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Rawls, John Franklin

Overview:

Our research uses multiple complementary approaches to understand how host-microbe interactions in the intestine regulate digestive physiology and energy balance. First, my lab uses genetic, gnotobiotic, and in vivo imaging approaches to determine how commensal microorganisms (microbiota) interact with vertebrate hosts to regulate their nutrition and immunity, as well as the mechanisms underlying assembly of intestinal microbial communities. We utilize the zebrafish as a host model in which host and microbial cells can be viewed and manipulated a transparent living vertebrate. We have pioneered the use of germ-free or gnotobiotic zebrafish to investigate the roles of microorganisms in vertebrate biology, and we are using these methods to investigate the bacterial signals and responsive host pathways that regulate host immunity, nutrition, and gene expression. Second, my laboratory is utilizing the zebrafish system to investigate mechanisms underlying the formation and function of adipose tissues. We have developed methods for in vivo imaging of zebrafish adipose tissue, and we are currently using these techniques to explore the developmental and environmental processes regulating adipose tissue growth and physiology. In both of these fields, we have effectively used zebrafish and mice to model key aspects of human physiology and pathophysiology, and to gain new insights into underlying mechanisms.

Positions:

Associate Professor of Molecular Genetics and Microbiology

Molecular Genetics and Microbiology
School of Medicine

Associate Professor in Medicine

Medicine, Gastroenterology
School of Medicine

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Education:

B.S. 1996

B.S. — Emory University

Ph.D. 2001

Ph.D. — Washington University

News:

Grants:

Summer Scholars Program in Genome Sciences and Medicine

Administered By
Duke Center for Applied Genomics and Precision Medicine
AwardedBy
National Institutes of Health
Role
Significant Contributor
Start Date
September 22, 2017
End Date
June 30, 2022

A comprehensive research resource to define mechanisms underlying microbial regulation of host metabolism in pediatric obesity and obesity-targeted therapeutics

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 25, 2016
End Date
August 31, 2021

Duke Training Grant in Digestive Diseases and Nutrition

Administered By
Medicine, Gastroenterology
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
July 01, 1988
End Date
June 30, 2021

NRT: Integrative Bioinformatics for Investigating and Engineering Microbiomes (IBIEM)

Administered By
Civil and Environmental Engineering
AwardedBy
National Science Foundation
Role
Co-Principal Investigator
Start Date
September 01, 2015
End Date
August 31, 2020

Microbial regulation of host nutrient metabolism

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
May 01, 2013
End Date
May 31, 2020

Regulation of Luminal Protein Uptake and Trafficking By Lysosome-Rich Enterocytes

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Co-Sponsor
Start Date
May 01, 2017
End Date
April 30, 2020

Host Innate Immune-Microbial Interactions in Intestinal Inflammation

Administered By
Molecular Genetics and Microbiology
AwardedBy
University of North Carolina - Chapel Hill
Role
Principal Investigator
Start Date
September 19, 2013
End Date
June 30, 2018

Organotin influences on assembly and obesogenic activity of the gut microbiota

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
March 21, 2014
End Date
August 31, 2017

Identifying genes required for digestive physiology and lipid metabolism

Administered By
Molecular Genetics and Microbiology
AwardedBy
Carnegie Institution of Washington
Role
Principal Investigator
Start Date
September 01, 2013
End Date
August 31, 2017

Immune Compromised Zebrafish for Cell Transplantation

Administered By
Molecular Genetics and Microbiology
AwardedBy
Massachusetts General Hospital
Role
Principal Investigator
Start Date
August 01, 2013
End Date
July 31, 2017

Lightsheet Imaging System

Administered By
Biology
AwardedBy
National Institutes of Health
Role
Major User
Start Date
March 15, 2016
End Date
March 14, 2017

Role of Developmental Signaling Pathways in Tuberculosis Pathogenesis

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Collaborating Investigator
Start Date
February 01, 2017
End Date
February 23, 2017

Microbial Regulation of Nuclear Receptor Activity in the Intestinal Epithelium

Administered By
Molecular Genetics and Microbiology
AwardedBy
Baylor College of Medicine
Role
Principal Investigator
Start Date
June 01, 2014
End Date
May 31, 2016

Microbial Ecology of the Zebrafish Intestine

Administered By
Molecular Genetics and Microbiology
AwardedBy
University of Oregon
Role
Principal Investigator
Start Date
May 01, 2013
End Date
November 30, 2015
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Awards:

Fellow. American Association for the Advancement of Science.

Type
National
Awarded By
American Association for the Advancement of Science
Date
January 01, 2016

Kavli Fellow. Kavli Frontiers of Science Program.

Type
National
Awarded By
Kavli Frontiers of Science Program
Date
January 01, 2009

Pew Scholar in the Biomedical Sciences. Pew Charitable Trust.

Type
National
Awarded By
Pew Charitable Trust
Date
January 01, 2008

Mentored Research Scientist Development Award. National Institutes of Health.

Type
National
Awarded By
National Institutes of Health
Date
January 01, 2006

Ruth L. Kirschstein Individual National Research Service Award. National Institutes of Health.

Type
National
Awarded By
National Institutes of Health
Date
January 01, 2002

Spencer T. and Ann W. Olin Medical Scientist Fellow. Washington University School of Medicine.

Type
School
Awarded By
Washington University School of Medicine
Date
January 01, 2001

Victor Hamburger Prize in Developmental Biology. Washington University School of Medicine.

Type
University
Awarded By
Washington University School of Medicine
Date
January 01, 2001

Publications:

Microbial colonization is required for normal neurobehavioral development in zebrafish.

Changes in resident microbiota may have wide-ranging effects on human health. We investigated whether early life microbial disruption alters neurodevelopment and behavior in larval zebrafish. Conventionally colonized, axenic, and axenic larvae colonized at 1 day post fertilization (dpf) were evaluated using a standard locomotor assay. At 10 dpf, axenic zebrafish exhibited hyperactivity compared to conventionalized and conventionally colonized controls. Impairment of host colonization using antibiotics also caused hyperactivity in conventionally colonized larvae. To determine whether there is a developmental requirement for microbial colonization, axenic embryos were serially colonized on 1, 3, 6, or 9 dpf and evaluated on 10 dpf. Normal activity levels were observed in axenic larvae colonized on 1-6 dpf, but not on 9 dpf. Colonization of axenic embryos at 1 dpf with individual bacterial species Aeromonas veronii or Vibrio cholerae was sufficient to block locomotor hyperactivity at 10 dpf. Exposure to heat-killed bacteria or microbe-associated molecular patterns pam3CSK4 or Poly(I:C) was not sufficient to block hyperactivity in axenic larvae. These data show that microbial colonization during early life is required for normal neurobehavioral development and support the concept that antibiotics and other environmental chemicals may exert neurobehavioral effects via disruption of host-associated microbial communities.

Authors
Phelps, D; Brinkman, NE; Keely, SP; Anneken, EM; Catron, TR; Betancourt, D; Wood, CE; Espenschied, ST; Rawls, JF; Tal, T
MLA Citation
Phelps, D, Brinkman, NE, Keely, SP, Anneken, EM, Catron, TR, Betancourt, D, Wood, CE, Espenschied, ST, Rawls, JF, and Tal, T. "Microbial colonization is required for normal neurobehavioral development in zebrafish." Scientific Reports 7.1 (September 11, 2017): 11244-.
PMID
28894128
Source
epmc
Published In
Scientific Reports
Volume
7
Issue
1
Publish Date
2017
Start Page
11244
DOI
10.1038/s41598-017-10517-5

Genomic dissection of conserved transcriptional regulation in intestinal epithelial cells.

The intestinal epithelium serves critical physiologic functions that are shared among all vertebrates. However, it is unknown how the transcriptional regulatory mechanisms underlying these functions have changed over the course of vertebrate evolution. We generated genome-wide mRNA and accessible chromatin data from adult intestinal epithelial cells (IECs) in zebrafish, stickleback, mouse, and human species to determine if conserved IEC functions are achieved through common transcriptional regulation. We found evidence for substantial common regulation and conservation of gene expression regionally along the length of the intestine from fish to mammals and identified a core set of genes comprising a vertebrate IEC signature. We also identified transcriptional start sites and other putative regulatory regions that are differentially accessible in IECs in all 4 species. Although these sites rarely showed sequence conservation from fish to mammals, surprisingly, they drove highly conserved IEC expression in a zebrafish reporter assay. Common putative transcription factor binding sites (TFBS) found at these sites in multiple species indicate that sequence conservation alone is insufficient to identify much of the functionally conserved IEC regulatory information. Among the rare, highly sequence-conserved, IEC-specific regulatory regions, we discovered an ancient enhancer upstream from her6/HES1 that is active in a distinct population of Notch-positive cells in the intestinal epithelium. Together, these results show how combining accessible chromatin and mRNA datasets with TFBS prediction and in vivo reporter assays can reveal tissue-specific regulatory information conserved across 420 million years of vertebrate evolution. We define an IEC transcriptional regulatory network that is shared between fish and mammals and establish an experimental platform for studying how evolutionarily distilled regulatory information commonly controls IEC development and physiology.

Authors
Lickwar, CR; Camp, JG; Weiser, M; Cocchiaro, JL; Kingsley, DM; Furey, TS; Sheikh, SZ; Rawls, JF
MLA Citation
Lickwar, CR, Camp, JG, Weiser, M, Cocchiaro, JL, Kingsley, DM, Furey, TS, Sheikh, SZ, and Rawls, JF. "Genomic dissection of conserved transcriptional regulation in intestinal epithelial cells." PLoS biology 15.8 (August 29, 2017): e2002054-.
Website
http://hdl.handle.net/10161/15397
PMID
28850571
Source
epmc
Published In
PLoS biology
Volume
15
Issue
8
Publish Date
2017
Start Page
e2002054
DOI
10.1371/journal.pbio.2002054

Microbiota regulate intestinal epithelial gene expression by suppressing the transcription factor Hepatocyte nuclear factor 4 alpha.

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.

Authors
Davison, JM; Lickwar, CR; Song, L; Breton, G; Crawford, GE; Rawls, JF
MLA Citation
Davison, JM, Lickwar, CR, Song, L, Breton, G, Crawford, GE, and Rawls, JF. "Microbiota regulate intestinal epithelial gene expression by suppressing the transcription factor Hepatocyte nuclear factor 4 alpha." Genome research 27.7 (July 2017): 1195-1206.
PMID
28385711
Source
epmc
Published In
Genome research
Volume
27
Issue
7
Publish Date
2017
Start Page
1195
End Page
1206
DOI
10.1101/gr.220111.116

A classification system for zebrafish adipose tissues.

The zebrafish model system offers significant utility for in vivo imaging of adipose tissue (AT) dynamics and for screening to identify chemical and genetic modifiers of adiposity. In particular, AT can be quantified accurately in live zebrafish using fluorescent lipophilic dyes. Although this methodology offers considerable promise, the comprehensive identification and classification of zebrafish ATs has not been performed. Here, we use fluorescent lipophilic dyes and in vivo imaging systematically to identify, classify and quantify the zebrafish AT pool. We identify 34 regionally distinct zebrafish ATs, including five visceral ATs and 22 subcutaneous ATs. For each of these ATs, we describe detailed morphological characteristics to aid their identification in future studies. Furthermore, we quantify the areas for each AT and construct regression models to allow prediction of expected AT size and variation across a range of developmental stages. Finally, we demonstrate the utility of this resource for identifying effects of strain variation and high-fat diet on AT growth. Altogether, this resource provides foundational information on the identity, dynamics and expected quantities of zebrafish ATs for use as a reference for future studies.

Authors
Minchin, JEN; Rawls, JF
MLA Citation
Minchin, JEN, and Rawls, JF. "A classification system for zebrafish adipose tissues." Disease models & mechanisms 10.6 (June 2017): 797-809.
PMID
28348140
Source
epmc
Published In
Disease models & mechanisms
Volume
10
Issue
6
Publish Date
2017
Start Page
797
End Page
809
DOI
10.1242/dmm.025759

In vivo imaging and quantification of regional adiposity in zebrafish.

Adipose tissues (ATs) are lipid-rich structures that supply and sequester energy-dense lipid in response to the energy status of an organism. As such, ATs provide an organism energetic insurance during periods of adverse physiological burden. ATs are deposited in diverse anatomical locations, and excessive accumulation of particular regional ATs modulates disease risk. Therefore, a model system that facilitates the visualization and quantification of regional adiposity holds significant biomedical promise. The zebrafish (Danio rerio) has emerged as a new model system for AT research in which the entire complement of regional ATs can be imaged and quantified in live individuals. Here we present detailed methods for labeling adipocytes in live zebrafish using fluorescent lipophilic dyes, and for identifying and quantifying regional zebrafish ATs.

Authors
Minchin, JEN; Rawls, JF
MLA Citation
Minchin, JEN, and Rawls, JF. "In vivo imaging and quantification of regional adiposity in zebrafish." Methods in cell biology 138 (January 2017): 3-27. (Review)
PMID
28129849
Source
epmc
Published In
Methods in cell biology
Volume
138
Publish Date
2017
Start Page
3
End Page
27
DOI
10.1016/bs.mcb.2016.11.010

Best practices for germ-free derivation and gnotobiotic zebrafish husbandry.

All animals are ecosystems with resident microbial communities, referred to as microbiota, which play profound roles in host development, physiology, and evolution. Enabled by new DNA sequencing technologies, there is a burgeoning interest in animal-microbiota interactions, but dissecting the specific impacts of microbes on their hosts is experimentally challenging. Gnotobiology, the study of biological systems in which all members are known, enables precise experimental analysis of the necessity and sufficiency of microbes in animal biology by deriving animals germ-free (GF) and inoculating them with defined microbial lineages. Mammalian host models have long dominated gnotobiology, but we have recently adapted gnotobiotic approaches to the zebrafish (Danio rerio), an important aquatic model. Zebrafish offer several experimental attributes that enable rapid, large-scale gnotobiotic experimentation with high replication rates and exquisite optical resolution. Here we describe detailed protocols for three procedures that form the foundation of zebrafish gnotobiology: derivation of GF embryos, microbial association of GF animals, and long-term, GF husbandry. Our aim is to provide sufficient guidance in zebrafish gnotobiotic methodology to expand and enrich this exciting field of research.

Authors
Melancon, E; Gomez De La Torre Canny, S; Sichel, S; Kelly, M; Wiles, TJ; Rawls, JF; Eisen, JS; Guillemin, K
MLA Citation
Melancon, E, Gomez De La Torre Canny, S, Sichel, S, Kelly, M, Wiles, TJ, Rawls, JF, Eisen, JS, and Guillemin, K. "Best practices for germ-free derivation and gnotobiotic zebrafish husbandry." Methods in cell biology 138 (January 2017): 61-100. (Review)
PMID
28129860
Source
epmc
Published In
Methods in cell biology
Volume
138
Publish Date
2017
Start Page
61
End Page
100
DOI
10.1016/bs.mcb.2016.11.005

Genomic sequencing-based mutational enrichment analysis identifies motility genes in a genetically intractable gut microbe.

A major roadblock to understanding how microbes in the gastrointestinal tract colonize and influence the physiology of their hosts is our inability to genetically manipulate new bacterial species and experimentally assess the function of their genes. We describe the application of population-based genomic sequencing after chemical mutagenesis to map bacterial genes responsible for motility in Exiguobacterium acetylicum, a representative intestinal Firmicutes bacterium that is intractable to molecular genetic manipulation. We derived strong associations between mutations in 57 E. acetylicum genes and impaired motility. Surprisingly, less than half of these genes were annotated as motility-related based on sequence homologies. We confirmed the genetic link between individual mutations and loss of motility for several of these genes by performing a large-scale analysis of spontaneous suppressor mutations. In the process, we reannotated genes belonging to a broad family of diguanylate cyclases and phosphodiesterases to highlight their specific role in motility and assigned functions to uncharacterized genes. Furthermore, we generated isogenic strains that allowed us to establish that Exiguobacterium motility is important for the colonization of its vertebrate host. These results indicate that genetic dissection of a complex trait, functional annotation of new genes, and the generation of mutant strains to define the role of genes in complex environments can be accomplished in bacteria without the development of species-specific molecular genetic tools.

Authors
Bae, S; Mueller, O; Wong, S; Rawls, JF; Valdivia, RH
MLA Citation
Bae, S, Mueller, O, Wong, S, Rawls, JF, and Valdivia, RH. "Genomic sequencing-based mutational enrichment analysis identifies motility genes in a genetically intractable gut microbe." Proceedings of the National Academy of Sciences of the United States of America 113.49 (December 2016): 14127-14132.
PMID
27911803
Source
epmc
Published In
Proceedings of the National Academy of Sciences of USA
Volume
113
Issue
49
Publish Date
2016
Start Page
14127
End Page
14132
DOI
10.1073/pnas.1612753113

Contribution of neutral processes to the assembly of gut microbial communities in the zebrafish over host development.

Despite their importance to host health and development, the communities of microorganisms associated with humans and other animals are characterized by a large degree of unexplained variation across individual hosts. The processes that drive such inter-individual variation are not well understood. To address this, we surveyed the microbial communities associated with the intestine of the zebrafish, Danio rerio, over developmental time. We compared our observations of community composition and distribution across hosts with that predicted by a neutral assembly model, which assumes that community assembly is driven solely by chance and dispersal. We found that as hosts develop from larvae to adults, the fit of the model to observed microbial distributions decreases, suggesting that the relative importance of non-neutral processes, such as microbe-microbe interactions, active dispersal, or selection by the host, increases as hosts mature. We also observed that taxa which depart in their distributions from the neutral prediction form ecologically distinct sub-groups, which are phylogenetically clustered with respect to the full metacommunity. These results demonstrate that neutral processes are sufficient to generate substantial variation in microbiota composition across individual hosts, and suggest that potentially unique or important taxa may be identified by their divergence from neutral distributions.

Authors
Burns, AR; Stephens, WZ; Stagaman, K; Wong, S; Rawls, JF; Guillemin, K; Bohannan, BJ
MLA Citation
Burns, AR, Stephens, WZ, Stagaman, K, Wong, S, Rawls, JF, Guillemin, K, and Bohannan, BJ. "Contribution of neutral processes to the assembly of gut microbial communities in the zebrafish over host development." The ISME Journal 10.3 (March 2016): 655-664.
PMID
26296066
Source
epmc
Published In
The ISME Journal
Volume
10
Issue
3
Publish Date
2016
Start Page
655
End Page
664
DOI
10.1038/ismej.2015.142

The composition of the zebrafish intestinal microbial community varies across development.

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.

Authors
Stephens, WZ; Burns, AR; Stagaman, K; Wong, S; Rawls, JF; Guillemin, K; Bohannan, BJM
MLA Citation
Stephens, WZ, Burns, AR, Stagaman, K, Wong, S, Rawls, JF, Guillemin, K, and Bohannan, BJM. "The composition of the zebrafish intestinal microbial community varies across development." The ISME journal 10.3 (March 2016): 644-654.
PMID
26339860
Source
epmc
Published In
The ISME Journal
Volume
10
Issue
3
Publish Date
2016
Start Page
644
End Page
654
DOI
10.1038/ismej.2015.140

The severity of nonalcoholic fatty liver disease is associated with gut dysbiosis and shift in the metabolic function of the gut microbiota.

Several animal studies have emphasized the role of gut microbiota in nonalcoholic fatty liver disease (NAFLD). However, data about gut dysbiosis in human NAFLD remain scarce in the literature, especially studies including the whole spectrum of NAFLD lesions. We aimed to evaluate the association between gut dysbiosis and severe NAFLD lesions, that is, nonalcoholic steatohepatitis (NASH) and fibrosis, in a well-characterized population of adult NAFLD. Fifty-seven patients with biopsy-proven NAFLD were enrolled. Taxonomic composition of gut microbiota was determined using 16S ribosomal RNA gene sequencing of stool samples. Thirty patients had F0/F1 fibrosis stage at liver biopsy (10 with NASH), and 27 patients had significant F≥2 fibrosis (25 with NASH). Bacteroides abundance was significantly increased in NASH and F≥2 patients, whereas Prevotella abundance was decreased. Ruminococcus abundance was significantly higher in F≥2 patients. By multivariate analysis, Bacteroides abundance was independently associated with NASH and Ruminococcus with F≥2 fibrosis. Stratification according to the abundance of these two bacteria generated three patient subgroups with increasing severity of NAFLD lesions. Based on imputed metagenomic profiles, Kyoto Encyclopedia of Genes and Genomes pathways significantly related to NASH and fibrosis F≥2 were mostly related to carbohydrate, lipid, and amino acid metabolism.NAFLD severity associates with gut dysbiosis and a shift in metabolic function of the gut microbiota. We identified Bacteroides as independently associated with NASH and Ruminococcus with significant fibrosis. Thus, gut microbiota analysis adds information to classical predictors of NAFLD severity and suggests novel metabolic targets for pre-/probiotics therapies.

Authors
Boursier, J; Mueller, O; Barret, M; Machado, M; Fizanne, L; Araujo-Perez, F; Guy, CD; Seed, PC; Rawls, JF; David, LA; Hunault, G; Oberti, F; Calès, P; Diehl, AM
MLA Citation
Boursier, J, Mueller, O, Barret, M, Machado, M, Fizanne, L, Araujo-Perez, F, Guy, CD, Seed, PC, Rawls, JF, David, LA, Hunault, G, Oberti, F, Calès, P, and Diehl, AM. "The severity of nonalcoholic fatty liver disease is associated with gut dysbiosis and shift in the metabolic function of the gut microbiota." Hepatology (Baltimore, Md.) 63.3 (March 2016): 764-775.
PMID
26600078
Source
epmc
Published In
Hepatology
Volume
63
Issue
3
Publish Date
2016
Start Page
764
End Page
775
DOI
10.1002/hep.28356

Got worms? Perinatal exposure to helminths prevents persistent immune sensitization and cognitive dysfunction induced by early-life infection.

The incidence of autoimmune and inflammatory diseases has risen dramatically in post-industrial societies. "Biome depletion" - loss of commensal microbial and multicellular organisms such as helminths (intestinal worms) that profoundly modulate the immune system - may contribute to these increases. Hyperimmune-associated disorders also affect the brain, especially neurodevelopment, and increasing evidence links early-life infection to cognitive and neurodevelopmental disorders. We have demonstrated previously that rats infected with bacteria as newborns display life-long vulnerabilities to cognitive dysfunction, a vulnerability that is specifically linked to long-term hypersensitivity of microglial cell function, the resident immune cells of the brain. Here, we demonstrate that helminth colonization of pregnant dams attenuated the exaggerated brain cytokine response of their offspring to bacterial infection, and that combined with post-weaning colonization of offspring with helminths (consistent with their mothers treatment) completely prevented enduring microglial sensitization and cognitive dysfunction in adulthood. Importantly, helminths had no overt impact on adaptive immune cell subsets, whereas exaggerated innate inflammatory responses in splenic macrophages were prevented. Finally, helminths altered the effect of neonatal infection on the gut microbiome; neonatal infection with Escherichia coli caused a shift from genera within the Actinobacteria and Tenericutes phyla to genera in the Bacteroidetes phylum in rats not colonized with helminths, but helminths attenuated this effect. In sum, these data point toward an inter-relatedness of various components of the biome, and suggest potential mechanisms by which this helminth might exert therapeutic benefits in the treatment of neuroinflammatory and cognitive disorders.

Authors
Williamson, LL; McKenney, EA; Holzknecht, ZE; Belliveau, C; Rawls, JF; Poulton, S; Parker, W; Bilbo, SD
MLA Citation
Williamson, LL, McKenney, EA, Holzknecht, ZE, Belliveau, C, Rawls, JF, Poulton, S, Parker, W, and Bilbo, SD. "Got worms? Perinatal exposure to helminths prevents persistent immune sensitization and cognitive dysfunction induced by early-life infection." Brain, behavior, and immunity 51 (January 2016): 14-28.
PMID
26162711
Source
epmc
Published In
Brain, Behavior, and Immunity
Volume
51
Publish Date
2016
Start Page
14
End Page
28
DOI
10.1016/j.bbi.2015.07.006

Baby, It's Cold Outside: Host-Microbiota Relationships Drive Temperature Adaptations.

When exposed to cold temperatures, mammals undergo remarkable physiological adaptations including thermogenesis, increased intake of dietary energy, and enhanced capacity for intestinal absorption. In a recent Cell paper, Chevalier, Stojanović, and colleagues reveal that these key adaptations to life in the cold are facilitated by the intestinal microbiota (Chevalier et al., 2015).

Authors
Gomez de la Torre Canny, S; Rawls, JF
MLA Citation
Gomez de la Torre Canny, S, and Rawls, JF. "Baby, It's Cold Outside: Host-Microbiota Relationships Drive Temperature Adaptations." Cell host & microbe 18.6 (December 2015): 635-636.
PMID
26651935
Source
epmc
Published In
Cell Host & Microbe
Volume
18
Issue
6
Publish Date
2015
Start Page
635
End Page
636
DOI
10.1016/j.chom.2015.11.009

Increasing severity of NAFLD is associated with gut dysbiosis and modification of the metabolic function of the gut microbiota

Authors
Boursier, J; Mueller, O; Barret, M; Machado, MV; Fizanne, L; Guy, CD; Rawls, JF; David, L; Hunault, G; Oberti, F; Cales, P; Diehl, AM
MLA Citation
Boursier, J, Mueller, O, Barret, M, Machado, MV, Fizanne, L, Guy, CD, Rawls, JF, David, L, Hunault, G, Oberti, F, Cales, P, and Diehl, AM. "Increasing severity of NAFLD is associated with gut dysbiosis and modification of the metabolic function of the gut microbiota." October 2015.
Source
wos-lite
Published In
Hepatology
Volume
62
Publish Date
2015
Start Page
1274A
End Page
1274A

Ontogenetic Differences in Dietary Fat Influence Microbiota Assembly in the Zebrafish Gut.

Gut microbiota influence the development and physiology of their animal hosts, and these effects are determined in part by the composition of these microbial communities. Gut microbiota composition can be affected by introduction of microbes from the environment, changes in the gut habitat during development, and acute dietary alterations. However, little is known about the relationship between gut and environmental microbiotas or about how host development and dietary differences during development impact the assembly of gut microbiota. We sought to explore these relationships using zebrafish, an ideal model because they are constantly immersed in a defined environment and can be fed the same diet for their entire lives. We conducted a cross-sectional study in zebrafish raised on a high-fat, control, or low-fat diet and used bacterial 16S rRNA gene sequencing to survey microbial communities in the gut and external environment at different developmental ages. Gut and environmental microbiota compositions rapidly diverged following the initiation of feeding and became increasingly different as zebrafish grew under conditions of a constant diet. Different dietary fat levels were associated with distinct gut microbiota compositions at different ages. In addition to alterations in individual bacterial taxa, we identified putative assemblages of bacterial lineages that covaried in abundance as a function of age, diet, and location. These results reveal dynamic relationships between dietary fat levels and the microbial communities residing in the intestine and the surrounding environment during ontogenesis.The ability of gut microbiota to influence host health is determined in part by their composition. However, little is known about the relationship between gut and environmental microbiotas or about how ontogenetic differences in dietary fat impact gut microbiota composition. We addressed these gaps in knowledge using zebrafish, an ideal model organism because their environment can be thoroughly sampled and they can be fed the same diet for their entire lives. We found that microbial communities in the gut changed as zebrafish aged under conditions of a constant diet and became increasingly different from microbial communities in their surrounding environment. Further, we observed that the amount of fat in the diet had distinct age-specific effects on gut community assembly. These results reveal the complex relationships between microbial communities residing in the intestine and those in the surrounding environment and show that these relationships are shaped by dietary fat throughout the life of animal hosts.

Authors
Wong, S; Stephens, WZ; Burns, AR; Stagaman, K; David, LA; Bohannan, BJM; Guillemin, K; Rawls, JF
MLA Citation
Wong, S, Stephens, WZ, Burns, AR, Stagaman, K, David, LA, Bohannan, BJM, Guillemin, K, and Rawls, JF. "Ontogenetic Differences in Dietary Fat Influence Microbiota Assembly in the Zebrafish Gut." mBio 6.5 (September 29, 2015): e00687-e00615.
PMID
26419876
Source
epmc
Published In
mBio
Volume
6
Issue
5
Publish Date
2015
Start Page
e00687
End Page
e00615
DOI
10.1128/mbio.00687-15

CPAG: software for leveraging pleiotropy in GWAS to reveal similarity between human traits links plasma fatty acids and intestinal inflammation.

Meta-analyses of genome-wide association studies (GWAS) have demonstrated that the same genetic variants can be associated with multiple diseases and other complex traits. We present software called CPAG (Cross-Phenotype Analysis of GWAS) to look for similarities between 700 traits, build trees with informative clusters, and highlight underlying pathways. Clusters are consistent with pre-defined groups and literature-based validation but also reveal novel connections. We report similarity between plasma palmitoleic acid and Crohn's disease and find that specific fatty acids exacerbate enterocolitis in zebrafish. CPAG will become increasingly powerful as more genetic variants are uncovered, leading to a deeper understanding of complex traits. CPAG is freely available at www.sourceforge.net/projects/CPAG/.

Authors
Wang, L; Oehlers, SH; Espenschied, ST; Rawls, JF; Tobin, DM; Ko, DC
MLA Citation
Wang, L, Oehlers, SH, Espenschied, ST, Rawls, JF, Tobin, DM, and Ko, DC. "CPAG: software for leveraging pleiotropy in GWAS to reveal similarity between human traits links plasma fatty acids and intestinal inflammation." Genome biology 16 (September 15, 2015): 190-.
Website
http://hdl.handle.net/10161/10609
PMID
26374098
Source
epmc
Published In
Genome Biology: biology for the post-genomic era
Volume
16
Publish Date
2015
Start Page
190
DOI
10.1186/s13059-015-0722-1

Plexin D1 determines body fat distribution by regulating the type V collagen microenvironment in visceral adipose tissue.

Genome-wide association studies have implicated PLEXIN D1 (PLXND1) in body fat distribution and type 2 diabetes. However, a role for PLXND1 in regional adiposity and insulin resistance is unknown. Here we use in vivo imaging and genetic analysis in zebrafish to show that Plxnd1 regulates body fat distribution and insulin sensitivity. Plxnd1 deficiency in zebrafish induced hyperplastic morphology in visceral adipose tissue (VAT) and reduced lipid storage. In contrast, subcutaneous adipose tissue (SAT) growth and morphology were unaffected, resulting in altered body fat distribution and a reduced VAT:SAT ratio in zebrafish. A VAT-specific role for Plxnd1 appeared conserved in humans, as PLXND1 mRNA was positively associated with hypertrophic morphology in VAT, but not SAT. In zebrafish plxnd1 mutants, the effect on VAT morphology and body fat distribution was dependent on induction of the extracellular matrix protein collagen type V alpha 1 (col5a1). Furthermore, after high-fat feeding, zebrafish plxnd1 mutant VAT was resistant to expansion, and excess lipid was disproportionately deposited in SAT, leading to an even greater exacerbation of altered body fat distribution. Plxnd1-deficient zebrafish were protected from high-fat-diet-induced insulin resistance, and human VAT PLXND1 mRNA was positively associated with type 2 diabetes, suggesting a conserved role for PLXND1 in insulin sensitivity. Together, our findings identify Plxnd1 as a novel regulator of VAT growth, body fat distribution, and insulin sensitivity in both zebrafish and humans.

Authors
Minchin, JEN; Dahlman, I; Harvey, CJ; Mejhert, N; Singh, MK; Epstein, JA; Arner, P; Torres-Vázquez, J; Rawls, JF
MLA Citation
Minchin, JEN, Dahlman, I, Harvey, CJ, Mejhert, N, Singh, MK, Epstein, JA, Arner, P, Torres-Vázquez, J, and Rawls, JF. "Plexin D1 determines body fat distribution by regulating the type V collagen microenvironment in visceral adipose tissue." Proceedings of the National Academy of Sciences of the United States of America 112.14 (April 2015): 4363-4368.
PMID
25831505
Source
epmc
Published In
Proceedings of the National Academy of Sciences of USA
Volume
112
Issue
14
Publish Date
2015
Start Page
4363
End Page
4368
DOI
10.1073/pnas.1416412112

Epigenetic control of intestinal barrier function and inflammation in zebrafish.

The intestinal epithelium forms a barrier protecting the organism from microbes and other proinflammatory stimuli. The integrity of this barrier and the proper response to infection requires precise regulation of powerful immune homing signals such as tumor necrosis factor (TNF). Dysregulation of TNF leads to inflammatory bowel diseases (IBD), but the mechanism controlling the expression of this potent cytokine and the events that trigger the onset of chronic inflammation are unknown. Here, we show that loss of function of the epigenetic regulator ubiquitin-like protein containing PHD and RING finger domains 1 (uhrf1) in zebrafish leads to a reduction in tnfa promoter methylation and the induction of tnfa expression in intestinal epithelial cells (IECs). The increase in IEC tnfa levels is microbe-dependent and results in IEC shedding and apoptosis, immune cell recruitment, and barrier dysfunction, consistent with chronic inflammation. Importantly, tnfa knockdown in uhrf1 mutants restores IEC morphology, reduces cell shedding, and improves barrier function. We propose that loss of epigenetic repression and TNF induction in the intestinal epithelium can lead to IBD onset.

Authors
Marjoram, L; Alvers, A; Deerhake, ME; Bagwell, J; Mankiewicz, J; Cocchiaro, JL; Beerman, RW; Willer, J; Sumigray, KD; Katsanis, N; Tobin, DM; Rawls, JF; Goll, MG; Bagnat, M
MLA Citation
Marjoram, L, Alvers, A, Deerhake, ME, Bagwell, J, Mankiewicz, J, Cocchiaro, JL, Beerman, RW, Willer, J, Sumigray, KD, Katsanis, N, Tobin, DM, Rawls, JF, Goll, MG, and Bagnat, M. "Epigenetic control of intestinal barrier function and inflammation in zebrafish." Proceedings of the National Academy of Sciences of the United States of America 112.9 (March 2015): 2770-2775.
PMID
25730872
Source
epmc
Published In
Proceedings of the National Academy of Sciences of USA
Volume
112
Issue
9
Publish Date
2015
Start Page
2770
End Page
2775
DOI
10.1073/pnas.1424089112

Alteration of the rat cecal microbiome during colonization with the helminth Hymenolepis diminuta.

The microbiome is now widely recognized as being important in health and disease, and makes up a substantial subset of the biome within the ecosystem of the vertebrate body. At the same time, multicellular, eukaryotic organisms such as helminths are being recognized as an important component of the biome that shaped the evolution of our genes. The absence of these macroscopic organisms during the early development and life of humans in Western culture probably leads to a wide range of human immunological diseases. However, the interaction between the microbiome and macroscopic components of the biome remains poorly characterized. In this study, the microbiome of the cecum in rats colonized for 2 generations with the small intestinal helminth Hymenolepis diminuta was evaluated. The introduction of this benign helminth, which is of considerable therapeutic interest, led to several changes in the cecal microbiome. Most of the changes were within the Firmicutes phylum, involved about 20% of the total bacteria, and generally entailed a shift from Bacilli to Clostridia species in the presence of the helminth. The results point toward ecological relationships between various components of the biome, with the observed shifts in the microbiome suggesting potential mechanisms by which this helminth might exert therapeutic effects.

Authors
McKenney, EA; Williamson, L; Yoder, AD; Rawls, JF; Bilbo, SD; Parker, W
MLA Citation
McKenney, EA, Williamson, L, Yoder, AD, Rawls, JF, Bilbo, SD, and Parker, W. "Alteration of the rat cecal microbiome during colonization with the helminth Hymenolepis diminuta." Gut microbes 6.3 (January 2015): 182-193.
PMID
25942385
Source
epmc
Published In
Gut Microbes
Volume
6
Issue
3
Publish Date
2015
Start Page
182
End Page
193
DOI
10.1080/19490976.2015.1047128

Microbiota modulate transcription in the intestinal epithelium without remodeling the accessible chromatin landscape.

Microbiota regulate intestinal physiology by modifying host gene expression along the length of the intestine, but the underlying regulatory mechanisms remain unresolved. Transcriptional specificity occurs through interactions between transcription factors (TFs) and cis-regulatory regions (CRRs) characterized by nucleosome-depleted accessible chromatin. We profiled transcriptome and accessible chromatin landscapes in intestinal epithelial cells (IECs) from mice reared in the presence or absence of microbiota. We show that regional differences in gene transcription along the intestinal tract were accompanied by major alterations in chromatin accessibility. Surprisingly, we discovered that microbiota modify host gene transcription in IECs without significantly impacting the accessible chromatin landscape. Instead, microbiota regulation of host gene transcription might be achieved by differential expression of specific TFs and enrichment of their binding sites in nucleosome-depleted CRRs near target genes. Our results suggest that the chromatin landscape in IECs is preprogrammed by the host in a region-specific manner to permit responses to microbiota through binding of open CRRs by specific TFs.

Authors
Camp, JG; Frank, CL; Lickwar, CR; Guturu, H; Rube, T; Wenger, AM; Chen, J; Bejerano, G; Crawford, GE; Rawls, JF
MLA Citation
Camp, JG, Frank, CL, Lickwar, CR, Guturu, H, Rube, T, Wenger, AM, Chen, J, Bejerano, G, Crawford, GE, and Rawls, JF. "Microbiota modulate transcription in the intestinal epithelium without remodeling the accessible chromatin landscape." Genome research 24.9 (September 2014): 1504-1516.
PMID
24963153
Source
epmc
Published In
Genome research
Volume
24
Issue
9
Publish Date
2014
Start Page
1504
End Page
1516
DOI
10.1101/gr.165845.113

Commensal microbiota stimulate systemic neutrophil migration through induction of serum amyloid A.

Neutrophils serve critical roles in inflammatory responses to infection and injury, and mechanisms governing their activity represent attractive targets for controlling inflammation. The commensal microbiota is known to regulate the activity of neutrophils and other leucocytes in the intestine, but the systemic impact of the microbiota on neutrophils remains unknown. Here we utilized in vivo imaging in gnotobiotic zebrafish to reveal diverse effects of microbiota colonization on systemic neutrophil development and function. The presence of a microbiota resulted in increased neutrophil number and myeloperoxidase expression, and altered neutrophil localization and migratory behaviours. These effects of the microbiota on neutrophil homeostasis were accompanied by an increased recruitment of neutrophils to injury. Genetic analysis identified the microbiota-induced acute phase protein serum amyloid A (Saa) as a host factor mediating microbial stimulation of tissue-specific neutrophil migratory behaviours. In vitro studies revealed that zebrafish cells respond to Saa exposure by activating NF-κB, and that Saa-dependent neutrophil migration requires NF-κB-dependent gene expression. These results implicate the commensal microbiota as an important environmental factor regulating diverse aspects of systemic neutrophil development and function, and reveal a critical role for a Saa-NF-κB signalling axis in mediating neutrophil migratory responses.

Authors
Kanther, M; Tomkovich, S; Xiaolun, S; Grosser, MR; Koo, J; Flynn, EJ; Jobin, C; Rawls, JF
MLA Citation
Kanther, M, Tomkovich, S, Xiaolun, S, Grosser, MR, Koo, J, Flynn, EJ, Jobin, C, and Rawls, JF. "Commensal microbiota stimulate systemic neutrophil migration through induction of serum amyloid A." Cellular microbiology 16.7 (July 2014): 1053-1067.
PMID
24373309
Source
epmc
Published In
Cellular Microbiology
Volume
16
Issue
7
Publish Date
2014
Start Page
1053
End Page
1067
DOI
10.1111/cmi.12257

Commensal microbiota stimulate systemic neutrophil migration through induction of Serum amyloid A

Summary: Neutrophils serve critical roles in inflammatory responses to infection and injury, and mechanisms governing their activity represent attractive targets for controlling inflammation. The commensal microbiota is known to regulate the activity of neutrophils and other leucocytes in the intestine, but the systemic impact of the microbiota on neutrophils remains unknown. Here we utilized in vivo imaging in gnotobiotic zebrafish to reveal diverse effects of microbiota colonization on systemic neutrophil development and function. The presence of a microbiota resulted in increased neutrophil number and myeloperoxidase expression, and altered neutrophil localization and migratory behaviours. These effects of the microbiota on neutrophil homeostasis were accompanied by an increased recruitment of neutrophils to injury. Genetic analysis identified the microbiota-induced acute phase protein serum amyloid A (Saa) as a host factor mediating microbial stimulation of tissue-specific neutrophil migratory behaviours. In vitro studies revealed that zebrafish cells respond to Saa exposure by activating NF-κB, and that Saa-dependent neutrophil migration requires NF-κB-dependent gene expression. These results implicate the commensal microbiota as an important environmental factor regulating diverse aspects of systemic neutrophil development and function, and reveal a critical role for a Saa-NF-κB signalling axis in mediating neutrophil migratory responses. © 2013 John Wiley & Sons Ltd.

Authors
Kanther, M; Tomkovich, S; Xiaolun, S; Grosser, MR; Koo, J; Flynn, EJ; Jobin, C; Rawls, JF
MLA Citation
Kanther, M, Tomkovich, S, Xiaolun, S, Grosser, MR, Koo, J, Flynn, EJ, Jobin, C, and Rawls, JF. "Commensal microbiota stimulate systemic neutrophil migration through induction of Serum amyloid A." Cellular Microbiology 16.7 (January 1, 2014): 1053-1067.
Source
scopus
Published In
Cellular Microbiology
Volume
16
Issue
7
Publish Date
2014
Start Page
1053
End Page
1067
DOI
10.1111/cmi.12257

Mucosal candidiasis elicits NF-κB activation, proinflammatory gene expression and localized neutrophilia in zebrafish.

The epithelium performs a balancing act at the interface between an animal and its environment to enable both pathogen killing and tolerance of commensal microorganisms. Candida albicans is a clinically important human commensal that colonizes all human mucosal surfaces, yet is largely prevented from causing mucosal infections in immunocompetent individuals. Despite the importance of understanding host-pathogen interactions at the epithelium, no immunocompetent vertebrate model has been used to visualize these dynamics non-invasively. Here we demonstrate important similarities between swimbladder candidiasis in the transparent zebrafish and mucosal infection at the mammalian epithelium. Specifically, in the zebrafish swimmbladder infection model, we show dimorphic fungal growth, both localized and tissue-wide epithelial NF-κB activation, induction of NF-κB -dependent proinflammatory genes, and strong neutrophilia. Consistent with density-dependence models of host response based primarily on tissue culture experiments, we show that only high-level infection provokes widespread activation of NF-κB in epithelial cells and induction of proinflammatory genes. Similar to what has been found using in vitro mammalian models, we find that epithelial NF-κB activation can occur at a distance from the immediate site of contact with epithelial cells. Taking advantage of the ability to non-invasively image infection and host signaling at high resolution, we also report that epithelial NF-κB activation is diminished when phagocytes control the infection. This is the first system to model host response to mucosal infection in the juvenile zebrafish, and offers unique opportunities to investigate the tripartite interactions of C. albicans, epithelium and immune cells in an intact host.

Authors
Gratacap, RL; Rawls, JF; Wheeler, RT
MLA Citation
Gratacap, RL, Rawls, JF, and Wheeler, RT. "Mucosal candidiasis elicits NF-κB activation, proinflammatory gene expression and localized neutrophilia in zebrafish." Disease models & mechanisms 6.5 (September 2013): 1260-1270.
PMID
23720235
Source
epmc
Published In
Disease models & mechanisms
Volume
6
Issue
5
Publish Date
2013
Start Page
1260
End Page
1270
DOI
10.1242/dmm.012039

Aquacultured rainbow trout (Oncorhynchus mykiss) possess a large core intestinal microbiota that is resistant to variation in diet and rearing density.

As global aquaculture fish production continues to expand, an improved understanding of how environmental factors interact in fish health and production is needed. Significant advances have been made toward economical alternatives to costly fishmeal-based diets, such as grain-based formulations, and toward defining the effect of rearing density on fish health and production. Little research, however, has examined the effects of fishmeal- and grain-based diets in combination with alterations in rearing density. Moreover, it is unknown whether interactions between rearing density and diet impact the composition of the fish intestinal microbiota, which might in turn impact fish health and production. We fed aquacultured adult rainbow trout (Oncorhynchus mykiss) fishmeal- or grain-based diets, reared them under high- or low-density conditions for 10 months in a single aquaculture facility, and evaluated individual fish growth, production, fin indices, and intestinal microbiota composition using 16S rRNA gene sequencing. We found that the intestinal microbiotas were dominated by a shared core microbiota consisting of 52 bacterial lineages observed across all individuals, diets, and rearing densities. Variations in diet and rearing density resulted in only minor changes in intestinal microbiota composition despite significant effects of these variables on fish growth, performance, fillet quality, and welfare. Significant interactions between diet and rearing density were observed only in evaluations of fin indices and the relative abundance of the bacterial genus Staphylococcus. These results demonstrate that aquacultured rainbow trout can achieve remarkable consistency in intestinal microbiota composition and suggest the possibility of developing novel aquaculture strategies without overtly altering intestinal microbiota composition.

Authors
Wong, S; Waldrop, T; Summerfelt, S; Davidson, J; Barrows, F; Kenney, PB; Welch, T; Wiens, GD; Snekvik, K; Rawls, JF; Good, C
MLA Citation
Wong, S, Waldrop, T, Summerfelt, S, Davidson, J, Barrows, F, Kenney, PB, Welch, T, Wiens, GD, Snekvik, K, Rawls, JF, and Good, C. "Aquacultured rainbow trout (Oncorhynchus mykiss) possess a large core intestinal microbiota that is resistant to variation in diet and rearing density." Appl Environ Microbiol 79.16 (August 2013): 4974-4984.
PMID
23770898
Source
pubmed
Published In
Applied and environmental microbiology
Volume
79
Issue
16
Publish Date
2013
Start Page
4974
End Page
4984
DOI
10.1128/AEM.00924-13

Oesophageal and sternohyal muscle fibres are novel Pax3-dependent migratory somite derivatives essential for ingestion.

Striated muscles that enable mouth opening and swallowing during feeding are essential for efficient energy acquisition, and are likely to have played a fundamental role in the success of early jawed vertebrates. The developmental origins and genetic requirements of these muscles are uncertain. Here, we determine by indelible lineage tracing in mouse that fibres of sternohyoid muscle (SHM), which is essential for mouth opening during feeding, and oesophageal striated muscle (OSM), which is crucial for voluntary swallowing, arise from Pax3-expressing somite cells. In vivo Kaede lineage tracing in zebrafish reveals the migratory route of cells from the anteriormost somites to OSM and SHM destinations. Expression of pax3b, a zebrafish duplicate of Pax3, is restricted to the hypaxial region of anterior somites that generate migratory muscle precursors (MMPs), suggesting that Pax3b plays a role in generating OSM and SHM. Indeed, loss of pax3b function led to defective MMP migration and OSM formation, disorganised SHM differentiation, and inefficient ingestion and swallowing of microspheres. Together, our data demonstrate Pax3-expressing somite cells as a source of OSM and SHM fibres, and highlight a conserved role of Pax3 genes in the genesis of these feeding muscles of vertebrates.

Authors
Minchin, JEN; Williams, VC; Hinits, Y; Low, S; Tandon, P; Fan, C-M; Rawls, JF; Hughes, SM
MLA Citation
Minchin, JEN, Williams, VC, Hinits, Y, Low, S, Tandon, P, Fan, C-M, Rawls, JF, and Hughes, SM. "Oesophageal and sternohyal muscle fibres are novel Pax3-dependent migratory somite derivatives essential for ingestion." Development (Cambridge, England) 140.14 (July 2013): 2972-2984.
PMID
23760954
Source
epmc
Published In
Development (Cambridge)
Volume
140
Issue
14
Publish Date
2013
Start Page
2972
End Page
2984
DOI
10.1242/dev.090050

Getting the Inside Tract: New Frontiers in Zebrafish Digestive System Biology

Authors
Sadler, KC; Rawls, JF; Farber, SA
MLA Citation
Sadler, KC, Rawls, JF, and Farber, SA. "Getting the Inside Tract: New Frontiers in Zebrafish Digestive System Biology." ZEBRAFISH 10.2 (June 2013): 129-131.
PMID
23738756
Source
wos-lite
Published In
Zebrafish
Volume
10
Issue
2
Publish Date
2013
Start Page
129
End Page
131
DOI
10.1089/zeb.2013.1500

Carbon monoxide and heme oxygenase-1 prevent intestinal inflammation in mice by promoting bacterial clearance.

Heme oxygenase-1 (HO-1) and its metabolic by-product, carbon monoxide (CO), protect against intestinal inflammation in experimental models of colitis, but little is known about their intestinal immune mechanisms. We investigated the interactions among CO, HO-1, and the enteric microbiota in mice and zebrafish.Germ-free, wild-type, and interleukin (Il)10(-/-) mice and germ-free zebrafish embryos were colonized with specific pathogen-free (SPF) microbiota. Germ-free or SPF-raised wild-type and Il10(-/-) mice were given intraperitoneal injections of cobalt(III) protoporphyrin IX chloride (CoPP), which up-regulates HO-1, the CO-releasing molecule Alfama-186, or saline (control). Colitis was induced in wild-type mice housed in SPF conditions by infection with Salmonella typhimurium.In colons of germ-free, wild-type mice, SPF microbiota induced production of HO-1 via activation of nuclear factor erythroid 2-related factor 2-, IL-10-, and Toll-like receptor-dependent pathways; similar observations were made in zebrafish. SPF microbiota did not induce HO-1 in colons of germ-free Il10(-/-) mice. Administration of CoPP to Il10(-/-) mice before transition from germ-free to SPF conditions reduced their development of colitis. In Il10(-/-) mice, CO and CoPP reduced levels of enteric bacterial genomic DNA in mesenteric lymph nodes. In mice with S typhimurium-induced enterocolitis, CoPP reduced the numbers of live S typhimurium recovered from the lamina propria, mesenteric lymph nodes, spleen, and liver. Knockdown of HO-1 in mouse macrophages impaired their bactericidal activity against E coli, E faecalis, and S typhimurium, whereas exposure to CO or overexpression of HO-1 increased their bactericidal activity. HO-1 induction and CO increased acidification of phagolysosomes.Colonic HO-1 prevents colonic inflammation in mice. HO-1 is induced by the enteric microbiota and its homeostatic function is mediated, in part, by promoting bactericidal activities of macrophages.

Authors
Onyiah, JC; Sheikh, SZ; Maharshak, N; Steinbach, EC; Russo, SM; Kobayashi, T; Mackey, LC; Hansen, JJ; Moeser, AJ; Rawls, JF; Borst, LB; Otterbein, LE; Plevy, SE
MLA Citation
Onyiah, JC, Sheikh, SZ, Maharshak, N, Steinbach, EC, Russo, SM, Kobayashi, T, Mackey, LC, Hansen, JJ, Moeser, AJ, Rawls, JF, Borst, LB, Otterbein, LE, and Plevy, SE. "Carbon monoxide and heme oxygenase-1 prevent intestinal inflammation in mice by promoting bacterial clearance." Gastroenterology 144.4 (April 2013): 789-798.
PMID
23266559
Source
epmc
Published In
Gastroenterology
Volume
144
Issue
4
Publish Date
2013
Start Page
789
End Page
798
DOI
10.1053/j.gastro.2012.12.025

Dwarfism and increased adiposity in the gh1 mutant zebrafish vizzini.

Somatic growth and adipogenesis are closely associated with the development of obesity in humans. In this study, we identify a zebrafish mutant, vizzini, that exhibits both a severe defect in somatic growth and increased accumulation of adipose tissue. Positional cloning of vizzini revealed a premature stop codon in gh1. Although the effects of GH are largely through igfs in mammals, we found no decrease in the expression of igf transcripts in gh1 mutants during larval development. As development progressed, however, we found overall growth to be progressively retarded and the attainment of specific developmental stages to occur at abnormally small body sizes relative to wild type. Moreover, both subcutaneous (sc) and visceral adipose tissues underwent precocious development in vizzini mutants, and at maturity, the sizes of different fat deposits were greatly expanded relative to wild type. In vivo confocal imaging of sc adipose tissue (SAT) expansion revealed that vizzini mutants exhibit extreme enlargement of adipocyte lipid droplets without a corresponding increase in lipid droplet number. These findings suggest that GH1 signaling restricts SAT hypertrophy in zebrafish. Finally, nutrient deprivation of vizzini mutants revealed that SAT mobilization was greatly diminished during caloric restriction, further implicating GH1 signaling in adipose tissue homeostasis. Overall, the zebrafish gh1 mutant, vizzini, exhibits decreased somatic growth, increased adipose tissue accumulation, and disrupted adipose plasticity after nutrient deprivation and represents a novel model to investigate the in vivo dynamics of vertebrate obesity.

Authors
McMenamin, SK; Minchin, JEN; Gordon, TN; Rawls, JF; Parichy, DM
MLA Citation
McMenamin, SK, Minchin, JEN, Gordon, TN, Rawls, JF, and Parichy, DM. "Dwarfism and increased adiposity in the gh1 mutant zebrafish vizzini." Endocrinology 154.4 (April 2013): 1476-1487.
PMID
23456361
Source
epmc
Published In
Endocrinology
Volume
154
Issue
4
Publish Date
2013
Start Page
1476
End Page
1487
DOI
10.1210/en.2012-1734

Zebrafish as a model to analyze macromolecule absorption in intestinal enterocytes

Authors
Cocchiaro, JL; Navis, A; Bagnat, M; Rawls, JF
MLA Citation
Cocchiaro, JL, Navis, A, Bagnat, M, and Rawls, JF. "Zebrafish as a model to analyze macromolecule absorption in intestinal enterocytes." April 2013.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
27
Publish Date
2013

Animals in a bacterial world, a new imperative for the life sciences.

In the last two decades, the widespread application of genetic and genomic approaches has revealed a bacterial world astonishing in its ubiquity and diversity. This review examines how a growing knowledge of the vast range of animal-bacterial interactions, whether in shared ecosystems or intimate symbioses, is fundamentally altering our understanding of animal biology. Specifically, we highlight recent technological and intellectual advances that have changed our thinking about five questions: how have bacteria facilitated the origin and evolution of animals; how do animals and bacteria affect each other's genomes; how does normal animal development depend on bacterial partners; how is homeostasis maintained between animals and their symbionts; and how can ecological approaches deepen our understanding of the multiple levels of animal-bacterial interaction. As answers to these fundamental questions emerge, all biologists will be challenged to broaden their appreciation of these interactions and to include investigations of the relationships between and among bacteria and their animal partners as we seek a better understanding of the natural world.

Authors
McFall-Ngai, M; Hadfield, MG; Bosch, TCG; Carey, HV; Domazet-Lošo, T; Douglas, AE; Dubilier, N; Eberl, G; Fukami, T; Gilbert, SF; Hentschel, U; King, N; Kjelleberg, S; Knoll, AH; Kremer, N; Mazmanian, SK; Metcalf, JL; Nealson, K; Pierce, NE; Rawls, JF; Reid, A; Ruby, EG; Rumpho, M; Sanders, JG; Tautz, D; Wernegreen, JJ
MLA Citation
McFall-Ngai, M, Hadfield, MG, Bosch, TCG, Carey, HV, Domazet-Lošo, T, Douglas, AE, Dubilier, N, Eberl, G, Fukami, T, Gilbert, SF, Hentschel, U, King, N, Kjelleberg, S, Knoll, AH, Kremer, N, Mazmanian, SK, Metcalf, JL, Nealson, K, Pierce, NE, Rawls, JF, Reid, A, Ruby, EG, Rumpho, M, Sanders, JG, Tautz, D, and Wernegreen, JJ. "Animals in a bacterial world, a new imperative for the life sciences." Proc Natl Acad Sci U S A 110.9 (February 26, 2013): 3229-3236. (Review)
PMID
23391737
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
110
Issue
9
Publish Date
2013
Start Page
3229
End Page
3236
DOI
10.1073/pnas.1218525110

Microgavage of zebrafish larvae.

The zebrafish has emerged as a powerful model organism for studying intestinal development(1-5), physiology(6-11), disease(12-16), and host-microbe interactions(17-25). Experimental approaches for studying intestinal biology often require the in vivo introduction of selected materials into the lumen of the intestine. In the larval zebrafish model, this is typically accomplished by immersing fish in a solution of the selected material, or by injection through the abdominal wall. Using the immersion method, it is difficult to accurately monitor or control the route or timing of material delivery to the intestine. For this reason, immersion exposure can cause unintended toxicity and other effects on extraintestinal tissues, limiting the potential range of material amounts that can be delivered into the intestine. Also, the amount of material ingested during immersion exposure can vary significantly between individual larvae(26). Although these problems are not encountered during direct injection through the abdominal wall, proper injection is difficult and causes tissue damage which could influence experimental results. We introduce a method for microgavage of zebrafish larvae. The goal of this method is to provide a safe, effective, and consistent way to deliver material directly to the lumen of the anterior intestine in larval zebrafish with controlled timing. Microgavage utilizes standard embryo microinjection and stereomicroscopy equipment common to most laboratories that perform zebrafish research. Once fish are properly positioned in methylcellulose, gavage can be performed quickly at a rate of approximately 7-10 fish/ min, and post-gavage survival approaches 100% depending on the gavaged material. We also show that microgavage can permit loading of the intestinal lumen with high concentrations of materials that are lethal to fish when exposed by immersion. To demonstrate the utility of this method, we present a fluorescent dextran microgavage assay that can be used to quantify transit from the intestinal lumen to extraintestinal spaces. This test can be used to verify proper execution of the microgavage procedure, and also provides a novel zebrafish assay to examine intestinal epithelial barrier integrity under different experimental conditions (e.g. genetic manipulation, drug treatment, or exposure to environmental factors). Furthermore, we show how gavage can be used to evaluate intestinal motility by gavaging fluorescent microspheres and monitoring their subsequent transit. Microgavage can be applied to deliver diverse materials such as live microorganisms, secreted microbial factors/toxins, pharmacological agents, and physiological probes. With these capabilities, the larval zebrafish microgavage method has the potential to enhance a broad range of research fields using the zebrafish model system.

Authors
Cocchiaro, JL; Rawls, JF
MLA Citation
Cocchiaro, JL, and Rawls, JF. "Microgavage of zebrafish larvae." Journal of visualized experiments : JoVE 72 (February 20, 2013): e4434-.
PMID
23463135
Source
epmc
Published In
Journal of Visualized Experiments
Issue
72
Publish Date
2013
Start Page
e4434
DOI
10.3791/4434

Glafenine-induced intestinal injury in zebrafish is ameliorated by μ-opioid signaling via enhancement of Atf6-dependent cellular stress responses.

Beside their analgesic properties, opiates exert beneficial effects on the intestinal wound healing response. In this study, we investigated the role of μ-opioid receptor (MOR) signaling on the unfolded protein response (UPR) using a novel zebrafish model of NSAID-induced intestinal injury. The NSAID glafenine was administered to zebrafish larvae at 5 days post-fertilization (dpf) for up to 24 hours in the presence or absence of the MOR-specific agonist DALDA. By analysis with histology, transmission electron microscopy and vital dye staining, glafenine-treated zebrafish showed evidence of endoplasmic reticulum and mitochondrial stress, with disrupted intestinal architecture and halted cell stress responses, alongside accumulation of apoptotic intestinal epithelial cells in the lumen. Although the early UPR marker BiP was induced with glafenine-induced injury, downstream atf6 and s-xbp1 expression were paradoxically not increased, explaining the halted cell stress responses. The μ-opioid agonist DALDA protected against glafenine-induced injury through induction of atf6-dependent UPR. Our findings show that DALDA prevents glafenine-induced epithelial damage through induction of effective UPR.

Authors
Goldsmith, JR; Cocchiaro, JL; Rawls, JF; Jobin, C
MLA Citation
Goldsmith, JR, Cocchiaro, JL, Rawls, JF, and Jobin, C. "Glafenine-induced intestinal injury in zebrafish is ameliorated by μ-opioid signaling via enhancement of Atf6-dependent cellular stress responses." Disease models & mechanisms 6.1 (January 2013): 146-159.
PMID
22917923
Source
epmc
Published In
Disease models & mechanisms
Volume
6
Issue
1
Publish Date
2013
Start Page
146
End Page
159
DOI
10.1242/dmm.009852

Microgavage of zebrafish larvae.

The zebrafish has emerged as a powerful model organism for studying intestinal development(1-5), physiology(6-11), disease(12-16), and host-microbe interactions(17-25). Experimental approaches for studying intestinal biology often require the in vivo introduction of selected materials into the lumen of the intestine. In the larval zebrafish model, this is typically accomplished by immersing fish in a solution of the selected material, or by injection through the abdominal wall. Using the immersion method, it is difficult to accurately monitor or control the route or timing of material delivery to the intestine. For this reason, immersion exposure can cause unintended toxicity and other effects on extraintestinal tissues, limiting the potential range of material amounts that can be delivered into the intestine. Also, the amount of material ingested during immersion exposure can vary significantly between individual larvae(26). Although these problems are not encountered during direct injection through the abdominal wall, proper injection is difficult and causes tissue damage which could influence experimental results. We introduce a method for microgavage of zebrafish larvae. The goal of this method is to provide a safe, effective, and consistent way to deliver material directly to the lumen of the anterior intestine in larval zebrafish with controlled timing. Microgavage utilizes standard embryo microinjection and stereomicroscopy equipment common to most laboratories that perform zebrafish research. Once fish are properly positioned in methylcellulose, gavage can be performed quickly at a rate of approximately 7-10 fish/ min, and post-gavage survival approaches 100% depending on the gavaged material. We also show that microgavage can permit loading of the intestinal lumen with high concentrations of materials that are lethal to fish when exposed by immersion. To demonstrate the utility of this method, we present a fluorescent dextran microgavage assay that can be used to quantify transit from the intestinal lumen to extraintestinal spaces. This test can be used to verify proper execution of the microgavage procedure, and also provides a novel zebrafish assay to examine intestinal epithelial barrier integrity under different experimental conditions (e.g. genetic manipulation, drug treatment, or exposure to environmental factors). Furthermore, we show how gavage can be used to evaluate intestinal motility by gavaging fluorescent microspheres and monitoring their subsequent transit. Microgavage can be applied to deliver diverse materials such as live microorganisms, secreted microbial factors/toxins, pharmacological agents, and physiological probes. With these capabilities, the larval zebrafish microgavage method has the potential to enhance a broad range of research fields using the zebrafish model system.

Authors
Cocchiaro, JL; Rawls, JF
MLA Citation
Cocchiaro, JL, and Rawls, JF. "Microgavage of zebrafish larvae." Journal of visualized experiments : JoVE 72 (2013): e4434-.
Source
scival
Published In
Journal of Visualized Experiments
Issue
72
Publish Date
2013
Start Page
e4434
DOI
10.3791/4434

Obese Humans With Nonalcoholic Fatty Liver Disease Display Alterations in Fecal Microbiota and Volatile Organic Compounds

Authors
Boursier, J; Rawls, JF; Diehl, AM
MLA Citation
Boursier, J, Rawls, JF, and Diehl, AM. "Obese Humans With Nonalcoholic Fatty Liver Disease Display Alterations in Fecal Microbiota and Volatile Organic Compounds." Clinical Gastroenterology and Hepatology 11.7 (2013): 876-878.
PMID
23628504
Source
scival
Published In
Clinical Gastroenterology and Hepatology
Volume
11
Issue
7
Publish Date
2013
Start Page
876
End Page
878
DOI
10.1016/j.cgh.2013.04.016

Increased rectal microbial richness is associated with the presence of colorectal adenomas in humans.

Differences in the composition of the gut microbial community have been associated with diseases such as obesity, Crohn's disease, ulcerative colitis and colorectal cancer (CRC). We used 454 titanium pyrosequencing of the V1-V2 region of the 16S rRNA gene to characterize adherent bacterial communities in mucosal biopsy samples from 33 subjects with adenomas and 38 subjects without adenomas (controls). Biopsy samples from subjects with adenomas had greater numbers of bacteria from 87 taxa than controls; only 5 taxa were more abundant in control samples. The magnitude of the differences in the distal gut microbiota between patients with adenomas and controls was more pronounced than that of any other clinical parameters including obesity, diet or family history of CRC. This suggests that sequence analysis of the microbiota could be used to identify patients at risk for developing adenomas.

Authors
Sanapareddy, N; Legge, RM; Jovov, B; McCoy, A; Burcal, L; Araujo-Perez, F; Randall, TA; Galanko, J; Benson, A; Sandler, RS; Rawls, JF; Abdo, Z; Fodor, AA; Keku, TO
MLA Citation
Sanapareddy, N, Legge, RM, Jovov, B, McCoy, A, Burcal, L, Araujo-Perez, F, Randall, TA, Galanko, J, Benson, A, Sandler, RS, Rawls, JF, Abdo, Z, Fodor, AA, and Keku, TO. "Increased rectal microbial richness is associated with the presence of colorectal adenomas in humans." The ISME journal 6.10 (October 2012): 1858-1868.
PMID
22622349
Source
epmc
Published In
The ISME Journal
Volume
6
Issue
10
Publish Date
2012
Start Page
1858
End Page
1868
DOI
10.1038/ismej.2012.43

Microbiota regulate intestinal absorption and metabolism of fatty acids in the zebrafish.

Regulation of intestinal dietary fat absorption is critical to maintaining energy balance. While intestinal microbiota clearly impact the host's energy balance, their role in intestinal absorption and extraintestinal metabolism of dietary fat is less clear. Using in vivo imaging of fluorescent fatty acid (FA) analogs delivered to gnotobiotic zebrafish hosts, we reveal that microbiota stimulate FA uptake and lipid droplet (LD) formation in the intestinal epithelium and liver. Microbiota increase epithelial LD number in a diet-dependent manner. The presence of food led to the intestinal enrichment of bacteria from the phylum Firmicutes. Diet-enriched Firmicutes and their products were sufficient to increase epithelial LD number, whereas LD size was increased by other bacterial types. Thus, different members of the intestinal microbiota promote FA absorption via distinct mechanisms. Diet-induced alterations in microbiota composition might influence fat absorption, providing mechanistic insight into how microbiota-diet interactions regulate host energy balance.

Authors
Semova, I; Carten, JD; Stombaugh, J; Mackey, LC; Knight, R; Farber, SA; Rawls, JF
MLA Citation
Semova, I, Carten, JD, Stombaugh, J, Mackey, LC, Knight, R, Farber, SA, and Rawls, JF. "Microbiota regulate intestinal absorption and metabolism of fatty acids in the zebrafish." Cell host & microbe 12.3 (September 2012): 277-288.
PMID
22980325
Source
epmc
Published In
Cell Host & Microbe
Volume
12
Issue
3
Publish Date
2012
Start Page
277
End Page
288
DOI
10.1016/j.chom.2012.08.003

Intestinal microbiota composition in fishes is influenced by host ecology and environment.

The digestive tracts of vertebrates are colonized by complex assemblages of micro-organisms, collectively called the gut microbiota. Recent studies have revealed important contributions of gut microbiota to vertebrate health and disease, stimulating intense interest in understanding how gut microbial communities are assembled and how they impact host fitness (Sekirov et al. 2010). Although all vertebrates harbour a gut microbiota, current information on microbiota composition and function has been derived primarily from mammals. Comparisons of different mammalian species have revealed intriguing associations between gut microbiota composition and host diet, anatomy and phylogeny (Ley et al. 2008b). However, mammals constitute <10% of all vertebrate species, and it remains unclear whether similar associations exist in more diverse and ancient vertebrate lineages such as fish. In this issue, Sullam et al. (2012) make an important contribution toward identifying factors determining gut microbiota composition in fishes. The authors conducted a detailed meta-analysis of 25 bacterial 16S rRNA gene sequence libraries derived from the intestines of different fish species. To provide a broader context for their analysis, they compared these data sets to a large collection of 16S rRNA gene sequence data sets from diverse free-living and host-associated bacterial communities. Their results suggest that variation in gut microbiota composition in fishes is strongly correlated with species habitat salinity, trophic level and possibly taxonomy. Comparison of data sets from fish intestines and other environments revealed that fish gut microbiota compositions are often similar to those of other animals and contain relatively few free-living environmental bacteria. These results suggest that the gut microbiota composition of fishes is not a simple reflection of the micro-organisms in their local habitat but may result from host-specific selective pressures within the gut (Bevins & Salzman 2011).

Authors
Wong, S; Rawls, JF
MLA Citation
Wong, S, and Rawls, JF. "Intestinal microbiota composition in fishes is influenced by host ecology and environment." Molecular ecology 21.13 (July 2012): 3100-3102.
PMID
22916346
Source
epmc
Published In
Molecular Ecology
Volume
21
Issue
13
Publish Date
2012
Start Page
3100
End Page
3102
DOI
10.1111/j.1365-294x.2012.05646.x

Special issue: gut microbial communities in health and disease.

Authors
Rawls, JF
MLA Citation
Rawls, JF. "Special issue: gut microbial communities in health and disease." Gut microbes 3.4 (July 2012): 277-278.
PMID
22688724
Source
epmc
Published In
Gut Microbes
Volume
3
Issue
4
Publish Date
2012
Start Page
277
End Page
278
DOI
10.4161/gmic.20485

Intronic cis-regulatory modules mediate tissue-specific and microbial control of angptl4/fiaf transcription.

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 β-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.

Authors
Camp, JG; Jazwa, AL; Trent, CM; Rawls, JF
MLA Citation
Camp, JG, Jazwa, AL, Trent, CM, and Rawls, JF. "Intronic cis-regulatory modules mediate tissue-specific and microbial control of angptl4/fiaf transcription." PLoS genetics 8.3 (January 2012): e1002585-.
PMID
22479192
Source
epmc
Published In
PLoS genetics
Volume
8
Issue
3
Publish Date
2012
Start Page
e1002585
DOI
10.1371/journal.pgen.1002585

Special issue: gut microbial communities in health and disease.

Authors
Rawls, JF
MLA Citation
Rawls, JF. "Special issue: gut microbial communities in health and disease." Gut microbes 3.4 (2012): 277-278.
Source
scival
Published In
Gut Microbes
Volume
3
Issue
4
Publish Date
2012
Start Page
277
End Page
278
DOI
10.4161/gmic.20485

Evidence for a core gut microbiota in the zebrafish.

Experimental analysis of gut microbial communities and their interactions with vertebrate hosts is conducted predominantly in domesticated animals that have been maintained in laboratory facilities for many generations. These animal models are useful for studying coevolved relationships between host and microbiota only if the microbial communities that occur in animals in lab facilities are representative of those that occur in nature. We performed 16S rRNA gene sequence-based comparisons of gut bacterial communities in zebrafish collected recently from their natural habitat and those reared for generations in lab facilities in different geographic locations. Patterns of gut microbiota structure in domesticated zebrafish varied across different lab facilities in correlation with historical connections between those facilities. However, gut microbiota membership in domesticated and recently caught zebrafish was strikingly similar, with a shared core gut microbiota. The zebrafish intestinal habitat therefore selects for specific bacterial taxa despite radical differences in host provenance and domestication status.

Authors
Roeselers, G; Mittge, EK; Stephens, WZ; Parichy, DM; Cavanaugh, CM; Guillemin, K; Rawls, JF
MLA Citation
Roeselers, G, Mittge, EK, Stephens, WZ, Parichy, DM, Cavanaugh, CM, Guillemin, K, and Rawls, JF. "Evidence for a core gut microbiota in the zebrafish." The ISME journal 5.10 (October 2011): 1595-1608.
PMID
21472014
Source
epmc
Published In
The ISME Journal
Volume
5
Issue
10
Publish Date
2011
Start Page
1595
End Page
1608
DOI
10.1038/ismej.2011.38

Microbial colonization induces dynamic temporal and spatial patterns of NF-κB activation in the zebrafish digestive tract.

BACKGROUND & AIMS: The nuclear factor κ-light-chain enhancer of activated B cells (NF-κB) transcription factor pathway is activated in response to diverse microbial stimuli to regulate expression of genes involved in immune responses and tissue homeostasis. However, the temporal and spatial activation of NF-κB in response to microbial signals have not been determined in whole living organisms, and the molecular and cellular details of these responses are not well understood. We used in vivo imaging and molecular approaches to analyze NF-κB activation in response to the commensal microbiota in transparent gnotobiotic zebrafish. METHODS: We used DNA microarrays, in situ hybridization, and quantitative reverse transcription polymerase chain reaction analyses to study the effects of the commensal microbiota on gene expression in gnotobiotic zebrafish. Zebrafish PAC2 and ZFL cells were used to study the NF-κB signaling pathway in response to bacterial stimuli. We generated transgenic zebrafish that express enhanced green fluorescent protein under transcriptional control of NF-κB, and used them to study patterns of NF-κB activation during development and microbial colonization. RESULTS: Bacterial stimulation induced canonical activation of the NF-κB pathway in zebrafish cells. Colonization of germ-free transgenic zebrafish with a commensal microbiota activated NF-κB and led to up-regulation of its target genes in intestinal and extraintestinal tissues of the digestive tract. Colonization with the bacterium Pseudomonas aeruginosa was sufficient to activate NF-κB, and this activation required a functional flagellar apparatus. CONCLUSIONS: In zebrafish, transcriptional activity of NF-κB is spatially and temporally regulated by specific microbial factors. The observed patterns of NF-κB-dependent responses to microbial colonization indicate that cells in the gastrointestinal tract respond robustly to the microbial environment.

Authors
Kanther, M; Sun, X; Mühlbauer, M; Mackey, LC; Flynn, EJ; Bagnat, M; Jobin, C; Rawls, JF
MLA Citation
Kanther, M, Sun, X, Mühlbauer, M, Mackey, LC, Flynn, EJ, Bagnat, M, Jobin, C, and Rawls, JF. "Microbial colonization induces dynamic temporal and spatial patterns of NF-κB activation in the zebrafish digestive tract." Gastroenterology 141.1 (July 2011): 197-207.
PMID
21439961
Source
pubmed
Published In
Gastroenterology
Volume
141
Issue
1
Publish Date
2011
Start Page
197
End Page
207
DOI
10.1053/j.gastro.2011.03.042

In vivo analysis of white adipose tissue in zebrafish.

White adipose tissue (WAT) is the major site of energy storage in bony vertebrates, and also serves central roles in the endocrine regulation of energy balance. The cellular and molecular mechanisms underlying WAT development and physiology are not well understood. This is due in part to difficulties associated with imaging adipose tissues in mammalian model systems, especially during early life stages. The zebrafish (Danio rerio) has recently emerged as a new model system for adipose tissue research, in which WAT can be imaged in a transparent living vertebrate at all life stages. Here we present detailed methods for labeling adipocytes in live zebrafish using fluorescent lipophilic dyes, and for in vivo microscopy of zebrafish WAT.

Authors
Minchin, JEN; Rawls, JF
MLA Citation
Minchin, JEN, and Rawls, JF. "In vivo analysis of white adipose tissue in zebrafish." Methods in cell biology 105 (January 2011): 63-86.
PMID
21951526
Source
epmc
Published In
Methods in cell biology
Volume
105
Publish Date
2011
Start Page
63
End Page
86
DOI
10.1016/b978-0-12-381320-6.00003-5

Study of host-microbe interactions in zebrafish.

All animals are ecosystems, home to diverse microbial populations. Animal-associated microbes play important roles in the normal development and physiology of their hosts, but can also be agents of infectious disease. Traditionally, mice have been used to study pathogenic and beneficial associations between microbes and vertebrate animals. The zebrafish is emerging as a valuable new model system for host-microbe interaction studies, affording researchers with the opportunity to survey large populations of hosts and to visualize microbe-host associations at a cellular level in living animals. This chapter provides detailed protocols for the analysis of zebrafish-associated microbial communities, the derivation and husbandry of germ-free zebrafish, and the modeling of infectious disease in different stages of zebrafish development via different routes of inoculation. These protocols offer a starting point for researchers to address a multitude of questions about animals' coexistence with microorganisms.

Authors
Milligan-Myhre, K; Charette, JR; Phennicie, RT; Stephens, WZ; Rawls, JF; Guillemin, K; Kim, CH
MLA Citation
Milligan-Myhre, K, Charette, JR, Phennicie, RT, Stephens, WZ, Rawls, JF, Guillemin, K, and Kim, CH. "Study of host-microbe interactions in zebrafish." Methods in cell biology 105 (January 2011): 87-116.
PMID
21951527
Source
epmc
Published In
Methods in cell biology
Volume
105
Publish Date
2011
Start Page
87
End Page
116
DOI
10.1016/b978-0-12-381320-6.00004-7

Molecular characterization of mucosal adherent bacteria and associations with colorectal adenomas

The human large bowel is colonized by complex and diverse bacterial communities. However, the relationship between commensal bowel bacteria and adenomas (colorectal cancer precursors) is unclear. This study aimed to characterize adherent bacteria in normal colon and evaluate differences in community composition associated with colorectal adenomas. We evaluated adherent bacteria in normal colonic mucosa of 21 adenoma and 23 non-adenoma subjects enrolled in a cross sectional study. Terminal restriction fragment length polymorphism, clone sequencing and fluorescent in-situ hybridization analysis of the 16S rRNA genes were used to characterize adherent bacteria. A total of 335 clones were sequenced and processed for phylogenetic and taxonomic analysis. Differences in bacterial composition between cases and controls were evaluated by UniFrac and analysis of similarity matrix. Overall, Firmicutes (62%), Bacteroidetes (26%) and Proteobacteria (11%) were the most dominant phyla. The bacterial composition differed significantly between cases and controls (UniFrac p < 0.001). We observed significantly higher abundance of Proteobacteria (p < 0.05) and lower abundance of Bacteroidetes (p < 0.05) in cases compared to controls. At the genus level, case subjects showed increased abundance of Dorea spp. (p < 0.005), Faecalibacterium spp. (p < 0.05) and lower proportions of Bacteroides spp. (p < 0.03) and Coprococcus spp. (p < 0.05) than controls. Cases had higher bacterial diversity and richness than controls. These findings reveal that alterations in bacterial community composition associated with adenomas may contribute to the etiology of colorectal cancer. Extension of these findings could lead to strategies to manipulate the microbiota to prevent colorectal adenomas and cancer as well as to identify individuals at high risk. © 2010 Landes Bioscience.

Authors
Shen, XJ; Rawls, JF; Randall, T; Burcal, L; Mpande, CN; Jenkins, N; Jovov, B; Abdo, Z; sandler, RS; Keku, TO
MLA Citation
Shen, XJ, Rawls, JF, Randall, T, Burcal, L, Mpande, CN, Jenkins, N, Jovov, B, Abdo, Z, sandler, RS, and Keku, TO. "Molecular characterization of mucosal adherent bacteria and associations with colorectal adenomas." Gut Microbes 1.3 (July 27, 2010): 1-10.
Source
scopus
Published In
Gut Microbes
Volume
1
Issue
3
Publish Date
2010
Start Page
1
End Page
10

Molecular characterization of mucosal adherent bacteria and associations with colorectal adenomas.

The human large bowel is colonized by complex and diverse bacterial communities. However, the relationship between commensal bowel bacteria and adenomas (colorectal cancer precursors) is unclear. This study aimed to characterize adherent bacteria in normal colon and evaluate differences in community composition associated with colorectal adenomas. We evaluated adherent bacteria in normal colonic mucosa of 21 adenoma and 23 non-adenoma subjects enrolled in a cross sectional study. Terminal restriction fragment length polymorphism, clone sequencing and fluorescent in-situ hybridization analysis of the 16S rRNA genes were used to characterize adherent bacteria. A total of 335 clones were sequenced and processed for phylogenetic and taxonomic analysis. Differences in bacterial composition between cases and controls were evaluated by UniFrac and analysis of similarity matrix. Overall, Firmicutes (62%), Bacteroidetes (26%) and Proteobacteria (11%) were the most dominant phyla. The bacterial composition differed significantly between cases and controls (UniFrac p < 0.001). We observed significantly higher abundance of Proteobacteria (p < 0.05) and lower abundance of Bacteroidetes (p < 0.05) in cases compared to controls. At the genus level, case subjects showed increased abundance of Dorea spp. (p < 0.005), Faecalibacterium spp. (p < 0.05) and lower proportions of Bacteroides spp. (p < 0.03) and Coprococcus spp. (p < 0.05) than controls. Cases had higher bacterial diversity and richness than controls. These findings reveal that alterations in bacterial community composition associated with adenomas may contribute to the etiology of colorectal cancer. Extension of these findings could lead to strategies to manipulate the microbiota to prevent colorectal adenomas and cancer as well as to identify individuals at high risk.

Authors
Shen, XJ; Rawls, JF; Randall, T; Burcal, L; Mpande, CN; Jenkins, N; Jovov, B; Abdo, Z; Sandler, RS; Keku, TO
MLA Citation
Shen, XJ, Rawls, JF, Randall, T, Burcal, L, Mpande, CN, Jenkins, N, Jovov, B, Abdo, Z, Sandler, RS, and Keku, TO. "Molecular characterization of mucosal adherent bacteria and associations with colorectal adenomas." Gut microbes 1.3 (May 13, 2010): 138-147.
PMID
20740058
Source
epmc
Published In
Gut Microbes
Volume
1
Issue
3
Publish Date
2010
Start Page
138
End Page
147
DOI
10.4161/gmic.1.3.12360

Host-microbe interactions in the developing zebrafish.

The amenability of the zebrafish to in vivo imaging and genetic analysis has fueled expanded use of this vertebrate model to investigate the molecular and cellular foundations of host-microbe relationships. Study of microbial encounters in zebrafish hosts has concentrated on developing embryonic and larval stages, when the advantages of the zebrafish model are maximized. A comprehensive understanding of these host-microbe interactions requires appreciation of the developmental context into which a microbe is introduced, as well as the effects of that microbial challenge on host ontogeny. In this review, we discuss how in vivo imaging and genetic analysis in zebrafish has advanced our knowledge of host-microbe interactions in the context of a developing vertebrate host. We focus on recent insights into immune cell ontogeny and function, commensal microbial relationships in the intestine, and microbial pathogenesis in zebrafish hosts.

Authors
Kanther, M; Rawls, JF
MLA Citation
Kanther, M, and Rawls, JF. "Host-microbe interactions in the developing zebrafish." Current opinion in immunology 22.1 (February 12, 2010): 10-19. (Review)
PMID
20153622
Source
epmc
Published In
Current Opinion in Immunology
Volume
22
Issue
1
Publish Date
2010
Start Page
10
End Page
19
DOI
10.1016/j.coi.2010.01.006

Tuberculous granuloma induction via interaction of a bacterial secreted protein with host epithelium.

Granulomas, organized aggregates of immune cells, are a hallmark of tuberculosis and have traditionally been thought to restrict mycobacterial growth. However, analysis of Mycobacterium marinum in zebrafish has shown that the early granuloma facilitates mycobacterial growth; uninfected macrophages are recruited to the granuloma where they are productively infected by M. marinum. Here, we identified the molecular mechanism by which mycobacteria induce granulomas: The bacterial secreted protein 6-kD early secreted antigenic target (ESAT-6), which has long been implicated in virulence, induced matrix metalloproteinase-9 (MMP9) in epithelial cells neighboring infected macrophages. MMP9 enhanced recruitment of macrophages, which contributed to nascent granuloma maturation and bacterial growth. Disruption of MMP9 function attenuated granuloma formation and bacterial growth. Thus, interception of epithelial MMP9 production could hold promise as a host-targeting tuberculosis therapy.

Authors
Volkman, HE; Pozos, TC; Zheng, J; Davis, JM; Rawls, JF; Ramakrishnan, L
MLA Citation
Volkman, HE, Pozos, TC, Zheng, J, Davis, JM, Rawls, JF, and Ramakrishnan, L. "Tuberculous granuloma induction via interaction of a bacterial secreted protein with host epithelium." Science (New York, N.Y.) 327.5964 (January 2010): 466-469.
PMID
20007864
Source
epmc
Published In
Science
Volume
327
Issue
5964
Publish Date
2010
Start Page
466
End Page
469
DOI
10.1126/science.1179663

Ontogeny and nutritional control of adipogenesis in zebrafish (Danio rerio).

The global obesity epidemic demands an improved understanding of the developmental and environmental factors regulating fat storage. Adipocytes serve as major sites of fat storage and as regulators of energy balance and inflammation. The optical transparency of developing zebrafish provides new opportunities to investigate mechanisms governing adipocyte biology, however zebrafish adipocytes remain uncharacterized. We have developed methods for visualizing zebrafish adipocytes in vivo by labeling neutral lipid droplets with Nile Red. Our results establish that neutral lipid droplets first accumulate in visceral adipocytes during larval stages and increase in number and distribution as zebrafish grow. We show that the cellular anatomy of zebrafish adipocytes is similar to mammalian white adipocytes and identify peroxisome-proliferator activated receptor gamma and fatty acid binding protein 11a as markers of the zebrafish adipocyte lineage. By monitoring adipocyte development prior to neutral lipid deposition, we find that the first visceral preadipocytes appear in association with the pancreas shortly after initiation of exogenous nutrition. Zebrafish reared in the absence of food fail to form visceral preadipocytes, indicating that exogenous nutrition is required for adipocyte development. These results reveal homologies between zebrafish and mammalian adipocytes and establish the zebrafish as a new model for adipocyte research.

Authors
Flynn, EJ; Trent, CM; Rawls, JF
MLA Citation
Flynn, EJ, Trent, CM, and Rawls, JF. "Ontogeny and nutritional control of adipogenesis in zebrafish (Danio rerio)." Journal of lipid research 50.8 (August 2009): 1641-1652.
PMID
19366995
Source
epmc
Published In
Journal of lipid research
Volume
50
Issue
8
Publish Date
2009
Start Page
1641
End Page
1652
DOI
10.1194/jlr.m800590-jlr200

Patterns and scales in gastrointestinal microbial ecology.

The body surfaces of humans and other animals are colonized at birth by microorganisms. The majority of microbial residents on the human body exist within gastrointestinal (GI) tract communities, where they contribute to many aspects of host biology and pathobiology. Recent technological advances have expanded our ability to perceive the membership and physiologic traits of microbial communities along the GI tract. To translate this information into a mechanistic and practical understanding of host-microbe and microbe-microbe relationships, it is necessary to recast our conceptualization of the GI tract and its resident microbial communities in ecological terms. This review depicts GI microbial ecology in the context of 2 fundamental ecological concepts: (1) the patterns of biodiversity within the GI tract and (2) the scales of time, space, and environment within which we perceive those patterns. We show how this conceptual framework can be used to integrate our existing knowledge and identify important open questions in GI microbial ecology.

Authors
Camp, JG; Kanther, M; Semova, I; Rawls, JF
MLA Citation
Camp, JG, Kanther, M, Semova, I, and Rawls, JF. "Patterns and scales in gastrointestinal microbial ecology." Gastroenterology 136.6 (May 7, 2009): 1989-2002. (Review)
PMID
19457423
Source
epmc
Published In
Gastroenterology
Volume
136
Issue
6
Publish Date
2009
Start Page
1989
End Page
2002
DOI
10.1053/j.gastro.2009.02.075

Methods for generating and colonizing gnotobiotic zebrafish.

Vertebrates are colonized at birth by complex and dynamic communities of microorganisms that can contribute significantly to host health and disease. The ability to raise animals in the absence of microorganisms has been a powerful tool for elucidating the relationships between animal hosts and their microbial residents. The optical transparency of the developing zebrafish and relative ease of generating germ-free (GF) zebrafish make it an attractive model organism for gnotobiotic research. Here we provide a protocol for generating zebrafish embryos; deriving and rearing GF zebrafish; and colonizing zebrafish with microorganisms. Using these methods, we typically obtain 80-90% sterility rates in our GF derivations with 90% survival in GF animals and 50-90% survival in colonized animals through larval stages. Obtaining embryos for derivation requires approximately 1-2 h, with a 3- to 8-h incubation period before derivation. Derivation of GF animals takes 1-1.5 h, and daily maintenance requires 1-2 h.

Authors
Pham, LN; Kanther, M; Semova, I; Rawls, JF
MLA Citation
Pham, LN, Kanther, M, Semova, I, and Rawls, JF. "Methods for generating and colonizing gnotobiotic zebrafish." Nature protocols 3.12 (January 2008): 1862-1875.
PMID
19008873
Source
epmc
Published In
Nature Protocols
Volume
3
Issue
12
Publish Date
2008
Start Page
1862
End Page
1875
DOI
10.1038/nprot.2008.186

Enteric infection and inflammation alter gut microbial ecology.

The complex microbial community residing within the intestine plays important roles in host defense. However, the impact of enteric infection and inflammation on this resident community has not been fully explored. In this issue of Cell Host & Microbe, Lupp and coworkers reveal that the composition of the intestinal microbiota changes in distinctive ways in response to infection and inflammation.

Authors
Rawls, JF
MLA Citation
Rawls, JF. "Enteric infection and inflammation alter gut microbial ecology." Cell host & microbe 2.2 (August 2007): 73-74.
PMID
18005720
Source
epmc
Published In
Cell Host & Microbe
Volume
2
Issue
2
Publish Date
2007
Start Page
73
End Page
74
DOI
10.1016/j.chom.2007.07.006

In vivo imaging and genetic analysis link bacterial motility and symbiosis in the zebrafish gut.

Complex microbial communities reside within the intestines of humans and other vertebrates. Remarkably little is known about how these microbial consortia are established in various locations within the gut, how members of these consortia behave within their dynamic ecosystems, or what microbial factors mediate mutually beneficial host-microbial interactions. Using a gnotobiotic zebrafish-Pseudomonas aeruginosa model, we show that the transparency of this vertebrate species, coupled with methods for raising these animals under germ-free conditions can be used to monitor microbial movement and localization within the intestine in vivo and in real time. Germ-free zebrafish colonized with isogenic P. aeruginosa strains containing deletions of genes related to motility and pathogenesis revealed that loss of flagellar function results in attenuation of evolutionarily conserved host innate immune responses but not conserved nutrient responses. These results demonstrate the utility of gnotobiotic zebrafish in defining the behavior and localization of bacteria within the living vertebrate gut, identifying bacterial genes that affect these processes, and assessing the impact of these genes on host-microbial interactions.

Authors
Rawls, JF; Mahowald, MA; Goodman, AL; Trent, CM; Gordon, JI
MLA Citation
Rawls, JF, Mahowald, MA, Goodman, AL, Trent, CM, and Gordon, JI. "In vivo imaging and genetic analysis link bacterial motility and symbiosis in the zebrafish gut." Proceedings of the National Academy of Sciences of the United States of America 104.18 (May 2007): 7622-7627.
PMID
17456593
Source
epmc
Published In
Proceedings of the National Academy of Sciences of USA
Volume
104
Issue
18
Publish Date
2007
Start Page
7622
End Page
7627
DOI
10.1073/pnas.0702386104

Reciprocal gut microbiota transplants from zebrafish and mice to germ-free recipients reveal host habitat selection.

The gut microbiotas of zebrafish and mice share six bacterial divisions, although the specific bacteria within these divisions differ. To test how factors specific to host gut habitat shape microbial community structure, we performed reciprocal transplantations of these microbiotas into germ-free zebrafish and mouse recipients. The results reveal that communities are assembled in predictable ways. The transplanted community resembles its community of origin in terms of the lineages present, but the relative abundance of the lineages changes to resemble the normal gut microbial community composition of the recipient host. Thus, differences in community structure between zebrafish and mice arise in part from distinct selective pressures imposed within the gut habitat of each host. Nonetheless, vertebrate responses to microbial colonization of the gut are ancient: Functional genomic studies disclosed shared host responses to their compositionally distinct microbial communities and distinct microbial species that elicit conserved responses.

Authors
Rawls, JF; Mahowald, MA; Ley, RE; Gordon, JI
MLA Citation
Rawls, JF, Mahowald, MA, Ley, RE, and Gordon, JI. "Reciprocal gut microbiota transplants from zebrafish and mice to germ-free recipients reveal host habitat selection." Cell 127.2 (October 2006): 423-433.
PMID
17055441
Source
epmc
Published In
Cell
Volume
127
Issue
2
Publish Date
2006
Start Page
423
End Page
433
DOI
10.1016/j.cell.2006.08.043

Gnotobiotic zebrafish reveal evolutionarily conserved responses to the gut microbiota.

Animals have developed the means for supporting complex and dynamic consortia of microorganisms during their life cycle. A transcendent view of vertebrate biology therefore requires an understanding of the contributions of these indigenous microbial communities to host development and adult physiology. These contributions are most obvious in the gut, where studies of gnotobiotic mice have disclosed that the microbiota affects a wide range of biological processes, including nutrient processing and absorption, development of the mucosal immune system, angiogenesis, and epithelial renewal. The zebrafish (Danio rerio) provides an opportunity to investigate the molecular mechanisms underlying these interactions through genetic and chemical screens that take advantage of its transparency during larval and juvenile stages. Therefore, we developed methods for producing and rearing germ-free zebrafish through late juvenile stages. DNA microarray comparisons of gene expression in the digestive tracts of 6 days post fertilization germ-free, conventionalized, and conventionally raised zebrafish revealed 212 genes regulated by the microbiota, and 59 responses that are conserved in the mouse intestine, including those involved in stimulation of epithelial proliferation, promotion of nutrient metabolism, and innate immune responses. The microbial ecology of the digestive tracts of conventionally raised and conventionalized zebrafish was characterized by sequencing libraries of bacterial 16S rDNA amplicons. Colonization of germ-free zebrafish with individual members of its microbiota revealed the bacterial species specificity of selected host responses. Together, these studies establish gnotobiotic zebrafish as a useful model for dissecting the molecular foundations of host-microbial interactions in the vertebrate digestive tract.

Authors
Rawls, JF; Samuel, BS; Gordon, JI
MLA Citation
Rawls, JF, Samuel, BS, and Gordon, JI. "Gnotobiotic zebrafish reveal evolutionarily conserved responses to the gut microbiota." Proceedings of the National Academy of Sciences of the United States of America 101.13 (March 19, 2004): 4596-4601.
PMID
15070763
Source
epmc
Published In
Proceedings of the National Academy of Sciences of USA
Volume
101
Issue
13
Publish Date
2004
Start Page
4596
End Page
4601
DOI
10.1073/pnas.0400706101

Temporal and molecular separation of the kit receptor tyrosine kinase's roles in zebrafish melanocyte migration and survival.

The Kit receptor tyrosine kinase is required by vertebrate melanocytes for their migration and survival. The relationship between these developmental roles of Kit, however, remains poorly understood. Here, we use two genetic approaches to demonstrate that Kit's roles in the migration and survival of embryonic melanocytes in the zebrafish (Danio rerio) are temporally and functionally independent. We use a temperature-sensitive kit mutation to show that kit promotes melanocyte migration and survival during distinct stages of development. These experiments additionally reveal that melanocyte migration is neither necessary nor sufficient for subsequent survival. We also identify kit alleles that molecularly separate kits roles in migration and survival. These results suggest that the melanocyte changes its response to Kit receptor signaling and function during development, first to promote migration, then to promote survival through distinct Kit-dependent mechanisms.

Authors
Rawls, JF; Johnson, SL
MLA Citation
Rawls, JF, and Johnson, SL. "Temporal and molecular separation of the kit receptor tyrosine kinase's roles in zebrafish melanocyte migration and survival." Developmental biology 262.1 (October 2003): 152-161.
PMID
14512025
Source
epmc
Published In
Developmental Biology
Volume
262
Issue
1
Publish Date
2003
Start Page
152
End Page
161
DOI
10.1016/s0012-1606(03)00386-5

Coupled mutagenesis screens and genetic mapping in zebrafish.

Forward genetic analysis is one of the principal advantages of the zebrafish model system. However, managing zebrafish mutant lines derived from mutagenesis screens and mapping the corresponding mutations and integrating them into the larger collection of mutations remain arduous tasks. To simplify and focus these endeavors, we developed an approach that facilitates the rapid mapping of new zebrafish mutations as they are generated through mutagenesis screens. We selected a minimal panel of 149 simple sequence length polymorphism markers for a first-pass genome scan in crosses involving C32 and SJD inbred lines. We also conducted a small chemical mutagenesis screen that identified several new mutations affecting zebrafish embryonic melanocyte development. Using our first-pass marker panel in bulked-segregant analysis, we were able to identify the genetic map positions of these mutations as they were isolated in our screen. Rapid mapping of the mutations facilitated stock management, helped direct allelism tests, and should accelerate identification of the affected genes. These results demonstrate the efficacy of coupling mutagenesis screens with genetic mapping.

Authors
Rawls, JF; Frieda, MR; McAdow, AR; Gross, JP; Clayton, CM; Heyen, CK; Johnson, SL
MLA Citation
Rawls, JF, Frieda, MR, McAdow, AR, Gross, JP, Clayton, CM, Heyen, CK, and Johnson, SL. "Coupled mutagenesis screens and genetic mapping in zebrafish." Genetics 163.3 (March 2003): 997-1009.
PMID
12663538
Source
epmc
Published In
Genetics
Volume
163
Issue
3
Publish Date
2003
Start Page
997
End Page
1009

How the zebrafish gets its stripes.

The study of vertebrate pigment patterns is a classic and enduring field of developmental biology. Knowledge of pigment pattern development comes from a variety of systems, including avians, mouse, and more recently, the zebrafish (Danio rerio). Recent analyses of the mechanisms underlying the development of the neural crest-derived pigment cell type common to all vertebrates, the melanocyte, have revealed remarkable similarities and several surprising differences between amniotes and zebrafish. Here, we summarize recent advances in the study of melanocyte development in zebrafish, with reference to human, mouse, and avian systems. We first review melanocyte development in zebrafish and mammals, followed by a summary of the molecules known to be required for their development. We then discuss several relatively unaddressed issues in vertebrate pigment pattern development that are being investigated in zebrafish. These include determining the relationships between genetically distinct classes of melanocytes, characterizing and dissecting melanocyte stem cell development, and understanding how pigment cells organize into a patterned tissue. Further analysis of zebrafish pigment pattern mutants as well as new generations of directed mutant screens promise to extend our understanding of pigment pattern morphogenesis.

Authors
Rawls, JF; Mellgren, EM; Johnson, SL
MLA Citation
Rawls, JF, Mellgren, EM, and Johnson, SL. "How the zebrafish gets its stripes." Developmental biology 240.2 (December 2001): 301-314. (Review)
PMID
11784065
Source
epmc
Published In
Developmental Biology
Volume
240
Issue
2
Publish Date
2001
Start Page
301
End Page
314
DOI
10.1006/dbio.2001.0418

Requirements for the kit receptor tyrosine kinase during regeneration of zebrafish fin melanocytes.

Embryonic neural crest-derived melanocytes and their precursors express the kit receptor tyrosine kinase and require its function for their migration and survival. However, mutations in kit also cause deficits in melanocytes that make up adult pigment patterns, including melanocytes that re-establish the zebrafish fin stripes during regeneration. As adult melanocytes in mice and zebrafish are generated and maintained by stem cell populations that are presumably established during embryonic development, it has been proposed that adult phenotypes in kit mutants result from embryonic requirements for kit. We have used a temperature-sensitive zebrafish kit mutation to show that kit is required during adult fin regeneration to promote melanocyte differentiation, rather than during embryonic stages to establish their stem cell precursors. We also demonstrate a transient role for kit in promoting the survival of newly differentiated regeneration melanocytes.

Authors
Rawls, JF; Johnson, SL
MLA Citation
Rawls, JF, and Johnson, SL. "Requirements for the kit receptor tyrosine kinase during regeneration of zebrafish fin melanocytes." Development (Cambridge, England) 128.11 (June 2001): 1943-1949.
PMID
11493518
Source
epmc
Published In
Development (Cambridge)
Volume
128
Issue
11
Publish Date
2001
Start Page
1943
End Page
1949

Mutational analysis of endothelin receptor b1 (rose) during neural crest and pigment pattern development in the zebrafish Danio rerio.

Pigment patterns of fishes are a tractable system for studying the genetic and cellular bases for postembryonic phenotypes. In the zebrafish Danio rerio, neural crest-derived pigment cells generate different pigment patterns during different phases of the life cycle. Whereas early larvae exhibit simple stripes of melanocytes and silver iridophores in a background of yellow xanthophores, this pigment pattern is transformed at metamorphosis into that of the adult, comprising a series of dark melanocyte and iridophore stripes, alternating with light stripes of iridophores and xanthophores. Although several genes have been identified in D. rerio that contribute to the development of both early larval and adult pigment patterns, comparatively little is known about genes that are essential for pattern formation during just one or the other life cycle phase. In this study, we identify the gene responsible for the rose mutant phenotype in D. rerio. rose mutants have wild-type early larval pigment patterns, but fail to develop normal numbers of melanocytes and iridophores during pigment pattern metamorphosis and exhibit a disrupted pattern of these cells. We show that rose corresponds to endothelin receptor b1 (ednrb1), an orthologue of amniote Ednrb genes that have long been studied for their roles in neural crest and pigment cell development. Furthermore, we demonstrate that D. rerio ednrb1 is expressed both during pigment pattern metamorphosis and during embryogenesis, and cells of melanocyte, iridophore, and xanthophore lineages all express this gene. These analyses suggest a phylogenetic conservation of roles for Ednrb signaling in the development of amniote and teleost pigment cell precursors. As murine Ednrb is essential for the development of all neural crest derived melanocytes, and D. rerio ednrb1 is required only by a subset of adult melanocytes and iridophores, these analyses also reveal variation among vertebrates in the cellular requirements for Ednrb signaling, and suggest alternative models for the cellular and genetic bases of pigment pattern metamorphosis in D. rerio.

Authors
Parichy, DM; Mellgren, EM; Rawls, JF; Lopes, SS; Kelsh, RN; Johnson, SL
MLA Citation
Parichy, DM, Mellgren, EM, Rawls, JF, Lopes, SS, Kelsh, RN, and Johnson, SL. "Mutational analysis of endothelin receptor b1 (rose) during neural crest and pigment pattern development in the zebrafish Danio rerio." Developmental biology 227.2 (November 2000): 294-306.
PMID
11071756
Source
epmc
Published In
Developmental Biology
Volume
227
Issue
2
Publish Date
2000
Start Page
294
End Page
306
DOI
10.1006/dbio.2000.9899

Zebrafish kit mutation reveals primary and secondary regulation of melanocyte development during fin stripe regeneration.

Fin regeneration in adult zebrafish is accompanied by re-establishment of the pigment stripes. To understand the mechanisms underlying fin stripe regeneration and regulation of normal melanocyte stripe morphology, we investigated the origins of melanocytes in the regenerating fin and their requirement for the kit receptor tyrosine kinase. Using pre-existing melanin as a lineage tracer, we show that most fin regeneration melanocytes develop from undifferentiated precursors, rather than from differentiated melanocytes. Mutational analysis reveals two distinct classes of regeneration melanocytes. First, an early regeneration class develops dependent on kit function. In the absence of kit function and kit-dependent melanocytes, a second class of melanocytes develops at later stages of regeneration. This late kit-independent class of regeneration melanocytes has little or no role in wild-type fin stripe development, thus revealing a secondary mode for regulation of fin stripes. Expression of melanocyte markers in regenerating kit mutant fins suggests that kit normally acts after mitf and before dct to promote development of the primary kit-dependent melanocytes. kit-dependent and kit-independent melanocytes are also present during fin stripe ontogeny in patterns similar to those observed during regeneration.

Authors
Rawls, JF; Johnson, SL
MLA Citation
Rawls, JF, and Johnson, SL. "Zebrafish kit mutation reveals primary and secondary regulation of melanocyte development during fin stripe regeneration." Development (Cambridge, England) 127.17 (September 2000): 3715-3724.
PMID
10934016
Source
epmc
Published In
Development (Cambridge)
Volume
127
Issue
17
Publish Date
2000
Start Page
3715
End Page
3724

Zebrafish sparse corresponds to an orthologue of c-kit and is required for the morphogenesis of a subpopulation of melanocytes, but is not essential for hematopoiesis or primordial germ cell development.

The relative roles of the Kit receptor in promoting the migration and survival of amniote melanocytes are unresolved. We show that, in the zebrafish, Danio rerio, the pigment pattern mutation sparse corresponds to an orthologue of c-kit. This finding allows us to further elucidate morphogenetic roles for this c-kit-related gene in melanocyte morphogenesis. Our analyses of zebrafish melanocyte development demonstrate that the c-kit orthologue identified in this study is required both for normal migration and for survival of embryonic melanocytes. We also find that, in contrast to mouse, the zebrafish c-kit gene that we have identified is not essential for hematopoiesis or primordial germ cell development. These unexpected differences may reflect evolutionary divergence in c-kit functions following gene duplication events in teleosts.

Authors
Parichy, DM; Rawls, JF; Pratt, SJ; Whitfield, TT; Johnson, SL
MLA Citation
Parichy, DM, Rawls, JF, Pratt, SJ, Whitfield, TT, and Johnson, SL. "Zebrafish sparse corresponds to an orthologue of c-kit and is required for the morphogenesis of a subpopulation of melanocytes, but is not essential for hematopoiesis or primordial germ cell development." Development (Cambridge, England) 126.15 (August 1999): 3425-3436.
PMID
10393121
Source
epmc
Published In
Development (Cambridge)
Volume
126
Issue
15
Publish Date
1999
Start Page
3425
End Page
3436

SCAR, a WASP-related protein, isolated as a suppressor of receptor defects in late Dictyostelium development.

G protein-coupled receptors trigger the reorganization of the actin cytoskeleton in many cell types, but the steps in this signal transduction cascade are poorly understood. During Dictyostelium development, extracellular cAMP functions as a chemoattractant and morphogenetic signal that is transduced via a family of G protein-coupled receptors, the cARs. In a strain where the cAR2 receptor gene is disrupted by homologous recombination, the developmental program arrests before tip formation. In a genetic screen for suppressors of this phenotype, a gene encoding a protein related to the Wiskott-Aldrich Syndrome protein was discovered. Loss of this protein, which we call SCAR (suppressor of cAR), restores tip formation and most later development to cAR2(-) strains, and causes a multiple-tip phenotype in a cAR2(+) strain as well as leading to the production of extremely small cells in suspension culture. SCAR-cells have reduced levels of F-actin staining during vegetative growth, and abnormal cell morphology and actin distribution during chemotaxis. Uncharacterized homologues of SCAR have also been identified in humans, mouse, Caenorhabditis elegans, and Drosophila. These data suggest that SCAR may be a conserved negative regulator of G protein-coupled signaling, and that it plays an important role in regulating the actin cytoskeleton.

Authors
Bear, JE; Rawls, JF; Saxe, CL
MLA Citation
Bear, JE, Rawls, JF, and Saxe, CL. "SCAR, a WASP-related protein, isolated as a suppressor of receptor defects in late Dictyostelium development." The Journal of Cell Biology 142.5 (September 1998): 1325-1335.
PMID
9732292
Source
epmc
Published In
The Journal of Cell Biology
Volume
142
Issue
5
Publish Date
1998
Start Page
1325
End Page
1335
DOI
10.1083/jcb.142.5.1325
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