Analysis of Jak2 signaling reveals resistance of mouse embryonic hematopoietic stem cells to myeloproliferative disease mutation.

The regulation of hematopoietic stem cell (HSC) emergence during development provides important information about the basic mechanisms of blood stem cell generation, expansion, and migration. We set out to investigate the role that cytokine signaling pathways play in these early processes and show here that the 2 cytokines interleukin 3 and thrombopoietin have the ability to expand hematopoietic stem and progenitor numbers by regulating their survival and proliferation. For this, they differentially use the Janus kinase (Jak2) and phosphatidylinositol 3-kinase (Pi3k) signaling pathways, with Jak2 mainly relaying the proproliferation signaling, whereas Pi3k mediates the survival signal. Furthermore, using Jak2-deficient embryos, we demonstrate that Jak2 is crucially required for the function of the first HSCs, whereas progenitors are less dependent on Jak2. The JAK2V617F mutation, which renders JAK2 constitutively active and has been linked to myeloproliferative neoplasms, was recently shown to compromise adult HSC function, negatively affecting their repopulation and self-renewal ability, partly through the accumulation of JAK2V617F-induced DNA damage. We report here that nascent HSCs are resistant to the JAK2V617F mutation and show no decrease in repopulation or self-renewal and no increase in DNA damage, even in the presence of 2 mutant copies. More importantly, this unique property of embryonic HSCs is stably maintained through ≥1 round of successive transplantations. In summary, our dissection of cytokine signaling in embryonic HSCs has uncovered unique properties of these cells that are of clinical importance.

Dlk1 is a negative regulator of emerging hematopoietic stem and progenitor cells.

The first mouse adult-repopulating hematopoietic stem cells emerge in the aorta-gonad-mesonephros region at embryonic day (E) 10.5. Their numbers in this region increase thereafter and begin to decline at E12.5, thus pointing to the possible existence of both positive and negative regulators of emerging hematopoietic stem cells. Our recent expression analysis of the aorta-gonad-mesonephros region showed that the Delta-like homologue 1 (Dlk1) gene is up-regulated in the region of the aorta-gonad-mesonephros where hematopoietic stem cells are preferentially located. To analyze its function, we studied Dlk1 expression in wild-type and hematopoietic stem cell-deficient embryos and determined hematopoietic stem and progenitor cell activity in Dlk1 knockout and overexpressing mice. Its role in hematopoietic support was studied in co-culture experiments using stromal cell lines that express varying levels of Dlk1. We show here that Dlk1 is expressed in the smooth muscle layer of the dorsal aorta and the ventral sub-aortic mesenchyme, where its expression is dependent on the hematopoietic transcription factor Runx1. We further demonstrate that Dlk1 has a negative impact on hematopoietic stem and progenitor cell activity in the aorta-gonad-mesonephros region in vivo, which is recapitulated in co-cultures of hematopoietic stem cells on stromal cells that express varying levels of Dlk1. This negative effect of Dlk1 on hematopoietic stem and progenitor cell activity requires the membrane-bound form of the protein and cannot be recapitulated by soluble Dlk1. Together, these data suggest that Dlk1 expression by cells of the aorta-gonad-mesonephros hematopoietic microenvironment limits hematopoietic stem cell expansion and is, to our knowledge, the first description of such a negative regulator in this tissue.

Signaling from the sympathetic nervous system regulates hematopoietic stem cell emergence during embryogenesis.

The first adult-repopulating hematopoietic stem cells (HSCs) emerge in the aorta-gonads-mesonephros (AGM) region of the embryo. We have recently identified the transcription factor Gata3 as being upregulated in this tissue specifically at the time of HSC emergence. We now demonstrate that the production of functional and phenotypic HSCs in the AGM is impaired in the absence of Gata3. Furthermore, we show that this effect on HSC generation is secondary to the role of Gata3 in the production of catecholamines, the mediators of the sympathetic nervous system (SNS), thus making these molecules key components of the AGM HSC niche. These findings demonstrate that the recently described functional interplay between the hematopoietic system and the SNS extends to the earliest stages of their codevelopment and highlight the fact that HSC development needs to be viewed in the context of the development of other organs.

Zebrafish based assays for the assessment of cardiac, visual and gut function--potential safety screens for early drug discovery.

INTRODUCTION: Safety pharmacology is integral to the non-clinical safety assessment of new chemical entities prior to first administration to humans. The zebrafish is a well established model organism that has been shown to be relevant to the study of human diseases. The potential role of zebrafish in safety pharmacology was evaluated using reference compounds in three models assessing cardiac, visual and intestinal function. METHODS: Compound toxicity was first established in zebrafish to determine the non toxic concentration of a blinded set of 16 compounds. In the cardiac assay, zebrafish larvae at 3 days post fertilisation (d.p.f.) were exposed to compounds for 3 h before measurement of the atrial and ventricular rates. To investigate visual function, the optomotor response was assessed in 8 d.p.f. larvae following a 5 day compound exposure. In the intestinal function assay, the number of gut contractions was measured in 7 d.p.f. larvae after a 1 h compound exposure. Finally, compound uptake was determined for 9 of the 16 compounds to measure the concentration of compound absorbed by the zebrafish larvae. RESULTS: Seven compounds out of nine produced an expected effect that was statistically significant in the cardiac and visual functions assays. In the gut contraction assay, six out of ten compounds showed a statistically significant effect that was also the expected result whilst two displayed anticipated but non-significant effects. The compound uptake method was used to determine larval tissue concentrations and allowed the identification of false negatives when compound was poorly absorbed into the zebrafish. DISCUSSION: Overall, results generated in three zebrafish larvae assays demonstrated a good correlation between the effects of compounds in zebrafish and the data available from other in vivo models or known clinical adverse effects. These results suggest that for the cardiac, intestinal and visual function, zebrafish assays have the potential to predict adverse drug effects and supports their possible role in early safety assessment of novel compounds.