Deep Convolutional Neural Networks for Annotating Gene Expression Patterns in the Mouse Brain

Background: Profiling gene expression in brain structures at various spatial and temporal scales is essential to understanding how genes regulate the development of brain structures. The Allen Developing Mouse Brain Atlas provides high-resolution 3-D in situ hybridization (ISH) gene expression patterns in multiple developing stages of the mouse brain. Currently, the ISH images are annotated with anatomical terms manually. In this paper, we propose a computational approach to annotate gene expression pattern images in the mouse brain at various structural levels over the course of development. Results: We applied deep convolutional neural network that was trained on a large set of natural images to extract features from the ISH images of developing mouse brain. As a baseline representation, we applied invariant image feature descriptors to capture local statistics from ISH images and used the bag-of-words approach to build image-level representations. Both types of features from multiple ISH image sections of the entire brain were then combined to build 3-D, brain-wide gene expression representations. We employed regularized learning methods for discriminating gene expression patterns in different brain structures. Results show that our approach of using convolutional model as feature extractors achieved superior performance in annotating gene expression patterns at multiple levels of brain structures throughout four developing ages. Overall, we achieved average AUC of 0.894 ± 0.014, as compared with 0.820 ± 0.046 yielded by the bag-of-words approach. Conclusions: Deep convolutional neural network model trained on natural image sets and applied to gene expression pattern annotation tasks yielded superior performance, demonstrating its transfer learning property is applicable to such biological image sets

Deep convolutional neural networks for annotating gene expression patterns in the mouse brain

Abstract Background Profiling gene expression in brain structures at various spatial and temporal scales is essential to understanding how genes regulate the development of brain structures. The Allen Developing Mouse Brain Atlas provides high-resolution 3-D in situ hybridization (ISH) gene expression patterns in multiple developing stages of the mouse brain. Currently, the ISH images are annotated with anatomical terms manually. In this paper, we propose a computational approach to annotate gene expression pattern images in the mouse brain at various structural levels over the course of development. Results We applied deep convolutional neural network that was trained on a large set of natural images to extract features from the ISH images of developing mouse brain. As a baseline representation, we applied invariant image feature descriptors to capture local statistics from ISH images and used the bag-of-words approach to build image-level representations. Both types of features from multiple ISH image sections of the entire brain were then combined to build 3-D, brain-wide gene expression representations. We employed regularized learning methods for discriminating gene expression patterns in different brain structures. Results show that our approach of using convolutional model as feature extractors achieved superior performance in annotating gene expression patterns at multiple levels of brain structures throughout four developing ages. Overall, we achieved average AUC of 0.894 ± 0.014, as compared with 0.820 ± 0.046 yielded by the bag-of-words approach. Conclusions Deep convolutional neural network model trained on natural image sets and applied to gene expression pattern annotation tasks yielded superior performance, demonstrating its transfer learning property is applicable to such biological image sets.http://deepblue.lib.umich.edu/bitstream/2027.42/111637/1/12859_2015_Article_553.pd

Altered metabolic activity in the developing brain of rats predisposed to high versus low depression-like behavior

Individual differences in human temperament can increase risk for psychiatric disorders like depression and anxiety. Our laboratory utilized a rat model of temperamental differences to assess neurodevelopmental factors underlying emotional behavior differences. Rats selectively bred for low novelty exploration (Low Responders, LR) display high levels of anxiety- and depression-like behavior compared to High Novelty Responder (HR) rats. Using transcriptome profiling, the present study uncovered vast gene expression differences in the early postnatal HR versus LR limbic brain, including changes in genes involved in cellular metabolism. These data led us to hypothesize that rats prone to high (versus low) anxiety/depression-like behavior exhibit distinct patterns of brain metabolism during the first weeks of life, which may reflect disparate patterns of synaptogenesis and brain circuit development. Thus, in a second experiment we examined activity of Cytochrome C Oxidase (COX), an enzyme responsible for ATP production and a correlate of metabolic activity, to explore functional energetic differences in HR/LR early postnatal brain. We found that HR rats display higher COX activity in the amygdala and specific hippocampal subregions compared to LRs during the first 2 weeks of life. Correlational analysis examining COX levels across several brain regions and multiple early postnatal time points suggested desynchronization in the developmental timeline of the limbic HR versus LR brain during the first two postnatal weeks. These early divergent COX activity levels may reflect altered circuitry or synaptic activity in the early postnatal HR/LR brain, which could contribute to the emergence of their distinct behavioral phenotypes

Cambios epigenómicos y transcriptómicos durante el envejecimiento en pez killi

El envejecimiento es un proceso universal que supone el declive de las funciones fisiológicas de los organismos. Es factor de riesgo para diversas enfermedades y su última consecuencia es la muerte. Al ser el cerebro un regulador clave de la homeostasis en organismos multicelulares, los efectos del envejecimiento en este tejido pueden afectar a otros procesos. Para el estudio del envejecimiento se han empleado organismos modelo invertebrados debido a la relativamente alta longevidad de organismos vertebrados. En los últimos años, ha aparecido un nuevo modelo vertebrado de vida corta que ha ganado interés en este campo, el pez killi (Nothobranchius furzeri). En este trabajo se ha estudiado cómo el envejecimiento es regulado en el cerebro del pez killi atendiendo a la información transcriptómica y epigenómica. Para ello se ha analizado genes y regiones accesibles de la cromatina usando las técnicas RNA-seq y ATAC-seq. Nuestros resultados sugieren que redes regulatorias relacionadas con el desarrollo aparecen alteradas durante el envejecimiento. Esto aporta evidencias en favor de la teoría del antagonismo pleiotrópico, donde se defiende que genes beneficiosos durante las primeras etapas de la vida pueden ser perjudiciales en etapas tardías

Analysis of candidate genes for behavioral differences in mice

Organisms have evolved different behavioral strategies for better survival and reproduction. However, the genetic basis for such traits remains still as a longstanding fascinating question in evolutionary biology. Mate choice strategy is one of the behavioral traits which can play an important role in the life history of organisms. A previous study had shown that mate choice preference between two populations of the Western house mouse (M. m. domesticus) is influenced by the genetic background of the fathers. Transcriptome analysis in a follow up study revealed an imprinted cluster on Chromosome 7, known as Prader-Willi Syndrome (PWS) region, and also Peg13 on Chromosome 15 as loci that are highly differentiated between mouse populations and therefore have been suggested as potential regions which may regulate this paternal preference in the house mouse. The present thesis was aimed to investigate the functional role of these two imprinted regions in Western house mice behavior. In the first chapter, I investigated the role of the PWS region on Chromosome 7 through a variety of techniques, including copy number variation analysis, behavioral correlations and transcriptomics. I found that two paternally imprinted tandemly repeated regulatory RNA coding genes (SNORD115 and SNORD116) are of special interest. Their copy number evolves very fast and correlates highly with personality traits between individuals. Further I found that the copy number variation influences the expression of more than 130 genes including genes involved in serotonin regulation, vocalization and bone development. The findings suggest a molecular mechanism for the generation and variability of personality traits in mammals. The second chapter focuses on the analysis of paternally imprinted Peg13 gene, which has so far not been functionally studied. This gene has been known as non-coding gene, but data from ribosomal profiling analysis revealed that this gene could indeed produce a small peptide and it is not a simple non-coding gene. By using knock out mice, I showed even a partial deletion at 3’ part of Peg13 could significantly change expression of hundreds genes in the brain and could thus influence various mouse behaviors. The phenotypic analysis showed a significant effect on mouse sexual and parental behavior. Since RNAseq analysis from mouse brain development timeline showed that this gene has highest expression in day 12.5, I propose that Peg13 may play important role in preoptic area development with possible direct role in sexual and mate choice behavior. 10 Overall the work presented in this thesis describes the first major molecular mechanism underlying mouse personality traits. It presents also the first functional study on the Peg13 gene in mice, which highlights its possible role in mouse brain development and sexual behavior.Contents Summary ................................................................................................................................................. 9 Zusammenfassung ................................................................................................................................. 11 General introduction ............................................................................................................................. 14 Behavior and evolution ..................................................................................................................... 14 Mate choice ....................................................................................................................................... 14 Assortative mating ............................................................................................................................ 15 House mouse and paternal mate choice preference .......................................................................... 16 1-Competitive Ability .................................................................................................................... 17 2-Personality ................................................................................................................................. 18 3-Ultrasonic Vocalization ................................................................................................................. 18 Genomic imprinting .......................................................................................................................... 19 Genomic imprinting and brain development .................................................................................... 19 Imprinted gene and mate choice preference in house mouse ............................................................ 20 Aim of the project ............................................................................................................................. 22 Chapter1 ................................................................................................................................................ 24 Involvement of the Prader-Willi Syndrome (PWS) region genes in mouse behavior .......................... 24 Introduction ....................................................................................................................................... 25 Analysis of SNORD Copy Number Variation .................................................................................. 28 SNORD115 and 116 target genes ..................................................................................................... 30 Link between SNORD copy number and personality ....................................................................... 34 Correlation between SNORD115 and 116 copy numbers ................................................................ 42 Inheritance of SNORD copy numbers .............................................................................................. 45 SNORD copy number differences across tissues .............................................................................. 50 Variation of personality in inbred mice ............................................................................................ 51 SNORD copy number influence on the brain transcriptome ............................................................ 54 SNORD116 copy number and craniofacial features ......................................................................... 60 Differential gene expression through SNORD CNV ........................................................................ 64 Discussion ......................................................................................................................................... 66 SNROD115/ 116 CNV and personality ............................................................................................ 66 Correlation of personality and cognitive ability ............................................................................... 67 Correlation of personality and metabolism ....................................................................................... 68 Correlation of personality traits and vocalization ............................................................................. 68 Personality and craniofacial features ................................................................................................ 69 SNOD115/ 116 and the Prader-Willi syndrome ............................................................................... 69 6 Inheritance of SNROD115 and 116 .................................................................................................. 70 Mice sample ...................................................................................................................................... 71 Mouse keeping .................................................................................................................................. 71 Mouse dissection ............................................................................................................................... 71 DNA extraction ................................................................................................................................. 71 mRNA extraction and cDNA synthesis ............................................................................................ 72 RNAseq analysis ............................................................................................................................... 73 Small RNA extraction, cDNA synthesis, library preparation and sequencing ................................. 73 Read mapping ................................................................................................................................... 74 snoRNA analysis ............................................................................................................................... 75 Droplet digital PCR ........................................................................................................................... 75 1- Finding a suitable reference gene ............................................................................................ 76 2-Primer designing ....................................................................................................................... 76 3-DNA digestion and ddPCR ........................................................................................................ 77 4-Copy Number Calculation ......................................................................................................... 78 Mouse Personality ............................................................................................................................. 81 Behavioral Tests................................................................................................................................ 81 Elevated Plus Maze ........................................................................................................................... 81 Open Field ......................................................................................................................................... 81 Dark/Light Box ................................................................................................................................. 81 Statistical Analysis ............................................................................................................................ 83 Shape phenotyping ............................................................................................................................ 85 Chapter 2 ............................................................................................................................................... 90 Functional analysis of the paternally expressed gene Peg13 ................................................................ 90 Introduction ....................................................................................................................................... 91 Results ............................................................................................................................................... 93 Peg13 and Trappc9 Expression ........................................................................................................ 93 Peg13 expression in the brain ........................................................................................................... 96 Peg13 expression during developmental stages ................................................................................ 97 A possible PEG13 protein ................................................................................................................. 98 Generation of Knock-out mice .......................................................................................................... 99 Peg13 3’-knockout mouse .............................................................................................................. 101 Behavioral tests on Peg13 3’-region knockout mice ...................................................................... 102 RNAseq analysis on Peg13 3’- region knockout mice ................................................................... 108 GO analysis ..................................................................................................................................... 109 7 Discussion ....................................................................................................................................... 109 Expression of Peg13, Kcnk9 and Trappc9 are co-regulated ........................................................... 109 Peg13 is not a simple non-coding RNA .......................................................................................... 110 Behavior of Peg13 3’-region knockout mice .................................................................................. 110 Anxiety behavior of Peg13 3’-KO mice ......................................................................................... 111 Conclusion ...................................................................................................................................... 112 Material and Methods ..................................................................................................................... 113 RNAseq analysis across different tissues ........................................................................................ 113 Ribosomal profiling data analysis ................................................................................................... 113 Generation of knock-out mice by genOway ................................................................................... 113 Novel Object Test ........................................................................................................................... 114 Other procedures ............................................................................................................................. 114 General Discussion ............................................................................................................................. 116 The general background underlying this PhD thesis....................................................................... 116 The PWS region may be associated to the paternal mate choice preference in Western house mouse by regulating mouse personality ..................................................................................................... 117 Evolution of personality .................................................................................................................. 117 1-Selective Neutrality .................................................................................................................. 117 2-Mutation-selection balance ..................................................................................................... 118 3-Balancing Selection ................................................................................................................. 122 Personality and imprinting .............................................................................................................. 123 1-Different recombination rate between male and female .......................................................... 123 2-Co-adapted gene expression .................................................................................................... 123 Peg13 could be involved in sexual behavior in mouse ................................................................... 124 Conclusion .......................................................................................................................................... 126 Reference ............................................................................................................................................ 128 Acknowledgements ............................................................................................................................. 151 Contributions to the thesis .................................................................................................................. 153 Declaration .......................................................................................................................................... 154 Curriculum Vitae ................................................................................................................................ 15

Normal cognitive and social development require posterior cerebellar activity

Cognitive and social capacities require postnatal experience, yet the pathways by which experience guides development are unknown. Here we show that the normal development of motor and nonmotor capacities requires cerebellar activity. Using chemogenetic perturbation of molecular layer interneurons to attenuate cerebellar output in mice, we found that activity of posterior regions in juvenile life modulates adult expression of eyeblink conditioning (paravermal lobule VI, crus I), reversal learning (lobule VI), persistive behavior and novelty-seeking (lobule VII), and social preference (crus I/II). Perturbation in adult life altered only a subset of phenotypes. Both adult and juvenile disruption left gait metrics largely unaffected. Contributions to phenotypes increased with the amount of lobule inactivated. Using an anterograde transsynaptic tracer, we found that posterior cerebellum made strong connections with prelimbic, orbitofrontal, and anterior cingulate cortex. These findings provide anatomical substrates for the clinical observation that cerebellar injury increases the risk of autism

Analysis of candidate genes for behavioral differences in mice

Animals show behavioral traits that can collectively be called personality and can be measured by standardized tests. We have studied the Prader-Willi Syndrom (PWS) gene region which includes two clusters of tandem repeats coding for small nucleolar RNAs, SNORD115 and SNORD116. SNORD115 is known to regulate splicing of the serotonin receptor Ht2cr and SNORD116 is predicted to interact with the transcript of the chromatin regulator Ankrd11. We show here that both snoRNA clusters display major copy number variation within and between populations and that this affects the expression of their specific target genes. Using a set of behavioral scores related to personality in mice, we find a strong correlation between the snoRNA copy number and these scores. Our results suggest that the SNORD clusters in the PWS region are major regulators of personality and correlated traits

The roles of podoplanin and clec-2 in the development and maintenance of the cerebral vasculature

The C-type lectin-like receptor, CLEC-2, is constitutively expressed on platelets, with reported expression on a number of leukocyte subsets in adult mice. Constitutive or platelet-specific deletion of CLEC-2 in mice induces cerebral haemorrhaging by midgestation. In this thesis, I investigated the basis of this defect, hypothesising that it is mediated by the loss of CLEC-2 activation by its endogenous ligand, podoplanin, expressed on the developing neural tube. Podoplaninfl/fl mice were crossed to mice expressing PGK-Cre to induce deletion of podoplanin at the two-cell stage. Developing blood vessels were visualized by 3-dimensional microscopy and found to be aberrantly patterned in CLEC-2- and podoplanin-deficient mice, culminating in widespread cerebral haemorrhaging by mid-gestation. Haemorrhages were also observed following Nestin-Cre driven deletion of podoplanin on neural progenitors and following deletion of the platelet integrin, αIIbβ3. Together these studies support that neuro-epithelial-derived podoplanin interacts with platelet-CLEC-2 to guide the maturation and integrity of the cerebral vasculature and to prevent haemorrhage by stimulating platelet aggregation. Using tamoxifen-inducible deletion of CLEC-2 in adult mice, the expression profile of CLEC-2 was investigated and shown to be restricted to platelets and circulating B-lymphocytes and CD11bhigh Gr1high myeloid cells. Furthermore, loss of CLEC-2 in adult mice was shown to be dispensable for maintaining blood-brain barrier permeability

The contribution of MAPKs to microglial immune responses, and a pathogen type comparison study to probe maternal immune activation effects on the foetal brain

Schizophrenia is a psychiatric disorder, and despite a rapid increase in understanding of the disease both clinically and preclinically, the aetiology of the disease remains somewhat uncertain. Inflammation is increasingly correlated with schizophrenia, with evidence from patients' serum and post-mortem brain samples. Moreover, in the brain, microglia are the primary cells to respond to immune stimulation; thus, their cellular processes in neuroinflammation in terms of contribution of schizophrenia-related pathological changes should be investigated. Regarding microglial immune responses, this study looks specifically at the mitogen-activated protein kinase (MAPK) pathways, including c-jun N-terminal kinase (JNK), because the evidence from genome-wide genetic association studies suggests that some of the genetic risk factors for schizophrenia are related to this pathway. Furthermore, as suggested from the neurodevelopmental theory, maternal stress, or infection during pregnancy, is one risk factor in the development of schizophrenia in the offspring. Indeed, Maternal Immune Activation (MIA) animal studies have suggested that prenatal immune stress could negatively affect CNS development and offspring behaviours. This thesis hypothesises that maternal immune activation caused by environmental challenges affects foetal microglial immunity via the MAPK pathway, and that differing responses are observed depending on the nature of the immune challenge. In order to examine the statement, this thesis introduces research to study the neurobiological functions of MAPKs in neuronal (primary mouse cortical cultured neurons) and microglial (SIM-A9 mouse cell line) cells, with preclinical studies of the impact of MIA in mice on immunological changes following the administration of different inflammatory agents. Results in cultured primary mouse cortical neurons showed that two different TLR agonists, LPS (TLR4 agonist), and resiquimod (TLR7/8 agonist), did not cause any significant changes in the level of activated phospho-JNKs (pJNKs); however, poly I:C (TLR3 agonist) stimulation showed potential effects on JNK activation. Interestingly, chemokine stimulation, with CXCL10 in particular, affected the level of pJNKs. Continuing the in vitro studies, microglial immune responses were investigated. Resiquimod significantly increased the activation levels of MAPKs. LPS also increased the activation levels of MAPKs; however, it required a longer time than resiquimod. Interestingly, poly I:C did not significantly increase activation levels of any MAPKs. LPS and resiquimod initiated microglial immune responses (measured by RT-qPCR and ELISA); however, protein levels did not always reflect mRNA level changes. Besides, MAPK pathway contributions to the microglial immune reactions were suggested via MAPK inhibitor experiments, but in a stimulusdependent manner. Interestingly, investigation of microglial markers, Tspo, Aif-1, and Tmem119, following TLR-mediated stimulations in microglial cells (SIM-A9) showed changes that did not necessarily reflect levels of microglial activity. Alongside these in vitro studies, MIA models, induced by dsRNA (poly I:C) and ssRNA (resiquimod) virus mimetics, were investigated. Initial data suggested that the level of CXCL12 in placental tissues was increased by maternal exposure to poly I:C, but placental CXCL10 was not affected, even though poly I:C administration meaningfully induced maternal inflammation. The data from the main MIA study suggested that administration of a TLR7/8 (resiquimod), but not a TLR3 (poly I:C), agonist, induced the upregulation of cytokine and chemokine expression in embryo brains, even though the evidence of inflammation caused by both poly I:C and resiquimod was detected in maternal serum and placentae. These findings suggest that MIA has the potential to alter foetus brain immune status and that the response could be pathogen dependent, with single-stranded RNA (ssRNA) virus exposure potentially producing greater effects on the foetus. Corresponding with the in vitro microglia experiment, microglial markers in foetal brains were changed by MIA; these findings indicated these markers are changed by environmental conditions rather than remaining stable. These data provide compelling evidence of the roles of MAPKs in microglial involved neuroinflammation, although the precise action of these signalling molecules on microglia related pathology in schizophrenia remains unclear. In addition to previous works, a significant impact of maternal ssRNA virus (resiquimod) exposure on the developing foetal brain, but the doublestranded RNA (dsRNA) (poly I:C) virus did not. This means that resiquimod is potentially more effective than imiquimod or poly I:C in terms of producing an immune response; therefore, an MIA model using resiquimod may be a good model for the study of environmental contribution to psychiatric disease risk. Further work into the interaction between environmental and genetic factors in the MIA, and associated behaviour changes at later developmental ages, will provide insight into how maternal immune reactions and foetal CNS immune reactions are related to foetal brain development

Behavioural and molecular characterisation of mice haploinsufficient for Map2k7, a schizophrenia risk gene

Schizophrenia is a serious psychiatric disorder characterised by a breakdown in thought, emotion and perception, which leads to alterations of normal behaviour and feelings, a withdrawal from reality and an impression of mental defragmentation. Of the positive, negative and cognitive symptoms, the positive symptoms are perhaps the most striking. However, it is the severity of cognitive deficits that are most closely associated with a patients’ functional outcome in the long-term. Despite this, the successful treatment of the cognitive deficits has been met with difficulty, partly due to a lack of suitable animal models. There is an urgent need for animal models with appropriate face, construct and predictive validity for schizophrenia so that improved drug targets can be identified, and new drugs tested. In 2012, Winchester et al. discovered that sequence variations in the Map2k7 gene were associated with increased risk for schizophrenia, and Map2k7 mRNA was decreased in the prefrontal cortex of the post mortem brains of patients. The primary aim of this thesis is to behaviourally and molecularly characterise mice which are heterozygous for Map2k7 (Map2k7+/- mice) as a potential animal model of relevance to schizophrenia. Sequence variants in the Map2k7 gene are moderately common in the population and they almost double the disease risk (OR~1.9); hence, alterations of the Map2k7 gene in mice represent an ideal basis for an animal model with good construct validity. The Map2k7 gene produces the MKK7 protein, a kinase within the stress-activated JNK pathway, and is involved in a diverse range of cellular processes, such as apoptosis, synaptic plasticity and regulation of the immune response. First and foremost, the components of the MKK7/JNK pathway were quantified in Map2k7+/- mice and MKK7γ was found to be significantly decreased in the prefrontal cortex compared to their wildtype (WT) littermates, a highly disrupted brain region in patients with schizophrenia. Map2k7+/- mice also exhibited behavioural phenotypes relevant to schizophrenia: hyperactivity in the open field and attentional dysfunction. Minocycline showed promise in alleviating the attentional deficits and hyperactivity in the open field, but did not influence protein levels of signalling pathway components. Map2k7+/- mice did not show a decrease in sensorimotor gating as many patients do; however, they exhibited signs of altered response to amphetamine administration just prior to testing of sensorimotor gating, compared to WT mice. Decision-making abilities were also investigated: Map2k7+/- mice showed normal learning and performance of the rodent gambling task. Additionally, all mice were able to alter their choice pattern to be more optimal when the task contingencies were subtly switched, which was the first time this has been shown in mice in the touchscreen apparatus. However, when the task demands were altered such that ‘punishment’ no longer featured as prominently, Map2k7+/- mice showed huge difficulty compared to their WT littermates in shifting their choice pattern to be more optimal, suggesting they have a deficit in aspects of cognitive flexibility. Finally, Map2k7+/- mice were investigated as a gene x environment interaction model, by injecting pregnant dams with Poly I:C and examining the resultant immune response in maternal serum and embryonic brain. Map2k7+/- dams exhibited an altered immune response to Poly I:C compared to WT dams; however, future experiments will be required to confirm whether this altered cytokine response is also present in embryonic brain. Overall, Map2k7+/- mice show utility for dissecting the cognitive deficits and some aspects of the positive symptoms of schizophrenia that could be targeted by novel compounds. This would be aimed at restoring the function of the MKK7/JNK pathway. Further molecular and behavioural characterisation will be required, particularly into the potential gene x environment interaction model. Although no mouse model can recapitulate the full symptom spectrum of a human neuropsychiatric disorder, Map2k7+/- mice exhibit an interesting accumulation of phenotypic abnormalities relevant to schizophrenia