Type of Culminating Activity
Thesis - Boise State University Access Only
Master of Science in Biology
Julia T. Oxford, Ph.D.
In vertebrates, the craniofacial skeleton is developed from migratory multi-potent cells, the neural crest cells. Neural crest cells also participate in the outflow formation of the heart. This cell population undergoes epithelial-mesenchymal transition (EMT) to reach its destinations from the neural ectoderm, where they are formed. This process is both contextual and quantitatively controlled by extrinsic and/or intrinsic signals. Disturbance during their journey would cause craniofacial and cardiac defects. As the most abundant extracellular molecules, collagens may be involved in this process.
Minor fibrillar collagens (type V and XI) are of less abundance compared to the major fibrillar collagens, type I, II and III. However, minor fibrillar collagens are recognized as the regulator of collagen fibrillogenesis. We postulate the hypothesis that fibrillar collagens play a role in neural crest migration and differentiation during craniofacial and cardiac development.
In this study, the conserved residues within each exon of the variable region (VR) in minor fibrillar collagens were identified. Predictions of intrinsic disorder, post-tranlational modification of the VR and possible microRNA regulation of expression have been made. To further investigate the functions of minor fibrillar collagens during early development, their zebrafish orthologs (col5a1, col5a3, col11a1 and col11a2) were identified. Their spatiotemporal expression patterns were extensively characterized by in situ hybridization (ISH). Generally, col11a1 and col11a2 as well as col5a1 were expressed in cartilaginous tissues at 72 hpf. However, additional expression sites were observed at earlier development stages. col5a1 was detectable in the neural crest, somites and eyes, whereas col5a3 was found predominantly in the notochord. col11a1 and col11a2 were both observed in notochord and developing otic vesicle. However, different expression sites were observed. The hindbrain expression and cephalic mesoderm were unique to col11a1 and col11a2, respectively.
To study their functions during early development, antisense morpholino oligonucleotides were used to knock down the expression of the entire transcripts and/or specific exons. Knockdown of the expression of col11a1 caused a decrease in size of Meckel’s cartilage, cardiac defects and otolith abnormalities, in addition to a shortened body length. Exon 1 and exon 6A knockdown produced more severe phenotypes. Exon 8 knockdown induced much milder phenotypes. Therefore, an exon-dependent function of col11a1 was indicated. Morphants of col5a1 knockdown exhibited mild defects at high dose and no detectable effect at low dose. By contrast, col11a1 knockdowns produced more severe phenotypes. In addition to these two genes, another potential col11a1, named col11a1*, was also studied. This gene was more similar to col5a1 than col11a1 in terms of expression patterns. However, the defects in the col11a1* knockdown morphants were more severe than those of col5a1. Observed defects of col11a1* knockdown morphants included hydrocephaly, abnormal otolith genesis and cardiac abnormalities in addition to the shortened body length. Disorientation of vertebral muscles was also seen in both the col5a1 and col11a1* knockdown morphants.
Fang, Ming, "The Role of Minor Fibrillar Collagens During Craniofacial Development in Zebrafish" (2010). Boise State University Theses and Dissertations. Paper 130.