SummaryLarge-scale phenotyping efforts have demonstrated that approximately 25-30% of mouse gene knockouts cause intra-uterine lethality. Analysis of these mutants has largely focussed on the embryo but not the placenta, despite the critical role of this extra-embryonic organ for developmental progression. Here, we screened 103 embryonic lethal and subviable mouse knockout lines from the Deciphering the Mechanisms of Developmental Disorders programme (https://dmdd.org.uk) for placental phenotypes. 68% of lines that are lethal at or after mid-gestation exhibited placental dys-morphologies. Early lethality (E9.5-E14.5) is almost always associated with severe placental malformations. Placental defects strongly correlate with abnormal brain, heart and vascular development. Analysis of mutant trophoblast stem cells and conditional knockouts suggests primary gene function in trophoblast for a significant number of factors that cause embryonic lethality when ablated. Our data highlight the hugely under-appreciated importance of placental defects in contributing to abnormal embryo development and suggest key molecular nodes governing placentation.

Dickkopf-1 (Dkk1) is a secreted protein that negatively modulates the Wnt/␤catenin pathway. Lack of Dkk1 function affects head formation in frog and mice, supporting the idea that Dkk1 acts as a "head inducer" during gastrulation. We show here that lack of Dkk1 function accelerates internalization and rostral progression of the mesendoderm and that gain of function slows down both internalization and convergence extension, indicating a novel role for Dkk1 in modulating these movements. The motility phenotype found in the morphants is not observed in embryos in which the Wnt/␤catenin pathway is overactivated, and that dominant-negative Wnt proteins are not able to rescue the gastrulation movement defect induced by absence of Dkk1. These data strongly suggest that Dkk1 is acting in a ␤catenin independent fashion when modulating gastrulation movements. We demonstrate that the glypican 4/6 homolog Knypek (Kny) binds to Dkk1 and that they are able to functionally interact in vivo. Moreover, Dkk1 regulation of gastrulation movements is kny dependent. Kny is a component of the Wnt/planar cell polarity (PCP) pathway. We found that indeed Dkk1 is able to activate this pathway in both Xenopus and zebrafish. Furthermore, concomitant alteration of the ␤catenin and PCP activities is able to mimic the morphant accelerated cell motility phenotype. Our data therefore indicate that Dkk1 regulates gastrulation movement through interaction with LRP5/6 and Kny and coordinated modulations of Wnt/␤catenin and Wnt/PCP pathways.[Keywords: Dickkopf-1; HSPG; Wnt/PCP; gastrulation movements] Supplemental material is available at http://www.genesdev.org.

We describe the molecular characterization and function of vielfä ltig (vfl ), a X-chromosomal gene that encodes a nuclear protein with six Krü ppel-like C2H2 zinc finger motifs. vfl transcripts are maternally contributed and ubiquitously distributed in eggs and preblastoderm embryos, excluding the germline precursor cells. Zygotically, vfl is expressed strongly in the developing nervous system, the brain, and in other mitotically active tissues. Vfl protein shows dynamic subcellular patterns during the cell cycle. In interphase nuclei, Vfl is associated with chromatin, whereas during mitosis, Vfl separates from chromatin and becomes distributed in a granular pattern in the nucleoplasm. Functional gain-offunction and lack-of-function studies show that vfl activity is necessary for normal mitotic cell divisions. Loss of vfl activity disrupts the pattern of mitotic waves in preblastoderm embryos, elicits asynchronous DNA replication, and causes improper chromosome segregation during mitosis. INTRODUCTIONZinc fingers constitute the most abundant structural motifs in the proteome predicted from the genome sequences of Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans (Rubin et al., 2000) as well as the draft human genomic sequences (Lander et al., 2001). Zinc finger proteins are best known as transcriptional regulators that participate in a variety of cellular activities such as development, differentiation, and tumor suppression. The most common form of zinc finger domain is the so called C2H2 domain, first identified in the basal transcription factor TFIIIA in Xenopus laevis (Miller et al., 1985) and subsequently found also in DNA-binding transcription factors regulating polymerase II-dependent gene expression (Rosenberg et al., 1985). The three-dimensional structure of the basic C2H2 zinc finger is a small domain composed of a ␤-hairpin followed by an ␣-helix, a structure that is held in place by a zinc ion. DNA-binding C2H2 zinc fingers generally occur as tandem arrays with a minimum of two fingers needed to specify the DNA-binding site (Choo and Klug, 1995). In addition to DNA-binding, zinc finger proteins have been implicated in RNA-binding, protein-protein interactions, and lipid binding (Lorick et al., 1999;Bach, 2000;Tucker et al., 2001).DNA-binding C2H2 zinc finger proteins contain proteinbinding domains that provide the basis for the assembly of regulatory complexes involved in chromatin remodeling and transcriptional regulation (Aasland et al., 1995;David et al., 1998). They are frequently expressed in distinct spatial and temporal patterns, and their subcellular localization was found to be regulated in a cell cycle-dependent manner. The entry of mitosis is characterized by the transcriptional shutdown (Prescott and Bender, 1962) that involves inactivation of the general transcription machinery (Segil et al., 1996;Bellier et al., 1997;Akoulitchev and Reinberg, 1998;Long et al., 1998) as well as gene-specific transcription factors during the G 2 /M transition during mitosis. F...

The thalamic complex is the major sensory relay station in the vertebrate brain and comprises three developmental subregions: the prethalamus, the thalamus and an intervening boundary region -the zona limitans intrathalamica (ZLI). Shh signalling from the ZLI confers regional identity of the flanking subregions of the ZLI, making it an important local signalling centre for regional differentiation of the diencephalon. However, our understanding of the mechanisms responsible for positioning the ZLI along the neural axis is poor. Here we show that, before ZLI formation, both Otx1l and Otx2 (collectively referred to as Otx1l/2) are expressed in spatially restricted domains. Formation of both the ZLI and the Irx1b-positive thalamus require Otx1l/2; embryos impaired in Otx1l/2 function fail to form these areas, and, instead, the adjacent pretectum and, to a lesser extent, the prethalamus expand into the mis-specified area. Conditional expression of Otx2 in these morphant embryos cell-autonomously rescues the formation of the ZLI at its correct location. Furthermore, absence of thalamic Irx1b expression, in the presence of normal Otx1l/2 function, leads to a substantial caudal broadening of the ZLI by transformation of thalamic precursors. We therefore propose that the ZLI is induced within the competence area established by Otx1l/2, and is posteriorly restricted by Irx1b.

BackgroundMonoclonal antibodies with high affinity and selectivity that work on wholemount fixed tissues are valuable reagents to the cell and developmental biologist, and yet isolating them remains a long and unpredictable process. Here we report a rapid and scalable method to select and express recombinant mouse monoclonal antibodies that are essentially equivalent to those secreted by parental IgG-isotype hybridomas.ResultsIncreased throughput was achieved by immunizing mice with pools of antigens and cloning - from small numbers of hybridoma cells - the functionally rearranged light and heavy chains into a single expression plasmid. By immunizing with the ectodomains of zebrafish cell surface receptor proteins expressed in mammalian cells and screening for formalin-resistant epitopes, we selected antibodies that gave expected staining patterns on wholemount fixed zebrafish embryos.ConclusionsThis method can be used to quickly select several high quality monoclonal antibodies from a single immunized mouse and facilitates their distribution using plasmids.