Physiological non-equivalence of the two isoforms of angiotensin-converting enzyme.

The structurally related somatic and germinal isoforms of angiotensin-converting enzyme (ACE) contain the same catalytic active center and are encoded by the same gene, whose disruption causes renal atrophy, hypotension, and male sterility. The reason for the evolutionary conservation of both isozymes is an enigma, because, in vitro, they have very similar enzymatic properties. Despite the common enzymatic properties, discrete expression of both isoforms is maintained in alternate cell types. We have previously shown that sperm-specific expression of transgenic germinal ACE in Ace -/- male mice restores fertility without curing their other abnormalities (Ramaraj, P., Kessler, S. P., Colmenares, C. & Sen, G. C. (1998) J. Clin. Invest. 102, 371-378). In this report we tested the biological equivalence of somatic ACE and germinal ACE utilizing an in vivo isozymic substitution approach. Here we report that restoration of male fertility was not achieved by the transgenic expression of enzymatically active, somatic ACE in the sperm of Ace -/- mice. Therefore, the requisite physiological functions of the two tissue-specific isozymes of ACE are not interchangeable.

Tissue-specific regulation of the WT1 locus.

The 11p13 Wilms' tumor locus consists of two coordinately regulated transcripts, WT1 and WIT-1. These genes are highly expressed in the developing urogenital system, beginning with the urogenital ridge at day 10.5, the metanephric blastema at day 11.5, and during glomerular formation at day 13.5, becoming ultimately restricted to the podocytes. Stromal cells of the gonad also show abundant expression. WT1 is expressed at lower levels in spleen, uterus, mesothelial linings of organs in the abdominal and thoracic cavities, and the ependymal layer of the ventral aspect of the spinal cord. WIT-1 mRNA is about 10-fold less abundant than WT1, but appears to be expressed in the same tissue-restricted manner. Expression of the WT1 protein is required for kidney development, although its physiological function remains to be determined. The function of WIT-1 is similarly unknown but one intriguing possibility is that it is an antisense regulator of WT1. An understanding of events controlling spatial and temporal regulation of these genes will greatly improve our ability to study the role of WT1 and WIT-1 in urogenital development. We have found that while chimeric reporter constructs containing 0.6-2.5 kb of the 5' region of the WT1 gene direct transcription in many different cell lines, we were unable to detect expression in 13.5-day mouse embryos. However, a cosmid containing about 42 kb encompassing this region was able to direct the expression of abundant levels of mRNA from the appropriate transcription initiation sites in both stable transfectants of mouse Leydig cells (TM3) or in transgenic embryos. We are currently localizing the DNA elements required for this expression.

In situ expression of the early growth response gene-1 during murine nephrogenesis.

WT1 maps to chromosome 11p13 and encodes a deoxyribonucleic acid (DNA) binding protein whose expression is necessary for normal urogenital development. The WT1 protein binds to some of the same DNA sequences as the early growth response gene-1 (EGR-1) protein, the latter being an immediate-early gene product that activates or represses transcription in a promoter and cell-specific manner. Transient transfection experiments have shown that WT1 can repress EGR-1 activated transcription from the EGR-1 promoter. To determine if WT1 is likely to be a physiologically important repressor of EGR-1 we performed ribonucleic acid (RNA) in situ hybridization of EGR-1 on sequential sagittal sections of murine embryos before and throughout nephrogenesis, and compared the results to our previous study of WT1 expression during murine embryogenesis. Prior to embryological day 9.5 WT1 messenger RNA expression is absent in the embryo proper but is expressed in the maternal uterus. With the initiation of organogenesis on embryological day 10.5 WT1 messenger RNA localizes within the pronephric and mesonephric tissues. By embryological day 11.5 the nephrogenic cord, urogenital ridge and metanephric tissue have WT1 hybridization signals and increasingly centripetal expression of WT1 in the kidney correlates with differentiation from embryological days 11.5 to 16.5. In contrast to previous reports of the tissue restricted expression of WT1, EGR-1 expression by in situ hybridization was apparent in all 3 germ layers and their derivatives throughout embryogenesis. Down-regulation of EGR-1 expression occurred in the maternal uterus as well as the metanephric blastema and its derivatives during renal development. This observation defines a spatial and temporal window during which WT1 competition for EGR-1 DNA binding sites may be involved in regulating EGR-1 expression.

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.

Antisense transcripts and protein binding motifs within the Wilms tumour (WT1) locus.

Transcription of the WT1 locus is restricted, both temporally and spatially, to a subset of epithelial cells in mammalian kidneys and gonads. WT1, one of the two divergent transcripts mapping to this locus encodes a zinc finger protein that is likely a transcriptional regulator. The other transcript, WIT1, encodes a product of unknown function that is subject to alternate splicing in the region immediately 5' of the WT1 gene. Analysis of the 5' end of this locus further revealed the presence of multiple transcriptional start sites for both genes, such that some of the WIT1 transcripts are encoded by the antisense strand of the first exon of WT1. The genomic region surrounding the transcriptional start sites appears to constitute part of a bi-directional promoter based on the ability of a DNA fragment derived from this region to direct expression of a chimeric CAT gene construct in transient transfection assays. Discrete sequences within the region are capable of interaction in vitro with nuclear extracts derived from a variety of rat and mouse tissues. Interestingly, recombinant WT1, representing the product of zinc finger region of the most abundant of the four alternatively spliced transcripts, is also capable of binding to sequences within this region.

Expression of the Wilms' tumor suppressor gene WT1 during mouse embryogenesis.

WT1 is a Wilms' tumor suppressor gene that maps to human chromosome 11p13 and encodes a putative transcription factor implicated in controlling normal urogenital development. Sporadic homozygous mutations in WT1 result in the development of Wilms' tumor (nephroblastoma), and heterozygous germline mutations can give rise to a phenotype which includes nephropathy and urogenital abnormalities (the Denys-Drash syndrome). Thus, inappropriate expression of WT1 results in developmental abnormalities affecting the urogenital system. To better define the temporal and spatial distribution of WT1 expression during embryogenesis, we have used in situ mRNA hybridization and immunohistochemistry to examine WT1 expression in murine embryos during the period prior to and throughout active organogenesis. Prior to embryological day 9.5 (E9.5), WT1 mRNA expression is absent in the embryo proper but is strongly expressed in the maternal uterus. During the initiation of organogenesis on E10.5, WT1 mRNA is localized within the pronephric and mesonephric tissues. By E11.5, the nephrogenic cord, urogenital ridge, and condensing metanephric tissue show intense WT1 hybridization signals, and increasingly centripetal expression of WT1 in the kidney correlates with renal differentiation from days E11.5 through E16.5. The stromal cell components in the developing gonad show expression of WT1 by E10.5, whereas in the remaining organs examined, WT1 expression is restricted to the uterus, spleen, abdominal wall musculature, and mesothelial lining of organs within the thoracic and abdominal cavities. Interestingly, there is also WT1 expression in the central nervous system which localizes to the ependymal layer of the ventral aspect of the spinal cord.(ABSTRACT TRUNCATED AT 250 WORDS)

Estrogen induction of alcohol dehydrogenase in the uropygial gland of mallard ducks.

Treatment of mallard ducks with estradiol, or a combination of estradiol and thyroxine, has been shown to result in the proliferation of peroxisomes and production of diesters of 3-hydroxy fatty acids, the female pheromones, in the uropygial gland of male and female mallard ducks. Such a treatment results in the induction of a unique set of proteins. A cDNA library enriched in hormone-induced transcripts was subjected to differential screening. The nucleotide sequence of one of the two unique cDNA clones, DGH1, had high similarity to the Human class I alcohol dehydrogenase (ADH) gamma subunit and represented the carboxy-terminus of the protein from amino acid 190-374. SDS/PAGE and Western blot analysis of the proteins indicated that the level of a 38-kDa protein that cross-reacted with antibodies prepared against the chicken ADH was increased 5-7-fold by hormone treatment. Assays for ADH activity in the uropygial gland extracts of male mallards showed a 5-7-fold induction of the enzyme by hormone treatment. The 1.9-kb ADH mRNA levels were increased 12-14-fold under these conditions. Of all the tissues tested, the uropygial gland had the highest levels of ADH mRNA. Induction of ADH by estradiol treatment occurred only in this tissue. Elevated levels of ADH were also observed in the glands of male mallards in eclipse, the post-nuptial condition when the hormonal balance is shifted to higher estrogen levels, suggesting that this enzyme is regulated by estrogens in this period. Estradiol treatment caused an 80% decrease in the NAD+/NADH ratio in the uropygial gland and a twofold increase in the fatty alcohol oxidation rate catalyzed by the gland extract. These observations could help explain how increased levels of ADH could contribute to the production of the diesters.