RIC8 in folliculogenesis and in the reproductive tract of adult mouse.

<p>RIC8 was visualized with RIC8 antibody (red), and cell nuclei were visualized with DAPI (blue). (<b>A-F</b>) Transversal cryosections of ovary with oocytes in different follicular stages starting from primordial follicle to Graafian follicle and (<b>G-I</b>) different regions of oviduct are shown. (<b>H</b>) Higher magnification of the region of ampulla and (<b>i</b>, indicated by white box) isthmus. Abbreviations: A, antrum; Amp, ampulla region of oviduct; Cb, basal layer of cilia; Cc, ciliated cell; Ci, cilia; Co, cumulus oophorus; Cr, corona radiata; Cx, cell cortex, Ec, epithelial cells; Fc, follicular cell; Gc, granulosa cells; Gf, Graafian follicle; GV, germinal vesicle; Ist, isthmus region of oviduct; Lp, lamina propria; Lu, lumen; Pc, peg cell; Pf, primary follicle; Pmf, primordial follicle; Po, primary oocyte; Sf, secondary follicle. Scale bars: 50 μm.</p

Localization pattern of RIC8, Gα_i1/2, LGN and NuMA at early cleavage stage of mouse embryo.

<p>(<b>A-C</b>) One-cell (1-cell) embryo at metaphase (Met) of first mitosis. (<b>D-L</b>) Two-cell (2-cell) mouse embryos. Embryos were double-labeled with RIC8 antibody (green) and Gα_i1/2, LGN or NuMA antibodies respectively (red). DNA was stained with DAPI (blue). (<b>d-i</b>) Higher magnification of overlapping regions (yellow to orange) of RIC8 and Gα_i1/2 or LGN in cortex area of blastomere (indicated with white arrowhead). Abbreviations: ms, mitotic spindle; pb, polar body. Scale bar: (<b>A-L</b>) 20 μm, (<b>d-i</b>) 10 μm.</p

Neurospecific deletion of <i>Ric8a</i> in mice.

<p>(<b>A</b>) Schematic representation of neuron-specific deletion of the <i>Ric8a</i> gene. First four exons of floxed <i>Ric8a</i> are removed by Cre-recombinase which expression is under the control of <i>Synapsin I</i> promoter (SynCre). Numbered boxes represent <i>Ric8a</i> exons and arrows <i>loxP</i> sites. (<b>B</b>) PCR-based genotyping of mice using DNA from tail samples to detect <i>SynCre</i> transgene, floxed <i>Ric8a</i> allele and <i>LacZ</i> allele respectively. <i>Ric8a</i>^<i>CKO</i> genotype is emphasized with dotted line. (<b>C</b>) Representative PCR showing the deletion of floxed <i>Ric8a</i> in <i>Ric8a</i>^<i>CKO</i> mouse nervous system and no deletion in non-neural organs. (<b>D</b> and <b>E</b>) Comparison of Cre-recombinase (in <i>SynCre</i>^<i>+/-</i><i>R26R</i>) and <i>Ric8a</i> (in <i>Ric8a</i>^{<i>lacZ/+</i>}) expression in E12.5 embryos by X-gal staining. (<b>F</b>) Down regulation of <i>Ric8a</i> mRNA expression in <i>Ric8a</i>^<i>CKO</i> mice relative to littermate control in hippocampus (HIP), spinal cord (SC), cardiac muscle (CM) and liver (LIV). (<b>G</b>) Deficiency of RIC8A protein in <i>Ric8a</i>^<i>CKO</i> (CKO) mice compared to littermate control (LM) in hippocampus (HIP), spinal cord (SC), spinal ganglia (SG) and cardiac muscle (CM). GAPDH was used as a reference. Abbreviations: del, PCR fragment from the deleted allele Drg, dorsal root ganglia; F, floxed allele; Hb, hindbrain; Mb, midbrain; Nt, neural tube; SC, spinal cord; SG, spinal ganglia; Syt, sympathetic trunk; Vno, vomeronasal organ; wt, PCR fragment from the wild-type allele; V, trigeminal ganglion; X, vagus ganglion; ** <i>P</i> < 0,01. Error bars represent mean ± SEM scores. Scale bars: (D and E) 1 mm.</p

Histological analysis of <i>Ric8a</i>^<i>CKO</i> mutants brains and comparison to littermate control mice.

<p>(<b>A</b>–<b>D</b>) Representative images of hematoxylin-eosin stained P3 coronal brain sections. No gross difference was detected in <i>Ric8a</i>^<i>CKO</i> brain. (<b>A</b> and <b>B</b>) Anterior sections of neonatal brains. (<b>a</b> and <b>b</b>) Magnified image of neocortex layers from black boxes in A and B respectively. (<b>C</b> and <b>D</b>) Posterior part of neonatal brains. (<b>c</b> and <b>d</b>) Magnified image of cerebellar cortex layers from white boxes in C and D respectively. (<b>E</b> and <b>F</b>) Representative spinal cord sections from thoracic region. Motor neurons are indicated with black arrowheads. (<b>e</b> and <b>f</b>) Magnified image of ventral horns from E and F respectively. Abbreviations: cc, <i>corpus callosum</i>; Ce, cerebellum; cp, <i>caudate putamen</i>; dg, dentate gyrus; EGL, external granular layer; GL, granular layer; Pc, Purkinje cell; PCL, Purkinje cell layer; I, II/III, IV/V, VI, neocortical cell layers. Scale bars: (A-D) 1 mm; (E and F) 200 µm; (a-f) 100 µm.</p

Histological analysis of the heart of <i>Ric8a</i>^<i>CKO</i> mutant mice compared to littermate control.

<p>(<b>A</b> and <b>B</b>) Representative images of hearts of P5 mice. (<b>B</b>) Heart of <i>Ric8a</i>^<i>CKO</i> mouse is markedly smaller. (<b>C</b>–<b>H</b>) Anterior-most part of the sinoatrial node extends to the branching level of main bronchi and to the level of transverse aortic arch. (<b>C</b> and <b>D</b>) The beginning of sinoatrial node (indicated by red arrowhead) is located more anteriorly in the heart of <i>Ric8a</i>^<i>CKO</i> mutant mice. We used separated bronchi (bps, bpd) and aorta (a) as reference. (<b>E</b> and <b>F</b>) The medio-ventral wall of superior vena cava (VCs) containing sinoatrial node (red arrowhead) is thinner in <i>Ric8a</i>^<i>CKO</i> mice compared to littermates. (<b>G</b> and <b>H</b>) The wall of the right ventricle (vd) is thinner and has less trabeculi in <i>Ric8a</i>^<i>CKO</i> heart (indicated by red line). Abbreviations: a, aorta; ad, <i>atrium dextrum</i>; bpd, <i>bronchus principalis dexter</i>; bps, <i>bronchus principalis sinister</i>; Oe, oesophagus; T, thymus; tp, <i>truncus pulmonalis</i>; VCs, superior <i>vena cava</i> ; vd, <i>ventriculus dexter</i>; vt, <i>valva tricuspidalis</i>. Scale bars: (A, B) 1 mm; (C,D, G, H) 200 µm; (E and F) 100 µm.</p

Ablation of RIC8A Function in Mouse Neurons Leads to a Severe Neuromuscular Phenotype and Postnatal Death

<div><p>Resistance to inhibitors of cholinesterase 8 (RIC8) is a guanine nucleotide exchange factor required for the intracellular regulation of G protein signalling. RIC8 activates different Gα subunits via non-canonical pathway, thereby amplifying and prolonging the G protein mediated signal. In order to circumvent the embryonic lethality associated with the absence of RIC8A and to study its role in the nervous system, we constructed <i>Ric8a</i> conditional knockout mice using Cre/loxP technology. Introduction of a synapsin I promoter driven Cre transgenic mouse strain (<i>SynCre</i>) into the floxed <i>Ric8a</i> (<i>Ric8a</i>^<i>F/F</i>) background ablated RIC8A function in most differentiated neuron populations. Mutant <i>SynCre</i>^<i>+/-</i><i>Ric8</i>^{<i>lacZ/F</i>} mice were born at expected Mendelian ratio, but they died in early postnatal age (P4-P6). The mutants exhibited major developmental defects, like growth retardation and muscular weakness, impaired coordination and balance, muscular spasms and abnormal heart beat. Histological analysis revealed that the deficiency of RIC8A in neurons caused skeletal muscle atrophy and heart muscle hypoplasia, in addition, the sinoatrial node was misplaced and its size reduced. However, we did not observe gross morphological changes in brains of <i>SynCre</i>^<i>+/-</i><i>Ric8a</i>^{<i>lacZ/F</i>} mutants. Our results demonstrate that in mice the activity of RIC8A in neurons is essential for survival and its deficiency causes a severe neuromuscular phenotype.</p> </div

Distribution of RIC8, Gα_i1/2, LGN and NuMA in the fertilized oocyte at pronuclear stages.

<p>(<b>A-I</b>) Mouse fertilized oocyte at pronuclear stages were double-labeled with RIC8 antibody (green) and NuMA, LGN or Gα_i1/2 antibodies respectively (red). DNA was stained with DAPI (blue). (<b>a-i</b>) Higher magnification of female or male (indicated by sex symbols) pronucleus. Yellow arrowheads point to small RIC8 foci localized in the nucleoplasm. White arrowheads point to the meiotic spindle. Dotted white line indicates the borders of oocyte. Abbreviations: ms, meiotic spindle; pb, polar body; PN, pronuclear stage. Scale bar: 10 μm.</p

RIC8 downregulation interferes with localization of Gα_i1/2 protein to the oocyte cortex.

<p>(<b>A, B</b>) RIC8 protein (green) expression in mouse oocytes was downregulated by microinjection of <i>Ric8</i> siRNA. DNA was stained with DAPI (blue). (<b>C</b>) Downregulation of <i>Ric8</i> mRNA expression with siRNA in microinjected oocytes quantified by qRT-PCR. (<b>D</b>) The relative intensity of RIC8 immunofluorescence signal measured by AutoQuant X3. (<b>E, F</b>) Localization of Gα_i1/2 (red) in mouse oocytes treated with <i>Ric8</i> and control siRNA at meiosis I. DNA was stained with DAPI (blue). (<b>G</b>) The relative intensity of Gα_i1/2 immunofluorescence signal measured by AutoQuant X3. Abbreviations: Cx, cortex. *<i>P</i> ˂ 0.05, **<i>P</i> ˂ 0.01; Students <i>t</i> test. Error Bars represent ±SEM scores. Scale bar: 10 μm.</p

Immunological detection of co-localization of RIC8 with Gα_i1/2, LGN and NuMA proteins during oocyte development.

<p>(<b>A-C</b>; <b>G-L</b>) Mouse oocytes at GV or (<b>D-F</b>) GVBD stages were double-labeled with RIC8 antibody (green) and NuMA, LGN or Gα_i1/2 antibodies respectively (red). DNA was stained with DAPI (blue). (<b>a-l</b>) Higher magnification of the region of chromatin. Dotted white line indicates the borders of oocyte. White arrowheads point RIC8 foci at nucleolus and on chromatin. Abbreviations: GV, germinal vesicle; GVBD, germinal vesicle breakdown. Scale bar: 10 μm.</p

GEF RIC8 co-localization with LGN during mouse oocyte maturation.

<p>(<b>A-F</b>) Mouse oocytes at meiosis I and (<b>G-I</b>) at metaphase block of meiosis II were double-labeled with RIC8 antibody (green) and LGN (red). DNA was stained with DAPI (blue). Localization of meiotic spindle is denoted with white arrows and yellow to orange colour in this area indicates the overlapping regions of RIC8 and LGN. Dotted white line indicates the borders of oocyte. Abbreviations: Ana/Tel I, anaphase/telophase of meiosis I; Met I or Met II, metaphase of meiosis I or meiosis II respectively; ms, meiotic spindle; pb, polar body. Scale bar: 10 μm.</p