Analysis of Quantitative Trait Loci for Behavioral Laterality in Mice

Corresponding author: Pierre L. Roubertoux, INPC.CNRS, 31 Chemin Joseph-Aiguier, 13402 Marseille Cedex 20, France., rouber{at}lnf.cnrs-mrs.fr (E-mail)

Communicating editor: J. B. WALSH

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Laterality is believed to have genetic components, as has been deduced from family studies in humans and responses to artificial selection in mice, but these genetic components are unknown and the underlying physiological mechanisms are still a subject of dispute. We measured direction of laterality (preferential use of left or right paws) and degree of laterality (absolute difference between the use of left and right paws) in C57BL/6ByJ (B) and NZB/BlNJ (N) mice and in their F₁ and F₂ intercrosses. Measurements were taken of both forepaws and hind paws. Quantitative trait loci (QTL) did not emerge for direction but did for degree of laterality. One QTL for forepaw (LOD score = 5.6) and the second QTL for hind paw (LOD score = 7.2) were both located on chromosome 4 and their peaks were within the same confidence interval. A QTL for plasma luteinizing hormone concentration was also found in the confidence interval of these two QTL. These results suggest that the physiological mechanisms underlying degree of laterality react to gonadal steroids.

TWENTY-SEVEN years ago, COLLINS 1975 characterized handedness as an "intriguing phenotype" and today both the genetic and the physiological pathways underlying left- or right-handed asymmetries remain unknown. Four decades of clinical and experimental work have produced an accumulation of contradictory results in genetic investigations of handedness. Although family studies indicate that the prevalence of left-handedness rises from 7% in Western populations to 21% in the offspring of probands (ANNETT 1996 ), suggesting that genes may have something to do with this phenotype, twin studies have not provided encouraging conclusions. About 15 twin studies have been published about handedness since 1924. Some showed left-handedness to be more frequent in twins compared to singletons (COREN 1994 ). Others failed to reveal any difference between the two types of twins (BISHOP 2001 ). Some of these studies, combining twin and family studies, concluded that inheritance of handedness followed a rather complicated model (ORLEBEKE et al.. 1996 ). Two genome scans performed for handedness helped find the quantitative trait loci (QTL; LAVAL et al. 1998 ; FRANCKS et al. 2002 ) but the LOD scores were low and chromosomal positions inconsistent despite extensive evidence for reproducibility of the method (ROUBERTOUX and LE ROY-DUFLOS 2001 ). Several other reasons could be presented as hypotheses to explain these inconsistencies.

1. All the authors referred to one of the two definitions of laterality. Most studies considered "direction" (the preferred left or right hand; FRANCKS et al. 2002 ) while others chose "relative hand skill" (deviation from the use of the right hand; LAVAL et al. 1998 ), and yet others referred to both direction and "degree" (absolute difference between the use of left and right hands; CARLIER et al. 1996 ).

2. Methods for measuring laterality differed from one study to another, but poor correlation in different laterality tests suggested that these tests measured different abilities (RIGAL 1992 ; DOYEN and CARLIER 2002 ), which correlated to different neuronal substrates that could involve different genes.

3. Depending on the acceptance in families of the use of the left hand, pressure in raising children may have produced a differential bias between individuals (CARLIER 1995 ).

High conservation of brain and motor asymmetries across species (VALLORTIGARA and ANDREW 1994 ; ZILLES et al. 1996 ; LAMENDOLA and BEVER 1997 ) including the mouse (COLLINS 1985 ) have been reported and this species was therefore chosen to elucidate the gene linked to laterality and corresponding physiological mechanisms.

Three hypotheses have attempted to explain individual differences in laterality. All three consider that an overdeveloped hemisphere of the brain means preferential use of the contralateral limbs (HECAEN 1984 ), the preference for the right hand corresponding to an overdevelopment of the left hemisphere, while the use of the left hand corresponds to less pronounced asymmetry between right and left hemisphere (GALABURDA et al.. 1978 ).

The first hypothesis sees brain asymmetry and consequent behavioral laterality as a specific case of visceral asymmetries, emerging as an output of genes implicated in the left-right body axis development in the embryo (RAMSDELL and YOST 1998 ; YOST 1998 ). Situs inversus, in mammals, and lefty and pitx2 in Danio rerio in zebra fish induce visceral asymmetries, affecting both the brain and the nodal gene modulating the right-left position of the adult pineal organ in zebra fish (CONCHA et al. 2000 ; LIANG et al. 2000 ). Functional asymmetry of the brain and behavioral laterality may be a pleiotropic effect of these genes. However, KENNEDY et al. 1999 and TANAKA et al. 1999 performed neurological investigations of situs invs. totalis patients and concluded that the left-right reversal in situs invs. did not involve functional brain asymmetries. No direct evidence appears to exist implicating nodal, lefty, and pitx2 in behavioral laterality.

The second hypothesis concerning dopamine involvement in motor behavior suggests the existence of dopaminergic asymmetries in the brain (GLICK and SHAPIRO 1985 ). Overfunctioning of the dopaminergic system in one hemisphere could induce increased skills of contralateral limbs. Although dopaminergic asymmetries have been reported in the brains of rodents (see CARLSON and GLICK 1992 for review), including asymmetry in dopamine uptake controlling the direction of rotation behavior (GORDON et al.. 1994 ), brain asymmetries of the dopaminergic system (uptake, concentration, receptor functioning) could not be related to a preferential use of left or right paws in mice (NEVEU 1996 ).

The third hypothesis suggests gonadal steroid involvement in laterality. In their pioneering article, GESCHWIND and GALABURDA 1985 suggested that high gonadal steroid levels slowed the growth of the left hemisphere, favoring the development of the right hemisphere and the consequent use of the contralateral limbs in humans. Recent data indicate less pronounced brain asymmetry in left-handed humans compared to right-handed subjects by brain magnetic resonance imaging (MRI; GESCHWIND et al.. 2002 ).

In mice, differences in direction could be the result of randomly distributed environmental events; two arguments support this hypothesis. First, intrastrain differences for direction cannot be attributed to residual genetic variation (COLLINS 1985 , COLLINS 1991 ). Second, direction did not respond to selection in a segregating population (COLLINS 1991 ). However, the "degree" of laterality, defined as the absolute difference between the preferences for the left or right paws, responded to selective breeding (COLLINS 1985 , COLLINS 1991 ), indicating that degree and not direction was inherited. Later, COLLINS et al. 1993 demonstrated that the differences between the selected lines were not due to differences in genetic heterogeneity. A low degree of laterality, i.e., an equal preference for the left or right paws, was associated with reduced asymmetry of the brain hemispheres (COLLINS 1985 ). Mice selected by Collins for a high degree of laterality showed more brain asymmetries than mice selected for a low degree of laterality (LIPP et al. 1984 ; WARD and COLLINS 1985 ; CASSELLS et al. 1990 ). Lines of evidence indicate that a high level of gonadal hormones is associated with a low degree of laterality in a wide range of species (COLLINS 1985 ; CLARK et al. 1996 ; WESTERGAARD et al. 2000 ). An excess of testosterone reduces brain asymmetry in several regions (WARD and COLLINS 1985 ; INASE and MACHIDA 1992 ; TABIBNIA et al. 1999 ). Taken together, these lines of evidence are compatible with an association between high concentration of gonadal hormones and a reduction of brain asymmetry producing a low degree of laterality.

This study reports the results of a wide genome scan for both direction and degree of laterality. Mice were successively subjected to two different tests of laterality to see whether the putative QTL were task dependent. We addressed the possibility of genes having an effect on left-right body axis development and the dopaminergic system. We therefore investigated chromosomal regions encompassing situs invs., nodal, lefty, and pitx2 as well as genes involved in the dopaminergic system. The gonadal hormone pathway was also examined. As direction and degree of laterality were reported in this study in both male and female mice, we selected plasma-luteinizing hormone concentration (PLHC), which is a common trigger for both male and female gonadotropic hormones.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Measuring laterality:
Laterality was measured with two independent tests, one for forepaw and the other for hind paw. We recorded the preferential use of right or left forepaw in a food-reaching task and the number of right or left hind paw slips during a bar-crossing test. Each mouse was subjected to the two measures, the interval between the two tests being between 17 and 33 days.

Laterality for forepaws was assessed according to COLLINS 1968 . Mice were deprived of food at 5:30 p.m. and tested 17 ± 2 hr later. Each mouse was placed in a chamber (10.5 x 6 x 6 cm), where its usual food was available in a tube located on the front wall at half height equally accessible from both the right and the left. The mouse could obtain the food by introducing only one of its forepaws into the tube. Each testing session consisted of observing 50 reaches and recording the sequence of paws used. Two values were calculated, "direction" and "degree." The number of right paw entries (RPE) during a session indicates the direction of laterality: the higher the score, the more right pawed the mouse. The degree of laterality was the absolute difference between the number of right paw entries and the number of left paw entries (LPE). The mice with the highest IRPE-LPEI were the most lateralized either to the right or to the left. Individual scores were transformed into logit (ln|RPE - LPE|) (COLLINS 1985 ) to ensure homoscedasticity in the nonsegregating generations.

Laterality of the hind paw was measured using a bar-crossing test (LIPP and WAHLSTEN 1992 ) modified by MAAROUF et al. (1999). A solid bar with a smooth surface was used for shaping. The mouse was first placed on the middle platform of the solid bar (50 x 5 x 5 cm) and trained to cross in periods lasting 2 or 3 min. When the mouse succeeded in crossing the solid bar fearlessly, it was placed on the middle platform of the carved bar for testing. The bar consisted of a small platform (5 x 5 cm) located in the middle of a carved wooden bar bridging a gap between two larger platforms (10 x 10 cm). The notched bar (100 x 5 x 5 cm) was formed by a series of regularly spaced notches 2 cm wide and 1.5 cm deep. The mouse was placed on the middle platform on the bar and had to reach one of the two end platforms (one trial). Two experimenters stood on either side of the bar, counting the number of times the animal slipped with either the right- (RPS) or the left-hind paw (LPS) during five trials. The bar had 11 notches and the mouse could therefore make 11 errors with each paw per trial, or a total of 55 errors over five trials. The direction of laterality was calculated as the number of right slips divided by the total number of slips [RPS/(RPS + LPS)]. The degree of laterality was the absolute difference between the number of slips with the left- and right-hind paws divided by the total number of slips [|RPS - LPS|/(RPS + LPS)] because the number of slips differed between mice.

We estimated the reliability of both direction and degree for the two tests. The reliabilities were estimated by split-half coefficients (r_tt), the split-half value being calculated as

where r_hh was the correlation between the half-tests (ANASTASI 1988 ). We computed the r_tt using the first 25 and the last 25 food reaches with a forepaw for direction and degree and between the first 50% and the last 50% of slips with the hind paw for both direction and degree.

Plasma luteinizing hormone concentration:
Mice were killed at 145 ± 5 days of age by cervical dislocation. PLHC was assayed by antibody radioimmunoassay. Blood was centrifuged and plasma frozen at -20° until assayed for PLHC. Because of homology between mouse and rat LH, the rat luteinizing hormone (rLH) [¹²⁵] assay system is used usually (SAITOH et al. 1991 ; TANG et al. 1993 ). We used the (rLH) [¹²⁵] provided by Amersham, which was calibrated against the National Institutes of Health rat LH RP-2 reference preparation. We assayed PLHC in duplicate. Assays were performed again when intraassay coefficients of variation were >10%. Results were expressed in terms of rat LH Rp-2 reference preparation as nanograms per milliliter of plasma.

Animals:
Identified breeders from B6 and N mice were purchased from the Jackson Laboratory (Bar Harbor, Maine) respectively at generations 190 and 156 of a brother x sister mating breeding protocol. Brother x sister mating was continued in the animal facility for another 4 generations before starting the experiment. The mice were maintained under standard rearing conditions: temperature, 23.5° ± 0.5°; photoperiod, 12/12 hr with lights on at 7:30 a.m.; food and water were available ad libitum; bedding, dust-free sawdust. Any females obviously close to parturition were isolated. Because the first litter from N mothers often dies, the first litter was discarded and the second litter was used for the experiment. Litters with only five to seven pups were chosen to reduce possible postnatal effects due to litter size. Litters of less than five were discarded and those with more than seven were culled to seven. There were no adoptions. Weaning took place at 28 ± 2 days of age. Females were housed in groups of four with same-sex littermates or females from NMRI H strain; males were housed alone with an NMRI female. The mice were tested between 110 and 130 days of age.

On the basis of a preliminary experiment with parental B6 and N strains and their reciprocal F₁'s showing no dominance in laterality measurements, an intercross design strategy was chosen for wide genome scanning using 33 B6, 31 N, 23 NB6F₁'s and 25 B6NF₁'s. Another 48 F₁ pairs were used to produce the 283 F₂ mice (68 NB6 x NB6F₂'s, 74 NB6 x B6NF₂'s, 71 B6N x NB6F₂'s, and 70 B6N x B6NF₂'s).

Statistics and QTL analysis:
Examination of variances in the nonsegregating generations showed heterogeneity, requiring raw data transformation. We selected logit for the forepaw and log 10 for both hind paw and PLHC, on the basis of a nonsignificant ² value with the Bartlett test. The transformed values from parental strains, reciprocal F₁'s, and F₂'s were used to compute heritability and to estimate the components of the mean differences in laterality and PLHC.

Heritability in the broad sense was estimated as

where

MATHER and JINKS's (1971) procedure was used for components of mean differences. Parameters were estimated and models fitting observed data were selected using Cavalli's least-squares fitting procedure. Several models fit observed values and we selected one model using the complementary method developed by KERBUSCH et al. 1981 . For this procedure, the best-fitting model has as few parameters as possible; a more complex model was accepted only if the fit was better than that for the simpler model. The lowest ² value indicates the best fit. Because of the number of generations, including reciprocal crosses in F₁ and F₂, seven parameters could be estimated: [m] mean, [d] additivity, [h] dominance, [i] interaction between homozygous loci, [j] interaction between homozygous and heterozygous loci, [l] interaction between heterozygous loci, and [cm] contribution of the mother.

Before performing the genome scan, we examined the number of segregating units, to establish whether one or more were associated with measures of laterality and PLHC. We used Collins's general nonparametric method for genetic analysis (COLLINS 1967 , COLLINS 1980 ) according to TULLY and HIRSCH 1982 and MICHARD and ROUBERTOUX 1986 for computations. For a variable and for one class of the phenotype continuum, it is possible to compute the theoretical values in segregating generations (F₂, B₁, B₂ ... ) from the observed values in nonsegregating generations (N, B, and their F₁). For a one-segregating-unit model and for class i, the Mendelian expression pi, in F₂ is

For each variable, the phenotype dimension was divided into five equal classes and the values for the phenotype dimension were reassigned to these classes. Theoretical and observed values were compared with a ² for accuracy of fit.

Genotyping was performed individually with the DNA from the 283 F₂'s mice using 67 single sequence length polymorphisms (SSLPs) as markers (average interval length, 22.5 cM) on the 20 chromosomes. At this stage, we used the chromosomal locations of the SSLPs reported in the consensus map provided by the MOUSE GENOME DATABASE (2002). Significant differences (P < 0.05 threshold) between the three genotypes N//N, N//B6, and B6//B6 were assessed. We used the Kruskal-Wallis test as the transformations providing homoscedasticity in the parental and F₁ populations did not necessarily produce normality in the distributions associated with the three genotypes in the F₂'s. In the second stage, when differences between the three genotypes were found with an SSLP, we selected other SSLPs on the chromosomal region displaying significant differences among the three genotypes. All the F₂ mice were individually genotyped for these additional SSLPs. The third stage produced a new SSLP map for the region based on distances found in the F₂'s. For this purpose, we anchored the most centromeric SSLP and computed the distances across the SSLPs. This new SSLP map, which was specific to our segregating population, was used then for likelihood ratios and LOD score computations. We estimated these values with the interval-mapping method (MapQTL-tm-version 3.0; VAN OOIJEN and MALIEPAARD 1996 ). The LOD score values and the chromosomal distances were compared to those obtained with composite interval mapping with cofactors (QTL Cartographer, model 6; ZENG 1994 ). After mapping QTL linked to laterality, a possible linkage with PLHC was investigated for the chromosomes where linkage with laterality had been detected. Confidence intervals were estimated with the method proposed by DARVASI and SOLLER 1997 .

Genotyping:
DNA was extracted from tails and stored at -80°. Genotyping was performed using SSLP that differed by at least 15 bases. Preparation of PCR was done with Beckmann 2000 and adapted for the robot and for each set of primers from general protocols. We used 3 pmol of each primer (Genetic Research, Alabama); 2.5 units of Taq polymerase and buffer, adjusted to 1 mM Mg²⁺ (Promega, Madison, WI); 200 ng of genomic DNA; and 0.2 mM of each dNTP in a total volume of 30 µl. Amplification included initial denaturation (94° for 3 min), and then 94° at 30 sec per cycle, annealing (1 min 15 sec from 42° to 55° according to the primers), extension (1 min 15 sec at 72°), and final extension (3 min). Electrophoresis was performed on an agarose gel. Each migration included DNA from N, B6, and F₂ and a molecular weight marker to determine the size of the alleles. Allele sizes were identified blind and independently by the first two authors with Transilluminator, the UVP PMW 20 computer system (4.5x magnification). Any discordant observation was followed by a second amplification.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The respective reliabilities with forepaw and hind paw were 0.97 and 0.95 for direction and 0.94 and 0.93 for degree. The reliability for preferential food reaching with forepaws was similar to those previously published on degree (0.92, COLLINS 1985 ; and 0.89, SIGNORE et al. 1991A ).

Components of mean differences:
The N and B6 mice did not differ for direction of laterality assessed either by preferential food reaching with the right forepaw or by the number of slips with the right hind paw during the bar-crossing test (data not shown), but did differ for the two corresponding indices of degree of laterality (Table 1). N strain mice were more ambidextrous (smaller absolute difference between right and left) than B6 for forepaw and hind paw and had a higher PLHC. Males and females were pooled for subsequent analyses as males and females did not differ for measurements of either laterality or PLHC.

F₁ values did not differ from midparent values for the two measures of degree of laterality and for PLHC (Table 1), suggesting that dominance did not contribute to these three phenotypes. This was confirmed by the analysis of the components of the mean differences. No model was able to fit for direction of laterality, but one model with additivity ([d] parameter) was the best fit for degree of laterality measured with the forepaw (² = 0.903, P < 0.52, [d] 0.39 ± 0.061) and hind paw (² = 0.527, P < 0.46, [d] 0.13 ± 0.06). The best-fitting model for PLHC was always additive (² = 0.712, P < 0.49, [d] 0.53 ± 0.074). With Collins's general nonparametric method, the one-segregating-unit model was not rejected for the two measurements of degree of laterality (² = 0.923, P < 0.63 for forepaw and ² = 0.5184, P < 0.91 for hind paw), but was rejected for PLHC (² = 9.126, P < 0.010).

In F₂, measures of degree of laterality with forepaw and hind paw were correlated (Bravais-Pearson product moment correlation; r = 0.31, P < 0.0005). Plasma luteinizing hormone levels correlated with degree of laterality for both forepaw (r = 0.35, P < 0.0001) and hind paw (r = 0.39, P < 0.0001).

QTL mapping:
The first genome scan was performed on the whole F₂ population with 67 SSLPs as markers covering all chromosomes. No significant differences between the three possible genotypes appeared for direction measured with either forepaw or hind paw. The degree of laterality was associated with SSLPs on chromosome 4: D4Mit205a (P < 0.0005) and D4Mit12 (P < 0.001) for forepaw and D4Mit205a (P < 0.00001) and D4Mit12 (P < 0.0002) for hind paw, suggesting an involvement of the central part of chromosome 4 in the two measurements. A total of 8 new SSLPs were therefore added onto this chromosome. The chromosomal positions of the 12 SSLPs were computed again for the F₂ population as described above and these positions were used for the final mapping with the MapQTL package (VAN OOIJEN and MALIEPAARD 1996 ). Chromosome 4 was scanned in the whole F₂ population with 12 SSLPs (Fig 1). One QTL linked to degree of laterality of the forepaw was mapped at 48 cM and the other linked to degree of laterality of the hind paw at 49.7 cM (Table 2). The overlapping was compatible with the significant correlation between forepaw and hind paw in the segregating F₂ generation (Table 1), indicating that the QTL found might encompass common genetic bases. The LOD scores (5.6 for forepaw, 7.2 for hind paw) met the criteria for highly significant linkage (LANDER and KRUGLYAK 1995 ).

View larger version (17K): In this window In a new window Download PPT slide   Figure 1. LOD plot of degree of laterality measured for forepaw, hind paw, and PLHC with MapQTL-(tm)-version 3.0. Genetic distances from centromere are indicated on the x-axis with SSLPs used as markers for maximum-likelihood tests. Underlining indicates those included in the first step (full-genome scan); the others were added in the second step for scanning chromosome 4. The LOD scores are plotted on the y-axis. The threshold 4.3 corresponding to a significant LOD score is indicated (horizontal line).

QTL mapping was performed for PLHC on chromosome 4 with the 12 SSLPs used for degree of laterality (Fig 1). We found a significant QTL with a LOD score of 4.4 at 48.8 cM from the centromere (MapQTL-tm-version 3.0) that became 3.7 with the QTL Cartographer, the corresponding distance being 44.3 cM. This QTL was included in the confidence intervals of each of the two QTL linked to degree of laterality. The lowest values for the two measurements of degree of laterality and the highest value for PLHC were linked to N genotypes (Table 2).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The lack of difference between N and B6 strains for direction measured with either the forepaw or the hind paw was not due to large sample errors as the two measurements had high reliability. In contrast, the difference between the two strains for degree was significant. The difference between their mean indexes was equivalent to the maximum difference between the 12 strains tested for this value (SIGNORE et al. 1991B ). Moreover, the difference is comparable to the difference in lines bred for differences in degree of laterality (COLLINS 1985 ); N mice record a score close to mice in the low selected line (ambidextrous), while B6 mice are close to mice in the high selected line (strongly lateralized). Highly significant QTL for degree of laterality and the absence of genetic components for direction indicate that direction and degree are different measurements of laterality. These results observed in the B6 and N population indicated a relationship between genetic variability and degree but not between genetic variability and direction. This result fits with Collins's view (COLLINS 1991 ), who rejected the hypothesis of a genetic control of direction from both his own experiments and phylogenic considerations.

For degree, each QTL contributed to part of the total variance, which approximated the respective heritabilities estimated in the measurements (30 vs. 28.9% for forepaw and 26.8 vs. 33% for hind paw). This point suggested that only a major QTL contributed to degree of laterality for each measurement in the population derived from B6 and N. This QTL might encompass several genes. Collins's general nonparametric method, which did not lead to rejection of the one-segregating-unit model for either of the two measurements of degree, did not support this last possibility in our data. As a consequence of finding exclusively an additive genetic component, we note that the nonsignificant effect of the "contribution of the mother" component tallies with previously published data showing that mitochondrial DNA did not contribute to degree of laterality measured for forepaw and hind paw (MAAROUF et al.. 1999 ). The contribution of genetic factors to the degree of laterality estimated by heritability, did, however, remain moderate, as has been widely reported for behavioral measurements in experimental genetics.

The measurements of degree of laterality recorded in the two tests were linked to the same chromosomal region. This suggests a linkage between the QTL that we discovered on the central part of chromosome 4 and a common physiological mechanism.

The three hypothetical mechanisms presented above as possibly involved in brain and behavioral laterality were tested. Our results led us to eliminate the implication of genes linked with left-right body axis development. Chromosome 4, where we detected the QTL for degree of laterality, did not include situs invs., nodal, lefty, and pitx2. Moreover, careful anatomical examination conducted according to previously defined protocols (YOKOYAMA et al. 1993 ) did not reveal situs invs. or similar phenotypes in N or B6 mice. For the dopaminergic hypothesis, none of the genes known as being associated with dopaminergic functioning were seen on chromosome 4 after inspecting the mouse genome map (MOUSE GENOME DATABASE 2002).

Much indirect evidence was compatible with the gonadal steroid hypothesis. An excess of perinatal testosterone favors left-handedness in Mongolian gerbils, among other species (CLARK et al.. 1996 ). Left-handedness has a higher prevalence in individuals with high plasma testosterone concentration (WESTERGAARD et al.. 2000 ). In humans, the QTL mapped by LAVAL et al. 1998 for degree was in the vicinity of the androgen receptor gene on the X chromosome. Mice carrying the Tfm mutation (impairment of the androgen receptor) were significantly less ambidextrous than their normal controls (NOSTEN et al. 1989 ). We investigated the population derived from N and B6 for testing a possible link between gonadal hormones and degree of laterality. The plasma testosterone concentration had appeared lower in N than in B6 males (CARLIER et al. 1990 ), suggesting that we should measure testosterone in the F₂'s. As we found no difference between males and females for degree of laterality in this population, we looked for a common trigger for both male and female gonadotropic hormones. We selected luteinizing hormone as it stimulates secretion of estrogen and estradiol and production of testosterone. The location of a QTL for PLHC in the confidence interval of the QTL linked to degree of laterality provided support for gonadal steroid implication in degree. However, genes involved in luteinizing hormone are not mapped on chromosome 4 but on chromosomes 7 and 17 (Lhb, luteinizing hormone ß, and Lhcgr, luteinizing hormone/chorionadotropin receptor, respectively; MOUSE GENOME DATABASE 2002). This result indicated that luteinizing hormone was probably not the prime mover involved in degree of laterality, but that the highest PLHC that we found in the ambidextrous mice was the consequence of mechanisms monitored by other genes. The leptin receptor gene (Lepr) might be one of the candidates. Its chromosomal location on chromosome 4 at 46.7 cM (MOUSE GENOME DATABASE 2002) is close to the QTL for degree that we found (between 48 and 49.7 cM). Lepr is implicated in the gonadal steroid cycle (CHEN et al. 1996 ; CIOFFI et al. 1996 ; CARRO et al. 1997 ) and in luteinizing hormone particularly. Leptin, which modulates luteinizing hormone and follicle-stimulating hormone, is also considered as a metabolic signal acting on the gonadotropin-releasing hormone system with consequences upon reproductive target organs (BARASH et al. 1996 ; ELMQUIST et al. 1998 ). As the male reproductive organ weight results from the contribution of testosterone during development (MCKINNEY and DESJARDIN 1973 ; JEAN-FAUCHER et al. 1983 ; ARGYROPOULOS and SHIRE 1989 ; HUTSON et al. 1994 ), it must be mentioned that the QTL that we found for both degree of laterality and PLHC correspond to the region of chromosome 4 where we had mapped one of the QTL linked with testes and seminal vesicle weights (47.5 and 48 cM, respectively; LE ROY et al. 2001 ).

The close linkage between degree of laterality and PLHC with Lepr is currently being examined by fine-mapping strategies using advanced intercrossed lines (DARVASI and SOLLER 1995 ). These results suggest that degree of laterality was associated with physiological mechanisms influenced by gonadal hormones and must be considered as specifically characterizing the population derived from the strains selected. Other genome scans with other genetic pools may reveal different QTL implicated in other physiological mechanisms. Many authors have claimed that laterality is implicated in motor or cognitive performances as well as in addictive behavior (CARLSON and GLICK 1992, among others). Given the present results, that measures of laterality (degree vs. direction in this study) have different genetic bases and are consequently associated with different physiological mechanisms, this implication should be revisited. On the basis of the present results, a specific pattern of characteristics for each measurement of laterality should be investigated.

	FOOTNOTES

¹ These authors contributed equally to this work.

	ACKNOWLEDGMENTS

We thank Michèle Carlier and Anne-Lise Doyen for their discussions and Robert Brush for his helpful comments on the manuscript. This study was supported by the Centre National de la Recherche Scientifique, the Ministry for Research and Technology, and the Fondation pour la Recherche Médicale.

Manuscript received June 21, 2002; Accepted for publication November 20, 2002.

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