Multimodal spatiotemporal phenotyping of human retinal organoid development.

Organoids generated from human pluripotent stem cells provide experimental systems to study development and disease, but quantitative measurements across different spatial scales and molecular modalities are lacking. In this study, we generated multiplexed protein maps over a retinal organoid time course and primary adult human retinal tissue. We developed a toolkit to visualize progenitor and neuron location, the spatial arrangements of extracellular and subcellular components and global patterning in each organoid and primary tissue. In addition, we generated a single-cell transcriptome and chromatin accessibility timecourse dataset and inferred a gene regulatory network underlying organoid development. We integrated genomic data with spatially segmented nuclei into a multimodal atlas to explore organoid patterning and retinal ganglion cell (RGC) spatial neighborhoods, highlighting pathways involved in RGC cell death and showing that mosaic genetic perturbations in retinal organoids provide insight into cell fate regulation.

Pyramidal neurons form active, transient, multilayered circuits perturbed by autism-associated mutations at the inception of neocortex.

Cortical circuits are composed predominantly of pyramidal-to-pyramidal neuron connections, yet their assembly during embryonic development is not well understood. We show that mouse embryonic Rbp4-Cre cortical neurons, transcriptomically closest to layer 5 pyramidal neurons, display two phases of circuit assembly in vivo. At E14.5, they form a multi-layered circuit motif, composed of only embryonic near-projecting-type neurons. By E17.5, this transitions to a second motif involving all three embryonic types, analogous to the three adult layer 5 types. In vivo patch clamp recordings and two-photon calcium imaging of embryonic Rbp4-Cre neurons reveal active somas and neurites, tetrodotoxin-sensitive voltage-gated conductances, and functional glutamatergic synapses, from E14.5 onwards. Embryonic Rbp4-Cre neurons strongly express autism-associated genes and perturbing these genes interferes with the switch between the two motifs. Hence, pyramidal neurons form active, transient, multi-layered pyramidal-to-pyramidal circuits at the inception of neocortex, and studying these circuits could yield insights into the etiology of autism.

Fast and highly sensitive full-length single-cell RNA sequencing using FLASH-seq.

We present FLASH-seq (FS), a full-length single-cell RNA sequencing (scRNA-seq) method with increased sensitivity and reduced hands-on time compared to Smart-seq3. The entire FS protocol can be performed in ~4.5 hours, is simple to automate and can be easily miniaturized to decrease resource consumption. The FS protocol can also use unique molecular identifiers (UMIs) for molecule counting while displaying reduced strand-invasion artifacts. FS will be especially useful for characterizing gene expression at high resolution across multiple samples.

Identification of LINE retrotransposons and long non-coding RNAs expressed in the octopus brain.

BACKGROUND: Transposable elements (TEs) widely contribute to the evolution of genomes allowing genomic innovations, generating germinal and somatic heterogeneity, and giving birth to long non-coding RNAs (lncRNAs). These features have been associated to the evolution, functioning, and complexity of the nervous system at such a level that somatic retrotransposition of long interspersed element (LINE) L1 has been proposed to be associated to human cognition. Among invertebrates, octopuses are fascinating animals whose nervous system reaches a high level of complexity achieving sophisticated cognitive abilities. The sequencing of the genome of the Octopus bimaculoides revealed a striking expansion of TEs which were proposed to have contributed to the evolution of its complex nervous system. We recently found a similar expansion also in the genome of Octopus vulgaris. However, a specific search for the existence and the transcription of full-length transpositionally competent TEs has not been performed in this genus. RESULTS: Here, we report the identification of LINE elements competent for retrotransposition in Octopus vulgaris and Octopus bimaculoides and show evidence suggesting that they might be transcribed and determine germline and somatic polymorphisms especially in the brain. Transcription and translation measured for one of these elements resulted in specific signals in neurons belonging to areas associated with behavioral plasticity. We also report the transcription of thousands of lncRNAs and the pervasive inclusion of TE fragments in the transcriptomes of both Octopus species, further testifying the crucial activity of TEs in the evolution of the octopus genomes. CONCLUSIONS: The neural transcriptome of the octopus shows the transcription of thousands of putative lncRNAs and of a full-length LINE element belonging to the RTE class. We speculate that a convergent evolutionary process involving retrotransposons activity in the brain has been important for the evolution of sophisticated cognitive abilities in this genus.

Cell Types of the Human Retina and Its Organoids at Single-Cell Resolution.

Human organoids recapitulating the cell-type diversity and function of their target organ are valuable for basic and translational research. We developed light-sensitive human retinal organoids with multiple nuclear and synaptic layers and functional synapses. We sequenced the RNA of 285,441 single cells from these organoids at seven developmental time points and from the periphery, fovea, pigment epithelium and choroid of light-responsive adult human retinas, and performed histochemistry. Cell types in organoids matured in vitro to a stable "developed" state at a rate similar to human retina development in vivo. Transcriptomes of organoid cell types converged toward the transcriptomes of adult peripheral retinal cell types. Expression of disease-associated genes was cell-type-specific in adult retina, and cell-type specificity was retained in organoids. We implicate unexpected cell types in diseases such as macular degeneration. This resource identifies cellular targets for studying disease mechanisms in organoids and for targeted repair in human retinas.

Trebonia kvetii gen. nov., sp. nov., an acidophilic actinobacterium, and proposal of the new actinobacterial family Treboniaceae fam. nov.

A novel actinobacterial strain, designated 15TR583T, was isolated from a waterlogged acidic soil collected near the town of Trebon, Czech Republic, and was subjected to a polyphasic taxonomic characterization. Phylogenetic analysis based on 16S rRNA gene and whole-genome sequences revealed that the organism forms an individual line of descent related to the order Streptosporangiales, class Actinomycetia. The strain shared highest 16S rRNA gene sequence similarity, yet of only 92.8%, with Actinocorallia aurea IFO 14752T. The strain grew in white colonies of aerobic, Gram-stain-positive, unbranching substrate mycelium bearing single spores at hyphae tips. The major fatty acids (>10%) were iso-C16 : 0, C16 : 0, iso-C17 : 1ω9 and 10-methyl-C17 : 0. The fatty acid pattern differed from all patterns currently described for actinobacterial genera. The organism contained as major menaquinones MK9(H6) and MK9(H8), which differentiated it from other actinobacterial families. Polar lipids were composed of six unidentified glycolipids, an unidentified phosphoglycolipid, two unidentified phospholipids and two unidentified aminolipids. Whole-cell sugars contained galactose, xylose and arabinose as major components. The peptidoglycan type was A1γ meso-diaminopimelic acid. The genomic DNA G+C content was 69.7 mol%. The distinct phylogenetic position and unusual combination of chemotaxonomic characteristics justify the proposal of Trebonia gen. nov., with the type species Trebonia kvetii sp. nov. (type strain 15TR583T=CCM 8942T=DSM 109105T), within Treboniaceae fam. nov.

MorphoSeq: Full Single-Cell Transcriptome Dynamics Up to Gastrulation in a Chordate.

Single-cell RNA sequencing (scRNA-seq) provides a leap forward in resolving cellular diversity and developmental trajectories but fails to comprehensively delineate the spatial organization and precise cellular makeup of individual embryos. Here, we reconstruct from scRNA-seq and light sheet imaging data a canonical digital embryo that captures the genome-wide gene expression trajectory of every single cell at every cell division in the 18 lineages up to gastrulation in the ascidian Phallusia mammillata. By using high-coverage scRNA-seq, we devise a computational framework that stratifies single cells of individual embryos into cell types without prior knowledge. Unbiased transcriptome data analysis mapped each cell's physical position and lineage history, yielding the complete history of gene expression at the genome-wide level for every single cell in a developing embryo. A comparison of individual embryos reveals both extensive reproducibility between symmetric embryo sides and a large inter-embryonic variability due to small differences in embryogenesis timing.

Apoptotic Cell Exclusion and Bias-Free Single-Cell Selection Are Important Quality Control Requirements for Successful Single-Cell Sequencing Applications.

Single-cell sequencing experiments are a new mainstay in biology and have been advancing science especially in the biomedical field. The high pressure to integrate the technology into daily laboratory live requires solid knowledge with respect to potential limitations and precautions to be taken care of before applying it to complex research questions. In the past, we have identified two issues with quality measures neglected by the growing community involving SmartSeq and droplet or micro-well-based scRNASeq methods (1) how to ensure that only single cells are introduced without biasing on light scattering when handling complex cell mixtures and organ preparations or (2) how best to control for (pro-)apoptotic cell contaminations in single-cell sequencing approaches. Sighting of concurrent literature involving single-cell sequencing technologies revealed that these topics are generally neglected or simply approached in silico but not at the bench before generating single-cell data sets. We fear that those important quality aspects are overlooked due to reduced awareness of their importance for guaranteeing the quality of experiments. In this Cytometry rigor issue, we provide experimentally supported guidance on how to circumvent those critical shortcomings in order to promote a better use of the fantastic single-cell sequencing toolbox in biology. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Adult sox10+ Cardiomyocytes Contribute to Myocardial Regeneration in the Zebrafish.

During heart regeneration in the zebrafish, fibrotic tissue is replaced by newly formed cardiomyocytes derived from preexisting ones. It is unclear whether the heart is composed of several cardiomyocyte populations bearing different capacity to replace lost myocardium. Here, using sox10 genetic fate mapping, we identify a subset of preexistent cardiomyocytes in the adult zebrafish heart with a distinct gene expression profile that expanded after cryoinjury. Genetic ablation of sox10+ cardiomyocytes impairs cardiac regeneration, revealing that these cells play a role in heart regeneration.

Nuclear factor of activated T-cells, NFATC1, governs FLT3ITD-driven hematopoietic stem cell transformation and a poor prognosis in AML.

BACKGROUND: Acute myeloid leukemia (AML) patients with a high allelic burden of an internal tandem duplication (ITD)-mutated FMS-like Tyrosine Kinase-3 (FLT3) have a dismal outcome. FLT3ITD triggers the proliferation of the quiescent hematopoietic stem cell (HSC) pool but fails to directly transform HSCs. While the inflammatory transcription factor nuclear factor of activated T-cells 2 (NFAT2, NFATC1) is overexpressed in AML, it is unknown whether it plays a role in FLT3ITD-induced HSC transformation. METHODS: We generated a triple transgenic mouse model, in which tamoxifen-inducible Cre-recombinase targets expression of a constitutively nuclear transcription factor NFATC1 to FLT3ITD positive HSC. Emerging genotypes were phenotypically, biochemically, and also transcriptionally characterized using RNA sequencing. We also retrospectively analyzed the overall survival of AML patients with different NFATC1 expression status. RESULTS: We find that NFATC1 governs FLT3ITD-driven precursor cell expansion and transformation, causing a fully penetrant lethal AML. FLT3ITD/NFATC1-AML is re-transplantable in secondary recipients and shows primary resistance to the FLT3ITD-kinase inhibitor quizartinib. Mechanistically, NFATC1 rewires FLT3ITD-dependent signaling output in HSC, involving augmented K-RAS signaling and a selective de novo recruitment of key HSC-transforming signaling pathways such as the Hedgehog- and WNT/B-Catenin signaling pathways. In human AML, NFATC1 overexpression is associated with poor overall survival. CONCLUSIONS: NFATC1 expression causes FLT3ITD-induced transcriptome changes, which are associated with HSC transformation, quizartinib resistance, and a poor prognosis in AML.

Development, Function, and Clinical Significance of Plasmacytoid Dendritic Cells in Chronic Myeloid Leukemia.

Plasmacytoid dendritic cells (pDC) are the main producers of a key T-cell-stimulatory cytokine, IFNα, and critical regulators of antiviral immunity. Chronic myeloid leukemia (CML) is caused by BCR-ABL, which is an oncogenic tyrosine kinase that can be effectively inhibited with ABL-selective tyrosine kinase inhibitors (TKI). BCR-ABL-induced suppression of the transcription factor interferon regulatory factor 8 was previously proposed to block pDC development and compromise immune surveillance in CML. Here, we demonstrate that pDCs in newly diagnosed CML (CML-pDC) develop quantitatively normal and are frequently positive for the costimulatory antigen CD86. They originate from low-level BCR-ABL-expressing precursors. CML-pDCs also retain their competence to maturate and to secrete IFN. RNA sequencing reveals a strong inflammatory gene expression signature in CML-pDCs. Patients with high CML-pDC counts at diagnosis achieve inferior rates of deep molecular remission (MR) under nilotinib, unless nilotinib therapy is combined with IFN, which strongly suppresses circulating pDC counts. Although most pDCs are BCR-ABL-negative in MR, a substantial proportion of BCR-ABL + CML-pDCs persists under TKI treatment. This could be of relevance, because CML-pDCs elicit CD8+ T cells, which protect wild-type mice from CML. Together, pDCs are identified as novel functional DC population in CML, regulating antileukemic immunity and treatment outcome in CML.Significance: CML-pDC originates from low-level BCR-ABL expressing stem cells into a functional immunogenic DC-population regulating antileukemic immunity and treatment outcome in CML. Cancer Res; 78(21); 6223-34. ©2018 AACR.

Vitamin A-Retinoic Acid Signaling Regulates Hematopoietic Stem Cell Dormancy.

Dormant hematopoietic stem cells (dHSCs) are atop the hematopoietic hierarchy. The molecular identity of dHSCs and the mechanisms regulating their maintenance or exit from dormancy remain uncertain. Here, we use single-cell RNA sequencing (RNA-seq) analysis to show that the transition from dormancy toward cell-cycle entry is a continuous developmental path associated with upregulation of biosynthetic processes rather than a stepwise progression. In addition, low Myc levels and high expression of a retinoic acid program are characteristic for dHSCs. To follow the behavior of dHSCs in situ, a Gprc5c-controlled reporter mouse was established. Treatment with all-trans retinoic acid antagonizes stress-induced activation of dHSCs by restricting protein translation and levels of reactive oxygen species (ROS) and Myc. Mice maintained on a vitamin A-free diet lose HSCs and show a disrupted re-entry into dormancy after exposure to inflammatory stress stimuli. Our results highlight the impact of dietary vitamin A on the regulation of cell-cycle-mediated stem cell plasticity. VIDEO ABSTRACT.

Endogenous Mouse Dicer Is an Exclusively Cytoplasmic Protein.

Dicer is a large multi-domain protein responsible for the ultimate step of microRNA and short-interfering RNA biogenesis. In human and mouse cell lines, Dicer has been shown to be important in the nuclear clearance of dsRNA as well as the establishment of chromatin modifications. Here we set out to unambiguously define the cellular localization of Dicer in mice to understand if this is a conserved feature of mammalian Dicer in vivo. To this end, we utilized an endogenously epitope tagged Dicer knock-in mouse allele. From primary mouse cell lines and adult tissues, we determined with certainty by biochemical fractionation and confocal immunofluorescence microscopy that endogenous Dicer is exclusively cytoplasmic. We ruled out the possibility that a fraction of Dicer shuttles to and from the nucleus as well as that FGF or DNA damage signaling induce Dicer nuclear translocation. We also explored Dicer localization during the dynamic and developmental context of embryogenesis, where Dicer is ubiquitously expressed and strictly cytoplasmic in all three germ layers as well as extraembryonic tissues. Our data exclude a direct role for Dicer in the nuclear RNA processing in the mouse.

Early transcriptional events linked to induction of diapause revealed by RNAseq in larvae of drosophilid fly, Chymomyza costata.

BACKGROUND: Diapause is a developmental alternative to direct ontogeny in many invertebrates. Its primary adaptive meaning is to secure survival over unfavourable seasons in a state of developmental arrest usually accompanied by metabolic suppression and enhanced tolerance to environmental stressors. During photoperiodically triggered diapause of insects, the ontogeny is centrally turned off under hormonal control, the molecular details of this transition being poorly understood. Using RNAseq technology, we characterized transcription profiles associated with photoperiodic diapause induction in the larvae of the drosophilid fly Chymomyza costata with the goal of identifying candidate genes and processes linked to upstream regulatory events that eventually lead to a complex phenotypic change. RESULTS: Short day photoperiod triggering diapause was associated to inhibition of 20-hydroxy ecdysone (20-HE) signalling during the photoperiod-sensitive stage of C. costata larval development. The mRNA levels of several key genes involved in 20-HE biosynthesis, perception, and signalling were significantly downregulated under short days. Hormonal change was translated into downregulation of a series of other transcripts with broad influence on gene expression, protein translation, alternative histone marking by methylation and alternative splicing. These changes probably resulted in blockade of direct development and deep restructuring of metabolic pathways indicated by differential expression of genes involved in cell cycle regulation, metabolism, detoxification, redox balance, protection against oxidative stress, cuticle formation and synthesis of larval storage proteins. This highly complex alteration of gene transcription was expressed already during first extended night, within the first four hours after the change of the photoperiodic signal from long days to short days. We validated our RNAseq differential gene expression results in an independent qRT-PCR experiment involving wild-type (photoperiodic) and NPD-mutant (non-photoperiodic) strains of C. costata. CONCLUSIONS: Our study revealed several strong candidate genes for follow-up functional studies. Candidate genes code for upstream regulators of a complex change of gene expression, which leads to phenotypic switch from direct ontogeny to larval diapause.

Sequencing of a patient with balanced chromosome abnormalities and neurodevelopmental disease identifies disruption of multiple high risk loci by structural variation.

Balanced chromosome abnormalities (BCAs) occur at a high frequency in healthy and diseased individuals, but cost-efficient strategies to identify BCAs and evaluate whether they contribute to a phenotype have not yet become widespread. Here we apply genome-wide mate-pair library sequencing to characterize structural variation in a patient with unclear neurodevelopmental disease (NDD) and complex de novo BCAs at the karyotype level. Nucleotide-level characterization of the clinically described BCA breakpoints revealed disruption of at least three NDD candidate genes (LINC00299, NUP205, PSMD14) that gave rise to abnormal mRNAs and could be assumed as disease-causing. However, unbiased genome-wide analysis of the sequencing data for cryptic structural variation was key to reveal an additional submicroscopic inversion that truncates the schizophrenia- and bipolar disorder-associated brain transcription factor ZNF804A as an equally likely NDD-driving gene. Deep sequencing of fluorescent-sorted wild-type and derivative chromosomes confirmed the clinically undetected BCA. Moreover, deep sequencing further validated a high accuracy of mate-pair library sequencing to detect structural variants larger than 10 kB, proposing that this approach is powerful for clinical-grade genome-wide structural variant detection. Our study supports previous evidence for a role of ZNF804A in NDD and highlights the need for a more comprehensive assessment of structural variation in karyotypically abnormal individuals and patients with neurocognitive disease to avoid diagnostic deception.

Heterozygous carriage of a dysfunctional Toll-like receptor 9 allele affects CpG oligonucleotide responses in B cells.

Toll-like receptors (TLR) are employed by the innate immune system to detect microbial pathogens based on conserved microbial pathogen molecules. For example, TLR9 is a receptor for CpG-containing microbial DNA, and its activation results in the production of cytokines and type I interferons from human B cells and plasmacytoid dendritic cells, respectively. Both are required for mounting an efficient antibacterial or antiviral immune response. These effects are mimicked by synthetic CpG oligodeoxynucleotides (ODN). Although several hyporesponsive TLR9 variants have been reported, their functional relevance in human primary cells has not been addressed. Here we report a novel TLR9 allele, R892W, which is hyporesponsive to CpG ODN and acts as a dominant-negative in a cellular model system. The R892W variant is characterized by increased MyD88 binding and defective co-localization with CpG ODN. Whereas primary plasmacytoid dendritic cells isolated from a heterozygous R892W carrier responded normally to CpG by interferon-α production, carrier B cells showed impaired IL-6 and IL-10 production. This suggests that heterozygous carriage of a hyporesponsive TLR9 allele is not associated with complete loss of TLR9 function but that TLR9 signals elicited in different cell types are regulated differently in human primary cells.

Distribution of Chlamydia trachomatis serotypes in clinical urogenital samples from north-eastern Croatia.

The purpose of this study was to determine prevalence of Chlamydia trachomatis (Ct) urogenital infection and its serotype distribution from clinical samples in north-eastern Croatia. During a 3-year period, 2,379 urogenital samples were analyzed by real-time polymerase chain reaction (A group), while 4,846 genital swabs were analyzed by direct fluorescent antibody test (B group). 132 Ct positive specimens were genotyped by omp1 gene sequencing. The prevalence rate of Ct was 3.2 % in A and 1 % in B group. The most prevalent chlamydial genotype was E (44 %), followed by F (33 %), K (11.5 %), G (8 %), J/UW (5.3 %), D-IC (4.4 %), D-B120 (1.8 %), and B/IU, J/IU, Ia/IU (0.9 % each) serotypes. Single-nucleotide polymorphisms (SNPs) of omp1 gene were detected in E, K, and G serotypes. Some of these SNPs (C/T at position 272 and G/A at position 813 in E strain; C/T at position 884 in D strain) might represent novel omp1 variants.

Down syndrome: parental origin, recombination, and maternal age.

The aims of the present study were to assess (1) the parental origin of trisomy 21 and the stage in which nondisjunction occurs and (2) the relationship between altered genetic recombination and maternal age as risk factors for trisomy 21. The study included 102 cases with Down syndrome from the Croatian population. Genotyping analyses were performed by polymerase chain reaction using 11 short tandem repeat markers along chromosome 21q. The vast majority of trisomy 21 was of maternal origin (93%), followed by paternal (5%) and mitotic origin (2%). The frequencies of maternal meiotic I (MI) and meiotic II errors were 86% and 14%, respectively. The highest proportion of cases with zero recombination was observed among those with maternal MI derived trisomy 21. A higher proportion of telomeric exchanges were presented in cases with maternal MI errors and cases with young mothers, although these findings were not statistically significant. The present study is the first report examining parental origin and altered genetic recombination as a risk factor for trisomy 21 in a Croatian population. The results support that trisomy 21 has a universal genetic etiology across different human populations.

Frequency Determination of α-1,3 Glucosyltransferase p.Y131H and p.F304S Polymorphisms in the Croatian Population Revealed Five Novel Single Nucleotide Polymorphisms in the hALG6 Gene.

The congenital disorder of glycosylation (CDG)-Ic (ALG6-CDG, CDG-Ic) is caused by mutations in the hALG6 gene that encodes the N-glycosylation pathway enzyme, α-1,3-glucosyltransferase (NP_037471.2). The aim of our study was to estimate the frequencies of ALG6-CDG related p.Y131H and p.F304S polymorphisms in the Croatian population. Genomic DNA was isolated from blood samples collected from 600 healthy individuals. Functional single-nucleotide polymorphisms rs35383149 and rs17856039 causing p.Y131H and p.F304S, respectively, were genotyped by the TaqMan method and direct sequencing. The frequency of p.F304S polymorphism in the studied cohort was shown to be similar to the frequencies found in other tested populations (27%), whereas the frequency of p.Y131H was found to be three times higher (6.7%). Five novel base substitutions in the hALG6 gene were also found: three in exon 5 (c.383T>C, c.390G>A, and c.429G>C) and two in a downstream intervening sequence (IVS5+17C/T and IVS5+34G/A).

Evaluation of the reliability of DNA typing in the process of identification of war victims in Croatia.

Aiming to estimate the frequency of various types of errors that can occur in the large-scale process of identification, we identified and compared genotypes of 911 parent-child pairs in the database of 3498 relatives of people that disappeared during the 1991/1992 war in Croatia. Genotypes of 891 pairs (97.8%) were matching, while 20 pairs did not match in one or more loci. Reanalysis of these samples revealed that out of 1822 analyzed genotypes, one genotype was completely wrong, and two genotypes had one wrong allele because of human errors. Five genotypes had a single wrong allele due to either polymerase chain reaction or electrophoresis errors. In five genotypes mutations were the cause of mismatch. Genetic inconsistencies with parentage were found in four "fathers" (4.2%) and three "mothers" (0.36%). As the majority of observed single-locus errors were caused by nonhuman errors, all databases produced with similar technology would probably have comparable level of errors.