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Fig. 1. Characterization of Xenopus Tes. (A) Amino acid sequence of Xtes with major domains highlighted. Underlined is the PET (Prickle, Espinas, Tes) domain.
Double underlined are the three LIM domain repeats. The critical cysteine and histidine residues for the double Zn+2 finger are indicated in bold small caps. (B)
Schematic drawings of the Tes family of proteins. The percent sequence identity of the PETand LIM domains from Xtes with other vertebrate orthologues is indicated.
Overall sequence identity is also indicated. Accession numbers are: X. laevis BC045027; chicken NM_204623; mouse NM_207176; human NM_152829; zebrafish
AAH75889. (C) Phylogenetic tree of the PET-LIM family. Additional accession numbers are: human dyxin (LMCD1) NM_014583; human LMO6 CAG33492; X.
laevis prickle A AF387815; X. laevis prickle B AY055473; zebrafish prickle1 NM_183342; Drosophila prickle (pk) AJ243708.
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Fig. 2. Expression of Xtes during early Xenopus development. (A) Real-time RTPCR
based expression profile of Xtes from stage 1 through to tail bud stage 32.
Values have been normalized to those of ornithine decarboxylase (ODC). (B–L)
In situ hybridization analysis of Xtes expression. (B) 4-cell stage embryo. (C)
Stage 18; arrows point to strong expression of Xtes next to the neural folds. (D)
Cross-section at neurula stage showing Xtes expression between the epithelium
and mesoderm. (E–H) Xtes expression is found in an anterior stream of cranial
neural crest. (E) White arrow marks the stream of Xtes expressing cells, whose
pattern is similar to the most rostral stream of XSlug positive neural crest cells (F;
white arrow). (G) Xtes expression ventral to the eye (black arrowhead)
corresponds to the most anterior cranial neural crest that is positive for Xtwist
expression (black arrowhead in panel H). White arrowhead and asterisks in
panel G denote Xkrox20 expression in rhombomeres 3 and 5 and the anterior
branchial crest, respectively. (I) Expression of Xtes in the head, in an embryo in
which BM purple stain development was stopped early; asterisks mark the three
lateral line placodes. (J) View of a whole tail bud embryo showing strong
expression in the notochord (nc). Staining was also evident in the otic vesicle
(arrow) and the rostral region of the dorsal fin (arrowhead). (K, L) If the BM
purple stain is allowed to develop longer, Xtes expression becomes evident
along the somitic boundaries (white arrowheads in the higher magnification
view in panel K).
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Fig. 3. Over-expression of Xtes RNA causes pigment disruption in the epithelium of the embryo. (A–C) Embryos were either (A) uninjected or (B, C) injected with
1 ng of Xtes RNA into one cell at the two-cell stage. Xtes-injected embryos developed normally save for small patches in which the pigmentation of the epithelium
became abnormal. These epithelial disruptions corresponded to the original site of injection of the two-cell embryo. A typical epithelial pigment disruption from one
embryo (white dotted box in panel B) has been enlarged in panel C. (D, E) Expression and localization of GFP-Xtes. Embryos were injected with (D) GFP or (E, F)
GFP-Xtes. At stage 8.5, animal caps were dissected from the embryos and cultured on fibronectin-coated coverslips until midgastrula stages. (D) Confocal image of a
GFP-injected cap, in which GFP expression was present throughout the cytoplasm. In contrast, GFP-Xtes was localized to the cell periphery (E). (F) Epifluorescent
image of the trunk of an embryo expressing GFP-Xtes. Peripheral localization of GFP-Xtes was evident in the epithelium (arrow) but not in the myotome (arrowhead);
inset shows higher magnification of white dotted box.
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Fig. 4. Antisense morpholino oligonucleotide depletion of Xtes protein. (A) Sequences of Xtes MOs aligned with the 5′-untranslated region (UTR) of Xtes mRNA. The
changed nucleotides in the MO1 control are in bold, and the ATG initiation codon is underlined. (B) Xtes MO inhibition of an in vitro transcription–translation of Xtes
RNA that includes the 5′-UTR sequences. Arrow indicates the band corresponding to Xtes protein. (C–H) Phenotypes of embryos either (C) uninjected or injected
with (D, E) MO1, (F) ConMO1, (G, H) MO2. Panels D and G are lateral views and panels E and H are anterior views.
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Fig. 5. Expression of anterior neural markers is unaffected in embryos lacking Xtes function. (A) Real-time PCR analysis of organizer gene expression following Xtes
depletion. Embryos were injected at the 1 cell stage with 80 ng of either Xtes MO1 or Xtes ConMO1, and then harvested for RNA analysis at stage 12. Values were
normalized relative to ODC and then expressed as a ratio of Xtes MO1:Xtes ConMO1. A value of 1 indicates that the expression was the same in Xtes-depleted
embryos and those injected with the control MO. (B–M) Xtes depletion does not alter anterior neural patterning. Embryos were injected with either (B, E, H, K) Con
MO1, or (C, F, I, L) Xtes MO1, or (D, G, J, M) Xtes MO2 at the 1 cell stage, and then expression of the anterior neural markers (B–D) Sox3, (E–G) XBF-1, (H–J) XRx1
and (K–M) En-2/Krox20 was analyzed by in situ hybridization. (J) Arrowheads denote the expression of Krox20 in rhombomeres r3 and r5, while En-2 at the midbrain
hindbrain isthmus is indicated with an arrow.
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Fig. 6. Cranial neural crest migration is altered in embryos lacking Xtes
function. Expression pattern of neural crest markers (A–C) Xslug and (D–F)
FoxD3 was normal in (A, D) Xtes ConMO1 (B, E) Xtes MO1, (C, F) Xtes MO2-
injected embryos. (G–I) Neural crest streams, marked by Xslug expression,
develop normally in Xtes MO-injected embryos. (G) Xtes ConMO1, (H) Xtes
MO1, (I) Xtes MO2. (J–M) Xtwist expression. One dorsal blastomere was
injected at the 4-cell stage with either (J, K) Xtes ConMO1 or (L, M) Xtes MO1.
β-gal RNA was injected with the MO as a lineage marker. (J, L) Control
uninjected sides; (K, M) injected sides. The arrowhead in panel M shows the
abnormal migration of Xtwist positive neural crest in the Xtes MO1-injected
embryo. (L–O) The role of Xtes in neural crest cell migration was further
explored by transplanting neural crest from stage 16 embryos that had been
injected with either (N) Xtes ConMO1 or (O) Xtes MO1. Fluorescent dextran
was added to the MOs to mark the transplant. (N) Cranial neural crest from an
Xtes ConMO1 donor migrated to occupy a region dorsal to the cement gland
(100%, n = 17). (O) There was a more limited migration of donor Xtes MO1
neural crest (31%, n = 19); note the limited signal ventral to the eye, and the
stream of labeled cells on the caudal rim of the developing eye (white
arrowhead).
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Fig. 7. Gain or loss of function of Xtes does not inhibit activin-dependent convergent extension movements in animal caps. (A–D) Embryos at the 1 cell stage were
either (A, B) uninjected or injected with 1 ng of either (C) Xtes or (D) Xspry2 RNA. At stage 8.5, animal caps were dissected and either (A) left untreated or (B–D)
treated with 16 U of activin until control embryos had reached stage 18. (E–H) In a separate experiment, effects of Xtes-depletion on convergent extension were tested
by treating animal caps from either (F) uninjected, (G) Xtes ConMO1 or (H) Xtes MO1-injected embryos with activin until stage 18 equivalent. Untreated control
uninjected embryos are shown in panel E. (I–L) Xtes depletion does not inhibit convergent extension in dorsal marginal zone (DMZ) explants. DMZs were excised
from early gastrula stage 10 embryos either (I) uninjected or injected with (J) Xtes ConMO1, (K) Xtes MO1 or (L) Xtes MO2. They were cultured to the equivalent of
stage 18.
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Fig. 8. Xtes MO depletion causes defects in somitogenesis and AP patterning. Axis elongation was examined by immunostaining for either (A–D) notochord using the
monoclonal antibody MZ15, which recognizes keratan sulfate, or (E–H) with 12–101, which recognizes differentiated muscle. Embryos were either (A, E) uninjected
or injected at the 1 cell stage with 80 ng of (B, F) Xtes ConMO1, (C–G) Xtes MO1 or (D–H) Xtes MO2. (I–K) 12–101 immunostaining of embryos injected with
40 ng of either (I) Xtes ConMO1 or (J, K) Xtes MO1. The 12 somites formed during gastrulation are indicated with a white arrowhead in panels I and J, with the 20th
somite also indicated in panel I. Xtes MO1-injected embryos displayed either (J) a weak patterning defect in the first 12 somites or (K) a complete loss of somitic
patterning, but in both cases (J, K) postgastrulation somitogenesis was disrupted. (L–W) The expression patterns of various genes expressed during tail bud stages (L,
P, T) FGF8, (M, Q, U) Xdelta-1, (N, O, R, S, V, W) and Xcad3 were determined by in situ hybridization at (L–N, P–R, T–V) stage 20 in embryos injected at the 1 cell
stage with either (L–N) Xtes ConMO1, (P–R) Xtes MO1 or (T–V) Xtes MO2. Note the loss of rostral FGF8 staining (white bracket) in (P) Xtes MO1 and (T) Xtes
MO2-injected embryos compared to (L) embryos injected with Xtes conMO1. White arrows in panels M, Q and U denote somitic expression of Xdelta-1. Expression
of Xcad3 in stage 28 embryos injected with either (O) Xtes conMO1, (S) Xtes MO1 or (W) Xtes MO2. The white arrowhead in panel O denotes the most rostral
expression of Xcad3 in the neural tube.
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Fig. 9. Xpk-depletion inhibits axis elongation. (A) Schematic drawing of Xtes and XpkA with % identities of the conserved PET and LIM domains indicated. (B)
Sequences of Xpk MOs aligned with the 5′-untranslated region (UTR) of XpkA mRNA. The ATG initiation codon is in bold in the cDNA and underlined in the two
Xpk MOs. XpkA is designed against the XpkA isoform, while XpkU denotes the morpholino designed by Takeuchi and colleagues (2003). (C) Inhibition of in vitro
transcription/translation of an Xpk expression construct that includes 5′ UTR sequences. Arrow indicates the band corresponding to Xpk protein. (D) The lengths of the
axes of embryos injected with Xpk MO are shorter than those of control uninjected embryos. * Indicates a significant difference in axis length between XpkA- and
XpkU-MO-injected embryos compared with control embryos (Student's t test; P < 0.05). ** Indicates no significant difference between XpkA and XpkU-injected
embryos. (E–I) Phenotypes of embryos injected with Xpk MO. Embryos were injected with either 40 ng of (E) XpkA MO, (F) XpkUMO or 80 ng of (H) XpkA MO or
(I) XpkU MO. Panel G shows uninjected control embryos.
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Fig. 10. Xtes and Xpk act synergistically to control axis elongation. (A–F) Phenotypes of embryos injected with Xtes and Xpk morpholinos. Embryos were injected at
the 1 cell stage with either (A) 80 ng of Xtes ConMO1, (B) 40 ng of XpkA MO, (C) 80 ng XpkA MO, (D) 40 ng Xtes MO1, (E) 40 ng each of Xtes MO1 and XpkA
MO, (F) 40 ng Xtes MO1 or 60 ng XpkA MO. (G–L) Depletion of the PET-LIM domain proteins Xtes or Xpk does not affect activin-dependent convergent extension.
Animal caps derived from the siblings of the embryos shown in panels A–F were either (G) untreated or (H–L) treated with 16 U of Activin and cultured until the
equivalent of stage 18. (G) Untreated control caps injected with 80 ng Xtes ConMO1. Caps treated with activin and derived from embryos injected with (H) 80 ng Xtes
ConMO1, (I) 40 ng Xtes MO1, (J) 80 ng XpkA MO, (K) 40 ng Xtes MO1/40 ng XpkA MO and (L) 40 ng Xtes MO1/60 ng XpkA MO. (M–P) Combined depletion of
Xtes and Xpk does not inhibit convergent extension in dorsal marginal zone explants. DMZs were excised from stage 10 embryos either (N) uninjected or injected with
either (O) 80 ng Xtes MO1, (P) 80 XpkA MO1 or (Q) 40 ng Xtes MO/60 ng XpkA MO and cultured to the equivalent of stage 18.
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tes (testis derived transcript (3 LIM domains)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 3, horizontal view, animal up.
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tes (testis derived transcript (3 LIM domains)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, dorsal view, anterior left.
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tes (testis derived transcript (3 LIM domains)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 36, lateral view, anterior left, dorsal up.
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