W. Zac Stephens

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.

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.

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.

Retinoic Acid Is Required for Endodermal Pouch Morphogenesis and Not for Pharyngeal Endoderm Specification

Because tissues from all three germ layers contribute to the pharyngeal arches, it is not surprising that all major signaling pathways are involved in their development. We focus on the role of retinoic acid (RA) signaling because it has been recognized for quite some time that alterations in this pathway lead to craniofacial malformations. Several studies exist that describe phenotypes observed upon RA perturbations in pharyngeal arch development; however, these studies did not address whether RA plays multiple roles at distinct time points during development. Here, we report the resulting phenotypes in the hindbrain, the neural crest–derived tissues, and the pharyngeal endoderm when RA synthesis is disrupted during zebrafish gastrulation and pharyngeal arch morphogenesis. Our results demonstrate that RA is required for the post‐gastrulation morphogenesis and segmentation of endodermal pouches, and that loss of RA does not affect the length of the pharyngeal ectoderm or medial endoderm along the anterior‐posterior axis. We also provide evidence that RA is not required for the specification of pharyngeal pouch endoderm and that the pharyngeal endoderm consists of at least two different cell populations, of which the pouch endoderm is sensitive to RA and the more medial pharyngeal endoderm is not. These results demonstrate that the developmental processes underlying pharyngeal arch defects differ depending on when RA signaling is disturbed during development. Developmental Dynamics 235:2695–2709, 2006.

Neuronal Neuregulin 1 type III directs Schwann cell migration

During peripheral nerve development, each segment of a myelinated axon is matched with a single Schwann cell. Tight regulation of Schwann cell movement, proliferation and differentiation is essential to ensure that these glial cells properly associate with axons. ErbB receptors are required for Schwann cell migration, but the operative ligand and its mechanism of action have remained unknown. We demonstrate that zebrafish Neuregulin 1 (Nrg1) type III, which signals through ErbB receptors, controls Schwann cell migration in addition to its previously known roles in proliferation and myelination. Chimera analyses indicate that ErbB receptors are required in all migrating Schwann cells, and that Nrg1 type III is required in neurons for migration. Surprisingly, expression of the ligand in a few axons is sufficient to induce migration along a chimeric nerve constituted largely of nrg1 type III mutant axons. These studies also reveal a mechanism that allows Schwann cells to fasciculate axons regardless of nrg1 type III expression. Time-lapse imaging of transgenic embryos demonstrated that misexpression of human NRG1 type III results in ectopic Schwann cell migration, allowing them to aberrantly enter the central nervous system. These results demonstrate that Nrg1 type III is an essential signal that controls Schwann cell migration to ensure that these glia are present in the correct numbers and positions in developing nerves.

MHC variation sculpts individualized microbial communities that control susceptibility to enteric infection

The presentation of protein antigens on the cell surface by major histocompatibility complex (MHC) molecules coordinates vertebrate adaptive immune responses, thereby mediating susceptibility to a variety of autoimmune and infectious diseases. The composition of symbiotic microbial communities (the microbiota) is influenced by host immunity and can have a profound impact on host physiology. Here we use an MHC congenic mouse model to test the hypothesis that genetic variation at MHC genes among individuals mediates susceptibility to disease by controlling microbiota composition. We find that MHC genotype significantly influences antibody responses against commensals in the gut, and that these responses are correlated with the establishment of unique microbial communities. Transplantation experiments in germfree mice indicate that MHC-mediated differences in microbiota composition are sufficient to explain susceptibility to enteric infection. Our findings indicate that MHC polymorphisms contribute to defining an individuals unique microbial fingerprint that influences health.

Investigating Bacterial-Animal Symbioses with Light Sheet Microscopy

Microbial colonization of the digestive tract is a crucial event in vertebrate development, required for maturation of host immunity and establishment of normal digestive physiology. Advances in genomic, proteomic, and metabolomic technologies are providing a more detailed picture of the constituents of the intestinal habitat, but these approaches lack the spatial and temporal resolution needed to characterize the assembly and dynamics of microbial communities in this complex environment. We report the use of light sheet microscopy to provide high-resolution imaging of bacterial colonization of the intestine of Danio rerio, the zebrafish. The method allows us to characterize bacterial population dynamics across the entire organ and the behaviors of individual bacterial and host cells throughout the colonization process. The large four-dimensional data sets generated by these imaging approaches require new strategies for image analysis. When integrated with other “omics” data sets, information about the spatial and temporal dynamics of microbial cells within the vertebrate intestine will provide new mechanistic insights into how microbial communities assemble and function within hosts.

Genome-Wide CRISPR-Cas9 Screen Identifies MicroRNAs That Regulate Myeloid Leukemia Cell Growth

Mammalian microRNA expression is dysregulated in human cancer. However, the functional relevance of many microRNAs in the context of tumor biology remains unclear. Using CRISPR-Cas9 technology, we performed a global loss-of-function screen to simultaneously test the functions of individual microRNAs and protein-coding genes during the growth of a myeloid leukemia cell line. This approach identified evolutionarily conserved human microRNAs that suppress or promote cell growth, revealing that microRNAs are extensively integrated into the molecular networks that control tumor cell physiology. miR-155 was identified as a top microRNA candidate promoting cellular fitness, which we confirmed with two distinct miR-155-targeting CRISPR-Cas9 lentiviral constructs. Further, we performed anti-correlation functional profiling to predict relevant microRNA-tumor suppressor gene or microRNA-oncogene interactions in these cells. This analysis identified miR-150 targeting of p53, a connection that was experimentally validated. Taken together, our study describes a powerful genetic approach by which the function of individual microRNAs can be assessed on a global level, and its use will rapidly advance our understanding of how microRNAs contribute to human disease.

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

ABSTRACT 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. IMPORTANCE 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. 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.

Loss of adenomatous polyposis coli (apc) results in an expanded ciliary marginal zone in the zebrafish eye.

The distal region of neural retina (ciliary marginal zone [CMZ]) contains stem cells that produce non‐neural and neuronal progenitors. We provide a detailed gene expression analysis of the eyes of apc mutant zebrafish where the Wnt/β‐catenin pathway is constitutively active. Wnt/β‐catenin signaling leads to an expansion of the CMZ accompanied by a central shift of the retinal identity gene sox2 and the proneural gene atoh7. This suggests an important role for peripheral Wnt/β‐catenin signaling in regulating the expression and localization of neurogenic genes in the central retina. Retinal identity genes rx1 and vsx2, as well as meis1 and pax6a act upstream of Wnt/β‐catenin pathway activation. Peripheral cells that likely contain stem cells can be identified by the expression of follistatin, otx1, and axin2 and the lack of expression of myca and cyclinD1. Our results introduce the zebrafish apc mutation as a new model to study signaling pathways regulating the CMZ. Developmental Dynamics 239:2066–2077, 2010.