Tetsuichiro Saito

Functionally Related Motor Neuron Pool and Muscle Sensory Afferent Subtypes Defined by Coordinate ETS Gene Expression

Motor function depends on the formation of selective connections between sensory and motor neurons and their muscle targets. The molecular basis of the specificity inherent in this sensory-motor circuit remains unclear. We show that motor neuron pools and subsets of muscle sensory afferents can be defined by the expression of ETS genes, notably PEA3 and ER81. There is a matching in PEA3 and ER81 expression by functionally interconnected sensory and motor neurons. ETS gene expression by motor and sensory neurons fails to occur after limb ablation, suggesting that their expression is coordinated by signals from the periphery. ETS genes may therefore participate in the development of selective sensory-motor circuits in the spinal cord.

Function of the 30 kd protein of tobacco mosaic virus: involvement in cell-to-cell movement and dispensability for replication

We have investigated the function of the 30 kd protein of tobacco mosaic virus (TMV) by a reverse genetics approach. First, a point mutation of TMV Ls1 (a temperature‐sensitive mutant defective in cell‐to‐cell movement), that causes an amino acid substitution in the 30 kd protein, was introduced into the parent strain, TMV L. The generated mutant showed the same phenotype as TMV Ls1, and therefore the one‐base substitution in the 30 kd protein gene adequately explains the defectiveness of TMV Ls1. Next, four kinds of frame‐shift mutants were constructed, whose mutations are located at three different positions of the 30 kd protein gene. All the frame‐shift mutants were replication‐competent in protoplasts but none showed infectivity on tobacco plants. From these observations the 30 kd protein was confirmed to be involved in cell‐to‐cell movement. To clarify that the 30 kd protein is not necessary for replication, two kinds of deletion mutants were constructed; one lacking most of the 30 kd protein gene and the other lacking both the 30 kd and coat protein genes. Both mutants replicated in protoplasts and the former still produced the subgenomic mRNA for the coat protein. These results clearly showed that the 30 kd protein, as well as the coat protein, is dispensable for replication and that no cis‐acting element for replication is located in their coding sequences. It is also suggested that the signal for coat protein mRNA synthesis may be located within about 100 nucleotides upstream of the initiation codon of the coat protein gene.

In vivo electroporation in the embryonic mouse central nervous system

This protocol describes a basic method for in vivo electroporation in the nervous system of embryonic mice. Delivery of electric pulses following microinjection of DNA into the brain ventricle or the spinal cord central canal enables efficient transfection of genes into the nervous system. Transfection is facilitated by forceps-type electrodes, which hold the uterus and/or the yolk sac containing the embryo. More than ten embryos in a single pregnant mouse can be operated on within 30 min. More than 90% of operated embryos survive and more than 90% of these survivors express the transfected genes appropriately. Gene expression in neurons persists for a long time, even at postnatal stages, after electroporation. Thus, this method could be used to analyze roles of genes not only in embryonic development but also in higher order function of the nervous system, such as learning.

Long-distance movement and viral assembly of tobacco mosaic virus mutants

Spreading of tobacco mosaic virus in infected plants is of two modes: cell-to-cell movement (to adjacent cells) and long-distance movement (to distant parts of the plant). Viral coat protein has been suggested to be involved in long-distance movement. To analyze the function of coat protein in the movement, we used mutants with modifications in the coat protein gene or in the assembly origin on the genomic RNA. A mutant which has the coding region for the C-terminal 5 amino acids of the protein deleted and mutants with 1 amino acid inserted after residue 101 or 152 of the protein retained both the abilities of long-distance movement and assembly into virus particles. Other mutants in the coat protein gene eliminated the two abilities. A mutant with modifications in the assembly origin displayed greatly reduced abilities of both the movement and assembly. These results suggest that both the coat protein with its ability to assemble into virus particles and the assembly origin are involved in long-distance movement, and that virus particles may play a pivotal role in the movement.

Progenitors resume generating neurons after temporary inhibition of neurogenesis by Notch activation in the mammalian cerebral cortex.

The mammalian cerebral cortex comprises six layers of neurons. Cortical progenitors in the ventricular zone generate neurons specific to each layer through successive cell divisions. Neurons of layer VI are generated at an early stage, whereas later-born neurons occupy progressively upper layers. The underlying molecular mechanisms of neurogenesis, however, are relatively unknown. In this study, we devised a system where the Notch pathway was activated spatiotemporally in the cortex by in vivo electroporation and Cre-mediated DNA recombination. Electroporation at E13.5 transferred DNA to early progenitors that gave rise to neurons of both low and upper layers. Forced expression of a constitutively active form of Notch (caNotch) at E13.5 inhibited progenitors from generating neurons and kept progenitors as proliferating radial glial cells. After subsequent transfection at E15.5 of a Cre expression vector to remove caNotch, double-transfected cells, in which caNotch was excised, migrated into the cortical plate and differentiated into neurons specific to upper layers. Bromodeoxyuridine-labeling experiments showed that the neurons were born after Cre transfection. These results indicate that cortical progenitors that had been temporarily subjected to Notch activation at an early stage generated neurons at later stages, but that the generation of low-layer neurons was skipped. Moreover, the double-transfected cells gave rise to upper-layer neurons, even after their transplantation into the E13.5 brain, indicating that the developmental state of progenitors is not halted by caNotch activity.

Molecular structure of cytoplasmic dynein 2 and its distribution in neuronal and ciliated cells

Cytoplasmic dynein is involved in a wide variety of cellular functions. In addition to the initially characterized form (MAP 1C/dynein 1), a second form of cytoplasmic dynein (dynein 2) has been identified and implicated in intraflagellar transport (IFT) in lower eukaryotes and in Golgi organization in vertebrates. In the current study, the primary structure of the full-length dynein 2 heavy chain (HC) was determined from cDNA sequence. The dynein 1 and dynein 2 sequences were similar within the motor region, and around the light intermediate chain (LIC)-binding site within the N-terminal stem region. The dynein 2 HC co-immunoprecipitated with LIC3, a homologue of dynein 1 LICs. Dynein 2 mRNA was abundant in the ependymal layer of the neural tube and in the olfactory epithelium. Antibodies to dynein 2 HC, LIC3 and a component of IFT particles strongly stained the ependymal layer lining the lateral ventricles. Both dynein 2 HC and LIC3 staining was also observed associated with connecting cilia in the retina and within primary cilia of non-neuronal cultured cells. These data support a specific role for dynein 2 in the generation and maintenance of cilia.

Identification by Differential RT-PCR of a Novel Paired Homeodomain Protein Specifically Expressed in Sensory Neurons and a Subset of Their CNS Targets

Sensory neurons are a major derivative of the neural crest for which there have been no definitive molecular markers in mammals. We have developed a method that combines differential hybridization with degenerate RT-PCR to rapidly screen gene families for members exhibiting differential expression among tissues or cell types. We used this approach to search for transcription factor-encoding genes specifically expressed in mammalian sensory neurons. A novel paired homeodomain protein, called DRG11, was identified. DRG11 is expressed in most sensory neurons, including trkA-expressing neurons, but not in glia or sympathetic neurons. Unexpectedly, it is also expressed in the dorsal horn of the spinal cord, a region to which NGF-dependent sensory neurons project. These data suggest that DRG11 is not only a useful marker for sensory neurons, but may also function in the establishment or maintenance of connectivity between some of these neurons and their central nervous system targets.

Consequence of the loss of Sox2 in the developing brain of the mouse

The transcription factor Sox2 is expressed at high levels in neural stem and progenitor cells. Here, we inactivated Sox2 specifically in the developing brain by using Cre–loxP system. Although mutant animals did not survive after birth, analysis of late gestation embryos revealed that loss of Sox2 causes enlargement of the lateral ventricles and a decrease in the number of neurosphere‐forming cells. However, although their neurogenic potential is attenuated, Sox2‐deficient neural stem cells retain their multipotency and self‐renewal capacity. We found that expression level of Sox3 is elevated in Sox2 null developing brain, probably mitigating the effects of loss of Sox2.

The Sox-2 Regulatory Regions Display Their Activities in Two Distinct Types of Multipotent Stem Cells

ABSTRACT The Sox-2 gene is expressed in embryonic stem (ES) cells and neural stem cells. Two transcription enhancer regions, Sox-2 regulatory region 1 (SRR1) and SRR2, were described previously based on their activities in ES cells. Here, we demonstrate that these regulatory regions also exert their activities in neural stem cells. Moreover, our data reveal that, as in ES cells, both SRR1 and SRR2 show their activities rather specifically in multipotent neural stem or progenitor cells but cease to function in differentiated cells, such as postmitotic neurons. Systematic deletion and mutation analyses showed that the same or at least overlapping DNA elements of SRR2 are involved in its activity in both ES and neural stem or progenitor cells. Thus, SRR2 is the first example of an enhancer in which a single regulatory core sequence is involved in multipotent-state-specific expression in two different stem cells, i.e., ES and neural stem cells.

The Sox2 Regulatory Region 2 Functions as a Neural Stem Cell-specific Enhancer in the Telencephalon

Sox2 is expressed at high levels in neuroepithelial stem cells and persists in neural stem/progenitor cells throughout adulthood. We showed previously that the Sox2 regulatory region 2 (SRR2) drives strong expression in these cells. Here we generated transgenic mouse strains with the β-geo reporter gene under the control of the SRR2 in order to examine the spatiotemporal function of this regulatory region. We show that the SRR2 functions specifically in neural stem/progenitor cells. However, unlike Nestin 2nd intronic enhancer, the SRR2 shows strong regional specificity functioning only in restricted areas of the telencephalon but not in any other portions of the central nervous system such as the spinal cord. We also show by in vitro clonogenic assay that at least some of these SRR2-functioning cells possess the hallmark properties of neural stem cells. In adult brains, we could detect strong β-geo expression in the subventricular zone of the lateral ventricle and along the rostral migrating stream where actively dividing cells reside. Chromatin immunoprecipitation assays reveal interactions of POU and Sox factors with SRR2 in neural stem/progenitor cells. Our data also suggest that the specific recruitment of these proteins to the SRR2 in the telencephalon defines the spatiotemporal activity of the enhancer in the developing nervous system.