Genome Editing Reveals Idiosyncrasy of CNGA2 Ion Channel-Directed Antibody Immunoreactivity Toward Oxytocin

Abstract

Presynaptic cGMP-gated ion (CNG) channels positively or negatively modulate neurotransmitter secretion as well as the strength of synaptic transmission. Zebrafish cGMP-gated ion channel, CNGA2a (a.k.a. CNGA5), was previously reported to be specifically enriched in synaptic terminals of zebrafish oxytocin (OXT) neurons. This conclusion was based on immunoreactivity of a monoclonal antibody (mAb) clone L55/54, which was directed against the carboxy terminal tail of the CNGA2a. To study the role of CNGA2a in oxytocin neurons function, we generated zebrafish mutants of cnga2a, cnga2b and oxt genes using clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing. We show that mAb L55/54 specifically recognizes CNGA2a protein when expressed in heterologous cell culture system. Surprisingly, anti-CNGA2a immunoreactivity was not eliminated following knockout of either cnga2a, cnga2b or both. However, knockout of oxt resulted in total loss of anti-CNGA2a mAb immunoreactivity despite the lack of sequence and structural similarities between OXT and CNGA2a proteins. Our results provide a noteworthy lesson of differences in antibody immunoreactivity, which could only be revealed using specific genetic tools.

Keywords: cGMP-gated ion channel; monoclonal antibody; neurohypophysis; neuropeptide; oxytocin.

FIGURE 1

Immunoreactivity of anti-CNGA2a mAb in larval and adult zebrafish. (A) Phylogram of the CNG channels protein sequences. Comparison of the CNGA2 protein homologues from zebrafish, human, and mouse species is enclosed in the blue box. The scale bar indicates 30% amino acid residues substitution. (B,C) Confocal images showing representative labeling of the OXT perikarya, neuronal projections, and neurohypophyseal axonal termini with anti-CNGA2a mAb (magenta) and anti-OXT Ab (gray scale) in the context of the EGFP-positive OXT-ergic population in oxt:egfp reporter (green). Immunohistochemical analysis show colocalization of EGFP+, OXT+, and CNGA2a+ moieties in the cell bodies, axons, and nerve termini in the neurohypophysis of 6-day-old larva (n = 30/30) (B) and dissected brain and pituitary from 3-month-old adult zebrafish (n = 3/3) (C). Scale bars: 10 μm.

FIGURE 2

Specificity of anti-CNGA2a mAb in heterologous expression system. (A) Western blot analysis of HEK293T cells transfected with cnga2a cDNA. HEK293T cells were transiently transfected with different amounts of cnga2a cDNA or a mock plasmid and were harvested 48 h post-transfection. Western blot analysis of equal amounts of protein extracts were performed using anti-CNGA2a mAb. The correct position of the doublet CNGA2a protein bands are marked by arrowheads (n = 2/2). (B) Confocal images of HEK293T cells transfected with cnga2a cDNA. HEK293T cells were transiently co-transfected with combinations of egfp cDNA either with cnga2a cDNA or an empty pcS2 plasmid. Forty-eight hours post-transfection the monolayer cultures were fixed in 3% paraformaldehyde (PFA), permeabilized with 0.5% Triton-X100/3% PFA, washed in PBS and fluorescently co-stained with anti-CNGA2a (magenta), anti-OXT (gray scale), and anti-GFP (green) antibodies (n = 4/4). Scale bars: 10 μm.

FIGURE 3

cnga2 isoforms genome editing using CRISPR/Cas9. (A) Schematic representation of the genetic structure of cnga2 isoforms. cnga2a and cnga2b. (B) Schematic representation of the predicted CNGA2a and CNGA2b protein products following sequence analysis of germline transmitted CRISPR-induced mutation alleles. (C) Embryos and adult fish were screened by PCR for germline transmission using gene-specific primers. PCR products were resolved in 1% LE agarose gels for cnga2a-del and cnga2b-del progenies and in 4% MetaPhor gel for cnga2a-stop and cnga2b-stop progenies.

FIGURE 4

Anti-CNGA2a immunoreactivity is not affected following KO of cnga2a and/or cnga2b. (A) Immunostaining and confocal imaging of 6-day-old larva NPO of the wild type zebrafish (n = 30/30), cnga2a (n = 20/20), cnga2b (n = 16/16), and double cnga2a/2b (n = 6/6) zebrafish mutant variants with anti-CNGA (magenta) and anti-OXT (gray scale) antibodies. (B) Anti-CNGA2a mAb immunostaining and confocal imaging of the hypothalamus and neurohypophysis of 6-day-old larva injected with different concentrations of cnga2a missense morpholino oligonucleotide (n = 10/10). Scale bars: 10 μm.

FIGURE 5

Anti-CNGA2a immunoreactivity is lost following KO of oxytocin. (A) Schematic representation of oxytocin (oxt) gene. OXT translation start site and sgRNA target site are both indicated with arrows. Embryos and adult fish were screened by PCR for germline transmission using gene-specific primers following PCR products separation in 4% MetaPhor gels. DNA sequence of mutant allele transmitted through the germline contained a 7 bp deletion. (B) Schematic representation of predicted translated products from oxt +/+ and oxt –/– alleles. (C) Confocal images showing representative anti-CNGA2a (magenta) and anti-OXT (gray scale) labeling of OXT cells and their neurohypophyseal axonal termini of OXT-KO 6-day-old larva in the background of a transgenic Tg(oxt:egfp-3′utr) reporter (green) (n = 12/12). Scale bars: 10 μm.