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In JoVE (1)
Other Publications (18)
- The Journal of Comparative Neurology
- Development (Cambridge, England)
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- Proceedings of the National Academy of Sciences of the United States of America
- Development, Growth & Differentiation
- Development (Cambridge, England)
- Development (Cambridge, England)
- PloS One
- Seminars in Cell & Developmental Biology
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- Nature Neuroscience
- Magnetic Resonance in Medicine : Official Journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine
- Genes to Cells : Devoted to Molecular & Cellular Mechanisms
- Development (Cambridge, England)
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- The Journal of Comparative Neurology
- The Journal of Comparative Neurology
- Nature Neuroscience
Articles by Tomomi Shimogori in JoVE
JoVE Neuroscience
Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection
Lab for Molecular Mechanisms of Thalamus Development, RIKEN Brain Science Institute
A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.
Other articles by Tomomi Shimogori on PubMed
Members of the Wnt, Fz, and Frp Gene Families Expressed in Postnatal Mouse Cerebral Cortex
The functions of Wingless-Int (Wnt) signaling, studied intensely in embryonic brain development, have been comparatively little investigated in the postnatal brain. We report remarkably patterned gene expression of Wnt signaling components in postnatal mouse cerebral cortex, lasting into young adulthood. Wnt genes are expressed in gene-specific regional and lamina patterns in each of the major subdivisions of the cerebral cortex: the olfactory bulb (OB), the hippocampal formation, and the neocortex. Genes encoding Frizzled (Fz) Wnt receptors, or secreted Frizzled-related proteins (sFrps), are also expressed in regional and lamina patterns. These findings suggest that Wnt signaling is active and regulated in the postnatal cortex and that different cortical cell populations have varying requirements for a Wnt signal. The OB, in particular, shows gene expression of a large variety of Wnt signaling components, making it a prime target for future functional studies. The penultimate components of the canonical Wnt pathway are the Tcf/Lef1 transcription factors, which regulate transcription of Wnt signaling target genes. Surprisingly, we found little Tcf/Lef1 expression in the postnatal neocortex. These observations suggest that noncanonical Wnt pathways predominate, which will require functional testing. However, Lef1 is widely expressed in the dorsal thalamus, and Wnt ligands and receptors are expressed, respectively, in cortical areas and thalamic nuclei that are interconnected. Thus, canonical Wnt signaling could be utilized in a major cortical input by Fz- and Lef1-expressing thalamic cells that innervate the Wnt-expressing cortex.
Embryonic Signaling Centers Expressing BMP, WNT and FGF Proteins Interact to Pattern the Cerebral Cortex
Recent findings implicate embryonic signaling centers in patterning the mammalian cerebral cortex. We used mouse in utero electroporation and mutant analysis to test whether cortical signaling sources interact to regulate one another. We identified interactions between the cortical hem, rich in Wingless-Int (WNT) proteins and bone morphogenetic proteins (BMPs), and an anterior telencephalic source of fibroblast growth factors (FGFs). Expanding the FGF8 domain suppressed Wnt2b, Wnt3a and Wnt5a expression in the hem. Next to the hem, the hippocampus was shrunken, consistent with its dependence for growth on a hem-derived WNT signal. Maintenance of hem WNT signaling and hippocampal development thus require a constraint on the FGF8 source, which is likely to be supplied by BMP activity. When endogenous BMP signaling is inhibited by noggin, robust Fgf8 expression appears ectopically in the cortical primordium. Abnormal signaling centers were further investigated in mice lacking the transcription factor EMX2, in which FGF8 activity is increased, WNT expression reduced, and the hippocampus defective. Suggesting that these defects are causally related, sequestering FGF8 in Emx2 homozygous mutants substantially recovered WNT expression in the hem and partially rescued hippocampal development. Because noggin can induce Fgf8 expression, we examined noggin and BMP signaling in the Emx2 mutant. As the telencephalic vesicle closed, Nog expression was expanded and BMP activity reduced, potentially leading to FGF8 upregulation. Our findings point to a cross-regulation of BMP, FGF, and WNT signaling in the early telencephalon, integrated by EMX2, and required for normal cortical development.
Fibroblast Growth Factor 8 Regulates Neocortical Guidance of Area-specific Thalamic Innervation
Thalamic innervation of each neocortical area is vital to cortical function, but the developmental strategies that guide axons to specific areas remain unclear. We took a new approach to determine the contribution of intracortical cues. The cortical patterning molecule fibroblast growth factor 8 (FGF8) was misexpressed in the cortical primordium to rearrange the area map. Thalamic axons faithfully tracked changes in area position and innervated duplicated somatosensory barrel fields induced by an ectopic source of FGF8, indicating that thalamic axons indeed use intracortical positional information. Because cortical layers are generated in temporal order, FGF8 misexpression at different ages could be used to shift regional identity in the subplate and cortical plate either in or out of register. Thalamic axons showed strikingly different responses in the two different conditions, disclosing sources of positional guidance in both subplate and cortical plate. Unexpectedly, axon trajectories indicated that an individual neocortical layer could provide not only laminar but also area-specific guidance. Our findings demonstrate that thalamocortical axons are directed by sequential, positional cues within the cortex and implicate FGF8 as an indirect regulator of thalamocortical innervation.
Possible Involvement of SINEs in Mammalian-specific Brain Formation
Retroposons, such as short interspersed elements (SINEs) and long interspersed elements (LINEs), are the major constituents of higher vertebrate genomes. Although there are many examples of retroposons' acquiring function, none has been implicated in the morphological innovations specific to a certain taxonomic group. We previously characterized a SINE family, AmnSINE1, members of which constitute a part of conserved noncoding elements (CNEs) in mammalian genomes. We proposed that this family acquired genomic functionality or was exapted after retropositioning in a mammalian ancestor. Here we identified 53 new AmnSINE1 loci and refined 124 total loci, two of which were further analyzed. Using a mouse enhancer assay, we demonstrate that one SINE locus, AS071, 178 kbp from the gene FGF8 (fibroblast growth factor 8), is an enhancer that recapitulates FGF8 expression in two regions of the developing forebrain, namely the diencephalon and the hypothalamus. Our gain-of-function analysis revealed that FGF8 expression in the diencephalon controls patterning of thalamic nuclei, which act as a relay center of the neocortex, suggesting a role for FGF8 in mammalian-specific forebrain patterning. Furthermore, we demonstrated that the locus, AS021, 392 kbp from the gene SATB2, controls gene expression in the lateral telencephalon, which is thought to be a signaling center during development. These results suggest important roles for SINEs in the development of the mammalian neuronal network, a part of which was initiated with the exaptation of AmnSINE1 in a common mammalian ancestor.
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.
Hes Genes and Neurogenin Regulate Non-neural Versus Neural Fate Specification in the Dorsal Telencephalic Midline
The choroid plexus in the brain is unique because it is a non-neural secretory tissue. It secretes the cerebrospinal fluid and functions as a blood-brain barrier, but the precise mechanism of specification of this non-neural tissue has not yet been determined. Using mouse embryos and lineage-tracing analysis, we found that the prospective choroid plexus region initially gives rise to Cajal-Retzius cells, specialized neurons that guide neuronal migration. Inactivation of the bHLH repressor genes Hes1, Hes3 and Hes5 upregulated expression of the proneural gene neurogenin 2 (Ngn2) and prematurely depleted Bmp-expressing progenitor cells, leading to enhanced formation of Cajal-Retzius cells and complete loss of choroid plexus epithelial cells. Overexpression of Ngn2 had similar effects. These data indicate that Hes genes promote specification of the fate of choroid plexus epithelial cells rather than the fate of Cajal-Retzius cells by antagonizing Ngn2 in the dorsal telencephalic midline region, and thus this study has identified a novel role for bHLH genes in the process of deciding which cells will have a non-neural versus a neural fate.
Fgf8 Controls Regional Identity in the Developing Thalamus
The vertebrate thalamus contains multiple sensory nuclei and serves as a relay station to receive sensory information and project to corresponding cortical areas. During development, the progenitor region of the diencephalon is divided into three parts, p1, p2 (presumptive thalamus) and p3, along its longitudinal axis. Besides the local expression of signaling molecules such as sonic hedgehog (Shh), Wnt proteins and Fgf8, the patterning mechanisms of the thalamic nuclei are largely unknown. Using mouse in utero electroporation to overexpress or inhibit endogenous Fgf8 at the diencephalic p2/p3 border, we revealed that it affected gene expression only in the p2 region without altering overall diencephalic size or the expression of other signaling molecules. We demonstrated that two distinctive populations in p2, which can be distinguished by Ngn2 and Mash1 in early embryonic diencephalon, are controlled by Fgf8 activity in complementary manner. Furthermore, we found that FGF activity shifts thalamic sensory nuclei on the A/P axis in postnatal brain. Moreover, gene expression analysis demonstrated that FGF signaling shifts prethalamic nuclei in complementary manner to the thalamic shift. These findings suggest conserved roles of FGF signaling in patterning along the A/P axis in CNS, and reveal mechanisms of nucleogenesis in the developing thalamus.
Prdm Proto-oncogene Transcription Factor Family Expression and Interaction with the Notch-Hes Pathway in Mouse Neurogenesis
Establishment and maintenance of a functional central nervous system (CNS) requires a highly orchestrated process of neural progenitor cell proliferation, cell cycle exit, and differentiation. An evolutionary conserved program consisting of Notch signalling mediated by basic Helix-Loop-Helix (bHLH) transcription factor activity is necessary for both the maintenance of neural progenitor cell character and the progression of neurogenesis; however, additional players in mammalian CNS neural specification remain largely unknown. In Drosophila we recently characterized Hamlet, a transcription factor that mediates Notch signalling and neural cell fate.
The Role of Fgf8 in Telencephalic and Diencephalic Patterning
Correct patterning of the developing brain is crucial importance for accurate wiring and function. Although the adult brain contains many complex structures, it begins with a simple structure-the neural tube. As it develops, the neural tube is divided into several regions, including the telencephalon, diencephalon, midbrain, and hindbrain. In each of these regions, signaling molecules are secreted from discrete zones, which establish positional information and regulate regional growth. There are many mechanistic questions that remain to be resolved about the action of these growth and differentiation factors. The cellular factors mediating patterning in response to these factors are largely unknown. Furthermore, identical differentiation factors are expressed in different regions of the brain and yet control significantly different patterning mechanisms, and the factors that control region-specific responses to these factors are mostly obscure. Furthermore, differentiation factors also show dramatically different expression patterns in different vertebrate species that may underlie changes in brain structure, but the mechanisms by which these changes in gene expression occur poorly understood. To address these issues, we discuss the role of Fgf8, which controls anterior/posterior patterning in different regions of the developing brain. We also discuss how modifications of Fgf8 expression in the diencephalon controlled by retrotransposons can change the shape and function of the brain in various species.
Segregation of Ipsilateral Retinal Ganglion Cell Axons at the Optic Chiasm Requires the Shh Receptor Boc
The pattern of contralaterally and ipsilaterally projecting retinal ganglion cell (RGC) axons at the optic chiasm is essential for the establishment of binocular vision. Contralateral axons cross the chiasm midline as they progress from the optic nerve to the optic tract. In contrast, ipsilateral axons deviate from the chiasm and continue in the ipsilateral optic tract, avoiding the chiasm midline. The molecular mechanism underlying this phenomenon is not completely understood. Here we show that the Sonic Hedgehog (Shh) receptor Boc is enriched in ipsilateral RGCs of the developing retina. Together with the presence of Shh at the midline, this complementary expression pattern led us to hypothesize that Shh might repel ipsilateral RGC axons at the chiasm. Consistent with this hypothesis, we found that only Boc-positive RGC axons retract in vitro in response to Shh and that this response is lost in Boc mutant RGCs. In vivo, we show that Boc is required for the normal segregation of ipsilateral axons at the optic chiasm and, conversely, that Boc expression in contralateral RGCs prevents their axons from crossing the optic chiasm. Together, these results suggest that Shh repels ipsilateral RGC axons at the optic chiasm via its receptor Boc. This work identifies a novel molecular pathway required for the segregation of axons at the optic chiasm.
A Genomic Atlas of Mouse Hypothalamic Development
The hypothalamus is a central regulator of many behaviors that are essential for survival, such as temperature regulation, food intake and circadian rhythms. However, the molecular pathways that mediate hypothalamic development are largely unknown. To identify genes expressed in developing mouse hypothalamus, we performed microarray analysis at 12 different developmental time points. We then conducted developmental in situ hybridization for 1,045 genes that were dynamically expressed over the course of hypothalamic neurogenesis. We identified markers that stably labeled each major hypothalamic nucleus over the entire course of neurogenesis and constructed a detailed molecular atlas of the developing hypothalamus. As a proof of concept of the utility of these data, we used these markers to analyze the phenotype of mice in which Sonic Hedgehog (Shh) was selectively deleted from hypothalamic neuroepithelium and found that Shh is essential for anterior hypothalamic patterning. Our results serve as a resource for functional investigations of hypothalamic development, connectivity, physiology and dysfunction.
Three-dimensional Diffusion Tensor Microimaging for Anatomical Characterization of the Mouse Brain
Diffusion tensor imaging is gaining increasing importance for anatomical imaging of the developing mouse brain. However, the application of diffusion tensor imaging to mouse brain imaging at microscopic levels is hindered by the limitation on achievable spatial resolution. In this study, fast diffusion tensor microimaging of the mouse brain, based on a diffusion-weighted gradient and spin echo technique with twin-navigator echo phase correction, is presented. Compared to echo planar and spin echo acquisition, the diffusion-weighted gradient and spin echo acquisition resulted in significant reduction in scan time and had minimal image distortion, thereby allowing acquisition at higher spatial resolution. In this study, three-dimensional diffusion tensor microimaging of the mouse brains at spatial resolutions of 50-60 microm revealed unprecedented anatomical details. Thin fiber bundles in the adult striatum and white matter tracts in the embryonic day 12 mouse brains were visualized for the first time. The study demonstrated that data acquired using the diffusion tensor microimaging technique allow three-dimensional mapping of gene expression data and can serve as a platform to study gene expression patterns in the context of neuroanatomy in the developing mouse brain.
Emergence of Mammals by Emergency: Exaptation
With the development of molecular embryology and the coming of the post-genomic era, the molecular mechanisms of morphological evolution have recently begun to be elucidated. Whole genome sequences of many vertebrate species have been determined, and comparative genomics has suggested that one source of biodiversity is conserved non-coding elements (CNEs), which may be involved in generating new networks of gene expression. Nishihara et al. (Genome Res. 2006; 16, 864) discovered retroposon (AmnSINE1s)-derived CNEs in the human genome, and suggested that the AmnSINE1s obtained their function (i.e., exapted) in a common mammalian ancestor and are involved in generating mammalian-specific morphology. Therefore, investigation of the function of AmnSINE1-derived CNEs in morphogenesis helps us understand the molecular events of how mammals obtained their specific morphological characters by exaptation that occurred when the first mammalian ancestor emerged about 250 Ma (million years ago). Because there are more than 100 AmnSINE1-derived CNE loci in the mammalian genome, a burst of exaptation of AmnSINE1s must have occurred, possibly triggered by the Permian-Triassic mass extinction 250 Ma. In this review, we discuss morphological evolution of the mammalian-specific characters including brain that were exapted after retrotransposition of AmnSINE1s by referring to two CNE loci described by Sasaki et al.
FGF8 Acts As a Classic Diffusible Morphogen to Pattern the Neocortex
Gain- and loss-of-function experiments have demonstrated that a source of fibroblast growth factor (FGF) 8 regulates anterior to posterior (A/P) patterning in the neocortical area map. Whether FGF8 controls patterning as a classic diffusible morphogen has not been directly tested. We report evidence that FGF8 diffuses through the mouse neocortical primordium from a discrete source in the anterior telencephalon, forms a protein gradient across the entire A/P extent of the primordium, and acts directly at a distance from its source to determine area identity. FGF8 immunofluorescence revealed FGF8 protein distributed in an A/P gradient. Fate-mapping experiments showed that outside the most anterior telencephalon, neocortical progenitor cells did not express Fgf8, nor were they derived from Fgf8-expressing cells, suggesting that graded distribution of FGF8 results from protein diffusion from the anterior source. Supporting this conclusion, a dominant-negative high-affinity FGF8 receptor captured endogenous FGF8 at a distance from the FGF8 source. New FGF8 sources introduced by electroporation showed haloes of FGF8 immunofluorescence indicative of FGF8 diffusion, and surrounding cells reacted to a new source of FGF8 by upregulating different FGF8-responsive genes in concentric domains around the source. Reducing endogenous FGF8 with the dominant-negative receptor in the central neocortical primordium induced cells to adopt a more posterior area identity, demonstrating long-range area patterning by FGF8. These observations support FGF8 as a classic diffusible morphogen in neocortex, thereby guiding future studies of neocortical pattern formation.
Molecular Pathways Controlling Development of Thalamus and Hypothalamus: from Neural Specification to Circuit Formation
The embryonic diencephalon gives rise to the vertebrate thalamus and hypothalamus, which play essential roles in sensory information processing and control of physiological homeostasis and behavior, respectively. In this review, we present new steps toward characterizing the molecular pathways that control development of these structures, based on findings in a variety of model organisms. We highlight advances in understanding how early regional patterning is orchestrated through the action of secreted signaling molecules such as Sonic hedgehog and fibroblast growth factors. We address the role of individual transcription factors in control of the regional identity and neural differentiation within the developing diencephalon, emphasizing the contribution of recent large-scale gene expression studies in providing an extensive catalog of candidate regulators of hypothalamic neural cell fate specification. Finally, we evaluate the molecular mechanisms involved in the experience-dependent development of both thalamo-cortical and hypothalamic neural circuitry.
Dynamic Spatiotemporal Gene Expression in Embryonic Mouse Thalamus
The anatomy of the mammalian thalamus is characterized by nuclei, which can be readily identified in postnatal animals. However, the molecular mechanisms that guide specification and differentiation of neurons in specific thalamic nuclei are still largely unknown, and few molecular markers are available for most of these thalamic subregions at early stages of development. We therefore searched for patterned gene expression restricted to specific mouse thalamic regions by in situ hybridization during the onset of thalamic neurogenesis (embryonic [E] days E10.5-E12.5). To obtain correct regional information, we used Shh as a landmark and compared spatial relationships with the zona limitans intrathalamica (Zli), the border of the p2 and p3 compartments of the diencephalon. We identified genes that are expressed specifically in the ventricular zone of the thalamic neuroepithelium and also identified a number of genes that already exhibited regional identity at E12.5. Although many genes expressed in the mantle regions of the thalamus at E12.5 showed regionally restricted patterns, none of these clearly corresponded to individual thalamic nuclei. We next examined gene expression at E15.5, when thalamocortical axons (TCAs) project from distinct regions of the thalamus and reach their targets in the cerebral cortex. Regionally restricted patterns of gene expression were again seen for many genes, but some regionally bounded expression patterns in the early postnatal thalamus had shifted substantially by E15.5. These findings reveal that nucleogenesis in the developing thalamus is associated with selective and complex changes in gene expression and provide a list of genes that may actively regulate the development of thalamic nuclei.
Region-specific Gene Expression in Early Postnatal Mouse Thalamus
Previous studies in the developing mouse thalamus have demonstrated that regional identity is established during early stages of development (Suzuki-Hirano et al. J. Comp. Neurol. 2011;519:528-543). However, the developing thalamus often shows little resemblance to the anatomical organization of the postnatal thalamus, making it difficult to identify genes that might mediate the organization of thalamic nuclei. We therefore analyzed the expression pattern of genes that we have identified as showing regional expression in embryonic thalamus on postnatal days (P) 6-8 by using in situ hybridization. We also identified several genes expressed only in the postnatal thalamus with restricted expression in specific nuclei. We first demonstrated the selective expression of neurotransmitter-related genes (vGlut2, vGAT, D2R, and HTR2C), identifying the neurotransmitter subtypes of cells in this region, and we also demonstrated selective expression of additional genes in the thalamus (Steel, Slitrk6, and AI852580). In addition, we demonstrated expression of genes specific to somatosensory thalamic nuclei, the ventrobasal posterior nuclei (VP); a visual thalamic nucleus, the dorsal lateral geniculate nucleus (dLGN); and an auditory thalamic nucleus, the medial geniculate body (MGB) (p57Kip, Nr1d1, and GFRα1). We also identified genes that are selectively expressed in multiple different nuclei (Foxp2, Chst2, and EphA8). Finally, we demonstrated that several bone morphogenetic proteins (BMPs) and their inhibitors are expressed in the postnatal thalamus in a nucleus-specific fashion, suggesting that BMPs play roles in the postnatal thalamus unrelated to their known role in developmental patterning. Our findings provide important information for understanding the mechanisms of nuclear specification and connectivity during development, as well as their maintenance in adult thalamus.
Scale: a Chemical Approach for Fluorescence Imaging and Reconstruction of Transparent Mouse Brain
Optical methods for viewing neuronal populations and projections in the intact mammalian brain are needed, but light scattering prevents imaging deep into brain structures. We imaged fixed brain tissue using Scale, an aqueous reagent that renders biological samples optically transparent but completely preserves fluorescent signals in the clarified structures. In Scale-treated mouse brain, neurons labeled with genetically encoded fluorescent proteins were visualized at an unprecedented depth in millimeter-scale networks and at subcellular resolution. The improved depth and scale of imaging permitted comprehensive three-dimensional reconstructions of cortical, callosal and hippocampal projections whose extent was limited only by the working distance of the objective lenses. In the intact neurogenic niche of the dentate gyrus, Scale allowed the quantitation of distances of neural stem cells to blood vessels. Our findings suggest that the Scale method will be useful for light microscopy-based connectomics of cellular networks in brain and other tissues.
