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The blood system is sustained by a pool of haematopoietic stem cells (HSCs) that are long-lived due to their capacity for self-renewal. A consequence of longevity is exposure to stress stimuli including reactive oxygen species (ROS), nutrient fluctuation and DNA damage (1,2). Damage that occurs within stressed HSCs must be tightly controlled to prevent either loss of function or the clonal persistence of oncogenic mutations that increase the risk of leukaemogenesis (3,4). Despite the importance of maintaining cell integrity throughout life, how the HSC pool achieves this and how individual HSCs respond to stress remain poorly understood. Many sources of stress cause misfolded protein accumulation in the endoplasmic reticulum (ER), and subsequent activation of the unfolded protein response (UPR) enables the cell to either resolve stress or initiate apoptosis (5,6). Here we show that human HSCs are predisposed to apoptosis through strong activation of the PERK branch of the UPR after ER stress, whereas closely related progenitors exhibit an adaptive response leading to their survival. Enhanced ER protein folding by overexpression of the cochaperone ERDJ4 (also called DNAJB9) increases HSC repopulation capacity in xenograft assays, linking the UPR to HSC function. Because the UPR is a focal point where different sources of stress converge, our study provides a framework for understanding how stress signalling is coordinated within tissue hierarchies and integrated with stemness. Broadly, these findings reveal that the HSC pool maintains clonal integrity by clearance of individual HSCs after stress to prevent propagation of damaged stem cells., The human haematopoietic hierarchy has recently been delineated at the single-cell level, enabling precise isolation of HSCs and progenitor cells (7-9) (Extended Data Table 1). Pathway analysis using gene expression [...]

TP53 is the most frequently mutated tumor suppressor gene in human cancer, with nearly 50% of all tumors exhibiting a loss-of-function mutation. To further elucidate the genetic pathways involving TP53 and cancer, we have exploited the zebrafish, a powerful vertebrate model system that is amenable to whole-genome forward-genetic analysis and synthetic-lethal screens. Zebrafish lines harboring missense mutations in the tp53 DNA-binding domain were identified by using a target-selected mutagenesis strategy. Homozygous mutant fish from two of these lines were viable and exhibited mutations similar to those found in human cancers ([tp53.sup.N168K] and [tp53.sup.M214K]). Although homozygous [tp53.sup.N168K] mutants were temperature-sensitive and suppressed radiation-induced apoptosis only at 37[degrees]C, cells in the [tp53.sup.M214K] embryos failed to undergo apoptosis in response to [gamma] radiation at both 28 and 37[degrees]C. Unlike wild-type control embryos, irradiated [tP53.sup.M214K] embryos also failed to up-regulate p21 and did not arrest at the [G.sub.1]/S checkpoint. Beginning at 8.5 months of age, 28% of [tp53.sup.M214K] mutant fish developed malignant peripheral nerve sheath tumors. In addition to providing a model for studying the molecular pathogenic pathways of malignant peripheral nerve sheath tumors, these mutant zebrafish lines provide a unique platform for modifier screens to identify genetic mutations or small molecules that affect tp53-related pathways, including apoptosis, cell-cycle delay, and tumor suppression.

Author(s): Adam F. L. Hurlstone [1, 2]; Anna-Pavlina G. Haramis [1, 2]; Erno Wienholds [1]; Harry Begthel [1]; Jeroen Korving [1]; Fredericus van Eeden [1]; Edwin Cuppen [1]; Danica Zivkovic [...]

The zebrafish has become a favorite organism for genetic analysis of vertebrate development, but methods for generating mutants by reverse genetic approaches have been lacking. We report a method to obtain stable mutants of a gene based on knowledge of the gene sequence only. Parental fish were mutagenized with N-ethyl-N-nitrosourea; in 2679 [F.sub.1] fish, the rag1 gene was analyzed for heterozygous mutations by resequencing. In total, we found 15 mutations: 9 resulted in amino acid substitutions and 1 resulted in a premature stop codon. This truncation mutant was found to be homozygous viable and defective in V(D)J joining. Although presumably immune deficient, these homozygous rag1 mutant fish are able to reach adulthood and are fertile. As sperm samples from all 2679 [F.sub.1] fish were collected and cryopreserved, we have in principle generated a mutant library from which mutants of most zebrafish genes can be isolated., Forward genetic screens in the zebrafish have identified thousands of mutants defective in many kinds of biological processes (1, 2). Cloning of the affected genes is accelerated by both an [...]

Tc1/mariner elements are able to transpose in species other than the host from which they were isolated. As potential vectors for insertional mutagenesis and transgenesis of the mouse, these cut-and-paste transposons were tested for their ability to transpose in the mouse germ line. First, the levels of activity of several Tc1/mariner elements in mammalian cells were compared; the reconstructed fish transposon Sleeping Beauty (SB) was found to be an order of magnitude more efficient than the other tested transposons. SB then was introduced into the mouse germ line as a two-component system: one transgene for the expression of the transposase in the male germ line and a second transgene carrying a modified transposon. In 20% of the progeny of double transgenic male mice the transposon had jumped from the original chromosomal position into another locus. Analysis of the integration sites shows that these jumps indeed occurred through the action of SB transposase, and that SB has a strong preference for intrachromosomal transposition. Analysis of the excision sites suggests that double-strand breaks in haploid spermatids are repaired via non-homologous end joining. The SB system may be a powerful tool for transposon mutagenesis of the mouse germ line.

MicroRNAs (miRNAs) are small noncoding RNAs, about 21 nucleotides in length, that can regulate gene expression by base-pairing to partially complementary mRNAs. Regulation by miRNAs can play essential roles in embryonic development. We determined the temporal and spatial expression patterns of 115 conserved vertebrate miRNAs in zebrafish embryos by microarrays and by in situ hybridizations, using locked-nucleic acid-modified oligonudeotide probes. Most miRNAs were expressed in a highly tissue-specific manner during segmentation and later stages, but not early in development, which suggests that their role is not in tissue fate establishment but in differentiation or maintenance of tissue identity., Current estimates of miRNA gene numbers in vertebrates are as high as 500 (1), of which many are conserved, and miRNAs may regulate up to 30% of genes (2). The [...]