REVIEW ARTICLE
Developmental Biology of Hematopoiesis
By Leonard I. Zon
T HE MYSTERIOUS GENESIS of hematopoietic stem
cells during early vertebrate development has intrigued
investigators for several centuries. The molecular steps that
lead to embryonic hematopoiesis remain to be determined,
although recent studies have begun to define early events
and migration patterns that regulate the hematopoietic pro-
gram. This review will provide a historical background of
the developmental biology of vertebrate hematopoiesis and
highlight the systems and methods that are being used to
study the regulation of the hematopoietic stem cell during
embryogenesis.
THE ONTOGENY OFPRIMITIVE AND DEFINITIVE
HEMATOPOIETIC CELLS
During vertebrate ontogeny, hematopoietic sites change
as new populations of stem cells emerge. In mammalian
development, stem cells sequentially occupy the embryonic
yolk sac, fetal liver, spleen, and adult bone marrow (BM).
The factors that regulate homing to hematopoietic sites have
not been identified, yet lower vertebrates provides some in-
formation about the process. The BM is first used in evolu-
tion as a site of adult hematopoiesis by certain amphibians
(Rana species but not Xenopus).’ Seasonal changes can
cause a shift in sites of hematopoiesis in some amphibians
(between the spleen and BM), possibly because of alterations
in circulating steroid levels.’ Although the yolk sac, fetal
liver, spleen, and BM are common sites of hematopoiesis in
many vertebrates, their use is not required, and some organ-
isms have developed unique characteristics of these hemato-
poietic sites. The chicken does not use the fetal liver as
a major erythropoietic organ but instead maintains blood
formation on the yolk sac and in the mesenteric regions
until BM hematopoiesis is established. In some organisms,
hematopoiesis occurs in unusual places such as the ovary,
testes, adrenal glands, and endocardium. Therefore, hemato-
poietic stem cells possess the ability to migrate within the
organism to a region that will support their existence and
allow subsequent differentiation.
In early studies of hematopoiesis, a model was suggested
in which fetal and adult hematopoietic stem cells are derived
from the original embryonic blood stem cells that occupy
the early yolk sac (Fig 1, top). In this model, a yolk-sac stem
cell migrates or “metastasizes” to a fetal hematopoietic site,
such as the liver, where it expresses a fetal program on
From the Division of Hematology, Children’s Hospital, Boston,
MA.
Submitted January 20, 1995; accepted June 6, 1995.
Supported by National Institutes of Health Grant No. HL48801.
L1.Z. is an Assistant Investigator of the Howard Hughes Medical
Institute.
Address reprint requests to Leonard I. Zon, MD, Division of He-
matology, Children’s Hospital 300 Longwood Ave, Boston, MA
02115.
0 1995 by The American Society of Hematology.
oooS-4971/95/8608-0011$3.00/0
2876
differentiati~n.~ Therefore, the activation of the fetal or adult
program relies predominantly on transcriptional regulation,
which could be triggered by environmental cues.
Although this modelhad certain appeal, other studies
showed that embryonic blood cells represent a distinct popu-
lation of cells from the fetal hematopoietic cells (Table 1).
For instance, embryonic erythrocytes morphologically are
larger in size, are nucleated, and have a more generous cyto-
plasm than fetal-adult blood cells. Embryonic hematopoietic
cells are mostly restricted to the erythroid lineage in vivo,
although this maypartly be due to characteristics of the
hematopoietic microenvironment. Embryonic cells are con-
stantly cycling during development and do not pause for
very long periods of time to differentiate, whereas fetal and
adult hematopoietic stem cells remain in Go for extensive
periods of time and are only activated occasionally, when
needed. Finally, embryonic, fetal, and adult hematopoietic
cells each synthesize distinct globins. These differences sug-
gest a model4-*(Fig 1, bottom) in which the embryonic
(“primitive”) and fetauadult (“definitive”) hematopoietic
stem cells are distinctly derived early in development. Primi-
tive cells undergo programmed cell death during fetal life,
and only defintive progenitors give rise to fetal and adult
hematopoiesis (Fig l, bottom). A corollary to the primitive-
definitive model suggests that globin switching is largely
governed by changes in cell populations rather than tran-
scriptional events.
INDUCTION AND MIGRATION OF HEMATOPOIETIC
STEM CELLS AND PROGENITORS
Hematopoietic Stem Cells Are Derived f r o m Ventral
Mesoderm
To comprehend the early events that lead to embryonic
hematopoiesis, it is necessary to understand pattern forma-
tion of the early vertebrate embryo. Classical studies in de-
velopmental biology have used amphibians to examine em-
bryogenesis (see Figs 2, 3, and 6), and the general principles
of embryonic development are maintained in higher organ-
isms. The unfertilized Xenopus egg contains an animal pole
(top hemisphere by convention) and a vegetal (bottom hemi-
sphere) p0le.’3’~ After fertilization, the dorsal axis of the
embryo will arise opposite the sperm entry point, and, by
the 32-ce11 stage, a single ventral cell (the C4 blastomere,
Fig 2) is fated to become blood. The early events of em-
bryogenesis are driven solely by maternal determinants de-
posited in the egg during oogenesis. The mid-blastula transi-
tion defines a developmental period, approximately 7 hours
after fertilization, when maternal mRNAs degrade and zy-
gotic transcription intiates. An early vertebrate embryo at
the blastula stage consists of two germ layers, ( l ) ectoderm,
which is located in the animal pole and is fated to become
skin and neural tissues, and (2) endoderm, which is located
in the vegetal pole and later becomes the gut. During gastru-
lation, morphogenetic movements (invagination just below
the equator of the embryo at the blastopore lip) brings ecto-
derm into apposition with endoderm. By the elaboration of
Biood, Vol 86,No 8 (October 15), 1995: pp 2876-2891
DEVELOPMENTALBIOLOGY OF HEMATOPOIESIS 2077

Hemo lobin Hemo lobin Table 1. Comparison of Embryonic, Fetal, and Adult Erythropoiesis
Swilch Swilch
Fetal/Prehatching/
P )D )D Embryonic Larval Adult

All
Lineages Erythroid All
Stem cell Cycling GO Go
Embryonic Site Fetal Site Adulb Site Erythroid site
H Yolk sac Liver BM
M Yolk sac Liver BM, spleen
Yolk sac, diffuse BM

. ....).iYI
C Yolk sac

(
b
! 4

Embryonic Site
--- - - - - - ----

Fetal Site Adult Site

X

Nucleated RBC
Ventral island

IM
Liver, (kidney)

Pronephros,
liver
Spleen, liver,
(BM)
Kidney, liver,
spleen

H Yes No No
Fig 1. Two developmental models for hematopoiesis. In the top M Yes NO No
model, embryonic hematopoietic stem cells (P) migrate from the yolk C Yes Yes Yes
sac t o the fetalliver, where they differentiateinto fetal hematopoietic X Yes Yes Yes
stem cells (D). Later, the second switch o w n when the fetal liver
F Yes Yes Yes
stem cells migrate e In the bottommodel, embryonic hema-
t o t hBM.
topoietic stem cells (P)exit the yolksac and undergo programmed a Globin
cell death. Another distinct populationof cells (D) migrates from the H
yolk sac t o the intra-embryonic dorsalmesentery or f e t a l liver and M
becomes the definitive pool. Some definitive cells from the fetal C
source can also migrate t o t h eembryonic site. P, primitive cells; D, X
definitive cells. p Globin
H
M
soluble peptide growth factors or by cell-cell interactions, C P. OH, P A PA

the endoderm induces ectoderm to form a third germ layer
known as mesoderm. This layer is initidly located between
the two other germ layers (ectoderm and endoderm) in a
middle layer called the marginal zone. Mesoderm is the un-
differentiated, self-renewing cells of the embryo that will
provide stem cells for the following tissues (from ventral to
dorsal across the marginal zone): blood, mesenchyme, kid-
ney, muscle, and notochord. merefore, blood development
involves the induction, proliferation, and differentiation Of
ventral mesoderm.
Yolk Sac Hematopoiesis
In most vertebrate organisms, hematopoietic stem cells
and vascular progenitors migrate to an extra-embryonic posi-
tion on the yolk sac (which is considered ventral by embyolo-
gists) to form the blood islands. Later, these cells enter the
embryo to form the primitive and definitive hematopoietic
lineages and a subset of the vasculature, respectively (Fig
4).5.11"5A blood island consists of at least three layers: an
endodermal layer that supports growth, a central core of
X PT1 PT1 PA
Only major globins are listed; eg, murine Pm globin is a low-abun-
dance variant of Ph1globin. The values shown in parentheses indicate
site(s) used by Some amphibian
Abbreviations: H, human; M, mice; C, chicken; X, Xenopus; F, fish
fteleostst; RBC, red blood ce,ls
hematopoietic stem cells that ultimately differentiates into
erythroblasts, and an endothelial layer that surrounds the
blood island as it is developing (Fig 4,bottom).16
Mammals. In the mouse, on day 7 'h, blood island forma-
tion begins in a central area of the egg cylinder. At day
7 there are regions of thickening of the inner mesodermal
layer in contact with the endoderm, which is thought to
support the growth of the hematopoietic tissue. Endothelial
cells subsequently differentiate and encompass the hemato-
poietic progenitors, which then budoff of the endodermal
base, forming a blood island. By day 9, there is a capillary
network, and mature erythroid cells enter circulation, but
yolk sacblood formation proceeds through day 12. In hu-

animalpole

ventral dor sal

Fig 2. A 32-cell fate map of the Xenopus embryo.
This was constructed by injection of various dyes
into a single cell (also called blastomere) of each tier
of the 32csll embryo and following dye the as devel-
opment proceeds.The C4 blastomere is the predomi-
nant cellfated to form blood (Reprinted with permis-
sion.'") vegetalpole
2878
Animal Pole
e$(+-) W
Vegetal Pole
Blastula
MK
Gastrula
mans, the process is similar to that for the mouse; hematopoi-
esis occurs initially at day 15 and continues for 6 weeks of
gestation (Fig 4). Mammalian embryonic erythroid cells are
nucleated, similar to the erythroid cells of lower vertebrates
including fish, frogs, reptiles, and chickens. Although most
progenitor populations are present in the yolk sac, the termi-
nal differentiation occurs primarily to the erythroid lin-
eage.'"19 Occasionally, monocytes and megakaryocytes have
been identified in blood islands.
Birds. The early embryology of blood formation has
been well-defined in the chicken (Fig 5A, B, and C)." The
boundary of the embryo is not distinct until after primitive
steak development, but the area opaca is considered extra-
embryonic even from early stages (Fig 5A). Cells originating
from the epiblast (the upper layer) invaginate through the
primitive streak and enter the area pellucida between the
epiblast and hypoblast (the lower layer) by 15 to 16 hours.
The cells are later separated into somatic and splanchnic
layers. Extra-embryonic splanchnoderm consists of two dif-
ferent layers, an endodermal layer and a mesodermal layer
that will ultimatelygive rise to blood islands in the yolk sac.
The vascular area invades the vitelline area, the two layers
become indistinct, and, eventually, the entire yolk sacis
vascularized. The initial blood islands are seen in the poste-
rior area opaca (Fig 5B) at the head-process or early somite
stages, at which time the posterior area pellucida becomes
vascularized. Hemoglobin can be detected as early as the 2-
4 somite stage." As the yolk sac spreads over the yolk sur-
face (Fig 5C), the central vascular area consists of blood
vessels, sinus marginalis, and blood islands. The yolk sac in
chickens will remain hematopoietic for most of embryonic
development.
Amphibians. Amphibians form blood in the ventral re-
gion of the embryo (Fig 6A). Brauns" described the prospec-
tive blood island region at early neurula stage as a monolay-
ered or bilayered sheets of cells extending along the entire
ventral region of the embryo. At first, these cells migrate
slowly in a ventral direction, then quickly migrate to form
the characteristic hematopoietic cords of blood island in a
Fig 4. Mammalian embryonic blood formation. Human embfyo at various stages. By day 16, cdls from the opiM.rt begin to invaginate.
and form the m d e r m a l layer. By day 18, mesoderm is pr-nt
the left demonstrates the typical appearonce of a blood island. Erythroblasts are surrounded by endothelial cells, and
to the endoderm. (Adapted and reprintedwith permiuion'"1
LEONARD I. ZON
Dorsal
Fig 3. Formation of the mesodermgermcell
layer. Signals from endoderm (arrows1 located
vegetal pole induce mesoderm, which is derived en-
from
tirely the animal
ectoderm.
pole The mesoderm
the in
.""
layer isalso called themarginal zone, whose cell fate
is organized in a ventral-to-dorsal pattern of blood
(B), mesenchyme (MI, kidney (K),somites (SI,and
notochord (NI.
"V" shape (Fig 6B). As ventral migration occurs, the lateral
plate mesoderm converges between the blood cells and skin
and yields the endothelium necessary for circulation. Exci-
sion of this region during neurula stages leads to tadpoles
with no circulating blood within a normal circulatory sys-
tem.22.23Bloodless tadpoles can live for over l month, indi-
cating that blood is not critical to early embryonic develop-
ment in amphibians.
In Xenopus, hemoglobinization occurs in an anterior-to-
posterior directionz4 starting at 36 hours after fertilization.
The island is anteriorly bounded by hepatic endoderm, which
is thought to secrete erythropoietin-like activity that accounts
for the anterior-to-posterior wave of hemoglobinization. The
heart starts beating at stage 33/34 (45 hours), the anterior
cells of the blood island enter the heart from the primitive
vasculature, and circulation is fully established at stage 351
36 (50 hours). The immune system begins to develop be-
tween stages 42-45 (80 hours), when the thymus becomes
histologically visible.
Fish. Bony fish (teleosts) form embryonic erythroid cells
in a distinct dorsal-lateral compartment of the embryo known
as the intercellular mass (IM) of Oellachef' (described in
1872; see Fig 7), and are, therefore, an exception to the
general vertebrate rule of embryonic hematopoiesis on the
yolk ~ a c . * ~The
- ~ *IM is derived from lateral mesoderm at
the posterior border of the embryo and is similar in position
to the initial yolk sac-derived hematopoietic mesoderm in
higher vertebrate^.^' Some Teleosts such as the angelfish,
killifish, and cartilaginous fish form embryonic blood both
on the yolk sac and in the IM.33
Dorsal Hematopoiesis
In addition to ventral embryonic (or yolk sac) hematopoie-
sis, most vertebrates have an additional intra-embryonic stem
cell population in the dorsal mesentery (also called the AGM
region for aorta, gonad, mesonephros region) that colonizes
later fetal (larval) sites of blood formation. In higher verte-
brates, this dorsal population predominantly gives rise to
*
in intra- and extra-embryonkIocdons. At day 'IS, the expmQd picture on
the islrnd is adb-nt
DEVELOPMENTAL BIOLOGY OF HEMATOPOIESIS 2879

Day I Day2 Day3 Duy4

Fertilization 2 Cell Stage Morula Blastocyst
Early
., .._. + _
_.* ._. :_. .

DuyS ,Maternal and Trophot)last Vessels

Late Blastocyst
piblast
,Amnion
-Ectoderm
- Endoderm
- Hypoblast
- Yolk sac

Duy 16 Duy 18
Primitive Streak"
Ectoderm
Mesoderm
(invaginattngl
Endoderm.
Yo1k sac

Duyf9
2880 LEONARD 1. ZON

7-8Hours 40-42 HOUKS

7Hensen's
Node --. ..,.L%Y

-Area Pellucida

B
-A Area Pellucida

Opaca

Fig 5. Avian embryonicblood formation. (A) Early

mesoderm inductionuntil 15 to 16 hours. The invagi-
nation of mesoderm through the primitive streak is
Islands depicted in a cross-section. (B)Chicken embryonic
hematopoiesisat the 7-somite stage. (Adapted and
reprinted with permission.') Note that theinitial he-
matopoietic progenitors arise in the area opaca.
DEVELOPMENTALBIOLOGY OF HEMATOPOIESIS 2881

C
/,
Area pellucida
l\

Vascular area
inus terminalis

Fig 5. (Cont'd) (C) Expansion of yolk sac hemato-

poiesis from day 1 (top embryo), day 2 (second), day
3 (third), and day 4 (bottom embryo). (Adapted and
reprinted with permission.")
definitive hematopoiesis, but in lower vertebrates both primi-
tive and definitive cells arise from these cells.
Studies using an in vitro embryo culture assay show that
the yolk sac is required for both primitive and definitive
hematopoiesis in mammals. Culture of day-7 (early 1-4 so-
mite) mouse embryos in tissue-culture plates showed normal
somite development, yolk sac hematopoiesis, and a beating
h e a t 3 Culture of day-7 yolk sac alone yielded abundant
hematopoietic colonies, but culture of day-7 embryos from
Yolk '
which the yolk sac has been removed develops without blood
in a normal circulatory system. In some of these embryos,
there was an absence of fetal liver hematopoietic progenitors,
suggesting that the genesis of the definitive hematopoietic
program requires the yolk sac structure. Therefore, the primi-
tiveand definitive lineages are likely to be derived from
common progenitor cells colocalized in the yolk sac (IM or
ventral blood island) region very early during embryogene-
SIS.
2882 LEONARD I. ZON

(3hours,32cellsl

B/ustu/u
(6hours;5000 cellsl
""_"""" """""""
Mid Blastula
~ .. -
~

Gusfru/u
(70 hours;3Q 000 cellsl

Neuru/u
(20hours; 90,000 celisl

Fig 6. Xenopus embryonic

blood formation. (A) Schematic
Epidermis view of hematopoietic induction
and development (Adapted and
System reprinted with permis~ion.'~')
7"Stuge The Xenopus egg contains an
(40hours; 200,000ceils Notocord Endoderm animal and a vegetal pole that
are distinguishable by pigment.
Lateral Plate (Mesenchyme) The blastomere of the em-
bryo that will become blood is
determined as early as the 32-
cellembryo (shaded). Presum-
ably, the vegetal blastomere
7udpo/e under this shaded region is re-
sponsible fortheinductionof
ventral type mesoderm. Meso-
derm is derived from the ecto-
dermal animal pole. T w o p r e
dominant signals have been
postulated t o affect the process
of mesoderm induction, the ven-
tral signal (V) and the dorsal sig-
nal (D). During gpstrula stages,
the Spemann organizer (arrow)
functions t o pattern mesoderm
across themarginal zone (the
centralregion of the embryo).
The commitment t o form blood
occurs in the ventral region of
the marginal zone. Later in neu-
rula and tailbudstages, blood is
the most ventral mesoderm, and
is locatedadjacent t o the epider-
mal layer. (B) Xenopus embryo
at stage 28 (about 34 hours after
fertilization). This embryo is
stained for embryonic @-globin
usingwholeembryoimmuno-
histochemistry analysis.
DEVELOPMENTALBIOLOGY OF HEMATOPOIESIS
18
somite
Fig 7. Zebrafish embryonichematopoiesis, a 24
schematic view. Blood is formed in the IM, a dorsal
intra-embryonic location. At 18 somites, the mass
consists of intermittent hematopoietic progenitors.
Undifferentiated cells will become vascular by the
24-somite stage. The posterior region of the mass
remains undifferentiated and may represent a stem
cell pool. (Adapted and reprinted with p e r m i s ~ i o n . ~ ~ l
The migration of hematopoietic progenitors during and
after neurula has been extensively studied in amphibians. By
transplanting a cytogenetically distinct ventral blood island
tissue into wild-type hosts, a small ventral portion of the
Xenopus neurula embryo has been shown to contain primi-
tive and definitive erythroid and T-cell/myeloid progenitors
(Fig gA).h.7.34-42This region in Rana pipiens contributes to
primitive erythropoiesis but does not contribute to T-cells
or myeloid cell populations>14h In Rana cutesbeiana, the
definitive progenitors of the dorsal mesentery colonize the
larval liver, whereas the primitive progenitors of this region
colonize the larval pronephro~."~ Thus, the distribution of
progenitor cells in the ventral or dorsal compartment of the
embyro is not conserved during vertebrate evolution, even
in closely related species. Cell transplantation studies have
also shown that T-cell progenitors are derived from the pos-
terior region of the embryo during neurula stages (Fig 8B),
and these cells migrate anteriorly across tissue planes to
eventually colonize the dorsal aorta region, the postcardinal
veins, and the thymus. The thymus may also be colonized
by circulating cells. The migratory pattern of hematopoietic
cells throughout amphibian development is schematically
drawn in Fig 8C.
The embryonic region to which the stem cells are trans-
planted influences the frequency of contribution to definitive
cell lineages (Fig 8D).3hLymphoid contribution is signifi-
cantly increased when cells are transplanted to a peripheral
location in the embryo, whereas erythroid contribution is
increased when cells are placed in a central region. Hence,
environment apparently exerts a directive effect on stem cells
of the embryo to form particular hematopoietic tissues.
The embryonic location of definitive hematopoietic progen-
itors in birds has been studied with interspeciesgrafts between
chickens and quails, so-called "yolk sac chimera^."^.' In these
experiments, age-matched chicken and quail eggs are incu-
bated for 30 to 36 hours (between8-12 somites), and the quail
embryonic body is grafted into the analogous position of a
chicken blastoderm. The quail embryo develops on a chick
yolk sac, and, at varying developmental stages, quail cells are
recognized by morphological differences or by markers such
as immunofluorescencewiththequail-specificmonoclonal
2883
Intermediate
Cell Mass
0hematopoietic
~undifferentiated
=vasculogenic
Intermediate
somite
antibodies, QH 14x or MB I >9 QHl and MB 1 recognize glyco-
proteins on quail hematopoietic, vascular, and germ cell lin-
eages, but not on chicken cells.
Between d3 and d5, chickens and quails have hematopoi-
etic foci on the ventral wall of the a ~ r t a . ~ .These
~ . ~ " foci may
be induced by endoderm that physically associates with the
ventral surface of the two dorsal aortae before they fuse."
By day 6-8, the definitive hematopoietic progenitors in
chicken and quail embryos are distributed differently in the
mesenchyme (Fig 9A). In the chicken, definitive hematopoi-
etic foci are found throughout themesenteryand around
visceral organs, including the dorsal region that will become
the thoracic duct. In the quail embryo, hematopoietic tissue
is found associated with the anterior cardinal vein system,
at the angle of the duct of Cuvier. These dorsal hematopoietic
cells are only separated from the vascular lumen by endothe-
lium and can infiltrate the wall oflarge venous vessels. Using
the QHI marker, cells from the dorsal regions can be fol-
lowed as they colonize the yolk sac or thymus by direct
migration or as they differentiate in situ.
The dorsal hematopoietic program of avians is regulated
by local environment. At day 3-4, mesodermal cells sur-
rounding the quail dorsal aorta can be reciprocally trans-
plantedto the analogous position in the chick embryo."
The donor quail cells contribute to blood-forming foci and,
occasionally, to endothelial structures based on QHI stain-
ing. When day 3-4 mesodermal cells are cultured with day
6.5 chick thymic rudiment, the cells stain with QHl and also
react with the CTl antibody (a T-cell marker). This suggests
that the mesoderm adopts a T-cell program in the environ-
ment of the thymus.
Recently, similar dorsal hematopoietic cell populations
have been shown for mammals (Fig 9).s2-s4 Spleen colony-
forming unit activity was shown in the region of the aorta,
gonad, and mesonephros from day-8 to day- 1 1 embryos. By
grafting intra-embryonic splanchnopleura from IO- to18-
somite embryos in SCID mice, IgM-secreting plasma cells
and the B 1a cell subset were reconstituted. Thus, the dorsal
Compartment in mice is ultimately capable of hematopoietic
development as documented for early spleen colony-forming
unit and B-cell development.
 LEONARD I. ZON

A General Hypothesis for Migration Pathways of
Hematopoietic Cells During Vertebrate Embryogenesis
During early gastrulation, induced ventral mesoderm mi-
grates to a lateral position on the yolk sac. As gastrulation
proceeds, these cells either migrate further onto the yolk
sac or return to an intra-embryonic location in the dorsal
mesentery. Most cells on the early yolk sac are primitive
progenitors that will form blood islands and eventually enter
the circulation. As the fetus develops, these primitive cells
undergo programmed cell death. Fetal-adult blood cells are
derived either from definitive progenitors that colonized the
yolk sac at the same time as the primitive progenitors or
from the dorsal mesenteric hematopoietic cells. The dorsal
mesenteric cells either migrate to the yolk sac or thymus or
can enter the circulation and colonize hematopoietic sites
such as the fetal liver. Thus, the prominent waves (from
early to late) of hematopoiesis in the embryo include (l)
primitive cells on the yolk sac, (2) definitive cells on the
yolk sac (which are likely derived from ventral mesoderm
that migrated with the primitive cells), and (3) early intra-
aortic and later para-aortic hematopoietic cells. Some of the
dorsal hematopoietic cells migrate to the yolk sac and form a
later wave of hematopoiesis on the yolk sac. These migratory
patterns of hematopoietic cells in the vertebrate embryo are
generally conserved throughout evolution.
Hematopoietic and Endothelial Development
The vascular and hematopoietic compartments are coordi-
nately regulated so that blood cells enter the circulation
as soon as terminal differentiation has occurred. A com-
mon progenitor called either a “hemangiocytoblast,”
‘‘hemangioblast,” or “angioblast”55~58 has been postulated
to exist during embryogenesis and has the potential to form
either vascular endothelial cells or hematopoietic cells in
the blood island region. Morphological data derived from
experiments with cultured chicken blastoderms support the
hypothesis that these bipotential cells can form either tissue.
In mice and humans, support for this concept is less apparent.
Support for the existence of a common bloodvascular pro-
genitor includes the finding of antigens (such as QH1)“~4X~‘’’
or molecular markers (such as GATA-2 and several tyrosine
kinases) that are common to early hematopoietic progenitors
and vascular progenitors. Recently, we have studied a zebra-
fish mutant with deficiencies of hematopoietic and endocar-
dial progenitors, potentially representing a defect in a com-
mon blood-vascular p r ~ g e n i t o r .Despite
~~ the above data
supporting the existence of a hemangioblast, the vasculature
and heart develop normally in bloodless amphibian embryos
from which the presumptive ventral blood island region has
been explanted during the neurula stage. It is possible that,
by early neurula, fates have been determined and the vascular
lineage becomes distinct from the hematopoietic lineage;
most ventral mesoderm becomes hematopoietic, whereas the
lateral mesodermal cells become the vascular progenitors.
STUDIES ON LOWERVERTEBRATES
We have used the fish and frog as model systems to study
the induction of the hematopoietic stem cell during ontogeny.
The embryos from these species are more accessible than
higher organisms, and their early development has been well-
characterized. The study of their hematopoietic program,
which may be more simple, will provide general principles
regarding hematopoiesis in vertebrates.
Zebrajish Hematopoiesis
We have characterized the expression of the zebrafish
GATA- 1 and GATA-2 during early development. These ’*
transcription factors are initially induced during gastrulation
and delineate cells that will form the hematopoietic IM (Fig
7). The posterior region of the IM contains early hematopoi-
etic progenitors (or “stem” cells) that express GATA-2, but
not GATA-l. Thus, the visibility of the zebrafish embryo
allows easy examination of early and late hematopoietic pop-
ulations.
Studies of genetic mutants for blood formation in the ze-
brafish system may provide insight into the induction events
that effect hematopoiesis. Using GATA-l and GATA-2 as
markers, we have studied three zebrafish mutants of blood

Fig 8. Studies of definitive hematopoiesis in amphibians. (A) Species-specificdifferences highlighted by transplantation experiments.
Reciprocal transplantation of ventral (V) or dorsal (D) regions of diploid embryos (Zn) into triploid1311) embryos at neurula stages(A, anterior;
P, posterior).The transplantedembryos areallowed to reach a premetamorphic stage to evaluate the primitive lineege anda postmetamorphic
stage when definitive hematopoiesis is occurring. Blood or thymic cells are analyzed by DNA flow cytometry to cletermine the contribution
of the grafted tissue to particular blood lineages. +, contribution to the lineage; -,does not contribute to the lineage. Myeloid blood cells are
derived from the same regions as T cells. Note the difference in T-cell and myeloid contribution in Rana and Xenopus; in Rana, the vantral
region only contributes to the red blood cell lineage previousto metamorphosis andnot to the early T-cell ormyeloid populations. (B) Studies
of thymus development by transplantation of diploid (2n) regions into triploid (3nl embryos during early neurula. The T-cell population is
derived from the posterior compartment of the embryo (top), but not from the posterior third ( m n d embryo). Anterior migration occurs
from the posterior region to the dorsal mesentery and, later, to the dewloping thymus. Grafting experiments ofthe pronephros and praumptive
ventral blood island regions showed that the thymus is derived from bothdorsal and ventral regions. (C) Cumulative summary of migratory
events of hematopoietic callsduring development in amphibians. The migratory pattern is thought to be similar for most vertebrate wecies.
The ventral blood island (VBI) contributes to the development of all hematopoietic lineages. Primitive cells form the blood island and enter
the circulation (1). During neurula stages, mesoderm aroundthe pronephric duct (PD) migrates anteriorly (2) to the region of the dorsel aorta
(DA; 3) and ducts of Cuvier(DC; 31 and the pronephros (P;4). These cells either colonizethe thymus (5) or enter circulation through the aorta
or ducts of Cuvier 16). Definitive celh within the VBI enter circulation also and eventually colonizethe thymus (T; 71 and the larval liver (LL;
81. Definitive cells also enter the circulation from the larval liver (9). In chickens and mice, the circulation is establiied before the yolk sac
structure is disrupted. Some definitive cells in the dorsal mesentery or in the circulation colonize the yolk sac. The chicken does not form
blood in the fetal liver (FLI, but has extensive hematopoiesis throughout the mesentery. The axis of the embryo is marked as follows: A,
anterior; P, posterior; D, dorsal; V, ventral. (D) Environmental influences ofdefmitive hematopoietic commitment. When the ventral t i i u e is
transplanted to a ventral central location. the donor tissue becomes erythroid, whereas transplant
a to a lateral location becomes T cells.
DEVELOPMENTAL BIOLOGY OF HEMATOPOIESIS 2885

Neurula D
C
A

J
-
p l
A

V
P

'-cell
.".L"I_l

INeurula I Primitive I Definitive I

Xenopus

Rana 1 1 -+ 1 ;I ;1 ;1
Dorsal

Dorsal

Xenopus Ventra I
- + + +

Ventral
View

Rana Ventral + - ? ?

B
Neurula (20 hrs)

D Thymus

A p +
60 hrs
V
V
2886
A
..
Chick Quail
B eny were mated individually to wild-type fish or to the par-
Fig 9. Schematic diagram of hematopoiesis in the dorsalpara-
aortic focusof birds (A) and mammals (B). This region has been
shown to bea site of hematopoiesisby morphologicalcriteria, benzi-
dene staining, and immunolocalizationwith QH1 antisera (in birds)
and by spleen colony assays and transplantation experiments. The
chicken has diffusedefinitive hematopoiesis, particularly around the
aorta (dark shaded region), whereas the quail has a more restricted
distribution of hematopoietic cells surrounding the Ducts of Cuvier
(darkshaded region). The hematopoietic progenitor cells in the
mouse have been studied functionally (see text) but have not been
morphologicallyorhistologically defined. DC,ductsofCuvier;A,
aorta; E, esophagus; B, bronchi. (Adapted and reprinted with permis-
sion.‘)
formation: bloodless, spadetail, and ~ I o c h e . ’ *The
~~~blood-
less mutation only affects hematopoiesis, whereas the spade-
tail mutation affects muscle andblood induction. Whole
embryo in situ analysis shows that bloodless (Fig 10) and
spadetail mutant embryos each lack GATA- 1 and GATA-2
expression, except for wild-type levels of GATA-2 expres-
sion in the posterior “stem” cells. Despite their similarities,
complementation analysis has shown that the bloodless and
spadetail mutations are in distinct genes. Thus, the self-
renewal or differentiation of early hematopoietic cells is af-
fected in the mutants.
The cloche mutation lacks the endocardial layer of the
heart and the blood cells in the IM region. Interestingly,
cloche mutants do not express GATA-2 or GATA- 1, even
in the posterior stem cells. Thus, the cloche mutation affects
LEONARD I. ZON
vascular cells and hematopoietic cells and, therefore, sup-
ports the existence of a bipotential “hemangioblast” during
development. Fate mapping experiments in wild-type em-
bryos at the 500-cell stage have shown that single ventral
blastomeres give rise to blood and blood vessel progenitors,
further suggesting a common origin. Therefore, the defect in
cloche effects the genesis of hematopoietic (and endothelial)
stem cells.
Two independent laboratories, Driever (Boston, MA) and
Nusslein-Volhard (Tubingen, Germany), have produced over
10,OOO zebrafish mutations using chemical mutagenesis.m.6’
In this method, fish were treated with ethyl nitroso urea
(ENU), a chemical that causes point mutations in the sperm
genome. These sperm were used to fertilize eggs, the prog-
ent, and a family was derived. Each family is then individu-
ally screened for mutant phenotypes, and over 30 mutants
affecting the blood system have been characterized.
The mutants can be used to genetically order a cascade
of steps for embryonic blood formation. This is a similar to
drosophila or yeast genetic epistasis analysis, which involves
paired matings of different mutants, thereby generating fish
that are mutant at two (or three) genetic loci. Assessment of
the phenotype of these double mutants allows a positioning
in a cascade of developmental events. These studies are cou-
pled with the examination of gene expression with specific
markers in mutant embryos andwith the analysis of the
effect of forced gene expression. A zebrafish genome map
consisting of over 400 random amplified polymorphic DNA
(RAPD)markers distributed throughout the genome has re-
cently been
and
we have started to map the
location of the genes affected in the mutant zebrafish. A
position-cloning strategy to isolate genes that are affected in
the mutants is then possible.
Xenopus Hematopoiesis
The induction of hematopoietic stem cells during em-
bryogenesis is likely to be regulated by signals that affect
mesoderm induction and patterning. These signals may not
function in the same manner as hematopoietic cytokines;
mesoderm-inducing factors are thought to induce distinct
tissues in a gradient rather than threshold mechanism. It is
possible that low doses of particular inducing factors would
stimulate blood formtion, but higher doses may inhibit hema-
topoiesis. Mesoderm induction has been so well-character-
izedin Xenopus that this species is ideal to characterize
factors that regulate the early events in hematopoiesis.
Assays in the frog for the induction of hematopoietc stem
cells. Transplantation studies have shown that Xenopus an-
imal-pole ectodermal cells can contribute to almost any tis-
sue of a developing embryo. As such, animal pole cells (also
called animal caps) are similar to murine embryonic stem
cells. Through the use of animal pole-vegetal pole “recombi-
nants” (see Fig 1 I), Nieuwkoop and colleague^^"^' have
shown that the dorsal-ventral organization of mesoderm is
determined by the vegetal pole. Culture of animal pole alone
in a simple salt solution results in ciliated epidermis (skin),
whereas culture of the vegetal pole yields undifferentiated
cells and gut (Fig 1 A recombinant of animal and
vegetal pole yields mesodermal tissues, including some
DEVELOPMENTAL BIOLOGY OF HEMATOPOIESIS
blood cells. During blastula stages, two predominant meso-
derm-inducing signals are thought to act on overlying equa-
torial cells. The ventral vegetal signal induces ventral meso-
derm such as blood and mesothelium and the dorsal vegetal
signal induces dorsal mesoderm including muscle and noto-
chord. Subsequently, during gastrulation, a dorsal region
called the Spemann organizer patterns mesoderm across the
marginal zone.- These data suggest that the endoderm elabo-
rates a signal required for the induction of blood, and that
the program is modulated by cell-cell interactions.
Surgical excision and culture of the dorsal marginal zone
(DMZ) yields mostly notochord and muscle, whereas ventral
marginal zone (VMZ) yields predominantly blood and mes-
enchyme (Fig 11, bottom).70 VMZs express globin after 40
hours in culture, and, thus, the inducers of blood are available
or programmed in this ventral region. A VMZ culture can
be viewed as equivalent to hematopoietic progenitor assays
with all of the authentic growth factors present.
Interspecies grafts between two different amphibian spe-
cies (Axolotl and Xenopus) have been used to characterize
the ventral and dorsal signals. Culture of Xenopus VMZ with
Axolotl DMZ yielded mostly Xenopus muscle and kidney
differentiation, showing the respecification of ventral meso-
derm by the dorsal signal. If the organizer region of the
DMZ is grafted onto the ventral region of an intact embryo
(a classic “Spemann Organizer” graft), a mirror-image du-
plication of the embryo is created.” Neither the primary nor
the secondary embryo is actually complete because they both
STME 8 a ”1
Aninal Cao
vegetal P&
LGut
Skh
8-
m a l Cap
RECOMBINANT Mesoderm hdwm
Vegetal Pole
Aninal Cap
+
Activin
(ie.
-
A)
Mesoderm
hductDn
””
Ventral
Marginal Zone Dorsal M a r m l Zone
Bbod,
c
Mesenchyme
i
k s c l e , Notocord
Fig 11. Mesoderm induction assays. (Top panel) The animal cap
of a blastula embryo (about8 hours] can be removed, cultured in a
pole can be
satt solution, andwill form skin (ectoderml. The vegetal
removed andwill form gut [endoderm). Recombination of the animal
cap (ectoderm) and vegetal pola (endodorm) leadsto the induction
of mesoderm entirely from the ectoderm. Animal caps can also be
incubated with purified growth factors that induce mesoderm. Ac-
Win and FGF induce mesoderm such as muscle, but blood is not
formed. (Bottom penel) The marginal zone of the gastrula embryo
contains the mesodermal proganitors. The type of mesodermis spa-
tially localized acrossthe marginal zone. For example, explant the
of
VMZ leads the induction of hematopoietic cells, whereas tha DMZ
does not form blood.
2007
lack blood islands. Thus, it is possible that the organizer
can reprogram ventral mesoderm and, thereby, functionally
repress blood formation.
The ventral and dorsal signals have been shown to be
soluble factors based on their ability to cross a millipore filter
placed between embryonic ex plant^.^'-^^ Very low levels of
globin expression are detected in animal-vegetal recombi-
nants, but not to the level in a VMZ e ~ p l a n t ? This ~ . ~ ~sug-
gests that globin production requires signals in the ventral-
most endoderm of the marginal zone?6
Studies of mesodermal inducing factors in Xenopus.
Activins are members of the transforming growth factor
(TGF- p) superfamily and are dimers of inhibin p chains.77
Activin is a very potent inducer of mesoderm in animal cap
assay^^^.^^" and can also induce differentiation of erythro-
leukemia cells.8sWhereas recombinants between animal and
vegetal poles yield all mesodermal components, activin A
(either from Xenous or mammalian sources) induces mostly
notochord, muscle, and kidney tubules. At low concentra-
tions of 0.1 to 1ng/mL, activin induces some blood-like
~ e l l s . ~However,
~ , ~ ’ the blood cells do not express hemoglo-
bin based on immunoflorescence with an anti-Xenopus glo-
bin antisera.
Fibroblast growth factor (FGF) treatment of animal pole
explants at low concentrations mimics the effects of the
ventral vegetal signal in V ~ V O . ~ &This
~ ’ consists of a concen-
tric arrangement of mesenchyme and mesothelium. Some
blood-like cells are present that do not express hemoglobin
but have a characteristic m ~ r p h o l o g y . ’Other ~ ~ ~ ~growth
~~~
factors effect the induction of hematopoietic mesoderm and
ventral patterning. BMP-4 is a member of the TGF-p super-
family and is capable of ventralizing whole embryos and
increasing the expression of g l ~ b i n ? ~ - ~ ~ a member
WNT-8,
of the wnt family, also can ventralize tissues when expressed
correctly after the onset of zygotic transcription96s97;
ever, if expressed earlier, WNT-8 predominantly dorsalizes
how-
tissues. More research is needed to determine the exact effect
these proteins have on hematopoietic induction, but it is clear
that dose and timing of exposure to stem cells will be critical
varibles to explore. There are also several dorsalizing factors
including TGF-P, wnr, and noggin98”* polypeptides that
have been described in Xenopus embryos that may function
to suppress hematopoiesis.
Studies of embryonic hematopoiesis in Xenopus. Using
whole embryo in situ analysis for GATA-1 and SCL RNA
expression, we have shown that the hematopoietic program
initiates at least as early as 11 hours after fertilization (during
gastrulation).“’ GATA-l RNA was not initially detected in
uninduced animal cap cells, but, after 17 hours of culture,
GATA-I was surprisingly expressed.”’ The level continued
to increase until 25 hours and then decreased. Later in CUI-
ture, embryonic a-globin was also expressed at a low level
in the animal cap cells. The expression of these genes is in
accord with the normal temporal pattern of expression.
MyoD, amuscle-specific transcription factor, is also ex-
pressed in the uninduced animal caps early in the culture
and is downregulated so that caps at 48 hours do not express
MyoD.’” Thus, the mesodermal programs (such as blood
and muscle) are evident in the animal cap cultures, but, in
the absence of induction, the programs are not maintained.
 Activin or FGF induction of animal pole explants leads to the
maintenance of MyoD expression and formation of muscle;
I however, neither factor rescues the blood program or main-
tains GATA-1 expression. As discussed above, the ventral
vegetal signal(s) present in VMZ explants is capable of in-
ducing and maintaining the blood program as defined by
GATA-1 and embryonic a-globin expression (Fig 12). The
animal cap itself has also been postulated to affect hemato-
poietic differentiation." When animal pole tissue is recom-
bined with isolated VMZ cells, an increase in hemoglobin

l is detected, indicating that the animal pole contains signals

that enhance globin expression. A focus of our laboratory is
to use these assays to isolate and define factors that partici-
pate in ventral axis patterning or mesoderm induction.

SUMMARY
The cellular and environmental regulation of hematopoie-
sis has been generally conserved throughout vertebrate evo-
lution, although subtle species differences exist. The factors
that regulate hematopoietic stem cell homeostasis may
closely resemble the inducers of embryonic patterning, rather
than the factors that stimulate hematopoietic cell prolifera-
tionand differentiation. Comparative study of embryonic
hematopoiesis in lower vertebrates can generate testable
hypotheses that similar mechanisms occur during hematopoi-
esis in higher species.

ACKNOWLEDGMENT
I appreciate thehelpful discussionswith Jim Turpen (Universityof
Nebraska, Lincoln,NB) and Bob Broyles (Universityof Oklahoma,
Norman, OK). I thank Todd Evans (University of Pittsburgh, Pitts-
burgh, PA), Gordon Keller (National Jewish Hospital, Denver, CO),
and Ramesh Shivdasani (Dana-Farber Cancer Institute, Boston, MA)
Fig 12. Localizationofhematopoieticmesoderm in the VMZ as and Merlin Crossley, Mitch Weiss,and Andrew Perkins (Children's
Hospital of Boston) for their critical review of the manuscript.
determined by globin expression. Three VMZs were explanted during
gastula stages and allowedto develop for 36 hours. These explants
(bottom)and the whole embryo(top) were subjectedto in situ analy- REFERENCES
sis with an antisense digoxigenin-labeled probeto embryonic a-glo-
bin. The expression is detected by an alkaline phosphatase-coupled
1. Turner RJ: Amphibians,inRowley AF RatcliffeNA (eds):
Vertebrate Blood Cells. Cambridge,UK,CambridgeUniversity,
digoxigenin-labeled antibody. Note that the VMZ expresses signifi-
1988, p 129
*
cant globin mRNA in a very localized region of the explant, sug
gesting that patterning factors affect the induction of blood from 2. Zapata AG, Varas A, TorrobaM: Seasonal variations in the
undifferentiated mesoderm. immune system of lower vertebrates. Immunol Today 13:142, 1992
DEVELOPMENTAL BIOLOGY OF HEMATOPOIESIS
3. Moore MA, Metcalf D: Ontogeny of the haemopoietic system:
Yolk sac origin of in vivo and in vitro colony forming cells in the
developing mouse embryo. Br J Haematol 18:279, 1970
4. Dieterlen LF, Martin C: Diffuse intraembryonic hemopoiesis
in normal and chimeric avian development. Dev Biol 88:180, 1981
5. Dieterlen LF, Beaupain D, Martin C: Origin of erythropoietic
stem cells in avian development: Shift from the yolk sac to an
intraembryonic site. Ann Immunol (Paris) 127:857, 1976
6. Maeno M, Tochinai S , Katagiri C: Differential participation of
ventral and dorsolateral mesoderms in the hemopoiesis of Xenopus,
as revealed in diploid-triploid or interspecific chimeras. Dev Biol
110:503, 1985
7. Maeno M, Todate A, Katagiri Ch: The localization of precursor
cells for l a r v a l and adult hemopoietic cells of Xenopus laevis in two
regions of embryos. Dev Growth Differ 27:137, 1985
8. Turpen JB, Knudson CM, Hoefen PS: The early ontogeny of
hematopoietic cells studied by grafting cytogenetically labeled tissue
anlagen: Localization of a prospective stem cell compartment. Dev
Biol 85:99, 1981
9. Ruiz I, Altaba A, Melton DA: Axial patterning and the estab-
lishment of polarity in the frog embryo. Trends Genet 657, 1990
10. Woodland HR: Mesoderm formation in Xenopus. Cell
59:767, 1989
11. Dieterlen LF, Pardanaud L, Yassine F, Cormier F: Early
haemopoietic stem cells in the avian embryo. J Cell Sci Suppl 10:29,
1988
12. Lassila 0, Martin C, Dieterlen LF, Nurmi TE, Eskola J, Toi-
vanen P: Is the yolk sac the primary origin of lymphoid stem cells?
Transplant Proc 11:1085, 1979
13. Lassila 0, Martin C, Dieterlen LF, Gilmour DC, Eskola J,
Toivanen P: Migration of prebursal stem cells from the early chicken
embryo to the yolk sac. Scand J Immunol 16:265, 1982
14. Le DN, Dieterlen LF, Oliver PD: Ontogeny of primary
lymphoid organs and lymphoid stem cells. Am J Anat 170:261, 1984
15. Toivanen P, Lassila 0, Eskola J, Martin C, Dieterlen LF,
Gilmour DC: Migration of erythropoietic and prebursal stem cells
from the early chicken embryo to the yolk sac. Adv Exp Med Biol
149:11, 1982
16. Tavassoli M: Embryonic and fetal hemopoiesis: An overview.
Blood Cells 17:269, 1991
17. Liu CP, Auerbach R: Ontogeny of murine T cells: Thymus-
regulated development of T cell receptor-bearing cells derived from
embryonic yolk sac. Eur J Immunol 21 :1849, 1991
18. Johnson GR, Barker DC: Erythroid progenitor cells and stim-
ulating factors during murine embryonic and fetal development. Exp
Hematol 13:200, 1985
19. Wong PMC, Chung SW, Chui DHK, Eaves CJ: Properties of
the earliest clonogenic hematopoietic precursors to appear inthe
developing murine yolk sac. Roc Natl Acad Sci USA 83:3851, 1986
20. Romanoff AL (ed): The Avian Embryo. New York, NY,Mac-
millan, 1960, p 569
2 1. Brauns A: Untersuchungen zur ermittlung der entstehung der
roten blutzellen in der embryonalentwicklung der urodelen. Roux’
Arch Entwicklungmech Organ 140741, 1940
22. Federici E: Recherches experimentales sur les potentialites
de 1’Ilot sanguin chez I’embryon de rana fusca. Arch de Biol36:465,
1926
23. Goss CM: Experimental removal of the blood island of am-
blystoma punctatum embryos. J Exp Zool 52:45, 1928
24. Mangia F, Procicchiami G , Manelli H: On the development
of the blood island in Xenopus laevis embryos: light and electron
microscope study. Acta Embryo1 Exp (Palermo) 2: 163, 1970
25. Oellacher J: Beitrage zur entwicklungsgeschichte der kno-
chenfische nach beobachtungen am bachforelleneie. Z Zool 23373,
1872
2889
26. Swaen A, Brachet A: Du meosblaste chez les poissons teleos-
teens. Arch Bioi 18:169, 1901
27. Colle-Vandevelde: Blood anlage in teleostei. Nature 1223,
1963
28. Wenckebach KF: The development of the blood-corpuscles
in the embryo of Perca Fluviatilis. J Anat Physiol 19:231, 1885
29. Ziegler HE: Die entstehung des blutes bei knochenfisch-em-
bryonen. Arch Mik Anat 30596, 1887
30. AI-Adhami MA, Kunz YW: Ontogenesis of haematopoietic
sites in brachydanio rerio. Dev Growth Differ 19:171, 1977
3 I . Stockard CR: The origin of blood and vascular endothelium
in embryos without a circulation of the bloodandin the normal
embryo. Am J Anat 18:227, 1915
32. Detrich HW,KieranMW, Chan FY, Barone LM, Yee K,
Rundstadler JA, Zon LI: Intra-embryonic hematopoietic cell migra-
tion during vertebrate development. Proc Natl Acad Sci USA 1995
(in press)
33. AI-AdhamiMA, Kunz YW: Hematopoietic centres in the
developing angelfish, Pterophyllum scalere (Cuvier and Valencien-
nes). Wilh Roux Arch 179:393, 1976
34. Smith PB, Flajnik MF, Turpen JB: Experimental analysis of
ventral blood island hematopoiesis in Xenopus embryonic chimeras.
DevBiol 131:302, 1989
35. Turpen JB, Smith PB: Analysis of hemopoietic lineage of
accessory cells in thedeveloping thymus of Xenopus laevis. J Immu-
no1 136:412, 1986
36. Turpen JB, Smith PB: Location of hemopoietic stem cells
influences frequency of lymphoid engraftment in Xenopus embryos.
J Immunol 143:3455, 1989
37. Turpen JB, Smith PB: Precursor immigration and thymocyte
succession during larval development and metamorphosis in Xeno-
pus. J Immunol 142:41, 1989
38. Turpen JB, Smith PB: Dorsal lateral plate mesoderm influ-
ences proliferation and differentiation of hemopoietic stem cells de-
rived from ventral lateral plate mesoderm during early development
of Xenopus laevis embryos. J Leukoc Biol 38:415, 1985
39. Kau CL, Turpen JB: Dual contribution of embryonic ventral
blood island and dorsal lateral plate mesoderm during ontogeny of
hemopoietic cells in Xenopus laevis. J Immunol 131:2262, 1983
40. Ohinata H, Tochinai S, Katagiri C: Occurrence of non-
lymphoid leukocytes that are not derived from blood islands in Xeno-
pus laevis larvae. Dev Biol 141:123, 1990
41. Ohinata H, Tochinai S, Katagiri C: Ontogeny and tissue distri-
bution of leukocyte-common antigen bearing cells during early de-
velopment of Xenopus laevis. Development 107:445, 1989
42. Katagiri C, Maeno M, Tochinai S : Differential commitment
of hemopoietic stem cells localized in distinct compartments of early
Xenopus embryos. Curr Top Dev Biol 20:315, 1986
43. Smith PB, Turpen JB: Differential contribution of dorsal and
ventral lateral plate mesoderm to hemopoiesis during Rana pipiens
embryogenesis. DevBiol104:497, 1984
44. Turpen JB, Turpen CJ, Flajnik M: Experimental analysis of
hematopoietic cell development in the liver of larval Rana pipiens.
Dev Biol 69:466, 1979
45. Smith PB, Turpen JB: Hemopoietic differentiation potential
of cultured lateral plate mesoderm explanted from Rana pipiens
embryos at successive developmental stages. Differentiation 28:244,
1985
46. Turpen JB, Knudson CM: Ontogeny of hematopoietic cells
in Rana pipiens: Precursor cell migration during embryogenesis. Dev
Biol 89:138, 1982
47. Broyles RH: Changes in the blood during amphibian meta-
morphosis, in Gilbert LI, Frieden E (eds): Metamorphosis: A Prob-
lem in Developmental Biology. New York, NY, Plenum, 1981, p
46 1
48. Pardanaud L, Altmann C, Kitos P, Dieterlen-Lievre F, Buck
2890
CA: Vasculogenesis in the early quail blastodisc as studied with
a monoclonal antibody recognizing endothelial cells. Development
100:339, 1987
49. Peault B, Thiery JP, LeDouarin NM: A surface marker for
the hemopoietic and endothelial cell lineage in the quail species
defined by a monoclonal antibody. Proc Natl AcadSci USA 80:2976,
1983
50. Miller AM: Histogenesis and morphogenesis of the thoracic
duct in the chick: Development of blood cells and their passage to
the blood stream via the thoracic duct. Am J Anat 15:131, 1913
5 1. Dieterlen-Lievre F: Hemopoiesis during avian ontogeny.
Poult Sci Rev 5:273, 1993
52. Medvinsky AL. Samoylina NL, Muller AM, Dzierzak EA:
An early pre-liver intraembryonic source of CFU-S in the developing
mouse. Nature 36454, 1993
53. Godin IE, Garcia-Porrere JA, Coutinho A, Dieterlen-Lievre
F, Marcos MAR: Para-aortic splanchnopleura from early mouse em-
bryos contains Bla cell progenitors. Nature 364:67, 1993
54. Muller AM, Mevinsky A, Strouboulis J, Grosveld F, Dzierzak
E: Development of hematopoietic stem cell activity in the mouse
embryo. Immunity 1:291, 1994
55. Flamme I, Risau W: Induction of vasculogenesis and hemato-
poiesis in vitro. Development 116:435, 1992
56. Sabin FR: Studies on the origin of blood vessels and of red
blood corpuscles as seen in the living blastoderm of chicks during
the second day of incubation. Contrib Embryol 9:213, 1920
57. Reagan FP: Experimenta studies on the origin of vascular
endothelium and of erythrocytes. Am J Anat 21:39, 1917
58. His W: Lecithoblast und angioblast der wirbelthiere. Abhandl
KS Ges Wiss Math-Phys 22:171, 1900
59. Stanier DYR, Weinstein BM, Detrich HW, Zon LI, Fishman
MC: Cloche, an early acting zebrafish gene, is required by both the
endothelial and hematopoietic lineages. Development (in press)
60. Solonica-Krezel L, Schier AF, Driever W: Efficient recovery
of ENU-induced mutations from the zebrafish germline. Genetics
136:1401, 1994
61. Mullins MC, Nusslein-Volhard C: Mutational approaches to
studying embryonic pattern formation in the zebrafish. Cum Opin
Genet Dev 3548, 1994
62. Postlethwait JH, Johnson SL, Midson CN, Talbot WS, Gates
M, Ballinger EW, Africa D, Andrews R, Carl T, Eisen JS, Home
S , Kimmel CB, Hutchinson M, Johnson M, Rodriguez A: A genetic
linkage map for the zebrafish. Science 264:699, 1994
63. Nieuwkoop PD, Faber J: Normal Table of Xenopus laevis
(Daudin). Amsterdam, the Netherlands, North-Holland, 1957, p 18
64. Nieuwkoop PD, Sutasurya LA: Embryological evidence for
a possible polyphyletic origin of the recent amphibians. J Embryol
Exp Morphol 35:159, 1976
65. Boterenbrood EC, Nieuwkoop PD: The formation of the
mesoderm in urodelean amphibians. Wilhelm Roux’ Arch 1173:319,
1973
66. Dale L, Smith JC, Slack JM: Mesoderm induction in Xenopus
laevis: A quantitative study using a cell lineage label and tissue-
specific antibodies. J Embryol Exp Morphol 89:289, 1985
67. Gurdon JB, Fairman S, Mohun TJ, Brennan S: Activation of
muscle-specific actin genes in Xenopus development by an induction
between animal and vegetal cells of a blastula. Cell 41:913, 1985
68. Jones EA, Woodland H R The development of animal cap
cells in Xenopus: a measure of the start of animal cap competence
to form mesoderm. Development 101557, 1987
69. Dale L, Slack JM: Regional specification within the meso-
derm of early embryos of Xenopus laevis. Development 100:279,
1987
70. Slack JM, Forman D: An interaction between dorsal and ven-
tral regions of the marginal zone in early amphibian embryos. J
Embryol Exp Morphol 56:283, 1980
LEONARD I. ZON
71. Yamada T, Takata K: A technique for testing macromolecular
samples in solution for morphogenetic efforts on the isolated ecto-
derm of the amphibian gastrula. Dev Biol 3:41 l , 1961
72. Grunz H, Tacke L: The inducing capacity of the presumptive
endoderm of Xenopus laevis studied by transfilter experiments.
Roux’s Arch Dev Biol 195:467, 1986
73. Gurdon JB: The localization of an inductive response. Devel-
opment 105:27, 1989
74. Cooke J, Smith JC: The midblastula cell cycle transition and
the character of mesoderm in UV-induced nonaxial Xenopus devel-
opment. Development 99:197, 1987
75. Green JB, Howes G, Symes K, Cooke J, Smith JC: The
biological effects of XTC-MIF: Quantitative comparison with Xeno-
pus bFGF. Development 108:173, 1990
76. Deparis P, Jaylet A: The role of endoderm inblood cell
ontogeny inthenewt Pleurodeles waltl. J Embryol Exp Morphol
81:37, 1984
77. New HV, Smith JC: Inductive interactions in early amphibian
development. Curr Opin Cell Biol 2:969, 1990
78. Green JB, Smith JC: Graded changes in dose of a Xenopus
activin A homologue elicit stepwise transitions in embryonic cell
fate [see comments]. Nature 347:391, 1990
79. Smith JC, Yaqoob M, Symes K: Purification, partial charac-
terization and biological effects of the XTC mesoderm-inducing fac-
tor. Development 103591, 1988
80. Smith JC: Mesoderm induction and mesoderm-inducing fac-
tors in early amphibian development. Development 105:665, 1989
81. Smith JC, Price BM, Van NK, Huylebroeck D: Identification
of a potent Xenopus mesoderm-inducing factor as a homologue of
activin A. Nature 345:729, 1990
82. Smith JC: A mesoderm-inducing factor is produced by a Xen-
opus cell line. Development 99:3, 1987
83. van den Eijnden-Van Raaij AJ, van Zoelent EJ, van Nimmen
K, Koster CH, Snoek GT, Durston AJ, Huylebroeck D: Activin-like
factor from a Xenopus laevis cell line responsible for mesoderm
induction. Nature 345:732, 1990
84. Sokol S, Wong GG, Melton DA: A mouse macrophage factor
induces head structures and organizes a body axis in Xenopus. Sci-
ence 249:561, 1990
85. Yu J, Shao LE, Lemas V, Yu AL, Vaugban J, Rivier J, Vale
W: Importance of FSH-releasing protein and inhibin in erythrodiffer-
entiation. Nature 330:765, 1987
86. Kimelman D, Kirschner M: Synergistic induction ofmeso-
derm by FGF and TGF-beta and the identification of an mRNA
coding for FGF in the early Xenopus embryo. Cell 51:869, 1987
87. Kimelman D, Abraham JA, Haaparanta T, Palisi m,
Kirschner MW: The presence of fibroblast growth factor in the frog
egg: Its role as a natural mesoderm inducer. Science 242: 1053, 1988
88. Slack JM, Darlington BG, Heath JK, Godsave SF: Mesoderm
induction in early Xenopus embryos by heparin-binding growth fac-
tors. Nature 326:197, 1987
89. Slack JM, Isaacs HV, Darlington BG: Inductive effects of
fibroblast growth factor and lithium ion on Xenopus blastula ecto-
derm. Development 103:581, 1988
90. Slack JM, Darlington BG, Gillespie LL, Godsave SF, Isaacs
HV, Paterno GD: The role of fibroblast growth factor in early Xeno-
pus development. Development 107:141, 1989 (suppl)
91. Slack JM, Darlington BG, Gillespie LL, Godsave SF, Isaacs
HV, Patemo GD: Mesoderm induction by fibroblast growth factor
in early Xenopus development. Philos Trans R SOCLond [Bioll
327175, 1990
92. Symes K, Yaqoob M, Smith JC: Mesoderm induction in Xen-
opus laevis: Responding cells must be in contact for mesoderm
formation but suppression of epidermal differentiation can occur in
single cells. Development 104:609, 1988
93. Koster M, Plessow S, Clement JH, Lorenz A, Tiedemann H,
DEVELOPMENTAL BIOLOGY OF HEMATOPOIESIS 2891

Knochel W: Bone morphogenetic protein 4 (BMP-4), a member of 100. Walmsley ME, Guille UT, Bertwistle D, Smith JC, Pizzey
the TGF-beta family, in early embryos of Xenopus laevis: analysis JA, Patient RK: Negative control of Xenopus GATA-2 by activin
of mesoderm inducing activity. Mech Dev 33:191, 1991 andnogginwith eventual expression in precursors of the ventral
94. Plessow S, Koster M, Knochel W: cDNA sequence of Xeno- blood islands. Development 1202519, 1994
pus laevis bone morphogenetic protein 2 (BMP-2). Biochim Biophys 101. Zon LI, Mather C, Burgess S , Bolce M, Harland R, Orkin
Acta 1089280, 1991 SH:Expression of GATA-binding proteins during embryonicdevel-
95. Jones CM, LyonsKM, Lapan PM, Wright CVE, Hogan BLM: opment in Xenopus laevis.Proc Natl Acad Sci USA 88: 10642, 1991
DVR-4 (bone morphogenetic protein-4) as a posterior-ventralizing 102. Kelley C, Yee K, Harland R, Zon L: Ventral expression of
factor in Xenopus mesoderm induction. Development 115:639,1992 GATA-1 and GATA-2 in the Xenopus embyro defines inductionof
96. Christian JL, McMahon JA, McMahon A P , Moon RT: Xwnt- hematopoietic mesoderm. Dev Biol 165:193, 1994
8, a Xenopus Wnt-lht-l related gene responsive to mesoderm in- 103. Rupp RAW, Weintraub H: Ubiquitous MyoD transcription
ducingfactors mayplay aroleinventralmesodermalpatterning atthemidblastulatransitionprecedesinduction-dependentMyoD
during embryogenesis. Development 11 1:1045, 1991 expression in presumptive mesodenn of X. laevis. Cell 65:927, 1991
97.Christian L,Moon RT:InteractionsbetweenXwnt-8and 104. Maeno M, Ong RC, Kung H: Positive and negative regula-
Spemann organizer signaling pathways generate dorsoventral patterntion of the differentiation of ventral mesoderm for erythrocytes in
in the embryonic mesoderm of Xenopus. Genes Dev 7:13, 1993 Xenopus laevis. Dev Growth Differ 34:567, 1992
98. Smith WC, HarlandRM: Expression cloningof noggin, a new 105. Dale L, Slack JM: Fate mapfor the 32-cell stageof Xenopus
dorsalizing factor localized to the Spemann organizer in Xenopus laevis. Development 99527, 1987
embryos. Cell 70829, 1992 106.Sadler T W (4) :
Langman’s MedicalEmbryology.Balti-
99. Smith WC, Knecht AK,Wu M, Harland RM: Secreted noggin more, MD, Williams & Wilkens, 1990, p 21
protein mimics the Spemann organizer in dorsalizing Xenopus meso- 107. Green JBA, Smith J C Growthfactors as morphogens.
derm. Nature 361:547. 1993 Trends Genet 7:245, 1991