Fig. 1. Sequence of the Xenopus
Thylacine 1 and Thylacine 2 genes.
(A) Xenopus Thylacine 1 cDNA
sequence and deduced amino acid
sequence. The amino acids in bold
represent the basic helix-loop-helix
(bHLH) domain. (B) Comparison of
the amino acid sequences of Thylacine
1 and Thylacine 2. Dots indicate
amino acid identity, dashes represent
gaps introduced to maximise the
alignment and black shading indicates
the bHLH domain. (C) Comparison of
Thylacine 1 and Thylacine 2 bHLH
domains with other members of the
bHLH family. Black shading indicates
amino acid identity with Thylacine 1.
Sequences were aligned using the
MegAlign program (DNASTAR Inc.).
GenBank accession numbers for the
Thylacine 1 and Thylacine 2
nucleotide sequences are Y14446 and
Fig. 2. Expression of Thylacine 1. The distribution of Thylacine 1
was analysed by RNase protection assay. (A) Distribution in Xenopus
embryos. Lane 1, undigested probes; lane 2, tRNA control; lanes 3-
11, unfertilised egg, stages 8, 10, 12.5, 14, 17, 20, 23, 34 embryo
RNA respectively. Two embryo equivalents of RNA (10 mg) was
used in each case. (B) Distribution in adult frog tissues. Lane 1,
undigested probes; lane 2, tRNA control; lanes 3-10, intestine,
stomach, gall bladder, spleen, skeletal muscle, lung, liver, stage 20
RNA respectively. 5 mg of total RNA was used in each assay. As an
internal control a probe for XMax2, which is expressed at constant
levels throughout early development and in adult tissues (Tonissen
and Krieg, 1994), was included in the Thylacine 1 assay.
Fig. 3. Localisation of Thylacine 1 transcripts in Xenopus embryos.
In this and other figures, embryos and sections are oriented with
anterior to the left. Embryos were stained in whole mount for the
expression of Thylacine 1 at neurula stages (stage 17: A,B; stage 19:
C,D), tailbud (stage 22/23: E,F; stage 26: G; stage 28: H) and
swimming tadpole (stage 35: I). Dorsal views are shown in A,C and
E, and lateral views in B,D,F,G,H and I.
Fig. 4. Comparison of Thylacine, X-Delta-2 and X-ESR5 expression patterns. Tailbud embryos (stage
27) were stained by whole-mount in situ hybridisation for Thylacine 1 (A,B), X-Delta-2 (C,D) or XESR5
(E,F) expression. Lateral views are shown in A,C and E, and sections of the same embryo
along the longitudinal axis are shown in B,D and F respectively, viewed from a dorsal aspect.
Expression is shown in dark blue and nuclei are counter-stained in red with Fuelgen. Brackets are
used to indicate the relative positions of somitomeres (numbered) and mature somites (labelled in
lower case letters). The forming somite (undergoing rotation) is designated somitomere 1. In all
cases, sections from 10-20 embryos were examined to confirm the precise positioning of the
transcripts relative to the somitomeres.
Fig. 5. Mapping the transcriptional activation domain of Thylacine 1.
Yeast strain Y190 was transformed with a yeast expression vector
encoding the indicated Gal 4-Thylacine 1 chimeras. Amino acids
contained in each mutant are indicated on the left. lacZ activity was
determined by liquid culture assay. Values are expressed as a
percentage of full length Thylacine 1 activity observed for each
mutant and are means ± standard errors of at least three experiments.
Fig. 6. Expression of Thylacine 1 alters the pattern of segmentation
in Xenopus embryos. Embryos were injected on one side, at the twocell
stage, with (A) nlacZ RNA or (B,C) a mixture of Thylacine 1
and nlacZ RNAs. At early tadpole stages, embryos were fixed and
stained in whole-mount with X-gal, which stains the nuclei blue, and
with the 12/101 antibody using HRP immunohistochemistry, which
stains the muscle cells brown. Representative longitudinal sections
are shown with the uninjected side oriented to the top in each case.
(A) Embryo injected with just the nlacZ tracer. (B) Embryo
expressing Thylacine 1 RNA with a ‘mild’ phenotype. (C) Embryo
expressing Thylacine 1 with a ‘strong’ phenotype.
Fig. 7. Ectopic Thylacine expression does not affect neurogenesis.
Embryos injected with (A) nlacZ, (B) a mixture of nlacZ and
Thylacine 1 or (C) a mixture of nlacZ and XNGNR1 RNAs were
fixed at early neurula stages and stained for N-tubulin expression
using whole-mount in situ hybridisation (dark blue-purple). To
determine the injected side, nlacZ expression was detected by X-gal
staining. In all cases dorsal views are shown with the uninjected side
of the embryo oriented to the top of the panel.
Fig. 8. Thylacine expression alters the expression of segmental
markers of the paraxial mesoderm. Embryos injected with (A,B,E,F)
nlacZ, or (C,D,G,H) a mixture of nlacZ and Thylacine 1 RNAs were
fixed at early neurula stages and stained for X-Delta-2 (A-D) or XESR5
(E-H) expression using whole-mount in situ hybridisation
(dark blue-purple). To determine the injected side, nlacZ expression
was detected by X-gal staining. In each pair of panels lateral views of
the uninjected and injected sides of the same embryo are shown.
Fig. 9. Thylacine 1 expression is altered by expression of XSu(
H)DBM and X-Su(H)/Ank. Embryos injected with (A,D)
nlacZ, (B,E) a mixture of nlacZ and X-Su(H)DBM, or (C,F) a
mixture of nlacZ and X-Su(H)/Ank RNAs were fixed at early
neurula stages and stained for either X-Delta-2 (A-C) or
Thylacine 1 (D-F) expression using whole-mount in situ
hybridisation (dark blue-purple). To determine the injected
side, nlacZ expression was detected by X-gal staining. In all
cases dorsal views are shown with the injected side of the
embryo oriented to the top of the panel.