Spatiotemporal pattern analysis of transcription factor 4 in the developing anorectum of the rat embryo with anorectal malformations.
OBJECTIVE
As a member of the transcription factors family, transcription factor 4(Tcf4) is known to influence gene expression in endodermally derived tissues including lung, liver, pancreas, stomach, and intestine. However, it remained unknown if this capability is active during anorectal development in the normal and anorectal malformations (ARM) rat embryos.


METHODS
In this study, ethylenethiourea (ETU)-induced ARM model was introduced to investigate the expression pattern of Tcf4 during anorectal development using immunohistochemical staining, reverse transcriptase polymerase chain reaction (RT-PCR), and Western blot analysis.


RESULTS
Immunostaining revealed that Tcf4 expression showed space-dependent changes in the developing anorectum: in normal embryos, Tcf4 protein is initially expressed in the dorsal endoderm of urorectal septum (URS) and hindgut on embryonic day 13 (E13). Additionally, separate expression domain develops intensively on the dorsal CM on E14. On E15, positive cells are then detected in the fused tissue of URS, and prominently in the anal membrane. In the ARM embryos, however, the epithelium of the cloaca, URS, and anorectum was negative or faint for Tcf4. In Western blot and RT-PCR, time-dependent changes of Tcf4 protein and mRNA expression were remarkable during the anorectal development: on E14, E14.5, and E15, the expression level reached the peak; after E16, Tcf4 expression gradually decreased. In contrast, in ARM embryos, spatiotemporal expression of Tcf4 was imbalanced during the anorectal morphogenesis from E13 to E16.


CONCLUSIONS
These data implied that the downregulation of Tcf4 at the time of cloacal separation into rectum and urethra might be related to the development of ARM.
Zhang
Tao
T
Department of Pediatric Surgery, Shengjing Hospital China Medical University, Heping District, Shenyang 110004, People's Republic of China.
Bai
Yu Zuo
YZ
Wang
Da Jia
da J
Jia
Hui Min
HM
Yuan
Zheng Wei
ZW
Wang
Wei Lin
WL
D001003:Anal Canal / Q000002:abnormalities* / Q000196:embryology; D000818:Animals; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D019521:Body Patterning; D004268:DNA-Binding Proteins / Q000032:analysis*; D004065:Digestive System Abnormalities / Q000196:embryology*; D004622:Embryo, Mammalian; D005031:Ethylenethiourea; D005786:Gene Expression Regulation / Q000502:physiology*; D051381:Rats; D012007:Rectum / Q000002:abnormalities* / Q000196:embryology; D013997:Time Factors; D014018:Tissue Distribution; D000073940:Transcription Factor 4; D014157:Transcription Factors / Q000032:analysis*
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C532096:TCF4 protein, human; D000073940:Transcription Factor 4; D014157:Transcription Factors; D005031:Ethylenethiourea