2. LITERATURE REVIEW
2.4. The role of the transcription factor Foxc1 in eye development
2.4.3. Mutations in Foxc1
Foxc1 is known to play specific key roles in somitic, cardiovascular, renal and ocular development, and also in tumorigenesis (Kume et al., 2000; Wang et al., 2001; Zarbalis et al., 2007). The important role of Foxc1 protein in the development of mesoderm-derived vertebrate
tissues (such as the kidney, eye, heart, bone and cartilage) has been demonstrated by studies targeting deletions of Foxc1 in the murine model (Cha et al., 2007; Tamimi et al., 2006). In mice, the loss of function associated with mutations in the gene encoding Foxc1 results in eye and heart defects, and lymphatic and germ cell migration abnormalities (Libby et al., 2003; Mattiske et al., 2006; Seo et al., 2006), whilst the decreased production of effective, viable Foxc1 proteins in zebrafish actively inhibits somitogenesis (Cha et al., 2007). Loss-of-function analyses show Foxc1 to be an essential transcription factor in Xenopus sp., necessary for the maintenance of cell-to-cell adhesion in the mesodermal germ layer during the early gastrula stage (Cha et al., 2007).
A wide spectrum of human FOXC1 mutations have been discovered to date; deletions, insertions, duplications, translocations, frameshift mutations and missense mutations are amongst the most well-known, with these anomalies being noted to result in haplo-insufficiency of the FOXC1 transcription factor protein (Komatireddy et al., 2003; Fuse et al., 2007; Saleem et al., 2003). It has also been noted that mutated residues within these altered genes remain highly conserved across species (Fuse et al., 2007). Studies by Strungaru et al. (2007) have shown that patients with FOXC1 gene mutations are more likely to manifest with systemic malformations than patients with FOXC1 duplication. Missense mutations have been characterized as being responsible for reducing FOXC1 protein stability by reducing wildtype conformations, limiting the ability of the tertiary structure to bind to DNA by inhibiting transcriptional initiation, or by causing reduced transactivation by FOXC1 even though the protein is able to bind to DNA (Figure 9) (Komatireddy et al., 2003; Saleem et al., 2003; Tamimi et al., 2006). Such studies on Foxc1 missense mutations show the extent to which mutations impair Foxc1 function and shed light onto the functional characteristics of manifesting defects (Saleem et al., 2003).
Figure 9: Effect of missense mutations in cell function. Image adapted from Vieira, 2008.
The abnormal expression of Foxc1 (brought about as a result of chromosomal translocations, deletions, duplications, and nonsense or missense mutations) has been implicated in the abnormal development of key ocular structures such as the iris, cornea and trabecular meshwork (Wang et al., 2001). A wide range of phenotypes of variable severity are caused by mutations in Foxc1;
this may be as a result of mutated proteins retaining partial function, causing milder phenotypes to manifest (Cvekl & Tamm, 2004) or as a result of Foxc2 expression compensating for loss in Foxc1 expression (Smith et al., 2000).
In humans, ASD is an umbrella term that refers to a range of structural deformities in the anterior eye. The current body of knowledge includes data from both human and mouse studies, and clearly defines anterior segment dysgenesis to encompass a complex spectrum of disorders (Gould et al., 2004). Heterozygous (Foxc1+/-) mice present with abnormalities that show analogy to those in patients presenting with ASD; these include (amongst others) iris hypoplasia, aberrantly developed trabecular meshwork, small or absent Schlemm’s canal, severely eccentric pupils, fusion of the lens to the cornea (Figure 10) and displaced Schwalbe’s line (Chakravarti, 2001; Kume et al., 2001; Wang et al., 2001; Saleem et al., 2003). These mice possess Foxc1 proteins with reduced stability, and have Foxc1 activity reduced to a degree that is just sufficient for early developmental events to proceed normally (Zarbalis et al., 2007). Mutant mice showing the absence of the fully functional gene (Foxc1-/-) die both pre- and peri-natally, with embryos manifesting outwardly with lethal, combined skeletal, ocular, and genito-urinary defects, and severe haemorrhagic hydrocephalus (Kume et al., 2001; Saleem et al., 2003; Gould et al., 2004;
Tamimi et al., 2006; Zarbalis et al., 2007).
Figure 10: Histology of heterozygous & homozygous congenital hydrocephalus (Mf1ch) (subsequently renamed Foxc1-/-) mouse eyes at E17. Images are original H&E stained sections of embryos by Dr.
Hans Gruneberg (1943). (A/C) Heterozygote (Foxc1+/-) shows closed eyelids (el) with distinct anterior chamber (*) & endothelial layer (en) along the posterior corneal margin. (B/D) Homozygous mutant (Foxc1-/-) with open eyelids & anterior chamber. Abbreviations: c=cornea; el=eyelid;
en=endothelium; le=lens; r=retina; s=sclera; tm=presumptive trabecular meshwork. Scale: A/B = 200 µm; C/D = 50 µm (Kidson et al., 1999).
Foxc1-/- Foxc1-/+
Whole mount preparations of the corneas of Foxc1-/- mutant eyes mice have a thickened epithelium, disorganized stroma and absent, undifferentiated corneal endothelium. The posterior mesenchyme in the developing cornea of Foxc1-/- null mutant mice fail to make the transition to corneal endothelium tissue. Frequently, the lens appears fused to the cornea (corneolenticular adhesions) resulting in the absence of a distinct anterior chamber (Figure 10) (Chakravarti, 2001;
Cvekl & Tamm, 2004; Saleem et al., 2003; Gould et al., 2004; Sommer et al., 2006; Zarbalis et al., 2007). Such studies implicate Foxc1 as a fundamental part of the signaling cascade responsible for the induction of and proper development of the endothelium and other anterior segment tissues (Chakravarti, 2001; Panicker et al., 2002).