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The process by which the pronephros develops was morphologically examined in chick embryos from Hamburger–Hamilton stage (ST) 8+ to ST34. The intermediate mesoderm, from which the pronephros arises, was first seen as a faint ridge of undifferentiated mesoderm between the segmental plate and lateral plate at ST8+. It formed a cell cord at the level of the 6th to the presumptive 13th somites at ST9 to ST10. This cell cord then separated into dorsal and ventral parts, the former becoming the nephric duct and the latter the tubules by ST14. The primordia of the external glomeruli (PEGs) appeared at ST15 through some epithelial cells protruding in the nephrostome (the opening of the nephric tubule into the body cavity). PEGs formed gradually in the caudal direction until ST18, while the pronephric tubules and PEGs in cranial locations disappeared. At this stage, only a few PEGs remained at the level of the 13th and 14th somites and these developed from ST23 to ST29 to become ultrastructurally similar to the glomeruli of the functional kidney. From these observations in the avian pronephros, we infer that the pronephric duct and tubules both form from a cell cord in the intermediate mesoderm and at the same time, but later develop differently.
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It is well known that during the development of the kidney in the avian embryo, the pronephros, mesonephros and metanephros appear in sequence in a manner similar to that seen in mammalian and reptilian embryos (Felix, 1906; Goodrich, 1958; Arey, 1965; Romer & Parsons, 1977). Some descriptions of the development of the pronephros in the avian embryo have been made over the years on the basis of observations of paraffin sections (Gasser, 1877; Sedgwick, 1881; Felix, 1906; Abdel-Malek, 1950; Davies, 1950; Hamilton, 1952). With regard to the initial development of the pronephros, however, two incompatible theories exist: (1) nephric (pronephric or Wolffian) tubules initially arise from the intermediate mesoderm (segment stalk), then fuse to form the nephric (pronephric or Wolffian) duct (Felix, 1906; Waddington, 1938; Abdel-Malek, 1950; Hamilton, 1952; Romanoff, 1960; Balinsky, 1981) or (2) the nephric duct arises first and the nephric tubules appear later (Gasser, 1877; Sedgwick, 1881; Davies, 1950). The pronephros of the avian embryo has attracted little recent interest because it was considered to be a rudimentary organ that disappears without ever functioning, in contrast to the situation in fish (Holmgren, 1950; Euler & Fänge, 1961; Ellis & Youson, 1989) and amphibians (Fraser, 1950; Fox, 1963; Jaffee, 1963; Christensen, 1964).
In the last two decades, the nephric duct has been investigated in detail using the electron microscope, both with regard to its early development and its posterior extension (Jacob et al. 1986, 1991, 1992; Jarzem & Meier, 1987; Bellairs et al. 1995). These observations suggested that, in avian embryos, the nephric duct arises as a protrusion of cells from the intermediate mesoderm between the paraxial mesoderm and the lateral plate at a time before the pronephric tubules appear. Although some authors studying paraffin sections have concluded that, in avian embryos, the pronephros is temporary during development and functionless – because the nephric tubules and external glomeruli (EGs) of the pronephros are rudimentary and degenerate at early stages (Sedgwick, 1881; Felix, 1906; Abdel-Malek, 1950; Davies, 1950; Hamilton, 1952) – others have proposed that the pronephros may perform particular functions (Needham, 1931; Waddington, 1938; Jacob et al. 1977). For example, Jacob et al. (1977), who observed the EGs in the chick embryo at the 5-day stage by scanning and transmission electron microscopy (SEM and TEM), confirmed that EGs consisted of epithelial cells with foot processes, a thin basement membrane and a fenestrated endothelium, and suggested that the pronephros in the avian embryo may be functional.
In avian embryos, the pronephros consists of the pronephric duct and tubules together with EGs projecting into the body cavity, as seen in both fish (Ellis & Youson, 1989) and amphibians (Fox, 1963; Christensen, 1964). In addition, there is an intermediate zone in which EGs and internal glomeruli (IGs) often coexist or are fused in both avian embryos (Sedgwick, 1881; Davies, 1950) and reptilian embryos (Wiedersheim, 1890; de Walsche, 1929; Tribe & Fisk, 1941; Davies, 1950). From the existence of IGs, some investigators have deduced that the pronephros and mesonephros overlap each other; however, the boundary between the pronephros and mesonephros has been described in a variety of ways (Abdel-Malek, 1950; Davies, 1950; Hamilton, 1952) and the details remain unclear. As the functional significance of the pronephros is controversial, we have tried to clarify the situation and investigate in detail the origin and early development of EGs in the pronephros using a battery of methods that include SEM, TEM, vascular casts and light microscopy (LM) of serial, semithin Epon sections.
Materials and methods
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- Materials and methods
Fertilized eggs of the domestic fowl, Gallus gallus domesticus, were supplied by the Poultry Station and the Livestock Center of Saitama Prefecture, and incubated at 38 °C. The chick embryos examined were from Hamburger–Hamilton stage (ST) 8+ to ST34 (Hamburger & Hamilton, 1951). Although Hamburger and Hamilton did not count the first somite after ST10 as it is rudimentary, we counted the first rudimentary somite at all observed stages, because a gap may otherwise arise in the counting of somite numbers. For example, although the embryo at ST12 has been said to have 16 somites, we call the last somite the 17th somite in the same embryo, because it really has 17 somites.
For SEM observations, the embryos were placed in plastic dishes and perfused via the heart with Locke solution (to wash out the blood) and then fixed with 2.5% glutaraldehyde mixed with 2% paraformaldehyde in 0.1 m cacodylate buffer at pH 7.6. The ectoderm or endoderm with the splanchnic mesoderm was removed in the fixative with the aid of tungsten needles. After a brief rinse in 0.1 m cacodylate buffer, these specimens were refixed in buffered 1% OsO4 for 1 or 2 h. They were then dehydrated through a graded ethanol series, critical-point dried in liquid CO2, sputter-coated with platinum–palladium (15–20 nm in thickness) and examined using an SEM (S-4100, Hitachi, Japan).
For LM and TEM examinations, the initial fixation was carried out using the same procedure as that used for SEM. After a brief rinse in 0.1 m cacodylate buffer, embryos were refixed in buffered 1% OsO4 for 1 or 2 h. They were then dehydrated through a graded ethanol series and embedded in Epon. The embryos were serially sectioned, either transversely or longitudinally, at 1-µm intervals through the pronephric region and stained with toluidine blue (for LM). The ultrathin sections for TEM were each mounted on a copper grid with a sufficiently large hole (c. 1 × 0.5 mm) to allow examination of the entire section. After staining with an aqueous solution of uranyl acetate and lead citrate, these sections were observed using a TEM (JEM-1010, JEOL, Japan).
For the preparation of vascular casts, the method of Hiruma & Hirakow (1995) was used. After the initial fixation had been carried out using the same procedure as that used for SEM, the embryos were injected manually with resin (Mercox, Dainippon Inc., Japan) via a syringe with a fine glass cannula. The injected specimens were then immersed in 15% KOH to remove tissues and washed with distilled water. After drying in air, the specimens were mounted on glass cover slips on aluminium stubs, sputter-coated with platinum–palladium (15–20 nm in thickness) and examined with an SEM microscope (S-4100 Hitachi, Japan).
Some embryos were fixed with 2.5% glutaraldehyde in 0.1 m cacodylate buffer at pH 7.6, the ectoderm removed in the fixative with the aid of tungsten needles and the embryos stained with haematoxylin and eosin after a brief rinse. After dehydration through a graded series of ethanols, whole-mount embryos on glass plates were observed using LM.
The number of external glomeruli (EGs) or their primordia (PEGs) that projected into the body cavity was counted in a total of 126 embryos (eight or more embryos per stage) from ST15 to ST34. On photographs enlarged four-fold from SEM images of EGs (250× original magnification), the long diameters of 804 EGs or PEGs were measured using vernier calipers (Table 1). We classified structures as EGs or PEGs depending on whether their epithelial cells did or did not have foot processes (Fig. 1).
Table 1. Number and size of EGs (or PEGs) in developing chick embryos
|Stage||Time of incubation||No. of embryos||No. of EGs (or PEGs)||Long diameter (µm)|
|St14|| || 8|| 0||0||–||–||–|
|ST15||55–63 h|| 8|| 30||1.9 ± 1.7||31.2 ± 11.3|| 69.8||18.3|
|ST16||60–68 h||12|| 90||3.8 ± 1.9||32.3 ± 11.7|| 65.8||13.1|
|ST17||65–72 h||14||152||5.4 ± 1.9||31.9 ± 10.3|| 65.0||14.1|
|ST18|| 3 days||10||131||6.6 ± 2.0||36.9 ± 14.8||112.8||14.6|
|ST19, 20|| 3–3.5 days|| 9|| 88||4.9 ± 1.8||43.4 ± 23.1||157.7||15.8|
|ST21, 22|| 3.5 days||10|| 91||4.6 ± 2.1||55.1 ± 28.5||163.5||20.5|
|ST23, 24|| 4 days||14||110||3.9 ± 1.7||73.0 ± 39.1||208.9||22.5|
|ST25, 26|| 5 days||13|| 61||2.4 ± 1.2||80.5 ± 44.1||192.7||23.7|
|ST27–29|| 6 days|| 9|| 25||1.6 ± 1.0||97.8 ± 44.2||184.0||34.0|
|ST30, 31|| 7 days||10|| 16||0.8 ± 1.0||93.3 ± 29.3||135.3||50.0|
|ST34|| 8 days|| 9|| 13||0.7 ± 0.7||89.2 ± 26.3||125.5||33.8|
Figure 1. Number of EGs (or PEGs) appearing in the body cavity on each side in chick embryos from ST15 to ST34. PEGs were considered to possess epithelial cells without foot processes (▪), EGs to possess epithelial cells (podocytes) with foot processes (□). The latter appear at ST21, whereas the former are poorly developed and disappear at an early stage.
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