XB-IMG-136085
Xenbase Image ID: 136085
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Figure 2. Expression of molecular markers
in wild-type and ASH3b RNA-injected
embryos. (A-C) Expression of ASH3a
mRNA in wild-type embryos, detected by
whole-mount in situ hybridization at three
representitive stages. (A) An early neural
plate embryo (stage 13.5) showing longitudinal
stripes of ASH3a expression within
prospective hindbrain and spinal cord. A
transverse stripe is also present in the anterior
of the future hindbrain (arrow). (B)
Expression persists as a continuous longitudinal
stripe (s) on each side of the spinal
cord and hindbrain in an early tail bud embryo
(stage 28), and ASH3a is also expressed
in the retina (r) and forebrain (f). A
second stripe of expression within the
hindbrain is also visible (open arrow). The
approximate extent of the hindbrain is
shown by the bar above the embryos in
both B and C. (C) The head of a later tail
bud embryo (stage 34) showing the loss of
ASH3a expression as neurons differentiate.
ASH3a expression is shown by purple
staining; brown dots are pigmented cells.
The pigment epithelium of the eye is also
brown. Note that expression in the retina
is reduced to a band at the margin, the location
of the retinal progenitors at this
stage. The extent of expression in the forebrain
and hindbrain is reduced, but the longitudinal
stripes remain in the hindbrain
and spinal cord (s). Anterior is toward the
right in B and C and toward the top in A
and D-M. Embryos in B, C, I, J, and M are
cleared. N-CAM mRNA expression in a
wild-type (D) and ASH3b RNA-injected embryo (E). A vertical bar shows the position of the midline and the injected side is labeled
inj in E. The N-CAM-positive CNS is expanded laterally on the injected side along its entire length. Arrows indicate the boundaries
of the prospective spinal cord; a double-headed arrow denotes the midline. The neural tube has failed to close because of the extra
tissue. The eye vesicle (e) is labeled in D but is hidden in E because of the open neural tube. The embryo shown in E and other injected
embryos shown in Figures 2-4 are examples of a strong but reproducible level of effect arising from ASH3b injection (see Materials and
methods). (F) twist mRNA is expressed in the cranial neural crest but is almost absent after ASH3b injection (G). The arrow points out
a small number of twist-positive cells that remain. N-CAM protein expression in an ASH3b-injected embryo treated with HUA (see
text) also shows an expansion of the CNS (H). Arrows denote the borders of the prospective spinal cord; a double-headed arrow shows
the midline. The injected sides of the embryos shown in H and I are marked by nuclear B-galactosidase expression (fine reddish dots).
en-2 mRNA is expressed at the midbrain-hindbrain boundary (I). Like N-CAM, en-2 expression in expanded after ASH3b RNA
injection (l). K and L show embryos injected with a DNA expression vector encoding an epitope-tagged ASH3b protein. The embryos
have been stained with an antibody to the epitope tag (see Materials and methods). (K) ASH3b expression is outside of the dorsal
ectoderm so the CNS does not show any enlargement. (L) ASH3b expression is present in the dorsal ectoderm and the future brain is
expanded (arrows; vertical bar indicates midline). Expression in the embryo in (L) is restricted to the ectoderm (not shown). (M) An
embryo injected with an ASH3b DNA expression vector showing the reduction of myosin heavy chain protein expression. Note that
the blastopore is displaced toward the injected side (arrow). The curved axis is indicated. Similar effects were also seen after ASH3b
RNA injection (not shown). Image published in: Turner DL and Weintraub H (1994) Copyright © 1994. Image reproduced on with permission of the Publisher, Cold Spring Harbor Laboratory Press. This is an Open Access article.
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