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Time-dependent patterning of the mesoderm and endoderm by Nodal signals in zebrafish.

Hagos EG, Dougan ST - BMC Dev. Biol. (2007)

Bottom Line: This permitted us to directly examine when Nodal signals specify cell types independently of dosage effects.Confirming this, cells exposed to a uniform Nodal dose adopt progressively more marginal fates with increasing lengths of exposure.We conclude that (1) Nodal signals are most active during the mid-to-late blastula stages, when nodal-related gene expression and the movement of responding cells are at their most dynamic; (2) Nodal signals specify cell fates along the animal-vegetal axis in a time-dependent manner; (3) cells respond to the total cumulative dose of Nodal signals to which they are exposed, as a function of distance from the source and duration of exposure.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cellular Biology, The University of Georgia, Athens, GA, USA. engda@uga.edu <engda@uga.edu>

ABSTRACT

Background: The vertebrate body plan is generated during gastrulation with the formation of the three germ layers. Members of the Nodal-related subclass of the TGF-beta superfamily induce and pattern the mesoderm and endoderm in all vertebrates. In zebrafish, two nodal-related genes, called squint and cyclops, are required in a dosage-dependent manner for the formation of all derivatives of the mesoderm and endoderm. These genes are expressed dynamically during the blastula stages and may have different roles at different times. This question has been difficult to address because conditions that alter the timing of nodal-related gene expression also change Nodal levels. We utilized a pharmacological approach to conditionally inactivate the ALK 4, 5 and 7 receptors during the blastula stages without disturbing earlier signaling activity. This permitted us to directly examine when Nodal signals specify cell types independently of dosage effects.

Results: We show that two drugs, SB-431542 and SB-505124, completely block the response to Nodal signals when added to embryos after the mid-blastula transition. By blocking Nodal receptor activity at later stages, we demonstrate that Nodal signaling is required from the mid-to-late blastula period to specify sequentially, the somites, notochord, blood, Kupffer's vesicle, hatching gland, heart, and endoderm. Blocking Nodal signaling at late times prevents specification of cell types derived from the embryo margin, but not those from more animal regions. This suggests a linkage between cell fate and length of exposure to Nodal signals. Confirming this, cells exposed to a uniform Nodal dose adopt progressively more marginal fates with increasing lengths of exposure. Finally, cell fate specification is delayed in squint mutants and accelerated when Nodal levels are elevated.

Conclusion: We conclude that (1) Nodal signals are most active during the mid-to-late blastula stages, when nodal-related gene expression and the movement of responding cells are at their most dynamic; (2) Nodal signals specify cell fates along the animal-vegetal axis in a time-dependent manner; (3) cells respond to the total cumulative dose of Nodal signals to which they are exposed, as a function of distance from the source and duration of exposure.

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SB-431542 rapidly blocks transcription of Nodal target genes. lefty1 expression in embryos treated with DMSO (A-C) or SB-431542 (D-F) at 4.3 h (dome stage), and fixed after 15 minutes (A, D), 30 minutes (B, E) or 45 minutes (D, F).
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Figure 2: SB-431542 rapidly blocks transcription of Nodal target genes. lefty1 expression in embryos treated with DMSO (A-C) or SB-431542 (D-F) at 4.3 h (dome stage), and fixed after 15 minutes (A, D), 30 minutes (B, E) or 45 minutes (D, F).

Mentions: To determine how quickly we could observe the effects of the drug, we examined the expression of the Nodal target gene lefty1 in a time course of embryos treated with SB-431542 at dome stage (4.3hpf) [44]. We found that transcription of Nodal target genes is normal 15 minutes after treatment (Fig. 2A, D; N = 11), but is severely reduced after 30 minutes (Fig. 2B, E; N = 24). No transcripts are detected 45 minutes after treatment (Fig. 2C, F; N = 21). Therefore, transcription of Nodal-dependent genes is rapidly blocked after drug treatment and the decrease in mRNA levels is apparent within 15–30 minutes.


Time-dependent patterning of the mesoderm and endoderm by Nodal signals in zebrafish.

Hagos EG, Dougan ST - BMC Dev. Biol. (2007)

SB-431542 rapidly blocks transcription of Nodal target genes. lefty1 expression in embryos treated with DMSO (A-C) or SB-431542 (D-F) at 4.3 h (dome stage), and fixed after 15 minutes (A, D), 30 minutes (B, E) or 45 minutes (D, F).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851950&req=5

Figure 2: SB-431542 rapidly blocks transcription of Nodal target genes. lefty1 expression in embryos treated with DMSO (A-C) or SB-431542 (D-F) at 4.3 h (dome stage), and fixed after 15 minutes (A, D), 30 minutes (B, E) or 45 minutes (D, F).
Mentions: To determine how quickly we could observe the effects of the drug, we examined the expression of the Nodal target gene lefty1 in a time course of embryos treated with SB-431542 at dome stage (4.3hpf) [44]. We found that transcription of Nodal target genes is normal 15 minutes after treatment (Fig. 2A, D; N = 11), but is severely reduced after 30 minutes (Fig. 2B, E; N = 24). No transcripts are detected 45 minutes after treatment (Fig. 2C, F; N = 21). Therefore, transcription of Nodal-dependent genes is rapidly blocked after drug treatment and the decrease in mRNA levels is apparent within 15–30 minutes.

Bottom Line: This permitted us to directly examine when Nodal signals specify cell types independently of dosage effects.Confirming this, cells exposed to a uniform Nodal dose adopt progressively more marginal fates with increasing lengths of exposure.We conclude that (1) Nodal signals are most active during the mid-to-late blastula stages, when nodal-related gene expression and the movement of responding cells are at their most dynamic; (2) Nodal signals specify cell fates along the animal-vegetal axis in a time-dependent manner; (3) cells respond to the total cumulative dose of Nodal signals to which they are exposed, as a function of distance from the source and duration of exposure.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cellular Biology, The University of Georgia, Athens, GA, USA. engda@uga.edu <engda@uga.edu>

ABSTRACT

Background: The vertebrate body plan is generated during gastrulation with the formation of the three germ layers. Members of the Nodal-related subclass of the TGF-beta superfamily induce and pattern the mesoderm and endoderm in all vertebrates. In zebrafish, two nodal-related genes, called squint and cyclops, are required in a dosage-dependent manner for the formation of all derivatives of the mesoderm and endoderm. These genes are expressed dynamically during the blastula stages and may have different roles at different times. This question has been difficult to address because conditions that alter the timing of nodal-related gene expression also change Nodal levels. We utilized a pharmacological approach to conditionally inactivate the ALK 4, 5 and 7 receptors during the blastula stages without disturbing earlier signaling activity. This permitted us to directly examine when Nodal signals specify cell types independently of dosage effects.

Results: We show that two drugs, SB-431542 and SB-505124, completely block the response to Nodal signals when added to embryos after the mid-blastula transition. By blocking Nodal receptor activity at later stages, we demonstrate that Nodal signaling is required from the mid-to-late blastula period to specify sequentially, the somites, notochord, blood, Kupffer's vesicle, hatching gland, heart, and endoderm. Blocking Nodal signaling at late times prevents specification of cell types derived from the embryo margin, but not those from more animal regions. This suggests a linkage between cell fate and length of exposure to Nodal signals. Confirming this, cells exposed to a uniform Nodal dose adopt progressively more marginal fates with increasing lengths of exposure. Finally, cell fate specification is delayed in squint mutants and accelerated when Nodal levels are elevated.

Conclusion: We conclude that (1) Nodal signals are most active during the mid-to-late blastula stages, when nodal-related gene expression and the movement of responding cells are at their most dynamic; (2) Nodal signals specify cell fates along the animal-vegetal axis in a time-dependent manner; (3) cells respond to the total cumulative dose of Nodal signals to which they are exposed, as a function of distance from the source and duration of exposure.

Show MeSH
Related in: MedlinePlus