Related Experiment Video
Updated: Nov 20, 2025
08:39
Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos
Published on: August 31, 2016
5.9K
Identification and Characterization of Zebrafish Tlr4 Coreceptor Md-2
Andrea N Loes1,2, Melissa N Hinman1,3, Dylan R Farnsworth3,4
1Institute of Molecular Biology, University of Oregon, Eugene, OR 97403.
Journal of Immunology (Baltimore, Md. : 1950)
|January 21, 2021
Summary
Researchers discovered a zebrafish gene for Md-2 (myeloid differentiation factor 2), identifying an ancient LPS-sensing TLR4/Md-2 complex in zebrafish innate immunity. This complex is likely ancestral, shared with mammals, not a new tetrapod invention.
Area of Science:
- Immunology
- Genetics
- Evolutionary Biology
- Zebrafish Model Organisms
Background:
- The innate immune system's lipopolysaccharide (LPS) sensing mechanism in zebrafish (Danio rerio) differs from amniotes.
- Amniotes utilize Toll-like receptor 4 (TLR4) and myeloid differentiation factor 2 (Md-2) for LPS recognition.
- Zebrafish were previously thought to lack Md-2 and orthologous Tlr4 genes.
Purpose of the Study:
- To re-evaluate the presence and function of Md-2 and Tlr4 in zebrafish innate immunity.
- To investigate the evolutionary origins of the LPS-sensing Tlr4/Md-2 complex.
- To understand the role of this complex in zebrafish immune responses.
Main Methods:
- Identification of a zebrafish Md-2 gene (ly96) using bioinformatics and single-cell RNA sequencing.
- Analysis of ly96 and Tlr4-like gene expression in innate immune cells.
- Functional assays involving zebrafish Md-2 and Tlr4ba complex formation and NF-κB activation in response to LPS.
- Assessment of LPS-induced cytokine production and toxicity in zebrafish ly96 loss-of-function mutants.
- Comparative genomic analysis of tlr4 gene context across jawed vertebrates.
Main Results:
- A zebrafish Md-2 gene (ly96) was identified and found to be co-expressed with Tlr4-like genes in innate immune cells, particularly macrophage-like cells.
- Zebrafish Md-2 and Tlr4ba form a functional complex that activates NF-κB signaling upon LPS stimulation.
- Loss-of-function mutations in ly96 impaired LPS-induced cytokine production but offered limited protection against LPS toxicity.
- Genomic analysis indicates that tlr4 predates the divergence of teleosts and tetrapods, suggesting an ancient origin.
Conclusions:
- Zebrafish possess a functional Tlr4/Md-2 complex capable of sensing LPS, challenging previous assumptions.
- The LPS-sensitive Tlr4/Md-2 complex is likely an ancestral feature of jawed vertebrates, shared by mammals and zebrafish.
- Zebrafish may retain an ancestral, lower-sensitivity Tlr4/Md-2 complex involved in specific innate immune cell responses to LPS.
