A human embryonic limb cell atlas resolved in space and time.

Human limbs emerge during the fourth post-conception week as mesenchymal buds, which develop into fully formed limbs over the subsequent months1. This process is orchestrated by numerous temporally and spatially restricted gene expression programmes, making congenital alterations in phenotype common2. Decades of work with model organisms have defined the fundamental mechanisms underlying vertebrate limb development, but an in-depth characterization of this process in humans has yet to be performed. Here we detail human embryonic limb development across space and time using single-cell and spatial transcriptomics. We demonstrate extensive diversification of cells from a few multipotent progenitors to myriad differentiated cell states, including several novel cell populations. We uncover two waves of human muscle development, each characterized by different cell states regulated by separate gene expression programmes, and identify musculin (MSC) as a key transcriptional repressor maintaining muscle stem cell identity. Through assembly of multiple anatomically continuous spatial transcriptomic samples using VisiumStitcher, we map cells across a sagittal section of a whole fetal hindlimb. We reveal a clear anatomical segregation between genes linked to brachydactyly and polysyndactyly, and uncover transcriptionally and spatially distinct populations of the mesenchyme in the autopod. Finally, we perform single-cell RNA sequencing on mouse embryonic limbs to facilitate cross-species developmental comparison, finding substantial homology between the two species.

A spatial human thymus cell atlas mapped to a continuous tissue axis.

T cells develop from circulating precursors, which enter the thymus and migrate throughout specialised sub-compartments to support maturation and selection. This process starts already in early fetal development and is highly active until the involution of the thymus in adolescence. To map the micro-anatomical underpinnings of this process in pre- vs. post-natal states, we undertook a spatially resolved analysis and established a new quantitative morphological framework for the thymus, the Cortico-Medullary Axis. Using this axis in conjunction with the curation of a multimodal single-cell, spatial transcriptomics and high-resolution multiplex imaging atlas, we show that canonical thymocyte trajectories and thymic epithelial cells are highly organised and fully established by post-conception week 12, pinpoint TEC progenitor states, find that TEC subsets and peripheral tissue genes are associated with Hassall's Corpuscles and uncover divergence in the pace and drivers of medullary entry between CD4 vs. CD8 T cell lineages. These findings are complemented with a holistic toolkit for spatial analysis and annotation, providing a basis for a detailed understanding of T lymphocyte development.

Knockout of the longevity gene Klotho perturbs aging- and Alzheimer's disease-linked brain microRNAs and tRNA fragments.

Overexpression of the longevity gene Klotho prolongs, while its knockout shortens lifespan and impairs cognition via altered fibroblast growth factor signaling that perturbs myelination and synapse formation; however, comprehensive analysis of Klotho's knockout consequences on mammalian brain transcriptomics is lacking. Here, we report the altered levels under Klotho knockout of 1059 long RNAs, 27 microRNAs (miRs) and 6 tRNA fragments (tRFs), reflecting effects upon aging and cognition. Perturbed transcripts included key neuronal and glial pathway regulators that are notably changed in murine models of aging and Alzheimer's Disease (AD) and in corresponding human post-mortem brain tissue. To seek cell type distributions of the affected short RNAs, we isolated and FACS-sorted neurons and microglia from live human brain tissue, yielding detailed cell type-specific short RNA-seq datasets. Together, our findings revealed multiple Klotho deficiency-perturbed aging- and neurodegeneration-related long and short RNA transcripts in both neurons and glia from murine and human brain.

High-throughput morphometric and transcriptomic profiling uncovers composition of naïve and sensory-deprived cortical cholinergic VIP/CHAT neurons.

Cortical neuronal networks control cognitive output, but their composition and modulation remain elusive. Here, we studied the morphological and transcriptional diversity of cortical cholinergic VIP/ChAT interneurons (VChIs), a sparse population with a largely unknown function. We focused on VChIs from the whole barrel cortex and developed a high-throughput automated reconstruction framework, termed PopRec, to characterize hundreds of VChIs from each mouse in an unbiased manner, while preserving 3D cortical coordinates in multiple cleared mouse brains, accumulating thousands of cells. We identified two fundamentally distinct morphological types of VChIs, bipolar and multipolar that differ in their cortical distribution and general morphological features. Following mild unilateral whisker deprivation on postnatal day seven, we found after three weeks both ipsi- and contralateral dendritic arborization differences and modified cortical depth and distribution patterns in the barrel fields alone. To seek the transcriptomic drivers, we developed NuNeX, a method for isolating nuclei from fixed tissues, to explore sorted VChIs. This highlighted differentially expressed neuronal structural transcripts, altered exitatory innervation pathways and established Elmo1 as a key regulator of morphology following deprivation.

Mapping the developing human immune system across organs.

Single-cell genomics studies have decoded the immune cell composition of several human prenatal organs but were limited in describing the developing immune system as a distributed network across tissues. We profiled nine prenatal tissues combining single-cell RNA sequencing, antigen-receptor sequencing, and spatial transcriptomics to reconstruct the developing human immune system. This revealed the late acquisition of immune-effector functions by myeloid and lymphoid cell subsets and the maturation of monocytes and T cells before peripheral tissue seeding. Moreover, we uncovered system-wide blood and immune cell development beyond primary hematopoietic organs, characterized human prenatal B1 cells, and shed light on the origin of unconventional T cells. Our atlas provides both valuable data resources and biological insights that will facilitate cell engineering, regenerative medicine, and disease understanding.

Cortical VIP+ /ChAT+ interneurons: From genetics to function.

One of the urgent tasks of neuroscience is to understand how neuronal circuits operate, what makes them fail, and how to repair them when needed. Achieving this goal requires identifying the principal circuitry elements and their interactions with one another. However, what constitutes 'an atom' of a neuronal circuit, a neuronal type, is a complex question. In this review we focus on a class of cortical neurons that are exclusively identified by the expression of vasoactive intestinal polypeptide (VIP) and choline acetyltransferase (ChAT). The genetic profile of these VIP+ /ChAT+ interneurons suggests that they can release both γ-aminobutyric acid (GABA) and acetylcholine (ACh). This hints to a specific potential role in the cortical circuitry. Yet the VIP+ /ChAT+ interneurons are sparse (a mere 0.5% of the cortical neurons), which raises questions about their potential to significantly affect the circuit function. In view of recent developments in genetic techniques that allow for direct manipulation of these neurons, we provide a thorough and updated picture of the properties of the VIP+ /ChAT+ interneurons. We discuss their genetic profile, their physiological and structural properties, and their input-output mapping in sensory cortices and the medial prefrontal cortex (mPFC). Then, we examine possible amplification mechanisms for mediating their function in the cortical microcircuit. Finally, we discuss directions for further exploration of the VIP+ /ChAT+ population, focusing on its function during behavioral tasks as compared to the VIP+ /ChAT- population.

Brain pathology and cerebellar purkinje cell loss in a mouse model of chronic neuronopathic Gaucher disease.

Gaucher disease (GD) is currently the focus of considerable attention due primarily to the association between the gene that causes GD (GBA) and Parkinson's disease. Mouse models exist for the systemic (type 1) and for the acute neuronopathic forms (type 2) of GD. Here we report the generation of a mouse that phenotypically models chronic neuronopathic type 3 GD. Gba-/-;Gbatg mice, which contain a Gba transgene regulated by doxycycline, accumulate moderate levels of the offending substrate in GD, glucosylceramide, and live for up to 10 months, i.e. significantly longer than mice which model type 2 GD. Gba-/-;Gbatg mice display behavioral abnormalities at ∼4 months, which deteriorate with age, along with significant neuropathology including loss of Purkinje neurons. Gene expression is altered in the brain and in isolated microglia, although the changes in gene expression are less extensive than in mice modeling type 2 disease. Finally, bone deformities are consistent with the Gba-/-;Gbatg mice being a genuine type 3 GD model. Together, the Gba-/-;Gbatg mice share pathological pathways with acute neuronopathic GD mice but also display differences that might help understand the distinct disease course and progression of type 2 and 3 patients.

A Parkinson's disease CircRNAs Resource reveals a link between circSLC8A1 and oxidative stress.

Circular RNAs (circRNAs) are brain-abundant RNAs of mostly unknown functions. To seek their roles in Parkinson's disease (PD), we generated an RNA sequencing resource of several brain region tissues from dozens of PD and control donors. In the healthy substantia nigra (SN), circRNAs accumulate in an age-dependent manner, but in the PD SN this correlation is lost and the total number of circRNAs reduced. In contrast, the levels of circRNAs are increased in the other studied brain regions of PD patients. We also found circSLC8A1 to increase in the SN of PD individuals. CircSLC8A1 carries 7 binding sites for miR-128 and is strongly bound to the microRNA effector protein Ago2. Indeed, RNA targets of miR-128 are also increased in PD individuals, suggesting that circSLC8A1 regulates miR-128 function and/or activity. CircSLC8A1 levels also increased in cultured cells exposed to the oxidative stress-inducing agent paraquat but were decreased in cells treated with the neuroprotective antioxidant regulator drug Simvastatin. Together, our work links circSLC8A1 to oxidative stress-related Parkinsonism and suggests further exploration of its molecular function in PD.

Barrel cortex VIP/ChAT interneurons suppress sensory responses in vivo.

Cortical interneurons expressing vasoactive intestinal polypeptide (VIP) and choline acetyltransferase (ChAT) are sparsely distributed throughout the neocortex, constituting only 0.5% of its neuronal population. The co-expression of VIP and ChAT suggests that these VIP/ChAT interneurons (VChIs) can release both γ-aminobutyric acid (GABA) and acetylcholine (ACh). In vitro physiological studies quantified the response properties and local connectivity patterns of the VChIs; however, the function of VChIs has not been explored in vivo. To study the role of VChIs in cortical network dynamics and their long-range connectivity pattern, we used in vivo electrophysiology and rabies virus tracing in the barrel cortex of mice. We found that VChIs have a low spontaneous spiking rate (approximately 1 spike/s) in the barrel cortex of anesthetized mice; nevertheless, they responded with higher fidelity to whisker stimulation than the neighboring layer 2/3 pyramidal neurons (Pyrs). Analysis of long-range inputs to VChIs with monosynaptic rabies virus tracing revealed that direct thalamic projections are a significant input source to these cells. Optogenetic activation of VChIs in the barrel cortex of awake mice suppresses the sensory responses of excitatory neurons in intermediate amplitudes of whisker deflections while increasing the evoked spike latency. The effect of VChI activation on the response was similar for both high-whisking (HW) and low-whisking (LW) conditions. Our findings demonstrate that, despite their sparsity, VChIs can effectively modulate sensory processing in the cortical microcircuit.

A lncRNA survey finds increases in neuroprotective LINC-PINT in Parkinson's disease substantia nigra.

Recent reports highlight regulatory functions of long noncoding RNAs (lncRNAs) in neurodegeneration and aging, but biomedical implications remain limited. Here, we report an rRNA-depletion-based long RNA-Sequencing Resource of 65 substantia nigra, amygdala, and medial temporal gyrus samples from Parkinson's disease (PD) and matched control brains. Using a lncRNA-focused analysis approach to identify functionally important transcripts, we discovered and prioritized many lncRNAs dysregulated in PD. Those included pronounced elevation of the P53-induced noncoding transcript LINC-PINT in the substantia nigra of PD patients, as well as in additional models of oxidative stress and PD. Intriguingly, we found that LINC-PINT is a primarily neuronal transcript which showed conspicuous increases in maturing primary culture neurons. LINC-PINT also accumulated in several brain regions of Alzheimer's and Huntington's disease patients and decreased with healthy brain aging, suggesting a general role in aging and neurodegeneration for this lncRNA. RNAi-mediated depletion of LINC-PINT exacerbated the death of cultured N2A and SH-SY5Y cells exposed to oxidative stress, highlighting a previously undiscovered neuroprotective role for this tumor-inducible lncRNA in the brains of patients with neurodegenerative disorders.

NEAT1 is overexpressed in Parkinson's disease substantia nigra and confers drug-inducible neuroprotection from oxidative stress.

Recent reports attribute numerous regulatory functions to the nuclear paraspeckle-forming long noncoding RNA, nuclear enriched assembly transcript 1 (NEAT1), but the implications of its involvement in Parkinson's disease (PD) remain controversial. To address this issue, we assessed NEAT1 expression levels and cell type patterns in the substantia nigra (SN) from 53 donors with and without PD, as well as in interference tissue culture tests followed by multiple in-house and web-available models of PD. PCR quantification identified elevated levels of NEAT1 expression in the PD SN compared with control brains, an elevation that was reproducible across a multitude of disease models. In situ RNA hybridization supported neuron-specific formation of NEAT1-based paraspeckles at the SN and demonstrated coincreases of NEAT1 and paraspeckles in cultured cells under paraquat (PQ)-induced oxidative stress. Furthermore, neuroprotective agents, including fenofibrate and simvastatin, induced NEAT1 up-regulation, whereas RNA interference-mediated depletion of NEAT1 exacerbated death of PQ-exposed cells in a leucine-rich repeat kinase 2-mediated manner. Our findings highlight a novel protective role for NEAT1 in PD and suggest a previously unknown mechanism for the neuroprotective traits of widely used preventive therapeutics.-Simchovitz, A., Hanan, M., Niederhoffer, N., Madrer, N., Yayon, N., Bennett, E. R., Greenberg, D. S., Kadener, S., Soreq, H. NEAT1 is overexpressed in Parkinson's disease substantia nigra and confers drug-inducible neuroprotection from oxidative stress.

Intensify3D: Normalizing signal intensity in large heterogenic image stacks.

Three-dimensional structures in biological systems are routinely evaluated using large image stacks acquired from fluorescence microscopy; however, analysis of such data is muddled by variability in the signal across and between samples. Here, we present Intensify3D: a user-guided normalization algorithm tailored for overcoming common heterogeneities in large image stacks. We demonstrate the use of Intensify3D for analyzing cholinergic interneurons of adult murine brains in 2-Photon and Light-Sheet fluorescence microscopy, as well as of mammary gland and heart tissues. Beyond enhancement in 3D visualization in all samples tested, in 2-Photon in vivo images, this tool corrected errors in feature extraction of cortical interneurons; and in Light-Sheet microscopy, it enabled identification of individual cortical barrel fields and quantification of somata in cleared adult brains. Furthermore, Intensify3D enhanced the ability to separate signal from noise. Overall, the universal applicability of our method can facilitate detection and quantification of 3D structures and may add value to a wide range of imaging experiments.

Pigment-Dispersing Factor-expressing neurons convey circadian information in the honey bee brain.

Pigment-Dispersing Factor (PDF) is an important neuropeptide in the brain circadian network of Drosophila and other insects, but its role in bees in which the circadian clock influences complex behaviour is not well understood. We combined high-resolution neuroanatomical characterizations, quantification of PDF levels over the day and brain injections of synthetic PDF peptide to study the role of PDF in the honey bee Apis mellifera We show that PDF co-localizes with the clock protein Period (PER) in a cluster of laterally located neurons and that the widespread arborizations of these PER/PDF neurons are in close vicinity to other PER-positive cells (neurons and glia). PDF-immunostaining intensity oscillates in a diurnal and circadian manner with possible influences for age or worker task on synchrony of oscillations in different brain areas. Finally, PDF injection into the area between optic lobes and the central brain at the end of the subjective day produced a consistent trend of phase-delayed circadian rhythms in locomotor activity. Altogether, these results are consistent with the hypothesis that PDF is a neuromodulator that conveys circadian information from pacemaker cells to brain centres involved in diverse functions including locomotion, time memory and sun-compass orientation.

miRNA-132 induces hepatic steatosis and hyperlipidaemia by synergistic multitarget suppression.

OBJECTIVE: Both non-alcoholic fatty liver disease (NAFLD) and the multitarget complexity of microRNA (miR) suppression have recently raised much interest, but the in vivo impact and context-dependence of hepatic miR-target interactions are incompletely understood. Assessing the relative in vivo contributions of specific targets to miR-mediated phenotypes is pivotal for investigating metabolic processes. DESIGN: We quantified fatty liver parameters and the levels of miR-132 and its targets in novel transgenic mice overexpressing miR-132, in liver tissues from patients with NAFLD, and in diverse mouse models of hepatic steatosis. We tested the causal nature of miR-132 excess in these phenotypes by injecting diet-induced obese mice with antisense oligonucleotide suppressors of miR-132 or its target genes, and measured changes in metabolic parameters and transcripts. RESULTS: Transgenic mice overexpressing miR-132 showed a severe fatty liver phenotype and increased body weight, serum low-density lipoprotein/very low-density lipoprotein (LDL/VLDL) and liver triglycerides, accompanied by decreases in validated miR-132 targets and increases in lipogenesis and lipid accumulation-related transcripts. Likewise, liver samples from both patients with NAFLD and mouse models of hepatic steatosis or non-alcoholic steatohepatitis (NASH) displayed dramatic increases in miR-132 and varying decreases in miR-132 targets compared with controls. Furthermore, injecting diet-induced obese mice with anti-miR-132 oligonucleotides, but not suppressing its individual targets, reversed the hepatic miR-132 excess and hyperlipidemic phenotype. CONCLUSIONS: Our findings identify miR-132 as a key regulator of hepatic lipid homeostasis, functioning in a context-dependent fashion via suppression of multiple targets and with cumulative synergistic effects. This indicates reduction of miR-132 levels as a possible treatment of hepatic steatosis.

Fear and C-reactive protein cosynergize annual pulse increases in healthy adults.

Recent international terror outbreaks notably involve long-term mental health risks to the exposed population, but whether physical health risks are also anticipated has remained unknown. Here, we report fear of terror-induced annual increases in resting heart rate (pulse), a notable risk factor of all-cause mortality. Partial least squares analysis based on 325 measured parameters successfully predicted annual pulse increases, inverse to the expected age-related pulse decline, in approximately 4.1% of a cohort of 17,380 apparently healthy active Israeli adults. Nonbiased hierarchical regression analysis among 27 of those parameters identified pertinent fear of terror combined with the inflammatory biomarker C-reactive protein as prominent coregulators of the observed annual pulse increases. In comparison, basal pulse primarily depended on general physiological parameters and reduced cholinergic control over anxiety and inflammation, together indicating that consistent exposure to terror threats ignites fear-induced exacerbation of preexisting neuro-immune risks of all-cause mortality.

Competing targets of microRNA-608 affect anxiety and hypertension.

MicroRNAs (miRNAs) can repress multiple targets, but how a single de-balanced interaction affects others remained unclear. We found that changing a single miRNA-target interaction can simultaneously affect multiple other miRNA-target interactions and modify physiological phenotype. We show that miR-608 targets acetylcholinesterase (AChE) and demonstrate weakened miR-608 interaction with the rs17228616 AChE allele having a single-nucleotide polymorphism (SNP) in the 3'-untranslated region (3'UTR). In cultured cells, this weakened interaction potentiated miR-608-mediated suppression of other targets, including CDC42 and interleukin-6 (IL6). Postmortem human cortices homozygote for the minor rs17228616 allele showed AChE elevation and CDC42/IL6 decreases compared with major allele homozygotes. Additionally, minor allele heterozygote and homozygote subjects showed reduced cortisol and elevated blood pressure, predicting risk of anxiety and hypertension. Parallel suppression of the conserved brain CDC42 activity by intracerebroventricular ML141 injection caused acute anxiety in mice. We demonstrate that SNPs in miRNA-binding regions could cause expanded downstream effects changing important biological pathways.