Figure 1.
Glomerulus development in medaka and zebrafish pronephros in comparison to mouse and rat metanephros.
A1-B5: Pronephric glomerulus development in 3 to 6 dpf medaka (A1-A5) and 34 hpf to 4 dpf zebrafish (B1-B5) by JB4 cross sections stained by hematoxylin and eosin. The glomerular primordium of medaka exhibited a C-shaped epithelial layer (A2, A3), which is similar to the mouse and rat S-shaped body stage (C3, D3), unlike in zebrafish (B2). The C-shaped primordium contained a characteristic balloon-like or sinusoidal capillary. The paired pronephric glomerulus was fused at the midline to form a glomerulus in zebrafish (B3), but remained separated into two parts by the interposition of an interglomerular mesangium in medaka (arrowheads in A2-A5). C1-D5: Metanephric glomerulus development in rat epoxy resin sections stained by toluidine blue (C1-C5) and in mouse E18.5 kidney sections stained with H&E (D1-D5). Cross section of rat and mouse metanephros shows renal vesicle (C1, D1), comma-shaped body (C2, D2), S-shaped body at (C3, D3), capillary loop at (C4, D4), and maturing glomerulus (C5, D5). Rat vascular cleft in C2 (arrowhead) and primitive podocyte layer in C3 (asterisk). Mouse vascular cleft in D2 and D3 (arrowheads). Scale bars = 10 µm.
Figure 2.
Temporal specification of the three glomerular components during glomerular development in medaka.
A1-A5: wt1a mRNA is expressed in the forming glomerulus from 3 dpf until 6 dpf. Cross sections show that wt1a positive layers of the paired glomerular primordium lie adjacent to each other throughout the pronephric development. B1-B5: Glomerular capillaries are visualized by detecting endogeneous alkaline phosphatase (AP) in endothelial cells. AP-positive endothelial cells are detected in the invaginated portion of nephron primordium from 3 dpf (arrowheads in B1). The balloon-like capillary starts forming at 4 dpf (B2), and then divides into a few smaller capillaries by mesangium by 4.5 dpf (B3). Capillaries continue to grow at 5 dpf (B4) and integrate with the glomerulus by 6 dpf (B5). Dorsal aorta (arrows in B1-B5). C1-C5: Glomerular basement membrane (GBM) and mesangial matrix are detected by periodic acid-methenamine-silver (PAM) stain. The invaginated portion of the basement membrane is densely labeled with PAM stain in comparison with other regions (arrowheads in C1). The PAM-positive matrix is formed at the interglomerular mesangial region (arrowhead in C2-C5) and has an amorphous appearance. Scale bars = 10 µm.
Figure 3.
Temporal specification of the three glomerular components during glomerular development in zebrafish.
A1-A4: wt1a mRNA is expressed in the forming glomerulus from 34 hpf to 4 dpf. Cross sections show wt1a-positive podocyte layers of the paired glomeruli at 34 hpf (A1). Unlike medaka, zebrafish wt1a-positive podocytes are merged at 2 dpf (A2). B1-B4: Glomerular capillaries are detected by AP stain. At 34 hpf, the flattened nephron primordia show proximity to the dorsal aorta (B1). Glomerular capillaries are found at 2 dpf (B2). Dorsal aorta (arrows in B1-B4). C1-C4: GBM and mesangial matrix are detected by PAM stain. The paired glomeruli fuse at the midline and the interglomerular mesangium is not formed (C2-C4). Scale bars = 10 µm.
Figure 4.
The progression of podocyte differentiation visualized by Podocalyxin immunostaining.
A1-A3: Cross sections of rat metanephric glomeruli. Throughout the rat metanephric glomeruli development, Podocalyxin is mainly localized at the apical membrane of podocytes. At the S-shaped body stage, Podocalyxin marks the apical membrane of the individual podocytes in a cap-shaped pattern (arrowheads in A1). By early capillary loop stage, Podocalyxin localizes in a U-shaped pattern in the individual podocytes (arrowheads A2). By the maturing glomerulus stage, Podocalyxin staining is detected along the entire surface of podocytes (A3). B1-B3: Cross sections of medaka pronephric glomeruli. Podocalyxin immunostaining is detected in the individual podocytes as a U-shaped pattern at 3 and 4 dpf (B1, B2). At 7 dpf, Podocalyxin staining is detected along the entire surface of podocytes (B3). Asterisks indicate blood cells in dorsal aorta. C1-C3: Cross sections of zebrafish pronephric glomeruli. At 34 hpf, Podocalyxin marks the apical membrane of individual podocytes at 34 hpf (C1). By 2 dpf (C2) and 5 dpf (C3), Podocalyxin immunostaining is expressed along entire surface of podocytes. Scale bars = 10 µm.
Figure 5.
Transmission electron microscopy of podocytes development in medaka pronephric glomerulus.
A-E: The ultrastructure of pronephric glomerulus cross sections. A: At 4.5 dpf, podocytes form a single columnar epithelium and are connected via intercellular junctions (arrowheads), which are located in the middle of the cell height. B: The intercellular junctions of podocytes are located in the vicinity of the GBM (arrows) at 5 dpf. Podocytes interdigitate at the cell periphery to form irregularly-shaped processes (arrowheads). C: The podocyte cell body is detached from the GBM to form a subpodocyte space (asterisks) by 6 dpf. The irregularly-shaped processes become flattened (arrowheads); however, foot processes with silt diaphragms have not formed yet. D: Foot processes connected by slit diaphragm are formed in some capillary walls (arrowheads) at 7 dpf. E: Foot processes are found in the most capillary walls (arrowheads) at 10 dpf. CL, capillary lumen; P, podocyte cell body: PE. Scale bars = 1 µm.
Figure 6.
Interglomerular mesangium in medaka pronephric glomeruli.
Ultrastructure of interglomerular mesangium of medaka pronephric glomeruli at 4 dpf (A1, A2), 8 dpf (B1, B2) and 10 dpf (C1, C2). Unlike zebrafish, the paired pronephric glomeruli are separated by interglomerular mesangium throughout medaka pronephric development (A1, B1, C1). The interglomerular mesangium is in close contact with the ventral surface of the dorsal aorta, which is located beneath the notochord or vertebra. The interglomerular mesangium consists of densely packed interglomerular mesangium cells and matrix. Most of the interglomerular mesangium cells contain granules which exhibit a variety of electron density (arrowheads in A2, B2, C2). Some interglomerular mesangium cells adhere to the afferent glomerular arteriole (AA) at 10 dpf (C2). CL, capillary lumen; DA, dorsal aorta; N, notochord; V, vertebra; IGM, interglomerular mesangium; IGMC, interglomerular mesangial cells; AA, afferent arteriole. Scale bars = 10 µm in A1, B1, C1; 1 µm in A2, B2, C2.
Figure 7.
renin mRNA expression in the Interglomerular mesangial region in medaka embryos.
Renin is expressed in the region between paired glomeruli, which are outlined by the yellow dotted lines, at 4 dpf (A) and 5 dpf (B). The location of the pronephric tubules are indicated by arrowheads. Scale bars = 10 µm.
Figure 8.
Summary of morphological processes during pronephric glomerulus development in medaka and zebrafish.
Medaka glomerulus 3 dpf (A1), 4 dpf (A2), 4.5 dpf (A3), 5 dpf (A4) and 6 dpf (A5). Zebrafish glomerulus 34 hpf (B1), 40 hpf (B2), 2 dpf (B3), 3 dpf (B4) and 4 dpf (B5). Individual components of pronephric glomerulus are indicated by blue (tubular primordia and pronephric tubules), green (podocytes), red (glomerular capillary), brown (mesangial cell, interglomerular mesangial cells) and black (interglomerular mesangial matrix).
Table 1.
Morphological types of pronephric glomeruli in teleost fishes.