BMC Dev Biol
November 15, 2011;
The response of early neural genes to FGF signaling or inhibition of BMP indicate the absence of a conserved neural induction module.
The molecular mechanism that initiates the formation of the vertebrate central nervous system
has long been debated. Studies in Xenopus and mouse demonstrate that inhibition of BMP signaling is sufficient to induce neural tissue
in explants or ES cells respectively, whereas studies in chick argue that instructive FGF signaling is also required for the expression of neural genes. Although additional signals may be involved in neural induction and patterning, here we focus on the roles of BMP inhibition and FGF8a
. To address the question of necessity and sufficiency of BMP inhibition and FGF signaling, we compared the temporal expression of the five earliest genes expressed in the neuroectoderm
and determined their requirements for induction at the onset of neural plate
formation in Xenopus. Our results demonstrate that the onset and peak of expression of the genes vary and that they have different regulatory requirements and are therefore unlikely to share a conserved neural induction regulatory module. Even though all require inhibition of BMP for expression, some also require FGF signaling; expression of the early-onset pan-neural genes sox2
and foxd5α requires FGF signaling while other early genes, sox3
and zicr1 are induced by BMP inhibition alone. We demonstrate that BMP inhibition and FGF signaling induce neural genes independently of each other. Together our data indicate that although the spatiotemporal expression patterns of early neural genes are similar, the mechanisms involved in their expression are distinct and there are different signaling requirements for the expression of each gene.
BMC Dev Biol
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Figure 1. Temporal expression of early neural genes and response to Noggin and FGF8a. Semi-quantitative RT-PCR of early neural genes in embryos over time (A, B) or in ectodermal explants (C, D). Explants are from uninjected (UI) embryos or those injected with mRNA coding for Noggin (Nog), FGF8a, or Nog + FGF8a. Embryos in (A) were collected at the times indicated at top (hours post fertilization = hpf) and explants were collected at stage 11.5 (C) or 17 (D). Ef1a expression is a loading control. Samples without MMLV RTase added were used as RT- controls (far right). (E) Immunohistochemistry for phosphorylated Histone H3 of ectodermal explants dissected from embryos injected with mRNA coding for Nog, FGF8a, or Nog+ FGF8a and cultured to stage 17. Explants injected with Nog+ FGF8a are up to 1.7 fold (cm) bigger than those from uninjected embryos. Graph is showing the number of cells marked with pH3 per mm2 expression in n = 10 explants. Nog+ FGF8a caps have 1.8 fold more pH3 per count area than uninjected explants (p=.0001). UI indicates explants that were dissected from uninjected embryos, WE is whole embryo.
Figure 2. BMP inhibition and FGF signaling prematurely induce the expression of early neural genes. (A-C) WISH for zicr1, foxd5α and sox2 of embryos injected with mRNA coding for Nog, FGF8a or Nog + FGF8a and lacZ mRNA (cyan) and collected at stage 8 (t = 6 hpf) and each subsequent hour after until stage 10.5 (t = 10 hpf) when cultured at room temperature. Red arrows indicate earliest onset of expression. All images are animal pole view with dorsal to the top. (D) RT-PCR of whole embryos dissected from uninjected embryos (UI) or embryos injected with Nog, FGF8a or Nog+FGF8a. Embryos were collected as in A-C. Genes analysed are indicated on left side, treatment on top, time of collection below panel. ODC used for loading control. All images are animal pole view with dorsal to the top.
Figure 3. FGF signaling is required for the induction of sox2 and foxd5α expression in the neural plate and the maintenance of other early neural genes. WISH for (A) sox2, (B) zicr1, (C) foxd5α, and (D) sox3 and gem of embryos that were either uninjected (UI) or injected with Noggin (Nog), dominant negative FGFR4a (Δ4a), or Nog + Δ4a and were collected at stages 10.5 and 11.5 (A-C) or 12.5 (D). All embryos are dorsal view with anterior to the top. The dashed line separates the domain of gem expression (right) from that inhibited by Δ4a (top left). (E) RT-PCR of ectodermal explants for genes indicated on the left side of panel. Explants were dissected from embryos that were either uninjected (UI) or injected with mRNA coding for Nog, Nog + dominant negative FGFR1 (XFD), Nog+delta4a, or Nog+XFD+delta4a.
Figure 4. FGF signaling induces neuron formation despite the presence of BMP signaling. (A) RT-PCR of embryos collected at the hpf indicated at the top. Primers are indicated on the left side of the panel. Ef1a was used as a loading control and samples without MMLV RTase added were used as RT- controls (far right). (B) RT-PCR of stage 17 ectodermal explants dissected from uninjected embryos (UI) or embryos injected with mRNA coding for Nog, FGF8a, or Nog+ FGF8a. WE is whole embryo control. Primers for markers of the neural plate border (slug, msx-1), epidermis (bmp4, vent2, vent1, epi-k) and mesoderm (xbra) and are shown on the left side of the panel. (C-I) WISH for (C) sox3, (D) foxd5α, (E) ngnr-1, (F) n-tub, (G, G') msx-1, (H) vent2 and (H) epi-k using embryos that were either uninjected (UI) or injected with either Nog, FGF8a or Nog+ FGF8a and lacZ mRNA. All stage 17 embryos are a dorsal view with anterior to the top (C-G, I) and G' embryos are a lateral view of embryos in G with anterior to the left. Embryos in H are stage 10.5 animal pole view with dorsal to the top. Asterisk indicates expansion in the deep layer of the ectoderm; white arrow indicates expansion in both the deep and superficial ectoderm and red arrow indicates expansion in the superficial layer only.
Albazerchi, A role for the hypoblast (AVE) in the initiation of neural induction, independent of its ability to position the primitive streak. 2007, Pubmed