Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
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
.
???displayArticle.abstract???
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.
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).
