The extracellular domain of Lrp5/6 inhibits noncanonical Wnt signaling in vivo.

Lrp5/6 are crucial coreceptors for Wnt/beta-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/beta-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.

Liver X receptors and oxysterols promote ventral midbrain neurogenesis in vivo and in human embryonic stem cells.

Control over progenitor proliferation and neurogenesis remains a key challenge for stem cell neurobiology and a prerequisite for successful stem cell replacement therapies for neurodegenerative diseases like Parkinson's disease (PD). Here, we examined the function of two nuclear receptors, liver X receptors (Lxralpha and beta) and their ligands, oxysterols, as regulators of cell division, ventral midbrain (VM) neurogenesis, and dopaminergic (DA) neuron development. Deletion of Lxrs reduced cell cycle progression and VM neurogenesis, resulting in decreased DA neurons at birth. Activation of Lxrs with oxysterol ligands increased the number of DA neurons in mouse embryonic stem cells (ESCs) and in wild-type but not Lxralphabeta(-/-) VM progenitor cultures. Likewise, oxysterol treatment of human ESCs (hESCs) during DA differentiation increased neurogenesis and the number of mature DA neurons, while reducing proliferating progenitors. Thus, Lxr ligands may improve current hESC replacement strategies for PD by selectively augmenting the generation of DA neurons.

Identification of midbrain floor plate radial glia-like cells as dopaminergic progenitors.

The floor plate (FP), a signaling center and a structure rich in radial glia-like cells, has been traditionally thought to be devoid of neurons and neuronal progenitors. However, in the midbrain, the FP contains neurons of the dopaminergic (DA) lineage that require contact with radial glia-like cells for their induction. We, therefore, decided to explore the interaction relationship between radial glia and neurons during DA neurogenesis. Taking advantage of a novel FP radial glia-like cell culture system and retroviruses, DA neurons were lineage traced in vitro. In utero BrdU pulse-chases extensively labeled the midbrain FP and traced DA neurons both in vivo and in FP cultures. Moreover, from E9.5 to E13.5 the midbrain FP contained dividing cells only in the most apical part of the neuroepithelium, in cells identified as radial glia-like cells. We, therefore, hypothesized that midbrain FP radial glia-like cells could be DA progenitors and tested our hypothesis in vivo. Lineage tracing of DA progenitors with EGFP in Tis21-EGFP knock-in mice, and genetic fate mapping in GLAST::CreERT2/ZEG mice identified the neuroepithelium of the midbrain FP, and specifically, GLAST+ radial glia-like cells as DA progenitors. Combined, our experiments support the concept that the midbrain FP differs from other FP regions and demonstrate that FP radial glia-like cells in the midbrain are neurogenic and give rise to midbrain DA neurons.

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.

Microarray analyses support a role for Nurr1 in resistance to oxidative stress and neuronal differentiation in neural stem cells.

Nurr1 is an orphan nuclear receptor required for the development of midbrain dopaminergic neurons. To better understand the molecular consequences of Nurr1 expression, we compared the transcriptomes of two independent control and Nurr1-expressing NSC lines using Affymetrix cDNA microarrays. These data reveal the regulation of genes involved in promoting cell survival (trophic/growth factors and stress response genes) and in preventing cell death (decreased caspase-3 and caspase-11 expression). We found that conditioned medium from Nurr1-expressing NSC lines enhanced the survival of midbrain dopaminergic neurons in primary cultures and that Nurr1-expressing NSC lines themselves were more resistant to oxidative stress. These findings are accompanied by a dynamic pattern of gene regulation that is consistent with a role for Nurr1 in promoting both the acquisition of brain-region-specific identity (Engrailed-1) and neuronal differentiation (tubulin beta III). Interestingly, our gene expression profiles suggested that tenascin-C was regulated by Nurr1 in developing dopaminergic neurons. This was further confirmed in vitro and in Nurr1 knockout mice where low levels of tenascin-C mRNA were observed. Analysis of tenascin-C-null mice revealed an increase in the number of Nurr1(+) cells that become tyrosine hydroxylase-positive (TH(+)) dopaminergic neurons at embryonic day 11.5, suggesting that tenascin-C normally delays the acquisition of TH by Nurr1(+) precursors. Thus, our results confirm the presence of both secreted and cell-intrinsic survival signals modulated by Nurr1 and suggest that Nurr1 is a key regulator of both survival and dopaminergic differentiation.

Abnormal development of mouse embryoid bodies lacking p27Kip1 cell cycle regulator.

Cultures of three-dimensional aggregates of embryonic stem cells (ESCs) called embryoid bodies (EBs) provide a valuable system for analyzing molecular mechanisms that regulate differentiation of this unique cell type. Cyclin-dependent kinase inhibitor p27Kip1 (p27) becomes elevated during the differentiation of mouse ESCs (mESCs). In this study, various aspects of differentiation of EBs produced from normal and p27-deficient mESCs were analyzed to address the biological significance of this elevation. It was found that EBs lacking p27 grew significantly bigger, but this was not accompanied by detect-able abnormalities in the activities of cyclin-dependent kinases (CDKs). In most EB cells, downregulation of activating cyclins rather than upregulation of inhibiting p27 is probably responsible for lowering the activity of their CDKs. Abnormalities in the development of specific cell lineages were also observed in p27-deficient EBs. These included elimination of cells positive for cytokeratin endo-A (TROMA-I) and increased proliferation and formation of cavities originating from cells positive for Lewis-X. Our data also suggest that although two different pools of Lewis-X-expressing cells, cluster forming (ESC-like) and cavity forming (neural progenitors), normally exist in EBs, the absence of p27 leads to the enhancement of only the neural pool. No failure was found when the neurogenic capacity of p27-deficient mESCs was tested using various protein markers. Together, our data point to a dual role of p27 in mESCs, with one role being in the regulation of proliferation and the other role in establishing some other aspects of a differentiated phenotype.

Region-specific effects of glia on neuronal induction and differentiation with a focus on dopaminergic neurons.

Radial glia (RG) are the first glial cell type to appear in the nervous system. Their broad distribution and apparent similarity hide important brain region-specific differences that are likely to be essential for development. However, recent evidence supports the stimulating concept that in addition to their classical function as neuroblast guides, RG are neuronal precursors (Malatesta et al. Development 127:5253-5263, 2000; Miyata et al. Neuron 31:727-741, 2001; Noctor et al. Nature 409:714-720, 2001; Skogh et al. Mol Cell Neurosci 17:811-820, 2001). We propose that RG not only generate and guide newborn neurons, but could also instruct their own neuronal progeny to adopt appropriate region-specific phenotypes.

Radial glia (RG) are the first glial cell type to appear in the nervous system. Their broad distribution and apparent similarity hide important brain region-specific differences that are likely to be essential for development. However, recent evidence supports the stimulating concept that in addition to their classical function as neuroblast guides, RG are neuronal precursors (Malatesta et al. Development 127:5253-5263, 2000; Miyata et al. Neuron 31:727-741, 2001; Noctor et al. Nature 409:714-720, 2001; Skogh et al. Mol Cell Neurosci 17:811-820, 2001). We propose that RG not only generate and guide newborn neurons, but could also instruct their own neuronal progeny to adopt appropriate region-specific phenotypes. GLIA 43:47-51, 2003.

Valproate regulates GSK-3-mediated axonal remodeling and synapsin I clustering in developing neurons.

Valproate (VPA) and lithium have been used for many years in the treatment of manic depression. However, their mechanisms of action remain poorly understood. Recent studies suggest that lithium and VPA inhibit GSK-3beta, a serine/threonine kinase involved in the insulin and WNT signaling pathways. Inhibition of GSK-3beta by high concentrations of lithium has been shown to mimic WNT-7a signaling by inducing axonal remodeling and clustering of synapsin I in developing neurons. Here we have compared the effect of therapeutic concentrations of lithium and VPA during neuronal maturation. VPA and, to a lesser extent, lithium induce clustering of synapsin I. In addition, lithium and VPA induce similar changes in the morphology of axons by increasing growth cone size, spreading, and branching. More importantly, both mood stabilizers decrease the level of MAP-1B-P, a GSK-3beta-phosphorylated form of MAP-1B in developing neurons, suggesting that therapeutic concentrations of these mood stabilizers inhibit GSK-3beta. In vitro kinase assays show that therapeutic concentrations of VPA do not inhibit GSK-3beta but that therapeutic concentrations of lithium partially inhibit GSK-3beta activity. Our results support the idea that both mood stabilizers inhibit GSK-3beta in developing neurons through different pathways. Lithium directly inhibits GSK-3beta in contrast to VPA, which inhibits GSK-3beta indirectly by an as-yet-unknown pathway. These findings may have important implications for the development of new strategies to treat bipolar disorders.