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Marten P Smidt - One of the best experts on this subject based on the ideXlab platform.
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Lmx1b Influences Correct Post-mitotic Coding of Mesodiencephalic Dopaminergic Neurons.
Frontiers in molecular neuroscience, 2019Co-Authors: Iris Wever, Elisa J. Hoekstra, Pablo Largo Barrientos, Marten P SmidtAbstract:The Lim Homeobox transcription factor 1 beta (LMX1b) has been identified as one of the transcription factors important for the development of mesodiencephalic dopaminergic (mdDA) neurons. During early development, Lmx1b is essential for induction and maintenance of the Isthmic Organizer (IsO), and genetic ablation results in the disruption of inductive activity from the IsO and loss of properly differentiated mdDA neurons. To study the downstream targets of Lmx1b without affecting the IsO, we generated a conditional model in which Lmx1b was selectively deleted in PITX3-expressing cells from embryonic day (E)13 onward. Supporting previous data, no significant changes could be observed in general dopamine (DA) marks, like Th, PITX3 and Vmat2 at E14.5. However, in depth analysis by means of RNA-sequencing revealed that Lmx1b is important for the mRNA expression level of survival factors En1 and En2 and for the repression of mdDA subset mark Ahd2 during (late) development. Interestingly, the regulation of Ahd2 by Lmx1b was found to be PITX3 independent, since PITX3 mRNA levels were not altered in Lmx1b conditional knock-outs (cKOs) and Ahd2 expression was also up-regulated in Lmx1b/PITX3 double mutants compared to PITX3 mutants. Further analysis of Lmx1b cKOs showed that post-mitotic deletion of Lmx1b additional leads to a loss of TH+ cells at 3 months age both in the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc). Remarkably, different cell types were affected in the SNc and the VTA. While TH+AHD2+ cells were lost the SNc, TH+AHD2- neurons were affected in the VTA, reflected by a loss of Cck expression, indicating that Lmx1b is important for the survival of a sub-group of mdDA neurons.
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Image_2_Lmx1b Influences Correct Post-mitotic Coding of Mesodiencephalic Dopaminergic Neurons.TIFF
2019Co-Authors: Iris Wever, Elisa J. Hoekstra, Pablo Largo-barrientos, Marten P SmidtAbstract:The Lim Homeobox transcription factor 1 beta (LMX1b) has been identified as one of the transcription factors important for the development of mesodiencephalic dopaminergic (mdDA) neurons. During early development, Lmx1b is essential for induction and maintenance of the Isthmic Organizer (IsO), and genetic ablation results in the disruption of inductive activity from the IsO and loss of properly differentiated mdDA neurons. To study the downstream targets of Lmx1b without affecting the IsO, we generated a conditional model in which Lmx1b was selectively deleted in PITX3-expressing cells from embryonic day (E)13 onward. Supporting previous data, no significant changes could be observed in general dopamine (DA) marks, like Th, PITX3and Vmat2 at E14.5. However, in depth analysis by means of RNA-sequencing revealed that Lmx1b is important for the mRNA expression level of survival factors En1 and En2 and for the repression of mdDA subset mark Ahd2 during (late) development. Interestingly, the regulation of Ahd2 by Lmx1b was found to be PITX3 independent, since PITX3 mRNA levels were not altered in Lmx1b conditional knock-outs (cKOs) and Ahd2 expression was also up-regulated in Lmx1b/PITX3 double mutants compared to PITX3 mutants. Further analysis of Lmx1b cKOs showed that post-mitotic deletion of Lmx1b additional leads to a loss of TH+ cells at 3 months age both in the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc). Remarkably, different cell types were affected in the SNc and the VTA. While TH+AHD2+ cells were lost the SNc, TH+AHD2- neurons were affected in the VTA, reflected by a loss of Cck expression, indicating that Lmx1b is important for the survival of a sub-group of mdDA neurons.
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Table_1_Lmx1b Influences Correct Post-mitotic Coding of Mesodiencephalic Dopaminergic Neurons.XLSX
2019Co-Authors: Iris Wever, Elisa J. Hoekstra, Pablo Largo-barrientos, Marten P SmidtAbstract:The Lim Homeobox transcription factor 1 beta (LMX1b) has been identified as one of the transcription factors important for the development of mesodiencephalic dopaminergic (mdDA) neurons. During early development, Lmx1b is essential for induction and maintenance of the Isthmic Organizer (IsO), and genetic ablation results in the disruption of inductive activity from the IsO and loss of properly differentiated mdDA neurons. To study the downstream targets of Lmx1b without affecting the IsO, we generated a conditional model in which Lmx1b was selectively deleted in PITX3-expressing cells from embryonic day (E)13 onward. Supporting previous data, no significant changes could be observed in general dopamine (DA) marks, like Th, PITX3and Vmat2 at E14.5. However, in depth analysis by means of RNA-sequencing revealed that Lmx1b is important for the mRNA expression level of survival factors En1 and En2 and for the repression of mdDA subset mark Ahd2 during (late) development. Interestingly, the regulation of Ahd2 by Lmx1b was found to be PITX3 independent, since PITX3 mRNA levels were not altered in Lmx1b conditional knock-outs (cKOs) and Ahd2 expression was also up-regulated in Lmx1b/PITX3 double mutants compared to PITX3 mutants. Further analysis of Lmx1b cKOs showed that post-mitotic deletion of Lmx1b additional leads to a loss of TH+ cells at 3 months age both in the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc). Remarkably, different cell types were affected in the SNc and the VTA. While TH+AHD2+ cells were lost the SNc, TH+AHD2- neurons were affected in the VTA, reflected by a loss of Cck expression, indicating that Lmx1b is important for the survival of a sub-group of mdDA neurons.
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PITX3 and En1 determine the size and molecular programming of the dopaminergic neuronal pool.
PloS one, 2017Co-Authors: Willemieke M. Kouwenhoven, Lars Von Oerthel, Marten P SmidtAbstract:Mesodiencephalic dopaminergic (mdDA) neurons are located in the ventral midbrain. These neurons form the substantia nigra (SNc) and the ventral tegmental area (VTA). Two transcription factors that play important roles in the process of terminal differentiation and subset-specification of mdDA neurons, are paired-like homeodomain transcription factor 3 (PITX3), and homeobox transcription factor Engrailed 1 (En1). We previously investigated the single PITX3KO and En1KO and observed important changes in the survival of mdDA neurons of the SNc and VTA as well as altered expression of pivotal rostral- and caudal-markers, Ahd2 and Cck, respectively. To refine our understanding of the regional-specific relationships between En1 and PITX3 and their (combined) role in the programming mdDA neurons on the rostral-to-caudal axis, we created double En1tm1Alj/tm1Alj;PITX3gfp/gfp (En1KO;PITX3GFP/GFP) animals. Here we report, that in absence of En1 and PITX3, only a limited number of mdDA neurons are present at E14.5. These mdDA neurons have a rudimentary dopaminergic cell fate, as they express Nurr1, Pbx3 and Otx2 but have lost their rostral or caudal subset identity. Furthermore, we report that the expression of Cck depends on En1 expression, while (in contrast) both PITX3 and En1 are involved in the initiation of Ahd2 expression. Thus we reveal in this manuscript that regulated levels of PITX3 and En1 control the size and rostral/caudal-identity of the mdDA neuronal population.
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identification of dlk1 ptpru and klhl1 as novel nurr1 target genes in meso diencephalic dopamine neurons
Development, 2009Co-Authors: Frank M. J. Jacobs, Annemarie J A Van Der Linden, Yuhui Wang, Lars Von Oerthel, Marten P SmidtAbstract:The orphan nuclear receptor Nurr1 is essential for the development of meso-diencephalic dopamine (mdDA) neurons and is required, together with the homeobox transcription factor PITX3, for the expression of genes involved in dopamine metabolism. In order to elucidate the molecular mechanisms that underlie the neuronal deficits in Nurr1 -/- mice, we performed combined gene expression microarrays and ChIP-on-chip analysis and thereby identified Dlk1, Ptpru and Klhl1 as novel Nurr1 target genes in vivo. In line with the previously described cooperativity between Nurr1 and PITX3, we show that the expression of Ptpru and Klhl1 in mdDA neurons is also dependent on PITX3. Furthermore, we demonstrate that Nurr1 interacts with the Ptpru promoter directly and requires PITX3 for full expression of Ptpru in mdDA neurons. By contrast, the expression of Dlk1 is maintained in PITX3 -/- embryos and is even expanded into the rostral part of the mdDA area, suggesting a unique position of Dlk1 in the Nurr1 and PITX3 transcriptional cascades. Expression analysis in Dlk1 -/- embryos reveals that Dlk1 is required to prevent premature expression of Dat in mdDA neuronal precursors as part of the multifaceted process of mdDA neuronal differentiation driven by Nurr1 and PITX3. Taken together, the involvement of Nurr1 and PITX3 in the expression of novel target genes involved in important neuronal processes such as neuronal patterning, axon outgrowth and terminal differentiation, opens up new avenues to study the properties of mdDA neurons during development and in neuronal pathology as observed in Parkinson9s disease.
Elena V Semina - One of the best experts on this subject based on the ideXlab platform.
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deletions of distant regulatory sequences upstream of zebrafish pitx2 result in a range of ocular phenotypes
bioRxiv, 2019Co-Authors: Elena Sorokina, Eric Weh, Kathryn Hendee, Doug B Gould, Elena V SeminaAbstract: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.
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mip aquaporin 0 represents a direct transcriptional target of PITX3 in the developing lens
PLOS ONE, 2011Co-Authors: Elena A Sorokina, Elena V Semina, Sanaa Muheisen, Nevin MlodikAbstract: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.
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identification of dominant foxe3 and pax6 mutations in patients with congenital cataract and aniridia
Molecular Vision, 2010Co-Authors: Dominique Bremondgignac, Jeffrey C Murray, Pierre Bitoun, Linda M Reis, Henri Copin, Elena V SeminaAbstract: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
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deletion in the promoter region and altered expression of PITX3 homeobox gene in aphakia mice
Human Molecular Genetics, 2000Co-Authors: Elena V Semina, Rebecca S Reiter, Ronald F Hrstka, Jochen GrawAbstract:Mouse aphakia (ak) is a recessive phenotype that spontaneously occurs in the 129/Sv-SlJ strain and is characterized by small eyes that lack a lens. We have recently identified a homeobox-containing gene, PITX3, and have shown that it is expressed in the developing lens and maps to chromosome 19 close to ak in mouse. Human PITX3 gene was found to underlie anterior segment dysgenesis and cataracts. We have now obtained the entire sequence of the mouse PITX3 gene including 10 kb of the 5' region and 5 kb of the 3' region. Of several microsatellite repeat regions identified within the PITX3 sequence, one was informative for linkage analysis. No recombination was observed between ak and the PITX3 marker, indicating that these two loci are closely linked (0.2 +/- 0.2 cM). Additionally, PITX3 transcripts were not detected in the ak/ak mice either in the lens placode or at later developmental stages of the lens by in situ hybridization. Since no differences were previously found between ak/ak and wild-type sequences in the PITX3 coding region, we hypothesized that an etiologic mutation is located in the promoter or other regulatory regions. To test this hypothesis we studied the 5' flanking region of the PITX3 gene. This analysis revealed a deletion of 652 bp located 2.5 kb upstream from the start point of the PITX3 5' UTR sequence in ak/ak mice. The deletion co-segregated with the ak mutation and was not detected in 16 samples from 10 different mouse strains including the founder strains. Analysis of the 652 bp region identified sequences similar to consensus binding sites for transcription factors AP-2 and Maf that were shown to play a critical role in lens determination. These lines of evidence suggest that the abnormal ocular development in the aphakia mouse is due to the deletion upstream of the PITX3 gene.
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antagonistic signals between bmp4 and fgf8 define the expression of pitx1 and pitx2 in mouse tooth forming anlage
Developmental Biology, 2000Co-Authors: Tara R St Amand, Jeffrey C Murray, Elena V Semina, Yanding Zhang, Xiang Zhao, Lan K Nguyen, Yiping ChenAbstract: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.
Jacques Drouin - One of the best experts on this subject based on the ideXlab platform.
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Specificity of PITX3-Dependent Gene Regulatory Networks in Subsets of Midbrain Dopamine Neurons
Molecular Neurobiology, 2017Co-Authors: Panojot Bifsha, Aurelio Balsalobre, Jacques DrouinAbstract: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.
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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, 2014Co-Authors: Aurore Lhonore, Jacques Drouin, Margaret Buckingham, Didier MontarrasAbstract: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.
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Redox regulation by Pitx2 and PITX3 is critical for fetal myogenesis.
Developmental Cell, 2014Co-Authors: Aurore L'honoré, Jacques Drouin, Jean-françois Ouimette, Pierre-henri Commère, Didier Montarras, Margaret BuckinghamAbstract: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.
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deletion of the pitx1 genomic locus affects mandibular tooth morphogenesis and expression of the barx1 and tbx1 genes
Developmental Biology, 2008Co-Authors: Thimios A Mitsiadis, Jacques DrouinAbstract: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.
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Formation and differentiation of multiple mesenchymal lineages during lung development is regulated by beta-catenin signaling.
Public Library of Science (PLoS), 2008Co-Authors: Stijn P. De Langhe, Jacques Drouin, Gianni Carraro, Denise Tefft, Yang Chai, Parviz Minoo, Mohammad K. Hajihosseini, Vesa KaartinenAbstract:The role of ss-catenin signaling in mesodermal lineage formation and differentiation has been elusive.To define the role of ss-catenin signaling in these processes, we used a Dermo1(Twist2)(Cre/+) line to target a floxed beta-catenin allele, throughout the embryonic mesenchyme. Strikingly, the Dermo1(Cre/+); beta-catenin(f/-) conditional Knock Out embryos largely phenocopy Pitx1(-/-)/Pitx2(-/-) double knockout embryos, suggesting that ss-catenin signaling in the mesenchyme depends mostly on the PITX family of transcription factors. We have dissected this relationship further in the developing lungs and find that mesenchymal deletion of beta-catenin differentially affects two major mesenchymal lineages. The amplification but not differentiation of Fgf10-expressing parabronchial smooth muscle progenitor cells is drastically reduced. In the angioblast-endothelial lineage, however, only differentiation into mature endothelial cells is impaired.Taken together these findings reveal a hierarchy of gene activity involving ss-catenin and PITX, as important regulators of mesenchymal cell proliferation and differentiation
Philip J. Gage - One of the best experts on this subject based on the ideXlab platform.
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the homeodomain transcription factor pitx2 is required for specifying correct cell fates and establishing angiogenic privilege in the developing cornea
Developmental Dynamics, 2014Co-Authors: Philip J. Gage, Chen Cha'o-kuang, Amanda L ZachariasAbstract: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.
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Sequential expression and redundancy of Pitx2 and PITX3 genes during muscle development.
Developmental biology, 2007Co-Authors: Aurore L'honoré, Philip J. Gage, Alexandre Marcil, Sally A Camper, Mélanie Lebel, Vincent Coulon, Julien Lafrance-vanasse, Jacques DrouinAbstract: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.
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extraocular muscle morphogenesis and gene expression are regulated by pitx2 gene dose
Investigative Ophthalmology & Visual Science, 2006Co-Authors: Adam G Diehl, Philip J. Gage, Sepideh Zareparsi, Min Qian, Ritu Khanna, Rowena AngelesAbstract: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.
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Expression of the homeobox gene Pitx2 in neural crest is required for optic stalk and ocular anterior segment development
Human Molecular Genetics, 2005Co-Authors: Amanda L. Evans, Philip J. GageAbstract: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.
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pitx2 distinguishes subtypes of terminally differentiated neurons in the developing mouse neuroepithelium
Developmental Biology, 2002Co-Authors: Donna M Martin, Philip J. Gage, Jennifer M Skidmore, Sally A CamperAbstract: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.
Jeffrey C Murray - One of the best experts on this subject based on the ideXlab platform.
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identification of dominant foxe3 and pax6 mutations in patients with congenital cataract and aniridia
Molecular Vision, 2010Co-Authors: Dominique Bremondgignac, Jeffrey C Murray, Pierre Bitoun, Linda M Reis, Henri Copin, Elena V SeminaAbstract: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
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antagonistic signals between bmp4 and fgf8 define the expression of pitx1 and pitx2 in mouse tooth forming anlage
Developmental Biology, 2000Co-Authors: Tara R St Amand, Jeffrey C Murray, Elena V Semina, Yanding Zhang, Xiang Zhao, Lan K Nguyen, Yiping ChenAbstract: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.
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a novel homeobox gene PITX3 is mutated in families with autosomal dominant cataracts and asmd
Nature Genetics, 1998Co-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 MurrayAbstract: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.
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isolation of a new homeobox gene belonging to the pitx rieg family expression during lens development and mapping to the aphakia region on mouse chromosome 19
Human Molecular Genetics, 1997Co-Authors: Elena V Semina, Rebecca S Reiter, Jeffrey C MurrayAbstract:We recently reported the positional cloning of a homeobox gene involved in the pathogenesis of Rieger syndrome, RIEG1 , and its mouse homolog, Rieg1 . Rieg1 (also independently described as Pitx2) is highly homologous to the Ptx1/Potx gene product, suggesting that there may be additional members of this novel Pitx family. The Pitx genes play an important role in eye, tooth, pituitary and umbilical region development as evidenced by Rieger syndrome and iris hypoplasia phenotypes, resulting from mutations in the RIEG1 gene and by expression studies. In order to characterize further the Pitx gene family we searched mouse cDNA libraries to identify additional members. A new gene was isolated which encodes a homeoprotein with strong homology to the other Pitx proteins and 97-100% identity in the homeodomain itself, suggesting that this is a third member of the family, PITX3 . In whole mount in situ hybridization on mouse embryos ranging from 8.5 to 11.5 days post-coitum (d.p.c.), PITX3 mRNA was seen only in the developing lens starting at day 11. Hybridization on cross-sections revealed strong signals in the lens vesicle in 11 d.p.c. embryos and throughout the lens, particularly in the anterior epithelium and equator region in 15 d.p.c. embryos. PITX3 was mapped close to aphakia on mouse chromosome 19. The aphakia homozygous mouse is characterized by small eyes lacking a lens, which fail to develop beyond 11 d.p.c. These data make PITX3 a strong candidate gene for the aphakia phenotype in the mouse and suggest a role for the human homolog in congenital lens malformations.