Blood stem cell-forming haemogenic endothelium in zebrafish derives from arterial endothelium.

Haematopoietic stem cells are generated from the haemogenic endothelium (HE) located in the floor of the dorsal aorta (DA). Despite being integral to arteries, it is controversial whether HE and arterial endothelium share a common lineage. Here, we present a transgenic zebrafish runx1 reporter line to isolate HE and aortic roof endothelium (ARE)s, excluding non-aortic endothelium. Transcriptomic analysis of these populations identifies Runx1-regulated genes and shows that HE initially expresses arterial markers at similar levels to ARE. Furthermore, runx1 expression depends on prior arterial programming by the Notch ligand dll4. Runx1-/- mutants fail to downregulate arterial genes in the HE, which remains integrated within the DA, suggesting that Runx1 represses the pre-existing arterial programme in HE to allow progression towards the haematopoietic fate. These findings strongly suggest that, in zebrafish, aortic endothelium is a precursor to HE, with potential implications for pluripotent stem cell differentiation protocols for the generation of transplantable HSCs.

Transforming Growth Factor ß Drives Hemogenic Endothelium Programming and the Transition to Hematopoietic Stem Cells.

Hematopoietic stem cells (HSCs) are self-renewing multipotent stem cells that generate mature blood lineages throughout life. They, together with hematopoietic progenitor cells (collectively known as HSPCs), emerge from hemogenic endothelium in the floor of the embryonic dorsal aorta by an endothelial-to-hematopoietic transition (EHT). Here we demonstrate that transforming growth factor ß (TGFß) is required for HSPC specification and that it regulates the expression of the Notch ligand Jagged1a in endothelial cells prior to EHT, in a striking parallel with the epithelial-to-mesenchymal transition (EMT). The requirement for TGFß is two fold and sequential: autocrine via Tgfß1a and Tgfß1b produced in the endothelial cells themselves, followed by a paracrine input of Tgfß3 from the notochord, suggesting that the former programs the hemogenic endothelium and the latter drives EHT. Our findings have important implications for the generation of HSPCs from pluripotent cells in vitro.

Analysis of Dll4 regulation reveals a combinatorial role for Sox and Notch in arterial development.

The mechanisms by which arterial fate is established and maintained are not clearly understood. Although a number of signaling pathways and transcriptional regulators have been implicated in arterio-venous differentiation, none are essential for arterial formation, and the manner in which widely expressed factors may achieve arterial-specific gene regulation is unclear. Using both mouse and zebrafish models, we demonstrate here that arterial specification is regulated combinatorially by Notch signaling and SoxF transcription factors, via direct transcriptional gene activation. Through the identification and characterization of two arterial endothelial cell-specific gene enhancers for the Notch ligand Delta-like ligand 4 (Dll4), we show that arterial Dll4 expression requires the direct binding of both the RBPJ/Notch intracellular domain and SOXF transcription factors. Specific combinatorial, but not individual, loss of SOXF and RBPJ DNA binding ablates all Dll4 enhancer-transgene expression despite the presence of multiple functional ETS binding sites, as does knockdown of sox7;sox18 in combination with loss of Notch signaling. Furthermore, triple knockdown of sox7, sox18 and rbpj also results in ablation of endogenous dll4 expression. Fascinatingly, this combinatorial ablation leads to a loss of arterial markers and the absence of a detectable dorsal aorta, demonstrating the essential roles of SoxF and Notch, together, in the acquisition of arterial identity.

VEGFA-dependent and -independent pathways synergise to drive Scl expression and initiate programming of the blood stem cell lineage in Xenopus.

The first haematopoietic stem cells share a common origin with the dorsal aorta and derive from putative adult haemangioblasts in the dorsal lateral plate (DLP) mesoderm. Here we show that the transcription factor (TF) stem cell leukaemia (Scl/Tal1) is crucial for development of these adult haemangioblasts in Xenopus and establish the regulatory cascade controlling its expression. We show that VEGFA produced in the somites is required to initiate adult haemangioblast programming in the adjacent DLP by establishing endogenous VEGFA signalling. This response depends on expression of the VEGF receptor Flk1, driven by Fli1 and Gata2. Scl activation requires synergy between this VEGFA-controlled pathway and a VEGFA-independent pathway controlled by Fli1, Gata2 and Etv2/Etsrp/ER71, which also drives expression of the Scl partner Lmo2. Thus, the two ETS factors Fli1 and Etv6, which drives the VEGFA expression in both somites and the DLP, sit at the top of the adult haemangioblast gene regulatory network (GRN). Furthermore, Gata2 is initially activated by Fli1 but later maintained by another ETS factor, Etv2. We also establish that Flk1 and Etv2 act independently in the two pathways to Scl activation. Thus, detailed temporal, epistatic measurements of key TFs and VEGFA plus its receptor have enabled us to build a Xenopus adult haemangioblast GRN.

Uncoupling VEGFA functions in arteriogenesis and hematopoietic stem cell specification.

VEGFA signaling is critical for endothelial and hematopoietic stem cell (HSC) specification. However, blood defects resulting from perturbation of the VEGFA pathway are always accompanied by impaired vascular/arterial development. Because HSCs derive from arterial cells, it is unclear whether VEGFA directly contributes to HSC specification. This is an important question for our understanding of how HSCs are formed and for developing their production in vitro. Through knockdown of the regulator ETO2 in embryogenesis, we report a specific decrease in expression of medium/long Vegfa isoforms in somites. This leads to absence of Notch1 expression and failure of HSC specification in the dorsal aorta (DA), independently of vessel formation and arterial specification. Vegfa hypomorphs and isoform-specific (medium/long) morphants not only recapitulate this phenotype but also demonstrate that VEGFA short isoform is sufficient for DA development. Therefore, sequential, isoform-specific VEGFA signaling successively induces the endothelial, arterial, and HSC programs in the DA.

Tel1/ETV6 specifies blood stem cells through the agency of VEGF signaling.

The regulation of stem cell ontogeny is poorly understood. We show that the leukemia-associated Ets transcription factor, Tel1/ETV6, specifies the first hematopoietic stem cells (HSCs) in the dorsal aorta (DA). In contrast, Tel1/ETV6 has little effect on embryonic blood formation, further distinguishing the programming of the long- and short-term blood populations. Consistent with the notion of concordance of arterial and HSC programs, we show that Tel1/ETV6 is also required for the specification of the DA as an artery. We further show that Tel1/ETV6 acts by regulating the transcription of VegfA in both the lateral plate mesoderm and also in the somites. Exogenous VEGFA rescues Tel1/ETV6 morphants, and depletion of VEGFA or its receptor, Flk1, largely phenocopies Tel1/ETV6 depletion. Few such links between intrinsic and extrinsic programming of stem cells have been reported previously. Our data place Tel1/ETV6 at the apex of the genetic regulatory cascade leading to HSC production.

The basic helix-loop-helix transcription factor, Tal2, marks the lateral floor plate of the spinal cord in zebrafish.

Basic helix-loop-helix (bHLH) transcription factors play key roles in the development of the central nervous system. Here we report the isolation of a zebrafish gene that encodes a homologue of the mammalian bHLH transcription factor, Tal2. In zebrafish embryos, tal2, like its mammalian homologue, is strongly expressed in the diencephalon and the mesencephalon, with the latter expression located in post-mitotic cells of the tectum. However, in addition to this conserved brain expression, we also detect expression in the floor plate of the spinal cord. By the location of this expression relative to other genes expressed in the floor plate and by analysing expression in a selection of midline mutants, we reveal that tal2 is expressed within the lateral floor plate as opposed to the medial floor plate, and also in more dorsal cells which are distinct from motorneurons and depend on either sonic hedgehog signalling or a signal coming from the lateral floor plate. This is to our knowledge the first report of a gene expressed specifically in lateral cells of the floor plate in the spinal cord.

Structures of CUG repeats in RNA. Potential implications for human genetic diseases.

Triplet repeats that cause human genetic diseases have been shown to exhibit unusual compact structures in DNA, and in this paper we show that similar structures exist in shorter "normal length" CNG RNA. CUG and control RNAs were made chemically and by in vitro transcription. We find that "normal" short CUG RNAs migrate anomalously fast on non-denaturing gels, compared with control oligos of similar base composition. By contrast, longer tracts approaching clinically relevant lengths appear to form higher order structures. The CD spectrum of shorter tracts is similar to triplex and pseudoknot nucleic acid structures and different from classical hairpin spectra. A model is outlined that enables the base stacking features of poly(r(G-C))(2).poly(r(U)) or poly(d(G-C))(2).poly(d(T)) triplexes to be achieved, even by a single 15-mer.