Ongoing Sonic Hedgehog Signaling is Required for Dorsal Midline Formation in the Developing Forebrain Monica Hayhurst, 1 Bryan B. Gore, 2,3 Marc Tessier-Lavigne, 3 Susan K. McConnell 1 1 Department of Biological Sciences, Stanford University, Stanford, California 94305 2 Neurosciences Program, Stanford University, Stanford, California 94305 3 Genentech Inc., 1 DNA Way, South San Francisco, California 94080 Received 7 May 2007; accepted 29 August 2007 ABSTRACT: The division of the mammalian fore- brain into distinct left and right hemispheres represents a critical step in neural development. Several signaling molecules including sonic hedgehog (SHH), fibroblast growth factor 8 (FGF8), and bone morphogenetic pro- teins (BMPs) have been implicated in dorsal midline de- velopment, and prior work suggests that the organizing centers from which these proteins are secreted mutually regulate one another during development. To explore the role of the ventral organizing center in the formation of two hemispheres, we assessed dorsal midline develop- ment in Shh mutant embryos and in wildtype embryos treated with the SHH signaling inhibitor HhAntag. Collectively, our findings demonstrate that SHH signal- ing plays an important role in maintaining the normal expression patterns of Fgf8 and Bmp4 in the developing forebrain. We further show that FGF8 can induce the expression of Zic2, which is normally expressed at the midline and is required in vivo for hemispheric cleavage, suggesting that FGF signaling may stimulate dorsal midline development by inducing Zic2 expression. ' 2007 Wiley Periodicals, Inc. Develop Neurobiol 68: 83–100, 2008 Keywords: FGF8; BMP; Zic2; holoprosencephaly; patterning; organizing center; HhAntag INTRODUCTION During mammalian brain development, the rostral neural tube bifurcates into the left and right cerebral hemispheres. This critical event in forebrain develop- ment involves the action of molecules secreted by at least three signaling centers that are critical for pat- terning (Furuta et al., 1997; Crossley et al., 2001; Fukuchi-Shimogori and Grove, 2001; Ohkubo et al., 2002). The dorsal signaling center expresses bone morphogenetic proteins (BMPs), which are critical for dorsal midline formation (Furuta et al., 1997; Pan- chision et al., 2001; He ´bert et al., 2002). Wnt proteins are also expressed at the dorsal midline; however, it appears that Wnts primarily regulate hippocampal de- velopment and proliferation (Lee et al., 2000). The rostral signaling center expresses several fibroblast growth factors (FGFs), including FGF8, which is im- portant for anterior forebrain development and corti- cal arealization (Meyers et al., 1998; Fukuchi-Shimo- gori and Grove, 2001; He ´bert et al., 2003a,b; Storm et al., 2003, 2006). The ventral signaling center expresses sonic hedgehog (SHH), which induces This article contains supplementary material available via the Internet at http://www.mrw.interscience.wiley.com/suppmat/1932- 8451/ suppmat. Correspondence to: S.K. McConnell (suemcc@stanford.edu). Contract grant sponsor: NIH; contract grant numbers: R01 MH65261, T32 HD007249. Contract grant sponsor: Lucille P. Markey Biomedical Fellow- ship. ' 2007 Wiley Periodicals, Inc. Published online 18 October 2007 in Wiley InterScience (www. interscience.wiley.com). DOI 10.1002/dneu.20576 83