Molecular cloning and expression analysis of ADAMs and cadherins during chicken embryonic development
Introduction: Cell adhesion molecules (CAMs) play various roles during embryonic development. Cadherins are a large superfamily of CAMs that mediate cell-cell adhesion and signaling transduction by a calcium-dependent mechanism. The ADAM family of genes can also be considered a family of CAMs, because their disintegrin domain binds integrin to mediate cell-cell or cell-matrix interactions, and their cysteine-rich domain and epidermal growth factor-like domain both are able to modify cell-cell adhesion. Moreover, many ADAMs are metalloproteases that shed different trans-membrane proteins, such as cadherins. However, the expression patterns and potential functions of most ADAMs and some cadherins remain poorly understood. In the present thesis, I cloned seven members of the ADAM family and a multiple cadherins from the chicken embryo, and investigated their molecular characteristics and expression profiles during chicken embryonic development. Methods: Fertilized eggs were incubated in a forced-draft incubator at 37°C and 65% humidity until the embryos reached the desired stage. Total RNA was isolated from different stages and partial or full-length cDNAs of the ADAMs and cadherins of interest were cloned by RT-PCR or RACE. The temporal expression profiles of each gene were analyzed with semi-quantitative RT-PCR during chicken embryonic development. Northern blot was performed to determine the amount and size of chicken cadherin-8 isoforms as well. Results from semi-quantitative RT-PCR and Northern blot were further analyzed by appropriate statistic methods. The temporal and spatial expression patterns of the cloned molecules were investigated using in situ hybridization of sections or whole mount specimens. Appropriate protein markers helped to confirm the identity of expression areas by immunostaining. To investigate the function of chicken ADAM17, an overexpression (gain-of-function) experiment was performed using in vivo (ex ovo) electroporation of the embryonic chicken tectum. Results: I obtained the novel full-length sequences of chicken ADAM12, ADAM13, ADAM22, Cdh8 and Cdh19. The expression pattern of ADAM13 was investigated in detail in the chicken embryo throughout the developing organism. The expression patterns of eight chicken cadherins (N-Cdh, R-Cdh, Cdh6, Cdh7, Cdh8, Cdh11, Cdh18 and Cdh20) were analyzed in the developing cochlea at older embryonic stages. I confirmed that 6. Summary 123 chicken Cdh8 possesses three isoforms from mRNA alternative splicing and analyzed their temporal and spatial expression in the chicken embryonic brain. Five ADAMs investigated (ADAM9, ADAM10, ADAM12, ADAM22, ADAM23) show different types of expression patterns in the developing chicken brain. Finally, ADAM17 overexpression (gain-offunction) resulted in a morphological change of the blood vessels in the developing tectum. This change coincided with an increase in the number of pericytes.