Pitx genes in development and disease

Abstract

Homeobox genes encode sequence-specific transcription factors (SSTFs) that recognize specific DNA sequences and regulate organogenesis in all eukaryotes. They are essential in specifying spatial and temporal cell identity and as a result, their mutations often cause severe developmental defects. Pitx genes belong to the PRD class of the highly evolutionary conserved homeobox genes in all animals. Vertebrates possess three Pitx paralogs, Pitx1, Pitx2, and Pitx3 while non-vertebrates have only one Pitx gene. The ancient role of regulating left-right (LR) asymmetry is conserved while new functions emerge to afford more complex body plan and functionalities. In mouse, Pitx1 regulates hindlimb tissue patterning and pituitary development. Pitx2 is essential for the development of the oral cavity and abdominal wall while regulates the formation and symmetry of other organs including pituitary, heart, gut, lung among others by controlling growth control genes upon activation of the Wnt/ß-catenin signaling pathway. Pitx3 is essential for lens development and migration and survival of the dopaminergic neurons of the substantia nigra. Pitx gene mutations are linked to various congenital defects and cancers in humans. Pitx gene family has the potential to offer a new approach in regenerative medicine and aid in identifying new drug targets.

Keywords: Development; Disease; Evolution; Homeobox genes; Pitx.

Fig. 1

The evolutionarily conserved Pitx gene family. a Pitx genes have been identified in most animal groups from the simplest to the most complex in the animal kingdom. Among the four basal phyla, pitx genes have not been determined in Porifera and Ctenophora. In Placozoa, pitx expression has been evidenced but no associated function has been reported. Pitx genes in members of the Cnidaria and Bilateria have been well characterized. In Protostomes, Bicoid is the homolog of Pitx. Only vertebrates possess Pitx1, Pitx2, and Pitx3. b Phylogenetic tree of the Pitx gene family in animal by protein sequence alignments. All genes are represented in human (H. sapiens), mouse (M. musculus), chick (G. gallus), zebra fish (D. rerio), fruit fly (D. melanogaster), jellyfish (C. hemisphaerica), sea vase (C. intestinalis), amphioxus (B. belcheri), starfish (P. pectinifera), and acorn worm (S. kowalevskii). There is an incredible conservation of PITX protein sequences of species belong to different groups of animals. There are two main duplication events. The first duplication gives rise to Pitx3 and a precursor of Pitx1/2 and the latter of which ultimately becomes Pitx1 and Pitx2 in the second duplication event. c Pitx in molecular pathway that regulates asymmetrical branching in hydra. The initial broad expression of Wnt/ß-Catenin (orange) induces a more localized expression of Nodal (yellow) around the budding site. Nodal activates the transcription of Pitx (green) that is restricted to budding site to initiate branching. d Anterior segment specification by bicoid in fruit fly (D. melanogaster). Two maternal genes that specify the anterior and posterior of the body plan of fruit fly are bicoid (green) and nanos (pink). Before fertilization, bicoid mRNA is most abundant in the anterior while nanos is highly expressed in the opposite pole of the egg. Upon fertilization, bicoid mRNA is translated to protein which enhances the expression of hunchback and other genes associated with the anterior segment while at the same time inhibit the transcription of genes regulating the posterior region. e Left–right asymmetry in non-vertebrate deuterostomes. The molecular pathway that determines the left-side morphology in invertebrates deuterostomes starts with asymmetric expression of Nodal (yellow) that activates the transcription of Pitx (green). PITX proteins then bind to the enhancer regions and activate downstream target genes that are responsible for regulating left–right asymmetry. f Left–right asymmetry in vertebrates. In chick embryo at stage 8, Pitx2 (blue) is expressed broadly in the left lateral plate mesoderm but is completely absent in the right counterpart. Pitx2 acts downstream of Nodal and Shh

Fig. 2

Genomic loci and alternative splicing of Pitx genes in mouse and human. a The Pitx1 locus is located in mouse chromosome 13 and human chromosome 5. Only one transcript coding for one isoform of PITX1 protein has been identified in both species. b The PITX2 protein possesses several different isoforms as a result of alternative splicing and translation sites. The DNA sequence coding for Pitx2 is located in mouse chromosome 3 and human chromosome 4. c The Pitx3 locus is found in mouse chromosome 19 and human chromosome 10 with only one transcript identified in both species. All PITX proteins possess the homeodomain (orange), OAR-motif (cyan) and a nuclear localization signal (green) located within the OAR

Fig. 3

Expression patterns of mouse Pitx genes during embryogenesis. The expression patterns of Pitx1 (red), Pitx2 (blue), and Pitx3 (green) are depicted in embryonic day 7.5 (E7.5) to E13.5. Pitx1 is first detected at E7.5 in the allantois and posterior lateral plate mesoderm. As embryogenesis progresses, the expression domain of Pitx1 is restricted to the head and hindlimb. Pitx2 is first detected at E8.0 in the somatopleure and oral cavity. At E9.5, it is strongly expressed in first and second branchial arches (BA) and left lateral plate mesoderm. By E13.5, Pitx2 is expressed in most facial and extraocular muscles, pituitary, tongue, tooth buds, myotomes and skeletal muscles of fore- and hindlimbs. Pitx3 is expressed at E10.5 in the eye lens and somites. By E12.5, Pitx3 expression domain includes the lens, ventral midbrain and their axonal projections, facial muscles and muscles of the trunk and fore- and hindlimbs

Fig. 4

Pitx genes in organogenesis. Pitx genes are essential in organogenesis. Pitx expressing adult brain and organs at E12.5 and of wild type and KO mice are depicted. Malformed organs and missing structures in mutants are depicted by a dashed line. Loss of Pitx2 and Pitx3 results in the absence of the subthalamic nuclei and cells of the substantia nigra. Both Pitx1 and Pitx2 are essential for the development of pituitary, tooth, cardiovascular system, and branchial arches-derived tissues. Although all Pitx genes are expressed in the hindlimb, only Pitx1 is crucial for the hindlimb patterning. Pitx2 and Pitx3 are indispensable in eye development. Loss of Pitx2 results in the absence of extraocular muscles, complete loss of the anterior chamber, and lack of corneal epithelium. Loss of Pitx3 causes abnormal lens development. The function of Pitx2 is also critical in heart, lung, stomach, and forelimb muscle development. Pitx2 is asymmetrically expressed in the heart and lungs and its loss results in multiple cardiac phenotypes, right lung isomerism and altered gut rotation. Pitx2 specifies facial myogenesis and regulates skeletal myogenesis

Fig. 5

Signaling pathways associated with Pitx2. a Pitx2 is induced by the Wnt/Dvl/ß-catenin pathway and is required for effective cell-type-specific proliferation by directly activating specific growth-regulating genes. Upon Wnt3 binding to Frizzled transmembrane protein (FZD), some of ß-catenin is translocated to the nucleus where it forms a complex with Tcf/LEF1 that displaces HDAC1 from the promoter region of Pitx2 and activate its transcription. Regulated exchange of HDAC1/ß-catenin converts Pitx2 from a repressor to activator, to serve as a competence factor required for the temporally ordered and growth factor-dependent recruitment of a series of specific coactivator complexes that prove necessary for induction of the growth control genes Ccnd2, Ccdn1, cjun and cmyc. b The expression of Pitx2 in the LPM is induced by the asymmetric expression of Nodal. Once released and cleaved into mature ligand, Nodal homodimer binds to the TGFRßI/TGFRßII heterodimer and results in a subsequent phosphorylation and dissociation of R-Smad from the TGFßR, followed by the trimerization of two R-Smads and Smad4. Smad trans-locates to the nucleus where it interacts with FoxH1 and/or Mixer on the promoters of Pitx2, Nodal, and Lefty