Proc Natl Acad Sci U S A
October 13, 2009;
BMP inhibition initiates neural induction via FGF signaling and Zic genes.
Neural induction is the process that initiates nervous system
development in vertebrates. Two distinct models have been put forward to describe this phenomenon in molecular terms. The default model states that ectoderm
cells are fated to become neural in absence of instruction, and do so when bone
morphogenetic protein (BMP) signals are abolished. A more recent view implicates a conserved role for FGF signaling that collaborates with BMP inhibition to allow neural fate specification. Using the Xenopus embryo
, we obtained evidence that may unite the 2 views. We show that a dominant-negative R-Smad, Smad5-somitabun-unlike the other BMP inhibitors used previously-can trigger conversion of Xenopus epidermis
into neural tissue
in vivo. However, it does so only if FGF activity is uncompromised. We report that this activity may be encoded by FGF4
, as its expression is activated upon BMP inhibition, and its knockdown suppresses endogenous, as well as ectopic, neural induction by Smad5-somitabun. Supporting the importance of FGF instructive activity, we report the isolation of 2 immediate early neural targets, zic3
. Conversely, we found that zic1
can be activated by BMP inhibition in the absence of translation. Finally, Zic1
are required together for definitive neural fate acquisition, both in ectopic and endogenous situations. We propose to merge the previous models into a unique one whereby neural induction is controlled by BMP inhibition, which activates directly, and, via FGF instructive activity, early neural regulators such as Zic genes.
Proc Natl Acad Sci U S A
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Fig. 1. In vivo neural induction by Smad5-sbn requires FGF activity. (A) Ventral views of stage 13 embryos injected at 16-cell stage in one AB4 blastomere with 2.5 ng FLDx alone or with 3 ng smad5-sbn mRNA, 1 ng dnFGFR4 mRNA, 23 ng FGF4 MO, or treated with 200 μM SU5402, as indicated. In this and the following figures, the orange staining reveals the presence of the lineage tracer FLDx. (B) Quantitative RT-PCR analysis of FGF4 expression at stage 10.5 of embryos injected in all cells at 4-cell stage with 1 ng/blastomere bmp4 mRNA, 1 ng/blastomere noggin mRNA, or 3 ng/blastomere smad5-sbn mRNA. For this and all following quantitative RT-PCR graphs, expression levels were normalized to levels of ornithine decarboxylase (ODC). (C) Quantitative RT-PCR analysis of FGF4 expression at stage 10 of animal caps taken at late blastula stage from embryos injected animally in all cells at 4-cell stage with FLDx alone or with 3 ng/blastomere smad5-sbn mRNA. (D) Top: Animal caps as in C. Bottom: Ventral views of stage 10 embryos injected with FLDx alone or with 3 ng smad5-sbn mRNA in AB4 at 16-cell stage. (E) Stage 13 animal caps taken at late blastula stage from embryos injected as in C, in the presence or absence of 23 ng/blastomere FGF4 MO. (F) Dorsal views of stage 10, stage 13, and stage 24 embryos injected marginally with 46 ng control MO or 46 ng FGF4 MO in one dorsal cell at 4-cell stage. The injected region is circled with a white dotted line. For the rescue assay, 350 pg of recombinant bFGF protein was injected in the blastocele at stage 8. In this and the following figures, the number of embryos or caps exemplified by the photograph over the total number analyzed is displayed.
Fig. 2. zic1, zic3, and foxD5a show differential regulation by BMP and FGF signals. (A) SU5402 treatment (200 μM) was from 4-cell stage to stage 10.5, and noggin mRNA (1 ng/blastomere) was injected in all cells at 4-cell stage. zic3 and foxD5a, but not zic1, activation by Noggin depends on FGF activity. For each marker, animal (Top) and vegetal views (Bottom) are shown. (B) Ventral views of stage 13 embryos injected in one AB4 blastomere with 2.5 ng FLDx alone or with 3 ng smad5-sbn mRNA, or treated with 200 μM SU5402. (C) Dorsal views of stage 10.5 embryos injected marginally with 46 ng control MO or 46 ng FGF4 MO in one of the 2 dorsal cells at 4-cell stage. For the rescue assay, 350 pg of bFGF protein was injected in the blastocele at stage 8. Dotted line represents midline; inj, injected side.
Fig. 3. zic3 and foxD5a are directs targets of FGF signaling, whereas zic1 is a direct target of BMP inhibition. (A) Animal views of stage 11.5 embryos injected at stage 10.5 in the blastocele with 40 ng BSA alone or combined with increasing amounts of bFGF protein, and treated with 10 μg/mL CHX or untreated. (B) Stage 8.5 embryos were injected in the blastocele with 40 ng BSA alone or combined with 36 ng Noggin protein in the presence or absence of CHX, and analyzed at stage 10. Animal views are shown for zic1 and zic3, vegetal views for foxD5a.
Fig. 4. Zic1 and Zic3 together are required for neural fate specification. (A–G) Ventral views of stage 13 embryos injected in one AB4 blastomere at 16-cell stage with 2.5 ng FLDx alone or with 3 ng smad5-sbn mRNA, and 23 ng control MO, 23 ng Zic1 MO, 23 ng Zic3 MO1, 23 ng Zic3 MO2, a mixture of 23 ng Zic1 MO and 23 ng Zic3 MO1, or a mixture of 23 ng Zic1 MO and 23 ng Zic3 MO2, as indicated. (H–W) Eight-cell embryos were injected in the 2 right animal blastomeres with 2.5 ng/blastomere FLDx and the indicated MOs (same amounts listed earlier). The combined knockdown of Zic1 and Zic3 leads to the suppression of sox2 expression at stage 10 (arrow in R), at stage 13 (L and P), and at stage 23 (S). Note the lack of sox2 staining in the posterior brain and spinal chord (asterisk in S). For rescue assays, 4-cell embryos were injected in the 2 right blastomeres with 500 pg zic3 (M) or zic1 (Q) mRNAs before MO injection. (H–Q) Dorso-anterior views at stage 13; (R and S) dorsal views; (T and U) anterior views at stage 15; and (V and W) posterior views at stage 15. (X) Four-cell embryos were injected with control or Zic MOs (same amounts as listed earlier in each of the 4 cells) and collected for quantitative RT-PCR analysis at stage 10.5.
Neural induction requires continued suppression of both Smad1 and Smad2 signals during gastrulation.