Haplo-insufficiency of the GATA3 gene causes hypoparathyroidism, sensorineural hearing loss, and renal disease (HDR) syndrome. Previous studies have shown that Gata3 is required for the development of the prosensory domain and spiral ganglion neurons (SGNs) of the mouse cochlea during embryogenesis. However, its role in supporting cells (SCs) after cell fate specification is largely unknown. In this study, we used tamoxifen-inducible Sox2CreERT2 mice to delete Gata3 in SCs of the neonatal mouse cochlea and showed that loss of Gata3 resulted in the proliferation of SCs, including the inner pillar cells (IPCs), inner border cells (IBCs), and lateral greater epithelium ridge (GER). In addition, loss of Gata3 resulted in the down-regulation of p27kip1, a cell cycle inhibitor, in the SCs of Gata3-CKO neonatal cochleae. Chromatin immunoprecipitation analysis revealed that GATA3 directly binds to p27kip1 promoter and could maintain the quiescent state of cochlear SCs by regulating p27kip1 expression. Furthermore, RNA-seq analysis revealed that loss of Gata3 function resulted in the change in the expression of genes essential for the development and function of cochlear SCs, including Tectb, Cyp26b1, Slitrk6, Ano1, and Aqp4.

Background Mesenchymal stem cells (MSCs) transplantation is a potential clinical therapy for cerebral ischemia. The therapeutic effects of MSCs primarily depends on the paracrine action by releasing exosomes (Exos). Exosomes derived from bone marrow mesenchymal stem cells (BMSC-Exos) could modulate target cell functions by transferring microRNAs (miRs) cargo. In this study, we aimed to investigate whether BMSC-Exos could promote angiogenesis via transfer of miR-21-5p after cerebral ischemia. Methods BMSC-Exos were isolated from conditioned medium of BMSCs by differential ultracentrifugation, and confirmed by transmission electron microscopy, nanoparticle tracking analysis, and western blot analysis. In mice with middle cerebral artery occlusion (MCAO), the neurological function was evaluated by Zea Longa’s method, and the infarct volume and microvessel density were detected by TTC staining and vWF immunofluorescence staining, respectively. The proangiogenic effects of BMSC-Exos were assessed via proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs) in vitro assays. The miR-21-5p expression was detected by qRT-PCR. The expression levels of VEGF, VEGFR2, Ang-1, and Tie-2 were determined by western blot. Results BMSC-Exos significantly improved neurological function and reduced infract volume after cerebral ischemia. Moreover, BMSC-Exos significantly upregulated the microvessel density and the expression levels of proangiogenic proteins VEGF, VEGFR2, Ang-1 and Tie-2 in the ischemic boundary region. MiR-21-5p expression was also dramatically increased after cerebral ischemia. In vitro assays revealed that BMSC-Exos enhanced HUVECs functions including proliferation, migration and tube formation, as well as increasing the expression of VEGF and VEGFR2. However, these proangiogenic effects of BMSC-Exos on HUVECs were reversed by miR-21-5p inhibitor. Conclusion Our study indicated that BMSC-Exos could promote angiogenesis and neurological function recovery via transfer of miR-21-5p. Therefore, the application of miR-21-5p-loaded BMSC-Exos might be an attractive treatment strategy of cerebral ischemia.

Tissue-specific inducible Cre recombinase mouse lines allow precise genetic manipulations in spatiotemporal manners and are pivotal for functional studies of genes during development and in adults. Growth factor independence 1 (GFI1) is an essential transcription factor expressed in the hair cells of mouse inner ear and Gfi1 locus serves as an excellent anchor site to drive the expression of inducible Cre recombinase in mouse inner hair cells. In this study, we have generated Gfi1-P2A-GFP-CreERT2 (Gfi1-GCE) knock-in mouse line by in-frame fusion of a selfcleaving GCE to the C-terminus of GFI1. We have shown that as predicted, the expression of GCE and GFI1 was detected specifically in the cytosol and nuclei of hair cells, respectively, of uninduced Gfi1-GCE mice, suggesting the successful cleavage and simultaneous expression of GFI1 and GCE. In addition, the in-frame fusion of the self-cleaving GCE does not interrupt the function of Gfi1 in the inner ear. Administration of tamoxifen leads to nuclear translocation of GCE and results in an efficient activation of tdTomato reporter gene expression specifically in most hair cells throughout development and in adults. Thus, this inducible Gfi1-GCE mouse line is a highly efficient Cre deleter and is suitable for gene manipulation in developing and adult inner ear hair cells.

LHX4 is a LIM-homeodomain transcription factor essential for the development of spinal cord and pituitary gland. Mice with homozygous Lhx4-null mutation suffer early postnatal death from lung defect. In this study, to facilitate the research on Lhx4 function, we designed a targeting construct to generate two novel Lhx4 mouse lines:Lhx4 loxP conditional knockout and Lhx4 tdT reporter knock-in mice. Lhx4 tdT/+ , Lhx4 loxP/+ , and Lhx4 loxP/loxP were viable, fertile, and did not display any gross abnormalities.By breeding Lhx4 loxP line with Cre-expressing mice, the Exon 3 of Lhx4 was efficiently removed, resulting in a shift in the reading frame and the inactivation of Lhx4. The expression of tdTomato knock-in reporter recapitulated the endogenous LHX4 expression and was detected in the retina, spinal cord, pituitary gland, and hindbrain of Lhx4 tdT mice. Thus, Lhx4 tdT and Lhx4 loxP mouse lines provide valuable tools for unraveling the tissue-specific role of Lhx4 at postnatal stages in mice. K E Y W O R D S homeobox, LIM-homeodomain, pituitary gland, retina, spinal cord