The Zinc Finger Transcription Factor RP58 Negatively Regulates Rnd2 for the Control of Neuronal Migration During Cerebral Cortical Development

Zhengdong Qu and Chiaki Ohtaka-Maruyamacontributed equally to this work.The zinc finger transcription factor RP58 (also known as ZNF238)regulates neurogenesis of the mouse neocortex and cerebellum(Okado et al. 2009; Xiang et al. 2011; Baubet et al. 2012; Ohtaka-Mar-uyama et al. 2013), but its mechanism of action remains unclear. Inthis study, we report a cell-autonomous function for RP58 during thedifferentiation of embryonic cortical projection neurons via its activi-ties as a transcriptional repressor. Disruption of RP58 expressionalters the differentiation of immature neurons and impairs theirmigration and positioning within the mouse cerebral cortex. Loss ofRP58 within the embryonic cortex also leads to elevated mRNA forRnd2, a member of the Rnd family of atypical RhoA-like GTPaseproteins important for cortical neuron migration (Heng et al. 2008).Mechanistically, RP58 repressestranscription of Rnd2 via binding to a3′-regulatory enhancer in a sequence-specific fashion. Using reporterassays, we found that RP58 repression of Rnd2 is competed by pro-neural basic helix–loop–helix transcriptional activators. Finally, ourrescueexperiments revealed that negative regulation ofRnd2 by RP58was important forcortical cell migration in vivo. Taken together, thesestudies demonstrate that RP58 is a key player in the transcriptionalcontrolofcellmigrationinthedevelopingcerebralcortex.Keywords: cerebral cortex, migration, pattern formation, transcription factorIntroductionDuring cortical development, projection neurons are born inthe germinal ventricular zone (VZ) and migrate through a tran-sitory intermediate zone (IZ) before settling within the corticalplate (CP) where they undergo terminal differentiation (Marinand Rubenstein 2003; Merot et al. 2009). The molecularcontrolof these events involves cell proliferation, cell cycle exit, andneural differentiation (Ge et al. 2006; Nguyen et al. 2006).However, to date, relatively few studies have explored the tran-sition from cell cycle exit to cell migration at the molecularlevel; or enquired whether or not these 2 processes arecoupled (Ge et al. 2006; Kawauchi et al. 2006; Nguyen et al.2006; Buchman etal. 2010; Singh et al. 2010).In contrast, the molecular control of proteins driving corticalneuron migration and maturation are better understood. For in-stance, we know how transcription factors such as Dlx1/2(Cobos et al. 2007 ), as well as members of the basic helix–loop–helix (bHLH) family, including Mash1 and Neurog2 (Hand et al.2005; Ge et al. 2006; Heng et al. 2008; Dixit et al. 2011; Pacaryet al. 2011), drive the maturation of embryonic cortical neurons.From these studies, we understand that corticogenesis iscarefully regulated at the transcriptional level, right from neur-onal migration (Handetal. 2005; Pacaryetal. 2011)toneurodif-ferentiation (Cobos et al. 2007) and acquisition of glutamatergic(Parras et al. 2002; Schuurmans et al. 2004; Molyneaux et al.2007)orγ-aminobutyric acidergic (Marin and Rubenstein 2001;Cobos et al. 2007 ) neuronal subidentities. Such studies demon-strate that timing, as well as gene dosage, regulates neuronalmigrationandterminaldifferentiation.In the present work, we focus on the zinc finger repressorRP58 (also known as Znf238 or Zfp238), which has recentlybeen found to be important for the development of multiple celllineageswithinthemouseembryo( Okadoetal.2009;Yokoyamaet al. 2009; Xiang et al. 2011; Baubet et al. 2012). Within thenervous system, loss of RP58 leads to abnormal neurodifferen-tiation and progenitor proliferation within the neocortex(Okado et al. 2009; Xiang et al. 2011) as well as the cerebellum(Okado et al. 2009; Baubet et al. 2012). Recently, it was also re-ported that RP58 controls cortical neurogenesis (Hirai et al.2012) and the early steps of cell migration within the embryo-nic mouse cortex through direct regulation of Neurog2, a pro-neural bHLH protein and a key regulator of cortical projectionneuron identity within the cortex (Ohtaka-Maruyama et al.2013). However, it is likely that RP58 has multiple downstreamtargets to direct the migration and differentiation of corticalneurons. In this study, we report that RP58 cell autonomouslyinfluences the migration and long-term positioning of corticalneurons and directly regulates the expression of Rnd2,amember of the Rnd family of atypical RhoA-like GTPases thatinfluences the migration of newborn cortical neurons (Handet al. 2005; Pacary et al. 2011). We demonstrate that RP58 med-iatesthe expression of Rnd2 throughitstranscriptionalrepressoractivity on a previously characterized 3′-regulatory enhancer(Henget al.2008). Finally,weperformrescueexperimentstode-monstrate that Rnd2 lies downstream of RP58 in the regulationof cell migration, including their multipolar-to-bipolar transitionwithin the embryonic cortex. Thus, RP58 directly influences thepositioningof cerebralcortical neuronsthrough atranscriptionalregulatorypathwayinvolvingthedownstreamtargetgeneRnd2.Materials and Methods