Mendelian randomization and genetic colocalization infer the effects of the multi-tissue proteome on 211 complex disease-related phenotypes

BACKGROUND: Human proteins are widely used as drug targets. Integration of large-scale protein-level genome-wide association studies (GWAS) and disease-related GWAS has thus connected genetic variation to disease mechanisms via protein. Previous proteome-by-phenome-wide Mendelian randomization (MR) studies have been mainly focused on plasma proteomes. Previous MR studies using the brain proteome only reported protein effects on a set of pre-selected tissue-specific diseases. No studies, however, have used high-throughput proteomics from multiple tissues to perform MR on hundreds of phenotypes. METHODS: Here, we performed MR and colocalization analysis using multi-tissue (cerebrospinal fluid (CSF), plasma, and brain from pre- and post-meta-analysis of several disease-focus cohorts including Alzheimer disease (AD)) protein quantitative trait loci (pQTLs) as instrumental variables to infer protein effects on 211 phenotypes, covering seven broad categories: biological traits, blood traits, cancer types, neurological diseases, other diseases, personality traits, and other risk factors. We first implemented these analyses with cis pQTLs, as cis pQTLs are known for being less prone to horizontal pleiotropy. Next, we included both cis and trans conditionally independent pQTLs that passed the genome-wide significance threshold keeping only variants associated with fewer than five proteins to minimize pleiotropic effects. We compared the tissue-specific protein effects on phenotypes across different categories. Finally, we integrated the MR-prioritized proteins with the druggable genome to identify new potential targets. RESULTS: In the MR and colocalization analysis including study-wide significant cis pQTLs as instrumental variables, we identified 33 CSF, 13 plasma, and five brain proteins to be putative causal for 37, 18, and eight phenotypes, respectively. After expanding the instrumental variables by including genome-wide significant cis and trans pQTLs, we identified a total of 58 CSF, 32 plasma, and nine brain proteins associated with 58, 44, and 16 phenotypes, respectively. For those protein-phenotype associations that were found in more than one tissue, the directions of the associations for 13 (87%) pairs were consistent across tissues. As we were unable to use methods correcting for horizontal pleiotropy given most of the proteins were only associated with one valid instrumental variable after clumping, we found that the observations of protein-phenotype associations were consistent with a causal role or horizontal pleiotropy. Between 66.7 and 86.3% of the disease-causing proteins overlapped with the druggable genome. Finally, between one and three proteins, depending on the tissue, were connected with at least one drug compound for one phenotype from both DrugBank and ChEMBL databases. CONCLUSIONS: Integrating multi-tissue pQTLs with MR and the druggable genome may open doors to pinpoint novel interventions for complex traits with no effective treatments, such as ovarian and lung cancers

Molecular Characterization of the Gastrula in the Turtle Emys orbicularis: An Evolutionary Perspective on Gastrulation

Due to the presence of a blastopore as in amphibians, the turtle has been suggested to exemplify a transition form from an amphibian- to an avian-type gastrulation pattern. In order to test this hypothesis and gain insight into the emergence of the unique characteristics of amniotes during gastrulation, we have performed the first molecular characterization of the gastrula in a reptile, the turtle Emys orbicularis. The study of Brachyury, Lim1, Otx2 and Otx5 expression patterns points to a highly conserved dynamic of expression with amniote model organisms and makes it possible to identify the site of mesoderm internalization, which is a long-standing issue in reptiles. Analysis of Brachyury expression also highlights the presence of two distinct phases, less easily recognizable in model organisms and respectively characterized by an early ring-shaped and a later bilateral symmetrical territory. Systematic comparisons with tetrapod model organisms lead to new insights into the relationships of the blastopore/blastoporal plate system shared by all reptiles, with the blastopore of amphibians and the primitive streak of birds and mammals. The biphasic Brachyury expression pattern is also consistent with recent models of emergence of bilateral symmetry, which raises the question of its evolutionary significance

Ipsographing the Dubject; or, The Contradictions of Twitter

Precise control of self-renewal and differentiation of progenitor cells into the cranial neural crest (CNC) pool ensures proper head development, guided by signaling pathways such as BMPs, FGFs, Shh and Notch. Here, we show that murine Sox2 plays an essential role in controlling progenitor cell behavior during craniofacial development. A "Conditional by Inversion" Sox2 allele (Sox2(COIN) ) has been employed to generate an epiblast ablation of Sox2 function (Sox2(EpINV) ). Sox2 (EpINV/+(H)) haploinsufficient and conditional (Sox2(EpINV/mosaic) ) mutant embryos proceed beyond gastrulation and die around E11. These mutant embryos exhibit severe anterior malformations, with hydrocephaly and frontonasal truncations, which could be attributed to the deregulation of CNC progenitor cells during their epithelial to mesenchymal transition. This irregularity results in an exacerbated and aberrant migration of Sox10(+) NCC in the branchial arches and frontonasal process of the Sox2 mutant embryos. These results suggest a novel role for Sox2 as a regulator of the epithelial to mesenchymal transitions (EMT) that are important for the cell flow in the developing head

Dynamic association with donor cell filopodia and lipid-modification are essential features of Wnt8a during patterning of the zebrafish neuroectoderm

BACKGROUND: Wnt proteins are conserved signaling molecules that regulate pattern formation during animal development. Many Wnt proteins are post-translationally modified by addition of lipid adducts. Wnt8a provides a crucial signal for patterning the anteroposterior axis of the developing neural plate in vertebrates. However, it is not clear how this protein propagates from its source, the blastoderm margin, to the target cells in the prospective neural plate, and how lipid-modifications might influence Wnt8a propagation and activity. RESULTS: We have dynamically imaged biologically active, fluorescently tagged Wnt8a in living zebrafish embryos. We find that Wnt8a localizes to membrane-associated, punctate structures in live tissue. In Wnt8a expressing cells, these puncta are found on filopodial cellular processes, from where the protein can be released. In addition, Wnt8a is found colocalized with Frizzled receptor-containing clusters on signal receiving cells. Combining in vitro and in vivo assays, we compare the roles of conserved Wnt8a residues in cell and non-cell-autonomous signaling activity and secretion. Non-signaling Wnt8 variants show these residues can regulate Wnt8a distribution in producing cell membranes and filopodia as well as in the receiving tissue. CONCLUSIONS: Together, our results show that Wnt8a forms dynamic clusters found on filopodial donor cell and on signal receiving cell membranes. Moreover, they demonstrate a differential requirement of conserved residues in Wnt8a protein for distribution in producing cells and receiving tissue and signaling activity during neuroectoderm patterning

Dental cell type atlas reveals stem and differentiated cell types in mouse and human teeth

Understanding cell types and mechanisms of dental growth is essential for reconstruction and engineering of teeth. Therefore, we investigated cellular composition of growing and non-growing mouse and human teeth. As a result, we report an unappreciated cellular complexity of the continuously-growing mouse incisor, which suggests a coherent model of cell dynamics enabling unarrested growth. This model relies on spatially-restricted stem, progenitor and differentiated populations in the epithelial and mesenchymal compartments underlying the coordinated expansion of two major branches of pulpal cells and diverse epithelial subtypes. Further comparisons of human and mouse teeth yield both parallelisms and differences in tissue heterogeneity and highlight the specifics behind growing and non-growing modes. Despite being similar at a coarse level, mouse and human teeth reveal molecular differences and species-specific cell subtypes suggesting possible evolutionary divergence. Overall, here we provide an atlas of human and mouse teeth with a focus on growth and differentiation. Unlike human teeth, mouse incisors grow throughout life, based on stem and progenitor cell activity. Here the authors generate single cell RNA-seq comparative maps of continuously-growing mouse incisor, non-growing mouse molar and human teeth, combined with lineage tracing to reveal dental cell complexity.Peer reviewe

Live Imaging of Xwnt5A-ROR2 Complexes

Secreted molecules of the Wnt family regulate key decisions in embryogenesis and adult tissue homeostasis by activating a complex network of Wnt signaling pathways. Although the different branches of Wnt signaling have been studied for more than 25 years, fluorophore tagged constructs for live cell imaging of Wnt molecules activating the Wnt/β-catenin pathway have become available only recently. We have generated a fluorophore tagged Wnt construct of the Xenopus Wnt5a protein (Xwnt5A) with the enhanced green fluorescent protein (EGFP), Xwnt5A-EGFP. This construct activates non-canonical Wnt pathways in an endocytosis dependent manner and is capable of compensating for the loss of endogenous Xwnt5A in Xenopus embryos. Strikingly, non-canonical Wnt pathway activation was restricted to short-range signaling while an inhibitory effect was observed in transwell cell cultures taken as long-range signaling model sytem. We used our Xwnt5A-EGFP construct to analyze in vivo binding of Wnt5A to its co-receptor ROR2 on the microscopic and on the molecular level. On the microscopic level, Xwnt5A-EGFP clusters in the membrane and recruits ROR2-mCherry to these clusters. Applying dual-colour dual-focus line-scanning fluorescence correlation spectroscopy on dorsal marginal zone explants, we identified membrane tethered Xwnt5A-EGFP molecules binding to ROR2-mCherry molecules. Our data favour a model, in which membrane-tethered Wnt-5A recruits ROR2 to form large ligand/receptor clusters and signals in an endocytosis-dependent manner

Cell-Sorting at the A/P Boundary in the Drosophila Wing Primordium: A Computational Model to Consolidate Observed Non-Local Effects of Hh Signaling

Non-intermingling, adjacent populations of cells define compartment boundaries; such boundaries are often essential for the positioning and the maintenance of tissue-organizers during growth. In the developing wing primordium of Drosophila melanogaster, signaling by the secreted protein Hedgehog (Hh) is required for compartment boundary maintenance. However, the precise mechanism of Hh input remains poorly understood. Here, we combine experimental observations of perturbed Hh signaling with computer simulations of cellular behavior, and connect physical properties of cells to their Hh signaling status. We find that experimental disruption of Hh signaling has observable effects on cell sorting surprisingly far from the compartment boundary, which is in contrast to a previous model that confines Hh influence to the compartment boundary itself. We have recapitulated our experimental observations by simulations of Hh diffusion and transduction coupled to mechanical tension along cell-to-cell contact surfaces. Intriguingly, the best results were obtained under the assumption that Hh signaling cannot alter the overall tension force of the cell, but will merely re-distribute it locally inside the cell, relative to the signaling status of neighboring cells. Our results suggest a scenario in which homotypic interactions of a putative Hh target molecule at the cell surface are converted into a mechanical force. Such a scenario could explain why the mechanical output of Hh signaling appears to be confined to the compartment boundary, despite the longer range of the Hh molecule itself. Our study is the first to couple a cellular vertex model describing mechanical properties of cells in a growing tissue, to an explicit model of an entire signaling pathway, including a freely diffusible component. We discuss potential applications and challenges of such an approach

Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M) and serine biosynthetic pathway genes are co-ordinately increased during anabolic agent-induced skeletal muscle growth

We aimed to identify novel molecular mechanisms for muscle growth during administration of anabolic agents. Growing pigs (Duroc/(Landrace/Large-White)) were administered Ractopamine (a beta-adrenergic agonist; BA; 20ppm in feed) or Reporcin (recombinant growth hormone; GH; 10mg/48hours injected) and compared to a control cohort (feed only; no injections) over a 27-day time course (1, 3, 7, 13 or 27-days). Longissimus Dorsi muscle gene expression was analyzed using Agilent porcine transcriptome microarrays and clusters of genes displaying similar expression profiles were identified using a modified maSigPro clustering algorithm. Anabolic agents increased carcass (p=0.002) and muscle weights (Vastus Lateralis: p<0.001; Semitendinosus: p=0.075). Skeletal muscle mRNA expression of serine/one-carbon/glycine biosynthesis pathway genes (Phgdh, Psat1 and Psph) and the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase-M (Pck2/PEPCK-M), increased during treatment with BA, and to a lesser extent GH (p<0.001, treatment x time interaction). Treatment with BA, but not GH, caused a 2-fold increase in phosphoglycerate dehydrogenase (PHGDH) protein expression at days 3 (p<0.05) and 7 (p<0.01), and a 2-fold increase in PEPCK-M protein expression at day 7 (p<0.01). BA treated pigs exhibit a profound increase in expression of PHGDH and PEPCK-M in skeletal muscle, implicating a role for biosynthetic metabolic pathways in muscle growth

A Comparative Analysis of Extra-Embryonic Endoderm Cell Lines

Prior to gastrulation in the mouse, all endodermal cells arise from the primitive endoderm of the blastocyst stage embryo. Primitive endoderm and its derivatives are generally referred to as extra-embryonic endoderm (ExEn) because the majority of these cells contribute to extra-embryonic lineages encompassing the visceral endoderm (VE) and the parietal endoderm (PE). During gastrulation, the definitive endoderm (DE) forms by ingression of cells from the epiblast. The DE comprises most of the cells of the gut and its accessory organs. Despite their different origins and fates, there is a surprising amount of overlap in marker expression between the ExEn and DE, making it difficult to distinguish between these cell types by marker analysis. This is significant for two main reasons. First, because endodermal organs, such as the liver and pancreas, play important physiological roles in adult animals, much experimental effort has been directed in recent years toward the establishment of protocols for the efficient derivation of endodermal cell types in vitro. Conversely, factors secreted by the VE play pivotal roles that cannot be attributed to the DE in early axis formation, heart formation and the patterning of the anterior nervous system. Thus, efforts in both of these areas have been hampered by a lack of markers that clearly distinguish between ExEn and DE. To further understand the ExEn we have undertaken a comparative analysis of three ExEn-like cell lines (END2, PYS2 and XEN). PYS2 cells are derived from embryonal carcinomas (EC) of 129 strain mice and have been characterized as parietal endoderm-like [1], END2 cells are derived from P19 ECs and described as visceral endoderm-like, while XEN cells are derived from blastocyst stage embryos and are described as primitive endoderm-like. Our analysis suggests that none of these cell lines represent a bona fide single in vivo lineage. Both PYS2 and XEN cells represent mixed populations expressing markers for several ExEn lineages. Conversely END2 cells, which were previously characterized as VE-like, fail to express many markers that are widely expressed in the VE, but instead express markers for only a subset of the VE, the anterior visceral endoderm. In addition END2 cells also express markers for the PE. We extended these observations with microarray analysis which was used to probe and refine previously published data sets of genes proposed to distinguish between DE and VE. Finally, genome-wide pathway analysis revealed that SMAD-independent TGFbeta signaling through a TAK1/p38/JNK or TAK1/NLK pathway may represent one mode of intracellular signaling shared by all three of these lines, and suggests that factors downstream of these pathways may mediate some functions of the ExEn. These studies represent the first step in the development of XEN cells as a powerful molecular genetic tool to study the endodermal signals that mediate the important developmental functions of the extra-embryonic endoderm. Our data refine our current knowledge of markers that distinguish various subtypes of endoderm. In addition, pathway analysis suggests that the ExEn may mediate some of its functions through a non-classical MAP Kinase signaling pathway downstream of TAK1