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1.  Mutations in vacuolar H+-ATPase subunits lead to biliary developmental defects in zebrafish 
Developmental Biology  2012;365(2):434-444.
Summary
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.
doi:10.1016/j.ydbio.2012.03.009
PMCID: PMC3337356  PMID: 22465374
2.  Plexin A3 and Turnout Regulate Motor Axonal Branch Morphogenesis in Zebrafish 
PLoS ONE  2013;8(1):e54071.
During embryogenesis motor axons navigate to their target muscles, where individual motor axons develop complex branch morphologies. The mechanisms that control axonal branching morphogenesis have been studied intensively, yet it still remains unclear when branches begin to form or how branch locations are determined. Live cell imaging of individual zebrafish motor axons reveals that the first axonal branches are generated at the ventral extent of the myotome via bifurcation of the growth cone. Subsequent branches are generated by collateral branching restricted to their synaptic target field along the distal portion of the axon. This precisely timed and spatially restricted branching process is disrupted in turnout mutants we identified in a forward genetic screen. Molecular genetic mapping positioned the turnout mutation within a 300 kb region encompassing eight annotated genes, however sequence analysis of all eight open reading frames failed to unambiguously identify the turnout mutation. Chimeric analysis and single cell labeling reveal that turnout function is required cell non-autonomously for intraspinal motor axon guidance and peripheral branch formation. turnout mutant motor axons form the first branch on time via growth cone bifurcation, but unlike wild-type they form collateral branches precociously, when the growth cone is still navigating towards the ventral myotome. These precocious collateral branches emerge along the proximal region of the axon shaft typically devoid of branches, and they develop into stable, permanent branches. Furthermore, we find that null mutants of the guidance receptor plexin A3 display identical motor axon branching defects, and time lapse analysis reveals that precocious branch formation in turnout and plexin A3 mutants is due to increased stability of otherwise short-lived axonal protrusions. Thus, plexin A3 dependent intrinsic and turnout dependent extrinsic mechanisms suppress collateral branch morphogenesis by destabilizing membrane protrusions before the growth cone completes navigation into the synaptic target field.
doi:10.1371/journal.pone.0054071
PMCID: PMC3549987  PMID: 23349787
3.  A Novel Role for Lh3 Dependent ECM Modifications during Neural Crest Cell Migration in Zebrafish 
PLoS ONE  2013;8(1):e54609.
During vertebrate development, trunk neural crest cells delaminate along the entire length of the dorsal neural tube and initially migrate as a non-segmented sheet. As they enter the somites, neural crest cells rearrange into spatially restricted segmental streams. Extracellular matrix components are likely to play critical roles in this transition from a sheet-like to a stream-like mode of migration, yet the extracellular matrix components and their modifying enzymes critical for this transition are largely unknown. Here, we identified the glycosyltransferase Lh3, known to modify extracellular matrix components, and its presumptive substrate Collagen18A1, to provide extrinsic signals critical for neural crest cells to transition from a sheet-like migration behavior to migrating as a segmental stream. Using live cell imaging we show that in lh3 null mutants, neural crest cells fail to transition from a sheet to a stream, and that they consequently enter the somites as multiple streams, or stall shortly after entering the somites. Moreover, we demonstrate that transgenic expression of lh3 in a small subset of somitic cells adjacent to where neural crest cells switch from sheet to stream migration restores segmental neural crest cell migration. Finally, we show that knockdown of the presumptive Lh3 substrate Collagen18A1 recapitulates the neural crest cell migration defects observed in lh3 mutants, consistent with the notion that Lh3 exerts its effect on neural crest cell migration by regulating post-translational modifications of Collagen18A1. Together these data suggest that Lh3–Collagen18A1 dependent ECM modifications regulate the transition of trunk neural crest cells from a non-segmental sheet like migration mode to a segmental stream migration mode.
doi:10.1371/journal.pone.0054609
PMCID: PMC3548841  PMID: 23349938
4.  The Tumor Suppressor Gene Retinoblastoma-1 Is Required for Retinotectal Development and Visual Function in Zebrafish 
PLoS Genetics  2012;8(11):e1003106.
Mutations in the retinoblastoma tumor suppressor gene (rb1) cause both sporadic and familial forms of childhood retinoblastoma. Despite its clinical relevance, the roles of rb1 during normal retinotectal development and function are not well understood. We have identified mutations in the zebrafish space cadet locus that lead to a premature truncation of the rb1 gene, identical to known mutations in sporadic and familial forms of retinoblastoma. In wild-type embryos, axons of early born retinal ganglion cells (RGC) pioneer the retinotectal tract to guide later born RGC axons. In rb1 deficient embryos, these early born RGCs show a delay in cell cycle exit, causing a transient deficit of differentiated RGCs. As a result, later born mutant RGC axons initially fail to exit the retina, resulting in optic nerve hypoplasia. A significant fraction of mutant RGC axons eventually exit the retina, but then frequently project to the incorrect optic tectum. Although rb1 mutants eventually establish basic retinotectal connectivity, behavioral analysis reveals that mutants exhibit deficits in distinct, visually guided behaviors. Thus, our analysis of zebrafish rb1 mutants reveals a previously unknown yet critical role for rb1 during retinotectal tract development and visual function.
Author Summary
Before an organism can execute necessary behavioral responses to environmental stimuli, the underlying neural circuits that regulate these behaviors must be precisely wired during embryonic development. A properly wired neural circuit is the product of a sophisticated collaboration of multiple genetic pathways that orchestrate cell type specification, the extension and growth of the cell processes that connect each circuit component, and the refinement of these connections. In an unbiased genetic screen designed to identify the genes required for proper circuit formation in developing zebrafish embryos, we identified a human disease causing mutation in the retinoblastoma-1 (rb1) gene that disrupts the formation of the zebrafish visual circuit. rb1 canonically functions to regulate the cell cycle, and when mutated the loss of rb1-mediated cell cycle control elicits childhood ocular tumor formation. Genetic models of rb1 have been developed to study the developmental role of rb1 in the retina; however, ectopic cell proliferation and death within the retina have largely precluded the ability to evaluate the formation and integrity of neural circuits connecting the retina with the brain. In this study, through genetic and cellular analysis of a zebrafish rb1 mutant, we reveal a novel role for rb1 in regulating the establishment and functionality of the visual circuitry.
doi:10.1371/journal.pgen.1003106
PMCID: PMC3510048  PMID: 23209449
5.  Zebrafish neurofibromatosis type 1 genes have redundant functions in tumorigenesis and embryonic development 
Disease Models & Mechanisms  2012;5(6):881-894.
SUMMARY
Neurofibromatosis type 1 (NF1) is a common, dominantly inherited genetic disorder that results from mutations in the neurofibromin 1 (NF1) gene. Affected individuals demonstrate abnormalities in neural-crest-derived tissues that include hyperpigmented skin lesions and benign peripheral nerve sheath tumors. NF1 patients also have a predisposition to malignancies including juvenile myelomonocytic leukemia (JMML), optic glioma, glioblastoma, schwannoma and malignant peripheral nerve sheath tumors (MPNSTs). In an effort to better define the molecular and cellular determinants of NF1 disease pathogenesis in vivo, we employed targeted mutagenesis strategies to generate zebrafish harboring stable germline mutations in nf1a and nf1b, orthologues of NF1. Animals homozygous for loss-of-function alleles of nf1a or nf1b alone are phenotypically normal and viable. Homozygous loss of both alleles in combination generates larval phenotypes that resemble aspects of the human disease and results in larval lethality between 7 and 10 days post fertilization. nf1-null larvae demonstrate significant central and peripheral nervous system defects. These include aberrant proliferation and differentiation of oligodendrocyte progenitor cells (OPCs), dysmorphic myelin sheaths and hyperplasia of Schwann cells. Loss of nf1 contributes to tumorigenesis as demonstrated by an accelerated onset and increased penetrance of high-grade gliomas and MPNSTs in adult nf1a+/−; nf1b−/−; p53e7/e7 animals. nf1-null larvae also demonstrate significant motor and learning defects. Importantly, we identify and quantitatively analyze a novel melanophore phenotype in nf1-null larvae, providing the first animal model of the pathognomonic pigmentation lesions of NF1. Together, these findings support a role for nf1a and nf1b as potent tumor suppressor genes that also function in the development of both central and peripheral glial cells as well as melanophores in zebrafish.
doi:10.1242/dmm.009779
PMCID: PMC3484870  PMID: 22773753
6.  Survival motor neuron (SMN) affects plastin 3 protein levels leading to motor defects 
The Journal of Neuroscience  2012;32(15):5074-5084.
The actin binding protein plastin 3 (PLS3) has been identified as a modifier of the human motoneuron disease spinal muscular atrophy (SMA). SMA is caused by decreased levels of the survival motor neuron protein (SMN) and in its most severe form causes death in infants and young children. To understand the mechanism of PLS3 in SMA, we have analyzed pls3 RNA and protein in zebrafish smn mutants. We show that Pls3 protein levels are severely decreased in smn−/− mutants without a reduction in pls3 mRNA levels. Moreover, we show that both pls3 mRNA and protein stability are unaffected when Smn is reduced. This indicates that SMN affects PLS3 protein production. We had previously shown that in smn mutants, the presynaptic protein SV2 is decreased at neuromuscular junctions (NMJs). Transgenically driving human PLS3 in motoneurons rescues the decrease in SV2 expression. To determine whether PLS3 could also rescue function, we performed behavioral analysis on smn mutants and found that they had a significant decrease in spontaneous swimming and turning. Driving PLS3 transgenically in motoneurons rescued both of these defects. These data show that PLS3 protein levels are dependent on SMN and that PLS3 is able to rescue the neuromuscular defects and corresponding movement phenotypes caused by low levels of Smn suggesting that decreased PLS3 contributes to SMA motor phenotypes.
doi:10.1523/JNEUROSCI.5808-11.2012
PMCID: PMC3355766  PMID: 22496553
7.  Netrin/DCC signaling guides olfactory sensory axons to their correct location in the olfactory bulb 
The Journal of Neuroscience  2012;32(13):4440-4456.
Olfactory sensory neurons expressing particular olfactory receptors project to specific reproducible locations within the bulb. The axonal guidance cues that organize this precise projection pattern are only beginning to be identified. To aid in their identification and characterization, we generated a transgenic zebrafish line, OR111-7:IRES:Gal4, in which a small subset of olfactory sensory neurons is labeled. Most sensory neurons expressing the OR111-7 transgene project to a specific location within the bulb, the central zone protoglomerulus, while a smaller number project to the LG1 protoglomerulus. Inhibiting netrin/DCC signaling perturbs the ability of OR111-7 expressing axons to enter the olfactory bulb and alters their patterns of termination within the bulb. The netrin receptor DCC is expressed in olfactory sensory neurons around the time that they elaborate their axons, netrin1a is expressed near the medial-most margin of the olfactory bulb, and netrin1b is expressed within the ventral region of the bulb. Loss of netrin/DCC signaling components causes some OR111-7 expressing sensory axons to wander posteriorly after exiting the olfactory pit, away from netrin expressing areas in the bulb. OR111-7 expressing axons that enter the bulb target the central zone less precisely than normal, spreading away from netrin expressing regions. These pathfinding errors can be corrected by the re-expression of DCC within OR111-7 transgene expressing neurons in DCC morphant embryos. These findings implicate netrins as the only known attractants for olfactory sensory neurons, first drawing OR111-7 expressing axons into the bulb and then into the ventromedially positioned central zone protoglomerulus.
doi:10.1523/JNEUROSCI.4442-11.2012
PMCID: PMC3356094  PMID: 22457493
8.  In vivo nerve-macrophage interactions following peripheral nerve injury 
The Journal of Neuroscience  2012;32(11):3898-3909.
In vertebrates, the peripheral nervous system has retained its regenerative capacity, enabling severed axons to reconnect with their original synaptic targets. While it is well documented that a favorable environment is critical for nerve regeneration, the complex cellular interactions between injured nerves with cells in their environment, as well as the functional significance of these interactions, have not been determined in vivo and in real time. Here we provide the first minute-by-minute account of cellular interactions between laser transected motor nerves and macrophages in live intact zebrafish. We show that macrophages arrive at the lesion site long before axon fragmentation, much earlier than previously thought. Moreover, we find that axon fragmentation triggers macrophage invasion into the nerve to engulf axonal debris, and that delaying nerve fragmentation in a Wlds model does not alter macrophage recruitment but induces a previously unknown ‘nerve scanning’ behavior, suggesting that macrophage recruitment and subsequent nerve invasion are controlled by separate mechanisms. Finally, we demonstrate that macrophage recruitment, thought to be dependent on Schwann cell derived signals, occurs independently of Schwann cells. Thus, live cell imaging defines novel cellular and functional interactions between injured nerves and immune cells.
doi:10.1523/JNEUROSCI.5225-11.2012
PMCID: PMC3324096  PMID: 22423110
zebrafish; peripheral nerve; motor axon; Wallerian degeneration; regeneration; Schwann cells; phagocytes; macrophage; Spi1; startle response
9.  Distinct retinal pathways drive spatial orientation behaviors in zebrafish navigation 
Current biology : CB  2010;20(4):381-386.
doi:10.1016/j.cub.2010.01.022
PMCID: PMC3412192  PMID: 20153194
navigation; phototaxis; orienting; behavioral choice; optic tectum; motor; control; zebrafish
10.  Molecular-Genetic Mapping of Zebrafish Mutants with Variable Phenotypic Penetrance 
PLoS ONE  2011;6(10):e26510.
Forward genetic screens in vertebrates are powerful tools to generate models relevant to human diseases, including neuropsychiatric disorders. Variability in phenotypic penetrance and expressivity is common in these disorders and behavioral mutant models, making their molecular-genetic mapping a formidable task. Using a ‘phenotyping by segregation’ strategy, we molecularly map the hypersensitive zebrafish houdini mutant despite its variable phenotypic penetrance, providing a generally applicable strategy to map zebrafish mutants with subtle phenotypes.
doi:10.1371/journal.pone.0026510
PMCID: PMC3198425  PMID: 22039502
11.  Determining the function of zebrafish epithalamic asymmetry 
As in many fishes, amphibians and reptiles, the epithalamus of the zebrafish, Danio rerio, develops with pronounced left–right (L–R) asymmetry. For example, in more than 95 per cent of zebrafish larvae, the parapineal, an accessory to the pineal organ, forms on the left side of the brain and the adjacent left habenular nucleus is larger than the right. Disruption of Nodal signalling affects this bias, producing equal numbers of larvae with the parapineal on the left or the right side and corresponding habenular reversals. Pre-selection of live larvae using fluorescent transgenic reporters provides a useful substrate for studying the effects of neuroanatomical asymmetry on behaviour. Previous studies had suggested that epithalamic directionality is correlated with lateralized behaviours such as L–R eye preference. We find that the randomization of epithalamic asymmetry, through perturbation of the nodal-related gene southpaw, does not alter a variety of motor behaviours, including responses to lateralized stimuli. However, we discovered significant deficits in swimming initiation and in the total distance navigated by larvae with parapineal reversals. We discuss these findings with respect to previous studies and recent work linking the habenular region with control of the motivation/reward pathway of the vertebrate brain.
doi:10.1098/rstb.2008.0234
PMCID: PMC2666080  PMID: 19064346
habenula; brain asymmetry; behaviour
12.  Wnt signals organize synaptic prepattern and axon guidance through the zebrafish unplugged/MuSK receptor 
Neuron  2009;61(5):721-733.
SUMMARY
Early during neuromuscular development acetylcholine receptors (AChRs) accumulate at the center of muscle fibers, precisely where motor growth cones navigate and synapses eventually form. Here, we show that Wnt11r binds to the zebrafish unplugged/MuSK ectodomain to organize this central muscle zone. In the absence of such zone, prepatterned AChRs fail to aggregate and, as visualized by live cell imaging, growth cones stray from their central path. Using inducible unplugged/MuSK transgenes we show that organization of the central muscle zone is dispensable for the formation of neural synapses, but essential for AChR prepattern and motor growth cone guidance. Finally, we show that blocking non-canonical dishevelled signaling in muscle fibers disrupts AChR prepatterning and growth cone guidance. We propose that Wnt ligands activate unplugged/MuSK signaling in muscle fibers to restrict growth cone guidance and AChR prepatterns to the muscle center through a mechanism reminiscent of the planar cell polarity pathway.
doi:10.1016/j.neuron.2008.12.025
PMCID: PMC2671566  PMID: 19285469
axonal guidance; motoneuron; synaptogenesis; muscle specific kinase; wnt11; neuromuscular junction; unplugged; zebrafish
13.  Temporal and Spatial Requirements of unplugged/MuSK Function during Zebrafish Neuromuscular Development 
PLoS ONE  2010;5(1):e8843.
One of the earliest events in neuromuscular junction (NMJ) development is the accumulation of acetylcholine receptor (AChR) at the center of muscle cells. The unplugged/MuSK (muscle specific tyrosine kinase) gene is essential to initiate AChR clustering but also to restrict approaching growth cones to the muscle center, thereby coordinating pre- and postsynaptic development. To determine how unplugged/MuSK signaling coordinates these two processes, we examined the temporal and spatial requirements of unplugged/MuSK in zebrafish embryos using heat-shock inducible transgenes. Here, we show that despite its expression in muscle cells from the time they differentiate, unplugged/MuSK activity is first required just prior to the appearance of AChR clusters to simultaneously induce AChR accumulation and to guide motor axons. Furthermore, we demonstrate that ectopic expression of unplugged/MuSK throughout the muscle membrane results in wildtype-like AChR prepattern and neuromuscular synapses in the central region of muscle cells. We propose that AChR prepatterning and axonal guidance are spatio-temporally coordinated through common unplugged/MuSK signals, and that additional factor(s) restrict unplugged/MuSK signaling to a central muscle zone critical for establishing mid-muscle synaptogenesis.
doi:10.1371/journal.pone.0008843
PMCID: PMC2809748  PMID: 20107509
14.  The neurogenetic frontier—lessons from misbehaving zebrafish 
One of the central questions in neuroscience is how refined patterns of connectivity in the brain generate and monitor behavior. Genetic mutations can influence neural circuits by disrupting differentiation or maintenance of component neuronal cells or by altering functional patterns of nervous system connectivity. Mutagenesis screens therefore have the potential to reveal not only the molecular underpinnings of brain development and function, but to illuminate the cellular basis of behavior. Practical considerations make the zebrafish an organism of choice for undertaking forward genetic analysis of behavior. The powerful array of experimental tools at the disposal of the zebrafish researcher makes it possible to link molecular function to neuronal properties that underlie behavior. This review focuses on specific challenges to isolating and analyzing behavioral mutants in zebrafish.
doi:10.1093/bfgp/eln039
PMCID: PMC2722256  PMID: 18836206
zebrafish; behavior; mutagenesis
15.  Unidirectional startle responses and disrupted left-right coordination of motor behaviors in robo3 mutant zebrafish 
Genes, brain, and behavior  2009;8(5):500-511.
The Roundabout (Robo) family of receptors and their Slit ligands play well-established roles in axonal guidance, including in humans where horizontal gaze palsy with progressive scoliosis (HGPPS) is caused by mutations in the robo3 gene. While significant progress has been made towards understanding the mechanism by which Robo receptors establish commissural projections in the central nervous system, less is known about how these projections contribute to neural circuits mediating behavior. Here we report cloning of the zebrafish behavioral mutant twitch twice and show that twitch twice encodes robo3. We demonstrate that in mutant hindbrains the axons of an identified pair of neurons, the Mauthner cells, fail to cross the midline. The Mauthner neurons are essential for the startle response, and in twitch twice/robo3 mutants misguidance of the Mauthner axons results in a unidirectional startle response. Moreover, we show that twitch twice mutants exhibit normal visual acuity but display defects in horizontal eye movements, suggesting a specific and critical role for twitch twice/robo3 in sensory guided behavior.
doi:10.1111/j.1601-183X.2009.00499.x
PMCID: PMC2752477  PMID: 19496826
robo3; zebrafish; behavior; axon guidance; Mauthner cell; optokinetic response
16.  Genomic structure and embryonic expression of zebrafish lysyl hydroxylase 1 and lysyl hydroxylase 2 
Collagen biosynthesis in both invertebrates and vertebrates is critically dependent upon the activity of lysyl hydroxylase (LH) enzymes. In humans, mutations in the genes encoding LH1 and LH2 have been shown to cause two distinct connective tissue disorders, Ehlers-Danlos (Type VIA) and Bruck syndromes. While the biochemical properties of these enzymes have been intensively studied, their embryonic patterns of expression and developmental roles remain unknown. We now present the cloning and analyses of the genes encoding LH1 and LH2 in the zebrafish, Danio rerio. We find these genes to be similarly organized to other vertebrate lh (plod) genes, including the presence of an alternatively spliced exon in lh2. We also examine the mRNA expression patterns of lh1 and lh2 during embryogenesis and find them to exhibit unique and dynamic patterns of expression. These results strongly suggest that LH enzymes are not merely housekeeping enzymes, but play distinct developmental roles. The identification of these genes in the zebrafish, a genetic model organism whose development is well characterized, now provides the basis for the establishment of the first animal models for both Ehlers-Danlos (Type VIA) and Bruck syndromes.
doi:10.1016/j.matbio.2006.09.007
PMCID: PMC1828209  PMID: 17056240
zebrafish; lysyl hydroxylase; lh; plod; extracellular matrix; collagen

Results 1-16 (16)