In 2007, the International Knockout Mouse Consortium (IKMC) made the ambitious promise to generate mutations in virtually every protein-coding gene of the mouse genome in a concerted worldwide action. Now, 5 years later, the IKMC members have developed high-throughput gene trapping and, in particular, gene-targeting pipelines and generated more than 17,400 mutant murine embryonic stem (ES) cell clones and more than 1,700 mutant mouse strains, most of them conditional. A common IKMC web portal (www.knockoutmouse.org) has been established, allowing easy access to this unparalleled biological resource. The IKMC materials considerably enhance functional gene annotation of the mammalian genome and will have a major impact on future biomedical research.

The ternary complex factors (TCFs) Net, Elk-1 and Sap-1 regulate immediate early genes through serum response elements (SREs) in vitro, but, surprisingly, their in vivo roles are unknown. Net is a repressor that is expressed in sites of vasculogenesis during mouse development. We have made gene-targeted mice that express a hypomorphic mutant of Net, Netd, which lacks the Ets DNA-binding domain. Strikingly, homozygous mutant mice develop a vascular defect and up-regulate an immediate early gene implicated in vascular disease, egr-1. They die after birth due to respiratory failure, resulting from the accumulation of chyle in the thoracic cage (chylothorax). The mice have dilated lymphatic vessels (lymphangiectasis) as early as E16.5. Interestingly, they express more egr-1 in heart and pulmonary arteries at E18.5. Net negatively regulates the egr-1 promoter and binds speci®-cally to SRE-5. Egr-1 has been associated with pathologies involving vascular stenosis (e.g. atherosclerosis), and here egr-1 dysfunction could possibly be associated with obstructions that ultimately affect the lymphatics. These results show that Net is involved in vascular biology and egr-1 regulation in vivo. Keywords: egr-1/Elk-3/ERP/Net/Sap-2 IntroductionThe ternary complex factors (TCFs) form a subfamily of Ets-domain transcription factors. The three TCFs, Elk-1, Sap-1 and Net/Sap-2/Erp/Elk-3 (Price et al., 1996;Wasylyk et al., 1998), have four conserved domains, A±D. A is the Ets DNA-binding domain (DBD). The B-box interacts with the serum response factor (SRF). C is a transcriptional activation domain that is stimulated by mitogen-activated protein (MAP) kinase phosphorylation. The D-domain is a MAP kinase-binding site and a nuclear localization signal. The TCFs are nuclear mediators of cellular responses to the activation of MAP kinase pathways. Net differs from the other TCFs in that in basal conditions, in which MAP kinases are not activated, it strongly inhibits transcription. Repression is mediated by two domains, the NID (Maira et al., 1996) and the CID (Criqui-Filipe et al., 1999). The TCFs form ternary complexes with SRF on serum response elements (SREs) of immediate early gene promoters, such as c-fos, egr-1 and jun-B. The SRE is constitutively occupied by factors, and extracellular signals are thought to lead to both phosphorylation of the complex and changes in its composition due to the exchange of TCFs.The in vivo role of the TCFs is poorly understood. They may regulate the expression of immediate early genes in response to various inductive stimuli. The TCFs are expressed in many cell types and tissues (Giovane et al., 1994;Lopez et al., 1994;Magnaghi-Jaulin et al., 1996;Nozaki et al., 1996;Sgambato et al., 1998), but their precise in vivo expression patterns are not well known. Net is expressed during mouse development at E7.5±8.5 in developing vascular primordia, including the yolk sac blood islands, allantoic vessels, heart endocardium and dorsal aortae (Ayadi et al., 2001). Vascular endothelial cell expression persists ...

Angiogenesis is fundamental to physiological and pathological processes. Despite intensive efforts, little is known about the intracellular circuits that regulate angiogenesis. The transcription factor Net is activated by phosphorylation induced by Ras, an indirect regulator of angiogenesis. Net is expressed at sites of vasculogenesis and angiogenesis during early mouse development, suggesting that it could have a role in blood vessel formation. We show here that down-regulation of Net inhibits angiogenesis and vascular endothelial growth factor (VEGF) expression in vivo, ex vivo, and in vitro. Ras-activated phosphorylated Net (P-Net) stimulates the mouse VEGF promoter through the −80 to −53 region that principally binds Sp1. P-Net and VEGF are coexpressed in angiogenic processes in wild-type mouse tissues and in human tumors. We conclude that Net is a regulator of angiogenesis that can switch to an activator following induction by pro-angiogenic molecules.

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The Net gene encodes an Ets transcription factor belonging to the ternary complex factor subfamily. We studied Net expression during mouse development (E7.5-E18.5) by in situ hybridization. Net is expressed at E7.5-8.5 in developing vascular primordia, including the allantoic vessels, heart endocardium and dorsal aortae. Vascular endothelial cell expression persists throughout development. Additional sites of expression appear at E9.5-E10.5, especially in facial, branchial arch and distal limb-bud mesenchyme. Later, expression is most conspicuous in developing cartilage and becomes progressively restricted to perichondrium. Net expression during mouse development correlates with vasculogenesis, angiogenesis and cartilage ontogeny.

The Ras signalling pathway targets transcription factors such as the ternary complex factors that are recruited by the serum response factor to form complexes on the serum response element (SRE) of the fos promoter. We have identified a new ternary complex factor, Net-b. We report the features of the net gene and show that it produces several splice variants, net-b and net-c. net-b RNA and protein are expressed in a variety of tissues and cell lines. net-c RNA is expressed at low levels, and the protein was not detected, raising the possibility that it is a cryptic splice variant. We have studied the composition of ternary complexes that form on the SRE of the fos promoter with extracts from fibroblasts (NIH 3T3) cultured under various conditions and pre-B cells (70Z/3) before and after differentiation with lipopolysaccharide (LPS). The fibroblast complexes contain mainly Net-b followed by Sap1 and Elk1. Net-b complexes, as well as Sap1 and Elk1, are induced by epidermal growth factor (EGF) stimulation of cells cultured in low serum. Pre-B-cell complexes contain mainly Sap1, with less of Net-b and little of Elk1. There is little change upon LPS-induced differentiation compared to the increase with EGF in fibroblasts. We have also found that Net-b is a nuclear protein that constitutively represses transcription. Net-b is not activated by Ras signalling, in contrast to Net, Sap1a, and Elk1. We have previously reported that down-regulation of Net proteins with antisense RNA increases SRE activity. The increase in SRE activity is observed at low serum levels and is even greater after serum stimulation, showing that the SRE is under negative regulation by Net proteins and the level of repression increases during induction. Net-b, the predominant factor in ternary complexes in fibroblasts, may both keep the activity of the SRE low in the absence of strong inducing conditions and rapidly shut the activity off after stimulation.

Cryptococcus neoformans and Cryptococcus gattii are pathogenic yeasts that cause cryptococcosis. These fungi were commonly associated with pigeon droppings and plant materials. The habitat of these pathogens has not been yet studied in Tunisia, although the ecology of these yeasts must be elucidated in order to establish surveillance programs and to prevent infections. The aim of this survey was to recover C. neoformans and C. gattii environmental isolates from pigeon droppings and plant materials in different areas of Sfax region, Tunisia. Nine hundred and fifty samples from leaves, wood, flowers, fruits and soil around trunk bases of 40 almond (Prunus dulcis) and 60 eucalyptus trees were collected as well as 250 pigeon droppings samples from different sites: buildings (n = 150), houses (n = 50) and zoo (n = 50). The identification of Cryptococcus neoformans complex was confirmed using the ID32C auxanogram panel (BioMérieux, Marcy l'Etoile, France); species were determined by multiplex PCR using the CN70 and CN49 primers, and mating type was determined by PCR. C. neoformans was recovered from 26 specimens of pigeon droppings (10.4%). This yeast was obtained more frequently from dry droppings (9.2%) than from moist droppings (1.2%). The mating type was determined. All the 31 environmental strains of C. neoformans and C. gattii were MATα. Out of 700 samples tested from 100 trees, only 5 isolates of Cryptococcus neoformans species complex were recovered (0.6%), two isolates of C. gattii and one isolate of C. neoformans were recovered from the wood of E. camaldulensis trees, and only two isolates of C. gattii were recovered from the wood of almond trees (Prunus dulcis Mill. var. zaaf and var. achek). These two Tunisian almond tree varieties were recorded for the first time in Africa as hosts for C. gattii. These results add new information to the ecology and epidemiology of C. neoformans species complex in Tunisia.

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