以斑馬魚為模式研究神經內分泌的新功能

Zebrafish an outstanding molecular model system to study novel physiological function of neuroendocrine related hormones

柯雪莉(Shelly Abad Cruz)

指導教授 ： 黃鵬鵬

國立臺灣大學/生命科學院/漁業科學研究所/博士(2011年)

https://doi.org/10.6342/NTU.2011.00149

摘要

章節一: 皮質醇藉由影響斑馬魚的轉錄因子Foxi3來控制表皮離子細胞的分化與增生
硬骨魚類在適應環境過程中，鰓以及皮膚上的離子細胞型態與增生深受皮質醇調控。在本研究中探討皮質醇影響離子細胞的背後分子機制，並探討皮質醇受器 (醣皮質醇受器)在魚類表皮離子細胞發育過程中的影響。此外，藉由使用斑馬魚當作模式動物，本實驗提供活體( in vivo) 以及體外 (in vitro) 的研究分析，同時研究促皮質醇釋放激素、腎上腺素及這些激素相關受器等在皮膚發育過程中的影響。
實驗結果發現，使用皮質醇去刺激斑馬魚受精卵會抑制醣類皮質醇受器基因轉錄，但是不會影響礦物性皮質醇受器基因轉錄。在皮質醇刺激下，斑馬魚的富含鈉幫浦細胞 (NaRCs) 數目、富含氫幫浦細胞 (HRCs) 數目、表皮鈣離子通道基因轉錄、gcm2基因轉錄、鈉氫交換蛋白 (nhe3b) 基因轉錄以及氫幫浦 (atp6v1a) 基因轉錄等皆會被向上調控。此外，使用原位雜交技術發現離子細胞分化指標基因 foxi3a跟 foxi3b的 mRNA表現會在皮質醇處理24到48小時後被刺激。但是在細胞分裂分析上則沒觀察到有被影響。在細胞凋亡的分析上則發現皮質醇在不同發育時間會有不同影響，在受精後48小時是減少但是受精後72小時是增加。
使用注射反義核苷酸剔除技術抑制醣類皮質醇受器的蛋白質轉譯，結果則發現醣類皮質醇受器的減少會降低富含鈉幫浦離子細胞以及富含氫幫浦離子細胞的數目。相反的，抑制礦物性皮質醇受器的蛋白質轉譯則不會影響表皮幹細胞跟離子細胞數目。在體外培養斑馬魚鰓的實驗上則發現，處理24 小時的皮質醇會刺激離子細胞數目跟分化指標基因foxi3a/b的轉錄。因此我們推論皮質醇是經由醣類皮質醇受器來調控foxi3a/b基因轉錄，進而影響前驅離子細胞的的特化與分化，並且皮質醇能延遲離子細胞凋亡。這樣的皮質醇作用最後促使成熟離子細胞的數目明顯增生。

章節二：促皮質醇釋放因子及其受器或相關蛋白質在斑馬魚皮膚發遇上的影響
下視丘-腦下垂體-間腎組織間軸 (HPI axis) 等系統性影響已經被證實在皮膚發育上扮演重要的角色。使用注射反義核苷酸技術抑制促皮質醇釋放因子及其第一型受器(CRFR1)蛋白質轉譯，發現會明顯影響表皮幹細胞跟離子細胞密度。CRFR1主要功能在於調節促皮質醇釋放因子功能，並且發現該受器在斑馬魚卵一開始受精後就存在。促皮質醇釋放因子第二型受器 (CRFR2)則未被發現能夠影響表皮幹細胞跟離子細胞密度。儘管如此，CRFR2在其他生理功能上的重要性仍不能夠被排除。
目前研究證實HPI axis能以系統性或是局部性的作用影響斑馬魚皮膚跟鰓的功能。促皮質醇釋放因子能夠以CRFR1來直接影響表皮幹細胞進而調控離子細胞分化，此外促皮質醇釋放因子也能夠經由影響皮質醇分泌，進而調控foxi3a/b基因轉錄來達到相同功能。未來研究需要再詳細去闡明神經內分泌系統在調控表皮離子細胞分化上的機制。

第二部分

章節一：斑馬魚飢餓激素 (Ghrelin)經由抑制腦中的胰島素或刺激昇糖激素(glucagon)來影響腦中糖類碳水化合物的代謝
糖類碳水化合物的代謝所產生能量對於中樞神經系統的能量供給非常重要。在胰臟，飢餓激素已被知道對於糖類碳水化合物代謝跟調控胰島素扮演非常明顯重要的角色。然而在除了胰臟的其他組織上，飢餓激素對於胰島素調控仍然不清楚。在這研究，我們使用斑馬魚成魚來探討斑馬魚飢餓激素在腦中的表現跟影響糖類碳水化合物代謝的功能。根據RT-PCR跟原位雜交技術的結果顯示：斑馬魚腦會表現飢餓激素與其受器 (GHS-R: zghs-r1a and zghs-r2a) 。 在蛋白質定位的證據更確認斑馬魚腦表現飢餓激素的事實。當注射合成的金魚飢餓激素peptide到斑馬魚成魚，結果發現會刺激斑馬魚腦中的zghs-r1a 跟 zghs-r2a轉錄，進一步分析發現，魚腦中的胰島素跟其受器會被明顯抑制，但是昇糖激素跟其受器則會被明顯刺激，在糖類碳水化合物代謝後，緊接而至的急迫肝醣感受反應將會平衡肝醣降解跟能量儲存。這些發現推論：在中樞神經系統，飢餓激素藉由調節其受器來控制胰島素合成，並且影響糖類碳水化合物代謝。

關鍵字

皮質醇

並列摘要

Chapter I Cortisol controls epidermal ionocyte differentiation and proliferation by targeting Foxi3 transcription factors in zebrafish (Danio rerio)
In teleost fish, skin/gill function, cell morphological changes and cell proliferation were greatly affected by cortisol during environmental acclimation. In the present study we examined the molecular mechanism behind cortisol action including its known receptor, glucocorticoid receptor (gr) in fish epidermal ionocyte progenitor development. Utilizing zebrafish for both in vivo and in vitro assay, together with the involvement of the neuroendocrine system in the skin development which includes the corticotrophin releasing factor (crf), crf receptor 1 (crfr1), crfr2 and melanocotropin 2 receptor (mc2r) or ACTH receptor.
Cortisol treatment of zebrafish newly fertilized eggs suppressed gr transcripts without affecting mr. Transcripts of ionocyte marker genes for Na+-K+-ATPase rich cells (NaRCs): epithelial Ca+ channel (ecac) and H+-ATPase rich cells (HRCs): glial cell missing 2 (gcm2), Na+-H+-exchanger 3b (nhe3b) (B), H+-ATPase A-subunit (atp6v1a) were all upregulated upon cortisol treatment. Immunocytochemistry confirmed that both NaRCs and HRCs density were significantly increased. In addition, ionocyte progenitor specification and differentiation marker genes foxi3a and foxi3b spatial mRNA expression were increased after 24-48 h treatment of cortisol via in situ hybridization. Cell division was not affected but cell apoptosis was decrease at 48 hpf and increased at 72 hpf in the cortisol-treated group.
Knock-down of gr by GR-ATG MO showed immense lowered NaRCs and HRCs numbers among GR morphants which is further confirmed by GR-SV MO. In contrast, loss of MR has no effect in epidermal stem cells and ionocyte density. In vitro, 24 h gill organ culture with cortisol treatment significantly increased NaRCs and HRCs numbers including upregulation of foxi3a/b transcripts. Hence, we propose that cortisol through gr targets foxi3a/b that regulates epidermal ionocyte progenitor specification/differentiation together with delayed apoptosis in which case caused proliferation of matured ionocytes as the apparent outcome.

Chapter II Corticotropin releasing factor (CRF), CRF-receptors and related proteins contribution on skin development of zebrafish (Danio rerio)
The systematic hypothalamus-pituitary-interrenal (HPI) axis was also demonstrated to play a critical role in the skin development. Loss of function assay by morpholino oligos showed crf and crfr1 significantly take part in the stem cell and ionocytes density. CRFR1 is the major receptor that could mediate CRF function. Both present maternally, these may suggest their importance during the early skin development in zebrafish. CRFR2 is also maternally deposited but it can be dispensable during skin development since there is no observed change in epidermal stem cells or ionocytes density and morphology upon crfr2 knock-down. Nevertheless, CRFR2 function cannot be ruled-out in other major physiological functions like in stress.
Altogether, the present results serve as a foundation of the HPI axis as a systematic event or locally present in the zebrafish skin and gills. In which crf initializes this event through crfr1, as a separate and local function or as a complete course of signaling events together with that of cortisol-gr-foxi3a/3b axis major effect in the epidermal ionocyte progenitor differentiation. More future work has to be done to clarify more issues and provide better understanding in the neuroendocrinology of the skin.

並列關鍵字

Cortisol

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