Oculomotor nerve guidance and terminal branching requires interactions with differentiating extraocular muscles.

Muscle function is dependent on innervation by the correct motor nerves. Motor nerves are composed of motor axons which extend through peripheral tissues as a compact bundle, then diverge to create terminal nerve branches to specific muscle targets. As motor nerves approach their targets, they undergo a transition where the fasciculated nerve halts further growth then after a pause, the nerve later initiates branching to muscles. This transition point is potentially an intermediate target or guidepost to present specific cellular and molecular signals for navigation. Here we describe the navigation of the oculomotor nerve and its association with developing muscles in mouse embryos. We found that the oculomotor nerve initially grew to the eye three days prior to the appearance of any extraocular muscles. The oculomotor axons spread to form a plexus within a mass of cells, which included precursors of extraocular muscles and other orbital tissues and expressed the transcription factor Pitx2. The nerve growth paused in the plexus for more than two days, persisting during primary extraocular myogenesis, with a subsequent phase in which the nerve branched out to specific muscles. To test the functional significance of the nerve contact with Pitx2+ cells in the plexus, we used two strategies to genetically ablate Pitx2+ cells or muscle precursors early in nerve development. The first strategy used Myf5-Cre-mediated expression of diphtheria toxin A to ablate muscle precursors, leading to loss of extraocular muscles. The oculomotor axons navigated to the eye to form the main nerve, but subsequently largely failed to initiate terminal branches. The second strategy studied Pitx2 homozygous mutants, which have early apoptosis of Pitx2-expressing precursor cells, including precursors for extraocular muscles and other orbital tissues. Oculomotor nerve fibers also grew to the eye, but failed to stop to form the plexus, instead grew long ectopic projections. These results show that neither Pitx2 function nor Myf5-expressing cells are required for oculomotor nerve navigation to the eye. However, Pitx2 function is required for oculomotor axons to pause growth in the plexus, while Myf5-expressing cells are required for terminal branch initiation.

Foxc1 and Foxc2 in the Neural Crest Are Required for Ocular Anterior Segment Development.

Purpose: The large Forkhead (Fox) transcription factor family has essential roles in development, and mutations cause a wide range of ocular and nonocular disease. One member, Foxc2 is expressed in neural crest (NC)-derived periocular mesenchymal cells of the developing murine eye; however, its precise role in the development, establishment, and maintenance of the ocular surface has yet to be investigated. Methods: To specifically delete Foxc2 from NC-derived cells, conditional knockout mice for Foxc2 (NC-Foxc2-/-) were generated by crossing Foxc2F mice with Wnt1-Cre mice. Similarly, we also generated compound NC-specific mutations of Foxc2 and a closely related gene, Foxc1 (NC-Foxc1-/-;NC-Foxc2-/-) in mice. Results: Neural crest-Foxc2-/- mice show abnormal thickness in the peripheral-to-central corneal stroma and limbus and displaced pupils with irregular iris. The neural crest-specific mutation in Foxc2 also leads to ectopic neovascularization in the cornea, as well as impaired ocular epithelial cell identity and corneal conjunctivalization. Compound, NC-specific Foxc1; Foxc2 homozygous mutant mice have more severe defects in structures of the ocular surface, such as the cornea and eyelids, accompanied by significant declines in the expression of another key developmental factor, Pitx2, and its downstream effector Dkk2, which antagonizes canonical Wnt signaling. Conclusions: The neural crest-Foxc2 mutation is associated with corneal conjunctivalization, ectopic corneal neovascularization, and disrupted ocular epithelial cell identity. Furthermore, Foxc2 and Foxc1 cooperatively function in NC-derived mesenchymal cells to ensure proper morphogenesis of the ocular surface via the regulation of Wnt signaling. Together, Foxc2 is required in the NC lineage for mesenchymal-epithelial interactions in corneal and ocular surface development.

A targeted approach to genome-wide studies reveals new genetic associations with central corneal thickness.

Purpose: To evaluate the ability of a targeted genome-wide association study (GWAS) to identify genes associated with central corneal thickness (CCT). Methods: A targeted GWAS was used to investigate whether ten candidate genes with known roles in corneal development were associated with CCT in two Singaporean populations. The single nucleotide polymorphisms (SNPs) within a 500 kb interval encompassing each candidate were analyzed, and in light of the resulting data, members of the Wnt pathway were subsequently screened using similar methodology. Results: Variants within the 500 kb interval encompassing three candidate genes, DKK1 (rs1896368, p=1.32×10-3), DKK2 (rs17510449, p=7.34×10-4), and FOXO1 (rs7326616, p=1.56×10-4 and rs4943785, p=1.19×10-3), were statistically significantly associated with CCT in the Singapore Indian population. DKK2 was statistically significantly associated with CCT in a separate Singapore Malaysian population (rs10015200, p=2.26×10-3). Analysis of Wnt signaling pathway genes in each population demonstrated that TCF7L2 (rs3814573, p=1.18×10-3), RYK (rs6763231, p=1.12×10-3 and rs4854785, p=1.11×10-3), and FZD8 (rs640827, p=5.17×10-4) were statistically significantly associated with CCT. Conclusions: The targeted GWAS identified four genes (DKK1, DKK2, RYK, and FZD8) with novel associations with CCT and confirmed known associations with two genes, FOXO1 and TCF7L2. All six participate in the Wnt pathway, supporting a broader role for Wnt signaling in regulating the thickness of the cornea. In parallel, this study demonstrated that a hypothesis-driven candidate gene approach can identify associations in existing GWAS data sets.

Heterozygous Pitx2 Null Mice Accurately Recapitulate the Ocular Features of Axenfeld-Rieger Syndrome and Congenital Glaucoma.

PURPOSE: The purpose of this analysis was to assess the utility of Pitx2+/- mice as a model for the ocular features of Axenfeld-Rieger Syndrome and for congenital glaucoma. METHODS: Eyes of Pitx2+/- and wild-type littermates were examined clinically using optical coherence tomography (OCT) and fundus photography. Intraocular pressures were measured using a TonoLab rebound tonometer. Eyes were examined histologically to assess PITX2 expression, structural integrity, and optic nerve and ganglion cell content. RESULTS: PITX2 is present postnatally in the corneal endothelium and stroma, iris stroma, trabecular meshwork, and Schlemm's canal. Reduced central corneal thickness, iris defects, and iridicorneal adhesions are all prevalent in Pitx2+/- eyes. Although optic nerve heads appear normal at postnatal day 7, IOP is elevated and optic nerve head cupping is fully penetrant in Pitx2+/- eyes by 3 weeks of age. Neurodegeneration is present in a significant percentage of optic nerves from Pitx2+/- mice by 3 weeks of age, and is fully penetrant by 2 months of age. Pitx2+/- eyes show significant reductions in specifically ganglion cell density in all four quadrants by 2 months of age. CONCLUSIONS: Pitx2+/- mice model the major ocular features of Axenfeld-Rieger Syndrome and will be an important resource for understanding the molecular mechanisms leading to anterior segment dysgenesis and a high prevalence of glaucoma in this disease. In addition, these mice may provide an efficient new model for assessing the molecular events in glaucoma more generally, and for developing and testing new treatment paradigms for this disease.

ß-catenin is required in the neural crest and mesencephalon for pituitary gland organogenesis.

BACKGROUND: The pituitary gland is a highly vascularized tissue that requires coordinated interactions between the neural ectoderm, oral ectoderm, and head mesenchyme during development for proper physiological function. The interactions between the neural ectoderm and oral ectoderm, especially the role of the pituitary organizer in shaping the pituitary precursor, Rathke's pouch, are well described. However, less is known about the role of head mesenchyme in pituitary organogenesis. The head mesenchyme is derived from definitive mesoderm and neural crest, but the relative contributions of these tissues to the mesenchyme adjacent to the pituitary are not known. RESULTS: We carried out lineage tracing experiments using two neural crest-specific mouse cre lines, Wnt1-cre and P0-cre, and determined that the head mesenchyme rostral to the pituitary gland is neural crest derived. To assess the role of the neural crest in pituitary development we ablated it, using Wnt1-cre to delete Ctnnb1 (ß-catenin), which is required for neural crest development. The Wnt1-cre is active in the neural ectoderm, principally in the mesencephalon, but also in the posterior diencephalon. Loss of ß-catenin in this domain causes a rostral shift in the ventral diencephalon, including the pituitary organizer, resulting in pituitary dysmorphology. The neural crest deficient embryos have abnormally dilated pituitary vasculature due to a loss of neural crest derived pericytes. CONCLUSIONS: ß-catenin in the Wnt1 expression domain, including the neural crest, plays a critical role in regulation of pituitary gland growth, development, and vascularization.

AP-2ß Is a Downstream Effector of PITX2 Required to Specify Endothelium and Establish Angiogenic Privilege During Corneal Development.

PURPOSE: The homeodomain transcription factor, PITX2, is at the apex of a genetic pathway required for corneal development, but the critical effector genes regulated by the PITX2 remain unknown. The purpose of this study was to discover and validate PITX2-dependent mechanisms required for specifying cell lineages and establishing angiogenic privilege within the developing cornea. METHODS: Microarrays were used to compare gene expression in corneas isolated from temporal Pitx2 knockout embryos and control littermates. Quantitative RT-PCR and immunohistochemistry was used to further validate Tfap2b expression differences in Pitx2 knockout versus control corneas. In situ hybridization and protein immunohistochemistry were used to assay eyes of a Tfap2b allelic series of embryos to identify differentiated cellular lineages in the cornea, blood vessel endothelium, or lymphatic vessel endothelium. RESULTS: We show that PITX2 is required for the expression of Tfap2b, encoding the AP-2ß transcription factor, in the neural crest during corneal development. Markers of differentiated corneal epithelium and stroma are expressed in the absence of AP-2ß. In contrast, markers of differentiated corneal endothelium are not expressed in the absence of AP-2ß. Endomucin+ blood vessels are present throughout the developing corneal stroma in the absence of AP-2ß, whereas LYVE1+ lymphatic vessels are not found. CONCLUSIONS: The AP-2ß transcription factor is an important effector of PITX2 function during corneal development, required for differentiation of corneal endothelium and establishment of angiogenic privilege. Unlike PITX2, AP-2ß is not required for the early expression of available lineage specific markers for the corneal epithelium and stroma during embryogenesis, nor establishment of lymphangiogenic privilege. Therefore, additional PITX2-dependent factors likely regulate these latter processes during embryonic development. These results extend our understanding of the genetic mechanisms regulating cornea development.

Mouse Models for the Dissection of CHD7 Functions in Eye Development and the Molecular Basis for Ocular Defects in CHARGE Syndrome.

PURPOSE: CHARGE syndrome (Coloboma of the eye, Heart defects, Atresia of the choanae, Retardation of growth and/or development, Genital and/or urinary tract abnormalities, and Ear abnormalities and deafness) is the second-leading cause of deaf-blindness after Usher syndrome. Heterozygous mutations in CHD7 cause CHARGE syndrome in 70% to 90% of patients. We tested the hypothesis that tissue-specific mutant mice provide models for molecularly dissecting CHD7 functions during eye development. METHODS: The conditional Chd7flox allele was mated together with tissue-specific Cre transgenes. Immunohistochemistry was used to determine the normal CHD7 pattern in the early eye primordia and to assess Chd7 mutants for expression of region-specific protein markers. RESULTS: CHD7 is present in the neural ectoderm and surface ectoderm of the eye. Deletion from neural and surface ectoderm results in severely dysmorphic eyes generally lacking recognizable optic cup structures and small lenses. Deletion from the neural ectoderm results in similar defects. Deletion from the surface ectoderm results in eyes with smaller lenses. Lens tissue and the major subdivisions of the neural ectoderm are present following conditional deletion of Chd7 from the neural ectoderm. Closure of the optic fissure depends on the Chd7 gene dose within the neural ectoderm. CONCLUSIONS: Eye development requires CHD7 in multiple embryonic tissues. Lens development requires CHD7 in the surface ectoderm, whereas optic cup and stalk morphogenesis require CHD7 in the neural ectoderm. CHD7 is not absolutely required for specification of the major subdivisions within the neural ectoderm. As in humans, normal eye development in mice is sensitive to Chd7 haploinsufficiency. These data indicate the Chd7 mutant mice are models for determining the molecular etiology of ocular defects in CHARGE syndrome.

Mutation of FOXC1 and PITX2 induces cerebral small-vessel disease.

Patients with cerebral small-vessel disease (CSVD) exhibit perturbed end-artery function and have an increased risk for stroke and age-related cognitive decline. Here, we used targeted genome-wide association (GWA) analysis and defined a CSVD locus adjacent to the forkhead transcription factor FOXC1. Moreover, we determined that the linked SNPs influence FOXC1 transcript levels and demonstrated that patients as young as 1 year of age with altered FOXC1 function exhibit CSVD. MRI analysis of patients with missense and nonsense mutations as well as FOXC1-encompassing segmental duplication and deletion revealed white matter hyperintensities, dilated perivascular spaces, and lacunar infarction. In a zebrafish model, overexpression or morpholino-induced suppression of foxc1 induced cerebral hemorrhage. Inhibition of foxc1 perturbed platelet-derived growth factor (Pdgf) signaling, impairing neural crest migration and the recruitment of mural cells, which are essential for vascular stability. GWA analysis also linked the FOXC1-interacting transcription factor PITX2 to CSVD, and both patients with PITX2 mutations and murine Pitx2-/- mutants displayed brain vascular phenotypes. Together, these results extend the genetic etiology of stroke and demonstrate an increasing developmental basis for human cerebrovascular disease.

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

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.

FGF9-Pitx2-FGF10 signaling controls cecal formation in mice.

Fibroblast growth factor (FGF) signaling to the epithelium and mesenchyme mediated by FGF10 and FGF9, respectively, controls cecal formation during embryonic development. In particular, mesenchymal FGF10 signals to the epithelium via FGFR2b to induce epithelial cecal progenitor cell proliferation. Yet the precise upstream mechanisms controlling mesenchymal FGF10 signaling are unknown. Complete deletion of Fgf9 as well as of Pitx2, a gene encoding a homeobox transcription factor, both lead to cecal agenesis. Herein, we used mouse genetic approaches to determine the precise contribution of the epithelium and/or mesenchyme tissue compartments in this process. Using tissue compartment specific Fgf9 versus Pitx2 loss of function approaches in the gut epithelium and/or mesenchyme, we determined that FGF9 signals to the mesenchyme via Pitx2 to induce mesenchymal Fgf10 expression, which in turn leads to epithelial cecal bud formation.

Pitx2 is an upstream activator of extraocular myogenesis and survival.

The transcription factors required to initiate myogenesis in branchial arch- and somite-derived muscles are known, but the comparable upstream factors required during extraocular muscle development have not been identified. We show Pax7 is dispensable for extraocular muscle formation, whereas Pitx2 is cell-autonomously required to prevent apoptosis of the extraocular muscle primordia. The survival requirement for Pitx2 is stage-dependent and ends following stable activation of genes for the muscle regulatory factors (e.g. Myf5, MyoD), which is reduced in the absence of Pitx2. Further, PITX2 binds and activates transcription of the Myf5 and MyoD promoters, indicating these genes are direct targets. Collectively, these data demonstrate that PITX2 is required at several steps in the development of extraocular muscles, acting first as an anti-apoptotic factor in pre-myogenic mesoderm, and subsequently to activate the myogenic program in these cells. Thus, Pitx2 is the first demonstrated upstream activator of myogenesis in the extraocular muscles.

Canonical Wnt/ß-catenin signaling is required for maintenance but not activation of Pitx2 expression in neural crest during eye development.

Pitx2 is a paired-like homeodomain gene that acts as a key regulator of eye development. Despite its significance, upstream regulation of Pitx2 expression during eye development remains incompletely understood. We use neural crest-specific ablation of Ctnnb1 to demonstrate that canonical Wnt signaling is not required for initial activation of Pitx2 in neural crest. However, canonical Wnt signaling is subsequently required to maintain Pitx2 expression in the neural crest. Eye development in Ctnnb1-null mice appears grossly normal early but significant phenotypes emerge following loss of Pitx2 expression. LEF-1 and ß-catenin bind Pitx2 promoter sequences in ocular neural crest, indicating a likely direct effect of canonical Wnt signaling on Pitx2 expression. Combining our data with previous reports, we propose a model wherein a sequential code of retinoic acid followed by canonical Wnt signaling are required for activation and maintenance of Pitx2 expression, respectively. Other key transcription factors in the neural crest, including Foxc1, do not require intact canonical Wnt signaling.

AP-2alpha knockout mice exhibit optic cup patterning defects and failure of optic stalk morphogenesis.

Appropriate development of the retina and optic nerve requires that the forebrain-derived optic neuroepithelium undergoes a precisely coordinated sequence of patterning and morphogenetic events, processes which are highly influenced by signals from adjacent tissues. Our previous work has suggested that transcription factor activating protein-2 alpha (AP-2alpha; Tcfap2a) has a non-cell autonomous role in optic cup (OC) development; however, it remained unclear how OC abnormalities in AP-2alpha knockout (KO) mice arise at the morphological and molecular level. In this study, we show that patterning and morphogenetic defects in the AP-2alpha KO optic neuroepithelium begin at the optic vesicle stage. During subsequent OC formation, ectopic neural retina and optic stalk-like tissue replaced regions of retinal pigment epithelium. AP-2alpha KO eyes also displayed coloboma in the ventral retina, and a rare phenotype in which the optic stalk completely failed to extend, causing the OCs to be drawn inward to the midline. We detected evidence of increased sonic hedgehog signaling in the AP-2alpha KO forebrain neuroepithelium, which likely contributed to multiple aspects of the ocular phenotype, including expansion of PAX2-positive optic stalk-like tissue into the OC. Our data suggest that loss of AP-2alpha in multiple tissues in the craniofacial region leads to severe OC and optic stalk abnormalities by disturbing the tissue-tissue interactions required for ocular development. In view of recent data showing that mutations in human TFAP2A result in similar eye defects, the current findings demonstrate that AP-2alpha KO mice provide a valuable model for human ocular disease.

Human PRKC apoptosis WT1 regulator is a novel PITX2-interacting protein that regulates PITX2 transcriptional activity in ocular cells.

Mutations in the homeobox transcription factor PITX2 result in Axenfeld-Rieger syndrome (ARS), which is associated with anterior segment dysgenesis and an increased risk of glaucoma. To understand the pathogenesis of the defects resulting from PITX2 mutations, it is essential to know the normal functions of PITX2 and its interaction with the network of proteins in the eye. Yeast two-hybrid screening was performed using a cDNA library from a human trabecular meshwork primary cell line to detect novel PITX2-interacting proteins and study their role in ARS pathogenesis. After screening of approximately 1 x 10(6) clones, one putative interacting protein was identified named PRKC apoptosis WT1 regulator (PAWR). This interaction was further confirmed by retransformation assay in yeast cells as well as co-immunoprecipitation in ocular cells and nickel pulldown assay in vitro. PAWR is reportedly a proapoptotic protein capable of selectively inducing apoptosis primarily in cancer cells. Our analysis indicates that the homeodomain and the adjacent inhibitory domain in PITX2 interact with the C-terminal leucine zipper domain of PAWR. Endogenous PAWR and PITX2 were found to be located in the nucleus of ocular cells and to co-localize in the mesenchyme of the iridocorneal angle of the developing mouse eye, consistent with a role in the development of the anterior segment of the eye. PAWR was also found to inhibit PITX2 transcriptional activity in ocular cells. These data suggest PAWR is a novel PITX2-interacting protein that regulates PITX2 activity in ocular cells. This information sheds new light in understanding ARS and associated glaucoma pathogenesis.

Signaling "cross-talk" is integrated by transcription factors in the development of the anterior segment in the eye.

Extracellular signaling "cross-talk" between tissues is an important requirement for development of many organs yet the underlying mechanisms generally remain poorly understood. The anterior segment of the eye, which is constructed from four embryonic lineages, provides a unique opportunity to genetically dissect developmental processes such as signaling "cross-talk" without fear of inducing lethality. In the current review, we summarize recent data showing that PITX2, a homeodomain transcription factor, integrates retinoic acid and canonical Wnt/beta-catenin signaling during anterior segment development. Because the requirements for retinoic acid signaling, canonical Wnt/beta-catenin signaling, and PITX2 are not unique to the eye, this newly identified pathway may have relevance elsewhere during development and in tissue homeostasis.

The canonical Wnt signaling antagonist DKK2 is an essential effector of PITX2 function during normal eye development.

Local control of cell signaling activity and integration of inputs from multiple signaling pathways are central for normal development but the underlying mechanisms remain poorly understood. Here we show that Dkk2, encoding an antagonist of canonical Wnt signaling, is an essential downstream target of the PITX2 homeodomain transcription factor in neural crest during eye development. Canonical Wnt signaling is ectopically activated in central ocular surface ectoderm and underlying mesenchyme in Pitx2- and Dkk2-deficient mice. General ocular surface ectoderm identity is maintained during development in Dkk2-deficient mice but peripheral fates, including conjunctival goblet cells and eyelash follicles, are ectopically permitted within more central structures and eyelids are hypomorphic. Loss of DKK2 results in ectopic blood vessels within the periocular mesenchyme and PITX2 expression remains persistently high, providing evidence for a negative feedback loop. Collectively, these data suggest that activation of Dkk2 by PITX2 provides a mechanism to locally suppress canonical Wnt signaling activity during eye development, a paradigm that may be a model for achieving local or transient inhibition of pathway activity elsewhere during embryogenesis. We further propose a model placing PITX2 as an essential integration node between retinoic acid and canonical Wnt signaling during eye development.

Myocardial Pitx2 differentially regulates the left atrial identity and ventricular asymmetric remodeling programs.

The Pitx2 gene regulates left-right (L/R) asymmetrical cardiac morphogenesis. Constitutive Pitx2 knock out (ko) mice die before birth and display, among other defects, right atrial isomerism, atrial and ventricular septal defects, and double outlet right ventricle. The myocardial role of the gene has not been dissected. In particular, how Pitx2 regulates the differential L/R cardiac identity program is not clear. Additionally, the relation between Pitx2 ko ventricular defects and the gene expression pattern is not understood. In this article we analyze Pitx2 myocardial function during mouse heart development. By in situ hybridization analysis we show that myocardial Pitx2 expression delineates the remodeling of the left atrioventricular canal, the inner curvature, the ventral part of the interventricular ring, and the ventral portion of the right and left ventricle. By genetic analysis using an allelic series of Pitx2 mutants, among which a myocardial specific ko (ko(myo)) we show it has a crucial role in this process. Pitx2 ko(myo) mutants survive to adulthood, when they present strong cardiac morphological and functional defects. Confocal analysis of embryonic Pitx2 ko(myo) hearts reveals delayed cardiomyocyte development in the ventricular but not in the atrial Pitx2 null areas. Conversely, selective left atrial BMP10 mRNA downregulation which normally occurs at fetal stages is not found in the Pitx2 ko(myo) mice. This is the first evidence for distinct Pitx2 action in mediating L/R atrial identity and asymmetrical ventricular remodeling.

Sequential expression and redundancy of Pitx2 and Pitx3 genes during muscle development.

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.

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.

Nestin-Cre mediated deletion of Pitx2 in the mouse.

Nestin-Cre mice are widely used to generate gene deletions in the developing brain. Surprisingly, fewNestin-Cre lines have been characterized for their temporal and brain region-specific recombination. In addition, some Nestin-Cre lines express Cre outside the central nervous system, making it difficult to choose appropriate lines for targeting genes with brain region-restricted expression. Here we describe the properties of a Nestin-Cre transgenic line and its use for conditional deletions of Pitx2, a paired-like homeodomain transcription factor. We report that Nestin-Cre conditional Pitx2 mutant mice have ocular and craniofacial defects consistent with the role of human PITX2 in Rieger syndrome. Conditional mutants exhibit defects in midbrain neuronal development similar to those in Pitx2 homozygous null embryos, but lack the abnormalities in subthalamic nucleus neurons that occur with complete loss of Pitx2 function. These data indicate that normal differentiation of midbrain neurons depends upon adequate Pitx2 function during the period of active neurogenesis.