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First published online May 5, 2004
doi: 10.1242/10.1242/dev.01133

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Patterning the forebrain: FoxA4a/Pintallavis and Xvent2 determine the posterior limit of Xanf1 expression in the neural plate

Natalia Martynova^1,*, Fedor Eroshkin^1,*, Galina Ermakova¹, Andrey Bayramov¹, Jessica Gray², Robert Grainger^2, and Andrey Zaraisky^1,

¹ Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences, Moscow, Russia
² University of Virginia, Department of Biology, Charlottesville, VA 22904, USA

View larger version (39K): [in a new window]   Fig. 1. A 14 bp regulatory element in the Xanf1 promoter restricts of Xanf1 expression to the presumptive forebrain. (A) Brain of a transgenic embryo bearing the double-reporter vector shown at the top demonstrates similar expression patterns of EGFP (green) and RFP (red) driven by promoter fragments -203 to +1 and -2200 to +1 respectively. (B) Deletion of a 14 bp promoter element (yellow), from the EGFP reporter cassette (top) results in the posterior shift in the EGFP reporter expression in the CNS of another transgenic embryo bearing this vector (bottom). RFP expression driven by the complete promoter fragment, -2200 to +1, is still localised in the forebrain (middle).

View larger version (67K): [in a new window]   Fig. 2. Expression domains of Xanf1, FoxA4a/Pintallavis and Xvent2 occupy complementary areas within the neural plate. Embryos at the early neurula stage are shown from the anterior, with the dorsal side upwards. (A) Expression of Xanf1 in the anterior neuroectoderm. (B) Expression of Xvent2 is localised within lateral parts of the neural plate, behind the expression domain of Xanf1 (marked by the broken red line). (C) FoxA4a/Pintallavis is expressed along the midline of the neural plate. The anterior tip of the FoxA4a/Pintallavis expression domain borders on the medial part of the posterior margin of the Xanf1 expression area (marked by the broken red line). (D,E) Double in situ hybridisation with probes to Xanf1 and Xvent2 (D) or Xanf1 and FoxA4a/Pintallavis (E) mRNAs demonstrates lack of gaps between expression domains of these genes. (F) Diagram of complementary areas occupied by the expression domains of Xanf1, FoxA4a/Pintallavis and Xvent2 within the neural plate.

View larger version (61K): [in a new window]   Fig. 3. Electrophoretic mobility shift assays demonstrating binding of FoxA4a/Pintallavis and Xvent2 with 14 and 22 bp elements from the Xanf1 promoter. (A,B) The reaction mixtures are as follows: lane 1, extract from oocytes microinjected with EGFP mRNA (control); lane 2, extract from oocytes microinjected with mRNA of FoxA4a/Pintallavis (A) or Xvent2 (B) diluted ten times with extract from oocytes microinjected with EGFP mRNA; lane 3, undiluted extract from oocytes microinjected with FoxA4a/Pintallavis (A) or Xvent2 (B) mRNA. (C) Lane 1, extract from oocytes microinjected with EGFP mRNA (control); lanes 2-4, extracts from oocytes microinjected with FoxA4a/Pintallavis mixed with normal (b) or two mutant (a,c) variants of the 14 bp element. Sequences of these variants are shown at the bottom of C. Sites of point mutations are underlined. (D) Core binding sites are indicated by the black and red lines for FoxA4a/Pintallavis and Xvent2, respectively; positions of binding sites located on the opposite DNA strand are underlined; possible binding site for FoxA4a/Pintallavis, which has one mismatch with the canonical binding site, is indicated by a broken line.

View larger version (108K): [in a new window]   Fig. 4. Effects exerted by ectopic expression of FoxA4a/Pintallavis and Xvent2, and repression and activation constructs on the expression of endogenous Xanf1. All embryos were microinjected with a mixture of FLD and mRNA. (AD) Ectopic expression of EnRFoxA4a/Pintallavis and EnRXvent fusions elicit inhibition of Xanf1 expression. Microinjection of mRNA: FoxA4a/Pintallavis (A,A'); Xvent2 (B,B'); EnR FoxA4a/Pintallavis (C,C'); EnRXvent2 (D,D'). (B1,B1',B2,B2') Representative histological sections at two sagittal levels (1 and 2) of the embryo shown in B and B' demonstrate that descendants of the microinjected blastomeres labelled with FLD are primarily localised in the neurectoderm. Only single labelled cells are found in underlying endomesoderm. (E-G) Ectopic expression of dominant-negative versions of FoxA4a/Pintallavis and Xvent2 result in posterior expansion of endogenous Xanf1 expression. Red and black arrows indicate position of the posterior border of the Xanf1 expression domain within microinjected and non-microinjected (normal) zones, respectively. Microinjection of mRNA: VP-16-FoxA4a/Pintallavis (E,E'); VP16-Xvent2 (F,F'); Xvent2-P40 (G,G'). (E1,E1') Representative histological section at level 1 of the embryo shown in E and E' illustrates that microinjected blastomeres primarly populate the neurectoderm and that endogenous Xanf1 is now expressed in these cells.

View larger version (68K): [in a new window]   Fig. 5. (A,B) Microinjections of anti-FoxA4a/Pintallavis (A,A') and anti-Xvent2 (B,B') morpholinos elicit short-range posterior expansion of the Xanf1 expression. In the case of the anti-sense to FoxA4a/Pintallavis, the expression of Xanf1 is expanded toward the posterior only along the midline of the neural plate, i.e. just in the region where the endogenous FoxA4a/Pintallavis is expressed (A, red arrows). No expansion of the Xanf1 expression was seen in the morpholino-containing cells occupying lateral parts of the neural plate (the upper rows of embryos in A and A'). By contrast, only lateral expansion of the Xanf1 expression was observed in embryos microinjected with the anti Xvent2 (B,B') morpholino and these embryos demonstrated normal expression of Xanf1 in the midline of the neural plate. (C) The mixture of anti-FoxA4a/Pintallavis morpholino and a synthetic FoxA4a/Pintallavis mRNA can interfere with the posterior expansion of endogenous Xanf1 expression elicited by the morpholino alone. (D) To monitor the effectiveness of CHX on suppression of protein synthesis, we microinjected Xenopus embryos at the two blasomere stage with plasmids expressing luciferase under the control of the CMV (commercial pGL3 vector, Promega) or the `full-length' Xanf1 promoter (Eroshkin et al., 2002). CHX was added to one half of the microinjected embryos at the midblastula transition (CMV-Luc) or at the midgastrula stage (Xanf1-Luc). After 2 hours of incubation, luciferase activity was measured in extracts from three independently collected groups of embryos (10 embryos in each group) treated and not treated with CHX. (E,F) Experiments with the hormone-inducible versions of FoxA4a/Pintallavis and Xvent2 under conditions of inhibition of protein synthesis by cycloheximide demonstrate that Xanf1 is the direct target of these transcription factors. The dexamethasone (DEX) treatment of embryos subsequently elicits activation of VP16-FoxA4a/Pintallavis-BDGR (E,E') or VP16-Xvent2-BDGR (F,F'), which under those conditions can activate transcription of only their own direct genetic targets, in particular, the transcription of Xanf1. Although only short-distance posterior expansion of the Xanf1 expression was observed in the case of VP16-FoxA4a/Pintallavis-BDGR (E, red arrows), much broader spreading of the expression was seen in embryos microinjected with VP16-Xvent2-BDGR mRNA (F, red arrows).

View larger version (18K): [in a new window]   Fig. 6. Xanf1 regulation byFoxA4a/Pintallavis and Xvent2 is consistent with Nieuwkoop's two-signal model of neural induction. (A) According to the Nieuwkoop model, anterior potentials induced during the first step of the neural induction throughout the neurectoderm are realised only in the anterior part of the neural plate (red) because of suppression by a transforming signal in the posterior part of the plate (grey). (B) Expression of Xanf1 is activated by Otx2, SoxD and possibly other transcription factors throughout the neural plate. However, during normal development the expression of Xanf1 is inhibited in the posterior part of the neural plate because of the inhibitory influence of FoxA4a/Pintallavis, Xvent2 and some as yet unidentified factors.