Squamates (snakes and lizards) exhibit a striking variety of phenotypes, with little known on their generative mechanisms. Studies aiming to understand the genetic basis of this wide diversity in morphology, physiology and ecology will greatly benefit from whole genome sequencing initiatives, as they provide the foundation for comparative analyses and improve our understanding of the evolution, development and diversification of traits. Here, we present the first draft genome of the corn snake Pantherophis guttatus, an oviparous snake that we promote as a particularly appropriate model species for evolutionary developmental studies in squamates. We sequenced 100-base paired-end reads from multiple individuals of a single family (parents and offspring) that produced a genome assembly of 1.53 gigabases (Gb), roughly covering 75% of the expected total genome size, and 297,768 scaffolds >1 Kb. We were able to fully retrieve 86, and partially another 106, of the 248 CEGMA core genes, indicating that a high genome completeness was achieved, even though the assembly is fragmented. Using MAKER2, we annotated 10,917 genes with high confidence (Annotation Edit Distance (AED)<1) and an additional 5,263 predicted genes matched with the species’ transcriptome. Numerous colour and colour pattern morphs exist in P. guttatus, making it an ideal model to study the genetic determinism, development, and evolution of adaptive colour traits in reptiles. Using our draft genome and a Single-Nucleotide Polymorphism (SNP) calling approach, we confirmed the interval with the causative mutation for the amelanistic phenotype, a result supported by a parallel exome-based study.

Significance: Reptiles exhibit a spectacular diversity of skin colors generated by interactions among black melanophores, red and yellow xanthophores, as well as iridophores producing structural colors. Here, we use the corn snake to investigate the generative mechanisms of skin colors beyond the zebrafish model. We perform sequencing and annotation of a nearly chromosome-quality genome of the corn snake, followed by mapping-by-sequencing and identification of a mutation in the lysosomal trafficking regulator gene ( LYST ) in the lavender variant with strongly affected coloration. Further analyses indicate that color-producing organelles of all chromatophores are substantially impacted in the LYST mutant, indicating that not only melanosomes, but also xanthosomes and iridosomes, are all lysosome-related organelles.

During reproduction, multiple species such as insects and all mammals undergo extensive physiological and morphological adaptions to ensure health and survival of the mother and optimal development of the offspring. Here we report that the intestinal epithelium undergoes expansion during pregnancy and lactation in mammals. This enlargement of the intestinal surface area results in a novel geometry of expanded villi. Receptor activator of nuclear factor-κΒ (RANK, encoded by TNFRSF11A) and its ligand RANKL were identified as a molecular pathway involved in this villous expansion of the small intestine in vivo in mice and in intestinal mouse and human organoids. Mechanistically, RANK–RANKL protects gut epithelial cells from cell death and controls the intestinal stem cell niche through BMP receptor signalling, resulting in the elongation of villi and a prominent increase in the intestinal surface. As a transgenerational consequence, babies born to female mice that lack Rank in the intestinal epithelium show reduced weight and develop glucose intolerance after metabolic stress. Whereas gut epithelial remodelling in pregnancy/lactation is reversible, constitutive expression of an active form of RANK is sufficient to drive intestinal expansion followed by loss of villi and stem cells, and prevents the formation of Apc^min-driven small intestinal stem cell tumours. These data identify RANK–RANKL as a pathway that drives intestinal epithelial expansion in pregnancy/lactation, one of the most elusive and fundamental tissue remodelling events in mammalian life history and evolution.

Despite the availability of deep-sequencing techniques, genomic and transcriptomic data remain unevenly distributed across phylogenetic groups. For example, reptiles are poorly represented in sequence databases, hindering functional evolutionary and developmental studies in these lineages substantially more diverse than mammals. In addition, different studies use different assembly and annotation protocols, inhibiting meaningful comparisons. Here, we present the “Reptilian Transcriptomes Database 2.0,” which provides extensive annotation of transcriptomes and genomes from species covering the major reptilian lineages. To this end, we sequenced normalized complementary DNA libraries of multiple adult tissues and various embryonic stages of the leopard gecko and the corn snake and gathered published reptilian sequence data sets from representatives of the four extant orders of reptiles: Squamata (snakes and lizards), the tuatara, crocodiles, and turtles. The LANE runner 2.0 software was implemented to annotate all assemblies within a single integrated pipeline. We show that this approach increases the annotation completeness of the assembled transcriptomes/genomes. We then built large concatenated protein alignments of single-copy genes and inferred phylogenetic trees that support the positions of turtles and the tuatara as sister groups of Archosauria and Squamata, respectively. The Reptilian Transcriptomes Database 2.0 resource will be updated to include selected new data sets as they become available, thus making it a reference for differential expression studies, comparative genomics and transcriptomics, linkage mapping, molecular ecology, and phylogenomic analyses involving reptiles. The database is available at www.reptilian-transcriptomes.org and can be enquired using a wwwblast server installed at the University of Geneva.

Organoids derived from human pluripotent stem (hPS) cells hold promise for therapeutic purposes. However, technological advances to overcome their massive production while ensuring differentiation fidelity are still lacking. Here we report a procedure sustaining the derivation of kidney organoids from hPS cells (hPSC-kidney organoids) using a scalable, reproducible and affordable approach that allows hPSC-kidney organoid differentiation into different renal cell types. Using single-cell RNA sequencing, confocal image analysis, metabolic assays and CRISPR–Cas9 engineering for generation of fluorescent reporters, we show that hPSC-kidney organoids exhibit transcriptional variety and cellular composition following cell-to-cell contact. We infuse human kidney organoids into ex vivo porcine kidneys using normothermic machine perfusion, and demonstrate in vivo engraftment of hPSC-kidney organoids. We further evaluate the immune response, confirming the feasibility and viability of the procedure. We identify cells of human origin after normothermic machine perfusion and in vivo transplantation by means of in situ hybridization, immunohistochemistry, confocal microscopy, image analysis and quantification, in vivo imaging, and flow cytometry. This work provides a foundation for using hPSC-kidney organoids for ex vivo cell-based therapies in clinical trials.

Corn snakes are emerging models for animal colouration studies. Here, we focus on the Terrazzo morph, whose skin pattern is characterized by stripes rather than blotches. Using genome mapping, we discover a disruptive mutation in the coding region of the Premelanosome protein (PMEL) gene. Our transcriptomic analyses reveal that PMEL expression is significantly downregulated in Terrazzo embryonic tissues. We produce corn snake PMEL knockouts, which present a comparable colouration phenotype to Terrazzo and the subcellular structure of their melanosomes and xanthosomes is also similarly impacted. Our single-cell expression analyses of wild-type embryonic dorsal skin demonstrate that all chromatophore progenitors express PMEL at varying levels. Finally, we show that in wild-type embryos PMEL-expressing cells are initially uniformly spread before forming aggregates and eventually blotches, as seen in the adults. In Terrazzo embryos, the aggregates fail to form. Our results provide insights into the mechanisms governing colouration patterning in reptiles.