The Molecular and Cellular Basis Study

    My study of the transcriptomes of regenerating adult feathers in chicken has significantly increased our understanding of the expression profiles of feather related genes. I examined the expression profiles of genes associated with the development of feather structure and compared the gene expression patterns in different types of feathers and different portions of a feather to advance our understanding of the molecular mechanisms of feather growth and the molecular basis of variation in feather structure. The results are a valuable resource for understanding the molecular mechanisms of avian feather development. My study produced abundant data for the analysis of gene expression during feather morphogenesis. Morphotype-specifically expressed genes were identified from five zones of feather filament epithelia. Some identified genes may be associated with the growth control during feather regeneration, the formation of special branching structures, or barb differentiation themselves. It provides a basis for future study of the complex molecular and cellular events during feather development. Many candidate genes were identified for growth control, morphogenesis, or the differentiation of specific structures of different feather types. I will mainly focus on cytoskeletons, cell junctions, and extracellular matrix that are differentially expressed among different types of feathers.


    I will start a new study to increase our understanding of the complex molecular and cellular events in feather development processes by investigating the roles of the candidate genes of cytoskeletons, cell junctions, and extracellular matrix I identified from the transcriptomes. To reveal cell type-specific functions as well as the mechanisms of feather genes by which mutations lead to diseases, it will be necessary to use conditional gene-targeting strategies and the introduction of mutated gene copies. The RCAS  (Replication-Competent ASLV long terminal repeat with a Splice acceptor) retroviral vector has been shown to be an efficient technique for functional study in the avian system. I have previously shown that the chicken feather is an excellent model to validate the cytological phenotypes of mutated human α-keratin genes. I also made antisense constructs to knockdown the gene expression of β-keratin genes in embryonic feathers. As far as we know, we are among the first to express antisense RNA reliably and stably using the RCAS vector.


    To study the function of candidate genes comprehensively, I propose to transfect RCAS vectors each carrying a specific α-keratin gene (cDNA) into chicken cell lines and collect the high titer virus for transgenesis. I will introduce the RCAS retrovirus in embryonic and adult feather follicles of various types of feathers. I propose to generate major mutations with dominant-negative phenotypes causing severe human diseases in our cloned genes and study the function of each gene in feather development and validate the potential effect of the mutations. Our preliminary result shows that by introducing mutated α-keratin genes, we not only can investigate the specific role of an α-keratin in feather development, but also can test if the mutations found in humans are able to generate mutant phenotypes using the feather as a model.


    Furthermore, alternative splicing (AS) has been proposed as an important source of genetic diversity. Birds possess diverse and distinct features, yet they share similar repertoires of coding genes with other reptiles and mammals. Feather evolved only in the lineage of feathered dinosaurs that led to birds, and the development of feather also employs many genes similar to the development of other organs and skin appendages. Does the development of feather purely utilize existing gene networks? Or new isoforms of proteins are also involved? Transcriptomes of chicken internal organs have been compared to those of mammals previously, providing a good database. My preliminary analysis of feather transcriptomes suggests that different AS isoforms of cytoskeleton, cell junction, and extracellular matrix genes are expressed in different types of feathers. Thus, it is interesting to find out how much AS contributes to feather protein diversity. 


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