The identification of additional zebrafish DICP genes reveals haplotype variation and linkage to MHC class I genes

Abstract

Bony fish encode multiple multi-gene families of membrane receptors that are comprised of immunoglobulin (Ig) domains and are predicted to function in innate immunity. One of these families, the diverse immunoglobulin (Ig) domain-containing protein (DICP) genes, maps to three chromosomal loci in zebrafish. Most DICPs possess one or two Ig ectodomains and include membrane-bound and secreted forms. Membrane-bound DICPs include putative inhibitory and activating receptors. Recombinant DICP Ig domains bind lipids with varying specificity, a characteristic shared with mammalian CD300 and TREM family members. Numerous DICP transcripts amplified from different lines of zebrafish did not match the zebrafish reference genome sequence suggesting polymorphic and haplotypic variation. The expression of DICPs in three different lines of zebrafish has been characterized employing PCR-based strategies. Certain DICPs exhibit restricted expression in adult tissues whereas others are expressed ubiquitously. Transcripts of a subset of DICPs can be detected during embryonic development suggesting roles in embryonic immunity or other developmental processes. Transcripts representing 11 previously uncharacterized DICP sequences were identified. The assignment of two of these sequences to an unplaced genomic scaffold resulted in the identification of an alternative DICP haplotype that is linked to a MHC class I Z lineage haplotype on zebrafish chromosome 3. The linkage of DICP and MHC class I genes also is observable in the genomes of the related grass carp (Ctenopharyngodon idellus) and common carp (Cyprinus carpio) suggesting that this is a shared character with the last common Cyprinidae ancestor.

Keywords: Haplotype; Immune receptor; Immunoglobulin domain; Innate immunity; Polymorphism.

Fig. 1. Overview of the oligonucleotide primer design employed for amplifying DICP sequences.

Primer pairs are listed on the left. Genes targeted by each primer pair and the overall genomic organization of these genes are listed to the right of each primer pair. Families of DICPs are defined by a number that denotes the DICP cluster (e.g., DICP1 cluster on chromosome 3) and gene names include a second number that denotes the order in which genes were identified (e.g., dicp1.1). Gray rectangles represent exons and black arrowheads approximate the relative location of each primer. Protein domains associated with each exon are indicated above the genomic organization (L: peptide leader sequence, D1: Ig domain, D2: Ig domain, LC: low complexity regions, TM: transmembrane domain, Cyt: cytoplasmic tail). Primer sequences are listed in Table 1.

Fig. 2. DICP gene expression in immune-related tissues.

DICP expression was evaluated using primer pairs listed in Table 1 and tissues from nine individual adult zebrafish of the TU, AB and EKW lines. RT-PCR amplicons shown were generated with Titanium Taq DNA polymerase and yellow rectangles indicate those products that were cloned and sequenced to confirm their identity. Orange rectangles indicate amplicons that subsequently were generated with a proofreading DNA polymerase (KAPA HiFi) for evaluation of sequence variation. The size and identity of recovered amplicons is listed on the right of the gel image with red text indicating nonfunctional transcripts. β-actin expression was used as a reference for cDNA quantity and quality.

Fig. 3. DICP gene expression in myeloid and lymphoid cells.

Kidney cells from five adult EKW zebrafish were pooled and lymphoid and myeloid cells sorted by flow cytometry. DICP transcripts were amplified by RT-PCR with Titanium Taq DNA polymerase and all amplicons detected were cloned and sequenced to confirm their identity. The size and identity of recovered amplicons is listed on the right of the gel image; red text indicates nonfunctional transcripts. RT-PCR of myeloperoxidase (mpx) provides a positive control for myeloid cells and TCR-α provides a positive control for T lymphocytes. β-actin expression was used as a reference for cDNA quantity and quality.

Fig. 4. DICP gene expression during zebrafish development.

RT-PCR was employed to detect DICP transcripts at different developmental stages from TU, AB and EKW zebrafish lines. Ten embryos were pooled for each cDNA template. RT-PCR was employed with Titanium Taq DNA polymerase. Yellow rectangles indicate amplicons that were cloned and sequenced to confirm their identity. The size and identity of recovered amplicons is listed on the right of the gel image; red text indicates nonfunctional transcripts. β-actin expression was used as a reference for cDNA quantity and quality.

Fig. 5. Phylogenetic comparison of newly identified DICP Ig domains with previously described sequences.

New DICP sequences that group with the DICP1 genes on chromosome 3 are in red text. New DICP sequences that group with the DICP3 genes on chromosome 16 are in blue text. DICP sequences encoded by the unplaced genomic scaffold NA310 (GRCz10 reference genome) are indicated by red triangles. All additional sequences were reported previously (Haire et al. 2012), including those predicted from genomic clones (BAC CH73–34H11 and BAC CH73–322B17), which are indicated by blue triangles. Nitr9 Ig V and I domains (Wei et al 2007; Yoder 2009) were used as an outgroup (bold characters). The percentage of replicate trees in which the associated taxa cluster together (bootstrap values) are shown next to the branches; values less than 50 are not shown.

Fig. 6. Exon-intron architecture of previously identified DICPs.

The exon-intron organization of transcript variants encoding previously described DICPs were compared with the DICP genes present in the reference genome. Sequence identifier numbers or GenBank accession numbers are listed to the right of each transcript. Red text indicates predicted non-functional transcripts. Details are provided in Online Resource 3 – DICP exon-intron architecture.

Fig. 7. Exon-intron architecture of newly identified DICPs.

The exon-intron organization of transcript variants encoding newly identified DICPs were predicted by comparison to DICP genes present in the reference genome and/or to similar DICP transcripts previously identified. Sequence identifier numbers or GenBank accession numbers are listed to the right of each transcript. Red text indicates predicted non-functional transcripts. Sequence identifiers shown in parentheses represent RACE clones. Details are provided in Online Resource 3 – DICP exon-intron architecture.

Fig. 8. Alternative haplotypes for the chromosome 3 DICP gene cluster.

(a) Relative chromosomal positions of the DICP1 genes on chromosome 3 (scaffold CTG10218) compared to the relative positions of the DICP1 genes found on unplaced scaffold NA310. Gray triangles represent a single DICP gene, except for dicp1.3–4 and dicp1.5–6 that are predicted to be encoded in single genes. Predicted pseudogenes are indicated with a “p” at the end of the gene symbol. Black triangles represent linked non-DICP genes. The ccdc134 or ccdc134l and the gimap8l or gimpa4l genes along with a member of the MHC class I Z lineage are present in both regions. A detailed sequence identity comparison is provided in Online Resource 3 – Fig. S10. (b) Genomic PCRs for dicp1.1, dicp1.22, mhc1zja and mhc1zka using genomic DNA from individual TU, AB and EKW zebrafish analyzed in Fig. 2 and predicted to be homozygous for one of the two haplotypes depicted in panel a. (c) Genomic PCRs for dicp1.1, dicp1.22, mhc1zja and mhc1zka using gDNA from individual TU zebrafish previously shown to be heterozygous for the two MHC class I Z gene haplotypes depicted in panel a (Dirscherl and Yoder 2014).