Characterization of Creb-regulated transcription coactivator 1 (Crtc1) during embryogenesis of Zebrafish (Danio rerio)

• Main Author: Sie, Lawrence Eng Kean
• Other Authors: Mark Stephen Featherstone
• Format: Theses and Dissertations
• Language: English
• Published: 2014
• Subjects: DRNTU::Science::Biological sciences::Molecular biology; DRNTU::Science::Biological sciences::Genetics
• Online Access: https://hdl.handle.net/10356/61877
• Institution: Nanyang Technological University

The function of homeobox (HOX) proteins depends on formation of higher order complexes with members of the three-amino-acid-loop-extension (TALE) family of cofactors such as Myeloid Ecotropic Insertion Site (MEIS) and Pre-B-Cell Leukemia Homeobox (PBX). The importance of such complexes is to impart selectivity and specificity to HOX proteins in homing in to their target genes and to confer repressor or activator activity depending on cellular and environmental cues. Furthermore, the function of HOX complexes is regulated by PKA signalling in a manner dependent on interaction with the cAMP responsive element binding (CREB) transcription factor and its co-activators CREB Regulated Transcription Coactivator (CRTC) and CREB Binding Protein (CBP). The biological significance of such interactions has been demonstrated in the context of leukemogenesis; however, much remains to be elucidated in term of embryonic development. Zebrafish is a vertebrate model system which has been extensively used by developmental biologists due to its embryological and genetic tractability. The availability of knockdown technology mediated by antisense morpholino oligonucleotides has been instrumental in the study of gene function during zebrafish embryonic development. Our lab is interested in the study of the cooperative action of HOX-MEIS-PBX complexes with CREB-CRTC during embryonic development. We aim to achieve this by elucidating the genetic interactions of the complex members, particularly MEIS1 and CRTC1. The N-terminal region of CRTC1 has been shown to interact with C-terminal transactivation domain of MEIS1, thus bridging HOX and CREB biological functions. Several labs have characterized the role of meis1 during zebrafish development. However, crtc1 function has not been explored in zebrafish. The aim of this thesis is to characterize the biological role of crtc1 during zebrafish development by a reverse genetics approach using morpholino technology. I have characterized the spatial and temporal expression of crtc1 and crtc1b during zebrafish development, and identified unique splicing of maternal and zygotic specific crtc1b transcripts. Also, I have demonstrated the co-activator function of zebrafish Crtc1 and Crtc1b by using CRE luciferase reporter assay in cell culture. Morpholino knockdown of crtc1 or crtc1b suggested partial functional redundancy of the crtc1 members. Knockdown of crtc1 function provoked a phenotype that overlaps with that of meis1 or creb morphants, such as reduction of the eye and brain. In addition, crtc1 morphants exhibited smaller body size and abnormal body shape (e.g. “curly-tail-down” phenotype). ISH results demonstrated reduction in cells expressing neural and retinal progenitor/differentiation markers in morphants. Other biological processes were shown to be affected by crtc1 knockdown, namely perturbed notochord maturation (as assessed by persistent expression of shha and ihhb), early dorsal-ventral patterning (as indicated by ventral expansion of gsc expression), and convergence movement (as demonstrated by reduced myod-expressing cell migration toward the dorsal midline). These results provide a platform for future work to elucidate the collaborative role of meis1 and creb-crtc1 during zebrafish embryonic development.