Asymmetric Functional Divergence of <i>alx4a</i> and <i>alx4b</i> in Iridophore Differentiation and Cranial Development in Nile Tilapia.

Overview

1. Integrative Science Center of Germplasm Creation in Western China (Chongqing) Science City, Key Laboratory of Freshwater Fish Reproduction and Development (Ministry of Education), Key Laboratory of Aquatic Science of Chongqing, School of Life Sciences, Southwest University, Chongqing 400715, China; (H.S.); (F.F.); (J.Y.); (S.J.)

Abstract

Highlights: The first teleost alx4a/alx4b double mutant model reveals unequal functional contributions of the two paralogs after genome duplication.

alx4a is the predominant contributor to iridophore-associated structural coloration, while alx4b provides a weaker, context-dependent contribution.

The two paralogs contribute unequally to cranial development, with double mutants showing reduced opercular calcein labeling.

Simple Summary: Neural crest cells give rise to both the craniofacial skeleton and several pigment cell types. Nile tilapia possess two paralogs of the developmental regulator Alx4, but their relative functional contributions remain poorly understood. We generated fish lacking either copy alone or both copies together. Loss of alx4a reduced reflective structural coloration in specific body regions and altered cranial morphology, whereas loss of alx4b caused no obvious phenotype under the conditions examined. Fish lacking both genes showed widespread loss of reflective coloration and severe cranial abnormalities. No significant genotype-dependent differences were detected in the measured abundance of other chromatophore lineages, and no obvious difference in gross dorsal-fin spine formation was observed under the conditions examined. Gene expression analyses were consistent with a role for alx4 in iridophore differentiation and maturation, although possible effects on iridophore survival cannot be excluded. These findings indicate that these two paralogous genes make unequal functional contributions, with alx4a playing the predominant role and alx4b retaining a weaker contribution, while both paralogs contribute unequally to cranial development. The study provides a genetic framework for understanding how duplicated developmental genes evolve and how neural-crest-derived pigmentation and skeletal traits are coordinated in teleost fishes.

Abstract: Neural crest cells give rise to the craniofacial skeleton and multiple pigment cell lineages, yet how duplicated developmental regulators partition their ancestral functions after teleost-specific whole-genome duplication remains unclear. Here, we employed CRISPR/Cas9 to generate alx4a and alx4b single and double mutants in Nile tilapia (Oreochromis niloticus). By integrating phenotype, skeleton, transcriptome, quantitative PCR, and AlphaFold-based structural modeling analyses, we revealed their functional divergence. Loss of alx4a caused a regionally restricted reduction in iridophore-derived reflectance and abnormal cranial morphology, whereas alx4b single mutants showed no obvious phenotype under the conditions examined. By contrast, double mutants exhibited an almost complete loss of iridophore-derived structural coloration and substantially more severe cranial defects, accompanied by reduced calcein labeling in the opercular region, consistent with altered cranial mineralization. Skin transcriptomic and quantitative PCR analyses revealed marked downregulation of pnp4a and tfec, which are associated with iridophore differentiation and coloration, whereas no significant expression differences were detected for the iridophore survival-related genes ltk and mpv17. AlphaFold2-assisted HDOCK protein–DNA modeling yielded more favorable docking metrics for Alx4a than for Alx4b with the pnp4a promoter, supporting a potential Alx4a–pnp4a promoter interaction that requires experimental validation. In contrast, no significant genotype-dependent differences were detected in the measured abundance of melanophores, xanthophores, or erythrophores, and no obvious difference in gross dorsal-fin spine formation was observed under the conditions examined. Together, these findings reveal unequal functional contributions of alx4a and alx4b, with alx4a acting as the dominant paralog in iridophore-associated structural coloration and both paralogs contributing unequally to cranial development, and support pnp4a as a candidate downstream gene associated with Alx4a activity.

Reproduced under the paper's license (CC BY), from the paper cited above.

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Data

Datasets cited

• geo:GSE343457 — at NCBI GEO; found in “Data Availability Statement”

Data Availability Statement

The RNA-seq dataset generated in this study has been deposited in the Gene Expression Omnibus (GEO) database under accession number GSE343457 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE343457 (accessed on 19 August 2026)). Other data supporting the findings of this study are available from the corresponding author upon reasonable request.

Reproduced under the paper's license (CC BY), from the paper cited above.

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Version 1, 27 September 2026: the first record

Recorded: type, language, journal, volume, issue, pages, dates, 6 authors, 4 keywords, 9 MeSH terms, 3 funders, 32 references.

Cite

This paper

Shi, H., Fang, F., Yao, J., Ju, S., Li, M., & Wang, D. (2026). Asymmetric Functional Divergence of <i>alx4a</i> and <i>alx4b</i> in Iridophore Differentiation and Cranial Development in Nile Tilapia. Cells, 15(17), 1512. https://doi.org/10.3390/cells15171512

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