Neuron-specific RNA-binding Protein Research Articles

HuD, a Neuronal-specific RNA-binding Protein, Increases thein Vivo Stability of MYCN RNA

MYCN amplification and consequent deregulated expression plays a crucial role in determining the clinical behavior of neuroblastoma. Enhanced expression of MYCN confers growth potential to neuroblastoma cells, and a direct link between MYCN expression and the development of neuroblastoma has been demonstrated in transgenic mice studies. Although the molecular pathways underlying the regulation of MYCN have not been fully elucidated, post-transcriptional mechanisms appear to be important. Previously, we reported that an embryonic lethal abnormal vision-like (ELAV) protein binds with high specificity to at least two AU-rich elements within the MYCN 3'-untranslated region. In this study, we characterized the ability of cis-acting elements within the MYCN 3'-untranslated region to destabilize mRNA in cells and examined the functional consequences of its interactions with the ELAV protein HuD. We show that at least 4 cis-acting elements within the MYCN 3'-untranslated region are able to signal the degradation of stable heterologous mRNA. Ectopic overexpression of HuD dramatically inhibits RNA decay mediated by the full-length MYCN 3'-untranslated region and cis-acting destabilizing elements that harbor HuD binding sites in vivo. HuD may contribute to the malignant phenotype of neuroblastoma cells by stabilizing MYCN mRNA, thereby enhancing steady-state levels of expression of this oncogene.

The neuron-specific RNA-binding protein ELAV regulates neuroglian alternative splicing in neurons and binds directly to its pre-mRNA.

Drosophila melanogaster neural-specific protein, ELAV, has been shown to regulate the neural-specific splicing of three genes: neuroglian (nrg), erect wing, and armadillo. Alternative splicing of the nrg transcript involves alternative inclusion of a 3'-terminal exon. Here, using a minigene reporter, we show that the nrg alternatively spliced intron (nASI) has all the determinants required to recreate proper neural-specific RNA processing seen with the endogenous nrg transcript, including regulation by ELAV. An in vitro UV cross-linking assay revealed that ELAV from nuclear extracts cross-links to four distinct sites along the 3200 nucleotide long nASI; one EXS is positioned at the polypyrimidine tract of the default 3' splice site. ELAV cross-linking sites (EXSs) have in common long tracts of (U)-rich sequence rather than a precise consensus; moreover, each tract has at least two 8/10U elements; their importance is validated by mutant transgene reporter analysis. Further, we propose criteria for ELAV target sequence recognition based on the four EXSs, sites within the nASI that are (U) rich but do not cross-link with ELAV, and predicted EXSs from a phylogenetic comparison with Drosophila virilis nASI. These results suggest that ELAV regulates nrg alternative splicing by direct interaction with the nASI.

Overexpression of HuD Accelerates Neurite Outgrowth and Increases GAP-43 mRNA Expression in Cortical Neurons and Retinoic Acid-Induced Embryonic Stem Cells in Vitro

The neuron-specific RNA-binding protein HuD binds to a U-rich regulatory element of the 3′ untranslated region (3′ UTR) of the GAP-43 mRNA and stabilizes the mRNA. We have previously shown that overexpression of HuD in PC12 cells increases GAP-43 protein expression and induces the spontaneous formation of multiple neurites (K. D. Anderson et al. 2000. J. Neurochem. 75: 1103–1114). In this study, we examined the effects of HuD overexpression on the initial stages of neurite outgrowth and on GAP-43 gene expression using two in vitro systems: E19 rat cortical neurons and retinoic acid (RA)-induced embryonic stem (ES) cells. Normal neurite outgrowth of cortical neurons in vitro occurs over a 3-day period with a concomitant increase in GAP-43 and HuD expression. Cortical cells were infected with a replication-deficient HSV-1 vector containing the HuD cDNA in the sense orientation (HSV-HuD). Overexpression of HuD accelerated the formation of neurites. Immunocytochemical analysis showed that excess HuD resulted in a threefold increase in the number of GAP-43-positive cells undergoing morphological differentiation after 24 h of treatment. Using in situ hybridization, we found that the increased HuD expression resulted in a twofold increase in the levels of GAP-43 mRNA. Similarly, overexpression of HuD in RA-induced embryonic stem cells was found to increase the number of GAP-43-positive cells undergoing process outgrowth. In conclusion, our results demonstrate that HuD functions in the initiation of neurite outgrowth in a manner due, at least in part, to its regulation of GAP-43 expression.

Chemical coding of intrinsic and extrinsic nerves in the guinea pig gallbladder: distributions of PACAP and orphanin FQ.

The complexity of the neural regulation of the gallbladder is reflected by the variety of neuroactive compounds that are found in the intrinsic and extrinsic nerves of the guinea pig gallbladder. The studies reported here used antisera to test for the presence of gallbladder nerves that are immunoreactive for the neuroactive peptides, pituitary adenylyl activating polypeptide (PACAP), and/or orphanin FQ (OFQ, also known as nociceptin). PACAP immunoreactivity was observed in nerve fibers of the paravascular plexus that were also immunoreactive for calcitonin gene-related peptide. These nerve fibers, which are also immunoreactive for substance P, could be followed into the ganglionated plexus. Within the ganglia, a small proportion of neurons was found to be immunoreactive for PACAP; these neurons were also immunoreactive for vasoactive intestinal peptide and nitric oxide synthase. Immunoreactivity for OFQ was observed in the perivascular plexus in nerve fibers that were also immunoreactive for tyrosine hydroxylase. These nerves were previously shown to be immunoreactive for neuropeptide Y. In the ganglionated plexus, immunoreactivity was observed in all gallbladder neurons, as demonstrated by double staining with antiserum directed against the neuron-specific RNA binding protein, Hu. OFQ immunoreactivity was also present in the small catecholaminergic neurons that are observed in a subset of the ganglia. These results further demonstrate the neurotransmitter diversity of the nerves of the gallbladder, and they provide an incentive for studies of the actions of these compounds in the gallbladder wall.

HuD RNA recognition motifs play distinct roles in the formation of a stable complex with AU-rich RNA.

Human neuron-specific RNA-binding protein HuD belongs to the family of Hu proteins and consists of two N-terminal RNA recognition motifs (RRM1 and -2), a hinge region, and a C-terminal RRM (RRM3). Hu proteins can bind to AU-rich elements in the 3' untranslated regions of unstable mRNAs, causing the stabilization of certain transcripts. We have studied the interaction between HuD and prototype mRNA instability elements of the sequence UU(AUUU)(n)AUU using equilibrium methods and real-time kinetics (surface plasmon resonance using a BIACORE). We show that a single molecule of HuD requires at least three AUUU repeats to bind tightly to the RNA. Deletion of RRM1 reduced the K(d) by 2 orders of magnitude and caused a decrease in the association rate and a strong increase in the dissociation rate of the RNA-protein complex, as expected when a critical RNA-binding domain is removed. In contrast, deletion of either RRM2 or -3, which only moderately reduced the affinity, caused marked increases in the association and dissociation rates. The slower binding and stabilization of the complex observed in the presence of all three RRMs suggest that a change in the tertiary structure occurs during binding. The individual RRMs bind poorly to the RNA (RRM1 binds with micromolar affinity, while the affinities of RRM2 and -3 are in the millimolar range). However, the combination of RRM1 and either RRM2 or RRM3 in the context of the protein allows binding with a nanomolar affinity. Thus, the three RRMs appear to cooperate not only to increase the affinity of the interaction but also to stabilize the formed complex. Kinetic effects, similar to those described here, could play a role in RNA binding by many multi-RRM proteins and may influence the competition between proteins for RNA-binding sites and the ability of RNA-bound proteins to be transported intracellularly.

A brain-enriched polypyrimidine tract-binding protein antagonizes the ability of Nova to regulate neuron-specific alternative splicing.

The Nova paraneoplastic antigens are neuron-specific RNA binding proteins that participate in the control of alternative splicing. We have used the yeast two-hybrid system to isolate Nova interacting proteins and identify an RNA binding protein that is closely related to the polypyrimidine tract-binding protein (PTB). The expression of this protein, brPTB, is enriched in the brain, where it is expressed in glia and neurons. brPTB interacts with Nova proteins in cell lines and colocalizes with Nova within neuronal nuclei. We previously found that Nova binds to a pyrimidine-rich RNA element present upstream of an alternatively spliced exon, E3A, in glycine receptor alpha2 (GlyRalpha2) pre-mRNA, and this binding is implicated in Nova-dependent regulation of splicing. Cotransfection assays with a GlyRalpha2 minigene demonstrate that brPTB antagonizes the action of Nova to increase utilization of GlyRalpha2 E3A. brPTB binds to a 90-nt GlyRalpha2 RNA adjacent to the Nova binding site, but with an affinity that is more than 10-fold lower than Nova. When a putative binding site for brPTB on the GlyRalpha2 RNA is mutated, binding is abolished and the inhibitory effect on Nova-dependent exon selection disappears. These results suggest that brPTB is a tissue-restricted RNA binding protein that interacts with and inhibits the ability of Nova to activate exon selection in neurons.

The neuron-enriched splicing pattern of Drosophila erect wing is dependent on the presence of ELAV protein.

Although the Drosophila melanogaster erect wing (ewg) gene is broadly transcribed in adults, an unusual posttranscriptional regulation involving alternative and inefficient splicing generates a 116-kDa EWG protein in neurons, while protein expression elsewhere or of other isoforms is below detection at this stage. This posttranscriptional control is important, as broad expression of EWG can be lethal. In this paper, we show that ELAV, a neuron-specific RNA binding protein, is necessary to regulate EWG protein expression in ELAV-null eye imaginal disc clones and that ELAV is sufficient for EWG expression in wing disc imaginal tissue after ectopic expression. Further, analysis of EWG expression elicited from intron-containing genomic transgenes and cDNA minitransgenes in ELAV-deficient eye discs shows that this regulation is dependent on the presence of ewg introns. Analyses of the ewg splicing patterns in wild-type and ELAV-deficient eye imaginal discs and in wild-type and ectopic ELAV-expressing wing imaginal discs, show that certain neuronal splice isoforms correspond to ELAV levels. The data presented in this paper are consistent with a mechanism in which ELAV increases the splicing efficiency of ewg transcripts in alternatively spliced regions rather than with a mechanism in which stability of specific splice forms is enhanced by ELAV. Additionally, we report that ELAV promotes a neuron-enriched splice isoform of Drosophila armadillo transcript. ELAV, however, is not involved in all neuron-enriched splice events.

Distribution and chemical coding of orphanin FQ/nociceptin-immunoreactive neurons in the myenteric plexus of guinea pig intestines and sphincter of Oddi.

Longitudinal muscle-myenteric plexus preparations of guinea pig intestines and sphincter of Oddi (SO) were immunostained for orphanin FQ/nociceptin. Orphanin FQ-immunoreactive (OFQ-IR) neurons and nerve fibers were relatively abundant in the SO, duodenum, ileum, cecum, and distal colon, with fewer neurons and nerve fibers observed in the proximal colon. Double staining with antibodies directed against the neuron-specific RNA binding protein Hu revealed that while the numbers of OFQ-IR neurons per ganglion decreased along the gut tube, similar proportions (7-9%) of neurons in these regions were OFQ-IR, whereas <1% of the neurons in the proximal colon were OFQ positive. In the ileum, where 8% of the myenteric neurons were OFQ-IR, all OFQ-IR neurons expressed choline acetyltransferase. In addition, multiple-label immunohistochemistry demonstrated that 58% of the OFQ-IR neurons were calretinin-IR, 52% were substance P-IR, and 28% were enkephalin-IR. Nitric oxide synthase immunoreactivity was observed in about 5% of OFQ-IR neurons, or 0.4% of the total population, and a similar proportion of the OFQ-IR neurons was positive for vasoactive intestinal peptide. No OFQ-IR neurons were immunoreactive for calbindin, somatostatin, or serotonin. These results, combined with previous studies of chemical coding and projection patterns in the guinea pig myenteric plexus, indicate that OFQ-IR is expressed preferentially in excitatory motor neurons projecting to the longitudinal and circular muscle layers, as well as a small subgroup of descending interneurons. Because OFQ is expressed by excitatory motor neurons, and because this peptide inhibits excitatory neurotransmission in the guinea pig ileum, it is likely that OFQ acts through a feedback autoinhibitory mechanism.

HuD, a neuronal-specific RNA-binding protein, is a putative regulator of N-myc pre-mRNA processing/stability in malignant human neuroblasts.

N-myc gene copy numbers and transcription rates are similar in N (neuroblastic, tumorigenic) and S (non-neuronal, non-tumorigenic) neuroblastoma cells with chromosomally integrated amplified N-myc genes. However, N cells show significantly higher N-myc mRNA levels than S cells. Therefore, post-transcriptional control of N-myc gene expression must differ between these cell types. Since no differences in N-myc mRNA half-life were found between N and S cells from two cell lines, steady-state levels of N-myc pre-mRNA processing intermediates were analysed. Results suggest that the differences in N-myc expression arise primarily at the nuclear post-transcriptional level. The neuronal-specific RNA-binding Hu proteins are present in cytoplasmic and nuclear fractions of N cells and one of them, HuD, binds specifically to both exonic and intronic N-myc RNA sequences. In sense and antisense HuD-transfected N cells, there are coordinate changes in HuD and N-myc expression levels. Thus, we propose that HuD plays a role in the nuclear processing/stability of N-myc pre-mRNA in N-type neuroblastoma cells.

Cis-Acting regulatory elements in the GAP-43 mRNA 3′-untranslated region can function in trans to suppress endogenous GAP-43 gene expression

The expression of the GAP-43 gene is controlled partly by changes in the stability of its mRNA, a process that is mediated by the interaction of specific sequences in the 3′-untranslated region (3′UTR) with neuronal-specific RNA-binding proteins. Limiting amounts of these trans-acting factors are available in the cell, thus we proposed that overexpression of the GAP-43 3′UTR could affect the levels of the endogenous mRNA via competitive binding to specific RNA-binding proteins. In this study, we show that chronic expression of GAP-43 3′UTR sequences in PC12 cells causes the depletion of the endogenous mRNA and consequent reduction of GAP-43 protein levels. The levels of the mRNAs for c- fos, the amyloid precursor protein (APP) and the microtubule associated protein tau, all three containing similar 3′UTR sequences, were not affected by the treatment. These results thus suggest that the effect of excess GAP-43 3′UTR is specific for its corresponding mRNA. We also used an HSV (herpes simplex virus)-1 vector and a mammalian expression vector with an inducible promoter to acutely express a 10 to 50 fold excess of 3′UTR sequences. Under these conditions, we found that transient expression of the GAP-43 3′UTR was effective in inhibiting both GAP-43 gene expression and neurite outgrowth in nerve growth factor (NGF)-treated PC12 cells and in primary neuronal cultures. These results underscore the role of 3′UTR sequences in the control of GAP-43 gene expression and suggest that overexpression of specific 3′UTR sequences could be used as a potential tool for probing the function of other post-transcriptionally-regulated proteins during neuronal differentiation.

Activation of the Drosophila C3G leads to cell fate changes and overproliferation during development, mediated by the RAS-MAPK pathway and RAP1.

The cellular signal transduction pathways by which C3G, a RAS family guanine nucleotide exchange factor, mediates v-crk transformation are not well understood. Here we report the identification of Drosophila C3G, which, like its human cognate, specifically binds to CRK but not DRK/GRB2 adaptor molecules. During Drosophila development, constitutive membrane binding of C3G, which also occurs during v-crk transformation, results in cell fate changes and overproliferation, mimicking overactivity of the RAS-MAPK pathway. The effects of C3G overactivity can be suppressed by reducing the gene dose of components of the RAS-MAPK pathway and of RAP1. These findings provide the first in vivo evidence that membrane localization of C3G can trigger activation of RAP1 and RAS resulting in the activation of MAPK, one of the hallmarks of v-crk transformation previously thought to be mediated through activation of SOS.

The neuronal RNA-binding protein Nova-2 is implicated as the autoantigen targeted in POMA patients with dementia.

Paraneoplastic opsoclonus myoclonus ataxia (POMA) is a neurologic disorder thought to be mediated by an autoimmune attack against onconeural disease antigens that are expressed by gynecologic or lung tumors and by neurons. One POMA disease antigen, termed Nova-1, has been identified as a neuron-specific KH-type RNA-binding protein. Nova-1 expression is restricted to specific regions of the central nervous system, primarily the hindbrain and ventral spinal cord, which correlate with the predominantly motor symptoms in POMA. However, POMA antisera recognize antigens that are widely expressed in both caudal and rostral regions of the central nervous system, and some patients develop cognitive symptoms. We have used POMA antisera to clone a cDNA encoding a second POMA disease antigen termed Nova-2. Nova-2 is closely related to Nova-1, and is expressed at high levels in neurons during development and in adulthood, and at lower levels in the adult lung. In the postnatal mouse brain, Nova-2 is expressed in a pattern that is largely reciprocal with Nova-1, including high levels of Nova-2 expression in the neocortex and hippocampus. Functional characterization of Nova-2 in RNA selection and nitrocellulose filter-binding assays reveals that Nova-2 binds RNA with high affinity and with sequence specificity that differs from Nova-1. Our results demonstrate that the immune response in POMA targets a family of highly related sequence-specific neuronal RNA-binding proteins. The expression pattern of the Nova-2 protein is likely to underlie the development of cognitive deficits in some POMA patients.

Mouse Chromosomal Locations of Nine Genes Encoding Homologs of Human Paraneoplastic Neurologic Disorder Antigens

The paraneoplastic neurologic disorders (PND) are a rare group of neurologic syndromes that arise when an immune response to systemic tumors expressing neuronal proteins (“onconeural antigens”) develops into an autoimmune neuronal degeneration. The use of patient antisera to clone the genes encoding PND antigens has led to new insight into the mechanism of these autoimmune disorders. The tumor antigens can now be grouped into three classes: (1) neuron-specific RNA-binding proteins, (2) nerve terminal vesicle-associated proteins, and (3) cytoplasmic signaling proteins. To understand better the evolutionary relatedness of these genes and to evaluate them as candidates for inherited neurological disorders, we have determined the mouse chromosomal locations of nine of these genes—Hua, Hub, Huc, Hud, Nova1, Nova2, Natpb, Cdr2,andCdr3.These data suggest that theHua–Hudgenes arose from gene duplication and dispersion, while the other genes are dispersed in the genome. We also predict the chromosomal locations of these genes in human and discuss the potential of these genes as candidates for uncloned mouse and human mutations.

HuD, a neuronal-specific RNA-binding protein, is a potential regulator of MYCN expression in human neuroblastoma cells.

HuD is one of a family of neural antigens recognised by the sera of patients with antibody-associated paraneoplastic encephalomyelitis. Localised exclusively to neurons, these proteins are among the earliest markers of the developing nervous system. Sequence analysis suggests that HuD is an RNA-binding protein. Hu protein levels were determined for the three cell types characterising human neuroblastoma cell lines: sympathoadrenal neuroblasts (N), substrate-adherent Schwann/glial/melanoblastic precursors (S) and stem cells (I) which can give rise to both N and S cells. Western blot analysis showed similar levels of protein in three N-type cell lines; S cells have no detectable Hu protein. Northern blot analysis indicated that N cells express all three Hu genes, HuD, HuC and Hel-N1. N cells, mostly from MYCN-amplified cell lines, have consistently higher steady-state levels of MYCN mRNA than S cell counterparts. Nuclear run-on and mRNA half-life experiments revealed no differences in transcription rate or mRNA stability between N and S cells from the LA-N-1 cell line, implicating differences in post-transcriptional regulation. HuD is postulated to be instrumental in splicing/processing and/or stabilisation of mRNAs involved in cell growth and neuronal differentiation. As determined by gel-mobility shift assays, HuD fusion protein binds to the 3'UTR of human MYCN mRNA. Analysis of HuD deletion mutants has demonstrated that the first and second RNA-recognition motifs (RRMs) are required for binding. Whether HuD regulates MYCN expression and thereby influences tumour aggressiveness is of major interest.

The neuronal RNA binding protein Nova-1 recognizes specific RNA targets in vitro and in vivo.

Nova-1, an autoantigen in paraneoplastic opsoclonus myoclonus ataxia (POMA), a disorder associated with breast cancer and motor dysfunction, is a neuron-specific nuclear RNA binding protein. We have identified in vivo Nova-1 RNA ligands by combining affinity-elution-based RNA selection with protein-RNA immunoprecipitation. Starting with a pool of approximately 10(15) random 52-mer RNAs, we identified long stem-loop RNA ligands that bind to Nova-1 with high affinity (Kd of approximately 2 nM). The loop region of these RNAs harbors a approximately 15-bp pyrimidine-rich element [UCAU(N)(0-2)]3 which is essential for Nova-1 binding. Mutagenesis studies defined the third KH domain of Nova-1 and the [UCAU(N)(0-2)]3 element as necessary for in vitro binding. Consensus [UCAU (N)(0-2)], elements were identified in two neuronal pre-mRNAs, one encoding the inhibitory glycine receptor alpha2 (GlyR alpha2) and a second encoding Nova-1 itself. Nova-1 protein binds these RNAs with high affinity and specificity in vitro, and this binding can be blocked by POMA antisera. Moreover, both Nova-1 and GlyR alpha2 pre-mRNAs specifically coimmunoprecipitated with Nova-1 protein from brain extracts. Thus, Nova-1 functions as a sequence-specific nuclear RNA binding protein in vivo; disruption of the specific interaction between Nova-1 and GlyR alpha2 pre-mRNA may underlie the motor dysfunction seen in POMA.

A Hierarchy of Hu RNA Binding Proteins in Developing and Adult Neurons

The Hu proteins are a group of antigens targeted in an immune-mediated neurodegenerative disorder associated with cancer. We have cloned and characterized four members of the Hu gene family from mouse. We find that the Hu genes encode a large number of alternatively spliced transcripts to produce a series of related neuron-specific RNA binding proteins. Despite this complexity, we have discerned several ordered features of Hu expression. In the embryo, specific Hu genes are expressed in a hierarchy during early neurogenesis. In the E16 developing cortex, mHuB is induced in very early postmitotic neurons exiting the ventricular zone, mHuD is expressed in migrating neurons of the intermediate zone, and mHuC is expressed in mature cortical plate neurons. Such a hierarchy suggests distinct functional roles for each gene in developing neurons. In the adult, all neurons express some set of Hu mRNA and protein. However, specific patterns are evident such that individual neuronal types in the hippocampus, cerebellum, olfactory cortex, neocortex, and elsewhere express from one to several Hu genes. The complexity of potential protein variants within a gene family and of different Hu family members within a neuron suggests a diverse array of function. Given the strong homologies among the Hu proteins, the Drosophila neurogenic gene elav, and the Drosophila splicing factor sxl, we predict that different combinations of Hu proteins determine different neuron-specific aspects of post-transcriptional RNA regulation. Our findings of specific developmental patterns of expression and the correlation between immune targeting of the Hu proteins and adult neurodegenerative disease suggest that the Hu proteins are critical in both the proper development and function of mature neurons.

ANOVA, a putative astrocytic RNA-binding protein gene that maps to chromosome 19q13.3.

Exon amplification from cosmids mapping to the glioma tumor suppressor gene candidate region on chromosome 19q13.3 yielded an exon with high homology to a portion of the NOVA1 gene, which encodes a neuron-specific RNA-binding protein recognized by the paraneoplastic syndrome antibody anti-Ri. Screening of a human brain cDNA library with this exon identified a 1.9 kb cDNA with extensive homology to NOVA1, including three nearly identical KH domains characteristic of a subtype of RNA-binding proteins. Northern blots demonstrated expression of a 2.5 kb mRNA in brain, but in no other tissues. In situ hybridization on human cerebral cortex showed mRNA expression restricted to astrocytes. We have therefore named the gene ANOVA, for astrocytic NOVA1-like gene. Southern blotting and single strand conformation polymorphism analyses did not show tumor-specific alterations of this gene in gliomas and RT-PCR studies showed expression in glioma cell lines, suggesting that ANOVA is not the chromosome 19q glioma tumor suppressor gene. Given that two cloned paraneoplastic antigens are neuronal RNA-binding proteins and that glial proteins may act as paraneoplastic antigens, the ANOVA product may be a target antigen in one of the undefined human paraneoplastic syndromes.

The HuD paraneoplastic protein shares immunogenic regions between PEM/PSN patients and several strains and species of experimental animals

Patients with small-cell carcinoma of the lung and paraneoplastic encephalomyelitis develop antibodies (anti-Hu) against HuD, a member of a family of neuronal specific RNA-binding proteins, which are also expressed by the tumor. In order to determine if the human HuD paraneoplastic antigen shares immunogenic regions between patients and several strains and species of animals, we immunized animals with HuD and several deletion constructs of this protein. All immunized animals developed high titers of anti-HuD antibodies, comparable to the antibody titers found in paraneoplastic patients. Using immunohistochemistry and Western blot analysis of human and mouse cerebral cortex, the pattern of reactivity of these antibodies could not be differentiated from the human paraneoplastic anti-Hu antibodies. None of the immunized animals developed neurologic symptoms. Western blot analysis of HuD deletion constructs demonstrated that the first and second RNA binding domains were the main immunodominant regions, and that the animal immune response was both strain and species dependent.

Zebrafish elav/HuC homologue as a very early neuronal marker

Drosophila ELAV, a neuron-specific RNA binding protein, is expressed in all neurons right after their birth. This specific pattern of expression has led to its use as a pan-neuronal marker. At least three members of the elav family, HuD, HuC/ple21 and Hel-N1, have been reported to be neuron-specific in vertebrates, although it is unknown which member of this family is expressed at the time for early neuronal determination. We have isolated a zebrafish elav/HuC homologue (zHuC) which has 89% homology to human HuC protein. It is first expressed in the neuronal percursor cells in the neural plate immediately after gastrulation, and then high expression levels persist in most regions of the nervous system. HuC, like elav in Drosophila, may be one of the earliest neuronal markers in zebrafish.

Zebrafish elav/HuC homologue as a very early neuronal marker

Drosophila ELAV, a neuron-specific RNA binding protein, is expressed in all neurons right after their birth. This specific pattern of expression has led to its use as a pan-neuronal marker. At least three members of the elav family, HuD, HuC/ple21 and Hel-N1, have been reported to be neuron-specific in vertebrates, although it is unknown which member of this family is expressed at the time for early neuronal determination. We have isolated a zebrafish elav/HuC homologue (zHuC) which has 89% homology to human HuC protein. It is first expressed in the neuronal percursor cells in the neural plate immediately after gastrulation, and then high expression levels persist in most regions of the nervous system. HuC, like elav in Drosophila, may be one of the earliest neuronal markers in zebrafish.