Ian G. Woods

Chromatin Signature of Embryonic Pluripotency Is Established during Genome Activation

After fertilization the embryonic genome is inactive until transcription is initiated during the maternal–zygotic transition. This transition coincides with the formation of pluripotent cells, which in mammals can be used to generate embryonic stem cells. To study the changes in chromatin structure that accompany pluripotency and genome activation, we mapped the genomic locations of histone H3 molecules bearing lysine trimethylation modifications before and after the maternal–zygotic transition in zebrafish. Histone H3 lysine 27 trimethylation (H3K27me3), which is repressive, and H3K4me3, which is activating, were not detected before the transition. After genome activation, more than 80% of genes were marked by H3K4me3, including many inactive developmental regulatory genes that were also marked by H3K27me3. Sequential chromatin immunoprecipitation demonstrated that the same promoter regions had both trimethylation marks. Such bivalent chromatin domains also exist in embryonic stem cells and are thought to poise genes for activation while keeping them repressed. Furthermore, we found many inactive genes that were uniquely marked by H3K4me3. Despite this activating modification, these monovalent genes were neither expressed nor stably bound by RNA polymerase II. Inspection of published data sets revealed similar monovalent domains in embryonic stem cells. Moreover, H3K4me3 marks could form in the absence of both sequence-specific transcriptional activators and stable association of RNA polymerase II, as indicated by the analysis of an inducible transgene. These results indicate that bivalent and monovalent domains might poise embryonic genes for activation and that the chromatin profile associated with pluripotency is established during the maternal–zygotic transition.

erbb3 and erbb2 Are Essential for Schwann Cell Migration and Myelination in Zebrafish

BACKGROUND Myelin is critical for efficient axonal conduction in the vertebrate nervous system. Neuregulin (Nrg) ligands and their ErbB receptors are required for the development of Schwann cells, the glial cells that form myelin in the peripheral nervous system. Previous studies have not determined whether Nrg-ErbB signaling is essential in vivo for Schwann cell fate specification, proliferation, survival, migration, or the onset of myelination. RESULTS In genetic screens for mutants with disruptions in myelinated nerves, we identified mutations in erbb3 and erbb2, which together encode a heteromeric tyrosine kinase receptor for Neuregulin ligands. Phenotypic analysis shows that both genes are essential for development of Schwann cells. BrdU-incorporation studies and time-lapse analysis reveal that Schwann cell proliferation and migration, but not survival, are disrupted in erbb3 mutants. We show that Schwann cells can migrate in the absence of DNA replication. This uncoupling of proliferation and migration indicates that erbb gene function is required independently for these two processes. Pharmacological inhibition of ErbB signaling at different stages reveals a continuing requirement for ErbB function during migration and also provides evidence that ErbB signaling is required after migration for proliferation and the terminal differentiation of myelinating Schwann cells. CONCLUSIONS These results provide in vivo evidence that Neuregulin-ErbB signaling is essential for directed Schwann cell migration and demonstrate that this pathway is also required for the onset of myelination in postmigratory Schwann cells.

The you Gene Encodes an EGF-CUB Protein Essential for Hedgehog Signaling in Zebrafish

Hedgehog signaling is required for many aspects of development in vertebrates and invertebrates. Misregulation of the Hedgehog pathway causes developmental abnormalities and has been implicated in certain types of cancer. Large-scale genetic screens in zebrafish have identified a group of mutations, termed you-class mutations, that share common defects in somite shape and in most cases disrupt Hedgehog signaling. These mutant embryos exhibit U-shaped somites characteristic of defects in slow muscle development. In addition, Hedgehog pathway mutations disrupt spinal cord patterning. We report the positional cloning of you, one of the original you-class mutations, and show that it is required for Hedgehog signaling in the development of slow muscle and in the specification of ventral fates in the spinal cord. The you gene encodes a novel protein with conserved EGF and CUB domains and a secretory pathway signal sequence. Epistasis experiments support an extracellular role for You upstream of the Hedgehog response mechanism. Analysis of chimeras indicates that you mutant cells can appropriately respond to Hedgehog signaling in a wild-type environment. Additional chimera analysis indicates that wild-type you gene function is not required in axial Hedgehog-producing cells, suggesting that You is essential for transport or stability of Hedgehog signals in the extracellular environment. Our positional cloning and functional studies demonstrate that You is a novel extracellular component of the Hedgehog pathway in vertebrates.

A Large-Scale Zebrafish Gene Knockout Resource for the Genome-Wide Study of Gene Function

With the completion of the zebrafish genome sequencing project, it becomes possible to analyze the function of zebrafish genes in a systematic way. The first step in such an analysis is to inactivate each protein-coding gene by targeted or random mutation. Here we describe a streamlined pipeline using proviral insertions coupled with high-throughput sequencing and mapping technologies to widely mutagenize genes in the zebrafish genome. We also report the first 6144 mutagenized and archived F1s predicted to carry up to 3776 mutations in annotated genes. Using in vitro fertilization, we have rescued and characterized ~0.5% of the predicted mutations, showing mutation efficacy and a variety of phenotypes relevant to both developmental processes and human genetic diseases. Mutagenized fish lines are being made freely available to the public through the Zebrafish International Resource Center. These fish lines establish an important milestone for zebrafish genetics research and should greatly facilitate systematic functional studies of the vertebrate genome.

Neuropeptidergic Signaling Partitions Arousal Behaviors in Zebrafish

Animals modulate their arousal state to ensure that their sensory responsiveness and locomotor activity match environmental demands. Neuropeptides can regulate arousal, but studies of their roles in vertebrates have been constrained by the vast array of neuropeptides and their pleiotropic effects. To overcome these limitations, we systematically dissected the neuropeptidergic modulation of arousal in larval zebrafish. We quantified spontaneous locomotor activity and responsiveness to sensory stimuli after genetically induced expression of seven evolutionarily conserved neuropeptides, including adenylate cyclase activating polypeptide 1b (adcyap1b), cocaine-related and amphetamine-related transcript (cart), cholecystokinin (cck), calcitonin gene-related peptide (cgrp), galanin, hypocretin, and nociceptin. Our study reveals that arousal behaviors are dissociable: neuropeptide expression uncoupled spontaneous activity from sensory responsiveness, and uncovered modality-specific effects upon sensory responsiveness. Principal components analysis and phenotypic clustering revealed both shared and divergent features of neuropeptidergic functions: hypocretin and cgrp stimulated spontaneous locomotor activity, whereas galanin and nociceptin attenuated these behaviors. In contrast, cart and adcyap1b enhanced sensory responsiveness yet had minimal impacts on spontaneous activity, and cck expression induced the opposite effects. Furthermore, hypocretin and nociceptin induced modality-specific differences in responsiveness to changes in illumination. Our study provides the first systematic and high-throughput analysis of neuropeptidergic modulation of arousal, demonstrates that arousal can be partitioned into independent behavioral components, and reveals novel and conserved functions of neuropeptides in regulating arousal.

nsf is essential for organization of myelinated axons in zebrafish.

BACKGROUND Myelinated axons are essential for rapid conduction of action potentials in the vertebrate nervous system. Of particular importance are the nodes of Ranvier, sites of voltage-gated sodium channel clustering that allow action potentials to be propagated along myelinated axons by saltatory conduction. Despite their critical role in the function of myelinated axons, little is known about the mechanisms that organize the nodes of Ranvier. RESULTS Starting with a forward genetic screen in zebrafish, we have identified an essential requirement for nsf (N-ethylmaleimide sensitive factor) in the organization of myelinated axons. Previous work has shown that NSF is essential for membrane fusion in eukaryotes and has a critical role in vesicle fusion at chemical synapses. Zebrafish nsf mutants are paralyzed and have impaired response to light, reflecting disrupted nsf function in synaptic transmission and neural activity. In addition, nsf mutants exhibit defects in Myelin basic protein expression and in localization of sodium channel proteins at nodes of Ranvier. Analysis of chimeric larvae indicates that nsf functions autonomously in neurons, such that sodium channel clusters are evident in wild-type neurons transplanted into the nsf mutant hosts. Through pharmacological analyses, we show that neural activity and function of chemical synapses are not required for sodium channel clustering and myelination in the larval nervous system. CONCLUSIONS Zebrafish nsf mutants provide a novel vertebrate system to investigate Nsf function in vivo. Our results reveal a previously unknown role for nsf, independent of its function in synaptic vesicle fusion, in the formation of the nodes of Ranvier in the vertebrate nervous system.

Neuropeptidergic Control of Sleep and Wakefulness

Sleep and wake are fundamental behavioral states whose molecular regulation remains mysterious. Brain states and body functions change dramatically between sleep and wake, are regulated by circadian and homeostatic processes, and depend on the nutritional and emotional condition of the animal. Sleep-wake transitions require the coordination of several brain regions and engage multiple neurochemical systems, including neuropeptides. Neuropeptides serve two main functions in sleep-wake regulation. First, they represent physiological states such as energy level or stress in response to environmental and internal stimuli. Second, neuropeptides excite or inhibit their target neurons to induce, stabilize, or switch between sleep-wake states. Thus, neuropeptides integrate physiological subsystems such as circadian time, previous neuron usage, energy homeostasis, and stress and growth status to generate appropriate sleep-wake behaviors. We review the roles of more than 20 neuropeptides in sleep and wake to lay the foundation for future studies uncovering the mechanisms that underlie the initiation, maintenance, and exit of sleep and wake states.

Targeted mutagenesis in zebrafish

Random mutagenesis of the zebrafish genome using chemicals, retroviruses or transposons has uncovered mutations in hundreds of genes1. The ability to engineer specific mutations, however, has remained elusive. Two papers in this issue, by Meng et al. 2 and Doyon et al. 3, introduce a method for targeted mutagenesis in zebrafish. Both studies employ zinc-finger nucleases (ZFNs)—chimeric molecules consisting of a DNA-binding zinc-finger domain and the FokI restriction endonuclease—to induce mutations in specific zebrafish genes (Fig. 1). This technique makes it possible to disrupt any gene of interest and may facilitate more sophisticated manipulations of the zebrafish genome. Figure 1 Targeted mutagenesis of zebrafish genes with zinc-finger nucleases (ZFNs) ZFNs induce targeted double-strand breaks in the genome4,5,6. The specificity of DNA cleavage is conferred by varying a ZFN’s repertoire of zinc fingers, each of which interacts with a particular triplet of DNA base pairs. Combining three or four zinc fingers allows specific binding to 9- or 12-bp motifs, respectively. Double-strand breaks occur when two ZFNs bind to target DNA, bringing their nuclease domains together. Active only as a dimer, the nuclease domains cleave the DNA between the bound ZFNs. The endogenous double-strand-break repair machinery can then edit the genome through two pathways. If a matching template sequence is available, repair can occur by homologous recombination. In the absence of a template, the DNA can be religated by nonhomologous end joining, often with the addition or deletion of bases. The ability of ZFNs to induce targeted double-strand breaks has been exploited in numerous applications, including the creation of knockouts in cell lines7 and invertebrates8 and gene editing in mammalian cells9. Both Meng et al. 2 and Doyon et al. 3 use ZFNs to generate mutations through nonhomologous end joining: mRNAs encoding two ZFNs are injected into fertilized eggs, and ZFN activity is assayed by PCR2 and phenotypic screening3 in the injected fish and their progeny (Fig. 1). Importantly, 30–50% of injected fish transmit ZFN-induced mutations to their progeny, and many (18% in ref. 2; 7% in ref. 3) of these progeny are mutant. These results indicate that screening for mutagenic events is very efficient. Both groups also show that ZFN-induced DNA cleavage is highly specific. Meng et al. 2, identify 41 regions of the zebrafish genome with sequences similar (differing by 1–4 nucleotides) to their intended ZFN target. Solexa sequencing of these regions in ZFN-injected embryos reveals that the rate of off-target cleavage is 1% in morphologically normal embryos and 5% in embryos with nonspecific “monster” phenotypes. Doyon et al. 3 analyze the five genomic regions with sequences most similar to their intended target in progeny of ZFN-injected fish and detect no off-target cleavage. Interestingly, Doyon et al. 3 observe that both copies of the targeted gene are disrupted in some cells of injected embryos, leading to mosaic mutant phenotypes. Meng et al. 2, however, do not report mosaic phenotypes. Although the reason for this difference is unclear, one possibility is that Doyon et al. 3 use ZFNs with four zinc fingers whereas Meng et al. 2 use ZFNs containing three zinc fingers. Increasing the number of zinc fingers enhances the target specificity of ZFNs and can reduce off-target cleavage of DNA4. Accordingly, embryos injected by Doyon et al. 3 tolerate nanogram amounts of injected ZFN mRNA, whereas 50-pg doses of ZFN mRNA are toxic to most embryos in the Meng et al. 2 study. Hence, the higher levels of ZFNs used by Doyon et al. 3 may be sufficient to disrupt both copies of the targeted gene. Another reason for the difference may be that some mutant cells are more readily observable in a wild-type background than others—Doyon et al. 3 score obvious pigment and body pattern phenotypes, whereas Meng et al.2 analyze more subtle vascular defects. Further work will clarify these issues. How easily can this technology be implemented in a standard zebrafish lab? Injection, genotyping and mutant analysis are well-established procedures. Therefore, the remaining obstacles involve the design, selection and validation of ZFNs. Indeed, both studies stem from collaborations between zebrafish researchers and ZFN experts. Web-based tools10 and published protocols11 are available to assist researchers in designing and synthesizing ZFNs. It should be noted that the construction of modular ZFNs based on individual zinc finger–DNA interactions has been generally unsuccessful unless the repertoire of zinc fingers is restricted to those with particularly well-validated target sequences12. A commercial source of ZFN expertise, design and optimization is under development3. Intriguingly, zebrafish embryos themselves might provide an excellent in vivo test and optimization system for ZFNs. The Meng et al. study2 exemplifies this potential: ZFNs were designed such that a restriction enzyme recognition sequence was situated between their binding targets, enabling embryos to be tested for ZFN activity by PCR and restriction digestion shortly after injection. A skilled zebrafish researcher can inject and assay hundreds of embryos in a single day, thus allowing multiple candidate ZFNs to be tested in parallel. It is likely that ZFNs will find widespread use in the zebrafish community and complement other approaches currently used to disrupt the function of specific genes1. For example, antisense morpholino oligonucleotides can block translation or splicing of specific RNAs but often induce off-target effects and are unsuited for phenotypic analyses at later stages of development. True genetic mutants can be generated through TILLING, in which large libraries of mutagenized fish are screened by PCR and sequenced for lesions in target genes. Although specific regions within genes can be analyzed for disruptions, the mutations obtained by TILLING are random; moreover, the required resources are beyond the scope of most laboratories. Retroviral insertions have also been successfully used to disrupt zebrafish genes. Because each insert can be mapped within the genome, large collections of insertions could in principle be created with inserts in nearly every gene. However, the potential to specifically edit the zebrafish genome is unique to ZFNs. In summary, Doyon et al.3 and Meng et al.2 convincingly demonstrate that ZFNs can induce mutations in zebrafish via nonhomologous end joining. The next step will be to coerce the DNA repair machinery to use homologous recombination, rather than nonhomologous end joining, to repair ZFN-induced double-strand breaks. Homologous recombination techniques would allow for exquisite control over mutagenesis and could also facilitate the introduction of transgenes that reflect endogenous gene expression and protein localization. Finally, the two studies suggest that ZFN technology can be applied to other organisms that have a sequenced genome and that are amenable to RNA injection. ZFNs might therefore become the major technology for genome manipulation.

Evolutionarily conserved regulation of hypocretin neuron specification by Lhx9

Loss of neurons that express the neuropeptide hypocretin (Hcrt) has been implicated in narcolepsy, a debilitating disorder characterized by excessive daytime sleepiness and cataplexy. Cell replacement therapy, using Hcrt-expressing neurons generated in vitro, is a potentially useful therapeutic approach, but factors sufficient to specify Hcrt neurons are unknown. Using zebrafish as a high-throughput system to screen for factors that can specify Hcrt neurons in vivo, we identified the LIM homeobox transcription factor Lhx9 as necessary and sufficient to specify Hcrt neurons. We found that Lhx9 can directly induce hcrt expression and we identified two potential Lhx9 binding sites in the zebrafish hcrt promoter. Akin to its function in zebrafish, we found that Lhx9 is sufficient to specify Hcrt-expressing neurons in the developing mouse hypothalamus. Our results elucidate an evolutionarily conserved role for Lhx9 in Hcrt neuron specification that improves our understanding of Hcrt neuron development. Summary: The transcription factor Lhx9 is sufficient to drive the specification of zebrafish and mouse hypocretin-expressing neurons, the neuron type that is affected in narcolepsy.

Touch responsiveness in zebrafish requires voltage-gated calcium channel 2.1b

The molecular and physiological basis of the touch-unresponsive zebrafish mutant fakir has remained elusive. Here we report that the fakir phenotype is caused by a missense mutation in the gene encoding voltage-gated calcium channel 2.1b (CACNA1Ab). Injection of RNA encoding wild-type CaV2.1 restores touch responsiveness in fakir mutants, whereas knockdown of CACNA1Ab via morpholino oligonucleotides recapitulates the fakir mutant phenotype. Fakir mutants display normal current-evoked synaptic communication at the neuromuscular junction but have attenuated touch-evoked activation of motor neurons. NMDA-evoked fictive swimming is not affected by the loss of CaV2.1b, suggesting that this channel is not required for motor pattern generation. These results, coupled with the expression of CACNA1Ab by sensory neurons, suggest that CaV2.1b channel activity is necessary for touch-evoked activation of the locomotor network in zebrafish.