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Fig. 1. Schematic of the blastomeres of a 32-cell stage Xenopus
embryo with each cell labeled according to Nakamura and Kishiyama
(1971).
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Fig. 2. Temporal analysis of xGAT-1 expression during Xenopus
development. The graph shows the mean fold change in xGAT-1
mRNA expression for the stages indicated as measured using quantitative
real-time PCR. xGAT-1 expression is first detectable above
background during late neurula stages (stage 20) and increases dramatically
over the course of development. Threshold cycle values were
normalized to control for input template amount and analyzed as
described in Materials and Methods. ue, unfertilized egg.
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Fig. 3. Whole mount xGAT-1 expression in the developing Xenopus
embryo. Insets show a stage-matched embryo hybridized with an
antisense GAD probe. A: xGAT-1 expression is first detected in the
spinal cord during late neurula stages (stage 21). B: xGAT-1 signal is
present in the hindbrain and forebrain by stage 23. C: By stage 26,
expression increases in the spinal cord and can be observed in all
parts of the developing brain. D–F: xGAT-1 expression continues to
strengthen during tailbud stages (D, stage 28; E, stage 32), and by
swimming tadpole stages (F, stage 40) is present at high levels
throughout the central nervous system and eye. G: Higher magnification
of the head of a stage 33 embryo showing distinct domains of
xGAT-1 expression in the forebrain, midbrain, and hindbrain. H: Dorsal
view of a tailbud stage embryo (stage 32). fb, forebrain; hb, hindbrain;
mb, midbrain; ol, olfactory placodes; sc, spinal cord. Scale bar
1 mm in A–H.
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Fig. 4. Histological analysis of xGAT-1 mRNA expression. A–H:
Serial (anterior to posterior) 10- m transverse sections of a larval
stage embryo (stage 34). Left insets show a stage-matched embryo
hybridized with the antisense GAD probe. A: Anteriormost xGAT-1
signal is concentrated in the lateral regions of the forebrain. B: In the
midbrain, expression is found in more ventral regions. Higher magnification
(right inset) demonstrates that xGAT-1 signal is present
immediately adjacent to the neurocoel. C,D: At the level of the hindbrain,
xGAT-1 signal is again concentrated in lateral regions midway
down the dorsal-ventral axis of the structure. E,F: This expression
pattern is maintained in the anterior spinal cord. G,H: xGAT-1
mRNA becomes localized to more distinct regions of the spinal cord
containing Kolmer-Agduhr cells and presumptive interneurons. I: By
swimming tadpole stages, xGAT-1 mRNA expression has increased
dramatically throughout the brain, and is detectable in the retina of
the eye. Arrows indicate areas of xGAT-1 mRNA expression. asterisk,
otic vesicle; cg, cement gland; e, eye; gu, gut; no, notochord. Scale
bar 0.5 mm in A–I.
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Fig. 5. Summary of GABAergic and glutamatergic fate mapping
experiments. Blastomere contributions to GABAergic (A) or glutamatergic
(B) cells in specific regions of the nervous system. Major regions
of the CNS are denoted in black as follows: FB, forebrain; MB, midbrain;
HB, hindbrain; SC, spinal cord. The specific regions of the
forebrain, midbrain, hindbrain, and spinal cord examined for expression
of xGAT-1 or xVGlut1 are indicated by lower case letters: d,
dorsal; i, intermediate; v, ventral. The peripheral structures examined
include the olfactory placodes (OL), the pineal gland (PG), and
the cranial ganglia: V, trigeminal; VII, geniculate; VIII, acousticus;
IX, glossopharyngeal. The approximate percentage of GABAergic or
glutamatergic tissue in a specific CNS region or peripheral structure
contributed by a given blastomere is indicated as follows: boldface
type, 26–50%; regular type, 10–25%; white type with black outline,
0–10%.
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Fig. 6. Colocalization of xGAT-1 and xVGlut1 signal with RLDX.
Representative sections of a B1-injected embryo demonstrating colocalization
of RLDX with xGAT-1 (A–D) or xVGlut1 (E–H) in situ
hybridization signals in the hindbrain. A,E: Brightfield images of
xGAT-1 or xVGlut1 expression with arrowheads indicating ISH staining.
B,F: RLDX fluorescence distribution in the same section. C,G:
Overlay of the brightfield and fluorescent images reveals overlap of
ISH signal with RLDX. D,H: A false-color merged image showing ISH
signal (blue), RLDX-labeled tissue (red), and colocalization of xGAT-1
or xVGlut1 with RLDX (yellow). Scale bar 0.25 mm in A (applies to
A–D), E (applies to E–H).
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Fig. 7. Summary of GABAergic and glutamatergic fate mapping
experiments by structure and blastomere. Blastomere contributions
to tissue expressing either xGAT-1 (left column) or xVGlut1 (right
column) in specific regions of the nervous system. The approximate
percentage of GABAergic or glutamatergic tissue in a specific structure
contributed by a given blastomere is denoted by shades of gray,
with darker shades representing a higher percentage contribution.
These percentages were calculated by averaging the contributions to
dorsal, intermediate, and ventral regions (or, in the case of the cranial
ganglia, each nerve) as described in Materials and Methods. FB,
forebrain; MB, midbrain; HB, hindbrain; SC, spinal cord; CG, cranial
ganglia.
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unnamed (hypothetical protein LOC100127682) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 21, lateral view, anterior right, dorsal up.
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unnamed (hypothetical protein LOC100127682) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior right, dorsal up.
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unnamed (hypothetical protein LOC100127682) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 40, lateral view, anterior right, dorsal up.
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