Live imaging of Runx1 expression in the dorsal aorta tracks the emergence of blood progenitors from endothelial cells.

Blood cells of an adult vertebrate are continuously generated by hematopoietic stem cells (HSCs) that originate during embryonic life within the aorta-gonad-mesonephros region. There is now compelling in vivo evidence that HSCs are generated from aortic endothelial cells and that this process is critically regulated by the transcription factor Runx1. By time-lapse microscopy of Runx1-enhanced green fluorescent protein transgenic zebrafish embryos, we were able to capture a subset of cells within the ventral endothelium of the dorsal aorta, as they acquire hemogenic properties and directly emerge as presumptive HSCs. These nascent hematopoietic cells assume a rounded morphology, transiently occupy the subaortic space, and eventually enter the circulation via the caudal vein. Cell tracing showed that these cells subsequently populated the sites of definitive hematopoiesis (thymus and kidney), consistent with an HSC identity. HSC numbers depended on activity of the transcription factor Runx1, on blood flow, and on proper development of the dorsal aorta (features in common with mammals). This study captures the earliest events of the transition of endothelial cells to a hemogenic endothelium and demonstrates that embryonic hematopoietic progenitors directly differentiate from endothelial cells within a living organism.

Zebrafish runx1 promoter-EGFP transgenics mark discrete sites of definitive blood progenitors.

The transcription factor Runx1 is essential for the development of definitive hematopoietic stem cells (HSCs) during vertebrate embryogenesis and is transcribed from 2 promoters, P1 and P2, generating 2 major Runx1 isoforms. We have created 2 stable runx1 promoter zebrafish-transgenic lines that provide insight into the roles of the P1 and P2 isoforms during the establishment of definitive hematopoiesis. The Tg(runx1P1:EGFP) line displays fluorescence in the posterior blood island, where definitive erythromyeloid progenitors develop. The Tg(runx1P2:EGFP) line marks definitive HSCs in the aorta-gonad-mesonephros, with enhanced green fluorescent protein-labeled cells later populating the pronephros and thymus. This suggests that a function of runx1 promoter switching is associated with the establishment of discrete definitive blood progenitor compartments. These runx1 promoter-transgenic lines are novel tools for the study of Runx1 regulation and function in normal and malignant hematopoiesis. The ability to visualize and isolate fluorescently labeled HSCs should contribute to further elucidating the complex regulation of HSC development.

G-quadruplex structures are stable and detectable in human genomic DNA.

The G-quadruplex is an alternative DNA structural motif that is considered to be functionally important in the mammalian genome for transcriptional regulation, DNA replication and genome stability, but the nature and distribution of G-quadruplexes across the genome remains elusive. Here, we address the hypothesis that G-quadruplex structures exist within double-stranded genomic DNA and can be explicitly identified using a G-quadruplex-specific probe. An engineered antibody is employed to enrich for DNA containing G-quadruplex structures, followed by deep sequencing to detect and map G-quadruplexes at high resolution in genomic DNA from human breast adenocarcinoma cells. Our high sensitivity structure-based pull-down strategy enables the isolation of genomic DNA fragments bearing single, as well as multiple G-quadruplex structures. Stable G-quadruplex structures are found in sub-telomeres, gene bodies and gene regulatory regions. For a sample of identified target genes, we show that G-quadruplex-stabilizing ligands can modulate transcription. These results confirm the existence of G-quadruplex structures and their persistence in human genomic DNA.

Osteogenic transcription factor Runx2 is a maternal determinant of dorsoventral patterning in zebrafish.

The maternal genome greatly influences vertebrate embryogenesis before activation of zygotic transcription. Dorsoventral patterning is initiated by maternal factors, but the molecular pathways involved are incompletely understood. In frogs and fish, localized zygotic domains are induced whereby cells either express dorsal or ventral genes. Wnt/beta-catenin signalling promotes expression of the earliest dorsal zygotic genes. Among key zygotic ventralizing factors are the bone morphogenetic proteins (Bmps) and the Vent homeodomain family; the latter act as repressors of dorsal organizer gene transcription. Here we show that Runx2, a transcription factor essential for bone formation, is an important maternal determinant of ventral zygotic genes in zebrafish. Depletion of maternal Runx2b type2 strongly dorsalizes embryos, due to loss of the earliest zygotic expression of vox, vent and ved, resulting in expansion of dorsal gene expression. To date, Runx2b is the only known regulator of vox, vent and ved at the onset of zygotic transcription; we show that this regulation is direct. Runx2 transcripts are processed in mature mouse oocytes and we show that murine Runx2 type2 can substitute for the zebrafish orthologue in its ventralizing function, suggesting that Runx2 may have an evolutionarily conserved role in axis formation.

A hierarchy of Runx transcription factors modulate the onset of chondrogenesis in craniofacial endochondral bones in zebrafish.

The Runx (runt-related) family of transcription factors are important regulators of cell fate decisions in early embryonic development, and in differentiation of tissues including blood, neurons, and bone. During skeletal development in mammals, while only Runx2 is essential for osteoblast differentiation, all family members seem to be involved in chondrogenesis. Runx2 and Runx3 control chondrocyte maturation. Both Runx1 and Runx2 are expressed early in mesenchymal condensations, but how they contribute to the initial stages of chondrocyte differentiation is unclear. Here we show that a hierarchy of Runx transcriptional regulation promotes the early program of chondrocyte differentiation from pre-cartilage mesenchyme in the zebrafish head skeleton. We have previously characterized the zebrafish orthologs for all Runx genes. Zebrafish runx2 is duplicated, but not runx1 or runx3. In the work presented here, we determined the early expression pattern of the runx genes in the craniofacial region. The earliest expression detected was that of runx3 in the pharyngeal endoderm, then runx2a and b in mesenchymal condensations, and later runx1 in the epithelium. Using antisense morpholino knockdown analysis, we examined their respective activities in early chondrogenesis. Depletion of runx2b (but not runx2a) and runx3 severely compromised craniofacial cartilage formation. Because runx2b expression was abolished in Runx3 morphants, we propose that endodermal Runx3 has a role in influencing signaling activities from the endoderm to promote chondrocyte differentiation. We also show that, in contrast to data from mouse studies, zebrafish Runx1 is not required in the initial steps of chondrogenesis leading to endochondral bone formation.