Integration and Evolution of Hypoxia Response Network: Systems Biology and Evolution Approach (III)

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基本資料

Project title

Code/計畫編號

MOST104-2627-B019-001

Translated Name/計畫中文名

低氧訊息網路的系統與演化生物學研究---斑馬魚低氧訊息網路系統的生物功能(總計畫及子計畫1)( III )

Project Coordinator/計畫主持人

Chin-Hwa Hu

Funding Organization/主管機關

National Science and Technology Council

Co-Investigator(s)/共同執行人

鄒文雄

Department/Unit

Department of Bioscience and Biotechnology

Website

https://www.grb.gov.tw/search/planDetail?id=11595986

Year

2015

Start date/計畫起

01-08-2015

Expected Completion/計畫迄

01-07-2016

Co-Investigator(s)

Wen-Shyong Tzou

Bugetid/研究經費

2680千元

ResearchField/研究領域

生物技術(理)
海洋科學

在胚胎發育過程中共有二階段的紅血球生成作用，在斑馬魚中第一階段的紅血球生成發生在中間細胞團組織中。在紅血球細胞分化成熟前，細胞內的GATA轉錄因子需要由GATA2轉換為GATA1，藉以啟動成熟紅血球細胞內的各種專一性蛋白質基因的表現。雖然已知GATA因子的轉換是透過負回饋的方式進行，但是詳細的機制尚不清楚。在本計畫中，我們發現低氧誘發蛋白HIF1α對於gata1基因的轉錄作用扮演非常重要的角色。將HIF1α基因敲落會大幅抑制gata1基因的轉錄作用，並且維持細胞內gata2基因高度的表現，使得紅血球細胞無法成熟。這結果造成紅血球內許多蛋白質無法生成，因而無法完成紅血球細胞的成熟。另一方面，外源性gata1a mRNA 可以協助大部分恢復胚胎的紅血球造血功能。利用pimonidazole染色，我們觀察到胚胎紅血球的造血組織處於明顯的缺氧微環境，因此可以讓紅血球內的HIFs蛋白質處於穩定的狀態。利用染色質免疫沉澱分析，我們發現在造血時期，胚胎內HIF1α會辨識gata1基因的後端低氧感應序列，顯示HIF1α直接參與了gata1基因的調控工作，亦即是紅血球細胞分化時的GATA因子轉換作用。我們的研究提供了新的線索證實細胞低氧訊息系統不但是協助生物進行低氧逆境生理適應的關鍵機制，也同時參與了正常胚胎內基本紅血球生成的作用。除了針對HIF1α在造血功能上的研究之外，我們亦針對HIF2α與Survivin在胚胎內的功能進行轉錄體的分析，結果發現hif2α與birc5a基因敲落均會增加foxo3以及其下游細胞凋亡相關基因（包括sesn3與 bcl6ab）的活性。這項結果符合了我們先前的觀察，發現將hif2α與birc5a基因敲落引起大量細胞凋亡的現象。我們推測hif2α與birc5a抑制後所引起的細胞凋亡可能與FOXO3的活化有密切的關係。此外我們亦利用Crispr-Cas9方式建立birc5a基因敲除品系斑馬魚，發現birc5a-/-胚胎呈現出與birc5a基因敲落胚胎相似的表徵，細胞大量凋亡，中樞神經發育嚴重受損的情況，顯示先前birc5a基因敲落具有基因的專一性。目前我們正利用此品系進行birc5a基因功能的探討。 In embryonic development, erythroid cells are generated by two waves of hematopoiesis. In zebrafish, the primitive wave occurs in the intermediate cell mass region. After cell lineage specification, expression of the GATA transcription factor in erythroid progenitor cells switches from GATA2 to GATA1. Although GATA2 is required to initiate GATA1 expression during erythroid differentiation, additional, as yet undefined, factors are involved in GATA1 transcription in hematopoietic stem cells. We found that HIF1α was critical for initiating gata1a transcription required for primitive erythroid differentiation in zebrafish embryos. HIF1α depletion by morpholino knockdown abrogated gata1a expression, and sustained a high level of gata2 transcription in primitive erythroid progenitor cells. Accompanying the decrease of GATA1a expression, numerous erythroid-specific genes were repressed, and primitive erythroid differentiation was inhibited in HIF1α morphant embryos. Ectopic gata1a mRNA ameliorated the defects of primitive erythropoiesis in HIF1α-morphant embryos. Results of pimonidazole staining indicated that the intermediate cell mass region was characterized by a hypoxic microenvironment, which may explain how HIF1α is stabilized in primitive erythroid progenitor cells. Results of chromatin immunoprecipitation assays showed that transcription of gata1a was initiated by HIF1α binding to the downstream region of gata1a during early stages of development. Unlike the conventional understanding that HIF only promotes hypoxic stress-induced erythropoiesis, here we demonstrate that HIF1 also plays an essential function in basal primitive erythropoiesis by facilitating gata1a transcription. In addition to the study of functions of HIF1α in primitive erythropoiesis, we have also analyzed functions of HIF2α and Survivin in CNS development by NGS transcriptome analysis. It appears depletion of either HIF2α or Survivin resulted in increasing transcriptions of foxo3b and its downstream target genes, including sesn3 and bcl6ab. This result is consistent with previous observation of increased apoptosis in hif2α and birc5a morphants. It appears that the cell death observed in hif2α and birc5a morphants was caused by induced FOXO3 activity.

Keyword(s)

低氧誘發蛋白
細胞凋亡
轉錄體分析
中樞神經發育
初階紅血球生成
GATA因子轉換
HIF1α
HIF2α
Survivin
GATA1
FOXO3
CNS development
primitive erythropoiesis
apoptosis
transcriptome

DSpace CRIS

Integration and Evolution of Hypoxia Response Network: Systems Biology and Evolution Approach (III)

基本資料

Description