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Dev Biol
1995 Oct 01;1712:641-54. doi: 10.1006/dbio.1995.1311.
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Initiation of anteriorhead-specific gene expression in uncommitted ectoderm of Xenopus laevis by ammonium chloride.
Mathers PH
,
Miller A
,
Doniach T
,
Dirksen ML
,
Jamrich M
.
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The role of homeobox-containing genes in the regional specification of the vertebrate embryo has been an area of intense research over the last decade. Whereas it appears that the homeobox genes of the Hox gene family play an important role in the specification of the trunk, the genes and processes involved in the specification of the head are less well understood. We have isolated a new head-specific homeobox gene, XANF-2, that appears to be involved in the regional specification of the anteriorhead of Xenopus embryos. This gene is initially expressed in the anterior dorsal region of early embryos and later exclusively in the primordium of the anterior pituitary gland. XANF-2 represents the earliest marker for the anterior pituitary lineage. Ammonium chloride is able to induce the expression of XANF-2 in uncommitted ectoderm. These and other data indicate that ammonium chloride is capable of inducing a large portion of the anterior dorsal region of the embryo which includes, but is not limited to, the anterior pituitary gland and cement gland anlagen. This implies that changes in intracellular ionic conditions play an important role in the formation of the anteriorhead region. In addition to NH4Cl, injection of follistatin RNA can induce transcription of XANF-2, suggesting that these two unrelated compounds can activate a chain of events leading to the formation of the amphibian head. Furthermore, we demonstrate that planar induction in Keller sandwiches can induce XANF-2 expression as well as the expression of the cement gland-specific gene, XCG 13, indicating that planar signaling can account for induction of even the most anterior regions of the embryo.
FIG. 3. Whole-mount in situ hybridization of XANF-2 to Xenopus embryos. (A) A stage 11 embryo with Xanf-2 staining in the dorsal ectoderm (right), but not in the area of the dorsal lip (arrow). (B) Section showing the dorsal region of a stage 11. embryo after hybridization with XANF-2. The demarcation between inner and outer ectodermal layers and involuting mesoderm is shown with dotted lines. The arrow points to the dorsal lip. (C) Embryos are oriented with the dorsal side up and latera] views have anterior to the left, Lateral view of a stage 14 embryo showing an intense hybridization signal in the anterior dorsal region. At this stage it can be seen that both the outer ectoderm and the inner ectoderm express XANF-2. (D Lateral view of a stage 18 embryo showing hybridization signal in the presumptive pituitary gland area. Ar this stage the entire signal is in the inner ectodermal layers. The outer ectodermal layer and the pharyngeal endoderm do not show any hybridization. (E) Lateral view of a stage 24 embryo with expression localized to the inner ectoderm lying between the forebrain and the cement gland. (F) Lateral view of a stage 32 embryo with XAMF-2 (purple) visualizing the pituitary gland cells migrating under the forebrain area and XCG7 green) showing the cement gland. (G) Anterior view of the stage 14 embryo from C. Expression occurs in a roughly uniform pattern, defining a field of cells termed the upper sensory plate. (H) Anterior view of a stage 16 embryo showing localization of XANF-2 signal, with the anterior dorsal region limited by two regions of more intense hybridization. The dorsal (posterior) limit corresponds to the anterior neural plate, while the ventral (anterior) limit is immediately adjacent to the future cement gland (see I). (I) Anterior view of a stage 19 embryo hybridized with XANF-2 and with a cement gland-specific marker XCG7 (Jamrich and Sato, 1989) showing the close proximity, without overlap, of expression for the two genes. At this stage, XCG 7 is expressed in the outer ectoderm while XANF-2 is expressed in the inner ectoderm, Purple, anteriorpituitary anlage; green, cement gland. (J) Anterior view of the stage 32 embryo from D hybridized with XANF-2 and XCG7. Purple, anteriorpituitary anlage; green, cement gland.
FIG. 8. Expression of XANF-2 in animal caps, Einsteck embryos, and Keller explants. (A) Whole mount in situ hybridization of an animal cap treated with NH4Cl for 6 hr and then cultured 20 hr in buffer. Explant was hybridized with the cement gland-specific probe, XCG 13. Note an individual Cell expressing XCG 13, surrounded by nonexpressing cells. (B) In situ hybridization on animal cap cultured for 6 hr in buffer alone with XANF-2 probe, Note the lack of hybridization. (C) In situ hybridization of animal cap treated for 6 hr in 10mM NH4Cl with XANF-2 probe. The inner ectoderm shows hybridization; the outer ectoderm is negative. (D) Animal cap treated for 6 hr with 20 mM NH4Cl and probed with XANF-2 cRNA. Note the increase in staining level and area in comparison with C. (E) Animal cap after injection with follistatin RNA. Ectoderm was isolated at the blastula stage and cultured in buffer for 16 hr, fixed, and then hybridized with XANF-2 probe. (F)Animal caps treated for 6 hr in lO mM NH4Cl were cut in half and placed into the blastocoel cavity of a host gastrulaembryo. The following day the embryo was fixed and hybridized with XCG 13 cRNA. The endogenous cement gland and the graft generated by this Einsteck method both show intense hybridization. (G) An Einsteck experiment equivalent to that shown in (F) but probed with XANF-2 cRNA. Both the graft and the anteriorpituitary area of the embryo hybridize. [J-1) A Keller sandwich (equivalent to stage 24) hybridized with XCG13 cRNA shows hybridization in the anteriorectoderm. (I) A Keller sandwich (control stage 21) probed with XANP-2 cRNA shows strong expression (closed circle). The explant was also probed with engrailed-2 cRNA (open circle) which is expressed in a more posterior position relative to XANF-2. The dark patches of staining in the animal pole region of the sandwich are artifacts. (J) An open-face Keller explant (control stage 16} hybridized with XANF-2 cRNA (arrows)
