To gain insight into the function of peroxisome proliferator-activated receptor (PPAR) isoforms in rodents, we disrupted the ligand-binding domain of the alpha isoform of mouse PPAR (mPPAR alpha) by homologous recombination. Mice homozygous for the mutation lack expression of mPPAR alpha protein and yet are viable and fertile and exhibit no detectable gross phenotypic defects. Remarkably, these animals do not display the peroxisome proliferator pleiotropic response when challenged with the classical peroxisome proliferators, clofibrate and Wy-14,643. Following exposure to these chemicals, hepatomegaly, peroxisome proliferation, and transcriptional-activation of target genes were not observed. These results clearly demonstrate that mPPAR alpha is the major isoform required for mediating the pleiotropic response resulting from the actions of peroxisome proliferators. mPPAR alpha-deficient animals should prove useful to further investigate the role of this receptor in hepatocarcinogenesis, fatty acid metabolism, and cell cycle regulation.

The thyroid-specific enhancer-binding protein (T/ebp) gene was disrupted by homologous recombination in embryonic stem cells to generate mice lacking T/EBP expression. Heterozygous animals developed normally, whereas mice homozygous for the disrupted gene were born dead and lacked the lung parenchyma. Instead, they had a rudimentary bronchial tree associated with an abnormal epithelium in their pleural cavities. Furthermore, the homozygous mice had no thyroid gland but had a normal parathyroid. In addition, extensive defects were found in the brain of the homozygous mice, especially in the ventral region of the forebrain. The entire pituitary, including the anterior, intermediate, and posterior pituitary, was also missing. In situ hybridization showed that the T/ebp gene is expressed in the normal thyroid, lung bronchial epithelium, and specific areas of the forebrain during early embryogenesis. These results establish that the expression of T/EBP, a transcription factor known to control thyroid-specific gene transcription, is also essential for organogenesis of the thyroid, lung, ventral forebrain, and pituitary.[Key Words: T/EBP; gene targeting; thyroid; lung; ventral forebrain; pituitary] Received August 22, 1995; revised version accepted November 10, 1995.Thyroid-specific enhancer-binding protein (T/EBP) binds to an enhancer element located -5.5 kb upstream of the human thyroid peroxidase gene transcription start site and regulates thyroid-specific gene expression (Kikkawa et al. 1990;Mizuno et al. 1991). T/EBP, also named thyroid-specific transcription factor 1 (TTF-1)or Nkx-2.1, was originally described to govern thyroid-specific expression of the rat thyroglobulin gene (Civitareale et al. 1989). Several studies have established the role of T/EBP in expression of genes encoding thyroid peroxidase (Kikkawa et al. 1990;Mizuno et al. 1991;Abramowicz et al. 1992;Francis-Lang et al. 1992), thyroglobulin (Civitareale et al. 1989), and the thyrotropin (TSH) receptor tCivitareale et al. 1993;Shimura et al. 1994). All three proteins are essential for thyroid hormone biosynthesis (DeGroot and Niepomniszcze 1977). T/EBP is also expressed in the lung (Guazzi et al. 1990;Mizuno et al. 1991), and it has been recently demonstrated that the expression of genes encoding the lung surfactant proteins A and B is regulated by this DNA-binding protein (Bohinski et al. 1994;Bruno et al. 1995). T/ebp ) is the first member of the mouse Nkx-2 gene family that is closely related to Drosophila NK-2 in their homeo domain sequences (68%-95% similarity) (Kim and Nirenberg 1989;Guazzi et al. 1990;Price et al. 1992;Lints et al. 1993). Members of the Nkx-2 family also share a highly conserved 17-aminoacid motif that is located on the carboxyl-terminal side of the homeo domain. From the six members of this family characterized to date, the expression patterns of three genes, T/ebp (Nkx-2.1), Nkx-2.2, and Nkx-2.5, have been studied. Lazzaro et al. (1991) have established T/ebp(Nkx-2.1) gene expression at -10.5 days postcoitum (El0.5) in the ...

The aryl hydrocarbon (Ah) receptor (AHR) mediates many carcinogenic and teratogenic effects of environmentally toxic chemicals such as dioxin. An AHR-deficient (Ahr-/-) mouse line was constructed by homologous recombination in embryonic stem cells. Almost half of the mice died shortly after birth, whereas survivors reached maturity and were fertile. The Ahr-/- mice showed decreased accumulation of lymphocytes in the spleen and lymph nodes, but not in the thymus. The livers of Ahr-/- mice were reduced in size by 50 percent and showed bile duct fibrosis Ahr-/- mice were also nonresponsive with regard to dioxin-mediated induction of genes encoding enzymes that catalyze the metabolism of foreign compounds. Thus, the AHR plays an important role in the development of the liver and the immune system.

Mammalian cells harbor three highly homologous and widely expressed members of the ras family (H-ras, N-ras, and K-ras), but it remains unclear whether they play specific or overlapping cellular roles. To gain insight into such functional roles, here we generated and analyzed H-ras null mutant mice, which were then also bred with N-ras knockout animals to ascertain the viability and properties of potential double null mutations in both loci. Mating among heterozygous H-ras ؉/؊ mice produced H-ras ؊/؊ offspring with a normal Mendelian pattern of inheritance, indicating that the loss of H-ras did not interfere with embryonic and fetal viability in the uterus. Homozygous mutant H-ras ؊/؊ mice reached sexual maturity at the same age as their littermates, and both males and females were fertile. Characterization of lymphocyte subsets in the spleen and thymus showed no significant differences between wild-type and H-ras ؊/؊ mice. Analysis of neuronal markers in the brains of knockout and wild-type H-ras mice showed that disruption of this locus did not impair or alter neuronal development. Breeding between our H-ras mutant animals and previously available N-ras null mutants gave rise to viable double knockout (H-ras ؊/؊ /N-ras ؊/؊ ) offspring expressing only K-ras genes which grew normally, were fertile, and did not show any obvious phenotype. Interestingly, however, lower-thanexpected numbers of adult, double knockout animals were consistently obtained in Mendelian crosses between heterozygous N-ras/H-ras mice. Our results indicate that, as for N-ras, H-ras gene function is dispensable for normal mouse development, growth, fertility, and neuronal development. Additionally, of the three ras genes, K-ras appears to be not only essential but also sufficient for normal mouse development.

We have analyzed the possible role of the aryl-hydrocarbon receptor (AHR) in the aging process of mice using a homozygous null mouse (Ahr-/-) line as a model. We studied 52 male and female Ahr-/- mice aged from 6-13 months. Forty-six percent died or were ill by 13 months of age. Ahr-/- mice developed age-related lesions in several organs, some of which were apparent after only 9 months of age. Cardiovascular alterations included cardiomyopathy (100%) with hypertrophy and focal fibrosis. Vascular hypertrophy and mild fibrosis were found in the portal areas of the liver (81%), and vascular hypertrophy and mineralization were common in the uterus (70%). Gastric hyperplasia that progressed with age into polyps was evident in the pylorus of 71% of the mice over 9 months of age. Ahr-/- mice had T-cell deficiency in their spleens but not in other lymphoid organs. The immune system deficiency described previously could be the origin for the rectal prolapse found in 48% of the null mice, associated with Helicobacter hepaticus infection. In the dorsal skin (53% incidence), severe, localized, interfollicular and follicular epidermal hyperplasia, with hyperkeratosis and acanthosis, and marked dermal fibrosis, associated with the presence of anagenic hair follicles, were also evident. None of these lesions were found in 42 control (Ahr +/+ or +/-) mice of similar ages. These observations suggest that the AHR protein, in the absence of an apparent exogenous (xenobiotic) ligand, plays an important role in physiology and homeostasis in major organs in mice, and further supports an evolutionary conserved role for this transcription factor.

Dihydropyrimidine dehydrogenase (DPD) deficiency constitutes an inborn error in pyrimidine metabolism associated with thymine-uraciluria in pediatric patients and an increased risk of toxicity in cancer patients receiving 5-fluorouracil (5-FU) treatment. The molecular basis for DPD deficiency in a British family having a cancer patient that exhibited grade IV toxicity 10 d after 5-FU treatment was analyzed. A 165-bp deletion spanning a complete exon of the DPYD gene was found in some members of the pedigree having low DPD catalytic activity. Direct sequencing of lymphocyte DNA from these subjects revealed the presence of a G to A point mutation at the 5 Ј -splicing site consensus sequence (GT to AT) that leads to skipping of the entire exon preceding the mutation during pre-RNA transcription and processing. A PCR-based diagnostic method was developed to determine that the mutation is found in Caucasian and Asian populations. This mutation was also detected in a Dutch patient with thymine-uraciluria and completely lacking DPD activity. A genotyping test for the G to A splicing point mutation could be useful in predicting cancer patients prone to toxicity upon administration of potentially toxic 5-FU and for genetic screening of heterozygous carriers and homozygous deficient subjects. ( J. Clin. Invest. 1996. 98:610-615.)

Traditionally considered as a critical intermediate in the toxic and carcinogenic response to dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD), the Aryl hydrocarbon/Dioxin receptor (AhR) has proven to be also an important regulator of cell physiology and organ homeostasis. AhR has become an interesting and actual area of research mainly boosted by a significant number of recent studies analyzing its contribution to the proper functioning of the immune, hepatic, cardiovascular, vascular and reproductive systems. At the cellular level, AhR establishes functional interactions with signaling pathways governing cell proliferation and cell cycle, cell morphology, cell adhesion and cell migration. Two exciting new aspects in AhR biology deal with its implication in the control of cell differentiation and its more than likely involvement in cell pluripotency and stemness. In fact, it is possible that AhR could help modulate the balance between differentiation and pluripotency in normal and transformed tumor cells. At the molecular level, AhR regulates an increasingly large array of physiologically relevant genes either by traditional transcription-dependent mechanisms or by unforeseen processes involving genomic insulators, chromatin dynamics and the transcription of mobile genetic elements. AhR is also closely related to epigenetics, not only from the point of view of target gene expression but also with respect to its own regulation by promoter methylation. It is reasonable to consider that deregulation of these many functions could have a causative role, or at least contribute to, human disease. Consequently, several laboratories have proposed that AhR could be a valuable tool as diagnostic marker and/or therapeutic target in human pathologies. An additional point of interest is the possibility of regulating AhR activity by endogenous non-toxic low weight molecules agonist or antagonist molecules that could be present or included in the diet. In this review, we will address these molecular and functional features of AhR biology within physiological and pathological contexts.

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