Main Research Goal    

The main research goals of my laboratory have been focused on gene regulatory mechanisms involved in zebrafish heart and GI tract development as well as using transgenic fish to study related human diseases. Methods including morpholino oligomer knock down, overexpression, and ENU mutagenesis are used to explore gene function. In situ hybridization, immunocytochemistry in combination with frozen and paraffin section are used to analyze gene expression pattern. Oligomer DNA microarray is used to further explore gene regulation during organogenesis. Stable transgenic fish lines are established to study specific gene function.

1. GI tract development

We study roles of transcription factors including Caudal-related homeobox protein, Kruppel-like factor, BMP4 growth factor, and Agr2 secreted protein in the GI tract development. Our results indicate that one of caudal-related homeobox gene is involved in early endoderm formation. In addition, the caudal-related homeobox gene and two Kruppel-like factors are involved in the differentiation of different intestinal cell types. Detailed molecular mechanisms involved are currently investigated. Agr2 is expressed in the mucous cells, olfactory bulb, otic vesicles, hatching gland, pharynx, esophagus, pneumatic duct, swim bladder, goblet cells in the mid intestine, and anus during different zebrafish embryonic developmental stages. In the future, its role in goblet cell differentiation will be further investigated.

2. Heart development

We obtain a segment of DNA element of BMP4 promoter and upstream region that can direct GFP expression in  the heart when analyzing tissue-specific BMP4 gene regulation.  Stable transgenic fish Tg(BMP4:EGFP) L line was constructed. GFP expression can be detected in both atrium and ventricle of L line of transgenic fish. We have also conducted ENU-mutagenesis in transgenic zebrafish line Tg(BMP4:EGFP) expressing GFP in the myocardium of heart and obtained two novel mutants by EP screening methods. Currently, we are conducting gene mapping and the characterization of mutant phenotype. In addition, we transiently expressed a proapoptotic protein, Nip3a, by heart-specific BMP4 promoter in zebrafish embryos and generated two variants of embryos with abnormal heart phenotypes (A and B). Embryos with phenotype A heart defects showed hypoplastic or elongated ventricles, elongated or enlarged atriums with no normal cardiac looping resulting a significant longer SV-BA distance, and bradycardia. Histological sections further revealed the absence of a proper atrioventricular boundary and no endocardial cells lining this region in both 48- and 72- hpf Nip3a-overexpressing embryos, implicating defective endocardial cushion formation. These phenotypes are reminiscent of atrioventricular canal defects (AVCD) in humans. Currently, we are establishing a transgenic fish line that can overexpress Nip3a in the heart under the control of tetracycline. In the future, we intend to use this transgenic fish line to study gene regulatory mechanisms that involved in atrioventricular canal differentiation and subsequent valve development.

主要的研究方向       

GFP expression in the heart of F1 embryos from a transgenic fish BMP4-GFP B line during zebrafish development. (D,G,J) 48 hpf, (E,H,K) 72 hpf, and (F,I,L) 100 hpf embryos are shown.

Reductions in respective gata5-, cas-, sox17-, and foxa2-expressing endodermal cell numbers in 85% epiboly cdx1b morphants are shown.

以斑馬魚為模式系統來探討胚胎發育過程中一些器官形態形成所參與的分子調控機制，目前所研究的基因調控機制有關於消化道及心臟的器官形成並利用基因轉殖魚來研究人類相關的疾病。所使用的方法包括以morpholino 寡核酸抑制，過度表現，建立 ENU 變種魚等方式來研究特定基因功能。以原位雜合反應或免疫化學並配合石臘及冷凍切片來研究特定基因在胚胎內的表現情形，以 oligomer DNA microarray來探討更多基因表現的變化情形等。另外亦製備一些基因轉殖魚進行特定器官形態形成基因調控的研究。