Generation of a Gal4-dependent gene recombination and illuminating mouse

Cell labeling technologies, including the Cre/loxP system, are powerful tools in developmental biology. Although the conventional Cre/loxP system has been extensively used to label the expression of specific genes, it is less frequently used for labeling protein-protein interactions owing to technical difficulties. In the present study, we generated a new Gal4-dependent transgenic reporter mouse line that expressed Cre recombinase and a nearinfrared fluorescent protein, miRFP670. To examine whether this newly generated transgenic mouse line is applicable in labeling of protein-protein interaction, we used a previously reported transgenic mouse lines that express Notch1 receptor with its intracellular domain replaced with a yeast transcription factor, Gal4. Upon the binding of this artificial Notch1 receptor and endogenous Notch1 ligands, Gal4 would be cleaved from the cell membrane to induce expression of Cre recombinase and miRFP670. Indeed, we observed miRFP670 signal in the mouse embryos (embryonic day 14.5). In addition, we examined whether our Cre recombinase was functional by using another transgenic mouse line that express dsRed after Cre-mediated recombination. We observed dsRed signal in small intestine epithelial cells where Notch1 signal was suggested to be involved in the crypt stem cell maintenance, suggesting that our Cre recombinase was functional. As our newly generated mouse line required only the functioning of Gal4, it could be useful for labeling several types of molecular activities in vivo.

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

Molecular cloning, genetic mapping, and expression analysis of four zebrafish c/ebp genes

Andrew Charles Oates

The CCAAT/enhancer binding protein family (C/EBP) are transcription factors that play integral roles in the development and function of many organ systems, including hematopoietic cells, adipose tissues, and liver. We have identified and characterized putative zebrafish orthologs of mammalian C/EBP α, β, γ, and δ using low-stringency hybridization screening and computer searches of the GenBank EST database. c/ebpa and g were mapped within 1 cM of each other on linkage group (LG) 7, syntenic with human CEBPA and G genes on chromosome 19. c/ebpb was mapped to LG8, and c/ebpd was mapped to LG24, on the same LG as a recently identified unique c/ebp in zebrafish, c/ebp1. The mapping of these genes established new syntenic relationships between LG8 and human chromosome 20, extended existing synteny between LG7 and human chromosome 19, and confirmed the synteny between LG24 and human chromosome 8. In addition, these syntenies between zebrafish and human chromosomes are also conserved in the mouse genome. To characterize the expression of these genes, RNA in situ hybridization in embryos of wild type and a hematopoietic mutant, cloche, was performed. The results showed that zebrafish c/ebpa, b, g, and d were expressed in many embryonic tissues. c/ebpa and b were expressed in a subset of hematopoietic cells in a region consistent with myeloid expression. In addition, there was expression of c/ebpa and b in the liver and c/ebpa, b, and d in regions of the gastrointestinal tract. The expression of the c/ebps may serve as important markers for analysis of myelopoiesis, hepatic development, and other developmental processes in the future. © 2001 Elsevier Science B.V. All rights reserved.

2001

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

Silvia Wirth

EPFL2014