Fig 1: Bulk RNA-seq analysis associated the Shh-related genes with lingual dorsal–ventral patterning. (A) Volcano plot displaying the DEGs between E13.5 WT dorsal and ventral tongues. (B) Venn diagram showed that there were 11 and 6 genes identified in the intersection of the dorsal–ventral patterning gene set, respectively, with the highly expressed genes in E13.5 WT dorsal (left) and ventral tongue (right). (C) The PPI network was constructed using the 184 Shh-related dorsal highly expressed genes, as shown in Supplementary Figure S3D. The violet nodes represent the genes in the Shh-related module. (D) HC-PIN in CytoCluster extracted the largest and most pivotal module, the Shh-related module, from the PPI network. (E) ANOVA assay on bulk RNA-seq showed that the transcription of Shh, Foxa2, Foxf1, and Gsc was much higher in WT dorsal than in the WT ventral tongues, but it was comparable between the WT ventral and Osr2-cre KI ;Rosa26R-Fgf8 ventral tongues. In contrast, Foxa2 transcription in Osr2-cre KI ;Rosa26R-Fgf8 dorsal tongues was significantly weaker than that in WT dorsal tongues, but it was more robust than those in WT ventral and Osr2-cre KI ;Rosa26R-Fgf8 ventral tongues. However, the transcription of Shh, Foxf1, and Gsc in Osr2-cre KI ;Rosa26R-Fgf8 dorsal tongues showed no significant difference from that in WT dorsal tongues, although it was still more robust than those in WT ventral and Osr2-cre KI ;Rosa26R-Fgf8 ventral tongues. (F) Immunofluorescence showed that the Foxa2 distribution in the dorsal epithelium and Foxf1 distribution in the lateral–dorsal mesenchyme of E13.5 WT tongue were reduced dorsally in the superficial mesenchyme underlying the dorsal epithelium, but it was still robust in the median mesenchyme of the E13.5 Osr2-cre KI ;Rosa26R-Fgf8 tongue. White arrows indicate the Foxa2 and Foxf1 boundary in the superficial dorsal mesenchyme. Similarly, the robust Gsc expression in the dorsal superficial mesenchyme of E13.5 WT tongue was absent in the superficial dorsal mesenchyme of the Osr2-cre KI ;Rosa26R-Fgf8 tongue (indicated by red asterisks), while the mild Gsc expression in the medial and ventral mesenchyme of E13.5 WT tongue was slightly impacted in the Osr2-cre KI ;Rosa26R-Fgf8 tongue. (G) Whole-mount in situ hybridization revealed suppressed Shh transcription in the E13.5 Osr2-cre KI ;Rosa26R-Fgf8 tongue. Scale bar: 200 µm.
Fig 2: Bulk RNA-seq revealed the Fgf8/Fgf18-Shh correlation in dorsal–ventral patterning in E13.5 WT and Osr2-cre KI ;Rosa26R-Fgf8 tongues. (A) Scatter plot showed 238 DEGs between E13.5 WT dorsal and Osr2-cre KI ;Rosa26R-Fgf8 dorsal tongues, among which 74 genes were upregulated (red dots) and 164 genes downregulated (blue dots) in the Osr2-cre KI ;Rosa26R-Fgf8 dorsal tongue. (B) The PPI network constructed using the 238 DEGs showed the largest and most pivotal module, in which Fgf8/Fgf18 and Shh acted as key nodes, extracted by HC-PIN in CytoCluster. The violet nodes represent the genes in the Fgf8/Fgf18-Shh related module. (C) Four-quadrant analysis of the Fgf8/Fgf18-Shh-related module in E13.5 WT dorsal and Osr2-cre KI ;Rosa26R-Fgf8 dorsal tongues. X-axis: E13.5 WT dorsal vs. Osr2-cre KI ;Rosa26R-Fgf8 dorsal tongues. Y-axis: E13.5 WT dorsal vs. WT ventral tongues. The genes in the boxes above the four-quadrant plot represent transcription factors. The green-labeled transcription factors were robustly expressed in the WT dorsal tongue, the yellow-labeled transcription factors were upregulated in the Osr2-cre KI ;Rosa26R-Fgf8 dorsal tongue, and the blue-labeled transcription factors were robustly expressed in the WT ventral tongue but downregulated in the Osr2-cre KI ;Rosa26R-Fgf8 dorsal tongue. The gray transcription factors, namely, Etv4, Etv5, Shox2, and Glis3, showed difference between the WT dorsal and ventral tongue but were upregulated or downregulated in the Osr2-cre KI ;Rosa26R-Fgf8 dorsal tongue. (D) The PPI network of the 12 transcription factors in the boxes above the four-quadrant plots showed that Fgf8 was the key node connecting Shh-related and Fgf8/Fgf18 modules in the Osr2-cre KI ;Rosa26R-Fgf8 tongue. (E) Boxplots from bulk RNA-seq showed that the expression of Etv4 and Etv5 was upregulated only in the E13.5 Osr2-cre KI ;Rosa26R-Fgf8 dorsal tongue but showed little difference among the E13.5 WT dorsal and ventral tongues and the Osr2-cre KI ;Rosa26R-Fgf8 ventral tongue. Boxplots also showed that the transcription of Lhx6 was significantly higher in the E13.5 Osr2-cre KI ;Rosa26R-Fgf8 dorsal tongue than in the WT dorsal tongue, but it was comparable in the Osr2-cre KI ;Rosa26R-Fgf8 ventral and WT ventral tongues. (F) Immunofluorescence showed that the Lhx6 distribution in the lateral–ventral mesenchyme of the E13.5 WT tongue was extended dorsally in the E13.5 Osr2-cre KI ;Rosa26R-Fgf8 tongue. White arrows indicate the Lhx6 boundary. Although the robust Etv4 expression in the medial mesenchyme of the E13.5 WT tongue was impacted slightly in the Osr2-cre KI ;Rosa26R-Fgf8 tongue, ectopic Etv4 activation was detected in the dorsal superficial mesenchyme and epithelium of the E13.5 Osr2-cre KI ;Rosa26R-Fgf8 tongue. The yellow boxes show the Etv4 distribution in the dorsal superficial mesenchyme and epithelium, which are amplified in the left and lower corners; the dashed lines delineate the boundary between the lingual epithelium and mesenchyme. Scale bar: 200 µm.
Fig 3: Suppression of Shh and Shh-related dorsal-specific gene expression by FGF8. (A) Whole-mount in situ hybridization revealed that the Shh transcription in the E13.0 WT tongue was suppressed by implanted FGF8-soaked agarose beads. (B–D) Immunofluorescence displayed that implanted FGF8-soaked agarose beads inhibited Gsc [red asterisk in (B)] and Scx expression [red asterisk in (C)] but not Foxa2 expression in the E13.0 WT tongue [white arrow in (B)]. In contrast, implanted FGF8-soaked agarose beads induced Lhx6 expression [red asterisk in (D)] in the E13.0 WT tongue. (E) Shh-cre;Rosa26R-mT/mG tongues exhibited Cre activity in the E11.5 (white arrows) and E12.5 lingual epithelium. (F) Whole-mount in situ hybridization showed significantly reduced Shh transcription in the E13.5 Shh-cre;Rosa26R-Fgf8 tongue. (G) Immunofluorescence showed that both Foxf1 (indicated by white arrows) and Foxa2 distribution were reduced to the medial dorsal region in the E12.5 shh-cre;Rosa26R-Fgf8 tongue; Gsc expression was almost reduced in the E13.5 Shh-cre;Rosa26R-Fgf8 tongue (indicated by red asterisks), while Lhx6 (indicated by white arrows) and Etv4 expressions were increased throughout the mesenchyme and epithelium of the E12.5 Shh-cre;Rosa26R-Fgf8 tongue, respectively. Scale bar: 200 µm.
Fig 4: Ex vivo culture of E13.0 WT tongues supplemented with FGF18 in the dorsal and SHH in the ventral mesenchyme. (A) Whole-mount in situ hybridization indicated suppressed Shh transcription in the E13.0 WT tongue by implanted FGF18-soaked agarose beads. (B–D) Immunofluorescence showed that implanted FGF18-soaked agarose beads inhibited Foxf1 expression [red arrows in (B)] but induced Lhx6 expression [red arrows in (C)]. Meanwhile, Foxa2 expression in the E13.0 WT tongue was impacted slightly by exogenous FGF18. (E–H) In contrast, immunofluorescence showed that the tongues with SHH-soaked agarose beads failed to repress Fgf18 [white arrows in (E)] and Lhx6 expression [white arrows in (F)] in the E13.0 lingual ventral mesenchyme. Similarly, SHH also failed to induce Foxf1 [white arrows in (G)] and Foxa2 [white arrows in (H)] expression in the E13.0 lingual ventral mesenchyme.
Supplier Page from MedChemExpress for SHH Protein, Mouse