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

Abstract

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 Dicer-deficient NSCs display abnormal differentiation and undergo cell death when mitogens are withdrawn.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.

Fig. 1.

Dicer-deficient NSCs can self-renew. (A,B) A great reduction of neurospheres derived from NSCs of Dicer^Cre/loxp (Ko) mice, as compared with controls (Ctrl), was observed. The primary neurosphere is termed as passage-0 (p-0). n=4 wells; **P<0.001. (C,D) The surviving Dicer-deficient NSCs produced numbers of neurospheres similar to those in controls after passaging, shown as a representative experiment at passage-2 (p-2). n=4 wells. (E) Dicer proteins were absent in Dicer-deficient neurospheres, shown as examples of passage-1 (p-1) and p-2, detected by western blotting assays. Actin served as a loading control. (F) miRNAs, for example miR-181b and miR-125b, were absent in p-1 Dicer-deficient neurospheres, as detected by northern blotting analyses. U6 served as a loading control.

Fig. 2.

Dicer-deficient NSCs express stem-cell markers. (A) Primary neurospheres (p-0) derived from Dicer-deficient NSCs (Ko) expressed nestin similarly to control NSCs (Ctrl). (B) More than 98% of Dicer-deficient NSCs (p-0) were BrdU positive, suggesting that they are dividing at the same rate as controls. (C) Nestin was expressed in the passaged neurospheres (p-2) derived from NSCs of KO and control cortices. The insets show neurosphere immunostaining without the primary antibodies for nestin. (D) More than 98% of Dicer-deficient NSCs (p-2) were BrdU positive. (E,F) Dissociated Dicer- deficient NSCs (p-2) expressed NSC markers such as nestin and SSEA-1. (G) Dicer-deficient NSCs (p-1) survived normally as control NSCs in a medium with mitogens. n>3 fields.

Fig. 3.

Surviving Dicer-deficient NSCs display abnormal differentiation. (A) Under the differentiation condition without mitogens, passaged (p-1) NSCs collected from Dicer knockout (Ko) cortices gave rise to cells expressing neuronal (Tuj1⁺) and glial (GFAP⁺ and O4⁺) markers. However, their morphology was abnormal, as shown with shorter neurites than controls (Ctrl). (B) The majority of Dicer-deficient NSCs underwent cell death, shown as an example of p-1 NSCs in differentiation condition cultured for 2 days in vitro. n=6 fields; **P<0.0008. (C) Dicer-deficient NSCs did not survive well in a differentiation culture medium without mitogens. Whereas control neurospheres (p-2) survived, most Dicer KO neurospheres died after 48 hours in culture. Many differentiated cells (arrows) migrated away from the control neurosphere but not from the Dicer-deficient neurosphere.

Fig. 4.

Surviving Dicer-deficient NSCs have normal karyotype and heterochromatin protein expression. (A,B) Karyotype analyses (metaphase spreads) of surviving Dicer-deficient (Ko) and control (Ctrl) NSCs collected from p-1. (C) The percentage of chromosome numbers among counted cells. More than 54% Dicer KO NSCs displayed 40 chromosomes, which is similar to the controls (63%); n=33 cells for either Ctrl or KO NSCs. (D) Immunohistochemistry for heterochromatin proteins in control and Dicer- deficient NSCs (p-1). Both H3K9me3 and HP1β were detected and coexpressed in Dicer-deficient NSCs. The insets in merged images show immunostaining without the primary antibodies for H3K9me3 and HP1β. (E) Almost 100% of control and Dicer-deficient NSCs were H3K9me3- and HP1β-positive. n>250 cells; three Dicer KO and three control animals were used for statistical analyses. (F) H3K9me3- and HP1β-expressing Dicer- deficient NSCs had elongated nuclei. (G) The intensity of H3K9me3 expression levels was similar between the control (blue line) and KO (red line) NSCs, as detected by the flow cytometric analysis. The gray area indicates an isotype-negative control. The representative result shown here is from three independent experiments.

Fig. 5.

Unbalanced cell-survival and cell-death pathways in Dicer-deficient NSCs. Signaling pathway analyses identified an increase of pro-cell-death proteins and a decrease of pro-survival proteins in Dicer-deficient NSCs.

Fig. 6.

Cell-death pathways affected by Dicer deletion in NSCs and progenitors. (A) An increased amount of apoptotic progenitor cells, detected by activated Casp-3 (arrowheads), was detected in E15.5 cortices of Dicer KO mice compared with controls (Ctrl). (B) Expression of PDCD4 was increased in Dicer-deficient NSCs, as detected by western blotting assays. Actin served as a loading control. Shown as one example experiment, relative levels of PDCD4 expression were normalized by actin expression between Ctrl and KO. (C,D) Decreased expression of SOD1 and increased expression of FMR1 were detected in Dicer-deficient NSCs, as analyzed by western blotting assays. Shown as example experiments; relative levels of SOD1 and FMR1 expression were normalized by actin expression between Ctrl and KO. The representative western blotting results shown here are from neurosphere cultures of at least three Dicer KO and three control animals. (E) A simplified network generated from proteins identified to be differentially expressed in NSCs of Dicer KO and Ctrl. It illustrates FMR1 as a hub protein in miRNA- and Dicer-dependent regulation of cell death and survival. The intensity of the node color indicates the degree of up- (red, representing >twofold higher in Ctrl compared with KO) or down- (green, representing >twofold higher in KO compared with Ctrl) regulation. The complete network is shown in supplementary material Fig. S7.