Itsuki Ajioka

Differentiated Horizontal Interneurons Clonally Expand to Form Metastatic Retinoblastoma in Mice

During neurogenesis, the progression from a progenitor cell to a differentiated neuron is believed to be unidirectional and irreversible. The Rb family of proteins (Rb, p107, and p130) regulates cell-cycle exit and differentiation during retinogenesis. Rb and p130 are redundantly expressed in the neurons of the inner nuclear layer (INL) of the retina. We have found that in the adult Rb;p130-deficient retinae p107 compensation prevents ectopic proliferation of INL neurons. However, p107 is haploinsufficient in this process. Differentiated Rb(-/-);p107(+/-);p130(-/-) horizontal interneurons re-entered the cell cycle, clonally expanded, and formed metastatic retinoblastoma. Horizontal cells were not affected in Rb(+/-);p107(-/-);p130(-/-) or Rb(-/-);p107(-/-);p130(+/-), retinae suggesting that one copy of Rb or p130 was sufficient to prevent horizontal proliferation. We hereby report that differentiated neurons can proliferate and form cancer while maintaining their differentiated state including neurites and synaptic connections.

Birth-date-dependent segregation of the mouse cerebral cortical neurons in reaggregation cultures

Cerebral cortical neurons form a six‐layered structure in which their position depends on their birth date. This developmental process requires the presence of Reelin, which is secreted by Cajal–Retzius cells in the cortical marginal zone (MZ). However, it is still unclear whether the migration from the ventricular zone (VZ) to beneath the MZ is essential for the neurons to segregate into layers. Previous transplantation studies of ferret cerebral cortical neurons suggested that their ultimate laminar fate is, at least to some extent, determined in the VZ but it is unknown how ‘laminar fate’ eventually positions cells in a specific layer. To explore the segregation properties of mouse cortical cells that have not yet arrived beneath the MZ, embryonic day (E)16 VZ and intermediate zone (IMZ) cells were dissociated and allowed to reaggregate for 1–4 days in vitro. The results suggested that the migrating neurons in the IMZ at E16 preferentially located near the centre of the aggregates, more than did the proliferative cells from the VZ. The birth‐date labelling followed by the dissociation–reaggregation culture suggested that the segregation properties of the E16 IMZ was characteristic of the E14‐born cells, which were migrating in the IMZ at E16, but they were not general properties of migrating IMZ cells. This birth‐date‐dependent segregation mechanism was also observed in the Reelin signalling‐deficient yotari cells. These findings suggest that cortical neurons acquire a birth‐date‐dependent segregation mechanism before their somas reach the MZ.

Detailed Expression Pattern of Aldolase C (Aldoc) in the Cerebellum, Retina and Other Areas of the CNS Studied in Aldoc-Venus Knock-In Mice

Aldolase C (Aldoc, also known as “zebrin II”), a brain type isozyme of a glycolysis enzyme, is expressed heterogeneously in subpopulations of cerebellar Purkinje cells (PCs) that are arranged longitudinally in a complex striped pattern in the cerebellar cortex, a pattern which is closely related to the topography of input and output axonal projections. Here, we generated knock-in Aldoc-Venus mice in which Aldoc expression is visualized by expression of a fluorescent protein, Venus. Since there was no obvious phenotypes in general brain morphology and in the striped pattern of the cerebellum in mutants, we made detailed observation of Aldoc expression pattern in the nervous system by using Venus expression in Aldoc-Venus heterozygotes. High levels of Venus expression were observed in cerebellar PCs, cartwheel cells in the dorsal cochlear nucleus, sensory epithelium of the inner ear and in all major types of retinal cells, while moderate levels of Venus expression were observed in astrocytes and satellite cells in the dorsal root ganglion. The striped arrangement of PCs that express Venus to different degrees was carefully traced with serial section alignment analysis and mapped on the unfolded scheme of the entire cerebellar cortex to re-identify all individual Aldoc stripes. A longitudinally striped boundary of Aldoc expression was first identified in the mouse flocculus, and was correlated with the climbing fiber projection pattern and expression of another compartmental marker molecule, heat shock protein 25 (HSP25). As in the rat, the cerebellar nuclei were divided into the rostrodorsal negative and the caudoventral positive portions by distinct projections of Aldoc-positive and negative PC axons in the mouse. Identification of the cerebellar Aldoc stripes in this study, as indicated in sample coronal and horizontal sections as well as in sample surface photos of whole-mount preparations, can be referred to in future experiments.

Expression profiles of EphA3 at both the RNA and protein level in the developing mammalian forebrain.

The ephrin/Eph system is well known to regulate various aspects of brain development. In this study, we analyzed the expression profiles of EphA3 at both the RNA and protein level in developing mouse forebrains. Although the EphA3 gene is known to encode two isoforms of the receptors, a full‐length transmembrane form, and a short, secretory form, only the full‐length isoform was detected in the developing forebrain. We found that, in the early developmental stages, while EphA3 mRNA was expressed in the dorsal thalamus and the cortical intermediate zone (IMZ), the EphA3 protein was detected in the IMZ and the internal capsule, but not in the dorsal thalamus. In the later stages the mRNA was expressed in the most superficial region of the cortical plate, while the protein was expressed in the IMZ. This discrepancy between the mRNA and protein expression patterns might be attributed to the possibility of the protein being transported to the axons to regulate the thalamocortical and corticofugal projection. The results of double‐immunostaining for L1 and EphA3 or TAG‐1 and EphA3 suggested that EphA3 protein was produced mainly in the thalamocortical axons and only partially in the corticofugal axons. In addition, the EphA3 protein was also detected in various other structures, such as the lateral olfactory tract, anterior commissure, and corpus callosum, suggesting the possibility that EphA3 might regulate the formation of various neuronal networks in the developing brain, including the TC projection and the commissural fibers. J. Comp. Neurol. 487:255–269, 2005.

Changes in retinoblastoma cell adhesion associated with optic nerve invasion

ABSTRACT In the 1970s, several human retinoblastoma cell lines were developed from cultures of primary tumors. As the human retinoblastoma cell lines were established in culture, growth properties and changes in cell adhesion were described. Those changes correlated with the ability of the human retinoblastoma cell lines to invade the optic nerve and metastasize in orthotopic xenograft studies. However, the mechanisms that underlie these changes were not determined. We used the recently developed knockout mouse models of retinoblastoma to begin to characterize the molecular, cellular, and genetic changes associated with retinoblastoma tumor progression and optic nerve invasion. Here we report the isolation and characterization of the first mouse retinoblastoma cell lines with targeted deletions of the Rb family. Our detailed analysis of these cells as they were propagated in culture from the primary tumor shows that changes in cadherin-mediated cell adhesion are associated with retinoblastoma invasion of the optic nerve prior to metastasis. In addition, the same changes in cadherin-mediated cell adhesion correlate with the invasive properties of the human retinoblastoma cell lines isolated decades ago, providing a molecular mechanism for these earlier observations. Most importantly, our studies are in agreement with genetic studies on human retinoblastomas, suggesting that changes in this pathway are involved in tumor progression.

Identification of ventricular-side-enriched molecules regulated in a stage-dependent manner during cerebral cortical development.

Radial glial cells are the main component of the embryonic cortical ventricular zone (VZ), producing deep‐layer excitatory neurons in the early stage and upper‐layer excitatory neurons in the late stage of development. Previous studies have suggested that the laminar fate of deep‐layer neurons might be determined by early‐stage‐specific secretory or transmembrane molecules (S/TMs) in the VZ. However, the different properties required to produce the different types of neurons in early‐stage and late‐stage VZ cells are largely unknown. Herein, we investigated the stage‐dependent transcriptional profiles of the ventricular side of the mouse cortex, which was manually dissected at embryonic day (E)12, E14 and E16, and identified 3985 ‘VZ‐enriched’ genes, regulated stage‐dependently, by GeneChip analysis. These molecules were classified into nine types based on stage‐dependent regulation patterns. Prediction programs for the S/TMs revealed 659 ‘VZ‐enriched’ S/TMs. In situ hybridization and real‐time PCR analysis for several of these molecules showed results consistent with the statistical analysis of the GeneChip experiments. Moreover, we identified 17 cell cycle‐related early‐stage and ‘VZ‐enriched’ molecules. These molecules included not only those involved in cell cycle progression, but also essential molecules for DNA double‐strand break repair, such as Rad51 and Rpa1. These results suggest that the early stage‐VZ cells, which produce both deep‐ and upper‐layer neurons, and the late‐stage VZ cells, which produce only upper‐layer neurons, are intrinsically different. The gene lists presented here will be useful for the investigation of stage‐dependent changes in VZ cells and their regulatory mechanisms in the developing cortex.

β1 integrin signaling promotes neuronal migration along vascular scaffolds in the post-stroke brain

Cerebral ischemic stroke is a main cause of chronic disability. However, there is currently no effective treatment to promote recovery from stroke-induced neurological symptoms. Recent studies suggest that after stroke, immature neurons, referred to as neuroblasts, generated in a neurogenic niche, the ventricular-subventricular zone, migrate toward the injured area, where they differentiate into mature neurons. Interventions that increase the number of neuroblasts distributed at and around the lesion facilitate neuronal repair in rodent models for ischemic stroke, suggesting that promoting neuroblast migration in the post-stroke brain could improve efficient neuronal regeneration. To move toward the lesion, neuroblasts form chain-like aggregates and migrate along blood vessels, which are thought to increase their migration efficiency. However, the molecular mechanisms regulating these migration processes are largely unknown. Here we studied the role of β1-class integrins, transmembrane receptors for extracellular matrix proteins, in these migrating neuroblasts. We found that the neuroblast chain formation and blood vessel-guided migration critically depend on β1 integrin signaling. β1 integrin facilitated the adhesion of neuroblasts to laminin and the efficient translocation of their soma during migration. Moreover, artificial laminin-containing scaffolds promoted neuroblast chain formation and migration toward the injured area. These data suggest that laminin signaling via β1 integrin supports vasculature-guided neuronal migration to efficiently supply neuroblasts to injured areas. This study also highlights the importance of vascular scaffolds for cell migration in development and regeneration.

Impaired striatal dopamine release in homozygous Vps35 D620N knock-in mice

Point mutations in the vacuolar protein sorting 35 gene (VPS35) have been associated with an autosomal dominant form of late-onset Parkinson disease (PARK17), but there has been considerable debate over whether it is caused by a loss- or gain-of-function mechanism and over the intracellular target site of neurotoxicity. To investigate the pathogenesis of PARK17 in vivo, we generated Vps35 D620N knock-in (KI) mice, expressing the homologous mutant protein with endogenous patterns of expression, simultaneously with Vps35 deletion 1 (Del1) mice, which carry 1bp deletion in the exon15 of Vps35, by CRISPR/Cas9-mediated genome engineering. Neither homozygous nor heterozygous Vps35 D620N KI mice suffered from premature death or developed clear neurodegeneration up to 70 weeks of age. Vps35 Del1 allele appeared to be a null or at least severely hypomorphic allele and homozygous Vps35 Del1 showed early embryonic lethality. Heterozygous crossings between Del1 and D620N knock-in mice revealed that the D620N/Del1 compound heterozygous mice, but not heterozygous Del1 mice, suffered from survival disadvantage. In vivo microdialysis showed that DA release evoked by 120 mM potassium chloride was significantly reduced in the caudate putamen of adult homozygous Vps35 D620N KI mice. Taken together, these results suggest that Vps35 D620N allele is a partial-loss-of-function allele and that such a genetic predisposition and age-related alterations in the nigrostriatal dopamine system cooperatively influence the pathogenesis of PARK17.

Brg1 coordinates multiple processes during retinogenesis and is a tumor suppressor in retinoblastoma.

Retinal development requires precise temporal and spatial coordination of cell cycle exit, cell fate specification, cell migration and differentiation. When this process is disrupted, retinoblastoma, a developmental tumor of the retina, can form. Epigenetic modulators are central to precisely coordinating developmental events, and many epigenetic processes have been implicated in cancer. Studying epigenetic mechanisms in development is challenging because they often regulate multiple cellular processes; therefore, elucidating the primary molecular mechanisms involved can be difficult. Here we explore the role of Brg1 (Smarca4) in retinal development and retinoblastoma in mice using molecular and cellular approaches. Brg1 was found to regulate retinal size by controlling cell cycle length, cell cycle exit and cell survival during development. Brg1 was not required for cell fate specification but was required for photoreceptor differentiation and cell adhesion/polarity programs that contribute to proper retinal lamination during development. The combination of defective cell differentiation and lamination led to retinal degeneration in Brg1-deficient retinae. Despite the hypocellularity, premature cell cycle exit, increased cell death and extended cell cycle length, retinal progenitor cells persisted in Brg1-deficient retinae, making them more susceptible to retinoblastoma. ChIP-Seq analysis suggests that Brg1 might regulate gene expression through multiple mechanisms. Summary: The SWI/SNF protein Brg1 controls cell cycle length, cell cycle exit and cell survival, and is required for cell differentiation and retinal lamination, in the developing mouse retina.

A new model of tumor susceptibility following tumor suppressor gene inactivation

Since the cloning of the first tumor suppressor gene 22 years ago, we have learned a great deal about the role of tumor suppressor pathways in human cancer. One general principle is that some tumor suppressor pathways (e.g., p53 and Rb pathways) are inactivated in virtually every human cancer. Thus, one might predict that inheritance of a genetic lesion in such a pathway would cause the rapid onset of tumors originating from different tissues. However, this is not true for the Rb pathway. Children with a defective copy of the RB1 gene show increased susceptibility to retinoblastoma but not to other developmental tumors of the nervous system. Moreover, after RB1 inactivation, certain retinal cell types are more susceptible to tumorigenesis than others. Our recent studies on the role of the Rb family of genes in retinal development and retinoblastoma have led to a new hypothesis that explains this paradox. We propose that cells that require the Rb family for their cell fate specification and/or differentiation are less susceptible to tumorigenesis than those that do not require the Rb family for these processes. If correct, this hypothesis would allow us to predict which cell types in the developing nervous system are susceptible to tumorigenesis after inactivation of the Rb family and may establish a general principle of tissue- and cell type–specific susceptibility to tumorigenesis. In this perspective, we discuss our recent findings that have changed our views on tumor initiation and progression following Rb family inactivation.