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???displayArticle.abstract??? Nodal and Nodal-related factors play fundamental roles in a number of developmental processes, including mesoderm and endoderm formation, patterning of the anterior neural plate, and determination of bilateral asymmetry in vertebrates. pitx2, a paired-like homeobox gene, has been proposed to act downstream of Nodal in the gene cascade providing left-right cues to the developing organs. Here, we report that pitx2 is required early in the Nodal signaling pathway for specification of the endodermal and mesodermal germ layers. We found that pitx2 is expressed very early during Xenopus and zebrafish development and in many regions where Nodal signaling is required, including the presumptive mesoderm and endoderm at the blastula and gastrula stages and the prechordal mesoderm at later stages. In Xenopus embryos, overexpression of pitx2 caused ectopic expression of goosecoid and sox-17 and interfered with mesoderm formation. Overexpression of pitx2 in Xenopus animal cap explants partially mimics the effects of Nodal overexpression, suggesting that pitx2 is a mediator of Nodal signaling during specification of the endoderm and prechordal plate, but not during mesoderm induction. We further demonstrate that pitx2 is induced by Nodal signaling in Xenopus animal caps and that the early expression of zebrafish pitx2 is absent when the Nodal signaling pathway is inactive. Inhibition of pitx2 function using a chimeric EnR-pitx2 blocked specification of the mesoderm and endoderm and caused severe embryonic defects resembling those seen when Nodal signaling is inhibited. Following inhibition of pitx2 function, the fate of ventral vegetal blastomeres was shifted from an endodermal to a more mesodermal fate, an effect that was reversed by wild-type pitx2. Finally, we show that inhibition of pitx2 function interferes with the response of cells to Nodal signaling. Our results provide direct evidence that pitx2 function is required for normal specification of the endodermal and mesodermal germ layers.
FIG. 1. Early expression of pitx2 transcripts during Xenopus development. (A) Deduced N-terminal sequences of three Pitx2 isoforms. The
sequence common to the different Pitx2 isoforms is in green. The junction between the variable and the common regions coincides with
the beginning of exon 5 in pitx2 (see Schweickert et al., 2000, for the genomic organization of the pitx2 locus). (B) Northern blot of total
RNA prepared at the indicated stages. The blot was probed with an antisense RNA probe derived from the 39 UTR of the cDNA. Similar
results were obtained with a probe containing the coding region. (C) RT-PCR analysis showing that in Xenopus, pitx2b, and pitx2d are
expressed maternally and zygotically while pitx2c is expressed only after MBT. RT-PCR was performed with total RNA prepared at the
indicated stages. (D) Expression of pitx2 in early Xenopus gastrulae (st 101/2) analyzed by dissection and RT-PCR. pitx2b and pitx2c are
expressed predominantly in the vegetal and marginal regions while pitx2d is expressed uniformly. Material from a single embryo was used
for the 1RT control. Five embryos were used for the animal, vegetal, and equatorial explants and two for the dorsal and ventral fragments.
(E–H) pitx2 in situ hybridizations showing an early broad expression at the blastula stage and expression in the dorsal mesoderm and
endoderm at the gastrula stage. In situ hybridizations of bisected embryos are shown in E and G and of whole embryos in F and H. (E) Side
view showing ubiquitous expression of pitx2 at the late blastula stage (stage 93/4). Note the punctate staining in the vegetal hemisphere. (F)
Midgastrula (stage 11) vegetal view, dorsal side at top. (G) Lateral view, dorsal side on the right. pitx2 transcripts are transiently enriched
in the involuting dorsal mesoderm. A slight dorsal–ventral gradient of pitx2 transcripts is apparent in the ectoderm, mesoderm, and, to a
lesser extent, endoderm. (H) Cleared neurula (stage 15). The most prominent expression of pitx2 is in the anteriorectoderm and prechordal
mesoderm.
FIG. 3. pitx2 expression in Xenopus is activated in response to
Nodal. (A) Animal cap assay showing induction of pitx2 expression
in response to Xnr-1 overexpression. Embryos at the four-cell stage
were microinjected at the animal pole in each blastomere with 50
pg of Xnr-1 mRNA. Animal caps were removed at stage 7, treated
continuously with 10 mg/ml cycloheximide, and analyzed at stage
9 in parallel with untreated control caps for the expression of Xbra,
goosecoid, pitx2b, pitx2c, and histone H4. While Xnr-1 induces
Xbra and goosecoid expression in absence of protein synthesis,
treatment with cycloheximide prevents the strong activation of
pitx2c and pitx2b expression.
FIG. 5. The response of Xenopus animal caps to microinjection of pitx2 mRNA partially mimics the response to Xnr-1. Embryos at the
eight-cell stage were microinjected at the animal pole in each blastomere with 25, 75, or 225 pg of pitx2c, pitx2b, or Xnr-1 mRNA. Animal
caps were removed at stage 9 and analyzed at stage 11 by RT-PCR for expression of the marker genes indicated. 2RT, control without
reverse transcription. pitx2 dose-dependently induces goosecoid, sox17b, cerberus, and antivin but not Xbra or chordin. These results are
representative of four independent experiments.
FIG. 6. Effects of overexpression of pitx2. (A) Induction of ectopic
cement gland and mesenchymal tissue by microinjection of pitx2
RNA into ventral blastomeres. Uninjected stage 34 embryo (top)
and two siblings injected with 300 pg of pitx2b RNA into one
ventral cell at the four-cell stage. Lumps of tissue containing
pigmented and secretory cement gland tissue (cg) are visible in
ventral/posterior regions. (B) Stage 41 embryos injected with 200 pg
of pitx2b into the two dorsal vegetal cells at the eight-cell stage
(uninjected embryo at top). Anterior and dorsal axial structures are
absent or greatly reduced, while some additional tail-like structures
have formed. (C) Embryos exposed to ultraviolet irradiation at
the one-cell stage. Injection of 200 pg of pitx2c RNA into one cell
at the four-cell stage induced formation of a cement gland and tail
structures but did not rescue dorsal axial structures. The lower
embryo was irradiated but not injected. (D) Animal cap explants
prepared from stage 9 embryos and cultured until stage 20. The cap
on the right was prepared from an embryo injected at the four-cell
stage with 50 pg of pitx2c RNA into the animal pole of each
blastomere. Extensive cement gland tissue was induced by pitx2c,
and a mass of large, yolky, nonadhesive cells (y) is also present. A
control explant, which has developed epithelial characteristics, is
shown on the left. (EâL) pitx2b RNA (200 pg) was injected into one
or two blastomeres at the four- or eight-cell stage and embryos were
fixed for whole-mount in situ hybridization at stage 10.5 or 11. (E,
F) chordin expression in the organizer region of uninjected (E) and
pitx2b-injected (F) embryos at stage 10.5. chordin expression is
strongly reduced following injection of pitx2 into the two dorsal
vegetal blastomeres at the eight-cell stage. (G, H) Vegetal views of
xbra expression in the presumptive mesoderm of an uninjected (G)
and pitx2b-injected (H) embryo at stage 10.5. Following injection of
pitx2b RNA into the two dorsal vegetal blastomeres at the eightcell
stage, half the Xbra expression has been abolished. (I, J) Lateral
views of sox-17b expression in the endoderm of an uninjected (I)
and a pitx2-injected (J) embryo at stage 11. sox-17b is expressed
ectopically following injection of pitx2 into one blastomere at the
four-cell stage. (K, L) Vegetal views of goosecoid expression in
uninjected embryo (K) or following injection of pitx2 RNA into the
two ventral vegetal blastomeres at the eight-cell stage. Ectopic
patches of goosecoid expression are visible in the vegetal and
marginal regions.
FIG. 7. pitx2-EnR RNA disrupts dorsoanterior development and prevents expression of many endoderm markers. (AâC) Phenotypes
caused by injection of 100 pg of the EnR-pitx2 RNA into the two dorsal vegetal cells at the eight-cell stage, including microcephaly,
reduction of the size of the cement gland, and mild cyclopia (A); mild cyclopia, shortening of the axis, and spinibifida (B); and severe anterior
and posterior truncations and presence of a collapsed blastocoele (b) (C). Uninjected embryos are included at the tops of AâC. (D) Embryos
injected radially with 50 pg of the EnR-pitx2 RNA into each vegetal cell at the eight-cell stage and cultured until stage 27. No epiboly has
occurred, gastrulation was prevented, and no dorsal or anterior structures have developed. (EâL) EnR-pitx2 RNA was injected into various
blastomeres at the four- or eight-cell stage and embryos were fixed for in situ hybridization at stage 10.5â11.5. (E) Xbra expression at stage
10.5 extinguished (arrow) in half the embryo following injection of 50 pg of EnR-pitx2 RNA into two vegetal cells at the eight-cell stage
(compare with Fig. 2F). (F) Similar injection with lineage tracing of the injected blastomere. Note the presence of cells that do not express
Xbra and do not contain the lineage label (arrow). (G) Bisected uninjected stage 11.5 embryos showing cerberus expression throughout the
endoderm, concentrated on the dorsal side. (H) Expression of cerberus is greatly reduced in siblings injected with 50 pg of EnR-pitx2 RNA
into the four vegetal cells at the eight-cell stage. (I) Bisected uninjected stage 10.5 embryos showing sox-17b expression throughout the
endoderm. (J) Siblings injected with 50 pg of EnR-pitx2 RNA into the four vegetal cells at the eight-cell stage showing greatly reduced
expression of sox-17b. (K) Bisected uninjected stage 11 embryo showing expression of sox-17b throughout the endoderm. (L) A sibling
injected with 50 pg of EnR-pitx2 mRNA into two vegetal cells at the eight-cell stage in which expression of sox-17b was abolished in half
of the endoderm territory.
FIG. 9. Change in the fate of vegetal blastomeres injected with low doses of EnR-pitx2 and reversal by wild-type pitx2 mRNA. Embryos
were injected into one ventral vegetal blastomere at the eight-cell stage with b-Gal RNA alone (A, D), b-Gal plus 75 pg EnR-pitx2 RNA (B,
E), b-Gal plus 200 pg wt pitx2 RNA (C), or b-Gal plus 75 pg EnR-pitx2 RNA plus 200 pg of wt pitx2 RNA (F) and processed for detection
of the lineage label around stage 34. The shift in position of the progeny of the injected cells caused by EnR-pitx2 is reversed by wild-type
pitx2. (D, E) Histological sections from equivalent posterior positions through embryos injected with b-Gal RNA (D) or with EnR-pitx2
RNA (E). Note that while in the control embryo b-Gal-positive cells populate mesodermal and endodermal structures, b-Gal-positive cells
are absent from the endoderm in the EnR-pitx2-injected embryo.
FIG. 10. EnR-pitx2 RNA blocks expression of sox-17b, goosecoid, and Xbra in response to Xnr-1. (A, B) Stage 11 embryos injected in two
blastomeres at the four-cell stage with 100 pg of Xnr-1 RNA alone or together with 200 pg of EnR-pitx2 RNA. (A) Ectopic sox-17b (purple)
induced by Xnr-1 (animal view). (B) Siblings in which 200 pg of EnR-pitx2 RNA was co-injected. Ectopic sox-17b transcripts are seen
surrounding sharply delimited patches of unstained cells (arrows). (C, D) Lateral views of embryos from a similar experiment but b-Gal
mRNA was included in the RNA mixture to allow lineage tracing (blue). (C) Injection of Xnr-1 alone induced sox-17b expression both in
the progeny of injected cells and in surrounding cells. (D) EnR-pitx2 mRNA blocked cell-autonomously the induction of sox-17b. (E, F)
Rescue of sox-17b expression by wild-type pitx2 RNA. (E) Cell-autonomous inhibition of induction caused by co-injecting 25 pg of
EnR-pitx2 RNA with 100 pg of Xnr-1 into one blastomere at the four-cell stage. (F) Reversal of the effect by co-injection of 25 pg of EnR-pitx2
RNA, 100 pg of Xnr-1, and 200 pg of wild-type pitx2 RNA. Expression of sox-17b in the clone of injected cells is rescued by co-injection
of wild-type pitx2 RNA. (G) Ectopic goosecoid induced by Xnr-1 mRNA. (H) Sibling in which 200 pg of EnR-pitx2 RNA was co-injected.
Lineage tracing of co-injected b-Gal RNA (blue) shows that ectopic goosecoid is not induced in the progeny of cells overexpressing Xnr-1
when the EnR-pitx2 RNA is present. (I) Ectopic Xbra induced at the periphery of the clone after injection of 50 pg of Xnr-1 into one
blastomere at the four-cell stage. The same dose caused induction of sox-17b in the center of the clone (not shown). (J) Almost complete
inhibition of Xbra induction at the border of the marked clone after co-injection of 50 pg of Xnr-1 and 200 pg of EnR-pitx2 mRNA.
