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doi:10.1006/mcne.2000.0880, available online at http://www.idealibrary.com on

Molecular and Cellular Neuroscience 16, 408–421 (2000)MCN

Perinatal Lethality of Microtubule-AssociatedProtein 1B-Deficient Mice Expressing AlternativeIsoforms of the Protein at Low Levels

Christian Gonzalez-Billault,* Evariste Demandt,*Francisco Wandosell,* Miguel Torres,†,‡ Paolo Bonaldo,‡

Anastasia Stoykova,‡ Kamal Chowdhury,‡ Peter Gruss,‡

Jesus Avila,* ,1 and Marina P. Sanchez* ,1

*Centro de Biologıa Molecular, and †Centro Nacional de Biotecnologıa, C.S.I.C., UniversidadAutonoma de Madrid, Cantoblanco, 28049 Madrid, Spain; and ‡Department of MolecularCell Biology, Max Planck Institute of Biophysical Chemistry, Am Fassberg,D-37077 Gottingen, Germany

Microtubule-associated protein 1B (MAP1B) has been im-plicated in axogenesis in cultured cells. To gain insightinto the functions that MAP1B plays in vivo, we analyzed astrain of Map1B mutant mice generated by a gene trap-ping approach. Homozygous mice die on the first dayafter birth, probably due to a severe abnormal develop-ment of the nervous system. They present alterations inthe structure of several brain regions. The normal Map1Bgene yields different protein isoforms from alternativelyspliced transcripts. The smaller isoforms were present inwild type, hetero-, and homozygous mice, but their ex-pression was higher in the mutants than in the wild-type.Moreover, trace amounts of MAP1B protein were alsoobserved in Map1B homozygous mutants, indicating analternative splicing around the gene trap insertion. Thus,the Map1B gene trapped mutation reported in this workdid not generated a null mutant, but a mouse with adrastic deficiency in MAP1B expression. Analyses ofthese mice indicate the presence of several neural de-fects and suggest the participation of MAP1B in neuronalmigration.

INTRODUCTION

Microtubules play an important role in the organiza-tion and maintenance of neuronal morphology. Duringdevelopment, microtubules are very dynamic struc-

1

To whom correspondence and reprint requests should be ad-ressed. Fax: (3491) 397-4799. E-mail: msanchez@cbm.uam.es or

avila@cbm.uam.es.

408

tures that are involved in neurite extension and in theestablishment of neuronal polarity (Avila et al., 1994;Kirschner and Mitchinson, 1986). Later on, they mustbecome stabilised to maintain the correct mature neuralconnections (Tanaka and Sabry, 1995). In neural cells,there are different microtubule associated proteins(MAPs) that contribute to the control of microtubulestabilisation (Matus, 1988). One of these MAPs, MAP1B(Bloom et al., 1985) is the first MAP, which is specificallyexpressed in neurons during nervous system develop-ment (Tucker and Matus, 1988; Tucker et al., 1989).

Functional MAP1B is a complex protein consisting ofa large subunit with a molecular weight of 255 kDa,known as the heavy chain, or simply MAP1B (Noble etal., 1989) and two smaller subunits, or light chains LC1(34 kD) and LC3 (19 kD) (Hammarback, 1997). Trans-fection of cDNAs expressing fragments of MAP1B (No-ble et al., 1989; Takemura et al., 1992) and studies ofchymotrypsin proteolysis (Kuznetsov et al., 1986; Ham-marback et al., 1991) indicates that the amino-terminalregion of the molecule contains a tubulin binding site.This region is structurally related to another MAP,MAP1A (Garner et al., 1990; Hammarback et al., 1991),and also contains the binding sites for LC1 and LC3(Hammarback, 1997). LC1 also has a microtubule bind-ing domain (Togel et al., 1998; Zauner et al., 1992).

Both MAP1B heavy chain and LC1 are translatedfrom a single mRNA, that yields a polyprotein which is

further proteolyzed to produce MAP1B and LC1 (Ham-marback et al., 1991; Togel et al., 1999). LC3 is expressed

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ldai1d

409MAP1B Mutants with Alternative Spliced Isoforms

from a different gene (Mann and Hammarback, 1994).Mouse MAP1B is encoded by a gene that is composedof seven (1–7) coding, and two noncoding (3U and 3A)exons (Kutschera et al., 1998). Two different alternativespliced isoforms, containing either 3U or 3A exons toexon 7, have been identified. These smaller transcriptsare expressed at a much lower level than the full-lengthtranscript (Kutschera et al., 1998).

Several functions have been suggested for MAP1B.Thus, in vitro studies using highly purified protein haveindicated that it facilitates microtubule nucleation (Van-decandelaere et al., 1996) and incorporation of tubulininto microtubules (Pedrotti and Islam, 1995a; Pedrottiand Islam, 1995b). Transfection studies have also indi-cated that MAP1B stabilises microtubules, but it is notclear if it also participates in the formation of microtu-bule bundles (Noble et al., 1989; Sato-Yoshitake et al.,1989; Takemura et al., 1992).

MAP1B function is regulated at different levels, byposttranslational modifications (proteolytic processing,light chain binding, and heavy chain phosphorylation),and by its differential expression during development.MAP1B can be regulated by two modes of phosphory-lation in the heavy chain (modes I and II), due toproline-directed protein kinases (GSK-3b and cdk5) andcasein kinase 2, respectively (Diaz-Nido et al., 1988;Gard and Kirschner, 1985; Garcia-Perez et al., 1998;Pigino et al., 1997; Ulloa et al., 1993a,b). During braindevelopment, MAP1B expression is regulated, beinghigher at the earliest developmental stages during neu-ritogenesis (Diaz-Nido et al., 1990; Riederer et al., 1986;Schoenfeld et al., 1989). Afterwards, it decreases duringthe period of synaptogenesis until reaching the lowadult levels (Fischer and Romano-Clarke, 1990; Rie-derer et al., 1990; Schoenfeld et al., 1989). However, insome brain regions where neuritogenesis persists intoadulthood, such as the olfactory system and photosen-sitive cells of the retina and, in areas with greater neuralplasticity potential, such as the hippocampus, high lev-els of MAP1B expression are maintained (Tucker andMatus, 1988; Viereck et al., 1989). Additional evidencefor the involvement of MAP1B in neuritogenesis hasbeen reported from antisense experiments designed todeplete MAP1B in neural-like cells (Brugg et al., 1993)and in cultured primary neurons (DiTella et al., 1996).

The generation of MAP1B mutant mice by gene tar-geting approaches in embryonic stem cells has pro-vided additional information about the function thatthis protein could play during the development of thenervous system. In two independent studies, mutation

of the Map1B gene leads to an impairment or retarda-tion of brain development (Edelmann et al., 1996; Takei

et al., 1997). However, the data obtained from these twomutant lines are somewhat controversial. In the firstreported model (Edelmann et al., 1996), Map1B homozy-gous mutants are not able to complete the embryonicdevelopment, and die before embryonic day 8. Thegene targeting approach that this group performedleads to the disruption of the Map1B gene at exon 5.However, the second reported Map1B homozygous mu-tant mice (Takei et al., 1997) show milder effect, eventhough the employed gene targeting approach intro-duces a stop codon in Map1B gene at exon 1. In fact,these Map1B homozygous mice reach adulthood, seemhealthy, and only show a mild retardation of the my-elination of certain nerves and tracts, and a slightlyreduced adult brain size, with no overt abnormalities intheir phenotypic development and behaviour (Takei etal., 1997). Here we report the characterisation of a newMap1B mutant mouse line that was obtained by a gene-trapping approach (Chowdhury et al., 1997). Insertionof the gene-trapping vector into the Map1B gene, atsome point between exons 2 and 3, leads to the expres-sion of a b-galactosidase marker and premature termi-nation of Map1B translation. Mutant mice die shortlyafter birth, probably due to the drastic defects observedin the development of the nervous system.

RESULTS

Generation and Genotyping of Map1B MutantMice

The gene trapping vector (IRESbgeo) was transfectedinto R1 ES cells by electroporation. The selected cloneswere aggregated into mouse morulla and the resultingchimeras were mated to NMRI mice until they trans-mitted the altered allele (Chowdhury et al., 1997). TheIRESbgeo vector contained the mouse En-2 splice ac-ceptor sequence, an IRES sequence (containing severalstop codons in the three reading frames), a promoter-less b-galactosidase gene (lacZ) fused with a neomycinresistance gene (neo) and the SV40 polyadenylation sig-nal. Insertion of the IRESbgeo vector into the Map1Bocus should finish transcription of the Map1B geneownstream of the codons corresponding to the first 95mino acids of the protein, as determined by sequenc-ng of RACE-PCR amplified cDNA (Chowdhury et al.,997). The insertion of the vector simultaneously pro-uced the expression of a fusion protein with b-galac-

tosidase and neomycin resistance activities, under the

control of the Map1B promoter (Fig. 1A).

To genotype mice, PCR amplification of genomic

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410 Gonzalez-Billault et al.

DNAs from mouse tails was performed using oligonu-cleotides corresponding to internal sequences of the neogene (Fig. 1B, left panel). The presence of a 238-bpamplified band allowed us to distinguish the heterozy-gous and homozygous animals from the wild-typemice. Genomic BamHI-digested DNAs were also ana-lysed by Southern blot with probes corresponding tocoding regions of both lacZ and Map1B genes (Fig. 1B,central panel). Comparison of hybridised band intensi-ties allowed us to distinguish between the animals car-rying one or two copies of lacZ, corresponding to het-ero- or homozygous mutants. Mouse genotypes werefurther verified by Western blot analysis using antibod-ies against MAP1B and b-galactosidase (Fig. 1B, rightpanel). MAP1B was present in wild-type and heterozy-

FIG. 1. IRESbgeo insertion into the Map1B gene and genotyping ofgene. The IRESbgeo vector was inserted downstream of the codonsequence is encoded by exons 1 and 2 of the Map1B gene, the insertion

as described by Kutschera et al. (1998) is also shown. 3U and 3A, nequence; IRES, internal ribosomal entry site; b-gal/neo, promoterless b

signal (Chowdhury et al., 1997). Alternative splicing giving rise to therise to 3U and 3A transcripts is indicated by dashed and dotted lines,amplification of neo gene from wild-type, hetero-, and homozygous ghetero-, and homozygous genomic DNAs with probes correspondinghetero-, and homozygous protein brain extracts with antibody 125 a

gous mice protein extracts but it was not observed inthe homozygous mice. Moreover, b-galactosidase was

present in both the heterozygous and homozygous pro-tein extracts, and it was expressed in the homozygotesat a double level compared with the heterozygotes, thusreflecting the presence of two copies of the gene trap-ping vector in homozygotes (Fig. 1B, right panel). Wild-type, hetero-, and homozygous mice were obtained atthe expected Mendelian ratio (25.1, 51.4, and 23.5%,respectively).

Analysis of the Expression of the Different MAP1BIsoforms in Mutant Mice

We used several antibodies directed against differentepitopes located along the MAP1B molecule to test forthe presence of the wild-type protein in Map1B mutants

B mutants. (A) Gene trapping insertion of IRESbgeo into the Map1Bsponding to the first 95 amino acids of the MAP1B protein. As thisred somewhere between exons 2 and 3. The Map1B genomic structureding exons; 1–7, Map1B coding exons 1–7; SA, En-2 splice acceptor

actosidase and neomycin resistance genes; pA, SV40 polyadenylationlength protein is indicated by filled lines. Alternative splicing givingctively. (B) Genotyping of Map1B mutants. Left panel shows the PCRic DNAs. Central panel shows a Southern blot analysis of wild-type,ap1B and lacZ genes. In the right panel, Western blots of wild-type,

t MAP1B, and b-galactosidase (b-gal) are shown.

Map1correoccuron co

-galfull

respe

(Fig. 2A). Using the antibody a1–15, raised against apeptide corresponding to the first 15 amino acids of the

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411MAP1B Mutants with Alternative Spliced Isoforms

MAP1B molecule, and antibodies 522, 531, 842, and 125,directed against different epitopes along the MAP1Bmolecule, we did not detect the full-length MAP1Bprotein in Map1B homozygous mutant mice. Moreover,LC1 protein was not detected in the homozygous ex-tracts with the antibodies used in this study (Fig. 2A).Additionally, Western blots with antibody a1–15 didnot detect the expected putative truncated product ofaround 10 kDa that would correspond to the first 95amino acids of the MAP1B molecule (Fig. 2B). How-ever, when blots with antibodies 522, 531, 842, and 125but not with a1–15 were over-exposed, a very faintband with a similar electrophoretic mobility to that ofMAP1B full-length protein was observed (i.e., see blotwith antibody 125, Fig. 3A). The protein correspondingto this faint band accounts for less than 5% of theprotein found in the wild-type, as determined by den-sitometric analyses. This band could correspond toother MAP1B alternative isoforms expressed from ex-ons 3A and 3U (Kutschera et al., 1998). These alternativetranscripts should encode a MAP1B protein lacking thefirst 122 amino acids. Although these isoforms wouldhave a predicted molecular mass around 12 kDa lessthan the full-length protein, the difference in their elec-trophoretic mobilities may be difficult to detect in thegel system used in this study. On the other hand, this

FIG. 2. Western blot analyses of MAP1B expression in wild-type,hetero-, and homozygous mice. (A) Western blot analyses of MAP1Bfull length expression in newborn mutant mice. Western blot analysesof spinal cord protein extracts from wild-type, hetero-, and homozy-gous animals with antibodies a1–15, 522, 531, 842, and 125, againstMAP1B, and with an antibody against LC1 protein. In the upperpanel, the location of the corresponding epitopes along the MAP1Bmolecule is indicated. (B) Western blot analysis of wild-type, hetero-,and homozygous spinal cord protein extracts with antibody a1–15used to detect a putative truncated product, corresponding to the first95 amino acids of the MAP1B protein, predicted as a possible expres-sion product of the Map1B gene containing the gene trapping vector.Lane C was used as a MW reference for a 10-kDa protein andcorresponds to a yeast ribosomal protein extract incubated with anantibody against ribosomal stalk proteins.

band could correspond to a full-length MAP1B proteinthat would be expressed if an alternative splicing event

ap

around the gene trap construct had occurred (Voss etal., 1998). To discern between these possibilities, North-ern blot analysis of E19 total brain RNAs from wild-type, hetero-, and homozygous mice was performed.Transcripts corresponding to a full-length isoform andto two alternative spliced isoforms were observed inwild-type and heterozygous total brain RNAs (Fig. 3B).However, a minor faint band corresponding to thewild-type Map1B transcript was also present in thehomozygous mice (Fig. 3B), suggesting that the spliceacceptors downstream of the gene trap vector are stillutilised, giving rise to a small amount of the full-lengthprotein in the homozygous mutant.

To further verify the presence of these transcripts inmutant mice, RT-PCR was performed from total brainRNAs, by using 59 specific primers corresponding tosequences contained in exons 1, 3A, and 3U, and acommon 39 downstream primer corresponding to exon5 (Fig. 3C; Kutschera et al., 1998). The PCR conditionsthat we selected allowed us to make an estimation ofthe relative amount of transcripts amplified in eachreaction. Primers from exons 1 and 5 amplified a 514-bpDNA fragment present in wild-type mice samples and,at a lower level, in heterozygous mice (Fig. 3D, upperpanel). A very faint band corresponding to the 514-bpDNA fragment was also present in the homozygouscDNA sample (Fig. 3D, upper panel). To better analyzethe presence of each of the Map1B cDNA species, South-ern blot hybridization was further performed on thePCR amplified DNAs (Fig. 3D, full-length, 3U and 3A,lower panels; Kutschera et al., 1998). The Map1B-specific

robe detected the 514-, 446-, and 360-bp PCR frag-ents in all three samples, confirming that they are

ndeed amplified from this gene. Curiously, the PCRragments amplified with primers from exons 3U, 3A,nd 5 were slightly more abundant in the hetero- andomozygous than in the wild-type brain samples (Figs.D, 3U, and 3A, lower panels; Kutschera et al., 1998).

nalysis of the Expression of Other MAPs andelated Proteins in Map1B Mutant Mice

We also studied the effects of the disruption of theap1B gene on the expression of other MAPs and re-

ated proteins in wild type, hetero-, and homozygousnimals, by Western blot analyses of spinal cord proteinxtracts (Fig. 4). Expression levels of the microtubulessociated proteins MAP1A, MAP2, MAP4, and tauere unaltered in MAP1B mutants, suggesting that no

ompensatory effects of these proteins were produced

s a result of the lack of MAP1B expression. Expressionatterns of a-tubulin, bII-tubulin, b-actin, and LC3 in

mMXes

wwafcu3ascc1fh l-length, 3U and 3A, lower panels). The Map1B-specific probe detected the5 e PCR fragments amplified with primers from exons 3U and 3A and exon5 in th

el

412 Gonzalez-Billault et al.

wild-type, hetero-, and homozygous animals were alsounmodified (Fig. 4). Thus, the only compensatory effectobserved in our Map1B homozygous mutant mice is theone previously indicated for the smaller Map1B iso-forms.

MAP1B Expression at Gestational Day 13 in inToto Heterozygous Mutant Embryos and GrossAnalysis of Map1B Mutant Mouse Phenotype

The presence of the gene encoding b-galactosidase inthe construct used to produce the Map1B gene trapping

utation allowed us to observe the activity of theap1B gene promoter by using lacZ as a reporter. In toto

-gal staining of heterozygous embryos showed the

FIG. 3. Expression of trace amounts of MAP1B protein and of Mild-type, hetero-, and homozygous Map1B mutant mice showing anith an electrophoretic mobility similar to that of the MAP1B full-l

nalysis of embryonic day 19 total brain RNAs from wild-type, hetull-length isoform and to two alternative spliced isoforms in wildorresponding to the wild-type Map1B transcript also present in thesed for PCR analysis, and predicted sizes of the PCR amplified cDNU or 3A exons have been identified. These transcripts are expressednd mice (Kutschera et al., 1998). (D) RT-PCR (upper panel) and Southamples from wild-type, hetero-, and homozygous total brain RNA. Rorresponding to sequences contained in exons 1, 3A, and 3U, andonditions that we selected allowed us to make an estimation of the re

and 5 amplified a 514-bp DNA fragment present in wild-type mull-length). A very faint band corresponding to the 514-bp DNA fraybridization was further performed on the PCR amplified DNAs (ful14-, 446-, and 360-bp PCR fragments in all three samples. Note that thwere slightly more abundant in the hetero- and homozygous than

ap1B alternative isoforms in mutant mice. (A) Western blot analysis ofover-exposed signal with antibody 125. Note the presence of a faint bandength protein in homozygous mouse protein extracts. (B) Northern blotero-, and homozygous mice showing the transcripts corresponding to a-type and heterozygous total brain RNAs. Note also a very faint bandhomozygous mice. (C) Map1B genomic organization, location of primersA fragments. Two different alternative spliced isoforms containing eitherat less than 10% of the level of the full-length transcript in wild-type ratern blot analysis (lower panels) of the Map1B transcripts present in cDNAT-PCR was performed from total brain RNAs, by using 59 specific primers

a common 39 downstream primer corresponding to exon 5. The PCRlative amount of transcripts amplified in each reaction. Primers from exonsice samples and, at a lower level, in heterozygous mice (upper panel,

gment was also present in the homozygous cDNA sample. Southern blot

xpression of MAP1B restricted to nervous systemtructures. At gestational day 13 (Fig. 5A), staining was

uoe

e wild-type brain samples (3U and 3A, lower panels).

FIG. 4. Western blot analyses of MAP1B related proteins. Westernblot analyses of high and low molecular weight microtubule associ-ated proteins MAP1A, MAP2, MAP4, and tau, and of a-tubulin,bII-tubulin, b-actin, and LC3, in wild-type, hetero-, and homozygousmbryos at gestational day 19 spinal cord protein extracts. Expressionevels of the microtubule-associated proteins related to MAP1B were

naltered in Map1B mutants, suggesting that no compensatory effectsf these proteins were produced as a result of the lack of MAP1Bxpression.

mtich

413MAP1B Mutants with Alternative Spliced Isoforms

present in the central and peripheral developing ner-vous system. Expression was observed in the neuraltube, cerebral vesicles, dorsal root, trigeminal and su-perior cervical ganglia, and nerve projections arisingfrom these ganglia.

Mutant Map1B homozygous mice die within theirfirst postnatal day of life. They exhibited an abnormallimb posture (compare Figs. 5B and 5D), and an absenceof activity in response to different physical stimuli ap-plied to their limbs.

Morphological Analysis of Newborn Map1B MutantMouse Brain

Analysis of histological brain sections from E19 ho-mozygous mice reflected variability in the mutant phe-notype, with quantitative and qualitative differences.Figures 6A–6D show the aspect of coronal sectionsfrom the whole head stained with cresyl violet in con-trol heterozygous (Figs. 6A and 6C) and homozygous(Figs. 6B and 6D) mutants.

Analyses at the caudate-putamen level (Figs. 6A and6B), showed an enlargement of lateral ventricles (LV) inhomozygous mutant sections (Fig. 6B, asterisk). Also, atthis level, the corpus callosum (cc) in the homozygoussample appeared interrupted (Fig. 6B, arrowheads).

Analyses of coronal sections at the hippocampal levelshowed that the third ventricle was enlarged too, and itwas not normally closed as compared with control lit-termates (Figs. 6C and 6D, asterisk). Concomitant withthis enlargement, a reduction of surrounding thalamicareas occurred. The abnormal ventricle area was quan-tified using the Metamorph/Metafluor system. Ho-mozygous ventricle area was increased around 60–70%(i.e., wild-type 335 mm2, homozygous 582 mm2). Statis-tical analyses of serial sections from four samples foreach group are represented in Fig. 6G.

Additionally, E19 mice showed an abnormal devel-opment of cerebellum. X-Gal staining of heterozygouscontrol and homozygous sagital sections (Figs. 6E and6F, respectively) revealed an alteration in the foliationand lobe organization pattern of the cerebellum. More-over, the wide band of Purkinje cells present at thisdevelopmental stage (Hatten and Heintz, 1995) in theheterozygous control (Fig. 6E, arrow) was absent inhomozygous mice (Fig. 6F, arrow-question mark). Inaddition, there were no apparent defects in the externalgerminal layer (EGL) in homozygous animals whencompared to control littermates (Figs. 6E and 6F).

Detailed analyses of the altered brain structures in

mutant mice were done at a higher resolution level (Fig.7). In this case X-Gal (Figs. 7A and 7D) and cresyl violet

aa

staining (Figs. 7B, 7C, 7E, and 7F) were done on sectionsfrom control heterozygous (Figs. 7A, 7B, 7D, and 7E)and homozygous mice (Figs. 7C and 7F).

In the control heterozygous cerebral cortex (i.e., at thelevel of somatosensorial cortical area, Figs. 7A–7C), cor-tical layers and subplate (sp) were easily distinguish-able. Additionally, the corpus callosum structure (cc)was found to be normal (Figs. 7A and 7B). However, inthe homozygous cerebral cortex, this pattern of neuro-nal cell lamination was completely absent, while anunusual number of cell bodies were located in the cor-pus callosum (Fig. 7C). Also, the subplate was notclearly observed (sp question mark). Variations in thethickness of cortical regions were also founded in ho-mozygous sections. At this respect, in the most drasticphenotype, the cerebral cortex of mutant mice was assmall as half of the size of their heterozygous litter-mates (compare Figs. 7B and 7C).

At the hippocampal level, brain sections stained withcresyl violet revealed the normal laminar organizationof the hippocampus at E19 (Fig. 7E). Homozygous mu-tant hippocampus also presented a lack of the normalcell laminar organization (Fig. 7F) and pyramidal neu-rons were scattered and separated from each other,throughout the distinct hippocampal areas (CA1, CA2,CA3, and dentate gyrus (DG), Fig. 7F). Moreover, thedentate gyrus also presented an abnormal cell organi-zation. Some differences were observed in olfactorybulb. Main, olfactory bulb shows disorganized layers, adisorganization that also occurs in the accessory olfac-tory bulb. While the olfactory bulb in control animalsshowed a cell lamination pattern clearly defined, inmost homozygous analyzed samples, the normal olfac-tory bulb lamination was absent, with the granular,external plexiform and glomerular layers disorganized(data not shown).

DISCUSSION

In the present study, we have examined a strain ofMap1B gene mutant mice generated by the insertion ofa gene trapping vector downstream of the sequenceencoding the first 95 amino acids of the protein(Chowdhury et al., 1997). Homozygous Map1B mutant

ice die during the first day of life, most likely due tohe abnormal nervous system development. Cell layersn the cortex and cerebellum were disorganized, indi-ating that some failure during cell migration couldave occurred. In addition, other brain structures such

s caudate-putamen, hippocampus, and thalamus arelso abnormally developed, and an anomalous enlarge-

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414 Gonzalez-Billault et al.

ment of the cerebral ventricles is detected in homozy-gous animals. The genetic background was contributedby R1 and NMRI strains. It has been described thatsome 129 mouse substrains and the ES-cells derivedfrom them may contain some genetic variations thatcould potentially alter the phenotype of targeted mu-tagenesis (Simpson et al., 1997). However, R1 and NMRIstrains have been previously used by Gruss’s group togenerate other gene targeting and gene trapping mod-els, and no defects in the corpus callosum and ventriclesize were found (Torres et al., 1997; Cecconi et al., 1998;

igle et al., 1999). Additionally, the corpus callosumnd the ventricle size defects here described were onlybserved in homozygous animals, while their wild-typend heterozygous littermates did not show these abnor-alities. Therefore, the phenotype of our mutants must

e considered as the result of the Map1b gene dysfunc-ion.

To determine whether the phenotype showed by ourutants is indeed due to the absence of MAP1B, we

erformed Western blot, Northern blot, and RT-PCRnalyses from neonatal whole brain and spinal cordxtracts. We observed trace amounts of the full-lengthap1B transcript, and a slightly up-regulated expres-

ion of two alternatives spliced isoforms in the homozy-ous mutants. By Western blot, it can be estimated thatomozygous mice contain 5% of the amount of MAP1Brotein present in their wild-type littermates. The ho-ozygous mice reported in this work are not, therefore

ull mutant mice, but deficient in Map1B gene expres-

FIG. 5. MAP1B expression revealed by in toto X-gal staining of a dmutant mice. (A) Lateral view of a heterozygous embryo at embryoniexpression pattern is restricted to the central and peripheral nervous sy(right) shows an abnormal limb appearance, as compared with a condevelopment. (D) Detailed view of homozygous mouse posterior lim

ion. Similar mechanisms giving rise to hypomorphicutated alleles have already been described for other

oi

ene trapping studies (Serafini et al., 1996; Voss et al.,1998; Yeo et al., 1997). In addition, a gene targetingpproach directed to disrupt the b-actin locus also gen-

erated a hypomorphic mutation (Shawlot et al., 1998).An important issue of this work was aimed to estab-

lish if a truncated form of MAP1B was acting in adominant negative fashion. This alternative is very im-probable because there are no phenotypic differencesbetween heterozygous and wild-type littermates. Adominant negative effect might be occurring if the al-tered allele in the heterozygous overcome the wild typecopy of the gene. However, in order to ascertain thisalternative, we used an antibody directed against thefirst 15 amino acids of the protein, and we could notdetect such a truncated product. Neither we detect anyfusion protein containing the N-terminal fragment ofMAP1B and b-galactosidase/neomycin phosphotrans-ferase. This possibility was indeed extremely unplau-sible since there are several stop codons, in the threereading frames, localized upstream of the IRES preced-ing lacZ/neor coding sequences. Furthermore, up-regu-ated alternative transcripts generated in hetero- andomozygous, should not be themselves deleterious be-ause they are present at a low level and there are nohenotypic differences between heterozygous and wild

ype littermates. Thus, homozygous phenotype shoulde due to the drastic reduction of MAP1B expression.Additionally, we rule out the possibility of the exis-

ence of other compensatory mechanisms involving theest of the known MAPs. Although redundant function

heterozygous embryo and newborn aspect of homozygous Map1B13 showing MAP1B expression as revealed by X-gal staining. Map1Bs. (B) Newborn Map1B mutant and control mice. Homozygous mousettermate (left). (C) Detail of the abnormal homozygous anterior limb

ay 13c day

f the tau protein on MAP1B function has been reportedn cell cultures (DiTella et al., 1996) we did not find an

FIG. 6. Morphological aspects at different brain areas of E19 control heterozygous and homozygous Map1B mutant, stained with X-Gal and cresylviolet. (A–D) Cresyl violet staining; (E and F) X-Gal staining; (A, C, and E) Control heterozygous; (B, D, and F) Homozygous Map1B mutant; (A andB) The brain at the caudate-putamen level; (C and D) The hippocampus area; (E and F) The cerebellum. CPu, caudate-putamen; Hi, hippocampus; LV,

lateral ventricle; PCL, Purkinje cell layer; EGL, external germinal layer. Asterisks (*) in B and D represent an enlargement of ventricles in homozygousmice. Arrowheads (B) indicate discontinuous corpus callosum in homozygous mice. Arrows (E and F) indicate Purkinje cell layer. (G) Statisticaldifferences between wild-type (wt) and homozygous (hom) lateral ventricle areas (P , 0.004). Scale bar, A, C, 10 mm; E, 50 mm.

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cF p1Bb CA1

416 Gonzalez-Billault et al.

up-regulation of tau expression that could compensatefor the lack of MAP1B function in our mutants.

The Map1B mutant mouse line that we describe inhis work will allow us to study the functions of MAP1Bn nervous system development. The brain abnormali-ies found in the patterning of all laminated structuresf the central nervous system, in particular those of theeocortex and cerebellum, suggest some failure in celligration during development. In addition to cell mi-

ration defects, the abnormal appearance of laminatedtructures in the brain could also be due to a reducedell proliferation or to an increased cell death. Theormal lamination of the olfactory bulb (Schambra etl., 1992), cerebral cortex (Angevine and Sidman, 1961;ltman and Bayer, 1990; Cobas et al., 1991), and cere-ellum (Kaufman, 1995; Schambra et al., 1992) is altered

n our homozygous mice. The abnormal pattern of cor-

FIG. 7. High magnification images of Map1B E19 control heterozygresyl violet. Sections of somatosensorial cortical area (A to C) and hip); from control heterozygous (A, B, D, and E) or from homozygous Maetween hippocampus and the basal layer of the cortex; CA1–3, fields

mm.

ical lamination is a feature also described for otherutant mouse lines. Thus, mice lacking cdk5 (Oshima

te

t al., 1996), p35 (Chae et al., 1997), Reelin (Caviness andakic, 1978; Rakic and Caviness, 1995), and mDab1

Howell et al., 1997; Ware et al., 1997) show alteredatterns of cortical cell lamination, although with someariations among them. Moreover, a recent report onhe phenotype of a double mutant mouse for the veryow density lipoprotein receptor (VLDL) and the apo-ipoprotein E receptor 2 (ApoER2) shows a similar cor-ical phenotype observed in reeler mice (Trommsdorff etl., 1999), and suggest a model implicating Reelin as aossible activator of VLDL and ApoER2 receptors, a

inkage to the cytoplasmic adapter mDab1 (Howell etl., 1997), and a subsequent tyrosine phosphorylation ofDab1, which may interact and recruit the cdk5/p35

inase complex (Trommsdorff et al., 1999). One stepurther in this model could consider that cdk5/p35

ight be controlling the MAP1B phosphorylation at the

nd homozygous Map1B mutant mice brains, stained with X-Gal andmpus (D to F) stained with X-Gal (A, D) or cresyl violet (B, C, E, and

mutants (C and F). cc, corpus callosum; sp, subplate; Hi, the boundary–3 of Ammon’s horn; DG, dentate gyrus. Scale bar: A, 100 mm; D, 50

ous apoca

ip of the growing axons where both of them are highlyxpressed (Delalle et al., 1997; Paglini et al., 1998).

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417MAP1B Mutants with Alternative Spliced Isoforms

Furthermore, differences in the genetic backgroundof the three Map1B generated mutants or the differentexpression of unconventional microtubule-associatedproteins such as the product of lis1 gene (Sapir et al.,997; Hirotsune et al., 1998) and doublecortin (Francis etl., 1999; Gleeson et al., 1999), could be producing someffect on the animal phenotypes. These unconventionalAPs have been related with neuronal migration in

uman and rodents. Thus, two human disorders linkedo impaired neural migration, lissencephaly or Miller–ieker Syndrome (Reiner and Sapir, 1998) and X-linked

issencephaly or double-cortex syndrom (Gleeson et al.,998) are related to disfunctions on lis1 and doublecortinene, respectively.Two different strains of Map1B knock-out mice have

lready been reported (Edelmann et al., 1996; Takei etl., 1997). Although both mutations suggest some roleor MAP1B during brain development, the severity ofhe phenotypic effects produced by those gene target-ng approaches is quite different in the two mouse lines.he first reported mutant line was produced by inser-

ion of a gene targeting construct, containing a stopodon and a polyadenylation signal, into exon 5 of the

ap1B gene (Edelmann et al., 1996). Homozygous miceor this mutation present an early embryonic lethality.owever, introduction of a stop codon and a polyade-ylation signal into exon 1 of the Map1B gene (Takei etl., 1997), produces only a very mild retardation of brainevelopment. As the two mutations were introduced atifferent points of the Map1B gene, they could be dif-

erentially affecting the expression of the putativeap1B alternative spliced isoforms. In our gene trappedap1B homozygous mutants, Map1B transcription and

ranslation was terminated by introducing several stopodons in the three reading frames and a polyadenyla-ion signal, between exons 2 and 3, resulting in perina-al death of mice, with failures during development ofortical and of other different brain structures. Thelight increase of alternative transcripts in our mutantould be in response to the almost complete absence ofhe full-length transcript or because of the presence ofhe trapping vector upstream of these transcripts thatould be affecting the regulatory elements of its expres-ion.

Thus, MAP1B function may be necessary in criticalteps of nervous system development and at a certainevel of expression. At around embryonic day 8 (Edel-

ann et al., 1996), certain minimal amounts of MAP1Bprotein could be required to allow the embryonic de-velopment to be completed, suggesting some role for

MAP1B protein in early cell differentiation processes.Additionally, both the exon 1 Map1B gene targeted

mutant (Takei et al., 1997) and our Map1B gene trappedutant could be producing enough amounts of MAP1B

rotein isoforms to compensate for the lack of MAP1Bild-type protein at this early developmental stage.owever, at a later developmental stage, around theate of birth, the requirements for MAP1B functionould be higher, and the trace amounts of MAP1B full-ength expression, together with the over-expressed

AP1B alternative isoforms present in our Map1B generapping mutant, would be not enough to facilitate theormal progress from this developmental step.

EXPERIMENTAL METHODS

Generation and Genotyping of Map1B MutantMice

The generation of heterozygous mice lacking a copyof Map1B by a gene trap approach, has been describedlsewhere (Chowdhury et al., 1997). The genetic back-

ground was contributed by R1 and NMRI strains(Chowdhury et al., 1997). The gene trapping vector(IRESbgeo) contained the En-2 splice acceptor sequence(SA), an IRES sequence, the lacZ gene, the neor gene andthe SV40 polyadenylation signal (pA).

To genotype our Map1B mutant mice, genomic DNAfrom mouse tails were isolated and analysed by PCRwith oligonucleotides corresponding to the neo se-quence (Chowdhury et al., 1997). The presence of a238-bp amplified band allowed us to distinguish be-tween the hetero- and homozygous animals, and thewild-type mice. Genomic DNAs were also analyzed bySouthern blot with probes corresponding to coding re-gions of both lacZ and Map1B genes. BamHI-digestedgenomic DNAs were separated in 1% agarose gels andtransferred to Hybond-N1 membranes (Amersham).Membranes were incubated in 0.5 M phosphate buffer,pH 7.0, containing 1% bovine serum albumin (BSA), 7%SDS, 15% formamide and 1 mM EDTA (Sambrook et al.,989). lacZ and Map1B cDNA probes were 32P-dCTPadiolabelled with Multiprime DNA labeling systemAmersham). Membranes were incubated with the ra-io labelled probes overnight at 65°C, washed, andxposed to a film overnight at 270°C.

Protein Extracts and Western Blots

Protein extracts were prepared from spinal cord andbrain homogenates in 20 mM Hepes, pH 7.4, containing

0.1 M NaCl, 10 mM NaF, 1 mM Na3VO4, 5 mM EDTA,1 mM okadaic acid, and protease inhibitors (2 mM

sB

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p

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418 Gonzalez-Billault et al.

PMSF and 10 mg/ml of aprotinin, leupeptin, and pep-tatin). Protein extract homogenates were quantified byradford assay and boiled in electrophoresis buffer.Protein samples were separated in SDS–polyacrylam-

de gels in parallel with prestained high moleculareight electrophoretic markers (Sigma). Proteins were

hen electroblotted onto nitrocellulose sheets, devel-ped with the ECL chemoluminescence system (Amer-ham), and quantified in a Molecular Dynamics Densi-ometer.

Antibodies used in this study included the MAP1Bolyclonal antibodies 522, 531, and 842 (Ulloa et al.,

1993b), and a1–15 (Johnstone et al., 1997, kindly giftedby Dr. Gordon-Weeks, King’s College London, UK); theMAP1B monoclonal antibody 125 (Ulloa et al., 1993b); amonoclonal antibody to b-galactosidase (Promega); theMAP1A monoclonal antibody HM1 (Sigma); the MAP2monoclonal antibody HM2 (Sigma); the rat monoclonalIF522 anti-MAP4 antibody (a kindly gift from Dr. J. B.Olmsted, Department of Biology, University of Roches-ter, NY); the monoclonal antibody 7.51, recognizing aTau phosphorylation-independent epitope (Novak etal., 1991, kindly gifted by Dr. C. M. Wischik, MRCCenter, Cambridge, UK); a monoclonal antibody to to-tal a-tubulin (Sigma); the polyclonal antibody 196 tobII-tubulin (kindly provided by Dr. R. Armas, Centrode Biologıa Molecular, Madrid, Spain); a monoclonalantibody to b-actin (Sigma); and two polyclonal anti-

odies to LC1 and LC3 (kindly gifted by Dr. J. Ham-arback, Department of Neurobiology, Wake Forestniversity School of Medicine, Winston Salem, NC).

orthern Blots and RT-PCR

Total RNA was isolated from embryonic brains at 19estation stage using Ultraspec RNA Isolation SystemBiotecx). For Northern blot analysis, 15 mg of totalrain RNA from wild-type, hetero-, and homozygousice were electrophoresed in a 0.66 M formaldehyde,

.1% agarose gel, and transferred to a Hybond-N1

membrane (Amersham). Membrane were incubated in53 SSC, 13 Denhardt’s, 50% formamide, 1% SDS, 20mM sodium phosphate, pH 6.6 (hybridization buffer),for 30 min at 42°C. The MAP1B probe was prepared byPCR amplification of a Map1B cDNA fragment withprimers corresponding to coding sequences containedin exons 1 and 5 (Kutschera et al., 1998) and radiola-beled with Multiprime DNA labeling system (Amer-

sham). After incubation, the membrane was washedtwice in 23 SSC, 0.1% SDS at room temperature, fol-

lowed by a 20-min wash in 0.23 SSC, 0.1% SDS at 42°C,and exposed to an autoradiography film overnight at270°C.

For RT-PCR, cDNA synthesis from 19 embryonic daytotal brain RNA was performed using the First StrandcDNA Synthesis kit (Pharmacia-Biotech). Wild-typetranscript encoding the full-length protein, 3A and 3Utranscripts were monitored using 59 specific primersocated in exons 1, 3A, and 3U, respectively, and a 39ownstream primer located in exon 5 (Fig. 3C)

Kutschera et al., 1998). Predicted sizes of wild-type, 3A,nd 3U fragments were 514, 360, and 446 bp, respec-ively. PCR amplified DNAs were then transferred to a

ybond-N1 membrane (Amersham) and further ana-lyzed by Southern blot. MAP1B specific probe was pre-pared as described for Northern blot analyses. In orderto make a relative estimation on the amount of thealternative transcripts expressed in wild-type, hetero-,and homozygous mice, we performed PCR reactionswith 25 cycles of 95°C 3 40 s, 60°C 3 1 min, and 72°C 3

min, with a final extension cycle of 72°C during 5 min.

istochemistry

For in toto X-gal staining, MAP1B heterozygous preg-ant females were sacrificed at gestation day 13 andmbryos were fixed by immersion in 4% paraformalde-yde overnight at 4°C. Embryos were then washed in00 mM potassium phosphate buffer, 5 mM EGTA, 2M MgCl2, pH 7.4 (KPP), containing 0.01% sodium

deoxycholate and 0.02% Nonidet P-40 (washing buffer).Embryos were then stained with 1 mg/ml X-Gal inwashing buffer containing 10 mM K3[Fe(CN)6], 10 mMK4[Fe(CN)6] overnight at 37°C. After washing, embryoswere fixed in 70% ethanol (Joyner, 1993).

For X-gal staining of brain sections, control and mu-tant E19 and newborn mice were sacrificed by decapi-tation and heads were fixed by immersion in 4% para-formaldehyde overnight at 4°C and cryoprotected byimmersion in 30% sucrose for 2 days at 4°C. Twenty-micrometer cryostat sections were treated with washingbuffer and then stained for 2–12 h at 37°C (Joyner,1993). Homozygous mice sections were stained abouthalf of the time of heterozygous mice, to obtain a sim-ilar stain level. Sections were washed in washing buffer,dehydrated, and mounted with DePeX medium. Forcresyl violet staining, 20-mm cryostat sections werepostfixed in 4% paraformaldehyde for 15 min at room

temperature, before staining. Sections were dehydratedand mounted with DePeX medium.

A

B

B

C

C

C

C

C

F

F

G

G

419MAP1B Mutants with Alternative Spliced Isoforms

Quantitation of Ventricular Width

Quantitation of ventricular volume was done usingthe Metamorph/Metafluor Software (Universal Imag-ing, West Chester, PA) and the NIH Scion Image (ScionCorp.). Both softwares have been widely used to ana-lyze morphological parameters (DiTella et al., 1996;Paglini et al., 1998; Ahmad et al., 2000). Serial sectionsimages of control and homozygous littermates wereused to measure the following ventricle parameters:area, perimeter, centroid X, centroid Y, width, height,and shape factor. The differences between groups werethen analyzed by the use of ANOVA and Student–Newman–Keuls test. An example of the measurementsis: Area, wild-type 702, homozygous 1325; Perimeter,wild-type 117.48, homozygous 166.6; Centroid X, wild-type 143.7, homozygous 135.4; Centroid Y, wild-type84.14, homozygous 87.7; Width, wild-type 33, homozy-gous 49; Height, wild-type 39, homozygous 53; Shapefactor, wild-type 0.63, homozygous 0.6. Values are inpixels and are then transformed in mm.

ACKNOWLEDGMENTS

We thank Drs. J. Dıaz-Nido, F. Lim, C. Sanchez, and J. Hammar-back for critical reading of the manuscript; Dr. A. Caceres for helpingus with ventricle area measurements and R. Cuadros, V. Cadenas,and P. Aganzo for technical assistance. We also thank personnel fromanimal care services, especially J. Palacın and M. A. Bordallo, andpersonnel from photographic facilities. This work was supported bygrants from the Spanish DGICYT, Comunidad de Madrid and Euro-pean Union. An A.E.C.I./I.C.I. predoctoral fellowship was awardedto C.G.-B., and Erasmus Programme fellowship to E.D., and a M.E.C./C.S.I.C contract to M.P.S. The MAP1B mutant mouse was generatedwith financial support from AMGEN and the Max Planck Society.

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Received April 12, 2000Revised June 6, 2000

Accepted June 7, 2000