MicroRNAs Establish Uniform Traits during the Architecture of Vertebrate Embryos.

Proper functioning of an organism requires cells and tissues to behave in uniform, well-organized ways. How this optimum of phenotypes is achieved during the development of vertebrates is unclear. Here, we carried out a multi-faceted and single-cell resolution screen of zebrafish embryonic blood vessels upon mutagenesis of single and multi-gene microRNA (miRNA) families. We found that embryos lacking particular miRNA-dependent signaling pathways develop a vascular trait similar to wild-type, but with a profound increase in phenotypic heterogeneity. Aberrant trait variance in miRNA mutant embryos uniquely sensitizes their vascular system to environmental perturbations. We discovered a previously unrecognized role for specific vertebrate miRNAs to protect tissue development against phenotypic variability. This discovery marks an important advance in our comprehension of how miRNAs function in the development of higher organisms.

In vivo mutagenesis of miRNA gene families using a scalable multiplexed CRISPR/Cas9 nuclease system.

A large number of microRNAs (miRNAs) are grouped into families derived from the same phylogenetic ancestors. miRNAs within a family often share the same physiological functions despite differences in their primary sequences, secondary structures, or chromosomal locations. Consequently, the generation of animal models to analyze the activity of miRNA families is extremely challenging. Using zebrafish as a model system, we successfully provide experimental evidence that a large number of miRNAs can be simultaneously mutated to abrogate the activity of an entire miRNA family. We show that injection of the Cas9 nuclease and two, four, ten, and up to twenty-four multiplexed single guide RNAs (sgRNAs) can induce mutations in 90% of the miRNA genomic sequences analyzed. We performed a survey of these 45 mutations in 10 miRNA genes, analyzing the impact of our mutagenesis strategy on the processing of each miRNA both computationally and in vivo. Our results offer an effective approach to mutate and study the activity of miRNA families and pave the way for further analysis on the function of complex miRNA families in higher multicellular organisms.

A Dicer-miR-107 Interaction Regulates Biogenesis of Specific miRNAs Crucial for Neurogenesis.

Dicer controls the biogenesis of microRNAs (miRNAs) and is essential for neurogenesis. Recent reports show that the levels and substrate selectivity of DICER result in the preferential biogenesis of specific miRNAs in vitro. However, how dicer expression levels and miRNA biogenesis are regulated in vivo and how this affects neurogenesis is incompletely understood. Here we show that during zebrafish hindbrain development dicer expression levels are controlled by miR-107 to tune the biogenesis of specific miRNAs, such as miR-9, whose levels regulate neurogenesis. Loss of miR-107 function stabilizes dicer levels and miR-9 biogenesis across the ventricular hindbrain zone, resulting in an increase of both proliferating progenitors and postmitotic neurons. miR-9 ectopic accumulation in differentiating neuronal cells recapitulated the excessive neurogenesis phenotype. We propose that miR-107 modulation of dicer levels in differentiating neuronal cells is required to maintain the homeostatic levels of specific miRNAs, whose precise accumulation is essential for neurogenesis.