Functional modifications associated with gastrointestinal tract organogenesis during metamorphosis in Atlantic halibut (Hippoglossus hippoglossus).

BACKGROUND: Flatfish metamorphosis is a hormone regulated post-embryonic developmental event that transforms a symmetric larva into an asymmetric juvenile. In altricial-gastric teleost fish, differentiation of the stomach takes place after the onset of first feeding, and during metamorphosis dramatic molecular and morphological modifications of the gastrointestinal (GI-) tract occur. Here we present the functional ontogeny of the developing GI-tract from an integrative perspective in the pleuronectiforme Atlantic halibut, and test the hypothesis that the multiple functions of the teleost stomach develop synchronously during metamorphosis. RESULTS: Onset of gastric function was determined with several approaches (anatomical, biochemical, molecular and in vivo observations). In vivo pH analysis in the GI-tract lumen combined with quantitative PCR (qPCR) of α and ß subunits of the gastric proton pump (H+/K+-ATPase) and pepsinogen A2 indicated that gastric proteolytic capacity is established during the climax of metamorphosis. Transcript abundance of ghrelin, a putative orexigenic signalling molecule produced in the developing stomach, correlated (p < 0.05) with the emergence of gastric proteolytic activity, suggesting that the stomach's role in appetite regulation occurs simultaneously with the establishment of proteolytic function. A 3D models series of the GI-tract development indicated a functional pyloric sphincter prior to first feeding. Observations of fed larvae in vivo confirmed that stomach reservoir function was established before metamorphosis, and was thus independent of this event. Mechanical breakdown of food and transportation of chyme through the GI-tract was observed in vivo and resulted from phasic and propagating contractions established well before metamorphosis. The number of contractions in the midgut decreased at metamorphic climax synchronously with establishment of the stomach's proteolytic capacity and its increased peristaltic activity. Putative osmoregulatory competence of the GI-tract, inferred by abundance of Na+/K+-ATPase α transcripts, was already established at the onset of exogenous feeding and was unmodified by metamorphosis. CONCLUSIONS: The functional specialization of the GI-tract was not exclusive to metamorphosis, and its osmoregulatory capacity and reservoir function were established before first feeding. Nonetheless, acid production and the proteolytic capacity of the stomach coincided with metamorphic climax, and also marked the onset of the stomach's involvement in appetite regulation via ghrelin.

Evidence for an ontogenetic change from pre-programmed to meal-responsive cck production in Atlantic herring, Clupea harengus L.

The effects of up to three days of food deprivation on the cholecystokinin (CCK)-producing cells in the Atlantic herring gut were assessed by quantifying the number of cells detected by in situ hybridization at three ontogenetic stages. In feeding larvae that still possessed yolk-sacs (2 and 8days after hatch, DAH), intestinal cck expression appeared to be maintained regardless of external nutritional conditions. In 30 DAH-old herring larvae with well-established exogenous feeding only, very few CCK-producing cells could be identified, indicating that cck production in the gut had shut down after three days of starvation. This suggests that cck transcription is pre-programmed by a local timer in the midgut during the yolk-sac stage, regardless of the nutritional status and presence of nutrients in the gut lumen; however, it becomes strongly influenced by the external nutritional conditions after the yolk has been completely absorbed. Our results suggest that CCK-producing cells in the gut develop "meal-responsiveness" later in post-hatch development.

Cholecystokinin mRNA in Atlantic herring, Clupea harengus--molecular cloning, characterization, and distribution in the digestive tract during the early life stages.

The mRNA of the peptide hormone cholecystokinin (CCK) was isolated from juvenile Atlantic herring, Clupea harengus, by RT-PCR. The open reading frame encodes a 137 amino acid-long precursor protein. The peptide sequence of herring CCK-8, DYMGWMDF, is identical to that of higher vertebrates and elasmobranchs, and contains methionine in the sixth position from the C-terminus, which has not been reported previously in teleosts. Expression analysis by in situ hybridization shows that positive endocrine-like cells were mainly located in the pyloric caeca and to a less extent in the rectum of the juvenile. A few positive cells were also found in the pyloric portion of the stomach and the intestine. CCK cells were present in all the larvae examined from the day of hatching onwards. Although the CCK cells were scattered throughout the whole midgut, no signals were detected in either the foregut or the hindgut. Since herring larvae have a straight gut, the distribution pattern of CCK cells seems to be reflected in the anatomy of the gut.

Reconstructed 3D models of digestive organs of developing Atlantic cod (Gadus morhua) larvae.

Six 3D models of the digestive system during ontogeny were reconstructed from histological sections of Atlantic cod larvae. The 3D models clearly visualize the following features: folding of the gut rotation; subdivision of digestive tract into foregut, midgut, and hindgut by sphincters; development of stomach and pyloric caeca from 39 dph; location of entrances of bile and pancreatic ducts in the medial plane of the anterior midgut; ontogeny of pancreas from a compact organ to an elongated and branched (but not diffuse) organ along the posterior midgut; one dominant islet of Langerhans until 39 dph and several smaller satellite islets also visible from 53 dph; the relatively large volume of the anterior midgut that probably increases residence time of ingested food mixing with secretions from pancreas and bile. Calculated volumes of each digestive organ demonstrate allometric changes during ontogeny. Interactive 3D models are available as QuickTime format downloadable files.

Oligopeptide transporter PepT1 in Atlantic cod (Gadus morhua L.): cloning, tissue expression and comparative aspects.

A novel full-length cDNA that encodes for the Atlantic cod (Gadus morhua L.) PepT1-type oligopeptide transporter has been cloned. This cDNA (named codPepT1) was 2,838 bp long, with an open reading frame of 2,190 bp encoding a putative protein of 729 amino acids. Comparison of the predicted Atlantic cod PepT1 protein with zebrafish, bird and mammalian orthologs allowed detection of many structural features that are highly conserved among all the vertebrate proteins analysed, including (1) a larger than expected area of hydrophobic amino acids in close proximity to the N terminus; (2) a single highly conserved cAMP/cGMP-dependent protein kinase phosphorylation motif; (3) a large N-glycosylation-rich region within the large extracellular loop; and (4) a conserved and previously undescribed stretch of 8-12 amino acid residues within the large extracellular loop. Expression analysis at the mRNA level indicated that Atlantic cod PepT1 is mainly expressed at intestinal level, but that it is also present in kidney and spleen. Analysis of its regional distribution along the intestinal tract of the fish revealed that PepT1 is ubiquitously expressed in all segments beyond the stomach, including the pyloric caeca, and through the whole midgut. Only in the last segment, which included the hindgut, was there a lower expression. Atlantic cod PepT1, the second teleost fish PepT1-type transporter documented to date, will contribute to the elucidation of the evolutionary and functional relationships among vertebrate peptide transporters. Moreover, it can represent a useful tool for the study of gut functional regionalization, as well as a marker for the analysis of temporal and spatial expression during ontogeny.

Distribution of cholecystokinin-immunoreactive cells in the digestive tract of the larval teleost, ayu, Plecoglossus altivelis.

The ontogenetic development of cholecystokinin-immunoreactive (CCK-IR) cells was studied in larval ayu, Plecoglossus altivelis. This species has a straight digestive tract during the larval phase. CCK-IR cells were present in all the larvae from the day of hatching (0 days after hatching, DAH). An immunoreaction to anti-trypsinogen antibody was also detected in the pancreas at this stage. The distribution pattern of the CCK-IR cells was quantified by recording the location of CCK-IR cells at 1, 16, and 76 DAH. Although the number of CCK-IR cells increased during development, the distribution pattern of CCK-IR cells did not change until 76 DAH. The CCK-IR cells were scattered throughout the midgut, with the exception of the regions adjacent to the pyloric and rectal sphincters. No CCK-IR cells were detected in the foregut or the hindgut. This distribution pattern differs from species with rotated digestive tracts, whose CCK-IR cells are only found in the anterior part of the midgut. CCK-IR cells seem to be located in regions where the ingested food is retained and thus can easily receive chemical signals from the food and the digestive process in order to control the release of the hormone.