Published online Feb 21, 2015. doi: 10.3748/wjg.v21.i7.2040
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
Processing time: 286 Days and 18.3 Hours
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.