人類的TMPRSS3是屬於第二型穿膜絲胺酸蛋白(TypeII Transmembrane Serine Protease，TTSPs)的超級家族之一。而TMPRSS3是第一個被提出與非症候群聽障相關的絲胺酸蛋白，先前我們實驗室針對台灣地區的非症候群聽障患者進行TMPRSS3基因的突變分析，並且在230位患有非症候群聽障的兒童中發現14位TMPRSS3基因發生突變，所占的比例約為6.09% (14/230)。為了建立一個動物模式來探討TMPRSS3基因的功能。先前實驗室利用生物資訊學的方法找到了在斑馬魚中與人類TMPRSS3和老鼠tmprss3相似的兩個基因tmprss4a和tmprss4b，也使用RT-PCR和全胚胎原位雜交(Whole-mount in situ hybridization WISH)技術觀察tmprss4a和tmprss4b在斑馬魚胚胎發育時期間的表現和基因表現位置。在RT-PCR結果顯示tmprss4a從1.5小時至120小時都有高度表現，而tmprss4b則是只有在早期和後期才有表現。先前實驗室利用WISH研究tmprss4a/b前期的基因表現，並且在tmprss4a觀察到有表現在耳囊和側線的位置，而在tmprss4b前期只有微量的表現，在本研究延續先前的結果使用WISH技術更進一步探討tmprss4a/b在中期、後期的基因表現，結果顯示在斑馬的tmprss4a在前、中、後期都是有看到強烈的訊號表現，而在tmprss4b只在早期和後期的發育時中有全身型的表現。同時我們也建構了tmprss4a和tmprss4b的質體，並且使用顯微注射(Micorinjection)方式去觀察我們設計的Morpholino (MO)是否能夠專一的去抑制tmprss4a/b這兩種基因表現，在本實驗結果也證實了Morpholino (MO) Knockdown(loss of function)是有明顯的專一抑制tmprss4a/b基因。綜合以上結果tmprss4a基因在胚胎發育時期中是有明顯表達在耳囊、側線、心臟的位置。而tmprss4b基因則只有表達在後期的耳囊、側線、心臟的位置。同時顯微注射Morpholino實驗更驗證了我們的MO是可以專一性抑制tmprss4a/b這兩種基因的表現。這些結果可以提供未來利用動物模式進一步探討TMPRSS3基因造成聽障機轉的重要參考。
Membrane-anchored TMPRSS, a kind of Type II transmembrane serine protease (TTSP), plays critical roles in the cellular development and the homeostasis of tissues and organs in muticullular organisms. We have found several mutations in the TMPRSS3 gene from 230 Taiwanese children with non-syndromic sensorineural deafness (14/230; 6.09％) and then have identified the effects of TMPRSS3 mutations using yeast and Xenopus models in previous studies. However, two zebrafish tmprss gene, named tmprss4a and tmprss4b, were likely co-orthologues to human and mouse TMPRSS3 using phylogenetic analysis. tmprss4a and tmprss4b were respectively expressed in different embryonic development stages of zebrafish as RT-PCR and whole-mount in situ hybridization (WISH) analysis. Meanwhile, either tmprss4a or tmprss4b morphants all exhibited bending or shorten phenotype and heart edema at 72 hpf. In this study, we conducted the previous studies to elucidate the physiological functions of tmprss4a and tmprss4b in the zebrafish embryonic development. According to previous RT-PCR results, we performed WISH analysis to examine the gene expression in the late embryonic stage (96 and 120 hpf). tmprss4a was robustly expressed in the otic vesicle, lateral line and heart at 96 and 120 hpf, whereas tmprss4b was only expressed at 96 hpf. In addition, we respectively cloned TagRFP-tagged tmprss4a or tmprss4b fusion protein construct and injected each plasmid into one-cell-stage embryos in the presence or absence of morpholinos. Both fusion proteins were almost expressed in the yolk of zebrafish. We also found that ATG-MO1 for tmprss4a or tmprss4b was indeed able to specifically knockdown the expression of each fusion protein, but morpholinos seemed to result in higher embryonic mortality than embryos injected plasmid only. Overall, zebrafish tmprss4 may be involved in the development of hearing, especially tmprss4a.
