Zimmerman K et al. (1993), XASH-3, a novel Xenopus achaete-scute homolog, ...

XB-ART-22250

Development 1993 Sep 01;1191:221-32.

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XASH-3, a novel Xenopus achaete-scute homolog, provides an early marker of planar neural induction and position along the mediolateral axis of the neural plate.

Zimmerman K , Shih J , Bars J , Collazo A , Anderson DJ .

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We have isolated a novel Xenopus homolog of the Drosophila achaete-scute genes, called XASH-3. XASH-3 expression is neural specific and is detected as early as stage 11 1/2, making it one of the earliest markers of neural induction so far described. Moreover, XASH-3 expression within the neural plate is regionally restricted. Transverse bands of XASH-3 mRNA mark discrete positions along the anteroposterior axis, while longitudinal bands mark a discrete position along the mediolateral axis. This latter site of XASH-3 expression appears to demarcate the prospective sulcus limitans, a boundary zone that later separates the functionally distinct dorsal (alar) and ventral (basal) regions of the spinal cord. In sandwich explants lacking any underlying mesoderm, XASH-3 is expressed in longitudinal stripes located lateral to the midline. This provides the first indication that planar or midline-derived inductive signals are sufficient to establish at least some aspects of positional identity along the mediolateral axis of the neural plate. By contrast, the transverse stripes of XASH-3 expression are not detected, suggesting that this aspect of anteroposterior neural pattern is lost or delayed in the absence of vertically passed signals. The restricted mediolateral expression of XASH-3 suggests that mediolateral patterning of the neural plate is an early event, and that this regionalization can be achieved in the absence of inducing signals derived from underlying mesoderm.

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Species referenced: Xenopus laevis
Genes referenced: ascl1 ascl2 cat.2 ncam1 tbx2 tcf3

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	Fig. 1. Comparison of XASH3 bHLH domain to previously characterized genes. Primers used in PCR amplifications are indicated by arrows. Stars represent conserved residues within the bHLH domain. Dashes indicate a gap in the protein alignment.
	Fig. 2. Deduced protein coding sequence of X3.A and X3.B cDNA clones. Residues upstream of the first methionine in the X3.A protein are indicated in italics as we are currently unable to determine if these residues are contained within the XASH3.A protein (see text). Stars indicate conserved residues. Dashes indicate a gap in the protein alignment. The nucleotide sequences are available from the GenBank data base, accession numbers L20214 (X3.A) and L20215 (X3.B).
	Fig. 3. XASH3 binds DNA and activates transcription of an MCK/CAT reporter construct in 10T cells. (A) Electrophoretic mobility shift assays were performed with in vitro translation extracts prepared with in vitro transcribed XASH3 (sense and antisense), MASH1 and E12 RNA transcripts. Extracts were combined as follows E12 (1), XASH3 (antisense) + E12 (2), XASH3 (sense) +12 (3), MASH1(sense) +E12 (4), mixed with a 32P-labelled, E-box containing oligonucleotide and analyzed by electrophoresis as previously described (Johnson et al., 1992a). Arrow indicates position of gel shifted band. (B) Cells were cotransfected with an MCK/CAT reporter construct, a CMV b-gal construct, and either an RSV luciferase construct (1), RSVMASH1 (2), RSVXASH3 in the antisense orientation (3), or RSVXASH3 in the sense orientation (4) construct. Samples were standardized for transfection efficiency by quantitation of b-gal activity prior to CAT enzymatic assay. Arrow indicates acetylated chloramphenicol reaction product.
	Fig. 4. XASH3 expression during mid-gastrula and neurula stages of Xenopus development. Whole-mount in situ analysis was performed at early stages of Xenopus development. Arrows mark specific aspects of XASH3 expression pattern: closed arrow (A-C) indicates stripes appearing at the midline of the mediolateral axis of the neural plate; in B and C these stripes parallel the anteroposterior axis of the embryo. Open arrow (A-C) indicates expression in transverse ‘eyebrow’ stripes within prospective hindbrain region. Arrowhead (C) marks anterior stripe of expression near hindbrain/midbrain junction. In all panels, anterior is to the right although the exact orientations are different.
	Fig. 5. Comparison of XASH3 and N-CAM expression in late-gastrula stage embryos. XASH3 (A,B,C) and N-CAM (D,E) expression in stage 14 embryos was determined by whole-mount in situ hybridization (A,D). Cross-sections of similarly staged embryos are also shown (B,C and E). Open arrow in A indicates XASH3 expression lateral to the neural plate; closed arrow indicates the mediolaterally restricted stripe revealed by crosssection in B, and the arrowhead indicates the transverse ‘eyebrow’ stripe revealed by cross-section in C. In A, anterior is towards 11 o’clock and, in D, it is towards 1 o’clock. Note the restricted expression of XASH3 along the mediolateral axis of the neural plate (B) in comparison to that of N-CAM (E).
	Fig. 6. Expression of XASH3 in embryos following neural tube closure. XASH3 expression as determined by whole-mount in situ analysis at stage 28 is shown in A; anterior is to the right. Cross sections of the spinal cord region from similarly staged embryo are shown in B and C; dorsal is up. Open and closed arrows in A indicate approximate planes of section in B and C, respectively. Closed arrows in B and C indicate the sulcus limitans; open arrow in C indicates expression adjacent to the floorplate.
	Fig. 7. XASH3 expression in explants. Closed-face Keller sandwiches were prepared as described in Materials and Methods, and analyzed at the equivalent of stage 17 for XASH3 expression by whole-mount in situ hybridization. For comparison, a normal stage 17 embryo hybridized with XASH3 is shown in A; closed arrows mark the mediolaterally restricted longitudinal stripes of XASH3 expression, open arrows mark the transverse ‘eyebrow’ stripes in the hindbrain (anterior is toward 11 o’clock). Explants in B-D are oriented with anterior to the right. Specimens in B and C have undergone symmetric convergent extension and show bilateral symmetric stripes of XASH3 expression displaced towards the lateral edges of the explants (arrows). In D, convergent extension was asymmetric and a single stripe of XASH3 is observed.
	Fig. 8. Models to explain the control of XASH3 expression in embryos (A-D) and in Keller sandwich explants (E-H). (A) The site of XASH3 expression along the mediolateral axis of the neural plate may be determined by a particular value of a single, midlinederived gradient (right), or by the point of intersection of two opposing gradients, one (shaded) derived from the lateral margins of the neural plate (left). These models apply specifically to the control of XASH3 expression and not necessarily to the determination of other spinal cord cell types. (B) Schematic of stage 11 neural plate showing sites of XASH3 expression relative to midline (black bar) and notochord (circle). (C) Following neural tube closure, the boundary zone defined by XASH3 expression may be important in converting graded, continuous signals deriving from the dorsal and ventral margins of the tube (stippled drawing on left) to discontinuous zones of gene expression within the forming alar and basal plates, perhaps by sharpening the transition from dorsal to ventral character of the spinal cord. (D) After subdivision of the neural tube into alar and basal zones, further steps lead to the differentiation of specific dorsal and ventral cell types such as Rohon Beard neurons (RB) and motorneurons (MN). (E) The position of the explanted dorsal marginal zone used in sandwich explants (arrows, bold line) is superimposed on the fate map of a stage 10+ gastrula (see Materials and Methods). Prospective forebrain (F), hindbrain (H), spinal cord (S) and sulcus limitans (S.L.) are indicated. Explanted tissue excludes the prospective sulcus limitans as determined by retrospective tracing of the midpoint of the neural tube from stage 13 (Eagleson and Harris, 1990) back to stage 10+ using time-lapse video records (Keller et al., 1992). Both the lines representing the explanted region and those defining the sulcus region were drawn by calculating the projected length of known angles; the bold line represents the 50° mark. (F-H) Model to explain why XASH3 is detected at the edges of the explants. (F) Postulated single or double gradients in the intact embryo (see A) are shown relative to the site of excision (dashed lines; see E). (G) Proportion regulation (lateralization) of the edges of the explant is suggested to occur either by weakening of the midline-derived signal (M, right), or by production of the lateral signal at the new edge of the neural plate created by excision (left, L¢). In the single-gradient model (G, right), as levels of the midline-derived signal fell (dashed arrow), the level of inducer activating XASH3 expression would first be achieved at the edge of the explant (H, right, arrow). In the double-gradient model, as the production of lateral signal increased (G, left, dashed arrows), it would first reach equivalency with the midline-derived signal at the edge (H, left). (A simultaneous readjustment of both midline- and lateral-margin-derived signals is also possible). Expression of XASH3 at the edge of the explant is indicated by shading (bottom). In F-H, midline-derived signals in explants may be produced exclusively by the mesoderm and diffuse or be propagated into the neuroepithelium, or they may derive from the notoplate (Ruiz i Altaba, 1992).