animals : the research was conducted in accordance with the declaration of 
 helsinki and was approved by the animal experiments committee of riken ( approved i d no . 
 h24 - 2 - 206 ) . the heterozygous rolling nagoya ( prod - rol/+ ) 
 strain was provided by the riken bioresource center ( tsukuba , japan ) , with support from the 
 national bioresource project of the ministry of education , culture , sports , science and 
 technology , japan .
c57bl/6j ( b6 ) mice were purchased from charles river japan ( yokohama , 
 japan ) . the homozygous rolling nagoya 
 ( prod - rol / rol ) and wild - type rolling 
 nagoya ( prod - s / s ) strains were bred by mating with 
 prod - rol/+ mice .
the mice 
 were allowed ad libitum access to water and food pellets ( crf-1 ; oriental 
 yeast , tokyo , japan ) and kept under conditions of controlled room temperature ( 23  1c ) and 
 humidity ( 55  5% ) with a 12:12-hr light  dark cycle ( lights on from 08:00 to 20:00 ) . 

reverse transcription - polymerase chain reaction ( rt - pcr ) : total rnas from 
 the brains of prod - s / s(male , female : n=10 , 10 ) , 
 prod - rol / rol ( male , female : n=10 , 10 ) and b6 ( male , female : n=10 , 10 ) 
 mice were isolated using trizol reagent ( invitrogen , burlington , canada ) .
first - strand 
 complementary dna ( cdna ) was synthesized using the superscript first - strand synthesis system 
 ( invitrogen ) .
the following pcr primer sets were used for amplification of 
 ednrb ( genbank i d : nm_007904.4 ) : forward , 5-gctagtgtgttttcagaggcttg-3 , 
 and reverse , 5-gactaagaatctaagtgcttccc-3. the rt - pcr products were sequenced using an abi 
 prism 3730 ( applied biosystems , foster city , ca , u.s.a . ) .
genomic structure analysis : to distinguish normal alleles from those with 
 insertion of a retroposon - like element in intron 1 of the ednrb gene 
 ( genbank i d : ab242436.1 ) , the pcr products were amplified with genomic dna from the tails 
 prod - s / s ( male , female : n=10 , 10 ) , prod - rol / rol ( male , 
 female : n=10 , 10 ) and b6 ( male , female : n=10 , 10 ) mice , and allele - specific primers designed 
 according to a previous report .
the pcr products 
 were sequenced and examined for the presence or absence of the insertion .
real - time quantitative rt - pcr ( real - time qrt - pcr ) : the levels of 
 ednrb and cacna1a mrna were measured using applied 
 biosystems taqman gene expression assays ( ednrb , assay i d : mm01224433_m1 ; 
 cacna1a , assay i d : mm00432190_m1 ) .
all data were normalized relative to 
 18s ribosomal rna ( assay i d : hs99999901_s1 , applied biosystems taqman gene expression 
 assay ) .
the pcr conditions were 94c for 10 min , followed by 40 cycles at 95c for 15 sec 
 and 60c for 1 min .
all male samples ( prod - s / s , 
 prod - rol / rol and b6 : n = each 10 ) were analyzed in duplicate , and the 
 threshold cycle ( ct ) value was calculated to determine the relative levels of expression 
 . in situ
hybridization : the male gastrointestinal tract 
 ( prod - s / s , prod - rol / rol and b6 : n = each 5 ) was dissected 
 after perfusion with saline followed by 4% paraformaldehyde , fixed with tissue fixative 
 ( gonostaff , co. , ltd . ,
tokyo , japan ) , embedded in paraffin and cut into section at a 
 thickness of 6 m for in situ hybridization .
the probes for the cav2.1 1 subunit cdna fragment 
 ( positions 60686748 ) were labeled with a digoxigenin rna labeling kit ( roche diagnostics , 
 mannheim , germany ) .
color reactions were performed with nbt / bcip solution ( sigma - aldrich , 
 st .
louis , mo , u.s.a . ) overnight and then washed with phosphate - buffered saline ( pbs ) .
the 
 sections were counterstained with kernechtrot stain solution ( mutoh pure chemicals , tokyo , 
 japan ) and mounted with cc / mount ( diagnostic biosystems inc . ,
microsatellite genotyping : to examine the origin of the 
 ednrb allele from genomic dna obtained from the tails of 
 prod - s / s ( male , female : n=10 , 10 ) , prod - rol / rol ( male , 
 female : n=10 , 10 ) and b6 ( male , female : n=10 , 10 ) mice , microsatellite markers 
 ( d14mit30 , d14mit193 , d14mit93 , 
 d14mit94 , d14mit170 , d14mit42 and 
 d14mit267 ) were typed by pcr amplification using the mouse microsatellite 
 data base of japan ( mmdbj , http://www.shigen.nig.ac.jp/mouse/mmdbj/ ) and the mouse genome 
 informatics ( mgi , http://www.informatics.jax.org/ ) .
statistical analysis : the data are presented as the means  standard error 
 of the mean ( sem ) .
statistical analyses were conducted using excel statistics 2006 ( ssri , 
 tokyo , japan ) .
the data were analyzed using dunnett s test between groups where appropriate . 
 in all analyses
gross observation of prod strain : prod - s / s and 
 prod - rol / rol mice had irregular white spots on the agouti coat ( fig . 1afig .
( a ) 
 representative photographs showing white spots on agouti coat of prod strain and 
 ataxia of prod - rol / rol mice are presented .
( b ) representative 
 photographs of the open abdomen of prod - s / s and 
 prod - rol / rol mice are shown . ) .
prod - rol / rol mice showed poor motor coordination and stiffness of 
 the hindlimbs .
neither prod - s / s nor prod - rol / rol mice 
 showed the megacolon phenotype ( fig .
( a ) 
 representative photographs showing white spots on agouti coat of prod strain and 
 ataxia of prod - rol / rol mice are presented .
( b ) representative 
 photographs of the open abdomen of prod - s / s and 
 prod - rol / rol mice are shown .
sequence analysis of the ednrb gene coding region : two c to t nucleotide 
 substitutions were found at nucleotides 159 and 1032 in the coding region of the 
 ednrb gene in prod - s / s mice ( fig .
( a ) exons are shown as 
 white boxes , and the retroposon - like region is shown as a black box .
white arrows 
 represent the primers used to amplify the normal ( wt ) and mutant ( mu ) alleles by pcr 
 as previously reported .
black arrows 
 represent the primers used to examine mrna expression using real - time qrt - pcr .
( c ) the expression levels of 
 ednrb mrna in the colon determined by real - time qrt - pcr .
the 
 ednrb mrna expression level for each strain was calculated relative 
 to that in b6 mice .
* p<0.05 compared to the appropriate control 
 ( dunnett s test ) . ) .
however , the amino sequence of ednrb in prod - s / s mice was not 
 different from the sequence of the b6 strain in the database .
these silent nucleotide 
 substitutions in the ednrb gene were detected in 
 s / s mice   and 
 jf1 mice   as a piebald mutation .
we found 
 nucleotide substitutions in the ednrb gene ( genbank i d : u32329.1 ) in both 
 male and female prod - s / s and prod - rol / rol mice ( data not 
 shown ) .
( a ) exons are shown as 
 white boxes , and the retroposon - like region is shown as a black box .
white arrows 
 represent the primers used to amplify the normal ( wt ) and mutant ( mu ) alleles by pcr 
 as previously reported .
black arrows 
 represent the primers used to examine mrna expression using real - time qrt - pcr .
( c ) the expression levels of 
 ednrb mrna in the colon determined by real - time qrt - pcr . the 
 ednrb mrna expression level for each strain
structure analysis of intron 1 of the ednrb gene : intron 1 has the 5.5-kb 
 retroposon - like element in s / s mice   and jf1 mice . the f and 
 r
2a ) and amplify a 225-base pair ( bp ) fragment as the wild type . 

the r - mu primer corresponds to a retroposon - like element in the 
 s / s mutant ( fig . 

2a ) , and the f and r - mu primers amplify a 318-bp fragment containing part of intron 
 1 of the ednrb gene and the retroposon - like element of the 
 s / s mutant type .
although a 225-bp fragment was 
 amplified from b6 mice , prod - s / s mice and prod - rol / rol 
 mice showed a 318-bp fragment ( fig .
sequence 
 analyses of the pcr products showed that the retroposon - like element was inserted in intron 
 1 ( data not shown ) .
we found the retroposon - like element in both male and female 
 prod - s / s and prod - rol / rol mice ( data not shown ) .
ednrb gene expression levels in the colon : real - time qrt - pcr analysis was 
 performed to determine the levels of ednrb mrna expression in enteric 
 neurons of prod - s / s , prod - rol / rol and b6 mice ( fig .
the relative expression level of 
 ednrb was not significantly different between prod - s / s 
 and prod - rol / rol mice , both of which showed about 25% lower levels of 
 ednrb expression in the colon compared to control b6 mice .
cacna1a gene expression patterns in the colon : to examine whether mutation 
 of the ednrb gene affects the cacna1a expression pattern 
 in the colon , we used in situ hybridization and real - time qrt - pcr analyses . 
 the results of in situ hybridization analysis indicated similar patterns of 
 cacna1a mrna expression in the colon of prod - s / s , 
 prod - rol / rol and b6 mice ( fig
3.expression patterns of cacna1a mrna in the colon of 
 prod - s / s , prod - rol / rol and b6 mice .
( a ) 
 representative photographs indicating the localization of cacna1a 
 mrna in the colon are shown .
( b ) the expression levels of cacna1a mrna in 
 the intestine determined using real - time qrt - pcr analysis are shown .
the 
 cacna1a mrna expression level for each strain was calculated 
 relative to that in b6 mice .
* p<0.05 compared to the appropriate 
 control ( dunnett s test ) . ) . using the antisense probe ,
no signals were seen in the colon using the 
 sense probe in any of these strains ( data not shown ) .
real - time qrt - pcr analysis was 
 performed to determine the levels of cacna1a mrna expression in enteric 
 neurons in the three mouse strains ( fig .
the 
 relative levels of cacna1a mrna were not significantly different between 
 prod - s / s and prod - rol / rol mice , but they were both 
 significantly lower than that in the colon of b6 mice .
expression patterns of cacna1a mrna in the colon of 
 prod - s / s , prod - rol / rol and b6 mice .
( a ) 
 representative photographs indicating the localization of cacna1a 
 mrna in the colon are shown .
( b ) the expression levels of cacna1a mrna in 
 the intestine determined using real - time qrt - pcr analysis are shown .
the 
 cacna1a mrna expression level for each strain was calculated 
 relative to that in b6 mice . *
analysis of microsatellite loci adjacent to the ednrb locus : to examine 
 the origin of the mutant ednrb gene in prod - s / s and 
 prod - rol / rol mice , we typed microsatellite loci on chromosome 14 .
( a ) linkage map of mouse chromosome 14 , including the 
 ednrb and microsatellite loci used in this study , is presented .
( b ) 
 genotyping results of the microsatellite makers are shown .. as shown in fig .
4b , fragments were 
 amplified from prod - s / s , prod - rol / rol and b6 mice . 
 according to mmdbj and mgi ,
the different sizes between jf1 and b6 strains are produced in 
 d14mit30 ( jf1 , b6 : size=116 bp , 154 bp ) , d14mit193 ( jf1 , 
 b6 : size=134 bp , 119 bp ) , d14mit93 ( jf1 , b6 : size=189 bp , 147 bp ) , 
 d14mit94 ( jf1 , b6 : size=108 bp , 104 bp ) , d14mit170 ( jf1 , 
 b6 : size=163 bp , 146 bp ) , d14mit42 ( jf1 , b6 : size=142 bp , 152 bp ) and 
 d14mit267 ( jf1 , b6 : size=97 bp , 114 bp ) loci .
amplified fragments of the 
 same length were obtained in both male and female prod - s / s and 
 prod - rol / rol mice ( data not shown ) .
( a ) linkage map of mouse chromosome 14 , including the 
 ednrb and microsatellite loci used in this study , is presented .
the results of the present study indicated that the rolling nagoya strain 
 prod - rol / rol , which exhibits white spots on the agouti 
 coat , has two mutations in the ednrb gene , i.e. , two c to t nucleotide 
 substitutions at positions 159 and 1032 in the coding region without amino acid 
 substitutions and a retroposon - like element insertion into intron 1 .
these mutations have 
 been reported to be piebald spotting ( s ) mutations [ 4 , 6 , 9 , 20 ] .
in addition to the coat pigment defect , piebald lethal 
 ( s / s ) mice , which carry a severe mutation at the 
 s locus and lack the full - length transcript of the ednrb 
 gene , show the megacolon phenotype .
the 
 s / s , 
 s / s , s / s 
 and s/+ mice , in which the relative ratios of 
 ednrb expression are about 0%:12.5%:25%:50% , show a  graded 
coat color 
 phenotype in the extent of white spotting and have a white coat on>95% , 4050% , ~20% and 
 0% of the body surface area , respectively .
the 
 megacolon phenotype occurs in s / s mice , but not in 
 s / s , s / s 
 or s/+ mice .
these 
 results indicate that the two neural crest - derived cell lineages require different minimal 
 threshold levels of ednrb gene expression .
more than 50% of the wild - type 
 level of ednrb density is likely needed for normal development of epidermal 
 melanocytes , whereas at least 12.5% of the wild - type level is required for a functional 
 enteric nervous system . in the present study ,
prod - rol / rol 
 and prod - s / s mice showed approximately 25% lower levels of 
 ednrb gene expression compared with b6 mice that have an intact 
 ednrb gene .
the white - spotted area on the body surface was detected in 
 prod- s / s and prod- rol / rol mice without 
 a megacolon .
regarding the origin of the laboratory mouse , while most regions of the chromosomes and 
 mitochondrial dna are of mus musculus domesticus origin , the y chromosome 
 and some particular regions of the autosomes are of m. m. musculus or 
 m. m. molossinus origin .
the 
 c to t nucleotide substitutions at position 1032 in the coding region of the 
 ednrb gene are unique to m. m. molossinus origin .
jf1 is thought to be derived from the japanese wild 
 mouse via m. m. molossinus .
jf1 
 mice show irregular black spots on the white coat body without a megacolon and have the 
 piebald s / s mutation .
the region between d14mit93 and 
 d14mit42 loci was identical to that of the m. m. 
 molossinus strain .
these results 
 indicate that the sequence around the s locus is derived from m. m. 
 molossinus . in the present study ,
microsatellite genotyping indicated that the 
 alleles of the d14mit193 , d14mit93 , 
 d14mit94 , d14mit170 and d14mit42 loci 
 were identical to those of the jf1 strain , but that d14mit30 and 
 d14mitw267 loci were identical to those of the b6 strain .
jf1 strain was 
 derived from fancy mice purchased at a market in denmark in 1987 and established as inbred 
 strain in 1993 .
prod strain was derived from the 
 descendants of ( siii x c57bl/6 ) f1 and discovered in 1969 .
although the genetic background of the siii strain has not been reported , our 
 results indicated that at least the region of chromosome 14 between the 
 d14mit193 and d14mit42 loci of the jf1 and prod strains 
 was derived from the japanese fancy piebald mouse via m. m. molossinus .
the results of this study indicate that prod - rol / rol is a 
 double - mutant strain with an amino acid change at r1262 g in the cacna1a 
 gene on chromosome 8 causing cav2.1 channel dysfunction and the classic piebald 
 mutation in the ednrb gene on chromosome 14 causing the coat pigment 
 defect .
expression analyses indicated that the mutation in the ednrb gene 
 did not affect the expression patterns of cav2.11 in the colon of 
 mice with the prod genetic background .
previous immunohistochemical experiments indicated 
 that jf1 mice showed a lower density of enteric neurons in the colon compared to b6 mice 
 . in the present study ,
the level of 
 cav2.11 expression in the prod strain was lower than that in the b6 
 strain , suggesting lower enteric neuronal density due to the classic piebald mutation .
acetylcholine release at the peripheral neuron 
 muscular junctions in the colon depends on calcium channels , including cav2.1 
 channels . to understand the functions of 
 cav2.1 in the rolling nagoya mouse strain , establishing a 
 strain with an intact ednrb gene will be important
 abstract ataxic 
 rolling nagoya ( prod - rol / rol ) mice , 
 which carry a mutation in the 1 subunit of the cav2.1 channel 
 ( cacna1a ) gene , were discovered in 1969 . 
 they show white spots on 
 agouti coat and have a mutation in the piebald spotting ( s ) locus . 
 
 however , mutation analysis of the s locus encoding the endothelin 
 receptor type b ( ednrb ) gene in 
 prod - rol / rol mice had not been performed . here , we 
 examined the genomic and mrna sequences of the ednrb gene in 
 prod - rol / rol and wild - type rolling 
 nagoya ( prod - s / s ) and studied the expression patterns of 
 ednrb and cacna1a genes in these mice in comparison 
 with c57bl/6j mice . 
 polymerase chain reaction analyses revealed two silent nucleotide 
 substitutions in the coding region and insertion of a retroposon - like element in intron 1 
 of the ednrb gene . 
 expression analyses demonstrated similar localizations 
 and levels of ednrb and cacna1a expression in the colon 
 between prod - rol / rol and 
 prod - s / s mice , but the expression levels of both genes 
 were diminished compared with c57bl/6j mice . 
 microsatellite genotyping showed that at 
 least particular regions of chromosome 14 proximal to the ednrb locus of 
 the prod strain were derived from japanese fancy piebald mice . 
 these results indicated 
 that prod - rol / rol mice have two mutant genes , 
 ednrb and cacna1a . 
 as no prod strain had an intact 
 ednrb gene , using congenic rolling mice would better serve to examine 
 rolling nagoya - type cav2.1 channel dysfunctions . 
MATERIALS AND METHODS
RESULTS
DISCUSSION