MicroRNA miR-9 modifies motor neuron columns by a tuning regulation of FoxP1 levels in developing spinal cords.

The precise organization of motor neuron subtypes in a columnar pattern in developing spinal cords is controlled by cross-interactions of multiple transcription factors and segmental expressions of Hox genes and their accessory proteins. Accurate expression levels and domains of these regulators are essential for organizing spinal motor neuron columns and axonal projections to target muscles. Here, we show that microRNA miR-9 is transiently expressed in a motor neuron subtype and displays overlapping expression with its target gene FoxP1. Overexpression or knockdown of miR-9 alters motor neuron subtypes, switches columnar identities, and changes axonal innervations in developing chick spinal cords. miR-9 modifies spinal columnar organization by specifically regulating FoxP1 protein levels, which in turn determine distinct motor neuron subtypes. Our findings demonstrate that miR-9 is an essential regulator of motor neuron specification and columnar formation. Moreover, the overlapping expression of miR-9 and its target FoxP1 further illuminates the importance of fine-tuning regulation by microRNAs in motor neuron development.

RNAase-III enzyme Dicer maintains signaling pathways for differentiation and survival in mouse cortical neural stem cells.

An important function of the RNAase-III enzyme Dicer is to process microRNA precursors into ~22-nucleotide non-coding small RNAs. But little is known about the role of Dicer in mammalian brain formation and neural stem cell (NSC) development. Here we show that Dicer plays a crucial role in controlling mouse cortical NSC development. We found that Dicer function is essential for expanding cortical neural progenitors and NSCs. We have identified a population of Dicer-deficient NSCs that can self-renew, and that display normal karyotype and heterochromatin protein expression levels but show enlarged nuclei. Dicer-deficient NSCs display abnormal differentiation and undergo cell death when mitogens are withdrawn. Dicer deletion affects the levels of many proteins, as revealed by a mass spectrometry proteomic approach. We have found that an increase of anti-survival and/or pro-apoptosis proteins and a decrease of pro-survival and/or anti-apoptosis proteins contribute to the cell death of Dicer-deficient NSCs, implying a general role for Dicer in protecting cells from apoptosis. Our results demonstrate important functions for Dicer in regulating NSC development by maintaining proper signaling pathways related to cell survival and differentiation.

Different timings of Dicer deletion affect neurogenesis and gliogenesis in the developing mouse central nervous system.

MicroRNAs, processed by the RNAase III enzyme Dicer, are approximately 22 nucleotide endogenous noncoding small RNAs. The function of Dicer in the mouse central nervous system (CNS) development is not well understood. Here, we show that specifically deleting Dicer expression in the CNS and in the cerebral cortex using two Cre lines results in reduced progenitor numbers, abnormal neuronal differentiation, and thinner cortical wall. Incomplete Dicer deletion during early embryonic stages contributes to normal development of early-born neurons in the cortex and motor neurons in the spinal cord. However, at late embryonic stages when Dicer is completely ablated in the CNS, the migration of late-born neurons in the cortex and oligodendrocyte precursor expansion and differentiation in the spinal cord are greatly affected. Our studies of different timings of Dicer deletion demonstrate the importance of the Dicer-mediated microRNA pathway in regulating distinct phases of neurogenesis and gliogenesis during the CNS development.

Upregulation of MicroRNA miR-9 Is Associated with Microcephaly and Zika Virus Infection in Mice.

Proper growth of the mammalian cerebral cortex, which is determined by expansion and survival of neural progenitors and mature neurons, is crucial for cognitive functions. Here, we show a role of the dosage of microRNA miR-9 in controlling brain size. Cortical-specific upregulation of miR-9 causes microcephalic defects in mice, due to apoptosis, reduced neural progenitor pool, and decreased neurogenesis. Glial cell-derived neurotrophic factor (GDNF) is a target of miR-9, and protects neural progenitors from miR-9-induced apoptosis. Furthermore, Zika virus (ZIKV) infection in embryonic mouse cortex causes reduced numbers in neural progenitors and newborn neurons, and results in upregulation of miR-9, downregulation of its target GDNF. Our studies indicate an association of altered levels of miR-9 and its target GDNF with microcephaly and ZIKV infection in mice.

An Optimized Sponge for microRNA miR-9 Affects Spinal Motor Neuron Development in vivo.

The specification of motor neuron (MN) subtypes and columnar organization in developing spinal cord is controlled by multiple transcription factors. FoxP1 drives specification of lateral motor neuron (LMN) subtypes, and we demonstrated in our previous work that FoxP1 expression levels are regulated by the microRNA miR-9. Here we show that ectopic FoxP1 expression in the chick spinal cord can rescue Lhx3 and Hb9 expression in MNs altered by miR-9 over-expression, demonstrating that FoxP1 is a critical functional interaction partner for miR-9 in LMN development. Moreover, we have optimized a technique called a miRNA sponge in vitro, to permit easy discovery of the role of individual miRNA in vivo using a loss-of-function approach. We here show that narrow spacing between binding sites, inclusion of a coding gene, and optimizing the number of miRNA binding sites can significantly increase the blocking ability of a sponge. We go on to show that a miR-9 sponge reduces detectable miR-9 in the ventral horn, preventing miR-9 silencing of FoxP1 in vivo, and in turn modifies MN subtypes in the spinal cord. Our designs for optimized sponges provide a knockdown tool that is ready to be used to study the function of miRNA in vivo, and in particular for generating transgenic animal models.

Retinal and olfactory bulb precursor cells show distinct responses to FGF-2 and laminin.

We analyzed whether the embryonic (E12.5-E14.5) mouse retina possesses genuine neural stem cells and how they respond to defined growth factors and extracellular matrix molecules. Whereas most combinations produced no or limited cell survival and proliferation in culture, FGF-2 plus heparin and laminin stimulated proliferation and the formation of aggregates composed, after two days, of 95.2% nestin-positive cells. However, cells in these aggregates could only be passaged poorly, lost nestin expression and proliferative capacity, and differentiated into neurons. Under the same conditions, olfactory bulb precursor cells divided efficiently and could be expanded. These data suggest that, in addition to FGF-2 and laminin, embryonic retinal neuroepithelial cells need additional extrinsic and/or intrinsic regulators to maintain cell proliferation and self-renewal.

Modulation of the PI 3-kinase-Akt signalling pathway by IGF-I and PTEN regulates the differentiation of neural stem/precursor cells.

Neural stem cells depend on insulin-like growth factor I (IGF-I) for differentiation. We analysed how activation and inhibition of the PI 3-kinase-Akt signalling affects the number and differentiation of mouse olfactory bulb stem cells (OBSCs). Stimulation of the pathway with insulin and/or IGF-I, led to an increase in Akt phosphorylated on residues Ser473 and Thr308 (P-Akt(Ser473) and P-Akt(Thr308), respectively) in proliferating OBSCs, and in differentiating cells. Conversely, P-Akt(Ser473) levels decreased by 50% in the OB of embryonic day 16.5-18.5 IGF-I knockout mouse embryos. Overexpression of PTEN, a negative regulator of the PI 3-kinase pathway, caused a reduction in the basal levels of P-Akt(Ser473) and P-Akt(Thr308) and a minor reduction in IGF-I-stimulated P-Akt(Ser473). Although PTEN overexpression decreased the proportion of neurons and astrocytes in the absence of insulin/IGF-I, it did not alter the proliferation or survival of OBSCs. Accordingly, overexpression of a catalytically inactive PTEN mutant promoted OBSCs differentiation. Inhibition of PI 3-kinase by LY294002 produced strong and moderate reductions in IGF-I-stimulated P-Akt(Ser473) and P-Akt(Thr308), respectively. Consequently, LY294002 reduced the proliferation of OBSCs and the number of neurons and astrocytes, and also augmented cell death. These findings indicate that OBSC differentiation is more sensitive to lower basal levels of P-Akt than proliferation or death. By regulating P-Akt levels in opposite ways, IGF-I and PTEN contribute to the fine control of neurogenesis in the olfactory bulb.