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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/17485

Title: 斑馬魚Cdx2基因與消化道及軟骨性頭骨發育之關係
cdx2 and zebrafish development
Authors: Pei-Yi Cheng
鄭珮宜
Contributors: NTOU:Institute of Bioscience and Biotechnology
國立台灣海洋大學:生物科技研究所
Keywords: Cdx2基因與消化道
cdx2 and zebrafish
Date: 2002
Issue Date: 2011-07-04
Abstract: 中文摘要 老鼠Cdx2基因,與果蠅caudal基因為同源性的基因,為含有homeodomain為其DNA-binding motif之轉錄因子。一些研究顯示在晚期的胚胎以及成體,Cdx2的表現侷限於前端大腸處之腸道上皮,同時Cdx2主導早期腸道的細胞增生、細胞分化以及細胞生長並保持腸道分化後的型態且與許多腸道癌症的形成有關,顯示了Cdx2對哺乳類腸道發育扮演著極端重要的角色。近年來斑馬魚為一新興的模式系統用來研究脊椎動物的胚胎發育過程,同時其與哺乳類可能具有相似的分子調節機制。因此本論文即以斑馬魚為研究材料來探討斑馬魚Cdx2同源基因是否亦在斑馬魚消化道的發育上扮演重要的角色。 利用斑馬魚基因庫- λgt10 cDNA library,得到可完整轉譯出255個胺基酸的斑馬魚Cdx2 cDNA,經由比對發現轉譯出的Cdx2蛋白質與西方有爪青蛙 (Silurana tropicalis) 的cad2有最高約69 % 氨基酸序列之相似度且包含靠近N-端的蛋白質活化區以及靠近C-端的保守區homeodomain。利用全覆式原位雜合反應來觀察Cdx2 mRNA在不同胚胎時期的表現,顯示Cdx2 mRNA首先出現於一細胞期,證實其為母體表現基因,表現持續至八細胞期、囊胚期、原腸胚期、尾芽體期至分節期,而原腸胚期時期起Cdx2 mRNA的胚胎表現主要集中於下胚層,且分節期在中後腦及部分體節有表現。受精後24小時,Cdx2 mRNA開始專一的表現於斑馬魚腸道之前端部分並逐漸向後端延伸,在48 hpf時表現於整條腸道。Cdx2 mRNA於腸道的表現在96 hpf之胚胎達到最高點,且其表現隨著胚胎發育至120 hpf及144 hpf有逐漸下降的趨勢。以48 hpf、72 hpf、96 hpf之斑馬魚冷凍切片來觀察此腸道專一性的表現,顯示其Cdx2 mRNA之表現主要集中於腸道頂端絨毛及基部細胞核等區域。 將反意寡核酸利用顯微注射入胚胎抑制Cdx2蛋白質的產生,在48 hpf時,注射過Cdx2專一性反意寡核酸的胚胎體成彎曲狀,且在120 hpf時可明顯發現軟骨性頭骨發育之不正常。進一步以全覆式原位雜合反應來觀察在注射過Cdx2專一性反意寡核酸的胚胎中各不同消化道標記基因表現的變異情形。如利用咽喉標記基因Shh及食道標記基因Dlx7來觀察兩者在咽喉與食道表現之變異情形,顯示了Cdx2基因功能之抑制會造成咽喉Shh mRNA之表現量及表現區有明顯的減少及不正常的現象且會造成Dlx7 mRNA在食道的表現量幾近於完全消失且使表現區有模糊不清且左右異位的現象;利用腸道標記基因Cdx2、IFABP及Gata6來觀察其在腸道表現的變異情形,顯示了Cdx2基因功能被抑制後會造成這些腸道標記基因的表現量大幅減少並使表現區域有不正常縮小且左右異位的現象;利用肝臟標記基因LFABP、HNF-4α、Gata6及Hhex來觀察其在肝臟表現的變異情形,顯示了Cdx2基因功能之抑制亦造成這些肝臟標記基因的表現量幾近消失並使表現區域沒有呈現正常新月型且左右異位的情形,而對另一個肝臟標記基因HNF-1α而言,HNF-1α mRNA在注射過胚胎中肝臟之表現沒有明顯的減少現象,但有左右異位的情形。利用外分泌性胰臟標記基因Trypsin及Gata6來觀察其在外分泌性胰臟表現的變異情形,同樣顯示了Cdx2基因功能的抑制會造成外分泌性胰臟標記基因的表現量減少並使表現侷限於一小區域及左右異位的情形;但利用內分泌性胰臟標記基因Insulin及Hhex來觀察其在內分泌性胰臟的表現,卻顯示了Cdx2基因功能的減少只造成內分泌胰臟的表現區域分散於兩處且有異位的情形產生但對其表現量是沒有影響。綜合上述,顯示Cdx2基因調控上述咽喉、食道、肝臟、外分泌性胰臟及腸道標記基因的表現並在這些消化器官發育左右不對稱性扮演著極端重要的角色。 為了了解Cdx2基因是否亦會調控其他左右不對稱發育的器官如:心臟的左右不對稱性分佈,進一步探討了表現於側板 (lateral plate) 中胚層左側之Lefty2基因以及表現於心臟的生長因子BMP4在被注射入Cdx2專一性反意寡核酸之胚胎中其在心臟表現的變異情形。所得結果亦顯示了Cdx2基因功能之抑制會造成心臟左右有異位的情形,綜上所知,Cdx2對斑馬魚器官左右不對稱之發育,扮演著上游調節性的重要角色。 Cdx2 基因亦為軟骨性頭骨正常發育所必需。以不同軟骨發育之標記基因來檢視注射過Cdx2專一性反意寡核酸的胚胎其軟骨性頭骨發育的過程。如利用檢視顱神經脊細胞遷移之標記基因Dlx2及Hoxa2來檢視顱神經脊細胞是否有正確的遷移情形,顯示Cdx2 基因功能的減少造成已遷移至菱腦原節鄰近與第二對及第三對咽弧軟骨之顱神經脊細胞中Dlx2及Hoxa2 mRNA的表現完全消失,證實了顱神經脊細胞沒有正確遷移的現象。利用Dlx7此標記基因來檢視顱神經脊細胞遷移至咽弧的過程,顯示Cdx2 基因功能之抑制對Dlx7 mRNA在咽弧的表現量並沒有影響,但其對第三至第七對咽弧表現區域之影響則顯示Cdx2 基因功能之喪失造成咽弧有融合在一起的現象;利用Chm1來檢視顱神經脊細胞遷移後分化成軟骨細胞之情形,結果亦顯示Chm1 mRNA在咽弧軟骨之表現量是不受影響的,證實了咽弧軟骨細胞之發育並沒有受到影響,但表現區域仍顯示Cdx2 基因功能之喪失造成咽弧有紊亂不正常的現象。另利用檢視軟骨細胞分化及提供軟骨發育所需膠原蛋白之標記基因Sox9a及Col2a1來檢視軟骨細胞是否有正常分化及型態形成的過程,顯示了Cdx2基因功能被抑制後其Sox9a及Col2a1 mRNA的表現量是正常的,證實了軟骨發育過程中軟骨細胞之分化現象及膠原蛋白的提供最後發育成為不同軟骨的過程是不受Cdx2基因功能喪失而有影響的,但其在軟骨性頭骨的表現區則顯示了Cdx2 基因功能之喪失造成咽弧軟骨不規則融合在一起且原來位於上方的髓顱軟骨和位於下方咽弧軟骨並沒有明顯的上下區隔而有髓顱軟骨與咽弧軟骨在下方重疊的現象。 綜合上述,顯示Cdx2 基因為髓顱及咽弧軟骨正常發育所必需,因為Cdx2 基因被抑制後會影響軟骨正常型態形成發育之過程並導致注射過的胚胎有咽弧軟骨不規則融合在一起且原來位於上方的髓顱軟骨和位於下方咽弧軟骨並沒有明顯的上下區隔而有髓顱軟骨與咽弧軟骨在下方重疊的現象。故Cdx2 基因之功能主要與顱神經脊細胞的遷移及遷移後於咽弧區域的定位有關,但對於顱神經脊遷移後咽弧軟骨細胞之形成、軟骨細胞的分化及軟骨基質的形成作用是沒有影響的。
Abstract Homeobox genes play essential roles in specifying the fates of different cell types during embryogenesis. In Drosophila, disturbing the expression of the homeobox gene caudal caused a severe disruption in body segmentation and global body patterning. In the later embryos and adult mouse, Cdx2 expression is confined to the intestinal epithelium, with highest expression in the proximal colon. Also, Cdx2 has been implicated in directing early processes in intestinal morphogenesis and in the maintenance of the differentiated phenotype. A study showed that Cdx2 homozygote null mutation was embryonically lethal, whereas Cdx2 heterozygote mice developed multiple intestinal polyps in the proximal colon in addition to developmental defects。 These results suggest that Cdx2 plays an important role in mammalian intestinal development. Recently, zebrafish became a model system to study vertebrate development and it may possess similar molecular regulatory mechanisms. Thus, we investigated possible roles of zebrafish Cdx2 on the development of digestive tract. Using zebrafish λgt10 cDNA library, we have isolated a caudal homologue Cdx2 cDNA from zebrafish. This gene encoded a 255-amino-acid long protein that sharing high sequence identity with Silurana tropicalis cad2 (69%). Cdx2 protein contains an N-terminal caudal like protein activation region and a highly conserved C-terminal homeodomain. Whole-mount in situ hybridization revealed that Cdx2 mRNA was first detected in the 1-cell stage, indicating that Cdx2 is a maternal expression gene. Cdx2 expression continued to 8-cells , blastula, gastrula, tail bud and segmentation stages. Cdx2 mRNA was mainly expressed in the hypoblast of gastrula stage embryos and in the midbrain, hindbrain and portion of somite of segmentation stage embryos. At 24 hour postfertilization (hpf), Cdx2 mRNA was only expressed in the anterior intestine. Cdx2 mRNA expression in the intestine extended posterior and covered the whole intestine at 48 hpf. Highest level of Cdx2 transcript in the intestine was detected at 96 hpf, whereas expression level decreased gradually from 120 hpf to 144 hpf. Transverse sections of 48 hpf, 72 hpf, and 96 hpf embryos demonstrated that Cdx2 mRNA was primarily expressed at the microvilli near the lumen and at the basal nuclei region. To investigate the function of Cdx2 on zebrafish development, we have microinjected specific antisense morpholino oligomer that complemented with the 5’-untranslated region including the translation initiation codon (ATG) of Cdx2 into blastomere at the 1-cell stage for gene knock-down analysis. Injection of Cdx2 antisense morpholino gave rise to embryos displaying curved body at 48 hpf and noticeable cartilage defect phenotype in head skeleton was observed at 120 hpf. To further characterize these phenotypes, we probed these embryos with different molecular markers specific for gastrointestinal tract and cartilage development. Marker genes used for different digestive organs included Shh and Dlx7, markers for pharynx and esophagus, respectively. Cdx2, IFABP and Gata6 were used to verify gut development. LFABP, HNF-4α, Gata6 and Hhex marker genes were used to assay liver development. Trypsin and Gata6 were used as exocrine pancreas markers. Finally, Insulin and Hhex were used to analyze endocrine pancreas development. In Cdx2-morpholino injected embryos, intensity and area of Shh expression in pharynx decreased obviously, while Dlx7 expression in esophagus was almost vanished. Abnormal decreased expression level of Cdx2, IFABP and Gata6 mRNA in gut were observed, in addition, some Cdx2 gene-knockdown embryos showed defects in left-right asymmetry of gut tube such that gut left-right asymmetry became fully reversal. In injected embryos, expression of liver markers LFABP, HNF-4α, Gata6 and Hhex did not reveal crescent-shaped liver structure and showed abnormal liver left-right asymmetry. Similar observations were made for the exocrine pancreas as revealed using Trypsin and Gata6 as probes. Surprisingly, Cdx2-morpholino injected embryos did contain normal-sized islet clusters as revealed by Insulin and Hhex markers, however some embryos showing shift of islet cells to reverse position that is similar to embryos having gut, liver and exocrine pancreas left-right asymmetry defect. These results suggest that Cdx2 plays important roles on pharynx, esophagus, gut, liver and exocrine pancreas development but plays a minimum role on islet development. In addition, Cdx2 also plays essential roles on the left-right asymmetry of these digestive organs. To further understand whether Cdx2 gene also regulates other left-right asymmetry organs distribution such as heart, we examined expression of Lefty2, which is expressed in lateral plate mesoderm and of a growth factor BMP4 in heart to characterize heart asymmetrical localization in Cdx2-morpholino injected embryos. Results demonstrated Cdx2 also functions on regulating asymmetric localization of heart. Overall, these experimental observations suggest that Cdx2 plays essential roles on establishing internal organ left-right asymmetry. Cdx2 gene is also essential for the development of cartilage skeleton in the neurocranium and pharyngeal aches. We used Dlx2 and Hoxa2 marker genes to examine migration of cranial neural crest cells. In Cdx2-morpholino injected embryos, whole-mount in situ hybridization results showed that Dlx2 and Hoxa2 expression levels in migrating crest cells that will migrate from the rhombomeric segments out to the second and third pharyngeal arches were strongly perturbed. These results suggest that Cdx2 is essential for the migration of cranial neural crest cells into the pharyngeal arches. To determine if postmigratory neural crest cells is properly specified with pharyngeal arches in Cdx2 gene-knockdown embryos, we have examined the expression pattern and level of Dlx7. Dlx7 expression level was similar in both wild type and Cdx2-morpholino injected embryos, however, Dlx7 expression in 3-7 pharyngeal arches were fused together. We have analyzed Chm1 expression in injected embryos. The results showed that the level of Chm1 expression was similar in both wild type and Cdx2-morpholino injected embryos, suggesting that differentiation of postmigratory cranial neural crest cells into chondrocytes is not affected. However, Chm1 expression pattern was random and out of regular order. Sox9a is essential for cartilage differentiation and Col2a1 encodes collagen protein that is a component of cartilage matrix. Whole-mount in situ hybridization results showed that the expression level of both Sox9a and Col2a1 was similar in both wild type and injected embryos. However, neurocranium and pharyngeal skeleton did not separate well and showed overlapped pattern in Cdx2- morpholino injected embryos. Overall, our results showed that Cdx2 is essential for the development of neurocranium and pharyngeal skeleton. Cdx2 is required for cranial neural crest cell migration and proper positioning in pharyngeal arch. However it is not required for the differentiation of postmigratory neural crest cells into chondrocytes, cartilage differentiation, and final cartilage matrix formation.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0000000206
http://ntour.ntou.edu.tw/ir/handle/987654321/17485
Appears in Collections:[生命科學暨生物科技學系] 博碩士論文

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