Spatiotemporal, optogenetic control of gene expression in organoids.

Organoids derived from stem cells have become an increasingly important tool for studying human development and modeling disease. However, methods are still needed to control and study spatiotemporal patterns of gene expression in organoids. Here we combined optogenetics and gene perturbation technologies to activate or knock-down RNA of target genes in programmable spatiotemporal patterns. To illustrate the usefulness of our approach, we locally activated Sonic Hedgehog (SHH) signaling in an organoid model for human neurodevelopment. Spatial and single-cell transcriptomic analyses showed that this local induction was sufficient to generate stereotypically patterned organoids and revealed new insights into SHH's contribution to gene regulation in neurodevelopment. With this study, we propose optogenetic perturbations in combination with spatial transcriptomics as a powerful technology to reprogram and study cell fates and tissue patterning in organoids.

miR-7 controls glutamatergic transmission and neuronal connectivity in a Cdr1as-dependent manner.

The circular RNA (circRNA) Cdr1as is conserved across mammals and highly expressed in neurons, where it directly interacts with microRNA miR-7. However, the biological function of this interaction is unknown. Here, using primary cortical murine neurons, we demonstrate that stimulating neurons by sustained depolarization rapidly induces two-fold transcriptional upregulation of Cdr1as and strong post-transcriptional stabilization of miR-7. Cdr1as loss causes doubling of glutamate release from stimulated synapses and increased frequency and duration of local neuronal bursts. Moreover, the periodicity of neuronal networks increases, and synchronicity is impaired. Strikingly, these effects are reverted by sustained expression of miR-7, which also clears Cdr1as molecules from neuronal projections. Consistently, without Cdr1as, transcriptomic changes caused by miR-7 overexpression are stronger (including miR-7-targets downregulation) and enriched in secretion/synaptic plasticity pathways. Altogether, our results suggest that in cortical neurons Cdr1as buffers miR-7 activity to control glutamatergic excitatory transmission and neuronal connectivity important for long-lasting synaptic adaptations.

The histone H4 lysine 20 demethylase DPY-21 regulates the dynamics of condensin DC binding.

Condensin is a multi-subunit structural maintenance of chromosomes (SMC) complex that binds to and compacts chromosomes. Here, we addressed the regulation of condensin binding dynamics using Caenorhabditis elegans condensin DC, which represses X chromosomes in hermaphrodites for dosage compensation. We established fluorescence recovery after photobleaching (FRAP) using the SMC4 homolog DPY-27 and showed that a well-characterized ATPase mutation abolishes DPY-27 binding to X chromosomes. Next, we performed FRAP in the background of several chromatin modifier mutants that cause varying degrees of X chromosome derepression. The greatest effect was in a null mutant of the H4K20me2 demethylase DPY-21, where the mobile fraction of condensin DC reduced from ∼30% to 10%. In contrast, a catalytic mutant of dpy-21 did not regulate condensin DC mobility. Hi-C sequencing data from the dpy-21 null mutant showed little change compared to wild-type data, uncoupling Hi-C-measured long-range DNA contacts from transcriptional repression of the X chromosomes. Taken together, our results indicate that DPY-21 has a non-catalytic role in regulating the dynamics of condensin DC binding, which is important for transcription repression.

Teashirt 1 (Tshz1) is essential for the development, survival and function of hypoglossal and phrenic motor neurons in mouse.

Feeding and breathing are essential motor functions and rely on the activity of hypoglossal and phrenic motor neurons that innervate the tongue and diaphragm, respectively. Little is known about the genetic programs that control the development of these neuronal subtypes. The transcription factor Tshz1 is strongly and persistently expressed in developing hypoglossal and phrenic motor neurons. We used conditional mutation of Tshz1 in the progenitor zone of motor neurons (Tshz1MNΔ) to show that Tshz1 is essential for survival and function of hypoglossal and phrenic motor neurons. Hypoglossal and phrenic motor neurons are born in correct numbers, but many die between embryonic day 13.5 and 14.5 in Tshz1MNΔ mutant mice. In addition, innervation and electrophysiological properties of phrenic and hypoglossal motor neurons are altered. Severe feeding and breathing problems accompany this developmental deficit. Although motor neuron survival can be rescued by elimination of the pro-apoptotic factor Bax, innervation, feeding and breathing defects persist in Bax-/-; Tshz1MNΔ mutants. We conclude that Tshz1 is an essential transcription factor for the development and physiological function of phrenic and hypoglossal motor neurons.

Alternative 3' UTRs direct localization of functionally diverse protein isoforms in neuronal compartments.

The proper subcellular localization of RNAs and local translational regulation is crucial in highly compartmentalized cells, such as neurons. RNA localization is mediated by specific cis-regulatory elements usually found in mRNA 3'UTRs. Therefore, processes that generate alternative 3'UTRs-alternative splicing and polyadenylation-have the potential to diversify mRNA localization patterns in neurons. Here, we performed mapping of alternative 3'UTRs in neurites and soma isolated from mESC-derived neurons. Our analysis identified 593 genes with differentially localized 3'UTR isoforms. In particular, we have shown that two isoforms of Cdc42 gene with distinct functions in neuronal polarity are differentially localized between neurites and soma of mESC-derived and mouse primary cortical neurons, at both mRNA and protein level. Using reporter assays and 3'UTR swapping experiments, we have identified the role of alternative 3'UTRs and mRNA transport in differential localization of alternative CDC42 protein isoforms. Moreover, we used SILAC to identify isoform-specific Cdc42 3'UTR-bound proteome with potential role in Cdc42 localization and translation. Our analysis points to usage of alternative 3'UTR isoforms as a novel mechanism to provide for differential localization of functionally diverse alternative protein isoforms.

Proton electrochemical gradient: Driving and regulating neurotransmitter uptake.

Accumulation of neurotransmitters in the lumen of synaptic vesicles (SVs) relies on the activity of the vacuolar-type H+ -ATPase. This pump drives protons into the lumen, generating a proton electrochemical gradient (ΔµH+ ) across the membrane. Recent work has demonstrated that the balance between the chemical (ΔpH) and electrical (ΔΨ) components of ΔµH+ is regulated differently by some distinct vesicle types. As different neurotransmitter transporters use ΔpH and ΔΨ with different relative efficiencies, regulation of this gradient balance has the potential to influence neurotransmitter uptake. Nevertheless, the underlying mechanisms responsible for this regulation remain poorly understood. In this review, we provide an overview of current neurotransmitter uptake models, with a particular emphasis on the distinct roles of the electrical and chemical gradients and current hypotheses for regulatory mechanisms.

Superpriming of synaptic vesicles as a common basis for intersynapse variability and modulation of synaptic strength.

Glutamatergic synapses show large variations in strength and short-term plasticity (STP). We show here that synapses displaying an increased strength either after posttetanic potentiation (PTP) or through activation of the phospholipase-C-diacylglycerol pathway share characteristic properties with intrinsically strong synapses, such as (i) pronounced short-term depression (STD) during high-frequency stimulation; (ii) a conversion of that STD into a sequence of facilitation followed by STD after a few conditioning stimuli at low frequency; (iii) an equalizing effect of such conditioning stimulation, which reduces differences among synapses and abolishes potentiation; and (iv) a requirement of long periods of rest for reconstitution of the original STP pattern. These phenomena are quantitatively described by assuming that a small fraction of "superprimed" synaptic vesicles are in a state of elevated release probability (p ∼ 0.5). This fraction is variable in size among synapses (typically about 30%), but increases after application of phorbol ester or during PTP. The majority of vesicles, released during repetitive stimulation, have low release probability (p ∼ 0.1), are relatively uniform in number across synapses, and are rapidly recruited. In contrast, superprimed vesicles need several seconds to be regenerated. They mediate enhanced synaptic strength at the onset of burst-like activity, the impact of which is subject to modulation by slow modulatory transmitter systems.

Functional analysis of PEX13 mutation in a Zellweger syndrome spectrum patient reveals novel homooligomerization of PEX13 and its role in human peroxisome biogenesis.

In humans, the concerted action of at least 13 different peroxisomal PEX proteins is needed for proper peroxisome biogenesis. Mutations in any of these PEX genes can lead to lethal neurometabolic disorders of the Zellweger syndrome spectrum (ZSS). Previously, we identified the W313G mutation located within the SH3 domain of the peroxisomal protein, PEX13. As this tryptophan residue is highly conserved in almost all known SH3 proteins, we investigated the pathogenic mechanism of the W313G mutation and its role in PEX13 interactions and functions in peroxisome biogenesis. Here, we report for the first time that human PEX13 interacts with itself in peroxisomes in living cells. We demonstrate that the import of PTS1 (peroxisomal targeting signal 1) proteins is specifically disrupted when homooligomerization of PEX13 is interrupted. Live cell FRET microscopy in living cells as well as co-immunoprecipitation experiments reveal that the highly conserved W313 residue is important for self-association of PEX13 but is not required for interaction with PEX14, a well-established interaction partner at the peroxisomal membrane. Experiments with truncated constructs indicate that although the W313G mutation resides in the C-terminal SH3 domain, the N-terminal half is necessary for peroxisomal localization, which in turn appears to be crucial for homooligomerization. Furthermore, rescue of homooligomerization in the W313G mutant cells through complementation with truncation constructs restores import of peroxisomal matrix proteins. Taken together, the thorough analyses of a ZSS patient mutation unraveled the general cell biological function of PEX13 and its mechanism in the import of peroxisomal matrix PTS1 proteins.

Calcium-buffering effects of gluconate and nucleotides, as determined by a novel fluorimetric titration method.

Significantly more Ca(2+) influx is required for eliciting release of neurotransmitter during whole cell patch clamp recording in the Calyx of Held, when gluconate with 3 mm free ATP is used as pipette filling solution, as compared to a methanesulfonate-based solution with excess Mg(2+). This reduction in efficiency of Ca(2+) in eliciting release is due to low-affinity Ca(2+) binding of both gluconate and ATP(2-) anions. To study these effects we developed a simple fluorimeteric titration procedure, which reports the dissociation constant, KD, of a given Ca(2+) indicator dye, multiplied by 1 plus the sum of Ca(2+) binding ratios of any anions, which act as low-affinity Ca(2+) ligands. For solutions without Ca(2+) binding anions we find KD values for Fura2FF ranging from 11.5 ± 1.7 to 15.6 ± 7.47 µm depending on the dominant anion used. For Fura6F and KCl-based solutions we find KD = 17.8 ± 1.3 µm. For solutions with gluconate as the main anion and for solutions that contain nucleotides, such as ATP and GTP, we find much higher values for the product. Assuming that the KD of the indicator dye is equal to that of KCl-based solutions we calculate the summed Ca(2+) binding ratios and find a value of 3.55 for a solution containing 100 mm potassium gluconate and 4 mm ATP. Gluconate contributes a value of 1.75 to this number, while the contribution of ATP depends strongly on the presence of Mg(2+) and varies from 0.8 (with excess Mg(2+)) to 13.8 (in the presence of 3 mm free ATP). Methanesulfonate has negligible Ca(2+) binding capacity. These results explain the reduced efficiency of Ca(2+) influx in the presence of gluconate or nucleotides, as these anions are expected to intercept Ca(2+) ions at short distance.

Heterodimerization of serotonin receptors 5-HT1A and 5-HT7 differentially regulates receptor signalling and trafficking.

Serotonin receptors 5-HT(1A) and 5-HT(7) are highly coexpressed in brain regions implicated in depression. However, their functional interaction has not been established. In the present study we show that 5-HT(1A) and 5-HT(7) receptors form heterodimers both in vitro and in vivo. Foerster resonance energy transfer-based assays revealed that, in addition to heterodimers, homodimers composed either of 5-HT(1A) or 5-HT(7) receptors together with monomers coexist in cells. The highest affinity for complex formation was obtained for the 5-HT(7)-5-HT(7) homodimers, followed by the 5-HT(7)-5-HT(1A) heterodimers and 5-HT(1A)-5-HT(1A) homodimers. Functionally, heterodimerization decreases 5-HT(1A)-receptor-mediated activation of G(i) protein without affecting 5-HT(7)-receptor-mediated signalling. Moreover, heterodimerization markedly decreases the ability of the 5-HT(1A) receptor to activate G-protein-gated inwardly rectifying potassium channels in a heterologous system. The inhibitory effect on such channels was also preserved in hippocampal neurons, demonstrating a physiological relevance of heteromerization in vivo. In addition, heterodimerization is crucially involved in initiation of the serotonin-mediated 5-HT(1A) receptor internalization and also enhances the ability of the 5-HT(1A) receptor to activate the mitogen-activated protein kinases. Finally, we found that production of 5-HT(7) receptors in the hippocampus continuously decreases during postnatal development, indicating that the relative concentration of 5-HT(1A)-5-HT(7) heterodimers and, consequently, their functional importance undergoes pronounced developmental changes.

MarShie: a clearing protocol for 3D analysis of single cells throughout the bone marrow at subcellular resolution.

Analyzing immune cell interactions in the bone marrow is vital for understanding hematopoiesis and bone homeostasis. Three-dimensional analysis of the complete, intact bone marrow within the cortex of whole long bones remains a challenge, especially at subcellular resolution. We present a method that stabilizes the marrow and provides subcellular resolution of fluorescent signals throughout the murine femur, enabling identification and spatial characterization of hematopoietic and stromal cell subsets. By combining a pre-processing algorithm for stripe artifact removal with a machine-learning approach, we demonstrate reliable cell segmentation down to the deepest bone marrow regions. This reveals age-related changes in the marrow. It highlights the interaction between CX3CR1+ cells and the vascular system in homeostasis, in contrast to other myeloid cell types, and reveals their spatial characteristics after injury. The broad applicability of this method will contribute to a better understanding of bone marrow biology.

Establishment of gastrointestinal assembloids to study the interplay between epithelial crypts and their mesenchymal niche.

The cellular organization of gastrointestinal crypts is orchestrated by different cells of the stromal niche but available in vitro models fail to fully recapitulate the interplay between epithelium and stroma. Here, we establish a colon assembloid system comprising the epithelium and diverse stromal cell subtypes. These assembloids recapitulate the development of mature crypts resembling in vivo cellular diversity and organization, including maintenance of a stem/progenitor cell compartment in the base and their maturation into secretory/absorptive cell types. This process is supported by self-organizing stromal cells around the crypts that resemble in vivo organization, with cell types that support stem cell turnover adjacent to the stem cell compartment. Assembloids that lack BMP receptors either in epithelial or stromal cells fail to undergo proper crypt formation. Our data highlight the crucial role of bidirectional signaling between epithelium and stroma, with BMP as a central determinant of compartmentalization along the crypt axis.

Modelling viral encephalitis caused by herpes simplex virus 1 infection in cerebral organoids.

Herpes simplex encephalitis is a life-threatening disease of the central nervous system caused by herpes simplex viruses (HSVs). Following standard of care with antiviral acyclovir treatment, most patients still experience various neurological sequelae. Here we characterize HSV-1 infection of human brain organoids by combining single-cell RNA sequencing, electrophysiology and immunostaining. We observed strong perturbations of tissue integrity, neuronal function and cellular transcriptomes. Under acyclovir treatment viral replication was stopped, but did not prevent HSV-1-driven defects such as damage of neuronal processes and neuroepithelium. Unbiased analysis of pathways deregulated upon infection revealed tumour necrosis factor activation as a potential causal factor. Combination of anti-inflammatory drugs such as necrostatin-1 or bardoxolone methyl with antiviral treatment prevented the damages caused by infection, indicating that tuning the inflammatory response in acute infection may improve current therapeutic strategies.

Prox2 and Runx3 vagal sensory neurons regulate esophageal motility.

Vagal sensory neurons monitor mechanical and chemical stimuli in the gastrointestinal tract. Major efforts are underway to assign physiological functions to the many distinct subtypes of vagal sensory neurons. Here, we use genetically guided anatomical tracing, optogenetics, and electrophysiology to identify and characterize vagal sensory neuron subtypes expressing Prox2 and Runx3 in mice. We show that three of these neuronal subtypes innervate the esophagus and stomach in regionalized patterns, where they form intraganglionic laminar endings. Electrophysiological analysis revealed that they are low-threshold mechanoreceptors but possess different adaptation properties. Lastly, genetic ablation of Prox2 and Runx3 neurons demonstrated their essential roles for esophageal peristalsis in freely behaving mice. Our work defines the identity and function of the vagal neurons that provide mechanosensory feedback from the esophagus to the brain and could lead to better understanding and treatment of esophageal motility disorders.

Quantitative intensity-based FRET approaches--a comparative snapshot.

Förster resonance energy transfer (FRET) has become an important tool for analyzing different aspects of interactions among biological macromolecules in their native environments. FRET analysis has also been successfully applied to study the spatiotemporal regulation of various cellular processes using genetically encoded FRET-based biosensors. A variety of procedures have been described for measuring FRET efficiency or the relative abundance of donor-acceptor complexes, based on analysis of the donor fluorescence lifetime or the spectrally resolved fluorescence intensity. The latter methods are preferable if one wants to not only quantify the apparent FRET efficiencies but also calculate donor-acceptor stoichiometry and observe fast dynamic changes in the interactions among donor and acceptor molecules in live cells. This review focuses on a comparison of the available intensity-based approaches used to measure FRET. We discuss their strengths and weaknesses in terms of FRET quantification, and provide several examples of biological applications.

Pregnancy-induced maternal microchimerism shapes neurodevelopment and behavior in mice.

Life-long brain function and mental health are critically determined by developmental processes occurring before birth. During mammalian pregnancy, maternal cells are transferred to the fetus. They are referred to as maternal microchimeric cells (MMc). Among other organs, MMc seed into the fetal brain, where their function is unknown. Here, we show that, in the offspring's developing brain in mice, MMc express a unique signature of sensome markers, control microglia homeostasis and prevent excessive presynaptic elimination. Further, MMc facilitate the oscillatory entrainment of developing prefrontal-hippocampal circuits and support the maturation of behavioral abilities. Our findings highlight that MMc are not a mere placental leak out, but rather a functional mechanism that shapes optimal conditions for healthy brain function later in life.

Pathogenic variants damage cell composition and single cell transcription in cardiomyopathies.

Pathogenic variants in genes that cause dilated cardiomyopathy (DCM) and arrhythmogenic cardiomyopathy (ACM) convey high risks for the development of heart failure through unknown mechanisms. Using single-nucleus RNA sequencing, we characterized the transcriptome of 880,000 nuclei from 18 control and 61 failing, nonischemic human hearts with pathogenic variants in DCM and ACM genes or idiopathic disease. We performed genotype-stratified analyses of the ventricular cell lineages and transcriptional states. The resultant DCM and ACM ventricular cell atlas demonstrated distinct right and left ventricular responses, highlighting genotype-associated pathways, intercellular interactions, and differential gene expression at single-cell resolution. Together, these data illuminate both shared and distinct cellular and molecular architectures of human heart failure and suggest candidate therapeutic targets.

Single synapse glutamate imaging reveals multiple levels of release mode regulation in mammalian synapses.

Mammalian central synapses exhibit vast heterogeneity in signaling strength. To understand the extent of this diversity, how it is achieved, and its functional implications, characterization of a large number of individual synapses is required. Using glutamate imaging, we characterized the evoked release probability and spontaneous release frequency of over 24,000 individual synapses. We found striking variability and no correlation between action potential-evoked and spontaneous synaptic release strength, suggesting distinct regulatory mechanisms. Subpixel localization of individual evoked and spontaneous release events reveals tight spatial regulation of evoked release and enhanced spontaneous release outside of evoked release region. Using on-stage post hoc immune-labeling of vesicle-associated proteins, Ca2+-sensing proteins, and soluble presynaptic proteins we were able to show that distinct molecular ensembles are associated with evoked and spontaneous modes of synaptic release.

Dual-view light-sheet imaging through a tilted glass interface using a deformable mirror.

Light-sheet microscopy has become indispensable for imaging developing organisms, and imaging from multiple directions (views) is essential to improve its spatial resolution. We combine multi-view light-sheet microscopy with microfluidics using adaptive optics (deformable mirror) which corrects aberrations introduced by the 45o-tilted glass coverslip. The optimal shape of the deformable mirror is computed by an iterative algorithm that optimizes the point-spread function in two orthogonal views. Simultaneous correction in two optical arms is achieved via a knife-edge mirror that splits the excitation path and combines the detection paths. Our design allows multi-view light-sheet microscopy with microfluidic devices for precisely controlled experiments and high-content screening.

Signal/noise analysis of FRET-based sensors.

Molecular sensors based on intramolecular Förster resonance energy transfer (FRET) have become versatile tools to monitor regulatory molecules in living tissue. However, their use is often compromised by low signal strength and excessive noise. We analyzed signal/noise (SNR) aspects of spectral FRET analysis methods, with the following conclusions: The most commonly used method (measurement of the emission ratio after a single short wavelength excitation) is optimal in terms of signal/noise, if only relative changes of this uncalibrated ratio are of interest. In the case that quantitative data on FRET efficiencies are required, these can be calculated from the emission ratio and some calibration parameters, but at reduced SNR. Lux-FRET, a recently described method for spectral analysis of FRET data, allows one to do so in three different ways, each based on a ratio of two out of three measured fluorescence signals (the donor and acceptor signal during a short-wavelength excitation and the acceptor signal during long wavelength excitation). Lux-FRET also allows for calculation of the total abundance of donor and acceptor fluorophores. The SNR for all these quantities is lower than that of the plain emission ratio due to unfavorable error propagation. However, if ligand concentration is calculated either from lux-FRET values or else, after its calibration, from the emission ratio, SNR for both analysis modes is very similar. Likewise, SNR values are similar, if the noise of these quantities is related to the expected dynamic range. We demonstrate these relationships based on data from an Epac-based cAMP sensor and discuss how the SNR changes with the FRET efficiency and the number of photons collected.