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

Painted turtles are remarkable for their freeze tolerance and supercooling ability along with their associated resilience to hypoxia/anoxia and oxidative stress, rendering them an ideal biomedical model for hypoxia-induced injuries (including strokes), tissue cooling during surgeries, and organ cryopreservation. Yet, such research is hindered by their seasonal reproduction and slow maturation. Here we developed and characterized adult stem cell-derived turtle liver organoids (3D self-assembled in vitro structures) from painted, snapping, and spiny softshell turtles spanning ~175My of evolution, with a subset cryopreserved. This development is, to the best of our knowledge, a first for this vertebrate Order, and complements the only other non-avian reptile organoids from snake venom glands. Preliminary characterization, including morphological, transcriptomic, and proteomic analyses, revealed organoids enriched in cholangiocytes. Deriving organoids from distant turtles and life stages demonstrates that our techniques are broadly applicable to chelonians, permitting the development of functional genomic tools currently lacking in herpetological research. Such platform could potentially support studies including genome-to-phenome mapping, gene function, genome architecture, and adaptive responses to climate change, with implications for ecological, evolutionary, and biomedical research.

Transcriptomic thermal plasticity underlying gonadal development in a turtle with ZZ/ZW sex chromosomes despite canalized genotypic sex determination.

Understanding genome-wide responses to environmental conditions during embryogenesis is essential for discerning the evolution of developmental plasticity and canalization, two processes generating phenotypic variation targeted by natural selection. Here, we present the first comparative trajectory analysis of matched transcriptomic developmental time series from two reptiles incubated under identical conditions, a turtle with a ZZ/ZW system of genotypic sex determination (GSD), Apalone spinifera, and a turtle with temperature-dependent sex determination (TSD), Chrysemys picta. Results from our genome-wide, hypervariate gene expression analysis of sexed embryos across five developmental stages revealed that substantial transcriptional plasticity in the developing gonads can persist for >145 Myr, long after the canalization of sex determination via the evolution of sex chromosomes, while some gene-specific thermal sensitivity drifts or evolves anew. Such standing thermosensitivity represents an underappreciated evolutionary potential harbored by GSD species that may be co-opted during future adaptive shifts in developmental programing, such as a GSD to TSD reversal, if favored by ecological conditions. Additionally, we identified novel candidate regulators of vertebrate sexual development in GSD reptiles, including sex-determining candidate genes in a ZZ/ZW turtle.

Karyotypic Evolution of Sauropsid Vertebrates Illuminated by Optical and Physical Mapping of the Painted Turtle and Slider Turtle Genomes.

Recent sequencing and software enhancements have advanced our understanding of the evolution of genomic structure and function, especially addressing novel evolutionary biology questions. Yet fragmentary turtle genome assemblies remain a challenge to fully decipher the genetic architecture of adaptive evolution. Here, we use optical mapping to improve the contiguity of the painted turtle (Chrysemys picta) genome assembly and use de novo fluorescent in situ hybridization (FISH) of bacterial artificial chromosome (BAC) clones, BAC-FISH, to physically map the genomes of the painted and slider turtles (Trachemys scripta elegans). Optical mapping increased C. picta's N50 by ~242% compared to the previous assembly. Physical mapping permitted anchoring ~45% of the genome assembly, spanning 5544 genes (including 20 genes related to the sex determination network of turtles and vertebrates). BAC-FISH data revealed assembly errors in C. picta and T. s. elegans assemblies, highlighting the importance of molecular cytogenetic data to complement bioinformatic approaches. We also compared C. picta's anchored scaffolds to the genomes of other chelonians, chicken, lizards, and snake. Results revealed a mostly one-to-one correspondence between chromosomes of painted and slider turtles, and high homology among large syntenic blocks shared with other turtles and sauropsids. Yet, numerous chromosomal rearrangements were also evident across chelonians, between turtles and squamates, and between avian and non-avian reptiles.