The proneural factors Ascl1a and Ascl1b contribute to the terminal differentiation of dopaminergic GABAergic dual transmitter neurons in zebrafish.

The proneural factor Ascl1 is involved in several steps of neurogenesis, from neural progenitor maintenance to initiation of terminal differentiation and neuronal subtype specification. In neural progenitor cells, Ascl1 initiates the cell-cycle exit of progenitors, and contributes to their differentiation into mainly GABAergic neurons. Several catecholaminergic neuron groups in the forebrain of zebrafish use GABA as co-transmitter, but a potential role of the two paralogues Ascl1a and Ascl1b in their neurogenesis is not understood. Here, we show that ascl1a, ascl1b double mutant embryos develop a significantly reduced number of neurons in all GABAergic and catecholaminergic dual transmitter neuron anatomical clusters in the fore- and hindbrain, while glutamatergic catecholaminergic clusters develop normally. However, none of the affected catecholaminergic cell clusters are lost completely, suggesting an impairment in progenitor pools, or a requirement of Ascl1a/b for differentiation of a subset of neurons in each cluster. Early progenitors which are dlx2a+, fezf2 + or emx2 + are not reduced whereas late progenitors and differentiating neurons marked by the expression of dlx5a, isl1 and arxa are severely reduced in ascl1a, ascl1b double mutant embryos. This suggests that Ascl1a and Ascl1b play only a minor or no role in the maintenance of their progenitor pools, but rather contribute to the initiation of terminal differentiation of GABAergic catecholaminergic neurons.

Neurog1 and Olig2 integrate patterning and neurogenesis signals in development of zebrafish dopaminergic and glutamatergic dual transmitter neurons.

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 during neurogenesis of A11-type dopaminergic clusters. 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.

ViBE-Z: a framework for 3D virtual colocalization analysis in zebrafish larval brains.

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.