斑馬魚Cdx1b通過調節Nodal信號調控大腦和內臟器官左右不對稱發育之研究

Abstract

左右不對稱發育對於一些雙邊對稱生物的內臟器官及神經系統發育正確與否是非常重要的。其中，Nodal 訊息路徑對於左右不對稱發育扮演了保守且重要的角色。在斑馬魚中，參與左右不對稱發育的Nodal 訊息路徑基本成員有：Ndr2、Ndr3二個配體，Lft1、Lft2 二個拮抗體以及下游轉錄因子Pitx2。經由Ndr 對自己的正向調控和對Lft1/2 的負向調控，使得Nodal 訊息路徑的表現時間和空間可以被精確地控制。 斑馬魚 Cdx1b 是homeobox 轉錄因子家族的一員。過去的研究已知Cdx1b 會調控Nodal signaling 的下游基因，並影響胚胎的內胚層細胞數量；另外，Cdx1b 也調控了腸道細胞的增生和分化。本篇研究論文發現，Cdx1b 藉由調控Nodal 訊息路徑，參與調控斑馬魚左右不對稱發育。這是一個在其他物種中尚未被報導的Cdx1b新功能。利用注射cdx1b 反義嗎啉基(antisense Morpholino)至斑馬魚胚胎中抑制cdx1b 轉譯，發現會造成胚胎上丘腦、心臟、肝臟、胰臟及腸的位置左右顛倒或是對稱發育。進一步研究發現，cdx1b 表現降低會導致胚胎Nodal 訊息路徑成員ndr3、lft1、lft2 和pitx2c 在左邊侧板中胚層的表現紊亂，以及ndr2、lft1、和pitx2c 在左邊背側間腦的表現喪失。降低cdx1b 表現也使初期建立左右不對稱的器官Kupffer’s vesicle 功能減低。此外cdx1b 的減少會使Nodal 訊息路徑成員ndr2、lft1和pitx2c 在前腹板(prechordal plate) 和頭端腹側神經外胚層(anterior ventral neuroectoderm)的表現降低，造成前腹板和頭端腹側神經外胚層的發育不良，更進一步影響上丘腦的左右不對稱發育。經由比對跨物種間保守序列以及染色質免疫沉澱法發現，ndr2 基因和lft1 基因5 端上游具有保守性Cdx1/Cdx2-結合基序列。 這顯示Cdx1b 可能會與這些基因上的保守性Cdx1/Cdx2-結合基序列結合，進而調控ndr2 和lft1 的表現而影響胚胎頭端腹側神經外胚層的發育以及上丘腦左右不對稱的發育。

Left-right asymmetric patterning is important for proper development of visceral organs and neurogenesis that is dependent on the appropriate activity of Nodal signaling. In zebrafish, after breaking the left-right symmetry by nodal flow inside Kupffer's vesicle (KV), ndr3 (Nodal-related 3) is asymmetrically expressed in the left lateral plate mesoderm (LPM) and turns on downstream genes encoding the Nodal ligands (ndr2 and ndr3 itself), the antagonists of Nodal ligands (lft1 and lft2), and the downstream effector of Nodal signaling (pitx2). This Self-Enhancement and Laterality Inhibition system of Nodal signaling are considered as a conserved molecular cascade across species to control their temporal and spatial expression precisely. Zebrafish caudal-related homeobox 1b (cdx1b) belongs to the caudal type homeobox (cdx) gene family, which has been implicated in anterior-posterior axial patterning and intestinal development across diverse bilaterians. Previous studies have shown that zebrafish Cdx1b regulates early endoderm formation and differentiation of various intestinal cell lineages. In this thesis, a new role of Cdx1b in left-right asymmetrical patterning was discovered. Knockdown of cdx1b by specific antisense morpholino oligonucleotides (MOs) caused the loss of left-laterality in the epithalamus, the failure of heart looping, and isomerism or situs inversus of visceral organs. In the cdx1b-knockdown embryos (cdx1b morphants), left-sided expression of Nodal signaling genes (ndr3, lft1, lft2, and pitx2c)were altered in the LPM, and expressions of ndr2, lft1 and pitx2c were not detected in the left dorsal diencephalon during the late somite and early pharygula stages (18-26 hpf). During the early- somite stages (4-6 somite stages), the cilia of KV were shortened and the Nodal flow was weakened in the cdx1b morphants. The symmetric right-sided expression of charon (the antagonist of Nodal around KV) was also altered in the cdx1b morphants. Knockdown of cdx1b reduced the expression of ndr2, lft1, and pitx2c in the prechordal plate and the anterior ventral neuroectoderm at the bud stage. In addition, decreased expression of gsc, a downstream target of Nodal signaling in the prechordal plate and ventral neuroectoderm, and shh, a structure marker of these tissues, were observed in the cdx1b morphants. These results indicate that Cdx1b is important for patterning of the Nodal-induced anterior mesendoderm and the anterior ventral neuroectoderm. Based on motif predictions by the JASPAR and UCSC websites, chromatin immunoprecipitation results showed that Cdx1b can bind on the conserved promotor/enhancer region of ndr2 (chr12:49,427,858-49,428,038) and lft1 (chr20:35,102,628-35,102,818). Together, these results suggest that Cdx1b may regulate transcription of ndr2 and lft1 to maintain proper activity of Nodal signaling that is essential for the mesendoderm induction, the anterior neurulation and subsequent establishment of laterality of epithalamus in the zebrafish embryo.