Fig. 1. Structure and expression of Xatv.
(A) Xatv is 66%, 37% and 35% identical to
zebrafish atv, mouse lefty-1 and lefty-2,
respectively (overline indicates hydrophobic
signal sequence; gray boxes, putative proteolytic
cleavage sites; dashes, identical residues; dots,
alignment gaps). Asterisks indicate cysteines of
the TGFb ligand ‘cysteine knot’; additional
cysteines residues conserved between lefty/atv
factors are indicated. (B) TGFb/BMP, lefty, and
Xatv pre-proproteins. Gray, black and white
boxes indicate signal sequence, pro region and
mature ligand peptide, respectively. (C) RT-PCR
analysis of Xatv expression during Xenopus
embryogenesis (stages indicated; 0.1 embryoequivalents/
lane). FGFR, loading control; MBT,
Fig. 2. Marginal zone and midline
expression of Xatv. (A) Stage 10 embryo,
dorsal view. Xatv transcripts are enriched
dorsally in the marginal zone. (B-D) Stage
10.5, 11 and 12. Dorsovegetal views, dorsal
up. Xatv expression within the forming
dorsal midline. Arrowhead, dorsal lip;
asterisk, yolk plug. (E) Stage 12,
longitudinal section, approximate location
shown in D. Xatv expression is seen in
posterior neuroectoderm (open arrow), more
anterior mesendoderm (solid arrow), and at
lower levels in newly involuting
mesendoderm (open arrowhead). Inset:
higher magnification of posterior midline;
Xatv is mostly expressed in deep
neuroectoderm (bracket; dorsal lip
indicated). (F) Stage 14. Midline expression,
dorsal view, anterior left. Bracket shows the
level of the section in G (arrowheads,
circumblastoporal expression). (G) Stage
14, transverse section: Xatv expression in
the prospective floorplate and underlying
mesendoderm. (H) A more posterior section
than in G shows Xatv expression only within
the prospective floorplate. (I) Stage 17.
Dorsal view, anterior left. (J) Stage 19.
Dorsal view, anterior left. Inset:
magnification of midline expression with
transient notochord expression bracketed.
(K,L) Stage 23 lateral view and stage 25
dorsal view, respectively (anterior left),
showing Xatv expression in left dorsal
endoderm (open arrowheads; see P for section), bilateral expression in the posterior dorsal endoderm (open arrows), and left LPM (solid
arrowhead). (M) Stage 23. An anterior transverse section; Xatv expression in floorplate and hypochord. (N) Stage 25. (O) Stage 29/30.
(P) Stage 25. Arrow, left dorsal endoderm expression. a, archenteron; b, blastocoel; fp, floorplate; n, notochord; h, hypochord.
Fig. 3. Asymmetric Xatv expression. (A-D) Uncleared embryos, (F-I) cleared embryos (stages indicated); lateral views, anterior left. Solid
arrowheads, Xatv expression in left LPM; open arrowheads, Xatv expression in left dorsal endoderm. (E) Transverse section of stage 25
embryo; approximate location indicated in C. Note expression in floorplate and hypochord. Solid arrowhead, Xatv expression in the left LPM.
(J,K) Stage 32. Sagittal and transverse section, respectively, of heart regions of an embryo double labeled for cardiac troponin I (pink) and Xatv
(purple), showing Xatv expression in the looping heart tube (bracket). *sectioning artifact. Epidermal coloration is normal embryo
pigmentation. c, cement gland; fp, floorplate; h, hypochord.
Fig. 4. Inhibition of Xnr2 and activin activity by Xatv. Animal caps
injected with Xatv plus or minus Xnr2 or activin RNA (pg/embryo
indicated) were assayed at stage 10.5 or stage 24. At 1:1 ratios (10 pg
each RNA), marker induction was similar to that caused by Xnr2
alone. In contrast, a 10:1 mix of Xatv:Xnr2 RNA suppressed
organizer-specific markers (cerberus, chordin, goosecoid and
noggin). At 5:1 or 50:1 ratios of Xatv:activin RNA, Xatv suppressed
all markers tested.
Fig. 5. Induction of Xatv in the LPM by Xnr1. pXEX/Xnr1 or
pCSKA/Xnr1 was injected into the right side of 4-cell embryos to
drive Xnr1 expression from late blastula or gastrula stages,
respectively, and Xatv expression analyzed at stage 24-25. Dorsal
views, anterior up. (A) Normal asymmetric expression of Xatv;
(B) bilateral expression; (C) right side expression and (D) no
expression. Bilateral expression predominated (Table 1). (E) Animal
caps injected with Xnr1, Xnr2, or activin RNA were assayed at stage
10.5 for Xatv and Xbra. Xbra, a pan-mesodermal marker, measured
induction efficiency. FGFR, loading control.
Fig. 6. Suppression of left LPM
Xnr1 and XPitx2 expression by
Xatv. pXEX/Xatv or
pCSKA/Xatv was injected into
the left side of 4-cell embryos
and (A-D) XPitx2, or (E,F) Xnr1
(A,B) Lateral views and (C,D)
dorsal views (anterior left).
(A,C) Normal XPitx2 expression
in left LPM (bracket) is (B,D)
suppressed by left-sided Xatv
overexpression. Bilateral XPitx2
expression in the eyes, head,
branchial arches and cement gland (arrowheads in A,B) remains relatively unaffected. (E,F) Dorsal views, anterior up. (E) Normal Xnr1
expression in left LPM is (F) suppressed by left-sided Xatv overexpression.
Fig. 7. Morphological analysis of Xatv-injected embryos. Plasmids
(100 pg) encoding Xnr1 or Xatv were injected into either the right
(R) or left (L) of 4-cell embryos and morphology analyzed at stage
43-45. (A,D,G,J,M) Lateral views; (B,E,H,K,N) corresponding
ventral views; anterior up. (C,F,I,L,O) Higher magnification of heart
region. (A-C) Uninjected embryo. (D-F) Reversed heart and gut situs
in right-side Xnr1 injections, for comparative purposes. (G-I) Normal
heart and gut situs in right-side Xatv-injected embryos. (J-O) Two
examples of embryos receiving Xatv on the left (percentage
incidences indicated; see Table 4 for details).
Fig. 8. A model for the role of
Xatv in the establishment of L-R
asymmetry. Time flows from top
to bottom. A hypothetical factor
‘X’ that is proposed to propagate
activates Xnr1 within the left
LPM during early tailbud stages.
Xnr1 activates XPitx2 expression
directly, or through intermediary
steps. Xnr1 also induces
asymmetric Xatv expression.
With a delay period, Xatv acts in
a negative feedback loop to
suppress Xnr1 expression.
Preliminary data suggest that
Xnr1 regulates its own
expression. In the experiments reported here, Xatv may inhibit
XPitx2 directly (dashed bar), or perhaps more likely by directly
suppressing Xnr1 (solid bar). The possibility of yet undiscovered
genes in Xenopus acting in a right-sided gene cascade, as in other
species (see text), is indicated.