Fig. 3
Spatiotemporal morphological assessment of the developing Hindbrain Choroid Plexus. A The illustration of the ChOP-CT-based measurement method of the total volume (cyan) and surface (green) is visualized on the frontal view of E13.5 Hindbrain Choroid Plexus. Scale bar: 1 mm. B Quantification of the total volume differences between the WT Hindbrain Choroid Plexuses of E13.5, E15.5 and E17.5 demonstrates a statistically significant increase in the total Hindbrain Choroid Plexus volume along the analyzed developing timeline. Note for B, C, E, F, J, N, O, V—the number of biological replicates within the particular group, n = 5, also indicated in the graph,. Error bars indicate the standard deviation, p-values—one-way ANOVA with Turkey´s multiple comparison test. Note for H, L, O, Q—the number of biological replicates within the particular group, n = 5, also indicated in the graph. Error bars indicate the standard deviation, p-values—two-way ANOVA with Turkey´s multiple comparison test. C Quantification of the surface area shifts between the WT Hindbrain Choroid Plexuses of E13.5, E15.5 and E17.5 displaying the statistically significant increase just between the first two analyzed samples. D Representation of the total Hindbrain Choroid Plexus volume (green) and the ventricle volume (yellow) measurement is visualized within the frontally positioned E13.5 embryo, which was stained by Lugol´s solution prior to µCT scanning. The data acquisition for this analysis was performed by the ChOP-CT tool. Scale bar: 0.65 mm. E Graphical representation of the gradual expansion of the 4th ventricle volume, which was observed every two days within our monitored period of embryonic development (from E13.5 to E17.5). F Quantification of the shift in the proportional test of the Hindbrain Choroid Plexus volume in the total volume of the 4th ventricle between the WT E13.5, E15.5 and E17.5. G Sagittally presented a 3D reconstructed ventricular system and 3D model of the E13.5 Hindbrain Choroid Plexus illustrating the manual outlining of the rostral (green) and caudal (blue) parts of the Hindbrain Choroid Plexus used for the volume quantification of these Hindbrain Choroid Plexus parts. Scale bar: 0.2 mm. H No significant shift was observed in the volume proportion of the rostral and caudal part of the WT Hindbrain Choroid Plexuses of E13.5, E15.5 and E17.5. I The illustration of the ChOP-CT-based measurement of the Hindbrain Choroid Plexus outgrowth angle into the 4th ventricle visualized on the sagittally positioned E13.5 3D Hindbrain Choroid Plexus and reconstructed brain ventricular system (yellow). Scale bar: 0.25 mm. J Quantification of the differences in Hindbrain Choroid Plexus outgrowth angle between the WT E13.5, E15.5 and E17.5, showing the significant inclination of the E15.5 Hindbrain Choroid Plexus to the above-lying cerebellum compared to other (E13.5 and E17.5) analyzed samples. K Transversally positioned 3D model of the E13.5 Hindbrain Choroid Plexus illustrating the manual measurement of the outgrowth angle of the individual (left and right) Hindbrain Choroid Plexus branches. Scale bar: 0.25 mm. L No disruption of the left–right symmetry between the WT Hindbrain Choroid Plexuses of E13.5, E15.5 and E17.5 in terms of outgrowth angle was detected by the quantifications of the outgrowth angle parameter. M Transversally positioned 3D model of the E13.5 Hindbrain Choroid Plexus model illustrating the manual measurement of the total length of the individual (left and right) Hindbrain Choroid Plexus branches. Scale bar: 0.25 mm. N Quantification of the Hindbrain Choroid Plexus length differences between the WT Hindbrain Choroid Plexuses of E13.5, E15.5 and E17.5 presenting the gradual expansion of the Hindbrain Choroid Plexus into the ventricular space. O Quantification of the variations in left and right Hindbrain Choroid Plexus branch length between the WT Hindbrain Choroid Plexuses of E13.5, E15.5 and E17.5. This manually measured analysis confirmed no breakage of the left–right symmetry in this morphological parameter. P The illustration of the length measurement of the ventricle (pink), the whole Hindbrain Choroid Plexus (blue) and the central Hindbrain Choroid Plexus part (white) using the E13.5 Hindbrain Choroid Plexus 3D model positioned in the segmented dorsal ventricular system. These body parts are presented within the embryo in the transverse plane. Scale bar: 0.35 mm. Q Quantification of the differences in the ventricle length, total Hindbrain Choroid Plexus length as well as the length of the central Hindbrain Choroid Plexus between the WT Hindbrain Choroid Plexuses of E13.5, E15.5 and E17.5 displaying the increasing proportion of lateral Hindbrain Choroid Plexus segments within the lateral recesses of the 4th ventricle as the total Choroid Plexus length is gradually increasing but the length of the Hindbrain Choroid Plexus central part does not. The analysis showed no differences between the left and right branches. R–T Frontal (left vertical panel), transverse (middle vertical panel) as well as sagittal (right vertical panel) views of the mean (green), minimal (grey) as well as maximal (red) models of the Hindbrain Choroid Plexus of E13.5 (S), E15.5 (T) and E17.5 (U). Scale bars can be found within the figures. U The illustration of the position of the landmarks within the E13.5 mean model of Hindbrain Choroid Plexus, which is positioned transversally. Scale bar: 0.35 mm. V Higher shape variability was detected within the group of E13.5 and E15.5 Hindbrain Choroid Plexuses compared to the E17.5 Hindbrain Choroid Plexus group as these two groups display greater procrusion distances between the landmarks placed within the individual Hindbrain Choroid Plexus 3D model and corresponding 3D mean models. Scale bars can be found in the figures. 3D three dimensional, µCT X-ray micro-computed tomography, E embryonic day, ChOP-CT ChOPping the ChOroid Plexus out of the µCT data, WT wild-type