A Tol2 Gateway-Compatible Toolbox for the Study of the Nervous System and Neurodegenerative Disease

Zebrafish are emerging as a powerful model system for studying neurodegenerative disease due to their genetic similarities with humans and ease of transgenesis (reviewed in Refs.^1,2). Transgenesis in zebrafish was revolutionized by the advent of the Tol2 transposon system in zebrafish^3,4 and the Multisite Gateway^®-based Tol2 kit for the standard format modular assembly of vectors required for transgenesis. ⁵ In addition, many transgenic lines have been generated through the use of promoter/enhancer sequences,^6,7 bacterial artificial chromosome transgenesis, ⁸ and gene-trap/enhancer detection screens.^4,9,10 These tools have allowed for the generation of countless transgenic lines expressing fluorescent proteins and genes of interest in cell and tissue-specific manners. Here, we build upon this work by presenting and sharing a Tol2kit-compatible toolbox that includes multiple neuronal and glial promoters and several compatible fluorophores for the flexible generation of transgenic lines for the study of the nervous system and neurodegenerative disease.

To streamline the production of zebrafish models for the study of the nervous system and neurodegenerative disease, we designed a Tol2 Gateway-compatible toolbox based on the original Tol2kit generated by the Chi-Bin Chien Lab. ⁵ We made use of the Tol2kit's flexibility to quickly assemble promoters, genes, and tags to generate a toolbox for the study of the nervous system and neurodegenerative disease. By utilizing the Tol2kit's standard format, the easy distribution and reuse of existing kit components are possible.

The neurodegenerative disease toolbox includes four promoters, six fluorophores (N- and C-terminal tags), and empty vectors with MultiSite Gateway (Invitrogen)-compatible cloning sites or multiple cloning sites for the easy insertion of genes of interest (Supplementary Tables S1–S5; Supplementary Data are available online at www.liebertpub.com/zeb). We have collated and adapted promoters for many key cell types of the nervous system that are implicated in neurodegenerative diseases, including promoters for motor neurons (-3mnx1/Hb9), pan neuronal (elavl3/HuC), microglial/macrophage (mpeg1.1), and astrocytic (gfap) expression. In addition, we have collated and adapted several fluorophores of nonoverlapping emission spectrums, such as mTagBFP (blue), TagRFPt (red), and EGFP (green) or mVenus (yellow), mCerulean3 (cyan), and mKOFP2 (orange). This will allow for the easy expression of a gene or fluorophore of interest in any of the key cell types of the nervous system.

Figure 1 shows an example of how our constructs can be used to create transgenic zebrafish lines expressing fluorescent reporters and diseased-linked genes. In this example, motor neurons are labeled in blue, a ubiquitously expressed neurodegenerative disease-linked protein (TDP-43) is labeled green and a ubiquitously secreted cell death reporter (ANNEXINV) is labeled in red (Fig. 1A, D–F). We first generated the Tg(-3mnx1:TagBFP) by selecting the p5E plasmid containing the motor neuron-specific promoter (p5E--3mnx1)^11,12 and combined it with the pME plasmid containing the mTagBFP fluorophore to create a fish line with blue motor neurons (Fig. 1B, C, E, G and Supplementary Data). To generate a zebrafish expressing a neurodegenerative disease-linked candidate gene, we choose to use the p5E plasmid containing the existing ubiquitous promoter (p5E-ubb) ¹³ and combined this with our pME-EGFP-NS and p3E-Hsa.TDP-43 plasmids to create a zebrafish expressing green TDP-43 in every cell [Tg(ubb:EGFP-NS-Hsa.TDP-43)] (Fig. 1B–D, H and Supplementary Data).

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FIG. 1.

An example of an ALS zebrafish model created with our toolbox to study the relationship between TDP-43 (an ALS-associated protein) and motor neuron degeneration. This zebrafish has blue motor neurons, green TDP-43, and a red cell death reporter (A). (B) Three constructs described here were generated by choosing a combination of 5′, middle and 3′ vectors from our toolbox and existing Tol2kit vectors and combined to form the final constructs pTol2pA2--3mnx1:mTagBFP (for blue motor neurons), pTol2pA2-ubb:EGFP-NS-Hsa.TDP-43 (for ubiquitous expression of TDP-43), and pTol2pA2-ubb:secHsa.ANNEXINV-mCherry (for a red cell death reporter). Each expression cassette has a three prime SV40 polyadenylation signal (pA) in either the 3′ vector or backbone to aid expression. (C) A traditional view of the constructs used to create the zebrafish in this example. (D–F) Representative zebrafish expressing TDP-43 tagged with EGFP quasi-ubiquitously (D) and mTagBFP selectively in motor neurons (E) injected with a construct carrying a secreted cell death reporter (ANNEXINV-mCherry) that will bind to cells undergoing cell death and fluoresce. Scale bar 1 mm. (G–J) We can then observe the effect of wild-type TDP-43 on motor neuron viability. Motor neurons are labeled in blue (G) co-expressing wild-type TDP-43 (H; green, J; white arrows), whereas cells undergoing apoptosis (I; red) recruit ANNEXINV-mCherry to the cell surface (J inset; *, arrow). Scale bar 10 μm. ALS, amyotrophic lateral sclerosis. Color images available online at www.liebertpub.com/zeb

To examine the effects of the neurodegenerative disease-linked protein, TDP-43, on the viability of motor neurons, we created a ubiquitously expressed cell death reporter by combining p5E-ubb with our pME plasmid containing secreted ANNEXINV with the existing C-terminal mCherry fluorophore ⁵ to generate a pTol2pA2—ubb:secHsa.ANNEXINV-mCherry construct (Fig. 1B, C, I, J and Supplementary Data). This construct, tagging a red protein to cells undergoing cell death, was injected into the compound transgenic Tg(-3mnx1:mTagBFP); Tg(ubb:EGFP-NS-Hsa.TDP-43) and live-imaging microscopy allowed us to examine whether the overexpression of our gene of interest induced motor neuron death in our animal model of neurodegenerative disease (Fig. 1J inset). In this way, sharing our Tol2 Gateway-compatible toolbox with the community allows for the flexibility and rapid generation of multiple zebrafish lines to study the biology of the nervous system and the processes of neurodegenerative disease.