Research

Investigating the molecular basis of visual system development

The study of eye development is an excellent model system both to elucidate the development of various tissues and to unravel the molecular and cellular basis of the coordinate growth of the different cell types. In the course of the last century, eye development has been studied in amphibians, avian embryos, fish and mammals mainly by employing classical embryological tools. Only recently, analyses of the genes responsible for eye mutations and cloning of genes demarcating domains within the developing eye have been instrumental in elucidating the molecular basis of tissue development and differentiation (Ashery-Padan and Gruss, 2001). Understanding the normal developmental regulation of the different eye structures is essential for understanding visual disorders and for designing treatments to ocular phenotypes including retinal degeneration, glaucoma and cataract, all of which are leading causes of blindness.

In our laboratory we focus on the function of the transcription factor Pax6 (Walther and Gruss, 1991) in the process of retina and lens development and in the adult eye. Pax6 is a transcription factor known as “master regulator” of eye development as this gene has an astonishing capacity to induce formation of ectopic eyes in flies and frogs upon miss-expression (Chow et al., 1999; Halder et al., 1995). Pax6 is invariably essential for formation of eyes in different organisms. In addition, the normal dosage of the protein is required for normal eye formation in vertebrates; carriers of Pax6 mutations suffer from severe eye phenotypes known as aniridia in humans and small eye in mice and rats (Hill et al., 1991; Hogan et al., 1986).

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Despite the obvious importance of this gene for eye development the study of Pax6 function in the eye has been until recently mostly precluded due to the complete absence of all eye structures in the homozygous for Pax6 mutation (Grindley et al., 1995). In order to circumvent this limitation we have employed the Cre/loxP recombination approach to mutate the gene in specific cell-types and define time points. We established the Pax6 floxed allele (Pax6flox) and two transgenic mouse lines in which the Cre expression is derived in part of Pax6 expression domain; in the Le-Cre line the Cre is expresses in the developing lens and pancreas, while the a-Cre line promotes recombination only in the distal neuroretina (Ashery-Padan et al., 2000; Marquardt et al., 2001).

Study of the eye phenotype in the Pax6flox/ Pax6flox/;Le-Cre have already revealed to us that Pax6 has a cell autonomous and essential function in the developing lens. The study further exposed the role of the lens in retinal morphology; the lens has been demonstrated to be essential for the correct number of cells and the positioning of the retina in the eye while the lens  seem not to be essential for cell differentiation in the retina (Ashery-Padan et al., 2000). To investigate the role of Pax6 in the developing retina we studied the eye phenotype in Pax6flox/Pax6flox /a-cre embryos. In this mutant Pax6 was eliminated in the retinal progenitor cells (RPCs) just prior to onset of cell differentiation (embryonic day 10.5: E10.5). The analysis revealed for the first time the role of Pax6 in RPCs: Pax6 is now recognized as being essential for normal proliferation of the RPCs and for maintaining the multipotency of RPCs (Marquardt et al., 2001). This study showed that the latter function is mediated by the requirement for Pax6 for the expression of bHLH transcription factors including; Math5, Mesh1 and Ngn2 (Marquardt et al., 2001).

In our current research we further study the in vivo cellular and molecular functions of the transcription factor Pax6. Furthermore we address the functions of the Notch signaling pathway in the developing eye. We focus on the following topics;

  1. Genetic dissection of the small eye and aniridia phenotypes in mice and humans.
  2. Elucidate the role of Pax6 in different cell types in the developing and in the adult eye.
  3. Identify the genes, which are differentially missexpressed in Pax6 deficient RPCs by using Gene Chip microarrays and PCR based subtraction approaches.
  4. Study the in vivo functions of Notch1 receptor in the developing eye in mammals.

Ashery-Padan, R. and Gruss, P. (2001). Pax6 lights-up the way for eye development. Curr Opin Cell Biol 13, 706-14.

Ashery-Padan, R., Marquardt, T., Zhou, X. and Gruss, P.(2000). Pax6 activity in the lens primordium is required for lens formation and for correct placement of a single retina in the eye.
Genes Dev
14, 2701-11.

Chow, R. L., Altmann, C. R., Lang, R. A. and Hemmati-Brivanlou, A.(1999). Pax6 induces ectopic eyes in a vertebrate.
Development
126, 4213-22.

Grindley, J. C., Davidson, D. R. and Hill, R. E. (1995). The role of Pax-6 in eye and nasal development.
Development
121, 1433-42.

Halder, G., Callaerts, P. and Gehring, W. J.(1995). Induction of ectopic eyes by targeted expression of the eyeless gene in Drosophila.
Science
267, 1788-92.

Hill, R. E., Favor, J., Hogan, B. L., Ton, C. C., Saunders, G. F., Hanson, I. M., Prosser, J., Jordan, T., Hastie, N. D. and van Heyningen, V.(1991). Mouse small eye results from mutations in a paired-like homeobox- containing gene.
Nature 354, 522-5.

Hogan, B. L., Horsburgh, G., Cohen, J., Hetherington, C. M., Fisher, G. and Lyon, M. F.(1986). Small eyes (Sey): a homozygous lethal mutation on chromosome 2 which affects the differentiation of both lens and nasal placodes in the mouse.
J Embryol Exp Morphol
97, 95-110.

Marquardt, T., Ashery-Padan, R., Andrejewski, N., Scardigli, R., Guillemot, F. and Gruss, P. (2001). Pax6 is required for the multipotent state of retinal progenitor cells.
Cell
105, 43-55.

Walther, C. and Gruss, P.(1991). Pax-6, a murine paired box gene, is expressed in the developing CNS.
Development 113, 1435-49.