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Whereas the zebrafish retina has long been an important model system for developmental and genetic studies, little is known about the responses of the inner retinal neurons. Here we report single-unit ganglion cell recordings from 5- to 6-day-old zebrafish larvae. In wild-type larvae we identify at least five subtypes of ganglion cell responses to full-field illumination, with ON-OFF and ON-type cells predominating. In the nrc mutant retina, in which the photoreceptor terminals develop abnormally, we observe normal OFF responses but abnormal ON-OFF responses and no ON responses. Previously characterized as blind, these mutants lack an optokinetic reflex (OKR), but in another behavioral assay nrc mutant fish have near-normal responses to the offset of light and slow and sluggish responses to the onset of light. Pharmacological block of the ON pathway mimics most of the nrc visual defects. We conclude that the abnormal photoreceptor terminals in nrc mutants predominantly perturb the ON pathway and that the ON pathway is necessary to drive the OKR in larval zebrafish.

The development of the embryonic vascular system into a highly ordered network requires precise control over the migration and branching of endothelial cells (ECs). We have previously identified angiomotin (Amot) as a receptor for the angiogenesis inhibitor angiostatin. Furthermore, DNA vaccination targeting Amot inhibits angiogenesis and tumor growth. However, little is known regarding the role of Amot in physiological angiogenesis. We therefore investigated the role of Amot in embryonic neovascularization during zebrafish and mouse embryogenesis. Here we report that knockdown of Amot in zebrafish reduced the number of filopodia of endothelial tip cells and severely impaired the migration of intersegmental vessels. We further show that 75% of Amot knockout mice die between embryonic day 11 (E11) and E11.5 and exhibit severe vascular insufficiency in the intersomitic region as well as dilated vessels in the brain. Furthermore, using ECs differentiated from embryonic stem (ES) cells, we demonstrate that Amot-deficient cells have intact response to vascular endothelial growth factor (VEGF) in regard to differentiation and proliferation. However, the chemotactic response to VEGF was abolished in Amot-deficient cells. We provide evidence that Amot is important for endothelial polarization during migration and that Amot controls Rac1 activity in endothelial and epithelial cells. Our data demonstrate a critical role for Amot during vascular patterning and endothelial polarization.

Huntington's disease (HD) is one of nine neurodegenerative disorders caused by expansion of CAG repeats encoding polyglutamine in their respective, otherwise apparently unrelated proteins. Despite these proteins having widespread and overlapping expression patterns in the brain, a specific and unique subset of neurons exhibits particular vulnerability in each disease. It has been hypothesized that perturbation of normal protein function contributes to the specificity of neuronal vulnerability; however, the normal biological functions of many of these proteins including the HD gene product, Huntingtin (Htt), are unclear. To explore the roles of Htt, we have used antisense morpholino oligonucleotides to observe the effects of Htt deficiency in early zebrafish development. Knockdown of Htt expression resulted in a variety of developmental defects. Most notably, Htt-deficient zebrafish had hypochromic blood due to decreased hemoglobin production, despite the presence of iron within blood cells. Furthermore, transferrin receptor 1 transcripts were increased, suggesting cellular iron starvation. Provision of iron to the cytoplasm in a bio-available form restored hemoglobin production in Htt-deficient embryos. Since erythroid cells acquire iron via receptor-mediated endocytosis of transferrin, these results suggest a role for Htt in making endocytosed iron accessible for cellular utilization. Iron is required for oxidative energy production, and defects in iron homeostasis and energy metabolism are features of HD pathogenesis that are most pronounced in the major region of neurodegeneration. It is therefore plausible that perturbation of Htt's normal role in the iron pathway (by polyglutamine tract expansion) contributes to HD pathology, and particularly to its neuronal specificity.

The Nodal-related subgroup of the TGFbeta superfamily of secreted cytokines regulates the specification of the mesodermal and endodermal germ layers during gastrulation. Two Nodal-related proteins - Squint (Sqt) and Cyclops (Cyc) - are expressed during germ-layer specification in zebrafish. Genetic sqt mutant phenotypes have defined a variable requirement for zygotic Sqt, but not for maternal Sqt, in midline mesendoderm development. However a comparison of phenotypes arising from oocytes or zygotes injected with Sqt antisense morpholinos has suggested a novel requirement for maternal Sqt in dorsal specification. In this study we examined maternal-zygotic mutants for each of two sqt alleles and we also compared phenotypes of closely related zygotic and maternal-zygotic sqt mutants. Each of these approaches indicated there is no general requirement for maternal Sqt. To better understand the dispensability of maternal and zygotic Sqt, we sought out developmental contexts that more rigorously demand intact Sqt signalling. We found that sqt penetrance is influenced by genetic modifiers, by environmental temperature, by levels of residual Activin-like activity and by Heat-Shock Protein 90 (HSP90) activity. Therefore, Sqt may confer an evolutionary advantage by protecting early-stage embryos against detrimental interacting alleles and environmental challenges.

Zebrafish Csrp1 is a member of the cysteine- and glycine-rich protein (CSRP) family and is expressed in the mesendoderm and its derivatives. Csrp1 interacts with Dishevelled 2 (Dvl2) and Diversin (Div), which control cell morphology and other dynamic cell behaviors via the noncanonical Wnt and JNK pathways. When csrp1 message is knocked down, abnormal convergent extension cell movement is induced, resulting in severe deformities in midline structures. In addition, cardiac bifida is induced as a consequence of defects in cardiac mesoderm cell migration. Our data highlight Csrp1 as a key molecule of the noncanonical Wnt pathway, which orchestrates cell behaviors during dynamic morphogenetic movements of tissues and organs.

During skeletal muscle differentiation, the actomyosin motor is assembled into myofibrils, multiprotein machines that generate and transmit force to cell ends. How expression of muscle proteins is coordinated to build the myofibril is unknown. Here we show that zebrafish Mef2d and Mef2c proteins are required redundantly for assembly of myosin-containing thick filaments in nascent muscle fibres, but not for the earlier steps of skeletal muscle fibre differentiation, elongation, fusion or thin filament gene expression. mef2d mRNA and protein is present in myoblasts, whereas mef2c expression commences in muscle fibres. Knockdown of both Mef2s with antisense morpholino oligonucleotides or in mutant fish blocks muscle function and prevents sarcomere assembly. Cell transplantation and heat-shock-driven rescue reveal a cell-autonomous requirement for Mef2 within fibres. In nascent fibres, Mef2 drives expression of genes encoding thick, but not thin, filament proteins. Among genes analysed, myosin heavy and light chains and myosin-binding protein C require Mef2 for normal expression, whereas actin, tropomyosin and troponin do not. Our findings show that Mef2 controls skeletal muscle formation after terminal differentiation and define a new maturation step in vertebrate skeletal muscle development at which thick filament gene expression is controlled.

Left-right (LR) asymmetry is regulated by early asymmetric signals within the embryo. Even though the role of the bone morphogenetic protein (BMP) pathway in this process has been reported extensively in various model organisms, opposing models for the mechanism by which BMP signaling operates still prevail. Here we show that in zebrafish embryos there are two distinct phases during LR patterning in which BMP signaling is required. Using transgenic lines that ectopically express either noggin3 or bmp2b, we show a requirement for BMP signaling during early segmentation to repress southpaw expression in the right lateral plate mesoderm and regulate both visceral and heart laterality. A second phase was identified during late segmentation, when BMP signaling is required in the left lateral plate mesoderm to regulate left-sided gene expression and heart laterality. Using morpholino knock down experiments, we identified Bmp4 as the ligand responsible for both phases of BMP signaling. In addition, we detected bmp4 expression in Kupffer's vesicle and show that restricted knock down of bmp4 in this structure results in LR patterning defects. The identification of these two distinct and opposing activities of BMP signaling provides new insight into how BMP signaling can regulate LR patterning.

In the skin of zebrafish embryo, the vacuolar H(+)-ATPase (V-ATPase, H(+) pump) distributed mainly in the apical membrane of H(+)-pump-rich cells, which pump internal acid out of the embryo and function similarly to acid-secreting intercalated cells in mammalian kidney. In addition to acid excretion, the electrogenic H(+) efflux via the H(+)-ATPases in the gill apical membrane of freshwater fish was proposed to act as a driving force for Na(+) entry through the apical Na(+) channels. However, convincing molecular physiological evidence in vivo for this model is still lacking. In this study, we used morpholino-modified antisense oligonucleotides to knockdown the gene product of H(+)-ATPase subunit A (atp6v1a) and examined the phenotype of the mutants. The H(+)-ATPase knockdown embryos revealed several abnormalities, including suppression of acid-secretion from skin, growth retardation, trunk deformation, and loss of internal Ca(2+) and Na(+). This finding reveals the critical role of H(+)-ATPase in embryonic acid -secretion and ion balance, as well.

Background: Identifying molecular pathways regulating the development of pacemaking and coordinated heartbeat is crucial for a comprehensive mechanistic understanding of arrhythmia-related diseases. Elucidation of these pathways has been complicated mainly by an insufficient definition of the developmental structures involved in these processes and the unavailability of animal models specifically targeting the relevant tissues. Here, we report on a highly restricted expression pattern of the homeodomain transcription factor Shox2 in the sinus venosus myocardium, including the sinoatrial nodal region and the venous valves., Methods and Results: To investigate its function in vivo, we have generated mouse lines carrying a targeted mutation of the Shox2 gene. Although heterozygous animals did not exhibit obvious defects, homozygosity of the targeted allele led to embryonic lethality at 11.5 to 13.5 dpc. Shox2-/- embryos exhibited severe hypoplasia of the sinus venosus myocardium in the posterior heart field, including the sinoatrial nodal region and venous valves. We furthermore demonstrate aberrant expression of connexin 40 and connexin 43 and the transcription factor Nkx2.5 in vivo specifically within the sinoatrial nodal region and show that Shox2 deficiency interferes with pacemaking function in zebrafish embryos., Conclusions: From these results, we postulate a critical function of Shox2 in the recruitment of sinus venosus myocardium comprising the sinoatrial nodal region.

In most eukaryotes, recombination of homologous chromosomes during meiosis is necessary for proper chromosome pairing and subsequent segregation. The molecular mechanisms of meiosis are still relatively unknown, but numerous genes are known to be involved, among which are many mismatch repair genes. One of them, mlh1, colocalizes with presumptive sites of crossing over, but its exact action remains unclear. We studied meiotic processes in a knockout line for mlh1 in zebrafish. Male mlh1 mutants are sterile and display an arrest in spermatogenesis at metaphase I, resulting in increased testis weight due to accumulation of prophase I spermatocytes. In contrast, females are fully fertile, but their progeny shows high rates of dysmorphology and mortality within the first days of development. SNP-based chromosome analysis shows that this is caused by aneuploidy, resulting from meiosis I chromosomal missegregation. Surprisingly, the small percentage of progeny that develops normally has a complete triploid genome, consisting of both sets of maternal and one set of paternal chromosomes. As adults, these triploid fish are infertile males with wild-type appearance. The frequency of triploid progeny of mlh1 mutant females is much higher than could be expected for random chromosome segregation. Together, these results show that multiple solutions exist for meiotic crossover/segregation problems.

We identified a novel secreted protein, fibin, in zebrafish, mice and humans. We inhibited its function in zebrafish embryos by injecting antisense fibin morpholino oligonucleotides. A knockdown of fibin function in zebrafish resulted in no pectoral fin bud initiation and abolished the expression of tbx5, which is involved in the specification of pectoral fin identification. The lack of pectoral fins in fibin-knockdown embryos was partially rescued by injection of fibin RNA. fibin was expressed in the lateral plate mesoderm of the presumptive pectoral fin bud regions. Its expression region was adjacent to that of tbx5. fibin expression temporally preceded tbx5 expression in presumptive pectoral fin bud regions, and not abolished in tbx5-knockdown presumptive fin bud regions. In contrast, fibin expression was abolished in retinoic acid signaling-inhibited or wnt2b-knockdown presumptive fin bud regions. These results indicate that fibin is a secreted signal essential for pectoral fin bud initiation in that it potentially acts downstream of retinoic acid and wnt signaling and is essential for tbx5 expression. The present findings have revealed a novel secreted lateral plate mesoderm signal essential for fin initiation in the lateral plate mesoderm.

The transcription factors Scl and Lmo2 are crucial for development of all blood. An important early requirement for Scl in endothelial development has also been revealed recently in zebrafish embryos, supporting previous findings in scl(-/-) embryoid bodies. Scl depletion culminates most notably in failure of dorsal aorta formation, potentially revealing a role in the formation of hemogenic endothelium. We now present evidence that the requirements for Lmo2 in zebrafish embryos are essentially the same as for Scl. The expression of important hematopoietic regulators is lost, reduced, or delayed, panendothelial gene expression is down-regulated, and aorta-specific marker expression is lost. The close similarity of the phenotypes for Scl and Lmo2 suggest that they perform these early functions in hemangioblast development within a multiprotein complex, as shown for erythropoiesis. Consistent with this, we find that scl morphants cannot be rescued by a non-Lmo2-binding form of Scl but can be rescued by non-DNA-binding forms, suggesting tethering to target genes through DNA-binding partners linked via Lmo2. Interestingly, unlike other hematopoietic regulators, the Scl/Lmo2 complex does not appear to autoregulate, as neither gene's expression is affected by depletion of the other. Thus, expression of these critical regulators is dependent on continued expression of upstream regulators, which may include cell-extrinsic signals.

The transcriptional Mediator (MED) is a multiprotein complex that transmits information from transcription factors to RNA polymerase II (PolII) to regulate transcription. At present, the role of distinct MED subunits in general transcription versus transcription stimulated by specific signaling pathways is unclear. By means of positional cloning, we reveal that the zebrafish mutant tennismatch is a hypomorphic allele of Med10, a conserved MED middle domain subunit. Using morpholino antisense oligonucleotides, we further demonstrate that reduction of Med10 levels led to an enhancement of the Wnt signaling pathway, while also suggesting a role for Med10 in mediating the Nodal signaling pathway. In contrast to the dual roles of Med10, reduction of Med12 and Med13 levels, two MED subunits in the regulatory domain, led to an enhancement of the Wnt signaling pathway but not the Nodal pathway, while reduction of Med15 levels, a MED subunit in the tail domain, suppressed the Nodal signaling pathway but not the Wnt signaling pathway. Thus, Med10 appears to be a unique MED subunit that differentially transduces information from distinct signaling pathways during zebrafish embryogenesis.

Mutations in DJ-1 lead to early onset Parkinson's disease (PD). The aim of this study was to elucidate further the underlying mechanisms leading to neuronal cell death in DJ-1 deficiency in vivo and determine whether the observed cell loss could be prevented pharmacologically. Inactivation of DJ-1 in zebrafish, Danio rerio, resulted in loss of dopaminergic neurons after exposure to hydrogen peroxide and the proteasome inhibitor MG132. DJ-1 knockdown by itself already resulted in increased p53 and Bax expression levels prior to toxin exposure without marked neuronal cell death, suggesting subthreshold activation of cell death pathways in DJ-1 deficiency. Proteasome inhibition led to a further increase of p53 and Bax expression with widespread neuronal cell death. Pharmacological p53 inhibition either before or during MG132 exposure in vivo prevented dopaminergic neuronal cell death in both cases. Simultaneous knockdown of DJ-1 and the negative p53 regulator mdm2 led to dopaminergic neuronal cell death even without toxin exposure, further implicating involvement of p53 in DJ-1 deficiency-mediated neuronal cell loss. Our study demonstrates the utility of zebrafish as a new animal model to study PD gene defects and suggests that modulation of downstream mechanisms, such as p53 inhibition, may be of therapeutic benefit.

Recent evidence indicates that growing blood-vessel sprouts consist of endothelial cells with distinct cell fates and behaviours; however, it is not clear what signals determine these sprout cell characteristics. Here we show that Notch signalling is necessary to restrict angiogenic cell behaviour to tip cells in developing segmental arteries in the zebrafish embryo. In the absence of the Notch signalling component Rbpsuh (recombining binding protein suppressor of hairless) we observed excessive sprouting of segmental arteries, whereas Notch activation suppresses angiogenesis. Through mosaic analysis we find that cells lacking Rbpsuh preferentially localize to the terminal position in developing sprouts. In contrast, cells in which Notch signalling has been activated are excluded from the tip-cell position. In vivo time-lapse analysis reveals that endothelial tip cells undergo a stereotypical pattern of proliferation and migration during sprouting. In the absence of Notch, nearly all sprouting endothelial cells exhibit tip-cell behaviour, leading to excessive numbers of cells within segmental arteries. Furthermore, we find that flt4 (fms-related tyrosine kinase 4, also called vegfr3) is expressed in segmental artery tip cells and becomes ectopically expressed throughout the sprout in the absence of Notch. Loss of flt4 can partially restore normal endothelial cell number in Rbpsuh-deficient segmental arteries. Finally, loss of the Notch ligand dll4 (delta-like 4) also leads to an increased number of endothelial cells within segmental arteries. Together, these studies indicate that proper specification of cell identity, position and behaviour in a developing blood-vessel sprout is required for normal angiogenesis, and implicate the Notch signalling pathway in this process.

The genetic study of titin has been notoriously difficult because of its size and complicated alternative splicing routes. Here, we have used zebrafish as an animal model to investigate the functions of individual titin isoforms. We identified 2 titin orthologs in zebrafish, ttna and ttnb, and annotated the full-length genomic sequences for both genes. We found that ttna, but not ttnb, is required for sarcomere assembly in the heart as well as the subsequent establishment of cardiac contractility. In fact, ttna is the earliest sarcomeric mRNA that is expressed in the heart, which makes it an early molecular marker for cardiomyocyte differentiation. Surprisingly, ttna is required for later steps of sarcomere assembly, including the assembly of Z-discs and A-bands, but not for early steps such as the assembly of Z-bodies and nonstriated myosin filaments. Reduction of individual titin isoforms in vivo using morpholino-modified antisense oligonucleotides indicated that (1) both N2B exon-containing and N2A exon-containing isoforms of ttna are required for sarcomere assembly in the heart; (2) N2A exon-containing isoforms of both ttna and ttnb are required for sarcomere assembly in the somites; and (3) the N2B exon-containing isoforms of ttnb are expressed later than other titin isoforms and are probably involved in modulating their expression; however, these isoforms of ttnb are not required for sarcomere assembly. Collectively, our results reveal distinct functions of different titin isoforms and suggest that various phenotypes in "titinopathies" may be attributable to the disruption of different titin isoforms.

The organizer, the signaling center that specifies vertebrate axial polarity and the nervous system, is a dorsal midline mesodermal domain in the gastrula that will form prechordal plate and anterior notochord. We show that in zebrafish the organizer is not a single domain when it first arises in the nascent mesoderm at the onset of gastrulation. Rather, in the presumptive prechordal plate region, the organizer is subdivided into two side-by-side cellular fields. Within minutes, concurrent medial and anterior cellular movements merge, or 'coalesce', the two fields to form the well-known singular midline field. Coalescence forms a symmetrical domain because the cell movements on the left and right sides initiate simultaneously and occur synchronously. However, in embryos with reduced function of the T-box transcription factor Tbx16 (Spadetail) or its genetic target paraxial protocadherin (Papc), synchrony is lost, coalesence is disrupted, and the midline domain is misshaped. Furthermore, with combined loss of Tbx16 and Wnt11 (Silberblick), coalesence is essentially absent. Possibly as a consequence, both the anterior movement of presumptive prechordal plate and organizer function, as assayed by eye-field separation, are disrupted. Our findings thus reveal that Tbx16, in combination with Wnt11, are critical components not only in morphogenesis but also in initial assembly of the organizer.

Mutations in the gene coding for selenoprotein N (SelN), a selenium containing protein of unknown function, cause different forms of congenital muscular dystrophy in humans. These muscular diseases are characterized by early onset of hypotonia which predominantly affect in axial muscles. We used zebrafish as a model system to understand the function of SelN in muscle formation during embryogenesis. Zebrafish SelN is highly homologous to its human counterpart and amino acids corresponding to the mutated positions in human muscle diseases are conserved in the zebrafish protein. The sepn1 gene is highly expressed in the somites and notochord during early development. Inhibition of the sepn1 gene by injection of antisense morpholinos does not alter the fate of the muscular tissue, but causes muscle architecture disorganization and greatly reduced motility. Ultrastructural analysis of the myotomes reveals defects in muscle sarcomeric organization and in myofibers attachment, as well as altered myoseptum integrity. These studies demonstrate the important role of SelN for muscle organization during early development. Moreover, alteration of myofibrils architecture and tendon-like structure in embryo deficient for SelN function provide new insights into the pathological mechanism of SelN-related myopathy.

The ribosomal proteins (RPs) form the majority of cellular proteins and are mandatory for cellular growth. RP genes have been linked, either directly or indirectly, to various diseases in humans. Mutations in RP genes are also associated with tissue-specific phenotypes, suggesting a possible role in organ development during early embryogenesis. However, it is not yet known how mutations in a particular RP gene result in specific cellular changes, or how RP genes might contribute to human diseases. The development of animal models with defects in RP genes will be essential for studying these questions. In this study, we knocked down 21 RP genes in zebrafish by using morpholino antisense oligos to inhibit their translation. Of these 21, knockdown of 19 RPs resulted in the development of morphants with obvious deformities. Although mutations in RP genes, like other housekeeping genes, would be expected to result in nonspecific developmental defects with widespread phenotypes, we found that knockdown of some RP genes resulted in phenotypes specific to each gene, with varying degrees of abnormality in the brain, body trunk, eyes, and ears at about 25 hours post fertilization. We focused further on the organogenesis of the brain. Each knocked-down gene that affected the morphogenesis of the brain produced a different pattern of abnormality. Among the 7 RP genes whose knockdown produced severe brain phenotypes, 3 human orthologs are located within chromosomal regions that have been linked to brain-associated diseases, suggesting a possible involvement of RP genes in brain or neurological diseases. The RP gene knockdown system developed in this study could be a powerful tool for studying the roles of ribosomes in human diseases.

Fibroblast growth factor (Fgf) and retinoic acid (RA) signals control the formation and anteroposterior patterning of posterior hindbrain. They are also involved in development processes in other regions of the embryo. Therefore, responsiveness to Fgf and RA signals must be controlled in a context-dependent manner. Inhibiting the caudal-related genes cdx1a and cdx4 in zebrafish embryos caused ectopic expression of genes that are normally expressed in the posterior hindbrain and anterior spinal cord, and ectopic formation of the hindbrain motor and commissure neurons in the posteriormost neural tissue. Combinational marker analyses suggest mirror-image duplication in the Cdx1a/4-defective embryos, and cell transplantation analysis further revealed that Cdx1a and Cdx4 repress a posterior hindbrain-specific gene expression cell-autonomously in the posterior neural tissue. Expression of fgfs and retinaldehyde dehydrogenase 2 suggested that in the Cdx1a/4-defective embryos, the Fgf and RA signaling activities overlap in the posterior body and display opposing gradients, compared with those in the hindbrain region. We found that Fgf and RA signals were required for ectopic expression. Expression of the posterior hox genes hoxb7a, hoxa9a or hoxb9a, which function downstream of Cdx1a/4, or activator fusion genes of hoxa9a or hoxb9a (VP16-hoxa9a, VP16-hoxb9a) suppressed this loss-of-function phenotype. These data suggest that Cdx suppresses the posterior hindbrain fate through regulation of the posterior hox genes; the posterior Hox proteins function as transcriptional activators and indirectly repress the ectopic expression of the posterior hindbrain genes in the posterior neural tissue. Our results indicate that the Cdx-Hox code modifies tissue competence to respond to Fgfs and RA in neural tissue.

Remarkable progress has been made in understanding the molecular mechanisms underlying left-right asymmetry in vertebrate animal models but little is known on left-right axis formation in humans. Previously, we identified SESN1 (also known as PA26) as a candidate gene for heterotaxia by positional cloning of the breakpoint regions of a de novo translocation in a heterotaxia patient. In this study, we show by means of a zebrafish sesn1-knockdown model that Sesn1 is required for normal embryonic left-right determination. In this model, developmental defects and expression data of genes implicated in vertebrate left-right asymmetry indicate a role for Sesn1 in mediating Nodal signaling. In the lateral plate mesoderm, Nodal signaling plays a central role in left-right axis formation in vertebrates and is mediated by FoxH1 transcriptional induction. In line with this, we show that Sesn1 physically interacts with FoxH1 or a FoxH1-containing complex. Mutation analysis in a panel of 234 patients with isolated heterotaxia did not reveal mutations, indicating that these are only exceptional causes of human heterotaxia. In this study, we identify SESN1 as an indispensable gene for vertebrate left-right asymmetry and a new player in mediating Nodal signaling.

A large fraction of homozygous zebrafish mutant belladonna (bel) larvae display a reversed optokinetic response (OKR) that correlates with failure of the retinal ganglion cells to cross the midline and form the optic chiasm. Some of these achiasmatic mutants display strong spontaneous eye oscillations (SOs) in the absence of motion in the surround. The presentation of a stationary grating was necessary and sufficient to evoke SO. Both OKR reversal and SO depend on vision and are contrast sensitive. We built a quantitative model derived from bel fwd (forward) eye behaviors. To mimic the achiasmatic condition, we reversed the sign of the retinal slip velocity in the model, thereby successfully reproducing both reversed OKR and SO. On the basis of the OKR data, and with the support of the quantitative model, we hypothesize that the reversed OKR and the SO can be completely attributed to RGC misrouting. The strong resemblance between the SO and congenital nystagmus (CN) seen in humans with defective retinotectal projections implies that CN, of so far unknown etiology, may be directly caused by a projection defect.

Hereditary spastic paraplegia (HSP) is a collection of neurological disorders characterized by developmental failure or degeneration of motor axons in the corticospinal tract and progressive lower limb spasticity. SPG4 mutations are the most common cause of autosomal dominant HSP and Spastin (the SPG4 gene product) is a microtubule severing protein that shares homology with katanin, the microtubule severing activity of which promotes axon growth in cultured neurons. Given the sequence and functional similarity between spastin and katanin, we hypothesized that spastin promotes the dynamic disassembly and remodelling of microtubules required for robust, properly directed motor axon outgrowth. To investigate this hypothesis, we cloned the zebrafish spg4 orthologue and used morpholino antisense oligonucleotides directed against the translation start site and the intron 7-8 splice donor site to knock down spastin function in the developing zebrafish embryo. Reduced spg4 function caused dramatic defects in motor axon outgrowth without affecting the events driving the initial specification of motor neurones. Other neuronal subtypes also exhibited a requirement for spg4 function, since spg4 knock down caused both widespread defects in neuronal connectivity and extensive CNS-specific apoptosis. Our results reveal a critical requirement for spastin to promote axonal outgrowth during embryonic development, and they validate the zebrafish embryo as a novel model system to dissect the pathogenetic mechanisms underlying HSP. Taken together with other recent studies, our findings suggest that axon outgrowth defects may be a common feature of childhood SPG3A and SPG4 cases.

Barth syndrome is an X-linked disorder characterized by cardiomyopathy, skeletal myopathy, neutropenia, organic aciduria, and growth retardation caused by mutations in tafazzin. The sequence similarity of tafazzin to acyltransferases suggests a role in mitochondrial phospholipid metabolism. To study the role of tafazzin in heart function and development, we created a knockdown zebrafish model. Zebrafish tafazzin mRNA is first evident at 7 hours post-fertilization (hpf). At 10 and 24 hpf, tafazzin mRNA is ubiquitous, with highest levels in the head. By 51 hpf, expression becomes cardiac restricted. The tafazzin knockdown created by antisense morpholino yolk injection resulted in dose-dependent lethality, severe developmental and growth retardation, marked bradycardia and pericardial effusions, and generalized edema, signs that resemble human Barth syndrome heart failure. This knockdown phenotype was rescued by concomitant injection of normal tafazzin mRNA. Abnormal cardiac development, with a linear, nonlooped heart, and hypomorphic tail and eye development proves that tafazzin is essential for overall zebrafish development, especially of the heart. The tafazzin knockdown zebrafish provides an animal model similar to Barth syndrome to analyze the severity of human mutants and to test potential treatments.

The vertebrate neural crest is a pluripotent cell population that generates a large variety of cell types, including peripheral neurons, cartilage and pigment cells. Mechanisms that control the patterning of the neural crest toward specific cell fates remain only partially understood. Zebrafish homozygous for the sympathetic mutation 1 (sym1) have defects in a subset of neural crest derivatives, such as peripheral neurons, glia and cartilage, but retain normal numbers of melanocytes. The sym1 mutation is a nucleotide deletion that disrupts the forkhead DNA-binding domain of the foxd3 gene, which encodes a conserved winged-helix transcription factor. We show that sym1 mutants have normal numbers of premigratory neural crest cells, but these cells express reduced levels of snai1b and sox10, implicating foxd3 as an essential regulator of these transcription factors in the premigratory neural crest. The onset of neural crest migration is also delayed in sym1 mutants, and there is a reduction in the number of migratory trunk neural crest cells, particularly along the medial migration pathway. TUNEL analysis revealed aberrant apoptosis localized to the hindbrain neural crest at the 15-somite stage, indicating a critical role for foxd3 in the survival of a subpopulation of neural crest cells. These results show that foxd3 selectively specifies premigratory neural crest cells for a neuronal, glial or cartilage fate, by inducing the expression of lineage-associated transcription factors in these cells and regulating their subsequent migration.

Gamma-secretase cleavage, mediated by a complex of presenilin, presenilin enhancer (Pen-2), nicastrin, and Aph-1, is the final proteolytic step in generating amyloid beta protein found in brains of Alzheimer's disease patients and Notch intracellular domain critical for proper neuronal development. Here, we employ the zebrafish model to study the role of Pen-2 in neuronal survival. We found that (i) knockdown of Pen-2 using antisense morpholino led to a reduction of islet-1 positive neurons, (ii) Notch signaling was reduced in embryos lacking Pen-2 or other gamma-secretase components, (iii) neuronal loss in Pen-2 knockdown embryos is not as a result of a lack of neuronal precursor cells or cell proliferation, (iv) absence of Pen-2 caused massive apoptosis in the whole animal, which could be suppressed by simultaneous knockdown of the tumor suppressor p53, (v) loss of islet-1 or acetylated tubulin positive neurons in Pen-2 knockdown embryos could be partially rescued by knockdown of p53. Our results demonstrate that knockdown of Pen-2 directly induces a p53-dependent apoptotic pathway that contributes to neuronal loss and suggest that Pen-2 plays an important role in promoting neuronal cell survival and protecting from apoptosis in vivo.

Arthrogryposis-renal dysfunction-cholestasis syndrome (ARC) is a rare cause of cholestasis in infants. Causative mutations in VPS33B, a gene that encodes a Class C vacuolar sorting protein, have recently been reported in individuals with ARC. We have identified a zebrafish vps33b-ortholog that is expressed in developing liver and intestine. Knockdown of vps33b causes bile duct paucity and impairs intestinal lipid absorption, thus phenocopying digestive defects characteristic of ARC. By contrast, neither motor axon nor kidney epithelial defects typically seen in ARC could be identified in vps33b-deficient larvae. Biliary defects in vps33b-deficient zebrafish larvae closely resemble the bile duct paucity associated with knockdown of the onecut transcription factor hnf6. Consistent with this, reduced vps33b expression was evident in hnf6-deficient larvae and in larvae with mutation of vhnf1, a downstream target of hnf6. Zebrafish vhnf1, but not hnf6, increases vps33b expression in zebrafish embryos and in mammalian liver cells. Electrophoretic mobility shift assays suggest that this regulation occurs through direct binding of vHnf1 to the vps33b promoter. These findings identify vps33b as a novel downstream target gene of the hnf6/vhnf1 pathway that regulates bile duct development in zebrafish. Furthermore, they show that tissue-specific roles for genes that regulate trafficking of intracellular proteins have been modified during vertebrate evolution.

Fibroblast growth factor (Fgf) signaling plays important roles in brain development. Fgf3 and Fgf8 are crucial for the formation of the forebrain and hindbrain. Fgf8 is also required for the midbrain to form. Here, we identified zebrafish Fgf19 and examined its roles in brain development by knocking down Fgf19 function. We found that Fgf19 expressed in the forebrain, midbrain and hindbrain was involved in cell proliferation and cell survival during embryonic brain development. Fgf19 was also essential for development of the ventral telencephalon and diencephalon. Regional specification is linked to cell type specification. Fgf19 was also essential for the specification of gamma-aminobutyric acid (GABA)ergic interneurons and oligodendrocytes generated in the ventral telencephalon and diencephalon. The cross talk between Fgf and Hh signaling is critical for brain development. In the forebrain, Fgf19 expression was down-regulated on inhibition of Hh but not of Fgf3/Fgf8, and overexpression of Fgf19 rescued partially the phenotype on inhibition of Hh. The present findings indicate that Fgf19 signaling is crucial for forebrain development by interacting with Hh and provide new insights into the roles of Fgf signaling in brain development.

Transcript elongation is a critical step in the production of mature messenger RNAs. Many factors have been identified that are required for transcript elongation, including Spt 5. Studies in yeast determined that spt 5 is required for cell viability, and analyses in Drosophila indicate Spt 5 is localized to sites of active transcription, suggesting it is required generally for transcription. However, the requirement for spt 5 for cell viability in a metazoan organism has not been addressed. We determined that zebrafish foggy/spt 5 is required cell-autonomously for the posterior migration of facial branchiomotor neurons from rhombomere 4 (r4) into r6 and r7 of the hindbrain. These genetic mosaics also give us the unique opportunity to determine whether spt 5 is required for mRNA transcription equivalently at all loci by addressing two processes within the same cell-neuronal migration and cell viability. In a wild-type host, spt 5 null facial branchiomotor neurons survive to at least 5 days postfertilization while failing to migrate posteriorly. This finding indicates that spt 5-dependent transcript elongation is required cell-autonomously for a complex cell migration but not for the survival of these same cells. This work provides evidence that transcript elongation is not a global mechanism equivalently required by all loci and may actually be under more strict developmental regulation., (Developmental Dynamics 234:651-658, 2005. (c) 2005 Wiley-Liss, Inc.)

shocked (sho) is a zebrafish mutation that causes motor deficits attributable to CNS defects during the first2dof development. Mutant embryos display reduced spontaneous coiling of the trunk, diminished escape responses when touched, and an absence of swimming. A missense mutation in the slc6a9 gene that encodes a glycine transporter (GlyT1) was identified as the cause of the sho phenotype. Antisense knock-down of GlyT1 in wild-type embryos phenocopies sho, and injection of wild-type GlyT1 mRNA into mutants rescues them. A comparison of glycine-evoked inward currents in Xenopus oocytes expressing either the wild-type or mutant protein found that the missense mutation results in a nonfunctional transporter. glyt1 and the related glyt2 mRNAs are expressed in the hindbrain and spinal cord in nonoverlapping patterns. The fact that these regions are known to be required for generation of early locomotory behaviors suggests that the regulation of extracellular glycine levels in the CNS is important for proper function of neural networks. Furthermore, physiological analysis after manipulation of glycinergic activity in wild-type and sho embryos suggests that the mutant phenotype is attributable to elevated extracellular glycine within the CNS.

Histone acetylation is an important epigenetic mechanism for the control of eukaryotic transcription. The histone deacetylase 1 (HDAC1) gene has been implicated in controlling the transcription of core cell cycle regulators, but the in vivo role of HDACs in cell cycle regulation is still poorly understood. Loss of HDAC1 activity causes underproliferation in several contexts during vertebrate development. In contrast, we show here that HDAC1 has the opposite effect in the zebrafish visual system, where loss of HDAC1 activity leads to failure of cells to exit the cell cycle in the retina and in the optic stalk. The effect of HDAC1 on cell cycle exit is cell-autonomous, and loss of HDAC1 in the retina leads to up-regulation of cyclin D and E transcripts. These results demonstrate that the in vivo role of HDAC1 in regulating cell cycle progression is region-specific, as HDAC1 promotes cell cycle exit in the retina but stimulates proliferation in other cellular contexts.

Blood and endothelial cells arise in close association in developing embryos, possibly from a shared precursor, the hemangioblast, or as hemogenic endothelium. The transcription factor, Scl/Tal1 (stem cell leukemia protein), is essential for hematopoiesis but thought to be required only for remodeling of endothelium in mouse embryos. By contrast, it has been implicated in hemangioblast formation in embryoid bodies. To resolve the role of scl in endothelial development, we knocked down its synthesis in zebrafish embryos where early precursors and later phenotypes can be more easily monitored. With respect to blood, the zebrafish morphants phenocopied the mouse knockout and positioned scl in the genetic hierarchy. Importantly, endothelial development was also clearly disrupted. Dorsal aorta formation was substantially compromised and gene expression in the posterior cardinal vein was abnormal. We conclude that scl is especially critical for the development of arteries where adult hematopoietic stem cells emerge, implicating scl in the formation of hemogenic endothelium.

Forward genetic screens in zebrafish have been used to identify mutations in genes with important roles in organogenesis. One of these mutants, small heart, develops a diminutive and severely malformed heart and multiple developmental defects of the brain, ears, eyes, and kidneys. Using a positional cloning approach, we identify that the mutant gene encodes the zebrafish Na+/K+-ATPase alpha1B1 protein. Disruption of Na+/K+-ATPase alpha1B1 function via morpholino "knockdown" or pharmacological inhibition with ouabain phenocopies the mutant phenotype, in a dose-dependent manner. Heterozygosity for the mutation sensitizes embryos to ouabain treatment. Our findings present novel genetic and morphological details on the function of the Na+/K+-ATPase alpha1B1 in early cardiac morphogenesis and the pathogenesis of the small heart malformation. We demonstrate that the reduced size of the mutant heart is caused by dysmorphic ventricular cardiomyocytes and an increase in ventricular cardiomyocyte apoptosis. This study provides a new insight that Na+/K+-ATPase alpha1B1 is required for maintaining ventricular cardiomyocyte morphology and viability.

fez-like (fezl) is a forebrain-expressed zinc finger gene required for the formation of the hypothalamic dopaminergic and serotonergic (monoaminergic) neurons in zebrafish. To reveal its function in mammals, we analyzed the expression of the mouse orthologue of fezl and generated fezl-deficient mice by homologous recombination. Mouse fezl was expressed specifically in the forebrain from embryonic day 8.5. At mid-gestation, fezl expression was detected in subdomains of the forebrain, including the dorsal telencephalon and ventral diencephalon. Unlike the zebrafish fezl mutant too few, the fezl-deficient mice displayed normal development of hypothalamic monoaminergic neurons, but showed abnormal "hyperactive" behavior. In fezl(-/-) mice, the thalamocortical axons (TCA) were reduced in number and aberrantly projected to the cortex. These mutants had a reduced number of subplate neurons, which are involved in guiding the TCA from the dorsal thalamus, although the subplate neurons were born normally. These results suggest that fezl is required for differentiation or survival of the subplate neurons, and reduction of the subplate neurons in fezl-deficient mice leads to abnormal development of the TCA, providing a possible link between the transcriptional regulation of forebrain development and hyperactive behavior., (Copyright 2004 Wiley-Liss, Inc.)

A role for the aryl hydrocarbon receptor (AHR) pathway in vascular maturation has been implicated by studies in Ahr-null mice. In this study the hypothesis that activation of AHR signaling by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters common cardinal vein (CCV) development in the zebrafish embryo was investigated. The CCV is a paired vessel that grows across the yolk, connecting to the heart. It is extensively remodeled and regresses as the heart migrates dorsally within the pericardium. TCDD significantly reduced CCV growth as early as 44 h post fertilization (hpf), and CCV area was reduced to 63% of control at 62 hpf. This vascular response to TCDD was at least as sensitive as previously defined endpoints of TCDD developmental toxicity in zebrafish. TCDD also blocked regression of the CCV (by 80 hpf), possibly contributing to the "string-like" heart phenotype seen in TCDD-exposed zebrafish larvae. Dependence of the block in CCV regression on zebrafish (zf) AHR2 was investigated using a zfahr2 specific morpholino to knock down expression of AHR2. The zfahr2 morpholino had no effect on CCV regression in the absence of TCDD, but did protect against the TCDD-induced block of CCV regression. This demonstrates that the TCDD-induced block in CCV regression is AHR2 dependent. It is significant that decreased CCV growth occurs before and inhibition of CCV regression occurs concurrent with overt signs of TCDD developmental toxicity. This suggests that alterations of vascular growth and remodeling may play a role in TCDD developmental toxicity in zebrafish.

By using retroviral insertional mutagenesis in zebrafish, we have identified a recessive lethal mutation in the not really started (nrs) gene. The nrs mutation disrupts a gene located in linkage group 3 that is highly homologous to the spinster gene identified in Drosophila and to spinster orthologs identified in mammals. In flies, spinster encodes a membrane protein involved in lysosomal metabolism and programmed cell death in the central nervous system and in the ovary. In nrs mutant fish embryos, we detect an opaque substance in the posterior yolk cell extension at approximately 1 day after fertilization. This material progressively accumulates and by 48 hr after fertilization fills the entire yolk. By day 3 of embryogenesis, mutant embryos are severely reduced in size compared with their wild-type siblings and they die a few hours later. By in situ hybridization, we show that the nrs mRNA is expressed in the yolk cell at the time the mutant phenotype becomes apparent. In wild-type embryos, nrs message is present maternally and zygotically throughout embryogenesis and is also detected in adult animals. In nrs homozygous mutant embryos, nrs transcripts are undetectable at the time the phenotype becomes apparent, indicating that the retroviral insertion has most likely abolished expression of the nrs gene. Finally, the nrs phenotype can be partially rescued by microinjection of nrs encoding DNA. These results suggest that the nrs mutation affects an essential gene encoding a putative membrane-bound protein expressed specifically in the yolk cell during zebrafish embryogenesis., (Copyright 2002 Wiley-Liss, Inc.)