Adaptive sequence evolution in a color gene involved in the formation of the characteristic egg-dummies of male haplochromine cichlid fishes

BACKGROUND: The exceptionally diverse species flocks of cichlid fishes in East Africa are prime examples of parallel adaptive radiations. About 80% of East Africa's more than 1 800 endemic cichlid species, and all species of the flocks of Lakes Victoria and Malawi, belong to a particularly rapidly evolving lineage, the haplochromines. One characteristic feature of the haplochromines is their possession of egg-dummies on the males' anal fins. These egg-spots mimic real eggs and play an important role in the mating system of these maternal mouthbrooding fish. RESULTS: Here, we show that the egg-spots of haplochromines are made up of yellow pigment cells, xanthophores, and that a gene coding for a type III receptor tyrosine kinase, colony-stimulating factor 1 receptor a (csf1ra), is expressed in egg-spot tissue. Molecular evolutionary analyses reveal that the extracellular ligand-binding and receptor-interacting domain of csf1ra underwent adaptive sequence evolution in the ancestral lineage of the haplochromines, coinciding with the emergence of egg-dummies. We also find that csf1ra is expressed in the egg-dummies of a distantly related cichlid species, the ectodine cichlid Ophthalmotilapia ventralis, in which markings with similar functions evolved on the pelvic fin in convergence to those of the haplochromines. CONCLUSION: We conclude that modifications of existing signal transduction mechanisms might have evolved in the haplochromine lineage in association with the origination of anal fin egg-dummies. That positive selection has acted during the evolution of a color gene that seems to be involved in the morphogenesis of a sexually selected trait, the egg-dummies, highlights the importance of further investigations of the comparative genomic basis of the phenotypic diversification of cichlid fishes

Three-dimensional super-resolution microscopy of the inactive X chromosome territory reveals a collapse of its active nuclear compartment harboring distinct Xist RNA foci

Background: A Xist RNA decorated Barr body is the structural hallmark of the compacted inactive X territory in female mammals. Using super resolution three-dimensional structured illumination microscopy (3D-SIM) and quantitative image analysis, we compared its ultrastructure with active chromosome territories (CTs) in human and mouse somatic cells, and explored the spatio-temporal process of Barr body formation at onset of inactivation in early differentiating mouse embryonic stem cells (ESCs). Results: We demonstrate that all CTs are composed of structurally linked chromatin domain clusters (CDCs). In active CTs the periphery of CDCs harbors low-density chromatin enriched with transcriptionally competent markers, called the perichromatin region (PR). The PR borders on a contiguous channel system, the interchromatin compartment (IC), which starts at nuclear pores and pervades CTs. We propose that the PR and macromolecular complexes in IC channels together form the transcriptionally permissive active nuclear compartment (ANC). The Barr body differs from active CTs by a partially collapsed ANC with CDCs coming significantly closer together, although a rudimentary IC channel system connected to nuclear pores is maintained. Distinct Xist RNA foci, closely adjacent to the nuclear matrix scaffold attachment factor-A (SAF-A) localize throughout Xi along the rudimentary ANC. In early differentiating ESCs initial Xist RNA spreading precedes Barr body formation, which occurs concurrent with the subsequent exclusion of RNA polymerase II (RNAP II). Induction of a transgenic autosomal Xist RNA in a male ESC triggers the formation of an `autosomal Barr body' with less compacted chromatin and incomplete RNAP II exclusion. Conclusions: 3D-SIM provides experimental evidence for profound differences between the functional architecture of transcriptionally active CTs and the Barr body. Basic structural features of CT organization such as CDCs and IC channels are however still recognized, arguing against a uniform compaction of the Barr body at the nucleosome level. The localization of distinct Xist RNA foci at boundaries of the rudimentary ANC may be considered as snap-shots of a dynamic interaction with silenced genes. Enrichment of SAF-A within Xi territories and its close spatial association with Xist RNA suggests their cooperative function for structural organization of Xi

STED Nanoscopy to Illuminate New Avenues in Cancer Research – From Live Cell Staining and Direct Imaging to Decisive Preclinical Insights for Diagnosis and Therapy

Molecular imaging is established as an indispensable tool in various areas of cancer research, ranging from basic cancer biology and preclinical research to clinical trials and medical practice. In particular, the field of fluorescence imaging has experienced exceptional progress during the last three decades with the development of various in vivo technologies. Within this field, fluorescence microscopy is primarily of experimental use since it is especially qualified for addressing the fundamental questions of molecular oncology. As stimulated emission depletion (STED) nanoscopy combines the highest spatial and temporal resolutions with live specimen compatibility, it is best-suited for real-time investigations of the differences in the molecular machineries of malignant and normal cells to eventually translate the acquired knowledge into increased diagnostic and therapeutic efficacy. This thesis presents the application of STED nanoscopy to two acute topics in cancer research of direct or indirect clinical interest. The first project has investigated the structure of telomeres, the ends of the linear eukaryotic chromosomes, in intact human cells at the nanoscale. To protect genome integrity, a telomere can mask the chromosome end by folding back and sequestering its single-stranded 3’-overhang in an upstream part of the double-stranded DNA repeat region. The formed t-loop structure has so far only been visualized by electron microscopy and fluorescence nanoscopy with cross-linked mammalian telomeric DNA after disruption of cell nuclei and spreading. For the first time, this work demonstrates the existence of t-loops within their endogenous nuclear environment in intact human cells. The identification of further telomere conformations has laid the groundwork for distinguishing cancerous cells that use different telomere maintenance mechanisms based on their individual telomere populations by a combined STED nanoscopy and deep learning approach. The population difference was essentially attributed to the promyelocytic leukemia (PML) protein that significantly perturbs the organization of a subpopulation of telomeres towards an open conformation in cancer cells that employ a telomerase-independent, alternative telomere lengthening mechanism. Elucidating the nanoscale topology of telomeres and associated proteins within the nucleus has provided new insight into telomere structure-function relationships relevant for understanding the deregulation of telomere maintenance in cancer cells. After understanding the molecular foundations, this newly gained knowledge can be exploited to develop novel or refined diagnostic and treatment strategies. The second project has characterized the intracellular distribution of recently developed prostate cancer tracers. These novel prostate-specific membrane antigen (PSMA) inhibitors have revolutionized the treatment regimen of prostate cancer by enabling targeted imaging and therapy approaches. However, the exact internalization mechanism and the subcellular fate of these tracers have remained elusive. By combining STED nanoscopy with a newly developed non-standard live cell staining protocol, this work confirmed cell surface clustering of the targeted membrane antigen upon PSMA inhibitor binding, subsequent clathrin-dependent endocytosis and endosomal trafficking of the antigen-inhibitor complex. PSMA inhibitors accumulate in prostate cancer cells at clinically relevant time points, but strikingly and in contrast to the targeted antigen itself, they eventually distribute homogenously in the cytosol. This project has revealed the subcellular fate of PSMA/PSMA inhibitor complexes for the first time and provides crucial knowledge for the future application of these tracers including the development of new strategies in the field of prostate cancer diagnostics and therapeutics. Relying on the photostability and biocompatibility of the applied fluorophores, the performance of live cell STED nanoscopy in the field of cancer research is boosted by the development of improved fluorophores. The third project in this thesis introduces a biocompatible, small molecule near-infrared dye suitable for live cell STED imaging. By the application of a halogen dance rearrangement, a dihalogenated fluorinatable pyridinyl rhodamine could be synthesized at high yield. The option of subsequent radiolabeling combined with excellent optical properties and a non-toxic profile renders this dye an appropriate candidate for medical and bioimaging applications. Providing an intrinsic and highly specific mitochondrial targeting ability, the radiolabeled analogue is suggested as a vehicle for multimodal (positron emission tomography and optical imaging) medical imaging of mitochondria for cancer diagnosis and therapeutic approaches in patients and biopsy tissue. The absence of cytotoxicity is not only a crucial prerequisite for clinically used fluorophores. To guarantee the generation of meaningful data mirroring biological reality, the absence of cytotoxicity is likewise a decisive property of dyes applied in live cell STED nanoscopy. The fourth project in this thesis proposes a universal approach for cytotoxicity testing based on characterizing the influence of the compound of interest on the proliferation behavior of human cell lines using digital holographic cytometry. By applying this approach to recently developed live cell STED compatible dyes, pronounced cytotoxic effects could be excluded. Looking more closely, some of the tested dyes slightly altered cell proliferation, so this project provides guidance on the right choice of dye for the least invasive live cell STED experiments. Ultimately, live cell STED data should be exploited to extract as much biological information as possible. However, some information might be partially hidden by image degradation due the dynamics of living samples and the deliberate choice of rather conservative imaging parameters in order to preserve sample viability. The fifth project in this thesis presents a novel image restoration method in a Bayesian framework that simultaneously performs deconvolution, denoising as well as super-resolution, to restore images suffering from noise with mixed Poisson-Gaussian statistics. Established deconvolution or denoising methods that consider only one type of noise generally do not perform well on images degraded significantly by mixed noise. The newly introduced method was validated with live cell STED telomere data proving that the method can compete with state-of-the-art approaches. Taken together, this thesis demonstrates the value of an integrated approach for STED nanoscopy imaging studies. A coordinated workflow including sample preparation, image acquisition and data analysis provided a reliable platform for deriving meaningful conclusions for current questions in the field of cancer research. Moreover, this thesis emphasizes the strength of iteratively adapting the individual components in the operational chain and it particularly points towards those components that, if further improved, optimize the significance of the final results rendering live cell STED nanoscopy even more powerful

Aneuploidy in Health and Disease

Aneuploidy means any karyotype that is not euploid, anything that stands outside the norm. Two particular characteristics make the research of aneuploidy challenging. First, it is often hard to distinguish what is a cause and what is a consequence. Secondly, aneuploidy is often associated with a persistent defect in maintenance of genome stability. Thus, working with aneuploid, unstable cells means analyzing an ever changing creature and capturing the features that persist. In the book Aneuploidy in Health and Disease we summarize the recent advances in understanding the causes and consequences of aneuploidy and its link to human pathologies

Analysis of the Barr body with super-resolution microscopy

X chromosome inactivation (XCI) in female mammalian cells is an ideal model system to study the relationship of epigenetic regulation and higher-order chromatin structure. However, light microscopic studies of chromosomal organization have long been limited by the diffraction barrier of optical resolution. Super-resolution 3D-structured illumination microscopy (3D-SIM) – one of several recent techniques that circumvent this limitation – enables multicolor optical sectioning of entire cells with eightfold-improved volumetric resolution compared to conventional fluorescence imaging methods. In the present work, 3D-SIM has been applied to analyze higher-order chromatin structure of the Barr body in mammalian nuclei, a characteristic hallmark of XCI, with yet unprecedented detail. First, the increased resolution prompted to reappraise the potential detrimental effect of the DNA-FISH procedure on chromatin structure. Comparative analyses revealed slight deteriorations at the resolution level of 3D-SIM, especially within more decondensed euchromatin sites within the nuclear interior. In contrast, overall nuclear morphology and the nuclear envelope as well as heterochromatic sites in general maintained well preserved. The results suggest that DNA-FISH studies can benefit from a combination with super-resolution microscopy. In particular, when keeping in mind the current developments of the FISH technique with increasingly small and higher-complexity probes. The compact shape of the Barr body led to the assumption of a contribution of this special higher-order chromatin structure to the establishment and maintenance of the silenced state in the inactive X chromosome (Xi). However, a confirmation of this view has always been hampered by the restrictions of conventional light microscopy. In this work, the 3D chromosomal organization of the Xi and autosomes has been investigated with 3D-SIM in various human and mouse somatic cells and in mouse embryonic stem cell (ESC) lines. The precise subchromosomal localization of a variety of factors involved in XCI in different developmental states was qualitatively and quantitatively assessed utilizing combined immunofluorescence, EdU- pulse and RNA-/DNA-FISH labeling protocols and novel data analysis tools customized for the special requirements of 3D-SIM. The results demonstrate that all autosomes are made of a three-dimensional interconnected network of chromatin domains (CDs, or topology associated domains, TADs) of highly-variable shape and dynamics. CDs/TADs are comprised of a compacted chromatin core enriched with repressive marks, which is collectively proposed to be the functionally passive chromatin compartment (PNC). This PNC is surrounded by a 50 – 150 nm locally defined, less compacted perichromatin region (PR) that is enriched with active histone modifications and pervaded by a three-dimensional interchromatin (IC) network. The PR and the IC are collectively referred to as being the functionally relevant active nuclear compartment (ANC) that harbors all major nuclear processes, including transcription and replication. 3D-SIM data revealed that the Barr body maintains this principle compartmentalization and that it is still pervaded by a narrow ANC network, which is able to fulfill its functional role as a hub for replication or rarely occurring expression of XCI-escape genes. Live-cell super-resolution imaging on HeLa H2B-GFP cells confirmed that the observed chromatin features do not reflect fixation artifacts. Xist RNA, the key factor of XCI, has been found to be preferentially located as distinct discernible foci within the ANC throughout the entire volume of the Barr body. Here, it is tightly associated with a Xi-specific form of the nuclear matrix protein SAF-A, which confirms a previously suggested role for this Xi-enriched protein in Xist RNA spreading. In contrast, Xist RNA shows no spatial correlation with repressive Xi-enriched histone marks that are found within compacted chromatin sites. This specific localization of Xist RNA reflects an intrinsic feature as it is already present during early spreading in differentiating female ESCs, where it precedes chromatin compaction concomitant with RNA Polymerase II exclusion. Its localization is further confirmed in a male ESC line carrying an inducible Xist transgene on an autosome, but where Xist RNA fails to form a true autosomal Barr body, which is less compacted and maintains transcriptional activity. Last, Xist RNA shows no direct association with PRC2, the mediator of H3K27me3, which is in contrast to the generally believed direct recruitment model of PRC2 to the Xi by Xist RNA. The data collected in this work reflects further support and a refinement of the not unequivocally accepted CT-IC (chromosome territory - interchromatin compartment) model of higher-order chromosome architecture. In addition, a first attempt has been made to integrate these findings with a recently growing number of studies using chromosome conformation capturing (3C)-based techniques and to complement them on the single-cell level. Finally, a novel model for Xist RNA function in XCI is presented, which proposes a sequence-independent structural role for gene silencing and the formation of a repressive chromatin compartment

Integrated Spatial Genomics Reveals Organizational Principles of Single-Cell Nuclear Architecture

Three-dimensional (3D) nuclear architecture plays key roles in many cellular processes such as gene regulation and genome replication. Recent sequencing-based and imaging-based single-cell studies have characterized a high variability of nuclear features in individual cells from a wide-range of measurement modalities, such as chromosome structures, subnuclear structures, chromatin states, and nascent transcription. However, the lack of technologies that allow us to interrelate those nuclear features simultaneously in the same single cells limits our understanding of nuclear architecture. To overcome this limitation, a technology that can examine 3D nuclear features across modalities from the same single cells is required. Here, we demonstrate integrated spatial genomics approaches, which enable genome-wide investigation of chromosome structures, subnuclear structures, chromatin states, and transcriptional states in individual cells. In Chapter 2, we introduce the "track first and identify later" approach, which enables multiplexed tracking of genomic loci in live cells by combining CRISPR/Cas9 live imaging and DNA sequential fluorescence in situ hybridization (DNA seqFISH) technologies. We demonstrate our approach by resolving the dynamics of 12 unique subtelomeric loci in mouse embryonic stem (ES) cells. In Chapter 3, we present the intron seqFISH technology, which enables transcriptome-scale gene expression profiling at their nascent transcription active sites in individual nuclei in mouse ES cells and fibroblasts, along with mRNA and lncRNA seqFISH and immunofluorescence. We show the transcription active sites position at the surfaces of chromosome territories with variable inter-chromosomal organization in individual nuclei. By building upon those technologies, in Chapter 4, we demonstrate integrated spatial genomics in mouse ES cells, which enables to image thousands of genomic loci by DNA seqFISH+, along with sequential immunofluorescence and RNA seqFISH in individual cells. We show "fixed loci" that are invariably associated with specific subnuclear structures across hundreds of single cells that can constrain nuclear architecture in individual nuclei. In addition, we find individual genomic loci appear to be pre-positioned to specific nuclear compartments with different frequencies, which are independent from nascent transcriptional states of single cells. Lastly, in Chapter 5, we demonstrate the integrated spatial genomics technology in the mouse brain cortex, enabling the investigation of single-cell nuclear architecture in a cell-type specific fashion as well as the exploration of common organizational principles of nuclear architecture across cell types. We reveal that inter-chromosomal organization and radial positioning of chromosomes are arranged with cell-type specific chromatin fixed loci and subnuclear structure organization in diverse cell types. We also uncover the variable organization of chromosome domain structures at the sub-megabase scale in individual cells, which can be obscured with bulk measurements. Together, these results demonstrate the ability of integrated spatial genomics to advance our overall understanding of single-cell nuclear architecture in various biological systems.</p

Subcellular Molecular Profiling of Midbrain Dopamine Neurons

Midbrain dopamine neurons play a critical role in motor function, motivation, reward, and cognition by providing modulatory input to cortical and basal ganglia circuits. Given the importance of dopamine neurotransmission and its dysregulation in disease, mechanistic insight into the molecular underpinnings of dopaminergic neuronal function is needed. This thesis seeks to advance our understanding of dopamine neuronal cell biology by developing and applying cutting edge molecular profiling methods to study the subcellular translatome and proteome of dopamine neurons in mice. Chapter 1 provides an overview of the anatomy and cell biology of midbrain dopamine systems, with a particular emphasis on dopamine neurotransmission, neuronal heterogeneity, and selective vulnerability in Parkinson’s disease. Chapter 2 focuses on methods for studying local translation in neurons and describes newly discovered artifacts associated with two of these methods. Chapter 3 describes a global analysis of ribosome and mRNA localization in dopamine neurons; the results suggest that local translation in dopaminergic dendrites, but not axons, regulates dopamine release. Chapter 4 presents a method for subcellular proteomic profiling of dopamine neurons in the mouse brain, revealing the somatodendritic and axonal polarization of proteins encoded by Parkinson’s disease-linked genes. Emerging data are presented on Synaptotagmin 17, a novel axonal protein identified in midbrain dopamine neurons. Finally, I synthesize key findings regarding the molecular organization underlying dopamine neuronal cell biology and highlight promising areas for future investigation