Olga Medina-Martinez

Severe defects in proliferation and differentiation of lens cells in Foxe3 null mice

ABSTRACT During mouse eye development, the correct formation of the lens occurs as a result of reciprocal interactions between the neuroectoderm that forms the retina and surface ectoderm that forms the lens. Although many transcription factors required for early lens development have been identified, the mechanism and genetic interactions mediated by them remain poorly understood. Foxe3 encodes a winged helix-forkhead transcription factor that is initially expressed in the developing brain and in the lens placode and later restricted exclusively to the anterior lens epithelium. Here, we show that targeted disruption of Foxe3 results in abnormal development of the eye. Cells of the anterior lens epithelium show a decreased rate of proliferation, resulting in a smaller than normal lens. The anterior lens epithelium does not properly separate from the cornea and frequently forms an unusual, multilayered tissue. Because of the abnormal differentiation, lens fiber cells do not form properly, and the morphogenesis of the lens is greatly affected. The abnormally differentiated lens cells remain irregular in shape, and the lens becomes vacuolated. The defects in lens development correlate with changes in the expression of growth and differentiation factor genes, including DNase II-like acid DNase, Prox1, p57, and PDGFα receptor. As a result of abnormal lens development, the cornea and the retina are also affected. While Foxe3 is also expressed in a distinct region of the embryonic brain, we have not observed abnormal development of the brain in Foxe3 −/− animals.

Foxe view of lens development and disease

The recent identification of a mutation in Foxe3 that causes congenital primary aphakia in humans marks an important milestone. Congenital primary aphakia is a rare developmental disease in which the lens does not form. Previously, Foxe3 had been shown to play a crucial role in vertebrate lens formation and this gene is one of the earliest integrators of several signaling pathways that cooperate to form a lens. In this review, we highlight recent advances that have led to a better understanding of the developmental processes and gene regulatory networks involved in lens development and disease.

Cell-Autonomous Requirement for Rx Function in the Mammalian Retina and Posterior Pituitary

Rx is a paired-like homeobox gene that is required for vertebrate eye formation. Mice lacking Rx function do not develop eyes or the posterior pituitary. To determine whether Rx is required cell autonomously in these tissues, we generated embryonic chimeras consisting of wild type and Rx−/− cells. We found that in the eye, Rx-deficient cells cannot participate in the formation of the neuroretina, retina pigment epithelium and the distal part of the optic stalk. In addition, in the ventral forebrain, Rx function is required cell autonomously for the formation of the posterior pituitary. Interestingly, Rx−/− and wild type cells segregate before the morphogenesis of these two tissues begins. Our observations suggest that Rx function is not only required for the morphogenesis of the retina and posterior pituitary, but also prior to morphogenesis, for the sorting out of cells to form distinct fields of retinal/pituitary cells.

Pitx3 controls multiple aspects of lens development

The transcription factor Pitx3 is critical for lens formation. Deletions in the promoter of this gene cause abnormal lens development in the aphakia (ak) mouse mutant, which has only rudimentary lenses. In this study, we investigated the role of Pitx3 in lens development and differentiation. We found that reduced expression of Pitx3 leads to changes in the proliferation, differentiation and survival of lens cells. The genetic interactions between Pitx3 and Foxe3 were investigated, as these two transcription factors are expressed at the same time in lens development and their absence has similar consequences for lens development. We found no evidence that these two genes genetically interact. In general, our study shows that the abnormal phenotype of the ak lenses is not due to just one molecular pathway, rather in the absence of Pitx3 expression multiple aspects of lens development are disrupted. Developmental Dynamics 238:2193–2201, 2009.

FOXE3 mutations predispose to thoracic aortic aneurysms and dissections.

The ascending thoracic aorta is designed to withstand biomechanical forces from pulsatile blood. Thoracic aortic aneurysms and acute aortic dissections (TAADs) occur as a result of genetically triggered defects in aortic structure and a dysfunctional response to these forces. Here, we describe mutations in the forkhead transcription factor FOXE3 that predispose mutation-bearing individuals to TAAD. We performed exome sequencing of a large family with multiple members with TAADs and identified a rare variant in FOXE3 with an altered amino acid in the DNA-binding domain (p.Asp153His) that segregated with disease in this family. Additional pathogenic FOXE3 variants were identified in unrelated TAAD families. In mice, Foxe3 deficiency reduced smooth muscle cell (SMC) density and impaired SMC differentiation in the ascending aorta. Foxe3 expression was induced in aortic SMCs after transverse aortic constriction, and Foxe3 deficiency increased SMC apoptosis and ascending aortic rupture with increased aortic pressure. These phenotypes were rescued by inhibiting p53 activity, either by administration of a p53 inhibitor (pifithrin-α), or by crossing Foxe3-/- mice with p53-/- mice. Our data demonstrate that FOXE3 mutations lead to a reduced number of aortic SMCs during development and increased SMC apoptosis in the ascending aorta in response to increased biomechanical forces, thus defining an additional molecular pathway that leads to familial thoracic aortic disease.

Expression of FoxE4 and Rx Visualizes the Timing and Dynamics of Critical Processes Taking Place during Initial Stages of Vertebrate Eye Development

Several transcription factors have a critical function during initial stages of vertebrate eye formation. In this paper, we discuss the role of the Rx subfamily of homeobox-containing genes in retinal development, and the role of the Foxe3 and FoxE4 subfamily of forkhead box-containing genes in lens development. Rx genes are expressed in the initial stages of retinal development and they play a critical role in eye formation. Elimination of Rx function in mice results in lack of eye formation. Abnormal eye development observed in the mouse mutation eyeless (ey1), the medakatemperature-sensitive mutation eyeless (el), and the zebrafish mutation chokh are caused by abnormal regulation or function of Rx genes. In humans, a mutation in Rx leads to anophthalmia. In contrast, Foxe3 and FoxE4 genes are expressed in the lens and they play an essential role in its formation. Mutations in the Foxe3 gene are the cause of the mouse mutation dysgenetic lens (dyl) and in humans, mutation in FOXE3 leads to anterior segment dysgenesis and cataracts. Since Rx and FoxE4 are expressed in the earliest stages of retina and lens development, their expression visualizes the timing and dynamics of the crucial processes that comprise eye formation. In this paper we present a model of eye development based on the expression pattern of these two genes.

Focus on Molecules: Pitx3

Pituitary homeobox 3 (Pitx3), (also known as Ptx3, CTPP4, MGC12766), was first described by Semina and coworkers in 1997 (Semina et al., 1997) (accession numbers for human sequences Nucleotide NG_008147, mRNA NM_005029, Protein NP_005020.1). It is a member of the RIEG/PITX homeobox gene family (paired-like bicoid class of homeodomain proteins, subclass Pitx), located on chromosome 10q25 in human and on chromosome 19C3 in mouse. Pitx3 encodes a 302 amino acid long protein that is highly conserved from yeast to man. Its structure is illustrated in Fig. 1. Members of the Pitx subfamily, including Pitx1, Pitx2, Pitx3 and the Drosophila cognate bicoid, encode transcription factors characterized by the presence of the conserved DNA binding K50 paired-like homeodomain and a 15 amino acid motif (R**SIA*LR*KA*EH), termed the OAR domain. Although it has been proposed that the OAR domain is important for the transactivation of transcription, DNA binding and/or proteineprotein interaction, its function is not fully understood. The Drosophila Pitx protein, bicoid, is highly similar to the vertebrate proteins in the homeodomain and the OAR sequence. Its DNA binding site, TAATCC, is also bound and transactivated by the vertebrate Pitx3 proteins.

Role of Fox Genes During Xenopus Embryogenesis

Fox genes encode a remarkably conserved family of nuclear proteins that can act as transcriptional activators or repressors. Their high level of conservation is probably due to the critical roles they play in embryonic pattern formation and tissue-specific gene expression (Dirksen and Jamrich 1992; Sasaki and Hogan 1993; Hatini et al. 1994; Dirksen and Jamrich 1995; Kaufmann and Knochel 1996; Martinez et al. 1997; Kenyon et al. 1999; Brownell et al. 2000; Carlsson and Mahlapuu 2002). Fox genes encode proteins that contain a highly conserved 110 amino acid long DNA-binding domain that was originally described in the Drosophila mutant fork head (Lai et al. 1990; Weigel and Jackie 1990). Because of this, they were called the forkhead genes. The structure of these proteins resembles a winged helix, and because of their structure, they are also referred to as winged helix proteins (Clark et al. 1993). Eventually, a unified nomenclature was established, and currently these genes are called Fox genes (Kaestner et al. 2000).