Roles of chromatin remodelers in maintenance mechanisms of multipotency of mouse trunk neural crest cells in the formation of neural crest-derived stem cells.

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Roles of chromatin remodelers in maintenance mechanisms of multipotency of mouse trunk neural crest cells in the formation of neural crest-derived stem cells

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Kyohei Fujita 1, Ryuhei Ogawa 1, Syunsaku Kawawaki, Kazuo Ito

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Department of Biological Sciences, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan

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A R T I C L E I N F O

A B S T R A C T

Article history:

We analyzed roles of two chromatin remodelers, Chromodomain Helicase DNA-binding

Received 5 February 2014

protein 7 (CHD7) and SWItch/Sucrose NonFermentable-B (SWI/SNF-B), and Bone

Received in revised form

Morphogenetic Protein (BMP)/Wnt signaling in the maintenance of the multipotency of

28 April 2014

mouse trunk neural crest cells, leading to the formation of mouse neural crest-derived

Accepted 2 May 2014

stem cells (mouse NCSCs). CHD7 was expressed in the undifferentiated neural crest cells

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and in the dorsal root ganglia (DRG) and sciatic nerve, typical tissues containing NCSCs. BMP/Wnt signaling stimulated the expression of CHD7 and participated in maintaining

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Keywords: Neural crest-derived stem cells Chromatin remodeler BMP/Wnt signaling Mouse

the multipotency of neural crest cells. Furthermore, the promotion of CHD7 expression maintained the multipotency of these cells. The inhibition of CHD7 and SWI/SNF-B expression significantly suppressed the maintenance of the multipotency of these cells. In addition, BMP/Wnt treatment promoted CHD7 expression and caused the increase of the percentage of multipotent cells in DRG. Thus, the present data suggest that the chromatin remodelers as well as BMP/Wnt signaling play essential roles in the maintenance of the multipotency of mouse trunk neural crest cells and in the formation of mouse NCSCs. 2014 Published by Elsevier Ireland Ltd.

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The neural crest is a transient embryonic structure in vertebrates. Neural crest cells arise within the neural fold, migrate ventrally and laterally, and contribute to a wide variety of cell types, such as peripheral neurons and their supportive cells, pigment cells, skeletal derivatives, and smooth muscle cells (Le Douarin and Kalcheim, 1999; Hall, 1999). Recent studies have revealed that some neural crest cells maintain multipotency and form neural crest-derived

Introduction

stem cells (NCSCs) (Nagoshi et al., 2009; Shakhova and Sommer, 2010; Achilleos and Trainor, 2012; Sieber-Blum, 2012). NCSCs have been shown to exist in late embryonic and adult tissues such as dorsal root ganglia (DRG) (Li et al., 2007; Nagoshi et al., 2008), sciatic nerve (Morrison et al., 1999; Joseph et al., 2004), gut (Kruger et al., 2002; Bixby et al., 2004), heart (Tomita et al., 2005), hair follicle (Sieber-Blum et al., 2004), and bone marrow (Nagoshi et al., 2008). However, little is known about the formation mechanisms of NCSCs.

* Corresponding author. Tel.: +81 6 6850 5807; fax: +81 6 6850 5817. E-mail address: [email protected] (K. Ito). 1 Both authors contributed equally to this paper. http://dx.doi.org/10.1016/j.mod.2014.05.001 0925-4773/ 2014 Published by Elsevier Ireland Ltd.

Please cite this article in press as: Fujita, K. et al, Roles of chromatin remodelers in maintenance mechanisms of multipotency of mouse trunk neural crest cells in the formation of neural crest-derived stem cells, Mech. Dev. (2014), http://dx.doi.org/10.1016/j.mod.2014.05.001

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Fig. 1 – Expression patterns of CHD7, Sox10, or p75 during mouse trunk neural crest development. The top, bottom, left, and right of each photograph correspond to the dorsal, ventral, proximal, and distal side of the embryo, respectively. (A–J) Transverse sections of a 28-somite stage embryo at the unsegmented level. (A–E) and (F–J) are neighboring sections. (A) Bright-field image. (B) Expression pattern of CHD7 in the same field as (A). (C) Expression pattern of Sox10 in the same field as (A). (D) DAPI nuclear stain of the same field as (A). (E) Merged image of (B–D). (F) Bright-field image. (G) Expression pattern of CHD7 in the same field as (F). (H) Expression pattern of p75 in the same field as (F). (I) DAPI nuclear stain of the same field as (F). (J) Merged image of (G–I). (K–T) Transverse sections of a 26-somite stage embryo at the 26-somite level. (K–O) and (P–T) are neighboring sections. (K) Bright-field image. (L) Expression pattern of CHD7 in the same field as (K). (M) Expression pattern of Sox10 in the same field as (K). (N) DAPI nuclear stain of the same field as (K). (O) Merged image of (L–N). (P) Bright-field image. (Q) Expression pattern of CHD7 in the same field as (P). (R) Expression pattern of p75 in the same field as (P). (S) DAPI nuclear stain of the same field as (P). (T) Merged image of (Q–S). (U–D 0 ) Oblique sections of a 26-somite stage embryo at the 4-somite level. (U–Y) and (Z–D 0 ) are neighboring sections. (U) Bright-field image. (V) Expression pattern of CHD7 in the same field as (U). (W) Expression pattern of Sox10 in the same field as (U). (X) DAPI nuclear stain of the same field as (U). (Y) Merged image of (V– X). (Z) Bright-field image. (A 0 ) Expression pattern of CHD7 in the same field as (Z). (B 0 ) Expression pattern of p75 in the same field as (Z). (C 0 ) DAPI nuclear stain of the same field as (Z). (D 0 ) Merged image of (A 0 –C 0 ). White arrows indicate the neural crest cells expressing both CHD7 and Sox10 or both CHD7 and p75. White arrowheads show a cell expressing both CHD7 and Sox10 in the dorsal neural tube that the premigratory neural crest cells exist. NT, neural tube. Scale bars = 20 lm.

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It has been shown that Bone Morphogenetic Protein (BMP) and Wnt are involved not only in neural crest induction in chick and Xenopus (Knecht and Brooner-Fraser, 2002; Steventon et al., 2009; Stuhlmiller and Garcıá-Castro, 2012) but also in the maintenance of the undifferentiated state of

neural crest cells in mouse and rat (Kle´ber et al., 2004). Moreover, a recent study has elucidated that two chromatin remodelers, Chromodomain Helicase DNA-binding protein 7 (CHD7) and SWItch/Sucrose NonFermentable-B (SWI/SNF-B), play important roles in neural crest induction by controlling


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Fig. 2 – Expression patterns of CHD7, Sox10, or p75 in mouse trunk neural crest cell cultures. Immunostaining with antiCHD7, anti-Sox10, and anti-p75 was performed on culture day 2, 4, and 6. (A and F) Bright-field images. (B and G) Anti-CHD7positive cells in the same fields as (A) and (F), respectively. (C and H) Anti-Sox10-positive cells in the same fields as (A) and (F), respectively. (D and I) DAPI nuclear stains of the same fields as (A) and (F), respectively. (E) Merged image of (B–D). (J) Merged image of (G–I). (K) Percentage of cells expressing CHD7 per total cells in a neural crest cell cluster (each cluster was derived from a neural tube explant). The statistically significant decrease (P < 0.05, t-test) in the percentage of anti-CHD7-positive cells was found on culture day 6 (asterisk). (L) Percentage of cells expressing Sox10 per total cells in a neural crest cell cluster. The statistically significant decrease (P < 0.05, t-test) in the percentage of anti-Sox10-positive cells was found on culture day 6 (asterisk). (M) Percentage of cells expressing both CHD7 and Sox10 per total cells in a neural crest cell cluster. The statistically significant decrease (P < 0.05, t-test) in the percentage of double-positive cells was observed on culture day 6 (asterisk). Data in (K–M) are expressed as the average (±SEM) of separate counts of 5–7 clusters (the number in a parenthesis on each bar). (N and S) Bright-field images. (O and T) Anti-CHD7-positive cells in the same fields as (N) and (S), respectively. (P and U) Anti-p75positive cells in the same fields as (N) and (S), respectively. (Q and V) DAPI nuclear stains of the same fields as (N) and (S), respectively. (R) Merged image of (O–Q). (W) Merged image of (T–V). (X) Percentage of cells expressing CHD7 per total cells in a neural crest cell cluster. The statistically significant decrease (P < 0.05, t-test) in the percentage of anti-CHD7-positive cells was found on culture day 6 (asterisk). (Y) Percentage of cells expressing p75 per total cells in a neural crest cell cluster. The statistically significant decreases (P < 0.05, t-test) in the percentage of anti-p75-positive cells were found on culture day 4 and 6 (asterisks). (Z) Percentage of cells expressing both CHD7 and p75 per total cells in a neural crest cell cluster. The statistically significant decreases (P < 0.05, t-test) in the percentage of double-positive cells were observed on culture day 4 and 6 (asterisks). Data in (X–Z) are expressed as the average (±SEM) of separate counts of 6–11 clusters (the number in a parenthesis on each bar). Scale bars = 50 lm.


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Fig. 3 – Effects of BMP and Wnt on the maintenance of the undifferentiated state. (A) After removal of neural tube explants, mouse trunk neural crest cells were exposed to BMP2/Wnt3a or BMP2/Wnt1 from 2 days to 6 days in culture. Immunostaining with anti-CHD7 and anti-Sox10 or with anti-CHD7 and anti-p75 was performed on culture day 6. (B) Bright-field image in the presence of BMP2 and Wnt3a. (C) Anti-CHD7-positive cells in the same field as (B). (D) Anti-Sox10-positive cells in the same field as (B). (E) DAPI nuclear stain of the same field as (B). (F) Merged image of (C–E). (G) Percentage of cells expressing CHD7 per total cells in a neural crest cell cluster (each cluster was derived from a neural tube explant). The asterisks indicate statistically significant differences (P < 0.05, t-test) from the proportion of anti-CHD7-positive cells in untreated cultures. (H) Percentage of cells expressing Sox10 per total cells in a neural crest cell cluster. The asterisks show statistically significant differences (P < 0.05, t-test) from the proportion of anti-Sox10-positive cells in untreated cultures. (I) Percentage of cells expressing both CHD7 and Sox10 per total cells in a neural crest cell cluster. The asterisks indicate statistically significant differences (P < 0.05, t-test) from the proportion of double-positive cells in untreated cultures. Data in (G–I) are expressed as the average (±SEM) of separate counts of 5–12 clusters (the number in a parenthesis on each bar). (J) Bright-field image in the presence of BMP2 and Wnt3a. (K) Anti-CHD7-positive cells in the same field as (J). (L) Anti-p75-positive cells in the same field as (J). (M) DAPI nuclear stain of the same field as (J). (N) Merged image of (K–M). (O) Percentage of cells expressing CHD7 per total cells in a neural crest cell cluster. The asterisks indicate statistically significant differences (P < 0.05, t-test) from the proportion of anti-CHD7-positive cells in untreated cultures. (P) Percentage of cells expressing p75 per total cells in a neural crest cell cluster. The asterisks indicate statistically significant differences (P < 0.05, t-test) from the proportion of anti-p75positive cells in untreated cultures. (Q) Percentage of cells expressing both CHD7 and p75 per total cells in a neural crest cell cluster. The asterisks show statistically significant differences (P < 0.05, t-test) from the proportion of double-positive cells in untreated cultures. Data in (O–Q) are expressed as the average (±SEM) of separate counts of 5–11 clusters (the number in a parenthesis on each bar). Scale bars = 50 lm.


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the expression of neural crest specifier genes (Bajpai et al., 2010). CHD7 also interacts with three master regulators of multipotency, Oct4, Sox2, and Nanog, in ES cells (Schnetz et al., 2010). The mutation of this remodeler gene causes the CHARGE syndrome by abnormal neural crest development (Siebert et al., 1985; Zentner et al., 2010; Patten et al., 2012). These findings suggest that chromatin remodelers as well as BMP/Wnt signaling may be involved in maintaining of the multipotency of neural crest cells and conduct them to mouse NCSCs. In the present study, therefore, we analyzed roles of two chromatin remodelers, CHD7 and SWI/SNF-B, and BMP/Wnt signaling in maintaining the multipotency of mouse trunk neural crest cells, leading to the formation of mouse NCSCs.

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2.1. CHD7 expression in trunk neural crest cells of mouse embryos

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Since there have been no reports of CHD7 expression in mouse neural crest cells, we examined the expression patterns of CHD7 in trunk neural crest cells of mouse embryos. No expression of CHD7, Sry-related HMG-BOX gene 10 (Sox10), and low-affinity neurotrophin receptor p75 (p75) were

Results

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observed at the unsegmented levels of the 28-somite stage embryos (Fig. 1A–J). At 26-somite levels of the 26-somite stage embryos, CHD7 expression was found in early migratory neural crest cells containing Sox10 or p75, markers of undifferentiated neural crest cells (Rao and Anderson, 1997; Kim et al., 2003) (white arrows in Fig. 1L, M, N, O, Q, R, S, and T). Furthermore, cells that express both CHD7 and Sox10 were observed in the dorsal neural tube containing premigratory neural crest cells (an arrowhead in Fig. 1L). In addition, there existed CHD7-expressing cells with both Sox10 and p75 at the 25-somite levels of the 28-somite stage embryos (data not shown). These results indicate that undifferentiated mouse trunk neural crest cells express CHD7. Furthermore, we examined the expression of CHD7, Sox10, and p75 during late migratory stages of the trunk neural crest cells. CHD7expressing cells containing Sox10 and/or p75 were observed (white arrows in Fig. 1U–D 0 ).

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2.2. Expression of CHD7, Sox10, and p75 in mouse trunk neural crest cell cultures

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Mouse trunk neural crest cells undergo temporally regulated differentiation even in cultures (Ito et al., 1993; Ito and Morita, 1995). Therefore, to examine the temporal changes in CHD7 expression, we used mouse trunk neural crest cell

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b Fig. 4 – Roles of Wnt/b-catenin pathway in the maintenance of the undifferentiated state. (A) After removal of neural tube explants, mouse trunk neural crest cells were exposed to BMP2, Wnt1, Wnt3a, noggin, chordin and/or BIO from 2 days to 6 days in culture. The DN b-catenin expression vectors, the DN TCF expression vectors, or the control vectors were applied from 2 days to 3 days in culture. Immunostaining with anti-CHD7 and anti-Sox10 was performed on culture day 6. (B) Percentage of cells expressing CHD7 per total cells in a neural crest cell cluster (each cluster was derived from a neural tube explant). The asterisks indicate statistically significant differences (P < 0.05, t-test) from the proportion of anti-CHD7-positive cells in untreated cultures. The daggers show statistically significant differences (P < 0.05, t-test) from the proportion of antiCHD7-positive cells in the cultures treated with BMP2/Wnt3a. (C) Percentage of cells expressing Sox10 per total cells in a neural crest cell cluster. The asterisks indicate statistically significant differences (P < 0.05, t-test) from the proportion of antiSox10-positive cells in untreated cultures. The daggers show statistically significant differences (P < 0.05, t-test) from the proportion of anti-Sox10-positive cells in the cultures treated with BMP2/Wnt3a. (D) Percentage of cells expressing both CHD7 and Sox10 per total cells in a neural crest cell cluster. The asterisks indicate statistically significant differences (P < 0.05, t-test) from the proportion of double-positive cells in untreated cultures. The daggers show statistically significant differences (P < 0.05, t-test) from the proportion of double-positive cells in the cultures treated with BMP2/Wnt3a. Data in (B–D) are expressed as the average (±SEM) of separate counts of 5–9 clusters (the number in a parenthesis on each bar). (E) Percentage of cells expressing CHD7 per total cells in a neural crest cell cluster. The asterisk indicates a statistically significant difference (P < 0.05, t-test) from the proportion of anti-CHD7-positive cells in the cultures treated with BMP2/Wnt3a. (F) Percentage of cells expressing Sox10 per total cells in a neural crest cell cluster. (G) Percentage of cells expressing both CHD7 and Sox10 per total cells in a neural crest cell cluster. The asterisks show statistically significant differences (P < 0.05, t-test) from the proportion of double-positive cells in the cultures treated with BMP2/Wnt3a. Data in (E–G) are expressed as the average (±SEM) of separate counts of 5–9 clusters. (H) Percentage of cells expressing CHD7 per total cells in a neural crest cell cluster. The asterisks indicate statistically significant differences (P < 0.05, t-test) from the proportion of anti-CHD7-positive cells in the cultures treated with BMP2/Wnt3a. The daggers show statistically significant differences (P < 0.05, t-test) from the proportion of anti-CHD7-positive cells in the cultures treated with the control vectors. (I) Percentage of cells expressing Sox10 per total cells in a neural crest cell cluster. The asterisks indicate statistically significant differences (P < 0.05, t-test) from the proportion of anti-Sox10-positive cells in the cultures treated with BMP2/Wnt3a. The daggers show statistically significant differences (P < 0.05, t-test) from the proportion of anti-Sox10-positive cells in the cultures treated with the control vectors. (J) Percentage of cells expressing both CHD7 and Sox10 per total cells in a neural crest cell cluster. The asterisks indicate statistically significant differences (P < 0.05, t-test) from the proportion of double-positive cells in the cultures treated with BMP2/Wnt3a. The daggers indicate statistically significant differences (P < 0.05, t-test) from the proportion of double-positive cells in the cultures treated with the control vectors. Data in (H–J) are expressed as the average (±SEM) of separate counts of 5– 9 clusters.


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cultures. After 2, 4, or 6 days in culture, we performed immunocytochemical analysis using anti-CHD7 and anti-Sox10. The percentage of cells expressing CHD7 and/or Sox10 per neural crest cell cluster (each cluster of neural crest cells was derived from a neural tube explant) was significantly lower at 6 days in culture than at 2 days in culture (Fig. 2A–M). Experiments using anti-CHD7 and anti-p75 also showed the same results (Fig. 2N–Z). CHD7 is likely to recognize undifferentiated mouse trunk neural crest cells along with Sox10 and p75.

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2.3. Actions of BMP and Wnt signaling on the maintenance of the undifferentiated state of mouse trunk neural crest cells

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It has been known that the concerted activity of BMP2 (10 ng/ml) and Wnt1 (unknown concentrations) maintains the undifferentiated state of neural crest cells (Kle´ber et al., 2004). Therefore, we examined whether these factors not only participate in the maintenance of the undifferentiated state of mouse trunk neural crest cells under our experimental conditions but also affect CHD7 expression in these cells. BMP2 and Wnt1 were added to the medium from 2 days to 6 days in culture (Fig. 3A). When Wnt1 was used at 10 ng/ ml, the expression of CHD7 and/or p75 was significantly maintained on culture day 6 (Fig. 3G and O–Q). The maintenance of Sox10 expression was less pronounced (Fig. 3H).

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However, in the treatment with 100 ng/ml Wnt1 and 10 ng/ ml BMP2, the expression of Sox10 drastically decreased (Fig. 3H). Furthermore, the percentage of double-positive cells expressing Sox10 and CHD7 was also suppressed (Fig. 3I). Therefore, we used Wnt3a, which is expressed in the dorsal neural tube in which neural crest cells are formed, similar to Wnt1 (Hollyday et al., 1995). The treatment with 10 ng/ml BMP2 and 10 ng/ml Wnt3a promoted the expression of CHD7, Sox10, or p75. The effects were unchanged even when Wnt3a was used at 100 ng/ml (Fig. 3). These results suggest that Wnt3a is more effective for the maintenance of the undifferentiated state of mouse trunk neural crest cells. Therefore, we examined the effects of BMP2, Wnt1 and Wnt3a in more detail. BMP2 or Wnt1 alone had almost no effects on CHD7 expression or inhibitory effects on Sox10 expression (Fig. 4B–D). These inhibitory effects may cause the decrease of Sox10 expression in BMP2/Wnt1 treatment, leading to the reduction of the proportion of cells expressing both Sox10 and CHD7. On the other hand, the treatment with Wnt3a alone, especially with 100 ng/ml Wnt3a, produced effects similar to those of the 10 ng/ml BMP2 and 10 ng/ml Wnt3a treatments on the maintenance of the undifferentiated state (Fig. 4B–D). We prevented BMP activities with two BMP inhibitors, noggin and chordin, to exclude the possibility that the effects of Wnt3a alone were caused by the BMP activities in fetal bovine serum (FBS) and/or extract of day-11 chick embryos (CEE) in the culture medium (Abzhanov et al.,

b Fig. 5 – Effects of the WT CHD7 expression vectors on the maintenance of the undifferentiated state. (A) After removal of neural tube explants, mouse trunk neural crest cells were exposed to the WT CHD7 expression vectors from 2 days to 3 days in culture. Immunostaining with anti-CHD7 and anti-Sox10 or with anti-CHD7 and anti-p75 was performed on culture day 6. (B) Bright-field image in the presence of the WT CHD7 expression vectors. (C) Anti-CHD7-positive cells in the same field as (B). (D) Anti-Sox10-positive cells in the same field as (B). (E) DAPI nuclear stain of the same field as (B). (F) Merged image of (C–E). (G) Percentage of cells expressing CHD7 per total cells in a neural crest cell cluster (each cluster was derived from a neural tube explant). The asterisks indicate statistically significant differences (P < 0.05, t-test) from the proportion of anti-CHD7-positive cells in untreated cultures. The daggers show statistically significant differences (P < 0.05, t-test) from the proportion of antiCHD7-positive cells in the cultures treated with BMP2/Wnt3a. (H) Percentage of cells expressing Sox10 per total cells in a neural crest cell cluster. The asterisks indicate statistically significant differences (P < 0.05, t-test) from the proportion of antiSox10-positive cells in untreated cultures. The daggers show statistically significant differences (P < 0.05, t-test) from the proportion of anti-Sox10-positive cells in the cultures treated with BMP2/Wnt3a. (I) Percentage of cells expressing both CHD7 and Sox10 per total cells in a neural crest cell cluster. The asterisks indicate statistically significant differences (P < 0.05, t-test) from the proportion of double-positive cells in untreated cultures. The daggers show statistically significant differences (P < 0.05, t-test) from the proportion of double-positive cells in the cultures treated with BMP2/Wnt3a. Data in (G–I) are expressed as the average (±SEM) of separate counts of 7–10 clusters (the number in a parenthesis on each bar). (J) Bright-field image in the presence of the WT CHD7 expression vectors. (K) Anti-CHD7-positive cells in the same field as (J). (L) Anti-p75positive cells in the same field as (J). (M) DAPI nuclear stain of the same field as (J). (N) Merged image of (K–M). (O) Percentage of cells expressing CHD7 per total cells in a neural crest cell cluster. The asterisks indicate statistically significant differences (P < 0.05, t-test) from the proportion of anti-CHD7-positive cells in untreated cultures. The daggers show statistically significant differences (P < 0.05, t-test) from the proportion of anti-CHD7-positive cells in the cultures treated with BMP2/ Wnt3a. (P) Percentage of cells expressing p75 per total cells in a neural crest cell cluster. The asterisks indicate statistically significant differences (P < 0.05, t-test) from the proportion of anti-p75-positive cells in untreated cultures. The daggers show statistically significant differences (P < 0.05, t-test) from the proportion of anti-p75-positive cells in the cultures treated with BMP2/Wnt3a. (Q) Percentage of cells expressing both CHD7 and p75 per total cells in a neural crest cell cluster. The asterisks indicate statistically significant differences (P < 0.05, t-test) from the proportion of double-positive cells in untreated cultures. The daggers indicate statistically significant differences (P < 0.05, t-test) from the proportion of double-positive cells in the cultures treated with BMP2/Wnt3a. Data in (O–Q) are expressed as the average (±SEM) of separate counts of 5–11 clusters (the number in a parenthesis on each bar). Scale bars = 50 lm.


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Fig. 6 – Effects of the DN CHD7 expression vectors or CHD7 siRNA on the maintenance of the undifferentiated state. (A) After removal of neural tube explants, mouse trunk neural crest cells were exposed to BMP2 and Wnt3a from 2 days to 6 days in culture. The DN CHD7 expression vectors or CHD7 siRNA were applied from 2 days to 3 days in culture. Immunostaining with anti-CHD7 and anti-Sox10 or with anti-CHD7 and anti-p75 was performed on culture day 6. (B) Percentage of cells expressing CHD7 per total cells in a neural crest cell cluster (each cluster was derived from a neural tube explant). The asterisks indicate statistically significant differences (P < 0.05, t-test) from the proportion of anti-CHD7-positive cells in untreated cultures. The daggers show statistically significant differences (P < 0.05, t-test) from the proportion of anti-CHD7-positive cells in the cultures treated with BMP2/Wnt3a. (C) Percentage of cells expressing Sox10 per total cells in a neural crest cell cluster. The asterisks indicate statistically significant differences (P < 0.05, t-test) from the proportion of anti-Sox10-positive cells in untreated cultures. The daggers show statistically significant differences (P < 0.05, t-test) from the proportion of anti-Sox10-positive cells in the cultures treated with BMP2/Wnt3a. (D) Percentage of cells expressing both CHD7 and Sox10 per total cells in a neural crest cell cluster. The asterisks indicate statistically significant differences (P < 0.05, t-test) from the proportion of double-positive cells in untreated cultures. The daggers show statistically significant differences (P < 0.05, t-test) from the proportion of double-positive cells in the cultures treated with BMP2/Wnt3a. Data in (B–D) are expressed as the average (±SEM) of separate counts of 4–9 clusters (the number in a parenthesis on each bar). (E) Percentage of cells expressing CHD7 per total cells in a neural crest cell cluster. The asterisks indicate statistically significant differences (P < 0.05, t-test) from the proportion of anti-CHD7-positive cells in untreated cultures. The daggers show statistically significant differences (P < 0.05, t-test) from the proportion of anti-CHD7-positive cells in the cultures treated with BMP2/Wnt3a. (F) Percentage of cells expressing p75 per total cells in a neural crest cell cluster. The asterisks indicate statistically significant differences (P < 0.05, t-test) from the proportion of anti-p75-positive cells in untreated cultures. The daggers show statistically significant differences (P < 0.05, t-test) from the proportion of anti-p75-positive cells in the cultures treated with BMP2/Wnt3a. (G) Percentage of cells expressing both CHD7 and Sox10 per total cells in a neural crest cell cluster. The asterisks indicate statistically significant differences (P < 0.05, t-test) from the proportion of double-positive cells in untreated cultures. The daggers show statistically significant differences (P < 0.05, t-test) from the proportion of double-positive cells in the cultures treated with BMP2/Wnt3a. Data in (E–G) are expressed as the average (±SEM) of separate counts of 4–11 clusters.


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Fig. 7 – Effects of SWI/SNF-B inhibition on the maintenance of the undifferentiated state. (A) Percentage of cells expressing CHD7 per total cells in a neural crest cell cluster (each cluster was derived from a neural tube explant). The asterisks indicate statistically significant differences (P < 0.05, t-test) from the proportion of anti-CHD7-positive cells in untreated cultures. The daggers show statistically significant differences (P < 0.05, t-test) from the proportion of anti-CHD7-positive cells in the cultures treated with BMP2/Wnt3a. (B) Percentage of cells expressing Sox10 per total cells in a neural crest cell cluster. The asterisks indicate statistically significant differences (P < 0.05, t-test) from the proportion of anti-Sox10-positive cells in untreated cultures. The daggers show statistically significant differences (P < 0.05, t-test) from the proportion of anti-Sox10positive cells in the cultures treated with BMP2/Wnt3a. (C) Percentage of cells expressing both CHD7 and Sox10 per total cells in a neural crest cell cluster. The asterisks indicate statistically significant differences (P < 0.05, t-test) from the proportion of double-positive cells in untreated cultures. The daggers show statistically significant differences (P < 0.05, t-test) from the proportion of double-positive cells in the cultures treated with BMP2/Wnt3a. Data are expressed as the average (±SEM) of separate counts of 5–11 clusters (the number in a parenthesis on each bar).

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2003). These inhibitors had no effects on the maintenance of CHD7 or Sox10 expression (Fig. 4E–G). We tried to identify the Wnt signaling pathway involved in the maintenance of the undifferentiated state. When the neural crest cell cultures were treated with 10 ng/ml BMP2 and 6-Bromoindirubin-3 0 -oxime (BIO), a reagent that activates the Wnt/b-catenin pathway (Meijer et al., 2003), effects similar to those of the 10 ng/ml BMP2 and 10 ng/ml Wnt3a

treatments were produced (Fig. 4H–J). In addition, the treatment with the dominant-negative (DN) b-catenin expression vectors or the DN T-cell factor (TCF) expression vectors drastically inhibited the effects of 10 ng/ml BMP2 and 10 ng/ml Wnt3a on CHD7 or Sox10 expression (Fig. 4H–J). Previous studies have shown that the treatment with 10 ng/ml BMP2 (Ota and Ito, 2006) or unknown concentrations of Wnt1 (Bronner-Fraser, 2004; Lee et al., 2004) promotes


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Fig. 8 – Proliferation and apoptosis of mouse trunk neural crest cells. After removal of neural tube explants, mouse trunk neural crest cells were exposed to BMP2 and Wnt3a from 2 days to 6 days in culture. The WT CHD7 expression vectors were applied from 2 days to 3 days in culture. BrdU incorporation and the double-labeling with anti-Sox10 and anti-BrdU or with anti-p75 and anti-BrdU were carried out on culture day 6. The double-staining with anti-Sox10 and anti-caspase-3 or with anti-p75 and anti-caspase-3 was also performed on culture day 6. (A) Bright-field image in the presence of the WT CHD7 expression vectors. (B) BrdU-labeled cells in the same field as (A). (C) Anti-Sox10-positive cells in the same field as (A). (D) DAPI nuclear stain of the same field as (A). (E) Merged image of (B–D). (F) Bright-field image of an untreated culture. (G) Caspase-3-labeled cells in the same field as (F). (H) Anti-Sox10-positive cells in the same field as (F). (I) DAPI nuclear stain of the same field as (F). (J) Merged image of (G–I). (K) Percentage of BrdU-incorporated cells per total cells in a neural crest cell cluster (each cluster was derived from a neural tube explant). The asterisks indicate statistically significant differences (P < 0.05, t-test) from the proportion of BrdU-labeled cells in untreated cultures. (L) Percentage of anti-Sox10- or anti-p75positive cells that incorporated BrdU per total Sox10- or p75-expressing cells in a neural crest cell cluster. The asterisks indicate statistically significant differences (P < 0.05, t-test) from the proportion of BrdU-labeled cells in untreated cultures. (M) Percentage of anti-caspase-3-positive cells per total cells in a neural crest cell cluster. Data are expressed as the average (±SEM) of separate counts of 5–19 clusters (the number in a parenthesis on each bar). Scale bars = 50 lm. 188 189 190 191 192 193 194 195 196

sensory neurogenesis of neural crest cells. Therefore, we investigated the effects of BMP2, Wnt1 and/or Wnt3a on sensory neurogenesis of mouse trunk neural crest cells under the present culture condition. The treatment with 10 ng/ml BMP2, Wnt1 (10 ng/ml or 100 ng/ml) and/or Wnt3a (10 ng/ml or 100 ng/ml) promoted the differentiation of sensory neurons containing Brn3a (data not shown), similar to previous reports. However, no synergistic effects of the treatment with both 10 ng/ml BMP2 and 10 ng/ml Wnt3a were found.

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2.4. Actions of CHD7 on the maintenance of the undifferentiated state of mouse trunk neural crest cells

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In this study, we showed that BMP/Wnt signaling not only maintained the undifferentiated state of mouse trunk neural crest cells but also activated CHD7 expression. It is possible

that CHD7 plays important roles in the maintenance of the undifferentiated state of mouse trunk neural crest cells. To examine this possibility, the neural crest cell cultures were treated with the wild-type (WT) CHD7, DN CHD7 expression vectors, or CHD7 small interfering RNA (siRNA) (Figs. 5A and 6A). The number of CHD7-positive cells increased dramatically by the addition of the WT CHD7 expression vectors (Fig. 5C, G, K, and O). This showed that the WT CHD7 expression vectors were effectively introduced to the neural crest cells. Furthermore, the WT CHD7 expression vectors maintained the expression of Sox10 or p75 (Fig. 5D, H, L, and P) and the co-expression of CHD7 and Sox10 (Fig. 5F and I) or of CHD7 and p75 (Fig. 5N and Q). When the DN CHD7 expression vectors were added, the number of CHD7-positive cells increased (Fig. 6B and E), indicating that the anti-CHD7 antibody used in this study recognizes the


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mutant CHD7, because this mutant protein changed only lysine 998 in the ATPase domain of CHD7 to arginine. However, treatment with the DN CHD7 expression vectors significantly suppressed the expression of Sox10 and p75 (Fig. 6C and F). This treatment also suppressed the co-expression of CHD7 and Sox10 or of CHD7 and p75 (Fig. 6D and G). Treatment with CHD7 siRNA dramatically suppressed the expression of CHD7, Sox10, and/or p75 (Fig. 6B–G).

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2.5. Actions of SWI/SNF-B on the maintenance of the undifferentiated state of mouse trunk neural crest cells

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It has been shown that SWI/SNF-B works co-operatively with CHD7 in neural crest induction (Bajpai et al., 2010). Therefore, we examined whether or not SWI/SNF-B participates in the maintenance of the undifferentiated state of mouse trunk neural crest cells. In knockdown experiments using siRNAs designed toward brahma-related gene 1 (BRG1), the catalytic subunit of SWI/SNF-B (Wu et al., 2009), and toward bromodomain-containing protein 7 (BRD7), which is the specific subunit of this chromatin remodeler (Kaeser et al., 2008), the maintenance of the undifferentiated state was significantly suppressed despite the presence of 10 ng/ ml BMP2 and 10 ng/ml Wnt3a (Fig. 7).

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2.6. Proliferation and apoptosis of mouse trunk neural crest cells by treatment with BMP2/Wnt3a or the WT CHD7 expression vectors

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Since the number of the undifferentiated neural crest cells was significantly increased by treatment with BMP2 (10 ng/ ml)/Wnt3a (10 ng/ml) or the WT CHD7 expression vectors, we examined the effects of these factors on the proliferation of mouse trunk neural crest cells using 5-bromo-2 0 -deoxyuridine (BrdU) incorporation method. The percentage of BrdUincorporated cells was increased by treatment with BMP2 (10 ng/ml)/Wnt3a (10 ng/ml) or the WT CHD7 expression vectors (Fig. 8K). Furthermore, these treatments increased the proportion of BrdU-incorporated cells per total Sox10- or p75-expressing cells (Fig. 8A–E and L). The 4 0 ,6-diamidino2-phenylindole (DAPI) nuclear-staining experiments showed that almost no cell death occurs under treatment with BMP2 (10 ng/ml)/Wnt3a (10 ng/ml) or the WT CHD7 expression vectors. Therefore, we measured apoptosis in these culture conditions. No significant differences of the proportion of apoptotic cell death were found among the culture conditions examined (Fig. 8M). Moreover, no cells expressing both caspase-3 and Sox10 (Fig. 8F–J) or both caspase-3 and p75 were observed under the culture conditions examined.

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2.7. Actions of CHD7 and BMP/Wnt signaling on the maintenance of the multipotency of mouse trunk neural crest cells

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We showed here that CHD7 was expressed in undifferentiated mouse trunk neural crest cells and involved in the maintenance of the undifferentiated state. Since undifferentiated neural crest cells are likely to be multipotential, CHD7 and BMP/Wnt may participate in maintaining the multipotency of mouse trunk neural crest cells. It has been known that

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multipotent trunk neural crest cells can differentiate into smooth muscle cells, neurons, and glia (Mundell and Labosky, 2011). Therefore, we investigated whether or not the activation of CHD7 or BMP/Wnt signaling triggers the increase in the number of multipotent mouse trunk neural crest cells that differentiate into smooth muscle cells, neurons, and glia. The trunk neural crest cells were treated with the WT CHD7 expression vectors or with BMP2 (10 ng/ml)/ Wnt3a (10 ng/ml) and colony cultures were subsequently performed in the presence or absence of 10 ng/ml fibroblast growth factor 2 (FGF2) (Fig. 9A). The presence of FGF2 was requisite for glial differentiation in colony cultures (Ota and Ito, 2006; Fujita et al., 2011). Four types of colonies appeared in the absence of FGF2: colonies containing both smooth muscle cells and neurons, colonies containing either smooth muscle cells or neurons, and colonies containing neither smooth muscle cells nor neurons (Fig. 9B–D). Furthermore, three types of colonies were observed in the presence of FGF2: colonies containing both smooth muscle cells and glia, colonies containing smooth muscle cells only, and colonies containing neither smooth muscle cells nor glia (Fig. 9F and G). The addition of WT CHD7 expression vectors or BMP2 (10 ng/ml)/ Wnt3a (10 ng/ml) significantly increased the proportion of colonies consisting of both smooth muscle cells and neurons or both smooth muscle cells and glia that originated from the multipotent trunk neural crest cells (Fig. 9E and H).

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2.8. Actions of BMP/Wnt signaling on the maintenance of the multipotency of mouse embryonic DRG cells

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Since rat embryonic DRG has been shown to contain mul300 tipotent NCSCs expressing Sox10 and/or p75 (Hagedorn et al., 301 1999; Kle´ber et al., 2004), we analyzed the expression of CHD7, 302 Sox10, and p75 in mouse embryonic DRG. Cells expressing Q2 303 both CHD7 and Sox10 (Fig. 10A–E 0 ) or both CHD7 and p75 304 (Fig. 10F–J 0 ) were found in the DRG. This in vivo result sug305 gested that the DRG contained NCSCs expressing CHD7. 306 Since our data showed that BMP/Wnt signaling partici307 pated in the maintenance of the multipotency of mouse trunk 308 neural crest cells by promoting CHD7 expression, we exam309 ined whether or not the treatment with BMP2 (10 ng/ml)/ 310 Wnt3a (10 ng/ml) affects the expression of CHD7, Sox10, and 311 p75 and increases the proportion of multipotent cells in 312 mouse embryonic DRG containing NCSCs. When the DRG 313 cells were treated with BMP2/Wnt3a, the expression of 314 CHD7 (Fig. 11C, G, I, K, O, and Q), Sox10 (Fig. 11D, H, and I), 315 and/or p75 (Fig. 11L, P, and Q) was significantly maintained, 316 similar to the case with the trunk neural crest cells. Further317 more, the DRG cells were treated with BMP2 (10 ng/ml)/Wnt3a 318 (10 ng/ml) and colony cultures were subsequently carried out 319 in the presence or absence of 10 ng/ml FGF2 to examine 320 whether or not this treatment is involved in the maintenance 321 of the multipotency of these cells. In the cultures, the differ322 entiation of smooth muscle cells were found as described pre323 viously (Hagedorn et al., 1999; Li et al., 2007; Nagoshi et al., 324 2008). In addition, BMP2/Wnt3a treatment increased the pro325 portion of colonies consisting of both smooth muscle cells 326 and neurons (Fig. 12B and E) or both smooth muscle cells 327 and glia (Fig. 12F and I) that were derived from NCSCs in the 328 DRG. 329


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Fig. 9 – Effects of the WT CHD7 expression vectors and BMP2/Wnt3a on the maintenance of the multipotency. (A) After removal of neural tube explants, mouse trunk neural crest cells were exposed to BMP2 and Wnt3a from 2 days to 6 days in culture. The WT CHD7 expression vectors were applied from 2 days to 3 days in culture. The colony cultures were started on culture day 6. Immunostaining with anti-SMA and anti-NF or with anti-SMA and anti-GFAP was performed on culture day 11. In colony cultures, FGF2 was added to the medium to stimulate the differentiation of anti-GFAP-positive glial cells. (B) A colony containing both anti-SMA (green)- and anti-NF (red)-positive cells in the presence of BMP2 and Wnt3a. (C) A colony containing anti-SMA-positive cells only in the presence of BMP2 and Wnt3a. (D) A colony containing anti-NF-positive cells only in the presence of BMP2 and Wnt3a. DAPI nuclear stain (blue). (E) Percentage of colonies containing cells expressing SMA and/or NF per total number of neural crest cell colonies under various culture conditions. The asterisks indicate statistically significant differences (P < 0.05, t-test) from the proportion of respective colonies in untreated cultures. Data are expressed as the average (±SEM) of separate counts of 3 independent experiments (the number in a parenthesis on each bar). (F) A colony containing both anti-SMA (green)- and anti-GFAP (red)-positive cells in the presence of WT CHD7 expression vectors. (G) A colony containing anti-SMA-positive cells only in the presence of WT CHD7 expression vectors. DAPI nuclear stain (blue). (H) Percentage of colonies containing cells expressing SMA and/or GFAP per total number of neural crest cell colonies under various culture conditions. The asterisks show statistically significant differences (P < 0.05, t-test) from the proportion of respective colonies in untreated cultures. Data are expressed as the average (±SEM) of separate counts of 3 independent experiments (the number in a parenthesis on each bar). Scale bars = 50 lm.

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2.9. Expression of CHD7 in mouse adult DRG and adult sciatic nerve

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Adult DRG and adult sciatic nerves have been known to contain NCSCs expressing Sox10 and/or p75 (Morrison et al., 1999; Joseph et al., 2004; Li et al., 2007; Nagoshi et al., 2008). Therefore, we analyzed the expression of CHD7, Sox10, and

p75 in adult DRG and adult sciatic nerves. Cells expressing CHD7, Sox10, or p75 were observed in the adult DRG (Fig. 13A–J) and the adult sciatic nerves (Fig. 13K–T). Moreover, the double-positive cells were found in all tissues examined (arrowheads in Fig. 13). However, the density of cells containing CHD7, Sox10, and/or p75 in the adult DRG was lower than that in the embryonic DRG.


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Fig. 10 – Expression patterns of CHD7, Sox10, or p75 in DRG derived from E12.5 mouse embryos. (A–J) Transverse sections of DRG. The top and bottom of each photograph correspond to the dorsal and ventral side of the embryo, respectively. (A) Brightfield image. (B) Expression pattern of CHD7 in the same field as (A). (C) Expression pattern of Sox10 in the same field as (A). (D) DAPI nuclear stain of the same field as (A). (E) Merged image of (B–D). (F) Bright-field image. (G) Expression pattern of CHD7 in the same field as (F). (H) Expression pattern of p75 in the same field as (F). (I) DAPI nuclear stain of the same field as (F). (J) Merged image of (G–I). (A 0 –E 0 ) and (F 0 –J 0 ) show enlarged images of white-edged squares in (A–E) and (F–J), respectively. NT, neural tube. Scale bars = 20 lm.

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Discussion

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3.1. Undifferentiated mouse trunk neural crest cells express CHD7

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In the present study, we analyzed roles of two chromatin remodelers, CHD7 (Vissers et al., 2004; Martin, 2010; Schnetz et al., 2010) and SWI/SNF-B (Ho and Crabtree, 2010; Wilson and Roberts, 2011), and BMP/Wnt signaling in maintaining the multipotency of mouse trunk neural crest cells. Chromatin remodeling plays important roles in development and differentiation (Mu¨ller and Leutz, 2001). It has been

shown that both of these chromatin remodelers control the expression of genes that characterize ES cells (Kaeser et al., 2008; Schnetz et al., 2010) and are involved in the induction of neural crest cells (Bajpai et al., 2010), which show multipotency like ES cells. Immunohistochemistry using mouse embryos showed that CHD7 was expressed in undifferentiated trunk neural crest cells containing Sox10 and p75, markers of undifferentiated neural crest cells (Rao and Anderson, 1997; Kim et al., 2003). Further, CHD7 expression was found in the dorsal neural tube. These in vivo data are consistent with the results of a previous study (Bajpai et al., 2010).


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Fig. 11 – Effects of BMP2 and Wnt3a on the maintenance of the undifferentiated state of mouse embryonic DRG cells. (A) DRG cells were exposed to BMP2 and Wnt3a from 0 days to 6 days in culture. Immunostaining with anti-CHD7 and anti-Sox10 or with anti-CHD7 and anti-p75 was performed on culture day 2, 4, and 6. (B) Bright-field image in the presence of BMP2 and Wnt3a on culture day 6. (C) Anti-CHD7-positive cells in the same field as (B). (D) Anti-Sox10-positive cells in the same field as (B). (E) DAPI nuclear stain of the same field as (B). (F) Merged image of (C–E). (G) Percentage of cells expressing CHD7 per total cells in a DRG cell cluster (each cluster was derived from a DRG explant). The asterisks indicate statistically significant differences (P < 0.05, t-test) from the proportion of anti-CHD7-positive cells in untreated cultures on culture day 2. (H) Percentage of cells expressing Sox10 per total cells in a DRG cell cluster. The asterisk shows a statistically significant difference (P < 0.05, t-test) from the proportion of anti-Sox10-positive cells in untreated cultures on culture day 2. (I) Percentage of cells expressing both CHD7 and Sox10 per total cells in a DRG cell cluster. The asterisks indicate statistically significant differences (P < 0.05, t-test) from the proportion of double-positive cells in untreated cultures on culture day 2. Data in (G-I) are expressed as the average (±SEM) of separate counts of 4–7 clusters (the number in a parenthesis on each bar). (J) Bright-field image in the presence of BMP2 and Wnt3a on culture day 6. (K) Anti-CHD7-positive cells in the same field as (J). (L) Anti-p75-positive cells in the same field as (J). (M) DAPI nuclear stain of the same field as (J). (N) Merged image of (K–M). (O) Percentage of cells expressing CHD7 per total cells in a DRG cell cluster. The asterisks indicate statistically significant differences (P < 0.05, t-test) from the proportion of anti-CHD7-positive cells in untreated cultures on culture day 2. (P) Percentage of cells expressing p75 per total cells in a DRG cell cluster. The asterisks indicate statistically significant differences (P < 0.05, t-test) from the proportion of anti-p75-positive cells in untreated cultures on culture day 2. (Q) Percentage of cells expressing both CHD7 and p75 per total cells in a DRG cell cluster. The asterisks show statistically significant differences (P < 0.05, t-test) from the proportion of double-positive cells in untreated cultures on culture day 2. Data in (O–Q) are expressed as the average (±SEM) of separate counts of 5–13 clusters (the number in a parenthesis on each bar). Scale bars = 50 lm.


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Fig. 12 – Effects of BMP/Wnt signaling on the maintenance of the multipotency of mouse embryonic DRG cells. (A) DRG cells were exposed to BMP2 and Wnt3a from 0 day to 6 days in culture. The colony cultures were started on culture day 6. Immunostaining with anti-SMA and anti-NF or with anti-SMA and anti-GFAP was performed on culture day 11. In colony cultures, FGF2 was added to the medium to stimulate the differentiation of anti-GFAP-positive glial cells. (B) A colony containing both anti-SMA (green)- and anti-NF (red)-positive cells in the presence of BMP2 and Wnt3a. (C) A colony containing anti-SMA-positive cells only in the presence of BMP2 and Wnt3a. (D) A colony containing anti-NF-positive cells only in the presence of BMP2 and Wnt3a. DAPI nuclear stain (blue). (E) Percentage of colonies containing cells expressing SMA and/or NF per total number of DRG cell colonies in untreated or BMP2/Wnt3a-treated cultures. The asterisk indicates a statistically significant difference (P < 0.05, t-test) from the proportion of respective colonies in untreated cultures. (F) A colony containing both anti-SMA (green)- and anti-GFAP (red)-positive cells in the presence of BMP2 and Wnt3a. (G) A colony containing antiSMA-positive cells only in the presence of BMP2 and Wnt3a. (H) A colony containing anti-GFAP-positive cells only in the presence of BMP2 and Wnt3a. DAPI nuclear stain (blue). (I) Percentage of colonies containing cells expressing SMA and/or GFAP per total number of DRG cell colonies in untreated or BMP2/Wnt3a-treated cultures. The asterisk shows a statistically significant difference (P < 0.05, t-test) from the proportion of respective colonies in untreated cultures. Data are expressed as the average (±SEM) of separate counts of 3 independent experiments (the number in a parenthesis on each bar). Scale bars = 50 lm.

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3.2. BMP/Wnt signaling affects CHD7 expression and the maintenance of the undifferentiated state of mouse trunk neural crest cells

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In mouse trunk neural crest cell cultures, the number of Sox10- or p75-positive undifferentiated neural crest cells decreased as the culture progressed. Similarly, CHD7 expression was also suppressed over time. These results suggest the hypothesis that CHD7 may participate in the maintenance of the undifferentiated state of neural crest cells. This hypothesis was examined by experiments using BMP and Wnt proteins. Kle´ber et al. (2004) has shown that undifferentiated neural crest cells expressing Sox10 and p75 are maintained by combined treatment with BMP2 (10 ng/ml) and

Wnt1 (unknown concentrations). In this study, BMP/Wnt treatment maintained CHD7 expression as well as Sox10/ p75 expression. Thus, it is conceivable that CHD7 not only affects neural crest induction but also plays important roles in the maintenance of the undifferentiated state of mouse trunk neural crest cells. Wnt1 and Wnt3a are expressed in partially overlapping domains in the dorsal neural tube in which neural crest cells are formed (Hollyday et al., 1995). When Wnt3a was added to mouse trunk neural crest cell cultures with BMP2, the expression of CHD7, Sox10, and p75 was maintained more effectively than the addition of Wnt1. Moreover, a high concentration of Wnt3a (100 ng/ml) independently maintained the expression of CHD7, Sox10, and p75, similar to


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Fig. 13 – Expression patterns of CHD7, Sox10, or p75 in DRG or sciatic nerve of adult mice. (A–J) Transverse sections of DRG in a 12-week old mouse. (A) Bright-field image. (B) Expression pattern of CHD7 in the same field as (A). (C) Expression pattern of Sox10 in the same field as (A). (D) DAPI nuclear stain of the same field as (A). (E) Merged image of (B–D). (F) Bright-field image. (G) Expression pattern of CHD7 in the same field as (F). (H) Expression pattern of p75 in the same field as (F). (I) DAPI nuclear stain of the same field as (F). (J) Merged image of (G–I). (K–T) Transverse sections of sciatic nerve in a 12-week old mouse. (K) Bright-field image. (L) Expression pattern of CHD7 in the same field as (K). (M) Expression pattern of Sox10 in the same field as (K). (N) DAPI nuclear stain of the same field as (K). (O) Merged image of (L–N). (P) Bright-field image. (Q) Expression pattern of CHD7 in the same field as (P). (R) Expression pattern of p75 in the same field as (P). (S) DAPI nuclear stain of the same field as (P). (T) Merged image of (Q–S). White arrowheads show cells expressing both CHD7 and Sox10 or both CHD7 and p75. Scale bars = 20 lm.

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the case with 10 ng/ml BMP and 10 ng/ml Wnt3a. This effect was unchanged even in the presence of noggin or chordin. These data suggest that the activation of Wnt signaling is a key requisite for the maintenance of CHD7 expression and thus for the maintenance of the undifferentiated state of neural crest cells, and that BMP signaling plays supportive roles in the maintenance of the undifferentiated state of these cells, as shown in the maintenance of the undifferentiated state of ES cells (Sato et al., 2004; Sokol, 2011; Tanaka et al., 2011). The addition of BIO, instead of Wnt, to neural crest cell cultures also maintained the undifferentiated state. Furthermore, the effects of BMP2 and Wnt3a were inhibited by the DN b-catenin or DN TCF expression vectors. These results suggest that the Wnt/b-catenin pathway is essential to the maintenance of the undifferentiated state of mouse trunk neural crest cells. Wnt signaling plays important roles in neural crest induction (Garcıá-Castro et al., 2002). Neurons derived from the proximal ganglion of cranial nerve IX are absent in the mutant mouse embryos lacking both Wnt1 and Wnt3a (Ikeya et al., 1997). Further, b-catenin mutant mouse embryos lack melanocytes and sensory neurons (Hari et al., 2002).

These in vivo data have shown that the Wnt/b-catenin pathway participates in neural crest induction and differentiation (Schmidt and Patel, 2005). In addition, it has been known that BMP signaling is essential for neural crest induction (Steventon et al., 2009) and BMP2 is especially required for migration of mouse neural crest cells (Correia et al., 2007). In the present study, BMP2 and/or Wnts also promoted sensory neurogenesis of mouse trunk neural crest cells. Thus, it is possible that BMP/Wnt signaling affect multiple aspects of neural crest development such as induction, migration, differentiation, the maintenance of the undifferentiated state.

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3.3. CHD7 and SWI/SNF-B participate in the maintenance of the undifferentiated state of mouse trunk neural crest cells

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When mouse trunk neural crest cell cultures were treated with the WT CHD7 expression vectors, the undifferentiated state of the trunk neural crest cells was maintained, similar to the case with BMP (10 ng/ml)/Wnt (10 ng/ml) treatment. The treatment with the DN CHD7 expression vectors or CHD7 siRNA significantly inhibited the effects of BMP/Wnt. Moreover, the inhibition of SWI/SNF-B suppressed the

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maintenance of the undifferentiated state of the neural crest cells despite the presence of BMP2 and Wnt3a. It is conceivable that CHD7 and SWI/SNF-B participate in the maintenance of the undifferentiated state of mouse trunk neural crest cells as well as in neural crest induction (Bajpai et al., 2010), similar to the case with BMP/Wnt signaling. BrdU-incorporated experiments showed that the proliferation of undifferentiated mouse trunk neural crest cells were significantly promoted by the treatment with BMP (10 ng/ ml)/Wnt (10 ng/ml) or WT CHD7 expression vectors. On the other hand, any selective cell death was not found in these treatments. In mouse neural crest cells, it has been shown that BRG1, the catalytic subunit of SWI/SNF-B, suppresses the apoptosis and promotes the proliferation (Li et al., 2013). Thus, the maintenance of the undifferentiated state of the trunk neural crest cells may be due to the promotion of the proliferation of the undifferentiated neural crest cells by BMP/Wnt signaling and the chromatin remodelers.

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3.4. CHD7 and BMP/Wnt signaling maintain the multipotency of mouse trunk neural crest cells and lead to the formation of mouse NCSCs

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The maintenance of the undifferentiated state of mouse trunk neural crest cells and mouse embryonic DRG cells may enable the maintenance of the multipotency of these cells. In fact, the results of colony cultures showed that multipotent cells were increased by the addition of WT CHD7 expression vectors or BMP (10 ng/ml)/Wnt (10 ng/ml). Furthermore, CHD7-containing cells expressing Sox10 or p75 were observed in the presumptive DRG, the embryonic DRG, and DRG and sciatic nerves of adult mice that had NCSCs (Li et al., 2007; Nagoshi et al., 2008). These results suggest that CHD7 participates in the maintenance of the multipotency of neural crest cells and conducts them to mouse NCSCs. Taken together, the present results suggest the possibility that the chromatin remodelers as well as BMP/Wnt signaling play essential roles in the maintenance of the multipotency of mouse trunk neural crest cells, leading to the formation of mouse NCSCs.

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Experimental procedures

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4.1.

Primary and colony cultures

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Primary cultures of mouse trunk neural crest cells were prepared from ddY mouse embryos at embryonic day (E) 9.5 (24- to 29-somite stages) (Ito et al., 1993; Ito and Morita, 1995; Ishikawa and Ito, 2009). Neural tubes were isolated from the dorsal trunk region at the last six-somite levels of E9.5 embryos. Neural tubes were explanted into 35-mm culture dishes coated with collagen gel (PureCol). The explants were scraped away with a tungsten needle after 48 h in culture, leaving the emigrated trunk neural crest cells on the substratum. Primary cultures of mouse embryonic DRG were performed by a modification of methods described previously (Singh et al., 2009). DRG was dissected from ddY mouse embryos at E12.5. Isolated DRG were explanted into 35-mm culture dishes coated with collagen gel.

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Colony cultures of the trunk neural crest cells were performed by a modification of methods described previously (Ito et al., 1993; Ijuin et al., 2008). Neural crest cells were resuspended by trypsinization after 6 days in culture. This essentially single cell suspension (>90% single cells) was diluted with culture medium, and 1 ml aliquots of this cell suspension were seeded at 500 cells/ml onto 35-mm culture dishes coated with a collagen gel and conditioned with culture medium containing 10 lg/ml plasma fibronectin (Itoham). Colony cultures of the DRG cells were carried out according to the methods of colony cultures of the trunk neural crest cells. The colony founder cells were identified at 8 h after seeding cells. The efficiency of colony formation was 5.5 (±1.5)%. The culture medium consisted of 85% a-modified minimum essential medium (a-MEM, Sigma), 10% FBS (Hyclone), 5% CEE, and 50 lg/ml gentamicin (Sigma). The cultures were incubated at 37 C in a humidified atmosphere containing 5% CO2. The culture medium was changed every other day. BMP2 (R&D Systems), Wnt1 (PEPROTECH), and Wnt3a (R&D Systems) were added to the medium at a concentration of 10 or 100 ng/ml. FGF2 (R&D Systems) was added to the medium at a concentration of 10 ng/ml. Chordin (R&D Systems), Noggin (R&D Systems), and BIO (Calbiochem) were added at concentrations of 1 lg/ml, 100 ng/ml, and 1 lM, respectively.

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4.2.

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Transfection of expression vectors

Mouse trunk neural crest cells were transfected with 1 lg of the following expression vectors: (1) pcDNA3 encoding the human DN TCF and a myc epitope (a gift from Dr. Jane B. Trepel; Tetsu and McCormick, 1999; Chung et al., 2002), (2) pcDNA4 encoding the mouse DN b-catenin (a gift from Dr. Jane B. Trepel; Tetsu and McCormick, 1999; Chung et al., 2002), (3) pcDNA3.1 encoding the human WT CHD7 and a Flag-6 · His tag (a gift from Dr. J. Wysocka; Bajpai et al., 2010), (4) pcDNA3.1 encoding the human DN CHD7 and a Flag-6 · His tag (a gift from Dr. J. Wysocka; Bajpai et al., 2010). pcDNA3.1 (Invitrogen) was used as the control vector. The transfection was performed by using Lipofectamine 2000 (Invitrogen) for 24 h immediately after the removal of the neural tubes. The transfection efficiency of the vectors encoding a myc epitope or a Flag tag was estimated by immunostaining using mouse anti-c-myc (9E10) (1:100; Santa Cruz Biotechnology) or mouse anti-Flag-M2 (1:20; Sigma). The transfection efficiency was presented as the proportion of cells expressing the myc epitope or the Flag tag per total cells in a neural crest cell colony. The percentage of cells expressing the myc epitope or the Flag tag reached 52.7 (±3.5) or 72.2 (±2.8)%.

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4.3.

535

siRNA preparation

siRNA was prepared as described previously (Sugiura and Ito, 2010; Fujita et al., 2011). The siRNA duplexes for CHD7 and two SWI/SNF-B subunits, BRD7 and BRG1, were designed on the basis of CHD7, BRD7, and BRG1 sequences published online [GenBank Accession Nos. NM001081417, NM012047, NM011417]. The sequences corresponded to (1) bases 4481– 4506 of the open reading frame of CHD7 mRNA: 5 0 -CAGGCTCAAGCTAGATGCCACAGAA (prepared by Invitrogen), (2)


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bases 1869–1894 of the open reading frame of BRD7 mRNA: 5 0 GAAGCUUGAUGAGACGACAAGAUUG-3 0 (prepared by Invitrogen), and (3) bases 1393–1418 of the open reading frame of BRG1 mRNA: 5 0 -UCGCACACAGAAUUCAGGAACUUGA-3 0 (prepared by Invitrogen). StealthTM RNAi Negative Control Medium GC Duplex #2 (Invitrogen) was used as the control for CHD7, BRD7, or BRG1 siRNA. Using Lipofectamine 2000, mouse trunk neural crest cells were transfected with 40 nM CHD7 siRNA, BRD7 siRNA, BRG1 siRNA, or RNAi Negative Control for 24 h immediately after the removal of the neural tubes. The transfection efficiency was examined by means of BLOCK-iT Fluorescent Oligo (Invitrogen). The percentage of cells showing a fluorescent signal per neural crest cell cluster reached 98.4 ± 0.49%.

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4.4.

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Mouse trunk neural crest cell cultures were fixed with 4% paraformaldehyde (PFA) for 1 h on ice at 4 C. The cultures were immunostained with the following primary antibodies: (1) rabbit anti-Sox10 (1:100; MILLIPORE), (2) rabbit anti-p75 (1:100; Promega), (3) goat anti-CHD7 (1:100; Santa Cruz), (4) rabbit anti-neurofilament L (anti-NF; 1:500; MILLIPORE), (5) mouse anti-a smooth muscle cell actin (anti-SMA; 1:800; Sigma), (6) rabbit anti-glial fibrillary acidic protein (anti-GFAP; 1:100; DAKO), and (7) mouse anti-Brn3a (1:100; Santa Cruz). Primary antibodies were applied for 16 h at 4 C. The cultures were treated with the following secondary antibodies at room temperature for 1 h: (1) Alexa-488-conjugated donkey antigoat IgG (1:500; Invitrogen), (2) Alexa-488-conjugated goat anti-mouse IgG (1:500; Invitrogen), and (3) Alexa-555-conjugated donkey anti-rabbit IgG (1:500; Invitrogen). E9.5 mouse embryos, DRG dissected from 11- to 12-weekold ddY mice, and sciatic nerve from L3 to L5 in 11- to 12week-old ddY mice were fixed with 4% PFA for 1 or 2 h on ice. Isolation of neural tubes with attached DRG from E12.5 mouse embryos were performed by a modification of methods described previously (Hall, 2006). Isolated neural tubes with attached DRG were fixed with 4% PFA for 1 h on ice. The fixed embryos and tissues were immersed in gradually increasing concentrations of sucrose solution and embedded in OCT compound (Miles). Cryostat sections were cut at 10 lm and mounted on albumin-coated glass slides. The sections were stained using the following primary antibodies: (1) rabbit anti-Sox10 (1:200), (2) rabbit anti-p75 (1:100), and (3) goat antiCHD7 (1:50). Primary antibodies were applied for 16 h at 4 C. The specimens were treated with the following secondary antibodies at room temperature for 1 h: (1) Alexa-488conjugated donkey anti-goat IgG (1:500) and (2) Alexa-555conjugated donkey anti-rabbit IgG (1:1000). Finally, the cultures and sections were nuclear-stained with 0.1 lg/ml DAPI (Dojindo). DAPI nuclear staining was particularly important for counting the exact number of immunoreactive cells in the neural crest cell cultures and for judging cell death.

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4.5.

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BrdU-labeling experiments were performed to estimate cell proliferation in mouse trunk neural crest cell cultures. BrdU (Wako) was prepared as a 1 mM stock solution in

Immunostaining

Measurement of cell proliferation and apoptosis

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19

a-MEM and added to the cultures at a final concentration of 50 lM. The cultures were exposed to BrdU for 90 min and subsequently fixed in 4% PFA on ice for 1 h. BrdUincorporated cells were detected with mouse anti-BrdU (1:10; Becton–Dickinson). In double-staining with antiSox10 and anti-BrdU or with anti-p75 and anti-BrdU, Alexa-555-conjugated donkey anti-rabbit IgG (1:500) and Alexa-488-conjugated donkey anti-mouse IgG (1:500) were used as secondary antibodies. Furthermore, we characterized the developmental cell death of the trunk neural crest cells by measuring the expression of the apoptotic marker, caspase-3. The cultures were fixed in 4% PFA on ice for 1 h and double-stained with anti-Sox10 and mouse anticaspase-3 (1:50; Santa Cruz) or with anti-p75 and anticaspase-3. Alexa-555-conjugated donkey anti-rabbit IgG (1:500) and Alexa-488-conjugated donkey anti-mouse IgG (1:500) were used as secondary antibodies. The cultures were nuclear-stained with 0.1 lg/ml DAPI for the total cell count.

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Acknowledgements

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We thank Drs. Jane B. Trepel for providing pcDNA3 encoding the human DN TCF and pcDNA4 encoding the mouse DN b-catenin, J. Wysocka for providing pcDNA3.1 encoding the human WT CHD7 and pcDNA3.1 encoding the human DN CHD7.

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Extended multipotency of neural crest cells and neural crest-derived cells.

Generating trunk neural crest from human pluripotent stem cells.

Roles of Hoxb5 in the development of vagal and trunk neural crest cells.

MycN Is Critical for the Maintenance of Human Embryonic Stem Cell-Derived Neural Crest Stem Cells.

Isolation and propagation of neural crest stem cells from mouse embryonic stem cells via cranial neurospheres.

Ultrastructure of cells of the neural crest.

Roles of imprinted genes in neural stem cells.

Exclusive multipotency and preferential asymmetric divisions in post-embryonic neural stem cells of the fish retina.

Alternative generation of CNS neural stem cells and PNS derivatives from neural crest-derived peripheral stem cells.

Slit molecules prevent entrance of trunk neural crest cells in developing gut.

Correction: MycN Is Critical for the Maintenance of Human Embryonic Stem Cell-Derived Neural Crest Stem Cells.

The origins of neural crest cells in the axolotl.

Neural crest cells in cardiovascular development.

Neural crest-derived cells sustain their multipotency even after entry into their target tissues.

Confetti clarifies controversy: neural crest stem cells are multipotent.

Identification of early neurogenic cells in the neural crest lineage.

Emerging roles of microglia cells in the regulation of adult neural stem cells.

Prospect of Human Pluripotent Stem Cell-Derived Neural Crest Stem Cells in Clinical Application.

FGF8 signaling sustains progenitor status and multipotency of cranial neural crest-derived mesenchymal cells in vivo and in vitro.

A potential inhibitory function of draxin in regulating mouse trunk neural crest migration.

Repression of SIRT1 promotes the differentiation of mouse induced pluripotent stem cells into neural stem cells.

Methods for Derivation of Multipotent Neural Crest Cells Derived from Human Pluripotent Stem Cells.

Engraftable neural crest stem cells derived from cynomolgus monkey embryonic stem cells.

Isolation of Mouse Cardiac Neural Crest Cells and Their Differentiation into Smooth Muscle Cells.