19 June 2005. This week, light from two very different angles was shone on the process of neurodevelopment. Writing in PNAS, researchers described a method to recapitulate subventricular zone (SVZ) neurogenesis in a culture dish, while a paper in Nature reveals that neuronal diversity within individuals could conceivably be due to active retrotransposons, sometimes called jumping genes.
The SVZ is one of only two areas where adult neurogenesis takes place, so understanding the process that goes on there could be an important step in developing cell-based therapies for neurodegenerative diseases. But to date, studying this process has meant isolating SVZ cells and then identifying precursors by their ability to undergo cell division, a process that fails to capture the dynamic nature of neurogenesis as differentiating cells migrate from the SVZ to their final destination. But now, using time-lapse, microscopic analysis of neuronal precursors in culture, Dennis Steindler and colleagues from the University of Florida, Gainesville, and from Cold Spring Harbor Laboratory, New York, have been able to identify, track, and characterize cells as they differentiate from one cell type into another.
Described in last week’s early online edition of PNAS, joint first authors Bjorn Scheffler and Noah Walton and their colleagues started off by plating single-cell suspensions of mouse SVZ cells in uncoated plastic dishes and growing them as monolayers. The researchers found that by bathing these cells with epidermal and basic fibroblast growth factors, they could maintain them for over 75 population doublings without any sign of senescence—an indication that they had isolated true stem cells.
To track differentiation of the cells, the authors removed the growth factors. Then, using various antibodies, time-lapse microscopic recordings, and electrical measurements, they watched and tested the cells that developed. They found that the appearance and disappearance of specific cell markers, such as glial fibrillary acid protein, βIII tubulin, nestin, and many more, closely followed patterns that have been described by previous in vivo experiments. The cells also responded to retinoic acid, which has been used to terminally differentiate neural precursors. “Taken together, these results suggest that it is possible to recapitulate and longitudinally track a heteromorphic differentiation process in vitro that temporally and phenotypically follows SVZ neurogenesis in vivo,” write the authors.
Jumping to the retrotransposon paper, Fred (Rusty) Gage and colleagues at the Salk Institute for Biological Studies, La Jolla, California, and the University of Michigan, Ann Arbor, reveal in an article in the June 16 Nature that one of the “hot” or active retrotransposons in the human genome, long interspersed nuclear element-1 (LINE-1, or L1), can retrotranspose in neural precursor cells. The finding hints that such transpositions might also take place during the development of the human brain and might partly explain individual differences in brain organization and function.
L1 elements are abundant, accounting for about 20 percent of the human genome. Most of them, however, are inactive, with only about 80-100 L1s considered retrotransposition-competent, and only about 10 percent of those considered highly active.
In studying human multipotent neural progenitor cells (NPCs), first author Alysson Muotri and colleagues found that L1 expression is upregulated in response to the addition of glycosylated cystatin C, which is commonly used in propagation of NPCs. To test if the mobile element might be activated in vivo, Muotri and colleagues engineered a reporter construct consisting of an L1 element that interrupts expression of enhanced green fluorescent protein (EGFP). The construct was devised such that retrotransposition of the element would trigger expression of EGFP, which could readily be detected by green fluorescence.
Muotri introduced the element into transgenic mice. The investigators found that GFP turned up in the mouse brain and always colocalized with neuronal markers, suggesting that retrotransposition occurs only in neuronal precursors and not precursors common to all brain cells. The authors also found that L1 can alter the expression of neuronal genes and effect cell fate in vitro.
As Eric Ostertag and Haig Kazazian from the University of Pennsylvania write in an accompanying News & Views, “if enough mobile DNA insertions occur in the brains of developing humans, then the outcome might be a change in their neuronal circuitry, for better or worse.” Whether this really happens or not remains to be seen. Ostertag and Kazazian also point out that it is impossible to determine directly the frequency of insertions that occur in vivo in human neuronal precursor cells. In future experiments, Gage and colleagues plan to focus on finding out if L1 retrotransposition naturally occurs in neural precursor cells, and if so, if the process has any developmental significance.—Tom Fagan.
Schefler B, Walton NM, Lin DD, Goetz AK, Enikolopov G, Roper SN, Steindler DA. Phenotypic and functional characterization of adult brain neuropoiesis. PNAS early edition. June 13, 2005. Abstract
Muotri AR, Chu VT, Marchetto MCN, Deng W, Moran JV, Gage FH. Somatic mosaicism in neuronal precursor cells mediated by L1 retrotransposition. Nature. June 16, 2005;435:903-910. Abstract
Ostertag EM, Kazazian HH. Genetics: LINEs in mind. Nature. June 16, 2005;435:890-891. Abstract