Abnormal enteric innervation identified without histopathologic staining in aganglionic colorectum from a mouse model of Hirschsprung's disease.
OBJECTIVE
The piebald lethal mouse with a deletion of endothelin-B receptor gene (EDNRB) is a model for Hirschsprung's disease (HD), whereas the SOX10 gene is vital for the development of intestinal neural crest-derived cells. Recently, we created a SOX10 transgenic mouse with intestinal neural crest-derived cells visible with enhanced green fluorescent protein (VENUS), that is, SOX10-VENUS(+)/EDNRB(sl/sl) to investigate intestinal innervation in HD.


METHODS
SOX10-VENUS(+)/EDNRB(sl/sl) (n = 30) were compared with wild-type littermates as controls (EDNRB(s/s), n = 30). Mice were killed on days 3, 7, or 12 of age. The entire colorectum was excised, fixed with 4% paraformaldehyde, and examined using fluorescence microscopy alone without staining.


RESULTS
In normoganglionic colorectum from controls, a grid network of nerve fibers/glial cells was visualized that connected smoothly with extrinsic nerve fibers running along the colorectal wall. In aganglionic colorectum from SOX10-VENUS(+)/EDNRB(sl/sl) mice, there was no grid network and more extrinsic nerve fibers than controls that invaded the colon wall becoming elongated with branching fibers. Normoganglionic colon from controls and SOX10-VENUS(+)/EDNRB(sl/sl) mice appeared the same. Innervation patterns did not change over time.


CONCLUSIONS
This is the first time for abnormal enteric innervation in aganglionic colon in a model for HD to be visualized without staining.
Department of Pediatric General and Urogenital Surgery, Juntendo University School of Medicine, Tokyo 113-8421, Japan. katsuo@juntendo.ac.jp
Katsumi
K
Miyahara
Yoshifumi
Y
Kato
Hiroyuki
H
Koga
Geoffrey J
GJ
Lane
Takayoshi
T
Inoue
Chihiro
C
Akazawa
Atsuyuki
A
Yamataka
D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics / Q000528:radiation effects; D019070:Cell Lineage; D003106:Colon / Q000294:innervation* / Q000473:pathology; D004195:Disease Models, Animal; D017615:Enteric Nervous System / Q000002:abnormalities* / Q000473:pathology; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes / Q000032:analysis / Q000528:radiation effects; D005726:Ganglia, Parasympathetic / Q000648:ultrastructure*; D006627:Hirschsprung Disease / Q000473:pathology*; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics / Q000528:radiation effects; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D008822:Mice, Transgenic; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009412:Nerve Fibers / Q000648:ultrastructure*; D009432:Neural Crest / Q000473:pathology; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D044023:Receptor, Endothelin B / Q000096:biosynthesis / Q000172:deficiency / Q000235:genetics; D012007:Rectum / Q000294:innervation* / Q000473:pathology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055757:SOXE Transcription Factors / Q000235:genetics; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D012333:RNA, Messenger; D044023:Receptor, Endothelin B; D055757:SOXE Transcription Factors; C527924:Sox10 protein, mouse; C054562:yellow fluorescent protein, Bacteria