Mice Lacking the Giant Protocadherin mFAT1 Exhibit Renal Slit Junction Abnormalities and a Partially Penetrant Cyclopia and Anophthalmia Phenotype

While roles in adhesion and morphogenesis have been documentedfor classical cadherins, the nonclassical cadherins are muchless well understood. Here we have examined the functions ofthe giant protocadherin FAT by generating a transgenic mouselacking mFAT1. These mice exhibit perinatal lethality, mostprobably caused by loss of the renal glomerular slit junctionsand fusion of glomerular epithelial cell processes (podocytes).In addition, some mFAT1^-/- mice show defects in forebrain development(holoprosencephaly) and failure of eye development (anophthalmia).In contrast to Drosophila, where FAT acts as a tumor suppressorgene, we found no evidence for abnormalities of proliferationin two tissues (skin and central nervous system [CNS]) containingstem and precursor cell populations and in which FAT is expressedstrongly. Our results confirm a necessary role for FAT1 in themodified adhesion junctions of the renal glomerular epithelialcell and reveal hitherto unsuspected roles for FAT1 in CNS development.

INTRODUCTION

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Introduction

Cell adhesion molecules are essential for the regulation ofcoordinated changes in cell shape and position that occur duringmorphogenesis and also for the maintenance of cell-cell interactionsonce development is complete. The best-studied families of celladhesion molecules are the cadherins. These were originallyidentified as Ca²⁺-dependent cell-cell adhesion proteins characterizedby five repeated cadherin-specific motifs in their extracellulardomain. This domain is an approximately 110-amino-acid peptidethat mediates homophilic interactions with other cadherin molecules,forming dimers which then interact with dimers on neighboringcells (41, 45, 46). The cytoplasmic domain of these so-calledclassical five-repeat cadherins is well conserved between differentmembers of the family, containing motifs required for interactionwith catenins that can then mediate linkage to the cytoskeleton(13, 17, 34, 42). In addition to the classical cadherins, atleast five other classes of cadherins have been identified:nonclassical cadherins, in which the first extracellular repeatdoes not contain the HAV (histidine-alanine-valine) cell adhesionsequence present in all classical cadherins, and four additionalfamilies. Two of these, the desmosomal adhesion molecules desmocollinsand desmogleins, all share the five extracellular cadherin repeatstructure with the sole exception of desmocollin-1. Two otherfamilies, the flamingo cadherins and protocadherins, have variablenumbers of cadherin repeats and additional motifs as well asdiffering cytoplasmic domain sequences (30, 44).

The functions of the protocadherin family, in particular, remainpoorly understood. The presence of many different protocadherins,many encoded by three gene clusters comprising 52 genes in thehuman (47), could provide the basis for specificity of intercellularinteractions based on differential adhesion. However, adhesiveproperties have been described only for Xenopus NF-protocadherin(the homologue of human pcdh7) (4), axial protocadherin (pcdh1)(21), and paraxial protocadherin (pcdh8) (19). In addition,the extracellular domain of pcdh2 shows homophilic adhesionproperties when fused to the cytoplasmic domain of the classicalcadherin (31). For the majority of protocadherins, therefore,no adhesive function has been documented, and the variabilityof structure within this class of molecules suggests that otherfunctions may well be significant.

One other such function suggested for protocadherins has beengrowth suppression. This conclusion arose from studies of FAT,the largest protocadherin, with 34 extracellular cadherin repeats(30). In Drosophila, loss-of-function mutations cause hyperplasiaof the pupal imaginal disks, suggesting a role as a tumor suppressorgene, while defects of epithelial morphogenesis are consistentwith an adhesive function (25). This giant cadherin has beensubsequently cloned in humans (10), rats (33), and mice (6).Structurally, these genes resemble the FAT gene of Drosophila,showing that FAT-like cadherins are conserved between vertebratesand invertebrates, and more recently two additional FAT genes(FAT2 and FAT3) have been identified in vertebrates (27, 29).The protein has a predicted size of 500 kDa, containing 5 epidermalgrowth factor (EGF) and 2 laminin-A-G domain repeats in additionto the 34 cadherin repeats in the extracellular domain. Thecytoplasmic domain contains two potential ß-cateninbinding regions (FC1 and FC2), although these are only weaklyconserved with classical cadherins (10). In addition, a putativePDZ domain binding sequence has been found at the C-terminaldomain of the vertebrate homologues (33), although this is notpresent in Drosophila FAT.

Expression studies show FAT mRNA to be expressed widely in epithelialtissues, including skin, neuroepithelium, and renal glomerularepithelial cells (podocytes), during development (6, 33), withupregulation reported in human epithelial tumors (6). We previouslyreported that FAT expression within the central nervous system(CNS) persisted in the adult in areas in which proliferativeneural stem cells remain throughout life (33). Given this localizationof a putative growth suppressor molecule, we reasoned that FATmight contribute to regulation of proliferation in the CNS andother tissues. To test this and to examine other functions ofthis molecule, we have generated an mFAT1-deficient mouse. Ourresults provide no support for a role of mFAT1 in the regulationof proliferation, but they do reveal novel roles in adhesionand cell-cell signaling.

MATERIALS AND METHODS

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Materials and Methods

Generation of Fat-deficient mice. A mouse genomic PAC library from a female 129/SvevTacfBr mousespleen was screened using probes from the 5' and 3' ends ofrat Fat cDNA (33). Three different genomic clones were identifiedand isolated. Restriction analysis and further Southern blottingusing as a probe the first 700 bp of the rat Fat cDNA (includingthe 5' untranslated region and the transcription start codon)identified a 7-kb fragment containing the predicted ATG startcodon for mFAT1. This fragment of 7 kb was then cloned in theEscherichia coli-yeast shuttle vector pRS414. Next, we modifieda second plasmid (pRay-1) containing a cassette comprising theneomycin resistance gene under the control of the thymidinekinase promoter, the yeast selection marker URA3, and the lacZreporter gene in front of these selection markers by inserting400-bp recombinogenic arms on either side of the cassette. Thesearms were generated by PCR from the mouse DNA clone and weredesigned to allow homologous recombination in yeast so as toinsert the lacZ gene in frame with the first ATG of FAT1, hencegenerating a targeting vector (43).

The entire cassette, now flanked by the two recombinogenic arms,was transferred into yeast cells together with the shuttle vectorpRS414 containing the 7-kb mouse DNA clone. Recombinant yeastcolonies in which homologous recombination had occurred wereselected by plating on Trp/URA dropout plates, requiring theURA3 marker gene for survival, and plasmids from the selectedcolonies were transferred to E. coli (Top 10; Stratgene) byelectroporation. Homologous recombination was confirmed usingrestriction analysis and sequencing. The final vector was linearizedand electroporated into 129Sv embryonic stem (ES) cells. Electroporatedcells were selected for 8 days in 250 µg of G418 (Gibco)per ml. Selected clones were expanded and analyzed by Southernblotting using an internal probe (lacZ) and an external probethat is adjacent to but not contained in the targeting construct(see Fig. 1A). Two of the three ES cell clones shown to haveundergone correct homologous recombination, HB-5 and FB-2, wereinjected into C57BL/6 mouse blastocysts, and the blastocystswere then implanted into pseudo-pregnant females. No chimericmice were born from HB-5-injected blastocysts. From FB-2-injectedblastocysts, nine mice with chimerism varying from 40 to 100%were born. One of these animals gave germ line transmissionof the mutant allele, and heterozygous progeny were intercrossedfor more than five generations and also backcrossed onto C57BL/6and 129Sv backgrounds. Genotyping of tail DNA was performedby either Southern blotting or PCR using two primer pairs flankingthe insertion site. For these PCR amplifications, the two senseprimers were 5' CAGCAGTTTGGAGACA 3' and 5' TAGCTTGGCTGGACGT3'and the antisense primer was 5' ACGAGGGGTCTAAGATGT 3', givingproducts of 150 nucleotides for the wild-type allele and 220nucleotides for the mutant allele.

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FIG. 1. (A) Schematic representation of the protocol used to mutate the mFAT1 locus, showing the wild-type (WT) locus (top), mFAT1 targeting vector (middle), and expected mutant allele after homologous recombination (bottom). A lacZ/Neo cassette was inserted in frame with the first ATG of the mFAT1 gene as described in Materials and Methods. The positive ES clones undergoing correct homologous recombination were identified by Southern blot analysis using a 3' external probe and an internal probe on the lacZ gene. (B) Southern blot analysis (using the 3' flanking probe) of EcoRI-digested tail genomic DNA from progeny of mFAT1 heterozygous intercrosses, showing the three possible genotypes obtained.

Analysis of mFAT1 expression. lacZ expression in embryos and sections (10 µm) from mFAT1transgenic mice was detected by using 5-bromo-4-chloro-3-indolyl-ß-D-galactopyronoside(X-Gal) staining solution containing 2 mM MgCl₂ and 5 mM K₄Fe(CN)₆in phosphate-buffered saline (PBS) plus 0.01% sodium deoxycholate,0.02% NP-40, and 1 mg of X-Gal per ml. The sections were stainedovernight at 37°C and then counterstained with Neutral Redbefore being mounted.

Western blotting. Embryos (18.5 days postcoitum or E18.5, with the plug date beingE0) were obtained by cesarean section following euthanasia ofthe pregnant female. The tail was removed for DNA extractionand genotyping. Skin was then removed, homogenized in lysisbuffer (50 mM Tris-HCl, 5 mM EDTA, 150 mM NaCl, 1% NP-40, 1%sodium dodecyl sulfate, 2 mM phenylmethylsulfonyl fluoride,1 µg of pepstatin A per ml, 2 mg of aprotinin per ml,5 mg of leupeptin per ml) on ice for 30 min, and centrifugedat 15,000 rpm in an Eppendorf Microfuge for 15 min. The amountof total protein in the extracts was determined using the Bio-Raddetergent-compatible protein assay with bovine serum albuminas a standard. Equal amounts of protein from each animal wereseparated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(7% polyacrylamide) under reducing conditions and electroblottedonto a nitrocellulose membrane (Hybond-C; Pharmacia). The membraneswere blocked for 1 h at room temperature in 5% nonfat dry milkin Tris-buffered saline containing 0.1% Tween 20 (TBS-T). Theblots were incubated overnight at 4°C in TBS-T with a rabbitantiseum raised against a fusion protein comprising the cytoplasmicdomain of rat FAT1 (to be described in detail elsewhere); thiswas followed by a 2-h incubation with an appropriate secondaryperoxidase-conjugated antibody (Amersham) in TBS-T. The immunoreactiveproteins were visualized using enhanced chemiluminescence (Amersham).

Proliferation assays. To measure proliferation in stem and precursor cells derivedfrom the CNS, neurospheres were grown from cells dissociatedfrom E18.5 CNS tissue and plated on nonadherent substrates aspreviously described (15), using different concentrations (20,2, and 0.2 ng/ml) of fibroblast growth factor 2 (FGF-2) or EGF.Proliferation was assessed by measuring bromodeoxyuridine (BrdU)incorporation with an immunofluorescence assay kit (Roche).BrdU was added to the cultures for 2 h after they had been grownfor 5 days in the appropriate growth factor concentration. Afterthe BrdU pulse, neurospheres were dissociated mechanically andcells were plated on poly-D-lysine-coated slides to ensure thatall cells attached prior to labeling. BrdU-labeled cells werevisualized using a mouse monoclonal anti-BrdU antibody followedby an anti-mouse fluorescein isothiocyanate-conjugated antibody.Before mounting, the slides were incubated for 10 min in propidiumiodide (20 µg/ml) in PBS, and the percentage of BrdU⁺cells was calculated. Three independent experiments were performedat each growth factor concentration, and in each case 500 cellsfrom five distinct fields were counted. To measure proliferationin skin, pregnant mFAT1^+/- females (previously crossed withmFAT1^+/- males) were injected twice with BrdU (100 µg/gof body weight) at 1-h intervals. At 1 h after the second injection,the animals were sacrificed and the embryos were extracted and,after euthanasia, were frozen in isopentane cooled by liquidnitrogen (-30 to -40°C). Frozen sections were cut, thawed,and fixed in 95% ethanol-5% acetic acid for 20 min at -20°C,washed in PBS, and incubated for 1 h in blocking solution containing10% normal goat serum (Sigma). For quantification of BrdU labeling,BrdU⁺ cells (detected as above) and propidium iodide-labeledcell nuclei were counted in five adjacent fields in the basallayer of the epidermis in skin of mutant embryos and their littermates,and the percentage of labeled cells was calculated for eachgenotype.

Electron microscopy. Tissue was fixed by immersion for 4 h at 4°C in 4% glutaraldehydein 0.1 M PIPES buffer containing 2.0 mM CaCl₂ at pH 7.4. Immediatelybefore use, H₂O₂ was added to the fixative to give a final concentrationof 0.1%. The tissue was rinsed in PIPES buffer, incubated in1% osmium ferricyanide for 1 h, rinsed in water, incubated in1% uranyl acetate for 1 h, rinsed again in water, and then dehydratedin an ascending series of ethanol solutions followed by tworinses in propylene oxide before being embedded in Spurr's resin.Thin sections (50 nm) were cut with an Ultracut UCT microtome(Leica, Vienna, Austria), mounted on copper grids, stained withuranyl acetate and lead citrate, and viewed in a CM100 electronmicroscope (Philips, Eindhoven, The Netherlands) operated at80 kV.

Immunofluorescence and TUNEL labeling in vivo. Cryostat sections (10 µm) from mFAT1^-/- mice were fixedfor 20 min in 4% paraformaldehyde and then permeabilized in0.1% Triton X-100 in 10% normal goat serum-4% bovine serum albuminfor 1 h at room temperature. Desmosomal cadherins were visualizedusing polyclonal antibodies (a kind gift of T. Magee), usedat 1/100, and a fluorescein isothiocyanate-conjugated secondaryantibody. ß-Catenin was identified using a mouse monoclonalantibody (Transduction Laboratories), used at 1/200, and a Texasred-conjugated secondary antibody. For TUNEL labeling, prefixedE13.5 mFAT1^-/- embryos were sectioned as above and labeled usingthe terminal deoxyribonucleotide transferase-mediated dUTP nick-endlabeling (TUNEL) method with a commercially available kit (ApoTag;Intergen).

RESULTS

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Results

To inactivate the mFAT1 gene in mice, we generated a targetingvector for homologous recombination by using the recombinationefficiency of yeast (43) to insert a lacZ marker-selection cassetteinto a 7-kb genomic clone containing the first ATG of the mFAT1gene as described in Materials and Methods. The targeting constructwas then introduced into ES cells, and clones showing homologousrecombination were identified by both genomic PCR and Southernblot analyses with an internal and an external 3' probe. A totalof 400 clones were screened, and the expected 3.4- and 4.2-kbEcoRI restriction fragments for the mutant and the wild-typeallele, respectively (Fig. 1A), were obtained with three ESclones. Chimeras were generated from two of these clones andcrossed with C57BL/6 mice, and the offspring were genotypedby Southern blotting (Fig. 1B) and PCR amplification (data notshown). One of these chimeric animals gave germ line transmission.Mice heterozygous for the mFAT1 mutant allele appeared normaland fertile and were indistinguishable from wild-type mice.Mice were then systematically backcrossed onto both C57BL/6and 129Sv backgrounds.

These heterozygous animals were first used for analysis of thepattern of expression of mFAT1, as determined by expressionof the lacZ reporter gene, during embryonic development. Theresults obtained were in close agreement with results of previousin situ hybridization studies of mice (6) and rats (33), withno evidence for expression of the transgene in areas not identifiedby these studies. We found mFAT1 expression early in postimplantationdevelopment, with detectable X-Gal staining at E6.5 in the epiblast(data not shown). At mid-gestation (E12.5), mFAT1 expressionwas observed in the limb buds, telenchephalon, developing eye,Rathke's pouch, and spinal cord (Fig. 2A, I to IV). Within thespinal cord, expression was observed in the floor plate cellsof the neural tube as well as in the notochord (Fig. 2A, V).At later embryonic stages, expression was also seen in the respiratoryepithelium, renal epithelial cells, cartilage, and basal layerof the skin (Fig. 2B, I to IV).

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FIG. 2. (A) The lacZ reporter gene was used to analyze the pattern of expression of the FAT1 gene in the embryo. Micrographs I to V show X-Gal staining in an intact (I) or sectioned (II to V) E12.5 embryo. The staining is strong in the forebrain (tel in panel I), mainly in the neocortex around the telencephalic vesicles (top part of panel II; note the lower level of staining in the ganglionic eminences in the lower part of the section), in the limb buds (panel I), in the developing eye (panel III), and in Rathke's pouch (panel IV). Panel V shows lacZ expression in the spinal cord of an E12.5 embryo. Note the staining in the region of the floor plate. e, eye; fp, floor plate; inf, infundibulum; lb, limb bud; ncx, neocortex; nl, neural layer; nc, notochord; os, optic stalk; pl, pigment layer; rp, Rathcke's pouch; tel, telencephalon. Bars indicates sizes in millimeters. (B) lacZ staining in newborn mFAT1^-/- pups. Epithelial cells in the lungs (I) and kidneys (II), cells surrounding developing cartilage (III), and the basal layer of the epidermis in the skin (IV) show a strong X-Gal staining. All sections were counter stained with Neutral Red. Bars indicate sizes in millimeters. (C) Hematoxylin-eosin staining of sections from lungs of wild-type and mFAT1^-/- mice (panels I and IV) and kidneys (panels II and V; shown at higher power in panels III and VI, respectively). Light microscopy does not reveal any differences between the different genotypes. Bars indicate sizes in millimeters. (D) Distribution of adhesion-related proteins in skin from wild-type and mFAT^-/- mice. No differences between the mutant and the wild-type animals were observed when using antibodies against the desmosomal proteins desmoglein (dsg), desmocollin (dsc), and desmoplakin (dp). The cellular distribution of ß-catenin also does not shown any alteration between the wild-type and mFAT1^-/- mice. (E) Anti-BrdU immunofluorescent labeling (green) and propidium iodide (red, to visualize nuclei) in the skin of wild-type and mutant mice after BrdU incorporation in vivo, as described in Materials and Methods. Labeled cells in the basal layer of the epidermis are indicated by white arrows, and no differences were observed in the labeling index in this cell layer. Note the lack of labeling in the more superficial epidermal cells. The labeled cells in the underlying dermis were not included in the analysis. (F) The holoprosencephaly phenotype seen in some mFAT1^-/- mice is illustratedin micrographs I and II, which show lateral and ventral views of an intact embryo with cyclopia (an eye in the centre of the face [arrowhead in panel I]). Micrograph III shows a cross-section of a newborn mutant mouse with holoprosencephaly, showing a proboscis protruding above the eye (arrowhead). The section has been stained with X-Gal, showing the expression of lacZ from the transgene. (G) The eye phenotype observed in many of the FAT1^-/- mice is shown. Micrographs I to IV are tissue sections from two different homozygous null embryos, one at E17.5 stained with hematoxylin and eosin (panels I and II) and one at E13.5 stained with X-Gal (panels III and IV). Note the complete loss of one eye in panels I and III (right arrowhead, with normal eye indicated by left arrowhead) and the presence of retinal pigment epithelium but not neural retina and lens in II and IV (arrowhead). Bars indicate sizes in millimeters. (H) TUNEL staining of an E13.5 embryo with an abnormal eye. The number of TUNEL-positive cells is dramatically increased in the abnormal eye (panels I and II, with panel I showing a 4',6-diamidino-2-phenylindole (DAPI)-labeled section to visualize tissue and panel II showing TUNEL-positive cells, with the eye indicated by the dashed line) compared with the normal eye (panel III and IV). Bars indicate size in millimeters.

We next crossed heterozygous animals to determine the homozygousphenotype. Genotyping of E18.5 embryos obtained from these crossesshowed no significant deviation from the expected Mendelianratio (1:2:1) of wild-type, heterozygous, and homozygous animals.After birth, however, we found dead pups within 48 h of birthand none of the surviving pups were homozygous for the mutatedmFAT1 allele (Fig. 3). Western blot analysis of skin from wild-type,heterozygous, and homozygous E18.5 mice, using a polyclonalaffinity-purified antibody raised against a glutathione S-transferasefusion protein comprising the cytoplasmic domain of mFAT1, confirmedthe complete loss of the mFAT1 protein in the homozygous animals(Fig. 3). We conclude, therefore, that mFAT1 is necessary forpostnatal survival.

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FIG. 3. (A) Genotype distribution of progeny from mFAT1^+/- intercrosses. Although the ratio of the three genotypes was normal at E18.5, no mFAT1^-/- animals were found postnatally. (B) Western blot analysis of skin from wild-type, heterozygous, and homozygous mutant mice confirms the complete loss of the mFAT1 protein.

To explain the perinatal death of the mFAT1 null mice, we examinedthe lungs and kidneys by using light and electron microscopy.These organs were chosen because FAT1 is expressed in epithelialcells within both organs (6, 10, 14, 33) and their functionis required only after birth, thus making dysfunction a possiblecause of perinatal death. The lungs (Fig. 2C, I and IV) andkidneys (Fig. 2C, II, III, V, and VI) of mFAT1 null E18.5 micewere analyzed by light microscopy and showed no abnormalitiescompared with heterozygous and wild-type littermate controls.In particular, renal glomerular size was similar in the differentgenotypes. While the epithelium in the lungs was also normalby electron microscopy (data not shown), in the kidney the footprocesses of the podocytes (the renal glomerular epithelialcells) were abnormal (Fig. 4). These processes are normallyjoined to each other adjacent to the glomerular basement membraneby slit junctions, specialized adhesion complexes that providea gap of 30 to 45 nm between processes, sufficient to allowfiltration from blood to renal tubules (36). In three null animalsexamined from two different litters, electron microscopy showedthat these gaps were obliterated, with the processes fused asshown in Fig. 4 to generate a sheet of cell processes overlyingthe basement membrane. By contrast, slit junctions between podocytefoot processes were normal in two heterozygotes and one wild-typeanimal from the same litters.

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FIG. 4. Abnormalities in the kidneys of mFAT1^-/- mice as seen by electron microscopy. The normal gaps (slit junctions [right panel]) between the podocyte (glomerular epithelial cell) foot processes (fp) adjacent to the glomerular basement membrane (gbm) have been lost, with flattening and fusion of the podocyte processes (left panel).

To determine the role of mFAT1 in other adhesion complexes,we examined skin, another tissue in which mFAT1 is expressedstrongly (Fig 2B, IV), from E18.5 mFAT1 null mice. Epidermalcells in the skin are connected by both adherence junctions,in which classical cadherin cytoplasmic domains are associatedwith ß-catenin (17), and desmosomes, in which thecytoplasmic domains of the cadherin family members desmocollinand desmoglein are associated with desmoplakin (11, 20). Wetherefore examined the distribution of these molecules by immunofluorescenceto determine whether loss of mFAT1 altered the distributionof adhesion complex-related proteins. As shown in Fig. 2D, thedistributions of desmoglein (I and IV), desmocollin (II andVI), desmopolakin (III and VII), and ß-catenin (IVand VIII) were not altered in the mFAT1 null mice compared withwild-type littermates. In addition, electron microscopic studiesof skin did not show any changes in cell morphology in nullanimals compared with wild-type and heterozygous littermates(not shown). We conclude, therefore, that mFAT1 is not essentialfor the formation and maintenance of intercellular adhesioncomplexes in the mouse skin.

In light of the tumor suppressor role for FAT in Drosophila(25), we next asked whether loss of mFAT1 was associated withany changes in cell proliferation in two tissues: skin and theneuroepithelium of the CNS. These tissue were chosen becausethey both contain stem and transit-amplifying precursor cellpopulations (8, 16). Moreover, mFAT1 expression persists inpostnatal animals in precisely those regions of these tissueswhere these cell populations are found, and cell proliferationwith the generation of new differentiated cells continues throughoutlife (33). To measure proliferation in neuroepithelial cells,we used a cell culture technique in which stem and precursorcells taken from embryonic CNS were grown as neurospheres: three-dimensionalaggregates of cells that can be grown in cell culture on nonadherentsubstrates (37, 38). These cells proliferate in response toeither FGF-2 or EGF, and when neurospheres were grown from heterozygousor null CNS tissue, no significant differences in the responsesto either growth factor (as measured by BrdU incorporation)were seen (Fig. 5). Proliferation in embryonic skin was measuredin vivo following BrdU injections into the pregnant mother latein the pregnancy. As expected, BrdU incorporation within theepidermis was seen only in the basal layer of cells from whichthe postmitotic keratinocytes arise. Once again, there wereno differences between heterozygous and null embryos (Fig. 2E),with the observed levels being 30 and 31% respectively.

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FIG. 5. BrdU proliferation assays on neural precursors grown in vitro. Neurospheres of the three different genotypes grown as described in Materials and Methods in different concentrations of the growth factors EGF and FGF-2 do not show any significant differences in the percentage of BrdU-positive cells.

Two unexpected phenotypes found in some of the mFAT1-deficientmice were holoprosencephaly and microphthalmia-anophthalmia.Holoprosencephaly was observed in 4 (6%) of 68 null embryos,with cyclopia (a single midline eye) and a midline proboscisbeing characteristic of this developmental abnormality of ventralforebrain induction (28) (Fig. 2F). Eye abnormalities were morecommon, with one or both eyes being either small or absent in27 (40%) of 68 embryos (Fig. 2G, I and III). Histological analysisshowed that the neural retinal layer and lens were absent inthese animals and that the retinal pigment layer was misshapenand greatly reduced in size (Fig. 2G, II and IV). Interestingly,the phenotype of the eye in null embryos was variable even withina single embryo, since we often saw a severe eye phenotype onone side and normal eye development on the other side (Fig.2G, I and III). Microphthalmia was also seen in one of threenull animals (all of which exhibited perinatal lathality) generatedwithin two litters of the transgenic line backcrossed five timesonto a 129Sv background. To determine whether the abnormalitiesof eye development reflected increased levels of apoptosis,we used TUNEL assays on sections of E13.5 mFAT1 null embryos.Animals with one normal and one abnormal eye were chosen soas to have an internal control in the experiment. The normaleye showed very few apoptotic cells (Fig. 2H, III and IV), whilethe abnormal eye contained a large number of these cells (Fig.2H, I and II), from which we conclude that the abnormal phenotypein mFAT1 null mice is associated with increased degenerationand apoptosis in the developing eye.

DISCUSSION

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Discussion

The abnormalities in the mFAT1 null mice described here confirmthe essential role of this protocadherin for correct developmentin the mouse. This result contrasts with the normal viabilityand fertility reported for mice lacking PAPC (pcd8), the onlyother protocadherin molecule to have been knocked out so far(48). Our analysis of the mFAT1 null mice reveals renal glomerularabnormalities as the most likely cause of the perinatal deathwe observed and which was also noted in a survey of transgenicmice generated using a gene trap approach for secreted and membraneproteins (26). Our results also provide evidence for a noveland unexpected role for FAT1 in forebrain and eye development.

The expression of mFAT1 at the slit junctions, the intracellularjunctions between podocytes in the glomerular filtration membrane,has been reported previously (14). Slit junctions are modifiedadhesion junctions, with the wide intercellular space allowingrenal filtration. Based on the large size of mFAT1 and colocalizationof the tight-junction protein ZO-1 with the FAT cytoplasmicdomain, it has been suggested that mFAT1 acts as both an adhesionmolecule and a spacer at the junction (14). As a consequence,mFAT1 generates the necessary intercellular adhesion betweenpodocyte processes while at the same time maintaining an extracellulargap wider than is normally found in adhesion junctions. Ourfindings with the mFAT1 null mice support this hypothesis. Inthe absence of mFAT1, the ordered array of slit junctions disappearsand the podocytes become closely apposed to one another, definingan essential role for mFAT1 as a slit junction adhesion moleculewith a role in spacing the processes. These abnormalities inpodocyte morphology are similar to those reported for mice lackingnephrin, another transmembrane molecule associated with theslit junction (35). In humans, nephrin mutations cause renalfiltration abnormalities, as evidenced by a congenital nephroticsyndrome (18, 22). Together, these observations in mice andhumans with nephrin mutations show that podocyte fusion, suchas we observed in the mice lacking mFAT1, is associated withsignificant abnormalities of renal function. We conclude, therefore,that this most probably explains the perinatal death of themFAT1 null mice.

Unlike the renal abnormalities, the defects in forebrain andeye development we observed in some of the mFAT1 null mice werecompletely unexpected on the basis of previous studies on thefunction of the protein. The partial or complete loss of theeye does not reflect a failure of the initial developmentalevents of optic vesicle formation from the forebrain, sinceremnants of pigment epithelium were present in the embryos.Since the development of the retinal layers requires a hierarchyof inductive interactions between optic vesicle and overlyingectoderm (40), these early inductive events must have occurrednormally. Rather, the phenotype suggests a subsequent degenerationof the eye, as demonstrated by increased apoptosis as well asby the remnants of some retinal cell layers. The mechanismslinking mFAT1 to the maintenance of the embryonic eye are unknown.mFAT1 is expressed in both the neural retina and the underlyingpigment epithelium and may therefore contribute to any adhesiveinteractions within and between these two cell layers necessaryfor development. The mFAT1 cytoplasmic domain contains bothsequences that bind ß-catenin in a two-hybrid assay(6) and a PDZ motif binding domain (33), thus allowing a numberof potential intracellular signaling pathways to be regulated.Characterization of these pathways requires further experiments,but the interesting observations that the eye phenotype of theBMP-7 null mouse is very similar to the one we observed in ourmFAT1 null mice (9) and that inhibition of BMP signaling inthe chick causes microphthalmia and other abnormalities of opticcup development (1) suggest that FAT1 may interact with theBMP signaling mechanisms.

Holoprosencephaly results from a failure of ventral inductivesignaling in forebrain development (12, 28), and the findingof this developmental abnormality in some of the mFAT1 nullmice demonstrates another important role for FAT in neural development.A failure of sonic hedgehog (Shh) signaling is a well-recognizedcause of holoprosencephaly in mice and humans (5, 39). However,we found no evidence for perturbation of Shh signaling in mFAT1^-/-mice, since experiments on the spinal cord did not show anyalterations in the expression pattern of genes known to be regulatedby the concentration gradient of Shh derived from ventral midlinesources such as the floorplate and notochord (L. Ciani, unpublishedobservations). Two other possible mechanisms for holoprosencepalyin the mice are suggested by previous work. First, in contrastto the spinal cord, where ventrally derived Shh is antagonizedby dorsally derived BMP signaling (23, 24), in forebrain developmentShh and BMP-7 are both produced ventrally by the prechordalmesoderm (or the prechordal plate) and signal cooperativelyto induce ventral midline cells in the overlying neuroectoderm(7). Any alteration in BMP-7 signaling, as suggested by theeye phenotype, could also therefore increase the likelihoodof holoprosencephaly. Second, an early event in forebrain development(before the formation of the prechordal plate) is the inductionof the anterior neural ectoderm by signals transmitted in planarfashion through the developing ectoderm from Hensen's node (32).Studies of Drosophila have shown that FAT is involved in planarsignaling in the eye (10a, 49), and a requirement for a similarrole in neural ectoderm could explain abnormalities of forebraindevelopment in mFAT1 null mice. Further studies of forebrainpatterning in the mFAT1 null mice are clearly required, butthese will be complicated by the low penetrance of the holoprosencephalyphenotype. However, at this stage our results clearly show thatFAT1 mutations are a potential cause of holoprosencephaly inhumans, with FAT1 being the first cell adhesion molecule tobe implicated in this relatively common congenital abnormality(28).

Our studies of the skin and neuroepithelium, both of which containa resident population of proliferating stem and precursor cellsand in which mFAT1 is highly expressed, provide no support forthe role for mFAT1 in growth suppression suggested by the Drosophilastudies (25). The presence of two other FAT genes and the potentialfor compensation in null mice make it impossible to concludethat mFAT1 does not play such a role in normal development.However, expression of these other FAT genes appears to be restrictedto the CNS (27), making a compensatory role in skin less likely.Generation of tissue- and stage-specific knockouts is requiredto examine this question of the role of FAT1 in proliferationmore fully. However, our results do show that significant functionaldifferences may be present between Drosophila FAT and vertebratemFAT1, a point also emphasized by the observation that DrosophilaFAT lacks the PDZ binding motif and may therefore interact witha different repertoire of downstream signaling molecules. Furthergenetic studies of FATs and other protocadherins will definethe roles of this large family of molecules in vertebrates.They may also, as illustrated by our findings on holoprosencephalyand by the association between a human deafness syndrome andmutations in pcdh15 (2, 3), provide further novel insights intohuman diseases.

ACKNOWLEDGMENTS

This work was funded by the Cancer Research Campaign (now CancerResearch UK) and the Wellcome Trust. C.F.-C. is a Wellcome TrustResearch Leave fellow, and A.P. was funded by a studentshipfrom Merck, Sharpe, and Dohme.

We are extremely grateful to Jeremy Skepper for help with theelectron microscopy, to Tony Magee for the gift of antibodiesand many helpful discussions throughout this work, and to IanHorseman for care of the mice.

FOOTNOTES

* Corresponding author. Mailing address: Department of Medical Genetics and Pathology, University of Cambridge, Tennis Court Rd., Cambridge CB2 1QP, United Kingdom. Phone: 44-1223-333723. Fax: 44-1223-333346. E-mail: cfc{at}mole.bio.cam.ac.uk.

REFERENCES

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