*3.4. Morphological Studies of the Intestine of Young and Old Animals*

*N. furzeri*, as a Cyprinodontiformes species, belongs to the group of agastric fish in which the intestine transits directly from the esophagus [48]. In adults, the intestine is folded into three sections: the rostral intestinal bulb, mid-intestine, and caudal intestine. We focused our analysis on the rostral intestinal bulb, which is known to likely serve a similar function to the mammalian stomach [49]. Sagittal sections of the rostral intestinal bulb reveal a simple architecture of a mucosa, submucosa, muscularis externa, and serosa layer (Figure 7A,B). The intestinal mucosa consists of columnar shaped enterocytes (Figure 7B). We observed that the intestine surface is covered by ridges that are oriented circumferentially across the intestine axis (Figure 7A,B). These ridges in the cross-section resemble the spatially separate villi in the mouse or human small intestine.

**Figure 7.** Transversal section showing localization of NUCB2B mRNA and Nesf-1 protein in the rostral intestinal bulb of *N. furzeri*. (**A**) Hematoxylin/eosin staining reveals a simple architecture of a mucosa, submucosa, muscularis externa, and serosa layer; (**B**) Higher magnification of A; (**C**) in young fish NUCB2B mRNA was detected in the lining epithelium; (**D**) Higher magnification of C; (**E**) in old fish, NUCB2B mRNA was detected in the lining epithelium; (**F**) Higher magnification of E; (**G**) in young subjects, Nesf-1-immuonoreactivity (ir) was detected within rounded or flask-shaped cells in submucosa, most of them scattered deep within the folds of the ridges; (**H**) Higher magnification of G; (**I**) in old fish, Nesf-1-ir cells were more abundant in the submucosa and in addition, some of the cells were dispersed along the apical regions of the ridges; (**J**) Higher magnification of I. Scale bars: A-C-E 100 μm, B-D-F-H-I-J 300 μm, G 200 μm.
