Analyzing in situ gene expression in the mouse brain with image registration, feature extraction and block clustering

Background: Many important high throughput projects use in situ hybridization and may require the analysis of images of spatial cross sections of organisms taken with cellular level resolution. Projects creating gene expression atlases at unprecedented scales for the embryonic fruit fly as well as the embryonic and adult mouse already involve the analysis of hundreds of thousands of high resolution experimental images mapping mRNA expression patterns. Challenges include accurate registration of highly deformed tissues, associating cells with known anatomical regions, and identifying groups of genes whose expression is coordinately regulated with respect to both concentration and spatial location. Solutions to these and other challenges will lead to a richer understanding of the complex system aspects of gene regulation in heterogeneous tissue. Results: We present an end-to-end approach for processing raw in situ expression imagery and performing subsequent analysis. We use a non-linear, information theoretic based image registration technique specifically adapted for mapping expression images to anatomical annotations and a method for extracting expression information within an anatomical region. Our method consists of coarse registration, fine registration, and expression feature extraction steps. From this we obtain a matrix for expression characteristics with rows corresponding to genes and columns corresponding to anatomical sub-structures. We perform matrix block cluster analysis using a novel row-column mixture model and we relate clustered patterns to Gene Ontology (GO) annotations. Conclusion: Resulting registrations suggest that our method is robust over intensity levels and shape variations in ISH imagery. Functional enrichment studies from both simple analysis and block clustering indicate that gene relationships consistent with biological knowledge of neuronal gene functions can be extracted from large ISH image databases such as the Allen Brain Atlas [1] and the Max-Planck Institute [2] using our method. While we focus here on imagery and experiments of the mouse brain our approach should be applicable to a variety of in situ experiments

Genome-wide analysis of DNA methylation topology to understand cell fate

DNA methylation is an epignetic modification associated with gene regulation. It has extensively been studied in the context of small regulatory regions. Yet, not so much is known about large domains characterized by fuzzy methylation patterns, termed Partially Methylated Domains (PMDs). The present thesis comprises PMD analyses in various contexts and provides several new aspects to study DNA methylation. First, a comprehensive analysis of PMDs across a large cohort of WGBS samples was performed, to identify structural and functional features associated with PMDs. A newly developed approach, ChromH3M, was proposed for the analysis and integration of a large spectrum of WGBS data sets. Second, PMDs were found to be indicators of the cellular proliferation history and segmented loss of DNA methylation in PMDs supports the sequential linear differentiation model of memory T-cells. Third, assessment of genome-wide methylation changes in PMDs of Multiple Sclerosis-discordant monozygotic co-twins did not show significant differences, but local changes (DMRs) were identified. Taken together, the outcomes of the presented studies shed light on a so far neglected aspect of DNA methylation, that is PMDs, in different contexts; lineage specialization, differentiation, replication, disease, chromatin organization and gene expression.Die DNA-Methylierung ist eine epigenetische Modi1kation, die funktionell mit der Genregulation verbunden ist. Sie wurde bereits ausführlich im Kontext kleiner regulatorischer Regionen untersucht. Es ist jedoch noch nicht sehr viel bekannt über große Domänen, welche erstmals in WGBS-Daten beschrieben wurden. Sie werden als partiell methylierte Regionen (PMDs) bezeichnet und sind durch das Vorhandensein variabler Methylierungsmuster charakterisiert. Die vorliegende Arbeit umfasst PMD-Analysen in unterschiedlichen Kontexten und liefert verschiedene neue Aspekte zur Untersuchung der DNA-Methylierung. Zuerst wurde eine umfassende Analyse von PMDs in einer großen Kohorte von WGBS-Proben durchgeführt, um strukturelle und funktionelle Merkmale zu identi 1zieren, die mit PMDs assoziert sind. Ein neu entwickelter Ansatz, ChromH3M, wurde für die Analyse und Integration einer großen Kohorte vonWGBS Datensätzen angewandt. Zweitens wurde festgestellt, dass PMDs Indikatoren für die Zellproliferationshistorie sind, und der zu beobachtende graduelle Verlust der globalen DNAMethylierung bei der Differenzierung von T-Gedächtniszellen unterstützt die Hypothese der sequenziellen linearen Differenzierung. Drittens zeigte die Bewertung der genomweiten Methylierungsänderungen in PMDs von Multiple Sklerose-diskordanten monozygoten Zwillingen keine signi1kanten Unterschiede, jedoch wurden lokale Änderungen (DMRs) identi1ziert. Insgesamt geben die Ergebnisse der vorgestellten Studien Aufschluss über einen bislang eher vernachlässigten Aspekt der DNA-Methylierung, d.h. PMDs, in verschiedenen Zusammenhängen: der Festlegung der Zell-entwicklungsbahnen, der Zelldifferenzierung, der Replikation, die Krankheit, der Organisation des Chromatins, sowie der Regulation der Genexpression

Gut development: the regulation, evolution and function of Bapx1

The NK homeobox gene family comprises an extensive collection of transcription factors which have been shown to play pivotal roles in cell-fate specification and organogenesis. Murine Bapxl (Nkx3.2) was isolated as an evolutionarily conserved homologue of Drosophila bagpipe (NK3) which specifies the visceral mesoderm giving rise to gut musculature. A role for Bapxl in gut development has been conserved to mammals, where it would appear to be required both to specify the identity and to direct asymmetric growth of the spleno-pancreatic mesenchyme. Epithelial-mesenchymal signalling is an essential part of gut organogenesis, however the precise role of the mesenchyme is poorly understood and critical time points for its presence remain unknown. Initial investigations to probe the importance of the mesenchyme focussed on attempts to ablate this specific cell population by directing a toxic phenotype to Bapxl -expressing cells. A recombineering approach using Bapxlcontaining PACs was employed to generate the targeting construct, however inability to modify the Bapxl locus necessitated adoption of an alternative approach.The regulatory elements responsible for directing tissue-specific Bapxl expression have not yet been determined, therefore to pursue investigation of the role of the mesenchyme in gut development would require elucidation of a gut-specific Bapxl enhancer. Comparative sequence analysis between human, mouse and fish identified two candidate control elements, termed ProxB and DistB, which reside 1 lkb and 18kb respectively downstream of Bapxl. ProxB constitutes a short region of high sequence conservation between human and mouse, within which 180bp was also found to be conserved to zebrafish. DistB encompasses a more extensive 8kb genomic region highly conserved in mammals. Both the ProxB putative regulatory element and a 2kb subfragment of DistB named DistB 1 were cloned into a LacZ reporter construct and assayed for enhancer activity in transgenic mice. Both elements exhibited tissue-specific enhancer capacities with ProxB-driven expression initially confined to the mandibular portion of the first branchial arch and later detected in Meckel's cartilage and the middle ear. Detailed analysis of the expression pattern and comparison to evolutionarily conserved expression domains provides good evidence that ProxB constitutes a regionspecific Bapxl regulatory element. DistB 1 appears to be capable of directing expression to several different tissues, notably including the digits where it closely resembles endogenous Bapxl expression. Further parallels between the DistB 1 and Bapxl expression domains suggest that this element may also contribute to the endogenous regulation of Bapxl.Phenotypic comparisons between Bapxl null mutant mice and their wildtype counterparts have contributed significantly to knowledge of the wildtype function of the gene. Within the developing gut, distinct molecular boundaries are apparent. Bapxl demarcates the domain of the posterior stomach and asplenic null mutants attest to its being essential for formation of the spleen. Various gut markers were employed to study the disruption of both gut development and defined molecular domains in the absence of Bapxl. A marked difference in Sonic hedgehog (Shh) expression was observed in mutant guts, revealing a fusion of the upper duodenum to the posterior stomach to contribute to the enlarged stomach phenotype. Furthermore, Shh expression was detected in an abnormal branch emanating from the duodenum and extending alongside the posterior stomach thus exposing a novel phenotypic manifestation of the mutation.A novel anatomical structure, the splanchnic mesothelial plate (SMP) has been implicated in leftward mesenchymal growth during spleen morphogenesis. Cells within the SMP express Bapxl, without which leftward growth is disrupted and asplenia results. Prompted by these observations, a description of spleen development was undertaken to closely examine the precise origin and subsequent morphogenesis of this organ. Expression of the spleen marker Hoxl 1 was followed in dissected guts within a defined developmental window. The marked morphological transformation from a small domain of Hoxl/-expressing cells posterior to the stomach to an extensive layer flanking the dorsal mesogastrium is described herein. This work prompts questions as to whether a wave of cell-cell signalling or cell migration is responsible for the observed development of the spleen

Cytoskeletal Regulation of Centromere Maintenance and Function in the Mammalian Cell Cycle

Equal partitioning of genetic materials of the chromosomes is key to the mitotic cell cycle, as unequal segregation of chromosomes during mitosis leads to aneuploidy, a hall mark of human cancer. Accurate chromosome segregation is directed by the kinetochore, a proteinaceous structure on each sister chromosome that physically connects the chromosome to the spindle microtubules. Kinetochore assembles at the centromere, a specialized chromosome region epigenetically defined by the histone H3 variant centromere protein A (CENP-A) in higher eukaryotes including mammals. In order to maintain centromere identity against CENP-A dilution caused by S phase genome replication, new CENP-A molecules are loaded at preexisting centromeres in G1 phase of the cell cycle. Despite of the several important stages and molecular components identified in CENP-A replenishment, little is known about how new CENP-A proteins become stably incorporated into centromeric nucleosomes. Here by using quantitative imaging, pulse-chase labeling, mutant analysis, cellular fractionation and computational simulations, I have identified the cytoskeleton protein diaphanous formin mDia2 to be essential for the essential for the stable incorporation of newly synthesized CENP-A at the centromere. The novel function of mDia2 depends on its nuclear localization and its actin nucleation activity. Furthermore, mDia2 functions downstream of a small GTPase molecular switch during CENP-A loading, and is responsible for the formation of dynamic and short actin filaments observed in early G1 nuclei. Importantly, the maintenance of centromeric CENP-A levels requires a pool of polymerizable actin inside the nucleus. Single particle tracking and quantitative analysis revealed that centromere movement in early G1 nuclei is relatively confined over the time scale of initial CENP-A loading, and the subdiffusive behavior was significantly altered upon mDia2 knockdown. Finally, knocking down mDia2 results in prolonged centromere association of Holliday junction recognition protein (HJURP), a chaperone required to undergo timely turnover to allow for new CENP-A loading at the centromere. Our findings suggest that diaphanous formin mDia2 forms a link between the upstream small GTPase signaling and the downstream confined viscoelastic nuclear environment, and therefore regulates the stable assembly of new CENP-A containing nucleosomes to mark centromeres’ epigenetic identity (Chapter 2 and 3). While centromere identity is essential for kinetochore assembly, once kinetochores are assembled, fine-tuned interactions between kinetochores and microtubules become important for a fully functioning mitotic spindle during chromosome segregation. It has been previously found that another diaphanous formin protein mDia3 and its interaction with EB1, a microtubule plus-end tracking protein, are essential for accurate chromosome segregation1. In Chapter 4 of this thesis, I found that knocking down mDia3 caused a compositional change at the microtubule plus-end attached to the kinetochores, marked by a loss of EB1 and a gain of CLIP-170 and the dynein light chain protein Tctex-1. Interestingly, this compositional change does not affect the release of cytoplasmic dynein from aligned kinetochores, suggesting a population of Tctex-1 can be recruited to the kinetochores without dynein. During mitosis, Tctex-1 associates with unattached kinetochores and is required for accurate chromosome segregation. Tctex-1 knockdown in cells does not affect the localization and function of dynein at the kinetochore, but produces a prolonged mitotic arrest with a few misaligned chromosomes, which are subsequently missegregated during anaphase. This function is independent of Tctex-1’s association with dynein. The kinetochore localization of Tctex-1 is independent of the ZW10-dynein pathway, but requires the Ndc80 complex. Thus, our findings reveal a dynein independent role of Tctex-1 at the kinetochore to enhance the stability of kinetochore-microtubule attachment. Together, these work suggest novel regulatory roles of the cytoskeletal systems in the maintenance as well as subsequent functions of the centromere/kinetochore, and provide mechanistic insights into the complex control principles of accurate chromosome segregation. Our findings provide a new model in understanding the epigenetic maintenance of genome integrity, and will have implications with regard to how aberrant cell divisions underlying aneuploidy can be targeted in the treatment of cancer

Evaluating disorders of cognition using integrative genomics

Integrative genomics embodies a collaborative approach that brings together various disciplines, merging genomic and computational methodologies to achieve a comprehensive understanding of complex biological systems. By integrating and analysing various omics datasets, researchers can uncover novel insights into biological processes, disease mechanisms, and molecular interactions. This thesis applies integrative genomic approaches to study the biological mechanisms of cognitive disorders resulting from neurodegenerative and neurodevelopmental impairments. It includes three studies utilising transcriptomic profiling to identify and assess these mechanisms. The final discussion chapter summarises the main findings, including shared and distinct mechanisms across different cognitive disorders, addressing technical limitations, and outlining future research directions. The first study focuses on Lewy body diseases, utilising single-nucleus transcriptomics. Differential expression analysis reveals widespread dysregulation in neurons and glial cell types, with similar gene expression profiles observed in Parkinson’s Disease Dementia (PDD) and Dementia with Lewy Bodies (DLB), while Parkinson’s Disease (PD) shows distinct transcriptional profiles. Heritability enrichment analysis highlights a genetic association between glial cell dysregulation and PD age of onset. A unique population of neurons associated with DLB, resembling medium spiny neurons, is also identified. The second study investigates early-stage abnormal tau species-related gene expression changes in Alzheimer’s Disease (AD). Analysis of gene expression at the single-cell level demonstrates distinct patterns between Tau-proximity Ligation Assay (tauPLA) positive and tauPLA negative brain tissues. Dysregulation of genes in various cell-types in tauPLA positive samples is observed in the absence of neurofibrillary tangles. Reactive astrocyte activation, even in the absence of neurofibrillary tangles, is also reported for the first time in a transcriptomic-based study, suggesting their role in AD progression. The third study examines the neurodevelopmental outcomes of valproate exposure. Transcriptomic analysis reveals significant gene expression changes in the brains of gestationally exposed pups, affecting synaptic function, neurodevelopment, and genes associated with schizophrenia, bipolar disorder, and IQ heritability. Differential splicing analysis suggests enduring effects on brain function through epigenetic encoding. Convergent and divergent mechanisms underlying various neurodegenerative disorders are identified across the studies, including pathways related to axonal degeneration, mRNA splicing, synaptic organisation, autophagy, neuron death, phosphorylation, memory, mitochondrial function, and vesicle-mediated transport regulation. This thesis contributes to understanding the biological basis of cognitive disorders through integrative genomic approaches, providing insights into shared and distinct mechanisms across different disorders. The findings have implications for the development of therapeutic interventions and underscore the importance of rigorous experimental design in transcriptomic investigations. Future research directions are outlined to further unravel the complex molecular mechanisms underlying cognitive disorders.Open Acces

NASA patent abstracts bibliography: A continuing bibliography. Section 1: Abstracts (supplement 42)

Abstracts are provided for 174 patents and patent applications entered into the NASA scientific and technical information system during the period July 1992 through December 1992. Each entry consists of a citation, an abstract, and in most cases, a key illustration selected from the patent or patent application

Forward vs. reverse genetics: a bovine perspective based on visible and hidden phenotypes of inherited disorders

In modern cattle production, we have seen a negative trend for decades in reproduction while productivity and performance have improved. Although considered genetically complex, part of these fecundity, fertility, and rearing success issues are caused by Mendelian monogenic disorders. Traditionally, such disorders are investigated opportunistically based on their sporadic occurrence and through subsequent targeted analysis of affected individuals. This approach is called the forward genetic approach (FGA). Modern genomic technologies, such as single nucleotide polymorphism (SNP) array genotyping and whole-genome sequencing (WGS), allow for straightforward locus mapping and the identification of candidate causal variants in affected individuals or families. Nevertheless, a major drawback is the arbitrary sampling and availability of well-phenotyped individuals for research, especially for mostly invisible defects affecting fecundity, early embryonic death, and abortions. Therefore, the reverse genetic approach (RGA) is applied to screen for underlying recessive lethal or sub-lethal variants. This approach requires the availability of massive population-wide genomic data. By applying a haplotype screen for a significant deviation of the Hardy-Weinberg equilibrium, genomic regions potentially harboring candidate causal variants are identified. The subsequent generation of WGS data of haplotype carriers allows for the mining for pathogenic variants potentially causing a reduction in homozygosity. In the first part of my thesis, I present 18 successful, 1 inconclusive example, and 1 example addressing co-dominant effects of a known disorder. These FGA analyzes include heritable skin (n=7), bone (n=7), neuromuscular (n=1), eye (n=2), as well as syndromic disorders (n=3) in various European cattle breeds. Missense and frameshift variants in the IL17RA, DSP, and FA2H genes were described in three recessive genodermatoses: immunodeficiency with psoriasis-like skin alterations, syndromic ichthyosis, and ichthyosis congenita, respectively. Hypohidrotic ectodermal dysplasia was described as X-linked disorder that is associated with a gross deletion in the EDA gene. In dominant genodermatoses, a missense variant in COL5A2 was shown to lead to classical Ehlers-Danlos syndrome, an in-frame deletion in KRT5 was shown to cause epidermolysis bullosa simplex, and results of a study using an individual case of juvenile angiomatosis remained inconclusive. A recessive disorder described as hemifacial macrosomia was associated with a missense variant in LAMB1. Chondrodysplasia in a single family was shown to be caused by a de novo mutation in the bull leading to a stop-loss of the gene FGFR3. De novo mutations (missense and large deletions) in the COL2A1 and COL1A1 genes were associated with achondrogenesis type II (bulldog calf syndrome), and osteogenesis imperfecta type II, respectively. Another mutation that we found to affect bone morphology was a trisomy in chromosome 29 leading to proportional dwarfism with facial dysplasia. Congenital neuromuscular channelopathy was for the first time associated with a missense variant in KCNG1. Furthermore, a de novo missense variant in ADAMTSL4 and a recessive missense variant in CNGB3 were shown to cause congenital cataract and achromatopsia, respectively. Additionally, cases of pulmonary hypoplasia and anasarca syndrome were analyzed and shown to be caused by trisomy 20 in two unrelated calves and a recessively inherited missense variant in ADAMTS3. Moreover, the fatal syndromic disorder skeletal-cardo-enteric dysplasia was described to be caused by a de novo missense variant in MAP2K2. Finally, I investigated the effects on blood cholesterol and triglyceride levels of heterozygous carriers of the previously described APOB-related cholesterol deficiency. In the second part of my thesis, I present the outcome of the RGA in four main Swiss populations, that was validated with the SWISScow custom array. In the Brown Swiss dairy population, 72 haplotype regions showed significant depletions in homozygosity. Four of these haplotypes (BH6, BH14, BH24, and BH34) were associated with missense and nonsense variants in different genes (MARS2, MRPL55, CPT1C, and ACSL5, respectively). In the Original Braunvieh population, eight haplotype regions were identified. Candidate causal variants included a missense variant in TUBGCP5 gene associated with haplotype OH2, and a splice site frameshift variant in LIG3 gene associated with haplotype OH4. In the Holstein population, 24 haplotype regions were identified with a significant reduction of homozygosity. Subsequently, four novel candidate variants were proposed: a nonsense variant in KIR2DS1 for haplotype HH13, in-frame deletion in the genes NOTCH3 for HH21 haplotype, and RIOX1 for HH25 haplotype, and finally, a missense variant in PCDH15 for HH35 haplotype. In the Simmental population, eleven haplotype regions were detected. The haplotype SH5 was associated with a frameshift variant in DIS3 gene and the haplotypes SH8 and SH9 with missense variants in the CYP2B6 and NUBPL genes, respectively. For the breeds Brown Swiss, Original Braunvieh, and Holstein, association studies were carried out including traits describing fertility, birth, growth, and survival. Thereby most of the described mentioned haplotypes show additive effects. Regardless of the approach, all the described candidate causal variants can be used as a tool of precision diagnostics and represent a step forward towards personalized medicine in cattle. Furthermore, these variants can be easily genotyped and allow for targeted breeding to reduce the number of risk matings, which would lead to a reduction of affected animals and significant improvement in animal health and welfare

Poising and connectivity of enhancers upon naïve-to-primed transition in human embryonic stem cells (hESCs)

Enhancers are non-coding DNAelements that play crucial roles in transcriptional control, particularly in development. Patterns of histone modifications at enhancers are commonly used to infer their activity states and, poised enhancers (PEs) in particular display a ’bivalent’ chromatin state: the ’active’ H3K4me1 and the ’repressive’, Polycombassociated H3K27me3. Typically observed in pluripotent stem cells (PSCs), it was shown that PEs are required for gene activation later during differentiation. However, the function of the poised state of enhancers remains largely unknown. To trace the emergence of PEs in early development, I have extensively optimized a recently developed low-cell number Capture Hi-C protocol to perform Poised Enhancer Capture Hi-C (PECHi-C) in PSCs, in time course upon the naïve-to-primed transition, which is known to associate with a major shift in the localisation of Polycomb proteins, from a broader to a highly focal pattern. PECHi-C revealed that the PE-mediated regulatory circuitry undergoes significant reorganization between the two states. In particular, I detected three predominant patterns of PE-mediated interactions: the UP, DOWN and CONSTANT interaction classes. Integrating these results with Cut&Tag data on histone modifications revealed an interplay between the acquisition of the poised state at enhancers and their interaction dynamics whereby, at least in some cases, the acquisition of the bivalent signature occurs in parallel to the acquisition of their contacts. Moreover, the analyses suggested that day 3 of the transition is a pivotal point of the naïve-to-primed transition for the emergence of PEs. Overall, this thesis provided further insights into the emergence of PE-mediated regulatory circuitry during early embryogenesis. The different patterns of PE connectivity suggest the presence of diverse regulatory mechanisms of PEs, further suggesting that PEs might play a role at earlier stages of embryogenesis, by ensuring the correct transition from the ground state of pluripotency to the primed state.Open Acces