Zebrafish Cdx1b regulates left-right asymmetry in the brain and visceral organs by modulating Nodal signaling

• Main Authors: Chun-Shiu Wu, 吳淳繡
• Other Authors: 黃銓珍
• Format: Others
• Language: en_US
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/5v46au

博士 === 國立臺灣大學 === 生化科學研究所 === 105 === 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.