Precise three-dimensional (3D) mapping of a large number of gene expression patterns, neuronal types and connections to an anatomical reference helps us to understand the vertebrate brain and its development. We developed the Virtual Brain Explorer (ViBE-Z), a software that automatically maps gene expression data with cellular resolution to a 3D standard larval zebrafish (Danio rerio) brain. ViBE-Z enhances the data quality through fusion and attenuation correction of multiple confocal microscope stacks per specimen and uses a fluorescent stain of cell nuclei for image registration. It automatically detects 14 predefined anatomical landmarks for aligning new data with the reference brain. ViBE-Z performs colocalization analysis in expression databases for anatomical domains or subdomains defined by any specific pattern; here we demonstrate its utility for mapping neurons of the dopaminergic system. The ViBE-Z database, atlas and software are provided via a web interface.
In the version of this article initially published, two items in the Online Methods section were incorrect. The MATLAB code in the 'ViBE-Z database file' section contained an extraneous semicolon, which appeared in the HTML only and has been corrected. In the section 'Stitching and dorsal-ventral alignment' , two formulas had a 'mapsto' symbol instead of an arrow. These errors have been corrected in the HTML and PDF versions of the article.
In this protocol we describe a method to produce multi-view confocal 3D datasets suitable to be processed by the Virtual Brain Explorer (ViBE-Z) software. The method is optimized for Zebrafish (Danio rerio) embryos and larvae from one to five days post fertilization, but may be used also for other small biological objects. Zebrafish larvae are stained using either fluorescent in situ hybridization or immunostaining. In addition, all samples are counterstained with a nuclear stain to generate information to be used for anatomical reference. Stained larval brains are imaged using standard laser scanning confocal microscopes. To properly represent regions of very high as well as very low signal intensity we generate image stacks at different laser intensities and merge them to high dynamic range datasets. Further, multiple views are recorded and merged into high resolution combined datasets. To reduce the loss of information by restricted optical depth as a result of absorption and light scattering occurring in thick samples, image stacks are recorded both from the dorsal and ventral side of larvae. Both dorsal and ventral recordings are fused using attenuation correction of the ViBE-Z software, leading to a data representation that significantly reduces absorption and diffraction artifacts typical for microscopy of tissues deep inside biological samples.
Dopaminergic neurons develop in distinct neural domains by integrating local patterning and neurogenesis signals. While the proneural proteins Neurog1 and Olig2 have been previously linked to development of dopaminergic neurons, their dependence on local prepatterning and specific contributions to dopaminergic neurogenesis are not well understood. Here, we show that both transcription factors are differentially required for the development of defined dopaminergic glutamatergic subpopulations in the zebrafish posterior tuberculum, which are homologous to A11 dopaminergic neurons in mammals. Both Olig2 and Neurog1 are expressed in otpa expressing progenitor cells and appear to act upstream of Otpa during dopaminergic neurogenesis. Our epistasis analysis confirmed that Neurog1 acts downstream of Notch signaling, while Olig2 acts downstream of Shh, but upstream and/or in parallel to Notch signaling. Furthermore, we identified Olig2 to be an upstream regulator of neurog1 in dopaminergic neurogenesis. This regulation occurs through Olig2-dependent repression of the proneural repressor and Notch target gene her2. Our study reveals how Neurog1 and Olig2 integrate local patterning signals, including Shh, with Notch neurogenic selection signaling, to specify the progenitor population and initiate neurogenesis and differentiation of A11-type dopaminergic neurons.
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