Modelling human genetic disorders in Xenopus tropicalis.

Recent progress in human disease genetics is leading to rapid advances in understanding pathobiological mechanisms. However, the sheer number of risk-conveying genetic variants being identified demands in vivo model systems that are amenable to functional analyses at scale. Here we provide a practical guide for using the diploid frog species Xenopus tropicalis to study many genes and variants to uncover conserved mechanisms of pathobiology relevant to human disease. We discuss key considerations in modelling human genetic disorders: genetic architecture, conservation, phenotyping strategy and rigour, as well as more complex topics, such as penetrance, expressivity, sex differences and current challenges in the field. As the patient-driven gene discovery field expands significantly, the cost-effective, rapid and higher throughput nature of Xenopus make it an essential member of the model organism armamentarium for understanding gene function in development and in relation to disease.

Report on the 2021 Aniridia North America symposium on PAX6, aniridia, and beyond.

The inaugural Aniridia North America (ANA) Symposium was held on the first weekend in November 2021 in Charlottesville, VA, at the University of Virginia. The purpose of this meeting was to bring together an international group of scientists, physicians, patient advocacy groups, and individuals with aniridia to discuss recent advances in knowledge about aniridia and other congenital eye diseases and the development of potential treatments for congenital eye disorders using personalized medicine. Leaders in several areas of eye research and clinical treatment provided a broad perspective on new research advances that impact an understanding of the causes of the damage to the eye associated with aniridia and the development of novel treatments for this and related disorders. Here we summarize the research discussed at the symposium.

Production of Transgenic F0 Animals and Permanent Lines by Sperm Nuclear Transplantation in Xenopus tropicalis.

Early efforts in the 1980s showed that DNA microinjected into Xenopus embryos could be integrated into the genome and transmitted through the germline at low efficiency. Subsequent studies revealed that transgenic lines, typically with multiple-copy inserts (e.g., to develop bright fluorescent protein-reporter lines), could be created via sperm nuclear injection protocols such as the one entitled restriction enzyme-mediated insertion, or REMI. Here we describe a refined sperm nuclear injection procedure, with a number of alterations, including elimination of a potential DNA-damaging restriction enzyme treatment, aimed at making F0 transgenic animals and transgenic lines in Xenopus tropicalis This protocol also uses an oocyte extract rather than the egg extract used in older protocols. These changes simplify and improve the efficiency of the procedure.

Best Practices for Xenopus tropicalis Husbandry.

Xenopus tropicalis has been adopted by laboratories as a developmental genetic system because of its diploid genome and short generation time, contrasting with Xenopus laevis, which is allotetraploid and takes longer to reach sexual maturity. Because X. tropicalis has been introduced more recently to many laboratories, some specific methods more appropriate for handling of eggs and embryos of X. tropicalis are still not widely known to researchers who use X. laevis Here we highlight some recommendations and opportunities possible with this model system that complement existing X. tropicalis procedures. Of particular importance, because of the value of generating genetically modified lines for researchers using X. tropicalis, we describe a procedure for sterilizing embryos, which could be applied to both species of Xenopus, but might be particularly useful for raising genetically modified animals in X. tropicalis This protocol will help ensure that a colony will have a high probability of being free of pathogens known to be serious threats to Xenopus health.

Homology-Directed Repair by CRISPR-Cas9 Mutagenesis in Xenopus Using Long Single-Stranded Donor DNA Templates via Simple Microinjection of Embryos.

We describe a step-by-step procedure to perform homology-directed repair (HDR)-mediated precise gene editing in Xenopus embryos using long single-stranded DNA (lssDNA) as a donor template for HDR in conjunction with the CRISPR-Cas9 system. A key advantage of this method is that it relies on simple microinjection of fertilized Xenopus eggs, resulting in high yield of healthy founder embryos. These embryos are screened for those animals carrying the precisely mutated locus to then generate homozygous and/or heterozygous mutant lines in the F1 generation. Therefore, we can avoid the more challenging "oocyte host transfer" technique, which is particularly difficult for Xenopus tropicalis, that is required for an alternate HDR approach. Several key points of this protocol are (1) to use efficiently active single-guide RNAs for targeting, (2) to use properly designed lssDNAs, and (3) to use 5'-end phosphorothioate-modification to obtain higher-efficiency HDR.

Gynogenetic Production of Embryos in Xenopus tropicalis Using a Cold Shock Procedure: Rapid Screening Method for Gene Editing Phenotypes.

Gynogenesis is a form of parthenogenesis in which eggs require sperm for fertilization but develop to adulthood without the contribution of paternal genome information, which happens naturally in some species. In Xenopus, gynogenetic diploid animals can be made experimentally. In mutagenesis strategies that only generate one allele of a recessive mutation, as might occur during gene editing, gynogenesis can be used to quickly reveal a recessive phenotype in eggs carrying a recessive mutation, thereby skipping one generation normally required to screen by conventional genetics. Xenopus oocytes do not complete meiosis until shortly after fertilization, and the second polar body is retained in fertilized eggs. Using ultraviolet (UV)-irradiated sperm, fertilization can be triggered without a genetic paternal contribution. Upon applying cold shock at the proper time to such embryos, ejection of the second polar body can be suppressed and both maternal sister chromatids are retained, leading to the development of gynogenetic diploid embryos. Because the genome of the resultant animals consists of recombined sister chromatids because of crossover events during meiosis, it is not completely homozygous throughout the whole genome. Nevertheless, the genome is homozygous at some loci proximal to the centromere that are unlikely to undergo recombination during meiosis and homozygous at reduced frequency if mutations are farther from the centromere, but still generally at a scorable level. Therefore, this technique is useful for rapid screening phenotypes of recessive mutations in such regions. We describe here a step-by-step protocol to achieve cold shock-mediated gynogenesis in Xenopus tropicalis.

Preparation of Intact Nuclei for Single-Nucleus Omics Using Frozen Cell Suspensions from Mutant Embryos of Xenopus tropicalis.

Single-cell omics such as single-cell RNA-sequencing (RNA-seq) have been used extensively to obtain single-cell genome-wide expression data. This technique can be used to compare mutant and wild-type embryos at predifferentiation stages when individual tissues are not yet formed (therefore requiring genotyping to distinguish among embryos), for example, to determine effects of mutations on developmental trajectories or congenital disease phenotypes. It is, however, hard to use single cells for this technique, because such embryos or cells would need to be frozen until genotyping is complete to capture a given developmental stage precisely, but intact cells cannot be isolated from frozen samples. We developed a protocol in which high-quality nuclei are isolated from frozen cell suspensions, allowing for genotyping individual embryos based on a small fraction of a single embryo suspension. The remaining suspension is frozen. After genotyping is complete, nuclei are isolated from embryo suspensions with the desired genotype and encapsulated in 10× Genomics barcoded gel beads for single-nucleus RNA-seq. We provide a step-by-step protocol that can be used for single transcriptomic analysis as well as single-nucleus chromatin accessibility assays such as ATAC-seq. This technique allows for high-quality high-throughput single-nucleus analysis of gene expression in genotyped embryos. This approach may also be valuable for collection of wild-type embryonic material, for example, when collecting tissue from a particular developmental stage. In addition, freezing of tissue suspensions allows precise staging of collected embryos or tissue that may be difficult to manage when collecting and processing cells from living embryos for single-cell RNA-seq.

Modeling Human Genetic Disorders with CRISPR Technologies in Xenopus.

Combining the power of Xenopus developmental biology with CRISPR-based technologies promises great discoveries in understanding and treating human genetic disorders. Here we provide a practical pipeline for how to go from known disease gene(s) or risk gene(s) of interest to methods for gaining functional insight into the contribution of these genes to disorder etiology in humans.

Elucidating the framework for specification and determination of the embryonic retina.

How cell determination is regulated remains a major unsolved problem in developmental biology. The early embryonic rudiments of many tissues and organs are difficult or impossible to identify, isolate and study at the time when determination occurs. We have examined the commitment process leading to retina formation in Xenopus laevis, where presumptive eye tissue can be identified and studied to assay its biological properties during the events leading up to determination. We find that for the retina, specification, the point at which a tissue placed in neutral culture medium can first properly differentiate, occurs during mid-gastrulation. By late gastrulation, determination, the final, irreversible step in commitment, has occurred. At this stage, the presumptive retina will differentiate and cannot be reprogrammed even if exposed to other active inducers, e.g. when challenged by transplantation to ectopic sites in the embryo. Key eye regulatory genes are initially expressed in the retinal field during specification and/or determination (e.g. rax, pax6, lhx2, and fzd5) potentially linking them, or genes that regulate them, to these processes. This study provides essential groundwork for defining the mechanisms for how these important developmental transitions occur.

Simple embryo injection of long single-stranded donor templates with the CRISPR/Cas9 system leads to homology-directed repair in Xenopus tropicalis and Xenopus laevis.

We report model experiments in which simple microinjection of fertilized eggs has been used to effectively perform homology-directed repair (HDR)-mediated gene editing in the two Xenopus species used most frequently for research: X. tropicalis and X. laevis. We have used long single-stranded DNAs having phosphorothioate modifications as donor templates for HDR at targeted genomic sites using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. First, X. tropicalis tyr mutant (i.e., albino) embryos were successfully rescued: partially pigmented tadpoles were seen in up to 35% of injected embryos, demonstrating the potential for efficient insertion of targeted point mutations. Second, in order to demonstrate the ability to tag genes with fluorescent proteins (FPs), we targeted the melanocyte-specific gene slc45a2.L of X. laevis to label it with the Superfolder green FP (sfGFP), seeing mosaic expression of sfGFP in melanophores in up to 20% of injected tadpoles. Tadpoles generated by these two approaches were raised to sexual maturity, and shown to successfully transmit HDR constructs through the germline with precise targeting and seamless recombination. F1 embryos showed rescue of the tyr mutation (X. tropicalis) and tagging in the appropriate pigment cell-specific manner of slc45a2.L with sfGFP (X. laevis).

Anterior chamber angle in aniridia with and without glaucoma.

PURPOSE: We performed a retrospective, comparative study to determine if patients with aniridia and glaucoma had open angles on high-resolution anterior segment optical coherence tomography (OCT) and clinical gonioscopy. PATIENTS AND METHODS: Forty-three patients (86 eyes) with aniridia had recorded anterior segment OCTs, gonioscopy, or both. Of these patients, 27 (54 eyes) were diagnosed with glaucoma and 16 (32 eyes) had no evidence of glaucoma. All patients had either anterior segment OCT, gonioscopy, or both. RESULTS: The 43 patients with aniridia had average age of 32±17 years, and 27 (62%) were female. Anterior segment OCT and gonioscopy were recorded in 25 (58%) of the patients and 18 (42%) of the patients had gonioscopy alone. Of the 54 eyes with aniridia and glaucoma, 4 (7%) eyes in 3 patients (11%) had partial or completely closed angles. Of the 32 eyes without glaucoma, all (100%) had open angles. The proportion of open angles in the aniridia with glaucoma eyes was not significantly different compared with the aniridia without glaucoma eyes (P=0.32). Of the 4 eyes with closed angles, all had a history of prior surgery for cataract, glaucoma, and/or keratopathy. The proportion of eyes with prior surgery was significantly higher in eyes with open-angle glaucoma and angle-closure glaucoma compared with eyes without glaucoma (P<0.001 and P=0.002, respectively). CONCLUSION: The majority of eyes with aniridia and glaucoma have open anterior chamber angles, similar to patients with aniridia without glaucoma. All eyes with aniridia and glaucoma that had closed angles had a prior history of ocular surgery.

Special Considerations for Making Explants and Transplants with Xenopus tropicalis.

Although Xenopus laevis is an important model organism for embryological experimentation, the smaller, more genetically tractable, and faster developing Xenopus tropicalis provides advantages for using genetic approaches to understand developmental mechanisms. Explant cultures and transplants of X. tropicalis embryonic tissues present unique opportunities to examine embryonic tissue determination in a simplified setting. Here we demonstrate preparation of explants and transplants of preplacodal head ectoderm in order to illustrate these approaches; however, these methods apply broadly to tissues throughout the embryo. We focus on technical adjustments to accommodate the differences in size, tissue character, and rate of development between X. laevis and X. tropicalis With only modest modifications, X. tropicalis embryos are quite amenable to the same kinds of experimental manipulations as X. laevis.

no privacy, a Xenopus tropicalis mutant, is a model of human Hermansky-Pudlak Syndrome and allows visualization of internal organogenesis during tadpole development.

We describe a novel recessive and nonlethal pigmentation mutant in Xenopus tropicalis. The mutant phenotype can be initially observed in tadpoles after stage 39/40, when mutant embryos display markedly reduced pigmentation in the retina and the trunk. By tadpole stage 50 almost all pigmented melanophores have disappeared. Most interestingly, those embryos fail entirely to make pigmented iridophores. The combined reduction/absence of both pigmented iridophores and melanophores renders these embryos virtually transparent, permitting one to easily observe both the developing internal organs and nervous system; accordingly, we named this mutant no privacy (nop). We identified the causative genetic lesion as occurring in the Xenopus homolog of the human Hermansky-Pudlak Syndrome 6 (HPS6) gene, combining several approaches that utilized conventional gene mapping and classical and modern genetic tools available in Xenopus (gynogenesis, BAC transgenesis and TALEN-mediated mutagenesis). The nop allele contains a 10-base deletion that results in truncation of the Hps6 protein. In humans, HPS6 is one of the genes responsible for the congenital disease HPS, pathological symptoms of which include oculocutaneous albinism caused by defects in lysosome-related organelles required for pigment formation. Markers for melanin-producing neural crest cells show that the cells that would give rise to melanocytes are present in nop, though unpigmented. Abnormalities develop at tadpole stages in the pigmented retina when overall pigmentation becomes reduced and large multi-melanosomes are first formed. Ear development is also affected in nop embryos when both zygotic and maternal hsp6 is mutated: otoliths are often reduced or abnormal in morphology, as seen in some mouse HPS mutations, but to our knowledge not described in the BLOC-2 subset of HPS mutations nor described in non-mammalian systems previously. The transparency of the nop line suggests that these animals will aid studies of early organogenesis during tadpole stages. In addition, because of advantages of the Xenopus system for assessing gene expression, cell biological mechanisms, and the ontogeny of melanosome and otolith formation, this should be a highly useful model for studying the molecular mechanisms underlying the acquisition of the HPS phenotype and the underlying biology of lysosome-related organelle function.

Functional Cloning Using a Xenopus Oocyte Expression System.

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in abundance, may not be secreted but tethered to the signaling cell(s), or may require the presence of binding partners or upstream regulatory molecules. Thus in a search for gene products capable of eliciting an early lens-inductive response in competent ectoderm, we utilized an expression cloning system that would allow identification of paracrine or juxtacrine factors as well as transcriptional or other regulatory proteins. Pools of mRNA were injected into Xenopus oocytes, and responding tissue placed directly on the oocytes and co-cultured. Following functional cloning of ldb1 from a neural plate stage cDNA library based on its ability to elicit the expression of the early lens placode marker foxe3 in lens-competent animal cap ectoderm, we characterized the mRNA expression pattern, and assayed developmental progression following overexpression or knockdown of ldb1. This system is suitable in a very wide variety of contexts where identification of an inducer or its upstream regulatory molecules is sought using a functional response in competent tissue.

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

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.

Cas9-based genome editing in Xenopus tropicalis.

Xenopus tropicalis has been developed as a model organism for developmental biology, providing a system offering both modern genetics and classical embryology. Recently, the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated (CRISPR/Cas) system for genome modification has provided an additional tool for Xenopus researchers to achieve simple and efficient targeted mutagenesis. Here, we provide insights into experimental design and procedures permitting successful application of this technique to Xenopus researchers, and offer a general strategy for performing loss-of-function assays in F0 and subsequently F1 embryos.

Early stages of induction of anterior head ectodermal properties in Xenopus embryos are mediated by transcriptional cofactor ldb1.

BACKGROUND: Specific molecules involved in early inductive signaling from anterior neural tissue to the placodal ectoderm to establish a lens-forming bias, as well as their regulatory factors, remain largely unknown. In this study, we sought to identify and characterize these molecules. RESULTS: Using an expression cloning strategy to isolate genes with lens-inducing activity, we identified the transcriptional cofactor ldb1. This, together with evidence for its nuclear dependence, suggests its role as a regulatory factor, not a direct signaling molecule. We propose that ldb1 mediates induction of early lens genes in our functional assay by transcriptional activation of lens-inducing signals. Gain-of-function assays demonstrate that the inductive activity of the anterior neural plate on head ectodermal structures can be augmented by ldb1. Loss-of-function assays show that knockdown of ldb1 leads to decreased expression of early lens and retinal markers and subsequently to defects in eye development. CONCLUSIONS: The functional cloning, expression pattern, overexpression, and knockdown data show that an ldb1-regulated mechanism acts as an early signal for Xenopus lens induction.

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

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.

Simple and efficient CRISPR/Cas9-mediated targeted mutagenesis in Xenopus tropicalis.

We have assessed the efficacy of the recently developed CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system for genome modification in the amphibian Xenopus tropicalis. As a model experiment, targeted mutations of the tyrosinase gene were verified, showing the expected albinism phenotype in injected embryos. We further tested this technology by interrupting the six3 gene, which is required for proper eye and brain formation. Expected eye and brain phenotypes were observed when inducing mutations in the six3 coding regions, as well as when deleting the gene promoter by dual targeting. We describe here a standardized protocol for genome editing using this system. This simple and fast method to edit the genome provides a powerful new reverse genetics tool for Xenopus researchers.