Groucho/transducin-like Enhancer of split (Gro/TLE) family proteins are corepressors found as part of multiple transcriptional complexes that play significant roles during many developmental processes, including neurogenesis. This thesis sought to characterize the molecular mechanisms underlying the biological activity of Gro/TLE1. More specifically, the aim was to clarify the contribution of different transcriptional cofactors, as well as phosphorylation events induced by cofactor binding, to Gro/TLE1 ability to inhibit neuronal differentiation from proliferating neural progenitor cells. / By characterizing specific point mutations within the C-terminal domain of Gro/TLE1, we were able to selectively impair binding of Gro/TLE1 to different classes of DNA-binding proteins and then assess the effect of those mutations on Gro/TLE1 anti-neurogenic function. These studies showed that the inhibition of cerebral cortex (cortical) neuron differentiation by Gro/TLE1 requires interaction with transcription factors that use short tetrapeptide sequences, WRP(W/Y), to recruit Gro/TLE1. In contrast, interactions with proteins that either interact with the C-terminal domain of Gro/TLE1 using a different type of binding sequence, termed engrailed homology 1 (Eh1) motif, or bind to the N-terminal part of the protein, are not required for Gro/TLE1 anti-neurogenic function. / Using a similar strategy based on mutation analysis, we characterized point mutations that block the hyperphosphorylation of Gro/TLE1 induced by transcription cofactor binding ("cofactor-activated phosphorylation") without impairing cofactor binding and transcriptional corepression ability. These mutations map at phosphorylatable serine residues, Ser-286, Ser-289, and Ser298. Mutation of those residues to alanine blocks/reduces both cofactor-activated phosphorylation and anti-neurogenic activity of Gro/TLE1, demonstrating that cofactor-activated phosphorylation is required for that function. Tandem mass spectroscopy analysis showed further that Ser-286 is phosphorylated. Taken together, these findings characterize the role of cofactor-activated phosphorylation and identify residues important for this mechanism. / Our studies also showed that homeodomain-interacting protein kinase 2 (HIPK2) mediates phosphorylation of Gro/TLE1 when the latter is complexed with transcriptional partners of the WRP(W/Y) motif family. However, HIPK2 is not involved in Gro/TLE1 cofactor-activated phosphorylation. Rather, HIPK2--mediated phosphorylation is antagonistic to the latter and decreases the ability of Gro/TLE1 to interact and repress transcription with WRP(W/Y) motif proteins. / Taken together, these results improve significantly our understanding of the mechanisms underlying the anti-neurogenic function of Gro/TLE1. This information provides new insight into the regulation of mammalian neuronal development and, possibly, other developmental processes controlled by Gro/TLE proteins.

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壓力感受器在調節血壓的壓力感受性反射中的作用已是眾所周知。兩個動脈壓力感受器，分別為主動脈壓力感受器和頸動脈壓力感受器。它們作為重要的感應器以檢測主要動脈血壓，並和孤束核溝通，從而調節血壓。然而，壓力感受器的機械力敏感元件的分子身份仍是奧秘。因為機械敏感的陽離子通道受機械力刺激時會增加的神經元活動, 所以機械敏感的陽離子通道是合適的候選人。 / 在本研究中，通過使用膜片鉗和動作電位的測量，瞬时受体电位通道C5(TRPC5)被確定在主動脈壓力感受器的機械傳感器中。透過在壓力感受器神經元的鈣測量實驗，證實TRPC5參與由拉伸引起的鈣離子([Ca²⁺]i)上升。TRPC5基因敲除小鼠出現壓力感受器功能受損, 表明了TRPC5在血壓控制的重要性。 / 比較主動脈壓力感受器或頸動脈壓力感受器的不同敏感度現時存有不少爭論。在本研究中，我發現主動脈壓力感受器比頸動脈壓力感受器對於壓力變化更加敏感。此外，我還發現了主動脈壓力感受器神經元比頸動脈壓力感受器神經元有一個相對較高的瞬时受体电位通道V4(TRPV4)表達。鈣測量研究表明TRPV4通道在主動脈壓力感受器神經元的靈敏度可能發揮著重要作用。 / Baroreceptors have been well known for its role in the baroreflex regulation of blood pressure. Two arterial baroreceptors, aortic and carotid baroreceptors, serve as the important sensors to detect blood pressure in main arteries, and they communicate with the solitary nucleus tract for blood pressure regulation. However, the molecular identity of the mechano-sensitive components in the baroreceptors is still mysteries. Mechano-sensitive cation channels are the fascinating candidates as they increase neuronal activities when stimulated by stretch. In the present study, with the use of patch clamp and action potential measurement, TRPC5 channels were identified to be the mechanical sensor in the aortic baroreceptor. Calcium measurement studies demonstrated that TRPC5 was involved in the stretch-induced [Ca2+]i rise in baroreceptor neurons. The importance of TRPC5 in blood pressure control was also studied in TRPC5 knockout mice, which displayed an impaired baroreceptor function. / There have been controversies as to whether aortic baroreceptors or carotid baroreceptors are more sensitive to the change in blood pressure. In the present study, aortic baroreceptor was found to be more sensitive to the pressure change than the carotid baroreceptor. Furthermore, I also found a relative higher expression of TRPV4, a mechanosensitive channel, in the aortic baroreceptor neurons than in the carotid baroreceptor neurons. Moreover, calcium measurement studies showed that TRPV4 channels should play an important role in governing the differential pressure sensitivity in these two types of baroreceptor neurons. / Taken together, the present study provided novel information on the role of TRPC5 and TRPV4 in baroreceptor mechanosensing. In future, it will be of interest to explore whether TRPC5 and/or TRPV4 dysfunction could contribute to human diseases that are related to blood pressure control. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Lau, On Chai Eva. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 133-152). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese. / Declaration --- p.i / Abstract of the thesis entitled --- p.ii / Acknowledgement --- p.vii / Abbreviation --- p.ix / Table of content --- p.xii / List of figures --- p.xv / List of table --- p.xvii / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- Baroreceptors --- p.1 / Chapter 1.1.2 --- Arterial baroreceptors --- p.2 / Chapter 1.1.2.1 --- Functions of arterial baroreceptors --- p.4 / Chapter 1.1.2.2 --- Sensitivity of the arterial baroreceptors --- p.6 / Chapter 1.1.3 --- Other baroreceptors --- p.8 / Chapter 1.1.4 --- The molecular identity of the mechanosensors in baroreceptor neurons --- p.9 / Chapter 1.2 --- Transient receptor potential ion channels (TRP channels) --- p.10 / Chapter 1.2.1 --- TRP channels superfamily --- p.10 / Chapter 1.2.2 --- Multimerization of TRP channels --- p.12 / Chapter 1.2.3 --- Physiological functions --- p.14 / Chapter 1.2.4 --- Mechanosensitive TRP channels --- p.16 / Chapter 1.2.5 --- Canonical transient receptor potential 5 (TRPC5) channels --- p.17 / Chapter 1.2.6 --- Vanilloid transient receptor potential 4 (TRPV4) channels Figures --- p.20 / Chapter Chapter 2: --- Objectives --- p.34 / Chapter Chapter 3: --- Materials and Methods --- p.35 / Chapter 3.1 --- Materials --- p.35 / Chapter 3.1.1 --- Chemicals and reagents --- p.35 / Chapter 3.1.2 --- Solutions --- p.36 / Chapter 3.1.2.1 --- Solutions for calcium imaging --- p.36 / Chapter 3.1.2.2 --- Solutions for electrophysiology study --- p.38 / Chapter 3.1.2.3 --- Solutions for agarose gel electrophoresis --- p.41 / Chapter 3.1.3 --- Primers for RT-PCR --- p.42 / Chapter 3.1.4 --- Animals --- p.43 / Chapter 3.2 --- Methods --- p.43 / Chapter 3.2.1 --- Total RNA isolation and RT-PCR --- p.43 / Chapter 3.2.2 --- Immunohistochemistry --- p.44 / Chapter 3.2.3 --- Neuron labeling by DiI --- p.45 / Chapter 3.2.4 --- Neuron culture --- p.46 / Chapter 3.2.5 --- [Ca²⁺]i measurement --- p.47 / Chapter 3.2.6 --- Electrophysiology --- p.48 / Chapter 3.2.7 --- Evaluation of baroreflex response --- p.49 / Chapter 3.2.8 --- Telemetric measurement of blood pressure --- p.50 / Chapter 3.2.9 --- Statistical analysis --- p.51 / Figures --- p.52 / Chapter Chapter 4: --- Functional role of TRPC5 channels in aortic baroreceptor --- p.56 / Chapter 4.1 --- Introduction --- p.56 / Chapter 4.2 --- Materials and Methods --- p.59 / Chapter 4.2.1 --- Animals --- p.59 / Chapter 4.2.2 --- Immunohistochemistry --- p.59 / Chapter 4.2.3 --- Neuron labeling by DiI --- p.61 / Chapter 4.2.4 --- Neuron culture --- p.62 / Chapter 4.2.5 --- [Ca²⁺]i measurement --- p.63 / Chapter 4.2.6 --- Electrophysiology --- p.63 / Chapter 4.2.7 --- Evaluation of baroreflex response --- p.64 / Chapter 4.2.8 --- Telemetric measurement of blood pressure --- p.66 / Chapter 4.2.9 --- Statistical analysis --- p.67 / Chapter 4.3 --- Results --- p.67 / Chapter 4.3.1 --- Endogenous expression of TRPC5 channels in aortic baroreceptor neurons --- p.67 / Chapter 4.3.2 --- Characterization on the pressure-sensitive component in aortic baroreceptors --- p.68 / Chapter 4.3.3 --- Involvement of TRPC5 in [Ca²⁺]i response in aortic baroreceptor neurons --- p.69 / Chapter 4.3.4 --- Participation of TRPC5 in pressure-induced action potential firing in cultured aortic baroreceptor neurons --- p.70 / Chapter 4.3.5 --- Role of TRPC5 in baroreceptor sensory nerve activity and baroreflex regulation --- p.71 / Chapter 4.3.6 --- Importance of TRPC5 in baroreceptor function --- p.72 / Chapter 4.4 --- Discussions --- p.74 / Figures --- p.79 / Table --- p.98 / Chapter Chapter --- 5: TRPV4 channels and baroreceptor sensitivity --- p.99 / Chapter 5.1 --- Introduction --- p.99 / Chapter 5.2 --- Materials and Methods --- p.101 / Chapter 5.2.1 --- Animals --- p.101 / Chapter 5.2.2 --- Neuron labeling by DiI --- p.101 / Chapter 5.2.3 --- Neuron culture --- p.102 / Chapter 5.2.4 --- Electrophysiology --- p.103 / Chapter 5.2.5 --- Immunohistochemistry --- p.104 / Chapter 5.2.6 --- [Ca²⁺]i measurement --- p.105 / Chapter 5.2.7 --- Statistical analysis --- p.105 / Chapter 5.3 --- Results --- p.106 / Chapter 5.3.1 --- Properties of the aortic and carotid baroreceptor neurons --- p.106 / Chapter 5.3.2 --- Stretch sensitivity of aortic and carotid baroreceptor neurons --- p.108 / Chapter 5.3.3 --- mRNA expression of mechanosensitive TRP channels in aortic and carotid baroreceptor neurons --- p.109 / Chapter 5.3.4 --- Protein expression of TRPV4 channels in aortic and carotid baroreceptor neurons --- p.109 / Chapter 5.3.5 --- Involvement of TRPV4 in stretch-induced [Ca²⁺]i response in baroreceptor neurons --- p.110 / Chapter 5.4 --- Discussions --- p.111 / Figures --- p.116 / Chapter Chapter 6: --- General conclusions and future directions --- p.124 / Figures --- p.128 / References --- p.133