a sox10-cre bacterial artificial chromosome ( bac ) construct was generated from the regulatory elements of sox10 within a well - characterized sox10 bac to obtain high levels of cre in nc derivatives .
the construct includes a nuclear localized cre sequence connected to a human growth hormone mrna stabilization sequence.14 , 15 loxp sites in the bac flanking arms were removed as described by boyle et al ( 2008 ) prior to microinjection of the bac into fertilized mouse eggs by standard procedures .
the resulting tg line , hereafter sox10-cre , was made congenic on the c3heb / fej background .
transgene - driven reporter expression mirrors known sox10 expression in nc - derived lineages ( rosebrock j , buehler dp , dekeyser jl , et  al .
experimental animals for nc - derived lineage quantification were from crosses between sox10 ; sox10-cre or sox10 ; sox10-cre / cre mice to r26r mice .
sox10 mice were identified by the presence of white feet and belly spotting as well as discernible hypoganglionosis and/or aganglionosis revealed by the absence of tdtomato fluorescent ganglia in the distal intestine .
the sox10 recapitulates hscr in humans with animals exhibiting varying lengths of aganglionosis within the bowel despite harboring the same hscr - causing mutation .
the presence of the sox10-cre transgene was verified by polymerase chain reaction ( pcr ) genotyping with primers specific for fragment amplification of the sp6 arm ( forward : reverse : ggcactttcatgttatctgagg ) , t7 arm ( forward : aagagcaagccttggaactg ; reverse : tcgagcttgacattgtaggac ) , and cre - recombinase ( forward : gcggcatggtgcaagttgaat ; reverse : cgttcaccggcatcaacgttt ) .
thermocycler conditions for all primers sets listed were as follows : 94c for 5 minutes [
0.5-second ramp up to 94c ) 35 times ] , 72c for 10 minutes , 4c indefinitely .
regions of the duodenum , ileum , and midcolon were collected from postnatal ( p ) 1519 day sox10 and sox10 littermates .
laminar muscle preparations containing myenteric plexus were isolated and subjected to immunohistochemical ( ihc ) analysis using the reagents described in table  1 , table  2 .
after incubation in primary antibodies , all tissues were rinsed in 1x phosphate - buffered saline ( pbs)/0.1%triton x-100 solution followed by incubation in secondary antibody dilution in block for 1 to 1.5 hours at room temperature .
rinses and incubation in a second secondary antibody dilution was repeated as previously described for double labeling .
after secondary antibody incubation , tissues were rinsed in 1x pbs/0.1%triton x-100 followed by rinses in 1x pbs .
tissue samples were stored in the dark in 1x pbs at 4c before being mounted onto slides with aqua - poly / mount mounting medium ( polysciences , warrington , pa ) .
z - stack images of samples were captured on a zeiss lsm 510 confocal microscope ( 20 objective magnification with 1.5 software zoom ; carl zeiss , thornwood , ny ) to quantify nc derivatives within the ganglia and primary connectives of the myenteric plexus .
image brightness and contrast were adjusted in adobe photoshop ( adobe systems , san jose , ca ) to aid in cell quantification .
we quantified n  = 56 samples per genotype for all duodenum and ileum nc lineage analyses and n  =  56 sox10 and n  = 1011 for sox10 samples for colon nc lineage analyses . to determine gastric emptying and small intestine transit rates , 4-week - old ( 2730 days ) or 6-week - old ( 4248 days ) mice
fifteen minutes after gavage , the stomach and 10 equal segments of small intestine were collected and homogenized separately . the fluorescence signal from the stomach and each intestinal segment was read on a molecular devices / ljl analyst ht ( molecular devices , union city , ca ) .
gastric emptying percent was determined by calculating the proportion of fluorescence that had left the stomach to total recovered fluorescence .
a small intestine transit score was assigned by determining the geometric mean of fluorescence within the 10 equal small intestine segments .
miller et  al originally described and validated this method in rats , and recent studies have used these assays in mice.19 , 20 , 21 to depict small intestine transit rates , the average percentage contribution of each individual intestine segment to the motility score was calculated .
a heat map was generated using matlab ( mathworks , natick , ma ) , where the intestine segment numbers contributing the most to the small intestine transit score ( higher fluorescent intensity ) are shown in darker red and those contributing little or none to the score ( lower fluorescent intensity ) in lighter red .
color intensity in between segment number hashes was interpolated to create each heat map ( n  = 68 mice per genotype , sex , and age ) .
transverse sections ( 5 m ) of entire colon from 6 week or older sox10 and sox10 littermates were stained with h&e .
an expert pathologist blinded to genotype scored the sample inflammation based on a previously developed scoring system using hirschsprung mouse models .
specifically , a final score of 07 was assigned by assessing the severity of inflammation ( 03 , with 0  = no inflammation or rare neutrophils ; 1  =  mild inflammatory infiltrates with no necrosis , 2  = moderate to marked inflammatory infiltrates and mucosal necrosis , and 3  = transmural necrosis ) and depth of inflammation ( 04 , with 0  = none , 1  = mucosa , 2  =  submucosa , 3  = muscularis propia ; 4  = subserosa / serosa ) . in this analysis ,
n  = 69 mice per genotype and per sex . for nc lineage quantification , five duodenal , three ileal , and two colonic images were manually counted for each animal . for sox10 mice with hypoganglionosis in the midcolon ,
additional images were quantified so that a comparable number of neurons were counted between sox10 and sox10 mice .
we tested for differences in cell type proportions , gastric emptying rates , small intestine transit rates , and inflammation scores using a student t test assuming unequal variance ( welch t test ) .
analysis of variance ( f test ) was used to test for statistical significance of slope and to determine the coefficients of determination ( r ) .
statistical analysis was performed using jmp software ( version 10 ; sas institute , cary , nc ) .
all study authors had access to study data , and all authors reviewed and approved the manuscript .
a sox10-cre bacterial artificial chromosome ( bac ) construct was generated from the regulatory elements of sox10 within a well - characterized sox10 bac to obtain high levels of cre in nc derivatives .
the construct includes a nuclear localized cre sequence connected to a human growth hormone mrna stabilization sequence.14 , 15 loxp sites in the bac flanking arms were removed as described by boyle et al ( 2008 ) prior to microinjection of the bac into fertilized mouse eggs by standard procedures .
the resulting tg line , hereafter sox10-cre , was made congenic on the c3heb / fej background .
transgene - driven reporter expression mirrors known sox10 expression in nc - derived lineages ( rosebrock j , buehler dp , dekeyser jl , et  al .
experimental animals for nc - derived lineage quantification were from crosses between sox10 ; sox10-cre or sox10 ; sox10-cre / cre mice to r26r mice .
sox10 mice were identified by the presence of white feet and belly spotting as well as discernible hypoganglionosis and/or aganglionosis revealed by the absence of tdtomato fluorescent ganglia in the distal intestine .
the sox10 recapitulates hscr in humans with animals exhibiting varying lengths of aganglionosis within the bowel despite harboring the same hscr - causing mutation .
the presence of the sox10-cre transgene was verified by polymerase chain reaction ( pcr ) genotyping with primers specific for fragment amplification of the sp6 arm ( forward : reverse : ggcactttcatgttatctgagg ) , t7 arm ( forward : aagagcaagccttggaactg ; reverse : tcgagcttgacattgtaggac ) , and cre - recombinase ( forward : gcggcatggtgcaagttgaat ; reverse : cgttcaccggcatcaacgttt ) .
thermocycler conditions for all primers sets listed were as follows : 94c for 5 minutes [
0.5-second ramp up to 94c ) 35 times ] , 72c for 10 minutes , 4c indefinitely .
regions of the duodenum , ileum , and midcolon were collected from postnatal ( p ) 1519 day sox10 and sox10 littermates .
laminar muscle preparations containing myenteric plexus were isolated and subjected to immunohistochemical ( ihc ) analysis using the reagents described in table  1 , table  2 .
after incubation in primary antibodies , all tissues were rinsed in 1x phosphate - buffered saline ( pbs)/0.1%triton x-100 solution followed by incubation in secondary antibody dilution in block for 1 to 1.5 hours at room temperature .
rinses and incubation in a second secondary antibody dilution was repeated as previously described for double labeling . after secondary antibody incubation
tissue samples were stored in the dark in 1x pbs at 4c before being mounted onto slides with aqua - poly / mount mounting medium ( polysciences , warrington , pa ) .
z - stack images of samples were captured on a zeiss lsm 510 confocal microscope ( 20 objective magnification with 1.5 software zoom ; carl zeiss , thornwood , ny ) to quantify nc derivatives within the ganglia and primary connectives of the myenteric plexus .
image brightness and contrast were adjusted in adobe photoshop ( adobe systems , san jose , ca ) to aid in cell quantification .
we quantified n  = 56 samples per genotype for all duodenum and ileum nc lineage analyses and n  =  56 sox10 and n  = 1011 for sox10 samples for colon nc lineage analyses .
to determine gastric emptying and small intestine transit rates , 4-week - old ( 2730 days ) or 6-week - old ( 4248 days ) mice were gavaged with a rhodamine dextran fluorescence containing meal .
fifteen minutes after gavage , the stomach and 10 equal segments of small intestine were collected and homogenized separately . the fluorescence signal from the stomach and each intestinal segment
was read on a molecular devices / ljl analyst ht ( molecular devices , union city , ca ) .
gastric emptying percent was determined by calculating the proportion of fluorescence that had left the stomach to total recovered fluorescence .
a small intestine transit score was assigned by determining the geometric mean of fluorescence within the 10 equal small intestine segments .
miller et  al originally described and validated this method in rats , and recent studies have used these assays in mice.19 , 20 , 21 to depict small intestine transit rates , the average percentage contribution of each individual intestine segment to the motility score was calculated .
a heat map was generated using matlab ( mathworks , natick , ma ) , where the intestine segment numbers contributing the most to the small intestine transit score ( higher fluorescent intensity ) are shown in darker red and those contributing little or none to the score ( lower fluorescent intensity ) in lighter red .
color intensity in between segment number hashes was interpolated to create each heat map ( n  = 68 mice per genotype , sex , and age ) .
transverse sections ( 5 m ) of entire colon from 6 week or older sox10 and sox10 littermates were stained with h&e .
an expert pathologist blinded to genotype scored the sample inflammation based on a previously developed scoring system using hirschsprung mouse models .
specifically , a final score of 07 was assigned by assessing the severity of inflammation ( 03 , with 0  = no inflammation or rare neutrophils ; 1  =  mild inflammatory infiltrates with no necrosis , 2  = moderate to marked inflammatory infiltrates and mucosal necrosis , and 3  = transmural necrosis ) and depth of inflammation ( 04 , with 0  = none , 1  = mucosa , 2  =  submucosa , 3  = muscularis propia ; 4  = subserosa / serosa ) . in this analysis ,
for nc lineage quantification , five duodenal , three ileal , and two colonic images were manually counted for each animal . for sox10 mice with hypoganglionosis in the midcolon , additional images
were quantified so that a comparable number of neurons were counted between sox10 and sox10 mice .
we tested for differences in cell type proportions , gastric emptying rates , small intestine transit rates , and inflammation scores using a student t test assuming unequal variance ( welch t test ) .
analysis of variance ( f test ) was used to test for statistical significance of slope and to determine the coefficients of determination ( r ) .
statistical analysis was performed using jmp software ( version 10 ; sas institute , cary , nc ) .
all study authors had access to study data , and all authors reviewed and approved the manuscript .
studies to assess alterations in enteric cell types in  vivo have largely focused on comparing total numbers of neurons and glia , although a few studies have assessed neuronal subtypes.20 , 23 while informative , ihc analysis alone may not comprehensively identify all nc derivatives in the ens and may miss alterations in patterning and distribution of enteric ganglia depending on the marker used . because sox10 is ubiquitously expressed in nc cells emerging from the neural tube , the use of sox10 regulatory regions linked to a cre driver enables extensive labeling of nc derivatives ( rosebrock j , buehler dp , dekeyser jl , et  al .
, in preparation).24 , 25 to visualize nc derivatives within enteric ganglia , we combined a sox10-cre bac transgene with a cre - dependent r26r reporter in crosses with sox10 mice .
analysis of sox10 mutants on a mixed background in these crosses is advantageous because this model recapitulates the variable penetrance and severity of aganglionosis seen in human hscr patients.4 , 26 in the resulting progeny , the cytoplasmic reporter tdtomato , which is concentrated in the cell soma and labels cellular processes , reveals ganglia structure , density , and connectives of cells in which cre is expressed .
we collected laminar gut muscle preparations in pups between postnatal ( p ) days 15 and 19 , before the majority of these animals succumb to megacolon at weaning .
we observed similar myenteric ganglia density , spacing between ganglia , and overall architecture of ganglia in the duodenum , ileum , and ganglionated regions of the colon in both sox10 and sox10 pups ( figure  1 ) .
although comparable ganglia patterning and density were seen , the possibility remained that total numbers of myenteric ncps might differ between sox10 and sox10 animals and could lead to altered gi function .
we compared the total numbers of enteric nc - derived cells between sox10 and sox10 pups .
this analysis revealed similar total numbers of nc - derived cells between sox10 and sox10 mice in both duodenum ( p  = .86 ) and ileum ( p  = .55 ) ( n  = 5 per genotype ) ( table  3 ) .
we did not compare the total number of nc - derived cells in the midcolon as many of the sox10 are clearly hypoganglionic in this region . in both in  vitro and in peripheral nervous structures outside the ens , timing and levels of sox10 expression affect neuronal and glial fate acquisition from nc progenitors.10 , 11 , 12 , 27 ,
28 however , the role of sox10 in neuronal and glial fate acquisition in the ens in  vivo has not been determined .
feasibly , disruption of sox10 could alter the ratio of these cell types and contribute to gi dysfunction by impacting motility or by contributing to inflammation secondary to perturbing enteric glia . to examine the possibility that ratios of enteric neurons and glia are abnormal in sox10 pups , we quantified the proportions of neurons and glia out of total nc derivatives within the myenteric plexus at p1517 . because different regions of the bowel have distinct functions and it is unknown
how proximal to regions of aganglionosis aberrancies in lineage segregation might occur , we assessed neuron and glia proportions in the duodenum , ileum , and midcolon .
enteric neurons ( hu+ ) and glia ( foxd3 + ) ( figure  2 ) were ihc labeled , counted , and their proportions out of the total nc derivatives calculated ( figure  3a ) .
proportions of neurons and glia were found to be comparable within the duodenum ( neurons p  =  .28 ; glia p  = .36 ) and the ileum ( neuron p  = .78 ; glia p  =  .50 ) between sox10 and sox10 animals ( figure  3b and c ) . although neuronal and glial proportions in the midcolon revealed no statistically significant difference between sox10 and sox10 animals ( neurons p  = .06 ; glia p  = .37 ) , we noted an overall decrease in neuron proportions and an overall increase in glia proportions in sox10 mice ( figure  3b and c ) .
given the variable length of aganglionosis present in hscr sox10 mice , which models that seen in hscr patients,4 , 26 we compared colonic neuronal and glial proportions against the length of colonic aganglionosis for each sox10 mouse .
this analysis detected a strong inverse correlation between neuron proportions and length of colonic aganglionosis ( p < .003 ; r  = 0.70 ) ( figure  3d ) .
conversely , a strong direct relationship is present between glia proportions and extent of colonic aganglionosis ( p  <  .005 ; r  = 0.66 ) ( figure  3d ) .
thus , although neuron and glia proportions in the sox10 small intestine are not altered and do not correlate with aganglionosis ; in the colon , neuronal numbers decrease in concert with an increase in glial numbers as the area of the bowel affected by aganglionosis increases .
correlation scores for cell type proportions and extent of aganglionosis for each region of the bowel examined are summarized in table  4 .
given the presence of multiple lineages , including myofibroblasts , in colonies grown from cultured enteric ncps in  vitro,11 , 12 , 30 , 31 we anticipated the possibility of identifying cells within the ens that express neither neuronal nor glial markers .
use of the sox10-cre transgene system revealed enteric nc derivatives that were not labeled by neuronal ( hu+ ) or glial ( foxd3 + ) immunoreagents .
however , these cells were extremely rare within myenteric ganglia and primary connectives with an average incidence of 1.5 cells in 1000 nc derivatives in the duodenum , 0.6 cells in 1000 in the ileum , and 6 cells in 1000 in the colon .
the presence of this cell type did not statistically significantly differ between sox10 and sox10 in any region of the intestine tested ( duodenum p  = .41 ; ileum p  = .90 ; colon p  = .23 ) ( figure  4a ) .
furthermore , the proportions of this cell type did not correlate with the length of aganglionosis ( table  4 ) .
concurrently , while evaluating neuron and glia proportions , we documented a relatively infrequent population of enteric nc - derived cells that expressed both neuronal ( hu+ ) and glial ( foxd3 + ) markers .
these cells were typically found in small clusters  mainly as couplets , triplets , or quadruplets  but occasionally found singularly ( figure  4b ) .
this cell type was very rare in both sox10 and sox10 mice , and its rate of appearance did not differ between sox10 and sox10 mice in the duodenum ( p  = .71 ) or ileum ( p  = .12 ) .
although they were still rare , sox10 mice had more of these double - positive cells within the colon ( p < .005 ) . on average ,
sox10 colon samples contained 40 hu+foxd3 + cells per 1000 nc derivatives whereas sox10 colons only contained 25 hu+foxd3 + cells per 1000 nc derivatives .
unlike neurons and glia , the proportion of this cell type did not correlate with disease severity in the colon of sox10 mice ( p  = .80 ; r = 0.01 ) .
additionally , no correlations were identified for this cell type in regions of the small intestine ( table  4 ) .
because clinicians have identified chronic constipation and incontinence as problematic long - term outcomes in hirschsprung patients , we examined the relative proportions of two resident neuronal subtypes in sox10 mutants with well - known roles in gi motility .
calretinin is expressed in cholinergic neurons , and its presence identifies excitatory muscle motor neurons within the myenteric plexus as well as some interneurons and intrinsic primary afferent neurons.1 , 32 therefore , alterations in calretinin - expressing ( calretinin+ ) neuron numbers could affect intestinal contraction dynamics and overall motility .
the proportion of calretinin+ neurons out of total neurons ( hu+ ) in the myenteric plexus was quantified through ihc analysis ( figure  5a ) .
the proportion of calretinin+ neurons in sox10 mice duodenum was statistically significantly greater compared with sox10 littermates ( p < .05 ) ( figure  5b ) .
calretinin+ neurons were also present in higher proportions in the ileum of sox10 mice ( p < .03 ) ( figure  5c ) . in sharp contrast to the findings in the small intestine , an overall decrease in the proportion of calretinin+ neurons was evident in the colon .
greater variance in colonic calretinin+ neuronal subtype proportions was present in sox10 mutants compared with sox10 samples , similar to the effect seen for colonic neuronal and glial proportions .
this analysis revealed that calretinin+ neuron proportions decrease dramatically as the length of aganglionosis increases ( p  <  .0001 , r  = 0.84 ) ( figure  5e ) .
this effect was limited to the colon as calretinin+ neuron proportions did not correlate with length of aganglionosis in the duodenum nor ileum ( table  4 ) . to further define alterations in other motor neuron types , we evaluated neuronal nitric oxide synthase ( nnos)-expressing neurons in sox10 mutants ( figure  6a ) .
nitrergic ( nnos+ ) neurons in the myenteric plexus are primarily inhibitory motor neurons responsible for the relaxation of intestinal smooth muscle and also include some interneurons .
interestingly , although calretinin+ neuron proportions were altered in the duodenum in sox10 mice , nnos+ neuron proportions were comparable between sox10 and sox10 animals ( p  = .68 ) ( figure  6b ) . similarly , nnos+ proportions were nearly identical in the ileum between the two genotypes ( p  = .51 ) ( figure  6c ) .
however , there was a trending increase in nnos+ neuron proportions in the colon of sox10 mice compared with sox10 controls ( p  = .099 ) ( figure  6d ) .
while calretinin+ neuron proportions in the colon decreased as length of aganglionosis increased , the opposite was true for nnos+ neurons .
animals with a greater extent of aganglionosis also exhibited a greater proportion of colonic nnos+ neurons ( p  = .0014 , r  = 0.74 ) ( figure  6e ) . like the majority of cell types we evaluated , correlations for nnos+ neurons were only detected in the colon and not the duodenum and ileum ( table  4 ) .
substantial numbers of hscr patients suffer gi dysfunction even after surgical resection of aganglionic regions .
given the skewing of neuron proportions observed in sox10 animals resulting from increased calretinin+ neurons in the duodenum and ileum , we examined gi motility dynamics proximal to the colon in sox10 mice . to evaluate gastric emptying and small intestine transit rates , we gavaged fasted mice with a nonabsorbable fluorescent meal .
gastric emptying was determined by calculating the proportion of fluorescent signal that had left the stomach to the total recovered fluorescence , and small intestine transit rates were determined by calculating the geometric mean of fluorescence within the small intestine.18 , 19 , 20 because distal intestinal obstruction and colonic distension can affect gi transit more proximally and pups with severe aganglionosis often succumb to megacolon around weaning , we chose to limit our analysis to more mature sox10 mice that survived past weaning to 4 weeks or 6 weeks of age .
the sox10 animals that survive to adulthood typically have a short length of aganglionosis , reach their full adult size , and feed and breed normally .
initially , gastric emptying and small intestine transit rates were compared in 4-week - old sox10 and sox10 animals . because hscr shows a sex bias in humans , with three in four hscr patients being male , we evaluated gi motility in both sox10 male and female mice . in 4-week - old animals , sox10 males had a statistically significantly slower small intestinal transit rate compared with sox10 males ( p < .04 ) ( figure  7a ) .
nearly identical results were obtained for females , with 4-week - old sox10 females also showing statistically significantly reduced small intestinal transit rates ( p < .006 ) ( figure  7b ) .
the gastric emptying rates were comparable between sox10 and sox10 mice for both sexes at this age ( males : p  = .96 ; females : p  = .68 ) ( figure  7a and b ) .
these findings are not surprising given the skew of neuronal subtypes we observed in the small intestine of sox10 and the known outcomes among hscr patients .
however , because neurally mediated traits are susceptible to change upon increased exposure to sex hormones,33 , 34 gastric emptying and small intestine transit rates were also evaluated in older , sexually mature ( 6-week+ ) animals .
for older male mice , we observed statistically comparable small intestine transit rates between sox10 males and their wild - type sox10 counterparts ( p  = .57 ) .
but , in contrast to the phenotype of 4-week - old males , we observed a marked increase in gastric emptying rates of 6-week - old sox10 males that was not observed in 6-week - old normal sox10 littermates ( p < .005 ) ( figure  7c ) .
small intestine transit rates were comparable between 6-week - old sox10 and sox10 females ( p  = .31 ) ( figure  7d ) . and , unlike the 6-week - old males , female 6-week - old sox10 and sox10 mice had comparable gastric emptying rates ( p  = .58 ) ( figure  7d ) .
to summarize , at an early age , both female and male sox10 mice have significant small intestine transit deficits ; however , in older sox10 animals , only males exhibit any type of motility defect .
many hirschsprung patients suffer from severe bouts of enterocolitis , and previous studies reported that some animals with an ednrb hscr mutation suffer from enterocolitis that can ultimately lead to bowel perforation , sepsis , and death.22 , 35 additionally , inflammatory processes within the bowel could potentially affect gi motility , making it difficult to determine whether gi motility deficits in sox10 mice result from changes in electrical properties due to ens deficits and/or influences of inflammatory processes on the bowel . to determine whether enterocolitis could be affecting gi motility in adult sox10 mice , we harvested entire colons from sox10 and sox10 6-week+ littermates .
colons were stained with h&e and scored for inflammation by a pathologist blinded to genotype .
we adopted a grading rubric for inflammation based on previous studies in hscr mouse mutants .
briefly , each mouse was assigned an inflammation score ( 07 ) based on the severity ( 03 ) and depth ( 04 ) of inflammation .
interestingly , sox10 mice had no or very little inflammation , comparable to their wild - type littermates ( p  = .99 ) ( figure  8a and b ) .
because males and females may differ in the levels of inflammation , we also compared inflammation scores by sex .
even when separated by sex , we observed similar levels of inflammation in sox10 and sox10 mice ( figure  8c and d ) .
other hallmark features of hirschsprung disease as reported in hscr patients , such as regions of hypoganglionosis proximal to aganglionosis and thickened muscularis propria , were evident ( figure  8a ) .
given the intestinal transit alterations in sox10 and the lack of inflammation in this hscr model , our results suggest the motility deficits in this hscr model are neurally mediated .
studies to assess alterations in enteric cell types in  vivo have largely focused on comparing total numbers of neurons and glia , although a few studies have assessed neuronal subtypes.20 , 23 while informative , ihc analysis alone may not comprehensively identify all nc derivatives in the ens and may miss alterations in patterning and distribution of enteric ganglia depending on the marker used . because sox10 is ubiquitously expressed in nc cells emerging from the neural tube , the use of sox10 regulatory regions linked to a cre driver enables extensive labeling of nc derivatives ( rosebrock j , buehler dp , dekeyser jl , et  al .
, in preparation).24 , 25 to visualize nc derivatives within enteric ganglia , we combined a sox10-cre bac transgene with a cre - dependent r26r reporter in crosses with sox10 mice .
analysis of sox10 mutants on a mixed background in these crosses is advantageous because this model recapitulates the variable penetrance and severity of aganglionosis seen in human hscr patients.4 , 26 in the resulting progeny , the cytoplasmic reporter tdtomato , which is concentrated in the cell soma and labels cellular processes , reveals ganglia structure , density , and connectives of cells in which cre is expressed .
we collected laminar gut muscle preparations in pups between postnatal ( p ) days 15 and 19 , before the majority of these animals succumb to megacolon at weaning .
we observed similar myenteric ganglia density , spacing between ganglia , and overall architecture of ganglia in the duodenum , ileum , and ganglionated regions of the colon in both sox10 and sox10 pups ( figure  1 ) .
although comparable ganglia patterning and density were seen , the possibility remained that total numbers of myenteric ncps might differ between sox10 and sox10 animals and could lead to altered gi function .
we compared the total numbers of enteric nc - derived cells between sox10 and sox10 pups .
this analysis revealed similar total numbers of nc - derived cells between sox10 and sox10 mice in both duodenum ( p  = .86 ) and ileum ( p  = .55 ) ( n  = 5 per genotype ) ( table  3 ) .
we did not compare the total number of nc - derived cells in the midcolon as many of the sox10 are clearly hypoganglionic in this region .
in both in  vitro and in peripheral nervous structures outside the ens , timing and levels of sox10 expression affect neuronal and glial fate acquisition from nc progenitors.10 , 11 , 12 , 27 , 28 however , the role of sox10 in neuronal and glial fate acquisition in the ens in  vivo has not been determined .
feasibly , disruption of sox10 could alter the ratio of these cell types and contribute to gi dysfunction by impacting motility or by contributing to inflammation secondary to perturbing enteric glia . to examine the possibility that ratios of enteric neurons and glia are abnormal in sox10 pups , we quantified the proportions of neurons and glia out of total nc derivatives within the myenteric plexus at p1517 . because different regions of the bowel have distinct functions and it is unknown
how proximal to regions of aganglionosis aberrancies in lineage segregation might occur , we assessed neuron and glia proportions in the duodenum , ileum , and midcolon .
enteric neurons ( hu+ ) and glia ( foxd3 + ) ( figure  2 ) were ihc labeled , counted , and their proportions out of the total nc derivatives calculated ( figure  3a ) .
proportions of neurons and glia were found to be comparable within the duodenum ( neurons p  =  .28 ; glia p  = .36 ) and the ileum ( neuron p  = .78 ; glia p  =  .50 ) between sox10 and sox10 animals ( figure  3b and c ) . although neuronal and glial proportions in the midcolon revealed no statistically significant difference between sox10 and sox10 animals ( neurons p  = .06 ; glia p  = .37 ) , we noted an overall decrease in neuron proportions and an overall increase in glia proportions in sox10 mice ( figure  3b and c ) .
given the variable length of aganglionosis present in hscr sox10 mice , which models that seen in hscr patients,4 , 26 we compared colonic neuronal and glial proportions against the length of colonic aganglionosis for each sox10 mouse .
this analysis detected a strong inverse correlation between neuron proportions and length of colonic aganglionosis ( p < .003 ; r  = 0.70 ) ( figure  3d ) .
conversely , a strong direct relationship is present between glia proportions and extent of colonic aganglionosis ( p  <  .005 ; r  = 0.66 ) ( figure  3d ) . thus , although neuron and glia proportions in the sox10 small intestine are not altered and do not correlate with aganglionosis ; in the colon , neuronal numbers decrease in concert with an increase in glial numbers as the area of the bowel affected by aganglionosis increases .
correlation scores for cell type proportions and extent of aganglionosis for each region of the bowel examined are summarized in table  4 .
given the presence of multiple lineages , including myofibroblasts , in colonies grown from cultured enteric ncps in  vitro,11 , 12 , 30 , 31 we anticipated the possibility of identifying cells within the ens that express neither neuronal nor glial markers .
use of the sox10-cre transgene system revealed enteric nc derivatives that were not labeled by neuronal ( hu+ ) or glial ( foxd3 + ) immunoreagents .
however , these cells were extremely rare within myenteric ganglia and primary connectives with an average incidence of 1.5 cells in 1000 nc derivatives in the duodenum , 0.6 cells in 1000 in the ileum , and 6 cells in 1000 in the colon .
the presence of this cell type did not statistically significantly differ between sox10 and sox10 in any region of the intestine tested ( duodenum p  = .41 ; ileum p  = .90 ; colon p  = .23 ) ( figure  4a ) .
furthermore , the proportions of this cell type did not correlate with the length of aganglionosis ( table  4 ) .
concurrently , while evaluating neuron and glia proportions , we documented a relatively infrequent population of enteric nc - derived cells that expressed both neuronal ( hu+ ) and glial ( foxd3 + ) markers .
these cells were typically found in small clusters  mainly as couplets , triplets , or quadruplets  but occasionally found singularly ( figure  4b ) .
this cell type was very rare in both sox10 and sox10 mice , and its rate of appearance did not differ between sox10 and sox10 mice in the duodenum ( p  = .71 ) or ileum ( p  = .12 ) .
although they were still rare , sox10 mice had more of these double - positive cells within the colon ( p < .005 ) . on average ,
sox10 colon samples contained 40 hu+foxd3 + cells per 1000 nc derivatives whereas sox10 colons only contained 25 hu+foxd3 + cells per 1000 nc derivatives .
unlike neurons and glia , the proportion of this cell type did not correlate with disease severity in the colon of sox10 mice ( p  = .80 ; r = 0.01 ) .
additionally , no correlations were identified for this cell type in regions of the small intestine ( table  4 ) .
because clinicians have identified chronic constipation and incontinence as problematic long - term outcomes in hirschsprung patients , we examined the relative proportions of two resident neuronal subtypes in sox10 mutants with well - known roles in gi motility .
calretinin is expressed in cholinergic neurons , and its presence identifies excitatory muscle motor neurons within the myenteric plexus as well as some interneurons and intrinsic primary afferent neurons.1 , 32 therefore , alterations in calretinin - expressing ( calretinin+ ) neuron numbers could affect intestinal contraction dynamics and overall motility .
the proportion of calretinin+ neurons out of total neurons ( hu+ ) in the myenteric plexus was quantified through ihc analysis ( figure  5a ) .
the proportion of calretinin+ neurons in sox10 mice duodenum was statistically significantly greater compared with sox10 littermates ( p < .05 ) ( figure  5b ) .
calretinin+ neurons were also present in higher proportions in the ileum of sox10 mice ( p < .03 ) ( figure  5c ) . in sharp contrast to the findings in the small intestine , an overall decrease in the proportion of calretinin+ neurons was evident in the colon .
greater variance in colonic calretinin+ neuronal subtype proportions was present in sox10 mutants compared with sox10 samples , similar to the effect seen for colonic neuronal and glial proportions .
this analysis revealed that calretinin+ neuron proportions decrease dramatically as the length of aganglionosis increases ( p  <  .0001 , r  = 0.84 ) ( figure  5e ) . this effect was limited to the colon as calretinin+ neuron proportions did not correlate with length of aganglionosis in the duodenum nor ileum ( table  4 ) . to further define alterations in other motor neuron types , we evaluated neuronal nitric oxide synthase ( nnos)-expressing neurons in sox10 mutants ( figure  6a ) .
nitrergic ( nnos+ ) neurons in the myenteric plexus are primarily inhibitory motor neurons responsible for the relaxation of intestinal smooth muscle and also include some interneurons .
interestingly , although calretinin+ neuron proportions were altered in the duodenum in sox10 mice , nnos+ neuron proportions were comparable between sox10 and sox10 animals ( p  = .68 ) ( figure  6b ) . similarly ,
nnos+ proportions were nearly identical in the ileum between the two genotypes ( p  = .51 ) ( figure  6c ) .
however , there was a trending increase in nnos+ neuron proportions in the colon of sox10 mice compared with sox10 controls ( p  = .099 ) ( figure  6d ) .
while calretinin+ neuron proportions in the colon decreased as length of aganglionosis increased , the opposite was true for nnos+ neurons .
animals with a greater extent of aganglionosis also exhibited a greater proportion of colonic nnos+ neurons ( p  = .0014 , r  = 0.74 ) ( figure  6e ) . like the majority of cell types we evaluated , correlations for nnos+ neurons were only detected in the colon and not the duodenum and ileum ( table  4 ) .
substantial numbers of hscr patients suffer gi dysfunction even after surgical resection of aganglionic regions .
given the skewing of neuron proportions observed in sox10 animals resulting from increased calretinin+ neurons in the duodenum and ileum , we examined gi motility dynamics proximal to the colon in sox10 mice . to evaluate gastric emptying and small intestine transit rates , we gavaged fasted mice with a nonabsorbable fluorescent meal .
gastric emptying was determined by calculating the proportion of fluorescent signal that had left the stomach to the total recovered fluorescence , and small intestine transit rates were determined by calculating the geometric mean of fluorescence within the small intestine.18 , 19 , 20 because distal intestinal obstruction and colonic distension can affect gi transit more proximally and pups with severe aganglionosis often succumb to megacolon around weaning , we chose to limit our analysis to more mature sox10 mice that survived past weaning to 4 weeks or 6 weeks of age .
the sox10 animals that survive to adulthood typically have a short length of aganglionosis , reach their full adult size , and feed and breed normally .
initially , gastric emptying and small intestine transit rates were compared in 4-week - old sox10 and sox10 animals . because hscr shows a sex bias in humans , with three in four hscr patients being male , we evaluated gi motility in both sox10 male and female mice . in 4-week - old animals ,
sox10 males had a statistically significantly slower small intestinal transit rate compared with sox10 males ( p < .04 ) ( figure  7a ) .
nearly identical results were obtained for females , with 4-week - old sox10 females also showing statistically significantly reduced small intestinal transit rates ( p < .006 ) ( figure  7b ) .
the gastric emptying rates were comparable between sox10 and sox10 mice for both sexes at this age ( males : p  = .96 ; females : p  = .68 ) ( figure  7a and b ) .
these findings are not surprising given the skew of neuronal subtypes we observed in the small intestine of sox10 and the known outcomes among hscr patients .
however , because neurally mediated traits are susceptible to change upon increased exposure to sex hormones,33 , 34 gastric emptying and small intestine transit rates were also evaluated in older , sexually mature ( 6-week+ ) animals . for older male mice , we observed statistically comparable small intestine transit rates between sox10 males and their wild - type sox10 counterparts ( p  = .57 ) . but , in contrast to the phenotype of 4-week - old males , we observed a marked increase in gastric emptying rates of 6-week - old sox10 males that was not observed in 6-week - old normal sox10 littermates ( p < .005 ) ( figure  7c ) .
small intestine transit rates were comparable between 6-week - old sox10 and sox10 females ( p  = .31 ) ( figure  7d ) . and , unlike the 6-week - old males , female 6-week - old sox10 and sox10 mice had comparable gastric emptying rates ( p  = .58 ) ( figure  7d ) . to summarize , at an early age , both female and male sox10 mice have significant small intestine transit deficits
; however , in older sox10 animals , only males exhibit any type of motility defect .
many hirschsprung patients suffer from severe bouts of enterocolitis , and previous studies reported that some animals with an ednrb hscr mutation suffer from enterocolitis that can ultimately lead to bowel perforation , sepsis , and death.22 , 35 additionally , inflammatory processes within the bowel could potentially affect gi motility , making it difficult to determine whether gi motility deficits in sox10 mice result from changes in electrical properties due to ens deficits and/or influences of inflammatory processes on the bowel . to determine whether enterocolitis could be affecting gi motility in adult sox10 mice , we harvested entire colons from sox10 and sox10 6-week+ littermates .
colons were stained with h&e and scored for inflammation by a pathologist blinded to genotype .
we adopted a grading rubric for inflammation based on previous studies in hscr mouse mutants .
briefly , each mouse was assigned an inflammation score ( 07 ) based on the severity ( 03 ) and depth ( 04 ) of inflammation .
interestingly , sox10 mice had no or very little inflammation , comparable to their wild - type littermates ( p  = .99 ) ( figure  8a and b ) . because males and females may differ in the levels of inflammation , we also compared inflammation scores by sex .
even when separated by sex , we observed similar levels of inflammation in sox10 and sox10 mice ( figure  8c and d ) .
other hallmark features of hirschsprung disease as reported in hscr patients , such as regions of hypoganglionosis proximal to aganglionosis and thickened muscularis propria , were evident ( figure  8a ) .
given the intestinal transit alterations in sox10 and the lack of inflammation in this hscr model , our results suggest the motility deficits in this hscr model are neurally mediated .
the sox10 hscr mutant is one of several hscr models frequently studied to better understand the nc deficits that give rise to aganglionosis.2 , 9 , 28 , 36 the sox10 allele disrupts migration of enteric ncps and contributes to extensive colonic aganglionosis.37 , 38 additionally , isolated sox10 enteric npcs do not produce a normal profile of cell lineages when cultured in  vitro . despite the insights gained from studying the fetal effects of sox10 mutations on ncp migration and consequent aganglionosis , no prior efforts have investigated postnatal consequences of sox10 mutations on ganglionated regions . in this study
, we undertook a comprehensive assessment of nc derivatives in the proximal , ganglionated intestine of sox10 mice to test the hypothesis that the sox10 mutation leads to imbalances in enteric nc - derived lineages and deficits in bowel function .
our analysis revealed effects of the sox10 mutation on neuronal - glial lineage segregation in the colon but not the small intestine .
interestingly , this study identified significant imbalances in proportions of excitatory and inhibitory enteric neurons accompanied by deficits in intestinal transit and gastric emptying .
calretinin+ neurons with roles in muscle contraction were significantly increased in the duodenum and ileum while nnos+ neurons with roles in muscle relaxation were unchanged in these regions .
sox10 effects on glial cell specification and ncp multipotency are well established in other aspects of the peripheral nervous system such as the dorsal root ganglion.10 , 27 , 28 , 39 surprisingly , our analysis indicated no effect of the sox10 mutation on neuronal - glial balance in the duodenum and ileum although there was a statistically significant correlation between the proportion of neurons and glia in the sox10 colon and the overall extent of aganglionosis .
it is possible that sox10 neurons and glia are born in equal proportions in the colon , with more neurons subsequently succumbing to apoptosis while glia persist .
however , increased apoptosis has not been observed in several ens mutants to date.12 , 40 , 41 moreover , cell death can not adequately account for the observed increases in colonic nnos+ neuron proportions to levels well above the wild - type average in sox10 mice with severe aganglionosis .
our study suggests that the timing of ncp lineage choice is likely a key factor in determining not only the length of aganglionosis but ultimately the proportions of enteric glia , neurons , and neuronal subtypes .
it has been postulated that premature neuronal differentiation contributes to hscr disease by depleting the enteric ncp pool .
premature ncp differentiation would not only exhaust the ncp pool , but may cause certain cell types to be born more or less often based on when ( temporally ) and where ( local environment ) lineage choice occurs .
this phenomenon could account for the region - specific imbalance of different nc - derived cell types we observed in sox10 mice .
such a possibility is corroborated by recent findings in the developing telencephalon , where oscillating or sustained expression of specific transcription factors controls whether neural progenitor cells go on to differentiate into specific cell types or continue to proliferate and give rise to daughter neural progenitor cells . given the diversity of cell types that can arise from cultured nc progenitors in  vitro , we were not surprised to identify nc - derived cells in the intestine that did not label with neuronal or glial immunoreagents .
these cells are infrequent and might represent a novel cell type or possibly cell turnover within ganglia . because proportions of these nc - derived cells were equivalent between p1519 sox10 and sox10 animals and because they were so rare , we did not attempt to characterize this lineage further . interestingly , in both sox10 and sox10 mice , we observed cells that double - labeled with neuronal ( hu+ ) and glial ( foxd3 + ) markers .
this cell type may represent either a novel lineage or a progenitor(s ) undergoing differentiation .
given their morphology and tendency to be found in clusters , these cells could also represent neural progenitors with adjacent daughter progenitors or daughter cells not fully committed to a specific cell type .
the latter is certainly feasible given that foxd3 is expressed in enteric ncps as well as enteric glia.29 , 44 enteric neural progenitors certainly exist in the adult intestine , and their exact location and nature are being actively investigated.21 , 24 , 31 however , no markers have been found that exclusively label neural stem cells in the intestine , convoluting efforts to characterize this cell type .
if truly an enteric neural stem cell , the increase in this cell type in the sox10 colon might represent a futile attempt by the remaining neural stem cells to populate the hypoganglionic or aganglionic areas of the distal intestine . because hscr is a multigenic disorder , whereby an independent variant in any one of several genes can produce aganglionosis , our findings are potentially of broad relevance to other hscr models .
hscr mouse models with deficiency of edn3 or ednrb were also found by sandgren et  al ( 2002 ) and zaitoun et  al ( 2013 ) , respectively , to exhibit increases in nnos+ expressing neurons in the colons of these models.23 , 45 however , nc - derived cell type proportions and their correlations with length of aganglionosis were not reported .
our analysis benefited from the varying levels of aganglionosis present with the sox10 mutation on a mixed background , and this type of analysis may not have been possible in other hscr mouse models where extent of aganglionosis is confined to a small region of distal colon .
this type of analysis is important , as similar perturbations of nc fate choice may be occurring in hscr patients and thus the length of aganglionosis could inform patient outcomes and care in the future .
furthermore , these other studies did not evaluate any regions of the intestine proximal to the ileum . for the first time , we report alterations in nc - derived proportions ( calretinin+ neurons specifically ) within the proximal small intestine ( duodenum ) of a hscr mouse model .
the mechanisms driving the altered proportions of nc derivates within hscr mutants are unclear at this time .
prenatal obstruction ( atresia ) has been shown to affect nc derivatives in rats , and the extent of aganglionosis in the sox10 hscr model may effectively be producing a varying length of obstruction .
we realize obstruction can not be excluded as a factor that impacts nc derivative choice ; however , sox10 mice are born in mendelian ratios relative to their wild - type littermates , and the majority survive to weaning , which suggests that obstruction does not play a large role prenatally .
we specifically elected to examine p1519 pups for nc - derived lineages to ensure that the ens was fully developed and at the same time to try to avoid influences of obstruction on the ens .
( the majority of sox10 mice that suffer from obstruction succumb to megacolon near or at weaning when they transition to solid food . )
mice with deficits in ens patterning or ncp lineage segregation can exhibit altered gi motility even when no aganglionosis is present.9 , 20 however , hscr models have not previously been evaluated for motility deficits within ganglionated regions of the small intestine .
many studies limit their analysis to male mice , but we chose to examine both males and females given the difference in incidence of hscr and other neurodevelopmental disorders between males and females . in the sox10 model , we documented slower small intestine transit rates in 4-week - old males and females . because sox10 mice show increases in excitatory motor neurons ( calretinin+ ) in the small intestine but no changes in inhibitory motor neurons ( nnos+ ) , we hypothesize that imbalances in motor neuron cell types cause changes in peristalsis coordination and/or neuron signaling .
because other neuronal subtypes also affect motility , we can not at this time directly attribute these changes in gi motility to our findings in calretinin+ and nnos+ neurons .
however , given that calretinin+ and nnos+ neurons comprise the majority of motor neurons in the myenteric plexus , our hypothesis provides a plausible and attractive explanation .
in contrast with our results in young animals , mature sox10 and sox10 mice showed comparable small intestine transit rates .
it could be that the sox10 intestine adapts or compensates for deficits as the ens matures or that we have selected for more mildly affected mice at this age , as many severely affected animals succumb to hscr megacolon near weaning .
however , we also observed increased gastric emptying in older sox10 males that we did not see in females , a result that was unexpected . increased gastric emptying in hscr mouse models has not previously been reported , and the underlying physiologic basis for this effect in males is not clear . for many neurodevelopment diseases ,
females are postulated to be protected or differentially affected due to circulating sex hormones while males are more susceptible.33 , 34 this tendency for males to be more severely affected by neurodevelopmental disorders may explain why males are afflicted more often with hscr than females ( 3:1 ) and could explain why 6-week - old sox10 males have increased gastric emptying rates , but not females . furthermore , increased gastric emptying rates may confound small intestine transit rates because gastric contents are more rapidly entering the small intestine .
therefore , it is possible that our sox10 males still have a decreased small intestine transit rate compared with their wild - type counterparts but this phenotype is being masked by increased gastric emptying .
our analysis is the first report of altered gastric emptying and small intestine transit in a hscr mouse model .
not surprisingly , given the presence of distal bowel aganglionosis , prior studies have reported altered or absent colonic migratory complexes in gdnf , edn3 , and ednrb hscr mutants.23 , 45 , 48 , 49 additionally , while most studies limit their analysis to male mice , our studies identified novel deficits in gastric emptying as well as small intestine transit for the sox10 hscr mouse model that are age and sex dependent . whether alterations in gastric emptying or small intestine transit are present among other hscr mutant alleles remains to be seen .
future analyses are required to determine the exact electrophysiologic changes within the ens in sox10 mice and other hscr mutants and to ascertain whether other cell types that contribute to motor activity ( such as serotonergic neurons or interstitial cells of cajal ) are perturbed .
similarities and disparities in outcomes between hscr mutant models should help elucidate exactly when and where hscr genes act within hscr gene pathways . despite the fact that infection and inflammatory processes are known to affect gi transit speed , we saw no difference in colonic inflammation between sox10 and sox10 adult mice .
this finding further suggests that the differences in gastric emptying and small intestine transit are most likely driven by neural mechanisms .
it remains to be seen whether sox10 mice are more or less susceptible to inflammation when challenged by surgery , infection , or chemical treatment .
some hscr patients and mouse models are susceptible to enterocolitis , but the mechanisms driving this susceptibility are still not well understood.7 , 22 , 35 collectively , this study demonstrates for the first time skewed enteric nc derivative proportions and altered gi motility in the small intestine of a hscr mouse model .
these findings demonstrate a role for sox10 in nc lineage specification in  vivo in the ens .
moreover , our results suggest a novel role for sox10 in neuronal subtype choice and demonstrate that perturbations in sox10 can affect gi transit in ganglionated regions of the intestine . different regions of the sox10 intestine were found to have distinct abnormalities , and gi transit assays revealed sex- and age - dependent effects , suggesting that timing and environment play a key role in not only nc lineage segregation but ultimately functional outcomes .
 background & aimsin hirschsprung disease ( hscr ) , neural crest - derived progenitors ( ncps ) fail to completely colonize the intestine so that the enteric nervous system is absent from distal bowel . despite removal of the aganglionic region , 
 many hscr patients suffer from residual intestinal dysmotility . to test the hypothesis that inappropriate lineage segregation of ncps in proximal ganglionated regions of the bowel could contribute to such postoperative disease , we investigated neural crest ( nc)-derived lineages and motility in ganglionated , postnatal intestine of the sox10dom/+ hscr mouse model.methodscre-mediated fate - mapping was applied to evaluate relative proportions of nc - derived cell types . 
 motility assays were performed to assess gastric emptying and small intestine motility while colonic inflammation was assessed by histopathology for sox10dom/+ mutants relative to wild - type controls.resultssox10dom/+ mice showed regional alterations in neuron and glia proportions as well as calretinin+ and neuronal nitric oxide synthase ( nnos)+ neuronal subtypes . in the colon , imbalance of enteric nc derivatives correlated with the extent of aganglionosis . 
 all sox10dom/+ mice exhibited reduced small intestinal transit at 4 weeks of age ; at 6 weeks of age , sox10dom/+ males had increased gastric emptying rates . 
 sox10dom/+ mice surviving to 6 weeks of age had little or no colonic inflammation when compared with wild - type littermates , suggesting that these changes in gastrointestinal motility are neurally mediated.conclusionsthe sox10dom mutation disrupts the balance of nc - derived lineages and affects gastrointestinal motility in the proximal , ganglionated intestine of adult animals . 
 this is the first report identifying alterations in enteric neuronal classes in sox10dom/+ mutants , which suggests a previously unrecognized role for sox10 in neuronal subtype specification . 
Materials and Methods
Animals
Immunohistochemistry
Gastrointestinal Transit Assays
Inflammation
Statistics
Results
Fate-Mapping Enteric NC Derivatives with a 
Correlation between Disease Severity and Neuron or Glia Proportions in the Colon of the 
Rare, Enteric Neural Crest-Derived Cell Types in 
Regional Alterations among Enteric Neuronal Subtypes in 
Gastric Emptying and Small Intestinal Transit in 
None
Discussion