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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2007.09.019 Byline: Matthew B. Veldman (a), Michael A. Bemben (b), Robert C. Thompson (a)(b)(c), Daniel Goldman (a)(b)(d) Keywords: Optic nerve; Regeneration; Retina; Gene expression; Microarray; Zebrafish; KLF6; KLF7; SOCS3; Sox11; Retinal ganglion cell Abstract: 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. Author Affiliation: (a) Neuroscience Program, University of Michigan, 5045 Biomedical Sciences Research Building, 109 Zina Pitcher Place, Ann Arbor, MI 48109, USA (b) Molecular and Behavioral Neuroscience Institute, University of Michigan, 5045 Biomedical Sciences Research Building, 109 Zina Pitcher Place, Ann Arbor, MI 48109, USA (c) Department of Psychiatry, University of Michigan, 5045 Biomedical Sciences Research Building, 109 Zina Pitcher Place, Ann Arbor, MI 48109, USA (d) Department of Biological Chemistry, University of Michigan, 5045 Biomedical Sciences Research Building, 109 Zina Pitcher Place, Ann Arbor, MI 48109, USA Article History: Received 12 July 2007; Revised 11 September 2007; Accepted 13 September 2007

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.03.024 Byline: Abhilasha Gulati-Leekha, Daniel Goldman Keywords: Tubulin; Transgenic; Mutagenesis; Neurogenesis; Cell cycle exit; Axonal branching; Zebrafish Abstract: [alpha]1-tubulin expression occurs in a neural-specific, temporally regulated, and regeneration-inducible fashion in zebrafish. A GFP reporter driven by the [alpha]1-tubulin promoter in transgenic zebrafish acts as a stable, in vivo molecular tag that follows neuronal development from birth/specification through postmitotic differentiation to axonal outgrowth and synaptogenesis. We exploited this transgenic system in a reporter expression-dependent (morphology-independent) mutagenesis screen to identify disruptions in genetic loci essential for neuronogenesis and axon elaboration, which would manifest as visually appreciable perturbations in GFP fluorescence. Thirty-two such recessive mutations were obtained, a subset of which was screened through a secondary RNA quantification-based assay to eliminate housekeeping gene defects. Three representative loci, when characterized in detail, were found to exhibit missteps in discrete, sequential stages of embryonic neuronal development. Mutation in sookshma panneurally diminishes the neural precursor pool by affecting cell proliferation in the developing embryo while patterning along the neuraxis remains unperturbed. Disruption of drishti on the other hand ameliorates the mitotic neural population by affecting cell cycle exit of progenitors and stalling their progression to the postmitotic neuronal stage, without impairing subsequent cell fate determination or differentiation. Finally, dhruva is required during neuronal differentiation for axonal branching and terminal innervation in spinal motoaxons and the retinotectal projection. Molecular identification of these loci and analysis of the remaining mutational repertoire will offer unique insights into the genetic inputs that go on to make a mature, differentiated neuron. Author Affiliation: Molecular and Behavioral Neuroscience Institute, Department of Biological Chemistry, University of Michigan, 205 Zina Pitcher Place, Ann Arbor, MI 48109-070, USA Article History: Received 26 October 2005; Revised 7 March 2006; Accepted 20 March 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.09.041 Byline: Emile G. Bruneau (a), Peter C. Macpherson (b), Daniel Goldman (b), Richard I. Hume (a), Mohammed Akaaboune (a) Keywords: C2C12; Myotube; Laminin; Agrin; Receptor turnover; Receptor insertion; Receptor removal; Receptor half-life Abstract: Using optical imaging assays, we investigated the dynamics of acetylcholine receptors (AChRs) at laminin-associated clusters on cultured myotubes in the absence or presence of the nerve-derived clustering factor, agrin. Using fluorescence recovery after photobleaching (FRAP) on fluorescent bungarotoxin-labeled receptors, we found that [approximately equal to]9% of original fluorescence was recovered after 8 h as surface AChRs were recruited into clusters. By quantifying the loss of labeled receptors and the recovery of fluorescence after photobleaching, we estimated that the half-life of clustered receptors was [approximately equal to]4.5 h. Despite the rapid removal of receptors, the accumulation of new receptors at clusters was robust enough to maintain receptor density over time. We also found that the AChR half-life was not affected by agrin despite its role in inducing the aggregation of AChRs. Interestingly, when agrin was added to myotubes grown on laminin-coated substrates, most new receptors were not directed into preexisting laminin-induced clusters but instead formed numerous small aggregates on the entire muscle surface. Time-lapse imaging revealed that the agrin-induced clusters could be seen as early as 1 h, and agrin treatment resulted in the complete dissipation of laminin-associated clusters by 24 h. These results reveal that while laminin and agrin are involved in the clustering of receptors they are not critical to the regulation of receptor metabolic stability at these clusters, and further argue that agrin is able to rapidly and fully negate the laminin substrate clustering effect while inducing the rapid formation of new clusters. Author Affiliation: (a) Department of Molecular, Cellular and Developmental Biology, University of Michigan, 830 N. University Ave., Ann Arbor, MI 48109, USA (b) Molecular and Behavioral Neuroscience Institute, University of Michigan Medical School, Ann Arbor, MI 48109, USA Article History: Received 18 May 2005; Revised 11 August 2005; Accepted 27 September 2005

The nucleotide sequence from -150 to -57 relative to the transcription start site on the gamma subunit gene of the nicotinic acetylcholine (ACh) receptor suppresses gene expression in the presence of electrical stimulation. During embryonic development, the ACh receptor gene is initially expressed throughout the muscle, but is subsequently localized to the synapse following muscle innervation.