 and STIM1 [85]. In summary, while 234 findings support a role for calcium signaling during mammalian left-right patterning, further studies are 235 required to fully identify the proteins involved and their specific contribution, both during nodal flow sensing 236 and in the transfer of asymmetric signals away from the node. 237 3.2 Heart and vascular system. Heart development commences at week three of human embryogenesis 238 (corresponding to E7.5 in the mouse), outcompeting all other organs in both its formation and function 239 (reviewed in [86,87]). Maintenance of calcium homeostasis is essential for the proper functioning and health 240 of the adult heart, as exemplified by the process of calcium-induced calcium release and its role in excitation- 241 contraction coupling, the perturbation of which has been linked to various cardiac pathologies [15,88]. It is, 242 therefore, unsurprising that calcium signaling should play an integral role during the development of the 243 embryonic heart. Indeed, requirements for oxygen and nutrient delivery to the growing embryo are met by the 244 developing cardiovascular system [86]. A number of studies have shown that calcium signaling proteins 245 known to play an important role in the adult heart—including members of the voltage-gated

246 family (e.g., Cavβ2) [89], NCX1 [53,90,91], and type 2 (RyR2) [63]—are essential for 247 murine embryonic heart development and embryonic cardiac function (Table 1). Calcium signaling proteins 248 not typically associated with normal adult cardiac function, such as the ubiquitous calcium binding protein 249 calreticulin, have also been identified as being essential for embryonic cardiac development [65]. Early studies 250 investigating the mode and onset of cardiac contractions in mouse embryos identified fluctuations in 7

251 intracellular calcium levels in explanted embryonic hearts, loaded with the ratiometric calcium indicator fura- 252 2, which increased in regularity and amplitude from E8 (the earliest time point assessed) [92]. More recently, 253 a study by Tyser et al. [93] revealed that spontaneous calcium oscillations in the forming cardiac crescent 254 (E7.75) precede discernable cardiac contractions in the early mouse embryo [93], supporting a role beyond 255 initiation of the first heartbeat. Using sensitive live imaging techniques combined with bulk loading of a 256 synthetic calcium indicator, CAL-520, the authors were able to visualize the progression of asynchronous 257 oscillations to coordinated calcium transients, culminating in the first heartbeat in cultured mouse embryos 258 [93]. Treatment of early mouse embryos with pharmacological inhibitors of NCX1 resulted in significant loss 259 of spontaneous calcium oscillations as well impaired heart development [93], supporting a role for NCX1- 260 mediated calcium signaling in early stage cardiac development and function. However, the selectivity of 261 NCX1 in regulating spontaneous calcium oscillations in the early mouse embryo appears to be temporally-

262 dependent, with nifedipine-mediated L-type calcium channel (Cav1.2) blockade demonstrating greater than or 263 equal inhibition of calcium transients in later stage embryos [93]. Using Ncx1 knockout mouse models, a 264 number of groups have confirmed an essential role for this channel during murine embryogenesis, particularly 265 with respect to the developing heart. However, variability regarding the ability of Ncx1 ablated embryos to 266 generate heartbeats and the degree of cardiac specific defects reported exists [64,91,94,95]. Further evidence 267 of complex and redundant roles of components of the calcium signaling toolkit during murine cardiogenesis 268 is provided by studies assessing the role of the intracellular calcium release channels inositol 1,4,5- 269 trisphosphate receptor -1 and -2 (InsP3R1 and -2) [96]. While embryos in which genetic disruption of either 270 InsP3R alone developed normally, InsP3R1 and -2 double mutants exhibited signs of impaired cardiogenesis 271 and lethality by E11.5, an effect proposed to be mediated in-part by disrupted NFAT signaling [96]. 272 Recently, the mechanosensitive calcium permeable ion channel, PIEZO1, was shown to be required 273 for normal development of the murine vascular system [56,61]. Mouse embryos with globally-disrupted 274 Piezo1 exhibit signs of impaired cardiovascular function, disrupted vascular architecture and lethality typically 275 by mid-gestation [56,61]. PIEZO1 has subsequently been shown to play a role in the regulation of lymphatic 276 valve formation in mice using lineage specific targeting of Piezo1 [97]. Importantly, PIEZO1 is activated in 277 response to shear stress [56,61], a physiologically relevant stimulus for this channel in developing vasculature 278 [98,99]. Thus, calcium signaling is likely to be a major player in the developing cardiovascular system, with 279 spontaneous calcium signals underpinning the first heartbeat to facilitate fluid flow that is sensed and 280 transduced by PIEZO1 in a calcium-dependent manner to drive the formation of the primitive vascular 281 network. Collectively, these findings highlight the complex and interconnected mechanisms by which the 282 calcium signal impacts cardiovascular development and function, thus further expanding our appreciation of 283 the importance of calcium signaling during mammalian embryogenesis. 284 3.3 Kidney development. In mammals, the kidney (metanephros) derives from the intermediate 285 mesoderm [100]. Two key lineages arising from the , the ureteric bud and the 286 metanephric mesenchyme, give rise to the cells of the collecting duct system and remainder of the kidney 8

287 (including nephrons, stromal and vascular elements), respectively [101]. Successful formation of the 288 metanephros is a complex process, requiring reciprocal interaction between the cells of the ureteric bud and 289 metanephric mesenchyme and an elaborate process of branching morphogenesis [100–102]. Relative to left- 290 right axis specification and heart formation, direct evidence of calcium signaling in mammalian embryonic 291 kidney development in the form of calcium imaging studies are lacking. However, a number of studies 292 indirectly support a role for the calcium ion as an intracellular messenger during various stages of kidney 293 formation [103–106]. Indeed, early studies identified distinct temporal and spatial patterns of expression of 294 various calcium-binding proteins, including calbindin-D28k and calbindin-D9k [105,106], during mammalian 295 kidney development [104]. The calcium/Nuclear Factor Activated in T cells (NFAT) signaling pathway has 296 been implicated in the regulation of numerous biological functions in vertebrates [107], including cardiac 297 valve formation in mice [108,109]. Calcineurin, a calcium/calmodulin-dependent serine phosphatase, 298 dephosphorylates NFAT proteins following cellular stimulation, allowing their nuclear translocation and 299 subsequent transcriptional activity [107]. Studies in which explanted embryonic mouse kidneys were treated 300 with the calcineurin inhibitor, Cyclosporin A, exhibit disrupted morphogenesis, providing further indirect 301 evidence of a role for calcium signaling in the regulation of early kidney development [103,110]. 302 As mentioned above, whole body knockout of the calcium permeable polycystin-2 (Pkd2) ion channel 303 in mice is embryonic lethal, resulting in laterality, cardiovascular and renal defects [60,76], with cyst 304 formation apparent from E15.5 in the developing kidney [60]. More recently, the calcium-permeable TRPM7 305 channel kinase was also shown to be essential for normal mouse development, with global Trpm7 disruption 306 resulting in early embryonic lethality [59] (Table 1). Lineage-specific deletion of Trpm7 has provided insight 307 into its role during the development of the mouse embryonic kidney. Selective Trpm7 knockout in cells of the 308 metanephric mesenchyme from E11.5 using a Pax3-Cre model resulted in a phenotype indicative of impaired 309 nephrogenesis, including reduced glomeruli number, presence of renal cysts and smaller kidney size, relative 310 to control animals [111]. In contrast, selective Trpm7 knockout in ureteric bud cells from the same stage of 311 embryonic development, using the HoxB7-Cre model, produced animals with kidneys of normal size and 312 histology [111]. While PKD2 and TRPM7 clearly play important roles during mammalian embryonic kidney 313 development, the question of their relationship to the calcium signal [given the potentially confounding kinase 314 activity (TRPM7) and permeability of non-selective ion channels to other divalent and monovalent cations], 315 and their endogenous mechanisms of activation in cells of the developing kidney remain to be answered 316 [82,112,113]. 317 Studies investigating direct intracellular calcium increases in the mammalian embryonic kidney are 318 limited in number, despite evidence of its role during renal development of other vertebrate systems (reviewed 319 in [114]). However, a role for spontaneous calcium oscillations in the regulation of branching morphogenesis 320 in explanted embryonic (E14) rat kidneys was recently investigated [115,116]. Using the calcium indicator 321 dye Oregon Green BAPTA-1/AM together with time-lapse imaging, spontaneous calcium oscillations were 322 observed in the cells of the outer kidney layer in which dye uptake occurred [115]. ER calcium store depletion 9

323 by pharmacological inhibition of SERCA using cyclopiazonic acid (CPA) reduced spontaneous calcium 324 activity. Rather paradoxically, however, calcium oscillations were not shown to run-down in the absence of 325 extracellular calcium [117]. Longer-term (24 h) exposure of kidney explants to low concentrations of CPA or 326 the intracellular calcium chelator BAPTA/AM negatively impacted ureteric branch tip and primitive glomeruli 327 number, suggestive of abnormal branching morphogenesis. The authors propose that spontaneous calcium 328 oscillations, together with stochastic factors, contribute to the process of branching morphogenesis in the 329 embryonic rat kidney [115]. However, given the inherent limitations of synthetic calcium indicators, including 330 the poor dye penetration of bulk-loaded kidney explants observed in this study, it would be valuable to 331 determine the extent and cell-type specificity of oscillatory behavior using transgenic animals engineered to 332 express a GECI in specific cell lineages of the kidney. Such a model would also allow for the long-term and 333 strategic assessment of the impact of pharmacological manipulation of calcium channels and pumps. 334 The examples provided above of calcium signaling during left-right axis patterning, heart and kidney 335 development illustrate just some of the ways in which this important intracellular second messenger may 336 contribute to mammalian embryonic development. Both established and emerging roles of the calcium signal 337 during embryogenesis have been discussed. Further studies, aimed at identifying the proteins that facilitate 338 calcium flux and specific downstream targets are required to more fully appreciate the role of calcium 339 signaling during normal mammalian embryonic development. 340 341 4.0 Postnatal development 342 By birth, most of the organs of placental mammals have reached functional competence, and postnatal 343 development is largely characterized by adaptation, maturation and allometric growth. Roles for calcium 344 signaling in differentiation and proliferation (key cellular events required for maturity and growth) are widely- 345 recognized and have been extensively reviewed elsewhere (see Refs [2,3,118]). There are, however, certain 346 organs including the mammary gland and prostate, which are not essential for early mammalian life, that 347 undergo a significant element of their development postnatally. Whilst mammary development commences in 348 the embryo, mammals are born with only a rudimentary mammary epithelial sprout [119,120]. It is not until 349 pubertal ductal morphogenesis that the arborized, bi-layered mammary epithelium is formed via proliferation 350 of lineage-restricted adult mammary stem and progenitor cells [121,122]. Additional postnatal development 351 and remodeling occur in the mammary gland with each pregnancy-lactation-involution cycle [120]. The 352 prostate, a male reproductive accessory organ responsible for producing key components of the seminal 353 plasma, also undergoes a phase of postnatal branching morphogenesis [123]. Prostatic branching commences 354 in the late rodent embryo and ductal elongation continues approximately two weeks after birth, with minor 355 side branching persisting in the juvenile and adolescent male prostate until the end of puberty [123]. Definitive 356 roles for calcium signaling in the postnatal development and morphogenesis of mammary and prostate 357 epithelia have not yet been investigated. Nevertheless, the calcium signal regulates adult mammary and 358 prostate physiology and pathology [46,124–126], and is hypothesized to play an important role in signal 10

359 transduction and integration in these developing ductal systems downstream of mechanical and/or growth 360 factor signaling [2]. 361 Fundamental advances in our understanding of and organogenesis can be used 362 to illuminate processes underpinning adult tissue regeneration. Varying degrees of regenerative potential have 363 been observed in adult mammalian tissues under physiological and/or non-physiological conditions, including 364 the mammary gland, [121,127], intestine [128], and liver [129]. The remarkable regenerative capacity of the 365 liver is highlighted by rodent models where a two-thirds partial hepatectomy stimulates regeneration and 366 restoration of the original liver mass approximately one-week post-surgery [129]. A recent study by 367 Hajnóczky and colleagues [130] using a liver-specific knockout of mitochondrial calcium uptake 1 (Micu1), 368 which serves as a gatekeeper of the mitochondrial calcium uniporter (MCU), has revealed an essential role for 369 mitochondrial calcium homeostasis in liver regeneration induced by surgical resection [130]. Whilst no 370 apparent histological differences were observed in liver tissue from control and Micu1-knockout mice under 371 non-stimulated conditions, marked differences were observed under conditions of tissue repair induced by 372 partial hepatectomy, including a complete absence of hepatocyte proliferation and evidence of necrosis [130]. 373 This phenotype was reversed by inhibition of the mitochondrial permeability transition pore, indicating that 374 adult liver regeneration under these experimental conditions is calcium-signal dependent (Fig. 1). In non- 375 mammalian vertebrate models, intracellular calcium signaling has been shown to regulate epimorphic fin 376 regeneration and is thought to be one of the signals responsible for diverting wound signals down a path of 377 regenerative growth versus scar tissue formation [131]. Transient increases in intracellular calcium signaling 378 have also been observed in numerous mammalian cell types following wounding, where they influence the 379 fate or phenotype of the surviving cells [132,133]. A greater understanding of how intracellular calcium 380 signaling may govern regenerative versus degenerative responses in mammalian organ-systems remains an 381 important aim for the future. 382 383 5.0 Conclusions and future perspectives 384 Calcium-mediated control of cell proliferation, differentiation, motility and death are well- 385 characterized in mammalian cell culture [2,3,118]. However, relatively little is known about how diverse 386 extracellular signals converge on calcium to orchestrate developmental programs in complex living 387 mammalian systems. The continued development and refinement of GECIs [134,135] offers developmental 388 biologists new opportunities to observe real-time changes in intracellular calcium in intact preparations, at 389 high cellular resolution and at precise developmental stages. Whole-tissue imaging with state-of-the-art GECIs 390 (with optimal dynamic range, affinity, selectivity and kinetics [136]) reduces experimental artifacts associated 391 with partial- or full- tissue digestion. Moreover, the strategic use of both GECIs and spectrally-distinct 392 reference fluorescent proteins, enables ratioing of 4D image sequences, minimizing motion artifacts [82]. 393 Leading the way in this pursuit are studies in non-mammalian vertebrate systems, where rapid genetic 394 manipulation, combined with the advantages of small and often transparent model organisms, has afforded 11

395 unprecedented insights into vertebrate development, ranging from the mapping of spontaneous neural activity 396 and its sensitivity to sensory input [137,138] to visualization of the injury-induced calcium flashes that precede 397 fin regeneration [131] in larval zebrafish. Major advances in light microscopy techniques, including lattice 398 light-sheet [139]—coupled with the improved biophysical properties of genetically-encoded sensors 399 [134,135], emerging platforms for 4-dimensional image analysis [140] and accelerated methods for 400 mammalian genome editing [141]—will undoubtedly yield exciting new insights into calcium-mediated 401 control of mammalian development in the coming years. 402

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403 Transparency document 404 The transparency document associated with this article can be found in the online version. 405 406 Acknowledgements 407 FMD is funded by the Australian National Health and Medical Research Council (1141008 and 1138214) and 408 The University of Queensland (UQ-FREA). FMD and TS are supported by the Mater Foundation. 409 410 Figure Legend 411 Fig. 1: Schematic representation highlighting some of the roles for the calcium signaling toolkit in the 412 reproduction and development of mammals. 413 414

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Table 1: Examples of genetic deletion models resulting in embryonic lethality

Model Developmental Ref. Notes Stage Cav1.2-null E14.5 [62,142] Normal cardiomyocyte function at E12.5; embryonic demise before E14.5 Cavβ2-null E10.5 [89] Diminished L-type currents (E9.5); growth restriction, pericardial effusion and lethality (E10.5) Cavβ2-/fl;MLC2a-Cre E13.5 [89] Deletion in cardiomyocytes at ~E8.5 produced lethality by E13.5; many E11.5-12.5 embryos showed morphological destruction Crt-null E12.5-18.0 [65] Morphological heart defects (E12.5); failed absorption of the umbilical hernia (E18.0) Mcu-null E11.5-13.5 [143] Lethality specific to embryos generated on a pure inbred C57BL/6 background; cause of lethality is unknown Ncx1-null E9.0-10.0 [64] Lack of obvious yolk sack blood vessels, enlarged pericardial sac and growth restricted, heartbeat absent or very slow/arrhythmic (E9.5) < E10.5 [144] Myocardial cell apoptosis (≥ 8.5); regions of embryo necrosis (≥ E9.0); growth restricted (E9.0-9.5); underdeveloped heart, weak contractions and low heart rate (E9.0); dilated pericardial sac (E9.5); embryonic demise before E10.5 E11.0 [90] Lack of discernible heartbeat and growth restricted (E10.0); cardiomyocytes exhibit signs of impaired contractile apparatus (E9.5); embryonic demise at ~ E11.5 Piezo1-null E9.5-14.5/16.5 [56,61] Lack of vessel organization (E9.5); growth retardation (from E9.5); pericardial effusion (E10.5) Piezo1fl/fl;Tie2-Cre nd#^ [56] Defects yolk sac vasculature (E9.5); growth retardation (E10.5); embryonic lethality observed within days of heart beating Pkd2-null E13.5-birth [60] Structural abnormalities in cardiac septation; cyst formation in kidneys and pancreas Pmca1-null nd# [26] Mating of heterozygous animals resulted in no homozygous progeny Ryr2-null E10.5-11.5 [63] Growth delay, morphological cardiac abnormalities but spontaneous contractions (E9.5); absence of heart beat and congestive peripheral tissues (E10.5) Trpm7-null < E7.5 [59] LacZ staining of TRPM7geo/+ embryos shows expression in embryonic heart (E9.5), ventral region (E10.5), with widespread distribution by E11.5-12.5 Trpm7fl/fl;CAGGCre- E7.0-9.0 [111] Prenatal lethality when gene deletion induced ERTM between E7.0-9.0; embryonic survival when induced at E14.5 Abbreviations: Crt, calreticulin; Mcu, mitochondrial uniporter; Ncx1; sodium-calcium exchanger; Pkd2, ; Pmca1, plasma membrane ca ATPase 1; Ryr2, ; Trpm7, transient receptor potential-melastatin-like 7 #nd, embryonic day not defined ^A recent study [97] of Tie2Cre;Piezo1cKO mice did not observe embryonic lethality (most pups died < P14); phenotype differences have been proposed to be dependent on genetic background by the authors 415 14

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NCX1, Cav1.2, Cavβ2, PIEZO1, RYR2, Calreticulin PKD2, TRPM7

ORAI1 MICU1 P2X1 [...] CATSPER PLCζ-InsP3-Ca2+ PMCA4 TRPM7 CACNA1H