Xenopus pax6 mutants affect eye development and other organ systems, and have phenotypic similarities to human aniridia patients

Abstract

SUMMARY Mutations in the Pax6 gene cause ocular defects in both vertebrate and invertebrate animal species, and the disease aniridia in humans. Despite extensive experimentation on this gene in multiple species, including humans, we still do not understand the earliest effects on development mediated by this gene. This prompted us to develop pax6 mutant lines in Xenopus tropicalis taking advantage of the utility of the Xenopus system for examining early development and in addition to establish a model for studying the human disease aniridia in an accessible lower vertebrate. We have generated mutants in pax6 by using Transcription Activator-Like Effector Nuclease (TALEN) constructs for gene editing in X. tropicalis. Embryos with putative null mutations show severe eye abnormalities and changes in brain development, as assessed by changes in morphology and gene expression. One gene that we found is downregulated very early in development in these pax6 mutants is myc, a gene involved in pluripotency and progenitor cell maintenance and likely a mediator of some key pax6 functions in the embryo. Changes in gene expression in the developing brain and pancreas reflect other important functions of pax6 during development. In mutations with partial loss of pax6 function eye development is initially relatively normal but froglets show an underdeveloped iris, similar to the classic phenotype (aniridia) seen in human patients with PAX6 mutations. Other eye abnormalities observed in these froglets, including cataracts and corneal defects, are also common in human aniridia. The frog model thus allows us to examine the earliest deficits in eye formation as a result of pax6 lesions, and provides a useful model for understanding the developmental basis for the aniridia phenotype seen in humans.

Using Xenopus to understand human disease and developmental disorders

“…Humanized mutations are easily introduced via expression vectors or established in founder lines, and the anatomy, function, and molecular regulation of most organs are highly similar across the tetrapods (amphibian, reptiles, avians and mammals). This approach has been used to investigate: aniridia (Nakayama et al, ); blood–brain barrier dysfunction (De Jesús Andino, Jones, Maggirwar, & Robert, ); congenital heart disease (Bhattacharya, Marfo, Li, Lane, & Khokha, ); demyelination diseases (Sekizar et al, ); holoprosencephaly (Nakayama et al, ); Huntington's disease (Haremaki, Deglincerti, & Brivanlou, ); myasthenia gravis (Yeo, Lim, Fukami, Yuki, & Lee, ); pneumonia (Walentek et al, ); and tumor progression (Hardwick & Philpott, ). We anticipate that similar contributions to human disease will rapidly expand due to the ease of performing these types of experiments in Xenopus .…”

The molecular mechanisms underlying lens fiber elongation

“…However, both chicken and rat lens explants do start the process when treated with factors known to drive lens fiber cell differentiation such as vitreous humor or bFGF (Beebe and Feagans, 1981; Iyengar et al, 2007; Menko and Boettiger, 1988; Musil, 2012; Piatigorsky et al, 1972; Zelenka et al, 2009), and could potentially be refined into quantitative models using modern imaging techniques. Alternatively, in vivo manipulation of early chick (Reza et al, 2007; Shestopalov and Bassnett, 2000) or frog embryos (Nakayama et al, 2015) could provide important insight into the mechanisms driving lens fiber cell elongation.…”

Effects of freezing and activation on membrane quality and DNA damage in Xenopus tropicalis and Xenopus laevis spermatozoa

“…This is increasingly important because a wide range of mutants is now available in this model organism (e.g. Fish et al 2014;Nakayama et al 2015;Shi et al 2015) that provides cost-effective access to a very wide range of experimental approaches while sharing much of the genome structure of humans (Hellsten et al 2010). More recent efforts to optimise cryopreservation of X. laevis spermatozoa based on a matrix devised to evaluate the various stages of the freezing protocol yielded encouraging results (Mansour et al 2009).…”