4943 RESEARCH ARTICLE INTRODUCTION microRNAs (miRNAs) are a fundamental class of biological molecules with a crucial role in development (Kloosterman and Plasterk, 2006), the dysfunction of which has been linked to cancer (Calin and Croce, 2006), among other biological processes. Genes encoding miRNAs, which are found in most eukaryotes, produce short (~22 nt) RNAs that bind to mRNA transcripts and downregulate their expression either through mRNA destabilization or translational repression (Bartel, 2009). How miRNAs recognize and bind their mRNA targets is still a subject of intense research (Bartel, 2009), although in most cases good complementary binding between the mRNA 3' untranslated region (UTR) and the first eight nucleotides of the miRNA is necessary, which we will refer to as the ‘seed’ site. miR-124 is one of the most abundant miRNAs expressed in the mouse brain (Lagos-Quintana et al., 2002). Subsequent studies showed that miR-124 is also expressed in the nervous systems of Drosophila (Aboobaker et al., 2005), C. elegans (Clark et al., 2010) and humans (Sempere et al., 2004). Its clinical importance has recently been revealed in a study showing that aberrant downregulation of miR-124 in humans is associated with the formation of brain tumors (Silber et al., 2008). In vertebrates, miR- 124 regulates two important anti-neural factors: SCP1 (small C-terminal domain phosphatase), a component of the REST/NRSF neuronal transcriptional repressor complex (Visvanathan et al., 2007), and PTBP1, which represses brain-specific alternative pre- mRNA splicing (Makeyev et al., 2007). A recent in vitro study using FACS-sorted stem cell astrocytes from the neurogenic subventricular zone of the mouse brain showed that miR-124 knockdown increased the number of dividing neural stem cells and reduced the number of postmitotic neurons, whereas conversely, miR-124 overexpression promoted cell cycle exit and increased the expression of postmitotic neuronal markers (Cheng et al., 2009). These results suggest a role of miR-124 in driving neuronal differentiation. However, aside from detailed studies of a few specific targets, the underlying genetic pathways by which miR- 124 may drive neuronal differentiation remain largely unexplored. Here, using computational target extraction with in vivo functional and transgenic assays, we show that, in the chordate ascidian Ciona intestinalis, miR-124 plays a multifaceted role in promoting neuronal development. As the sister group of vertebrates (Delsuc et al., 2006), ascidians possess a simplified chordate nervous system. The central nervous system (CNS) has ~100 neurons and consists of a sensory vesicle and a dorsal nerve cord, whereas the peripheral nervous system (PNS) consists primarily of epidermal sensory neurons (ESNs) (Imai and Meinertzhagen, 2007a; Imai and Meinertzhagen, 2007b; Passamaneck and Di Gregorio, 2005). Early in development, specification of the ascidian CNS requires FGF signaling (Akanuma and Nishida, 2004; Bertrand et al., 2003) and is likely to utilize Hedgehog signaling as well (Takatori et al., 2002). In the PNS, both FGF and BMP signaling are required early, and, subsequently, Notch signaling resolves the pattern of sensory neurons within the midline of the larval tail (Pasini et al., 2006). Using our sensor assay, we demonstrate that miR-124 can downregulate genes from all of Development 138, 4943-4953 (2011) doi:10.1242/dev.068049 © 2011. Published by The Company of Biologists Ltd 1 Computational Science Research Center, 2 Department of Biology, and 3 Center for Applied and Experimental Genomics, San Diego State University, 5500 Campanile Drive, San Diego, CA 92182, USA. *Author for correspondence (rzeller@sciences.sdsu.edu) Accepted 19 September 2011 SUMMARY The nervous system-enriched microRNA miR-124 is necessary for proper nervous system development, although the mechanism remains poorly understood. Here, through a comprehensive analysis of miR-124 and its gene targets, we demonstrate that, in the chordate ascidian Ciona intestinalis, miR-124 plays an extensive role in promoting nervous system development. We discovered that feedback interaction between miR-124 and Notch signaling regulates the epidermal-peripheral nervous system (PNS) fate choice in tail midline cells. Notch signaling silences miR-124 in epidermal midline cells, whereas in PNS midline cells miR-124 silences Notch, Neuralized and all three Ciona Hairy/Enhancer-of-Split genes. Furthermore, ectopic expression of miR-124 is sufficient to convert epidermal midline cells into PNS neurons, consistent with a role in modulating Notch signaling. More broadly, genome-wide target extraction with validation using an in vivo tissue-specific sensor assay indicates that miR-124 shapes neuronal progenitor fields by downregulating non-neural genes, notably the muscle specifier Macho-1 and 50 Brachyury- regulated notochord genes, as well as several anti-neural factors including SCP1 and PTBP1. 3'UTR conservation analysis reveals that miR-124 targeting of SCP1 is likely to have arisen as a shared, derived trait in the vertebrate/tunicate ancestor and targeting of PTBP1 is conserved among bilaterians except for ecdysozoans, while extensive Notch pathway targeting appears to be Ciona specific. Altogether, our results provide a comprehensive insight into the specific mechanisms by which miR-124 promotes neuronal development. KEY WORDS: miR-124, microRNA targets, Notch signaling, Macho-1, SCP1, Notochord, Ciona intestinalis, PTBP1 miR-124 function during Ciona intestinalis neuronal development includes extensive interaction with the Notch signaling pathway Jerry S. Chen 1,2 , Matthew San Pedro 1,2 and Robert W. Zeller 1,2,3, * DEVELOPMENT