Fig 2: Craniofacial skeleton abnormalities of Trps1−/− mice compared to WT. Measurements were done on E18.5 head skeletal preparations stained with Alcian blue and Alizarin red. Representative images are shown. (A) Landmarks used for head measurements. (B) Comparison of E18.5 WT (left) and Trps1−/− (right) lateral skull (top panel) and mandible (middle panel: lateral view; bottom panel: intraoral view). Coronoid process, condylar cartilage, and angular cartilage of Trps1−/− mice were hypoplastic (dotted arrows). The mandibular incisors have not erupted in Trps1−/− mandible (solid arrow). Meckel’s cartilage and molar crypts were present in both WT and knockout phenotypes. (C) There was no significant difference in total length of the head between the two groups but the nose was significantly shorter and downward-sloping in the Trps1−/− mice. The Trps1−/− mandible was also significantly shorter than WT. Red dashes represent the mean value in each data set. Solid data points represent individual samples, data points with white circle within indicate two samples with the same measurement and data points with white cross within indicate three samples with the same measurement. Error bars indicate the standard deviation of the sample. Asterisk indicates statistically significant differences between the WT and Trps1−/− groups (p < 0.05). Scale bar: 2 mm. ag – angular process, cr – coronoid process, cd – condylar process, fr – frontal bone, in – incisor, mc – Meckel’s cartilage, mlc – molar crypt, n – nasal bone, nc – nasal cartilage, so – supraoccipital.

Fig 3: Zygomatic arch and cranial base abnormalities in Trps1−/− mice. Skeletal staining of E18.5 WT (left) and Trps1−/− (right) mice. (A,B) Inferior view of skull demonstrating hypoplastic zygomatic process (black arrows) of the maxillary bone in Trps1−/− mice. (C,D) Magnified view of the nasal area illustrating underdeveloped vomer bones in Trps1−/− mice (yellow arrows). (E,F) The basioccipital bone is unaffected in Trps1−/− mice, while the basisphenoid bone is smaller and the cleft palate is evident with the unfused palatal shelves. (G,H) Removal of palatine bones revealed the absence of presphenoid bone in Trps1−/− mice (dotted arrow). (I,J) Cranial vault bones showed no apparent differences between the two groups. Scale bar: 2 mm. bo – basioccipital, bs – basisphenoid, p – palatine bone, ps – presphenoid.

Fig 4: Trps1 is required for the initiation of the palatal shelves fusion and expression of CSPG on palatal and nasal septum surfaces. (A,B) Stereoscope imaging and (C,D) H&E staining of palatal shelves isolated from E13.5 mice embryos and cultured ex vivo for 48 h. Of note, palatal shelves remained in contact with each other during ex vivo culture; however, Trps1−/− palatal shelves separated during histological processing, therefore only one palatal shelf is present on the image. Fusion of palatal shelves (red arrow) was initiated in all WT palatal shelves (n = 11) (C) while Trps1−/− palatal shelves (D) were unable to initiate fusion (red arrowhead, n = 11). (E–H) Immunohistochemistry for CSPG on fusion surfaces (white arrows) and mesenchyme (yellow arrows) of palatal shelves and nasal septum in E14.5 WT (E,G) and Trps1−/− (F,H) mice. CSPG was absent on the epithelial surface of Trps1−/− mice palatal shelves and nasal septum (white arrowheads), while the mesenchymal expression appeared unaffected. Scale bar: 50 μm. NS – nasal septum, PS – palatal shelf, T – tongue.

Fig 5: No difference in apoptosis or proliferation between E14.5 WT and Trps1−/− mice palatal shelves. Top: H&E staining of frontal head sections. Boxed areas indicate palatal shelves areas magnified on the images in the middle and bottom panels. Middle and bottom: TUNEL staining and immunofluorescent staining for Ki-67 of WT and Trps1−/− mice demonstrated no apparent difference in apoptosis or proliferation, respectively, between the two groups. Scale bar: 50 μm. MN – mandible, NS – nasal septum, PS – palatal shelf, T – tongue.