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Abstract

Alpha1 tubulin gene expression is induced in the developing and regenerating CNS of vertebrates. Therefore, alpha1 tubulin gene expression may serve as a good probe for mechanisms underlying CNS development and regeneration. One approach to identify these mechanisms is to work backwards from the genome. This requires identification of alpha1 tubulin DNA sequences that mediate its developmental and regeneration-dependent expression pattern. Therefore, we generated transgenic zebrafish harboring a fragment of the alpha1 tubulin gene driving green fluorescent protein expression (GFP). In these fish, and similar to the endogenous gene, transgene expression was dramatically induced in the developing and regenerating nervous system. Although transgene expression generally declined during maturation of the nervous system, robust GFP expression was maintained in progenitor cells in the retinal periphery, lining brain ventricles and surrounding the central canal of the spinal cord. When these cells were cultured in vitro they divided and gave rise to new neurons. We also show that optic nerve crush in adult fish re-induced transgene expression in retinal ganglion cells. These studies identified a relatively small region of the alpha1 tubulin promoter that mediates its regulated expression pattern in developing and adult fish. This promoter will be extremely useful to investigators interested in targeting gene expression to the developing or regenerating nervous system. As adult transgenic fish maintain transgene expression in neural progenitors, these fish also provide a valuable resource of labeled adult neural progenitor cells that can be studied in vivo or in vitro. Finally, these fish should provide a unique in vivo system for investigating mechanisms mediating CNS development and regeneration.

The Circadian System Is a Target and Modulator of Prenatal Cocaine Effects

“…Consistent with the original observations [12], [14], α1-tubulin-associated GFP fluorescence in control α1-TGFP zebrafish embryos was first detected 12–14 hours post fertilization (hpf) and, within a few hours, highlighted the entire central nervous system (CNS), thereafter peaking in different regions in a predictable pattern. The control group showed highly similar levels of fluorescence (Fig.…”

“…Clarifying this issue and studying microscopic changes associated with the observed alterations in the expression of the α1-tubulin-associated reporter will be a subject of further investigation. Considering that neuron-specific α1-tubulin transcription spans the entire period of neuron development, from the neuroprogenitor stage to the completion of axonogenesis [12], [31], [32], alteration in all or some of these stages could result in changed intensity or patterns of fluorescence.…”

“…This and genetically encoded fluorescent reporters (e.g., green fluorescent protein, GFP) in transgenic zebrafish provide a unique opportunity for studying expression of specific genes throughout development. To assess potential differences in zebrafish brain development in response to cocaine, we have used α1-T-GFP transgenic zebrafish, with a GFP reporter driven by the 1.696-kb goldfish α1-tubulin promoter fragment [12]. α1-Tubulin is a neural-specific isoform of the α-tubulin family of proteins, forming the microtubule cytoskeleton in developing axons and dendrites [for review 13].…”

“…α1-Tubulin is a neural-specific isoform of the α-tubulin family of proteins, forming the microtubule cytoskeleton in developing axons and dendrites [for review 13]. The reporter recapitulates α1-tubulin endogenous gene expression and thus is present in committed neuroprogenitors and actively developing neurons, allowing qualitative and quantitative evaluation of the developing brain and spinal cord [12]–[14].…”

Characterization of a Muscle-specific Enhancer in Human MuSK Promoter Reveals the Essential Role of Myogenin in Controlling Activity-dependent Gene Regulation

“…Zebrafish Embryo Injection for Transient in Vivo Expression-Zebrafish embryo injection was performed as reported previously (31). For these experiments, zebrafish embryos, at the 1-2-cell stage, were injected with ϳ300 pl of a solution containing 25-50 ng/l wild-type or mutant expression vector DNA using a glass micropipette and picopump.…”