High-Resolution, 3D Imaging of the Zebrafish Gill-Associated Lymphoid Tissue (GIALT) Reveals a Novel Lymphoid Structure, the Amphibranchial Lymphoid Tissue

The zebrafish is extensively used as an animal model for human and fish diseases. However, our understanding of the structural organization of its immune system remains incomplete, especially the mucosa-associated lymphoid tissues (MALTs). Teleost MALTs are commonly perceived as diffuse and scattered populations of immune cells throughout the mucosa. Yet, structured MALTs have been recently discovered in Atlantic salmon (Salmo salar L.), including the interbranchial lymphoid tissue (ILT) in the gills. The existence of the ILT was only recently identified in zebrafish and other fish species, highlighting the need for in-depth characterizations of the gill-associated lymphoid tissue (GIALT) in teleosts. Here, using 3-D high-resolution microscopy, we analyze the GIALT of adult zebrafish with an immuno-histology approach that reveals the organization of lymphoid tissues via the labeling of T/NK cells with an antibody directed to a highly conserved epitope on the kinase ZAP70. We show that the GIALT in zebrafish is distributed over at least five distinct sub-regions, an organization found in all pairs of gill arches. The GIALT is diffuse in the pharyngeal part of the gill arch, the interbranchial septum and the filaments/lamellae, and structured in two sub-regions: the ILT, and a newly discovered lymphoid structure located along each side of the gill arch, which we named the Amphibranchial Lymphoid Tissue (ALT). Based on RAG2 expression, neither the ILT nor the ALT constitute additional thymi. The ALT shares several features with the ILT such as presence of abundant lymphoid cells and myeloid cells embedded in a network of reticulated epithelial cells. Further, the ILT and the ALT are also a site for T/NK cell proliferation. Both ILT and ALT show structural changes after infection with Spring Viraemia of Carp Virus (SVCV). Together, these data suggest that ALT and ILT play an active role in immune responses. Comparative studies show that whereas the ILT seems absent in most neoteleosts (“Percomorphs”), the ALT is widely present in cyprinids, salmonids and neoteleosts, suggesting that it constitutes a conserved tissue involved in the protection of teleosts via the gills.

. General organization of the zebrafish GIALT, other specimens.
Representative deconvolved confocal images of gills from 3 adult zebrafish acquired from an oblique longitudinal (A,C) and transversal orientations (B). Images were acquired from 30 μm whole-body cryosections stained with phalloidin (green) and DAPI (blue) and where T / NK cells are labeled with anti-ZAP70 antibody (red hot). The gills from the 3 different specimens displayed a distribution of ZAP70 positive cells similar to the Figure 1 with a segmentation into five subregions (1-5) (ILT, interlamellar region-lamellae-efferent aspect of filaments, interbranchial septum, gill arch, T cell clusters at the base of filaments on each side of the gill arch). (A-C) Images are maximum intensity projections (MIP). Annotations: Aa, Afferent artery; Aaa, Afferent arch artery; Bc, Branchial cavity; C, Cartilage; dILT, distal Interbranchial Lymphoid Tissue; Eaa, Efferent arch artery; Ea, Efferent artery; F, Filament; Ga, Gill arch; Gr, Gill raker; La, Lamellae; pILT, proximal Interbranchial Lymphoid Tissue; S, Septum; Sw, Septum wall. Scale bars: 50 μm (A) and 100 μm (B,C). Representative deconvolved confocal high-resolution images of fli:GFP adult zebrafish ALT acquired with a transversal (A,B) and sagittal orientations (C). Images were acquired from 30 μm whole-body cryosections stained with phalloidin (green) and DAPI (blue). Endothelial cells from blood and lymphatic vessels express GFP (Red hot). (A,B) The ALT is in direct contact with the vascular compartment of the most basal lamellae of each filaments (cyan arrows). In addition, the ALT is close to many vessels located on the nearby connective tissue (cyan arrowheads). (C) Occasionally, the ALT lie on anastomotic vessels (cyan star) that connect two successive efferent arteries (green arrowheads). Images are maximum intensity projections. Annotations: ALT, Amphibranchial Lymphoid Tissue; Bc, Branchial cavity; Ct, Connective tissue; Ea, Efferent artery; Ga, Gill arch; La, Lamellae and Rc, Rodlet cells. Scale bars: 20 μm (A-C).

Figure S5. Adhesion of immune cells to the endothelium of vascular blebs.
Representative deconvolved confocal high-resolution images of adult zebrafish gills acquired with a coronal orientation. (A-A') 30 μm whole-body cryosection from mhc2:GFP (green) zebrafish stained with phalloidin (Red hot) and DAPI (blue). Many mhc2 expressing cells bind to the endothelium of the vascular bleb (cyan arrowheads), right beneath the ILT. Some actin-rich GFP negative circulating cells also adhere to the endothelium of vascular blebs (yellow stars).  Representative deconvolved confocal images of an adult zebrafish infected with SVCV. The images were acquired from 30 μm whole-body cryosections. Unless specified, the sections were stained with phalloidin (green) and DAPI (blue), and SVCV particles were labeled with anti-SVCV-N antibody (magenta hot). (A) Afferent arch artery of an infected fish showing circulating infected cells (red) with fragmented nuclei (cyan hot) (yellow arrowhead) and circulating viral particles (white arrowhead). Although only a few endothelial cells bear an anti-SVCV-N signal, a high SVCV-N signal was found in the connective tissue beyond the smooth muscle layers of major blood vessels. For an optimal quality, we recommend to download the files to watch them.
Video S1. Organization of the zf-GIALT from a single branchial cavity.
Video of a representative 3D deconvolved confocal image of an adult zebrafish branchial cavity displaying the four gill arches with a transversal orientation. The video was acquired from a 30 μm whole-body cryosections stained with phalloidin (red) and DAPI (blue), and where T / NK cells are labeled with anti-ZAP70 antibody (white). At the beginning of the video, the pharynx is at the bottom, the skin at the top, teeth to the left and the mouth is toward to right.

Video S2. Interbranchial lymphoid tissue
Video of a representative 3D deconvolved confocal image of an adult zebrafish proximal interbranchial lymphoid tissue seen with a coronal orientation. The video was acquired from a 30 μm whole-body cryosections stained with phalloidin (red) and DAPI (blue), and where T / NK cells are labeled with anti-ZAP70 antibody (white). Note the contrast of the ZAP70 labeling between the interbranchial lymphoid tissue in the middle and the rest of the filaments.

Video S3. Organization of the zf-GIALT at the gill arch
Video of a representative 3D deconvolved confocal image of an adult zebrafish gill arch seen with a transversal orientation. The video was acquired from a 30 μm whole-body cryosections stained with phalloidin (red) and DAPI (blue), and where T / NK cells are labeled with anti-ZAP70 antibody (white). Note the presence of the two T cell cluster on the sides of the gill arch, at the base of filaments.

Video S4. Organization of the zf-GIALT at the pharyngeal side of the gill arch.
Video of a representative 3D deconvolved confocal image of an adult zebrafish pharyngeal side of a gill arch seen with a transversal orientation. The video was acquired from a 30 μm whole-body cryosections stained with phalloidin (red) and DAPI (blue), and where T / NK cells are labeled with anti-ZAP70 antibody (white). Note the presence of the two T cell clusters on the sides of the gill arch, at the base of filaments.
9 Video S5. Vascular bleb Video of a representative 3D deconvolved confocal image of a fli:GFP adult zebrafish vascular blebs seen with a transversal orientation. The video was acquired from a 30 μm whole-body cryosections stained with phalloidin (green) and DAPI (blue), and where endothelial cells are fluorescent (red hot). The video goes through the optical sections of the 3D acquisition. The vascular bleb are at the bottom right and are located at the uppermost region of the septum. The vascular blebs represent radial enlargement of the afferent artery of each filament. Note that the vascular bleb from two successive filaments does not directly connect with each other's. Lamellae are seen at the bottom right of the video, only the cells on the edge of the vascular compartment express GFP.

Video S6. Vascular bleb does not connect with each other's but forms radial pits.
Video of a representative 3D deconvolved confocal image of a fli:GFP adult zebrafish vascular blebs seen with a transversal orientation. The video was acquired from a 30 μm whole-body cryosections stained with phalloidin (green) and DAPI (blue), and where endothelial cells are fluorescent (red hot). The video goes through the optical sections of the 3D acquisition. The vascular bleb is at the middle left of the video, to the right is an afferent artery. Through the optical sections, the vascular bleb is getting closed, indicating it is forming a pit and not a tube directly interconnecting with the vascular blebs of other filaments.

Video S7. 3D architecture of the amphibranchial lymphoid tissue.
Video of a representative 3D deconvolved confocal image of an adult zebrafish gill arch seen with a sagittal orientation. The video was acquired from a wholemount gill arch stained with phalloidin (red) and DAPI (blue), and where T / NK cells are labeled with anti-ZAP70 antibody (white). The ALT is a continuous lymphoid structure along the sides of gill arches. The ALT is at its thinnest at the level of efferent arteries and at its thickest when in-between filaments. At the start of the video, the gill rakers are at the bottom and the filaments are at the top. To note an interesting morphological peculiarity, a filament to the right is segmented in two.

Video S8. Neuromast/taste-bud like structure of the ALT.
Video of a representative 3D deconvolved confocal image of an adult zebrafish ALT displaying a neuromast/taste-bud like structure. The video was acquired from a 30 μm whole-body cryosections stained with phalloidin (green) and DAPI (blue). These structures are protruding from the ALT in a similar fashion as taste-buds and neuromasts.

Video S9. Highly SVCV infected cell contained in the lamellae blood compartment.
Video of a representative 3D deconvolved confocal image of an SVCV infected adult zebrafish lamellae displaying an infected cells contained in the blood of a lamellae. The video was acquired from a 30 μm whole-body cryosections stained with phalloidin (green) and DAPI (blue), and where SVCV particles are labeled with an anti-SVCV-N antibody (red hot). These highly infected cells have a fragmented nuclei and appears stuck between pillar cells, which are the cells displaying a ring like phalloidin pattern.