The Functional Roles of Single-Minded Gene and Toxicities of Environmental Hormones in Zebrafish Head Skeleton Development (III)

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Project title

Code/計畫編號

NSC93-2313-B019-005

Translated Name/計畫中文名

Single-Minded基因與環境荷爾蒙戴奧辛對斑馬魚頭骨發育的影響(III)

Project Coordinator/計畫主持人

Chin-Hwa Hu

Funding Organization/主管機關

National Science and Technology Council

Department/Unit

Department of Bioscience and Biotechnology

Website

https://www.grb.gov.tw/search/planDetail?id=990750

Year

2004

Start date/計畫起

01-08-2004

Expected Completion/計畫迄

01-07-2005

Bugetid/研究經費

1317千元

ResearchField/研究領域

漁業
生物技術(理)

脊椎動物頭背部神經脊細胞在發育過程中會自背側向前端側遷移並同時與週遭細胞相互作用進細胞分化，最後到達終點後逐漸形成頭骨結構。在整個過程中，任何的差錯將會造成顱面的各種缺陷。在頭骨發育過程中各細胞的命運是由細胞內的各項轉因子與細胞外界的訊息傳遞相互作用造成。在本項計畫中，我們用反意morpholino核酸抑制胚胎內的arnt2a基因的功能，觀察到對斑馬魚的頭骨產生非常嚴重的抑制效果, 包括初始的神經脊細胞產生與遷移，以及其後內皮鰓囊的形成，與最後骨細胞的分化等等。全覆式RNA雜交染色分析發現這些經過arnt2a反意核酸注射的胚胎內，多個與頭骨發育相關的神經脊細胞標基因均受到抑制或者無法表現，其中包括snail2, fkd6, edn1, shh, sox9a, sox9b and col2a1等等。注射人工合成的sox9am RNA可回部分的頭骨造骨功能，顯示ARNT2的相關細胞訊息傳遞系統對sox9a╱9b基因有調控的機制。這項結果首次確ARNT2與其相關的bHLH-PAS蛋白在脊椎動物頭骨發育中所扮演的多重角色。（本項研究報告內容摘自王文德博士文部份，將投稿至國際學術期刊） Cranial neural crest cells in the vertebrate embryo migrate and interact with surrounding cells to shape the skull, and defects in these processes cause various cranial syndrome. During craniofacial development, the fate of cranial neural crest cells is determined by the cooperative interactions of multiple intrinsic factors and various environmental signals. Here we present that loss the function of zebrafish arnt2a╱2b╱2c by antisense morpholino oligonucleotide caused multiple defects in cranial cartilage development, including the neural crest induction and migration, endodermal pouches formation, pharyngeal chondorgenesis and neurocranium formation. Whole-mount in situ hybridization analyses revealed that the cranial neural crest markers, including snail2, fkd6, edn1, shh, sox9a, sox9b and col2a1, were all repressed or eliminated in arnt2a╱2b╱2c-knockdown embryos. Injection of sox9a mRNA could partially rescue cranial col2a1 transcription in those arnt2a╱2b╱2c loss-of-function embryos. It implies that the arnt2a╱2b╱2c-related signaling pathways act upstream of sox9a╱9b in cranial cartilage development. Our results establish a vital role for ARNT2 factor in vertebrate cranial cartilage differentiation. The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that can be activated by a diverse synthetic and naturally-occurring chemicals, such as the halogenated aromatic hydrocarbons (HAHs) and the non-halogenated polycyclic aromatic hydrocarbons (PAHs). The liganded AHR modulates the genetic activity of a variety of xenobiotic-responsive genes, including cytochrome P4501A1 (CYP1A1). The tyrosinase inhibitor 1-phenyl-2-thiourea (PTU) is widely used in zebrash research to suppress pigmentation in developing embryos╱fry. Here we showed that 0.2 mM PTU induced a basal level of CYP1A1 transcription in zebrash embryonic integument as early as 24 h postfertilization (hpf) stage. Subsequently, PTU induced CYP1A1 transcription in blood vessels at 36 hpf. During larval stage, the liver and all pharyngeal arch vessels of PTU-treated embryos exhibited CYP1A1 transcription as well. Comparing to TCDD, PTU induces CYP1A1 transcription with much lower efcacy in zebrash embryos. Coincubating the embryos with PTU and TCDD led to repressing TCDD-induced CYP1A1 transcription. Mechanistic studies indicated that both of PTU- and TCDD- mediated CYP1A1 transcriptions are modulated by the same AHR-ARNT signaling pathway. # 2004 Elsevier Inc. All rights reserved. In mammals, CYP3A isozymes collectively comprise the largest portion of the liver and small intestinal CYP protein. They are involved in the metabolism of an extensive range of endogenous substrates and xenobiotics and make a significant contribution to the termination of the action of steroid hormones. A full-length cDNA of CYP3A gene, named CYP3A65, was cloned from zebrafish by RT-PCR. The CYP3A65 mRNA was initially transcribed only in the liver and intestine upon hatching of the zebr

Keyword(s)

Phenylthiourea (PTU)
CYP1A1
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)
Aryl hydrocarbon receptor 2 (AHR2)
Aryl hydrocarbon receptor nuclear translocator (ARNT)
Zebrash xenobiotics
CYP3A
Zebrafish larva

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The Functional Roles of Single-Minded Gene and Toxicities of Environmental Hormones in Zebrafish Head Skeleton Development (III)

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