Ccdc85c Encoding a Protein at Apical Junctions of Radial Glia Is Disrupted in Hemorrhagic Hydrocephalus (hhy) Mice
2011; Elsevier BV; Volume: 180; Issue: 1 Linguagem: Inglês
10.1016/j.ajpath.2011.09.014
ISSN1525-2191
AutoresNobuko Mori, Mitsuru Kuwamura, Natsuki Tanaka, Ryuji Hirano, Mikoto Nabe, Masato Ibuki, Jyoji Yamate,
Tópico(s)Genetic and Kidney Cyst Diseases
ResumoCortical heterotopia, a malformation of the developing cortex, are a major cause of epilepsy and mental retardation in humans. Hemorrhagic hydrocephalus (hhy) mutation on mouse chromosome 12 results in subcortical heterotopia and nonobstructive hydrocephalus with frequent brain hemorrhage. Here, we show that coiled-coil domain-containing 85C (Ccdc85c), consisting of 6 exons that encode a 420 amino acid protein, is disrupted by replacement of a 3.2-kb sequence, including exon 2 in Ccdc85c by a 1.5-kb retrotransposon-like repeat sequence in the hhy mutant. Immunoreactivity to Ccdc85C was detected predominantly at the apical junctions of radial glia in the wall of lateral ventricles of the developing brain. In the hhy brain at embryonic (E) day 18 (E18), radial glial demise followed by agenesis of the ependymal layer lining the neonatal cortex and accumulation of neuronal specific nuclear protein (NeuN)-positive postmigratory neurons in the subcortical area occurred. Accumulation of E15-born, but not of E13-born, 5-bromo-2′-deoxyuridine labeled neurons expressing special AT-rich sequence binding protein 2 was detected in both heterotopia and the superficial layers of the hhy neocortex at postnatal day 7. Ccdc85c deficiency permitted radial scattering of paired box gene 6-positive neural progenitors in the ventricular zone, likely resulting in reduced self-renewal of the progenitors in the developing hhy cortex. These findings indicate an important role of Ccdc85C in cortical development and provide a mouse model to study pathogenesis of subcortical heterotopia and hydrocephalus. Cortical heterotopia, a malformation of the developing cortex, are a major cause of epilepsy and mental retardation in humans. Hemorrhagic hydrocephalus (hhy) mutation on mouse chromosome 12 results in subcortical heterotopia and nonobstructive hydrocephalus with frequent brain hemorrhage. Here, we show that coiled-coil domain-containing 85C (Ccdc85c), consisting of 6 exons that encode a 420 amino acid protein, is disrupted by replacement of a 3.2-kb sequence, including exon 2 in Ccdc85c by a 1.5-kb retrotransposon-like repeat sequence in the hhy mutant. Immunoreactivity to Ccdc85C was detected predominantly at the apical junctions of radial glia in the wall of lateral ventricles of the developing brain. In the hhy brain at embryonic (E) day 18 (E18), radial glial demise followed by agenesis of the ependymal layer lining the neonatal cortex and accumulation of neuronal specific nuclear protein (NeuN)-positive postmigratory neurons in the subcortical area occurred. Accumulation of E15-born, but not of E13-born, 5-bromo-2′-deoxyuridine labeled neurons expressing special AT-rich sequence binding protein 2 was detected in both heterotopia and the superficial layers of the hhy neocortex at postnatal day 7. Ccdc85c deficiency permitted radial scattering of paired box gene 6-positive neural progenitors in the ventricular zone, likely resulting in reduced self-renewal of the progenitors in the developing hhy cortex. These findings indicate an important role of Ccdc85C in cortical development and provide a mouse model to study pathogenesis of subcortical heterotopia and hydrocephalus. Hydrocephalus is a disease characterized by the accumulation of cerebrospinal fluid (CSF) in the brain cavities, resulting in ventricular dilatation. Hydrocephalus is caused by abnormalities in the CSF flow within the ventricular system or production/resorption of CSF by the choroid plexus and subarachnoid space. With or without aqueductal stenosis, hydrocephalus is classified into two types: obstructive hydrocephalus and communicating (nonobstructive) hydrocephalus. Congenital hydrocephalus is a frequent birth defect in humans, as well as mice. A significant portion of the human cases is genetic in origin, but molecular genetics of this disease is poorly understood.1Zang J. Williams M.A. Rigamonti D. Genetics of human hydrocephalus.J Neurol. 2006; 253: 1255-1266Crossref PubMed Scopus (136) Google Scholar In mice, a number of hydrocephalus mutations have been reported. Some of these hydrocephalus mutations recently identified are implicated in ependymal malfunction, in particular, cilia dysfunction.2Davy B.E. Robinson M.L. Congenital hydrocephalus in hy3 mice is caused by a frameshift mutation in Hydin, a large novel gene.Hum Mol Genet. 2003; 12: 1163-1170Crossref PubMed Scopus (132) Google Scholar, 3Ibanez-Tallon I. Gorokhova S. Heintz N. Loss of function of axonemal dynein Mdnah5 causes primary ciliary dyskinesia and hydrocephalus.Hum Mol Genet. 2002; 11: 715-721Crossref PubMed Scopus (184) Google Scholar, 4Ibanez-Tallon I. Pagenstecher A. Fliegauf M. Olbrich H. Kispert A. Ketelsen U.-P. North A. Heintz N. Omran H. Dysfunction of axonemal dynein heavy chain Mdnah5 inhibits ependymal flow and reveals a novel mechanism for hydrocephalus formation.Hum Mol Genet. 2004; 13: 2133-2141Crossref PubMed Scopus (273) Google Scholar, 5Banizs B. Pike M.M. Millican C.L. Ferguson W.B. Komlosi P. Sheetz J. Bell P.D. Schwiebert E.M. Yoder B.K. Dysfunctional cilia lead to altered ependyma and choroid plexus function, and result in the formation of hydrocephalus.Development. 2005; 132: 5329-5339Crossref PubMed Scopus (279) Google Scholar, 6Baas D. Meiniel A. Benadiba C. Bonnafe E. Meiniel O. Reith W. Durand B. A deficiency in RFX3 causes hydrocephalus associated with abnormal differentiation of ependymal cells.Eur J Neurosci. 2006; 24: 1020-1030Crossref PubMed Scopus (93) Google Scholar, 7Del Bigio M.R. Ependymal cells: biology and pathology.Acta Neuropathol. 2010; 119: 55-73Crossref PubMed Scopus (234) Google Scholar, 8Takaki E. Fujimoto M. Nakahari T. Yonemura S. Miyata Y. Hayashida N. Yamamoto K. Vallee R.B. Mikuriya T. Sugahara K. Yamashita H. Inouye S. Nakai A. Heat shock transcription factor 1 is required for maintenance of ciliary beating in mice.J Biol Chem. 2007; 282: 37285-37292Crossref PubMed Scopus (52) Google Scholar, 9Wodarczyk C. Rowe I. Charavalli M. Pema M. Qian F. Boletta A. A novel mouse model reveals that polycystin-1 deficiency in ependyma and choroid plexus results in dysfunctional cilia and hydrocehalus.PLoS One. 2009; 4: e7137Crossref PubMed Scopus (71) Google Scholar However, molecular mechanisms underlying the development of hydrocephalus remain obscure. Cortical heterotopia are results of a malformation of the developing cortex. Patients with heterotopia show characteristic disorders such as epilepsy and developmental delay.10Pang T. Atefy R. Sheen V. Malformations of cortical development.Neurologist. 2008; 14: 181-191Crossref PubMed Scopus (111) Google Scholar Heterotopia in humans are classified into two subtypes, nodules of neurons lining the lateral ventricles, namely periventricular heterotopia (PH), and heterotopic neurons arrested under the normal cerebral cortex (ie, subcortical band heterotopia [SBH]). So far, X-linked dominant mutations in the gene for actin-binding phosphoprotein filamin A (FLNA) are associated with PH in humans.11Fox J.W. Lamperti E.D. Ekşioğlu Y.Z. Hong S.E. Feng Y. Graham D.A. Scheffer I.E. Dobyns W.B. Hirsch B.A. Radtke R.A. Berkovic S.F. Huttenlocher P.R. Walsh C.A. Mutations in filamin 1 prevent migration of cerebral cortical neurons in human periventricular heterotopia.Neuron. 1998; 21: 1315-1325Abstract Full Text Full Text PDF PubMed Scopus (701) Google Scholar Human SBH is caused by X-linked dominant mutations in the microtubule-associated doublecortin (DCX) gene.12Gleeson J.G. Allen K.M. Fox J.W. Lamperti E.D. Bercovic S. Scheffer I. Cooper E.C. Dobyns W.B. Minnerath S.R. Ross M.E. Walsh C.A. Doublecortin, a brain-specific gene mutated in human X-linked lissencephaly and double cortex syndrome, encodes a putative signaling protein.Cell. 1998; 92: 63-72Abstract Full Text Full Text PDF PubMed Scopus (884) Google Scholar SBH is usually observed in females; males with DCX mutations develop lissencephaly. Impairment of neuronal migration, in particular cell motility, has been implicated in both types of heterotopia13Gleeson J.G. Lin P.T. Flanagan L.A. Walsh C.A. Doublecortin is a microtubule-associated protein and is expressed widely by migrating neurons.Neuron. 1999; 23: 257-271Abstract Full Text Full Text PDF PubMed Scopus (1063) Google Scholar, 14Walsh C.A. Goffinet A.M. Potential mechanisms of mutations that affect neuronal migration in man and mouse.Curr Opin Genet Dev. 2000; 10: 270-274Crossref PubMed Scopus (84) Google Scholar, 15Pilz D. Stoodley N. Golden J.A. Neuronal migration disorders, genetics, and epileptogenesis.J Child Neurol. 2002; 20: 287-299Google Scholar. PH likely represents a disorder of the initiation of migration in a small portion of neurons, whereas SBH arrested neurons in the course of migration. More recently, autosomal recessive mutations in the vesicle trafficking regulator ADP-ribosylation factor guanine exchange factor 2 (ARFGEF2) in humans and a mutation in Napa encoding the soluble NSF-attachment protein (α-SNAP) in the congenital hydrocephalus (hyh) mouse were identified as responsible genes for PH, elucidating defects in the vesicle trafficking of polarized protein as a possible mechanism of PH formation16Sheen V.L. Ganesh V.S. Topcu M. Sebire G. Bodell A. Hill R.S. Grant P.E. Shugart Y.Y. Imitola J. Khoury S.J. Guerrini R. Walsh C.A. Mutations in ARFGEF2 implicate vesicle trafficking in neural progenitor proliferation and migration in the human cerebral cortex.Nat Genet. 2004; 36: 69-76Crossref PubMed Scopus (293) Google Scholar, 17Chae T.H. Kim S. Marz K.E. Hanson P.I. Walsh C.A. The hyh mutation uncovers roles for αSnap in apical protein localization and control of neural cell fate.Nat Genet. 2004; 36 (364–270)PubMed Google Scholar, 18Hong H.-K. Chakravarti A. Takahashi J.S. The gene for soluble N-ethylmaleimide sensitive factor attachment protein α is mutated in hydrocephaly with hop gait (hyh) mice.Proc Natl Acad Sci USA. 2004; 101: 1748-1753Crossref PubMed Scopus (53) Google Scholar, 19Ferland R.J. Batiz L.F. Neal J. Lian G. Bundock E. Lu J. Hsiao Y.-C. Diamond R. Mei D. Banham A.H. Brown P.J. Vanderburg C.R. Joseph J. Hecht J.L. Folkerth R. Guerrini R. Walsh C.A. Rodriguez E.M. Sheen V.L. Disruption of neural progenitors along the ventricular and subventricular zones in periventricular heterotopia.Hum Mol Genet. 2009; 18: 497-516Crossref PubMed Scopus (147) Google Scholar. In contrast, molecular genetics underlying SBH is poorly understood. Congenital hydrocephalus is the consequence of abnormal brain development. We identified an autosomal recessive hemorrhagic hydrocephalus (hhy) mutation on mouse chromosome 12.20Kuwamura M. Kinoshita A. Okumoto M. Yamate J. Mori N. Hemorrhagic hydrocephalus (hhy): a novel mutation on mouse chromosome 12.Brain Res Dev Brain Res. 2004; 152: 69-72Crossref PubMed Scopus (10) Google Scholar The mutant animals develop nonobstructive hydrocephalus with a high penetrance (nearly 100%). A clinical sign of hydrocephalus is a dome-shaped head, which becomes apparent in the hhy mutant, mostly within 2 weeks of birth. Neither the choroid plexus nor the subarachnoid space of the mutant shows any pathological abnormalities, precluding overproduction or impaired resorption of CSF as a mechanism of hydrocephalus. The structure and orientations of brain vessels are both normal in the mutant, whereas affected animals frequently show brain hemorrhage. More importantly, animals homozygous for the hhy mutation all have subcortical heterotopia analogous to SBH below the normally laminated cortex, suggesting that the hhy gene plays a role in cortical development. In the present study, we demonstrate that a gene for coiled-coil domain-containing protein 85C (Ccdc85c) with unknown subcellular functions is disrupted in the hhy mutant. Using an antibody raised against the Ccdc85C protein, we found a considerable part of immunoreactivity to Ccdc85C at the apical junctions of radial glia (ie, neural progenitors) in the wall of the lateral ventricles. We further show that Ccdc85c deficiency causes premature depletion of radial glia in the developing hhy cortex. Thus, Ccdc85C may play an important role in cortical development, especially in the maintenance of radial glia. The hhy mutation arising in the BALB/cOpu (BALB/c) genome has been maintained as hhy heterozygous (hhy/+) carriers on the MSM/Opu (MSM) background (M-hhy/+) at the animal facility of Osaka Prefecture University, Osaka, Japan. M-hhy/+ mice were intercrossed at N3–N6, and their offspring were used in fine mapping, histology, immunohistochemistry, and scanning electron microscopy analysis. Progeny from an intercross of hhy/+ mice on the BALB/c and MSM heterozygous background (C/M-hhy/+) were used in 5-bromo-2′-deoxyuridine (BrdU)-labeling and immunofluorescent analysis. hhy/hhy mice were referred to as hhy, and hhy/+, or +/+ mice, as the control in our pathological studies. Animal experiments were conducted according to the guidelines of Osaka Prefecture University. Genotyping of microsatellite markers was performed by PCR and polyacrylamide gel electrophoresis as previously described.21Okumoto M. Song C.-W. Tabata K. Ishibashi M. Mori N. Park Y.-G. Kominami R. Matsumoto Y. Takamori Y. Esaki K. Putative tumor suppressor gene region within 0.85 cM on chromosome 12 in radiation-induced murine lymphomas.Mol Carcinog. 1998; 22: 175-181Crossref PubMed Scopus (17) Google Scholar Information of the microsattelite markers D12Mit28, D12Mit53, D12Mit181, D12Mit141, D12Nds2, and MCA1 used is available on the website (Mouse Genome Informatics at http://www.informatics.jax.org). Primers for newly developed microsatellite markers (Opu series) are listed in Table 1. After identification of the hhy mutation, we genotyped Ccdc85c using the primers listed in the Table 1 with the cycling conditions: 1 cycle of 94°C for 30 seconds, 30 cycles of 94°C for 30 seconds, 57°C for 30 seconds, and 72°C for 1 minute. PCR products were analyzed by electrophoresis in 2% agarose gel.Table 1List of Primer SequencesPrimersForwardReverseMicrosatellite genotyping D12Opu85′-CCGGTTGTCAAGTAGCTGTT-3′5′-GACGCTCTAGCTTGACTCTGG-3′ D12Opu25′-CTGTGATCATGTGTTTGCTTCAG-3′5′-CTCCAAGCAGCCAAGAGACT-3′ D12Opu145′-CAGCTCATTCCATAATCTGGTG-3′5′-CAGCGTTGAGCAGTTTGAGA-3′ D12Opu175′-GGGCAGAGGAACAGAGGAA-3′5′-CATGCTCCTGGCTCTTGTTT-3′ D12Opu55′-GCACGAGCACATACACAAGC-3′5′-CAATCCACAACGGAACAGAA-3′Ccdc85c genotyping Common5′-CTGCAGTTCCCATCCCTGCT-3′ Wild type5′-CTCTCATGAGATCTGACGTCC-3′ hhy5′-CGTGTGCTACAAAGGGCCTG-3′RT-PCRCcdc85c5′-TCTCTGGATGACCTGTCAGC-3′5′-GGCCCCTTGAACTGATTACC-3′ B2m5′-CTGGTGCTTGTCTCACTGAC-3′5′-AATGTGAGGCGGGTGGAACT-3′Shuttle PCR for restriction digestion analysisCcdc85c (9.8 kb)5′-TGAAAGGTCCTGTCTGATGGTGACCCTGGTGC-3′5′-TGAGAGGCAACACCACGATGCCAACTGGCTC-3′Ccdc85c (3.8 kb)5′-GAAGGGACAGATGCCTTATCGTCCACTGCAG-3′5′-GAACTGAGCTGCAGTAATTCAGCCTGTGAGC-3′ Open table in a new tab Phenotype determination was performed by a gross pathological inspection. When their heads became dome-shaped before weaning (a clinical sign of hhy hydrocephalus), they were sacrificed and inspected for ventricular dilatation. Animals without clinical signs before weaning were subsequently reared for 1 to 5 months, and were pathologically inspected for ventricular dilatation. Total RNA was isolated from the hhy/hhy, hhy/+, and BALB/c (+/+) brains using a TAKARA FastPure RNA Kit (Takara Bio Inc., Kyoto, Japan). Reverse transcription was performed using SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA) on 1 μg of total RNA, according to the manufacturer's protocol. PCR amplification of Ccdc85c and β-2 microglobulin (β2m) cDNA was performed using the primers listed in Table 1. The cycling conditions were 1 cycle of 95°C for 3 minutes and 35 cycles of 95°C for 20 seconds, 55°C for 30 seconds, and 72°C for 45 seconds for Ccdc85c, and 1 cycle of 94°C for 3 minutes and 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute for β2m. PCR products were analyzed by electrophoresis in 2% or 3% agarose gels. PCR products for Ccdc85c cDNA from +/+ and hhy/hhy animals were sequenced directly or cloned using the pGEM-T Easy Vector System (Promega, Madison, WI) and then sequenced. Sequence analysis was performed by Bio Matrix Research (Nagareyama-shi, Chiba, Japan). Ccdc85c genomic DNA from BALB/c (+/+) and hhy/hhy mice was amplified by TAKARA LA-Taq polymerase (Takara Bio Inc.) using the primers listed in Table 1 with a shuttle PCR program: 1 cycle of 94°C for 1 minute and 30 cycles of 98°C for 20 seconds, and 68°C for 10 minutes (or 2 minutes). The PCR products were purified using BD CHROMA SPIN Columns (BD Biosciences Clontech, Mountain View, CA), digested using EcoRV, BamHI, XbaI, and XhoI (Takara Bio Inc.), and analyzed in a 1% agarose gel. The 2.0-kb hhy DNA was cloned and sequenced as described. Pregnant animals were intraperitoneally injected by 100 mg/kg body weight of BrdU dissolved in physiological saline at gestational days 13 and 15. hhy homozygous pups born to the BrdU-injected mothers were sacrificed at postnatal (P) day 7 (P7), and their brains were frozen for immunofluorescence analysis. hhy mice from embryonic (E) day 14 (E14) to P7 and age-matched controls were used. Brains from at least two animals were examined for each genotype at each age. Fresh frozen brains were coronally sliced into 10-μm sections. The sections were fixed in the Zamboni's fixative containing 0.21% picric acid and 2% paraformaldehyde in 0.15M phosphate buffer (pH 7.3) at 4°C for 15 minutes and treated with normal goat serum (1:10; Sigma-Aldrich, St. Louis, MO) at room temperature for 30 minutes. The following rabbit polyclonal antibodies were applied to the sections: Ccdc85C (1:1000), ZO-1 (1:1000; Invitrogen), paired box gene 6 (Pax6) (1:1000; MBL), Tbr1 (1:500; Abcam, Inc., Cambridge, MA), Tbr2 (1:500; Abcam), neuronal specific nuclear protein (NeuN) (1:500; Millipore), Ki-67 (1:500; Abcam), and BrdU (Dako, 1: 200). We produced the C-terminal 172 amino acid peptide of Ccdc85C using a standard GST-fusion protein Esherichia coli expression system (GE Healthcare, Uppsala, Sweden). The Ccdc85C-specific antibody (rabbit IgG, polyclonal) was generated by Japan Bio Service, Co. Ltd. (Saitama-shi, Saitama). Before staining for BrdU, frozen sections were immersed in 2N HCl for 20 minutes. The sections were incubated with the primary antibodies at room temperature for 1 hour or at 4°C overnight. After washing with PBS, the sections were incubated with the Alexa Fluor 488-labeled secondary antibody against rabbit IgG (1:1000; Invitrogen) at room temperature for 45 minutes or for BrdU staining, with the Cy3-labeled secondary antibody against rabbit IgG (1:1000; Invitrogen). After washing with PBS, sections were mounted by Mounting medium with DAPI (Vector Laboratories Inc., Burlingame, CA). Signals were detected with a confocal imaging system C1Si (Nikon, Tokyo, Japan). Brains were excised from E14 to P7 hhy animals and age-matched controls. At least two mutants at each age were used. Brains were fixed using the Methacarn fixative (methanol:chloroform:acetic acid = 6:3:1) at 4°C for 1 hour and embedded in paraffin. Paraffin-embedded tissues were cut into 4-μm sections and were deparaffinized in serial xylene-ethanol washes. Following antigen retrieval by microwaving in 10 mmol/L citrate-buffer pH 6.0 for 5 to 20 minutes, the sections were incubated with an anti-nestin antibody (1:200; Chemicon), an anti-NeuN antibody (1:500; Millipore) or anti-Satb2 antibody (1: 250; Abcam) at 4°C overnight, and then incubated with a horseradish peroxidase-labeled antibody against mouse IgG (Histofine Simple Stain Kit; Nichirei Biosciences, Inc., Tokyo, Japan) for 1 hour at room temperature. Positive reaction was developed using the DAB Substrate Kit (Vector Laboratories), and the nuclei were counterstained using H&E. For the histopathological evaluations, paraffin sections were stained by H&E. P0 and P14 hhy animals (3 at each point) and age-matched controls were used. P0 mice were euthanized and their brains were excised. P14 animals were subjected to intracardiac perfusion with phosphate-buffered 4% paraformaldehyde before brain excision. Coronally sliced sections were fixed by immersion in 2% glutaraldehyde at 4°C overnight, further fractionated, and postfixed in 2% osmium. After dehydration and critical point drying, the tissue blocks were coated with platinum-palladium vapor deposition and observed under a scanning electron microscope (Hitachi Research and Development, Yokohama, Japan). Previously, we localized the hhy mutation arising in the BALB/c (C) mouse genome to a 7-centimorgan (cM) region (the hhy region) between D12Mit28 (52 cM) and D12Nds2 (59 cM) on chromosome 12.20Kuwamura M. Kinoshita A. Okumoto M. Yamate J. Mori N. Hemorrhagic hydrocephalus (hhy): a novel mutation on mouse chromosome 12.Brain Res Dev Brain Res. 2004; 152: 69-72Crossref PubMed Scopus (10) Google Scholar The hhy heterozygous (hhy/+) carriers were crossed to MSM (M) mice, and the F1 hybrids bearing the hhy mutation on BALB/c chromosome 12 were backcrossed to MSM mice to maintain the mutation in the MSM background (M-hhy/+ mice). At N3 to N6 generations, we intercrossed the M-hhy/+ mice with the BALB/c (C) and MSM (M) heterozygous (C/M) alleles at microsatellite markers ranging D12Mit28–D12Mit53 (52 cM)–D12Mit181 (53 cM)–D12Mit141 (55 cM)–D12Nds2 in the hhy region. To conduct fine mapping of hhy, we analyzed 50 offspring carrying the homozygous BALB/c (C/C) genotype at any of the markers previously enumerated. Of these, 29 having dome-shaped heads, a clinical sign of hhy hydrocephalus, were diagnosed as affected animals, and other 21 showing neither clinical sign of hydrocephalus nor ventricular dilatation in pathological inspection were diagnosed as nonaffected animals. Haplotypes of these animals are shown (Figure 1A). Of 29 affected animals, 23 had the C/C allele throughout the hhy region, and 6 harbored recombination in the hhy region: 1 recombinant had the C/C genotype at the markers D12Mit28, D12Mit53, D12Mit181, and D12Mit141, and non-C/C genotype at D12Nds2; 5 recombinants had the C/C alleles at D12Mit28, D12Mit53, and D12Mit181, and non-C/C alleles at the markers D12Mit141 and D12Nds2. Accordingly, the hhy-containing region was restricted to the 3-cM segment between D12Mit28 and D12Mit141. Subsequently, we analyzed 21 nonaffected recombinants. Numbers of animals with the C/C allele only at D12Nds2, at markers D12Mit141 and D12Nds2, at D12Mit28 and D12Mit53, and at D12Mit28 only were 1, 7, 9, and 4, respectively. Conversely, these nonaffected recombinants all had the C/M or M/M (non-C/C) genotype at D12Mit181. Hence, D12Mit181 was the marker the nearest to the hhy mutation. We further examined the haplotypes of 5 affected and 16 nonaffected animals, which had recombination between D12Mit181 and either of the markers next to D12Mit181 (ie, D12Mit53 or D12Mit141), using newly developed polymorphic microsatellite markers (Table 1) and a microsatellite marker MCA1. The results indicated that 16 nonaffected recombinants commonly had the non-C/C genotype in a 1-Mb region between MCA1 (108.4 Mb) and D12Opu2 (109.4 Mb), in which five affected recombinants had the C/C genotype (Figure 1B). In the 1-Mb region critical for the hhy mutation, 14 genes (Bcl11b, Setd3, Ccnk, Ccdc85c, Hipl1, Cyp46a1, Eml1, Evl, Degs2, Yy1, Slc25a29, AI132487, Wars, and Wdr25) are located. We examined the expression of the 14 genes situated in the 1-Mb region critical for the hhy mutation in the wild-type (BALB/c) and mutant brains using RT-PCR. These genes were all expressed in both wild-type and mutant brains. Among them, the mutant band for coiled-coil domain containing 85C (Ccdc85c) was small in size compared to the wild-type band (Figure 2A). The band patterns for other 13 genes showed no obvious difference between the wild-type and mutant samples. The Ccdc85c gene consists of 6 exons spanning a 69-kilobase (kb) genomic interval, encoding a 420 amino acid protein with unknown functions (NC_000078.5; NCBI). We amplified the mutant and wild-type Ccdc85c transcripts by RT-PCR using primers for a 518-base-pair sequence extending over exons 1–6 in the wild-type transcript and analyzed the sequences. A 74 base-pair sequence equivalent to exon 2 was absent in all (8 of 8) mutant clones (Figure 2, B and C), while present in the wild-type clone (Figure 2D). These mutant clones all contained an irregular exon 1–3 splicing at the normal splicing signal sequence in 3 clones (Figure 2B), or one frame inside the exon 3 sequence in 5 clones (Figure 2C). These irregular transcripts both result in frame shifts generating a premature stop codon (p.Asp266AlafsX74 and p.Asp266GlyfsX73). To search for genomic mutation, we conducted a series of long-range PCRs using primers for the sequences in intron 1 and 3 in Ccdc85c, and identified an 8.1-kb band from the hhy mutant, which was 1.7 kb shorter in size than the 9.8-kb wild-type counterpart (Figure 2E). The PCR products were digested by EcoRV, BamHI, XbaI, or XhoI. A digestion pattern and the restriction maps determined by the analysis are shown (Figure 2, F and G). Digestion with EcoRV or XhoI generated 4.05- or 5.7-kb mutant bands, respectively, each of which was 1.7 kb shorter than their wild-type counterparts (5.75 kb or 7.4 kb, respectively), suggesting that a deletion mutation occurred within the EcoRV–XhoI segment. Moreover, the mutant DNA remained uncut after the XbaI digestion, whereas the wild-type DNA was cut by XbaI at the expected site. Furthermore, in the lane for BamHI-digested mutant DNA, two similar-sized bands (approximately 3.3 and 3.1 kb) appeared instead of a large single band (expected size, 6.37 kb). These results indicated that the exon 2-containing EcoRV-XhoI region was rearranged in the mutant. By sequencing the 2.0-kb hhy fragment, we found that a 3.2-kb sequence extending over exon 2 and XbaI site was replaced by a 1.5-kb retrotransposon-like repeat sequence (RTLR) in the hhy mutant (g.52662–55846delinsRTLR; GenBank accession no. AB560874). A novel BamHI site was at the expected position in the insert. Altogether, Ccdc85c is genetically disrupted by a recombination involving exon 2 in the hhy mutant. Undergoing irregular exon 1–3 splicing, mutant Ccdc85c transcripts, although expressed, contained frame shifts starting at the exon 1–3 junction. Ccdc85C is a member of the DUF2216 superfamily with a conserved uncharacterized domain in the N-terminal half. Because the N-terminus of Ccdc85C showed a high sequence homology to Ccdc85A, a DUF superfamily member, we produced an anti-Ccdc85C antibody against a 172 (serine-247–glycine-418) amino acid sequence in the C-terminal half of the predicted 420-amino acid Ccdc85C protein. Using this antibody, we immunostained brain sections from the wild-type and hhy mice. The walls of the lateral and third ventricles were predominantly immunoreactive to Ccdc85C in the control at E18 (Figure 3A). The subventricular zone (SVZ) and the pial surface of the normal brain were modestly positive for Ccdc85C. In contrast to this, the E18 hhy homozygous brain showed scarcely any immunoreactivity, when stained by the antibody (Figure 3B). Thus, our Ccdc85C antibody reacts specifically with mouse Ccdc85C. The lateral ventricles wall of the normal brains at E15 (Figure 3C), P0 (Figure 3D), as well as P5 (Figure 3E) was positive for Ccdc85C, whereas the reactivity was drastically reduced at P5. Double-staining for Ccdc85C and zonula occludens-1 (ZO-1), an apical junction marker for radial glial cells in the developing brain,22Bauer H. Zweimueller-Mayer J. Steinbacher P. Lametschwandtner A. Bauer HC The dual role of zonula occludens (ZO) proteins.J Biomed Biotechnol Epub. 2010; Google Scholar, 23Fanning A.S. Anderson J.M. Zonula occludens-1 and -2 are cytosolic scaffolds that regulate the assembly of cellular junctions.Ann NY Acad Sci. 2009; 1165: 113-120Crossref PubMed Scopus (286) Google Scholar indicated that immunoreactivities to Ccdc85C and ZO-1 largely overlapped in the normal brain at E15 (Figure 3, F–H). Of note, developing blood vessels and the choroid plexus outlined by ZO-1 immunoreactivity were faintly positive for Ccdc85C in these panels. In micrographs of high magnification (Figure 3, I–K), there was considerable overlap of Ccdc85C immunoreactivity on the meshwork-like structure of adherens junctions visualized by immunostaining for ZO-1 on the lateral ventricles wall. hhy mutants show heterotopia in the subcortical area of the brain.20Kuwamura M. Kinoshita A. Okumoto M. Yamate J. Mori N. Hemorrhagic hydrocephalus (hhy): a novel mutation on mouse chromosome 12.Brain Res Dev Brain Res. 2004; 152: 69-72Crossref PubMed Scopus (10) Google Scholar To define the pathological phenotype of heterotopia in the hhy mice, we stained coronally and parasagittally sliced brain sections from hhy and control mice at postnatal ages from P0 to P7 by H&E. Heterotopia were distributed with a mediolateral span in the subcortical area that extended from parietal to occipital lobes of the hhy brain at P0 (Figure 4, A and B). The boxed area in Figure 4B was expanded in Figure 4C and compared to a normal equivalent (Figure 4D). The cortical layer of the hhy brain at P0 did not significantly differ from the control. It is of note that the cellularity of the germinal zone of the hhy brain was considerably poor. Subcortical accumulation of heterotopic cells and denudation of the ventricular wall surface became evident at P4 (Figure 4E). Ventricular dilatation, a diagnostic criterion for hydrocephalus, was pathologically appreciable as early as P0, and became evident at P4. The hhy cortex at P7 appeared degenerated under the pressure of accumulating CSF (Figure 4G). Between E13 and E16, cortical radial glial cells in the dorsal wall of the la
Referência(s)