Lis1 Is Necessary for Normal Non-Radial Migration of Inhibitory Interneurons
2004; Elsevier BV; Volume: 165; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)63340-8
ISSN1525-2191
AutoresMatthew F. McManus, Ilya M. Nasrallah, Maclean M. Pancoast, Anthony Wynshaw‐Boris, Jeffrey A. Golden,
Tópico(s)Microtubule and mitosis dynamics
ResumoType I lissencephaly is a central nervous system (CNS) malformation characterized by mental retardation and epilepsy. These clinical features suggest a deficit in inhibitory neurons may, in part, underlie the pathogenesis of this disorder. Mutations in, or deletions of, LIS1 are the most commonly recognized genetic anomaly associated with type I lissencephaly. The pathogenesis of type I lissencephaly is believed to be a defect in radial neuronal migration, a process requiring LIS1. In contrast the inhibitory neurons migrate non-radially from the basal forebrain to the neocortex and hippocampus. Given that Lis1 is expressed in all neurons, we hypothesized that Lis1 also functions in non-radial migrating inhibitory neurons. To test this hypothesis we used a combination of in vivo and in vitro studies with Lis1 mutant mice and found non-radial cell migration is also affected. Our data indicate Lis1 is required for normal non-radial neural migration and that the Lis1 requirement is primarily cell autonomous, although a small cell non-autonomous effect could not be excluded. These data indicate inhibitory neuron migration is slowed but not absent, similar to that found for radial cell migration. We propose that the defect in non-radial cell migration is likely to contribute to the clinical phenotype observed in individuals with a LIS1 mutation. Type I lissencephaly is a central nervous system (CNS) malformation characterized by mental retardation and epilepsy. These clinical features suggest a deficit in inhibitory neurons may, in part, underlie the pathogenesis of this disorder. Mutations in, or deletions of, LIS1 are the most commonly recognized genetic anomaly associated with type I lissencephaly. The pathogenesis of type I lissencephaly is believed to be a defect in radial neuronal migration, a process requiring LIS1. In contrast the inhibitory neurons migrate non-radially from the basal forebrain to the neocortex and hippocampus. Given that Lis1 is expressed in all neurons, we hypothesized that Lis1 also functions in non-radial migrating inhibitory neurons. To test this hypothesis we used a combination of in vivo and in vitro studies with Lis1 mutant mice and found non-radial cell migration is also affected. Our data indicate Lis1 is required for normal non-radial neural migration and that the Lis1 requirement is primarily cell autonomous, although a small cell non-autonomous effect could not be excluded. These data indicate inhibitory neuron migration is slowed but not absent, similar to that found for radial cell migration. We propose that the defect in non-radial cell migration is likely to contribute to the clinical phenotype observed in individuals with a LIS1 mutation. Lissencephaly is a central nervous system (CNS) malformation characterized by the loss of normal gyri and sulci on the surface of the brain and a thickened and disorganized cerebral cortex. Patients with this malformation exhibit variable degrees of mental retardation and epilepsy, along with other neurological signs.1Walsh CA Genetic malformations of the human cerebral cortex.Neuron. 1999; 23: 19-29Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar Lissencephaly has traditionally been separated into two types, type I and type II, or classical and cobblestone, respectively. Only type I lissencephalies have been clearly linked to a deficit in cell migration, in contrast, type II lissencephalies are primarily an over-migration defect. To date, mutations in four genes have been causally associated with type I lissencephaly. The first identified was LIS1.2Hattori M Adachi H Tsujimoto M Arai H Inoue K Miller-Dieker lissencephaly gene encodes a subunit of brain platelet-activating factor acetylhydrolase.Nature. 1994; 370: 216-218Crossref PubMed Scopus (448) Google Scholar Heterozygous deletions of 17p13.3, containing the LIS1 gene, or point mutations therein can lead to either isolated lissencephaly sequence (ILS) or the Miller-Dieker syndrome (MDS), MDS having a more severe phenotype than ILS and is likely the result of a contiguous gene deletion syndrome involving the linked gene 14–3-3ε.3Cardoso C Leventer RJ Ward HL Toyo-Oka K Chung J Gross A Martin CL Allanson J Pilz DT Olney AH Mutchinick OM Hirotsune S Wynshaw-Boris A Dobyns WB Ledbetter DH Refinement of a 400-kb critical region allows genotypic differentiation between isolated lissencephaly, Miller-Dieker syndrome, and other phenotypes secondary to deletions of 17p13.3.Am J Hum Genet. 2003; 72: 918-930Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 4Dobyns WB Lissencephaly Overview.www.geneclinics.orgDate: 2000Google Scholar, 5Reiner O Carrozzo R Shen Y Wehnert M Faustinella F Dobyns WB Caskey CT Ledbetter DH Isolation of a Miller-Dieker lissencephaly gene containing G protein beta-subunit-like repeats.Nature. 1993; 364: 717-721Crossref PubMed Scopus (883) Google Scholar Mutations in doublecortin (DCX)6des Portes V Pinard JM Billuart P Vinet MC Koulakoff A Carrie A Gelot A Dupuis E Motte J Berwald-Netter Y Catala M Kahn A Beldjord C Chelly J 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 (658) Google Scholar, 7Gleeson JG Allen KM Fox JW Lamperti ED Berkovic S Scheffer I Cooper EC Dobyns WB Minnerath SR Ross ME Walsh CA 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 (862) Google Scholar and ARX,8Kitamura K Yanazawa M Sugiyama N Miura H Iizuka-Kogo A Kusaka M Omichi K Suzuki R Kato-Fukui Y Kamiirisa K Matsuo M Kamijo S Kasahara M Yoshioka H Ogata T Fukuda T Kondo I Kato M Dobyns WB Yokoyama M Morohashi K Mutation of ARX causes abnormal development of forebrain and testes in mice and X-linked lissencephaly with abnormal genitalia in humans.Nat Genet. 2002; 32: 359-369Crossref PubMed Scopus (569) Google Scholar both X-linked genes, also lead to type I lissencephaly in males and distinct malformations in females. Finally, mutations in REELIN have been linked to AR lissencephaly.9Hong SE Shugart YY Huang DT Al Shahwan S Grant PE Hourihane JOB Martin NDT Walsh CA Autosomal recessive lissencephaly with cerebellar hypoplasia (LCH) is associated with human reelin gene mutations.Nat Genet. 2000; 26: 93-96Crossref PubMed Scopus (694) Google Scholar Data from patients and animal models indicate that each of these mutations is associated with at least a radial cell migration defect. LIS1 mutations are the most commonly identified genetic basis for lissencephaly. The LIS1 gene product is a 45-kd, ubiquitously expressed protein that is found in a particularly high concentration in neurons.10Hirotsune S Fleck MW Gambello MJ Bix GJ Chen A Clark GD Ledbetter DH McBain CJ Wynshaw-Boris A Graded reduction of Pafah1b1 (Lis1) activity results in neuronal migration defects and early embryonic lethality.Nat Genet. 1998; 19: 333-339Crossref PubMed Scopus (482) Google Scholar, 11Smith DS Niethammer M Ayala R Zhou Y Gambello MJ Wynshaw-Boris A Tsai LH Regulation of cytoplasmic dynein behaviour and microtubule organization by mammalian Lis1.Nat Cell Biol. 2000; 2: 767-775Crossref PubMed Scopus (328) Google Scholar LIS1 is the non-catalytic subunit of platelet-activating factor acetyl hydrolase (PAFAH) 1B.2Hattori M Adachi H Tsujimoto M Arai H Inoue K Miller-Dieker lissencephaly gene encodes a subunit of brain platelet-activating factor acetylhydrolase.Nature. 1994; 370: 216-218Crossref PubMed Scopus (448) Google Scholar Homologues of LIS1 are known to exist in fungi,12Xiang X Osmani AH Osmani SA Xin M Morris NR NudF, a nuclear migration gene in Aspergillus nidulans, is similar to the human LIS-1 gene required for neuronal migration.Mol Biol Cell. 1995; 6: 297-310Crossref PubMed Scopus (288) Google Scholar yeast,13Geiser JR Schott EJ Kingsbury TJ Cole NB Totis LJ Bhattacharyya G He L Hoyt MA Saccharomyces cerevisiae genes required in the absence of the CIN8-encoded spindle motor act in functionally diverse mitotic pathways.Mol Biol Cell. 1997; 8: 1035-1050Crossref PubMed Scopus (174) Google Scholar Drosophila,14Liu Z Steward R Luo L Drosophila Lis1 is required for neuroblast proliferation, dendritic elaboration, and axonal transport.Nat Cell Biol. 2000; 2: 776-783Crossref PubMed Scopus (179) Google Scholar and mice10Hirotsune S Fleck MW Gambello MJ Bix GJ Chen A Clark GD Ledbetter DH McBain CJ Wynshaw-Boris A Graded reduction of Pafah1b1 (Lis1) activity results in neuronal migration defects and early embryonic lethality.Nat Genet. 1998; 19: 333-339Crossref PubMed Scopus (482) Google Scholar, 15Peterfy M Hozier JC Hall B Gyuris T Peterfy K Takecs L Localization of the mouse lissencephaly-1 gene to mouse chromosome 11B3, in close proximity to D11Mit65.Somat Cell Mol Genet. 1995; 21: 345-349Crossref PubMed Scopus (4) Google Scholar and function to stabilize cytoplasmic dynein and microtubules.11Smith DS Niethammer M Ayala R Zhou Y Gambello MJ Wynshaw-Boris A Tsai LH Regulation of cytoplasmic dynein behaviour and microtubule organization by mammalian Lis1.Nat Cell Biol. 2000; 2: 767-775Crossref PubMed Scopus (328) Google Scholar, 16Faulkner NE Dujardin DL Tai CY Vaughan KT O'Connell CB Wang Y Vallee RB A role for the lissencephaly gene LIS1 in mitosis and cytoplasmic dynein function.Nat Cell Biol. 2000; 2: 784-791Crossref PubMed Scopus (370) Google Scholar, 17Sapir T Elbaum M Reiner O Reduction of microtubule catastrophe events by LIS1, platelet-activating factor acetylhydrolase subunit.EMBO J. 1997; 16: 6977-6984Crossref PubMed Scopus (260) Google Scholar This results in Lis1 or its homologues playing an important role in cytoplasmic dynamics important for cell division and movement. In yeast, the LIS1 homologous protein PAC1 (33%) is required for segregation of chromosomes during mitosis and for nuclear orientation.13Geiser JR Schott EJ Kingsbury TJ Cole NB Totis LJ Bhattacharyya G He L Hoyt MA Saccharomyces cerevisiae genes required in the absence of the CIN8-encoded spindle motor act in functionally diverse mitotic pathways.Mol Biol Cell. 1997; 8: 1035-1050Crossref PubMed Scopus (174) Google Scholar Similarly, in Aspergillus nidulans, NUDF, which is 42% identical to LIS1, is necessary for the distribution and migration of nuclei.18Morris NR Efimov VP Xiang X Nuclear migration, nucleokinesis, and lissencephaly.Trends Cell Biol. 1998; 8: 467-470Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar Drosophila Lis1, dLis1, is 70% identical to LIS1 and is necessary for the normal development of egg chambers and for germline cell division.14Liu Z Steward R Luo L Drosophila Lis1 is required for neuroblast proliferation, dendritic elaboration, and axonal transport.Nat Cell Biol. 2000; 2: 776-783Crossref PubMed Scopus (179) Google Scholar In mice, LIS1 is localized to the centrosome19Feng Y Olson EC Stukenberg PT Flanagan LA Kirschner MW Walsh CA LIS1 regulates CNS lamination by interacting with mNudE, a central component of the centrosome.Neuron. 2000; 28: 665-679Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar and is involved in interkinetic nuclear migration, neuroblast proliferation, and programmed cell death of cortical ventricular zone neuroblasts.20Cahana A Escamez T Nowakowski RS Hayes NL Giacobini M von Holst A Shmueli O Sapir T McConnell SK Wurst W Martinez S Reiner O Targeted mutagenesis of Lis1 disrupts cortical development and LIS1 homodimerization.Proc Natl Acad Sci USA. 2001; 98: 6429-6434Crossref PubMed Scopus (130) Google Scholar, 21Gambello MJ Darling DL Yingling J Tanaka T Gleeson JG Wynshaw-Boris A Multiple dose-dependent effects of Lis1 on cerebral cortical development.J Neurosci. 2003; 23: 1719-1729Crossref PubMed Google Scholar Additionally, mice heterozygous for a null allele of Lis1 exhibit abnormal cerebral cortical, hippocampal, cerebellar, and olfactory bulb development, as well as impaired radial neuronal migration and hippocampal electrophysiological abnormalities.10Hirotsune S Fleck MW Gambello MJ Bix GJ Chen A Clark GD Ledbetter DH McBain CJ Wynshaw-Boris A Graded reduction of Pafah1b1 (Lis1) activity results in neuronal migration defects and early embryonic lethality.Nat Genet. 1998; 19: 333-339Crossref PubMed Scopus (482) Google Scholar, 21Gambello MJ Darling DL Yingling J Tanaka T Gleeson JG Wynshaw-Boris A Multiple dose-dependent effects of Lis1 on cerebral cortical development.J Neurosci. 2003; 23: 1719-1729Crossref PubMed Google Scholar, 22Fleck MW Hirotsune S Gambello MJ Phillips-Tansey E Suares G Mervis RF Wynshaw-Boris A McBain CJ Hippocampal abnormalities and enhanced excitability in a murine model of human lissencephaly.J Neurosci. 2000; 20: 2439-2450PubMed Google Scholar In vitro experiments have shown that cerebellar granule cells containing only one functional copy of Lis1 exhibit cell autonomous migration defects.10Hirotsune S Fleck MW Gambello MJ Bix GJ Chen A Clark GD Ledbetter DH McBain CJ Wynshaw-Boris A Graded reduction of Pafah1b1 (Lis1) activity results in neuronal migration defects and early embryonic lethality.Nat Genet. 1998; 19: 333-339Crossref PubMed Scopus (482) Google Scholar, 21Gambello MJ Darling DL Yingling J Tanaka T Gleeson JG Wynshaw-Boris A Multiple dose-dependent effects of Lis1 on cerebral cortical development.J Neurosci. 2003; 23: 1719-1729Crossref PubMed Google Scholar Radial migration from the ventricular zone out to the surface of the developing brain, perhaps the best-characterized migratory pathway for neurons,23Rakic P Principles of neural cell migration.Experientia. 1990; 46: 882-891Crossref PubMed Scopus (568) Google Scholar, 24Cajal R: Histologie du Systeme Nerveux de l'Homme et des Vertebres. 1952 MadridGoogle Scholar is deficient in Lis1 heterozygous mice.10Hirotsune S Fleck MW Gambello MJ Bix GJ Chen A Clark GD Ledbetter DH McBain CJ Wynshaw-Boris A Graded reduction of Pafah1b1 (Lis1) activity results in neuronal migration defects and early embryonic lethality.Nat Genet. 1998; 19: 333-339Crossref PubMed Scopus (482) Google Scholar A second pathway of migration, perpendicular to radial migration, has more recently been identified. This tangential or, more accurately, non-radial cell migration (NRCM) pathway has been described at nearly all levels of the developing nervous system,25Cepko C Golden J Szele F Lin J Cowen W Jessell T Zipursky S Lineage analysis in the vertebrate central nervous system. Neuronal Development. Oxford University Press, Oxford1997Google Scholar, 26Marin O Rubenstein JL A long, remarkable journey: tangential migration in the telencephalon.Nat Rev Neurosci. 2001; 2: 780-790Crossref PubMed Scopus (816) Google Scholar, 27Pilz D Stoodley N Golden JA Neuronal migration, cerebral cortical development, and cerebral cortical anomalies.J Neuropathol Exp Neurol. 2002; 61: 1-11PubMed Google Scholar and plays a significant role in the migration of GABAergic interneurons from the ganglionic eminence to the cerebral cortex and hippocampus.28Anderson SA Eisenstat DD Shi L Rubenstein JL Interneuron migration from basal forebrain to neocortex: dependence on Dlx genes.Science. 1997; 278: 474-476Crossref PubMed Scopus (1209) Google Scholar, 29Anderson SA Marin O Horn C Jennings K Rubenstein JL Distinct cortical migrations from the medial and lateral ganglionic eminences.Development. 2001; 128: 353-363PubMed Google Scholar, 30Pleasure SJ Anderson S Hevner R Bagri A Marin O Lowenstein DH Rubenstein JL Cell migration from the ganglionic eminences is required for the development of hippocampal GABAergic interneurons.Neuron. 2000; 28: 727-740Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar Significantly, the loss or compromise of inhibitory interneuron function has been associated with human epileptogenesis.31Ferrer I Pineda M Tallada M Oliver B Russi A Oller L Noboa R Zujar MJ Alcantara S Abnormal local-circuit neurons in epilepsia partialis continua associated with focal cortical dysplasia.Acta Neuropathol (Berl). 1992; 83: 647-652Crossref PubMed Scopus (123) Google Scholar, 32Treiman DM GABAergic mechanisms in epilepsy.Epilepsia. 2001; 42: 8-12Crossref PubMed Google Scholar Lis1 is expressed in all neurons, and defects in inhibitory neurons are a cause of epilepsy (one of the clinical features in patients with lissencephaly). We, therefore, hypothesized that mutations in LIS1 would similarly affect NRCM. To investigate this hypothesis, we compare the characteristics of GABAergic NRCM in Lis1 +/− and wild-type mice and have studied the number of inhibitory interneurons in several patients with MDS. Our data indicate that Lis1 is required for normal NRCM in mice, and that there is both a cell autonomous and a smaller cell non-autonomous effect on NRCM. We propose that the NRCM defect contributes to the CNS anomalies and clinical manifestations in patients with lissencephaly associated with a LIS1 deletion or mutation. The background of all Lis1 mice used was a mixture of 129SvEvTac and NIH Black Swiss. All mice have been backcrossed to Black Swiss at least eight generations, making this the predominant background. Timed-pregnant mice were considered to be embryonic day 0.5 (E0.5) on the morning a vaginal plug was found, in addition all embryos were morphologically staged.33Theiler K The house mouse: atlas of embryonic development. Springer-Verlag, New York1989: 178Google Scholar Genotyping was conducted using embryo tongues and the following primers for pcr: null and wild-type allele forward 5′-GTGTGGGATTATGAGACTGG-3′; Lis1-Neo (null) allele reverse 5′-GATCTCTCGTGGGATCATTG-3′; and Lis1-wt wild-type control reverse 5′-CCAGATGGTTTAAGTATGAGTC-3′ (positive control for the wild-type allele). Timed-pregnant Lis1 mice were bred in our animal facility. All animal breeding, handling, and experimental procedures were approved by the institution animal care and use committee. Embryos were collected in ice-cold Hanks Balanced Salt Solution/Streptomycin/Penicillin (HanksSP). For immunohistochemistry, brains were dissected in ice cold HanksSP, and after the meninges removed, they were fixed in 4% paraformaldehyde (PFA) or 4% PFA/0.25% gluteraldehyde (for anti-GABA staining) in phosphate-buffered saline (PBS), embedded in 2% agarose (SeaKem LE) and vibratome sectioned at 50 μm. Immunofluorescence was performed as previously described.34Heffron DS Golden JA DM-GRASP is necessary for non-radial cell migration during chick diencephalic development.J Neurosci. 2000; 20: 2287-2294Crossref PubMed Google Scholar Briefly, sections were blocked in 10% normal goat serum for 1 hour at room temperature (RT) with 0.1% Triton-100x. Primary antibodies used were diluted in PBS and included: anti-calretinin, 1:2000 (Swant, Bellonzona, Switzerland); and anti-GABA, 1:1500 (Sigma, St. Louis, MO). Sections were incubated in 2 to 10% normal goat serum with primary antibody and 0.01% to 0.3% Triton-100x from 1.5 days (anti-GABA) to 3 days (anti-calretinin and -calbindin) at 4°C. Secondary antibodies used (2 to 10% normal goat serum, 1 to 4 hours at RT) were biotinylated goat anti-rabbit; biotinylated goat anti-rat and biotinylated goat anti-mouse (Jackson ImmunoResearch, West Grove, PA). Biotinylated secondary antibodies were subsequently incubated with streptavidin-conjugated Cy3 (1:500, Jackson ImmunoResearch). Nuclei were counterstained with DAPI (1:1000, Molecular Probes, Eugene, OR). For immunohistochemistry, sections were incubated with biotinylated secondary antibodies and detected according to the Vector ABC detection system standard protocol (Vector Laboratories, Burlingame, CA) and counterstained with hematoxylin and eosin. Images were obtained using a Leica DMR microscope equipped with epifluorescence and either a Hamamatsu C5180 camera or a Hamamatsu ORCA-ER C4742–95 and OpenLab 3.0.8 or a Nikon Eclipse TE300 equipped with a Hamamatsu C4742–95 using Phase 3 Imaging software for image acquisition. Images were then analyzed using either Phase 3 Imaging or Adobe Photoshop 7.0 software. All distances were calculated, using Phase 3 Imaging software, from the cortico-striatal notch in a straight-line distance to the center of the immunolabeled cells' somata. Animals from three litters were used for GABAergic and calretinin studies. A minimum of seven sections were counted and averaged for each animal. For the in vitro migration studies, embryonic mouse brains were dissected as described above, embedded in 2% low-melt agarose (Fischer, Morris Plains, NJ), sectioned on a vibratome in ice-cold HanksSP at 250 μm and transferred onto 12-mm Millicell-CM cell culture inserts (Millipore, Billerica, MA) in 45%DMEM/45% F-12/10% fetal calf serum (HiClone, Logan, UT)/1X PS/6.5g/L glucose (DFS medium). For initial migration assays (no transplantation), DiI crystals (Molecular Probes) of approximately equal size were placed at the dorsal boundary of the striatum, and the slice was surrounded by matrigel (BD Bioscience, Bedford, MA). Cultures were then incubated for 2 hours in DFS before being change to DMEM/1X N2 medium supplement (Invitrogen, Carlsbad, CA)/1X PS/6.5g/L glucose (DM) and cultured for 48 to 60 hours at 37°C, 5% CO2 before fixing in 4% PFA for 2 hours at 4°C. Cortical transplantations were performed by incising from the cortical notch ventro-laterally to the piriform cortex after vibratome sectioning. Cortices and ganglionic eminences were then re-apposed randomly and cultured as above (see Figure 6 below). All migration analyses were conducted without knowledge of the animal's genotype. For DiI implant studies without transplantation, a total of 35 animals were taken from five separate litters. For transplantation studies, a total of 36 animals from six separate litters were used. All distances were calculated, using Phase 3 Imaging software, from the dorso-lateral edge of the DiI crystal in a straight-line distance to the center of the labeled cells' somata. Comparisons were made using two-tailed Student's t-tests. Radial cell migration is slowed but not absent in Lis1 +/− mice.35Wynshaw-Boris A Gambello MJ LIS1 and dynein motor function in neuronal migration and development.Genes Dev. 2001; 15: 639-651Crossref PubMed Scopus (140) Google Scholar We hypothesized a similar defect would exist for NRCM. To determine whether an in vivo defect exists in NRCM in the Lis1 +/− mice, we studied the expression of inhibitory interneuron markers at various developmental stages spanning the period during which NRCM occurs. Inhibitory interneurons were identified by the expression of several markers including GABA and calretinin. While GABA labels the total set of interneurons, calretinin only labels a subset of interneurons.36Xu Q Cobos I De La Cruz E Rubenstein JL Anderson SA Origins of cortical interneuron subtypes.J Neurosci. 2004; 24: 2612-2622Crossref PubMed Scopus (500) Google Scholar, 37Xu Q de la Cruz E Anderson SA Cortical interneuron fate determination: diverse sources for distinct subtypes?.Cereb Cortex. 2003; 13: 670-676Crossref PubMed Scopus (76) Google Scholar We found GABA and calretinin labeling gave proportionately similar results despite calretinin only labeling a subset of the interneuron population (see Figure 1, Figure 2). Although calretinin represents only a subpopulation of interneurons, calretinin labeling of individual cells is much clearer than GABA labeling (Figure 1). As a result, calretinin was used in many of the subsequent studies to represent the interneuron population, although recognizably not the entire population. While GABAergic cells can be found leaving the ganglionic eminence (GE) in transit to the cortex as early as E1329Anderson SA Marin O Horn C Jennings K Rubenstein JL Distinct cortical migrations from the medial and lateral ganglionic eminences.Development. 2001; 128: 353-363PubMed Google Scholar in both wild-type and mutant animals (data not shown), we focused on E14 to E14.5 animals to allow cells time to migrate sufficiently far to assess differences between mutants and wild types, yet before the leading cells migrated past the dorsal cortex (by E15.5, data not shown), which would complicate quantification and interpretation. At E14.5, inhibitory interneurons orientation non-radially with morphologies typical of migrating cells are observed from the GE into and through the neocortex in both wild-type and mutant embryos (Figure 1). We focused exclusively on non-radially migrating cells in the intermediate zone and subventricular zone, as cells migrating in layer 1 (molecular layer) are a mixed population of glutamatergic, calretinin-positive neurons (Cajal-Reitzius cells), and GABAergic interneurons.38Hevner RF Neogi T Englund C Daza RA Fink A Cajal-Retzius cells in the mouse: transcription factors, neurotransmitters, and birthdays suggest a pallial origin.Brain Res Dev Brain Res. 2003; 141: 39-53Crossref PubMed Scopus (160) Google Scholar, 39Ang Jr, ES Haydar TF Gluncic V Rakic P Four-dimensional migratory coordinates of GABAergic interneurons in the developing mouse cortex.J Neurosci. 2003; 23: 5805-5815PubMed Google ScholarFigure 2Total distance and percentage of distance from GE to dorsum of cortex migrated in Lis1 +/− and wild-type E14.5 embryos. The averaged distance migrated by all calretinin-positive cells past the cortico-striatal notch is significantly greater in wild-type animals by E14.5 (A). When only the leading GABAergic cells are examined, the differences between mutant and wild-type animals is greater in magnitude, as are the total distances traveled (B). Comparing the percentage of the distance from the cortico-striatal notch to the dorsum of the cortex in both cases reveals that Lis1 +/− animals also traverse a significantly smaller portion of the developing brain than do wild-type age-matched controls (C and D)View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine whether mutant cells lagged behind wild-type cells, we quantified the average distance traveled past the cortico-striatal notch both by all calretinin-positive cells and also by the leading GABAergic cells. In all cases we assessed only those cells with a non-radial orientation and excluded those in the marginal zone. The average distance traveled by all calretinin-positive cells past the border to the GE in E14.5 animals was calculated for both the Lis1 +/− and the wild-type animals (245 μm versus 564 μm, respectively). Similarly, the distance traveled by the leading 12 cells, representing the cell migrating front, was calculated for E14 and E14.5 Lis1 +/− and Lis +/+ littermates (629 μm versus 1045 μm, respectively, in the E14.5 animals). These differences were significant at both ages (Figure 2, A, B, and data not shown). As the level of section varies slightly within an animal and differences in total brain size were possible between heterozygous and wild-type animals, the percent distance traveled from the notch to the dorsal-most aspect of the neocortex was also calculated to normalize for differences in forebrain size. Using this measure, mutant cells still progressed a significantly shorter distance than did wild-type cells (Figure 2, C and D). These findings indicate that differences in distance traveled are not due to variations in brain size between or within genotypes. Having data from two time points in development allowed us to make an approximate calculation of the average migration speed. Unfortunately, the staging at the time points we used in mice is imprecise. We used morphological staging to separate out younger (E14) and older embryos (E14.5). Younger embryos harvested at E14 were defined as those with identifiable digits with interdigital webs whereas those harvested at E14.5 and used in these studies had no interdigital webs but a radial ray of digits on the hindlimb.33Theiler K The house mouse: atlas of embryonic development. Springer-Verlag, New York1989: 178Google Scholar While we recognize this is not a perfect system, it allowed us to separate embryos and calculate an approximate time difference for speed calculations. Using these criteria, we calculated the change in the average distance traveled for the leading 12 GABAergic cells and divided by the average time of 12 hours from E14 and E14.5 to generate approximate speeds of cell migration. Our data indicate wild-type cells move at approximately 43.8 μm/hr, while mutants progress an average of only 23 μm/hour. Due to the difficulty in staging these embryos, these numbers should be used as approximate values but show a clear difference between wild-type and Lis1 +/− animals. The in vivo data are consistent with a decrease in the rate of cell migration; alternatively, a delay in initiation of migration from the progenitor zone could account for the observed in vivo data. To determine whether non-radially migrating cells in Lis1 +/− mice migrate more slowly than wild-type cells, inhibitory interneurons were labeled in vitro and assayed for distance migrated over time. GABAergic cells have already begun migrating in both wild-type and mutant animals by E14.5 (see above), placing this time point approximately midway in the migratory process. Therefore, any observed differences in labeled cell positions should be due to varied speeds of migration and not to a delay in the start of migration. To follow the migration of inhibitory neurons from the GE in vitro, the LGE of coronal forebrain slices were implanted with DiI crystals to label migrating cells. After 48 to 60 hours, cells are observed migrating out from the site of crystal implantation in both wild-type (F
Referência(s)