Brain Research, 564 (1991) 194-202 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 000689939117102H

194

BRES 17102

Reimpressed selective breeding for lateralization of handedness in mice Robert L. Collins The Jackson Laboratory, Bar Harbor, ME 04609 (U.S.A.) (Accepted 4 June 1991) Key words: Lateralization; Asymmetry; Cerebral dominance; Handedness; Selective breeding; Mouse

Eleven generations of bidirectional selection for lateralization produced 2 lines of mice that differ markedly in degree of asymmetry for hand preference. The foundation population was derived from 6 distantly related inbred strains and 2 stocks of wild mice, M. castaneus. HI line matings were made using mice that exhibited consistent right or left paw use in a food reaching task and LO line matings were made using mice with little overall paw preference. All matings were made without regard to the expressed directions of asymmetry. Line differences emerged at the third generation and increased thereafter. Selection was relaxed at generation 12 and the lines were maintained by random within-line mating. At generation 28 selective breeding was reimpressed for 3 generations. Results indicated that between-line divergence in degree of lateralization had remained high during 17 generations of relaxed selection. Mice of the HI line are more strongly lateralized than mice of the unselected HET population. Mice of the LO line are more weakly lateralized than controls. The selected lines may provide a useful mammalian genetic resource for studying the neurobiology of cerebral lateralization. INTRODUCTION

mice 10. O n average, right-handed females were more

Asymmetries of structure and function, like mathematical vectors, possess both a dimension of directional-

dextral than right-handed males, and left-handed-females were more sinistral than left-handed males. A similar pattern has b e e n reported for other postural/motor asymmetries in laboratory animals 43. This sex difference pro-

ity (right/left or clockwise/anticlockwise) and a dimension of magnitude (strongly lateralized/weakly lateralized). Although much effort has focused on studying the inheritance of asymmetry, results from genetic studies of directionality remain surprisingly enigmatic. For example, we found that approximately half the mice of the C57BL/6J strain, inbred for more than 100 generations, were right-handed, and half, left-handed s. This indicates that near m a x i m u m phenotypic variability is observed in mice possessing m i n i m u m genetic heterogeneity. Furthermore, we were unable to increase the proportions of dextral or sinistral mice following 3 generations of selective breeding for right- or left-handedness 9. Thus, the variation of expressed handedness in inbred C57BL/6J mice does not appear to be maintained by a residue of genetic variation remaining unfixed after prolonged inbreeding. It is noted that the hand preferences of mice are highly reliable when measured on repeated testing after several days or several months s'36'38. However, we did observe a possible genetic effect on degree of asymmetry. In observing paw preference patterns in a large sample of C57BL/6J mice, we found that female mice were more strongly lateralized than male

vided the motivation to initiate a bidirectional selective breeding experiment for lateralization itself. This report summarizes results obtained from the initial eleven generations of bidirectional selection for lateralization in mice, and presents information on the stability of line differences from results o n 3 generations of reimpressed selection. MATERIALS AND METHODS A genetically heterogeneous foundation population was created using the following 6 inbred strains and 2 partially inbred stocks of wild mice: BALB/cJ, C57BL/6J, DBA/2J, LP/J, RF/J, SM/J, Mus molossinus, and Mus castaneus, designated C, B6, D2, LP, RF, SM, Mol and Cas, respectively. These inbred strains were chosen because they showed remote common ancestry and exhibited wide differences at polymorphic loci (T.H. Roderick, personal communication). Characteristics of the wild stocks are reviewed elsewhere 12. The 8-way cross foundation population was generated in 2 steps. First, 4 sets of F t crosses were made: B6 x SM, Mol x C, Cas x RF, and LP x D2. These F l's then were intercrossed systematically in a diallel mating plan. This produced 16 ceils in which the diagonal elements comprised 4 segregating F2 crosses and the 2 sets of 6 off-diagonal elements were generations segregating for the genetic contributions of 4 progenitors. Progeny from this intercross-

Correspondence: R.L. Collins, The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609, U.S.A. Fax: (1) (207) 288-5079.

195 ing became the foundation population, G o. A total of 327 mice from 41 parental pairs in G Owere tested for handedness using procedures developed in this laboratory. Briefly, mice aged 6-8 weeks were singly housed and food restricted for 18 h prior to testing; all had access to water. They were then placed into test cubicles (inside dimensions 3.8 cm wide by 5.5 em deep by 11.5 cm high) to which a 9-mm OD cylindrical food tube was attached on the front wall equidistant from the 2 sides. The tube was partially filled with rolled wheat (Maypo). An observer recorded 50 reaches for food for every mouse. From these data 2 primary measures of lateralization were derived: the number of right paw entries in 50 reaches (RPE score), and the number of 'preferred paw entries' (PPE score). The PPE score ranges from 25 to 50 and indicates the degree of asymmetry without regard to its directionality: PPE = abs(RPE - 25) + 25. For example, RPE scores of 10 for a left-handed mouse and of 40 for a right-handed mouse are both PPE scores of 40. For statistical analysis PPE scores were transformed using the logit: LPPE = 0.5 In [(PPE + 1/6)/(50 - PPE + 1/6)] (see ref. 34). The high lateralization line (HI) was formed by mating G o mice with PPE scores of 48-50. The low lateralization line (LO) was formed using mice showing little overall paw preference, PPE scores of 25---40. Mice scoring in the region of 41-47 PPEs were not used for breeding. All matings were made without regard to the left/right directionality of laterality and were subject only to avoidance of brother-sister inbreeding. From generation G 1 onward behavioral testing and selection followed this pattern except that in response to selection gains, the upper cutoff point for retaining mice for LO line matings was reduced from the 40 PPE ceiling to 33 PPE at Glo. Each generation required an 11-13 week sequence of testing during which mice were coded and tested blindly. During the first 2 generations, entire litters were randomized and tested in contiguous lots. From G 3 onward the testing sequence of individual mice within and between litters was randomized by computer. We strove to maintain an average sample size of 200 tested mice per line per generation (range 140-234 mice). All HI and LO line matings were made within lines, In terms of the PPE measure, both lines were subjected to directional selection. In terms of the RPE measure, the HI line could be considered subject to disrup-

tive selection, and the LO line to stabilizing selection. A control population of genetically heterogeneous mice (HET) was also established using the diallel intercrossing plan. It is maintained by random mating with avoidance of inbreeding and without selection for lateralization. Selective breeding continued for 11 consecutive generations and was relaxed at G12. The lines were then propagated by random within-line mating using about 25 parental pairs per line per generation. At G28 bidirectional selection was again impressed for 3 consecutive generations. Procedures followed those used originally. During generations G15, 616 and G33 , mice from HI, LO, and H E r reference lines were tested for handedness in a challenge paradigm 1°. Briefly, mice were tested first in the standard unbiased or 'U-worid' and then twice later in worlds biased opposite to their expressed handedness: sequences 'U-R-R' for left-handed mice, or 'U-L-L' for right-handers. Whereas the unbiased world apparatus had the food tube placed on the midline of the front wall enabling equal accessibility to right and left paws, in biased worlds the feeding tube was located flush against either the right wall as faced by the mouse (R-world) or against the left wall (L-world). This made it inconvenient for a mouse to continue reaching with its preferred paw. Challenge tests were conducted to assess whether average lateralization of both selected lines was differentiated with respect to that of the control line, and whether line differences remained divergent in the absence of continued selection. RESULTS T h e d i s t r i b u t i o n o f R P E scores for 327 m i c e o f t h e G O f o u n d a t i o n p o p u l a t i o n is p r e s e n t e d in Fig. 1. T h e distrib u t i o n is b i m o d a i a n d has a p r o n o u n c e d ' U ' - s h a p e . A p p r o x i m a t e l y 42% o f m i c e c o u l d b e d e s i g n a t e d dextral ( R P E scores g r e a t e r t h a n 25), a n d 55% w e r e sinistral ( R P E scores less t h a n 25). T w o p e r c e n t o f m i c e h a d R P E scores o f 25. This R P E distribution for the g e n e t i c a l l y h e t e r o g e n e o u s f o u n d a t i o n p o p u l a t i o n is similar to t h o s e o f g e n e t i c a l l y h o m o g e n e o u s m i c e o f i n b r e d strains obs e r v e d in this l a b o r a t o r y . Fig. 2 shows the distribution

N % rel freq

of P P E scores for G o m i c e , and indicates the P P E r a n g e s

2818"6 26 8.0

that f o r m e d the basis for selective b r e e d i n g . D u r i n g 11 g e n e r a t i o n s o f b i d i r e c t i o n a l s e l e c t i o n m o r e t h a n 4500 m i c e w e r e t e s t e d for h a n d e d n e s s . C h a n g e s in

2 1

L P P E a v e r a g e s t h r o u g h o u t selective b r e e d i n g are s h o w n in Fig. 3. A t g e n e r a t i o n G 1 t h e r e was a non-significant r e v e r s a l of m e a n l a t e r a l i z a t i o n (cp. Gimelfarb25). This

1 1

c o r r e c t e d itself by G 2. A t G3 the s e l e c t e d lines first dif-

1

f e r e d statistically in d e g r e e o f l a t e r a l i z a t i o n (F1.403 = 9.66; P = 0.002). In t e r m s o f effect size 7, a v e r a g e later-

1

alization o f G 3 H I and L O lines d i f f e r e d by 0.31 stand a r d d e v i a t i o n s (SDs). F r o m G4 o n w a r d the s e l e c t e d lines c o n t i n u e d to d i v e r g e . B y G l o the line d i f f e r e n c e in 0.000

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Fig. 1. Distribution of right paw entries (RPE) in 50 food reaches for 327 mice of the foundation population, G o. The abscissa is divided into 51 intervals ranging from 0 RPE (all left-hand reaches) to 50 RPE (all right-hand reaches). The ordinate shows the number and relative frequency of G O mice observed in each RPE interval. Note that the distribution of RPE scores is markedly 'U'shaped. Most mice were either strongly left-handed, or strongly right-handed, whereas few were ambilateral.

d e g r e e o f a s y m m e t r y was p r o n o u n c e d (F1,42 a = 120.4; P < 0.0001). M e a n L P P E scores o f G l o lines d i f f e r e d by 1.12 SDs. M i c e o f G l l w e r e u s e d in a n o t h e r e x p e r i m e n t and t h e i r scores are n o t available. T h e 2 r e g r e s s i o n lines s h o w n in Fig. 3 w e r e c a l c u l a t e d for e a c h s e l e c t e d line using d a t a f r o m G1 t h r o u g h Glo and p r o j e c t e d to Go. This p r o j e c t i o n indicates t h a t t h e a v e r a g e l a t e r a l i z a t i o n o b s e r v e d in t h e f o u n d a t i o n p o p u -

196 N */* rel freq

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52~159 Go foundation population

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Fig. 4. Response to selection based upon the proportion of mice exhibiting strong expressed lateralization, scores of 48-50 PPE. By Glo 44.7% of HI line mice and only 8.0% of LO line mice scored in this region.

*4.900

Fig. 2. Distribution of paw entries (PPE) for 327 mice of the foundation population, G0. The PPE score ranges from 25 to 50 and indicates the strength of lateralization for handedness without regard to its directionality. Blackened regions indicate PPE intervals used for initial selective breeding. Mice scoring in the region of 25-40 PPE were used to propagate the LO line. Mice scoring in the region of 48-50 PPE were used to form the HI line. Mice in the region of 41-47 PPE were not used for breeding.

ization (PPE scores of 48-50). A t Gt0 44.7% of H I line mice showed extreme lateralization compared to 8.0% of LO line mice (Z2 = 74.9; P < 0.0001). Fig. 5 presents similar information on the proportions of mice showing weak lateralization (PPE scores of 25-40). By G10 67.7% of L O line mice scored in this region compared to only 27.7% of H I l i n e mice (Z2 = 66.0; P < 0.0001). The

lation was somewhat higher than expected. This may be attributable to having used F 2 generations of the diallel

proportion of Glo H I line mice scoring 48-50 P P E exceeded that in G O (Z2 = 9.38; P < 0.005). The propor-

cross directly for Go without allowing for one or more preliminary generations of r a n d o m mating. The regression lines also indicate that the average selection gains for H I and L O lines were approximately equal. Fig. 4 presents information on the changes of proportions of mice of each line with strong expressed lateral-

tion of Glo L O line mice within 25"40 PPEs exceeded that observed in Go (Z2 = 56.0; P < 0.0001). The first objective of the research program was met. Results indicated that the degree of functional asymmetry was heritable and that extreme expression of lateralization could be achieved through selective breeding. The second objective was to establish divergent lines of mice that might usefully serve research needs for studies of lateralization in the nervous system. To meet this goal

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Fig. 3. Response to selective breeding for ,degree of lateralization of handedness for mice of the HI line (upper triangles) and LO line (lower triangles) for GOto G~o. The ordinate indicates the logit transformed average degree of lateralization (LPPE). Standard error bars depict -+ 1 S.E.M. Selected lines first differed statistically at G3, and diverged increasingly thereafter. Two regression lines (dotted lines) were plotted using data from G1 to Glo and back projected to GO. These indicate that the overall rates of change for HI and LO lines were similar. They also indicate that average lateralization of the foundation population was somewhat higher than expected.

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Fig. 5. Response to selection based on the proportion o f mice ex-

hibiting weak expressed lateralization, scores of 25-40 PPE. By G lo 67.7% of LO line mice and only 27.7% of HI line mice scored in this region.

197 G15-G16

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Fig. 6. Average LPPE scores to biased world challenge for HI and LO lines (solid lines) of G15 and G16 and for the HET control line (dashed line). Mice were tested first in the standard unbiased world and then twice later in test cubicles biased opposite to their original expressed laterality: test sequences 'U-R-R' or 'U-L-L'. HI line mice tended to retain their strong original hand preference, whereas LO line mice tended to change their laterality. HET reference line mice showed an intermediate pattern. Standard error bars indicate --- 1 S.E.M.

it was necessary to show that the selected lines retain their distinctive behavior in the absence of further selection. Selection was relaxed at G12. From G15 and G16 sel e c t e d lines and the H E T control line 361 mice were tested in the unbiased world and twice later in worlds biased opposite to their hand preference. Preferred paw entry scores in biased world tests are defined in a slightly different way from that used previously. These PPE scores were referenced to the mouse's original laterality observed in the unbiased world and could range from 50

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Fig. 7. Response to 3 generations of reimpressed selection during G28-G30 compared to original selection gains. Mean LPPE score for G28 HI line mice was lower than that for HI line mice of G 10. By 629 the HI line had regained former levels. Mean LPPE scores for G ~ and G29 LO line were comparable to LO line mice of G 10, whereas performance of G30 LO line exhibited the lowest average LPPE score of any previous generation. Standard errors bars show -+ 1 S.E.M.

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i (330

Fig. 8. Effects of sex on line differences of mean LPPE score for Ga-Glo and G28-G30 (female mice: solid line; male mice: dotted line). Note that the initial decline of G28 LPPE scores is attributable to reduced male performance. The degree of lateralization for female G28 HI line is comparable to Ga-Glo and G29-G30 averages. Standard error bars show 4- 1 S.E.M.

to 0. For example, a left-handed mouse with a 'U-R-R' sequence of 2, 15, and 40 RPE would have PPE scores of 48, 35, and 10. PPE scores less than 25 indicate that a mouse was exhibiting a laterality opposite to that observed originally in the 'U'-world. Fig. 6 illustrates changes in the average LPPE scores for the challenge test. HI line mice exhibited stronger lateralization in the 'U'-world test and were resistant to change when confronted with a biased environmental challenge. HI line resistance to biased world challenge was noteworthy. Their median PPE scores slightly increased on the last biased world test (45.0, 45.0 and 46.0 PPE). LO mice displayed both lessened lateralization in the 'U'-world and a greater responsiveness to challenge. Mice of the H E T reference stock showed intermediate lateralization. Repeated measures A N O V A of LPPE scores for line and sex treatments indicated a main effect across the experiment for line (F2.350 = 23.6; P < 0.0001). There was no significant effect for sex or its interaction with line. In the unbiased world test, HI and LO line means differed by 0.78 SDs. Dunnett's simultaneous comparison procedure 47 was employed to contrast mean LPPE scores of the selected lines against the HET control average for each test condition (P < 0.05; onetailed test). In the unbiased world the LO line was less lateralized than HET, but the HI line was not more lateralized than the HET line. For the first biased world test, both selected lines differed from HET. For the last biased world test, the HI line was more lateralized than controls, but the L O - H E T comparison did not differ. The maximum discrimination of the selected lines against the H E T population occurred in the first biased world test. Results of the challenge experiment indicate that the selected lines remained divergent in degree of lateraliza-

198 HET line. Thus each selected line might serve as a useful lateralization resource in its own right. From 617 until Ga8 HI and LO lines were maintained by random within-line mating using about 25 parental pairs per line per generation. During this time we focused on studying behavioral 12'44, neural 6'32'46, and immunological 1'13'23"24 characters potentially associated with variable lateralization. Although we measured lateralization in samples of mice of the selected and control lines, full-time handedness testing could not be continued. Concern arose as to whether the divergence between lines had been reduced and whether lateralization of selected lines had regressed to average values. To address these we began a full-scale reimpression of selection beginning at Gz8. This continued for 3 generations. The protocol and procedures followed those used during original selection, except that the observer was new. A total of 1123 mice were tested. Fig. 7 presents the mean LPPE scores for G28-G3o mice. Two-way A N O V A for line and sex confirmed significant line differences for G2s (F1,327 = 44.1), for G29 (F1,4o8 = 119.4), and for G30 (F1,376 = 148.8). In each case P < 0.0001. In terms of effect size, mean LPPE scores of HI and LO lines differed by 0.74 standard deviations at G28, 1.09 SDs at G29 and 1.25 SDs at G30. G28 HI line mice showed a lower degree of lateraliza-

G 33 1.6. 1.41.2-

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Fig. 9. Average LPPE scores to biased world challenge for HI and LO lines of G33 (solid lines) and for the HET control population (dashed line). Mice were tested first in the standard unbiased world and then twice later in test cubicles biased opposite to their original expressed lateraiity: test sequences 'U-R-R' or 'U-L-L'. Whereas HI line mice tended to maintain their original directionality of handedness and strong lateralization, LO line mice increasingly changed their handedness in response to environmental challenge. Response of HET control line mice was intermediate between the 2 divergent selected lines. Standard error bars show + 1 S.E.M.

tion following 4 or 5 generations of relaxed selection. In addition, these results indicate that both selected lines differed from average lateralization of the unselected

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199 tion compared to G10 and subsequent generations. Although this coutd indicate counterselection for average lateralization during the preceding generations of relaxed selection, we believe that this was due to other causes. Lessened lateralization of the G28 HI line was observed only in male mice (1.04 --- 0.115 LPPE), whereas G28 HI line female scores were comparable to earlier and later generations (1.36 --- 0.106 LPPE). In addition, HI line male scores recovered completely the next generation (Fig. 8). If genes favoring average lateralization had accumulated during the 17 generations of random mating we would not expect their effect to be limited to one sex only, or that their influence would be reversed completely in the next generation of selective breeding. The selected lines were maintained by random mating following G30. At G33 293 mice from HI, LO, and H E T lines were tested in the 'U-R-R' or 'U-L-L' challenge test using the same procedures employed at G~5 and 616. Fig. 9 illustrates the trend in LPPE averages. As observed during the previous challenge, median PPE scores of HI line mice remained flat across biased world tests (46.0, 46.0, 45.5 PPE). Two-way ANOVA indicated statistically significant line differences (F2,287 = 26.54; P < 0.0001). There was no effect of sex or its interaction with line. For the initial 'U'-world test, HI and LO line averages differed by 1.16 SDs. Dunnett's procedure was again applied to contrast mean performance of selected and control lines at each test (P < 0.05; one-tailed test). In both the 'U'-world and the first biased world test, mice of the HI line were more strongly lateralized than H E T controls, and mice of the LO line were less lateralized than controls. For the second biased world test, LO line mice were less lateralized than controls, whereas the H I - H E T contrast did not differ. As observed previously, maximum discrimination between the selected and H E T reference lines occurred in the first biased world challenge test.

DISCUSSION Bidirectional artificial selection for degree of lateralization for handedness produced 2 lines of mice divergent for functional asymmetry. Mice of the HI line exhibit extreme right- and left-handedness and resist environmental challenges to change preferred paw usage. Mice of the LO line exhibit weak lateralization and are malleable to environmental pressures to increase use of the non-preferred paw. Mice of the unselected HET reference line show an intermediate pattern of lateralization. The success of artificial selection for degree of asymmetry contrasts with the difficulty of inducing lasting changes in the directionality of handedness in rats 39 and

mice 2'9 using similar methods. The seemingly random relationship between the directionality of asymmetry and genetic treatments is not limited to experiments using laboratory rodents. For example, there appears to be non-genetic individuality in isogenic bacteria for smooth swimming and tumbling to chemical attractants 45. These behaviors are driven by an asymmetric flagellar motor in which counterclockwise rotation is associated with smooth swimming and clockwise rotation with tumbling 29. Right- and left-mirror image arrangements of cell-surface structures of Tetrahymena thermophila do not depend upon differences in nuclear genes 35. The directionality of foliar spiraling in the coconut palm, Cocos nucifera, a characteristic associated with differences in yield and disease resistance, was unaffected after application of selective breeding t5'16. Although human children tend to resemble their parents in handedness, the association of laterality in human monozygous and dizygous twins is little more than would be expected on the basis of random pairing 11. The radial direction of hairwhorls of human twins behaves in the same way n. Nevertheless, there is some evidence that continues to implicate genetic influences in the control of expressed directionality. The inheritance of the body spiral asymmetry in the gastropod Limnaea peregra seems to follow a maternal pattern of inheritance with dextrality dominant to sinistrality 3'1s'22. The asymmetry of the starry flounder (Platichthys stellatus) in which the position of the eyes is shifted to one side of the head implicates a pattern of both maternal and paternal genetic inheritance 4°'41. Sims inversus in mice is inherited in a single locus recessive pattern 28. The wild type allele ( + / + and +/iv) leads to normal visceral development, whereas the iv~iv genotype is associated with random sims; approximately one-half iv/iv mice have their stomachs on the left or normal side, and half on the right side. The/v-locus has been recently genetically mapped to distal Chromosome 12, and is located between Aat (a ~-antitrypsin gene complex) and Igh-C (immunoglobulin heavy-chain variable-region gene complex) 5. Considerations of linkage conservation between mouse and man suggest that a human homolog of iv may exist on human chromosome

14q5. Taken together these findings are challenging to either extreme view of the genetics of asymmetry. They present apparent counterexamples to the hypothesis that genes do not, or cannot, code for the directions of asymmetry 10'14'33, as well as to the hypothesis that alternative genetic alleles strictly code one or both senses of asymmetry 3°. Nevertheless it is possible to retain the stochastic generation of senses of asymmetry while allowing for an apparent inheritance of uniform directionality. This obtains in models in which the stochastic outcomes in-

200 teract with external gradients of asymmetry or world biases 12. Brown and Wolpert 4 recently advanced an attractive developmental model for the generation of left/right asymmetry in which an inherent molecular asymmetry plays a key role. By contrast, the degree of asymmetry or lateralization itself may be more well-behaved genetically. The success of bidirectional selection in mice for extreme expression of lateralization for handedness suggests that genetic mechanisms are responsible for this heritability. However, it must be emphasized that the observation of a realized heritability for phenotypic forms does not in itself prove the genetic hypothesislk Selective breeding was suspended at G~2 and line differences remained stable during a period of 17 generations of random mating until the reimpression of further selection. Line differences did not regress away from extreme expression to return toward original levels. This failure to regress at first seemed surprising, for such effects have been observed widely, for example in behavioral experiments for geotaxis in lines of Drosophila melanogaster2°, and for geotaxis and phototaxis in lines of Drosophila pseudoobscura~9. In the latter case, the speed of regression of negative phototaxis actually exceeded the rate of forward selection. Such regression can be considered an effect of genetic homeostasis, defined by Lerner as "the property of the population to equilibrate its genetic composition and to resist sudden changes "at . Essential to this notion is that if the target character is related to reproductive fitness, and if natural selection favors, for example, an intermediate optimum, then natural selection will tend to oppose gains made by artificial selection. The suspension of selection itself has been considered by Falconer to be a 'perturbation experiment' in which a character may be regarded as neutral if the mean does not revert to original values, or does so only slowly21. One way to test the neutrality hypothesis is to examine ratios of effective to expected selection differentials during the course of original selection. The selection differential measures the average phenotypic superiority of parents relative to the mean of their generation and indicates the degree of selection applied. The expected or unweighted selection differential considers that parental pairs contribute progeny equally to the next generation. The effective or weighted selection differential adjusts for the number of offspring contributed by the parents. The ratio of effective to expected selection differential may indicate whether differences in reproductive success are related to parental phenotypic values, and whether natural selection is operating to help or hinder changes in the target character 21. Ratios less than 1.00 suggest that natural selection is working against artificial selection, whereas ratios greater than 1.00 suggest that natu-

ral selection was favoring response. Fig. 10 presents these ratios for both H I and LO lines during the first 10 generations of selection. For the H I line, these ratios averaged 0.998 (range 0.993-1.006), and the least squares regression line indicated no change with advancing generation. For the LO line, the ratios also averaged 0.998 (range 0.967-1.012). Again, there was no significant regression of ratios with generation number. The pattern of results suggests that natural selection was neutral with respect to both high and low degree of lateralization for handedness. Our interest in possible regression effects is motivated by both practical and theoretical concerns. It is of practical importance to know whether line divergence remains stable or whether it is necessary to repeatedly reimpress selection to maintain line differences. Secondly, there is considerable interest in knowing whether strong, weak, or intermediate lateralization is optimum for an organism. Several workers have suggested that there is not a monotonic relationship between lateralization and organismic advantage, but rather an inverted U-shaped function in which moderate lateralization is optimal and extremes to either side disadvantageous 26'37. The results observed are sufficient to address the first question, but not necessarily the second. The between line divergence observed is approximately 1 SD; the maximum difference was 1.25 SD observed at G33. This is considerably less than the 4 or more SD differences obtained in other selection experiments for behavioral characters 27, for example, selection for open-field activity in mice 17. Although the RPE distributions of H I and LO lines are dearly visually different, bimodal versus unimodal, and mean lateralization differences are statistically highly significant, yet there remains complete overlap in the range of RPE scores, 0-50. Furthermore a 1 SD mean difference in PPE score could be considered to be still within the normal range of the original population. Accordingly, this suggests that in employing selection based upon 50 reaches, the floor and ceiling may have remained too close and a more discriminating test procedure should be employed in future work. For example, we could base further selection on the results of 2 or more 50-reach sessions, or use an index selection procedure conditioned upon performance in the 'U'world and the first biased world challenge test. In results reported here we have twice observed greater betweenline discrimination in the first biased world challenge than in the prior unbiased world test. The evidence obtained thus far does not suggest that the selected lines have reached a plateau in performance, and encourages us to seek greater gains. Selective breeding experiments provide useful information on the realized heritability of the phenotype of

201 i n t e r e s t and t h e y m a y g e n e r a t e d i v e r g e n t lines o f a n i m a l s

s e a r c h strategies

that b e n e f i t studies o f b i o l o g i c a l p r o c e s s e s c o r r e l a t e d

substrates o f v a r i a b l e lateralization.

to b e t t e r

understand

the biological

w i t h d i f f e r e n c e s in t h e t a r g e t c h a r a c t e r . T h e success o f s e l e c t i v e b r e e d i n g for v a r i a t i o n in d e g r e e o f a s y m m e t r y for h a n d e d n e s s in m i c e indicates clearly t h e heritability o f s t r o n g and w e a k l a t e r a l i z a t i o n . T h i s is n o t e w o r t h y bec a u s e l a t e r a l i z a t i o n is a d i m e n s i o n o f a s y m m e t r y that has so far r e c e i v e d l i m i t e d s u s t a i n e d a t t e n t i o n . T h e selective b r e e d i n g p r o c e s s has also p r o d u c e d lines o f m i c e t h a t w i t h c a r e in i n t e r p r e t a t i o n 27 m a y p r o v e useful in re-

Acknowledgements. This research was supported by Grant GM 23618 from the National Institutes of Health. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care. I especially thank research assistants Dirck W. Bradt, Martha M. Davis, Lisa Burton, and Kathryn Walsh who patiently monitored paw reaching performance. I also thank Donald W. Bailey, James F. Crow, Larry E. Mobraaten and Paul E. Neumann for critical reading of the manuscript.

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