Neuronal Neuregulin 1 type III directs Schwann cell migration

During peripheral nerve development, each segment of a myelinated axon is matched with a single Schwann cell. Tight regulation of Schwann cell movement, proliferation and differentiation is essential to ensure that these glial cells properly associate with axons. ErbB receptors are required for Schwann cell migration, but the operative ligand and its mechanism of action have remained unknown. We demonstrate that zebrafish Neuregulin 1 (Nrg1) type III, which signals through ErbB receptors, controls Schwann cell migration in addition to its previously known roles in proliferation and myelination. Chimera analyses indicate that ErbB receptors are required in all migrating Schwann cells, and that Nrg1 type III is required in neurons for migration. Surprisingly, expression of the ligand in a few axons is sufficient to induce migration along a chimeric nerve constituted largely of nrg1 type III mutant axons. These studies also reveal a mechanism that allows Schwann cells to fasciculate axons regardless of nrg1 type III expression. Time-lapse imaging of transgenic embryos demonstrated that misexpression of human NRG1 type III results in ectopic Schwann cell migration, allowing them to aberrantly enter the central nervous system. These results demonstrate that Nrg1 type III is an essential signal that controls Schwann cell migration to ensure that these glia are present in the correct numbers and positions in developing nerves.     

Protein-Trap Insertional Mutagenesis Uncovers New Genes Involved in Zebrafish Skin Development,Including a Neuregulin 2a-Based ErbB Signaling Pathway Required during Median Fin Fold Morphogenesis

Skin disorders are widespread, but available treatments are limited. A more comprehensive understanding of skin development mechanisms will drive identification of new treatment targets and modalities. Here we report the Zebrafish Integument Project (ZIP), an expression-driven platform for identifying new skin genes and phenotypes in the vertebrate model Danio rerio (zebrafish). In vivo selection for skin-specific expression of gene-break transposon (GBT) mutant lines identified eleven new, revertible GBT alleles of genes involved in skin development. Eight genes—fras1, grip1, hmcn1, msxc, col4a4, ahnak, capn12, and nrg2a—had been described in an integumentary context to varying degrees, while arhgef25b, fkbp10b, and megf6a emerged as novel skin genes. Embryos homozygous for a GBT insertion within neuregulin 2a (nrg2a) revealed a novel requirement for a Neuregulin 2a (Nrg2a) – ErbB2/3 – AKT signaling pathway governing the apicobasal organization of a subset of epidermal cells during median fin fold (MFF) morphogenesis. In nrg2a mutant larvae, the basal keratinocytes within the apical MFF, known as ridge cells, displayed reduced pAKT levels as well as reduced apical domains and exaggerated basolateral domains. Those defects compromised proper ridge cell elongation into a flattened epithelial morphology, resulting in thickened MFF edges. Pharmacological inhibition verified that Nrg2a signals through the ErbB receptor tyrosine kinase network. Moreover, knockdown of the epithelial polarity regulator and tumor suppressor lgl2 ameliorated the nrg2a mutant phenotype. Identifying Lgl2 as an antagonist of Nrg2a – ErbB signaling revealed a significantly earlier role for Lgl2 during epidermal morphogenesis than has been described to date. Furthermore, our findings demonstrated that successive, coordinated ridge cell shape changes drive apical MFF development, making MFF ridge cells a valuable model for investigating how the coordinated regulation of cell polarity and cell shape changes serves as a crucial mechanism of epithelial morphogenesis.     

Investigating Nrg1 Signaling in the Regenerating Axolotl Spinal Cord Using Multiplexed FISH

Amputation of a salamander tail leads to functional spinal cord regeneration through activation of endogenous stem cells. Identifying the signaling pathways that control cell proliferation in these neural stem cells will help elucidate the mechanisms underlying the salamander’s regenerative ability. Here, we show that neuregulin 1 (Nrg1)/ErbB2 signaling is an important pathway in the regulation of neural stem cell proliferation in the spinal cord of the axolotl salamander (Ambystoma mexicanum). Simultaneous localization of nrg1 mRNA and Nrg1 protein was performed by utilizing a hybridization chain reaction fluorescence in situ hybridization (FISH) methodology in tissue sections. Multiplexed FISH also permitted the phenotyping of multiple cell types on a single fixed section allowing the characterization of mRNA expression, protein expression, and tissue architecture. Pharmacological inhibition of ErbB2 showed that intact Nrg1/ErbB2 signaling is critical for adult homeostatic regeneration as well as for injury‐induced spinal cord regeneration. Overall, our results highlight the importance of the NRG1/ErbB2 signaling pathway in neural stem cell proliferation in the axolotl.     

Neuregulin 1-β regulates cell adhesion molecule L1 expression in the cortex and hippocampus of mice

Neuregulin 1 (Nrg1) functions in neuronal migration, survival and differentiation as well as synaptogenesis during ontogenetic development and maintenance of synaptic functions in the adult mammalian brain. The neural adhesion molecule L1 (L1CAM) functions in similar overlapping, but also non-overlapping roles in the nervous system. In the present study, we therefore investigated some aspects of the functional relationship between Nrg1 and L1 in mammalian neural cells. Nrg1 regulates the expression of L1 in cultures of both human neuroblastoma SK-N-SH cells and mouse cortical and hippocampal neurons. To analyze the role of Nrg1 on L1 expression in vivo, young adult male mice received intraperitoneal injections of Nrg1 or PBS (vehicle control). The correlation between Nrg1 and L1 expression was tested by qPCR, Western blot analysis, and immunocytology. Our data indicate that neuregulin 1-β (Nrg1β) increases L1 expression in neurons of the cerebral cortex, and decreases expression in neurons of the hippocampus in vitro and in vivo. In addition, Nrg1 induces phosphorylation of its receptors, ErbB2 and ErbB4, the predominant ErbB receptors in the nervous system. These results show that Nrg1β affects expression of L1 in the central nervous system and in parallel activates the ErbB receptors for Nrg1, suggesting a crosstalk between molecules that are of prime importance for nervous system functions.     

Zebrafish heart as a model for human cardiac electrophysiology

The zebrafish (Danio rerio) has become a popular model for human cardiac diseases and pharmacology including cardiac arrhythmias and its electrophysiological basis. Notably, the phenotype of zebrafish cardiac action potential is similar to the human cardiac action potential in that both have a long plateau phase. Also the major inward and outward current systems are qualitatively similar in zebrafish and human hearts. However, there are also significant differences in ionic current composition between human and zebrafish hearts, and the molecular basis and pharmacological properties of human and zebrafish cardiac ionic currents differ in several ways. Cardiac ionic currents may be produced by non-orthologous genes in zebrafish and humans, and paralogous gene products of some ion channels are expressed in the zebrafish heart. More research on molecular basis of cardiac ion channels, and regulation and drug sensitivity of the cardiac ionic currents are needed to enable rational use of the zebrafish heart as an electrophysiological model for the human heart.     

<Emphasis Type="Italic">Engrailed</Emphasis> is expressed in larval development and in the radial nervous system of <Emphasis Type="Italic">Patiriella</Emphasis> sea stars

We documented expression of the pan-metazoan neurogenic gene engrailed in larval and juvenile Patiriella sea stars to determine if this gene patterns bilateral and radial echinoderm nervous systems. Engrailed homologues, containing conserved En protein domains, were cloned from the radial nerve cord. During development, engrailed was expressed in ectodermal (nervous system) and mesodermal (coeloms) derivatives. In larvae, engrailed was expressed in cells lining the larval and future adult coeloms. Engrailed was not expressed in the larval nervous system. As adult-specific developmental programs were switched on during metamorphosis, engrailed was expressed in the central nervous system and peripheral nervous system (PNS), paralleling the pattern of neuropeptide immunolocalisation. Engrailed was first seen in the developing nerve ring and appeared to be up-regulated as the nervous system developed. Expression of engrailed in the nerve plexus of the tube feet, the lobes of the hydrocoel along the adult arm axis, is similar to the reiterated pattern of expression seen in other animals. Engrailed expression in developing nervous tissue reflects its conserved role in neurogenesis, but its broad expression in the adult nervous system of Patiriella differs from the localised expression seen in other bilaterians. The role of engrailed in patterning repeated PNS structures indicates that it may be important in patterning the fivefold organisation of the ambulacrae, a defining feature of the Echinodermata.     

Ndrg4 is required for normal myocyte proliferation during early cardiac development in zebrafish

NDRG4 is a novel member of the NDRG family (N-myc downstream-regulated gene). The roles of NDRG4 in development have not previously been evaluated. We show that, during zebrafish embryonic development, ndrg4 is expressed exclusively in the embryonic heart, the central nervous system (CNS) and the sensory system. Ndrg4 knockdown in zebrafish embryos causes a marked reduction in proliferative myocytes and results in hypoplastic hearts. This growth defect is associated with cardiac phenotypes in morphogenesis and function, including abnormal heart looping, inefficient circulation and weak contractility. We reveal that ndrg4 is required for restricting the expression of versican and bmp4 to the developing atrioventricular canal. This constellation of ndrg4 cardiac defects phenocopies those seen in mutant hearts of heartstrings (hst), the tbx5 loss-of-function mutants in zebrafish. We further show that ndrg4 expression is significantly decreased in hearts with reduced tbx5 activities. Conversely, increased expression of tbx5 that is due to tbx20 knockdown leads to an increase in ndrg4 expression. Together, our studies reveal an essential role of ndrg4 in regulating proliferation and growth of cardiomyocytes, suggesting that ndrg4 may function downstream of tbx5 during heart development and growth.     

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.     

Defective adult oligodendrocyte and Schwann cell development, pigment pattern, and craniofacial morphology in puma mutant zebrafish having an alpha tubulin mutation

The processes of myelination remain incompletely understood but are of profound biomedical importance owing to the several dysmyelinating and demyelinating disorders known in humans. Here, we analyze the zebrafish puma mutant, isolated originally for pigment pattern defects limited to the adult stage. We show that puma mutants also have late-arising defects in Schwann cells of the peripheral nervous system, locomotor abnormalities, and sex-biased defects in adult craniofacial morphology. Using methods of positional cloning, we identify a critical genetic interval harboring two alpha tubulin loci, and we identify a chemically induced missense mutation in one of these, tubulin alpha 8-like 3a (tuba8l3a). We demonstrate tuba8l3a expression in the central nervous system (CNS), leading us to search for defects in the development of oligodendrocytes, the myelinating cells of the CNS. We find gross reductions in CNS myelin and oligodendrocyte numbers in adult puma mutants, and these deficits are apparent already during the larval-to-adult transformation. By contrast, analyses of embryos and early larvae reveal a normal complement of oligodendrocytes that nevertheless fail to localize normal amounts of myelin basic protein (mbp) mRNA in cellular processes, and fail to organize these processes as in the wild-type. This study identifies the puma mutant as a valuable model for studying microtubule-dependent events of myelination, as well as strategies for remyelination in the adult.     

Regulation of cardiomyocyte proliferation during development and regeneration

The regulation of cardiomyocyte proliferation is important for heart development and regeneration. The proliferation patterns of cardiomyocytes are closely related to heart morphogenesis, size, and functions. The proliferation levels are high during early embryogenesis; however, mammalian cardiomyocytes exit the cell cycle irreversibly soon after birth. The cell cycle exit inhibits cardiac regeneration in mammals. On the other hand, cardiomyocytes of adult zebrafish and probably newts can proliferate after cardiac injury, and the hearts can be regenerated. Therefore, the ability to reproliferate determines regenerative ability. As in other cells, the relationship between proliferation and differentiation is very interesting, and is closely related to cardiac development, regeneration and homeostasis. In this review, these topics are discussed.     

Differential Requirement for irf8 in Formation of Embryonic and Adult Macrophages in Zebrafish

Interferon regulatory factor 8 (Irf8) is critical for mammalian macrophage development and innate immunity, but its role in teleost myelopoiesis remains incompletely understood. In particular, genetic tools to analyze the role of Irf8 in zebrafish macrophage development at larval and adult stages are lacking. We generated irf8 null mutants in zebrafish using TALEN-mediated targeting. Our analysis defines different requirements for irf8 at different stages. irf8 is required for formation of all macrophages during primitive and transient definitive hematopoiesis, but not during adult-phase definitive hematopoiesis starting at 5-6 days postfertilization. At early stages, irf8 mutants have excess neutrophils and excess cell death in pu.1-expressing myeloid cells. Macrophage fates were recovered in irf8 mutants after wildtype irf8 expression in neutrophil and macrophage lineages, suggesting that irf8 regulates macrophage specification and survival. In juvenile irf8 mutant fish, mature macrophages are present, but at numbers significantly reduced compared to wildtype, indicating an ongoing requirement for irf8 after embryogenesis. As development progresses, tissue macrophages become apparent in zebrafish irf8 mutants, with the possible exception of microglia. Our study defines distinct requirement for irf8 in myelopoiesis before and after transition to the adult hematopoietic system.     

Reconstitution of defective protein trafficking rescues Long-QT syndrome in zebrafish

Inherited cardiac arrhythmias are caused by genetic defects in ion channels and associated proteins. Mutations in these channels often do not affect their biophysical properties, but rather interfere with their trafficking to the cell membrane. Accordingly, strategies that could reroute the mutated channels to the membrane should be sufficient to restore the electrical properties of the affected cells, thereby suppressing the underlying arrhythmia. We identified here both, embryonic and adult zebrafish breakdance (bre) as a valuable model for human Long-QT syndrome. Electrocardiograms of adult homozygous bre mutants exhibit significant QT prolongation caused by delayed repolarization of the ventricle. We further show that the bre mutation (zERG^I59S) disrupts ERG protein trafficking, thereby reducing the amount of active potassium channels on the cell membrane. Interestingly, improvement of channel trafficking by cisapride or dimethylsulfoxid is sufficient to reconstitute ERG channels on the cell membrane in a manner that suffices to suppress the Long-QT induced arrhythmia in breakdance mutant zebrafish. In summary, we show for the first time that therapeutic intervention can cure protein trafficking defects and the associated cardiac arrhythmia in vivo.     

Na,K-ATPase is essential for embryonic heart development in the zebrafish

Na,K-ATPase is an essential gene maintaining electrochemical gradients across the plasma membrane. Although previous studies have intensively focused on the role of Na,K-ATPase in regulating cardiac function in the adults, little is known about the requirement for Na,K-ATPase during embryonic heart development. Here, we report the identification of a zebrafish mutant, heart and mind, which exhibits multiple cardiac defects, including the primitive heart tube extension abnormality, aberrant cardiomyocyte differentiation, and reduced heart rate and contractility. Molecular cloning reveals that the heart and mind lesion resides in the alpha1B1 isoform of Na,K-ATPase. Blocking Na,K-ATPase alpha1B1 activity by pharmacological means or by morpholino antisense oligonucleotides phenocopies the patterning and functional defects of heart and mind mutant hearts, suggesting crucial roles for Na,K-ATPase alpha1B1 in embryonic zebrafish hearts. In addition to alpha1B1, the Na,K-ATPase alpha2 isoform is required for embryonic cardiac patterning. Although the alpha1B1 and alpha2 isoforms share high degrees of similarities in their coding sequences, they have distinct roles in patterning zebrafish hearts. The phenotypes of heart and mind mutants can be rescued by supplementing alpha1B1, but not alpha2, mRNA to the mutant embryos, demonstrating that alpha1B1 and alpha2 are not functionally equivalent. Furthermore, instead of interfering with primitive heart tube formation or cardiac chamber differentiation, blocking the translation of Na,K-ATPase alpha2 isoform leads to cardiac laterality defects.     

Isolation and characterization of two new negative regulatory mutants for nitrate assimilation inChlamydomonas reinhardtii obtained by insertional mutagenesis

Plasmid DNA carrying either the nitrate reductase (NR) gene or the argininosuccinate lyase gene as selectable markers and the correspondingChlamydomonas reinhardtii mutants as recipient strains have been used to isolate regulatory mutants for nitrate assimilation by insertional mutagenesis. Identification of putative regulatory mutants was based on their chlorate sensitivity in the presence of ammonium. Among 8975 transformants, two mutants, N1 and T1, were obtained. Genetic characterization of these mutants indicated that they carry recessive mutations at two different loci, namedNrg1 andNrg2. The mutation in N1 was shown to be linked to the plasmid insertion. Two copies of the nitrate reductase plasmid, one of them truncated, were inserted in the N1 genome in inverse orientation. In addition to the chlorate sensitivity phenotype in the presence of ammonium, these mutants expressed NR, nitrite reductase and nitrate transport activities in ammonium-nitrate media. Kinetic constants for ammonium (¹⁴C-methylammonium) transport, as well as enzymatic activities related to the ammonium-regulated metabolic pathway for xanthine utilization, were not affected in these strains. The data strongly suggest thatNrg1 andNrg2 are regulatory genes which specifically mediate the negative control exerted by ammonium on the nitrate assimilation pathway inC. reinhardtii.     

Zebrafish con/disp1 reveals multiple spatiotemporal requirements for Hedgehog-signaling in craniofacial development

Background  

The vertebrate head skeleton is derived largely from cranial neural crest cells (CNCC). Genetic studies in zebrafish and mice have established that the Hedgehog (Hh)-signaling pathway plays a critical role in craniofacial development, partly due to the pathway's role in CNCC development. Disruption of the Hh-signaling pathway in humans can lead to the spectral disorder of Holoprosencephaly (HPE), which is often characterized by a variety of craniofacial defects including midline facial clefting and cyclopia [1, 2]. Previous work has uncovered a role for Hh-signaling in zebrafish dorsal neurocranium patterning and chondrogenesis, however Hh-signaling mutants have not been described with respect to the ventral pharyngeal arch (PA) skeleton. Lipid-modified Hh-ligands require the transmembrane-spanning receptor Dispatched 1 (Disp1) for proper secretion from Hh-synthesizing cells to the extracellular field where they act on target cells. Here we study chameleon mutants, lacking a functional disp1(con/disp1).