Ca²⁺ is a crucial second messenger utilized in a diverse range of cellular processes in the development of multicellular organisms 12. Since changes in cytosolic Ca²⁺ trigger calcium-dependent cellular responses by activating signaling and transcriptional cascades mediated by Ca²⁺-dependent proteins 34, the levels of cytosolic Ca²⁺ need to be highly regulated to effect appropriate developmental signaling responses. The major intracellular store of Ca²⁺ is the endoplasmic reticulum (ER) from where Ca²⁺ is mobilized for signaling processes 5. Developmental regulation of Ca²⁺ release from the ER is critical for patterning the body plan of the vertebrate embryo 4. In dorso-ventral patterning, cytosolic Ca²⁺ activates protein kinase C and calcium/calmodulin-dependent kinase II enzymes that promote ventral fate through activation of the transcriptional regulator NFAT 67, and inhibits dorsal fate by antagonising components of the canonical Wnt/β-catenin pathway 8. The developmental regulation of the Ca²⁺ flux between the ER lumen and the cytosol, and the replenishment of Ca²⁺ stores from extracellular Ca²⁺ is thus likely to play a significant role in the signaling pathways that specify changes in cell phenotype.

While the IP₃ receptor-mediated release of Ca²⁺ from the ER into the cytosol and the SERCA pumps that regulate Ca²⁺ influx back into the ER are well understood 9, the molecular nature of the components that regulate the replenishment of Ca²⁺ stores from the extracellular space after depletion of ER stores have only recently been elucidated 1011. STIM1 and Orai1 (CRACM1) have been identified as critical regulators of ER Ca²⁺ homeostasis in mammalian cells 1213141516. STIM1 is predominantly localized in the ER membrane where it functions as the essential molecular sensor of ER Ca²⁺ levels. When levels are sufficiently depleted STIM1 triggers Ca²⁺ entry through highly selective store-operated Ca²⁺(SOC) channels in the plasma membrane, also referred to as Ca²⁺-release activated channels (CRAC) in lymphocytes 17. Ca²⁺-depletion induces rapid aggregation of STIM1 polypeptides within the ER membrane and their subsequent translocation to specific regions of the ER membrane juxtaposed to the plasma membrane 1819202122. At these sites STIM1 activates Ca²⁺ entry through SOC channels by interacting with the cytosolic domains of Orai1 proteins, the key molecular subunits of SOC channels 232425. The structurally related family member STIM2 appears to independently regulate basal ER Ca²⁺ levels 26 and can also antagonise the function of STIM1 27. Both STIM1 and STIM2 have also been implicated in store-independent regulation of Ca²⁺ entry 2829, suggesting that STIM proteins may have broad roles in modulating store-operated and store-independent Ca²⁺entry pathways in a variety of cell types.

STIM1 and STIM2 are known to be widely expressed in embryonic and adult tissues 3031, and recent genetic knockout analyses in mice indicate they have overlapping but not identical functions, and that both are required for survival beyond the first few weeks of life 32. The important physiological role of STIM-mediated Ca²⁺ entry through SOC (CRAC) channels has been demonstrated in T lymphocyte activation and contractile function of skeletal muscle 3233. STIM1 and STIM2 expression are both critical for SOC influx and NFAT nuclear translocation and cytokine production in mouse T lymphocytes, as well as for the development of regulatory T cells 32. Defective SOC entry in human lymphocytes results in severe combined immunodeficiency 12. In murine skeletal muscle STIM1 expression is also required for NFAT-mediated activation of gene expression during myogenesis, with STIM1-deficiency causing skeletal myopathy 33. These data suggest that STIM proteins might participate in calcium signaling networks by activating calcineurin/NFAT pathways that intersect with other signaling pathways to regulate a wide range of developmental processes 3435 and are frequently deregulated in tumorigenesis 36. While NFATc proteins are restricted to vertebrates, calcineurin genes are found in invertebrate organisms such as Drosophila melanogaster where they, like their mammalian counterparts, have important roles in muscle development and regulation of the immune response 3537.

In this study we have utilized Drosophila as a model to investigate the role of dSTIM in developmental processes in vivo, and to identify potential signaling pathways that intersect with dSTIM function. The single Drosophila STIM homologue is structurally similar to both STIM1 and STIM2 31 and was identified as an essential regulator of SOC entry independently of mammalian STIMs 15. dSTIM interacts with a single dOrai protein (olf186-F) to activate Ca²⁺ entry through SOC channels in a similar way to the mammalian counterparts 1238. We have generated transgenic Drosophila expressing full-length UAS-dSTIM cDNA and UAS-dSTIM RNAi constructs to determine the effects of STIM1 overexpression and STIM1 knockdown, respectively, in tissues expressing Gal4 under the influence of developmentally regulated cell type-specific promoters 39. We demonstrate an essential role for dSTIM in larval development and survival, and a tissue-specific role in cell fate specification of mechanosensory bristles in the notum and in specification of wing vein thickness. These studies are the first to demonstrate an important developmental role for the Ca²⁺ entry regulator STIM1 in tissue patterning.

These studies demonstrate that while dSTIM is ubiquitously expressed in Drosophila embryonic and larval tissues, these endogenous levels of dSTIM expression are not essential for many developmental processes. RNAi-mediated posttranscriptional knockdown of dSTIM expression in the early embryo had no observable effects on growth or patterning of larvae, but did cause lethality by early 3^rd instar stages. Reduced mobility of larvae suggested functional defects in myogenic and/or neurogenic lineages that could affect nutrient intake and metabolism. This late embryo lethal phenotype was also observed when dSTIM was overexpressed at early stages, indicating that normal tissue function is dependent on the maintenance of correct levels of dSTIM expression and thus highly regulated Ca²⁺ entry at these stages. STIM1-deficiency in mouse embryos influences the development and contractile function of skeletal muscle 33, and deficiencies in either STIM1 or STIM2 cause perinatal lethality but do not interfere with early processes of embryonic growth and development 32. Likewise, STIM1-knockdown in the C. elegans nematode does not result in embryonic lethality, but reduces the contraction of sheath cells that trigger spermathecal opening for ovulation, and results in complete sterility 60. Together these studies indicate that many cell types in developing invertebrate and mammalian embryos are not reliant on high levels of STIM expression for survival, proliferation and differentiation, and may regulate Ca²⁺ influx through STIM-independent mechanisms. Since RNAi did not cause complete knockout of dSTIM gene expression generation of flies having a null mutation in dSTIM will be necessary to determine the absolute requirement of dSTIM in developing Drosophila tissues, as will an analysis of the phenotype of double STIM1/STIM2 knockout mice.

Using RNAi-mediated knockdown in imaginal discs we have shown that dSTIM does have an essential and very specific developmental role in tissue patterning and cell fate specification in the wing and the notum, but not in the eye. Absence of an eye phenotype could indicate either inadequate knockdown of dSTIM expression with any of the Gal4 drivers used or that dSTIM is indeed not required for normal eye development. Knockdown of dSTIM expression in the developing wing resulted in significant vein thickening with no other observable patterning defects, while dSTIM-deficiency in the mesothoracic disc resulted in loss of mechanosensory bristles (microchaetes) on the notum. Notch signaling plays a key role in these two independent developmental processes and we have demonstrated a genetic interaction between dSTIM and Notch function in the wing. Notch signaling normally acts to prevents cells adopting a vein cell fate by inhibiting activation of the Egfr pathway and subsequent activation of the Dpp pathway 61, such that inhibition of Notch signaling promotes the differentiation of provein territory into vein tissues and gives rise to abnormally thickened veins 6263. We observed enhancement of vein thickening when Notch deficiency was combined with dSTIM knockdown, and showed that the activated Notch allele, TN^ΔBRV, an allele known to rescue most Notch hypomorphic alleles 64, was able to suppress the RNAi-mediated dSTIM deficiency. These results suggest that dSTIM function specifically intersects with regulators of Notch signaling during regulation of vein differentiation.

The phenotypic effects of dSTIM knockdown and dSTIM overexpression in the mesothorax indicate a role for dSTIM in cell fate specification in the mechanosensory bristle cell lineage, a process also regulated by Notch signaling. The absence of mechanosensory bristles (microchaetes) on the notum in flies expressing dSTIM^RNAi is consistent with a neurogenic phenotype, representing preferential neuronal specification of both daughter cells from the sensory organ precursor (SOP) 65. Overexpression of dSTIM resulted in exactly the opposite phenotype, with the development of twin-shafted or duplicated bristles. While the underlying cellular basis for these phenotypes was not ascertained in this study, the data are consistent with defects either in the asymmetric division of SOP cells, or the process of lateral inhibition that specifies SOP cells within each proneural cluster in the notum, both of which are principally regulated by Notch signaling 65. Loss-of-function mutations in the Notch receptor and in positive regulators of Notch cause a 'bald' neurogenic phenotype due to aberrant division and specification of daughter cells 6667. A similar neurogenic phenotype is caused by gain-of-function mutations in the Notch antagonist, Numb, while loss-of-function mutations in Numb result in the formation of duplicated bristles 68. Asymmetric segregation of Numb during SOP cell division determines the level of Notch signaling in the daughter cells 69. Numb is a target of the Ca²⁺ -dependent kinase CamKII 70, and its polarized distribution has been shown to be regulated by aPKC phosphorylation 71, suggesting that Numb localization and function may be regulated by Ca²⁺ -dependent mechanisms. Our data suggest that STIM-mediated Ca²⁺ entry may potentially influence the cellular localization and/or activity of Numb or other Notch regulators. It is interesting that Notch signaling in mammalian cells has been linked to a calcineurin-dependent mechanism that activates NFAT 72, a pathway that is regulated by mammalian STIM1 3233. While Ca²⁺ -regulated NFAT homologues have not been identified in Drosophila 73, genetic screens have identified dSTIM as a regulator of nuclear translocation and dephosphorylation of NFAT 12 and Drosophila calcineurin homologues regulate other target proteins, such as Egfr 74. Our results suggest that Notch and dSTIM may intersect at a functional level through activation of calcineurin independently of NFATc proteins in Drosophila, and further studies are now required to elucidate the molecular nature of these interactions.

Apart from development of the mechanosensory bristle cell lineage in the mesothorax, overexpression of dSTIM did not have opposite developmental effects to those of dSTIM deficiency. DSTIM overexpression caused the misorientation of the ommatidial lattice in the eye and inhibited the formation of interommatidial bristles, and disrupted development of the wing margin, processes that were not influenced by dSTIM deficiency but are known to be regulated by Notch signaling. A secreted dominant-negative form of the Notch ligand Serrate (SerS), and the glycosyl transferase Fringe (Fng) are both negative regulators of Notch signaling, and their expression in the early eye results in reduced eye size and square rather than hexagonal ommatidial facets 7576. In addition, overexpression of SerS in the wing results in wing margin loss and downregulation of Wg and Cut at the wing margin 76. Loss-of-function mutations in either the Notch receptor or in activators of Notch signaling result in loss of interommatidial bristles in the eye 77. The similarity of these eye and wing phenotypes to those resulting from dSTIM overexpression support the proposal that dSTIM overexpression negatively regulates Notch signaling at these sites.

The developmental defects associated with dSTIM overexpression are also consistent with dominant effects of abnormally increased intracellular Ca²⁺ levels causing enhanced activity of Ca²⁺-dependent processes. A major effect of increased intracellular Ca²⁺ in many cell types is the induction of apoptotic cell death 578, and smaller eyes and wings were seen in flies overexpressing dSTIM, with increased apoptotic cell death observed in the wing disc. Observations of increased growth arrest and apoptosis in some mammalian cell lines overexpressing STIM1 led to the initial hypothesis that STIM1 functions as a tumour suppressor 3179. In addition to induction of apoptosis we have demonstrated that wing margin defects caused by overexpression of dSTIM in the wing disc were associated with decreased expression of Arm (β-catenin) and other Wg signalling outputs at the wing margin, demonstrating inhibition of canonical Wg signaling. While these effects may simply reflect a dependence on Notch activity at the wing margin 545556, a direct interaction between dSTIM and Wg signaling is also possible. Intracellular Ca²⁺ is an important second messenger in non-canonical Wg/Wnt signaling pathways that primarily modulate cell polarity and movement, including the vertebrate Wnt/Ca²⁺ pathway that activates PKC and CamKII, and the planar cell polarity (PCP) pathway that activates Jun-N-terminal kinase (JNK) 8081. Induction of Ca²⁺ release by non-canonical Wnt ligands activates PKC and CamKII enzymes that can antagonize components of β-catenin signaling 80, as observed in our study. The Drosophila PCP pathway plays a fundamental role in the orientation of hairs, bristles and ommatidia 82, and development of multiple wing hairs 83 which are all observed with overexpression of dSTIM. Planar polarity defects are generated by abnormal segregation or random dispersion of proteins, including the non-canonical Wg components Fz, and Dsh, that normally provide the cell with polarity cues 84. These data support a potential role of dSTIM in providing Ca²⁺ dependent mechanisms for determining asymmetric protein localization within the cell. While STIM proteins are major regulators of Ca²⁺ entry through activation of SOC channels, there is increasing evidence for their role in store-independent Ca²⁺ entry mechanisms 2829. Identification of the single Drosophila homologue of the SOC channel subunit protein Orai 1238 will now allow a detailed analysis of SOC function during Drosophila development. Comparison of phenotypes caused by mutations in dSTIM and in dOrai will indicate whether the effects of dSTIM knockdown observed in this study are due specifically to a reduction in SOC-mediated Ca²⁺ entry or whether dSTIM plays a role in developmental processes by mediating other pathways of Ca²⁺ entry. Preliminary evidence from a genetic screen to identify molecules that affect vein patterning in the Drosophila wing suggests that ectopic expression of dOrai may have similar patterning effects (loss of vein and wing margin) to ectopic dSTIM 85.

Aberrant expression of STIM1 and STIM2 proteins has been associated with human cancer 7986 but their direct role in tumorigenic events has yet to be established. However, a recent study has identified STIM2 as a putative target of β-catenin/TCF in colorectal cancer (A. Villanueva, personal communication). Deregulation of Ca²⁺ homeostasis and Ca²⁺-mediated signaling events have been implicated in tumorigenesis 87, and using Drosophila as a model organism to determine how regulators of Ca²⁺ homeostasis interact with developmental pathways such as Wnt and Notch will reveal potential mechanisms that can be further analysed in mammalian systems.

The results presented here are the first to describe a developmental function of Ca²⁺ entry mediated by dSTIM. While the precise molecular mechanisms cannot be elucidated from these studies we have identified Notch signaling as a potential pathway to be dependent on dSTIM-mediated Ca²⁺ entry in certain cell types and Wg signaling to be influenced by dSTIM overexpression. The recent association of STIM2 with colorectal cancer also highlights the relevance of this family of proteins to molecular pathways that regulate cell proliferation and differentiation. A crucial aspect of Ca²⁺ signaling is the necessity to generate localized transient elevations in Ca²⁺ concentrations at important target sites within the cells. It is noteworthy that the developmental processes affected by alterations in dSTIM expression involve asymmetric cellular distribution of signaling components that establish spatial patterns and determine cell fate. The localization and translocation of STIM proteins in specialized regions of the ER place it in a good position to regulate highly localized Ca²⁺ entry through the plasma membrane. Analysis of the co-localization of dSTIM activity with Ca²⁺ dependent signaling will be a major step forward in determining whether dSTIM has an important role in regulating localized Ca²⁺-dependent signaling events that are critical for normal development, and can be aberrant in tumorigenesis.

JPE carried out and designed studies and participated in drafting the manuscript. AMA participated in design and analysis of studies. HR assisted in analysis of wing studies and drafting the manuscript. GRH and MD conceived the project, participated in its design and coordination, and drafted the manuscript.