The N-Myc downstream-regulated gene 4 is required for enteric nervous system development and function

Abstract

Background: We recently described that the N- Myc Downstream- Regulated Gene 4 (NDRG4), an established early- detection DNA- methylation marker for colorectal cancer (CRC), is specifically expressed by enteric neurons. So far, our studies focused on how enteric neuronal NDRG4 is involved in the development and pro-gression of CRC. However, the role of NDRG4 in the development and function of the enteric nervous system (ENS) during gastrointes-tinal homeostasis also remains to be elucidated.Methods: To explore the role of ndrg4 in ENS morphogenesis and intestinal motility, we used a (i) morpholino (MO) and (ii) CRISPR- Cas9 knockdown approach in transgenic zebrafish (Danio rerio) ex-pressing the fluorescent- kaede- protein in enteric precursor cells and enteric neurons (Tg(- 8.3phox2b:kaede) zebrafish). We quantified the number of enteric neurons in the distal intestine and created spati-otemporal maps from video recordings to examine intestinal motility in wild- type and ndrg4- knockdown (ndrg4−/−) zebrafish larvae (5 and 7 days post- fertilization (dpf), respectively).Results: In agreement with mouse and human expression studies, we found that ndrg4 is also expressed within the GI- tract of zebrafish larvae. In 5dpf zebrafish, loss of ndrg4 is associated with a significant reduction in the number of enteric neurons: (i) average number 102.4 control- injected vs. 67.4 ndrg4- MO injected (P < 0.001) and (ii) aver-age number 110.9 ndrg4+/+ vs. 97.8 ndrg4−/− zebrafish (P = 0.028). Although the frequency, velocity and contraction interval of peri-staltic waves were not affected in 7dpf ndrg4−/− zebrafish, their travel distance was significantly shorter in ndrg4−/− as compared to ndrg4+/+ zebrafish (P = 0.040).Conclusion: This is the first study describing ndrg4 expression in the zebrafish intestinal tract. Our data suggest that loss of ndrg4 has consequences for ENS development and function.