Building the house aroundthe plumbingBrant Weinstein

SummarySignalingbetweengrowingbloodvessels and the tissuesthat they innervate has traditionally beenviewedasaone-way conversation, with organs and tissues supplyingimportant cues for the growth and anatomical patterningof the blood vessels supplying them, but not vice-versa.Two recent papers(1,2) now provide evidence that bloodvessels can have an important role in promoting the as-sembly of organs and tissues. These papers show thatproper formation of the pancreas(1) and liver(2) and induc-tion of endocrine and hepatic cell types in these endo-dermal organs requires inductive signals from bloodvessels. BioEssays 24:397–400, 2002.Published 2002 Wiley Periodicals, Inc.

Building the house around the plumbing?

Most of us would consider indoor plumbing an essential fea-

ture ofmodern life, but it is not usually the first thing assembled

when a new house is constructed. A foundation comes first,

followed by the structural framework, roof, and walls. Proper

installation and interconnection of pipes to bring fresh water

and remove wastewater is essential, but the layout of the

plumbing is subordinate to the overall architectural plan. Like

the plumbing in a house, the complex and intricate vascular

network of vertebrates plays a vital functional role, supplying

oxygen and nutrients, removing wastes, and serving as the

conduit for transport of immune and hormonal cells and

factors. Also like plumbing, the anatomical pattern of the vas-

culature does seem to follow from the requirements of the

tissues that it serves and the overall anatomy of the animal.

Two recent papers in Science test the limits of this analogy,

however, by showing that blood vessels help direct the

assembly of at least some internal organs.

Cues guiding vascular patterning

during development

In mammalian and avian embryos, the first vascular networks

form by aggregation of free vascular endothelial progenitors,

or angioblasts. These give rise to initially relatively uniform

plexuses of interconnected small vascular segments on the

extraembryonic yolk sac, and intraembryonically within the

developing trunk and other locales. This process of de novo

aggregation of angioblast progenitors into vascular networks

has been termed vasculogenesis.(3–5) The vascular plexuses

are remodeled and expanded in later steps via enlargement

or coalescence of some channels, regression and loss of

others, division (intussusception) and branching of vessels,

and sprouting and elongation of new vessels. During these

later steps, the final morphological pattern of the vasculature

and the defined positions of arteries and veins and their

interconnections become established. Collectively, this later

process of remodeling and further elaboration of the vascu-

lature is termed angiogenesis.(4) Blood vessels also form in

adult life during wound healing, tumor formation, and other

processes. These vessels appear to form primarily by angio-

genesis, although recent evidence suggests that vasculo-

genic-like vessel formation also occurs postnatally,(6) and that

the distinctions between these two processes may not be so

sharply defined.

What molds dispersed angioblasts or apparently ‘‘naive’’

vasculogenic vascular plexuses into the exquisitely patterned

form of the postnatal vasculature? The classical view of blood

vessel development is that it is largely self-organizing process

driven by metabolic requirements of local tissues and hemo-

dynamic forces, which act upon the primitive vascular plexus

to strengthen and enlarge active channels and reduce or

eliminate inactive channels. Eventually this results in an opti-

mized configuration of blood vessels, which further elaborates

via continued vascular growth and remodeling in response to

the needsof local tissues. This paradigmdoes comfortably fit a

number of well-studied models of vascular development, in-

cluding the avian and murine yolk sac, chorioallantoic mem-

brane,(7) and retina.(8) In these tissues, vessel anatomy does

not follow a rigidly fixed pattern, and does seem to be depen-

dent on local needs and flow dynamics. Vascularization of

the retina, for example, is highly dependent on oxygenation.

Hypoxia leads to upregulation of vascular endothelial growth

factor (vegf), an important vascular signaling molecule that

stimulates blood vessel formation in unvascularized portions

of the retina.(8) Hyperoxia during retinal development results in

undervascularization of the retina, such as occurs in prema-

ture infants kept in hyperoxygenated incubators. This under-

vascularization leads to retinal hypoxia and damage when the

infants are later returned to normoxic conditions, a syndrome

called retinopathy of prematurity.(9)

BioEssays 24:397–400, Published 2002 Wiley Periodicals, Inc. BioEssays 24.5 397This article is a US Government work, and as such, is in the public

domain in the United States of America

Correspondence to: Brant Weinstein, Unit of Vertebrate Organo-

genesis, Laboratory of Molecular Genetics, NICHD, NIH Building 6B,

Room 309, 6 Center Drive, Bethesda, MD 20892.

E-mail: bmw7@mail.nih.gov

DOI 10.1002/bies.10090

Published online in Wiley InterScience (www.interscience.wiley.com).

What the papers say

Although some vessels do seem to self-organize, the deve-

lopment of major intraembryonic blood vessels appears more

‘‘hard-wired.’’ These vessels form with a reproducible and

evolutionarily conserved pattern, and are not appreciably

affected by the metabolic needs of local tissues. Moreover,

in vertebrates such as fish and amphibians many of these

vessels form de novo as distinct, single tubes, suggesting that

vascular plexus formation and its subsequent remodeling are

not obligatory steps. Evidence has also emerged for gene-

tically programmed cues directing vascular patterning and

differentiation of some of these major vascular highways.

Formation of the dorsal aorta and acquisition of its arterial

identity are directed by cues from adjacent axial mesoderm

and other tissues, cues that include the well-studied hedge-

hog, vegf, andnotchsignalingpathways.(10–14)Theanatomyof

blood vessels supplying internal organs is also generally

reproducible and evolutionarily conserved, and functionally

critical. In the kidney, bloodvessels are closely associatedwith

podocytes, the renal cells that perform the filtering function of

this organ, and the podocytes’ glomerular basement mem-

brane is notmaintainedproperly in a zebrafishmutant deficient

in endothelium called cloche.(15,16) The identification of an

endocrine endothelial-specific angiogenic factor indicates that

patterning of bloodvessels in different organs canbemediated

by organ-specific vascular factors.(17) Vascularization of the

brain and other neural tissues may also involve genetically

programmed patterning cues. Blood vessels and nerve tracts

frequently follow adjacent tracks, and neural blood vessel

formationmay be directed by the same sorts of molecules that

guide axonal patterning (reviewed in Ref. 18).

Vascular development and organogenesis—

a two-way conversation?

But whatever guides vessel formation, the prevailing dogma

has been that the signaling is one-way; that vessels are

patterned by cues from the tissues and organs that they

innervate but not vice-versa.Now, newevidencesuggests that

there can in fact be a two-way conversation, and that blood

vessels can in turn provide important cues for the development

of the organs that they serve. In back-to-back papers in

Science, two groups investigated the role of blood vessels in

formation of the pancreas(1) and liver.(2) The liver, pancreas,

and other endodermal organs emerge from different regions

of the primitive gut tube by budding and growing into the

surrounding mesenchyme. The findings in these two papers

indicate that vascular endothelial cells play important inductive

roles in the formation of these organs- in a sense, building the

house around the plumbing.

Vascular cells participate

in pancreatic induction

In the first paper, Lammert et al.(1) investigated whether blood

vessels play an inductive role in the formation of the pancreas.

Pancreatic bud emergence and later differentiation of the

endocrine pancreatic islets both take place in close proximity

to blood vessels (Fig. 1). At 9.0 days post coitus (dpc) pan-

creatic precursor cells expressing the transcription factor pdx1

(pancreatic duodenal homeobox 1) are found adjacent to and

between the fusing dorsal aortae. Pdx1 initially marks portions

of the primitive gut tube that include regions fated to give rise to

the pancreas, and is also expressed at later stages in cells that

give rise to the duodenum and stomach. By birth it becomes

restricted to insulin-producing endocrine cells of the pan-

creatic islets. Islet cells also express the important vascular

signaling molecule, vascular endothelial growth factor (vegf),

and are surrounded by capillary blood vessels expressing

vascular endothelial growth factor receptor 2 (vegfr2, or flk1)

and platelet endothelial cell adhesion molecule (PECAM, or

CD31, Ref. 19). Thus not only do pancreatic endocrine cells

develop in close proximity to endothelial cells, they express an

important factor required for endothelial development. To

examine whether association with endothelial cells is impor-

tant for the formation of the pancreas or pancreatic islets, the

authors made use of three different experimental systems.

They used explant culture to show that vascular endothelium

has the capacity to induce endoderm to undergo pancreatic

differentiation ex vivo. Mouse endoderm co-cultured with

dorsal aorta explants formed numerous buds and expressed

pdx1 and insulin adjacent toPECAM-expressing endothelium.

Endoderm cultured alone did not form buds or express

pancreatic marker genes, but formed an undifferentiated gut-

tube-like structure. Co-culture with umbilical artery or me-

senchyme explants also resulted in induction of some pdx1

and insulin expression. These results suggested that en-

dothelial cells provide an inductive cue for pancreatic

Figure 1. A,B: As yet unidentified signals from the dorsal

aorta (red) help induce the pancreas (orange) and pancreatic

endocrine cells (purple) from the primitive gut tube (green). C:At later stages of pancreatic development, blood vessels (red)

are found in close association with endocrine pancreatic islets

(purple).

What the papers say

398 BioEssays 24.5

differentiation in vitro. The ability of mesenchyme to substitute

for blood vessel explants likely reflects the presence of endo-

thelial cell progenitors or angioblasts, which are known to be

widely distributed in mesenchyme, although some inductive

capacity for nonvascular mesenchymal cell types was not

ruled out in these experiments.

To determine whether endothelial signals are required for

pancreatic differentiation in vivo, dorsal aorta formation was

blocked in Xenopus laevis embryos by surgically excising the

lateral mesodermal tissues that give rise to dorsal aorta

progenitors. Manipulated animals showed a dramatic reduc-

tion in insulin expression aswell as in pancreatic expression of

neuroD and pax6. Other nearby tissues, including the noto-

chord, gut tube, and floor plate, appeared unaffected by this

procedure. In a few cases, the dorsal aorta regenerated in

surgically manipulated animals due to immigration of vascular

progenitors from elsewhere in the embryo. In these ‘‘rescued’’

embryos, pancreaticmarkers were again expressed, suggest-

ing that lack of pancreatic induction in the other manipulated

animals was due to lack of an inductive signal from vascular

endothelium and not due to a lack of lateral mesoderm per se.

This inductive effect was confirmed independently using

transgenicmice expressing vascular endothelial growth factor

under the control of the pdx1 promoter. These mice express

excess vegf early in pancreatic development, driving hyper-

vascularization of the pancreas. The pancreas in these

transgenic animals was cystic and showed islet hyperplasia

as well as reduction in the amount of surrounding pancreatic

acinar tissue, suggesting that endocrine tissues were pro-

moted at the expense of acinar cell types. As noted above,

pdx1 expression expands to include the posterior part of

the stomach and duodenum at later stages of development

(by 11.5 dpc). In the transgenic mice, the stomach was

prematurely hypervascularized and, in close proximity to

the ectopically induced endothelial cells, there were ectopic

insulin-expressing cells. In normal nontransgenic animals,

insulin was never expressed in the stomach.

Taken together, the results of the explant, surgical ablation,

and transgenic expression experiments performed by Lam-

mert et al. strongly support the idea that thedevelopment of the

pancreas and insulin-producing pancreatic islets requires

inductive signals from vascular endothelial cells.

Vascular cells participate in liver induction

In a second paper, Matsumoto and colleagues(2) used vegfr2

(flk1) knockout mice and a liver explant system to examine the

role of endothelial cells in liver development, arriving at similar

conclusions to those of Lammert et al. flk�/� embryos are

deficient in both blood vessels and blood cells, dying by E10.5.

Differentiated endothelial cells do not formandprogenitor cells

fail to migrate to the locations of normal early intraembryonic

vessel formation.(20) flkþ/� heterozygous mice develop nor-

mally, however, and heterozygotes with lacZ ‘‘knocked in’’

downstream from the flk1 promoter permit convenient visuali-

zation of developing blood vessels.(20)

Formation of the murine liver begins with a thickening of the

primitive gut tube at E8.5–E9.0.(21) At approximately E9.5

these hepatic epithelial cells migrate into the surrounding

mesenchyme to form the liver bud. Endothelial cells, visua-

lized using either PECAM or b-galactosidase (in flkþ/� lacZ

knockin mice), are found in close association with hepatic

progenitors throughout this time. Isolated angioblasts or

endothelial cells surrounded the thickening hepatic epithelium

of the gut tube at E8.5–E9.0, while nascent blood vessels

were interspersed throughout the forming hepatic tissue at

E9.5–E10.5.

To examine whether endothelial cells play a role in promoting

hepatic morphogenesis, liver development was examined in

flk�/� knockout mice. Despite lack of any detectable angio-

blasts or endothelial cells around the endoderm, initial hepatic

induction did take place in these animals, as evidenced by a

thickened hepatic primordium expressing liver markers

albumin, transthyretin, andHex atE9.0. However, subsequent

proliferation of the hepatic rudiment and migration of hepatic

cells into the surrounding mesenchyme did not occur. This

suggests that endothelial cells are necessary for liver bud

outgrowth but are not required for the initial induction of the

endodermal hepatic rudiment.

To more directly test the hepatic induction capacity of

endothelium, and to separate the direct effects of the flk�/�

mutant on vascular endothelium fromsecondary effects on the

growth and vitality of knockout animals, the authors developed

a liver bud explant system that could support liver vasculogen-

esis in vitro. Liver buds explanted from wild-type or flkþ/�

heterozygous mice underwent a 15-fold increase in surface

area over 3 days in vitro, with increases evident in both hepatic

(albumin-positive) and vascular (PECAM-positive) tissues.

AlbuminmRNAexpressionwasgenerally confined to themore

highly vascularized regions of flkþ/� explants. Liver bud ex-

plants from flk�/� mice also increased in size approximately

15-fold after three days in culture but, in contrast to wild-type

explants, these contained fewer albumin-positive hepatic cells

(5% versus 20% in wild-type or flkþ/� heterozygous explants).

Most of the proliferating cells in flk�/� explants appeared to be

fibroblastic and the primary, thick tissue mass of the explant

(the presumptive hepatic rudiment) remained small. A similar

result was obtained when wild-type explants were treated with

the angiogenesis inhibitor NK4 to inhibit the growth and

development of endothelial cells, suggesting that the conti-

nued presence of endothelial cells is required for hepatic

differentiation.

Conclusion

Obviously, intensive effort will now be focused on uncovering

the molecular basis for the inductive signals provided by

endothelial cells or angioblasts. Although it remains to be

What the papers say

BioEssays 24.5 399

determined whether signals from endothelium help direct the

formation of organs other than the liver and pancreas, blood

vessels clearly play critical and highly integrated roles inmany

other organs and it seems likely that this is the case. In the end,

vascular development and the anatomy of the circulatory

system probably depends on a complex interplay between

metabolic needs, hemodynamics, programmed patterning

cues from tissues and organs to angioblasts and endothelial

cells, as well as cues from vascular cells to surrounding

tissues. Uncovering all of these factors, and understanding the

rules governing how they work together to fashion the

stereotypic anatomy of the adult vertebrate vasculature, will

undoubtedly keep vascular biologists occupied formany years

to come.

Acknowledgment

The author thanks Mildred Pack for reading this manuscript.

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What the papers say

400 BioEssays 24.5