Cilia are microtubule-based organelles that project into the extracellular space and have various functions including transducing sensory information, regulating developmental signalling pathways, and generating directed fluid flow, making them important regulators of vertebrate development and homeostasis. Despite their importance, there are many aspects of cilia formation and function that remain poorly understood. The planar cell polarity (PCP) pathway is a branch of Wnt signalling that provides positional information to cells and is required for polarized morphogenic cell movements. Previous studies of PCP effector proteins suggested that PCP signalling was required for cilia formation. However, these proteins are not specific to the PCP pathway and are shared with other branches of Wnt signalling. To determine the role of a core and specific PCP regulator on ciliogenesis, I examined maternal-zygotic (MZ) vangl2 zebrafish mutants using an in vivo marker of cilia, Arl13b-GFP. Analysis of MZvangl2 mutants revealed that PCP is not required for cilia formation but is required for the posterior tilting and posterior positioning of motile cilia, essential for directed fluid flow. A parallel branch of studies suggested that cilia are actually required to regulate PCP signalling because defects in PCP-mediated morphogenic movements were observed with the knockdown of certain proteins that localize at or near cilia or basal bodies. To determine whether cilia were required to establish PCP, I generated MZ-intraflagellar transport-88 (IFT88) mutants, where ciliogenesis is completely abolished. Analysis of MZift88 mutants revealed that cilia are not directly required for PCP-mediated morphogenic movements. However, I observed that MZift88 mutants had defects in oriented cell divisions (OCD) occurring during gastrulation. Remarkably, these divisions occur prior to cilia formation, suggesting a cilia-independent role for IFT proteins in cell divisions, which may have important consequences on the interpretation of the role of cilia in disease.

Cilia are microtubule-based organelles that project into the extracellular space and have various functions including transducing sensory information, regulating developmental signalling pathways, and generating directed fluid flow, making them important regulators of vertebrate development and homeostasis. Despite their importance, there are many aspects of cilia formation and function that remain poorly understood. The planar cell polarity (PCP) pathway is a branch of Wnt signalling that provides positional information to cells and is required for polarized morphogenic cell movements. Previous studies of PCP effector proteins suggested that PCP signalling was required for cilia formation. However, these proteins are not specific to the PCP pathway and are shared with other branches of Wnt signalling. To determine the role of a core and specific PCP regulator on ciliogenesis, I examined maternal-zygotic (MZ) vangl2 zebrafish mutants using an in vivo marker of cilia, Arl13b-GFP. Analysis of MZvangl2 mutants revealed that PCP is not required for cilia formation but is required for the posterior tilting and posterior positioning of motile cilia, essential for directed fluid flow. A parallel branch of studies suggested that cilia are actually required to regulate PCP signalling because defects in PCP-mediated morphogenic movements were observed with the knockdown of certain proteins that localize at or near cilia or basal bodies. To determine whether cilia were required to establish PCP, I generated MZ-intraflagellar transport-88 (IFT88) mutants, where ciliogenesis is completely abolished. Analysis of MZift88 mutants revealed that cilia are not directly required for PCP-mediated morphogenic movements. However, I observed that MZift88 mutants had defects in oriented cell divisions (OCD) occurring during gastrulation. Remarkably, these divisions occur prior to cilia formation, suggesting a cilia-independent role for IFT proteins in cell divisions, which may have important consequences on the interpretation of the role of cilia in disease.

Engineering epithelium with correct structure is essential for generating functional tissue. During tissue development, cells organize in defined patterns through cellular signalling. Artificial generation of the signalling that organizes cells within the tissue offers a novel approach for engineering tissues with appropriate structure. Planar cell polarity (PCP) is a cellular signalling pathway involved in the organization of epithelial cells. Our goal is to study the effect that co-culturing genetically distinct populations of epithelial cells, with variable levels of one of the core PCP proteins, has in epithelial cell sheet organization. MDCK cells transduced with a tagged PCP core protein (GFP-Vangl2) and wild type MDCK cells were co-cultured side-by-side. The effect of tight junction and cilia formation, and localization of the GFP-Vangl2 protein were evaluated. The results suggest that tight junction and cilia formation are not affected. On the other hand, the GFP-Vangl2 protein seems to be affected at some level.

Engineering epithelium with correct structure is essential for generating functional tissue. During tissue development, cells organize in defined patterns through cellular signalling. Artificial generation of the signalling that organizes cells within the tissue offers a novel approach for engineering tissues with appropriate structure. Planar cell polarity (PCP) is a cellular signalling pathway involved in the organization of epithelial cells. Our goal is to study the effect that co-culturing genetically distinct populations of epithelial cells, with variable levels of one of the core PCP proteins, has in epithelial cell sheet organization. MDCK cells transduced with a tagged PCP core protein (GFP-Vangl2) and wild type MDCK cells were co-cultured side-by-side. The effect of tight junction and cilia formation, and localization of the GFP-Vangl2 protein were evaluated. The results suggest that tight junction and cilia formation are not affected. On the other hand, the GFP-Vangl2 protein seems to be affected at some level.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21262号 / 医博第4380号 / 新制||医||1029(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 平井 豊博, 教授 伊達 洋至, 教授 渡邊 直樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The Wnt signaling network has critical roles in embryonic development and is implicated in human disease. One of the outputs of the Wnt network, called the planar cell polarity (PCP) pathway, regulates tissue polarity and directs cell migration. Core PCP components (Frizzled, Dishevelled, Prickle, Vangl, Celsr) localize asymmetrically in polarized cells and establish polarity across the tissue through protein interactions between adjacent cells. The core PCP component activate tissue-specific "effectors" which translate the signal into morphological changes. PCP is related to several disease conditions, including neural tube defects, cystic kidney disease, and cance metastasis. However, mechanisms of the PCP underlying physiological and disease-related conditions are not well understood. Here, I explore functions of the core PCP component Pk, and its relationship to disease, in the zebrafish model system. Mutations in Pk1 and Pk2 have been identified in human progressive myoclonic epilepsy patients. Pk coodinate cell movement, neuronal migration and axonal outgrowth during embryonic development. Yet, how dysfunctions of pk relates to epilepsy is unknown. Here, I show that knockdown of pk1a sensitizes the zebrafish larva to convulsant drug. To model the defects in central nervous system, I examine neurogenesis in the retina and find that both pk1a and pk2 are required for proper dendritic outgrowth in the retinal inner plexiform layer. Furthermore, I characterize the epilepsy-related mutant forms of Pk1a and Pk2. The mutant Pk1a forms show reduced ability to suppress the retinal neurogenesis defects compared to the wild-type, as well as differential ubiquitination levels. Pk2 mutant forms also show differential activities in overexpression assays and seemingly more stable proteins relative to the wild-type. Taken together, pk1a and pk2 may contribute to epilepsy by affecting neuronal patterning and thus signal processing. Another aspect of PCP function has been implicated in cilia and cilia-related disorders, also called ciliopathy. PCP effectors have been shown to modulate ciliogenesis and core PCP proteins (Vang and Dvl) regulate cilia orientation. On the other hand, cilia are not required for PCP signaling, especially asymmetric core PCP protein localization. These findings leave open the question what is the precise relationship between PCP and cilia. The Bardet Biedl Syndrome (BBS) is a type of ciliopathy that leads to obesity, retinitis pigmentosa, polydactyly, mental retardation and other symptons. A subset of BBS genes share similar knockdown phenotype in cell migration as seen in PCP knockdown embryos. Shared pehnotypes have led some to proposethat PCP and bbs genes may interact. Yet a direct relationship has yet to be established. I examine the interaction between pk2 and a central Bbs gene, bbs7. By analyzing shared phenotypes in double knockdown embryos, I find no synergistic interaction between the two, suggesting they act in distinct pathways. Bbs regulate ciliary trafficking and in zebrafish, knockdown of bbs genes leads to delayed retrograde melanosome transport. Interestingly, I find knockdown of pk2 suppresses this retrograde transport delay. Additionally, pk2 knockdown embryos show a delay in anterograde melanosome transport. These findings highlight a new role for pk2 in intracellular transport and clarifies the relationship between PCP and BBS. In summary, my work here strengthens the link between pk mutations and human epilepsy and identifies functions of pk in retinal neurogenesis and in intracellular transport. To what extent the role of neurogenesis and intracellular transport are related is worth future study. Yet, this new information provides insights into potential mechanisms of epilepsy and the relationship between PCP and BBS.

During neuronal development, the axonal and dendritic projections are polarized and oriented along specific body axis. To further explore the molecular basis of neuritogenesis in vivo, we used the nematode Caenorhabditis elegans as a developmental model and performed a forward genetic screen to identify genes that specify the polarity of neurite outgrowth. We examined the VC4 and VC5 neurons, members of the six VC motor neurons using the Pcat-1::gfp transgene cyIs4. The VC motor neurons are ventrally located neurons that extend two processes. VC1, VC2, VC3, and VC6 extend axons along the anterior-posterior (A/P) axis; VC4 and VC5 extend axons around the vulva along a mediolateral left-right (L/R) axis perpendicular to the A/P axis. We identified and showed that vang-1/Van Gogh, a core component of planar cell polarity (PCP) signalling pathway, acts cell-autonomously in VC4 and VC5 neurons and non-autonomously from the epithelial cells to restrict neurite formation along the A/P axis. vang-1 mutant animals display ectopic neurites along the A/P axis. Using a candidate gene approach, we further identified and revealed two additional core members of PCP signalling, Prickle (PRKL-1) and Dishevelled (DSH-1), to play a role in A/P-directed neurite suppression. We also showed prkl-1 and dsh-1 genetically interact with vang-1 and VANG-1 is required to suppress A/P-directed neurite outgrowth from larval stage 4 to adulthood. Overexpression of VANG-1 results in a loss-of-function (lof) phenotype, suggesting that an appropriate level of VANG-1 activity is important. Additionally, vang-1/prkl-1, and dsh-1 may interact in parallel pathways. Our findings implicate PCP genes to play a previously unidentified role in maintaining polarized neuronal morphology by inhibiting neuronal outgrowth responses to environmental cues.

Endothelial cells (ECs) respond to flow but the exact mechanism producing alignment is not completely understood. We characterized EC alignment in microfluidic channels, 4 mm wide by 350 um high, to generate shear of 20 dynes / cm2 across the cell surface. In microchannels, ECs aligned perpendicular under flow. Analytical tools were developed to quantify nuclear alignment at 67% for human umbilical vein endothelial cells (HUVECs); cell elongation under shear flow shifted aspect ratio from 2.41 to 2.86. We next sought to probe the mechanism through which ECs communicate during realignment. The planar cell polarity (PCP) signaling pathway is involved in cell organization and coordination during development. A number of genes are known to affect the formation and organization of cellular structures through PCP signaling in human ECs. Higher expression of Vangl1 and Dvl1 proteins did not alter cell reorganization; knockdown of Vangl1 expression decreased EC alignment.

Endothelial cells (ECs) respond to flow but the exact mechanism producing alignment is not completely understood. We characterized EC alignment in microfluidic channels, 4 mm wide by 350 um high, to generate shear of 20 dynes / cm2 across the cell surface. In microchannels, ECs aligned perpendicular under flow. Analytical tools were developed to quantify nuclear alignment at 67% for human umbilical vein endothelial cells (HUVECs); cell elongation under shear flow shifted aspect ratio from 2.41 to 2.86. We next sought to probe the mechanism through which ECs communicate during realignment. The planar cell polarity (PCP) signaling pathway is involved in cell organization and coordination during development. A number of genes are known to affect the formation and organization of cellular structures through PCP signaling in human ECs. Higher expression of Vangl1 and Dvl1 proteins did not alter cell reorganization; knockdown of Vangl1 expression decreased EC alignment.

During neuronal development, the axonal and dendritic projections are polarized and oriented along specific body axis. To further explore the molecular basis of neuritogenesis in vivo, we used the nematode Caenorhabditis elegans as a developmental model and performed a forward genetic screen to identify genes that specify the polarity of neurite outgrowth. We examined the VC4 and VC5 neurons, members of the six VC motor neurons using the Pcat-1::gfp transgene cyIs4. The VC motor neurons are ventrally located neurons that extend two processes. VC1, VC2, VC3, and VC6 extend axons along the anterior-posterior (A/P) axis; VC4 and VC5 extend axons around the vulva along a mediolateral left-right (L/R) axis perpendicular to the A/P axis. We identified and showed that vang-1/Van Gogh, a core component of planar cell polarity (PCP) signalling pathway, acts cell-autonomously in VC4 and VC5 neurons and non-autonomously from the epithelial cells to restrict neurite formation along the A/P axis. vang-1 mutant animals display ectopic neurites along the A/P axis. Using a candidate gene approach, we further identified and revealed two additional core members of PCP signalling, Prickle (PRKL-1) and Dishevelled (DSH-1), to play a role in A/P-directed neurite suppression. We also showed prkl-1 and dsh-1 genetically interact with vang-1 and VANG-1 is required to suppress A/P-directed neurite outgrowth from larval stage 4 to adulthood. Overexpression of VANG-1 results in a loss-of-function (lof) phenotype, suggesting that an appropriate level of VANG-1 activity is important. Additionally, vang-1/prkl-1, and dsh-1 may interact in parallel pathways. Our findings implicate PCP genes to play a previously unidentified role in maintaining polarized neuronal morphology by inhibiting neuronal outgrowth responses to environmental cues.