碩士 / 國立臺灣海洋大學 / 生物科技研究所 / 94 / Previously we obtained a zebrafish cdx2 cDNA and named it cad2 from a zebrafish cDNA library screening. Cad2 encodes a 255 amino acids long polypeptide that containing a caudal like activation domain near N-terminal region and a 60 amino acid homeobox DNA binding domain near the C-terminus. In Pei-Yi Cheng master thesis, embryos that have been injected with antisense morpholino oligomer-1 (cad2-MO1), which complemented with the 5’-untranslated region including the translation initiation codon (ATG) of cad2 showed swollen pericardiac chamber at 48 hour post fertilization (hpf). Therefore, the main goal of this thesis is to explore the role of cad2 in zebrafish heart development. Morphant embryos injected with cad2-MO1 (8 ng/embryo) showed curved tail, smaller eyes, malformation of the midbrain/hindbrain boundary (MHB), and reduction in the midbrain ventricle at 24 hpf. Some cad2 morphant embryos showed swollen pericardiac chamber, and some of them showed fused eyes at 48 hpf. Pericardiac chamber swelling was more seriously and the presence of linear heart tube in cad2 72hpf morphant embryos. Cad2 72 hpf morphant hearts displayed four phenotypes including normal D-looping, non-looping, L-looping and D-looping with defect. In cad2 72 hpf morphants with non-looping heart phenotype showed linear heart tube with slower beating rate than wild type. To gain a clear observation about heart defects in morphant embryos, we probed them with heart chamber markers, amhc and cmlc2, specific for cardiac myosin. Whole-mount in situ hybridization results indicated that respective percentages of four heart phenotypes observed in cad2 72 hpf morphants are similar as that obtained by observation directly under the microscope. However, expression levels of amhc and cmlc2 were the same as that in wild type embryos. These results suggest that cad2 may regulate heart looping process but not cardiac muscle development.
Heart looping is one of left-right axis asymmetric morphogenetic processes. cad2 is expressed in the prechordal plate and notochord during late gastrulation, which is similar to expression patterns of genes involved in the left-right axis asymmetry. Therefore, we further characterized effects on genes involved in L-R axis asymmetry in cad2-MO1 injected embryos using a nodal signaling pathway downstream gene (pitx2), an antagonist of nodal (lefty1), and midline development related genes including gsc, flh and ntl., Expression levels and domains of pitx2, lefty1, and gsc in prechordal plate were decreased in cad2-MO1 injected 85% or 60% epiboly embryos. While expression levels and domains of ntl as well as expression domains of flh in the notochord were reduced respectively in cad2 85% epiboly morphant embryos. Overall, our results suggest that cad2 may regulate left-right axis asymmetry to affect heart looping process.
In order to examine cad2-MO1 specificity, we prepared cad2 polyclonal antibody. Antigen region does not contain conserved homeobox DNA binding domain. Results from western blot analyses showed that cad2 polyclonal antibody can’t recognize putative 28 kDa cad2 protein in either 72 hpf wild type embryos, embryos that have been injected with pcDNA-cad2 overexpression plasmid, or COS-1 cells that have been transfected with pcDNA-cad2 plasmid. Therefore, we injected cad2 dominant-negative plasmid, which containing only putative NLS and homeobox DNA binding domain into zebrafish 1-cell zygote and analyze expression pattern of IFABP at 72hpf. Results showed a decrease in IFABP expression level in the intestine or altered expression of IFABP in the intestinal bulb locating at the right site or in the middle of embryos that have been injected with cad2 dominant-negative plasmid. In the future, we can further analyze effects on genes involved in L-R axis asymmetry in embryos that have been injected with cad2 dominant-negative plasmid. These results will be complementary with our previous observation in cad2-MO1 injected embryos to support the idea that cad2 can regulate genes involved in L-R axis asymmetry to affect heart looping process.
| Identifer | oai:union.ndltd.org:TW/094NTOU5111015 |
| Date | January 2006 |
| Creators | Chun-Yen Tsai, 蔡君彥 |
| Contributors | Sheng-Ping L. Hwang, 黃聲蘋 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 102 |
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