Non‐core subunit eIF3h of translation initiation factor eIF3 regulates zebrafish embryonic development

Eukaryotic translation initiation factor eIF3, which plays a central role in translation initiation, consists of five core subunits that are present in both the budding yeast and higher eukaryotes. However, higher eukaryotic eIF3 contains additional (non‐core) subunits that are absent in the budding yeast. We investigated the role of one such non‐core eIF3 subunit eIF3h, encoded by two distinct genes—eif3ha and eif3hb, as a regulator of embryonic development in zebrafish. Both eif3h genes are expressed during early embryogenesis, and display overlapping yet distinct and highly dynamic spatial expression patterns. Loss of function analysis using specific morpholino oligomers indicates that each isoform has specific as well as redundant functions during early development. The morphant phenotypes correlate with their spatial expression patterns, indicating that eif3h regulates development of the brain, heart, vasculature, and lateral line. These results indicate that the non‐core subunits of eIF3 regulate specific developmental programs during vertebrate embryogenesis. Developmental Dynamics 239:1632–1644, 2010. © 2010 Wiley‐Liss, Inc.     

A mutation of SCN1B associated with GEFS+ causes functional and maturation defects of the voltage‐dependent sodium channel

Voltage‐dependent sodium channels are responsible of the rising phase of the action potential in excitable cells. These integral membrane proteins are composed of a pore‐forming α‐subunit, and one or more auxiliary β subunits. Mutation p.Asp25Asn (D25N; c.73G > A) of the β1 subunit, coded by the gene SCN1B, has been reported in a patient with generalized epilepsy with febrile seizure plus type 1 (GEFS+). In human embryonic kidney 293 (HEK) cells, the heterologous coexpression of D25N‐β1 subunit with Nav1.2, Nav1.4, and Nav1.5 α subunits, representative of brain, skeletal muscle, and heart voltage gated sodium channels, determines a reduced sodium channel functional expression and a negative shift of the activation and inactivation steady state curves. The D25N mutation of the β1 subunit causes a maturation (glycosylation) defect of the protein, leading to a reduced targeting to the plasma membrane. Also the β1‐dependent gating properties of the sodium channels are abolished by the mutation, suggesting that D25N is no more able to interact with the α subunit. Our work underscores the role played by the β1 subunit, highlighting how a defective interaction between the sodium channel constituents could lead to a disabling pathological condition, and opens the possibility to design a mutation‐specific GEFS+ treatment based on protein maturation.     

The Repertoire of Na,K-ATPase α and β Subunit Genes Expressed in the Zebrafish, Danio rerio

We have identified a cohort of zebrafish expressed sequence tags encoding eight Na,K-ATPase alpha subunits and five beta subunits. Sequence comparisons and phylogenetic analysis indicate that five of the zebrafish alpha subunit genes comprise an alpha1-like gene subfamily and two are orthologs of the mammalian alpha3 subunit gene. The remaining alpha subunit clone is most similar to the mammalian alpha2 subunit. Among the five beta subunit genes, two are orthologs of the mammalian beta1 isoform, one represents a beta2 ortholog, and two are orthologous to the mammalian beta3 subunit. Using zebrafish radiation hybrid and meiotic mapping panels, we determined linkage assignments for each alpha and beta subunit gene. Na,K-ATPase genes are dispersed in the zebrafish genome with the exception of four of the alpha1-like genes, which are tightly clustered on linkage group 1. Comparative mapping studies indicate that most of the zebrafish Na,K-ATPase genes localize to regions of conserved synteny between zebrafish and humans. The expression patterns of Na,K-ATPase alpha and beta subunit genes in zebrafish are quite distinctive. No two alpha or beta subunit genes exhibit the same expression profile. Together, our data imply a very high degree of Na,K-ATPase isoenzyme heterogeneity in zebrafish, with the potential for 40 structurally distinct alpha/beta subunit combinations. Differences in expression patterns of alpha and beta subunits suggest that many of the isoenzymes are also likely to exhibit differences in functional properties within specific cell and tissue types. Our studies form a framework for analyzing structure function relationships for sodium pump isoforms using reverse genetic approaches.     

Molecular characterization of the zebrafish P2X receptor subunit gene family

P2X receptors are non-selective cation channels gated by extracellular ATP and are encoded by a family of seven subunit genes in mammals. These receptors exhibit high permeabilities to calcium and in the mammalian nervous system they have been linked to modulation of neurotransmitter release. Previously, three complementary DNAs (cDNAs) encoding members of the zebrafish gene family have been described. We report here the cloning and characterization of an additional six genes of this family. Sequence analysis of all nine genes suggests that six are orthologs of mammalian genes, two are paralogs of previously described zebrafish subunits, and one remains unclassified. All nine subunits were physically mapped onto the zebrafish genome using radiation hybrid analysis. Of the nine gene products, seven give functional homo-oligomeric receptors when recombinantly expressed in human embryonic kidney cell line 293 cells. In addition, these subunits can form hetero-oligomeric receptors with phenotypes distinct from the parent subunits. Analysis of gene expression patterns was carried out using in situ hybridization, and seven of the nine genes were found to be expressed in embryos at 24 and 48 h post-fertilization. Of the seven that were expressed, six were present in the nervous system and four of these demonstrated considerable overlap in cells present in the sensory nervous system. These results suggest that P2X receptors might play a role in the early development and/or function of the sensory nervous system in vertebrates.     

Sequence and expression of the zebrafish alpha‐actinin gene family reveals conservation and diversification among vertebrates

alpha‐actinins are actin microfilament crosslinking proteins. Vertebrate actinins fall into two classes: the broadly‐expressed actinins 1 and 4 (actn1 and actn4) and muscle‐specific actinins, actn2 and actn3. Members of this family have numerous roles, including regulation of cell adhesion, cell differentiation, directed cell motility, intracellular signaling, and stabilization of f‐actin at the sarcomeric Z‐line in muscle. Here we identify five zebrafish actinin genes including two paralogs of ACTN3. We describe the temporal and spatial expression patterns of these genes through embryonic development. All zebrafish actinin genes have unique expression profiles, indicating specialization of each gene. In particular, the muscle actinins display preferential expression in different domains of axial, pharyngeal, and cranial musculature. There is no identified avian actn3 and approximately 16% of humans are null for ACTN3. Duplication of actn3 in the zebrafish indicates that variation in actn3 expression may promote physiological diversity in muscle function among vertebrates. Developmental Dynamics 238:2936–2947, 2009. © 2009 Wiley‐Liss, Inc.     

Electrophysiology and beyond: Multiple roles of Na channel β subunits in development and disease

Voltage-gated Na⁺ channel (VGSC) β Subunits are not “auxiliary.” These multi-functional molecules not only modulate Na⁺ current (I_Na), but also function as cell adhesion molecules (CAMs)—playing roles in aggregation, migration, invasion, neurite outgrowth, and axonal fasciculation. β subunits are integral members of VGSC signaling complexes at nodes of Ranvier, axon initial segments, and cardiac intercalated disks, regulating action potential propagation through critical intermolecular and cell–cell communication events. At least in vitro, many β subunit cell adhesive functions occur both in the presence and absence of pore-forming VGSC α subunits, and in vivo β subunits are expressed in excitable as well as non-excitable cells, thus β subunits may play important functional roles on their own, in the absence of α subunits. VGSC β1 subunits are essential for life and appear to be especially important during brain development. Mutations in β subunit genes result in a variety of human neurological and cardiovascular diseases. Moreover, some cancer cells exhibit alterations in β subunit expression during metastasis. In short, these proteins, originally thought of as merely accessory to α subunits, are critical players in their own right in human health and disease. Here we discuss the role of VGSC β subunits in the nervous system.     

Mutations Causing Slow‐Channel Myasthenia Reveal That a Valine Ring in the Channel Pore of Muscle AChR is Optimized for Stabilizing Channel Gating

We identify two novel mutations in acetylcholine receptor (AChR) causing a slow‐channel congenital myasthenia syndrome (CMS) in three unrelated patients (Pts). Pt 1 harbors a heterozygous βV266A mutation (p.Val289Ala) in the second transmembrane domain (M2) of the AChR β subunit (CHRNB1). Pts 2 and 3 carry the same mutation at an equivalent site in the ε subunit (CHRNE), εV265A (p.Val285Ala). The mutant residues are conserved across all AChR subunits of all species and are components of a valine ring in the channel pore, which is positioned four residues above the leucine ring. Both βV266A and εV265A reduce the amino acid size and lengthen the channel opening bursts by fourfold by enhancing gating efficiency by approximately 30‐fold. Substitution of alanine for valine at the corresponding position in the δ and α subunit prolongs the burst duration four‐ and eightfold, respectively. Replacing valine at ε codon 265 either by a still smaller glycine or by a larger leucine also lengthens the burst duration. Our analysis reveals that each valine in the valine ring contributes to channel kinetics equally, and the valine ring has been optimized in the course of evolution to govern channel gating.     

The embryonic expression patterns and the knockdown phenotypes of zebrafish ADP‐ribosylation factor‐like 6 interacting protein gene

ADP‐ribosylation factor‐like 6 (Arl6) mutation is linked to human disease and Arl6 interacts with Arl6 interacting protein (Arl6ip). However, the expression pattern and function of Arl6ip during embryogenesis are unknown. To confirm whether abnormal Arl6ip function might result in embryonic defects in zebrafish, we examined the expression patterns of arl6ip during embryogenesis, and they were maternally expressed and exhibited in the brain, optic primordia, hypochord, spinal cord, myotome, heart, fin‐bud, kidney, trunk, and retina. Knockdown of Arl6ip revealed the following phenotypic defects: microphthalmia, disorganized pigment pattern, flat head, defective tectum, deficient pectoral fins, abnormal pneumatic duct, pericardial edema, and deformed trunk. Particularly, histological dissection of the retinae of arl6ip‐morphants revealed that neuronal differentiation is severely delayed, resulting in no formation of retinal layers. We further confirmed that opsins of arl6ip‐morphants were not transcribed. Based on this evidence, Arl6ip may play important roles in zebrafish ocular, heart, and fin‐bud development. Developmental Dynamics 238:232–240, 2009. © 2008 Wiley‐Liss, Inc.     

Loss of the β1 subunit of the sodium pump during lymphocyte differentiation

The Na, K-ATPase, or sodium pump, is responsible for maintaining cellular volume and is involved in receptor-mediated endocytosis; it is a ubiquitous transmembrane enzyme in higher eukaryotes and consists of an α and a β subunit. In the mouse, two isotypes of β with no known function have been identified: β1 and β2. We have studied the expression of β1 and β2 in lymphocytes from bone marrow, spleen, peripheral blood, and thymus. The β2 subunit is not expressed in any of the lymphocytes tested. Pre-B lymphocytes and the majority of mature, resting B cells in the bone marrow express the β1 subunit, as do all pre-T cells and mature thymocytes. In the spleen and in blood, β1 expression defines subsets of T and B lymphocytes. Mitogen-stimulated T and B cells lose β1 expression and do not express β2. While there is no indication that there is a change in α subunit isoform expression as a result of lymphocyte activation or that it is expressed in smaller amounts, there is a switch in the expression of the β isoform.     

Regulation of the activity of voltage-gated potassium channels by β subunits

Voltage-gated potassium channels may consist of two distinct types of subunits assembled in heteromultimeric complexes. One type of subunit, the α subunits, are sufficient to form the ion channel pore and the principal channel gating machinery. The second type of subunit, the β subunits, may have an auxiliary function, in particular in mediating rapid inactivation of voltage-gated potassium channels. Therefore, different α/β subunit compositions may generate voltage-gated potassium channels with properties distinct from the ones which are expressed by α subunits alone. The primary sequences of α subunits are structurally related and belong to a gene superfamily which may have evolved from a primordial ion channel gene. Similarly, β-subunit-related primary sequences have been identified in plants as well as in procaryotes. Possibly, primordial potassium channels were already assembled from α and βsubunits.     

Identification and expression patterns of members of the protease‐activated receptor (par) gene family during zebrafish development

Protease‐activated receptors (PARs) play critical roles in hemostasis in vertebrates including zebrafish. However, the zebrafish gene classification appears to be complex, and the expression patterns of par genes are not established. Based on analyses of genomic organization, phylogenetics, protein primary structure, and protein internalization, we report the identification of four zebrafish PARs: par1, par2a, par2b, and par3. This classification differs from one reported previously. We also show that these genes have distinct spatiotemporal expression profiles in embryos and larvae, with par1, par2a, and par2b expressed maternally and ubiquitously during gastrula stages and their expression patterns refined at later stages, and par3 expressed only in 3‐day‐old larvae. Notably, the expression patterns of zebrafish par1 and par2b resemble those of their mammalian counterparts, suggesting that receptor function is conserved among vertebrates. This conservation is supported by our findings that Par1 and Par2b are internalized following exposure to thrombin and trypsin, respectively. Developmental Dynamics, 2011. © 2010 Wiley‐Liss, Inc.     

Role of α2/δ subunit in the development of morphine-induced rewarding effect and behavioral sensitization

Previous data demonstrate that L-type voltage-gated calcium channel (VGCC) blockers, which bind to α₁ subunits of VGCC to suppress Ca²⁺ entry into cells, inhibit the development of psychological dependence on drugs of abuse, suggesting the upregulation of L-type VGCC in the development of psychological dependence. However, there are few available data on changes of the auxiliary subunit α₂/δ modifying L-type VGCC under such conditions. We therefore investigated here the role of α₂/δ subunits of VGCCs in the brain of mouse after repeated treatment with morphine. The treatment with morphine increased α₂/δ subunit expression in the frontal cortex and the limbic forebrain of mice showing rewarding effect and sensitization to hyperlocomotion by morphine. The morphine-induced behavioral sensitization and place preference were also suppressed by gabapentin, which binds to an exofacial epitope of the α₂/δ auxiliary subunits of VGCCs. These findings indicate that the upregulation of α₂/δ subunit as well as α₁ subunits of VGCC in the frontal cortex and the limbic forebrain plays a critical role in development of morphine-induced rewarding effect and behavioral sensitization following neuronal plasticity.     

Neuron‐specific expression of atp6v0c2 in zebrafish CNS

Vacuolar ATPase (V‐ATPase) is a multi‐subunit enzyme that plays an important role in the acidification of a variety of intracellular compartments. ATP6V0C is subunit c of the V₀ domain that forms the proteolipid pore of the enzyme. In the present study, we investigated the neuron‐specific expression of atp6v0c2, a novel isoform of the V‐ATPase c‐subunit, during the development of the zebrafish CNS. Zebrafish atp6v0c2 was isolated from a genome‐wide analysis of the zebrafish mib^ta52b mutant designed to identify genes differentially regulated by Notch signaling. Whole‐mount in situ hybridization revealed that atp6v0c2 is expressed in a subset of CNS neurons beginning several hours after the emergence of post‐mitotic neurons. The ATP6V0C2 protein is co‐localized with the presynaptic vesicle marker, SV2, suggesting that it is involved in neurotransmitter storage and/or secretion in neurons. In addition, the loss‐of‐function experiment suggests that ATP6V0C2 is involved in the control of neuronal excitability. Developmental Dynamics 239:2501–2508, 2010. © 2010 Wiley‐Liss, Inc.