Nicholas W. Plummer

A role for Orai in TRPC-mediated Ca2+ entry suggests that a TRPC:Orai complex may mediate store and receptor operated Ca2+ entry

TRPC and Orai proteins have both been proposed to form Ca2+-selective, store-operated calcium entry (SOCE) channels that are activated by store-depletion with Ca2+ chelators or calcium pump inhibitors. In contrast, only TRPC proteins have been proposed to form nonselective receptor-operated calcium entry (ROCE) cation channels that are activated by Gq/Gi-PLCβ signaling, which is the physiological stimulus for store depletion. We reported previously that a dominant negative Orai1 mutant, R91W, inhibits Ca2+ entry through both SOCE and ROCE channels, implicating Orai participation in both channel complexes. However, the argument for Orai participating in ROCE independently of store depletion is tenuous because store depletion is an integral component of the ROCE response, which includes formation of IP3, a store-depleting agent. Here we show that the R91W mutant also blocks diacylglycerol (DAG)-activated Ca2+ entry into cells that stably, or transiently, express DAG-responsive TRPC proteins. This strongly suggests that Orai and TRPC proteins form complexes that participate in Ca2+ entry with or without activation of store depletion. To integrate these results with recent data linking SOCE with recruitment of Orai and TRPCs to lipid rafts by STIM, we develop the hypothesis that Orai:TRPC complexes recruited to lipid rafts mediate SOCE, whereas the same complexes mediate ROCE when they are outside of lipid rafts. It remains to be determined whether the molecules forming the permeation pathway are the same when Orai:TRPC complexes mediate ROCE or SOCE.

Developmental origins of central norepinephrine neuron diversity

Central norepinephrine-producing neurons comprise a diverse population of cells differing in anatomical location, connectivity, function and response to disease and environmental insult. The mechanisms that generate this diversity are unknown. Here we elucidate the lineal relationship between molecularly distinct progenitor populations in the developing mouse hindbrain and mature norepinephrine neuron subtype identity. We have identified four genetically separable subpopulations of mature norepinephrine neurons differing in their anatomical location, axon morphology and efferent projection pattern. One of the subpopulations showed an unexpected projection to the prefrontal cortex, challenging the long-held belief that the locus coeruleus is the sole source of norepinephrine projections to the cortex. These findings reveal the embryonic origins of central norepinephrine neurons and provide multiple molecular points of entry for future study of individual norepinephrine circuits in complex behavioral and physiological processes including arousal, attention, mood, memory, appetite and homeostasis.

Allelic mutations of the sodium channel SCN8A reveal multiple cellular and physiological functions

Allelic mutations of Scn8a in the mouse have revealed the range of neurological disorders that can result from alternations of one neuronal sodium channel. Null mutations produce the most severe phenotype, with motor neuron failure leading to paralysis and juvenile lethality. Two less severe mutations cause ataxia, tremor, muscle weakness, and dystonia. The electrophysiological effects have been studied at the cellular level by recording from neurons from the mutant mice. The data demonstrate that Scn8a is required for the complex spiking of cerebellar Purkinje cells and for persistent sodium current in several classes of neurons, including some with pacemaker roles. The mouse mutations of Scn8a have also provided insight into the mode of inheritance of channelopathies, and led to the identification of a modifier gene that affects transcript splicing. These mutations demonstrate the value of mouse models to elucidate the pathophysiology of human disease.

Expanding the power of recombinase-based labeling to uncover cellular diversity

Investigating the developmental, structural and functional complexity of mammalian tissues and organs depends on identifying and gaining experimental access to diverse cell populations. Here, we describe a set of recombinase-responsive fluorescent indicator alleles in mice that significantly extends our ability to uncover cellular diversity by exploiting the intrinsic genetic signatures that uniquely define cell types. Using a recombinase-based intersectional strategy, these new alleles uniquely permit non-invasive labeling of cells defined by the overlap of up to three distinct gene expression domains. In response to different combinations of Cre, Flp and Dre recombinases, they express eGFP and/or tdTomato to allow the visualization of full cellular morphology. Here, we demonstrate the value of these features through a proof-of-principle analysis of the central noradrenergic system. We label previously inaccessible subpopulations of noradrenergic neurons to reveal details of their three-dimensional architecture and axon projection profiles. These new indicator alleles will provide experimental access to cell populations at unprecedented resolution, facilitating analysis of their developmental origin and anatomical, molecular and physiological properties. Summary: New fluorescent indicator alleles utilize Cre, Flp and Dre recombinases to label previously inaccessible cell populations. The complete architecture of two cell populations can be visualized in the same mouse.

Development of the mammalian axial skeleton requires signaling through the Gαi subfamily of heterotrimeric G proteins

129/SvEv mice with a loss-of-function mutation in the heterotrimeric G protein α-subunit gene Gnai3 have fusions of ribs and lumbar vertebrae, indicating a requirement for Gαi (the “inhibitory” class of α-subunits) in somite derivatives. Mice with mutations of Gnai1 or Gnai2 have neither defect, but loss of both Gnai3 and one of the other two genes increases the number and severity of rib fusions without affecting the lumbar fusions. No myotome defects are observed in Gnai3/Gnai1 double-mutant embryos, and crosses with a conditional allele of Gnai2 indicate that Gαi is specifically required in cartilage precursors. Penetrance and expressivity of the rib fusion phenotype is altered in mice with a mixed C57BL/6 × 129/SvEv genetic background. These phenotypes reveal a previously unknown role for G protein-coupled signaling pathways in development of the axial skeleton.

DAG‐sensitive and Ca2+ permeable TRPC6 channels are expressed in dentate granule cells and interneurons in the hippocampal formation

Members of the transient receptor potential (TRP) cation channel family play important roles in several neuronal functions. To understand the precise role of these channels in information processing, their presence on neuronal elements must be revealed. In this study, we investigated the localization of TRPC6 channels in the adult hippocampal formation. Immunostainings with a specific antibody, which was validated in Trpc6 knockout mice, showed that in the dentate gyrus, TRPC6 channels are strongly expressed in granule cells. Immunogold staining revealing the subcellular localization of TRPC6 channels clarified that these proteins were predominantly present on the membrane surface of the dendritic shafts of dentate granule cells, and also in their axons, often associated with intracellular membrane cisternae. In addition, TRPC6 channels could be observed in the dendrites of some interneurons. Double immunofluorescent staining showed that TRPC6 channels were present in the dendrites of hilar interneurons and hippocampal interneurons with horizontal dendrites in the stratum oriens expressing mGlu1a receptors, whereas parvalbumin immunoreactivity was revealed in TRPC6‐expressing dendrites with radial appearance in the stratum radiatum. Electron microscopy showed that the immunogold particles depicting TRPC6 channels were located on the surface membranes of the interneuron dendrites. Our results suggest that TRPC6 channels are in a key position to alter the information entry into the trisynaptic loop of the hippocampal formation from the entorhinal cortex, and to control the function of both feed‐forward and feed‐back inhibitory circuits in this brain region.

An essential role for Gα(i2) in Smoothened-stimulated epithelial cell proliferation in the mammary gland.

Gαi2 mediates proliferation stimulated by Smoothened in the mammary gland. Smoothened signals through G proteins The Hedgehog signaling pathway promotes the expression of genes that are critical during development through regulation of the Gli family of transcription factors. Because this pathway is subverted by cancer cells, and because the gene encoding the Hedgehog effector Smoothened (SMO) is aberrantly expressed in some types of breast cancers, various chemotherapeutics have been developed to target SMO. Villanueva et al. (see also the Focus by Ogden) showed that SMO promoted cell proliferation in the mammary glands of mice through the G protein Gαi2, rather than through Gli-mediated changes in gene expression. These results confirm that SMO can act as a G protein–coupled receptor in mammals and suggest that SMO-targeting drugs should also be screened for their ability to inhibit Gαi2 activation. Hedgehog (Hh) signaling is critical for organogenesis, tissue homeostasis, and stem cell maintenance. The gene encoding Smoothened (SMO), the primary effector of Hh signaling, is expressed aberrantly in human breast cancer, as well as in other cancers. In mice that express a constitutively active form of SMO that does not require Hh stimulation in mammary glands, the cells near the transgenic cells proliferate and participate in hyperplasia formation. Although SMO is a seven-transmembrane receptor like G protein–coupled receptors (GPCRs), SMO-mediated activation of the Gli family of transcription factors is not known to involve G proteins. However, data from Drosophila and mammalian cell lines indicate that SMO functions as a GPCR that couples to heterotrimeric G proteins of the pertussis toxin (PTX)–sensitive Gαi class. Using genetically modified mice, we demonstrated that SMO signaling through G proteins occurred in the mammary gland in vivo. SMO-induced stimulation of proliferation was PTX-sensitive and required Gαi2, but not Gαi1, Gαi3, or activation of Gli1 or Gli2. Our findings show that activated SMO functions as a GPCR to stimulate proliferation in vivo, a finding that may have clinical importance because most SMO-targeted agents have been selected based largely on their ability to block Gli-mediated transcription.

A knock‐in allele of En1 expressing Dre recombinase

Engrailed 1 (En1) is a homeobox‐containing transcription factor expressed during development in diverse tissues, including the embryonic midbrain and anterior hindbrain. To facilitate investigation of genetic and developmental heterogeneity among cells with a history of En1 expression, we have generated En1Dre, a knock‐in allele expressing Dre recombinase. En1Dre can be used with existing Cre and Flp recombinase lines for genetic intersectional labeling, fate mapping, and functional manipulation of subpopulations of cells characterized by transient expression of En1. To avoid disrupting En1 function, the Dre cDNA is inserted at the 3′ end of the En1 coding sequence, together with a viral 2A peptide to mediate translation of separate EN1 and Dre proteins. Consequently, viable and fertile En1Dre homozygotes can be used to increase the proportion of useful genotypes produced in complex crosses. The pattern of Dre expression from En1Dre is indistinguishable from wild‐type En1 expression in mid‐gestation mouse embryos, and En1Dre controls Dre‐responsive indicator alleles by efficiently recombining rox sites in vivo. Through the application of genetic tools that allow manipulation of cells based on combinatorial expression of multiple distinct recombinases, En1Dre will significantly extend the ability to target important subpopulations of neurons and other cells within the broader En1 expression domain. genesis 54:447–454, 2016. Published 2016. This article is a US Government work and is in the public domain in the USA.

Uncovering diversity in the development of central noradrenergic neurons and their efferents.

Uncovering the mechanisms that underlie central noradrenergic neuron heterogeneity is essential to understanding selective subtype vulnerability to disease and environmental insult. Using recombinase-based intersectional genetic fate mapping we have previously demonstrated that molecularly distinct progenitor populations give rise to mature noradrenergic neurons differing in their anatomical location, axon morphology and efferent projection pattern. Here we review the findings from our previous study and extend our analysis of the noradrenergic subpopulation defined by transient developmental expression of Hoxb1. Using a combination of intersectional genetic fate mapping and analysis of a targeted loss of function mutation in Hoxb1, we have now uncovered additional heterogeneity based on the requirement of some noradrenergic neurons for Hoxb1 expression. By comparing the distribution of noradrenergic neurons derived from the Hoxb1 expression domain in wild-type and mutant mice, we demonstrate that Hoxb1 expression is required by a subset of neurons in the pons. Additional fate mapping, using a Hoxb1 enhancer element that drives Cre recombinase expression exclusively in rhombomere 4 of the hindbrain, reveals the existence of a subpopulation of noradrenergic neurons in the pons with more restricted axonal targets than the full Hoxb1-derived subpopulation. The unique projection profile of this newly defined subpopulation suggests that it may be functionally distinct. These analyses shed new light on the molecular determinants of noradrenergic identity in the pons and the overall complexity of the central noradrenergic system. This article is part of a Special Issue entitled SI: Noradrenergic System.

A new mouse line for cell ablation by diphtheria toxin subunit A controlled by a Cre-dependent FLEx switch

Recombinase responsive mouse lines expressing diphtheria toxin subunit A (DTA) are well established tools for targeted ablation of genetically defined cell populations. Here we describe a new knock‐in allele at the Gt(Rosa)26Sor locus that retains the best features of previously described DTA alleles—including a CAG promoter, attenuated mutant DTA cDNA, and ubiquitous EGFP labeling—with the addition of a Cre‐dependent FLEx switch for tight control of expression. The FLEx switch consists of two pairs of antiparallel lox sites requiring Cre‐mediated recombination for inversion of the DTA to the proper orientation for transcription. We demonstrate its utility by Cre‐dependent ablation of both a broad domain in the embryonic nervous system and a discrete population of cells in the fetal gonads. We conclude that this new DTA line is useful for targeted ablation of genetically‐defined cell populations.