The ciliary zonules provide a pathway for immune cells to populate the avascular lens during eye development

Animals

All animal studies performed are in compliance with the Institutional Animal Care and Use Committee (IACUC) guidelines, approved by Thomas Jefferson University, and with the Association of Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Vision Research. Day 5, 8–10, 12, and 15 chick embryos were used in these studies and the embryonated eggs were obtained from Poultry Futures, Lititz, PA, USA. These studies also included embryonic day 14 and adult mice on the mixed genetic background 129Sv/C57B1/6J.

Sample preparation for cryosectioning

Whole embryonic chick eyes between E8 and E15 were removed after decapitation. Whole heads were used for studies of E5 chick embryo eyes. Samples were fixed in 4% paraformaldehyde overnight at 4°C, cryoprotected in 30% sucrose in Dulbecco’s phosphate-buffered saline (DPBS) with calcium and magnesium for 24 h at 4°C. E14 mouse heads and adult mouse eyes were removed after sacrificing, fixed, and cryoprotected with the same protocol. Samples were then placed in optimal cutting temperature (OCT) freezing media (Polyscience, Niles, IL, USA) and flash frozen using dry ice and 100% ethanol; 30-µm-thick cryosections were cut using a Microm HM 550 Cryostat.

Immunofluorescence labeling of tissue sections

Cryosections were permeabilized in 0.5% Triton-X100 (BP151-100, Fisher) for 1 h and then incubated in block buffer (3% bovine serum albumin [BSA], 5% goat/donkey serum, 0.5% Triton-X100) for 2 h at room temperature prior to incubating with primary antibodies diluted in the block buffer at 37^oC overnight. The sections were washed and incubated for 1 h at 37°C in fluorescent-conjugated secondary antibody (Jackson ImmunoResearch Laboratories) diluted in block buffer containing fluorescent-conjugated phalloidin (Invitrogen) and 4′,6-diamidino-2-phenylindole (DAPI) (Biolegend, 422,801). Sections were washed and mounted in ProLong Diamond Antifade Mountant (Invitrogen, P36970). Primary antibodies used for the embryonic chick studies included fibrillin-2 (JB3, DSHB), tenascin-C (AB19013, Millipore), fibronectin (PA1-23693, ThermoFisher), collagen I (NBP1-300054, Novus), KUL-01 (MCA5770, BioRad), and CD45 (MCA2413GA, BioRad). Primary antibodies used for the mouse studies included thrombospondin (SC-393504, SantaCruz), MAGP1 (gift from Dr Robert Mecham, Washington University School of Medicine in St. Louis), and fibrillin-2 (gift from Dr Robert Mecham).

Antigen retrieval

For studies with the fibronectin antibody the cryosections were treated with an antigen retrieval protocol prior to immunolabeling. The cryosections were brought to room temperature, washed in 1× Tris-buffered saline with 0.1% Tween 20 detergent (TBST) (1× TBS + 0.05% Triton-X100) for 15 min, boiled in sodium citrate buffer (95°C–100°C) for 20 min, and allowed to cool in sodium citrate buffer for 10 min. Sections were blocked in 10% goat serum diluted in 1× TBST for 1 h at room temperature and prior to immunolabeling with the fibronectin antibody diluted in 1% BSA.

RAW 264.7 cell culture

RAW 264.7 (ATCC #TIB-71) is a mouse monocyte/macrophage cell line. They are cultured in complete media (Dulbecco’s modified eagle medium [DMEM] [ATCC #30-2002]) supplemented with 10% fetal bovine serum (FBS), penicillin/streptomycin, and l-glutamine in a 37°C incubator with 5% CO₂.

Human non-pigmented ciliary epithelial cell culture

Human non-pigmented ciliary epithelial cells (HNPCEpiCs) (ScienCell #6580) were cultured in epithelial cell medium (EpiCM) (ScienCell #4101), supplemented with 2% FBS (#0010), 1% epithelial cell growth supplement (EpiCGS) (#4152), and penicillin/streptomycin (#0503). Primary HNPCEpiCs are received frozen, thawed in a 37°C water bath, gently resuspended, and plated in a T-75 flask coated with 10 mg/mL poly-l-lysine (ScienCell #0413) in complete media in a 37°C incubator with 5% CO₂. The cells were grown to 90–100% confluency before passaging and splitting into 35 mm culture dishes (Corning #430165) or six-well culture plates (Corning #3516). Once cells reach 100% confluency, the culture dishes/wells were decellularized using 20 mM ammonia hydroxide (ThermoFisher #390030010) in phosphate-buffered saline (PBS) plus 0.5% Triton-X100 for 5 min at 37°C, leaving behind the ciliary zonule protein-containing extracellular matrix substrates. The matrix substrates were washed twice with DPBS without calcium and magnesium (Corning #21-031-CV), dried down, and dishes stored at 4°C.

Time lapse and cell tracking

RAW 264.7 (1 × 10⁴) cells per well were plated in six-well plates with rehydrated HNPCEpiC-produced matrix substrates or tissue culture plastic alone in complete media (DMEM [ATCC #30-2002] supplemented with 10% FBS, penicillin/streptomycin, and l-glutamine). The cultures were incubated for 2 h at 37°C to allow the RAW 264.7 cells to attach to the HNPCEpiC matrix substrates or the uncoated tissue culture wells. Time-lapse imaging was performed with a Nikon Eclipse Ti microscope with an attached Tokai Hit incubation system that maintains the cultures at 37°C and 5% CO₂. Using NIS-Elements AR 5.11.03 64-bit software, a 24-h time-lapse video was acquired. RAW cell tracking and movement parameters were determined from the time-lapse images using Imaris Software version 9.7.0. Cell tracks were evaluated using the “spot” tool and default settings (“Track Spots (over time),” “Classify Spots,” and “Object-Object Statistics”). Various statistical values were determined, including track length, track displacement, mean speed, and straightness of the path of movement. Bar graphs were created using GraphPad Prism 9 and the significance determined comparing the parameters analyzed for RAW 264.7 cell migration on the HNPCEpiC matrix to tissue culture plastic.

Immunofluorescence staining of HNPCEpiC-produced matrix substrates

Decellularized HNPCEpiC culture substrates prepared in 35 mm tissue culture dishes were treated with 0.5% Triton-X100 for 30 min at room temperature and then blocked in 5% goat serum for 30 min at room temperature. The substrates were incubated in primary antibody diluted in 0.1% Tween20 (FisherScientific #BP337-500) overnight at 37°C. Primary antibodies included (tenascin-C [Millipre #AB19013], fibrillin-1 [abcam ab68444], and fibronectin [abcam PA1-23693]). Dishes were then washed two times with PBS, followed by a 1-h incubation at 37°C in secondary antibody diluted in 0.1% Tween20, washed twice in PBS and then mounted in ProLong Diamond Antifade Mountant (Invitrogen, P36970).

Confocal image analysis and Imaris image analysis

Images were acquired using Zeiss LSM800 confocal microscope. Confocal images were obtained at either 5× or 40× and then analyzed using Zeiss Zen Software. To provide an overview of the whole eye, 5× tiles were used. For the studies at 40×, z-stacks were collected, each optical slice at either 0.33 or 0.50 µm. Three-dimensional surface renderings were generated using the Imaris software (version 9.7) 3D View tool. The entire z-stack is generated as a 3D surface structure that can be rotated and zoomed in without distortion of the original image.

Identification of matrix compartments in the developing eye that could guide immune cell migration to the embryonic lens

We have examined the development-state-specific expression and organization of cell migration-promoting matrix molecules in three different structures of the chick embryo eye whose proximity to the lens makes them potential paths for the delivery of lens tissue-resident immune cells. They included: region 1—the ciliary zonules that link the highly vascularized ciliary body to the avascular lens; region 2—the vitreous humor that contacts the posterior lens capsule; and region 3—the matrix associated with the pecten oculi, a vascular structure that extends through the vitreous from the posterior retina to the ciliary body, as modeled in Figure 1. Cryosections prepared from chick embryo heads at embryonic day 5 (E5) and chick embryo eyes at both E9 and E15 were co-immunolabeled for fibrillin-2 (Figure 2(A), (C), (D), (F), (G), and (I)) and tenascin-C (Figure 2(B), (C), (E), (F), (H), and (I)). Nuclei were labeled with DAPI, and the sections imaged by confocal microscopy at low magnification. This approach provided an overview of the distribution of fibrillin-2 and tenascin-C matrices across the entire eye during development. The results showed that tenascin-C co-localized with fibrillin-2 in the ciliary zonules forming between the developing ciliary body and lens as early as E5 and were extended posteriorly into the vitreal compartment (Figure 2(A) to (C)). Both fibrillin-2 and tenascin-C remained components of the ciliary zonules and vitreous throughout development (Figure 2(D) to (I)). At E15, they were also highly localized to the matrix associated with the surface of the pecten oculi (Figure 2(G) to (I)). These results showed that fibrillin-2/tenascin-C-rich fibrillar matrices had already begun to organize adjacent to the equatorial and posterior aspects of the lens very early in development, prior to our earliest time point of E5, and continued to expand over time.

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Figure 1.

Diagram of the embryonic chick eye, illustrating the three regions of interest that were examined as potential pathways by which resident immune cells could gain access to the avascular lens. (A) Region 1 shows an immune cell path across the ciliary zonules (CZs) that link the highly vascularized ciliary body (CB) to the lens, where we have identified the presence of immune cells in the adult mouse eye. (B) Region 2 identifies the vitreous, a matrix component bounded by the posterior zonules, the posterior lens, and the retina, which is a source of hyalocytes. (C) Region 3 shows the pecten oculi, a vascular-rich structure in avian species that extends from the retina to the ciliary body. (A color version of this figure is available in the online journal.)

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Figure 2.

Overview of tenascin-C/fibrillin-2 fibril organization during development of the chick embryo eye; 5× confocal tiles were acquired of cryosections of embryonic day 5 (A to C), 9 (D to F), and 15 (G to I) chick eyes that were co-immunolabeled for fibrillin-2 (green) and tenascin-C (red), and co-labeled for nuclei with DAPI (blue). Mag. bars: E5 = 100 µm, E9 = 500 µm, E15 = 1000 µm. (A color version of this figure is available in the online journal.)

Cell migration–promoting matrix proteins tenascin-C and fibronectin are key molecules of the developing ciliary zonule fibrils linking the ciliary body to the lens

Using high-resolution confocal microscopy, we examined the development-state-specific organization and distribution of migration-promoting matrix molecules associated with the ciliary zonules fibrils. These studies were focused on determining the presence of these molecules in developing zonule fibers and their potential to provide a substrate for the movement of immune cells from the ciliary body to the lens. These questions were examined by immunolabeling cryosections of E5 chick embryo heads, and both E9 and E15 chick embryo eyes for the zonule backbone protein fibrillin-2 and co-immunolabeling for either tenascin-C (Figure 3) or fibronectin (Figure 4), both key molecules in the promotion of cell adhesion and migration.^{32 –35} Studies using the fibronectin antibody required performing an antigen retrieval protocol prior to immunolabeling, and the results are presented as fibrillin-2/fibronectin overlays (Figure 4), with separate images for each of these matrix molecules included together with the overlays in Supplemental Figure 1. All sections were co-labeled for nuclei with DAPI. Confocal microscopy imaging showed that as early in eye development as E5, at which time the presumptive ciliary body is located in very close proximity to the equator of the developing lens, fibrillin-2-rich zonule fibrils were already present in the region between the ciliary body and the lens. Many of these early zonule fibrils had a fine, feathery appearance (Figure 3(A)), and there was a high degree of co-localization of tenascin-C with fibrillin-2 in this wispy matrix meshwork (Figure 3(Ad)). The use of the antigen retrieval protocol for fibronectin labeling revealed a subset of fibrillin-rich zonules with a more highly developed fibrillar morphology that are co-labeled with fibronectin (Figure 4(A)). Fibronectin was also detected in close vicinity to but not directly associated with the fibrillin-2-labeled fibrils, possibly reflecting the reported role of fibronectin in the assembly of fibrillin. ³⁶

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Figure 3.

Tenascin-C is highly co-localized to fibrillin-2-rich zonule fibrils during their developmental-state-specific formation. Cryosections of chick embryo heads at E5 (A), and whole chick embryo eyes at E9 (B) and E15 (C) were co-immunolabeled for the matrix proteins fibrillin-2 (green) and tenascin-C (red). Nuclei were labeled with DAPI (blue); 40× confocal z-stacks were acquired and shown as projection images. At E9 and E15, images were acquired in three distinct regions of the ciliary zonules including where the upper ciliary zonules extend along the anterior region of the lens (Ba–d; Ca–d), the upper zonules that extend anteriorly from the lens equator (Be–h; Ce–h), and the lower zonules that extend posteriorly from the lens equator (Bi–l; Ci–l). Mag. bars = 20 µm. (A color version of this figure is available in the online journal.)

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Figure 4.

Fibronectin highly co-localizes to fibrilin-2-rich fibrillar structures of the developing zonules. Cryosections of chick embryo heads at E5 (A), and whole chick embryo eyes at E9 (B) and E15 (C) were co-immunolabeled for the matrix proteins fibrillin-2 (green) and fibronectin (red) following antigen retrieval. Nuclei were labeled with DAPI (blue); 40× confocal z-stacks were acquired and shown as projection images of the overlay between fibrillin-2 and fibronectin. At E9 and E15, images were acquired in three distinct regions of the ciliary zonules, including where the upper ciliary zonules extend along the anterior region of the lens (Ba, b; Ca, b), the upper zonules that extend anteriorly from the lens equator (Bc, d; Cc, d), and the lower zonules that extend posteriorly from the lens equator (Be, f; Ce, f). Mag. bars = 20 µm. (A color version of this figure is available in the online journal.)

As the eye continues to develop, the fibrillin-2-based zonule fibers between the ciliary body and the lens become more extensive and are increasingly organized into thicker, more linear fibrillar structures (Figure 3(Bb, f, j) and (Cb, f, j)). By E9, the three distinct ciliary zonule regions characteristic of adults could be identified. These regions include: (1) the lower zonules that extend posteriorly along the lens equator (Figures 3(Bi), (Ci) and 4(Be), (Ce)); (2) the upper zonules that extend anteriorly along the lens equator (Figures 3(Be), (Ce) and 4(Bc), (Cc)); and (3) a partial extension of the upper zonule fibrils along the anterior surface of the lens (Figures 3(Ba), (Ca) and 4(Ba), (Ca)). Immunolabeling for fibrillin-2 at E9 revealed that the upper zonule fibers no longer had a wispy appearance and had begun to acquire a more distinct fibrillar morphology (Figure 3(Bf)). The fibrils of the upper zonules continued to thicken by E15, their fibrillar structure becoming even more pronounced (Figure 3(Cf)). Also by E9, the upper zonule fibers have extended along the edge of the anterior surface of the lens (Figure 3(Bb)) and were maintained similarly throughout eye development (Figure 3(Cb)). In contrast to the upper zonule fibrils, the morphology of the fibrillin-2-rich lower zonule fibrils appeared finer and less organized at both E9 and E15 (Figure 3(Bj) and (Cj)).

In the studies presented above, fibrillin-2 had been co-immunolabeled with antibody to tenascin-C. This extracellular matrix protein co-localized with fibrillin-2 along most but not all of the upper zonule fibrils at E9 and E15 (Figure 3(Bg, h) and (Cg, h)), and had a high level of coincidence in the ciliary zonule fibrils extended along the anterior surface of the lens (Figure 3(Bb–d) and (Cb–d)). In the fibrillar meshwork that characterized the lower zonule fibrils at these developmental times much of the tenascin-C labeling was also coincident with fibrillin-2 (Figure 3(Bj–l) and (Cj–l)). Note that, in addition to its presence in the ciliary zonules, tenascin-C has been identified as an integral component of the lens capsule. ²² Studies performed after antigen retrieval that were co-immunolabeled for fibronectin and fibrillin-2 highlighted the co-localization of these two matrix proteins in a subset of the upper zonule fibers that had already formed a distinct linear, fibrillar structure (Figure 4(Bd) and (Cd)). Fibronectin, like tenascin-C, localized to the fibrillin-2 containing zonule fibrils extending anteriorly along the lens surface (Figure 4(Bb) and (Cb)), and, as previously shown, also is a component of the lens capsule. ²² Using the antigen retrieval protocol required for immunolabeling with the fibronectin antibody revealed that there is a significant subset of lower zonules that had become organized into more structurally distinct zonule fibrils that were positive for both fibronectin and fibrillin-2 (Figure 4(Bf) and (Cf)). Separate images for each of these matrix molecules included together with the overlays are presented in Supplemental Figure 1.

As a comparison, we examined cryosections of E14 mouse eyes that were co-immunolabeled for fibrillin-2 and thrombospondin-1 (Supplemental Figure 2(B) to (D)) or for the fibrillin binding protein microfibril-associated glycoprotein-1 (MAGP1) and thrombospondin 1 (Supplemental Figure 2(E) to (G)). The pattern of organization of these ciliary zonule molecules in the E14 mouse eye confirmed that the developing ciliary zonules in the embryonic mouse are similar in appearance to those of the developing chick eye. We also performed co-immunolabeling for MAGP1 and thrombospondin-1 in adult mouse eyes (Supplemental Figure 2(I) to (K) and (M) to (O)). The labeling for MAGP1 showed that the zonules were mostly organized as very thick, rope-like structures and that thrombospondin-1 was primarily associated with thinner zonule fibrils still present in the adult (Supplemental Figure 2(E) to (G)).

Tenascin-C and fibronectin are components of fibrillin-2 fibrillar network in the vitreous of embryonic eyes

In addition to forming the backbone of the ciliary zonules, fibrillin has been identified as a matrix component of the vitreous. ³⁷ Using high-resolution confocal microscopy, we examined the development-state-specific organization of fibrillin-2 in the vitreous and its association with other matrix components that could support immune cell migration (Figure 5). By E5, a highly interwoven network of structurally fine fibrillin-2 fibrils had been assembled throughout the vitreous (Figure 5(B) and (C)). This fibrillar meshwork extended from a region adjacent to the posterior lens capsule, which had been previously shown to contain fibrillin-2, ²² to the retina. The structure and organization of the fibrillin-rich fibrils of the vitreous remained similar when examined at E9 (Figure 5(E) and (F)) and E15 (Figure 5(H) and (I)). Low-magnification imaging of whole eye cryosections co-immunolabeled for fibrillin-2 and tenascin-C at E9 showed many areas of the vitreous in which these two matrix proteins are coincident, as well as indicated that tenascin-C was present in regions outside of the fibrillin-2 fibrils (Figure 2(D) to (F)). High-magnification, high-resolution confocal imaging of the vitreous in cryosections of E9 eyes co-immunolabeled for tenascin-C and fibrillin-2 support this conclusion (Figure 6(B) and (E)). In this image, a single cell is present that highly expresses fibrillin-2, suggesting that the fibrillin-2 matrix is created by cells localized within the vitreous. We also found that fibronectin is a matrix element of the vitreous in the developing eye (Figure 6(C) and (F)). Like fibrillin-2, its structural organization is fibrillar, with co-immunolabeling for fibronectin and fibrillin-2 showing that the distribution of fibronectin was highly coincident with fibrillin-2 (Figure 6(F)). However, there were also fibrillin-2-rich fibrils that were not also labeled with fibronectin. We also examined the coincidence of fibrillin-2 with collagen I, the latter being a principal component of the adult vitreous. Co-immunolabeling for collagen I and fibrillin-2 revealed that there is a high level of co-localization of these two matrix proteins in the vitreous during development (Figure 6(D) and (G)).

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Figure 5.

Fibrilliin-2-rich fibrils extend throughout the vitreous during development. Cryosections of chick embryo heads at E5 (A to C), and whole chick embryo eyes at E9 (D to F) and E15 (G to I) were immunolabeled for the matrix protein fibrillin-2 (green) and co-labeled with fluorescent-conjugated phalloidin (white); 40× confocal z-stacks, each image representing a single optical slice of 0.33 µm, were acquired in the region along the posterior lens capsule (B, E, and H) and in more posterior aspects of the vitreous (C, F, and I), as indicated in the diagrams (A, D, and G). Mag. bars = 20 µm. (A color version of this figure is available in the online journal.)

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Figure 6.

Tenascin-C, fibronectin, and collagen I co-localize with the fibrillin-2 fibrils of the developing vitreous. Cryosections of chick embryo eyes at E9 were co-immunolabeled for fibrillin-2 (E to G, green) with either tenascin-C (B, E), fibronectin (C, F), or collagen I (D, G). Nuclei were labeled with DAPI (blue); 40× confocal z-stacks, each image representing a single optical slice of 0.33 µm, were acquired in the center of the vitreous, as indicated in the diagram (A). Mag. bars = 20 µm. (A color version of this figure is available in the online journal.)

The ciliary zonule fibrils provide a path for immune cells to travel from the ciliary body to the lens during development

Our findings above show that both fibronectin and tenascin-C are associated with the fibrillin-2-rich zonule fibrils that link the ciliary body to the lens from early stages of eye development. Among their many properties, these matrix proteins play key roles in promoting both the attachment and migration of immune cells.^{20,21,25 –27} Our previous studies showed the presence of immune cells migrating within the ciliary zonule fibrils at E15. ⁹ However, by this time in eye development, we also showed that resident immune cells had already populated the lens. ⁹ We now examined whether immune cells become associated with the zonule fibrils from earlier times of eye development. For these studies, the zonule fibers at E5, E9, and E15 were immunolabeled using antibody to tenascin-C and the sections co-immunolabeled with KUL-01, an antibody specific to chick monocytes and macrophages. Nuclei were labeled using DAPI and the sections imaged by high-resolution confocal microscopy along the zonule fibers (Figures 7 to 9).

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Figure 7.

Immune cells populate the ciliary zonules as early as E5. Cryosections of E5 heads were co-immunolabeled for tenascin-C (red) to highlight the ciliary zonules and KUL-01 (green), which is expressed by immune cells of the monocyte/macrophage lineage. Nuclei were labeled with DAPI (blue); 40× confocal z-stacks were acquired of the ciliary zonule region as identified in the diagram (A). Projection images at low magnification are shown with just the tenascin-C and DAPI labels (B, C) and the regions denoted by asterisks shown at high magnification including the KUL-01 labeling for the immune cells (Bi–iii; Ci–iii). Immune cells were identified along the zonule fibers. Mag. bars: (B) and (C) = 20 µm, (Bi–iii) and (Ci–iii) = 5 µm. (A color version of this figure is available in the online journal.)

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Figure 8.

Immune cells emerging from the ciliary body travel along the ciliary zonules during development. Cryosections of whole eyes at E9, diagrammed in (A) were co-immunolabeled for tenascin-C (red) to highlight the ciliary zonules and KUL-01 (green) expressed by immune cells of the monocyte/macrophage lineage (B to D). Nuclei were labeled with DAPI (blue); 40× confocal z-stacks were acquired along different regions of the ciliary zonules. Projection images at low magnification are shown with just the tenascin-C and DAPI labels (B to D) and the regions denoted by asterisks shown at high magnification including the KUL-01 labeling for the immune cells (Bi to iii; Ci; Di). Dashed line boxed areas in (C) and (D) are shown at higher magnification and including the KUL-01 label in the respective inserts. At this stage of development, immune cells were identified emerging from the ciliary body and along the zonule fibers. Mag. bars: (B) to (D) = 20 µm, and (Bi–iii), (Ci), and (Di) = 5 µm. (A color version of this figure is available in the online journal.)

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Figure 9.

Immune cells emerging from the ciliary body, travel along the ciliary zonules and migrate across the lens capsule during development. Cryosections of whole eyes at E15, diagrammed in (A) were co-immunolabeled for tenascin-C (red) to highlight the ciliary zonules and KUL-01 (green) expressed by immune cells of the monocyte/macrophage lineage (B to E). Nuclei were labeled with DAPI (blue); 40× confocal z-stacks were acquired along different regions of the ciliary zonules and along the matrix capsule that surrounds the lens. Projection images at low magnification are shown with just the tenascin-C and DAPI labels (B to E) and the regions denoted by asterisks are shown at high magnification including the KUL-01 labeling for the immune cells (Bi; Ci, ii; Di, ii, Ei–iii). The dashed line boxed area in (B) is shown at higher magnification and including the KUL-01 label in (Bii), with the individual cells denoted by 1, 2, and 3. At this stage of development, immune cells were identified emerging from the ciliary body, along the zonule fibers, and migrating across the lens capsule. Mag. bars: (B) to (E) = 20 mm and (Bi), (Bii), (Ci), (Cii), (Di), (Dii), and (Ei–iii) = 5 µm. (A color version of this figure is available in the online journal.)

Image analysis of the developing zonules linking the presumptive ciliary body to the lens at E5 (diagramed, Figure 7(A)) revealed that by this early stage of development immune cells have already populated the zonules (Figure 7(B) and (C)). Their localization along the zonules is shown in the low-magnification images by their nuclear label (Figure 7(B) and (C)). The immune cell identity of these zonule-linked cells, denoted by asterisks, is shown in higher magnification images that include the KUL-01 monocyte/macrophage label together with labeling for tenascin-C and DAPI (Figure 7(Bi–iii) and (Ci–iii)). The findings show that immune cells localized all along the zonule fibrils at E5, from positions near the developing ciliary body to where the zonules insert into the lens capsule. These findings provide the first evidence that immune cells traverse the ciliary zonule fibrils at very early stages of lens development. Later in development, E9, when the zonule fibers are thicker, more distinct and more widely distributed, KUL-01+ immune cells are observed migrating along individual zonule fibers (Figure 8(Bii, iii)), and are often located within the zonule fibril networks (Figure 8(Bi), (Ci), and (Di)). The localization of these immune cells along the zonule fibers between the ciliary body and the lens is denoted by the asterisks in the accompanying low-magnification images (Figure 8(B) to (D)). The boxed regions in Figure 8(C) and (D) are shown as inserts within the figures, highlighting the presence of KUL-01+ immune cells that are located both within the ciliary body (Figure 8(C)) and migrating out of the ciliary body to become linked to the zonules (Figure 8(D)). As we previously reported, ⁹ monocyte/macrophage immune cell populations continued to migrate between the ciliary body and the lens at E15 (Figure 9(A) to (E), low-magnification images showing overlay of tenascin-C and cell nuclei). Here, we show KUL-01+ immune cells in a high-magnification image of the boxed area of Figure 9(A) at three stages of their egress from the ciliary body to the zonules (Figure 9(Bii)), and additional examples of immune cells located within the ciliary zonules just outside of the ciliary body (Figure 9(Bi), (Ci, ii), and (Eii)). In addition to showing immune cells traveling across the ciliary zonule fibers at E15 (Figure 9(Bi), (Di, ii), and (Ei)), we now provide evidence of an immune cell crossing the lens capsule (Figure 9(Eiii)). These results support the conclusion that immune cells originating from the ciliary body migrate across the ciliary zonules to populate the lens beginning at early stages of eye development.

Immune cell migration on ciliary zonule protein matrices was examined by performing time-lapse imaging of RAW 264.7 cell migration, a monocyte/macrophage cell line, on a HNPCEpiC-conditioned substrate that mimics the protein composition and organization of ciliary zonule fibers. ³⁸ Non-pigmented ciliary epithelial cells, which in vivo are located along the inner surface of the ciliary processes, are the source of the ciliary zonule proteins that link the ciliary body to the lens. ¹⁷ For these studies, primary HNPCEpiCs are grown to confluence (Figure 10(A)) prior to decellularization. The presence of ciliary zonule matrix molecules in the HNPCEpiC-conditioned matrix on the culture substrate, including those examined in our developmental studies, is confirmed by immunolocalization analysis. Fibrillin, the backbone of the zonule fibrils (Figure 10(C)), fibronectin (Figure 10(B)), and tenascin-C (Figure 10(D)) are all found to be primary components of the HNPCEpiC-conditioned matrix, and to associate with fibrillar matrix structures. RAW 264.7 cells are plated on decellularized HNPCEpiC-conditioned matrix substrates or control tissue culture substrates and time-lapse imaging performed over 24 h in eight spots/well. Imaris imaging software was used to trace the migration track of 126 individual cells on the HNPCEpiC substrate and 114 individual cells on tissue culture plastic. Representative tracks of cells on the HNPCEpiC substrate (Figure 10(E)) and the tissue culture substrate (Figure 10(F)) are shown with purple denoting the time 0 start site and red the cells’ position at 24 h. Imaris image analytics were applied to the tracked cells to quantify the total average distance the cells moved (Figure 10(G), track length), how far the cells moved from their site of origin at time 0 (Figure 10(H), track displacement), how fast the cells moved (Figure 10(I), track speed), and the straightness of the cell migration track (Figure 10(J), track straightness). While the immune cells had a similar speed and overall track length, there were significant differences in how far the cells moved and their ability to move in a directional manner, with the HNPCEpiC-produced matrix substrate found to be an excellent matrix environment for promoting immune cell migration.

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Figure 10.

Ciliary zonule-like protein matrix promotes migration of immune cells. (A) Phase contrast image of primary cultures of human non-pigmented ciliary epithelial cells (HNPCEpiCs), the ciliary body cell type that produces the proteins that comprise the ciliary zonules (CZ). (B to D) Confocal microscopy imaging of HNPCEpiC-conditioned matrix substrates immunolabeled for (B) fibronectin, (C) fibrillin-1, or (D) tenascin-C. (E, F) Representative tracks of cells traced with Imaris software from 24-h time-lapse imaging of RAW 264.7 cells migrating on (E) HNPCEpiC-conditioned substrates and (F) tissue culture substrates. (Ei, Fi) phase contrast images of the position of cells at time 0 of the time-lapse study overlaid with the track of the cells with purple denoting the time 0 start site and red the cells’ position at 24 h. (Eii, Fii) Images showing the traced cell paths alone. (G to J) Bar graphs quantifying Imaris image analytics of 126 cells tracked on HNPCEpiC-conditioned substrates as compared to 114 cells tracked on tissue culture plastic to determine (G) track length, (H) track displacement, (I) track speed, and (J) track straightness. While the immune cells had a similar speed and overall track length, there were significant differences in how far the cells moved and their ability to move in a directional manner, with the HNPCEpiC-produced matrix substrate promoting directional immune cell migration. (A color version of this figure is available in the online journal.)

Immune cells associate with the fibrillar matrix of the vitreous during eye development

The vitreous has been identified as a site populated by hyalocytes, a monocyte/macrophage cell type with properties of tissue-resident innate immune cells, and which have been linked to both physiological and pathophysiological functions in the eye.^{28,39 –41} In this study, we examined whether the vitreous might also be a source of the resident immune cells that populate the lens during development. For these studies, cryosections prepared from E5 chick embryo heads, E10 chick embryo eyes, and E12 chick embryo eyes were immunolabeled for tenascin-C and co-immunolabeled for immune cells using either the KUL-01 antibody (Figure 11(A) and (Bb)) or the pan-leukocyte antibody CD45 (Figure 11(Bc) and (C)). The sections were co-labeled with DAPI to detect cell nuclei, and fluorescent-conjugated phalloidin which binds to F-actin. Confocal microscopy images were acquired in different regions of the vitreous as indicted in the diagrams (Figure 11(Aa) to (Ca)). While immune cells were detected among the tenascin-rich fibrils of the vitreous, including regions close to the retina (Figure 11(Ac) to (Cc)), we did not detect immune cells in the region where the vitreous contacts the posterior lens capsule, or in association with or crossing the posterior lens capsule (Figure 11(Ab) to (Cb)). These results do not exclude the possibility that immune cells from the vitreous compartment of the developing eye may provide resident immune cells to the lens at a stage of development we did not examine here. Note that the punctate labeling observed for the KUL-01 and CD45 immune cell labels may represent the ectodomain of these receptors left behind on the matrix as the cells migrate through the vitreous.

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Figure 11.

Immune cells associate with the vitreous matrix during eye development. Cryosections of (A) E5 chick embryo heads, (B) E10 chick embryo eyes, and (C) E12 chick embryo eyes were immunolabeled for KUL-01 or CD45 (green), co-immunolabeled for tenascin-C (red), and co-labeled for F-actin with fluorescent-conjugated phalloidin (white) and for nuclei with DAPI (blue).Two regions within the vitreous of the eye were analyzed for the presence of immune cells by confocal microscopy, both in close proximity to the lens (Ab, Bb, and Cb) and further down within the vitreous (Ac, Bc, and Cc) as illustrated in the diagrams (Aa, Ba, and Ca). While at early stages of development, immune cells were identified among the tenascin C–rich fibrils of the vitreous in a region close to the retina, at later stages, immune cells were detected throughout the vitreous matrix. No immune cells were found in direct contact with the posterior lens capsule at any stage analyzed (Ab, Bb, and Cb). Insets represent high magnification of immune cells marked by asterisks in the lower magnification panels. (Ab, c, Cb, c) are projection images; (Bb, c) represent a single optical plane. Mag. bars: (Ab–c), (Bb–c), (Cb–c) = 20 µm and 5 µm for insets. AqH: aqueous humor; CB: ciliary body; CZ: ciliary zonules; ONH: optic nerve head; PCB: presumptive ciliary body; PO: pecten oculi; VH: vitreous humor. (A color version of this figure is available in the online journal.)

Development of the pecten oculi, a vascular structure of the avian eye

Avian species develop a unique vasculature called the pecten oculi.^30,31 It is a pigmented, pleated structure, with the folds of the pleats joined together in a fan-like shape. During development, the pecten oculi begins to form at the head of the optic nerve, extends through the vitreous to a region adjacent to the ciliary body, and persists in the adult. Because of the potential that this vascular structure could be a source of immune cells that populate the developing avian lens, we examined its formation during development by labeling lens cryosections for F-actin with fluorescent-conjugated phalloidin at embryonic days 8, 9, 12, and 15, and imaging the sections by confocal microscopy (Figure 12). To determine whether there was a matrix link between the tip of the pecten oculi and the ciliary body, cryosections were also immunolabeled for tenascin-C and the results shown in higher magnification insets at E12 and E15 (Figure 12(C) and (Db)). The pecten oculi was shown to emerge as a thin projection from the posterior retina into the vitreous by E8 (Figure 12(A)). By E9, the developing pecten oculi was extended anteriorly toward the developing ciliary body (Figure 12(B), inset showing boxed area near the ciliary body at higher magnification). The classic pecten oculi pleats had formed by E12 (Figure 12(C)). Higher magnification at E12 showed that at this time in development the F-actin-labeled cells of the pecten oculi are very close to the ciliary body (Figure 12(Ci), with tenascin-C-rich fibrils providing a link between these two structures (Figure 12(Cii)). By E15, the structure of the pecten oculi was established, with its size and positioning in the vitreous requiring several sections to view its different regions (Figure 12(Da, b)). Higher magnification insets of the boxed region at E15 highlighted the tenascin-rich matrix fibrils that linked the pecten oculi to the ciliary body (Figure 12(Dbi, ii)).

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Figure 12.

Tenascin-C matrix fibrils link the developing vasculature of the pecten oculi with the ciliary body. Cryosections of E8, E9, E12, and E15 chick eyes were labeled with fluorescent-conjugated phalloidin (white) to visualize the development of the chick pecten oculi. At E8 (A), the pecten oculi is identified as a thin projection from the optic nerve head (ONH) into the vitreous. By E9 (B), the pecten oculi extends anteriorly through the vitreous to the ciliary body, the region in the dashed box shown at higher magnification as an inset (Bi). At E12 (C), the pleated structure of the pecten has begun to develop, and is more extensive by E15 (Da, b). Higher magnification insets in C and D are from consecutive sections that are also immmunolabeled for tenascin-C (Ci, Di—F-actin [white]; Cii, Dii—tenascin-C [red]). Tenascin C–rich fibrils are shown to provide a link between the pectin oculi and the ciliary body. Arrowheads in Ci and Di indicate the same positions in (Ci) or (Dbi). Mag. bars: (A) to (D) = 500 and 50 µm for the insets. C: cornea; CB: ciliary body; L: lens; PO: pecten oculi; R: retina. (A color version of this figure is available in the online journal.)

Immune cells populate the matrix-rich regions adjacent to the pecten oculi during development

It has been shown previously that hyalocytes, immune cells that are located in the vitreous, accumulate along the surface of the pecten oculi pleats. ⁴² We have examined the localization of immune cells associated with the pecten oculi during development. For these studies, E12 whole eye cryosections were co-immunolabeled for CD45 and tenascin-C and co-labeled for F-actin and nuclei (Figures 13 and 14). At the posterior aspect of the pecten oculi where it emerges from the retina, CD45+ immune cells were detected both within (Figure 13(Ab, bii)) and along the outside edge of this structure (Figure 13(Ab, bi)). The higher magnification images highlight the different microenvironments of these immune cells (Figure 13(Abi, bii)), and that the immune cell along the outside edge of the pecten oculi is entwined within tenascin-C-rich microfibrils (Figure 13(Abi)). CD45+ immune cells were more abundant in the pleated region (Figure 13(B)). While CD45+ immune cells were detected within the pecten oculi and along its external surfaces (Figure 13(Bc, ci)), immune cells were most highly associated with the vitreal fibrillar matrix that is located between the pleats (Figure 13(Bb, bi, d)). A 3D surface rendering created from a z-stack of the boxed region in Figure 13(Bd) revealed the relationship of these immune cells with the tenascin-C fibrils of the vitreous and their migratory morphology including the extension of processes within the tenascin-C-rich fibrous matrix (Figure 13(Bdi, dii)). We also discovered the presence of immune cells at the apical tip of the pecten oculi at E12 (Figure 14). Here, CD45+ immune cells were located within the pecten oculi tip (Figure 14(Ba, ai, aii)) and associated with the tenascin-C-rich fibrous matrix that lines this tissue’s outer surface and connects the pecten oculi to the ciliary body (Figure 14(Ca, ai, aii)).

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Figure 13.

Immune cells are highly concentrated in the vitreous matrix between the pleats of the pecten oculi. Cryosections of E12 chick eyes were co-immunolabeled with the pan-leukocyte marker CD45 (green). Nuclei were labeled with DAPI (blue). Confocal images were obtained in two regions as illustrated in the diagrams including the base of the optic nerve head (ONH) (Aa), and within the pleats (Ba) of the pecten oculi. Boxed in regions illustrate where each image was acquired. Immune cells are detected within the base of the ONH (Ab and Abii) and found entwined with tenascin-C fibrils on the outer edge of this structure (Ab and Abi). Immune cells were found to densely populate the pleated region of the pecten oculi, specifically the inner and outer surface of the folds (Bb, Bbi), in the matrix within the gaps between the folds (Bd), and more rarely within the fold itself (Bc, Bci). Migratory phenotype of immune cells entangled in matrix between folds is highlighted in 3D surface renderings of a confocal Z-stack from which (Bd) was taken with solid (Bdi) and transparent (Bdii) tenascin-C. Asterisks indicate immune cells at higher magnification from selected regions. Mag. Bars: Ab, Bb–d = 20 µm, Abi, Abii, Bbi, Bbii = 5 µm, Bdi, Bdii = 3 µm. PO, pecten oculi; ONH, optic nerve head; VH, vitreous humor. (Ab, Bb–c) Projection image; (Bd) single optical plane; (Bdi, Bdii) 3D surface structure. (A color version of this figure is available in the online journal.)

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Figure 14.

The pecten oculi is a likely source of immune delivery to the ciliary body. (A) Diagram of an E12 chick eye with boxed areas indicating the regions imaged, both adjacent to the pleats (B) and at the apex of the pecten (C). E12 eyes were co-immunolabeled for CD45 (green) and tenascin-c (red), and co-labeled for F-actin (white), and nuclei (blue). CD45+ cells were detected within the tissue of the pecten oculi leading to the apex (Ba). Three-dimensional structural renderings were constructed of the boxed area in (Ba) with F-actin both solid (Bai) and transparent (Baii). Immune cells (single and double asterisks) are shown present within the pectin oculi apex and emerging from the apical tip of the pecten oculi associated with the tenascin-C fibrils connecting this structure and the matrix-rich lining of the ciliary body (Ca). Three-dimensional renderings of boxed area in Ca with tenascin-C solid (Cai) and transparent (Caii) highlight processes of the immune cell interacting with the surrounding matrix. (Ba) Single optical plane; (Bai, Baii, Cai, Caii) 3D surface structure and (Ca) projection image. Mag. bars: Ba, Ca = 20 µm, Bai, Baii = 3 µm, Cai, Caii = 2 µm. AqH: aqueous humor; CB: ciliary body; CZ: ciliary zonules; PO: pecten oculi; VH: vitreous humor. (A color version of this figure is available in the online journal.)