Ventricular Zone Precursor Research Articles

Identification ofin vivoDNA-binding mechanisms of Pax6 and reconstruction of Pax6-dependent gene regulatory networks during forebrain and lens development

The transcription factor Pax6 is comprised of the paired domain (PD) and homeodomain (HD). In the developing forebrain, Pax6 is expressed in ventricular zone precursor cells and in specific subpopulations of neurons; absence of Pax6 results in disrupted cell proliferation and cell fate specification. Pax6 also regulates the entire lens developmental program. To reconstruct Pax6-dependent gene regulatory networks (GRNs), ChIP-seq studies were performed using forebrain and lens chromatin from mice. A total of 3514 (forebrain) and 3723 (lens) Pax6-containing peaks were identified, with ∼70% of them found in both tissues and thereafter called ‘common’ peaks. Analysis of Pax6-bound peaks identified motifs that closely resemble Pax6-PD, Pax6-PD/HD and Pax6-HD established binding sequences. Mapping of H3K4me1, H3K4me3, H3K27ac, H3K27me3 and RNA polymerase II revealed distinct types of tissue-specific enhancers bound by Pax6. Pax6 directly regulates cortical neurogenesis through activation (e.g. Dmrta1 and Ngn2) and repression (e.g. Ascl1, Fezf2, and Gsx2) of transcription factors. In lens, Pax6 directly regulates cell cycle exit via components of FGF (Fgfr2, Prox1 and Ccnd1) and Wnt (Dkk3, Wnt7a, Lrp6, Bcl9l, and Ccnd1) signaling pathways. Collectively, these studies provide genome-wide analysis of Pax6-dependent GRNs in lens and forebrain and establish novel roles of Pax6 in organogenesis.

Pax6 Interactions with Chromatin and Identification of Its Novel Direct Target Genes in Lens and Forebrain

Pax6 encodes a specific DNA-binding transcription factor that regulates the development of multiple organs, including the eye, brain and pancreas. Previous studies have shown that Pax6 regulates the entire process of ocular lens development. In the developing forebrain, Pax6 is expressed in ventricular zone precursor cells and in specific populations of neurons; absence of Pax6 results in disrupted cell proliferation and cell fate specification in telencephalon. In the pancreas, Pax6 is essential for the differentiation of α-, β- and δ-islet cells. To elucidate molecular roles of Pax6, chromatin immunoprecipitation experiments combined with high-density oligonucleotide array hybridizations (ChIP-chip) were performed using three distinct sources of chromatin (lens, forebrain and β-cells). ChIP-chip studies, performed as biological triplicates, identified a total of 5,260 promoters occupied by Pax6. 1,001 (133) of these promoter regions were shared between at least two (three) distinct chromatin sources, respectively. In lens chromatin, 2,335 promoters were bound by Pax6. RNA expression profiling from Pax6+/− lenses combined with in vivo Pax6-binding data yielded 76 putative Pax6-direct targets, including the Gaa, Isl1, Kif1b, Mtmr2, Pcsk1n, and Snca genes. RNA and ChIP data were validated for all these genes. In lens cells, reporter assays established Kib1b and Snca as Pax6 activated and repressed genes, respectively. In situ hybridization revealed reduced expression of these genes in E14 cerebral cortex. Moreover, we examined differentially expressed transcripts between E9.5 wild type and Pax6−/− lens placodes that suggested Efnb2, Fat4, Has2, Nav1, and Trpm3 as novel Pax6-direct targets. Collectively, the present studies, through the identification of Pax6-direct target genes, provide novel insights into the molecular mechanisms of Pax6 gene control during mouse embryonic development. In addition, the present data demonstrate that Pax6 interacts preferentially with promoter regions in a tissue-specific fashion. Nevertheless, nearly 20% of the regions identified are accessible to Pax6 in multiple tissues.

Gap Junctions/Hemichannels Modulate Interkinetic Nuclear Migration in the Forebrain Precursors

During mitotic division in the telencephalic proliferative ventricular zone (VZ), the nuclei of the neural precursors move basally away from the ventricular surface for DNA synthesis, and apically return to the surface for mitotic division; a process known as interkinetic migration or "to-and-fro" nuclear translocation. The cell, which remains attached to the ventricular surface, either continues cycling, or exits the cycle and migrates to the subventricular zone or the developing cortical plate. Although gap junctions/hemichannels are known to modulate DNA synthesis via Ca(2+) waves, the role of Ca(+) oscillations and the mechanism of nuclear translocation in the VZ precursors are unclear. Here, we provide evidence that, during apical nuclear migration, VZ precursors display dynamic spontaneous Ca(2+) transients, which depend on functional gap junctions/hemichannels via ATP release and Ca(2+)-mobilizing messenger diffusion. Furthermore, we found that blocking gap junctions/hemichannels or short hairpin RNA-mediated knockdown of Cx43 (connexin 43) retards the apically directed interkinetic nuclear migration accompanied with changes in the nuclear length/width ratio. In addition, we demonstrated that blocking functional gap junctions/hemichannels induces phosphorylation of small GTPase cdc42 in the VZ precursors. The basal phase of interkinetic migration is much slower and appears to be mediated passively by mechanical forces after cell division. Our findings indicate that functional interference with gap junctions/hemichannels during embryonic development may lead to abnormal corticogenesis and dysfunction of the cerebral cortex in adult organisms.

Cortical Neural Precursors Inhibit Their Own Differentiation via N-Cadherin Maintenance of β-Catenin Signaling

Little is known about the architecture of cellular microenvironments that support stem and precursor cells during tissue development. Although adult stem cell niches are organized by specialized supporting cells, in the developing cerebral cortex, neural stem/precursor cells reside in a neurogenic niche lacking distinct supporting cells. Here, we find that neural precursors themselves comprise the niche and regulate their own development. Precursor-precursor contact regulates beta-catenin signaling and cell fate. In vivo knockdown of N-cadherin reduces beta-catenin signaling, migration from the niche, and neuronal differentiation in vivo. N-cadherin engagement activates beta-catenin signaling via Akt, suggesting a mechanism through which cells in tissues can regulate their development. These results suggest that neural precursor cell interactions can generate a self-supportive niche to regulate their own number.

Cerebellar GABAergic progenitors adopt an external granule cell-like phenotype in the absence of Ptf1a transcription factor expression

We report in this study that, in the cerebellum, the pancreatic transcription factor Ptf1a is required for the specific generation of Purkinje cells (PCs) and interneurons. Moreover, granule cell progenitors in the external GCL (EGL) appear to be unaffected by deletion of Ptf1a. Cell lineage analysis in Ptf1a(Cre/Cre) mice was used to establish that, in the absence of Ptf1a expression, ventricular zone progenitors, normally fated to produce PCs and interneurons, aberrantly migrate to the EGL and express typical markers of these cells, such as Math1, Reelin, and Zic1/2. Furthermore, these cells have a fine structure typical of EGL progenitors, indicating that they adopt an EGL-like cell phenotype. These findings indicate that Ptf1a is necessary for the specification and normal production of PCs and cerebellar interneurons. Moreover, our results suggest that Ptf1a is also required for the suppression of the granule cell specification program in cerebellar ventricular zone precursors.

Cell-Autonomous β-Catenin Signaling Regulates Cortical Precursor Proliferation

Overexpression of beta-catenin, a protein that functions in both cell adhesion and signaling, causes expansion of the cerebral cortical precursor population and cortical surface area enlargement. Here, we find that focal elimination of beta-catenin from cortical neural precursors in vivo causes premature neuronal differentiation. Precursors within the cerebral cortical ventricular zone exhibit robust beta-catenin-mediated transcriptional activation, which is downregulated as cells exit the ventricular zone. Targeted inhibition of beta-catenin signaling during embryonic development causes cortical precursor cells to prematurely exit the cell cycle, differentiate into neurons, and migrate to the cortical plate. These results show that beta-catenin-mediated transcriptional activation functions in the decision of cortical ventricular zone precursors to proliferate or differentiate during development, and suggest that the cell-autonomous signaling activity of beta-catenin can control the production of cortical neurons and thus regulate cerebral cortical size.

Molecular and Morphological Heterogeneity of Neural Precursors in the Mouse Neocortical Proliferative Zones

The proliferative ventricular zone (VZ) is the main source of projection neurons for the overlying cerebral neocortex. The number and diversity of neocortical neurons is determined, in part, by factors controlling the proliferation and specification of VZ cells during embryonic development. We used a variety of methods, including in utero electroporation with specific cellular markers, computer-assisted serial EM cell reconstruction, and time-lapse multiphoton imaging to characterize the molecular and morphological characteristics of the VZ constituents and to capture their behavior during cell division. Our analyses reveal at least two types of dividing cells in the VZ: (1) radial glial cells (RGCs) that span the entire neocortical wall and maintain contact both at the ventricular and pial surfaces throughout mitotic division, and (2) short neural precursors (SNPs) that possess a ventricular endfoot and a basal process of variable length that is retracted during mitotic division. These two precursor cell classes are present concomitantly in the VZ, but their relative number changes over the course of cortical neurogenesis. Moreover, the SNPs are morphologically, ultrastructurally and molecularly distinct from dividing RGCs. For example, SNPs are marked by their preferential expression of the tubulin alpha-1 promoter whereas RGCs instead express the glutamate-aspartate transporter and brain lipid binding protein promoters. In contrast to recent studies that suggest that RGCs are the sole type of VZ precursor, the present study indicates that the VZ in murine dorsal telencephalon is similar to that in human and nonhuman primates, because it contains multiple types of neuronal precursors.

CNTF/LIF/gp130 receptor complex signaling maintains a VZ precursor differentiation gradient in the developing ventral forebrain

The extrinsic signaling pathways responsible for the formation and maintenance of the unique laminar organization of the forebrain germinal zones are largely unknown. In the present study, we asked whether ciliary neurotrophic factor (CNTF)/leukemia inhibitory factor (LIF)/gp130 signaling plays a role in the development of the germinal layers in the lateral ganglionic eminence. We found that CNTF/LIF/gp130 receptor signaling promotes the self-renewal/expansion of a subpopulation of fibroblast growth factor-responsive ventricular zone (VZ) precursors in the ventral forebrain. Analysis of Lifr-/- mice suggests that CNTF/LIF/gp130 signaling maintains a subpopulation of GSH2+ VZ precursors, which are necessary for normal growth of the early ventral forebrain and for maintaining a gradient of VZ precursor differentiation in the lateral ganglionic eminence, as defined by GSH2, MASH1 and DLX2 expression. Furthermore, addition of exogenous CNTF to embryonic forebrain explant cultures deprived of choroid plexus-derived CNTF, was sufficient to promote a VZ differentiation gradient. In contrast to the forebrain, CNTF/LIF/gp130 signaling reduced, rather than enhanced, precursor self-renewal/expansion in the spinal cord. These results demonstrate a novel region-specific role for CNTF/LIF/gp130 signaling in the development of the germinal layers of the embryonic telencephalon.

Generation of Dopaminergic Neurons in the Adult Brain from Mesencephalic Precursor Cells Labeled with anestin-GFPTransgene

Mesencephalic precursor cells may one day provide dopaminergic neurons for the treatment of Parkinson's disease. However, the generation of dopaminergic neurons from mesencephalic precursors has been difficult to follow, partly because an appropriate means for recognizing mesencephalic ventricular zone precursors has not been available. To visualize and isolate mesencephalic precursor cells from a mixed population, we used transgenic mice and rats carrying green fluorescent protein (GFP) cDNA under the control of the nestin enhancer. nestin-driven GFP was detected in the mesencephalic ventricular zone, and it colocalized with specific markers for neural precursor cells. In addition, data from flow-cytometry indicated that Prominin/CD133, a cell-surface marker for ventricular zone cells, was expressed specifically in these GFP-positive (GFP(+)) cells. After sorting by fluorescence-activated cell sorting, the GFP(+) cells proliferated in vitro and expressed precursor cell markers but not neuronal markers. Using clonogenic sphere formation assays, we showed that this sorted population was enriched in multipotent precursor cells that could differentiate into both neurons and glia. Importantly, many neurons generated from nestin-GFP-sorted mesencephalic precursors developed a dopaminergic phenotype in vitro. Finally, nestin-GFP(+) cells were transplanted into the striatum of a rat model of Parkinson's disease. Bromodeoxyuridine-tyrosine hydroxylase double-labeling revealed that the transplanted cells generated new dopaminergic neurons within the host striatum. The implanted cells were able to restore dopaminergic function in the host striatum, as assessed by a behavioral measure: recovery from amphetamine-induced rotation. Together, these findings indicate that precursor cells harvested from the embryonic ventral mesencephalon can generate dopaminergic neurons able to restore function to the chemically denervated adult striatum.

Temporally and spatially regulated expression of a candidate G-protein-coupled receptor during cerebral cortical development.

Genes expressed in layer-specific patterns in the mammalian cerebral cortex may play a role in specifying the identity of different cortical layers. Using PCR-differential display, we identified a cDNA that encodes rCNL3, a gene cloned previously by sequence homology to G-protein-coupled receptors. rCNL3 is expressed predominantly in layers 2-4 of the young rat cortex and in the developing and adult striatum. Cortical expression of rCNL3 begins postnatally at P3 and continues at high levels until around P15, while striatal expression begins at E20 and continues through adulthood. rCNL3 expression is not detectable in the ventricular zone precursors that generate the neurons of layers 2-4. The expression pattern of rCNL3 in the developing cortex suggests that rCNL3 is not involved in the initial specification of laminar fate, but rather may be involved with later differentiation events within the superficial cortical layers.

Newly generated neurons of the adult songbird brain become functionally active in long-term culture

The vocal control nucleus, HVc, of the songbird forebrain undergoes neurogenesis in adulthood, as ventricular zone precursor cells divide and their daughter cells migrate into the subjacent forebrain, where they differentiate into neurons. We have previously demonstrated that the migration and development of these new neurons can proceed in vitro, in HVc ventricular zone explant cultures derived from the adult canary HVc. By a combination of electron microscopy and electrophysiology, we now report that these newly produced neurons become functionally mature and synaptically competent in culture. These cells developed synaptic contacts which became morphologically evident during the second week in culture, and which preceded the development of both stimulus-evoked and spontaneous action potentials during the second and third weeks in vitro. Thus, the newly generated neurons of the adult avian forebrain can form structurally complex, electrically interactive networks in long-term culture.

Neuronal development and migration in explant cultures of the adult canary forebrain

The vocal control nucleus (HVc) of the songbird forebrain undergoes neurogenesis in adulthood, as ventricular zone precursor cells divide, and their daughter cells migrate into the subjacent forebrain and differentiate into neurons. A procedure is now described by which the migration and development of these new neurons can be directly observed in vitro, in explant cultures of the adult canary forebrain. Cultured explants have yielded extensive outgrowth of new neurons from 2 neurogenic regions of the adult songbird brain: the HVc and its adjacent dorsomediocaudal neostriatum. Immunocytochemically identified neurons were determined by 3H-thymidine autoradiography to have been newly generated in vivo, in the days preceding explantation. These cells were immunoreactive for a variety of neuron-selective antigens, including MAP-1B, MAP-2, neuron-specific enolase, synaptic vesicle protein-2, N-CAM, and the tetanus toxin and A2B5 ligands. Combined immunostaining and autoradiography of cultures derived from HVcs exposed to 3H-thymidine in vivo revealed a population of newly generated, MAP-2/3H-thymidine-positive neurons. The neurons identified in these adult explant outgrowths comprised new, recently generated postmitotic cells which had arisen in the neostriatal ventricular zone and were in the early stages of their parenchymal migration at the time of tissue explantation. Accordingly, nonneurogenic control regions, including the anterior hyperstriatum, cerebellum, and optic tectum, did not display neuronal outgrowth in culture. Only forebrain regions exhibiting neurogenesis in vivo manifested neuronal outgrowth and maturation in vitro.