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1.  gdf6a Is Required for Cone Photoreceptor Subtype Differentiation and for the Actions of tbx2b in Determining Rod Versus Cone Photoreceptor Fate 
PLoS ONE  2014;9(3):e92991.
Functional vision restoration is within reach via stem cell therapy, but one of the largest obstacles is the derivation of colour-sensitive cone photoreceptors that are required for high-acuity daytime vision. To enhance progress made using nocturnal murine models, we instead utilize cone-rich zebrafish and herein investigate relationships between gdf6a and tbx2b in cone photoreceptor development. Growth/differentiation factor 6a (gdf6a), a bone morphogenetic protein family ligand, is an emerging factor in photoreceptor degenerative diseases. The T-box transcription factor tbx2b is required to specify UV cone photoreceptor fate instead of rod photoreceptor fate. Interactions between these factors in cone development would be unanticipated, considering the discrete phenotypes in their respective mutants. However, gdf6a positively modulates the abundance of tbx2b transcript during early eye morphogenesis, and we extended this conclusion to later stages of retinal development comprising the times when photoreceptors differentiate. Despite this, gdf6a−/− larvae possess a normal relative number of UV cones and instead present with a low abundance of blue cone photoreceptors, approximately half that of siblings (p<0.001), supporting a differential role for gdf6a amongst the spectral subtypes of cone photoreceptors. Further, gdf6a−/− larvae from breeding of compound heterozygous gdf6a+/−;tbx2b+/− mutants exhibit the recessive lots-of-rods phenotype (which also shows a paucity of UV cones) at significantly elevated rates (44% or 48% for each of two tbx2b alleles, χ2 p≤0.007 for each compared to expected Mendelian 25%). Thus the gdf6a−/− background sensitizes fish such that the recessive lots-of-rods phenotype can appear in heterozygous tbx2b+/− fish. Overall, this work establishes a novel link between tbx2b and gdf6a in determining photoreceptor fates, defining the nexus of an intricate pathway influencing the abundance of cone spectral subtypes and specifying rod vs. cone photoreceptors. Understanding this interaction is a necessary step in the refinement of stem cell-based restoration of daytime vision in humans.
PMCID: PMC3969374  PMID: 24681822
2.  Prepulse Inhibition of the Acoustic Startle Reflex in High Functioning Autism 
PLoS ONE  2014;9(3):e92372.
High functioning autism is an autism spectrum disorder that is characterized by deficits in social interaction and communication as well as repetitive and restrictive behavior while intelligence and general cognitive functioning are preserved. According to the weak central coherence account, individuals with autism tend to process information detail-focused at the expense of global form. This processing bias might be reflected by deficits in sensorimotor gating, a mechanism that prevents overstimulation during the transformation of sensory input into motor action. Prepulse inhibition is an operational measure of sensorimotor gating, which indicates an extensive attenuation of the startle reflex that occurs when a startling pulse is preceded by a weaker stimulus, the prepulse.
In the present study, prepulse inhibition of acoustic startle was compared between 17 adults with high functioning autism and 17 sex-, age-, and intelligence-matched controls by means of electromyography.
Results indicate that participants with high functioning autism exhibited significantly higher startle amplitudes than the control group. However, groups did not differ with regard to PPI or habituation of startle.
These findings challenge the results of two previous studies that reported prepulse inhibition deficits in high-functioning autism and suggest that sensorimotor gating is only impaired in certain subgroups with autism spectrum disorder.
PMCID: PMC3958534  PMID: 24643088
3.  The Role of inab in Axon Morphology of an Identified Zebrafish Motoneuron 
PLoS ONE  2014;9(2):e88631.
The ability of an animal to move and to interact with its environment requires that motoneurons correctly innervate specific muscles. Although many genes that regulate motoneuron development have been identified, our understanding of motor axon branching remains incomplete. We used transcriptional expression profiling to identify potential candidate genes involved in development of zebrafish identified motoneurons. Here we focus on inab, an intermediate filament encoding gene dynamically expressed in a subset of motoneurons as well as in an identified interneuron. We show that inab is necessary for proper axon morphology of a specific motoneuron subtype.
PMCID: PMC3922942  PMID: 24533123
4.  Impaired embryonic motility in dusp27 mutants reveals a developmental defect in myofibril structure 
Disease Models & Mechanisms  2013;7(2):289-298.
An essential step in muscle fiber maturation is the assembly of highly ordered myofibrils that are required for contraction. Much remains unknown about the molecular mechanisms governing the formation of the contractile apparatus. We identified an early embryonic motility mutant in zebrafish caused by integration of a transgene into the pseudophosphatase dual specificity phosphatase 27 (dusp27) gene. dusp27 mutants exhibit near complete paralysis at embryonic and larval stages, producing extremely low levels of spontaneous coiling movements and a greatly diminished touch response. Loss of dusp27 does not prevent somitogenesis but results in severe disorganization of the contractile apparatus in muscle fibers. Sarcomeric structures in mutants are almost entirely absent and only rare triads are observed. These findings are the first to implicate a functional role of dusp27 as a gene required for myofiber maturation and provide an animal model for analyzing the mechanisms governing myofibril assembly.
PMCID: PMC3917250  PMID: 24203884
Zebrafish; dusp27; Motility; Myofibrillogenesis; Muscle
5.  The Nogo-C2/Nogo Receptor Complex Regulates the Morphogenesis of Zebrafish Lateral Line Primordium through Modulating the Expression of dkk1b, a Wnt Signal Inhibitor 
PLoS ONE  2014;9(1):e86345.
The fish lateral line (LL) is a mechanosensory system closely related to the hearing system of higher vertebrates, and it is composed of several neuromasts located on the surface of the fish. These neuromasts can detect changes in external water flow, to assist fish in maintaining a stationary position in a stream. In the present study, we identified a novel function of Nogo/Nogo receptor signaling in the formation of zebrafish neuromasts. Nogo signaling in zebrafish, like that in mammals, involves three ligands and four receptors, as well as three co-receptors (TROY, p75, and LINGO-1). We first demonstrated that Nogo-C2, NgRH1a, p75, and TROY are able to form a Nogo-C2 complex, and that disintegration of this complex causes defective neuromast formation in zebrafish. Time-lapse recording of the CldnB::lynEGFP transgenic line revealed that functional obstruction of the Nogo-C2 complex causes disordered morphogenesis, and reduces rosette formation in the posterior LL (PLL) primordium during migration. Consistent with these findings, hair-cell progenitors were lost from the PLL primordium in p75, TROY, and Nogo-C2/NgRH1a morphants. Notably, the expression levels of pea3, a downstream marker of Fgf signaling, and dkk1b, a Wnt signaling inhibitor, were both decreased in p75, TROY, and Nogo-C2/NgRH1a morphants; moreover, dkk1b mRNA injection could rescue the defects in neuromast formation resulting from knockdown of p75 or TROY. We thus suggest that a novel Nogo-C2 complex, consisting of Nogo-C2, NgRH1a, p75, and TROY, regulates Fgf signaling and dkk1b expression, thereby ensuring stable organization of the PLL primordium.
PMCID: PMC3897714  PMID: 24466042
6.  Properties and Expression of Na+/K+-ATPase α-Subunit Isoforms in the Brain of the Swamp Eel, Monopterus albus, Which Has Unusually High Brain Ammonia Tolerance 
PLoS ONE  2013;8(12):e84298.
The swamp eel, Monopterus albus, can survive in high concentrations of ammonia (>75 mmol l−1) and accumulate ammonia to high concentrations in its brain (∼4.5 µmol g−1). Na+/K+-ATPase (Nka) is an essential transporter in brain cells, and since NH4+ can substitute for K+ to activate Nka, we hypothesized that the brain of M. albus expressed multiple forms of Nka α-subunits, some of which might have high K+ specificity. Thus, this study aimed to clone and sequence the nka α-subunits from the brain of M. albus, and to determine the effects of ammonia exposure on their mRNA expression and overall protein abundance. The effectiveness of NH4+ to activate brain Nka from M. albus and Mus musculus was also examined by comparing their Na+/K+-ATPase and Na+/NH4+-ATPase activities over a range of K+/NH4+ concentrations. The full length cDNA coding sequences of three nkaα (nkaα1, nkaα3a and nkaα3b) were identified in the brain of M. albus, but nkaα2 expression was undetectable. Exposure to 50 mmol l−1 NH4Cl for 1 day or 6 days resulted in significant decreases in the mRNA expression of nkaα1, nkaα3a and nkaα3b. The overall Nka protein abundance also decreased significantly after 6 days of ammonia exposure. For M. albus, brain Na+/NH4+-ATPase activities were significantly lower than the Na+/K+-ATPase activities assayed at various NH4+/K+ concentrations. Furthermore, the effectiveness of NH4+ to activate Nka from the brain of M. albus was significantly lower than that from the brain of M. musculus, which is ammonia-sensitive. Hence, the (1) lack of nkaα2 expression, (2) high K+ specificity of K+ binding sites of Nkaα1, Nkaα3a and Nkaα3b, and (3) down-regulation of mRNA expression of all three nkaα isoforms and the overall Nka protein abundance in response to ammonia exposure might be some of the contributing factors to the high brain ammonia tolerance in M. albus.
PMCID: PMC3877266  PMID: 24391932
7.  Ribbon Synapse Plasticity in the Cochleae of Guinea Pigs after Noise-Induced Silent Damage 
PLoS ONE  2013;8(12):e81566.
Noise exposure at low levels or low doses can damage hair cell afferent ribbon synapses without causing permanent threshold shifts. In contrast to reports in the mouse cochleae, initial damage to ribbon synapses in the cochleae of guinea pigs is largely repairable. In the present study, we further investigated the repair process in ribbon synapses in guinea pigs after similar noise exposure. In the control samples, a small portion of afferent synapses lacked synaptic ribbons, suggesting the co-existence of conventional no-ribbon and ribbon synapses. The loss and recovery of hair cell ribbons and post-synaptic densities (PSDs) occurred in parallel, but the recovery was not complete, resulting in a permanent loss of less than 10% synapses. During the repair process, ribbons were temporally separated from the PSDs. A plastic interaction between ribbons and postsynaptic terminals may be involved in the reestablishment of synaptic contact between ribbons and PSDs, as shown by location changes in both structures. Synapse repair was associated with a breakdown in temporal processing, as reflected by poorer responses in the compound action potential (CAP) of auditory nerves to time-stress signals. Thus, deterioration in temporal processing originated from the cochlea. This deterioration developed with the recovery in hearing threshold and ribbon synapse counts, suggesting that the repaired synapses had deficits in temporal processing.
PMCID: PMC3857186  PMID: 24349090
8.  Deep brain photoreceptors control light seeking behavior in zebrafish larvae 
Current biology : CB  2012;22(21):2042-2047.
Most vertebrates process visual information using elaborately structured photosensory tissues including the eyes and pineal. However there is strong evidence that other tissues can detect and respond to photic stimuli [1, 2, 3]. Many reports suggest that photosensitive elements exist within the brain itself and influence physiology and behavior, however a long standing puzzle has been the identity of the neurons and photoreceptor molecules involved [4, 5]. We tested whether light cues influence behavior in zebrafish larvae through deep brain photosensors. We found that larvae lacking eyes and pineal perform a simple light-seeking behavior triggered by loss of illumination (`dark photokinesis'). Neuroanatomical considerations prompted us to test orthopedia (otpa) deficient fish which showed a profound reduction in dark photokinesis. Using targeted genetic ablations, we narrowed the photosensitive region to neurons in the preoptic area. Neurons in this region express several photoreceptive molecules, but expression of the melanopsin opn4a is selectively lost in otpa mutants, suggesting that opn4a mediates dark photokinesis. Our findings shed light on the identity and function of deep brain photoreceptors and suggest that otpa specifies an ancient population of sensory neurons that mediate behavioral responses to light.
PMCID: PMC3494761  PMID: 23000151
9.  Aldh1-Expressing Endocrine Progenitor Cells Regulate Secondary Islet Formation in Larval Zebrafish Pancreas 
PLoS ONE  2013;8(9):e74350.
Aldh1 expression is known to mark candidate progenitor populations in adult and embryonic mouse pancreas, and Aldh1 enzymatic activity has been identified as a potent regulator of pancreatic endocrine differentiation in zebrafish. However, the location and identity of Aldh1-expressing cells in zebrafish pancreas remain unknown. In this study we demonstrate that Aldh1-expressing cells are located immediately adjacent to 2F11-positive pancreatic ductal epithelial cells, and that their abundance dramatically increases during zebrafish secondary islet formation. These cells also express neurod, a marker of endocrine progenitor cells, but do not express markers of more mature endocrine cells such as pax6b or insulin. Using formal cre/lox-based lineage tracing, we further show that Aldh1-expressing pancreatic epithelial cells are the direct progeny of pancreatic notch-responsive progenitor cells, identifying them as a critical intermediate between multi-lineage progenitors and mature endocrine cells. Pharmacologic manipulation of Aldh1 enzymatic activity accelerates cell entry into the Aldh1-expressing endocrine progenitor pool, and also leads to the premature maturation of these cells, as evidenced by accelerated pax6b expression. Together, these findings suggest that Aldh1-expressing cells act as both participants and regulators of endocrine differentiation during zebrafish secondary islet formation.
PMCID: PMC3798260  PMID: 24147152
10.  Weight-Bearing Locomotion in the Developing Opossum, Monodelphis domestica following Spinal Transection: Remodeling of Neuronal Circuits Caudal to Lesion 
PLoS ONE  2013;8(8):e71181.
Complete spinal transection in the mature nervous system is typically followed by minimal axonal repair, extensive motor paralysis and loss of sensory functions caudal to the injury. In contrast, the immature nervous system has greater capacity for repair, a phenomenon sometimes called the infant lesion effect. This study investigates spinal injuries early in development using the marsupial opossum Monodelphis domestica whose young are born very immature, allowing access to developmental stages only accessible in utero in eutherian mammals. Spinal cords of Monodelphis pups were completely transected in the lower thoracic region, T10, on postnatal-day (P)7 or P28 and the animals grew to adulthood. In P7-injured animals regrown supraspinal and propriospinal axons through the injury site were demonstrated using retrograde axonal labelling. These animals recovered near-normal coordinated overground locomotion, but with altered gait characteristics including foot placement phase lags. In P28-injured animals no axonal regrowth through the injury site could be demonstrated yet they were able to perform weight-supporting hindlimb stepping overground and on the treadmill. When placed in an environment of reduced sensory feedback (swimming) P7-injured animals swam using their hindlimbs, suggesting that the axons that grew across the lesion made functional connections; P28-injured animals swam using their forelimbs only, suggesting that their overground hindlimb movements were reflex-dependent and thus likely to be generated locally in the lumbar spinal cord. Modifications to propriospinal circuitry in P7- and P28-injured opossums were demonstrated by changes in the number of fluorescently labelled neurons detected in the lumbar cord following tracer studies and changes in the balance of excitatory, inhibitory and neuromodulatory neurotransmitter receptors’ gene expression shown by qRT-PCR. These results are discussed in the context of studies indicating that although following injury the isolated segment of the spinal cord retains some capability of rhythmic movement the mechanisms involved in weight-bearing locomotion are distinct.
PMCID: PMC3741377  PMID: 23951105
11.  Does the Hearing Sensitivity in Thorny Catfishes Depend on Swim Bladder Morphology? 
PLoS ONE  2013;8(6):e67049.
Thorny catfishes exhibit large variations in swim bladder morphology. These organs are of different sizes, forms and may have simple or branched diverticula. The swim bladder plays an important role in otophysans because it enhances their hearing sensitivity by transmitting sound pressure fluctuations via ossicles to the inner ear.
Methodology/Principal Findings
To investigate if a form-function relationship exists, the swim bladder morphology and hearing ability were analyzed in six species. The morphology was quantified by measuring the length, width and height and calculating a standardized swim bladder length (sSBL), which was then used to calculate the relative swim bladder length (rSBL). Hearing was measured using the auditory evoked potential (AEP) recording technique. Two species had simple apple-shaped and four species heart-shaped (cordiform) bladders. One of the latter species had short unbranched diverticula on the terminal margin, two had a secondary bladder and two had many long, branched diverticula. The rSBL differed significantly between most of the species. All species were able to detect frequencies between 70 Hz and 6 kHz, with lowest thresholds found between 0.5 and 1 kHz (60 dB re 1 µPa). Hearing curves were U-shaped except in Hemidoras morrisi in which it was ramp-like. Mean hearing thresholds of species possessing smaller rSBLs were slightly lower (maximum 8.5 dB) than those of species having larger rSBLs.
The current findings reveal a relationship between swim bladder form and its function among thorny catfishes. Relatively smaller swim bladders resulted in relatively better hearing. This is in contrast to a prior inter-familial study on catfishes in which species with large unpaired bladders possessed higher sensitivity at higher frequencies than species having tiny paired and encapsulated bladders.
PMCID: PMC3692464  PMID: 23825615
12.  Two ENU-Induced Alleles of Atp2b2 Cause Deafness in Mice 
PLoS ONE  2013;8(6):e67479.
Over 120 loci are known to cause inherited hearing loss in humans. The deafness gene has been identified for only half of these loci. With the aim of identifying some of the remaining deafness genes, we performed an ethylnitrosourea mutagenesis screen for deaf mice. We isolated two mutants with semi-dominant hearing loss, Deaf11 and Deaf13. Both contained causative mutations in Atp2b2, which encodes the plasma membrane calcium ATPase 2. The Atp2b2Deaf11 mutation leads to a p. I1023S substitution in the tenth transmembrane domain. The Atp2b2Deaf13 mutation leads to a p. R561S substitution in the catalytic core. Mice homozygous for these mutations display profound hearing loss. Heterozygotes display mild to moderate, progressive hearing loss.
PMCID: PMC3691321  PMID: 23826306
13.  Mutations in vacuolar H+-ATPase subunits lead to biliary developmental defects in zebrafish 
Developmental Biology  2012;365(2):434-444.
We identified three zebrafish mutants with defects in biliary development. One of these mutants, pekin (pn), also demonstrated generalized hypopigmentation and other defects, including disruption of retinal cell layers, lack of zymogen granules in the pancreas, and dilated Golgi in intestinal epithelial cells. Bile duct cells in pn demonstrated an accumulation of electron dense bodies. We determined that the causative defect in pn was a splice site mutation in the atp6ap2 gene that leads to an inframe stop codon. atp6ap2 encodes a subunit of the vacuolar H+-ATPase (V-H+-ATPase), which modulates pH in intracellular compartments. The Atp6ap2 subunit has also been shown to function as an intracellular renin receptor that stimulates fibrogenesis. Here we show that mutants and morphants involving other V-H+-ATPase subunits also demonstrated developmental biliary defects, but did not demonstrate the inhibition of fibrogenic genes observed in pn. The defects in pn are reminiscent of those we and others have observed in class C VPS (vacuolar protein sorting) family mutants and morphants, and we report here that knockdown of atp6ap2 and vps33b had an additive negative effect on biliary development. Our findings suggest that pathways important in modulating intracompartmental pH lead to defects in digestive organ development, and support previous studies demonstrating the importance of intracellular sorting pathways in biliary development.
PMCID: PMC3337356  PMID: 22465374
14.  The dorsal raphe modulates sensory responsiveness during arousal in zebrafish 
During waking behavior animals adapt their state of arousal in response to environmental pressures. Sensory processing is regulated in aroused states and several lines of evidence imply that this is mediated at least partly by the serotonergic system. However there is little information directly showing that serotonergic function is required for state-dependent modulation of sensory processing. Here we find that zebrafish larvae can maintain a short-term state of arousal during which neurons in the dorsal raphe modulate sensory responsiveness to behaviorally relevant visual cues. Following a brief exposure to water flow, larvae show elevated activity and heightened sensitivity to perceived motion. Calcium imaging of neuronal activity after flow revealed increased activity in serotonergic neurons of the dorsal raphe. Genetic ablation of these neurons abolished the increase in visual sensitivity during arousal without affecting baseline visual function or locomotor activity. We traced projections from the dorsal raphe to a major visual area, the optic tectum. Laser ablation of the tectum demonstrated that this structure, like the dorsal raphe, is required for improved visual sensitivity during arousal. These findings reveal that serotonergic neurons of the dorsal raphe have a state-dependent role in matching sensory responsiveness to behavioral context.
PMCID: PMC3535275  PMID: 23100441
15.  Quantitative Trait Loci for Interhemispheric Commissure Development and Social Behaviors in the BTBR T+ tf/J Mouse Model of Autism 
PLoS ONE  2013;8(4):e61829.
Autism and Agenesis of the Corpus Callosum (AgCC) are interrelated behavioral and anatomic phenotypes whose genetic etiologies are incompletely understood. We used the BTBR T+ tf/J (BTBR) strain, exhibiting fully penetrant AgCC, a diminished hippocampal commissure, and abnormal behaviors that may have face validity to autism, to study the genetic basis of these disorders.
We generated 410 progeny from an F2 intercross between the BTBR and C57BL/6J strains. The progeny were phenotyped for social behaviors (as juveniles and adults) and commisural morphology, and genotyped using 458 markers. Quantitative trait loci (QTL) were identified using genome scans; significant loci were fine-mapped, and the BTBR genome was sequenced and analyzed to identify candidate genes.
Six QTL meeting genome-wide significance for three autism-relevant behaviors in BTBR were identified on chromosomes 1, 3, 9, 10, 12, and X. Four novel QTL for commissural morphology on chromosomes 4, 6, and 12 were also identified. We identified a highly significant QTL (LOD score = 20.2) for callosal morphology on the distal end of chromosome 4.
We identified several QTL and candidate genes for both autism-relevant traits and commissural morphology in the BTBR mouse. Twenty-nine candidate genes were associated with synaptic activity, axon guidance, and neural development. This is consistent with a role for these processes in modulating white matter tract development and aspects of autism-relevant behaviors in the BTBR mouse. Our findings reveal candidate genes in a mouse model that will inform future human and preclinical studies of autism and AgCC.
PMCID: PMC3626795  PMID: 23613947
16.  Validation of the Zebrafish Pentylenetetrazol Seizure Model: Locomotor versus Electrographic Responses to Antiepileptic Drugs 
PLoS ONE  2013;8(1):e54166.
Zebrafish have recently emerged as an attractive in vivo model for epilepsy. Seven-day-old zebrafish larvae exposed to the GABAA antagonist pentylenetetrazol (PTZ) exhibit increased locomotor activity, seizure-like behavior, and epileptiform electrographic activity. A previous study showed that 12 out of 13 antiepileptic drugs (AEDs) suppressed PTZ-mediated increases in larval movement, indicating the potential utility of zebrafish as a high-throughput in vivo model for AED discovery. However, a question remained as to whether an AED-induced decrease in locomotion is truly indicative of anticonvulsant activity, as some drugs may impair larval movement through other mechanisms such as general toxicity or sedation. We therefore carried out a study in PTZ-treated zebrafish larvae, to directly compare the ability of AEDs to inhibit seizure-like behavioral manifestations with their capacity to suppress epileptiform electrographic activity. We re-tested the 13 AEDs of which 12 were previously reported to inhibit convulsions in the larval movement tracking assay, administering concentrations that did not, on their own, impair locomotion. In parallel, we carried out open-field recordings on larval brains after treatment with each AED. For the majority of AEDs we obtained the same response in both the behavioral and electrographic assays. Overall our data correlate well with those reported in the literature for acute rodent PTZ tests, indicating that the larval zebrafish brain is more discriminatory than previously thought in its response to AEDs with different modes of action. Our results underscore the validity of using the zebrafish larval locomotor assay as a rapid first-pass screening tool in assessing the anticonvulsant and/or proconvulsant activity of compounds, but also highlight the importance of performing adequate validation when using in vivo models.
PMCID: PMC3544809  PMID: 23342097
17.  Evolution and Functional Characterisation of Melanopsins in a Deep-Sea Chimaera (Elephant Shark, Callorhinchus milii) 
PLoS ONE  2012;7(12):e51276.
Non-visual photoreception in mammals is primarily mediated by two splice variants that derive from a single melanopsin (OPN4M) gene, whose expression is restricted to a subset of retinal ganglion cells. Physiologically, this sensory system regulates the photoentrainment of many biological rhythms, such as sleep via the melatonin endocrine system and pupil constriction. By contrast, melanopsin exists as two distinct lineages in non-mammals, opn4m and opn4x, and is broadly expressed in a wide range of tissue types, including the eye, brain, pineal gland and skin. Despite these findings, the evolution and function of melanopsin in early vertebrates are largely unknown. We, therefore, investigated the complement of opn4 classes present in the genome of a model deep-sea cartilaginous species, the elephant shark (Callorhinchus milii), as a representative vertebrate that resides at the base of the gnathostome (jawed vertebrate) lineage. We reveal that three melanopsin genes, opn4m1, opn4m2 and opn4x, are expressed in multiple tissues of the elephant shark. The two opn4m genes are likely to have arisen as a result of a lineage-specific duplication, whereas “long” and “short” splice variants are generated from a single opn4x gene. By using a heterologous expression system, we suggest that these genes encode functional photopigments that exhibit both “invertebrate-like” bistable and classical “vertebrate-like” monostable biochemical characteristics. We discuss the evolution and function of these melanopsin pigments within the context of the diverse photic and ecological environments inhabited by this chimaerid holocephalan, as well as the origin of non-visual sensory systems in early vertebrates.
PMCID: PMC3522658  PMID: 23251480
18.  The α-Tocopherol Transfer Protein Is Essential for Vertebrate Embryogenesis 
PLoS ONE  2012;7(10):e47402.
The hepatic α-tocopherol transfer protein (TTP) is required for optimal α-tocopherol bioavailability in humans; mutations in the human TTPA gene result in the heritable disorder ataxia with vitamin E deficiency (AVED, OMIM #277460). TTP is also expressed in mammalian uterine and placental cells and in the human embryonic yolk-sac, underscoring TTP’s significance during fetal development. TTP and vitamin E are essential for productive pregnancy in rodents, but their precise physiological role in embryogenesis is unknown. We hypothesize that TTP is required to regulate delivery of α-tocopherol to critical target sites in the developing embryo. We tested to find if TTP is essential for proper vertebrate development, utilizing the zebrafish as a non-placental model. We verify that TTP is expressed in the adult zebrafish and its amino acid sequence is homologous to the human ortholog. We show that embryonic transcription of TTP mRNA increases >7-fold during the first 24 hours following fertilization. In situ hybridization demonstrates that Ttpa transcripts are localized in the developing brain, eyes and tail bud at 1-day post fertilization. Inhibiting TTP expression using oligonucleotide morpholinos results in severe malformations of the head and eyes in nearly all morpholino-injected embryos (88% compared with 5.6% in those injected with control morpholinos or 1.7% in non-injected embryos). We conclude that TTP is essential for early development of the vertebrate central nervous system.
PMCID: PMC3471827  PMID: 23077608
19.  A Three-Dimensional Stereotaxic MRI Brain Atlas of the Cichlid Fish Oreochromis mossambicus 
PLoS ONE  2012;7(9):e44086.
The African cichlid Oreochromis mossambicus (Mozambique tilapia) has been used as a model system in a wide range of behavioural and neurobiological studies. The increasing number of genetic tools available for this species, together with the emerging interest in its use for neurobiological studies, increased the need for an accurate hodological mapping of the tilapia brain to supplement the available histological data. The goal of our study was to elaborate a three-dimensional, high-resolution digital atlas using magnetic resonance imaging, supported by Nissl staining. Resulting images were viewed and analysed in all orientations (transverse, sagittal, and horizontal) and manually labelled to reveal structures in the olfactory bulb, telencephalon, diencephalon, optic tectum, and cerebellum. This high resolution tilapia brain atlas is expected to become a very useful tool for neuroscientists using this fish model and will certainly expand their use in future studies regarding the central nervous system.
PMCID: PMC3439461  PMID: 22984463
20.  Relationship between Swim Bladder Morphology and Hearing Abilities–A Case Study on Asian and African Cichlids 
PLoS ONE  2012;7(8):e42292.
Several teleost species have evolved anterior extensions of the swim bladder which come close to or directly contact the inner ears. A few comparative studies have shown that these morphological specializations may enhance hearing abilities. This study investigates the diversity of swim bladder morphology in four Asian and African cichlid species and analyzes how this diversity affects their hearing sensitivity.
Methodology/Principal Findings
We studied swim bladder morphology by dissections and by making 3D reconstructions from high-resolution microCT scans. The auditory sensitivity was determined in terms of sound pressure levels (SPL) and particle acceleration levels (PAL) using the auditory evoked potential (AEP) recording technique. The swim bladders in Hemichromis guttatus and Steatocranus tinanti lacked anterior extensions and the swim bladder was considerably small in the latter species. In contrast, Paratilapia polleni and especially Etroplus maculatus possessed anterior extensions bringing the swim bladder close to the inner ears. All species were able to detect frequencies up to 3 kHz (SPL) except S. tinanti which only responded to frequencies up to 0.7 kHz. P. polleni and E. maculatus showed significantly higher auditory sensitivities at 0.5 and 1 kHz than the two species lacking anterior swim bladder extensions. The highest auditory sensitivities were found in E. maculatus, which possessed the most intimate swim bladder-inner ear relationship (maximum sensitivity 66 dB re 1 µPa at 0.5 kHz).
Our results indicate that anterior swim bladder extensions seem to improve mean absolute auditory sensitivities by 21–42 dB (SPLs) and 21–36 dB (PALs) between 0.5 and 1 kHz. Besides anterior extensions, the size of the swim bladder appears to be an important factor for extending the detectable frequency range (up to 3 kHz).
PMCID: PMC3413697  PMID: 22879934
21.  Distinct retinal pathways drive spatial orientation behaviors in zebrafish navigation 
Current biology : CB  2010;20(4):381-386.
PMCID: PMC3412192  PMID: 20153194
navigation; phototaxis; orienting; behavioral choice; optic tectum; motor; control; zebrafish
22.  Mutation of pescadillo Disrupts Oligodendrocyte Formation in Zebrafish 
PLoS ONE  2012;7(2):e32317.
In vertebrates, the myelin sheath is essential for efficient propagation of action potentials along the axon shaft. Oligodendrocytes are the cells of the central nervous system that create myelin sheaths. During embryogenesis, ventral neural tube precursors give rise to oligodendrocyte progenitor cells, which divide and migrate throughout the central nervous system. This study aimed to investigate mechanisms that regulate oligodendrocyte progenitor cell formation.
Methodology/Principal Findings
By conducting a mutagenesis screen in transgenic zebrafish, we identified a mutation, designated vu166, by an apparent reduction in the number of oligodendrocyte progenitor cells in the dorsal spinal cord. We subsequently determined that vu166 is an allele of pescadillo, a gene known to play a role in ribosome biogenesis and cell proliferation. We found that pescadillo function is required for both the proper number of oligodendrocyte progenitors to form, by regulating cell cycle progression, and for normal levels of myelin gene expression.
Our data provide evidence that neural precursors require pes function to progress through the cell cycle and produce oligodendrocyte progenitor cells and for oligodendrocyte differentiation.
PMCID: PMC3285679  PMID: 22384214
23.  Modeling Activity and Target-Dependent Developmental Cell Death of Mouse Retinal Ganglion Cells Ex Vivo 
PLoS ONE  2012;7(2):e31105.
Programmed cell death is widespread during the development of the central nervous system and serves multiple purposes including the establishment of neural connections. In the mouse retina a substantial reduction of retinal ganglion cells (RGCs) occurs during the first postnatal week, coinciding with the formation of retinotopic maps in the superior colliculus (SC). We previously established a retino-collicular culture preparation which recapitulates the progressive topographic ordering of RGC projections during early post-natal life. Here, we questioned whether this model could also be suitable to examine the mechanisms underlying developmental cell death of RGCs. Brn3a was used as a marker of the RGCs. A developmental decline in the number of Brn3a-immunolabelled neurons was found in the retinal explant with a timing that paralleled that observed in vivo. In contrast, the density of photoreceptors or of starburst amacrine cells increased, mimicking the evolution of these cell populations in vivo. Blockade of neural activity with tetrodotoxin increased the number of surviving Brn3a-labelled neurons in the retinal explant, as did the increase in target availability when one retinal explant was confronted with 2 or 4 collicular slices. Thus, this ex vivo model reproduces the developmental reduction of RGCs and recapitulates its regulation by neural activity and target availability. It therefore offers a simple way to analyze developmental cell death in this classic system. Using this model, we show that ephrin-A signaling does not participate to the regulation of the Brn3a population size in the retina, indicating that eprhin-A-mediated elimination of exuberant projections does not involve developmental cell death.
PMCID: PMC3281910  PMID: 22363559
24.  Brain selective transgene expression in zebrafish using an NRSE derived motif 
Transgenic technologies enable the manipulation and observation of circuits controlling behavior by permitting expression of genetically encoded reporter genes in neurons. Frequently though, neuronal expression is accompanied by transgene expression in non-neuronal tissues, which may preclude key experimental manipulations, including assessment of the contribution of neurons to behavior by ablation. To better restrict transgene expression to the nervous system in zebrafish larvae, we have used DNA sequences derived from the neuron-restrictive silencing element (NRSE). We find that one such sequence, REx2, when used in conjunction with several basal promoters, robustly suppresses transgene expression in non-neuronal tissues. Both in transient transgenic experiments and in stable enhancer trap lines, suppression is achieved without compromising expression within the nervous system. Furthermore, in REx2 enhancer trap lines non-neuronal expression can be de-repressed by knocking down expression of the NRSE binding protein RE1-silencing transcription factor (Rest). In one line, we show that the resulting pattern of reporter gene expression coincides with that of the adjacent endogenous gene, hapln3. We demonstrate that three common basal promoters are susceptible to the effects of the REx2 element, suggesting that this method may be useful for confining expression from many other promoters to the nervous system. This technique enables neural specific targeting of reporter genes and thus will facilitate the use of transgenic methods to manipulate circuit function in freely behaving larvae.
PMCID: PMC3531662  PMID: 23293587
transgenesis; enhancer trap; ablation; NRSE; rest; zebrafish
25.  Adeno-Associated Virus-Mediated Rescue of the Cognitive Defects in a Mouse Model for Angelman Syndrome 
PLoS ONE  2011;6(12):e27221.
Angelman syndrome (AS), a genetic disorder occurring in approximately one in every 15,000 births, is characterized by severe mental retardation, seizures, difficulty speaking and ataxia. The gene responsible for AS was discovered to be UBE3A and encodes for E6-AP, an ubiquitin ligase. A unique feature of this gene is that it undergoes maternal imprinting in a neuron-specific manner. In the majority of AS cases, there is a mutation or deletion in the maternally inherited UBE3A gene, although other cases are the result of uniparental disomy or mismethylation of the maternal gene. While most human disorders characterized by severe mental retardation involve abnormalities in brain structure, no gross anatomical changes are associated with AS. However, we have determined that abnormal calcium/calmodulin-dependent protein kinase II (CaMKII) regulation is seen in the maternal UBE3A deletion AS mouse model and is responsible for the major phenotypes. Specifically, there is an increased αCaMKII phosphorylation at the autophosphorylation sites Thr286 and Thr305/306, resulting in an overall decrease in CaMKII activity. CaMKII is not produced until after birth, indicating that the deficits associated with AS are not the result of developmental abnormalities. The present studies are focused on exploring the potential to rescue the learning and memory deficits in the adult AS mouse model through the use of an adeno-associated virus (AAV) vector to increase neuronal UBE3A expression. These studies show that increasing the levels of E6-AP in the brain using an exogenous vector can improve the cognitive deficits associated with AS. Specifically, the associative learning deficit was ameliorated in the treated AS mice compared to the control AS mice, indicating that therapeutic intervention may be possible in older AS patients.
PMCID: PMC3235088  PMID: 22174738

Results 1-25 (31)