Ermakova GV et al. (1999), The homeobox gene, Xanf-1, can control both neu...

XB-ART-12285

Development 1999 Oct 01;12620:4513-23. doi: 10.1242/dev.126.20.4513.

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The homeobox gene, Xanf-1, can control both neural differentiation and patterning in the presumptive anterior neurectoderm of the Xenopus laevis embryo.

Ermakova GV, Alexandrova EM, Kazanskaya OV, Vasiliev OL, Smith MW, Zaraisky AG.

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From the onset of neurectoderm differentiation, homeobox genes of the Anf class are expressed within a region corresponding to the presumptive telencephalic and rostral diencephalic primordia. Here we investigate functions of the Xenopus member of Anf, Xanf-1, in the differentiation of the anterior neurectoderm. We demonstrate that ectopic Xanf-1 can expand the neural plate at expense of adjacent non-neural ectoderm. In tadpoles, the expanded regions of the plate developed into abnormal brain outgrowths. At the same time, Xanf-1 can inhibit terminal differentiation of primary neurones. We also show that, during gastrula/neurula stages, the exogenous Xanf-1 can downregulate four transcription regulators, XBF-1, Otx-2, Pax-6 and the endogenous Xanf-1, that are expressed in the anterior neurectoderm. However, during further development, when the exogenous Xanf-1 was presumably degraded, re-activation of XBF-1, Otx-2 and Pax-6 was observed in the abnormal outgrowths developed from blastomeres microinjected with Xanf-1 mRNA. Other effects of the ectopic Xanf-1 include cyclopic phenotype and inhibition of the cement gland, both by Otx-2-dependent and -independent mechanisms. Using fusions of Xanf-1 with the repressor domain of Drosophila engrailed or activator domain of herpes virus VP16 protein, we showed that most of the observed effects of Xanf-1 were probably elicited by its functioning as a transcription repressor. Altogether, our data indicate that the repressor function of Xanf-1 may be necessary for regulation of both neural differentiation and patterning in the presumptive anterior neurectoderm.

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Species referenced: Xenopus laevis
Genes referenced: ag1 foxg1 hesx1 ncam1 nog nppa otogl2 otx2 pax6 twist1

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	Fig. 1. (A) At the late gastrula stage, Xanf-1 is expressed in the anterior neurectoderm. Xenopus embryo hybridized in whole-mount is shown from the anterior, dorsal side up. (B) Before DEX application, GFP-Xanf-1-BDGR is uniformly distributed into the ectodermal cells of the early gastrula embryo. (C) 30 minutes after DEX treatment of the embryo shown in B, GFP-Xanf-1-BDGR is accumulated into cell nuclei. (D) Expansion of the neural plate in cranial region of the midneurula embryo microinjected with GFP-Xanf-1- BDGR mRNA. The expanded area at the right half of the neural plate (left to the viewer) is occupied by PGLC containing the hybrid protein. Note lateral displacement of the cranial neural fold (arrowheads) at the microinjected side. Lateral borders of the neural plate are marked by dotted lines. (E) Whole-mount in situ hybridization with NCAM probe. The neural plate is expanded at the right side of the midneurula embryo microinjected with GFP-Xanf-1-BDGR mRNA. (F) The same embryo as in E, shown alive under epifluorescence. (G) Transverse section of embryo shown in E demonstrates enlargement of the right half of the neural plate. (H) Inhibition of the expression of Xtwist in the cranial neural fold (arrow) of the midneurula embryo microinjected with GFP-Xanf-1-BDGR mRNA. (I) The dorsal limit of expression of epidermal marker, Xep-1, is shifted ventrally at the right side of the embryo microinjected in the right animal blastomere with GFP-Xanf-1-BDGR mRNA and treated by DEX. The position of the midline is marked by dashed line. (J,K) Animal cap explants of embryos microinjected with GFP-Xanf-1-BDGR mRNA or noggin mRNA and hybridized with NCAM probe at the 32 stage equivalent. No signals were observed in GFP-Xanf-1-BDGR-containing explants treated with DEX (J), in comparison with those microinjected with noggin mRNA (K). (L) In situ hybridization with type II b- tubulin mRNA probe showing suppression of primary neurones (arrowhead) on the microinjected side of the late neurula embryo.
	Fig. 2. (A) Brain of stage-47 normal embryo. Dorsal view. Abbreviations: tel, telencephalon; di, diencephalon; mes, mesencephalon; hind, hindbrain. The telencephalic (B) and mesencephalic (C) outgrowths (arrowheads) developed in embryos microinjected with GFP-Xanf-1-BDGR mRNA and treated by DEX. (D,E) GFP label in the telencephalic outgrowth (arrowhead) of the stage-47 embryo microinjected with the mixture of GFP and GFPXanf- 1-BDGR mRNAs and treated by DEX under white and UV light, respectively. (F,G) No brain abnormalities were observed in the embryo microinjected with the same mixture, but not treated by DEX, despite high concentration of the exogenous proteins in brain cells (arrowhead).
	Fig. 3. (A-H). Whole-mount in situ hybridization with digoxigenin-labelled probes of Xanf-1 (A-C), Otx-2 (D-F), Pax-6 (G-I) and XBF-1 (J-L) mRNAs. Embryos shown from the anterior, dorsal side up, are at the late gastrula (stage 12-12.5), midneurula (stage 14-15) and late neurula (stage 18-19), respectively. External and internal borders of the neural folds of embryos at the 15 stage are marked by dotted lines.
	Fig. 4. (A,B) Expression of Xanf-1 at the late gastrula and midneurula stages, respectively, as seen on sagittal sections. Dorsal side up, anterior to the right. At both stages, the Xanf-1 expression domain in the outer layer of neurectoderm has a very sharp boundary (arrowhead) with the Xanf-1 non-expression domain. (C,D) Expression of Otx-2 at the late gastrula and midneurula stages, respectively. Whereas at the end of gastrulation Otx-2 is expressed throughout the anterior neurectoderm (C), by the midneurula stage its expression is ceased in the region superimposed with the Xanf-1 expression domain. (E) At the end of gastrulation, XBF-1 is expressed in a single band of cells, just beneath the superficial layer of the anterior neurectoderm. (F) During early neurula stage, the expression of XBF-1 is extended posteriorly in the superficial cell layer, where the second expression band is formed. (G) Schematic diagram of the Xanf-1, Otx-2 and XBF-1 expression patterns on sagittal sections of the midneurula.
	Fig. 5. (A,B) Inhibition of the Otx-2 expression in the early neurula embryo microinjected with GFP-Xanf-1-BDGR mRNA. The same embryo is shown after whole-mount in situ hybridization with Otx-2 mRNA probe (A), and alive under epi-fluorescence (B). Note, that the band-shaped area where Otx-2 expression is abnormally repressed (arrowhead) corresponds to the location of PGLC. (C-J) Ectodermal grafts shown by arrowheads were homotopically transplanted at the early gastrula stage from the embryo microinjected with mixture of GFPXanf- 1-BDGR and GFP mRNAs to the non-injected embryo. (C) Note much lower levels of Pax-6 expression in the graft in comparison with the symmetrical zone in the left side of embryo. (D) The same embryo as in C is shown alive under epi-fluorescence. The contour of the embryo is shown by dashed line. (E) In the embryo untreated by DEX, cells of the graft intensively express Pax-6. (F) The same embryo as in E is shown under epi-fluorescence. (G,H) Inhibition of the endogenous Xanf-1 in the grafted cells and epi-fluorescent view of the graft, respectively. (I) No inhibition of endogenous Xanf-1 is seen in the graft of the embryo untreated by DEX. (J) Inhibition of XBF-1 in the graft after DEX treatment. (K,L) Though XBF-1 is downregulated within the territories occupied by PGLC during neurulation, in further development its expression is reactivated (L), to the level even higher than that of the untreated embryos (K). Embryos hybridized at stage 26 with XBF-1 probe are shown from the anterior, dorsal side up. In the DEX-treated embryo (L), the expression of XBF-1 is expanded ventrally, over the cement gland territory. In this embryo, the cement gland differentiation was suppressed by exogenous Xanf-1 (see Fig. 8 for the details), and only three small patches of cement gland cells remain.
	Fig. 6. Exogenous Xanf-1 can impose telencephalic identity on cells of the dorsal part of abnormal diencephalic outgrowths, but not on cells of the outgrowths located in more posterior regions. All embryos were microinjected at the 8-cell stage in the right dorsal blastomere with mixture of GFP-Xanf-1-BDGR and GFP mRNA. DEX was applied at the midgastrula stage. (A) In the DEX-untreated embryo, XBF-1 is expressed exclusively in the telencephalon. (B) In the DEX-treated embryo, an additional site of the XBF-1 expression (arrowhead) is seen in the dorsal part of the abnormal diencephalic outgrowth. (C) In contrast, no expression of XBF-1 is seen in the mesencephalic outgrowth (arrowhead). (D-F) Hybridization with the probe for another telencephalic marker, Emx-1 also demonstrates that exogenous Xanf-1 is able to induce telencephalic differentiation in the diencephalic outgrowth (E, arrowhead), but not in the mesencephalic one (F, arrowhead). (G,H) The expression of Pax-6, which is normally observed in the diencephalon and telencephalon (G), but not in the mesencephalon, is not induced by exogenous Xanf-1 in the mesencephalic outgrowth (H, arrowhead). (I-L) Otx-2, which is normally expressed in the di- and mesencephalon (I), in microinjected embryos is also intensively expressed in the mesencephalic outgrowth (L, arrowhead). (J,K) The mesencephalic outgrowth shown in L is composed of PGLC derived from the graft containing GFP-Xanf-1-BDGR mRNA.
	Fig. 7. (A,B). The cyclopic phenotype, characterised by eye fusion and reduction of the forebrain, is observed in the tadpoles when PGLC containing GFP-Xanf-1-BDGR mRNA cross the anterior neural fold (A). (C) The control embryo in which PGLC did not cross the anterior neural fold. (D-F) Transplantations of small ectodermal grafts from microinjected to non-injected embryos reveal that the cyclopic phenotype develops only when grafts occupy the anterior margin of the anterior neural fold (E), but not if they are located more posteriorly (D) or anteriorly (F).
	Fig. 8. Exogenous Xanf-1 can inhibit differentiation of the cement gland. All embryos at the tailbud stage are shown from ventral side, anterior to the top. (A-E) Inhibition of the cement gland in the embryos microinjected at the 8-cell stage with GFP-Xanf-1-BDGR mRNA in both dorsal blastomeres. (A) Almost complete suppression of the cement gland differentiation in the embryo whose head area is occupied by PGLC. (B) A very small patch of the cement gland cells is still formed just in the place where GFP labelling is absent (arrow). (C,D) In the control embryo microinjected with GFP-Xanf-1-BDGR mRNA but untreated by DEX, no signs of the cement gland inhibition are seen (C) despite wide expansion of PGLC in head area (D). (E) Hybridization with cement gland marker XAG-1 probe shows that several small patches of the cement gland cells differentiated in the embryo microinjected with GFP-Xanf-1-BDGR mRNA and treated by DEX (left), instead of a big single cement gland in the DEX-untreated embryo (right). (F,G) Transplantations of small ectodermal grafts from embryos microinjected with GFP-Xanf-1-BDGR mRNA to non-injected embryos. (F) The cement gland differentiation is inhibited in the central part of the normal cement gland area occupied by the DEX-treated graft. Two small cement glands (arrowheads) develop from those parts of the presumptive cement gland territory that are not occupied by the graft. (G) Epi-fluorescent view of the embryo shown on F reveals grafted cells. (H) Hybridization with the epidermal marker Xep-1 probe demonstrates that in the embryo shown in F and G, cells of the graft acquire epidermal fate. (I-L) Xanf-1 can suppress the cement gland differentiation induced by Otx-2. (I). The cement gland differentiation in the control embryo is revealed by XAG-1 probe. (J) Activation of GFP-Xanf-1-BDGR in embryos microinjected with the mixture of Otx-2 and GFP-Xanf-1-BDGR mRNAs results in severe reduction of the XAG-1 expression, despite the presence of Otx-2. (K,L) In animal caps isolated at the late blastula stage from embryos microinjected with the mixture of Otx-2 and GFP-Xanf-1-BDGR mRNAs, exogenous Xanf-1 demonstrates the same epistatic effect over Otx-2 as observed in the whole embryos. (K) An intense expression of XAG-1 under the influence of Otx-2 in the animal caps not treated by DEX. (L) Activation of GFP-Xanf-1-BDGR in treated caps represses the XAG-1 expression.
	Fig. 9. (A-F) Microinjections of EnR-Xanf-1-BDGR mRNA (mixed with GFP mRNA) elicit the effects similar to those caused by exogenous Xanf-1. (A) Hybridization with NCAM probe demonstrates expansion of the neural plate on the microinjection side. (B) The abnormal diencephalic outgrowth in the microinjected embryo contains cells expressing the telencephalic marker, XBF-1 (arrow). (C,D) Inhibition of the Otx-2 expression (C) within the territory occupied by PGLC (D). (E,F) Inhibition of the cement gland differentiation in the medial region of cement gland territory occupied by PGLC, results in splitting into two separate cement glands. (G-L) In contrast to EnR-Xanf-1-BDGR, VP16-Xanf-1-BDGR injected into the right side of the embryo produces some effects opposite to those elicited by Xanf-1 alone: downregulation of NCAM (arrowhead G), expansion of the right Xtwist expression domain (arrowhead H), the ectopic expression of Otx-2 (arrowheads I,J), cement gland differentiation (K,L) observed in the regions occupied by cells containing the activated VP16-Xanf-1-BDGR.