Neurophysiological Defects and Neuronal Gene Deregulation in Drosophila mir-124 Mutants

Abstract

miR-124 is conserved in sequence and neuronal expression across the animal kingdom and is predicted to have hundreds of mRNA targets. Diverse defects in neural development and function were reported from miR-124 antisense studies in vertebrates, but a nematode knockout of mir-124 surprisingly lacked detectable phenotypes. To provide genetic insight from Drosophila, we deleted its single mir-124 locus and found that it is dispensable for gross aspects of neural specification and differentiation. On the other hand, we detected a variety of mutant phenotypes that were rescuable by a mir-124 genomic transgene, including short lifespan, increased dendrite variation, impaired larval locomotion, and aberrant synaptic release at the NMJ. These phenotypes reflect extensive requirements of miR-124 even under optimal culture conditions. Comparison of the transcriptomes of cells from wild-type and mir-124 mutant animals, purified on the basis of mir-124 promoter activity, revealed broad upregulation of direct miR-124 targets. However, in contrast to the proposed mutual exclusion model for miR-124 function, its functional targets were relatively highly expressed in miR-124–expressing cells and were not enriched in genes annotated with epidermal expression. A notable aspect of the direct miR-124 network was coordinate targeting of five positive components in the retrograde BMP signaling pathway, whose activation in neurons increases synaptic release at the NMJ, similar to mir-124 mutants. Derepression of the direct miR-124 target network also had many secondary effects, including over-activity of other post-transcriptional repressors and a net incomplete transition from a neuroblast to a neuronal gene expression signature. Altogether, these studies demonstrate complex consequences of miR-124 loss on neural gene expression and neurophysiology.

microRNA target prediction programs predict many false positives

“…5A and Supplemental Fig. S6; see also Farh et al [2005]; Sun et al [2012]). Note that the P CT compensates for 3 ′ UTR overall conservation (binding sites located in highly conserved UTRs have to be even more conserved themselves to attain the same P CT than binding sites in poorly conserved UTRs).…”

MicroRNAs: regulators of neuronal fate

“…While most loss-of-function and overexpression studies in vertebrates supports miR-124 as a promoter of neuronal differentiation and an inhibitor of progenitor self-renewal (discussed further below), independent reports that studied miR-124 knockout in Drosophila showed that it is grossly dispensable for neuronal differentiation, but rather required for proliferation of neural stem cells [34-36]. Several possibilities could underlie the apparent divergence in the function of miR-124.…”

“…Several possibilities could underlie the apparent divergence in the function of miR-124. Notably in flies miR-124 is expressed in proliferating neuroblasts, whereas in vertebrates, its expression starts when double-cortin positive neurons appear [34,35,37,38]. Similarly, in human embryonic stem cell to neuron differentiation system, miR-124 expression was largely restricted to differentiated neurons [39].…”

Can the ‘neuron theory’ be complemented by a universal mechanism for generic neuronal differentiation

“…Somewhat surprisingly, however, mutational inactivation in C. elegans (Miska et al 2007) and D. melanogaster (Weng and Cohen 2012;Sun et al 2012), which in contrast to mammals possess only one gene coding for miR-124, leaves homozygous mutants viable and without gross abnormalities. In Drosophila, a range of mutant phenotypes can be found, notably compromised of neuronal progenitor proliferation with a decreased number of Elav-positive cells per type I neuroblast-derived clone (Weng and Cohen 2012), and incomplete transition from neuroblast to neuronal gene expression signature (Sun et al 2012), but no complete failure of neuron generation.…”

“…In Drosophila, a range of mutant phenotypes can be found, notably compromised of neuronal progenitor proliferation with a decreased number of Elav-positive cells per type I neuroblast-derived clone (Weng and Cohen 2012), and incomplete transition from neuroblast to neuronal gene expression signature (Sun et al 2012), but no complete failure of neuron generation. miR-9: a critical link to vertebrate proneural gene function Similar to miR-124, miR-9 is initially identified as a brainenriched microRNA (see Coolen et al 2013 for review).…”