HairyE/(spl)-related (Her) genes are central components of the segmentation oscillator and display redundancy with the Delta/Notch signaling pathway in the formation of anterior segmental boundaries in the zebrafish

We have examined the expression of a Hairy/E(spl)-related (Her) gene, her7, in the zebrafish and show that its expression in the PSM cycles similarly to her1 and deltaC. A decrease in her7 function generated by antisense oligonucleotides disrupts somite formation in the posterior trunk and tail, and disrupts the dynamic expression domains of her1 and deltaC, suggesting that her7 plays a role in coordinating the oscillations of neighboring cells in the presomitic mesoderm. This phenotype is reminiscent of zebrafish segmentation mutants with lesions in genes of the Delta/Notch signaling pathway, which also show a disruption of cyclic her7 expression. The interaction of HER genes with the Delta/Notch signaling system was investigated by introducing a loss of her7 function into mutant backgrounds. This leads to segmental defects more anterior than in either condition alone. Combining a decrease of her7 function with reduction of her7 function results in an enhanced phenotype that affects all the anterior segments, indicating that Her functions in the anterior segments are also partially redundant. In these animals, gene expression does not cycle at any time, suggesting that a complete loss of oscillator function had been achieved. Consistent with this, combining a reduction of her7 and her1 function with a Delta/Notch mutant genotype does not worsen the phenotype further. Thus, our results identify members of the Her family of transcription factors that together behave as a central component of the oscillator, and not as an output. This indicates, therefore, that the function of the segmentation oscillator is restricted to the positioning of segmental boundaries. Furthermore, our data suggest that redundancy between Her genes and genes of the Delta/Notch pathway is in part responsible for the robust formation of anterior somites in vertebrates.

Coordination of symmetric cyclic gene expression during somitogenesis by Suppressor of Hairless involves regulation of retinoic acid catabolism

Andrew Charles Oates

Vertebrate embryos faithfully produce bilaterally symmetric somites that give rise to repetitive body structures such as vertebrae and skeletal muscle. Body segmentation is regulated by a cyclic gene expression system, containing the Delta-Notch pathway and targets, which generates bilaterally symmetric oscillations across the Pre-Somitic Mesoderm (PSM). The position of the forming somite boundary is controlled by interaction of this oscillator with a determination front comprised of opposing gradients of FGF and retinoic acid (RA) signalling. Disruption of RA production leads to asymmetries in cyclic gene expression, but the link between RA and the oscillator is unknown. In somitogenesis, Notch signalling activates target genes through the transcription factor Suppressor of Hairless (Su(H)). Here, we report that two Su(H) genes coordinate bilaterally symmetric positioning of somite boundaries in the zebrafish embryo. Combined Su(H) gene knockdown caused defects in visceral left/right asymmetry, neurogenic lateral inhibition, and symmetrical failure of the segmentation oscillator. However, by selectively down-regulating Su(H)2 or Su(H)1 function using specific antisense morpholinos, we observed asymmetric defects in anterior or posterior somite boundaries, respectively. These morphological abnormalities were reflected by underlying asymmetric cyclic gene expression waves in the presomitic mesoderm, indicating a key role for Su(H) in coordinating the left-right symmetry of this process. Strikingly, expression of the RA-degrading enzyme cyp26a1 in the tailbud was controlled by Su(H) activity, and morpholino knockdown of cyp26a1 alone caused asymmetric cyclic dlc expression, suggesting that excess RA in the tailbud may contribute to the cyclic asymmetries. Indeed, exogenous RA was sufficient to generate asymmetric expression of all cyclic genes. Our observations indicate that one element of the Notch signalling pathway, Su(H), is required for control of RA metabolism in the tailbud and that this regulation is involved in bilateral symmetry of cyclic gene expression and somitogenesis. © 2006 Elsevier Inc. All rights reserved.

2007

Generation of segment polarity in the paraxial mesoderm of the zebrafish through a T-box-dependent inductive event

Andrew Charles Oates, Laurel Ann Rohde

The first morphological sign of vertebrate postcranial body segmentation is the sequential production from posterior paraxial mesoderm of blocks of cells termed somites. Each of these embryonic structures is polarized along the anterior/posterior axis, a subdivision first distinguished by marker gene expression restricted to rostral or caudal territories of forming somites. To better understand the generation of segment polarity in vertebrates, we have studied the zebrafish mutant fused somites (fss), because its paraxial mesoderm lacks segment polarity. Previously examined markers of caudal half-segment identity are widely expressed, whereas markers of rostral identity are either missing or dramatically down-regulated, suggesting that the paraxial mesoderm of the fss mutant embryo is profoundly caudalized. These findings gave rise to a model for the formation of segment polarity in the zebrafish in which caudal is the default identity for paraxial mesoderm, upon which is patterned rostral identity in an fss-dependent manner. In contrast to this scheme, the caudal marker gene ephrinA1 was recently shown to be down-regulated in fss embryos. We now show that notch5, another caudal identity marker and a component of the Delta/Notch signaling system, is not expressed in the paraxial mesoderm of early segmentation stage fss embryos. We use cell transplantation to create genetic mosaics between fss and wild-type embryos in order to assay the requirement for fss function in notch5 expression. In contrast to the expression of rostral markers, which have a cell-autonomous requirement for fss, expression of notch5 is induced in fss cells at short range by nearby wild-type cells, indicating a cell-non-autonomous requirement for fss function in this process. These new data suggest that segment polarity is created in a three-step process in which cells that have assumed a rostral identity must subsequently communicate with their partially caudalized neighbors in order to induce the fully caudalized state. © 2005 Elsevier Inc. All rights reserved.

2005

Assessing the role of Hes1 and identification of target genes in human and murine T-ALL

Silvia Wirth

The Notch signalling pathway is an ancient cell signalling mechanism that enables short-range communications between cells and controls a broad spectrum of cell fates and developmental processes. In the haematopoietic system Notch signalling has been linked to physiological, but also to pathological phenotypes of T cell development. Identification of numerous activating mutations within the NOTCH1 receptor in human T cell acute lymphoblastic leukaemia (T-ALL) patient samples demonstrated that aberrant activation of this signalling pathway is a frequent event in T-ALL. Active Notch signalling leads to the expression of a plethora of genes that might be drivers or passengers in tumorigenesis including Hes1, which encodes a transcriptional repressor. We have therefore studied Hes1 in the context of T-ALL in mouse models that mimic human disease and in human T-ALL cell lines with the aim to evaluate whether Hes1 affects disease development and disease maintenance and to characterize its function on a molecular level. The first part of this study is dedicated to the functional characterisation of Hes1 in vivo in T-ALL mouse models and in vitro in human T-ALL cell lines. Using a retroviral bone marrow transplant model of T-ALL in which progenitor cells are transduced with a retrovirus expressing constitutive active Notch1 intracellular domain (NICD) we could show that Hes1 is not required in established, late stage T-ALL cells, but that it promotes tumour progression in the early stages of the disease. We have also investigated the function of HES1 in human T-ALL and can show that the overexpression of dominant negative versions of Hes1 in two patient derived cell lines (T-ALL1, CUTLL-1) does not affect proliferation or apoptosis, emphasising that HES1 does not affect cell viability once cells are fully transformed to the leukemic state. In the second part of the study we set out to systematically delineate the gene regulatory networks down-stream of Hes1 by combining RNA-seq and ChIP-seq analysis. Comparing the gene expression of murine premalignant, early stage NICD induced CD4+CD8+ DP cells in the presence and absence of Hes1 by RNA-seq, we unexpectedly found a predominant upregulation of genes in cells expressing Hes1 indicating that Hes1 might rather act as a positive regulator of transcription than as a transcriptional repressor of distinct target genes. Hes1 might influence transcription through secondary or indirect effects as combinatorial analysis of RNA-seq and ChIP-seq led to the identification of only a small number of potential direct target genes that are transcriptionally regulated by Hes1. In summary, the role of Hes1 in promoting the early stages of the disease appears to be highly complex and is likely to involve both direct and indirect control of gene regulatory networks.

EPFL2014