903 Introduction The highly ordered cytoarchitecture and the relative simplicity of cerebellar development make it one of the best studied systems for neurogenesis. Most of the cerebellar neurons (e.g. Purkinje cells, deep cerebellar nuclei and interneurons) arise at a ventricular zone located at the edge of the fourth ventricle (Hatten and Heintz, 1995). Precursors of the cerebellar granule cells (CGC) are born in a second proliferative zone, the rhombic lip, where they proliferate and later migrate via a rostral movement over the surface of the embryonic cerebellum (Altman and Bayer, 1997; Gilthorpe et al., 2002; Wingate, 2001). Consequently, these CGC precursors yield the external granule/germinate layer (EGL) of the cerebellum, a displaced germinal zone, where proliferation continues and peaks at postnatal day 7 (P7) in mouse (Altman and Bayer, 1997; Hatten et al., 1997; Hatten and Heintz, 1995). Postmitotic cells congregate in the inner EGL, and then migrate into the cerebellar cortex along Bergman radial glia towards their final destination: the cerebellar internal granule layer (IGL) (Edmondson et al., 1988; Fishman and Hatten, 1993; Hatten and Heintz, 1995). The later stages of CGC development – EGL formation and migration towards the IGL – have been extensively studied (reviewed by Goldowitz and Hamre, 1998; Hatten and Heintz, 1995; Millen et al., 1999; Wang and Zoghbi, 2001), in contrast to the earlier stages of precursor specification and differentiation, which are less characterized. Math1 (Atoh1 – Mouse Genome Informatics) encodes a murine basic helix-loop-helix (bHLH) transcription activator (Akazawa et al., 1995; Ben-Arie et al., 1996), orthologous to the Drosophila atonal. In the developing cerebellum, Math1 is expressed in mitotic CGC at the rhombic lip and in the outer EGL (Akazawa et al., 1995; Ben-Arie et al., 1997; Ben-Arie et al., 2000; Ben-Arie et al., 1996; Helms et al., 2000). Genomic disruption has proven that Math1 is essential for proper development of CGC, as Math1 null mice lack the EGL (Ben-Arie et al., 1997; Ben-Arie et al., 2000). However, overexpression of Math1 resulted in cerebellar abnormalities without extra neurogenesis (Helms et al., 2001; Isaka et al., Cerebellar granule cells (CGC) are the most abundant neurons in the mammalian brain, and an important tool for unraveling molecular mechanisms underlying neurogenesis. Math1 is a bHLH transcription activator that is essential for the genesis of CGC. To delineate the effects of Math1 on CGC differentiation, we generated and studied primary cultures of CGC progenitors from Math1/lacZ knockout mice. Rhombic lip precursors appeared properly positioned, expressed CGC-specific markers, and maintained Math1 promoter activity in vivo and in vitro, suggesting that Math1 is not essential for the initial stages of specification or survival of CGC. Moreover, the continuous activity of Math1 promoter in the absence of MATH1, indicated that MATH1 was not necessary for the activation of its own expression. After 6, but not 3, days in culture, Math1 promoter activity was downregulated in control cultures, but not in cells from Math1 null mice, thus implying that Math1 participates in a negative regulatory feedback loop that is dependent on increased levels of MATH1 generated through the positive autoregulatory feedback loop. In addition, Math1 null CGC did not differentiate properly in culture, and were unable to extend processes. All Notch signaling pathway receptors and ligands tested were expressed in the rhombic lip at embryonic date 14, with highest levels of Notch2 and Jag1. However, Math1-null rhombic lip cells presented conspicuous downregulation of Notch4 and Dll1. Moreover, of the two transcriptional repressors known to antagonize Math1, Hes5 (but not Hes1) was downregulated in Math1- null rhombic lip tissue and primary cultures, and was shown to bind MATH1, thus revealing a negative regulatory feedback loop. Taken together, our data demonstrate that CGC differentiation, but not specification, depends on Math1, which acts by regulating the level of multiple components of the Notch signaling pathway. Key words: Rhombic lip, Cerebellum, Cerebellar granule cells, Neurite, Notch, Delta, Jagged, Hes, Knockout, Mouse Summary Math1 controls cerebellar granule cell differentiation by regulating multiple components of the Notch signaling pathway Roi Gazit 1, * ,† , Valery Krizhanovsky 1, * and Nissim Ben-Arie 1,2,‡ 1 Cell and Animal Biology, The Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel 2 Roland Center for Neurodegenerative Diseases, The Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel *These authors contributed equally to the work † Present address: The Lautenberg Center for General and Tumor Immunology, Hadassah Medical School, Jerusalem, Israel ‡ Author for correspondence (e-mail: nbenarie@vms.huji.ac.il) Accepted 12 November 2003 Development 131, 903-913 Published by The Company of Biologists 2004 doi:10.1242/dev.00982 Research article