Sonic hedgehog and FGF8 collaborate to induce dopaminergic phenotypes in the Nurr1-overexpressing neural stem cell

Neural stem cells are self-renewing cells capable of differentiating into all neural lineage cells in vivo and in vitro. In the present study, coordinated induction of midbrain dopaminergic phenotypes in an immortalized multipotent neural stem cell line can be achieved by both overexpression of nuclear receptor Nurr1, and fibroblast growth factor-8 (FGF-8), and sonic hedgehog (Shh) signals. Nurr1 overexpression induces neuronal differentiation and confers competence to respond to extrinsic signals such as Shh and FGF-8 that induce dopaminergic fate in a mouse neural stem cell line. Our findings suggest that immortalized NSCs can serve as an excellent model for understanding mechanisms that regulate specification of ventral midbrain DA neurons and as an unlimited source of DA progenitors for treating Parkinson disease patients by cell replacement.     

Proliferation of Murine Midbrain Neural Stem Cells Depends upon an Endogenous Sonic Hedgehog (Shh) Source

The Sonic Hedgehog (Shh) pathway is responsible for critical patterning events early in development and for regulating the delicate balance between proliferation and differentiation in the developing and adult vertebrate brain. Currently, our knowledge of the potential role of Shh in regulating neural stem cells (NSC) is largely derived from analyses of the mammalian forebrain, but for dorsal midbrain development it is mostly unknown. For a detailed understanding of the role of Shh pathway for midbrain development in vivo, we took advantage of mouse embryos with cell autonomously activated Hedgehog (Hh) signaling in a conditional Patched 1 (Ptc1) mutant mouse model. This animal model shows an extensive embryonic tectal hypertrophy as a result of Hh pathway activation. In order to reveal the cellular and molecular origin of this in vivo phenotype, we established a novel culture system to evaluate neurospheres (nsps) viability, proliferation and differentiation. By recreating the three-dimensional (3-D) microenvironment we highlight the pivotal role of endogenous Shh in maintaining the stem cell potential of tectal radial glial cells (RGC) and progenitors by modulating their Ptc1 expression. We demonstrate that during late embryogenesis Shh enhances proliferation of NSC, whereas blockage of endogenous Shh signaling using cyclopamine, a potent Hh pathway inhibitor, produces the opposite effect. We propose that canonical Shh signaling plays a central role in the control of NSC behavior in the developing dorsal midbrain by acting as a niche factor by partially mediating the response of NSC to epidermal growth factor (EGF) and fibroblast growth factor (FGF) signaling. We conclude that endogenous Shh signaling is a critical mechanism regulating the proliferation of stem cell lineages in the embryonic dorsal tissue.     

Ectopic Wnt/beta-catenin signaling induces neurogenesis in the spinal cord and hindbrain floor plate

The most ventral structure of the developing neural tube, the floor plate (FP), differs in neurogenic capacity along the neuraxis. The FP is largely non-neurogenic at the hindbrain and spinal cord levels, but generates large numbers of dopamine (mDA) neurons at the midbrain levels. Wnt1, and other Wnts are expressed in the ventral midbrain, and Wnt/beta catenin signaling can at least in part account for the difference in neurogenic capacity of the FP between midbrain and hindbrain levels. To further develop the hypothesis that canonical Wnt signaling promotes mDA specification and FP neurogenesis, we have generated a model wherein beta-catenin is conditionally stabilized throughout the FP. Here, we unambiguously show by fate mapping FP cells in this mutant, that the hindbrain and spinal cord FP are rendered highly neurogenic, producing large numbers of neurons. We reveal that a neurogenic hindbrain FP results in the altered settling pattern of neighboring precerebellar neuronal clusters. Moreover, in this mutant, mDA progenitor markers are induced throughout the rostrocaudal axis of the hindbrain FP, although TH+ mDA neurons are produced only in the rostral aspect of rhombomere (r)1. This is, at least in part, due to depressed Lmx1b levels by Wnt/beta catenin signaling; indeed, when Lmx1b levels are restored in this mutant, mDA are observed not only in rostral r1, but also at more caudal axial levels in the hindbrain, but not in the spinal cord. Taken together, these data elucidate both patterning and neurogenic functions of Wnt/beta catenin signaling in the FP, and thereby add to our understanding of the molecular logic of mDA specification and neurogenesis.     

Cell-autonomous FGF signaling regulates anteroposterior patterning and neuronal differentiation in the mesodiencephalic dopaminergic progenitor domain

The structure and projection patterns of adult mesodiencephalic dopaminergic (DA) neurons are one of the best characterized systems in the vertebrate brain. However, the early organization and development of these nuclei remain poorly understood. The induction of midbrain DA neurons requires sonic hedgehog (Shh) from the floor plate and fibroblast growth factor 8 (FGF8) from the isthmic organizer, but the way in which FGF8 regulates DA neuron development is unclear. We show that, during early embryogenesis, mesodiencephalic neurons consist of two distinct populations: a diencephalic domain, which is probably independent of isthmic FGFs; and a midbrain domain, which is dependent on FGFs. Within these domains, DA progenitors and precursors use partly different genetic programs. Furthermore, the diencephalic DA domain forms a distinct cell population, which also contains non-DA Pou4f1(+) cells. FGF signaling operates in proliferative midbrain DA progenitors, but is absent in postmitotic DA precursors. The loss of FGFR1/2-mediated signaling results in a maturation failure of the midbrain DA neurons and altered patterning of the midbrain floor. In FGFR mutants, the DA domain adopts characteristics that are typical for embryonic diencephalon, including the presence of Pou4f1(+) cells among TH(+) cells, and downregulation of genes typical of midbrain DA precursors. Finally, analyses of chimeric embryos indicate that FGF signaling regulates the development of the ventral midbrain cell autonomously.     

FGF8 can activate Gbx2 and transform regions of the rostral mouse brain into a hindbrain fate.

The mid/hindbrain junction region, which expresses Fgf8, can act as an organizer to transform caudal forebrain or hindbrain tissue into midbrain or cerebellar structures, respectively. FGF8-soaked beads placed in the chick forebrain can similarly induce ectopic expression of mid/hindbrain genes and development of midbrain structures (Crossley, P. H., Martinez, S. and Martin, G. R. (1996) Nature 380, 66-68). In contrast, ectopic expression of Fgf8a in the mouse midbrain and caudal forebrain using a Wnt1 regulatory element produced no apparent patterning defects in the embryos examined (Lee, S. M., Danielian, P. S., Fritzsch, B. and McMahon, A. P. (1997) Development 124, 959-969). We show here that FGF8b-soaked beads can not only induce expression of the mid/hindbrain genes En1, En2 and Pax5 in mouse embryonic day 9.5 (E9.5) caudal forebrain explants, but also can induce the hindbrain gene Gbx2 and alter the expression of Wnt1 in both midbrain and caudal forebrain explants. We also show that FGF8b-soaked beads can repress Otx2 in midbrain explants. Furthermore, Wnt1-Fgf8b transgenic embryos in which the same Wnt1 regulatory element is used to express Fgf8b, have ectopic expression of En1, En2, Pax5 and Gbx2 in the dorsal hindbrain and spinal cord at E10.5, as well as exencephaly and abnormal spinal cord morphology. More strikingly, Fgf8b expression in more rostral brain regions appears to transform the midbrain and caudal forebrain into an anterior hindbrain fate through expansion of the Gbx2 domain and repression of Otx2 as early as the 7-somite stage. These findings suggest that normal Fgf8 expression in the anterior hindbrain not only functions to maintain development of the entire mid/hindbrain by regulating genes like En1, En2 and Pax5, but also might function to maintain a metencephalic identity by regulating Gbx2 and Otx2 expression.     

Retinoids control anterior and dorsal properties in the developing forebrain

We have previously shown that retinoic acid (RA) synthesized by the retinaldehyde dehydrogenase 2 (RALDH2) is required in forebrain development. Deficiency in RA due to inactivation of the mouse Raldh2 gene or to complete absence of retinoids in vitamin-A-deficient (VAD) quails, leads to abnormal morphogenesis of various forebrain derivatives. In this study we show that double Raldh2/Raldh3 mouse mutants have a more severe phenotype in the craniofacial region than single null mutants. In particular, the nasal processes are truncated and the eye abnormalities are exacerbated. It has been previously shown that retinoids act mainly on cell proliferation and survival in the ventral forebrain by regulating SHH and FGF8 signaling. Using the VAD quail model, which survives longer than the Raldh-deficient mouse embryos, we found that retinoids act in maintaining the correct position of anterior and dorsal boundaries in the forebrain by modulating FGF8 anteriorly and WNT signaling dorsally. Furthermore, BMP4 and FGF8 signaling are affected in the nasal region and BMP4 is ventrally expanded in the optic vesicle. At the optic cup stage, Pax6, Tbx5 and Bmp4 are ectopically expressed in the presumptive retinal pigmented epithelium (RPE), while Otx2 and Mitf are not induced, leading to a dorsal transdifferentiation of RPE to neural retina. Therefore, besides being required for survival of ventral structures, retinoids are involved in restricting anterior identity in the telencephalon and dorsal identity in the diencephalon and the retina.     

FGF17b and FGF18 have different midbrain regulatory properties from FGF8b or activated FGF receptors

Early patterning of the vertebrate midbrain and cerebellum is regulated by a mid/hindbrain organizer that produces three fibroblast growth factors (FGF8, FGF17 and FGF18). The mechanism by which each FGF contributes to patterning the midbrain, and induces a cerebellum in rhombomere 1 (r1) is not clear. We and others have found that FGF8b can transform the midbrain into a cerebellum fate, whereas FGF8a can promote midbrain development. In this study we used a chick electroporation assay and in vitro mouse brain explant experiments to compare the activity of FGF17b and FGF18 to FGF8a and FGF8b. First, FGF8b is the only protein that can induce the r1 gene Gbx2 and strongly activate the pathway inhibitors Spry1/2, as well as repress the midbrain gene Otx2. Consistent with previous studies that indicated high level FGF signaling is required to induce these gene expression changes, electroporation of activated FGFRs produce similar gene expression changes to FGF8b. Second, FGF8b extends the organizer along the junction between the induced Gbx2 domain and the remaining Otx2 region in the midbrain, correlating with cerebellum development. By contrast, FGF17b and FGF18 mimic FGF8a by causing expansion of the midbrain and upregulating midbrain gene expression. This result is consistent with Fgf17 and Fgf18 being expressed in the midbrain and not just in r1 as Fgf8 is. Third, analysis of gene expression in mouse brain explants with beads soaked in FGF8b or FGF17b showed that the distinct activities of FGF17b and FGF8b are not due to differences in the amount of FGF17b protein produced in vivo. Finally, brain explants were used to define a positive feedback loop involving FGF8b mediated upregulation of Fgf18, and two negative feedback loops that include repression of Fgfr2/3 and direct induction of Spry1/2. As Fgf17 and Fgf18 are co-expressed with Fgf8 in many tissues, our studies have broad implications for how these FGFs differentially control development.     

Sonic hedgehog controls stem cell behavior in the postnatal and adult brain

Sonic hedgehog (Shh) signaling controls many aspects of ontogeny, orchestrating congruent growth and patterning. During brain development, Shh regulates early ventral patterning while later on it is critical for the regulation of precursor proliferation in the dorsal brain, namely in the neocortex, tectum and cerebellum. We have recently shown that Shh also controls the behavior of cells with stem cell properties in the mouse embryonic neocortex, and additional studies have implicated it in the control of cell proliferation in the adult ventral forebrain and in the hippocampus. However, it remains unclear whether it regulates adult stem cell lineages in an equivalent manner. Similarly, it is not known which cells respond to Shh signaling in stem cell niches. Here we demonstrate that Shh is required for cell proliferation in the mouse forebrain's subventricular zone (SVZ) stem cell niche and for the production of new olfactory interneurons in vivo. We identify two populations of Gli1+ Shh signaling responding cells: GFAP+ SVZ stem cells and GFAP- precursors. Consistently, we show that Shh regulates the self-renewal of neurosphere-forming stem cells and that it modulates proliferation of SVZ lineages by acting as a mitogen in cooperation with epidermal growth factor (EGF). Together, our data demonstrate a critical and conserved role of Shh signaling in the regulation of stem cell lineages in the adult mammalian brain, highlight the subventricular stem cell astrocytes and their more abundant derived precursors as in vivo targets of Shh signaling, and demonstrate the requirement for Shh signaling in postnatal and adult neurogenesis.     

Regulation of the neural patterning activity of sonic hedgehog by secreted BMP inhibitors expressed by notochord and somites

The secretion of Sonic hedgehog (Shh) from the notochord and floor plate appears to generate a ventral-to-dorsal gradient of Shh activity that directs progenitor cell identity and neuronal fate in the ventral neural tube. In principle, the establishment of this Shh activity gradient could be achieved through the graded distribution of the Shh protein itself, or could depend on additional cell surface or secreted proteins that modify the response of neural cells to Shh. Cells of the neural plate differentiate from a region of the ectoderm that has recently expressed high levels of BMPs, raising the possibility that prospective ventral neural cells are exposed to residual levels of BMP activity. We have examined whether modulation of the level of BMP signaling regulates neural cell responses to Shh, and thus might contribute to the patterning of cell types in the ventral neural tube. Using an in vitro assay of neural cell differentiation we show that BMP signaling markedly alters neural cell responses to Shh signals, eliciting a ventral-to-dorsal switch in progenitor cell identity and neuronal fate. BMP signaling is regulated by secreted inhibitory factors, including noggin and follistatin, both of which are expressed in or adjacent to the neural plate. Conversely, follistatin but not noggin produces a dorsal-to-ventral switch in progenitor cell identity and neuronal fate in response to Shh both in vitro and in vivo. These results suggest that the specification of ventral neural cell types depends on the integration of Shh and BMP signaling activities. The net level of BMP signaling within neural tissue may be regulated by follistatin and perhaps other BMP inhibitors secreted by mesodermal cell types that flank the ventral neural tube.     

Generation of regionally specified neural progenitors and functional neurons from human embryonic stem cells under defined conditions

To model human neural-cell-fate specification and to provide cells for regenerative therapies, we have developed a method to generate human neural progenitors and neurons from human embryonic stem cells, which recapitulates human fetal brain development. Through the addition of a small molecule that activates canonical WNT signaling, we induced rapid and efficient dose-dependent specification of regionally defined neural progenitors ranging from telencephalic forebrain to posterior hindbrain fates. Ten days after initiation of differentiation, the progenitors could be transplanted to the adult rat striatum, where they formed neuron-rich and tumor-free grafts with maintained regional specification. Cells patterned toward a ventral midbrain (VM) identity generated a high proportion of authentic dopaminergic neurons after transplantation. The dopamine neurons showed morphology, projection pattern, and protein expression identical to that of human fetal VM cells grafted in parallel. VM-patterned but not forebrain-patterned neurons released dopamine and reversed motor deficits in an animal model of Parkinson's disease.     

Neural subtype specification of fertilization and nuclear transfer embryonic stem cells and application in parkinsonian mice

Existing protocols for the neural differentiation of mouse embryonic stem (ES) cells require extended in vitro culture, yield variable differentiation results or are limited to the generation of selected neural subtypes. Here we provide a set of coculture conditions that allows rapid and efficient derivation of most central nervous system phenotypes. The fate of both fertilization- and nuclear transfer-derived ES (ntES) cells was directed selectively into neural stem cells, astrocytes, oligodendrocytes or neurons. Specific differentiation into gamma-aminobutyric acid (GABA), dopamine, serotonin or motor neurons was achieved by defining conditions to induce forebrain, midbrain, hindbrain and spinal cord identity. Neuronal function of ES cell-derived dopaminergic neurons was shown in vitro by electron microscopy, measurement of neurotransmitter release and intracellular recording. Furthermore, transplantation of ES and ntES cell-derived dopaminergic neurons corrected the phenotype of a mouse model of Parkinson disease, demonstrating an in vivo application of therapeutic cloning in neural disease.     

Brain area-specific effect of TGF-beta signaling on Wnt-dependent neural stem cell expansion

Regulating the choice between neural stem cell maintenance versus differentiation determines growth and size of the developing brain. Here we identify TGF-beta signaling as a crucial factor controlling these processes. At early developmental stages, TGF-beta signal activity is localized close to the ventricular surface of the neuroepithelium. In the midbrain, but not in the forebrain, Tgfbr2 ablation results in ectopic expression of Wnt1/beta-catenin and FGF8, activation of Wnt target genes, and increased proliferation and horizontal expansion of neuroepithelial cells due to shortened cell-cycle length and decreased cell-cycle exit. Consistent with this phenotype, self-renewal of mutant neuroepithelial stem cells is enhanced in the presence of FGF and requires Wnt signaling. Moreover, TGF-beta signal activation counteracts Wnt-induced proliferation of midbrain neuroepithelial cells. Thus, TGF-beta signaling controls the size of a specific brain area, the dorsal midbrain, by antagonizing canonical Wnt signaling and negatively regulating self-renewal of neuroepithelial stem cells.