July 15, 2007;
The homeodomain factor Xanf represses expression of genes in the presumptive rostral forebrain that specify more caudal brain regions.
Early development of the rostral forebrain
(RF) in vertebrates is accompanied by the inhibition of two homeobox regulators, Otx2
in the rostral sector of the anterior
neural plate, further giving rise to the RF. However, the precise molecular mechanism and meaning of this inhibition is still obscure. We now demonstrate that the activity of the Anf
homeodomain protein is necessary and sufficient for the anterior
inhibition of Otx2
. Specifically, we show that knockdown of the Xenopus laevis Anf
, by antisense morpholino oligonucleotides results in the anterior
expansion of Otx2
expression into the presumptive RF territory. Furthermore, by overexpressing hormone-inducible activator- and repressor-fused variants of Xanf
in the absence of protein synthesis, we present evidence that Xanf
can directly downregulate Otx2
but not the more rostrally expressed Bf1
, Bf2, Fgf8
and Nkx2.4. These results explain how the inhibitory activity of Xanf
can discriminate RF regulators in favor of posterior forebrain
ones. Assuming that the Anf
type of homeobox is specific for vertebrates, our data suggest that the emergence of Anf
in evolution could be a critical event for RF development in vertebrates through the elimination of homologues of modern posterior forebrain
regulators from the rostral sector of the anterior
[+] show captions
Fig. 1. Effects of anti-Xanf MO on expression of different marker genes in the neural plate of Xenopus laevis midneurula embryos (stage 14). (A–J) Expression of Hox9, Wnt8, Otx2, Pax6, Six3 and Engrailed2 (in situ hybridization with the mixture of probes), Xanf, Bf2 and Engrailed2 (in situ hybridization with the mixture of probes), Bf1, Fgf8 and Xanf respectively in control embryos. (A1–J1) Expression of the aforementioned markers in embryos microinjected with anti-Xanf MO. Note the anterior expansion of expression of the posterior forebrain markers (Otx2 and Pax6) in contrast to inhibition of the rostral ones (Bf1, Bf2, Fgf8). (A1′–J1′) Distribution of FLD tracer in embryos shown in panels A1–J1 is shown in fluorescence. (A2–J2 and A3–J3) Schematic diagrams of expression domains (purple) of the aforementioned marker genes in control (A2–J2) and experimental (A3–J3) embryos are shown in comparison with the normal expression domain of Xanf (pink). All embryos are shown from the anterior, dorsal side up. Approximate position of the anterior margin of the neural plate is indicated by the red dotted line.
Fig. 5. Expression of different markers in brains of tadpoles (stages 46–47) developed from embryos microinjected with anti-Xanf MO. Note the significant reduction of the telencephalon and the anterior (ventral) diencephalic structures, which include infundibulum (inf), hypothalamus (hth), prethalamus (pth) and zona limitance intrathalamica (zli). At the same time, the more posterior brain regions, including tectum (t) and thalamus (th), appear normal.
Fig. 2. Abnormal expression of the posterior and anterior forebrain markers is preserved in late tailbud-stage embryos (stages 26–28) microinjected with anti-Xanf MO. (A–E) Expression of Otx2, Pax6, Bf1, Nkx2.4 and Xanf in control embryos. (F) Expression of Nkx2.4 and Xanf in the same control embryo as revealed by double in situ hybridization with mixed probes to Nkx2.4 and Xanf mRNA. (A1 and B1) Expression of Otx2 (A1) and Pax6 (B1) in embryos microinjected with anti-Xanf MO demonstrates an anterior expansion that is especially pronounced in the case of Otx2. (C1 and D1) Expression of Bf1 (C1) and Nkx2.4 (D1) is inhibited in embryos microinjected with anti-Xanf MO. (E1) In contrast to Bf1 and Nkx2.4, Xanf appears to be overexpressed when its translation is inhibited by anti-Xanf MO. (F1) Anti-Xanf MO exerts an opposite effect on the expression of Nkx2.4 and Xanf. (A1′–F1′) Distribution of FLD tracer in embryos shown in panels A1–F1. All embryos are shown from the anterior, dorsal side up. Red dotted line indicates rostral margin of the neural tube as seen from the anterior. Abbreviations: cg—cement gland; p—pituitary; tel—telencephalon.
Fig. 6. Anterior expansion of Otx2 expression elicits abnormalities similar to those generated by anti-Xanf MO. (A, C and E) Expression of Bf1, Fgf8 and Pax6 in control midneurula embryos (stage 14). (B, B′, D, D′ and F, F′) In embryos microinjected with Otx2 mRNA into animal dorsal blastomeres at the 8-cell stage, Bf1 and Fgf8 are strongly inhibited, while expression of Pax6 is expanded towards the medial sector of the anterior neural plate. (G and J) In contrast to the control (J), the experimental (G) tadpole (stages 46–47) that developed from the embryo microinjected with Otx2 mRNA has a phenotype resembling the phenotype of tadpoles which developed from embryos microinjected with anti-Xanf MO (compare with Fig. 4B). (H) Transgenic embryo (stage 14) bearing the double-cassette vector (pXanf-Otx2-pCardActin-RFP) that targets expression of Otx2 towards the area, in which expression of the endogenous Otx2 is normally inhibited under the influence of Xanf, demonstrates the reduction of the telencephalic marker, Bf1, expression. (K) Control transgenic embryo (stage 14) expressing RFP under the control of the cardiac actin promoter demonstrates normal expression of Bf1. Note that RFP expression corresponding to two longitudinal stripes of somite mesoderm and revealed by in situ hybridization, along with the expression of Bf1 in embryos shown in panels H and K, confirm transgenic status of both embryos. (I and L) Tadpole (stages 46–47) that developed from transgenic embryos bearing pXanf-Otx2-pCardActin-RFP demonstrates a phenotype (I) similar to that of tadpoles that developed from embryos microinjected with anti-Xanf MO or Otx2 mRNA (compare with Fig. 4B and panel G of this figure). At the same time, control transgenic embryos bearing pCardActin-RFP construct demonstrate a normal phenotype (L).