Poznanski A and Keller R (1997), The role of planar and early vertical signaling...

XB-ART-16622

Dev Biol 1997 Apr 15;1842:351-66. doi: 10.1006/dbio.1996.8500.

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The role of planar and early vertical signaling in patterning the expression of Hoxb-1 in Xenopus.

Poznanski A , Keller R .

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In this paper we examine the contributions of planar and vertical signaling to the patterning of gene expression in neural development and we examine the routes of this neural induction. We have examined how the expression of Xenopus homeobox gene, Hoxb-1, is regulated by instruction from the mesoderm and/or endoderm and ask whether this instruction is by the vertical or planar routes. We investigated normal expression patterns of Hoxb-1 during early Xenopus development and Hoxb-1 expression in sandwich explants of the dorsal marginal zone, which putatively allow only planar signals to pass from the mesodermal and endodermal tissue (Spemann's organizer) to the prospective neural tissue. In the latter case we found significant variability of expression. Observations during dissections suggested that variable degrees of invasion of the mesodermal-endodermal tissue at the leading edge of the mesodermal mantle might be the cause of this variability. Alternatively, differing lengths of time that the prospective neural region spends in planar contact with tissues of the lateral or ventral regions of the embryo could also contribute to this variability. Analysis of staged Keller sandwich explants, "skewered" sandwiches, in which the degree of contact with underlying, involuted mesoderm-endodermal tissues was marked, and "over-the-pole" and "giant" sandwich explants, in which the degree of planar contact with lateral or ventral tissues was normalized, suggests that both planar and vertical signals are involved in induction and patterning of Hoxb-1 expression. The shift in Hoxb-1 expression from a broad, diffuse pattern to a local, focused pattern, characteristic of the ultimate expression pattern in vivo, does not reflect variable degrees of contact with ventral or lateral tissues, but rather reflects early vertical contact with underlying mesodermal-endodermal tissues. We observed such contact at early gastrula stages (stages 10 to 10+), stages commonly assumed not to have the potential for vertical signaling. As the bottle cells first begin to form, at stage 10-, a massive rotation of the lower involuting marginal zone occurs around an internal lip ("levre interne," Nieuwkoop and Florschutz, 1950). This rotation initiates the formation of the Cleft of Brachet from the floor of the blastocoele and brings the prospective mesoderm and endoderm at the leading edge of the marginal zone into vertical apposition with the prospective neural region quite early in gastrulation. The consequence and importance of recognizing these early internal rearrangements are that it pushes backward the time at which potential vertical inductive interactions between mesoderm and neurectoderm can occur. This means that a purely planar inductive situation can cease to exist as early as the inception of bottle cell formation and that neural patterning through vertical induction starts at the very beginning of gastrulation.

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Species referenced: Xenopus
Genes referenced: egr2 hoxb1 hoxd1 mtor

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	FIG. 1. Diagrams show the routes of planar and vertical neural inducing signals in whole embryos, shown in sagittal section, dorsal to the left (A) and in explants (B). At the onset of gastrulation (A, left), prior to involution of the headmesoderm/pharyngeal endoderm (heavily shaded), only planar signals (arrow, A) pass from the Organizer to the prospective neural ectoderm (shaded). Shortly after gastrulation begins (A, right), the head mesoderm/pharyngeal endoderm has involuted beneath the prospective neural ectoderm and can provide vertical neural inducing signals (arrow). In sandwich explants, only planar signals can pass from the Organizer to the prospective neural ectoderm (B, left), unless the mesoderm is allowed to involute (B, right).
	FIG. 2. Diagrams show the method of making the standard ‘‘Keller’’ sandwich explant (A); the ‘‘over the pole’’ sandwich explant (B), which includes ventral vegetal endoderm (cross-hatched); the ‘‘giant,’’ 3607 sandwich explant (C); and the ‘‘pita’’ sandwich explant (D). The dashed lines indicate the positions of cuts, shown in vegetal view of the embryo with dorsal at the top (top row) and in midsagittal view, dorsal to the left (second row). See the text for a detailed, step-by-step description of the operations.
	FIG. 3. Homeobox amino acid sequence comparison between various members of the Hoxb-1-related genes, showing location of the helical regions (I, II, and III underlined) and the variable amino acids (boxed), shows that the homeobox sequence we amplified in this paper, designated here as X-B1, is most closely related to Hoxb-1 of mouse and chick and not the paralogs Hoxa-1 (Xhoxlab2) and Hoxd- 1 (Xhoxlab1, Xlab).
	FIG. 4. The normal pattern of expression of Hoxb-1 by whole-mount in situ hybridization. (A) At stage 14 Hoxb-1 is expressed in the anterior lateral neural plate and folds (arrows). (B) At stage 16–17 expression is stronger and in the shape of a comma whose tail lies in the prospective neural crest (arrow). Faint expression can also be seen posteriorly. (C) At stage 23 (bottom) and stage 27 (top) expression in rhombomere 4 (arrows) and in the second branchial arch (arrowheads) can be seen. (D) By stage 27 expression is focused in rhombomere 4 (R4) and in the neural crest which has migrated into the second branchial arch (arrowhead). Note that Hoxb-1 is not expressed in the floorplate or roofplate of the neural tube (arrow). (E) Hoxb-1 expression in rhombomere 4 (magenta) is located between Krox-20 expression in rhombomeres 3 and 5 (blue). (F) Hoxb-1 is expressed autonomously in a neural plate isolated at stage 10.5. (G) Hoxb-1 is expressed autonomously in a pita explant made at stage 10.5. N, notochord.
	FIG. 5. Light micrographs of external (A, E, I) and of internal (B, F, J) morphology, and corresponding cutaway diagrams of tissues and movements (C, G, K) are correlated with in situ Hoxb-1 expression patterns in Keller sandwich explants (D, H, L). These are shown for stage 100 (A– D), stage 10 (E– H), and stage 10/ (I–L) embryos. A, E, and I are exterior vegetal views of the forming bottle cells. In B, F, and J the view is from the animal pole looking vegetally down onto the floor of the blastocoel with dorsal toward the tops of the pictures. In C, G, and K the embryos are viewed from the right sides with dorsal to the left, the animal pole at the top, and the vegetal pole below. The head mesoderm is indicated by stippling and the arrows indicate movements (F, forebrain; Bl, blastocoel; BC, bottle cells). In stage 100 and 10 sandwiches (D and H) only neural regions are present; the mesodermal regions of these sandwiches were broken off during processing. An intact explant can be seen in the stage 10/ sandwiches (L) where the notochord expresses Hoxb-1 in addition to its expression in the hindbrain region. About half of the explants made at stage 10 (5 of 11) showed the diffuse pattern, while about half (6 of 11) showed focusing of expression to a bar pattern in the approximate R4 region.
	FIG. 6. An SEM of a stage 10/ embryo in midsagittal fracture (A) shows the proximity of head mesoderm/pharyngeal endoderm (HM/ PE) to the overlying prospective neural ectoderm (NE) across the Cleft of Brachet (CB). The animal pole is at the top and vegetal pole is at the bottom. The asterisk indicates the point of view for (B), looking down at the ledge of the blastocoel floor where the head mesoderm (below) can be seen bridging the Cleft of Brachet and contacting the underside of the prospective neural ectoderm (above).
	FIG. 7. The design of the ‘‘skewer’’ experiment is shown, documenting the location of head mesoderm/pharyngeal endoderm contact with overlying tissue across the Cleft of Brachet (A, B). The point of contact of the involuting tissue with the overlying tissue is marked with a skewer as described in the text. Hoxb-1 is expressed in a ‘‘comet’’ pattern in an explant skewered in the involuting marginal zone (arrow), cultured to stage 27 (C). Hoxb-1 is expressed in a ‘‘bar’’ pattern in an explant skewered in the anterior neural region (arrow), cultured to stage 27 (D). Expression of Krox-20 (blue, arrowhead) lies anterior to the tail of the ‘‘comet’’ in the middle of Hoxb-1 (magenta) expression in an explant skewered in the involuting marginal zone, cultured to stage 27 (E). Expression of Krox-20 (blue) lies posterior to the Hoxb-1 (magenta) expression in an explant skewered in the anterior neural region (arrow), cultured to stage 27 (F). A and B are reproduced from Keller and others (1996) with permission.
	FIG. 8. Hoxb-1 (b-1) and Krox-20 (kr-20) expression are shown in an explant with head mesoderm attached at the anterior end of the mesodermal/endodermal axis (A, B). Persistent presence of head mesoderm, indicated by the expression of Hoxb-1, in a Keller sandwich explant made at stage 100, is unable to focus the neural expression of Hoxb-1 (A). Krox-20 is expressed, although in an abnormal geometry under these conditions (B). Hoxb-1 expression in ‘‘over-the-pole’’ explants made at stages 100 (C, left, diffuse) and 10/ (C, right, focused) shows that the persistent presence of ventral tissue to stage 10.5 is unable to focus Hoxb-1 expression in explants made at stage 100. The ventral vegetal endoderm of the over-the-pole explants was cut off at stage 10.5 and all explants were cultured to the same final tailbud stage. Hoxb-1 expression in ‘‘giant’’ explants made at stages 100 (D, left, diffuse) and 10/ (D, right, focused) shows that the persistent presence of ventral and lateral tissue is unable to focus Hoxb-1 expression in explants made at stage 100. Overlapping of Hoxb- 1 (magenta) and Krox-20 (blue) expression in the neural region of a Keller sandwich explant shows that planar signals alone are unable to refine pattern (E). A gap in Hoxb-1 expression (pointer, F) occurs between the concentrated bar above and diffuse expression below in the hindbrain and spinal cord of Keller sandwich explants made at stage 10 (F). Tor 70 immunostaining for notochord (pointer, left explant in G) shows the relation between proximity of notochord tissue and a split in the Hoxb-1 expression pattern, in contrast to the solid bar expression pattern in an explant without notochord (right explant, G). Tor 70 immunostaining of Keller sandwich explants made at stage 10/ (H) shows the location of the notochord (N) in relation to Hoxb-1 expression (b-1). The notochord in the left explant is curled above, not along the neural axis. Hoxb-1 is expressed in the notochord of Keller sandwich explants (I, pointer, bottom specimen) but only faintly in the notochord of control embryos (I, top specimen).