The Spinster Homolog, Two of Hearts, Is Required for Sphingosine 1-Phosphate Signaling in Zebrafish

SummaryThe bioactive lipid sphingosine 1-phosphate (S1P) and its G protein-coupled receptors play critical roles in cardiovascular, immunological, and neural development and function [1–6]. Despite its importance, many questions remain about S1P signaling, including how S1P, which is synthesized intracellularly, is released from cells. Mutations in the zebrafish gene encoding the S1P receptor Miles Apart (Mil)/S1P2 disrupt the formation of the primitive heart tube [5]. We find that mutations of another zebrafish locus, two of hearts (toh), cause phenotypes that are morphologically indistinguishable from those seen in mil/s1p2 mutants. Positional cloning of toh reveals that it encodes a member of the Spinster-like family of putative transmembrane transporters. The biological functions of these proteins are poorly understood, although phenotypes of the Drosophila spinster and zebrafish not really started mutants suggest that these proteins may play a role in lipid trafficking [7, 8]. Through gain- and loss-of-function analyses, we show that toh is required for signaling by S1P2. Further evidence indicates that Toh is involved in the trafficking or cellular release of S1P

Cse1l Is a Negative Regulator of CFTR-Dependent Fluid Secretion

Transport of chloride through the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel is a key step in regulating fluid secretion in vertebrates[1, 2]. Loss of CFTR function leads to cystic fibrosis (CF)[1, 3, 4], a disease that affects the lungs, pancreas, liver, intestine and vas deferens. Conversely, un-controlled activation of the channel leads to increased fluid secretion and plays a major role in several diseases and conditions including cholera[5, 6] and other secretory diarrheas [7] as well as Polycystic Kidney Disease (PKD)[8–10]. Understanding how CFTR activity is regulated in vivo has been limited by the lack of a genetic model. Here, we used a forward genetic approach in zebrafish to uncover CFTR regulators. We report the identification, isolation and characterization of a mutation in the zebrafish cse1l gene that leads to the sudden and dramatic expansion of the gut tube. We show that this phenotype results from a rapid accumulation of fluid due to the un-controlled activation of the CFTR channel. Analyses in zebrafish embryos and mammalian cells indicate that Cse1l is a negative regulator of CFTR-dependent fluid secretion. This work demonstrates the importance of fluid homeostasis in development and establishes the zebrafish as a much needed model system to study CFTR regulation in vivo

Non-steric-zipper models for pathogenic α-synuclein conformers

Parkinson's disease neurodegenerative brain tissue exhibits two biophysically distinct α-synuclein fiber isoforms—single stranded fibers that appear to be steric-zippers and double-stranded fibers with an undetermined structure. Herein, we describe a β-helical homology model of α-synuclein that exhibits stability in probabilistic and Monte Carlo simulations as a candidate for stable prional dimer conformers in equilibrium with double-stranded fibers and cytotoxic pore assemblies. Molecular models of β-helical pore assemblies are consistent with α-synucleinA53T transfected rat immunofluorescence epitope maps. Atomic force microscopy reveals that α-synuclein peptides aggregate into anisotropic fibrils lacking the density or circumference of a steric-zipper. Moreover, fibrillation was blocked by mutations designed to hinder β-helical but not steric-zipper conformations. β-helical species provide a structural basis for previously described biophysical properties that are incompatible with a steric-zipper, provide pathogenic mechanisms for familial human α-synuclein mutations, and offer a direct cytotoxic target for therapeutic development

Disruption of pdgfra alters endocardial and myocardial fusion during zebrafish cardiac assembly

Cardiac development in vertebrates is a finely tuned process regulated by a set of conserved signaling pathways. Perturbations of these processes are often associated with congenital cardiac malformations. Platelet-derived growth factor receptor α (PDGFRα) is a highly conserved tyrosine kinase receptor, which is essential for development and organogenesis. Disruption of Pdgfrα function in murine models is embryonic lethal due to severe cardiovascular defects, suggesting a role in cardiac development, thus necessitating the use of alternative models to explore its precise function. In this study, we generated a zebrafish pdgfra mutant line by gene trapping, in which the Pdgfra protein is truncated and fused with mRFP (Pdgfra-mRFP). Our results demonstrate that pdgfra mutants have defects in cardiac morphology as a result of abnormal fusion of myocardial precursors. Expression analysis of the developing heart at later stages suggested that Pdgfra-mRFP is expressed in the endocardium. Further examination of the endocardium in pdgfra mutants revealed defective endocardial migration to the midline, where cardiac fusion eventually occurs. Together, our data suggests that pdgfra is required for proper medial migration of both endocardial and myocardial precursors, an essential step required for cardiac assembly and development

Integrating glycomics and genomics uncovers SLC10A7 as essential factor for bone mineralization by regulating post-Golgi protein transport and glycosylation

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NANS-mediated synthesis of sialic acid is required for brain and skeletal development

We identified biallelic mutations in NANS, the gene encoding the synthase for N-acetylneuraminic acid (NeuNAc; sialic acid), in nine individuals with infantile-onset severe developmental delay and skeletal dysplasia. Patient body fluids showed an elevation in N-acetyl-D-mannosamine levels, and patient-derived fibroblasts had reduced NANS activity and were unable to incorporate sialic acid precursors into sialylated glycoproteins. Knockdown of nansa in zebrafish embryos resulted in abnormal skeletal development, and exogenously added sialic acid partially rescued the skeletal phenotype. Thus, NANS-mediated synthesis of sialic acid is required for early brain development and skeletal growth. Normal sialylation of plasma proteins was observed in spite of NANS deficiency. Exploration of endogenous synthesis, nutritional absorption, and rescue pathways for sialic acid in different tissues and developmental phases is warranted to design therapeutic strategies to counteract NANS deficiency and to shed light on sialic acid metabolism and its implications for human nutrition