Disruption of the mouse Necdin gene results in hypothalamic and

Published by Oxford University Press Human Molecular Genetics, 2000, Vol. 9, No. 20 3101–3110

Disruption of the mouse Necdin gene results inhypothalamic and behavioral alterations reminiscent ofthe human Prader–Willi syndromeFrançoise Muscatelli+, Djoher Nora Abrous1, Annick Massacrier, Irène Boccaccio,Michel Le Moal1, Pierrre Cau and Harold Cremer2

INSERM U491/IBDM, Faculté de Médecine, 27 Boulevard Jean Moulin, F-13385 Marseille Cedex 5, France,1INSERM U259, Laboratoire des Comportements Adaptatifs, Institut François Magendie, Rue Camille Saint-Saëns,F-33077 Bordeaux, France and 2LGPD/IBDM/Université de Méditerranée, Campus de Luminy, Marseille Cedex 8,France

Received 2 October 2000; Revised and Accepted 9 October 2000

Prader–Willi syndrome (PWS) is a complex neuro-genetic disorder with considerable clinical variabilitythat is thought in large part to be the result of ahypothalamic defect. PWS results from the absenceof paternal expression of imprinted genes localizedin the 15q11–q13 region; however, none of the char-acterized genes has so far been shown to be involvedin the etiology of PWS. Here, we provide a detailedinvestigation of a mouse model deficient for Necdin.Linked to the mutation, a neonatal lethality of vari-able penetrance is observed. Viable Necdin mutantsshow a reduction in both oxytocin-producing andluteinizing hormone-releasing hormone (LHRH)-producing neurons in hypothalamus. This repre-sents the first evidence of a hypothalamic deficiencyin a mouse model of PWS. Necdin-deficient mice alsodisplay increased skin scraping activity in the openfield test and improved spatial learning and memoryin the Morris water maze. The latter features are remi-niscent of the skin picking and improved spatialmemory that are characteristics of the PWS pheno-type. These striking parallels in hypothalamic struc-ture, emotional and cognitive-related behaviorsstrongly suggest that NECDIN is responsible for atleast a subset of the multiple clinical manifestationsof PWS.

INTRODUCTION

Prader–Willi syndrome (PWS) is a neurogenetic disorderwhich occurs in 1 in 10 000–15 000 births (1). Diagnosis forthis disease is based on a scoring system established from a listof ∼20 major and minor criteria, differing in infancy fromthose in child- and adulthood (2) (OMIM 176270). The majorfindings include a transient infantile hypotonia and a failure tothrive during the newborn period. From 2 years of age, PWSindividuals display hyperphagia leading to severe obesity.

They also present a global developmental delay, typical facialfeatures, hypogonadotropic hypogonadism, mild to moderatemental retardation, learning profiles with particular strengthsand weaknesses and characteristic behavioral problems.Furthermore, a variety of less frequent findings have beendescribed, for example respiratory distress at birth (3). Thus,PWS is a complex disorder with considerable clinical varia-bility (1). Many of the manifestations are thought to be due tohypothalamic deficiency (1,4).

PWS is a classical example of a human disease that revealsthe regulation of the 15q11–q13 chromosomal region bygenomic imprinting, a process by which a subset of autosomalgenes is differentially expressed depending on parent of origin.All of the PWS patients examined have genetic abnormalities(large paternal deletion, maternal disomy or imprinting muta-tion) resulting in the inactivation of paternally expressedgenes. Furthermore, there is no reported case of a normalpaternal copy of 15q11–q13 with an inheritance consistentwith a single mutated gene. These data imply that PWS is amultigenic syndrome (5–8).

The identification of genes involved in PWS is difficult,requiring the isolation of candidate genes among all the pater-nally expressed sequences in a region spanning >1.5 Mb. Oneapproach is to characterize all the human candidates in theregion and to clone their mouse orthologs. This allows thecreation of animal models in order to determine the role ofeach gene in the etiology of PWS (8).

Is the mouse an appropriate model for PWS? The mouse 7Cchromosomal region is a region of conserved synteny to thehuman chromosome 15q11–q13 region (9). Four mousemodels have so far been reported as Prader–Willi models,resulting in the deficiency of paternal gene expression in the7C chromosomal (10–13). The phenotype observed is lethalityduring the first post-natal week, associated with poor feeding(10,11) or postnatal lethality before weaning associated withrespiratory distress (12) or hypotonia, growth retardation andlethality in 80% of newborn mice (13). Based only on thesedata, these mouse phenotypes were correlated with the feedingdifficulties and failure to thrive that characterize PWS infants.

+To whom correspondence should be addressed. Tel: +33 4 9178 6894; Fax: +33 4 9180 4319; Email: [email protected]

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Furthermore, such models do not allow the discrimination ofthe role of each single gene in the PWS phenotype.

Six imprinted genes have so far been described in humans(Fig. 1a): SNURF-SNRPN (14), ZNF127 (15), IPW (16),MAGEL2 (17) and NDN (18,19) (Fig. 1a). All of these geneshave orthologs in the mouse 7C chromosomal region, with thesame imprinted status. Mice deficient for Snrpn (11), Snurf(13), Zfp127 (20) and Ipw (20) have been independentlycreated but no role in the etiology of PWS could be ascribed toany of these genes. Two lines of Ndn-deficient mice have beendescribed. In one model, an early postnatal lethality probablydue to respiratory distress was observed, although the pene-trance of this mutation was highly variable depending on thegenetic background (21). The second model was presentedonly as viable, fertile without any sign of obesity (22).

As described above, the features of PWS are complex.However, none of the mouse models has been studied forsubtle morphological modifications or changes in behavior.

The human NDN gene is a strong candidate to be involved inthe etiology of PWS, due to its expression in the nervoussystem and the observation that it is inactivated in PWSpatients (19). To investigate the biological function of Necdinand its role in the etiology of PWS, we generated Ndn-deficientmice and performed a detailed histological and behavioralanalysis. Our study revealed strong parallels in terms ofmorphological, behavioral and cognitive modifications,between the effects of Ndn mutation in mice and key featuresof PWS.

RESULTS

Generation of Ndn-deficient mice

To investigate the in vivo function of Necdin, we inactivatedthe Ndn gene in the mouse germ line by conventional genetargeting (Fig. 1b). The promoter and the first two-thirds of theNdn coding sequence were replaced with the HSV-TK-Neocassette flanked by loxP sites, which allows subsequent CRE-dependent deletion of the marker gene. Chimeric males,derived from three independent recombinant embryonic stem(ES) cell clones, were mated with either wild-type C57Bl6/Jfemales or C57Bl6/J females expressing CRE recombinase(23). Two lines of mutant N1 progeny were thereby produced.In the first one, the Ndn paternal allele was replaced with Neo(+Neo), which enabled us to study the imprinting state of Neoin this genetic context. In the second line, the Neo marker wasdeleted, in order to investigate the consequences of Ndn defi-ciency in the absence of putative interference between the Neocassette and the normal transcription of neighboring genes(24).

In both lines, matings between Ndn heterozygotes producedN2 progenies of wild-type, heterozygous (+m/–p or –m/+p) andhomozygous mutant (–/–) genotypes as determined bygenomic Southern blot analysis (Fig. 2a). Northern blotanalysis (Fig. 2b and c) or RT–PCR analysis (Fig. 3a) wasperformed to verify the complete absence of Ndn transcripts inhomozygous mutants as well as heterozygous animals in which

Figure 1. Physical map of the Prader–Willi region and gene targeting of Ndn. (a) Physical map of the 15q11–q13 region including the PWS and Angelman syn-drome (AS) critical regions, the typical 4 Mb deletion breakpoints (), genes encoding proteins (closed circles), genes encoding transcripts only (open circles) andthe imprinting center (IC). Note that MAGEL2 is located 40 kb centromeric to NDN. (b) Strategy for the targeted inactivation of the mouse Ndn gene. (Top)Genomic structure and partial restriction map of the Ndn locus (B, BamHI; S, SacII; X, XbaI; HincII). Ndn is monoexonic, with the coding sequence shown inblack. A 1.3 kb XbaI fragment was used as the 5′ external probe for screening of recombinant embryonic stem cell clones. (Bottom) Vector construct used to targetthe Ndn locus: A loxP-flanked Neo cassette was introduced into the Ndn locus by homologous recombination, thereby replacing the promoter and the first two-thirds of Ndn coding sequence. Neo is transcribed in the opposite direction to Ndn. The integration of Neo-loxP introduced a new HincII site at the 5′ end of Ndn,generating the diagnostic 5.5 kb HincII fragment (5.9 kb in the wild-type) used for the identification of recombinant clones. TK, HSV-thymidine kinase gene.

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the paternal allele was deleted (–m/–p and +m/–p). In addition,these analyses demonstrated the complete absence of Ndnexpression from the maternal allele in the neonatal or adultbrain as well as in other tissues (data not shown). The absenceof Ndn expression was also verified at the protein level bywestern blot analysis on whole brain homogenates (Fig. 2d),using a peptide-specific antibody against the C-terminal regionof Necdin.

The imprinting state of Neo was studied using RT–PCR onRNA extracted from mouse brains with a maternally or pater-nally derived Neo allele (Fig. 3a). In no case were Neo tran-scripts detected. Such a transcriptional inhibition of Neo couldresult from the presence of the viral HSV-TK promoter, whichmight be inactive in this context. Alternatively, regulatoryelements of the Ndn promoter could be necessary to allow anactive brain transcription (25) or an imprinting control of thislocus. Both hypotheses are supported by the results obtainedby Gérard et al. (21). These authors disrupted the Ndn gene byreplacing exclusively the coding part with the PGK-Neo

cassette and found that PGK-Neo is imprinted, being expressedexclusively from the paternal allele.

To generate Necdin mutant mice, mice in which the Neomarker was entirely deleted, chimeric males were crossed withC57Bl6/J females expressing CRE in the oocyte to produce theN1 generation. In this generation 34.5% instead of the expectedNdn 50% mutants were identified. A second backcrossbetween N1 +m/–p males and C57Bl6 wild-type femalesproduced N2 offspring. Genotyping revealed 18% mutants inthis generation (expected 50%; Table 1). In both generations,lack of mutants was observed in males and females in equalproportions. The observed ratio was estimated after geno-typing at weaning. To investigate whether the lethality due toNdn deficiency is a pre- or postnatal event, we genotyped 40mice 2 h after birth. At this time point we observed a 50% ratiobetween mutant and wild-type mice, suggesting that lethalityoccurred postnatally. In addition, we followed a cohort of 24newborn mice over a 48 h interval and found that five animalsdied during this period; they appeared cyanotic and showedsigns of respiratory distress. This phenotype appears similar tothe phenotype described by Gérard et al. (21). In conclusion,we observed a partial early postnatal lethality due to the Ndnmutation. Furthermore, the penetrance of this phenotype isapparently increased in the C57Bl6/J background. Survivingmice lacking Necdin expression were viable, fertile and showno sign of obesity until 18 months of age, the latest time pointobserved. Dissection of paternal-deficient and wild-type micerevealed no gross morphological abnormalities.

Figure 2. Genomic analysis and expression pattern of mice lacking Ndn.(a) Southern blot of tail DNA from offspring of matings between Ndn heterozy-gotes hybridized to the 5′ probe as indicated in Figure 1b, revealing a 5.9 kbwild-type and a 5.5 kb mutant HincII fragment. Among these N2 progeny miceof wild-type (+/+), heterozygous (+/–p or –m/+) and homozygous mutant (–/–)genotypes were identified. (b and c) Northern blot analysis on total RNAs usinga probe from the 3′-UTR of Ndn confirm the complete absence of Ndn tran-scripts in homozygous mutants (data not shown) as well as heterozygous ani-mals in which the paternal allele was deleted (+/–p) at postnatal day 1 (P1) (b)as well as in the adult (c). Note the complete absence of Ndn expression fromthe maternal allele in the neonatal or adult brain. (d) Western blot analysis onwhole brain homogenates using a peptide-specific antibody against the C-terminal region. Necdin-specific immunoreactivity (at 47 kDa) was visible inwild-type or –m/+ heterozygous mice, whereas homozygous mutants andheterozygous animals (+/–p) showed no necdin expression. Note the strongbackground due to non-specific binding of the antibody.

Figure 3. Imprinting state of Neo and expression of neighboring genes.(a) RT–PCR on total RNAs extracted from mouse adult brains carrying amaternal or paternal Neo allele was used to investigate the imprinting state ofNeo. In no case were Neo transcripts detected whereas Ndn expression showedthe expected pattern. (b) RT–PCR analysis of genes neighboring the Ndn locusindicating that Magel2, Snrpn and Zfp127 are normally expressed in (–Neo)animals carrying a targeted Ndn allele in neonatal as well as adult mice.

Table 1. Genotypes of Ndn mice at weaning and at birth

Mating Male 129SV+/– × female C57BL5/J+/– Male N1 +m/–p (129SV × C57B16/J) × female C57BL6/J+/+

Progeny N1 adults % (n) N2 adults % (n) N2 PO % (n)

Mutants (+/–p) 34.5 (47) 18 (37) 45 (18)

Wild-type (+/+) 50 (68) 50 (101) 50 (20)


To check that the introduced mutation has no influence onthe genomic environment, we analyzed the integrity of tran-scription of neighboring genes by RT–PCR. This analysisrevealed that Snrpn, Zfp127 and Magel2 (a member of theMage/Ndn gene family) (Figs 1a and 3b) show no differencesin their transcription patterns, as expected.

In the following studies, the analyses of Ndn mutants wereperformed on an N2 population of adult male mice issued fromthe following backcross: [+m/–p male N1 (chimeric 129SV–C57Bl6/J-CRE) × wild-type female C57Bl6/J] and comparedpaternally deficient mice for Ndn (+m/–p) with wild-typelittermates (+m/+p). We chose this population in order to avoidinterference with the female reproductive cycle and to get allthe results on the same genetic background as used for thebehavioral studies. Due to the strong influence of the geneticstrain background on mouse behaviors such as learning,aggression and anxiety, we followed the recommendationsconcerning the genetic background on behavioral analysis(26).

Ndn is expressed in a subpopulation of postmitotic neuronsin newborn and adult brain

PWS patients show no obvious abnormalities in brainmorphology (27). In accord with this finding, general histo-logical markers, like Nissl blue staining and MAP2 immuno-labeling on brain sections, revealed no obvious differences inbrain structure in Ndn mutant mice (data not shown).

In order to focus the analysis of Ndn-deficient mice on spe-cific brain structures, we first performed in situ hybridizationhistochemistry (ISHH) using a specific probe to the 3′ untrans-lated region (3′-UTR) of Ndn on wild-type mouse brain. Wefound that Ndn expression was restricted to postmitoticneurons in specific regions of the adult brain. For example, astrong expression is detected in the locus coeruleus, whereasno neurons were labeled in the cerebellar cortex (Fig. 4a).Importantly, even in the hypothalamus, where general expres-sion is the highest, Ndn is not expressed in all postmitoticneurons (Fig. 4b and c).

In conclusion, we find that the expression pattern of Ndndefines regionally distinct subpopulations of neurons in adultbrain; therefore, Ndn is not a pan-neuronal marker of post-mitotic neurons, as previously reported (28).

The number of hypothalamic oxytocin- and luteinizinghormone-releasing hormone (LHRH)-producing neuronsis decreased in Ndn mutant mice

Hypothalamic deficiencies have been suggested to underlie anumber of symptoms in PWS (4). In this regard, we found thatNdn is highly expressed in this area during embryogenesis and inadulthood (A. Massacrier et al., in preparation). Histologicalexamination of the hypothalamic structure in adult Ndn (+m/–p)mutant mice revealed no obvious abnormalities (data notshown). However, since Ndn is expressed only in a subpopula-tion of postmitotic neurons, subtle changes could be expected.

In PWS patients, a significantly reduced number ofoxytocin-expressing neurons was described in the para-ventricular nucleus (PVN) (4). To investigate this cell popula-tion in Ndn mutant mice we counted oxytocin-producingneurons in consecutive sections of the hypothalamus (Fig. 5a).In lateral parts of the PVN (Fig. 5b and c) we observed a

significant decrease of 29% in the number of oxytocin-expressing neurons (Ndn +m/–p: 224.75 ± 16, mean ± SD, n =4; wild-type: 313 ± 45.4, n = 4; P = 0.009).

In PWS patients, alterations in LHRH neurons are thought tobe responsible for the decreased levels of sex hormones,resulting in non-descended testes and undersized sex organs(4). LHRH neurons are consistently found within the preopticarea (POA) where Ndn is highly expressed (Figs 4c–f and 5d).Using double labeling (ISHH for Ndn and immunocyto-chemistry for LHRH), we demonstrated cellular colocalizationof Ndn mRNA and LHRH protein expression (Fig. 5e–g).Based on the overall distribution of LHRH cells in the fore-brain of the adult rat (29), we counted all the LHRH neuronslocated within a 1.5 mm span covering the medial POA(MPOA), the region containing the majority of these cells (Fig.5a and d). LHRH neurons were identified using an anti-LHRH(LR1) antibody (Fig. 5d and e), allowing the detection of bothtypes of LHRH cell. Counting of all labeled neurons onconsecutive sections revealed a statistically significant loss of∼25% of LHRH neurons in the Ndn mutants compared with thewild-type (knockout +m/–p: 205.8 ± 32, n = 7; wild-type:276.7 ± 49, n = 7; P = 0.0163).

Behavioral studies

Besides a motor delay, PWS patients present behavioraldisorders often related to emotional problems like tempertantrums, stubbornness, obsessive–compulsive characteristicsand difficulty in adapting to a change in routine (30,31).Furthermore, up to 95% of the patients manifest a skin pickingbehavior resulting from incessant scraping (30,32). PWSpatients exhibit cognitive impairments (most patients aremildly retarded) with, however, some strengths in specificcognitive patterns. Typically, a relative strength in reading,particular visual–spatial skills, excellent long-term memory aswell as a common unusual skill to perform jigsaw puzzles havebeen described. Weaknesses have been observed in arithmetic,sequential processing and short-term memory (1,31,33,34).

To investigate whether Ndn-deficiency is associated withbehavioral changes resembling the alterations found in PWSpatients, we studied motor capacities, emotional behavior andcognitive functions in paternal deficiency (N2 males +m/–p,n = 14) and wild-type littermates (+m/+p, n = 18).

Spontaneous behavior: skin scraping is increased in Ndnmutant mice. The Ndn +m/–p mutant mice show normalmotor-related behavior as measured in the inclined plane,beam balance test and the extension reflex task (data notshown). Furthermore, the Ndn +m/–p mice were not differentfrom wild-type mice in their daytime activity measured incircular alleys (Fig. 6a) [F(1,30) = 2.80, P > 0.05], in theelevated plus maze test [analysis performed for the time spentin the open and closed arms: F(1,29) = 1.65, P > 0.05] and inthe open field (Fig. 6b) (P > 0.05). These data also indicatedthat locomotion–exploratory activity was not modified.

In the elevated plus maze test (Fig. 6c), wild-type and mutantmice spent the same amount of time in the closed and openarms [interaction arm × group effect: F(1,29) = 0.339, P <0.05], indicating that the anxiety level was not modified; theindex of anxiety levels was not different among both groups


(P > 0.05). Initial latency was also not different among groups(P < 0.05).

In the open field test, fear-associated parameters (faeces,urination and toilets) (Fig. 6d) were not altered by the mutation

(P > 0.05) with the exception, however, of the latency to quitthe first central square which was higher in mutant micecompared with the wild-type (P < 0.05). Interestingly, skinscraping was significantly increased in paternal-deficient mice

Figure 4. Expression of Ndn in the adult nervous system. (a). Cresyl violet staining of the mouse cerebellum (CB) and neighboring structures including the locuscoeruleus (lc). (b) In situ hybridization on successive sections revealed the specific expression of Ndn in the locus coeruleus whereas the cerebellum appearsentirely negative. (c) Coronal section of the mouse brain at the level of the preoptic area stained with cresyl violet. cc, corpus callosum; ct, cortex; HYP, hypotha-lamus; lsn, lateralis septum nucleus; lv, lateral ventricle. (d) In situ hybridization performed on the successive section showing specific labeling in the preoptic area.(e and f) Higher magnifications of (d) demonstrate that only subpopulations of neurons are positive. A large number of cells appear entirely devoid on Ndn tran-scripts [arrow in (f)]. Scale bars: (a and b) 100 µm; (c and d) 200 µm; (e and f) 50 µm.


(P < 0.01) (Fig. 6d). This characteristic observed in Ndn-deficient mice could be seen as analogous to the skin-pickingbehavior described in PWS patients.

Cognitive function: spatial learning is improved in Ndnmutant mice. Spatial learning was assessed in the Morris watermaze (MWM) in which animals have to locate a hidden plat-form using spatial cues. This paradigm was used since visualprocessing tasks and long-term memory, two parameters that

are tested in the water maze, are well described aspects of thecognitive profile of PWS patients (2,31). After 9 days oftraining, both groups of mice improved their performance asindicated by the decreasing escape latencies [days effect:F(8,240) = 11.36, P < 0.001] (Fig. 7a) and the decreasing pathlength (data not shown) [days effect: F(8,240) = 12.57, P <0.001] over the course of training. However, there were signif-icant differences in the rate of acquisition between groups. Thelatency to find the hidden platform [F(1,30) = 4.38, P < 0.05]and the distance covered during the search was reduced (datanot shown) [F(1,30) = 4.75, P < 0.05] were significantlydecreased in the Ndn mutants mice compared with the wild-type. The observation that both groups of mice showed compa-rable behavioral performances during the first days of testingindicates that differences in learning were not related to differ-ences in emotional status. In the probe trial, given on the 7thday of testing (before training), the Ndn mutant mice enteredmore often and spent more time in the former target (P < 0.05).These data reflect a higher ability of the mutant to rememberaccurately the location of the platform.

Altogether, our study showed that spatial learning andmemory capabilities in the MWM were improved in Ndn-deficient mice.

DISCUSSION

Our analyses show an abnormal phenotype of mice deficientfor Ndn. Lack of Ndn is correlated to an early postnatallethality with a partial penetrance. The surviving mutants havebeen investigated for subtle modifications of the hypo-thalamus, which showed a reduced number of oxytocin- andLHRH-producing neurons. Finally, Ndn-deficient mice presenta characteristic behavioral profile with higher skin scraping as

Figure 5. Reduction of LHRH and of oxytoxin neurons in Ndn mutant mice.(a) Schematic representation of the adult mouse brain in sagittal section.LHRH neurons were counted in serial coronal sections from region A [1.5 mmwide; see (d–g)]. Oxytocin neurons were counted in serial coronal sectionsfrom region B [1 mm wide; see (b and c)]. (b) Coronal section of Ndn mutantmouse brain. Immunoreactive oxytocin neurons were present in the paraven-tricular (pv) nucleus and in the lateral hypothalamus. (c) Magnification of thelateral hypothalamic area [rectangle in (b)] showing an oxytocin neuronoriented perpendicular to oxytocinergic axons of the hypothalamo-neuro hypophyseal tract. Knockout animals showed a statistically significant loss (29%)of oxytocin neurons compared with wild-type mice. (d) Coronal sectionthrough the medial preoptic area (mpoa) showing the area where LHRH neu-rons have been counted. (e–g) Colocalization of LHRH using immunodetec-tion with FITC-coupled secondary antibody (e) and Ndn mRNA (alkalinephosphatase) (f) in the same neuron [merge in (g)]. Knockout animals exhib-ited a statistically significant loss of 25% of LHRH neurons compared withwild-type mice. ar, arcuate nucleus; ca, anterior commissure; cc, corpus callo-sum; dmh, dorsomedial hypothalamus; f, fornix; hp, hippocampus; ip, interpe-duncular nucleus; me, median eminence; mm, mammilary nucleus; mot,medial olfactory tract; mpoa, medial preoptic area; oc, optic chiasma; so,supraoptic nucleus; vmh, ventromedial hypothalamus. Scale bars: (b and d) 1mm; (c) 10 µm; (e–g) 5 µm.

Figure 6. Spontaneous behavior of Ndn-deficient mice. Daytime activitymeasured in circular alleys (a), time spent in the open and closed arms of anelevated plus maze (b) and total activity in an open field (c) were unaffected inNdn-deficient mice, indicating that locomotion/exploratory activities werealtered by the mutation with the exception, however, that the latency to quit thefirst central square was higher in mutants. Furthermore, scraping was signifi-cantly increased in paternal-deficient mice (d) (P < 0.01).


well as an improvement in particular cognitive functions asso-ciated with spatial learning and memory capability. This is thefirst report of hypothalamic or behavioral alterations in amouse model deficient for Ndn. Such a model is of greatinterest with regard to the features of PWS.

An abrogation of the mouse Ndn gene has been previouslyreported by two different groups. Tsai et al. (22) observed noabnormal phenotype in Ndn-deficient mice, whereas Gérard etal. (21) observed postnatal respiratory distress leading tolethality in the first 30 h after birth. The penetrance of thisphenotype was dependent on the genetic background. A highpenetrance was observed in offspring of C57BL/6 mothers, inwhich males were more affected than females (95 versus 45%).The discrepancy between both reports could be attributed todifferences between the mouse strains used for embryonicstem cell generation and breeding or to an unexpected contri-bution of the targeting construct to the phenotype (8). Here wereport a third model of Ndn deficiency. We observed an earlypostnatal lethality on the 129Sv–C57Bl6/J genetic back-ground. The penetrance of this phenotype varied according tothe content of the C57Bl6/J genetic background, beingincreased in the N2 generation. Our data confirm the observa-tions made by Gérard et al. (21) and support the hypothesis thatmodifier genes influence the phenotype of Ndn deficiency inthe different mouse strains. However, we found a lower pene-trance of lethality in the absence of sex-linked difference.These discrepancies might result from a divergence in the 129and C57Bl/6 substrains used in both laboratories. Such a diver-gence has been suggested by Simpson et al. (35). Therefore,

the lack of Ndn expression results in respiratory distress andneonatal lethality that resemble the respiratory problems oftenobserved in PWS (3) and the failure to thrive of PWS patients(2).

Considering the relatively ‘mild phenotype’ associated withPWS, specifically the absence of obvious structural abnormal-ities in the brain, we did not expect gross defects in the absenceof Ndn in mice. Thus, a detailed phenotypic analysis in searchfor more subtle changes was undertaken in order to define thebiological role of the protein. In situ hybridization analysisshowed an expression of Ndn in subpopulations of hypo-thalamic neurons. Quantification of hypothalamic oxytocin-and LHRH-producing neurons revealed a reduction of ∼29 and25% of the populations analyzed, respectively. These dataappear interesting with regard to the symptoms of PWS;obstetric problems and insatiable hunger have been suggestedto be due to a lack of oxytocin (4) and hypogonadism to LHRHdeficiency. A highly significant decrease in the number ofoxytocin-expressing neurons (42%) was found in all fivepatients with PWS studied (36). LHRH-expressing neuronswere not investigated in these patients. Preliminary data revealundersized testes in Ndn-deficient males, which might be aconsequence of reduction of LHRH neurons.

Specific behavioral and cognitive profiles represent impor-tant criteria of PWS (2,30,31,37). A general behavioral studywas performed, blind to the genotype. These experimentsdemonstrated that Ndn deficiency does not affect behavioralresponses related to motor coordination, exploratory activity,anxiety or stress. However, skin scraping activity in the openfield was significantly elevated in mutants. Given that skinpicking resulting from incessant scraping represents one of themain criteria described in PWS patients (32,38), this observa-tion represents an interesting and important parallel.

The use of the MWM revealed that the abrogation of Ndnimproves spatial learning and memory whereas it does notmodify other behavioral responses affecting performance inthis task. In order to understand how the inactivation of onegene may result in an improvement in learning capacities, oneshould keep in mind that the hidden platform version of MWMtests exclusively spatial learning and memory abilities (39).Consequently, better performance in this task does not test fora general improvement of cognitive function. Human geneticdisorders, for example autism, Williams syndrome or PWS, areno longer considered as global disabilities, but as specificcognitive profiles with particular strengths and weaknesses.Consistent with these ideas, one of the strengths in PWS isvisual–spatial integration and visual memory including anunusual skill to perform jigsaw puzzles (30,31,33,37).

An improvement in learning capacities due to the geneticmanipulation of mice have so far been reported in few cases(40). Interestingly, enhanced performance in the MWM hasbeen observed in animals following administration of a peptidehormone (39) or in mice lacking the neurotransmitter receptor(40). The latter findings imply that a disregulation in neuro-peptide housekeeping can have consequences for spatiallearning and memory. Thus, it can be hypothesized that Ndndeficiency may improve cognitive functions, at least in theMWM, through changes in neuropeptide levels and/or neuro-transmitter activity, which remain to be determined.

A final important consideration is whether Ndn-deficientmice reflect the role of NDN deficiency in human PWS. Our

Figure 7. Spatial learning in an MWM. (a) Latency (s) to find a hidden plat-form during the acquisition stage. Results are expressed as a mean score (mean± SEM) over four trials per day. Spatial learning in the knockout mice wereimproved compared with the wild-type. (b) Number of entries in the formertarget quadrant and (c) percentage of time spent in this quadrant during theprobe trial. Knockout mice remembered more accurately the location of theplatform when compared with the wild-type (*P < 0.05; **P < 0.01).


analysis revealed striking parallels between the effects of thismutation in mice and phenotypic manifestations in PWSpatients. However, it is clear that PWS is a multigenic diseaseshowing a large variability of clinical features. The modelpresented here may consequently be representative of only asubset of these features although we cannot exclude that ouranalysis missed other important manifestations. More exten-sive anatomical, physiological and behavioral investigationswill be necessary to determine whether there are further paral-lels between Ndn deficiency and PWS. In any event, Ndn-deficient mice represent a good model to investigate particularaspects of the molecular and physiological basis of respiratorydistress, hypothalamic function and cognitive capacity and willbe useful to investigate the role of Necdin in the centralnervous system.

MATERIALS AND METHODS

Gene targeting and generation of Ndn-deficient mice

A mouse Ndn cDNA clone was used to screen the 129/Svgenomic DNA library of bacteriophage artificial chromosomes(BACs) produced by Research Genetics (Huntsville, AL). Thepositive BAC 143C10 was subcloned in pBluescript-SK afterXbaI and BamHI digestion. The targeting vector was designedto delete 1.2 kb from XbaI to SacII, which contains thepromoter and two-thirds of the coding part of Ndn. Theplasmid contained a neo-selectable marker flanked by loxPsites on each side and an HSV-TK– selectable marker at the endof the construct. Using gancyclovir in the culture medium, thislatter marker allows the elimination of clones in which arandom integration occurred.

Vector DNA (15 µg) was linearized with NotI and electro-porated into CK35-ES (129Sv genetic background) cells asdescribed previously (42). G418-resistant clones werescreened by Southern blotting using a 5′ flanking probe corre-sponding to the XbaI fragment (Fig. 1b). Three positive clonesamong ∼1000 were expanded and injected into blastocysts bystandard procedure (42).

Chimeric males were then bred to wild-type C57Bl6/Jfemales or to females expressing CRE on C57Bl6/J geneticbackground (a gift from K. Rajewsky, Cologne, Germany).Southern blot analyses were performed as previously described(17).

Western blot analysis

Proteins were isolated with Trizol reagent (Life TechnologieTech-Line, Cergy Pontoise, France) from total frozen brain.Aliquots of 35 µg proteins were run in a 10% SDS–polyacryl-amide gel, blotted over with transfer buffer (25 mM Tris base,192 mM glycine, 20% methanol) at 4°C.

Immunodetection was performed with a 1:1000 dilution ofpolyclonal antipeptide AC2 antibody in Tween-20 [Tween–phosphate-buffered saline (PBS)] for 1 h at room temperature.The filters were washed three times for 15 min in 1× PBS,0.1% Tween-20 and then incubated with a 1:5000 dilution ofanti-rabbit Ig–horseradish peroxidase secondary antibody

(Amersham Pharmacia Biotech, Orsay, France) in Tween–PBS for 1 h at room temperature.

Two polyclonal antibodies directed against the samesynthetic peptides (C2 and N1) as designed by Aizawa et al.(28) were produced by Neosystem (Strasbourg, France). Achemiluminescence kit (ECL; Amersham) was used for visual-ization.

Northern and RT–PCR analysis

RNAs and cDNAs were prepared as previously described (18).Necdin PCR was performed using Nec3 (5′-TCTGGAGCAG-GCCAGAGCTC-3′) and Nec4 (5′-TGCTAAGTGCCTACA-CTGAG-3′) primers and a 562 bp fragment was amplified.PCR conditions were 95°C for 2 min followed by 30 cycles of95°C for 30 s, 50°C for 30 s and 72°C for 40 s. Neo PCR wasperformed using primers Neo1 (5′-TTTGTCAAGAACGAC-CTGTC-3′) and Neo2 (5′-CGATACCGTAAAGCACGAGG-3′) and a 598 bp fragment was amplified. PCR conditions were95°C for 2 min followed by 30 cycles of 95°C for 45 s, 56°Cfor 45 s and 72°C for 45 s. Magel2 PCR was performed asdescribed by Boccaccio et al. (17).

Snrpn PCR was performed using primers Snrpn1 (5′-GAGT-AGCAAGATGCTGCAGC-3′) and Snrpn2 (5′-GCCTCCCA-ACTCCTCTGACAG-3′) and a 392 bp fragment was ampli-fied. PCR conditions were 95°C for 2 min followed by 35cycles of 95°C for 20 s, 55°C for 30 s and 72°C for 1 min.

Zfp127 PCR was performed using primers Zfp1 (5′-GTTCT-TCCTTCTCTGATGAC-3′) and Zfp2 (5′-CACAAGTTAAC-AAGTGCAC-3′) and a 558 bp fragment was amplified. PCRconditions were 95°C for 2 min followed by 45 cycles of 95°Cfor 20 s, 49°C for 30 s and 72°C for 30 s.

In situ hybridization

Knockout (+m/–p) and wild-type mice were perfused intra-cardially using 4% paraformaldehyde in PBS. Brain wasremoved, frozen and stored at –80°C until use. Serial sagittalsections (14 µm) were obtained using a Microm cryostat andthen collected onto silanized slides. Digoxigenin-labeledcRNA probes, specific for the 3′-UTR (from nucleotide 1174after the ATG to nucleotide 1466) of Ndn, were obtained by invitro transcription, Ndn mRNAs were detected as previouslydescribed (18,43).

Immunolabeling and counting LHRH- and oxytocin-producing neurons

The general protocol was described by Yoshida et al. (44).LR1 anti-LHRH polyclonal antibodies were diluted 1:30 000and polyclonal anti-oxytoxin antibodies were diluted 1:10 000.

LHRH neurons were counted in knockout (+m/–p) and wild-type mice using 70 µm sagittal vibratome serial sectionsexploring 1.5 mm span containing the MPOA (Fig. 5).Oxytocin neurons were counted using 50 µm sagittalvibratome serial sections exploring 1 mm span beginning at theanterior part of the PVN and including supraoptic and acces-sory magnocellular nuclei located in lateral hypothalamus.Statistical analysis (Mann–Whitney non-parametric U-test)was performed using GB-STAT program 5.0.4 (DynamicMicrosystems, Silver Spring, MD).


Colocalization of LHRH protein and Ndn mRNAs inhypothalamic neurons

Double labeling experiments were performed using 50 µmvibratome slices. Floating sections were first subjected to in situhybridization using our general protocol (see below). After thepost-hybridization rinses, sections were incubated overnight at4°C with LR1 anti-LHRH antibodies. After a rinse, sectionswere incubated with goat anti-digoxigenin antibodies coupled toalkaline phosphatase according to the supplier’s recommenda-tions (Boehringer/Interchim/Roche, Meylan, France). Visualiza-tion of enzyme was performed using NBT and BCIP for 6 h. Thesections were then incubated with anti-rabbit IgG conjugated toFITC, mounted in Mowiol and then placed on coverslips.Sections were viewed using a Leica DMR microscope. Digitizedfluorescent or brightfield pictures were taken using a PrincetonCoolSNAP camera (Roper Scientific, Trenton, NJ) and IPLabprogram 3.5 (Scananalytics, Fairfax, VA) running in a G4Macintosh.

Spontaneous locomotor activity

Daytime activity was measured in a circular alley (outer diam-eter 20 cm; inner diameter 10 cm) equipped with eight photo-cells spaced evenly around the periphery. The number ofinterruptions of the photocell beams per 60 min was recordedautomatically. The animals were placed in the apparatus at10:00 h and were retrieved at 11:00 h. The sampling time was5 min.

Elevated plus maze

The apparatus was made of four black metallic arms, two openarms (29 × 8 cm) and two enclosed arms (29 × 8 × 17 cm) thatformed a cross shape with two arms opposite to each other. Themaze was elevated 55 cm above the floor and placed in a dimlyilluminated room (20 lux). Mice were placed individually onthe central platform and allowed to explore the apparatus for 5min. Anxiety was assessed by comparing the time spent in theopen versus the enclosed arms. Furthermore, as an index ofanxiety we used the ratio of (time spent in the open arms):(timespent in the open + closed arms). The latency for the beginningof exploration was also recorded.

Open field

The apparatus consisted of a white wooden box (0.8 × 0.8 ×0.5 m), the floor of which was divided into 25 squares. Thearena was well illuminated (130 lux). Mice were placed on thecentral square and allowed to explore for 20 min. The samplingtime was 5 min. Horizontal activity was the sum of the numberof outer squares (those adjacent to the walls) crossed (outerlocomotion) and of the number of inner squares crossed (innerlocomotion). Horizontal activity and vertical activity (numberof rearings) were computed as total activity. The latency to quitthe first central square and the numbers of defecations, urina-tions, groomings and scrapings were also registered.

MWM

Spatial learning capacities were measured in an MWM (46).The apparatus consisted of a circular swimming pool built ofgray plastic (180 cm diameter × 60 cm height), which was

filled with water at room temperature and made opaque by theaddition of milk. Four points on the rim of the pool divided thesurface of the pool into four equal quadrants. During the test,the mice could escape onto a transparent perspex platform(10 × 10 cm) located in the middle of one of the quadrants.Starting points were chosen randomly in one of the threeremaining quadrants. Spatial cues were placed in the room andwere not moved during the experiment. The behavior of eachanimal was monitored and recorded by a videotrack system(View point, Lyon, France). Phase 1 involved no platform.During this habituation phase, animals were given one trial of60 s per day over 3 days. Phase 2 involved a hidden platform.The mice were trained daily, for four consecutive trials of 90 sduring 9 days, to escape onto a platform hidden just below thesurface of the liquid (1.5 cm). The platform was kept in aconstant position for the duration of this phase. The first trialwas initiated by putting the animal on the platform for 30 s. Ifthe animal jumped from the platform, it was replaced on theplatform until it remained there for 30 s. The animal was thenplaced in one of the three quadrants. The trial ended when themouse had climbed onto the platform where it was left for 30 s.If the animal did not find the platform in 90 s, it was placedthere for 30 s. After this period of time, a new trial began.Phase 3 (probe trial). On the 7th day, a probe trial wasperformed. The platform was removed and animals wereallowed to explore for 60 s. The number of entries in the plat-form zone and the time spent in the quadrant were recorded.Data were compared using Student’s t-test and, when neces-sary with an analysis of variance.

SUPPLEMENTARY MATERIAL

Supplementary material relating to this paper is available athttp://www.hmg.oupjournals.org .

ACKNOWLEDGEMENTS

We thank Marc Lalande, Christo Goridis, Willy Mayo forhelpful discussions and support, Roger Keynes for criticalreading of the manuscript; Nathalie Roëckel, Saïd Ech-Chadiand Elodie Drapeau for technical assistance; Charles Babinetand Chantal Kress (Pasteur Institute, France) for the gift of theCK35-ES cells; Robert Benoit (Montreal General Hospital,Montreal, France) for the gift of LR1 antibody; Gérard Tramu(Bordeaux, France) for the gift of oxytocin antibody; RobertJeffard (Bordeaux, France) for the use of circular alleys mate-rial. This work was supported by grants from the AssociationFrançaise contre les Myopathies (AFM), the Association pourla Recherche sur le Cancer (ARC), INSERM and ComitéMixte Inter-Universitaire Franco-Marocain.

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