Primary microRNA cleavage by the Drosha-Dgcr8 'Microprocessor' complex is critical for microRNA biogenesis. Yet, the Microprocessor may also cleave other nuclear RNAs in a nonspecific manner. We studied Microprocessor function using mathematical modeling and experiments in mouse and human tissues. We found that the autoregulatory feedback on Microprocessor expression is instrumental for balancing the efficiency and specificity of its activity by effectively tuning Microprocessor levels to those of its pri-miRNA substrate.

DiGeorge syndrome (DGS), characterized genetically by a deletion within chromosome 22q11.2, is associated with a constellation of congenital heart defects. DiGeorge critical region 8 (Dgcr8), a gene that maps to the common deletion region for DGS, encodes a double stranded RNA-binding protein that is essential for miRNA biogenesis. To address the potential contribution of Dgcr8 insufficiency to cardiovascular development, we have inactivated Dgcr8 in cardiac neural crest cells (cNCCs). Dgcr8 mutants displayed a wide spectrum of malformations, including persistent truncus arteriosus (PTA) and ventricular septal defect (VSD). Interestingly, Dgcr8-null cNCCs that properly migrated into the cardiac outflow tract (OFT) proliferate normally and differentiate into vascular smooth muscle cells. However, loss of Dgcr8 causes a significant portion of the cNCCs to undergo apoptosis, causing a decrease in the pool of progenitors required for OFT remodeling. Our data uncover a new role of Dgcr8 in cardiovascular morphogenesis, plausibly as part of transmission mechanism for FGF-dependent survival cue for migrating cNCCs.

The etiology and pathophysiology of anxiety and mood disorders is linked to inappropriate regulation of the central stress response. To determine whether microRNAs have a functional role in the regulation of the stress response, we inactivated microRNA processing by a lentiviral-induced local ablation of the Dicer gene in the central amygdala (CeA) of adult mice. CeA Dicer ablation induced a robust increase in anxiety-like behavior, whereas manipulated neurons survive and appear to exhibit normal gross morphology in the time period examined. We also observed that acute stress in wild-type mice induced a differential expression profile of microRNAs in the amygdala. Bioinformatic analysis identified putative gene targets for these stress-responsive microRNAs, some of which are known to be associated with stress. One of the prominent stress-induced microRNAs found in this screen, miR-34c, was further confirmed to be upregulated after acute and chronic stressful challenge and downregulated in Dicer ablated cells. Lentivirally mediated overexpression of miR34c specifically within the adult CeA induced anxiolytic behavior after challenge. Of particular interest, one of the miR-34c targets is the stress-related corticotropin releasing factor receptor type 1 (CRFR1) mRNA, regulated via a single evolutionary conserved seed complementary site on its 3' UTR. Additional in vitro studies demonstrated that miR-34c reduces the responsiveness of cells to CRF in neuronal cells endogenously expressing CRFR1. Our results suggest a physiological role for microRNAs in regulating the central stress response and position them as potential targets for treatment of stress-related disorders.

The patterning of multicellular organisms is robust to environmental, genetic, or stochastic fluctuations. Mathematical modeling is instrumental in identifying mechanisms supporting this robustness. The principle of lateral inhibition, whereby a differentiating cell inhibits its neighbors from adopting the same fate, is frequently used for selecting a single cell out of a cluster of equipotent cells. For example, Sensory Organ Precursors (SOP) in the fruit-fly Drosophila implement lateral inhibition by activating the Notch-Delta pathway. We discuss parameters affecting the rate of errors in this process, and the mechanism (inhibitory cis interaction between Notch and Delta) predicted to reduce this error.

The specific program that enables the stereotypic differentiation of specialized cartilages, including the trachea, must be intrinsically distinct from the program that gives rise to growth plate hypertrophic chondrocytes. For example, Snail1 is an effector of FGF signaling in growth plate pre-hypertrophic chondrocytes, but it derails the normal program of permanent chondrocytes, repressing the transcription of Aggrecan and Collagen type 2a1 (Col2a1). Here we show that miRNA activity is essential for normal trachea development and that miR-125b and miR-30a/c keep Snail1 at low levels, thus enabling full functional differentiation of Col2a1 tracheal chondrocytes. Specific inhibition of miR-125b and miR-30a/c in chondrocytes or Dicer1 knockout in the trachea, de-repress Snail1. As a consequence, the transcription of Aggrecan and Col2a1 is hampered and extracellular matrix deposition is decreased. Our data reveals a new miRNA pathway that is safekeeping the specific genetic program of differentiated and matrix-producing tracheal chondrocytes from acquisition of unwanted signals. This pathway may improve understanding of human primary tracheomalacia and improve protocols for cartilage tissue engineering.

Commitment of progenitors in the dermomyotome to myoblast fate is the first step in establishing the body musculature. Pax3 is a crucial transcription factor, important for skeletal muscle development and expressed in myogenic progenitors in the dermomyotome of developing somites and in migratory muscle progenitors that populate the limb buds. Down-regulation of Pax3 is essential to ignite the myogenic program, including up-regulation of myogenic regulators, Myf-5 and MyoD. MicroRNAs (miRNAs) confer robustness to developmental timing by posttranscriptional repression of genetic programs that are related to previous developmental stages or to alternative cell fates. Here we demonstrate that the muscle-specific miRNAs miR-1 and miR-206 directly target Pax3. Antagomir-mediated inhibition of miR-1/miR-206 led to delayed myogenic differentiation in developing somites, as shown by transient loss of myogenin expression. This correlated with increased Pax3 and was phenocopied using Pax3-specific target protectors. Loss of myogenin after antagomir injection was rescued by Pax3 knockdown using a splice morpholino, suggesting that miR-1/miR-206 control somite myogenesis primarily through interactions with Pax3. Our studies reveal an important role for miR-1/miR-206 in providing precision to the timing of somite myogenesis. We propose that posttranscriptional control of Pax3 downstream of miR-1/miR-206 is required to stabilize myoblast commitment and subsequent differentiation. Given that mutually exclusive expression of miRNAs and their targets is a prevailing theme in development, our findings suggest that miRNA may provide a general mechanism for the unequivocal commitment underlying stem cell differentiation.

HASH(0x8e04ca0)

Here, we detail a protocol to design and introduce sequence-specific cholesterol-conjugated antisense oligonucleotides into mouse organ culture. We review design principles for "antagomirs", antisense oligos with a cholesterol-moiety modification at the 3', and present an optimized method to apply them onto 3D cultured embryonic pancreas. The method offers an approach to study the developmental functions of individual miRNAs and to evaluate miRNA targets, which is significantly faster and simpler than comparable genetics-based approaches.

MicroRNAs (miRNAs) were shown to be important for pancreas development, yet their roles in differentiated β-cells remain unclear. Here, we show that miRNA inactivation in β-cells of adult mice results in a striking diabetic phenotype. While islet architecture is intact and differentiation markers are maintained, Dicer1-deficient β-cells show a dramatic decrease in insulin content and insulin mRNA. As a consequence of the change in insulin content, the animals become diabetic. We provide evidence for involvement of a set of miRNAs in regulating insulin synthesis. The specific knockdown of miR-24, miR-26, miR-182 or miR-148 in cultured β-cells or in isolated primary islets downregulates insulin promoter activity and insulin mRNA levels. Further, miRNA-dependent regulation of insulin expression is associated with upregulation of transcriptional repressors, including Bhlhe22 and Sox6. Thus, miRNAs in the adult pancreas act in a new network that reinforces insulin expression by reducing the expression of insulin transcriptional repressors.