Expression Pattern and Localization Dynamics of Guanine Nucleotide Exchange Factor RIC8 during Mouse Oogenesis.

Targeting of G proteins to the cell cortex and their activation is one of the triggers of both asymmetric and symmetric cell division. Resistance to inhibitors of cholinesterase 8 (RIC8), a guanine nucleotide exchange factor, activates a certain subgroup of G protein α-subunits in a receptor independent manner. RIC8 controls the asymmetric cell division in Caenorhabditis elegans and Drosophila melanogaster, and symmetric cell division in cultured mammalian cells, where it regulates the mitotic spindle orientation. Although intensely studied in mitosis, the function of RIC8 in mammalian meiosis has remained unknown. Here we demonstrate that the expression and subcellular localization of RIC8 changes profoundly during mouse oogenesis. Immunofluorescence studies revealed that RIC8 expression is dependent on oocyte growth and cell cycle phase. During oocyte growth, RIC8 is abundantly present in cytoplasm of oocytes at primordial, primary and secondary preantral follicle stages. Later, upon oocyte maturation RIC8 also populates the germinal vesicle, its localization becomes cell cycle dependent, and it associates with chromatin and the meiotic spindle. After fertilization, RIC8 protein converges to the pronuclei and is also detectable at high levels in the nucleolus precursor bodies of both maternal and paternal pronucleus. During first cleavage of zygote RIC8 localizes in the mitotic spindle and cell cortex of forming blastomeres. In addition, we demonstrate that RIC8 co-localizes with its interaction partners Gαi1/2:GDP and LGN in meiotic/mitotic spindle, cell cortex and polar bodies of maturing oocytes and zygotes. Downregulation of Ric8 by siRNA leads to interferred translocation of Gαi1/2 to cortical region of maturing oocytes and reduction of its levels. RIC8 is also expressed at high level in female reproductive organs e.g. oviduct. Therefore we suggest a regulatory function for RIC8 in mammalian gametogenesis and fertility.

Deletion of RIC8A in neural precursor cells leads to altered neurogenesis and neonatal lethality of mouse.

RIC8A is a noncanonical guanine nucleotide exchange factor for a subset of Gα subunits. RIC8A has been reported in different model organisms to participate in the control of mitotic cell division, cell signalling, development and cell migration. Still, the function of RIC8A in the mammalian nervous system has not been sufficiently analysed yet. Adult mice express RIC8A in the brain regions involved in the regulation of memory and emotional behaviour. To elucidate the role of RIC8A in mammalian neurogenesis we have inactivated Ric8a in neural precursor cells using Cre/Lox system. As a result, the conditional knockout mice were born at expected Mendelian ratio, but died or were cannibalized by their mother within 12 h after birth. The cerebral cortex of the newborn Nes;Ric8a(CKO) mice was thinner compared to littermates and the basement membrane was discontinuous, enabling migrating neurons to invade to the marginal zone. In addition, the balance between the planar and oblique cell divisions was altered, influencing the neuron production. Taken together, RIC8A has an essential role in the development of mammalian nervous system by maintaining the integrity of pial basement membrane and modulating cell division.

Ablation of RIC8A function in mouse neurons leads to a severe neuromuscular phenotype and postnatal death.

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 Ric8a conditional knockout mice using Cre/loxP technology. Introduction of a synapsin I promoter driven Cre transgenic mouse strain (SynCre) into the floxed Ric8a (Ric8a (F/F) ) background ablated RIC8A function in most differentiated neuron populations. Mutant SynCre (+/-) Ric8 (lacZ/F) 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 SynCre (+/-) Ric8a (lacZ/F) 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.

Nucleotide exchange factor RIC-8 is indispensable in mammalian early development.

The guanine nucleotide exchange factor RIC-8 is a conserved protein essential for the asymmetric division in the early embryogenesis in different organisms. The function of RIC-8 in mammalian development is not characterized so far. In this study we map the expression of RIC-8 during the early development of mouse. To elucidate the RIC-8 function we used Ric-8(-/-) mutant embryos. The Ric-8(-/-) embryos reach the gastrulation stage but do not develop further and die at E6.5-E8.5. We characterized the Ric-8(-/-) embryonic phenotype by morphological and marker gene analyses. The gastrulation is initiated in Ric-8(-/-) embryos but their growth is retarded, epiblast and mesoderm disorganized. Additionally, the basement membrane is defective, amnion folding and the formation of allantois are interfered, also the cavitation. Furthermore, the orientation of the Ric-8(-/-) embryo in the uterus was abnormal. Our study reveals that the activity of RIC-8 protein is irreplaceable for the correct gastrulation of mouse embryo.

Lack of Gata3 results in conotruncal heart anomalies in mouse.

The transcription factor Gata3 is an important regulator of the development of thymus, the nervous system, ear, kidney, and adrenal glands. This study analyzes the role of Gata3 in the developing heart using a mouse strain containing an nlsLacZ reporter gene fused in frame to the Gata3 gene by homologous recombination. Using in situ hybridization, RT-PCR and Gata3-LacZ histochemistry, Gata3 expression was shown in various cardiac structures up to newborn stage. During looping stages (E9.5-E11.5) Gata3-LacZ activity recapitulated endogenous Gata3 and was abundantly expressed in the endocardial ridges and endothelium of distal outflow tract. Strong reporter gene expression was also noted in the mesenchyme of ventral branchial arches, and in the epithelium. In the atrioventricular canal expression was relatively lower. In the four-chambered heart stages (E13.5-E17.5) the LacZ-staining did not recapitulate the endogenous Gata3 transcript and showed rather lineage tracing of formerly Gata3-expressing cells in the hearts. beta-Galactosidase activity was detected in the cusps of semilunar valves, aorta, pulmonary trunk, innominate and common carotid arteries, and faintly in the atrioventricular valves. Gata3-null embryos die normally between E11 and E12. Pharmacological treatment with sympathomimetic beta-adrenergic receptor agonist lengthens the survival up to E18 when malformations of the heart such as ventricular septal defect (VSD), double-outlet of right ventricle (DORV), anomalies of the aortic arch (AAA) and persistent truncus arteriosus (PTA) were detected. The specified malformations correlate with the normal developmental pattern of Gata3-LacZ expression. The short outflow tract and insufficient rotation of truncus arteriosus during looping stages might be the main reasons underlying these malformations.

Gata3 is required for early morphogenesis and Fgf10 expression during otic development.

Inner ear develops from an induced surface ectoderm placode that invaginates and closes to form the otic vesicle, which then undergoes a complex morphogenetic process to form the membranous labyrinth. Inner ear morphogenesis is severely affected in Gata3 deficient mouse embryos, but the onset and basis of the phenotype has not been known. We show here that Gata3 deficiency leads to severe and unique abnormalities during otic placode invagination. The invagination problems are accompanied often by the formation of a morphological boundary between the dorsal and ventral otic cup and by the precocious appearance of dorsal endolymphatic characteristics. In addition, the endolymphatic domain often detaches from the rest of the otic epithelium during epithelial closure. The expression of several cell adhesion mediating genes is altered in Gata3 deficient ears suggesting that Gata3 controls adhesion and morphogenetic movements in early otic epithelium. Inactivation of Gata3 leads also to a loss of Fgf10 expression in otic epithelium and auditory ganglion demonstrating that Gata3 is an important regulator of Fgf-signalling during otic development.

Cat odour exposure decreases exploratory activity and alters neuropeptide gene expression in CCK(2) receptor deficient mice, but not in their wild-type littermates.

An attempt was made to establish whether the anxiogenic effect of cat odour differs in female wild-type and CCK(2) receptor deficient mice, having different exploratory activity in the elevated plus-maze. The exposure of wild-type and homozygous CCK(2) receptor deficient mice to cat odour did not reveal substantial differences between the two genotypes. The number of contacts with the cat odour impregnated cloth was reduced and the frequency of stretch-attend postures was increased similarly in wild-type and homozygous mice. However, the following exposure of mice to the elevated plus-maze established differences as homozygous mice displayed increased exploratory activity in the plus-maze. The cat odour exposure significantly reduced exploratory activity only in homozygous mice. Together with the increased exploratory activity we established in homozygous mice significantly increased expression of the Oprm1 gene in the frontal cortex and mesencephalon. The exposure of mice to cat odour caused only minor changes in the gene expression of wild-type mice, whereas in homozygous animals a significantly increased expression of the Mc3r gene in the frontal cortex and temporal lobe, and the Pomc1 gene in the temporal lobe, mesencephalon and mesolimbic area was established. In conclusion, CCK(2) receptor deficient mice displayed reduced anxiety compared to their wild-type littermates in the plus-maze test. This behavioural effect seems to be related, at least partly, to an increased tone of opioid system in the brain. Moreover, homozygous mice respond to the exposure of cat odour with an increased anxiety. This effect seems to be related to the increased function of the melanocortin system in the brain structures of genetically modified mice.

Heterozygous mice with Ric-8 mutation exhibit impaired spatial memory and decreased anxiety.

Ric-8 is a guanine nucleotide exchange factor for a subset of Galpha proteins and it is required to maintain Galpha(q) and the Galpha(s) pathways in functional state. In adult mice Ric-8 is expressed in regions involved in the regulation of behavior (neocortex, cingulate cortex and hippocampus). As Ric-8 is shown to regulate neuronal transmitter release, the aim of present study was to perform behavioral analysis of ric-8 mutant. Homozygous (-/-) ric-8 mutant mice are not viable and die in early embryonic development, therefore for behavioral analysis heterozygous (+/-) ric-8 mutant mice were used. We found decreased anxiety of ric-8 heterozygous mice in light-dark compartment test where mutant mice significantly avoided the light compartment. In spatial learning paradigm (Morris water maze) the performance of ric-8 (+/-) mice was impaired. Namely, in the reversal test, ric-8 (+/-) mice exhibited significant delay to find the hidden platform compared to wild-type (wt) littermates. We did not find differences in the behavioral tests reflecting the motor abilities of mice (motor activity, rota-rod). Therefore, described alterations seem to be specific for anxiety and spatial learning. Based on these results we can conclude the importance of ric-8 in the regulation of memory and emotional behavior.

Gene expression analysis of Gata3-/- mice by using cDNA microarray technology.

Transcription factor Gata3 is implicated in the formation of autosomal dominant hypoparathyroidism, sensorineural deafness, and renal anomaly (HDR) syndrome. We pursued to identify the potential Gata3 target genes by profiling the gene expression pattern in E9.5 Gata3-/- mouse embryos. Altogether four independent microarray hybridizations were carried out on NIA Mouse15K cDNA arrays. We discovered two hundred and sixty one genes that are downregulated in Gata3 mutant embryos at E9.5 (with a minimal 2.0-fold change). The majority of the differentially expressed genes belong to two functional groups--genes involved in transcription regulation and cellular signaling. One of the genes discovered to be downregulated in Gata3 mutant embryos was tumor suppressor gene Disabled 2. The validity of this finding was checked by using the whole mount in situ hybridization technology. This study revealed that the sites, where Dab2 is downregulated in the mutant embryos partly overlap with the Gata3 expression domains, including the mid-embryo region, branchial arches and facio-acoustic (VII-VIII) neural crest complex. This is the first time when tumor supressor gene Dab2 is shown to be implicated in the defective phenotype of Gata3 mutant mice.

Alterations in opioid system of the rat brain after cat odor exposure.

The effect of cat odor exposure was studied on morphine-induced increase of exploratory behavior and on the expression of opioid genes in forebrain structures of male Wistar rats. Treatment with morphine (1 mg/kg) induced a significant increase in exploratory behavior in an unfamiliar environment in rats. Previous exposure of animals to cat odor completely abolished this stimulating action of mu-opioid receptor agonist on exploratory activity. Cat odor exposure induced a significant increase in the expression of pro-opio-melanocortin (POMC) and mu-opioid receptor (MOR) genes in the brain structures related to anxiety and motivation. This study clearly demonstrates that cat odor exposure increases the activity of opioid system in rat forebrain structures.

Partially overlapping expression of Gata2 and Gata3 during inner ear development.

Gata2 and Gata3 belong to the Gata family of transcription factors in vertebrates that bind to a consensus "GATA" DNA sequence. The Gata3 gene is one of the earliest markers for the developing mouse inner ear. Ear morphogenesis is blocked in Gata3-deficient embryos, whereas nothing was known of the role of Gata2 in mouse inner ear. Here, we have compared the expression patterns of Gata2 and Gata3 during normal inner ear development and investigated their relationship in mice where either Gata3 or Gata2 has been inactivated. The expression of the two Gata genes is highly overlapping at embryonic day (E)10.5 but becomes increasingly distinct later. Whereas Gata2 is predominantly expressed in the dorsal vestibular system, Gata3 was detected mainly in the ventral cochlear duct and ganglion. No phenotypic abnormalities were observed in the inner ear of Gata2-/- embryos before lethality at E10.5 and Gata3 expression was unchanged. In contrast, a delay and strong reduction of Gata2 expression was detected in Gata3-/- otic epithelium.

Hearing loss following Gata3 haploinsufficiency is caused by cochlear disorder.

Patients with HDR syndrome suffer from hypoparathyroidism, deafness, and renal dysplasia due to a heterozygous deletion of the transcription factor GATA3. Since GATA3 is prominently expressed in both the inner ear and different parts of the auditory nervous system, it is not clear whether the deafness in HDR patients is caused by peripheral and/or central deficits. Therefore, we have created and examined heterozygous Gata3 knockout mice. Auditory brainstem response (ABR) thresholds of alert heterozygous Gata3 mice, analyzed from 1 to 19 months of age, showed a hearing loss of 30 dB compared to wild-type littermates. Neither physiological nor morphological abnormalities were found in the brainstem, cerebral cortex, the outer or the middle ear. In contrast, cochleae of heterozygous Gata3 mice showed significant progressive morphological degeneration starting with the outer hair cells (OHCs) at the apex and ultimately affecting all hair cells and supporting cells in the entire cochlea. Together, these findings indicate that hearing loss following Gata3 haploinsufficiency is peripheral in origin and that this defect is detectable from early postnatal development and maintains through adulthood.

A tissue-specific knockout reveals that Gata1 is not essential for Sertoli cell function in the mouse.

The transcription factor Gata1 is essential for the development of erythroid cells. Consequently, Gata1 null mutants die in utero due to severe anaemia. Outside the haematopoietic system, Gata1 is only expressed in the Sertoli cells of the testis. To elucidate the function of Gata1 in the testis, we made a Sertoli cell-specific knockout of the Gata1 gene in the mouse. We deleted a normally functioning 'floxed' Gata1 gene in pre-Sertoli cells in vivo through the expression of Cre from a transgene driven by the Desert Hedgehog promoter. Surprisingly, Gata1 null testes developed to be morphologically normal, spermatogenesis was not obviously affected and expression levels of putative Gata1 target genes, and other Gata factors, were not altered. We conclude that expression of Gata1 in Sertoli cells is not essential for testis development or spermatogenesis in the mouse.

Expression of ric-8 (synembryn) gene in the nervous system of developing and adult mouse.

Recent biochemical studies revealed that ric-8A encodes a guanine nucleotide exchange factor for a subset of Galpha proteins. Ric-8 is a key component of a signaling network in C. elegans that regulates neurotransmitter secretion and also plays a role in centrosome-mediated events during early embryogenesis. Here we show that during the early development in mice (E9.5-E12.0) ric-8 (synembryn) is expressed in the developing nervous system such as the cranial ganglia, neural tube, sympathetic chain and dorsal root ganglia. Ric-8 is also found in the lens, vomeronasal organ, and endolymphatic sac. In adult brain, it is expressed in the neocortex, hippocampus, and cerebellum as well as in the pineal gland and ependymal layer.