Elena V. Semina

Cloning and characterization of a novel bicoid-related homeobox transcription factor gene, RIEG, involved in Rieger syndrome

Rieger syndrome (REG) is an autosomal–dominant human disorder that includes anomalies of the anterior chamber of the eye, dental hypoplasia and a protuberant umbilicus. We report the human cDNA and genomic characterization of a new homeobox gene, RIEG, causing this disorder. Six mutations in RIEG were found in individuals with the disorder. The cDNA sequence of Rieg, the murine homologue of RIEG, has also been isolated and shows strong homology with the human sequence. In mouse embryos Rieg mRNA localized in the periocular mesenchyme, maxillary and mandibular epithelia, and umbilicus, all consistent with RIEG abnormalities. The gene is also expressed in Rathkes pouch, vitelline vessels and the limb mesenchyme. RIEG characterization provides opportunities for understanding ocular, dental and umbilical development and the pleiotropic interactions of pituitary and limb morphogenesis.

Pitx2. a bicoid-type homeobox gene, is involved in a lefty-signaling pathway in determination of left-right asymmetry

Signaling molecules such as Activin, Sonic hedgehog, Nodal, Lefty, and Vg1 have been found to be involved in determination of left-right (L-R) asymmetry in the chick, mouse, or frog. However, a common signaling pathway has not yet been identified in vertebrates. We report that Pitx2, a bicoid-type homeobox gene expressed asymmetrically in the left lateral plate mesoderm, may be involved in determination of L-R asymmetry in both mouse and chick. Since Pitx2 appears to be downstream of lefty-1 in the mouse pathway, we examined whether mouse Lefty proteins could affect the expression of Pitx2 in the chick. Our results indicate that a common pathway from lefty-1 to Pitx2 likely exists for determination of L-R asymmetry in vertebrates.

A novel homeobox gene PITX3 is mutated in families with autosomal-dominant cataracts and ASMD

We report here the identification of a new human homeobox gene, PITX3, and its involvement in anterior segment mesenchymal dysgenesis (ASMD) and congenital cataracts in humans. The PITX3 gene is the human homologue of the mouse Pitx3 gene and is a member of the RIEG/PITX homeobox gene family. The protein encoded by PITX3 shows 99% amino-acid identity to the mouse protein, with 100% identity in the homeodomain and approximately 70% overall identity to other members of this family. We mapped the human PITX3 gene to 10q25 using a radiation-hybrid panel. A collection of 80 DNA samples from individuals with various eye anomalies was screened for mutations in the PITX3 gene. We identified two mutations in independent patients. A 17-bp insertion in the 3´-end of the coding sequence, resulting in a frame shift, occured in a patient with ASMD and cataracts, and a G→A substitution, changing a codon for serine into a codon for asparagine, in the 5´-end of the gene occured in a patient with congenital cataracts. Both mutations cosegregate with the disease phenotype in families, and neither were found in up to 300 control individuals studied. Further expression analysis of Pitx3in the mouse supports a unique role in early ocular development, with later expression extending to the midbrain, tongue, incisors, sternum, vertebrae and limbs. These data strongly suggest a role for PITX3 in ASMD and cataracts and provide new evidence of the contribution of the RIEG/PITX gene family to the developmental program underpinning normal eye formation.

Selective loss of dopaminergic neurons in the substantia nigra of Pitx3-deficient aphakia mice

Dopaminergic (DA) neurons in the ventral midbrain nuclei, substantia nigra pars compacta (SNc, A9) and ventral tegmental area (VTA, A10), play important roles in the control of movement, emotion, cognition, and reward related behavior. Although several transcription factors have been shown to be critical for midbrain DA neuron development, there has been no report of factor(s) that differentially regulate individual DA neuronal groups. Based on its highly restricted expression in the SNc and VTA in the brain, we hypothesize that the homeobox transcription factor Pitx3 may critically regulate the development of ventral midbrain DA neurons. In this study, we report that in Pitx3-deficient ak/ak mice, DA neurons in the SNc and the nigrostriatal pathway fail to develop properly, and DA levels are reduced to 10% of the wild type mice in the dorsal striatum. On the contrary, A10 neurons are intact in ak/ak mice and DA levels within their projection areas are not affected. This region-specific defect was already evident in newborn mice, suggesting that the defect had occurred during the early stages of mouse development. Taken together, our results indicate that Pitx3 is the first known transcription factor that may critically and selectively control proper development of A9 DA neurons and the nigrostriatal pathway. This observation is of great importance in understanding the mechanisms of DA neuron development and may also help us to understand the mechanism of selective degeneration of A9 DA neurons in Parkinsons disease and to devise novel therapeutic approaches for the disorder.

Generation of embryonic stem cells and transgenic mice expressing green fluorescence protein in midbrain dopaminergic neurons

We have generated embryonic stem (ES) cells and transgenic mice with green fluorescent protein (GFP) inserted into the Pitx3 locus via homologous recombination. In the central nervous system, Pitx3‐directed GFP was visualized in dopaminergic (DA) neurons in the substantia nigra and ventral tegmental area. Live primary DA neurons can be isolated by fluorescence‐activated cell sorting from these transgenic mouse embryos. In culture, Pitx3–GFP is coexpressed in a proportion of ES‐derived DA neurons. Furthermore, ES cell‐derived Pitx3–GFP expressing DA neurons responded to neurotrophic factors and were sensitive to DA‐specific neurotoxin N‐4‐methyl‐1, 2, 3, 6‐tetrahydropyridine. We anticipate that the Pitx3–GFP ES cells could be used as a powerful model system for functional identification of molecules governing mDA neuron differentiation and for preclinical research including pharmaceutical drug screening and transplantation. The Pitx3 knock‐in mice, on the other hand, could be used for purifying primary neurons for molecular studies associated with the midbrain‐specific DA phenotype at a level not previously feasible. These mice would also provide a useful tool to study DA fate determination from embryo‐ or adult‐derived neural stem cells.

Autosomal dominant iris hypoplasia is caused by a mutation in the rieger syndrome (rieg/pitx2) gene

PURPOSE To determine whether autosomal dominant iris hypoplasia is caused by mutations in the newly described gene for Rieger syndrome (RIEG/PITX2). METHOD Mutation screening and sequence analysis was performed in a single family. RESULTS A novel mutation in the RIEG/PITX2 gene was found in all affected but no unaffected individuals. This mutation would be expected to result in an arginine to tryptophan amino acid change in the homeodomain of solurshin, the RIEG/ITX2 gene product. CONCLUSION Autosomal dominant iris hypoplasia is caused by a defect in the same gene that is defective in many cases of Rieger syndrome.

The Molecular Basis of Rieger Syndrome ANALYSIS OF PITX2 HOMEODOMAIN PROTEIN ACTIVITIES

Rieger syndrome is an autosomal-dominant developmental disorder that includes glaucoma and mild craniofacial dysmorphism in humans. Mutations in the Pitx2 homeobox gene have been linked to Rieger syndrome. We have characterized wild type and mutant Pitx2 activities using electrophoretic mobility shift assays, protein binding, and transient transfection assays. Pitx2 preferentially binds the bicoid homeodomain binding site and transactivates reporter genes containing this site. The combination of Pitx2 and another homeodomain protein, Pit-1, yielded a synergistic 55-fold activation of the prolactin promoter in transfection assays. Addition of Pit-1 increased Pitx2 binding to the bicoidelement in electrophoretic mobility shift assays. Furthermore, we demonstrate specific binding of Pit-1 to Pitx2 in vitro. Thus, wild type Pitx2 DNA binding activity is modulated by protein-protein interactions. We next studied two Rieger mutants. A threonine to proline mutation (T68P) in the second helix of the homeodomain retained DNA binding activity with the same apparentK D and only about a 2-fold reduction in theB max. However, this mutant did not transactivate reporter genes containing the bicoid site. The mutant Pitx2 protein binds Pit-1, but there was no detectable synergism on the prolactin promoter. A second mutation (L54Q) in a highly conserved residue in helix 1 of the homeodomain yielded an unstable protein. Our results provide insights into the potential mechanisms underlying the developmental defects in Rieger syndrome.

The Pitx2 protein in mouse development

The Rieger syndrome, an autosomal dominant disorder involving ocular, dental, and umbilical defects is caused by mutations in PITX2, a Bicoid‐type homeobox protein. Mouse Pitx2 mRNA is expressed in eye, tooth and umbilicus consistent with the human Riegers phenotype. Moreover, Pitx2 is involved in the Nodal/Sonic hedgehog pathway that determines left/right polarity. In this report we demonstrate a 32‐kDa polypeptide on Western blots of nuclear extracts from a rat pituitary cell line, using a Pitx2 specific antibody (designated P2R10). We describe also for the first time expression of the Pitx2 protein in mouse. Pitx2 protein immunostaining was detectable during the development of the eye, tooth, umbilicus, and also in the pituitary, heart, gut, and limb. We demonstrate for the first time directly that Pitx2 is asymmetrically expressed in early heart, gut, and lung development. Dev Dyn;218:195–200.

Rieger syndrome: a clinical, molecular, and biochemical analysis.

Abstract. Rieger syndrome (RIEG 1; MIM 180500) is an autosomal dominant disorder of morphogenesis. It is a phenotypically heterogeneous disorder characterized by malformations of the eyes, teeth, and umbilicus. RIEG belongs to the Axenfeld-Rieger group of anomalies, which includes Axenfeld anomaly and Rieger anomaly (or Rieger eye malformation), which display ocular features only. Recently, mutations in the homeodomain transcription factor, PITX2, have been shown to be associated with Rieger syndrome. This review discusses the clinical manifestations of Rieger syndrome and how they correlate with the current molecular and biochemical studies on this human disorder.

Current molecular understanding of Axenfeld–Rieger syndrome

Axenfeld-Rieger syndrome (ARS) is a rare autosomal dominant inherited disorder affecting the development of the eyes, teeth and abdomen. The syndrome is characterised by complete penetrance but variable expressivity. The ocular component of the ARS phenotype has acquired most clinical attention and has been dissected into a spectrum of developmental eye disorders, of which open-angle glaucoma represents the main challenge in terms of treatment. Mutations in several chromosomal loci have been implicated in ARS, including PITX2, FOXC1 and PAX6. Full-spectrum ARS is caused primarily by mutations in the PITX2 gene. The homeobox transcription factor PITX2 is produced as at least four different transcriptional and splicing isoforms, with different biological properties. Intriguingly, PITX2 is also involved in left-right polarity determination, although asymmetry defects are not a feature of ARS. In experimental animal models and in cell culture experiments using PITX2, abundant evidence indicates that a narrow window of expression level of this gene is vital for its correct function.