March 1, 1999;
The homeobox gene Pitx2: mediator of asymmetric left-right signaling in vertebrate heart and gut looping.
asymmetry in vertebrates is controlled by activities emanating from the left lateral
plate. How these signals get transmitted to the forming organs is not known. A candidate mediator in mouse, frog and zebrafish embryos is the homeobox gene Pitx2
. It is asymmetrically expressed in the left lateral plate mesoderm
, tubular heart
and early gut
tube. Localized Pitx2
expression continues when these organs undergo asymmetric looping morphogenesis. Ectopic expression of Xnr1
in the right lateral
plate induces Pitx2
transcription in Xenopus. Misexpression of Pitx2
affects situs and morphology of organs. These experiments suggest a role for Pitx2
in promoting looping of the linear heart
[+] show captions
Fig. 1. The Pitx family of homeobox genes.
Alignment of amino acid sequences of the homeo
domains of vertebrate Pitx2 (identical in human,
mouse, chick, zebrafish, and Xenopus) with
Drosophila Pitx (Dpitx), mouse Pitx1 (mPtx1) and mouse goosecoid (mgsc). Only positions that differ from Pitx2 are shown. The lysine
residue characteristic of the bicoid group of paired type homeoboxes is shown in bold face. Note that the homeo domain of Drosophila Pitx
displays higher homology to vertebrate Pitx2 than to Pitx1.
Fig. 2. Left-sided Pitx2 expression is conserved
between mouse, Xenopus and zebrafish.
(A-D) Pitx2 expression in the mouse from E7.0 to
E9.0. (A) Earliest localized expression of Pitx2 is
seen in two patches in the prospective head
mesenchyme. Anterior view of an E7.0 embryo.
(B) Pitx2 is asymmetrically expressed in the left
LPM and in the left myocardium of the linear
heart tube. Ventral view of a E8.0 embryo (6
somites). hf, headfolds; h, heart; l, left; r, right.
(C,D) Left-sided expression of Pitx2 in the tubular
gut at E9.0. Plane of section in D is indicated by
the line in C. bw, body wall; g, gut; lsp, left
splanchnopleura; rsp, right splanchnopleura; va,
vitelline artery. (E-H) Pitx2 expression in Xenopus
from stage 12 to stage 26. (E-G) From stage 12 to
18 sickle-shaped expression is seen in the cement
gland anlage (E-G) and in two patches of head
mesenchyme (G). (H) At stage 26 strong
expression persists in the cement gland.
Asymmetric expression is seen in the left (top) but
not in the right (bottom) lateral plate mesoderm. l,
left; r, right. (I-L) Pitx2 expression in the zebrafish
embryo from 90% epiboly to 23 hour in the
polster (I-K) and left LPM (L). (I) Animal pole
view of an embryo at 90% epiboly (9 hours).
(J) Animal pole view of a 3 somite embryo (10
hours). (K) Animal pole view of a 11 somite
embryo (14.5 hours). (L) Dorsal view of a 23 hour
embryo. Note that the staining is confined to the
left LPM. l, left; r, right.
Fig. 3. Pitx2 expression in the Xenopus heart and
gut. (A) At stage 30 left-sided Pitx2 expression
(right embryo) extends into the heart region.
Staining of an embryo with the myocardial marker
cardiac troponin I (cTnI; left) is shown for
comparison. Approximate planes of sections in B-E
are indicated by transverse lines. (B-E) Histological
sections of the embryos shown in (A). Note that
Pitx2 expression is restricted to the left
myocardium (C,D) and LPM (E). (F,G) Left-sided
Pitx2 expression in the looping gut at stage 42
(arrowheads in F), and along the outside of the gut
spiral at stage 45 (G). (H) Diagram of a stage 45
embryo and a schematic drawing of gut looping
(Niewkoop and Faber, 1967). Pitx2 mRNA
expression is indicated in red.
Fig. 6. Induction of Pitx2 in the right LPM by Xnr1 and activin.
CMV-lacZ, CMV-activin and synthetic Xnr1 mRNA were injected
into defined blastomeres of Xenopus embryos at the 8-cell stage (AC)
and Pitx2 transcripts were analyzed by whole mount in situ
hybridization at stage 29 (A¢-C¢). (A,A¢) Left-sided Pitx2 expression
in control injected embryos. (B,B¢) Pitx2 expression in the left and
right LPM following bilateral Xnr1 injection. (C,C¢) Bilateral Pitx2
expression following activin injection into dorsal-right blastomeres.
Fig. 7. Induction of Pitx2 by Xnr1 and activin in animal cap explants.
Embryos were injected with goosecoid, activin, Xnr1, eFGF or
BMP-4 RNA into animal blastomeres at the 4-8 cell stage, animal
caps were cut at stage 8, cultured until gastrula (stage 10.5-11), and
assayed for the presence of XPitx2, EF1-a, Xnr1, Xbra and Xvent1
RNA by RT-PCR. Note that Pitx2 mRNA was specifically induced
by activin and Xnr1. Co., control uninjected embryos.
Fig. 8. Alterations of heart and gut laterality and morphology
following misexpression of Pitx2 in Xenopus embryos.
(A) Experimental design. Xenopus embryos were injected at the 8-
cell stage with a Pitx2 DNA expression construct into both dorsal
right blastomeres and cultured up to stage 45-50. Laterality of heart
and gut was assessed in live embryos; heart morphology was
analyzed following isolation of malformed hearts. (B-G) Ventral
view of uninjected control embryos (B,D) with normal situs of the
heart (B) and the gut (D) and Pitx2-injected specimen (C,E)
displaying situs inversion of the heart (C) and the gut (E). Dots
outline the shape of the heart in B and C; arrows indicate direction of
gut looping in D and E. (F) Aberrant gut morphology of Pitx2-
injected embryos. Note that the gut stayed almost linear in these two
embryos. (G) Comparison of normal heart morphology (co) and
three malformed hearts (e1-e3) resulting from Pitx2 injections.
Dorsal is to the right, and ventral to the left. The outflow tract in e2
was disconnected during preparation. Note that experimental hearts
were hypertrophic and showed misalignment of atrium (a), ventricle
(v) and outflow tract (ot).