Abstract
The developing vertebrate nervous system contains a remarkable array of neural cells organized into complex, evolutionarily conserved structures. The labeling of living cells in these structures is key for the understanding of brain development and function, yet the generation of stable lines expressing reporter genes in specific spatio-temporal patterns remains a limiting step. In this study we present a fast and reliable pipeline to efficiently generate a set of stable lines expressing a reporter gene in multiple neuronal structures in the developing nervous system in medaka. The pipeline combines both the accurate computational genome-wide prediction of neuronal specific cis-regulatory modules (CRMs) and a newly developed experimental setup to rapidly obtain transgenic lines in a cost-effective and highly reproducible manner. 95% of the CRMs tested in our experimental setup show enhancer activity in various and numerous neuronal structures belonging to all major brain subdivisions. This pipeline represents a significant step towards the dissection of embryonic neuronal development in vertebrates.
Highlights
Recent years are witnessing a flood of new discoveries in neuroscience largely resulting from the ability to monitor living cells in the context of the developing nervous system using reporter gene expression [1]
The algorithm first identifies individual transcription factor binding sites (TFBS) based on a set of 402 high quality position-weight matrices (PWMs), from manually curated databases of known TFBS (Transfac [30], Jaspar [31]) and results from ChIP data [32] (Figure 1 and methods)
It assesses conservation of the predicted TFBS by comparing the medaka sequence to the orthologous sequences in Tetraodon nigroviridis, Takifugu rubripes and Gasterosteus aculeatus
Summary
Recent years are witnessing a flood of new discoveries in neuroscience largely resulting from the ability to monitor living cells in the context of the developing nervous system using reporter gene expression [1]. Exciting development in engineering new proteins has extended current barriers to allow monitoring and manipulating the activity of specific pathways within living cells [2]–[5]. These techniques rely heavily on the ability to drive gene expression to specific developmental stages, brain structures and cell types in a stable and reproducible way. The most widely used strategy to express reporters in anatomical structures relies on the use of regulatory elements, often promoters of genes known to be expressed in the desired structures (promoter bashing). In mouse [12] and zebrafish [13],[14], enhancer assays have been developed essentially to test genomic elements for enhancer activity
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