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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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Nodal signals pattern the ventrolateral mesoderm along the animal-vegetal axis in a time-dependent manner. Heart myocardial cell fates were examined in embryos treated with DMSO (A1-C1), or with SB-431542 at various time points (A2-C5). (A2-C2) Embryos treated at 4 h, express small amounts of amhc and cmlc2, but not vmhc (arrows). (A3-3) cmlc2, amhc and vmhc are bilaterally expressed in embryos treated at 4.3 h. (A4-C5) All heart markers are expressed at the midline in embryos treated at 4.7 h. Images are dorsal views at 24 h, anterior to the left.
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Figure 7: Nodal signals pattern the ventrolateral mesoderm along the animal-vegetal axis in a time-dependent manner. Heart myocardial cell fates were examined in embryos treated with DMSO (A1-C1), or with SB-431542 at various time points (A2-C5). (A2-C2) Embryos treated at 4 h, express small amounts of amhc and cmlc2, but not vmhc (arrows). (A3-3) cmlc2, amhc and vmhc are bilaterally expressed in embryos treated at 4.3 h. (A4-C5) All heart markers are expressed at the midline in embryos treated at 4.7 h. Images are dorsal views at 24 h, anterior to the left.

Mentions: We next asked if Nodal signals act similarly in the ventrolateral margin. The teleost heart is composed of two chambers, the atrium and ventricle, which express atrial myosin heavy chain (amhc) and ventricular myosin heavy chain (vmhc), respectively (Fig. 7B1, C1) [49]. Both chambers express cardiac myosin light chain 2 (cmlc2) (Fig. 7A1). Despite a large region of overlap, atrial myocardial precursors can be found in more animal locations and ventricular myocardial precursors are located closer to the margin. We found that cmlc2 and amhc expression are first detected when Nodal signaling is blocked at 4 h (Fig. 7A2, B2 arrows; N = 16/20 and N = 18/21, respectively). vmhc expression is never observed at this time point, indicating that specification of atrial myocardium precedes specification of ventricular myocardium (Fig. 7C2; N = 0/19). vmhc expression is first observed in embryos treated at 4.3 h (Fig. 7C3, N = 24/27). The short delay between specification of atrial myocardial precursors and ventricular myocardium is consistent with the small number of atrial progenitors located in animal cells where ventricle myocardial is not found [7]. The bilateral expression of heart myocardial genes in embryos treated at 4.3 h is consistent with the lack of endoderm at this stage (Fig. 7A3-C3) [50]. Myocardial precursors fuse into a tube at the midline when Nodal signals are blocked at 4.7 h, when we first observe beating hearts in live embryos (Fig. 7A4-C4; Fig. 5). We conclude that within the presumptive mesoderm and endoderm, marginal cell types require longer periods of Nodal signaling than other cell types.


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

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

Nodal signals pattern the ventrolateral mesoderm along the animal-vegetal axis in a time-dependent manner. Heart myocardial cell fates were examined in embryos treated with DMSO (A1-C1), or with SB-431542 at various time points (A2-C5). (A2-C2) Embryos treated at 4 h, express small amounts of amhc and cmlc2, but not vmhc (arrows). (A3-3) cmlc2, amhc and vmhc are bilaterally expressed in embryos treated at 4.3 h. (A4-C5) All heart markers are expressed at the midline in embryos treated at 4.7 h. Images are dorsal views at 24 h, anterior to the left.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Nodal signals pattern the ventrolateral mesoderm along the animal-vegetal axis in a time-dependent manner. Heart myocardial cell fates were examined in embryos treated with DMSO (A1-C1), or with SB-431542 at various time points (A2-C5). (A2-C2) Embryos treated at 4 h, express small amounts of amhc and cmlc2, but not vmhc (arrows). (A3-3) cmlc2, amhc and vmhc are bilaterally expressed in embryos treated at 4.3 h. (A4-C5) All heart markers are expressed at the midline in embryos treated at 4.7 h. Images are dorsal views at 24 h, anterior to the left.
Mentions: We next asked if Nodal signals act similarly in the ventrolateral margin. The teleost heart is composed of two chambers, the atrium and ventricle, which express atrial myosin heavy chain (amhc) and ventricular myosin heavy chain (vmhc), respectively (Fig. 7B1, C1) [49]. Both chambers express cardiac myosin light chain 2 (cmlc2) (Fig. 7A1). Despite a large region of overlap, atrial myocardial precursors can be found in more animal locations and ventricular myocardial precursors are located closer to the margin. We found that cmlc2 and amhc expression are first detected when Nodal signaling is blocked at 4 h (Fig. 7A2, B2 arrows; N = 16/20 and N = 18/21, respectively). vmhc expression is never observed at this time point, indicating that specification of atrial myocardium precedes specification of ventricular myocardium (Fig. 7C2; N = 0/19). vmhc expression is first observed in embryos treated at 4.3 h (Fig. 7C3, N = 24/27). The short delay between specification of atrial myocardial precursors and ventricular myocardium is consistent with the small number of atrial progenitors located in animal cells where ventricle myocardial is not found [7]. The bilateral expression of heart myocardial genes in embryos treated at 4.3 h is consistent with the lack of endoderm at this stage (Fig. 7A3-C3) [50]. Myocardial precursors fuse into a tube at the midline when Nodal signals are blocked at 4.7 h, when we first observe beating hearts in live embryos (Fig. 7A4-C4; Fig. 5). We conclude that within the presumptive mesoderm and endoderm, marginal cell types require longer periods of Nodal signaling than other cell types.

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