Martin Gering

The SCL gene specifies haemangioblast development from early mesoderm

The SCL gene encodes a basic helix–loop–helix (bHLH) transcription factor that is essential for the development of all haematopoietic lineages. SCL is also expressed in endothelial cells, but its function is not essential for specification of endothelial progenitors and the role of SCL in endothelial development is obscure. We isolated the zebrafish SCL homologue and show that it was co‐expressed in early mesoderm with markers of haematopoietic, endothelial and pronephric progenitors. Ectopic expression of SCL mRNA in zebrafish embryos resulted in overproduction of common haematopoietic and endothelial precursors, perturbation of vasculogenesis and concomitant loss of pronephric duct and somitic tissue. Notochord and neural tube formation were unaffected. These results provide the first evidence that SCL specifies formation of haemangioblasts, the proposed common precursor of blood and endothelial lineages. Our data also underline the striking similarities between the role of SCL in haematopoiesis/vasculogenesis and the function of other bHLH proteins in muscle and neural development.

Lmo2 and Scl/Tal1 convert non-axial mesoderm into haemangioblasts which differentiate into endothelial cells in the absence of Gata1.

The LIM domain protein Lmo2 and the basic helix-loop-helix transcription factor Scl/Tal1 are expressed in early haematopoietic and endothelial progenitors and interact with each other in haematopoietic cells. While loss-of-function studies have shown that Lmo2 and Scl/Tal1 are essential for haematopoiesis and angiogenic remodelling of the vasculature, gain-of-function studies have suggested an earlier role for Scl/Tal1 in the specification of haemangioblasts, putative bipotential precursors of blood and endothelium. In zebrafish embryos, Scl/Tal1 can induce these progenitors from early mesoderm mainly at the expense of the somitic paraxial mesoderm. We show that this restriction to the somitic paraxial mesoderm correlates well with the ability of Scl/Tal1 to induce ectopic expression of its interaction partner Lmo2. Co-injection of lmo2 mRNA with scl/tal1 dramatically extends its effect to head, heart, pronephros and pronephric duct mesoderm inducing early blood and endothelial genes all along the anteroposterior axis. Erythroid development, however, is expanded only into pronephric mesoderm, remaining excluded from head, heart and somitic paraxial mesoderm territories. This restriction correlates well with activation of gata1 transcription and co-injection of gata1 mRNA along with scl/tal1 and lmo2 induces erythropoiesis more broadly without ventralising or posteriorising the embryo. While no ectopic myeloid development from the Scl/Tal1-Lmo2-induced haemangioblasts was observed, a dramatic increase in the number of endothelial cells was found. These results suggest that, in the absence of inducers of erythroid or myeloid haematopoiesis, Scl/Tal1-Lmo2-induced haemangioblasts differentiate into endothelial cells.

Novel binding partners of Ldb1 are required for haematopoietic development.

Ldb1, a ubiquitously expressed LIM domain binding protein, is essential in a number of tissues during development. It interacts with Gata1, Tal1, E2A and Lmo2 to form a transcription factor complex regulating late erythroid genes. We identify a number of novel Ldb1 interacting proteins in erythroleukaemic cells, in particular the repressor protein Eto-2 (and its family member Mtgr1), the cyclin-dependent kinase Cdk9, and the bridging factor Lmo4. MO-mediated knockdowns in zebrafish show these factors to be essential for definitive haematopoiesis. In accordance with the zebrafish results these factors are coexpressed in prehaematopoietic cells of the early mouse embryo, although we originally identified the complex in late erythroid cells. Based on the change in subcellullar localisation of Eto-2 we postulate that it plays a central role in the transition from the migration and expansion phase of the prehaematopoietic cells to the establishment of definitive haematopoietic stem cells.

Hedgehog and Bmp Polarize Hematopoietic Stem Cell Emergence in the Zebrafish Dorsal Aorta

Hematopoietic stem cells (HSCs) are first detected in the floor of the embryonic dorsal aorta (DA), and we investigate the signals that induce the HSC program there. We show that while continued Hedgehog (Hh) signaling from the overlying midline structures maintains the arterial program characteristic of the DA roof, a ventral Bmp4 signal induces the blood stem cell program in the DA floor. This patterning of the DA by Hh and Bmp is the mirror image of that in the neural tube, with Hh favoring dorsal rather than ventral cell types, and Bmp favoring ventral rather than dorsal. With the majority of current data supporting a model whereby HSCs derive from arterial endothelium, our data identify the signal driving this conversion. These findings are important for the study of the production of HSCs from embryonic stem cells and establish a paradigm for the development of adult stem cells.

Regulation of the stem cell leukemia (SCL) gene: A tale of two fishes

The stem cell leukemia (SCL) gene encodes a tissue-specific basic helix–loop–helix (bHLH) protein with a pivotal role in hemopoiesis and vasculogenesis. Several enhancers have been identified within the murine SCL locus that direct reporter gene expression to subdomains of the normal SCL expression pattern, and long-range sequence comparisons of the human and murine SCL loci have identified additional candidate enhancers. To facilitate the characterization of regulatory elements, we have sequenced and analyzed 33 kb of the SCL genomic locus from the pufferfish Fugu rubripes, a species with a highly compact genome. Although the pattern of SCL expression is highly conserved from mammals to teleost fish, the genes flanking pufferfish SCL were unrelated to those known to flank both avian and mammalian SCL genes. These data suggest that SCL regulatory elements are confined to the region between the upstream and downstream flanking genes, a region of 65 kb in human and 8.5 kb in pufferfish. Consistent with this hypothesis, the entire 33-kb pufferfish SCL locus directed appropriate expression to hemopoietic and neural tissue in transgenic zebrafish embryos, as did a 10.4-kb fragment containing the SCL gene and extending to the 5′ and 3′ flanking genes. These results demonstrate the power of combining the compact genome of the pufferfish with the advantages that zebrafish provide for studies of gene regulation during development. Furthermore, the pufferfish SCL locus provides a powerful tool for the manipulation of hemopoiesis and vasculogenesis in vivo.

Hey2 acts upstream of Notch in hematopoietic stem cell specification in zebrafish embryos.

Hematopoietic stem cells (HSCs) are essential for homeostasis and injury-induced regeneration of the vertebrate blood system. Although HSC transplantations constitute the most common type of stem cell therapy applied in the clinic, we know relatively little about the molecular programming of HSCs during vertebrate embryogenesis. In vertebrate embryos, HSCs form in close association with the ventral wall of the dorsal aorta. We have shown previously that in zebrafish, HSC formation depends on the presence of a signaling cascade that involves Hedgehog, vascular endothelial growth factor, and Notch signaling. Here, we reveal that Hey2, a hairy/enhancer-of-split-related basic helix-loop-helix transcription factor often believed to act downstream of Notch, is also required for HSC formation. In dorsal aorta progenitors, Hey2 expression is induced downstream of cloche and the transcription factor Scl/Tal1, and is maintained by Hedgehog and vascular endothelial growth factor signaling. Whereas knockdown of Hey2 expression results in a loss of Notch receptor expression in dorsal aorta angioblasts, activation of Notch signaling in hey2 morphants rescues HSC formation in zebrafish embryos. These results establish an essential role for Hey2 upstream of Notch in HSC formation.

Blood flow suppresses vascular Notch signalling via dll4 and is required for angiogenesis in response to hypoxic signalling

Aims The contribution of blood flow to angiogenesis is incompletely understood. We examined the effect of blood flow on Notch signalling in the vasculature of zebrafish embryos, and whether blood flow regulates angiogenesis in zebrafish with constitutively up-regulated hypoxic signalling. Methods and results Developing zebrafish (Danio rerio) embryos survive via diffusion in the absence of circulation induced by knockdown of cardiac troponin T2 or chemical cardiac cessation. The absence of blood flow increased vascular Notch signalling in 48 h post-fertilization old embryos via up-regulation of the Notch ligand dll4. Despite this, patterning of the intersegmental vessels is not affected by absent blood flow. We therefore examined homozygous vhl mutant zebrafish that have constitutively up-regulated hypoxic signalling. These display excessive and aberrant angiogenesis from 72 h post-fertilization, with significantly increased endothelial number, vessel diameter, and length. The absence of blood flow abolished these effects, though normal vessel patterning was preserved. Conclusion We show that blood flow suppresses vascular Notch signalling via down-regulation of dll4. We have also shown that blood flow is required for angiogenesis in response to hypoxic signalling but is not required for normal vessel patterning. These data indicate important differences in hypoxia-driven vs. developmental angiogenesis.

Notch signalling and haematopoietic stem cell formation during embryogenesis.

The Notch signalling pathway is repeatedly employed during embryonic development and adult homeostasis of a variety of tissues. In particular, its frequent involvement in the regulation of stem and progenitor cell maintenance and proliferation, as well as its role in binary fate decisions in cells that are destined to differentiate, is remarkable. Here, we review its role in the development of haematopoietic stem cells during vertebrate embryogenesis and put it into the context of Notchs functions in arterial specification, angiogenic vessel sprouting and vessel maintenance. We further discuss interactions with other signalling cascades, and pinpoint open questions and some of the challenges that lie ahead. J. Cell. Physiol. 222:11–16, 2010.

5-hydroxymethyl-cytosine enrichment of non-committed cells is not a universal feature of vertebrate development

5-hydroxymethyl-cytosine (5-hmC) is a cytosine modification that is relatively abundant in mammalian pre-implantation embryos and embryonic stem cells (ESC) derived from mammalian blastocysts. Recent observations imply that both 5-hmC and Tet1/2/3 proteins, catalyzing the conversion of 5-methyl-cytosine to 5-hmC, may play an important role in self renewal and differentiation of ESCs. Here we assessed the distribution of 5-hmC in zebrafish and chick embryos and found that, unlike in mammals, 5-hmC is immunochemically undetectable in these systems before the onset of organogenesis. In addition, Tet1/2/3 transcripts are either low or undetectable at corresponding stages of zebrafish development. However, 5-hmC is enriched in later zebrafish and chick embryos and exhibits tissue-specific distribution in adult zebrafish. Our findings show that 5-hmC enrichment of non-committed cells is not a universal feature of vertebrate development and give insights both into evolution of embryonic pluripotency and the potential role of 5-hmC in its regulation.

Wallerian Degeneration Is Executed by an NMN-SARM1-Dependent Late Ca2+ Influx but Only Modestly Influenced by Mitochondria

Axon injury leads to rapid depletion of NAD-biosynthetic enzyme NMNAT2 and high levels of its substrate, NMN. We proposed a key role for NMN in Wallerian degeneration but downstream events and their relationship to other mediators remain unclear. Here, we show, in vitro and in vivo, that axotomy leads to a late increase in intra-axonal Ca(2+), abolished by pharmacological or genetic reduction of NMN levels. NMN requires the pro-degenerative protein SARM1 to stimulate Ca(2+) influx and axon degeneration. While inhibition of NMN synthesis and SARM1 deletion block Ca(2+) rise and preserve axonal integrity, they fail to prevent early mitochondrial dynamic changes. Furthermore, depolarizing mitochondria does not alter the rate of Wallerian degeneration. These data reveal that NMN and SARM1 act in a common pathway culminating in intra-axonal Ca(2+) increase and fragmentation and dissociate mitochondrial dysfunctions from this pathway, elucidating which steps may be most effective as targets for therapy.