Abstract
The specification of motor neuron (MN) subtypes and columnar organization in developing spinal cord is controlled by multiple transcription factors. FoxP1 drives specification of lateral motor neuron (LMN) subtypes, and we demonstrated in our previous work that FoxP1 expression levels are regulated by the microRNA miR-9. Here we show that ectopic FoxP1 expression in the chick spinal cord can rescue Lhx3 and Hb9 expression in MNs altered by miR-9 over-expression, demonstrating that FoxP1 is a critical functional interaction partner for miR-9 in LMN development. Moreover, we have optimized a technique called a miRNA sponge in vitro, to permit easy discovery of the role of individual miRNA in vivo using a loss-of-function approach. We here show that narrow spacing between binding sites, inclusion of a coding gene, and optimizing the number of miRNA binding sites can significantly increase the blocking ability of a sponge. We go on to show that a miR-9 sponge reduces detectable miR-9 in the ventral horn, preventing miR-9 silencing of FoxP1 in vivo, and in turn modifies MN subtypes in the spinal cord. Our designs for optimized sponges provide a knockdown tool that is ready to be used to study the function of miRNA in vivo, and in particular for generating transgenic animal models.
Highlights
In developing spinal cords, motor neurons (MNs) are specified from progenitors under the control of cross-interactions of multiple transcription factors (Jessell, 2000; Goulding, 2009)
Our own work indicates that microRNA miR-9 plays a role in optimizing FoxP1 expression levels and subsequently modifying MN subtypes (Otaegi et al, 2011)
FoxP1 IS A SPECIFIC TARGET FOR miR-9 IN MOTOR NEURON DEVELOPMENT Our previous work showed that miR-9 regulates MN subtypes by targeting chick FoxP1 via two miR-9 binding sites in its miR-9 sponge-28 nt miR-9 sponge-51 nt miR-9 sponge-64 nt Forward: gacACTAGTtcatacagctagtgaccaaagaGAATATtcatacagctagtgaccaaagaTCTAGAcag Reverse: ctgTCTAGAtctttggtcactagctgtatgaATATTCtctttggtcactagctgtatgaACTAGTgtc Forward: gacACTAGTtcatacagctagtgaccaaagaGTTCAGTAATTTCCAAACTCAGAATATAAtctagaCAG Reverse: CTGtctagaTTATATTCTGAGTTTGGAAATTACTGAACtctttggtcactagctgtatgaACTAGTgtc Forward: gacACTAGTtcatacagctagtgaccaaagaGAAACATCATAGTATTGCTCGTAATTGGAGATATATTTAACAtctagaGAC Reverse: GTCtctagaTGTTAAATATATCTCCAATTACGAGCAATACTATGATGTTTCtctttggtcactagctgtatgaACTAGTgtc
Summary
Motor neurons (MNs) are specified from progenitors under the control of cross-interactions of multiple transcription factors (Jessell, 2000; Goulding, 2009). Medial motor column (MMC) neurons innervate axial muscles, and lateral motor columns (LMC) innervate limb muscles (Dasen et al, 2003, 2005; Shah et al, 2004; Wu et al, 2008; Jung et al, 2010). Previous studies have shown that proper expression levels of FoxP1 are critical for determining LMC or MMC MN fate (Dasen et al, 2008; Rousso et al, 2008). Our own work indicates that microRNA (miRNA) miR-9 plays a role in optimizing FoxP1 expression levels and subsequently modifying MN subtypes (Otaegi et al, 2011). Emerging studies have shown that miRNAs have a broad impact on embryogenesis (Huang et al, 2011; Suh and Blelloch, 2011), tumor formation (Esquela-Kerscher and Slack, 2006; Zhang et al, 2007), and are implicated in numerous human diseases (Huang et al, 2011)
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