Intronic cis-regulatory modules mediate tissue-specific and microbial control of angptl4/fiaf transcription.
ABSTRACT: The intestinal microbiota enhances dietary energy harvest leading to increased fat storage in adipose tissues. This effect is caused in part by the microbial suppression of intestinal epithelial expression of a circulating inhibitor of lipoprotein lipase called Angiopoietin-like 4 (Angptl4/Fiaf). To define the cis-regulatory mechanisms underlying intestine-specific and microbial control of Angptl4 transcription, we utilized the zebrafish system in which host regulatory DNA can be rapidly analyzed in a live, transparent, and gnotobiotic vertebrate. We found that zebrafish angptl4 is transcribed in multiple tissues including the liver, pancreatic islet, and intestinal epithelium, which is similar to its mammalian homologs. Zebrafish angptl4 is also specifically suppressed in the intestinal epithelium upon colonization with a microbiota. In vivo transgenic reporter assays identified discrete tissue-specific regulatory modules within angptl4 intron 3 sufficient to drive expression in the liver, pancreatic islet ?-cells, or intestinal enterocytes. Comparative sequence analyses and heterologous functional assays of angptl4 intron 3 sequences from 12 teleost fish species revealed differential evolution of the islet and intestinal regulatory modules. High-resolution functional mapping and site-directed mutagenesis defined the minimal set of regulatory sequences required for intestinal activity. Strikingly, the microbiota suppressed the transcriptional activity of the intestine-specific regulatory module similar to the endogenous angptl4 gene. These results suggest that the microbiota might regulate host intestinal Angptl4 protein expression and peripheral fat storage by suppressing the activity of an intestine-specific transcriptional enhancer. This study provides a useful paradigm for understanding how microbial signals interact with tissue-specific regulatory networks to control the activity and evolution of host gene transcription.
Project description:Gender is one of the factors influencing the intestinal microbial composition in mammals, but whether fish also have gender-specific intestinal microbial patterns remains unknown. In this decade, endocrine disrupting chemicals in surface and ground water of many areas and increasing observation of freshwater male fish displaying female sexual characteristics have been reported. Here we identified the difference in intestinal microbiota between male and female zebrafish, and revealed the influence of endocrine disrupting chemicals on zebrafish intestinal microbiota by using high-throughput sequencing. The results indicated that Fusobacteria, Bacteroidetes and Proteobacteria were dominant in the gut of zebrafish and there were no obvious gender-specific intestinal microbial patterns. Two endocrine disrupting chemicals, Estradiol (E2) and Bisphenol A (BPA), were selected to treat male zebrafish for 5 weeks. E2 and BPA increased vitellogenin expression in the liver of male zebrafish and altered the intestinal microbial composition with the abundance of the phylum CKC4 increased significantly. Our results suggested that because of the developmental character and living environment, gender did not influence the assembly of intestinal microbiota in zebrafish as it does in mammals, but exposure extra to endocrine disrupting chemicals disturbed the intestinal microbial composition, which may be related to changes in host physiological metabolism.
Project description:Dysbiosis of the intestinal microbial community is considered a risk factor for development of chronic intestinal inflammation as well as other diseases such as diabetes, obesity and even cancer. Study of the innate and adaptive immune pathways controlling bacterial colonization has however proven difficult in rodents, considering the extensive cross-talk between bacteria and innate and adaptive immunity. Here, we used the zebrafish to study innate and adaptive immune processes controlling the microbial community. Zebrafish lack a functional adaptive immune system in the first weeks of life, enabling study of the innate immune system in the absence of adaptive immunity. We show that in wild type zebrafish, the initial lack of adaptive immunity associates with overgrowth of Vibrio species (a group encompassing fish and human pathogens), which is overcome upon adaptive immune development. In Rag1-deficient zebrafish (lacking adaptive immunity) Vibrio abundance remains high, suggesting that adaptive immune processes indeed control Vibrio species. Using cell transfer experiments, we confirm that adoptive transfer of T lymphocytes, but not B lymphocytes into Rag1-deficient recipients suppresses outgrowth of Vibrio. In addition, ex vivo exposure of intestinal T lymphocytes to Rag1-deficient microbiota results in increased interferon-gamma expression by these T lymphocytes, compared to exposure to wild type microbiota. In conclusion, we show that T lymphocytes control microbial composition by effectively suppressing the outgrowth of Vibrio species in the zebrafish intestine.
Project description:Understanding how intestinal microbiota alters energy homeostasis and lipid metabolism is a critical process in energy balance and health. However, the exact role of intestinal microbiota in the regulation of lipid metabolism in fish remains unclear. Here, we used two zebrafish models (germ-free and antibiotics-treated zebrafish) to identify the role of intestinal microbiota in lipid metabolism. Conventional and germ-free zebrafish larvae were fed with egg yolk. Transmission electron microscopy was used to detect the presence of lipid droplets in the intestinal epithelium. The results showed that, microbiota increased lipid accumulation in the intestinal epithelium. The mRNA sequencing technology was used to assess genes expression level. We found majority of the differentially expressed genes were related to lipid metabolism. Due to the limitation of germ-free zebrafish larvae, antibiotics-treated zebrafish were also used to identify the relationship between the gut microbiota and the host lipid metabolism. Oil-red staining showed antibiotics-treated zebrafish had less intestinal lipid accumulation than control group. The mRNA expression of genes related to lipid metabolism in liver and intestine was also quantified by using real-time PCR. The results indicated that apoa4, hsl, cox15, slc2a1a, and lss were more related to intestinal bacteria in fish, while the influence of intestinal microbiota on the activity of fabp6, acsl5, cd36, and gpat2 was different between the liver and intestine. This study identified several genes regulated by intestinal microbiota. Furthermore, the advantages and disadvantages of each model have been discussed. This study provides valuable information for exploring host-microbiota interactions in zebrafish in future.
Project description:Recently, Streptococcus agalactiae has become a major pathogen leading to Streptococcosis. To understand the physiological responses of zebrafish (Danio rerio) to S. agalactiae, the intestinal microbiota composition of the intestine (12 and 24 h post-infection, hpi, respectively) in zebrafish infected with S. agalactiae were investigated. The intestinal bacterial composition was analyzed using PacBio high-throughput full-length 16S rRNA gene sequencing. The most predominant bacteria in the zebrafish intestine were the Fusobacteria phylum and Sphingomonas genus. S. agalactiae infection affected the composition of partially intestinal microbiota. At the species level, the relative abundance of the pathogenic intestinal bacteria Aeromonas veronii, S. agalactiae, and Clostridium tarantellae significantly increased after S. agalactiae infection (p < 0.05), while that of the beneficial intestinal bacteria Bacillus licheniformis, Comamonas koreensis, and Romboutsia ilealis significantly decreased (p < 0.05), showing that S. agalactiae infection aggravates the zebrafish disease through promoting abundance of other intestinal pathogenic bacteria. This study is the first PacBio analyses of the zebrafish intestinal microbiota community under pathogenic infection. Results suggest that the S. agalactiae infection alters the intestinal flora structure in zebrafish.
Project description:The assembly of resident microbial communities is an important event in animal development; however, the extent to which this process mirrors the developmental programs of host tissues is unknown. Here we surveyed the intestinal bacteria at key developmental time points in a sibling group of 135 individuals of a model vertebrate, the zebrafish (Danio rerio). Our survey revealed stage-specific signatures in the intestinal microbiota and extensive interindividual variation, even within the same developmental stage. Microbial community shifts were apparent during periods of constant diet and environmental conditions, as well as in concert with dietary and environmental change. Interindividual variation in the intestinal microbiota increased with age, as did the difference between the intestinal microbiota and microbes in the surrounding environment. Our results indicate that zebrafish intestinal microbiota assemble into distinct communities throughout development, and that these communities are increasingly different from the surrounding environment and from one another.
Project description:Nkx2.2 is a homeodomain-containing transcription factor essential for pancreatic islet cell specification. In this study we investigate the role of Nkx2.2 within the small intestine. We have determined that Nkx2.2 is expressed at the onset of intestinal epithelial cell differentiation in specific intestinal cell populations, including a subset of enteroendocrine cells. Similar to its role in the pancreatic islet, Nkx2.2 regulates cell fate choices within the intestinal enteroendocrine population; in the Nkx2.2 null mice, several hormone-producing enteroendocrine cell populations are absent or reduced and the ghrelin-producing cell population is upregulated. The remaining intestinal cell populations, including the paneth cells, goblet cells, and enterocytes appear to be unaffected by the loss of Nkx2.2. Furthermore, similar to the pancreatic islet, Nkx2.2 appears to function upstream of Pax6 in regulating intestinal cell fates; Pax6 mRNA and protein expression is decreased in the Nkx2.2 null mice. These studies identify a novel role for Nkx2.2 in intestinal endocrine cell development and reveal the regulatory similarities between cell type specification in the pancreatic islet and in the enteroendocrine population of the intestine.
Project description: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 ?-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 ?-catenin signaling by deletion of the ?-catenin coactivator tcf4 partially decreases the proliferation-promoting capacity of A. veronii. We show that numbers of intestinal epithelial cells with cytoplasmic ?-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 ?-catenin in intestinal epithelial cells and promote cell proliferation in the developing vertebrate intestine.
Project description:Host-intestine microbiota interactions have been widely studied in aquatic animals, but these interactions in the intestine regeneration process of the sea cucumber Apostichopus japonicus have been rarely investigated. To understand how intestine regeneration impacts the developing intestinal microbiome composition and function, we performed a case study to characterize the intestinal microbial composition and functional genes of A. japonicus during intestine regeneration stages. High-throughput 16S rRNA gene sequencing revealed significantly different intestine microbiota compositions in different regeneration stages. The phylogenetic diversity and composition of the intestinal microbiota changed significantly in the early regeneration stage and tended to recover in the end stage. During the regeneration process, the abundance of Bacteroidetes and Rhodobacterales increased significantly. A network analysis revealed that Rhodobacteraceae and Flavobacteriaceae may function as keystone taxa in the intestinal microbial community of A. japonicus during intestine regeneration. Metagenomic analyses of representative samples revealed that the microbiomes of regenerating intestines were enriched in genes facilitating cell proliferation, digestion and immunity. The increased abundance of Bacteroidetes elevated the enrichment of genes associated with carbohydrate utilization. Some functional features in the subsystem category changed in a pattern that was consistent with the changing pattern of microbiota composition during intestine regeneration. Our results revealed that seemingly regular alterations in the intestinal microbiome composition and function are associated with intestine regeneration stages. Intestinal microbiota can increase the abundance of beneficial bacterial members and upregulate related functional genes to adapt to intestine regeneration and reconstruct a stable community structure. This study provides a new insight into the mechanism of the host-microbiota interaction response to organ regeneration.
Project description:<h4>Background</h4>The early-life microbiota exerts a profound and lifelong impact on host health. Longitudinal studies in humans have been informative but are mostly based on the analysis of fecal samples and cannot shed direct light on the early development of mucosa-associated intestinal microbiota and its impact on GI function. Using piglets as a model for human infants, we assess here the succession of mucosa-associated microbiota across the intestinal tract in the first 35?days after birth.<h4>Results</h4>Although sharing a similar composition and predicted functional profile at birth, the mucosa-associated microbiome in the small intestine (jejunum and ileum) remained relatively stable, while that of the large intestine (cecum and colon) quickly expanded and diversified by day 35. Among detected microbial sources (milk, vagina, areolar skin, and feces of sows, farrowing crate, and incubator), maternal milk microbes were primarily responsible for the colonization of the small intestine, contributing approximately 90% bacteria throughout the first 35?days of the neonatal life. Although maternal milk microbes contributed greater than 90% bacteria to the large intestinal microbiota of neonates upon birth, their presence gradually diminished, and they were replaced by maternal fecal microbes by day 35. We found strong correlations between the relative abundance of specific mucosa-associated microbes, particularly those vertically transmitted from the mother, and the expression levels of multiple intestinal immune and barrier function genes in different segments of the intestinal tract.<h4>Conclusion</h4>We revealed spatially specific trajectories of microbial colonization of the intestinal mucosa in the small and large intestines, which can be primarily attributed to the colonization by vertically transmitted maternal milk and intestinal microbes. Additionally, these maternal microbes may be involved in the establishment of intestinal immune and barrier functions in neonates. Our findings strengthen the notion that studying fecal samples alone is insufficient to fully understand the co-development of the intestinal microbiota and immune system and suggest the possibility of improving neonatal health through the manipulation of maternal microbiota.
Project description:Microbiota influence diverse aspects of intestinal physiology and disease in part by controlling tissue-specific transcription of host genes. However, host genomic mechanisms mediating microbial control of intestinal gene expression are poorly understood. Hepatocyte nuclear factor 4 (HNF4) is the most ancient family of nuclear receptor transcription factors with important roles in human metabolic and inflammatory bowel diseases, but a role in host response to microbes is unknown. Using an unbiased screening strategy, we found that zebrafish Hnf4a specifically binds and activates a microbiota-suppressed intestinal epithelial transcriptional enhancer. Genetic analysis revealed that zebrafish hnf4a activates nearly half of the genes that are suppressed by microbiota, suggesting microbiota negatively regulate Hnf4a. In support, analysis of genomic architecture in mouse intestinal epithelial cells disclosed that microbiota colonization leads to activation or inactivation of hundreds of enhancers along with drastic genome-wide reduction of HNF4A and HNF4G occupancy. Interspecies meta-analysis suggested interactions between HNF4A and microbiota promote gene expression patterns associated with human inflammatory bowel diseases. These results indicate a critical and conserved role for HNF4A in maintaining intestinal homeostasis in response to microbiota.