Fuyi Chen

Knockdown of the Dyslexia-Associated Gene Kiaa0319 Impairs Temporal Responses to Speech Stimuli in Rat Primary Auditory Cortex

One in 15 school age children have dyslexia, which is characterized by phoneme-processing problems and difficulty learning to read. Dyslexia is associated with mutations in the gene KIAA0319. It is not known whether reduced expression of KIAA0319 can degrade the brains ability to process phonemes. In the current study, we used RNA interference (RNAi) to reduce expression of Kiaa0319 (the rat homolog of the human gene KIAA0319) and evaluate the effect in a rat model of phoneme discrimination. Speech discrimination thresholds in normal rats are nearly identical to human thresholds. We recorded multiunit neural responses to isolated speech sounds in primary auditory cortex (A1) of rats that received in utero RNAi of Kiaa0319. Reduced expression of Kiaa0319 increased the trial-by-trial variability of speech responses and reduced the neural discrimination ability of speech sounds. Intracellular recordings from affected neurons revealed that reduced expression of Kiaa0319 increased neural excitability and input resistance. These results provide the first evidence that decreased expression of the dyslexia-associated gene Kiaa0319 can alter cortical responses and impair phoneme processing in auditory cortex.

A method for stable transgenesis of radial glia lineage in rat neocortex by piggyBac mediated transposition.

Methods that combine lineage tracing with cellular transgenesis are needed in order to determine mechanisms that specify neural cell types. Currently available methods include viral infection and Cre-mediated recombination. In utero electroporation (IUE) has been used in multiple species to deliver multiple transgenes simultaneously into neural progenitors. In standard IUE, most plasmids remain episomal, are lost during cell division, and so transgenes are not expressed in the complete neural lineage. Here we combine IUE with a binary piggyBac transposon system (PB-IUE), and show that unlike conventional IUE, a single embryonic transfection of neocortical radial glia with a piggyBac transposon system results in stable transgene expression in the neural lineage of radial glia: cortical neurons, astrocytes, oligodendrocytes, and olfactory bulb interneurons. We also developed a modular toolkit of donor and helper plasmids with different promoters that allows for shRNA, bicistronic expression, and trangenesis in subsets of progenitors. As a demonstration of the utility of the toolkit we show that transgenesis of epidermal growth factor receptor (EGFR) expands the number of astrocytes and oligodendrocyrtes generated from progenitors. The relative ease of implementation and experimental flexibility should make the piggyBac IUE method a valuable new tool for tracking and manipulating neural lineages.

Mutual antagonism between Sox10 and NFIA regulates diversification of glial lineages and glioma subtypes

Lineage progression and diversification is regulated by the coordinated action of unique sets of transcription factors. Oligodendrocytes (OL) and astrocytes (AS) comprise the glial sub-lineages in the CNS, and the manner in which their associated regulatory factors orchestrate lineage diversification during development and disease remains an open question. Sox10 and NFIA are key transcriptional regulators of gliogenesis associated with OL and AS. We found that NFIA inhibited Sox10 induction of OL differentiation through direct association and antagonism of its function. Conversely, we found that Sox10 antagonized NFIA function and suppressed AS differentiation in mouse and chick systems. Using this developmental paradigm as a model for glioma, we found that this relationship similarly regulated the generation of glioma subtypes. Our results describe the antagonistic relationship between Sox10 and NFIA that regulates the balance of OL and AS fate during development and demonstrate for the first time, to the best of our knowledge, that the transcriptional processes governing glial sub-lineage diversification oversee the generation of glioma subtypes.

Tracking and transforming neocortical progenitors by CRISPR/Cas9 gene targeting and piggyBac transposase lineage labeling

The ability to induce targeted mutations in somatic cells in developing organisms and then track the fates of those cells is a powerful approach both for studying neural development and for modeling human disease. The CRISPR/Cas9 system allows for such targeted mutagenesis, and we therefore tested it in combination with a piggyBac transposase lineage labeling system to track the development of neocortical neural progenitors with targeted mutations in genes linked to neurodevelopmental disruptions and tumor formation. We show that sgRNAs designed to target PTEN successfully decreased PTEN expression, and led to neuronal hypertrophy and altered neuronal excitability. Targeting NF1, by contrast, caused increased astrocytogenesis at the expense of neurogenesis, and combined targeting of three tumor suppressors (PTEN, NF1 and P53) resulted in formation of glioblastoma tumors. Our results demonstrate that CRISPR/Cas9 combined with piggyBac transposase lineage labeling can produce unique models of neurodevelopmental disruption and tumors caused by somatic mutation in neural progenitors. Summary: The CRISPR/Cas9 system is used to target three tumor suppressor genes (PTEN, NF1 and P53), alone and in combination with piggyBac transposase lineage marking, to analyze their function in the developing mouse brain.

Mechanistic Studies of the Genetically Encoded Fluorescent Protein Voltage Probe ArcLight

ArcLight, a genetically encoded fluorescent protein voltage probe with a large ΔF/ΔV, is a fusion between the voltage sensing domain of the Ciona instestinalis voltage sensitive phosphatase and super ecliptic pHluorin carrying a single mutation (A227D in the fluorescent protein). Without this mutation the probe produces only a very small change in fluorescence in response to voltage deflections (∼1%). The large signal afforded by this mutation allows optical detection of action potentials and sub-threshold electrical events in single-trials in vitro and in vivo. However, it is unclear how this single mutation produces a probe with such a large modulation of its fluorescence output with changes in membrane potential. In this study, we identified which residues in super ecliptic pHluorin (vs eGFP) are critical for the ArcLight response, as a similarly constructed probe based on eGFP also exhibits large response amplitude if it carries these critical residues. We found that D147 is responsible for determining the pH sensitivity of the fluorescent protein used in these probes but by itself does not result in a voltage probe with a large signal. We also provide evidence that the voltage dependent signal of ArcLight is not simply sensing environmental pH changes. A two-photon polarization microscopy study showed that ArcLights response to changes in membrane potential includes a reorientation of the super ecliptic pHluorin. We also explored different changes including modification of linker length, deletion of non-essential amino acids in the super ecliptic pHluorin, adding a farnesylation site, using tandem fluorescent proteins and other pH sensitive fluorescent proteins.

Fate Mapping by PiggyBac Transposase Reveals That Neocortical GLAST+ Progenitors Generate More Astrocytes Than Nestin+ Progenitors in Rat Neocortex

Progenitors within the neocortical ventricular zone (VZ) first generate pyramidal neurons and then astrocytes. We applied novel piggyBac transposase lineage tracking methods to fate-map progenitor populations positive for Nestin or glutamate and aspartate transpoter (GLAST) promoter activities in the rat neocortex. GLAST+ and Nestin+ progenitors at embryonic day 13 (E13) produce lineages containing similar rations of neurons and astrocytes. By E15, the GLAST+ progenitor population diverges significantly to produce lineages with 5-10-fold more astrocytes relative to neurons than generated by the Nestin+ population. To determine when birth-dated progeny within GLAST+ and Nestin+ populations diverge, we used a Cre/loxP fate-mapping system in which plasmids are lost after a cell division. By E18, birth-dated progeny of GLAST+ progenitors give rise to 2-3-fold more neocortical astrocytes than do Nestin+ progenitors. Finally, we used a multicolor clonal labeling method to show that the GLAST+ population labeled at E15 generates astrocyte progenitors that produce larger, spatially restricted, clonal clusters than the Nestin+ population. This study provides in vivo evidence that by mid-corticogenesis (E15), VZ progenitor populations have significantly diversified in terms of their potential to generate astrocytes and neurons.

Speech sound processing deficits and training-induced neural plasticity in rats with dyslexia gene knockdown.

In utero RNAi of the dyslexia-associated gene Kiaa0319 in rats (KIA-) degrades cortical responses to speech sounds and increases trial-by-trial variability in onset latency. We tested the hypothesis that KIA- rats would be impaired at speech sound discrimination. KIA- rats needed twice as much training in quiet conditions to perform at control levels and remained impaired at several speech tasks. Focused training using truncated speech sounds was able to normalize speech discrimination in quiet and background noise conditions. Training also normalized trial-by-trial neural variability and temporal phase locking. Cortical activity from speech trained KIA- rats was sufficient to accurately discriminate between similar consonant sounds. These results provide the first direct evidence that assumed reduced expression of the dyslexia-associated gene KIAA0319 can cause phoneme processing impairments similar to those seen in dyslexia and that intensive behavioral therapy can eliminate these impairments.

Knockdown of Dyslexia-Gene Dcdc2 Interferes with Speech Sound Discrimination in Continuous Streams.

Dyslexia is the most common developmental language disorder and is marked by deficits in reading and phonological awareness. One theory of dyslexia suggests that the phonological awareness deficit is due to abnormal auditory processing of speech sounds. Variants in DCDC2 and several other neural migration genes are associated with dyslexia and may contribute to auditory processing deficits. In the current study, we tested the hypothesis that RNAi suppression of Dcdc2 in rats causes abnormal cortical responses to sound and impaired speech sound discrimination. In the current study, rats were subjected in utero to RNA interference targeting of the gene Dcdc2 or a scrambled sequence. Primary auditory cortex (A1) responses were acquired from 11 rats (5 with Dcdc2 RNAi; DC−) before any behavioral training. A separate group of 8 rats (3 DC−) were trained on a variety of speech sound discrimination tasks, and auditory cortex responses were acquired following training. Dcdc2 RNAi nearly eliminated the ability of rats to identify specific speech sounds from a continuous train of speech sounds but did not impair performance during discrimination of isolated speech sounds. The neural responses to speech sounds in A1 were not degraded as a function of presentation rate before training. These results suggest that A1 is not directly involved in the impaired speech discrimination caused by Dcdc2 RNAi. This result contrasts earlier results using Kiaa0319 RNAi and suggests that different dyslexia genes may cause different deficits in the speech processing circuitry, which may explain differential responses to therapy. SIGNIFICANCE STATEMENT Although dyslexia is diagnosed through reading difficulty, there is a great deal of variation in the phenotypes of these individuals. The underlying neural and genetic mechanisms causing these differences are still widely debated. In the current study, we demonstrate that suppression of a candidate-dyslexia gene causes deficits on tasks of rapid stimulus processing. These animals also exhibited abnormal neural plasticity after training, which may be a mechanism for why some children with dyslexia do not respond to intervention. These results are in stark contrast to our previous work with a different candidate gene, which caused a different set of deficits. Our results shed some light on possible neural and genetic mechanisms causing heterogeneity in the dyslexic population.

Contribution of Tumor Heterogeneity in a New Animal Model of CNS Tumors

The etiology of central nervous system (CNS) tumor heterogeneity is unclear. To clarify this issue, a novel animal model was developed of glioma and atypical teratoid/rhabdoid-like tumor (ATRT) produced in rats by nonviral cellular transgenesis initiated in utero. This model system affords the opportunity for directed oncogene expression, clonal labeling, and addition of tumor-modifying transgenes. By directing HRasV12 and AKT transgene expression in different cell populations with promoters that are active ubiquitously (CAG promoter), astrocyte-selective (glial fibrillary acidic protein promoter), or oligodendrocyte-selective (myelin basic protein promoter) we generated glioblastoma multiforme and anaplastic oligoastrocytoma, respectively. Importantly, the glioblastoma multiforme and anaplastic oligoastrocytoma tumors were distinguishable at both the cellular and molecular level. Furthermore, proneural basic helix-loop-helix (bHLH) transcription factors, Ngn2 (NEUROG2) or NeuroD1, were expressed along with HRasV12 and AKT in neocortical radial glia, leading to the formation of highly lethal ATRT like tumors. This study establishes a unique model in which determinants of CNS tumor diversity can be parsed out and reveals that both mutation and expression of neurogenic bHLH transcription factors contribute to CNS tumor diversity. Implications: A novel CNS tumor model reveals that oncogenic events occurring in disparate cell types and/or molecular contexts lead to different tumor types; these findings shed light on the sources of brain tumor heterogeneity. Mol Cancer Res; 12(5); 742–53. ©2014 AACR.

Abstract 359: Systematic manipulation of brain tumor diversity using piggybac transposon mediated transgenesis approach in rat.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Animal models are important for interrogating the native biology of tumor formation, invasiveness, and may lead to novel therapeutic approaches. Here we describe the development of a rat glioma model which provides a fast and convenient approach to reproducibly induce brain tumors of different type in rat. We utilize a piggybac transposon system delivered by in utero electroporation (PB-IUE) to express oncogenic HRasV12 in different cell types in CNS. When expression of HRasV12 was driven by ubiquitous CAG promoter in radial glia cells, glioblastoma multiforme (GBM) was induced. When we specifically express HRasV12 in astrocytes using a mouse GFAP promoter, malignant astroglioma resembling GBM was induced in rat brains. In contrast, anaplastic oligoastrocytoma was induced when HRasV12 were expressed in oligodendrocytes under the control a rat MBP promoter. Surprisingly when co-expressing a basic helix loop helix protein Neurogenin2 (Ngn2) with HRasV12 both driven by CAG promoter, primitive neuroectodermal tumor (PNET) like tumors were generated. Molecular classification of tumors induced by CAG and GFAP HRasV12 matched mesenchymal GBM subtypes. Our data demonstrate the induction of different tumor types using a non-viral piggyBac transposon mediated transgenesis approach. The binary plasmid system we developed provides a method to direct different oncogenetic signals to different cell types. The models generated with this system should facilitate our understanding of the sources of tumor diversity. Citation Format: Fuyi Chen, Albert Becker, Joseph LoTurco. Systematic manipulation of brain tumor diversity using piggybac transposon mediated transgenesis approach in rat. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 359. doi:10.1158/1538-7445.AM2013-359