Embryonic origin and lineage of juxtaglomerular cells.

To define the embryonic origin and lineage of the juxtaglomerular (JG) cell, transplantation of embryonic kidneys between genetically marked and wild-type mice; labeling studies for renin, smooth muscle, and endothelial cells at different developmental stages; and single cell RT-PCR for renin and other cell identity markers in prevascular kidneys were performed. From embryonic kidney day 12 to day 15 (E12 to E15), renin cells did not yet express smooth muscle or endothelial markers. At E16 renin cells acquired smooth muscle but not endothelial markers, indicating that these cells are not related to the endothelial lineage, and that the smooth muscle phenotype is a later event in the differentiation of the JG cell. Prevascular genetically labeled E12 mouse kidneys transplanted into the anterior chamber of the eye or under the kidney capsule of adult mice demonstrated that renin cell progenitors originating within the metanephric blastema differentiated in situ to JG cells. We conclude that JG cells originate from the metanephric mesenchyme rather than from an extrarenal source. We propose that renin cells are less differentiated than (and have the capability to give rise to) smooth muscle cells of the renal arterioles.

Ren1d and Ren2 cooperate to preserve homeostasis: evidence from mice expressing GFP in place of Ren1d.

To distinguish the contributions of Ren1(d) and Ren2 to kidney development and blood pressure homeostasis, we placed green fluorescent protein (GFP) under control of the Ren1(d) renin locus by homologous recombination in mice. Homozygous Ren1(d)-GFP animals make GFP mRNA in place of Ren1(d) mRNA in the kidney and maintain Ren2 synthesis in the juxtaglomerular (JG) cells. GFP expression provides an accurate marker of Ren1(d) expression during development. Kidneys from homozygous animals are histologically normal, although with fewer secretory granules in the JG cells. Blood pressure and circulating renin are reduced in Ren1(d)-GFP homozygotes. Acute administration of losartan decreases blood pressure further, suggesting a role for Ren2 protein in blood pressure homeostasis. These studies demonstrate that, in the absence of Ren1(d), Ren2 preserves normal kidney development and prevents severe hypotension. Chronic losartan treatment results in compensation via recruitment of both Ren1(d)- and Ren2-expressing cells along the preglomerular vessels. This response is achieved by metaplastic transformation of arteriolar smooth muscle cells, a major mechanism to control renin bioavailability and blood pressure homeostasis.

Molecular cloning of KS, a novel rat gene expressed exclusively in the kidney.

BACKGROUND: We aimed to identify genes with kidney specific, developmentally regulated expression. Here we report the cDNA sequence and expression pattern of KS, a novel kidney-specific rat gene. METHODS: A partial cDNA was identified by differential display polymerase chain reaction (PCR) of a renal cell fraction enriched for proximal tubular and renin-expressing cells. Using the partial cDNA as a probe, a rat kidney cDNA library was screened. The full-length KS sequence was obtained by PCR amplification of cDNA ends. The expression pattern of KS was investigated by Northern blot. RNA was extracted from several organs of newborn and adult rats, as well as from the kidneys of rats with altered tubular function, that is, rats that had undergone unilateral nephrectomy, unilateral ureteral obstruction, neonatal losartan treatment, and the appropriate control animals. The expression of KS was also investigated in the kidneys of rats with spontaneous or renovascular hypertension. RESULTS: The KS cDNA (2426 bp) contained one open reading frame encoding a predicted 572 amino acid protein. The derived peptide sequence displayed approximately 70% similarity to the hypertension-related SA gene product and approximately 50% similarity to prokaryotic and eukaryotic acetyl-CoA synthases (EC 6. 2.1.1). KS was expressed in the kidney and not in any other organ assayed. KS RNA was not detected in fetal and newborn rat kidney but became apparent after one week of postnatal life. Gene expression was downregulated in rat models of altered tubular function. KS expression was decreased in spontaneously hypertensive rats but not in renovascular hypertension. CONCLUSION: KS, a novel rat gene, exhibits a unique tissue-specific expression exclusively in mature kidneys. The data suggest KS may encode an adenosine monophosphate binding enzyme.

Angiotensin-dependent gene expression in the developing rat kidney.

We aimed to identify genes involved in the growth effects of angiotensin II (Ang II) during kidney development. In rats treated from birth with the Ang II type-1 receptor blocker losartan, expression of transforming growth factor beta1 (TGF-beta1), platelet-derived growth factor B (PDGF-B), vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF), as measured by Northern blot, did not change significantly (N = 4 to 6 per group each). Differential display methods, used to identify genes with Ang II-dependent expression, produced mostly false positives. We identified one novel rat partial cDNA, termed AD.5, that is related to a human orphan receptor. AD.5 was expressed in a developmentally regulated pattern and may be involved in kidney development and/or the trophic actions of Ang II.

Renin-expressing cells are associated with branching of the developing kidney vasculature.

To define the relationship between renal vascular development and renin distribution during kidney ontogeny, the complete renal arterial tree of Sprague Dawley rats during fetal (20 d) and postnatal (1 to 90 d) life was microdissected and immunostained for renin. A shift in renin distribution from interlobar and arcuate arteries in the fetus to the afferent arterioles in the adult was observed. In addition, seven types of renin distribution along the afferent arterioles were identified. In type I, renin was distributed continuously along the whole length of the afferent vessel. This pattern was most frequently observed in the fetus. In type II, renin extended upstream from the glomerulus but did not occupy the whole length of the arteriole. This type was relatively constant throughout postnatal life. In type III, renin was present as bands along the afferent vessel; it was most frequently observed in the fetal and early perinatal periods. In type IV, renin was restricted to the "classical" juxtaglomerular localization. It was the most frequent type observed in the adult rat. In type V, no renin was found in the arteriole. It was the second most frequent type observed in the adult rat. In addition, two "mixed" patterns, type III/IV and type III/II, were occasionally observed. The distribution of renin-expressing cells was spatially and temporally associated with the development of blood vessels. Development of a new arterial branch was preceded by the appearance of renin-expressing cells at the point of branching. This was followed by an outpouching of the arterial wall that progressively elongated to form a new arteriole. During this process, renin-expressing cells were distributed along the whole of the newly formed vessel. As the vessel matured, renin-expressing cells became restricted to the juxtaglomerular portion of the afferent arteriole. It is concluded that throughout life and within each individual arterial tree, expression of renin is heterogeneous, following patterns that are unique for each developmental stage. Furthermore, the association of renin-expressing cells with branching of renal arterioles suggests a role for these cells in the development of the kidney vasculature.

Zis: a developmentally regulated gene expressed in juxtaglomerular cells.

Renal juxtaglomerular (JG) cells are specialized myoepithelioid cells located in the afferent arteriole at the entrance to the glomerulus. Their main function and distinctive feature is the synthesis and release of renin, the key hormone-enzyme of the renin-angiotensin system that regulates arterial blood pressure. Despite their relevance to health and disease, not much is known about factors that confer and/or maintain JG cell identity. To identify genes uniquely expressed in JG cells, we used a cell culture model and RNA differential display. JG cells cultured for 2 days express renin and renin mRNA, but after 10 days in culture they no longer contain or release renin and renin mRNA is reduced 700-fold. We report one cDNA differentially expressed in the 2-day JG cell culture that detects a 2.6-kb mRNA expressed at higher levels in newborn than adult kidney. Screening a 2-day culture JG cell cDNA library yielded clones representing differentially spliced transcripts. These cDNAs encode one unique protein (Zis) containing zinc fingers and domains characteristic of splicing factors and RNA binding proteins. Northern blot analysis confirmed Zis mRNA expression in differentiated JG cells, and identified an additional unique 1.5-kb transcript. The Zis transcripts are developmentally regulated in kidney and a number of other organs. The features of the Zis protein and its organ distribution suggest a possible role in regulation of transcription and/or splicing, both important steps for controlling developmentally expressed genes.

Biomechanical coupling in renin-releasing cells.

The renin-angiotensin system is a major regulatory system controlling extracellular fluid volume and blood pressure. The rate-limiting enzyme in this hormonal cascade is renin, which is synthesized and secreted into the circulation by renal juxtaglomerular (JG) cells. The renal baroreceptor is a key physiologic regulator of renin secretion, whereby a change in renal perfusion pressure is sensed by these cells and results in a change in renin release. However, the mechanism, direct or indirect, underlying pressure transduction is unknown. We studied the direct application of mechanical stretch to rat JG cells and human renin-expressing (CaLu-6) cells on the release of renin. JG cells released a low level of baseline renin, comprising < 5% of their total renin content. By contrast, renin secretion from CaLu-6 cells comprised approximately 30% of cellular stores, yet was also stimulated twofold by 10 microM forskolin (P

The genetic and molecular organization of the Dopa decarboxylase gene cluster of Drosophila melanogaster.

We report the complete molecular organization of the Dopa decarboxylase gene cluster. Mutagenesis screens recovered 77 new Df(2L)TW130 recessive lethal mutations. These new alleles combined with 263 previously isolated mutations in the cluster to define 18 essential genes. In addition, seven new deficiencies were isolated and characterized. Deficiency mapping, restriction fragment length polymorphism (RFLP) analysis and P-element-mediated germline transformation experiments determined the gene order for all 18 loci. Genomic and cDNA restriction endonuclease mapping, Northern blot analysis and DNA sequencing provided information on exact gene location, mRNA size and transcriptional direction for most of these loci. In addition, this analysis identified two transcription units that had not previously been identified by extensive mutagenesis screening. Most of the loci are contained within two dense subclusters. We discuss the effectiveness of mutagens and strategies used in our screens, the variable mutability of loci within the genome of Drosophila melanogaster, the cytological and molecular organization of the Ddc gene cluster, the validity of the one band-one gene hypothesis and a possible purpose for the clustering of genes in the Ddc region.

Ontogeny of renin and AT1 receptor in the rat.

The enzyme renin and the angiotensin II (Ang II), subtype I receptor (ATI) are developmentally regulated in a tissue-specific manner. In early life, renin is expressed widely along the renal vasculature. As maturation progresses, there is a decrease in renin mRNA levels and a shift in the localization of renin close to the glomerulus. In addition, in the newborn rat, the number of renin-secreting cells is higher than in the adult rat. Exposure of neonatal and adult cells to Ang II results in a decrease of similar magnitude in the number of renin-secreting cells. These findings suggest that the high levels of renin observed in immature animals are due to increased renin synthesis and release rather than to a blunted response to Ang II. Expression of the ATI gene is also developmentally regulated in a tissue-specific manner. With maturation, ATI mRNA levels decrease in the kidney while they increase in the liver. The localization of ATI transcripts in precursor cells of the nephrogenic cortex suggests a role for this receptor in nephron growth and development. Inhibition of ATI with DUP753 results in delayed kidney and somatic growth and in increased renin mRNA levels and recruitment of renin-containing cells. These observations suggest that Ang II exerts a tonic negative feedback on renin gene expression via the ATI receptor subtype. Further studies are necessary to delineate the molecular and cellular signals mediating these developmental changes.

Drosophila melanogaster diphenol oxidase A2: gene structure and homology with the mouse mast-cell tum- transplantation antigen, P91A.

The Drosophila melanogaster diphenol oxidase (DOX) A2-encoding gene (Dox-A2) is involved in catecholamine metabolism, melanin formation and sclerotization of the cuticle. Insect phenol oxidases (POX) are well studied biochemically, but not genetically and molecularly. The Dox-A2 (2-53.9) gene is the first insect POX-encoding gene to be cloned and sequenced. It encodes a protein product unique among currently known POX. The deduced protein, however, exhibits extensive similarity (58-81%) to the mouse mast cell tum- antigen, P91A [Lurquin et al., Cell 58 (1989) 293-303] and may identify the normal mouse protein as a DOX.

Mutations affecting phenol oxidase activity in Drosophila: quicksilver and tyrosinase-1.

The complex enzyme phenol oxidase plays a major role in sclerotization and melanization of cuticle in insects. Production of active enzyme from the inactive proenzyme involves at least six protein components in Drosophila. We examine here the biochemical phenotype of two loci that affect phenol oxidase activity--quicksilver (qs; 1-39.5) and tyrosinase-1 (tyr-1; 2-54.5). Three mutations isolated by different procedures in three different laboratories are alleles at the quicksilver locus. The effects of these mutations have been monitored by means of enzyme assays in vitro and in polyacrylamide gels and by measurement of catecholamine pool sizes. The activity of all three active enzyme components (A1, A2, and A3) is reduced in qs mutants. The activated enzyme of one qs allele is thermolabile, while its activator is normal. Deletion and genetic mapping place tyr-1 near purple (pr; 2-54.5). Enzyme activity is reduced to 10% of normal but is not thermolabile and the activator is normal. The activity of all three A components is reduced. The diphenol oxidase activity in double mutant combinations shows that these mutations and Dox-A2 (Pentz et al., 1986) affect this enzyme in different ways.

The alpha methyl dopa hypersensitive gene, 1(2)amd, and two adjacent genes in Drosophila melanogaster: physical location and direct effects of amd on catecholamine metabolism.

The dopa decarboxylase gene (Ddc) is located in a very dense cluster of genes many of whose functions appear to be related to the physiological role of dopa decarboxylase (DDC) in catecholamine metabolism. In Drosophila melanogaster catecholamine metabolism is involved in the production of neurotransmitters and in the synthesis of cross-linking agents for cuticular sclerotization. In this report we consider three loci near Ddc that affect cuticle formation. The alpha methyl dopa hypersensitive gene, 1(2)amd, is definitively assigned to a transcriptional unit 2 kb distal to Ddc. The assignment of 1(2) 37 Bd and 1(2)37 Cc to coding regions in the immediate vicinity of amd and Ddc is examined. amd+ gene activity performs a vital function essential for the formation of insect cuticle and also determines the level of sensitivity to the DDC analogue inhibitor, alpha methyl dopa. We present data that provide direct evidence that the amd+ gene product is required for a step in the metabolism of dopa to one or more novel catecholamines involved in the colorless sclerotization of cuticle.

A diphenol oxidase gene is part of a cluster of genes involved in catecholamine metabolism and sclerotization in Drosophila. II. Molecular localization of the Dox-A2 coding region.

Mutations at the Dox-A2 (2-53.9) locus alter the A2 component of diphenol oxidase, an enzyme having an important role in cuticle formation. This locus is in the dopa decarboxylase, Df(2L)TW130 region, which contains a cluster of at least 14 genes involved in catecholamine metabolism and the formation, sclerotization and melanization of cuticle in Drosophila. The region is subdivided by deficiencies, and localization of breakpoints in cloned DNA reveals a dense subcluster of six genes in the 23 kb proximal to Ddc. Five lethal loci distal to Ddc comprise a second such subcluster. The proximal breakpoints of deficiencies Df(2L)hk18 and Df(2L)OD15 define a 14.3- to 16.8-kb region containing Dox-A2 and l(2)37Bb, and those of Df(2L)OD15 and Df(2L)TW203 define a 9.3- to 12.1-kb region containing l(2)37Ba, l(2)37Bc and l(2)37Be. Southern blots show two of the Dox-A2 mutations are small deletions (0.1 and 1.1 kb). The Dox-A2 locus mRNA is 1.7 kb. cDNA clones indicate that the 3' end is centromere proximal and that the coding region contains at least one small intron. The Dox-A2 locus is within 3.4 to 4.4 kb of the Df(2L)OD15 breakpoint, placing four of the vital loci within a maximum of 15.5 kb. The location of Dox-A2 in a cluster of genes affecting cuticle formation is discussed.

A diphenol oxidase gene is part of a cluster of genes involved in catecholamine metabolism and sclerotization in drosophila. I. Identification of the biochemical defect in Dox-A2 [l(2)37Bf] mutants.

Phenol oxidase, a complex enzyme, plays a major role in the processes of sclerotization and melanization of cuticle in insects. Several loci have been reported to affect levels of phenol oxidase activity, but to date only one structural locus has been identified [Dox-3F (2-53.1+)]. Recently isolated Dox-A2 mutations (2-53.9) are recessive, early larval lethals, which as heterozygotes reduce phenol oxidase activity. A homozygous mutant escaper had weak, completely unpigmented cuticle and unpigmented bristles. Enzyme assays show that Dox-A2 heterozygotes have diphenol oxidase activity reduced to 47-79% of wild type, whereas monophenol oxidase activity, at 94-106% of wild type, is normal. Elevated pool sizes of the diphenol oxidase substrates DOPA, dopamine, and N-acetyldopamine are observed in the mutant, confirming the enzyme assay results. Separation of the three phenol oxidase A component activities on polyacrylamide gels shows that Dox-A2 mutations reduce the activity of only the A2 component. Dox-A2 may identify a structural locus for the A2 component of the diphenol oxidase enzyme system. The Dox-A2 locus is one of 18 loci in the dopa decarboxylase, Df (2L)TW130 region of the second chromosome, at least 14 of which affect the formation, melanization or sclerotization of cuticle in some way. These loci form an apparent cluster of functionally related genes.

Multiple Allele Approach to the Study of Genes in DROSOPHILA MELANOGASTER That Are Involved in Imaginal Disc Development.

The phenotypes of five different lethal mutants of Drosophila melanogaster that have small imaginal discs were analyzed in detail. From these results, we inferred whether or not the observed imaginal disc phenotype resulted exclusively from a primary imaginal disc defect in each mutant. To examine the validity of these inferences, we employed a multiple-allele method. Lethal alleles of the five third-chromosome mutations were identified by screening EMS-treated chromosomes for those which fail to complement with a chromosome containing all five reference mutations. Twenty-four mutants were isolated from 13,197 treated chromosomes. Each of the 24 was then tested for complementation with each of the five reference mutants. There was no significant difference in the mutation frequencies at these five loci. The stage of lethality and the imaginal disc morphology of each mutant allele were compared to those of its reference allele in order to examine the range of defects to be found among lethal alleles of each locus. In addition, hybrids of the alleles were examined for intracistronic complementation. For two of the five loci, we detected no significant phenotypic variation among lethal alleles. We infer that each of the mutant alleles at these two loci cause expression of the null activity phenotype. However, for the three other loci, we did detect significant phenotypic variation among lethal alleles. In fact, one of the mutant alleles at each of these three loci causes no detectable imaginal disc defect. This demonstrates that attempting to assess the developmental role of a gene by studying a single mutant allele may lead to erroneous conclusions. As a byproduct of the mutagenesis procedure, we have isolated two dominant, cold-sensitive mutants.