Conditional knockout of TMEM16A/anoctamin1 abolishes the calcium-activated chloride current in mouse vomeronasal sensory neurons.

Pheromones are substances released from animals that, when detected by the vomeronasal organ of other individuals of the same species, affect their physiology and behavior. Pheromone binding to receptors on microvilli on the dendritic knobs of vomeronasal sensory neurons activates a second messenger cascade to produce an increase in intracellular Ca2+concentration. Here, we used whole-cell and inside-out patch-clamp analysis to provide a functional characterization of currents activated by Ca2+in isolated mouse vomeronasal sensory neurons in the absence of intracellular K+. In whole-cell recordings, the average current in 1.5 \u3bcM Ca2+and symmetrical Cl-was -382 pA at -100 mV. Ion substitution experiments and partial blockade by commonly used Cl-channel blockers indicated that Ca2+activates mainly anionic currents in these neurons. Recordings from inside-out patches from dendritic knobs of mouse vomeronasal sensory neurons confirmed the presence of Ca2+-activated Cl-channels in the knobs and/or microvilli. We compared the electrophysiological properties of the native currents with those mediated by heterologously expressed TMEM16A/anoctamin1 or TMEM16B/anoctamin2 Ca2+-activated Cl-channels, which are coexpressed in microvilli of mouse vomeronasal sensory neurons, and found a closer resemblance to those of TMEM16A. We used the Cre-loxP system to selectively knock out TMEM16A in cells expressing the olfactory marker protein, which is found in mature vomeronasal sensory neurons. Immunohistochemistry confirmed the specific ablation of TMEM16A in vomeronasal neurons. Ca2+-activated currents were abolished in vomeronasal sensory neurons of TMEM16A conditional knockout mice, demonstrating that TMEM16A is an essential component of Ca2+-activated Cl-currents in mouse vomeronasal sensory neurons

Calcium-activated chloride channels in the apical region of mouse vomeronasal sensory neurons

The rodent vomeronasal organ plays a crucial role in several social behaviors. Detection of pheromones or other emitted signaling molecules occurs in the dendritic microvilli of vomeronasal sensory neurons, where the binding of molecules to vomeronasal receptors leads to the influx of sodium and calcium ions mainly through the transient receptor potential canonical 2 (TRPC2) channel. To investigate the physiological role played by the increase in intracellular calcium concentration in the apical region of these neurons, we produced localized, rapid, and reproducible increases in calcium concentration with flash photolysis of caged calcium and measured calcium-activated currents with the whole cell voltage-clamp technique. On average, a large inward calcium-activated current of -261 pA was measured at -50 mV, rising with a time constant of 13 ms. Ion substitution experiments showed that this current is anion selective. Moreover, the chloride channel blockers niflumic acid and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid partially inhibited the calcium-activated current. These results directly demonstrate that a large chloride current can be activated by calcium in the apical region of mouse vomeronasal sensory neurons. Furthermore, we showed by immunohistochemistry that the calcium-activated chloride channels TMEM16A/anoctamin1 and TMEM16B/anoctamin2 are present in the apical layer of the vomeronasal epithelium, where they largely colocalize with the TRPC2 transduction channel. Immunocytochemistry on isolated vomeronasal sensory neurons showed that TMEM16A and TMEM16B coexpress in the neuronal microvilli. Therefore, we conclude that microvilli of mouse vomeronasal sensory neurons have a high density of calcium-activated chloride channels that may play an important role in vomeronasal transduction. \ua9 2012 Dibattista et al

A multivesicular body-like organelle mediates stimulus-regulated trafficking of olfactory ciliary transduction proteins

Stimulus transduction in cilia of olfactory sensory neurons is mediated by odorant receptors, Gαolf, adenylate cyclase-3, cyclic nucleotide-gated and chloride ion channels. Mechanisms regulating trafficking and localization of these proteins in the dendrite are unknown. By lectin/immunofluorescence staining and in vivo correlative light-electron microscopy (CLEM), we identify a retinitis pigmentosa-2 (RP2), ESCRT-0 and synaptophysin-containing multivesicular organelle that is not part of generic recycling/degradative/exosome pathways. The organelle's intraluminal vesicles contain the olfactory transduction proteins except for Golf subunits Gγ13 and Gβ1. Instead, Gβ1 colocalizes with RP2 on the organelle’s outer membrane. The organelle accumulates in response to stimulus deprivation, while odor stimuli or adenylate cyclase activation cause outer membrane disintegration, release of intraluminal vesicles, and RP2/Gβ1 translocation to the base of olfactory cilia. Together, these findings reveal the existence of a dendritic organelle that mediates both stimulus-regulated storage of olfactory ciliary transduction proteins and membrane-delimited sorting important for G protein heterotrimerization

Decreased proliferation and change in physical properties of mutant optic vesicle.

<p>(A) Immunohistochemical analysis for the presence of BrdU-labelled cells in the prospective neural retina on a coronal section of an E10.5 control embryo (left panel) and an <i>Lhx2-Cre:β-catenin^flox/flox</i> embryo (right panel). The part of the prospective neural retina that was analysed for BrdU⁺ cells in the control and mutant embryos are outlined. (B) Immunohistochemical analysis for the presence apoptotic cells as revealed by the presence of activated Casp-3⁺ cells on coronal section of an E10.5 control embryo (left panel) and a <i>Lhx2-Cre:β-catenin^flox/flox</i> embryo (right panel). (C) Immunohistochemical analyses for the presence of phosphorylated myosin light chain 2 (pMLC2) on a coronal section of an E10.5 control embryo. (D) Immunohistochemical analyses for the presence of pMLC2 on a coronal section on an E10.5 <i>Lhx2-Cre:β-catenin^flox/flox</i> embryo. Insets in C and D are a magnification of the areas indicated by the squares. Scale bars: (A, B and C, D) 100 µm.</p

β-catenin remains associated with N-cadherin and F-actin at the apical side of the cells in the mutant optic vesicle.

<p>(A–D) Immunohistochemical analyses for cellular localisation of the indicated proteins on coronal sections of E10 (ss30–31) optic vesicles from control embryos (left panels) and <i>Lhx2-Cre:β-catenin^flox/flox</i> embryos (right panels). (C) is a merge of (A) and (B) to show co-localisation (yellow) in the apical side of the cells in the optic vesicle on both control and mutant embryos (arrows). (D) A serial section of the same embryo as in (A–C) revealing F-actin protein at the apical side of the cells in both control and mutant optic vesicles (arrows). Scale bars: (A–C and D) 50 µm.</p

Canonical Wnt/β-catenin signalling is required for RPE cell commitment.

<p>(A–C) <i>In situ</i> hybridization analyses of the indicated genes on coronal sections of E9.25 somite stage 21–23 (ss21–23) control embryos (left panels) and <i>Lhx2-Cre:β-catenin^flox/flox</i> embryos (right panels). (D–I) <i>In situ</i> hybridization analyses of the indicated genes on coronal sections of E9.75 (ss26–28) control embryos (left panels) and <i>Lhx2-Cre:β-catenin^flox/flox</i> embryos (right panels). (J–O) <i>In situ</i> hybridization analyses (K, M–O) or immunohistochemical analyses (J, L) for gene expression of the indicated genes on coronal sections of E10.5 (ss32–34) control embryos (left panels) and <i>Lhx2-Cre:β-catenin^flox/flox</i> embryos (right panels). β-catenin protein is indicated by red labelling in J and L while Mitf protein is indicated by green labelling in L. Arrows indicate the area where <i>β-catenin</i> has been inactivated in A, D and J. Arrow heads indicate the area of Axin2 expression in E and K. Dorsal-ventral (D–V) orientation for all panels is indicated in A. Scale bars: (A–C and D–O) 100 µm.</p

The few RPE cells that develop in the mutant embryos are derived from eye committed progenitor cells in the anterior neural plate that escape <i>β-catenin</i> inactivation.

<p>(A) Immunohistochemical analyses of Mitf (green) and β-catenin (red) on coronal sections of an E13.5 optic rudiment in an <i>Lhx2-Cre:β-catenin^flox/</i>⁻ embryo. RPE cells are identified by pigment (pseudocoloured blue) and Mitf (green). (B) Immunohistochemical analysis of β-catenin and DAPI labelling of the same coronal section as in (A) to reveal that both β-catenin⁺ and β-catenin⁻ cells are present in the optic rudiment that develops in mutant embryos. * indicates the area of β-catenin⁻ non-RPE cells and the arrow head indicates the area of β-catenin⁺ non-RPE cells in the optic rudiment. (C–E) is a magnification of the area indicated by a rectangle in (A). (C) and (D) are merged in (E) to show co-localisation of β-catenin, Mitf and pigment. (F) Immunohistochemical analysis of Mitf (green) and β-catenin (red) on a coronal section of an E12.5 optic rudiment in a <i>Lhx2-Cre:β-catenin^flox/flox:ROSA26R</i> embryo. (G) Immunohistochemical analysis of Mitf (green) and β-Gal (red) on a serial section following (F) revealing that all cells in the optic rudiment, including the RPE cells, are β-Gal⁺ and hence derived from Cre⁺ progenitor cells in the anterior neural plate. (H–J) is a magnification of the area indicated by a rectangle in (G). (H) and (I) are merged in (J) to show co-localisation of Mitf, β-Gal and pigment. Scale bars: (A, B, and H–J) 50 µm. (C–E) 25 µm. (F, G) 100 µm.</p

β-catenin is important for maintenance of dorsoventral patterning of the retina.

<p>(A–D) <i>In situ</i> hybridization analyses (A, C, D) and immunohistochemical analysis (B) on coronal sections of E9.5 (ss25–26) optic vesicles from control (left panels) and on <i>Lhx2-Cre:β-catenin^flox/flox</i> embryos (right panels). (E–H) In situ hybridization analyses (E, G, H) and immunohistochemical analysis (F) on coronal sections of E10.5 (ss32–34) optic cups from control (left panels) and <i>Lhx2-Cre:β-catenin^flox/flox</i> embryos (right panels). Black arrow heads in A and E, and white arrow heads in B and F, indicate the boundaries of the area where β-catenin has been inactivated. (I, J) <i>In situ</i> hybridization analyses on coronal sections of E10.5 (ss32–34) optic cups from control (left panel) and <i>Lhx2-Cre:β-catenin^flox/flox</i> embryos (right panels). (K, L) <i>In situ</i> hybridization analyses on coronal sections of an eye from E12.5 control (left panels) and <i>Lhx2-Cre:β-catenin^flox/flox</i> embryos. Dorsal-ventral (D–V) orientation of all panels is indicated in A. Scale bars: (A–E, G–J, F and K, L) 100 µm.</p

Dorsal shift in expression of retina-specific eye field transcription factors in the mutant optic vesicle.

<p>(A–E) <i>In situ</i> hybridization analyses on coronal sections of E10.5 optic cups from control embryos (left panels) and <i>Lhx2-Cre:β-catenin^flox/flox</i> mutant embryos (right panels). Arrows indicate the dorsal shift in expression of the retina-specific transcription factors <i>Six3</i>, <i>Six6</i> and <i>Rx</i> in mutant embryos (C–E). Scale bar: 100 µm.</p

β-catenin mutant embryos are anophthalmic.

<p>(A–C) The left panels show lateral views of E16.5 embryos of control (A), <i>Lhx2-Cre:β-catenin^flox/flox</i> (β-cat^f/f) (B) and <i>Lhx2-Cre:β-catenin^flox/</i>⁻ (<i>β-cat^f/</i>⁻) (C) embryos. Arrow heads indicate where the eye should be located in the mutant embryos. The right panels show hematoxylin/eosin staining of coronal tissue sections of the same embryos. Black arrows indicate the optic rudiment surrounded by RPE cells (red arrow heads) that can develop in the mutant embryos. NR: neural retina. L: lens. RPE: retinal pigment epithelium. (D) Relative size of the optic vesicle-derived structure in the <i>Lhx2-Cre:β-catenin^flox/flox</i> (<i>f/f</i>) embryos and <i>Lhx2-Cre:β-catenin^flox/</i>⁻ (<i>f/−</i>) embryos compared with control embryos (c) at the indicated embryonic age and postnatal day 1 (P1). To get an approximate value of the relative eye size for the different genotypes, the largest diameter of the control eyes and the mutant optic rudiment at different ages were measured and compared. The diameter of the eye in control embryos at each developmental stage is arbitrarily defined as 1.0. The number of eyes analysed for each genotype at the respective age is indicated for each group. SD is indicated when applicable. Scale bar: 200 µm.</p