Morphological and molecular evidence for functional organization along the rostrocaudal axis of the adult zebrafish intestine

Abstract

Background: The zebrafish intestine is a simple tapered tube that is folded into three sections. However, whether the intestine is functionally similar along its length remains unknown. Thus, a systematic structural and functional characterization of the zebrafish intestine is desirable for future studies of the digestive tract and the intestinal biology and development.

Results: To characterize the structure and function of the adult zebrafish intestine, we divided the intestine into seven roughly equal-length segments, S1-S7, and systematically examined the morphology of the mucosal lining, histology of the epithelium, and molecular signatures from transcriptome analysis. Prominent morphological features are circumferentially-oriented villar ridges in segments S1-S6 and the absence of crypts. Molecular characterization of the transcriptome from each segment shows that segments S1-S5 are very similar while S6 and S7 unique. Gene ontology analyses reveal that S1-S5 express genes whose functions involve metabolism of carbohydrates, transport of lipids and energy generation, while the last two segments display relatively limited function. Based on comparative Gene Set Enrichment Analysis, the first five segments share strong similarity with human and mouse small intestine while S6 shows similarity with human cecum and rectum, and S7 with human rectum. The intestinal tract does not display the anatomical, morphological, and molecular signatures of a stomach and thus we conclude that this organ is absent from the zebrafish digestive system.

Conclusions: Our genome-wide gene expression data indicate that, despite the lack of crypts, the rostral, mid, and caudal portions of the zebrafish intestine have distinct functions analogous to the mammalian small and large intestine, respectively. Organization of ridge structures represents a unique feature of zebrafish intestine, though they produce similar cross sections to mammalian intestines. Evolutionary lack of stomach, crypts, Paneth cells and submucosal glands has shaped the zebrafish intestine into a simpler but unique organ in vertebrate intestinal biology.

Figure 1

Anatomical features of adult zebrafish intestine. (A) A partially dissected 6-month-old zebrafish to show the folding of the three portion of intestine in vivo: rostral intestinal bulb (RIB), mid-intestine (MI) and caudal intestine (CI). Liver, ovary, anus, swimbladder anterior (SBa) and posterior (SBp) chambers are indicated. (B) An isolated zebrafish intestine in vitro after removal of the surrounding mesentery. The isolated intestine was divided into seven roughly equal-length segments as indicated by green lines: S1-S2 from RIB, S3-S4 from MI and S5-S7 from CI. The associated gall bladder is indicated. (C - I) Surface views of segments S1-S7 showing the folding of the mucosal surface into circumferential ridges. Scale bars, 500 μm.

Figure 2

Histological features of adult zebrafish intestine along the seven anterior-posterior segments. (A-G) Representative cross sections of intestine from segments S1-S7 respectively. All sections were stained by Hematoxylin/Eosin/alcian blue. Segments S1-S6 contain three tissue layers: mucosa, muscularis externa and serosa, while S7 has a simple epithelium directly adjacent to the muscularis externa. Goblet cells (stained blue) are interspersed among the absorptive cells. Examples of enterocytes (e) and goblet cells (g) are indicated in Panels (A) and (G). Scale bars: 50 μm.

Figure 3

Anayses of genes differentially expressed along the anterior-posterior intestine. (A) Hierarchical clustering of segments S1-S7 by differentially expressed genes selected by ANOVA analysis. Segments S1 to S5 are clustered as one group; segments S6 and S7 are clustered as another group. (B) Overlap analysis of up-regulated genes in adjacent segments. The number and percentage of overlapping genes are indicated within and below the intersection respectively.

Figure 4

Expression patterns of selected intestinal genes. (A) Expression patterns of selected genes based on microarray data. The genes were selected based on their known function in the digestive tract and/or from their expression profiles. (B-I) qRT-PCR validated expression pattern of selected genes. The histograms show the relative changes of the gene expression levels compared with their respective levels of the housekeeping gene bactin2. Gene names are indicated in each panel.

Figure 5

Phylogenetical analysis of zebrafish genes encoding aspartic proteases. The amino acid sequences of zebrafish digestive proteases were compared with those from other species, including mammals, amphibians and fishes. The parasite aspartic protease (Haemonchus contortus), CAA96571, is used as the outgroup. * indicates a candidate hypothetical protein product.