# Synergistic Regulation of Pigment Cell Precursors’ Differentiation and Migration by ednrb1a and ednrb2 in Nile Tilapia

## TL;DR

This study shows that two genes, ednrb1a and ednrb2, work together to control the development and movement of pigment cells in Nile tilapia, leading to a transparent fish phenotype.

## Contribution

The study reveals the synergistic roles of ednrb1a and ednrb2 in pigment cell development and establishes a mechanism for generating transparent embryos in tilapia.

## Key findings

- ednrb1a and ednrb2 synergistically regulate iridophore and erythrophore development in Nile tilapia.
- Double mutants exhibit near-complete loss of iridophores and a transparent phenotype without affecting guanine synthesis.
- mitfa mRNA injection rescues the phenotype of ednrb1a−/−;ednrb2−/− mutants, identifying mitfa as a key downstream effector.

## Abstract

ednrb1a−/−; ednrb2−/− mutants exhibit complete iridophore loss, similarly to mpv17 mutants.ednrb mutants display no defects in the guanine synthesis pathway in tilapia.ednrb1a and ednrb2 synergistically regulate iridophore and erythrophore development.mitfa mRNA injection rescues the phenotype of ednrb1a−/−;ednrb2−/− mutants.

ednrb1a−/−; ednrb2−/− mutants exhibit complete iridophore loss, similarly to mpv17 mutants.

ednrb mutants display no defects in the guanine synthesis pathway in tilapia.

ednrb1a and ednrb2 synergistically regulate iridophore and erythrophore development.

mitfa mRNA injection rescues the phenotype of ednrb1a−/−;ednrb2−/− mutants.

The evolutionary loss of ednrb2 in specific vertebrate lineages, such as mammals and cypriniform fish, raises fundamental questions about its functional necessity and potential redundancy or synergy with paralogous endothelin receptors in pigment cell development. In teleosts possessing both ednrb1a and ednrb2 (e.g., Nile tilapia), their respective and combined roles in regulating neural crest-derived pigment cell precursors remains unresolved. Using CRISPR/Cas9, we generated single and double ednrb mutants to dissect their functions. We demonstrated that ednrb1a and ednrb2 synergistically govern the differentiation and migration of iridophore precursors. While ednrb1a is broadly essential for iridophore development, ednrb2 plays a unique and indispensable role in the colonization of iridophores in the dorsal iris. Double mutants exhibit near-complete iridophore loss; severe depletion of melanophores, xanthophores, and erythrophores; and a striking, fertile, transparent phenotype. Crucially, this iridophore deficiency does not impair systemic guanine synthesis pathways. mRNA rescue experiments confirmed mitfa as a key downstream effector within the Ednrb signaling cascade. This work resolves the synergistic regulation of pigment cell fates by Ednrb receptors and establishes a mechanism for generating transparent ermplasm.