Characterization of Pipefish Immune Cell Populations Through Single-Cell Transcriptomics

Abstract

Teleost adaptive immune systems have evolved with more flexibility than previously assumed. A particularly enigmatic system to address immune system modifications in the evolutionary past is represented by the Syngnathids, the family of pipefishes, seahorses and seadragons. These small fishes with their unique male pregnancy have lost the spleen as an important immune organ as well as a functional major histocompatibility class II (MHC II) pathway. How these evolutionary changes have impacted immune cell population dynamics have up to this point remained unexplored. Here, we present the first immune cell repertoire characterization of a syngnathid fish (Syngnathus typhle) using single-cell transcriptomics. Gene expression profiles of individual cells extracted from blood and head-kidney clustered in twelve putative cell populations with eight belonging to those with immune function. Upregulated cell marker genes identified in humans and teleosts were used to define cell clusters. While the suggested loss of CD4+ T-cells accompanied the loss of the MHC II pathway was supported, the upregulation of specific subtype markers within the T-cell cluster indicates subpopulations of regulatory T-cells (il2rb) and cytotoxic T-cells (gzma). Utilizing single-cell RNA sequencing this report is the first to characterize immune cell populations in syngnathids and provides a valuable foundation for future cellular classification and experimental work within the lineage.

Keywords: cell profiling; immune cell; immunity; pipefish; single-cell transcriptomics.

Figure 1

(A) Visual representation of isolated blood and head kidney extracted cell types of Syngnathus typhle using Uniform manifold approximation projection (UMAP). Cell clusters characterized by differentially expressed gene markers associated with specific cell sub-types. All markers associated with immune function previously reported in humans and/or fish species. Two erythrocyte clusters are represented with the second highlighted as such (2). Full names for all clusters are as follows: 1. Hematopoietic cells, 2 and 3. Erythrocytes, 4. Natural killer cells, 5. Neutrophils, 6. Macrophages, 7. B-cells, 8. Basophils, 9. Thrombocytes, 10. T-cells, 11. Connective tissue cells, 12. Eosinophils. (B) Differential gene expression heatmap highlighting top 10 marker genes, with genes representing rows and cells representing columns.

Figure 2

Dot plot visualization of deduced cell markers used to define cell clusters in Syngnathus typhle. Dot size denotes the percentage of cells expressing the gene within each cluster and the mean expression level of active expressional cells is indicated by the color intensity.

Figure 3

Feature plot highlighting Syngnathus typhle blood and head kidney cells expressing selected genes characterizing selected immune cell clusters within Uniform manifold approximation projection (UMAP). Increased cell color intensity indicates increased gene expression.

Figure 4

Feature plot highlighting Syngnathus typhle blood and head kidney cells expressing the hcls1 gene within a Uniform manifold approximation projection (UMAP). Increased cell color intensity indicates increased gene expression.