Mol. Cells 2017; 40(6): 426-433
Published online June 13, 2017
https://doi.org/10.14348/molcells.2017.0052
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: scpark@sookmyung.ac.kr
Keywords bHLH transcription factor, EphA, ephrin-A5, mesencephalon
Ephrin-A5 has a broad range of biological effects during embryonic brain development due to the fact it binds members of both the EphA and EphB receptor families (Kullander and Klein, 2002; Pasquale, 2005).
It appears that gradients in the expression of Eph receptors and ephrin ligands are key regulators of brain morphogenesis and wiring (Coulthard et al., 2002; Klein, 2012). However, understanding how these gradients of expression are regulated at the gene level during brain development has received little attention, except for the development of the neural retina. The opposing gradients of expression shown by ephrin-As and EphAs along the nasotemporal axis of the neural retina are regulated by chick brain factors 1 (CBF1, an orthologue of forkhead box G1 (FOXG1) in mice) and 2 (CBF2, an orthologue of FOXD1 in mice), respectively. CBF1 is a nasal-specific winged-helix transcription factor. Altered expression of CBF1 in the temporal retina was shown to repress the expression of CBF2 and EphA3, whereas it promoted the expression of ephrin-A2 and ephrin-A5 (Polleux et al., 2007; Takahashi et al., 2003). In contrast, CBF2/FOXD1 is expressed in the temporal retina and the knocking-out of FOXD1 was found to increase the expression of ephrin-A5 (Carreres et al., 2011). The opposing gradients of expression for ephrin-Bs and EphBs along the dorsal-ventral axis of the neural retina are regulated by T-box transcription factor 5 (TBX5) and ventral anterior homeo-box 2 (VAX2), respectively. In the dorsal retina, TBX5 was shown to repress the expression of VAX2 and was sufficient to induce the expression of ephrin-B1 and ephrin-B2 (Koshiba-Takeuchi et al., 2000). Meanwhile, VAX2 was shown to regulate the expression of EphB2 and EphB3 in the ventral retina (Mui et al., 2002; Schulte et al., 1999). Overall, these findings suggest that the combination of various expression patterns of transcription factors along the naso-temporal and dorso-ventral axis of the retina determines the graded expression patterns of ephrin ligands and Eph receptors. These processes and patterns underlie embryo morphogenesis and brain wiring. However, many questions remain unaddressed. In particular, what are the transcriptional mechanisms that determine the gradients of expression for ephrin ligands and Eph receptors along the developing axis of the midbrain?
In this study, we determined that a bHLH transcription factor binding site in the 800 bp mesencephalon-specific
Homologous arms for modifying 4O15, 211L12, 375B7, and 23O22 BACs were amplified via PCR by using the following primer sets. For the A arm, forward 5′-GGTGTATGTCTCCC CGCAG-3′ and reverse 5′-GGAGAGATCGGGGATCCAG-3′ and for the B arm, forward 5′-TGCTCTTTCTGGTGCTCT-3′ and reverse 5′-CTTTCCACTCACCCATCCCT-3′. For PCR amplification of the B arm to generate the 385B7del30.4 BAC, the primers were forward 5′-TAGCACTCTCATCTGTCTCTCGG -3′ and reverse 5′-GGCACTTGTGGTCACAGAAATAG-3′. Homologous A and B arms were sub-cloned into a reporter vector containing a LacZ reporter gene and a SV40 poly (A) signal on a pGEM11Z vector backbone (Promega). Homologous A and B arms were cloned into the
C1, C2, and C3 fragments were synthesized via PCR by using the following primer sets. For C1 5′-TAAAGCACTGAAA AGTAGACCCAAG-3′ and 5′-ATTTACTTCAGTGTGAAGCCA CTGT-3′, for C2 5′-GTTTTAAGGCTGCAGTAATCACTGT-3′ and 5′-TCTGAAAATTCTACAAAGTGCCCTA-3′, and for C3 (5′-ATCCATATCCTCTCTTACCTCAGGA-3′ and 5′-CCGAGA GACAGATGAGAGTGCTA-3′. Each amplifying fragment was cloned into the
For C3delECR1, a 240 bp fragment was amplified with primers 5′-GGTTAACTCTTGCTTGGGCATGTG-3′ and 5′-GCATTCTGCCTTCCAAGAGGCAACACACAGTGG-3′, and a 320 bp fragment was amplified with primers 5′-CTGTGTGTT GCCTCTTGGAAGGCAGAATGCTGA-3′ and 5′-GCAGCAGC CAACAGAATTGCACCC-3′. The two partially complementary PCR fragments that were generated were annealed and used as the templates in a second PCR with primers 5′-GGTTAACTCTTGCTTGGGCATGTG-3′ and 5′-GCAGCAGCC AACAGAATTGCACCC-3′. The resulting 540 bp product was digested with
BAC DNA was purified using a Qiagen Large-Construct kit (Qiagen). Recombinant BACs for microinjection were diluted in injection buffer and then injected into 200 fertilized oocyte pronuclei from C57BL/6 mice, as described previously (Kim et al., 2007). Each BAC transgenic embryo was identified by PCR genotyping, using the primers 5′-GTTACAATAA AGCAATAGCATCACA-3′ and 5′-GGTTGCTGCTGTTCCAGT AGAC-3′. Transgenic embryos carrying plasmid vector were identified via PCR genotyping by using the following primer sets. For C1, 5′-GTTACAATAAAGCAATAGCATCACA-3′ and 5′-AATACCTGGCCCATAGTAGACACTTTA-3′, for C2, 5′-GTT ACAATAAAGCAATAGCATCACA-3′ and 5′-ATTTACTTCAGT GTGAAGCCACTGT-3′, and for C3, 5′-GTTACAATAAAGCA ATAGCATCACA-3′ and 5′-ATTTTATTGTGTACTGAAGAGAT TGGG-3′.
For X-gal staining, embryos were dissected in phosphate buffered saline (PBS), fixed in 0.2% glutaraldehyde for 10 min, and finally washed and stained using procedures described previously (Park et al., 2013).
Proteins for DNA binding assays were produced using a T7 TNT-coupled
HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium, supplemented with L-glutamine, 10% fetal bovine serum, and penicillin/streptomycin. A total of 0.9 μg of DNA containing 0.4 μg of luciferase reporter construct and 0.5 μg of expression construct (pcDNA-Ascl1-tether-E47 was gift by Dr. Guillemot) was transfected using Lipofectamine (Invitrogen). A co-transfected Renilla luciferase reporter (18 ng per transfection) was used to normalize transfection efficiency. For generating eA5-E6, eA5-Em6, and Dll1-E6 constructs, each concatemeric oligonucleotide containing six tandem repeats of E box (indicated in Fig. 4D) was sub-cloned into a pTAL-Luc vector (Promega). Cells were lysed 24 h after transfection, and the luciferase activity of each extract was assayed using a Dual Luciferase Assay Kit (Promega).
The mouse
To further restrict the possible locations of the mesencephalon-specific enhancer within the first intron, the 375B7 BAC was modified using a different targeting vector. This vector was identical to the targeting vector described for Fig. 1, except the 1.1 kb B arm consisted of the region +30.5 kb to +31.6 kb from the TSS. The resulting BAC (375B7del30.4) was verified based on the deletion of the genomic region spanning +0.1 kb to +30.5 kb (Fig. 2A). Importantly, transgenic embryos carrying this recombinant BAC did not show significant LacZ expression in the mesencephalon (Fig. 2C, first panel from the left). This suggested that the deleted 30.4 kb region included the mesencephalon-specific enhancer of ephrin-A5. As the 4O15 BAC did not show enhancer activity in transgenic embryos, it was concluded that the enhancer most likely resides in a smaller 18.9 kb region spanning +11.6 kb to +30.5 kb from the TSS (Fig. 2A). This 18.9 kb genomic region was further divided into three regions, designated C1, C2, and C3 (Fig. 2B). Each genomic fragment was amplified by PCR, sub-cloned, and then intensively analyzed by DNA sequencing. Validated genomic DNA was selected and inserted into a site immediately upstream of a 0.4 kb
To search for the mesencephalon-specific
It has been reported that Ascl1, a bHLH TF, is expressed in the mesencephalon. Additionally, mice with an
To further assess whether Ascl1-E47 fusion protein directly induces transcriptional activation via interaction with
In this study, we have presented
(A) A physical map displaying
(A) Schematic of BAC clones 4O15, 375B7, and 375B7del30.4. The dashed line in 375B7del30.4 BAC represents a genomic portion deleted by BAC recombination. (B) Schematic of C1 (from +11.6 kb to 18.3 kb), C2 (from +18.1 kb to 25.1 kb), and C3 (from +25 kb to 30.5 kb) fragments amplified via PCR. (C) Analysis of LacZ expression using transgenic embryos. Transgenic embryos carrying 375B7del30.4, C1, and C2 do not show robust LacZ expression in the posterior part of the mesencephalon (first panel, E9.5; second panel, E8.5; fourth panel, E10.5; fifth panel, E8.5). Only C3 transgenic embryos show LacZ expression in the mesencephalon (third panel, E8.5; sixth panel, E9.5). Scale bar, 500 μm.
(A) Pair-wise alignment of mouse
(A) DNA sequences of the oligonucleotides used for EMSA. bHLH transcription factor binding sites are underlined in bold. (B and C) Electrophoretic mobility shift assay demonstrating the binding of Ascl1-E47 to the
Mol. Cells 2017; 40(6): 426-433
Published online June 30, 2017 https://doi.org/10.14348/molcells.2017.0052
Copyright © The Korean Society for Molecular and Cellular Biology.
Eunjeong Park, Hyuna Noh, and Soochul Park*
Department of Biological Sciences, Sookmyung Women’s University, Seoul 04310, Korea
Correspondence to:*Correspondence: scpark@sookmyung.ac.kr
Keywords: bHLH transcription factor, EphA, ephrin-A5, mesencephalon
Ephrin-A5 has a broad range of biological effects during embryonic brain development due to the fact it binds members of both the EphA and EphB receptor families (Kullander and Klein, 2002; Pasquale, 2005).
It appears that gradients in the expression of Eph receptors and ephrin ligands are key regulators of brain morphogenesis and wiring (Coulthard et al., 2002; Klein, 2012). However, understanding how these gradients of expression are regulated at the gene level during brain development has received little attention, except for the development of the neural retina. The opposing gradients of expression shown by ephrin-As and EphAs along the nasotemporal axis of the neural retina are regulated by chick brain factors 1 (CBF1, an orthologue of forkhead box G1 (FOXG1) in mice) and 2 (CBF2, an orthologue of FOXD1 in mice), respectively. CBF1 is a nasal-specific winged-helix transcription factor. Altered expression of CBF1 in the temporal retina was shown to repress the expression of CBF2 and EphA3, whereas it promoted the expression of ephrin-A2 and ephrin-A5 (Polleux et al., 2007; Takahashi et al., 2003). In contrast, CBF2/FOXD1 is expressed in the temporal retina and the knocking-out of FOXD1 was found to increase the expression of ephrin-A5 (Carreres et al., 2011). The opposing gradients of expression for ephrin-Bs and EphBs along the dorsal-ventral axis of the neural retina are regulated by T-box transcription factor 5 (TBX5) and ventral anterior homeo-box 2 (VAX2), respectively. In the dorsal retina, TBX5 was shown to repress the expression of VAX2 and was sufficient to induce the expression of ephrin-B1 and ephrin-B2 (Koshiba-Takeuchi et al., 2000). Meanwhile, VAX2 was shown to regulate the expression of EphB2 and EphB3 in the ventral retina (Mui et al., 2002; Schulte et al., 1999). Overall, these findings suggest that the combination of various expression patterns of transcription factors along the naso-temporal and dorso-ventral axis of the retina determines the graded expression patterns of ephrin ligands and Eph receptors. These processes and patterns underlie embryo morphogenesis and brain wiring. However, many questions remain unaddressed. In particular, what are the transcriptional mechanisms that determine the gradients of expression for ephrin ligands and Eph receptors along the developing axis of the midbrain?
In this study, we determined that a bHLH transcription factor binding site in the 800 bp mesencephalon-specific
Homologous arms for modifying 4O15, 211L12, 375B7, and 23O22 BACs were amplified via PCR by using the following primer sets. For the A arm, forward 5′-GGTGTATGTCTCCC CGCAG-3′ and reverse 5′-GGAGAGATCGGGGATCCAG-3′ and for the B arm, forward 5′-TGCTCTTTCTGGTGCTCT-3′ and reverse 5′-CTTTCCACTCACCCATCCCT-3′. For PCR amplification of the B arm to generate the 385B7del30.4 BAC, the primers were forward 5′-TAGCACTCTCATCTGTCTCTCGG -3′ and reverse 5′-GGCACTTGTGGTCACAGAAATAG-3′. Homologous A and B arms were sub-cloned into a reporter vector containing a LacZ reporter gene and a SV40 poly (A) signal on a pGEM11Z vector backbone (Promega). Homologous A and B arms were cloned into the
C1, C2, and C3 fragments were synthesized via PCR by using the following primer sets. For C1 5′-TAAAGCACTGAAA AGTAGACCCAAG-3′ and 5′-ATTTACTTCAGTGTGAAGCCA CTGT-3′, for C2 5′-GTTTTAAGGCTGCAGTAATCACTGT-3′ and 5′-TCTGAAAATTCTACAAAGTGCCCTA-3′, and for C3 (5′-ATCCATATCCTCTCTTACCTCAGGA-3′ and 5′-CCGAGA GACAGATGAGAGTGCTA-3′. Each amplifying fragment was cloned into the
For C3delECR1, a 240 bp fragment was amplified with primers 5′-GGTTAACTCTTGCTTGGGCATGTG-3′ and 5′-GCATTCTGCCTTCCAAGAGGCAACACACAGTGG-3′, and a 320 bp fragment was amplified with primers 5′-CTGTGTGTT GCCTCTTGGAAGGCAGAATGCTGA-3′ and 5′-GCAGCAGC CAACAGAATTGCACCC-3′. The two partially complementary PCR fragments that were generated were annealed and used as the templates in a second PCR with primers 5′-GGTTAACTCTTGCTTGGGCATGTG-3′ and 5′-GCAGCAGCC AACAGAATTGCACCC-3′. The resulting 540 bp product was digested with
BAC DNA was purified using a Qiagen Large-Construct kit (Qiagen). Recombinant BACs for microinjection were diluted in injection buffer and then injected into 200 fertilized oocyte pronuclei from C57BL/6 mice, as described previously (Kim et al., 2007). Each BAC transgenic embryo was identified by PCR genotyping, using the primers 5′-GTTACAATAA AGCAATAGCATCACA-3′ and 5′-GGTTGCTGCTGTTCCAGT AGAC-3′. Transgenic embryos carrying plasmid vector were identified via PCR genotyping by using the following primer sets. For C1, 5′-GTTACAATAAAGCAATAGCATCACA-3′ and 5′-AATACCTGGCCCATAGTAGACACTTTA-3′, for C2, 5′-GTT ACAATAAAGCAATAGCATCACA-3′ and 5′-ATTTACTTCAGT GTGAAGCCACTGT-3′, and for C3, 5′-GTTACAATAAAGCA ATAGCATCACA-3′ and 5′-ATTTTATTGTGTACTGAAGAGAT TGGG-3′.
For X-gal staining, embryos were dissected in phosphate buffered saline (PBS), fixed in 0.2% glutaraldehyde for 10 min, and finally washed and stained using procedures described previously (Park et al., 2013).
Proteins for DNA binding assays were produced using a T7 TNT-coupled
HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium, supplemented with L-glutamine, 10% fetal bovine serum, and penicillin/streptomycin. A total of 0.9 μg of DNA containing 0.4 μg of luciferase reporter construct and 0.5 μg of expression construct (pcDNA-Ascl1-tether-E47 was gift by Dr. Guillemot) was transfected using Lipofectamine (Invitrogen). A co-transfected Renilla luciferase reporter (18 ng per transfection) was used to normalize transfection efficiency. For generating eA5-E6, eA5-Em6, and Dll1-E6 constructs, each concatemeric oligonucleotide containing six tandem repeats of E box (indicated in Fig. 4D) was sub-cloned into a pTAL-Luc vector (Promega). Cells were lysed 24 h after transfection, and the luciferase activity of each extract was assayed using a Dual Luciferase Assay Kit (Promega).
The mouse
To further restrict the possible locations of the mesencephalon-specific enhancer within the first intron, the 375B7 BAC was modified using a different targeting vector. This vector was identical to the targeting vector described for Fig. 1, except the 1.1 kb B arm consisted of the region +30.5 kb to +31.6 kb from the TSS. The resulting BAC (375B7del30.4) was verified based on the deletion of the genomic region spanning +0.1 kb to +30.5 kb (Fig. 2A). Importantly, transgenic embryos carrying this recombinant BAC did not show significant LacZ expression in the mesencephalon (Fig. 2C, first panel from the left). This suggested that the deleted 30.4 kb region included the mesencephalon-specific enhancer of ephrin-A5. As the 4O15 BAC did not show enhancer activity in transgenic embryos, it was concluded that the enhancer most likely resides in a smaller 18.9 kb region spanning +11.6 kb to +30.5 kb from the TSS (Fig. 2A). This 18.9 kb genomic region was further divided into three regions, designated C1, C2, and C3 (Fig. 2B). Each genomic fragment was amplified by PCR, sub-cloned, and then intensively analyzed by DNA sequencing. Validated genomic DNA was selected and inserted into a site immediately upstream of a 0.4 kb
To search for the mesencephalon-specific
It has been reported that Ascl1, a bHLH TF, is expressed in the mesencephalon. Additionally, mice with an
To further assess whether Ascl1-E47 fusion protein directly induces transcriptional activation via interaction with
In this study, we have presented
(A) A physical map displaying
(A) Schematic of BAC clones 4O15, 375B7, and 375B7del30.4. The dashed line in 375B7del30.4 BAC represents a genomic portion deleted by BAC recombination. (B) Schematic of C1 (from +11.6 kb to 18.3 kb), C2 (from +18.1 kb to 25.1 kb), and C3 (from +25 kb to 30.5 kb) fragments amplified via PCR. (C) Analysis of LacZ expression using transgenic embryos. Transgenic embryos carrying 375B7del30.4, C1, and C2 do not show robust LacZ expression in the posterior part of the mesencephalon (first panel, E9.5; second panel, E8.5; fourth panel, E10.5; fifth panel, E8.5). Only C3 transgenic embryos show LacZ expression in the mesencephalon (third panel, E8.5; sixth panel, E9.5). Scale bar, 500 μm.
(A) Pair-wise alignment of mouse
(A) DNA sequences of the oligonucleotides used for EMSA. bHLH transcription factor binding sites are underlined in bold. (B and C) Electrophoretic mobility shift assay demonstrating the binding of Ascl1-E47 to the
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(A) A physical map displaying
(A) Schematic of BAC clones 4O15, 375B7, and 375B7del30.4. The dashed line in 375B7del30.4 BAC represents a genomic portion deleted by BAC recombination. (B) Schematic of C1 (from +11.6 kb to 18.3 kb), C2 (from +18.1 kb to 25.1 kb), and C3 (from +25 kb to 30.5 kb) fragments amplified via PCR. (C) Analysis of LacZ expression using transgenic embryos. Transgenic embryos carrying 375B7del30.4, C1, and C2 do not show robust LacZ expression in the posterior part of the mesencephalon (first panel, E9.5; second panel, E8.5; fourth panel, E10.5; fifth panel, E8.5). Only C3 transgenic embryos show LacZ expression in the mesencephalon (third panel, E8.5; sixth panel, E9.5). Scale bar, 500 μm.
|@|~(^,^)~|@|A bHLH transcription factor binding site is critical for the(A) Pair-wise alignment of mouse
(A) DNA sequences of the oligonucleotides used for EMSA. bHLH transcription factor binding sites are underlined in bold. (B and C) Electrophoretic mobility shift assay demonstrating the binding of Ascl1-E47 to the