physiology is directed by complex interactions between factors encoded
in the animal’s genome and those encountered in their environment. The
impact of these interactions on animal health is most evident in the
intestine, where digestion and absorption of dietary nutrients occur in
the presence of complex communities of microorganisms (microbiota).
Interactions between diet, microbiota, and animal hosts regulate immune
and metabolic homeostasis and also contribute to a spectrum of human
diseases, including the inflammatory bowel diseases, obesity, and
malnutrition. Our research interests are focused on understanding how
environmental factors such as the intestinal microbiota and diet
interact with host genome-encoded processes to influence host
physiology and pathophysiology. We are using the zebrafish as a
vertebrate model system for this research. The small size and optical
transparency of the zebrafish facilitate high-resolution in
imaging as well as genetic and chemical manipulations that complement
the technical limitations of mammalian models. Extensive anatomic,
physiologic, and genomic homologies between zebrafish and mammals
permit translation of insights gained in zebrafish into advances in
human medicine. To facilitate our research, we have developed methods
for rearing zebrafish under germ-free conditions and for introducing
selected microbial communities and sterilized diets into germ-free
fish. We are currently using zebrafish and mouse models to investigate
how microbial communities are assembled in the intestine and how
microbes and dietary nutrients regulate host metabolism and immunity.
We have also established methods for in
of adipose tissues in zebrafish, and we are using that experimental
platform to elucidate the mechanisms underlying adipose tissue
physiology and obesity-associated metabolic disease. The overall
objective of our work is to improve our understanding of vertebrate
physiology as a complex and dynamic integration of genome-encoded and
environmental factors, which is expected to yield new strategies for
promoting health in humans and other animals.
ecology in the intestine
at birth, the vertebrate intestine is colonized by
communities that contribute significantly to host physiology and
disease. Recent advances in high-throughput DNA sequencing have fueled
a marked expansion in our understanding of gut microbial community
However, the ecological and physiologic principles that govern the
and persistence of gut microbial communities remain poorly
understood. To address these gaps, we are using zebrafish as vertebrate
host models because their transparency permits high resolution in
imaging and their small size facilitates scaling of sample number. We
have used 16S rRNA gene sequencing to define the membership of the
zebrafish gut microbiota. We found that the same bacterial phyla
dominate the gut microbiota of zebrafish, humans, and mice, although
relative phylum abundance and the specific bacteria in those
By transplanting gut microbiotas from zebrafish or mouse donors into
germ-free mouse or zebrafish recipients, we discovered that differences
in community structure between zebrafish and mice arise in part from
distinct selective pressures imposed within the gut habitat of each host.
In accord, we found striking similarities between gut bacterial
communities from zebrafish collected recently from their natural
habitat and those domesticated for generations in lab facilities,
including a shared “core” gut microbiota. These results suggest that
lab-reared domesticated zebrafish can serve as a valid model for
investigating coevolved host-microbe relationships that occur in their
We have also found that feeding increases the abundance of the
bacterial phylum Firmicutes in the zebrafish intestine.
Diet-dependent enrichment of Firmicutes has also been observed
humans and mice in the context of obesity[7-11],
suggesting this might be a conserved ecological theme in the vertebrate
intestine. To facilitate direct observation of gut microbial
communities, we exploited the transparency of the zebrafish to develop
techniques for real-time in
of fluorescently labeled bacteria within the intact intestine.
Our current research in this area seeks to understand the physiologic
and nutritional mechanisms underlying host selection of microbial
community assembly in the intestine. This work is expected to provide
an improved understanding of the principles underlying the assembly and
maintenance of vertebrate gut microbial communities, which will be
essential to the design of therapeutic strategies to safely and
effectively promote beneficial gut microbial communities and prevent or
correct pathogenic ones.
regulation of intestinal physiology
intestinal microbiota has been identified as an important environmental
factor that contributes to many aspects of human health and disease[1,2],
which has prompted considerable interest in understanding the
underlying microbial signals and responsive host signal transduction
mechanisms. One of the most powerful experimental approaches for this
work is to rear animals under conditions in which all microbial life
forms are either excluded or known (gnotobiotic). We have developed
zebrafish as a gnotobiotic host model because its amenability to in
plus genetic and gnotobiotic manipulation provides a useful
complement to existing mammalian models.
We used these methods to conduct functional genomic comparisons of gene
expression in digestive tracts of zebrafish reared germ-free (GF) to
ex-GF animals colonized with a conventional microbiota
(conventionalized of CONVD) or to animals reared from birth with a
microbiota (conventionally raised or CONV-R). This study revealed
zebrafish genes regulated by the microbiota including many that are
conserved in the mouse intestine and involved in stimulation of
epithelial proliferation, promotion of nutrient metabolism, and innate
To test the specificity of these responses, we performed reciprocal
transplantations of gut microbiotas from CONV-R zebrafish or mouse
donors into GF zebrafish or mouse recipients. In both of zebrafish and
mouse recipients, metabolic responses were stimulated by microbiota
donated from either species. In contrast, immune responses in each
were induced only by their respective normal microbiota,
suggesting that immune and metabolic responses to the microbiota are
evoked by distinct bacterial signals. We also identified individual
culturable representatives of the zebrafish and mouse gut microbiotas
that elicit conserved responses, providing candidates for mechanistic
Based on these results, we used Pseudomonas
as a genetically manipulatable model gut bacterium to define the
mechanisms by which members of the microbiota colonize and elicit
conserved responses in zebrafish hosts. Using in
imaging in the zebrafish intestine, we found that bacteria display
complex patterns of localization and behavior, including
flagella-mediated directional motility. Genetic analysis in P.
revealed that loss of flagellar function results in attenuation of
conserved host innate immune responses but not of conserved metabolic
Current research in our lab seeks to use gnotobiotic zebrafish and
mouse models to understand the mechanisms underlying conserved innate
immune responses to members of the microbiota. Once available, this
information could be used to develop novel approaches for reducing
inflammation in the context of chronic inflammatory diseases such as
IBD or for enhancing immunity to prevent opportunistic infections.
is currently intense interest in understanding the physiologic
mechanisms by which the gut microbiota regulates host nutrient
metabolism and energy balance. It is known that microbial fermentation
of otherwise indigestible polysaccharides into short chain fatty acids
improves digestive efficiency and promotes positive energy balance. In
contrast, the contributions of the microbiota to metabolism of other
dietary nutrient classes such as energy-rich lipids remain unresolved.
We used in
of fluorescent fatty acid (FA) analogs delivered into
gnotobiotic zebrafish hosts to reveal that the microbiota stimulates FA
uptake and lipid droplet (LD) formation in the intestinal epithelium
and liver. We found that the Firmicutes bacterium Exiguobacterium
its products were sufficient to increase epithelial LD number,
whereas LD size was increased by other bacterial types. Therefore,
different members of the intestinal microbiota promote lipid absorption
via distinct mechanisms.
To further explore mechanisms of nutrient absorption, we have developed
methods for micro-gavage of zebrafish larvae with selected materials.
We are currently investigating the bacterial factors that promote
intestinal absorption of lipids and other nutrients, and the responsive
host mechanisms that mediate these effects on nutrient absorption. Once
identified, these factors would represent promising targets for new
therapies designed to address human diseases such as malnutrition,
obesity, and associated metabolic diseases.
responses in eukaryotic cells are often mediated by changes in gene
transcription that are specific to the cell type and the stimulus. The
specificity of the transcriptional response is determined by targeted
alterations in chromatin accessibility and the resulting interactions
between specific transcription factors (TFs) and open chromatin at DNA
cis-regulatory modules (CRMs).
A fundamental challenge in the study of host-microbiota interactions is
to understand how host cells interpret dynamic microbial signals within
the context of tissue-specific regulatory networks to evoke appropriate
transcriptional responses. The amenability of the zebrafish to in
transgenesis, and gnotobiotic manipulations provides exciting
opportunities to address these questions. The NF-kB transcriptional
control pathway is known to mediate microbial stimuli that regulate
expression of diverse host genes.
However, the temporal and spatial activation of NF-kB in response to
microbial signals had not been determined in whole living organisms. We
generated transgenic zebrafish that express fluorescent protein under
the transcriptional control of NF-kB and used them to show that
distinct cell types in the digestive tract and other tissues display
robust and dynamic responses to variations in the microbial environment.
The capacity of the microbiota to promote fat storage is caused in part
by the microbial suppression of intestinal epithelial transcription of
Angiopoietin-like 4 (Angptl4/Fiaf), a circulating inhibitor of
lipoprotein lipase (LPL) that inhibits hydrolysis of serum
triglycerides for storage in adipose tissues[18,19].
We used in
reporter assays to identify discrete tissue-specific CRMs at
drive expression in intestinal enterocytes and other tissues.
Strikingly, the microbiota suppressed the transcriptional activity of
the intestine-specific CRM similar to the endogenous angptl4
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
We are currently seeking to identify the TFs that mediate the tissue
specificity and microbial sensitivity of this and other CRMs. We are
also using functional genomic methods in intestinal epithelial cells
from gnotobiotic animals to elucidate global transcriptional regulatory
networks underlying physiologic responses to the microbiota. The
expected outcomes of this work include fundamental new insights into
the genomic foundations of host-microbe commensalism and novel
molecular targets for the manipulation of intestinal physiology and
pathophysiology in humans and other vertebrates.
tissue physiology and metabolic disease
current epidemic of obesity and obesity-associated metabolic diseases
represents major public health challenges. Obesity is caused by
prolonged positive energy balance resulting in storage of excess energy
as neutral lipid in adipocytes within white adipose tissues (AT). AT
forms in several distinct anatomic regions and expands through addition
of new adipocytes from undifferentiated precursors (hyperplasia) or by
increase in adipocyte size (hypertrophy). The regional distribution and
cellular morphology of AT have been identified as critical factors
linking obesity to metabolic disease. Specifically, selective expansion
of visceral AT and hypertrophic AT morphology are associated with
increased risk for metabolic diseases such as insulin resistance (IR)[21-24].
However, the mechanisms governing regional distribution and cellular
morphology of AT remain poorly understood. To address these gaps, we
have pioneered the use of the zebrafish model for investigating AT
development and physiology. We developed methods for vital labeling
imaging of zebrafish AT,
and used them to reveal extensive molecular, cellular, and
physiological homologies between zebrafish and mammalian white
Using these methods, we found that zebrafish growth
mutants exhibit delayed somatic growth, increased AT accumulation, and
disrupted adipose plasticity during nutrient deprivation.
We are currently exploring evolutionarily conserved mechanisms by which
the vascular system and fibrotic programs determine regional AT
morphology. This work could lead to new strategies for preventing human
obesity and associated metabolic diseases by selectively controlling
distinct aspects of AT development and physiology, including AT
distribution and cellular morphology.
Camp, J. G., Kanther, M., Semova, I. & Rawls, J. F. Patterns
and scales in gastrointestinal microbial ecology. Gastroenterology
Clemente, J. C., Ursell, L. K., Parfrey, L. W. & Knight, R. The
impact of the gut microbiota on human health: an integrative view. Cell
Rawls, J. F., Samuel, B. S. & Gordon, J. I. Gnotobiotic
reveal evolutionarily conserved responses to the gut microbiota. Proc.
Natl. Acad. Sci. U. S. A.
Rawls, J. F., Mahowald, M. A., Ley, R. E. & Gordon, J. I.
Reciprocal gut microbiota transplants from zebrafish and mice to
germ-free recipients reveal host habitat selection. Cell
Roeselers, G., Mittge, E. K., Stephens, W. Z., Parichy, D. M.,
Cavanaugh, C. M., Guillemin, K. & Rawls, J. F. Evidence for a
gut microbiota in the zebrafish. ISME
Semova, I., Carten, J. D., Stombaugh, J., Mackey, L. C., Knight, R.,
Farber, S. A. & Rawls, J. F. Microbiota regulate intestinal
absorption and metabolism of fatty acids in the zebrafish. Cell
Ley, R. E., Turnbaugh, P. J., Klein, S. & Gordon, J. I.
Microbial ecology: human gut microbes associated with obesity. Nature
Turnbaugh, P. J., Bäckhed, F., Fulton, L. & Gordon, J. I.
Diet-induced obesity is linked to marked but reversible alterations in
the mouse distal gut microbiome. Cell
Crawford, P. A., Crowley, J. R., Sambandam, N., Muegge, B. D.,
Costello, E. K., Hamady, M., Knight, R. & Gordon, J. I.
of myocardial ketone body metabolism by the gut microbiota during
nutrient deprivation. Proc
Natl Acad Sci U S A
Hildebrandt, M. A., Hoffmann, C., Sherrill-Mix, S. A., Keilbaugh, S.
A., Hamady, M., Chen, Y. Y., Knight, R., Ahima, R. S., Bushman, F.
& Wu, G. D. High-fat diet determines the composition of the
gut microbiome independently of obesity. Gastroenterology
1716-1724 e1711-1712, (2009).
Jumpertz, R., Le, D. S., Turnbaugh, P. J., Trinidad, C., Bogardus, C.,
Gordon, J. I. & Krakoff, J. Energy-balance studies reveal
associations between gut microbes, caloric load, and nutrient
absorption in humans. Am
J Clin Nutr
Rawls, J. F., Mahowald, M. A., Goodman, A. L., Trent, C. M. &
Gordon, J. I. In
and genetic analysis link bacterial motility and symbiosis in the
zebrafish gut. Proc.
Natl. Acad. Sci. U. S. A.
Pham, L. N., Kanther, M., Semova, I. & Rawls, J. F. Methods for
generating and colonizing gnotobiotic zebrafish. Nat
Kanther, M., Sun, X., Muhlbauer, M., Mackey, L. C., Flynn, E. J., 3rd,
Bagnat, M., Jobin, C. & Rawls, J. F. Microbial colonization
dynamic temporal and spatial patterns of NF-kappaB activation in the
zebrafish digestive tract. Gastroenterology,
.Cocchiaro, J. L. & Rawls, J. F. Microgavage of zebrafish
Maston, G. A., Evans, S. K. & Green, M. R. Transcriptional
regulatory elements in the human genome. Annu
Rev Genomics Hum Genet
Karrasch, T. & Jobin, C. NF-kappaB and the intestine: friend or
Bäckhed, F., Ding, H., Wang, T., Hooper, L. V., Koh, G. Y., Nagy, A.,
Semenkovich, C. F. & Gordon, J. I. The gut microbiota as an
environmental factor that regulates fat storage. Proc
Natl Acad Sci U S A
Bäckhed, F., Manchester, J. K., Semenkovich, C. F. & Gordon, J.
Mechanisms underlying the resistance to diet-induced obesity in
germ-free mice. Proc
Natl Acad Sci U S A
Camp, J. G., Jazwa, A. L., Trent, C. M. & Rawls, J. F. Intronic
cis-regulatory modules mediate tissue-specific and microbial control of
angptl4/fiaf transcription. PLoS
Fox, C. S., Massaro, J. M., Hoffmann, U., Pou, K. M., Maurovich-Horvat,
P., Liu, C. Y., Vasan, R. S., Murabito, J. M., Meigs, J. B., Cupples,
visceral and subcutaneous adipose tissue compartments:
association with metabolic risk factors in the Framingham Heart Study. Circulation
Gallagher, D., Kelley, D. E., Yim, J. E., Spence, N., Albu, J., Boxt,
L., Pi-Sunyer, F. X. & Heshka, S. Adipose tissue distribution
different in type 2 diabetes. Am
J Clin Nutr
Penders, J., Thijs, C., Vink, C., Stelma, F. F., Snijders, B.,
Kummeling, I., van den Brandt, P. A. & Stobberingh, E. E.
influencing the composition of the intestinal microbiota in early
Hoffstedt, J., Arner, E., Wahrenberg, H., Andersson, D. P., Qvisth, V.,
Lofgren, P., Ryden, M., Thorne, A., Wiren, M., Palmer, M.
Regional impact of adipose tissue morphology on the metabolic profile
in morbid obesity. Diabetologia
Minchin, J. E. & Rawls, J. F. In vivo analysis of white adipose
tissue in zebrafish. Methods
Flynn, E. J., 3rd, Trent, C. M. & Rawls, J. F. Ontogeny and
nutritional control of adipogenesis in zebrafish (Danio rerio). J
McMenamin, S. K., Minchin, J. E., Gordon, T. N., Rawls, J. F. &
Parichy, D. M. Dwarfism and Increased Adiposity in the gh1 Mutant
Zebrafish vizzini. Endocrinology