Expression of the Von Hippel-Lindau Tumor Suppressor Gene, VHL, in Human Fetal Kidney and During Mouse Embryogenesis

Abstract

Background

Von Hippel-Lindau (VHL) disease is a familial cancer syndrome that has a dominant inherited pattern which predisposes affected individuals to a variety of tumors. The most frequent tumors are hemangioblastomas of the central nervous system and retina, renal cell carcinoma (RCC), and pheochromocytoma. The recent identification and characterization of the VHL gene on human chromosome 3p and mutational analyses confirms the VHL gene functions as a classical tumor suppressor. Not only are mutations in this gene responsible for the VHL syndrome, but mutations are also very frequent in sporadic RCC.

Materials and Methods

VHL expression in human kidney and during embryogenesis, was analyzed by in situ mRNA hybridization with ³⁵S-labeled antisense VHL probes, derived from human and mouse cDNAs, on cryosections of human fetal kidney and paraffin sections of murine embryos.

Results

In human fetal kidney, there was enhanced expression of VHL within the epithelial lining of the proximal tubules. During embryogenesis, VHL expression was ubiquitous in all three germ cell layers and their derivatives. Expression occurred in the cerebral cortex, midbrain, cerebellum, retina, spinal cord, and postganglionic cell bodies. All organs of the thoracic and abdominal cavities expressed VHL, but enhanced expression was most apparent in the epithelial components of the lung, kidney, and eye.

Conclusions

In human fetal kidney, the enhanced epithelial expression of the VHL gene is consistent with the role of this gene in RCC. There is widespread expression of the VHL gene during embryogenesis, but this is pronounced in areas associated with VHL phenotypes. These findings provide a histological framework for investigating the physiological role of the VHL gene and as basis for further mutational analysis.

Introduction

Von Hippel-Lindau (VHL) disease is an autosomal dominant cancer syndrome characterized by the development of a variety of benign and malignant tumors affecting tissues of varying embryonic origin. Affected individuals develop tumors of the central nervous system including hemangioblastomas of the spinal cord and cerebellum, retinal angiomas, pancreatic cysts, renal cell carcinomas, and pheochromocytomas. Renal cell carcinoma is the most common cause of death, with life expectancy reduced to a median of 49 years of age (1).

The VHL tumor suppressor gene was recently isolated by positional cloning methods. Fluorescence in situ hybridization (FISH) maps the gene to a single locus at chromosome 3p25 (2). The complete coding sequence of the VHL gene has not yet been delineated, but the cloned partial cDNA encodes three exons encompassing 852 nucleotides. This sequence encodes a short open reading frame of 284 amino acids. The single copy gene is highly conserved across species as diverse as mammals, drosophila, and sea urchin, suggesting it encodes a protein with fundamental cellular function. VHL is expressed in all human tissues tested by Northern analysis. Two transcripts are expressed in a spatial and temporal manner and represent alternative spliced forms of the VHL mRNA (2,3).

Subsequent studies have focused on somatic mutations within the VHL tumor suppressor gene in renal cell carcinoma. RCC can be subdivided into two categories: tumors associated with or without chromosome 3 abnormalities. Most VHL and familial renal tumors as well as sporadic RCC are of the clear cell type associated with chromosome 3p abnormalities (4,5). Two separate investigations have recently identified VHL somatic mutations in 57 and 56% of sporadic clear cell renal carcinomas studies (3,6,7). Comparison of advanced primary or metastatic renal carcinoma to localized clear cell carcinoma show that VHL mutations occur in the early stages of kidney tumorigenesis. Additionally, VHL mutations were identified in a familial RCC carrying a constitutional translocation. This form of familial cancer, as well as mutations found in both localized and advanced sporadic RCC, indicates that the VHL gene has a critical role in development of renal carcinoma (3).

In an attempt to further characterize the role of the VHL gene in differentiation and development, we have examined its expression pattern in the human fetal kidney and throughout murine embryogenesis. The VHL gene is expressed in virtually all human tissues tested by Northern hybridization, however, its exact expression pattern in regard to tissue and cellular localization remains unknown. To better define the temporal and spatial distribution of VHL expression, we have performed in situ mRNA hybridization with ³⁵S-labeled antisense VHL probes on frozen sections of a human fetal kidney and on paraffin-embedded sections of murine embryos at different stages of development.

Materials and Methods

Embryo Collection

Embryos of Swiss Webster matings were recovered at various stages of postimplantation development. Day 0.5 postconception was determined by the presence of a vaginal plug. Embryos were harvested on embryological days 7.5 to 17.5, then fixed in buffered formalin and paraffin embedded. For the in situ hybridization analyses, the mouse embryos were sectioned at 4 µm.

Human Fetal Kidney

Human fetal kidneys were resected from a 14-week-old aborted fetus and then embedded in OCT compound (Tissue Tek, Miles, Inc., Elkhart, IN, U.S.A.). For further analysis, the fetal kidneys were sectioned at 7 µm on a cryostat microtome.

Cloning of the Human VHL cDNA

The g7 partial cDNA clone was isolated from a lambda gt11 teratocarcinoma cDNA library (2). The NotI-XbaI fragment used for the in situ analysis was subcloned from this cDNA into pBluescript II KS⁺ (Stratagene, La Jolla, CA, U.S.A.). (Genbank Accession number L15409.)

Cloning of the Mouse VHL cDNA

The Ml partial cDNA (1390 bp) was isolated from a mouse brain cDNA made in lambda ZA-PII. The NotI-AccI (294 bp) fragment of the M1 clone used in in situ analysis was subcloned into pBluescript II KS⁺ (Stratagene, La Jolla, CA, U.S.A.). The homology of the M1 VHL cDNA to the human VHL cDNA is 52% at the nucleotide level over both cDNAs. The homology between the probes used for in situ hybridization analysis is 89% at the nucleotide level. (Genbank accession number U12570.)

In Situ RNA Hybridizations

In Situ hybridizations on sections of mouse embryos as well as the human fetal kidney sections were adapted from methods previously described by Sassoon and Rosenthal and Rackley et al. (8,9). The 594-bp antisense probe used for the human fetal kidney sections was generated by transcribing the Notl-XbaI fragment of the g7 cDNA from the T3 promoter. The sense probe of the 594 bp was synthesized from the same NotI-XbaI fragment using the T7 promoter. Both human and mouse RNA probes were labeled with ³⁵S UTP 400 Ci/mmole, Amersham Corp., Arlington Heights, IL, U.S.A.. The mouse probes were generated by transcription of the NotI-AccI fragment of the M1 cDNA from the T3 promoter to give an antisense probe of 335 bp. The sense probe was transcribed from the T7 promoter generating a 328-bp probe. RNA probes were hydrolyzed by alkali treatment to approximately 150 nt in length prior to hybridization. After hybridization at 50°C, washing procedures were carried out as described by Wilkinson et al. (10). Following washing, the slides were dehydrated and dipped into NTB-2 emulsion (Eastman Kodak, Rochester, NY, U.S.A.) and exposed for 2 weeks. After developing, the sections were stained with 0.5% toluidene blue O and mounted with Permount (Fischer Scientific, Pittsburgh, PA, U.S.A.) for observation under both brightfield and dark-field illumination using a Nikon Microphot-FXA.

Results

Localization of VHL Expression During Human Fetal Nephrogenesis

In situ localization of VHL expression of 14 week gestation human kidney revealed expression throughout the renal cortex and medulla (Fig. 1 A and B). Specifically, in the renal cortex, moderate levels of VHL expression were noted in the metanephric blastema. In contrast, high levels of VHL expression existed within the epithelial lining of the proximal tubule. Of note, while VHL expression existed in the distal convoluted tubules this expression was much less than that of the proximal tubules. Thus, while VHL expression is noted throughout stromal components of the developing kidney, there is an obvious differential expression that exists in the proximal tubules.

Fig. 1

Photomicrographs of VHL expression in the human fetal kidney and in murine embryogenesis

(A and B) Expression of VHL in the human fetal kidney at 14 weeks gestation shown in toluidene blue O-stained brightfield photomicrographs and the corresponding darkfield images. VHL expression is evident in the proximal tubules (pt), distal tubules (dt), and metanephric blastema (mb) (A, B: ×60), however, a prominent difference is noted in proximal tubular expression when compared with the other structures. (C, D, E, and F) Expression of VHL in murine embryogenesis (E = 13.5 days). Light and corresponding darkfield photomicrographs of thorax and abdominal development revealing expression of VHL in the atrium (a) and ventricle (v) of the heart. Expression is more prominent in the developing liver (li), pancreas (p), brachial arch (ba), and the urogenital ridge (ur) and cephalad, the adjacent neuroepithelium (ne) lining the developing brain structures (C, D: ×60; E, F: ×60). (G, H, I, and J) Higher magnification views of light and corresponding darkfield photomicrographs of murine VHL expression in the abdomen (E = 13.5 days) showing moderate expression in the intestines (in), and more intense expression in the developing urogenital ridge (ur), gonad (gd), and mesonephric tubules (mt). The kidney (k) and pancreas (p) show high levels of VHL expression with lower levels of expression in the adjacent adrenal gland (ad), vertebral body (vb), ganglion (gl), and pelvic organs of the uterus (ut), bladder (bl), and rectum (r) (G, H: ×120; I, J: ×50).

Localization of VHL Expression During Murine Abdominal and Thoracic Organogenesis

To determine whether the VHL gene is involved in specific developmental events during embryogenesis, we studied VHL expression in a murine model of embryogenesis between embryologiC Days 10.5 and 16.5. Following the stages of gastrulation and neurulation, the murine embryo enters the embryonic and fetal phase of organ development (11). By E 10.5 rudimentary organogenesis has progressed in a craniocaudal direction. At this stage of development, VHL expression was noted throughout the derivatives of the three germ cell layers within the pleuropericardial (thoracic) and abdominal regions (Fig. 1 C–J). In particular, VHL expression was evident in the following structures: peripheral and central nervous system, thymus, heart, primordial lung bud, liver, spleen, stomach, pancreas, kidney, gonads, uterus, bladder, adrenal gland, intestine, somatic muscle, and cartilage (Fig. 1 C–J).

THORAX. Early in lung development, VHL expression was evident in a diffuse pattern. As the lung underwent further differentiation, a differential pattern of expression was apparent within the epithelial lining of the bronchioles. Within the developing pericardial cavity, expression of VHL was noted in the pericardium and the heart musculature (Figs. 1 and 2). Minimal expression occurred within the developing thyroid gland and thymic rudiment (data not shown).

Fig. 2

VHL murine thoracic and abdominal organogenesis in toluidene blue O-stained brightfield photomicrographs and corresponding darkfield images (E = 16.5 days)

(A and B) Light and darkfield images of thorax development revealing expression in the lung (1), atrium (a), ventricle (v), and the liver (li) and vertebral body (vb) (A, B: ×30). (C–H) Higher magnification views of light and darkfield images of abdominal and pelvic organogenesis revealing low level VHL expression in the stomach (st), spleen (sp), and adrenal gland (ad), and high VHL expression in the kidney (k), liver (li), testis (t), and epididymis (ep). Panels E and F demonstrate pancreatic (p) expression localizable to developing epithelial structures. Panels G and H reveal moderate expression in the ovary (ov) and uterus (ut) (C, D: ×30; E, F: ×120; G, H: ×100).

ABDOMEN. Hepatic expression of VHL was diffuse in nature and with embryologic development, VHL expression appeared to be concentrated within the vascular components of the liver (Fig. 2 E and F). The VHL expression noted in the primordial pancreas was unique compared with other organs since it revealed both stromal and epithelial expression as compared with the predominant epithelial expression pattern in other areas often affected in VHL disease (Figs. 1 I and J, 2 E and F). The spleen, intestine, and adrenal gland showed low VHL expression. Specifically in the adrenal gland, no differential expression existed between the cortex as compared with the medulla (Figs. 1 I and J, 2 C and D). This finding was interesting as the cortex is of mesodermal derivation and the medulla is derived from neural crest ectoderm.

GENITOURINARY SYSTEM. By E 10.5, the urogenital ridge was easily recognized (Fig. 1 C, D, G, and H). The bilateral nephrogenic cords produce longitudinal bulges referred to as urogenital ridges, which are the origins of both nephric and gonadal structures (11). Within the urogenital ridge, mesonephric structures revealed differential VHL expression. The epithelial lining of the mesonephric tubules appear to express VHL to a higher degree than the surrounding stromal tissue (Fig. 1 G and H). Within the caudal portion of the retroperitoneum, condensation of metanephric mesoderm around the ureteric bud signals the formation of the permanent kidney. During this early metanephric renal development no differential renal expression was noted (Fig. 1 E and F). By E 13.5, the well-differentiated metanephric kidney revealed a differential pattern of VHL expression (Figs. 1 I and J, 2 C and D). During this time period of metanephric nephrogenesis, the testis, ovary, and supporting structures reveal interesting VHL expression. Specifically, the seminiferous tubules of the testis show higher VHL expression as compared with the surrounding supporting stroma. In addition, the Wolffian duct derivatives consisting of the epididymis and the vas deferens all express VHL. In comparison, the ovary and Muellarian duct derivatives exhibit low levels of VHL expression (Fig. 2 G and H). The developing bladder reveals minimal VHL expression.

Localization of VHL During Murine Nervous System Development

The VHL expression pattern in both the central and peripheral nervous systems corroborates well with the varied clinical presentations seen in this syndrome. By E 10.5 diffuse VHL expression existed within the developing nervous system. While both the neuroepithelium of the lateral ventricles and the cephalic mesenchyme exhibit VHL expression, there was a higher degree of expression in the structures of the developing central nervous system (Fig. 3 A–D).

Fig. 3

VHL expression in the developing murine central nervous system (E = 16.5 days) with light and corresponding darkfield images

(A and B) Prominent VHL expression in the neuroepithelium (ne) and nasal process (np) is evident with lower levels of VHL expression in the brachial arch (ba) (A, B: ×30). (C and D) Higher power views of the same structures revealing differential expression in the brachial arch (ba), neuroepithelium (ne), and nasal process (np) (C, D: ×60). (E–H) Low and high power views of the midbrain (mb), choroid plexus (cp), cerebellum (cb), pons (po), neuroepithelium (ne), and fourth ventricle (v) demonstrating elevated levels of VHL in the cerebellum and choriod plexus (cp) compared with other adjacent structures (E, F: ×60; G, H: ×100). (I and J) Views of the mid and anterior brain demonstrating the thalamus (th), midbrain (mb), and pituitary (pt), all with minimal levels of VHL expression (I, J: ×100). (K–O) Low and high power views of the developing eye revealing intense VHL expression in the retina (rt) and its outer neuroblastic layer (onl) in contrast to the lower levels of expression in the lens (le), vitreous humor (vh), and cornea (c). Panel O represents sense probe (K, L: ×125; M, N, O: ×250).

By E 13.5, the well-developed central nervous system reveals an easily recognizable third and fourth ventricles, thalamus, midbrain, and cerebellum. In addition to the neuroepithelial VHL expression in the above mentioned structures, the cerebellum and choroid plexus of the fourth ventricle appear to contain a higher degree of VHL gene activity (Fig. 3 E–H). In the peripheral nervous system, the cranial nerves of the trigeminal ganglion (V) and the vestibulocochlear ganglion (VIII) as well as the paraspinal ganglions exhibited high levels of VHL expression (data not shown).

The most impressive VHL expression pattern in the central nervous system was noted in the E 16.5 embryo in the structures of the eye. While both the inner and outer neuroblastic layers of the retina and lens contain high VHL expression, the adjacent structures reveal minimal activity. A control sense probe shows no VHL expression indicating the specificity of the expression pattern (Fig. 3O).

Discussion

The Von Hippel-Lindau disease represents a spectrum of phakomatoses whose inherent features include those of an autosomal dominant penetrance, as well as developing lesions with a propensity towards malignant degeneration. The most common lesions in VHL include those of hemangioblastomas of the retina and cerebellum as well as angiomas of spinal cord and cystic lesions of solid organs. These cystic areas tend to affect the pancreas and epididymis and, to a lesser extent, the liver and lungs (12). Furthermore, several urologic manifestations of VHL represent a significant portion of the overall disease process and include pheochromocytoma, renal cysts, renal angiomas, and renal cell carcinomas. With the wide spectrum of anomalies in this disease process, we thought it important to further characterize the VHL expression pattern throughout embryogenesis to further gain insight into normal and abnormal organogenesis, and the developmental potential of benign and malignant degeneration based on VHL misregulation.

VHL expression was clearly evident in all derivatives of the three germ cell layers of the murine embryo model. While specific areas known to undergo abnormal differentiation as part of the VHL syndrome revealed developmental expression of VHL, many areas not previously reported as developing pathologic changes in this syndrome also demonstrated VHL expression. These areas included the thymus and thyroid gland, spleen, heart, bladder, skeletal muscle, uterus, ovary, and testes.

An interesting observation apparent from this in situ investigation was the differential developmental VHL expression within epithelial cells derived from mesoderm (kidney and epididymis), endoderm (lung and pancreas), and ectoderm (eye). As demonstrated in human and murine nephrogenesis, VHL expression was ubiquitous within the metanephric, ureteric, and stromal components of the developing kidney. Higher expression was noted within the well-differentiated epithelial cells of the proximal tubules which are derived from metanephric blastema of mesodermal origin (13). This localization of VHL expression is consistent with the histopathologic studies of the origin of the clear cell phenotype of renal cell carcinoma and their associated VHL mutations and/or methylation (3,14,15). The developing lung also showed a higher differential expression within the endodermally derived cuboidal epithelial lining of the terminal and respiratory bronchioles. Most impressive was the VHL expression within the epithelial cells of the lens which derive from the ectoderm.

Hemangioblastomas of the central and peripheral nervous system as well as angiomas of solid organs are a central feature of the VHL syndrome. While in situ expression of VHL was demonstrated in the characteristic areas where these lesions develop such as the cerebellum and retina, no obvious distinctive cellular features regarding blood vessel expression were localized in these areas. The clinical development of hemangiomas and angiomas has recently been shown to be secondary to VHL mutations which suggest that normal VHL expression may be required for the differentiation of mesenchyme into angioblasts and endothelial cells (6).

Having characterized the pattern of VHL expression throughout murine embryogenesis and human nephrogenesis, how several of the structures with high levels of VHL gene expression undergo malignant transformation remains to be determined. Conversely, why some organ systems with developmental expression of VHL reveal no phenotypic changes in the VHL syndrome is also unclear. Renal cell carcinoma has been studied with respect to various mutations and other changes in the VHL genome that may provide mechanisms for its carcinogenesis. In a recent study by Herman et al., they demonstrated hypermethylation of a normally un-methylated CpG island in the 5′ region of the VHL gene in 5 of 26 tumors examined for this change (15). Another study on renal cell carcinomas confirmed a loss of heterozygosity (LOH) in greater than 97% of sporadic tumors with 57% having VHL mutations (3). As we have shown differential expression of the VHL gene in several areas of the developing embryo, it remains plausible that abnormal methylation of cytosine residues, mutations with or without LOH may convert this normal expression pattern into either a benign or malignant degenerative pattern, or may suppress functional developmental expression following terminal differentiation. Therefore, there appear to be several factors which function as initiating or regulating events of the VHL gene in normal organogenesis as well as the pathogenesis of benign and malignant transformation associated with the VHL syndrome.

In summary, we have characterized a unique pattern of VHL expression in the human fetal kidney and murine embryo model. The differential expression patterns seen in several of the developing tissue structures corroborate with the known postnatal developmental defects that are a part of the spectrum seen in patients with von Hippel-Lindau disease but need to be confirmed by immunohistochemical localization when suitable antibodies become available. However, this work clearly provides a temporal and spatial framework for further identifying and characterizing the deleterious effects of the VHL gene in a variety of disease states not previously reported in the clinical spectrum of the VHL syndrome. In addition, further investigation of the developmental regulation of the VHL gene is warranted based on the unique epithelial induction of VHL expression demonstrated by this in situ hybridization analysis.

Note

The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. The publisher or recipient acknowledges right of the U.S. Government to retain a nonexclusive, royalty-free license in and to any copyright covering the article. Animal care was provided in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 86–23, 1985).