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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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Related in: MedlinePlus

Single, triple, and quadruple knockdowns of sox2/3/19a/19b.Bright-field images of live embryos observed at the indicated time points. All are lateral views. (A) Uninjected control (Ctr) embryos. (B) Single knockdowns of sox2/3/19a/19b. A 1∶1 mixture of two MOs (0.9 ng each) targeting one of the four B1 sox genes was injected for a single KD. The percentage of embryos in the same morphological class is indicated in each panel. (C) Triple knockdowns of sox2/3/19a/19b. A mixture of indicated combinations of MOs (i.e., a mixture of six MOs, 5.4 ng in total) was used to simultaneously knockdown three out of the four B1 sox genes. The major classes of morphological defects are shown with the percentage of occurrence. The remaining embryos showed either milder or more severe defects. (D) Uninjected control embryos (a–f) and sox2/3/19a/19b quadruple knockdown (QKD) embryos injected with a mixture of MOs targeting the four B1 sox genes (i.e., a mixture of eight MOs, 7.2 ng in total) (a’–f’). The QKD caused very severe developmental abnormalities: a delay in epiboly, a shortened anterior-posterior axis, and impairment of CNS development (61%). The remaining embryos showed either milder defects (7%), more severe defects (24%) or lethality (8%). The aberrant movement of the anterior prechordal plate (arrows in d and e’) suggests a decreased adhesion of ectodermal cells as well as defects in convergence and extension movements in the QKD embryos. The broken lines (e’ and f’) indicate the dorsal trunk regions where cell dissociation was observed. (g, h) Dose-dependent effects of the MOs used for QKD were examined by injecting reduced amounts of the mixture of MOs targeting the four B1 sox genes (3.6 ng in total [g] and 1.8 ng in total [h]). (i) As a negative control, a mixture of 5-base-mismatch control MOs (i.e., a mixture of eight 5mis-MOs, 7.2 ng in total) was injected. (j) The coinjection of a p53-MO (2 ng) had no impact on the neural defects in the QKD embryos.
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pgen-1000936-g001: Single, triple, and quadruple knockdowns of sox2/3/19a/19b.Bright-field images of live embryos observed at the indicated time points. All are lateral views. (A) Uninjected control (Ctr) embryos. (B) Single knockdowns of sox2/3/19a/19b. A 1∶1 mixture of two MOs (0.9 ng each) targeting one of the four B1 sox genes was injected for a single KD. The percentage of embryos in the same morphological class is indicated in each panel. (C) Triple knockdowns of sox2/3/19a/19b. A mixture of indicated combinations of MOs (i.e., a mixture of six MOs, 5.4 ng in total) was used to simultaneously knockdown three out of the four B1 sox genes. The major classes of morphological defects are shown with the percentage of occurrence. The remaining embryos showed either milder or more severe defects. (D) Uninjected control embryos (a–f) and sox2/3/19a/19b quadruple knockdown (QKD) embryos injected with a mixture of MOs targeting the four B1 sox genes (i.e., a mixture of eight MOs, 7.2 ng in total) (a’–f’). The QKD caused very severe developmental abnormalities: a delay in epiboly, a shortened anterior-posterior axis, and impairment of CNS development (61%). The remaining embryos showed either milder defects (7%), more severe defects (24%) or lethality (8%). The aberrant movement of the anterior prechordal plate (arrows in d and e’) suggests a decreased adhesion of ectodermal cells as well as defects in convergence and extension movements in the QKD embryos. The broken lines (e’ and f’) indicate the dorsal trunk regions where cell dissociation was observed. (g, h) Dose-dependent effects of the MOs used for QKD were examined by injecting reduced amounts of the mixture of MOs targeting the four B1 sox genes (3.6 ng in total [g] and 1.8 ng in total [h]). (i) As a negative control, a mixture of 5-base-mismatch control MOs (i.e., a mixture of eight 5mis-MOs, 7.2 ng in total) was injected. (j) The coinjection of a p53-MO (2 ng) had no impact on the neural defects in the QKD embryos.

Mentions: No gross abnormalities were observed in the embryo morphology when any one of sox2/3/19a/19b was knocked down (single KD, Figure 1B), although the development of the CNS may be slightly perturbed and 75% of the sox2 morphants showed an up-turned tail phenotype (Figure 1Ba). When any three of sox2/3/19a/19b were simultaneously knocked down (triple KD), a range of morphological abnormalities was observed depending on the combination of KD targets (Figure 1C). Triple KDs of sox2/19a/19b and sox2/3/19b caused only mild morphological defects (Figure 1Cc and 1Cd), presumably because the remaining sox3 and sox19a genes, respectively, mostly cover the B1 sox expression domains. sox3/19a/19b morphants often showed stronger yet variable defects in their posterior structures (Figure 1Ca and 1Cb), presumably reflecting the weak sox2 expression in the posterior neuroectoderm. sox2/3/19a morphants appeared normal during gastrulation, but later developed morphological abnormalities (Figure 1Ce), likely because sox19b expression decreases in later stages.


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)

Single, triple, and quadruple knockdowns of sox2/3/19a/19b.Bright-field images of live embryos observed at the indicated time points. All are lateral views. (A) Uninjected control (Ctr) embryos. (B) Single knockdowns of sox2/3/19a/19b. A 1∶1 mixture of two MOs (0.9 ng each) targeting one of the four B1 sox genes was injected for a single KD. The percentage of embryos in the same morphological class is indicated in each panel. (C) Triple knockdowns of sox2/3/19a/19b. A mixture of indicated combinations of MOs (i.e., a mixture of six MOs, 5.4 ng in total) was used to simultaneously knockdown three out of the four B1 sox genes. The major classes of morphological defects are shown with the percentage of occurrence. The remaining embryos showed either milder or more severe defects. (D) Uninjected control embryos (a–f) and sox2/3/19a/19b quadruple knockdown (QKD) embryos injected with a mixture of MOs targeting the four B1 sox genes (i.e., a mixture of eight MOs, 7.2 ng in total) (a’–f’). The QKD caused very severe developmental abnormalities: a delay in epiboly, a shortened anterior-posterior axis, and impairment of CNS development (61%). The remaining embryos showed either milder defects (7%), more severe defects (24%) or lethality (8%). The aberrant movement of the anterior prechordal plate (arrows in d and e’) suggests a decreased adhesion of ectodermal cells as well as defects in convergence and extension movements in the QKD embryos. The broken lines (e’ and f’) indicate the dorsal trunk regions where cell dissociation was observed. (g, h) Dose-dependent effects of the MOs used for QKD were examined by injecting reduced amounts of the mixture of MOs targeting the four B1 sox genes (3.6 ng in total [g] and 1.8 ng in total [h]). (i) As a negative control, a mixture of 5-base-mismatch control MOs (i.e., a mixture of eight 5mis-MOs, 7.2 ng in total) was injected. (j) The coinjection of a p53-MO (2 ng) had no impact on the neural defects in the QKD embryos.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1000936-g001: Single, triple, and quadruple knockdowns of sox2/3/19a/19b.Bright-field images of live embryos observed at the indicated time points. All are lateral views. (A) Uninjected control (Ctr) embryos. (B) Single knockdowns of sox2/3/19a/19b. A 1∶1 mixture of two MOs (0.9 ng each) targeting one of the four B1 sox genes was injected for a single KD. The percentage of embryos in the same morphological class is indicated in each panel. (C) Triple knockdowns of sox2/3/19a/19b. A mixture of indicated combinations of MOs (i.e., a mixture of six MOs, 5.4 ng in total) was used to simultaneously knockdown three out of the four B1 sox genes. The major classes of morphological defects are shown with the percentage of occurrence. The remaining embryos showed either milder or more severe defects. (D) Uninjected control embryos (a–f) and sox2/3/19a/19b quadruple knockdown (QKD) embryos injected with a mixture of MOs targeting the four B1 sox genes (i.e., a mixture of eight MOs, 7.2 ng in total) (a’–f’). The QKD caused very severe developmental abnormalities: a delay in epiboly, a shortened anterior-posterior axis, and impairment of CNS development (61%). The remaining embryos showed either milder defects (7%), more severe defects (24%) or lethality (8%). The aberrant movement of the anterior prechordal plate (arrows in d and e’) suggests a decreased adhesion of ectodermal cells as well as defects in convergence and extension movements in the QKD embryos. The broken lines (e’ and f’) indicate the dorsal trunk regions where cell dissociation was observed. (g, h) Dose-dependent effects of the MOs used for QKD were examined by injecting reduced amounts of the mixture of MOs targeting the four B1 sox genes (3.6 ng in total [g] and 1.8 ng in total [h]). (i) As a negative control, a mixture of 5-base-mismatch control MOs (i.e., a mixture of eight 5mis-MOs, 7.2 ng in total) was injected. (j) The coinjection of a p53-MO (2 ng) had no impact on the neural defects in the QKD embryos.
Mentions: No gross abnormalities were observed in the embryo morphology when any one of sox2/3/19a/19b was knocked down (single KD, Figure 1B), although the development of the CNS may be slightly perturbed and 75% of the sox2 morphants showed an up-turned tail phenotype (Figure 1Ba). When any three of sox2/3/19a/19b were simultaneously knocked down (triple KD), a range of morphological abnormalities was observed depending on the combination of KD targets (Figure 1C). Triple KDs of sox2/19a/19b and sox2/3/19b caused only mild morphological defects (Figure 1Cc and 1Cd), presumably because the remaining sox3 and sox19a genes, respectively, mostly cover the B1 sox expression domains. sox3/19a/19b morphants often showed stronger yet variable defects in their posterior structures (Figure 1Ca and 1Cb), presumably reflecting the weak sox2 expression in the posterior neuroectoderm. sox2/3/19a morphants appeared normal during gastrulation, but later developed morphological abnormalities (Figure 1Ce), likely because sox19b expression decreases in later stages.

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