Bourguignon C et al. (1998), XBF-1, a winged helix transcription factor with...

XB-ART-13990

Development 1998 Dec 01;12524:4889-900. doi: 10.1242/dev.125.24.4889.

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XBF-1, a winged helix transcription factor with dual activity, has a role in positioning neurogenesis in Xenopus competent ectoderm.

Bourguignon C , Li J , Papalopulu N .

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Neuronal differentiation in the vertebrate nervous system is temporally and spatially controlled by mechanisms which are largely unknown. Here we investigate the role of XBF-1, an anterior neural plate-specific winged helix transcription factor, in controlling the pattern of neurogenesis in Xenopus ectoderm. We show that, in the anterior neural plate of normal embryos, prospective neurogenesis is positioned at the anterior boundary of the XBF-1 expression domain. By misexpressing XBF-1 in the posterior neural plate we show that a high dose of XBF-1 has a dual effect; it suppresses endogenous neuronal differentiation in high expressing cells and induces ectopic neuronal differentiation in adjacent cells. In contrast, a low dose of XBF-1 does not suppress but instead, expands the domain of neuronal differentiation in the lateral and ventral sides of the embryo. XBF-1 regulates the expression of XSox3, X-ngnr-1, X-Myt-1 and X-&Dgr;-1 suggesting that it acts early in the cascade leading to neuronal differentiation. A fusion of XBF-1 to a strong repressor domain (EnR) mimics most of the XBF-1 effects suggesting that the wild type XBF-1 is a transcriptional repressor. However, fusion of XBF-1 to a strong activation domain (E1A) specifically suppresses neuronal differentiation suggesting that XBF-1 may also work as a transcriptional activator. Based on these findings, we propose that XBF-1 is involved in positioning neuronal differentiation by virtue of its concentration dependent, dual activity, as a suppressor and an activator of neurogenesis.

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Species referenced: Xenopus
Genes referenced: dll1 en2 foxg1 gal.2 igh mst1 myc neurog2 otx2 sox3 tubb2b

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	Fig. 2. Expression pattern of XBF-1, in relation to the expression of N-tubulin, X-Delta-1 and X- ngnr-1. (A) Expression of XBF-1 in stage 15, 32 and 35 embryos, lateral views, dorsal up. (B) Lateral view and (C) horizontal section, (plane of section indicated by a broken line in B), of a stage 35 embryo hybridised with N- tubulin (magenta) and XBF-1 (light blue). High XBF-1 expression is restricted to the telencephalon and olfactory placodes. N-tubulin expression is localised towards the mantle while XBF-1 is localised towards the ventricular area of the neural tube. (D-H) Expression on the anterior neural plate of (D) X-Delta-1, (E) XBF- 1, (F) XBF-1 (light blue) and X-Delta-1 (magenta, arrow), (G) X-ngnr-1 (arrow) and En-2 (arrowhead) (H) XBF-1(light blue) and X-ngnr-1 (magenta, arrow). Expression of X-Delta-1 in the anterior neural plate occurs in an anterior and a posterior curved stripe and that of X-ngnr-1 in two bilateral patches. The anterior stripe of X-Delta-1 (arrow) and the patches of Xngnr-1 expression (arrow) are positioned around the anterior edge of the XBF-1 expression domain. di, diencephalon; FB, forebrain; HB, hindbrain; MB, midbrain; op, olfactory placode; tel, telencephalon.
	Fig. 3. Misexpression of XBF-1 and qin suppresses endogenous and induces ectopic primary neurogenesis. Embryos were injected with XBF-1/lacZ (A-G,K), qin/lacZ (H-J) RNA, or were uninjected (L) and were processed for X-gal staining (light blue) and whole-mount in situ hybridisation for N-tubulin (purple). Dorsal (A,C,I,L) and side (B,D,E,J,K) views are shown, anterior to the right. Black arrowheads connect dorsal and lateral views of the same embryos. The lateral views show that the ectopic N-tubulin forms far from the dorsal midline at the lateral and even ventral side of the embryo, outside the XBF-1/lacZ-expressing ectoderm but at the boundary of the high expressing ectoderm. Similarly, XBF-1/lacZ injection of one blastomere of a 32 cell stage embryo (K), produces ectopic N-tubulin (arrow in K) at the boundary of the high XBF- 1/lacZ-expressing patch. (F) The XBF-1-injected side and (G) the uninjected control side of a tadpole stage embryo. In E and F, note that the ectopic N-tubulin stripe follows the boundary of X-gal staining and has formed perpendicular, rather than the normal parallel, to the antero- posterior axis. (H) A high magnification view of the lateral side of an embryo similar to the one shown in (D); it shows that there is no overlap between the high XBF-1/lacZ-expressing cells (blue) and the N-tubulin-expressing cells (brown/purple). The X-gal staining is nuclear while the in situ signal is cytoplasmic. In L a white broken line indicates the dorsal midline of the neural plate and separates the three bilaterally symmetrical stripes of N-tubulin expression. a, anterior; p, posterior.
	Fig. 4. A high concentration of XBF-1 suppresses endogenous and induces ectopic N-tubulin while a low concentration only induces additional N-tubulin. Embryos were injected with XBF-1 RNA, lacZ RNA (control), a mixture of XBF-1 and lacZ RNA or XBF-1-myc RNA at a high (0.5 ng) or low (90 pg) concentration and processed by in situ hybridisation for N-tubulin (purple), either alone (XBF-1), or combined with X-gal staining, (light blue; XBF-1/lacZ and lacZ) or with myc antibody staining, (orange; XBF-1-myc). In all panels, anterior is to the right and injected side towards the lower end. The left and right panels show dorsal views while the middle panel shows lateral views of the embryos shown on the left. At high concentrations ectopic tubulin forms at the boundary of the XBF-1- expressing ectoderm (light blue in embryos co-injected with lacZ and orange in embryos injected with XBF-1-myc) while at low concentrations ectopic (i.e. supernumerary) N-tubulin forms within the XBF-1-expressing ectoderm.
	Fig. 5. XBF-1 regulates the expression of X-Myt-1 and X-ngnr-1. Embryos injected with XBF-1/lacZ (A-F) and qin/lacZ (G,H) or lacZ (J) RNA and analysed for X-Myt-1 (A-E and G-J) and X-ngnr-1 expression (F). Black arrowheads connect dorsal and lateral or ventral views of the same embryos. (A,B,D,E,G,H) represent the igh dosephenotype, where X-Myt-1 is suppressed over the X-gal stained ectoderm but ectopically induced in adjacent cells. In G and J a black dotted line indicates the dorsal midline. The lateral stripe of X-Myt-1 expression (arrow in G and J) appears at a great distance from the dorsal midline in experimental embryos (G) compared to controls (J). A lateroventral view (H) of the embryo shown in (G) shows that X-Myt-1 expression formed along the boundary of the ectoderm that stained highly and uniformly with X-gal and in a punctate pattern outside it. C and F represent the low dose phenotype, where the normal expression domain of X-Myt-1 and X- ngnr-1 is greatly expanded on the injected side. The expression of X- ngnr-1 at high concentrations of XBF-1 or qin was not determined.
	Fig. 6. High XBF-1-expressing cells do not overlap with N-tubulin-positive cells. (A-H) Transverse sections through embryos injected with high and low concentrations of XBF-1/lacZ of XBF-1-myc as indicated. Staining for X-gal (light blue) and myc (orange) is nuclear while the in situ signal for N-tubulin (purple) is cytoplasmic. In all panels a solid line passes through the notochord and indicates the dorsal midline. (B,D,F,H) High magnification views of the lateroventral side of the embryo, therefore N-tubulin shown in these frames is ectopic. At the high XBF-1 concentration (B), broken lines delimit a cluster of ectopic N- tubulin cells formed adjacent to the ectoderm that shows detectable X-gal staining. At high XBF-1 concentration (A,B,E,F) N-tubulin cells do not express detectable lacZ or myc. By contrast at low XBF-1 concentration (C,D,G,H) some cells that express ectopic N-tubulin have detectable levels of nuclear X-gal or myc staining (arrows) while others do not (arrowhead). See text for details. Note that ectopic N-tubulin forms in the deep layer of the ectoderm, where the endogenous N-tubulin is also located.
	Fig. 7. XBF-1 and qin induce ectopic XSox3. Upper panel (A-F) shows embryos injected with a high dose of XBF-1/lacZ (A,D,E) or qin/lacZ (C,F) and with lacZ alone (B) and were processed for X-gal staining and in situ hybridisation for XSox3. (D,E) Transverse sections at low (D) and high (E) magnification through an embryo similar to the one shown in A). The lateral ectoderm is thickened and expresses ectopic XSox3, over the area that also misexpresses XBF- 1/lacZ. (F) A high magnification view of ectopic XSox3-expressing ectoderm located laterally on an embryo similar to the one shown in C. It shows that ectopic XSox3 expression is largely coincident with X-gal staining, in contrast to the expression of N-tubulin, shown in Fig. 2H. Lower panels (G-L) show embryos injected with XBF-1 (H,I,K,L), or were uninjected (G,J) and processed with double in situ hybridisation for N-tubulin (magenta) and XSox3 (light blue). All embryos are shown with anterior to the right and black arrowheads connect dorsal and side views of the same embryo. (H,K) The high dose phenotype, (I,L) the low dose phenotype. The control embryo (G), shows that there is no overlap between XSox3 and the lateral most stripe of N-tubulin expression; the experimental embryos (H,I,K,L) show that ectopic XSox3 and ectopic N-tubulin are mutually exclusive (injected side towards the lower end of the panel). The embryo shown in H and K formed ectopic N-tubulin on the lateral side, outside an expanded XSox3 expression domain. The embryo shown in I and L formed a greatly expanded lateral N-tubulin stripe that did not express ectopic XSox3.
	Fig. 8. Lateral inhibition is activated by XBF-1 and qin and contributes to the dispersed pattern of ectopic N-tubulin expression. (A) Embryos were injected with 0.5 ng qin or XBF-1 RNA, co- injected with lacZ RNA and assayed for X-gal staining (light blue) and X-Delta-1 expression (purple). In the qin-injected embryo, the injected side (shown on the left) expresses ectopic X-Delta-1 (arrow) while the control side (shown on the right) does not. In the XBF-1- injected embryo, the left panel represents a ventral view and the right panel a section through the ventral side of the embryo under high magnification, both showing ectopic X-Delta-1 expression in purple. (B) Embryos were injected with qin, XBF-1, qin/X-Delta-1 stu (a dominant negative form of X-Delta-1), XBF-1/X-Delta-1 stu or X- Delta-1 stu RNA, co-injected with lacZ RNA and assayed for X-gal staining (light blue) and N-tubulin expression (purple). In the qin- and XBF-1-injected embryos, the pattern of ectopic neuronal differentiation is less dispersed in the presence of X-Delta-1 stu suggesting that lateral inhibition limits the number of cells that adopt a neuronal fate in response to qin or XBF-1. However, neuronal differentiation is not observed in areas that express high levels of qin (identified by strong uniform X-gal staining, middle panels), even in the presence of X-Delta-1 stu. A white dotted line indicates the dorsal midline, for comparison between the injected and uninjected side. Note that both the high dose uppression of endogenous-ectopic inductionphenotype (top two frames, middle panel) and the low dose expansion of endogenousN-tubulin phenotype (lower frame, middle panel) are affected by co-expression of X-Delta-1stu.
	Fig. 9. Misexpression of XBF-1-E1A versus XBF-1-EnR has opposite effects on primary neurogenesis. (A,B) Injection of XBF-1-E1A completely suppresses N-tubulin (purple) on the injected side, identified by X-gal staining (A) or anti-myc staining (B). C shows that XBF-1-E1A suppresses N-tubulin (magenta) but does not affect XSox3 expression (light blue). None of the embryos in A-C showed any N-tubulin on the lateral or ventral side. Injection of XBF-1-EnR results in dispersed and ectopic N-tubulin expression (purple in E-G). In some XBF-1-EnR embryos, ectopic N-tubulin is also found anteriorly (I), while XBF-1-E1A-injected (J) and control (H) embryos show no N-tubulin in the anterior neural plate. Injected areas are identified by X-gal staining (light blue) in E and anti-myc staining (light brown) in F,G,I and J.
	Fig. 10. Misexpression of XBF-1-E1A versus XBF-1-EnR and XBF-1 has opposite effects on anterior development. Embryos were injected with various RNAs as indicated on the side of each set of panels and were analysed by double in situ hybridisation for N-tubulin (magenta) and XOtx2 (light blue), or by in situ hybridisation for XOtx2 (purple) and X-gal staining (light blue), as indicated. Dorsal (left panels) and anterior (middle and right panels; injected side to the left) views are shown. XBF-1-EnR reduces XOtx2 expression while XBF-1-E1A expands XOtx2 expression locally. XBF-1 also reduces XOtx2 expression (bottom right). The XBF-1-injected embryo shown in the middle panel has normal XOtx2 expression, presumably because it did not receive RNA anteriorly. Some XBF-1- EnR embryos show ectopic N-tubulin anteriorly, in the area where XOtx2 is suppressed (second row from top).