Fig. 7: Gene expression changes upon multiple Cdh gene mutations are revealed to be minimal by means of RNA-sequencing (seq).

a Summarized are sequential steps to select Fgf15 by analyzing the RNA-seq dataset between the WT_midbrain (Mb) and Cdh6/8/11 triple knockout (TKO)_Mb. b Selection processes for Fgf15 are outlined by the volcano plots. Each step to narrow down those plots is explained in a, and the selected number of plots are also specified by the same color used in a. In brief, the upper most volcano plots indicate all transcripts that are differentially expressed between the WT_Mb and Cdh6/8/11 TKO_Mb. In the volcano plots, y and x axis show the negative log₁₀ of the adjusted p value and log₂ fold changes, respectively. The horizontal dashed black line represents the significance threshold (Benjamin–Hochberg adjusted p value (FDR) < 0.05) and those plots under adjusted p value are cut off (painted by light pink) to select 100 plots (orange in a). In the middle plots, those plots similarly fluctuating between the Cdh8/11 DKO_Mb and Cdh6/8/11 TKO_Mb are selected (dark pink in a). As the next step, those plots also expressed and fluctuated in the Fb and showing similar fluctuating property with the Mb are excluded to select seven plots in the lower-most panel (purple in a). Note that those seven molecules harbor statistical significance, but they do not fluctuate drastically among genotypes. Finally, we focus on Fgf15 which is the only candidate gene reported to be involved in the neural plate/tube patterning (dark purple in a). c Seven candidate genes specifically fluctuated in the Mb are additionally evaluated by the box plots. Left and right panels show fragments per kilobase of exon per million reads mapped (FPKM) in the Fb and Mb, respectively. Boxes indicate individual data from WT (colored by gray), Cdh8/11 DKO_cranial defect (CD) (colored by dark blue), Cdh8/11 DKO_exencephaly (Ex) (colored by dark green) and Cdh6/8/11 TKO_Ex (colored by dark magenta) mice, respectively. *FDR < 0.05, **FDR <0.01, ***FDR < 0.001. d, g Section levels of an E9.0 e, f or an E9.5 h–j embryo are depicted. e, f, h–j ISH for Fgf15 mRNA is performed in WT, non-excencephalic (EX) Cdh8/11 DKO and EX Cdh8/11 DKO mice. The phenotypic penetrance for EX or non-EX is indicated at the both sides of f. Although the dorsal midline in WT or non-EX Cdh8/11 DKO is completely negative for Fgf15 (white arrowheads), the neural ridge in the exencephalic Mb continuously expresses Fgf15 mRNA (black arrowheads). Scale bar: 100 μm.