Mechanisms of Wnt signaling: from embryonic stem cells to dopaminergic neurons

The ability of a cell to respond in a specific way to certain signals represents key biological phenomena governing development of multicellular organism. Cellular signaling regulates all aspects of cell biology such as proliferation, migration, differentiation, and death. Detailed understanding of mechanisms by which various signals are interpreted into certain cellular responses is crucial in order to efficiently manipulate these processes. Guiding a stem cell via specific cues to a cell type of interest, such as dopaminergic (DA) neurons, is a necessary prerequisite for cell replacement therapy (CRT) of diseases, such as Parkinson’s disease (PD), where DA neurons are progressively lost. This thesis examines molecular mechanisms of action of Wnts, a group of factors providing such cues, and their functional role in midbrain development and DA neuron differentiation. In our first study we manipulated Wnt/β-catenin signaling pathway in mouse embryonic stem cells (mESCs) to analyze its impact on mESC differentiation into DA neurons. We show that pathway impairment at the ligand (Wnt1) or receptor (LRP6) level enhances neuronal and DA differentiation of mESCs. Similarly, application of Dkk1 (Wnt/β-catenin pathway inhibitor) also increased the yield of mESC-derived DA neurons. Combined, our data demonstrate that Wnt1 and LRP6 are dispensable for mESC DA differentiation, that mESC differentiation into DA neurons is facilitated by attenuated Wnt/β-catenin signaling, and that inhibitors of Wnt/β-catenin pathway can be used to increase efficiency of DA differentiation protocols. Earlier reports from our lab demonstrated enhancement of DA differentiation by Wnt5a, an activator of Wnt/β-catenin-independent pathways in DA cells. Thus, we focused on mechanisms of Wnt/β-catenin-independent signaling and its functional aspects in our following studies, as these were not elucidated before this thesis. We show by analyses of Wnt5a -/- mice embryos the importance of Wnt5a for proper midbrain morphogenesis. Moreover, absence of Wnt5a led to increase in proliferation of DA progenitors, accumulation of Nurr1+ precursors and attenuated differentiation of these precursors into TH+ DA neurons. To characterize Wnt5a-mediated effect on DA differentiation we analyzed possible activation of putative downstream pathway components. We demonstrate that Wnt5a effects on DA differentiation are mediated via small GTPase Rac1, which is a downstream effector of Wnt5a/Dvl signaling in DA cells. Subsequently, we examined molecular aspects of the Wnt5a/Dvl/Rac signaling in closer detail. We demonstrate that β-arrestin is a crucial component of Wnt5a/Dvl/Rac signaling route and we show its critical role in regulation of CE movements during Xenopus gastrulation. Moreover, we found that specification of Wnt-mediated signaling at the level of Dvl is further controlled by phosphorylation of Dvl by casein kinases CK1 and CK2. Therefore, CK1 and CK2 act as switches between distinct branches of Wnt/β-cateninindependent signaling. Next, to get further insight into Wnt5a/Dvl-mediated activation of Rac1 we analyzed the Dvl-Rac1 interaction and performed a proteomic screen for Dvl-binding regulators of Rac1 activity. We show that Dvl and Rac1 form a complex, and the N-terminal part of Dvl mediates this interaction. Further, we demonstrate that Tiam1, a novel Dvl-binding partner found in our study, is required both for Rac1 activation in the Wnt5a/Dvl/Rac signaling branch and for DA neuron differentiation. Collectively, we identified β-arrestin, CK1, CK2, and Tiam1 as novel regulators of Wnt5a-induced signaling. In sum, data in the presented thesis describes molecular mechanisms and functional consequences of Wnt-driven signaling pathways and pinpoints the modulation of Wnt signaling as a possible tool to improve PD therapies

Wnt5a Regulates Ventral Midbrain Morphogenesis and the Development of A9–A10 Dopaminergic Cells In Vivo

Wnt5a is a morphogen that activates the Wnt/planar cell polarity (PCP) pathway and serves multiple functions during development. PCP signaling controls the orientation of cells within an epithelial plane as well as convergent extension (CE) movements. Wnt5a was previously reported to promote differentiation of A9–10 dopaminergic (DA) precursors in vitro. However, the signaling mechanism in DA cells and the function of Wnt5a during midbrain development in vivo remains unclear. We hereby report that Wnt5a activated the GTPase Rac1 in DA cells and that Rac1 inhibitors blocked the Wnt5a-induced DA neuron differentiation of ventral midbrain (VM) precursor cultures, linking Wnt5a-induced differentiation with a known effector of Wnt/PCP signaling. In vivo, Wnt5a was expressed throughout the VM at embryonic day (E)9.5, and was restricted to the VM floor and basal plate by E11.5–E13.5. Analysis of Wnt5a−/− mice revealed a transient increase in progenitor proliferation at E11.5, and a precociously induced NR4A2+ (Nurr1) precursor pool at E12.5. The excess NR4A2+ precursors remained undifferentiated until E14.5, when a transient 25% increase in DA neurons was detected. Wnt5a−/− mice also displayed a defect in (mid)brain morphogenesis, including an impairment in midbrain elongation and a rounded ventricular cavity. Interestingly, these alterations affected mostly cells in the DA lineage. The ventral Sonic hedgehog-expressing domain was broadened and flattened, a typical CE phenotype, and the domains occupied by Ngn2+ DA progenitors, NR4A2+ DA precursors and TH+ DA neurons were rostrocaudally reduced and laterally expanded. In summary, we hereby describe a Wnt5a regulation of Wnt/PCP signaling in the DA lineage and provide evidence for multiple functions of Wnt5a in the VM in vivo, including the regulation of VM morphogenesis, DA progenitor cell division, and differentiation of NR4A2+ DA precursors

The E3 ubiquitin ligase Mib1 regulates Plk4 and centriole biogenesis

Centrioles function as core components of centrosomes and as basal bodies for the formation of cilia and flagella. Thus, effective control of centriole numbers is essential for embryogenesis, tissue homeostasis, and genome stability. In mammalian cells, the centriole duplication cycle is governed by Polo-like kinase 4 (Plk4). Here we identify the E3 ubiquitin ligase Mind bomb (Mib1) as a novel interaction partner of Plk4. We show that Mib1 localizes to centriolar satellites but redistributes to centrioles in response to conditions that induce centriole amplification. The E3 ligase activity of Mib1 triggers ubiquitination of Plk4 on multiple sites, causing the formation of Lys11-, Lys29- and Lys48-ubiquitin linkages. These modifications control the abundance of Plk4 and its ability to interact with centrosomal proteins, thus counteracting centriole amplification induced by excess Plk4. Collectively, these results identify the interaction between Mib1 and Plk4 as a novel important element in the control of centriole homeostasis

Inactivation of PLK4-STIL Module Prevents Self-Renewal and Triggers p53-Dependent Differentiation in Human Pluripotent Stem Cells

Summary: Centrioles account for centrosomes and cilia formation. Recently, a link between centrosomal components and human developmental disorders has been established. However, the exact mechanisms how centrosome abnormalities influence embryogenesis and cell fate are not understood. PLK4-STIL module represents a key element of centrosome duplication cycle. We analyzed consequences of inactivation of the module for early events of embryogenesis in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). We demonstrate that blocking of PLK4 or STIL functions leads to centrosome loss followed by both p53-dependent and -independent defects, including prolonged cell divisions, upregulation of p53, chromosome instability, and, importantly, reduction of pluripotency markers and induction of differentiation. We show that the observed loss of key stem cells properties is connected to alterations in mitotic timing and protein turnover. In sum, our data define a link between centrosome, its regulators, and the control of pluripotency and differentiation in PSCs. : Recently a link has been established between centrosome and developmental disorders, yet the mechanisms connecting centrosome and cell fate are not understood. Cajanek and colleagues analyzed consequences of centrosome loss using hESCs/hiPSCs. They demonstrated that PLK4/STIL inhibition-mediated centrosome removal leads to loss of key stem cell properties connected to alterations in protein turnover and mitotic timing, which triggers p53-dependent differentiation. Keywords: centrosome, centriole, stem cell, differentiation, self-renewal, cell cycle, pluripotency, acentrosoma

KIF14 controls ciliogenesis via regulation of Aurora A and is important for Hedgehog signaling

International audiencePrimary cilia play critical roles in development and disease. Their assembly and disassembly are tightly coupled to cell cycle progression. Here, we present data identifying KIF14 as a regulator of cilia formation and Hedgehog (HH) signaling. We show that RNAi depletion of KIF14 specifically leads to defects in ciliogenesis and basal body (BB) biogenesis, as its absence hampers the efficiency of primary cilium formation and the dynamics of primary cilium elongation, and disrupts the localization of the distal appendage proteins SCLT1 and FBF1 and components of the IFT-B complex. We identify deregulated Aurora A activity as a mechanism contributing to the primary cilium and BB formation defects seen after KIF14 depletion. In addition, we show that primary cilia in KIF14-depleted cells are defective in response to HH pathway activation, independently of the effects of Aurora A. In sum, our data point to KIF14 as a critical node connecting cell cycle machinery, effective ciliogenesis, and HH signaling

Molecular basis of tubulin transport within the cilium by IFT74 and IFT81

Intraflagellar transport (IFT) of ciliary precursors such as tubulin from the cytoplasm to the ciliary tip is involved in the construction of the cilium, a hairlike organelle found on most eukaryotic cells. However, the molecular mechanisms of IFT are poorly understood. Here, we found that the two core IFT proteins IFT74 and IFT81 form a tubulin-binding module and mapped the interaction to a calponin homology domain of IFT81 and a highly basic domain in IFT74. Knockdown of IFT81 and rescue experiments with point mutants showed that tubulin binding by IFT81 was required for ciliogenesis in human cells

Wnt2 Regulates Progenitor Proliferation in the Developing Ventral Midbrain*

Wnts are secreted, lipidated proteins that regulate multiple aspects of brain development, including dopaminergic neuron development. In this study, we perform the first purification and signaling analysis of Wnt2 and define the function of Wnt2 in ventral midbrain precursor cultures, as well as in Wnt2-null mice in vivo. We found that purified Wnt2 induces the phosphorylation of both Lrp5/6 and Dvl-2/3, and activates β-catenin in SN4741 dopaminergic cells. Moreover, purified Wnt2 increases progenitor proliferation, and the number of dopaminergic neurons in ventral midbrain precursor cultures. In agreement with these findings, analysis of the ventral midbrain of developing Wnt2-null mice revealed a decrease in progenitor proliferation and neurogenesis that lead to a decrease in the number of postmitotic precursors and dopaminergic neurons. Collectively, our observations identify Wnt2 as a novel regulator of dopaminergic progenitors and dopaminergic neuron development

KIF14 controls ciliogenesis via regulation of Aurora A and is important for Hedgehog signaling

International audiencePrimary cilia play critical roles in development and disease. Their assembly and disassembly are tightly coupled to cell cycle progression. Here, we present data identifying KIF14 as a regulator of cilia formation and Hedgehog (HH) signaling. We show that RNAi depletion of KIF14 specifically leads to defects in ciliogenesis and basal body (BB) biogenesis, as its absence hampers the efficiency of primary cilium formation and the dynamics of primary cilium elongation, and disrupts the localization of the distal appendage proteins SCLT1 and FBF1 and components of the IFT-B complex. We identify deregulated Aurora A activity as a mechanism contributing to the primary cilium and BB formation defects seen after KIF14 depletion. In addition, we show that primary cilia in KIF14-depleted cells are defective in response to HH pathway activation, independently of the effects of Aurora A. In sum, our data point to KIF14 as a critical node connecting cell cycle machinery, effective ciliogenesis, and HH signaling

The Extracellular Domain of Lrp5/6 Inhibits Noncanonical Wnt Signaling In Vivo

Lrp5/6 are crucial coreceptors for Wnt/β-catenin signaling, a pathway biochemically distinct from noncanonical Wnt signaling pathways. Here, we examined the possible participation of Lrp5/6 in noncanonical Wnt signaling. We found that Lrp6 physically interacts with Wnt5a, but that this does not lead to phosphorylation of Lrp6 or activation of the Wnt/β-catenin pathway. Overexpression of Lrp6 blocks activation of the Wnt5a downstream target Rac1, and this effect is dependent on intact Lrp6 extracellular domains. These results suggested that the extracellular domain of Lrp6 inhibits noncanonical Wnt signaling in vitro. In vivo, Lrp6−/− mice exhibited exencephaly and a heart phenotype. Surprisingly, these defects were rescued by deletion of Wnt5a, indicating that the phenotypes resulted from noncanonical Wnt gain-of-function. Similarly, Lrp5 and Lrp6 antisense morpholino-treated Xenopus embryos exhibited convergent extension and heart phenotypes that were rescued by knockdown of noncanonical XWnt5a and XWnt11. Thus, we provide evidence that the extracellular domains of Lrp5/6 behave as physiologically relevant inhibitors of noncanonical Wnt signaling during Xenopus and mouse development in vivo