The Dlx5 and Foxg1 transcription factors, linked via miRNA-9 and -200, are required for the development of the olfactory and GnRH system

Abstract

During neuronal development and maturation, microRNAs (miRs) play diverse functions ranging from early patterning, proliferation and commitment to differentiation, survival, homeostasis, activity and plasticity of more mature and adult neurons. The role of miRs in the differentiation of olfactory receptor neurons (ORNs) is emerging from the conditional inactivation of Dicer in immature ORN, and the depletion of all mature miRs in this system. Here, we identify specific miRs involved in olfactory development, by focusing on mice null for Dlx5, a homeogene essential for both ORN differentiation and axon guidance and connectivity. Analysis of miR expression in Dlx5−/− olfactory epithelium pointed to reduced levels of miR-9, miR-376a and four miRs of the -200 class in the absence of Dlx5. To functionally examine the role of these miRs, we depleted miR-9 and miR-200 class in reporter zebrafish embryos and observed delayed ORN differentiation, altered axonal trajectory/targeting, and altered genesis and position of olfactory-associated GnRH neurons, i.e. a phenotype known as Kallmann syndrome in humans. miR-9 and miR-200-class negatively control Foxg1 mRNA, a fork-head transcription factor essential for development of the olfactory epithelium and of the forebrain, known to maintain progenitors in a stem state. Increased levels of z-foxg1 mRNA resulted in delayed ORN differentiation and altered axon trajectory, in zebrafish embryos. This work describes for the first time the role of specific miR (-9 and -200) in olfactory/GnRH development, and uncovers a Dlx5–Foxg1 regulation whose alteration affects receptor neuron differentiation, axonal targeting, GnRH neuron development, the hallmarks of the Kallmann syndrome.

Generation and analysis of an improved Foxg1-IRES-Cre driver mouse line

“…This targeting strategy was designed to generate a bicistronic messenger RNA (mRNAs), encoding both endogenous Foxg1 and the transgenic Cre . Moreover, the targeting strategy was designed to insert the IRES-Cre and frt-flanked PGKneo cassettes 27bp after the stop codon of the full length Foxg1 gene, thus maintaining the previously described miRNAs targeting sites in 3′UTR of Foxg1 gene (e.g., miR-9 and miR-200 ) (Choi et al, 2008; Garaffo et al, 2015; Shibata et al., 2011, 2008). Importantly, this strategy did not incorporate an exogenous polyadenylation (polyA) sequence, which causes abnormal miRNA regulation in the Foxg1-Cre line due to the deletion of Foxg1 3′UTR from the Cre coding mRNA (Miyoshi and Fishell, 2012).…”

“…The dysregulation of expression in the Foxg1-Cre line precludes its use as a telencephalon specific Cre driver line, as ectopic Cre activity is invariably observed in regions around the mid- and hindbrain (Achim et al, 2012; Fuccillo et al, 2004; Hébert and McConnell, 2000; Kasberg et al, 2013; Li et al, 2008, 2012; Ma et al, 2002; Zembrzycki et al, 2007). The reasons behind these inconsistent Cre activities are not completely known but could be due in part by deletion of functional endogenous microRNAs (miRNAs) sites in the Foxg1 3′UTR from the Cre coding messenger RNA (mRNA) (Miyoshi and Fishell, 2012) that would normally repress Foxg1 expression (Choi et al, 2008; Garaffo et al, 2015; Shibata et al, 2011, 2008). In addition, the Foxg1-Cre allele contains a PGK-neo cassette, which in some case has been shown to result in unpredictable Cre activity (Iwasato et al, 2004; Pham et al, 1996).…”

A screen for deeply conserved non-coding GWAS SNPs uncovers a MIR-9-2 functional mutation associated to retinal vasculature defects in human

“…Many studies previously reported and validated the efficiency and specificity of the miR-9 MO (45,48–51). Embryos were injected at the one-cell stage with 2 pmole of the miR-9 or control MO.…”

The Functions of MicroRNA-200 Family in Ovarian Cancer: Beyond Epithelial-Mesenchymal Transition

“…Table 1 summarizes the results of a myriad of studies, which suggest that miR-200 members play a critical role in the differentiation and proliferation of neurons [8,9,10], podocyte differentiation [11], taste bud formation [12], insulin signaling pathway regulation in the control of fat body and body size [7], and for the hormonal regulation of endometrial stromal decidualization [13,14] during embryo implantation [15]. …”