Glutamate regulates retinal progenitors cells proliferation during development Rodrigo A. P. Martins, 1,2 Rafael Linden 2 and Michael A. Dyer 1,3 1 Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA 2 Laborato ´rio de Neuroge ˆ nese, Instituto de Biofı ´sica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil 3 Department of Ophthalmology, University of Tennessee Health Science Center, Memphis, TN 38163, USA Keywords: AMPA ⁄ kainate, cyclin E ⁄ Cdk2, neurotransmitters, NMDA, retrovirus Abstract The precise coordination of cell cycle exit and cell fate specification is essential for generating the correct proportion of retinal cell types during development. The decision to exit the cell cycle is regulated by intrinsic and extrinsic cues. There is growing evidence that neurotransmitters can regulate cell proliferation and cell fate specification during the early stages of CNS development prior to the formation of synaptic connections. We found that the excitatory neurotransmitter glutamate regulates retinal progenitor cell proliferation during embryonic development of the mouse. AMPA ⁄ kainate and N-methyl-d-aspartate receptors are expressed in embryonic retinal progenitor cells. Addition of exogenous glutamate leads to a dose-dependent decrease in cell proliferation without inducing cell death or activating the p53 pathway. Activation of AMPA ⁄ kainate receptors induced retinal progenitor cells to prematurely exit the cell cycle. Using a replication-incompetent retrovirus to follow the clonal expansion of individual retinal progenitor cells, it was observed that blockade of AMPA ⁄ kainate receptors increased the proportion of large clones, showing that modulation of endogenous glutamatergic activity can have long-term consequences on retinal cell proliferation. Real time reverse transcriptase- polymerase chain reaction and immunoblot analyses demonstrated that glutamate does not alter the levels of the mRNA and proteins that regulate the G1 ⁄ S-phase transition. Instead, the activity of the Cdk2 kinase is reduced in the presence of glutamate. These data indicate that glutamate regulates retinal progenitor cell proliferation by post-translational modulation of cyclin ⁄ Cdk2 kinase activity. Introduction The mammalian retina is composed of six neuronal types (rod, cone, horizontal, bipolar, amacrine and ganglion cells) and one glial cell type. During development, multipotent progenitor cells generate those different cell types in a conserved birth order (Young, 1985). The proliferation and the cell cycle exit of these progenitor cells must be precisely regulated to generate a functional retina that contains the appropriate number and proportion of each cell type (Dyer & Cepko, 2001b). Deregulated proliferation of progenitor cells during retinal development can lead to blindness as a result of degeneration (Ma et al., 1998), dysplasia (Nakayama et al., 1996; Dyer & Cepko, 2000a; Levine et al., 2000) or retinoblastoma (Zhang et al., 2004a). Both cell-intrinsic and -extrinsic factors regulate progenitor cell proliferation during CNS development (Dyer & Cepko, 2001a). Prox1, Rb, Six3 and Chx10 are examples of intrinsic transcriptional regulators that contribute to proliferation regulation during retinal development (Zhu et al., 2002; Dyer, 2003; Marquardt, 2003; Zhang et al., 2004b). Similarly, extracellular molecules such as growth factors and neurotransmitters have been implicated in the extrinsic regulation of cell proliferation in the developing CNS, including the retina (Carey et al., 2002; Pearson et al., 2002, 2005; Nguyen et al., 2003). Interestingly, a role of neurotransmitters in cell fate regulation was also proposed from studies in developing mouse retina (Young & Cepko, 2004). The excitatory neurotransmitter glutamate regulates cell prolifera- tion in different regions of the CNS. In the developing cerebral cortex, glutamate has an anti-proliferative effect upon neuronal progenitors through the AMPA ⁄ kainate subtype of ionotropic receptor (LoTurco et al., 1995; Haydar et al., 2000). Interestingly, it was reported that N-methyl-d-aspartate (NMDA) receptor activation has the opposite effect upon neuronal progenitor cells of the striatum (Luk et al., 2003). This finding suggests that signaling through different classes of glutamatergic receptors may have distinct effects upon the cell cycle machinery that controls neuronal progenitors cells proliferation. Advances have been made in the understanding of the intracellular machinery that regulates cell cycle progression during retinal devel- opment (Dyer & Cepko, 2001a). It was shown that the cyclin- dependent kinases inhibitors p19 (Cdkn2d), p27 (Cdkn1b) and p57 (Cdkn1c) control cell cycle exit of retinal progenitor cells (Levine et al., 2000; Dyer & Cepko, 2001b; Cunningham et al., 2002). The importance of cyclin D1 (Ccnd1) as a positive regulator of cell cycle progression in the retina has been demonstrated in related studies (Fantl et al., 1995; Sicinski et al., 1995; Ma et al., 1998). Some studies indicated mechanisms of how an extrinsic cue communicates with the cell cycle machinery during the development of the retina (Close et al., 2005). However, it is still unclear whether neurotransmitters regulate Correspondence: Dr M.A. Dyer, 1 Department of Development Neurobiology, as above. E-mail: michael.dyer@stjude.org Received 7 February 2006, revised 9 May 2006, accepted 14 May 2006 European Journal of Neuroscience, Vol. 24, pp. 969–980, 2006 doi:10.1111/j.1460-9568.2006.04966.x ª The Authors (2006). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd