doi: 10.3389/fncel.2016.00047.
eCollection 2016.
Function of Armcx3 and Armc10/SVH Genes in the Regulation of Progenitor Proliferation and Neural Differentiation in the Chicken Spinal Cord
Affiliations
- PMID: 26973462
- PMCID: PMC4776218
- DOI: 10.3389/fncel.2016.00047
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Function of Armcx3 and Armc10/SVH Genes in the Regulation of Progenitor Proliferation and Neural Differentiation in the Chicken Spinal Cord
Front Cell Neurosci.
.
Abstract
The eutherian X-chromosome specific family of Armcx genes has been described as originating by retrotransposition from Armc10/SVH, a single Arm-containing somatic gene. Armcx3 and Armc10/SVH are characterized by high expression in the central nervous system and they play an important role in the regulation of mitochondrial distribution and transport in neurons. In addition, Armcx/Arm10 genes have several Armadillo repeats in their sequence. In this study we address the potential role of this gene family in neural development by using the chick neural tube as a model. We show that Armc10/SVH is expressed in the chicken spinal cord, and knocking-down Armc10/SVH by sh-RNAi electroporation in spinal cord reduces proliferation of neural precursor cells (NPCs). Moreover, we analyzed the effects of murine Armcx3 and Armc10 overexpression, showing that both proteins regulate progenitor proliferation, while Armcx3 overexpression also specifically controls neural maturation. We show that the phenotypes found following Armcx3 overexpression require its mitochondrial localization, suggesting a novel link between mitochondrial dynamics and regulation of neural development. Furthermore, we found that both Armcx3 and Armc10 may act as inhibitors of Wnt-β-catenin signaling. Our results highlight both common and differential functions of Armcx/Armc10 genes in neural development in the spinal cord.
Keywords: Armc10 gene; Armcx gene cluster; mitochondria; neuronal differentiation; progenitor proliferation; spinal cord development.
Figures
Armcx3 overexpression reduces progenitor proliferation. (A–D) Representative transverse sections of neural tubes from embryos electroporated at HH stage 12 with pCIG and pCIGArmcx3 vectors and analyzed at 24hpe with the indicated immunostaining. GFP, Sox2 (red) and HuC/D (blue) stain, respectively, the electroporated cells, the neural progenitors, and the differentiating neurons; Armcx3 overexpressing cells show a lateral distribution from the lumen to the MZ of the neural tube. (E,F) Ectopic expression of Armcx3 leads to a dramatic increase in the percentage of HuC/D/GFP-positive cells, concomitant to a decrease in Sox2/GFP-positive cells. (G,H) The percentage of GFP-positive electroporated cells positive for PH3 or BrDU decreases in pCIGArmcx3 electroporated embryos. Data represent the mean ± s.e.m. (*p < 0.05, **p < 0.01).
Armcx3 overexpression promotes neural differentiation. (A–E) Representative transverse sections of neural tubes from embryos electroporated at HH stage 12 with pCIG and pCIGArmcx3 vectors and analyzed at 48hpe with the indicated immunostaining. Armcx3 overexpressing cells show a lateral distribution from the VZ to the MZ of the neural tube. (F) Ectopic expression of Armcx3 leads to an increase in the percentage of HuC/D/GFP-positive cells and a decrease in Sox2/GFP-positive cells (anti-HuC/D, blue; anti-Sox2, red). (G) The HuC/D+ areas corresponding to the MZ (formed by the differentiating neurons) were defined using ImageJ processing. (H) The areas measured for the electroporated side (EP) were standardized to their contralateral controls (CNTRL) and are presented as ratios of the area of MZ (HuC/D+ Area); the widths of the Tuj-1-marked region for the electroporated side were standardized to their contralateral controls and are presented as ratios of the size of MZ (Tuj+ Size). (I) Histogram showing the percentage of electroporated cells (GFP+) positive for Tuj-1. Data represent the mean ± s.e.m. (*p < 0.05, **p < 0.01).
Armcx3 effects on progenitor proliferation depend on its mitochondrial localization. (A–D,F,H) Representative transverse sections of chick neural tubes from embryos electroporated at HH stage 12 with the indicated plasmids and processed and analyzed 24hpe as described in Figure 1. Overexpressed Arcmx3 is localized at the mitochondria of neural progenitors as indicated by its colocalization with the mitochondrial marker CoxIV. By contrast, a truncated form of Armcx3 which lacks its mitochondrial targeting sequence (Armcx3Δ1-12) displays a cytoplasmic non-mitochondrial localization (A). Arrows in (A) label mitochondria co-localizing with Armcx3 (left panels) but not with the Armcx3Δ(1-12) construct lacking the mitochondrial targeting sequence (right panels). The overexpression of this truncated form neither induced a lateral distribution of progenitors (B,E) nor modified the percentage of Sox2/GFP-positive or HuCD/GFP-positive cells (D–G). Consistently, the non-mitochondrial Armcx3 form did not induce changes in the percentage of GFP/PH3 or GFP/BrdU positive cells (I). Data represent the mean ± s.e.m.
Armcx3 effects on neural differentiation depend on its mitochondrial localization. (A,C,E) Representative transverse sections of chick neural tubes from embryos electroporated at HH stage 12 with the indicated plasmids and processed and analyzed 48hpe as described in Figure 2. In contrast to the full length Armcx3 (see Figure 2), the overexpression of a non-mitochondrial truncated version of Arcmx3 (Armcx3Δ1-12) did not induce apparent changes in the lateral distribution of progenitors (A–D) or modify the percentage of Sox2/GFP or HuCD/GFP positive cells. (F) Similarly, Armcx3Δ1-12 overexpression has no impact on the HuCD + area (G,H), the width of Tuj-1 positive region (G,H), or the percentage of electroporated cells positive for Tuj-1 (I). Data represent the mean ± s.e.m.
Armc10 overexpression reduces progenitors proliferation. Representative transverse sections of chick neural tubes from embryos electroporated at HH stage 12 with the indicated plasmids and processed and analyzed 24hpe as described in Figure 1. As is seen with Arcmx3, Armc10 protein is localized at the mitochondria of neural progenitors as indicated by its colocalization with the mitochondrial marker CoxIV (A). (C) Schematic representation illustrating the distributions of the VZ and MZ at HH12+24hpe. Armc10 overexpression did not induce changes in the lateral distribution of electroporated neural progenitors (B,E) or in the percentage of GFP/Sox2 or GFP/HuCD positive cells (D,F,G). However, it induced a reduction in the percentage of PH3/GFP and BrdU/GFP positive cells (H,I). Data represent the mean ± s.e.m. (*p < 0.05).
Armc10 overexpression does not promote neural differentiation. Representative transverse sections of chick neural tubes from embryos electroporated at HH stage 12 with the indicated plasmids and processed and analyzed 48hpe as described in Figure 2. The overexpression of Armc10 did not induce changes in the lateral distribution of electroporated progenitors (A) or modify the percentage of Sox2/GFP or HuCD/GFP positive cells (C,D). By contrast, Armc10 overexpression reduced the HuCD area and the width of Tuj-1 positive region (G,H). The percentage of electroporated cells positive for Tuj-1 was not different in embryos where Armc10 was overexpressed with respect to controls (pCIG) (I). Data represent the mean ± s.e.m. (*p < 0.05, **p < 0.01).
Endogenous Armc10 is expressed in chicken spinal cord. (A) Structure and domains of the two different isoforms of chicken Armc10 protein predicted by in silico analysis. They differ in the N-terminal region, containing a mitochondrial targeting peptide, an N-terminal transmembrane region, and a predicted cleavage site. The C-terminal domain consists of up to six Arm-like tandem repeats. (B) Both Armc10 isoforms share a strong sequence homology with mouse Armc10. (C) HH stage 12, 19, and 24 showing expression of chick Armc10. (D) Efficiency of Armc10 gene silencing using shRNA in chicken neural tube, as measured with RT-qPCR. Armc10 mRNA expression was calculated relative to that of control shRNA (shControl; error bars indicate the SD).
Endogenous Armc10 silencing inhibits progenitor proliferation. (A–D) Representative transverse sections of neural tubes from embryos electroporated at HH stage 12 with shControl and shArmc10 vectors and analyzed at 24hpe with the indicated immunostaining. No changes in the distribution of shArmc10 expressing cells are observed. (E,F) Armc10 silencing does not affect the percentage of HuC/D/GFP or Sox2/GFP-positive cells with respect to the control. (G,H) The percentage of GFP-positive electroporated cells positive for BrDU decreases in shArmc10 electroporated embryos; no changes are observed in the percentage of GFP-positive electroporated cells positive for PH3 with respect to the control. Data represent the mean ± s.e.m. (*p < 0.05).
Endogenous Armc10 silencing inhibits progenitor proliferation but does not affect neural maturation. (A–F) Representative transverse sections of neural tubes from embryos electroporated at HH stage 12 with shControl and shArmc10 plasmids and analyzed at 48hpe with the indicated immunostaining. No changes in the distribution of shArmc10 expressing cells (D) or in the percentage of HuC/D/GFP and Sox2/GFP-positive cells (F) are observed (anti-HuC/D, blue; anti-Sox2, red). (G,H) The HuC/D+ areas corresponding to the MZ (formed by the differentiating neurons) were defined using ImageJ processing (G). The areas measured for the electroporated side (EP) were standardized to their contralateral controls (CNTRL) and are presented as ratios of the area of MZ (HuC/D+Area); the widths of the Tuj-1-marked region for the electroporated side were standardized to their contralateral controls and are presented as ratios of the size of MZ (Tuj+ Size) (H). (I) Histogram showing the percentage of electroporated cells (GFP+) positive for Tuj-1. Data represent the mean ± s.e.m. (*p < 0.05).
Armcx3 and Armc10 expression levels regulate β-catenin-induced Tcf/LEF transcriptional activity without affecting dorsoventral patterning. (A,B) Armcx3 and Armc10 inhibit the Wnt pathway upstream from the TCF activity. HH stage 12 embryos were co-electroporated with the pCIG empty vector, β-cateninCA or TCF-VP16 together with Armcx3 or Armc10. Analysis of the TOP-Flash reporter activity shows that both Armcx3 and Armc10 inhibit β-cateninCA-mediated transcriptional activity but not TCF activation. (C) Inhibition of endogenous Armc10 activates Wnt pathway. A similar assay electroporating TOP-Flash reporter together with shArmc10 constructs shows an increase in β-cateninCA-mediated transcriptional activity. (D,E) Alterations in Tcf/LEF transcriptional activity mediated by changes in Armcx3 or Armc10 expression levels do not affect dorsoventral patterning organization. Representative sections of HH12 neural tubes electroporated with the indicated vectors and processed 24hpe for immunostaining analysis against the dorsoventral markers Pax7 or Nk6.1 (red). Anti-GFP or anti-Armcx3 (green) antibodies were used to report the transgene expression. Data represent the mean ± s.e.m. (*P < 0.05; **P < 0.01; ***P < 0.001).
