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©The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.
Depletion of the IKBKAP ortholog in zebrafish leads to hirschsprung disease-like phenotype
William Wai-Chun Cheng, Clara Sze-Man Tang, Hong-Sheng Gui, Man-Ting So, Vincent Chi-Hang Lui, Paul Kwong-Hang Tam, Maria-Mercè Garcia-Barcelo
William Wai-Chun Cheng, Man-Ting So, Vincent Chi-Hang Lui, Paul Kwong-Hang Tam, Maria-Mercè Garcia-Barcelo, Department of Surgery, Li Ka Shing Faculty of Medicine, the University of Hong Kong, Hong Kong, China
Clara Sze-Man Tang, Department of Psychiatry, Li Ka Shing Faculty of Medicine, the University of Hong Kong, Hong Kong, China
Hong-Sheng Gui, Center for Genomic Sciences of the Li Ka Shing Faculty of Medicine, the University of Hong Kong, Hong Kong, China
Vincent Chi-Hang Lui, Paul Kwong-Hang Tam, Maria-Mercè Garcia-Barcelo, Centre for Reproduction, Development and Growth, Li Ka Shing Faculty of Medicine, the University of Hong Kong, Hong Kong, China
Author contributions: Cheng WWC, So MT, Gui HS and Tang CSM performed the experiments; Cheng WWC, Tang CSM, Lui VCH, Tam PKH and Garcia-Barcelo MM conceived and designed the research and analysed the data; Cheng WWC wrote the manuscript; Lui VCH and Garcia-Barcelo MM revised the manuscript.
Supported by Small Project Funding, the University of Hong Kong, No. 201209176125 to Cheng WWC; Hong Kong Research Grants Council HKU No. 778610M to Tam PKH; Health and Medical Research Fund No. 01121326 to Lui VCH and The University of Hong Kong Genomics Strategic Research Theme.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Maria-Mercè Garcia-Barcelo, Associate Professor, Department of Surgery, Li Ka Shing Faculty of Medicine, the University of Hong Kong, 9/F Faculty of Medicine Building, 21 Sassoon Road, Pokfulam, Hong Kong, China. mmgarcia@hku.hk
Telephone: +852-2-8315073 Fax: +852-2-8199623
Received: April 30, 2014
Peer-review started: May 2, 2014
First decision: May 29, 2014
Revised: August 1, 2014
Accepted: September 12, 2014
Article in press: September 16, 2014
Published online: February 21, 2015
AIM: To investigate the role of IKBKAP (inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase complex-associated protein) in the development of enteric nervous system (ENS) and Hirschsprung disease (HSCR).
METHODS: In this study, we injected a morpholino that blocked the translation of ikbkap protein to 1-cell stage zebrafish embryos. The phenotype in the ENS was analysed by antibody staining of the pan-neuronal marker HuC/D followed by enteric neuron counting. The mean numbers of enteric neurons were compared between the morphant and the control. We also studied the expressions of ret and phox2bb, which are involved in ENS development, in the ikbkap morpholino injected embryos by quantitative reverse transcriptase polymerase chain reaction and compared them with the control.
RESULTS: We observed aganglionosis (χ2, P < 0.01) and a reduced number of enteric neurons (38.8 ± 9.9 vs 50.2 ± 17.3, P < 0.05) in the zebrafish embryos injected with ikbkap translation-blocking morpholino (morphant) when compared with the control embryos. Specificity of the morpholino was confirmed by similar results obtained using a second non-overlapping morpholino that blocked the translation of ikbkap. We further studied the morphant by analysing the expression levels of genes involved in ENS development such as ret, phox2bb and sox10, and found that phox2bb, the ortholog of human PHOX2B, was significantly down-regulated (0.51 ± 0.15 vs 1.00 ± 0, P < 0.05). Although we also observed a reduction in the expression of ret, the difference was not significant.
CONCLUSION: Loss of IKBKAP contributed to HSCR as demonstrated by functional analysis in zebrafish embryos.
Core tip: To investigate the functional role of IKBKAP in enteric nervous system (ENS) development, we knocked down the zebrafish ortholog ikbkap using a translation blocking antisense morpholino. Loss of ikbkap caused aganglionosis and a reduced number of enteric neurons, indicating that IKBKAP is important for proper ENS development.
