Automated deep-phenotyping of the vertebrate brain.

Amin Allalou,Yuelong Wu,Mostafa Ghannad-Rezaie,Mehmet Fatih Yanik,Peter M Eimon

doi:10.7554/elife.23379

Amin Allalou, Yuelong Wu + Show 3 more

Open Access

https://doi.org/10.7554/elife.23379

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Abstract

Here, we describe an automated platform suitable for large-scale deep-phenotyping of zebrafish mutant lines, which uses optical projection tomography to rapidly image brain-specific gene expression patterns in 3D at cellular resolution. Registration algorithms and correlation analysis are then used to compare 3D expression patterns, to automatically detect all statistically significant alterations in mutants, and to map them onto a brain atlas. Automated deep-phenotyping of a mutation in the master transcriptional regulator fezf2 not only detects all known phenotypes but also uncovers important novel neural deficits that were overlooked in previous studies. In the telencephalon, we show for the first time that fezf2 mutant zebrafish have significant patterning deficits, particularly in glutamatergic populations. Our findings reveal unexpected parallels between fezf2 function in zebrafish and mice, where mutations cause deficits in glutamatergic neurons of the telencephalon-derived neocortex.

Highlights

Systematic initiatives such as the Zebrafish Mutation Project (ZMP) aim to identify disruptive alleles in all of the more than 26,000 genes in the vertebrate genome and make these available to the scientific community (Kettleborough et al, 2013)
Since Alcian blue is limited to detecting cartilage, we sought to make optical projection tomography (OPT) applicable to all anatomical structures or genes of interest by adapting our platform to embryos stained using chromogenic whole mount in situ hybridization (WISH)
We developed a process for fabricating a tapered transparent insert from refractive index (RI)-matched optical adhesive within the capillary to accommodate embryos from multiple developmental stages and ensure stability during high-speed rotational imaging (Figure 1B, Figure 1—figure supplement 1A–C; details in Materials and methods)

Summary

Introduction

Systematic initiatives such as the Zebrafish Mutation Project (ZMP) aim to identify disruptive alleles in all of the more than 26,000 genes in the vertebrate genome and make these available to the scientific community (Kettleborough et al, 2013). The advent of next-generation genome editing tools is enabling researchers to efficiently target virtually any genomic locus of interest for inactivation or precision alteration. These technologies, combined with prior large-scale mutagenesis screens, have created rapidly growing publicly available collections of zebrafish mutant lines that are in need of detailed characterization. Useful for preliminary screening, these criteria are too general to provide mechanistic insight into alleles that produce obvious defects and completely overlook those with interesting but subtle phenotypes

Methods

Results

Conclusion