XB-ART-13523Development. March 1, 1999; 126 (6): 1225-34.
The homeobox gene Pitx2: mediator of asymmetric left-right signaling in vertebrate heart and gut looping.
Left-right 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 and gut.
PubMed ID: 10021341
Article link: Development.
Genes referenced: bmp4 fgf4 gsc nodal1 pitx1 pitx2 tbxt tnni3
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|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).|