Morpholino oligonucleotide knockdown of the extracellular calcium-sensing receptor impairs early skeletal development in zebrafish.

The complex vertebrate skeleton depends on regulated cell activities to lay down protein matrix and mineral components of bone. As a distinctive vertebrate characteristic, bone is a storage site for physiologically-important calcium ion. The extracellular calcium-sensing receptor (CaSR) is linked to homeostatic regulation of calcium through its expression in endocrine glands that secrete calcium homeostatic hormones, in Ca(2+)- and ion-transporting epithelia, and in skeleton. Since CaSR is restricted in its presence to the chordate-vertebrate evolutionary lineage, we propose there to be important functional ties between CaSRs and vertebrate skeleton in the context of that group's characteristic form of calcium-mineralized skeleton. Since little is known about CaSR in the skeletal biology of non-mammalian vertebrates, reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization and immunohistochemistry were applied to adult and embryonic zebrafish to reveal CaSR transcript and protein expression in several tissues, including, among these, chondrocytes and developing bone and notochord as components in skeletal development. Morpholino oligonucleotide (MO) knockdown technique was used to probe CaSR role(s) in the zebrafish model system. By RT-PCR assessment, injection of a splice-inhibiting CaSR MO reduced normally-spliced Casr gene transcript expression measured at 2days postfertilization (dpf). Corresponding to the knockdown of normally-spliced mRNA by the CaSR MO, we observed a morphant phenotype characterized by stunted growth and disorganization of the notochord and axial skeleton by 1dpf. We conclude that, like its critically important role in normal bone development in mammals, CaSR is essential in skeletogenesis in fishes.

Vertebrate extracellular calcium-sensing receptor evolution: selection in relation to life history and habitat.

Ionic calcium (Ca(2+)) supports essential functions within physiological systems, and consequently its concentration is homeostatically regulated within narrow bounds in the body fluids of animals through endocrine effects at ion-transporting osmoregulatory tissues. In vertebrates, extracellular Ca(2+) is detected at the cell surface by the extracellular calcium-sensing receptor (CaSR), a member of the G protein-coupled receptor (GPCR) superfamily. Interestingly, the taxonomic distribution of CaSRs is restricted to vertebrates, with some CaSR-like receptors apparently present in non-vertebrate chordates. Since bone is a known Ca(2+) storage site and is characteristically restricted to the vertebrate lineage, we hypothesized a functional association of CaSR with vertebrate skeleton that may have an ancient origin. Protein sequence alignment and phylogenetic analysis of vertebrate CaSRs and related GPCRs of the glutamate receptor-like family expose similarities and indel differences among these receptors, and reveal the evolutionary history of CaSRs. Evolutionary selection was tested statistically by evaluating the relationship between non-synonymous (replacement, dN) versus synonymous (silent, dS) amino acid substitution rates (as dN/dS) of protein-coding DNA sequences among branches of the estimated protein phylogeny. On a background of strong purifying selection (dN/dS<1) in the CaSR phylogeny, statistical evidence for adaptive evolution (dN/dS>1) was detected on some branches to major clades in the CaSR phylogeny, especially to the tetrapod vertebrate CaSRs and chordate CaSR-like branches. Testing also revealed overall purifying selection at the codon level. At some sites relaxation from strong purifying selection was seen, but evidence for adaptive evolution was not detected for individual sites. The results suggest purifying selection of CaSRs, and of adaptive evolution among some major vertebrate clades, reflecting clade specific differences in natural history and organismal biology, including skeletal involvement in calcium homeostasis.

Skeletal tissues in Mozambique tilapia (Oreochromis mossambicus) express the extracellular calcium-sensing receptor.

Molecular phylogenetic analysis suggests that the extracellular calcium-sensing receptor (CaSR) emerged evolutionarily in association with the chordate-vertebrate lineage. Our studies overall explore the evolution of CaSRs, and the possible historical linkage of CaSRs to vertebrate skeleton as functional components of calcium homeostasis through regulated storage and/or release. We applied both reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) to evaluate Casr gene and CaSR protein expression, respectively, in skeletal tissues of a cichlid teleost, the Mozambique tilapia (Oreochromis mossambicus). By RT-PCR, CaSR gene (Casr) expression was observed in skull and vertebral column (including notochordal tissues). Relative to skeleton, IHC revealed CaSR protein expression in notochordal sheath cells within the vertebral canal, in scleroblasts associated with body surface scales, and in chondrocytes within hyaline cartilage. Although closely apposed cells border the acellular bone in tilapia, these cells were only weakly immunostained. We conclude, therefore, that CaSR is expressed in skeletal tissues of tilapia, an advanced teleost fish, and that Casr may be part of a genetic network associated with vertebrate skeletal system. Our immunohistochemical examination also newly revealed CaSR protein expression in epidermis and red muscle of fishes.

Extracellular calcium-sensing receptor distribution in osmoregulatory and endocrine tissues of the tilapia.

The extracellular calcium-sensing receptor (CaSR) serves an important detector function in vertebrate Ca(2+) homeostasis. In this study, we surveyed using immunohistochemistry the tissue and cellular distribution of the CaSR protein in the Mozambique tilapia (Oreochromis mossambicus) and the Japanese eel (Anguilla japonica). Specifically, we examined receptor expression in ion-transporting barrier tissues that may be directly responsive to extracellular Ca(2+) levels, and in tissues that are implicated in endocrine signaling to homeostatic effectors such as Ca(2+)-transporting epithelia. In tilapia osmoregulatory tissues, CaSR protein is strongly expressed in proximal segments of renal tubule, but not in distal segments (where Na(+),K(+)-ATPase is prominently expressed) or in glomeruli. The receptor was also localized in the ion-transporting mitochondria-rich cells of gill and in ion- and nutrient-transporting epithelia of middle and posterior intestine. Consistent with our earlier RT-PCR assessment of mRNA expression in tilapia, CaSR protein expression was salinity dependent in some osmoregulatory tissues. In tilapia pituitary gland, CaSR expression was observed in the rostral pars distalis (containing prolactin-secreting cells, and in the pars intermedia (containing somatolactin-secreting and melanocyte-stimulating hormone-secreting cells), with notably greater expression in the latter. In the eel, weak immunostaining was seen in the stanniocalcin-secreting cells of the corpuscles of Stannius. Olfactory lobe CaSR expression suggests an environment-sensing role for the receptor. Altogether, these findings support the involvement of CaSR in piscine Ca(2+) homeostasis at the levels of environmental sensing, of integrative endocrine signaling through both hypercalcemic (prolactin, and perhaps somatolactin) and hypocalcemic (stanniocalcin) hormones, and of direct local regulation of Ca(2+)-transporting tissues.

Extracellular calcium-sensing receptors in fishes.

The extracellular calcium-sensing receptor (CaSR) in fishes, like the CaSRs of tetrapod vertebrates, is a dimeric seven transmembrane, G protein-coupled receptor. The receptor is expressed on the plasma membranes of a variety of tissues and cells where it functions as a sensor of extracellular calcium concentration ([Ca(2+)](o)) in the physiological range. In the context of systemic calcium homeostasis, CaSR expressed in endocrine tissues that secrete calciotropic and other hormones (pituitary gland and corpuscles of Stannius) may play a central role in global integrative signaling, whereas receptor expressed in ion-transporting tissues (kidney, intestine, gills, and elasmobranch rectal gland) may have local direct effects on monovalent and divalent ion transport that are independent of endocrine signaling. In fishes, specifically, CaSR expression at the body surface (at the gills and olfactory tissues, for example) may permit direct sensing of environmental Ca(2+) and Mg(2+) concentrations, especially in the marine environment. Additionally, CaSRs may have other widespread and diverse roles in extracellular Ca(2+) sensing related both to organismal calcium homeostasis and to intercellular Ca(2+) signaling. As a consequence of the broad spectrum of recognized ligands, including polyvalent cations and amino acids, and of binding site shielding by monovalent cations, additional receptor functionalities related to salinity and nutrient detection are proposed for CaSRs. CaSR expression in the gastrointestinal tract may be multifunctional as a sensor for polyvalent cations and amino acids. Structural and phylogenetic analyses reveal strongly conserved features among CaSRs, and suggest that calcium sensing by mammalian parathyroid gland-type CaSR proteins may be restricted to chordates. Comparative functional and genomic studies that include piscine CaSRs can be useful model systems for testing existing hypotheses regarding receptor function, and will shed light on the evolutionary developmental history of calcium homeostasis in the vertebrates.

cDNA cloning and functional expression of a Ca2+-sensing receptor with truncated C-terminal tail from the Mozambique tilapia (Oreochromis mossambicus).

The complete cDNA sequence of the tilapia extracellular Ca(2+)-sensing receptor (CaR) was determined. The transcript length of tilapia CaR (tCaR) is 3.4 kbp and encodes a 940-amino acid, 7-transmembrane domain protein that is consistent in its structural features with known mammalian and piscine CaRs. The tCaR extracellular domain includes a characteristic hydrophobic segment, conserved cysteine residues that are implicated in receptor dimerization (Cys(129) and Cys(131)) and in coupling to the transmembrane domain (nine conserved cysteine residues), and conserved serine residues (Ser(147) and Ser(169-171)) that are linked to receptor binding of Ca(2+) and L-amino acid-mediated potentiation of function. mRNA expression of tCaR was strong in kidney, brain, and gill. Weaker expression was observed in pituitary, stomach, intestine, urinary bladder, and heart. This distribution is consistent with possible physiological roles in endocrine cells, excitable tissues, and ion-transporting barrier epithelia. Expression of tCaR mRNA in kidney and intestine was salinity-dependent, suggesting a role for the receptor in iono-/osmoregulation in this euryhaline teleost species. Human embryonic kidney-293 cells transiently transfected with tCaR cDNA demonstrated dose-dependent phospholipase C activation in response to elevations in the extracellular Ca(2+) concentration ([Ca(2+)](o)). Functional activation of the mitogen-activated protein kinase cascade by high [Ca(2+)](o) was also confirmed in these cells despite the naturally occurring truncation of the receptor's intracellular tail, which removes segments variably linked in mammalian CaRs to filamin-coupled activation of mitogen-activated protein kinase cascades. Sensitivity of phospholipase C activation to [Ca(2+)](o) was dependent on the ionic strength of the bathing medium, supporting a role in salinity sensing.

Development of Intracellular Membrane Systems in the Columnar Epithelium of the Urinary Bladder of the Euryhaline Goby Gillichthys mirabilis: (urinary bladder/intracellular membranes/euryhaline teleost/prolactin/osmoregulation).

The membrane systems in "columnar cells" of the goby urinary bladder were studied after staining with ferrocyanide-reduced osmium tetroxide (Karnovsky). In addition to the endoplasmic reticulum, two distinct systems of membranes were observed: 1) a vesiculotubular system made up of vesicles and short tubules located between the Golgi area and the apical membrane and 2) well-developed infoldings of the laterobasal plasma membrane which form either complete or fenestrated sheets. Adaptation to 5% seawater or prolactin exposure of seawater fish induces a proliferation of these membrane systems and, in particular, of the complete infoldings of the laterobasal plasma membrane. These observations suggest high activity of these bladder cells in osmoregulatory adjustments to hypotonic environments. The divergence between cytological and physiological indicators of activity is considered.