Notch signalling is required for cyclic expression of the hairy-like gene HES1 in the presomitic mesoderm

Somitic segmentation provides the framework on which the segmental pattern of the vertebrae, some muscles and the peripheral nervous system is established. Recent evidence indicates that a molecular oscillator, the 'segmentation clock', operates in the presomitic mesoderm (PSM) to direct periodic expression of c-hairy1 and lunatic fringe (l-fng). Here, we report the identification and characterisation of a second avian hairy-related gene, c-hairy2, which also cycles in the PSM and whose sequence is closely related to the mammalian HES1 gene, a downstream target of Notch signalling in vertebrates. We show that HES1 mRNA is also expressed in a cyclic fashion in the mouse PSM, similar to that observed for c-hairy1 and c-hairy2 in the chick. In HES1 mutant mouse embryos, the periodic expression of l-fng is maintained, suggesting that HES1 is not a critical component of the oscillator mechanism. In contrast, dynamic HES1 expression is lost in mice mutant for Delta1, which are defective for Notch signalling. These results suggest that Notch signalling is required for hairy-like genes cyclic expression in the PSM.     

Cyclic expression of esr9 gene in Xenopus presomitic mesoderm

During somitogenesis, the cycling expression of members of the Notch signalling cascade is involved in a segmentation clock that regulates the periodic budding of somites in chicken, mouse, and zebrafish. In frog, genes with cycling expression in the presomitic mesoderm have not been reported. Here, we describe the expression of Xenopus esr9 and esr10, two new members of the Hairy/Enhancer of split related family of bHLH proteins. We show that they are expressed in a highly dynamic fashion, with their mRNA levels oscillating periodically in the presomitic mesoderm during somitogenesis. This dynamic expression is independent of de novo protein synthesis. Thus, expression of esr9 and esr10 is an indicator of the segmentation clock in the amphibian embryo. This confirms the evolutionary conservation of a molecular pathway involved in vertebrate segmentation clock.     

Homologues of c-hairy1 (her9) and lunatic fringe in zebrafish are expressed in the developing central nervous system, but not in the presomitic mesoderm

A number of genes that are involved in somitogenesis in vertebrates are cyclically expressed in the presomitic mesoderm. These include homologues of the Drosophila genes fringe and hairy. We have analysed here two genes that belong to these classes in the zebrafish, namely the apparent orthologues of lunatic fringe (l-fng) and of c-hairy1 (called her9). However, unlike the respective mouse and chicken genes, they are not expressed cyclically in the presomitic mesoderm. Instead, both genes are mainly expressed in the central nervous system. her9 is predominantly expressed in the fore- and midbrain, and transiently in the hindbrain. Thus, the previously identified and only very distantly related her1 gene of zebrafish has more similarities to the expression of the c-hairy1 gene than its apparent orthologue her9, indicating that sequence similarity and similarity of function are not necessarily linked in this case. l-fng expression is found in alternating pre-rhombomeres, comparable to the equivalent mouse gene expression and in the anterior compartments of the mature somites, which was also shown for the chicken l-fng gene. The latter expression indicates that it might be involved in boundary definition and cell fate decision processes, rather than in pre-patterning of the somites. Interestingly, a similar role has previously been inferred for the grasshopper homologue of l-fng. This suggests that the function of l-fng in boundary definition of the somites might be ancestral, while its recruitment to the pre-patterning process of the somites might be a derived feature in higher vertebrates.     

Combinatorial gene regulation by Bmp and Wnt in zebrafish posterior mesoderm formation

Combinatorial signaling is an important mechanism that allows the embryo to utilize overlapping signaling pathways to specify different territories. In zebrafish, the Wnt and Bmp pathways interact to regulate the formation of the posterior body. In order to understand how this works mechanistically, we have identified tbx6 as a posterior mesodermal gene activated by both of these signaling pathways. We isolated a genomic fragment from the tbx6 gene that recapitulates the endogenous tbx6 expression, and used this to ask how the Bmp and Wnt signaling pathways combine to regulate gene expression. We find that the tbx6 promoter utilizes distinct domains to integrate the signaling inputs from each pathway, including multiple Tcf/LEF sites and a novel Bmp-response element. Surprisingly, we found that overexpression of either signaling pathway can activate the tbx6 promoter and the endogenous gene, whereas inputs from both pathways are required for the normal pattern of expression. These results demonstrate that both Bmp and Wnt are present at submaximal levels, which allows the pathways to function combinatorially. We present a model in which overlapping Wnt and Bmp signals in the ventrolateral region activate the expression of tbx6 and other posterior mesodermal genes, leading to the formation of posterior structures.     

From dynamic expression patterns to boundary formation in the presomitic mesoderm

The segmentation of the vertebrate body is laid down during early embryogenesis. The formation of signaling gradients, the periodic expression of genes of the Notch-, Fgf- and Wnt-pathways and their interplay in the unsegmented presomitic mesoderm (PSM) precedes the rhythmic budding of nascent somites at its anterior end, which later develops into epithelialized structures, the somites. Although many in silico models describing partial aspects of somitogenesis already exist, simulations of a complete causal chain from gene expression in the growth zone via the interaction of multiple cells to segmentation are rare. Here, we present an enhanced gene regulatory network (GRN) for mice in a simulation program that models the growing PSM by many virtual cells and integrates WNT3A and FGF8 gradient formation, periodic gene expression and Delta/Notch signaling. Assuming Hes7 as core of the somitogenesis clock and LFNG as modulator, we postulate a negative feedback of HES7 on Dll1 leading to an oscillating Dll1 expression as seen in vivo. Furthermore, we are able to simulate the experimentally observed wave of activated NOTCH (NICD) as a result of the interactions in the GRN. We esteem our model as robust for a wide range of parameter values with the Hes7 mRNA and protein decays exerting a strong influence on the core oscillator. Moreover, our model predicts interference between Hes1 and HES7 oscillators when their intrinsic frequencies differ. In conclusion, we have built a comprehensive model of somitogenesis with HES7 as core oscillator that is able to reproduce many experimentally observed data in mice.     

Cooperation of polarized cell intercalations drives convergence and extension of presomitic mesoderm during zebrafish gastrulation

During vertebrate gastrulation, convergence and extension (C&E) movements narrow and lengthen the embryonic tissues, respectively. In zebrafish, regional differences of C&E movements have been observed; however, the underlying cell behaviors are poorly understood. Using time-lapse analyses and computational modeling, we demonstrate that C&E of the medial presomitic mesoderm is achieved by cooperation of planar and radial cell intercalations. Radial intercalations preferentially separate anterior and posterior neighbors to promote extension. In knypek;trilobite noncanonical Wnt mutants, the frequencies of cell intercalations are altered and the anteroposterior bias of radial intercalations is lost. This provides evidence for noncanonical Wnt signaling polarizing cell movements between different mesodermal cell layers. We further show using fluorescent fusion proteins that during dorsal mesoderm C&E, the noncanonical Wnt component Prickle localizes at the anterior cell edge, whereas Dishevelled is enriched posteriorly. Asymmetrical localization of Prickle and Dishevelled to the opposite cell edges in zebrafish gastrula parallels their distribution in fly, and suggests that noncanonical Wnt signaling defines distinct anterior and posterior cell properties to bias cell intercalations.     

Collagen and collagenase gene expression in three-dimensional collagen lattices are differentially regulated by alpha 1 beta 1 and alpha 2 beta 1 integrins

The reorganization of extracellular matrix (ECM) is an important function in many biological and pathophysiological processes. Culture of fibroblasts in a three-dimensional collagenous environment represents a suitable system to study the underlying mechanisms resulting from cell-ECM interaction, which leads to reprogramming of fibroblast biosynthetic capacity. The aim of this study was to identify receptors that transduce ECM signals into cellular events, resulting in reprogramming of connective tissue metabolism. Our data demonstrate that in human skin fibroblasts alpha 1 beta 1 and alpha 2 beta 1 integrins are the major receptors responsible for regulating ECM remodeling: alpha 1 beta 1 mediates the signals inducing downregulation of collagen gene expression, whereas the alpha 2 beta 1 integrin mediates induction of collagenase (MMP-1). Applying mAb directed against different integrin subunits resulted in triggering the heterodimeric receptors and enhancing the normal biochemical response to receptor ligation. Different signal transduction inhibitors were tested for their influence on gel contraction, expression of alpha 1(I) collagen and MMP-1 in fibroblasts within collagen gels. Ortho-vanadate and herbimycin A displayed no significant effect on any of these three processes. In contrast, genistein reduced lattice contraction, and completely inhibited induction of MMP-1, whereas type I collagen down- regulation was unaltered. Calphostin C inhibited only lattice contraction. Taken together, these data indicate a role of tyrosine- specific protein kinases in mediating gel contraction and induction of MMP-1, as well as an involvement of protein kinase C in the contraction process. The data presented here indicate that different signaling pathways exist leading to the three events discussed here, and that these pathways do not per se depend upon each other.     

Endoderm-derived Sonic hedgehog and mesoderm Hand2 expression are required for enteric nervous system development in zebrafish

The zebrafish enteric nervous system (ENS), like those of all other vertebrate species, is principally derived from the vagal neural crest cells (NCC). The developmental controls that govern the migration, proliferation and patterning of the ENS precursors are not well understood. We have investigated the roles of endoderm and Sonic hedgehog (SHH) in the development of the ENS. We show that endoderm is required for the migration of ENS NCC from the vagal region to the anterior end of the intestine. We show that the expression of shh and its receptor ptc-1 correlate with the development of the ENS and demonstrate that hedgehog (HH) signaling is required in two phases, a pre-enteric and an enteric phase, for normal ENS development. We show that HH signaling regulates the proliferation of vagal NCC and ENS precursors in vivo. We also show the zebrafish hand2 is required for the normal development of the intestinal smooth muscle and the ENS. Furthermore we show that endoderm and HH signaling, but not hand2, regulate gdnf expression in the intestine, highlighting a central role of endoderm and SHH in patterning the intestine and the ENS.     

Discovering tightly regulated and differentially expressed gene sets in whole genome expression data

MOTIVATION: Recently, a new type of expression data is being collected which aims to measure the effect of genetic variation on gene expression in pathways. In these datasets, expression profiles are constructed for multiple strains of the same model organism under the same condition. The goal of analyses of these data is to find differences in regulatory patterns due to genetic variation between strains, often without a phenotype of interest in mind. We present a new method based on notions of tight regulation and differential expression to look for sets of genes which appear to be significantly affected by genetic variation. RESULTS: When we use categorical phenotype information, as in the Alzheimer's and diabetes datasets, our method finds many of the same gene sets as gene set enrichment analysis. In addition, our notion of correlated gene sets allows us to focus our efforts on biological processes subjected to tight regulation. In murine hematopoietic stem cells, we are able to discover significant gene sets independent of a phenotype of interest. Some of these gene sets are associated with several blood-related phenotypes. AVAILABILITY: The programs are available by request from the authors.     

Cell coherence during production of the presomitic mesoderm and somitogenesis in the mouse embryo

In this study, we investigated (in the early mouse embryo) the clonal properties of precursor cells which contribute to the segmented myotome, a structure derived from the somites. We used the laacZ method of single cell-labelling to visualise clones born before segmentation and bilateralisation. We found that clones which contribute to several segments both unilateral and bilateral were regionalised along the mediolateral axis and that their mediolateral position was maintained in successive adjacent segments. Furthermore, clones contributed to all segments, from their most anterior to their most posterior borders. Therefore, it appears that mediolateral regionalisation of myotomal precursor cells is a property established before bilateralisation of the presomitic mesoderm and that coherent clonal growth accompanies cell dispersion along both the mediolateral and anteroposterior axes. These findings in the mouse correlate well with what is known in the chick, suggesting conservation of the mode of production and distribution of the cells of the presomitic mesoderm. However, in addition, we also found that the mediolateral contribution of a clone is already determined in the pool of self-renewing cells that produces the myotomal precursor cells and thus that this pool is itself regionalised. Finally, we found that bilateral clones exhibit symmetry in right and left sides in the embryo at all levels of the mediolateral axis of the myotome. All these properties indicate synchrony and symmetry of formation of the presomitic mesoderm on both sides of the embryo leading to formation of a static embryonic structure with few cell movements. We suggest that sequential production of groups of cells with an identical clonal origin for both sides of the embryo from a single pool of self-renewing cells, coupled with acquisition of static cell behaviour, could play a role in colinearity of expression of Hox genes and in the segmentation system of higher vertebrates.     

Transgenic zebrafish reveal stage-specific roles for Bmp signaling in ventral and posterior mesoderm development

Bone morphogenetic protein (Bmp) signaling is crucial for the formation and patterning of zebrafish ventral and posterior mesoderm. Mutants defective in the Bmp pathway have expanded trunk muscle, abnormal tails and severely impaired development of ventral mesodermal derivatives such as vasculature, blood and pronephros. As Bmps continue to be expressed in the ventral and posterior mesoderm after gastrulation, it is likely that Bmp signaling continues to play an important developmental role during outgrowth of the posterior body. However, because Bmp signaling plays an essential role during the gastrula stages, it has not been possible with mutants or standard disruption techniques to determine the later functions of the Bmp pathway. To study the role of Bmp signaling in the ventral and posterior mesoderm during trunk and tail outgrowth, we generated a transgenic zebrafish line containing a heatshock-inducible dominant-negative Bmp receptor-GFP fusion. Our data show that Bmps are important for tail organizer formation and for patterning the ventral mesoderm during early gastrulation. However, from mid-gastrulation to the early somitogenesis stages, Bmp signaling is important for ventral tail fin development and for preventing secondary tail formation. We conclude that the role of Bmp signaling in the ventral and posterior mesoderm changes as gastrulation proceeds.