Search
Close this search box.

Establishment and characterization of turtle liver organoids provides a potential model to decode their unique adaptations – Communications Biology

  • Rhodin, A. G. J. et al. Global Conservation Status of Turtles and Tortoises (Order Testudines). Chelonian Conserv. Biol. 17, 135–161 (2018).

  • Lovich, J. E., Ennen, J. R., Agha, M. & Whitfield Gibbons, J. Where have all the turtles gone, and why does it matter. BioScience 68, 771–781 (2018).


    Google Scholar
     

  • Valenzuela, N. The Painted Turtle, Chrysemys picta: A Model System for Vertebrate Evolution, Ecology, and Human Health. Cold Spring Harb. Protoc. 2009, pdb.emo124 (2009).

    PubMed 

    Google Scholar
     

  • Chaousis, S., Leusch, F. D. L., Nouwens, A., Melvin, S. D. & van de Merwe, J. P. Changes in global protein expression in sea turtle cells exposed to common contaminants indicates new biomarkers of chemical exposure. Sci. Total Environ. 751, 141680 (2021).

    ADS 
    CAS 
    PubMed 

    Google Scholar
     

  • Bista, B. & Valenzuela, N. Turtle insights into the evolution of the reptilian karyotype and the genomic architecture of sex determination. Genes 11, 416 (2020).

    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Congdon, J. D., Buhlmann, K. A. & Gibbons, J. W. Comparing Life Histories of the Shortest-Lived Turtle Known (Chicken Turtles, Deirochelys reticularia) with Long-Lived Blanding’s Turtles (Emydoidea blandingii). Chelonian Conserv. Biol. 21, 28–36 (2022).


    Google Scholar
     

  • Lee, L. S. et al. Karyotypic evolution of sauropsid vertebrates illuminated by optical and physical mapping of the painted turtle and slider turtle genomes. Genes 11, 1–20 (2020).


    Google Scholar
     

  • Mizoguchi, B., Topping, N. E., Lavin, A. M. & Valenzuela, N. Cadmium Ecotoxic Effects on Embryonic Dmrt1 and Aromatase Expression in Chrysemys picta Turtles May Implicate Changes in DNA Methylation. Genes 13, 1318 (2022).

    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Montiel, E. E. et al. Cytogenetic Insights into the Evolution of Chromosomes and Sex Determination Reveal Striking Homology of Turtle Sex Chromosomes to Amphibian Autosomes. Cytogenet Genome Res. 148, 292–304 (2016).

    CAS 
    PubMed 

    Google Scholar
     

  • Sabath, N. et al. Sex determination, longevity, and the birth and death of reptilian species. Ecol. Evol. 6, 5207–5220 (2016).

    PubMed 
    PubMed Central 

    Google Scholar
     

  • Thépot, D. Sex chromosomes and master sex-determining genes in turtles and other reptiles. Genes 12, 1822 (2021). volPreprint at.

    PubMed 
    PubMed Central 

    Google Scholar
     

  • Rasys, A. M. et al. CRISPR-Cas9 Gene Editing in Lizards through Microinjection of Unfertilized Oocytes. Cell Rep. 28, 2288–2292.e3 (2019).

    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Lozito, T. P., Londono, R., Sun, A. X. & Hudnall, M. L. Introducing dorsoventral patterning in adult regenerating lizard tails with gene-edited embryonic neural stem cells. Nat. Commun. 12, 6010 (2021).

    ADS 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Sato, T. et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009).

    ADS 
    CAS 
    PubMed 

    Google Scholar
     

  • Lehmann, R. et al. Human organoids: A new dimension in cell biology. Mol. Biol. Cell 30, 1129–1137 (2019).

    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • De Souza, N. Organoids. Nat. Methods 15, 23 (2018).


    Google Scholar
     

  • Fatehullah, A., Tan, S. H. & Barker, N. Organoids as an in vitro model of human development and disease. Nat. Cell Biol. 18, 246–254 (2016).

    PubMed 

    Google Scholar
     

  • Gabriel, V. et al. Standardization and Maintenance of 3D Canine Hepatic and Intestinal Organoid Cultures for Use in Biomedical Research. J. Vis. Exp. 1–28 https://doi.org/10.3791/63515 (2022).

  • Ambrosini, Y. M. et al. Recapitulation of the accessible interface of biopsy-derived canine intestinal organoids to study epithelial-luminal interactions. PLoS One 15, 1–17 (2020).


    Google Scholar
     

  • Chandra, L. et al. Derivation of adult canine intestinal organoids for translational research in gastroenterology. BMC Biol. 17, 33 (2019).

    PubMed 
    PubMed Central 

    Google Scholar
     

  • Minkler, S. et al. Emerging roles of urine-derived components for the management of bladder cancer: One man’s trash is another man’s treasure. Cancers 13, 1–13 (2021).


    Google Scholar
     

  • Mochel, J. P. et al. Intestinal Stem Cells to Advance Drug Development, Precision, and Regenerative Medicine: A Paradigm Shift in Translational Research. AAPS J. 20, 1–17 (2018).

    CAS 

    Google Scholar
     

  • Nantasanti, S. et al. Disease modeling and gene therapy of copper storage disease in canine hepatic organoids. Stem Cell Rep. 5, 895–907 (2015).

    CAS 

    Google Scholar
     

  • Penning, L. C. & van den Boom, R. Companion animal organoid technology to advance veterinary regenerative medicine. Front Vet. Sci. 10, 1032835 (2023).

    PubMed 
    PubMed Central 

    Google Scholar
     

  • Shoji, J. Y., Davis, R. P., Mummery, C. L. & Krauss, S. Global Meta-Analysis of Organoid and Organ-on-Chip Research. Adv. Healthc. Mater. 21, e2301067 (2023).


    Google Scholar
     

  • Chen, B., Slocombe, R. F. & Georgy, S. R. Advances in organoid technology for veterinary disease modeling. Front. Vet. Sci. 10, 1234628 (2023).

    PubMed 
    PubMed Central 

    Google Scholar
     

  • Zhao, D. et al. From crypts to enteroids: establishment and characterization of avian intestinal organoids. Poult. Sci. 101, 101642 (2022).

    CAS 
    PubMed 

    Google Scholar
     

  • Post, Y. et al. Snake Venom Gland Organoids. Cell 180, 233–247.e21 (2020).

    CAS 
    PubMed 

    Google Scholar
     

  • Storey, K. B. & Storey, J. M. Molecular physiology of freeze tolerance in vertebrates. Physiol. Rev. 97, 623–665 (2017).

    CAS 
    PubMed 

    Google Scholar
     

  • Bradley Shaffer, H. et al. The western painted turtle genome, a model for the evolution of extreme physiological adaptations in a slowly evolving lineage. Genome Biol. 14, R28 (2013).

    PubMed 
    PubMed Central 

    Google Scholar
     

  • Krivoruchko, A. & Storey, K. B. Turtle anoxia tolerance: Biochemistry and gene regulation. Biochim Biophys. Acta Gen. Subj. 1850, 1188–1196 (2015).

    CAS 

    Google Scholar
     

  • Dinkelacker, S. A., Costanzo, J. P. & Lee, R. E. Anoxia tolerance and freeze tolerance in hatchling turtles. J. Comp. Physiol. B 175, 209–217 (2005).

    CAS 
    PubMed 

    Google Scholar
     

  • Storey, K. B. Reptile freeze tolerance: Metabolism and gene expression. Cryobiology 52, 1–16 (2006).

    CAS 
    PubMed 

    Google Scholar
     

  • Roth, K. J. & Copple, B. L. Role of Hypoxia-Inducible Factors in the Development of Liver Fibrosis. CMGH 1, 589–597 (2015). volPreprint at.

    PubMed 
    PubMed Central 

    Google Scholar
     

  • Moon, J.-O., Welch, T. P., Gonzalez, F. J. & Copple, B. L. Reduced liver fibrosis in hypoxia-inducible factor-1-deficient mice. Am. J. Physiol. Gastrointest. Liver Physiol. 296, 582–592 (2009).


    Google Scholar
     

  • Kaltenmeier, C. et al. Role of Immuno-Inflammatory Signals in Liver Ischemia-Reperfusion Injury. Cells 11, 2222 (2022).

    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Jia, J. et al. A systematic review and meta-analysis of machine perfusion vs. Static cold storage of liver allografts on liver transplantation outcomes: The future direction of graft preservation. Front. Med. 7, 135 (2020).


    Google Scholar
     

  • Huelsz-Prince, G., Devries, A. L., Bakker, H. J., Zon, J. S. V. & Meister, K. Effect of antifreeze glycoproteins on organoid survival during and after hypothermic storage. Biomolecules 9, 1–9 (2019).


    Google Scholar
     

  • Hofer, M. & Lutolf, M. P. Engineering organoids. Nat. Rev. Mater. 6, 402–420 (2021).

    ADS 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Choi, J. H. & Ro, J. Y. The Recent Advances in Molecular Diagnosis of Soft Tissue Tumors. Int. J. Mol. Sci. 24, 5934 (2023).

    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Moura, L. R. et al. Morphological aspects of the liver of the freshwater turtle phrynops geoffroanus schweigger, 1812 (testudines, Chelidae). Braz. J. Morphol. Sci. 26, 129–134 (2009).

  • Rogers, A. B. & Dintzis, R. Z. 13 – Hepatobiliary System. In Comparative Anatomy and Histology (Second Edition) (eds. Treuting, P. M., Dintzis, S. M. & Montine, K. S.) 229–239 (Academic Press, 2018), https://doi.org/10.1016/B978-0-12-802900-8.00013-0.

  • Knotkova, Z., Dorrestein, G. M., Jekl, V., Janouskova, J. & Knotek, Z. Fasting and postprandial serum bile acid concentrations in 10 healthy female red-eared terrapins (Trachemys scripta elegans). Vet. Rec. 163, 510–514 (2008).

    CAS 
    PubMed 

    Google Scholar
     

  • Stremmel, W., Lukasova, M. & Weiskirchen, R. The neglected biliary mucus and its phosphatidylcholine content: a putative player in pathogenesis of primary cholangitis—a narrative review article. Ann. Transl. Med. 9, 738–738 (2021).

    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Zarei, K. et al. Early pathogenesis of cystic fibrosis gallbladder disease in a porcine model. Lab. Investig. 100, 1388–1399 (2020).

    CAS 
    PubMed 

    Google Scholar
     

  • Meyerholz, D. K., Stoltz, D. A., Pezzulo, A. A. & Welsh, M. J. Pathology of gastrointestinal organs in a porcine model of cystic fibrosis. Am. J. Pathol. 176, 1377–1389 (2010).

    PubMed 
    PubMed Central 

    Google Scholar
     

  • Zarei, K., Meyerholz, D. K. & Stoltz, D. A. Early intrahepatic duct defects in a cystic fibrosis porcine model. Physiol. Rep. 9, e14978 (2021).

    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Johnson, J. C. et al. Characterization of Testudine Melanomacrophage Linear, Membrane Extension Processes: Cablepodia: By Phase and Atomic Force Microscopy. Vitr. Cell Dev. Biol. Anim. 41, 225–231 (2005).


    Google Scholar
     

  • Das, D. et al. Draft genome of the common snapping turtle, chelydra serpentina, a model for phenotypic plasticity in reptiles. G3 Genes Genomes Genet. 10, 4299–4314 (2020).

    CAS 

    Google Scholar
     

  • Henderson, S. J., Weitz, J. I. & Kim, P. Y. Fibrinolysis: strategies to enhance the treatment of acute ischemic stroke. J. Thrombosis Haemost. 16, 1932–1940 (2018).

    CAS 

    Google Scholar
     

  • Myrka, A. & Buck, L. Cytoskeletal arrest: An anoxia tolerance mechanism. Metabolites 11, 561 (2021). volPreprint at.

    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Franko, A. et al. Identification of the secreted proteins originated from primary human hepatocytes and HepG2 cells. Nutrients 11, 1795 (2019).

    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Zhang, W., Niu, C., Chen, B. & Storey, K. B. Digital Gene Expression Profiling reveals transcriptional responses to acute cold stress in Chinese soft-shelled turtle Pelodiscus sinensis juveniles. Cryobiology 81, 43–56 (2018).

    CAS 
    PubMed 

    Google Scholar
     

  • Hassan, F. M. et al. Identification of RPL5 gene variants and the risk of hepatic vein thrombosis in Saudi patients. Saudi Med. J. 42, 969–974 (2021).

    PubMed 
    PubMed Central 

    Google Scholar
     

  • Keenan, S. W., Hill, C. A., Kandoth, C., Buck, L. T. & Warren, D. E. Transcriptomic responses of the heart and brain to anoxia in the western painted turtle. PLoS One 10, e0131669 (2015).

    PubMed 
    PubMed Central 

    Google Scholar
     

  • Kondo, R. et al. Downregulation of protein disulfide-isomerase A3 expression inhibits cell proliferation and induces apoptosis through STAT3 signaling in hepatocellular carcinoma. Int J. Oncol. 54, 1409–1421 (2019).

    CAS 
    PubMed 

    Google Scholar
     

  • Wei, F. Y. & Tomizawa, K. tRNA modifications and islet function. Diabetes Obes. Metab. 20, 20–27 (2018).

    CAS 
    PubMed 

    Google Scholar
     

  • Yang, S. et al. Parallel comparative proteomics and phosphoproteomics reveal that cattle myostatin regulates phosphorylation of key enzymes in glycogen metabolism and glycolysis pathway. Oncotarget 9, 11352–11370 (2018).

    PubMed 
    PubMed Central 

    Google Scholar
     

  • Yang, W. et al. Phosphorylase kinase β represents a novel prognostic biomarker and inhibits malignant phenotypes of liver cancer cell. Int J. Biol. Sci. 15, 2596–2606 (2019).

    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • MacParland, S. A. et al. Single cell RNA sequencing of human liver reveals distinct intrahepatic macrophage populations. Nat. Commun. 9, 4383 (2018).

    ADS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Racapé, M. et al. The involvement of SMILE/TMTC3 in endoplasmic reticulum stress response. PLoS One 6, e19321 (2011).

    ADS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Takahashi, A. et al. The CCR4-NOT complex maintains liver homeostasis through mRNA deadenylation. Life Sci. Alliance 3, e201900494 (2020).

    PubMed 
    PubMed Central 

    Google Scholar
     

  • Charlton, M. R. Protein metabolism and liver disease. Baillieres Clin. Endocrinol. Metab. 10, 617–635 (1996).

    CAS 
    PubMed 

    Google Scholar
     

  • Sinturel, F. et al. Diurnal Oscillations in Liver Mass and Cell Size Accompany Ribosome Assembly Cycles. Cell 169, 651–663.e14 (2017).

    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Mello, A. et al. Soluble Epoxide Hydrolase Hepatic Deficiency Ameliorates Alcohol-Associated Liver Disease. CMGH 11, 815–830 (2021).

    CAS 
    PubMed 

    Google Scholar
     

  • Kim, J., Koo, B. K. & Knoblich, J. A. Human organoids: model systems for human biology and medicine. Nat. Rev. Mol. Cell Biol. 21, 571–584 (2020).

    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Cornet, C., Di Donato, V. & Terriente, J. Combining Zebrafish and CRISPR/Cas9: Toward a more efficient drug discovery pipeline. Front. Pharmacol. 9, 703 (2018).

    PubMed 
    PubMed Central 

    Google Scholar
     

  • Russell, J. J. et al. Non-model model organisms. BMC Biol. 15, 55 (2017).

    PubMed 
    PubMed Central 

    Google Scholar
     

  • Alföldi, J. et al. The genome of the green anole lizard and a comparative analysis with birds and mammals. Nature 477, 587–591 (2011).

    ADS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Janes, D. E., Organ, C. & Valenzuela, N. New resources inform study of genome size, content, and organization in nonavian reptiles. Integr. Comp. Biol. 48, 447–453 (2008).

    PubMed 
    PubMed Central 

    Google Scholar
     

  • Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

    ADS 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • WareJoncas, Z. et al. Precision gene editing technology and applications in nephrology. Nat. Rev. Nephrol. 14, 663–677 (2018).

    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Rhie, A. et al. Towards complete and error-free genome assemblies of all vertebrate species. Nature 592, 737–746 (2021).

    ADS 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Bista, B., Wu, Z., Literman, R. & Valenzuela, N. Thermosensitive sex chromosome dosage compensation in ZZ/ZW softshell turtles, Apalone spinifera. Philos. Trans. R. Soc. B Biol. Sci. 376, 20200101 (2021).

    CAS 

    Google Scholar
     

  • Huch, M. et al. In vitro expansion of single Lgr5 + liver stem cells induced by Wnt-driven regeneration. Nature 494, 247–250 (2013).

    ADS 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Huch, M. et al. Long-term culture of genome-stable bipotent stem cells from adult human liver. Cell 160, 299–312 (2015).

    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Tomofuji, K. et al. Liver ductal organoids reconstruct intrahepatic biliary trees in decellularized liver grafts. Biomaterials 287, 121614 (2022).

    CAS 
    PubMed 

    Google Scholar
     

  • Gebril, M. et al. Bio-modulated mice epithelial endometrial organoids by low-level laser therapy serves as an invitro model for endometrial regeneration. Reprod. Biol. 21, 100564 (2021).

    PubMed 

    Google Scholar
     

  • Ye, S. et al. A Chemically Defined Hydrogel for Human Liver Organoid Culture. Adv. Funct. Mater. 30, 200089 (2020).


    Google Scholar
     

  • Schweigger, P. & Simone, D. Morphological aspects of the liver of the freshwater turtle. J. Morphological Sci. 1812, 129–134 (2005).

  • Chakrabarti, J., Koh, V., So, J. B. Y., Yong, W. P. & Zavros, Y. A Preclinical Human-Derived Autologous Gastric Cancer Organoid/Immune Cell Co-Culture Model to Predict the Efficacy of Targeted Therapies. J. Vis. Exp. 2021, https://doi.org/10.3791/61443 (2021).

  • Kasprzak, A. & Adamek, A. Mucins: The old, the new and the promising factors in hepatobiliary carcinogenesis. Int. J. Mol. Sci. 20, https://doi.org/10.3390/ijms20061288 (2019).

  • Andrews, T. S. et al. Single-Cell, Single-Nucleus, and Spatial RNA Sequencing of the Human Liver Identifies Cholangiocyte and Mesenchymal Heterogeneity. Hepatol. Commun. 6, 2022 (2021).


    Google Scholar
     

  • Aktas, R. G. et al. Long-Term Characteristics of Human-Derived Biliary Organoids under a Single Continuous Culture Condition. Cells 11, 3797 (2022).

    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Haque, A., Guild, J. & Revzin, A. Chapter 36 – Designing Stem Cell Niche for Liver Development and Regeneration. in Biology and Engineering of Stem Cell Niches (eds. Vishwakarma, A. & Karp, J. M.) 581–600 (Academic Press), https://doi.org/10.1016/B978-0-12-802734-9.00036-6 (2017)

  • Fanter, C. E. et al. Development-specific transcriptomic profiling suggests new mechanisms for anoxic survival in the ventricle of overwintering turtles. J. Exp. Biol. 223, jeb213918 (2020).

    PubMed 

    Google Scholar
     

  • Gessler, T. B., Wu, Z. & Valenzuela, N. Transcriptomic thermal plasticity underlying gonadal development in a turtle with ZZ/ZW sex chromosomes despite canalized genotypic sex determination. Ecol. Evol. 13, e9854 (2023).

    PubMed 
    PubMed Central 

    Google Scholar
     

  • Czerwinski, M., Natarajan, A., Barske, L., Looger, L. L. & Capel, B. A timecourse analysis of systemic and gonadal effects of temperature on sexual development of the red-eared slider turtle Trachemys scripta elegans. Dev. Biol. 420, 166–177 (2016).

    CAS 
    PubMed 

    Google Scholar
     

  • Alderman, S. L., Riggs, C. L., Bullingham, O. M. N., Gillis, T. E. & Warren, D. E. Cold acclimation induces life stage-specific responses in the cardiac proteome of western painted turtles (Chrysemys picta bellii): Implications for anoxia tolerance. J. Exp. Biol. 224, jeb242387 (2021).

    PubMed 

    Google Scholar
     

  • Radhakrishnan, S., Literman, R., Neuwald, J., Severin, A. & Valenzuela, N. Transcriptomic responses to environmental temperature by turtles with temperaturedependent and genotypic sex determination assessed by RNAseq inform the genetic architecture of embryonic gonadal development. PLoS One 12, e017204 (2017).


    Google Scholar
     

  • Shiota, J., Zaki, N. H. M., Merchant, J. L., Samuelson, L. C. & Razumilava, N. Generation of Organoids from Mouse Extrahepatic Bile Ducts. J. Vis. Exper., https://doi.org/10.3791/59544 (2019).

  • Tysoe, O. C. et al. Isolation and propagation of primary human cholangiocyte organoids for the generation of bioengineered biliary tissue. Nat. Protoc. 14, 1884–1925 (2019).

    CAS 
    PubMed 

    Google Scholar
     

  • Bronikowski, A. M. et al. Sex-specific aging in animals: Perspective and future directions. Aging Cell 21, e13542 (2022). volPreprint at.

    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Bartlett, A. P., Harman, R. M., Weiss, J. R. & Van de Walle, G. R. Establishment and characterization of equine mammary organoids using a method translatable to other non-traditional model species. Development 149, dev200412 (2022).

    CAS 
    PubMed 

    Google Scholar
     

  • Sage, R. F. Global change biology: A primer. Glob. Change Biol. 26, 3–30 (2020).

    ADS 

    Google Scholar
     

  • Laloë, J. O., Cozens, J., Renom, B., Taxonera, A. & Hays, G. C. Climate change and temperature-linked hatchling mortality at a globally important sea turtle nesting site. Glob. Chang Biol. 23, 4922–4931 (2017).

    ADS 
    PubMed 

    Google Scholar
     

  • Jensen, M. P. et al. Environmental Warming and Feminization of One of the Largest Sea Turtle Populations in the World. Curr. Biol. 28, 154–159.e4 (2018).

    CAS 
    PubMed 

    Google Scholar
     

  • Hawkes, L. A., Broderick, A. C., Godfrey, M. H. & Godley, B. J. Investigating the potential impacts of climate change on a marine turtle population. Glob. Chang. Biol. 13, 923–932 (2007).

  • Merchant-Larios, H., Díaz-Hernández, V. & Cortez, D. Molecular and Cellular Mechanisms Underlying Temperature-Dependent Sex Determination in Turtles. Sex. Dev. 15, 38–46 (2021).

    CAS 
    PubMed 

    Google Scholar
     

  • Badenhorst, D., Stanyon, R., Engstrom, T. & Valenzuela, N. A ZZ/ZW microchromosome system in the spiny softshell turtle, Apalone spinifera, reveals an intriguing sex chromosome conservation in Trionychidae. Chromosome Res. 21, 137–147 (2013).

    CAS 
    PubMed 

    Google Scholar
     

  • Shaw, R. C., Greggor, A. L. & Plotnik, J. M. The Challenges of Replicating Research on Endangered Species. Anim. Behav. Cogn. 8, 240–246 (2021).


    Google Scholar
     

  • Russell, W. M. S. & Burch, R. L. The principles of humane experimental technique (Methuen, 1959).

  • Forsythe, S. D. et al. Environmental toxin screening using human-derived 3D bioengineered liver and cardiac organoids. Front Public Health 6, 103 (2018).

    PubMed 
    PubMed Central 

    Google Scholar
     

  • Votanopoulos, K. I. et al. Model of Patient-Specific Immune-Enhanced Organoids for Immunotherapy Screening: Feasibility Study. Ann. Surg. Oncol. 27, 1956–1967 (2020).

    PubMed 

    Google Scholar
     

  • Huo, J., Dong, A., Yan, J. & Dong, A. Effects of cadmium on the activities of ALT and AST as well as the content of TP in plasma of freshwater turtle Mauremys reevesii. Environ. Sci. Pollut. Res. 27, 18025–18028 (2020).

    CAS 

    Google Scholar
     

  • Huo, J., Dong, A., Yan, J. & Dong, A. Effects of cadmium on the gene transcription of the liver in the freshwater turtle (Chinemys reevesii). Environ. Sci. Pollut. Res. 27, 8431–8438 (2020).

    CAS 

    Google Scholar
     

  • Valenzuela, N., Badenhorst, D., Montiel, E. E. & Literman, R. Molecular cytogenetic search for cryptic sex chromosomes in painted turtles Chrysemys picta. Cytogenet Genome Res. 144, 39–46 (2014).

    PubMed 

    Google Scholar
     

  • Yntema, C. L. A series of stages in the embryonic development of Chelydra serpentina. J. Morphol. 125, 219–251 (1968).

    CAS 
    PubMed 

    Google Scholar
     

  • Ewert, M. A., Jackson, D. R. & Nelson, C. E. Patterns of temperature-dependent sex determination in turtles. J. Exp. Zool. 270, 3–15 (1994).


    Google Scholar
     

  • Bull, J. J. & Vogt, R. C. Temperature-Dependent Sex Determination in Turtles. Science 206, 1186–1188 (1979).

    ADS 
    CAS 
    PubMed 

    Google Scholar
     

  • Literman, R. et al. Development of sexing primers in Glyptemys insculpta and Apalone spinifera turtles uncovers an XX/XY sex-determining system in the critically-endangered bog turtle Glyptemys muhlenbergii. Conserv Genet Resour. 9, 651–658 (2017).


    Google Scholar
     

  • Literman, R., Badenhorst, D. & Valenzuela, N. qPCR-based molecular sexing by copy number variation in rRNA genes and its utility for sex identification in soft-shell turtles. Methods Ecol. Evol. 5, 872–880 (2014).


    Google Scholar
     

  • Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).

    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Andrews, S. FastQC: a quality control tool for high throughput sequence data. https://scholar.google.com/scholar?cluster=6536267464756235244&hl=en&as_sdt=80005&sciodt=0,11 (2010).

  • Wu, T. D. & Watanabe, C. K. GMAP: A genomic mapping and alignment program for mRNA and EST sequences. Bioinformatics 21, 1859–1875 (2005).

    CAS 
    PubMed 

    Google Scholar
     

  • Wu, T. D. & Nacu, S. Fast and SNP-tolerant detection of complex variants and splicing in short reads. Bioinformatics 26, 873–881 (2010).

    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    PubMed 
    PubMed Central 

    Google Scholar
     

  • Pertea, M. et al. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat. Biotechnol. 33, 290–295 (2015).

    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

    PubMed 
    PubMed Central 

    Google Scholar
     

  • R Core Team, Rf. R: A language and environment for statistical computing. https://ropensci.org/blog/2021/11/16/how-to-cite-r-and-r-packages/ (2018).

  • Camacho, C. et al. BLAST+: Architecture and applications. BMC Bioinforma. 10, 421 (2009).


    Google Scholar
     

  • Consortium, T. U. UniProt: a worldwide hub of protein knowledge. Nucleic Acids Res. 47, D506–D515 (2019).


    Google Scholar
     

  • Pertea, M. & Pertea, G. GFF Utilities: GffRead and GffCompare. F1000Res. 9, ISCB Comm J-304 (2020).

  • Thomas, P. D. et al. PANTHER: Making genome-scale phylogenetics accessible to all. Protein Sci. 31, 8–22 (2022).

    CAS 
    PubMed 

    Google Scholar
     

  • Mi, H. et al. Protocol Update for large-scale genome and gene function analysis with the PANTHER classification system (v.14.0). Nat. Protoc. 14, 703–721 (2019).

    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Soneson, C., Love, M. I. & Robinson, M. D. Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences. F1000Res. 4, 1521 (2015).

    PubMed 

    Google Scholar
     

  • Ashburner, M. et al. Gene Ontology: tool for the unification of biology. Nat. Genet. 25, 25–29 (2000).

    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Carbon, S. et al. The Gene Ontology resource: Enriching a GOld mine. Nucleic Acids Res. 49, D325–D334 (2021).

    CAS 

    Google Scholar
     

  • Supek, F., Bošnjak, M., Škunca, N. & Šmuc, T. Revigo summarizes and visualizes long lists of gene ontology terms. PLoS One 6, e21800 (2011).

    ADS 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Tennekes, M. & Ellis, P. treemap: Treemap visualization. R. Package Version 2, 4 (2017).


    Google Scholar
     

  • Zheng, G. X. Y. et al. Massively parallel digital transcriptional profiling of single cells. Nat. Commun. 8, 14049 (2017).

    ADS 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Hao, Y. et al. Integrated analysis of multimodal single-cell data. Cell 184, 3573–3587.e29 (2021).

    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Wiśniewski, J. R., Zougman, A., Nagaraj, N. & Mann, M. Universal sample preparation method for proteome analysis. Nat. Methods 6, 359–362 (2009).

    PubMed 

    Google Scholar
     

  • Masuda, T., Tomita, M. & Ishihama, Y. Phase transfer surfactant-aided trypsin digestion for membrane proteome analysis. J. Proteome Res. 7, 731–740 (2008).

    CAS 
    PubMed 

    Google Scholar
     

  • Orsburn, B. C. Proteome discoverer-a community enhanced data processing suite for protein informatics. Proteomes 9, 15 (2021).

    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Ison, J. et al. Tools and data services registry: A community effort to document bioinformatics resources. Nucleic Acids Res. 44, D38–D47 (2016).

    CAS 
    PubMed 

    Google Scholar
     

  • Valenzuela, N. ValenzuelaLab/ZdyrskiEtAl2023_CommBiol_TurtleLiver Organoids: Code_ZdyrskiEtAl2023_CommBiol_TurtleLiverOrganoids. https://doi.org/10.5281/zenodo.10359908 (2023).