Limits...
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.

Show MeSH

Related in: MedlinePlus

Cell fate specification is delayed squint mutants. Cell fates were examined in sqt mutant embryos treated with DMSO (A1-D1), or with SB-431542 at various time points. (A1-7) flh was first expressed at the midline in embryos treated at 5 h (A5). (B2-7) gsc expression is first detected in embryos treated at 6 h (B6). (C2-7) sox17 expression is first detected when embryos are treated at 7 h (C7). (D1-7) cmlc2 expression was first detected in embryos treated 4.7 h (D4, arrowhead). Dorsal views of 10 h (A1-B7), 8 h (C1-C7) or 24 h (D1-D6). In D1-D6, anterior is up. The embryos in Figs. 8 and 9 are from the same clutch and weretreated in parallel, along with wild type controls (not shown).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC1851950&req=5

Figure 8: Cell fate specification is delayed squint mutants. Cell fates were examined in sqt mutant embryos treated with DMSO (A1-D1), or with SB-431542 at various time points. (A1-7) flh was first expressed at the midline in embryos treated at 5 h (A5). (B2-7) gsc expression is first detected in embryos treated at 6 h (B6). (C2-7) sox17 expression is first detected when embryos are treated at 7 h (C7). (D1-7) cmlc2 expression was first detected in embryos treated 4.7 h (D4, arrowhead). Dorsal views of 10 h (A1-B7), 8 h (C1-C7) or 24 h (D1-D6). In D1-D6, anterior is up. The embryos in Figs. 8 and 9 are from the same clutch and weretreated in parallel, along with wild type controls (not shown).

Mentions: We have shown that Nodal signals specify different cell types within the mesoderm and endoderm at different times, although there is a period during which they specify multiple tissues simultaneously. This could be explained if the responding cells have fixed time windows during which they need to be exposed to Nodal signals in order to adopt particular fates. If so, then mesoderm and endodermal cell fates will be specified at the same time as wild type even when the Nodal dose is reduced. To test this, we asked when cell fates are specified in sqt mutants, which have reduced levels of Nodal signaling. We found that flh expression at the midline was only observed when Nodal signaling was blocked at 5 h in sqt mutants (Fig. 8A5; N = 15), as opposed to 4 hr in wild type (Fig. 4B6). Thus, notochord specification is delayed by an hour in sqt mutants. Specification of the prechordal plate and endoderm are also delayed in sqt mutants. gsc expression is only apparent in sqt mutants treated at the onset of gastrulation (6 h) (Fig. 8B6, N = 21; compare with Fig. 6C4), and sox17 expression is first apparent in embryos treated at 7 h (Fig. 8C7, N = 16/20; compare with Fig. 6E5). We also observed a delay in specification of ventrolateral cell types in sqt mutants, since cmlc2 expression is only apparent in embryos treated at 4.7 h (Fig. 8D4, arrowhead, N = 12/16; compare with Fig. 7A2). These results rule out the possibility that presumptive mesoderm and endodermal cells have discrete windows of competence that determine their response to Nodal signals.


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

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

Cell fate specification is delayed squint mutants. Cell fates were examined in sqt mutant embryos treated with DMSO (A1-D1), or with SB-431542 at various time points. (A1-7) flh was first expressed at the midline in embryos treated at 5 h (A5). (B2-7) gsc expression is first detected in embryos treated at 6 h (B6). (C2-7) sox17 expression is first detected when embryos are treated at 7 h (C7). (D1-7) cmlc2 expression was first detected in embryos treated 4.7 h (D4, arrowhead). Dorsal views of 10 h (A1-B7), 8 h (C1-C7) or 24 h (D1-D6). In D1-D6, anterior is up. The embryos in Figs. 8 and 9 are from the same clutch and weretreated in parallel, along with wild type controls (not shown).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Cell fate specification is delayed squint mutants. Cell fates were examined in sqt mutant embryos treated with DMSO (A1-D1), or with SB-431542 at various time points. (A1-7) flh was first expressed at the midline in embryos treated at 5 h (A5). (B2-7) gsc expression is first detected in embryos treated at 6 h (B6). (C2-7) sox17 expression is first detected when embryos are treated at 7 h (C7). (D1-7) cmlc2 expression was first detected in embryos treated 4.7 h (D4, arrowhead). Dorsal views of 10 h (A1-B7), 8 h (C1-C7) or 24 h (D1-D6). In D1-D6, anterior is up. The embryos in Figs. 8 and 9 are from the same clutch and weretreated in parallel, along with wild type controls (not shown).
Mentions: We have shown that Nodal signals specify different cell types within the mesoderm and endoderm at different times, although there is a period during which they specify multiple tissues simultaneously. This could be explained if the responding cells have fixed time windows during which they need to be exposed to Nodal signals in order to adopt particular fates. If so, then mesoderm and endodermal cell fates will be specified at the same time as wild type even when the Nodal dose is reduced. To test this, we asked when cell fates are specified in sqt mutants, which have reduced levels of Nodal signaling. We found that flh expression at the midline was only observed when Nodal signaling was blocked at 5 h in sqt mutants (Fig. 8A5; N = 15), as opposed to 4 hr in wild type (Fig. 4B6). Thus, notochord specification is delayed by an hour in sqt mutants. Specification of the prechordal plate and endoderm are also delayed in sqt mutants. gsc expression is only apparent in sqt mutants treated at the onset of gastrulation (6 h) (Fig. 8B6, N = 21; compare with Fig. 6C4), and sox17 expression is first apparent in embryos treated at 7 h (Fig. 8C7, N = 16/20; compare with Fig. 6E5). We also observed a delay in specification of ventrolateral cell types in sqt mutants, since cmlc2 expression is only apparent in embryos treated at 4.7 h (Fig. 8D4, arrowhead, N = 12/16; compare with Fig. 7A2). These results rule out the possibility that presumptive mesoderm and endodermal cells have discrete windows of competence that determine their response to Nodal signals.

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