Signaling pathways
1.Signaling pathways during development.
Diagnostics, and molecules for intervention strategies, for multifactorial disease can be realized by the identification of the signaling pathways involved in development and differentiation. Within the embryo, the normal differentiation of specific cell types such as those in the intestine, vascular system, myelinating cells of the nervous system, stem cells for the hematopoietic system, the signaling centers involved in the development of the face, limbs or external genitalia are known to involve molecules within the Wnt, Hedgehog, TGFb and FGF signaling pathways. Included within these signaling pathways are modulators, intracellular localization molecules and transcription factors. Recently, collaborating groups in this proposal (Clevers, Fodde) have provided evidence that the Wnt signal transduction pathway induces the nuclear translocation of b-catenin and plays a key role in cell-fate determination. Some of the key components of this signaling pathway currently under study include: the tumor suppressor APC and its close relative APC2, the Tcf transcription factors and the transcriptional co-activator b-catenin, the secreted Wnt factors and one of their receptors, Frizzled-5. Since the role of the Wnt/b-catenin signaling pathway is thought be in the maintenance of stem cell compartments, both during development and in the adult organism, we request support to examine the expression profiles of these molecules in stem cells and tissues as they are generated in the embryo (WP2).
The developing mouse embryo can be used as a paradigm to understand the molecular genetics of signaling cascades in growth and patterning. Recent evidence points to a critical role for a limited set of signaling pathways that regulate the decision to proliferate (self-renewal) or enter into differentiation, the crucial key to the balance between growth and patterning during development and disease. A major developmental signalling pathway is the Wnt pathway, and important target gene families in this respect are the well-conserved Homeobox (Hox and Cdx) gene families which determine the positional identity and polarity of body parts and organs. We aim at elucidating the cascades of genetic activities involving Hox and Cdx genes, underlying fundamental decisions in embryonic axis formation and patterning (Deschamps), most of which involve genetic interactions with the Wnt and Fgf signalling cascades. We are studying the prerequisites to initial Hox and Cdx gene expression at early stages during gastrulation and differentiation of the epiblast, mimicked by in vitro cultured embryonic stem (ES) cells. Wnt signals are essential for primitive streak formation and gastrulation, and we have recently shown that they initially induce Hox genes (Deschamps). We also study the mechanism of action of Hox and Cdx genes in later processes during axial extension, partterning and organogenesis. Wnt mutants exhibit severe posterior truncations, which we have observed in Cdx null mutants as well (Deschamps), again pointing to genetic interactions between the transcription factor-encoding genes and Wnt signalling during tissue generation and patterning. We aim to identify the molecules controling of the crucial balance between self-renewal and differentiation of stem cells present in the posterior aspect of developing embryos during axis elongation, focussing on the role of Wnt signalling (WP3). One of our long-term goals is to shed light on the cell to cell signalling interactions by which organisers control vertebrate organogenesis (Meijlink). The vertebrate limb is an excellent model, as the signalling centres that control limb morphogenesis are well defined. Signalling by AER and polarising region controls patterning of several 100,000 limb bud mesenchymal cells. To analyse the mechanisms by which large numbers of cells are organised to form tissues and organs, we are focusing on the roles of aristaless-related genes, Prx and Alx, in studies of craniofacial development. As is the common theme, controlled organogenesis requires a limited set of signaling pathways. In particular, the Sonic hedgehog pathway is central to embryonic development as its disruption in higher vertebrates and humans causes devastating congenital malformations affecting limbs (loss of hand and feet), craniofacial structures, the neural tube (cyclopia) and other morphogenetic processes. For these studies, we combine mouse molecular genetics (gene ablation, gain-of-function, gene regulation studies in vivo) with genome-wide screening approaches. Our recent research addressed the genetic cascades that control establishment of the limb bud organiser (polarising region) and activation of Shh signalling in the posterior mesenchyme. These studies indicated that polarisation of the early limb bud not only depends on posterior activation but also on anterior repression. Gli3 and Alx4 are expressed anteriorly in the limb bud and loss of function of either gene leads to extra anterior digits. We aim to establish the genetic hierarchies and identify the interactions of this anterior, restrictive cascade (WP3). Our approach includes (1) genetic studies of relevant double mutants to establish hierarchies within the cascade; (2) promoter studies: we identified a 350-bp promoter fragment that is sufficient, when linked to a reporter, to reproduce the characteristic Alx4 limb bud expression. This should enable us to identify direct regulators of Alx4. Finally (3), we have recently performed a genome-wide screen based on Prx1/Prx2 mutant limbs. This resulted in the identification of a series of downstream targets of these and probably other aristaless-related genes. Our aim within this programme is therefore to further characterise the anterior restrictive cascade both upstream and downstream from the Alx4 gene. Similarly, the Hedgehog signaling pathway has been implicated in the development of the gonads, external genitalia and the peripheral nervous system, and in hematopoietic and vascular fate determination. In mammals three Hedgehog proteins exist: Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh). Dhh plays an important role during gonadal somatic stem-cell proliferation, development of testicular architecture and maintenance of the germ cell lineage. As a result, Dhh mutant mice show dysregulation of testis development and are infertile. Ihh mutant mice show defects in vascular and hematopoietic development and Dhh is further known to be expressed in the Schwann cells of the peripheral nervous system, where it plays an important role in the development of the protective nerve sheath (Meijer). The transmembrane proteins Smoothened (Smo) and Patched (Ptc) control Hedgehog-dependent activation of specific intracellular transcriptional effectors, Gli1, Gli2, and Gli3, and some of these molecules show restricted expression patterns in gonadal cells and cells of the hematopoietic system. Furthermore, during testis development and ovarian folliculogenesis a network of interactions between pre-Sertoli cells, pre-Leydig cells, pre-granulosa cells and pre-thesa cells is highly important in the life-long maintenance and differentiation of gonada, somatic and germ cells, suggesting a specific microenvironment and gradient of inducing factors (Grootegoed). Hence, we aim to study Hedgehog signaling in (dys)regulation of gonadal development, germ cell maintenance and external genitalia development (hypospadias), as well as in other tissue systems (WP3). The above common themes allow for synergy in the rapid analysis of all the signaling pathways implicated in stem cell and tissue development. We ask support to produce a new biochemical tool, which will allow us to screen simultaneously for all the molecules involved in the Wnt, Hedgehog, TGFb and FGF signaling pathways (and others as they are discovered) (WP1). We will generate a dedicated probe set and expression chip that will allow a rapid assessment whether molecules in these developmental signaling pathways are involved in the developing/differentiating tissue/stem cell under study. It is anticipated that all members of the initiative, as well as the international community and industry will be interested in such expression profile libraries. Moreover, common intracellular signaling pathways converge on the nucleus to regulate specific programs of gene expression that drive the stable differentiation of cells and more importantly keep cells undifferentiated. The genetic programs that are controlled by the Wnt, Hedgehog and other pathways (as they are found) in several developmental tissue systems will also be evaluated. The molecular expression profiling studies of normal or dysregulated/mutant tissues from mouse models will be performed in the context of existing facilities (NKI, Leiden, Utrecht, Rotterdam). For example, concerning the Wnt signaling pathway, we aim to unravel the Wnt/b-catenin transcriptional program in the developing intestine by gene expression analysis utilizing Apc and Tcf4 knockout mice (Clevers, Fodde) (WP2). Comparisons of such profiles produced in the different laboratories, particularly those screening stem cells (Mummery, Dzierzak), will be performed (WP7). The combination and integration of this data will reveal a complete picture of downstream targets allowing the discrimination between cell lineage specific versus common targets of these signaling pathways (new bioinformatics resources). Validation of the role of these molecules in each developmental/differentiation system will be by transgenic, gene targeting strategies and cre-lox technology in mice, but also take advantage of state of the art biotechnologic strategies involving transposition, RNAi and single chain monoclonal antibodies (see WPs in Theme 2). Moreover, intergration of these results with our industrial partner Semaia Pharmaceuticals BV will lead to the production of a wide range of novel molecules for intervention in signaling pathways, which in turn could lead to useful therapeutic applications.
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