INTRODUCTION"Einstecks" refers to a procedure for placing a piece of tissue into the blastocoel of an early gastrula, in order to assess the inductive potential of the introduced tissue. The foreign tissue adheres to surrounding tissue and becomes incorporated into the host embryo. This simple transplant procedure, which is described here, has been used to assess which types of axial tissue can be induced by different regions of the mesendoderm or ectoderm, and how forced expression of genes or treatment with various factors can alter this potential.

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Xenopus is ideal for systematic decoding of cis-regulatory networks because its evolutionary position among vertebrates allows one to combine comparative genomics with efficient transgenic technology in one system. Here, we have identified and analyzed the major enhancer of FoxE3 (Lens1), a gene essential for lens formation that is activated in the presumptive lens ectoderm (PLE) when commitment to the lens fate occurs. Deletion and mutation analyses of the enhancer based on comparison of Xenopus and mammalian sequences and in vitro and in vivo binding assays identified two essential transcriptional regulators: Otx2,a homeodomain protein expressed broadly in head ectoderm including the PLE,and Su(H), a nuclear signal transducer of Notch signaling. A Notch ligand,Delta2, is expressed in the optic vesicle adjacent to the PLE, and inhibition of its activity led to loss, or severe reduction, of FoxE3 expression followed by failure of placode formation. Ectopic activation of Notch signaling induced FoxE3 expression within head ectoderm expressing Otx2, and additional misexpression of Otx2 in trunk ectoderm extended the Notch-induced FoxE3 expression posteriorly. These data provide the first direct evidence of the involvement of Notch signaling in lens induction. The obligate integration of inputs of a field-selector (Otx2)and localized signaling (Notch) within target cis-regulatory elements might be a general mechanism of organ-field specification in vertebrates (as it is in Drosophila). This concept is also consistent with classical embryological studies of many organ systems involving a `multiple-step induction'.

In this report we describe an easy, highly efficient transgenesis method for Xenopus. The method is very simple; a commercially available meganuclease, I-SceI, is incubated with a transgene construct carrying its recognition sites, and is subsequently microinjected into fertilized eggs. Approximately 30% (in Xenopus tropicalis) or 20% (in Xenopus laevis) of injected embryos exhibit non-mosaic, promoter-dependent transgene expression, and transgenes from the founder animals are transmitted to offspring. The method is compatible with mRNA or antisense morpholino oligonucleotide injection, and these secondary reagents can be introduced simultaneously or sequentially with a transgene to test their interaction. This high-throughput transgenic technique will be a powerful tool for studying the complex wiring of regulatory networks at the genome-wide level, as well as for facilitating genetic studies in the rapidly breeding diploid frog, X. tropicalis.

A small, fast-breeding, diploid relative of the frog Xenopus laevis, Xenopus tropicalis, has recently been adopted for research in developmental genetics and functional genomics. X. tropicalis shares advantages of X. laevis as a classic embryologic system, but its simpler genome and shorter generation time make it more convenient for multigenerational genetic, genomic, and transgenic approaches. Its embryos closely resemble those of X. laevis, except for their smaller size, and assays and molecular probes developed in X. laevis can be readily adapted for use in X. tropicalis. Genomic manipulation techniques such as gynogenesis facilitate genetic screens, because they permit the identification of recessive phenotypes after only one generation. Stable transgenic lines can be used both as in vivo reporters to streamline a variety of embryologic and molecular assays, or to experimentally manipulate gene expression through the use of binary constructs such as the GAL4/ UAS system. Several mutations have been identified in wild-caught animals and during the course of generating inbred lines. A variety of strategies are discussed for conducting and managing genetic screens, obtaining mutations in specific sequences, achieving homologous recombination, and in developing and taking advantage of the genomic resources for Xenopus tropicalis.