and Miguel Torres Laura Carramolino, Joana Fuentes, Clara García ...

Platelets Play an Essential Role in Separating the Blood and Lymphatic Vasculatures
During Embryonic Angiogenesis
Laura Carramolino, Joana Fuentes, Clara García-Andrés, Valeria Azcoitia, Dieter Riethmacher
and Miguel Torres
Circ Res. 2010;106:1197-1201; originally published online March 4, 2010;
doi: 10.1161/CIRCRESAHA.110.218073
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2010 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circres.ahajournals.org/content/106/7/1197
Data Supplement (unedited) at:
http://circres.ahajournals.org/content/suppl/2010/03/04/CIRCRESAHA.110.218073.DC1.html
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published
in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the
Editorial Office. Once the online version of the published article for which permission is being requested is
located, click Request Permissions in the middle column of the Web page under Services. Further information
about this process is available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Circulation Research is online at:
http://circres.ahajournals.org//subscriptions/
Downloaded from
http://circres.ahajournals.org/ by guest on April 17, 2013

Brief UltraRapid Communication
Platelets Play an Essential Role in Separating the Blood and
Lymphatic Vasculatures During Embryonic Angiogenesis
Laura Carramolino, Joana Fuentes, Clara García-Andrés, Valeria Azcoitia,
Dieter Riethmacher, Miguel Torres
Rationale: Several mutations that impair the development of blood lineages in the mouse also impair the formation
of the lymphatic vasculature and its separation from the blood vasculature. However, the basis for these defects
has remained unknown because the mutations characterized affect more than one blood lineage.
Objective: We tested the hypothesis that megakaryocytes/platelets are required for the formation of the lymphatic
vasculature and its separation from the blood vascular system.
Methods and Results: We characterized the vascular patterning defects of mice deficient for the homeodomain
transcription factor Meis1 (myeloid ecotropic viral integration site 1), which completely lack megakaryocyte/
platelets. Meis1 null embryos fail to separate the blood and lymphatic vasculature, showing blood-filled
primary lymphatic sacs and superficial lymphatic vessels. To test the involvement of megakaryocytes/platelets
in this phenotype, we generated megakaryocyte/platelet-specific deficient mice by targeted lineage
ablation, without affecting other blood lineages. This model reproduces the lymphatic/blood vasculature
separation defects observed in Meis1 mutants. A similar phenotype was induced by antibody-mediated
ablation of circulating platelets in wild type mice. Strong association of platelets with vascular endothelium
at regions of contact between lymphatic sacs and veins confirmed a direct role of platelets in the separation
of the 2 vasculatures.
Conclusions: In addition to their known protective function in the response accidental vascular injury, platelets are
also required during embryonic lymphangiogenesis for the separation of the nascent lymphatic vasculature from
blood vessels. (Circ Res. 2010;106:1197-1201.)
Key Words: megakaryocytes platelets lymphatics Meis1 angiogenesis lymphangiogenesis
The mammalian lymphatic vasculature develops at
midgestation from lymphendothelial precursors produced
from embryonic veins. 1 Endothelial cells in different
regions of the cardinal veins start to express the lymphatic
endothelial cell master gene Prox1 at embryonic development
day (E)10.5. 2 This activates expression of lymphaticspecific
molecules and the formation of lymphatic sacs
from the vein-derived lymphendothelial precursors, stimulated
by vascular endothelial growth factor-C/vascular
endothelial growth factor receptor 3. 3,4 The mature postnatal
lymphatic vasculature is completely independent of
the blood vascular system, except for the connections of
the thoracic and right ducts with the subclavian veins,
through which lymph drains into the blood circulation. At
these junctions, specialized valves prevent blood reflux
into the lymphatic vessels. 5 However, during development,
connections between the venous system and the forming
lymphatic vasculature lack valves and therefore additional
mechanisms must ensure their separation. Mutations in Syk
and Slp-76 6,7 and Runx1, 1 all affecting the development of
blood lineages, block separation of the blood and lymphatic
vasculature; however, the nature of the cell lineages
involved and the mechanisms by which they control
blood/lymphatic vessel separation remains unclear, because
all mutations studied affect more than one blood
lineage. Meis1 (myeloid ecotropic viral integration site 1)
encodes a homeodomain transcription factor important for
definitive hematopoietic stem cell development in the
Original received January 29, 2010; revision received February 15, 2010; accepted February 22, 2010.
From the Departamento de Biología del Desarrollo Cardiovascular (L.C., J.F., C.G.-A., V.A., M.T.), Centro Nacional de Investigaciones
Cardiovasculares, Instituto de Salud Carlos III, Madrid, Spain; and Division of Human Genetics (D.R.), School of Medicine, University of Southampton,
United Kingdom.
Brief UltraRapid Communications are designed to be a format for manuscripts that are of outstanding interest to the readership, report definitive
observations, but have a relatively narrow scope. Less comprehensive than Regular Articles but still scientifically rigorous, BURCs present seminal
findings that have the potential to open up new avenues of research. A decision on BURCs is rendered within 7 days of submission.
Correspondence to Miguel Torres, Centro Nacional de Investigaciones Cardiovasculares, Instituto de Salud Carlos III, 3, Melchor Fernández Almagro,
28029 Madrid, Spain. E-mail mtorres@cnic.es
© 2010 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.110.218073
Downloaded from
http://circres.ahajournals.org/ 1197 by guest on April 17, 2013

1198 Circulation Research April 16, 2010
Non-standard Abbreviations and Acronyms
E embryonic day
Meis1 myeloid ecotropic viral integration site 1
PF4 platelet factor 4
embryo and essential for megakaryocyte lineage development.
8 Here, through characterization of Meis1-deficient
mice and targeted ablation of megakaryocytes/platelets, we
demonstrate an essential role for platelets in the separation
of the blood and lymphatic vasculature.
Methods
Immunohistochemistry and immunofluorescence were performed
as previously described. 8 Primary antibodies were as follows:
anti-CD31 (553370) and anti-CD41 (553847) (BD Biosciences-
Pharmingen); anti-CD61 (NB100–79980CC, Novus Biologicals);
anti–Lyve-1 (103-PA50S ReliaTech); and anti-Meis1. 8 Secondary
antibodies were as follows: biotinylated goat antirat (Ab7096,
Abcam) and goat anti-rabbit biotin (111-066-003, Jackson Immunoresearch),
followed by Streptavidin-Alexa 488 (s-11223, Invitrogen)
or -Cy3 (016-160-084, Jackson Immunoresearch). For
immunohistochemistry, we used Vectastain ABC (Vector Laboratories)
with alkaline phosphatase (AK-5000) and developed
with FastRed (11496549001, Roche Diagnostic).
Whole mount in situ hybridization was performed using standard
procedures. Riboprobes were obtained by SP6 polymerase transcription
from cDNA PCR-amplified fragments.
Meis1 mutant mice 8 were backcrossed to C57BL/6J mice
for more than 20 generations. Platelet factor (PF)4-Cre 9 and
R26:lacZbpAfloxDTA 10 were backcrossed to the C57BL/6J background
for more than 10 generations.
To transiently deplete platelets during gestation, pregnant
C57BL/6J females received IP injections twice, at E11.5 postcoitum
and 9 hours later, with 125 g (100 L) of rabbit anti-mouse
thrombocyte antibody (AIA31440 Accurate Chemical).
Animals were housed in accordance with Spanish bioethical
regulations for laboratory animals.
Results and Discussion
Meis1-deficient embryos accumulate blood at ectopic foci,
resulting eventually in generalized edema with internal
hemorrhage. 8 The distribution of ectopic blood foci
matches the pattern of nascent lymphatic vessels at E12.5
and E13.5 (Figure 1a through 1d), 1 and analysis of endothelial
and lymphendothelial markers identified the ectopic
blood foci as the recently formed lymphatic sacs, which
were filled with erythrocytes (Figure 1e through 1g and 1i
through 1k). Meis1 is not expressed in blood vascular
endothelial cells 8 or lymphatic endothelium (Figure 1h and
1i), suggesting a nonautonomous involvement of nonvascular
cells. We therefore explored the involvement of
Meis1 blood lineage defects in this phenotype. At the stage
when the first lymphatic defects are observed in Meis1
mutant embryos the only blood cells severely affected are
the megakaryocytes. 8 Expression of the megakaryocytespecific
marker cxcl7 is first detected in WT embryos at
E9.5, in cells distributed throughout the embryo, but not in
the yolk sac (Figure 2a). From E10.5, the yolk sac and the
Figure 1. Meis1 plays a nonautonomous role in the separation
of the lymphatic and blood vasculatures. a through
d, Ectopic blood foci (arrowheads) are visible in Meis1deficient
fetuses in the region of primary lymphatic sac formation
at E12.5 and E13.5. e through g and i through k,
Immunofluorescent and immunohistochemical detection of
Lyve-1, PECAM, and DAPI identifies these ectopic blood foci
as the primary lymphatic sacs. Asterisks mark the lumen of
lymphatic sacs. The jugular lymphatic sac of Meis1-deficient
specimens (i through k), but not WT (e through g), is filled
with primitive erythrocytes (note the highly condensed nuclei
of primitive erythrocytes). h and i, Meis1 (red) is not
expressed in WT lymphatic endothelial cells (Lyve-1) (green,
arrowhead).
incipient liver primordium show colonization by cxcl7expressing
cells, which intensifies by E11.5 and is accompanied
by expression of cxcl4 (Figure 2b through 2d).
Circulating platelets are detected from E10.5, coinciding
with megakaryocyte liver colonization. 11 By E12.5
megakaryocytes in WT liver have increased considerably
in size and intensity of marker expression, including the
platelet antigen CD41 (Figure 2e). In contrast, Meis1deficient
embryos lack cells expressing megakaryocyte
markers at all stages analyzed (Figure 2f through 2j).
Although these results suggest involvement of
megakaryocytes/platelets in the separation of the lymphatic
and blood vasculatures, they are not definitive
because Meis1 mutants also show defective generation of
hematopoietic stem cells. 8 We therefore generated
megakaryocyte-deficient mice by targeted ablation of this
lineage, using the Rosa26R-LacZbpa-DTA mouse line,
which conditionally expresses the diphtheria toxin from
the Rosa26 locus upon Cre recombination. 10 The inducer
strain was the PF4-Cre line, which specifically expresses
Cre recombinase in the megakaryocyte lineage (Figure
3a). 9 Early megakaryocyte development in the DTA model
appeared normal up to E11.5 (data not shown), but E12.5
embryos showed strongly reduced megakaryocyte marker
expression (Figure 3d and 3g). Megakaryocyte lineage
deletion takes place later in the DTA model than in
Meis1-deficient mice (Figure 3c and 3f). Megakaryocytedeficient
mice reproduced the blood-filled lymphatic sacs
seen in Meis1-deficient mice at E13.5 (Figure 3h, 3i, 3l,
and 3p). However, unlike Meis1-deficient embryos,
Downloaded from
http://circres.ahajournals.org/ by guest on April 17, 2013

megakaryocyte-depleted fetuses survive to E15.5 and do
not show liver hypoplasia (Figure 3h through 3k; Figure 1b
and 1d). Blood-filled peripheral lymphatic vessels are still
evident at these later stages (Figure 3i through 3k, 3m
through 3o, and 3q through 3s).
To test whether platelets were involved in the observed
defects, we injected pregnant females twice, at 11.5 and 12
days postcoitum, with anti-thrombocyte antibody. Treated
fetuses examined at E12.5 showed ectopic blood foci
similar to those observed in Meis1-null and PF4-deleted
embryos (Figure 4a through 4c; N6/8), and histological
analysis confirmed correspondence of these foci to the
nascent lymphatic sacs (Figure 4d through 4g). Consistently,
circulating platelets, which are abundant in untreated
E12.5 WT embryos, were undetectable in Meis1null
and PF4-deleted embryos (Figure 4h through 4m).
Carramolino et al Platelets Separate Blood and Lymphatic Vasculature 1199
Figure 2. Megakaryocyte
development is abrogated in
Meis1 mutants. Whole mount
in situ hybridization detection
of cxcl7 (pro-platelet basic protein)
(a through c; f through
h) and cxcl4 (PF4) (d and i) in
WT (a through d) and Meis1deficient
embryos (f through i).
The presence of positive cells
in peripheral tissues of WT
embryos is evident from E9.5
(see high-magnification in inset
in a) and in the liver from
E10.5 (b through d). Expression
is abolished in Meis1deficient
embryos (f through i).
Arrowheads mark the liver primordium.
e and j, Immunofluorescent
detection of CD41 (red) in sections of WT (e) and Meis1-deficient E12.5 livers (j). YS indicates yolk sac.
CD41 staining of anti-platelet–treated embryos confirmed
that the megakaryocyte cell population was unaffected
(Figure 4o), but that circulating platelets were agglutinated
in large aggregates. Analysis of the junctions between
primary lymphatic sacs and cardinal veins detected platelets
adhering specifically to the lymphatic and venous
endothelia at the sites where the 2 vasculatures meet
(Figure 4r, 4s, and 4u), but not to endothelia outside this
region (Figure 4r, 4h, and 4i).
These results demonstrate a morphogenetic role for
platelets during the process that separates the blood and
lymphatic vasculatures. Adhesion of platelets to the vascular
wall suggests that they are activated by contact with
the endothelium at the lymphatic/venous interface. Platelets
are not components of lymph and therefore might be
activated on contact with lymphendothelial-specific sur-
Figure 3. Targeted ablation of the megakaryocyte lineage disrupts the separation of the lymphatic and blood vasculatures.
a, Genetic strategy used to delete the megakaryocyte lineage. b through g, Immunohistochemical detection of CD41 and CD61
expression in E12.5 livers of WT, Meis1-deficient (Meis1KO), and PF4-Cre; Rosa26R-LacZbpa-DTA mice. h through k, Ectopic
blood foci (arrowheads) are evident in PF4-Cre; Rosa26R-LacZbpa-DTA specimens at E13.5, whereas littermates (h) are normal.
At E14.5 and E15.5, blood-filled vasculature reveals the typical arrangement of peripheral lymphatic vessels (i through k). Ectopic
blood is found in the primary jugular lymphatic sacs of E12.5 PF4-Cre; Rosa26R-LacZbpa-DTA fetuses (p and l). At E13.5,
ectopic blood is found in the subcutaneous lymphatic vessels of Meis1-deficient fetuses (n and r) and PF4-Cre; Rosa26R-
LacZbpa-DTA fetuses (o and s) but not WT fetuses (m and q). Asterisks mark the lumen of lymphatic sacs and vessels.
Downloaded from
http://circres.ahajournals.org/ by guest on April 17, 2013

1200 Circulation Research April 16, 2010
face molecules, thus preventing connections between the
lymphatic and venous vasculatures by forming a structural
barrier. This model is strongly supported by evidence that
platelets are activated by contact with the lymphendothelial
molecule podoplanin and by the occurrence of blood–
lymphatic separation defects in podoplanin-deficient
mice, 12 although additional local signaling roles of platelets
cannot be discarded. We suggest that the known
involvement of Syk and Slp-76 in platelet activation 13 and
the defective megakaryocyte differentiation in Runx1 mutants
14 contribute to the blood/lymphatics separation defects
in these mutants. These results indicate that platelets,
in addition to their role in repairing accidental vascular
injuries, play a morphogenetic role during angiogenesis
that allows the proper separation of blood and lymphatic
circulation.
Acknowledgments
We thank Radek Skoda (Basel) for the PF4-Cre mice; Tamara
Córdoba and Virginia García for mouse care; Silvia Vela for
mouse genotyping; Roisin Doohan for histology; José Manuel
Ligos, Mariano Vitón, and Raquel Nieto for FACS; and Simon
Bartlett for text editing.
Sources of Funding
The Centro Nacional de Investigaciones Cardiovasculares is
supported by the Spanish Ministry of Science and Innovation
and the Pro-CNIC Foundation. This work was supported by
Spanish Ministry of Science and Innovation (RD06/0010/0008
and BFU2009-08331/BMC) and the EU COST program
(COST-BM0805).
None.
Disclosures
Figure 4. Platelets are required for the
separation of the lymphatic and blood
vasculatures. a through c, Ectopic blood
foci (arrowheads) in the primary lymphatic
sacs of E12.5 fetuses after anti-Platelet
treatment. d through g, Immunohistochemical
detection of Lyve-1 and DAPI staining
confirms ectopic blood in lymphatic sacs of
anti-platelet–treated fetuses. h through m,
Immunohistochemical detection of CD41 in
large vessels of E12.5 fetuses shows abundant
platelets in the circulation of WT specimens
(h and i) and their absence in Meis1deficient
specimens (j and k) and PF4-Cre;
Rosa26R-LacZbpa-DTA specimens (l and
m). o and p, CD41 staining in E12.5 antiplatelet–treated
fetuses demonstrates preservation
of the liver megakaryocyte population
(o) and aggregation of circulating
platelets (p). q through s, Immunofluorescence
detection of PECAM, Lyve-1, and
CD41 identifies association between platelets
(arrowheads) and regions of contact
between the lymphatic and venous endothelia.
CV indicates cardinal vein; LS, lymphatic
sac. s shows a high-magnification view;
arrowheads mark platelets. t through v,
CD41 and DAPI staining at the venous–lymphatic
junction indicates that the CD41 signal
corresponds to platelets (arrowheads)
and not to nucleated CD41-expressing cells.
References
1. Srinivasan RS, Dillard ME, Lagutin OV, Lin FJ, Tsai S, Tsai MJ,
Samokhvalov IM, Oliver G. Lineage tracing demonstrates the venous
origin of the mammalian lymphatic vasculature. Genes Dev. 2007;21:
2422–2432.
2. Wigle JT, Oliver G. Prox1 function is required for the development of the
murine lymphatic system. Cell. 1999;98:769–778.
3. Karkkainen MJ, Haiko P, Sainio K, Partanen J, Taipale J, Petrova TV,
Jeltsch M, Jackson DG, Talikka M, Rauvala H, Betsholtz C, Alitalo K.
Vascular endothelial growth factor C is required for sprouting of the
first lymphatic vessels from embryonic veins. Nat Immunol. 2004;5:
74–80.
4. Wigle JT, Harvey N, Detmar M, Lagutina I, Grosveld G, Gunn MD,
Jackson DG, Oliver G. An essential role for Prox1 in the induction of
the lymphatic endothelial cell phenotype. EMBO J. 2002;21:
1505–1513.
5. Oliver G. Lymphatic vasculature development. Nat Rev. 2004;4:35–45.
6. Abtahian F, Guerriero A, Sebzda E, Lu MM, Zhou R, Mocsai A, Myers
EE, Huang B, Jackson DG, Ferrari VA, Tybulewicz V, Lowell CA,
Lepore JJ, Koretzky GA, Kahn ML. Regulation of blood and lymphatic
vascular separation by signaling proteins SLP-76 and Syk. Science. 2003;
299:247–251.
7. Sebzda E, Hibbard C, Sweeney S, Abtahian F, Bezman N, Clemens G,
Maltzman JS, Cheng L, Liu F, Turner M, Tybulewicz V, Koretzky GA,
Kahn ML. Syk and Slp-76 mutant mice reveal a cell-autonomous hematopoietic
cell contribution to vascular development. Dev Cell. 2006;11:
349–361.
8. Azcoitia V, Aracil M, Martinez AC, Torres M. The homeodomain protein
Meis1 is essential for definitive hematopoiesis and vascular patterning in
the mouse embryo. Dev Biol. 2005;280:307–320.
9. Tiedt R, Schomber T, Hao-Shen H, Skoda RC. Pf4-Cre transgenic mice
allow the generation of lineage-restricted gene knockouts for studying
megakaryocyte and platelet function in vivo. Blood. 2007;109:
1503–1506.
10. Brockschnieder D, Pechmann Y, Sonnenberg-Riethmacher E, Riethmacher
D. An improved mouse line for Cre-induced cell ablation due to
diphtheria toxin A, expressed from the Rosa26 locus. Genesis. 2006;44:
322–327.
11. Tober J, Koniski A, McGrath KE, Vemishetti R, Emerson R, de
Mesy-Bentley KK, Waugh R, Palis J. The megakaryocyte lineage
originates from hemangioblast precursors and is an integral com-
Downloaded from
http://circres.ahajournals.org/ by guest on April 17, 2013

ponent both of primitive and of definitive hematopoiesis. Blood.
2007;109:1433–1441.
12. Uhrin P, Zaujec J, Breuss JM, Olcaydu D, Chrenek P, Stockinger H,
Fuertbauer E, Moser M, Haiko P, Fassler R, Alitalo K, Binder BR,
Kerjaschki D. Novel function for blood platelets and podoplanin in
developmental separation of blood and lymphatic circulation. Blood.
January 2010; doi: 10.1182/blood-2009-04-216069.
What Is Known?
● The lymphatic vasculature emerges during mammalian embryonic
development from preexisting blood vessels through a centripetal
sprouting process.
● The separation of the blood and lymphatic circulations is linked to the
differentiation of the blood lineages, but until now, it was unknown
which lineage controls blood/lymphatic vessel separation.
What New Information Does This Article Contribute?
● Using 3 independent methods to eliminate the megakaryocyte/platelet
lineage in mice, we show that platelets are required for the
separation of the blood and lymphatic vasculatures.
● This novel action of platelets involves their specific activation and
adhesion with the endothelium at the junctions between blood and
lymphatic vasculatures.
Carramolino et al Platelets Separate Blood and Lymphatic Vasculature 1201
Novelty and Significance
13. Samaha FF, Kahn ML. Novel platelet and vascular roles for immunoreceptor
signaling. Arterioscler Thromb Vasc Biol. 2006;26:
2588–2593.
14. Ichikawa M, Asai T, Saito T, Seo S, Yamazaki I, Yamagata T, Mitani K, Chiba
S, Ogawa S, Kurokawa M, Hirai H. AML-1 is required for megakaryocytic
maturation and lymphocytic differentiation, but not for maintenance of hematopoietic
stem cells in adult hematopoiesis. Nat Med. 2004;10:299–304.
● These findings identify a previously unknown morphogenetic role for
platelets and hint at a general role in vascular morphogenesis and
remodeling
Separation of the blood and lymphatic vasculatures is disrupted
by mutations that affect blood lineage differentiation, but the
specific lineages and mechanisms involved were unknown. We
show that specific elimination of the megakaryocyte/platelet
lineage results in blood-filled lymphatic vessels, indicating a
failure to separate the blood and lymphatic circulations. Adhesion
of platelets at the junctions between blood and lymphatic
vasculatures indicates that local activation of platelets is involved
in this process. These results identify a previously
unknown morphogenetic role for platelets during lymphangiogenesis
and suggest a general role of platelets in vascular
morphogenesis and remodelling potentially relevant in vascular
disease.
Downloaded from
http://circres.ahajournals.org/ by guest on April 17, 2013

Methods
Antibody staining
Staged mouse embryos were washed with PBS and fixed in 4% PFA overnight.
Embryos were paraffin embedded or cryopreserved in OCT or sucrose-gelatin mix.
Primary antibodies used were rat-anti mouse CD31 (553370) and CD41 (553847) (BD
Biosciences-Pharmingen); rabbit anti human CD61 (NB100-79980CC, Novus
Biologicals, Littleton, CO); rabbit anti mouse Lyve1 (103-PA50S ReliaTech,
Wolfenbüttel, Germany). Rabbit anti-mouse Meis1 was generated in house 1 .
Secondary antibodies were biotinylated goat anti-rat (Ab7096, Abcam, Cambridge, UK)
and goat anti rabbit biotin (111-066-003. Jackson Immunoresearch) followed by
Streptavidin-Alexa 488 (s-11223, invitrogene) or -Cy3 (016-160-084, Jackson
Immunoresearch). For immunohistochemistry we used the Vectastain ABC detection
system (Vector Laboratories) with alkaline phosphatase (AK-5000), followed by signal
development with FastRed (11496549001. Roche Diagnosticc GmbH, Manheim,
Germany)
Whole-mount in situ hybridization
Whole mount in situ hybridyzation was carried out manually as described 2 or in an
Insitu Pro VS robot (Intavis AG Bioanalytical Instruments, Köln, Germany). In both
cases riboprobe was obtained by SP6 polymerase replication of a PCR fragment from
E12.5 liver cDNA. Oligonucleotides were as follows: PBPP (CXCL7) 1U, GCC TGC
CCA CTT CAT AAC CT; PBPP 1L, ATT TAG GTG ACA CTA TAG AAG CGA AAA
CTG CTT GAC TCC; Pf4 1L, AGC CCT AGA CCC ATT TCC TC; PF4.SP6 1R, ATT
TAG GTG ACA CTA TAG ATA TAG GGG TGC TTG CCG.
Mice
Animals were housed in accordance with Spanish bioethical regulations for laboratory
animals. Meis1 mutant mice 1 were maintained in heterozygosity by crossing with
C57BL/6J mice (Harlan, UK). More than 20 backcrosses to the C57BL/6J background
were done before analysis. Meis1-deficient and WT control embryos were generated
by crossing Meis1-deficient heteroygous mice. Progeny were genotyped as described 1 .
PF4-Cre 3 and R26:lacZbpAfloxDTA 4 mice were both in the C57BL/6J background for
more than 10 generations. Embryos obtained from these crosses were genotyped for
both genotypes with oligonucleotides described in the cited references.
To transiently deplete platelets during gestation we used an in vivo antibody treatment
as previously described 5 . Pregnant C57BL/6J females were injected i.p. twice with 125
µgr (diluted to 100 µl in PBS) of rabbit anti-mouse thrombocyte antibody (AIA31440
Accurate Chemical), at E11.5 p.c. and again nine hours later. Embryos were obtained
from CO2 euthanasied females at E12.5 and fixed with 4% PFA overnight. As reported
before 5 , antibody treatment induced strong depletion of platelet counts and only minor
alterations in other blood cell counts.
References to Methods
1. Azcoitia V, Aracil M, Martinez AC, Torres M. The homeodomain protein Meis1
is essential for definitive hematopoiesis and vascular patterning in the mouse
embryo. Developmental biology. 2005;280:307-320.
Downloaded from
http://circres.ahajournals.org/ by guest on April 17, 2013

2. Wilkinson DG, Nieto MA. Detection of messenger RNA by in situ hybridization
to tissue sections and whole mount. Methods Enzymol. 1993;225:361-373.
3. Tiedt R, Schomber T, Hao-Shen H, Skoda RC. Pf4-Cre transgenic mice allow
the generation of lineage-restricted gene knockouts for studying megakaryocyte
and platelet function in vivo. Blood. 2007;109:1503-1506.
4. Brockschnieder D, Pechmann Y, Sonnenberg-Riethmacher E, Riethmacher D.
An improved mouse line for Cre-induced cell ablation due to diphtheria toxin A,
expressed from the Rosa26 locus. Genesis. 2006;44:322-327.
5. Hidalgo A, Chang J, Jang JE, Peired AJ, Chiang EY, Frenette PS. Heterotypic
interactions enabled by polarized neutrophil microdomains mediate
thromboinflammatory injury. Nature medicine. 2009;15:384-391.
Downloaded from
http://circres.ahajournals.org/ by guest on April 17, 2013