Recent advances in stem cell technology offer hope that cell therapy and tissue engineering may one day replace renal function lost as a consequence of maldevelopment or disease. Prerequisite to tissue engineering is the understanding of the mechanisms underlying embryonic kidney induction and development. The pronephros arises directly from intermediate mesoderm (IM) and represents the first stage of vertebrate kidney induction. Paraxial mesoderm (PM), which gives rise to somites, induces the pronephros, but the cellular and molecular mechanisms underlying pronephros induction by the PM have not been defined. Somitogenesis and pronephros induction are closely linked to vascular development. The pronephros lies sandwiched between the post-cardinal vein and the aorta in the early embryo, and the PM contains angioblasts. Angioblast-derived signals induce the embryonic heart, pancreas, and liver, suggesting that this may be a general inductive mechanism. Pilot studies showed that inhibition of vasculogenesis decreased expression of the kidney marker, pax-2, in the IM, and suggested that the timing of vascular disruption is important. We hypothesize that molecules secreted by resident angioblasts regulate pronephros induction by the paraxial mesoderm. We know that angioblasts meet the temporal and spatial criteria for a pronephros inducer.
In Aim 1, we will define the temporal and spatial appearance of blood vessel precursors in relation to pronephros induction using high-resolution fate mapping. Time-lapse videomicroscopy will follow the development of PM, IM angioblasts, and blood vessels. We will determine whether ectopically induced somites and pronephroi form in association with ectopic blood vessels. Results of these studies will help us understand the details of interactions between angioblasts, PM and IM during their specification.
Aim 2 will determine if blood vessel precursors are required for pronephros induction by PM, thereby fulfilling the second criterion for an inducer. We will determine if PM obtained from VEGFR2 (-/-) mice, which lack blood vessels, can induce the pronephros in competent (able to form pronephros) quail mesoderm. Soluble VEGF-R1 will be injected into quail mesoderm to disrupt vasculogenesis, and secondarily, pronephros induction.
Aim 3 will determine if blood vessels are sufficient for pronephros induction and thus fulfill the third criterion for an inducer. We will examine the effect of ectopic or excessive blood vessel formation on pronephros induction, and we will recombine embryonic arteries and veins with uninduced competent mesoderm in collagen gel cultures to determine if pronephros induction is specific to a particular vascular type, or is a general feature of blood vessels and angioblasts. The experiments described in this proposal will combine the ease of experimental manipulation of the avian model with the genetic power of mutant mice to define the molecular relationships between blood vessel formation, somitogenesis and pronephros induction. ? ?
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