The role of Tc-foxQ2 in the central brain development in Tribolium castaneum
by Bicheng He
Date of Examination:2018-12-12
Date of issue:2019-01-24
Advisor:Prof. Dr. Gregor Bucher
Referee:Prof. Dr. Gregor Bucher
Referee:Prof. Dr. Ernst Wimmer
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Abstract
English
The brain is the most complex organ in animals. It controls and coordinates operations like physiology and behavior. The insect brain is built by a conserved set of neuropils which arise from the neuroblasts (NBs). The central complex (CX) is an assembly of neuropils spanning the brain midline, which consists of the protocerebral bridge (PB), central body (CB) with upper and lower unit and the noduli (NO). Drosophila melanogaster and Schistocerca gregaria, are the most studied insect model systems for brain development. Some NBs and lineages contributing to the CX were identified in Drosophila and Schistocerca. However, the molecular and genetic investigations are hampered by the lack of a sequenced genome and the limited potential of the RNAi technique in the latter. Drosophila is not well suited for studying the embryonic signals required for CX development, since the CX is not detected during the embryogenesis. The red flour beetle Tribolium castaneum, with respect to functional genetics, has more tools than most other insects apart from Drosophila. Most importantly, the CB partially forms during embryogenesis. Tc-foxQ2 has shown to be a key regulatory factor in the anterior median head. It is exclusively expressed in the anterior protocerebral neuroectoderm. Further, Tc-foxQ2 knock-down was shown to result in central brain phenotypes in Tribolium. However, the embryonic development of the anterior brain is poorly studied. Nothing is known about the function of this gene in Drosophila. In order to get more insights into these processes, I used Tribolium as a model organism to study the role of Tc-foxQ2 in the early brain development, focusing on the CX and central brain development. In this study, I wanted to study the contribution of Tc-foxQ2 positive cells to the brain and characterize the function of the Tc-foxQ2 gene. To this end I generated a Tc-FoxQ2 antibody and an imaging line to mark Tc-foxQ2 positive cells by using CRISPR/Cas9 system. With this imaging line, NBs and at least subsets of their lineages contributing to the central brain could be visualized and traced. I identified Tc-FoxQ2 positive NBs at different stages in the embryo and demonstrated that the transcription factors Tc-six3, Tc-six4, Tc-chx, Tc-rx, Tc-ey, Tc-scro and Tc-fez1 are co-expressed in these Tc-FoxQ2 positive NBs. Further, I was able to show that Tc-FoxQ2 positive cells projected to the contralateral side through the initial axonal commissure of the brain, but did not pioneer the primary brain commissure. Further, I found that the identified FAM2 NB generated a median lineage and the A-PD NB generated a lateral lineage, which projected axon fascicles into the central brain primordium in the late embryo. In the adult brain, the median lineage and lateral lineage generated axon bundles projecting into the PB. Moreover, Tc-foxQ2 is required for the formation of midline crossing projections of a number of cell groups. knock-down of Tc-foxQ2 leads to the aberrant formation of the brain commissures at later stage, but did not affect the formation of the initial brain commissures at an earlier stage. Additionally, I found a medial fusion of the brain hemispheres as described before and the reduction of a number of cells which contribute to the central brain. Finally, the reduction of EGFP signal and number of marked cells in Tc-foxQ2 RNAi in the Tc-foxQ2 enhancer trap line indicated a self-regulatory function of this gene. Taken together, this study reveals an important function of Tc-foxQ2 in central brain formation.
Keywords: Tribolium castaneum; foxQ2; central brain development; enhancer trap; CRISPR/Cas9; neuroblast; neural lineage; RNAi