Antagonistic interactions safeguard mitotic propagation of genetic and epigenetic information in zebrafish.

The stability of cellular phenotypes in developing organisms depends on error-free transmission of epigenetic and genetic information during mitosis. Methylation of cytosine residues in genomic DNA is a key epigenetic mark that modulates gene expression and prevents genome instability. Here, we report on a genetic test of the relationship between DNA replication and methylation in the context of the developing vertebrate organism instead of cell lines. Our analysis is based on the identification of hypomorphic alleles of dnmt1, encoding the DNA maintenance methylase Dnmt1, and pole1, encoding the catalytic subunit of leading-strand DNA polymerase epsilon holoenzyme (Pole). Homozygous dnmt1 mutants exhibit genome-wide DNA hypomethylation, whereas the pole1 mutation is associated with increased DNA methylation levels. In dnmt1/pole1 double-mutant zebrafish larvae, DNA methylation levels are restored to near normal values, associated with partial rescue of mutant-associated transcriptional changes and phenotypes. Hence, a balancing antagonism between DNA replication and maintenance methylation buffers against replicative errors contributing to the robustness of vertebrate development.

The chilling origin of germinal centers.

Germinal center-like structures have been identified in ectothermic vertebrates, establishing germinal centers as a universal component of humoral immunity (see related Research Article by Shibasaki et al.).

Recombinant human diamine oxidase prevents hemodynamic effects of continuous histamine infusion in guinea pigs.

OBJECTIVE: To test whether recombinant human diamine oxidase (rhDAO) with a mutated heparin-binding motif (mHBM), which shows an increased alpha-distribution half-life, prevents histamine-induced hemodynamic effects. MATERIAL: Thirty-eight female guinea pigs were either pretreated with rhDOA_mHBM or buffer. TREATMENT AND METHODS: Guinea pigs received a continuous infusion of histamine. Heart rate (HR), body core temperature and mean arterial pressure (MAP) were measured and blood was collected. RESULTS: Continuous intravenous infusion of 8 µg/kg/min histamine increased mean peak plasma histamine levels from 5 (± 0.3 SEM) to 28 ng/mL (± 4.9 SEM) after 30 min but had no effect on oxygen saturation. Guinea pigs pretreated with 4 mg/kg rhDAO_mHBM showed lower mean HR (p = 0.008), histamine plasma concentrations (p = 0.002), and higher body core temperatures at the end of the histamine challenge (p = 0.02) compared to controls. Cessation of histamine infusion led to a rebound increase in MAP, but this hemodynamic instability was prevented by rhDAO_mHBM. Pretreatment with 4 mg/kg rhDAO_mHBM reduced urinary histamine (p = 0.004) and 1-Methylhistamine (p < 0.0001) concentrations compared to controls. CONCLUSIONS: Prophylactic infusion of rhDAO_mHBM prevents hemodynamic effects in a guinea pig model of continuous histamine infusion. These findings might help in the translation from animals to humans and in the selection of the optimal dosing of rhDAO_mHBM during human histamine challenge studies.

Foxn1 is not essential for T-cell development in teleosts.

In mammals, T-cell development depends on the activity of the Foxn1 transcription factor in the thymic epithelium; mutations in the vertebrate-specific Foxn1 gene are associated with profound T-cell lymphopenia and fatal immunodeficiency. Here, we examined the extent of T-cell development in teleosts lacking a functional foxn1 gene. In zebrafish carrying a deleterious internal deletion of foxn1, reduced but robust lymphopoietic activity is maintained in the mutant thymus. Moreover, pseudogenization or loss of foxn1 in the genomes of deep-sea anglerfishes is independent of the presence or absence of the canonical signatures of the T-cell lineage. Thus, in contrast to the situation in mammals, the teleost thymus can support foxn1-independent lymphopoiesis, most likely through the activity of the Foxn4, an ancient metazoan paralog of Foxn1. Our results imply that during the early stages of vertebrate evolution, genetic control of thymopoiesis was functionally redundant and thus robust; in mammals, the genetic network was reorganized to become uniquely dependent on the FOXN1 transcription factor.

Lymphocyte pathway analysis using naturally lymphocyte-deficient fish.

Comparative phylogenetic analyses are of potential value to establish the essential components of genetic networks underlying physiological traits. For species that naturally lack particular lymphocyte lineages, we show here that this strategy readily distinguishes trait-specific actors from pleiotropic components of the genetic network governing lymphocyte differentiation. Previously, three of the four members of the DNA polymerase X family have been implicated in the junctional diversification process during the somatic assembly of antigen receptors. Our phylogenetic analysis indicates that the presence of terminal deoxynucleotidyl transferase is strictly associated with the facility of V(D)J recombination, whereas PolL and PolM genes are retained even in species lacking Rag-mediated somatic diversification of antigen receptor genes.

Evolution of two distinct variable lymphocyte receptors in lampreys: VLRD and VLRE.

Jawless vertebrates possess an alternative adaptive immune system in which antigens are recognized by variable lymphocyte receptors (VLRs) generated by combinatorial assembly of leucine-rich repeat (LRR) cassettes. Three types of receptors, VLRA, VLRB, and VLRC, have been previously identified. VLRA- and VLRC-expressing cells are T cell-like, whereas VLRB-expressing cells are B cell-like. Here, we report two types of VLRs in lampreys, VLRD and VLRE, phylogenetically related to VLRA and VLRC. The germline VLRD and VLRE genes are flanked by 39 LRR cassettes used in the assembly of mature VLRD and VLRE, with cassettes from chromosomes containing the VLRA and VLRC genes also contributing to VLRD and VLRE assemblies. VLRD and VLRE transcription is highest in the triple-negative (VLRA-/VLRB-/VLRC-) population of lymphocytes, albeit also detectable in VLRA+ and VLRC+ populations. Tissue distribution studies suggest that lamprey VLRD+ and VLRE+ lymphocytes comprise T-like sublineages of cells.

A survey of the adaptive immune genes of the polka-dot batfish Ogcocephalus cubifrons.

BACKGROUND: The anglerfish, belonging to the teleost order Lophiiformes, are a diverse and species-rich group of fish that are known to exhibit a number of unique morphological, reproductive and immunological adaptations. Work to date has identified the loss of specific adaptive immune components in two of the five Lophiiformes sub-orders (Lophioidei and Ceratioidei), while no anomalies have been identified to date in two other sub-orders, Antennaroidei and Chaunacoidei. The immunogenome of the fifth sub-order, Ogcocephaloidei has not yet been investigated, and we have therefore used whole genome shotgun sequencing, combined with RNA-seq, to survey the adaptive immune capabilities of the polka-dot batfish, O. cubifrons, as a representative of this as yet unexplored sub-order. RESULTS: We find that the O. cubifrons genome encodes the core genes needed to mount adaptive T and B cell responses. These genes include those necessary for rearranging and editing antigen receptors, the antigen receptors themselves; as well as the co-receptors, signalling molecules, and antigen presenting molecules (both class I and class II) needed for B cell and T cell development and activation. CONCLUSIONS: From an immune perspective, the polka-dot batfish has a canonical complement of adaptive immune genes, and does not exhibit any of the adaptive immune changes previously identified in monkfish and oceanic anglerfish.

Incubation of protonated NADH or NADPH with ortho-aminobenzaldehyde generates a novel fluorescent nicotinamide dihydroquinazoline condensate.

Incubation of reduced nicotinamide adenine dinucleotide (NADH) but not oxidized NAD+ with ortho-aminobenzaldehyde (oABA) generated an uncharacterized chromophore with an absorption peak characteristic of a dihydroquinazoline condensate. This chromophore is responsible for a non-specific signal in a diamine oxidase (DAO) activity assay based on the generation of fluorescent dihydroquinazoline structures directly from DAO substrates. Herein we show that at pH values below 3.0 the glycosidic bond of NADH/NADPH is broken releasing double protonated dihydro-nicotinamide (dihydro-NAM), which consequently condensates with oABA to a novel dihydroquinazoline chromophore and fluorophore, namely the 6- or 8-carbamoyl-5H,7H,8H,9H-10λ5-pyrido[2,1-b]quinazolin-10-ylium isomer (CMPQ). The second protonation event closely correlates with the pKa of the N1 nitrogen of C5-protonated dihydro-NAM and fluorophore stability. The fusion partner of oABA is likely the iminium of the primary acid product of dihydro-NAM after glycosidic bond hydrolysis and before irreversible cyclization. Trapping of protonated dihydro-NAM from NADH or NADPH with oABA allows quantification of these dinucleotides. Despite almost a century of research studying acid-catalyzed molecular rearrangements of NADH and NADPH, new and surprising details can be discovered.

Generation of extracellular fluids from first-trimester decidual tissues and their validation by detecting tissue-specific secreted proteins.

The human placenta comes in direct contact with maternal cells and blood at two interfaces. The syncytiotrophoblast layer is surrounded by maternal blood at the intervillous space, and extravillous trophoblasts breach the vascular endothelial cells layer upon spiral artery remodeling and invasion of decidual veins. However, little knowledge exists about EVT-derived secreted factors, which may serve as predictive markers for obstetrical syndromes or shape the local environment at the maternal-fetal interface. Here, we define secreted EVT-associated genes and describe a method that yields interstitial fluids from patient-matched first-trimester decidua basalis and parietalis tissues.

Origin and evolutionary malleability of T cell receptor α diversity.

Lymphocytes of vertebrate adaptive immune systems acquired the capability to assemble, from split genes in the germline, billions of functional antigen receptors1-3. These receptors show specificity; unlike the broadly tuned receptors of the innate system, antibodies (Ig) expressed by B cells, for instance, can accurately distinguish between the two enantiomers of organic acids4, whereas T cell receptors (TCRs) reliably recognize single amino acid replacements in their peptide antigens5. In developing lymphocytes, antigen receptor genes are assembled from a comparatively small set of germline-encoded genetic elements in a process referred to as V(D)J recombination6,7. Potential self-reactivity of some antigen receptors arising from the quasi-random somatic diversification is suppressed by several robust control mechanisms8-12. For decades, scientists have puzzled over the evolutionary origin of somatically diversifying antigen receptors13-16. It has remained unclear how, at the inception of this mechanism, immunologically beneficial expanded receptor diversity was traded against the emerging risk of destructive self-recognition. Here we explore the hypothesis that in early vertebrates, sequence microhomologies marking the ends of recombining elements became the crucial targets of selection determining the outcome of non-homologous end joining-based repair of DNA double-strand breaks generated during RAG-mediated recombination. We find that, across the main clades of jawed vertebrates, TCRα repertoire diversity is best explained by species-specific extents of such sequence microhomologies. Thus, selection of germline sequence composition of rearranging elements emerges as a major factor determining the degree of diversity of somatically generated antigen receptors.

Tnpo3 controls splicing of the pre-mRNA encoding the canonical TCR α chain of iNKT cells.

Unconventional T cells, such as innate natural killer T cells (iNKT) cells, are an important part of vertebrate immune defences. iNKT recognise glycolipids through a T cell receptor (TCR) that is composed of a semi-invariant TCR α chain, paired with a restricted set of TCR ß chains. Here, we show that splicing of the cognate Trav11-Traj18-Trac pre-mRNA encoding the characteristic Vα14Jα18 variable region of this semi-invariant TCR depends on the presence of Tnpo3. The Tnpo3 gene encodes a nuclear transporter of the ß-karyopherin family whose cargo includes various splice regulators. The block of iNKT cell development in the absence of Tnpo3 can be overcome by transgenic provision of a rearranged Trav11-Traj18-Trac cDNA, indicating that Tnpo3 deficiency does not interfere with the development of iNKT cells per se. Our study thus identifies a role for Tnpo3 in regulating the splicing of the pre-mRNA encoding the cognate TCRα chain of iNKT cells.

MHC I tetramer staining tends to overestimate the number of functionally relevant self-reactive CD8 T cells in the preimmune repertoire.

Previous studies that used peptide-MHC (pMHC) tetramers (tet) to identify self-specific T cells have questioned the effectiveness of thymic-negative selection. Here, we used pMHCI tet to enumerate CD8 T cells specific for the immunodominant gp33 epitope of lymphocytic choriomeningitis virus glycoprotein (GP) in mice transgenically engineered to express high levels of GP as a self-antigen in the thymus. In GP-transgenic mice (GP+ ), monoclonal P14 TCR+ CD8 T cells that express a GP-specific TCR could not be detected by gp33/Db -tet staining, indicative of their complete intrathymic deletion. By contrast, in the same GP+ mice, substantial numbers of polyclonal CD8 T cells identifiable by gp33/Db -tet were present. The gp33-tet staining profiles of polyclonal T cells from GP+ and GP-negative (GP- ) mice were overlapping, but mean fluorescence intensities were ∼15% lower in cells from GP+ mice. Remarkably, the gp33-tet+ T cells in GP+ mice failed to clonally expand after lymphocytic choriomeningitis virus infection, whereas those of GP- mice did so. In Nur77GFP -reporter mice, dose-dependent responses to gp33 peptide-induced TCR stimulation revealed that gp33-tet+ T cells with high ligand sensitivity are lacking in GP+ mice. Hence, pMHCI tet staining identifies self-specific CD8 T cells but tends to overestimate the number of truly self-reactive cells.

Genetic conflicts and the origin of self/nonself-discrimination in the vertebrate immune system.

Genetic conflicts shape the genomes of prokaryotic and eukaryotic organisms. Here, we argue that some of the key evolutionary novelties of adaptive immune systems of vertebrates are descendants of prokaryotic toxin-antitoxin (TA) systems. Cytidine deaminases and RAG recombinase have evolved from genotoxic enzymes to programmable editors of host genomes, supporting the astounding discriminatory capability of variable lymphocyte receptors of jawless vertebrates, as well as immunoglobulins and T cell receptors of jawed vertebrates. The evolutionarily recent lymphoid lineage is uniquely sensitive to mutations of the DNA maintenance methylase, which is an orphaned distant relative of prokaryotic restriction-modification systems. We discuss how the emergence of adaptive immunity gave rise to higher order genetic conflicts between genetic parasites and their vertebrate host.

Simple, fast and inexpensive quantification of glycolate in the urine of patients with primary hyperoxaluria type 1.

In primary hyperoxaluria type 1 excessive endogenous production of oxalate and glycolate leads to increased urinary excretion of these metabolites. Although genetic testing is the most definitive and preferred diagnostic method, quantification of these metabolites is important for the diagnosis and evaluation of potential therapeutic interventions. Current metabolite quantification methods use laborious, technically highly complex and expensive liquid, gas or ion chromatography tandem mass spectrometry, which are available only in selected laboratories worldwide. Incubation of ortho-aminobenzaldehyde (oABA) with glyoxylate generated from glycolate using recombinant mouse glycolate oxidase (GO) and glycine leads to the formation of a stable dihydroquinazoline double aromatic ring chromophore with specific peak absorption at 440 nm. The urinary limit of detection and estimated limit of quantification derived from eight standard curves were 14.3 and 28.7 µmol glycolate per mmol creatinine, respectively. High concentrations of oxalate, lactate and L-glycerate do not interfere in this assay format. The correlation coefficient between the absorption and an ion chromatography tandem mass spectrometry method is 93% with a p value < 0.00001. The Bland-Altmann plot indicates acceptable agreement between the two methods. The glycolate quantification method using conversion of glycolate via recombinant mouse GO and fusion of oABA and glycine with glyoxylate is fast, simple, robust and inexpensive. Furthermore this method might be readily implemented into routine clinical diagnostic laboratories for glycolate measurements in primary hyperoxaluria type 1.

Stage-specific and cell type-specific requirements of ikzf1 during haematopoietic differentiation in zebrafish.

The zinc finger transcription factor Ikaros1 (Ikzf1) is required for lymphoid development in mammals. Four zinc fingers constitute its DNA binding domain and two zinc fingers are present in the C-terminal protein interaction module. We describe the phenotypes of zebrafish homozygous for two distinct mutant ikzf1 alleles. The IT325 variant lacks the C-terminal two zinc fingers, whereas the fr105 variant retains only the first zinc finger of the DNA binding domain. An intact ikzf1 gene is required for larval T cell development, whereas low levels of adult lymphoid development recover in the mutants. By contrast, the mutants exhibit a signature of increased myelopoiesis at larval and adult stages. Both mutations stimulate erythroid differentiation in larvae, indicating that the C-terminal zinc fingers negatively regulate the extent of red blood cell production. An unexpected differential effect of the two mutants on adult erythropoiesis suggests a direct requirement of an intact DNA binding domain for entry of progenitors into the red blood cell lineage. Collectively, our results reinforce the biological differences between larval and adult haematopoiesis, indicate a stage-specific function of ikzf1 in regulating the hierarchical bifurcations of differentiation, and assign distinct functions to the DNA binding domain and the C-terminal zinc fingers.

Human thymoma-associated mutation of the GTF2I transcription factor impairs thymic epithelial progenitor differentiation in mice.

Few human tumours present with a recurrent pathognomonic mutation in a transcription factor. Thymomas are an exception, with the majority of some subtypes exhibiting a distinct somatically acquired missense mutation in the general transcription factor GTF2I. Co-dominant expression of wild-type and mutated forms of Gtf2i in the mouse thymic epithelium is associated with aberrant thymic architecture and reduced thymopoietic activity. Phenotypic and molecular characterization of the mutant epithelium indicates that medullary differentiation is particularly affected as a result of impaired differentiation of bi-potent epithelial progenitors. The resulting gene expression signature is dominated by that of immature cortex-like thymic epithelial cells. TCR repertoire analysis of the cytopenic T cell compartment indicates efficient intrathymic selection; hence, despite marked homeostatic proliferation of T cell clones, autoimmunity is not observed. Thus, our transgenic mouse model recapitulates some aspects of the pathophysiology of a genetically defined type of human thymoma.

Clonal dynamics underlying the skewed CD4/CD8 ratio of mouse thymocytes revealed by TCR-independent barcoding.

T cell differentiation in the thymus generates CD4+ helper and cytotoxic CD8+ cells as the two principal T cell lineages. Curiously, at the end of this complex selection process, CD4+ cells invariably outnumber CD8+ cells. Here, we examine the dynamics of repertoire formation and the emergence of the skewed CD4/CD8 ratio using high-resolution endogenous CRISPR/Cas9 barcoding that indelibly marks immature T cells at the DN2/DN3 pre-TCR stage. In wild-type mice, greater clone size of CD4+ cells and an intrinsically greater probability of Tcr ß clonotypes for pMHCII interactions are major contributors to the skewed CD4/CD8 ratio. Clonal perturbations of thymocyte differentiation following the precocious expression of a rearranged iNKT invariant TCR α chain are due to loss of Tcr ß clonotypes from the CD4 lineage-committed pre-selection repertoire. The present barcoding scheme offers a novel means to examine the clonal dynamics of lymphocyte differentiation orthogonal to that using TCR clonotypes.