Conditional Deletion Of Dicer Research Articles

Conditional Deletion of Dicer in Adult Mice Impairs Skeletal Muscle Regeneration.

Skeletal muscle has a remarkable regenerative capacity, which is orchestrated by multiple processes, including the proliferation, fusion, and differentiation of the resident stem cells in muscle. MicroRNAs (miRNAs) are small noncoding RNAs that mediate the translational repression or degradation of mRNA to regulate diverse biological functions. Previous studies have suggested that several miRNAs play important roles in myoblast proliferation and differentiation in vitro. However, their potential roles in skeletal muscle regeneration in vivo have not been fully established. In this study, we generated a mouse in which the Dicer gene, which encodes an enzyme essential in miRNA processing, was knocked out in a tamoxifen-inducible way (iDicer KO mouse) and determined its regenerative potential after cardiotoxin-induced acute muscle injury. Dicer mRNA expression was significantly reduced in the tibialis anterior muscle of the iDicer KO mice, whereas the expression of muscle-enriched miRNAs was only slightly reduced in the Dicer-deficient muscles. After cardiotoxin injection, the iDicer KO mice showed impaired muscle regeneration. We also demonstrated that the number of PAX7+ cells, cell proliferation, and the myogenic differentiation capacity of the primary myoblasts did not differ between the wild-type and the iDicer KO mice. Taken together, these data demonstrate that Dicer is a critical factor for muscle regeneration in vivo.

Dicer promotes endothelial recovery and limits lesion formation after vascular injury through miR-126-5p

BackgroundThe recovery of endothelial cells (ECs) after vascular injury is mainly mediated by the proliferation of resident ECs, thereby reducing neointima formation. The RNase Dicer processes microRNAs (miRNAs) and regulates EC function by controlling miRNA-mediated regulation of gene expression. This study aimed to investigate the impact of miRNA biogenesis in ECs on endothelial repair during lesion formation after vascular injury. Methods and resultsTo study the effect of Dicer on ECs during neointima formation, conditional deletion of Dicer was induced in Apoe−/− mice (EC-Dicerflox) by tamoxifen injection. Following wire-induced injury to carotid arteries of EC-Dicerflox mice, the EC recovery was impaired and the neointima formation and lesional macrophage accumulation was increased. Moreover, conditional deletion of Dicer in ECs diminished the expression of miR-126-5p in EC-Dicerflox mice. Notably, reconstitution of miR-126-5p in the injured arteries of EC-Dicerflox mice using miR-126-5p mimic, prevented the impaired endothelial recovery and increased lesion formation observed in EC-Dicerflox mice. ConclusionsDeficiency of endothelial Dicer diminished endothelial recovery and promoted neointima formation probably due to impaired miR-126-5p expression. Treatment with miR-126-5p mimics promotes endothelial recovery and thereby limits neointima formation. Thus, miR-126-5p therapy represents a potential approach to improve endothelial recovery and prevent restenosis following vascular injury.

Dicer generates a regulatory microRNA network in smooth muscle cells that limits neointima formation during vascular repair.

MicroRNAs (miRNAs) coordinate vascular repair by regulating injury-induced gene expression in vascular smooth muscle cells (SMCs) and promote the transition of SMCs from a contractile to a proliferating phenotype. However, the effect of miRNA expression in SMCs on neointima formation is unclear. Therefore, we studied the role of miRNA biogenesis by Dicer in SMCs in vascular repair. Following wire-induced injury to carotid arteries of Apolipoprotein E knockout (Apoe -/-) mice, miRNA microarray analysis revealed that the most significantly regulated miRNAs, such as miR-222 and miR-21-3p, were upregulated. Conditional deletion of Dicer in SMCs increased neointima formation by reducing SMC proliferation in Apoe -/- mice, and decreased mainly the expression of miRNAs, such as miR-147 and miR-100, which were not upregulated following vascular injury. SMC-specific deletion of Dicer promoted growth factor and inflammatory signaling and regulated a miRNA-target interaction network in injured arteries that was enriched in anti-proliferative miRNAs. The most connected miRNA in this network was miR-27a-3p [e.g., with Rho guanine nucleotide exchange factor 26 (ARHGEF26)], which was expressed in medial and neointimal SMCs in a Dicer-dependent manner. In vitro, miR-27a-3p suppresses ARHGEF26 expression and inhibits SMC proliferation by interacting with a conserved binding site in the 3' untranslated region of ARHGEF26 mRNA. We propose that Dicer expression in SMCs plays an essential role in vascular repair by generating anti-proliferative miRNAs, such as miR-27a-3p, to prevent vessel stenosis due to exaggerated neointima formation.

Abstract 146: Inducible Conditional Deletion of Dicer in Neural Progenitor Cells Induces Cognitive Impairment

Background: MicroRNAs regulate adult neurogenesis. Conditional deletion of Dicer in neural stem cells (NSCs) causes postnatal death. The functional role of Dicer in adult neurogenesis remains unknown. Using mice with inducible conditional knock-down of Dicer in adult NSCs, we investigated the effect of ablation of Dicer on neurogenesis and cognitive function. Methods and Results: Young adult Ascl1-CreER:Dicerflox/flox mice (Dicer Cko, n= 32) were administered (i.p) with tamoxifen daily for 5 consecutive days and age matched wild-type litters (n= 30) were used as control. The mice were sacrificed at 2, 14 or 30 days after injection. Immunstaining was performed to detect phenotypes of subventricular zone (SVZ) cells. An array of cognitive tests including Morris Water Maze, odor-based novelty recognition, and sociability test were performed. Primary NPCs were isolated from the SVZ for vitro studies. Compared to control, Dicer CKo had 54% reduction of Dicer protein in NPCs. Cognitive tests showed that CKo spent 35% less time in the correct quadrant (p<0.05) of Morris Water Maze, significantly reduced time exploring new odor objects, 44 ±10% (p<0.05) in Cko animals compared with 77 ±10% in control, and significantly less time with other mice when they encountered a strange mouse during the sociability test (70 ± 9 vs 118 ±7 seconds, p<0.05). CKo significantly (p<0.05) reduced BrdU+ (16±2% vs 24±3%), and Ki67+ NPCs (25±3% vs 33±3%), doublecortin (DCX)+ neuroblasts (2± 0.6% vs 6± 1%), and Ng2+ oligodendrocyte progenitor cells (OPCs, 8±1% vs 12± 2%) in the SVZ. However, Dicer CKo animals exhibited a significant increase (p<0.05) of apoptotic NPCs (14±0.5% vs 10 ±0.3%). These in vivo findings were consistent with data from cultured NPCs. Dicer CKo also showed significant (p<0.05) reduction DCX+ neuroblasts in the rostral migratory stream and APC+ mature oligodendrocytes in the corpus callosum. Conclusion: Our data demonstrated that inducible conditional ablation of Dicer in Ascl1 lineage NPCs impairs neurogenesis and oligodendrogenesis in adult SVZ niche and white matter, and induces cognitive impairment, indicating that Dicer in adult NPCs is essential for maintaining neurogenesis and oligodendrogenesis and is important for cognitive function.

MicroRNA-124 Regulates Cell Specification in the Cochlea through Modulation of Sfrp4/5.

The organ of Corti, the auditory organ of the mammalian inner ear, contains sensory hair cells and supporting cells that arise from a common sensory progenitor. The molecular bases allowing the specification of these progenitors remain elusive. In the present study, by combining microarray analyses with conditional deletion of Dicer in the developing inner ear, we identified that miR-124 controls cell fate in the developing organ of Corti. By targeting secreted frizzled-related protein 4 (Sfrp4) and Sfrp5, two inhibitors of the Wnt pathway, we showed that miR-124 controls the β-catenin-dependent and also the PCP-related non-canonical Wnt pathways that contribute to HC differentiation and polarization in the organ of Corti. Thus, our work emphasizes the importance of miR-124 as an epigenetic safeguard that fine-tunes the expression of genes critical for cell patterning during cochlear differentiation.

Oral Lining Mucosa Development Depends on Mesenchymal microRNAs

The oral mucosa plays critical roles in protection, sensation, and secretion and can be classified into masticatory, lining, and specialized mucosa that are known to be functionally, histologically, and clinically distinct. Each type of oral mucosa is believed to develop through discrete molecular mechanisms, which remain unclear. MicroRNAs (miRNAs) are 19 to 25nt non-coding small single-stranded RNAs that negatively regulate gene expression by binding target mRNAs. miRNAs are crucial for fine-tuning of molecular mechanisms. To investigate the role of miRNAs in oral mucosa development, we examined mice with mesenchymal (Wnt1Cre;Dicerfl/fl) conditional deletion of Dicer. Wnt1Cre;Dicerfl/fl mice showed trans-differentiation of lining mucosa into an epithelium with masticatory mucosa/ skin-specific characteristics. Up-regulation of Fgf signaling was found in mutant lining mucosal epithelium that was accompanied by an increase in Fgf7 expression in mutant mesenchyme. Mesenchyme miRNAs thus have an indirect effect on lining mucosal epithelial cell growth/differentiation.

Deletion of miRNA processing enzyme Dicer in POMC-expressing cells leads to pituitary dysfunction, neurodegeneration and development of obesity

MicroRNAs (miRNAs) have recently emerged as key regulators of metabolism. However, their potential role in the central regulation of whole-body energy homeostasis is still unknown. In this study we show that the expression of Dicer, an essential endoribonuclease for miRNA maturation, is modulated by nutrient availability and excess in the hypothalamus. Conditional deletion of Dicer in POMC-expressing cells resulted in obesity, characterized by hyperphagia, increased adiposity, hyperleptinemia, defective glucose metabolism and alterations in the pituitary-adrenal axis. The development of the obese phenotype was paralleled by a POMC neuron degenerative process that started around 3 weeks of age. Hypothalamic transcriptomic analysis in presymptomatic POMCDicerKO mice revealed the downregulation of genes implicated in biological pathways associated with classical neurodegenerative disorders, such as MAPK signaling, ubiquitin-proteosome system, autophagy and ribosome biosynthesis. Collectively, our results highlight a key role for miRNAs in POMC neuron survival and the consequent development of neurodegenerative obesity.

Distinct roles of MicroRNAs in epithelium and mesenchyme during tooth development

Tooth development is known to be mediated by the cross-talk between signaling pathways, including Shh, Fgf, Bmp, and Wnt. MicroRNAs (miRNAs) are 19- to 25-nt noncoding small single-stranded RNAs that negatively regulate gene expression by binding target mRNAs, which is believed to be important for the fine-tuning signaling pathways in development. To investigate the role of miRNAs in tooth development, we examined mice with either mesenchymal (Wnt1Cre/Dicer(fl/fl)) or epithelial (ShhCre/Dicer(fl/fl)) conditional deletion of Dicer, which is essential for miRNA processing. By using a CD1 genetic background for Wnt1Cre/Dicer(fl/fl), we were able to examine tooth development, because the mutants retained mandible and maxilla primordia. Wnt1Cre/Dicer(fl/fl) mice showed an arrest or absence of teeth development, which varied in frequency between incisors and molars. Extra incisor tooth formation was found in ShhCre/Dicer(fl/fl) mice, whereas molars showed no significant anomalies. Microarray and in situ hybridization analysis identified several miRNAs that showed differential expression between incisors and molars. In tooth development, miRNAs thus play different roles in epithelium and mesenchyme, and in incisors and molars.

Conditional deletion of Dicer in vascular smooth muscle cells leads to the developmental delay and embryonic mortality

Dicer is a RNAase III enzyme that cleaves double stranded RNA and generates small interfering RNA (siRNA) and microRNA (miRNA). The goal of this study is to examine the role of Dicer and miRNAs in vascular smooth muscle cells (VSMCs). We deleted Dicer in VSMCs of mice, which caused a developmental delay that manifested as early as embryonic day E12.5, leading to embryonic death between E14.5 and E15.5 due to extensive hemorrhage in the liver, brain, and skin. Dicer KO embryos showed dilated blood vessels and a disarray of vascular architecture between E14.5 and E15.5. VSMC proliferation was significantly inhibited in Dicer KOs. The expression of VSMC marker genes were significantly downregulated in Dicer cKO embryos. The vascular structure of the yolk sac and embryo in Dicer KOs was lost to an extent that no blood vessels could be identified after E15.5. Expression of most miRNAs examined was compromised in VSMCs of Dicer KO. Our results indicate that Dicer is required for vascular development and regulates vascular remodeling by modulating VSMC proliferation and differentiation.

Distinct Requirements of MicroRNAs in NK Cell Activation, Survival, and Function

MicroRNAs (miRNAs) are small noncoding RNAs that have recently emerged as critical regulators of gene expression within the immune system. In this study, we used mice with conditional deletion of Dicer and DiGeorge syndrome critical region 8 (Dgcr8) to dissect the roles of miRNAs in NK cell activation, survival, and function during viral infection. We developed a system for deletion of either Dicer or Dgcr8 in peripheral NK cells via drug-induced Cre activity. We found that Dicer- and Dgcr8-deficient NK cells were significantly impaired in survival and turnover, and had impaired function of the ITAM-containing activating NK cell receptors. We further demonstrated that both Dicer- and Dgcr8-dependent pathways were indispensable for the expansion of Ly49H(+) NK cells during mouse cytomegalovirus infection. Our data indicate similar phenotypes for Dicer- and Dgcr8-deficient NK cells, which strongly suggest that these processes are regulated by miRNAs. Thus, our findings indicate a critical role for miRNAs in controlling NK cell homeostasis and effector function, with implications for miRNAs regulating diverse aspects of NK cell biology.

The microRNA-processing enzyme dicer maintains juxtaglomerular cells.

Juxtaglomerular cells are highly specialized myoepithelioid granulated cells located in the glomerular afferent arterioles. These cells synthesize and release renin, which distinguishes them from other cells. How these cells maintain their identity, restricted localization, and fate is unknown and is fundamental to the control of BP and homeostasis of fluid and electrolytes. Because microRNAs may control cell fate via temporal and spatial gene regulation, we generated mice with a conditional deletion of Dicer, the RNase III endonuclease that produces mature microRNAs in cells of the renin lineage. Deletion of Dicer severely reduced the number of juxtaglomerular cells, decreased expression of the renin genes (Ren1 and Ren2), lowered plasma renin concentration, and decreased BP. As a consequence of the disappearance of renin-producing cells, the kidneys developed striking vascular abnormalities and prominent striped fibrosis. We conclude that microRNAs maintain the renin-producing juxtaglomerular cells and the morphologic integrity and function of the kidney.

Loss of MicroRNA Leads to Abnormal Function and Development of the Adult Mouse Uterus.

MicroRNAs (miRNA) are 19-22 nucleotide long, non-coding RNA molecules that post-transcriptionally regulate gene expression by base-pairing with the 3′untranslated region of complementary messenger RNA targets. To date, over 700 miRNAs have been identified in animal cells and have been reported to play a role in numerous vital processes, including: embryonic development, cellular differentiation and proliferation, and apoptosis. Our laboratory recently demonstrated that conditional deletion of Dicer in ovarian granulosa cells and derivatives of the Mullerian duct (i.e. oviduct, uterus, and cervix) by Cre/loxP recombination (Dicerfl/fl;Amhr2-Cre, Dicer-cKO) led to female infertility. This was shown to be the result of a severe oviductal developmental defect that prevented fertilized embryos from entering the uterus. While the uterus of Dicer-cKO females was also disrupted, as evidenced by shortened uterine horns and reduced numbers of uterine glands, its functional ability to establish and maintain pregnancy is not yet known. We first evaluated whether the uterus of pseudopregnant mice could undergo a decidual reaction. To test uterine decidualization, sesame oil was injected into one uterine horn of 3.5 day pseudopregnant mice, while the contralateral horn served as the non-injected control. We next completed an embryo transfer experiment, where 5 blastocysts collected from naturally mated CD-1 mice on day 3.5 (day 0.5 = plug) were transferred into each uterine horn of day 2.5 pseudopregnant adult female Dicer-cKO (n=13) and wild-type mice (Dicerfl/fl or Dicerfl/wt littermates lacking the Amhr2-Cre allele, n=7). Recipient females were then sacrificed on day 7 of pregnancy and the number of implantation sites per mouse recorded. Results of the decidualization experiment indicated that uteri of Dicer-cKO mice (n=11) failed to exhibit a normal decidualization response when compared to wild-type mice (n=7), which exhibited a 3.6-fold increase (p<0.05) in uterine wet weight (mg) after body weight (g) correction. The uteri were 11.8 ± 4.3 mg and 19.7 ± 3.8 mg for the non-injected and 15.0 ± 6.1 mg and 70.8 ± 28.0 mg for sesame oil injected, for the Dicer cKO and wild-type mice, respectively. In the embryo transfer experiment, implantation occurred in 4 of 13 Dicer-cKO recipient females. Interestingly, all implantation sites in the pregnant Dicer-cKO mice were located at the cervical end of the uterine horns. Implantation sites in Dicer-cKO mice appeared less vascular, as evidenced by a blanched color in comparison to the dark red implantation sites observed in wild-type mice. Embryos failed to space appropriately in the two Dicer-cKO recipients with multiple embryos. Histological analysis, however, indicated that the uteri of the pregnant Dicer-cKO mice were able to mount what appeared to be a normal decidualization response with normal embryonic development. We next examined adult female Dicer-cKO uterine tissue histology at 60, 180, 230, and 330 days of age. Uteri from Dicer-cKO mice exhibited adenomyosis, a condition of abnormal endometrial growth within the myometrial layer, which progressively worsened as these animals aged. In conclusion, uterine function in Dicer-cKO mice was compromised and miRNA mediated post-transcriptional gene regulation appears to play a significant role in normal uterine development and function. (poster)