Biosynthesis and cellular localization of functional polyketides in the gastropod mollusc Scaphander lignarius.

Opisthobranchs belong to a subclass of highly evolved and specialised marine gastropods that rely on the use of secondary metabolites for their survival. Here we report the full elucidation of the biosynthesis of aromatic metabolites, lignarenones, in one of these gastropods, the cephalaspidean Scaphander lignarius. Feeding experiments with ²H- and ¹³C-labelled precursors revealed a mixed acetate/propionate polyketide pathway primed by benzoic acid. Phenylalanine ammonia lyase (PAL), unprecedented in animals, is central to the synthesis of this aromatic precursor by oxidative deamination of L-phenylalanine to cinnamic acid. Lignarenones are synthesised in the cytoplasm of specialised eukaryotic cells named Blochmann's glands, which are distributed in biosynthetic tissue localised in the vulnerable mantle of the mollusc. This result supports the hypothesis that this lineage of gastropods has acquired the genetic information to produce the chemical substances that they use for their survival.

Rhes is involved in striatal function.

The development and the function of central nervous system depend on thyroid hormones. In humans, the lack of thyroid hormones causes cretinism, a syndrome of severe mental deficiency. It is assumed that thyroid hormones affect the normal development and function of the brain by activating or suppressing target gene expression because several genes expressed in the brain have been shown to be under thyroid hormone control. Among these, the Rhes gene, encoding a small GTP-binding protein, is predominantly expressed in the striatal region of the brain. To clarify the role of Rhes in vivo, we disrupted the Rhes gene by homologous recombination in embryonic stem cells and generated mice homozygous for the Rhes null mutation (Rhes(-/-)). Rhes(-/-) mice were viable but weighed less than wild-type mice. Furthermore, they showed behavioral abnormalities, displaying a gender-dependent increase in anxiety levels and a clear motor coordination deficit but no learning or memory impairment. These results suggest that Rhes disruption affects selected behavioral competencies.

Missense mutation in the transcription factor NKX2-5: a novel molecular event in the pathogenesis of thyroid dysgenesis.

CONTEXT: Congenital hypothyroidism (CH) is a common endocrine disorder with an incidence of 1:3000-4000 at birth. In 80-85% of cases, CH is caused by defects in thyroid organogenesis, resulting in absent, ectopically located, and/or severely reduced gland [thyroid dysgenesis (TD)]. Mutations in genes controlling thyroid development have demonstrated that in a few cases, TD is a Mendelian trait. However, accumulating evidence supports the view that the genetics of TD are complex, possibly with a polygenic/multifactorial basis. A higher prevalence of congenital heart disease has been documented in children with CH than in the general population. Such an association suggests a possible pathogenic role of genes involved in both heart and thyroid development. NKX2-5 encodes a homeodomain-containing transcription factor with a major role in heart development, and mutations affecting this gene have been reported in individuals with congenital heart disease. OBJECTIVE: In the present work we investigated the possible involvement of NKX2-5 mutations in TD. RESULTS: Our results indicate that Nkx2-5(-/-) embryos exhibit thyroid bud hypoplasia, providing evidence that NKX2-5 plays a role in thyroid organogenesis and that NKX2-5 mutations contribute to TD. NKX2-5 mutational screening in 241 patients with TD allowed the identification of three heterozygous missense changes (R25C, A119S, and R161P) in four patients with TD. Functional characterization of the three mutations demonstrated reduced DNA binding and/or transactivation properties, with a dominant-negative effect on wild-type NKX2-5. CONCLUSION: Our results suggest a previously unknown role of NKX2-5 in the pathogenesis of TD.

A mouse model demonstrates a multigenic origin of congenital hypothyroidism.

Congenital hypothyroidism with thyroid dysgenesis (TD) is a frequent human condition characterized by elevated levels of TSH in response to reduced thyroid hormone levels. Congenital hypothyroidism is a genetically heterogeneous disease. In the majority of cases studied, no causative mutations have been identified and very often the disease does not show a Mendelian transmission. However, in approximately 5% of cases, it can be a consequence of mutations in genes encoding the TSH receptor or the transcription factors TITF1, FOXE1, or PAX8. We report here that in mouse models, the combination of partial deficiencies in the Titf1 and Pax8 genes results in an overt TD phenotype that is absent in either of the singly deficient, heterozygous mice. The disease is characterized by a small thyroid gland, elevated levels of TSH, reduced thyroglobulin biosynthesis, and high occurrence of hemiagenesis. The observed phenotype is strain specific, and the pattern of transmission indicates that at least two other genes, in addition to Titf1 and Pax8, are necessary to generate the condition. These results show that TD can be of multigenic origin in mice and strongly suggest that a similar pathogenic mechanism may be observed in humans.

Induction of tissue factor in the arterial wall during recurrent thrombus formation.

OBJECTIVE: Tissue factor (TF) is normally expressed at low levels in the media of blood vessels, but it is readily induced after vessel injury. It is not known whether vascular damage per se or thrombus formation is responsible for this phenomenon. METHODS AND RESULTS: Cyclic flow variations (CFVs), attributable to recurrent thrombus formation, were induced in stenotic rabbit carotid arteries with endothelial injury. CFVs were observed for 30 minutes and 2, 4, and 8 hours in different groups of animals. Another group of rabbits pretreated with hirudin before inducing arterial damage to inhibit thrombus formation was observed for 8 hours. Arterial sections were immunostained for TF. Undamaged arteries served as controls. In additional rabbits, in situ hybridization experiments were performed. No TF expression was observed in the media of control vessels, whereas a progressive increase in TF mRNA and protein expression was observed in carotid arteries as CFVs progressed. No increase in TF expression was observed in animals pretreated with hirudin. In vitro experiments demonstrated that TF mRNA is induced in smooth muscle cells stimulated with activated platelets as well as with some platelet-derived mediators. CONCLUSIONS: This phenomenon may contribute to sustain intravascular thrombus formation after the initial thrombogenic stimulus.

Diverse human aldolase C gene promoter regions are required to direct specific LacZ expression in the hippocampus and Purkinje cells of transgenic mice.

Aldolase C is selectively expressed in the hippocampus and Purkinje cells in adult mammalian brain. The gene promoter regions governing cell-specific aldolase C expression are obscure. We show that aldolase C messenger expression in the hippocampus is restricted to CA3 neurons. The human distal promoter region (-200/-1200 bp) is essential for beta-galactosidase (beta-gal) expression in CA3 neurons and drives high stripe-like beta-gal expression in Purkinje cells. The 200 bp proximal promoter region is sufficient to drive low brain-specific and stripe-like beta-gal expression in Purkinje cells. Thus, the human aldolase C gene sequences studied drive endogenous-like expression in the brain.

Replacement of K-Ras with H-Ras supports normal embryonic development despite inducing cardiovascular pathology in adult mice.

Ras proteins are highly related GTPases that have key roles in regulating growth, differentiation and tumorigenesis. Gene-targeting experiments have shown that, out of the three mammalian ras genes, only K-ras is essential for normal mouse embryogenesis, and that mice deprived of H-ras and/or N-ras show no major phenotype. We generated mice (HrasKI) in which the K-ras gene had been modified to encode H-Ras protein. HrasKI mice produce undetectable amounts of K-Ras but-in contrast to mice homozygous for a null K-ras allele-they are born at the expected mendelian frequency, indicating that H-Ras can be substituted for K-Ras in embryonic development. However, adult HrasKI mice show dilated cardiomyopathy associated with arterial hypertension. Our results show that K-Ras can be replaced by H-Ras in its essential function in embryogenesis, and indicate that K-Ras has a unique role in cardiovascular homeostasis.

Distribution of the titf2/foxe1 gene product is consistent with an important role in the development of foregut endoderm, palate, and hair.

Titf2/foxe1 is a forkhead domain-containing gene expressed in the foregut, in the thyroid, and in the cranial ectoderm of the developing mouse. Titf2 null mice exhibit cleft palate and either a sublingual or completely absent thyroid gland. In humans, mutations of the gene encoding for thyroid transcription factor-2 (TTF-2) result in the Bamforth syndrome, characterized by thyroid agenesis, cleft palate, spiky hair, and choanal atresia. Here, we report a detailed expression pattern of TTF-2 protein during mouse embryogenesis and show its presence in structures where it has not been described yet. At embryonic day (E) 10.5, TTF-2 is expressed in Rathke's pouch, in thyroid, and in the epithelium of the pharyngeal wall and arches, whereas it is absent in the epithelium of the pharyngeal pouches. According to this expression, at E13.5, TTF-2 is present in endoderm derivatives, such as tongue, palate, epiglottis, pharynx, and oesophagus. Later in embryogenesis, we detect TTF-2 in the choanae and whiskers. This pattern of expression helps to define the complex phenotype displayed by human patients. Finally, we show that TTF-2 is a phosphorylated protein. These results help to characterize the domains of TTF-2 expression, from early embryogenesis throughout organogenesis, providing more detail on the potential role of TTF-2 in the development of endoderm and ectoderm derived structures.

A preservation method that allows recovery of intact RNA from tissues dissected by laser capture microdissection.

We report a novel method for preparing samples for laser capture microdissection. The procedure described here permits extraction of intact RNA while preserving morphology, thus being suitable both for identification of specific cells and for analysis of their gene expression. The method is applicable to both mouse embryos and human tumors and may improve the preparation of cDNA libraries from specific cell types without interfering with histological diagnosis.

An integrated regulatory network controlling survival and migration in thyroid organogenesis.

The thyroid gland originates from the ventral floor of the foregut as a thickening of the endodermal cell layer. The molecular mechanisms underlying the early steps of thyroid morphogenesis are not known. Gene targeting experiments have contributed to the identification of several transcription factors, in general playing a role in the proliferation, survival, and migration of the thyroid cell precursors. The experiments reported here analyze the expression of the transcription factors Titf1, Hhex, Pax8, and Foxe1 in the thyroid primordium of null mutants of each of them. We found that most of these transcription factors are linked in an integrated regulatory network, each of them controlling the presence of other members of the network. The expression of Foxe1 is regulated in an intriguing fashion as it is strongly dependent on the presence of Pax8 in thyroid precursor cells, while the expression of the same gene in the pharyngeal endoderm surrounding the primordium is dependent on Sonic hedgehog (Shh)-derived signaling. Moreover, by the generation of mouse mutants expressing Foxe1 exclusively in the thyroid primordium, we provide a better understanding of the role of Foxe1 in these cells in order to acquire the competence to migrate into the underlying mesenchyme. In conclusion, we provide the first evidence of gene expression programs, controlled by a hierarchy of transcription factors expressed in the thyroid presumptive gut domain and directing the progression of thyroid morphogenesis.