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Sally A Camper - One of the best experts on this subject based on the ideXlab platform.

  • Sequential expression and redundancy of Pitx2 and Pitx3 genes during muscle development.
    Developmental biology, 2007
    Co-Authors: Aurore L'honoré, Philip J. Gage, Alexandre Marcil, Sally A Camper, Mélanie Lebel, Vincent Coulon, Julien Lafrance-vanasse, Jacques Drouin
    Abstract:

    The myogenic program is controlled by different groups of transcription factors acting during muscle development, including bHLH muscle regulatory factors (MRFs), the paired factors Pax3 and Pax7 and the homeobox factors Six1 and Six4. This program is critically dependent on MRFs that target downstream muscle-specific genes. We now report the expression of Pitx2 and Pitx3 transcription factors throughout muscle development. Pitx2 is first expressed in muscle progenitor cells of the dermomyotome and myotome. The onset of myoblast differentiation is concomitant with expression of Pitx3; its expression is maintained in all skeletal muscles while Pitx2 expression decreases thereafter. We have generated Pitx3 mutant mice and this deficiency does not significantly perturb muscle development but it is completely compensated by the maintenance of Pitx2 expression in all skeletal muscles. These experiments suggest that Pitx genes are important for myogenesis and that Pitx2 and Pitx3 may have partly redundant roles.

  • PITX1 and pitx2 are required for development of hindlimb buds
    Development, 2003
    Co-Authors: Alexandre Marcil, Michel Chamberland, Emilie Dumontier, Sally A Camper, Jacques Drouin
    Abstract:

    Two closely related homeobox transcription factors, PITX1 and Pitx2, have been implicated in patterning of lateral plate mesoderm derivatives: PITX1 for specification of hindlimb identity and Pitx2 for determination of laterality. We show that, together, PITX1 and Pitx2 are required for formation of hindlimb buds and, when present in limited doses, for development of proximal (femur) and anterior (tibia and digit 1) hindlimb structures. Although PITX1 is expressed throughout developing hindlimb buds, Pitx2 is not expressed in limb bud mesenchyme itself, but is co-expressed with PITX1 in the presumptive hindlimb field before bud growth. Thus, PITX1 and Pitx2 genes are required for sustained hindlimb bud growth and formation of hindlimbs.

  • pitx2 distinguishes subtypes of terminally differentiated neurons in the developing mouse neuroepithelium
    Developmental Biology, 2002
    Co-Authors: Donna M Martin, Philip J. Gage, Jennifer M Skidmore, Sally A Camper
    Abstract:

    Abstract Pitx2, a homeodomain transcription factor, is essential for normal development of pituitary, eyes, heart, and teeth. In the developing mouse brain, Pitx2 (Rieg, Ptx2, Otlx2, Brx1) mRNA is expressed in discrete regions of the diencephalon, mesencephalon, and rhombencephalon. While prior reports have provided an overview of the temporal and regional specificity of Pitx2 mRNA expression in the brain, the precise cell types that express PITX2 are not known. In this study, we analyzed Pitx2 mRNA and PITX2 protein expression in individual cells of the developing e10.5–e14.5 mouse CNS using multiple markers of cellular proliferation and differentiation. We identified Pitx2 expression in nestin-positive neural progenitors and in postmitotic, developing neurons. In the diencephalon, PITX2 is expressed in neurons of the zona limitans intrathalamica and mammillary region and in γ-aminobutyric acid (GABA)-producing neurons of the zona incerta. In the mesencephalon, PITX2-labeled nuclei also appear in differentiated neurons, some of which are GABAergic and destined to occupy superior colliculus. Our results suggest that PITX2 expression in postmitotic neurons may contribute to development of GABAergic and other differentiated neuronal phenotypes.

  • pitx2 is required at multiple stages of pituitary organogenesis pituitary primordium formation and cell specification
    Development, 2002
    Co-Authors: Hoonkyo Suh, Philip J. Gage, Jacques Drouin, Sally A Camper
    Abstract:

    Analysis of an allelic series in mice revealed that the Pitx2 homeobox gene is required at multiple stages of pituitary development. It is necessary for initiating expansion of Rathke's pouch and maintaining expression of the fetal-specific transcription factors Hesx1 and Prop1. At later stages Pitx2 is necessary for specification and expansion of the gonadotropes and Pit1 lineage within the ventral and caudomedial anterior pituitary. Mechanistically, this is due to the dependence of several critical lineage-specific transcription factors, Pit1, Gata2, Egr1 and Sf1, on a threshold level of PITX2. The related PITX1 gene has a role in hormone gene transcription, and it is important late in ontogeny for the final expansion of the differentiated cell types. PITX1 and Pitx2 have overlapping functions in the expansion of Rathke's pouch, revealing the sensitivity of pituitary organogenesis to the dosage of the PITX family. The model developed for PITX gene function in pituitary development provides a better understanding of the etiology of Rieger syndrome and may extend to other PITX-sensitive developmental processes.

  • pituitary homeobox 2 a novel member of the bicoid related family of homeobox genes is a potential regulator of anterior structure formation
    Human Molecular Genetics, 1997
    Co-Authors: Philip J. Gage, Sally A Camper
    Abstract:

    Genetic analysis of mouse mutants has demonstrated the importance of the homeobox genes Rpx, Lhx3 and Pit1 for anterior pituitary gland development. Pit1 mutations have also been identified in several human families with multiple pituitary hormone deficiencies. To identify additional homeobox regulators of pituitary development, we screened an adult pituitary gland cDNA library for homeobox sequences. Here, we report the identification of a novel bicoid-related homeodomain gene expressing two alternatively spliced mRNA products, which encode proteins of 271 and 317 amino acids, respectively. The proteins have been named Ptx2a and Ptx2b since they are highly related to Ptx1/P-OTX. Ptx2 is expressed in both developing and adult pituitary gland, eye and brain tissues, suggesting an important role in development and maintenance of anterior structures. Ptx2 was mapped close to Egf on mouse chromosome 3, in a region having extensive synteny homology with HSA 4q. These data make the human Ptx2 homologue a candidate gene for Rieger syndrome, an autosomal-dominant disorder with variable craniofacial, dental, eye and pituitary anomalies.

Philip J. Gage - One of the best experts on this subject based on the ideXlab platform.

  • the homeodomain transcription factor pitx2 is required for specifying correct cell fates and establishing angiogenic privilege in the developing cornea
    Developmental Dynamics, 2014
    Co-Authors: Philip J. Gage, Chen Cha'o-kuang, Amanda L Zacharias
    Abstract:

    Background: 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. Results: 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. Conclusions: Together, these data provide important insights into the mechanisms regulating cornea development. Developmental Dynamics 243:1391–1400, 2014. © 2014 Wiley Periodicals, Inc.

  • Sequential expression and redundancy of Pitx2 and Pitx3 genes during muscle development.
    Developmental biology, 2007
    Co-Authors: Aurore L'honoré, Philip J. Gage, Alexandre Marcil, Sally A Camper, Mélanie Lebel, Vincent Coulon, Julien Lafrance-vanasse, Jacques Drouin
    Abstract:

    The myogenic program is controlled by different groups of transcription factors acting during muscle development, including bHLH muscle regulatory factors (MRFs), the paired factors Pax3 and Pax7 and the homeobox factors Six1 and Six4. This program is critically dependent on MRFs that target downstream muscle-specific genes. We now report the expression of Pitx2 and Pitx3 transcription factors throughout muscle development. Pitx2 is first expressed in muscle progenitor cells of the dermomyotome and myotome. The onset of myoblast differentiation is concomitant with expression of Pitx3; its expression is maintained in all skeletal muscles while Pitx2 expression decreases thereafter. We have generated Pitx3 mutant mice and this deficiency does not significantly perturb muscle development but it is completely compensated by the maintenance of Pitx2 expression in all skeletal muscles. These experiments suggest that Pitx genes are important for myogenesis and that Pitx2 and Pitx3 may have partly redundant roles.

  • reduced human and murine corneal thickness in an axenfeld rieger syndrome subtype
    Investigative Ophthalmology & Visual Science, 2006
    Co-Authors: Mika Asaicoakwell, Philip J. Gage, Christopher J Backhouse, Ronald J Casey, Ordan J Lehmann
    Abstract:

    Purpose Axenfeld-Rieger malformations of the anterior segment are clinically heterogeneous, and up to 50% of cases are attributable to PITX2 or FOXC1 mutation. In view of PITX2's contribution to corneal development and the altered CCT in some FOXC1-related cases, this study was undertaken to investigate whether a related phenotype is associated with the PITX2/Pitx2 mutation. Methods Central corneal thickness (CCT) was measured in patients and mice with PITX2/Pitx2 mutations. CCT in affected individuals and unaffected first-degree relatives from a large PITX2 mutation pedigree was measured with ultrasonic pachymetry. For murine measurements, the optical coherence tomogram (OCT) was calibrated against plastic films whose thickness had been determined with scanning electron microscopy (SEM). Subsequently, CCT was measured in ex vivo eyes from Pitx2(+/-) and wild-type murine littermates by using OCT. Results CCT in individuals with the PITX2 mutation (mean 484 microm; range, 425-519; n = 8) was significantly lower than in their unaffected first-degree relatives (mean 582 microm; range, 550-590; n = 5; P = 0.0002, t-test). Scanning electron microscopy (SEM) and OCT measurements of reference films correlated closely (r = 0.9995) and subsequent OCT analysis of murine eyes revealed a significant reduction in CCT in Pitx2(+/-) compared with wild-type littermates (Pitx2(+/-): mean, 72 microm; range, 57-87, n = 6; wt: mean, 88 microm; range, 63-100; n = 6, P = 0.035, t-test). Conclusions The results show that PITX2/Pitx2 mutation results in reduced corneal thickness and provides the first example of reduced CCT in a genetic subtype of glaucoma. These data will facilitate management of developmental glaucoma and offer potential for guiding molecular genetic testing in patients with Axenfeld-Rieger. The similar CCT reduction observed in patients and mice with comparable mutations emphasizes the utility of this murine model. The technical advance of optical murine CCT measurement also provides scope for serial in vivo imaging of the developing anterior segment and determining the effects of altered CCT on measured IOP.

  • extraocular muscle morphogenesis and gene expression are regulated by pitx2 gene dose
    Investigative Ophthalmology & Visual Science, 2006
    Co-Authors: Adam G Diehl, Philip J. Gage, Sepideh Zareparsi, Min Qian, Ritu Khanna, Rowena Angeles
    Abstract:

    PURPOSE. PITX2 gene dose plays a central role in Axenfeld-Rieger syndrome. The purpose of this study was to test the hypothesis that the effects of Pitx2 gene dose on eye development can be molecularly dissected in available Pitx2 mutant mice. METHODS. A panel of mice with Pitx2 gene dose ranging from wild-type (+/+) to none (-/-) was generated. Eye morphogenesis was assessed in animals with each Pitx2 gene dose. We also compared global gene expression in eye primordia taken from e12.5 Pitx2 +/+ , Pitx2 +/- , Pitx2 -/- embryos using gene microarrays. The validity of microarray results was confirmed by qRT-PCR. RESULTS. Morphogenesis of all extraocular muscle bundles correlated highly with Pitx2 gene dose, but there were some differences in sensitivity among muscle groups. Superior and inferior oblique muscles were most sensitive and disappeared before the four rectus muscles. Expression of muscle-specific genes was globally sensitive to Pitx2 gene dose, including the muscle-specific transcription factor genes Myf5, Myog, Myodi, Smydl, Msc, and Csrp3. CONCLUSIONS. Pitx2 gene dose regulates both morphogenesis and gene expression in developing extraocular muscles. The expression of key muscle-specific transcription factor genes is regulated by Pitx2 gene dose, suggesting that sufficient levels of PITX2 protein are essential for early initiation of the myogenic regulatory cascade in extraocular muscles. These results document the first ocular tissue affected by Pitx2 gene dose in a model organism, where the underlying mechanisms can be analyzed, and provide a paradigm for future experiments designed to elucidate additional effects of Pitx2 gene dose during eye development.

  • Expression of the homeobox gene Pitx2 in neural crest is required for optic stalk and ocular anterior segment development
    Human Molecular Genetics, 2005
    Co-Authors: Amanda L. Evans, Philip J. Gage
    Abstract:

    Heterozygous mutations in the homeobox gene, PITX2, result in ocular anterior segment defects and a high incidence of early-onset glaucoma. Pitx2 is expressed in both the neural crest and the mesoderm-derived precursors of the periocular mesenchyme. Complete loss of function in mice results in agenesis or severe disruption of periocular mesenchyme structures and extrinsic defects in early optic nerve development. However, the specific requirements for Pitx2 in neural crest versus mesoderm could not be determined using these mice, and only roles in the initial stages of eye development could be assessed due to early embryonic lethality. To determine the specific roles of Pitx2 in the neural crest precursor pool, we generated neural crest-specific Pitx2 knockout mice (Pitx2-ncko). Because Pitx2-nkco mice are viable, we also analyzed gene function in later eye development. Pitx2 is intrinsically required in neural crest for specification of corneal endothelium, corneal stroma and the sclera. Pitx2 function in neural crest is also required for normal development of ocular blood vessels. Pitx2-ncko mice exhibit a unique optic nerve phenotype in which the eyes are progressively displaced towards the midline until they are directly attached to the ventral hypothalamus. As Pitx2 is not expressed in the optic stalk, an essential function of PITX2 protein in neural crest is to regulate an extrinsic factor(s) required for development of the optic nerve. We propose a revised model of optic nerve development and new mechanisms that may underlie the etiology of glaucoma in Axenfeld‐ Rieger patients.

Jacques Drouin - One of the best experts on this subject based on the ideXlab platform.

  • shh signaling influences the phenotype of PITX1 hindlimbs
    Developmental Biology, 2018
    Co-Authors: Stephen Nemec, Aurelie Huang Sung, Jacques Drouin
    Abstract:

    Forelimbs (FLs) and hindlimbs (HLs) develop under the instructive and integrated guidance of signaling centers and transcription factor (TF) action. The development of structures specific to each limb type depends on the limb-specific modulation of these integrated components. PITX1 is a transcription factor gene expressed in HL, absent in FL, and required for HL-specific patterning and development, in particular for formation of anterior HL skeletal elements. PITX1 achieves this function by direct TF action on the core limb program, which is largely shared between FL and HL. Shh signaling plays a crucial role in anterior-posterior (AP) patterning in both FL and HL. The present work assessed the relationship between Shh signaling and PITX1 action for AP patterning. We found that reducing the gene dosage of Shh in the context of the PITX1-/- HL decreases the severity of the PITX1-/- phenotype, in particular, the loss of anterior limb structures and the shortening of femur length. However, this did not rescue HL-specific patterning features. Thus, PITX1 action integrates Shh signaling but not for limb-type-specific patterning.

  • Specificity of Pitx3-Dependent Gene Regulatory Networks in Subsets of Midbrain Dopamine Neurons
    Molecular Neurobiology, 2017
    Co-Authors: Panojot Bifsha, Aurelio Balsalobre, Jacques Drouin
    Abstract:

    Dysfunction of midbrain dopaminergic (mDA) neurons is involved in Parkinson’s disease (PD) and neuropsychiatric disorders. Pitx3 is expressed in mDA neuron subsets of the substantia nigra compacta (SNc) and of the ventral tegmental area (VTA) that are degeneration-sensitive in PD. The genetic network(s) and mode(s) of action of Pitx3 in these mDA neurons remain poorly characterized. We hypothesized that, given their distinct neuronal identities, Pitx3-expressing neurons of SNc and VTA should differ in their Pitx3-controlled gene expression networks and this may involve subset-specific co-regulators. Expression profiling of purified mDA neuronal subsets indicates that Pitx3 regulates different sets of genes in SNc and VTA, such as activating the expression of primary cilium gene products specifically in VTA neurons. Interaction network analysis pointed to the participation of differentially expressed Lhx/Lmo family members in the modulation of Pitx3 action in SNc and VTA mDA neurons. Conversely, global binding patterns of Pitx3 on genomic DNA of human dopaminergic cells revealed that Pitx3 is often co-recruited to regions that foster the formation of GATA-bHLH-BRN complexes, which usually involve Lmo co-regulatory proteins. We focused on Lmo3 for its preferential expression in SNc neurons and demonstrated that it functions as a transcriptional co-activator of Pitx3 by enhancing its activity on genomic regulatory elements. In summary, we defined the SN and VTA-specific programs of Pitx3-dependent gene expression and identified Lmo3 as a SN-specific co-regulator of Pitx3-dependent transcription.

  • pitx2 and pitx3 transcription factors two key regulators of the redox state in adult skeletal muscle stem cells and muscle regeneration
    Free Radical Biology and Medicine, 2014
    Co-Authors: Aurore Lhonore, Jacques Drouin, Margaret Buckingham, Didier Montarras
    Abstract:

    Adult tissue homeostasis and regeneration rely on tissue stem cell populations that generate committed precursors and differentiated cells while maintaining a pool of stem cells. In adult skeletal muscle, such cells, called satellite cells, remain quiescent at the periphery of muscle fibers. Upon injury they undergo activation, proliferation and differentiation to replace damaged fibers and also self-renew to reconstitute the muscle stem cell pool. During regeneration, the transition from a quiescent muscle stem cell to a differentiated fiber is accompanied by major metabolic changes. Such changes, and notably the switch from a glycolytic proliferative progenitor state to an oxidative post-mitotic differentiated state, require extensive mitochondrial biogenesis that takes place at the onset of differentiation and leads to increased ROS production. However, it is unclear whether this enhanced ROS production/mitochondrial content reflects an adaptation to the rising energy demand or whether it constitutes an essential regulation element of the differentiation program.To investigate the potential role of this metabolic switch and more specifically of reactive oxygen species during muscle regeneration, we took advantage of mouse mutants for Pitx2 and Pitx3 genes. Both genes are involved in foetal myogenesis where they have been identified as key regulators of the redox state preventing excessive ROS levels and DNA damage as cells undergo differentiation. We have now analyzed adult single and double Pitx2:Pitx3 conditional mutant mouse lines targeted to the muscle stem cell compartment. Double mutant satellite cells undergo senescence with impaired regeneration after injury, whereas in single Pitx3 mutants, premature differentiation occurs. We show that these effects are directly linked to dose-dependent changes in ROS levels and can be reversed by lowering ROS with the N-acetylcystein, supporting the notion that a controlled increase in ROS is required for differentiation of muscle stem cells.

  • Redox regulation by Pitx2 and Pitx3 is critical for fetal myogenesis.
    Developmental Cell, 2014
    Co-Authors: Aurore L'honoré, Jacques Drouin, Jean-françois Ouimette, Pierre-henri Commère, Didier Montarras, Margaret Buckingham
    Abstract:

    During development, major metabolic changes occur as cells become more specialized within a lineage. In the case of skeletal muscle, differentiation is accompanied by a switch from a glycolytic proliferative progenitor state to an oxidative postmitotic differentiated state. Such changes require extensive mitochondrial biogenesis leading to increased reactive oxygen species (ROS) production that needs to be balanced by an antioxidant system. Our analysis of double conditional Pitx2/3 mouse mutants, both in vivo during fetal myogenesis and ex vivo in primary muscle cell cultures, reveals excessive upregulation of ROS levels leading to DNA damage and apoptosis of differentiating cells. This is a consequence of downregulation of Nrf1 and genes for antioxidant enzymes, direct targets of Pitx2/3, leading to decreased expression of antioxidant enzymes, as well as impairment of mitochondrial function. Our analysis identifies Pitx2 and Pitx3 as key regulators of the intracellular redox state preventing DNA damage as cells undergo differentiation.

  • deletion of the PITX1 genomic locus affects mandibular tooth morphogenesis and expression of the barx1 and tbx1 genes
    Developmental Biology, 2008
    Co-Authors: Thimios A Mitsiadis, Jacques Drouin
    Abstract:

    PITX1 is a bicoid-related homeodomain factor that exhibits preferential expression in the developing hindlimb, mandible, pituitary gland and teeth. PITX1 gene-deleted mice exhibit striking abnormalities in morphogenesis and growth of both hindlimb and mandible, suggesting a proliferative defect in these two structures. Here, we studied the expression and regulation of PITX1 in both mandible and developing teeth and analyzed tooth morphology, cell proliferation, apoptosis and expression of Pitx2, Barx1 and Tbx1 in dental tissues of PITX1-/- mouse embryos. PITX1 expression is restricted to the epithelium of the growing tooth anlagen. Tissue recombination and bead implantation experiments demonstrated that bone morphogenetic protein-4 down-regulates PITX1 expression in both mandibular mesenchyme and dental epithelium. Deletion of the PITX1 locus results in micrognathia and abnormal morphology of the mandibular molars. Although Pitx2 expression in teeth of PITX1-/- embryos is not altered, expression of Barx1 decreased in the mesenchyme of the mandibular molars. Furthermore, PITX1 deletion results in suppression of Tbx1 expression in dental epithelium. Taken together, these results indicate that independent genetic pathways in mandibular and maxillary processes determine tooth development and morphology.

Elena V Semina - One of the best experts on this subject based on the ideXlab platform.

  • deletions of distant regulatory sequences upstream of zebrafish pitx2 result in a range of ocular phenotypes
    bioRxiv, 2019
    Co-Authors: Elena Sorokina, Eric Weh, Kathryn Hendee, Doug B Gould, Elena V Semina
    Abstract:

    Development of the anterior segment of the vertebrate eye is a highly coordinated process. Genetic mutations in factors guiding this process result in Anterior Segment Dysgenesis (ASD), a spectrum of disorders affecting the iris, cornea, trabecular meshwork and/or other iridocorneal angle structures and associated with glaucoma. One of the first factors linked to ASD in humans was PITX2, a homeodomain containing transcription factor with a role in Axenfeld-Rieger syndrome (ARS). In addition to pathogenic alleles within the coding region of PITX2, deletions affecting the distant upstream region, but not PITX2 itself, have also been reported in ARS. Consistent with this, the distant upstream region was shown to contain multiple conserved elements (CE) with pitx2-related enhancer activity identified through studies in zebrafish. The two smallest human deletions reported to date encompass conserved elements 5-11 ({Delta}CE5-11) or 5-7 ({Delta}CE5-7). We previously reported the generation of{Delta} CE5-11 in zebrafish and we have now replicated the smallest deletion,{Delta} CE5-7, in the same model and studied the associated phenotype, expression, and DNA methylation profiles; we also performed further phenotypic examinations of the pitx2{Delta}CE5-11 fish. We show that the expression changes and phenotypes observed in the two lines are variable but that the severity generally correlates with the size of the deletion and the number of affected CEs; pitx2 promoter and a nearby region were hypermethylated in the pitx2{Delta}CE5-7 embryonic eyes. In addition, a subset of pitx2{Delta}CE5-11 animals were found to have a severe retinal phenotype suggesting that additional factors may modify the effects of this allele. These data provide further insight into functional sequences in the PITX2/pitx2 genomic region that coordinate PITX2/pitx2 expression during eye development and provide the basis for future studies into PITX2/pitx2 upstream regulators and modifiers.

  • mip aquaporin 0 represents a direct transcriptional target of pitx3 in the developing lens
    PLOS ONE, 2011
    Co-Authors: Elena A Sorokina, Elena V Semina, Sanaa Muheisen, Nevin Mlodik
    Abstract:

    The PITX3 bicoid-type homeodomain transcription factor plays an important role in lens development in vertebrates. PITX3 deficiency results in a spectrum of phenotypes from isolated cataracts to microphthalmia in humans, and lens degeneration in mice and zebrafish. While identification of downstream targets of PITX3 is vital for understanding the mechanisms of normal ocular development and human disease, these targets remain largely unknown. To isolate genes that are directly regulated by PITX3, we performed a search for genomic sequences that contain evolutionarily conserved bicoid/PITX3 binding sites and are located in the proximity of known genes. Two bicoid sites that are conserved from zebrafish to human were identified within the human promoter of the major intrinsic protein of lens fiber, MIP/AQP0. MIP/AQP0 deficiency was previously shown to be associated with lens defects in humans and mice. We demonstrate by both chromatin immunoprecipitation and electrophoretic mobility shift assay that PITX3 binds to MIP/AQP0 promoter region in vivo and is able to interact with both bicoid sites in vitro. In addition, we show that wild-type PITX3 is able to activate the MIP/AQP0 promoter via interaction with the proximal bicoid site in cotransfection experiments and that the introduction of mutations disrupting binding to this site abolishes this activation. Furthermore, mutant forms of PITX3 fail to produce the same levels of transactivation as wild-type when cotransfected with the MIP/AQP0 reporter. Finally, knockdown of pitx3 in zebrafish affects formation of a DNA-protein complex associated with mip1 promoter sequences; and examination of expression in pitx3 morphant and control zebrafish revealed a delay in and reduction of mip1 expression in pitx3-deficient embryos. Therefore, our data suggest that PITX3 is involved in direct regulation of MIP/AQP0 expression and that the alteration of MIP/AQP0 expression is likely to contribute to the lens phenotype in cataract patients with PITX3 mutations.

  • identification of dominant foxe3 and pax6 mutations in patients with congenital cataract and aniridia
    Molecular Vision, 2010
    Co-Authors: Dominique Bremondgignac, Jeffrey C Murray, Pierre Bitoun, Linda M Reis, Henri Copin, Elena V Semina
    Abstract:

    Purpose: Aniridia and congenital cataract represent rare but severe developmental ocular conditions. We examined 33 probands from France for mutations in several transcription factors associated with these phenotypes, the forkhead box E3 (FOXE3), paired box gene 6 (PAX6), paired-like homeodomain transcription factor 2 (PITX2), and paired-like homeodomain transcription factor 3 (PITX3) genes. Methods: Out of 33 probands, 27 were affected with congenital cataract while the remaining six were affected with aniridia (with or without cataract). The coding regions of FOXE3, PAX6, PITX2, and PITX3 were examined by direct DNA sequencing of gene-specific PCR products. Results: A novel dominant mutation at the stop codon of FOXE3, c.959G>C (p.X320SerextX72), was identified in a patient with congenital cataract. Another novel FOXE3 sequence change, c.571–579dup (p.Tyr191_Pro193dup), was identified in a patient with aniridia, mild lens opacities, and some additional ocular defects; this patient was also found to carry a nonsense mutation in PAX6. PAX6 mutations were identified in two additional probands with aniridia and cataracts. None of the observed sequence alterations were found in normal controls. No mutations were identified in PITX2 or PITX3. Conclusions: The p.X320SerextX72 mutation is only the fourth FOXE3 allele associated with a dominant phenotype since the majority of FOXE3 mutations appear to be recessive with no phenotype observed in heterozygous carriers. The encoded protein is predicted to contain a complete normal sequence followed by seventy-two erroneous amino acids; the position and effect of this mutation are similar to two of the previously reported dominant changes, suggesting a common mechanism for dominant alleles. The p.Tyr191_Pro193dup is predicted to result in an in-frame duplication of three amino acids; however, the contribution of this mutation to the phenotype is unclear since the affected patient also carries a nonsense mutation in PAX6 which acts upstream of FOXE3 in the molecular pathway. The identified PAX6 mutations correspond to the two most commonly observed mutant alleles and demonstrate phenotypes that are consistent with the previously reported spectrum. Aniridia and congenital cataract represent rare but severe ocular conditions reflecting abnormal development of the anterior segment of the eye. Aniridia is characterized by the congenital absence of the iris and is most commonly inherited as an autosomal-dominant disorder. Aniridia is a panocular disease often associated with additional ocular symptoms such as visual impairment and nystagmus [1]. Congenital cataract is an opacification of the ocular lens present at birth. Congenital cataract is usually hereditary and can be transmitted as a dominant or a recessive trait. Congenital cataracts are frequently accompanied by other ocular defects, particularly anterior segment anomalies including corneal

  • antagonistic signals between bmp4 and fgf8 define the expression of PITX1 and pitx2 in mouse tooth forming anlage
    Developmental Biology, 2000
    Co-Authors: Tara R St Amand, Jeffrey C Murray, Elena V Semina, Yanding Zhang, Xiang Zhao, Lan K Nguyen, Yiping Chen
    Abstract:

    Members of the Pitx/RIEG family of homeodomain-containing transcription factors have been implicated in vertebrate organogenesis. In this study, we examined the expression and regulation of PITX1 and Pitx2 during mouse tooth development. PITX1 expression is detected in early development in a widespread pattern, in both epithelium and mesenchyme, covering the tooth-forming region in the mandible, and is then maintained in the dental epithelium from the bud stage to the late bell stage. Pitx2 expression, on the other hand, is restricted to the dental epithelium throughout odontogenesis. Interestingly, from E9.5 to E10.5, the expression domains of PITX1 and Pitx2, in the developing mandible, overlap with that of Fgf8 but are exclusive to the zone of Bmp4 expression. Bead implantation experiments demonstrate that ectopic expression of Fgf8 can induce/maintain the expression of both PITX1 and Pitx2 at E9.5. In contrast, Bmp4-expressing tissues and BMP4-soaked beads were able to repress PITX1 expression in mandibular mesenchyme and Pitx2 expression in the presumptive dental epithelium, respectively. However, the effects of FGF8 and BMP4 are transient. It thus appears that the early expression patterns of PITX1 and Pitx2 in the developing mandible are regulated by the antagonistic effects of FGF8 and BMP4 such that the PITX1 and Pitx2 expression patterns are defined. These results indicate that the epithelial-derived signaling molecules are responsible not only for restricting specific gene expression in the dental mesenchyme, but also for defining gene expression in the dental epithelium.

  • a novel homeobox gene pitx3 is mutated in families with autosomal dominant cataracts and asmd
    Nature Genetics, 1998
    Co-Authors: Elena V Semina, Rebecca S Reiter, Robert E Ferrell, Helen A Mintzhittner, Pierre Bitoun, Wallace L M Alward, Carrie Funkhauser, Sandra Daackhirsch, Jeffrey C Murray
    Abstract:

    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.

Daniel J Bernard - One of the best experts on this subject based on the ideXlab platform.

  • novel forms of paired like homeodomain transcription factor 2 pitx2 generation by alternative translation initiation and mrna splicing
    BMC Molecular Biology, 2008
    Co-Authors: Pankaj Lamba, Tord A. Hjalt, Daniel J Bernard
    Abstract:

    Background Members of the Paired-like homeodomain transcription factor (PITX) gene family, particularly PITX1 and PITX2, play important roles in normal development and in differentiated cell functions. Three major isoforms of PITX2 were previously reported to be produced through both alternative mRNA splicing (PITX2A and PITX2B) and alternative promoter usage (PITX2C). The proteins derived from these mRNAs contain identical homeodomain and carboxyl termini. Differences in the amino-termini of the proteins may confer functional differences in some contexts.

  • novel forms of paired like homeodomain transcription factor 2 pitx2 generation by alternative translation initiation and mrna splicing
    BMC Molecular Biology, 2008
    Co-Authors: Pankaj Lamba, Tord Hjalt, Daniel J Bernard
    Abstract:

    Members of the Paired-like homeodomain transcription factor (PITX) gene family, particularly PITX1 and PITX2, play important roles in normal development and in differentiated cell functions. Three major isoforms of PITX2 were previously reported to be produced through both alternative mRNA splicing (PITX2A and PITX2B) and alternative promoter usage (PITX2C). The proteins derived from these mRNAs contain identical homeodomain and carboxyl termini. Differences in the amino-termini of the proteins may confer functional differences in some contexts. Here, we report the identification of two novel PITX2 isoforms. First, we demonstrate that the Pitx2c mRNA generates two protein products, PITX2Cα and PITX2Cβ, via alternative translation initiation. Second, we identified a novel mRNA splice variant, Pitx2b2, which uses the same 5' splice donor in intron 2 as Pitx2b (hereafter referred to as Pitx2b1), but employs an alternative 3' splice acceptor, leading to an in-frame deletion of 39 base pairs relative to Pitx2b1. Pitx2b2 mRNA is expressed in both murine and human pituitary. The data show that in a murine gonadotrope cell line and adult murine pituitary what was previously thought to be PITX2B1 is actually PITX2Cβ, or perhaps PITX2B2. PITX2B1 is expressed at lower levels than previously thought. PITX2Cβ and PITX2B2 activate gonadotrope-specific gene promoter-reporters similarly to known PITX2 isoforms. We have identified and characterized two novel isoforms of PITX2, generated by alternative translation initiation (PITX2Cβ) and alternative mRNA splicing (PITX2B2). These proteins show similar DNA binding and trans-activation functions as other PITX2 isoforms in vitro, though their conservation across species suggests that they may play distinct, as yet unidentified, roles in vivo.