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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.06.017 Byline: Hannah Thompson, David Barker, Olivier Camand, Lynda Erskine Keywords: Axon guidance; Slit; Robo; Optic tract; Retinal ganglion cell; Growth cone Abstract: 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. Author Affiliation: Divisions of Visual Science and Molecular Genetics, Institute of Ophthalmology, University College London, 11-43 Bath Street, London, EC1V 9EL, UK Article History: Received 3 May 2006; Revised 8 June 2006; Accepted 10 June 2006

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.