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The pitx2 gene is a member of the bicoid-homeodomain class of transcription factors that has been implicated in the control of left-right asymmetry during organogenesis. Here we demonstrate that in zebrafish there are two pitx2 isoforms, pitx2a and pitx2c, which show distinct expression patterns and have non-overlapping functions during mesendoderm and asymmetric organ development. pitx2c is expressed symmetrically in presumptive mesendoderm during late blastula stages and in the prechordal plate during late gastrulation. pitx2a expression is first detected at bud stage in the anterior prechordal plate. The regulation of early mesendodermpitx2c expression is dependent on one-eyed pinhead (EGF-CFC-related gene) and spadetail (tbx-transcription factor) and can be induced by ectopic goosecoid expression. Maintenance of pitx2c midline expression is dependent on cyclops (nodal) and schmalspur, but not no tail (brachyury). Ectopic expression of pitx2 isoforms results in distinct morphological and molecular phenotypes, indicating that pitx2a and pitx2c have divergent regulatory functions. Both isoforms downregulate goosecoid on the dorsal side, but in contrast to earlier reports that nodal and lefty are upstream of pitx2, ectopic pitx2c in other regions induces cyclops, lefty2 and goosecoid expression. Asymmetric isoform expression occurs in non-overlapping domains, with pitx2c in left dorsal diencephalon and developing gut and pitx2a in leftheartprimordium. Targeted asymmetric expression in Xenopus shows that both isoforms can alter left-right development, but pitx2a has a slightly stronger effect on heart laterality. Our results indicate that distinct genetic pathways regulate pitx2a and pitx2c isoform expression, and each isoform regulates different downstream pathways during mesendoderm and asymmetric organ development.
Fig. 1. pitx2 isoforms are conserved in vertebrate evolution. (A) The human pitx2 gene uses two promoters (arrows) to generate pitx2a and
pitx2c isoforms that differ in their coding potential in 5¢ regions (Arakawa et al., 1998). The zebrafish genomic structure is unknown. Here, the
Pitx2 proteins predicted from cDNA sequences are compared across vertebrate species; conservation of the amino acid sequence for both
zebrafish isoforms suggests a genomic structure similar to that in humans. Black boxes represent regions encoding homeodomain. (B) The
zebrafish (z) Pitx2a unique region compared to Pitx2a from mouse (m), the human (h) Pitx2a homolog, Arp1a, and Xenopus (xl) Pitx2b. The
nomenclature of the Xenopus isoforms does not conform with the nomenclature of other species. (C) The zebrafish Pitx2c unique region
compared to a similar region in other species including chicken (c). (D) The bicoid-like homeodomain and C-terminal region of zebrafish Pitx2
compared to Pitx2 from other species. The homeodomain region is underlined. Dark gray boxes show regions of identity, while light gray show
conservative amino acid substitutions.
Fig. 7. Ectopic expression of either pitx2a or pitx2c affects heart and
gut morphogenesis, including laterality, in Xenopus embryos.
Embryos were injected at the 4-cell stage on the right side.
(A) Normal heart morphology in an uninjected Xenopus embryo.
(B) Reversed heart in an embryo injected with pitx2a DNA.
(C) Malrotated heart with normal laterality in an embryo injected
with pitx2c DNA. Embryos were fixed at stage 46 and processed for
immunocytochemistry with an anti-cardiac troponin T antibody.
Arrowheads indicate the direction of the heart outflow tract. a,
atrium, v, ventricle. (D) Normal coiling of the gut in a uninjected
embryo. Diagram depicts the counterclockwise coiling of the gut.
(E) Reversed gut coil in an embryo injected with pitx2a.
(F) Abnormal and reversed gut coiling in an embryo injected with
pitx2a. Insets in E and F show improper initiation of coiling.
Fig. 8. Model of genetic interactions involving pitx2 isoforms during
early mesendoderm and prechordal plate development in zebrafish.
Red arrows indicate associations deduced from analysis of mutant
zebrafish embryos. Blue arrows show relationships where increased
expression of pitx2 isoforms induces ectopic expression of targets.
Green lines represent negative regulation of endogenous expression
as a result of ectopic expression. Black lines indicate decisions in cell
fate. (A) Regulation of pitx2c. (B) Regulation of pitx2a. Antagonistic
interactions between cyc and lft and lft regulation of pitx2 were
shown by Bisgrove et al. (1999). In addition to showing that pitx2c is
reduced in cyc and oep mutants (this study; blue arrow), the
regulation of pitx2 by ectopic expression of nodal (Xnr1 in Xenopus
or cyc in fish) was shown by Campione et al. (1999) (red arrow).
