Interhost dispersal alters microbiome assembly and can overwhelm host innate immunity in an experimental zebrafish model.

The diverse collections of microorganisms associated with humans and other animals, collectively referred to as their "microbiome," are critical for host health, but the mechanisms that govern their assembly are poorly understood. This has made it difficult to identify consistent host factors that explain variation in microbiomes across hosts, despite large-scale sampling efforts. While ecological theory predicts that the movement, or dispersal, of individuals can have profound and predictable consequences on community assembly, its role in the assembly of animal-associated microbiomes remains underexplored. Here, we show that dispersal of microorganisms among hosts can contribute substantially to microbiome variation, and is able to overwhelm the effects of individual host factors, in an experimental test of ecological theory. We manipulated dispersal among wild-type and immune-deficient myd88 knockout zebrafish and observed that interhost dispersal had a large effect on the diversity and composition of intestinal microbiomes. Interhost dispersal was strong enough to overwhelm the effects of host factors, largely eliminating differences between wild-type and immune-deficient hosts, regardless of whether dispersal occurred within or between genotypes, suggesting dispersal can independently alter the ecology of microbiomes. Our observations are consistent with a predictive model that assumes metacommunity dynamics and are likely mediated by dispersal-related microbial traits. These results illustrate the importance of microbial dispersal to animal microbiomes and motivate its integration into the study of host-microbe systems.

Correction: A MultiSite Gateway Toolkit for Rapid Cloning of Vertebrate Expression Constructs with Diverse Research Applications.

[This corrects the article DOI: 10.1371/journal.pone.0159277.].

A MultiSite Gateway Toolkit for Rapid Cloning of Vertebrate Expression Constructs with Diverse Research Applications.

Recombination-based cloning is a quick and efficient way to generate expression vectors. Recent advancements have provided powerful recombinant DNA methods for molecular manipulations. Here, we describe a novel collection of three-fragment MultiSite Gateway cloning system-compatible vectors providing expanded molecular tools for vertebrate research. The components of this toolkit encompass a broad range of uses such as fluorescent imaging, dual gene expression, RNA interference, tandem affinity purification, chemically-inducible dimerization and lentiviral production. We demonstrate examples highlighting the utility of this toolkit for producing multi-component vertebrate expression vectors with diverse primary research applications. The vectors presented here are compatible with other Gateway toolkits and collections, facilitating the rapid generation of a broad range of innovative DNA constructs for biological research.

Lhx3 and Lhx4 suppress Kolmer-Agduhr interneuron characteristics within zebrafish axial motoneurons.

A central problem in development is how fates of closely related cells are segregated. Lineally related motoneurons (MNs) and interneurons (INs) express many genes in common yet acquire distinct fates. For example, in mouse and chick Lhx3 plays a pivotal role in the development of both cell classes. Here, we utilize the ability to recognize individual zebrafish neurons to examine the roles of Lhx3 and its paralog Lhx4 in the development of MNs and ventral INs. We show that Lhx3 and Lhx4 are expressed by post-mitotic axial MNs derived from the MN progenitor (pMN) domain, p2 domain progenitors and by several types of INs derived from pMN and p2 domains. In the absence of Lhx3 and Lhx4, early-developing primary MNs (PMNs) adopt a hybrid fate, with morphological and molecular features of both PMNs and pMN-derived Kolmer-Agduhr' (KA') INs. In addition, we show that Lhx3 and Lhx4 distinguish the fates of two pMN-derived INs. Finally, we demonstrate that Lhx3 and Lhx4 are necessary for the formation of late-developing V2a and V2b INs. In conjunction with our previous work, these data reveal that distinct transcription factor families are deployed in post-mitotic MNs to unequivocally assign MN fate and suppress the development of alternative pMN-derived IN fates.

Zebrafish Mnx proteins specify one motoneuron subtype and suppress acquisition of interneuron characteristics.

BACKGROUND: Precise matching between motoneuron subtypes and the muscles they innervate is a prerequisite for normal behavior. Motoneuron subtype identity is specified by the combination of transcription factors expressed by the cell during its differentiation. Here we investigate the roles of Mnx family transcription factors in specifying the subtypes of individually identified zebrafish primary motoneurons. RESULTS: Zebrafish has three Mnx family members. We show that each of them has a distinct and temporally dynamic expression pattern in each primary motoneuron subtype. We also show that two Mnx family members are expressed in identified VeLD interneurons derived from the same progenitor domain that generates primary motoneurons. Surprisingly, we found that Mnx proteins appear unnecessary for differentiation of VeLD interneurons or the CaP motoneuron subtype. Mnx proteins are, however, required for differentiation of the MiP motoneuron subtype. We previously showed that MiPs require two temporally-distinct phases of Islet1 expression for normal development. Here we show that in the absence of Mnx proteins, the later phase of Islet1 expression is initiated but not sustained, and MiPs become hybrids that co-express morphological and molecular features of motoneurons and V2a interneurons. Unexpectedly, these hybrid MiPs often extend CaP-like axons, and some MiPs appear to be entirely transformed to a CaP morphology. CONCLUSIONS: Our results suggest that Mnx proteins promote MiP subtype identity by suppressing both interneuron development and CaP axon pathfinding. This is, to our knowledge, the first report of transcription factors that act to distinguish CaP and MiP subtype identities. Our results also suggest that MiP motoneurons are more similar to V2 interneurons than are CaP motoneurons.

Turning gene function ON and OFF using sense and antisense photo-morpholinos in zebrafish.

To understand the molecular mechanisms of development it is essential to be able to turn genes on and off at will and in a spatially restricted fashion. Morpholino oligonucleotides (MOs) are very common tools used in several model organisms with which it is possible to block gene expression. Recently developed photo-activated MOs allow control over the onset of MO activity. However, deactivation of photo-cleavable MO activity has remained elusive. Here, we describe photo-cleavable MOs with which it is possible to activate or de-activate MO function by UV exposure in a temporal and spatial manner. We show, using several different genes as examples, that it is possible to turn gene expression on or off both in the entire zebrafish embryo and in single cells. We use these tools to demonstrate the sufficiency of no tail expression as late as tailbud stage to drive medial precursor cells towards the notochord cell fate. As a broader approach for the use of photo-cleavable MOs, we show temporal control over gal4 function, which has many potential applications in multiple transgenic lines. We demonstrate temporal manipulation of Gal4 transgene expression in only primary motoneurons and not secondary motoneurons, heretofore impossible with conventional transgenic approaches. In another example, we follow and analyze neural crest cells that regained sox10 function after deactivation of a photo-cleavable sox10-MO at different time points. Our results suggest that sox10 function might not be critical during neural crest formation.

An A257V mutation in the bacillus subtilis response regulator Spo0A prevents regulated expression of promoters with low-consensus binding sites.

In Bacillus species, the master regulator of sporulation is Spo0A. Spo0A functions by both activating and repressing transcription initiation from target promoters that contain 0A boxes, the binding sites for Spo0A. Several classes of spo0A mutants have been isolated, and the molecular basis for their phenotypes has been determined. However, the molecular basis of the Spo0A(A257V) substitution, representative of an unusual phenotypic class, is not understood. Spo0A(A257V) is unusual in that it abolishes sporulation; in vivo, it fails to activate transcription from key stage II promoters yet retains the ability to repress the abrB promoter. To determine how Spo0A(A257V) retains the ability to repress but not stimulate transcription, we performed a series of in vitro and in vivo assays. We found unexpectedly that the mutant protein both stimulated transcription from the spoIIG promoter and repressed transcription from the abrB promoter, albeit twofold less than the wild type. A DNA binding analysis of Spo0A(A257V) showed that the mutant protein was less able to tolerate alterations in the sequence and arrangement of its DNA binding sites than the wild-type protein. In addition, we found that Spo0A(A257V) could stimulate transcription of a mutant spoIIG promoter in vivo in which low-consensus binding sites were replaced by high-consensus binding sites. We conclude that Spo0A(A257V) is able to bind to and regulate the expression of only genes whose promoters contain high-consensus binding sites and that this effect is sufficient to explain the observed sporulation defect.

Bacillus subtilis RNA polymerase recruits the transcription factor Spo0A approximately P to stabilize a closed complex during transcription initiation.

The Bacillus subtilis response regulator Spo0A approximately P activates transcription from the spoIIG promoter by stimulating a rate-limiting transition between the initial interaction of RNA polymerase with the promoter and initiation of RNA synthesis. Previous work showed that Spo0A exerts its effect on RNA polymerase prior to the formation of an open complex in which the DNA strands at the initiation site have been separated. To isolate the effect of Spo0A approximately P on events prior to DNA strand separation at spoIIG we studied RNA polymerase binding to DNA fragments that were truncated to contain only promoter sequences 5' to the -10 element by electrophoretic mobility shift assays. RNA polymerase bound to these fragments readily though highly reversibly, and polymerase-promoter complexes recruited Spo0A approximately P. Sequence-independent interactions between the RNA polymerase and the DNA upstream of the core promoter were important for RNA polymerase binding and essential for Spo0A approximately P recruitment, while sequence-specific Spo0A approximately P-DNA interactions positioned and stabilized RNA polymerase binding to the DNA. Spo0A approximately P decreased the dissociation rate of the complexes formed with truncated promoter templates which could contribute to the means by which Spo0A approximately P stimulates spoIIG expression.

The Bacillus subtilis response regulator Spo0A stimulates sigmaA-dependent transcription prior to the major energetic barrier.

At the spoIIG promoter phosphorylated Spo0A (Spo0A approximately P) binds 0A boxes overlapping the -35 element, interacting with RNA polymerase to facilitate open complex formation. We have compared in vitro transcription from a series of heteroduplex templates containing denatured regions within the promoters. Transcription from heteroduplex templates with 12, 8, or 6 base pairs denatured was independent of Spo0A approximately P, but heteroduplexes with 4 or 2 base pairs denatured required Spo0A approximately P for maximal levels of transcription. Investigation of the thermal dependence of transcription suggested that strand separation was the primary thermodynamic barrier to transcription initiation but indicated that Spo0A approximately P does not reduce this energetic barrier. Kinetic assays revealed that Spo0A approximately P stimulated both the rate of formation of initiated complexes as well as increasing the number of complexes capable of initiating transcription. These results imply that Spo0A approximately P stimulates transcription at least in part by stabilizing the RNA polymerase-spoIIG complex until contacts between RNA polymerase and the -10 element induce strand separation.