Functional analysis of zebrafish NF-YC (nuclear factor Y subunit C) during early embryonic development
| Autor: | Hui-Chuan Fu, 傅慧娟 |
|---|---|
| Rok vydání: | 2011 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 99 Nuclear factor-Y (NF-Y) is a CCAAT-box-binding transcription factor which is composed of three subunits (NF-YA, NF-YB, and NF-YC). In this study, we used zebrafish as an animal model to study their roles during early developmental stage. First, we cloned two types of zebrafish NF-YC genes (nfyc-tv1 and nfyc-tv2) which revealed high sequence similarity with homologs from other species (zebrafish nfyc-tv1 (nfyc-tv2) polypeptide shares sequence similarities of 86%(81%), 87%(81%), 87%(81%), 87%(81%), 87%(81%) and 84%(79%) with the reported nfyc-tv1 (nfyc-tv2) of human, rat, mouse, chicken, bovine and Xenopus, respectively). We observed the expression of nfyc in zebrafish embryos using whole-mount in situ hybridization. In 24-hpf embryos, nfyc expression was found in brain, eyes and neural tube. Immunostaining with NF-YC antibody revealed the expression of NF-YC in primary head sinus, heart and intersegmental vessel. While endogenous nfyc was knocked down by antisense morpholino of nfyc (nfyc-MO), a reduction in eye size was observed in nfyc-MO-injected embryos (0.25 ± 0.02 mm, n=30) compared with wild-type embryos (0.60 ± 0.01 mm, n=30). Immunostaining with neuron-specific antibodies (Zn8 and Zn12) revealed that nfyc-MO affected nfyc expression in ganglion cell layer (GCL), inner plexiform layer (IPL) and outer nuclear layer (OPL), suggesting that nfyc is associated with the development of retinal neurons. Based on immunostaining with phosphor-histone H3 (PH3) antibody, a decrease in proliferating cells was found in eyes and heart of nfyc-MO-injected embryos. TUNEL assay results revealed the apoptosis in head and eyes of nfyc-MO-injected embryos. Our in situ hybridization data showed an increase in retinal homeobox 1 (rx1) accompanied by a decrease in cone-rod homeobox (crx), suggesting an impaired cell differentiation in nfyc-MO-injected embryos. Furthermore, we used transgenic zebrafish Tg(fli1:EGFP)×Tg(gata1:RFP) as a model to observe the intersegmental vessel and blood cells, and found knockdown of nfyc led to damaged blood vessels and slowed blood flow. Taken together, our results suggested that zebrafish nfyc may affect the development of retinal neurons and blood vessels, and further affect zebrafish eye development. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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