Mouse genetic manipulation has provided an excellent system to characterize gene function in numerous contexts. A number of mutants have been produced by using transgenic, gene knockout, and mutagenesis techniques. Nevertheless, one limitation is that it is difficult to express a gene in vivo in a restricted manner (i.e., spatially and temporally), because the number of available enhancers and promoters which can confine gene expression is limited. We have developed a novel method to introduce DNA into in/exo utero embryonic mouse brains at various stages by using electroporation. More than 90% of operated embryos survived, and more than 65% of these expressed the introduced genes in restricted regions of the brain. Expression was maintained even after birth, 6 weeks after electroporation. The use of fluorescent protein genes clearly visualized neuronal morphologies in the brain. Moreover, it was possible to transfect three different DNA vectors into the same cells. Thus, this method will be a powerful tool to characterize gene function in various settings due to its high efficiency and localized gene expression.

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.

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.

Using monoclonal antibodies, we have examined the expression pattern of MASH1, a basic helix-loop-helix protein that is a mammalian homolog of the Drosophila achaete-scute proteins. In Drosophila, achaete-scute genes are required for the determination of a subset of neurons. In the rat embryo, MASH1 expression is confined to subpopulations of neural precursor cells. The induction of MASH1 precedes, but is extinguished upon, overt neuronal differentiation. MASH1 is expressed in the forebrain by spatially restricted domains of neuroepithelium and in the peripheral nervous system exclusively by precursors of sympathetic and enteric neurons. The features of early and transient expression, in spatially restricted subpopulations of neural precursors, are similar to those observed for achaete-scute. Thus, the amino acid sequence conservation between MASHI and achaete-scute is reflected in a parallel conservation of cell type specificity of expression, similar to the case of mammalian MyoD and Drosophila nautilus. These data support the idea that helix-loop-helix proteins may represent an evolutionarily conserved family of cell-type determination genes, of which MASH1 is the first neural-specific member identified in vertebrates.

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.

The MASH genes are vertebrate homologues of achaete-scute, genes required for neuronal determination in Drosophila. The sequence of MASH1 and MASH2 contains a basic helix-oop-helix (bHLH) motif that is present in other transcriptional regulators such as MyoD and E12. In the absence of an authentic target for the MASH proteins, we examined their DNA binding and transcriptional regulatory activity by using a binding site (the E box) from the muscle creatine kinase (MCK) gene, a target of MyoD. Like myogenic bHLH proteins, the MASH proteins form heterooligomers with E12 that bind the MCK E box with high affinity in vitro.Unexpectedly, however, MASH1 and MASH2 also activate transcription of both exogenous and endogenous MCK in transfected C3H/1OTI/2 fibroblasts. However, they do not induce myogenesis. Myogenic activity is not exclusively a property of the MyoD basic region, as substitution of this domain fails to confer myogenic activity on MASH1. These data suggest that different bHLH proteins may activate overlapping but distinct sets of target genes in the same cell type.The MASH genes are mammalian homologues of the neuronal determination genes of the Drosophila achaete-scute complex (1). Like its Drosophila counterparts, MASH) is specifically expressed in the developing nervous system (2). The MASH gene products are members of the basic helixloop-helix (bHLH) family of transcription factors, whose members include the myc gene products (3) and MyoD (for review see ref. 4) and which appear to be involved in the control ofproliferation and cell type determination. Members ofthe bHLH family share a common basic region required for DNA binding and a helix-loop-helix domain required for homo-and heterodimer formation (5, 6).Heterodimers between E12/E47 and MyoD (or other myogenic bHLH proteins) bind a core consensus site, CANNTG, termed the E box (6-9). The E box is present in the IgH enhancer-like element of the muscle-specific creatine kinase (MCK) enhancer (10), as well as in the E2 element ofthe IgK enhancer (5), which appear to be downstream targets of E12/E47 and myogenic bHLH proteins, respectively. Heterodimers of E12 and the Drosophila achaete-scute protein T3 will bind the E-box sequences in the IgK enhancer in vitro (7).We wished to determine whether, as predicted by their amino acid sequences, the MASH genes encode DNAbinding and transcriptional regulatory proteins. In the absence of an authentic target for the MASH proteins, we used the MCK E box, which has been shown to bind heterodimers of Drosophila scute (T4) and E12 in vitro (6). We found that both MASH1 and MASH2 form high-affinity E-box-binding complexes as heterooligomers with E12, confirming that they are DNA-binding proteins. Since DNA binding is a necessary but not sufficient condition for transcriptional regulation (6, 11), we also tested the ability of the MASH genes to activate transcription of the MCK enhancer. Unlike Drosophila scute and E12, which bind the MCK E box but do not activate transcription from this site (6, 11), the M...

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.

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.