flk-1, an flt-related receptor tyrosine kinase is an early marker for endothelial cell precursors

We have used RT-PCR to screen pluripotent murine embryonic stem cells to identify receptor tyrosine kinases (RTKs) potentially involved in the determination or differentiation of cell lineages during early mouse development. Fourteen different tyrosine kinase sequences were identified. The expression patterns of four RTKs have been examined and all are expressed in the mouse embryo during, or shortly after, gastrulation. We report here the detailed expression pattern of one such RTK, the flt-related gene flk-1. In situ hybridization analysis of the late primitive streak stage embryo revealed that flk-1 was expressed in the proximal-lateral embryonic mesoderm; tissue fated to become heart. By headfold stages, staining was confined to the endocardial cells of the heart primordia as well as to the blood islands of the visceral yolk sac and the developing allantois. Patchy, speckled staining was detected in the endothelium of all the major embryonic and extraembryonic blood vessels as they formed. During early organogenesis, expression was detected in the blood vessels of highly vascularized tissues such as the brain, liver, lungs and placenta. Since flk-1 was expressed in early mesodermal cells prior to any morphological evidence for endothelial cell differentiation (vasculogenesis), as well as in cells that form blood vessels from preexisting ones (angiogenesis), it appears to be a very early marker of endothelial cell precursors. We have previously reported that another novel RTK, designated tek, was expressed in differentiating endothelial cells. We show here that flk-1 transcripts are expressed one full embryonic day earlier than the first tek transcripts. The expression of these two RTKs appear to correlate with the specification and early differentiation of the endothelial cell lineage respectively, and therefore may play important roles in the establishment of this lineage.

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Hex: a homeobox gene revealing peri-implantation asymmetry in the mouse embryo and an early transient marker of endothelial cell precursors

Development ◽

10.1242/dev.125.1.85 ◽

1998 ◽

Vol 125 (1) ◽

pp. 85-94 ◽

Cited By ~ 14

Author(s):

P.Q. Thomas ◽

A. Brown ◽

R.S. Beddington

Keyword(s):

Endothelial Cell ◽

Mouse Embryo ◽

Expression Patterns ◽

Homeobox Gene ◽

Definitive Endoderm ◽

Visceral Endoderm ◽

Primitive Endoderm ◽

Mouse Development ◽

Expression Domain ◽

Early Marker

The divergent homeobox gene Hex exhibits three notable expression patterns during early mouse development. Initially Hex is expressed in the primitive endoderm of the implanting blastocyst but by 5.5 dpc its transcripts are present only in a small patch of visceral endoderm at the distal tip of the egg cylinder. Lineage analysis shows that these cells move unilaterally to assume an anterior position while continuing to express Hex. The primitive streak forms on the opposite side of the egg cylinder from this anterior Hex expression domain approximately 24 hours after the initial anterior movement of the distal visceral endoderm. Thus, Hex expression marks the earliest unequivocal molecular anteroposterior asymmetry in the mouse embryo and indicates that the anteroposterior axis of the embryo develops from conversion of a proximodistal asymmetry established in the primitive endoderm lineage. Subsequently, Hex is expressed in the earliest definitive endoderm to emerge from the streak and its expression within the gut strongly suggests that the ventral foregut is derived from the most anterior definitive endoderm and that the liver is probably the most anterior gut derivative. Hex is also an early marker of the thyroid primordium. Within the mesoderm, Hex is transiently expressed in the nascent blood islands of the visceral yolk sac and later in embryonic angioblasts and endocardium. Comparison with flk-1 (T. P. Yamaguchi et al., Development 118, 489–498, 1993) expression indicates that Hex is also an early marker of endothelial precursors but its expression in this progenitor population is much more transient than that of flk-1, being downregulated once endothelial cell differentiation commences.

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CD34 Expression Patterns During Early Mouse Development Are Related to Modes of Blood Vessel Formation and Reveal Additional Sites of Hematopoiesis

Blood ◽

10.1182/blood.v90.6.2300 ◽

1997 ◽

Vol 90 (6) ◽

pp. 2300-2311 ◽

Cited By ~ 142

Author(s):

Heather B. Wood ◽

Gillian May ◽

Lyn Healy ◽

Tariq Enver ◽

Gillian M. Morriss-Kay

Keyword(s):

Blood Vessel ◽

Blood Vessels ◽

Expression Patterns ◽

Hematopoietic Cells ◽

Surface Glycoprotein ◽

Mouse Development ◽

Cell Surface Glycoprotein ◽

Blood Vessel Formation ◽

Vessel Formation ◽

Stem And Progenitor Cells

Abstract CD34 is a cell surface glycoprotein that is selectively expressed within the human hematopoietic system on stem and progenitor cells, and in early blood vessels. To elucidate its functions during early blood vessel formation and hematopoiesis, we analyzed the expression patterns, in day 8 to day 10 mouse embryos, of CD34 RNA by in situ hybridization and protein by immunohistochemistry using the monclonal antibody RAM 34. Levels of expression in embryonic blood vessels were correlated with the mode of vessel formation, being high in pre-endothelial cells and in vessels forming by vasculogenesis (particularly the dorsal aortae) or angiogenesis, but low in vessels forming by coalescence (the cardinal veins). CD34+ erythroid cells, presumably of yolk sac origin, were present in the liver of day 10 embryos; at the same stage, putative definitive hematopoietic cells, strongly CD34+, were present in the para-aortic mesenchyme. Possible sites of hemangioblastic differentiation were detected in the form of CD34+ endothelium-attached hematopoietic cells in the dorsal aorta and in two previously unreported locations, the proximal umbilical and vitelline arteries. These observations suggest functions for CD34 in relation to specific modes of blood vessel formation, and a hemangioblastic role in both embryonic and extraembryonic sites.

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Tyrosine kinase signalling in embryonic stem cells

Clinical Science ◽

10.1042/cs20070388 ◽

2008 ◽

Vol 115 (2) ◽

pp. 43-55 ◽

Cited By ~ 20

Author(s):

Cecilia Annerén

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Tyrosine Kinase ◽

Tyrosine Kinases ◽

Es Cells ◽

Embryonic Stem ◽

Cell Types ◽

Self Renewal ◽

Mouse Es Cells ◽

Wide Range

Pluripotent ES (embryonic stem) cells can be expanded in culture and induced to differentiate into a wide range of cell types. Self-renewal of ES cells involves proliferation with concomitant suppression of differentiation. Some critical and conserved pathways regulating self-renewal in both human and mouse ES cells have been identified, but there is also evidence suggesting significant species differences. Cytoplasmic and receptor tyrosine kinases play important roles in proliferation, survival, self-renewal and differentiation in stem, progenitor and adult cells. The present review focuses on the role of tyrosine kinase signalling for maintenance of the undifferentiated state, proliferation, survival and early differentiation of ES cells.

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A primary requirement for nodal in the formation and maintenance of the primitive streak in the mouse

Development ◽

10.1242/dev.120.7.1919 ◽

1994 ◽

Vol 120 (7) ◽

pp. 1919-1928 ◽

Cited By ~ 88

Author(s):

F.L. Conlon ◽

K.M. Lyons ◽

N. Takaesu ◽

K.S. Barth ◽

A. Kispert ◽

...

Keyword(s):

Direct Evidence ◽

Primitive Streak ◽

Morphological Evidence ◽

Axis Formation ◽

Tgf Beta ◽

Loss Of Function ◽

Primitive Endoderm ◽

Mouse Development ◽

Mature Node ◽

Embryonic Ectoderm

The 413.d insertional mutation arrests mouse development shortly after gastrulation. nodal, a novel TGF beta-related gene, is closely associated with the locus. The present study provides direct evidence that the proviral insertion causes a loss of function mutation. nodal RNA is initially detected at day 5.5 in the primitive ectoderm. Concomitant with gastrulation, expression becomes restricted to the proximal posterior regions of the embryonic ectoderm. nodal RNA is also expressed in the primitive endoderm overlying the primitive streak. A few hours later, expression is strictly confined to the periphery of the mature node. Interestingly 413.d mutant embryos show no morphological evidence for the formation of a primitive streak. Nonetheless, about 25% of mutant embryos do form randomly positioned patches of cells of a posterior mesodermal character. Data presented in this report demonstrate the involvement of a TGF beta-related molecule in axis formation in mammals.

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Heat shock protein 90 and tyrosine kinase regulate eNOS NO· generation but not NO· bioactivity

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00736.2003 ◽

2004 ◽

Vol 286 (2) ◽

pp. H561-H569 ◽

Cited By ~ 36

Author(s):

Jingsong Ou ◽

Jason T. Fontana ◽

Zhijun Ou ◽

Deron W. Jones ◽

Allan W. Ackerman ◽

...

Keyword(s):

Endothelial Cell ◽

Heat Shock ◽

Heat Shock Protein ◽

Tyrosine Kinase ◽

Tyrosine Kinases ◽

Heat Shock Protein 90 ◽

No Production ◽

Endothelial Cell Function ◽

Enos Activity ◽

Text Production

An increase in the association of heat shock protein 90 (HSP90) with endothelial nitric oxide (NO) synthase (eNOS) is well recognized for increasing NO (NO·) production. Despite the progress in this field, the mechanisms by which HSP90 modulates eNOS remain unclear due, in part, to the fact that geldanamycin (GA) redox cycles to generate superoxide anion ([Formula: see text]) and the fact that inhibiting HSP90 with GA or radicicol (RAD) destabilizes tyrosine kinases that rely on the chaperone for maturation. In this report, we determine the extent to which these side effects alter vascular and endothelial cell function in physiologically relevant systems and in cultured endothelial cells. Vascular endothelial growth factor (VEGF)-stimulated vascular permeability, as measured by Evans blue leakage in the ears of male Swiss mice in vivo, and acetylcholine-induced vasodilation of isolated, pressurized mandibular arterioles from male C57BL6 mice ex vivo were attenuated by Nω-nitro-l-arginine methyl ester (l-NAME), GA, and RAD. Z-1[ N-(2-aminoethyl)- N-(2-ammonoethyl)amino]diazen-1-ium-1,2-dioate (DETA-NONOate), a slow releasing NO· donor, increased vasodilation of arterioles pretreated with GA, RAD, and l-NAME equally well except at 10–5 M, the highest concentration used, where vasodilation was greater in pressurized arterioles treated with l-NAME than in arterioles pretreated with GA or RAD alone. Both GA and RAD reduced NO· release from stimulated endothelial cell cultures and increased [Formula: see text] production in the endothelium of isolated aortas by an l-NAME-inhibitable mechanism. Pretreatment with RAD increased stimulated [Formula: see text] production from eNOS, whereas pretreatment with genistein (GE), a broad-spectrum tyrosine kinase inhibitor, did not; however, pretreatment with GE + RAD resulted in a super-induced state of uncoupled eNOS activity upon stimulation. These data suggest that the tyrosine kinases, either directly or indirectly, and HSP90-dependent signaling pathways act in concert to suppress uncoupled eNOS activity.

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Vasculogenesis in the day 6.5 to 9.5 mouse embryo

Blood ◽

10.1182/blood.v95.5.1671.005k39_1671_1679 ◽

2000 ◽

Vol 95 (5) ◽

pp. 1671-1679 ◽

Cited By ~ 278

Author(s):

Christopher J. Drake ◽

Paul A. Fleming

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Blood Vessels ◽

Mouse Embryo ◽

Expression Patterns ◽

Hematopoietic Cells ◽

Sequential Pattern ◽

Temporal Expression ◽

Hematopoietic Lineage ◽

Associated Proteins

The process of vasculogenesis was characterized in the 6.5- to 9.5-day mouse embryo and in allantoic culture by analysis of spatial and temporal expression patterns of the endothelial or hematopoietic lineage-associated proteins, TAL1, Flk1, platelet/endothelial cell adhision molecule (PECAM), CD34, VE-cadherin, and Tie2. The study establishes that: (1) TAL1 and Flk1 are coexpressed in isolated mesodermal cells that give rise to endothelial cells and thus can be defined as angioblasts; (2) hematopoietic cells of blood islands express TAL1, but not Flk1; (3) vasculogenesis in the embryo proper is initiated by mesoderm fated to give rise to the endocardium; (4) the maturation/morphogenesis of blood vessels can be defined in terms of a sequential pattern of expression in which TAL1 and Flk1 are expressed first followed by PECAM, CD34, VE-cadherin, and later Tie2; and (5) TAL1 expression is down-regulated in endothelial cells of mature vessels.

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A novel nonreceptor tyrosine kinase, Srm: cloning and targeted disruption.

Molecular and Cellular Biology ◽

10.1128/mcb.14.10.6915 ◽

1994 ◽

Vol 14 (10) ◽

pp. 6915-6925 ◽

Cited By ~ 34

Author(s):

N Kohmura ◽

T Yagi ◽

Y Tomooka ◽

M Oyanagi ◽

R Kominami ◽

...

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Kinases ◽

Structural Characteristics ◽

Sequence Similarity ◽

Embryonic Stem ◽

Structural Features ◽

Mutant Mice ◽

Chromosome 2 ◽

Nonreceptor Tyrosine Kinase ◽

Nonreceptor Tyrosine Kinases

We have isolated a novel nonreceptor tyrosine kinase, Srm, that maps to the distal end of chromosome 2. It has SH2, SH2', and SH3 domains and a tyrosine residue for autophosphorylation in the kinase domain but lacks an N-terminal glycine for myristylation and a C-terminal tyrosine which, when phosphorylated, suppresses kinase activity. These are structural features of the recently identified Tec family of nonreceptor tyrosine kinases. The Srm N-terminal unique domain, however, lacks the structural characteristics of the Tec family kinases, and the sequence similarity is highest to Src in the SH region. The expression of two transcripts is rather ubiquitous and changes according to tissue and developmental stage. Mutant mice were generated by gene targeting in embryonic stem cells but displayed no apparent phenotype as in mutant mice expressing Src family kinases. These results suggest that Srm constitutes a new family of nonreceptor tyrosine kinases that may be redundant in function.

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Geometrical confinement guides Brachyury self-patterning in embryonic stem cells

10.1101/138354 ◽

2017 ◽

Cited By ~ 2

Author(s):

Blin Guillaume ◽

Catherine Picart ◽

Manuel Thery ◽

Michel Puceat

Keyword(s):

Embryonic Stem ◽

Primitive Streak ◽

The Self ◽

Mouse Development ◽

Specific Population ◽

Reciprocal Regulation ◽

Asymmetric Pattern ◽

Tissue Geometry ◽

Robust Process ◽

Low Cell Density

AbstractDuring embryogenesis, signaling molecules initiate cell diversification, sometimes via stochastic processes, other times via the formation of long range gradients of activity which pattern entire fields of cells. Such mechanisms are not insensitive to noise (Lander, 2011), yet embryogenesis is a remarkably robust process suggesting that multiple layers of regulations secure patterning during development. In the present study, we present a proof of concept according to which an asymmetric pattern of gene expression obtained from a spatially disorganised population of cells can be guided by the geometry of the environment in a reproducible and robust manner. We used ESC as a model system whithin which multiple developmental cell states coexist (MacArthur and Lemischka, 2013; Smith, 2017; Torres-Padilla and Chambers, 2014). We first present evidence that a reciprocal regulation of genes involved in the establishment of antero-posterior polarity during peri-implantation stages of mouse development is spontaneously occuring within ESC. We then show that a population of cells with primitive streak characteristics localise in regions of high curvature and low cell density. Finally, we show that this patterning did not depend on self-organised gradients of morphogen activity but instead could be attributed to positional rearrangements. Our findings unveil a novel role for tissue geometry in guiding the self-patterning of primitive streak cells and provide a framework to further refine our understanding of symmetry breaking events occuring in ESC aggregates. Finally, this work demonstrates that the self-patterning of a specific population of ESC, Brachyury positive cells in this case, can be directed by providing engineered external geometrical cues.

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Control of lymphopoiesis by p50csk, a regulatory protein tyrosine kinase.

Journal of Experimental Medicine ◽

10.1084/jem.181.2.463 ◽

1995 ◽

Vol 181 (2) ◽

pp. 463-473 ◽

Cited By ~ 23

Author(s):

J A Gross ◽

M W Appleby ◽

S Chien ◽

S Nada ◽

S H Bartelmez ◽

...

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Kinases ◽

Protein Tyrosine Kinase ◽

Fetal Liver ◽

Regulatory Protein ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cell ◽

B Cell Differentiation ◽

Blood Leukocytes ◽

Protein Tyrosine

The csk gene encodes a nonreceptor protein tyrosine kinase that acts in part by regulating the activity of src-family protein tyrosine kinases. Since the src-family kinases p56lck and p59fyn play pivotal roles during lymphocyte development, it seemed plausible that p50csk might contribute to these regulatory circuits. Using a gene targeting approach, mouse embryonic stem cell lines lacking functional csk genes were generated. These csknull embryonic stem cells proved capable of contributing to many adult tissues, notably heart and brain. However, although csknull progenitors colonized the developing thymus, T and B cell differentiation were both blocked at very early stages. This represented a relatively selective interdiction of lymphocyte maturation, since csknull hematopoietic progenitors supported the development of normal-appearing MAC-1+ blood leukocytes, and the successful maturation of granulocyte/macrophage-colony-forming units from fetal liver progenitors. We conclude that p50csk regulates normal lymphocyte differentiation, but that it almost certainly does so by acting on targets other than p56lck and p59fyn.

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