Control of cardiac contractions using Cre-lox and degron strategies in zebrafish.

Cardiac contractions and hemodynamic forces are essential for organ development and homeostasis. Control over cardiac contractions can be achieved pharmacologically or optogenetically. However, these approaches lack specificity or require direct access to the heart. Here, we compare two genetic approaches to control cardiac contractions by modulating the levels of the essential sarcomeric protein Tnnt2a in zebrafish. We first recombine a newly generated tnnt2a floxed allele using multiple lines expressing Cre under the control of cardiomyocyte-specific promoters, and show that it does not recapitulate the tnnt2a/silent heart mutant phenotype in embryos. We show that this lack of early cardiac contraction defects is due, at least in part, to the long half-life of tnnt2a mRNA, which masks the gene deletion effects until the early larval stages. We then generate an endogenous Tnnt2a-eGFP fusion line that we use together with the zGRAD system to efficiently degrade Tnnt2a in all cardiomyocytes. Using single-cell transcriptomics, we find that Tnnt2a depletion leads to cardiac phenotypes similar to those observed in tnnt2a mutants, with a loss of blood and pericardial flow-dependent cell types. Furthermore, we achieve conditional degradation of Tnnt2a-eGFP by splitting the zGRAD protein into two fragments that, when combined with the cpFRB2-FKBP system, can be reassembled upon rapamycin treatment. Thus, this Tnnt2a degradation line enables non-invasive control of cardiac contractions with high spatial and temporal specificity and will help further understand how they shape organ development and homeostasis.

Genetic compensation triggered by mutant mRNA degradation.

Genetic robustness, or the ability of an organism to maintain fitness in the presence of harmful mutations, can be achieved via protein feedback loops. Previous work has suggested that organisms may also respond to mutations by transcriptional adaptation, a process by which related gene(s) are upregulated independently of protein feedback loops. However, the prevalence of transcriptional adaptation and its underlying molecular mechanisms are unknown. Here, by analysing several models of transcriptional adaptation in zebrafish and mouse, we uncover a requirement for mutant mRNA degradation. Alleles that fail to transcribe the mutated gene do not exhibit transcriptional adaptation, and these alleles give rise to more severe phenotypes than alleles displaying mutant mRNA decay. Transcriptome analysis in alleles displaying mutant mRNA decay reveals the upregulation of a substantial proportion of the genes that exhibit sequence similarity with the mutated gene's mRNA, suggesting a sequence-dependent mechanism. These findings have implications for our understanding of disease-causing mutations, and will help in the design of mutant alleles with minimal transcriptional adaptation-derived compensation.

Corrigendum: Cloche is a bHLH-PAS transcription factor that drives haemato-vascular specification.

This corrects the article DOI: 10.1038/nature18614.

Genome-wide strategies reveal target genes of Npas4l associated with vascular development in zebrafish.

The development of a vascular network is essential to nourish tissues and sustain organ function throughout life. Endothelial cells (ECs) are the building blocks of blood vessels, yet our understanding of EC specification remains incomplete. Zebrafish cloche/npas4l mutants have been used broadly as an avascular model, but little is known about the molecular mechanisms of action of the Npas4l transcription factor. Here, to identify its direct and indirect target genes, we have combined complementary genome-wide approaches, including transcriptome analyses and chromatin immunoprecipitation. The cross-analysis of these datasets indicates that Npas4l functions as a master regulator by directly inducing a group of transcription factor genes that are crucial for hematoendothelial specification, such as etv2, tal1 and lmo2 We also identified new targets of Npas4l and investigated the function of a subset of them using the CRISPR/Cas9 technology. Phenotypic characterization of tspan18b mutants reveals a novel player in developmental angiogenesis, confirming the reliability of the datasets generated. Collectively, these data represent a useful resource for future studies aimed to better understand EC fate determination and vascular development.

Cloche is a bHLH-PAS transcription factor that drives haemato-vascular specification.

Vascular and haematopoietic cells organize into specialized tissues during early embryogenesis to supply essential nutrients to all organs and thus play critical roles in development and disease. At the top of the haemato-vascular specification cascade lies cloche, a gene that when mutated in zebrafish leads to the striking phenotype of loss of most endothelial and haematopoietic cells and a significant increase in cardiomyocyte numbers. Although this mutant has been analysed extensively to investigate mesoderm diversification and differentiation and continues to be broadly used as a unique avascular model, the isolation of the cloche gene has been challenging due to its telomeric location. Here we used a deletion allele of cloche to identify several new cloche candidate genes within this genomic region, and systematically genome-edited each candidate. Through this comprehensive interrogation, we succeeded in isolating the cloche gene and discovered that it encodes a PAS-domain-containing bHLH transcription factor, and that it is expressed in a highly specific spatiotemporal pattern starting during late gastrulation. Gain-of-function experiments show that it can potently induce endothelial gene expression. Epistasis experiments reveal that it functions upstream of etv2 and tal1, the earliest expressed endothelial and haematopoietic transcription factor genes identified to date. A mammalian cloche orthologue can also rescue blood vessel formation in zebrafish cloche mutants, indicating a highly conserved role in vertebrate vasculogenesis and haematopoiesis. The identification of this master regulator of endothelial and haematopoietic fate enhances our understanding of early mesoderm diversification and may lead to improved protocols for the generation of endothelial and haematopoietic cells in vivo and in vitro.

Tissue- and stage-dependent dosage compensation on the neo-X chromosome in Drosophila pseudoobscura.

Sex chromosome dosage compensation (DC) is widely accepted in various organisms. This concept is mostly supported by comparisons of gene expression between chromosomes and between sexes. However, genes on the X chromosome and autosomes are mostly not homologous, and the average gene expression level on these chromosomes may not be the same even under DC, which complicates comparisons between chromosomes. Many genes with sex-biased expression also make comparisons between sexes difficult. To overcome these issues, we investigated DC by comparing the expression of neo-X-linked genes in Drosophila pseudoobscura with those of their autosomal orthologs in other Drosophila species. The ratio of the former to the latter in males would be 1 under DC, whereas it becomes 0.5 without DC. We found that the ratio was ∼0.85 for adult whole bodies, indicating that the DC is incomplete on the neo-X chromosome in adults as a whole. The ratio (∼0.90) was also significantly less than 1 for adult bodies without gonads, whereas it was ∼1.0 for adult heads. These results indicate that DC varies among tissues. Our sliding-window analysis of the ratio also revealed that the upregulation of neo-X-linked genes in males occurred chromosome wide in all tissues analyzed, indicating global upregulation mechanisms. However, we found that gene functions also affected the levels of DC. Furthermore, most of the genes recently moved to the X were already under DC at the larval stage but not at the adult stage. These results suggest that DC in Drosophila species operates in a tissue/stage-dependent manner.

The developing epicardium regulates cardiac chamber morphogenesis by promoting cardiomyocyte growth.

The epicardium, the outermost layer of the heart, is an important regulator of cardiac regeneration. However, a detailed understanding of the crosstalk between the epicardium and myocardium during development requires further investigation. Here, we generated three models of epicardial impairment in zebrafish by mutating the transcription factor genes tcf21 and wt1a, and ablating tcf21+ epicardial cells. Notably, all three epicardial impairment models exhibited smaller ventricles. We identified the initial cause of this phenotype as defective cardiomyocyte growth, resulting in reduced cell surface and volume. This failure of cardiomyocyte growth was followed by decreased proliferation and increased abluminal extrusion. By temporally manipulating its ablation, we show that the epicardium is required to support cardiomyocyte growth mainly during early cardiac morphogenesis. By transcriptomic profiling of sorted epicardial cells, we identified reduced expression of FGF and VEGF ligand genes in tcf21-/- hearts, and pharmacological inhibition of these signaling pathways in wild type partially recapitulated the ventricular growth defects. Taken together, these data reveal distinct roles of the epicardium during cardiac morphogenesis and signaling pathways underlying epicardial-myocardial crosstalk.

Parental mutations influence wild-type offspring via transcriptional adaptation.

Transgenerational epigenetic inheritance (TEI) is mostly discussed in the context of physiological or environmental factors. Here, we show intergenerational and transgenerational inheritance of transcriptional adaptation (TA), a process whereby mutant messenger RNA (mRNA) degradation affects gene expression, in nematodes and zebrafish. Wild-type offspring of animals heterozygous for mRNA-destabilizing alleles display increased expression of adapting genes. Notably, offspring of animals heterozygous for nontranscribing alleles do not display this response. Germline-specific mutations are sufficient to induce TA in wild-type offspring, indicating that, at least for some genes, mutations in somatic tissues are not necessary for this process. Microinjecting total RNA from germ cells of TA-displaying heterozygous zebrafish can trigger TA in wild-type embryos and in their progeny, suggesting a model whereby mutant mRNAs in the germline trigger a TA response that can be epigenetically inherited. In sum, this previously unidentified mode of TEI reveals a means by which parental mutations can modulate the offspring's transcriptome.

Protein tyrosine phosphatase SHP2 is involved in Semaphorin 4D-induced axon repulsion.

Semaphorin-4D (Sema4D), a member of class 4 membrane-bound Semaphorins, acts as a chemorepellant to the axons of retinal ganglion cells and hippocampal neurons. Plexin-B1, a neuronal Sema4D receptor, associates with either one of receptor tyrosine kinases, c-Met or ErbB2, to mediate Sema4D-signaling. In contrast to this significance, the involvement of protein tyrosine phosphatases in Semaphorin-signaling remains unknown. We here show that Src homology 2-containing protein-tyrosine phosphatase 2 (SHP2) participates in Sema4D-signaling. SHP2 was localized in the growth cones of chick embryonic retinal ganglion neurons. Phenylarsine oxide, a protein tyrosine phosphatase inhibitor, suppressed Sema4D-induced contractile response in COS-7 cells expressing Plexin-B1. Ectopic expression of a phosphatase-inactive mutant of SHP2 in the retinal ganglion cells attenuated Sema4D-induced growth cone collapse response. A SHP1/2 specific inhibitor, 8-hydroxy-7-(6-sulfonaphthalen-2-yl)diazenyl-quinoline-5-sulfonic acid (NSC-87877), also suppressed this collapse response. These results suggest that SHP2-mediated tyrosine dephosphorylation is an important step in Sema4D-induced axon repulsion.

HHEX is a transcriptional regulator of the VEGFC/FLT4/PROX1 signaling axis during vascular development.

Formation of the lymphatic system requires the coordinated expression of several key regulators: vascular endothelial growth factor C (VEGFC), its receptor FLT4, and a key transcriptional effector, PROX1. Yet, how expression of these signaling components is regulated remains poorly understood. Here, using a combination of genetic and molecular approaches, we identify the transcription factor hematopoietically expressed homeobox (HHEX) as an upstream regulator of VEGFC, FLT4, and PROX1 during angiogenic sprouting and lymphatic formation in vertebrates. By analyzing zebrafish mutants, we found that hhex is necessary for sprouting angiogenesis from the posterior cardinal vein, a process required for lymphangiogenesis. Furthermore, studies of mammalian HHEX using tissue-specific genetic deletions in mouse and knockdowns in cultured human endothelial cells reveal its highly conserved function during vascular and lymphatic development. Our findings that HHEX is essential for the regulation of the VEGFC/FLT4/PROX1 axis provide insights into the molecular regulation of lymphangiogenesis.

Evolutionary Transitions of MicroRNA-Target Pairs.

How newly generated microRNA (miRNA) genes are integrated into gene regulatory networks during evolution is fundamental in understanding the molecular and evolutionary bases of robustness and plasticity in gene regulation. A recent model proposed that after the birth of a miRNA, the miRNA is generally integrated into the network by decreasing the number of target genes during evolution. However, this decreasing model remains to be carefully examined by considering in vivo conditions. In this study, we therefore compared the number of target genes among miRNAs with different ages, combining experiments with bioinformatics predictions. First, we focused on three Drosophila miRNAs with different ages. As a result, we found that an older miRNA has a greater number of target genes than a younger miRNA, suggesting the increasing number of targets for each miRNA during evolution (increasing model). To further confirm our results, we also predicted all target genes for all miRNAs in D. melanogaster, considering co-expression of miRNAs and mRNAs in vivo The results obtained also do not support the decreasing model but are reasonably consistent with the increasing model of miRNA-target pairs. Furthermore, our large-scale analyses of currently available experimental data of miRNA-target pairs also showed a weak but the same trend in humans. These results indicate that the current decreasing model of miRNA-target pairs should be reconsidered and the increasing model may be more appropriate to explain the evolutionary transitions of miRNA-target pairs in many organisms.

No association between Helicobacter pylori infection and diabetes mellitus among a general Japanese population: a cross-sectional study.

Several case-control studies have reported that patients with diabetes mellitus (DM) had a higher prevalence of Helicobacter pylori infection than those without DM, but these findings remain equivocal. Additionally, there are few studies examining associations between East Asian CagA-positive H. pylori and DM. This cross-sectional study aimed to investigate whether H. pylori infection was a possible risk factor for DM in a general Japanese population. The study included 5165 subjects (1467 men, 3698 women) aged 35-69 years from the Daiko Study, part of the Japan Multi-Institutional Collaborative Cohort Study. A urinary anti-H. pylori antibody was used to detect H. pylori infection. The medical history of physician-diagnosed DM was confirmed by self-administered questionnaire. The odds ratios (ORs) and their 95 % confidence intervals (CIs) for DM (current and former) were calculated using unconditional logistic regression analysis, adjusting for age, sex, educational status, alcohol use, smoking status, body mass index, energy intake, and physical activity. The prevalence of DM was 4.6 % (95 % CI 3.7-5.6 %) among 1878 participants with H. pylori infection and 3.2 % (2.6-3.8 %) among 3287 without the infection (p = 0.009). The crude, age-adjusted, and multivariate-adjusted ORs for DM in those with the infection relative to those without were 1.47 (95 % CI 1.10-1.97), 1.02 (0.76-1.38), and 0.97 (0.71-1.32), respectively. We found a significantly higher DM prevalence among those with H. pylori infection than among those without. However, almost all the difference in prevalence could be explained by the older age of those infected. Our findings did not support an association between H. pylori infection and DM.

A cross-sectional study to find out the relationship of methylenetetrahydrofolate reductase (MTHFR) C677T genotype with plasma levels of folate and total homocysteine by daily folate intake in Japanese.

In those with the methylenetetrahydrofolate reductase (MTHFR) 677TT genotype, enzyme activity is lowered. Therefore, these individuals might require an increased intake of folate to maintain or control blood levels of plasma folate or total homocysteine (tHcy). We examined associations of dietary folate intake with fasting plasma folate and total homocysteine (tHcy) according to genotype among 554 Japanese (207 men and 347 women aged 39-89 y) recruited in 2009. Intake of folate was estimated with a food frequency questionnaire. The MTHFR polymorphism was genotyped by a polymerase chain reaction with confronting two-pair primers. The log-transformed concentration of folate or tHcy was regressed on energy-adjusted folate intake in a linear regression analysis. Higher folate intake was associated with higher plasma folate among those with the CC (ß=0.165, p=0.066) or CT (ß=0.248, p<0.001) genotypes, and with lower tHcy levels only among those with the CC (ß=-0.141, p=0.013) genotype. Plasma folate was significantly and inversely associated with tHcy, irrespective of MTHFR genotype. When the analysis was restricted to those with tHcy levels higher than the reference range (≥13.5 nmol/mL, n=20), these significant associations were not found. The interaction between folate intake or plasma folate and genotype was not significant in any analysis. In conclusion, dietary folate intake was positively associated with plasma folate among those with the CC or CT genotypes and inversely associated with tHcy among those with the CC genotype, but the associations were not clear among those with higher levels of tHcy.

Common variants of cGKII/PRKG2 are not associated with gout susceptibility.

OBJECTIVE: Recently, genetic analyses indicated the association between gout and cGMP-dependent protein kinase 2 (cGKII/PRKG2) gene in a Fukien-Taiwanese heritage population. However, no replication study has been reported in other ancestries. Therefore, we investigated this association in a Japanese population. METHODS: Genotyping of 4 variants (rs11736177, rs10033237, rs7688672, and rs6837293) of cGKII was performed in 741 male gout patients and 1302 male controls. RESULTS: cGKII variants have no association with gout. CONCLUSION: Our replication study suggests that cGKII is not involved in gout susceptibility.