Identification of four new PITX2 gene mutations in patients with Axenfeld-Rieger syndrome.

PURPOSE: Axenfeld Rieger syndrome (ARS) is an autosomal dominant inherited disorder affecting development of the ocular anterior chamber, abdomen, teeth and facial structures. The PITX2 gene is a major gene encoding a major transcription factor associated with ARS. METHODS: ARS patients were collected from six unrelated families. Patients and their families were ophthalmologically phenotyped and their blood was collected for DNA extraction. We screened the coding region of human PITX2 gene by direct sequencing. The consequences of the mutations described were investigated by generating crystallographic representations of the amino acid changes. In order to better understand the occurrence of glaucoma in ARS patients, we studied the PITX2 gene expression in human embryonic and fetal ocular tissue sections. RESULTS: We identified four novel PITX2 genetic alterations in four unrelated families with ARS. These mutations included two nonsense mutations (E55X and Y121X), an eight nucleotides insertion (1251 ins CGACTCCT) and a substitution (F58L), in familial and sporadic cases of ARS. We also showed for the first time that PITX2 is expressed at early stages of the human embryonic and fetal periocular mesenchyme, as well as at later stages of human development in the fetal ciliary body, ciliary processes, irido corneal angle and corneal endothelium. The human fetal eye PITX2 gene expression pattern reported here for the first time provides a strong basis for explaining the frequent occurrence of glaucoma in patients affected by PITX2 gene mutations. CONCLUSIONS: Two mutations identified affect the homeodomain (E55X and F58L). The E55X nonsense mutation is likely to alter dramatically the DNA-binding capabilities of the PITX2 homeodomain. Furthermore, there is a complete loss of the carboxy-terminal part of the PITX2 protein beyond the site of the mutation. The phenylalanine F58 is known to contribute to the hydrophobic network of the homeodomain. The crystallographic representations of the mutation F58L show that this mutation may change the conformation of the helical core. The F58L mutation is very likely to modify the homeodomain conformation and probably alters the DNA binding properties of PITX2. The other mutations (Y121X and the eight-nucleotide insertion (1251 ins CGA CTC CT) CGA CTC CT, at position 224 in PITX2A) result in partial loss of the C-terminal domain of PITX2. Pitx2 synergistically transactivates the prolactin promoter in the presence of the POU homeodomain protein Pit-1. Pitx2 activity is regulated by its own C-terminal tail. This region contains a highly conserved 14-amino-acid element involved in protein-protein interactions. The C-terminal 39-amino-acid tail represses DNA binding activity and is required for Pitx2 interactions with other transcription factors, for Pitx2-Pit-1 interaction and Pit-1synergism. Pit-1 interaction with the Pitx2 C terminus masks the inhibitory effect and promotes increased DNA binding activity. Thus, the partial or complete loss of the C terminus tail can lead to decreased or absent DNA binding activity and trigger severe ARS phenotypes. Our in situ hybridization results obtained on human embryonic and fetal ocular tissue sections constitute the first molecular histological data providing an explanation for the occurrence of precocious glaucoma in human patients affected by ARS caused by PITX2 mutations. Further structural and biochemical studies are needed for understanding the wide spectrum of clinical phenotypes caused by the increasing number of new PITX2 mutations found in ARS affected patients.

Slit proteins regulate distinct aspects of retinal ganglion cell axon guidance within dorsal and ventral retina.

An early step in the formation of the optic pathway is the directed extension of retinal ganglion cell (RGC) axons into the optic fiber layer (OFL) of the retina in which they project toward the optic disc. Using analysis of knock-out mice and in vitro assays, we found that, in the mammalian retina, Slit1 and Slit2, known chemorepellents for RGC axons, regulate distinct aspects of intraretinal pathfinding in different regions of the retina. In ventral and, to a much lesser extent, dorsal retina, Slits help restrict RGC axons to the OFL. Additionally, within dorsal retina exclusively, Slit2 also regulates the initial polarity of outgrowth from recently differentiated RGCs located in the retinal periphery. This regional specificity occurs despite the fact that Slits are expressed throughout the retina, and both dorsal and ventral RGCs are responsive to Slits. The gross morphology and layering of the retina of the slit-deficient retinas is normal, demonstrating that these distinct guidance defects are not the result of changes in the organization of the tissue. Although displaced or disorganized, the aberrant axons within both dorsal and ventral retina exit the eye. We also have found that the lens, which because of its peripheral location within the developing eye is ideally located to influence the initial direction of RGC axon outgrowth, secretes Slit2, suggesting this is the source of Slit regulating OFL development. These data demonstrate clearly that multiple mechanisms exist in the retina for axon guidance of which Slits are an important component.

Slits contribute to the guidance of retinal ganglion cell axons in the mammalian optic tract.

RGC axons extend in the optic tracts in a manner that correlates with the expression in the hypothalamus and epithalamus of a soluble factor inhibitory to RGC axon outgrowth. Additionally, although the RGC axons extend adjacent to the telencephalon, they do not normally grow into this tissue. Here, we show that slit1 and slit2, known chemorepellents for RGC axons expressed in specific regions of the diencephalon and telencephalon, help regulate optic tract development. In mice lacking slit1 and slit2, a subset of RGC axons extend into the telencephalon and grow along the pial surface but not more deeply into this tissue. Surprisingly, distinct guidance errors occur in the telencephalon of slit1 -/-; slit2 +/- and slit1/2 -/- embryos, suggesting that the precise level of Slits is critical for determining the path followed by individual axons. In mice lacking both slit1 and slit2, a subset of RGC axons also project aberrantly into the epithalamus, pineal and across the dorsal midline. However, many axons reach their primary target, the superior colliculus. This demonstrates that Slits play an important role in directing the guidance of post-crossing RGC axons within the optic tracts but are not required for target innervation.

Pleiotropic and diverse expression of ZFHX1B gene transcripts during mouse and human development supports the various clinical manifestations of the "Mowat-Wilson" syndrome.

ZFHX1B encodes Smad-interacting protein 1, a transcriptional corepressor involved in the transforming growth factors beta (TGFbeta) signaling pathway. ZFHX1B mutations cause a complex developmental phenotype characterized by severe mental retardation (MR) and multiple congenital defects. We compared the distribution of ZFHX1B transcripts during mouse and human embryogenesis as well as in adult mice and humans. This showed that this gene is strongly transcribed at an early stage in the developing peripheral and central nervous systems of both mice and humans, in all neuronal regions of the brains of 25-week human fetuses and adult mice, and at varying levels in numerous nonneural tissues. Northern blot analysis suggested that ZFHX1B undergoes tissue-specific alternative splicing in both species. These results strongly suggest that ZFHX1B determines the transcriptional levels of target genes in various tissues through the combinatorial interactions of its isoforms with different Smad proteins. Thus, as well as causing neural defects, ZFHX1B mutations may also cause other malformations.

Mutational analysis of the OA1 gene in ocular albinism.

Ocular albinism type 1 (OA1) is an X-linked disorder, mainly characterized by a severe reduction in visual acuity, foveal hypoplasia, nystagmus, hypopigmentation of the retina, the presence of macromelanosomes in the skin and eyes, and the misrouting of optic pathways, resulting in the loss of stereoscopic vision. We screened the OA1 gene for mutations in three unrelated Canadian and French families and in two isolated patients with OA1. We found three different missense mutations and two different nonsense mutations, three of which were novel. To date, 41 mutations (including missense mutations, insertions, and deletions) have been reported in the OA1 gene. Mutation and polymorphism data for this gene are available from the international albinism center albinism database website: http://www.cbc.umn.edu/tad/oa1map.htm.

Embryonic expression of the leptin receptor gene in mesoderm-derived tissues.

Leptin acts on the hypothalamus to reduce food intake and on a number of non-neuronal tissues via specific receptors (Lepr). The use of in situ hybridisation to map the Lepr gene in pre-natal mice revealed transcripts in the yolk sac in various structures of the central nervous system and in mesoderm-derived tissues, such as cartilage/bone primordia and musculoaponeurotic laminae. At later stages, significant amounts of Lepr were expressed in the region surrounding the developing eye of the embryo. Lepr was also found to be expressed in the choroid, sclera and connective tissues of the limbus in the adult eye. In conclusion, we have identified new targets for leptin action during embryogenesis and adulthood.