Research Article Widespread Dispersion of Neuronal Clones Across Functional Regions of the Cerebral Cortex CHRISTOPHER WALSH AND CONSTANCE L. CEPKO* The cerebral cortex of the mammalian brain has expanded rapidly during the course of evolution and acquired structurally distinguishable areas devoted to separate functions. In some brain regions, topographic restrictions to cell intermixing occur during embryonic development. As a means of examining experimentally whether such restrictions occur during formation of functional subdi- visions in the rat neocortex, clonally related neocortical cells were marked by retroviral-mediated transfer of a histochemical marker gene. Clonal boundaries were de- termined by infection of the developing brain with a library of genetically distinct viruses and amplification of single viral genomes by the polymerase chain reaction. Many clonally related neurons in the cerebral cortex became widely dispersed across functional areas of the cortex. Specification of cortical areas therefore occurs after neurogenesis. T HE CEREBRAL CORTEX REACHES ITS GREATEST SIZE AND functional complexity in primates. The cortex covers the forebrain as a folded sheet, subdivided into areas devoted to vision, sensation, audition, or other functions. Each area shows regional specialization of a basic cellular structure, comprising six neuronal layers. Furthermore, each area can be divided physiologi- cally into smaller units, or columns, in which the six layers share anatomical connections and physiological properties. A more primitive part of the brain, the hindbrain, develops according to a pattern analogous to the segmental development of the Drosophila embryo (1). The anlage of the future hindbrain forms segmental bulges called rhombomeres. Rhombomeres form do- mains that confine clones of cells (2) and domains of differential gene expression (3). It is not yet clear whether there are similar cell lineage restrictions during early development of the neocortex. Several lines of evidence suggest that there is substantial inter- mingling of clones of cortical neurons during development. In experiments on chimeric mice, formed by fusion of cells from distinguishable species or strains, intermingling of cells was seen between radially oriented columns (4). Retroviral marking studies of the mammalian cortex have confirmed that cortical cells migrate along radial glia (5), as had been shown by other methods (6). C. Walsh is in the Department of Genetics, Harvard Medical School, 25 Shattuck Street, Boston, MA 02115, and the Department of Neurology, Massachusetts General Hospital, Fruit Street, Boston, MA 02114. C. L. Cepko is in the Department of Genetics, Harvard Medical School, 25 Shattuck Street, Boston, MA 02115. *To whom correspondence should be addressed. Although Luskin and co-workers (7) suggested that clones remain substantially clustered, we and others have noted significant disper- sion among clonally related cells during their migration (8-10). Dispersion seemed to be greatest in the medial-lateral plane during fetal development and continued after birth. In postnatal and adult brains, cells labeled by retroviral marking formed unpredictable, dispersed patterns, and clonal boundaries were impossible to deter- mine accurately (8, 10). It is not possible to definitely determine clonal boundaries in the adult cortex by marking cells with retroviruses because these vectors do not provide a means for distinguishing marked cells that are part of different clones (11). We now describe a method that allows clonal boundaries to be determined independently of patterns of migration; and we have applied this method to test the functional specification of progenitors in the cortex. Retroviral library construction and clonal analysis by the polymerase chain reaction (PCR). A library of retroviral vectors was constructed by inserting fragments of DNA into BAG (12), a viral vector that contains the lacZ gene of Escherichia coli, which encodes 3-galactosidase. These inserts function as genetic tags that can precisely identify each member of the library (13) (Fig. 1). Amplification of 120 constructs by PCR followed by simultaneous digestion of these PCR products with five restriction enzymes (Cfo I, Alu I, Rsa I, Msp I, and Mse I) allowed the selection of 100 viral constructs with tags that were easily distinguished from each other BAG library Packaging nE~ZI~1LTR cells 7 \ Viral library Fetal rat FLTR ~Z LRFI: FLTR c T LTRFw---- LTR1 100 Inserts (different sizes, different restriction patterns) Fig. 1. Strategy for the preparation of the retroviral library. Each vector contains the lacZ gene and the neomycin resistance gene, as well as a short piece of unique DNA as a genetic tag. One hundred plasmid constructs were transfected pairwise to produce 50 viral supernatants. A mixture of these supernatants was injected into the brain of developing rats to label the progenitors of cerebral cortical cells and their progeny. Sites for the PCR primers are indicated by arrowheads. 434 SCIENCE, VOL. 255 This content downloaded from 128.103.147.149 on Tue, 19 Oct 2021 18:45:45 UTC All use subject to https://about.jstor.org/terms