Identification Of N-Terminal Extracellular Domain Determinants In Nicotinic Acetylcholine Receptor (Nachr) α6 Subunits That Influence Effects Of Wild-Type Or Mutant β3 Subunits On Function Of α6β2*- Or α6β4*-Nachr

Despite the apparent function of naturally expressed mammalian α6*-nicotinic acetylcholine receptors (α6*-nAChR; where*indicates the known or possible presence of additional subunits), their functional and heterologous expression has been difficult. Here, we report that coexpression with wild-type β3 subunits abolishes the small amount of function typically seen for all-human or all-mouse α6β4*-nAChR expressed in Xenopus oocytes. However, levels of function and agonist potencies are markedly increased, and there is atropine-sensitive blockade of spontaneous channel opening upon coexpression of α6 and β4 subunits with mutant β3 subunits harboring valine-to-serine mutations at 9′- or 13′-positions. There is no function when α6 and β2 subunits are expressed alone or in the presence of wild-type or mutant β3 subunits. Interestingly, hybrid nAChR containing mouse α6 and human (h) β4 subunits have function potentiated rather than suppressed by coexpression with wild-type hβ3 subunits and potentiated further upon coexpression with hβ3 V9,S subunits. Studies using nAChR chimeric mouse/human α6 subunits indicated that residues involved in effects seen with hybrid nAChR are located in the α6 subunit N-terminal domain. More specifically, nAChR hα6 subunit residues Asn-143 and Met-145 are important for dominant-negative effects of nAChR hβ3 subunits on hα6hβ4-nAChR function. Asn-143 and additional residues in the N-terminal domain of nAChR hα6 subunits are involved in the gain-of-function effects of nAChR hβ3 V9,S subunits on α6β2*-nAChR function. These studies illuminate the structural bases for effects of β3 subunits on α6*-nAChR function and suggest that unique subunit interfaces involving the complementary rather than the primary face of α6 subunits are involved. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc

Modulation Of Recombinant α2* α3* Or α4*-Nicotinic Acetylcholine Receptor (Nachr) Function By Nachr β3 Subunits

The nicotinic acetylcholine receptor (nAChR) β3 subunit is thought to serve an accessory role in nAChR subtypes expressed in dopaminergic regions implicated in drug dependence and reward. When β3 subunits are expressed in excess, they have a dominant-negative effect on function of selected nAChR subtypes. In this study, we show, in Xenopus oocytes expressing α2, α3 or α4 plus either β2 or β4 subunits, that in the presumed presence of similar amounts of each nAChR subunit, co-expression with wild-type β3 subunits generally (except for α3*-nAChR) lowers amplitudes of agonist-evoked, inward peak currents by 20-50% without having dramatic effects (≤ 2-fold) on agonist potencies. By contrast, co-expression with mutant β subunits generally (except for α4β2*-nAChR) increases agonist potencies, consistent with an expected gain-of-function effect. This most dramatically demonstrates formation of complexes containing three kinds of subunit. Moreover, for oocytes expressing nAChR containing any α subunit plus β4 and β subunits, there is spontaneous channel opening sensitive to blockade by the open channel blocker, atropine. Collectively, the results indicate that β3 subunits integrate into all of the studied receptor assemblies and suggest that natural co-expression with β3 subunits can influence levels of expression and agonist sensitivities of several nAChR subtypes. © 2012 International Society for Neurochemistry

Phenotypically silent Cre recombination within the postnatal ventricular conduction system

<div><p>The cardiac conduction system (CCS) is composed of specialized cardiomyocytes that initiate and maintain cardiac rhythm. Any perturbation to the normal sequence of electrical events within the heart can result in cardiac arrhythmias. To understand how cardiac rhythm is established at the molecular level, several genetically modified mouse lines expressing Cre recombinase within specific CCS compartments have been created. In general, Cre driver lines have been generated either by homologous recombination of Cre into an endogenous locus or Cre expression driven by a randomly inserted transgene. However, haploinsufficiency of the endogenous gene compromises the former approach, while position effects negatively impact the latter. To address these limitations, we generated a Cre driver line for the ventricular conduction system (VCS) that preserves endogenous gene expression by targeting the Contactin2 (Cntn2) 3’ untranslated region (3’UTR). Here we show that <i>Cntn2</i>^{<i>3’UTR-IRES-Cre-EGFP/+</i>} mice recombine floxed alleles within the VCS and that Cre expression faithfully recapitulates the spatial distribution of Cntn2 within the heart. We further demonstrate that Cre expression initiates after birth with preservation of native Cntn2 protein. Finally, we show that <i>Cntn2</i>^{<i>3’UTR-IRES-Cre-EGFP/+</i>} mice maintain normal cardiac mechanical and electrical function. Taken together, our results establish a novel VCS-specific Cre driver line without the adverse consequences of haploinsufficiency or position effects. We expect that our new mouse line will add to the accumulating toolkit of CCS-specific mouse reagents and aid characterization of the cell-autonomous molecular circuitry that drives VCS maintenance and function.</p></div

Genomic architecture of the <i>Cntn2</i>^{<i>3’UTR-IRES-Cre-EGFP/+</i>} allele.

<p>a) Schematic of the endogenous <i>Contactin-2 (Cntn2)</i> locus comprising 23 exons as indicated by blue numbered blocks. Green block denotes the 3’UTR of the <i>Cntn2</i> gene located in exon 23. The black right-handed arrow indicates the <i>Cntn2</i> transcriptional start site driven by endogenous regulatory elements. The <i>IRES-Cre-EGFP-FRT-neo-PGK-FRT</i> KI cassette was targeted to the 3’ UTR of the <i>Cntn2</i> locus to ensure unperturbed bi-cistronic expression of endogenous Cntn2 protein and a Cre-EGFP fusion protein under the control of the endogenous regulatory elements upon FLP recombination. b) PCR genotyping of mice before FLP-mediated recombination using the indicated primer sets to amplify the 5’ (i) and 3’ (ii) insertion sites. In this example, 4 out of 5 pups were positive by F1-R1 genotyping, and these 4 were subjected to the second round of genotyping using F2-R2 primer sets. C) PCR genotyping of mice after FLP-mediated recombination using the indicated primer sets to amplify across the deleted <i>Neo</i>^<i>R</i> cassette (i) and the <i>Cre</i> coding sequence (ii). In this example, 4 out of 6 pups were positive by F3-R3 genotyping, and these 4 were subjected to the second round of genotyping using F4-R4 primer sets.</p

tdTomato co-localizes with VCS markers in <i>Cntn2</i>^{<i>3’UTR-IRES-Cre-EGFP/+</i>}<i>; R26R</i>^{<i>tdTomato/+</i>} mice.

<p>(a-d) <i>Cntn2</i>^{<i>3’UTR-IRES-Cre-EGFP/+</i>} mice were crossed with <i>R26R</i>^{<i>tdTomato/tdTomato</i>} reporter mice, and P42 heart cryosections were imaged by confocal microscopy. (a, a’, a”, a”‘) High resolution confocal images of the AVN region demonstrates co-localization of tdTomato (a’, red), endogenous Cntn2 (a”, green), and endogenous Hcn4 (a”‘, magenta) with the merged image shown in (a). (b, b’, b”, b”‘) High specificity and complete overlap (b: merged signal) of native tdTomato (b’, red), endogenous Cntn2 (b”, green), and endogenous Hcn4 (b’”, magenta) expression upon Cre recombination in the AVB and BB. (c, c’, c”, c”‘) Confocal images of mouse heart sections showing that Cre recombined tdTomato cells of left PFs (c’, red) co-localize with endogenous Cntn2 (c”, green) and Hcn4 (c”‘, magenta). (d, d’, d”, d”‘) Images of serial <i>Cntn2</i>^{<i>3’UTR-IRES-Cre-EGFP/+</i>}<i>; R26R</i>^{<i>tdTomato/+</i>} mice heart sections verified co-expression of endogenous Cntn2 (d”, green) and Hcn4 (d”‘, magenta) in recombined tdTomato cells (d’, red) of right PFs. Blue signal indicates nuclear counterstaining with DAPI. The cardiac anatomical location for each confocal micrograph is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174517#pone.0174517.s002" target="_blank">S2A Fig</a>. Scale bar: 100 μm.</p

Cardiac mechanical function is preserved in <i>Cntn2</i>^{<i>3’UTR-IRES-Cre-EGFP/+</i>} mice.

<p>(a-b) M-mode echocardiography was performed in conscious mice to analyze heart function parameters in P42 (a) wild-type (WT) and (b) <i>Cntn2</i>^{<i>3’UTR-IRES-Cre-EGFP/+</i>} mice. Representative traces show normal heart function in both groups. (c) Heart Rate (HR) in <u>B</u>eats <u>P</u>er <u>M</u>inute (BPM), (d) Ejection Fraction (EF) as a percentage, (e) Fractional Shortening (FS) as a percentage, and (f) Cardiac Output (CO) in milliliters per minute were calculated for n = 9 animals in both groups. Black circles represent individual WT and <i>Cntn2</i>^{<i>3’UTR-IRES-Cre-EGFP/+</i>} mice used in this study. Blue and red bars represent mean values of each parameter in WT and <i>Cntn2</i>^{<i>3’UTR-IRES-Cre-EGFP/+</i>} mice, respectively. No statistically significant differences were observed between groups. ns, not significant.</p

Cre-mediated recombination in <i>Cntn2</i>^{<i>3’UTR-IRES-Cre-EGFP/+</i>}<i>; R26R</i>^{<i>tdTomato/+</i>} mice is only evident after birth.

<p>(a, a’) Whole mount fluorescent image of an E18.5 double heterozygous embryo with robust expression of the recombined reporter protein in the cerebrum. tdTomato expression in the brain was also seen at E16.5 (data not shown). (b, b’) Following microdissection of the heart at E18.5, native tdTomato signal was not observed. (c, c’) Double heterozygous P0 mouse heart after dissection revealed bright reporter protein expression in the AVB, right and left BB, and right and left PF network. P0 was the earliest point at which reporter expression was observed in the heart. (d,d’) Micro-dissected P7 double heterozygous hearts express tdTomato protein in the identical anatomical regions as described in (c,c’). The cardiac PF network appears fully developed by P7 based on fluorescent reporter expression. At least 8 hearts were dissected per group of animals at each time point. Scale bar: 500 μm.</p

Cardiac electrical function is preserved in <i>Cntn2</i>^{<i>3’UTR-IRES-Cre-EGFP/+</i>} mice.

<p>(a-j) Serial surface lead II ECGs were recorded on both groups of mice (n = 9 per group) under mild anesthesia at P7, P14, P21, P28, and P42. Exemplary ECG traces for P42 (a) WT and (b) <i>Cntn2</i>^{<i>3’UTR-IRES-Cre-EGFP/+</i>} mice are shown. Scale size: 40 milliseconds (ms). (c) PR interval (in seconds), (d) QRS width (in seconds), (e) RR interval (in seconds), and (f) PR/RR ratio were measured at P7, P14, P21, and P42. At P28, (g) PR interval (in seconds), (h) QRS width (in seconds), (i) RR interval (in seconds), and (j) PR/RR ratio were measured pre- and post-isoproterenol (-/+ Iso) injection. Black circles represent individual WT and <i>Cntn2</i>^{<i>3’UTR-IRES-Cre-EGFP/+</i>} mice used in this study. Blue and red bars represent mean values for each parameter in WT and <i>Cntn2</i>^{<i>3’UTR-IRES-Cre-EGFP/+</i>} mice, respectively. No statistically significant differences were observed between groups. ns, not significant.</p

<i>Cntn2</i>^{<i>3’UTR-IRES-Cre-EGFP/+</i>}<i>; R26R</i>^{<i>tdTomato/+</i>} mice display robust reporter expression in the VCS.

<p>(a-e) <i>Cntn2</i>^{<i>3’UTR-IRES-Cre-EGFP/+</i>} mice were bred with <i>R26R</i>^{<i>tdTomato/tdTomato</i>} reporter mice to characterize the <i>Cntn2</i>^{<i>3’UTR-IRES-Cre-EGFP/+</i>} allele by monitoring tdTomato expression at P28 by whole mount fluorescence imaging (a and a’) in brain (dorsal view, used as a positive control) where endogenous Contactin 2 is known to be expressed abundantly and (b-e) in heart. We monitored tdTomato expression in at least 15 independent <i>Cntn2</i>^{<i>3’UTR-IRES-Cre-EGFP/+</i>}<i>; R26R</i>^{<i>tdTomato/+</i>} mice from multiple litters in comparison to 12 WT control littermates. (b and b’) Native tdTomato fluorescence was visualized at the Atrio-Ventricular Junction (AVJ) by whole-mount microscopy following Cre recombination of the <i>R26R</i> locus. (c-e) A P28 mouse heart was sectioned grossly in the four-chamber orientation to visualize native tdTomato expression in the VCS. (c and c’) Robust tdTomato fluorescence was observed in the Atrio-Ventricular Bundle (AVB), right and left Bundle Branches (BB), and right and left Purkinje Fiber (PF) network. Faint speckled fluorescent signal was also observed in the Right Atrium (RA) (not visible in the image). (a-c) Scale bar: 500 μM. (d and e) Higher magnification images of (d) right inlet in (c’) and (e) left inlet in (c’) to visualize the intricate structures of AVB and highly branched PF network in the adult mouse heart. Scale bar: 250 μm. LA, Left Atrium; LV, Left Ventricle; RV, Right Ventricle.</p

PAN-INTACT enables direct isolation of lineage-specific nuclei from fibrous tissues.

Recent studies have highlighted the extraordinary cell type diversity that exists within mammalian organs, yet the molecular drivers of such heterogeneity remain elusive. To address this issue, much attention has been focused on profiling the transcriptome and epigenome of individual cell types. However, standard cell type isolation methods based on surface or fluorescent markers remain problematic for cells residing within organs with significant connective tissue. Since the nucleus contains both genomic and transcriptomic information, the isolation of nuclei tagged in specific cell types (INTACT) method provides an attractive solution. Although INTACT has been successfully applied to plants, flies, zebrafish, frogs, and mouse brain and adipose tissue, broad use across mammalian organs remains challenging. Here we describe the PAN-INTACT method, which can be used to isolate cell type specific nuclei from fibrous mouse organs, which are particularly problematic. As a proof-of-concept, we demonstrate successful isolation of cell type-specific nuclei from the mouse heart, which contains substantial connective tissue and harbors multiple cell types, including cardiomyocytes, fibroblasts, endothelial cells, and epicardial cells. Compared to established techniques, PAN-INTACT allows more rapid isolation of cardiac nuclei to facilitate downstream applications. We show cell type-specific isolation of nuclei from the hearts of Nkx2-5Cre/+; R26Sun1-2xsf-GFP-6xmyc/+ mice, which we confirm by expression of lineage markers. Furthermore, we perform Assay for Transposase Accessible Chromatin (ATAC)-Seq to provide high-fidelity chromatin accessibility maps of Nkx2-5+ nuclei. To extend the applicability of PAN-INTACT, we also demonstrate successful isolation of Wt1+ podocytes from adult kidney. Taken together, our data suggest that PAN-INTACT is broadly applicable for profiling the transcriptional and epigenetic landscape of specific cell types. Thus, we envision that our method can be used to systematically probe mechanistic details of cell type-specific functions within individual organs of intact mice