Mir-17-3p controls spinal neural progenitor patterning by regulating Olig2/Irx3 cross-repressive loop

Abstract

Neural patterning relies on transcriptional cross-repressive interactions that ensure unequivocal assignment of neural progenitor identity to proliferating cells. Progenitors of spinal motor neurons (pMN) and V2 interneurons (p2) are specified by a pair of cross-repressive transcription factors, Olig2 and Irx3. Lineage tracing revealed that many p2 progenitors transiently express the pMN marker Olig2 during spinal cord development. Here we demonstrate that the repression of Olig2 in p2 domain is controlled by mir-17-3p microRNA-mediated silencing of Olig2 mRNA. Mice lacking all microRNAs or just the mir-17∼92 cluster manifest a dorsal shift in pMN/p2 boundary and impairment in the production of V2 interneurons. Our findings suggest that microRNA-mediated repression of Olig2 mRNA plays a critical role during the patterning of ventral spinal progenitor domains by shifting the balance of cross-repressive interactions between Olig2 and Irx3 transcription factors.

Figure 1. Transient Olig2 Expression in p2 Progenitors

(A) Five cardinal progenitor domains (p0~p2, pMN and p3) are defined in the ventral spinal cord by the combinatorial expression of transcription factors. Each progenitor domain generates different set of ventral spinal interneurons or motor neurons. For more details, see Briscoe et al., 2000; Novitch et al., 2001. (B) Schematic illustration of Olig2 lineage tracing. Ventral progenitor cells that express Olig2 and all their progeny are indelibly marked by YFP expression in Olig2^Cre/+; ROSA26-loxp-STOP-loxp-YFP embryos. (C–H) Analysis of Olig2 lineage (YFP^on cells) in Olig2^Cre/+; ROSA26-loxp-STOP-loxp-YFP E11.5 spinal cord sections. YFP is expressed in a broad ventral domain that includes p3 progenitors and a subset of p2 progenitors (Olig2^off, Irx3^on and Nkx6.1^on ) ventral and dorsal to the pMN (Olig2^on ) domain. (I and J) A subset of V2a (Chx10^on ) and V2b (Gata3^on ) interneurons derived from p2 domain expresses YFP. For quantification see supplementary Figure S1. (K) Olig2 is transiently expressed in three ventral spinal progenitor domains. Olig2 expression is selectively down-regulated in p2 and p3 progenitors during spinal cord patterning.

Figure 2. Ectopic Olig2 Expression in the p2 Domain in Dicer^−/− Embryoid Bodies and Embryos

(A) CAGG-Cre; Dicer^WT/loxp and CAGG:Cre; Dicer^loxp/loxp ES cell lines (referred to as Dicer^+/− and Dicer^−/− after 4-hydroxytamoxifen (4OH-TM) treatment). ES cells are differentiated with RA (1 μM) and lo[Hh] (5 nM) or hi[Hh] (500 nM) on day 2 and spinal progenitor identities are determined by immunostaining embryoid body sections on day 4. (B–E) Increase in the fraction of cells expressing pMN identity (Nkx6.1^on, Olig2^on, Irx3^off ) and decrease in the number of p2 progenitors (Nkx6.1^on, Olig2^off, Irx3^on ) in Dicer^−/− embryoid bodies under lo[Hh] condition. (G–K) The pMN (Olig2^on) and p3 (Nkx2.2^on ) progenitors are not affected in Dicer^−/− embryoid bodies under hi[Hh] condition. (F and K) Quantification of progenitor cells (percentage of total cells, mean ± SD; n=3 independent experiments) reveals decrease in p2 (p < 0.01) and increase in pMN (p < 0.01) progenitors in Dicer^−/− embryoid bodies under lo[Hh] condition, but no change in p3 and pMN fractions under the hi[Hh] conditions. (L) To study the function of Dicer gene in vivo, pregnant Dicer^loxp/loxp mice mated with CAGG-Cre; Dicer^WT/loxp males were injected with Tamoxifen (TM) on E5.5 and embryos were analyzed for patterning defects on E9.5 (M–T) Dorsal expansion of Olig2 cells is apparent in the Dicer^−/− E9.5 embryonic spinal cord sections. In contrast, the size of p2 progenitor domain (Nkx6.1^on, Olig2^off, Irx3^on) is diminished. The positions of dorsal boundaries of Nkx6.1 (p2/p1 boundary) or Nkx2.2 (p3/pMN boundary) are not changed. (U) Quantification of p2 (Nkx6.1^on, Irx3^on), pMN (Olig2^on) and p3 (Nkx2.2^on) ventral progenitors (number of positive cells per 15 μm cervical spinal cord hemisection) in control and Dicer mutant embryos (mean ± SD, n = 5 embryos), reveals an increase in the number of pMN and decrease of p2 progenitors (p < 0.01). (V) Summary of phenotypes in the ventral neural tube of Dicer mutant embryos. Olig2 is repressed in prospective p3 and p2 domains in control spinal cord. Deletion of Dicer function results in expansion of Olig2 expression into the p2 domain resulting in a diminished number of p2 progenitors.

Figure 3. Identification of lo[Hh] Enriched miRNAs Predicted to Target Olig2

(A) Small RNAs were isolated from day 4 embryoid bodies differentiated under lo[Hh] or hi[Hh] conditions. Expression levels of miRNAs were analyzed by rodent TaqMan Low Density Arrays (TLDA). (B and C) A set of miRNAs exhibiting differential expression levels between lo[Hh] and hi[Hh] conditions (n = 2 independent experiments). miRNAs shown in red (B) are the candidates enriched in lo[Hh] progenitors predicted to target to Olig2 by TargetScan, miRanda and MicroCosm target prediction algorithms (C). (D) Verification of TLDA data by qPCR in independent differentiation experiments. mir-17-3p and mir-302b show >2 fold increase in RQ (relative quantity or 2^−ΔΔCt) in lo[Hh] embryoid bodies when compared to hi[Hh] condition. Data represent three independent experiments (n = 3) performed in triplicate. Error bars indicate SD. (E) The predicted target sites of mir-17-3p and mir-302b miRNAs within the 3′UTR of Olig2. mir-17-5p and mir-302b share similar seed sequence (highlighted in red).

Figure 4. Regulatory Interactions Between mir-17 and Olig2/Irx3 Cross-repressive Loop

(A) Expression of mir-17-3p examined by in situ hybridization on E9.5 spinal cord section. (B) Expression of Irx3, and Olig2 revealed by immunocytochemistry on adjacent spinal cord section. (C) The design of inducible “Tet-On” ES cell lines expressing Olig2 or Irx3 under the doxycycline (Dox) regulated promoter. In the presence of Dox, the reverse tTA (rtTA) activator is recruited to the TRE (Tetracycline Response Element), thereby initiating the transcription of the downstream gene. (D–G) Expression of Irx3 is repressed in inducible Olig2 (iOlig2) day 4 embryoid bodies differentiated under lo[Hh] condition and treated with Dox on day 3 (D–E). Conversely, expression of pMN marker Olig2 is extinguished upon the induction of Irx3 (iIrx3) expression by Dox treatment of day 3 embryoid bodies differentiated under hi[Hh] condition (F–G). (H) The expression levels of mir-16, mir-17-5p, and mir-17-3p were analyzed by qPCR after induction of Olig2 or Irx3 in lo[Hh] or hi[Hh] treated embryoid bodies, respectively. Data were normalized to expression levels in control lo[Hh] embryoid bodies and represent three independent experiments (n = 3) performed in triplicate. Changes in Olig2/Irx3 status of differentiating cells result in corresponding changes in mir-17-5p and mir-17-3p expression levels. (I) Generation of Dox inducible iMir-17 ES cell line in which mir-17 hairpin is inserted into 3′UTR of GFP. The mir-17-3p sequence is marked in red. (J) Induction of mir-17 in embryoid bodies differentiated under med[Hh] on day 3 results in a decrease in Olig2 expressing pMN and an increase in Irx3/Nkx6.1 double positive p2 progenitors. (K) Induction of mir-17 results in a significant decrease in the fraction of Nkx6.1^on cells expressing Olig2 and increase in the fraction of cells expressing Irx3 upon induction of mir-17 expression (p < 0.01, mean ± SD, n = 3 independent experiments).

Figure 5. Direct Silencing of Olig2 by mir-17-3p

(A) Luciferase reporters were constructed with either a control Olig2 3′UTR or the 3′UTR sequence in which the two potential target sites of mir-17-3p were mutated (red), the two potential target sites of mir-17-5p were mutated (green), or all four targets sites were mutated (5p/3p Mut). (B) Co-expression of luciferase construct with mir-17 in HeLa cells silences reporter carrying intact mir-17-3p target sites (WT and 5p-Mut), while mir-17 fails to silence 3p-Mut and 3p/5p-Mut luciferase constructs (n=3 independent experiments, mean ± SD, p<0.01). (C) Generation of inducible ES cell lines expressing either mir-17-3p or mir-17-5p (marked in red) as artificial hairpins flanked by mir-30 backbone (sequence in black) inserted into GFP 3′ UTR (Stegmeier et al., 2005). ES cells were differentiated under hi[Hh] (500 nM) condition with or without Dox treatment on day 3 of differentiation. (D) Expression of Olig2 and Nkx6.1 in embryoid bodies composed of a mixture of control (GFP negative) and inducible (GFP positive) cells. Induction of mir-17 or mir-17-3p on day 3 of differentiation under hi[Hh] condition results in silencing of Olig2 expression, while GFP^on cells maintain expression of Nkx6.1. In contrast, induction of mir-17-5p has no discernible effect on Olig2 expression.

Figure 6. Loss of mir-17~92 Cluster Results in a Deficit in p2 progenitors and V2 interneurons in vitro

(A–E) Expression of Olig2, Irx3 and Nkx6.1 in control and mir-17~92^−/− embryoid bodies differentiated under lo[Hh] condition. Loss of mir-17~92 results in an increase in the number of Olig2 positive cells (p < 0.01) per section as well as an increase in the fraction of Nkx6.1 positive cells expressing Olig2 (p<0.001), mean ± SD, n = 3 independent experiments. (N–R) Reduction of V2 (Chx10^on ) interneurons in day 7 miR-17~92^−/− embryoid bodies cultured under lo[Hh] condition (n = 3, p < 0.01). The numbers of V1 (En1^on ) and V0 (Evx1^on ) interneurons remain unchanged. Data are quantified as percentage of Chx10, Evx1 and En1 positive cells (mean ± SD; n = 3 independent experiments).

Figure 7. Loss of mir-17~92 Cluster Results in a Deficit in p2 progenitors and V2 interneurons in vivo

(A–H) Dorsal expansion of pMN (Olig2^on) progenitor domain is apparent in the mir-17~92^−/− E9.5 embryonic spinal cord sections. In contrast, the size of p2 progenitor domain (Nkx6.1^on, Olig2^off, Irx3^on ) is diminished (E, F). Domains expressing Nkx6.1 or Nkx2.2 are not changed. The location of Hb9^on motor neurons is expanded dorsally in the mir-17~92^−/− embryos (G, H). (I) Quantification of p2, pMN and p3 ventral progenitors (number of positive cells per 15 μm cervical spinal cord hemisection) in control and mir-17~92 mutant embryos, mean ± SD reveals a decrease in the number of p2 and an increase in the number of Olig2^on pMN progenitors and Hb9^on motor neurons (p < 0.01; n = 6 embryos). (J–O) Immunostaining of E11.5 spinal cord sections reveals dorsal shift in the distribution of Hb9^on motor neurons (M, arrowheads) and decrease in Chx10^on V2a and Gata3^on V2b interneurons in miR-17~92 mutant embryos. In contrast, Evx1^on V0 interneurons appear to be unchanged. (P) Quantification of ventral postmitotic V0, V2a and V2b interneurons, and motor neurons (MNs) (number of positive cells per 15 μm brachial spinal cord hemisection) in control and mir-17~92 mutant embryos (mean ± SD) reveals a decrease in the number of V2a and V2b interneurons (p < 0.01, n = 3 embryos).

Figure 8. Model of Ventral Spinal Patterning in the Presence and Absence of mir-17-3p

A proposed model of dynamic changes in progenitor marker expression in wild type and Dicer ^−/− or mir-17~92^−/− embryos. In early stages, Nkx6.1 and Olig2 are co-expressed in a broad ventral domain spanning the prospective p3, pMN and part of p2 domains. Subsequently, Nkx2.2 induced by sustained Shh signaling represses Olig2 in the p3 domain (Dessaud et al., 2007) and mir-17-3p induced by Irx3 silences Olig2 in the p2 domain, forming the normal p2, pMN, and p3 progenitor domains. In Dicer and mir-17~92 mutant embryos, Olig2 is not efficiently silenced in the prospective p2 domain, resulting in a dorsal shift in the p2/pMN boundary and a deficit in V2 interneurons (Chx10^on and Gata3^on).