Angiogenesis is required for a wide variety of physiological and pathological processes. The endothelial cell-specific mitogen vascular endothelial growth factor (VEGF) is a major mediator of pathological angiogenesis. Also, the expression of VEGF and its two receptors, Flt-1 and Flk-1/KDR, is related to the formation of blood vessels in mouse and rat embryos. Mice homozygous for mutations that inactivate either receptor die in utero between days 8.5 and 9.5. However, ligand(s) other than VEGF might activate such receptors. To assess the role of VEGF directly, we disrupted the VEGF gene in embryonic stem cells. Here we report the unexpected finding that loss of a single VEGF allele is lethal in the mouse embryo between days 11 and 12. Angiogenesis and blood-island formation were impaired, resulting in several developmental anomalies. Furthermore, VEGF-null embryonic stem cells exhibit a dramatically reduced ability to form tumours in nude mice.

Approximately one third of all mammalian genes are essential for life. Phenotypes resulting from mouse knockouts of these genes have provided tremendous insight into gene function and congenital disorders. As part of the International Mouse Phenotyping Consortium effort to generate and phenotypically characterize 5000 knockout mouse lines, we have identified 410 lethal genes during the production of the first 1751 unique gene knockouts. Using a standardised phenotyping platform that incorporates high-resolution 3D imaging, we identified novel phenotypes at multiple time points for previously uncharacterized genes and additional phenotypes for genes with previously reported mutant phenotypes. Unexpectedly, our analysis reveals that incomplete penetrance and variable expressivity are common even on a defined genetic background. In addition, we show that human disease genes are enriched for essential genes identified in our screen, thus providing a novel dataset that facilitates prioritization and validation of mutations identified in clinical sequencing efforts.

Glial cell-line derived neurotrophic factor (GDNF) is a potent survival factor for embryonic midbrain dopaminergic, spinal motor, cranial sensory, sympathetic, and hindbrain noradrenergic neurons, and is available to these cells in vivo. It is therefore considered a physiological trophic factor and a potential therapeutic agent for Parkinson's disease, amyotrophic lateral sclerosis, and Alzheimer's disease. Here we show that at postnatal day 0 (P0), GDNF-deficient mice have deficits in dorsal root ganglion, sympathetic and nodose neurons, but not in hindbrain noradrenergic or midbrain dopaminergic neurons. These mice completely lack the enteric nervous system (ENS), ureters and kidneys. Thus GDNF is important for the development and/or survival of enteric, sympathetic and sensory neurons and the renal system, but is not essential for catecholaminergic neurons in the central nervous system (CNS).

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Interleukin-8 (IL-8) is a proinflammatory cytokine that specifically attracts and activates human neutrophils. A murine gene with a high degree of homology to the two known human IL-8 receptors was cloned and then deleted from the mouse genome by homologous recombination in embryonic stem (ES) cells. These mice, although outwardly healthy, had lymphadenopathy, resulting from an increase in B cells, and splenomegaly, resulting from an increase in metamyelocytes, band, and mature neutrophils. Thus, this receptor may participate in the expansion and development of neutrophils and B cells. This receptor was the major mediator of neutrophil migration to sites of inflammation and may provide a potential therapeutic target in inflammatory disease.

Tumour necrosis factor (TNF) elicits multiple biological effects through two distinct cell surface receptors, TNF-R1 (p55) and TNF-R2 (p75). Most TNF-mediated biological responses, such as cell death, gene induction, antiviral activity and cytokine production, have been attributed to TNF-R1 (refs 1-5). Gene targeting of this receptor confirms its role in the lethality attributable to low doses of lipopolysaccharide after sensitization with D-galactosamine; surprisingly, the toxicity of high doses of lipopolysaccharide was unaffected. The function of TNF-R2 is less well understood, although there are data supporting a role in T-cell development and the proliferation of cytotoxic T lymphocytes. To clarify the physiological role of TNF-R2, we have generated mice deficient in this receptor by gene targeting. The TNF-R2-/- mice show normal T-cell development and activity, but we find that they have increased resistance to TNF-induced death. Additionally, such mice injected subcutaneously with TNF show a dramatic decrease in tissue necrosis, indicating that this receptor plays a role in the necrotic effects of TNF.

Glial cell line-derived neurotrophic factor (GDNF) is a distant member of the TGFbeta protein family that is essential for neuronal survival and renal morphogenesis. We show that mice who are deficient in the glycosyl-phosphatidyl inositol (GPI) -linked protein GFRalpha1 (GDNFRalpha) display deficits in the kidneys, the enteric nervous system, and spinal motor and sensory neurons that are strikingly similar to those of the GDNF- and Ret-deficient mice. GFRalpha1-deficient dopaminergic and nodose sensory ganglia neurons no longer respond to GDNF or to the structurally related protein neurturin (NTN) but can be rescued when exposed to GDNF or neurturin in the presence of soluble GFRalpha1. In contrast, GFRalpha1-deficient submandibular parasympathetic neurons retain normal response to these two factors. Taken together with the available genetic and biochemical data, these findings support the idea that GFRalpha1 and the transmembrane tyrosine kinase Ret are both necessary receptor components for GDNF in the developing kidney and nervous system, and that GDNF and neurturin can mediate some of their activities through a second receptor.