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Involvement of SF-1 in neurogenesis and neuronal migration in the developing neocortex
Neuroscience Letters 600 (2015) 85–90
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Involvement of SF-1 in neurogenesis and neuronal migration in the developing neocortex Munekazu Komada, Mifumi Takahashi, Yayoi Ikeda ∗ Department of Anatomy, School of Dentistry, Aichi Gakuin University, Nagoya, Japan
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SF-1 was expressed in the dorsal telencephalon at E15.5–E18.5, but not in adulthood. In SF-1 KO embryos, neurons in the IZ/SVZ were increased, in the CP decreased. The APCs increased and radial fibers showed abnormal morphology in SF-1 KO embryos. Cell cycle duration was shortened and exit inhibited in SF-1 KO stem/progenitor cells. Expression of ESR˛ was up- and of Cyp19a1 down-regulated in SF-1 KO embryos.
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Article history: Received 9 March 2015 Received in revised form 2 June 2015 Accepted 3 June 2015 Available online 9 June 2015 Keywords: Steroidogenic factor-1 Dorsal telencephalon Neurogenesis Proliferation Cell cycle Estrogen signaling
a b s t r a c t The nuclear receptor steroidogenic factor-1 (SF-1) plays essential roles in the development and function of the endocrine and reproductive systems. During embryogenesis, SF-1 is expressed in the ventromedial hypothalamic nucleus (VMH) and regulates the migration and terminal differentiation of the VMH neurons. Additionally, in situ hybridization data indicated SF-1 expression in the dorsal telencephalon at embryonic day (E) 13.5. In this study, we investigated the neocortical development in SF-1 knockout (KO) mouse embryos. The number of neurons was increased in the intermediate/subventricular zones and decreased in the cortical plate in the SF-1 KO embryos. SF-1 KO embryos produced more neural stem/progenitor cells, especially apical progenitor cells, and showed abnormal radial glial fiber morphology. The increase in neural stem/progenitor cells was caused by an increased S-phase fraction in the proliferative cells and the inhibition of cell cycle exit in these cells. The mRNA expression of the estrogen receptor ESR˛ was up-regulated and that of the estrogen synthetase Cyp19a1 was down-regulated in the dorsal telencephalon of SF-1 KO embryos. We showed that SF-1 is expressed in the dorsal telencephalon at E15.5 and E18.5, but not in adult animals. Our data demonstrated that SF-1 is involved in cell cycle regulation, neurogenesis, and neuronal migration via controlling the estrogen signaling for proper neocortical development. © 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Steroidogenic factor 1 (SF-1), which is encoded by the NR5A1 gene, is a member of the nuclear hormone receptor family [1]. SF-1 is required for adrenal and gonadal steroidogenesis, control-
Abbreviations: APC, apical progenitor cell; CP, cortical plate; DCX, doublecortin; E, embryonic day; ESR, estrogen receptor; GXD, gene expression database; IdU, iododeoxyuridine; IPC, intermediate progenitor cell; IZ, intermediate zone; KO, knockout; SF-1, steroidogenic factor-1; SVZ, subventricular zone; VMH, ventromedial hypothalamic nucleus; VZ, ventricular zone. ∗ Corresponding author at: 1–100 Kusumoto-cho, Chikusa-ku, Nagoya, Aichi 464–8650, Japan. Fax: +81 52 757 6755. E-mail address: [email protected] (Y. Ikeda). http://dx.doi.org/10.1016/j.neulet.2015.06.005 0304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.
ling the expression of a number of key target genes including those encoding cytochrome P450 steroid hydroxylase and aromatase in adulthood [2]. It is expressed in the gonads, adrenal cortex, pituitary, and hypothalamus during embryogenesis [3,4], and SF-1 knockout (KO) mice exhibit agenesis of the adrenal gland and gonads, deficiency of pituitary gonadotropin secretion, and abnormal structure of the ventromedial hypothalamic nucleus (VMH), suggesting that SF-1 plays critical roles in the development of the tissues at all the levels of the hypothalamus-pituitarygonad/adrenal axis [5,6]. The disorganization of the VMH was shown to be due to the accumulation of immature neurons in the proliferative zone of the diencephalon, which was caused by migration defects in these neurons [7]. In addition, misexpression of VMH markers and loss of neuronal projections to the bed nucleus of
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the stria terminalis and the amygdala in SF-1 KO mice have been reported [8]. These data suggest that SF-1 is involved in the regulation of cell fate determination, neuronal migration, and terminal differentiation during the development of the VMH. During neocortical development, apical progenitor cells (APCs, neural stem cells) in the ventricular zone (VZ) and intermediate progenitor cells (IPCs, neural progenitor cells) in the subventricular zone (SVZ) self-renew and generate the neurons [9]. APCs and IPCs are distinguished by their distribution, self-renewal ability, capacity for neurogenesis, and expression of transcription factors [10]. The division patterns of APCs and IPCs affect the number of neurons and determine the neocortical size [11]. Their proliferation and neurogenesis are orchestrated by a signaling network of factors including morphogens, transcription factors, and growth factors. For example, estrogen receptors and aromatase that are expressed in the developing dorsal telencephalon control the proliferation, migration of neural stem/progenitor cells, and neurogenesis [12,13]. In situ hybridization data available in the Gene Expression Database (GXD), a publicly available resource for gene expression information from laboratory mice, indicated that SF-1 is expressed in the dorsal telencephalon at E13.5 [14]. However, the role of SF1 in this region remains unknown. In this study, we investigated the roles of SF-1 in the morphogenesis of the neocortex using SF-1 KO embryos. First, we analyzed the histological changes and the changes in expression of markers for neurogenesis, proliferation, and migration of neural stem/progenitor cells using immunostaining. Next, the cell cycle kinetics of neural stem/progenitor cells were examined by iododeoxyuridine (IdU)-labeling and double immunostaining for Ki67 and IdU. Finally, we compared the expression of genes involved in estrogen signaling and neurogenesis during the development of the neocortex in wild-type (WT) and SF-1 KO embryos. The results suggested that SF-1 is involved in the regulation of neurogenesis, proliferation, and neuronal migration in the developing neocortex, as well as in the hypothalamus. 2. Materials and methods 2.1. Immunofluorescence Immunofluorescence staining of the sections was performed as described previously [15,16]. The antibodies used in the study and detailed methods are described in the Supplemental methods. Primary and secondary antibody incubations were conducted overnight and for 3 h, respectively, at room temperature. Nuclei were counterstained with DAPI (D9542, Sigma–Aldrich, St Louis, MO, USA; 1:1000) for 3 h at RT. Six sections from three embryos per genotype were used for immunofluorescence analysis of each marker.
2.3. Quantitative immunofluorescence analysis Immunofluorescence staining was quantified in selected areas of the dorsal telencephalon by manually counting the number of cells and measuring the areas of expression within the selected area or by measuring the positively stained area in six anatomically matched sections from three embryos using the Adobe Photoshop CS4 software (Adobe, San Jose, CA, USA) as previously described [15,16,18]. NeuN+ and DAPI+ cells were manually counted in the CP and intermediate zone (IZ). Ki67+, Tbr2+, IdU+, and DAPI+ cells were manually counted and Pax6+ and DAPI+ areas were measured in the VZ, SVZ, and IZ. DCX+ and DAPI+ areas were manually measured in sections in a frame with a 100 m width covering a region from the ventricle to the pial surface of the dorsal telencephalon. The frames used for the counting and measuring are indicated in Fig. 1B–F and A–C. The ratios of positive cells (number of marker+ cells per number of DAPI + cells × 100) and positive areas (DCX and Pax6: marker + area per DAPI + area × 100) were calculated. 2.4. Semi-quantitative RT-PCR The primer sequences are listed in Supplementary Table S1. Semi-quantification of mRNA expression was performed using Adobe Photoshop CS4 and ImageJ (NIH, Bethesda, MD, USA). Gene expression of the target genes was normalized to -actin expression. Three animals per genotype and per developmental stage were analyzed. 3. Results 3.1. SF-1 KO embryos have a reduced number of neurons in the cortical plate of the dorsal telencephalon We performed a histological analysis at E15.5 to evaluate whether the dorsal telencephalon of SF-1 KO embryos had an anomalous structure. H&E staining showed no structural differences between WT and SF-1 KO embryos (Fig. 1A, Fig. S1). The effects on neurogenesis and migration of neural stem/progenitor cells in the dorsal telencephalon in the SF-1 KO embryos were investigated by immunostaining of the neuronal markers DCX and NeuN. The DCX+ area in the CP was decreased (WT: 23.7 ± 0.97%, KO: 19.5 ± 0.91%, p = 0.005), while the DCX+ area in the SVZ/IZ was increased in the SF-1 KO compared to the WT embryos (WT: 44.8 ± 1.12%, KO: 49.5 ± 0.56%, p = 0.003) (Fig. 1B and G). The number of NeuN+ neurons in the SVZ/IZ was increased in the SF-1 KO compared to the WT embryos (WT: 40.0 ± 0.87%, KO: 42.4 ± 0.51%, p = 0.012) (Fig. 1C and H). 3.2. Apical progenitor cells, but not intermediate progenitor cells, are increased in the SF-1 KO mice
2.2. IdU incorporation and cell cycle kinetics For in vivo labeling of the S-phase cells and cell cycle exiting cells, IdU (I7125, Sigma–Aldrich, 50 mg/kg) was intraperitoneally injected into the pregnant mice at 1 h and 24 h prior to sacrificing embryos at E15.5, respectively. To estimate the cell cycle duration, we counted the number of Ki67+, proliferative cells that were labeled by a 1 h pulse of IdU. The fraction of IdU+/Ki67+ cells among all Ki67+ cells provides a rough estimate of the neural stem/progenitor cell cycle duration; a smaller population of IdU+ cells among the Ki67+ cells is indicative of an increased cell cycle length [17,16]. The percentage of cells in the dorsal telencephalon exiting cell cycle was estimated from the ratio of IdU+/Ki67- (postmitotic) and IdU+/Ki67+ (cell cycle non-exiting) cells to all IdU+ cells labeled by a 24 h pulse of IdU.
Next, we analyzed the proliferation of neural stem/progenitor cells by immunostaining at E15.5. The number of cells that were stained positively for Ki67, a marker of proliferative cells, was decreased in the SF-1 KO embryos (WT: 52.8 ± 0.86%, KO: 58.5 ± 1.78%, p = 0.007) (Fig. 1D and I). To estimate which neural stem/progenitor cell population (APCs or IPCs) was affected in the SF-1 KO embryos, the sections were immunostained for Pax6 and Tbr2, which are markers for APCs and IPCs, respectively. Since we could distinguish individual Pax6+ cells to count, we measured the Pax6+ area and performed the statistical analysis. Pax6+ area and Tbr2+ cells were detected in the VZ and SVZ, respectively (Fig. 1E and F). The Pax6+ area was significantly expanded in the SF-1 KO compared to the WT embryos (WT: 28.5 ± 2.13%, KO: 40.0 ± 1.49%, p = 0.003) (Fig. 1E and J). There was no significant difference in the
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Fig. 1. The neuronal distribution and the number of APCs are affected in the dorsal telencephalon of SF-1 KO embryos. (A) Parasagittal sections of E.15.5 dorsal telencephalon from WT and SF-1 KO embryos stained with H&E. (B–F) Parasagittal sections of E15.5 dorsal telencephalon immunostained with antibodies against DCX, NeuN, Ki67, Tbr2, and Pax6. (G and H) At E15.5, the positive area of DCX and the ratio of NeuN+ neurons were increased in the SVZ/IZ, while the DCX+ area and the ratio of NeuN+ neurons was decreased in the CP of SF-1 KO compared to WT embryos. (I and J) The numbers of Ki67+ neural stem/progenitor cells and the area of Pax6+ neural stem/progenitor cells were increased in the SF-1 KO compared to WT embryos. (K) The number of Tbr2+ cells was not significantly affected. The expression area and number of cells were determined in 100 m-wide rectangular areas selected to include VZ, SVZ, and IZ (selected areas are framed in B–F). CP: cortical plate, SVZ: subventricular zone, IZ: intermediate zone. *p < 0.05 (Student’s t-test). Scale bar = 100 m. High-quality magnified images of A–F are shown in Supplemental Fig. S1.
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Fig. 2. S-phase cells are increased and neurogenesis is suppressed in the neural stem/progenitor cells of SF-1 KO embryos. (A) Parasagittal sections of E15.5 dorsal telencephalon immunostained with anti-Nestin. The Nestin+ radial fibers displayed morphological aberrations in the dorsal telencephalon of SF-1 KO embryos. Scale bar = 10 m. Parasagittal sections of E15.5 dorsal telencephalon immunostained with anti-Ki67 and anti-IdU after a 1 h IdU pulse (B) and after a 24 h IdU pulse (C). Cell cycle exit was determined as the ratio of cells that exited cell cycle (red, IdU+/Ki67-, post-mitotic) and cell cycle non-exiting cells (yellow, IdU+/Ki67+) to all IdU-labeled cells (red and yellow) after labeling for 24 h. (D) Ratio of IdU+/Ki67+ cells. In the SF-1 KO embryos, the labeling index after a 1 h pulse of IdU was significantly increased compared to that in WT, indicating a shortened cell cycle. (E) Fraction of cells exiting cell cycle shown as ratio of IdU+/Ki67- (red) to IdU+ (red and yellow) cells. In the SF-1 KO embryos, the ratio was significantly decreased compared to that in WT. (F) The fraction of cells that did not exit cell cycle is shown as the ratio of IdU+/Ki67+ (yellow) to IdU+ (red and yellow) cells. In the SF-1 KO embryos, the ratio was significantly increased compared to that in WT. The number of cells was determined in 100 m-wide rectangular areas (selected areas are framed in B and C). *p < 0.05 (Student’s t-test). Scale bar = 100 m, except in (A) as indicated. High-quality magnified images of A–F are shown in Supplemental Fig. S3.
number of Tbr2+ cells between the SF-1 KO and WT embryos (WT: 25.8 ± 2.17%, KO: 26.3 ± 0.85%) (Fig. 1F and K). APCs display apico-basal polarity with a radially oriented fiber, extending from the VZ to the pial surface of the telencephalon. Immunostaining for Nestin, a marker for APC radial fibers, revealed morphological abnormalities in the fibers in the dorsal telencephalon of SF-1 KO compared to WT embryos (Fig. 2A).
for Ki67 and IdU. The cell cycle length of neural stem/progenitor cells was determined as the ratio of the total number of neural stem/progenitor cells to the number of S-phase cells, which were labeled by IdU 1 h after injection (Fig. 2B). In the SF-1 KO embryos, the ratio of Ki67+/IdU+ to total Ki67+ cells was significantly increased compared to WT embryos at E15.5 (WT: 25.8 ± 1.84%, KO: 32.1 ± 1.98%, p = 0.015) (Fig. 2B and D).
3.3. S-phase cells are increased in the dorsal telencephalon of SF-1 KO mice
3.4. Neurogenesis is inhibited in the dorsal telencephalon by the knockout of SF-1
To investigate the cause of the increase in neural stem/progenitor cells, we estimated the S-phase fraction in proliferative cells using IdU injection and double immunostaining
We next examined cell cycle exiting using IdU-labeling. At 24 h after IdU injection at E15.5, post-mitotic cells were detected as Ki67-/IdU+ cells in the CP and IZ (Fig. 2C). As shown in Fig. 2E, the
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Fig. 3. ESR˛ expression is up-regulated and Cyp19a1 expression down-regulated in the dorsal telencephalon of SF-1 KO embryos. (A) Expression of ESR␣, ESR, and Cyp19a1 in the dorsal telencephalon at E15.5, E18.5, and adult stage in WT embryos. SF-1 was expressed at E15.5 and E18.5 in the dorsal telencephalon, but not in the adult neocortex in WT embryos. (B) Expression of SF-1 in the dorsal telencephalon at E15.5 in SF-1 KO and WT embryos. (C) Gene expression levels as determined by semi-quantitative RT-PCR and normalized to -actin expression in the dorsal telencephalon of WT and SF-1 KO embryos at E15.5. The expression of ESR␣ was up-regulated and Cyp19a1 was down-regulated in the SF-1 KO embryos, respectively (C). Expression levels of ESR, NeuroD, and Ngn2 were not significantly different between SF-1 KO and WT embryos. *p < 0.05 (Student’s t-test).
number of post-mitotic cells in the CP and IZ of SF-1 KO embryos was significantly reduced compared to that in WT embryos, indicating that cell cycle exit of neural stem/progenitor cells was inhibited by SF-1 KO (WT: 48.6 ± 1.69%, KO: 44.8 ± 1.67%, p = 0.049) (Fig. 2C and E). Consequently, the number of cell cycle non-exiting cells of SF-1 KO embryos was significantly increased compared to that in WT embryos (WT: 51.4 ± 1.69%, KO: 55.2 ± 1.67%, p = 0.049). 3.5. Expression of ESR˛ and Cyp19a1 in the dorsal telencephalon is affected in SF-1 KO embryos Estrogen signaling is important for proper neurogenesis by controlling neuronal migration and differentiation [12,13]. To determine whether estrogen signaling is involved in the abnormal development of the neocortex by the SF-1 deletion, we investigated the mRNA expression of estrogen receptors ESR˛, ESRˇ, and of Cyp19a1, the gene encoding the steroidogenic aromatase enzyme, in the dorsal telencephalon at E15.5, E18.5, and in adulthood by using RT-PCR (Fig. 3A). In WT embryos, SF-1 mRNA was detected in the dorsal telencephalon at E15.5 and E18.5, but not in the neocortex in adulthood (Fig. 3A). In the SF-1 KO embryos, SF-1 expression was not detected in the dorsal telencephalon at E15.5 (Fig. 3B). To compare the mRNA expression levels of ESR˛, ESRˇ, Cyp19a1, and the neurogenesis-related genes NeuroD and Ngn2 between
WT and SF-1 KO embryos, we performed semi-quantitative RTPCR. The expression level of ESR˛ was significantly increased (WT: 1.00 ± 0.002, KO: 1.60 ± 0.236, p = 0.012), whereas that of Cyp19a1 was decreased (WT: 1.00 ± 0.040, KO: 0.78 ± 0.010, p = 0.0008) in SF-1 KO compared to WT embryos (Fig. 3C). The expression levels of ESRˇ (WT: 1.00 ± 0.234, KO: 1.11 ± 0.110), NeuroD (WT: 1.00 ± 0.027, KO: 0.97 ± 0.003), and Ngn2 (WT: 1.00 ± 0.027, KO: 0.97 ± 0.003) did not differ between the WT and SF-1 KO embryos (Fig. 3C). These data suggest that SF-1 deletion affects the expression of ESR˛ and Cyp19a1, but not that of NeuroD and Ngn2, two important factors for neurogenesis and neuronal migration. 4. Discussion Previous studies have suggested that SF-1 is required for the organization of the VMH [7,8]. In the present study, although we did not detect any obvious morphological changes in the dorsal telencephalon of SF-1 KO embryos at E15.5 by H&E staining, immunostaining of DCX and NeuN revealed that the number of immature neurons was reduced in the CP and increased in the SVZ by the SF-1 deficiency. In addition, immunostaining of Ki67 and Pax6 showed an increase in APCs in the VZ of SF-1 KO embryos. To quantify the fraction of S-phase cells, we counted the percentage of Ki67+ progenitor cells that were labeled by a single 1 h pulse of
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BrdU. Because in neural stem cells the length of S-phase remains relatively constant while the length of G1 regulates proliferation, the labeling index provides an estimation of cell cycle length [17]. IdU-labeling and the labeling index indicated an increase in the number of APCs accompanied by shortened cell cycle duration in the dorsal telencephalon of SF-1 KO embryos. Furthermore, immunostaining of Nestin showed that the morphology of APC radial fibers was irregular in the SF-1 KO compared to the WT embryos. Because the radial fibers act as foothold for the immature neurons during migration from the ventricle toward the pial surface, these results suggested that neuronal migration was affected by the SF-1 deficiency. The Nestin enhancer contains SF-1 binding sites and SF-1 mediates basal Nestin expression in undifferentiated P19EC cells [19]. As Nestin is a marker of the differentiation stage of neural stem cells, it is possible that SF-1 controls the neuronal differentiation of APCs in the dorsal telencephalon, thereby mediating its morphological characteristics. Estrogen signaling and SF-1, which are highly expressed in the hypothalamus, are important for sexual differentiation [6]. In our study, no gender differences were observed in the developing neocortex in male and female embryos. Sexual differentiation of the brain takes place in the critical period around birth; therefore, the stages analyzed in this study (E15 and E18) were too early to analyze the roles of SF-1 for sexual differentiation of brain. Although the majority of studies on SF-1 using immunostaining and in situ hybridization detected SF-1 expression only in the VMH in the brain [5,4], GDX data indicated that SF-1 is expressed in the dorsal telencephalon at E13.5 [14]. In the present study, we detected faint expression of SF-1 in the dorsal telencephalon of WT embryos but not in SF-1 KO embryos at E15.5 by RT-PCR, indicating that the SF-1 expression level in the dorsal telencephalon is much lower than that in the hypothalamus during embryogenesis. Previous studies have shown that aromatase and ESR␣ are present in the APCs and IPCs during neocortical development, respectively [12]. ESR KO embryos showed neuronal deficit during corticogenesis [13]. In addition, aromatase, ESR␣, and ESR play crucial roles in the development of the neocortex [12,13]. Our results confirmed that Cyp19a1, ESR˛, and ESRˇ are expressed in the developing and adult neocortex. In addition, ESR˛ and Cyp19a1 expression were affected by the SF-1 deficiency in the dorsal telencephalon. As Cyp19a1 is a target gene of SF-1 [20], the reduction of Cyp19a1 expression in the SF-1 KO dorsal telencephalon might be due to the SF-1 deficiency. The reduction in aromatase expression led to reduced estrogen production in the neurons and neural stem/progenitor cells of the dorsal telencephalon [12]. ESR˛ is not a direct target gene of SF-1 [21,22]. It is possible that insufficient levels of estrogen due to the reduction of aromatase in the dorsal telencephalon and the absence of gonads in SF-1 KO embryos, led to increased ESR˛ expression. Therefore, the indirect regulation of ESR˛ expression by SF-1 might induce changes in the expression of target genes downstream of SF-1 and in the estrogen signaling in SF-1 KO embryos. Although the underlying mechanisms are unknown, the disruption of estrogen signaling might be related to the inhibition of neurogenesis and neuronal migration in the SF-1 KO embryos. In conclusion, our results suggest that the weak expression of SF1 in the dorsal telencephalon is involved in the cell cycle regulation, neurogenesis, and neuronal migration to ensure proper neocortical development, by controlling the estrogen signaling.
Acknowledgements We thank A. Tagami for technical assistance and S. Takahashi for administrative assistance. This work was supported by JSPS KAKENHI grant number 26460258, 90523994.
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Research article
Involvement of SF-1 in neurogenesis and neuronal migration in the developing neocortex Munekazu Komada, Mifumi Takahashi, Yayoi Ikeda ∗ Department of Anatomy, School of Dentistry, Aichi Gakuin University, Nagoya, Japan
h i g h l i g h t s • • • • •
SF-1 was expressed in the dorsal telencephalon at E15.5–E18.5, but not in adulthood. In SF-1 KO embryos, neurons in the IZ/SVZ were increased, in the CP decreased. The APCs increased and radial fibers showed abnormal morphology in SF-1 KO embryos. Cell cycle duration was shortened and exit inhibited in SF-1 KO stem/progenitor cells. Expression of ESR˛ was up- and of Cyp19a1 down-regulated in SF-1 KO embryos.
a r t i c l e
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Article history: Received 9 March 2015 Received in revised form 2 June 2015 Accepted 3 June 2015 Available online 9 June 2015 Keywords: Steroidogenic factor-1 Dorsal telencephalon Neurogenesis Proliferation Cell cycle Estrogen signaling
a b s t r a c t The nuclear receptor steroidogenic factor-1 (SF-1) plays essential roles in the development and function of the endocrine and reproductive systems. During embryogenesis, SF-1 is expressed in the ventromedial hypothalamic nucleus (VMH) and regulates the migration and terminal differentiation of the VMH neurons. Additionally, in situ hybridization data indicated SF-1 expression in the dorsal telencephalon at embryonic day (E) 13.5. In this study, we investigated the neocortical development in SF-1 knockout (KO) mouse embryos. The number of neurons was increased in the intermediate/subventricular zones and decreased in the cortical plate in the SF-1 KO embryos. SF-1 KO embryos produced more neural stem/progenitor cells, especially apical progenitor cells, and showed abnormal radial glial fiber morphology. The increase in neural stem/progenitor cells was caused by an increased S-phase fraction in the proliferative cells and the inhibition of cell cycle exit in these cells. The mRNA expression of the estrogen receptor ESR˛ was up-regulated and that of the estrogen synthetase Cyp19a1 was down-regulated in the dorsal telencephalon of SF-1 KO embryos. We showed that SF-1 is expressed in the dorsal telencephalon at E15.5 and E18.5, but not in adult animals. Our data demonstrated that SF-1 is involved in cell cycle regulation, neurogenesis, and neuronal migration via controlling the estrogen signaling for proper neocortical development. © 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Steroidogenic factor 1 (SF-1), which is encoded by the NR5A1 gene, is a member of the nuclear hormone receptor family [1]. SF-1 is required for adrenal and gonadal steroidogenesis, control-
Abbreviations: APC, apical progenitor cell; CP, cortical plate; DCX, doublecortin; E, embryonic day; ESR, estrogen receptor; GXD, gene expression database; IdU, iododeoxyuridine; IPC, intermediate progenitor cell; IZ, intermediate zone; KO, knockout; SF-1, steroidogenic factor-1; SVZ, subventricular zone; VMH, ventromedial hypothalamic nucleus; VZ, ventricular zone. ∗ Corresponding author at: 1–100 Kusumoto-cho, Chikusa-ku, Nagoya, Aichi 464–8650, Japan. Fax: +81 52 757 6755. E-mail address: [email protected] (Y. Ikeda). http://dx.doi.org/10.1016/j.neulet.2015.06.005 0304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.
ling the expression of a number of key target genes including those encoding cytochrome P450 steroid hydroxylase and aromatase in adulthood [2]. It is expressed in the gonads, adrenal cortex, pituitary, and hypothalamus during embryogenesis [3,4], and SF-1 knockout (KO) mice exhibit agenesis of the adrenal gland and gonads, deficiency of pituitary gonadotropin secretion, and abnormal structure of the ventromedial hypothalamic nucleus (VMH), suggesting that SF-1 plays critical roles in the development of the tissues at all the levels of the hypothalamus-pituitarygonad/adrenal axis [5,6]. The disorganization of the VMH was shown to be due to the accumulation of immature neurons in the proliferative zone of the diencephalon, which was caused by migration defects in these neurons [7]. In addition, misexpression of VMH markers and loss of neuronal projections to the bed nucleus of
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the stria terminalis and the amygdala in SF-1 KO mice have been reported [8]. These data suggest that SF-1 is involved in the regulation of cell fate determination, neuronal migration, and terminal differentiation during the development of the VMH. During neocortical development, apical progenitor cells (APCs, neural stem cells) in the ventricular zone (VZ) and intermediate progenitor cells (IPCs, neural progenitor cells) in the subventricular zone (SVZ) self-renew and generate the neurons [9]. APCs and IPCs are distinguished by their distribution, self-renewal ability, capacity for neurogenesis, and expression of transcription factors [10]. The division patterns of APCs and IPCs affect the number of neurons and determine the neocortical size [11]. Their proliferation and neurogenesis are orchestrated by a signaling network of factors including morphogens, transcription factors, and growth factors. For example, estrogen receptors and aromatase that are expressed in the developing dorsal telencephalon control the proliferation, migration of neural stem/progenitor cells, and neurogenesis [12,13]. In situ hybridization data available in the Gene Expression Database (GXD), a publicly available resource for gene expression information from laboratory mice, indicated that SF-1 is expressed in the dorsal telencephalon at E13.5 [14]. However, the role of SF1 in this region remains unknown. In this study, we investigated the roles of SF-1 in the morphogenesis of the neocortex using SF-1 KO embryos. First, we analyzed the histological changes and the changes in expression of markers for neurogenesis, proliferation, and migration of neural stem/progenitor cells using immunostaining. Next, the cell cycle kinetics of neural stem/progenitor cells were examined by iododeoxyuridine (IdU)-labeling and double immunostaining for Ki67 and IdU. Finally, we compared the expression of genes involved in estrogen signaling and neurogenesis during the development of the neocortex in wild-type (WT) and SF-1 KO embryos. The results suggested that SF-1 is involved in the regulation of neurogenesis, proliferation, and neuronal migration in the developing neocortex, as well as in the hypothalamus. 2. Materials and methods 2.1. Immunofluorescence Immunofluorescence staining of the sections was performed as described previously [15,16]. The antibodies used in the study and detailed methods are described in the Supplemental methods. Primary and secondary antibody incubations were conducted overnight and for 3 h, respectively, at room temperature. Nuclei were counterstained with DAPI (D9542, Sigma–Aldrich, St Louis, MO, USA; 1:1000) for 3 h at RT. Six sections from three embryos per genotype were used for immunofluorescence analysis of each marker.
2.3. Quantitative immunofluorescence analysis Immunofluorescence staining was quantified in selected areas of the dorsal telencephalon by manually counting the number of cells and measuring the areas of expression within the selected area or by measuring the positively stained area in six anatomically matched sections from three embryos using the Adobe Photoshop CS4 software (Adobe, San Jose, CA, USA) as previously described [15,16,18]. NeuN+ and DAPI+ cells were manually counted in the CP and intermediate zone (IZ). Ki67+, Tbr2+, IdU+, and DAPI+ cells were manually counted and Pax6+ and DAPI+ areas were measured in the VZ, SVZ, and IZ. DCX+ and DAPI+ areas were manually measured in sections in a frame with a 100 m width covering a region from the ventricle to the pial surface of the dorsal telencephalon. The frames used for the counting and measuring are indicated in Fig. 1B–F and A–C. The ratios of positive cells (number of marker+ cells per number of DAPI + cells × 100) and positive areas (DCX and Pax6: marker + area per DAPI + area × 100) were calculated. 2.4. Semi-quantitative RT-PCR The primer sequences are listed in Supplementary Table S1. Semi-quantification of mRNA expression was performed using Adobe Photoshop CS4 and ImageJ (NIH, Bethesda, MD, USA). Gene expression of the target genes was normalized to -actin expression. Three animals per genotype and per developmental stage were analyzed. 3. Results 3.1. SF-1 KO embryos have a reduced number of neurons in the cortical plate of the dorsal telencephalon We performed a histological analysis at E15.5 to evaluate whether the dorsal telencephalon of SF-1 KO embryos had an anomalous structure. H&E staining showed no structural differences between WT and SF-1 KO embryos (Fig. 1A, Fig. S1). The effects on neurogenesis and migration of neural stem/progenitor cells in the dorsal telencephalon in the SF-1 KO embryos were investigated by immunostaining of the neuronal markers DCX and NeuN. The DCX+ area in the CP was decreased (WT: 23.7 ± 0.97%, KO: 19.5 ± 0.91%, p = 0.005), while the DCX+ area in the SVZ/IZ was increased in the SF-1 KO compared to the WT embryos (WT: 44.8 ± 1.12%, KO: 49.5 ± 0.56%, p = 0.003) (Fig. 1B and G). The number of NeuN+ neurons in the SVZ/IZ was increased in the SF-1 KO compared to the WT embryos (WT: 40.0 ± 0.87%, KO: 42.4 ± 0.51%, p = 0.012) (Fig. 1C and H). 3.2. Apical progenitor cells, but not intermediate progenitor cells, are increased in the SF-1 KO mice
2.2. IdU incorporation and cell cycle kinetics For in vivo labeling of the S-phase cells and cell cycle exiting cells, IdU (I7125, Sigma–Aldrich, 50 mg/kg) was intraperitoneally injected into the pregnant mice at 1 h and 24 h prior to sacrificing embryos at E15.5, respectively. To estimate the cell cycle duration, we counted the number of Ki67+, proliferative cells that were labeled by a 1 h pulse of IdU. The fraction of IdU+/Ki67+ cells among all Ki67+ cells provides a rough estimate of the neural stem/progenitor cell cycle duration; a smaller population of IdU+ cells among the Ki67+ cells is indicative of an increased cell cycle length [17,16]. The percentage of cells in the dorsal telencephalon exiting cell cycle was estimated from the ratio of IdU+/Ki67- (postmitotic) and IdU+/Ki67+ (cell cycle non-exiting) cells to all IdU+ cells labeled by a 24 h pulse of IdU.
Next, we analyzed the proliferation of neural stem/progenitor cells by immunostaining at E15.5. The number of cells that were stained positively for Ki67, a marker of proliferative cells, was decreased in the SF-1 KO embryos (WT: 52.8 ± 0.86%, KO: 58.5 ± 1.78%, p = 0.007) (Fig. 1D and I). To estimate which neural stem/progenitor cell population (APCs or IPCs) was affected in the SF-1 KO embryos, the sections were immunostained for Pax6 and Tbr2, which are markers for APCs and IPCs, respectively. Since we could distinguish individual Pax6+ cells to count, we measured the Pax6+ area and performed the statistical analysis. Pax6+ area and Tbr2+ cells were detected in the VZ and SVZ, respectively (Fig. 1E and F). The Pax6+ area was significantly expanded in the SF-1 KO compared to the WT embryos (WT: 28.5 ± 2.13%, KO: 40.0 ± 1.49%, p = 0.003) (Fig. 1E and J). There was no significant difference in the
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Fig. 1. The neuronal distribution and the number of APCs are affected in the dorsal telencephalon of SF-1 KO embryos. (A) Parasagittal sections of E.15.5 dorsal telencephalon from WT and SF-1 KO embryos stained with H&E. (B–F) Parasagittal sections of E15.5 dorsal telencephalon immunostained with antibodies against DCX, NeuN, Ki67, Tbr2, and Pax6. (G and H) At E15.5, the positive area of DCX and the ratio of NeuN+ neurons were increased in the SVZ/IZ, while the DCX+ area and the ratio of NeuN+ neurons was decreased in the CP of SF-1 KO compared to WT embryos. (I and J) The numbers of Ki67+ neural stem/progenitor cells and the area of Pax6+ neural stem/progenitor cells were increased in the SF-1 KO compared to WT embryos. (K) The number of Tbr2+ cells was not significantly affected. The expression area and number of cells were determined in 100 m-wide rectangular areas selected to include VZ, SVZ, and IZ (selected areas are framed in B–F). CP: cortical plate, SVZ: subventricular zone, IZ: intermediate zone. *p < 0.05 (Student’s t-test). Scale bar = 100 m. High-quality magnified images of A–F are shown in Supplemental Fig. S1.
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Fig. 2. S-phase cells are increased and neurogenesis is suppressed in the neural stem/progenitor cells of SF-1 KO embryos. (A) Parasagittal sections of E15.5 dorsal telencephalon immunostained with anti-Nestin. The Nestin+ radial fibers displayed morphological aberrations in the dorsal telencephalon of SF-1 KO embryos. Scale bar = 10 m. Parasagittal sections of E15.5 dorsal telencephalon immunostained with anti-Ki67 and anti-IdU after a 1 h IdU pulse (B) and after a 24 h IdU pulse (C). Cell cycle exit was determined as the ratio of cells that exited cell cycle (red, IdU+/Ki67-, post-mitotic) and cell cycle non-exiting cells (yellow, IdU+/Ki67+) to all IdU-labeled cells (red and yellow) after labeling for 24 h. (D) Ratio of IdU+/Ki67+ cells. In the SF-1 KO embryos, the labeling index after a 1 h pulse of IdU was significantly increased compared to that in WT, indicating a shortened cell cycle. (E) Fraction of cells exiting cell cycle shown as ratio of IdU+/Ki67- (red) to IdU+ (red and yellow) cells. In the SF-1 KO embryos, the ratio was significantly decreased compared to that in WT. (F) The fraction of cells that did not exit cell cycle is shown as the ratio of IdU+/Ki67+ (yellow) to IdU+ (red and yellow) cells. In the SF-1 KO embryos, the ratio was significantly increased compared to that in WT. The number of cells was determined in 100 m-wide rectangular areas (selected areas are framed in B and C). *p < 0.05 (Student’s t-test). Scale bar = 100 m, except in (A) as indicated. High-quality magnified images of A–F are shown in Supplemental Fig. S3.
number of Tbr2+ cells between the SF-1 KO and WT embryos (WT: 25.8 ± 2.17%, KO: 26.3 ± 0.85%) (Fig. 1F and K). APCs display apico-basal polarity with a radially oriented fiber, extending from the VZ to the pial surface of the telencephalon. Immunostaining for Nestin, a marker for APC radial fibers, revealed morphological abnormalities in the fibers in the dorsal telencephalon of SF-1 KO compared to WT embryos (Fig. 2A).
for Ki67 and IdU. The cell cycle length of neural stem/progenitor cells was determined as the ratio of the total number of neural stem/progenitor cells to the number of S-phase cells, which were labeled by IdU 1 h after injection (Fig. 2B). In the SF-1 KO embryos, the ratio of Ki67+/IdU+ to total Ki67+ cells was significantly increased compared to WT embryos at E15.5 (WT: 25.8 ± 1.84%, KO: 32.1 ± 1.98%, p = 0.015) (Fig. 2B and D).
3.3. S-phase cells are increased in the dorsal telencephalon of SF-1 KO mice
3.4. Neurogenesis is inhibited in the dorsal telencephalon by the knockout of SF-1
To investigate the cause of the increase in neural stem/progenitor cells, we estimated the S-phase fraction in proliferative cells using IdU injection and double immunostaining
We next examined cell cycle exiting using IdU-labeling. At 24 h after IdU injection at E15.5, post-mitotic cells were detected as Ki67-/IdU+ cells in the CP and IZ (Fig. 2C). As shown in Fig. 2E, the
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Fig. 3. ESR˛ expression is up-regulated and Cyp19a1 expression down-regulated in the dorsal telencephalon of SF-1 KO embryos. (A) Expression of ESR␣, ESR, and Cyp19a1 in the dorsal telencephalon at E15.5, E18.5, and adult stage in WT embryos. SF-1 was expressed at E15.5 and E18.5 in the dorsal telencephalon, but not in the adult neocortex in WT embryos. (B) Expression of SF-1 in the dorsal telencephalon at E15.5 in SF-1 KO and WT embryos. (C) Gene expression levels as determined by semi-quantitative RT-PCR and normalized to -actin expression in the dorsal telencephalon of WT and SF-1 KO embryos at E15.5. The expression of ESR␣ was up-regulated and Cyp19a1 was down-regulated in the SF-1 KO embryos, respectively (C). Expression levels of ESR, NeuroD, and Ngn2 were not significantly different between SF-1 KO and WT embryos. *p < 0.05 (Student’s t-test).
number of post-mitotic cells in the CP and IZ of SF-1 KO embryos was significantly reduced compared to that in WT embryos, indicating that cell cycle exit of neural stem/progenitor cells was inhibited by SF-1 KO (WT: 48.6 ± 1.69%, KO: 44.8 ± 1.67%, p = 0.049) (Fig. 2C and E). Consequently, the number of cell cycle non-exiting cells of SF-1 KO embryos was significantly increased compared to that in WT embryos (WT: 51.4 ± 1.69%, KO: 55.2 ± 1.67%, p = 0.049). 3.5. Expression of ESR˛ and Cyp19a1 in the dorsal telencephalon is affected in SF-1 KO embryos Estrogen signaling is important for proper neurogenesis by controlling neuronal migration and differentiation [12,13]. To determine whether estrogen signaling is involved in the abnormal development of the neocortex by the SF-1 deletion, we investigated the mRNA expression of estrogen receptors ESR˛, ESRˇ, and of Cyp19a1, the gene encoding the steroidogenic aromatase enzyme, in the dorsal telencephalon at E15.5, E18.5, and in adulthood by using RT-PCR (Fig. 3A). In WT embryos, SF-1 mRNA was detected in the dorsal telencephalon at E15.5 and E18.5, but not in the neocortex in adulthood (Fig. 3A). In the SF-1 KO embryos, SF-1 expression was not detected in the dorsal telencephalon at E15.5 (Fig. 3B). To compare the mRNA expression levels of ESR˛, ESRˇ, Cyp19a1, and the neurogenesis-related genes NeuroD and Ngn2 between
WT and SF-1 KO embryos, we performed semi-quantitative RTPCR. The expression level of ESR˛ was significantly increased (WT: 1.00 ± 0.002, KO: 1.60 ± 0.236, p = 0.012), whereas that of Cyp19a1 was decreased (WT: 1.00 ± 0.040, KO: 0.78 ± 0.010, p = 0.0008) in SF-1 KO compared to WT embryos (Fig. 3C). The expression levels of ESRˇ (WT: 1.00 ± 0.234, KO: 1.11 ± 0.110), NeuroD (WT: 1.00 ± 0.027, KO: 0.97 ± 0.003), and Ngn2 (WT: 1.00 ± 0.027, KO: 0.97 ± 0.003) did not differ between the WT and SF-1 KO embryos (Fig. 3C). These data suggest that SF-1 deletion affects the expression of ESR˛ and Cyp19a1, but not that of NeuroD and Ngn2, two important factors for neurogenesis and neuronal migration. 4. Discussion Previous studies have suggested that SF-1 is required for the organization of the VMH [7,8]. In the present study, although we did not detect any obvious morphological changes in the dorsal telencephalon of SF-1 KO embryos at E15.5 by H&E staining, immunostaining of DCX and NeuN revealed that the number of immature neurons was reduced in the CP and increased in the SVZ by the SF-1 deficiency. In addition, immunostaining of Ki67 and Pax6 showed an increase in APCs in the VZ of SF-1 KO embryos. To quantify the fraction of S-phase cells, we counted the percentage of Ki67+ progenitor cells that were labeled by a single 1 h pulse of
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BrdU. Because in neural stem cells the length of S-phase remains relatively constant while the length of G1 regulates proliferation, the labeling index provides an estimation of cell cycle length [17]. IdU-labeling and the labeling index indicated an increase in the number of APCs accompanied by shortened cell cycle duration in the dorsal telencephalon of SF-1 KO embryos. Furthermore, immunostaining of Nestin showed that the morphology of APC radial fibers was irregular in the SF-1 KO compared to the WT embryos. Because the radial fibers act as foothold for the immature neurons during migration from the ventricle toward the pial surface, these results suggested that neuronal migration was affected by the SF-1 deficiency. The Nestin enhancer contains SF-1 binding sites and SF-1 mediates basal Nestin expression in undifferentiated P19EC cells [19]. As Nestin is a marker of the differentiation stage of neural stem cells, it is possible that SF-1 controls the neuronal differentiation of APCs in the dorsal telencephalon, thereby mediating its morphological characteristics. Estrogen signaling and SF-1, which are highly expressed in the hypothalamus, are important for sexual differentiation [6]. In our study, no gender differences were observed in the developing neocortex in male and female embryos. Sexual differentiation of the brain takes place in the critical period around birth; therefore, the stages analyzed in this study (E15 and E18) were too early to analyze the roles of SF-1 for sexual differentiation of brain. Although the majority of studies on SF-1 using immunostaining and in situ hybridization detected SF-1 expression only in the VMH in the brain [5,4], GDX data indicated that SF-1 is expressed in the dorsal telencephalon at E13.5 [14]. In the present study, we detected faint expression of SF-1 in the dorsal telencephalon of WT embryos but not in SF-1 KO embryos at E15.5 by RT-PCR, indicating that the SF-1 expression level in the dorsal telencephalon is much lower than that in the hypothalamus during embryogenesis. Previous studies have shown that aromatase and ESR␣ are present in the APCs and IPCs during neocortical development, respectively [12]. ESR KO embryos showed neuronal deficit during corticogenesis [13]. In addition, aromatase, ESR␣, and ESR play crucial roles in the development of the neocortex [12,13]. Our results confirmed that Cyp19a1, ESR˛, and ESRˇ are expressed in the developing and adult neocortex. In addition, ESR˛ and Cyp19a1 expression were affected by the SF-1 deficiency in the dorsal telencephalon. As Cyp19a1 is a target gene of SF-1 [20], the reduction of Cyp19a1 expression in the SF-1 KO dorsal telencephalon might be due to the SF-1 deficiency. The reduction in aromatase expression led to reduced estrogen production in the neurons and neural stem/progenitor cells of the dorsal telencephalon [12]. ESR˛ is not a direct target gene of SF-1 [21,22]. It is possible that insufficient levels of estrogen due to the reduction of aromatase in the dorsal telencephalon and the absence of gonads in SF-1 KO embryos, led to increased ESR˛ expression. Therefore, the indirect regulation of ESR˛ expression by SF-1 might induce changes in the expression of target genes downstream of SF-1 and in the estrogen signaling in SF-1 KO embryos. Although the underlying mechanisms are unknown, the disruption of estrogen signaling might be related to the inhibition of neurogenesis and neuronal migration in the SF-1 KO embryos. In conclusion, our results suggest that the weak expression of SF1 in the dorsal telencephalon is involved in the cell cycle regulation, neurogenesis, and neuronal migration to ensure proper neocortical development, by controlling the estrogen signaling.
Acknowledgements We thank A. Tagami for technical assistance and S. Takahashi for administrative assistance. This work was supported by JSPS KAKENHI grant number 26460258, 90523994.
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