The ability to generate specific genetic modifications in mice provides a powerful approach to assess gene function. When genetic modifications have been generated in the germ line, however, the resulting phenotype often only reflects the first time a gene has an influence on - or is necessary for - a particular biological process. Therefore, systems allowing conditional genetic modification have been developed (for a review, see [1]); for example, inducible forms of the Cre recombinase from P1 phage have been generated that can catalyse intramolecular recombination between target recognition sequences (loxP sites) in response to ligand [2] [3] [4] [5]. Here, we assessed whether a tamoxifen-inducible form of Cre recombinase (Cre-ERTM) could be used to modify gene activity in the mouse embryo in utero. Using the enhancer of the Wnt1 gene to restrict the expression of Cre-ERTM to the embryonic neural tube, we found that a single injection of tamoxifen into pregnant mice induced Cre-mediated recombination within the embryonic central nervous system, thereby activating expression of a reporter gene. Induction was ligand dependent, rapid and efficient. The results demonstrate that tamoxifen-inducible recombination can be used to effectively modify gene function in the mouse embryo.

Hypertension, a major cardiovascular risk factor and cause of mortality worldwide, is thought to arise from primary renal abnormalities. However, the etiology of most cases of hypertension remains unexplained. Vascular tone, an important determinant of blood pressure, is regulated by nitric oxide, which causes vascular relaxation by increasing intracellular cGMP and activating cGMPdependent protein kinase I (PKGI). Here we show that mice with a selective mutation in the N-terminal protein interaction domain of PKGI␣ display inherited vascular smooth muscle cell abnormalities of contraction, abnormal relaxation of large and resistance blood vessels, and increased systemic blood pressure. Renal function studies and responses to changes in dietary sodium in the PKGI␣ mutant mice are normal. These data reveal that PKGI␣ is required for normal VSMC physiology and support the idea that high blood pressure can arise from a primary abnormality of vascular smooth muscle cell contractile regulation, suggesting a new approach to the diagnosis and therapy of hypertension and cardiovascular diseases.cyclic nucleotides ͉ hypertension ͉ nitric oxide ͉ vascular biology ͉ vascular smooth muscle E levated blood pressure is a major risk factor for cardiovascular diseases and is responsible for widespread morbidity and mortality (1). Blood pressure is regulated by a variety of complex neurohumoral and mechanical signals that together determine systemic vascular tone and resistance (2, 3). The prevailing model for elevated blood pressure states that renal abnormalities of sodium handling cause volume expansion, increased systemic vascular resistance, and hypertension, and a large number of physiologic and genetic studies support this model and the central role of the renal renin-angiotensinaldosterone system in blood pressure regulation (4-8). Changes in vascular morphology and tone can increase vascular resistance and blood pressure (5), but the hypothesis that primary abnormalities of vascular smooth muscle tone can cause hypertension has not been sufficiently tested (6).Vascular smooth muscle contraction is initiated by both calcium-dependent and -independent mechanisms. Increases in intracellular calcium from receptor-or ion channel-activated pathways (2) lead to activation of myosin light chain kinase, which phosphorylates myosin light chains, activating myosin ATPase and increasing vascular smooth muscle cell (VSMC) contraction and vascular tone. The central calcium-independent pathway regulating VSMC tension is mediated by the GTPase RhoA and Rho kinase, which promote VSMC differentiation, stress fiber formation, and contraction, also increasing vascular tone (2, 7). Conversely, VSMC relaxation is mediated by activation of myosin light chain phosphatase (MLCP), which dephosphorylates myosin light chains to cause relaxation. The relative proportion of phosphorylated and dephosphorylated myosin light chains thus determines the state of VSMC tone (reviewed in ref.2). Nitric oxide, the most important endogenous vasodilator, cause...

The TGFβ superfamily plays diverse and essential roles in kidney development. Gdf11 and Bmp4 are essential for outgrowth and positioning of the ureteric bud, the inducer of metanephric mesenchyme. During nephrogenesis, Bmp7 is required for renewal of the mesenchyme progenitor population. Additionally, in vitro studies demonstrate inhibitory effects of BMPs and TGFβs on collecting duct branching and growth. Here,we explore the predicted models of TGFβ superfamily function by cell-specific inactivation of Smad4, a key mediator of TGFβsignaling. Using a HoxB7cre transgene expressed in ureteric bud and collecting duct, we find that development of the collecting duct is Smad4 independent. By contrast, removal of Smad4 in nephrogenic mesenchyme using the Bmp7cre/+ allele leads to disorganization of the nephrogenic mesenchyme and impairment of mesenchyme induction. Smad4-deficient metanephric mesenchyme does not display defects in inducibility in LiCl or spinal cord induction assays. However, in situ hybridization and lineage analysis of Smad4 null mesenchyme cells at E11.5 show that the nephrogenic mesenchyme does not aggregate tightly around the ureteric bud tips, but remains loosely associated, embedded within a population of cells expressing markers of both nephrogenic mesenchyme and peripheral stroma. We conclude that the failure of recruitment of nephrogenic mesenchyme leaves a primitive population of mesenchyme at the periphery of the kidney. This population is gradually depleted, and by E16.5 the periphery is composed of cells of stromal phenotype. This study uncovers a novel role for TGFβ superfamily signaling in the recruitment and/or organization of the nephrogenic mesenchyme at early time-points of kidney development. Additionally, we present conclusive genetic lineage mapping of the collecting duct and nephrogenic mesenchyme.

Site specific recombinases have provided the experimental strategy necessary to modulate the expression of gene products in the mouse embryo. In this study we have exploited Cre recombinase to develop a widely applicable cell marking system which functions efficiently even at early post-implantation embryonic stages. Importantly, the techniques and reagents derived in this study are generally applicable to any recombinase driven approach, including strategies to temporally and spatially modulate endogenous or ectopic gene expression in the embryo. The cell marking scheme has two essential components which were derived as separate mouse lines. The first line carries a universal conditional lacZ reporter (UCR) locus which was prepared by using gene targeting in a novel approach to modify a ubiquitously expressed retroviral lacZ promoter trap insertion. The UCR locus is silent until it undergoes a Cre mediated DNA rearrangement to restore lacZ expression. To generate the Cre expressing allele, we outline a flexible strategy which requires the introduction of a novel IRES-Cre cassette into exon sequence of an endogenous locus by gene targeting. We successfully demonstrate this approach by generating a Cre expressing allele of the EphA2 gene, an Eph receptor protein tyrosine kinase expressed early in development. Analysis of double heterozygote embryos clearly demonstrates that Cre recombinase is expressed in vivo from the EphA2 IRES-Cre allele, and that the conditional reporter locus is efficiently restored in EphA2-expressing cells as early as 7.5 dpc. This cell marking experiment establishes the feasibility of expressing Cre recombinase from a single copy allele in the embryo and demonstrates the utility of the conditional reporter mouse which can be used in the analysis of any Cre expressing allele.