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Neural induction by cell-cell signaling was discovered a century ago by the organizer transplantations of Spemann and Mangold in amphibians. Spemann later found that early dorsal blastopore lips induced heads and late organizers trunk-tail structures. Identifying region-specific organizer signals has been a driving force in the progress of animal biology. Head induction in the absence of trunk is designated archencephalic differentiation. Two specific head inducers, Cerberus and Insulin-like growth factors (IGFs), that induce archencephalic brain but not trunk-tail structures have been described previously. However, whether these two signals interact with each other had not been studied to date and was the purpose of the present investigation. It was found that Cerberus, a multivalent growth factor antagonist that inhibits Nodal, BMP and Wnt signals, strongly cooperated with IGF2, a growth factor that provides a positive signal through tyrosine kinase IGF receptors that activate MAPK and other pathways. The ectopic archencephalic structures induced by the combination of Cerberus and IGF2 are of higher frequency and larger than either one alone. They contain brain, a cyclopic eye and multiple olfactory placodes, without trace of trunk structures such as notochord or somites. A dominant-negative secreted IGF receptor 1 blocked Cerberus activity, indicating that endogenous IGF signals are required for ectopic brain formation. In a sensitized embryonic system, in which embryos were depleted of β-catenin, IGF2 did not by itself induce neural tissue while in combination with Cerberus it greatly enhanced formation of circular brain structures expressing the anterior markers Otx2 and Rx2a, but not spinal cord or notochord markers. The main conclusion of this work is that IGF provides a positive signal initially uniformly expressed throughout the embryo that potentiates the effect of an organizer-specific negative signal mediated by Cerberus. The results are discussed in the context of the history of neural induction.
Fig. 1. IGF2 and Cerberus mRNAs cooperate in ectopic head induction in Xenopus embryos.
Embryos were microinjected into the ventral marginal zone of a single blastomere at the 4- to 8-cell stage. (A) Uninjected control sibling at early tailbud stage (n = 81). (B) A single ventral injection of IGF2 mRNA caused a small ectopic head protrusion with a pigmented cement gland on the belly (n = 86, 68 % with ectopic structures). (C) Cerberus mRNA induced a secondary head-like structure (n = 78, 94 % with ectopic heads). (D) Co-injection of IGF2 and Cerberus mRNAs induced a large ectopic head with an expanded cement gland (n = 90, 98 % with ectopic heads). (E–H) Panoramic views of control and injected embryos. Injected mRNA doses per embryo were: Cerberus, 100 pg; IGF2, 2 ng. Results from two experiments. Scale bars are 500 μm (A-D) and 2 mm (E-H).
Fig. 2. Ectopic heads induced by IGF2 and Cerberus developed a cyclopic eye.
(A) Uninjected control tadpole at stage 46. (B) Ectopic head with an eye (arrow) bigger than the endogenous eyes induced by the combination of IGF2 and Cerberus mRNAs, shown at stage 46. (C–E) Uninjected control tadpole at day 40. (F–H) Three magnifications of an ectopic head structure of induced by coinjection of IGF2 and Cerberus in tadpole undergoing metamorphosis. Arrows indicate the ectopic eye. Injected mRNA doses per embryo were: Cerberus, 100 pg; IGF2, 2 ng. Scale bars are 1 mm (A, B), 2 mm (D, G) and 500 μm (E, H).
Fig. 3. Histological analysis of the ectopic head induced by IGF2 and Cerberus in 40-day old Xenopus tadpole embryos.
(A) Hematoxylin and eosin staining of sectioned ectopic head at low power showing brain, eye and olfactory placodes. (B) Brain at higher magnification showing gray matter (GM) consisting of neuronal bodies on the outside and white matter (WM) consisting of myelinated projections in the inside. (C) Eye including neural retina (NR) and surrounded by retinal pigmented epithelium (RPE). (D) Olfactory placode (OP) imaged at higher magnification, see scale bar. Injected mRNA doses per embryo were: Cerberus 100 pg, IGF2 2 ng. Scale bars are 200 μm (A), 50 μm (B) and 20 μm (C, D).
Fig. 4. In β-catenin depleted embryos, 4× coinjection of Cerberus mRNA or in combination with IGF2 mRNA induced circular radial brain structures; IGF2 and Cerberus cooperated in brain marker induction but did not induce notochord markers at stage 12 late gastrula.
(A) Control embryo at stage 22 (n = 55). (B) Radial 4× β-catenin MO injection resulted in complete ventralization (n = 15). (C) Embryo with 4× microinjection of β-catenin MO together with IGF2 mRNA (n = 35, 83 % completely ventralized, 17 % with very weak axis). (D) β-catenin MO and Cerberus radial coinjection induced radial cement gland and brain (n = 17, all radially dorsalized). (E) Embryos injected 4× with a combination of β-catenin MO, IGF2 mRNA and Cerberus mRNA with enlarged radial cement gland and brain (n = 30, all with radial phenotype). Arrowheads indicate the position of blastopore. Scale bar, 500 μm. qRT-PCR results at late gastrula of siblings (three embryos per sample in triplicate). (F) Anterior neural marker Otx2. (G) pan-neural marker Sox2, (H–I) The trunk mesodermal midline and late organizer markers Chordin and ADMP at stage 12. Error bars denote SD (n = 3) (*P < 0.05 and **P < 0.01). Coinjections were performed four times radially at the 2- to 4-cell stage with the following doses per embryo: β-catenin MO, 34 ng total; IGF2 mRNA, 8 ng total; Cerberus mRNA, 400 pg total.
Fig. 5. In β-catenin MO ventralized embryos a single injection of IGF2 and Cerberus induced circular brains; IGF2 alone did not induce neural markers at early tailbud but cooperated with Cerberus inducing anterior markers while spinal cord or posteriortrunkmesoderm markers were not induced.
(A) Uninjected control at tailbud stage 22 (n = 100). (B) 4× β-catenin MO injected embryos (n = 40, all completely ventralized). (C) Radial β-catenin MO followed by 1-time ventral injection of IGF2 and Cerberus plus β-catenin MO resulted in ectopic radial cement gland and brain structures (n = 46, all radial brain). (D) β-catenin MO did not have any discernable effect on wild type embryos injected 1 time ventrally with IGF2 mRNA and Cerberus mRNA (n = 91, all with ectopic head). Arrowheads indicate the position of blastopore. Insets show embryos from the same experiment photographed one day later at stage 34. Scale bar, 500 μm. qRT-PCR results for (F) eye marker Rx2a, (G) anterior neural marker Otx2, (H) spinal cord marker HoxB9/XlHbox6, and (I) trunk mesodermal midline and late organizer marker ADMP at stage 22. The qRT-PCR experiments represent biological triplicates. Error bars denote SD (n = 3) (**P < 0.01 and P < 0.1). Doses per injection were: β-catenin MO, 8.5 ng; IGF2 mRNA, 2 ng; Cerberus mRNA, 100 pg.
Fig. 6. Dominant-negative IGF receptor 1 blocked ectopic head formation by Cerberus mRNA in single ventral injections.
(A) Control embryo at stage 24 injected with LacZ (100 pg) mRNA but not stained for β-galactosidase (n = 50). (B) DN-IGFR (600 pg) and LacZ injected embryos (n = 20, all normal). (C) Cerberus (100 pg) injected embryos with ectopic heads (n = 56, 96 % ectopic heads). (D) DN-IGFR blocked Cerberus ectopic heads (n = 33, 88 % with no ectopic structures, 12 % with small cement glands). (E–F) LacZ staining for (A–D). Scale bar, 500 μm.
Fig. 7. Diagram of signaling pathways involved in brain induction in Xenopus embryos.
Cerberus is a multivalent inhibitor of BMP, Nodal and xWnt8 growth factors, inhibiting their binding to plasma membrane receptors. FGF8 and IGF2 are important positive signals that activate MAPK activity. Smads are transcription factors activated by BMP or Nodal/TGF-β carboxy terminal phosphorylation. They are also phosphorylated by MAPK, which primes phosphorylation by GSK3 resulting in the net inhibition of Smad4. The receptor Smads 1/5/8 and 2/3 also have linker inhibitory MAPK/GSK3 sites. Linker phosphorylations provide a hard-wired mechanism for the integration of signaling pathways at the protein level. Note that FGF8 and IGF2 also have Smad-independent positive mechanisms for neural induction and trunk inhibition. Figure drawn using BioRender.
Supplemental Fig. S1. Suboptimal levels of IGF2 with almost no inductive activity could synergize with Cerberus in ectopic head induction.
(A) Uninjected control embryo at tailbud stage 30 (n=37). (B) IGF2 injection (400 pg) had no effect (n=50, all normal). (C) Ectopic head induced by Cerberus (100 pg) (n=37, 14% with ectopic structures in this experiment). (D) IGF2 and Cerberus coinjection caused higher frequency of ectopic heads (n=64, 89% with ectopic heads). (E) Control embryo at stage 34 (n=70). (F) IGF2 injection (1 ng) with a small ectopic cement gland (n=56, 17.8%). (G) Cerberus (100 pg) injection showing archencephalic induction in the ventral region (n=25, 36% with ectopic heads). (H) IGF2 and Cerberus coinjected embryo with large ectopic head and enlarged cement gland (n=29, 100% with phenotypes). (I) Control embryo at stage 32 (n=27). (J) IGF2 injection (2 ng) resulted in ectopic head formation (n=55, 53% with ectopic heads). (K) Cerberus (100 pg) injected embryos with ectopic head structure (n=55, 96% with ectopic heads). (L) IGF2 and Cerberus coinjection induced a large ectopic head (n=55, 100% with ectopic heads). Scale bar, 500 µm.
Supplemental Fig. S2. Cerberus mRNA induces radial brains in β-catenin MO ventralized embryos after single or radial injection.
(A) Uninjected control embryo at stage 29 (n=60). (B) Sibling after 4 times radial injection of β-catenin MO (34 ng total) showing a completely ventralized phenotype (n=37), arrowhead indicates the blastopore. (C) Radial β-catenin MO followed by a single ventral injection of Cerberus mRNA (100 pg) generated an embryo with brain and a circular cement gland at the opposite end of the closed blastopore (arrowhead) (n=29, all radially dorsalized). (D) Radial injection of β-catenin MO (34 ng total) together with Cerberus mRNA (400 pg total) induced radial structures surrounded by cement glands (n=17, all radially dorsalized). Scale bar, 500 µm.
