Mol. Cells 2021; 44(10): 723-735
Published online October 25, 2021
https://doi.org/10.14348/molcells.2021.0055
© The Korean Society for Molecular and Cellular Biology
Correspondence to : jbkim@hallym.ac.kr (JK); ejlee@hallym.ac.kr (UL)
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
Spemann organizer is a center of dorsal mesoderm and itself retains the mesoderm character, but it has a stimulatory role for neighboring ectoderm cells in becoming neuroectoderm in gastrula embryos. Goosecoid (Gsc) overexpression in ventral region promotes secondary axis formation including neural tissues, but the role of gsc in neural specification could be indirect. We examined the neural inhibitory and stimulatory roles of gsc in the same cell and neighboring cells contexts. In the animal cap explant system, Gsc overexpression inhibited expression of neural specific genes including foxd4l1.1, zic3, ncam, and neurod. Genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) and promoter analysis of early neural genes of foxd4l1.1 and zic3 were performed to show that the neural inhibitory mode of gsc was direct. Site-directed mutagenesis and serially deleted construct studies of foxd4l1.1 promoter revealed that Gsc directly binds within the foxd4l1.1 promoter to repress its expression. Conjugation assay of animal cap explants was also performed to demonstrate an indirect neural stimulatory role for gsc. The genes for secretory molecules, Chordin and Noggin, were up-regulated in gsc injected cells with the neural fate only achieved in gsc uninjected neighboring cells. These experiments suggested that gsc regulates neuroectoderm formation negatively when expressed in the same cell and positively in neighboring cells via soluble factors. One is a direct suppressive circuit of neural genes in gsc expressing mesoderm cells and the other is an indirect stimulatory circuit for neurogenesis in neighboring ectoderm cells via secreted BMP antagonizers.
Keywords chordin, dorsal organizer, Gsc, Gsc response element, neuroectoderm, Noggin, transcriptional regulation, Xenopus
Spemann organizer has been established as a center of dorsal mesoderm in the early embryo; this is demonstrated by the organizer transplantation into the ventral side of another embryo, resulting in duplication of the body axis including the head and trunk (Cho et al., 1991; De Robertis et al., 2000; Nieto, 1999). In the same experiment, the duplicated complete axis contained well-organized body patterning of all three germ layers. This structural patterning of Spemann organizer was considered to be due to instructive signaling to induce neuroectoderm formation from the ectoderm. However, search for such instructive signal(s) led to the unexpected finding that instead of providing instructive signals, inhibitory signals emanate from the organizer. The main organizer genes including
Consistent with the conserved natured of the organizer across species, a conserved group of genes have been identified associated with the organizer. One of the first genes identified in the organizer was the homeobox transcription factor (TF)
Gsc is also one of the organizer genes responsible for dorso-ventral patterning (Niehrs et al., 1993). Introduction of
Foxd4l1.1 is a forkhead/winged helix TF that functions in a variety of differentiation processes (Jackson et al., 2010; Katoh et al., 2013; Katoh and Katoh, 2004; Pohl and Knochel, 2005).
In the present study, we hypothesized that organizer gene
This animal study was conducted in accordance with the regulations of the Institutional Animal Care and Use Committee (IACUC) of Hallym University (Hallym 2019-79, 2019-80). All the research members attended both the educational and training courses for the appropriate care and use of experimental animals at our institution in order to receive an animal use permit. Adult
All mRNA used for this study were synthesized by linearizing the target vectors with the appropriate restriction enzymes, including Sp6/SacII
Cloning of
The 5’-flanking region of reporter construct of
DNBR mRNA (0.5 ng/embryo),
Antisense morpholinos (MOs) for
Embryos were injected with mRNAs as indicated and subsequently processed for whole-mount
Relative promoter activities were measured using a luciferase assay system according to the manufacturer’s instructions (Promega) and were performed as previously described (Yoon et al., 2014). 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 primer oligonucleotides in accordance with the manufacturer’s instructions (Table 3).
Chromatin immunoprecipitation assay was performed as described previously (Blythe et al., 2009). Embryos were injected at one-cell stage with mRNA encoding 3Flag-Gsc (1 ng/embryo). Injected embryos were collected at stage 11 (100 embryos/sample) and processed according to 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 immune-precipitate the chromatin. ChIP-PCR was performed with the immune-precipitated chromatin using
The Gsc mRNA (0.5 ng/embryo) was injected at the one-cell stage. Approximately 1,000 embryos were harvested at stage 11. The ChIP assay was performed accordingly to a previously described method (Blythe et al., 2009). Total immunoprecipitated chromatin was sequenced by Macrogen (Korea), and raw data with short reads were received in FASTA format. Galaxy (https://usegalaxy.org), an online tool, was used for data analysis as described previously (Zhang et al., 2008). MACS call peak data were used for visualization, and Gsc coverage within the
AC explants were dissected at stages 8 and conjugation of the two required AC explants together was in 30% MMR containing 50 mg/ml gentamycin. The recombinants were cultured for 40 min at 16°C to heal the torn ends of the tissue explants before being transferred to fresh L-15 media.
Data were analyzed by unpaired two-tailed Student’s
Previous reports demonstrate that
Several independent studies demonstrate that Gsc has strong repressor activity through direct binding on cis-acting response element within target genes to block their transcription (Cho et al., 1991; Christian and Moon, 1993; Latinkic and Smith, 1999; Yasuo and Lemaire, 2001). To identify the neural specific gene(s) (
As shown above, ChIP-seq results indicated putative GREs within
During early development, Gsc is known as a neural stimulator, probably through induction of
In the present study, we sought to define the role of the homeobox repressor
We have previously reported on various mechanisms existing in reciprocally exclusive germ-layer specifications in early vertebrate embryogenesis (ectoderm, mesoderm and neuroectoderm) (Kumar et al., 2020). In the ventral mesoderm and ectoderm region, neural repressor
From our preliminary findings, Gsc promoter also contained the direct binding response element for Foxd4l1.1 (from Chip-seq and reporter assay of Gsc promoter) (unpublished data). Whether a reciprocal repression of
In this study, we adopted
Gsc as an organizer specific repressor TF has been studied in the context of dorso-ventral specification as well as a factor in protecting the organizer by repression of signaling molecules including BMP and Wnt8 (Yao and Kessler, 2001). Gsc has been proposed as a central TF involved in mesoderm patterning (Niehrs et al.,1994). Although showing a direct reciprocal repression between dorsal and ventral specific homeobox genes
Additional studies have mainly focused on neural stimulatory effect of Gsc through induction of neural stimulatory factor
In the present paper, we sought to explain the dual roles of the organizer in neuroectoderm specification in the context of the functional activity of the organizer specific TF
This article was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), which is funded by the Ministry of Education, Science, and Technology of Korea (2016R1D1A1B02008770, 2018M3C7A1056285, and 2021M3H9A1097557).
Z.U. and V.K. performed the experiments and wrote the primary manuscript. J.K. and U.L. designed and supervised the study. R.S.G. and S.K. contributed to the data analysis and revision of the manuscript.
The authors have no potential conflicts of interest to disclose.
Primers used for serially-deleted FoxD5b reporter gene constructs
Primer | Primer name | Sequence |
---|---|---|
Upstream primer | Foxd4l1.1(–1551) | 5’-CCGGTACCTAGAGGTTGGATAAAGTCAATTGC-3’ |
Foxd4l1.1(–1316) | 5’-CCGGTACCTATATGCAGAGCTGCTAATAGTC-3’ | |
Foxd4l1.1(–1016) | 5’-CCGGTACCTATATGCAGAGCTGCTAATAGTC-3’ | |
Foxd4l1.1(–816) | 5’-CCGGTACCTAGAATTCCAGTTCCCATAATC-3’ | |
Foxd4l1.1(–301) | 5’-CCGGTACCTTGGATTGCAAGTTAGTGGCTC-3’ | |
Foxd4l1.1(–186) | 5’-GGGGTACCTTCATTCAGCAAAAGCACAGCC-3’ | |
Foxd4l1.1(–78) | 5’-GGGGTACCAATTCAAGTGCAGATGACTGCC-3’ | |
Downstream primer | Foxd4l1.1-R | 5’-ATCTCGAGGCTTGGTTGGCAGTAAGTAG-3’ |
Primers used for RT-PCR amplification
Gene name | Sequence | Annealing temperature (°C) | Cycle |
---|---|---|---|
F5’-TCTCAGGATCTGAACACCT-3’ | 45 | 28 | |
R5’-CCCTATAAGACAAGGAATAC-3’ | |||
F5’-ACTCTATCAGGCACAACCTGTC-3’ | 50 | 30 | |
R5’-GGTCTGTAGTAAGGCAGAGAGT-3’ | |||
F5’-GGATCGTTATCACCTCTG-3’ | 57 | 25 | |
R5’-GTGTAGTCTGTAGCAGCA-3’ | |||
F5’-CTGGTGACCGACCAACTAAG-3’ | 55 | 28 | |
R5’-TGCGAACTCTGCTTCCAAAC-3’ | |||
F5’-ATGTGCGGAGGCTGCGTC-3’ | 60 | 27 | |
R5’-CGTGGGTCATCGGGTAGAAC-3’ | |||
F5’-GAGCTGATGAGGTGCAAGAG-3’ | 60 | 27 | |
R5’-TTTGCTCATCTTCTTGTTGG-3’ | |||
F5’-CACAGTTCCACCAAATGC-3’ | 57 | 29 | |
R5’-GGAATCAAGCGGTACAGA-3’ | |||
F5’-GGATGGTGCTGCTACCGTGCGAGTACC-3’ | 65 | 30 | |
R5’-CAAGCGCAGAGTTCAGGTTGTGCATGC-3’ | |||
F5’-GGATGGATTTGTTGCACCAGTC-3’ | 57 | 27 | |
R5’-CACTCTCCCAGCTCACTTCTC-3’ | |||
F5’-TGGTGTTGAACAAGTGCAGG-3’ | 57 | 25 | |
R5’-ACCTCCTCGACAATGGTCTT-3’ | |||
F5’-AACCGCCCCAGTAAGACC-3’ | 57 | 28 | |
R5’-GTGTCAGCCTGTCCTGTTAG-3’ | |||
F5’-GTGAAATCCCAATAGACACC-3’ | 57 | 28 | |
R5’-TTCCCCATATCTAAAGGCAG-3’ | |||
F5’-TACTTACGGGCTTGGCTGGA-3’ | 68 | 26 | |
R5’-AGCGTGTAACCAGTTGGCTG-3’ | |||
F5’-GCTGATTCCACCAGTGCCTCACCAG-3’ | 60 | 30 | |
R5’-GGTCCTGTGCCTCCTCCTCCTCCTG-3’ | |||
F5’-AGTTGCAGATGTGGCTCT-3’ | 57 | 27 | |
R5’-AGTCCAAGAGTCTGAGCA-3’ | |||
F5’-TTAGAGAGGAGAGCAACTCGGGCAAT-3’ | 57 | 25 | |
R5’-GTGCTCCTGTTGCGAAACTCTACAGA-3’ | |||
F5’-CATCATGATTCCTGGTAACCGA-3’ | 57 | 25 | |
R5’-CTCCATGCTGATATCGTGCAG-3’ | |||
F5’-CCTTCAGCATGGTTCAACAG-3’ | 57 | 26 | |
R5’-CATCCTTCTTCCTTGGCATC-3’ | |||
F5’-CCTGAATCACCCAGGCCAGATTGTG-3’ | 57 | 19 | |
R5’-GAGGGTACTCTGAGAAAGCTCTCCACG-3’ | |||
F5’-GTCAATGATGGAGTGTATGGATC-3’ | 55 | 25 | |
R5’-TCCATTCCGCTCTCCTGAGCAC-3’ |
Primers used for site-directed mutagenesis
Mutated site | Primer name | Sequence |
---|---|---|
GRE | Foxd4l1.1mGRE | F5’-GGACCCTCTCACGTGGGAGCTTATCTGATAR-3’ |
R5’-TATCAGATAAGCTCCCACGTGAGAGGGTCC-3’ |
Primers used for ChIP-PCR assay
Prime name | Sequence | Annealing temperature (°C) | Cycle |
---|---|---|---|
Foxd4l1.1ChIP-GRE | F5’-ACCTTGTTGGACTACAGATTC-3’ | 52 | 30 |
R5’-CAGTCATCTGCACTTGAATTGG-3’ |
Mol. Cells 2021; 44(10): 723-735
Published online October 31, 2021 https://doi.org/10.14348/molcells.2021.0055
Copyright © The Korean Society for Molecular and Cellular Biology.
Zobia Umair1,3,4 , Vijay Kumar1,4
, Ravi Shankar Goutam1
, Shiv Kumar1
, Unjoo Lee2,*
, and Jaebong Kim1,*
1Department of Biochemistry, Institute of Cell Differentiation and Aging, College of Medicine, Hallym University, Chuncheon 24252, Korea, 2Department of Electrical Engineering, Hallym University, Chuncheon 24252, Korea, 3Department of Molecular Medicine, School of Medicine, Gachon University, Incheon 21999, Korea, 4These authors contributed equally to this work.
Correspondence to:jbkim@hallym.ac.kr (JK); ejlee@hallym.ac.kr (UL)
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
Spemann organizer is a center of dorsal mesoderm and itself retains the mesoderm character, but it has a stimulatory role for neighboring ectoderm cells in becoming neuroectoderm in gastrula embryos. Goosecoid (Gsc) overexpression in ventral region promotes secondary axis formation including neural tissues, but the role of gsc in neural specification could be indirect. We examined the neural inhibitory and stimulatory roles of gsc in the same cell and neighboring cells contexts. In the animal cap explant system, Gsc overexpression inhibited expression of neural specific genes including foxd4l1.1, zic3, ncam, and neurod. Genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) and promoter analysis of early neural genes of foxd4l1.1 and zic3 were performed to show that the neural inhibitory mode of gsc was direct. Site-directed mutagenesis and serially deleted construct studies of foxd4l1.1 promoter revealed that Gsc directly binds within the foxd4l1.1 promoter to repress its expression. Conjugation assay of animal cap explants was also performed to demonstrate an indirect neural stimulatory role for gsc. The genes for secretory molecules, Chordin and Noggin, were up-regulated in gsc injected cells with the neural fate only achieved in gsc uninjected neighboring cells. These experiments suggested that gsc regulates neuroectoderm formation negatively when expressed in the same cell and positively in neighboring cells via soluble factors. One is a direct suppressive circuit of neural genes in gsc expressing mesoderm cells and the other is an indirect stimulatory circuit for neurogenesis in neighboring ectoderm cells via secreted BMP antagonizers.
Keywords: chordin, dorsal organizer, Gsc, Gsc response element, neuroectoderm, Noggin, transcriptional regulation, Xenopus
Spemann organizer has been established as a center of dorsal mesoderm in the early embryo; this is demonstrated by the organizer transplantation into the ventral side of another embryo, resulting in duplication of the body axis including the head and trunk (Cho et al., 1991; De Robertis et al., 2000; Nieto, 1999). In the same experiment, the duplicated complete axis contained well-organized body patterning of all three germ layers. This structural patterning of Spemann organizer was considered to be due to instructive signaling to induce neuroectoderm formation from the ectoderm. However, search for such instructive signal(s) led to the unexpected finding that instead of providing instructive signals, inhibitory signals emanate from the organizer. The main organizer genes including
Consistent with the conserved natured of the organizer across species, a conserved group of genes have been identified associated with the organizer. One of the first genes identified in the organizer was the homeobox transcription factor (TF)
Gsc is also one of the organizer genes responsible for dorso-ventral patterning (Niehrs et al., 1993). Introduction of
Foxd4l1.1 is a forkhead/winged helix TF that functions in a variety of differentiation processes (Jackson et al., 2010; Katoh et al., 2013; Katoh and Katoh, 2004; Pohl and Knochel, 2005).
In the present study, we hypothesized that organizer gene
This animal study was conducted in accordance with the regulations of the Institutional Animal Care and Use Committee (IACUC) of Hallym University (Hallym 2019-79, 2019-80). All the research members attended both the educational and training courses for the appropriate care and use of experimental animals at our institution in order to receive an animal use permit. Adult
All mRNA used for this study were synthesized by linearizing the target vectors with the appropriate restriction enzymes, including Sp6/SacII
Cloning of
The 5’-flanking region of reporter construct of
DNBR mRNA (0.5 ng/embryo),
Antisense morpholinos (MOs) for
Embryos were injected with mRNAs as indicated and subsequently processed for whole-mount
Relative promoter activities were measured using a luciferase assay system according to the manufacturer’s instructions (Promega) and were performed as previously described (Yoon et al., 2014). 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 primer oligonucleotides in accordance with the manufacturer’s instructions (Table 3).
Chromatin immunoprecipitation assay was performed as described previously (Blythe et al., 2009). Embryos were injected at one-cell stage with mRNA encoding 3Flag-Gsc (1 ng/embryo). Injected embryos were collected at stage 11 (100 embryos/sample) and processed according to 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 immune-precipitate the chromatin. ChIP-PCR was performed with the immune-precipitated chromatin using
The Gsc mRNA (0.5 ng/embryo) was injected at the one-cell stage. Approximately 1,000 embryos were harvested at stage 11. The ChIP assay was performed accordingly to a previously described method (Blythe et al., 2009). Total immunoprecipitated chromatin was sequenced by Macrogen (Korea), and raw data with short reads were received in FASTA format. Galaxy (https://usegalaxy.org), an online tool, was used for data analysis as described previously (Zhang et al., 2008). MACS call peak data were used for visualization, and Gsc coverage within the
AC explants were dissected at stages 8 and conjugation of the two required AC explants together was in 30% MMR containing 50 mg/ml gentamycin. The recombinants were cultured for 40 min at 16°C to heal the torn ends of the tissue explants before being transferred to fresh L-15 media.
Data were analyzed by unpaired two-tailed Student’s
Previous reports demonstrate that
Several independent studies demonstrate that Gsc has strong repressor activity through direct binding on cis-acting response element within target genes to block their transcription (Cho et al., 1991; Christian and Moon, 1993; Latinkic and Smith, 1999; Yasuo and Lemaire, 2001). To identify the neural specific gene(s) (
As shown above, ChIP-seq results indicated putative GREs within
During early development, Gsc is known as a neural stimulator, probably through induction of
In the present study, we sought to define the role of the homeobox repressor
We have previously reported on various mechanisms existing in reciprocally exclusive germ-layer specifications in early vertebrate embryogenesis (ectoderm, mesoderm and neuroectoderm) (Kumar et al., 2020). In the ventral mesoderm and ectoderm region, neural repressor
From our preliminary findings, Gsc promoter also contained the direct binding response element for Foxd4l1.1 (from Chip-seq and reporter assay of Gsc promoter) (unpublished data). Whether a reciprocal repression of
In this study, we adopted
Gsc as an organizer specific repressor TF has been studied in the context of dorso-ventral specification as well as a factor in protecting the organizer by repression of signaling molecules including BMP and Wnt8 (Yao and Kessler, 2001). Gsc has been proposed as a central TF involved in mesoderm patterning (Niehrs et al.,1994). Although showing a direct reciprocal repression between dorsal and ventral specific homeobox genes
Additional studies have mainly focused on neural stimulatory effect of Gsc through induction of neural stimulatory factor
In the present paper, we sought to explain the dual roles of the organizer in neuroectoderm specification in the context of the functional activity of the organizer specific TF
This article was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), which is funded by the Ministry of Education, Science, and Technology of Korea (2016R1D1A1B02008770, 2018M3C7A1056285, and 2021M3H9A1097557).
Z.U. and V.K. performed the experiments and wrote the primary manuscript. J.K. and U.L. designed and supervised the study. R.S.G. and S.K. contributed to the data analysis and revision of the manuscript.
The authors have no potential conflicts of interest to disclose.
Primers used for serially-deleted FoxD5b reporter gene constructs
Primer | Primer name | Sequence |
---|---|---|
Upstream primer | Foxd4l1.1(–1551) | 5’-CCGGTACCTAGAGGTTGGATAAAGTCAATTGC-3’ |
Foxd4l1.1(–1316) | 5’-CCGGTACCTATATGCAGAGCTGCTAATAGTC-3’ | |
Foxd4l1.1(–1016) | 5’-CCGGTACCTATATGCAGAGCTGCTAATAGTC-3’ | |
Foxd4l1.1(–816) | 5’-CCGGTACCTAGAATTCCAGTTCCCATAATC-3’ | |
Foxd4l1.1(–301) | 5’-CCGGTACCTTGGATTGCAAGTTAGTGGCTC-3’ | |
Foxd4l1.1(–186) | 5’-GGGGTACCTTCATTCAGCAAAAGCACAGCC-3’ | |
Foxd4l1.1(–78) | 5’-GGGGTACCAATTCAAGTGCAGATGACTGCC-3’ | |
Downstream primer | Foxd4l1.1-R | 5’-ATCTCGAGGCTTGGTTGGCAGTAAGTAG-3’ |
Primers used for RT-PCR amplification
Gene name | Sequence | Annealing temperature (°C) | Cycle |
---|---|---|---|
|
F5’-TCTCAGGATCTGAACACCT-3’ | 45 | 28 |
R5’-CCCTATAAGACAAGGAATAC-3’ | |||
|
F5’-ACTCTATCAGGCACAACCTGTC-3’ | 50 | 30 |
R5’-GGTCTGTAGTAAGGCAGAGAGT-3’ | |||
|
F5’-GGATCGTTATCACCTCTG-3’ | 57 | 25 |
R5’-GTGTAGTCTGTAGCAGCA-3’ | |||
|
F5’-CTGGTGACCGACCAACTAAG-3’ | 55 | 28 |
R5’-TGCGAACTCTGCTTCCAAAC-3’ | |||
|
F5’-ATGTGCGGAGGCTGCGTC-3’ | 60 | 27 |
R5’-CGTGGGTCATCGGGTAGAAC-3’ | |||
|
F5’-GAGCTGATGAGGTGCAAGAG-3’ | 60 | 27 |
R5’-TTTGCTCATCTTCTTGTTGG-3’ | |||
|
F5’-CACAGTTCCACCAAATGC-3’ | 57 | 29 |
R5’-GGAATCAAGCGGTACAGA-3’ | |||
|
F5’-GGATGGTGCTGCTACCGTGCGAGTACC-3’ | 65 | 30 |
R5’-CAAGCGCAGAGTTCAGGTTGTGCATGC-3’ | |||
|
F5’-GGATGGATTTGTTGCACCAGTC-3’ | 57 | 27 |
R5’-CACTCTCCCAGCTCACTTCTC-3’ | |||
|
F5’-TGGTGTTGAACAAGTGCAGG-3’ | 57 | 25 |
R5’-ACCTCCTCGACAATGGTCTT-3’ | |||
|
F5’-AACCGCCCCAGTAAGACC-3’ | 57 | 28 |
R5’-GTGTCAGCCTGTCCTGTTAG-3’ | |||
|
F5’-GTGAAATCCCAATAGACACC-3’ | 57 | 28 |
R5’-TTCCCCATATCTAAAGGCAG-3’ | |||
|
F5’-TACTTACGGGCTTGGCTGGA-3’ | 68 | 26 |
R5’-AGCGTGTAACCAGTTGGCTG-3’ | |||
|
F5’-GCTGATTCCACCAGTGCCTCACCAG-3’ | 60 | 30 |
R5’-GGTCCTGTGCCTCCTCCTCCTCCTG-3’ | |||
|
F5’-AGTTGCAGATGTGGCTCT-3’ | 57 | 27 |
R5’-AGTCCAAGAGTCTGAGCA-3’ | |||
|
F5’-TTAGAGAGGAGAGCAACTCGGGCAAT-3’ | 57 | 25 |
R5’-GTGCTCCTGTTGCGAAACTCTACAGA-3’ | |||
|
F5’-CATCATGATTCCTGGTAACCGA-3’ | 57 | 25 |
R5’-CTCCATGCTGATATCGTGCAG-3’ | |||
|
F5’-CCTTCAGCATGGTTCAACAG-3’ | 57 | 26 |
R5’-CATCCTTCTTCCTTGGCATC-3’ | |||
|
F5’-CCTGAATCACCCAGGCCAGATTGTG-3’ | 57 | 19 |
R5’-GAGGGTACTCTGAGAAAGCTCTCCACG-3’ | |||
|
F5’-GTCAATGATGGAGTGTATGGATC-3’ | 55 | 25 |
R5’-TCCATTCCGCTCTCCTGAGCAC-3’ |
Primers used for site-directed mutagenesis
Mutated site | Primer name | Sequence |
---|---|---|
GRE | Foxd4l1.1mGRE | F5’-GGACCCTCTCACGTGGGAGCTTATCTGATAR-3’ |
R5’-TATCAGATAAGCTCCCACGTGAGAGGGTCC-3’ |
Primers used for ChIP-PCR assay
Prime name | Sequence | Annealing temperature (°C) | Cycle |
---|---|---|---|
Foxd4l1.1ChIP-GRE | F5’-ACCTTGTTGGACTACAGATTC-3’ | 52 | 30 |
R5’-CAGTCATCTGCACTTGAATTGG-3’ |
. Primers used for serially-deleted FoxD5b reporter gene constructs .
Primer | Primer name | Sequence |
---|---|---|
Upstream primer | Foxd4l1.1(–1551) | 5’-CCGGTACCTAGAGGTTGGATAAAGTCAATTGC-3’ |
Foxd4l1.1(–1316) | 5’-CCGGTACCTATATGCAGAGCTGCTAATAGTC-3’ | |
Foxd4l1.1(–1016) | 5’-CCGGTACCTATATGCAGAGCTGCTAATAGTC-3’ | |
Foxd4l1.1(–816) | 5’-CCGGTACCTAGAATTCCAGTTCCCATAATC-3’ | |
Foxd4l1.1(–301) | 5’-CCGGTACCTTGGATTGCAAGTTAGTGGCTC-3’ | |
Foxd4l1.1(–186) | 5’-GGGGTACCTTCATTCAGCAAAAGCACAGCC-3’ | |
Foxd4l1.1(–78) | 5’-GGGGTACCAATTCAAGTGCAGATGACTGCC-3’ | |
Downstream primer | Foxd4l1.1-R | 5’-ATCTCGAGGCTTGGTTGGCAGTAAGTAG-3’ |
. Primers used for RT-PCR amplification .
Gene name | Sequence | Annealing temperature (°C) | Cycle |
---|---|---|---|
F5’-TCTCAGGATCTGAACACCT-3’ | 45 | 28 | |
R5’-CCCTATAAGACAAGGAATAC-3’ | |||
F5’-ACTCTATCAGGCACAACCTGTC-3’ | 50 | 30 | |
R5’-GGTCTGTAGTAAGGCAGAGAGT-3’ | |||
F5’-GGATCGTTATCACCTCTG-3’ | 57 | 25 | |
R5’-GTGTAGTCTGTAGCAGCA-3’ | |||
F5’-CTGGTGACCGACCAACTAAG-3’ | 55 | 28 | |
R5’-TGCGAACTCTGCTTCCAAAC-3’ | |||
F5’-ATGTGCGGAGGCTGCGTC-3’ | 60 | 27 | |
R5’-CGTGGGTCATCGGGTAGAAC-3’ | |||
F5’-GAGCTGATGAGGTGCAAGAG-3’ | 60 | 27 | |
R5’-TTTGCTCATCTTCTTGTTGG-3’ | |||
F5’-CACAGTTCCACCAAATGC-3’ | 57 | 29 | |
R5’-GGAATCAAGCGGTACAGA-3’ | |||
F5’-GGATGGTGCTGCTACCGTGCGAGTACC-3’ | 65 | 30 | |
R5’-CAAGCGCAGAGTTCAGGTTGTGCATGC-3’ | |||
F5’-GGATGGATTTGTTGCACCAGTC-3’ | 57 | 27 | |
R5’-CACTCTCCCAGCTCACTTCTC-3’ | |||
F5’-TGGTGTTGAACAAGTGCAGG-3’ | 57 | 25 | |
R5’-ACCTCCTCGACAATGGTCTT-3’ | |||
F5’-AACCGCCCCAGTAAGACC-3’ | 57 | 28 | |
R5’-GTGTCAGCCTGTCCTGTTAG-3’ | |||
F5’-GTGAAATCCCAATAGACACC-3’ | 57 | 28 | |
R5’-TTCCCCATATCTAAAGGCAG-3’ | |||
F5’-TACTTACGGGCTTGGCTGGA-3’ | 68 | 26 | |
R5’-AGCGTGTAACCAGTTGGCTG-3’ | |||
F5’-GCTGATTCCACCAGTGCCTCACCAG-3’ | 60 | 30 | |
R5’-GGTCCTGTGCCTCCTCCTCCTCCTG-3’ | |||
F5’-AGTTGCAGATGTGGCTCT-3’ | 57 | 27 | |
R5’-AGTCCAAGAGTCTGAGCA-3’ | |||
F5’-TTAGAGAGGAGAGCAACTCGGGCAAT-3’ | 57 | 25 | |
R5’-GTGCTCCTGTTGCGAAACTCTACAGA-3’ | |||
F5’-CATCATGATTCCTGGTAACCGA-3’ | 57 | 25 | |
R5’-CTCCATGCTGATATCGTGCAG-3’ | |||
F5’-CCTTCAGCATGGTTCAACAG-3’ | 57 | 26 | |
R5’-CATCCTTCTTCCTTGGCATC-3’ | |||
F5’-CCTGAATCACCCAGGCCAGATTGTG-3’ | 57 | 19 | |
R5’-GAGGGTACTCTGAGAAAGCTCTCCACG-3’ | |||
F5’-GTCAATGATGGAGTGTATGGATC-3’ | 55 | 25 | |
R5’-TCCATTCCGCTCTCCTGAGCAC-3’ |
. Primers used for site-directed mutagenesis .
Mutated site | Primer name | Sequence |
---|---|---|
GRE | Foxd4l1.1mGRE | F5’-GGACCCTCTCACGTGGGAGCTTATCTGATAR-3’ |
R5’-TATCAGATAAGCTCCCACGTGAGAGGGTCC-3’ |
. Primers used for ChIP-PCR assay .
Prime name | Sequence | Annealing temperature (°C) | Cycle |
---|---|---|---|
Foxd4l1.1ChIP-GRE | F5’-ACCTTGTTGGACTACAGATTC-3’ | 52 | 30 |
R5’-CAGTCATCTGCACTTGAATTGG-3’ |
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