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We have previously shown that the Gsx family homeobox gene Gsh2 is part of the regulatory network specifying dorsoventral pattern of primary neurons in the developing amphibian embryo. Here, we investigate the role of Gsx transcription factors in regulating the transcription of Iroquois family homeobox genes in the amphibian neural plate. Iroquois genes are key regulators of neural patterning and their expression is coincident with that of the Gsx genes during open neural plate stages. We show that Gsx proteins repress Iroquois expression in the embryo and conversely, inhibition of Gsx activity with either antisense morpholino oligos or an anti-morphic Gsx protein up-regulates Iroquois expression. These data indicate that Gsx factors act as negative regulators of Iroquois gene expression in the amphibian neural plate and support a model in which the Gsx proteins promote neuronal differentiation by repressing the expression of known inhibitors of neuronal differentiation such as Iro3.
Fig. 1. Phenotypic effects of Gsh2 and Gsh2 fusion proteins. A-C: Phenotypes at stage 41 of X. tropicalis control embryos (A), and embryos injected with (B) 10 pg Gsh2, or (C) 50 pg Gsh2-EnR mRNA. D,E: Phenotypes at stage 33 of (D) control embryos, and (E) embryos injected into the leftventralblastomere with 15 pg of Gsh2-VP16 mRNA. All embryos are with anterior to the left and dorsal to the top. F, G: Section of an embryo from (E), at the level of Gsh2-VP16-induced partial secondary axis, histologically stained with borax carmine and picro blue-black. Nuclei stain red, cells green, and axons turquoise. nc, notochord; nt, neural tube. White arrows indicate partial secondary axes. Red arrows indicate ectopic neural tissue.
Fig. 2. Effects of Gsx proteins on bone morphogenetic protein (BMP) signaling. A: Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of expression of BMP4 and BMP7 in stage 14 X. laevis control embryos or embryos injected with mRNAs encoding Gsh2 or Gsh2-VP16 as indicated. B,C: Western blot analysis of stage 11 X. laevis embryos injected with mRNAs as indicated, and blotted with antibodies against PhosphoSmad1/5/8 and GAPDH as a loading control. B: Gsh2 and Gsh2 fusion proteins. C: Gsh1 and Gsh1 fusion proteins. Noggin is used as a control for BMP signaling inhibition.
Fig. 3. Repression of Iroquois gene expression by Gsx proteins. A: Quantitative-polymerase chain reaction(Q-PCR) analysis of Gsh2 expression during early development. Levels are normalized to ornithine decarboxylase (ODC) expression and are shown relative to expression at Nieuwkoop and Faber (NF) stage 16. B: Western blot analysis of stage 11 X. laevis embryos injected with mRNAs as indicated, and blotted with antibodies against PhosphoSmad1/5/8 and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as a loading control. C-F: In situ hybridization analysis of (C,D) Iro2 and (E,F) Iro3 expression in neurula stage 14 X. tropicalis embryos unilaterally injected with (C,E) 20 pg of Gsh1 mRNA and (D,F) 5 pg Gsh2,mRNA. Dorsal views, anterior to left, injected side at top and marked with asterisk). Injected region indicated by b-galactosidase co-injection and red-gal staining (pink). G-J: Expression of (G,H) Iro2 and (I,J) Iro3 in animal cap explants from (G,I) control X. laevis embryos and (H,J) embryos injected with 40 pg Gsh2-VP16 mRNA. K-N: In situ hybridization analysis of (K,M) Iro2 and (L,N) Iro3 expression in neurula stage 14 X. tropicalis embryos unilaterally injected with (K,L) 15pg Gsh2-VP16-GR mRNA with dexamethasone treatment with stage 11or (M,N) Gsh2- and Gsh1-antisense morpholinos. O: Reverse transcriptase-PCR (RT-PCR) analysis of expression of Iro3 in animal cap explants taken from uninjected X. laevis control embryos or embryos injected with 20 pg of Gsh2-VP16-GR, and treated with cycloheximide (CHX) and/or dexamethasone (DEX) as indicated.
Fig. 4. A model for promotion of neuronal differentiation by Gsx proteins.
