Molecular mechanism that controls early morphogenesis of the vertebrate respiratory system

(1) Fgf10的表达域局限于原代支气管内胚层突起的中胚层。这种限制发生在分级Hoxb-6表达域的前表达边界。从功能得失实验中，我们发现在Hoxb-6表达边界两侧诱导异位Fgf10表达。这表明Fgf10的表达是由Hoxb-6依赖细胞诱导的。精算我们发现Hoxb-6抑制EphA4表达，激活efnb1表达。EphA4或efnb1的异位表达在其表达边界诱导Fgf10的表达，表明这些分子参与了Fgf10的表达。此外，我们发现Hoxb-6的表达边界具有信号中心的作用，并诱导呼吸原体后区初级支气管的成对形态发生。(2)通过转基因小鼠系统鉴定肺特异性Fgf10基因增强子。我们发现多个肺特异性增强子存在于携带肢体芽特异性增强子的7kb基因内DNA中。肺特异性增强子与Fgf10启动子和/或5'上游序列中存在的肺特异性表达序列相互合作。(3)我们尝试通过微阵列分析和WISH鉴定肺原中特异性表达的基因。我们发现可能参与控制Fgf10的转录因子，如Isl1和Tbx20，被确定为候选基因。有趣的是，这些基因中的大多数也在心脏中表达，这表明在早期发育过程中，心脏和肺原素的诱导存在类似的机制。Wnt2的表达很早就在肺原组织中发现，表明Fgf10的表达是通过Wnt/ β -连环蛋白系统参与的。

(1) The expression domain of Fgf10 become restricted the mesoderm lining the endodermal protruding of the primary bronchus. This restriction takes place at the anterior expression boundary of graded Hoxb-6 expression domain. From gain-and loss-of-function experiment, we found that ectopic Fgf10 expression was induced at both side of the Hoxb-6 expression boundary. This indicates that Fgf10 expression is induced by Hoxb-6 dependent cell to tell communication. Actuary we found that Hoxb-6 represses EphA4 expression and activates efnb1 expression. Ectopic EphA4 or efnb1 expression induces Fgf10 expression at their expression boundary, indicating that these molecules are involved in the Fgf10 expression. In addition, we found that the expression boundary of Hoxb-6 has a role of the signaling center and induces the paired morphogenesis of the primary bronchus at the posterior region of the respiratory anlagen. (2) We tried to identify the lung specific enhancer of Fgf10 gene by transgenic mouse system. We found that the multiple lung specific enhancers are present in the introngenic 7kb DNA that also carries the limb bud specific enhancer. The lung specific enhancers cooperate each other with the sequence present in the Fgf10 promoter and/or 5' upstream sequence for lung specific expression. (3) We tried to identify the genes that are specifically expressed in the lung anlagen by microarray analysis followed by WISH. We found that transcription factors potentially involved in the control of Fgf10, for example Isl1 and Tbx20, are identified as a candidate gene. Interestingly, majority of these genes are also expressed in the heart, indicating that similar mechanism is present for induction both heart and lung anlagen at early development. Wnt2 expression was found from very early in the lung anlagen, indicating the involvement of Fgf10 expression through Wnt/beta-catenin system.