Whole-mount in situ hybridization (WISH) optimized for gene expression analysis in mouse embryos and embryoid bodies.

Whole-mount in situ hybridization (WISH) is a technique widely used in developmental biology to study the localization of RNA sequences in intact tissues or whole organisms. In this chapter we present a detailed protocol that was optimized for gene expression analysis in early stage mouse embryos (5.5-10.5 days post-coitum) and embryoid bodies formed by differentiating embryonic stem cells and can be used for the detection of up to two distinct RNA sequences simultaneously. The initial steps of the procedure are the generation of the labeled riboprobe(s) and the embryo or embryoid body preparation, which can be completed in less than 2 days. The actual WISH procedure, comprised of the hybridization, the post-hybridization washes, and the immunological staining, can be completed in 3 days.

Wnt-induced proteolytic targeting.

Misregulation of the Wnt pathway is a common route to cancer, including primary breast cancers. In this issue of Genes & Development, Miranda-Carboni and colleagues (3121-3134) demonstrate that the cyclin-dependent kinase inhibitor p27(Kip1) is ubiquitylated for proteasomal degradation in Wnt10b-induced mammary tumors exclusively by the Cul4A E3 ligase, which is strongly induced by Wnt signaling. The discovery of a new Wnt-induced proteolytic targeting system has important implications for the mechanism of Wnt-initiated tumorigenesis.

An optimized procedure for whole-mount in situ hybridization on mouse embryos and embryoid bodies.

Determining the precise expression pattern of a gene of interest at various stages of development is essential to understanding its biological function in embryology. This protocol describes a sensitive method for whole-mount in situ hybridization (WISH) to mouse embryos, using cRNA probes. Adaptations are provided that allow the protocol to be applied to embryonic stages ranging from blastocysts to postimplantation stage embryos, and to embryoid bodies. We also describe an in situ method for differential detection of two probes. Probe labeling and dissection and preparation of the embryos can be performed in 2 d. The actual WISH procedure can be completed in another 3 d.

Expression of Frizzled5, Frizzled7, and Frizzled10 during early mouse development and interactions with canonical Wnt signaling.

Wnt signaling has been shown to be important in the patterning of the gastrulating mouse embryo, especially in axis formation. To this date, there is no clear indication that the Wnt receptors, Frizzleds (Fzds), are involved in such early specification. Moreover, at the gastrulation stage, the only Fzd with a known characterized expression pattern is Fzd8, which is expressed in the anterior visceral endoderm (aVE) (Lu et al. [2004] Gene Expr Patterns 4:569-572). Following a real time RT-PCR study to evaluate Fzd expression in the gastrulating embryo, we used whole-mount in situ hybridization to reveal new expression domains for Fzd5, Fzd7, and Fzd10. Fzd5 is expressed in the aVE and Fzd7 expression is restricted to the epiblast of the gastrulating embryo. The expression pattern of Fzd10 in the primitive streak of the gastrula suggests it has a role in mesoderm induction. We also show that the purified, secreted forms of the extracellular cysteine-rich domains (CRDs) of FZD5, Fzd7, and Fzd8 can antagonize Wnt3a-induced beta-Catenin accumulation in L-cells, whereas in mouse embryonic stem cells, these CRDs can inhibit spontaneous mesoderm formation and promote neural differentiation. Our data demonstrate that Fzd5, Fzd7, and Fzd10 are expressed in distinct domains of the gastrulating embryo, and that the CRDs of FZD5, Fzd7, and Fzd8 can regulate Wnts, indicating that Fzds interpret Wnt signals during embryonic mesoderm and neural induction.

The transcription factor hepatocyte nuclear factor-6 controls the development of pancreatic ducts in the mouse.

BACKGROUND & AIMS: A number of hereditary polycystic diseases are associated with formation of cysts within the pancreatic ducts. The cysts result from abnormal tubulogenesis, but how normal pancreatic duct development is controlled remains poorly understood. Here, we investigate the transcriptional mechanisms that control pancreatic duct development by addressing the role of the transcription factor hepatocyte nuclear factor (HNF)-6. METHODS: Using immunostaining, we have determined the expression pattern of HNF-6 in pancreatic ducts during mouse development. Hnf6 null mice at various stages of development were studied by immunolocalization methods to assess the morphology, differentiation, and proliferation status of ductal cells. The expression of genes involved in hereditary polycystic diseases was determined by real-time, reverse-transcription polymerase chain reaction (RT-PCR). RESULTS: We show that HNF-6 is expressed in the pancreatic duct epithelium throughout development and that, in the absence of HNF-6, duct morphogenesis is perturbed. Although development of the intercalated ducts is normal, cysts appear within the interlobular and intralobular ducts. This is associated with abnormal development of primary cilia at the apical pole of the duct cells and with reduced expression of a set of genes involved in polycystic diseases, namely those coding for HNF-1beta and for the cilium-associated proteins polyductin/fibrocystin and cystin. CONCLUSIONS: We identify HNF-6 as the first transcriptional regulator of pancreatic duct development and reveal the existence of different regulatory mechanisms in distinct duct compartments. HNF-6 controls a network of genes involved in cilium formation and in hereditary polycystic diseases. Finally, HNF-6 deficiency represents a genetically defined model of pancreatic cystic disease.