Towards the function of the PDZ-binding domain of Delta1.

The evolutionarily conserved Notch-signal transduction has been described as a cell-to-cell signaling pathway mediated by the transmembrane proteins Delta, Jagged and Notch that interact in a ligand-receptor like manner. Notch signaling regulates cell fate decisions and cellular differentiation in various tissues and has essential functions in embryonic patterning and tumorigenesis. Whereas signal transduction to Notch expressing cells and the role of the Notch intracellular domain has been described in detail, the cytoplasmic part of the ligands is less well understood. Several studies demonstrated that also the ligands undergo regulated cleavages leading to the release of the intracellular domain of the ligand into the cytoplasm. Recently, we reported on the identification of proteins interacting with the intracellular domain of Delta1 (Dll1cyto) (Pfister et al., 2003). We have identified several PDZ-domain containing proteins by in vitro and in vivo systems, e.g. from the MAGUK family, that interact with the C-terminal PDZ-binding motif of Delta1. To address these interactions in more detail we generated a conditional mouse mutant targeting the PDZ-binding motif of Delta1 (Dll1DPDZ-bm) and a mouse mutant targeting the Dll1cyto interacting protein Magi2. Here, we will present our newest results regarding the role of the PDZ-binding motif of Delta1

Node and midline defects are associated with left-right development in Delta1 mutant embryos.

Axes formation is a fundamental process of early embryonic development. In addition to the anteroposterior and dorsoventral axes, the determination of the left-right axis is crucial for the proper morphogenesis of internal organs and is evolutionarily conserved in vertebrates. Genes known to be required for the normal establishment and/or maintenance of left-right asymmetry in vertebrates include, for example, components of the TGF-β family of intercellular signalling molecules and genes required for node and midline function. We report that Notch signalling, which previously had not been implicated in this morphogenetic process, is required for normal left-right determination in mice. We show, that the loss-of-function of the delta 1 (Dll1) gene causes a situs ambiguous phenotype, including randomisation of the direction of heart looping and embryonic turning. The most probable cause for this left-right defect in Dll1 mutant embryos is a failure in the development of proper midline structures. These originate from the node, which is disrupted and deformed in Dll1 mutant embryos. Based on expression analysis in wild-type and mutant embryos, we suggest a model, in which Notch signalling is required for the proper differentiation of node cells and node morphology

Modeling coexistence of oscillation and Delta/Notch-mediated lateral inhibition in pancreas development and neurogenesis.

During pancreas development, Neurog3 positive endocrine progenitors are specified by Delta/Notch (D/N) mediated lateral inhibition in the growing ducts. During neurogenesis, genes that determine the transition from the proneural state to neuronal or glial lineages are oscillating before their expression is sustained. Although the basic gene regulatory network is very similar, cycling gene expression in pancreatic development was not investigated yet, and previous simulations of lateral inhibition in pancreas development excluded by design the possibility of oscillations. To explore this possibility, we developed a dynamic model of a growing duct that results in an oscillatory phase before the determination of endocrine progenitors by lateral inhibition. The basic network (D/N + Hes1 + Neurog3) shows scattered, stable Neurog3 expression after displaying transient expression. Furthermore, we included the Hes1 negative feedback as previously discussed in neurogenesis and show the consequences for Neurog3 expression in pancreatic duct development. Interestingly, a weakened HES1 action on the Hes1 promoter allows the coexistence of stable patterning and oscillations. In conclusion, cycling gene expression and lateral inhibition are not mutually exclusive. In this way, we argue for a unified mode of D/N mediated lateral inhibition in neurogenic and pancreatic progenitor specification

The Notch gene regulatory network during somitogenesis.

The Delta/Notch gene regulatory network (Notch – GRN) is a cell-to-cell signalling pathway with pleiotropic and essential functions during mammalian development and adult homeostasis. The GSF – Institutes of Experimental Genetics and of Biomathematics and Biometry have initiated an interdisciplinary approach combining the expertises of theoretical and experimental biologists to begin to model the Notch – GRN during mesodermal patterning. In this embryogenic process, we regard the Notch - GRN as a generic thematic unit to begin to study regulatory systems at a whole. Our current mathematical model simulates the in situ expression of selected Notch pathway genes in each of thousands of presomitic cells in 3D. The further development of the E-cells model requires the integration of quantitative data and the identification of novel factors that are co-regulated within the Notch – GRN. The presentation will thus focus on the biological background of somitogenesis and our recent experimental findings. We have been studying the regulation of the Notch ligand Delta1 (Dll1) at the cis-regulatory level, the functional requirement of Dll1 signalling during patterning and differentiation of the paraxial mesoderm, the identification of novel direct interactors of the Dll1 protein and the identification of novel Notch target genes. The data resulting from these experimental studies are an indispensable basis for our collaborative effort to extend and refine our in silico mathematical simulations of the Notch - GRN

Interaction of the MAGUK family member Acvrinp1 and the cytoplasmic domain of the notch ligand Delta1.

The evolutionarily conserved Notch signal transduction pathway regulates cell fate and cellular differentiation in various tissues and has essential functions in embryonic patterning and tumorigenesis. Cell-cell signaling by the Notch pathway is mediated by the interaction of the transmembrane receptor Notch with its ligands Delta or Jagged presented on adjacent cells. Whereas signal transduction to Notch expressing cells has been described, it is unclear whether Delta-dependent signaling may exist within the Delta-expressing cell. Here, we report on the identification of Acvrinp1, a MAGUK family member, interacting with the intracellular domain of Delta1 (Dll1). We confirmed the interaction between Dll1 and Acvrinp1 by pull-down experiments in vitro and in a mammalian two-hybrid system in vivo. We delimited the fourth PDZ domain of Acvrinp1 and the PDZ-binding domain of Dll1 as major interacting domains. In situ expression analyses in mouse embryos revealed that Dll1 and Acvrinp1 show partly overlapping but distinct expression patterns, for example, in the central nervous system and the vibrissae buds. Further, we found that expression of Acvrinp1 is altered in Dll1 loss-of-function mouse embryos. © 2003 Elsevier Ltd. All rights reserved

<em>In vitro</em> analysis of bone phenotypes in <em>Col1a1</em> and<em> Jagged1</em> mutant mice using a standardized osteoblast cell culture system.

The mouse is a valuable model organism for studying bone biology and for unravelling pathological processes in skeletal disorders. In vivo methods like X-ray analysis, DXA measurements, pQCT and &mu;CT are available to investigate the bone phenotype of mutant mice. However, the descriptive nature of such methods does not provide insights into the cellular and molecular bases of the observed bone alterations. Thus, first-line investigations might be complemented by cell culture-based methods to characterize the pathological processes at the cellular level independent from systemic influences. By combining well-established assays, we designed a comprehensive test system to investigate the cellular and molecular phenotype of primary calvarial osteoblasts in mutant mice compared to wild-type controls as a first-line phenotyping method. The compilation of 9 different quantifiable assays allows assessment of general properties of cell growth and investigation of bone-specific parameters at the functional, protein and RNA level in a kinetic fashion throughout a 3-week culture period, thus maximizing the chance to discover and explain new phenotypes in mutant mice. By analyzing mutant mouse lines for Col1a1 and Jag1 (Delta-Notch pathway) that both showed clear alterations in several bone-related parameters we could demonstrate the usefulness of our cell culture system to discriminate between primary (Col1a1) and secondary effects (Jag1) in osteoblasts

Wer hat an der Uhr gedreht? Regulationsmechanismen der Segmentierung im Wirbeltierembryo.

Die Somitogenese ist ein Prozess in der Embryonalentwicklung aller Vertebraten, bei der die Grundlage f&uuml;r die Segmentierung des Wirbeltierk&ouml;rpers gelegt wird. Vom mesenchymalen Gewebe seitlich des Neuralrohrs spalten sich am Vorderende kleine Zellballen (Somiten) ab, aus denen sp&auml;ter die Wirbel hervorgehen. Dies geschieht mit einer speziesspezifischen Periodizit&auml;t. Die molekularen Grundlagen dieser Somitogenese-Uhr sind noch immer nicht restlos aufgekl&auml;rt. Um diese Mechanismen besser verstehen zu k&ouml;nnen, haben wir ein Computermodell entwickelt, das es erlaubt, diese Vorg&auml;nge durch Differentialgleichungen zu beschreiben, f&uuml;rviele Zellen eines Gewebes zu berechnen und Genexpressionen anschaulich darzustellen

DLL1- and DLL4-mediated Notch signaling is essential for adult pancreatic islet homeostasis.

Genes of the Notch signaling pathway are expressed in different cell types and organs at different time points during embryonic development and adulthood. The Notch ligand Delta-like 1 (DLL1) controls the decision between endocrine and exocrine fates of multipotent progenitors in the developing pancreas, and loss of Dll1 leads to premature endocrine differentiation. However, the role of Delta-Notch signaling in adult tissue homeostasis is not well understood. Here, we describe the spatial expression pattern of Notch pathway components in adult murine pancreatic islets and show that DLL1 and DLL4 are specifically expressed in beta-cells, whereas JAGGED1 is expressed in alpha-cells. We show that mice lacking both DLL1 and DLL4 in adult beta-cells display improved glucose tolerance, increased glucose-stimulated insulin secretion, and hyperglucagonemia. In contrast, overexpression of the intracellular domain of DLL1 in adult murine pancreatic beta-cells results in impaired glucose tolerance and reduced insulin secretion, both in vitro and in vivo. These results suggest that Notch ligands play specific roles in the adult pancreas and highlight a novel function of the Delta/Notch pathway in beta-cell insulin secretion