Bellefroid EJ et al. (1998), Xiro3 encodes a Xenopus homolog of the Drosophi...

XB-ART-15410

EMBO J 1998 Jan 02;171:191-203. doi: 10.1093/emboj/17.1.191.

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Xiro3 encodes a Xenopus homolog of the Drosophila Iroquois genes and functions in neural specification.

Bellefroid EJ , Kobbe A , Gruss P , Pieler T , Gurdon JB , Papalopulu N .

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We have identified in Xenopus and in the mouse two highly related genes, Xiro3 and Irx3 respectively, that encode a Drosophila Iroquois-related homeobox transcription factor. Xiro3 in Xenopus and Irx3 in the mouse are expressed early in the prospective neural plate in a subset of neural precursor cells. In Xenopus, injection of Xiro3 mRNA expands the neural tube and induces ectopic neural tissue in the epidermis, based on the ectopic expression of early neural markers such as Xsox3. In contrast, the differentiation of the early forming primary neurons, as revealed by the expression of the neuronal marker N-tubulin, is prevented by Xiro3 expression. Activation of Xiro3 expression itself requires the combination of a neural inducing (noggin) and a posteriorizing signal (basic fibroblast growth factor). These results suggest that Xiro3 activation constitutes one of the earliest steps in the development of the neural plate and that it functions in the specification of a neural precursor state.

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Species referenced: Xenopus
Genes referenced: actc1 actl6a ascl2 dll1 fgf2 gata2 irx3 krt12.4 myt1 neurog2 nog nrp1 otx2 sox3 tubb2b twist1

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	Fig. 2. Xiro3 expression during early embryogenesis. (A) Embryos at the indicated stages are analysed by in situ hybridization with probes for Xiro3 (A) and XASH3 (G). Note that Xiro3 expression within the prospective neural plate is first detected at stage 11 in the form of two wide symmetrical patches (B) and that later Xiro3 and XASH3 are similarly expressed in the neural plate in the form of two symmetrical longitudinal stripes and two transverse anterior bands. In addition, expression of Xiro3 also occurs in the ectoderm lateral to the anterior neural plate (asterisk). (J) Double in situ hybridization of Xiro3 (red) and N-tubulin (purple) in a stage 15 embryo (m, medial; i, intermediate; l, lateral). (K) Cross-sections of a stage 15 Xiro3/N-tubulin-stained embryo showing that Xiro3 is expressed in regions corresponding to the medial and intermediate stripes of N-tubulin and that it is detected in cells located more superficially than those expressing N-tubulin. (L) Cross-section of a stage 15 Xiro3/N-tubulin- stained embryo at a more posterior position. Note that Xiro3 expression appears in both the superficial and deep layer of the neurectoderm. Xiro3 is also expressed in anterior lateral mesoderm (arrowheads). (M) Double in situ hybridization of XSox3 (blue) and N-tubulin (purple). (N) Cross-section of the XSox3/N-tubulin-stained embryo shown in (M). Note that XSox3 is expressed like Xiro3 in the posterior neural plate in superficial layers of the neurectoderm in between the intermediate and medial N-tubulin stripes. (O) Lateral view of the head of a tadpole embryo. (P) Transverse section at the level of the otic vesicle. All embryos are shown in a dorsal view, with anterior to the top, except in (J), (M) and (O).
	Fig. 4. Neural expansion in Xiro3-injected embryos. The injected side of the embryos is indicated and can be visualized by nuclear LacZ staining. Anterior is toward the right. Embryos are viewed from the dorsal or dorso-lateral side in (A), (B), (C) and (F) and from the the ventral side in (G) and (H). (A) Stage 30 embryo with increased XSox3 expression in the expanded neural tube. (B) Stage 15 embryo showing ectopic XSox3 expression in the ectoderm. (C) Ectopic expression of nrp-1 in the lateral ectoderm. (D) A section through the hindbrain region of a stage 30 embryo labelled for XSox3 expression. (E) A section through the hindbrain region of a Hoechst-stained stage 30 embryo showing that the number of cells in the expanded neural tube is increased. (F) X-twist is expressed in cranial neural crest on the control side but is absent on the injected side of the embryos. (G) Epidermal keratin is expressed outside the neural plate and is blocked in the Xiro3-injected area. (H) Like epidermal keratin, GATA-2 is expressed in the non-neural ectoderm and is absent after Xiro3 injection. (I) A section through the hindbrain of a stage 30 embryo showing that the formation of the somites as revealed by cardiac actin expression is affected on the injected side (n, notochord; s, somite).
	Fig. 5. Xiro3 blocks the differentiation of the primary neurons and expands the expression of XASH3. All embryos are viewed from the dorsal side except in (B) (lateral view). In (A and G), embryos are viewed with the anterior end toward the right. The injected side of the embryos is marked by blue staining for nuclear LacZ expression. (A) Stage 15 embryo injected with Xiro3 mRNA. Note that the lateral stripe of N-tubulin expression is suppressed on the injected side. (B) Stage 15 embryo injected with Xiro3 mRNA showing disorganized N-tubulin expression at the border of the injected area. (C) Stage 15 embryo injected with Xiro3 mRNA showing that the lateral and intermediate stripes of N-tubulin expression are moved to a more ventral position. (D) Stage 14 embryo injected with Xiro3 mRNA. Expression of X-MyT1 is blocked on the injected side of the embryo. (E) Section through the hindbrain region of a stage 30 embryo labelled for N-tubulin expression. (F) Cross-section through a stage 30 embryo injected with Xiro3 in the lateral ectoderm. Note that Xiro3 induces extra tissue in the lateral epidermis of the embryo that is partially N-tubulin positive. (G) Stage 14 embryo injected with Xiro3 showing ectopic expression of X-Delta-1 over the injected area. (H) Stage 14 embryos injected with a combination of Xiro3 and a dominant-negative form of X-Delta-1, X-Delta-1stu that block lateral inhibition. No rescue of the blocking of N-tubulin expression is observed. (I) Control embryos injected with X-Delta-1stu alone showing an increase of density of N-tubulin-positive cells within the stripes. (J) Stage 16 embryos injected with Xiro3. The expression of XASH3 is slightly expanded on the injected side.
	Fig. 6. Xiro3 induces early neural genes in animal caps. Xiro3 mRNA was injected in both cells of two-cell stage embryos and animal caps were dissected at stages 90. Explants were cultured to stage 16 and then analysed by whole-mount in situ hybridization. For cardiac actin expression, explants were cultured to tadpole stage. Note that Xiro3 injection induces the expression of all neural, but not neuronal markers tested.
	Fig. 7. Regulation of Xiro3 expression. (A) N-tubulin expression is strongly induced in embryos injected unilaterally with X-ngnr-1 mRNA. (B) Xiro3 expression, in contrast to N-tubulin, is blocked by overexpression of X-ngnr-1. (C) Xiro3 expression (purple) in a Keller explant is detected in the form of a stripe overlapping with that of engrailed (blue). A second band of expression (asterisk) is also detected in the cephalic mesendoderm. (D) Xiro3 is not expressed in noggin-injected animal caps. (E) Otx2 expression is induced in noggin-injected animal caps. (F) Xiro3 expression in animal caps treated with bFGF. (G) Xiro3 is induced in the form of a transverse band in elongated animal caps injected with noggin and treated with bFGF. (H) Xiro3 is not induced in noggin-injected animal caps treated with RA. (I) Otx2 expression is down-regulated in noggin-injected animal caps treated with RA. (J) Xiro3 expression in a stage 15 embryo injected at the two-cell stage in each blastomere with the dominant-negative form of the FGF receptor.
	ascl2 ( achaete-scute family bHLH transcription factor 2 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 16, dorsal view, anterior up.

References [+] :

Amaya, Expression of a dominant negative mutant of the FGF receptor disrupts mesoderm formation in Xenopus embryos. 1991, Pubmed, Xenbase