DEV005066 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Expression of class 3 semaphorin in the developing SMGs. (A) Semi-quantitative RT-PCR analysis of transcripts of class 3 semaphorins on the SMGs from different developmental stages. Almost all tested class 3 semaphorins were detectable in the E13 and E17 SMGs. Bar graph shows normalized ratio for the signal from each class 3 semaphorin against the signal from the house-keeping gene Gapdh. (B) RNA in situ hybridization analysis of class 3 semaphorins in the E15.5 mouse embryos. Sema3A and Sema3C were abundantly expressed in the SMGs. Scale bar: 50 μm.

• Supplemental Figure S2 -

  Fig. S2. Sema3A-mediated branching activity does not cause changes of the proliferation activity in the SMG. (A) Partial inhibition of cell proliferation by tunicamycin (1.75 ng/ml) did not interfere with Sema3A-promoted budding activity (n=4 for bar graph). Scale bar: 100 μm. (B) The proliferation activities in the SMG explants were compared by performing BrdU incorporation assay in the Sema3A-treated and mock-treated co-cultures. No significant changes in proliferation activity were detected in either epithelium or mesenchyme analyzed by MetaMorph software (n=5 for bar graph). Scale bar: 100 μm.

• Supplemental Figure S3 -

  Fig. S3. Expression pattern studies of all members of plexin family in the developing SMGs. (A) Semi-quantitative RT-PCR analysis of transcripts of members of plexin family on the SMGs from different developmental stages. Only plexin A1, A2 and D1 were detectable in the E13 SMGs. Members of plexin B family were detectable at late embryonic stages and after birth. Bar graph showing normalized ratio for the signal from each plexin against the signal from the house-keeping gene Gapdh. (B) RNA in situ hybridization analysis of all plexins in the E15.5 mouse embryos. At this stage, plexin A1, plexin A2 and plexin D1 were abundantly expressed in the SMGs. H&E: Hematoxylin and Eosin stain. Scale bar: 50 μm.

• Supplemental Figure S4 -

  Fig. S4. The expression of plexin A2 and plexin D1 in the SMG is developmentally regulated. (A) Plexin D1 was abundantly expressed in the E15.5 and E17.5 SMGs. The transcripts were reduced after birth and were not detectable in the adult SMG. The SMG in b (boxed) is magnified in b′. Dashed area, salivary gland. Scale bar: 50 μm. (B) Plexin A2 was highly expressed in the E15.5 SMG but was significantly reduced at E17.5. No plexin A2 signal could be detected after birth. The SMG in a (boxed) is magnified in a′. Area within dashed lines, salivary gland; SC, spinal cord; SCG, superior cervical ganglion; SMG, submandibular gland. Scale bar: 50 μm. (C) Plexin D1 transcripts were localized in the epithelial buds of the SMG. The signals colocalized with those of the epithelium marker E-cadherin (b and b′), but not with those of the mesenchymal marker fibronectin (c and c′). ISH, RNA in situ hybridization; IF, immunofluorescence. Scale bar: 100 μm.