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• W2149695265 abstract "The development of the mammalian brain is dependent on extensive neuronal migration. Mutations in mice and humans that affect neuronal migration result in abnormal lamination of brain structures with associated behavioral deficits. Here, we report the identification of a hyperactive N-ethyl-N-nitrosourea (ENU)-induced mouse mutant with abnormalities in the laminar architecture of the hippocampus and cortex, accompanied by impaired neuronal migration. We show that the causative mutation lies in the guanosine triphosphate (GTP) binding pocket of α-1 tubulin (Tuba1) and affects tubulin heterodimer formation. Phenotypic similarity with existing mouse models of lissencephaly led us to screen a cohort of patients with developmental brain anomalies. We identified two patients with de novo mutations in TUBA3, the human homolog of Tuba1. This study demonstrates the utility of ENU mutagenesis in the mouse as a means to discover the basis of human neurodevelopmental disorders. The development of the mammalian brain is dependent on extensive neuronal migration. Mutations in mice and humans that affect neuronal migration result in abnormal lamination of brain structures with associated behavioral deficits. Here, we report the identification of a hyperactive N-ethyl-N-nitrosourea (ENU)-induced mouse mutant with abnormalities in the laminar architecture of the hippocampus and cortex, accompanied by impaired neuronal migration. We show that the causative mutation lies in the guanosine triphosphate (GTP) binding pocket of α-1 tubulin (Tuba1) and affects tubulin heterodimer formation. Phenotypic similarity with existing mouse models of lissencephaly led us to screen a cohort of patients with developmental brain anomalies. We identified two patients with de novo mutations in TUBA3, the human homolog of Tuba1. This study demonstrates the utility of ENU mutagenesis in the mouse as a means to discover the basis of human neurodevelopmental disorders. IntroductionThe development of the mammalian brain depends on extensive neuronal migration resulting in the formation of a number of highly laminar structures, most notably the cortex and hippocampus. This process involves a large number of neurons that originate in the proliferative ventricular zones and migrate radially to their final locations past previously formed neurons (Gupta et al., 2002Gupta A. Tsai L.H. Wynshaw-Boris A. Life is a journey: a genetic look at neocortical development.Nat. Rev. Genet. 2002; 3: 342-355Crossref PubMed Scopus (305) Google Scholar). Genetic studies in mice and humans have identified some of the molecular determinants of neuronal migration: mutations in doublecortin (DCX) (des Portes et al., 1998des Portes V. Pinard J.M. Billuart P. Vinet M.C. Koulakoff A. Carrie A. Gelot A. Dupuis E. Motte J. Berwald-Netter Y. et al.A novel CNS gene required for neuronal migration and involved in X-linked subcortical laminar heterotopia and lissencephaly syndrome.Cell. 1998; 92: 51-61Abstract Full Text Full Text PDF PubMed Scopus (667) Google Scholar, Gleeson et al., 1998Gleeson J.G. Allen K.M. Fox J.W. Lamperti E.D. Berkovic 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 (872) Google Scholar) and LIS1 (Reiner et al., 1993Reiner O. Carrozzo R. Shen Y. Wehnert M. Faustinella F. Dobyns W.B. Caskey C.T. Ledbetter D.H. Isolation of a Miller-Dieker lissencephaly gene containing G protein beta-subunit-like repeats.Nature. 1993; 364: 717-721Crossref PubMed Scopus (894) Google Scholar) have been shown to impair migration and cause type 1 lissencephaly in humans, a disease characterized by a four-layered cortex with an absence or diminution of gyri and sulci (Dobyns and Truwit, 1995Dobyns W.B. Truwit C.L. Lissencephaly and other malformations of cortical development: 1995 update.Neuropediatrics. 1995; 26: 132-147Crossref PubMed Scopus (304) Google Scholar). In females, mutations in DCX can also cause subcortical laminar heterotopia (SCLH), in which an additional layer of misplaced heterotopic neurons is found in the white matter. Perturbations of the laminar architecture of the cortex also result from mutations in the very low density lipoprotein receptor (VLDLR) (Boycott et al., 2005Boycott K.M. Flavelle S. Bureau A. Glass H.C. Fujiwara T.M. Wirrell E. Davey K. Chudley A.E. Scott J.N. McLeod D.R. Parboosingh J.S. Homozygous deletion of the very low density lipoprotein receptor gene causes autosomal recessive cerebellar hypoplasia with cerebral gyral simplification.Am. J. Hum. Genet. 2005; 77: 477-483Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar) and the extracellular matrix protein Reelin (Hong et al., 2000Hong S.E. Shugart Y.Y. Huang D.T. Shahwan S.A. Grant P.E. Hourihane J.O. Martin N.D. Walsh C.A. Autosomal recessive lissencephaly with cerebellar hypoplasia is associated with human RELN mutations.Nat. Genet. 2000; 26: 93-96Crossref PubMed Scopus (706) Google Scholar). Together, mutations in DCX, LIS1, VLDLR, and reelin are responsible for ∼70% of the cases of type 1 lissencephaly. The pathogenic mutations responsible for the remaining cases are unknown (Leventer, 2005Leventer R.J. Genotype-phenotype correlation in lissencephaly and subcortical band heterotopia: the key questions answered.J. Child Neurol. 2005; 20: 307-312Crossref PubMed Scopus (29) Google Scholar).Impaired neuronal migration affects behavior as well as brain structure. Humans with lissencephaly are mentally retarded and frequently suffer from epilepsy (Francis et al., 2006Francis F. Meyer G. Fallet-Bianco C. Moreno S. Kappeler C. Socorro A.C. Tuy F.P. Beldjord C. Chelly J. Human disorders of cortical development: from past to present.Eur. J. Neurosci. 2006; 23: 877-893Crossref PubMed Scopus (117) Google Scholar). Similarly, the reeler mouse mutant and mice with loss of function mutations in Dcx and Lis1 exhibit varying degrees of cognitive dysfunction (Corbo et al., 2002Corbo J.C. Deuel T.A. Long J.M. LaPorte P. Tsai E. Wynshaw-Boris A. Walsh C.A. Doublecortin is required in mice for lamination of the hippocampus but not the neocortex.J. Neurosci. 2002; 22: 7548-7557Crossref PubMed Google Scholar, Paylor et al., 1999Paylor R. Hirotsune S. Gambello M.J. Yuva-Paylor L. Crawley J.N. Wynshaw-Boris A. Impaired learning and motor behavior in heterozygous Pafah1b1 (Lis1) mutant mice.Learn. Mem. 1999; 6: 521-537Crossref PubMed Scopus (81) Google Scholar, Qiu et al., 2005Qiu S. Korwek K.M. Pratt-Davis A.R. Peters M. Bergman M.Y. Weeber E.J. Cognitive disruption and altered hippocampus synaptic function in Reelin haploinsufficient mice.Neurobiol. Learn. Mem. 2005; 85: 228-242Crossref PubMed Scopus (166) Google Scholar). Mice with mutations that cause behavioral abnormalities are currently being identified as part of a large-scale mutagenesis screen in the UK (Nolan et al., 2000Nolan P.M. Peters J. Strivens M. Rogers D. Hagan J. Spurr N. Gray I.C. Vizor L. Brooker D. Whitehill E. et al.A systematic, genome-wide, phenotype-driven mutagenesis programme for gene function studies in the mouse.Nat. Genet. 2000; 25: 440-443Crossref PubMed Scopus (537) Google Scholar). In this dominant screen, N-ethyl-N-nitrosourea (ENU) is injected into the peritoneum of BALB/cAnN male mice and causes random mutations in spermatogonia. Mutagenized males are mated with C3H/HeH females and their offspring assessed for variation in locomotor activity. Here we describe a hyperactive ENU-induced mouse mutant with cortical and hippocampal abnormalities due to impaired neuronal migration. We show that mutations in the human homolog of this gene cause cortical brain malformations, including lissencephaly.ResultsIdentification and Mapping of a Novel Behavioral MutantWe screened 9216 mice from the Harwell ENU mutagenesis program. The total distance each mouse travelled in 35 min was assessed. Mice with levels of activity more than three standard deviations outside the mean were retested 7 days later (n = 87) (Figure 1A). We excluded mice with abnormal vestibular, skeletal, or neuromuscular phenotypes that might also influence locomotor behavior. Ten outliers were subject to heritability testing by backcrossing to C3H/HeH. A single line that we named Jenna (Jna) was identified with a semidominant hyperactive phenotype.We mapped variation in locomotor activity to the distal end of chromosome 15 by treating the phenotype quantitatively and applying standard methods for quantitative trait locus (QTL) mapping (Zeng, 1994Zeng Z.B. Precision mapping of quantitative trait loci.Genetics. 1994; 136: 1457-1468PubMed Google Scholar). To generate sufficient animals to map the mutation, we employed in vitro fertilization, raising a total of 89 animals on a C3H/HeH background. We attempted to intercross hyperactive animals but failed to generate any pregnancies.We identified a QTL on chromosome 15 with a logP (negative logarithm of the P value, base 10) of 12.6. This was the only locus that exceeded the genome-wide 5% significance threshold of logP 4.1 (Churchill and Doerge, 1994Churchill G.A. Doerge R.W. Empirical threshold values for quantitative trait mapping.Genetics. 1994; 138: 963-971Crossref PubMed Google Scholar). No other loci were detected with a logP greater than 2.0. The presence of a relatively large QTL segregating on chromosome 15 suggested that we had mapped the causative mutation (Figure 1B), but we could not exclude the possibility that the genetic effect arose from a naturally occurring polymorphism between BALB/cAnN and C3H/HeH.We reasoned that the presence of pleiotropy would distinguish the effect of the ENU-induced mutation from a naturally occurring polymorphism. Unlike the latter, engineered and induced mutations with an effect on behavior frequently display additional phenotypic abnormalities. We found that mice from the mapping population could be divided into two groups by variation in weight and activity, the smaller mice exhibiting higher levels of activity (Figure 1C). We then fine-mapped the mutation to a 1.3 Mb region between D15Mit43 and a single nucleotide polymorphism rs32344030 (Figure 1D). This region contains 41 annotated genes (http://www.ensembl.org).The coding and exon-flanking sequences of all 41 genes was sequenced in affected animals (n = 4), a wild-type control, and the two mapping strains, C3H/HeH and BALB/cAnN. All the variants identified were present in the two mapping strains, except for a T to C transition in exon 4 of α-1 tubulin (Tuba1) (Figure 1E). We sequenced exon 4 of α-1 tubulin from the inbred strains AKR/J, A/J, C57BL6/J, RIII, DBA/2J, IS, FVB, and 129 and did not observe this polymorphism.We extracted whole brain mRNA from Jna/+ animals, sequenced the cDNA, and confirmed the presence of the mutation in the brain Tuba1 transcript. The mutation causes an amino acid change from serine to glycine at residue 140 (S140G). This amino acid is located in the T4 loop of Tuba1, forming part of the N-site, a highly conserved glycine-rich motif that binds GTP (Lowe et al., 2001Lowe J. Li H. Downing K.H. Nogales E. Refined structure of alpha beta-tubulin at 3.5 A resolution.J. Mol. Biol. 2001; 313: 1045-1057Crossref PubMed Scopus (982) Google Scholar) (Figures 1F and 7B). GTP bound at the N site does not undergo hydrolysis and is thought to act as a structural cofactor stabilizing the α/β heterodimer (Spiegelman et al., 1977Spiegelman B.M. Penningroth S.M. Kirschner M.W. Turnover of tubulin and the N site GTP in Chinese hamster ovary cells.Cell. 1977; 12: 587-600Abstract Full Text PDF PubMed Scopus (93) Google Scholar).The S140G Mutation Reduces GTP Binding and Native Heterodimer FormationWe hypothesized that the S140G mutation might affect the ability of Tuba1 to bind GTP at the N site. We therefore conducted in vitro folding assays containing α-32P-GTP. Equal amounts of urea-unfolded wild-type or mutant Tuba1 probes, quantitated by Coomassie blue staining, were introduced by sudden dilution into a buffer containing ATP, radio-labeled GTP, and cytosolic chaperonin (CCT). CCT is the first chaperone required in a cascade of interacting proteins that act in concert to facilitate the formation of native tubulin heterodimers (Figure 2A) (Lewis et al., 1996Lewis S.A. Tian G. Vainberg I.E. Cowan N.J. Chaperonin-mediated folding of actin and tubulin.J. Cell Biol. 1996; 132: 1-4Crossref PubMed Scopus (98) Google Scholar). CCT undergoes multiple rounds of ATP-dependent interaction with unfolded α-tubulin polypeptides, forming chaperonin bound quasi-native tubulin folding intermediates that contain a GTP binding pocket (Tian et al., 1995Tian G. Vainberg I.E. Tap W.D. Lewis S.A. Cowan N.J. Quasi-native chaperonin-bound intermediates in facilitated protein folding.J. Biol. Chem. 1995; 270: 23910-23913Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar).Figure 2Tuba1 S140G Has a Reduced Ability to Incorporate GTP, Resulting in a Marked Decline in the Efficiency of Tubulin Heterodimer FormationShow full caption(A) The tubulin heterodimer assembly pathway. Newly synthesized α/β-tubulin polypeptides undergo one or more rounds of ATP-dependent interaction with cytosolic chaperonin (CCT). Quasi-native intermediates formed as a result of this interaction are then captured and stabilized by a series of tubulin-specific chaperones termed TBCA–TBCE. α-tubulin intermediates interact with TBCB and TBCE, while β-tubulin intermediates interact with TBCA and TBCD. TBCEα (Eα) and TBCDβ (Dβ) together form a supercomplex. Entry of TBCC into this supercomplex triggers GTP hydrolysis by β-tubulin; this acts as a switch for the discharge of the newly assembled α/β heterodimer. This is based on Tian et al., 2006Tian G. Huang M.C. Parvari R. Diaz G.A. Cowan N.J. Cryptic out-of-frame translational initiation of TBCE rescues tubulin formation in compound heterozygous HRD.Proc. Natl. Acad. Sci. USA. 2006; 103: 13491-13496Crossref PubMed Scopus (20) Google Scholar.(B) Incorporation of labeled GTP into CCT bound folding intermediates. Reaction products, analyzed by nondenaturing gel electrophoresis, show an approximately 5-fold reduction in GTP bound intermediates (CCT/α) in the case of the S140G mutant.(C) Incorporation of labeled GTP in fully reconstituted in vitro tubulin-folding reactions containing CCT, ATP, GTP, TBCB, TBCC, TBCD, and TBCE; native brain tubulin; and equal amounts of wild-type or S140G mutant α-tubulin. Products analyzed by nondenaturing gel electrophoresis show a reduction in the level of native α/β-tubulin heterodimers.(D) Incorporation of 35S-labeled wild-type or mutant (S140G) Tuba1 in fully reconstituted in vitro reactions confirm a reduction in the production of native α/β-tubulin heterodimers in the case of the S140G mutant.(E and F) In vitro transcription and translation in rabbit reticulocyte lysate in the presence of 35S-methionine. Products analyzed by SDS (E) or nondenaturing gel electrophoresis (F) demonstrate that the mutation does not affect translation of the protein (E) in contrast to the diminished yield of heterodimers in (F). In (E) molecular mass markers (in kDa) are shown on the left.(G) Microtubule polymerization/depolymerization. Cycling of 35S-labeled, in vitro translated wild-type and mutant (S140G) tubulin with native bovine brain microtubules demonstrates that wild-type and mutant heterodimers are equally capable of incorporation into microtubules.(H) The S140G mutant α-1 tubulin incorporates into microtubules in vivo. C-terminally FLAG-tagged wild-type or mutant Tuba1 were transfected into HeLa cells and the microtubule network visualized by staining with a polyclonal anti-β-tubulin antibody (shown in green) and a monoclonal anti-FLAG antibody (shown in red). Calibration bar shows 10 μm. Arrows in panels (D), (E), (F), and G denote the location of the α-tubulin/CCT binary complex (CCT/α) or the native tubulin heterodimer (α/β).View Large Image Figure ViewerDownload Hi-res image Download (PPT)We found that the S140G mutation decreased the ability of CCT bound quasi-native α-tubulin folding intermediates to incorporate GTP by approximately 5-fold (Figure 2B). We then assessed what effect this would have on native tubulin heterodimer formation. To do this, we repeated the assay, including the additional components required for de novo heterodimer formation. We found that the yield of labeled heterodimers was significantly less in reactions carried out with the mutant protein compared to the wild-type protein (Figure 2C).To show that the reduced incorporation of GTP in mutant heterodimers is reflected in a reduced yield of native dimerized protein, we repeated our in vitro folding experiments using mutant and wild-type unfolded 35S-methionine labeled Tuba1 probes. Consistent with the data obtained in our GTP labeling experiments, we found that the S140G mutation reduced the efficiency of de novo heterodimer formation under conditions where equal amounts of 35S-labeled protein were analyzed (Figure 2D). These data are not artifacts of in vitro reconstitution, because we observed the same reduced yield of tubulin heterodimers in parallel experiments in which the identical cloned sequences used for the generation of probes for in vitro folding experiments were translated in a cell free system from rabbit reticulocytes (Figures 2E and 2F). We then investigated whether the heterodimers produced in these in vitro experiments were able to polymerize. The products of the reactions shown in Figure 2D were mixed with depolymerized brain microtubules and taken through two successive cycles of polymerization/depolymerization. At the end of each cycle, an aliquot of the depolymerized material was removed and analyzed. While heterodimer formation was reduced in all our assays carried out with the S140G mutant, the ability of mutant heterodimers to cocycle with native tubulin in vitro was not affected (Figure 2G).Consistent with these data we found that a FLAG-tagged mutant Tuba1 incorporated into the normal interphase microtubule network upon overexperession in cultured cells (Figure 2H) and that these microtubules behaved in the same manner as their wild-type counterparts in a regrowth assay following depolymerization with nocodazole (Figure S1). These experiments demonstrate the ability of the S140G tubulin, once folded, to assemble into heterodimers that can copolymerize into dynamic microtubules in vivo. We conclude that the S140G mutation results in a diminished efficiency of polymerization-competent tubulin heterodimer formation as a result of a compromised GTP binding pocket. Our data indicate that the S140G substitution is a partial loss-of-function mutation, suggesting that the phenotype in Jna/+ mice is due to haploinsufficiency.Genetic Rescue of the Hyperactive Phenotype in Jna/+ MiceWe tested whether delivery of additional copies of α-1 tubulin by BAC transgenesis would rescue the phenotype in Jna/+ mice. We selected a BAC clone (RP23-3124H4) that contained the Tuba1 gene for this experiment. Purified BAC DNA was injected into the pronucleus of C3H/HeH embryos, and these embryos transferred into recipient females. Four transgenic lines were identified that carried the BAC (H2, H8, H17, and H41). We established germline transmission in two of these (H8 and H41). Quantitative PCR analysis of the transgene indicated that four copies of the clone had been incorporated into the H41 line and a single copy into the H8 line (data not shown). Behavioral analysis of the H41 line revealed no significant differences in measures of activity with control animals (F[1,16] < 1; P > 0.5), demonstrating that delivery of additional copies of Tuba1, by itself, has no effect on locomotor behavior (Figure 5A). The H41 line was selected for rescue and was crossed with Jna/+ mice. There was a significant reduction in the locomotor behavior of transgenic animals with the S140G mutation (Jna/+/BAC) in comparison to nontransgenic mutants (F[1,14] = 18.7; P < 0.005) and no significant difference from controls (F[1,16] = 1.2; P > 0.1) (Figure 5A).The S140G Mutation Results in Abnormal Hippocampal and Cortical Morphology in Jna/+ MiceWe undertook a complete histological examination of Jna/+ mice (n = 4). Haematoxylin and eosin staining revealed no gross anatomical defects in somatic organs or tissues (data not shown). We focused on the neuroanatomical features of the Jna/+ mice. Brains from Jna/+ mice were smaller than wild-type (∼85%), but this is probably because mutant animals weigh ∼30% less than littermate controls. Examination of coronal sections of Jna/+ mice (n = 6), littermate controls (n = 6), and rescued brains (n = 6) from animals aged 8 weeks revealed abnormal lamination of pyramidal cells in the hippocampus of mutant animals (Figures 3A–3I). Staining with cresyl violet and with the neuronal marker NeuN showed hippocampal disorganization with an additional layer of pyramidal cells in the stratum oriens that extended throughout the pyramidal cell subfields into the subiculum, which was most marked in the CA3 region where the neurons were loosely packed. The abnormality was also apparent in mutant animals aged 8 months (n = 3) (data not shown). Staining with calbindin revealed fewer calbindin-positive pyramidal neurons in the CA1 region and a disorganized mossy fiber tract extending from the dentate gyrus to CA3. No gross morphological abnormalities were apparent in the dentate gyrus of Jna/+ mice, the hilus appearing intact. There were no detectable abnormalities in the hippocampal morphology of Jna/+/BAC mice (Figures 3C, 3F, and 3I).Figure 3Abnormal Hippocampal and Cortical Morphology in Jna/+ MiceShow full caption(A–I) Coronal sections of the hippocampus from littermate controls, heterozygote (Jna/+) and rescued (Jna/+/BAC) animals aged 8 weeks when stained with cresyl violet (A–C), NeuN (D–F), and calbindin (G–I). These stains reveal a fractured pyramidal cell layer (shown with an arrow) that is most severe in the CA3, fewer calbindin-positive pyramidal neurons in the CA1 region, and a disorganized mossy fiber tract (arrowed).(J–ZD) Coronal sections of the visual cortex when stained with anti-sera for NeuN (J–L, Y–ZA), calbindin (M–O), Cux-1 (P–R, ZB–ZD), Er81 (S–U), and FOXP2 (V–X) from mice aged 8 weeks. These stains showed that the laminar structure of the cortex is preserved; however, on closer examination of NeuN (Y–ZA) and Cux-1 (ZB–ZD) staining, wave-like perturbations in layers II/III and IV (arrowed) can be seen. The calibration bar for the hippocampus shows 500 μm. The calibration bars for the cortex show 200 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We looked for laminar abnormalities in the cortex, staining with antisera for NeuN, Cux-1 (a layer II/III/IV marker), calbindin (a layer II/III and V marker), Er81 (a layer V marker), FOXP2 (a layer VI marker) (Figure 3 J-X), Brn-1 (expressed predominantly in late cortical plate neurons, Figure S4), and cresyl violet (Figure S2). These experiments showed that the laminar cytoarchitecture of the cortex was intact in Jna/+ mice, but closer examination of NeuN, Cux-1, and Nissl stains revealed wave-like perturbations in layer IV (Figure 3, Y-ZA and ZB-ZD; Figure S2). While layer IV still consisted of the characteristic granular cells that were of similar size and shape to those observed in littermate controls, they were organized into three to four cellular fronts, each containing a single row of cells. This perturbation was evident in the visual, auditory, and somatosensory cortices but was not observed in the motor or retrosplenial cortices (Figure S2). It was observed primarily in layer IV but extended into layers II/III in the posterior and dorsal medial regions of the cortex as revealed by Cux-1 staining. Jna/+/BAC mice were indistinguishable from wild-type mice for all stains (Figure 3, L, O, R, U, and X). Analysis of Golgi- stained sections revealed no significant differences in the percentage of misorientated (θ > 8°) or inverted (θ > 90°) apical dendrites in the visual, somatosensory, or motor cortices between littermate controls (n = 3) and Jna/+ mice (n = 3) (Demyanenko et al., 2004Demyanenko G.P. Schachner M. Anton E. Schmid R. Feng G. Sanes J. Maness P.F. Close homolog of L1 modulates area-specific neuronal positioning and dendrite orientation in the cerebral cortex.Neuron. 2004; 44: 423-437Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) (Figure S3). No anatomical abnormalities were seen in the cerebellum or amygdala (Figure S4).Defective Neuronal Migration in Jna/+ MiceWe considered whether abnormal radial migration might be responsible for the unusual hippocampal and cortical architecture observed in the Jna/+ mice. We injected pregnant females with BrdU at three time points (E12.5, E14.5, and E16.5) and counted labeled cells at the day of birth (P0), dividing the cortex into ten equal bins extending from the intermediate zone to the cortical surface (Figures 4A–4C). We tested for an interaction between genotype and the mean percentage of cells in each bin. There was no significant difference between littermate controls and Jna/+ mutants when BrdU was injected at E12.5 (F[9,189] < 1; P > 0.05), however there was a highly significant difference when injected at E14.5 (F[9,198] = 4.75; P < 0.0001), and at E16.5 (F[9,270] = 13.3; P < 0.0001). At these time points, a higher percentage of BrdU-positive cells are observed in bins 1, 2, and 3 in wild-type littermates. These results are consistent with our anatomical observations that showed perturbations in layers II/III and IV of the cortex, as the majority of cells that populate these layers are born between E14.5 and E16.5 (Takahashi et al., 1999Takahashi T. Goto T. Miyama S. Nowakowski R.S. Caviness Jr., V.S. Sequence of neuron origin and neocortical laminar fate: relation to cell cycle of origin in the developing murine cerebral wall.J. Neurosci. 1999; 19: 10357-10371Crossref PubMed Google Scholar).Figure 4Abnormal Neuronal Migration in Jna/+ MiceShow full caption(A–I) Staining for BrdU in the cortex and hippocampus of littermate controls and mutant pups harvested at P0, after injection of BrdU at E12.5 (A and B), E14.5 (D and E) and E16.5 (G and H). The cortex was divided into ten equal bins (shown on the left), extending from the intermediate zone to the molecular layer and BrdU-positive cells counted blind to the genotype. Panels (C), (F), and (I) show the mean percentage of BrdU-labeled cells in bins 1 to 10 for wild-type littermates (white) and Jna/+ mutants (gray). Error bars show the SEM. Test of the interaction between genotype and the distribution of cells across bins were as follows: E12.5 (F[9,189] < 1; P > 0.05); E14.5 (F[9,198] = 4.75; P < 0.0001), and E16.5 (F[9,270] = 13.3; P < 0.0001).(J and K) BrdU-positive cells in the hippocampus of a littermate control (J) and Jna/+ mouse (K) following injection of BrdU at E14.5. Jna/+ mice show disorganization of BrdU-positive cells in Ammon's horn, affecting both CA1 and CA3 regions (arrowed). Scale bar shows 200 μm (J and K).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Examination of hippocampal sections following injection of BrdU at E14.5, a time at which most pyramidal neurons are born (Angevine, 1965Angevine Jr., J.B. Time of neuron origin in the hippocampal region. An autoradiographic study in the mouse.Exp. Neurol. 1965; : 1-70Google Scholar), revealed disorganization in Ammon's horn affecting both CA1 and CA3 regions (Figures 4D and 4E). Several layers of BrdU-positive cells could be seen, lacking structure when compared to wild-type littermates. The dentate gyrus, unlike the phenotype in the adult, also appeared chaotic, suggesting a delay in its development. This result, together with our analysis of BrdU staining in the cortex, is consistent with impaired radial migration in Jna/+ mice.Jna/+ Mice Show Impaired Spatial Working Memory, Reduced Anxiety, and Abnormal Nesting Consistent with a Hippocampal DeficitGiven the lamination defect in the hippocampus, we tested whether hippocampal-dependent behaviors are altered in Jna/+ mice (Deacon et al., 2002Deacon R.M. Croucher A. Rawlins J.N. Hippocampal cytotoxic lesion effects on species-typical behaviours in mice.Behav. Brain Res. 2002; 132: 203-213Crossref PubMed Scopus (229) Google Scholar). A number of behaviors are known that require an intact hippocampus. Effects on memory are well documented, but lesions also result in increased activity, reduced anxiety, and disruption of a number of species-typical behaviors (Deacon et al., 2002Deacon R.M. Croucher A. Rawlins J.N. Hippocampal cytotoxic lesion effects on species-typical behaviours in mice.Behav. Brain Res. 2002; 132: 203-213Crossref PubMed Scopus (229) Google Scholar). We were unable to transfer the S140G mutation into a C57BL/6 background for behavioral testing, so all phenotyping was conducted on a C3H/HeH background.We assessed the Jna/+ mice for spontaneous alternation in a T-maze, a working memory task that does not require vision (Buhot et al., 2001Buhot M.C. Dubayle D. Malleret G. Javerzat S. Segu L. Exploration, anxiety, and spatial memory in transgenic anophthalmic mice.Behav. Neurosci. 2001; 115: 455-467Crossref" @default.
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• W2149695265 title "Mutations in α-Tubulin Cause Abnormal Neuronal Migration in Mice and Lissencephaly in Humans" @default.
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