Visualizing spatiotemporal dynamics of multicellular cell-cycle progression.

The cell-cycle transition from G1 to S phase has been difficult to visualize. We have harnessed antiphase oscillating proteins that mark cell-cycle transitions in order to develop genetically encoded fluorescent probes for this purpose. These probes effectively label individual G1 phase nuclei red and those in S/G2/M phases green. We were able to generate cultured cells and transgenic mice constitutively expressing the cell-cycle probes, in which every cell nucleus exhibits either red or green fluorescence. We performed time-lapse imaging to explore the spatiotemporal patterns of cell-cycle dynamics during the epithelial-mesenchymal transition of cultured cells, the migration and differentiation of neural progenitors in brain slices, and the development of tumors across blood vessels in live mice. These mice and cell lines will serve as model systems permitting unprecedented spatial and temporal resolution to help us better understand how the cell cycle is coordinated with various biological events.

Axonal projections of mechanoreceptive dorsal root ganglion neurons depend on Ret.

Establishment of connectivity between peripheral and central organs is essential for sensory processing by dorsal root ganglion (DRG) neurons. Using Ret as a marker for mechanoreceptive DRG neurons, we show that both central and peripheral projections of mechanoreceptive neurons are severely impaired in the absence of Ret. Death of DRG neurons in Ret-deficient mice can be rescued by eliminating Bax, although their projections remain disrupted. Furthermore, ectopic expression of the Ret ligand neurturin, but not Gdnf, in the spinal cord induces aberrant projection of mechanoreceptive afferents. Our results demonstrate that Ret expression in DRG neurons is crucial for the neurturin-mediated formation of precise axonal projections in the central nervous system.

Ptch1-mediated dosage-dependent action of Shh signaling regulates neural progenitor development at late gestational stages.

Sonic hedgehog (Shh) signaling regulates cell differentiation and proliferation during brain development. However, the role of Shh in neurogenesis during late gestation (embryonic day 13.5-18.5) remains unclear. Herein, we used a genetic approach and in utero electroporation to investigate the role of mouse Shh and patched homolog 1 (Ptch1), the putative receptor for Shh. Proliferating cortical intermediate (basal) progenitor cells (IPCs) were severely reduced in Shh mutant mice, suggesting that endogenous Shh signaling could play an essential role in cortical IPC development. During cortical neurogenesis, strong upregulation of Shh signaling enhanced the transition from ventricular zone (VZ) progenitors to ventralized IPCs, while low levels of signaling enhanced the generation and proliferation of cortical IPCs in the subventricular zone. The effects of Shh upregulation in this study were consistent with a phenotype of conditional loss of function of Ptch1, and the phenotype of a hypomorphic allele of Ptch1, respectively. These data indicated that endogenous Ptch1 mediates the broad effects of Shh on the transition from VZ progenitors to IPCs and activation of proliferation of the IPCs in the cortex during late gestational stages.

SNAP23 deficiency causes severe brain dysplasia through the loss of radial glial cell polarity.

In the developing brain, the polarity of neural progenitor cells, termed radial glial cells (RGCs), is important for neurogenesis. Intercellular adhesions, termed apical junctional complexes (AJCs), at the apical surface between RGCs are necessary for cell polarization. However, the mechanism by which AJCs are established remains unclear. Here, we show that a SNARE complex composed of SNAP23, VAMP8, and Syntaxin1B has crucial roles in AJC formation and RGC polarization. Central nervous system (CNS)-specific ablation of SNAP23 (NcKO) results in mice with severe hypoplasia of the neocortex and no hippocampus or cerebellum. In the developing NcKO brain, RGCs lose their polarity following the disruption of AJCs and exhibit reduced proliferation, increased differentiation, and increased apoptosis. SNAP23 and its partner SNAREs, VAMP8 and Syntaxin1B, are important for the localization of an AJC protein, N-cadherin, to the apical plasma membrane of RGCs. Altogether, SNARE-mediated localization of N-cadherin is essential for AJC formation and RGC polarization during brain development.

DSCAM deficiency causes loss of pre-inspiratory neuron synchroneity and perinatal death.

Down syndrome cell adhesion molecule (DSCAM) is a neural adhesion molecule that plays diverse roles in neural development. We disrupted the Dscam locus in mice and found that the null mutants (Dscam(-/-)) died within 24 h after birth. Whole-body plethysmography showed irregular respiration and lower ventilatory response to hypercapnia in the null mutants. Furthermore, a medulla-spinal cord preparation of Dscam(-/-) mice showed that the C4 ventral root activity, which drives diaphragm contraction for inspiration, had an irregular rhythm with frequent apneas. Optical imaging of the preparation using voltage-sensitive dye revealed that the pre-inspiratory neurons located in the rostral ventrolateral medulla and belonging to the rhythm generator for respiration, lost their synchroneity in Dscam(-/-) mice. Dscam(+/-) mice, which survived to adulthood without any overt abnormalities, also showed irregular respiration but milder than Dscam(-/-) mice. These results suggest that DSCAM plays a critical role in central respiratory regulation in a dosage-dependent manner.

The transcriptional repressor RP58 is crucial for cell-division patterning and neuronal survival in the developing cortex.

The neocortex and the hippocampus comprise several specific layers containing distinct neurons that originate from progenitors at specific development times, under the control of an adequate cell-division patterning mechanism. Although many molecules are known to regulate this cell-division patterning process, its details are not well understood. Here, we show that, in the developing cerebral cortex, the RP58 transcription repressor protein was expressed both in postmitotic glutamatergic projection neurons and in their progenitor cells, but not in GABAergic interneurons. Targeted deletion of the RP58 gene led to dysplasia of the neocortex and of the hippocampus, reduction of the number of mature cortical neurons, and defects of laminar organization, which reflect abnormal neuronal migration within the cortical plate. We demonstrate an impairment of the cell-division patterning during the late embryonic stage and an enhancement of apoptosis of the postmitotic neurons in the RP58-deficient cortex. These results suggest that RP58 controls cell division of progenitor cells and regulates the survival of postmitotic cortical neurons.

Twisting of neocortical progenitor cells underlies a spring-like mechanism for daughter-cell migration.

The mammalian neocortical wall thickens extensively during embryogenesis via proliferation of progenitor cells [1-4] and migration of daughter cells toward the pial surface [5-8]. Time-lapse imaging and functional experiments were carried out so that the possible involvement of mechanical forces in these processes could be examined. When bipolar cells connecting the ventricular and pial surfaces of the mouse cerebral wall lose their ventricular attachment, they undergo somal translocation toward the outer zones, which contain differentiated neurons. The pial process of these transitioning unipolar cells exhibits a coiled or hairpin-loop morphology, suggesting that twisting and stretching of the pial process establishes a spring-like mechanism that propels the daughter cell toward the pial surface upon ventricular detachment. This model is supported by morphological changes observed in microsurgically transected pial processes. Pharmacological experiments further reveal the involvement of intermediate filaments in twisting of pial processes. These results uncover a novel mechanism for cellular migration and provide valuable tools for the detailed study of the role of mechanical forces in 3D brain development.

Periventricular notch activation and asymmetric Ngn2 and Tbr2 expression in pair-generated neocortical daughter cells.

To understand the cellular and molecular mechanisms regulating cytogenesis within the neocortical ventricular zone, we examined at high resolution the spatiotemporal expression patterns of Ngn2 and Tbr2. Individually DiI-labeled daughter cells were tracked from their birth in slice cultures and immunostained for Ngn2 and Tbr2. Both proteins were initially absent from daughter cells during the first 2 h. Ngn2 expression was then initiated asymmetrically in one of the daughter cells, with a bias towards the apical cell, followed by a similar pattern of expression for Tbr2, which we found to be a direct target of Ngn2. How this asymmetric Ngn2 expression is achieved is unclear, but gamma-secretase inhibition at the birth of daughter cells resulted in premature Ngn2 expression, suggesting that Notch signaling in nascent daughter cells suppresses a Ngn2-Tbr2 cascade. Many of the nascent cells exhibited pin-like morphology with a short ventricular process, suggesting periventricular presentation of Notch ligands to these cells.

Loss of pre-inspiratory neuron synchroneity in mice with DSCAM deficiency.

Down syndrome cell adhesion molecule (DSCAM) is a neural adhesion molecule that plays diverse roles in neural development. We disrupted the Dscam locus in mice and found that the null mutants (Dscam (-/-)) died within 24 hours after birth. Whole body plethysmography showed irregular respiration and lower ventilatory response to hypercapnia in the null mutants. Further, a medulla-spinal cord preparation of Dscam (-/-) mice showed that the C4 ventral root activity, which drives diaphragm contraction for inspiration, had an irregular rhythm with frequent apneas. Optical imaging of the preparation using voltage-sensitive dye revealed that the pre-inspiratory (Pre-I) neurons located in the rostral ventrolateral medulla (RVLM) and belonging to the rhythm generator for respiration, lost their synchroneity in Dscam (-/-) mice. Dscam (+/-) mice, which survived to adulthood without any overt abnormalities, also showed irregular respiration but milder than Dscam (-/-) mice. These results suggest that DSCAM plays a critical role in central respiratory regulation in a dosage-dependent manner. These results have been published (Amano et al. 2009).

Zic deficiency in the cortical marginal zone and meninges results in cortical lamination defects resembling those in type II lissencephaly.

The formation of the highly organized cortical structure depends on the production and correct placement of the appropriate number and types of neurons. The Zic family of zinc-finger transcription factors plays essential roles in regulating the proliferation and differentiation of neuronal progenitors in the medial forebrain and the cerebellum. Examination of the expression of Zic genes demonstrated that Zic1, Zic2, and Zic3 were expressed by the progenitor cells in the septum and cortical hem, the sites of generation of the Cajal-Retzius (CR) cells. Immunohistochemical studies have revealed that Zic proteins were abundantly expressed in the meningeal cells and that the majority of the CR cells distributed in the medial and dorsal cortex also expressed Zic proteins in the mid-late embryonic and postnatal cortical marginal zones. During embryonic cortical development, Zic1/Zic3 double-mutant and hypomorphic Zic2 mutant mice showed a reduction in the number of CR cells in the rostral cortex, whereas the cell number remained unaffected in the caudal cortex. These mutants also showed mislocalization of the CR cells and cortical lamination defects, resembling the changes noted in type II (cobblestone) lissencephaly, throughout the brain. In the Zic1/3 mutant, reduced proliferation of the meningeal cells was observed before the thinner and disrupted organization of the pial basement membrane (BM) with reduced expression of the BM components and the meningeal cell-derived secretory factor. These defects correlated with the changes in the end feet morphology of the radial glial cells. These findings indicate that the Zic genes play critical roles in cortical development through regulating the proliferation of meningeal cells and the pial BM assembly.

FGF8 signaling patterns the telencephalic midline by regulating putative key factors of midline development.

FGF8 has been reported to act as a primary regulator of neocortical patterning along the anteroposterior (AP) axis in the mouse telencephalon, and disruption of FGF signaling causes distortion of molecular arealization along the AP axis. Since hypoplasia of midline structures is observed in Fgf8 mutant mice, FGF8 is also postulated to be involved in telencephalic midline development. In this study we analyzed the role of FGF8 in midline development by means of gain-of-function and loss-of-function experiments. The results showed that FGF8 up-regulates the expression of transcription factor (TF) genes, including putative key factors involved in midline development. Although FGF8 had been thought to act downstream of SHH signaling, ectopic FGF8 up-regulates the expression of midline TF genes in Shh null mice, suggesting that FGF signaling acts as an upstream positive regulator of midline TFs during midline development independently of SHH.

Signaling via immunoglobulin Fc receptors induces oligodendrocyte precursor cell differentiation.

Dramatic changes in morphology and myelin protein expression take place during the differentiation of oligodendrocyte precursor cells (OPCs) into myelinating oligodendrocytes. Fyn tyrosine kinase was reported to play a central role in the differentiation process. Molecules that could induce Fyn signaling have not been studied. Such molecules are promising therapeutic targets in demyelinating diseases. We provide evidence that the common gamma chain of immunoglobulin Fc receptors (FcRgamma) is expressed in OPCs and has a role in triggering Fyn signaling. FcRgamma cross-linking by immunoglobulin G on OPCs promotes the activation of Fyn signaling and induces rapid morphological differentiation with upregulation of myelin basic protein (MBP) expression levels. Mice deficient in FcRgamma are hypomyelinated, and a significant reduction in MBP content is evident. Our findings indicate that the FcRgamma-Fyn-MBP cascade is pivotal during the differentiation of OPCs into myelinating oligodendrocytes, revealing an unexpected involvement of immunological molecules.

Regulation of apoptosis and neurite extension by FKBP38 is required for neural tube formation in the mouse.

FKBP38 (also known as FKBP8) is a transmembrane chaperone protein that inhibits apoptosis by recruiting the anti-apoptotic proteins Bcl-2 and Bcl-x(L) to mitochondria. We have now generated mice harboring a loss-of-function mutation in Fkbp38. The Fkbp38(-/-) mice die soon after birth manifesting defects in neural tube closure in the thoraco-lumbar-sacral region (spina bifida) as well as skeletal defects including scoliosis, rib deformities, club foot and curled tail. The neuroepithelium is disorganized and that formation of dorsal root ganglia is defective in Fkbp38(-/-) embryos, likely as a result of an increased frequency of apoptosis and aberrant migration of neuronal cells. Furthermore, the extension of nerve fibers in the spinal cord is abnormal in the mutant embryos. To explore the mechanisms underlying these characteristics, we screened for proteins that interact with FKBP38 in the yeast two-hybrid system and thereby identified protrudin, a protein that promotes process formation by regulating membrane trafficking. Protrudin was found to be hyperphosphorylated in the brain of Fkbp38(-/-) mice, suggesting that FKBP38 regulates protrudin-dependent membrane recycling and neurite outgrowth. Together, our findings suggest that FKBP38 is required for neuroectodermal organization during neural tube formation as a result of its anti-apoptotic activity and regulation of neurite extension.

Unusual patch-matrix organization in the retrosplenial cortex of the reeler mouse and Shaking rat Kawasaki.

The rat granular retrosplenial cortex (GRS) is a simplified cortex, with distinct stratification and, in the uppermost layers, distinct modularity. Thalamic and cortical inputs are segregated by layers and in layer 1 colocalize, respectively, with apical dendritic bundles originating from neurons in layers 2 or 5. To further investigate this organization, we turned to reelin-deficient reeler mouse and Shaking rat Kawasaki. We found that the disrupted lamination, evident in Nissl stains in these rodents, is in fact a patch-matrix mosaic of segregated afferents and dendrites. Patches consist of thalamocortical connections, visualized by vesicular glutamate transporter 2 (VGluT2) or AChE. The surrounding matrix consists of corticocortical terminations, visualized by VGluT1 or zinc. Dendrites concentrate in the matrix or patches, depending on whether they are OCAM positive (matrix) or negative (patches). In wild-type rodents and, presumably, mutants, OCAM(+) structures originate from layer 5 neurons. By double labeling for dendrites (filled by Lucifer yellow in fixed slice) and OCAM immunofluorescence, we ascertained 2 populations in reeler: dendritic branches either preferred (putative layer 5 neurons) or avoided (putative supragranular neurons) the OCAM(+) matrix. We conclude that input-target relationships are largely preserved in the mutant GRS and that dendrite-dendrite interactions involving OCAM influence the formation of the mosaic configuration.

Gene application with in utero electroporation in mouse embryonic brain.

Mouse genetic manipulations, such as the production of gene knock-out, knock-in, and transgenic mice, have provided excellent systems for analysis of numerous genes functioning during development. Nevertheless, the lack of specific promoters and enhancers that control gene expression in specific regions and at specific times, limits usage of these techniques. However, progress in in utero systems of electroporation into mouse embryos has opened a new window, permitting new approaches to answering important questions. Simple injection of plasmid DNA solution and application of electrical current to mouse embryos results in transient area- and time-dependent transfection. Further modification of the technique, arising from variations in types of electrodes used, has made it possible to control the relative size of the region of transfection, which can vary from a few cells to entire tissues. Thus, this technique is a powerful means not only of characterizing gene function in various settings, but also of tracing the migratory routes of cells, due to its high efficiency and the localization of gene expression it yields. We summarize here some of the potential uses and advantages of this technique for developmental neuroscience research.

Runx3 controls the axonal projection of proprioceptive dorsal root ganglion neurons.

Dorsal root ganglion (DRG) neurons specifically project axons to central and peripheral targets according to their sensory modality. The Runt-related genes Runx1 and Runx3 are expressed in DRG neuronal subpopulations, suggesting that they may regulate the trajectories of specific axons. Here we report that Runx3-deficient (Runx3(-/-)) mice displayed severe motor uncoordination and that few DRG neurons synthesized the proprioceptive neuronal marker parvalbumin. Proprioceptive afferent axons failed to project to their targets in the spinal cord as well as those in the muscle. NT-3-responsive Runx3(-/-) DRG neurons showed less neurite outgrowth in vitro. However, we found no changes in the fate specification of Runx3(-/-) DRG neurons or in the number of DRG neurons that expressed trkC. Our data demonstrate that Runx3 is critical in regulating the axonal projections of a specific subpopulation of DRG neurons.

The c-Jun N-terminal kinase activator dual leucine zipper kinase regulates axon growth and neuronal migration in the developing cerebral cortex.

Mammalian corticogenesis substantially depends on migration and axon projection of newborn neurons that are coordinated by a yet unidentified molecular mechanism. Dual leucine zipper kinase (DLK) induces activation of c-Jun N-terminal kinase (JNK), a molecule that regulates morphogenesis in various organisms. We show here, using gene targeting in mice, that DLK is indispensable for establishing axon tracts, especially those originating from neocortical pyramidal neurons of the cerebrum. Direct and quantitative analysis of radial migration of pyramidal neurons using slice culture and a time-lapse imaging system revealed that acceleration around the subplate was affected by DLK gene disruption and by administration of a JNK inhibitor. Phosphorylation of JNK substrates, including c-Jun and doublecortin, and of JNK itself at the activation loop were partially affected in brains of DLK-deficient mouse embryos. These data suggest that DLK plays a significant role in the coordinated regulation of radial migration and axon projection by modulating JNK activity.

Transformation of pin-like ventricular zone cells into cortical neurons.

Recent imaging studies revealed that neocortical neurons undergo multiple modes of migration, but their earliest morphologies and behaviors remain unclear. We found that some neurons generated at the ventricular surface initially adopt a pin-like morphology and generally lack a leading process. During abventricular nuclear movement, their centrosomes are localized to the ventricular endfoot. Their detachment from the germinal zone is accomplished by retraction of the ventricular process, which is frequently accompanied by an adventricular somal movement, suggesting that a pulling force within the process might function to contract the cell. Subsequently, they become multipolar upon entry into the subventricular zone.

Modulation of Reelin signaling by Cyclin-dependent kinase 5.

The Reelin signaling and Cyclin-dependent kinase 5 (Cdk5) both regulate neuronal positioning in the developing brain. Using double-transgenic mice, we have previously shown that these two signaling pathways lie in parallel fashion and have a genetic interaction. Disabled-1 (Dab1), an adapter protein, mediates Reelin signaling and becomes tyrosine-phosphorylated on the binding of Reelin to its receptors. Several isoforms of Dab1 are expressed in embryonic mouse brain, and p80 [Dab1(555)] is the major protein translated. In the present study, we investigated whether Cdk5-mediated phosphorylation of Dab1 modulates Reelin signaling. Cdk5 phosphorylates p80 Dab1 at multiple sites in its carboxyl-terminal region, and tyrosine phosphorylation of p80 Dab1 by Fyn tyrosine kinase is attenuated by this Cdk5-mediated phosphorylation in vitro. Tyrosine phosphorylation of p80 Dab1 induced by exogenous Reelin is enhanced in Cdk5-deficient neurons, corroborating the inhibitory effect of Cdk5-mediated Ser/Thr phosphorylation on tyrosine phosphorylation of p80 Dab1. Another isoform, p45 Dab1 [Dab1(271)], however, is phosphorylated by Cdk5 at one serine residue within a unique carboxyl-terminal region, and its serine phosphorylation enhances tyrosine phosphorylation by Fyn and results in progressive degradation of p45 Dab1. These results indicate that Cdk5 modulates Reelin signaling through the Ser/Thr phosphorylation of Dab1 differently in an isoform-specific manner.

Cyclin-dependent kinase 5/p35 contributes synergistically with Reelin/Dab1 to the positioning of facial branchiomotor and inferior olive neurons in the developing mouse hindbrain.

Cyclin-dependent kinase 5 (Cdk5)/p35 is a serine/threonine kinase, and its activity is detected primarily in postmitotic neurons. Mice lacking Cdk5/p35 display migration defects of the cortical neurons in the cerebrum and cerebellum. In this study, we demonstrate that although most brainstem nuclei are found in their proper positions, the motor nucleus of the facial nerve is ectopically located and neurons of the inferior olive fail to position correctly, resulting in the lack of their characteristic structures in the hindbrain of Cdk5-/- mice. Despite the defective migration of these neurons, axonal exits of the facial nerve from brainstem and projections of the inferior cerebellar axons appear unchanged in Cdk5-/- mice. Defective neuronal migration in Cdk5-/- hindbrain was rescued by the neuron-specific expression of Cdk5 transgene. Because developmental defects of these structures have been reported in reeler and Dab1 mutant mice, we analyzed the double-null mutants of p35 and Dab1 and found more extensive ectopia of VII motor nuclei in these mice. These results indicate that Cdk5/p35 and Reelin signaling regulates the selective mode of neuronal migration in the developing mouse hindbrain.