Xenopus mutant reveals necessity of rax for specifying the eye field which otherwise forms tissue with telencephalic and diencephalic character

Abstract

SUMMARY The retinal anterior homeobox (rax) gene encodes a transcription factor necessary for vertebrate eye development. rax transcription is initiated at the end of gastrulation in Xenopus, and is a key part of the regulatory network specifying anterior neural plate and retina. We describe here a Xenopus tropicalis rax mutant, the first mutant analyzed in detail from a reverse genetic screen. As in other vertebrates, this nonsense mutation results in eyeless animals, and is lethal peri-metamorphosis. Tissue normally fated to form retina in these mutants instead forms tissue with characteristics of diencephalon and telencephalon. This implies that a key role of rax, in addition to defining the eye field, is in preventing alternative forebrain identities. Our data highlight that brain and retina regions are not determined by the mid-gastrula stage but are by the neural plate stage. An RNA-Seq analysis and in situ hybridization assays for early gene expression in the mutant revealed that several key eye field transcription factors (e.g. pax6, lhx2 and six6) are not dependent on rax activity through neurulation. However, these analyses identified other genes either up- or down-regulated in mutant presumptive retinal tissue. Two neural patterning genes of particular interest that appear up-regulated in the rax mutant RNA-seq analysis are hesx1 and fezf2. These genes were not previously known to be regulated by rax. The normal function of rax is to partially repress their expression by an indirect mechanism in the presumptive retina region in wildtype embryos, thus accounting for the apparent up-regulation in the rax mutant. Knock-down experiments using antisense morpholino oligonucleotides directed against hesx1 and fezf2 show that failure to repress these two genes contributes to transformation of presumptive retinal tissue into non-retinal forebrain identities in the rax mutant.

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

“…unless otherwise described. Genotyping of rax mutant and wild-type embryos was performed as described (Fish et al, 2014). …”

“…Whole-mount in situ hybridization was carried out essentially according to the procedure in (Sive et al, 2000), though modified when genotyping was required, omitting acetic anhydride and post-color fixation steps as described (Fish et al, 2014). A minimum of 3 (up to 5) mutant embryos were examined by in situ hybridization for alteration of expression of downstream genes, showing highly consistent results.…”

“…All antisense probes were labeled with digoxigenin (Roche) and detected by BCIP/NBT (Roche) or BM Purple (Roche). Probes for myc and pax6 were prepared as described previously (Fish et al, 2014). mab21l1 cDNA in pCS107 vector was obtained from The Wellcome Trust Sanger Institute and digested with Eco RI and used as template for T7 in vitro transcription to make the probe.…”

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).…”

Cas9-Based Genome Editing in Xenopus tropicalis

“…However, the number of characterized mutants to date is small. Mutational screens, including directed approaches such as Targeting Induced Local Lesions In Genomes (e.g., Fish et al, 2014) are laborious, and new approaches to efficiently edit the genome are urgently needed. Recent technological advances have allowed researchers to readily perform targeted gene editing in many organisms.…”