Piebaldism and chromatophore development in reptiles are linked to the tfec gene

Abstract

Reptiles display great diversity in color and pattern, yet much of what we know about vertebrate coloration comes from classic model species such as the mouse and zebrafish.^1,2,3,4 Captive-bred ball pythons (Python regius) exhibit a remarkable degree of color and pattern variation. Despite the wide range of Mendelian color phenotypes available in the pet trade, ball pythons remain an overlooked species in pigmentation research. Here, we investigate the genetic basis of the recessive piebald phenotype, a pattern defect characterized by patches of unpigmented skin (leucoderma). We performed whole-genome sequencing and used a case-control approach to discover a nonsense mutation in the gene encoding the transcription factor tfec, implicating this gene in the leucodermic patches in ball pythons. We functionally validated tfec in a lizard model (Anolis sagrei) using the gene editing CRISPR/Cas9 system and TEM imaging of skin. Our findings show that reading frame mutations in tfec affect coloration and lead to a loss of iridophores in Anolis, indicating that tfec is required for chromatophore development. This study highlights the value of captive-bred ball pythons as a model species for accelerating discoveries on the genetic basis of vertebrate coloration.

Keywords: Mendelian phenotype; ball python; color morph; genomics; pigmentation.

Figure 1.. A small sample of the phenotypic variation found in captive-bred ball pythons (Python regius)

(A) Wild type, (B) piebald, (C) banana piebald, (D) pastel piebald, (E) pastel HRA enhancer, (F) ultramel clown, and (G) banana champagne. Photo credit: pethelpful.com (A) and Designing Morphs (B–G).

Figure 2.. Inheritance patterns and genomic differentiation

(A) Clutch records (2008–2018) from a commercial breeder (KINOVA) indicate piebald has a recessive mode of inheritance. (B) F_ST plot between piebald and non-piebald samples using a chromosome-length genome assembly. The F_ST peak on chromosome 7 delineates the region of interest containing the putative causal gene for the piebald phenotype.

Figure 3.. Phenotypic comparisons of Anolis sagrei

Wild type (A) and F0 tfec mutant (B). The mutant showed reduced body coloration, particularly in the snout, forelimbs, and hindlimbs (arrows).

Figure 4.. tfec is required for iridophore development in Anolis sagrei

Presented are eye and skin samples from wild type (A, D, G, and J) and mutants with reading frame mutations in tyr (B, E, H, and K) and tfec (C, F, I, and L). (A–C) Anterior view of hatchling eyes. (D–F) Dissected skin from the trunk of hatchlings. For these panels, anterior surface is up, and the posterior surface is down. Ventral surface is on the left side of the image and the dorsal surface is on the right. The dorsal stripe can be seen in (D) and (E) while (F) exhibits a lack of this back pattern. (G–L) TEM images of individual dorsal scales (G–I) and higher-magnification images of melanophores and iridophores (J–L). Melanophores hold pigmented melanosomes while iridophore reflectiveness arises from guanine crystals. For tyr^−/− note the absence of melanosomes and the presence of guanine crystals. For tfec^−/− note the presence of melanosomes and the absence of guanine crystals. tyr samples are from F2 lizards; tfec samples are from F1 lizards. Asterisks show melanosomes while arrowheads point to guanine crystals. Scale bars, (A–F) 500 μm, (G–I) 6 μm, (J–L) 2 μm.