Mol. Cells 2018; 41(12): 1061-1071
Published online November 26, 2018
https://doi.org/10.14348/molcells.2018.0341
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: jbkim@hallym.ac.kr (JBK); ejlee@hallym.ac.kr (UJL)
From
Keywords neurogenesis, transcriptional regulation, Ventx1.1,
BMP4 is a member of the transforming growth factor beta (TGF-β) superfamily which facilitates multiple biological phenomena, including axis patterning, germ-layer specification, differentiation, hematopoiesis, etc. (Dale and Jones, 1999; Munoz-Sanjuan and Brivanlou, 2002). Previous studies have documented BMP4/Smad1 leads the ectoderm and ventral mesoderm formation (VMZ) in
Zic3 has been documented as being a member of the zinc finger transcriptional activator and one of the earliest neural genes actively involved in neural tissue specification, including for neural crest and neural plate formation (Houtmeyers et al., 2013; Nakata et al., 1998). A recent study demonstrated Activin/Smad2/3 positively regulating
In the present study, Ventx1.1 inhibited the Engrailed repressor domain-fused FoxD5b (FoxD5b-En)-induced mRNA expression level of early and late neural genes, including
An approval by the Institutional Animal Care and Use Committee (IACUC) is currently not required for the experimental use of amphibians or reptiles in Korea. However, all our research members attended both the educational and training courses for appropriate care and usage of experimental animals at our institutions. Adult
All mRNA used for this study were synthesized by linearizing the target vectors with the appropriate restriction enzymes, including FoxD5b-En: T7, BamHI, Xcad2: Sp6, Asp718 and Flag-Ventx1.1: Sp6, SacII. Each vector was linearized with the appropriate restriction enzyme and used for in-vitro transcription using the MEGA script kit according to manufacturer’s instructions (Ambion, USA). Synthetic mRNAs were quantified by a spectrophotometer at 260/280nm (SpectraMax, Molecular Devices, USA).
Cloning of
The 1.8 kb of 5′-flanking region of reporter construct was further sub-cloned into the pGL3-basic plasmid (Promega) by using specific restriction sites of KpnI/HindIII. Serially-deleted
Ventx1.1 mRNA (500 pg/embryo) and FoxD5b-En mRNA (280 pg/embryo) were injected into the animal pole at the one or 2-cell stage of
Relative promoter activities were measured using a luciferase assay system according to the manufacturer’s instructions (Promega). Five different groups of embryos (3 embryos per group) were harvested and homogenized in 10 μl lysis buffer per embryo. Embryo homogenates at 10 μl each were assayed with 40 μl luciferase substrate and the reporter gene activity was read by an illuminometer (Berthold Technologies, Germany). All experiments were repeated at least three times for independently derived sample sets.
Mutagenesis was performed by a site-directed mutagenesis kit (Muta-Direct, iNtRON Biotechnology, Korea) using the primer oligonucleotides in accordance with the manufacturer’s instructions (Table 3).
Chromatin immunoprecipitation assay was performed as described (Blythe et al., 2009). Embryos were injected at the one-cell stage with mRNA encoding 3xFlag-Ventx1.1 (500 pg/embryo). Injected embryos were collected at stage 11 (100 embryos/sample) and processed according to the protocol. Anti-Flag monoclonal antibodies (F-1804, Sigma) or normal mouse IgG (SC-2025, Santa Cruz Biotechnology, USA) were then added to the cell lysates to immuneprecipitate the chromatin. ChIP-PCR was performed with the immune-precipitated chromatin using
The
Data were analyzed by unpaired two-tailed Student’s t test using GraphPad Prism4 (GraphPad, USA). Asterisks designations are ** for
Ventx1.1 is a well-known transcriptional repressor of the Xvent family and is one of the direct downstream targets of BMP4/Smad1 signaling (Lee, Lee et al., 2011). Studies have shown that ectopic expression of Ventx1.1 strongly inhibits neurogenesis and organizer expansion in
Previous studies have demonstrated that Ventx1.1 is one of the endogenous neural inhibitors of the Xvent family in
To identify the functionally active Ventx1.1 binding response elements (VREs) within the
Mapping of the
Previously, FoxD5b-En increased
Previous study has demonstrated that Xcad2 is one of the positive regulators of BMP4 signaling and a homeobox transcriptional activator which induces the expression of
BMP4/Smad1 is a crucial signaling circuit determining the fate of ectoderm cells for ectoderm and neuro-ectoderm as well as for ventral and dorsal mesoderm in the early embryogenesis of vertebrates (Ault et al., 1996; Dale and Jones, 1999; Hwang et al., 2003). Both gradients of BMP4/Smad1 from ventral to dorsal mesoderm and from ectoderm to endoderm are important for primordial germ layer specification of mid-blastula embryos (Cha et al., 2008; Maeno et al., 1996; Sasai, 1998). A high concentration of instructive BMP4 signal is required to induce the ventral mesoderm and ectoderm (Maeno et al., 1996; Suzuki et al., 1997a; Wilson et al., 1997), but on the other hand, inhibition of BMP4 signal is crucial for generating the dorsal mesoderm (Spemann organizer) for blastula embryos and the neuro-ectoderm for the gastrula embryos (Delaune et al., 2005; De Robertis and Kuroda, 2004; Sasai et al., 1995; Xu et al., 1995). At the mid-blastula stage, a BMP4/Smad1 signal starts to induce transcription of a set of immediate early genes (maternal to zygotic transition genes), including
From the recent completion of the
Previously, it was reported that downstream target genes of BMP4/Smad1 such as
Ventx1.1 as one of the direct downstream targets of BMP4/Smad1 signaling directly represses expression of
Xcad2 is one of the positive regulators of BMP4 signaling and is a homeobox transcriptional activator which induces the expression of
We observe that VRE1-mutated
The first main event in neural tissue formation including for the central and peripheral nerve system is neuro-ectoderm formation from the naive ectoderm. In the ectoderm, high BMP4/Smad1 signal gradient induces the expression of the immediate early transcription factors (MZT genes), including
Primers used for serially-deleted reporter gene constructs
Primers | Primer Name | Sequences (5′ → 3′) |
---|---|---|
Upstream primers | −1805 | GCGGGTACCTTTTTTACACTTTGTAGGTAT |
−910 | GCGGGTACCTTATGCTGCACGGAAAGG | |
−517 | GCGGGTACCCTCATGAACTGCAAAGCTG | |
−297 | GCGGGTACCAACATCTTTCCACAATCCAT | |
Downstream primer | GCGAAGCTTCCTCTCCAACTGAGTAATGTC |
Primers used for RT-PCR amplification
Gene Name | Sequences | Annealing Temp (°C) | Cycles |
---|---|---|---|
Zic3 | F5′-TTCTCAGGATCTGAACACCT-3′ | 45 | 28 |
R5′-CCCTATAAGACAAGGAATAC-3′ | |||
FoxD5b | F5′-ACTCTATCAGGCACAACCTGTC-3′ | 50 | 30 |
R5′-GGTCTGTAGTAAGGCAGAGAGT-3′ | |||
Xbra | F5′-GGATCGTTATCACCTCTG-3′ | 57 | 25 |
R5′-GTGTAGTCTGTAGCAGCA-3′ | |||
EF1α | F5′-CCTGAATCACCCAGGCCAGATTGTG-3′ | 57 | 19 |
R5′-GAGGGTACTCTGAGAAAGCTCTCCACG-3′ | |||
NCAM | F5′-CACAGTTCCACCAAATGC-3′ | 57 | 29 |
R5′-GGAATCAAGCGGTACAGA-3′ | |||
Xngnr1 | F5′-GGATGGTGCTGCTACCGTGCGAGTACC-3′ | 65 | 30 |
R5′-CAAGCGCAGAGTTCAGGTTGTGCATGC-3′ | |||
Otx2 | F5′-GGATGGATTTGTTGCACCAGTC-3′ | 57 | 27 |
R5′-CACTCTCCCAGCTCACTTCTC-3′ | |||
Xk81 | F5′-TGGTGTTGAACAAGTGCAGG-3′ | 57 | 25 |
R5′-ACCTCCTCGACAATGGTCTT-3′ | |||
Krox20 | F5′-AACCGCCCCAGTAAGACC-3′ | 57 | 28 |
R5′-GTGTCAGCCTGTCCTGTTAG-3′ | |||
NeuroD | F5′-GTGAAATCCCAATAGACACC-3′ | 57 | 28 |
R5′-TTCCCCATATCTAAAGGCAG-3′ |
Primers used for site-directed mutagenesis gene constructs
Mutated Site | Primer Name | Sequence |
---|---|---|
mVRE1 | F5′-AATGTGTGGGAAAATA | |
mVRE2 | F5′-TTACAATTAGCCATC |
Primers used for ChIP-PCR assay
Primer Name | Sequences | Annealing Temp (°C) | Cycles |
---|---|---|---|
F5′-TTCTCAGGATCTGAACACCT-3′ | 52 | 30 | |
F5′-CTTGATCAGATTTTATGTTTC-3′ | 46 | 25 |
Mol. Cells 2018; 41(12): 1061-1071
Published online December 31, 2018 https://doi.org/10.14348/molcells.2018.0341
Copyright © The Korean Society for Molecular and Cellular Biology.
Zobia Umair1, Shiv Kumar1, Daniel H. Kim3, Khezina Rafiq1, Vijay Kumar1, SungChan Kim1, Jae-Bong Park1, Jae-Yong Lee1, Unjoo Lee2,*, and Jaebong Kim1,*
1Department of Biochemistry, Institute of Cell Differentiation and Aging, College of Medicine, Hallym University, Gangwon-Do 24252, Korea, 2Department of Electrical Engineering, Hallym University, Gangwon-Do 24252, Korea, 3Department of Chemical and Biomolecular Engineering, Yonsei University, Seoul 03722, Korea
Correspondence to:*Correspondence: jbkim@hallym.ac.kr (JBK); ejlee@hallym.ac.kr (UJL)
From
Keywords: neurogenesis, transcriptional regulation, Ventx1.1,
BMP4 is a member of the transforming growth factor beta (TGF-β) superfamily which facilitates multiple biological phenomena, including axis patterning, germ-layer specification, differentiation, hematopoiesis, etc. (Dale and Jones, 1999; Munoz-Sanjuan and Brivanlou, 2002). Previous studies have documented BMP4/Smad1 leads the ectoderm and ventral mesoderm formation (VMZ) in
Zic3 has been documented as being a member of the zinc finger transcriptional activator and one of the earliest neural genes actively involved in neural tissue specification, including for neural crest and neural plate formation (Houtmeyers et al., 2013; Nakata et al., 1998). A recent study demonstrated Activin/Smad2/3 positively regulating
In the present study, Ventx1.1 inhibited the Engrailed repressor domain-fused FoxD5b (FoxD5b-En)-induced mRNA expression level of early and late neural genes, including
An approval by the Institutional Animal Care and Use Committee (IACUC) is currently not required for the experimental use of amphibians or reptiles in Korea. However, all our research members attended both the educational and training courses for appropriate care and usage of experimental animals at our institutions. Adult
All mRNA used for this study were synthesized by linearizing the target vectors with the appropriate restriction enzymes, including FoxD5b-En: T7, BamHI, Xcad2: Sp6, Asp718 and Flag-Ventx1.1: Sp6, SacII. Each vector was linearized with the appropriate restriction enzyme and used for in-vitro transcription using the MEGA script kit according to manufacturer’s instructions (Ambion, USA). Synthetic mRNAs were quantified by a spectrophotometer at 260/280nm (SpectraMax, Molecular Devices, USA).
Cloning of
The 1.8 kb of 5′-flanking region of reporter construct was further sub-cloned into the pGL3-basic plasmid (Promega) by using specific restriction sites of KpnI/HindIII. Serially-deleted
Ventx1.1 mRNA (500 pg/embryo) and FoxD5b-En mRNA (280 pg/embryo) were injected into the animal pole at the one or 2-cell stage of
Relative promoter activities were measured using a luciferase assay system according to the manufacturer’s instructions (Promega). Five different groups of embryos (3 embryos per group) were harvested and homogenized in 10 μl lysis buffer per embryo. Embryo homogenates at 10 μl each were assayed with 40 μl luciferase substrate and the reporter gene activity was read by an illuminometer (Berthold Technologies, Germany). All experiments were repeated at least three times for independently derived sample sets.
Mutagenesis was performed by a site-directed mutagenesis kit (Muta-Direct, iNtRON Biotechnology, Korea) using the primer oligonucleotides in accordance with the manufacturer’s instructions (Table 3).
Chromatin immunoprecipitation assay was performed as described (Blythe et al., 2009). Embryos were injected at the one-cell stage with mRNA encoding 3xFlag-Ventx1.1 (500 pg/embryo). Injected embryos were collected at stage 11 (100 embryos/sample) and processed according to the protocol. Anti-Flag monoclonal antibodies (F-1804, Sigma) or normal mouse IgG (SC-2025, Santa Cruz Biotechnology, USA) were then added to the cell lysates to immuneprecipitate the chromatin. ChIP-PCR was performed with the immune-precipitated chromatin using
The
Data were analyzed by unpaired two-tailed Student’s t test using GraphPad Prism4 (GraphPad, USA). Asterisks designations are ** for
Ventx1.1 is a well-known transcriptional repressor of the Xvent family and is one of the direct downstream targets of BMP4/Smad1 signaling (Lee, Lee et al., 2011). Studies have shown that ectopic expression of Ventx1.1 strongly inhibits neurogenesis and organizer expansion in
Previous studies have demonstrated that Ventx1.1 is one of the endogenous neural inhibitors of the Xvent family in
To identify the functionally active Ventx1.1 binding response elements (VREs) within the
Mapping of the
Previously, FoxD5b-En increased
Previous study has demonstrated that Xcad2 is one of the positive regulators of BMP4 signaling and a homeobox transcriptional activator which induces the expression of
BMP4/Smad1 is a crucial signaling circuit determining the fate of ectoderm cells for ectoderm and neuro-ectoderm as well as for ventral and dorsal mesoderm in the early embryogenesis of vertebrates (Ault et al., 1996; Dale and Jones, 1999; Hwang et al., 2003). Both gradients of BMP4/Smad1 from ventral to dorsal mesoderm and from ectoderm to endoderm are important for primordial germ layer specification of mid-blastula embryos (Cha et al., 2008; Maeno et al., 1996; Sasai, 1998). A high concentration of instructive BMP4 signal is required to induce the ventral mesoderm and ectoderm (Maeno et al., 1996; Suzuki et al., 1997a; Wilson et al., 1997), but on the other hand, inhibition of BMP4 signal is crucial for generating the dorsal mesoderm (Spemann organizer) for blastula embryos and the neuro-ectoderm for the gastrula embryos (Delaune et al., 2005; De Robertis and Kuroda, 2004; Sasai et al., 1995; Xu et al., 1995). At the mid-blastula stage, a BMP4/Smad1 signal starts to induce transcription of a set of immediate early genes (maternal to zygotic transition genes), including
From the recent completion of the
Previously, it was reported that downstream target genes of BMP4/Smad1 such as
Ventx1.1 as one of the direct downstream targets of BMP4/Smad1 signaling directly represses expression of
Xcad2 is one of the positive regulators of BMP4 signaling and is a homeobox transcriptional activator which induces the expression of
We observe that VRE1-mutated
The first main event in neural tissue formation including for the central and peripheral nerve system is neuro-ectoderm formation from the naive ectoderm. In the ectoderm, high BMP4/Smad1 signal gradient induces the expression of the immediate early transcription factors (MZT genes), including
. Primers used for serially-deleted reporter gene constructs.
Primers | Primer Name | Sequences (5′ → 3′) |
---|---|---|
Upstream primers | −1805 | GCGGGTACCTTTTTTACACTTTGTAGGTAT |
−910 | GCGGGTACCTTATGCTGCACGGAAAGG | |
−517 | GCGGGTACCCTCATGAACTGCAAAGCTG | |
−297 | GCGGGTACCAACATCTTTCCACAATCCAT | |
Downstream primer | GCGAAGCTTCCTCTCCAACTGAGTAATGTC |
. Primers used for RT-PCR amplification.
Gene Name | Sequences | Annealing Temp (°C) | Cycles |
---|---|---|---|
Zic3 | F5′-TTCTCAGGATCTGAACACCT-3′ | 45 | 28 |
R5′-CCCTATAAGACAAGGAATAC-3′ | |||
FoxD5b | F5′-ACTCTATCAGGCACAACCTGTC-3′ | 50 | 30 |
R5′-GGTCTGTAGTAAGGCAGAGAGT-3′ | |||
Xbra | F5′-GGATCGTTATCACCTCTG-3′ | 57 | 25 |
R5′-GTGTAGTCTGTAGCAGCA-3′ | |||
EF1α | F5′-CCTGAATCACCCAGGCCAGATTGTG-3′ | 57 | 19 |
R5′-GAGGGTACTCTGAGAAAGCTCTCCACG-3′ | |||
NCAM | F5′-CACAGTTCCACCAAATGC-3′ | 57 | 29 |
R5′-GGAATCAAGCGGTACAGA-3′ | |||
Xngnr1 | F5′-GGATGGTGCTGCTACCGTGCGAGTACC-3′ | 65 | 30 |
R5′-CAAGCGCAGAGTTCAGGTTGTGCATGC-3′ | |||
Otx2 | F5′-GGATGGATTTGTTGCACCAGTC-3′ | 57 | 27 |
R5′-CACTCTCCCAGCTCACTTCTC-3′ | |||
Xk81 | F5′-TGGTGTTGAACAAGTGCAGG-3′ | 57 | 25 |
R5′-ACCTCCTCGACAATGGTCTT-3′ | |||
Krox20 | F5′-AACCGCCCCAGTAAGACC-3′ | 57 | 28 |
R5′-GTGTCAGCCTGTCCTGTTAG-3′ | |||
NeuroD | F5′-GTGAAATCCCAATAGACACC-3′ | 57 | 28 |
R5′-TTCCCCATATCTAAAGGCAG-3′ |
. Primers used for site-directed mutagenesis gene constructs.
Mutated Site | Primer Name | Sequence |
---|---|---|
mVRE1 | F5′-AATGTGTGGGAAAATA | |
mVRE2 | F5′-TTACAATTAGCCATC |
. Primers used for ChIP-PCR assay.
Primer Name | Sequences | Annealing Temp (°C) | Cycles |
---|---|---|---|
F5′-TTCTCAGGATCTGAACACCT-3′ | 52 | 30 | |
F5′-CTTGATCAGATTTTATGTTTC-3′ | 46 | 25 |
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