Membrane potential drives the exit from pluripotency and cell fate commitment via calcium and mTOR.

Transitioning from pluripotency to differentiated cell fates is fundamental to both embryonic development and adult tissue homeostasis. Improving our understanding of this transition would facilitate our ability to manipulate pluripotent cells into tissues for therapeutic use. Here, we show that membrane voltage (Vm) regulates the exit from pluripotency and the onset of germ layer differentiation in the embryo, a process that affects both gastrulation and left-right patterning. By examining candidate genes of congenital heart disease and heterotaxy, we identify KCNH6, a member of the ether-a-go-go class of potassium channels that hyperpolarizes the Vm and thus limits the activation of voltage gated calcium channels, lowering intracellular calcium. In pluripotent embryonic cells, depletion of kcnh6 leads to membrane depolarization, elevation of intracellular calcium levels, and the maintenance of a pluripotent state at the expense of differentiation into ectodermal and myogenic lineages. Using high-resolution temporal transcriptome analysis, we identify the gene regulatory networks downstream of membrane depolarization and calcium signaling and discover that inhibition of the mTOR pathway transitions the pluripotent cell to a differentiated fate. By manipulating Vm using a suite of tools, we establish a bioelectric pathway that regulates pluripotency in vertebrates, including human embryonic stem cells.

PPIL4 is essential for brain angiogenesis and implicated in intracranial aneurysms in humans.

Intracranial aneurysm (IA) rupture leads to subarachnoid hemorrhage, a sudden-onset disease that often causes death or severe disability. Although genome-wide association studies have identified common genetic variants that increase IA risk moderately, the contribution of variants with large effect remains poorly defined. Using whole-exome sequencing, we identified significant enrichment of rare, deleterious mutations in PPIL4, encoding peptidyl-prolyl cis-trans isomerase-like 4, in both familial and index IA cases. Ppil4 depletion in vertebrate models causes intracerebral hemorrhage, defects in cerebrovascular morphology and impaired Wnt signaling. Wild-type, but not IA-mutant, PPIL4 potentiates Wnt signaling by binding JMJD6, a known angiogenesis regulator and Wnt activator. These findings identify a novel PPIL4-dependent Wnt signaling mechanism involved in brain-specific angiogenesis and maintenance of cerebrovascular integrity and implicate PPIL4 gene mutations in the pathogenesis of IA.

A convergent molecular network underlying autism and congenital heart disease.

Patients with neurodevelopmental disorders, including autism, have an elevated incidence of congenital heart disease, but the extent to which these conditions share molecular mechanisms remains unknown. Here, we use network genetics to identify a convergent molecular network underlying autism and congenital heart disease. This network is impacted by damaging genetic variants from both disorders in multiple independent cohorts of patients, pinpointing 101 genes with shared genetic risk. Network analysis also implicates risk genes for each disorder separately, including 27 previously unidentified genes for autism and 46 for congenital heart disease. For 7 genes with shared risk, we create engineered disruptions in Xenopus tropicalis, confirming both heart and brain developmental abnormalities. The network includes a family of ion channels, such as the sodium transporter SCN2A, linking these functions to early heart and brain development. This study provides a road map for identifying risk genes and pathways involved in co-morbid conditions.

Functional testing for variant prioritization in a family with long QT syndrome.

Next-generation sequencing platforms are being increasingly applied in clinical genetic settings for evaluation of families with suspected heritable disease. These platforms potentially improve the diagnostic yield beyond that of disease-specific targeted gene panels, but also increase the number of rare or novel genetic variants that may confound precise diagnostics. Here, we describe a functional testing approach used to interpret the results of whole exome sequencing (WES) in a family presenting with syncope and sudden death. One individual had a prolonged QT interval on electrocardiogram (ECG) and carried a diagnosis of long QT syndrome (LQTS), but a second individual did not meet criteria for LQTS. Filtering WES results for uncommon variants with arrhythmia association identified four for further analyses. In silico analyses indicated that two of these variants, KCNH2 p.(Cys555Arg) and KCNQ1 p.(Arg293Cys), were likely to be causal in this family's LQTS. We subsequently performed functional characterization of these variants in a heterologous expression system. The expression of KCNQ1-Arg293Cys did not show a deleterious phenotype but KCNH2-Cys555Arg demonstrated a loss-of-function phenotype that was partially dominant. Our stepwise approach identified a precise genetic etiology in this family, which resulted in the establishment of a LQTS diagnosis in the second individual as well as an additional asymptomatic family member, enabling personalized clinical management. Given its ability to aid in the diagnosis, the application of functional characterization should be considered as a value adjunct to in silico analyses of WES.

Genes and mechanisms of heterotaxy: patients drive the search.

Heterotaxy, a disorder in which visceral organs, including the heart, are mispatterned along the left-right body axis, contributes to particularly severe forms of congenital heart disease that are difficult to mitigate even despite surgical advances. A higher incidence of heterotaxy among individuals with blood kinship and the existence of rare monogenic disease forms suggest the existence of a genetic component, but the genetic and phenotypic heterogeneity of the disease have rendered gene discovery challenging. Next generation genomics in patients with syndromic, but also non-syndromic and sporadic heterotaxy, have recently helped to uncover new candidate disease genes, expanding the pool of genes already identified via traditional animal studies. Further characterization of these new genes in animal models has uncovered fascinating mechanisms of left-right axis development. In this review, we will discuss recent findings on the functions of heterotaxy genes with identified patient alleles.

Candidate Heterotaxy Gene FGFR4 Is Essential for Patterning of the Left-Right Organizer in Xenopus.

Congenital heart disease (CHD) is the most common birth defect, yet its genetic causes continue to be obscure. Fibroblast growth factor receptor 4 (FGFR4) recently emerged in a large patient exome sequencing study as a candidate disease gene for CHD and specifically heterotaxy. In heterotaxy, patterning of the left-right (LR) body axis is compromised, frequently leading to defects in the heart's LR architecture and severe CHD. FGF ligands like FGF8 and FGF4 have been previously implicated in LR development with roles ranging from formation of the laterality organ [LR organizer (LRO)] to the transfer of asymmetry from the embryonic midline to the lateral plate mesoderm (LPM). However, much less is known about which FGF receptors (FGFRs) play a role in laterality. Here, we show that the candidate heterotaxy gene FGFR4 is essential for proper organ situs in Xenopus and that frogs depleted of fgfr4 display inverted cardiac and gut looping. Fgfr4 knockdown causes mispatterning of the LRO even before cilia on its surface initiate symmetry-breaking fluid flow, indicating a role in the earliest stages of LR development. Specifically, fgfr4 acts during gastrulation to pattern the paraxial mesoderm, which gives rise to the lateral pre-somitic portion of the LRO. Upon fgfr4 knockdown, the paraxial mesoderm is mispatterned in the gastrula and LRO, and crucial genes for symmetry breakage, like coco, xnr1, and gdf3 are subsequently absent from the lateral portions of the organizer. In summary, our data indicate that FGF signaling in mesodermal LRO progenitors defines cell fates essential for subsequent LR patterning.

Activation of zebrafish Src family kinases by the prion protein is an amyloid-ß-sensitive signal that prevents the endocytosis and degradation of E-cadherin/ß-catenin complexes in vivo.

BACKGROUND: Prions and amyloid-ß (Aß) oligomers trigger neurodegeneration by hijacking a poorly understood cellular signal mediated by the prion protein (PrP) at the plasma membrane. In early zebrafish embryos, PrP-1-dependent signals control cell-cell adhesion via a tyrosine phosphorylation-dependent mechanism. RESULTS: Here we report that the Src family kinases (SFKs) Fyn and Yes act downstream of PrP-1 to prevent the endocytosis and degradation of E-cadherin/ß-catenin adhesion complexes in vivo. Accordingly, knockdown of PrP-1 or Fyn/Yes cause similar zebrafish gastrulation phenotypes, whereas Fyn/Yes expression rescues the PrP-1 knockdown phenotype. We also show that zebrafish and mouse PrPs positively regulate the activity of Src kinases and that these have an unexpected positive effect on E-cadherin-mediated cell adhesion. Interestingly, while PrP knockdown impairs ß-catenin adhesive function, PrP overexpression enhances it, thereby antagonizing its nuclear, wnt-related signaling activity and disturbing embryonic dorsoventral specification. The ability of mouse PrP to influence these events in zebrafish embryos requires its neuroprotective, polybasic N-terminus but not its neurotoxicity-associated central region. Remarkably, human Aß oligomers up-regulate the PrP-1/SFK/E-cadherin/ß-catenin pathway in zebrafish embryonic cells, mimicking a PrP gain-of-function scenario. CONCLUSIONS: Our gain- and loss-of-function experiments in zebrafish suggest that PrP and SFKs enhance the cell surface stability of embryonic adherens junctions via the same complex mechanism through which they over-activate neuroreceptors that trigger synaptic damage. The profound impact of this pathway on early zebrafish development makes these embryos an ideal model to study the cellular and molecular events affected by neurotoxic PrP mutations and ligands in vivo. In particular, our finding that human Aß oligomers activate the zebrafish PrP/SFK/E-cadherin pathway opens the possibility of using fish embryos to rapidly screen for novel therapeutic targets and compounds against prion- and Alzheimer's-related neurodegeneration. Altogether, our data illustrate PrP-dependent signals relevant to embryonic development, neuronal physiology and neurological disease.

Conserved roles of the prion protein domains on subcellular localization and cell-cell adhesion.

Analyses of cultured cells and transgenic mice expressing prion protein (PrP) deletion mutants have revealed that some properties of PrP -such as its ability to misfold, aggregate and trigger neurotoxicity- are controlled by discrete molecular determinants within its protein domains. Although the contributions of these determinants to PrP biosynthesis and turnover are relatively well characterized, it is still unclear how they modulate cellular functions of PrP. To address this question, we used two defined activities of PrP as functional readouts: 1) the recruitment of PrP to cell-cell contacts in Drosophila S2 and human MCF-7 epithelial cells, and 2) the induction of PrP embryonic loss- and gain-of-function phenotypes in zebrafish. Our results show that homologous mutations in mouse and zebrafish PrPs similarly affect their subcellular localization patterns as well as their in vitro and in vivo activities. Among PrP's essential features, the N-terminal leader peptide was sufficient to drive targeting of our constructs to cell contact sites, whereas lack of GPI-anchoring and N-glycosylation rendered them inactive by blocking their cell surface expression. Importantly, our data suggest that the ability of PrP to homophilically trans-interact and elicit intracellular signaling is primarily encoded in its globular domain, and modulated by its repetitive domain. Thus, while the latter induces the local accumulation of PrPs at discrete punctae along cell contacts, the former counteracts this effect by promoting the continuous distribution of PrP. In early zebrafish embryos, deletion of either domain significantly impaired PrP's ability to modulate E-cadherin cell adhesion. Altogether, these experiments relate structural features of PrP to its subcellular distribution and in vivo activity. Furthermore, they show that despite their large evolutionary history, the roles of PrP domains and posttranslational modifications are conserved between mouse and zebrafish.

PrPs: Proteins with a purpose: Lessons from the zebrafish.

The best-known attribute of the prion protein (PrP) is its tendency to misfold into a rogue isoform. Much less understood is how this misfolded isoform causes deadly brain illnesses. Neurodegeneration in prion disease is often seen as a consequence of abnormal PrP function yet, amazingly little is known about the normal, physiological role of PrP. In particular, the absence of obvious phenotypes in PrP knockout mice has prevented scientists from answering this important question. Using knockdown approaches, we previously produced clear PrP loss-of-function phenotypes in zebrafish embryos. Analysis of these phenotypes revealed that PrP can modulate E-cadherin-based cell-cell adhesion, thereby controlling essential morphogenetic cell movements in the early gastrula. Our data also showed that PrP itself can elicit homophilic cell-cell adhesion and trigger intracellular signaling via Src-related kinases. Importantly, these molecular functions of PrP are conserved from fish to mammals. Here we discuss the use of the zebrafish in prion biology and how it may advance our understanding of the roles of PrP in health and disease.