<em>MicroRNA-9a</em> ensures the precise specification of sensory organ precursors in<em>Drosophila</em>
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MicroRNAs (miRNAs) have been implicated in regulating various aspects of animal development, but their functions in neurogenesis are largely unknown. Here we report that loss of miR-9a function in the Drosophila peripheral nervous system leads to ectopic production of sensory organ precursors (SOPs), whereas overexpression of miR-9a results in a severe loss of SOPs. We further demonstrate a strong genetic interaction between miR-9a and senseless (sens) in controlling the formation of SOPs in the adult wing imaginal disc. Moreover, miR-9a suppresses Sens expression through its 3′ untranslated region. miR-9a is expressed in epithelial cells, including those adjacent to SOPs within proneural clusters, suggesting that miR-9a normally inhibits neuronal fate in non-SOP cells by down-regulating Sens expression. These results indicate that miR-9a ensures the generation of the precise number of neuronal precursor cells during development.
The peripheral nervous system (PNS) is essential for animals to detect and relay environmental stimuli to central neurons for information processing. It is largely unknown what ensures the development of precise numbers of sensory organs for a particular external sensory cue. The Drosophila PNS has been used as an excellent model system for dissecting the genetic programs that control sensory organ formation (Ghysen and Dambly Chaudiere 1993; Jan and Jan 1993; Modolell 1997). In embryonic abdominal segments, external sensory (ES) organs and chordotonal (CH) organs contain single-dendrite neurons associated with support cells and function in receiving mechanical stimuli (Campos-Ortega and Hartenstein 1985; Ghysen et al. 1986). In contrast, multidendritic (MD) neurons elaborate highly branched dendrites underneath the epidermis (Bodmer and Jan 1987; Gao et al. 1999) that probably function as stretch, touch, or other sensory receptors (Ainsley et al. 2003; Liu et al. 2003; Tracey et al. 2003). In adult flies, most external sensory organs have one neuron, one hair cell, and a few support cells (Campos-Ortega and Hartenstein 1985).
Precise numbers of sensory organs are generated through similar developmental processes in embryos and adults (Campos-Ortega and Hartenstein 1985). For example, in embryonic/larval abdominal segments, each dorsal cluster contains four ES organs and eight MD neurons. Some ES neurons are generated from sensory organ precursors (SOPs) that also produce MD neurons (ES– MD lineage), while other lineages produce ES but not MD neurons (Brewster and Bodmer 1995; Vervoort et al. 1997). Adult flies have four macrochaetes on the notum and a well-defined number of sensory bristles on the wing margin. All cells in each adult sensillium are generated from a single SOP in two or three rounds of asymmetric cell division (Hartenstein and Posakony 1989; Bardin et al. 2004).
The selection of SOPs from early ectoderm begins with the proneural cluster, which consists of a small number of cells that express proneural genes encoding the basic helix–loop–helix (bHLH) proteins (Achaete, Scute, Asense, Atonal, and Amos), rendering those cells competent to develop into SOPs (Romani et al. 1989; Cubas et al. 1991; Skeath and Carroll 1991; Jarman et al. 1993; Ruiz-Gomez and Ghysen 1993; Goulding et al. 2000; Huang et al. 2000). It is thought that the cells with the highest level of proneural proteins are selected as SOPs, which express a higher level of Delta and activate Notch in neighboring cells (Goriely et al. 1991; Artavanis-Tsakonas et al. 1999). Activation of Notch initiates a signaling cascade in which Suppressor of hairless [Su(H)] and Enhancer of split [E(spl)] complexes are involved to suppress neuronal fate in non-SOP cells (Knust et al. 1992; Schweisguth and Posakony 1992).
One of the genes activated by bHLH proteins, senseless (sens), encodes a transcription factor with four zinc fingers whose expression is dynamically regulated within the proneural clusters (Nolo et al. 2000). Within the proneural cluster, a high level of Sens is required to up-regulate and maintain the proneural gene expression in SOPs (Nolo et al. 2000), while a low level of Sens represses the transcription of proneural genes in adjacent cells (Jafar-Nejad et al. 2003). This dual function of Sens as an activator or suppressor suggests that the proper level of Sens is required to control the formation of the precise number of SOPs. However, how differential expression levels of Sens in SOPs and adjacent cells are ensured remains unknown.
In recent years, post-transcriptional regulation by microRNAs (miRNAs) has emerged as an important mechanism for controlling gene expression in animal development (Carrington and Ambros 2003; Bartel 2004; He and Hannon 2004; Alvarez-Garcia and Miska 2005; Carthew 2006). miRNAs are endogenous noncoding small RNAs 21–23 nucleotides (nt) in length. Several miRNAs have been implicated in nervous system development, with roles ranging from regulating left/right neuronal asymmetry in Caenorhabditis elegans, photo-receptor formation in Drosophila, and brain morphogenesis in zebrafish, to neuronal differentiation in mammals (Johnston and Hobert 2003; Chang et al. 2004; Giraldez et al. 2005; Li and Carthew 2005; Vo et al. 2005; Krichevsky et al. 2006; Schratt et al. 2006). However, the precise roles of miRNAs in early neurogenesis have not been studied yet. Moreover, only a few miRNAs have been studied using loss-of-function approaches in Drosophila (e.g., Brennecke et al. 2003; Xu et al. 2003; Kwon et al. 2005; Li and Carthew 2005; Sokol and Ambros 2005; Teleman et al. 2006).
Here, through both loss-of-function and gain-of-function in vivo analyses, we found that miR-9a is required to ensure the generation of precise numbers of sensory organs in Drosophila embryos and adults. To accomplish this regulatory function, miR-9a down-regulates the expression of Sens through its 3′ untranslated region (UTR) to ensure the differential expression of Sens in SOPs and adjacent epithelial cells. These findings provide new evidence that miRNAs function at the translational level to ensure the appropriate level of gene expression during different developmental processes.
We thank H. Bellen, the Bloomington Stock Center, and the Developmental Hybridoma Bank for reagents and fly lines. We thank S. Ordway and G. Howard for editorial assistance, K. Nyuyen for manuscript preparation, and L.-P. Chang for help with the UAS–pre-miR-9a construct. We also thank T. Kornberg, Y. Hong, and laboratory members for discussions and comments during the course of this work. This study was supported by an NIH training grant (T32 AG00278 to F.W.), the Korea Research Foundation (KRF: M01-2005-000-10157-0 to J.L.), and the Esther A. and Joseph Klingenstein Fund, the McKnight Endowment Fund for Neuroscience, FRAXA Foundation, Pfizer/ AFAR, and the NIH (to F.-B.G.).
Supplemental material is available at http://www.genesdev.org.
Article published online ahead of print. Article and publication date are online at http://www.genesdev.org/cgi/doi/10.1101/gad.1466306.