Figure 2

Application of Atlas Toolkit to mapping nuclear protein levels against Optic Vesicle morphology for eighteen stage HH10 chick embryos.

(a) Overview of method workflow with blue boxes highlighting the steps performed by Atlas Toolkit. (b) Method for relative quantification of nuclear protein levels used by the tool ‘Extract Nuclear Signal’. (c–f) Method for projecting nuclear signal onto surface morphology used by the tool ‘Project to Segment Label’: (c) 3D reconstruction of cell nuclei segmented from an HH12 Optic Vesicle, colour-coded according to phospho-ERK1/2 protein levels (cold colours = low signal; warm colours = high signal). (d) Local averaging is used to ‘fill-in’ intra-nuclear space: the image volume is sub-divided into 12 μm target voxels, each of which is assigned the mean nuclear signal level from its surrounding 36 μm ‘sample voxel’. (e) Result of local averaging applied to cell nuclei in (c).(f) Surface morphology is restored by cropping the locally averaged volume in (e) using the segmentation label. (g–l) Group-wise 3D registration of Optic Vesicles segmented from 18 stage HH10 chick embryos stained with six different antibodies (three embryos per antibody). The nuclear signal levels were quantified and projected onto Optic Vesicle morphology prior to registration. The Optic Vesicle morphology shown in (g–l) is the consensus of all 18 embryos, while each antibody stain is the mean average of three embryos (normalised to background): (g) nuclear phospho-Smad1/5/8 (n = 3); (h) nuclear Smad2 (n = 3); (i) nuclear Smad3 (n = 3); (j) nuclear phospho-ERK1/2 (n = 3); (k) nuclear β-catenin (n = 3); (l) nuclear Pax6 (n = 3).