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Here, we describe an computerized platform ideal for large-scale deep-phenotyping of

Here, we describe an computerized platform ideal for large-scale deep-phenotyping of zebrafish mutant lines, which uses optical projection tomography to quickly picture brain-specific gene manifestation patterns in 3D at mobile quality. al., 2004). Knockout mice show 57852-57-0 IC50 a striking loss of corticospinal motor neurons and other closely related subcerebral projection neurons in cortical layer 5, which are replaced by callosal projection neurons (Molyneaux et al., 2005; Chen et al., 2005a, 2005b, 2008). Although developmental expression of is similar in fish and mammals (Berberoglu et al., 2009; Blechman et al., 2007; Levkowitz et al., 2003; Yang et al., 2001; Chen et al., 2005a, 2005b; Hirata et al., 2004; Molyneaux et al., 2005), previously reported loss-of-function phenotypes differ in important aspects. The function of in zebrafish has been extensively studied using the mutant (also known as defects are seen in the glutamatergic projection neurons of the telencephalon-derived neocortex, early developmental deficits in zebrafish embryos have only been reported in diencephalon, particularly in dopaminergic (DA), serotonergic (5-HT), oxytocinergic-like, and GABAergic populations (Blechman et al., 2007; Rink and Guo, 2004; Yang et al., 2012; Guo et al., 1999; Levkowitz et al., 2003). These differences raise the possibility that function has Rabbit polyclonal to HIP diverged substantially over time, particularly in regions of the telencephalon giving rise to the mammalian neocortex. Alternatively, 57852-57-0 IC50 important but subtle aspects of the phenotype may have been overlooked during zebrafish development. Support for the latter possibility comes from the finding that adult mutant zebrafish have significantly smaller telencephalons, in spite of the fact that early developmental defects are reported in the diencephalon (Berberoglu et al., 2014). Although the telencephalon appears grossly normal at two weeks post fertilization, we speculated that deep-phenotyping might uncover earlier abnormalities. We therefore analyzed mutants at two?and three days post fertilization (dpf) using 57852-57-0 IC50 a diverse in situ riboprobe library to detect progenitor populations, differentiated neuron subtypes, and brain regions. Our custom image analysis algorithms allowed us to automatically detect and quantify areas of significantly altered gene expression throughout the entire brain. In addition to previously reported defects in the ventral diencephalon, we found a second phenotypic hotspot in the telencephalon. Detailed analysis shows that mutants exhibit a variety of novel forebrain abnormalities as early as 2 dpf. Notably, we report for the first time that mutants display a dramatic loss of glutamatergic neurons in the pallium of the telencephalon, although telencephalic DA and GABAergic populations appear to be relatively normal. This phenotype is usually accompanied by a reduction in telencephalon volume. Our findings show that this zebrafish phenotype corresponds much more closely to mammalian deficits than previously assumed. Our data suggest a model in which controls two distinct developmental programs in the zebrafish brain: (1) a glutamatergic program in the telencephalon with similarities to mammalian function, and (2) a broader neurogenic program in the diencephalon. Results High-resolution 3D imaging of gene expression patterns using high-throughput OPT We have previously exhibited a high-throughput OPT platform for fast 3D imaging of non-embedded zebrafish embryos at micrometer quality (Pardo-Martin et al., 2013, 2010). Because formaldehyde fixation makes clear zebrafish embryos optically thick normally, high-resolution OPT needs tissue clearing ways to decrease light scattering and increase transparency. Inside our prior publication, clearing was attained utilizing a trypsin option on embryos stained using the histological dye Alcian blue. Since Alcian blue is bound to discovering cartilage, we searched for to create OPT applicable to all or any anatomical buildings or genes appealing by adapting our system to embryos stained using chromogenic Desire. This required an alternative solution tissues clearing technique, as trypsin is usually ineffective on WISH-stained samples. Immersion in a mixture of benzyl alcohol and benzyl benzoate (BABB) is usually a simple and quick solvent-based clearing technique. BABBs hydrophobicity and high refractive index (RI; 1.559) render samples optically transparent, making deeper structures accessible for high-resolution 3D imaging.