ΔSCOPE: A new method to quantify 3D biological structures and identify differences in zebrafish forebrain development

Abstract

Research in the life sciences has traditionally relied on the analysis of clear morphological phenotypes, which are often revealed using increasingly powerful microscopy techniques analyzed as maximum intensity projections (MIPs). However, as biology turns towards the analysis of more subtle phenotypes, MIPs and qualitative approaches are failing to adequately describe these phenotypes. To address these limitations and quantitatively analyze the three-dimensional (3D) spatial relationships of biological structures, we developed the computational method and program called ∆SCOPE (Changes in Spatial Cylindrical Coordinate Orientation using PCA Examination). Our approach uses the fluorescent signal distribution within a 3D data set and reorients the fluorescent signal to a relative biological reference structure. This approach enables quantification and statistical analysis of spatial relationships and signal density in 3D multichannel signals that are positioned around a well-defined structure contained in a reference channel. We validated the application of ∆SCOPE by analyzing normal axon and glial cell guidance in the zebrafish forebrain and by quantifying the commissural phenotypes associated with abnormal Slit guidance cue expression in the forebrain. Despite commissural phenotypes which display disruptions to the reference structure, ∆SCOPE was able to detect subtle, previously uncharacterized changes in zebrafish forebrain midline crossing axons and glia. This method has been developed as a user-friendly, open source program. We propose that ∆SCOPE is an innovative approach to advancing the state of image quantification in the field of high resolution microscopy, and that the techniques presented here are of broad applications to the life science field.

Characterizing the diverse cells that associate with the developing commissures of the zebrafish forebrain

“…Although, little documentation currently exists to support the existence of astroglial subtypes in zebrafish, the no-isthmus (noi; pax2a) mutants have a loss of reticulate astrocytes in the glia limitans at the optic nerve, which results in pathfinding defects and impaired fasciculation by axons of the optic chiasm and POC (Macdonald et al, 1997). As alluded to previously, Gfap+ astroglial fibers develop into condensed midline bridges in the diencephalon and telencephalon, and directly interact with commissural and retinal axons during and after midline crossing (Barresi et al, 2005;Schwartz et al, 2020).…”

“…• C and staged in hours post-fertilization (hpf) (Kimmel, Ballard, et al, 1995). Immunoctyochemistry was carried out as described previously (Johnson et al 2014;Johnson et al, 2016;Schwartz et al, 2020). Briefly, embryos were fixed with 4% paraformaldehyde with 10% dimethylsulfoxide (DMSO) for 3 hrs at room temp or 1 hour at room temp followed by 16 hours at 4…”

“…The quantitative distribution of the anti-Zrf immunolabeling was analyzed with ∆ Scope as described in Schwartz et al 2020. In brief, following imaging on the confocal microscope, data from each anti-Zrf labeled sample was first cropped to focus on the POC and the background signal eliminated, after which the image converted to an HDF5 (.h5) file type using the HDF5 plugin with Fiji (The HDF Group, 1997).…”

“…AT and Zrf1-4 channels were then processed using ilastik to improve the signal to noise ratios and generate signal probability values for each pixel. Output ilastik files for Zrf1-4 experimental groups were finally processed using the ∆ Scope workflow identical to (Schwartz et al, 2020).…”

“…Prior to commissure formation, processes of these Gfap+ cells appear to ubiquitously cover the basal or superficial most extents of the forebrain, however Gfap labeling soon condenses into midline spanning swaths of astroglia corresponding to the presumptive diencephalic and telencephalic commissural regions (Barresi et al, 2005). Moreover, axonal growth is quantitatively correlated with the movement of these astroglial cells over the course of commissure formation, strongly suggesting that these "glial bridges" provide a supportive substrate for pathfinding commissural axons (Barresi et al, 2005;Schwartz et al, 2020).…”