Ikari Tatsuya, Hiraki Akimitsu, Seki Katsuhiro, Sugiura Tsuyoshi, Matsumoto Kunio, Shirasuna Kanemitsu
Department of Oral and Maxillofacial Surgery, Graduate School of Dental Science, Kyushu University, Fukuoka, Japan.
Dev Dyn. 2003 Oct;228(2):173-84. doi: 10.1002/dvdy.10377.
We investigated the involvement of hepatocyte growth factor (HGF) in salivary gland (SG) branching morphogenesis. The mouse submandibular gland (SMG) starts to develop at embryonic day 11.5-12 (E11.5-E12), and branching morphogenesis occurs in the area between the mandibular bone and tongue between E14 and E16.5. Real-time reverse transcriptase-polymerase chain reaction showed that the expression of the c-met/HGF receptor gene in SMG increased and peaked between E14 and E16.5, concomitant with epithelial branching, and high levels of HGF mRNA were detected in the surrounding mesenchyme at E14-E15.5. Although strong expression of the HGF and c-met transcripts was observed in the tongue muscles, this expression was limited at E13.5-E14.5. Serum-free organ cultures were established, in which SG rudiments that contained SMG and sublingual gland (SLG) primordia (explant 1) and SMG/SLG rudiments with peripheral tissue that included part of the tongue muscle (explant 2) were isolated from E13.5 or E14 embryos. Mesenchyme-free SMG epithelium was obtained by the removal of mesenchymal tissue from explant 1. In the explant 1 and 2 organ cultures, SMG/SLG rudiments showed growth and branching morphogenesis, while mesenchyme-free epithelium failed to grow. When E13.5 or E14 mesenchyme-free epithelium and a recombinant human HGF (rh-HGF) -soaked bead were placed on Matrigel, the epithelium migrated toward the bead and formed branches, while the E13 epithelium failed to branch. The exogenous application of rh-HGF and anti-HGF antibody to the SMG/SLG rudiment cultures resulted in stimulation and inhibition, respectively, of branching morphogenesis. However, the response of E13.5 SMG to rh-HGF was very weak, while the branching of E14 SMG was enhanced strongly by rh-HGF. The branching morphogenesis of SMG was also inhibited by the addition of either antisense HGF or c-met oligodeoxynucleotides to the cultures. The development of SMG in explant 2, which was significantly better than in explant 1, was comparable to that seen in vivo. Moreover, the expression of both HGF and c-Met in the SMG of explant 2 was higher than in the SMG of explant 1. These findings provide the first demonstration that the branching morphogenesis of SMG is regulated by interactions with the surrounding mesenchyme-derived HGF and c-met expression in SMG, which occur concomitant with epithelial branching. The present data also suggest that the HGF that is released transiently from tongue muscles may contribute to the rapid development of SMG at the branching stage.
我们研究了肝细胞生长因子（HGF）在唾液腺（SG）分支形态发生中的作用。小鼠下颌下腺（SMG）在胚胎第11.5 - 12天（E11.5 - E12）开始发育，在E14至E16.5期间，下颌骨和舌头之间的区域发生分支形态发生。实时逆转录 - 聚合酶链反应显示，SMG中c - met/HGF受体基因的表达在E14至E16.5期间增加并达到峰值，与上皮分支同时出现，并且在E14 - E15.5时在周围间充质中检测到高水平的HGF mRNA。虽然在舌肌中观察到HGF和c - met转录本的强表达，但这种表达在E13.5 - E14.5时受到限制。建立了无血清器官培养体系，从E13.5或E14胚胎中分离出含有SMG和舌下腺（SLG）原基的SG原基（外植体1）以及包含部分舌肌的外周组织的SMG/SLG原基（外植体2）。通过从外植体1中去除间充质组织获得无间充质的SMG上皮。在外植体1和2的器官培养中，SMG/SLG原基显示出生长和分支形态发生，而无间充质上皮未能生长。当将E13.5或E14的无间充质上皮和重组人HGF（rh - HGF）浸泡的珠子置于基质胶上时，上皮细胞向珠子迁移并形成分支，而E13上皮未能分支。向SMG/SLG原基培养物中外源应用rh - HGF和抗HGF抗体分别导致分支形态发生的刺激和抑制。然而，E13.5的SMG对rh - HGF的反应非常弱，而rh - HGF强烈增强了E14的SMG的分支。向培养物中添加反义HGF或c - met寡脱氧核苷酸也抑制了SMG的分支形态发生。外植体2中SMG的发育明显优于外植体1，与体内观察到的情况相当。此外，外植体2的SMG中HGF和c - Met的表达均高于外植体1的SMG。这些发现首次证明，SMG的分支形态发生受与周围间充质来源的HGF相互作用以及SMG中c - met表达的调节，这与上皮分支同时发生。目前的数据还表明，从舌肌中短暂释放的HGF可能有助于SMG在分支阶段的快速发育。
