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Abstract

The contribution of extrinsic and genetic mechanisms in determining areas of the mammalian neocortex has been a contested issue. This study analyzes the roles of the regulatory genesEmx2 and Pax6, which are expressed in opposing gradients in the neocortical ventricular zone, in specifying areas. Changes in the patterning of molecular markers and area-specific connections between the cortex and thalamus suggest that arealization of the neocortex is disproportionately altered in Emx2 andPax6 mutant mice in opposing manners predicted from their countergradients of expression: rostral areas expand and caudal areas contract in Emx2 mutants, whereas the opposite effect is seen in Pax6 mutants. These findings suggest thatEmx2 and Pax6 cooperate to regulate arealization of the neocortex and to confer area identity to cortical cells.

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Embryonic day 18.5 (E18.5) embryos were obtained from timed-pregnant matings of Emx2 heterozygous mice maintained on a C57BL/6J background (E0.5 = noon of day of vaginal plug detection). Animal care was in accordance with institutional guidelines. Littermate sets of cortices from wild type, heterozygote, and homozygous mutants were analyzed to control for differences in developmental rate. Whole-mount in situ hybridization of cortices {modified from work by D. Henrique et al., [Nature 375, 787 (1995)]} used digoxygenin-labeled riboprobes encoding Cad6 and Cad8. Emx2 mice were genotyped by polymerase chain reaction on tail DNA. Whole-mount in situ hybridizations were quantified with the Image program from the National Institutes of Health. Quantification was done blinded to genotype.
14
Whole-mount in situ hybridization on E18.5 Pax6 mutant brains was performed as in (13). Embryos were obtained from matings of heterozygous Sey mice maintained on a C57BL/6J × DBA/2J background. Sey mice were genotyped by examination of the eyes: homozygous mutants lack eyes, and heterozygotes have reduced external eye size and a pronounced size reduction of the developing lens at E18.5. Analysis was as in (13), done blinded to genotype.
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23
A delay in overall development of the cortex cannot account for our findings. The mutant patterns of cadherin expression and thalamocortical projections do not resemble patterns observed at earlier stages in wild types. For example, the domains of cadherin expression do not exhibit significant proportional expansions or contractions during normal embryonic cortical development (6); axons from VP neurons do not transiently invade the visual area at early stages and the somatosensory area later, and vice versa for dLG axons; and the distribution of corticothalamic projection neurons is area specific, even at the earliest times that they can be labeled (16). Emx2 and Pax6 mutants also show opposite changes in cadherin expression patterns, which cannot be accounted for by a delay in cortical development in both mutants.
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Independent regulation of Emx2 and Pax6 is suggested by their temporal coexpression and the findings that Emx2 is expressed in the Pax6 mutant and that Pax6 is expressed in the Emx2 mutant in graded patterns in the developing neocortex similar to those in wild-type mice [
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We are grateful to P. Gruss for the gift of Emx2 mice, M. Goulding for Sey mice and Pax6 plasmid, and Y. Nakagawa for Cad6, Cad8, and Gbx2 plasmids and helpful discussions. This work was supported by NIH grant NS31558 (D.D.M.O'L.), the Natural Sciences and Engineering Research Council of Canada (K.M.B.), and the Max-Planck Society and European Commission grant BI04-CT96-0378 (G.G.).

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Published In

Science
Volume 288 | Issue 5464
14 April 2000

Submission history

Received: 4 October 1999
Accepted: 22 February 2000
Published in print: 14 April 2000

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Authors

Affiliations

Kathie M. Bishop
Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA.
Guy Goudreau
Max-Planck-Institute for Biophysical Chemistry, Department of Molecular Cell Biology, Am Fassberg 11, 37077 Göttingen, Germany.
Dennis D. M. O'Leary*
Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA.

Notes

*
To whom correspondence should be addressed. E-mail: [email protected]

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