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Figure 1. RT-PCR analysis of pfdn6a and tcp1α expression during Xenopus development.
(A) pfdna6 and tcp1α expression during embryonic development. cDNA derived from Xenopus
laevis embryos at different stages (indicated at the top of the panel) was amplified using primers
specific for pfdn6a, tcp1α and ornithine decarboxylase gene (odc). The cDNAs were normalized to
odc. For control reactions, reverse transcriptase was omitted (–RT). (B) Histogram representing the
optical integrated density ratios of pfdn6a, tcp1α and odc RT-PCR bands. The optical density was
calculated using the ImageJ software. Values are means of 3 independent experiments.
Significance: p-value<0.05: *; p-value<0.001: **.
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Figure 2. Whole mount in situ hybridization analysis of pfdn6a and tcp1α during
segmentation and gastrulation. Stages of embryos are indicated at the bottom right corner of each
panel. (st.: stage), while the analyzed gene is indicated to left of each row. (A, C) Lateral views of
stage 7 embryos; animal pole to the top, vegetal pole to the bottom. (A’, C’) Sagittal sections of the
hybridized embryos shown in (A) and (C), respectively; the insets in (A’, C’), indicated by the
dashed line square in each panel, show the intracellular localization of the two transcripts in animal
blastomeres; the white arrow in (C’) shows absence of the tcp1α transcript in subcortical portion of
cytoplasm; white arrowheads in (A’, C’) indicate Hoechst-stained nuclei. (B, D) Lateral view of
stage 101/4 embryos; animal pole to the top, vegetal pole to the bottom. (B’, D’) Sagittal sections of
the hybridized embryos shown in (B) and (D), respectively; black arrows point to the blastopore lip,
square brackets indicate the involuting mesoderm. Scale bars = 250μm.
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Figure 3. Whole mount in situ hybridization analysis of pfdn6a and tcp1α during
neurulation. (A-C, F-H) show frontal views (dorsal to the top) of hybridized embryos at the
indicated neurula stages; white arrowheads indicate neural crests. (D, I) Dorsal view of stage 18/19
embryos injected unilaterally with Rx1 RNA and nuclear LacZ RNA as a tracer (red staining).
Arrows point at regions displaying stronger repression. (E, J) Lateral views of hybridized stage 22-
23 embryos; anterior is to the left; nc: neural crests. Scale bars = 250μm.
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Figure 4. Whole mount in situ hybridization analysis of pfdn6a expression in tadpole
embryos. Stages of embryos are indicated at the bottom right corner of each panel. (B), (D), (J), (L)
are magnified views of (A), (C), (I), and (K), respectively. In panels (A-D, I-L) anterior is to the
left. Dashed lines in (C) point to the planes of the sections shown in (E-H). Dashed lines in (K)
indicate the planes of the sections shown in (M-P). Light blue arrowheads in (G, H) point out a
hybridization signal in lateral plate mesoderm at stage 30. Blue arrowheads point to pfdn6aexpressing
ventral regions of stage 30 (H) and stage 35-36 embryos (O, P). Black arrow in (K)
indicates pronephric developing duct. ba: brachial arches; e: eye; f: forebrain; h:hindbrain; le: lens;
m: midbrain; me: mesencephalon; n: notochord; ov: otic vesicle; p: pronephros; pr: prosencephalon;
rh: rhomboencephalon; r: retina; s: somite; sc: spinal cord; tb: tailbud. Scale bars in (A, C, I, K)=
500 μm; in (B, D, J, L)= 250 μm; in (E-H, M-P)= 200 μm;
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Figure 5. Cryosections of stage 42 embryos hybridized with pfdn6a (A-E) and tcp1α probes
(F-J). The red arrow in (F) denotes absence of tcp1α signal in a dorsal region of prosencephalon.
Black arrowheads in (A, F) indicate a region located above the pharynx, labeled with pfdn6a and
tcp1α, respectively. The asterisks in (B, C, G, H) indicate white matter at the level of diencephalon
(B, G) and rhomboencephalon (C, H). Black arrowheads in (B, G) indicate the Jacobson’s organ.
The white arrowhead in (I) points to the outer plexiform layer of retina. The black arrowheads in (J)
indicate facial muscles. cmz: ciliary marginal zone; ga: ganglia; le: lens; op: olfactory pits; ph:
pharynx; pr: prosencephalon; rh: rhomboencephalon; vp: velar plate. Scale bars in (A-C, E, F-H,
J)= 100 μm; in (D, I)= 50 μm.
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pfdn6 (prefoldin subunit 6) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 16, anterior view, dorsal up.
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pfdn6 (prefoldin subunit 6) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 23, lateral view, anterior left, dorsal up.
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pfdn6 (prefoldin subunit 6) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 33, lateral view, anterior left, dorsal up.
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tcp1 (t-complex 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage, horizontal/lateral view, animal up.
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tcp1 (t-complex 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 13, anterior view, dorsal up.
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tcp1 (t-complex 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, lateral view, anterior left, dorsal up.
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