Work Package 1

Rapid screening for developmental pathways

Introduction
Developmental Biological studies have in the past led to identification of many signaling pathways that control organogenesis at fetal and adult stages. Hedgehog signalling and Wnt signalling are examples of molecular-genetic pathways that were initially shown to be crucial to fruitfly embryology, and were subsequently linked to cancer. Our understanding of these and other signaling cascades is incomplete: many of the genes involved are yet to be uncovered. Additionally, while many genes are linked to important functions in organogenesis by mutational studies, it remains unclear how they relate to the known molecular genetic pathways. Collaborating groups in this proposal have provided evidence for novel functions of the Wnt signal transduction pathway in development and disease. These include fundamental developmental functions (e.g. embryonic axis formation), cell fate determination and maintenance of stem-cell compartments in the adult gut. This work established a link between Wnt signaling and colorectal cancer in humans (Clevers, Fodde). Similarly, the Hedgehog signaling pathway has been implicated in extremely diverse functions including development of the gonads, blood vessels, limbs, face and peripheral nervous system (Grootegoed, Meijer), while its disruption has more recently been shown to lead to basal cell carcinoma. Prx/Alx genes and Gli3(Meijlink) have been shown to be part of elusive pathways that interact with the Hedgehog pathway in the craniofacial structure and the limb bud. Hox and Cdx genes are involved in embryonic body axis extension and patterning and have additional functions throughout development (Deschamps); they depend on the FGF and Wnt pathways. Alx, Hox, Gli3 and Cdx genes encode transcription factors and all these molecules have been implicated in human disease.

Objective
We will apply existing methodology and generate novel tools to extend our knowledge of these hierarchical networks of interacting genes. We will make use of unique mutants available to and generated by the collaborating groups. For example, in the Wnt signaling cascade, mouse mutants with inactivated Apc, Tcf1 and Tcf4 genes will serve to provide the material for identification of novel downstream targets. In other mouse mutants it remains currently elusive which signaling cascades are affected.

Approach
We will develop a signaling pathway probe set, in which the included genes are known to be involved in the Wnt, Hedgehog, TGFb/BMP and FGF and other signaling pathways. This tool will be used to characterize existing mutants (Apc mutants, Fodde; Cdx and Hox mutants, Deschamps; Prx/Alx mutants, Gli3, Meijlink; Oct 6, Meijer) as well as mutants yet to be generated, to establish association with these pathways. This will allow a rapid assessment whether genes in these developmental signaling pathways are involved in the tissue or cells under study. This methodology is also applicable to analysis of human material, stem cells, differentiating cells and tumors. At the same time we will (continue to) use gene-chips, cDNA arrays and antibody arrays representing essentially all available genes for an unbiased analysis of mutants. This will identify direct and indirect targets of the mutated genes; in addition we will be able to recognize signaling cascades involved and possibly discover novel ones.

Key-words: gene expression, molecular-genetic pathways, Wnt, Hh, signaling, Hox genes.

Links with other parts of the program: Interactions are anticipated especially with WP2 (downstream targets of Wnt signaling), WP3 (signaling centers) and WP7 (identification of stem cell vs. differentiated cell genetic programs).

Participants: Deschamps, Meijlink, Grootegoed , Clevers, Fodde.