Homeodomain Transcription Factor Pitx2 Research Articles

Genetically Defined Functional Modules for Spatial Orienting in the Mouse Superior Colliculus.

SummaryIn order to explore and interact with their surroundings, animals need to orient toward specific positions in space. Throughout the animal kingdom, head movements represent a primary form of orienting behavior. The superior colliculus (SC) is a fundamental structure for the generation of orienting responses, but how genetically distinct groups of collicular neurons contribute to these spatially tuned behaviors remains largely to be defined. Here, through the genetic dissection of the murine SC, we identify a functionally and genetically homogeneous subclass of glutamatergic neurons defined by the expression of the paired-like homeodomain transcription factor Pitx2. We show that the optogenetic stimulation of Pitx2ON neurons drives three-dimensional head displacements characterized by stepwise, saccade-like kinematics. Furthermore, during naturalistic foraging behavior, the activity of Pitx2ON neurons precedes and predicts the onset of spatially tuned head movements. Intriguingly, we reveal that Pitx2ON neurons are clustered in an orderly array of anatomical modules that tile the entire intermediate layer of the SC. Such a modular organization gives origin to a discrete and discontinuous representation of the motor space, with each Pitx2ON module subtending a defined portion of the animal’s egocentric space. The modularity of Pitx2ON neurons provides an anatomical substrate for the convergence of spatially coherent sensory and motor signals of cortical and subcortical origins, thereby promoting the recruitment of appropriate movement vectors. Overall, these data support the view of the superior colliculus as a selectively addressable and modularly organized spatial-motor register.

Requirement of Pitx2 for skeletal muscle homeostasis

Skeletal muscle is generated by the successive incorporation of primary (embryonic), secondary (fetal), and tertiary (adult) fibers into muscle. Conditional excision of Pitx2 function by an MCKCre driver resulted in animals with histological and ultrastructural defects in P30 muscles and fibers, respectively. Mutant muscle showed severe reduction in mitochondria and FoxO3-mediated mitophagy. Both oxidative and glycolytic energy metabolism were reduced. Conditional excision was limited to fetal muscle fibers after the G1-G0 transition and resulted in altered MHC, Rac1, MEF2a, and alpha-tubulin expression within these fibers. The onset of excision, monitored by a nuclear reporter gene, was observed as early as E16. Muscle at this stage was already severely malformed, but appeared to recover by P30 by the expansion of adjoining larger fibers. Our studies demonstrate that the homeodomain transcription factor Pitx2 has a postmitotic role in maintaining skeletal muscle integrity and energy homeostasis in fetal muscle fibers.

The homeodomain transcription factor PITX2 is required for specifying correct cell fates and establishing angiogenic privilege in the developing cornea.

Correct specification of cell lineages and establishing angiogenic privilege within the developing cornea are essential for normal vision but the mechanisms controlling these processes are poorly understood. We show that the homeodomain transcription factor PItX2 is expressed in mesenchymal cells of the developing and mature cornea and use a temporal gene knockout approach to demonstrate that PITX2 is required for corneal morphogenesis and the specification of cell fates within the surface ectoderm and mesenchymal primordia. PITX2 is also required to establish angiogenic privilege in the developing cornea. Further, the expression of Dkk2 and suppression of canonical Wnt signaling activity levels are key mechanisms by which PITX2 specifies ocular surface ectoderm as cornea. In contrast, specifying the underlying mesenchyme to corneal fates and establishing angiogenic privilege in the cornea are less sensitive to DKK2 activity. Finally, the cellular expression patterns of FOXC2, PITX1, and BARX2 in Pitx2 and Dkk2 mutants suggest that these transcription factors may be involved in specifying cell fate and establishing angiogenic privilege within the corneal mesenchyme. However, they are unlikely to play a role in specifying cell fate within the corneal ectoderm. Together, these data provide important insights into the mechanisms regulating cornea development.

Pitx2 Expression Promotes p21 Expression and Cell Cycle Exit in Neural Stem Cells

Cortical development is a complex process that involves many events including proliferation, cell cycle exit and differentiation that need to be appropriately synchronized. Neural stem cells (NSCs) isolated from embryonic cortex are characterized by their ability of self-renewal under continued maintenance of multipotency. Cell cycle progression and arrest during development is regulated by numerous factors, including cyclins, cyclin dependent kinases and their inhibitors. In this study, we exogenously expressed the homeodomain transcription factor Pitx2, usually expressed in postmitotic progenitors and neurons of the embryonic cortex, in NSCs with low expression of endogenous Pitx2. We found that Pitx2 expression induced a rapid decrease in proliferation associated with an accumulation of NSCs in G1 phase. A search for potential cell cycle inhibitors responsible for such cell cycle exit of NSCs revealed that Pitx2 expression caused a rapid and dramatic (≉20-fold) increase in expression of the cell cycle inhibitor p21 (WAF1/Cip1). In addition, Pitx2 bound directly to the p21 promoter as assessed by chromatin immunoprecipitation (ChIP) in NSCs. Surprisingly, Pitx2 expression was not associated with an increase in differentiation markers, but instead the expression of nestin, associated with undifferentiated NSCs, was maintained. Our results suggest that Pitx2 promotes p21 expression and induces cell cycle exit in neural progenitors.

Prediction of gene network models in limb muscle precursors

The ventrolateral dermomyotome gives rise to all muscles of the limbs through the delamination and migration of cells into the limb buds. These cells proliferate and form myoblasts, withdraw from the cell cycle and become terminally differentiated. The myogenic lineage colonizes pre-patterned regions to form muscle anlagen as muscle fibers are assembled. The regulatory mechanisms that control the later steps of this myogenic program are not well understood. The homeodomain transcription factor Pitx2 is expressed in the muscle lineage from the migration of precursors to adult muscle. Ablation of Pitx2 results in distortion, rather than loss, of limb muscle anlagen, suggesting that its function becomes critical during the colonization of, and/or fiber assembly in, the anlagen. Gene expression arrays were used to identify changes in gene expression in flow-sorted migratory muscle precursors, labeled by Lbx1EGFP, which resulted from the loss of Pitx2. Target genes of Pitx2 were clustered using the “David Bioinformatics Functional Annotation Tool” to bin genes according to enrichment of gene ontology keywords. This provided a way to both narrow the target genes and identify potential gene families regulated by Pitx2. Representative target genes in the most enriched bins were analyzed for the presence and evolutionary conservation of Pitx2 consensus binding sequence, TAATCY, on the −20kb, intronic, and coding regions of the genes. Fifteen Pitx2 target genes were selected based on the above analysis and were identified as having functions involving cytoskeleton organization, tissue specification, and transcription factors. Data from these studies suggest that Pitx2 acts to regulate cell motility and expression of muscle specific genes in the muscle precursors during forelimb muscle development. This work provides a framework to develop the gene network leading to skeletal muscle development, growth and regeneration.

Regulation of Motility of Myogenic Cells in Filling Limb Muscle Anlagen by Pitx2

Cells of the ventrolateral dermomyotome delaminate and migrate into the limb buds where they give rise to all muscles of the limbs. The migratory cells proliferate and form myoblasts, which withdraw from the cell cycle to become terminally differentiated myocytes. The myogenic lineage colonizes pre-patterned regions to form muscle anlagen as muscle fibers are assembled. The regulatory mechanisms that control the later steps of this myogenic program are not well understood. The homeodomain transcription factor Pitx2 is expressed specifically in the muscle lineage from the migration of precursors to adult muscle. Ablation of Pitx2 results in distortion, rather than loss, of limb muscle anlagen, suggesting that its function becomes critical during the colonization of, and/or fiber assembly in, the anlagen. Microarrays were used to identify changes in gene expression in flow-sorted migratory muscle precursors, labeled by Lbx1EGFP/+, which resulted from the loss of Pitx2. Very few genes showed changes in expression. Many small-fold, yet significant, changes were observed in genes encoding cytoskeletal and adhesion proteins which play a role in cell motility. Myogenic cells from genetically-tagged mice were cultured and subjected to live cell-tracking analysis using time-lapse imaging. Myogenic cells lacking Pitx2 were smaller, more symmetrical, and had more actin bundling. They also migrated about half of the total distance and velocity. Decreased motility may prevent myogenic cells from filling pre-patterned regions of the limb bud in a timely manner. Altered shape may prevent proper assembly of higher-order fibers within anlagen. Pitx2 therefore appears to regulate muscle anlagen development by appropriately balancing expression of cytoskeletal and adhesion molecules.

Distinct populations of GABAergic neurons in mouse rhombomere 1 express but do not require the homeodomain transcription factor PITX2

Hindbrain rhombomere 1 (r1) is located caudal to the isthmus, a critical organizer region, and rostral to rhombomere 2 in the developing mouse brain. Dorsal r1 gives rise to the cerebellum, locus coeruleus, and several brainstem nuclei, whereas cells from ventral r1 contribute to the trochlear and trigeminal nuclei as well as serotonergic and GABAergic neurons of the dorsal raphe. Recent studies have identified several molecular events controlling dorsal r1 development. In contrast, very little is known about ventral r1 gene expression and the genetic mechanisms regulating its formation. Neurons with distinct neurotransmitter phenotypes have been identified in ventral r1 including GABAergic, serotonergic, and cholinergic neurons. Here we show that PITX2 marks a distinct population of GABAergic neurons in mouse embryonic ventral r1. This population appears to retain its GABAergic identity even in the absence of PITX2. We provide a comprehensive map of markers that places these PITX2-positive GABAergic neurons in a region of r1 that intersects and is potentially in communication with the dorsal raphe.

Pitx2-dependent Occupancy by Histone Deacetylases Is Associated with T-box Gene Regulation in Mammalian Abdominal Tissue

The homeodomain transcription factor Pitx2 and the T-box transcription factors are essential for organogenesis. Pitx2 and T-box genes are induced by growth factors and function as transcriptional activators or repressors. Gene expression analyses on abdominal tissue were used to identify seven of the T-box genes of the genome as Pitx2 target genes in the abdomen at embryonic day.10.5. Pitx2 activated Tbx4, Tbx15, and Mga and repressed Tbx1, Tbx2, Tbx5, and Tbx6 expression. As expected, activated genes showed reduced expression patterns, and repressed T-box genes showed increased expression patterns in the abdomen of Pitx2 mutants. Pitx2 occupied chromatin sites near all of these T-box genes. Co-occupancy by coactivators, corepressors, and histone acetylation at these sites was frequently Pitx2-dependent. Genes repressed by Pitx2 generally showed increased histone acetylation and decreased histone deacetylase (HDAC)/corepressor occupancy in Pitx2 mutants. The lower N-CoR, HDAC1, and HDAC3 occupancy observed at multiple sites along Tbx1 chromatin in mutants is consistent with the model that increased histone acetylation and gene expression of Tbx1 may result from a loss of recruitment of corepressors by Pitx2. Genes activated by Pitx2 showed less consistent patterns in chromatin analyses. Reduced H4 acetylation and increased HDAC1/nuclear receptor corepressor (N-CoR) occupancy at some Tbx4 sites were accompanied by increased H3 acetylation and reduced HDAC3 occupancy at the same or other more distal chromatin sites in mutants. Pitx2-dependent occupancy by corepressors resulted in alteration of the acetylation levels of several T-box genes, whereas Pitx2-dependent occupancy by coactivators was more site-localized. These studies will provide the basic scientific underpinning to understand abdominal wall syndromes.

Pitx2 Prevents Osteoblastic Transdifferentiation of Myoblasts by Bone Morphogenetic Proteins

Muscle cells are often exposed to bone morphogenetic proteins (BMPs) in pathological muscle and/or bone conditions. Because BMPs function as strong bone inducers as well as myogenesis inhibitors, certain molecules likely prevent muscle cells from converting into pathologic bone; without these molecules, de novo bone would form as observed in myositis ossificans traumatica. When C2C12 myoblasts are exposed to BMPs, they differentiate into osteoblastic cells but cannot mature into bone cells. As the Osterix gene, a transcription factor for osteoblast differentiation, is only transiently induced upon BMP stimulation in C2C12 cells, we hypothesized that unknown transcriptional repressor(s) inhibit Osterix expression and prevent complete osteoblastic differentiation. Gene microarray analyses were performed to identify putative inhibitors for osteoblastic differentiation, and the paired-like homeodomain transcription factor Pitx2 (also termed Rieg), which plays an important regulatory role in left-right asymmetry, was identified. Pitx2 was induced 2 days after BMP stimulation in C2C12 cells in concert with Osterix down-regulation. Overexpression of Pitx2 repressed Osterix expression and subsequent osteoblastic differentiation, whereas Runx2, the most upstream regulator of osteogenesis, was unaffected. Conversely, the induction of short hairpin RNA for Pitx2 in C2C12 cells enhanced Osterix expression and osteoblastic maturation upon BMP stimulation. Moreover, mouse embryonic fibroblasts containing myoblasts from Pitx2-null embryos showed enhanced Osterix expression upon BMP stimulation. These findings suggest that Pitx2 suppresses osteogenic signals induced by BMPs in myoblasts to prevent their osteoblastic conversion.

DNA-methylation of the homeodomain transcription factor PITX2 reliably predicts risk of distant disease recurrence in tamoxifen-treated, node-negative breast cancer patients – Technical and clinical validation in a multi-centre setting in collaboration with the European Organisation for Research and Treatment of Cancer (EORTC) PathoBiology group

Our aim was to identify and validate DNA-methylation markers associated with very good outcome in node negative, hormone receptor positive breast cancer patients after adjuvant endocrine therapy which might allow identifying patients who could be spared the burden of adjuvant chemotherapy. Using a methylation microarray, we analysed 117 candidate genes in hormone receptor-positive tumours from 109 breast cancer patients treated by adjuvant tamoxifen. Results were validated in an independent cohort ( n = 236, 5 centres). Independent methodological validation was achieved by a real-time polymerase chain reaction (PCR)-based technique. DNA methylation of PITX2 showed the strongest correlation with distant recurrence. Its impact on patient outcome was validated in the independent cohort: 86% of patients with low PITX2 methylation were metastasis-free after 10 years, compared to 69% with elevated PITX2 methylation. Moreover, PITX2 methylation added significant independent information to established clinical factors. All clinical and technical findings were confirmed by quantitative DNA-methylation PCR. These results provide strong evidence that DNA-methylation analysis allows clinically relevant risk assessment in tamoxifen-treated primary breast cancer. Based on PITX2 methylation, about half of hormone receptor-positive, node-negative breast cancer patients receiving adjuvant tamoxifen monotherapy can be considered low-risk regarding development of distant recurrences and may thus be spared adjuvant chemotherapy. In addition, these low-risk postmenopausal patients seem to respond sufficiently well to tamoxifen so that they may not require up-front aromatase inhibitor therapy.

Expression pattern of the homeodomain transcription factor Pitx2 during muscle development

Late-stage Pitx2 +/LacZ mouse embryos stained with x-gal appeared to have blue muscles, suggesting that Pitx2 expression specifically marks some phase of the myogenic progression or muscle anlagen formation. Detailed temporal and spatial analyses were undertaken to determine the extent and onset of Pitx2 expression in muscle. Pitx2 was specifically expressed in the vast majority of muscles of the head and trunk in late embryos and adults. Early Pitx2 expression in the cephalic mesoderm, first branchial arch and somatopleure preceded specification of head muscle. In contrast, Pitx2 expression appeared to follow muscle specification events in the trunk. However, Pitx2 expression was rapidly upregulated in these myogenic structures by E10.5. Upregulation correlated tightly with the apposition of a non-myogenic, Pitx2-expressing, cell cluster lateral to the dermomyotome. This cluster first appeared at the forelimb level at E10.25, gradually elongated in the posterior direction, appeared to aggregate from delaminated cells emanating from the ventrally located somatopleure, and was named the dorsal somatopleure. Immunohistochemistry on appendicular sections after E10.5 demonstrated that Pitx2 neatly marked the areas of muscle anlagen, that Pax3, Lbx1, and the muscle regulatory factors (MRFs) stained only subsets of Pitx2 + cells within these areas, and that virtually all Pitx2 + cells in these areas express at least one of these known myogenic markers. Taken together, the results demonstrate that, within muscle anlagen, Pitx2 marks the muscle lineage more completely that any of the known markers, and are consistent with a role for Pitx2 in muscle anlagen formation or maintenance.

Tbx1 affects asymmetric cardiac morphogenesis by regulatingPitx2in the secondary heart field

Individuals with 22q11 deletion syndrome (22q11DS; DiGeorge/velo-cardio-facial syndrome) have multiple congenital malformations, including cardiovascular defects. Most individuals with this syndrome possess 1.5-3.0 Mb hemizygous 22q11.2 deletions. The T-box transcription factor TBX1, lies within the nested 1.5 Mb interval and is a strong candidate for its etiology. Inactivation of Tbx1 in the mouse results in neonatal lethality owing to the presence of a single cardiac outflow tract. One important goal is to understand the molecular pathogenesis of cardiovascular defects in this syndrome. However, the molecular pathways of Tbx1 are still largely unexplored. Here, we show that Tbx1 is co-expressed with the bicoid-like homeodomain transcription factor Pitx2 in secondary heart field cells in the pharyngeal mesenchyme. In situ hybridization studies in Tbx1(-/-) mouse embryos revealed downregulation of Pitx2 in these cells. To test for a possible genetic interaction, we intercrossed Tbx1(+/-) and Pitx2(+/-) mice. Tbx1(+/-); Pitx2(+/-) mice died perinatally with cardiac defects, including double outlet right ventricle, and atrial and ventricular septal defects, all occurring with variable penetrance. An enhancer located between exons 4 and 5 in which a putative T-half site was identified near an Nkx2.5-binding site regulates asymmetric expression of Pitx2. We show using in vitro studies that Tbx1 binds to this site and activates the Pitx2 enhancer with the synergistic action of Nkx2.5. The results presented in this study unravel a novel Tbx1-Pitx2 pathway linking Tbx1 to asymmetric cardiac morphogenesis.

Cardiovascular development: Toward biomedical applicability

Cardiovascular development: Toward biomedical applicability