Dense 4D nanoscale reconstruction of living brain tissue.

Three-dimensional (3D) reconstruction of living brain tissue down to an individual synapse level would create opportunities for decoding the dynamics and structure-function relationships of the brain's complex and dense information processing network; however, this has been hindered by insufficient 3D resolution, inadequate signal-to-noise ratio and prohibitive light burden in optical imaging, whereas electron microscopy is inherently static. Here we solved these challenges by developing an integrated optical/machine-learning technology, LIONESS (live information-optimized nanoscopy enabling saturated segmentation). This leverages optical modifications to stimulated emission depletion microscopy in comprehensively, extracellularly labeled tissue and previous information on sample structure via machine learning to simultaneously achieve isotropic super-resolution, high signal-to-noise ratio and compatibility with living tissue. This allows dense deep-learning-based instance segmentation and 3D reconstruction at a synapse level, incorporating molecular, activity and morphodynamic information. LIONESS opens up avenues for studying the dynamic functional (nano-)architecture of living brain tissue.

Strategies to maximize performance in STimulated Emission Depletion (STED) nanoscopy of biological specimens.

Super-resolution fluorescence microscopy has become an important catalyst for discovery in the life sciences. In STimulated Emission Depletion (STED) microscopy, a pattern of light drives fluorophores from a signal-emitting on-state to a non-signalling off-state. Only emitters residing in a sub-diffraction volume around an intensity minimum are allowed to fluoresce, rendering them distinguishable from the nearby, but dark fluorophores. STED routinely achieves resolution in the few tens of nanometers range in biological samples and is suitable for live imaging. Here, we review the working principle of STED and provide general guidelines for successful STED imaging. The strive for ever higher resolution comes at the cost of increased light burden. We discuss techniques to reduce light exposure and mitigate its detrimental effects on the specimen. These include specialized illumination strategies as well as protecting fluorophores from photobleaching mediated by high-intensity STED light. This opens up the prospect of volumetric imaging in living cells and tissues with diffraction-unlimited resolution in all three spatial dimensions.

Genome amplification and cellular senescence are hallmarks of human placenta development.

Genome amplification and cellular senescence are commonly associated with pathological processes. While physiological roles for polyploidization and senescence have been described in mouse development, controversy exists over their significance in humans. Here, we describe tetraploidization and senescence as phenomena of normal human placenta development. During pregnancy, placental extravillous trophoblasts (EVTs) invade the pregnant endometrium, termed decidua, to establish an adapted microenvironment required for the developing embryo. This process is critically dependent on continuous cell proliferation and differentiation, which is thought to follow the classical model of cell cycle arrest prior to terminal differentiation. Strikingly, flow cytometry and DNAseq revealed that EVT formation is accompanied with a genome-wide polyploidization, independent of mitotic cycles. DNA replication in these cells was analysed by a fluorescent cell-cycle indicator reporter system, cell cycle marker expression and EdU incorporation. Upon invasion into the decidua, EVTs widely lose their replicative potential and enter a senescent state characterized by high senescence-associated (SA) ß-galactosidase activity, induction of a SA secretory phenotype as well as typical metabolic alterations. Furthermore, we show that the shift from endocycle-dependent genome amplification to growth arrest is disturbed in androgenic complete hydatidiform moles (CHM), a hyperplastic pregnancy disorder associated with increased risk of developing choriocarinoma. Senescence is decreased in CHM-EVTs, accompanied by exacerbated endoreduplication and hyperploidy. We propose induction of cellular senescence as a ploidy-limiting mechanism during normal human placentation and unravel a link between excessive polyploidization and reduced senescence in CHM.

Endothelin-1 down-regulates matrix metalloproteinase 14 and 15 expression in human first trimester trophoblasts via endothelin receptor type B.

STUDY QUESTION: Does endothelin-1 (ET-1) regulate matrix metalloproteinase (MMP) 14 and 15 production and invasion of human first trimester trophoblasts? SUMMARY ANSWER: ET-1 in pathophysiological concentrations down-regulates MMP14 and MMP15 expression via endothelin receptor (ETR) type B and decreases trophoblast migration and invasion. WHAT IS KNOWN ALREADY: MMP14 and MMP15 are involved in trophoblast invasion. Impairment of invasion has been linked to pregnancy complications such as pre-eclampsia (PE). ET-1 is up-regulated in PE. STUDY DESIGN, SIZE, DURATION: In vitro study using primary human trophoblasts from 50 first trimester placentas (gestational week 7-12). PARTICIPANTS/MATERIALS, SETTING, METHODS: Trophoblasts were cultured in the absence or presence of 10-100 nM ET-1. MMP14 and MMP15 mRNA and protein were quantified by RT-qPCR and Western blotting, respectively. Selective antagonists for ETRA (BQ-123) or ETRB (BQ-788) were used to identify ETR subtypes involved. Functional ET-1 effects were tested in first trimester chorionic villous explants and transwell invasion assays. The roles of tumor necrosis factor (TNF)-α (25 ng/ml) and oxygen (1%) in ET-1 regulation of MMP14 and 15 expression were assessed by Western blotting. MAIN RESULTS AND THE ROLE OF CHANCE: ET-1 down-regulated MMP14 and MMP15 mRNA (-21% and -26%, respectively, P < 0.05) and protein levels (-18% and -22%, respectively, P < 0.05). This effect was mediated via ETRB. ET-1 decreased trophoblast outgrowth in placental explants (-24%, P < 0.05) and trophoblast invasion (-26%, P ≤ 0.01). TNF-α enhanced ET-1 mediated MMP15 down-regulation (by 10%, P < 0.05), whereas hypoxia abolished the effect of ET-1 on both MMPs. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Only primary trophoblasts were used in this study. Since trophoblast yield from first trimester placental material is limited, further aspects of MMP14 and 15 regulation could not be characterized. Other anti-invasive factors may be altered by ET-1 in trophoblasts and, thus, contribute to the reduced invasion, but have not been investigated. Oxygen levels similar to those found in the decidua (5-8% O2) were not analyzed in this study. WIDER IMPLICATIONS OF THE FINDINGS: ET-1 modifies placental function already during the first trimester of pregnancy, the time-window when the placental changes implicated in PE occur. Thus, our results improve the understanding of the placental mechanisms underlying trophoblast invasion and PE. STUDY FUNDING/COMPETING INTERESTS: The study was funded by the Oesterreichische Nationalbank (Anniversary Fund, project number: 14796) and the Herzfelder'sche Familienstiftung (to J.P.; number: 00685). AMM received funding from the Austrian Science Fund FWF (W1241) and the Medical University Graz through the PhD Program Molecular Fundamentals of Inflammation (DK-MOLIN). The authors have no conflict of interest.

Extravillous trophoblast-associated ADAM12 exerts pro-invasive properties, including induction of integrin beta 1-mediated cellular spreading.

ADAM12, consisting of a membrane-bound (ADAM12L) and a secreted (ADAM12S) form, is expressed exclusively in regenerating and developing tissue as well as in certain cancer types. Strong ADAM12 expression levels have been noticed in the human placenta, and deregulated ADAM12S levels were associated with various pregnancy-related disorders including pre-eclampsia and intrauterine growth restriction. However, the role of ADAM12 in trophoblast motility has not been investigated so far. Hence, the present study aimed to investigate the specific function of the protease by using different primary trophoblast cell models. Immunofluorescence and Western blot analyses of first trimester placental tissue and differentiating primary first trimester cytotrophoblasts (CTBs) indicated strong upregulation of both of the ADAM12 isoforms during extravillous trophoblast differentiation. Functional assays involving short interfering RNA (siRNA)-mediated knockdown studies in primary CTBs and first trimester explant cultures revealed a significant repression of trophoblast motility upon partial loss of ADAM12. Conversely, isoform-specific overexpression in the ADAM12-negative trophoblast cell line SGHPL-5 enhanced the invasive capacity of these cells. We further confirmed proteolytic activity of trophoblast-derived ADAM12S by demonstrating its potential to degrade insulin-like growth factor-binding protein 3. Finally, we suggest that ADAM12S exerts its pro-migratory function in trophoblasts by inducing integrin beta 1-mediated cellular spreading.

Chiral and nematic phases of flexible active filaments.

The emergence of large-scale order in self-organized systems relies on local interactions between individual components. During bacterial cell division, FtsZ-a prokaryotic homologue of the eukaryotic protein tubulin-polymerizes into treadmilling filaments that further organize into a cytoskeletal ring. In vitro, FtsZ filaments can form dynamic chiral assemblies. However, how the active and passive properties of individual filaments relate to these large-scale self-organized structures remains poorly understood. Here we connect single-filament properties with the mesoscopic scale by combining minimal active matter simulations and biochemical reconstitution experiments. We show that the density and flexibility of active chiral filaments define their global order. At intermediate densities, curved, flexible filaments organize into chiral rings and polar bands. An effectively nematic organization dominates for high densities and for straight, mutant filaments with increased rigidity. Our predicted phase diagram quantitatively captures these features, demonstrating how the flexibility, density and chirality of the active filaments affect their collective behaviour. Our findings shed light on the fundamental properties of active chiral matter and explain how treadmilling FtsZ filaments organize during bacterial cell division.

Imaging brain tissue architecture across millimeter to nanometer scales.

Mapping the complex and dense arrangement of cells and their connectivity in brain tissue demands nanoscale spatial resolution imaging. Super-resolution optical microscopy excels at visualizing specific molecules and individual cells but fails to provide tissue context. Here we developed Comprehensive Analysis of Tissues across Scales (CATS), a technology to densely map brain tissue architecture from millimeter regional to nanometer synaptic scales in diverse chemically fixed brain preparations, including rodent and human. CATS uses fixation-compatible extracellular labeling and optical imaging, including stimulated emission depletion or expansion microscopy, to comprehensively delineate cellular structures. It enables three-dimensional reconstruction of single synapses and mapping of synaptic connectivity by identification and analysis of putative synaptic cleft regions. Applying CATS to the mouse hippocampal mossy fiber circuitry, we reconstructed and quantified the synaptic input and output structure of identified neurons. We furthermore demonstrate applicability to clinically derived human tissue samples, including formalin-fixed paraffin-embedded routine diagnostic specimens, for visualizing the cellular architecture of brain tissue in health and disease.

A direct excitatory projection from entorhinal layer 6b neurons to the hippocampus contributes to spatial coding and memory.

The mammalian hippocampal formation (HF) plays a key role in several higher brain functions, such as spatial coding, learning and memory. Its simple circuit architecture is often viewed as a trisynaptic loop, processing input originating from the superficial layers of the entorhinal cortex (EC) and sending it back to its deeper layers. Here, we show that excitatory neurons in layer 6b of the mouse EC project to all sub-regions comprising the HF and receive input from the CA1, thalamus and claustrum. Furthermore, their output is characterized by unique slow-decaying excitatory postsynaptic currents capable of driving plateau-like potentials in their postsynaptic targets. Optogenetic inhibition of the EC-6b pathway affects spatial coding in CA1 pyramidal neurons, while cell ablation impairs not only acquisition of new spatial memories, but also degradation of previously acquired ones. Our results provide evidence of a functional role for cortical layer 6b neurons in the adult brain.

Advantages of Acute Brain Slices Prepared at Physiological Temperature in the Characterization of Synaptic Functions.

Acute brain slice preparation is a powerful experimental model for investigating the characteristics of synaptic function in the brain. Although brain tissue is usually cut at ice-cold temperature (CT) to facilitate slicing and avoid neuronal damage, exposure to CT causes molecular and architectural changes of synapses. To address these issues, we investigated ultrastructural and electrophysiological features of synapses in mouse acute cerebellar slices prepared at ice-cold and physiological temperature (PT). In the slices prepared at CT, we found significant spine loss and reconstruction, synaptic vesicle rearrangement and decrease in synaptic proteins, all of which were not detected in slices prepared at PT. Consistent with these structural findings, slices prepared at PT showed higher release probability. Furthermore, preparation at PT allows electrophysiological recording immediately after slicing resulting in higher detectability of long-term depression (LTD) after motor learning compared with that at CT. These results indicate substantial advantages of the slice preparation at PT for investigating synaptic functions in different physiological conditions.

Pregnancy-associated diamine oxidase originates from extravillous trophoblasts and is decreased in early-onset preeclampsia.

Human extravillous trophoblast (EVT) invasion of the pregnant uterus constitutes a pivotal event for the establishment of the maternal-fetal interface. Compromised EVT function manifesting in inadequate arterial remodeling is associated with the severe pregnancy disorder early-onset preeclampsia (eoPE). Recent studies suggest that EVTs invade the entire uterine vasculature including arteries, veins and lymphatics in the first trimester of pregnancy. We therefore hypothesized that EVT-derived factors accumulate in the circulation of pregnant women early in gestation and may serve to predict eoPE. In contrast to published literature, we demonstrate that placenta-associated diamine oxidase (DAO) is not expressed by maternal decidual cells but solely by EVTs, especially when in close proximity to decidual vessels. Cultures of primary EVTs express and secret large amounts of bioactive DAO. ELISA measurements indicate a pregnancy-specific rise in maternal DAO plasma levels around gestational week (GW) 7 coinciding with vascular invasion of EVTs. Strikingly, DAO levels from eoPE cases were significantly lower (40%) compared to controls in the first trimester of pregnancy but revealed no difference at mid gestation. Furthermore, DAO-containing pregnancy plasma rapidly inactivates pathophysiologically relevant histamine levels. This study represents the first proof of concept suggesting EVT-specific signatures as diagnostic targets for the prediction of eoPE.

Chymase-producing cells of the innate immune system are required for decidual vascular remodeling and fetal growth.

Intrauterine growth restriction (IUGR) is caused by insufficient remodeling of spiral arteries (SAs). The mechanism underlying the relevance of natural killer cells (NKs) and mast cells (MCs) for SA remodeling and its effects on pregnancy outcome are not well understood. We show that NK depletion arrested SA remodeling without affecting pregnancy. MC depletion resulted in abnormally remodeled SAs and IUGR. Combined absence of NKs and MCs substantially affected SA remodeling and impaired fetal growth. We found that α-chymase mast cell protease (Mcpt) 5 mediates apoptosis of uterine smooth muscle cells, a key feature of SA remodeling. Additionally, we report a previously unknown source for Mcpt5: uterine (u) NKs. Mice with selective deletion of Mcpt5+ cells had un-remodeled SAs and growth-restricted progeny. The human α-chymase CMA1, phylogenetic homolog of Mcpt5, stimulated the ex vivo migration of human trophoblasts, a pre-requisite for SA remodeling. Our results show that chymases secreted by uMCs and uNKs are pivotal to the vascular changes required to support pregnancy. Understanding the mechanisms underlying pregnancy-induced vascular changes is essential for developing therapeutic options against pregnancy complications associated with poor vascular remodeling.

Function and control of human invasive trophoblast subtypes: Intrinsic vs. maternal control.

The establishment of a functional placenta is pivotal for normal fetal development and the maintenance of pregnancy. In the course of early placentation, trophoblast precursors differentiate into highly invasive trophoblast subtypes. These cells, referred to as extravillous trophoblasts (EVTs), penetrate the maternal uterus reaching as far as the inner third of the myometrium. One of the most fundamental functions of EVTs is the transformation of spiral arteries to establish the uteroplacental blood circulation assuring an adequate nutrient and gas supply to the developing fetus. To achieve this, specialized EVT subpopulations interact with maternal immune cells, provoke elastolysis in the arterial wall and replace the endothelial cells lining the spiral arteries to induce intraluminal vascular remodeling. These and other trophoblast-mediated processes are tightly controlled by paracrine signals from the maternal decidua and furthermore underlie an intrinsic cell-type specific program. Various severe pregnancy complications such as preeclampsia or intrauterine growth retardation are associated with abnormal EVT function, shallow invasion, and decreased blood flow to the placenta. Hence a better understanding of human trophoblast invasion seems mandatory to improve therapeutic intervention. This approach, however, requires a profound knowledge of the human placenta, its various trophoblast subtypes and in particular a better understanding of the regulatory network that controls the invasive phenotype of EVTs.

Wingless ligand 5a is a critical regulator of placental growth and survival.

The maternal uterine environment is likely critical for human placental morphogenesis and development of its different trophoblast subtypes. However, factors controlling growth and differentiation of these cells during early gestation remain poorly elucidated. Herein, we provide evidence that the ligand Wnt5a could be a critical regulator of trophoblast proliferation and survival. Immunofluorescence of tissues and western blot analyses of primary cultures revealed abundant Wnt5a expression and secretion from first trimester decidual and villous stromal cells. The ligand was also detectable in decidual glands, macrophages and NK cells. Wnt5a increased proliferation of villous cytotrophoblasts and cell column trophoblasts, outgrowth on collagen I as well as cyclin A and D1 expression in floating explant cultures, but suppressed camptothecin-induced apoptosis. Similarly, Wnt5a stimulated BrdU incorporation and decreased caspase-cleaved cytokeratin 18 neo-epitope expression in primary cytotrophoblasts. Moreover, Wnt5a promoted activation of the MAPK pathway in the different trophoblast models. Chemical inhibition of p42/44 MAPK abolished cyclin D1 expression and Wnt5a-stimulated proliferation. Compared to controls, MAPK phosphorylation and proliferation of cytotrophoblasts declined upon supplementation of supernatants from Wnt5a gene-silenced decidual or villous stromal cells. In summary, non-canonical Wnt5a signalling could play a role in early human trophoblast development by promoting cell proliferation and survival.

Notch2 controls prolactin and insulin-like growth factor binding protein-1 expression in decidualizing human stromal cells of early pregnancy.

Decidualization, the transformation of the human uterine mucosa into the endometrium of pregnancy, is critical for successful implantation and embryonic development. However, key regulatory factors controlling differentiation of uterine stromal cells into hormone-secreting decidual cells have not been fully elucidated. Hence, we herein investigated the role of the Notch signaling pathway in human decidual stromal cells (HDSC) isolated from early pregnancy samples. Immunofluorescence of first trimester decidual tissues revealed expression of Notch2 receptor and its putative, membrane-anchored interaction partners Jagged1, Delta-like (DLL) 1 and DLL4 in stromal cells whereas other Notch receptors and ligands were absent from these cells. During in vitro differentiation with estrogen/progesterone (E2P4) and/or cyclic adenosine monophosphate (cAMP) HDSC constitutively expressed Notch2 and weakly downregulated Jagged1 mRNA and protein, measured by quantitative PCR (qPCR) and Western blotting, respectively. However, increased levels of DLL1 and DLL4 were observed in the decidualizing cultures. Transfection of a Notch luciferase reporter and qPCR of the Notch target gene hairy and enhancer of split 1 (HES1) revealed an induction of canonical Notch activity during in vitro differentiation. In contrast, treatment of HDSC with a chemical Notch/γ-secretase inhibitor decreased cAMP/E2P4-stimulated Notch luciferase activity, HES1 transcript levels and mRNA expression of the decidual marker genes prolactin (PRL) and insulin-like growth factor binding protein 1 (IGFBP1). Similarly, siRNA-mediated gene silencing or antibody-mediated blocking of Notch2 diminished HES1, PRL and IGFBP1 mRNA levels as well as secreted PRL protein. In summary, the data suggest that canonical, Notch2-dependent signaling plays a role in human decidualization.

Notch signaling plays a critical role in motility and differentiation of human first-trimester cytotrophoblasts.

Failures in human extravillous trophoblast (EVT) development could be involved in the pathogenesis of pregnancy diseases. However, the underlying mechanisms have been poorly characterized. Here, we provide evidence that Notch signaling could represent a key regulatory pathway controlling trophoblast proliferation, motility, and differentiation. Immunofluorescence of first-trimester placental tissues revealed expression of Notch receptors (Notch2 and Notch3) and membrane-anchored ligands (delta-like ligand [DLL] 1 and -4 and Jagged [JAG] 1 and -2) in villous cytotrophoblasts (vCTBs), cell column trophoblasts (CCTs), and EVTs. Notch4 and Notch1 were exclusively expressed in vCTBs and in CCTs, respectively. Both proteins decreased in Western blot analyses of first-trimester, primary cytotrophoblasts (CTBs) differentiating on fibronectin. Luciferase reporter analyses suggested basal, canonical Notch activity in SGHPL-5 cells and primary cells that was increased upon seeding on DLL4-coated dishes and diminished in the presence of the Notch/γ-secretase inhibitors N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT) or L-685,458. Bromodeoxyuridine labeling, cyclin D1 mRNA expression, and cell counting indicated that chemical inhibition of Notch signaling elevated proliferation in the different primary trophoblast model systems. Notch inhibition also increased motility of SGHPL-5 cells through uncoated and fibronectin-coated Transwells, motility of primary CTBs, as well as migration in villous explant cultures on collagen I. Accordingly, small interfering RNA-mediated gene silencing of Notch1 also elevated SGHPL-5 cell migration. In contrast, motility of primary cultures and SGHPL-5 cells was diminished in the presence of DLL4. Moreover, DAPT increased markers of differentiated EVT, ie, human leukocyte antigen G1, integrin α5, and T-cell factor 4, whereas DLL4 provoked the opposite. In summary, the data suggest that canonical Notch signaling impairs motility and differentiation of first-trimester CTBs.

Control of human endometrial stromal cell motility by PDGF-BB, HB-EGF and trophoblast-secreted factors.

Human implantation involves extensive tissue remodeling at the fetal-maternal interface. It is becoming increasingly evident that not only trophoblast, but also decidualizing endometrial stromal cells are inherently motile and invasive, and likely contribute to the highly dynamic processes at the implantation site. The present study was undertaken to further characterize the mechanisms involved in the regulation of endometrial stromal cell motility and to identify trophoblast-derived factors that modulate migration. Among local growth factors known to be present at the time of implantation, heparin-binding epidermal growth factor-like growth factor (HB-EGF) triggered chemotaxis (directed locomotion), whereas platelet-derived growth factor (PDGF)-BB elicited both chemotaxis and chemokinesis (non-directed locomotion) of endometrial stromal cells. Supernatants of the trophoblast cell line AC-1M88 and of first trimester villous explant cultures stimulated chemotaxis but not chemokinesis. Proteome profiling for cytokines and angiogenesis factors revealed neither PDGF-BB nor HB-EGF in conditioned media from trophoblast cells or villous explants, while placental growth factor, vascular endothelial growth factor and PDGF-AA were identified as prominent secretory products. Among these, only PDGF-AA triggered endometrial stromal cell chemotaxis. Neutralization of PDGF-AA in trophoblast conditioned media, however, did not diminish chemoattractant activity, suggesting the presence of additional trophoblast-derived chemotactic factors. Pathway inhibitor studies revealed ERK1/2, PI3 kinase/Akt and p38 signaling as relevant for chemotactic motility, whereas chemokinesis depended primarily on PI3 kinase/Akt activation. Both chemotaxis and chemokinesis were stimulated upon inhibition of Rho-associated, coiled-coil containing protein kinase. The chemotactic response to trophoblast secretions was not blunted by inhibition of isolated signaling cascades, indicating activation of overlapping pathways in trophoblast-endometrial communication. In conclusion, trophoblast signals attract endometrial stromal cells, while PDGF-BB and HB-EGF, although not identified as trophoblast-derived, are local growth factors that may serve to fine-tune directed and non-directed migration at the implantation site.