探討Wntless蛋白在斑馬魚胚胎發育中的功能

Abstract

Wnt 蛋白由一個細胞分泌後，結合至目標細胞後的訊息傳導扮演很重要的角色，如:體軸生成、神經發育與器官生成。從果蠅與哺乳類細胞突變篩選分析中找到的Wntless(Wls)蛋白具有將Wnt輸送到細胞膜使其分泌的功能。在果蠅與線蟲的研究中發現如果缺少Wls ，Wnt無法被運送；小鼠如果缺少Wnt則產生顱骨與腦部缺陷。然而，wls基因與Wls蛋白在脊椎動物胚胎發育中所扮演的角色仍有許多未知。 wls突變的斑馬魚具有內耳(inner ear)、下顎軟骨(jaw cartilage)與韁核(habenular nuclei) 的缺陷。先前我們實驗室發現dickkopf1b (dkk1b)在出生後兩天wls突變的斑馬魚的內耳與咽弓(pharyngeal arch)表現量下降。為了釐清Wls功能與dkk1b表現量的關係，於是我進行尋找dkk1b 啟動子(promoter)的工作。我複製了dkk1b上游2055鹼基對，黏合至帶有報導基因的Tol2質體。顯微注射(microinjection) 的結果顯示這段序列可以啟動GFP報導基因，表現位置也與出生後一天與兩天大胚胎dkk1b表現位置相似。為了了解哪些Wls蛋白區段負責其在斑馬魚胚胎發與中的功能，我製作了被刪除序列的wls，顯微注射經過轉錄後的mRNA進入wls突變魚魚卵。結果顯示Wls胺基酸221-420 與 421-542在活體中有絕對重要性。

Secreted Wnt molecules bind to receptors on receiving cells play pivotal roles in body axis formation, neural patterning and organogenesis. Wntless (Wls), identified from mutagenesis screens of Drosophila and cultured mammalian cells is suggested to function in promoting Wnt molecules to secrete out from their producing cells. Studies from Drosophila melanogaster and Caenorhabditis elegans showed that lack of Wls activities results in failure of Wnt processing, and lack of Wls activities in mice caused craniofacial and brain defects. However, the genetic and molecular roles of Wls in vertebrate embryonic development are still largely unknown. In zebrafish, null mutations in wls locus result in jaw cartilage malformation and defects in habenular nuclei development. Previously, our laboratory discovered that dickkopf1b (dkk1b) expression in the inner ear and pharyngeal arch is greatly reduced in 2 days post-fertilization (dpf) wls mutants. In order to understand the relationship between the function of Wls and dkk1b expression, it is required to identify dkk1b promoter that drives endogenous dkk1b expression. I have cloned the 5'-upstream 2055 base pairs fragment from dkk1b gene locus into Tol2 reporter vectors and the microinjection results showed that this fragment is able to drive reporter GFP gene expression in tissues that are similar to the tissue domains where endogenous dkk1b are expressed in 1 dpf and 2 dpf embryos. In order to understand the roles of different protein domains that are required for the biological function of Wls, I generated truncated Wls constructs carrying deletions of various domains and injected in vitro transcribed mRNAs into wls-/- embryos to examine which domain(s) is responsible for Wls in vivo function during zebrafish embryonic development. My results suggest that the domain containing amino acids 221 to 420 and 421 to 542 of Wls is crucial for its function in vivo.