Transcription Factor Pitx2 Research Articles

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.

Pitx1 expression in developing and regenerating axolotl limbs

The expression of the homeobox transcription factor Pitx1 was investigated in the Mexican axolotl (Ambystoma mexicanum) during limb development and regeneration by whole-mount mRNA in situ hybridizations. This clone shares high amino acid identity with Pitx1 from other vertebrates (92% Xenopus; 87% chick; 75% human and mouse) within the region isolated. In the developing limbs, Pitx1 was expressed in hindlimb mesenchyme, as has been reported in other species. The expression pattern in the hindlimb might have been conserved during evolution. In the regenerating limbs, Pitx1 was expressed in both fore- and hindlimb blastemas. Our observations suggest two roles of Pitx1 in the axolotl: one is to determine the hindlimb pattern during development, and the other that relates to proliferation of regenerating tissues without regard to fore- or hindlimb.

Flow-Induced Asymmetry

Mammalian embryos initially develop symmetrically; then, in response to Nodal signaling and the action of the transcription factor Pitx2, organ asymmetry occurs. For example, during embryonic development, two blood vessels on the right side--the right sixth branchial arch artery and the dorsal aorta--undergo complete regression, resulting in the formation of the asymmetrical aortic arch and the pulmonary trunk (see Snider and Conway). Yashiro et al . determined that although Pitx2 activity was required for regression of the sixth branchial arch artery, which is the first vessel to regress, expression of this gene was not required in the artery itself. Instead, Pitx2 was required for a morphological change, the formation of a spiral structure of the myocardial left outflow tract, a structure through which blood leaves the heart. In animals in which the outflow tract did not spiral, regression of neither the right sixth branchial arch artery nor the right dorsal aorta occurred. Echocardiography showed that after the formation of the spiral structure, the blood flow to the right side vessels destined for regression was reduced, whereas blood flow to the left side vessels was greater than before the morphological change. Furthermore, this change in blood flow was lost in embryos with a promoter-mutated Pitx2 (Pitx ΔASE/ΔASE ), which also lacked a spiral form to the outflow tract and had persistent right vessels. To confirm that decreased blood flow was the signal for regression, the authors ligated the left sixth branchial arch artery prior to the formation of the spiral structure by the outflow tract and found that the left-side vessels (sixth branchial arch artery and dorsal aorta) regressed and the right, unligated vessels persisted. In the persistent left vessels in wild-type animals, there was increased expression of the gene encoding platelet-derived growth factor-A (PDGF-A) and increased phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) compared with that in the right vessels after the morphological change. Again, this difference was lost in the Pitx ΔASE/ΔASE embryos. Simultaneous pharmacological inhibition of PDGFR and VEGFR triggered bilateral loss of the sixth branchial arch arteries. Thus, the authors suggest that the Nodal-Pitx2 pathway triggers a morphological change that increases blood flow on the left side and decreases blood flow on the right side, producing an asymmetric signal of growth factor signaling, allowing the side with the higher blood flow to survive and the side with less to regress. P. Snider, S. J. Conway, The power of blood. Nature 450 , 180-181 (2007). [PubMed] K. Yashiro, H. Shiratori, H. Hamada, Haemodynamics determined by a genetic programme govern asymmetric development of the aortic arch. Nature 450 , 285-288 (2007). [PubMed]

Left Right Asymmetry, the Pulmonary Vein, and A-Fib

See related article, pages 902–909 Atrial fibrillation (AF), a common adult cardiac arrhythmia, involves abnormal atrial contractions. As every medical student is taught, it is easily diagnosed as an irregularly irregular pulse and loss of organized atrial activity on an ECG. Clinical interest in AF is considerable because AF is the most common adult arrhythmia that increases in prevalence with age, with 5% of the over 65 population having AF. Moreover, patients with AF have a significantly increased risk of stroke.1 Electrical impulses, critical for a coordinated and physiologic heartbeat, are normally initiated in the sinoatrial (SA) node. However, in AF the disorganized atrial activity overrides normal SA node function resulting in irregular conduction of impulses to the ventricles. In the majority of cases, ectopic electrical activity originates in the pulmonary veins.1 In this issue of Circulation Research , Mommersteeg and colleagues2 make a significant advance in our understanding of pulmonary vein development by making a strong connection between AF and embryonic axis determination. Moreover, their work fits nicely with exciting new data from human genetic studies implicating the same genetic pathways in familial AF.3 Mommersteeg and colleagues make a number of fundamental observations regarding pulmonary vein development. Previous work suggested that pulmonary myocardium derives from atrial myocardium.4 Using lineage tracing, the authors convincingly rule out this possibility. Moreover, they show other data revealing that pulmonary myocardium is derived from Isl1 -positive second heart field and has a distinct origin from the systemic venous circulation.5 Together, these findings suggest that the pulmonary vein myocardium forms by differentiation of mesenchyme around the pulmonary vein. To address this hypothesis, the authors use a Pitx2 mutant mouse model that had been shown to have defects in pulmonary vein development.6 The Pitx (Pituitary homeobox) family of …

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.

Pancreatic β Cell Proliferation Is Stimulated by Wnt Signaling

Both type 1 and type 2 diabetes result from a loss of pancreatic β cell mass. There is considerable interest, therefore, in understanding the mechanisms responsible for β cell proliferation. Previous studies have shown that various components of the Wnt signaling pathway are found in the pancreas and that dysregulation of this pathway can result in reduced islet mass, but the exact mechanisms involved are unknown. Wnts are small proteins that signal though cell surface receptors called Frizzleds, leading to the stabilization and accumulation of β-catenin. This protein then translocates to the nucleus, where it interacts with transcription factors to control the transcription of target genes. Rulifson et al . investigated this link between the Wnt pathway and β cell proliferation by treating cultured mouse pancreatic islets with purified Wnt3a. Immunohistochemical analysis demonstrated increased amounts of Ki67, a marker of cell proliferation. Treatment of islets with Wnt3a also increased the abundance of the mRNAs for the transcription factor Pitx2, a known downstream target of β-catenin, and cyclin D2, a protein involved in control of the cell cycle. Chromatin immunoprecipitation assays revealed that Wnt3a directed the association of Pitx2 with the cyclin D2 gene. Mice with pancreas-specific expression of a constitutively active form of β-catenin had increased β cell proliferation and higher serum insulin levels than did wild-type mice. In contrast, transgenic mice with pancreas-specific inducible expression of Axin, a negative regulator of β-catenin, had reduced islet mass, lower insulin production, and lower abundance of Pitx2 and cyclin D2 than did wild-type mice. This study provides mechanistic evidence of the importance of Wnt signaling for β cell proliferation. I. C. Rulifson, S. K. Karnik, P. W. Heiser, D. ten Berge, H. Chen, X. Gu, M. M. Taketo, R. Nusse, M. Hebrok, S. K. Kim, Wnt signaling regulates pancreatic β cell proliferation. Proc. Natl. Acad. Sci. USA 104 , 6247-6252 (2007). [Abstract] [Full Text]

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.

Pitx1 determines the morphology of muscle, tendon, and bones of the hindlimb

The vertebrate forelimb and hindlimb are serially homologous structures; however, their distinctive morphologies suggest that different mechanisms are associated with each limb type to give rise to limb-type identity. Three genes have been implicated in this process; T-box transcription factors Tbx5 and Tbx4, which are expressed in the forelimb and hindlimb, respectively, and a paired-type homeodomain transcription factor Pitx1, expressed in the hindlimb. To explore the roles of Pitx1 and Tbx4 in patterning the hindlimb, we have ectopically misexpressed these genes in the mouse forelimb using transgenic methods. We have developed a novel technique for visualising the structure and organisation of tissues in limbs in 3D using optical projection tomography (OPT). This approach provides unparalleled access to understanding the relationships between connective tissues during development of the limb. Misexpression of Pitx1 in the forelimb results in the transformation and translocation of specific muscles, tendons, and bones of the forelimb so that they acquire a hindlimb-like morphology. Pitx1 also upregulates hindlimb-specific factors in the forelimb, including Hoxc10 and Tbx4. In contrast, misexpression of Tbx4 in the forelimb does not result in a transformation of limb-type morphology. These results demonstrate that Pitx1, but not Tbx4, determines the morphological identity of hindlimb tissues.

Analysis of expression of regulatory genes Pax6, Prox1, and Pitx2 in differentiating eye cells in human fetus

Expression of transcription factors PAX6, PROX1, and PITX2 was evaluated in eye tissues after 9.5 and 22.0 weeks of human fetus development using polymerase chain reaction. Pax6, Prox1 and Pitx2 expression has been revealed in the cornea, lens, retina, and eye membranes (total preparation of the pigment epithelium, choroid, and sclera) after 9.5 weeks of prenatal development with the maximum expression of Pax6 gene in all studied tissues. After 22.0 weeks of development, Pax6 expression increased in the retina and lens but decreased in the cornea. Insignificant levels of Pax6 transcription have been detected in the eye membranes. Prox1 expression was apparent in the cornea, lens, retina, and eye membranes after 9.5 weeks. After 22.0 weeks, Prox1 expression increased in the lens and retina, decreased in the cornea, and was undetectable in the eye membranes. High level of Pitx2 expression has been revealed in all studied eye tissues after 9.5 weeks with the lowest transcription level observed in the retina. After 22.0 weeks, Pitx2 expression notably decreased in the lens and cornea and became undetectable in the retina and eye membranes. The differential pattern of Pax6, Prox1 and Pitx2 expression in developing human eye tissues after 9.5 and 22.0 weeks of development agrees with our histological data.

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

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.

Screening for Tumor Suppressors

Two separate groups, Westbrook et al. and Kolfschoten et al. , used RNA interference (RNAi)-based screening in human cell lines to identify novel tumor suppressor genes. Westbrook et al. infected immortalized human mammary epithelial cells (TLM-HMECs) with a retroviral library of short-hairpin RNAs (shRNAs) in which each shRNA was tagged with a unique 60-nucleotide sequence (DNA "bar code") and looked for clones that showed anchorage-independent growth (indicative of malignant transformation). Combining sequencing of the proviral shRNA of individual clones with bar-code microarray analysis of pooled clones, the authors identified genes associated with formation of anchorage-independent colonies. Transforming growth factor-β receptor II (TGF-βRII), a known tumor suppressor, was identified in the screen, and shRNA knockdown of the tumor suppressor PTEN also elicited anchorage-independent cell growth. Array-based comparative genomic hybridization analysis indicated that two genes identified through RNAi screening, TGFBR2 and REST (for RE1-silencing transcription factor, also known as NRSF for neuron-restrictive silencing factor, a transcriptional repressor), were frequently deleted in colorectal cancers. Microdeletions involving REST were found in a primary colon cancer and a colon cancer cell line, and further analysis revealed a dominant-negative REST mutant in another colon cancer line. REST knockdown promoted signaling through the phosphoinositide 3-kinase (PI3K) pathway, whereas expression of a dominant-negative form of the PI3K regulatory subunit inhibited REST-shRNA-induced transformation. Thus, the authors conclude that REST is a good candidate for a tumor suppressor that may act by suppressing PI3K signaling. Using a similar approach, Kolfschoten et al. infected immortalized human fibroblasts that can be transformed by oncogenic Ras with a retroviral RNAi library. Among the genes identified as targets of RNAi whose knockdown could substitute for oncogenic RAS signaling was the homeodomain pituitary transcription factor PITX1. PITX1 knockdown enhanced RAS signaling without affecting RAS abundance and produced a phenotype similar to that seen with overexpression of activated RAS. PITX1 expression was reduced compared with control tissues in human prostate and bladder cancers as well as in colon cancers that expressed wild-type RAS. When PITX1 was expressed in Caco-2 cells (a colon cancer line with reduced endogenous abundance of PITX1), it inhibited cell proliferation, growth in soft agar, and tumorigenicity in nude mice. Sequence analysis indicated that the promoter of the RAS-GAP (GTPase activating protein) RASAL1 contained a PITX1 binding site, and chromatin immunoprecipitation analysis indicated that PITX1 associated with the RASAL1 promoter. Transfection of human embryonic kidney (HEK) 293 cells with PITX1 enhanced RASAL1 mRNA abundance, whereas PITX1 knockdown reduced RASAL1 mRNA expression. Moreover, RASAL1 knockdown antagonized a reduction in ERK (extracellular signal-regulated kinase) phosphorylation produced by PITX1 overexpression and itself stimulated anchorage-independent cell growth. Thus, PITX1 appears to function as a tumor suppressor that acts through RASAL1 to repress RAS signaling. Downward discusses both articles in a Preview. T. F. Westbrook, E. S. Martin, M. S. Schlabach, Y. Leng, A. C. Liang, B. Feng, J. J. Zhao, T. M. Roberts, G. Mandel, G. J. Hannon, R. A. DePinho, L. Chin, S. J. Elledge, A genetic screen for candidate tumor suppressors identifies REST. Cell 121 , 837-848 (2005). [PubMed] I. G. M. Kolfschoten, B. van Leeuwen, K. Berns, J. Mullenders, R. L. Beijersbergen, R. Bernards, P. M. Voorhoeve, R. Agami, A genetic screen identifies PITX1 as a suppressor of RAS activity and tumorigenicity. Cell 121 , 849-858 (2005). [PubMed] J. Downward, RNA interference libraries prove their worth in hunt for tumor suppressor genes. Cell 121 , 813-815 (2005). [PubMed]

Compound developmental eye disorders following inactivation of TGFβ signaling in neural-crest stem cells

BackgroundDevelopment of the eye depends partly on the periocular mesenchyme derived from the neural crest (NC), but the fate of NC cells in mammalian eye development and the signals coordinating the formation of ocular structures are poorly understood.ResultsHere we reveal distinct NC contributions to both anterior and posterior mesenchymal eye structures and show that TGFβ signaling in these cells is crucial for normal eye development. In the anterior eye, TGFβ2 released from the lens is required for the expression of transcription factors Pitx2 and Foxc1 in the NC-derived cornea and in the chamber-angle structures of the eye that control intraocular pressure. TGFβ enhances Foxc1 and induces Pitx2 expression in cell cultures. As in patients carrying mutations in PITX2 and FOXC1, TGFβ signal inactivation in NC cells leads to ocular defects characteristic of the human disorder Axenfeld-Rieger's anomaly. In the posterior eye, NC cell-specific inactivation of TGFβ signaling results in a condition reminiscent of the human disorder persistent hyperplastic primary vitreous. As a secondary effect, retinal patterning is also disturbed in mutant mice.ConclusionIn the developing eye the lens acts as a TGFβ signaling center that controls the development of eye structures derived from the NC. Defective TGFβ signal transduction interferes with NC-cell differentiation and survival anterior to the lens and with normal tissue morphogenesis and patterning posterior to the lens. The similarity to developmental eye disorders in humans suggests that defective TGFβ signal modulation in ocular NC derivatives contributes to the pathophysiology of these diseases.

Developmental Regulation and Pathogenesis of Pituitary Corticotroph Function

The different cell lineages of the pituitary gland arise sequentially during organogenesis, which, in the mouse, takes place between days 10 and 18 (e10–e18) of fetal development. The corticotroph lineage is the first to appear with the first POMC‐positive cells being detected at e12.5. Differentiation of this lineage has now been linked to a number of organ‐ or cell‐restricted transcription factors. In corticotrophs, the homeobox transcription factor Pitx1 (Ptx1), the bHLH factor BETA2/NeuroD1, and the Tbox factor Tpit all play essential roles for cell‐specific transcription of the POMC gene and for development. Pitx1, together with Pitx2, marks the embryological origin of the pituitary in the anterior surface ectoderm and it is an important regulator in all pituitary lineages. Importantly, Pitx1 is required for protein:protein interactions with cell‐restricted factors, such as NeuroD1 and Tpit. NeuroD1 is transiently‐expressed in fetal corticotrophs where it is important for POMC expression. Tpit is a highly cell‐restricted transcription factor since it is only present in mouse corticotrophs and melanotrophs. Tpit is important for terminal differentiation of corticotrophs, but it is not required for commitment to this lineage. Tpit‐deficient mice present with an isolated deficiency of pituitary ACTH (IAD), which results in adrenal insufficiency and hypoplasia. This phenotype is extremely similar to a neonatal onset form of IAD that we defined in the course of a search for human TPIT gene mutations. So far, at least 13 different mutations have been found in the human TPIT gene within more than 20 different families of various ethnic origins. These patients define a very homogenous clinical presentation of neonatal IAD. It is possible that this entity was not previously recognized because of the neonatal lethality associated with it.

New emerging roles of transcription factor Pitx1 and Reg growth factors in osteoarthritis pathogenesis

New emerging roles of transcription factor Pitx1 and Reg growth factors in osteoarthritis pathogenesis

The Wnt/β-Catenin→Pitx2 Pathway Controls the Turnover of Pitx2 and Other Unstable mRNAs

The Wnt/β-catenin pathway rapidly induces the transcription of the cell-type-restricted transcription factor Pitx2 that is required for effective cell-specific proliferation activating growth-regulating genes. Here we report that Pitx2 mRNA displays a rapid turnover rate and that activation of the Wnt/β-catenin pathway stabilizes Pitx2 mRNA as well as other unstable mRNAs, including c-Jun, Cyclin D1, and Cyclin D2, encoded by critical transcriptional target genes of the same pathway. Our data indicate that Pitx2 mRNA stabilization is due to a reduced interaction of Pitx2 3′UTR with the destabilizing AU-rich element (ARE) binding proteins (BPs) KSRP and TTP as well as to an increased interaction with a stabilizing ARE-BP, HuR. Pitx2 itself is a mediator of Wnt/β-catenin-induced mRNA stabilization. Our previous and present data support the hypothesis that a single pathway can coordinately regulate sequential transcriptional and posttranscriptional events leading to an integrated functional gene regulatory network.

Pitx2c attenuation results in cardiac defects and abnormalities of intestinal orientation in developing Xenopus laevis

The experimental manipulation of early embryologic events, resulting in the misexpression of the homeobox transcription factor pitx2, is associated with subsequent defects of laterality in a number of vertebrate systems. To clarify the role of one pitx2 isoform, pitx2c, in determining the left–right axis of amphibian embryos, we examined the heart and gut morphology of Xenopus laevis embryos after attenuating pitx2c mRNA levels using chemically modified antisense oligonucleotides. We demonstrate that the partial depletion of pitx2c mRNA in these embryos results in alteration of both cardiac morphology and intestinal coiling. The most common cardiac abnormality seen was a failure of rightward migration of the outflow tract, while the most common intestinal laterality phenotype seen was a full reversal in the direction of coiling, each present in 23% of embryos injected with the pitx2c antisense oligonucleotide. An abnormality in either the heart or gut further predisposed to a malformation in the other. In addition, a number of other cardiac anomalies were observed after pitx2c mRNA attenuation, including abnormalities of atrial septation, extracellular matrix restriction, relative atrial–ventricular chamber positioning, and restriction of ventricular development. Many of these findings correlate with cardiac defects previously reported in pitx2 null and hypomorphic mice, but can now be assigned specifically to attenuation of the pitx2c isoform in Xenopus.

Pitx Factors Are Involved in Basal and Hormone-regulated Activity of the Human Prolactin Promoter

The pituitary-specific POU homeodomain factor Pit-1 likely interacts with other factors for cell-specific expression of prolactin. Here we identify the paired-like homeobox transcription factors Pitx1 and Pitx2 as factors functionally activating the proximal human prolactin promoter (hPRL-164luc). Using in vitro binding assays and a series of site-specific mutations of the proximal hPRL promoter, we mapped the B1 and B2 bicoid sites involved in Pitx-mediated transactivation of the hPRL-164luc construct. In somatolactotroph GH4C1 cells, basal proximal hPRL promoter activity was inhibited by a Pitx2 dominant-negative form in a dose-dependent manner, whereas binding disruptive mutations in the Pitx sites significantly reduced basal activity of the promoter. We also show that synergistic activation of hPRL-164luc by Pitx2 and Pit-1 requires the integrity of the B2 Pitx binding site, and at least one of the P1 and P2 Pit-1 response elements. In addition, mutation in the B2 Pitx site results in attenuation of the promoter's responsiveness to forskolin, thyrotropin-releasing hormone, and epidermal growth factor. Conversely, Pitx1 or Pitx2 overexpression in GH4C1 cells leads to an enhancement of the drugs stimulatory effects. Altogether, these results suggest that full responsiveness to several signaling pathways regulating the hPRL promoter requires the B2 Pitx binding site and that Pitx factors may be part of the proteic complex involved in these regulations. Finally, in situ hybridization analysis showing coexpression of the PRL and Pitx2 genes in rat and human lactotroph cells corroborates the physiological relevance of these results.

Identification of a Wnt/Dvl/β-Catenin → Pitx2 Pathway Mediating Cell-Type-Specific Proliferation during Development

Understanding the cell type-specific molecular mechanisms by which distinct signaling pathways combinatorially control proliferation during organogenesis is a central issue in development and disease. Here, we report that the bicoid-related transcription factor Pitx2 is rapidly induced by the Wnt/Dvl/β-catenin pathway and is required for effective cell-type-specific proliferation by directly activating specific growth-regulating genes. Regulated exchange of HDAC1/β-catenin converts Pitx2 from repressor to activator, analogous to control of TCF/LEF1. Pitx2 then serves 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 Cyclin D2 gene induction. The molecular strategy underlying interactions between the Wnt and growth factor-dependent signaling pathways in cardiac outflow tract and pituitary proliferation is likely to be prototypic of cell-specific proliferation strategies in other tissues.