Supplementary Material for: Exogenous Fibroblast Growth Factor-10 Induces Cystic Lung Development with Altered Target Gene Expression in the Presence of Heparin in Cultures of Embryonic Rat Lung

Objectives: Signaling by fibroblast growth factor (FGF) receptor (FGFR) 2IIIb regulates branching morphogenesis in the mammalian lung. FGFR2IIIb is primarily expressed in epithelial cells, whereas its ligands, FGF-10 and keratinocyte growth factor (KGF; FGF-7), are expressed in mesenchymal cells. FGF-10 null mice lack lungs, whereas KGF null animals have normal lung development, indicating that FGF-10 regulates lung branching morphogenesis. In this study, we determined the effects of FGF-10 on lung branching morphogenesis and accompanying gene expression in cultures of embryonic rat lungs. Methods: Embryonic day 14 rat lungs were cultured with FGF-10 (0–250 ng/ml) in the absence or presence of heparin (30 ng/ml) for 4 days. Gene expression profiles were analyzed by Affymetrix microchip array including pathway analysis. Some of these genes, functionally important in FGF-10 signaling, were further analyzed by Northern blot, real-time PCR, in situ hybridization and immunohistochemistry. Results: Exogenous FGF-10 inhibited branching and induced cystic lung growth only in cultures containing heparin. In total, 252 upregulated genes and 164 downregulated genes were identified, and these included Spry1 (Sprouty-1), Spry2 (Sprouty-2), Spred-1, Bmp4 (bone morphogenetic protein-4, BMP-4), Shh (sonic hedgehog, SHH), Pthlh (parathyroid hormone-related protein, PTHrP), Dusp6 (MAP kinase phosphatase-3, MKP-3) and Clic4 (chloride intracellular channel-4, CLIC-4) among the upregulated genes and Igf1 (insulin-like growth factor-1, IGF-1), Tcf21 (POD), Gyg1 (glycogenin 1), Sparc (secreted protein acidic and rich in cysteine, SPARC), Pcolce (procollagen C-endopeptidase enhancer protein, Pro CEP) and Lox (lysyl oxidase) among the downregulated genes. Gsk3β and Wnt2, which are involved in canonical Wnt signaling, were up- and downregulated, respectively. Conclusions: Unlike FGF-7, FGF-10 effects on lung branching morphogenesis are heparin-dependent. Sprouty-2, BMP-4, SHH, IGF-1, SPARC and POD are known to regulate branching morphogenesis; however, potential roles of CLIC-4 and MKP-3 in lung branching morphogenesis remain to be investigated. FGF-10 may also function in regulating branching morphogenesis or inducing cystic lung growth by inhibiting Wnt2/β-catenin signaling.

研究目的：成纤维细胞生长因子（fibroblast growth factor, FGF）受体2IIIb（fibroblast growth factor receptor 2IIIb, FGFR2IIIb）可调控哺乳动物肺部的分支形态发生。FGFR2IIIb主要表达于上皮细胞，而其配体FGF-10与角质形成细胞生长因子（keratinocyte growth factor, KGF; FGF-7）则表达于间质细胞。FGF-10基因缺失小鼠无法形成肺部，而KGF基因缺失动物的肺部发育正常，这表明FGF-10可调控肺部分支形态发生。本研究旨在探究FGF-10对胚胎大鼠肺组织体外培养中肺部分支形态发生及伴随的基因表达的影响。
实验方法：将胚胎第14天的大鼠肺组织在添加或不添加肝素（30 ng/ml）的条件下，用浓度范围为0~250 ng/ml的FGF-10培养4天。通过Affymetrix基因芯片分析基因表达谱，并进行通路分析。对其中部分在FGF-10信号通路中发挥关键功能的基因，进一步采用Northern印迹、实时荧光定量PCR、原位杂交及免疫组化进行验证分析。
实验结果：外源性FGF-10仅在添加肝素的培养体系中抑制分支形成并诱导囊性肺组织生长。最终共鉴定出252个上调基因与164个下调基因，其中上调基因包括Spry1（Sprouty-1）、Spry2（Sprouty-2）、Spred-1、骨形态发生蛋白4（bone morphogenetic protein-4, BMP-4）、音猬因子（sonic hedgehog, SHH）、甲状旁腺激素相关蛋白（parathyroid hormone-related protein, PTHrP）、双特异性磷酸酶6（MAP激酶磷酸酶3, MKP-3）及细胞内氯离子通道4（chloride intracellular channel-4, CLIC-4）；下调基因则包括胰岛素样生长因子1（insulin-like growth factor-1, IGF-1）、转录因子21（transcription factor 21, POD）、糖原蛋白1（glycogenin 1）、富含半胱氨酸酸性分泌蛋白（secreted protein acidic and rich in cysteine, SPARC）、前胶原C端肽酶增强蛋白（procollagen C-endopeptidase enhancer protein, Pro CEP）及赖氨酰氧化酶（lysyl oxidase, Lox）。参与经典Wnt信号通路的Gsk3β与Wnt2分别出现上调与下调。
研究结论：与FGF-7不同，FGF-10对肺部分支形态发生的调控作用依赖于肝素。已知Sprouty-2、BMP-4、SHH、IGF-1、SPARC及POD均可调控分支形态发生，但CLIC-4与MKP-3在肺部分支形态发生中的潜在作用仍有待进一步探究。FGF-10或可通过抑制Wnt2/β-连环蛋白信号通路，参与调控肺部分支形态发生或诱导囊性肺组织生长。