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

Treatment with 800 μM SB-431542 or 50 μM SB-505124 at MBT prevents formation of mesoderm and endoderm. (A-F) Images of live embryos at 24hpf treated at 2.75 h with DMSO (A-C), SB-431542 (D-F; J-S), or SB-505124 (G-I). Embryos treated with SB-431542 (D-F) or SB-505124 (G-I) lack derivatives of the mesoderm and endoderm in the head and trunk, display severe cyclopia and lack Kupffer's vesicle. (J-P) Images of embryos treated with DMSO (J-N) or SB-431542 at MBT (O-S) and processed to reveal expression of markers for derivatives of dorsal mesoderm (ntl: J, O; flh: L, Q), paraxial mesoderm (myoD: K, P), intermediate mesoderm (pax2.1: M, R), and ventral neurectoderm (shhb: N, S). Dorsal views of embryos fixed at 10 h (J, L, N, P, Q, S)or 14 h (K, M, P, R). Arrowhead in (C) is the Kupffer's vesicle.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Treatment with 800 μM SB-431542 or 50 μM SB-505124 at MBT prevents formation of mesoderm and endoderm. (A-F) Images of live embryos at 24hpf treated at 2.75 h with DMSO (A-C), SB-431542 (D-F; J-S), or SB-505124 (G-I). Embryos treated with SB-431542 (D-F) or SB-505124 (G-I) lack derivatives of the mesoderm and endoderm in the head and trunk, display severe cyclopia and lack Kupffer's vesicle. (J-P) Images of embryos treated with DMSO (J-N) or SB-431542 at MBT (O-S) and processed to reveal expression of markers for derivatives of dorsal mesoderm (ntl: J, O; flh: L, Q), paraxial mesoderm (myoD: K, P), intermediate mesoderm (pax2.1: M, R), and ventral neurectoderm (shhb: N, S). Dorsal views of embryos fixed at 10 h (J, L, N, P, Q, S)or 14 h (K, M, P, R). Arrowhead in (C) is the Kupffer's vesicle.

Mentions: To determine when Sqt and Cyc signals induce and pattern the germ layers, we developed a drug-based strategy that permits us to block endogenous Nodal signals at different stages after the mid-blastula transition (MBT). SB-431542 binds competitively to the ATP binding sites of the ALK 4, 5 and 7 receptors, preventing their kinase activity [39]. This drug has been used previously on zebrafish embryos during the cleavage stages, but did not completely block Nodal signals when added after MBT [40,41]. Therefore, we developed a protocol to use SB-431542 to block zygotic Nodal signals in whole embryos between MBT (2.75 h) and the onset of gastrulation (6 h) (see Methods). Control embryos had a normal morphology at 24 h, indicating that our manipulations did not affect early embryogenesis (Fig. 1A–C). By contrast, embryos treated with 800 μM SB-431542 display severe cyclopia and lack all derivatives mesoderm and endoderm in the head and trunk, including the somites, notochord, blood, heart and Kupffer's vesicle (Fig. 1D–F). These defects strongly resemble those previously described for sqt; cyc double mutants [19]. Like sqt; cyc double mutants, SB-431542 treated embryos lack axial expression of the pan-mesendodermal marker no-tail (ntl) and the notochord marker floating head (flh) (Fig. 1J, L, O, Q). Interestingly, flh expression in the neurectoderm is greatly expanded in drug treated embryos, suggesting an expanded epiphysis (Fig. 1Q) [42]. Drug treated embryos also lack MyoD expression at 14 h (Fig. 1K, P). Since tail somites do not form until later stages, this indicates that trunk somites are missing [43]. The prechordal plate and pronephros are also missing in these embryos, as indicated by the lack of goosecoid (gsc) and pax2.1 expression, respectively (Fig. 1R, M; Fig. 3C, D). Drug treated embryos also lack expression of sonic hedgehog b (shhb), indicating the absence of floorplate (Fig. 1N, S). Because high concentrations of the drug were necessary to produce these defects, we next asked if we could achieve similar results with SB-505124, a more potent and bioactive inhibitor of the ALK 4/5/7 receptors than SB-431542 [38]. 30–50 μM of SB-505124 is sufficient to phenocopy sqt; cyc mutants when added at MBT (Fig. 1G–I). The ability of both drugs to phenocopy sqt; cyc mutants when added to 2.75 h embryos indicates that they reduce ALK 4/5/7 receptor activity to levels as low as that in zygotic mutants for nodal-related gene function. Subsequent experiments were performed with SB-431542 and confirmed with SB-505124 as indicated.


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

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

Treatment with 800 μM SB-431542 or 50 μM SB-505124 at MBT prevents formation of mesoderm and endoderm. (A-F) Images of live embryos at 24hpf treated at 2.75 h with DMSO (A-C), SB-431542 (D-F; J-S), or SB-505124 (G-I). Embryos treated with SB-431542 (D-F) or SB-505124 (G-I) lack derivatives of the mesoderm and endoderm in the head and trunk, display severe cyclopia and lack Kupffer's vesicle. (J-P) Images of embryos treated with DMSO (J-N) or SB-431542 at MBT (O-S) and processed to reveal expression of markers for derivatives of dorsal mesoderm (ntl: J, O; flh: L, Q), paraxial mesoderm (myoD: K, P), intermediate mesoderm (pax2.1: M, R), and ventral neurectoderm (shhb: N, S). Dorsal views of embryos fixed at 10 h (J, L, N, P, Q, S)or 14 h (K, M, P, R). Arrowhead in (C) is the Kupffer's vesicle.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Treatment with 800 μM SB-431542 or 50 μM SB-505124 at MBT prevents formation of mesoderm and endoderm. (A-F) Images of live embryos at 24hpf treated at 2.75 h with DMSO (A-C), SB-431542 (D-F; J-S), or SB-505124 (G-I). Embryos treated with SB-431542 (D-F) or SB-505124 (G-I) lack derivatives of the mesoderm and endoderm in the head and trunk, display severe cyclopia and lack Kupffer's vesicle. (J-P) Images of embryos treated with DMSO (J-N) or SB-431542 at MBT (O-S) and processed to reveal expression of markers for derivatives of dorsal mesoderm (ntl: J, O; flh: L, Q), paraxial mesoderm (myoD: K, P), intermediate mesoderm (pax2.1: M, R), and ventral neurectoderm (shhb: N, S). Dorsal views of embryos fixed at 10 h (J, L, N, P, Q, S)or 14 h (K, M, P, R). Arrowhead in (C) is the Kupffer's vesicle.
Mentions: To determine when Sqt and Cyc signals induce and pattern the germ layers, we developed a drug-based strategy that permits us to block endogenous Nodal signals at different stages after the mid-blastula transition (MBT). SB-431542 binds competitively to the ATP binding sites of the ALK 4, 5 and 7 receptors, preventing their kinase activity [39]. This drug has been used previously on zebrafish embryos during the cleavage stages, but did not completely block Nodal signals when added after MBT [40,41]. Therefore, we developed a protocol to use SB-431542 to block zygotic Nodal signals in whole embryos between MBT (2.75 h) and the onset of gastrulation (6 h) (see Methods). Control embryos had a normal morphology at 24 h, indicating that our manipulations did not affect early embryogenesis (Fig. 1A–C). By contrast, embryos treated with 800 μM SB-431542 display severe cyclopia and lack all derivatives mesoderm and endoderm in the head and trunk, including the somites, notochord, blood, heart and Kupffer's vesicle (Fig. 1D–F). These defects strongly resemble those previously described for sqt; cyc double mutants [19]. Like sqt; cyc double mutants, SB-431542 treated embryos lack axial expression of the pan-mesendodermal marker no-tail (ntl) and the notochord marker floating head (flh) (Fig. 1J, L, O, Q). Interestingly, flh expression in the neurectoderm is greatly expanded in drug treated embryos, suggesting an expanded epiphysis (Fig. 1Q) [42]. Drug treated embryos also lack MyoD expression at 14 h (Fig. 1K, P). Since tail somites do not form until later stages, this indicates that trunk somites are missing [43]. The prechordal plate and pronephros are also missing in these embryos, as indicated by the lack of goosecoid (gsc) and pax2.1 expression, respectively (Fig. 1R, M; Fig. 3C, D). Drug treated embryos also lack expression of sonic hedgehog b (shhb), indicating the absence of floorplate (Fig. 1N, S). Because high concentrations of the drug were necessary to produce these defects, we next asked if we could achieve similar results with SB-505124, a more potent and bioactive inhibitor of the ALK 4/5/7 receptors than SB-431542 [38]. 30–50 μM of SB-505124 is sufficient to phenocopy sqt; cyc mutants when added at MBT (Fig. 1G–I). The ability of both drugs to phenocopy sqt; cyc mutants when added to 2.75 h embryos indicates that they reduce ALK 4/5/7 receptor activity to levels as low as that in zygotic mutants for nodal-related gene function. Subsequent experiments were performed with SB-431542 and confirmed with SB-505124 as indicated.

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