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BACKGROUND: The goal of this study was to identify and characterize cell-cell interactions that facilitate endothelial tip cell fusion downstream of BMP (bone morphogenic protein)-mediated venous plexus formation. METHODS: High resolution and time-lapse imaging of transgenic reporter lines and loss-of-function studies were carried out to study the involvement of mesenchymal stromal cells during venous angiogenesis. RESULTS: BMP-responsive stromal cells facilitate timely and precise fusion of venous tip cells during developmental angiogenesis. CONCLUSIONS: Stromal cells are required for anastomosis of venous tip cells in the embryonic caudal hematopoietic tissue.

Characterizing the precise three-dimensional morphology and anatomical context of neurons is crucial for neuronal cell type classification and circuitry mapping. Recent advances in tissue clearing techniques and microscopy make it possible to obtain image stacks of intact, interweaving neuron clusters in brain tissues. As most current 3D neuronal morphology reconstruction methods are only applicable to single neurons, it remains challenging to reconstruct these clusters digitally. To advance the state of the art beyond these challenges, we propose a fast and robust method named G-Cut that is able to automatically segment individual neurons from an interweaving neuron cluster. Across various densely interconnected neuron clusters, G-Cut achieves significantly higher accuracies than other state-of-the-art algorithms. G-Cut is intended as a robust component in a high throughput informatics pipeline for large-scale brain mapping projects., Most neuronal reconstruction software can automatically trace single neuronal morphologies but tracing multiple, densely interwoven neurons is much more challenging. Here the authors develop G-Cut, a computational approach for accurate segmentation of densely interconnected neuron clusters.

The right optic nerve of adult, 6 month to 1 year old, female and male Danio rerio were crushed and collected three days after. Matching controls of uninjured left optic nerves were also collected. The tissue was dissected from euthanized fish and frozen on dry ice. Samples were pooled for each category (female crush, female control, male crush, male control) n = 24 to obtain sufficient tissue for analysis. The brain from one male fish was also collected for control/calibration. Lipid extraction was done with the Bligh and Dyer [1] method, followed by untargeted liquid chromatography-mass spectrometry (LC MS-MS) lipid profiling using a Q-Exactive Orbitrap instrument coupled with Vanquish Horizon Binary UHPLC LC-MS system. The lipids were identified and quantified with LipidSearch 4.2.21 and the statistical analysis was conducted through Metaboanalyst 5.0. This data is available at Metabolomics Workbench, Study ID ST001725.

Ever since the seminal findings of Ramon y Cajal, dendritic and axonal morphology has been recognized as a defining feature of neuronal types. Yet our knowledge concerning the diversity of neuronal morphologies, in particular distal axonal projection patterns, is extremely limited. To systematically obtain single neuron full morphology on a brain-wide scale, we established a platform with five major components: sparse labeling, whole-brain imaging, reconstruction, registration, and classification. We achieved sparse, robust and consistent fluorescent labeling of a wide range of neuronal types by combining transgenic or viral Cre delivery with novel transgenic reporter lines. We acquired high-resolution whole-brain fluorescent images from a large set of sparsely labeled brains using fluorescence micro-optical sectioning tomography (fMOST). We developed a set of software tools for efficient large-volume image data processing, registration to the Allen Mouse Brain Common Coordinate Framework (CCF), and computer-assisted morphological reconstruction. We reconstructed and analyzed the complete morphologies of 1,708 neurons from the striatum, thalamus, cortex and claustrum. Finally, we classified these cells into multiple morphological and projection types and identified a set of region-specific organizational rules of long-range axonal projections at the single cell level. Specifically, different neuron types from different regions follow highly distinct rules in convergent or divergent projection, feedforward or feedback axon termination patterns, and between-cell homogeneity or heterogeneity. Major molecularly defined classes or types of neurons have correspondingly distinct morphological and projection patterns, however, we also identify further remarkably extensive morphological and projection diversity at more fine-grained levels within the major types that cannot presently be accounted for by preexisting transcriptomic subtypes. These insights reinforce the importance of full morphological characterization of brain cell types and suggest a plethora of ways different cell types and individual neurons may contribute to the function of their respective circuits.

Dendritic and axonal morphology reflects the input and output of neurons and is a defining feature of neuronal types1,2, yet our knowledge of its diversity remains limited. Here, to systematically examine complete single-neuron morphologies on a brain-wide scale, we established a pipeline encompassing sparse labelling, whole-brain imaging, reconstruction, registration and analysis. We fully reconstructed 1,741 neurons from cortex, claustrum, thalamus, striatum and other brain regions in mice. We identified 11 major projection neuron types with distinct morphological features and corresponding transcriptomic identities. Extensive projectional diversity was found within each of these major types, on the basis of which some types were clustered into more refined subtypes. This diversity follows a set of generalizable principles that govern long-range axonal projections at different levels, including molecular correspondence, divergent or convergent projection, axon termination pattern, regional specificity, topography, and individual cell variability. Although clear concordance with transcriptomic profiles is evident at the level of major projection type, fine-grained morphological diversity often does not readily correlate with transcriptomic subtypes derived from unsupervised clustering, highlighting the need for single-cell cross-modality studies. Overall, our study demonstrates the crucial need for quantitative description of complete single-cell anatomy in cell-type classification, as single-cell morphological diversity reveals a plethora of ways in which different cell types and their individual members may contribute to the configuration and function of their respective circuits., Sparse labelling and whole-brain imaging are used to reconstruct and classify brain-wide complete morphologies of 1,741 individual neurons in the mouse brain, revealing a dependence on both brain region and transcriptomic profile.

The right optic nerve of adult, 6 month to 1 year old, female and male Danio rerio were crushed and collected three days after. Matching controls of uninjured left optic nerves were also collected. The tissue was dissected from euthanized fish and frozen on dry ice. Samples were pooled for each category (female crush, female control, male crush, male control) n = 24 to obtain sufficient tissue for analysis. The brain from one male fish was also collected for control/calibration. Lipid extraction was done with the Bligh and Dyer [1] method, followed by untargeted liquid chromatography-mass spectrometry (LC MS-MS) lipid profiling using a Q-Exactive Orbitrap instrument coupled with Vanquish Horizon Binary UHPLC LC-MS system. The lipids were identified and quantified with LipidSearch 4.2.21 and the statistical analysis was conducted through Metaboanalyst 5.0. This data is available at Metabolomics Workbench, Study ID ST001725.

Author(s): Yan, Guanrong; Yan, Ruibin; Chen, Cheng; Chen, Cheng; Zhao, Yanqiu; Qin, Wei; Veldman, Matthew B; Li, Song; Lin, Shuo

Dendritic and axonal morphology is a defining feature of neuronal types and their connectivity. Yet our knowledge concerning the diversity of neuronal morphology is extremely limited. To systematically obtain single neuron full morphology on a brain-wide scale in mice, we established a pipeline that encompasses five major components: sparse labeling, whole-brain imaging, reconstruction, registration, and classification. We achieved sparse, robust and consistent fluorescent labeling by combining transgenic or viral Cre delivery with novel transgenic reporter lines, and generated whole-brain fluorescent imaging datasets containing tens of thousands of reconstructable neurons. We developed software tools for large-volume image data processing and computer-assisted morphological reconstruction. For a proof-of-principle, we reconstructed the full morphologies of 96 neurons from the claustrum and cortex that belong to a single transcriptomically-defined subclass, and classified them into multiple morphological types, suggesting that they work in a targeted and coordinated manner to process cortical information over a large region.

Cajal recognized that the elaborate shape of neurons is fundamental to their function in the brain. However, there are no simple and generalizable genetic methods to study neuronal or glial cell morphology in the mammalian brain. Here, we describe four mouse lines conferring Cre-dependent sparse cell labeling based on mononucleotide repeat frameshift (MORF) as a stochastic translational switch. Notably, the optimized MORF3 mice, with a membrane-bound multivalent immunoreporter, confer Cre-dependent sparse and bright labeling of thousands of neurons, astrocytes, or microglia in each brain, revealing their intricate morphologies. MORF3 mice are compatible with imaging in tissue-cleared thick brain sections and with immuno-EM. An analysis of 151 MORF3-labeled developing retinal horizontal cells reveals novel morphological cellclusters and axonal maturation patterns. Our study demonstrates a conceptually novel, simple, generalizable, and scalable mouse genetic solution to sparsely label and illuminate the morphology of genetically defined neurons and glia in the mammalian brain.

Huntington's disease (HD), a dominantly inherited neurodegenerative disease, is defined by its genetic cause, a CAG-repeat expansion in the HTT gene, its motor and psychiatric symptomology and primary loss of striatal medium spiny neurons (MSNs). However, the molecular mechanisms from genetic lesion to disease phenotype remain largely unclear. Mouse models of HD have been created that exhibit phenotypes partially recapitulating those in the patient, and specifically, cortico-striatal disconnectivity appears to be a shared pathogenic event shared by HD mouse models and patients. Molecular studies have begun to unveil converging molecular and cellular pathogenic mechanisms that may account for cortico-striatal miscommunication in various HD mouse models. Systems biological approaches help to illuminate synaptic molecular networks as a nexus for HD cortio-striatal pathogenesis, and may offer new candidate targets to modify the disease.

In this issue of Neuron, Gasset-Rosa et al. (2017) and Grima et al. (2017) describe defects in the nuclear pore complex and impaired nucleocytoplasmic transport in Huntington's disease (HD). The findings suggest that erosion of nuclear gatekeeping function, which is found in normal brain aging, may play an important role in the pathogenesis of multiple neurodegenerative disorders, including HD.

Rationale: Endothelial cells are developmentally derived from angioblasts specified in the mesodermal germ cell layer. The transcription factor etsrp/etv2 is at the top of the known genetic hierarchy for angioblast development. The transcriptional events that induce etsrp expression and angioblast specification are not well understood. Objective: We generated etsrp:gfp transgenic zebrafish and used them to identify regulatory regions and transcription factors critical for etsrp expression and angioblast specification from mesoderm. Methods and Results: To investigate the mechanisms that initiate angioblast cell transcription during embryogenesis, we have performed promoter analysis of the etsrp locus in zebrafish. We describe three enhancer elements sufficient for endothelial gene expression when place in front of a heterologous promoter. The deletion of all 3 regulatory regions led to a near complete loss of endothelial expression from the etsrp promoter. One of the enhancers, located 2.3 kb upstream of etsrp contains a consensus FOX binding site that binds Foxc1a and Foxc1b in vitro by EMSA and in vivo using ChIP. Combined knockdown of foxc1a/b , using morpholinos, led to a significant decrease in etsrp expression at early developmental stages as measured by quantitative reverse transcriptase–polymerase chain reaction and in situ hybridization. Decreased expression of primitive erythrocyte genes scl and gata1 was also observed, whereas pronephric gene pax2a was relatively normal in expression level and pattern. Conclusions: These findings identify mesodermal foxc1a/b as a direct upstream regulator of etsrp in angioblasts. This establishes a new molecular link in the process of mesoderm specification into angioblast.

We report that knockdown of the alpha1 tubulin isoform Tuba1a, but not the highly related Tuba1b, dramatically impedes nervous system formation during development and RGC axon regeneration following optic nerve injury in adults. Within the tuba1a promoter, a G/C-rich element was identified that is necessary for tuba1a induction during RGC differentiation and optic axon regeneration. KLF6a and 7a, which we previously reported are essential for optic axon regeneration (Veldman et al., 2007), bind this G/C-rich element and transactivate the tuba1a promoter. In vivo knockdown of KLF6a and 7a attenuate regeneration-dependent activation of the endogenous tuba1a and p27 genes. These results suggest tuba1a expression is necessary for CNS development and regeneration and that KLF6a and 7a mediate their effects, at least in part, via transcriptional control of tuba1a promoter activity.

Background Although the genetic cause for Huntington’s disease (HD) has been known for over 20 years, the mechanisms that cause the neurotoxicity and behavioral symptoms of this disease are not well understood. One hypothesis is that N-terminal fragments of the HTT protein are the causative agents in HD and that peptide sequences adjacent to the poly-glutamine (Q) repeats modify its toxicity. Here we test the function of the N-terminal 17 amino acids (N17) in the context of the exon 1 fragment of HTT in a novel, inducible zebrafish model of HD. Results Deletion of N17 coupled with 97Q expansion (mHTT-ΔN17-exon1) resulted in a robust, rapidly progressing movement deficit, while fish with intact N17 and 97Q expansion (mHTT-exon1) have more delayed-onset movement deficits with slower progression. The level of mHTT-ΔN17-exon1 protein was significantly higher than mHTT-exon1, although the mRNA level of each transgene was marginally different, suggesting that N17 may regulate HTT protein stability in vivo. In addition, cell lineage specific induction of the mHTT-ΔN17-exon1 transgene in neurons was sufficient to recapitulate the consequences of ubiquitous transgene expression. Within neurons, accelerated nuclear accumulation of the toxic HTT fragment was observed in mHTT-ΔN17-exon1 fish, demonstrating that N17 also plays an important role in sub-cellular localization in vivo. Conclusions We have developed a novel, inducible zebrafish model of HD. These animals exhibit a progressive movement deficit reminiscent of that seen in other animal models and human patients. Deletion of the N17 terminal amino acids of the huntingtin fragment results in an accelerated HD-like phenotype that may be due to enhanced protein stability and nuclear accumulation of HTT. These transgenic lines will provide a valuable new tool to study mechanisms of HD at the behavioral, cellular, and molecular levels. Future experiments will be focused on identifying genetic modifiers, mechanisms and therapeutics that alleviate polyQ aggregation in the nucleus of neurons. Electronic supplementary material The online version of this article (doi:10.1186/s13024-015-0063-2) contains supplementary material, which is available to authorized users.

Background: Endothelial cells line the luminal surface of blood vessels and form a barrier between the blood and other tissues of the body. Ets variant 2 (ETV2) is transiently expressed in both zebrafish and mice and is necessary and sufficient for vascular endothelial cell specification. Overexpression of this gene in early zebrafish and mouse embryos results in ectopic appearance of endothelial cells. Ectopic expression of ETV2 in later development results in only a subset of cells responding to the signal. Findings: We have examined the expression pattern of ETV2 in differentiating human embryonic stem cells (ESCs) to determine when the peak of ETV2 expression occurs. We show that overexpression of ETV2 in differentiating human ESC is able to increase the number of endothelial cells generated when administered during or after the endogenous peak of gene expression. Conclusions: Addition of exogenous ETV2 to human ESCs significantly increased the number of cells expressing angioblast genes without arterial or venous specification. This may be a viable solution to generate in vitro endothelial cells for use in research and in the clinic.

Anxiety and amygdalar stimulation may induce or exacerbate compulsions triggered by cortical-limbic hyperactivity, as in human obsessive-compulsive disorder (OCD). We previously created transgenic mice that exhibit OCD-like biting, movement and behavioral perseverance abnormalities. These behaviors are caused by expression of a neuro-potentiating cholera toxin (CT) transgene in dopamine D1 receptor-expressing (D1+) neurons within the amygdalar intercalated nucleus (ICN) and within cortical areas that project to orbitofrontal cortex and striatum. Here we tested whether anxiety and increased amygdalar stimulation may play a role in eliciting or exacerbating such behaviors. D1CT mice exhibited increased thigmotaxis (tendency of mice to remain along the perimeter of open areas) in the open field assay, and increased latency to first transit and reduced transit number in the light-dark assay. These studies indicate that the D1CT mice exhibit a significant increase in behavioral indicators of anxiety. Furthermore, yohimbine, a drug that induces both amygdalar stimulation and behavioral indicators of anxiety, exacerbated abnormal leaping in D1CT mice but failed to exacerbate their abnormal behavioral perseverance. These data suggest that chronic potentiation of D1+ neurons in the amygdalar ICN increases anxiety and facilitates particular compulsive behaviors.

Inflammatory pathways protect the body from infection and promote healing following injury. Recent reports demonstrate the surprising involvement of these pathways during hematopoietic stem cell emergence from the hemogenic endothelium in both zebrafish and mice.

Polo-like kinase 1 (PLK1), one of the key regulators of mitosis, is a target for cancer therapy due to its abnormally high activity in several tumors. Plk1 is highly conserved and shares a nearly identical 3-D structure between zebrafish and humans. The initial 10 mitoses of zebrafish embryonic cleavages occur every∼30 minutes, and therefore provide a rapid assay to evaluate mitosis inhibitors including those targeting Plk1. To increase efficiency and specificity, we first performed a computational virtual screen of∼60000 compounds against the human Plk1 3-D structure docked to both its kinase and Polo box domain. 370 candidates with the top free-energy scores were subjected to zebrafish assay and 3 were shown to inhibit cell division. Compared to general screen for compounds inhibiting zebrafish embryonic cleavage, computation increased the efficiency by 11 folds. One of the 3 compounds, named I2, was further demonstrated to effectively inhibit multiple tumor cell proliferation in vitro and PC3 prostate cancer growth in Xenograft mouse model in vivo. Furthermore, I2 inhibited Plk1 enzyme activity in a dose dependent manner. The IC50 values of I2 in these assays are compatible to those of ON-01910, a Plk1 inhibitor currently in Phase III clinic trials. Our studies demonstrate that zebrafish assays coupled with computational screening significantly improves the efficiency of identifying specific regulators of biological targets. The PLK1 inhibitor I2, and its analogs, may have potential in cancer therapeutics.

Promoters with high levels of ubiquitous expression are of significant utility in the production of transgenic animals and cell lines. One such promoter is derived from the human cytomegalovirus immediate early (CMV-IE) gene. We sought to ascertain if the simian CMV-IE promoter (sCMV), used extensively in non-mammalian vertebrate research, also directs intense, widespread expression when stably introduced into zebrafish. Analysis of sCMV-driven expression revealed a temporal and spatial pattern not predicted by studies using the hCMV promoter in other transgenic animals or by observations of early F0 embryos expressing injected sCMV-reporter plasmids. Unexpectedly, in transgenic fish produced by both integration of linearized plasmid or Tol2-mediated transgenesis, sCMV promoter expression was generally observed in a small population of cells in telencephalon and spinal cord between days 2 and 7, and was thereafter confined to discrete regions of CNS that included the olfactory bulb, retina, cerebellum, spinal cord, and lateral line. In skeletal muscle, intense transgene expression was not observed until well into adulthood (>2–3 months post-fertilization). One final unexpected characteristic of the sCMV promoter in stable transgenic fish was tissue-specific responsiveness of the promoter to heat shock at both embryonic and adult stages. These data suggest that, in the context of stable transgenesis, the simian CMV-IE gene promoter responds differently to intracellular regulatory forces than other characterized CMV promoters.

Zebrafish has many advantages as a model of human pediatric research. Given the physical and ethical problems with performing experiments on human patients, biomedical research has focused on using model organisms to study biologic processes conserved between humans and lower vertebrates. The most common model organisms are small mammals, usually rats and mice. Although these models have significant advantages, they are also expensive to maintain, difficult to manipulate embryonically, and limited for large-scale genetic studies. The zebrafish model nicely complements these deficiencies in mammalian experimental models. The low cost, small size, and external development of zebrafish make it an excellent model for vertebrate development biology. Techniques for large-scale genome mutagenesis and gene mapping, transgenesis, protein overexpression or knockdown, cell transplantation and chimeric embryo analysis, and chemical screens have immeasurably increased the power of this model organism. It is now possible to rapidly determine the developmental function of a gene of interest in vivo, and then identify genetic and chemical modifiers of the processes involved. Discoveries made in zebrafish can be further validated in mammals. With novel technologies being regularly developed, the zebrafish is poised to significantly improve our understanding of vertebrate development under normal and pathologic conditions.

Neuromuscular synaptogenesis is initiated by the release of agrin from motor neurons and the activation of the receptor tyrosine kinase, MuSK, in the postsynaptic membrane. MuSK gene expression is regulated by nerve-derived agrin and muscle activity. Agrin stimulates synapse-specific MuSK gene expression by activating GABP(alphabeta) transcription factors in endplate-associated myonuclei. In contrast, the mechanism by which muscle activity regulates MuSK gene expression is not known. We report on a 60-bp MuSK enhancer that confers promoter regulation by muscle differentiation, changes in intracellular calcium, and muscle activity. Within this enhancer, we identified a single E-box that is essential for this regulation. This E-box binds myogenin, and we showed that myogenin is necessary for not only MuSK but also nAChR gene regulation by muscle activity. Surprisingly, the same E-box functions in vivo to mediate muscle-specific and differentiation-dependent gene induction in zebrafish, suggesting an evolutionary conserved mechanism of regulation of synaptic protein gene expression.

Seizures can be induced by systemic dopamine D1 receptor agonists or by cortical-limbic neurostimulation non-selectively. Seizures are also often associated with tics and compulsions, which likewise involve cortical-limbic hyperactivity. To determine if selective potentiation of cortical-limbic D1 receptor-expressing (D1+) neurons increases seizure susceptibility, we administered pentylenetetrazole (PTZ) to mice that express a neuropotentiating transgene only in a glutamatergic, cortical-limbic subset of D1+ neurons (D1CT-7 line). These mice exhibited increased PTZ-dependent seizure incidence, onset rate and intensity. Because D1CT-7 mice also exhibit tic+compulsion-like behaviors, this implies that glutamatergic hyperactivity induced by cortical-limbic D1+ neuropotentiation facilitates not only epilepsy but also tics and compulsions. This suggests a dopamine-regulated glutamatergic basis for all three states and may explain why they often co-exist in humans.

Etv2, a master regulator of endothelial cell fate, can induce fast skeletal muscle cells to transdifferentiate into endothelial cells in the zebrafish embryo., Etsrp/Etv2 (Etv2) is an evolutionarily conserved master regulator of vascular development in vertebrates. Etv2 deficiency prevents the proper specification of the endothelial cell lineage, while its overexpression causes expansion of the endothelial cell lineage in the early embryo or in embryonic stem cells. We hypothesized that Etv2 alone is capable of transdifferentiating later somatic cells into endothelial cells. Using heat shock inducible Etv2 transgenic zebrafish, we demonstrate that Etv2 expression alone is sufficient to transdifferentiate fast skeletal muscle cells into functional blood vessels. Following heat treatment, fast skeletal muscle cells turn on vascular genes and repress muscle genes. Time-lapse imaging clearly shows that muscle cells turn on vascular gene expression, undergo dramatic morphological changes, and integrate into the existing vascular network. Lineage tracing and immunostaining confirm that fast skeletal muscle cells are the source of these newly generated vessels. Microangiography and observed blood flow demonstrated that this new vasculature is capable of supporting circulation. Using pharmacological, transgenic, and morpholino approaches, we further establish that the canonical Wnt pathway is important for induction of the transdifferentiation process, whereas the VEGF pathway provides a maturation signal for the endothelial fate. Additionally, overexpression of Etv2 in mammalian myoblast cells, but not in other cell types examined, induced expression of vascular genes. We have demonstrated in zebrafish that expression of Etv2 alone is sufficient to transdifferentiate fast skeletal muscle into functional endothelial cells in vivo. Given the evolutionarily conserved function of this transcription factor and the responsiveness of mammalian myoblasts to Etv2, it is likely that mammalian muscle cells will respond similarly., Author Summary The endothelial cell is a specialized cell type that lines blood vessels. These cells are involved in normal cardiovascular function and become damaged in cardiovascular disease states such as atherosclerosis and stroke. We have discovered that developing muscle cells in the zebrafish embryo can be converted into endothelial cells by the expression of a transcription factor called Etv2. Etv2 normally functions during embryonic development to specify blood and blood vessels. When expressed in muscle cells, Etv2 induces the expression of genes that are normally expressed in endothelial cells; it also represses muscle gene expression. On expressing Etv2, muscle cells change shape and go on to form lumenized blood vessels that connect to the existing circulatory system and support blood flow. The Wnt and VEGF signaling pathways are required for this fate transformation. Our results suggest that muscle cells may be a viable source for the de novo generation of endothelial cells for use in transplantation therapies and they highlight signalling pathways that might be manipulated to improve the efficiency of this process in mammalian cells.

The transcription factor etsrp/Er71/Etv2 is a master control gene for vasculogenesis in all species studied to date. It is also required for hematopoiesis in zebrafish and mice. Several novel genes expressed in vasculature have been identified through transcriptional profiling of zebrafish embryos overexpressing etsrp by microarrays. Here we re-examined this transcriptional profile by Illumina RNA-sequencing technology, revealing a substantially increased number of candidate genes regulated by etsrp. Expression studies of 50 selected candidate genes from this dataset resulted in the identification of 39 new genes that are expressed in vascular cells. Regulation of these genes by etsrp was confirmed by their ectopic induction in etsrp overexpressing and decreased expression in etsrp deficient embryos. Our studies demonstrate the effectiveness of the RNA-sequencing technology to identify biologically relevant genes in zebrfish and produced a comprehensive profile of genes previously unexplored in vascular endothelial cell biology.

Unlike mammals, teleost fish are able to mount an efficient and robust regenerative response following optic nerve injury. Although it is clear that changes in gene expression accompany axonal regeneration, the extent of this genomic response is not known. To identify genes involved in successful nerve regeneration, we analyzed gene expression in zebrafish retinal ganglion cells (RGCs) regenerating their axons following optic nerve injury. Microarray analysis of RNA isolated by laser capture microdissection from uninjured and 3-day post-optic nerve injured RGCs identified 347 up-regulated and 29 down-regulated genes. Quantitative RT-PCR and in situ hybridization were used to verify the change in expression of 19 genes in this set. Gene ontological analysis of the data set suggests regenerating neurons up-regulate genes associated with RGC development. However, not all regeneration-associated genes are expressed in differentiating RGCs indicating the regeneration is not simply a recapitulation of development. Knockdown of six highly induced regeneration-associated genes identified two, KLF6a and KLF7a, that together were necessary for robust RGC axon re-growth. These results implicate KLF6a and KLF7a as important mediators of optic nerve regeneration and suggest that not all induced genes are essential to mount a regenerative response.