Trk signaling regulates neural precursor cell proliferation and differentiation during cortical development

Katarzyna Bartkowska 1 2 Annie Paquin 0 2 Andre S. Gauthier 1 2 4 David R. Kaplan 0 1 4 Freda D. Miller 0 2 3 4 0 Institute of Medical Sciences, University of Toronto , Toronto , Canada 1 Cell Biology Programs, Hospital for Sick Children, University of Toronto , Toronto , Canada 2 Developmental and Stem Cell Biology 3 Physiology, University of Toronto , Toronto , Canada 4 Departments of Molecular and Medical Genetics Increasing evidence indicates that development of embryonic central nervous system precursors is tightly regulated by extrinsic cues located in the local environment. Here, we asked whether neurotrophin-mediated signaling through Trk tyrosine kinase receptors is important for embryonic cortical precursor cell development. These studies demonstrate that inhibition of TrkB (Ntrk2) and/or TrkC (Ntrk3) signaling using dominant-negative Trk receptors, or genetic knockdown of TrkB using shRNA, caused a decrease in embryonic precursor cell proliferation both in culture and in vivo. Inhibition of TrkB/C also caused a delay in the generation of neurons, but not astrocytes, and ultimately perturbed the postnatal localization of cortical neurons in vivo. Conversely, overexpression of BDNF in cortical precursors in vivo promoted proliferation and enhanced neurogenesis. Together, these results indicate that neurotrophin-mediated Trk signaling plays an essential, cell-autonomous role in regulating the proliferation and differentiation of embryonic cortical precursors and thus controls cortical development at earlier stages than previously thought. INTRODUCTION Development of the cerebral cortex is achieved through a common pool of precursor cells that sequentially generate neurons and glial cells. Emerging evidence indicates that whereas intrinsic cues are important in cortical precursor development, differences in the availability of growth factors determine precursor survival, proliferation and the appropriate timed genesis of neurons versus astrocytes (Miller and Gauthier, 2007). The neurotrophins are growth factors that are best known for regulating the biology of central nervous system (CNS) neurons, but that also regulate development of at least some precursor cells (Huang and Reichardt, 2003). At least two members of the neurotrophin family, BDNF and NT3 (also known as Ntf3 Mouse Genome Informatics), along with their preferred tyrosine kinase receptors, TrkB and TrkC (Ntrk2 and Ntrk3, respectively Mouse Genome Informatics), are expressed in the cortical ventricular/ subventricular zones (VZ/SVZ) during the period of cortical neurogenesis (Maisonpierre et al., 1990; Tessarollo et al., 1993; Behar et al., 1997; Fukumitsu et al., 1998; Fukumitsu et al., 2006). Moreover, culture work indicates that: (1) NT3 selectively regulates cell cycle exit and neuronal differentiation in cortical progenitors (Ghosh and Greenberg, 1995; Lukaszewicz et al., 2002); and (2) cortical precursors themselves synthesize and secrete the neurotrophins BDNF and NT3, which promote their survival and differentiation in an autocrine/paracrine fashion by activating TrkB/TrkC receptors (Barnab-Heider and Miller, 2003). However, an in vivo role for the Trk receptors in cortical precursor biology has not yet been established. *These authors contributed equally Author for correspondence (e-mail: ) Here, we have asked whether Trk signaling is important for embryonic cortical precursor development in vivo, by performing in utero electroporation with dominant-negative TrkB and TrkC or with TrkB shRNA to acutely, and in a cell-autonomous fashion, disrupt Trk signaling. In this regard, the TrkB receptor can be activated by BDNF, NT3 and NT4 (also known as Ntf5 Mouse Genome Informatics) (Huang and Reichardt, 2003), and whereas previous work (Jones et al., 1994; Alcantara et al., 1997; Ringstedt et al., 1998; Xu et al., 2000; Lotto et al., 2001; Medina et al., 2004) has indicated that BDNF-mediated TrkB activation is important for cortical development in vivo, these studies concluded that any observed perturbations were a consequence of altered TrkB signaling in cortical neurons. The TrkC receptor is only activated by NT3, and previous work on Nt3/ and TrkC/ mice has primarily focused upon the profound deficits observed in the peripheral nervous system (Ernfors et al., 1994; Wilkinson et al., 1996; Klein et al., 1994; Tessarollo et al., 1994), or on perturbations in the biology of committed CNS glia or neurons (Minichiello and Klein, 1996; Martinez et al., 1998; Kahn et al., 1999; Ma et al., 2002; von Bohlen und Halbach et al., 2003). As both BDNF and NT3 are known to be expressed in precursor cells of the cortical neuroepithelium (Maisonpierre et al., 1990; Fukumitsu et al., 1998; Behar et al., 1997; Barnab-Heider and Miller, 2003; Fukumitsu et al., 2006), and as cortical precursors express both of these receptors (Tessarollo et al., 1993; Behar et al., 1997; Barnab-Heider and Miller, 2003), we have chosen to disrupt signaling via these two receptors both individually and together. These studies demonstrate that TrkB and TrkC receptor activation, presumably in response to BDNF and NT3, are necessary for the appropriate proliferation and differentiation of embryonic cortical precursors, and thus play an important early role in cortical development. MATERIALS AND METHODS Cultures and transfections Cortical precursors were cultured as described previously (Barnab-Heider and Miller, 2003; Barnab-Heider et al., 2005; Gauthier et al., 2007). Cell density was 125,000 cells/well for four-well chamber slides. For transfections, 2 to 4 hours after plating, 1 g DNA and 1.5 l Fugene 6 (Roche, Welwyn Garden City, UK) mixed with 100 l of Opti-MEM (Invitrogen) were incubated at room temperature for 40 minutes and then added to each well. The rat dnTrkB mutant consisted of a single mutation (K538N) in the ATP-binding site that rendered it kinase-dead (Atwal et al., 2000), whereas the rat dnTrkC mutant contained three mutated tyrosines (Y705N, Y709N and Y710N) within catalytic subdomain VIII (the kind gift of Pantelis Tsoulfas, University of Miami, Miami, FL). Trk constructs were subcloned into the pEF-GM expression vector (Paquin et al., 2005). The two TrkB shRNA constructs targeted two different regions on the TrkB mouse mRNA sequence. The sequence for TrkB shRNA1 was 5 TTGTGGATTCCGGCTTAAATTCAAGAGATTTAAGCCGGAATCCACAA-3 , for TrkB shRNA2 was 5 -CCTTGTAGGAGAAGATCAATTCAAGAGATTGATCTTCTCCTACAAGG-3 , and for the control shRNA was 3 -TTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAA-3 . This control shRNA was mismatched to known human and mouse genes (EZBiolab, Westfield, IN). The dnAkt mutant contains a point mutation (K179M) within its ATP-binding site (Songyang et al., 1997). In utero electroporation In utero electroporation was performed as described with the square electroporator CUY21 EDIT (TR Tech, Japan), delivering five 50 ms pulses of 40-50 V with 950 ms intervals (Barnab-Heider et al., 2005; Paquin et al. (...truncated)