Leukolectins support lifelong innate immunity in lower vertebrates, and reveal dichotomies of several leukocytic lineages.

Innate immunity is vital for animal homeostasis and survival. First-line immuno-defense for fish larvae involves mucus enriched with leukolectin (LL) secreted by dermal lectocytes. Later during the critical transition from yolk-nutrition to feeding, additional larval immuno-protection in zebrafish (zF) is provided by macrophages containing LL (lectophages). This work investigated new LL-expression in embryos and in blood, structures of fish leukocytic LL and LL-genes, and LL-presence in chicken leukocytes. In zF-embryos, lectophages appear ∼10 hpf, while later, cells co-expressing myeloperoxidase- and LL-mRNA were detected (∼19 hpf). Furthermore, protein-extracts of Atlantic salmon (Ssal) leukocytes contained LL-proteins, compartmentalized in the cytosol. Cloning and sequencing revealed 94 % nt-sequence identity between variants of Ssal-leukolectins. Highly conserved LLs allowed production of epitope-specific anti-LL IgGs. Immuno-fluorescence-analysis demonstrated that most Ssal-bloodcells were LL-negative, but both some large cells with protrusions and some small, rounded cells did express LL. Immunoperoxidase-staining method confirmed LL-expression in some Ssal-leukocytes, identified as macrophages, PMN-leukocytes, thrombocytes and dendritic cells. However, closer examination revealed a dichotomy of these cell-categories into either LL-positive, or LL-negative variants. In situ hybridization demonstrated profuse LL-expression in Ssal head kidney interstitial tissue, while LL-transcripts were absent in large kidney tubules. Both hematopoietic (non-pigmented) marrow cells and melano-macrophages expressed LL-mRNA, implying that leukolectins provide lifelong innate immuno-protection. PCR-amplification using Ssal-leukocytic DNA as template, and direct sequencing yielded a leukocytic ll-gene. Some cells in salmon, cod, halibut, oikopleura and zebrafish embryos express LL-proteins and/or LL-mRNA, and LL-mRNA is detected in salmon, cod and chicken leukocytes. However, current genomes for these species lack recognizable LL-loci except the Ssal_v3.1 Genome-assembly. The data demonstrate an unexpected dichotomy of some leukocyte lineages into LL-positive or LL-negative cell-variants. Such dichotomies suggest exploring differential impacts from the duplicated leukocyte-lineages in health and disease.

Leukolectin is expressed in lectophages, a distinct population of zebrafish embryonic macrophages.

Leukolectins (LL) belong to the tectonin-family of proteins, with functions in innate immunity. Fish larvae compensating for loss of maternal chorionic protection post-hatching, provide a model-system for studying how lectins contribute to immunity. Atlantic salmon (Ssal) LL-proteins function after secretion in mucus from dermal lectocytes, as this mucus envelops embryos and larvae. The Ssalll-gene possesses multiple putative binding sites for diverse transcription-factors, suggestive of LL-functions in non-epithelial cells. Since zebrafish (zF) perivitelline fluid (PVF) contains LL-proteins, this study aims to characterize zF-leukolectins, their cellular origin, expression and gene structure. Extracts of (10 hpf) zF-embryos contained LL-proteins, and whole mount immuno-histochemistry revealed dispersed LL-positive cells including zF-lectocytes, accounting for exocrine LL-secretion by embryos. Lectocytes are lcp1-negative, but other zF-cells co-expressed LL-proteins and lcp1-transcripts, which (at this stage) identified such non-lectocytes as early macrophages (termed lectophages). In sections, LL-expression characterized large macrophage-progenitors and smaller colonizing macrophages. RT- and RACE-PCR yielded zF-LLcDNA including parts of untranslated regions. ORF encoded 255 AAs including (19 AA) signal peptide. Processing of a primary LL-transcript to (∼1.300 nt) LL-mRNA was suggested by Northern blots. Most zebrafish-egg lectins (zFELs) possess four TECPR-domains, while five TECPR-domains were predicted for zF-LL. Minor sequence variations suggested nearly identical zF-LL isoforms. Alignment of zFEL-proteins predicted a zFEL-tree with a separate leukolectin-branch. LL-amplification using zF-DNA, revealed five exons and four introns. Predicted structures of zF- and Ssal-leukolectins showed strong structural conservation (92% sequence-identity) with shorter zF-introns 2&4, but identical introns 1&3. Non-lectocytic LL-functions were investigated further by dual in situ hybridization, revealing that only some embryonic lcp1-expressing cells in early zF-embryos co-expressed LL-transcripts. Macrophages from erythro-myeloid progenitor (EMP) are known to colonize zebrafish tissues as resident macrophages (TRM), e.g. nervous system (CNS) and epiderm. Unlike Ssal-larvae relying on yolk for months, zF-larvae switch within days to nutrition from the digestive-tract, necessitating additional immuno-protection possibly from TRMs. EMP also gives rise to microglia, the TRM of CNS. The neural tube of zF-embryos exhibited numerous small, LL-positive cells, presumably stemming from lectophage-progenitors. Functions of these LL-positive embryonic microglia (lectoglia) appear more relevant for tissue remodelling than for pathogenic threats. Lectoglia sustaining CNS-neurons suggests physiological LL-roles relevant for adult health and disease. The data focus the need for resolving whether lectophages represent an unrecognized myelogenic lineage, or whether instead, LL-expression occurs in a subpopulation of the early macrophage-lineage.

Leukolectin-proteins are synthesized and secreted by lectocytes, a distinct category of fish embryonic mucus cells.

Fish perivitelline fluid (PVF) is a vital extra-embryonic compartment. At hatching, PVF-contents dissolve into the hatching fluid (HF). Analysis of Atlantic salmon HF reveals nearly a hundred distinct proteins, most of which were identified by advanced mass-spectrometry. However, one entity with an apparent molecular weight 26 kDa, necessitated identification from its tryptic peptides. Subsequent cloning and sequencing revealed novel leukolectin-proteins. From bioinformatic analysis, leukolectins (LL) belong in the tectonin protein-family, with recognized functions in innate immunity. This study aims to identify LL-expressing cells in diverse fish species, and to characterize the LL-gene in order to predict bio-functions of leukolectins. LL-proteins were detected in HF from several fish species and one invertebrate, using polyclonal LL-specific IgGs. Embryonic LL-immunoreactive cells were numerous in Atlantic salmon, rainbow trout, fewer in Atlantic cod, and rare in Atlantic halibut and Oikopleura dioica. LL-immunoreactive cells were termed lectocytes, which corresponded to peridermal mucuscells stained by PAS, but unstained by eosin. Hence, lectocytes and hatching-gland cells were clearly distinguished. Northern blots revealed two salmon LL-transcripts at mid-embryogenesis. Such transcripts were detected in epithelial cells of the periderm, gills and oral cavity. LL-transcripts predominated in the periderm, while choriolysin-transcripts were dominant in the gills. No co-expression of choriolysins and LL-transcripts was detected. BAC-library screening yielded salmon LL's genestructure with 4 introns, 5 exons, TATA-box, multiple upstream putative transcription-factor bindingsites and polyadenylation site. LL-gene location on chromosome ssa17 was identified in Ssal_v3.1, the 2021version of the salmon genome. In conclusion, larvae from several fish species are outfitted with mucus enriched by LL-proteins. Mucus cells are present in embryos of all fishes, but embryonic lectocyte-numbers are far higher in species with near total larval survival. When (maternal) chorionic first-line immuno-defence is lost at hatching, leukolectin-enriched mucus may provide vital protection for larvae.

Choriolytic and non-choriolytic proteases during hatching of Atlantic Salmon embryos.

Fish embryonic hatching glands (HGs) secrete choriolysin-zymogens, which when activated degrade the chorion, allowing larval exit. Thus, hatching encompasses two dissimilar choriolysin-processes: pre-choriolysis including activated choriolysins accessing the perivitelline space (PVS), followed by choriolysis. Discovery of serine-proteolytic zonase in Atlantic salmon hatching fluid (HF) raises questions concerning its participation in hatching. This work aims to identify salmon choriolysins, and evaluate their role and that of zonase during hatching. Precocious salmon hatching occurs under alkaline conditions, producing an HF containing similar metallo- and serine- proteolytic activities. Purified zonase is selectively inhibited by peFabloc, whose MW (580 Da) allows diffusion through the chorion into the PVS. Without apparent toxicity, brief peFabloc-pretreatment (2 mM) of salmon eggs reduced precocious hatching substantially, compatible with a zonase-relevance for hatching. Atlantic salmon differed from other fishes since their HGs were not immuno-stained by polyclonal antibodies against pike choriolysins. However, cloning and sequencing experiments revealed a single low choriolytic enzyme (LCE) of 69% identity to pike LCE. Similar experiments with high choriolytic enzymes (HCEs) revealed two types (HCE-1 and HCE-2) with respectively 71% and 91% identity to pike HCE-1 & HCE-2. In situ hybridization revealed typical HGs. However, zebrafish-choriolysis is achieved by HCE-class choriolysins alone. Another zebrafish choriolysin (HE2) was not expressed. Peptide-bond hydrolysis by non-choriolytic zonase mimicks HCE-action generating hydrophilic sites for LCE-choriolytic catalysis. Ultimately, precise definitions of choriolytic and pre-choriolytic catalysis requires developmental genetics. Our data are compatible with a complex pre-choriolytic hatching-stage in Atlantic salmon, before LCE-choriolysis degrades the chorion.