Nephroblastomatosis and nephroblastoma in nonhuman primates

Wilms' tumors, or nephroblastomas, are renal embryonal malignancies with a high incidence in humans. Nephroblastomas are uncommon in nonhuman primates. This report describes three cases of spontaneous proliferative renal tumors in young monkeys: two cases of unilateral kidney nephroblastomas in baboons and a nephroblastomatosis in a cynomolgus macaque. Histologically, both baboon tumors were typical of Wilms' tumors found in humans, with proliferative epithelial cells forming tubules and aborted glomeruli, nephrogenic rests and proliferative fibrovascular tissue. The left kidney of the macaque was markedly enlarged and histologically similar to the baboon tumors, although normal kidney architecture was completely effaced by primitive tubules and occasional glomeruli surrounded by edematous stromal tissue. Cytogenetic analysis did not detect any macaque or baboon equivalents to human Wilms' tumor chromosomal abnormalities. By human pathology classification, the diffuse nature of the macaque tumor is more consistent with nephroblastomatosis than nephroblastoma. This differentiation is the first to be reported in a species other than human. The nephroblastomas described here are the first nephroblastomas to be reported in baboons. Our observations indicate that nonhuman primate nephroblastomatosis and nephroblastomas develop in a similar way to Wilms' tumors in humans, although no genetic marker has been associated with nephroblastomas of nonhuman primates thus far.     

The N-myc proto-oncogene and IGF-II growth factor mRNAs are expressed by distinct cells in human fetal kidney and brain

We studied the expression of the N-myc proto-oncogene and the insulin-like growth factor-II (IGF-II) gene in human fetuses of 16-19 gestational wk. Both genes have specific roles in the growth and differentiation of embryonic tissues, such as the kidney and neural tissue. Since continued expression of N-myc and IGF-II mRNAs is also a characteristic feature of Wilms' tumor, a childhood neoplasm of probable fetal kidney origin, we were particularly interested in the possibility that their expression might be linked or coordinately regulated in the developing kidney. Expression of N-myc mRNA was observed in the brain and in the kidney by Northern hybridization analysis. In in situ hybridization of the kidney, N-myc autoradiographic grains were primarily located over epithelially differentiating mesenchyme while most of the mesenchymal stromal cells showed only a background signal with the N-myc probe. N-myc mRNA was detectable throughout the developing brain with a slight accentuation in the intermediate zone cells in between the subependymal and cortical layers. Thus, even postmitotic neuroepithelial cells of the fetal cerebrum expressed N-myc mRNA. In Northern hybridization, IGF-II mRNA signal was abundant in the kidney but much weaker, though definite, in the brain. The regional distribution of IGF-II mRNA in the kidney was largely complementary to that of N-myc. IGF-II autoradiographic grains were located predominantly over the stromal and blastemal cells with a relative lack of hybridization over the epithelial structures. In the brain, IGF-II mRNA was about two- to threefold more abundant in the subependymal and intermediate layers than in the cortical plate and ependymal zone, respectively. The fetal expression patterns of the N-myc and IGF-II mRNAs are reflected by the types of tumors known to express the corresponding genes during postnatal life such as Wilms' tumor. However, the apparent coexpression of the IGF-II and N-myc genes in immature kidneys occurs largely in distinct cell types.     

Coordinate expression of Wilms' tumor genes correlates with Wilms' tumor phenotypes.

The cloning and molecular characterization of two putative tumor genes, WT1 and WIT1, from the chromosome 11p13 region has provided a means of evaluating their role in the generation of Wilms' tumor heterogeneity. A series of 29 tumors were analyzed for WT1 and WIT1 expression by Northern blot or RNase protection analyses, and results were compared with tumor histopathology. Tumors were scored for the percentage of mesenchymal and epithelial derived tissue components. Homotypic tumors comprised blastema, tubular epithelium, and a fibroblast-like mesenchyme. In addition to these tissue components, the group of tumors designated as heterotypic also contained ectopic cell phenotypes such as muscle and squamous epithelium. The analyses suggest that heterotypic differentiation patterns occur when WT1 and WIT1 expression is low relative to normal fetal kidney. In situ hybridization using antisense RNA probes showed that WT1 and WIT1 were concordantly expressed in normal fetal kidney and in the blastema of tumors. The ratio of WT1:WIT1 expression remained relatively constant in homotypic tumors but deviated significantly in heterotypic tumors. These results suggest that expression patterns of the WT1 and WIT1 genes can be closely correlated to Wilms' tumor histopathology.     

Evaluation of metanephric maturation in a human fetal kidney explant model

Summary We have developed a unique human fetal kidney explant model to study the role of the insulinlike growth factor (IGF) system in metanephric development. Kidneys from 10–18 wk gestation human abortuses were maintained in serum-free conditions and defined medium, which was shown to support the induction and differentiation of the viable metanephric blastema. Histologically the tissue remained viable to 192 h of serum-free culture, while metanephric differentiation, reflected by a shrinking nephrogenic zone and the formation of maturing S-shape and glomerular forms, was accelerated and occurred between 48 and 96 h. In the nephrogenic zone, a significant decrease in IGF-II gene expression occurred, which reflected the differentiation of the metanephric blastema cell mass. IGF-II expression persisted, however, in the expanded interstitial mesenchyme. With differentiation over 48 h an increase in IGFBP-2 and WT1 gene expression by Northern blot analysis occurred, and was localized by in situ hybridization to the differentiating glomerular epithelial cell mass. Analysis of the explant-conditioned media by Western ligand blot demonstrated an increase in the rate of IGF binding protein (IGFBP)-2 peptide production by the differentiating explant, consistent with an increase in IGFBP-2 gene expression and with metanephric differentiation. This pattern of temporal and spatial gene expression closely approximates that of normal in vivo fetal renal development and of glomerular epithelial cell differentiation.     

Allelic expression of the putative tumor suppressor gene p73 in human fetal tissues and tumor specimens

p73, a proposed tumor suppressor, shares significant amino acid sequence homology with p53. However, p73 is rarely mutated in tumors but it has been suggested that p73 is monoallelically expressed in some tissues. This latter feature would predispose p73 to gene inactivation because a single genetic 'hit' or the loss of the expressed parental allele would leave the cell without p73 activity. We examined the allelic expression of p73 in normal fetal tissues and in ovarian cancer and Wilms' tumor. We found that p73 was biallelically expressed in all fetal tissues, except in brain, where differential expression of the two parental alleles was observed. Biallelic expression of p73 was also observed in paired samples of ovary cancer and Wilms' tumor. Loss of heterozygosity of p73 occurred at relatively low rates in tumors: one of 11 informative samples (9.1%) of ovarian cancer and two of 19 (10.1%) Wilms' tumors. These data demonstrate that p73 is biallelically expressed in most tissues, thus excluding genomic imprinting as a molecular mechanism to predispose to allelic inactivation of p73 in human tumors.     

Renal ontogeny in the rhesus monkey (Macaca mulatta) and directed differentiation of human embryonic stem cells towards kidney precursors

The development of the metanephric kidney was studied immunohistochemically across gestation in monkeys to identify markers of cell specification, and to aid in developing experimental paradigms for renal precursor differentiation from human embryonic stem cells (hESC). PAX2, an important kidney developmental marker, was expressed at the tips of the ureteric bud, in the surrounding condensing mesenchyme, and in the renal vesicle. Vimentin, a mesenchymal and renal marker, was strongly expressed in the metanephric blastema then found to be limited to the glomerulus and interstitial cells of the medulla and cortex. A model of gene expression based on human and nonhuman primate renal ontogeny was developed and incorporated into studies of hESC differentiation. Spontaneous hESC differentiation revealed markers of metanephric mesenchyme (OSR1, PAX2, SIX2, WT1) that increased over time, followed by upregulation of kidney precursor markers (EYA1, LIM1, CD24). Directed hESC differentiation was also evaluated with the addition of retinoic acid, Activin-A, and BMP-4 or BMP-7, and using different culture substrate conditions. Of the culture substrates studied, gelatin most closely recapitulated the anticipated directed developmental pattern of renal gene expression. No differences were found when BMP-4 and BMP-7 were compared with baseline conditions. PAX2 and Vimentin immunoreactivity in differentiating hESC was also similar to the renal precursor patterns reported for human fetal kidneys and findings described in rhesus monkeys. The results of these studies are as follows: (1) provide additional data to support that rhesus monkey kidney development parallels that of humans, and (2) provide a useful model for hESC directed differentiation towards renal precursors.     

Expression of the Von Hippel-Lindau tumor suppressor gene,VHL, in human fetal kidney and during mouse embryogenesis.

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 tumours. 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.     

Eel WT1 sequence and expression in spontaneous nephroblastomas in Japanese eel

Nephroblastomas spontaneously developing in Japanese eel reared at farms for 5 to 9months after collection from the wild [Masahito et al., Cancer Res., 52 (1992) 2575-2579] were investigated to cast light on the role of Wilms' tumor 1 gene (WT1) in eel kidney tumorigenesis. Cloning of the WT1 counterpart, EWT1, revealed that conservation of an alternative splice II site, located between the third and fourth zinc fingers, was conserved. The zinc finger domain was highly conserved. The transregulator region, sequences corresponding to exons 4 and 5 in WT1, were lacking in EWT1 cDNA. EWT1 was found to be expressed in kidney, testis and spleen and in situ hybridization revealed dark-stained immature cells in elver kidney to be positive. Although no EWT1 gene mutations were found in 38 eel nephroblastomas, 26 polymorphic nucleic acid changes were observed. Aberrant WT1 expression was noted in epithelial (12 out of 27; 44%) and nephroblastic cell histological types (three out of five; 60%) of eel nephroblastomas. On in situ hybridization the EWT1 expressive cells resembled human blastema cells, similar to those in human Wilms' tumor. These data demonstrated strong signals that the EWT1 protein may function in the development of eel kidney and play a role in genesis of nephroblastomas as in mammals.     

Genome-wide loss of maternal alleles in a nephrogenic rest and Wilms' tumour from a BWS patient

A patient with Beckwith-Wiedemann syndrome (BWS) presented with Wilms' tumour. Examination of the nephrectomy specimen showed, in addition to the tumour, the presence of nephrogenic rests. Nephrogenic rests are thought to be precursor lesions from which a Wilms' tumour may develop. A molecular analysis examining the loss of constitutional heterozygosity (LOCH), initially for chromosome 11, was performed on peripheral blood, the normal kidney, nephrogenic rest and tumour material. The study was extended to include markers from all 23 chromosomes. At each informative, locus, LOCH of the maternal allele was shown in the nephrogenic rest and tumour material. In addition, the normal kidney displayed allele imbalance. It would appear from these results that either extensive LOCH across the genome was an early genetic event in the development of malignancy in this patient or that the tumour and rest developed from cells containing no maternal chromosomes. The apparent LOCH seen in the normal kidney sample implies that full reduction to homozygosity is consistent with a histologically normal appearance. Putative mechanisms to explain this phenomenon are discussed.     

Temporal changes in the expression of the insulin-like growth factor II gene associated with tissue maturation in the human fetus

The insulin-like growth factors are broadly distributed in the human conceptus and are thought to play a role in the growth and differentiation of tissues during development. Using in situ hybridization we have shown that a wide variety of specific cell types within tissues express the gene for insulin-like growth factor II at times of development from 18 days to 14 weeks of gestation. Examination of blastocysts produced by in vitro fertilization showed no expression, thus bracketing the time of first accumulation of IGF-II mRNA to between 5 and 18 days postfertilization. The pattern of IGF-II expression shows specific age-related differences in different tissues. In the kidney, for example, expression is found in the cells of the metanephric blastema which is dramatically reduced as the blastema differentiates. The reverse is also seen, and we have noted an increase in expression of IGF-II in the cytotrophoblast layer of the placenta with gestational age. The sites of expression do not correlate with areas of either high mitotic activity or specific types of differentiation, but the observed pattern of expression in the kidney, adrenal glands and liver suggests an explanation for the abnormally high IGF-II mRNA expression in developmental tumours such as Wilms' tumour.     

配对盒基因家族——发育过程中重要的转录因子

在大量的脊椎和无脊椎动物中发现的配对盒转录因子(paired box,PAX)及其同源物,在胚胎发育的许多阶段发挥着关键的作用.该基因家族因其具有保守的成对结构域而得名,除此之外其还具有八肽和同源域.根据结构域的组成和序列的同源性,该基因家族主要分为4个亚家族:PAX1/9(PAX1、PAX9),PAX2/5/8(PA...