Fbw7 repression by hes5 creates a feedback loop that modulates Notch-mediated intestinal and neural stem cell fate decisions.

FBW7 is a crucial component of an SCF-type E3 ubiquitin ligase, which mediates degradation of an array of different target proteins. The Fbw7 locus comprises three different isoforms, each with its own promoter and each suspected to have a distinct set of substrates. Most FBW7 targets have important functions in developmental processes and oncogenesis, including Notch proteins, which are functionally important substrates of SCF(Fbw7). Notch signalling controls a plethora of cell differentiation decisions in a wide range of species. A prominent role of this signalling pathway is that of mediating lateral inhibition, a process where exchange of signals that repress Notch ligand production amplifies initial differences in Notch activation levels between neighbouring cells, resulting in unequal cell differentiation decisions. Here we show that the downstream Notch signalling effector HES5 directly represses transcription of the E3 ligase Fbw7ß, thereby directly bearing on the process of lateral inhibition. Fbw7(Δ/+) heterozygous mice showed haploinsufficiency for Notch degradation causing impaired intestinal progenitor cell and neural stem cell differentiation. Notably, concomitant inactivation of Hes5 rescued both phenotypes and restored normal stem cell differentiation potential. In silico modelling suggests that the NICD/HES5/FBW7ß positive feedback loop underlies Fbw7 haploinsufficiency. Thus repression of Fbw7ß transcription by Notch signalling is an essential mechanism that is coupled to and required for the correct specification of cell fates induced by lateral inhibition.

Experiments, measurements, and mathematical modeling to decipher time signals in development.

Good progress has been made in identifying key signaling molecules and explaining how they are used to generate spatial patterns during embryonic development. In contrast, little is known about the control of timing or how cells use time signals in the developing embryo. In this review, I describe how direct measurements from the embryo combined with mathematical modeling could bring new insights. To illustrate this point, I discuss three examples: the Dpp gradient during growth of the Drosophila wing imaginal disc; the Polycomb-based epigenetic silencing during vernalization in plants; and the Notch-dependent somite segmentation clock.

Absence of an external germinal layer in zebrafish and shark reveals a distinct, anamniote ground plan of cerebellum development.

The granule cell layer of the cerebellum comprises the largest population of neurons in the vertebrate CNS. In amniotes, its precursors undergo a unique phase of transit amplification, regulated by Sonic hedgehog. They do so within a prominent but transient secondary proliferative epithelium, the external germinal layer, which is formed by tangential migration of precursor cells from the rhombic lip. This behavior is a hallmark of bird and mammal cerebellum development. Despite its significance for both development and disease, it is unclear whether an external germinal layer is a requirement for granule cell production or an expedient of transit amplification. Evidence for its existence in more basal vertebrates is contradictory. We therefore examined cerebellum development in the zebrafish, specifically in relation to the expression of the basic helix-loop-helix gene Atonal 1, which definitively characterizes granule cell precursors. The expression of Atoh1a-Atoh1c, in combination with patterns of proliferation and fate maps, define precursor pools at the rhombic lip and cerebellar midline but demonstrate that an external germinal layer is absent. Sonic hedgehog signaling is correspondingly absent in the zebrafish cerebellum. Sustained roof-plate-derived signals suggest that, in the absence of transit amplification, primary granule cell precursor pools are maintained throughout development. To determine whether this pattern is specific to zebrafish or reflects a more general anamniote organization, we examined the expression of similar genes in the dogfish, Scylliorhinus canicula. We show that these anamniotes share a common ground plan of granule cell production that does not include an external germinal layer.

Cloning and embryonic expression of five distinct sfrp genes in the zebrafish Danio rerio.

Recently, a new member of the secreted frizzled-related protein (sFRP) family, named tlc, has been identified as expressed by the anterior neural border (ANB) cells in the zebrafish Danio rerio. Tlc plays an important role in telencephalic induction and patterning. In absence of Tlc, formation of the telencephalon is severely delayed, but not abolished. This prompted us to clone the other zebrafish sfrp family members and analyse their expression patterns, in search of a family member that may partly functionally overlap with Tlc. Except sizzled, expression profile of sfrp genes in zebrafish has not been reported so far. Here, we describe the cloning of full-length cDNA for sfrp1a, sfrp1b, sfrp2, sfrp3 and sfrp5 gene transcripts and we examine their expression at different embryonic stages. Only sfrp1a is expressed in the anterior neural plate including the ANB cells where and when tlc is expressed. Interestingly, compared to both tlc and sfrp1a, wnt genes are complementary expressed more posteriorly in the neural plate. Later, both sfrp1a and sfrp5 expression profiles are overlapping, in particular at pharyngula stage these genes are expressed in the ventral part of the forebrain, midbrain and hindbrain. sfrp1b, sfrp2 and sfrp3 are mainly expressed in mesodermal and endodermal embryonic tissues. Expression profiles of these different genes in zebrafish gave interesting clues on the possible function and evolution of sFRPs in zebrafish and other organisms.

H-NS in Gram-negative bacteria: a family of multifaceted proteins.

DNA-packaging and the control of gene expression constitute a major challenge for bacteria to survive and adapt to environmental changes. The use of multiple strategies to solve these problems could explain the presence of various nucleoid-associated proteins in bacteria. H-NS, one of these proteins, has been extensively studied in Escherichia coli, and a variety of phenotypes have been associated with a mutation in its structural gene. However, the role of H-NS in bacterial physiology and its mechanism of action are still a matter of debate. The expanding number of H-NS-related proteins identified in Gram-negative bacteria reveals interesting clues about their structure-function-evolution relationship.

A Novel H-NS-like protein from an antarctic psychrophilic bacterium reveals a crucial role for the N-terminal domain in thermal stability.

We describe here new members of the H-NS protein family identified in a psychrotrophic Acinetobacter spp. bacterium collected in Siberia and in a psychrophilic Psychrobacter spp. bacterium collected in Antarctica. Both are phylogenetically closely related to the HvrA and SPB Rhodobacter transcriptional regulators. Their amino acid sequence shares 40% identity, and their predicted secondary structure displays a structural and functional organization in two modules similar to that of H-NS in Escherichia coli. Remarkably, the Acinetobacter protein fully restores to the wild-type H-NS-dependent phenotypes, whereas the Psychrobacter protein is no longer able to reverse the effects of H-NS deficiency in an E. coli mutant strain above 30 degrees C. Moreover, in vitro experiments demonstrate that the ability of the Psychrobacter H-NS protein to bind curved DNA and to form dimers is altered at 37 degrees C. The construction of hybrid proteins containing the N- or the C-terminal part of E. coli H-NS fused to the C- or N-terminal part of the Psychrobacter protein demonstrates the role of the N-terminal domain in this process. Finally, circular dichroism analysis of purified H-NS proteins suggests that, as compared with the E. coli and Acinetobacter proteins, the alpha-helical domain displays weaker intermolecular interactions in the Psychrobacter protein, which may account for the low thermal stability observed at 37 degrees C.

Effect of mild acid pH on the functioning of bacterial membranes in Vibrio cholerae.

In this paper, we initiated the first two-dimensional electrophoresis map of Vibrio cholerae, the aetiological agent of cholera disease. In this pathogen the efficient adaptation to detrimental conditions plays an important role in its survival in both the aquatic reservoir and human intestine. By proteome analysis we investigated the effect of mild acid treatment on the physiology of V. cholerae. More than 50 proteins were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and database searching. Amongst them, pH regulated proteins belong to various functional classes such as intermediary metabolism and bacterial envelope. Several proteins whose accumulation level was decreased in response to acidic pH are known to be involved in the organization and the functioning of membranes, including lipopolysaccharide. Consistent with this, we observed an increased susceptibility to hydrophobic drugs, a loss of motility and a reduction in the ability to form a biofilm in cells grown at pH 6. Our results suggest that V. cholerae is able to sense a moderate decrease in pH and to modify accordingly its structure and physiology.