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In animal species that have the capability of regenerating tissues and limbs, cell proliferation is enhanced after wound healing and is essential for the reconstruction of injured tissue. Although the ability to induce cell proliferation is a common feature of such species, the molecular mechanisms that regulate the transition from wound healing to regenerative cell proliferation remain unclear. Here, we show that upon injury, InhibinβA and JunB cooperatively function for this transition during Xenopus tadpole tail regeneration. We found that the expression of inhibin subunit beta A (inhba) and junB proto-oncogene (junb) is induced by injury-activated TGF-β/Smad and MEK/ERK signaling in regenerating tails. Similarly to junb knockout (KO) tadpoles, inhba KO tadpoles show a delay in tail regeneration, and inhba/junb double KO (DKO) tadpoles exhibit severe impairment of tail regeneration compared with either inhba KO or junb KO tadpoles. Importantly, this impairment is associated with a significant reduction of cell proliferation in regenerating tissue. Moreover, JunB regulates tail regeneration via FGF signaling, while InhibinβA likely acts through different mechanisms. These results demonstrate that the cooperation of injury-induced InhibinβA and JunB is critical for regenerative cell proliferation, which is necessary for re-outgrowth of regenerating Xenopus tadpole tails., (© 2024. The Author(s).)

Genetic triggers for sex determination are frequently co-inherited with other linked genes that may also influence one or more sex-specific phenotypes. To better understand how sex-limited regions evolve and function, we studied a small W chromosome-specific region of the frog Xenopus laevis that contains only three genes (dm-w, scan-w, ccdc69-w) and that drives female differentiation. Using gene editing, we found that the sex-determining function of this region requires dm-w but that scan-w and ccdc69-w are not essential for viability, female development, or fertility. Analysis of mesonephros+gonad transcriptomes during sexual differentiation illustrates masculinization of the dm-w knockout transcriptome, and identifies mostly non-overlapping sets of differentially expressed genes in separate knockout lines for each of these three W-specific gene compared to wildtype sisters. Capture sequencing of almost all Xenopus species and PCR surveys indicate that the female-determining function of dm-w is present in only a subset of species that carry this gene. These findings map out a dynamic evolutionary history of a newly evolved W chromosome-specific genomic region, whose components have distinctive functions that frequently degraded during Xenopus diversification, and evidence the evolutionary consequences of recombination suppression., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Cauret et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The first steps of vision take place within a stack of tightly packed disc-shaped membranes, or 'discs', located in the outer segment compartment of photoreceptor cells. In rod photoreceptors, discs are enclosed inside the outer segment and contain deep indentations in their rims called 'incisures'. The presence of incisures has been documented in a variety of species, yet their role remains elusive. In this study, we combined traditional electron microscopy with three-dimensional electron tomography to demonstrate that incisures are formed only after discs become completely enclosed. We also observed that, at the earliest stage of their formation, discs are not round as typically depicted but rather are highly irregular in shape and resemble expanding lamellipodia. Using genetically manipulated mice and frogs and measuring outer segment protein abundances by quantitative mass spectrometry, we further found that incisure size is determined by the molar ratio between peripherin-2, a disc rim protein critical for the process of disc enclosure, and rhodopsin, the major structural component of disc membranes. While a high perpherin-2 to rhodopsin ratio causes an increase in incisure size and structural complexity, a low ratio precludes incisure formation. Based on these data, we propose a model whereby normal rods express a modest excess of peripherin-2 over the amount required for complete disc enclosure in order to ensure that this important step of disc formation is accomplished. Once the disc is enclosed, the excess peripherin-2 incorporates into the rim to form an incisure., Competing Interests: TL, SP, CC, KK, KC, WT, YH, NS, MH, ME, VA No competing interests declared, (© 2023, Lewis et al.)

Precisely regulated thyroid hormone (TH) signaling within tissues during frog metamorphosis gives rise to the organism-wide coordination of developmental events among organs required for survival. This TH signaling is controlled by multiple cellular mechanisms, including TH transport across the plasma membrane. A highly specific TH transporter has been identified, namely monocarboxylate transporter 8 (MCT8), which facilitates uptake and efflux of TH and is differentially and dynamically expressed among tissues during metamorphosis. We hypothesized that loss of MCT8 would alter tissue sensitivity to TH and affect the timing of tissue transformation. To address this, we used CRISPR/Cas9 to introduce frameshift mutations inslc16a2, the gene encoding MCT8, inXenopus laevis. We produced homozygous mutant tadpoles with a 29-bp mutation in the l-chromosome and a 20-bp mutation in the S-chromosome. We found that MCT8 mutants survive metamorphosis with normal growth and development of external morphology throughout the larval period. Consistent with this result, the expression of the pituitary hormone regulating TH plasma levels (tshb) was similar among genotypes as was TH response gene expression in brain at metamorphic climax. Further, delayed initiation of limb outgrowth during natural metamorphosis and reduced hindlimb and tail TH sensitivity were not observed in MCT8 mutants. In sum, we did not observe an effect on TH-dependent development in MCT8 mutants, suggesting compensatory TH transport occurs in tadpole tissues, as seen in most tissues in all model organisms examined., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Asymmetric signalling centres in the early embryo are essential for axis formation in vertebrates. These regions (e.g. amphibian dorsal morula, mammalian anterior visceral endoderm) require stabilised nuclear β-catenin, but the role of localised Wnt ligand signalling activity in their establishment remains unclear. In Xenopus, dorsal β-catenin is initiated by vegetal microtubule-mediated symmetry breaking in the fertilised egg, known as 'cortical rotation'. Localised wnt11b mRNA and ligand-independent activators of β-catenin have been implicated in dorsal β-catenin activation, but the extent to which each contributes to axis formation in this paradigm remains unclear. Here, we describe a CRISPR-mediated maternal-effect mutation in Xenopus laevis wnt11b.L. We find that wnt11b is maternally required for robust dorsal axis formation and for timely gastrulation, and zygotically for left-right asymmetry. Importantly, we show that vegetal microtubule assembly and cortical rotation are reduced in wnt11b mutant eggs. In addition, we show that activated Wnt coreceptor Lrp6 and Dishevelled lack behaviour consistent with roles in early β-catenin stabilisation, and that neither is regulated by Wnt11b. This work thus implicates Wnt11b in the distribution of putative dorsal determinants rather than in comprising the determinants themselves. This article has an associated 'The people behind the papers' interview., Competing Interests: Competing interests D.W.H. is director of the Developmental Studies Hybridoma Bank and serves on the Xenbase advisory board; M.E.H. is director of the National Xenopus Resource., (© 2022. Published by The Company of Biologists Ltd.)

The diploid anuran Xenopus tropicalis has emerged as a key research model in cell and developmental biology. To enhance the usefulness of this species, we developed methods for generating immortal cell lines from Nigerian strain (NXR_1018, RRID:SCR_013731) X. tropicalis embryos. We generated 14 cell lines that were propagated for several months. We selected four morphologically distinct lines, XTN-6, XTN-8, XTN-10 and XTN-12 for further characterization. Karyotype analysis revealed that three of the lines, XTN-8, XTN-10 and XTN-12 were primarily diploid. XTN-6 cultures showed a consistent mixed population of diploid cells, cells with chromosome 8 trisomy, and cells containing a tetraploid content of chromosomes. The lines were propagated using conventional culture methods as adherent cultures at 30°C in a simple, diluted L-15 medium containing fetal bovine serum without use of a high CO 2 incubator. Transcriptome analysis indicated that the four lines were distinct lineages. These methods will be useful in the generation of cell lines from normal and mutant strains of X. tropicalis as well as other species of Xenopus .

The global amphibian declines are compounded by infections with members of the Ranavirus genus such as Frog Virus 3 (FV3). Premetamorphic anuran amphibians are believed to be significantly more susceptible to FV3 while this pathogen targets the kidneys of both pre- and postmetamorphic animals. Paradoxically, FV3-challenged Xenopus laevis tadpoles exhibit lower kidney viral loads than adult frogs. Presently, we demonstrate that X. laevis tadpoles are intrinsically more resistant to FV3 kidney infections than cohort-matched metamorphic and postmetamorphic froglets and that this resistance appears to be epigenetically conferred by endogenous retroviruses (ERVs). Using a X. laevis kidney-derived cell line, we show that enhancing ERV gene expression activates cellular double-stranded RNA-sensing pathways, resulting in elevated mRNA levels of antiviral interferon (IFN) cytokines and thus greater anti-FV3 protection. Finally, our results indicate that large esterase-positive myeloid-lineage cells, rather than renal cells, are responsible for the elevated ERV/IFN axis seen in the tadpole kidneys. This conclusion is supported by our observation that CRISPR-Cas9 ablation of colony-stimulating factor-3 results in abolished homing of these myeloid cells to tadpole kidneys, concurrent with significantly abolished tadpole kidney expression of both ERVs and IFNs. We believe that the manuscript marks an important step forward in understanding the mechanisms controlling amphibian antiviral defenses and thus susceptibility and resistance to pathogens like FV3. IMPORTANCE Global amphibian biodiversity is being challenged by pathogens like the Frog Virus 3 (FV3) ranavirus, underlining the need to gain a greater understanding of amphibian antiviral defenses. While it was previously believed that anuran (frog/toad) amphibian tadpoles are more susceptible to FV3, we demonstrated that tadpoles are in fact more resistant to this virus than metamorphic and postmetamorphic froglets. We showed that this resistance is conferred by large myeloid cells within the tadpole kidneys (central FV3 target), which possess an elevated expression of endogenous retroviruses (ERVs). In turn, these ERVs activate cellular double-stranded RNA-sensing pathways, resulting in a greater expression of antiviral interferon cytokines, thereby offering the observed anti-FV3 protection.

Genome editing simplifies the generation of new animal models for congenital disorders. However, the detailed and unbiased phenotypic assessment of altered embryonic development remains a challenge. Here, we explore how deep learning (U-Net) can automate segmentation tasks in various imaging modalities, and we quantify phenotypes of altered renal, neural and craniofacial development in Xenopus embryos in comparison with normal variability. We demonstrate the utility of this approach in embryos with polycystic kidneys (pkd1 and pkd2) and craniofacial dysmorphia (six1). We highlight how in toto light-sheet microscopy facilitates accurate reconstruction of brain and craniofacial structures within X. tropicalis embryos upon dyrk1a and six1 loss of function or treatment with retinoic acid inhibitors. These tools increase the sensitivity and throughput of evaluating developmental malformations caused by chemical or genetic disruption. Furthermore, we provide a library of pre-trained networks and detailed instructions for applying deep learning to the reader's own datasets. We demonstrate the versatility, precision and scalability of deep neural network phenotyping on embryonic disease models. By combining light-sheet microscopy and deep learning, we provide a framework for higher-throughput characterization of embryonic model organisms. This article has an associated 'The people behind the papers' interview., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Amphibians such as Xenopus tropicalis exhibit a remarkable capacity for tissue regeneration after traumatic injury. Although transforming growth factor-β (TGF-β) receptor signaling is known to be essential for tissue regeneration in fish and amphibians, the role of TGF-β ligands in this process is not well understood. Here, we show that inhibition of TGF-β1 function prevents tail regeneration in Xenopus tropicalis tadpoles. We found that expression of tgfb1 is present before tail amputation and is sustained throughout the regeneration process. CRISPR-mediated knock-out (KO) of tgfb1 retards tail regeneration; the phenotype of tgfb1 KO tadpoles can be rescued by injection of tgfb1 mRNA. Cell proliferation, a critical event for the success of tissue regeneration, is downregulated in tgfb1 KO tadpoles. In addition, tgfb1 KO reduces the expression of phosphorylated Smad2/3 (pSmad2/3) which is important for TGF-β signal-mediated cell proliferation. Collectively, our results show that TGF-β1 regulates cell proliferation through the activation of Smad2/3. We therefore propose that TGF-β1 plays a critical role in TGF-β receptor-dependent tadpole tail regeneration in Xenopus., Competing Interests: Declaration of competing interest To the best of our knowledge, the named authors have no conflict of interest, financial or otherwise., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The embryos of the African clawed frog, Xenopus laevis , are a powerful substrate for the study of complex fundamental biological and disease mechanisms in neurobiology, physiology, molecular biology, cell biology, and developmental biology. A simple and straightforward technique for generating a large number of developmentally synchronized embryos is in vitro fertilization (IVF). IVF permits simultaneous fertilization of thousands of eggs but requires the death of the parental male, which may not be feasible if the male comes from a stock of precious animals. An alternative to euthanizing a precious male is to use a natural mating, which allows for the collection of many embryos with minimal preparation but with the potential loss of the experimental advantage of developmental synchronization. Here we present both strategies for obtaining X. laevis embryos., (© 2021 Cold Spring Harbor Laboratory Press.)

Modular recirculating animal aquaculture systems incorporate UV sterilization and biological, mechanical, and activated carbon filtration, creating a nearly self-contained stable housing environment for Xenopus tropicalis Nonetheless, minimal water exchange is necessary to mitigate accumulation of metabolic waste, and regular weekly, monthly, and yearly maintenance is needed to ensure accurate and efficient operation. This protocol describes the methods for establishing a new recirculating system and the necessary maintenance, as well as water quality parameters, required for keeping Xenopus tropicalis ., (© 2020 Cold Spring Harbor Laboratory Press.)

In many species, sexual differentiation is a vital prelude to reproduction, and disruption of this process can have severe fitness effects, including sterility. It is thus interesting that genetic systems governing sexual differentiation vary among-and even within-species. To understand these systems more, we investigated a rare example of a frog with three sex chromosomes: the Western clawed frog, Xenopus tropicalis. We demonstrate that natural populations from the western and eastern edges of Ghana have a young Y chromosome, and that a male-determining factor on this Y chromosome is in a very similar genomic location as a previously known female-determining factor on the W chromosome. Nucleotide polymorphism of expressed transcripts suggests genetic degeneration on the W chromosome, emergence of a new Y chromosome from an ancestral Z chromosome, and natural co-mingling of the W, Z, and Y chromosomes in the same population. Compared to the rest of the genome, a small sex-associated portion of the sex chromosomes has a 50-fold enrichment of transcripts with male-biased expression during early gonadal differentiation. Additionally, X. tropicalis has sex-differences in the rates and genomic locations of recombination events during gametogenesis that are similar to at least two other Xenopus species, which suggests that sex differences in recombination are genus-wide. These findings are consistent with theoretical expectations associated with recombination suppression on sex chromosomes, demonstrate that several characteristics of old and established sex chromosomes (e.g., nucleotide divergence, sex biased expression) can arise well before sex chromosomes become cytogenetically distinguished, and show how these characteristics can have lingering consequences that are carried forward through sex chromosome turnovers., Competing Interests: The authors have declared that no competing interests exist.

Modular recirculating animal aquaculture systems incorporate UV sterilization and biological, mechanical, and activated carbon filtration, creating a nearly self-contained stable housing environment for Xenopus laevis Nonetheless, minimal water exchange is necessary to mitigate accumulation of metabolic waste, and regular weekly, monthly, and yearly maintenance is needed to ensure accurate and efficient operation. This protocol describes the methods for establishing a new recirculating system and the necessary maintenance, as well as water quality parameters, required for keeping Xenopus laevis ., (© 2020 Cold Spring Harbor Laboratory Press.)

CRISPR/Cas9 genome editing has revolutionized functional genomics in vertebrates. However, CRISPR/Cas9 edited F 0 animals too often demonstrate variable phenotypic penetrance due to the mosaic nature of editing outcomes after double strand break (DSB) repair. Even with high efficiency levels of genome editing, phenotypes may be obscured by proportional presence of in-frame mutations that still produce functional protein. Recently, studies in cell culture systems have shown that the nature of CRISPR/Cas9-mediated mutations can be dependent on local sequence context and can be predicted by computational methods. Here, we demonstrate that similar approaches can be used to forecast CRISPR/Cas9 gene editing outcomes in Xenopus tropicalis, Xenopus laevis, and zebrafish. We show that a publicly available neural network previously trained in mouse embryonic stem cell cultures (InDelphi-mESC) is able to accurately predict CRISPR/Cas9 gene editing outcomes in early vertebrate embryos. Our observations can have direct implications for experiment design, allowing the selection of guide RNAs with predicted repair outcome signatures enriched towards frameshift mutations, allowing maximization of CRISPR/Cas9 phenotype penetrance in the F 0 generation.

Nighttime surges in melatonin levels activate melatonin receptors, which synchronize cellular activities with the natural light/dark cycle. Melatonin receptors are expressed in several cell types in the retina, including the photon-sensitive rods and cones. Previous studies suggest that long-term photoreceptor survival and retinal health is in part reliant on melatonin orchestration of circadian homeostatic activities. This scenario would accordingly envisage that disruption of melatonin receptor signaling is detrimental to photoreceptor health. Using in vivo CRISPR/Cas9 genomic editing, we discovered that a small deletion mutation of the Mel1a melatonin receptor (mtnr1a) gene causes a loss of rod photoreceptors in retinas of developing Xenopus tropicalis heterozygous, but not homozygous mutant tadpoles. Cones were relatively spared from degeneration, and the rod loss phenotype was not obvious after metamorphosis. Localization of Mel1a receptor protein appeared to be about the same in wild type and mutant retinas, suggesting that the mutant protein is expressed at some level in mutant retinal cells. The severe impact on early rod photoreceptor viability may signify a previously underestimated critical role in circadian influences on long-term retinal health and preservation of sight. These data offer evidence that disturbance of homeostatic, circadian signaling, conveyed through a mutated melatonin receptor, is incompatible with rod photoreceptor survival.

Fragile-X mental retardation autosomal homologue-1 (FXR1) is a muscle-enriched RNA-binding protein. FXR1 depletion is perinatally lethal in mice, Xenopus, and zebrafish; however, the mechanisms driving these phenotypes remain unclear. The FXR1 gene undergoes alternative splicing, producing multiple protein isoforms and mis-splicing has been implicated in disease. Furthermore, mutations that cause frameshifts in muscle-specific isoforms result in congenital multi-minicore myopathy. We observed that FXR1 alternative splicing is pronounced in the serine- and arginine-rich intrinsically disordered domain; these domains are known to promote biomolecular condensation. Here, we show that tissue-specific splicing of fxr1 is required for Xenopus development and alters the disordered domain of FXR1. FXR1 isoforms vary in the formation of RNA-dependent biomolecular condensates in cells and in vitro. This work shows that regulation of tissue-specific splicing can influence FXR1 condensates in muscle development and how mis-splicing promotes disease., (© 2020 Smith et al.)

Xenopus tropicalis tadpoles can regenerate an amputated tail, including spinal cord, muscle and notochord, through cell proliferation and differentiation. However, the molecular mechanisms that regulate cell proliferation during tail regeneration are largely unknown. Here we show that JunB plays an important role in tail regeneration by regulating cell proliferation. The expression of junb is rapidly activated and sustained during tail regeneration. Knockout (KO) of junb causes a delay in tail regeneration and tissue differentiation. In junb KO tadpoles, cell proliferation is prevented before tissue differentiation. Furthermore, TGF-β signaling, which is activated just after tail amputation, regulates the induction and maintenance of junb expression. These findings demonstrate that JunB, a downstream component of TGF-β signaling, works as a positive regulator of cell proliferation during Xenopus tail regeneration., Competing Interests: Declaration of competing interest To the best of our knowledge, the named authors have no conflict of interest, financial or otherwise., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Codevelopment of the lungs and heart underlies key evolutionary innovations in the transition to terrestrial life. Cardiac specializations that support pulmonary circulation, including the atrial septum, are generated by second heart field (SHF) cardiopulmonary progenitors (CPPs). It has been presumed that transcription factors required in the SHF for cardiac septation, e.g., Tbx5 , directly drive a cardiac morphogenesis gene-regulatory network. Here, we report instead that TBX5 directly drives Wnt ligands to initiate a bidirectional signaling loop between cardiopulmonary mesoderm and the foregut endoderm for endodermal pulmonary specification and, subsequently, atrial septation. We show that Tbx5 is required for pulmonary specification in mice and amphibians but not for swim bladder development in zebrafish. TBX5 is non-cell-autonomously required for pulmonary endoderm specification by directly driving Wnt2 and Wnt2b expression in cardiopulmonary mesoderm. TBX5 ChIP-sequencing identified cis -regulatory elements at Wnt2 sufficient for endogenous Wnt2 expression domains in vivo and required for Wnt2 expression in precardiac mesoderm in vitro. Tbx5 cooperated with Shh signaling to drive Wnt2b expression for lung morphogenesis. Tbx5 haploinsufficiency in mice, a model of Holt-Oram syndrome, caused a quantitative decrement of mesodermal-to-endodermal Wnt signaling and subsequent endodermal-to-mesodermal Shh signaling required for cardiac morphogenesis. Thus, Tbx5 initiates a mesoderm-endoderm-mesoderm signaling loop in lunged vertebrates that provides a molecular basis for the coevolution of pulmonary and cardiac structures required for terrestrial life., Competing Interests: The authors declare no conflict of interest.

Xenopus laevis embryos are an established model for studying kidney development. The nephron structure and genetic pathways that regulate nephrogenesis are conserved between Xenopus and humans, allowing for the study of human disease-causing genes. Xenopus embryos are also amenable to large-scale screening, but studies of kidney disease-related genes have been impeded because assessment of kidney development has largely been limited to examining fixed embryos. To overcome this problem, we have generated a transgenic line that labels the kidney. We characterize this cdh17 :eGFP line, showing green fluorescent protein (GFP) expression in the pronephric and mesonephric kidneys and colocalization with known kidney markers. We also demonstrate the feasibility of live imaging of embryonic kidney development and the use of cdh17 :eGFP as a kidney marker for secretion assays. Additionally, we develop a new methodology to isolate and identify kidney cells for primary culture. We also use morpholino knockdown of essential kidney development genes to establish that GFP expression enables observation of phenotypes, previously only described in fixed embryos. Taken together, this transgenic line will enable primary kidney cell culture and live imaging of pronephric and mesonephric kidney development. It will also provide a simple means for high-throughput screening of putative human kidney disease-causing genes., Competing Interests: The authors declare no conflict of interest. The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Studying genes involved in organogenesis is often difficult because many of these genes are also essential for early development. The allotetraploid frog, Xenopus laevis , is commonly used to study developmental processes, but because of the presence of two homeologs for many genes, it has been difficult to use as a genetic model. Few studies have successfully used CRISPR in amphibians, and currently there is no tissue-targeted knockout strategy described in Xenopus The goal of this study is to determine whether CRISPR/Cas9-mediated gene knockout can be targeted to the Xenopus kidney without perturbing essential early gene function. We demonstrate that targeting CRISPR gene editing to the kidney and the eye of F0 embryos is feasible. Our study shows that knockout of both homeologs of lhx1 results in the disruption of kidney development and function but does not lead to early developmental defects. Therefore, targeting of CRISPR to the kidney may not be necessary to bypass the early developmental defects reported upon disruption of Lhx1 protein expression or function by morpholinos, antisense RNA, or dominant negative constructs. We also establish a control for CRISPR in Xenopus by editing a gene ( slc45a2 ) that when knocked out results in albinism without altering kidney development. This study establishes the feasibility of tissue-specific gene knockout in Xenopus , providing a cost-effective and efficient method for assessing the roles of genes implicated in developmental abnormalities that is amenable to high-throughput gene or drug screening techniques., (Copyright © 2018 by the Genetics Society of America.)

Robust and efficient protocols for fertilization and early embryo care of Xenopus laevis and Xenopus tropicalis are essential for experimental success, as well as maintenance and propagation of precious animal stocks. The rapid growth of the National Xenopus Resource has required effective implementation and optimization of these protocols. Here, we discuss the procedures used at the National Xenopus Resource, which we found helpful for generation and early upkeep of Xenopus embryos and tadpoles.

Maintenance of optimal conditions such as water parameters, diet, and feeding is essential to a healthy Xenopus laevis and Xenopus tropicalis colony and thus to the productivity of the lab. Our prior husbandry experience as well as the rapid growth of the National Xenopus Resource has given us a unique insight into identifying and implementing these optimal parameters into our husbandry operations. Here, we discuss our standard operating procedures that will be of use to both new and established Xenopus facilities.

Completion of the Xenopus laevis genome sequence from inbred J strain animals has facilitated the generation of germline mutant X. laevis using targeted genome editing. In the last few years, numerous reports have demonstrated that TALENs are able to induce mutations in F0 Xenopus embryos, but none has demonstrated germline transmission of such mutations in X. laevis. In this report we used the oocyte host-transfer method to generate mutations in both tyrosinase homeologs and found highly-penetrant germline mutations; in contrast, embryonic injections yielded few germline mutations. We also compared the distribution of mutations in several F0 somatic tissues and germ cells and found that the majority of mutations in each tissue were different. These results establish that X. laevis J strain animals are very useful for generating germline mutations and that the oocyte host-transfer method is an efficient technique for generating mutations in both homeologs., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Injection of human Chorionic Gonadotropin (hCG) directly into the dorsal lymph sac of Xenopus is a commonly used protocol for induction of ovulation, but recent shortages in the stocks of commercially available hCG as well as lack of a well tested alternative have resulted in frustrating experimental delays in laboratories that predominantly use Xenopus in their research. Mammalian Luteinizing Hormones (LH) share structural similarity, functional equivalency, and bind the same receptor as hCG; this suggests that LH may serve as a good alternative to hCG for promoting ovulation in Xenopus. LH has been found to induce maturation of Xenopus oocytes in vitro, but whether it can be used to induce ovulation in vivo has not been examined. Here we compared the ability of four mammalian LH proteins, bovine (bLH), human (hLH), ovine (oLH), porcine (pLH), to induce ovulation in Xenopus when injected into the dorsal lymph sac of sexually mature females. We find that both ovine and human LH, but not bovine or porcine, are good substitutes for hCG for induction of ovulation in WT and J strain Xenopus laevis and Xenopus tropicalis., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

The amphibian model Xenopus, has been used extensively over the past century to study multiple aspects of cell and developmental biology. Xenopus offers advantages of a non-mammalian system, including high fecundity, external development, and simple housing requirements, with additional advantages of large embryos, highly conserved developmental processes, and close evolutionary relationship to higher vertebrates. There are two main species of Xenopus used in biomedical research, Xenopus laevis and Xenopus tropicalis; the common perception is that both species are excellent models for embryological and cell biological studies, but only Xenopus tropicalis is useful as a genetic model. The recent completion of the Xenopus laevis genome sequence combined with implementation of genome editing tools, such as TALENs (transcription activator-like effector nucleases) and CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated nucleases), greatly facilitates the use of both Xenopus laevis and Xenopus tropicalis for understanding gene function in development and disease. In this paper, we review recent advances made in Xenopus laevis and Xenopus tropicalis with TALENs and CRISPR-Cas and discuss the various approaches that have been used to generate knockout and knock-in animals in both species. These advances show that both Xenopus species are useful for genetic approaches and in particular counters the notion that Xenopus laevis is not amenable to genetic manipulations., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

We characterize the genetic diversity of Xenopus laevis strains using RNA-seq data and allele-specific analysis. This data provides a catalogue of coding variation, which can be used for improving the genomic sequence, as well as for better sequence alignment, probe design, and proteomic analysis. In addition, we paint a broad picture of the genetic landscape of the species by functionally annotating different classes of mutations with a well-established prediction tool (PolyPhen-2). Further, we specifically compare the variation in the progeny of four crosses: inbred genomic (J)-strain, outbred albino (B)-strain, and two hybrid crosses of J and B strains. We identify a subset of mutations specific to the B strain, which allows us to investigate the selection pressures affecting duplicated genes in this allotetraploid. From these crosses we find the ratio of non-synonymous to synonymous mutations is lower in duplicated genes, which suggests that they are under greater purifying selection. Surprisingly, we also find that function-altering ("damaging") mutations constitute a greater fraction of the non-synonymous variants in this group, which suggests a role for subfunctionalization in coding variation affecting duplicated genes., (Copyright © 2017 Elsevier Inc. All rights reserved.)

A heterozygous nonsense variant was identified in dapper, antagonist of beta-catenin, 1 (DACT1) via whole-exome sequencing in family members with imperforate anus, structural renal abnormalities, genitourinary anomalies, and/or ear anomalies. The DACT1 c.1256G>A;p.Trp419 * variant segregated appropriately in the family consistent with an autosomal dominant mode of inheritance. DACT1 is a member of the Wnt-signaling pathway, and mice homozygous for null alleles display multiple congenital anomalies including absent anus with blind-ending colon and genitourinary malformations. To investigate the DACT1 c.1256G>A variant, HEK293 cells were transfected with mutant DACT1 cDNA plasmid, and immunoblotting revealed stability of the DACT1 p.Trp419 * protein. Overexpression of DACT1 c.1256G>A mRNA in Xenopus embryos revealed a specific gastrointestinal phenotype of enlargement of the proctodeum. Together, these findings suggest that the DACT1 c.1256G>A nonsense variant is causative of a specific genetic syndrome with features overlapping Townes-Brocks syndrome., (© 2017 WILEY PERIODICALS, INC.)

The Western clawed frog, Xenopus tropicalis, is a highly promising model amphibian, especially in developmental and physiological research, and as a tool for understanding disease. It was originally found in the West African rainforest belt, and was introduced to the research community in the 1990s. The major strains thus far known include the Nigerian and Ivory Coast strains. However, due to its short history as an experimental animal, the genetic relationship among the various strains has not yet been clarified, and establishment of inbred strains has not yet been achieved. Since 2003 the Institute for Amphibian Biology (IAB), Hiroshima University has maintained stocks of multiple X. tropicalis strains and conducted consecutive breeding as part of the National BioResource Project. In the present study we investigated the inbreeding ratio and genetic relationship of four inbred strains at IAB, as well as stocks from other institutions, using highly polymorphic microsatellite markers and mitochondrial haplotypes. Our results show successive reduction of heterozygosity in the genome of the IAB inbred strains. The Ivory Coast strains clearly differed from the Nigerian strains genetically, and three subgroups were identified within both the Nigerian and Ivory Coast strains. It is noteworthy that the Ivory Coast strains have an evolutionary divergent genetic background. Our results serve as a guide for the most effective use of X. tropicalis strains, and the long-term maintenance of multiple strains will contribute to further research efforts.

Diseases affecting endodermal organs like the pancreas, lung and gastrointestinal (GI) tract have a substantial impact on human welfare. Since many of these are congenital defects that arise as a result of defects during development broad efforts are focused on understanding the development of these organs so as to better identify risk factors, disease mechanisms and therapeutic targets. Studies implementing model systems, like the amphibian Xenopus, have contributed immensely to our understanding of signaling (e.g. Wnt, FGF, BMP, RA) pathways and gene regulation (e.g. hhex , ptf1a , ngn3 ) that underlie normal development as well as disease progression. Recent advances in genome engineering further enhance the capabilities of the Xenopus model system for pursuing biomedical research, and will undoubtedly result in a boom of new information underlying disease mechanisms ultimately leading to advancements in diagnosis and therapy.

Background: Mass spectrometry-based proteomics enables the global identification and quantification of proteins and their posttranslational modifications in complex biological samples. However, proteomic analysis requires a complete and accurate reference set of proteins and is therefore largely restricted to model organisms with sequenced genomes., Results: Here, we demonstrate the feasibility of deep genome-free proteomics by using a reference proteome derived from heterogeneous mRNA data. We identify more than 11,000 proteins with 99% confidence from the unfertilized Xenopus laevis egg and estimate protein abundance with approximately 2-fold precision. Our reference database outperforms the provisional gene models based on genomic DNA sequencing and references generated by other methods. Surprisingly, we find that many proteins in the egg lack mRNA support and that many of these proteins are found in blood or liver, suggesting that they are taken up from the blood plasma, together with yolk, during oocyte growth and maturation, potentially contributing to early embryogenesis., Conclusion: To facilitate proteomics in nonmodel organisms, we make our platform available as an online resource that converts heterogeneous mRNA data into a protein reference set. Thus, we demonstrate the feasibility and power of genome-free proteomics while shedding new light on embryogenesis in vertebrates., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Pancreas-specific transcription factor 1a (Ptf1a), a bHLH transcription factor, has two temporally distinct functions during pancreas development; initially it is required for early specification of the entire pancreas, while later it is required for proper differentiation and maintenance of only acinar cells. The importance of Ptf1a function was revealed by the fact that loss of Ptf1a leads to pancreas agenesis in humans. While Ptf1a is one of the most important pancreatic transcription factors, little is known about the differences between the regulatory networks it controls during initial specification of the pancreas as opposed to acinar cell development, and to date no comprehensive analysis of its downstream targets has been published. In this article, we use Xenopus embryos to identify putative downstream targets of Ptf1a. We isolated anterior endoderm tissue overexpressing Ptf1a at two early stages, NF32 and NF36, and compared their gene expression profiles using microarrays. Our results revealed that Ptf1a regulates genes with a wide variety of functions, providing insight into the complexity of the regulatory network required for pancreas specification., (Copyright © 2012 Wiley Periodicals, Inc.)

Xenopus is an essential vertebrate model system for biomedical research that has contributed to important discoveries in many disciplines, including cell biology, molecular biology, physiology, developmental biology, and neurobiology. However, unlike other model systems no central repository/stock center for Xenopus had been established until recently. Similar to mouse, zebrafish, and fly communities, which have established stock centers, Xenopus researchers need to maintain and distribute rapidly growing numbers of inbred, mutant, and transgenic frog strains, along with DNA and protein resources, and individual laboratories struggle to accomplish this efficiently. In the last 5 years, two resource centers were founded to address this need: the European Xenopus Resource Center (EXRC) at the University of Portsmouth in England, and the National Xenopus Resource (NXR) at the Marine Biological Laboratory in Woods Hole, MA. These two centers work together to provide resources and support to the Xenopus research community. The EXRC and NXR serve as stock centers and acquire, produce, maintain and distribute mutant, inbred and transgenic Xenopus laevis and Xenopus tropicalis lines. Independently, the EXRC is a repository for Xenopus cDNAs, fosmids, and antibodies; it also provides oocytes and wild-type frogs within the United Kingdom. The NXR will complement these services by providing research training and promoting intellectual interchange through hosting mini-courses and workshops and offering space for researchers to perform short-term projects at the Marine Biological Laboratory. Together the EXRC and NXR will enable researchers to improve productivity by providing resources and expertise to all levels, from graduate students to experienced PIs. These two centers will also enable investigators that use other animal systems to take advantage of Xenopus' unique experimental features to complement their studies., (Copyright © 2012 Wiley Periodicals, Inc.)

Defining the regulatory molecular networks involved in patterning the developing anterior endoderm is essential to understand how the pancreas, liver, stomach, and duodenum are discretely specified from each other. In this study, we analyzed the expression and function of the double-stranded RNA-binding protein Staufen2 in Xenopus laevis endoderm. We found that staufen2 was broadly expressed within the developing endoderm beginning at gastrulation becoming localized to the anterior endoderm at later stages. Through morpholino-mediated knockdown, we demonstrate that Staufen2 function is required for proper formation of the stomach, liver, and pancreas. We define that its function is required during gastrulation for proper patterning of the dorsal-ventral axis and that it acts to regulate expression of BMP signaling components., (Copyright © 2011 Wiley Periodicals, Inc.)

Mutations in rfx6 were recently associated with Mitchell-Riley syndrome, which involves neonatal diabetes, and other digestive system defects. To better define the function of Rfx6 in early endoderm development we cloned the Xenopus homologue. Expression of rfx6 begins early, showing broad expression throughout the anterior endoderm; at later stages rfx6 expression becomes restricted to the endocrine cells of the gut and pancreas. Morpholino knockdown of rfx6 caused a loss of pancreas marker expression, as well as other abnormalities. Co-injection of exogenous wild-type rfx6 rescued the morpholino phenotype in Xenopus tadpoles, whereas attempts to rescue the loss-of-function phenotype using mutant rfx6 based on Mitchell-Riley patients were unsuccessful. To better define the pleiotropic effects, we performed microarray analyses of gene expression in knockdown foregut tissue. In addition to pancreatic defects, the microarray analyses revealed downregulation of lung, stomach and heart markers and an upregulation of kidney markers. We verified these results using RT-PCR and in situ hybridization. Based on the different rfx6 expression patterns and our functional analyses, we propose that rfx6 has both early and late functions. In early development Rfx6 plays a broad role, being essential for development of most anterior endodermal organs. At later stages however, Rfx6 function is restricted to endocrine cells., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Developmental control of proliferation relies on tight regulation of protein expression. Although this has been well studied in early embryogenesis, how the cell cycle is regulated during organogenesis is not well understood. Bruno-Like RNA binding proteins bind to consensus sequences in the 3'UTR of specific mRNAs and repress protein translation, but much of this functional information is derived from studies on mainly two members, Drosophila Bruno and vertebrate BrunoL2 (CUGBP1). There are however, six vertebrate and three Drosophila Bruno family members, but less is known about these other family members, and none have been shown to function in the endoderm. We recently identified BrunoL1 as a dorsal pancreas enriched gene, and in this paper we define BrunoL1 function in Xenopus endoderm development. We find that, in contrast to other Bruno-Like proteins, BrunoL1 acts to enhance rather than repress translation. We demonstrate that BrunoL1 regulates proliferation of endoderm cells through translational control of cyclin A2 mRNA. Specifically BrunoL1 enhanced translation of cyclin A2 through binding consensus Bruno Response Elements (BREs) in its 3'UTR. We compared the ability of other Bruno-Like proteins, both vertebrate and invertebrate, to stimulate translation via the cyclin A2 3'UTR and found that only Drosophila Bru-3 had similar activity. In addition, we also found that both BrunoL1 and Bru-3 enhanced translation of mRNAs containing the 3'UTRs of Drosophila oskar or cyclin A, which have been well characterized to mediate repression. Lastly, we show that it is the Linker region of BrunoL1 that is both necessary and sufficient for this activity. These results are the first example of BRE-dependent translational enhancement and are the first demonstration in vertebrates of Bruno-Like proteins regulating translation through BREs., ((c) 2010 Elsevier Inc. All rights reserved.)

In mammals, it has been well established that gastrointestinal and pancreatic endocrine cells are specified by a cascade of different transcription factors, but whether these same pathways (or linear relationships) operate in Xenopus is currently unknown. We recently identified the endocrine-specific zinc finger transcription factor insulinoma associated protein 1 (insm1) as a dorsal-enriched gene. We found that insm1 is expressed in the dorsal pancreas as early as NF28, making it one of the earliest markers to be localized to the dorsal pancreas. Through morpholino-mediated knockdown, we demonstrate that insm1 is essential for proper specification of both gastrointestinal and pancreatic endocrine cells. In addition, we place insm1 downstream of ngn3 and upstream of pax6 and neuroD in the endocrine cell transcription factor cascade. These are the first results showing that the endodermal endocrine cell development in Xenopus uses the same transcriptional cascade as in mammals.

Understanding how the pancreas develops is vital to finding new treatments for a range of pancreatic diseases, including diabetes and pancreatic cancer. Xenopus is a relatively new model organism for the elucidation of pancreas development, and has already made contributions to the field. Recent studies have shown benefits of using Xenopus for understanding both early patterning and lineage specification aspects of pancreas organogenesis. This review focuses specifically on Xenopus pancreas development, and covers events from the end of gastrulation, when regional specification of the endoderm is occurring, right through metamorphosis, when the mature pancreas is fully formed. We have attempted to cover pancreas development in Xenopus comprehensively enough to assist newcomers to the field and also to enable those studying pancreas development in other model organisms to better place the results from Xenopus research into the context of the field in general and their studies specifically. Developmental Dynamics 238:1271-1286, 2009. (c) 2009 Wiley-Liss, Inc.

During embryogenesis, the pancreas develops from separate dorsal and ventral buds, which fuse to form the mature pancreas. Little is known about the functional differences between these two buds or the relative contribution of cells derived from each region to the pancreas after fusion. To follow the fate of dorsal or ventral bud derived cells in the pancreas after fusion, we produced chimeric Elas-GFP transgenic/wild-type embryos in which either dorsal or ventral pancreatic bud cells expressed GFP. We found that ventral pancreatic cells migrate extensively into the dorsal pancreas after fusion, whereas the converse does not occur. Moreover, we found that annular pancreatic tissue is composed exclusively of ventral pancreas-derived cells. To identify ventral pancreas-specific genes that may play a role in pancreatic bud fusion, we isolated individual dorsal and ventral pancreatic buds, prior to fusion, from NF38/39 Xenopus laevis tadpoles and compared their gene expression profiles (NF refers to the specific stage of Xenopus development). As a result of this screen, we have identified several new ventral pancreas-specific genes, all of which are expressed in the same location within the ventral pancreas at the junction where the two ventral pancreatic buds fuse. Morpholino-mediated knockdown of one of these ventral-specific genes, transmembrane 4 superfamily member 3 (tm4sf3), inhibited dorsal-ventral pancreatic bud fusion, as well as acinar cell differentiation. Conversely, overexpression of tm4sf3 promoted development of annular pancreas. Our results are the first to define molecular and behavioral differences between the dorsal and ventral pancreas, and suggest an unexpected role for the ventral pancreas in pancreatic bud fusion.

Insulin-producing beta-cells are present as single cells or in small clusters distributed throughout the pancreas of the Xenopus laevis tadpole. During metamorphic climax when the exocrine pancreas dedifferentiates to progenitor cells, the beta-cells undergo two changes. Insulin mRNA is down regulated at the beginning of metamorphic climax (NF62) and reexpressed again near the end of climax. Secondly, the beta-cells aggregate to form islets. During climax the increase in insulin cluster size is not caused by cell proliferation or by acinar-to-beta-cell transdifferentiation, but rather is due to the aggregation of pre-existing beta-cells. The total number of beta-cells does not change during the 8 days of climax. Thyroid hormone (TH) induction of premetamorphic tadpoles causes an increase in islet size while prolonged treatment of tadpoles with the goitrogen methimazole inhibits this increase. Expression of a dominant negative form of the thyroid hormone receptor (TRDN) driven by the elastase promoter not only protects the exocrine pancreas of a transgenic tadpole from TH-induced dedifferentiation but also prevents aggregation of beta-cells at climax. These transgenic tadpoles do however undergo normal loss and resynthesis of insulin mRNA at the same stage as controls. In contrast transgenic tadpoles with the same TRDN transgene driven by an insulin promoter do not undergo down regulation of insulin mRNA, but do aggregate beta-cells to form islets like controls. These results demonstrate that TH controls the remodeling of beta-cells through cell-cell interaction with dedifferentiating acinar cells and a cell autonomous program that temporarily shuts off the insulin gene.

Determining the functional attributes of pancreatic transcription factors is essential to understand how the pancreas is specified distinct from other endodermal organs, such as liver, stomach and duodenum, and to direct the differentiation of other cell types into pancreas. Previously, we demonstrated that Pdx1-VP16 was sufficient to convert liver to pancreas. In this paper, we characterize the functional ability of another pancreatic transcription factor, Ptf1a, in promoting ectopic pancreatic fates at early stages throughout the endoderm and later during organogenesis. Using the transthyretin promoter to drive expression in the early liver region/bud of transgenic Xenopus tadpoles, we find that Ptf1a-VP16 is able to convert liver to pancreas. Overexpression of the unmodified Ptf1a on the other hand has no effect in liver but is able to convert stomach and duodenum to pancreas. When overexpressed at earlier embryonic stages throughout the endoderm, Ptf1a activity is similarly limited, whereas Ptf1a-VP16 has increased activity. Interestingly, in all instances we find that Ptf1a-VP16 is only capable of promoting acinar cell fates, whereas Ptf1a promotes both acinar and endocrine fates. Lastly, we demonstrate that, similar to mouse and zebrafish, Xenopus Ptf1a is essential for the initial specification of both endocrine and exocrine cells during normal pancreas development.

Unlike other organs, the adult heart has limited regenerative potential owing to the inability of postnatal cardiomyocytes to undergo proliferative growth. As a result, ischemic heart disease continues to be a major cause of morbidity and mortality worldwide. Elucidating the molecular pathways of cardiomyocyte differentiation and proliferation holds great promise for human health. In a recent paper we employed a multidisciplinary approach to identify a novel pathway required for cardiomyocyte growth and differentiation. Starting with the dissection of a new regulatory sequence required for cardiac specific expression, we identified the cognate DNA binding protein as KLF13, a tissue-restricted member of the newly identified KLF family of zinc-finger proteins. We took advantage of the ease in manipulating Xenopus embryos to genetically alter KLF13 levels thus demonstrating a requirement for KLF13 in cardiac progenitor cell proliferation and heart morphogenesis. Furthermore, we combined biochemical approaches with genetic manipulations in Xenopus to show that KLF13 is a GATA4 interacting protein and a genetic modifier of GATA4 function. Cyclin D1 was identified as a direct transcriptional target for KLF13 that may account for the proliferation defects observed in embryos with downregulated KLF13 levels. Thus, tissue-specific regulators of the cell cycle may be potential congenital heart disease causing genes in humans.

In humans, congenital heart defects occur in 1-2% of live birth, but the molecular mechanisms and causative genes remain unidentified in the majority of cases. We have uncovered a novel transcription pathway important for heart morphogenesis. We report that KLF13, a member of the Krüppel-like family of zinc-finger proteins, is expressed predominantly in the heart, binds evolutionarily conserved regulatory elements on cardiac promoters and activates cardiac transcription. KLF13 is conserved across species and knockdown of KLF13 in Xenopus embryos leads to atrial septal defects and hypotrabeculation similar to those observed in humans or mice with hypomorphic GATA-4 alleles. Physical and functional interaction with GATA-4, a dosage-sensitive cardiac regulator, provides a mechanistic explanation for KLF13 action in the heart. The data demonstrate that KLF13 is an important component of the transcription network required for heart development and suggest that KLF13 is a GATA-4 modifier; by analogy to other GATA-4 collaborators, mutations in KLF13 may be causative for congenital human heart disease.

The amphibian embryo is a highly successful model system with great promise for organogenesis research. Since the late 1800s, amphibians have been employed to understand vertebrate development and since the 1950s, the African clawed frog Xenopus laevis has been the amphibian of choice. In the past two decades, Xenopus has led the way forward in, among other things, identifying transcription factors, gene regulatory networks and inter- and intracellular signaling pathways that control early development (from fertilization through gastrulation and neurulation). Perhaps the best measure of how successful Xenopus has been as a model for early mammalian development is the observation that much of the knowledge gleaned from Xenopus studies has subsequently directly translated to discoveries of similar mechanisms operating in mouse development. Despite this great success in early development, research on organogenesis in Xenopus has lagged behind the mouse. However, recent technical advances now make Xenopus amenable for studies on later development, including organogenesis. Here, we discuss why Xenopus is well suited for such research and, we believe, permits addressing questions that have been difficult to approach using other model systems. We also highlight how Xenopus researchers have already begun studying a number of major organs, pancreas, liver, kidney and heart, and suggest how Xenopus might contribute more to these areas in the near future.

We have characterised the transdifferentiation of human HepG2 (hepatoma) cells to pancreatic cells following introduction of an activated version of the pancreatic transcription factor Pdx1 (XlHbox8-VP16). The following questions are addressed: (1) are all types of pancreatic cells produced? (2) is the requirement for expression of the transgene temporary or permanent? (3) are the transdifferentiated beta-cells responsive to physiological stimuli? The results showed that both pancreatic exocrine cells (by detection of amylase protein), and endocrine cells (by detecting insulin, glucagon and somatostatin proteins) are induced after XlHbox8VP16 transfection. Moreover, the hepatic phenotype becomes suppressed during transdifferentiation of hepatocytes to pancreatic cells. Requirement for the transgene is only temporary and it is no longer required once the pancreatic differentiation program is activated. Finally, we provided results to suggest that the transdifferentiated cells are functional by detecting: (1) functional markers for pancreatic beta-cells including prohormone convertase 1/3 (PC1/3), insulin C-peptide and glucagon-like peptide 1 receptor (GLP-1R), (2) increased insulin mRNA expression after treatment of cells with GLP-1 and betacellulin, physiological stimuli that regulate pancreatic function and (3) elevated insulin secretion after glucose challenge. The transdifferentiation of hepatic to pancreatic cells represents one possible source of beta-cells for human islet transplantation and this study shows that such a transdifferentiation can be achieved in vitro.

Pdx1 has been shown to convert hepatocytes into both exocrine and endocrine pancreatic cells in mice, but it fails to selectively convert hepatocytes into pure insulin-producing cells (IPCs). The molecular mechanisms underlying the transdifferentiation remain unclear. In this study, we generated a stably transfected rat hepatic cell line named WB-1 that expresses an active form of Pdx1 along with a reporter gene, RIP-eGFP. Our results demonstrate that Pdx1 induces the expression of multiple genes related to endocrine pancreas development and islet function in these liver cells. We do not however find any expression of the late-stage genes (Pax4, Pax6, Isl-1, and MafA) related to beta-cell development, and the cells do not secrete insulin upon the glucose challenge. Yet when WB-1 cells are transplanted into diabetic NOD-scid mice, these genes become activated and hyperglycemia is completely reversed. Detailed comparison of gene expression profiles between pre- and posttransplanted WB-1 cells demonstrates that the WB-1 cells have similar properties as that seen in pancreatic beta-cells. In addition, in vitro culture in high-glucose medium is sufficient to induce complete maturation of WB-1 cells into functional IPCs. In summary, we find that Pdx1-VP16 is able to selectively convert hepatic cells into pancreatic endocrine precursor cells. However, complete transdifferentiation into functional IPCs requires additional external factors, including high glucose or hyperglycemia. Thus, transdifferentiation of hepatocytes into functional IPCs may serve as a viable therapeutic option for patients with type 1 diabetes.

Mammalian studies have implicated important roles for the basic helix-loop-helix transcription factor Ptf1a-p48 in the development of both exocrine and endocrine pancreas. We have cloned the Ptf1a-p48 ortholog in Danio rerio. Early zebrafish ptf1a expression is observed in developing hindbrain and in endodermal pancreatic precursors. Analysis of ptf1a and insulin expression reveals a population of exocrine precursors that, throughout early development, are temporally and spatially segregated from endocrine elements. Morpholino-mediated knockdown of ptf1a confirms early divergence of these endocrine and exocrine lineages. Ptf1a morphants lack differentiated exocrine pancreas, but maintain normal differentiation and organization of the principal islet. In addition to the exocrine phenotype, ptf1a knockdown also reduces the prevalence of a small population of anterior endocrine cells normally found outside the principal islet. Together, these findings suggest the presence of distinct ptf1a-dependent and ptf1a-independent precursor populations in developing zebrafish pancreas.

Background: The liver and the pancreas arise from adjacent regions of endoderm in embryonic development. Pdx1 is a key transcription factor that is essential for the development of the pancreas and is not expressed in the liver. The aim of this study was to determine whether a gene overexpression protocol based on Pdx1 would be able to cause conversion of liver to pancreas., Results: We show that a modified form of Pdx1, carrying the VP16 transcriptional activation domain, can cause conversion of liver to pancreas, both in vivo and in vitro. Transgenic Xenopus tadpoles carrying the construct TTR-Xlhbox8-VP16:Elas-GFP were prepared. Xlhbox8 is the Xenopus homolog of Pdx1, the TTR (transthyretin) promoter directs expression to the liver, and the GFP is under the control of an elastase promoter and provides a real-time visible marker of pancreatic differentiation. In the transgenic tadpoles, part or all of the liver is converted to pancreas, containing both exocrine and endocrine cells, while liver differentiation products are lost from the regions converted to pancreas. The timing of events is such that the liver is differentiating by the time Xlhbox8-VP16 is expressed, so we consider this a transdifferentiation event rather than a reprogramming of embryonic development. Furthermore, this same construct will bring about transdifferentiation of human hepatocytes in culture, with formation of both exocrine and endocrine cells., Conclusions: We consider that the conversion of liver to pancreas could be the basis of a new type of therapy for insulin-dependent diabetes. Although expression of the transgene is transient, once the ectopic pancreas is established, it persists thereafter.

Transdifferentiation is the name used to describe the direct conversion of one differentiated cell type into another. Cells which have the potential to interconvert by transdifferentiation generally arise from adjacent regions in the developing embryo. For example, the liver and pancreas arise from the same region of the endoderm. The transdifferentiation of pancreas to liver (and vice versa) has been observed in animal experiments and in certain human pathologies. Understanding transdifferentiation is important to developmental biologists because it will help elucidate the cellular and molecular differences that distinguish neighbouring regions of the embryo. While the in vivo models for the transdifferentiation of liver to pancreas have been valuable, it is more difficult to extrapolate from these studies to individual changes at the cellular or molecular levels. The recent development of two in vitro systems (AR42J cells and embryonic pancreatic cultures) for the transdifferentiation of pancreas to liver has shown that an environmental change in the form of an exogenous glucocorticoid can cause the conversion of pancreatic exocrine cells into hepatocytes. The AR42J cell system has been used to elucidate the cell lineage and the molecular basis of transdifferentiation of pancreas to liver.

To better understand the relationship between the endocrine and exocrine cell types in the Xenopus pancreas, we have cloned the Xenopus amylase cDNA and compared its expression profile with that of four other pancreatic markers: insulin, glucagon, elastase and trypsinogen. Our results demonstrate that the first pancreatic marker to be expressed is insulin, exclusively in the dorsal pancreas. These insulin-expressing cells form small groups which resemble islets, but no insulin is detected in the ventral pancreas until stage 47. In contrast, the exocrine markers, amylase, elastase and trypsinogen are first expressed only in the ventral pancreas beginning at stage 41; by stage 45 their expression extends into the dorsal pancreas. Glucagon, on the other hand, is not expressed in the pancreas until stage 45. In the endocrine cell clusters we do not find glucagon-expressing cells surrounding insulin-expressing cells, either in the tadpole or in the mature frog pancreas.