Eugene Bell Center for Regenerative Biology and Tissue Engineering
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The ability of many animals to spontaneously regenerate their body parts has intrigued scientific observers for centuries. Although humans share the same basic genes and pathways, we have somehow lost these regenerative capacities, which leads to significant health costs. An understanding of tissue and organ regeneration in lower animals holds great promise for translating to medical treatments for serious human conditions, including spinal cord injury, diabetes, organ failure, and degenerative neural diseases such as Alzheimer’s.
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Browsing Eugene Bell Center for Regenerative Biology and Tissue Engineering by Subject "Xenopus"
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ArticleAge-associated DNA methylation changes in Xenopus frogs(Taylor and Francis Group, 2023-04-06) Morselli, Marco ; Bennett, Ronan ; Shaidani, Nikko-Ideen ; Horb, Marko ; Peshkin, Leonid ; Pellegrini, MatteoAge-associated changes in DNA methylation have been characterized across various animals, but not yet in amphibians, which are of particular interest because they include widely studied model organisms. In this study, we present clear evidence that the aquatic vertebrate speciesdisplays patterns of age-associated changes in DNA methylation. We have generated whole-genome bisulfite sequencing (WGBS) profiles from skin samples of nine frogs representing young, mature, and old adults and characterized the gene- and chromosome-scale DNA methylation changes with age. Many of the methylation features and changes we observe are consistent with what is known in mammalian species, suggesting that the mechanism of age-related changes is conserved. Moreover, we selected a few thousand age-associated CpG sites to build an assay based on targeted DNA methylation analysis (TBSseq) to expand our findings in future studies involving larger cohorts of individuals. Preliminary results of a pilot TBSeq experiment recapitulate the findings obtained with WGBS setting the basis for the development of an epigenetic clock assay. The results of this study will allow us to leverage the unique resources available forto study how DNA methylation relates to other hallmarks of ageing.
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ArticleDeep learning is widely applicable to phenotyping embryonic development and disease(The Company of Biologists, 2021-11-05) Naert, Thomas ; Çiçek, Özgün ; Ogar, Paulina ; Bürgi, Max ; Shaidani, Nikko-Ideen ; Kaminski, Michael M. ; Xu, Yuxiao ; Grand, Kelli ; Vujanovic, Marko ; Prata, Daniel ; Hildebrandt, Friedhelm ; Brox, Thomas ; Ronneberger, Olaf ; Voigt, Fabian F. ; Helmchen, Fritjof ; Loffing, Johannes ; Horb, Marko E. ; Rankin Willsey, Helen ; Lienkamp, Soeren S.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.
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ArticleDeveloping immortal cell lines from Xenopus embryos, four novel cell lines derived from Xenopus tropicalis(The Royal Society, 2022-07-06) Gorbsky, Gary J. ; Daum, John R. ; Sapkota, Hem ; Summala, Katja ; Yoshida, Hitoshi ; Georgescu, Constantin ; Wren, Jonathan D. ; Peshkin, Leonid ; Horb, Marko E.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 CO2 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.
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ArticleMaternal Wnt11b regulates cortical rotation during Xenopus axis formation: analysis of maternal-effect wnt11b mutants(The Company of Biologists, 2022-09-08) Houston, Douglas W. ; Elliott, Karen L. ; Coppenrath, Kelsey ; Wlizla, Marcin ; Horb, Marko E.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.
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PreprintMicroarray analysis of Xenopus endoderm expressing Ptf1a( 2012-07) Bilogan, Cassandra K. ; Horb, Marko E.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 paper, 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.
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ArticleAn optimized method for cryogenic storage of Xenopus sperm to maximise the effectiveness of research using genetically altered frogs(Elsevier, 2017-01-17) Pearl, Esther J. ; Morrow, Sean ; Noble, Anna ; Lerebours, Adelaide ; Horb, Marko E. ; Guille, MatthewCryogenic storage of sperm from genetically altered Xenopus improves cost effectiveness and animal welfare associated with their use in research; currently it is routine for X. tropicalis but not reliable for X. laevis. Here we compare directly the three published protocols for Xenopus sperm freeze-thaw and determine whether sperm storage temperature, method of testes maceration and delays in the freezing protocols affect successful fertilisation and embryo development in X. laevis. We conclude that the protocol is robust and that the variability observed in fertilisation rates is due to differences between individuals. We show that the embryos made from the frozen-thawed sperm are normal and that the adults they develop into are reproductively indistinguishable from others in the colony. This opens the way for using cryopreserved sperm to distribute dominant genetically altered (GA) lines, potentially saving travel-induced stress to the male frogs, reducing their numbers used and making Xenopus experiments more cost effective.
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ArticleTransgenic Xenopus laevis line for in vivo labeling of nephrons within the kidney(MDPI AG, 2018-04-06) Corkins, Mark E. ; Hanania, Hannah L. ; Krneta-Stankic, Vanja ; DeLay, Bridget D. ; Pearl, Esther J. ; Lee, Moonsup ; Ji, Hong ; Davidson, Alan J. ; Horb, Marko E. ; Miller, Rachel K.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.
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PreprintXenopus as a model for GI/pancreas disease( 2015-03) Salanga, Matthew C. ; Horb, Marko E.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.