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First published online 11 July 2007
doi: 10.1242/dev.005066

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Semaphorin signaling facilitates cleft formation in the developing salivary gland

Ling Chung¹, Tsung-Lin Yang^1,*, Hsiu-Ru Huang^1,*, Su-Ming Hsu^1,2, Hwai-Jong Cheng^3, and Pei-Hsin Huang^1,2,,

¹ Graduate Institute of Pathology, College of Medicine, National Taiwan University, Taipei, Taiwan.
² Department of Pathology, National Taiwan University Hospital, Taipei, Taiwan.
³ Center for Neuroscience, University of California, Davis, CA 95618, USA.

View larger version (112K): [in this window] [in a new window]   Fig. 1. Neuropilin 1 is transiently expressed in the developing mouse SMG. (A) Semi-quantitative RT-PCR analysis of neuropilins on the cDNA samples prepared from SMGs at the indicated developmental stage. Npn1, but not Npn2, was transiently detected in the embryonic SMG. Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) transcript is shown as an internal control. The amount of Npn1 transcript was normalized against the internal control at each stage for comparison. (B) RNA in situ hybridization analysis of Npn1 transcript on tissue sections. Npn1 was detectable in the rudimentary SMG at E12.5 (a). The expression of Npn1 transcript peaked at E15.5 (b), and was dramatically decreased at E17.5 (c), P1 (d) and adult mouse (e). No signals were detectable in the SMG by using antisense Npn2 or sense Npn1 probes. The boxed areas in a and b are magnified and shown in a' and b',b'', (original magnification 400x), respectively. Areas within dashed lines: salivary gland. (C) Colocalization of Npn1 transcript with E-cadherin, but not with fibronectin, in E15.5 SMG cultured ex vivo. (D) Semi-quantitative RT-PCR analysis of the cDNA samples prepared from either SMG epithelium or the surrounding mesenchyme. Npn1 was mainly expressed in the SMG epithelium. Fgfr2 and Fgf7 were controls for specific expression in Epi and in M, respectively. Scale bars: 50 µm in B; 100 µm in C. DA, descending aorta; DRG, dorsal root ganglion; Epi, epithelium; GAPDH, Gapdh internal control; IF, immunofluorescence staining; ISH, RNA in situ hybridization; M, mesenchyme; Nc, nasal cavity; PC, positive control; SC, spinal cord; SCG, superior cervical ganglion; SMG, submandibular gland.

View larger version (65K): [in this window] [in a new window]   Fig. 2. Neuropilin 1 is required for SMG branching morphogenesis in mice. (A) Npn1-neutralizing antibody inhibited SMG branching in a concentration-dependent manner. (a) Representative photographs at the indicated time point of the SMG explants treated with either control antibody or different concentrations of anti-Npn1 antibodies. (b) The numbers of terminal buds in each cultured SMG explant were counted and summarized (from five independent experiments). Paired t-test: *, P<0.05; **, P<0.01. (B) ODNs against Npn1 mRNA specifically blocked SMG branch formation. (a) Top panels: representative photographs of the SMG explants cultured 48 hours after treatment with 2 µM Npn1 antisense ODNs, scrambled ODNs or Npn1-sense ODNs. Middle panels: RNA in situ hybridization indicated that the Npn1 transcript decreased in the explant treated with Npn1 antisense ODNs. Bottom panels: BrdU (bromodeoxyuridine) labeling revealed that the rates of cell proliferation were not significantly changed in each experimental condition. (b) The numbers of terminal buds in each SMG explant cultured for 48 hours were counted and are summarized as a bar graph (from five independent experiments). Paired t-test: *, P<0.05. (c) Quantification of the proliferative activity labeled by BrdU is shown by bar graph as the green fluorescence intensity relative to DAPI-stained blue fluorescence intensity per unit area analyzed by MetaMorph software (n=12). (C) Semi-quantitative RT-PCR indicated that Npn1 transcripts were significantly reduced in the SMG treated with Npn1 antisense ODNs. Scale bars: 100 µm. AS, Npn1 antisense ODN; C, scrambled sequence; HGF, hepatocyte growth factor; S, Npn1-sense ODN.

View larger version (90K): [in this window] [in a new window]   Fig. 3. Sema3A and Sema3C additively promote SMG cleft formation. (A) Sema3A and Sema3C are the only class 3 semaphorins that could promote cleft formation in the SMG co-culture assays. Representative photographs of the growth of SMG explants co-cultured with class 3 semaphorins were shown. The bar graph summarizes the ratios of the number of terminal buds in each co-culture to the number of the terminal buds in the control culture (n7). The number of terminal buds at the presence of Sema3A or Sema3C was almost doubled. (B) Treatment of antisense ODNs against Sema3A or Sema3C specifically reduced the number of terminal buds in the SMG cultured ex vivo. Representative explants are shown. The bar graph summarizes five independent experiments. The terminal bud number was significantly further reduced when both Sema3A antisense ODNs and Sema3C antisense ODNs were added together into the culture. Paired t-test: *, P<0.05; **, P<0.01. (C) Sema3A and Sema3C additively promoted bud formation in a concentration-dependent manner. In the SMG co-culture experiments, Sema3-transfected COS cells were serially diluted to test the synergistic effects. Fold dilution in the co-culture is indicated as the Sema3-transfected COS cells diluted with mock-transfected COS cells. (a) Additive effects of Sema3A and Sema3C were most obvious at the lower concentrations of semaphorins. The effects were saturated at 1:1 dilution. (b) The amounts of semaphorin proteins present in each condition were assayed by western blotting. Gradual decreases of Sema3A or Sema3C in the serial dilutions were observed. Tubulin: internal loading control. (c) The bar graph summarizes four independent experiments. Scale bars: 100 µm. C, scrambled sequence.

View larger version (79K): [in this window] [in a new window]   Fig. 4. Functional Npn1 is required for the Sema3A- and Sema3C-mediated SMG branching morphogenesis in mice. (A) Anti-Npn1 antibody dose-dependently abolished SMG cleft formation promoted by Sema3A or Sema3C in the SMG co-cultures. Complete inhibition could be achieved in the presence of 5 µg/ml neutralizing antibody. Representative explants are shown and the summary of six independent experiments is shown in the bar graph. Paired t-test: *, P<0.05; **, P<0.01. (B) Npn1-AP fusion proteins, but not AP proteins, blocked Sema3A-mediated branching activity in the SMG co-cultures. Note that Npn1-AP fusion protein alone could block the endogenous branching activities (Mock) (upper panels). By contrast, application of Npn2-Fc fusion proteins in the co-cultures had no effects on the SMG branching activity (lower panels). Representative explants were shown and the summary of five independent experiments is shown in the bar graph. Paired t-test: *, P<0.05; **, P<0.01. (C) Sema3A-AP bound the epithelial buds in the SMG cultured ex vivo for 24 hours. As a control, when the Sema3A-AP conditioned medium was depleted of the AP-fusion proteins by pre-incubation with Npn1-transfected COS cells, the binding activity on the epithelial buds was greatly reduced. (D) Sema3A mRNA was detected in the SMG epithelial buds cultured ex vivo (a) and in E15.5 embryonic SMG (b). The expression was distributed as a gradient with the highest level at the front end of the terminal bud. Immunofluorescence staining of E-cadherin highlighted the epithelial buds in a. Area within dashed line, epithelial bud. (E) Semi-quantitative RT-PCR analysis confirmed that Sema3A transcript was mainly in the SMG epithelium. Scale bars: 100 µm. C, mock-transfected COS cells; Epi, epithelium; IF, immunofluorescence; ISH, RNA in situ hybridization; M, mesenchyme.

View larger version (46K): [in this window] [in a new window]   Fig. 5. VEGF is not required for Npn1-mediated cleft formation in mice. (A) Whole-mount immunohistochemistry showed that VEGF and its receptors, Flt1 and Flk1, did not express in the epithelial buds of the SMG. By contrast, Fgfr2 was expressed in the SMG epithelial buds as previously reported. (B) Neither exogenous VEGF nor VEGF antibody (VEGF Ab) affected normal branching morphogenesis of the SMG cultured ex vivo. The bar graph summarizes five independent experiments. (C) VEGF had no effects on the branching activities promoted by Sema3A. The bar graph summarizes four independent experiments. (D) VEGF could not change effects caused by application of Npn1-neutralizing antibody at sub-optimal concentration (2 µg/ml) in the SMG ex vivo cultures. The bar graph summarizes five independent experiments. Scale bars: 100 µm.

View larger version (65K): [in this window] [in a new window]   Fig. 6. Plexin A2 and plexin D1 are required for class 3 semaphorin-mediated cleft formation. (A) SMG cleft formation was reduced in the SMG ex vivo cultures treated with antisense ODNs against plexin A2 or plexin D1 for 48 hours. No effects were observed in the SMG explants dissected from knockout mouse mutants of plexin A3 or plexin A4, or in the SMG explants treated with antisense ODNs against other plexins. Representative explants are shown in a and the summary of five independent experiments is shown as a bar graph in b. Paired t-test: *, P<0.05 while compared with the controls. (B) SMG cleft formation promoted by Sema3A in the SMG co-cultures was partially abolished by the treatment of antisense ODNs against plexin A2 or plexin D1, but not by sense ODNs. Representative photographs were taken from 24-hour co-cultures (a). A summary of seven independent experiments is shown in b. Scale bars: 100 µm. AS, antisense ODNs; KO, knockout mutants; S, sense ODNs.

View larger version (56K): [in this window] [in a new window]   Fig. 7. FGF-induced SMG branching morphogenesis is independent of Npn1 function in mice. (A) Semi-quantitative RT-PCR analysis indicated that the expression of Fgf10, Fgf7 and Fgfr2 in the SMG explants were not altered when the SMG cultures were treated with Npn1 antisense ODNs. Gapdh is an internal control. C, no treatment; S, SMG cultures treated with Npn1 sense ODNs. (B) Fgf7 (500 ng/ml) and Fgf10 (1000 ng/ml) still effectively promoted SMG branching formation in the presence of Npn1 antisense ODNs (a). Representative photographs were taken from 24-hour cultures. A summary of five independent experiments is shown in b. Scale bar: 100 µm. AS, treated with Npn1 antisense ODNs; C, control peptide; S, treated with Npn1 sense ODNs.

View larger version (22K): [in this window] [in a new window]   Fig. 8. Sema3A restricts migration of cultured SMG epithelial cells. (A) The migration of mouse epithelial cells in mesenchyme-free SMG explant culture was restricted by synthetic Sema3A peptide (100 ng/ml). (B) The extent of epithelial cell migration is summarized at three time points, as indicated. The migration ratios were obtained from dividing the area measured at each time point by the area measured at 0 hours. (C) Sema3A caused cytoplasmic constriction of the SMG epithelial cells. Dissociated epithelial cells from E13.5 SMG were cultured for 2 days and then treated with synthetic Sema3A or scrambled peptide for another 1 or 2 days. Cells were fixed with paraformaldehyde and stained with rhodamine phalloidin. Scale bar: 100 µm.

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