Establishment And Functional Studies Of FoxP2Gene Knockdown Bat Model By Lentivirus&Analysis Of Small RNA Transcriptome Of Miniopterus Fuliginosus

1. Forkhead box P2(FoxP2) is a member of the winged helix/forkhead class of transcription factors. Mutations of the FoxP2gene in human cause severe speech and language disorders. FoxP2is also found to be connected to song learning and adult song stability in songbird. Recently it has been shown that it underwent accelerated evolution in bats and may play a role in the evolution and development of echo location. To figure out the relevance between FoxP2and the complex high frequency vocal behavior of bat, we studied the difference of FoxP2expression in three echolocating bats(Hipposideros armiger, Rhinolophus ferrumequinum, and Myotis ricketti) and two non-echo locating bats (Rousettus leschenaultii and Cynopterus sphinx) at both mRNA and protein level, using in situ hybridization and immunohistochemistry. The results showed that the region of anterior cingulate cortex (ACC) exhibits a different FoxP2expression between the two kinds of bats. Then we report the loss of function study of FoxP2gene in bat brain tissue for the first time, based on RNAi mediated by lentivirus. We designed three shRNAs which can express short hairpin RNA (shRNA) targeting Hipposideros armiger FoxP2, one of the echolocating bats which can produce high frequency sound and perform Doppler-shift compensation (DSC). One of the three shRNAs was demonstrated to knock down FoxP2fusion protein efficiently in293T cell line. Then the pseudovirus particles that contain interference sequence were administered via stereotactic intracerebral microinjection into the anterior cingulate cortex of Hipposideros armiger to construct a FoxP2knockdown bat model. We examined the vocal behavior of FoxP2knockdown animals and found that they can still perform normal high frequency sound but the pulse interval at rest increased. Besides, knockdown of FoxP2expression in the ACC significantly altered DSC by decreasing the max compensation depth and increasing the pulse interval. Overall, the results suggest the expression of FoxP2 in ACC of Hipposideros armiger may play an important role in the precise, rapid audio-vocal integration and coordination.2. MicroRNAs (miRNAs) are a class of endogenous small noncoding RNAs that widely exist in eukaryotic organism and play a key role in diverse biological processes. Mature miRNAs are generated through two sequential cleavages of the primary transcripts by Drosha and Dicer. This kind of small RNAs can integrate into RNA-induced silence complex (RISC) and regulate the target gene at post-transcriptional level either by inhibiting the mRNA translation or degrading the target gene directly according to the extent of base pairing. miRNA is a kind of fine-tuning RNA molecule that can regulate various functions. Recently, the solexa sequencing technology has become a convincing strategy for identifying miRNA transcriptome, more and more miRNAs were identified in different species. However, the miRNA informations of Chiroptera were still unknown. In this study, we constructed and sequenced a miRNA library from Miniopterus fuliginosus. We identified229miRNA precursors and338mature miRNAs (conserved miRNA) after aligning the sequencing data with known miRNAs registered in miRBase and the genome information of Myotis lucifugus and Pteropus vampyrus, two relatives of Miniopterus fuliginosus. We also discovered33bat specific novel miRNA precursors. Then we deeply analyzed the characteristics and variations of the conserved miRNAs. Among the total conserved miRNAs,14mature miRNAs have undergone seed shifting and27miRNAs have sequence changes (mutation, insert or delete). According to the genome loci of these miRNAs, we found90miRNAs distributed as gene clusters, belonging to26miRNA clusters. The arm usage was investigated and the results showed that5miRNAs occurred arm switching and7miRNAs had different arm usage compared to other vertebrates but failed to have any effect due to the mature miRNA sequences are identical ("nonsense switching").In addition, the expression patterns of miRNAs in embryo and adult brain were also investigated using comprehensive microarray profiling. Finally, we found51miRNAs showed a divergent expression pattern, which implied these miRNAs may play a role in brain development. The identification of miRNA transcriptome of Miniopterus fuliginosus definitely advances our knowledge of miRNA populations of bat and more importantly it would provide a clue for better understanding of their roles in brain development.