Analysis of the HPE phenotype in cdKO embryos.

(A) Scanning electron microscopy (SEM) images of the frontal anterior view of embryos at 8.25 dpc, from Tgif1;Tgif2 conditional double intercrosses with epiblast specific deletion of the conditional Tgif1 allele (referred to as cdKO), Shh mutant intercrosses and a stage matched control are shown. The genotype of the control embryos is not indicated as they are representative of normal embryos from these crosses. The arrow indicates the separation of ventral lips of the cephalic folds in the control, that is defective in the cdKO and Shh null (marked by asterisks). Note, the conditional Shh null allele is referred to as ‘r’, for recombined. (B) SEM images of the frontal view of the forebrain of control and cdKO embryos at 8.75 and 9.25 dpc are shown. (C and D) Whole-mount images and hematoxylin and eosin (H&E) stained coronal section of fixed and paraffin-embedded control and cdKO embryos at 9.0 (C) and control, cdKO and Shhr/r at 10.0 dpc (D). The white lines indicate the plane of the coronal sections through the forebrain vesicle. Embryos are representative of at least 3 analyzed. In D, the division of the nasal field by the neuroepithelium is marked by an arrow. Note the continuous thickened layer of surface ectoderm in the mutants. (E) Whole mount images and H&E stained sections of fixed and paraffin-embedded control, cdKO and Shh null embryos at 12.5 dpc are shown. The two planes of section are indicated in the upper panels, and a magnified view of the eye is shown at the bottom (boxed region in section ii). Only two cdKO embryos were identified at this stage. Scale bars: 100 µm in A and B; 250 µm for whole-mount and 100 µm for section in C; 500 µm for whole-mount and 200 µm for sections in D; 2 mm for whole-mount, 250 µm for i, 500 µm for ii and 100 µm for ii-zoom in E.



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