The rSC begins to express <i>Prox1</i> and to acquire a tube-like morphology.

<p>Z-projection of confocal stacks encompassing the rSC from a <i>Prox1-GFP</i> eye at P4.5. VECAD immunostaining shows a region with branching of the rudimentary vessel (block arrows, equivalent to stage D in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001912#pbio-1001912-g011" target="_blank">Figure 11</a>). <i>Prox1</i> is first expressed in the rSC at late P4. The branching rSC begins to acquire <i>Prox1</i> expression as indicated by GFP⁺ cells (arrowhead). Higher <i>Prox1</i> expression correlates with transition of rSC morphology from a sprouting chain of cells to a flattened tube (arrows). (Inset) DIC merged image showing that the tubular regions of the rSC have pockets of space filled with RBCs (black arrows). Scale bar, 50 µm.</p

Summary of early SC development.

<p>The images show VECAD (yellow) immunolabeled cells in the IZ in the plane of the future SC at the developmental ages listed. The cartoons represent the corresponding stages. The development of SC starts by sprouting from the LVP and RV. RV and their sprouts have been left out of the cartoons for the sake of simplicity and in no way are meant to suggest lesser importance of RV. (A) As in angiogenesis, sprouts led by tip cells penetrate into the IZ all around the limbus. (B) However, in contrast to angiogenesis, multiple tip cells interact and form tip cell clusters (TCCs). (C) These local clusters of cells connect with each other to form a continuous structure encircling the entire limbus. We call this structure the rSC. (D) The number of cells in the rSC increases and the cellular chain branches. Scale bar, 20 µm.</p

Macrophages are present at sites of tip cell interactions mediated by filopodia.

<p>(A) Tip cells in the IZ interact through their filopodia, and macrophages are present at sites of tip cell interaction. All images are Z-projections of the future SC plane of a P2 eye. The numbers correspond to their location in that eye in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001912#pbio.1001912.s010" target="_blank">Figure S10</a>. (Left) Endomucin staining of tip cells and their filopodia (arrowheads). (Middle) IBA1 staining marks macrophages. (Right) A merge of the endomucin and IBA1 staining. The interacting filopodia clearly have associated macrophages. (B) Interacting filopodia between two tip cells that are coated with VECAD. The filopodia are intertwined. The numbers label distinct cells. Image was captured using the highly sensitive photon-counting mode on the confocal microscope. The cartoon represents these tip cell–macrophage interactions. RV and their sprouts are not shown for the sake of simplicity and in no way meant to suggest lesser importance of RV. Scale bar, (A) 20 µm and (B) 5 µm.</p

Cellular proliferation occurs during SC development.

<p>(A–C) Z-projections of the presumptive SC plane at the indicated ages. The top panels at each age show a merged image of endomucin and Ki67 staining. The endomucin staining delineates the developing SC. Ki67 labels dividing cells, labeling both the developing SC and other cells. Bottom panels show endomucin and Ki67 double-labeled cells in the developing SC in blue (voxel overlap of endomucin and Ki67 in a Z-projection). The double-positive proliferating cells closely track the developing SC. Thus, SC growth involves cell proliferation. Scale bar, 50 µm.</p

Blocking KDR function has a sustained disrupting effect on SC development.

<p>Whole mounts from P12 mice injected with the control (IgG) or inhibitory (DC101) antibodies. Images are Z-projections of confocal stacks encompassing the SC. Multiple Z stacks were first stitched together to obtain a panoramic view of a region of the whole mounts. (Left) Control. The control IgG-treated eyes were indistinguishable from untreated eyes, with a robust and complex morphology. (Right) DC101 treated. The KDR inhibiting antibody had a profound effect on SC development. Representative examples from different mice are shown. Scale bar, 100 µm.</p

Lineage tracing using a <i>Lyve1-Cre</i> mouse shows SC does not originate from lymphatics.

<p>(A) Outcome of a genetic cross between a <i>Lyve1-Cre</i> mouse and the <i>Cre</i> reporter <i>ROSA26R-mtmG</i> (<i>mTmG</i>) mouse. <i>Cre</i> expressed from the <i>Lyve1</i> promoter is found early in developing lymphatic tissue and in macrophages. CRE-mediated recombination indelibly labels lymphatics and macrophages with GFP fluorescence in a backdrop of red fluorescent cells that are not of these lineages. Because this recombination is irreversible, tissue derived from <i>Lyve1</i>-expressing cells will always be green fluorescent. Cells that have never expressed <i>Lyve1-Cre</i> and whose ancestral cells never expressed <i>Lyve1-Cre</i> are red fluorescent. (B) SC does not originate from lymphatics. Corresponding confocal planes of adult SC at the levels of the labeled tissues are shown for a <i>Lyve1-Cre mTmG</i> mouse. The lymphatics (Ly, arrowheads) and macrophages (arrows) are green fluorescent, indicating <i>Lyve1-Cre</i>–mediated recombination occurred as expected. Blood vessels (BV) of the LVP and the corneoscleral tissue are red fluorescent, showing that <i>Lyve1-Cre</i> was never expressed in these tissues. SC cells are also red (GFP/tdTomato panel). SC is outlined by a dotted line to help distinguish it from the corneoscleral tissue that surrounds it. The form of SC is clearly demonstrated by VECAD immunolabeling (bottom). The green cells are macrophages associated with SC and not SC itself (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001912#pbio.1001912.s006" target="_blank">Figure S6</a>). Scale bar, 100 µm.</p

Development from rSC to mature morphology.

<p>(Left, A–E) Z-projections of confocal stacks encompassing the developing SC in <i>Prox1-GFP</i> eyes. (A) At P4.5 VECAD labeling shows multicellular sprouts (*) at the sides of the GFP + tubular rSC. These sprouts have no detectable <i>Prox1</i> expression. (B) By P5, remodeling has formed a central core of PROX1⁺ flattened cells without any discernible internal space or RBCs. Further development continues by sprouting (*, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001912#pbio.1001912.s013" target="_blank">Figure S13</a>) from the central core. Dotted line delineates developing SC from autofluorescence arising from nearby tissue. (C) At P10, developing SC has expanded considerably in size, and maturation with lumen formation and polarization of PROX1 expression to the inner wall have begun (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001912#pbio-1001912-g015" target="_blank">Figure 15</a>). The developing SC remains connected to the LVP (c) but no connections to the RV are detected after P5. (D) SC looks mature at P17 but continues to grow. (E) Adult SC. (Right) The cartoons distill the essential points of each stage. At P5 PROX1^- sprouts are shown in magenta. At all developmental stages, the SC is connected to the LVP. The adult stage SC cartoon attached to the LVP is seen from the inner wall perspective, where the thin cells are a darker green to depict strong <i>Prox1</i> expression. The SC shown below is from the outer wall perspective, with the paler green large cells having weak or no detectable PROX1 expression. BV, blood vessels; c, collector channels. Scale bar, 50 µm.</p

SC visualized in 3D using whole mounts.

<p>(A) Enface view of adult SC and other limbal vessels. (Left) DIC image of a whole mount prepared as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001912#pbio.1001912.s001" target="_blank">Figure S1</a>. SC and limbal vessels are located in the limbus, just inside the dark pigmented band (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001912#pbio.1001912.s001" target="_blank">Figure S1</a>). (Middle) Localization of the limbal vessels stained with endomucin in a Z-projection of confocal limbal stacks encompassing the entire whole mount. The whole-mount stacks were Z-depth color-coded (ICE LUT, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001912#pbio.1001912.s001" target="_blank">Figure S1</a>). Color scale shows depth code colors of all structures stained with endomucin from inside (in) the eye to outside (out), with the SC being the most internal coded structure. (Right) Overlay of DIC and immunofluorescence image. (B) Higher magnification showing SC in relation to blood vessels of the LVP. BV, blood vessel; cc, collector channel. The blood vessels (magenta) are closer to the external ocular surface than SC (cyan). (C, D) Imaged whole mounts with the basement membrane marker collagen IV (COLIV) used to highlight SC in enface (XY) and conventional (XZ) orientations. (C) XY view; the icon in the lower left corner indicates the orientation of SC (also used in subsequent figures). The inner wall (IW) is closer to the reader, and the outer wall (OW) is away from the reader. (D) XZ view through the plane indicated by yellow lines in (C). In this XZ orientation, the lumen of SC (*) is evident between the COLIV-labeled inner and outer walls (compare to E). (E) Frozen section with COLIV labeling overlaid on its DIC image. The similarity between the XZ-represented whole mount and frozen section is clear. Note the characteristic bulbous undulations of the inner wall protruding into the lumen (arrowheads). Scale bar, (A) 500 µm, (B) 100 µm, and (C–E) 20 µm.</p

Sprouts have leading tip cells and following stalk cells, as is characteristic of angiogenesis.

<p>(A–B) Tip cells from the LVP penetrating into the deeper limbal tissue. (A) Depth coded maximum intensity 3D rendering of limbal region of a P2 eye stained with endomucin. The deepest, endomucin-positive tissue is colored cyan and the more superficial tissue is redder. (Left) Low-magnification image with tip cells emanating from the LVP. Their cyan color indicated that they are in the plane of the future SC. (Middle) The region bounded by the white box in the left panel at higher digital zoom. (Right) The region bounded by the white box in middle panel at higher digital zoom with the superficial layer electronically removed in Imaris to show the tip cells in greater detail. The color key indicates the depth code in relation to relative depth from the outside of the eye (In, closer to inside of the eye; out, closer to outside of the eye; see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001912#pbio.1001912.s001" target="_blank">Figure S1</a>). (B) (Left) Tip cell region from middle panel of (A) rendered using blend mode of Imaris. This provides density so that the characteristic tip cell filipodial structures are clearly evident. (Middle and Right) Progressively, the tip cell in greater detail. (C) Cartoon representing this stage of development but ignoring the RV and their sprouts for the sake of simplicity. This is in no way meant to suggest lesser importance of RV. Scale bar, (A and B, Left images), 20 µm and (A and B, middle and right images) 10 µm.</p

Schematic showing the stages of SC development by the novel process of canalogenesis.

<p>Cartoons have been drawn for clarity and are not intended to suggest that most early sprouts arise from the LVP.</p