Philip J. Gage

The bicoid -related Pitx gene family in development

The important roles of homeobox genes in development of the hindbrain and axial body are well established. More recently, it has become clear that certain subfamilies of homeobox genes play particularly important roles in the development of more anterior structures. These have included the paired gene family in the eye (Gehring, 1996; Hanson and Van Heyningen, 1995; Macdonald and Wilson, 1996; Wehr and Gruss, 1996), the orthodenticle and distalless gene families in the foreand midbrains (Acampora et al., 1996; Acampora et al., 1995; Price et al., 1991; Simeone et al., 1994; Williams, 1998), and the Lhx gene family in the pituitary gland (Sheng et al., 1997; Sheng et al., 1996). This review summarizes the newly identified Pitx gene family and its role in development. This family includes three vertebrate paralogues that have been cloned in multiple organisms, and a fly cognate. Mutations in two members of this gene family lead to human disease or birth defects affecting anterior structures. The nomenclature for this gene family has been complicated by the fact that members have been cloned and uniquely named by more than one laboratory (Table 1). The first member of this family, mouse Ptx1 (pituitary homeobox 1) was isolated as a transcription factor involved in pro-opiomelanocortin gene transcription in anterior pituitary corticotropes (Lamonerie et al., 1996). However, since some pentaxin genes in mouse and human had previously been assigned the Ptx gene symbol, the gene symbols for the three mouse paralogues for this new homeobox gene family are Pitx1, Pitx2, and Pitx3 (Mouse Genome Database). In this review, we have adopted the official nomenclature of the MGD and propose that, for clarity, this nomenclature be adopted for other organisms. Three vertebrate paralogues, Pitx1, Pitx2, and Pitx3, have all been cloned from mouse and human (Table 1 and references therein). Some paralogues have also been cloned from chicken (Pitx1 and Pitx2), xenopus and zebrafish (Pitx2), and rat (Pitx3) (Table 1 and references therein). In two reports, mouse Pitx1 was cloned in functional assays: in a two-hybrid screen using Pit-1 as bait (Szeto et al., 1996) and as noted above. Human PITX2 was identified by positional cloning of the Rieger Syndrome gene (Semina et al., 1996). In the other reports, cloning was the result of using degenerate PCR or low stringency hybridization to detect expressed homeobox sequences in a variety of embryonic and adult tissues. The difficulty in cloning Pitx1 from xenopus and zebrafish has suggested that this orthologue may not be as widely distributed in nature as Pitx2 (Kitamura et al., 1997). However, the recent identification of a fly Pitx gene during a chromosome walk demonstrates that this gene family arose prior to the divergence of vertebrates and invertebrates (Vorbruggen et al., 1997). Each vertebrate paralogue has been mapped genetically in mouse and human (Table 1). The Pitx proteins all belong to the bicoid-related subclass of homeodomain proteins because they encode the defining lysine at residue 50 within the homeodomain. This residue, at residue 9 within the recognition helix of the homeodomain, is the major determinant of DNA binding specificity (Gehring et al., 1994; Hanes and Brent, 1989). Several members of this small subfamily are essential for axis and pattern formation (Ang et al., 1996). Pitx2 expresses multiple protein isoforms as a result of alternative splicing (Gage and Camper, 1997; Kitamura et al., 1997) and the use of different promoters (P. Gage and E. Semina, unpublished results) (Fig. 1). The three vertebrate paralogues are all highly conserved at the amino acid level (Fig. 1). For example, in mouse the Pitx2 and Pitx3 homeodomains are identical while Pitx1 differs by only two amino acids. The paralogues are also conserved Cterminal to the homeodomain (55–70%). In contrast, the N-termini of these proteins are essentially unrelated. The vertebrate orthologues are even more highly conserved. For example, there the mouse and chicken Pitx2a proteins are 96% identical with only ten amino acid substitutions between them. The Drosophila Pitx protein shows high conservation to the vertebrate proteins within the homeodomain (90–93%) and a short region near C-terminus that has been termed the OAR sequence (Furukawa et al., 1997) or the C-peptide (Kitamura et al., 1997). This sequence is present in several homeobox genes. In Pitx2, this domain appears to function as an intrinsic inhibitor of DNA binding activity whose function can be modulated by protein-protein interactions (Amendt et al., 1998). The vertebrate Pitx genes each have unique developmental and tissue-specific expression patterns (Fig. 2 and Table 2). However, there are several significant overlaps in expression pattern (Fig. 2). The most significant may be in the eye, where both Pitx2 and Pitx3 are expressed in the mesenchyme and its derivatives (Semina et al., 1998; Semina et al., 1996; Smidt et al., 1997). Demonstration in humans that mutations to Pitx2 result in Rieger’s Syndrome (Semina et al., 1996) and mutations to Pitx3 result in anterior segment mesenchymal dysgenesis and dominant cataracts (Semina et al., 1998) confirmed the importance of these genes in eye development. These autosomal-dominant conditions each affect the development or maintenance of anterior structures of the eye. Interestingly, mouse Pitx3 maps near aphakia, a recessive mutation resulting in small eyes that lack lenses and fail to develop beyond 11 days of gestation (Semina et al., 1997). Rieger’s Syndrome patients frequently show defects in dental and umbilical development in addition to their ocular defects (Feingold et al., 1969; Rieger, 1935), and subsets of patients also present with isolated growth insufficiency (Feingold et al., 1969). Several observations suggest that Pitx genes are also important for the development and function of other organs. The stomodeum is an ectoderm-derived layer of epithelium that derives from the anterior neural ridge and forms the earliest mouth structures (Couly and Le Douarin, 1985). Pitx1 expression defines the stomodeum and continues within stomodial derivatives, including the nasal pit and Rathke’s pouch (Lanctot et al., 1997). Pitx1 is also expressed more caudally in the posterior lateral plate and extraCorrespondence to: P.J. Gage Mammalian Genome 10, 197–200 (1999).

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 (komyo) we show it has a crucial role in this process. Pitx2 komyo mutants survive to adulthood, when they present strong cardiac morphological and functional defects. Confocal analysis of embryonic Pitx2 komyo 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 komyo mice. This is the first evidence for distinct Pitx2 action in mediating L/R atrial identity and asymmetrical ventricular remodeling.

AP-2α 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.

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.

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.

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/β‐catenin signaling during anterior segment development. Because the requirements for retinoic acid signaling, canonical Wnt/β‐catenin signaling, and PITX2 are not unique to the eye, this newly identified pathway may have relevance elsewhere during development and in tissue homeostasis. Developmental Dynamics 238:2149–2162, 2009.

Canonical Wnt/β‐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 β‐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. Developmental Dynamics 239:3215–3225, 2010.

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

BackgroundThe 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.ResultsWe 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 (β-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 β-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.Conclusionsβ-catenin in the Wnt1 expression domain, including the neural crest, plays a critical role in regulation of pituitary gland growth, development, and vascularization.

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.