Prof Roger Patient

Technology Exchange: In situ hybridisation, In vivo imaging, Microscopy (Confocal), Microscopy (Video), Transcript profiling and Transgenesis
Web Links:
Blood stem cells (purple) emerging in the floor of the dorsal aorta independently of the circulating primitive red cells in zebrafish embryos

Blood stem cells (purple) emerging in the floor of the dorsal aorta independently of the ...

Blood and cardiovascular development, with a particular focus on stem cells.  Transcriptional network assembly in response to embryonic signals.  Xenopus and zebrafish models.

There are no collaborations listed for this principal investigator.

Long HK, King HW, Patient RK, Odom DT, Klose RJ. 2016. Protection of CpG islands from DNA methylation is DNA-encoded and evolutionarily conserved. Nucleic Acids Res, 44 (14), pp. 6693-6706. | Show Abstract | Read more

DNA methylation is a repressive epigenetic modification that covers vertebrate genomes. Regions known as CpG islands (CGIs), which are refractory to DNA methylation, are often associated with gene promoters and play central roles in gene regulation. Yet how CGIs in their normal genomic context evade the DNA methylation machinery and whether these mechanisms are evolutionarily conserved remains enigmatic. To address these fundamental questions we exploited a transchromosomic animal model and genomic approaches to understand how the hypomethylated state is formed in vivo and to discover whether mechanisms governing CGI formation are evolutionarily conserved. Strikingly, insertion of a human chromosome into mouse revealed that promoter-associated CGIs are refractory to DNA methylation regardless of host species, demonstrating that DNA sequence plays a central role in specifying the hypomethylated state through evolutionarily conserved mechanisms. In contrast, elements distal to gene promoters exhibited more variable methylation between host species, uncovering a widespread dependence on nucleotide frequency and occupancy of DNA-binding transcription factors in shaping the DNA methylation landscape away from gene promoters. This was exemplified by young CpG rich lineage-restricted repeat sequences that evaded DNA methylation in the absence of co-evolved mechanisms targeting methylation to these sequences, and species specific DNA binding events that protected against DNA methylation in CpG poor regions. Finally, transplantation of mouse chromosomal fragments into the evolutionarily distant zebrafish uncovered the existence of a mechanistically conserved and DNA-encoded logic which shapes CGI formation across vertebrate species.

Short S, Peterkin T, Guille M, Patient R, Sharpe C. 2015. Short linear motif acquisition, exon formation and alternative splicing determine a pathway to diversity for NCoR-family co-repressors. Open Biol, 5 (8), pp. 150063-150063. | Show Abstract | Read more

Vertebrate NCoR-family co-repressors play central roles in the timing of embryo and stem cell differentiation by repressing the activity of a range of transcription factors. They interact with nuclear receptors using short linear motifs (SLiMs) termed co-repressor for nuclear receptor (CoRNR) boxes. Here, we identify the pathway leading to increasing co-repressor diversity across the deuterostomes. The final complement of CoRNR boxes arose in an ancestral cephalochordate, and was encoded in one large exon; the urochordates and vertebrates then split this region between 10 and 12 exons. In Xenopus, alternative splicing is prevalent in NCoR2, but absent in NCoR1. We show for one NCoR1 exon that alternative splicing can be recovered by a single point mutation, suggesting NCoR1 lost the capacity for alternative splicing. Analyses in Xenopus and zebrafish identify that cellular context, rather than gene sequence, predominantly determines species differences in alternative splicing. We identify a pathway to diversity for the NCoR family beginning with the addition of a SLiM, followed by gene duplication, the generation of alternatively spliced isoforms and their differential deployment.

Gu W, Monteiro R, Zuo J, Simões FC, Martella A, Andrieu-Soler C, Grosveld F, Sauka-Spengler T, Patient R. 2015. A novel TGFβ modulator that uncouples R-Smad/I-Smad-mediated negative feedback from R-Smad/ligand-driven positive feedback. PLoS Biol, 13 (2), pp. e1002051. | Show Abstract | Read more

As some of the most widely utilised intercellular signalling molecules, transforming growth factor β (TGFβ) superfamily members play critical roles in normal development and become disrupted in human disease. Establishing appropriate levels of TGFβ signalling involves positive and negative feedback, which are coupled and driven by the same signal transduction components (R-Smad transcription factor complexes), but whether and how the regulation of the two can be distinguished are unknown. Genome-wide comparison of published ChIP-seq datasets suggests that LIM domain binding proteins (Ldbs) co-localise with R-Smads at a substantial subset of R-Smad target genes including the locus of inhibitory Smad7 (I-Smad7), which mediates negative feedback for TGFβ signalling. We present evidence suggesting that zebrafish Ldb2a binds and directly activates the I-Smad7 gene, whereas it binds and represses the ligand gene, Squint (Sqt), which drives positive feedback. Thus, the fine tuning of TGFβ signalling derives from positive and negative control by Ldb2a. Expression of ldb2a is itself activated by TGFβ signals, suggesting potential feed-forward loops that might delay the negative input of Ldb2a to the positive feedback, as well as the positive input of Ldb2a to the negative feedback. In this way, precise gene expression control by Ldb2a enables an initial build-up of signalling via a fully active positive feedback in the absence of buffering by the negative feedback. In Ldb2a-deficient zebrafish embryos, homeostasis of TGFβ signalling is perturbed and signalling is stably enhanced, giving rise to excess mesoderm and endoderm, an effect that can be rescued by reducing signalling by the TGFβ family members, Nodal and BMP. Thus, Ldb2a is critical to the homeostatic control of TGFβ signalling and thereby embryonic patterning.

Ciau-Uitz A, Monteiro R, Kirmizitas A, Patient R. 2014. Developmental hematopoiesis: ontogeny, genetic programming and conservation. Exp Hematol, 42 (8), pp. 669-683. | Show Abstract | Read more

Hematopoietic stem cells (HSCs) sustain blood production throughout life and are of pivotal importance in regenerative medicine. Although HSC generation from pluripotent stem cells would resolve their shortage for clinical applications, this has not yet been achieved mainly because of the poor mechanistic understanding of their programming. Bone marrow HSCs are first created during embryogenesis in the dorsal aorta (DA) of the midgestation conceptus, from where they migrate to the fetal liver and, eventually, the bone marrow. It is currently accepted that HSCs emerge from specialized endothelium, the hemogenic endothelium, localized in the ventral wall of the DA through an evolutionarily conserved process called the endothelial-to-hematopoietic transition. However, the endothelial-to-hematopoietic transition represents one of the last steps in HSC creation, and an understanding of earlier events in the specification of their progenitors is required if we are to create them from naïve pluripotent cells. Because of their ready availability and external development, zebrafish and Xenopus embryos have enormously facilitated our understanding of the early developmental processes leading to the programming of HSCs from nascent lateral plate mesoderm to hemogenic endothelium in the DA. The amenity of the Xenopus model to lineage tracing experiments has also contributed to the establishment of the distinct origins of embryonic (yolk sac) and adult (HSC) hematopoiesis, whereas the transparency of the zebrafish has allowed in vivo imaging of developing blood cells, particularly during and after the emergence of HSCs in the DA. Here, we discuss the key contributions of these model organisms to our understanding of developmental hematopoiesis.

Chatfield J, O'Reilly MA, Bachvarova RF, Ferjentsik Z, Redwood C, Walmsley M, Patient R, Loose M, Johnson AD. 2014. Stochastic specification of primordial germ cells from mesoderm precursors in axolotl embryos. Development, 141 (12), pp. 2429-2440. | Show Abstract | Read more

A common feature of development in most vertebrate models is the early segregation of the germ line from the soma. For example, in Xenopus and zebrafish embryos primordial germ cells (PGCs) are specified by germ plasm that is inherited from the egg; in mice, Blimp1 expression in the epiblast mediates the commitment of cells to the germ line. How these disparate mechanisms of PGC specification evolved is unknown. Here, in order to identify the ancestral mechanism of PGC specification in vertebrates, we studied PGC specification in embryos from the axolotl (Mexican salamander), a model for the tetrapod ancestor. In the axolotl, PGCs develop within mesoderm, and classic studies have reported their induction from primitive ectoderm (animal cap). We used an axolotl animal cap system to demonstrate that signalling through FGF and BMP4 induces PGCs. The role of FGF was then confirmed in vivo. We also showed PGC induction by Brachyury, in the presence of BMP4. These conditions induced pluripotent mesodermal precursors that give rise to a variety of somatic cell types, in addition to PGCs. Irreversible restriction of the germ line did not occur until the mid-tailbud stage, days after the somatic germ layers are established. Before this, germline potential was maintained by MAP kinase signalling. We propose that this stochastic mechanism of PGC specification, from mesodermal precursors, is conserved in vertebrates.

Pouget C, Peterkin T, Simões FC, Lee Y, Traver D, Patient R. 2014. FGF signalling restricts haematopoietic stem cell specification via modulation of the BMP pathway. Nat Commun, 5 pp. 5588. | Show Abstract | Read more

Haematopoietic stem cells (HSCs) are produced during embryogenesis from the floor of the dorsal aorta. The localization of HSCs is dependent on the presence of instructive signals on the ventral side of the vessel. The nature of the extrinsic molecular signals that control the aortic haematopoietic niche is currently poorly understood. Here we demonstrate a novel requirement for FGF signalling in the specification of aortic haemogenic endothelium. Our results demonstrate that FGF signalling normally acts to repress BMP activity in the subaortic mesenchyme through transcriptional inhibition of bmp4, as well as through activation of two BMP antagonists, noggin2 and gremlin1a. Taken together, these findings demonstrate a key role for FGF signalling in establishment of the developmental HSC niche via its regulation of BMP activity in the subaortic mesenchyme. These results should help inform strategies to recapitulate the development of HSCs in vitro from pluripotent precursors.

Gorsi B, Liu F, Ma X, Chico TJA, Shrinivasan A, Kramer KL, Bridges E, Monteiro R, Harris AL, Patient R, Stringer SE. 2014. The heparan sulfate editing enzyme Sulf1 plays a novel role in zebrafish VegfA mediated arterial venous identity Angiogenesis, 17 (1), pp. 77-91. | Show Abstract | Read more

Arterial and venous specification is critical for establishing and maintaining a functioning vascular system, and defects in key arteriovenous signaling pathways including VEGF (vascular endothelial growth factor) lead to congenital arteriopathies. The activities of VEGF, are in part controlled by heparan sulfate (HS) proteoglycans, significant components of the endothelial glycocalyx. The level of 6-O sulfation on HS polysaccharide chains, that mediate the interaction between HS and VEGFA, is edited at the cell surface by the enzyme SULF1. We investigated the role of sulf1 in vascular development. In zebrafish sulf1 is expressed in the head and tail vasculature, corresponding spatially and temporally with vascular development. Targeted knockdown of sulf1 by antisense morpholinos resulted in severe vascular patterning and maturation defects. 93 % of sulf1 morphants show dysmorphogenesis in arterial development leading to occlusion of the distal aorta and lack of axial and cranial circulation. Co-injection of vegfa 165 mRNA rescued circulatory defects. While the genes affecting haematopoiesis are unchanged, expression of several arterial markers downstream of VegfA signalling such as notch and ephrinB2 are severely reduced in the dorsal aorta, with a concomitant increase in expression of the venous markers flt4 in the dorsal aorta of the morphants. Furthermore, in vitro, lack of SULF1 expression downregulates VEGFA-mediated arterial marker expression, confirming that Sulf1 mediates arterial specification by regulating VegfA 165 activity. This study provides the first in vivo evidence for the integral role of the endothelial glycocalyx in specifying arterial-venous identity, vascular patterning and arterial integrity, and will help to better understand congenital arteriopathies. © 2013 Springer Science+Business Media Dordrecht.

Ciau-Uitz A, Wang L, Patient R, Liu F. 2013. ETS transcription factors in hematopoietic stem cell development Blood Cells, Molecules, and Diseases, 51 (4), pp. 248-255. | Show Abstract | Read more

Hematopoietic stem cells (HSCs) are essential for the maintenance of the hematopoietic system. However, these cells cannot be maintained or created in vitro, and very little is known about their generation during embryogenesis. Many transcription factors and signaling pathways play essential roles at various stages of HSC development. Members of the ETS ('E twenty-six') family of transcription factors are recognized as key regulators within the gene regulatory networks governing hematopoiesis, including the ontogeny of HSCs. Remarkably, although all ETS transcription factors bind the same DNA consensus sequence and overlapping tissue expression is observed, individual ETS transcription factors play unique roles in the development of HSCs. Also, these transcription factors are recurrently used throughout development and their functions are context-dependent, increasing the challenge of studying their mechanism of action. Critically, ETS factors also play roles under pathological conditions, such as leukemia and, therefore, deciphering their mechanism of action will not only enhance our knowledge of normal hematopoiesis, but also inform protocols for their creation in vitro from pluripotent stem cells and the design of new therapeutic approaches for the treatment of malignant blood cell diseases. In this review, we summarize the key findings on the roles of ETS transcription factors in HSC development and discuss novel mechanisms by which they could control hematopoiesis. © 2013 Elsevier Inc.

Gorsi B, Liu F, Ma X, Chico TJ, v A, Kramer KL, Bridges E, Monteiro R, Harris AL, Patient R, Stringer SE. 2014. The heparan sulfate editing enzyme Sulf1 plays a novel role in zebrafish VegfA mediated arterial venous identity. Angiogenesis, 17 (1), pp. 77-91. | Show Abstract | Read more

Arterial and venous specification is critical for establishing and maintaining a functioning vascular system, and defects in key arteriovenous signaling pathways including VEGF (vascular endothelial growth factor) lead to congenital arteriopathies. The activities of VEGF, are in part controlled by heparan sulfate (HS) proteoglycans, significant components of the endothelial glycocalyx. The level of 6-O sulfation on HS polysaccharide chains, that mediate the interaction between HS and VEGFA, is edited at the cell surface by the enzyme SULF1. We investigated the role of sulf1 in vascular development. In zebrafish sulf1 is expressed in the head and tail vasculature, corresponding spatially and temporally with vascular development. Targeted knockdown of sulf1 by antisense morpholinos resulted in severe vascular patterning and maturation defects. 93 % of sulf1 morphants show dysmorphogenesis in arterial development leading to occlusion of the distal aorta and lack of axial and cranial circulation. Co-injection of vegfa165 mRNA rescued circulatory defects. While the genes affecting haematopoiesis are unchanged, expression of several arterial markers downstream of VegfA signalling such as notch and ephrinB2 are severely reduced in the dorsal aorta, with a concomitant increase in expression of the venous markers flt4 in the dorsal aorta of the morphants. Furthermore, in vitro, lack of SULF1 expression downregulates VEGFA-mediated arterial marker expression, confirming that Sulf1 mediates arterial specification by regulating VegfA165 activity. This study provides the first in vivo evidence for the integral role of the endothelial glycocalyx in specifying arterial-venous identity, vascular patterning and arterial integrity, and will help to better understand congenital arteriopathies.

Cited:

36

Scopus

Nimmo R, Ciau-Uitz A, Ruiz-Herguido C, Soneji S, Bigas A, Patient R, Enver T. 2013. MiR-142-3p controls the specification of definitive hemangioblasts during ontogeny Developmental Cell, 26 (3), pp. 237-249. | Show Abstract | Read more

Hematopoietic stem cells (HSCs) emerge during embryogenesis from hemogenic endothelium, but it remains unclear how the HSC lineage is initially established from mesoderm during ontogeny. In Xenopus, the definitive hemangioblast precursors of the HSC lineage have been identified in dorsal lateral plate (DLP) mesoderm, an d a transcriptional gene regulatory network (GRN) controlling hemangioblast programming has been elucidated. Herein, we identify an essential role for microRNAs (miRNAs) in establishing the mesodermal lineage leading to both HSC emergence and vasculogenesis and determine that a single miRNA, miR-142-3p, is primarily responsible for initiation of definitive hemangioblast specification. miR-142-3p forms a double-negative gate unlocking entry into the hemangioblast program, in part by inhibiting TGFβ signaling. Our results table miR-142-3p as a master regulator of HSC lineage specification, sitting at the apex of the hierarchy programming the adult hemangioblast, thus illustrating that miRNAs can act as instructive determinants of cell fate during development. © 2013 Elsevier Inc.

Cited:

39

Scopus

Masiero M, Simões FC, Han HD, Snell C, Peterkin T, Bridges E, Mangala LS, Wu SY-Y, Pradeep S, Li D et al. 2013. A Core Human Primary Tumor Angiogenesis Signature Identifies the Endothelial Orphan Receptor ELTD1 as a Key Regulator of Angiogenesis Cancer Cell, 24 (2), pp. 229-241. | Show Abstract | Read more

Limited clinical benefits derived from anti-VEGF therapy have driven the identification of new targets involved in tumor angiogenesis. Here, we report an integrative meta-analysis to define the transcriptional program underlying angiogenesis in human cancer. This approach identified ELTD1, an orphan G-protein-coupled receptor whose expression is induced by VEGF/bFGF and repressed by DLL4 signaling. Extensive analysis of multiple cancer types demonstrates significant upregulation of ELTD1 in tumor-associated endothelial cells, with a higher expression correlating with favorable prognosis. Importantly, ELTD1 silencing impairs endothelial sprouting and vessel formation invitro and invivo, drastically reducing tumor growth and greatly improving survival. Collectively, these results provide insight into the regulation of tumor angiogenesis and highlight ELTD1 as key player in blood vessel formation. © 2013 The Authors.

Nimmo R, Ciau-Uitz A, Ruiz-Herguido C, Soneji S, Bigas A, Patient R, Enver T. 2013. MiR-142-3p controls the specification of definitive hemangioblasts during ontogeny. Dev Cell, 26 (3), pp. 237-249. | Show Abstract | Read more

Hematopoietic stem cells (HSCs) emerge during embryogenesis from hemogenic endothelium, but it remains unclear how the HSC lineage is initially established from mesoderm during ontogeny. In Xenopus, the definitive hemangioblast precursors of the HSC lineage have been identified in dorsal lateral plate (DLP) mesoderm, and a transcriptional gene regulatory network (GRN) controlling hemangioblast programming has been elucidated. Herein, we identify an essential role for microRNAs (miRNAs) in establishing the mesodermal lineage leading to both HSC emergence and vasculogenesis and determine that a single miRNA, miR-142-3p, is primarily responsible for initiation of definitive hemangioblast specification. miR-142-3p forms a double-negative gate unlocking entry into the hemangioblast program, in part by inhibiting TGFβ signaling. Our results table miR-142-3p as a master regulator of HSC lineage specification, sitting at the apex of the hierarchy programming the adult hemangioblast, thus illustrating that miRNAs can act as instructive determinants of cell fate during development.

Masiero M, Simões FC, Han HD, Snell C, Peterkin T, Bridges E, Mangala LS, Wu SY, Pradeep S, Li D et al. 2013. A core human primary tumor angiogenesis signature identifies the endothelial orphan receptor ELTD1 as a key regulator of angiogenesis. Cancer Cell, 24 (2), pp. 229-241. | Show Abstract | Read more

Limited clinical benefits derived from anti-VEGF therapy have driven the identification of new targets involved in tumor angiogenesis. Here, we report an integrative meta-analysis to define the transcriptional program underlying angiogenesis in human cancer. This approach identified ELTD1, an orphan G-protein-coupled receptor whose expression is induced by VEGF/bFGF and repressed by DLL4 signaling. Extensive analysis of multiple cancer types demonstrates significant upregulation of ELTD1 in tumor-associated endothelial cells, with a higher expression correlating with favorable prognosis. Importantly, ELTD1 silencing impairs endothelial sprouting and vessel formation in vitro and in vivo, drastically reducing tumor growth and greatly improving survival. Collectively, these results provide insight into the regulation of tumor angiogenesis and highlight ELTD1 as key player in blood vessel formation.

ElOmari K, Hoosdally SJ, Tuladhar K, Karia D, Hall-Ponselé E, Platonova O, Vyas P, Patient R, Porcher C, Mancini EJ. 2013. Structural Basis for LMO2-Driven Recruitment of the SCL: E47bHLH Heterodimer to Hematopoietic-Specific Transcriptional Targets Cell Reports, 4 (1), pp. 135-147. | Show Abstract | Read more

Cell fate is governed by combinatorial actions of transcriptional regulators assembling into multiprotein complexes. However, the molecular details of how these complexes form are poorly understood. One such complex, which contains the basic-helix-loop-helix heterodimer SCL:E47 and bridging proteins LMO2:LDB1, critically regulates hematopoiesis and induces Tcell leukemia. Here, we report the crystal structure of (SCL:E47) bHLH :LMO2:LDB1 LID bound to DNA, providing a molecular account of the network of interactions assembling this complex. This reveals an unexpected role for LMO2. Upon binding to SCL, LMO2 induces new hydrogen bonds in SCL:E47, thereby strengthening heterodimer formation. This imposes a rotation movement onto E47 that weakens the heterodimer:DNA interaction, shifting the main DNA-binding activity onto additional protein partners. Along with biochemical analyses, this illustrates, at an atomic level, how hematopoietic-specific SCL sequesters ubiquitous E47 and associated cofactors and supports SCL'sreported DNA-binding-independent functions. Importantly, this work will drive the design of small molecules inhibiting leukemogenic processes. © 2013 The Authors.

El Omari K, Hoosdally SJ, Tuladhar K, Karia D, Hall-Ponselé E, Platonova O, Vyas P, Patient R, Porcher C, Mancini EJ. 2013. Structural basis for LMO2-driven recruitment of the SCL:E47bHLH heterodimer to hematopoietic-specific transcriptional targets. Cell Rep, 4 (1), pp. 135-147. | Show Abstract | Read more

Cell fate is governed by combinatorial actions of transcriptional regulators assembling into multiprotein complexes. However, the molecular details of how these complexes form are poorly understood. One such complex, which contains the basic-helix-loop-helix heterodimer SCL:E47 and bridging proteins LMO2:LDB1, critically regulates hematopoiesis and induces T cell leukemia. Here, we report the crystal structure of (SCL:E47)bHLH:LMO2:LDB1LID bound to DNA, providing a molecular account of the network of interactions assembling this complex. This reveals an unexpected role for LMO2. Upon binding to SCL, LMO2 induces new hydrogen bonds in SCL:E47, thereby strengthening heterodimer formation. This imposes a rotation movement onto E47 that weakens the heterodimer:DNA interaction, shifting the main DNA-binding activity onto additional protein partners. Along with biochemical analyses, this illustrates, at an atomic level, how hematopoietic-specific SCL sequesters ubiquitous E47 and associated cofactors and supports SCL's reported DNA-binding-independent functions. Importantly, this work will drive the design of small molecules inhibiting leukemogenic processes.

Sacilotto N, Monteiro R, Fritzsche M, Becker PW, Sanchez-Del-Campo L, Liu K, Pinheiro P, Ratnayaka I, Davies B, Goding CR et al. 2013. Analysis of Dll4 regulation reveals a combinatorial role for Sox and Notch in arterial development. Proc Natl Acad Sci U S A, 110 (29), pp. 11893-11898. | Show Abstract | Read more

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.

Loose M, Patient R, Fang X, Lei H. 2013. Gene Regulatory Networks in the Genomics Era Genomics, Proteomics and Bioinformatics, 11 (3), pp. 133-134. | Read more

Ciau-Uitz A, Pinheiro P, Kirmizitas A, Zuo J, Patient R. 2013. VEGFA-dependent and -independent pathways synergise to drive Scl expression and initiate programming of the blood stem cell lineage in Xenopus. Development, 140 (12), pp. 2632-2642. | Show Abstract | Read more

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.

Wang L, Liu T, Xu L, Gao Y, Wei Y, Duan C, Chen GQ, Lin S, Patient R, Zhang B et al. 2013. Fev regulates hematopoietic stem cell development via ERK signaling. Blood, 122 (3), pp. 367-375. | Show Abstract | Read more

Reprogramming of somatic cells to desired cell types holds great promise in regenerative medicine. However, production of transplantable hematopoietic stem cells (HSCs) in vitro by defined factors has not yet been achieved. Therefore, it is critical to fully understand the molecular mechanisms of HSC development in vivo. Here, we show that Fev, an ETS transcription factor, is a pivotal regulator of HSC development in vertebrates. In fev-deficient zebrafish embryos, the first definitive HSC population was compromised and fewer T cells were found in the thymus. Genetic and chemical analyses support a mechanism whereby Fev regulates HSC through direct regulation of ERK signaling. Blastula transplant assay demonstrates that Fev regulation of HSC development is cell autonomous. Experiments performed with purified cord blood show that fev is expressed and functions in primitive HSCs in humans, indicating its conserved role in higher vertebrates. Our data indicate that Fev-ERK signaling is essential for hemogenic endothelium-based HSC development.

Leung A, Ciau-Uitz A, Pinheiro P, Monteiro R, Zuo J, Vyas P, Patient R, Porcher C. 2013. Uncoupling VEGFA functions in arteriogenesis and hematopoietic stem cell specification. Dev Cell, 24 (2), pp. 144-158. | Show Abstract | Read more

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.

Ciau-Uitz A, Wang L, Patient R, Liu F. 2013. ETS transcription factors in hematopoietic stem cell development. Blood Cells Mol Dis, 51 (4), pp. 248-255. | Show Abstract | Read more

Hematopoietic stem cells (HSCs) are essential for the maintenance of the hematopoietic system. However, these cells cannot be maintained or created in vitro, and very little is known about their generation during embryogenesis. Many transcription factors and signaling pathways play essential roles at various stages of HSC development. Members of the ETS ('E twenty-six') family of transcription factors are recognized as key regulators within the gene regulatory networks governing hematopoiesis, including the ontogeny of HSCs. Remarkably, although all ETS transcription factors bind the same DNA consensus sequence and overlapping tissue expression is observed, individual ETS transcription factors play unique roles in the development of HSCs. Also, these transcription factors are recurrently used throughout development and their functions are context-dependent, increasing the challenge of studying their mechanism of action. Critically, ETS factors also play roles under pathological conditions, such as leukemia and, therefore, deciphering their mechanism of action will not only enhance our knowledge of normal hematopoiesis, but also inform protocols for their creation in vitro from pluripotent stem cells and the design of new therapeutic approaches for the treatment of malignant blood cell diseases. In this review, we summarize the key findings on the roles of ETS transcription factors in HSC development and discuss novel mechanisms by which they could control hematopoiesis.

Loose M, Patient R, Fang X, Lei H. 2013. Gene regulatory networks in the genomics era. Genomics Proteomics Bioinformatics, 11 (3), pp. 133-134. | Read more

Long HK, Sims D, Heger A, Blackledge NP, Kutter C, Wright ML, Grützner F, Odom DT, Patient R, Ponting CP, Klose RJ. 2013. Epigenetic conservation at gene regulatory elements revealed by non-methylated DNA profiling in seven vertebrates. Elife, 2 (2), pp. e00348. | Show Abstract | Read more

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.

Cited:

25

Scopus

Leung A, Ciau-Uitz A, Pinheiro P, Monteiro R, Zuo J, Vyas P, Patient R, Porcher C. 2013. Uncoupling VEGFA Functions in Arteriogenesis and Hematopoietic Stem Cell Specification Developmental Cell, 24 (2), pp. 144-158. | Show Abstract | Read more

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.

Zhang C, Patient R, Liu F. 2013. Hematopoietic stem cell development and regulatory signaling in zebrafish Biochimica et Biophysica Acta - General Subjects, 1830 (2), pp. 2370-2374. | Show Abstract | Read more

Background: Hematopoietic stem cells (HSCs) are a population of multipotent cells that can self-renew and differentiate into all blood lineages. HSC development must be tightly controlled from cell fate determination to self-maintenance during adulthood. This involves a panel of important developmental signaling pathways and other factors which act synergistically within the HSC population and/or in the HSC niche. Genetically conserved processes of HSC development plus many other developmental advantages make the zebrafish an ideal model organism to elucidate the regulatory mechanisms underlying HSC programming. Scope of review: This review summarizes recent progress on zebrafish HSCs with particular focus on how developmental signaling controls hemogenic endothelium-derived HSC development. We also describe the interaction of different signaling pathways during these processes. Major conclusions: The hematopoietic stem cell system is a paradigm for stem cell studies. Use of the zebrafish model to study signaling regulation of HSCs in vivo has resulted in a great deal of information concerning HSC biology in vertebrates. General significance: These new findings facilitate a better understanding of molecular mechanisms of HSC programming, and will provide possible new strategies for the treatment of HSC-related hematological diseases, such as leukemia. This article is part of a Special Issue entitled Biochemistry of Stem Cells. © 2012 Elsevier B.V.

Bridge G, Monteiro R, Henderson S, Emuss V, Lagos D, Georgopoulou D, Patient R, Boshoff C. 2012. The microRNA-30 family targets DLL4 to modulate endothelial cell behavior during angiogenesis. Blood, 120 (25), pp. 5063-5072. | Show Abstract | Read more

Delta-like 4 (DLL4), a membrane-bound ligand belonging to the Notch signaling family, plays a fundamental role in vascular development and angiogenesis. We identified a conserved microRNA family, miR-30, which targets DLL4. Overexpression of miR-30b in endothelial cells led to increased vessel number and length in an in vitro model of sprouting angiogenesis. Microinjection of miR-30 mimics into zebrafish embryos resulted in suppression of dll4 and subsequent excessive sprouting of intersegmental vessels and reduction in dorsal aorta diameter. Use of a target protector against the miR-30 site within the dll4 3'UTR up-regulated dll4 and synergized with Vegfa signaling knockdown to inhibit angiogenesis. Furthermore, restoration of miR-30b or miR-30c expression during Kaposi sarcoma herpesvirus (KSHV) infection attenuated viral induction of DLL4. Together these results demonstrate that the highly conserved molecular targeting of DLL4 by the miR-30 family regulates angiogenesis.

van Riel B, Pakozdi T, Brouwer R, Monteiro R, Tuladhar K, Franke V, Bryne JC, Jorna R, Rijkers EJ, van Ijcken W et al. 2012. A novel complex, RUNX1-MYEF2, represses hematopoietic genes in erythroid cells. Mol Cell Biol, 32 (19), pp. 3814-3822. | Show Abstract | Read more

RUNX1 is known to be an essential transcription factor for generating hematopoietic stem cells (HSC), but much less is known about its role in the downstream process of hematopoietic differentiation. RUNX1 has been shown to be part of a large transcription factor complex, together with LDB1, GATA1, TAL1, and ETO2 (N. Meier et al., Development 133:4913-4923, 2006) in erythroid cells. We used a tagging strategy to show that RUNX1 interacts with two novel protein partners, LSD1 and MYEF2, in erythroid cells. MYEF2 is bound in undifferentiated cells and is lost upon differentiation, whereas LSD1 is bound in differentiated cells. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) and microarray expression analysis were used to show that RUNX1 binds approximately 9,000 target sites in erythroid cells and is primarily active in the undifferentiated state. Functional analysis shows that a subset of the target genes is suppressed by RUNX1 via the newly identified partner MYEF2. Knockdown of Myef2 expression in developing zebrafish results in a reduced number of HSC.

Zhang C, Patient R, Liu F. 2013. Hematopoietic stem cell development and regulatory signaling in zebrafish. Biochim Biophys Acta, 1830 (2), pp. 2370-2374. | Show Abstract | Read more

BACKGROUND: Hematopoietic stem cells (HSCs) are a population of multipotent cells that can self-renew and differentiate into all blood lineages. HSC development must be tightly controlled from cell fate determination to self-maintenance during adulthood. This involves a panel of important developmental signaling pathways and other factors which act synergistically within the HSC population and/or in the HSC niche. Genetically conserved processes of HSC development plus many other developmental advantages make the zebrafish an ideal model organism to elucidate the regulatory mechanisms underlying HSC programming. SCOPE OF REVIEW: This review summarizes recent progress on zebrafish HSCs with particular focus on how developmental signaling controls hemogenic endothelium-derived HSC development. We also describe the interaction of different signaling pathways during these processes. MAJOR CONCLUSIONS: The hematopoietic stem cell system is a paradigm for stem cell studies. Use of the zebrafish model to study signaling regulation of HSCs in vivo has resulted in a great deal of information concerning HSC biology in vertebrates. GENERAL SIGNIFICANCE: These new findings facilitate a better understanding of molecular mechanisms of HSC programming, and will provide possible new strategies for the treatment of HSC-related hematological diseases, such as leukemia. This article is part of a Special Issue entitled Biochemistry of Stem Cells.

Wilkinson RN, Koudijs MJ, Patient RK, Ingham PW, Schulte-Merker S, van Eeden FJ. 2012. Hedgehog signaling via a calcitonin receptor-like receptor can induce arterial differentiation independently of VEGF signaling in zebrafish. Blood, 120 (2), pp. 477-488. | Show Abstract | Read more

Multiple signaling pathways control the specification of endothelial cells (ECs) to become arteries or veins during vertebrate embryogenesis. Current models propose that a cascade of Hedgehog (Hh), vascular endothelial growth factor (VEGF), and Notch signaling acts instructively on ECs to control the choice between arterial or venous fate. Differences in the phenotypes induced by Hh, VEGF, or Notch inhibition suggest that not all of the effects of Hh on arteriovenous specification are mediated by VEGF. We establish that full derepression of the Hh pathway in ptc1;ptc2 mutants converts the posterior cardinal vein into a second arterial vessel that manifests intact arterial gene expression, intersegmental vessel sprouting, and HSC gene expression. Importantly, although VEGF was thought to be absolutely essential for arterial fates, we find that normal and ectopic arterial differentiation can occur without VEGF signaling in ptc1;ptc2 mutants. Furthermore, Hh is able to bypass VEGF to induce arterial differentiation in ECs via the calcitonin receptor-like receptor, thus revealing a surprising complexity in the interplay between Hh and VEGF signaling during arteriovenous specification. Finally, our experiments establish a dual function of Hh during induction of runx1(+) HSCs.

Blackledge NP, Long HK, Zhou JC, Kriaucionis S, Patient R, Klose RJ. 2012. Bio-CAP: a versatile and highly sensitive technique to purify and characterise regions of non-methylated DNA. Nucleic Acids Res, 40 (4), pp. e32. | Show Abstract | Read more

Across vertebrate genomes methylation of cytosine residues within the context of CpG dinucleotides is a pervasive epigenetic mark that can impact gene expression and has been implicated in various developmental and disease-associated processes. Several biochemical approaches exist to profile DNA methylation, but recently an alternative approach based on profiling non-methylated CpGs was developed. This technique, called CxxC affinity purification (CAP), uses a ZF-CxxC (CxxC) domain to specifically capture DNA containing clusters of non-methylated CpGs. Here we describe a new CAP approach, called biotinylated CAP (Bio-CAP), which eliminates the requirement for specialized equipment while dramatically improving and simplifying the CxxC-based DNA affinity purification. Importantly, this approach isolates non-methylated DNA in a manner that is directly proportional to the density of non-methylated CpGs, and discriminates non-methylated CpGs from both methylated and hydroxymethylated CpGs. Unlike conventional CAP, Bio-CAP can be applied to nanogram quantities of genomic DNA and in a magnetic format is amenable to efficient parallel processing of samples. Furthermore, Bio-CAP can be applied to genome-wide profiling of non-methylated DNA with relatively small amounts of input material. Therefore, Bio-CAP is a simple and streamlined approach for characterizing regions of the non-methylated DNA, whether at specific target regions or genome wide.

Luc S, Luis TC, Boukarabila H, Macaulay IC, Buza-Vidas N, Bouriez-Jones T, Lutteropp M, Woll PS, Loughran SJ, Mead AJ et al. 2012. The earliest thymic T cell progenitors sustain B cell and myeloid lineage potential. Nat Immunol, 13 (4), pp. 412-419. | Show Abstract | Read more

The stepwise commitment from hematopoietic stem cells in the bone marrow to T lymphocyte-restricted progenitors in the thymus represents a paradigm for understanding the requirement for distinct extrinsic cues during different stages of lineage restriction from multipotent to lineage-restricted progenitors. However, the commitment stage at which progenitors migrate from the bone marrow to the thymus remains unclear. Here we provide functional and molecular evidence at the single-cell level that the earliest progenitors in the neonatal thymus had combined granulocyte-monocyte, T lymphocyte and B lymphocyte lineage potential but not megakaryocyte-erythroid lineage potential. These potentials were identical to those of candidate thymus-seeding progenitors in the bone marrow, which were closely related at the molecular level. Our findings establish the distinct lineage-restriction stage at which the T cell lineage-commitment process transits from the bone marrow to the remote thymus.

Wang L, Zhang P, Wei Y, Gao Y, Patient R, Liu F. 2011. A blood flow-dependent klf2a-NO signaling cascade is required for stabilization of hematopoietic stem cell programming in zebrafish embryos. Blood, 118 (15), pp. 4102-4110. | Show Abstract | Read more

Blood flow has long been thought to be important for vessel development and function, but its role in HSC development is not yet fully understood. Here, we take advantage of zebrafish embryos with circulation defects that retain relatively normal early development to illustrate the combinatorial roles of genetic and hemodynamic forces in HSC development. We show that blood flow is not required for initiation of HSC gene expression, but instead is indispensable for its maintenance. Knockdown of klf2a mimics the silent heart (sih/tnnt2a) phenotype while overexpression of klf2a in tnnt2a morphant embryos can rescue HSC defects, suggesting that klf2a is a downstream mediator of blood flow. Furthermore, the expression of NO synthase (nos) was reduced in klf2a knockdown embryos, and ChIP analysis showed that endogenous Klf2a is bound to the promoters of nos genes in vivo, indicating direct gene regulation. Finally, administration of the NO agonist S-nitroso N-acetylpenicillamine (SNAP) can restore HSC development in tnnt2a and klf2a morphants, suggesting that NO signaling is downstream of Klf2a which is induced by hemodynamic forces. Taken together, we have demonstrated that blood flow is essential for HSC development and is mediated by a klf2a-NO signaling cascade in zebrafish.

Simões FC, Peterkin T, Patient R. 2011. Fgf differentially controls cross-antagonism between cardiac and haemangioblast regulators. Development, 138 (15), pp. 3235-3245. | Show Abstract | Read more

Fibroblast growth factor (Fgf) has been implicated in the control of heart size during development, although whether this is by controlling cell fate, survival or proliferation has not been clear. Here, we show that Fgf, without affecting survival or proliferation, acts during gastrulation to drive cardiac fate and restrict anterior haemangioblast fate in zebrafish embryos. The haemangioblast programme was thought to be activated before the cardiac programme and is repressive towards it, suggesting that activation by Fgf of the cardiac programme might be via suppression of the haemangioblast programme. However, we show that the cardiac regulator nkx2.5 can also repress the haemangioblast programme and, furthermore, that cardiac specification still requires Fgf signalling even when haemangioblast regulators are independently suppressed. We further show that nkx2.5 and the cloche candidate gene lycat are expressed during gastrulation and regulated by Fgf, and that nkx2.5 overexpression, together with loss of the lycat targets etsrp and scl can stably induce expansion of the heart. We conclude that Fgf controls cardiac and haemangioblast fates by the simultaneous regulation of haemangioblast and cardiac regulators. We propose that elevation of Fgf signalling in the anterior haemangioblast territory could have led to its recruitment into the heart field during evolution, increasing the size of the heart.

Monteiro R, Pouget C, Patient R. 2011. The gata1/pu.1 lineage fate paradigm varies between blood populations and is modulated by tif1γ. EMBO J, 30 (6), pp. 1093-1103. | Show Abstract | Read more

Lineage fate decisions underpin much of development as well as tissue homeostasis in the adult. A mechanistic paradigm for such decisions is the erythroid versus myeloid fate decision controlled by cross-antagonism between gata1 and pu.1 transcription factors. In this study, we have systematically tested this paradigm in blood-producing populations in zebrafish embryos, including the haematopoietic stem cells (HSCs), and found that it takes a different form in each population. In particular, gata1 activity varies from autostimulation to autorepression. In addition, we have added a third member to this regulatory kernel, tif1γ (transcription intermediate factor-1γ). We show that tif1γ modulates the erythroid versus myeloid fate outcomes from HSCs by differentially controlling the levels of gata1 and pu.1. By contrast, tif1γ positively regulates both gata1 and pu.1 in primitive erythroid and prodefinitive erythromyeloid progenitors. We therefore conclude that the gata1/pu.1 paradigm for lineage decisions takes different forms in different cellular contexts and is modulated by tif1γ.

Noseda M, Peterkin T, Simões FC, Patient R, Schneider MD. 2011. Cardiopoietic factors: extracellular signals for cardiac lineage commitment. Circ Res, 108 (1), pp. 129-152. | Show Abstract | Read more

Cardiac muscle creation during embryogenesis requires extracellular instructive signals that are regulated precisely in time and space, intersecting with intracellular genetic programs that confer or fashion the ability of the cells to respond. Unmasking the essential signals for cardiac lineage decisions has paramount importance for cardiac development and regenerative medicine, including the directed differentiation of progenitor and stem cells to a cardiac muscle fate.

Cited:

38

Scopus

Wang L, Zhang P, Wei Y, Gao Y, Patient R, Liu F. 2011. Ablood flow-dependent klf2a-NO signaling cascade is required for stabilization of hematopoietic stem cell programming in zebrafish embryos Blood, 118 (15), pp. 4102-4110. | Show Abstract | Read more

Blood flow has long been thought to be important for vessel development and function, but its role in HSC development is not yet fully understood. Here, we take advantage of zebrafish embryos with circulation defects that retain relatively normal early development to illustrate the combinatorial roles of genetic and hemodynamic forces in HSC development. We show that blood flow is not required for initiation of HSC gene expression, but instead is indispensable for its maintenance. Knockdown of klf2a mimics the silent heart (sih/tnnt2a) phenotype while overexpression of klf2a in tnnt2a morphant embryos can rescue HSC defects, suggesting that klf2a is a downstream mediator of blood flow. Furthermore, the expression of NO synthase (nos) was reduced in klf2a knockdown embryos, and ChIP analysis showed that endogenous Klf2a is bound to the promoters of nos genes in vivo, indicating direct gene regulation. Finally, administration of the NO agonist S-nitroso N-acetylpenicillamine (SNAP) can restore HSC development in tnnt2a and klf2a morphants, suggesting that NO signaling is downstream of Klf2a which is induced by hemodynamic forces. Taken together, we have demonstrated that blood flow is5 essential for HSC development and is mediated by a klf2a-NO signaling cascade in zebrafish. © 2011 by The American Society of Hematology.

Cited:

43

Scopus

Monteiro R, Pouget C, Patient R. 2011. The gata1/pu.1 lineage fate paradigm varies between blood populations and is modulated by tif1γ EMBO Journal, 30 (6), pp. 1093-1103. | Show Abstract | Read more

Lineage fate decisions underpin much of development as well as tissue homeostasis in the adult. A mechanistic paradigm for such decisions is the erythroid versus myeloid fate decision controlled by cross-antagonism between gata1 and pu.1 transcription factors. In this study, we have systematically tested this paradigm in blood-producing populations in zebrafish embryos, including the haematopoietic stem cells (HSCs), and found that it takes a different form in each population. In particular, gata1 activity varies from autostimulation to autorepression. In addition, we have added a third member to this regulatory kernel, tif1β 3 (transcription intermediate factor-1γ 3). We show that tif1β 3 modulates the erythroid versus myeloid fate outcomes from HSCs by differentially controlling the levels of gata1 and pu.1. By contrast, tif1γ 3 positively regulates both gata1 and pu.1 in primitive erythroid and prodefinitive erythromyeloid progenitors. We therefore conclude that the gata1/pu.1 paradigm for lineage decisions takes different forms in different cellular contexts and is modulated by tif1γ 3. © 2011 European Molecular Biology Organization | All Rights Reserved.

Miller LC, Freter S, Liu F, Taylor JS, Patient R, Begbie J. 2010. Separating early sensory neuron and blood vessel patterning. Dev Dyn, 239 (12), pp. 3297-3302. | Show Abstract | Read more

The anatomical association between sensory nerves and blood vessels is well recognised in the adult, and interactions between the two are important during development. Here we have examined the relationship between developing blood vessels and sensory neuronal cell bodies, which is less well understood. We show in the chick that the nascent dorsal root ganglia (DRG) lie dorsal to the longitudinal anastomosis, adjacent to the developing neural tube at the level of the sulcus limitans. Furthermore, the blood vessel is present prior to the neurons suggesting that it may play a role in positioning the DRG. We use the zebrafish cloche mutation to analyse DRG formation in the absence of blood vessels and show that the DRG are positioned normally. Thus, despite their close anatomical relationship, the patterning of the blood vessel and DRG alongside the neural tube is separable rather than interdependent.

El Omari K, Hoosdally SJ, Tuladhar K, Karia D, Vyas P, Patient R, Porcher C, Mancini EJ. 2011. Structure of the leukemia oncogene LMO2: implications for the assembly of a hematopoietic transcription factor complex. Blood, 117 (7), pp. 2146-2156. | Show Abstract | Read more

The LIM only protein 2 (LMO2) is a key regulator of hematopoietic stem cell development whose ectopic expression in T cells leads to the onset of acute lymphoblastic leukemia. Through its LIM domains, LMO2 is thought to function as the scaffold for a DNA-binding transcription regulator complex, including the basic helix-loop-helix proteins SCL/TAL1 and E47, the zinc finger protein GATA-1, and LIM-domain interacting protein LDB1. To understand the role of LMO2 in the formation of this complex and ultimately to dissect its function in normal and aberrant hematopoiesis, we solved the crystal structure of LMO2 in complex with the LID domain of LDB1 at 2.4 Å resolution. We observe a largely unstructured LMO2 kept in register by the LID binding both LIM domains. Comparison of independently determined crystal structures of LMO2 reveals large movements around a conserved hinge between the LIM domains. We demonstrate that such conformational flexibility is necessary for binding of LMO2 to its partner protein SCL/TAL1 in vitro and for the function of this complex in vivo. These results, together with molecular docking and analysis of evolutionarily conserved residues, yield the first structural model of the DNA-binding complex containing LMO2, LDB1, SCL/TAL1, and GATA-1.

Ciau-Uitz A, Pinheiro P, Gupta R, Enver T, Patient R. 2010. Tel1/ETV6 specifies blood stem cells through the agency of VEGF signaling. Dev Cell, 18 (4), pp. 569-578. | Show Abstract | Read more

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.

Ciau-Uitz A, Liu F, Patient R. 2010. Genetic control of hematopoietic development in Xenopus and zebrafish. Int J Dev Biol, 54 (6-7), pp. 1139-1149. | Show Abstract | Read more

Blood development has been highly conserved during evolution. Hematopoietic cells in amphibian and fish embryos, as in mammalian embryos, emerge and progressively differentiate in several locations. Hematopoiesis, including of the immune system, is similar in the amphibian, Xenopus, to mammals and the embryos are ideal for tissue transplantation and lineage labelling experiments, which have enabled the elucidation of the distinct origins of embryonic and adult hematopoietic cells, as well as their migration pathways and organ colonisation behaviours. The zebrafish hematopoietic system is less well understood, but these embryos have recently emerged as a powerful system for both genetic analysis and imaging. In this review, we summarise our current knowledge of the cellular and genetic basis of ontogeny of the hematopoietic system in Xenopus and zebrafish embryos.

Gering M, Patient R. 2010. Notch signalling and haematopoietic stem cell formation during embryogenesis. J Cell Physiol, 222 (1), pp. 11-16. | Show Abstract | Read more

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 Notch's 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.

Wilkinson RN, Pouget C, Gering M, Russell AJ, Davies SG, Kimelman D, Patient R. 2009. Hedgehog and Bmp polarize hematopoietic stem cell emergence in the zebrafish dorsal aorta. Dev Cell, 16 (6), pp. 909-916. | Show Abstract | Read more

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.

Peterkin T, Gibson A, Patient R. 2009. Common genetic control of haemangioblast and cardiac development in zebrafish. Development, 136 (9), pp. 1465-1474. | Show Abstract | Read more

Over the past few years it has become clear that over half of the mammalian heart derives from outside the heart field as originally defined. Such a second heart field, however, has not been described in zebrafish, which could explain its smaller, two-chambered heart. Instead, zebrafish have a population of haemangioblasts, which is absent in mammalian embryos, raising the possibility that these cells represent the evolutionary ancestor of the second heart field. Here, we show for the first time that the genetic programmes of these anterior haemangioblasts and the adjacent heart field are co-regulated, by transcription factors previously associated with heart but not blood or endothelial development. We demonstrate that gata4, gata5 and gata6 are essential for anterior haemangioblast specification, and for subsequent myelopoiesis, acting as early as cloche and upstream of scl. The requirement for gata4, gata5 and gata6 in myeloid, endothelial and cardiac specification is in the mesoderm, but these factors also control, from within the endoderm and the yolk syncytial layer, the migration of the cardiac precursors as they differentiate. This genetic link between the blood/endothelial and cardiac programmes supports the notion that this haemangioblast population in zebrafish is an evolutionary antecedent of the second heart field, and has implications for the differentiation of haemangioblasts and cardiomyocytes from pluripotent cells, and for the origins of stem cells in the adult heart.

Liu F, Patient R. 2008. Genome-wide analysis of the zebrafish ETS family identifies three genes required for hemangioblast differentiation or angiogenesis. Circ Res, 103 (10), pp. 1147-1154. | Show Abstract | Read more

ETS domain transcription factors have been linked to hematopoiesis, vasculogenesis, and angiogenesis. However, their biological functions and the mechanisms of action, remain incompletely understood. Here, we have performed a systematic analysis of zebrafish ETS domain genes and identified 31 in the genome. Detailed gene expression profiling revealed that 12 of them are expressed in blood and endothelial precursors during embryonic development. Combined with a phylogenetic tree assay, this suggests that some of the coexpressed genes may have redundant or additive functions in these cells. Loss-of-function analysis of 3 of them, erg, fli1, and etsrp, demonstrated that erg and fli1 act cooperatively and are required for angiogenesis possibly via direct regulation of an endothelial cell junction molecule, VE-cadherin, whereas etsrp is essential for primitive myeloid/endothelial progenitors (hemangioblasts) in zebrafish. Taken together, these results provide a global view of the ETS genes in the zebrafish genome during embryogenesis and provide new insights on the functions and biology of erg, fli1, and etsrp, which could be applicable to higher vertebrates, including mice and humans.

Afouda BA, Martin J, Liu F, Ciau-Uitz A, Patient R, Hoppler S. 2008. GATA transcription factors integrate Wnt signalling during heart development. Development, 135 (19), pp. 3185-3190. | Show Abstract | Read more

Cardiogenesis is inhibited by canonical Wnt/beta-catenin signalling and stimulated by non-canonical Wnt11/JNK signalling, but how these two signalling pathways crosstalk is currently unknown. Here, we show that Wnt/beta-catenin signalling restricts cardiogenesis via inhibition of GATA gene expression, as experimentally reinstating GATA function overrides beta-catenin-mediated inhibition and restores cardiogenesis. Furthermore, we show that GATA transcription factors in turn directly regulate Wnt11 gene expression, and that Wnt11 is required to a significant degree for mediating the cardiogenesis-promoting function of GATA transcription factors. These results demonstrate that GATA factors occupy a central position between canonical and non-canonical Wnt signalling in regulating heart muscle formation.

Liu F, Walmsley M, Rodaway A, Patient R. 2008. Fli1 acts at the top of the transcriptional network driving blood and endothelial development. Curr Biol, 18 (16), pp. 1234-1240. | Show Abstract | Read more

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.

Dee CT, Hirst CS, Shih Y-H, Tripathi VB, Patient RK, Scotting PJ. Sox3 regulates both neural fate and differentiation in the zebrafish ectoderm Developmental Biology, | Show Abstract | Read more

Little is known of the first transcriptional events that regulate neural fate in response to extracellular signals such as Bmps and Fgfs. Sox3 is one of the earliest transcription factors to be expressed in the developing CNS and has been shown to be regulated by these signalling pathways. We have used both gain- and loss-of-function experiments in zebrafish to elucidate the role of Sox3 in determining neural fate. Ectopic Sox3 caused induction of neural tissue from a very early stage of cell specification in the ectoderm and this effect was maintained such that large domains of additional CNS were apparent, including almost complete duplications of the CNS. Knock-down of Sox3 using morpholinos resulted in a reduction in the size of the CNS, ears and eyes and subsequent inhibition of some aspects of neurogenesis. Our data also suggest that the pro-neural effects of Sox3 can compensate for inhibition of Fgf signalling in inducing neural tissue but it is not sufficient to maintain neural fate, suggesting the presence of Sox3-independent roles of Fgf at later stages. © 2008 Elsevier Inc. All rights reserved.

Gering M, Patient R. 2008. Notch in the niche. Cell Stem Cell, 2 (4), pp. 293-294. | Show Abstract | Read more

Notch signaling is essential for hematopoietic stem cell (HSC) formation during embryogenesis, and hitherto it was also thought to be required for HSC maintenance. However, in this issue of Cell Stem Cell, Maillard et al. (2008) demonstrate rather conclusively that inactivation of the Notch pathway in HSCs does not interfere with their self-renewal.

Monteiro R, van Dinther M, Bakkers J, Wilkinson R, Patient R, ten Dijke P, Mummery C. 2008. Two novel type II receptors mediate BMP signalling and are required to establish left-right asymmetry in zebrafish. Dev Biol, 315 (1), pp. 55-71. | Show Abstract | Read more

Ligands of the transforming growth factor beta (TGFbeta) superfamily, like Nodal and bone morphogenetic protein (BMP), are pivotal to establish left-right (LR) asymmetry in vertebrates. However, the receptors mediating this process are unknown. Here we identified two new type II receptors for BMPs in zebrafish termed bmpr2a and bmpr2b that induce a classical Smad1/5/8 response to BMP binding. Morpholino-mediated knockdown of bmpr2a and bmpr2b showed that they are required for the establishment of concomitant cardiac and visceral LR asymmetry. Expression of early laterality markers in morphants indicated that bmpr2a and bmpr2b act upstream of pitx2 and the nodal-related southpaw (spaw), which are expressed asymmetrically in the lateral plate mesoderm (LPM), and subsequently regulate lefty2 and bmp4 in the left heart field. We demonstrated that bmpr2a is required for lefty1 expression in the midline at early segmentation while bmpr2a/bmpr2b heteromers mediate left-sided spaw expression in the LPM. We propose a mechanism whereby this differential interpretation of BMP signalling through bmpr2a and bmpr2b is essential for the establishment of LR asymmetry in the zebrafish embryo.

Walmsley M, Cleaver D, Patient R. 2008. Fibroblast growth factor controls the timing of Scl, Lmo2, and Runx1 expression during embryonic blood development. Blood, 111 (3), pp. 1157-1166. | Show Abstract | Read more

To program pluripotent cells into blood, a knowledge of the locations of precursors during their journey through the embryo and the signals they experience would be informative. The anterior (a) and posterior (p) ventral blood islands (VBIs) in Xenopus are derived from opposite sides of the pregastrula embryo. The aVBI goes through a "hemangioblast" state, characterized by coexpression of blood and endothelial genes at neurula stages, whereas the pVBI expresses these genes in a nonoverlapping fashion several hours later, after commitment to either a blood or an endothelial fate. We describe a novel role for fibroblast growth factor (FGF) in controlling the timing of Scl, Lmo2, and Runx1 expression in the 2 VBI compartments. Blocking FGF signaling during gastrulation expands expression at neurula stages into posterior regions. We show, by lineage labeling, explant analysis, and targeted blocking of FGF signaling, that this is due to the pVBI prematurely expressing these genes with the timing of the aVBI. In contrast, overexpression of FGF in aVBI precursors eliminates the anterior hemangioblast program. Using this information, we have recapitulated the anterior hemangioblast program in pluripotent cells in vitro by inhibiting FGF signaling in anterior mesoderm induced by activin and exposed to bone morphogenetic protein (BMP) signaling.

Patient R, Hourioux C, Roingeard P. 2008. Morphogenesis of the hepatitis B virus Virologie, 12 (6), pp. 453-464. | Read more

Dee CT, Hirst CS, Shih YH, Tripathi VB, Patient RK, Scotting PJ. 2008. Sox3 regulates both neural fate and differentiation in the zebrafish ectoderm. Dev Biol, 320 (1), pp. 289-301. | Show Abstract | Read more

Little is known of the first transcriptional events that regulate neural fate in response to extracellular signals such as Bmps and Fgfs. Sox3 is one of the earliest transcription factors to be expressed in the developing CNS and has been shown to be regulated by these signalling pathways. We have used both gain- and loss-of-function experiments in zebrafish to elucidate the role of Sox3 in determining neural fate. Ectopic Sox3 caused induction of neural tissue from a very early stage of cell specification in the ectoderm and this effect was maintained such that large domains of additional CNS were apparent, including almost complete duplications of the CNS. Knock-down of Sox3 using morpholinos resulted in a reduction in the size of the CNS, ears and eyes and subsequent inhibition of some aspects of neurogenesis. Our data also suggest that the pro-neural effects of Sox3 can compensate for inhibition of Fgf signalling in inducing neural tissue but it is not sufficient to maintain neural fate, suggesting the presence of Sox3-independent roles of Fgf at later stages.

Peterkin T, Gibson A, Patient R. 2007. Redundancy and evolution of GATA factor requirements in development of the myocardium. Dev Biol, 311 (2), pp. 623-635. | Show Abstract | Read more

The transcription factors, GATA4, 5 and 6, recognize the same DNA sequence and are all expressed in the developing myocardium. However, knockout studies in the mouse have indicated that none of them are absolutely required for the specification of the myocardium. Here we present evidence for redundancy in this family for the first time. Using morpholinos in both Xenopus and zebrafish embryos, we show that GATA4 knockdown, for example, only affects cardiac marker expression in the absence of either GATA5 or GATA6. A similar situation pertains for GATA5 in Xenopus whereas, in zebrafish, GATA5 (faust) plays a major role in driving the myocardial programme. This requirement for GATA5 in zebrafish is for induction of the myocardium, in contrast to the GATA6 requirement in both species, which is for differentiation. This early role for GATA5 in zebrafish correlates with its earlier expression and with an earlier requirement for BMP signalling, suggesting that a mutual maintenance loop for GATA, BMP and Nkx expression is the evolutionarily conserved entity.

Loose M, Swiers G, Patient R. 2007. Transcriptional networks regulating hematopoietic cell fate decisions. Curr Opin Hematol, 14 (4), pp. 307-314. | Show Abstract | Read more

PURPOSE OF REVIEW: We provide a summary of the temporal cascade of transcriptional networks giving rise to the hematopoietic stem cell (HSC) and controlling differentiation of the erythroid lineage from it. We focus on the mechanisms by which cell fate decisions are made and comment on recent developments and additions to the networks. RECENT FINDINGS: A role for an SCL/LMO2 complex in HSC emergence, as well as in subsequent erythroid differentiation, has received support. Connections between the transcriptional networks and signaling molecules are being made but more work is needed in this area. Evidence that transcriptional cross-antagonistic switches underlie the choice between lineage pathways is increasing, and we highlight how the dynamics of earlier lineage decisions can influence later ones. Mathematical models are being built and reveal a surprising degree of power in these simple motifs to explain lineage choices. SUMMARY: New links in the transcriptional networks underlying cell-fate decisions are constantly emerging, and their incorporation into the evolving networks will make mathematical modeling more precise in its predictions of cell behavior, which can be tested experimentally.

Lok CY, Merryweather-Clarke AT, Brakspear K, Pinheiro P, Patterson L, Patient R, Robson KJH. 2007. Expression of haemochromatosis-associated genes in the zebrafish AMERICAN JOURNAL OF HEMATOLOGY, 82 (6), pp. 577-577.

Smith J, Wardle F, Loose M, Stanley E, Patient R. 2007. Germ layer induction in ESC--following the vertebrate roadmap. Curr Protoc Stem Cell Biol, Chapter 1 pp. Unit-1D.1. | Show Abstract | Read more

Controlled differentiation of pluripotential cells takes place routinely and with great success in developing vertebrate embryos. It therefore makes sense to take note of how this is achieved and use this knowledge to control the differentiation of embryonic stem cells (ESCs). An added advantage is that the differentiated cells resulting from this process in embryos have proven functionality and longevity. This unit reviews what is known about the embryonic signals that drive differentiation in one of the most informative of the vertebrate animal models of development, the amphibian Xenopus laevis. It summarizes their identities and the extent to which their activities are dose-dependent. The unit details what is known about the transcription factor responses to these signals, describing the networks of interactions that they generate. It then discusses the target genes of these transcription factors, the effectors of the differentiated state. Finally, how these same developmental programs operate during germ layer formation in the context of ESC differentiation is summarized.

Soneji S, Huang S, Loose M, Donaldson IJ, Patient R, Göttgens B, Enver T, May G. 2007. Inference, validation, and dynamic modeling of transcription networks in multipotent hematopoietic cells. Ann N Y Acad Sci, 1106 (1), pp. 30-40. | Show Abstract | Read more

Identifying the transcription factor interactions that are responsible for cell-specific gene expression programs is key to understanding the regulation of cell behaviors, such as self-renewal, proliferation, differentiation, and death. The rapidly increasing availability of microarray-derived global gene expression data sets, coupled with genome sequence information from multiple species, has driven the development of computational methods to reverse engineer and dynamically model genetic regulatory networks. An understanding of the architecture and behavior of transcriptional networks should lend insight into how the huge number of potential gene expression programs is constrained and facilitates efforts to direct or redirect cell fate.

Patterson LJ, Gering M, Eckfeldt CE, Green AR, Verfaillie CM, Ekker SC, Patient R. 2007. The transcription factors Scl and Lmo2 act together during development of the hemangioblast in zebrafish. Blood, 109 (6), pp. 2389-2398. | Show Abstract | Read more

The transcription factors Scl and Lmo2 are crucial for development of all blood. An important early requirement for Scl in endothelial development has also been revealed recently in zebrafish embryos, supporting previous findings in scl(-/-) embryoid bodies. Scl depletion culminates most notably in failure of dorsal aorta formation, potentially revealing a role in the formation of hemogenic endothelium. We now present evidence that the requirements for Lmo2 in zebrafish embryos are essentially the same as for Scl. The expression of important hematopoietic regulators is lost, reduced, or delayed, panendothelial gene expression is down-regulated, and aorta-specific marker expression is lost. The close similarity of the phenotypes for Scl and Lmo2 suggest that they perform these early functions in hemangioblast development within a multiprotein complex, as shown for erythropoiesis. Consistent with this, we find that scl morphants cannot be rescued by a non-Lmo2-binding form of Scl but can be rescued by non-DNA-binding forms, suggesting tethering to target genes through DNA-binding partners linked via Lmo2. Interestingly, unlike other hematopoietic regulators, the Scl/Lmo2 complex does not appear to autoregulate, as neither gene's expression is affected by depletion of the other. Thus, expression of these critical regulators is dependent on continued expression of upstream regulators, which may include cell-extrinsic signals.

Dee CT, Gibson A, Rengifo A, Sun SK, Patient RK, Scotting PJ. 2007. A change in response to Bmp signalling precedes ectodermal fate choice. Int J Dev Biol, 51 (1), pp. 79-84. | Show Abstract | Read more

Bone morphogenetic protein (Bmp) signalling plays a central role in the decision of ectoderm to adopt either neural or non-neural fates. The effects of this signalling are seen at mid-gastrulation in the activation of genes such as the Gata factors and the repression of genes such as the SoxB1 transcription factors in the non-neural regions. Using zebrafish embryos, we show that this Bmp signalling does not repress the expression of these same neural markers just 2-3 hours earlier. Since expression of the Bmp signalling effector, Smad1, only begins during early gastrulation, we tested the role of Smad1 and Smad5 (which is maternally expressed) in controlling gene expression both before and during gastrulation. This showed that the absence of Smad1 does not explain the lack of response of neural genes to Bmp signalling at early stages. However, these experiments showed that expression of the non-neural marker, gata2, is mediated by Smad5 in the absence of Smad1 at early stages, but is dependent upon Smad1 at later stages. Hence, we have shown a dynamic change in the molecular machinery underlying the Bmp response in the ectoderm during gastrulation stages of development.

Meier N, Krpic S, Rodriguez P, Strouboulis J, Monti M, Krijgsveld J, Gering M, Patient R, Hostert A, Grosveld F. 2006. Novel binding partners of Ldb1 are required for haematopoietic development. Development, 133 (24), pp. 4913-4923. | Show Abstract | Read more

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.

Fletcher G, Jones GE, Patient R, Snape A. 2006. A role for GATA factors in Xenopus gastrulation movements. Mech Dev, 123 (10), pp. 730-745. | Show Abstract | Read more

Gastrulation movements in Xenopus laevis are becoming increasingly well characterised, however the molecular mechanisms involved are less clear. Active migration of the leading edge mesendoderm across the fibronectin-coated blastocoel roof is necessary for further development of tissues such as head mesoderm, heart, blood and liver. The zinc finger transcription factors GATA4 and GATA6 are expressed in this migratory tissue during gastrulation, but their role here is unknown. This study further characterises the expression of GATA4 and 6 during gastrulation, and investigates their function in migratory behaviour. Gain-of-function experiments with these GATA factors induce cell spreading, polarisation and migration in non-motile presumptive ectoderm cells. Expression of a dominant-interfering form of GATA6, which inhibits transactivation of GATA targets, severely impairs the ability of dorsal leading edge mesendoderm to spread and translocate on fibronectin. Mosaic inhibition of GATA activity indicates that GATA factors function cell autonomously to induce cell spreading and movement in dorsal mesendoderm. Knockdown of specific GATA factors using anti-sense morpholinos indicates that GATA4 and GATA6 both contribute to dorsal mesendoderm migration in vitro. GATA4 and GATA6 are known to be involved in cell-specification of mesoderm and endoderm-derived tissues, but this is the first description of an additional role for these factors in cell migration.

Loose M, Patient R. 2006. Global genetic regulatory networks controlling hematopoietic cell fates. Curr Opin Hematol, 13 (4), pp. 229-236. | Show Abstract | Read more

PURPOSE OF REVIEW: The gene expression profile of a cell is a consequence of transcription factor activities, which, in turn, are controlled by extra-cellular signals. The relationships between all these regulators constitute a genetic regulatory network, which can be used to predict the behavior of the cell in changing environments. We outline the progress being made to identify Genetic Regulatory Networks for hematopoiesis, using gene-by-gene approaches or emerging genomic technologies. RECENT FINDINGS: The construction of genetic regulatory networks for single and multicellular organisms has inspired the building of genetic regulatory networks for erythropoiesis and B-cell differentiation. genetic regulatory networks are 'scale-free', whereby some genes have many connections while others have very few. The well connected genes, or hubs, correspond to master regulators of the networks, acting to integrate signals and control the sequential passage of the cells through the differentiation process. Lineage decisions are governed by cross-antagonism between two hubs. Large datasets from genome-wide analyses support the concept of multilineage priming and will increasingly refine the network topologies. SUMMARY: As the underlying genetic regulatory networks for hematopoiesis continue to emerge, the program for lineage choice and differentiation will be revealed. More large-scale datasets identifying network components are needed alongside continued gene-by-gene analyses.

Swiers G, Patient R, Loose M. 2006. Genetic regulatory networks programming hematopoietic stem cells and erythroid lineage specification. Dev Biol, 294 (2), pp. 525-540. | Show Abstract | Read more

Erythroid cell production results from passage through cellular hierarchies dependent on differential gene expression under the control of transcription factors responsive to changing niches. We have constructed Genetic Regulatory Networks (GRNs) describing this process, based predominantly on mouse data. Regulatory network motifs identified in E. coli and yeast GRNs are found in combination in these GRNs. Feed-forward motifs with autoregulation generate forward momentum and also control its rate, which is at its lowest in hematopoietic stem cells (HSCs). The simultaneous requirement for multiple regulators in multi-input motifs (MIMs) provides tight control over expression of target genes. Combinations of MIMs, exemplified by the SCL/LMO2 complexes, which have variable content and binding sites, explain how individual regulators can have different targets in HSCs and erythroid cells and possibly also how HSCs maintain stem cell functions while expressing lineage-affiliated genes at low level, so-called multi-lineage priming. MIMs combined with cross-antagonism describe the relationship between PU.1 and GATA-1 and between two of their target genes, Fli-1 and EKLF, with victory for GATA-1 and EKLF leading to erythroid lineage specification. These GRNs are useful repositories for current regulatory information, are accessible in interactive form via the internet, enable the consequences of perturbation to be predicted, and can act as seed networks to organize the rapidly accumulating microarray data.

Priddle H, Jones DR, Burridge PW, Patient R. 2006. Hematopoiesis from human embryonic stem cells: overcoming the immune barrier in stem cell therapies. Stem Cells, 24 (4), pp. 815-824. | Show Abstract | Read more

The multipotency and proliferative capacity of human embryonic stem cells (hESCs) make them a promising source of stem cells for transplant therapies and of vital importance given the shortage in organ donation. Recent studies suggest some immune privilege associated with hESC-derived tissues. However, the adaptability of the immune system makes it unlikely that fully differentiated tissues will permanently evade immune rejection. One promising solution is to induce a state of immune tolerance to a hESC line using tolerogenic hematopoietic cells derived from it. This could provide acceptance of other differentiated tissues from the same line. However, this approach will require efficient multilineage hematopoiesis from hESCs. This review proposes that more efficient differentiation of hESCs to the tolerogenic cell types required is most likely to occur through applying knowledge gained of the ontogeny of complex regulatory signals used by the embryo for definitive hematopoietic development in vivo. Stepwise formation of mesoderm, induction of definitive hematopoietic stem cells, and the application of factors key to their self-renewal may improve in vitro production both quantitatively and qualitatively.

Patterson LJ, Patient R. 2006. The "Ets" factor: Vessel formation in zebrafish - The missing link? PLOS BIOLOGY, 4 (1), pp. 21-24. | Read more

Patterson LJ, Patient R. 2006. The "Ets" factor: Vessel formation in zebrafish - The missing link? PLoS Biology, 4 (1), pp. 0021-0024. | Read more

Patterson LJ, Patient R. 2006. The "Ets" factor: vessel formation in zebrafish--the missing link? PLoS Biol, 4 (1), pp. e24. | Read more

Alexandrovich A, Arno M, Patient RK, Shah AM, Pizzey JA, Brewer AC. 2006. Wnt2 is a direct downstream target of GATA6 during early cardiogenesis. Mech Dev, 123 (4), pp. 297-311. | Show Abstract | Read more

The GATA4, 5 and 6 subfamily of transcription factors are potent transactivators of transcription expressed within the precardiac mesoderm. However, little is known of the immediate downstream targets of GATA-factor regulation during the earliest stages of cardiogenesis. Using the P19-CL6 embryonal carcinoma (EC) cell line as an in vitro model of cardiogenesis, we show that GATA6 is the most abundantly expressed of the GATA factors in presumptive cardiac cells. Consequently, we performed a microarray screen comparing mRNA from control EC cells, early in the cardiac differentiation pathway, with those in which GATA6 had been overexpressed. These studies identified 103 genes whose expression changed significantly and this was verified in a representative array of these genes by real-time RT-PCR. We show that early cardiac expression of one of these genes, Wnt2, mirrors that of GATA6 in vitro and in vivo. In addition, its upregulation by GATA6 in differentiating EC cells is mediated by the direct binding of GATA-factor(s) to the cognate Wnt2 promoter, suggesting Wnt2 is an immediate downstream target of GATA-factor regulation during early cardiogenesis.

Rengifo A, Gibson A, Dee C, Sun S-K, Patient RK, Scotting PJ. 2005. Expression of SOX and GATA demonstrates change in sensitivity to BMP signalling at the onset of Gastrulation MECHANISMS OF DEVELOPMENT, 122 pp. S33-S33.

Gibson A, Patient R. 2005. Gata4, 5 or 6 are essential for heart and blood development in the zebrafish MECHANISMS OF DEVELOPMENT, 122 pp. S168-S169.

Patterson LJ, Gering M, Patient R. 2005. Scl is required for dorsal aorta as well as blood formation in zebrafish embryos. Blood, 105 (9), pp. 3502-3511. | Show Abstract | Read more

Blood and endothelial cells arise in close association in developing embryos, possibly from a shared precursor, the hemangioblast, or as hemogenic endothelium. The transcription factor, Scl/Tal1 (stem cell leukemia protein), is essential for hematopoiesis but thought to be required only for remodeling of endothelium in mouse embryos. By contrast, it has been implicated in hemangioblast formation in embryoid bodies. To resolve the role of scl in endothelial development, we knocked down its synthesis in zebrafish embryos where early precursors and later phenotypes can be more easily monitored. With respect to blood, the zebrafish morphants phenocopied the mouse knockout and positioned scl in the genetic hierarchy. Importantly, endothelial development was also clearly disrupted. Dorsal aorta formation was substantially compromised and gene expression in the posterior cardinal vein was abnormal. We conclude that scl is especially critical for the development of arteries where adult hematopoietic stem cells emerge, implicating scl in the formation of hemogenic endothelium.

Gering M, Patient R. 2005. Hedgehog signaling is required for adult blood stem cell formation in zebrafish embryos. Dev Cell, 8 (3), pp. 389-400. | Show Abstract | Read more

Studies with embryonic explants and embryonic stem cells have suggested a role for Hedgehog (Hh) signaling in hematopoiesis. However, targeted deletion of Hh pathway components in the mouse has so far failed to provide in vivo evidence. Here we show that zebrafish embryos mutant in the Hh pathway or treated with the Hh signaling inhibitor cyclopamine display defects in adult hematopoietic stem cell (HSC) formation but not in primitive hematopoiesis. Hh is required in the trunk at three consecutive stages during vascular development: for the medial migration of endothelial progenitors of the dorsal aorta (DA), for arterial gene expression, and for the formation of intersomitic vessel sprouts. Interference with Hh signaling during the first two stages also interferes with HSC formation. Furthermore, HSC and DA formation also share Vegf and Notch requirements, which further distinguishes them from primitive hematopoiesis and underlines their close relationship during vertebrate development.

Afouda BA, Ciau-Uitz A, Patient R. 2005. GATA4, 5 and 6 mediate TGFbeta maintenance of endodermal gene expression in Xenopus embryos. Development, 132 (4), pp. 763-774. | Show Abstract | Read more

The individual contributions of the three vertebrate GATA factors to endoderm formation have been unclear. Here we detail the early expression of GATA4, 5 and 6 in presumptive endoderm in Xenopus embryos and their induction of endodermal markers in presumptive ectoderm. Induction of HNF3beta by all three GATA factors was abolished when protein synthesis was inhibited, showing that these inductions are indirect. In contrast, whereas induction of Sox17alpha and HNF1beta by GATA4 and 5 was substantially reduced when protein synthesis was inhibited, induction by GATA6 was minimally affected, suggesting that GATA6 is a direct activator of these early endodermal genes. GATA4 induced GATA6 expression in the same assay and antisense morpholino oligonucleotides (MOs), designed to knock down translation of GATA6, blocked induction of Sox17alpha and HNF1beta by GATA4, suggesting that GATA4 induces these genes via GATA6 in this assay. All three GATA factors were induced by activin, although GATA4 and 6 required lower concentrations. GATA MOs inhibited Sox17alpha and HNF1beta induction by activin at low and high concentrations in the order: GATA6>GATA4>GATA5. Together with the timing of their expression and the effects of GATA MOs in vivo, these observations identify GATA6 as the predominant GATA factor in the maintenance of endodermal gene expression by TGFbeta signaling in gastrulating embryos. In addition, examination of gene expression and morphology in later embryos, revealed GATA5 and 6 as the most critical for the development of the gut and the liver.

Peterkin T, Gibson A, Loose M, Patient R. 2005. The roles of GATA-4, -5 and -6 in vertebrate heart development. Semin Cell Dev Biol, 16 (1), pp. 83-94. | Show Abstract | Read more

The transcription factors GATA-4, -5 and -6 are expressed very early in heart tissue. Essential GATA sites have been detected in several cardiac genes and the cardiac GATA factors interact with a wide variety of cofactors which synergistically increase gene expression. These multi-protein transcriptional complexes confer promoter-specificity on the GATA factors and also on the more broadly expressed cofactors. Here we summarise the data on these interactions and represent the conclusions as a GATA factor-based genetic regulatory network for the heart. Of the three cardiac GATAs, GATA-4 is by far the most extensively studied, however, loss-of-function data question its presumed dominance during heart development as opposed to hypertrophy.

Brewer AC, Alexandrovich A, Mjaatvedt CH, Shah AM, Patient RK, Pizzey JA. 2005. GATA factors lie upstream of Nkx 2.5 in the transcriptional regulatory cascade that effects cardiogenesis. Stem Cells Dev, 14 (4), pp. 425-439. | Show Abstract | Read more

Members of the GATA-4, -5, and -6 subfamily of transcription factors are co-expressed with the homeoprotein Nkx 2.5 in the precardiac mesoderm during the earliest stages of its specification and are known to be important determinants of cardiac gene expression. Ample evidence suggests that GATA factors and Nkx 2.5 cross-regulate each other's expression; however, the temporal order of the expression of these transcription factors in vivo remains unresolved, and thus precise definition of the role of the products of the genes they transcribe in early development has been difficult to assess. We employed P19 CL6 mouse embryonic carcinoma cells as a model to investigate this problem, because these cells, like embryonic stem cells, can be induced to differentiate along multiple lineages. Here we demonstrate that when P19 CL6 cells are induced to differentiate to a cardiogenic lineage, the expression of GATA-4 and GATA-6 is up-regulated prior to the transcriptional activation of Nkx 2.5. Moreover, over-expression of GATA-4 or -6 at the time of Nkx 2.5 induction results in a significant up-regulation of endogenous Nkx 2.5 transcription. Finally, it is known that a Nkx-dependent enhancer is necessary for GATA-6 expression within cardiomyocytes of the developing mouse embryo. We demonstrate that within undifferentiated P19 CL6 cells, GATA-6 expression is subject to active repression by a novel upstream element that possesses binding sites for factors involved in transcriptional repression that are conserved between mammalian species.

Walmsley M, Ciau-Uitz A, Patient R. 2005. Tracking and programming early hematopoietic cells in Xenopus embryos. Methods Mol Med, 105 pp. 123-136. | Show Abstract

The fates of lineage labeled hematopoietic precursor populations in Xenopus embryos are followed by use of in situ hybridization, looking for overlap between lineage labeled cells and in situ probes specific for known cell populations or states of differentiation. By coinjection of dominant interfering constructs, it also is possible to define the environmental cues or signals required for specification and/or maintenance of the hematopoietic program at different times and locations in the early embryo. As a lineage trace, we use beta-galactosidase, which is injected as in vitro synthesized ribonucleic acid (RNA) in to Xenopus embryos at early cleavage stages. Because the interfering constructs we use also are in the form of injected RNA, the use of beta-galactosidase RNA as a lineage trace assures accurate determination of the cells expressing the dominant negative construct. Embryos are cultured to desired developmental stages, fixed briefly and processed for the beta-galactosidase reaction. Embryos are then analyzed by whole mount in situ hybridization, embedded in wax, and sectioned. Alternatively, after the beta-galactosidase reaction, embryos can be fixed long term in paraformaldehyde, mounted in wax, sectioned, and probed by in situ hybridization directly on sections.

Abu-Daya A, Steer WM, Trollope AF, Friedeberg CE, Patient RK, Thorne AW, Guille MJ. 2005. Zygotic nucleosome assembly protein-like 1 has a specific, non-cell autonomous role in hematopoiesis. Blood, 106 (2), pp. 514-520. | Show Abstract | Read more

Nucleosome assembly proteins (NAPs) bind core histones, facilitate chromatin remodeling, and can act as transcriptional coactivators. We previously described the isolation of a Xenopus NAP1-like (xNAP1L) cDNA, which encodes a member of this protein family. Its zygotic expression is restricted to neural cells, the outer cells of the ventral blood island (VBIs), and the ectoderm overlying the blood precursors. Here, we report that depletion of zygotic xNAP1L in embryos produces no obvious morphologic phenotype, but ablates alpha-globin mRNA expression in the VBIs. Transcript levels of the hematopoietic precursor genes SCL and Xaml (Runx-1) are also reduced in the VBIs. SCL expression can be rescued by injection of xNAP1L mRNA into the ectoderm, showing that the effect of xNAP1L can be non-cell autonomous. Fli1 and Hex, genes expressed in hemangioblasts but subsequently endothelial markers, were unaffected, suggesting that xNAP1L is required for the hematopoietic lineage specifically. Our data are consistent with a requirement for xNAP1L upstream of SCL, and injection of SCL mRNA into xNAP1L-depleted embryos rescues alpha-globin expression. Thus, xNAP1L, which belongs to a family of proteins previously believed to have general roles, has a specific function in hematopoiesis.

Bachvarova RF, Masi T, Drum M, Parker N, Mason K, Patient R, Johnson AD. 2004. Gene expression in the axolotl germ line: Axdazl, Axvh, Axoct-4, and Axkit. Dev Dyn, 231 (4), pp. 871-880. | Show Abstract | Read more

Primordial germ cells (PGCs) in embryos of mammals and urodele amphibians are formed by induction in the absence of germ plasm. We describe expression of four germ cell-related genes through the germ cell cycle of the axolotl. The orthologs of vasa and daz-like are up-regulated in PGCs of tail bud embryos before the gonad forms and are expressed throughout the female germ cell cycle. Mammalian Oct-4 is a marker of pluripotency in embryonic cells. Axolotl Oct-4 has higher homology to Oct-4 than that found in other vertebrates. It is expressed in the equivalent of the mouse epiblast, in the posterior mesoderm of late gastrulae that gives rise to PGCs, and in diplotene growing oocytes, but not in presumptive PGCs after gastrulation. Finally, a c-kit homolog is expressed in gonadal oogonia and growing oocytes as in mice but is also not found in PGCs. The expression pattern in urodele gonadal germ cells is similar to that of other vertebrates, although the pattern in pregonadal PGCs is distinctly different from that of mice. We conclude that PGCs are restricted to the germ line later in urodeles than in mice or lack migration and proliferation programs.

Loose M, Patient R. 2004. A genetic regulatory network for Xenopus mesendoderm formation. Dev Biol, 271 (2), pp. 467-478. | Show Abstract | Read more

We have constructed a genetic regulatory network (GRN) summarising the functional relationships between the transcription factors (TFs) and embryonic signals involved in Xenopus mesendoderm formation. It is supported by a relational database containing the experimental evidence and both are available in interactive form via the World Wide Web. This network highlights areas for further study and provides a framework for systematic interrogation of new data. Comparison with the equivalent network for the sea urchin identifies conserved features of the deuterostome ancestral pathway, including positive feedback loops, GATA factors, SoxB, Brachyury and a previously underemphasised role for beta-catenin. In contrast, some features central to one species have not yet been found in the other, for example, Krox and Otx in sea urchin, and Mix and Nodal in Xenopus. Such differences may represent evolved features or may eventually be resolved. For example, in Xenopus, Nodal-related genes are positively regulated by beta-catenin and at least one of them is repressed by Sox3, as is the uncharacterised early signal (ES) inducing endomesoderm in the sea urchin, suggesting that ES may be a Nodal-like TGF-beta. Wider comparisons of such networks will inform our understanding of developmental evolution.

Pinheiro P, Gering M, Patient R. 2004. The basic helix-loop-helix transcription factor, Tal2, marks the lateral floor plate of the spinal cord in zebrafish. Gene Expr Patterns, 4 (1), pp. 85-92. | Show Abstract | Read more

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.

Johnson AD, Crother B, White ME, Patient R, Bachvarova RF, Drum M, Masi T. 2003. Regulative germ cell specification in axolotl embryos: a primitive trait conserved in the mammalian lineage. Philos Trans R Soc Lond B Biol Sci, 358 (1436), pp. 1371-1379. | Show Abstract | Read more

How germ cells are specified in the embryos of animals has been a mystery for decades. Unlike most developmental processes, which are highly conserved, embryos specify germ cells in very different ways. Curiously, in mouse embryos germ cells are specified by extracellular signals; they are not autonomously specified by maternal germ cell determinants (germ plasm), as are the germ cells in most animal model systems. We have developed the axolotl (Ambystoma mexicanum), a salamander, as an experimental system, because classic experiments have shown that the germ cells in this species are induced by extracellular signals in the absence of germ plasm. Here, we provide evidence that the germ cells in axolotls arise from naive mesoderm in response to simple inducing agents. In addition, by analysing the sequences of axolotl germ-cell-specific genes, we provide evidence that mice and urodele amphibians share a common mechanism of germ cell development that is ancestral to tetrapods. Our results imply that germ plasm, as found in species such as frogs and teleosts, is the result of convergent evolution. We discuss the evolutionary implications of our findings.

Peterkin T, Gibson A, Patient R. 2003. GATA-6 maintains BMP-4 and Nkx2 expression during cardiomyocyte precursor maturation. EMBO J, 22 (16), pp. 4260-4273. | Show Abstract | Read more

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.

Brewer A, Nemer G, Gove C, Rawlins F, Nemer M, Patient R, Pizzey J. 2003. Erratum: Widespread expression of an extended peptide sequence of GATA-6 during murine embryo-genesis and non-equivalence of RNA and protein expression domains (Gene Expression Patterns (2002) vol. 2 (123-131)) Gene Expression Patterns, 3 (4), pp. 543. | Read more

Gering M, Yamada Y, Rabbitts TH, Patient RK. 2003. Lmo2 and Scl/Tal1 convert non-axial mesoderm into haemangioblasts which differentiate into endothelial cells in the absence of Gata1. Development, 130 (25), pp. 6187-6199. | Show Abstract | Read more

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.

Walmsley M, Ciau-Uitz A, Patient R. 2002. Adult and embryonic blood and endothelium derive from distinct precursor populations which are differentially programmed by BMP in Xenopus. Development, 129 (24), pp. 5683-5695. | Show Abstract | Read more

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.

Brewer A, Nemer G, Gove C, Rawlins F, Nemer M, Patient R, Pizzey J. 2002. Widespread expression of an extended peptide sequence of GATA-6 during murine embryogenesis and non-equivalence of RNA and protein expression domains. Mech Dev, 119 Suppl 1 (SUPPL. 1), pp. S121-S129. | Show Abstract | Read more

The transcription factor GATA-6 is known to be a critical determinant of early vertebrate development. We have shown previously that mammalian GATA-6 genes have the potential to encode two protein isoforms, resulting from alternative, in-frame, initiator methionine codons. We have generated GATA-6 antibodies, including one specific to the longer form of GATA-6, and by immunohistochemical analysis we demonstrate here that the longer protein, which is the more potent transcriptional transactivator, is widely expressed in vivo. In accordance with previous RNA expression studies, GATA-6 protein was found to be abundant within regions of the gut and pulmonary systems, in addition to the heart myocardium. We also report novel GATA-6 expression within sites of chondrogenesis derived from cranial neural crest and sclerotomes. Surprisingly however, levels of GATA-6 protein were substantially reduced within the endocardial cushions and outflow tract of the heart. These are regions which express the highest levels of GATA-6 RNA within the heart.

Brewer A, Nemer G, Gove C, Rawlins F, Nemer M, Patient R, Pizzey J. 2002. Widespread expression of an extended peptide sequence of GATA-6 during murine embryogenesis and non-equivalence of RNA and protein expression domains. Gene Expr Patterns, 2 (1-2), pp. 123-131. | Show Abstract | Read more

The transcription factor GATA-6 is known to be a critical determinant of early vertebrate development. We have shown previously that mammalian GATA-6 genes have the potential to encode two protein isoforms, resulting from alternative, in-frame, initiator methionine codons. We have generated GATA-6 antibodies, including one specific to the longer form of GATA-6, and by immunohistochemical analysis we demonstrate here that the longer protein, which is the more potent transcriptional transactivator, is widely expressed in vivo. In accordance with previous RNA expression studies, GATA-6 protein was found to be abundant within regions of the gut and pulmonary systems, in addition to the heart myocardium. We also report novel GATA-6 expression within sites of chondrogenesis derived from cranial neural crest and sclerotomes. Surprisingly however, levels of GATA-6 protein were substantially reduced within the endocardial cushions and outflow tract of the heart. These are regions which express the highest levels of GATA-6 RNA within the heart.

Göttgens B, Nastos A, Kinston S, Piltz S, Delabesse EC, Stanley M, Sanchez MJ, Ciau-Uitz A, Patient R, Green AR. 2002. Establishing the transcriptional programme for blood: the SCL stem cell enhancer is regulated by a multiprotein complex containing Ets and GATA factors. EMBO J, 21 (12), pp. 3039-3050. | Show Abstract | Read more

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.

Patient RK, McGhee JD. 2002. The GATA family (vertebrates and invertebrates). Curr Opin Genet Dev, 12 (4), pp. 416-422. | Show Abstract | Read more

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.

Barton LM, Gottgens B, Gering M, Gilbert JG, Grafham D, Rogers J, Bentley D, Patient R, Green AR. 2001. Regulation of the stem cell leukemia (SCL) gene: a tale of two fishes. Proc Natl Acad Sci U S A, 98 (12), pp. 6747-6752. | Show Abstract | Read more

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.

Rodaway A, Patient R. 2001. Mesendoderm. an ancient germ layer? Cell, 105 (2), pp. 169-172. | Read more

Ciau-Uitz A, Walmsley M, Gering M, Loose M, Patient R. 2000. Origins and programming of hematopoietic stem cells in fish and frog embryos. BLOOD CELLS MOLECULES AND DISEASES, 26 (5), pp. 501-501.

Ciau-Uitz A, Walmsley M, Patient R. 2000. Distinct origins of adult and embryonic blood in Xenopus. Cell, 102 (6), pp. 787-796. | Show Abstract | Read more

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.

Weber H, Symes CE, Walmsley ME, Rodaway AR, Patient RK. 2000. A role for GATA5 in Xenopus endoderm specification. Development, 127 (20), pp. 4345-4360. | Show Abstract

The endoderm gives rise to the gut and tissues that develop as outgrowths of the gut tube, including the lungs, liver and pancreas. Here we show that GATA5, a zinc-finger transcription factor, is expressed in the yolk-rich vegetal cells of Xenopus embryos from the early gastrula stage onwards, when these cells become committed to form endoderm. At mid-gastrula stages, GATA5 is restricted to the sub-blastoporal endoderm and is the first molecular marker for this subset of endodermal cells so far identified. We show that GATA4 and GATA5 are potent inducers of endodermal marker genes in animal cap assays, while other GATA factors induce these genes only weakly, if at all. When injected into the dorsal marginal zone, GATA5 respecifies prospective mesoderm towards an endodermal fate, thereby disrupting the convergence and extension movements normally undergone by the dorsal mesoderm. The resulting phenotype is very similar to those seen after injection of dominant negative versions of the FGF-receptor or the T-box transcription factor, Xbra and can be rescued by eFGF. The ability of GATA5 to respecify ectodermal and mesodermal cells towards endoderm suggests an important role for GATA5 in the formation of this germlayer. In animal cap assays, GATA5 is induced by concentrations of activin above those known to induce dorsal mesoderm and heart, in an FGF-independent manner. These data indicate that the emerging view for endodermal induction in general, namely that it is specified by high levels of TGF-beta in the absence of FGF signalling, is specifically true for sub-blastoporal endoderm.

Cited:

203

Scopus

Brown LA, Rodaway ARF, Schilling TF, Jowett T, Ingham PW, Patient RK, Sharrocks AD. 2000. Insights into early vasculogenesis revealed by expression of the ETS-domain transcription factor Fli-1 in wild-type and mutant zebrafish embryos Mechanisms of Development, 90 (2), pp. 237-252. | Show Abstract | Read more

Fli-1 is an ETS-domain transcription factor whose locus is disrupted in Ewing's Sarcoma and F-MuLV induced erythroleukaemia. To gain a better understanding of its normal function, we have isolated the zebrafish homologue. Similarities with other vertebrates, in the amino acid sequence and DNA binding properties of Fli-1 from zebrafish, suggest that its function has been conserved during vertebrate evolution. The initial expression of zebrafish fli-1 in the posterior lateral mesoderm overlaps with that of gata2 in a potential haemangioblast population which likely contains precursors of blood and endothelium. Subsequently, fli-1 and gata2 expression patterns diverge, with separate fli-1 and gata2 expression domains arising in the developing vasculature and in sites of blood formation respectively. Elsewhere in the embryo, fli-1 is expressed in sites of vasculogenesis. The expression of fli-1 was investigated in a number of zebrafish mutants, which affect the circulatory system. In cloche, endothelium is absent and blood is drastically reduced. In contrast to the blood and endothelial markers that have been studied previously, fli-1 expression was initiated normally in cloche embryos, indicating that induction of fli-1 is one of the earliest indicators of haemangioblast formation. Furthermore, although fli-1 expression in the trunk was not maintained, the normal expression pattern in the anterior half of the embryo was retained. These anterior cells did not, however, condense to form blood vessels. These data indicate that cloche has previously unsuspected roles at multiple stages in the formation of the vasculature. Analysis of fli-1 expression in midline patterning mutants floating head and squint, confirms a requirement for the notochord in the formation of the dorsal-aorta. The formation of endothelium in one-eyed pinhead, cyclops and squint embryos indicates a novel role for the endoderm in the formation of the axial vein. The phenotype of sonic-you mutants implies a likely role for Sonic Hedgehog in mediating these processes. Copyright (C) 2000 Elsevier Science Ireland Ltd.

Brewer A, Gove C, Davies A, McNulty C, Barrow D, Koutsourakis M, Farzaneh F, Pizzey J, Bomford A, Patient R. 1999. The human and mouse GATA-6 genes utilize two promoters and two initiation codons. J Biol Chem, 274 (53), pp. 38004-38016. | Show Abstract | Read more

GATA-6 has been implicated in the regulation of myocardial differentiation during cardiogenesis. To determine how its expression is controlled, we have characterized the human and mouse genes. We have mapped their transcriptional start sites and demonstrate that two alternative promoters and 5' noncoding exons are utilized. Both transcript isoforms are expressed in the same tissue-specific and developmental stage-specific pattern, and their ratio appears similar wherever examined. The more upstream noncoding exon showed a substantial degree of homology between the two mammalian species, suggesting a conserved regulatory function. Moreover, in transfection assays we show that elements within this exon act to promote its transcription. Positive regulatory elements that effect transcription from the more downstream exon were not apparent in this assay, revealing a regulatory distinction between the two promoters. We also demonstrate alternative initiator codon usage in both the human and mouse GATA-6 genes. Both isoforms of the protein are synthesized in vitro regardless of which 5' noncoding exon is present in the RNA, although the larger protein has greater transcriptional activation potential in transfection assays. Thus, GATA-6 function in the cell is controlled by a complex interplay of transcriptional and translational regulation.

Modelling blood stem cell development in vitro

Over the past decade, numerous efforts have been deployed to produce the elusive blood (haematopoietic) stem cell (HSC) in vitro from pluripotent stem cells (PSCs) for mechanistic and therapeutic purposes. However, none of the differentiation cultures developed so far have been able to produce long-term repopulating HSCs, the cells with self-renewal and multilineage potentialities that give rise to the entire blood system when injected into a host organism. This is likely to reflect the lack of ...

View project

1521