Paula M. Oliver

TGFα deficiency results in hair follicle and eye abnormalities in targeted and waved-1 mice

To explore the physiological roles of transforming growth factor alpha (TGF alpha), we disrupted the mouse gene by homologous recombination in embryonic stem cells. Homozygous mutant mice were viable and fertile, but displayed pronounced waviness of the whiskers and fur, accompanied by abnormal curvature, disorientation, and misalignment of the hair follicles. Homozygous and, to a lesser extent, heterozygous mice displayed eye abnormalities of variable incidence and severity, including open eyelids at birth, reduced eyeball size, and superficial opacity. Histological examination revealed eyelid and anterior segment dysgenesis, corneal inflammation and scarring, and lens and retinal defects. Although TGF alpha deficiency affected skin and eyes, wound healing in these tissues was not impaired. Similar hair and eye defects have been previously associated with the recessive mutation waved-1 (wa-1), and Northern analysis revealed reduced expression of TGF alpha in wa-1 mice. Crosses between wa-1 homozygotes and TGF alpha-targeted mice confirmed that wa-1 and TGF alpha are allelic.

Hypertension Associated with Decreased Testosterone Levels in Natriuretic Peptide Receptor-A Gene-Knockout and Gene-Duplicated Mutant Mouse Models*

Mice lacking the gene (Npr1) encoding the natriuretic peptide receptor A (NPRA) have hypertension with elevated blood pressure and cardiac hypertrophy. In particular, Npr1 gene-deficient male mice exhibit lethal vascular events similar to those seen in untreated human hypertensive patients. Serum testosterone levels tend to be lower in hypertensive male humans than in normal males without hypertension, but the genetic basis for this tendency remains unknown. To determine whether Npr1 gene function affects the testosterone level, we measured serum testosterone in male hypertensive mice lacking a functional Npr1 gene, wild-type animals with two copies, and the gene-duplicated littermates expressing four copies of the gene. In the Npr1 gene-knockout (zero-copy) mice, the serum testosterone level was 62% lower than that in the two-copy control mice (80 ± 10 vs. 120 ± 14 ng/ml, respectively; P < 0.005). Serum testosterone in the four-copy mice was 144% (P < 0.005) of that in the two-copy wild-type control mice...

Evidence for a novel natriuretic peptide receptor that prefers brain natriuretic peptide over atrial natriuretic peptide.

Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) exert their physiological actions by binding to natriuretic peptide receptor A (NPRA), a receptor guanylate cyclase (rGC) that synthesizes cGMP in response to both ligands. The family of rGCs is rapidly expanding, and it is plausible that there might be additional, as yet undiscovered, rGCs whose function is to provide alternative signalling pathways for one or both of these peptides, particularly given the low affinity of NPRA for BNP. We have investigated this hypothesis, using a genetically modified (knockout) mouse in which the gene encoding NPRA has been disrupted. Enzyme assays and NPRA-specific Western blots performed on tissues from wild-type mice demonstrate that ANP-activated cGMP synthesis provides a good index of NPRA protein expression, which ranges from maximal in adrenal gland, lung, kidney, and testis to minimal in heart and colon. In contrast, immunoreactive NPRA is not detectable in tissues isolated from NPRA knockout animals and ANP- and BNP-stimulatable GC activities are markedly reduced in all mutant tissues. However, testis and adrenal gland retain statistically significant, high-affinity responses to BNP. This residual response to BNP cannot be accounted for by natriuretic peptide receptor B, or any other known mammalian rGC, suggesting the presence of a novel receptor in these tissues that prefers BNP over ANP.

Genomic structure, organization, and promoter region analysis of murine guanylyl cyclase/atrial natriuretic peptide receptor-A gene.

We have determined the complete genomic nucleotide sequence and analyzed the promoter region of murine guanylyl cyclase/natriuretic peptide receptor-A gene (Npr1,coding for NPRA). The gene spans about 17.8 kb and contains 22 exons interrupted by 21 introns. All the exon-intron boundaries possess the consensus GT/AG splice junctions. Four different types of short interspersed nuclear elements (ten mouse B1 elements, seven mouse B2-B4 elements, one ID and one MIR element) and one medium reiteration frequency repeats have been found in the non-coding regions of the gene. Eleven tandem repeats, including three in the promoter region of the gene, have been identified. The transcription start site, 362 bp upstream from the start codon, was determined by 5- rapid amplification of cDNA ends. The 1.98 kb 5-flanking region contains three potential SP1 binding sites and one inverted CCAAT box but lacks the TATA box. This region also contains several putative cis-acting motifs known to bind kidney specific nuclear protein HFH-3, cAMP-responsive element binding protein (CREB) and AP-4. In addition, the binding sites for a variety of transcription factors: AML-1 alpha, SRY, Nkx-2.5, LyF-1, p300, GATA-1/2, HNF-3 beta, c/EBP alpha/beta and USF have been localized in the promoter region of Npr1 gene. The analyses and characterization of the genomic structure of murine Npr1 gene should yield important insights into the species-specific regulation of this important gene family.