Transcriptional dynamics during Rhodococcus erythropolis infection with phage WC1.

BACKGROUND: Belonging to the Actinobacteria phylum, members of the Rhodococcus genus thrive in soil, water, and even intracellularly. While most species are non-pathogenic, several cause respiratory disease in animals and, more rarely, in humans. Over 100 phages that infect Rhodococcus species have been isolated but despite their importance for Rhodococcus ecology and biotechnology applications, little is known regarding the molecular genetic interactions between phage and host during infection. To address this need, we report RNA-Seq analysis of a novel Rhodococcus erythopolis phage, WC1, analyzing both the phage and host transcriptome at various stages throughout the infection process. RESULTS: By five minutes post-infection WC1 showed upregulation of a CAS-4 family exonuclease, putative immunity repressor, an anti-restriction protein, while the host showed strong upregulation of DNA replication, SOS repair, and ribosomal protein genes. By 30 min post-infection, WC1 DNA synthesis genes were strongly upregulated while the host showed increased expression of transcriptional and translational machinery and downregulation of genes involved in carbon, energy, and lipid metabolism pathways. By 60 min WC1 strongly upregulated structural genes while the host showed a dramatic disruption of metal ion homeostasis. There was significant expression of both host and phage non-coding genes at all time points. While host gene expression declined over the course of infection, our results indicate that phage may exert more selective control, preserving the host's regulatory mechanisms to create an environment conducive for virion production. CONCLUSIONS: The Rhodococcus genus is well recognized for its ability to synthesize valuable compounds, particularly steroids, as well as its capacity to degrade a wide range of harmful environmental pollutants. A detailed understanding of these phage-host interactions and gene expression is not only essential for understanding the ecology of this important genus, but will also facilitate development of phage-mediated strategies for bioremediation as well as biocontrol in industrial processes and biomedical applications. Given the current lack of detailed global gene expression studies on any Rhodococcus species, our study addresses a pressing need to identify tools and genes, such as F6 and rpf, that can enhance the capacity of Rhodococcus species for bioremediation, biosynthesis and pathogen control.

Trpm4 ion channels in pre-Bötzinger complex interneurons are essential for breathing motor pattern but not rhythm.

Inspiratory breathing movements depend on pre-Bötzinger complex (preBötC) interneurons that express calcium (Ca2+)-activated nonselective cationic current (ICAN) to generate robust neural bursts. Hypothesized to be rhythmogenic, reducing ICAN is predicted to slow down or stop breathing; its contributions to motor pattern would be reflected in the magnitude of movements (output). We tested the role(s) of ICAN using reverse genetic techniques to diminish its putative ion channels Trpm4 or Trpc3 in preBötC neurons in vivo. Adult mice transduced with Trpm4-targeted short hairpin RNA (shRNA) progressively decreased the tidal volume of breaths yet surprisingly increased breathing frequency, often followed by gasping and fatal respiratory failure. Mice transduced with Trpc3-targeted shRNA survived with no changes in breathing. Patch-clamp and field recordings from the preBötC in mouse slices also showed an increase in the frequency and a decrease in the magnitude of preBötC neural bursts in the presence of Trpm4 antagonist 9-phenanthrol, whereas the Trpc3 antagonist pyrazole-3 (pyr-3) showed inconsistent effects on magnitude and no effect on frequency. These data suggest that Trpm4 mediates ICAN, whose influence on frequency contradicts a direct role in rhythm generation. We conclude that Trpm4-mediated ICAN is indispensable for motor output but not the rhythmogenic core mechanism of the breathing central pattern generator.

Crowdsourcing biocuration: The Community Assessment of Community Annotation with Ontologies (CACAO).

Experimental data about gene functions curated from the primary literature have enormous value for research scientists in understanding biology. Using the Gene Ontology (GO), manual curation by experts has provided an important resource for studying gene function, especially within model organisms. Unprecedented expansion of the scientific literature and validation of the predicted proteins have increased both data value and the challenges of keeping pace. Capturing literature-based functional annotations is limited by the ability of biocurators to handle the massive and rapidly growing scientific literature. Within the community-oriented wiki framework for GO annotation called the Gene Ontology Normal Usage Tracking System (GONUTS), we describe an approach to expand biocuration through crowdsourcing with undergraduates. This multiplies the number of high-quality annotations in international databases, enriches our coverage of the literature on normal gene function, and pushes the field in new directions. From an intercollegiate competition judged by experienced biocurators, Community Assessment of Community Annotation with Ontologies (CACAO), we have contributed nearly 5,000 literature-based annotations. Many of those annotations are to organisms not currently well-represented within GO. Over a 10-year history, our community contributors have spurred changes to the ontology not traditionally covered by professional biocurators. The CACAO principle of relying on community members to participate in and shape the future of biocuration in GO is a powerful and scalable model used to promote the scientific enterprise. It also provides undergraduate students with a unique and enriching introduction to critical reading of primary literature and acquisition of marketable skills.

Xenopus embryos show a compensatory response following perturbation of the Notch signaling pathway.

As an essential feature of development, robustness ensures that embryos attain a consistent phenotype despite genetic and environmental variation. The growing number of examples demonstrating that embryos can mount a compensatory response to germline mutations in key developmental genes has heightened interest in the phenomenon of embryonic robustness. While considerable progress has been made in elucidating genetic compensation in response to germline mutations, the diversity, mechanisms, and limitations of embryonic robustness remain unclear. In this work, we have examined whether Xenopus laevis embryos are able to compensate for perturbations of the Notch signaling pathway induced by RNA injection constructs that either upregulate or inhibit this signaling pathway. Consistent with earlier studies, we found that at neurula stages, hyperactivation of the Notch pathway inhibited neural differentiation while inhibition of Notch signaling increases premature differentiation as assayed by neural beta tubulin expression. However, surprisingly, by hatching stages, embryos begin to compensate for these perturbations, and by swimming tadpole stages most embryos exhibited normal neuronal gene expression. Using cell proliferation and TUNEL assays, we show that the compensatory response is, in part, mediated by modulating levels of cell proliferation and apoptosis. This work provides an additional model for addressing the mechanisms of embryonic robustness and of genetic compensation.

Mercury delays cerebellar development in a model songbird species, the zebra finch.

Mercury exposure can disrupt development of the cerebellum, part of the brain essential for coordination of movement through a complex environment, including flight. In precocial birds, such as fowl, the cerebellum develops embryonically, and the chick is capable of leaving the nest within hours of hatching. However, most birds, including all songbirds, are altricial, and spend weeks in the nest between hatching and fledging. The objective of this study was to describe the normal development of the cerebellum in a model altricial songbird so as to determine the effect of exposure to mercury on cerebellar maturation. Adult zebra finch (Taeniopygia guttata) pairs were fed either a control diet, or a diet augmented with one of four treatment-levels of methylmercury (0.3-2.4 µg/g wet weight), and their offspring, the subjects of this study, were fed the same diet by parents. We documented, for the first time, the schedule of cerebellar development in an altricial bird, and compared stages of development among methylmercury-exposed groups. For all treatments of methylmercury, the age of completion of cellular migration was later than for control zebra finches, indicating a delay in cerebellar maturation. Displaced (heterotopic) Purkinje neurons, a pathology typical of methylmercury exposure in developing vertebrate brains, were more numerous in methylmercury-exposed birds, and persisted at least until the age of independence. Delays in maturation of the cerebellum could delay fledging in altricial bird species, with potential serious implications for the fitness of exposed individuals, as predation rates in the nest are often very high.

Calcium Activity Dynamics Correlate with Neuronal Phenotype at a Single Cell Level and in a Threshold-Dependent Manner.

Calcium is a ubiquitous signaling molecule that plays a vital role in many physiological processes. Recent work has shown that calcium activity is especially critical in vertebrate neural development. Here, we investigated if calcium activity and neuronal phenotype are correlated only on a population level or on the level of single cells. Using Xenopus primary cell culture in which individual cells can be unambiguously identified and associated with a molecular phenotype, we correlated calcium activity with neuronal phenotype on the single-cell level. This analysis revealed that, at the neural plate stage, a high frequency of low-amplitude spiking activity correlates with an excitatory, glutamatergic phenotype, while high-amplitude spiking activity correlates with an inhibitory, GABAergic phenotype. Surprisingly, we also found that high-frequency, low-amplitude spiking activity correlates with neural progenitor cells and that differentiating cells exhibit higher spike amplitude. Additional methods of analysis suggested that differentiating marker tubb2b-expressing cells exhibit relatively persistent and predictable calcium activity compared to the irregular activity of neural progenitor cells. Our study highlights the value of using a range of thresholds for analyzing calcium activity data and underscores the importance of employing multiple methods to characterize the often irregular, complex patterns of calcium activity during early neural development.

Deciphering the Calcium Code: A Review of Calcium Activity Analysis Methods Employed to Identify Meaningful Activity in Early Neural Development.

The intracellular and intercellular flux of calcium ions represents an ancient and universal mode of signaling that regulates an extensive array of cellular processes. Evidence for the central role of calcium signaling includes various techniques that allow the visualization of calcium activity in living cells. While extensively investigated in mature cells, calcium activity is equally important in developing cells, particularly the embryonic nervous system where it has been implicated in a wide variety array of determinative events. However, unlike in mature cells, where the calcium dynamics display regular, predictable patterns, calcium activity in developing systems is far more sporadic, irregular, and diverse. This renders the ability to assess calcium activity in a consistent manner extremely challenging, challenges reflected in the diversity of methods employed to analyze calcium activity in neural development. Here we review the wide array of calcium detection and analysis methods used across studies, limiting the extent to which they can be comparatively analyzed. The goal is to provide investigators not only with an overview of calcium activity analysis techniques currently available, but also to offer suggestions for future work and standardization to enable informative comparative evaluations of this fundamental and important process in neural development.

Sequence analysis of Discoknowium, an A5 mycobacteriophage.

Discoknowium is a temperate A5 bacteriophage that infects the bacterial host Mycobacterium smegmatis. Isolated from a rat fecal sample, Discoknowium's genome is 50,222 bp in length, contains 84 genes and 1 tRNA, and shares 82%-98% nucleotide identity with other A5 subcluster phages.

Anthropogenic noise is associated with reductions in the productivity of breeding Eastern Bluebirds (Sialia sialis).

Although previous studies have related variations in environmental noise levels with alterations in communication behaviors of birds, little work has investigated the potential long-term implications of living or breeding in noisy habitats. However, noise has the potential to reduce fitness, both directly (because it is a physiological stressor) and indirectly (by masking important vocalizations and/or leading to behavioral changes). Here, we quantified acoustic conditions in active breeding territories of male Eastern Bluebirds (Sialia sialis). Simultaneously, we measured four fitness indicators: cuckoldry rates, brood growth rate and condition, and number of fledglings produced (i.e., productivity). Increases in environmental noise tended to be associated with smaller brood sizes and were more strongly related to reductions in productivity. Although the mechanism responsible for these patterns is not yet clear, the breeding depression experienced by this otherwise disturbance-tolerant species indicates that anthropogenic noise may have damaging effects on individual fitness and, by extraction, the persistence of populations in noisy habitats. We suggest that managers might protect avian residents from potentially harmful noise by keeping acoustically dominant anthropogenic habitat features as far as possible from favored songbird breeding habitats, limiting noisy human activities, and/or altering habitat structure in order to minimize the propagation of noise pollution.

Cloning and characterization of GABAA α subunits and GABAB subunits in Xenopus laevis during development.

Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the adult nervous system, acts via two classes of receptors, the ionotropic GABA(A) and metabotropic GABA(B) receptors. During the development of the nervous system, GABA acts in a depolarizing, excitatory manner and plays an important role in various neural developmental processes including cell proliferation, migration, synapse formation, and activity-dependent differentiation. Here we describe the spatial and temporal expression patterns of the GABA(A) and GABA(B) receptors during early development of Xenopus laevis. Using in situ hybridization and qRT-PCR, GABA(A) α2 was detected as a maternal mRNA. All other α-subunits were first detected by tailbud through hatching stages. Expression of the various subunits was seen in the brain, spinal cord, cranial ganglia, olfactory epithelium, pineal, and pituitary gland. Each receptor subunit showed a distinctive, unique expression pattern, suggesting these receptors have specific functions and are regulated in a precise spatial and temporal manner.

Tissue Rotation of the Xenopus Anterior-Posterior Neural Axis Reveals Profound but Transient Plasticity at the Mid-Gastrula Stage.

The establishment of anterior-posterior (AP) regional identity is an essential step in the appropriate development of the vertebrate central nervous system. An important aspect of AP neural axis formation is the inherent plasticity that allows developing cells to respond to and recover from the various perturbations that embryos continually face during the course of development. While the mechanisms governing the regionalization of the nervous system have been extensively studied, relatively less is known about the nature and limits of early neural plasticity of the anterior-posterior neural axis. This study aims to characterize the degree of neural axis plasticity in Xenopus laevis by investigating the response of embryos to a 180-degree rotation of their AP neural axis during gastrula stages by assessing the expression of regional marker genes using in situ hybridization. Our results reveal the presence of a narrow window of time between the mid- and late gastrula stage, during which embryos are able undergo significant recovery following a 180-degree rotation of their neural axis and eventually express appropriate regional marker genes including Otx, Engrailed, and Krox. By the late gastrula stage, embryos show misregulation of regional marker genes following neural axis rotation, suggesting that this profound axial plasticity is a transient phenomenon that is lost by late gastrula stages.

Transcriptomes of electrophysiologically recorded Dbx1-derived respiratory neurons of the preBötzinger complex in neonatal mice.

Breathing depends on interneurons in the preBötzinger complex (preBötC) derived from Dbx1-expressing precursors. Here we investigate whether rhythm- and pattern-generating functions reside in discrete classes of Dbx1 preBötC neurons. In a slice model of breathing with ~ 5 s cycle period, putatively rhythmogenic Type-1 Dbx1 preBötC neurons activate 100-300 ms prior to Type-2 neurons, putatively specialized for output pattern, and 300-500 ms prior to the inspiratory motor output. We sequenced Type-1 and Type-2 transcriptomes and identified differential expression of 123 genes including ionotropic receptors (Gria3, Gabra1) that may explain their preinspiratory activation profiles and Ca2+ signaling (Cracr2a, Sgk1) involved in inspiratory and sigh bursts. Surprisingly, neuropeptide receptors that influence breathing (e.g., µ-opioid and bombesin-like peptide receptors) were only sparsely expressed, which suggests that cognate peptides and opioid drugs exert their profound effects on a small fraction of the preBötC core. These data in the public domain help explain the neural origins of breathing.

Single cell transcriptome sequencing of inspiratory neurons of the preBötzinger complex in neonatal mice.

Neurons in the brainstem preBötzinger complex (preBötC) generate the rhythm and rudimentary motor pattern for inspiratory breathing movements. We performed whole-cell patch-clamp recordings from inspiratory neurons in the preBötC of neonatal mouse slices that retain breathing-related rhythmicity in vitro. We classified neurons based on their electrophysiological properties and genetic background, and then aspirated their cellular contents for single-cell RNA sequencing (scRNA-seq). This data set provides the raw nucleotide sequences (FASTQ files) and annotated files of nucleotide sequences mapped to the mouse genome (mm10 from Ensembl), which includes the fragment counts, gene lengths, and fragments per kilobase of transcript per million mapped reads (FPKM). These data reflect the transcriptomes of the neurons that generate the rhythm and pattern for inspiratory breathing movements.

Optimizing Calcium Detection Methods in Animal Systems: A Sandbox for Synthetic Biology.

Since the 1970s, the emergence and expansion of novel methods for calcium ion (Ca2+) detection have found diverse applications in vitro and in vivo across a series of model animal systems. Matched with advances in fluorescence imaging techniques, the improvements in the functional range and stability of various calcium indicators have significantly enhanced more accurate study of intracellular Ca2+ dynamics and its effects on cell signaling, growth, differentiation, and regulation. Nonetheless, the current limitations broadly presented by organic calcium dyes, genetically encoded calcium indicators, and calcium-responsive nanoparticles suggest a potential path toward more rapid optimization by taking advantage of a synthetic biology approach. This engineering-oriented discipline applies principles of modularity and standardization to redesign and interrogate endogenous biological systems. This review will elucidate how novel synthetic biology technologies constructed for eukaryotic systems can offer a promising toolkit for interfacing with calcium signaling and overcoming barriers in order to accelerate the process of Ca2+ detection optimization.

The tweety Gene Family: From Embryo to Disease.

The tweety genes encode gated chloride channels that are found in animals, plants, and even simple eukaryotes, signifying their deep evolutionary origin. In vertebrates, the tweety gene family is highly conserved and consists of three members-ttyh1, ttyh2, and ttyh3-that are important for the regulation of cell volume. While research has elucidated potential physiological functions of ttyh1 in neural stem cell maintenance, proliferation, and filopodia formation during neural development, the roles of ttyh2 and ttyh3 are less characterized, though their expression patterns during embryonic and fetal development suggest potential roles in the development of a wide range of tissues including a role in the immune system in response to pathogen-associated molecules. Additionally, members of the tweety gene family have been implicated in various pathologies including cancers, particularly pediatric brain tumors, and neurodegenerative diseases such as Alzheimer's and Parkinson's disease. Here, we review the current state of research using information from published articles and open-source databases on the tweety gene family with regard to its structure, evolution, expression during development and adulthood, biochemical and cellular functions, and role in human disease. We also identify promising areas for further research to advance our understanding of this important, yet still understudied, family of genes.

Cloning and characterization of voltage-gated calcium channel alpha1 subunits in Xenopus laevis during development.

Voltage-gated calcium channels play a critical role in regulating the Ca2+ activity that mediates many aspects of neural development, including neural induction, neurotransmitter phenotype specification, and neurite outgrowth. Using Xenopus laevis embryos, we describe the spatial and temporal expression patterns during development of the 10 pore-forming alpha1 subunits that define the channels' kinetic properties. In situ hybridization indicates that CaV1.2, CaV2.1, CaV2.2, and CaV3.2 are expressed during neurula stages throughout the neural tube. These, along with CaV1.3 and CaV2.3, beginning at early tail bud stages, and CaV3.1 at late tail bud stages, are detected in complex patterns within the brain and spinal cord through swimming tadpole stages. Additional expression of various alpha1 subunits was observed in the cranial ganglia, retina, olfactory epithelium, pineal gland, and heart. The unique expression patterns for the different alpha1 subunits suggests they are under precise spatial and temporal regulation and are serving specific functions during embryonic development.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis.

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.

Expression patterns of glycine transporters (xGlyT1, xGlyT2, and xVIAAT) in Xenopus laevis during early development.

Glycine, a major inhibitory neurotransmitter in the vertebrate nervous system, not only functions in synaptic signaling, but has also been implicated in regulating neuronal differentiation, neuronal proliferation, synaptic modeling, and neural network stability. Elements of the glycinergic phenotype include the membrane-bound glycine transporters (GLYT1 and GLYT2), which remove glycine from the synaptic cleft, and the vesicular inhibitory amino acid transporter (VIAAT or VGAT), which sequesters both glycine and GABA into synaptic vesicles. Here, we describe the spatial and temporal expression patterns of xGlyT1, xGlyT2, and xVIAAT during early developmental stages of Xenopus laevis. In situ hybridization reveals that xGlyT1 is first expressed in early tailbud stages in the midbrain, hindbrain, and anterior spinal cord; it extends posteriorly through the spinal cord and appears in the forebrain, retina, between the somites, and in the blood islands by swimming tadpole stages. xGlyT2 and xVIAAT initially appear in late neurula stages in the anterior spinal cord. By swimming tadpole stages, the expression of these genes appears in the forebrain, midbrain, and hindbrain and extends posteriorly through the spinal cord; xVIAAT is also expressed in the retina. Confocal analysis of multiplex fluorescent in situ hybridization signal in the spinal cord reveals that xGlyT1 and xGlyT2 share little cellular colocalization. While there is significant coexpression between xVIAAT and xGlyT2, xVIAAT and the GABAergic marker glutamic acid decarboxylase (xGAD67), and xGlyT2 and xGAD67, each gene also appears to have discrete, non-colocalized areas of expression.

Histological Observation of Teratogenic Phenotypes Induced in Frog Embryo Assays.

Amphibian embryos have long served as an ideal model for teratogenicity testing. While whole-mount embryo observations can be utilized, histological observation of teratogenic phenotypes provides a wealth of additional information that can lead to mechanistic insights. In this chapter, detailed protocols for two methods of sectioning embryos as well as a guide for histological analysis is provided.

Transcriptome of Xenopus andrei, an octoploid frog, during embryonic development.

Although polyploidy occurs throughout the fish and amphibian lineages, the Xenopus genus exhibits a high incidence of polyploidy, with 25 out of the 26 known species being polyploid. However, transcriptomic information is currently available for only one of these species, the tetraploid Xenopus laevis. Xenopus andrei, an octoploid species within the Xenopus genus, offers an opportunity for assessing a novel polyploid transcriptome during vertebrate development. RNA-Seq data was generated at nine different developmental stages ranging from unfertilized eggs through swimming tadpole stages and raw FASTQ files were deposited in the NCBI SRA database (accession number SRP134281). Additionally, RNA-seq data from all nine stages were pooled to create a de novo assembly of the transcriptome using Trinity and has been deposited in the NCBI GEO database (accession number GSE111639). To our knowledge, this represents the first published assembly of an octoploid vertebrate transcriptome. In total, 849 Mb were assembled, which led to the identification of 1,650,048 transcripts in the assembly with a contig N50 of 630 bases. This RNA-Seq and transcriptome data will be valuable for comparing polyploid transcriptomes across Xenopus species, as well as understanding evolutionary implications of whole-genome duplication and polyploidy in vertebrates.