Citation: Kabangu, M.; Cecil, R.; Strohl, L., II; Timoshevskaya, N.; Smith, J.J.; Voss, S.R. Leukocyte Tyrosine Kinase (Ltk) Is the Mendelian Determinant of the Axolotl Melanoid Color Variant. Genes 2023, 14, 904. https://doi.org/10.3390/ genes14040904 Academic Editor: Alexander V. Ereskovsky Received: 23 February 2023 Revised: 2 April 2023 Accepted: 10 April 2023 Published: 13 April 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). genes G C A T T A C G G C A T Article Leukocyte Tyrosine Kinase (Ltk) Is the Mendelian Determinant of the Axolotl Melanoid Color Variant Mirindi Kabangu 1,2 , Raissa Cecil 1 , Lloyd Strohl II 3 , Nataliya Timoshevskaya 4 , Jeramiah J. Smith 4 and Stephen R. Voss 1, * 1 Department of Neuroscience, Spinal Cord and Brain Injury Research Center, and Ambystoma Genetic Stock Center, University of Kentucky, Lexington, KY 40536, USA 2 Indiana University School of Medicine, Indianapolis, IN 46202, USA 3 Independent Researcher, Vevay, IN 47043, USA 4 Department of Biology, University of Kentucky, Lexington, KY 40506, USA * Correspondence: srvoss@uky.edu Abstract: The great diversity of color patterns observed among amphibians is largely explained by the differentiation of relatively few pigment cell types during development. Mexican axolotls present a variety of color phenotypes that span the continuum from leucistic to highly melanistic. The melanoid axolotl is a Mendelian variant characterized by large numbers of melanophores, proportionally fewer xanthophores, and no iridophores. Early studies of melanoid were influential in developing the single-origin hypothesis of pigment cell development, wherein it has been proposed that all three pigment cell types derive from a common progenitor cell, with pigment metabolites playing potential roles in directing the development of organelles that define different pigment cell types. Specifically, these studies identified xanthine dehydrogenase (XDH) activity as a mechanism for the permissive differentiation of melanophores at the expense of xanthophores and iridophores. We used bulked segregant RNA-Seq to screen the axolotl genome for melanoid candidate genes and identify the associated locus. Dissimilar frequencies of single-nucleotide polymorphisms were identified between pooled RNA samples of wild-type and melanoid siblings for a region on chromosome 14q. This region contains gephyrin (Gphn), an enzyme that catalyzes the synthesis of the molybdenum cofactor that is required for XDH activity, and leukocyte tyrosine kinase (Ltk), a cell surface signaling receptor that is required for iridophore differentiation in zebrafish. Wild-type Ltk crispants present similar pigment phenotypes to melanoid, strongly implicating Ltk as the melanoid locus. In concert with recent findings in zebrafish, our results support the idea of direct fate specification of pigment cells and, more generally, the single-origin hypothesis of pigment cell development. Keywords: genetic linkage analysis; axolotl; mutant; pigmentation; neural crest 1. Introduction The Mexican axolotl has a deep and colorful history. Thirty-three axolotls were famously imported from Mexico to Europe in 1863 to establish a population at the Jardin des Plantes in Paris [1]. Descendants from this population were distributed throughout the world to found laboratory populations that remain in existence today. All of the original Paris axolotls, except for one, presented approximately olive-green wild-type axolotl coloration, owing to the intermingling of black melanophores, yellow xanthophores, and more sparsely distributed iridescent iridophores (Figure 1). The lone non-wild-type axolotl in the original Paris collection presented a leucistic phenotype (white) characterized by few to no melanophores. White axolotls proved to be valuable in early embryological studies because they provided a non-pigmented background to monitor the behaviors of grafted, melanin-containing cells from wild-type axolotl donors. Today, white axolotls are preferred over more darkly pigmented wild-type axolotls for visualization of fluorescent probes and making transgenics with fluorescent reporter constructs. While the white Genes 2023, 14, 904. https://doi.org/10.3390/genes14040904 https://www.mdpi.com/journal/genes