Genome-wide association study meta-analysis of dizygotic twinning illuminates genetic regulation of female fecundity.

STUDY QUESTION: Which genetic factors regulate female propensity for giving birth to spontaneous dizygotic (DZ) twins? SUMMARY ANSWER: We identified four new loci, GNRH1, FSHR, ZFPM1, and IPO8, in addition to previously identified loci, FSHB and SMAD3. WHAT IS KNOWN ALREADY: The propensity to give birth to DZ twins runs in families. Earlier, we reported that FSHB and SMAD3 as associated with DZ twinning and female fertility measures. STUDY DESIGN, SIZE, DURATION: We conducted a genome-wide association meta-analysis (GWAMA) of mothers of spontaneous dizygotic (DZ) twins (8265 cases, 264 567 controls) and of independent DZ twin offspring (26 252 cases, 417 433 controls). PARTICIPANTS/MATERIALS, SETTING, METHODS: Over 700 000 mothers of DZ twins, twin individuals and singletons from large cohorts in Australia/New Zealand, Europe, and the USA were carefully screened to exclude twins born after use of ARTs. Genetic association analyses by cohort were followed by meta-analysis, phenome wide association studies (PheWAS), in silico and in vivo annotations, and Zebrafish functional validation. MAIN RESULTS AND THE ROLE OF CHANCE: This study enlarges the sample size considerably from previous efforts, finding four genome-wide significant loci, including two novel signals and a further two novel genes that are implicated by gene level enrichment analyses. The novel loci, GNRH1 and FSHR, have well-established roles in female reproduction whereas ZFPM1 and IPO8 have not previously been implicated in female fertility. We found significant genetic correlations with multiple aspects of female reproduction and body size as well as evidence for significant selection against DZ twinning during human evolution. The 26 top single nucleotide polymorphisms (SNPs) from our GWAMA in European-origin participants weakly predicted the crude twinning rates in 47 non-European populations (r = 0.23 between risk score and population prevalence, s.e. 0.11, 1-tail P = 0.058) indicating that genome-wide association studies (GWAS) are needed in African and Asian populations to explore the causes of their respectively high and low DZ twinning rates. In vivo functional tests in zebrafish for IPO8 validated its essential role in female, but not male, fertility. In most regions, risk SNPs linked to known expression quantitative trait loci (eQTLs). Top SNPs were associated with in vivo reproductive hormone levels with the top pathways including hormone ligand binding receptors and the ovulation cycle. LARGE SCALE DATA: The full DZT GWAS summary statistics will made available after publication through the GWAS catalog (https://www.ebi.ac.uk/gwas/). LIMITATIONS, REASONS FOR CAUTION: Our study only included European ancestry cohorts. Inclusion of data from Africa (with the highest twining rate) and Asia (with the lowest rate) would illuminate further the biology of twinning and female fertility. WIDER IMPLICATIONS OF THE FINDINGS: About one in 40 babies born in the world is a twin and there is much speculation on why twinning runs in families. We hope our results will inform investigations of ovarian response in new and existing ARTs and the causes of female infertility. STUDY FUNDING/COMPETING INTEREST(S): Support for the Netherlands Twin Register came from the Netherlands Organization for Scientific Research (NWO) and The Netherlands Organization for Health Research and Development (ZonMW) grants, 904-61-193, 480-04-004, 400-05-717, Addiction-31160008, 911-09-032, Biobanking and Biomolecular Resources Research Infrastructure (BBMRI.NL, 184.021.007), Royal Netherlands Academy of Science Professor Award (PAH/6635) to DIB, European Research Council (ERC-230374), Rutgers University Cell and DNA Repository (NIMH U24 MH068457-06), the Avera Institute, Sioux Falls, South Dakota (USA) and the National Institutes of Health (NIH R01 HD042157-01A1) and the Genetic Association Information Network (GAIN) of the Foundation for the National Institutes of Health and Grand Opportunity grants 1RC2 MH089951. The QIMR Berghofer Medical Research Institute (QIMR) study was supported by grants from the National Health and Medical Research Council (NHMRC) of Australia (241944, 339462, 389927, 389875, 389891, 389892, 389938, 443036, 442915, 442981, 496610, 496739, 552485, 552498, 1050208, 1075175). L.Y. is funded by Australian Research Council (Grant number DE200100425). The Minnesota Center for Twin and Family Research (MCTFR) was supported in part by USPHS Grants from the National Institute on Alcohol Abuse and Alcoholism (AA09367 and AA11886) and the National Institute on Drug Abuse (DA05147, DA13240, and DA024417). The Women's Genome Health Study (WGHS) was funded by the National Heart, Lung, and Blood Institute (HL043851 and HL080467) and the National Cancer Institute (CA047988 and UM1CA182913), with support for genotyping provided by Amgen. Data collection in the Finnish Twin Registry has been supported by the Wellcome Trust Sanger Institute, the Broad Institute, ENGAGE-European Network for Genetic and Genomic Epidemiology, FP7-HEALTH-F4-2007, grant agreement number 201413, National Institute of Alcohol Abuse and Alcoholism (grants AA-12502, AA-00145, AA-09203, AA15416, and K02AA018755) and the Academy of Finland (grants 100499, 205585, 118555, 141054, 264146, 308248, 312073 and 336823 to J. Kaprio). TwinsUK is funded by the Wellcome Trust, Medical Research Council, Versus Arthritis, European Union Horizon 2020, Chronic Disease Research Foundation (CDRF), Zoe Ltd and the National Institute for Health Research (NIHR) Clinical Research Network (CRN) and Biomedical Research Centre based at Guy's and St Thomas' NHS Foundation Trust in partnership with King's College London. For NESDA, funding was obtained from the Netherlands Organization for Scientific Research (Geestkracht program grant 10000-1002), the Center for Medical Systems Biology (CSMB, NVVO Genomics), Biobanking and Biomolecular Resources Research Infrastructure (BBMRI-NL), VU University's Institutes for Health and Care Research (EMGO+) and Neuroscience Campus Amsterdam, University Medical Center Groningen, Leiden University Medical Center, National Institutes of Health (NIH, ROI D0042157-01A, MH081802, Grand Opportunity grants 1 RC2 Ml-1089951 and IRC2 MH089995). Part of the genotyping and analyses were funded by the Genetic Association Information Network (GAIN) of the Foundation for the National Institutes of Health. Computing was supported by BiG Grid, the Dutch e-Science Grid, which is financially supported by NWO. Work in the Del Bene lab was supported by the Programme Investissements d'Avenir IHU FOReSIGHT (ANR-18-IAHU-01). C.R. was supported by an EU Horizon 2020 Marie Sklodowska-Curie Action fellowship (H2020-MSCA-IF-2014 #661527). H.S. and K.S. are employees of deCODE Genetics/Amgen. The other authors declare no competing financial interests. TRIAL REGISTRATION NUMBER: N/A.

In Vivo Fast Nonlinear Microscopy Reveals Impairment of Fast Axonal Transport Induced by Molecular Motor Imbalances in the Brain of Zebrafish Larvae.

Cargo transport by molecular motors along microtubules is essential for the function of eukaryotic cells, in particular neurons in which axonal transport defects constitute the early pathological features of neurodegenerative diseases. Mainly studied in motor and sensory neurons, axonal transport is still difficult to characterize in neurons of the brain in absence of appropriate in vivo tools. Here, we measured fast axonal transport by tracing the second harmonic generation (SHG) signal of potassium titanyl phosphate (KTP) nanocrystals (nanoKTP) endocytosed by brain neurons of zebrafish (Zf) larvae. Thanks to the optical translucency of Zf larvae and to the perfect photostability of nanoKTP SHG, we achieved a high scanning speed of 20 frames (of ≈90 µm × 60 µm size) per second in Zf brain. We focused our study on endolysosomal vesicle transport in axons of known polarization, separately analyzing kinesin and dynein motor-driven displacements. To validate our assay, we used either loss-of-function mutations of dynein or kinesin 1 or the dynein inhibitor dynapyrazole and quantified several transport parameters. We successfully demonstrated that dynapyrazole reduces the nanoKTP mobile fraction and retrograde run length consistently, while the retrograde run length increased in kinesin 1 mutants. Taking advantage of nanoKTP SHG directional emission, we also quantified fluctuations of vesicle orientation. Thus, by combining endocytosis of nanocrystals having a nonlinear response, fast two-photon microscopy, and high-throughput analysis, we are able to finely monitor fast axonal transport in vivo in the brain of a vertebrate and reveal subtle axonal transport alterations. The high spatiotemporal resolution achieved in our model may be relevant to precisely investigate axonal transport impairment associated with disease models.

Evolutionary divergence of locomotion in two related vertebrate species.

Locomotion exists in diverse forms in nature; however, little is known about how closely related species with similar neuronal circuitry can evolve different navigational strategies to explore their environments. Here, we investigate this question by comparing divergent swimming pattern in larval Danionella cerebrum (DC) and zebrafish (ZF). We show that DC displays long continuous swimming events when compared with the short burst-and-glide swimming in ZF. We reveal that mesencephalic locomotion maintenance neurons in the midbrain are sufficient to cause this increased swimming. Moreover, we propose that the availability of dissolved oxygen and timing of swim bladder inflation drive the observed differences in the swim pattern. Our findings uncover the neural substrate underlying the evolutionary divergence of locomotion and its adaptation to their environmental constraints.

Bi-allelic variants in IPO8 cause a connective tissue disorder associated with cardiovascular defects, skeletal abnormalities, and immune dysregulation.

Dysregulated transforming growth factor TGF-ß signaling underlies the pathogenesis of genetic disorders affecting the connective tissue such as Loeys-Dietz syndrome. Here, we report 12 individuals with bi-allelic loss-of-function variants in IPO8 who presented with a syndromic association characterized by cardio-vascular anomalies, joint hyperlaxity, and various degree of dysmorphic features and developmental delay as well as immune dysregulation; the individuals were from nine unrelated families. Importin 8 belongs to the karyopherin family of nuclear transport receptors and was previously shown to mediate TGF-ß-dependent SMADs trafficking to the nucleus in vitro. The important in vivo role of IPO8 in pSMAD nuclear translocation was demonstrated by CRISPR/Cas9-mediated inactivation in zebrafish. Consistent with IPO8's role in BMP/TGF-ß signaling, ipo8-/- zebrafish presented mild to severe dorso-ventral patterning defects during early embryonic development. Moreover, ipo8-/- zebrafish displayed severe cardiovascular and skeletal defects that mirrored the human phenotype. Our work thus provides evidence that IPO8 plays a critical and non-redundant role in TGF-ß signaling during development and reinforces the existing link between TGF-ß signaling and connective tissue defects.

Bilateral visual projections exist in non-teleost bony fish and predate the emergence of tetrapods.

In most vertebrates, camera-style eyes contain retinal ganglion cell neurons that project to visual centers on both sides of the brain. However, in fish, ganglion cells were thought to innervate only the contralateral side, suggesting that bilateral visual projections appeared in tetrapods. Here we show that bilateral visual projections exist in non-teleost fishes and that the appearance of ipsilateral projections does not correlate with terrestrial transition or predatory behavior. We also report that the developmental program that specifies visual system laterality differs between fishes and mammals, as the Zic2 transcription factor, which specifies ipsilateral retinal ganglion cells in tetrapods, appears to be absent from fish ganglion cells. However, overexpression of human ZIC2 induces ipsilateral visual projections in zebrafish. Therefore, the existence of bilateral visual projections likely preceded the emergence of binocular vision in tetrapods.

An interhemispheric neural circuit allowing binocular integration in the optic tectum.

Binocular stereopsis requires the convergence of visual information from corresponding points in visual space seen by two different lines of sight. This may be achieved by superposition of retinal input from each eye onto the same downstream neurons via ipsi- and contralaterally projecting optic nerve fibers. Zebrafish larvae can perceive binocular cues during prey hunting but have exclusively contralateral retinotectal projections. Here we report brain activity in the tectal neuropil ipsilateral to the visually stimulated eye, despite the absence of ipsilateral retinotectal projections. This activity colocalizes with arbors of commissural neurons, termed intertectal neurons (ITNs), that connect the tectal hemispheres. ITNs are GABAergic, establish tectal synapses bilaterally and respond to small moving stimuli. ITN-ablation impairs capture swim initiation when prey is positioned in the binocular strike zone. We propose an intertectal circuit that controls execution of the prey-capture motor program following binocular localization of prey, without requiring ipsilateral retinotectal projections.

Dynactin1 depletion leads to neuromuscular synapse instability and functional abnormalities.

BACKGROUND: Dynactin subunit 1 is the largest subunit of the dynactin complex, an activator of the molecular motor protein complex dynein. Reduced levels of DCTN1 mRNA and protein have been found in sporadic amyotrophic lateral sclerosis (ALS) patients, and mutations have been associated with disease, but the role of this protein in disease pathogenesis is still unknown. METHODS: We characterized a Dynactin1a depletion model in the zebrafish embryo and combined in vivo molecular analysis of primary motor neuron development with live in vivo axonal transport assays in single cells to investigate ALS-related defects. To probe neuromuscular junction (NMJ) function and organization we performed paired motor neuron-muscle electrophysiological recordings and GCaMP calcium imaging in live, intact larvae, and the synapse structure was investigated by electron microscopy. RESULTS: Here we show that Dynactin1a depletion is sufficient to induce defects in the development of spinal cord motor neurons and in the function of the NMJ. We observe synapse instability, impaired growth of primary motor neurons, and higher failure rates of action potentials at the NMJ. In addition, the embryos display locomotion defects consistent with NMJ dysfunction. Rescue of the observed phenotype by overexpression of wild-type human DCTN1-GFP indicates a cell-autonomous mechanism. Synaptic accumulation of DCTN1-GFP, as well as ultrastructural analysis of NMJ synapses exhibiting wider synaptic clefts, support a local role for Dynactin1a in synaptic function. Furthermore, live in vivo analysis of axonal transport and cytoskeleton dynamics in primary motor neurons show that the phenotype reported here is independent of modulation of these processes. CONCLUSIONS: Our study reveals a novel role for Dynactin1 in ALS pathogenesis, where it acts cell-autonomously to promote motor neuron synapse stability independently of dynein-mediated axonal transport.

Redox Signaling via Lipid Peroxidation Regulates Retinal Progenitor Cell Differentiation.

Reactive oxygen species (ROS) and downstream products of lipid oxidation are emerging as important secondary messengers in tissue homeostasis. However, their regulation and mechanism of action remain poorly studied in vivo during normal development. Here, we reveal that the fine regulation of hydrogen peroxide (H2O2) levels by its scavenger Catalase to mediate the switch from proliferation to differentiation in retinal progenitor cells (RPCs) is crucial. We identify 9-hydroxystearic acid (9-HSA), an endogenous downstream lipid peroxidation product, as a mediator of this effect in the zebrafish retina. We show that the 9-HSA proliferative effect is due to the activation of Notch and Wnt pathways through the inhibition of the histone deacetylase 1. We show that the local and temporal manipulation of H2O2 levels in RPCs is sufficient to trigger their premature differentiation. We finally propose a mechanism that links H2O2 homeostasis and neuronal differentiation via the modulation of lipid peroxidation.

An Attractive Reelin Gradient Establishes Synaptic Lamination in the Vertebrate Visual System.

A conserved organizational and functional principle of neural networks is the segregation of axon-dendritic synaptic connections into laminae. Here we report that targeting of synaptic laminae by retinal ganglion cell (RGC) arbors in the vertebrate visual system is regulated by a signaling system relying on target-derived Reelin and VLDLR/Dab1a on the projecting neurons. Furthermore, we find that Reelin is distributed as a gradient on the target tissue and stabilized by heparan sulfate proteoglycans (HSPGs) in the extracellular matrix (ECM). Through genetic manipulations, we show that this Reelin gradient is important for laminar targeting and that it is attractive for RGC axons. Finally, we suggest a comprehensive model of synaptic lamina formation in which attractive Reelin counter-balances repulsive Slit1, thereby guiding RGC axons toward single synaptic laminae. We establish a mechanism that may represent a general principle for neural network assembly in vertebrate species and across different brain areas.

Deletion of a kinesin I motor unmasks a mechanism of homeostatic branching control by neurotrophin-3.

Development and function of highly polarized cells such as neurons depend on microtubule-associated intracellular transport, but little is known about contributions of specific molecular motors to the establishment of synaptic connections. In this study, we investigated the function of the Kinesin I heavy chain Kif5aa during retinotectal circuit formation in zebrafish. Targeted disruption of Kif5aa does not affect retinal ganglion cell differentiation, and retinal axons reach their topographically correct targets in the tectum, albeit with a delay. In vivo dynamic imaging showed that anterograde transport of mitochondria is impaired, as is synaptic transmission. Strikingly, disruption of presynaptic activity elicits upregulation of Neurotrophin-3 (Ntf3) in postsynaptic tectal cells. This in turn promotes exuberant branching of retinal axons by signaling through the TrkC receptor (Ntrk3). Thus, our study has uncovered an activity-dependent, retrograde signaling pathway that homeostatically controls axonal branching.

CRISPR/Cas9-mediated conversion of eGFP- into Gal4-transgenic lines in zebrafish.

Here we present a protocol for the conversion of eGFP-transgenic zebrafish lines into lines expressing Gal4 from the same locus. This conversion allows the in-depth analysis of the former eGFP-expressing cell population; with the Gal4-upstream activating sequence (UAS) system, diverse UAS transgenes can be transactivated. Site-specific targeting of the gene encoding eGFP is achieved using the clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) system. A single-guide RNA (sgRNA) that targets eGFP is injected into embryos together with a donor vector containing an optimized version of Gal4 (KalTA4) to trigger integration of the donor into the targeted eGFP genomic location. To enable screening for successful integration events, injection is performed in a UAS:RFP transgenic background; fish showing mosaic eGFP-to-RFP conversion are raised to adulthood. The progeny of these adult fish are then screened for stable germline transmission, and converted progeny are used to generate stable lines. We have been able to generate two stably converted transgenic lines within 4 months.

Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair.

Sequence-specific nucleases like TALENs and the CRISPR/Cas9 system have greatly expanded the genome editing possibilities in model organisms such as zebrafish. Both systems have recently been used to create knock-out alleles with great efficiency, and TALENs have also been successfully employed in knock-in of DNA cassettes at defined loci via homologous recombination (HR). Here we report CRISPR/Cas9-mediated knock-in of DNA cassettes into the zebrafish genome at a very high rate by homology-independent double-strand break (DSB) repair pathways. After co-injection of a donor plasmid with a short guide RNA (sgRNA) and Cas9 nuclease mRNA, concurrent cleavage of donor plasmid DNA and the selected chromosomal integration site resulted in efficient targeted integration of donor DNA. We successfully employed this approach to convert eGFP into Gal4 transgenic lines, and the same plasmids and sgRNAs can be applied in any species where eGFP lines were generated as part of enhancer and gene trap screens. In addition, we show the possibility of easily targeting DNA integration at endogenous loci, thus greatly facilitating the creation of reporter and loss-of-function alleles. Due to its simplicity, flexibility, and very high efficiency, our method greatly expands the repertoire for genome editing in zebrafish and can be readily adapted to many other organisms.

Characterization of the calcium binding protein family in zebrafish.

Calcium Binding Proteins (CaBPs), part of the vast family of EF-Hand-domain containing proteins, modulate intracellular calcium levels. They thereby contribute to a broad spectrum of biological processes - amongst others cell migration, gene expression and neural activity. In this study we identified twelve members of this protein family in zebrafish including one gene (cabp4b) currently not present in the zebrafish genome assembly. To gain insight into their biological functions, we carried out a detailed analysis of the expression patterns of these genes during zebrafish late embryonic and early larval development. We detected specific transcription for most of them in different neuronal cell populations including the neuroretina, the inner ear and the notochord. Our data supports potential roles for CaBPs during neuronal development and function and provides a starting point for genetic studies to examine CaBPs' function in these tissues and organs.

Inhibition of PP1 phosphatase activity by HBx: a mechanism for the activation of hepatitis B virus transcription.

The regulatory protein HBx is essential for hepatitis B virus (HBV) replication in vivo and for transcription of the episomal HBV genome. We previously reported that in infected cells HBx activates genes targeted by the transcription factor CREB [cyclic adenosine monophosphate (cAMP) response element-binding protein]. cAMP induces phosphorylation and activation of CREB, and CREB inactivation is promoted by protein phosphatase 1 (PP1), which binds to CREB through histone deacetylase 1 (HDAC1). We showed that CREB was recruited to HBV DNA. Phosphorylation induced by cAMP had a longer half-life when CREB was bound to the episomal HBV genome compared to when it was bound to the promoter of a host target gene not regulated by HBx, suggesting that the virus has developed a mechanism to favor its own transcription. This mechanism required HBx, which interacted with and inhibited PP1 to extend the half-life of CREB phosphorylation. Silencing of PP1 rescued replication of an HBx-deficient HBV genome, suggesting that HBx enhances viral transcription in part by neutralizing PP1 activity. Our results illustrate a previously unknown mechanism of HBV transcriptional activation by HBx in which HBx interferes with the inactivation of CREB by the PP1 and HDAC1 complex.

Stem cell-like micro-RNA signature driven by Myc in aggressive liver cancer.

Myc activation has been implicated in the pathogenesis of hepatoblastoma (HB), a rare embryonal neoplasm derived from liver progenitor cells. Here, microRNA (miR) expression profiling of 65 HBs evidenced differential patterns related to developmental stage and Myc activity. Undifferentiated aggressive HBs overexpressed the miR-371-3 cluster with concomitant down-regulation of the miR-100/let-7a-2/miR-125b-1 cluster, evoking an ES cell expression profile. ChIP and Myc inhibition assays in hepatoma cells demonstrated that both miR clusters are regulated by Myc in an opposite manner. We show that the two miR clusters exert antagonistic effects on cell proliferation and tumorigenicity. Moreover, their combined deregulation cooperated in modulating the hepatic tumor phenotype, implicating stem cell-like regulation of Myc-dependent miRs in poorly differentiated HBs. Importantly, a four-miR signature representative of these clusters efficiently stratified HB patients, and when applied to 241 hepatocellular carcinomas (HCCs), it identified invasive tumors with a poor prognosis. Our data argue that Myc-driven reprogramming of miR expression patterns contributes to the aggressive phenotype of liver tumors originating from hepatic progenitor cells.