Transcriptomic Analysis Identifies Candidate Genes for Differential Expression during Xenopus laevis Inner Ear Development.

Background: The genes involved in inner ear development and maintenance of the adult organ have yet to be fully characterized. Previous genetic analysis has emphasized the early development that gives rise to the otic vesicle. This study aimed to bridge the knowledge gap and identify candidate genes that are expressed as the auditory and vestibular sensory organs continue to grow and develop until the systems reach postmetamorphic maturity. Methods: Affymetrix microarrays were used to assess inner ear transcriptome profiles from three Xenopus laevis developmental ages where all eight endorgans comprise mechanosensory hair cells: larval stages 50 and 56, and the post-metamorphic juvenile. Pairwise comparisons were made between the three developmental stages and the resulting differentially expressed X. laevis Probe Set IDs (Xl-PSIDs) were assigned to four groups based on differential expression patterns. DAVID analysis was undertaken to impart functional annotation to the differentially regulated Xl-PSIDs. Results: Analysis identified 1510 candidate genes for differential gene expression in one or more pairwise comparison. Annotated genes not previously associated with inner ear development emerged from this analysis, as well as annotated genes with established inner ear function, such as oncomodulin, neurod1, and sp7. Notably, 36% of differentially expressed Xl-PSIDs were unannotated. Conclusions: Results draw attention to the complex gene regulatory patterns that characterize Xenopus inner ear development, and underscore the need for improved annotation of the X. laevis genome. Outcomes can be utilized to select candidate inner ear genes for functional analysis, and to promote Xenopus as a model organism for biomedical studies of hearing and balance.

Total RNA Isolation from Separately Established Monolayer and Hydrogel Cultures of Human Glioblastoma Cell Line.

Astrocytoma is an invasive carcinoma occurring in the nervous system and currently lacks effective treatment options. A deeper understanding of the mechanisms of tumorigenesis and tumor progression is needed in order to develop novel therapeutic strategies. Recent advances in in vitro culture systems have demonstrated that the use of three-dimensional (3D) culture models could be more relevant for this purpose as compared to monolayer or two-dimensional (2D) models due to their resemblance to in vivo cancer pathology. High-throughput techniques such as RNA sequencing, microarray analyses and cloning could provide useful insights into the relevance of these systems to the native tissue. Previous studies have reported RNA extraction protocols needed for such applications. We have modified these protocols to suit the isolation of total RNA from monolayer and hydrogel cultures of astrocytoma established using basement membrane matrix, Geltrex™. We have used this method to demonstrate the differences in the expression of genes involved in autophagy, a process deregulated in many cancer types, in monolayer and hydrogel cultures using quantitative polymerase chain reaction (qPCR). This protocol can be adopted by the researchers who wish to understand the molecular basis of gene expression in hydrogel cultures of normal as well as cancer cell lines.

The NIH BRAIN Initiative: Integrating Neuroethics and Neuroscience.

The NIH Brain Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative is focused on developing new tools and neurotechnologies to transform our understanding of the brain, and neuroethics is an essential component of this research effort. Coordination with other brain projects around the world will help maximize success.

Expression analysis of RNA sequencing data from human neural and glial cell lines depends on technical replication and normalization methods.

BACKGROUND: The potential for astrocyte participation in central nervous system recovery is highlighted by in vitro experiments demonstrating their capacity to transdifferentiate into neurons. Understanding astrocyte plasticity could be advanced by comparing astrocytes with stem cells. RNA sequencing (RNA-seq) is ideal for comparing differences across cell types. However, this novel multi-stage process has the potential to introduce unwanted technical variation at several points in the experimental workflow. Quantitative understanding of the contribution of experimental parameters to technical variation would facilitate the design of robust RNA-Seq experiments. RESULTS: RNA-Seq was used to achieve biological and technical objectives. The biological aspect compared gene expression between normal human fetal-derived astrocytes and human neural stem cells cultured in identical conditions. When differential expression threshold criteria of |log2 fold change| > 2 were applied to the data, no significant differences were observed. The technical component quantified variation arising from particular steps in the research pathway, and compared the ability of different normalization methods to reduce unwanted variance. To facilitate this objective, a liberal false discovery rate of 10% and a |log2 fold change| > 0.5 were implemented for the differential expression threshold. Data were normalized with RPKM, TMM, and UQS methods using JMP Genomics. The contributions of key replicable experimental parameters (cell lot; library preparation; flow cell) to variance in the data were evaluated using principal variance component analysis. Our analysis showed that, although the variance for every parameter is strongly influenced by the normalization method, the largest contributor to technical variance was library preparation. The ability to detect differentially expressed genes was also affected by normalization; differences were only detected in non-normalized and TMM-normalized data. CONCLUSIONS: The similarity in gene expression between astrocytes and neural stem cells supports the potential for astrocytic transdifferentiation into neurons, and emphasizes the need to evaluate the therapeutic potential of astrocytes for central nervous system damage. The choice of normalization method influences the contributions to experimental variance as well as the outcomes of differential expression analysis. However irrespective of normalization method, our findings illustrate that library preparation contributed the largest component of technical variance.

Morphometric analysis of a triple negative breast cancer cell line in hydrogel and monolayer culture environments.

Triple negative breast cancer (TNBC) is a belligerent carcinoma that is unresponsive to targeted receptor therapies. Development of new treatment strategies would benefit from an expanded repertoire of in vitro cell culture systems, such as those that support tridimensional growth in the presence of hydrogel scaffolds. To this end, we established protocols for maintenance of the TNBC cell line HCC70 in monolayer culture and in a commercially available basement membrane matrix hydrogel. We evaluated the general morphology of cells grown in both conditions with light microscopy, and examined their subcellular organization using transmission electron microscopy (TEM). Phase contrast and confocal microscopy showed the prevalence of irregularly shaped flattened cells in monolayer cultures, while cells maintained in hydrogel organized into multi-layered spheroids. A quantitative ultrastructural analysis comparing cells from the two culture conditions revealed that cells that formed spheroids comprised a greater number of mitochondria, autophagic vacuoles and intercellular junctions than their monolayer counterparts, within the equivalent area of sampled tissue. These observations suggest that triple negative breast cancer cells in culture can alter their organelle content, as well as their morphology, in response to their microenvironment. Methods presented here may be useful for those who intend to image cell cultures with TEM, and for investigators who seek to implement diverse in vitro models in the search for therapeutic molecular targets for TNBC.

Hydrogel Environment Supports Cell Culture Expansion of a Grade IV Astrocytoma.

Malignant astrocytomas are aggressive cancers of glial origin that can develop into invasive brain tumors. The disease has poor prognosis and high recurrence rate. Astrocytoma cell lines of human origin are an important tool in the experimental pathway from bench to bedside because they afford a convenient intermediate system for in vitro analysis of brain cancer pathogenesis and treatment options. We undertook the current study to determine whether hydrogel culture methods could be adapted to support the growth of astrocytoma cell lines, thereby facilitating a system that may be biologically more similar to in vivo tumor tissue. Our experimental protocols enabled maintenance of Grade IV astrocytoma cell lines in conventional monolayer culture and in the extracellular matrix hydrogel, Geltrex™. Light and fluorescence microscopy showed that hydrogel environments promoted cellular reorganization from dispersed cells into multilayered aggregates. Transmission electron microscopy revealed the prevalence of autophagy and nuclear membrane distortions in both culture systems. Analysis of microarray Gene Expression Omnibus (GEO) DataSets highlighted expression of genes implicated in pathways for cancer progression and autophagy. A pilot quantitative polymerase chain reaction (qPCR) analysis of the autophagic biomarkers, Beclin 1 (BECN1) and microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B), with two reference genes (beta actin, ACTB; glyceraldehyde 3-phosphate dehydrogenase, GAPDH), uncovered a relative increase of BECN1 and LC3B in hydrogel cultures of astrocytoma as compared to the monolayer. Taken together, results establish that ultrastructural and molecular characteristics of autophagy are features of this astrocytoma cell line, and that hydrogel culture systems can afford novel opportunities for in vitro studies of glioma.

Post-Translational Tubulin Modifications in Human Astrocyte Cultures.

The cytoskeletal protein tubulin plays an integral role in the functional specialization of many cell types. In the central nervous system, post-translational modifications and the expression of specific tubulin isotypes in neurons have been analyzed in greater detail than in their astrocytic counterparts. In this study, we characterized post-translational specifications of tubulin in human astrocytes using the normal human astrocyte (NHA; Lonza) commercial cell line of fetal origin. Immunocytochemical techniques were implemented in conjunction with confocal microscopy to image class III ß-tubulin (ßIII-tubulin), acetylated tubulin, and polyglutamylated tubulin using fluorescent antibody probes. Fluorescent probe intensity differences and colocalization were quantitatively assessed with the 'EBImage' package for the statistical programming language R. Colocalization analysis revealed that, although both acetylated tubulin and polyglutamylated tubulin showed a high degree of correlation with ßIII-tubulin, the correlation with acetylated tubulin was stronger. Quantification and statistical analysis of fluorescence intensity demonstrated that the fluorescence probe intensity ratio for acetylated tubulin/ßIII-tubulin was greater than the ratio for polyglutamylated tubulin/ßIII-tubulin. The open source GEODATA set GSE819950, comprising RNA sequencing data for the NHA cell line, was mined for the expression of enzymes responsible for tubulin modifications. Our analysis uncovered greater expression at the mRNA level for enzymes reported to function in acetylation and deacetylation as compared to enzymes implicated in glutamylation and deglutamylation. Taken together, the results represent a step toward unraveling the tubulin isotypic expression profile and post-translational modification patterns in astrocytes during human brain development.

Hydrogel scaffolds promote neural gene expression and structural reorganization in human astrocyte cultures.

Biomaterial scaffolds have the potential to enhance neuronal development and regeneration. Understanding the genetic responses of astrocytes and neurons to biomaterials could facilitate the development of synthetic environments that enable the specification of neural tissue organization with engineered scaffolds. In this study, we used high throughput transcriptomic and imaging methods to determine the impact of a hydrogel, PuraMatrix™, on human glial cells in vitro. Parallel studies were undertaken with cells grown in a monolayer environment on tissue culture polystyrene. When the Normal Human Astrocyte (NHA) cell line is grown in a hydrogel matrix environment, the glial cells adopt a structural organization that resembles that of neuronal-glial cocultures, where neurons form clusters that are distinct from the surrounding glia. Statistical analysis of next generation RNA sequencing data uncovered a set of genes that are differentially expressed in the monolayer and matrix hydrogel environments. Functional analysis demonstrated that hydrogel-upregulated genes can be grouped into three broad categories: neuronal differentiation and/or neural plasticity, response to neural insult, and sensory perception. Our results demonstrate that hydrogel biomaterials have the potential to transform human glial cell identity, and may have applications in the repair of damaged brain tissue.

RNA Extraction from Xenopus Auditory and Vestibular Organs for Molecular Cloning and Expression Profiling with RNA-Seq and Microarrays.

The amphibian Xenopus offers a unique model system for uncovering the genetic basis of auditory and vestibular function in an organism that is well-suited for experimental manipulation during animal development. However, many procedures for analyzing gene expression in the peripheral auditory and vestibular systems mandate the ability to isolate intact RNA from inner ear tissue. Methods presented here facilitate preparation of high-quality inner ear RNA from larval and post-metamorphic Xenopus specimens that can be used for a variety of purposes. We demonstrate that RNA isolated with these protocols is suitable for microarray analysis and Illumina-Solexa sequencing (RNA-Seq) of inner ear organs, and for cloning of large transcripts, such as those for ion channels. Genetic sequences cloned with these procedures can be used for transient transfection of Xenopus kidney cell lines with fluorescent protein fusion constructs.

RNA-Seq and microarray analysis of the Xenopus inner ear transcriptome discloses orthologous OMIM(®) genes for hereditary disorders of hearing and balance.

BACKGROUND: Auditory and vestibular disorders are prevalent sensory disabilities caused by genetic and environmental (noise, trauma, chemicals) factors that often damage mechanosensory hair cells of the inner ear. Development of treatments for inner ear disorders of hearing and balance relies on the use of animal models such as fish, amphibians, reptiles, birds, and non-human mammals. Here, we aimed to augment the utility of the genus Xenopus for uncovering genetic mechanisms essential for the maintenance of inner ear structure and function. RESULTS: Using Affymetrix GeneChip(®) X. laevis Genome 2.0 Arrays and Illumina-Solexa sequencing methods, we determined that the transcriptional profile of the Xenopus laevis inner ear comprises hundreds of genes that are orthologous to OMIM(®) genes implicated in deafness and vestibular disorders in humans. Analysis of genes that mapped to both technologies demonstrated that, with our methods, a combination of microarray and RNA-Seq detected expression of more genes than either platform alone. CONCLUSIONS: As part of this study we identified candidate scaffold regions of the Xenopus tropicalis genome that can be used to investigate hearing and balance using genetic and informatics procedures that are available through the National Xenopus Resource (NXR), and the open access data repository, Xenbase. The results and approaches presented here expand the viability of Xenopus as an animal model for inner ear research.

Probing the Xenopus laevis inner ear transcriptome for biological function.

BACKGROUND: The senses of hearing and balance depend upon mechanoreception, a process that originates in the inner ear and shares features across species. Amphibians have been widely used for physiological studies of mechanotransduction by sensory hair cells. In contrast, much less is known of the genetic basis of auditory and vestibular function in this class of animals. Among amphibians, the genus Xenopus is a well-characterized genetic and developmental model that offers unique opportunities for inner ear research because of the amphibian capacity for tissue and organ regeneration. For these reasons, we implemented a functional genomics approach as a means to undertake a large-scale analysis of the Xenopus laevis inner ear transcriptome through microarray analysis. RESULTS: Microarray analysis uncovered genes within the X. laevis inner ear transcriptome associated with inner ear function and impairment in other organisms, thereby supporting the inclusion of Xenopus in cross-species genetic studies of the inner ear. The use of gene categories (inner ear tissue; deafness; ion channels; ion transporters; transcription factors) facilitated the assignment of functional significance to probe set identifiers. We enhanced the biological relevance of our microarray data by using a variety of curation approaches to increase the annotation of the Affymetrix GeneChip(®) Xenopus laevis Genome array. In addition, annotation analysis revealed the prevalence of inner ear transcripts represented by probe set identifiers that lack functional characterization. CONCLUSIONS: We identified an abundance of targets for genetic analysis of auditory and vestibular function. The orthologues to human genes with known inner ear function and the highly expressed transcripts that lack annotation are particularly interesting candidates for future analyses. We used informatics approaches to impart biologically relevant information to the Xenopus inner ear transcriptome, thereby addressing the impediment imposed by insufficient gene annotation. These findings heighten the relevance of Xenopus as a model organism for genetic investigations of inner ear organogenesis, morphogenesis, and regeneration.

Optimization of gene delivery methods in Xenopus laevis kidney (A6) and Chinese hamster ovary (CHO) cell lines for heterologous expression of Xenopus inner ear genes.

The Xenopus inner ear provides a useful model for studies of hearing and balance because it shares features with the mammalian inner ear, and because amphibians are capable of regenerating damaged mechanosensory hair cells. The structure and function of many proteins necessary for inner ear function have yet to be elucidated and require methods for analysis. To this end, we seek to characterize Xenopus inner ear genes outside of the animal model through heterologous expression in cell lines. As part of this effort, we aimed to optimize physical (electroporation), chemical (lipid-mediated; Lipofectamine™ 2000, Metafectene® Pro), and biological (viral-mediated; BacMam virus Cellular Lights™ Tubulin-RFP) gene delivery methods in amphibian (Xenopus; A6) cells and mammalian (Chinese hamster ovary (CHO)) cells. We successfully introduced the commercially available pEGFP-N3, pmCherry-N1, pEYFP-Tubulin, and Cellular Lights™ Tubulin-RFP fluorescent constructs to cells and evaluated their transfection or transduction efficiencies using the three gene delivery methods. In addition, we analyzed the transfection efficiency of a novel construct synthesized in our laboratory by cloning the Xenopus inner ear calcium-activated potassium channel ß1 subunit, then subcloning the subunit into the pmCherry-N1 vector. Every gene delivery method was significantly more effective in CHO cells. Although results for the A6 cell line were not statistically significant, both cell lines illustrate a trend towards more efficient gene delivery using viral-mediated methods; however the cost of viral transduction is also much higher. Our findings demonstrate the need to improve gene delivery methods for amphibian cells and underscore the necessity for a greater understanding of amphibian cell biology.

Imaging heterostructured quantum dots in cultured cells with epifluorescence and transmission electron microscopy.

Quantum dots (QDs) are semiconductor nanocrystals with extensive imaging and diagnostic capabilities, including the potential for single molecule tracking. Commercially available QDs offer distinct advantages over organic fluorophores, such as increased photostability and tunable emission spectra, but their cadmium selenide (CdSe) core raises toxicity concerns. For this reason, replacements for CdSe-based QDs have been sought that can offer equivalent optical properties. The spectral range, brightness and stability of InP QDs may comprise such a solution. To this end, LANL/CINT personnel fabricated moderately thick-shell novel InP QDs that retain brightness and emission over time in an aqueous environment. We are interested in evaluating how the composition and surface properties of these novel QDs affect their entry and sequestration within the cell. Here we use epifluorescence and transmission electron microscopy (TEM) to evaluate the structural properties of cultured Xenopus kidney cells (A6; ATCC) that were exposed either to commercially available CdSe QDs (Qtracker® 565, Invitrogen) or to heterostructured InP QDs (LANL). Epifluorescence imaging permitted assessment of the general morphology of cells labeled with fluorescent molecular probes (Alexa Fluor® ® phalloidin; Hoechst 33342), and the prevalence of QD association with cells. In contrast, TEM offered unique advantages for viewing electron dense QDs at higher resolution with regard to subcellular sequestration and compartmentalization. Preliminary results show that in the absence of targeting moieties, InP QDs (200 nM) can passively enter cells and sequester nonspecifically in cytosolic regions whereas commercially available targeted QDs principally associate with membranous structures within the cell. Supported by: NIH 5R01GM084702.

Strategies for enhanced annotation of a microarray probe set.

We aim to determine the biological relevance of genes identified through microarray-mediated transcriptional profiling of Xenopus sensory organs and brain. Difficulties with genetic data analysis arise because of limitations in probe set annotation and the lack of a universal gene nomenclature. To overcome these impediments, we used sequence based and semantic linking methods in combination with computational approaches to augment probe set annotation on a commercially available microarray. Our curation efforts enabled linkage of probe sets and expression data to public databases, increased the biological significance of our microarray data, and assisted with the tentative identification of unidentified probe sets.

RNA isolation from Xenopus inner ear sensory endorgans for transcriptional profiling and molecular cloning.

The amphibian Xenopus offers a unique model system for uncovering the genetic basis of auditory and vestibular function in an organism that is well-suited for experimental manipulation during animal development. However, many procedures for analyzing gene expression in the peripheral auditory and vestibular systems mandate the ability to isolate intact RNA from inner ear tissue. Methods presented here facilitate preparation of high quality inner ear RNA from larval and post-metamorphic Xenopus specimens that can be used for a variety of purposes. We demonstrate that RNA isolated with these protocols is suitable for microarray analysis of inner ear organs, and for cloning of large transcripts, such as those for ion channels. Genetic sequences cloned with these procedures can be used for transient transfection of Xenopus kidney cell lines with GFP fusion constructs.

Cell proliferation during the early compartmentalization of the Xenopus laevis inner ear.

The auditory and vestibular endorgans of the inner ear which are essential for the senses of hearing and balance form early during development when the otocyst undergoes a period of rapid growth and compartmentalization. Here we show the spatial and temporal patterns of proliferating cells in the Xenopus laevis inner ear as this organ develops from an otic vesicle at stage 31 until stage 47, an age at which compartmentalization and the initial appearance of sensory structures are evident. Sites of new cell production were identified in specimens at stages 31, 37, 42, 45 and 47 using immunohistochemical methods to detect bromodeoxyuridine (BrdU) incorporation three hours after exposure to this thymidine analogue. Cells undergoing terminal mitosis at stages 37, 42 and 45 were detected by exposing specimens at these stages to BrdU and permitting development to proceed until stage 47. Our results show that while newly replicating cells are uniformly distributed throughout the stage 31 otic vesicle, they are spatially restricted in stages 37 through 45, with few dividing cells visible in the central patches of the emerging sensory epithelia. In contrast, no clear proliferative pattern was discerned at stage 47. BrdU-positive cells that had undergone terminal mitosis at stage 37, 42 and 45 were detected in the central regions of nascent sensory epithelia at stage 47. These findings are consistent with a developmental mechanism in which cells undergoing terminal mitosis during early X. laevis stages contribute to sensory epithelia and in which cell mixing and migration are features of inner ear compartmentalization.

Tissue and species differences in the application of quantum dots as probes for biomolecular targets in the inner ear and kidney.

Quantum dots (QDs) are useful biological probes because of the increased photostability and quantum efficiency they offer over organic fluorophores. However, toxicity concerns arise because the QD core is composed of cadmium and selenium, metals known to be unsafe for humans and animals. We investigated the feasibility of quantum dots as biological labels for imaging studies of inner ear and kidney, tissues that share a polarized epithelial arrangement and drug susceptibility. We found that methods for labeling the actin cytoskeleton of monolayers of cultured amphibian kidney cells (Xenopus A6) with 565 nm QD conjugates were not feasible with large Xenopus inner ear organs. We then compared the uptake of 565 nm cationic peptide-targeted and nontargeted QDs in live kidney cell lines (amphibian, A6 and XLK-WG; human, HEK-293). Results showed that targeted QDs are internalized by all three kidney cell lines, and that nontargeted CdSe nanocrystals are sequestered only by human kidney cells. CellTracker Red CMTPX confirmed the membrane integrity and viability of HEK-293 cells that internalized QDs. Our results demonstrate species and tissue differences in QD uptake and labeling, and underscore the need for long-term studies of QD toxicity and fate in cells.

Inner ear formation during the early larval development of Xenopus laevis.

The formation of the eight independent endorgan compartments (sacculus, utricle, horizontal canal, anterior canal, posterior canal, lagena, amphibian papilla, and basilar papilla) of the Xenopus laevis inner ear is illustrated as the otic vesicle develops into a complex labyrinthine structure. The morphology of transverse sections and whole-mounts of the inner ear was assessed in seven developmental stages (28, 31, 37, 42, 45, 47, 50) using brightfield and laser scanning confocal microscopy. The presence of mechanosensory hair cells in the sensory epithelia was determined by identification of stereociliary bundles in cryosectioned tissue and whole-mounts of the inner ear labeled with the fluorescent F-actin probe Alexa-488 phalloidin. Between stages 28 and 45, the otic vesicle grows in size, stereociliary bundles appear and increase in number, and the pars inferior and pars superior become visible. The initial formation of vestibular compartments with their nascent stereociliary bundles is seen by larval stage 47, and all eight vestibular and auditory compartments with their characteristic sensory fields are present by larval stage 50. Thus, in Xenopus, inner ear compartments are established between stages 45 and 50, a 2-week period during which the ear quadruples in length in the anteroposterior dimension. The anatomical images presented here demonstrate the morphological changes that occur as the otic vesicle forms the auditory and vestibular endorgans of the inner ear. These images provide a resource for investigations of gene expression patterns in Xenopus during inner ear compartmentalization and morphogenesis.