Mol. Cells 2014; 37(3): 220-225
Published online March 6, 2014
https://doi.org/10.14348/molcells.2014.2302
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: jbkim@hallym.ac.kr
Suppression of bone morphogenetic protein (BMP) signaling induces neural induction in the ectoderm of developing embryos. BMP signaling inhibits eural induction
Keywords BMP, FoxD5b, neurogenesis, PV.1,
During the development of vertebrate embryos, bone morphogenetic protein (BMP) signaling negatively regulates neural induction (Dale and Jones, 1999; Hawley et al., 1995; Wilson and Hemmati-Brivanlou, 1995). Ectopic expression of dominant-negative BMP receptors (DNBR) or dissociation results in down-regulation of BMP signaling and leads to neural induction in ectodermal explants (Xu et al., 1995). This phenomenon is known as default neurogenesis (Kuroda et al., 2005). BMPs are a subfamily of the transforming growth factor beta (TGF-β) superfamily and modulate various biological processes
Previous research has shown that the Xvent family of proteins modulates dorso-ventral specification (Friedle and Knochel, 2002; Gawantka et al., 1995). BMP-4 signaling directly induces the expression of Xvent and suppresses neural and dorsal mesodermal fate (Gawantka et al., 1995). PV.1 is a transcription factor that belongs to the Xvent gene family. PV.1 contains a homeodomain and acts as repressor
FoxD5 is a forkhead/winged helix transcription factor that functions in a variety of differentiation processes (Jackson et al., 2010; Katoh and Katoh, 2004; Katoh et al., 2012; Pohl and Knochel, 2005). During early
Here, we confirmed that FoxD5b expression is negatively regulated by BMP signaling. The over-expression of PV.1 (one of the target genes of BMP) indicated that PV.1 directly suppressed FoxD5b expression. Additionally, a promoter assay revealed that PV.1 might regulate FoxD5b expression indirectly via Hox genes. These results suggest that BMP signaling suppressed FoxD5b expression
Embryos were injected with mRNAs as indicated and subsequently processed for whole-mount
For qRT-PCR, total RNA was prepared using the TRIzol reagent (Tel-Test, Inc., USA), and cDNA was synthesized using the SuperScript pre-amplification system (Invitrogen). The PCR primers and cycling conditions are described in the
The PV.1 and DNBR mRNAs used for microinjection were produced by
The level of luciferase activity was measured using a luciferase assay system according to the manufacturer’s instruction (Promega, USA). Five or six groups of animal caps (5 animal caps per group) were harvested and homogenized in 30 μl of lysis buffer. A luminometer was used to measure 40 μl of luciferase substrate and 10 μl of whole cell lysate (Promega, USA). All experiments were repeated at least three times using independently derived sample sets.
Mutagenesis of -301_(m)Hox was performed using a Site-Directed Mutagenesis Kit (Intronbio, KR) according to the manufacturer’s instructions. PCR primers were (upstream) 5′-CA TCACATAGATGCGCGAGACTTAATTATTGG-3′ and (downstream) 5′-CCAATAATTAAGTCTCGCGCATCTATGTGATG-3′.
All experiments were independently performed more than three times. The data are presented as the means ± the SEs.
PV.1 is a downstream target gene of BMP signaling (Ault et al., 1996; Lee et al., 2011) and has a ventralizing effect in
Taken together, these data suggest that PV.1 regulates FoxD5b expression in blastula embryos.
To investigate whether PV.1 directly or indirectly decreases FoxD5b expression, the transcriptional levels of FoxD5b were examined in cyclohexamide (CHX)-treated ectodermal explants by RT-PCR. As shown in Fig. 2A, suppression of BMP signaling induced FoxD5b expression, but co-injection of PV.1 significantly decreased the FoxD5b expression that was induced by DNBR in the control animal cap explants. Interestingly, co-injection of DNBR and PV.1 also led to same result in the cyclohexamide-treated animal cap explants. These data suggests that PV.1 directly reduced FoxD5b expression. Additionally, we also explored whether the expression of Zic3, one of the neural-specific genes, was regulated by PV.1. Our data showed that Zic3 was also directly suppressed by PV.1 (Fig. 2B). Taken together, these results suggest that PV.1 directly suppressed neural gene expression.
To investigate how PV.1 negatively regulates FoxD5b expression, a promoter assay was performed with the 5′-flanking region of the FoxD5b promoter. Over-expression of PV.1 decreased the luciferase activity of the FoxD5b promoter (Fig. 3A). To identify the PV.1-response element, serial truncated FoxD5b promoters were analyzed. Our previous studies have shown that the AP-1 binding site, which acts as a positive regulatory element, is located in between -1336 and -1316. Additionally, we found that two promoter regions of FoxD5a and FoxD5b were highly conserved as shown in Fig. 3B. Truncation of the AP-1 binding site in the FoxD5b promoter decreased luciferase activity because the positive regulatory element was eliminated. Interestingly, over-expression of PV.1 decreased the luciferase activity of the serially truncated FoxD5b promoter, but the activity of the -186 construct was not reduced by PV.1.
These results suggest that the PV.1 response element is located in between -301 and -186, which is a conserved region in the FoxD5 and FoxD5b promoters.
We have previously shown that PV.1 negatively regulates FoxD5b expression and that the response element is located in the FoxD5b promoter between -301 and -186. Thus, we next analyzed promoter sequences to identify the Xvent/PV.1 binding site (CAAATAA) (Taylor et al., 2006). However, the putative Vent/PV.1 binding site was not present, and chromate immunoprecipitation (ChIP) analysis also demonstrated that PV.1 could not bind in this region (data not shown). To confirm if the PV.1 response element is present in the region between -301 and -186, the expression of the luciferase gene, which is encoded by the FoxD5b promoter, was measured using cyclohexamide-treated animal cap explants and RT-PCR. As shown in Fig. 4A, the expression level of luciferase mRNA, which is encoded by the FoxD5b promoter, was increased by the DNBR. Co-injection of PV.1 with DNBR decreased luciferase expression in the DMSO-treated animal cap explants. However, over-expression of PV.1 did not change the expression of the luciferase gene in the cyclohexamide-treated animal cap explants. These results suggest that our FoxD5b promoter construct did not contain the direct PV.1 regulatory element; rather the indirect regulatory response element exists in this region. Interestingly, we found a putative Hox binding site in the region between -301 and -186.
Hox genes are also BMP-target genes and are involved in ventral fate specification (Wacker et al., 2004). Thus, we generated the a -301_(m)Hox construct that contained a mutation in the Hox binding site. We compared the promoter activity of -301 and -301_(m)Hox in whole embryos as indicated in Fig. 4C. The decreased reporter activity of the wild type promoter by PV.1 was not found when -301-(m)Hox was co-injected with PV.1. Thus, our data suggest that the putative Hox binding site mediates the suppression of FoxD5b expression due to the over-expression of PV.1. Additionally, the over-expression of PV.1 revealed that the expression of some Hox genes, includeing HoxB4 and HoxC6, were increased (Fig. 4D).
Taken together, we interpret our data to suggest that the suppression of FoxD5b expression by PV.1 may be mediated by a Hox gene.
BMP signaling is involved in various cell fate specification in of vertebrate embryogenesis (Dale and Wardle, 1999; Dosch et al., 1997; Glinka et al., 1997; Hawley et al., 1995; Wilson and Hemmati-Brivanlou, 1995). During the early development of
Our previous research has shown that the FoxD5a and FoxD5b promoters have two highly conserved regions. The suppression of BMP signaling induces FoxD5b expression through AP-1c-Jun/FosB. Interestingly, the AP-1 binding site is a conserved region. Furthermore, we also found that the over-expression of BMP4 strongly reduced the activity of the FoxD5b promoter in the entire embryo (data not shown). These data suggest that the FoxD5b promoter has a negative response element that is regulated by BMP signaling.
PV.1 has roles in ventralization and inhibition of neural induction that are mediated by BMP signaling
Although the over-expression of BMP downstream target genes, including MSX1, GATA and Vents, is sufficient for the suppression of neural fate, no data exists that suggests that the knock-down of these genes induces neural tissue. In this study, we also examined whether the knock down of PV.1 induced neural induction. The micro-injection of PV.1 morpholino oligos slightly increased the expression of Zic3 and FoxD5b at stage 10, but these treatments did not induce any neural marker at stage 24. This result indicates that the knock-down of PV.1 alone was not sufficient to induce neurogenesis. In other words, PV.1 and some BMP down-stream target genes cooperatively suppress the neural gene expression.
Mol. Cells 2014; 37(3): 220-225
Published online March 31, 2014 https://doi.org/10.14348/molcells.2014.2302
Copyright © The Korean Society for Molecular and Cellular Biology.
Jaeho Yoon1,2, Jung-Ho Kim1,2, Sung Chan Kim1, Jae-Bong Park1, Jae-Yong Lee1, and Jaebong Kim1,*
1Department of Biochemistry, Institute of Cell Differentiation and Aging, College of Medicine, Hallym University, Chuncheon 200-702, Korea, 2These authors contributed equally to this work.
Correspondence to:*Correspondence: jbkim@hallym.ac.kr
Suppression of bone morphogenetic protein (BMP) signaling induces neural induction in the ectoderm of developing embryos. BMP signaling inhibits eural induction
Keywords: BMP, FoxD5b, neurogenesis, PV.1,
During the development of vertebrate embryos, bone morphogenetic protein (BMP) signaling negatively regulates neural induction (Dale and Jones, 1999; Hawley et al., 1995; Wilson and Hemmati-Brivanlou, 1995). Ectopic expression of dominant-negative BMP receptors (DNBR) or dissociation results in down-regulation of BMP signaling and leads to neural induction in ectodermal explants (Xu et al., 1995). This phenomenon is known as default neurogenesis (Kuroda et al., 2005). BMPs are a subfamily of the transforming growth factor beta (TGF-β) superfamily and modulate various biological processes
Previous research has shown that the Xvent family of proteins modulates dorso-ventral specification (Friedle and Knochel, 2002; Gawantka et al., 1995). BMP-4 signaling directly induces the expression of Xvent and suppresses neural and dorsal mesodermal fate (Gawantka et al., 1995). PV.1 is a transcription factor that belongs to the Xvent gene family. PV.1 contains a homeodomain and acts as repressor
FoxD5 is a forkhead/winged helix transcription factor that functions in a variety of differentiation processes (Jackson et al., 2010; Katoh and Katoh, 2004; Katoh et al., 2012; Pohl and Knochel, 2005). During early
Here, we confirmed that FoxD5b expression is negatively regulated by BMP signaling. The over-expression of PV.1 (one of the target genes of BMP) indicated that PV.1 directly suppressed FoxD5b expression. Additionally, a promoter assay revealed that PV.1 might regulate FoxD5b expression indirectly via Hox genes. These results suggest that BMP signaling suppressed FoxD5b expression
Embryos were injected with mRNAs as indicated and subsequently processed for whole-mount
For qRT-PCR, total RNA was prepared using the TRIzol reagent (Tel-Test, Inc., USA), and cDNA was synthesized using the SuperScript pre-amplification system (Invitrogen). The PCR primers and cycling conditions are described in the
The PV.1 and DNBR mRNAs used for microinjection were produced by
The level of luciferase activity was measured using a luciferase assay system according to the manufacturer’s instruction (Promega, USA). Five or six groups of animal caps (5 animal caps per group) were harvested and homogenized in 30 μl of lysis buffer. A luminometer was used to measure 40 μl of luciferase substrate and 10 μl of whole cell lysate (Promega, USA). All experiments were repeated at least three times using independently derived sample sets.
Mutagenesis of -301_(m)Hox was performed using a Site-Directed Mutagenesis Kit (Intronbio, KR) according to the manufacturer’s instructions. PCR primers were (upstream) 5′-CA TCACATAGATGCGCGAGACTTAATTATTGG-3′ and (downstream) 5′-CCAATAATTAAGTCTCGCGCATCTATGTGATG-3′.
All experiments were independently performed more than three times. The data are presented as the means ± the SEs.
PV.1 is a downstream target gene of BMP signaling (Ault et al., 1996; Lee et al., 2011) and has a ventralizing effect in
Taken together, these data suggest that PV.1 regulates FoxD5b expression in blastula embryos.
To investigate whether PV.1 directly or indirectly decreases FoxD5b expression, the transcriptional levels of FoxD5b were examined in cyclohexamide (CHX)-treated ectodermal explants by RT-PCR. As shown in Fig. 2A, suppression of BMP signaling induced FoxD5b expression, but co-injection of PV.1 significantly decreased the FoxD5b expression that was induced by DNBR in the control animal cap explants. Interestingly, co-injection of DNBR and PV.1 also led to same result in the cyclohexamide-treated animal cap explants. These data suggests that PV.1 directly reduced FoxD5b expression. Additionally, we also explored whether the expression of Zic3, one of the neural-specific genes, was regulated by PV.1. Our data showed that Zic3 was also directly suppressed by PV.1 (Fig. 2B). Taken together, these results suggest that PV.1 directly suppressed neural gene expression.
To investigate how PV.1 negatively regulates FoxD5b expression, a promoter assay was performed with the 5′-flanking region of the FoxD5b promoter. Over-expression of PV.1 decreased the luciferase activity of the FoxD5b promoter (Fig. 3A). To identify the PV.1-response element, serial truncated FoxD5b promoters were analyzed. Our previous studies have shown that the AP-1 binding site, which acts as a positive regulatory element, is located in between -1336 and -1316. Additionally, we found that two promoter regions of FoxD5a and FoxD5b were highly conserved as shown in Fig. 3B. Truncation of the AP-1 binding site in the FoxD5b promoter decreased luciferase activity because the positive regulatory element was eliminated. Interestingly, over-expression of PV.1 decreased the luciferase activity of the serially truncated FoxD5b promoter, but the activity of the -186 construct was not reduced by PV.1.
These results suggest that the PV.1 response element is located in between -301 and -186, which is a conserved region in the FoxD5 and FoxD5b promoters.
We have previously shown that PV.1 negatively regulates FoxD5b expression and that the response element is located in the FoxD5b promoter between -301 and -186. Thus, we next analyzed promoter sequences to identify the Xvent/PV.1 binding site (CAAATAA) (Taylor et al., 2006). However, the putative Vent/PV.1 binding site was not present, and chromate immunoprecipitation (ChIP) analysis also demonstrated that PV.1 could not bind in this region (data not shown). To confirm if the PV.1 response element is present in the region between -301 and -186, the expression of the luciferase gene, which is encoded by the FoxD5b promoter, was measured using cyclohexamide-treated animal cap explants and RT-PCR. As shown in Fig. 4A, the expression level of luciferase mRNA, which is encoded by the FoxD5b promoter, was increased by the DNBR. Co-injection of PV.1 with DNBR decreased luciferase expression in the DMSO-treated animal cap explants. However, over-expression of PV.1 did not change the expression of the luciferase gene in the cyclohexamide-treated animal cap explants. These results suggest that our FoxD5b promoter construct did not contain the direct PV.1 regulatory element; rather the indirect regulatory response element exists in this region. Interestingly, we found a putative Hox binding site in the region between -301 and -186.
Hox genes are also BMP-target genes and are involved in ventral fate specification (Wacker et al., 2004). Thus, we generated the a -301_(m)Hox construct that contained a mutation in the Hox binding site. We compared the promoter activity of -301 and -301_(m)Hox in whole embryos as indicated in Fig. 4C. The decreased reporter activity of the wild type promoter by PV.1 was not found when -301-(m)Hox was co-injected with PV.1. Thus, our data suggest that the putative Hox binding site mediates the suppression of FoxD5b expression due to the over-expression of PV.1. Additionally, the over-expression of PV.1 revealed that the expression of some Hox genes, includeing HoxB4 and HoxC6, were increased (Fig. 4D).
Taken together, we interpret our data to suggest that the suppression of FoxD5b expression by PV.1 may be mediated by a Hox gene.
BMP signaling is involved in various cell fate specification in of vertebrate embryogenesis (Dale and Wardle, 1999; Dosch et al., 1997; Glinka et al., 1997; Hawley et al., 1995; Wilson and Hemmati-Brivanlou, 1995). During the early development of
Our previous research has shown that the FoxD5a and FoxD5b promoters have two highly conserved regions. The suppression of BMP signaling induces FoxD5b expression through AP-1c-Jun/FosB. Interestingly, the AP-1 binding site is a conserved region. Furthermore, we also found that the over-expression of BMP4 strongly reduced the activity of the FoxD5b promoter in the entire embryo (data not shown). These data suggest that the FoxD5b promoter has a negative response element that is regulated by BMP signaling.
PV.1 has roles in ventralization and inhibition of neural induction that are mediated by BMP signaling
Although the over-expression of BMP downstream target genes, including MSX1, GATA and Vents, is sufficient for the suppression of neural fate, no data exists that suggests that the knock-down of these genes induces neural tissue. In this study, we also examined whether the knock down of PV.1 induced neural induction. The micro-injection of PV.1 morpholino oligos slightly increased the expression of Zic3 and FoxD5b at stage 10, but these treatments did not induce any neural marker at stage 24. This result indicates that the knock-down of PV.1 alone was not sufficient to induce neurogenesis. In other words, PV.1 and some BMP down-stream target genes cooperatively suppress the neural gene expression.
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