Gap junction Delta-2b (/Cx35.1) depletion causes hyperopia and visual-motor deficiencies in the zebrafish.

The zebrafish is a powerful model to investigate the developmental roles of electrical synapses because many signaling pathways that regulate the development of the nervous system are highly conserved from fish to humans. Here, we provide evidence linking the mammalian connexin-36 (Cx36) ortholog /Cx35.1, a major component of electrical synapses in the zebrafish, with a refractive error in the context of morphological, molecular, and behavioral changes of zebrafish larvae.

Panx1 channels promote both anti- and pro-seizure-like activities in the zebrafish via p2rx7 receptors and ATP signaling.

The molecular mechanisms of excitation/inhibition imbalances promoting seizure generation in epilepsy patients are not fully understood. Evidence suggests that Pannexin1 (Panx1), an ATP release channel, modulates the excitability of the brain. In this report, we performed electrophysiological, behavioral, and molecular phenotyping experiments on zebrafish larvae bearing genetic or pharmacological knockouts of Panx1a and Panx1b channels, each homologous to human PANX1.

Panx1b Modulates the Luminance Response and Direction of Locomotion in the Zebrafish.

Pannexin1 (Panx1) can form ATP-permeable channels that play roles in the physiology of the visual system. In the zebrafish two ohnologs of Panx1, Panx1a and Panx1b, have unique and shared channel properties and tissue expression patterns. Panx1a channels are located in horizontal cells of the outer retina and modulate light decrement detection through an ATP/pH-dependent mechanisms and adenosine/dopamine signaling.

Convergent NMDA receptor-Pannexin1 signaling pathways regulate the interaction of CaMKII with Connexin-36.

Ca/calmodulin-dependent protein kinase II (CaMKII) binding and phosphorylation of mammalian connexin-36 (Cx36) potentiate electrical coupling. To explain the molecular mechanism of how Cx36 modifies plasticity at gap junctions, we investigated the roles of ionotropic N-methyl-D-aspartate receptors and pannexin1 (Panx1) channels in regulating Cx36 binding to CaMKII. Pharmacological interference and site-directed mutagenesis of protein interaction sites shows that NMDA receptor activation opens Cx36 channels, causing the Cx36- CaMKII binding complex to adopt a compact conformation.

Visuomotor deficiency in panx1a knockout zebrafish is linked to dopaminergic signaling.

Pannexin 1 (Panx1) forms ATP-permeable membrane channels that play roles in the nervous system. The analysis of roles in both standard and pathological conditions benefits from a model organism with rapid development and early onset of behaviors. Such a model was developed by ablating the zebrafish panx1a gene using TALEN technology.

Tubulin-Dependent Transport of Connexin-36 Potentiates the Size and Strength of Electrical Synapses.

Connexin-36 (Cx36) electrical synapses strengthen transmission in a calcium/calmodulin (CaM)/calmodulin-dependent kinase II (CaMKII)-dependent manner similar to a mechanism whereby the N-methyl-D-aspartate (NMDA) receptor subunit NR2B facilitates chemical transmission. Since NR2B-microtubule interactions recruit receptors to the cell membrane during plasticity, we hypothesized an analogous modality for Cx36. We determined that Cx36 binding to tubulin at the carboxy-terminal domain was distinct from Cx43 and NR2B by binding a motif overlapping with the CaM and CaMKII binding motifs.

A Potential Compensatory Role of Panx3 in the VNO of a Panx1 Knock Out Mouse Model.

Pannexins (Panx) are integral membrane proteins, with Panx1 being the best-characterized member of the protein family. Panx1 is implicated in sensory processing, and knockout (KO) animal models have become the primary tool to investigate the role(s) of Panx1 in sensory systems. Extending previous work from our group on primary olfaction, the expression patterns of Panxs in the vomeronasal organ (VNO), an auxiliary olfactory sense organ with a role in reproduction and social behavior, were compared.

Structural and Functional Consequences of Connexin 36 (Cx36) Interaction with Calmodulin.

Functional plasticity of neuronal gap junctions involves the interaction of the neuronal connexin36 with calcium/calmodulin-dependent kinase II (CaMKII). The important relationship between Cx36 and CaMKII must also be considered in the context of another protein partner, Ca loaded calmodulin, binding an overlapping site in the carboxy-terminus of Cx36. We demonstrate that CaM and CaMKII binding to Cx36 is calcium-dependent, with Cx36 able to engage with CaM outside of the gap junction plaque.

Differential expression of astrocytic connexins in a mouse model of prenatal alcohol exposure.

Maternal alcohol consumption during gestation can cause serious injury to the fetus, and may result in a range of physiological and behavioral impairments, including increased seizure susceptibility, that are collectively termed fetal alcohol spectrum disorder (FASD). The cellular mechanisms underlying increased seizure susceptibility in FASD are not well understood, but could involve altered excitatory coupling of neuronal populations mediated by gap junction proteins. We utilized a mouse model of the prenatal alcohol exposure (PAE) to study the expression pattern of connexin (Cx) major components of gap junctions, and pannexin proteins, which form membrane channels, in the brain of 2-3weeks old PAE and control postnatal offspring.

Investigation of olfactory function in a Panx1 knock out mouse model.

Pannexin 1 (Panx1), the most extensively investigated member of a channel-forming protein family, is able to form pores conducting molecules up to 1.5 kDa, like ATP, upon activation. In the olfactory epithelium (OE), ATP modulates olfactory responsiveness and plays a role in proliferation and differentiation of olfactory sensory neurons (OSNs).

Pannexin1 channel proteins in the zebrafish retina have shared and unique properties.

In mammals, a single pannexin1 gene (Panx1) is widely expressed in the CNS including the inner and outer retinae, forming large-pore voltage-gated membrane channels, which are involved in calcium and ATP signaling. Previously, we discovered that zebrafish lack Panx1 expression in the inner retina, with drPanx1a exclusively expressed in horizontal cells of the outer retina. Here, we characterize a second drPanx1 protein, drPanx1b, generated by whole-genome duplications during teleost evolution.

Molecular diversity of connexin and pannexin genes in the retina of the zebrafish Danio rerio.

Gap junctions are among the most widely distributed cell structures involved in cell-to-cell communication. Recently completed genome sequencing projects including species from all major phyla have demonstrated the existence of three distinct gene families, the connexins, pannexins, and innexins, as molecular building blocks of gap junctional communication. In the present study, the authors have addressed the molecular complexity of gap junction gene expression in the zebrafish retina, a remarkably complex sensory organ built by diverse neuronal subtypes.

Identification and characterization of ZFP-57, a novel zinc finger transcription factor in the mammalian peripheral nervous system.

To isolate new zinc finger genes expressed at early stages of peripheral nerve development, we have used PCR to amplify conserved zinc finger sequences. RNA from rat embryonic day 12 and 13 sciatic nerves, a stage when nerves contain Schwann cell precursors, was used to identify several genes not previously described in Schwann cells. One of them, zinc finger protein (ZFP)-57, proved to be the homologue of a mouse gene found in F9 teratocarcinoma cells.

Molecular cloning and functional expression of zfCx52.6: a novel connexin with hemichannel-forming properties expressed in horizontal cells of the zebrafish retina.

Gap junction-mediated electrical coupling contributes to synchronous oscillatory activities of neurons, and considerable progress has been made in defining the molecular composition of gap junction channels. In particular, cloning and functional expression of gap junction proteins (connexins (Cx)) from zebrafish retina have shown that this part of the brain possesses a high degree of connexin diversity that may account for differential functional properties of electrical synapses. Here, we report the cloning and functional characterization of a new connexin, designated zebrafish Cx52.

Molecular evidence for local denervation of paraspinal muscles in failed-back surgery/postdiscotomy syndrome.

A study was performed to analyze whether local denervation of the medial branch of the dorsal ramus of the lumbar spinal nerve occurs in a patient with postoperative failed-back surgery syndrome/postdiscotomy syndrome (FBSS/PDS). We investigated the effect of the loss of innervation of the multifidus muscle on neuronal nitrite oxide synthetase (n-NOS) and endothelial nitrite oxide synthetase (e-NOS) applying realtime RT-PCR and immunohistochemistry. Our study demonstrates a substantial reduction of n-NOS expression, supporting the view that local denervation of the multifidus is involved in the pathology of FBSS.

Evidence for a role of the N-terminal domain in subcellular localization of the neuronal connexin36 (Cx36).

The expression and functional properties of connexin36 (Cx36) have been investigated in two neuroblastoma cell lines (Neuro2A, RT4-AC) and primary hippocampal neurons transfected with a Cx36-enhanced green fluorescent protein (EGFP) expression vector. Transfected cells express Cx36-EGFP mRNA, and Cx36-EGFP protein is localized in the perinuclear area and cell membrane. Upon differentiation of cell lines, Cx36-EGFP protein was detectable in processes with both axonal and dendritic characteristics.

Loss of HNF1alpha function in human renal cell carcinoma: frequent mutations in the VHL gene but not the HNF1alpha gene.

Human renal cell carcinoma (RCC) is a common malignant disease of the kidney characterized by dedifferentiation of renal epithelial cells. Our previous experiments showed that most RCCs have a loss of function of the tissue-specific transcription factor hepatocyte nuclear factor (HNF) 1alpha. Detailed analyses of the 10 exons encoding HNF1alpha in 32 human RCCs by single-strand conformation polymorphism analysis and direct DNA sequencing revealed no tumor-associated mutation, whereas with the same probes we frequently found mutations in the von Hippel-Lindau tumor suppressor gene.

Helix-loop-helix proteins in Schwann cells: a study of regulation and subcellular localization of Ids, REB, and E12/47 during embryonic and postnatal development.

Although basic helix-loop-helix (bHLH) proteins play an important role in transcriptional control in many cell types, the role of HLH proteins in Schwann cells has yet to be assessed. In this study, we have analyzed the expression of the dominant negative HLH genes, Id1 to Id4 and the class A gene REB, during Schwann cell development. We found that mRNA derived from these genes was present in the Schwann cell lineage throughout development including embryonic precursors and mature cells.

Identification of transcriptionally regulated mRNAs from mouse Schwann cell precursors using modified RNA fingerprinting methods.

We have adopted RNA fingerprinting methods to screen for genes that are rapidly up- or down-regulated during normal mammalian development, comparing mRNA from early (embryo day 12) to late (embryo day 13) mouse Schwann cell precursors. The use of total RNA, a reduction of cDNA template for amplification, the detection of RT-PCR products with a sensitive DNA stain and polyacrylamide gel electrophoresis and rigid selection criteria involving three screening steps are significant improvements on previous methods. Of 19 differentially displayed bands, 15 represented novel genes.

P0 is constitutively expressed in the rat neural crest and embryonic nerves and is negatively and positively regulated by axons to generate non-myelin-forming and myelin-forming Schwann cells, respectively.

We show that in the rat, the major gene of PNS myelin, P0, is expressed long before myelination in the neural crest, Schwann cell precursors, and embryonic Schwann cells irrespective of whether they will myelinate or not. This myelin-independent P0 expression is constitutive and likely to serve as a specific marker for the Schwann cell lineage. The much higher P0 expression accompanying myelination is therefore not new gene expression but strong up-regulation of preexisting basal levels.

Regulation and function of the tissue-specific transcription factor HNF1 alpha (LFB1) during Xenopus development.

We review the data available on the structure, developmental appearance and embryonic regulation of the tissue-specific transcription factor HNF1 alpha (LFB1) in Xenopus. The expression of the HNF1 alpha gene starts early in embryogenesis shortly after mid-blastula transition and the protein accumulates in the region of the embryo where liver, pronephros and gut--tissues that contain HNF1 alpha in the adult--are developing. The cofactor DCoH, known to stabilize dimer formation of HNF1 alpha, is present as a maternal factor in the egg and has a partially distinct tissue distribution compared to HNF1 alpha.

Lowered amounts of the tissue-specific transcription factor LFB1 (HNF1) correlate with decreased levels of glutathione S-transferase alpha messenger RNA in human renal cell carcinoma.

Human renal cell carcinoma is characterized by the loss of differentiation markers such as glutathione S-transferase alpha (GST-alpha). In this paper we show that the promoter of a GST-alpha gene contains a functional binding site for the cell-specific transcription factor LFB1 (HNF1). To investigate the potential role of LFB1 in the down-regulation of GST-alpha expression, we have compared the amount and the binding activity of the LFB1 protein between normal kidney and tumor tissue.

Genomic structure of the Xenopus laevis liver transcription factor LFB1.

Liver factor B1 [LFB1, also called hepatocyte nuclear factor 1 (HNF1)] is a tissue-specific vertebrate transcription factor that is present in the liver, intestine, stomach and kidney. The LFB1 protein contains an unusual homeobox that is characterized by an insertion of 21 amino acids (aa) not found in any other homeodomain protein. We have isolated and characterized the genomic sequences encoding the LFB1 of Xenopus laevis.

Elements and factors involved in tissue-specific and embryonic expression of the liver transcription factor LFB1 in Xenopus laevis.

LFB1 (HNF1) is a tissue-specific transcription factor found in the livers, stomachs, intestines, and kidneys of vertebrates. By analyzing the promoter of the Xenopus LFB1 gene, we identified potential autoregulation by LFB1 and regulation by HNF4, a transcription factor with a tissue distribution similar to that of LFB1. Injection of LFB1 promoter-chloramphenicol acetyltransferase constructs into Xenopus eggs revealed embryonic activation that is restricted to the region of the developing larvae expressing endogeneous LFB1.