Roger Patient

The GATA family (vertebrates and invertebrates)

Over the past year, vertebrate GATA factors have been found to participate directly in several signal-transduction pathways. Smad3, phosphorylated by TGF-beta signalling, interacts with GATA3 to induce differentiation of T helper cells. Hypertrophic stimuli act through RhoA GTPase and ROCK kinase to activate GATA4 in cardiac myocytes. In the liver, GATA4 is elevated by BMP and FGF signalling, and is able to bind to chromatin targets. Invertebrate GATA factors play a central role in specifying the mesendoderm.

Establishing the transcriptional programme for blood: the SCL stem cell enhancer is regulated by a multiprotein complex containing Ets and GATA factors

Stem cells are a central feature of metazoan biology. Haematopoietic stem cells (HSCs) represent the best‐characterized example of this phenomenon, but the molecular mechanisms responsible for their formation remain obscure. The stem cell leukaemia (SCL) gene encodes a basic helix–loop–helix (bHLH) transcription factor with an essential role in specifying HSCs. Here we have addressed the transcriptional hierarchy responsible for HSC formation by characterizing an SCL 3′ enhancer that targets expression to HSCs and endothelium and their bipotential precursors, the haemangioblast. We have identified three critical motifs, which are essential for enhancer function and bind GATA‐2, Fli‐1 and Elf‐1 in vivo. Our results suggest that these transcription factors are key components of an enhanceosome responsible for activating SCL transcription and establishing the transcriptional programme required for HSC formation.

Distinct Origins of Adult and Embryonic Blood in Xenopus

Whether embryonic and adult blood derive from a single (yolk sac) or dual (yolk sac plus intraembryonic) origin is controversial. Here, we show, in Xenopus, that the yolk sac (VBI) and intraembryonic (DLP) blood compartments derive from distinct blastomeres in the 32-cell embryo. The first adult hematopoietic stem cells (HSCs) are thought to form in association with the floor of the dorsal aorta, and we have detected such aortic clusters in Xenopus using hematopoietic markers. Lineage tracing shows that the aortic clusters derive from the blastomere that gives rise to the DLP. These observations indicate that the first adult HSCs arise independently of the embryonic lineage.

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.

Mesendoderm: An Ancient Germ Layer?

including the posterior portion of the pharynx, an organ University of Nottingham that has homologies to the heart, coelomocytes, which Queens Medical Centre resemble macrophages, and a subset of body wall mus-Nottingham NG7 2UH cle. The two GATA factors therefore occupy a pivotal United Kingdom position as zygotic factors specifying a single precursor that gives rise to both mesoderm and endoderm. In much closer relatives of the vertebrates, the sea ur-Formation of the three primary germ layers, ectoderm, chins (echinodermata, a sister phylum to the chordates), mesoderm, and endoderm, is an early distinction be-the macromeres of the 16-cell embryo, and their daugh-tween groups of cells in developing embryos. Our under-ters in the 32-cell embryo, are also mesendodermal, in standing of their generation in vertebrates has benefit-that they give rise to both the endoderm and the majority ted from the classical experiments of Nieuwkoop and of the mesoderm (referenced in Angerer and Angerer, his colleagues (referenced in Nieuwkoop, 1997), in which 2000). What, then, is the evidence that vertebrates de-explants of tissue from the animal hemisphere of am-velop along similar lines? phibian embryos (fated to form ectoderm) apposed to Vertebrate Mesendoderm explants of vegetal tissue (fated to form endoderm) were Labeling of single cells at the mid-blastula stage in ze-induced to form mesoderm. These results have been brafish has shown that endoderm and mesoderm, in-widely interpreted as indicating that mesoderm forms cluding blood, heart, and muscle, derive from cells in the at the interface between presumptive endoderm and 3–4 tiers nearest to the vegetal margin of the blastoderm presumptive ectoderm as a consequence of inductive (Warga and Nusslein-Volhard, 1999 and references therein). signals from the former to the latter. However, recent We will refer to these cells as mesendoderm (Figure 2A). data from nematodes and zebrafish suggest that endo-The mesendodermal nature of these cells is also sug-derm and some portion of the mesoderm may derive gested, in the late blastula, by the coexpression of genes from a bipotential layer of cells, called the mesendo-expressed by and active in the formation of mesoderm derm. In addition, the genes involved in this process and endoderm: the transcription factors, brachyury (bra, may be conserved. also known as no tail (ntl) in the zebrafish) and gata5 In C. elegans, a nematode worm, the endoderm and (faust in the zebrafish) (Rodaway et al., 1999). Perturba-much of the mesoderm derive from the mesendodermal tion of signaling at …

Fli1 Acts at the Top of the Transcriptional Network Driving Blood and Endothelial Development

Blood and endothelium arise in close association during development, possibly from a common precursor, the hemangioblast [1-4]. Genes essential for blood and endothelial development contain functional ETS binding sites, and binding and expression data implicate the transcription factor, friend leukaemia integration 1 (Fli1) [5-10]. However, loss-of-function phenotypes in mice, although suffering both blood and endothelial defects, have thus far precluded the conclusion that Fli1 is essential for these two lineages [11, 12]. By using Xenopus and zebrafish embryos, we show that loss of Fli1 function results in a substantial reduction or absence of hemangioblasts, revealing an absolute requirement. TUNEL assays show that the cells are eventually lost by apoptosis, but only after the regulatory circuit has been disrupted by loss of Fli1. In addition, a constitutively active form of Fli1 is sufficient to induce expression of key hemangioblast genes, such as Scl/Tal1, Lmo2, Gata2, Etsrp, and Flk1. Epistasis assays show that Fli1 expression is induced by Bmp signaling or Cloche, depending on the hemangioblast population, and in both cases Fli1 acts upstream of Gata2, Scl, Lmo2, and Etsrp. Taken together, these results place Fli1 at the top of the transcriptional regulatory hierarchy for hemangioblast specification in vertebrate embryos.

Adult and embryonic blood and endothelium derive from distinct precursor populations which are differentially programmed by BMP in Xenopus.

Blood and blood vessels develop in close association in vertebrate embryos and loss-of-function mutations suggest common genetic regulation. By the criteria of co-expression of blood and endothelial genes, and lineage tracing of progeny, we locate two distinct populations of progenitors for blood and endothelial cells in developing Xenopus embryos. The first population is located immediately posterior to the cement gland during neurula stages and gives rise to embryonic blood and vitelline veins in the anterior ventral blood island (aVBI), and to the endocardium of the heart. The second population resides in the dorsal lateral plate mesoderm, and contains precursors of adult blood stem cells and the major vessels. Both populations differentiate into endothelial cells in situ but migrate to new locations to differentiate into blood, suggesting that their micro-environments are unsuitable for haematopoietic differentiation. Both require BMP for their formation, even the Spemann organiser-derived aVBI, but individual genes are affected differentially. Thus, in the embryonic population, expression of the blood genes, SCL and GATA2, depend on BMP signalling while expression of the endothelial gene, Xfli1, does not. By contrast, Xfli1 expression in the adult, DLP population does require BMP. These results indicate that both adult and the anterior component of embryonic blood in Xenopus embryos derive from populations of progenitors that also give rise to endothelial cells. However, the two populations give rise to distinct regions of the vasculature and are programmed differentially by BMP.

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.

Epigenetic conservation at gene regulatory elements revealed by non-methylated DNA profiling in seven vertebrates

Two-thirds of gene promoters in mammals are associated with regions of non-methylated DNA, called CpG islands (CGIs), which counteract the repressive effects of DNA methylation on chromatin. In cold-blooded vertebrates, computational CGI predictions often reside away from gene promoters, suggesting a major divergence in gene promoter architecture across vertebrates. By experimentally identifying non-methylated DNA in the genomes of seven diverse vertebrates, we instead reveal that non-methylated islands (NMIs) of DNA are a central feature of vertebrate gene promoters. Furthermore, NMIs are present at orthologous genes across vast evolutionary distances, revealing a surprising level of conservation in this epigenetic feature. By profiling NMIs in different tissues and developmental stages we uncover a unifying set of features that are central to the function of NMIs in vertebrates. Together these findings demonstrate an ancient logic for NMI usage at gene promoters and reveal an unprecedented level of epigenetic conservation across vertebrate evolution. DOI: http://dx.doi.org/10.7554/eLife.00348.001

GATA-6 maintains BMP-4 and Nkx2 expression during cardiomyocyte precursor maturation

GATA‐6 is expressed in presumptive cardiac mesoderm before gastrulation, but its role in heart development has been unclear. Here we show that Xenopus and zebrafish embryos, injected with antisense morpholino oligonucleotides designed specifically to knock‐down translation of GATA‐6 protein, are severely compromised for heart development. Injected embryos express greatly reduced levels of contractile machinery genes and, at the same stage, of regulatory genes such as bone morphogenetic protein‐4 (BMP‐4) and the Nkx2 family. In contrast, initial BMP and Nkx2 expression is normal, suggesting a maintenance role for GATA‐6. Endoderm is critical for heart formation in several vertebrates including Xenopus, and separate perturbation of GATA‐6 expression in the deep anterior endoderm and in the overlying heart mesoderm shows that GATA‐6 is required in both for cardiogenesis. The GATA‐6 requirement in cardiac mesoderm was confirmed in zebrafish, an organism in which endoderm is thought not to be necessary for heart formation. We therefore conclude that proper maturation of cardiac mesoderm requires GATA‐6, which functions to maintain BMP‐4 and Nkx2 expression.