Efficient Gene Transfer into the Embryonic Mouse Brain Using in Vivo Electroporation

AbstractMouse 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

Induction and repression of mammalian achaete-scute homologue (MASH) gene expression during neuronal differentiation of P19 embryonal carcinoma cells

MASH1 and MASH2, mammalian homologues of the Drosophila neural determination genes achaete-scute, are members of the basic helix-loop-helix (bHLH) family of transcription factors. We show here that murine P19 embryonal carcinoma cells can be used as a model system to study the regulation and function of these genes. MASH1 and MASH2 display complementary patterns of expression during the retinoic-acid-induced neuronal differentiation of P19 cells. MASH1 mRNA is undetectable in undifferentiated P19 cells but is induced to high levels by retinoic acid coincident with neuronal differentiation. In contrast, MASH2 mRNA is expressed in undifferentiated P19 cells and is repressed by retinoic acid treatment. These complementary expression patterns suggest distinct functions for MASH1 and MASH2 in development, despite their sequence homology. In retinoic-acid-treated P19 cells, MASH1 protein expression precedes and then overlaps expression of neuronal markers. However, MASH1 is expressed by a smaller proportion of cells than expresses such markers. MASH1 immunoreactivity is not detected in differentiated cells displaying a neuronal morphology, suggesting that its expression is transient. These features of MASH1 expression are similar to those observed in vivo, and suggest that P19 cells represent a good model system in which to study the regulation of this gene. Forced expression of MASH1 was achieved in undifferentiated P19 cells by transfection of a cDNA expression construct. The transfected cells expressing exogenous MASH1 protein contained E-box-binding activity that could be super-shifted by an anti-MASH1 antibody, but exhibited no detectable phenotypic changes. Thus, unlike myogenic bHLH genes, such as MyoD, which are sufficient to induce muscle differentiation, expression of MASH1 appears insufficient to promote neurogenesis

Identification of Novel Paired Homeodomain Protein Related to C. elegans unc-4 as a Potential Downstream Target of MASH1

A novel paired homeodomain protein, PHD1, that is most closely related toC. elegansunc-4 has been identified by a differential RT–PCR method.PHD1is expressed in a narrow layer adjacent to the ventricular zone of the dorsal spinal cord, immediately following expression of MASH1 but preceding overt neuronal differentiation. Some cells coexpressing MASH1 andPHD1can be seen, suggesting that these two genes are sequentially activated within the same lineage. In the olfactory sensory epithelium,PHD1expression not only follows but is dependent upon MASH1 function, suggesting that PHD1 acts downstream of MASH1. A sequential action of bHLH and paired homeodomain proteins is apparent in other neurogenic lineages and may be a general feature of both vertebrate and invertebate neurogenesis

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

Mammalian BarH Homologue Is a Potential Regulator of Neural bHLH Genes

Vertebrate neurogenesis involves sequential actions of transcription factors.neurogenins,encoding Atonal-related bHLH transcription factors, function as neuronal determination genes inXenopus. neurogeninsand another bHLH factor gene,Mash1,are expressed in distinct subsets or areas of cells giving rise to neurons, suggesting that these genes play important roles to generate distinct populations of neurons. A mammalian homologue of BarH(MBH1) is expressed in a complementary pattern to Mash1expression in the developing nervous system likeneurogenins.Forced expression of MBH1down-regulates expression of Mash1and up-regulatesneurogenin2/Math4A, a member of neurogenins,in P19 cells during neuronal differentiation. This suggests thatMBH1is a potential regulator of mammalian neural bHLH genes, thereby establishing distinct pathways of neuronal differentiation

Dendritic complexity was not affected in MCs of <i>Maml1</i> mutants at E15.5.

<p>(<b>A, B</b>) Representative images (<b>A</b>) and dendritic tracing (<b>B</b>) of MCs at E15.5. Scale bar: 20 μm. (<b>C–E</b>) Number of branch points (<b>C</b>), total dendrite length (<b>D</b>) and length of the longest dendrite (<b>E</b>) of MCs: <i>Maml1</i>^+/+, <i>n</i> = 25 from 6 mice; <i>Maml1</i>^+/−, <i>n</i> = 23 from 5 mice; <i>Maml1</i>^−/−, <i>n</i> = 22 from 5 mice. MCs that were in the dorsomedial OB and located at 300 to 600 μm from the rostral tip of the OB were analyzed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006514#pgen.1006514.g001" target="_blank">Fig 1</a>. There were no significant differences in the number of branch points, total dendrite length or length of the longest dendrite among the genotypes (P ranged from 0.181 to 0.728).</p