Wednesday, 2 April 2014

Gnotobiotic zebrafish reveal evolutionarily conserved responses to the gut microbiota.

Gnotobiotic zebrafish reveal evolutionarily conserved responses to the gut microbiota

NB: Gnotobiotic refers to an organism in which only known strains of microorganisms are present. This definition technically includes ‘germ-free’ organisms, aka organisms who do not harbour any microorganisms.

Some background knowledge: The zebrafish is a stomach-less teleost; using its oesophagus/proximal intestine (segment 1) for lipid absorption, proximal intestine (segment 2) for absorption of other macromolecules, and proximal intestine (segment 3) for water/ion transport. In larval and adult stomach-less teleosts, macromolecules (absorbed from segment 2 of the lumen) are transported through a network of superficial invaginations and vesicles in enterocytes to large supranuclear vacuoles, where they are stored and/or degraded. A similar tubulovesicular network exists in neonatal mammalian enterocytes but this disappears at the initiation of protein digestion. Finally, zebrafish (like mice and humans) constantly renew their intestinal epithelium throughout their life span.

The main purpose of this study was to compare mouse microbiota with zebrafish microbiota, but there were several key aspects that I think tie in with this module.

Initially the authors raised control zebrafish (aka conventionally raised aka CONR) and GF (germ-free) zebrafish. One can create GF zebrafish by killing an adult breeding pair, sterilize their bodies and remove their egg/sperm in sterile conditions. Eggs may then be fertilized in vitro, before being washing in sterile water and placed in gnotobiotic isolators. Once GF zebrafish begin feeding, all food must be autoclaved in order to destroy any potential microbes.
Authors also raised conventionalised zebrafish (CONV), that is GF zebrafish who were exposed to zebrafish water (water other zebrafish has been raised in, which therefore contained a selection of the normal microbiotia commonly found in zebrafish) at either 3dpf (days post fertilization) or 6dpf


·        GF zebrafish remained macroscopically identical to CONR zebrafish up till 8dpf, with both CONR and GF zebrafish beginning to feed normally at 5dpf.
·        At 9dpf GF zebrafish began to develop a stereotyped, rapidly progressive epidermal degeneration phenotype manifested by epidermal opacity, loss of epidermal integrity and the sloughing off of epidermal cells.
o   Interestingly, if GF zebrafish were not fed they did not exhibit this phenotype (up to 12dpf) suggesting that this phenotype results from exposure to autoclaved feed, the negative effects of which are normally negated by the presence of normal microbiota
o   GF zebrafish show a consistent morphological phenotype in their segment 2 enterocytes (the large suprauclear vacuoles were filled with clear electron-lucent material; whereas in CONV and CONR zebrafish this material was eosinophilic and electron dense)
·        By 20dpf GF zebrafish suffered an 100% mortality rate (n=824)
·        CONV zebrafish also didn’t exhibit the mortality observed in GF zebrafish, proving that mortality of GF zebrafish was not due to irreversible changes acquired during early development. However, comparisons of the digestive tract microbiota of 6dpf CONV vrs CONR zebrafish showed community structural differences (CONV zebrafish were  made up of Aeromonas (61%), and Vibrio (12%) species whereas CONV zebrafish were made up of Aeromonas (0.3%) and Vibrio (57%) species were found in CONV zebrafish)
·        GF zebrafish and 3dpf CONV zebrafish showed a similar gross morphology at 6dpf. Additionally by 6dpf GF zebrafish shared the same body size as CONV and CONR larvae
·        A broad, functional genomics-based analysis of gene expression in the digestive tracts of 6-dpf GF, CONV and CONR was conducted using DNA microarrays. Results from this indicated that 212 genes exhibited differential expression in both GF vrs CONV and GF vrs CONR comparisons.
o   These genes were then compared to a previous DNA microarray dataset of genes differentially expressed in the GI tracts (small intestine, colon or liver) of adult GF mice vrs ex-GF mice (GF mice, further colonised with normal mouse intestinal microbiota). 66 homologous were found, 54 of which changed in the same expression (up or down) in both species. These genes are well researched in mice, which made it possible to loosely identify what these genes were doing in zebrafish.
·        Epithelial proliferation genes: the increased epithelial proliferation associated with the microbiota was manifested by the increased expression of 15 genes involved in DNA replication and cell division. General results indicate the absence of a microbiota in zebrafish is associated with a compromised ability to use nutrients, and the assumption of a metabolic state that shares features associated with fasting. Additionally microbiota appears to modulates cholesterol metabolism and trafficking in both mice and zebrafish.
Finally 6dpf GF zebrafish up-regulate certain transporters involved in the transport of amino acids or peptides, which was hypothesised to compensate for an inability to efficiently process ingested proteins

·        Xenobiotic metabolism genes: the GF zebrafish digestive tract may be less competent to detoxify dietary constituents and other environmental components. There is a hypothesis that xenobiotic components found in autoclaved ‘chow’ may be toxic to GF larvae. Some species of bacterium ID’ed in normal zebrafish microbiota have been previously shown to help control the growth and establishment of pathogens in other fish species (e.g. Lactococcus lactis and Pseudomonas spp. can inhibit the growth of Vibrio anguillarum). As zebrafish do not show an adaptive immune response during their larval/juvenile stages it is possible that these bacteria are key in maintaining zebrafish health during the initial free-living period.

Rawls, J. F., Samuel, B. S., & Gordon, J. I. (2004). Gnotobiotic zebrafish reveal evolutionarily conserved responses to the gut microbiota. Proceedings of the National Academy of Sciences of the United States of America101(13), 4596-4601.

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