A simple slice culture system for the imaging of nerve development in embryonic mouse.

Newborn neurons elaborate an axon that undertakes a complicated journey to find its ultimate target in the brain or periphery. Although major progress in the study of this process has been made by analysis of dissociated neurons in vitro, one would like to observe and manipulate axonal outgrowth and pathfinding as it occurs in situ, as fasciculated nerves growing within the tissue itself. Here, we present a simple technique to do this, through cultivation of embryonic mouse slices expressing enhanced green fluorescent protein (EGFP) specifically in newborn neurons. This system allows for imaging of outgrowth of peripheral nerves into structures such as the developing limb. We demonstrate a reproduction of normal innervation patterns by spinal nerves derived from spinal cord motor neurons and sensory neurons of the dorsal root ganglia. The slices can be manipulated pharmacologically as well as genetically, by crossing the EGFP-expressing line with lines containing targeted mutations in genes of interest.

Cre recombinase specificity defined by the tau locus.

We generated a transgenic mouse line (tau::Cre) by targeting the Cre to the tau locus (Mapt). Based on previous reports on the expression of Tau during development, we expected the Cre recombinase to be expressed in a neuron-specific and pan-neuronal manner. However, intercrosses between the tau::Cre and the Cre-activatable reporter animals resulted in offspring with recombination either restricted to the nervous system or throughout the entire conceptus, indicating expression of Tau early in development. The percentage of neuron-specific excision was dependent on the Cre reporter used representing different Cre target sites in the mouse genome. In spite of the observed variability, our data suggest that the tau::Cre mouse line can be used for pan-neuronal recombination of floxed alleles when it is used with caution.

Developmental potential of defined neural progenitors derived from mouse embryonic stem cells.

The developmental potential of a uniform population of neural progenitors was tested by implanting them into chick embryos. These cells were generated from retinoic acid-treated mouse embryonic stem (ES) cells, and were used to replace a segment of the neural tube. At the time of implantation, the progenitors expressed markers defining them as Pax6-positive radial glial (RG) cells, which have recently been shown to generate most pyramidal neurons in the developing cerebral cortex. Six days after implantation, the progenitors generated large numbers of neurons in the spinal cord, and differentiated into interneurons and motoneurons at appropriate locations. They also colonized the host dorsal root ganglia (DRG) and differentiated into neurons, but, unlike stem cell-derived motoneurons, they failed to elongate axons out of the DRG. In addition, they neither expressed the DRG marker Brn3a nor the Trk neurotrophin receptors. Control experiments with untreated ES cells indicated that when colonizing the DRG, these cells did elongate axons and expressed Brn3a, as well as Trk receptors. Our results thus indicate that ES cell-derived progenitors with RG characteristics generate neurons in the spinal cord and the DRG. They are able to respond appropriately to local cues in the spinal cord, but not in the DRG, indicating that they are restricted in their developmental potential.

Differentiation of mouse embryonic stem cells into a defined neuronal lineage.

Although it has long been known that cultured embryonic stem cells can generate neurons, the lineage relationships with their immediate precursors remain unclear. We report here that selection of highly proliferative stem cells followed by treatment with retinoic acid generated essentially pure precursors that markers identified as Pax-6-positive radial glial cells. As they do in vivo, these cells went on to generate neurons with remarkably uniform biochemical and electrophysiological characteristics.

Tau EGFP embryonic stem cells: an efficient tool for neuronal lineage selection and transplantation.

Pluripotency and the capacity for continuous self-renewal make embryonic stem (ES) cells an attractive donor source for cell-replacement strategies. A key prerequisite for a therapeutic application of ES cells is the generation of defined somatic cell populations. Here we demonstrate that a targeted insertion of the EGFP gene into the tau locus permits efficient fluorescence-activated cell sorting (FACS)-based lineage selection of ES cell-derived neurons. After in vitro differentiation of heterozygous tau EGFP ES cells into multipotent neural precursors, EGFP is selectively induced in postmitotic neurons of various neurotransmitter phenotypes. By using FACS, ES cell-derived neurons can be enriched to purities of more than 90%. Because neuron-specific EGFP fluorescence is also observed upon transplantation of ES cell-derived neural precursors, the tau EGFP mutant represents a useful tool for the in vivo analysis of grafted ES cell-derived neurons.

Glial cells generate neurons: the role of the transcription factor Pax6.

Radial glial cells, ubiquitous throughout the developing CNS, guide radially migrating neurons and are the precursors of astrocytes. Recent evidence indicates that radial glial cells also generate neurons in the developing cerebral cortex. Here we investigated the role of the transcription factor Pax6 expressed in cortical radial glia. We showed that radial glial cells isolated from the cortex of Pax6 mutant mice have a reduced neurogenic potential, whereas the neurogenic potential of non-radial glial precursors is not affected. Consistent with defects in only one neurogenic lineage, the number of neurons in the Pax6 mutant cortex in vivo is reduced by half. Conversely, retrovirally mediated Pax6 expression instructs neurogenesis even in astrocytes from postnatal cortex in vitro. These results demonstrated an important role of Pax6 as intrinsic fate determinant of the neurogenic potential of glial cells.