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).