Gill developmental program in the teleost mandibular arch

  1. Mathi Thiruppathy
  2. Peter Fabian
  3. J Andrew Gillis
  4. J Gage Crump  Is a corresponding author
  1. Eli and Edythe Broad California Institute for Regenerative Medicine Center for Regenerative Medicine and Stem Cell Research, Department of Stem Cell Biology and Regenerative Medicine, University of Southern California Keck School of Medicine, United States
  2. Marine Biological Laboratory, United States
  3. Department of Zoology, University of Cambridge, United Kingdom
4 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
The zebrafish pseudobranch derives from mandibular arch mesenchyme and first pouch epithelia.

(a), Schematic showing the pseudobranch (arrows), gill filaments (branched green structures) connected to gill bars (blue), teeth (purple), vasculature (pink), and jaw and jaw-support skeleton (gray). (b) Hematoxylin and Eosin-stained sections show emergence of the pseudobranch bud at 4 dpf (adapted from https://bio-atlas.psu.edu/zf/view.php?atlas=5&s=41), five filaments at 17 dpf (adapted from https://bio-atlas.psu.edu/zf/view.php?atlas=65&s=1738), and the fused pseudobranch at 90 dpf (adapted from https://bio-atlas.psu.edu/zf/view.php?atlas=29&s=312). (c) Dissected adult pseudobranch shows the ophthalmic artery connecting it to the eye. (d) Alcian staining shows five cartilage rods in the pseudobranch and similar cartilage in gill primary filaments. (e) Photoconverted kikGR-expressing mesenchyme (red) from the dorsal first arch (numbered) at 1.5 dpf contributes to the palatoquadrate cartilage (pq) and pseudobranch mesenchyme (arrow) at 3.5 dpf. Photoconverted dorsal second arch cells do not contribute to the pseudobranch. In green, fli1a:GFP labels the vasculature and neural crest-derived mesenchyme, with mesenchyme also labeled by unconverted sox10:kikGR. (f) In fgf10:nEOS embryos, photoconversion of first pouch endoderm (numbered) at 1.5 dpf labels the pseudobranch epithelium (arrow) at 5 dpf. n numbers denote experimental replicates in which similar contributions were observed. Scale bars, 50 µm.

Figure 1—figure supplement 1
Development of zebrafish pseudobranch and lineage analysis of gill filament epithelia.

(a) In Sox10:Cre; acta2:loxP-BFP-Stop-loxP-dsRed fish at 5 dpf, the developing pseudobranch (white arrow) and gill buds (yellow arrow) consist of Cre-converted dsRed+ neural crest-derived mesenchyme (magenta) and unconverted BFP+ epithelia (gray). (b) At 6 dpf, kdrl:mCherry labeling of vasculature reveals a branch of the first aortic arch in the position of the pseudobranch (white arrow), and branches of the posterior aortic arches in the positions of the gills (yellow arrow). (c) Endoderm is labeled in red by adding 4OH-tamoxifen to sox17:CreERT2; ubb:loxP-Stop-loxP-mCherry fish at 6.5 hr post-fertilization to induce Cre recombination that removes the Stop cassette and allows mCherry expression (mCherry channel alone shown in inset). Co-localization shows fgf10b:nEOS expression (green) in endodermal pouches. (d), Endoderm labeling by addition of 4OH-tamoxifen to sox17:CreERT2; ubb:loxP-Stop-loxP-mCherry fish at 6.5 hr post-fertilization results in contribution to cdh1:mlanYFP+ pseudobranch epithelium at 5 dpf. (e,f) In fgf10:nEOS embryos, photoconversion of first pouch endoderm (and some more ventral mandibular cells) at 1.5 dpf labels pseudobranch (white arrow) but not gill epithelia (yellow arrow) at 5 dpf in (e), and conversion of third pouch cells labels the first gill filament epithelium (yellow arrow, boxed region magnified to right and shown in merged and red-only channels) in (f). Scale bars, 50 µM.

Shared regulatory program for pseudobranch and gill development.

(a-c) In the pseudobranch (white arrows) and gill filaments (yellow arrows), gata3-p1:GFP labels growing buds, ucmaa-p1:GFP labels cellular cartilage (distinct from hyaline cartilage, arrowhead), and irx5a-p1:GFP labels pillar cells. sox10:dsRed labels cartilage for reference. Images in (b) and (c) are confocal projections, with magnified regions shown below in single sections for gata3-p1:GFP and ucmaa-p1:GFP. Scale bars, 50 µM.

Figure 3 with 2 supplements
Pseudobranch and gill development requires gata3 function.

(a) Similar expression of gata3 and gata2a in developing pseudobranch (white arrows) and gill regions (yellow arrows). (b) Sox10:Cre; acta2:loxP-BFP-Stop-loxP-dsRed labels Cre-converted dsRed+ neural crest-derived mesenchyme (magenta) and unconverted BFP+ epithelia (gray). (c) gata3-p1:GFP labels pseudobranch and gill filament buds, and sox10:dsRed labels cartilage. For both (b) and (c), 3/3 gata3 mutants displayed reduced formation of the pseudobranch (white arrows) and gill filaments (yellow arrows), compared to 3 controls each. Scale bars, 50 µM.

Figure 3—figure supplement 1
Pseudobranch shares gene expression with gill filaments.

(a) Expression of ucmaa in pseudobranch cartilage from one-year-old fish. (b,c) Developing pseudobranch (white arrows) and gill buds (yellow arrows) express gata3 and gata2a at 5 dpf. DAPI labels nuclei in blue (a) or white (b,c). Scale bars, 50 µM.

Figure 3—figure supplement 2
The pseudobranch shares irx5a-p1 pillar cell enhancer activity with gill filaments.

(a) An intergenic irx5a-p1 region displays accessible chromatin specifically in pillar cells at 60 dpf. (b) irx5a-p1 drives GFP expression in pillar cells of the developing gills (yellow arrows) and pseudobranch (insets). (c,d) Dissected pseudobranch from a 60 dpf irx5a-p1:GFP adult shows GFP-positive pillar cells adjacent to a core of filament cartilage. The boxed region magnified below depicts an individual pillar cell (white arrow) flanked by characteristic lacunae (yellow arrows). Cartilage is labeled by sox10:dsRed in (b) and (c). Scale bars, 50 µM.

Author response image 1

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene
(Danio rerio)
ucmaaEnsembl: ENSDARG00000027799
Gene
(Danio rerio)
gata3Ensembl: ENSDARG00000016526
Gene
(Danio rerio)
gata2aEnsembl: ENSDARG00000059327
Gene
(Danio rerio)
irx5aEnsembl: ENSDARG00000034043
Genetic reagent (Danio rerio)TübingenZIRCRRID:ZIRC_ZL57Wildtype strain of zebrafish
Genetic reagent (Danio rerio)Tg(fli1a:eGFP)y1Lawson and Weinstein, 2002
Genetic reagent (Danio rerio)Tg(sox10:kikGR)el2Balczerski et al., 2012
Genetic reagent (Danio rerio)Tg(ucmaa_p1:GFP, cryaa:Cerulean)el851Fabian et al., 2022
Genetic reagent (Danio rerio)Tg(gata3_p1:GFP, cryaa:Cerulean)el858Fabian et al., 2022
Genetic reagent (Danio rerio)Tg(fgf10b:nEOS)el865Fabian et al., 2022
Genetic reagent (Danio rerio)Tg(–3.5ubb:loxP-STOP-loxP-mCherry)el818Fabian et al., 2020
Genetic reagent (Danio rerio)Tg(Mmu.Sox10-Mmu.Fos:Cre)zf384Kague et al., 2012
Genetic reagent (Danio rerio)Tg(actab2:loxP-BFP-STOP-loxP-dsRed)sd27Kobayashi et al., 2014
Genetic reagent (Danio rerio)Tg(−6.5kdrl:mCherry)ci5Proulx et al., 2010
Genetic reagent (Danio rerio)Tg(–5.0sox17:Cre-ERT2,myl7:DsRed)sid1TgHockman et al., 2017
Genetic reagent (Danio rerio)Tg(cdh1:mlanYFP)xt17TgCronan and Tobin, 2019
Genetic reagent (Danio rerio)gata3b1075Sheehan-Rooney et al., 2013
Genetic reagent (Danio rerio)Tg(irx5a-p1:GFP, cryaa:Cerulean)el859This paperSee Materials and Methods, Section Zebrafish Lines
Recombinant DNA reagentPCS2FA-transposaseTol2KitPUBMED: 17937395
396.pCS2-transposase
Recombinant DNA reagentpDestTol2AB2-irx5a-p1-E1B:GFP_pAThis paperSee Materials and Methods, Section Zebrafish Lines
Sequence-based reagentucmaa RNAScope probe (Danio rerio); Channel 1ACD Bio
Sequence-based reagentgata2a RNAScope probe (Danio rerio); Channel 1ACD Bio
Sequence-based reagentgata3 RNAScope probe (Danio rerio); Channel 2ACD Bio
Commercial assay or kitIn-Fusion HD Cloning PlusTakaraTakara:638,910
Commercial assay or kitRNAScope Multiplex Fluorescent v2 AssayACD BioACD Bio:323,100
OtherDraq5 nuclear dyeAbcamAbcam:Ab108410See Materials and Methods, Section Imaging

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  1. Mathi Thiruppathy
  2. Peter Fabian
  3. J Andrew Gillis
  4. J Gage Crump
(2022)
Gill developmental program in the teleost mandibular arch
eLife 11:e78170.
https://doi.org/10.7554/eLife.78170