Thesis Title: Search for novel regulators of brain circulation and their functional analysis
Brain circulation has attracted renewed interest as an active signalling milieu that regulates brain development, homeostasis, and disease. The vertebrate brain ventricular system is a series of connected cavities which are filled with cerebrospinal fluid (CSF) and surrounded by neuroepithelium. CSF serves as a fluid envelop that protects the central nervous system. Due to its immediate contact with neural stem cells in the developing and adult brain, it also plays an essential role in regulating neuronal functioning. While the gross anatomy of ventricular system and CSF hydrodynamics are well documented, the molecular mechanisms underlying their development are poorly understood. Identification of novel molecular regulators of brain circulation will help to uncover more details of this important process. In this study, a zebrafish forward genetic screen was carried out using Tol2 transposon-mediated gene-breaking insertional mutagenesis strategy. Among all the mutants generated, a kcng4b mutant, which lacks voltage-gated potassium channel Kv6.4b silent subunit function, displayed developmental defects in embryonic brain ventricles, including neuroepithelial cells delamination, ventricle dilation and thus hydrocephalus. Functional analyses of the mutant provide the first in vivo evidence that the silent Kv6.4b subunits, play an essential role in regulating embryonic brain lumen inflation by modulating neuroepithelial cell proliferation and maintaining neuroepithelium integrity, presumably through the formation of Kv2.1/Kv6.4b heterotetramers at the embryonic brain ventricular zone. These results provide a functional basis for the modulation of Kv2.1 by silent Kv6.4b subunits during vertebrate embryonic brain ventricle morphogenesis.
Figure legend: Neuroepithelium of kcng4b mutants displayed hyper-proliferation. A-D. Dorsal view of embryos stained with anti-phosphohistone H3 antibody (red) and DAPI (blue) to reveal cells in M-phase of cell cycle and cell nuclei. A,B. 24hpf, C,D. 48hpf; A,C. Controls, B,D. kcng4b mutant embryos. E,F. proliferative index.
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