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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.

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Rescue of the QKD phenotype by exogenous B1 sox mRNAs.(A, B) Uninjected control (Ctr) embryos (A) and the QKD embryos (B). Live embryos were observed at 10–11 (a), 15–16 (b) and 30–31 (c) hpf. Expression of hesx1, pax2a and hoxb1b (d), dlx3b, hgg1 and ntl (e), and neurog1 (f) was visualized by whole-mount in situ hybridization. Lateral views (a–c); dorsal views with anterior to the top (d–f). (C) The QKD phenotype is similarly rescued by an exogenous supply of any B1 sox mRNA. The MOs for QKD were coinjected with the indicated mRNAs. In the B1 sox mRNA-coinjected embryos, the expression of hesx1, dlx3b and nuerog1 was recovered; patterning of the neural plate marked by pax2a and hoxb1b was normalized; and the expression patterns of hgg1, ntl and dlx3b reflecting C&E movements were also restored. Blue bracket, gap between the hgg1 and ntl expression domains; white dotted line, neural plate border. (D) Recovery of the AP axis elongation in QKD embryos injected with one of the B1 sox mRNAs. The length of the embryos at 15–16 hpf along the AP axis between the arrowheads (Ab, Bb, Cb) was measured for the uninjected control (Ctr) (n = 7), the QKD (n = 9) and QKD with B1 sox mRNA injection (sox2, n = 9; sox3, n = 6; sox19a, n = 7; sox19b, n = 6). The average AP axis lengths with standard errors are shown.
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pgen-1000936-g002: Rescue of the QKD phenotype by exogenous B1 sox mRNAs.(A, B) Uninjected control (Ctr) embryos (A) and the QKD embryos (B). Live embryos were observed at 10–11 (a), 15–16 (b) and 30–31 (c) hpf. Expression of hesx1, pax2a and hoxb1b (d), dlx3b, hgg1 and ntl (e), and neurog1 (f) was visualized by whole-mount in situ hybridization. Lateral views (a–c); dorsal views with anterior to the top (d–f). (C) The QKD phenotype is similarly rescued by an exogenous supply of any B1 sox mRNA. The MOs for QKD were coinjected with the indicated mRNAs. In the B1 sox mRNA-coinjected embryos, the expression of hesx1, dlx3b and nuerog1 was recovered; patterning of the neural plate marked by pax2a and hoxb1b was normalized; and the expression patterns of hgg1, ntl and dlx3b reflecting C&E movements were also restored. Blue bracket, gap between the hgg1 and ntl expression domains; white dotted line, neural plate border. (D) Recovery of the AP axis elongation in QKD embryos injected with one of the B1 sox mRNAs. The length of the embryos at 15–16 hpf along the AP axis between the arrowheads (Ab, Bb, Cb) was measured for the uninjected control (Ctr) (n = 7), the QKD (n = 9) and QKD with B1 sox mRNA injection (sox2, n = 9; sox3, n = 6; sox19a, n = 7; sox19b, n = 6). The average AP axis lengths with standard errors are shown.

Mentions: The earliest detectable morphological abnormality of the QKD embryos was a delay in epiboly, notably after the shield stage (Figure 1D). At 10 hour post-fertilization (hpf), when normal embryos reach the tailbud stage, the QKD embryos were still in late epiboly. The thickening of the anterior head region was less prominent in the QKD embryos (Figure 1Dc and 1Dd), suggesting impairment of CNS development. Impaired CNS development was also indicated by the loss of hesx1 expression in the anterior-most neuroectoderm (Figure 2Bd; see also Figure 3Ca) and by the anterolateral displacement of the pax2a expression domains that mark the midbrain-hindbrain boundary (MHB) (Figure 2Bd). An early phase of neurogenesis was also affected in the QKD embryos as indicated by the loss of proneural neurog1 expression (Figure 2Bf). The QKD embryos further displayed a shortened anterior-posterior (AP) axis with a broadened neural plate (marked by hoxb1b) and broadened mesodermal structures including notochord (marked by ntl) (Figure 1D and Figure 2B). Consistently, the gap between the prechordal plate (marked by hgg1) and notochord was reduced in the QKD embryos (Figure 2Be). These abnormalities commonly occur in zebrafish embryos when C&E movements are impaired during gastrulation [31], [32].


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)

Rescue of the QKD phenotype by exogenous B1 sox mRNAs.(A, B) Uninjected control (Ctr) embryos (A) and the QKD embryos (B). Live embryos were observed at 10–11 (a), 15–16 (b) and 30–31 (c) hpf. Expression of hesx1, pax2a and hoxb1b (d), dlx3b, hgg1 and ntl (e), and neurog1 (f) was visualized by whole-mount in situ hybridization. Lateral views (a–c); dorsal views with anterior to the top (d–f). (C) The QKD phenotype is similarly rescued by an exogenous supply of any B1 sox mRNA. The MOs for QKD were coinjected with the indicated mRNAs. In the B1 sox mRNA-coinjected embryos, the expression of hesx1, dlx3b and nuerog1 was recovered; patterning of the neural plate marked by pax2a and hoxb1b was normalized; and the expression patterns of hgg1, ntl and dlx3b reflecting C&E movements were also restored. Blue bracket, gap between the hgg1 and ntl expression domains; white dotted line, neural plate border. (D) Recovery of the AP axis elongation in QKD embryos injected with one of the B1 sox mRNAs. The length of the embryos at 15–16 hpf along the AP axis between the arrowheads (Ab, Bb, Cb) was measured for the uninjected control (Ctr) (n = 7), the QKD (n = 9) and QKD with B1 sox mRNA injection (sox2, n = 9; sox3, n = 6; sox19a, n = 7; sox19b, n = 6). The average AP axis lengths with standard errors are shown.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1000936-g002: Rescue of the QKD phenotype by exogenous B1 sox mRNAs.(A, B) Uninjected control (Ctr) embryos (A) and the QKD embryos (B). Live embryos were observed at 10–11 (a), 15–16 (b) and 30–31 (c) hpf. Expression of hesx1, pax2a and hoxb1b (d), dlx3b, hgg1 and ntl (e), and neurog1 (f) was visualized by whole-mount in situ hybridization. Lateral views (a–c); dorsal views with anterior to the top (d–f). (C) The QKD phenotype is similarly rescued by an exogenous supply of any B1 sox mRNA. The MOs for QKD were coinjected with the indicated mRNAs. In the B1 sox mRNA-coinjected embryos, the expression of hesx1, dlx3b and nuerog1 was recovered; patterning of the neural plate marked by pax2a and hoxb1b was normalized; and the expression patterns of hgg1, ntl and dlx3b reflecting C&E movements were also restored. Blue bracket, gap between the hgg1 and ntl expression domains; white dotted line, neural plate border. (D) Recovery of the AP axis elongation in QKD embryos injected with one of the B1 sox mRNAs. The length of the embryos at 15–16 hpf along the AP axis between the arrowheads (Ab, Bb, Cb) was measured for the uninjected control (Ctr) (n = 7), the QKD (n = 9) and QKD with B1 sox mRNA injection (sox2, n = 9; sox3, n = 6; sox19a, n = 7; sox19b, n = 6). The average AP axis lengths with standard errors are shown.
Mentions: The earliest detectable morphological abnormality of the QKD embryos was a delay in epiboly, notably after the shield stage (Figure 1D). At 10 hour post-fertilization (hpf), when normal embryos reach the tailbud stage, the QKD embryos were still in late epiboly. The thickening of the anterior head region was less prominent in the QKD embryos (Figure 1Dc and 1Dd), suggesting impairment of CNS development. Impaired CNS development was also indicated by the loss of hesx1 expression in the anterior-most neuroectoderm (Figure 2Bd; see also Figure 3Ca) and by the anterolateral displacement of the pax2a expression domains that mark the midbrain-hindbrain boundary (MHB) (Figure 2Bd). An early phase of neurogenesis was also affected in the QKD embryos as indicated by the loss of proneural neurog1 expression (Figure 2Bf). The QKD embryos further displayed a shortened anterior-posterior (AP) axis with a broadened neural plate (marked by hoxb1b) and broadened mesodermal structures including notochord (marked by ntl) (Figure 1D and Figure 2B). Consistently, the gap between the prechordal plate (marked by hgg1) and notochord was reduced in the QKD embryos (Figure 2Be). These abnormalities commonly occur in zebrafish embryos when C&E movements are impaired during gastrulation [31], [32].

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