Spatiotemporal Regulation of Gene Expression during Plant Meristem Development

Plants are products of meristems. The root apical meristem (RAM) gives rise to the below-ground root system, while the shoot apical meristem (SAM) generates all the above-ground organs. The RAM and SAM contain highly organized stem cell niches, characterized by the presence of distinct cell types at various developmental stages. Meristems are thus ideal model tissues to study molecular regulatory mechanisms during development, via the generation of high-resolution expression atlases. In this dissertation, I aim to study the spatiotemporal regulation of gene expression during plant meristem development at a genome-wide level, including transcriptional regulation and post-transcriptional regulation. In my first study, high-throughput single cell RNA sequencing (scRNA-Seq) was used to build a cellular resolution gene expression atlas of the Arabidopsis root that includes all major cell types. In total, 4,727 single cell profiles were generated and analyzed. Developmental trajectories along root development were built. These depict a cascade of developmental progressions from stem cell to final differentiation. New regulators and downstream genes that define cell types or control cell state transition during the development were identified. This study demonstrates the power of applying scRNA- Seq to plants, and provides a unique spatiotemporal perspective of root cell differentiation. In my second study, a high-resolution maize shoot apex expression atlas in appendix II (Knauer et al., 2019) was used to investigate the spatiotemporal action of microRNAs (miRNAs) during development. Expression patterns of miRNA precursors and mature miRNA accumulation were examined, revealing that miRNA accumulation is regulated at both transcriptional and post-transcriptional level. Examples of the latter included effects on miRNA processing and/or stability in the vasculature and the stem cell population at the SAM tip, as well as the movement of miRNA within developing leaf primordia. By integrating data from RNA-Seq and degradome-Seq, a system was devised to predict the regulatory mechanism employed by miRNAs on their targets. This study provides a first comprehensive investigation of how the activity of the miRNAs that are critical to developmental pattern are regulated across space and time, revealing inputs from processes of regulating transcription, processing, stability, mobilities and miRNA efficacy

Modeling the Transcriptional Landscape of in vitro Neuronal Differentiation and ALS Disease

The spinal cord is a complex structure responsible for processing sensory inputs and motor outputs. As such, the developmental and spatial organization of cells is highly organized. Diseases affecting the spinal cord, such as Amyotrophic Lateral Sclerosis (ALS), result in the disruption of normal cellular function and intercellular interactions, culminating in neurodegeneration. Deciphering disease mechanisms requires a fundamental understanding of both the normal development of cells within the spinal cord as well as the homeostatic environment that allows for proper function. Biological processes such as cellular differentiation, maturation, and disease progression proceed in an asynchronous and cell type-specific manner. Until recently, bulk measurements of a mixed population of cells have been key in understanding these events. However, bulk measurements can obscure the molecular mechanisms involved in branched or coinciding processes, such as differential transcriptional responses occurring between subpopulations of cells. Measurements in individual cells have largely been restricted to 4 color immunofluorescence assays, which provide a solid but limited view of molecular-level changes. Recently, developments in single cell RNA-sequencing (scRNA-Seq) have provided an avenue of accurately profiling the RNA expression levels of thousands of genes concomitantly in an individual cell. With this increased experimental precision comes the ability to explore pathways that are differentially activated in subpopulations of cells, and to determine the transcriptional programs that underlie complex biological processes. In this dissertation, I will first review the key features of scRNA-Seq and downstream analysis. I will then discuss two applications of scRNA-seq: 1) the in vitro differentiation of mouse embryonic stem cells into motor neurons, and 2) the effect of the ALS-associated gene SOD1G93A expression on cultured motor neurons in a cellular model of ALS

Pathways linking atherosclerosis to aortic stenosis

Cardiovascular disease is the most common cause of death world-wide where atherosclerosis is the main culprit and aortic valve disease accounts about two percent of all CVD deaths. Atherosclerosis is a lipid and inflammation driven disease that share many features with aortic valve stenosis (AVS). Globally, the prevalence of AVS has been estimated to over 10 million patients and the incidence to over 12 500 new cases annually which is likely increasing due to increased longevity, yet no medical treatment is available. A link between atherosclerosis and AVS has previously been established by overlapping prevalence and common pathobiological hallmarks including lipid infiltration, inflammation, and calcification. Recent genetic studies have demonstrated several loci in which single nucleotide polymorphisms are associated with both diseases. However, there is also evidence pointing to separate etiologies including disease specific genetic risk factors, histopathological differences, and isolated clinical presentation. The aim of this thesis was to establish the interplay between atherosclerosis and AVS. A physiologic part was covered in Article I, specific mechanisms in Article II-IV and molecular epidemiology in Article IV. In Article I, arterial stiffness was determined in a cohort with ascending aortic dilatation and/or aortic valve disease before and after cardiac surgery. Arterial stiffness correlates with atherosclerotic cardiovascular disease and aggravates the increased left ventricular stress in AVS. Cardio-ankle vascular index (CAVI) measures arterial stiffness from the heart to the ankle and was lower in subjects with AVS compared with aortic regurgitation and ascending aortic dilation, before surgery, despite being older. In contrast, aortic stiffness assessed by carotid femoral pulse wave velocity (cfPWV) was not different between the groups. After surgery, CAVI but not cfPWV increased in patients with AVS but remained unchanged in patients undergoing aortic surgery. Age, diabetes, lower body mass index, decreased ejection time and lower preoperative CAVI was associated with an increased CAVI after surgery. The results suggest that AVS may mask an increased arterial stiffness if peripheral arteries are included in the measurement. Also, ejection time emerged as an important variable to account for when measuring arterial stiffness in aortic valve disease patients. Future work should aim to establish if arterial stiffness may be used to risk-stratify AVS patients. In Article II, the impact of a single nucleotide polymorphism (SNP) within FADS1 on aortic valve gene expression and fatty acid composition was identified. Fatty acid desaturase (FADS)1 and FADS2 encode rate limiting enzymes in the metabolism of omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) and the SNP within this locus is associated with lower risk of both AVS and CAD. The SNP rs17547 was associated with FADS2 mRNA expression in calcified aortic valve tissue and the enzymatic activity of both FADS1 and FADS2. In addition, the aortic valve omega-3 PUFA docosahexaenoic acid proportion was higher in non-calcified compared with calcified tissue and positively correlated with the SNP. The results indicate that the protective effects of the SNP might be mediated via an increased DHA proportion in the aortic valve and/or possibly via downstream mediators from DHA such as specialized pro-resolving mediators which have been shown to dampen inflammation. Further pathophysiological evidence of shared pathways between CAD and AVS was obtained in Article III. The presence of antiphospholipid antibodies (aPL) in the general population is higher in patients with a recent myocardial infarction. Positivity for antibodies against β2-glycoprotein I and/or cardiolipin of IgG isotype was identified to be 8-fold higher in AVS patients compared with matched controls. In aortic valve tissue, aPL positivity was associated with downregulated interferon pathways and upregulated pathways related to mechanosensory signaling. Importantly, the differentially expressed genes could predict resilient (healthy), thickened (fibrotic) and calcified aortic valve tissue with high accuracy using supervised machine learning models suggesting a tight relationship between aPL related genes and local disease progression. The overall results imply that aPL IgG in the general population (without rheumatic disease) could be a risk factor for AVS and may potentially be used guide AVS precision medicine. In Article IV, CAD associated gene expression in aortic valve tissue was identified. First, the prevalence of CAD in a contemporary surgical tricuspid AVS cohort was established at 49% and was associated with claudication, smoking, male sex, and diabetes. An exploratory analysis of aortic valve transcriptomic data from 74 patients revealed that severe CAD, affecting 2 or 3 vessel territories, was associated with the most prominent difference in gene expression. The differentially expressed genes were primarily found in non-calcified tissue and were enriched in pathways related to oxidative stress, inflammation, and lipids. Furthermore, a supervised machine learning model could predict if aortic valve tissue stemmed from patients with severe CAD, at high accuracy. The most important gene predictors of severe CAD could further be used to predict atherosclerotic or macroscopically normal carotid artery tissue. The results suggest that AVS patients with concomitant severe CAD exhibit more atherosclerosis related mechanisms in non-calcified tissue, ultimately leading to a common end-stage disease with severe AVS. In summary, the results in this thesis demonstrate that AVS may be a cause of masked systemic arterial stiffness. Furthermore, pathways related to fatty acid metabolism and aPL are implicated in the pathophysiology of AVS and patients with severe CAD exhibit upregulated pathways related to atherosclerosis in the aortic valve. Collectively, pathways linking and differentiating aortic valve and vascular atherosclerotic disease were unraveled which open up for novel precision treatment regiments to halt AVS

The Molecular-enriched Functional Circuits Underlying Consciousness and Cognition

Homo Sapiens consist of trillions of atoms, each inanimate, yet somehow collectively constituting a conscious being. The fundamental question of how organisms are organised to beget consciousness and cognition has largely been approached through independent examination of the structure and function of the nervous system at varying levels of granularity. As neuroscience progresses, it has thus increasingly fragmented into separate streams of research which study the brain at these different scales. This has resulted in the field becoming “data rich, but theory poor”, which is largely attributable to the paucity of methods which bridge these levels of analysis to provide novel trans-hierarchical insights and inform unified theories. The research in this doctoral thesis therefore aims to explore how a specific type of multimodal analysis - Receptor-Enriched Analysis of functional Connectivity by Targets (REACT) – can begin to bridge the theoretic void between molecular level mechanisms and systems levels dynamics to provide novel perspectives on the function and dysfunction of the brain. First, I provide a narrative synthesis of the challenges precluding a meaningful understanding of the human brain utilising conventional functional neuroimaging and outlining how incorporation of molecular information may help overcome these limitations. Specifically, by embedding functional dynamics in the molecular landscape of the brain, we can begin to move from the simple characterisation of “where” cognitive phenomena may be within the brain towards mechanistic accounts of “how” they are produced. Additionally, this offers enticing opportunities to link pharmacological treatments to novel molecular-network based biomarkers. Second, I explore how networks enriched with the spatial configurations of serotonergic and dopaminergic receptor subtypes are modulated by lysergic acid diethylamide (LSD) as compared to placebo in healthy participants. The results highlight the challenges of disentangling pharmacodynamics of drugs exhibiting rich pharmacology as well as identifying differential relationship between serotonergic and dopaminergic networks and phenomenological sub- components of psychedelic state. Third, I expand the remit of molecular-enriched network analyses beyond pure psychopharmacology to examine the direct and indirect actions of propofol anaesthesia on inhibitory and modulatory neurotransmission at both rest as well as during a naturalistic listening task. This work demonstrates for the first time that these molecular-networks can capture broader perceptual and cognitive-driven network reconfigurations as well as indirect pharmacological actions on neuromodulatory systems. Moreover, it provides evidence that the effects of propofol on consciousness are enacted through both direct inhibitory as well as indirect neuromodulatory mechanisms.Finally, I produce normative models of networks enriched with the principal neuromodulatory, excitatory, and inhibitory transmitter systems, testing their capacity to characterise neural dysfunction within and across several neuropsychiatric disorders. This work provides a computational foundation for large scale integration of molecular mechanisms and functional imaging to provide novel individualised biomarkers for neuropsychiatric disorders. Collectively, this thesis offers methodological and theoretical progress towards a trans-hierarchical characterisation of the human brain, providing insights into the neural correlates of both conscious contents and level as well as the perturbations underlying key neuropsychiatric conditions

Biologically informed risk scoring in schizophrenia based on genome-wide omics data

Extensive efforts in characterizing the biological architecture of schizophrenia have moved psychiatric research closer towards clinical application. As our understanding of psychiatric illness is slowly shifting towards a conceptualization as dimensional constructs that cut across traditional diagnostic boundaries, opportunities for personalized medicine applications that are afforded by the application of advanced data science methods on the increasingly available, large-scale and multimodal data repositories are starting to be more broadly recognized. A particularly intriguing phenomenon is the discrepancy between the high heritability of schizophrenia and the difficulty in identifying predictive genetic signatures, for which polygenic risk scores of common variants that explain approximately 18% of illness-associated variance remain the gold standard. A substantial body of research points towards two lines of investigation that may lead to a significant advance, resolve at least in part the ‘missing heritability’ phenomenon, and potentially provide the basis for more predictive, personalized clinical tools. First, it is paramount to better understand the impact of environmental factors on illness risk and elucidate the biology underlying their impact on altered brain function in schizophrenia. This thesis aims to close a major gap in our understanding of the multivariate, epigenetic landscape associated with schizophrenia, its interaction with polygenic risk and its association with DLPFC-HC connectivity, a well-established and robust neural intermediate phenotype of schizophrenia. As a basis for this, we have developed a novel biologically-informed machine learning framework by incorporating systems-level biological domain knowledge, i.e., gene ontological pathways, entitled ‘BioMM’ using genome-wide DNA methylation data obtained from whole blood samples. An epigenetic poly-methylation score termed ‘PMS’ was estimated at the individual level using BioMM, trained and validated using a total of 2230 whole-blood samples and 244 post-mortem brain samples. The pathways contributing most to this PMS were strongly associated with synaptic, neural and immune system-related functions. The identified PMS could be successfully validated in two independent cohorts, demonstrating the robust generalizability of the identified model. Furthermore, the PMS could significantly differentiate patients with schizophrenia from healthy controls when predicted in DLPFC post-mortem brain samples, suggesting that the epigenetic landscape of schizophrenia is to a certain extent shared between the central and peripheral tissues. Importantly, the peripheral PMS was associated with an intermediate neuroimaging phenotype (i.e., DLPFC-HC functional connectivity) in two independent imaging samples under the working memory paradigm. However, we did not find sufficient evidence for a combined genetic and epigenetic effect on brain function by integrating PRS derived from GWAS data, which suggested that DLPFC-HC coupling was predominantly impacted by environmental risk components, rather than polygenic risk of common variants. The epigenetic signature was further not associated with GWAS-derived risk scores implying the observed epigenetic effect did likely not depend on the underlying genetics, and this was further substantiated by investigation of data from unaffected first-degree relatives of patients with SCZ, BD, MDD and autism. In summary, the characterization of PMS through the systems-level integration of multimodal data elucidates the multivariate impact of epigenetic effects on schizophrenia-relevant brain function and its interdependence with genetic illness risk. Second, the limited predictive value of polygenic risk scores and the difficulty in identifying associations with heritable neural differences found in schizophrenia may be due to the possibility that the manifestation of the functional consequences of genetic risk is modulated by spatio-temporal as well as sex-specific effects. To address this, this thesis identifies sex-differences in the spatio-temporal expression trajectories during human development of genes that showed significant prefrontal co-expression with schizophrenia risk genes during the fetal phase and adolescence, consistent with a core developmental hypothesis of schizophrenia. More specifically, it was found that during these two time-periods, prefrontal expression was significantly more variable in males compared to females, a finding that could be validated in an independent data source and that was specific for schizophrenia compared to other psychiatric as well as somatic illnesses. Similar to the epigenetic differences described above, the genes underlying the risk-associated gene expression differences were significantly linked to synaptic function. Notably, individual genes with male-specific variability increases were distinct between the fetal phase and adolescence, potentially suggesting different risk associated mechanisms that converge on the shared synaptic involvement of these genes. These results provide substantial support to the hypothesis that the functional consequences of genetic risk show spatiotemporal specificity. Importantly, the temporal specificity was linked to the fetal phase and adolescence, time-periods that are thought to be of predominant importance for the brain-functional consequences of environmental risk exposure. Therefore, the presented results provide the basis for future studies exploring the polygenic risk architecture and its interaction with environmental effects in a multivariate and spatiotemporally stratified manner. In summary, the work presented in this thesis describes multivariate, multimodal approaches to characterize the (epi-)genetic basis of schizophrenia, explores its association with a well-established neural intermediate phenotype of the illness and investigates the spatio-temporal specificity of schizophrenia-relevant gene expression effects. This work expands our knowledge of the complex biology underlying schizophrenia and provides the basis for the future development of more predictive biological algorithms that may aid in advancing personalized medicine in psychiatry

ALK1 SIGNALING IS REQUIRED FOR DIRECTED ENDOTHELIAL CELL MIGRATION IN THE PREVENTION OF ARTERIOVENOUS MALFORMATIONS

ALK1, a TGF-β type I receptor serine/threonine kinase, is critical for proper vascular development. Heterozygous loss of ALK1 results in the vascular disorder, hereditary hemorrhagic telangiectasia type 2 (HHT2), which is characterized by the development of arteriovenous malformations (AVMs) and affects 1:8000 people worldwide. alk1-/- zebrafish develop embryonic lethal AVMs which form via a two-step mechanism. First, loss of alk1 results in an increase in endothelial cell number in cranial arteries, which results in increased vessel caliber. In the second step, normally transient connections between arteries and veins are maintained as an adaptive mechanism to cope with an increased hemodynamic load. Using zebrafish as a tool to study the AVM formation due to loss of Alk1 signaling, I have found that Alk1 is required for directed arterial endothelial cell migration in opposition to blood flow. Embryos lacking alk1 experience a redistribution of cells, with endothelial cells failing to efficiently migrate against the direction of blood flow and accumulating in more distal regions of alk1-dependent arteries. This altered cellular distribution causes an increase in arterial caliber and consequent retention of downstream arteriovenous connections, resulting in fatal AVMs. Notch and ALK1 have been implicated in arterial specification and loss of function of either pathway causes AVMs. Furthermore, ALK1 can cooperate with Notch to upregulate expression of Notch target genes in cultured endothelial cells. These findings have led to the hypothesis that Notch and ALK1 collaboratively program arterial identity and prevent AVMs. I modulated Notch and Alk1 activities in zebrafish embryos and examined effects on Notch target gene expression and vascular morphology. Results demonstrate that control of Notch targets is context-dependent, with gene-specific and region-specific requirements for Notch and Alk1. Although loss of alk1 increases expression of dll4, which encodes a Notch ligand, and enhanced Notch signaling causes AVMs, AVMs in alk1 mutants could neither be phenocopied by Notch activation nor rescued by Notch inhibition. In conclusion, Alk1 is dispensable for acquisition and maintenance of arterial identity, and perturbations in Notch signaling cannot account or AVM development in alk1 mutants

Cell Type-specific Analysis of Human Interactome and Transcriptome

Cells are the fundamental building block of complex tissues in higher-order organisms. These cells take different forms and shapes to perform a broad range of functions. What makes a cell uniquely eligible to perform a task, however, is not well-understood; neither is the defining characteristic that groups similar cells together to constitute a cell type. Even for known cell types, underlying pathways that mediate cell type-specific functionality are not readily available. These functions, in turn, contribute to cell type-specific susceptibility in various disorders

Transcriptional and post-transcriptional regulation of leaf development in Arabidopsis thaliana

Plant growth follows a strict developmental program but needs to incorporate also environmental cues to adapt to the encountered conditions. This requires a complex regulatory network to ensure an appropriate response to changing conditions. We used the first leaf pair of Arabidopsis thaliana as a model system to study the regulation of organ development. Leaf growth can be divided in subsequent phases according to the major process driving it. In a young leaf primordium cells divide continuously and cell size homeostasis is ensured by matching rates of cell expansion. Next, cell division ceases and cell expansion becomes the driving force for growth. When the leaf has attained its final size, maturity is reached. In this thesis, I studied the regulation of leaf development at two regulatory levels. At the gene level, we analyzed the function of the CYCA2 core cell cycle regulatory gene family. We also studied the function of two new proliferation specific gene families putatively involved in cell cycle regulation. On the other hand, we profiled small RNA sequences during development and linked this with the occurrence of DNA methylation. The core machinery of the cell cycle in plants has been thoroughly studied, but our knowledge on how developmental and environmental signals impinge on cell division is still limited. CYCA2s are known core cell cycle regulators, involved in G2-to-M transition. Here, we studied the functional requirement of this gene family and showed that transcriptional repression is required for specific differentiation processes. Members of the CYCA2 protein family function in vascular development and differentiation of guard cells. For the latter process, we demonstrated that FOUR LIPS and MYB88, two transcription factors involved in stomatal development, directly repress CYCA2;3 expression, thus ensuring correct guard cell differentiation. Next to known ‘core’ cell cycle regulating genes, we also selected proliferation specific genes with unknown function, assuming them to be involved in the cell division process. We focused on two small gene families: three genes with four transmembrane domains (4TMs) and two genes containing three High Mobility Group (HMG) domains (3xHMG-box). Expression analysis and localization of transcriptional fusions with a fluorescent marker confirmed for both gene families the highly proliferation-specific expression pattern. Moreover, both families are highly induced in the M-phase of the cell cycle in synchronized cell cultures. The 4TMs localize to the cell plate during mitosis and we observed defects in cell plate formax tion upon overexpression and depletion of these genes. Therefore, we hypothesize that the 4TM genes are involved in formation of the cell plate. Profiling of small RNAs (sRNAs) in plants has thusfar mainly been focused on inflorescence tissue or whole seedlings. Here, we studied sRNAs during the different phases of development. Early in development, microRNAs implicated in nutrient stress response are upregulated, suggesting that at this phase nutrient availability is limiting for growth. We showed that specifically 24-nt sRNAs increase in expression during development. This class of sRNAs is known to be involved in RNA-dependent DNA methylation (RdDM) and can thus silence both transposons and genes. In general, the expression of sRNAs matching the coding sequences of protein-coding genes is positively correlated to the mRNA expression of this gene. We specifically selected genes that do not show this correlation, which were highly enriched in two categories: targets of microRNAs and trans-acting siRNAs, which generate phased sRNAs upon cleavage, and genes for which the sRNA profile is enriched for 24-nt sRNAs. This latter category is likely regulated through RdDM as this subset of genes shows increased DNA methylation in the gene body. This suggests that sRNA regulation could play an important role in regulating the leaf developmental process not only by preserving genome integrity by repressing transposon activity but also through silencing of protein-coding genes