Functional analysis of cnil ; an intra-cellular transporter of EGF-repeat-containing growth factors

在向外周迁移的过程中，脑神经脊细胞(NCC)在与菱形核(R)3和5相邻的间质中被ErbB4依赖的信号(S)排斥，菱形核(R)3和5是节段性的后脑神经聚体。ERBB4具有多种配体，但目前还不清楚哪些配体在上述系统中起作用。在此，我们发现在发育中的雏鸡R3和R5中表达了一个角质样蛋白/角质同源物2(CNIL/CNIH2)基因。在细胞培养系统中，其产品促进了ErbB4的配体之一--肝素结合的表皮生长因子样生长因子(HB-EGF)的分泌。当截短形式的CNIL强迫表达扰乱鸡胚胎的CNIL功能时，NCCs的分布会受到影响，从而导致颅感觉神经节之间异常的神经纤维连接。此外，用siRNAs敲除CNIL或HB-EGF也产生了类似的表型。这种表型与ErbB4基因敲除(KO)小鼠胚胎非常相似。由于HB-EGF在胚胎后脑中统一表达，CNIL似乎将HB-EGF的作用部位限制在与ErbB4一致的R3和R5。

During their migration to the periphery, cranial neural crest cells (NCCs) are repulsed by an ErbB4-dependent cue(s) in the mesenchyme adjoining rhombomeres (r) 3 and 5, which are segmented hindbrain neuromeres. ErbB4 has many ligands, but which ligand functions in the above system has not yet been clearly determined. Here we found that a cornichon-like protein/cornichon homolog 2 (CNIL/CNIH2) gene was expressed in the developing chick r3 and r5. In a cell culture system, its product facilitated the secretion of heparin-binding epidermal growth factor-like growth factor (HB-EGF), one of the ligands of ErbB4. When CNIL function was perturbed in chick embryos by forced expression of a truncated form of CNIL, the distribution of NCCs was affected, which resulted in abnormal nerve fiber connections among the cranial, sensory ganglia. Also, knockdown of CNIL or HB-EGF with siRNAs yielded a similar phenotype. This phenotype closely resembled that of ErbB4 knockout (KO) mouse embryos. Since HB-EGF was uniformly expressed in the embryonic hindbrain, CNIL seems to confine the site of HB-EGF action to r3 and r5 in concert with ErbB4.