Collagen Organization Does Not Influence T-Cell Distribution in Stroma of Human Pancreatic Cancer.

The dominant intrastromal T-cell infiltration in pancreatic cancer is mainly caused by the contact guidance through the excessive desmoplastic reaction and could represent one of the obstacles to an effective immune response in this tumor type. This study analyzed the collagen organization in normal and malignant pancreatic tissues as well as its influence on T-cell distribution in pancreatic cancer. Human pancreatic tissue was analyzed using immunofluorescence staining and multiphoton and SHG microscopy supported by multistep image processing. The influence of collagen alignment on activated T-cells was studied using 3D matrices and time-lapse microscopy. It was found that the stroma of malignant and normal pancreatic tissues was characterized by complex individual organization. T-cells were heterogeneously distributed in pancreatic cancer and there was no relationship between T-cell distribution and collagen organization. There was a difference in the angular orientation of collagen alignment in the peritumoral and tumor-cell-distant stroma regions in the pancreatic ductal adenocarcinoma tissue, but there was no correlation in the T-cell densities between these regions. The grade of collagen alignment did not influence the directionality of T-cell migration in the 3D collagen matrix. It can be concluded that differences in collagen organization do not change the spatial orientation of T-cell migration or influence stromal T-cell distribution in human pancreatic cancer. The results of the present study do not support the rationale of remodeling of stroma collagen organization for improvement of T-cell-tumor cell contact in pancreatic ductal adenocarcinoma.

A PRDX1-p38α heterodimer amplifies MET-driven invasion of IDH-wildtype and IDH-mutant gliomas.

The Peroxiredoxin 1 (PRDX1) gene maps to chromosome arm 1p and is hemizygously deleted and epigenetically silenced in isocitrate dehydrogenase 1 or 2 (IDH)-mutant and 1p/19q-codeleted oligodendroglial tumors. In contrast, IDH-wildtype astrocytic gliomas including glioblastomas mostly lack epigenetic silencing and express PRDX1 protein. In our study, we investigated how PRDX1 contributes to the infiltrative growth of IDH-wildtype gliomas. Focusing on p38α-dependent pathways, we analyzed clinical data from 133 patients of the NOA-04 trial cohort to look for differences in the gene expression profiles of gliomas with wildtype or mutant IDH. Biochemical interaction studies as well as in vitro and ex vivo migration studies were used to establish a biological role of PRDX1 in maintaining pathway activity. Whole-brain high-resolution ultramicroscopy and survival analyses of pre-clinical mouse models for IDH-wildtype gliomas were then used for in vivo confirmation. Based on clinical data, we found that the absence of PRDX1 is associated with changes in the expression of MET/HGF signaling components. PRDX1 forms a heterodimer with p38α mitogen-activated protein kinase 14 (MAPK14), stabilizing phospho-p38α in glioma cells. This process amplifies hepatocyte growth factor (HGF)-mediated signaling and stimulates actin cytoskeleton dynamics that promote glioma cell migration. Whole-brain high-resolution ultramicroscopy confirms these findings, indicating that PRDX1 promotes glioma brain invasion in vivo. Finally, reduced expression of PRDX1 increased survival in mouse glioma models. Thus, our preclinical findings suggest that PRDX1 expression levels may serve as a molecular marker for patients who could benefit from targeted inhibition of MET/HGF signaling.

Neural stem cells induce the formation of their physical niche during organogenesis.

Most organs rely on stem cells to maintain homeostasis during post-embryonic life. Typically, stem cells of independent lineages work coordinately within mature organs to ensure proper ratios of cell types. Little is known, however, on how these different stem cells locate to forming organs during development. Here we show that neuromasts of the posterior lateral line in medaka are composed of two independent life-long lineages with different embryonic origins. Clonal analysis and 4D imaging revealed a hierarchical organisation with instructing and responding roles: an inner, neural lineage induces the formation of an outer, border cell lineage (nBC) from the skin epithelium. Our results demonstrate that the neural lineage is necessary and sufficient to generate nBCs highlighting self-organisation principles at the level of the entire embryo. We hypothesise that induction of surrounding tissues plays a major role during the establishment of vertebrate stem cell niches.

Sequential organogenesis sets two parallel sensory lines in medaka.

Animal organs are typically formed during embryogenesis by following one specific developmental programme. Here, we report that neuromast organs are generated by two distinct and sequential programmes that result in parallel sensory lines in medaka embryos. A ventral posterior lateral line (pLL) is composed of neuromasts deposited by collectively migrating cells whereas a midline pLL is formed by individually migrating cells. Despite the variable number of neuromasts among embryos, the sequential programmes that we describe here fix an invariable ratio between ventral and midline neuromasts. Mechanistically, we show that the formation of both types of neuromasts depends on the chemokine receptor genes cxcr4b and cxcr7b, illustrating how common molecules can mediate different morphogenetic processes. Altogether, we reveal a self-organising feature of the lateral line system that ensures a proper distribution of sensory organs along the body axis.

Early Commissural Diencephalic Neurons Control Habenular Axon Extension and Targeting.

Most neuronal populations form on both the left and right sides of the brain. Their efferent axons appear to grow synchronously along similar pathways on each side, although the neurons or their environment often differ between the two hemispheres [1-4]. How this coordination is controlled has received little attention. Frequently, neurons establish interhemispheric connections, which can function to integrate information between brain hemispheres (e.g., [5]). Such commissures form very early, suggesting their potential developmental role in coordinating ipsilateral axon navigation during embryonic development [4]. To address the temporal-spatial control of bilateral axon growth, we applied long-term time-lapse imaging to visualize the formation of the conserved left-right asymmetric habenular neural circuit in the developing zebrafish embryo [6]. Although habenular neurons are born at different times across brain hemispheres [7], we found that elongation of habenular axons occurs synchronously. The initiation of axon extension is not controlled within the habenular network itself but through an early developing proximal diencephalic network. The commissural neurons of this network influence habenular axons both ipsilaterally and contralaterally. Their unilateral absence impairs commissure formation and coordinated habenular axon elongation and causes their subsequent arrest on both sides of the brain. Thus, habenular neural circuit formation depends on a second intersecting commissural network, which facilitates the exchange of information between hemispheres required for ipsilaterally projecting habenular axons. This mechanism of network formation may well apply to other circuits, and has only remained undiscovered due to technical limitations.

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System.

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.

The ventral habenulae of zebrafish develop in prosomere 2 dependent on Tcf7l2 function.

BACKGROUND: The conserved habenular neural circuit relays cognitive information from the forebrain into the ventral mid- and hindbrain. In zebrafish, the bilaterally formed habenulae in the dorsal diencephalon are made up of the asymmetric dorsal and symmetric ventral habenular nuclei, which are homologous to the medial and lateral nuclei respectively, in mammals. These structures have been implicated in various behaviors related to the serotonergic/dopaminergic neurotransmitter system. The dorsal habenulae develop adjacent to the medially positioned pineal complex. Their precursors differentiate into two main neuronal subpopulations which differ in size across brain hemispheres as signals from left-sided parapineal cells influence their differentiation program. Unlike the dorsal habenulae and despite their importance, the ventral habenulae have been poorly studied. It is not known which genetic programs underlie their development and why they are formed symmetrically, unlike the dorsal habenulae. A main reason for this lack of knowledge is that the vHb origin has remained elusive to date. RESULTS: To address these questions, we applied long-term 2-photon microscopy time-lapse analysis of habenular neural circuit development combined with depth color coding in a transgenic line, labeling all main components of the network. Additional laser ablations and cell tracking experiments using the photoconvertible PSmOrange system in GFP transgenic fish show that the ventral habenulae develop in prosomere 2, posterior and lateral to the dorsal habenulae in the dorsal thalamus. Mutant analysis demonstrates that the ventral habenular nuclei only develop in the presence of functional Tcf7l2, a downstream modulator of the Wnt signaling cascade. Consistently, photoconverted thalamic tcf7l2exl/exl mutant cells do not contribute to habenula formation. CONCLUSIONS: We show in vivo that dorsal and ventral habenulae develop in different regions of prosomere 2. In the process of ventral habenula formation, functional tcf7l2 gene activity is required and in its absence, ventral habenular neurons do not develop. Influenced by signals from parapineal cells, dorsal habenular neurons differentiate at a time at which ventral habenular cells are still on their way towards their final destination. Thus, our finding may provide a simple explanation as to why only neuronal populations of the dorsal habenulae differ in size across brain hemispheres.

Habenula circuit development: past, present, and future.

The habenular neural circuit is attracting increasing attention from researchers in fields as diverse as neuroscience, medicine, behavior, development, and evolution. Recent studies have revealed that this part of the limbic system in the dorsal diencephalon is involved in reward, addiction, and other behaviors and its impairment is associated with various neurological conditions and diseases. Since the initial description of the dorsal diencephalic conduction system (DDC) with the habenulae in its center at the end of the nineteenth century, increasingly sophisticated techniques have resolved much of its anatomy and have shown that these pathways relay information from different parts of the forebrain to the tegmentum, midbrain, and hindbrain. The first part of this review gives a brief historical overview on how the improving experimental approaches have allowed the stepwise uncovering much of the architecture of the habenula circuit as we know it today. Our brain distributes tasks differentially between left and right and it has become a paradigm that this functional lateralization is a universal feature of vertebrates. Moreover, task dependent differential brain activities have been linked to anatomical differences across the left-right axis in humans. A good way to further explore this fundamental issue will be to study the functional consequences of subtle changes in neural network formation, which requires that we fully understand DDC system development. As the habenular circuit is evolutionarily highly conserved, researchers have the option to perform such difficult experiments in more experimentally amenable vertebrate systems. Indeed, research in the last decade has shown that the zebrafish is well suited for the study of DDC system development and the phenomenon of functional lateralization. We will critically discuss the advantages of the zebrafish model, available techniques, and others that are needed to fully understand habenular circuit development.

Protein diffusion in mammalian cell cytoplasm.

We introduce a new method for mesoscopic modeling of protein diffusion in an entire cell. This method is based on the construction of a three-dimensional digital model cell from confocal microscopy data. The model cell is segmented into the cytoplasm, nucleus, plasma membrane, and nuclear envelope, in which environment protein motion is modeled by fully numerical mesoscopic methods. Finer cellular structures that cannot be resolved with the imaging technique, which significantly affect protein motion, are accounted for in this method by assigning an effective, position-dependent porosity to the cell. This porosity can also be determined by confocal microscopy using the equilibrium distribution of a non-binding fluorescent protein. Distinction can now be made within this method between diffusion in the liquid phase of the cell (cytosol/nucleosol) and the cytoplasm/nucleoplasm. Here we applied the method to analyze fluorescence recovery after photobleach (FRAP) experiments in which the diffusion coefficient of a freely-diffusing model protein was determined for two different cell lines, and to explain the clear difference typically observed between conventional FRAP results and those of fluorescence correlation spectroscopy (FCS). A large difference was found in the FRAP experiments between diffusion in the cytoplasm/nucleoplasm and in the cytosol/nucleosol, for all of which the diffusion coefficients were determined. The cytosol results were found to be in very good agreement with those by FCS.

Parvovirus induced alterations in nuclear architecture and dynamics.

The nucleus of interphase eukaryotic cell is a highly compartmentalized structure containing the three-dimensional network of chromatin and numerous proteinaceous subcompartments. DNA viruses induce profound changes in the intranuclear structures of their host cells. We are applying a combination of confocal imaging including photobleaching microscopy and computational methods to analyze the modifications of nuclear architecture and dynamics in parvovirus infected cells. Upon canine parvovirus infection, expansion of the viral replication compartment is accompanied by chromatin marginalization to the vicinity of the nuclear membrane. Dextran microinjection and fluorescence recovery after photobleaching (FRAP) studies revealed the homogeneity of this compartment. Markedly, in spite of increase in viral DNA content of the nucleus, a significant increase in the protein mobility was observed in infected compared to non-infected cells. Moreover, analysis of the dynamics of photoactivable capsid protein demonstrated rapid intranuclear dynamics of viral capsids. Finally, quantitative FRAP and cellular modelling were used to determine the duration of viral genome replication. Altogether, our findings indicate that parvoviruses modify the nuclear structure and dynamics extensively. Intranuclear crowding of viral components leads to enlargement of the interchromosomal domain and to chromatin marginalization via depletion attraction. In conclusion, parvoviruses provide a useful model system for understanding the mechanisms of virus-induced intranuclear modifications.

Mapping eGFP oligomer mobility in living cell nuclei.

Movement of particles in cell nuclei can be affected by viscosity, directed flows, active transport, or the presence of obstacles such as the chromatin network. Here we investigate whether the mobility of small fluorescent proteins is affected by the chromatin density. Diffusion of inert fluorescent proteins was studied in living cell nuclei using fluorescence correlation spectroscopy (FCS) with a two-color confocal scanning detection system. We first present experiments exposing FCS-specific artifacts encountered in live cell studies as well as strategies to prevent them, in particular those arising from the choice of the fluorophore used for calibration of the focal volume, as well as temperature and acquisition conditions used for fluorescence fluctuation measurements. After defining the best acquisition conditions, we show for various human cell lines that the mobility of GFP varies significantly within the cell nucleus, but does not correlate with chromatin density. The intranuclear diffusional mobility strongly depends on protein size: in a series of GFP-oligomers, used as free inert fluorescent tracers, the diffusion coefficient decreased from the monomer to the tetramer much more than expected for molecules free in aqueous solution. Still, the entire intranuclear chromatin network is freely accessible for small proteins up to the size of eGFP-tetramers, regardless of the chromatin density or cell line. Even the densest chromatin regions do not exclude free eGFP-monomers or multimers.