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In vertebrates, the formation of the nervous system starts at gastrulation with a process called neural induction. This process requires, at least in part, the inhibition of BMP signalling in the ectoderm by noggin, as well as FGF receptor activation and Ca2+ signalling. Our studies with Xenopus embryos suggest that an increase in intracellular Ca2+ concentration ([Ca2+]i), via dihydropyridine-sensitive Ca2+ channels (DHP-sensitive Ca2+ channels) is necessary and sufficient to direct the ectodermal cells toward a neural fate, and that Ca2+ directly controls the expression of neural genes. The mechanism by which the DHP-sensitive Ca2+ channels are activated during neural induction remains unknown. One possible mechanism is via the activation of FGF signalling. Using isolated ectodermtissue, here we demonstrated that FGF-4 depolarises the membrane of ectodermal cells and induces an increase in [Ca2+]i. This Ca2+ increase can be blocked by SU5402, an FGF receptor inhibitor, and by DHP-sensitive Ca2+ channel antagonists. These inhibitors also block the induction of neural genes. We discuss a possible gating mechanism for the activation of DHP-sensitive Ca2+ channels via the FGF signalling pathway, which involves arachidonic acid and TRPC1 channel activation.
Fig. 1.
FGF signalling is required for the noggin–induced Ca2+ increase. (A) The expression of two pan neural markers, Zic3 and Sox2 in 10 animal caps was measured by RT-PCR. Animal caps were pre-incubated (+) or not (-) for 30 min with 80 μM of the FGFR inhibitor, SU5402 prior to incubation with noggin (2 μg/mL). The RNA from one sibling embryo at stage 13 served as positive control and PCR on RNA without reverse transcription was performed to check the absence of genomic DNA. ODC was used as loading control. (B) Whole-mount in situ hybridization of animal caps with the Zic3 probe. Animal caps from blastula stage embryos were either treated with 2 μg/mL of noggin (right) or pre-incubated for 30 min with SU5402 (80 μM) prior to the addition of noggin (left). All animal caps were cultured to stage 13. Scale bar, 500 μm. (C-E) Representative examples of PMT traces obtained from cp-aequorin-loaded animal caps (data sampled at a rate of one measure each 10 seconds). Light emission, expressed in photons per second and reflecting [Ca2+]i was recorded upon: (C) The addition of noggin (2 μg/mL); (D) Pre-incubation in 80 μM SU5402 prior to the addition of noggin; (E) The addition of noggin and followed (at the peak of the emitted light) by the addition of SU5402 (80 μM).
Fig. 2.
FGF-4 induces an increase in intracellular Ca2+. (A-C) Representative examples of PMT traces obtained from aequorin-loaded animal caps (data sampled at a rate of one measure each 10 seconds). Light emission, expressed in photons per second and reflecting an increase in [Ca2+]i was recorded upon: (A) The addition of FGF-4 (100 ng/mL); (B) Pre-incubation with 80 μM SU5402 prior to the addition of FGF-4; (C) The addition of FGF-7 (100 ng/mL), followed by the addition of FGF-4.
Fig. 3.
The FGF-induced increase in Ca2+ involves voltage-gated Ca2+ channels in animal caps. (A) A representative example of a PMT trace obtained from cp-aequorin-loaded animal caps (data sampled at a rate of one measure each 10 seconds). Light emission, expressed in photons per second and reflecting [Ca2+]i was recorded upon pre-incubation with 300 μM nifedipine prior to the addition of FGF-4. (B-D) Representative examples of the relative change in fluorescent intensity (F/F0), reflecting changes in membrane potential, recorded upon: (B) The addition of noggin (2 μg/mL); (C) The addition of FGF-4 (100 ng/mL); and (D) Pre-incubation with 80 μM SU5402 prior to the addition of FGF-4.
Fig. 4.
Effect of blocking Ca2+ influx by La3+. (A) The expression of Zic3 and Xngnr-1a in 10 animal caps was measured by RT-PCR. Animal caps were pre-incubated (+) (or not (-)) for 30 min with 50 μM or 250 μM La3+ prior to incubation with noggin (2 μg/mL). RNA from one sibling embryo at stage 13 served as positive control and PCR on RNA without reverse transcription was performed to check the absence of genomic DNA. ODC was used as loading control. (B) A representative example of a PMT trace obtained from cp-aequorin-loaded animal caps (data sampled at a rate of one measure each 10 seconds). Light emission, expressed in photons per second and reflecting [Ca2+]i was recorded upon pre-incubation in 250 μM La3+ prior to the addition of FGF-4. (C) Temporal distribution of XTRPC1 RNA expression in adult tissues and during embryogenesis. RT-PCR analysis of RNA extracted from adult tissues, heart (H) and brain (B); oocyte (Oo), embryos at blastula (8) and gastrula (101/2) stages, and in animal caps dissected from embryos of blastula (8, 9) and gastrula (101/2) stages.
Fig. 5.
Arachidonic acid induces an increase in intracellular Ca2+ and the expression of neural gene. (A) A representative example of a PMT trace obtained from cp-aequorin-loaded animal caps (data sampled at a rate of one measure each 10 seconds). Light emission, expressed in photons per second and reflecting [Ca2+]i was recorded upon addition of arachidonic acid (200 μM). (B) The expression of a pan neural marker, Zic3, in 10 animal caps was measured by RT-PCR. Animal caps were incubated (+) or not (-) with either noggin (2 μg/mL) or arachidonic acid (200 μM). The RNA from one sibling embryo at stage 13 served as positive control and PCR on RNA without reverse transcription was performed to check the absence of genomic DNA. ODC was used as loading control.
Fig. 6.
Hypothetical model of DHP-sensitive Ca2+ channel activation during neural induction in Xenopus laevis. Neural induction requires the inhibition of BMP signalling by noggin, the activation of FGF pathway and Ca2+ signalling. Our model illustrates possible interactions between these pathways. (1) FGFR activation leads to the stimulation of various signal transduction pathways. FGFR activation is involved in neuralization of dissociated ectoderm by the inhibition of Smad1 activity through a Ras/MAPK pathway [84]. It can also elicit the production of arachidonic acid (AA). AA itself or its metabolites have been shown to act on transient potential receptor (TRP) Ca2+ channels. (2) The initial Ca2+ increase through TRP channels triggers cell membrane depolarisation (3) and the subsequent influx of Ca2+ through DHP-sensitive Ca2+ channels, a process shown to be necessary and sufficient for neuralization of the ectoderm [21]. The possible interaction between the BMPRII and TRP channels is also suggested.
