Limits...
B1 SOX coordinate cell specification with patterning and morphogenesis in the early zebrafish embryo.

Okuda Y, Ogura E, Kondoh H, Kamachi Y - PLoS Genet. (2010)

Bottom Line: Chromatin immunoprecipitation analysis of the her3, hesx1, neurog1, pcdh18a, and cyp26a1 genes further suggests a direct regulation of these genes by B1 SOX.We also found an interesting overlap between the early phenotypes of the B1 sox quadruple knockdown embryos and the maternal-zygotic spg embryos that are devoid of pou5f1 activity.These findings indicate that the B1 SOX proteins control a wide range of developmental regulators in the early embryo through partnering in part with Pou5f1 and possibly with other factors, and suggest that the B1 sox functions are central to coordinating cell fate specification with patterning and morphogenetic processes occurring in the early embryo.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Frontier Biosciences, Osaka University, Suita, Japan.

ABSTRACT
The B1 SOX transcription factors SOX1/2/3/19 have been implicated in various processes of early embryogenesis. However, their regulatory functions in stages from the blastula to early neurula remain largely unknown, primarily because loss-of-function studies have not been informative to date. In our present study, we systematically knocked down the B1 sox genes in zebrafish. Only the quadruple knockdown of the four B1 sox genes sox2/3/19a/19b resulted in very severe developmental abnormalities, confirming that the B1 sox genes are functionally redundant. We characterized the sox2/3/19a/19b quadruple knockdown embryos in detail by examining the changes in gene expression through in situ hybridization, RT-PCR, and microarray analyses. Importantly, these phenotypic analyses revealed that the B1 SOX proteins regulate the following distinct processes: (1) early dorsoventral patterning by controlling bmp2b/7; (2) gastrulation movements via the regulation of pcdh18a/18b and wnt11, a non-canonical Wnt ligand gene; (3) neural differentiation by regulating the Hes-class bHLH gene her3 and the proneural-class bHLH genes neurog1 (positively) and ascl1a (negatively), and regional transcription factor genes, e.g., hesx1, zic1, and rx3; and (4) neural patterning by regulating signaling pathway genes, cyp26a1 in RA signaling, oep in Nodal signaling, shh, and mdkb. Chromatin immunoprecipitation analysis of the her3, hesx1, neurog1, pcdh18a, and cyp26a1 genes further suggests a direct regulation of these genes by B1 SOX. We also found an interesting overlap between the early phenotypes of the B1 sox quadruple knockdown embryos and the maternal-zygotic spg embryos that are devoid of pou5f1 activity. These findings indicate that the B1 SOX proteins control a wide range of developmental regulators in the early embryo through partnering in part with Pou5f1 and possibly with other factors, and suggest that the B1 sox functions are central to coordinating cell fate specification with patterning and morphogenetic processes occurring in the early embryo.

Show MeSH

Related in: MedlinePlus

Direct regulatory targets of the B1 SOX proteins in the zebrafish embryo.(A) ChIP analysis showing direct association of B1 SOX with regulatory sequences of the downstream genes. (a) Potential binding sites for SOX and POU within the analyzed genomic regions are schematically shown. (b) ChIP-PCR analysis using anti-SOX2 antibody. ChIP experiments were performed using zebrafish embryos at the 70–80%E and tailbud to 2-somite stages. ChIP-PCR analysis using anti-SOX2 antibody that weakly cross-reacts with SOX3/19A/19B revealed specific binding of B1 SOX to the regulatory sequences of the hesx1, her3, pcdh18a, cyp26a1 and neurog1 genes in the zebrafish embryo. bactin2 was used as a negative control. (B) B1 SOX-dependent activities of the regulatory sequences of hesx1, cyp26a1 and pcdh18a. (a) The Venusluc fusion reporter (Venus plus firefly luciferase) constructs containing either of the promoters for hesx1 or cyp26a1 or the upstream conserved sequence of pcdh18a with the HSV TK promoter are schematically shown. (b) The Venusluc reporters were injected into embryos with or without the MOs for QKD together with the reference vector TK-Renilla luciferase. More than 20 injected embryos per sample were collected at the tailbud stage, and luciferase assays were performed. The normalized luciferase activity generated by TK-Venusluc was arbitrarily assigned a value of 1. Data are shown as the average values of four independent injection experiments with standard errors.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2865518&req=5

pgen-1000936-g007: Direct regulatory targets of the B1 SOX proteins in the zebrafish embryo.(A) ChIP analysis showing direct association of B1 SOX with regulatory sequences of the downstream genes. (a) Potential binding sites for SOX and POU within the analyzed genomic regions are schematically shown. (b) ChIP-PCR analysis using anti-SOX2 antibody. ChIP experiments were performed using zebrafish embryos at the 70–80%E and tailbud to 2-somite stages. ChIP-PCR analysis using anti-SOX2 antibody that weakly cross-reacts with SOX3/19A/19B revealed specific binding of B1 SOX to the regulatory sequences of the hesx1, her3, pcdh18a, cyp26a1 and neurog1 genes in the zebrafish embryo. bactin2 was used as a negative control. (B) B1 SOX-dependent activities of the regulatory sequences of hesx1, cyp26a1 and pcdh18a. (a) The Venusluc fusion reporter (Venus plus firefly luciferase) constructs containing either of the promoters for hesx1 or cyp26a1 or the upstream conserved sequence of pcdh18a with the HSV TK promoter are schematically shown. (b) The Venusluc reporters were injected into embryos with or without the MOs for QKD together with the reference vector TK-Renilla luciferase. More than 20 injected embryos per sample were collected at the tailbud stage, and luciferase assays were performed. The normalized luciferase activity generated by TK-Venusluc was arbitrarily assigned a value of 1. Data are shown as the average values of four independent injection experiments with standard errors.

Mentions: To further explore whether the B1 SOX proteins directly regulate the potential downstream genes described above, we searched for possible B1 SOX binding sites (containing the consensus sequence CATTGTT [21], [59] or closely related sequences) in the regulatory regions of these genes. We identified potential SOX-binding sites in the regulatory sequences of her3 [47], hesx1, cyp26a1 [60] and neurog1 [61] and also in the conserved non-coding sequences upstream of pcdh18a (Figure 7Aa). To investigate the direct interaction of the B1 SOX proteins with these genomic sequences in vivo, ChIP experiments were performed using zebrafish embryos at the 70–80%E and tailbud to 2-somite stages. ChIP analysis using anti-SOX2 antibody that weakly cross-reacts with SOX3/19A/19B revealed specific binding of B1 SOX to these regulatory sequences in the zebrafish embryo (Figure 7Ab). Similar results were obtained with anti-SOX3 antibody (data not shown). It is thus likely that these genes are direct downstream targets of B1 SOX.


B1 SOX coordinate cell specification with patterning and morphogenesis in the early zebrafish embryo.

Okuda Y, Ogura E, Kondoh H, Kamachi Y - PLoS Genet. (2010)

Direct regulatory targets of the B1 SOX proteins in the zebrafish embryo.(A) ChIP analysis showing direct association of B1 SOX with regulatory sequences of the downstream genes. (a) Potential binding sites for SOX and POU within the analyzed genomic regions are schematically shown. (b) ChIP-PCR analysis using anti-SOX2 antibody. ChIP experiments were performed using zebrafish embryos at the 70–80%E and tailbud to 2-somite stages. ChIP-PCR analysis using anti-SOX2 antibody that weakly cross-reacts with SOX3/19A/19B revealed specific binding of B1 SOX to the regulatory sequences of the hesx1, her3, pcdh18a, cyp26a1 and neurog1 genes in the zebrafish embryo. bactin2 was used as a negative control. (B) B1 SOX-dependent activities of the regulatory sequences of hesx1, cyp26a1 and pcdh18a. (a) The Venusluc fusion reporter (Venus plus firefly luciferase) constructs containing either of the promoters for hesx1 or cyp26a1 or the upstream conserved sequence of pcdh18a with the HSV TK promoter are schematically shown. (b) The Venusluc reporters were injected into embryos with or without the MOs for QKD together with the reference vector TK-Renilla luciferase. More than 20 injected embryos per sample were collected at the tailbud stage, and luciferase assays were performed. The normalized luciferase activity generated by TK-Venusluc was arbitrarily assigned a value of 1. Data are shown as the average values of four independent injection experiments with standard errors.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2865518&req=5

pgen-1000936-g007: Direct regulatory targets of the B1 SOX proteins in the zebrafish embryo.(A) ChIP analysis showing direct association of B1 SOX with regulatory sequences of the downstream genes. (a) Potential binding sites for SOX and POU within the analyzed genomic regions are schematically shown. (b) ChIP-PCR analysis using anti-SOX2 antibody. ChIP experiments were performed using zebrafish embryos at the 70–80%E and tailbud to 2-somite stages. ChIP-PCR analysis using anti-SOX2 antibody that weakly cross-reacts with SOX3/19A/19B revealed specific binding of B1 SOX to the regulatory sequences of the hesx1, her3, pcdh18a, cyp26a1 and neurog1 genes in the zebrafish embryo. bactin2 was used as a negative control. (B) B1 SOX-dependent activities of the regulatory sequences of hesx1, cyp26a1 and pcdh18a. (a) The Venusluc fusion reporter (Venus plus firefly luciferase) constructs containing either of the promoters for hesx1 or cyp26a1 or the upstream conserved sequence of pcdh18a with the HSV TK promoter are schematically shown. (b) The Venusluc reporters were injected into embryos with or without the MOs for QKD together with the reference vector TK-Renilla luciferase. More than 20 injected embryos per sample were collected at the tailbud stage, and luciferase assays were performed. The normalized luciferase activity generated by TK-Venusluc was arbitrarily assigned a value of 1. Data are shown as the average values of four independent injection experiments with standard errors.
Mentions: To further explore whether the B1 SOX proteins directly regulate the potential downstream genes described above, we searched for possible B1 SOX binding sites (containing the consensus sequence CATTGTT [21], [59] or closely related sequences) in the regulatory regions of these genes. We identified potential SOX-binding sites in the regulatory sequences of her3 [47], hesx1, cyp26a1 [60] and neurog1 [61] and also in the conserved non-coding sequences upstream of pcdh18a (Figure 7Aa). To investigate the direct interaction of the B1 SOX proteins with these genomic sequences in vivo, ChIP experiments were performed using zebrafish embryos at the 70–80%E and tailbud to 2-somite stages. ChIP analysis using anti-SOX2 antibody that weakly cross-reacts with SOX3/19A/19B revealed specific binding of B1 SOX to these regulatory sequences in the zebrafish embryo (Figure 7Ab). Similar results were obtained with anti-SOX3 antibody (data not shown). It is thus likely that these genes are direct downstream targets of B1 SOX.

Bottom Line: Chromatin immunoprecipitation analysis of the her3, hesx1, neurog1, pcdh18a, and cyp26a1 genes further suggests a direct regulation of these genes by B1 SOX.We also found an interesting overlap between the early phenotypes of the B1 sox quadruple knockdown embryos and the maternal-zygotic spg embryos that are devoid of pou5f1 activity.These findings indicate that the B1 SOX proteins control a wide range of developmental regulators in the early embryo through partnering in part with Pou5f1 and possibly with other factors, and suggest that the B1 sox functions are central to coordinating cell fate specification with patterning and morphogenetic processes occurring in the early embryo.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Frontier Biosciences, Osaka University, Suita, Japan.

ABSTRACT
The B1 SOX transcription factors SOX1/2/3/19 have been implicated in various processes of early embryogenesis. However, their regulatory functions in stages from the blastula to early neurula remain largely unknown, primarily because loss-of-function studies have not been informative to date. In our present study, we systematically knocked down the B1 sox genes in zebrafish. Only the quadruple knockdown of the four B1 sox genes sox2/3/19a/19b resulted in very severe developmental abnormalities, confirming that the B1 sox genes are functionally redundant. We characterized the sox2/3/19a/19b quadruple knockdown embryos in detail by examining the changes in gene expression through in situ hybridization, RT-PCR, and microarray analyses. Importantly, these phenotypic analyses revealed that the B1 SOX proteins regulate the following distinct processes: (1) early dorsoventral patterning by controlling bmp2b/7; (2) gastrulation movements via the regulation of pcdh18a/18b and wnt11, a non-canonical Wnt ligand gene; (3) neural differentiation by regulating the Hes-class bHLH gene her3 and the proneural-class bHLH genes neurog1 (positively) and ascl1a (negatively), and regional transcription factor genes, e.g., hesx1, zic1, and rx3; and (4) neural patterning by regulating signaling pathway genes, cyp26a1 in RA signaling, oep in Nodal signaling, shh, and mdkb. Chromatin immunoprecipitation analysis of the her3, hesx1, neurog1, pcdh18a, and cyp26a1 genes further suggests a direct regulation of these genes by B1 SOX. We also found an interesting overlap between the early phenotypes of the B1 sox quadruple knockdown embryos and the maternal-zygotic spg embryos that are devoid of pou5f1 activity. These findings indicate that the B1 SOX proteins control a wide range of developmental regulators in the early embryo through partnering in part with Pou5f1 and possibly with other factors, and suggest that the B1 sox functions are central to coordinating cell fate specification with patterning and morphogenetic processes occurring in the early embryo.

Show MeSH
Related in: MedlinePlus