Zebrafish Intestine Research Articles

Expression of neuropeptides and anoctamin 1 in the embryonic and adult zebrafish intestine, revealing neuronal subpopulations and ICC-like cells

This immunohistochemical study in zebrafish aims to extend the neurochemical characterization of enteric neuronal subpopulations and to validate a marker for identification of interstitial cells of Cajal (ICC). The expression of neuropeptides and anoctamin 1 (Ano1), a selective ICC marker in mammals, was analyzed in both embryonic and adult intestine. Neuropeptides were present from 3 days postfertilization (dpf). At 3 dpf, galanin-positive nerve fibers were found in the proximal intestine, while calcitonin gene-related peptide (CGRP)- and substance P-expressing fibers appeared in the distal intestine. At 5 dpf, immunoreactive fibers were present along the entire intestinal length, indicating a well-developed peptidergic innervation at the onset of feeding. In the adult intestine, vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP), galanin, CGRP and substance P were detected in nerve fibers. Colchicine pretreatment enhanced only VIP and PACAP immunoreactivity. VIP and PACAP were coexpressed in enteric neurons. Colocalization stainings revealed three neuronal subpopulations expressing VIP and PACAP: a nitrergic noncholinergic subpopulation, a serotonergic subpopulation and a subpopulation expressing no other markers. Ano1-immunostaining revealed a 3-dimensional network in the adult intestine containing multipolar cells at the myenteric plexus and bipolar cells interspersed between circular smooth muscle cells. Ano1 immunoreactivity first appeared at 3 dpf, indicative of the onset of proliferation of ICC-like cells. It is shown that the Ano1 antiserum is a selective marker of ICC-like cells in the zebrafish intestine. Finally, it is hypothesized that ICC-like cells mediate the spontaneous regular activity of the embryonic intestine.

Zebrafish as a model to analyze macromolecule absorption in intestinal enterocytes

Intestinal absorption and transcellular transport of macromolecules is important for neonatal passive immunity and development of oral tolerance to dietary antigens. The purpose of this study was to develop tools for live analysis of macromolecule absorption in the zebrafish intestine. Using a novel method for zebrafish gavage, we directly introduced macromolecules including immunoglobulin (IgG), dextran, and transferrin (Tnf) into the intestinal lumen of zebrafish larvae and performed live confocal microscopy to examine uptake and sorting within enterocytes. All macromolecules were initially absorbed into apical vesicles but subsequently partitioned within a larger supranuclear vacuole. In transgenic zebrafish expressing Lamp2‐RFP fusion, we found that Lamp2 was targeted to the vacuole membranes, and IgG and Tnf accumulated within these Lamp2‐ringed structures. Initial colocalization of macromolecules within the same apical vesicles suggested a non‐specific bulk endocytic mechanism; however, pretreatment of larvae with a panel of endocytosis inhibitors prior to gavage revealed that IgG and Tnf absorption were specifically blocked by chlorpromazine and phenylarsine oxide, inhibitors of clathrin‐mediated endocytosis. These results provide a zebrafish system to study the physiology and environmental regulation of macromolecule absorption and transport in the intestine. Research funded by NIDDK F32‐DK094592 to JLC and NIH RO1‐DK073695 awarded to JFR.

Fibroblast Growth Factor Receptor 2c Signaling Is Required for Intestinal Cell Differentiation in Zebrafish

BackgroundThere are four cell lineages derived from intestinal stem cells that are located at the crypt and villus in the mammalian intestine the non-secretory absorptive enterocytes, and the secretory cells, which include mucous-secreting goblet cells, regulatory peptide-secreting enteroendocrine cells and antimicrobial peptide-secreting Paneth cells. Although fibroblast growth factor (Fgf) signaling is important for cell proliferation and differentiation in various tissues, its role in intestinal differentiation is less well understood.Methodology/Principal FindingsWe used a loss of function approach to investigate the importance of Fgf signaling in intestinal cell differentiation in zebrafish; abnormal differentiation of goblet cells was observed when Fgf signaling was inhibited using SU5402 or in the Tg(hsp70ldnfgfr1-EGFP) transgenic line. We identified Fgfr2c as an important receptor for cell differentiation. The number of goblet cells and enteroendocrine cells was reduced in fgfr2c morphants. In addition to secretory cells, enterocyte differentiation was also disrupted in fgfr2c morphants. Furthermore, proliferating cells were increased in the morphants. Interestingly, the loss of fgfr2c expression repressed secretory cell differentiation and increased cell proliferation in the mibta52b mutant that had defective Notch signaling.Conclusions/SignificanceIn conclusion, we found that Fgfr2c signaling derived from mesenchymal cells is important for regulating the differentiation of zebrafish intestine epithelial cells by promoting cell cycle exit. The results of Fgfr2c knockdown in mibta52b mutants indicated that Fgfr2c signaling is required for intestinal cell differentiation. These findings provide new evidences that Fgf signaling is required for the differentiation of intestinal cells in the zebrafish developing gut.

The bHLH transcription factor Ascl1a is essential for the specification of the intestinal secretory cells and mediates Notch signaling in the zebrafish intestine

Notch signaling has a fundamental role in stem cell maintenance and in cell fate choice in the intestine of different species. Canonically, Notch signaling represses the expression of transcription factors of the achaete-scute like (ASCL) or atonal related protein (ARP) families. Identifying the ARP/ASCL genes expressed in the gastrointestinal tract is essential to build the regulatory cascade controlling the differentiation of gastrointestinal progenitors into the different intestinal cell types. The expression of the ARP/ASCL factors was analyzed in zebrafish to identify, among all the ARP/ASCL factors found in the zebrafish genome, those expressed in the gastrointestinal tract. ascl1a was found to be the earliest factor detected in the intestine. Loss-of-function analyses using the pia/ascl1a mutant, revealed that ascl1a is crucial for the differentiation of all secretory cells. Furthermore, we identify a battery of transcription factors expressed during secretory cell differentiation and downstream of ascl1a. Finally, we show that the repression of secretory cell fate by Notch signaling is mediated by the inhibition of ascl1a expression. In conclusion, this work identifies Ascl1a as a key regulator of the secretory cell lineage in the zebrafish intestine, playing the same role as Atoh1 in the mouse intestine. This highlights the diversity in the ARP/ASCL family members acting as cell fate determinants downstream from Notch signaling.

L’administration postopératoire de furosémide réduit les taux de transfusion pour la chirurgie de la scaphocéphalie

The present study was carried out to examine the dose–response of microcystin-LR (MC-LR) and microcystin-RR (MC-RR) toxicity in adult Danio rerio (zebrafish) under balneation conditions at various time points. The differential responses of superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione-S-transferase (GST) as biomarkers were assessed for oxygen mediated toxicity in liver, gills, intestine and brain tissues of zebrafish exposed to dissolved MC-LR and MC-RR (0.1–10.0 μg l−1). To investigate the time related response of biomarkers, fish were sampled after 4, 7 and 15 days of exposure. Responses varied (i) between MC-LR and MC-RR (for certain groups), (ii) for different enzymes at all time points, and (iii) for different tissues. In general, most of the enzymes followed a bell shaped curve, with an abrupt increase in activity at a particular concentration. It was observed that upon exposure to MC-LR and MC-RR, some enzymes showed an adaptive response after the first time point wherein the enzyme activity increased in some tissues. The increase in enzyme activity is suggestive of their cellular and metabolic adaptations to the continued stress and toxin exposure. Enzyme activities in general increased at lower concentrations (≤5.0 μg l−1) and decreased at higher concentrations (≥5.0 μg l−1). An abrupt change in enzyme activities was observed at a particular concentration in all the tissue enzymes. For GPx and GR, there was a differential response in the case of fish exposed to MC-LR and MC-RR, which could be due to the difference in toxicity potentials of these cyanotoxins. In general, initial stress conditions were observed in most of the tissue enzymes following the exposure to microcystins (MCs). This observation suggests that MCs found in trace levels are likely to have deleterious effects on aquatic organisms and can trigger a variety of biochemical responses depending on their specific toxicity.

Smooth Muscle Tension Induces Invasive Remodeling of the Zebrafish Intestine

Author SummaryThe epithelial cells lining the digestive tract are separated from the connective tissue stroma by a thin layer of extracellular matrix called the basement membrane. During cell invasion, as occurs during cancer metastasis, epithelial cells breach the basement membrane and invade the tissue stroma. The proteases used by invasive cells to degrade basement membrane in vitro are localized in specialized plasma membrane protrusions known as invadopodia. It is not known, however, whether invadopodia are required for cell invasion in vivo or what triggers their formation. Here, we show that epithelial cells in the intestine of the zebrafish mutant meltdown form invadopodia-like protrusions and invade the tissue stroma in response to unregulated contractile tone in the surrounding smooth muscle layer. The invadopodia-like protrusions that form in response to this physical signal are required for epithelial cell invasion in this in vivo model, and they can be induced when unregulated smooth muscle contraction is induced by oxidative stress. These findings provide the first direct evidence that invadopodia play a role in tissue cell invasion in vivo and identify a novel inducible physical signaling mechanism that can drive this process.

RETRACTED: Visualization of lipid metabolism in the zebrafish intestine reveals a relationship between NPC1L1-mediated cholesterol uptake and dietary fatty acid

The small intestine is the primary site of dietary lipid absorption in mammals. The balance of nutrients, microorganisms, bile, and mucus that determine intestinal luminal environment cannot be recapitulated ex vivo, thus complicating studies of lipid absorption. We show that fluorescently labeled lipids can be used to visualize and study lipid absorption in live zebrafish larvae. We demonstrate that the addition of a BODIPY-fatty acid to a diet high in atherogenic lipids enables imaging of enterocyte lipid droplet dynamics in real time. We find that a lipid-rich meal promotes BODIPY-cholesterol absorption into an endosomal compartment distinguishable from lipid droplets. We also show that dietary fatty acids promote intestinal cholesterol absorption by rapid re-localization of NPC1L1 to the intestinal brush border. These data illustrate the power of the zebrafish system to address longstanding questions in vertebrate digestive physiology.

Ultra-structural identification of interstitial cells of Cajal in the zebrafish Danio rerio

The interstitial cells of Cajal (ICCs) are important mediators of gastrointestinal (GI) motility because of their role as pacemakers in the GI tract. In addition to their function, ICCs are also structurally distinct cells most easily identified by their ultra-structural features and expression of the tyrosine kinase receptor c-KIT. ICCs have been described in mammals, rodents, birds, reptiles, and amphibians, but there are no reports at the ultra-structural level of ICCs within the GI tract of an organism from the teleost lineage. We describe the presence of cells in the muscularis of the zebrafish intestine; these cells have similar features to ICCs in other vertebrates. The ICC-like cells are associated with the muscularis, are more electron-dense than surrounding smooth muscle cells, possess long cytoplasmic processes and mitochondria, and are situated opposing enteric nervous structures. In addition, immunofluorescent and immunoelectron-microscopic studies with antibodies targeting the zebrafish ortholog of a putative ICC marker, c-KIT (kita), showed c-kit immunoreactivity in zebrafish ICCs. Taken together, these data represent the first ultra-structural characterization of cells in the muscularis of the zebrafish Danio rerio and suggest that ICC differentiation in vertebrate evolution dates back to the teleost lineage.

Smooth muscle caldesmon modulates peristalsis in the wild type and non‐innervated zebrafish intestine

The high molecular weight isoform of the actin-binding protein Caldesmon (h-CaD) regulates smooth muscle contractile function by modulating cross-bridge cycling of myosin heads. The normal inhibitory activity of h-CaD is regulated by the enteric nervous system; however, the role of h-CaD during intestinal peristalsis has never been studied. We identified a zebrafish paralog of the human CALD1 gene that encodes an h-CaD isoform expressed in intestinal smooth muscle. We examined the role of h-CaD during intestinal peristalsis in zebrafish larvae by knocking down the h-CaD protein using an antisense morpholino oligonucleotide. We also developed transgenic zebrafish that express inhibitory peptides derived from the h-CaD myosin and actin-binding domains, and examined their effect on peristalsis in wild-type zebrafish larvae and sox10 (colourless) mutant larvae that lack enteric nerves. Genomic analyses identified two zebrafish Caldesmon paralogs. The cald1a ortholog encoded a high molecular weight isoform generated by alternative splicing whose intestinal expression was restricted to smooth muscle. Propulsive intestinal peristalsis was increased in wild-type zebrafish larvae by h-CaD knockdown and by expression of transgenes encoding inhibitory myosin and actin-binding domain peptides. Peristalsis in the non-innervated intestine of sox10 (colourless) larvae was partially restored by h-CaD knockdown and expression of the myosin-binding peptide. Disruption of the normal inhibitory function of h-CaD enhances intestinal peristalsis in both wild-type zebrafish larvae and mutant larvae that lack enteric nerves, thus confirming a physiologic role for regulation of smooth muscle contraction at the actin filament.

Bacterial Community Assembly and Turnover within the Intestines of Developing Zebrafish

BackgroundThe majority of animal associated microorganisms are present in digestive tract communities. These intestinal communities arise from selective pressures of the gut habitats as well as host's genotype are regarded as an extra ‘organ’ regulate functions that have not evolved wholly on the host. They are functionally essential in providing nourishment, regulating epithelial development, and influencing immunity in the vertebrate host. As vertebrates are born free of microorganisms, what is poorly understood is how intestinal bacterial communities assemble and develop in conjunction with the development of the host.Methodology/Principal FindingsSet within an ecological framework, we investigated the bacterial community assembly and turnover within the intestinal habitats of developing zebrafish (from larvae to adult animals). Spatial and temporal species-richness relationships and Mantel and partial Mantel tests revealed that turnover was low and that richness and composition was best predicted by time and not intestinal volume (habitat size) or changes in food diet. We also observed that bacterial communities within the zebrafish intestines were deterministically assembled (reflected by the observed low turnover) switching to stochastic assembly in the later stages of zebrafish development.Conclusions/SignificanceThis study is of importance as it provides a novel insight into how intestinal bacterial communities assemble in tandem with the host's development (from early to adult stages). It is our hope that by studying intestinal microbiota of this vertebrate model with such or some more refined approaches in the future could well provide ecological insights for clinical benefit. In addition, this study also adds to our still fledgling knowledge of how spatial and temporal species-richness relationships are shaped and provides further mounting evidence that bacterial community assembly and dynamics are shaped by both deterministic and stochastic considerations.

Imaging Bacterial Colonization of the Zebrafish Gut with Selective Plane Illumination

Microbes are a major component of living animals, outnumbering the human cells in humans, for example, by an order of magnitude. The mechanisms by which resident microbial communities assemble, however, are very poorly understood, in large part due to the difficulty of imaging microbes inside animals and to the scarcity of tractable model systems. Selective plane illumination microscopy (SPIM), also known as light sheet fluorescence microscopy, is a recently developed imaging technique that is able to acquire high-speed three-dimensional images over long periods of time with high resolution, a wide field of view, and low levels of phototoxicity. Zebrafish (Danio rerio) are well-studied model vertebrates that are transparent during larval life when their intestines are first colonized by bacteria. We can rear germ-free zebrafish that can then be inoculated with particular bacterial species expressing fluorescent proteins. Using a home-built SPIM microscope, we have begun to investigate the dynamics of bacterial colonization of the larval zebrafish intestine. We provide here the first demonstration that SPIM has the resolution and speed necessary to image individual bacteria as well as larger colonies over periods of several hours. Imaging colonies allows quantification of changes in the spatial distribution of bacterial populations, while imaging individuals makes it possible to explore the motility of these organisms in more natural settings than conventional culture-based assays allow.

Biochemical response of diverse organs in adult Danio rerio (zebrafish) exposed to sub-lethal concentrations of microcystin-LR and microcystin-RR: A balneation study

The present study was carried out to examine the dose–response of microcystin-LR (MC-LR) and microcystin-RR (MC-RR) toxicity in adult Danio rerio (zebrafish) under balneation conditions at various time points. The differential responses of superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione-S-transferase (GST) as biomarkers were assessed for oxygen mediated toxicity in liver, gills, intestine and brain tissues of zebrafish exposed to dissolved MC-LR and MC-RR (0.1–10.0μgl−1). To investigate the time related response of biomarkers, fish were sampled after 4, 7 and 15 days of exposure. Responses varied (i) between MC-LR and MC-RR (for certain groups), (ii) for different enzymes at all time points, and (iii) for different tissues. In general, most of the enzymes followed a bell shaped curve, with an abrupt increase in activity at a particular concentration. It was observed that upon exposure to MC-LR and MC-RR, some enzymes showed an adaptive response after the first time point wherein the enzyme activity increased in some tissues. The increase in enzyme activity is suggestive of their cellular and metabolic adaptations to the continued stress and toxin exposure. Enzyme activities in general increased at lower concentrations (≤5.0μgl−1) and decreased at higher concentrations (≥5.0μgl−1). An abrupt change in enzyme activities was observed at a particular concentration in all the tissue enzymes. For GPx and GR, there was a differential response in the case of fish exposed to MC-LR and MC-RR, which could be due to the difference in toxicity potentials of these cyanotoxins. In general, initial stress conditions were observed in most of the tissue enzymes following the exposure to microcystins (MCs). This observation suggests that MCs found in trace levels are likely to have deleterious effects on aquatic organisms and can trigger a variety of biochemical responses depending on their specific toxicity.

The spatio-temporal patterning of Hoxa9 and Hoxa13 in the developing zebrafish enteric nervous system

Hirschsprung's disease is characterised by the absence of ganglion cells in the distal bowel, a process which is controlled by complex genetic pathways. Homeobox genes have a major role in gut development and this is depicted by the enteric Hox code which describes the different spatial and temporal expression of Hox genes. Hoxa9 and Hoxa13 mutations have been discovered in patients with Hirschsprung's disease (HD). The aim of this study was to determine the spatio-temporal pattern of Hoxa9 and Hoxa13 in enteric nervous system (ENS) development using the zebrafish model. Purified plasmids that contained the gene of interest were obtained and inoculated into culture medium to exponentially increase the number of bacteria containing the plasmid. Cells were then harvested by centrifugation and plasmid DNA was extracted, which was then linearised and precipitated. RNA digoxigenin-labelled probes were made by in vitro transcription reaction. In situ hybridisation was carried out using these probes on zebrafish embryos which were collected from 24 to 120 h post fertilisation (hpf), by which time the zebrafish intestine is fully developed. Embryos were then mounted in glycerol and imaged using an Olympus B40 microscope and images were taken using an Olympus Super F1.8 digital camera. At 24 hpf, Hoxa9 expression is seen in the forebrain and hindbrain and also in the very distal myotome whereas Hoxa13 expression, however, is seen only at the forebrain and hindbrain. At 48 hpf, Hoxa9- and Hoxa13-labelled cells are seen migrating distally from the forebrain into the notochord and spinal cord. At 72 hpf, Hoxa9-labelled cells can be seen throughout the spinal cord whereas Hoxa13 positive cells are seen migrating down from the spinal cord and in the proximal gut. By 96 hpf, Hoxa9- and Hoxa13-labelled cells have migrated down the full length of the spinal cord and along the proximal and mid intestine. By 120 hpf, Hoxa9 and Hoxa13 positive cells can be seen along the entire length of the zebrafish intestine. These results show further evidence that Hoxa9 and Hoxa13 are involved in the early and organised patterning of ENS development in the zebrafish model.

Zebrafish Krüppel-Like Factor 4a Represses Intestinal Cell Proliferation and Promotes Differentiation of Intestinal Cell Lineages

BackgroundMouse krüppel-like factor 4 (Klf4) is a zinc finger-containing transcription factor required for terminal differentiation of goblet cells in the colon. However, studies using either Klf4−/− mice or mice with conditionally deleted Klf4 in their gastric epithelia showed different results in the role of Klf4 in epithelial cell proliferation. We used zebrafish as a model organism to gain further understanding of the role of Klf4 in the intestinal cell proliferation and differentiation.Methodology/Principal FindingsWe characterized the function of klf4a, a mammalian klf4 homologue by antisense morpholino oligomer knockdown. Zebrafish Klf4a shared high amino acid similarities with human and mouse Klf4. Phylogenetic analysis grouped zebrafish Klf4a together with both human and mouse Klf4 in a branch with high bootstrap value. In zebrafish, we demonstrate that Klf4a represses intestinal cell proliferation based on results of BrdU incorporation, p-Histone 3 immunostaining, and transmission electron microscopy analyses. Decreased PepT1 expression was detected in intestinal bulbs of 80- and 102-hours post fertilization (hpf) klf4a morphants. Significant reduction of alcian blue-stained goblet cell number was identified in intestines of 102- and 120-hpf klf4a morphants. Embryos treated with γ-secretase inhibitor showed increased klf4a expression in the intestine, while decreased klf4a expression and reduction in goblet cell number were observed in embryos injected with Notch intracellular domain (NICD) mRNA. We were able to detect recovery of goblet cell number in 102-hpf embryos that had been co-injected with both klf4a and Notch 1a NICD mRNA.Conclusions/SignificanceThis study provides in vivo evidence showing that zebrafih Klf4a is essential for the repression of intestinal cell proliferation. Zebrafish Klf4a is required for the differentiation of goblet cells and the terminal differentiation of enterocytes. Moreover, the regulation of differentiation of goblet cells in zebrafish intestine by Notch signaling at least partially mediated through Klf4a.

Physiological response to a metal-contaminated invertebrate diet in zebrafish: Importance of metal speciation and regulation of metal transport pathways

Dietary metal uptake in fish is determined by metal bioavailability in prey and the metal requirements of the fish. In this study zebrafish were fed the intertidal polychaete worm Nereis diversicolor (3% wet weight day −1) collected from Ag, Cd and Cu-impacted Restronguet Creek (RC) or a reference site, Blackwater estuary (BW), for 21 days. On days 0, 7, 14 and 21 fish were fed a single meal of RC or BW N. diversicolor labeled with 110mAg or 109Cd for measurements of metal assimilation efficiency (AE). Zebrafish intestines were also taken for mRNA expression analysis of copper transporter 1 ( ctr1), divalent metal transporter 1 ( dmt1) and metallothionein 2 ( mt2). No significant difference was observed in the AE of 109Cd in metal naïve fish at day 0 between RC and BW worms, 11.8 ± 2.1 and 15.3 ± 2.8%, respectively. However, AE of 110mAg was significantly greater in fish fed worms from BW compared to RC, 5 ± 1.2% and 1.6 ± 0.5%, respectively at day 0. Fractionation analysis of radiolabeled metal partitioned in N. diversicolor from RC revealed a greater proportion of Ag (40 ± 1.1%) in a fraction containing protein and organelle bound metal, associated with high trophic availability, compared to BW polychaetes (24 ± 2.5%). Lower AE of 110mAg from RC polychaetes is therefore unlikely due to speciation of 110mAg in N. diversicolor from RC, but to the high concentration of Cu, a potential Ag antagonist. Exposure to RC polychaetes significantly increased the AE of 110mAg (6.2 ± 1%), but not 109Cd, from RC worms, after 21 days. AE of 110mAg and 109Cd was unaffected by pre-exposure to BW. Elevated concentration of intestinal Cu and increased expression of ctr1, dmt1 and mt2 after 14 days exposure in fish fed worms from RC suggest altered Cu handling strategy of these fish which may increase AE of Ag via shared Ag and Cu transport pathways. These data suggest metal exposure history of invertebrates may affect metal bioavailability to fish, and fish may alter intestinal uptake physiology during chronic dietary exposure with implications for the assimilation and toxicity of dietary metals.

Chitosan coated carbon fiber microelectrode for selective in vivo detection of neurotransmitters in live zebrafish embryos

We report the development of a chitosan modified carbon fiber microelectrode for in vivo detection of serotonin. We find that chitosan has the ability to reject physiological levels of ascorbic acid interferences and facilitate selective and sensitive detection of in vivo levels of serotonin, a common catecholamine neurotransmitter. Presence of chitosan on the microelectrode surface was investigated using scanning electron microscopy (SEM) and cyclic voltammetry (CV). The electrode was characterized using differential pulse voltammetry (DPV). A detection limit of 1.6 nM serotonin with a sensitivity of 5.12 nA/μM, a linear range from 2 to 100 nM and a reproducibility of 6.5% for n = 6 electrodes were obtained. Chitosan modified microelectrodes selectively measure serotonin in presence of physiological levels of ascorbic acid. In vivo measurements were performed to measure concentration of serotonin in the live embryonic zebrafish intestine. The sensor quantifies in vivo intestinal levels of serotonin while successfully rejecting ascorbic acid interferences. We demonstrate that chitosan can be used as an effective coating to reject ascorbic acid interferences at carbon fiber microelectrodes, as an alternative to Nafion, and that chitosan modified microelectrodes are reliable tools for in vivo monitoring of changes in neurotransmitter levels.

Topographical distribution of antimicrobial genes in the zebrafish intestine

The zebrafish is increasingly being utilized to study aspects of the conserved innate intestinal immunity of vertebrates. In mammals, some antimicrobial proteins are synthesised by specialised immune cells that appear to have no equivalent in zebrafish. To delineate foci of antimicrobial protein production along the zebrafish intestine, we examined the antero-posterior expression gradients of antimicrobial genes. Quantitative PCR revealed distinct expression gradient profiles, with the mid-intestine exhibiting elevated expression of several genes such as dual oxidase and the defensin beta-like and peptidoglycan recognition protein families. This region also presented with the most numbers of leukocytes and endocytic cells, supporting a specialised immunological role. Conversely, expression of the Dr-RNase family was prominent in the anterior intestine. Expression of the zebrafish β-defensin family was examined in adult zebrafish tissues. Strong expression of defensin beta-like 1 was detected in the swim bladder of zebrafish from the larval stage of development through to adults.

STORM: a general model to determine the number and adaptive changes of epithelial stem cells in teleost, murine and human intestinal tracts.

Intestinal stem cells play a pivotal role in the epithelial tissue renewal, homeostasis and cancer development. The lack of a general marker for intestinal stem cells across species has hampered analysis of stem cell number in different species and their adaptive changes upon intestinal lesions or during development of cancer. Here a two-dimensional model, named STORM, has been developed to address this issue. By optimizing epithelium renewal dynamics, the model examines the epithelial stem cell number by taking experimental input information regarding epithelium proliferation and differentiation. As the results suggest, there are 2.0–4.1 epithelial stem cells on each pocket section of zebrafish intestine, 2.0–4.1 stem cells on each crypt section of murine small intestine and 1.8–3.5 stem cells on each crypt section of human duodenum. The model is able to provide quick results for stem cell number and its adaptive changes, which is not easy to measure through experiments. Its general applicability to different species makes it a valuable tool for analysis of intestinal stem cells under various pathological conditions.

Epithelial cell proliferation in the developing zebrafish intestine is regulated by the Wnt pathway and microbial signaling via Myd88

Rates of cell proliferation in the vertebrate intestinal epithelium are modulated by intrinsic signaling pathways and extrinsic cues. Here, we report that epithelial cell proliferation in the developing zebrafish intestine is stimulated both by the presence of the resident microbiota and by activation of Wnt signaling. We find that the response to microbial proliferation-promoting signals requires Myd88 but not TNF receptor, implicating host innate immune pathways but not inflammation in the establishment of homeostasis in the developing intestinal epithelium. We show that loss of axin1, a component of the β-catenin destruction complex, results in greater than WT levels of intestinal epithelial cell proliferation. Compared with conventionally reared axin1 mutants, germ-free axin1 mutants exhibit decreased intestinal epithelial cell proliferation, whereas monoassociation with the resident intestinal bacterium Aeromonas veronii results in elevated epithelial cell proliferation. Disruption of β-catenin signaling by deletion of the β-catenin coactivator tcf4 partially decreases the proliferation-promoting capacity of A. veronii. We show that numbers of intestinal epithelial cells with cytoplasmic β-catenin are reduced in the absence of the microbiota in both WT and axin1 mutants and elevated in animals' monoassociated A. veronii. Collectively, these data demonstrate that resident intestinal bacteria enhance the stability of β-catenin in intestinal epithelial cells and promote cell proliferation in the developing vertebrate intestine.

Neurochemical coding of enteric neurons in adult and embryonic zebrafish (Danio rerio)

Although the morphology and development of the zebrafish enteric nervous system have been extensively studied, the precise neurochemical coding of enteric neurons and their proportional enteric distribution are currently not known. By using immunohistochemistry, we determined the proportional expression and coexpression of neurochemical markers in the embryonic and adult zebrafish intestine. Tyrosine hydroxylase (TH), vasoactive intestinal peptide (VIP), and pituitary adenylate cyclase-activating peptide (PACAP) were observed only in nerve fibers, whereas other markers were also detected in neuronal cell bodies. Calretinin and calbindin had similar distributions. In embryos, all markers, except for choline acetyltransferase (ChAT) and TH, were present from 72 hours postfertilization. Nitrergic neurons, evenly distributed and remaining constant in time, constituted the major neuronal subpopulation. The neuronal proportions of the other markers increased during development and were characterized by regional differences. In the adult, all markers examined were expressed in the enteric nervous system. A large percentage of enteric neurons displayed calbindin and calretinin, and serotonin was the only marker showing significant distribution differences in the three intestinal regions. Colocalization studies showed that serotonin was not coexpressed with any of the other markers. At least five neuronal subpopulations were determined: a serotonergic, a nitrergic noncholinergic, two cholinergic nonnitrergic subpopulations along with one subpopulation expressing both ChAT and neuronal nitric oxide synthase. Analysis of nerve fibers revealed that nitrergic neurons coexpress VIP and PACAP, and that nitrergic neurons innervate the tunica muscularis, whereas serotonergic and cholinergic nonnitrergic neurons innervate the lamina propria and the tunica muscularis.