基因調控網路與 Sox32 和Sox17 之內胚層斑馬魚發育次基因調控網路

Gene Regulatory Networks and Subcircuits of Sox32 and Sox17 in Zebrafish Endoderm Development

詹子民(Tzu-Min Chan)

指導教授 ： 喻秋華 ； 汪宏達

國立清華大學/生命科學院/生物科技研究所/博士(2009年)

https://doi.org/10.6843/NTHU.2009.00025

摘要

The process of development is a result of cascades made by gene regulated interaction,
which operates mainly through the regulated expression of genes encoding transcription factors
and signaling pathways. In this thesis, I first integrated preexist bioinformatics data, combining
gene expression pattern from ZFIN website and the interrelationship between genes from
previous literatures. We established a database, and then constructed the genomic regulatory
networks in zebrafish embryogenesis by using BioTapestry. We focused on the endoderm
formation and dorsoanterior–ventroposterior patterning. This is the first part of this thesis.
In vertebrate development, the process of gastrulation leads to three germ layers: ectoderm,
endoderm, and mesoderm. The endoderm originates from the most marginal blastomeres of
blastula stage embryos in zebrafish (Danio rerio). The most important signaling molecule in the
endoderm formation is Nodal. Upon binding to its receptors, Nodal leads to the activation of
gata5, bon and og9x, which then activate sox32 to promote the expression of sox17 and activate
the endoderm differentiation. In the second part of this thesis, we established the subcircuits of
sox32 and sox17 using morpholinos against sox32 and sox17, measured certain gene expression
profiles by real time RT-PCR and validation by in situ hybridization. We identified several
interesting functional motifs that are important building blocks in zebrafish developmental gene
regulatory networks (GRNs). At the early stage, Sox32 and sox17 provide an autoregulatory lock
on the endodermal fate of cells. Early activation turns to late repression for Sox32 to sox32 itself,
and for Otx2 to sox17. Late transcription factors repress early activators: e.g., Gata5 activates
sox17 and then Sox17 represses gata5; Gbx1 activates sox32 and then Sox32 represses gbx1. We
found Sox32 and Sox17 repress many early transcription factors such as foxh1, and sox17
represses itself at a later stage. This interaction network extends from the two important
endoderm transcription factors and provides in-depth understanding of the complex regulatory
architecture in early embryonic development.
In the third part of this thesis, we further analyzed cis-regulatory element and directly tested
for the existence of functional modules. The sox17 gene is a key marker of endodermal cell in
the zebrafish. According to the predictions of the GRNs, based on perturbation experiment and
literature search, the sox17 gene is engaged with two other regulatory genes, sox32 and pou5f1,however the regulatory inputs of sox17 at the genomic sequence level are not known. I analyzed
the regulatory modules and transcription factor binding sites on the sox17 gene, and discovered
three evolutionary conserved region, A, B, and C, are positive regulatory modules with a
synergistic effect among them. I revealed the functionality for Pou5f1-transcription factor
binding site on the B module, and Sox32-transcription factor binding site on the C module, and
those two transcription factors work synergistically to positively regulate sox17. Furthermore, an
evolutionarily non-conserved R module exhibits a repressive effect on both the ventral and dorsal
side of the ectoderm. My research provides new insight into the complexity of endoderm
formation. This is the first elucidated node in the zebrafish endoderm GRN`s which has been
proved directly by their structural and functional relationships. It may serve as a landmark for
decipher the complete endoderm gene regulatory network.

關鍵字

斑馬魚 ； 背腹側生成 ； 內中胚分化 ； 基因調控網路 ； 轉錄因子 ； 斑馬魚內胚層發育

並列摘要

發育過程是轉錄因子(transcription factors) 及訊號傳遞路徑(signaling pathways) 相
互作用的結果。在這份論文研究，首先我利用整合生物資訊的方法，結合ZFIN 基因表現
的資訊以及過去研究所產生出來的基因與基因之間關係的資料，統整成資料庫並建構出胚
胎發育調控網路，尤其是著重內胚層的發育及背腹的區別之基因調控網路，構成本論文的
第一部分。
在脊椎動物發育過程當中，經過原腸期(gastrulation) 分化出外胚層、內胚層和中胚
層，而在斑馬魚胚胎囊胚(blastula) 階段內胚層源自於胚葉細胞(blastomeres) 最邊緣。內
胚層形成中最重要的訊息分子是Nodal 訊號傳遞路徑，經由Nodal 與它的接受體(receptor)
結合之後活化gata5、bon 和og9x，然後活化sox32 進而促成sox17 的表現和內胚層的分
化(differentiation)。內胚層形成中最重要的轉錄因子為sox32 及sox17。因此為了清楚了解
這两個基因如何影響內胚層基因表現，第二部分的論文以實驗方法建立sox32 和sox17 次
調控網路(subcircuits)。我們使用嗎琳代低聚核甘酸(morpholinos oligonucleotides) 專一性
的抑制sox32 和sox17 基因的轉譯，然後利用即時定量聚合酶鏈反應(real time RT-PCR)
測量下游基因表現量的改變， 並利用原位雜交(in situ hybridization)的方法驗證，以建立
sox32 和sox17 次調控網路(subcircuits)。其中我們發現許多在斑馬魚發育之基因調控網路
中重要的功能性群組。在早期的階段，sox32 和sox17 提供一個自我調控的循環位於將來
會分化成內胚層區域。Sox32 早期活化但是晚期卻抑制sox32 它自己，並且Otx2 早期活
化sox17 而晚期被Sox17 所抑制。另外還有一些相同調控的轉錄因子，如Gata5 早期活化
sox17 而晚期被Sox17 所抑制和Gbx1 早期活化sox32 而晚期被Sox32 所抑制。我們也發
現在晚期Sox32 和Sox17 會抑制早期的轉錄因子諸如foxh1，而Sox17 會抑制它自己。
Sox32 及Sox17 這兩個重要的轉錄因子所組成的次調控網路關係，讓我們對於胚胎發育過
程中複雜調控機制有了更深入的瞭解。
第三部分的論文進一步在斑馬魚的內胚層發育基因調控網路，直接證明關鍵的基因調
控之結構和功能性上的關係。在斑馬魚sox17 基因是一個內胚層細胞之關鍵性的標誌，根據來自於擾動實驗(perturbation experiment) 和文獻搜尋的基因調控網路，顯示Sox32 和
Pou5f1 這兩個重要的轉錄因子對於sox17 基因的調控扮演非常重要的角色，但在DNA 序
列層次上，sox17 基因同側調控元素(cis-regulatory elements)並不是很清楚。我分析sox17 的
調控模組（regulatory module）及基因調控子序列（transcription factor binding site），發現
sox17 基因上含有三個演化保守區域A、B、C，在功能上是具有協同作用的正調控模組。
並證明B 模組上含有Pou5f1 調控子序列，且C 模組上含有Sox32 調控子序列，而Pou5f1
和Sox32 互相作用對於正調控sox17 基因具有增效作用。此外，sox17 基因上非演化保守
區域R 模組具有抑制sox17 基因表現在腹側及背側外胚層的作用。我的研究對於內胚層形
成的複雜度提供一個新的見解，這是第一個斑馬魚的內胚層發育基因調控網路中，被研究
透徹的網點(node)，對於完整的內胚層基因調控網路的譯解具有指標性意義。

並列關鍵字

Zebrafish ； Dorsal–ventral polarity ； Mesendoderm specification ； Gene regulatory network ； Transcription regulation ； Zebrafish endoderm development

參考文獻

structural, thermodynamic and clustering patterns of conserved residues in DNA-binding

proteins. Nucleic Acids Res 36, 5922-5932.

Alexander, J., and Stainier, D. Y. (1999). A molecular pathway leading to endoderm

Amsterdam, A., and Hopkins, N. (2006). Mutagenesis strategies in zebrafish for identifying

genes involved in development and disease. Trends Genet 22, 473-478.