February 1, 1996;
The Xenopus laevis homeobox gene Xgbx-2 is an early marker of anteroposterior patterning in the ectoderm.
In a search for homeobox genes expressed during early Xenopus development, we have isolated a gene which appears to be the Xenopus cognate of the mouse Gbx-2 gene. Expression of Xgbx-2
is first detectable by in situ hybridization at the midgastrula stage when it is predominantly expressed in the dorsolateral ectoderm
, with a gap in expression at the dorsal midline. By the end of gastrulation and during neurulation, Xgbx-2
is expressed dorsolaterally in the neural ectoderm
and laterally and ventrally in the epidermis
with sharp anterior
expression borders in both tissues. The anteriormost expression in the neural ectoderm
persists throughout the early stages of development, and was mapped to the region of rhombomere 1
, with an anterior
expression border in the region of the midbrain-hindbrain boundary
. Thus Xgbx-2
is expressed anterior
to the Hox genes. Xgbx-2
expression is induced by retinoic acid (RA) in animal caps, and RA treatment of whole embryos expands and enhances Xgbx-2
expression in the ectoderm
. We suggest a role for Xgbx-2
in establishing the midbrain-hindbrain boundary
, which appears to separate early neurectodermal regions expressing genes that are positively and negatively regulated by RA.
[+] show captions
Fig. 3. Spatial expression of Xgbx-2 during early development. The expression was analyzed by whole mount in situ hybridization of albino embryos. In all pictures, anterior is to the left. (A) Dorsal view of a stage 11 embryo. Note the wide gap at the dorsal midline and the distance of the expression from the blastopore lip (b). (B) Dorsolateral view of a stage 11 embryo. (C) Dorsal view of a stage 13-14 embryo. The gap in expression at the dorsal midline has narrowed. Note the sharp anterior expression border in the neuroectoderm (n). (D) Lateral view of a stage 13-14 embryo. Xgbx-2 is expressed in the neuroectoderm (n) and in epidermal ectoderm (e). (E) Dorsal view of a stage 15 embryo. The neurectodermal expression is concentrated in a pair of anterior stripes (n). The epidermal expression has a sharp anterior border (e). There is additional expression in the presumptive posterior spinal cord (s). (F) Dorsal view of a stage 19-20 embryo. Labeling as in (E). (G) Lateral view of a stage 19-20 embryo. The lateral epidermal expression (e) shows a sharp anterior border and gradually tapers off towards the posterior end. Labeling as in (E). (H) Dorsal view of a cleared stage 22-23 embryo. Xgbx-2 is expressed in the anterior neuroectoderm (n), in the otic vesicle (o), in a pair of lateral stripes anterior to the otic vesicle, in the neural tube (nt) posterior to the otic vesicle, in the posterior spinal cord (s), and in the future branchial arches (lateral to the otic vesicle). (I) Dorsal view of an uncleared stage 22-23 embryo. Expression is in the region of the neural crest (arrowhead) ventrally and posterioventrahy to the otic vesicle. (J) Dorsal view of a cleared stage 31 embryo. Note the crescent-shaped expression in the dorso-medial part of the otic vesicle (0). Labeling as in (H). (K) Lateral view of a cleared stage 31 embryo. Note expression in the dorsal and ventral part of the anterior hindbrain (n). Them is weak expression in the region of the visceral arches (va). Labeling as in (H). (L) Dorsolateral view of a cleared stage 31-32 embryo, showing Xgbx-2 expression in the neural tube dorsal to the eye (arrowhead).
Fig. 4. Comparison of the expression of Xgbx-2 to the expression of Xbra. All three embryos are stage 11 (midgastrula stage). The same magnification is used in all photographs. Shown are dorsovegetal views; future anterior is to the top. (A) Embryo hybridized for Xgbx-2 alone. (B) Embryo hybridized for Xgbx-2 and Xbra. (C) Embryo hybridized for Xbra alone.
Fig. 5. Histological analysis of Xgbx-2 expression. Embryos were processed for whole-mount in situ hybridization, embedded in Paraplast, and 16pm (A,B) or 10pm (C-E) sections were cut. Dorsal is at the top in all sections except in (A). (A) Horizontal section of a stage 14-15 embryo which is cut anterior to the hindbrain. Note that the anterior neural plate is bent ventralwards at this stage, so that the horizontal section passes through the future forebrain and midbrain, and the lateral epidermis. Anterior is at the top. The anterior border of the epidermal expression (e) lies in the region of the posterior border of the anterior neural plate. (B) Transverse section of a stage 14-15 embryo at a level near the anterior expression border in the neural plate. The section is slightly oblique, such that the left side is more anterior than the tight side. Staining is observed in the thickened neural plate (n) and in the epidermis (e). There is no expression in the dorsal midline. At this stage, the most anterior epidermal expression (left side) appeared to be restricted to the inner (sensorial) layer of the epidermis. Epidermal expression is observed across the ventral midline. (C-E) Transverse sections of the same stage 31 embryo. (C) Section at the level of the anterior hindbrain. Staining is observed in the dorsal part of the neural tube, but not at the dorsal midline. The section is slightly oblique, such that the ventrolateral expression in the anterior hindbrain (Fig. 3K) is not visible. (D) Section at the level of the otic vesicle. Expression is found along the medial (inner) side of the dorsal part of the otic epithelium. The notochord (no) is labeled. (E) Section through the posterior hindbrain. There is staining in two bilaterally symmetric spots in the neural tube.
Fig. 6. Localization of Xgbx-2 expression in the hindbrain. Embryos were hybridized alone or in combinations with Xgbx-2, Krox-20, or En-2. For all embryos, a lateral view is shown with dorsal at the top and anterior to the left. The magnification is the same in all photographs. All embryos are cleared. Arrowhead in A, B, D-F: anterior border of Xgbx-2 expression. (A-C) Stage 21. (A) Embryo hybridized with Xgbx-2 alone. Xgbx-2 is also expressed in the head endoderm (arrow). (B) Embryo cohybridized with Xgbx-2 and Krox-20. Note the gap between the Xgbx-2 stripe and the rhombomere 3-stripe (3) of Krox-20, which is a length of approximately one rhombomere. Krox-20 is also expressed in neural crest (nc) migrating into the third visceral arch (Bradley et al., 1992). (C) Embryo hybridized with Krox-20 alone. (DG) Stage 31. (D) Embryo cohybridized with Xgbx-2 and Krox-20. The anterior Xgbx-2 expression border (arrowhead) lies at least 2 rhombomere lengths anterior to the rhombomere 2/3 boundary. (E) Embryo hybridized with Xgbx-2 alone. (F) Embryo cohybridized with En-2 and Xgbx-2. The En-2 stripe does not appear to be widened compared to (G). The anterior border of Xgbx-2 expression (arrowhead) would be predicted to lie approximately halfway into the En-2 expressing region (compare to E). (G) Embryo hybridized with En-2 alone. En-2 is also expressed in the mandibular arch (arrow).
Fig. 7. Treatment with retinoic acid causes an expansion and enhancement of ectodermal Xgbx-2 expression in whole embryos. Pigmented embryos were treated for 30 min at stage 9 with 1OpM all-rrans RA, fixed when controls reached stage 12.5-13, and processed for whole mount in situ hybridization with Xgbx-2 antisense probe. Embryos that were only treated with ethanol, the RA solvent, looked the same as untreated control embryos (not shown). For all embryos, anterior is to the left, and the magnification is the same. (A) Untreated control embryo, dorsal view. (B) The same control embryo, lateral view. (C) RA-treated embryo, dorsal view. (D) RA-treated embryo, lateral view.