817

IntroductionThe Notch gene family encodes transmembrane receptors thatare highly conserved evolutionarily (Kimble and Simpson,1997; Lewis, 1998; Artavanis-Tsakonis et al., 1999). Inmammals, there are four Notch receptors (Notch1-4)(Weinmaster et al., 1991; Weinmaster et al., 1992; Franco delAmo et al., 1992; Lardelli and Lendahl, 1993; Lardelli et al.,1994; Uyttendaele et al., 1996), and two families of ligands,Deltalike1 (Dll1) (Bettenhausen et al., 1996), Dll3 (Dunwoodieet al., 1997) and Dll4 (Shutter et al., 2000), and Jagged1(Lindsell et al., 1995) and Jagged2 (Shawber et al., 1997). Thereceptors and ligands have overlapping expression patterns inmany tissues (Lardelli and Lendahl, 1993; Williams et al.,1995; Lindsell et al., 1996; Johnson et al., 2001). Activation ofNotch by ligand binding triggers cleavage of the receptor in aprocess known as regulated intramembrane proteolysis (RIP),generating the Notch intracellular domain (NotchIC) that thentranslocates to the nucleus (Schroeter et al., 1996; Kopan et al.,1996; Blaumueller et al., 1997; Logeat et al., 1998). In thenucleus, Notch forms transcriptional complexes with CSLtranscription factors and activates the expression ofdownstream target genes (Tamura et al., 1995; Hsieh et al.,1996; Struhl and Adachi, 1998). These target genes are twofamilies of basic helix-loop-helix (bHLH) proteins referred toas hairy enhancer of split (Hes), and a related family variouslyreferred to as Hesr, HRT, Hey, CHF, and Gridlock (Jarriault etal., 1995; Jarriault et al., 1998; Ohtsuka et al., 1999;Nakagowa, 1999; Nakagowa, 2000; Kokubo et al., 1999; Chinet al., 2000; Maier and Gessler, 2000; Zhong et al., 2000).

These proteins have been implicated in the repression of tissue-specific gene transcription (Jarriault et al., 1995; Jarriault et al.,1998; Ohtsuka et al., 1999; Hsieh et al., 1999; Nakagawa et al.,2000; Chin et al., 2000).

The interaction between Notch and its ligands is modulatedby O-linked fucose moieties that are added to the EGF repeatsof the extracellular domain. Usually fucose is unaltered whenit is added to proteins; however, on the Notch receptors fucoseis modified with N-acetylglucosamine (GlcNac) added by theFringe proteins (Moloney et al., 2000; Bruckner et al., 2000).The Fringe proteins are Golgi-localized and belong to a largefamily of β1,3-N-acetylglucosaminyl transferases (Moloney etal., 2000; Bruckner et al., 2000; Schwientek et al., 2002).Enzymes in this family have strong substrate and targetspecificity and diverse functions. The only known targets of themammalian fringe proteins are the Notch receptors(Schwientek et al., 2000). In mammals, there are three fringeproteins, radical (Rfng), manic (Mfng) and lunatic fringe(Lfng) (Johnston et al., 1997). Modification of the extracellulardomain of Notch by Lfng can potentiate or inhibit theinteraction between a particular Notch receptor-ligand pair. Forexample, Lfng potentiates the interaction between Notch1 andDll1, but inhibits Notch1-Jagged1 interactions. However, Lfngpotentiates both Dll1 and Jagged1 mediated activation ofNotch2 (Hicks et al., 2000). Lfng and Mfng reportedly modifydifferent sites in the extracellular domain of Notch2 (Shimizuet al., 2001), indicating they may have different roles to playin regulating Notch signaling.

In Drosophila, two Golgi localized β1,3-N-

We have demonstrated that Notch genes are expressed indeveloping mammalian ovarian follicles. Lunatic fringe isan important regulator of Notch signaling. In this study,data are presented that demonstrate that radical fringe andlunatic fringe are expressed in the granulosa cells ofdeveloping follicles. Lunatic fringe null female micewere found to be infertile. Histological analysis of thelunatic fringe-deficient ovary demonstrated aberrantfolliculogenesis. Furthermore, oocytes from these mutants

did not complete meiotic maturation. This is a novelobservation because this is the first report describing ameiotic defect that results from mutations in genes that areexpressed in the somatic granulosa cells and not theoocytes. This represents a new role for the Notch signalingpathway and lunatic fringe in mammalian folliculogenesis.

Key words: Lunatic fringe, Notch, Ovary, Follicle, Meiosis, Fertility

Summary

Lunatic fringe null female mice are infertile due to defects in meioticmaturationKatherine L. Hahn1,4, Joshua Johnson2,*, Brian J. Beres2,4, Sheena Howard3,4 and Jeanne Wilson-Rawls1,2,4,†

1Molecular and Cellular Graduate Program, Arizona State University, Tempe, AZ 85284-4501, USA2Biology Graduate Program, Arizona State University, Tempe, AZ 85284-4501, USA3Minority Access to Research Careers (MARC) Program at ASU, Arizona State University, Tempe, AZ 85284-4501, USA4School of Life Sciences, Box 4501, Arizona State University, Tempe, AZ 85284-4501, USA*Present address: Vincent Center for Reproductive Biology, Department of Obstetrics and Gynecology, Massachusetts General Hospital, Harvard Medical School,Room 6607, Building 149, 149 13th Street, Charlestown, MA 02129, USA†Author for correspondence (e-mail: nrawls@imap4.asu.edu)

Accepted 1 December 2004

Development 132, 817-828Published by The Company of Biologists 2005doi:10.1242/dev.01601

Research article

Dev

elop

men

t

818

acetylglucosamine transferases, Fringe and Brainiac, playimportant roles in oogenesis and folliculogenesis (Goode et al.,1996a; Goode et al., 1996b; Hicks et al., 2000; Bruckner et al.,2000; Munro and Freeman, 2000; Schwientek, 2002). Brainiacactivity is needed in the germ line for proper organization ofthe follicle (Goode, 1996). Fringe, the homolog of Lfng, isnecessary for specification of the polar cells (Grammont andIrvine, 2001). Brainiac has been demonstrated to modifyglycosphingolipids by adding GlcNac residues to mannose andgalactose moieties on ceramide (Schwientek et al., 2002). Inmice, a null mutation of the murine homolog of brainiacdemonstrated that this protein is important for very earlydevelopment, as braniac–/– embryos die prior to implantation(Vollrath et al., 2001). No role for this family of proteins inmammalian folliculogenesis has been described.

It has been demonstrated that Lfng is an important regulatorof Notch signaling. For example, Lfng null mutants havesegmentation defects that are similar to those seen in nullmutations of Notch1 and Dll1 (Evrard et al., 1997; Zhang andGridley, 1997). In somites, where Lfng is the only familymember expressed, Notch receptors and ligands are expressednormally in Lfng–/– mutants, but the Notch downstream targetgene Hes5 was not detected, indicating a lack of Notchactivation in the presence of ligand. However, Hes5 wasexpressed normally in the neural tube and developing brain ofLfng null mutants, probably due to expression of Mfng andRfng in these tissues (Evrard et al., 1997). Interestingly, Rfng-deficient mice had no phenotype and Rfng/Lfng double nullmutants had only defects associated with a lack of Lfng (Zhanget al., 2000).

Folliculogenesis is the process by which oocytes develop inresponse to hormonal cues. This requires the coordination ofthe proliferation and differentiation of granulosa cells and thegrowth and maturation of the oocyte. Primordial folliclesconsist of a small oocyte surrounded by squamous somaticcells. When recruited to develop, the granulosa cells proliferateand become cuboidal. As these cells continue to proliferate,layers develop around the growing oocyte. Once a follicle hasseveral layers of cells a fluid filled space, the antrum, will beginto form. The antrum spatially separates the two functionallydistinct granulosa cell populations, cumulus and mural. Duringthis time the oocyte has grown and at the time of antrumformation it becomes competent to resume meiosis in responseto luteinizing hormone (LH). Resumption of meiosis is markedby the breakdown of the germinal vesicle (GVB). Meiosiscontinues to metaphase II (MII), and oocytes are blocked atthis stage until fertilization. Studies done in mice havedemonstrated that reciprocal signaling between the oocyte andthe granulosa cells is necessary for the differentiation of thecumulus granulosa cells and meiotic maturation of the oocyte(Rodgers et al., 1999; Erickson and Shimasaki, 2000; Eppig,2001; Matzuk et al., 2002).

We have previously shown that Notch2, Notch3 and Jagged2are expressed in granulosa cells, and Jagged1 is expressed inthe oocytes of developing mammalian follicles (Johnson et al.,2001). Furthermore, transcripts of the Notch downstream targetgenes, Hes1, Hes5, Hesr1, Hesr2 and Hesr3 also were detectedin the granulosa cells of follicle types 3b-8 (Johnson et al.,2001), indicating that Notch signaling was active. As all threemammalian fringe proteins can modify the Notch receptorswhen expressed in the same cell (Bruckner et al., 2000;

Moloney et al., 2000; Hicks et al., 2000), we hypothesized thatthe fringe genes would also be expressed in the granulosa cells,and furthermore, that they would have a role in regulatingfolliculogenesis through Notch2 and Notch3. In this study, wedemonstrate that Lfng is expressed in the granulosa cells andtheca of developing follicles from primary to preovulatory insize. Rfng is expressed briefly in granulosa cells of early antralfollicles. Mfng is only detected in the vasculature. Nullmutations of the Notch receptors and ligands result inembryonic lethal phenotypes (Swiatek et al., 1994; Conlon etal., 1995; Hrabé de Angelis et al., 1997; Jiang et al., 1998;Hamada et al., 1999; Xue et al., 1999; McCright et al., 2001).Some Lfng–/– mice survive to adulthood, therefore weexamined folliculogenesis in these mutants. Female Lfng nullmutant mice have many aberrant follicles. When induced toovulate they released oocytes into the oviduct, but only a smallpercentage could be fertilized in vitro. Examination of theseoocytes demonstrated that cumulus expansion occurred inresponse to exogenous hormones, but the oocytes were not atmetaphase of meiosis II, and had not completed meioticmaturation. Mutations that block the progression of meiosishave been described, but they are all germ-cell-specific genes.These are novel observations because the disregulation ofmeiosis is caused by a change in the somatic cells. Thisrepresents a new regulatory pathway in folliculogenesis and anew role for Notch signaling in mammals.

Materials and methodsMating studyEleven-week-old heterozygous male, and 8-week old null andheterozygous female, mice were paired. Each morning females wereexamined for the presence of a copulatory plug. If a plug was present,the female was removed and a new female introduced to the malecage. If after 6 days no copulatory plug was detected, the female wasplaced with a new male. Copulatory plugs and litter numbers wererecorded and the genotype of the offspring was determined.

Whole-mount thick section in situ hybridization (ISH)Whole-mount ISH was done on thick sections according to Johnsonet al. (Johnson et al., 2001). Briefly, ovaries were fixed in 10% neutral-buffered formalin (NBF) (Richard-Allen Scientific, Kalamazoo, MI),and embedded in paraffin wax after stepwise dehydration in ethanol.Thirty micron (µm) sections were cut perpendicular to the axis ofentry of ovarian blood vessels. Sections were dewaxed, rehydrated andantisense digoxigenin-labeled gene-specific RNA probes werehybridized. Transcripts were identified using anti-digoxigeninantibody (Roche, Indianapolis, IN) conjugated to alkalinephosphatase and the BM purple substrate (Roche, Indianapolis,IN). Replicates were performed on sections from at least 3ovaries/genotype and probes were checked for specificity by ISH onembryos.

HistologyTissues were fixed as above and sectioned to 10 µm. Sections wereprepared by standard procedures and stained with Hematoxylin andEosin.

Bone and cartilage preparationThe mice were skinned and eviscerated. The carcasses were placed inAlcian Blue (Sigma A3157) for 48 hours to stain the cartilage,followed by 2% KOH for 48 hours. Skeletons were then placed inAlizarin Red (Sigma A5533) to stain the bone for 72 hours.

Development 132 (4) Research article

Dev

elop

men

t

819Lfng null females are infertile

Hormone treatment and isolation of OCC and oocytesMice were injected intraperitoneally (ip) with 5 international units(IU) of pregnant mare’s serum gonadotropin (PMSG) (Calbiochem,Carlsbad, CA) and 48 hours later, were injected ip with 5 IU of humanchorionic gonadotropin (hCG) (Calbiochem, Carlsbad, CA). Oocytecumulus complexes (OCC) were harvested from the oviduct 16 hourslater. OCC and ovaries were collected in KSOM (Specialty Media, LaJolla, CA) with 10% FBS. The OCC were incubated in KSOMcontaining hyaluronidase (300 µg/ml) for 30 seconds, then washed inKSOM, and fixed as described in LeMaire-Adkins et al. (LeMaire-Adkins et al., 1997). Briefly, oocytes were fixed in 2% formaldehyde,1% Triton X-100, 0.1 mM PIPES, 5 mM MgCl2, 1 mM DTT and 2.5mM EGTA in D2O (Sigma, St Louis, MO) containing aprotinin(Sigma, St Louis, MO) and taxol (Sigma, St Louis, MO) at 37°C.Oocytes were washed in 0.1% normal goat serum (NGS) in PBS(GIBCO/BRL, Gaithersburg MD) and blocked at 37°C in PBScontaining 10% NGS and 0.1% Triton X-100. Oocytes were stored inthis at 4°C until staining was performed. For staining, oocytes weretransferred to 1% Triton X-100 in PBS at room temperature thenincubated with a monoclonal anti-α-tubulin (clone DM 1A, Sigma, StLouis, MO) conjugated to FITC at a 1:50 dilution. The oocytes werewashed in PBS, incubated in PBS containing 1 µg/ml Hoechst 33258(Molecular Probes, Eugene, OR). Oocytes were washed in PBS andmounted in glycerol with p-phenylenediamine and visualized byconfocal microscopy. Confocal analysis was done using a Leica TCSNT, final magnification of 800�. The FITC was visualized using anAr laser, and Hoechst 33258 was visualized using an UV laser.

In vitro fertilization (IVF)OCC were collected after hormone administration as above. Spermwere collected from the vas deferens and cauda epididymis in humantubal fluid (HTF) and capacitated for 2 hours at 37°C. Sperm (1�106)were added to each OCC sample and fertilization allowed to proceedfor 2 hours at 37°C. Eggs were washed three times in sperm freeKSOM and incubated at 37°C. Eggs were scored as fertilized by thepresence of two pronuclei and embryogenesis was scored daily.

Immunohistochemistry (IHC)Sections were heated at 80°C for 30 minutes, cooled to roomtemperature, followed by xylenes, rehydrated through graded alcoholsto 70% ethanol. Slides were incubated in water, then PBS, placed in0.1 M sodium citrate (pH 6) and epitope retrieval done in themicrowave. The sections were cooled to room temperature, rinsed inPBS, and incubated in 3% H2O2 in 60% methanol to destroyendogenous peroxidases. IHC was performed using the HistostainSPkit according to the manufacturer’s instructions (Zymed Labs, SanFrancisco, CA), and primary antibodies were diluted according to thisprotocol except for the following: polyclonal anti-c-Kit antibody (Ab-1, Calbiochem, Carlsbad, CA) was diluted 1:25, and anti-connexin43(Santa Cruz Biotechnology, Santa Cruz CA), 1:50. Proteins weredetected with alkaline-phosphatase-conjugated anti-rabbit secondaryantibody and exposed to colour reagent. No primary antibody controlswere included in each experiment.

Reverse transcription polymerase chain reaction (RT-PCR)Total ovary RNA was isolated using TRIzol (Life Technologies,Gaithersburg, MD), according to the manufacturer’s directions, from3 different animals/genotype. For oocytes, 15 oocytes per sample weredenuded using hyaluronidase and total RNA extracted. cDNA wassynthesized using Superscript III (Invitrogen, Carlsbad CA),according to the manufacturer’s protocol. For each gene examined bysemi-quantitative (sq) RT-PCR, 3 sets of samples comprising all threegenotypes and no RT controls were amplified using α-[32P]dATP(Perkin-Elmer Life and Analytical Sciences, Boston, MA). For eachgene-specific primer pair the minimum number of cycles to the linearrange was determined and used for all subsequent experiments. Allprimer sets span at least one intron. Control experiments were done

using total embryo RNA. All cDNA samples were normalized usingthe ribosomal gene L7 (Meyuhas et al., 1990), and quantified using aStorm 860 PhosphorImager and ImageQuant software (MolecularDynamics, Sunnyvale, CA). To detect the presence of transcripts inoocytes, a qualitative PCR protocol and amplification beyond thelinear range after normalization was used (Münsterberg and Lassar,1995).

Kinase assaysKinase assays were carried out as described in Svoboda et al.(Svoboda et al., 2000). Single eggs were transferred in 1.5 µl ofKSOM into 3.5 µl of double kinase lysis buffer (10 µg/ml aprotinin,10 µg/ml leupeptin, 10 µM p-nitrophenyl phosphate, 20 mM β-glycerophosphate, 0.1 mM sodium orthovanadate, 5 mM EGTA) andimmediately frozen in liquid nitrogen, then stored at –80°C until theassay was performed. The kinase reaction was initiated by the additionof 5 µl of double kinase buffer (24 mM p-nitrophenyl phosphate, 90mM β-glycerophosphate, 24 mM MgCl2, 24 mM EGTA, 0.2 mMEDTA, 4.6 mM sodium orthovanadate, 4 mM NaF, 1.6 mMdithiothreitol, 60 µg/ml aprotinin, 60 µg/ml leupeptin, 2 mg/mlpolyvinyl alcohol, 2.2 mM protein kinase A inhibitor peptide (Sigma),40 mM 3-(nmorpholino) propanesulfonic acid (MOPS), pH 7.2, 0.6mM ATP, 2 mg/ml histone (type III-S, Sigma), 0.5 mg/ml MBP with500 mCi/ml γ-[32P]ATP (3000 Ci/mmol) (Perkin-Elmer Life andAnalytical Sciences). To determine the background level ofphosphorylation, 5 µl of double kinase lysis buffer was added insteadof egg lysate. Reactions were incubated for 30 minutes at 30°C, andterminated by the addition of 10 µl 2�SDS-PAGE sample buffer andboiling for 3 minutes. Following 15% SDS-PAGE, the gel was driedand exposed to a phosphorimager screen and quantified. The meanvalue of the control samples was set to one and all others expressedas fold activity of control.

Quantifying cumulus expansionOCC were collected from the oviducts of Lfng+/– and Lfng–/– mice(n=5/genotype) post-hormone administration. OCC werephotographed at 70� and photomicrographs printed the same size.The widest diameter of each OCC was measured in mm and meandiameter±s.d. was determined.

Statistical analysisAnalysis was carried out using the SAS system and the FREQprocedure. Our data was found to be significant (P<0.0001) by Chi-square, likelihood ratio Chi-square, Continuity-adjusted Chi-squareand Mantel-Haenszel Chi-square analysis.

ResultsExpression of lunatic fringe in the murine ovaryIn mammals, the three Fringe proteins are often expressed inoverlapping and distinct patterns within the same tissue(Johnston et al., 1997; Ishii et al., 2000). Lfng modification ofthe Notch extracellular domain is known to potentiateinteractions between Notch2 and jagged 1 (Hicks et al., 2000).However, its effect on the interactions between Notch2 andjagged 2, or between any ligand and Notch3 is uncharacterized.Rfng has been shown to be completely redundant to Lfng(Zhang et al., 2000). The role of Mfng is not well known, butit modifies different sites on the extracellular domain of Notch1to Lfng (Shimizu et al., 2001). Knowing the importance of thefringe proteins in modulating Notch signaling, and havingdemonstrated the expression of Notch2 and Notch3, the jaggedligands and the downstream target genes of Notch in developing

Dev

elop

men

t

820

follicles (Johnson et al., 2001), it was logical to examinewhether the fringe genes were also expressed in the ovary.

In order to determine which cells in the ovary expressed Lfng,whole-mount thick section in situ hybridization (ISH) wascarried out using antisense digoxigenin-labeled RNA probes, asdescribed by Johnson et al. (Johnson et al., 2001). Using twodifferent probes, one that included 3′ untranslated sequencesand another that only included the coding region, Lfng mRNAwas detected in the granulosa cells of follicles from type 3 tothose preovulatory in size (Fig. 1A,B). Interestingly, in smallgrowing follicles, Lfng transcripts clearly demarcate the outeredge of the follicle (Fig. 1B, inset). In antral follicles, Lfngexpression in the theca was evident (Fig. 1A,B). Furthermore,this gene was expressed in blood vessels (Fig. 1B, red arrow),so it is possible that the expression noted in the theca is also inthe vascular component. Lfng was not expressed in oocytes,primordial or primary follicles, or corpus luteum (CL). The lackof Lfng transcripts in oocytes was confirmed by RT-PCRperformed using total RNA from denuded germinal vesicle(GV) stage oocytes and ovulated MII eggs (Fig. 1F).

Using RT-PCR of total ovary RNA, both Rfng and Mfngtranscripts were detected (Fig. 1E). ISH done with an antisenseMfng probe demonstrated that transcripts for this gene weredetected only in the vasculature (Fig. 1D). Rfng transcripts weredetected in the vasculature and transiently in granulosa cells ofearly antral follicles (Fig. 1C). These data demonstrate that Lfngand, briefly Rfng, overlap in expression with the Notch receptorsin the granulosa cells. The transient expression of Rfng, the factthat Rfng null mutants have no phenotype, and the fact thatRfng/Lfng double null mutants have only defects associatedwith a lack of Lfng (Zhang et al., 2000), indicate that Lfng is

likely to be the more important family member expressed in theadult mouse ovary.

Lunatic fringe-deficient ovaries have aberrantfolliclesThe role of Notch signaling in the ovary is unknown, and nullmutants of most of the Notch receptors and Jagged ligands haveembryonic or perinatal lethal phenotypes (Swiatek et al., 1994;Conlon et al., 1995; Hrabé de Angelis et al., 1997; Jiang et al.,1998; Hamada et al., 1999; Xue et al., 1999; McCright et al.,2001). Lfng is an important modifier of Notch signaling (delBarco Barrantes et al., 1999; Hicks et al., 2000; Moloney et al.,2000); in its absence, Notch signaling in the somites wasimpaired, as determined by the lack of Hes gene expression inLfng–/– embryos (Evrard et al., 1997). Furthermore, Lfng nullmutants have segmentation defects that are consistent withmutations in Dll1 and Notch1 (Zhang and Gridley, 1997; Evrardet al., 1997). Interestingly, of the two different Lfng–/–

mutations, one results in complete embryonic lethality ofLfng–/– offspring (Zhang and Gridley, 1997), whereas the otherhas a 25% survival rate (Evrard et al., 1997); we studied the roleof Lfng and the Notch signaling pathway in folliculogenesisusing the latter mutant.

Initially studies were carried out to determine whether femaleLfng-deficient mice would mate and produce litters. Lfng nulland heterozygous mice were paired with Lfng+/– male mice.All heterozygous females (n=11) mated within 6 days, asdetermined by the presence of a copulatory plug. There were26 litters from heterozygous pairings; by comparison, over thesame time, null females demonstrated neither copulatory plugsnor pregnancies. These observations indicated the possibility of

Development 132 (4) Research article

Fig. 1. Lunatic fringe is expressed in thegranulosa cells and theca. Whole-mountthick section ISH of wild-type 42-day-oldovary was carried out using antisensedigoxigenin-labeled RNA gene-specificprobes. (A) Lfng-specific probes including3′ untranslated sequences and Lfngtranscripts were detected in follicles fromtype 3 to preovulatory in granulosa cells,but not in oocytes or interstitial cells.(B) Lfng exon only probe. Transcriptswere detected in the theca and in thevasculature (red arrow indicates positiveblood vessel), a band of Lfng transcriptssurrounding small follicles was observed(inset). (C) Rfng was expressed transientlyin the granulosa cells of antral follicles(arrow). (D) Mfng transcripts were onlydetected in the vasculature (red arrow).(E) RT-PCR from total ovary RNAdemonstrates the presence of Mfng andRfng transcripts. E9.5 represents total E9.5embryo control; L+/+ represents totalovary RNA from a 42-day-old wild-typeanimal; mock represents no RT control.(F) RT-PCR of Lfng and Gdf9 transcriptsfrom total RNA from GV and MII stageoocytes. E9.5, total embryo control; MW,100 bp ladder. Scale bars: 100 µm.

Dev

elop

men

t

821Lfng null females are infertile

a fertility defect. However these mice haveabnormalities of their axial skeletons, includingfusions of the vertebrae and kyphosis, which maymake lordosis and mating impossible (Evrard etal., 1997; Zhang and Gridley, 1997) (see Fig. S1in the supplementary material).

In order to determine whether infertility wasdue to either defects in ovary development orfolliculogenesis in the Lfng–/– mice, grossmorphological and histological examination ofovaries from neonatal and adult Lfng–/– and Lfng+/–

mice was done. At the gross morphological level,the ovaries and reproductive tracts of 4- and 7-week-old Lfng null mice were smaller thanheterozygous littermates, but no abnormalitieswere noted. Protuberances on the surface of theovaries indicated the presence of developingfollicles (Fig. 2A,B). Lfng null mice have spinaldefects that result in a shortened body axis, sosmaller organs are not unexpected. The ovarianhistomorphology of neonatal Lfng null mutantswas disorganized compared with heterozygouslittermates (Fig. 2, compare C with D), butprimordial follicles were clearly present. Ovariesfrom sexually mature Lfng–/– mice had developingfollicles of all sizes, but there were many abnormalfollicles present. For example, there werepolyovular follicles (Fig. 2F-H, red arrows in F).Furthermore, there were follicles that lacked a complete layerof theca developing next to the theca of other follicles or sharingtheca, but not truly polyovular (Fig. 2E,F, black arrows in F).Many of these follicles appeared atretic. CL were noted, but,there were also many large lutealinized follicles with trappedoocytes (Fig. 2I,J).

As there were many oocytes trapped in lutealinized folliclesin the Lfng-deficient mice, and we never detected copulatoryplugs, it was possible that these mice did not ovulate.Alternatively, if they did ovulate, the oocytes might not becompetent for fertilization and subsequent developmentbecause folliculogenesis was aberrant. In order to examinethese questions, 42-day-old Lfng null and control littermates

were given PMSG and hCG to induce ovulation, and OCCwere harvested from the oviduct. The OCC were incubatedwith capacitated heterozygous sperm. Only fertilized eggs, asdetermined by the presence of two pronuclei, were kept, andembryogenesis was scored daily.

Lfng null mice released approximately the same number ofeggs with cumulus cells as controls in response to exogenoushormones, and cumulus expansion was evident (Table 1, Fig.3C). After fertilization, 48.8% of wild-type eggs became two-cell embryos, but only 9.7% of null eggs did. Furthermore,whereas 41.9% of heterozygous embryos continued to developto the four- to eight-cell stage, and 31.4% became blastocysts,only 2% of the null embryos became four- to eight-cell

Fig. 2. Histological and morphological examination ofLfng–/– ovaries. (A) Ovaries from 4-week-old Lfng+/–

and Lfng–/– mice; null ovaries are smaller anddeveloping follicles are obvious in both. (B) Thereproductive tracts of 7-week-old Lfng+/– and Lfng–/–

mice; all structures were present and no grossabnormalities were noted. (C,D) Histological sectionsof neonatal ovaries stained with Hematoxylin andEosin. Primordial follicles are evident; however, theLfng null ovary (C) is smaller than the Lfng+/– ovary (D)and the morphology is not as well organized.(E-J) Histological sections of ovaries from 42-day-oldLfng–/– mice. Many abnormal follicles were noted.Some large follicles had smaller follicles within theirboundaries (E, black arrow). There were follicles thatshared theca or had incomplete theca (F, blackarrowheads). There were polyovular follicles containingtwo or three oocytes (F, red arrows; G,H). There werealso many large lutealinized follicles with trappedoocytes (I,J). Scale bars: 100 µm.

Dev

elop

men

t

822

embryos, and none developed into blastocysts (Table 1). TheLfng null mutants can respond to exogenous hormone, but theyhad a very low fertilization rate and there may be a block inearly development. A lack of fertilization and developmentsuggests a defect in folliculogenesis.

Meiotic maturation is compromised in Lfng femalenull miceOocyte maturation occurs in antral follicles in response to thesurge of LH from the pituitary. The first step of maturation isGVB, after which the first meiotic spindle forms. The spindleis accentric at the oocyte cortex, resulting in conserved, limitedcytokinesis with the outside spindle pole protruding throughthe oocyte surface into the perivitelline space. At the end of thefirst meiotic division, one set of homologous chromosomesremains within the oocyte and the other set is abstricted in the

first polar body. After formation of the second meiotic spindle,meiotic maturation is completed (Robker and Richards, 1998;Erickson and Shimasaki, 2000; Matzuk et al., 2002). Eggsreleased into the oviduct should be at metaphase II (MII), sowe determined which stage of meiosis Lfng null eggs hadreached by ovulation.

Exogenous hormones were used to induce ovulation in Lfngnull and control littermates, and OCC were harvested from theoviduct, as described above. Eggs were fixed and stained withanti-α-tubulin antibody conjugated to FITC; the chromatin wasstained with Hoechst 33258. We found that 77.8% and 88.2%of heterozygous and wild-type eggs, respectively, were in MII,as determined by the presence of a barrel-shaped meioticspindle with chromosomes on the metaphase plate and a polarbody, but only 5% of Lfng–/– eggs were in MII (P<0.0001, nullcompared with controls). Most of the oocytes from Lfng–/–

mice were at metaphase I (MI), anaphase/telophase I, or hadmultiple bodies with chromatin fragments throughout (Fig.3A). These eggs were not characteristically parthenogenic,there were no obvious nuclei, nor polar bodies, none were twocells (Hirao and Eppig, 1997), and the diffuse chromatin wasindicative of apoptosis. These data indicated that in Lfng-deficient follicles, oocytes were not completing meioticmaturation prior to induced ovulation. Interestingly, theseoocytes have a normally expanded cumulus (Fig. 3C): themean diameter of the OCC from Lfng heterozygous andnull mice was not different when compared (mean diameter

Development 132 (4) Research article

Fig. 3. Lfng–/– females ovulate in response toexogenous hormones, but oocytes are not at meioticmetaphase II. (A) After hormone administration, OCCwere collected from the oviduct. Oocytes were stainedwith anti-α-tubulin-FITC and Hoechst 33258, andvisualized using confocal microscopy at 800� finalmagnification. Genotypes are as indicated. In Lfng+/+

and Lfng+/– oocytes, note the presence of the polarbodies (white arrowhead) and chromosomes alignedon a metaphase spindle. Many Lfng–/– oocytescontained one or more bodies with scattered chromatin(white arrowheads). Some Lfng–/– oocytes were atanaphase/telophase I; note the lack of a polar body.(B) Representative kinase assays analyzed on 15%SDS-PAGE and visualized using a phosphorimager.Lanes are as marked. MBP, myelin basic protein.(C) Lfng+/– and Lfng–/– mice were induced to ovulateand OCC were collected from the oviducts. OCCdemonstrated normal cumulus expansion in Lfng nullmutant females, under light microscopy at 70�magnification. (D) Graphs show relative kinaseactivity of MPF and CSF, the kinase assays werequantified and expressed as fold activity over control(n=9/genotype) ±s.d.

Table 1. Embryonic development of Lfng–/– eggsGenotype Lfng+/– Lfng–/–

Total eggs 86 124Percent two-cell embryos (number/total) 48.8% (42/86) 9.7% (12/124)Percent four- to eight-cell embryos 41.9% (36/86) 1.6% (2/124)

(number/total)Percent blastocysts (number/total) 31.4% (27/86) 0% (0/124)

Eggs were harvested from the oviducts after hormone administration andIVF was performed with 1�106 heterozygous sperm. Fertilized eggs wereallowed to develop to blastocysts in culture. Development was scored daily.

Dev

elop

men

t

823Lfng null females are infertile

heterozygous: 3.8±0.4 mm; null: 3.4±0.4 mm; P=0.1) (see Fig.S2 in the supplementary material).

To further examine oocyte maturation in the Lfng null ovary,the level of maturation promoting factor (MPF) and cytostaticfactor (CSF) kinase activities was determined. MPF isnecessary for GVB, and its activation precedes CSF activation.Mos activates MAP kinase, which is a component of CSF andis necessary for the MII block. MPF and CSF activity both peakin MII eggs (Zhao et al., 1991; Verlhac et al., 1993; Gebauerand Richter, 1997; Sagata, 1997). Eggs were collected fromoviducts post-hormone administration, and kinase activitydetermined in null and control eggs (n=9, representative datain Fig. 3B). Both MPF and CSF kinase activity was evidentin null oocytes, but it was less than in controls (Fig. 3D).Consistently, 75% of Lfng+/– (n=364) and 74.9% of Lfng–/–

(n=355) (P=1) oocytes, underwent GVB in vitro within 2hours, thus null oocytes resume meiosis normally.

It is possible that null oocytes have a defect that blocksprogression through meiosis, so GV stage oocytes werecollected from large follicles post-PMSG administration andallowed to mature in vitro, as described by LeMaire-Adkins etal. (LeMaire-Adkins et al., 1997). Only oocytes that underwentGVB within 2 hours were followed, in order to have asynchronous cohort. After 10, 12 and 16 hours in culture,oocytes were fixed and stained and the stage of meiosis wasdetermined. Oocytes from heterozygous ovaries progressedthrough meiosis comparably to previously published data(LeMaire-Adkins et al., 1997), with 40.4% at MII at 10 hours,57% at 12 hours and 93.6% at 16 hours (Table 2). Oocytes fromnull ovaries reached MI quickly (75% at 10 hours), but fewprogressed to MII. At early timepoints, there were oocytesprogressing through telophase/anaphase I, but by 16 hours inculture this stage was not detected; possibly these cells diequickly if progression to MII does not occur. We did not detectan increase in parthenogenically activated oocytes (4.3% ofcontrol and 6.5% of null, P=0.13).

Notch family gene expression in Lunatic fringe-deficient folliclesIn the somites of Lfng–/– embryos, the expression of Notch1and Dll1 were not altered; however, the Notch downstreamtarget gene Hes5 was not expressed (Evrard et al., 1997). Thus,a lack of Lfng blocked Notch activation in the presence ofligand in the somites. Also, disruption of the somites andsomite-derived tissues in Lfng-deficient mice was consistentwith the phenotypes of Notch1–/– and Dll1–/– mutants (Evrardet al., 1997; Zhang and Gridley, 1997). As Lfng is thepredominant family member expressed in granulosa cells, it

was logical to ask whether Notch signaling pathway geneswere altered in expression and whether Notch signaling wasinhibited in the Lfng-deficient granulosa cells.

To examine the effect of a lack of Lfng on the Notch pathwayin the ovary, the expression of Notch receptor and ligand geneswas determined by sqRT-PCR, using whole ovary RNA, andcompared with the results obtained from ISH. RepresentativesqRT-PCR from three replicates is presented in Fig. 4. Notch2and Jagged2 demonstrated no change in expression level (Fig.4), and no change in their cell or follicle stage-specific patternof expression when examined by ISH (data not shown). Notch3had a slightly reduced level of expression by sqRT-PCR, butno change in expression was detected by ISH (Figs 4, 5).Jagged1 was expressed at greatly reduced levels in Lfng-deficient ovaries. This gene was still oocyte restricted, but wasexpressed only in very small follicles (Figs 4, 5). No change inthe expression level of Notch2 and Jagged2, and a relativelysmall reduction in Notch3, is consistent with observationsreported in the somites of Lfng–/– embryos (Evrard et al., 1997).

Lfng has been demonstrated to potentiate interactionsbetween Notch2 and Jagged1 and Dll1 (Hicks et al., 2000). Asstated previously, in the somites, where Lfng is the only fringe

Table 2. In vitro maturation of oocytesTime in culture 10 hours 12 hours 16 hours

Stage of meiosis +/– –/– +/– –/– +/– –/–

Prophase 0 6.5% 10% 0 0 16.9%Metaphase I 48.9% 75% 25% 79.3% 2.1% 64.6%Telophase/anaphase I 10.6% 5% 10% 1.7% 4.3% 0Metaphase II 40.4% 14% 57% 19% 93.6% 18.5%n 47 107 60 58 47 65

P<0.001 P<0.001 P<0.001 P<0.001 P<0.001 P<0.001

GV stage oocytes were collected from 23-day-old mice 48 hours post-PMSG administration by fine-needle aspiration of large follicles. Oocytes were fixed andstained, and stage of meiosis evaluated by confocal microscopy.

Fig. 4. sqRT-PCR examination ofgene expression in Lfng–/– ovaries.sqRT-PCR with gene-specificprimers was performed to detecttranscripts in total ovary RNAfrom 42-day-old mice of all threegenotypes (Lfng+/+, Lfng+/– andLfng–/–). Three samples pergenotype were normalized againstthe ribosomal gene L7 andrepresentative data are presented.N2, Notch2; N3, Notch3; J1,Jagged1; J2, Jagged2; EP2,prostaglandin type 2 receptor(Ptger2 – Mouse GenomeInformatics); Fshr, folliclestimulating hormone receptor.

Dev

elop

men

t

824

gene expressed, the downstream target gene Hes5 was notexpressed. No data regarding Hes1 or the Hesr family of Notchtarget genes is available. As the ovary primarily expresses Lfng(Fig. 1), the expression of the downstream target genes wasassessed as a measure of Notch signaling activity. Thedownstream target genes Hes1, Hes5, Hesr1 and Hesr2 weregreatly reduced by sqRT-PCR and were undetectable by ISH(Figs 4, 5). Interestingly, Hesr3 was unchanged in its expressionlevel (Fig. 4), and there was no change in its expression pattern(data not shown).

Expression of other follicle genes in the Lfng –/–

ovaryReciprocal signaling between the oocyte and granulosacells is crucial for folliculogenesis and oocytematuration (Gosden et al., 1997; Erickson andShimasaki, 2000; Su et al., 2002; Su et al., 2003). Lfng-deficient oocytes do not mature normally, so possiblyother important signaling pathways may be altered inthis mutant. Kit ligand (KL) is expressed in granulosacells and the Kit receptor (previously known as c-Kit) isexpressed in oocytes, in follicles from primordial topreovulatory in size. In antral follicles, KL is onlyexpressed in the mural population. Signaling throughthis pathway has been implicated in differentiation ofthe theca, proliferation of granulosa cells and meioticmaturation (for a review, see Rawls et al., 2001). KL didnot change in expression, and there was no changedetected in Kit receptor expression in the Lfng nullovary when compared with controls (Fig. 6).

Signaling through gap junctions is crucial forfolliculogenesis (Anderson and Albertini, 1976; Gilula etal., 1978; Simon et al., 1997; Juneja et al., 1999; Ackertet al., 2001). Connexin43 (Cx43) is the major connexin ingap junctions between granulosa cells and betweengranulosa cells and oocytes, and folliculogenesis isarrested very early in Cx43 null mutant mice (Juneja et al.,1999; Ackert et al., 2001). An anti-Cx43 antibody wasused for IHC and Cx43 was detected in the granulosa cellsand oocytes of follicles of both Lfng+/– and Lfng–/– ovaries(Fig. 6), indicating that gap junctions are present. Thepresence of antral follicles in the mutant ovaries and theobservation of ovulation in response to exogenoushormones would further indicate that the gap junctions arefunctional.

Signaling through the FSH receptor (Fshr), which isexpressed by every granulosa cell, is necessary for normaldevelopment of the ovary and for normal sexualmaturation. Mice with null mutations in the Fshr gene(FORKO) demonstrate hypergonadotropic hypogonadism(Danilovich et al., 2000). When expression of Fshr wasexamined by sqRT-PCR, no change was detected (Fig. 4).Because the FORKO mice demonstrate only primary andsmall preantral follicles and no CL (Danilovich et al.,2000), these data are consistent. As there was no changein the expression of many genes that are important forfolliculogenesis, we conclude that the defects are due to alack of Lfng and decreased Notch signaling in the folliclesof these mutants.

DiscussionWe determined that Lfng had overlapping expression withNotch2 and Notch3 in granulosa cells, but not in oocytes, ofstages 3-8 follicles (Fig. 1). There is no information regardingthe role of Lfng in interactions between any Notch receptor andJagged2, or between Notch3 and any ligand. But, with theexception of Hesr3, all of the downstream target genes of Notchwere downregulated in the absence of Lfng (Figs 4, 5). Hesr3expression may be induced by the transient expression of Rfngin follicles or through non-Lfng-dependent Notch signaling. Theearly downregulation of Jagged1 expression in oocytes may

Development 132 (4) Research article

Fig. 5. Expression of Notch familygenes in the Lfng–/– ovary by ISH.Gene-specific digoxigenin labeledprobes were used, as indicated;representative data from aminimum of three replicates ofLfng+/– and Lfng–/– are presented.Notch3 demonstrated no change infollicle stage or cell-type-specificexpression in null ovaries; black arrows indicate granulosa cell staining andthe red arrowhead indicates expression in an early CL. Jagged1 was limitedto oocytes of small growing follicles in the Lfng–/– ovary (arrowhead).Expression of Notch downstream target genes Hes5, Hes1, Hesr1 andHesr2 was not detected in the Lfng–/– follicles (only representative familymembers Hes5 and Hesr2 are presented). Note positive follicles indicated byarrows in +/– and compare with similarly sized follicles indicated byarrowheads in –/–.

Dev

elop

men

t

825Lfng null females are infertile

indicate that a lack of Notch signaling either activates aninhibitory signal that turns off Jagged1 or that Notch activity isnecessary to maintain expression of Jagged1. It has beendemonstrated that signaling between Notch and its ligandspositively regulates the expression of both genes, i.e. an increasein Notch and the ligand in their respective cells (Piddini andVincent, 2003).

We have demonstrated that Lfng–/– female mice are infertile.Furthermore, there is an interesting disconnection betweencumulus expansion and GVB and completion of meioticmaturation. Observations of null oocytes indicated a lack ofprogression from MI to MII (Table 2; Fig. 3). These data indicatean important role for Lfng, and thereby for Notch signaling, infolliculogenesis.

Lfng deficiency results in morphological defects offolliclesThe defects noted in developing follicles are unusual, andindicate that there is a problem with the definition of bordersbetween follicles. In mice, early germ cells are found in cystswith interconnecting ring canals. At embryonic day (E) 13.5 toE19.5, germ cells are found in cysts of eight cells or more, bypostnatal day 3, almost all germ cells are single oocytes inprimordial follicles (Pepling and Spradling, 1998; Pepling andSpradling, 2001). In the process of becoming single oocytes, thecysts break down through apoptosis of single germ cells. Thecurrent model posits that this results in cysts of three, and eachof these germ cells will become a primordial follicle. Thesomatic cells of the ovary are important for ring canal breakdown

and formation of primordial follicles (Pepling andSpradling, 2001). In Lfng-deficient ovaries there arepolyovular follicles, this may indicate a role forNotch signaling in the organization of primordialfollicles. Furthermore, the ring of Lfng transcriptsthat we observed around small growing follicles (Fig.1) may indicate a process of early boundaryformation that is not occurring correctly in mutantfollicles. Similarly, during oogenesis in Drosophila,the polar cells are required to separate germ cell cystsand enclose them appropriately in somatic cells.Fringe-deficient mutants had compound folliclescontaining more than one egg. They also had adisorganized polar epithelium; instead of a singlelayer there were multiple layers of cells (Grammontand Irvine, 2001). Thus, these data point toevolutionary conservation of function for thispathway in the development of follicles.

Signaling through the Notch pathway isnecessary for completion of meioticmaturationIt is possible that the trapped oocytes in lutealinizedfollicles reflect a pituitary defect. However, in thehypogonadal (hpg) mouse, FSH and LH are notsecreted by the pituitary, and there is a lack ofpostnatal ovary and follicle development (Halpin etal., 1986a; Halpin et al., 1986b). Grafting neuraltissue or providing exogenous hormones to hpgfemale mice completely reverses this phenotype;folliculogenesis is initiated and results in MII eggsthat are competent for fertilization and embryonicdevelopment (Charlton, 1987; Hashizume et al.,1995). The hpg phenotype is significantly differentthan the Lfng null females in several ways. First, Lfngnull mice demonstrated postnatal ovary developmentand had developing, albeit abnormal, follicles (Fig.2). Second, providing exogenous hormone to Lfng–/–

females induced ovulation, cumulus expansion andGVB, but did not induce complete meioticmaturation, and these eggs had a low fertilization rate(Tables 1, 2; Fig. 3). These observations indicate thatLfng–/– mice have a defect at the level of the follicle.

Reciprocal signaling between the oocyte and thesomatic granulosa cells is necessary for meioticmaturation of the oocyte and differentiation of thecumulus cells. Work by several groups has

Fig. 6. Expression of other follicle-specific genes. Connexin43 (Cx43)was detected by IHC using an alkalinephosphatase-conjugated secondaryantibody and DAB substrate, asindicated by the red stain in granulosacells and oocytes. There was noalteration in Cx43 expression. Kitligand is expressed normally in Lfng–/–

granulosa cells. Kit ligand transcriptswere detected by ISH, note the purplestain in granulosa cells, dark fieldmicroscopy. Kit receptor (c-Kit) was also unaffected in Lfng null follicles. Kitreceptor was detected by IHC, arrowhead in +/– indicates a group of primaryfollicles with positive oocytes. Controls included no primary antibody, note thelack of red stain.

Dev

elop

men

t

826

demonstrated that these events are regulated by the interplay ofseveral different pathways. For example, recombinant Gdf9 caninduce the expression of has2 and cox2, and cumulus expansion,but not GVB (Elvin et al., 1999; Su et al., 2002; Su et al., 2003).Furthermore, gonadotropin induced activation of ERK1/2 ingranulosa cells is necessary for cumulus expansion and meioticmaturation, and yet both of these events also require a signalfrom the oocyte (Su et al., 2002; Su et al., 2003). This suggeststhat there is some signal that is switched on or off in oocytesduring the final stages of folliculogenesis that must activate asignal(s) from the cumulus cells to the oocyte that is necessaryfor GVB and meiotic maturation (Su et al., 2003). It is possiblethat activation of Notch, in the granulosa cells, by Jagged1 in theoocyte, regulates some of these signals. This idea is furthersupported by the observation that oocytes from preantral folliclescultured with cumulus cells can activate ERK1/2, but thatcumulus expansion does not occur, whereas fully grown oocytescan activate ERK1/2 and induce expression of cox2 and cumulusexpansion when cultured with cumulus cells (Vanderhyden et al.,1990; Joyce et al., 2001). Normally, expression of Jagged1 inthe oocyte is downregulated as follicles develop an antrum(Johnson et al., 2001). This is the point at which the oocyte isacquiring the competence to resume meiosis and cumulus cellswould begin differentiating. But, in oocytes from Lfng nullmutants, the expression of Jagged1 is restricted to very smallgrowing follicles, indicating a change in the reciprocal signalingbetween the germ and somatic cells (Fig. 5).

The meiosis defect and trapped oocytes reflect alterations inthe granulosa cells caused by a lack of Notch signalling, whichleads to temporal changes in, or a lack of, some signal(s) fromthese cells. As Notch signaling can regulate lineage decisions, itis possible that the defects observed are due to alterations in muraland cumulus cell populations in Lfng-deficient follicles. In theabsence of normal Notch signalling, the granulosa cells couldbecome predominantly cumulus at the expense of mural cells, asexpansion occurs in response to hormone. Alternatively, it couldbe a temporal change in cell differentiation: a lack of Notchsignaling may allow for differentiation of the cumulus lineage insmall growing follicles instead of early antral follicles. This couldalter the timing of a crucial signal, and the oocyte may be unableto appropriately detect or respond to it. This would be consistentwith the noted loss of Jagged1 expression in small growinginstead of early antral follicles that we observed (Figs 4, 5). Thisis also consistent with data from other systems, changes in lineagechoice mediated by Lfng alteration of Notch signaling has beendemonstrated in lymphoid progenitors. Transgenic expression ofLfng inhibited Notch1 activation and caused the cells to choosethe B lineage at the expense of the T lineage (Koch et al., 2001).Future studies will be necessary to determine whether there arechanges in the mural and cumulus populations.

We determined that oocytes from Lfng-deficient ovariesresume meiosis in response to exogenous hormones, but do notcomplete it. MPF and CSF kinase activity were decreased withrespect to control oocytes. However, there is enough MPFactivity for meiosis to resume (Fig. 3, Table 2). There are no datathat demonstrate that a reduction of CSF activity will blockprogression of meiosis. On the contrary, mos null oocytes do notarrest at MII, they continue to divide and activateparthenogenically. Mos is a kinase that is necessary for MAPKand CSF activity in mouse oocytes (O’Keefe et al., 1989; Zhaoet al., 1991;Verlhac et al., 1993; Verlhac et al., 1996; Colledge

et al., 1994; Hashimoto et al., 1994). Other germ-cell-specificgenes also have roles in progression through meiosis. Forexample, cks2, a Cdk1 binding protein, is necessary for entry toanaphase I, and cks2–/– oocytes do not progress past MI (Sprucket al., 2003). There are many genes necessary for progression ofmeiosis that participate in synapsis and recombination (Hunt andHassold, 2002). Importantly, in all cases these are oocyte-specific genes, Lfng is not expressed in the oocyte (Fig. 1) it isexpressed in granulosa cells, as are Notch2 and Notch3, itstargets. Our data indicate a fundamental change in the signalingbetween somatic granulosa cells and oocytes in Lfng-deficientfollicles. Disregulation of meiosis through a somatic-cell-specific signal is a novel observation. These data reinforce theimportance of reciprocal signaling between the granulosa cellsand the oocyte during folliculogenesis. Altered Notch signalingresults in the loss of normal meiotic maturation, but not cumulusexpansion and GVB. Collectively, these data demonstrate thatLfng and the Notch signaling pathway play important roles inmammalian folliculogenesis.

We thank Drs Barbara Vanderhyden, Patricia Hunt, RobertMcGaughey and Alan Rawls for helpful discussions and advice, DrsRandy Johnson and Shirley Tilghman for the Lfng mice, CarolynChristel for help with statistical analysis and Dr Barbara Vanderhydenfor the c-KIT probe. Confocal microscopy was done in the W. M. KeckBioimaging Laboratory at ASU. This work was supported in part by agrant from the NIH to J.W.-R. Authors have no competing financialinterests. All animal work was done in accordance with AALAACstandards.

Supplementary materialSupplementary material for this article is available athttp://dev.biologists.org/cgi/content/full/132/4/817/DC1

ReferencesAckert, C. L., Gittens, J. E. I., O’Brien, M. J., Eppig, J. J. and Kidder, G.

M. (2001). Intercellular communication via Connexin43 gap junctions isrequired for ovarian folliculogenesis in the mouse. Dev. Biol. 233, 258-270.

Anderson, E. and Albertini, D. F. (1976). Gap junctions between the oocyteand companion follicle cells in the mammalian ovary. J. Cell Biol. 71, 680-686.

Artavanis-Tsakonis, S., Rand, M. D. and Lake, R. J. (1999). Notch signaling:cell fate control and signal integration in development. Science 284, 770-776.

Bettenhausen, B., Hrabe de Angelis, M., Guenet, J. L. and Gosler, A. (1995).Transient and restricted expression during mouse embryogenesis of Dll1, amurine gene closely related to Drosophila Delta. Development 21, 2407-2418.

Bruckner, K., Peruz, L., Clausen, H. and Cohen, S. (2000).Glycosyltransferase activity of Fringe modulates Notch-Delta interactions.Nature 406, 411-415.

Charlton, H. M. (1987). Neural grafts and the restoration of pituitary andgonadal function in hypogonadal (HPG) mice. Ann. Endocrinol. 48, 378-384.

Chin, M. T., Maemura, K., Fukumoto, S., Jain, M. K., Layne, M. D.,Watanabe, M., Hsieh, C. M. and Lee, M. E. (2000). Cardiovascular basichelix loop helix factor 1, a novel transcriptional repressor expressedpreferentially in the developing and adult cardiovascular system. J. Biol.Chem. 275, 6381-6387.

Cohen, B., Bashirullah, A., Dagnino, L., Campbell, C., Fisher, W. W., Leow,C. C., Whiting, E., Ryan, D., Zinyk, D., Boulianne, G. et al. (1997). Fringeboundaries coincide with Notch-dependent patterning centres in mammalsand alter Notch-dependent development in Drosophila. Nat. Genet. 16, 283-288.

Colledge, W. H., Carlton, M. B. L., Udy, G. B. and Evans, M. J. (1994).Disruption of c-mos causes parthenogenetic development of unfertilizedmouse eggs. Nature 370, 65-67.

Conlon, R. A., Reaume, A. G. and Rossant, J. (1995). Notch1, is required forthe coordinate segmentation of somites. Development 121, 1533-1545.

Danilovich, N., Babu, P. S., Xing, W., Gerdes, M., Krishnamurthy, H. and

Development 132 (4) Research article

Dev

elop

men

t

827Lfng null females are infertile

Sairam, M. R. (2000). Estrogen deficiency, obesity, and skeletalabnormalities in follicle-stimulating hormone receptor knockout (FORKO)female mice. Endocrinology 141, 4295-4308.

del Barco Barrantes, I., Elia, A. J., Wünsch, K., Hrabe De Angelis, M., Mak,T. W., Rossant, J., Conlon, R. A., Gossler, A. and de la Pompa, J. L. (1999).Interaction between Notch signalling and Lunatic fringe during somiteboundary formation in the mouse. Curr. Biol. 9, 470-480.

Dunwoodie, S. L., Henrique, D., Harrison, S. M. and Beddington, R. S.(1997). Mouse Dll3: a novel divergent Delta gene which may complement thefunction of other Delta homologues during early pattern formation in themouse embryo. Development 16, 3065-3076.

Eppig, J. J. (2001). Oocyte control of ovarian follicular development andfunction in mammals. Reproduction 122, 829-838.

Erickson, G. F. and Shimasaki, S. (2000). The role of the oocyte infolliculogenesis. Trends Endocrinol. Med. 11, 193-198.

Evrard, Y. A., Lun, Y., Aulehla, A., Gan, L. and Johnson, R. L. (1998).Lunatic fringe is an essential mediator of somite segmentation and patterning.Nature 394, 377-381.

Franco Del Amo, F., Smith, D. E., Swiatek, P. J., Gendron-Maguire, M.,Greenspan, R. J., McMahon, A. P. and Gridley, T. (1992). Expressionpattern of Motch, a mouse homolog of Drosophila Notch, suggests animportant role in early postimplantation mouse development. Development115, 737-744.

Gebauer, F. and Richter, J. D. (1997). Synthesis and function of Mos: thecontrol switch of vertebrate oocyte meiosis. BioEssays 19, 23-28.

Gilula, N. B., Epstein, M. L. and Beers, W. H. (1978). Cell-to-cellcommunication and ovulation: a study of the cumulus-oocyte complex. J. CellBiol. 78, 58-75.

Goode S., Morgan, M., Liang, Y. P. and Mahowald, A. P. (1996). Brainiacencodes a novel, putative secreted protein that cooperates with Grk TGF alphain the genesis of the follicular epithelium. Dev. Biol. 178, 35-50.

Goode, S., Melnick, M., Chou, T.-B. and Perrimon, N. (1996). The neurogenicgenes egghead and brainiac define a novel signaling pathway essential forepithelial morphogenesis during Drosophila oogenesis. Development 122,3863-3879.

Gosden, R., Krapez, J. and Briggs, D. (1997). Growth and development of themammalian oocyte. BioEssays 19, 875-882.

Grammont, M. and Irvine, K. D. (2001). Fringe and Notch specify polar cellfate during Drosophila oogenesis. Development 128, 2243-2253.

Halpin, D. M., Charlton, H. M. and Faddy, M. J. (1986a). Effects ofgonadotrophin deficiency on follicular development in hypogonadal (hpg)mice. J. Reprod. Fertil. 78, 119-125.

Halpin, D. M., Jones, A., Fink, G. and Charlton, H. M. (1986b). Postnatalovarian follicle development in hypogonadal (hpg) and normal mice andassociated changes in the hypothalamic-pituitary ovarian axis. J. Reprod.Fertil. 77, 287-296.

Hamada, Y., Kadokawa, Y., Okabe, M., Ikawa, M., Coleman, J. R. andTsujimoto, Y. (1999). Mutation in ankyrin repeats of the mouse Notch2 geneinduces early embryonic lethality. Development 126, 3415-3424.

Hashimoto, N., Watanabe, N., Fauruta, Y., Tamemoto, H., Sagata, N.,Yokoyama, M., Okazaki, K., Nagayoshi, M., Takeda, N., Ikawa, Y. andAizawa, S. (1994). Parthenogenetic activation of oocytes in c-mos-deficientmice. Nature 370, 68-71.

Hashizume, K., Tsujii, H. and Rokutanda, M. (1995). Effects of gonadotropinadministration on follicular growth and in vitro fertilization in femalehypogonadal mice. Exp. Anim. 44, 241-244.

Hicks, C., Johnston, S. H., diSibio, G., Collazo, A., Vogt, T. F. andWeinmaster, G. (2000). Fringe differentially modulates Jagged1 and Delta1signalling through Notch1 and Notch2. Nat. Cell Biol. 2, 515-520.

Hirao, Y. and Eppig, J. J. (1997). Parthenogenetic development of Mos-deficient mouse oocytes. Mol. Reprod. Devel. 48, 391-396.

Hojo, M., Ohtsuka, T., Hashimoto, N., Gradwohl, G., Guillemot, F. andKageyama, R. (2000). Glial cell fate specification modulated by the bHLHgene Hes5 in mouse retina. Development 127, 2515-2522.

Hrabe de Angelis, M., McIntyre, J., II and Gossler, A. (1997). Maintenanceof somite borders in mice requires the Delta homologue Dll1. Nature 386,717-721.

Hsieh, J. J. D., Henkel, T., Salmon, P., Robey, E., Peterson, M. G. andHayward, S. D. (1996). Truncated mammalian Notch1 activates CBF1/RBP-Jk-repressed genes by a mechanism resembling that of Epstein-Barr virusEBNA2. Mol. Cell. Biol. 16, 952-959.

Hunt, P. A. and Hassold, T. J. (2002). Sex matters in meiosis. Science 296,2181-2183.

Ishii, Y., Nakamura, S. and Osumi, N. (2000). Demarcation of early

mammalian cortical development by differential expression of fringe genes.Brain Res. Dev. Brain Res. 7, 307-320.

Jarriault, S., Brou, C., Logeat, F., Schroeter, E. H., Kopan, R. T. and Israel,A. (1995). Signaling downstream of activated mammalian Notch. Nature 377,355-358.

Jarriault, S., Le Bail, O., Hirsinger, E., Pourquie, O., Logeat, F., Strong, C.F., Brou, C., Seidah, N. G. and Israel, A. (1998). Delta-1 activation of notch-1 signaling results in HES-1 transactivation. Mol. Cell. Biol. 18, 7423-7431.

Jaleco, A. C., Neves, H., Hooijberg, E., Gameiro, P., Clode, N., Haury, M.,Henrique, D. and Parreira, L. (2001). Differential effects of Notch ligandsDelta-1 and Jagged-1 in human lymphoid differentiation. J. Exp. Med. 194,991-1002.

Jiang, R., Lan, Y., Chapman, H. D., Shawber, C., Norton, C. R., Serreze, D.V., Weinmaster, G. and Gridley, T. (1998). Defects in limb, craniofacial, andthymic development in Jagged2 mutant mice. Genes Dev. 12, 1046-1057.

Johnson, J., Espinoza, T., McGaughey, R. W., Rawls, A. and Wilson-Rawls,J. (2001). Notch pathway genes are expressed in mammalian ovarian follicles.Mech. Dev. 109, 355-361.

Johnston, S. H., Rauskolb, C., Wilson, R., Prabhakaran, B., Irvine, K. D.and Vogt, T. F. (1997). A family of mammalian Fringe genes implicated inboundary determination and the Notch pathway. Development 124, 2245-2254.

Joyce, I. M., Pendola, F. L., O’Brien, M. and Eppig, J. J. (2001). Regulationof prostaglandin-endoperoxide synthase 2 messenger ribonucleic acidexpression in mouse granulosa cells during ovulation. Endocrinology 142,3187-3197.

Kimble, J. and Simpson, P. (1997). The lin-12/Notch signaling pathway andits regulation. Annu. Rev. Cell. Dev. Biol. 13, 333-361.

Koch, U., Lacombe, T. A., Holland, D., Bowman, J. L., Cohen, B. L., Egan,S. E. and Guidos, C. J. (2001). Subversion of the T/B lineage decision in thethymus by Lunatic Fringe mediated inhibition of Notch-1. Immunity 15, 225-236.

Kokubo, H., Lun, Y. and Johnson, R. L. (1999). Identification and expressionof a novel family of bHLH cDNAs related to Drosophila Hairy and Enhancerof Split. Biochem. Biophys. Res. Commun. 260, 459-465.

Kopan, R., Schroeter, E. H., Weintraub, H. and Nye, J. S. (1996). Signaltransduction by activated mNotch: importance of proteolytic processing andits regulation by the extracellular domain. Proc. Natl. Acad. Sci. USA 93,1683-1688.

Lanford, P. J., Lan, Y., Jiang, R., Lindsell, C., Weinmaster, G., Gridley, T.and Kelley, M. W. (1999). Notch signaling pathway mediates hair celldevelopment in mammalian cochlea. Nat. Genet. 21, 289-292.

Lardelli, M. and Lendahl, U. (1993). MotchA and MotchB- Two mouse Notchhomologues coexpressed in a wide variety of tissues. Exp. Cell Res. 204, 364-372.

Lardelli, M., Dahlstrand, J. and Lendahl, U. (1994). The novel Notchhomologue mouse Notch3 lacks specific epidermal growth factor-repeats andis expressed in proliferating neuroepithelium. Mech. Dev. 46, 123-136.

Leimeister, C., Externbrink, A., Klamt, B. and Gessler, M. (1999). Heygenes: a novel subfamily of hairy- and Enhancer of split related genesspecifically expressed during mouse embryogenesis. Mech. Dev. 85, 173-177.

LeMaire-Adkins, R., Radke, K. and Hunt, P. A. (1997). Lack of checkpointcontrol at the metaphase/anaphase transition: A mechanism of meioticnondisjunction in mammalian females. J. Cell Biol. 139, 1611-1619.

Lewis, J. (1998). Notch signaling and the control of cell fate choices invertebrates. Semin. Cell Dev. Biol. 9, 583-589.

Lindsell, C. E., Shawber, C. J., Boulter, J. and Weinmaster, G. (1995).Jagged: a mammalian ligand that activates Notch1. Cell 80, 909-917.

Lindsell, C. E., Boulter, J., diSibio, G., Gossler, A. and Weinmaster, G.(1996). Expression patterns of Jagged, Delta1, Notch1, Notch2, and Notch3genes identify ligand-receptor pairs that may function in neural development.Mol. Cell. Neurosci. 8, 14-27.

Logeat, F., Bessia, C., Brou, C., LeBail, O., Jarriault, S., Seiday, N. andIsrael, A. (1998). The Notch 1 receptor is cleaved constitutively by a furin-like convertase. Proc. Natl. Acad. Sci. USA 95, 8108-8112.

Maier, M. M. and Gessler, M. (2000). Comparative analysis of the human andmouse Hey1 promoter Hey genes are new Notch target genes. Biochem.Biophys. Res. Commun. 275, 652-660.

Matzuk, M. M., Burns, K. H., Viveiros, M. M. and Eppig, J. J. (2002).Intercellular communication in the mammalian ovary: oocytes carry theconversation. Science 296, 2178-2180.

McCright, B., Gao, X., Shen, L., Lozier, J., Lan, Y., Maguire, M., Herzlinger,D., Weinmaster, G., Jiang, R. and Gridley, T. (2001). Defects in

Dev

elop

men

t

828

development of the kidney, heart and eye vasculature in mice homozygous fora hypomorphic Notch2 mutation. Development 128, 491-502.

Meyuhas, O. and Klein, A. (1990). The mouse ribosomal protein L7 gene. Itsprimary structure and functional analysis of the promoter. J. Biol. Chem. 265,11465-11473.

Moloney, D. J., Panin, V. M., Johnston, S. H., Chen, J., Shao, L., Wilson, R.,Wang, Y., Stanley, P., Irvine, K. D., Haltiwanger R. S. and Vogt, T. F.(2000). Fringe is a glycosyltransferase that modifies Notch. Nature 406, 369-375.

Morrison, S. J., Perez, S. E., Qiao, Z., Verdi, J. M., Hicks, C., Weinmaster,G. and Anderson, D. J. (2000). Transient Notch activation initiates anirreversible switch from neurogenesis to gliogenesis by neural crest stem cells.Cell 101, 499-510.

Münsterberg, A. E. and Lassar, A. B. (1995). Combinatorial signals from theneural tube, floor plate and notochord induce myogenic bHLH geneexpression in the somite. Development 121, 651-660.

Nakagawa, O., Nakagawa, M., Richardson, J. A., Olson, E. N. andSrivasatava, D. (1999). HRT1, HRT2, and HRT3: a new subclass of bHLHtranscription factors marking specific cardiac, somitic, and pharyngeal archsegments. Dev. Biol. 26, 72-84.

Nakagawa, O., McFadden, D. G., Nakagawa, M., Yanagisawa, H., Hu, T.,Srivastava, D. and Olson, E. N. (2000). Members of the HRT family of basichelix-loop-helix proteins act as transcriptional repressors downstream ofNotch signaling. Proc. Natl. Acad. Sci. USA 25, 13655-13660.

O’Keefe, S. J., Wolfes, J., Kiessling, A. A. and Cooper, G. M. (1989).Microinjection of antisense c-mos oligonucleotides prevents meiosis II in thematuring mouse egg. Proc. Natl. Acad. Sci. USA 86, 7038-7042.

Ohtsuka, T., Ishibiashi, M., Gradwohl, F., Nakanishi, S., Guillemot, F. andKageyama, R. (1999). Hes1 and Hes5 as Notch effectors in mammalianneuronal differentiation. Embo J. 18, 2196-2207.

Pepling, M. E. and Spradling, A. C. (1998). Female mouse germ cells formsynchronously dividing cysts. Development 125, 3323-3328.

Pepling, M. E. and Spradling, A. C. (2001). Mouse ovarian germ cell cystsundergo programmed breakdown to form primordial follicles. Dev. Biol. 234,339-351.

Pederson, T. and Peters, H. (1968). Proposal for the classification of oocytesand follicles in the mouse ovary. J. Reprod. Fertil. 17, 555-557.

Piddini, E. and Vincent, J.-P. (2003). Modulation of developmental signals byendocytosis:different means and many ends. Curr. Opin. Cell Biol. 15, 474-481.

Rand, M. D., Grimm, L. M., Artavanis-Tsakonis, S., Patriub, V., Blacklow,S. C., Sklar, J. and Aster, J. C. (2000). Calcium depletion dissociates andactivates heterodimeric notch receptors. Mol. Cell. Biol. 20, 1825-1835.

Rawls, A., McGaughey, R. W. and Wilson-Rawls, J. (2001). Developmentalhistory of the mammalian oocyte: Insight from mouse mutations. Front.Biosci. 6, d1173-d1185.

Robker, R. L. and Richards, J. S. (1998). Hormone-induced proliferation anddifferentiation of granulosa cells: a coordinated balance of the cell cycleregulators cyclin D2 and p27Kip1. Mol. Endocrinol. 12, 924-940.

Rodgers, R. J., Lavranos, T. C., van Wezel, I. L. and Irving-Rodgers, H. F.(1999). Development of the ovarian follicular epithelium. Mol. Cell.Endocrinol. 151, 171-179.

Sagata, N. (1997). What does Mos do in oocytes and somatic cells? BioEssays19, 13-21.

Schroeter, E. H., Kisslinger, J. A. and Kopan, R. (1996). Notch1 signalingrequires ligand induced proteolytic release of the intracellular domain. Nature393, 382-386.

Schwientek, T., Keck, B., Levery, S. B., Jensen, M. A., Pedersen, J. W.,Wandall, H. H., Stroud, M., Cohen, S. M., Amado, M. and Clausen, H.(2002). The Drosophila gene brainiac encodes a glycosyltransferaseputatively involved in glycosphingolipid Synthesis. J. Biol. Chem. 277, 32421-32429.

Shawber, C., Boulter, J., Lindsell, C. E. and Weinmaster, G. (1996). Jagged2:a serrate-like gene expressed during rat embryogenesis. Dev. Biol. 180, 370-376.

Shimizu, K., Chiba, S., Saito, T., Kimano, K., Takahashi, T. and Hirai, H.(2001). Manic fringe and lunatic fringe modify different sites of the Notch2extracellular region, resulting in different signaling modulation. J. Biol. Chem.276, 25753-25758.

Shutter, J. R., Scully, S., Fan, W., Richards, W. G., Kitajewski, J., Deblander,G. A., Kintner, C. R. and Stark, K. L. (2000). Dll4, a novel Notch ligandexpressed in arterial endothelium. Genes Dev. 14, 1313-1318.

Spruck, C. H., de Miguel, M. P., Smith, A. P. L., Ryan, A., Stein, P., Schultz,R. M., Lincoln, A. J., Donovan, P. J. and Reed, S. I. (2003). Requirement

of Cks2 for the first metaphase/anaphase transition of mammalian meiosis.Science 300, 647-650.

Struhl, G. and Adachi, A. (1998). Nuclear access and action of Notch in vivo.Cell 93, 649-660.

Su, Y.-Q., Wigglesworth, K., Pendola, F. L., O’Brien M. J. and Eppig, J. J.(2002). Mitogen-activated protein kinase (MAPK) activity in cumulus cells isessential for gonadotropin-induced oocyte meiotic resumption and cumulusexpansion in the mouse. Endocrinology 143, 2221-2232.

Su, Y.-Q., Denegre, J. M., Wigglesworth, K., Pendola, F. L., O’Brien, M. J.and Eppig, J. J. (2003). Oocyte-dependent activation of mitogen-activatedprotein kinase (ERK1/2) in cumulus cells is required for the maturation of themouse oocyte-cumulus cell complex. Dev. Biol. 263, 126-138.

Svoboda, P., Stein, P., Hayashi, H. and Schultz, R. M. (2000). Selectivereduction of dormant maternal mRNAs in mouse oocytes by RNAinterference. Development 127, 4147-4156.

Swiatek, P. J., Lindsell, C. E., Franco del Amo, F., Weinmaster, G. andGridley, T. (1994). Notch1 is essential for postimplantation development inmice. Genes Dev. 8, 707-719.

Tamura, K., Taniguchi, Y., Minoguchi, S., Sakai, T., Tin, Furukawa, T. andHonjo, T. (1995). Physical interaction of a novel domain of the notch receptorwith the RBP-Jk transcription factor. Curr. Biol. 5, 1416-1423.

Uyttendaele, H., Marazzi, G., Wu, G., Yan, Q., Sassoon, D. and Kitajewski,J. (1996). Notch4/int-3, a mammary proto-oncogene, is an endothelial cell-specific mammalian Notch gene. Development 122, 2251-2259.

Vanderhyden, B. C., Caron, P. J., Buccione, R. and Eppig, J. J. (1990).Developmental pattern of the secretion of cumulus-expansion enabling factorby mouse oocytes and the role of oocytes in promoting granulosa celldifferentiation. Dev. Biol. 140, 307-317.

Verlhac, M.-H., De Pennart, H., Maro, B., Cobb, M. H. and Clarke, H. J.(1993). MAP kinase becomes stably activated at metaphase and is associatedwith microtubule-organizing centers during meiotic maturation of mouseoocytes. Dev. Biol. 158, 330-340.

Verlhac, M.-H., Kubiak, J. Z., Weber, M., Geraud, G., Colledge, W. H.,Evans, M. J. and Maro, B. (1996). Mos is required for MAP kinase activationand is involved in microtubule organization during meiotic maturation in themouse. Development 122, 815-822.

Vollrath, B., Pudney, J., Asa, S., Leder, P. and Fitzgerald, K. (2001). Isolationof a murine homologue of the Drosophila neuralized gene, a gene requiredfor axonemal integrity in spermatozoa and terminal maturation of themammary gland. Mol. Cell. Biol. 21, 7481-7494.

Wharton, K. A., Johansen, K. M., Xu, T. and Artavanis-Tsakonis, S. (1985).Nucleotide sequence from the neurogenic locus Notch implies a gene productthat shares homology with proteins containing EGF-like repeats. Cell 43, 567-581.

Weinmaster, G., Roberts, V. J. and Lemke, G. (1991). A homolog ofDrosophila Notch expressed during mammalian development. Development113, 199-205.

Weinmaster, G., Roberts, V. J. and Lemke, G. (1992). Notch2: a secondmammalian Notch gene. Development 116, 931-941.

Weinmaster, G. (2000). Notch signal transduction: a real RIP and more. Curr.Opin. Gen. Dev. 10, 363-369.

Williams R., Lendahl, U. and Lardelli, M. (1995). Complementary andcombinatorial patterns of Notch gene family expression during early mousedevelopment. Mech. Dev. 53, 357-368.

Xue, Y., Gao, X., Lindsell, C. E., Norton, C. R., Chang, B., Hicks, C.,Gendron-Maguire, M., Rand, E. B., Weinmaster, G. and Gridley, T.(1999). Embryonic lethality and vascular defects in mice lacking the Notchligand Jagged1. Hum. Mol. Genet. 8, 723-730.

Zhang, N. A. and Gridley, T. (1998). Defects in somite formation in lunaticfringe deficient mice. Nature 394, 374-377.

Zhang, N., Norton, C. R. and Gridley, T. (2002). Segmentation defects ofNotch pathway mutants and absence of a synergistic phenotype in lunaticfringe/radical fringe double mutant mice. Genesis 33, 21-28.

Zhao, X., Singh, B. and Batton, B. E. (1991). The role of c-mos proto-oncoprotein in mammalian meiotic maturation. Oncogene 6, 43-49.

Zhong, T. P., Rosenber, M., Mohideen, M. A., Weinstein, B. and Fishman,M. C. (2000). Gridlock, an HLH gene required for assembly of the aorta inzebrafish. Science 287, 1820-1824.

Zine, A., Van De Water, T. R. and de Ribaupierre, F. (2000). Notch signalingregulates the pattern of auditory hair cell differentiation in mammals.Development 127, 3373-3383.

Development 132 (4) Research article

Dev

elop

men

t