The SCL gene encodes a basic helix-loop-helix transcription factor which is expressed in early haematopoietic progenitors throughout ontogeny and is essential for the normal development of blood and blood vessels. Transgenic studies have characterised spatially distinct 5′ enhancers which direct lacZ expression to subdomains of the normal SCL expression pattern, but the same elements failed to produce appropriate haematopoietic expression. We now describe an SCL 3′ enhancer with unique properties. It directed lacZ expression in transgenic mice to extra-embryonic mesoderm and subsequently to both endothelial cells and to a subset of blood cells at multiple sites of embryonic haematopoiesis including the yolk sac, para-aortic splanchnopleura and AGM region. The 3′ enhancer also targeted expression to haematopoietic progenitors in both foetal liver and adult bone marrow. Purified lacZ(+)cells were highly enriched for clonogenic myeloid and erythroid progenitors as well as day-12 spleen colony forming units (CFU-S). Within the total gated population from bone marrow, 95% of the myeloid and 90% of the erythroid colony-forming cells were contained in the lacZ(+) fraction, as were 98% of the CFU-S. Activation of the enhancer did not require SCL protein. On the contrary, transgene expression in yolk sacs was markedly increased in an SCL−/− background, suggesting that SCL is subject to negative autoregulation. Alternatively the SCL−/− environment may alter differentiation of extra-embryonic mesoderm and result in an increased number of cells capable of expressing high levels of the transgene. Our data represents the first description of an enhancer that integrates information necessary for expression in developing endothelium and early haematopoietic progenitors at distinct times and sites throughout ontogeny. This enhancer provides a potent tool for the manipulation of haematopoiesis and vasculogenesis in vivo.

Reference

Anagnostou
A.
,
Liu
Z.
,
Steiner
M.
,
Chin.
K.
,
Lee
E. S. N. K.
,
Noguchi
C. T.
(
1994
)
Erythropoietin receptor mRNA expression in human endothelial cells.
Proc. Natl. Acad. Sci. USA
26
,
3974
3978
Aplan
P. D.
,
Begley
C. G.
,
Bertness
V.
,
Nussmeier
M. A.
,
Ezquerra
A.
,
Coligan
J.
,
Kirsch
I. R.
(
1990
)
The SCL gene is formed from a transcriptionally complex locus.
Mol. Cell Biol
10
,
6426
6435
Aplan
P. D.
,
Jones
C. A.
,
Chervinsky
D. S.
,
Zhao
X. F.
,
Ellsworth
M.
,
Wu
C. Z.
,
McGuire
E. A.
,
Gross
K. W.
(
1997
)
An scl gene product lacking the transactivation domain induces bony abnormalities and cooperates with LMO1 to generate T-cell malignancies in transgenic mice.
EMBO J
16
,
2408
2419
Baldwin
H. S.
,
Shen
H. M.
,
Yan
H. C.
,
DeLisser
H. M.
,
Chung
A.
,
Mickanin
C.
,
Trask
T.
,
Kirschbaum
N. E.
,
Newman
P. J.
,
Albelda
, et al. 
(
1994
)
Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31): alternatively spliced, functionally distinct isoforms expressed during mammalian cardiovascular development.
Development
120
,
2539
2553
Begley
C. G.
,
Robb
L.
,
Rockman
S.
,
Visvader
J.
,
Bockamp
E.-O.
,
Chan
Y. S.
,
Green
A. R.
(
1994
)
Structure of the gene encoding the murine SCL protein.
Gene
138
,
93
99
Bernard
O.
,
Azogui
O.
,
Lecointe
N.
,
Mugneret
F.
,
Berger
R.
,
Laszenc
J.
,
Mathieu-Mahul
D.
(
1992
)
A third tal-1 promoter is specifically used in human T cell leukaemia.
J. Exp. Med
176
,
919
925
Bienz
M.
,
Tremml
G.
(
1988
)
Domain of Ultrabithorax expression in Drosophila visceral mesoderm from autoregulation and exclusion.
Nature
333
,
576
578
Bockamp
E.-O.
,
McLaughlin
F.
,
Murrell
A. M.
,
Göttgens
B.
,
Robb
L.
,
Begley
C. G.
,
Green
A. R.
(
1995
)
Lineage-restricted regulation of the murine SCL/TAL-1 promoter.
Blood
86
,
1502
1514
Bockamp
E. O.
,
Fordham
J. L.
,
Göttgens
B.
,
Murrell
A. M.
,
Sanchez
M.-J.
,
Green
A. R.
(
1998
).
Transcriptional regulation of the stem cell leukemia gene by PU.1 and Elf-1.
J. Biol. Chem
273
,
29032
29042
Bockamp
E. O.
,
McLaughlin
F.
,
Göttgens
B.
,
Murrell
A. M.
,
Elefanty
A. G.
,
Green
A. R.
(
1997
)
Distinct mechanisms direct SCL/tal-1 expression in erythroid cells and CD34 positive primitive myeloid cells.
J. Biol. Chem
272
,
8781
8790
Caprioli
A.
,
Jaffredo
T.
,
Gautier
R.
,
Dubourg
C.
,
Dieterlen-Lievre
F.
(
1998
)
Blood borne seeding by haematopoietic and endothelial precursors from the allantois.
Proc. Natl. Acad. Sci. USA
95
,
1641
1646
Cheng
T. C.
,
Tseng
B. S.
,
Merlie
J. P.
,
Klein
W. H.
,
Olson
E. N.
(
1995
)
Activation of the myogenin promoter during mouse embryogenesis in the absence of positive autoregulation.
Proc. Natl. Acad. Sci. USA
92
,
561
565
Choi
K.
,
Kennedy
M.
,
Kazarov
A.
,
Papadimitriou
J. C.
,
Keller
G.
(
1998
)
A common precursor for hematopoietic and endothelial cells.
Development
125
,
725
732
Codias
E. K.
,
Cray
C.
,
Baler
R. D.
,
Levy
R. B.
,
Malek
T. R.
(
1989
)
Expression of Ly-6A/E alloantigens in thymocyte and T-lymphocyte subsets: variability related to the Ly-6a and Ly-6b haplotypes.
Immunogenetics
29
,
98
107
Cross
M. A.
,
Heyworth
C. M.
,
Murrell
A. M.
,
Bockamp
E.-O.
,
Cobley
U. T.
,
Dexter
T. M.
,
Green
A. R.
(
1994
)
Expression of lineage restricted transcription factors precedes lineage specific differentiation inamultipotent haemopoietic progenitor cell line.
Oncogene
9
,
3013
3016
Cumano
A.
,
Dieterlen-Lievre
F.
,
Godin
I.
(
1996
)
Lymphoid potential, probed before circulation in mouse, is restricted to caudal intraembryonic splanchnopleura.
Cell
86
,
907
916
Dieterlen-Lievre
F.
(
1975
)
On the origin of haemopoietic stem cells in the avian embryo: an experimental approach.
J. Embryol. Exp. Morph
33
,
607
619
Dieterlen-Lievre
F.
,
Martin
C.
(
1981
)
Diffuse intraembryonic hemopoeisis in normal and chimeric avian development.
Dev. Biol
88
,
180
191
Drake
C. J.
,
Brandt
S. J.
,
Trusk
T. C.
,
Little
C. D.
(
1997
)
TAL1/SCL is expressed in endothelial progenitor cells/angioblasts and defines a dorsal-to-ventral gradient of vasculogenesis.
Dev. Biol
192
,
17
30
Dzierzak
E.
,
Medvinsky
A.
(
1995
)
Mouse embryonic hematopoiesis.
Trends Genet
11
,
359
366
Elefanty
A. G.
,
Begley
C. G.
,
Metcalf
D.
,
Barnett
L.
,
Kontgen
F.
,
Robb
L.
(
1998
)
Characterization of hematopoietic progenitor cells that express the transcription factor SCL, using a lacZ ‘knock-in’ strategy.
Proc. Natl. Acad. Sci. USA
95
,
11897
11902
Garcia-Porrero
J. A.
,
Godin
I. E.
,
Dieterlen-Lievre
F.
(
1995
)
Potential intraembryonic hemogenic sites at pre-liver stages in the mouse.
Anat. Embryol
192
,
425
435
Gering
M.
,
Rodaway
A. R. F.
,
Göttgens
B.
,
Patient
R. K.
,
Green
A. R.
(
1998
)
The SCL gene specifies haemangioblast development from early mesoderm.
EMBO J
17
,
4029
4045
Godin
I.
,
Dieterlen-Lievre
F.
,
Cumano
A.
(
1995
).
Emergence of mutipotent hemopoietic cells in the yolk sac and paraaortic splanchnopleura in mouse embryos, beginiing at 8.5 days postcoitus.
Proc. Natl. Acad. Sci. USA
92
,
773
777
Godin
I. E.
,
Garcia-Porrero
J. A.
,
Coutinho
A.
,
Dieterlen-Lievre
F.
,
Marcos
M. A. R.
(
1993
)
Para-aortic splanchnopleura from early mouse embryos contains B1a cell progenitors.
Nature
364
,
67
70
Göttgens
B.
,
McLaughlin
F.
,
Bockamp
E. O.
,
Fordham
J. L.
,
Begley
C. G.
,
Kosmopoulos
K.
,
Elefanty
A. G.
,
Green
A. R.
(
1997
)
Transcription of the SCL gene in erythroid and CD34 positive primitive myeloid cells is controlled by a complex network of lineage-restricted chromatin-dependent and chromatin-independent regulatory elements.
Oncogene
15
,
2419
2428
Graubert
T. A.
,
Hug
B. A.
,
Wesselschmidt
R.
,
Hsieh
C.-L.
,
Ryan
T. M.
,
Townes
T. M.
,
Ley
T. J.
(
1998
)
Stochastic, stage-specific mechanisms account for the variegation of a human globin transgene.
Nucl. Acids Res
26
,
2849
2858
Green
A. R.
,
Salvaris
E.
,
Begley
C. G.
(
1991
)
Erythroid expression of the ‘helix-loop-helix’ gene, SCL.
Oncogene
6
,
475
479
Green
A. R.
,
Visvader
J.
,
Lints
T.
,
Harvey
R.
,
Begley
C. G.
(
1992
)
SCL is co-expressed with GATA-1 in haemopoietic cells but is also expressed in developing brain.
Oncogene
7
,
653
660
Hannon
R.
,
Evans
R.
,
Felsenfeld
G.
,
Gould
H.
(
1991
)
Structure and promoter activity of the gene for the erythroid transcription factor GATA-1.
Proc. Natl. Acad. Sci. USA
88
,
3004
3008
He
X. Y.
,
Cockerill
P. N.
,
Cen
D.
,
Davis
B. R.
(
1994
)
Transcriptional regulation and chromatin structure of the human CD34 gene promoter region.
Blood
83
,
1822
1830
Hwang
L.-Y.
,
Siegelman
M.
,
Davis
L.
,
Oppenheimer-Marks
N.
,
Baer
R.
(
1993
)
Expression of the TAL-1 proto-oncogene in cultured endothelial cells and blood vessels of the spleen.
Oncogene
8
,
3043
3046
Jaffredo
T.
,
Gautier
R.
,
Eichmann
A.
,
Dieterlen-Lievre
F.
(
1998
)
Intraaortic hemopoietic cells are derived from endothelial cells during ontogeny.
Development
125
,
4575
4583
Kaiho
S. I.
,
Mizuno
K.
(
1985
)
Sensitive assay systems for detection of hemoglobin with 2,7-diaminofluorene: histochemistry and colorimetry for erythrodifferentiation.
Analyt. Biochem
149
,
117
120
Kallianpur
A. R.
,
Jordan
J. E.
,
Brandt
S. J.
(
1994
)
The SCL/TAL-1 gene is expressed in progenitors of both the hematopoietic and vascular systems during embryogenesis.
Blood
83
,
1200
1208
Kamiura
S.
,
Nolan
C. M.
,
Meruelo
D.
(
1992
)
Long-range physical map of the Ly-6 complex: mapping the Ly-6 multigene family by field-inversion and two-dimensional gel electrophoresis.
Genomics
12
,
89
105
Lawrence
P. A.
,
Johnston
P.
,
Macdonald
P.
,
Struhl
G.
(
1987
)
Borders of parasegments in Drosophila embryos are delimited by the fushi tarazu and even-skipped genes.
Nature
328
,
440
442
Lecointe
N.
,
Bernard
O.
,
Naert
K.
,
Joulin
V.
,
Larsen
C. J.
,
Romeo
P. H.
,
Mathieu-Mahul
D.
(
1994
)
GATA-and SP1-binding sites are required for the full activity of the tissue-specific promoter of the tal-1 gene.
Oncogene
9
,
2623
2632
Lee
J. E.
,
Hollenberg
S. M.
,
Snider
L.
,
Turner
D. L.
,
Lipnick
N.
,
Weintraub
H.
(
1995
)
Conversion of Xenopus ectoderm into neurons by NeuroD, a basic helix-loop-helix protein.
Science
268
,
836
844
Leroy-Viard
K.
,
Vinit
M.-A.
,
Lecointe
N.
,
Mathieu-Mahul
D.
,
Romeo
P.-H.
(
1994
)
Distinct DNase-I hypersensitive sites are assocaited with TAL-1 transcription in erythroid and T-cell lines.
Blood
84
,
3619
3827
Liao
E. C.
,
Paw
B. H.
,
Oates
A. C.
,
Pratt
S. J.
,
Postlethwait
J. H.
,
Zon
L. I.
(
1998
)
SCL/Tal-1 transcription factor acts downstrem of cloche to specify hematopoietic and vascular progenitors in zebrafish.
Genes Dev
12
,
621
626
Liao
W.
,
Bisgrove
B. W.
,
Sawyer
H.
,
Hug
B.
,
Bell
B.
,
Peters
K.
,
Grunwald
D. J.
,
Stainier
D. Y. R.
(
1997
)
The zebrafish gene cloche acts upstream of a flk-1 homologue to regulate endothelial cell differentiation.
Development
124
,
381
389
May
G.
,
Enver
T.
(
1995
)
Targeting gene expression to haemopoietic stem cells: a chromatin-dependent upstream element mediates cell type specific expression of the stem cell antigen CD34.
EMBO J
14
,
564
574
McDevitt
M. A.
,
Fujiwara
Y.
,
Shivdasani
R. A.
,
Orkin
S. H.
(
1997
)
An upstream, DNase 1 hypersensitive region of the hematopoietic-expressed transcription factor GATA-1 gene confers developmental specificity in transgenic mice.
Proc. Natl. Acad. Sci. USA
94
,
7976
7981
McNagny
K.
,
Pettersson
I.
,
Rossi
F.
,
Flamme
I.
,
Shevchenko
A.
,
Mann
M.
,
Graf
T.
(
1997
)
Thrombomucine, a novel cell surface protein that defines thrombocytes and multypotent hematopoietic progenitors.
J. Cell Biol
138
,
1395
1407
Medvinsky
A.
,
Dzierzak
E.
(
1996
)
Definitive hematopoiesis is autonomously initiated by the AGM region.
Cell
86
,
897
906
Medvinsky
A. L.
,
Samoylina
N. L.
,
Muller
A. M.
,
Dzierzak
E. A.
(
1993
)
An early pre-liver intra-embryonic source of CFU-S in the developing mouse.
Nature
364
,
64
67
Miles
C.
,
Sanchez
M.-J.
,
Sinclair
A.
,
Dzierzak
E.
(
1997
).
Expression of the Ly-6E.1 (Sca-1) transgene in adult haematopoietic stem cells and the developing mouse embryo.
Development
124
,
537
547
Morel
F.
,
Szilvassy
S. J.
,
Travis
M.
,
Chen
B.
,
Galy
A.
(
1996
)
Primitive haematopoietic cells in murine bone marrow express the CD34 antigen.
Blood
88
,
3774
3784
Mouthon
M.-A.
,
Bernard
O.
,
Mitjavila
M.-T.
,
Romeo
P.-H.
,
Vainchenker
W.
,
Mathieu-Mahul
D.
(
1993
)
Expression of Tal-1 and GATA-binding proteins during human hematopoiesis.
Blood
81
,
647
655
Muller
A. M.
,
Medvinsky
A.
,
Strouboulis
J.
,
Grosveld
F.
,
Dzierzak
E.
(
1994
)
Development of hematopoietic stem cell activity in the mouse embryo.
Immunity
1
,
291
301
Murray
P. D. F.
(
1932
)
The development in vitro of the blood of the early chick embryo.
Proc. Roy. Soc. B
111
,
497
521
Nicolis
S.
,
Bertini
C.
,
Ronchi
A.
,
Crotta
S.
,
Lanfranco
L.
,
Moroni
E.
,
Giglioni
B.
,
Ottolenghi
S.
(
1991
)
An erythroid specific enhancer upstream to the gene encoding the cell-type specific transcription factor GATA-1.
Nucl. Acids Res
11
,
5285
5291
Nishikawa
S. I.
,
Nishikawa
S.
,
Kawamoto
H.
,
Yoshida
H.
,
Kizumoto
M.
,
Kataoka
H.
,
Katsura
Y.
(
1998
)
In vitro generation of lymphohematopoietic cells from endothelial cells purified from murine embryos.
Immunity
8
,
761
769
Okada
S.
,
Nakauchi
H.
,
Ngayoshi
K.
,
Nishikawa
S.
,
Nishikawa
S.-I.
,
Miura
Y.
,
Suda
T.
(
1991
)
Enrichment and characterisation of murine hematopoietic stem cells that express c-kit molecule.
Blood
78
,
1706
1717
Pardanaud
L.
,
Altmann
C.
,
Kitos
P.
,
Dieterlen-Lievre
F.
,
Buck
C. A.
(
1987
)
Vasculogenesis in the early quail blastodics as studies with a monoclonal antibodiy recognizing endothelial cells.
Development
100
,
339
349
Pardanaud
L.
,
Luton
D.
,
Prigent
M.
,
L-M
B.
,
Catala
M.
,
Dieterlen-Lievre
F.
(
1996
)
Two distinct endothelial lineages in ontogeny, one of them related to hemopoiesis.
Development
122
,
1363
–.
Porcher
C.
,
Swat
W.
,
Rockwell
K.
,
Fujiwara
Y.
,
Alt
F. W.
,
Orkin
S. H.
(
1996
)
The T cell leukemia oncoprotein SCL/tal-1 is essential for development of all hematopoietic lineages.
Cell
86
,
47
57
Pulford
K.
,
Lecointe
N.
,
Leroy-Viard
K.
,
Jones
M.
,
Mathieu-Mahul
D.
,
Mason
D. Y.
(
1995
)
Expression of TAL-1 proteins in human tissues.
Blood
85
,
675
684
Rhodes
S. J.
,
Chen
R.
,
DiMattia
G. E.
,
Scully
K. M.
,
Kalla
K. A.
,
Lin
S. C.
,
Yu
V. C.
,
Rosenfeld
M. G.
(
1993
)
A tissue-specific enhancer confers Pit-1-dependent morphogen inducibility and autoregulation on the pit-1 gene.
Genes Dev
7
,
913
932
Robb
L.
,
Elwood
N. J.
,
Elefanty
A. G.
,
Köntgen
F.
,
Li
R.
,
Barnett
L. D.
,
Begley
C. G.
(
1996
)
The SCL gene product is required for the generation of all hematopoietic lineages in the adult mouse.
EMBO J
15
,
4123
4129
Robb
L.
,
Lyons
I.
,
Li
R.
,
Hartley
L.
,
Kontgen
F.
,
Harvey
R. P.
,
Metcalf
D.
,
Begley
C. G.
(
1995
)
Absence of yolk sac hematopoiesis from mice with a targeted disruption of the scl gene.
Proc. Natl. Acad. Sci. USA
92
,
7075
7079
Sanchez
M.-J.
,
Holmes
A.
,
Miles
C.
,
Dzierzak
E.
(
1996
)
Characterization of the first definitive haematopoietic stem cells in the AGM and liver of the mouse embryo.
Immunity
5
,
513
525
Shalaby
F.
,
Ho
J.
,
Stanford
W. L.
,
Fischer
K.-D.
,
Schuh
A. C.
,
Schwartz
L.
,
Bernstein
A.
,
Rossant
J.
(
1997
)
A requirement for Flk1 in primitive and definitive hematopoiesis and vasculogenesis.
Cell
89
,
981
990
Shalaby
F.
,
Rossant
J.
,
Yamaguchi
T. P.
,
Gertsenstein
M.
,
Wu
X. F.
,
Breitman
M. L.
,
Schuh
A. C.
(
1995
)
Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice.
Nature
376
,
62
66
Shivdasani
R. A.
,
Mayer
E. L.
,
Orkin
S. H.
(
1995
)
Absence of blood formation in mice lacking the T-cell leukemia oncoprotein tal-1/SCL.
Nature
373
,
432
434
Silver
L.
,
Palis
J.
(
1997
)
Initiation of murine embryonic erythropoiesis: A spatial analysis.
Blood
89
,
1154
1164
Spangrude
G. J.
,
Heimfeld
S.
,
Weissman
I. L.
(
1988
)
Purification and characterisation of mouse hematopoietic stem cells.
Science
241
,
58
62
Tavian
M.
,
Coulombel
L.
,
Luton
D.
,
San Clemente
H.
,
Dieterlen-Lievre
F.
,
Peault
B.
(
1996
)
Aorta-associated CD34+ hematopoietic cells in the early human embryo.
Blood
87
,
67
72
Tewari
R.
,
Gillemans
N.
,
Harper
A.
,
Wijgerde
M.
,
Zafarana
G.
,
Drabek
D.
,
Grosveld
F.
,
Philipsen
S.
(
1996
)
The human -globin locus control region confers an early embryonic erythroid-specific expression pattern to a basic promoter driving the bacterial lacZ gene.
Development
122
,
3991
3999
Tsai
S. F.
,
Strauss
E.
,
Orkin
S. H.
(
1991
)
Functional analysis and in vivo footprinting implicate the erythroid transcription factor GATA-1 as a positive regulator of its own promoter.
Genes Dev
56
,
919
931
Visvader
J.
,
Begley
C. G.
,
Adams
J. M.
(
1991
)
Differential expression of the LYL, SCL and E2A helix-loop-helix genes within the hemopoietic system.
Oncogene
6
,
195
204
Visvader
J. E.
,
Fujiwara
Y.
,
Orkin
S. H.
(
1998
)
Unsuspected role for the T-cell leukemia protein SCL/tal-1 in vascular development.
Genes & Devel
12
,
473
479
Weintraub
H.
,
Davis
R.
,
Tapscott
S.
,
Thayer
M.
,
Krause
M.
,
Benezra
R.
,
Blackwell
T. K.
,
Turner
D.
,
Rupp
R.
,
Hollenberg
S.
, et al. 
(
1991
)
The myoD gene family: nodal point during specification of the muscle cell lineage.
Science
251
,
761
766
Wood
H. B.
,
May
G.
,
Healy
L.
,
Enver
T.
,
Morriss-Kay
G. M.
(
1997
)
CD34 expression patterns during early mouse development are related to modes of blood vessel formation and reveal additional sites of hematopoiesis.
Blood
90
,
2300
2311
Yamamoto
K.
,
Tojo
A.
,
Aoki
N.
,
Shibuya
M.
(
1993
)
Characterization of the promoter region of the human c-kit proto-oncogene.
Jpn. J. Cancer Res
84
,
1136
1144
Yasuda
H.
,
Galli
S. J.
,
Geissler
E. N.
(
1993
)
Cloning and functional analysis of the mouse c-kit promoter.
Biochem. Biophys. Res. Commun
191
,
893
901
Yoder
M.
,
Hiatt
K.
,
Dutt
P.
,
Mukherjee
P.
,
Bodine
D. M.
,
Orlic
D.
(
1997
)
Characterization of definitive lymphohematopoietic stem cells in the day 9 murine yolk sac.
Immunity
7
,
335
344
Young
P. E.
,
Baumhueter
S.
,
Lasky
L. A.
(
1995
)
The sialumucin CD34 is expressed on hematopoietic cells and blood vessels during murine development.
Blood
85
,
96
105
Zernika-Goetz
M.
,
Pines
J.
,
Ryan
K.
,
Siemering
K. R.
,
Haseloff
J.
,
Evans
M. J.
,
Gurdon
J. B.
(
1996
)
An indelible lineage marker for Xenopus using a mutated green fluorescent protein.
Development
122
,
3719
3724
This content is only available via PDF.