Zebrafish has emerged as a admired archetypal to abstraction alive cellular processes15,16 due to its sequenced genome, accuracy through aboriginal adulthood, and acquiescence to high-throughput screens17,18. To actuate if zebrafish is a advantageous in vivo archetypal for the abstraction of fungus physiology, we searched its genome for genes with affinity to buried mucins from added vertebrates. We focused on polymeric buried mucins, the aloft gel-forming architectonics blocks of the fungus barrier19,20,21. One appropriate of gel-forming mucins is the accedence of two protein domains, the Proline, Threonine and Serine (PTS) domain, which is the capital armpit of O-linked glycosylation on the protein, and the Von Willebrand Factor D (VWD) domain, which contributes to the polymerization of mucins. Application ahead characterized computational tools22 we articular bristles accepted mucin genes in zebrafish that accommodate coding regions for both PTS and VWD domains (Supplementary Fig. S1). Based on the consecutive assay (see below) we called the bristles accepted mucin genes muc5.1 (Ensembl ID ENSDARG00000070331), muc5.2 (ENSDARG00000058556), muc5.3 (ENSDARG00000089847), muc2.1 (ENSDARG00000074142) and muc2.2 (ENSDARG00000078994).
The alignment of the protein domains in the articular zebrafish mucins is apparent in Figure 1a. The better-studied beastly polymerizing mucins MUC2, MUC5AC and MUC5B are characterized by an adjustment of four VWD domains with an all-encompassing PTS area amid the third and fourth VWD domains. In addition, they accommodate CysD domains that are interspersed in the PTS domains, and a cystine bond at the C terminus; both CysD domains and the cystine bond are complex in polymerization. The beastly MUC6 mucin is agnate in architectonics but lacks the CysD domains as able-bodied as a fourth VWD area at its C terminus23. Our abstracts actualization that two articular associates of the zebrafish Muc5-family, Muc5.1 and Muc5.2, accommodate the appropriate adjustment of four VWD domains and a PTS area localized amid the third and fourth VWD domains (Figure 1a, Supplementary Figs. S1, S2, S3). Muc5.3 is altered in that it appears to accommodate alone the aboriginal three VWD domains. For the associates of the Muc2-family (Muc2.1 and Muc2.2) we had beneath arrangement advice accessible and therefore, action a added basic interpretation. For Muc2.1 we articular three VWD domains and a truncated PTS area in the predicted N-terminal allocation of the protein. For Muc2.2, alone a abbreviate Ensembl archetype was available, which was the base for the depicted C-terminal VWD area (Fig. 1a). In addition, muc2.1 mRNA was acclimated as a adviser to adumbrate the exon/intron anatomy of the muc2.2 gene from the accessible genomic sequence. From the constant muc2.2 gene archetypal two added VWD domains were articular at the N-terminus of Muc2.2. In the genomic anticipation we additionally begin affirmation for a PTS arena (Supplementary Fig. S1). The PTS arena was not included in the protein area analogy because it was absent from the Ensembl archetype archetypal at the time (Fig. 1a). However, should approaching adapted archetype models accommodate the arrangement for the PTS, Muc2.2 would accept the accepted architectonics of a MUC2 blazon mucin. For added capacity apropos the genomic alignment and protein affinity of the zebrafish mucins, as able-bodied as advice on added mucin-like transcripts, the clairvoyant is referred to Supplementary Figs. S1 and S3, and Supplementary Table S1.
(a) – Analogy of predicted mucin protein area architectures. Two associates of the Muc5 ancestors (Muc5.1 and Muc5.2) are composed of three alternating VWD domains, followed by a PTS area and a fourth VWD area at the C-terminus. This architectonics is archetypal for beastly gel-forming buried mucins. The third Muc5 ancestors member, Muc5.3, has a agnate predicted area agreement but lacks the fourth VWD area at the C-terminus. For the Muc2 ancestors members, Muc2.1 and Muc2.2, regions of the protein arrangement are missing as the accepted genome accumulation is incomplete. The Muc2.2 protein area lacks a PTS area because it was absent from the Ensembl archetype prediction, admitting such area was begin at the genomic akin (Supplementary Fig. S1). VWD: Von Willebrand Factor blazon D domain; PTS: Proline, Threonine and Serine domain; CysD and Cys-knot are cysteine affluent domains. All area structures except the N-terminal allocation of Muc2.2 were articular based on Ensembl transcripts. The sequences acclimated for the architecture of the depicted protein models are listed in Supplementary Add. S1. (b) – Phylogenetic timberline comparing zebrafish mucins with craven and animal polymeric buried mucins from N-terminal portions of the mucins absolute the three aboriginal VWD domains. The numbers at the branches represent afterwards probabilities. The timberline shows that the articular mucins accumulation with MUC5 and MUC2, but not with MUC6, from craven and human. (c) – Tissue administration of the mucin transcripts as detected by RT-PCR. The muc5 ancestors of mucins is bidding in respiratory organs (skin, gills, pharynx and esophagus). muc2.1 announcement is detected in the digestive system, predominantly in the gut which is archetypal for MUC2 mucins in mammals. muc2.2 announcement is detected in changeable organs.
Based on the N-terminal portions of the mucin proteins that accommodate the aboriginal three VWD domains we complete phylogenetic copse (Fig. 1b: MrBayes, Supplementary Fig. S2: neighbor-joining tree). The after-effects actualization that Muc5.1, Muc5.2 and Muc5.3 accumulation with the animal and craven MUC5AC and MUC5B. Muc5.1 and Muc5.2 arise carefully accompanying to anniversary other. Muc2.1 and Muc2.2 accumulation with the animal and craven MUC2 mucins. Our abstracts additionally advance that none of the advised zebrafish genes are accompanying to the bearcat MUC6 mucin.
From the genomic anatomy we acquired that the genes muc5.1, muc5.2 and muc2.2 are localized in a array on chromosome 25 (Supplementary Fig. S1). muc2.1 currently has an unassigned position in the genome, Zv9_NA774, with about two thirds of its breadth unknown. The muc5.3 gene, which is amid on chromosome 7, has the gene tollip as its actual neighbor. The accident of zebrafish mucin genes in a cluster, as able-bodied as the genomic synteny with the tollip gene, is evocative of the mucin gene alignment in added vertebrates, including humans24.
To actuate the tissue administration of the accepted zebrafish mucin gene transcripts we performed RT-PCR from tissue abandoned from developed zebrafish (Fig. 1c). Our abstracts actualization that muc5.1 and muc5.2 are both bidding in the skin, the aspect and the pharynx/esophagus, while muc5.3 announcement appears to be belted to the pharynx/esophagus. By in situ admixture on developed zebrafish sections we were able to added specify the administration of muc5.1 and muc5.2 to the pharynx and muc5.3 to the esophagus (Supplementary Fig. S4). In addition, by amid the gill lamellae from the gill arches we saw that alone muc5.1 is bidding in the lamellar allotment (Supplementary Fig. S5) and hence, appears to represent a bona fide respiratory mucin. muc2.1 is predominantly begin in the gut, while muc2.2 is bidding in testes and ovaries (Fig. 1c). Together, the announcement arrangement of the zebrafish muc5 ancestors appears evocative of the animal MUC5 family, which is begin in the respiratory and the aerial digestive tracts25. Moreover, muc2.1 in zebrafish shares its tissue localization with animal MUC2, the aloft mucin in the gut. The outlier is muc2.2, which is bidding in the changeable organs in zebrafish; in humans, MUC5AC, MUC5B and MUC6 are begin in the macho urogenital tract26 and changeable endocervix. We additionally empiric that announcement of the mucin genes during zebrafish development correlates with the admission of development of the corresponding organs, in which mucins are begin in the developed fish27,28 (Supplementary Fig. S6).
The aloft arrangement advice on mucin genes was acclimated to actualize a beaming anchorman of mucin action in zebrafish. The complete accessible account frames of buried mucins is difficult to tag due to its ample size. As a consequence, appropriately far alone one abounding breadth mucin, MUC5AC from the mouse, has been auspiciously fluorescently tagged and bidding beneath a basal promoter29. Our ambition actuality was to accomplish a anchorman in zebrafish that is bidding beneath the autogenous mucin promoter, which would accredit the absolute time tracking of mucin assembly in acknowledgment to physiological changes. To accomplish this, we excised from the BAC CH211-19808 9.8 kb of genomic arrangement of muc5.1 that comprises 4.6 kb upstream and 5.2 kb afterwards of the mucin alpha codon ATG, and cloned it into the pBSII KS( ) vector. Our ambition was to accurate the Red Beaming Protein (RFP) in anatomy with the ATG and the muc5.1 secretory signaling arrangement to accredit beard of the mucin reporter. We amid a Tag-RFP targeting cassette at 93 bp afterwards from the mucin ATG, application Lambda-Red akin recombination (Supplementary Fig. S7). In this construct, the RFP is concluded with the stop codon and hence, does not accommodate the mucin coding arrangement above the signaling sequence.
To analysis for the functionality of the mucin reporter, the assemble was linearized, injected into one-cell date zebrafish embryos, and the animals were aloft to adolescence and buried for antibody band transmission. In antibody band transgenics of the muc5.1:S-RFP reporter, fluorescence became apparent at two canicule post-fertilization (dpf) as dots on the bark (Fig. 2a). At day four, the beaming arresting was axiomatic forth the anatomy arbor including in the aperture of the larva (Fig. 2a). At two weeks column fertilization, the beaming arresting was broadcast throughout the bark at an boilerplate of 2500 dots/mm2 (Fig. 2b). Such dotted administration has been appear for mucus-secreting beef in the zebrafish civil and in the animal respiratory epithelium30,31. A afterpiece analysis of the beaming loci by confocal microscopy shows that the mucin anchorman is stored in almost ample granules central the cells, which resemble secretory vesicles artlessly produced by mucus-secreting beef (Fig. 2c)32. The cellular membranes in Fig. 2c were visualized with GFP alloyed to a CAAX motif, which targets the GFP to the film and which is bidding beneath the adjustment of the muc5.1 promoter. The GFP-CAAX anchorman delineates the cellular and vesicular membranes of the muc5.1 bearing beef (Fig. 2c). Together these abstracts advance that the mucin anchorman is produced in secretory beef and compartmentalized in granules, as is accepted for buried mucins.
(a) –Visualization of embryos at 2, 4, 7 and 14 canicule column fertilization (dpf) by fluorescence (top row) and ablaze acreage (bottom row) microscopy shows that the mucin anchorman expresses in audible loci broadcast beyond the bark of the fish. Scale confined are 0.5 mm. (b) –Live decision of the beaming mucin anchorman in the arch (top) and block (bottom) of 14 dpf fish. Scale bar is 200 μm. (c) – Confocal angel of a muc5.1:S-RFP-positive locus shows that the mucin anchorman is arranged central secretory vesicles in beef aural the skin. The corpuscle membranes are labeled with GFP that was bidding beneath the apostle of muc5.1 and targeted to the film via a CAAX motif. Scale bar is 5 μm. (d) – Acknowledgment of angle to LPS after-effects in the fractional accident of muc5.1:S-RFP loci and a accompanying actualization of fluorescence in the actual surrounding of the fish, suggesting the beard of the mucin reporter. The ablaze acreage angel (bottom) shows that angle abide complete during this treatment. (e) – Altitude of beaming loci aural a constant arena (approximately amid the arch and top of the trunk) in ten alone angle afore and afterwards acknowledgment to LPS. The red aboveboard is the beggarly of 10 ascendancy angle (no LPS addition) at t = 0, 5, and 10 minutes. The beggarly amount is 35.7 beyond all three time points. For the LPS addition, anniversary point represents the cardinal of beaming loci in the aforementioned angle at the assorted time credibility (10 angle total). The absurdity confined announce accepted deviation. One * indicates p < 0.05 amid 0 account and 5 account application commutual two-tailed T-test. Two ** indicates p < 0.01 application the aforementioned analysis amid 0 account and 10 minutes. In best fish, a abundant admeasurement of loci are absent on analysis with LPS, suggesting that the mucin-reporter is buried on this stimulus.
To analysis if muc5.1:S-RFP can be expelled from the cells, appropriately acceptance the ascertainment of alive secretion, we acclimated lipopolysaccharide (LPS) from E. coli as a characterized stimulant33. Our abstracts actualization that in 4 dpf fish, on acknowledgment to 0.5 μg/μl LPS, the majority of muc5.1:S-RFP-producing beef lose fluorescence aural account while the appear RFP collects as a aura about the angle (Fig. 2d). A altitude of beef that belch muc5.1:S-RFP reveals that almost 40% of the accepted beaker beef per angle absolution the fluorescence aural bristles account afterwards consecration with LPS, with added arresting accident aural the aing bristles account (Fig. 2d, 2e). 10 account afterwards antecedent accession of LPS the anesthetized angle displayed accustomed baby and circulation, suggesting that the banishment of the mucin-reporter is not due to toxicity.
In summary, we actualization affirmation for bristles gel-forming buried mucin genes in zebrafish with a aerial amount of affinity to added bearcat mucins in their genomic and protein area organization, as able-bodied as their tissue specific expression. We developed a action to body a beaming mucin anchorman bidding beneath built-in authoritative elements and actualization that its absolution can be triggered and quantified in the alive fish. Together, our assignment offers a advantageous set of accoutrement to abstraction the dynamics of mucin beard and announcement in the assertive angle and aloft pathogenic, pharmacological or abiogenetic challenges. Our achievement is that this beginning arrangement may acquiesce for screening of altitude that not aloof activate fungus secretion, but account abiding furnishings of beaker corpuscle adverse apparent as metaplasia/hyperplasia in mucosal diseases.
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