A Novel Taxon of Monosegmented Double-Stranded RNA Viruses Endemic to Triclad Flatworms.

Freshwater planarians, flatworms from order Tricladida, are experimental models of stem cell biology and tissue regeneration. An aspect of their biology that remains less well studied is their relationship with viruses that may infect them. In this study, we identified a taxon of monosegmented double-stranded RNA (dsRNA) viruses in five planarian species, including the well-characterized model Schmidtea mediterranea Sequences for the S. mediterranea virus (abbreviated SmedTV for S. mediterranea tricladivirus) were found in public transcriptome data from multiple institutions, indicating that SmedTV is prevalent in S. mediterranea lab colonies, though without causing evident disease. The presence of SmedTV in discrete cells was shown through in situ hybridization methods for detecting the viral RNA. SmedTV-staining cells were found to be concentrated in neural structures (eyes and brain) but were also scattered in other worm tissues as well. In contrast, few SmedTV-staining cells were seen in stem cell compartments (also consistent with RNA sequencing data) or early blastema tissue. RNA interference (RNAi) targeted to the SmedTV sequence led to apparent cure of infection, though effects on worm health or behavior were not observed. Efforts to transmit SmedTV horizontally through microinjection were unsuccessful. Based on these findings, we conclude that SmedTV infects S. mediterranea in a persistent manner and undergoes vertical transmission to progeny worms during serial passage in lab colonies. The utility of S. mediterranea as a regeneration model, coupled with the apparent capacity of SmedTV to evade normal host immune/RNAi defenses under standard conditions, argues that further studies are warranted to explore this newly recognized virus-host system.IMPORTANCE Planarians are freshwater flatworms, related more distantly to tapeworms and flukes, and have been developed as models to study the molecular mechanisms of stem cell biology and tissue regeneration. These worms live in aquatic environments, where they are likely to encounter a variety of viruses, bacteria, and eukaryotic organisms with pathogenic potential. How the planarian immune system has evolved to cope with these potential pathogens is not well understood, and only two types of planarian viruses have been described to date. Here, we report discovery and inaugural studies of a novel taxon of dsRNA viruses in five different planarian species. The virus in the best-characterized model species, Schmidtea mediterranea, appears to persist long term in that host while avoiding endogenous antiviral or RNAi mechanisms. The S. mediterranea virus-host system thus seems to offer opportunity for gaining new insights into host defenses and their evolution in an important lab model.

An optimized QF-binary expression system for use in zebrafish.

The zebrafish model organism has been of exceptional utility for the study of vertebrate development and disease through the application of tissue-specific labelling and overexpression of genes carrying patient-derived mutations. However, there remains a need for a binary expression system that is both non-toxic and not silenced over animal generations by DNA methylation. The Q binary expression system derived from the fungus Neurospora crassa is ideal, because the consensus binding site for the QF transcription factor lacks CpG dinucleotides, precluding silencing by CpG-meditated methylation. To optimize this system for zebrafish, we systematically tested several variants of the QF transcription factor: QF full length; QF2, which lacks the middle domain; QF2w, which is an attenuated version of QF2; and chimeric QFGal4. We found that full length QF and QF2 were strongly toxic to zebrafish embryos, QF2w was mildly toxic, and QFGal4 was well tolerated, when injected as RNA or expressed ubiquitously from stable transgenes. In addition, QFGal4 robustly activated a Tg(QUAS:GFPNLS) reporter transgene. To increase the utility of this system, we also modified the QF effector sequence termed QUAS, which consists of five copies of the QF binding site. Specifically, we decreased both the CpG dinucleotide content, as well as the repetitiveness of QUAS, to reduce the risk of transgene silencing via CpG methylation. Moreover, these modifications to QUAS removed leaky QF-independent neural expression that we detected in the original QUAS sequence. To demonstrate the utility of our QF optimizations, we show how the Q-system can be used for lineage tracing using a Cre-dependent Tg(ubi:QFGal4-switch) transgene. We also demonstrate that QFGal4 can be used in transient injections to tag and label endogenous genes by knocking in QFGal4 into sox2 and ubiquitin C genes.

Mespaa can potently induce cardiac fates in zebrafish.

The Mesp family of transcription factors have been implicated in the early formation and migration of the cardiac lineage, although the precise molecular mechanisms underlying this process remain unknown. In this study we examine the function of Mesp family members in zebrafish cardiac development and find that Mespaa is remarkably efficient at promoting cardiac fates in normally non-cardiogenic cells. However, Mespaa is dispensable for normal cardiac formation. Despite no overt defects in cardiovascular specification, we find a consistent defect in cardiac laterality in mespaa null embryos. This is further exacerbated by the depletion of other mesp paralogues, highlighting a conserved role for the mesp family in left-right asymmetry, distinct from a function in cardiac specification. Despite an early requirement for mespaa to promote cardiogenesis, cells over-expressing mespaa are found to both exhibit unique cellular behaviors and activate the transcription of gata5 only after the completion of gastrulation. We propose that while mespaa remains capable of driving cardiac progenitor formation in zebrafish, it may not play an essential role in the cardiac regulatory network. Furthermore, the late activation of migration and cardiac gene transcription in mespaa over-expressing cells challenges previous studies on the timing of these events and provides intriguing questions for future study.

Med14 cooperates with brg1 in the differentiation of skeletogenic neural crest.

BACKGROUND: An intricate gene regulatory network drives neural crest migration and differentiation. How epigenetic regulators contribute to this process is just starting to be understood. RESULTS: We found that mutation of med14 or brg1 in zebrafish embryos resulted in a cluster of neural crest cell-related defects. In med14 or brg1 mutants, neural crest cells that form the jaw skeleton were specified normally and migrated to target sites. However, defects in their subsequent terminal differentiation were evident. Transplantation experiments demonstrated that med14 and brg1 are required directly in neural crest cells. Analysis of med14; brg1 double mutant embryos suggested the existence of a strong genetic interaction between members of the Mediator and BAF complexes. CONCLUSIONS: These results suggest a critical role for Mediator and BAF complex function in neural crest development, and may also clarify the nature of defects in some craniofacial abnormalities.

Anisotropic stress orients remodelling of mammalian limb bud ectoderm.

The physical forces that drive morphogenesis are not well characterized in vivo, especially among vertebrates. In the early limb bud, dorsal and ventral ectoderm converge to form the apical ectodermal ridge (AER), although the underlying mechanisms are unclear. By live imaging mouse embryos, we show that prospective AER progenitors intercalate at the dorsoventral boundary and that ectoderm remodels by concomitant cell division and neighbour exchange. Mesodermal expansion and ectodermal tension together generate a dorsoventrally biased stress pattern that orients ectodermal remodelling. Polarized distribution of cortical actin reflects this stress pattern in a ß-catenin- and Fgfr2-dependent manner. Intercalation of AER progenitors generates a tensile gradient that reorients resolution of multicellular rosettes on adjacent surfaces, a process facilitated by ß-catenin-dependent attachment of cortex to membrane. Therefore, feedback between tissue stress pattern and cell intercalations remodels mammalian ectoderm.

An in vivo requirement for the mediator subunit med14 in the maintenance of stem cell populations.

The Mediator complex has recently been shown to be a key player in the maintenance of embryonic and induced pluripotent stem cells. However, the in vivo consequences of loss of many Mediator subunits are unknown. We identified med14 as the gene affected in the zebrafish logelei (log) mutant, which displayed a morphological arrest by 2 days of development. Surprisingly, microarray analysis showed that transcription was not broadly affected in log mutants. Indeed, log cells transplanted into a wild-type environment were able to survive into adulthood. In planarians, RNAi knockdown demonstrated a requirement for med14 and many other Mediator components in adult stem cell maintenance and regeneration. Multiple stem/progenitor cell populations were observed to be reduced or absent in zebrafish med14 mutant embryos. Taken together, our results show a critical, evolutionarily conserved, in vivo function for Med14 (and Mediator) in stem cell maintenance, distinct from a general role in transcription.

Identification of myopia-related marker proteins in tilapia retinal, RPE, and choroidal tissue following induced form deprivation.

PURPOSE: Experimentally induced myopia is characterized by axial elongation of the eye. The molecular pathways leading to this condition are largely unknown, even though many candidate proteins have been proposed to be involved in this process. This study has identified proteins that were differentially expressed in myopic and control combined retina, retinal pigment epithelium (RPE), and choroidal tissue in tilapia (Oreochromis niloticus). METHODS: Form deprivation was used to induce myopia in tilapia (n = 3). In this initial study on tilapia retina, RPE and choroid, 2-D differential in gel electrophoresis (DIGE) and mass spectrometry were used to identify differentially expressed proteins. Homology-based gene cloning was used to obtain full sequence data for one of the identified proteins. RESULTS: A total of 18 protein spots separated by 2-D electrophoresis exhibited statistically significant differences in expression between the myopic and contralateral control combined retinal, RPE, and choroidal tissue. Three proteins were identified at a significance level of p < 0.05, as annexin A5 (down-regulated 47%), Gelsolin (down-regulated 27%), and TCP-1 (CCT) (down-regulated 54%). DNA sequencing of tilapia annexin A5 shows an amino acid sequence identity of 84.5% with the homologous Japanese ricefish annexin max2. CONCLUSIONS: A proteomics approach has been used to identify differentially expressed proteins in form-deprived combined retinal, RPE, and choroidal tissue from myopic versus normal eyes. The identified proteins may be components of pathways involved in myopia pathogenesis.