IMMUNOHISTOCHEMICAL LOCALIZATION OF NEP AND ENDO-
3.1 INTRODUCTION
3.3.2 Immunolocalization of NEP in the developing rat head and face
Having established that rat embryos contained both enzymes under study the next step was to establish their spatio-temporal distributions. Localization of NEP was assessed by immunoperoxidase staining of frozen sections obtained from postimplantation rat embryos at gestational ages BIO to E16. These results have been summarized in Table 3.1. All positive staining was abolished by pre-absorption of RAHE and PHM-6 with the adult kidney membrane preparation which contained NEP. Staining in adult rat kidney, the positive control tissue, was confined to the brush-border lining the luminal surface of the proximal convoluted tubules and the Bowman’s capsule (Fig. 3B).
At ElO, the craniofacial region exhibited no distinct regions of positive staining. At E12, NEP was detected in the mesenchymal component of the medial and lateral nasal processes (Fig. 3E), throughout the notochord (Fig. 3H) and to a lesser extent on the luminal surface of the otocyst epithelium and of the branchial arch arteries. The craniofacial vasculature exhibited moderately positive staining. At E14, there was strong staining in the choroid plexus in both the mesenchyme and along the luminal/ventricular surface (Fig. 3V). The craniofacial vasculature, the pigmented retina (Fig. 3T) and the inner ear primordia also stained strongly.
At E l6, distinct patterns of positive staining were observed within the secondary palatal shelves (Fig. 3J), and the lower jaw particularly surrounding Meckel’s cartilage (Figs. 3L and M) and in the base of the tongue (31). The choroid plexus, inner ear (Fig. 3K) and the arytenoid cartilages all exhibited strong positive NEP staining. The craniofacial vasculature, cartilage and bone were negative, however there was a distinct population of positively-stained cells in a perichondrial layer enveloping Meckel’s cartilage, nasal septum and other craniofacial skeletal elements (Figs. 3L-N).
Fig. 3B. Immunolocalization of Endo-2 in an adult rat kidney section. Counterstained with haematoxylin (blue). Positive staining (brown) is confined to the brush border of the epithelia lining the proximal convoluted tubules. Distal tubules are negative. Bar = 50 microns.
Fig. 3C. Immunolocalization of NEP in an adult rat kidney section. Counterstained with haematoxylin (blue). Positive staining (brown) is evident on the brush border of the epithelia lining the proximal convoluted tubules and on the Bowman’s capsule (arrow) surrounding the glomeruli. The distal tubules, glomeruli and blood vessels are all negative.
Bar = 50 microns.
Fig. 3D. Localization of NEP mRNA in an adult rat kidney section following in situ
hybridization with a radiolabelled antisense probe. NEP in evident on the proximal convoluted tubules.
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►_ - ■ %,< L y : ' » s rFig. 3E. The lateral nasal process m esenchym e (arrow) o f the E12 rat em bryo, shown here in sagittal section, exhibits strong N EP localization (blue/black). T he epithelia on the rostral side o f the first branchial arch and the lum inal surface o f the first branchial arch artery dem onstrate m oderate N EP staining. In this parasagittal section the anterior/ventral surface o f the em bryo is to the left, and the dorsal surface is to the right. C ounterstained w ith neutral red.
Bar = 50 m icrons.
Fig. 3F. At ElO , the dorsal lum inal surface o f the gut (arrow ), shown here in transverse section, stains positively for N EP (blue/black). C ounterstained with neutral red.
Bar = 50 m icrons.
Fig. 3G. The lum inal surface o f the epithelia lining the mesonephric tubules (arrows) at E12 are strongly positive for N EP (blue/black). C ounterstained with neutral red.
Bar = 50 m icrons.
Fig. 3H. The notochord (arrow) at E12, shown here in longitudinal section, is positive for N EP. The neural tube (asterisk) is negative. C ounterstained with neutral red.
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F ig. 31. At E16, N E P (brow n) is localized in the base o f the tongue (asterisk). M eckel’s cartilage (m) is negative. No counterstain. Bar = 50 m icrons.
F ig. 3 J . C entral regions o f the palatal shelf m esenchym e (asterisks) display positive N EP localization (brown) at E16. The nasal septum (n) is negative. No counterstain.
Bar = 50 m icrons.
F ig. 3 K . N EP (brow n) is localized on the lum inal surface o f the inner ear epithelia (arrow ) at E l 6. Counterstained with haem atoxylin. Bar = 50 m icrons.
F ig. 3 L . At E I6 , the perichondrium (arrow heads) around the mandibular prim ordium , M eckel’s cartilage (m ), stains positively for N EP (brow n). C ounterstained with haem atoxylin.
Bar = 50 m icrons.
F ig. 3M . H igher m agnification detailing the N EP perichondrial staining (brow n). Counterstained with haem atoxylin. Bar = 10 m icrons.
F ig. 3N . T he perichondria around the other craniofacial skeletal elements (asterisk) exhibit strong N E P staining (brown) at E16. Counterstained with haem atoxylin.
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Fig. 3 0 . Sagittal section through the heart at E l 4. N E P is localized (brown) on the septum (s) and to a lesser extent on the surface o f the myocardial cells. Counterstained with haem atoxylin.
Bar = 50 m icrons.
Fig. 3P . T he apical surface o f the E l 4 gut endoderm stains positively (brown) for N EP (arrow heads). C ounterstained with haem atoxylin. Bar = 10 m icrons.
F ig . 3 Q . T he notochord (arrow ), is strongly positive for N EP at E14, shown here in longitudinal section. C ounterstained with haem atoxylin. Bar = 10 m icrons.
F ig. 3 R . T he apical surface o f the E14 lung (arrow heads) exhibits strong N EP staining (brow n). Counterstained with haem atoxylin. Bar = 10 m icrons.
F ig. 3S. The perichondria (arrow s) around the E14 lateral vertebral processes and the derm is (arrow heads) show strong N E P staining (brow n). The dorsal root ganglia (d) are negative. C ounterstained with haem atoxylin.
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Fig. 3T. The pigm ented retina (arrow ) at E14, exhibits strong N EP staining (brow n), w hilst the neural retina and lens (asterisk) are negative. Counterstained with haem atoxylin.
Bar = 10 microns.
Fig. 3U. Positive N EP staining (brow n) is evident in the mesenchymal stroma and on the apical surface o f the epithelia (arrow ) covering the choroid plexus at E14. C ounterstained with haem atoxylin.
Bar = 50 microns.
Fig. 3V. At E14, the lum inal surface o f the choroid plexus epithelia (arrow) exhibits strongly positive Endo-2 staining (brow n), and in contrast to the N EP im m unoreactivity, the strom a is negative. Counterstained with haem atoxylin.
Bar = 50 microns.
Fig. 3W. At E14, the lum inal surface o f the epithelia lining R athke’s pouch (asterisks) exhibit strong Endo-2 staining (brow n). Counterstained with haem atoxylin.