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doi: 10.1242/10.1242/dev.00172

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Gene expression profiles of mouse submandibular gland development: FGFR1 regulates branching morphogenesis in vitro through BMP- and FGF-dependent mechanisms

Matthew P. Hoffman*, Benjamin L. Kidder, Zachary L. Steinberg, Saba Lakhani, Susan Ho, Hynda K. Kleinman and Melinda Larsen

Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, 30 Convent Drive, MSC 4370, Bethesda, MD 20892-4370, USA



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  		Fig. 1. (A) Developmental stages of mouse SMGs used for array analysis. eb, epithelial bud; m, mesenchyme; tb, terminal bud; d, duct; bv, blood vessel; a, acini; sd, striated duct; gct, granular convoluted tubule. (B) A portion of the Atlas 1.2 cDNA arrays shows five examples of developmentally regulated genes during gland formation.

 


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  		Fig. 3. The expression profiles of several developmentally regulated genes were confirmed using RT-PCR. (A) The profile of gene expression of the selected genes from the Atlas 1.2 arrays is shown with relative pixel density on the y-axis. The profiles of gene expression were obtained using Atlas Navigator 1.0 software as described for Fig. 2. (B) The RT-PCR results confirm the profile of gene expression. The cDNAs were normalized to expression levels of Gapd and Actb (ß-actin) for the PCR.

 


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  		Fig. 4. FGFR isoforms and FGFs are developmentally regulated during SMG formation. RT-PCR analysis of FGFR family isoforms (A) and ligands (B) during submandibular gland development is shown with quantitation of the changes shown in the bar graph. The results include receptor/ligand isoforms not present on the Atlas arrays and cDNA from E12 and E13 mouse SMGs. The number of PCR cycles was optimized for each gene so that no products were saturated. Differences in expression levels were quantitated.

 


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  		Fig. 2. (A) The expression data from the Atlas arrays were clustered into eight groups of genes with similar expression profiles using Atlas Navigator software (Clontech Laboratories, Inc., Palo Alto, CA). A total of 468 genes were identified in at least one stage of SMG development. The number of genes in each cluster is written in parentheses. (B) Ten examples from each group are listed in order of their Atlas array coordinates (#). The table also includes their common name and a GenBank accession number (GB).

 


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  		Fig. 5. (A) Antisense oligonucleotides to Fgfr1 decrease branching morphogenesis of E12 SMGs; a representative photograph of each group is shown. (B) At least 6 glands at each timepoint were photographed and the number of terminal buds was counted. An unpaired t-test was used to compare branching. There was a significant decrease in the number of buds in the antisense-treated glands (**P<0.003). (C) Antisense oligonucleotide uptake into the mesenchyme (M) and the epithelial bud (E) was detected at 44 hours in a 7 µm optical section taken by confocal microscopy. (D) Antisense oligonucleotides to Fgfr1 decrease Fgfr1c expression by ~75% and Fgfr11b expression by~25%. The expression of Fgfr11b and Fgfr1c were normalized to expression levels of Gapd.

 


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  		Fig. 6. An inhibitor of FGFR1 tyrosine phosphorylation, SU5402, inhibits branching morphogenesis of E13 SMGs. (A) A time-course of SU5402 treatment reveals that at 20 hours (T20) the epithelial buds do not enlarge and cleft. The major duct continues to enlarge and undergo lumen formation (arrowhead). (B) Dose response of SU5402 treatment at 20 hours. The number of buds is expressed as a ratio of the number of buds at 20 hours/the number of buds at 2 hours (T20/T2).

 


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  		Fig. 7. SU5402 treatment decreases epithelial cell proliferation but does not cause apoptosis of the epithelial buds. (A-D) BrdU incorporation at 44 hours was detected with fluorescently labeled BrdU. The proliferating cell nuclei appear as green punctate spots. Peanut lectin-rhodamine stains the epithelium red and anti-perlecan stains mesenchyme and the basement membrane blue. The images in B, D, E and G are compressed stacks of optical sections through the entire gland. (A) Light micrographs of control gland. (B) Control gland shows BrdU labeling concentrated on the epithelial buds and at the periphery of the mesenchyme. (C) Light micrographs of SU5402-treated gland. (D) SU5402 treatment results in less BrdU labeling on the epithelial buds. Proliferating cells are still apparent in the mesenchyme. (E-I) SU5402 treatment does not cause increased apoptosis at 20 or 44 hours. Apoptosis of mesenchyme cells at the edges and on the surface of the glands in culture was detected with TUNEL staining in red. FITC-peanut lectin stains the epithelium green and anti-perlecan stains mesenchyme and the basement membrane blue. (E) Low power whole-mount view of control gland cultured for 20 hours shows red apoptotic nuclei in the mesenchyme. (F) Higher power section showing apoptotic nuclei in the mesenchyme, but none are detected in the epithelium. (G) Low power whole-mount view of gland treated for 20 hours with SU5402. (H) Higher power section showing apoptotic nuclei in the mesenchyme. (I) The fluorescent BrdU and apoptosis staining were quantitated using the MetaMorph image analysis program. The total fluorescent pixels were expressed as a ratio of the area of the gland. At least 5 glands/condition were used for quantitation and the experiments were repeated three times. *P<0.05, **P<0.01, ns, not significant.

 


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  		Fig. 8. (A) Fgfr, Fgf and Bmp gene expression was localized to either epithelium or mesenchyme. E13 submandibular glands (n=20) were micro-dissected into epithelium (E) and mesenchyme (M) and the light micrographs show a typical gland before and after separation. Gene expression was analyzed by RT-PCR with GAPDH and S29 as controls. The experiment was repeated three times with similar results. (B) SU5402 treatment modulates gene expression of FGFRs, FGFs and BMPs. Expression of FGFs, FGFRs and BMPs was analyzed by real time PCR at 2, 6, and 20 hours of SU5402 treatment. E13 SMGs (n=6) were cultured with either 5 µM of SU5402 (black triangle) or an equal volume of DMSO (white square) for 2, 6 or 20 hours. Real time PCR was performed using SYBR Green PCR Master Mix and a TaqManTM 7700 thermocycler. The results are expressed as fold increase in gene expression compared to the control glands after 2 hours of culture. The reactions were run in triplicate, the experiment repeated three times, and the results were combined to generate the graphs.

 


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  		Fig. 9. (A) Exogenous FGFs and BMPs modulate SMG morphology. A range of concentrations of exogenous growth factors were added to glands and cultured for 20 hours. An example of each is shown, FGF1 (100 ng/ml), FGF2 (100 ng/ml), FGF7 (200 ng/ml), FGF10 (500 ng/ml), BMP7 (100 ng/ml), and BMP4 (100 ng/ml), were added to glands and cultured for 20 hours. The gland with BMP4 is also shown after 42 hours in culture, and resembles a SU5402-treated gland. (B) FGF7, FGF10 and BMP7 rescue SMGs treated with SU5402 (1.5 µM ~ IC50). The addition of FGF7 (500 ng/ml), FGF10 (500 ng/ml), or BMP7 (100 ng/ml) rescues SMGs treated with SU5402 (1.5 µM=IC 50). FGF1 (100 ng/ml) is not able to rescue the glands. The number of buds=(T42/T2), the number of buds at 42 hours/the number of buds at 2 hours. (C) An image of a gland from each group is shown after 44 hours of culture.

 

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© The Company of Biologists Ltd 2002