pbio.1000246 1..14 - Vermot2009p6492Plos

Reversing Blood Flows Act through

klf2a

to Ensure

Normal Valvulogenesis in the Developing Heart

Julien Vermot

1¤

, Arian S. Forouhar

1,2

, Michael Liebling

1,3

, David Wu

1,2

, Diane Plummer

, Morteza

Gharib

, Scott E. Fraser

1,2

Biological Imaging Center, Beckman Institute, California Institute of Technology, Pasadena, California, United States of America,

Option in Bioengineering, California

Institute of Technology, Pasadena, California, United States of America,

Electrical and Computer Engineering, University of California Santa Barbara, Santa Barbara,

California, United States of America

Abstract

Heart valve anomalies are some of the most common congenital heart defects, yet neither the genetic nor the epigenetic

forces guiding heart valve development are well understood. When functioning normally, mature heart valves prevent

intracardiac retrograde blood flow; before valves develop, there is considerable regurgitation, resulting in reversing (or

oscillatory) flows between the atrium and ventricle. As reversing flows are particularly strong stimuli to endothelial cells in

culture, an attractive hypothesis is that heart valves form as a developmental response to retrograde blood flows through

the maturing heart. Here, we exploit the relationship between oscillatory flow and heart rate to manipulate the amount of

retrograde flow in the atrioventricular (AV) canal before and during valvulogenesis, and find that this leads to arrested valve

growth. Using this manipulation, we determined that

klf2a

is normally expressed in the valve precursors in response to

reversing flows, and is dramatically reduced by treatments that decrease such flows. Experimentally knocking down the

expression of this shear-responsive gene with morpholine antisense oligonucleotides (MOs) results in dysfunctional valves.

Thus,

klf2a

expression appears to be necessary for normal valve formation. This, together with its dependence on

intracardiac hemodynamic forces, makes

klf2a

expression an early and reliable indicator of proper valve development.

Together, these results demonstrate a critical role for reversing flows during valvulogenesis and show how relatively subtle

perturbations of normal hemodynamic patterns can lead to both major alterations in gene expression and severe valve

dysgenesis.

Citation:

Vermot J, Forouhar AS, Liebling M, Wu D, Plummer D, et al. (2009) Reversing Blood Flows Act through

klf2a

to Ensure Normal Valvulogenesis in the

Developing Heart. PLoS Biol 7(11): e1000246. doi:10.1371/journal.pbio.1000246

Academic Editor:

Hiroshi Hamada, Osaka University, Japan

Received

April 1, 2009;

Accepted

October 9, 2009;

Published

November 17, 2009

ß

2009 Vermot et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits

unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding:

Support received through a National Institutes of Health (NIH) grant to SEF (P01HD037105). JV was supported by a fellowship from the Human Frontier

Science Program (HFSP), AF by a fellowship from the NIH, ML by a fellowship from the Swiss National Science Foundation (PA002-111433), DW by the NIH Med

ical

Scientist Training Program at UCLA/Caltech, and DP by a fellowship from the Summer Undergraduate Research Fellowship (SURF). The funders had no role

study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests:

The authors have declared that no competing interests exist.

Abbreviations:

AV, atrioventricular; hpf, hours postfertilization; ISH, in situ hybridization; MO, morpholine oligonucleotide; RFF, retrograde flow fraction;

WSS,

wall shear stress.

* E-mail: sefraser@caltech.edu

¤ Current address: IGBMC (Institut de Ge

tique et de Biologie Mole

culaire et Cellulaire), Department of Cell Biology and Development, Inserm-U964, CNRS-

UMR7104, Universite

de Strasbourg, Illkirch, F-67400 France

Introduction

Formation of valves is a critical step in the development of a

functionally mature heart, yet little is known about the

mechanisms that initiate valve formation in vivo. In vertebrates,

valves form from the endothelial cell layer located at the border

between the atrium and the ventricle [1–3]. In fish, this region is

called atrioventricular (AV) canal [1,4,5] and defines the

endothelial ring [6]. The expression of genes specific to this

territory depends on the activity of molecules secreted in the

subjacent AV myocardium and on an elaborate combination of

signaling pathways between the two cell layers, including Wnt/

b

catenin, bone morphogenetic protein (BMP), and Notch signaling

[4,7–10]. Not surprisingly, aberrant patterning of the myocardial

layer of the early heart can lead to valve defects, as the

specification of the AV canal is impaired [6]. The analysis of

zebrafish mutants has led to the identification of several cellular

changes happening in the endothelial cell precursors during the

process of valvulogenesis [1], and it has been shown that some of

these changes are associated with physical stimuli provided by

blood flow [11]. Interestingly, valve morphogenesis is clearly

dependant on the geometry of the beating heart chambers, further

suggesting that the physical environment near the developing

valves plays a critical role for their development [11]. Along with

previous observations demonstrating the importance of intracar-

diac fluid flow for cardiogenesis [4,12–14], this offers the exciting

possibility that the genetic programs that govern valve formation

in vivo depend on intracardiac hemodynamics. Harvesting this

possibility has been challenging, as some attempts to uncouple

contractility and flow have been taken to suggest that they play

opposing roles in modulating cell shape within the developing

heart [12]; other studies have suggested that flow forces regulate

looping, cell size and shape in the heart chambers, and the

formation of trabeculae [5,12–14]. A recent publication highlights

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November 2009 | Volume 7 | Issue 11 | e1000246

the uncertainty concerning the role of flow during heart valve

development, since it reports a cardiac contractility mutant that

can form normal valves [15]; thus, something more than the mere

presence (or absence) of flow or contractility must be involved in

directing valve development.

The predominant model to explain endothelial cell response to

flow envisions that the shear stress, which directly depends on the

viscosity and the velocity of the blood, is the main physical

stimulus. More recently,

disturbed flow

has been used as a general

term to group abnormal flow patterns (including low flow,

oscillatory flow, flow separation, gradients, turbulence, and

reversing flows), potentially leading to atherogenic stimulus for

endothelial cells [16–18]. This hypothesis is indirectly supported

by observation that, in vitro, endothelial cells can be responsive to

disturbed flows [19,20], leading to an atherogenic-like cell

response [21]. Thus, an attractive hypothesis is that heart valves

form as a developmental response to disturbed blood flows. A key

prediction from this model is that altering flow patterns within the

heartbeat cycle should directly affect valvulogenesis. In vitro

approaches have so far been unsuccessful in addressing this

question, possibly due to the absence of specific valve markers

usable in vitro and to the difficulty to mimic in vitro the

complexity of flow patterns observed in vivo.

To circumvent these limitations, we characterized embryonic

zebrafish heart flow in vivo to identify a critical feature of the flow

pattern associated with valve specification and tested its impor-

tance using a set of experimental manipulations including both

genetic and pharmacological approaches. Taking advantage of

high-speed imaging, we quantified the flow patterns generated in

the beating heart and compared them with anatomical landmarks

of the heart specified by expression patterns of known genes. Using

antisense morpholine oligonucleotides (MOs) and drugs to alter

these flow patterns in zebrafish, we show that reversing flow is

essential to trigger flow-responsive genes in the AV canal and for

initiating valvulogenesis. Our findings validate a key prediction of

a specific and local role for reversing flows during cardiogenesis.

Results

Reversing Flows Are Higher in the AV Canal

In order to better understand the roles played by blood flow in

heart and valve development, we have developed imaging

techniques to capture cardiac motion and analyze blood flow.

Imaging with these tools reveals dramatic changes in intracardiac

blood flow patterns during cardiac development: as the heart

enlarges, blood flow becomes increasingly bidirectional until the

stage at which functional valve leaflets emerge at the boundary

between the atrium and the ventricle (Figure S1, Video S1, and

unpublished data, see also [11,22]). Although reversing blood

flows are at times visible in the atrium and ventricle, reversing

flows are most pronounced at the AV canal in the second and

third days of development (Figure 1A–1C, Video S2). We

quantified the degree of reversing flow by measuring the fraction

of the cardiac cycle during which there is retrograde flow, and

term this the retrograde flow fraction (RFF). RFF is largest at the

AV canal at embryonic stages that precede valve formation. Our

ability to observe intracardiac blood flow simultaneously with

heart pumping dynamics and morphogenetic changes provides a

direct means to assess the proposal that the presence of particular

patterns of intracardiac blood flow play a critical role in heart

valve development.

To better understand how reversing flow relates to valve

development at the molecular level, we analyzed the expression

pattern by in situ hybridization (ISH) of three known shear-related

genes at the AV canal:

notch1b

, a zebrafish Notch homolog

[23–25],

klf2a

, a transcription factor from the Kruppel-like factor

(Klf) family [26], and

bmp4

, a secreted growth factor of the bone

morphogenetic protein (Bmp) family [27]. Notch is essential for

valve formation [28], and the Notch pathway is activated by shear

stress in HUVEC cells [29–31].

klf2a

and

bmp4

are expressed in the

zebrafish conduction system [4,21,25]. Our analysis concentrated

on the AV canal during its specification (between 22 and 48 hours

postfertilization [hpf]) [4,25] as well as slightly before valve leaflet

formation (58 hpf) [11] (Figure 1G–1O). Both

notch1b

and

klf2a

were expressed in the endothelium (Figure 1G–1L; Figure S2,

Video S3). In contrast,

bmp4

was expressed in the myocardium of

the heart tube, starting around 20–22 hpf and later became

restricted to the AV canal between 36 and 58 hpf (Figure 1M–

1O). Strikingly, expression of

notch1b

klf2a

, and

bmp4

became

restricted to the region of high reversing flow we identified in the

AV canal as the heart matured.

Reversing Flows Control Valve Morphogenesis in

Addition to Shear Stress

In the developing zebrafish heart, where the Reynolds numbers

are much less than one [14], flow patterns are dominated by the

relationship between viscous forces and pressure gradients [32].

Thus, two methods of altering the reversing intracardiac blood

flows in vivo are to: (1) manipulate blood viscosity, or (2) modulate

pacemaker activity in order to change intracardiac pressure

gradients [33]. To alter blood viscosity, we lowered the hematocrit

by targeting two genes controlling early hematopoiesis in

zebrafish,

gata1

and

gata2

[34], with MOs. Embryos injected with

gata1

MO are completely devoid of circulating blood cells [34],

have a lower blood viscosity (reduced by

,

90%, see Materials and

Methods), and display an increased RFF compared to controls

(Video S4;

,

RFF: 45%

6

12% in

gata1

morphants, compared to

35%

6

7% in controls, Figure 2A and 2B). Embryos injected with

gata2

MO contain fewer circulating blood cells in comparison to

wild-type embryos (72% fewer blood cells, Figure 2L, Video S5),

have a reduced viscosity (

,

70% lower than controls), and display

Author Summary

The growth and development of vertebrates are critically

dependent on efficient cardiac output to drive blood

circulation. An essential step of heart development is the

formation of heart valves, whose leaflets are made through

a complex set of cellular rearrangements of endothelial

cells. Endothelial cells experience high flow forces as blood

circulates. Moreover, heart valves and associated structures

can be malformed when flow forces are abnormal,

suggesting that these flow forces are in fact required for

proper valve formation. Whether it is the force of the

blood flow, its directionality (forward or reverse), or both

that are important is not clear. We studied the interplay

during valve development between key genes known to

be involved in the process and epigenetic influences such

as flow forces. Using zebrafish, whose optical clarity allows

analyzing blood flow patterns at high resolution, we

identified the presence of reversing flows specifically at the

level of valve precursors. By manipulating blood flow

patterns, we show that reversing flows are essential for

valve morphogenesis. Specifically, we show that the

expression of the gene

klf2a

depends on the presence of

reversing flows and is required for valve development. We

predict that by influencing levels of

klf2a

, reversing flows

constitute an important stimulus controlling the appropri-

ate biological responses of endothelial cells during valve

formation.

Reversing Blood Flows during Valvulogenesis

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November 2009 | Volume 7 | Issue 11 | e1000246

Figure 1. Transvalvular oscillatory flow patterns change during heart valve morphogenesis and gene expression in the AV canal.

(A–C) Average transvalvular flow direction as a function of time for wild-type hearts as seen in the AV canal (light magenta box) or atrium (light blue

box) between 36, 48, and 58 hpf in the area highlighted in the heart drawings (ventral view, anterior to the top). Anterograde flow from the atrium to

ventricle is shown in black, retrograde flow from the ventricle to the atrium in red, and no flow between the chambers is shown in white. The

sequence of time segments with retrograde, anterograde, and no-flow fractional periods are depicted in red, black, and white, respectively. The

retrograde flow fraction (RFF) is the fraction of the cardiac cycle that is red. (D–F)

cmlc2

expression reveals changes in heart morphology.

cmlc2

expressed in the heart tube in the anterior region at 36 hpf (D), and is expressed strongly in the ventricle and weakly in the atrium at 48 and 54 hpf (E

and F). (G–O) Expression of

notch1b

klf2a

, and

bmp4

progressively becomes localized to the AV canal during valve specification. mRNA distribution of

notch1b

(G–I),

klf2a

(J–L),

bmp4

(M–O) at 36 hpf (D, G, J, and M), 48 hpf (E, H, K, and N), and 56 hpf (F, I, L, and O).

notch1b

is found in the anterior part

of the heart tube at 36 hpf (G), and becomes stronger in the AV canal and in the ventricle at 48 hpf (H). At 54 hpf,

notch1b

expression becomes

restricted in the AV canal and the outflow tract (I).

klf2a

expression is found throughout the heart tube at 36 hpf (J) and becomes stronger in the AV

canal and in the atrium at 48 hpf (K). At 56 hpf,

klf2a

is exclusively expressed in the AV canal and the outflow tract, displaying an expression pattern

Reversing Blood Flows during Valvulogenesis

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November 2009 | Volume 7 | Issue 11 | e1000246

a strongly reduced RFF (17%

6

4% of the heart cycle, Figure 2C;

Video S4) in a majority of embryos (

= 13, 54%), highlighting the

nonlinear relationship between heartbeat frequency and viscosity.

When analyzed at 96 hpf, a majority of

gata1

embryos had normal

valves (77% of embryos had normal valves,

= 13; Figure 2F and

2L); in contrast, the majority of

gata2

morphants displayed severe

valve defects (64% of the embryos displayed abnormal valves,

= 14; Figure 2G and 2L). To make sure that the abnormal valve

development was related to the lower RFF and not to other

functions of

gata2

, we analyzed the effect of simultaneously

inactivating

gata1

and

gata2

. This treatment further reduced blood

viscosity, restored the RFF to 50% (Figure 2D, Video S4), and

rescued valve formation (87% of embryos had normal valves,

= 8; Figure 2H and 2L). We also confirmed that lack of blood

cells does not affect heart chamber patterning and vascular

development (Figure S3). Because shear force depends directly

upon viscosity, the reduced blood viscosity resulting from the

gata1

gata1/2

MOs reduces the magnitude of the shear forces

throughout the cardiovascular system with respect to normal or

gata2

morphants (Figure S4). Thus, the normal valve development

of the

gata1

and

gata1/2

morphants, and the abnormal valve

development in the

gata2

morphants show that reversing flows,

rather than magnitude of shear stress alone, are critical for valve

leaflet formation (Figure 2M).

To better define the effects of RFF alteration in the

gata2

morphants, we used quantitative reverse transcriptase PCR (qRT-

PCR) to study a set of flow-responsive genes. We compared

expression of

bmp4

klf2a

notch1b

neuregulin1

(

nrg1

), and

endothelin1

(

edn1

) in wild-type,

gata1

, and

gata1/2

morphant embryos

(Figure 2I–2K). Their expression levels in the

gata1

morphants

remained close to the control baseline (Figure 2I), as did their

levels in

gata1/2

morphants, except for a slight decrease in

bmp4

expression (about 2-fold, Figure 2K). In

gata2

morphants, two

genes were significantly down-regulated:

klf2a

(about 5-fold

reduction) and

notch1b

(about 2.5-fold reduction);

edn1

and

nrg1

display a mild reduction (about 1.5-fold reduction, Figure 2J).

Since wall shear stress (WSS) is a major stimulus for endothelial

cell response in vitro, we explored whether it is also associated with

the developmental changes we observe in vivo. Blood cell velocity

measurements were used to estimate the WSS generated in the AV

canal in control and altered flow conditions (summarized in Figure

S4). In all

gata

morphants, the WSS is decreased due to the

reduced blood viscosity. Interestingly, although

gata2

and

gata1

morphants display comparable amounts of WSS, they have

opposite valve phenotypes. Thus, WSS magnitude cannot be the

only determining factor for valvulogenesis. To explore this

relationship further, we analyzed four heart contractility mutants

(

cx36.7

myh6

ttna

, and

sih

; Figure S5A–S5E), and find that they

have widely varying RFFs (Video S6). Furthermore, the mutants

exhibiting a decreased RFF demonstrate both reduced

klf2a

expression (Figure S5F–S5H) and increased valve dysgenesis

(Figure S5C, S5D, S5I, and S5J). Thus, results from animals with

reduced blood viscosity and with reduced heart contractility

suggest that, for normal development of valves, the reversing

nature of the WSS is more important than its magnitude.

klf2a

Is Modulated by Low RFF in the AV Canal

We further explored the relationship between RFF and valve

development by using lidocaine, a sodium channel blocker, to

decrease heart rate [35], as well as increased temperature to

increase heart rate [33]. Lidocaine increases the time from

ventricular contraction to the atrial contraction of the next

heartbeat, thus lengthening the period between the onset of the E

wave (early diastolic filling due to ventricular suction) and A wave

(ventricular filling due to atrial contraction). Slowing the heart rate

by only 30% reduced the RFF by as much as 60% (Figure 3A).

Similarly, warming the animal by 2–4

u

C sped up the heart and

reduced the RFF (Video S7). Because lidocaine is easily applied

and rinsed out, we could decrease the RFF for defined periods to

find the stages at which oscillatory flow is critical for valve

development. Starting at 24, 36, or 48 h of development, we

incubated fish in lidocaine for either 12 or 24 h, after which the

fish were returned to normal medium (Figure 3B). When scored at

96 hpf, valve leaflets were evident in all control embryos (no

lidocaine exposure; Figure 3C; Video S8). In contrast, fish in

which lidocaine reduced the RFF displayed a range of valve

defects (Figure 3B, blue bars and red bars). Similar defects were

observed after reducing the RFF with elevated temperature

(Figure 3B, yellow bars). In the subtlest defect manifestations,

valve leaflets did not form (Figure 3D; Figure S3B; Video S8). In

more extreme cases, the heart retained an immature tubular shape

(17%,

= 36; Figure S6). The defects cannot be from a side effect

of the lidocaine, as slight warming of the animals to restore heart

rate, and thereby RFF, to normal rescued valve leaflet formation

(Figure 3E, 3H, and 3K, Video S8). The highest proportion of

valve defects resulted when 12- or 24-h lidocaine treatments were

initiated at 36 hpf, suggesting the greatest sensitivity to decreased

RFF from 36–48 hpf (Figure 3B). Interestingly, this time window

corresponds to the period when

bmp4

notch1b

, and

klf2a

normally

become restricted to the AV canal.

To explore the timing relationships between the flow-responsive

genes, we analyzed their expression by ISH after a 5- or 15-h

lidocaine treatment, starting at 31 hpf.

klf2a

expression signifi-

cantly decreased in as little as 5 h of treatment (Figure 3, compare

3F and 3G), indicating that

klf2a

may be an immediate target of

the mechanism(s) that sense RFF. In contrast, expression of

notch1b

was normal after the short lidocaine treatment, but was decreased

after 15 h of treatment (Figure S7). Quantitative PCR studies

show that

klf2a

edn1

, and

notch1b

were strongly down-regulated

after 10 h of lidocaine treatment, started at 36 h, (about 5-fold

reduction compared to controls); whereas

nrg1

and

bmp4

expression levels were almost normal (Figure 3L and 3M). Shorter

treatments (6 h) led to a significant decrease in

klf2a

and

nrg1

mRNA levels (about 2.5- and 2-fold reduction, respectively),

suggesting that these two genes may be primary targets of

retrograde flow (Figure 3L). The strong dependence of

klf2a

expression on the presence of oscillatory flow during both the 6- or

10-h treatments, as well as the similarity of its expression kinetics

to those observed in cell culture [36], makes

klf2a

an excellent

candidate as a key component in mediating the effects of

oscillatory flow on valve specification, validating the proposed

involvement of this gene in vertebrate cardiogenesis [37–39].

klf2a

Knock Down Affects Valvulogenesis

We tested whether

klf2a

is required for valve formation by

knocking down its expression using MOs, and obtained AV valve

dysgenesis phenotypes that were remarkably similar to those of

embryos exposed to reduced oscillatory shear stress (scored at

very similar to

notch1b

(L).

bmp4

expression is found in the anterior part of the heart tube at 36 hpf (M) and becomes progressively concentrated at

the level of the AV canal from 48 to 54 hpf (N and O). Anterior to the top, white arrows point to the AV canal, black arrows to the outflow tract. Scale

bar indicates 50

m

doi:10.1371/journal.pbio.1000246.g001

Reversing Blood Flows during Valvulogenesis

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November 2009 | Volume 7 | Issue 11 | e1000246

Figure 2. Decreased retrograde flow via lowered blood viscosity affects valve morphogenesis.

(A–D) Flow pattern at 48 hpf in (A)

control and after (B)

gata1

, (C)

gata2

, and (D)

gata1/2

knock down.

gata2

inactivation leads to a dramatic decrease in the RFF, whereas

gata1

and

gata1/2

knock downs exhibit increased RFF compared to the control. (E–H) Confocal sections of the valve-forming region in (E) control, (F)

gata1

, (G)

gata2

, and (H)

gata1/2

morphants. Only

gata2

morphants at 96 hpf show valve dysgenesis. Scale bar indicates 50

m

m. (I–K) Quantitative RT-PCR

showing the expression level of several flow-responsive genes after

gata1

gata2

,or

gata1/2

knock down. **

0.01, ANOVA. (L) Percentage of

embryos displaying valve malformation at 96 hpf (red bar), hematocrit level (yellow bar), and RFF (blue bar) observed in morphants and controls at

48 hpf. The proportions were significantly different at a 10% level of significance (

a

= 0.1). (M) Outline summarizing the experimental outcome of

manipulating oscillatory flow by decreasing circulating blood cells. The color code for gene expression is the same as in (I).

doi:10.1371/journal.pbio.1000246.g002

Reversing Blood Flows during Valvulogenesis

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November 2009 | Volume 7 | Issue 11 | e1000246

96 hpf; Figure 4A–4D); 52% of

klf2a

MO-treated embryos (

= 36)

revealed valve dysgenesis; none of the sham- or control-injected

embryos (

= 45) showed abnormal valve development (Figure 4A–

4D). This similarity in phenotypes suggests that expression of

klf2a

is a key part of the genetic program that makes valve development

responsive to normal oscillatory flow (Figure S8). In mouse, loss of

Klf2

is associated with heart failure and altered cardiac output

[38]. In our studies, the zebrafish

klf2a

morphants displayed a

heart rate similar to that of the control embryos at 48 hpf (1.7 Hz;

Figure 4E and 4F), and had normal flow patterns within the AV

canal (

= 5, Figure 4E and 4F). We found that atrial and

ventricular fates are properly assigned in the

klf2a

morphants,

because the chamber-specific expression of

nppa

bmp4

, and

cmlc2

appear normal (Figure 4G and 4H; Figure S9A–S9D). Thus, the

effects of our MO experiments are not secondary to an alteration

in heart structure or blood flow. ISH revealed that the first

apparent molecular defects in the

klf2a

morphants are a decrease

notch1b

expression at 36 hpf and a lack of

notch1b

expression at

the AV boundary of the heart at 46 hpf (Figure 4L and Figure S9E

and S9F), consistent with previous work showing that

klf2

lies

upstream of Notch in HUVEC cells [40]. In contrast,

bmp4

expression is normal at 36 hpf and slightly decreased at 46 hpf

(Figure 4I and 4J and Figure S9C and S9D). When measured by

qRT-PCR, the expression levels of

bmp4

edn1

, and

nrg1

were lower

than normal by at least a factor of two (Figure 4M); the strong

decrease in

notch1b

expression seen by ISH corresponds to a 10-

fold reduction compared to controls (Figure 4M). Together with

the fact that

klf2a

expression is a primary target of oscillatory flow,

these data indicate that

klf2a

functions upstream of many known

flow-induced genes in the process of AV valve formation in

response to oscillatory flow.

Cell Shape Is Affected by Decreased Reversing Flows

during Valve Invagination

Zebrafish valves emerge from the endothelium through the

combined actions of cell rearrangements and cell shape changes

[1,11]. To characterize the leaflet phenotype in the different

mutants exhibiting altered RFF, we analyzed cell number and cell

Figure 3. Decreased oscillatory flow decreases

klf2a

expression.

(A) RFF is decreased by alterations in heart rate. The highest RFF is seen at

the control heart rate (

30% between 1.5 and 2 Hz) at 48 hpf. Raising fish at lowered or elevated temperatures slows or speeds heart rate and

significantly decreases RFF. Lidocaine treatment decreases heart rate and RFF (blue data point). The decreased heart rate and RFF is rescued by

elevating the temperature to 34

u

C (red data point). (B) Decreased RFF from treatment with lidocaine or with high-temperature (34

u

C) leads to valve

defects. The maximal effect is observed when treatment is initiated at 36 h. (C–E) Valve formation in normal and lidocaine treated embryos. (C)

Embryos that were raised in control conditions have valve leaflets (white arrows). (D) Embryos in which RFF was decreased by lidocaine treatment

from 31 to 55 hpf have endocardial tissue thickening (asterisk) but no valve leaflets are apparent (50%,

= 36). (E) Heart valve dysgenesis in fish

exposed to 0.15% lidocaine for 24 h is rescued by incubating it at 34

u

C to restore normal RFF. Heart valve leaflets are present and function normally

(white arrows). All embryos are imaged at 96 hpf. A, atrium; V, ventricle. (F–H)

klf2a

expression in 46-hpf-old embryos is altered by lidocaine

treatment. (F)

klf2a

expression is localized at the AV boundary in control embryos. (G)

klf2a

expression decreases after 15-h lidocaine treatment (90%,

= 67). (H) Restoring heart rate and RFF to normal by raising the fish at 34

u

C restores

klf2a

expression (90%,

= 45). Anterior to the top. (I–K)

nppa

expression remains largely unaffected by lidocaine treatment and temperature rescue. (L) Quantitative RT-PCR showing the expression level of

several flow-responsive genes after lidocaine treatment.

klf2a

expression is significantly decreased after 6 and 10 h of treatment and is restored by

incubation at 34

u

C; 100% of expression corresponds to a normal expression level. *

0.05; **

0.01, ANOVA. (M) Outline summarizing the

experimental outcome of decreasing oscillatory flow by decreasing heart rate. The color code for gene expression is the same as in (L).

doi:10.1371/journal.pbio.1000246.g003

Reversing Blood Flows during Valvulogenesis

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Figure 4. Morpholine antisense oligonucleotide treatment decreases expression of the flow-responsive gene

klf2a

results in valve

dysgenesis.

(A–D) Valve leaflets scored at 96 hpf show effects of

klf2a

MO. (A and C) Sham-injected embryos form normal heart valves. (B and D)

klf2a

MO-treated embryos display valve dysgenesis, often with a complete absence of valve leaflets. (C and D) Detailed views of valve morphology.

klf2a

MO-treated embryo has no valve leaflets forming from the

endocardium (arrow) (52%,

= 46). The proportions were significantly different at a level of significance

a

= 0.01. Scale bars indicate 50

m

m. (E and F)

Average flow pattern at 48 hpf in controls (E) and

klf2a

morphants (F) showing that the RFF is unaffected in the mutants but that the heart rate is

slightly decreased. (G–L) Expression of three marker genes at 48 hpf in normal and

klf2a

morphants. (G–J)

nppa

expression is normal in the

klf2a

morphants, showing that chamber specification occurs independently of

klf2a

. (I and J)

bmp4

mRNA distribution at 48 hpf showing that expression is

decreased in the MO-treated embryo in the AV node region at 48 hpf (

= 23, 40%; compare expression at arrow in panels [I and J]). (K and L)

notch1b

expression at 48 hpf decreases in the AV boundary of the

klf2a

morphants (

= 45, 71%). Arrows point to the AV boundary in all panels (G–L). (M)

Summary of quantitative RT-PCR showing the expression level of flow-responsive genes in

klf2a

morphants. Expression of all genes decreases

significantly, confirming the down-regulation of

bmp4

and

notch1b

observed by ISH. **

0.01, ANOVA. (N) Summary diagram of

klf2a

function

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November 2009 | Volume 7 | Issue 11 | e1000246

shape using the

Tg(flk1:gfp)

fish line [41] at 72 hpf, a stage at which

the valve invagination is clearly visible [11]. In this line, the GFP

accumulates in endothelial cells, but the fluorescence level is

different from cell to cell. The inherent brightness variation allows

us to count and assess the shape of every endothelial cell in the

heart. Focusing our analysis on the AV canal, we found that the

endothelial ring forms in every morphant and lidocaine-treated

embryo (Figure 5A–5E). Strikingly,

gata1

and

gata1/2

morphants

display normal cell numbers in the AV canal (Figure 5A and 5B

and unpublished data); however, all treatments that disrupt the

invagination of valve leaflets (

gata2

MO,

klf2a

MO, and lidocaine)

exhibit decreased endothelial cell number in the AV canal

compared to the controls. Three-dimensional volumetric mea-

surement of the endothelial AV cells reveals that wild-type controls

as well as the

gata1

and

gata1/2

morphants possess endothelial cells

that are cuboidal (Figure 5A and 5B, 5F–5G, and 5Q; Video S9);

in contrast, the endothelial cells remain flat and elongated in

gata2

morphants,

klf2a

morphants, and lidocaine-treated embryos

(Figure 5C–5E, 5H–5J, and 5Q; Video S9). These differences

precede the absence of valve invagination in embryos with

decreased RFF and suggest that cell remodeling is important for

leaflet morphogenesis. Taken together, our results show that the

loss of

klf2a

expression, lack of invagination, decreased endothelial

cell number, and abnormal endothelial cell shape characterize the

effects of decreased RFF.

Discussion

The beating heart is a highly dynamic structure. Its contraction

generates multiple types of forces at different scales: Although both

the myocardial and endocardial cells undergo a compression-

stretching sequence during each contraction at the tissue scale,

individual endothelial cells directly experience shear stress and

oscillatory flows generated by moving blood. Although it is well

established that both cell types are responsive to mechanical cues

[17,42–44], it has been difficult to clearly state which is the

mechanical stimulus activating endothelial cells to respond to flow.

To address this question, we have applied fast imaging on live

embryos to carefully describe the flow patterns generated at the

earliest stages of the valve development. Based on analysis of both

live embryos and gene expression in fixed tissues, we find that the

specific accumulation of

klf2a

transcripts within the valve precursor

correlates with the presence of reversing flow in the AV canal and

that altering flow patterns in the AV canal affects gene expression

patterns in the endothelial cell layer. The differential response of

endothelial cells to the presence or absence of reversing flows gives

rise to an area prone for valvulogenesis. This response gets

reinforced as reversing flows gradually concentrate in the AV

canal. This phenomenon can be explained by the progressive

reduction of the AV diameter as the atrium and ventricle loop, and

the endothelial ring develops. Our results show that valvulogenesis

results through the combination of a complex set of morphoge-

netic changes and are in full agreement with the different studies

on the subject [5,6,11–14,45].

The Morphogenesis of Valve Leaflets Depends on Blood

Flows in Zebrafish

In zebrafish, the first step of valvulogenesis involves the

clustering of endothelial cells at the AV boundary. Cells coalesce

to form an endothelial ring lining the AV canal between 24 and

48 hpf [6]. As seen previously [6], we found that flow is not

necessary for endocardial ring formation. However, blood flow is

critical for cell shape change and leaflet invagination. Knocking

down

klf2a

does not affects endothelial ring formation, confirming

that

klf2a

function starts when its expression becomes detectable in

the AV canal. Our data together with those of others [11] show

that the endothelial ring is assembled in a region coinciding with

klf2a

expression, and that reversing flows progressively increase in

amplitude specifically at the AV canal after the endothelial ring

forms. This timing suggests that the effects of blood flow act after

an initial patterning that is guided by a genetic program,

reminiscent of the process acting in vascular development [46].

Thus, it appears that the earliest steps of heart development can be

considered as genetically hardwired, but that secondary events,

such as valvulogenesis, are contingent on the presence of reversing

flows.

Zebrafish valve development appears to be somewhat divergent

from the process described in amniote vertebrates. In chicken and

mice, valve leaflets arise from a mesenchymal cushion; in

zebrafish, valves emerge directly through an invagination of the

AV endothelium [11]. The origins of this morphogenetic process

are unclear, but it allows the maturation of a functional valve in

less than 96 h of embryonic development [11,22]. Our results

show that this morphogenetic mechanism is dependent on

reversing flow forces. Interestingly, the absence of invagination

correlates with a lack of cell shape change that would normally

occur during this process. Many observations using endothelial cell

culture have shown that the presence of flow activates signaling

pathways implicated in cytoskeletal remodeling [17,31]. It is thus

tempting to speculate that reversing flows initiate the invagination

process by stimulating the necessary movements and cytoskeletal

rearrangements of endothelial cells in the AV canal to build a

functional valve.

Necessity and Modulation of

klf2a

Expression for Normal

Valve Formation Highlights the Genetic Link between

Biomechanical Stimulus and Cell Response to Reversing

flows

The formation of heart valves allows unidirectional flow to be

sustained as the peripheral vasculature develops and the increase

in systemic resistance reduces the net flow of the valveless heart

that results in the appearance of retrograde flow. The RFF is

greater in the AV canal than in the rest of the heart or the

cardiovascular system. The AV canal, a constriction, is exposed to

high hemodynamic forces due to the higher velocities generated in

areas with reduced cross section. Our studies clearly show that,

although the drop in WSS magnitude affects gene expression levels

in the heart, they are not sufficient to explain the abnormality in

valve formation. Another aspect of the WSS, namely its oscillating

directionality due to reversing flows, has to be included to

understand the apparition of valve abnormalities in the

gata2

morphants and not in the

gata1

morphants where the shear forces

are the lowest. Our quantitative imaging analysis strongly suggests

that reversing flows are the proper stimulus controlling valve

formation. Reversing flows have been observed in the developing

cardiovascular system of many vertebrates ([47] and S. E. Fraser,

unpublished data) and could be involved in other important steps

during heart valve formation.

klf2a

acts as a transcriptional relay between the reversing flow generated by the circulating blood cells at the AV canal

and several genes activated in the AV endothelial cells (such as

notch1b

neuregulin1

, and

endothelin1

klf2a

also affects the expression of

bmp4

revealing a possible interaction between myocardium and endothelium essential for valve morphogenesis.

doi:10.1371/journal.pbio.1000246.g004

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Figure 5. Comparison of the valve phenotype between the different treatments affecting valvulogenesis in transgenic

Tg(flk1:EGFP)

zebrafish at 72 hpf.

GFP is expressed in the endothelial cell layer and highlights the developing valves. (A, F, and F’) control embryo, (B, G, and G’)

gata1

morphant, (C, H, and H’) lidocaine treated, (D, I, and I’)

gata2

morphant, and (E, J, and J’)

klf2a

morphant. Each treatment lead to an incomplete

ingression of the endothelial cells in order to make a functional leaflet except in

gata1

morphants. (F’–J’) Schematic representation of the panels (F–J)

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November 2009 | Volume 7 | Issue 11 | e1000246

of cardiovascular development. Among the many genes responsive

to flow,

klf2a

seems to be specifically responsive to disturbed flows

as observed both in vivo (this study) and in vitro [36]. Although, a

direct involvement of

klf2

(the homolog of

klf2a

in mouse) during

valve development remains to be uncovered in higher vertebrates,

this study should stimulate investigation of subtler valve alterations

in these mutants [38]. Genetic evidence also suggests that

klf2

has

atheroprotective roles in adult mice [48] and humans [21], further

suggesting that

klf2

, reversing flows, and cardiac physiology and

development are tightly interconnected and that

klf2

could also be

implicated in the flow response during these processes.

klf2a

stands

out as a possible early indicator of defective valve development.

Nevertheless, it is clear that a number of other genes are mediating

the response of endothelial cells to flow and that more

investigations will be required to isolate them all as well as

determine their interconnections.

Reversing Flows Constitute a Unique Physical Stimulus

for Valve Development

Given that heart-pumping activity and blood content constantly

change as the heart develops, a patterning mechanism based on

flow sensing provides a very practical way to coordinate the timing

of valve formation with the pumping efficiency of the heart. In the

context of valvulogenesis, reversing flows constitute an efficient

signal by providing specific stimuli that dynamically locate the

valve forming area. This hypothesis is fully consistent with

emerging models arising from studies addressing the role of

biomechanical stimuli during embryogenesis, which suggest that

extrinsic forces and intrinsic hardwired programs are intercon-

nected into feedback loops [49,50]. The advantage of such a

mechanogenetic interplay is that it offers the opportunity for cells

to locally adjust to the rapid environmental changes occurring in

dynamic environments in conjunction with organizing centers

[51]. In such systems, cells can directly react to the dynamics of the

organ and can properly adapt at the single-cell level to organize as

a coherently growing tissue. In conclusion, we have demonstrated

that heart rate and blood viscosity can modulate the duration of

oscillatory flow in vivo and have presented a set of useful methods

to control hemodynamic forces during cardiogenesis. Together,

these simple approaches offer powerful tools for predicting and

potentially treating dysgenesis of cardiac valves and broaden the

array of mechanisms to consider for explaining the origins of

congenital cardiac malformation.

Materials and Methods

Confocal Imaging

The Zeiss LSM 510 was used to image

Tg(flk1:gfp)

and

BODIPY-ceramide (Molecular Probes) stained embryos to

visualize valve structure. Embryos were anesthetized prior to

imaging in 0.0175% tricaine and placed in agarose wells. All

images were taken with a 40

6

/1.1 LD C-Apochromat water

immersion lens. For high-speed imaging, the Zeiss LSM 5 LIVE

was used to image BODIPY-ceramide–stained embryos and to

visualize valve formation and flow patterns. Images of 256

6

256

pixels were acquired at 151 frames per second.

High-Speed Video Microscopy

Brightfield images were taken with a Basler A602f CMOS

camera mounted on a home-built microscope equipped with an

Olympus 0.5 NA 10

6

air objective coupled with a 300-mm focal

length tube lens. Images were acquired at 216 frames per second.

Transvalvular Flow Characterization

Transvalvular blood flow was characterized as positive,

negative, or absent (no flow) by analyzing blood cell motions

across the developing valve leaflets. For embryos lacking blood

cells, the plasma was labeled by injecting microbeads (Bangs

Laboratories) into the yolk sac. The region of interest was defined

relative to the atrium and ventricle and moved with the valve

plane during the cardiac cycle. Blood flow direction was marked in

every frame taken during the cardiac cycle, and the retrograde

flow fraction (RFF) was determined by dividing the total number

of frames exhibiting retrograde flow by the total number of frames

per cycle. For each treatment, five to 15 embryos were analyzed.

The boxes represented in Figures 1, 2, and 4 represent the average

flow observed during a minimum of ten heartbeats.

Shear Stress Estimates

Instantaneous blood cell velocity as a function of heart cycle

time in the developing heart was assessed at 48 hpf by tracking

blood cells manually in the AV canal over an average of four

frames. Two heartbeats were analyzed in each condition. Shear

stress was calculated as in [14]. The velocity of blood in the heart

was modeled as

ðÞ

~

where

is the centerline velocity,

is the half-width of the region

of interest (that is, the radius), and

is the distance from the wall.

The shear stress is

~

L

ðÞ

L

~

where

is the dynamic viscosity of the fluid with units g

We measured the AV canal diameter every ten frames to set

(on

average

m

m).

The force exerted on a cell of surface area

~

~

:

We assumed that the size of a cell in the AV canal was constant

using 10

m

as its exposed surface area. The energy expenditure

during one cardiac cycle (

, in dyne

cm) on a single cell is

therefore given by

~

|

|

RFF

|

{

underlining the endothelial cells within valve-forming region (yellow) and the heart lumen (white). A, atrium; V, ventricle. (K–O) Three-dimensio

nal

reconstruction of 10

m

m depth of the AV area in control (K), gata1 morphants (L), lidocaine-treated embryo (M), and

gata2

(N) and

klf2a

(O)

morphants. The white arrows point to the cell that has been reconstructed in three dimensions and which is presented in (P–T). (P–T) Side view (left)

and top view (right) of a reconstructed cell of the AV canal. (U) Schematic drawing showing the approach used to define cuboidal versus non-

cuboidal cell shape. (V) Graph summarizing the number of cells counted in the AV canal (corresponding to the yellow cells in [F’–J’]) (blue bars), and

the ratio between cuboidal versus non cuboidal cell shape (red bars). Yellow arrows in (A–E) point to the endocardial ring.

doi:10.1371/journal.pbio.1000246.g005

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where RFF is the retrograde flow fraction and

is the heart

rate (s

Hematocrit Count

Blood cells were imaged within the eye capillary. We counted

the number of cells crossing a virtual line during the same time

window in controls and MO-treated embryos (Video S5). Blood

viscosity in

gata

morphants was estimated using the plot of relative

viscosity versus particle volume fraction [52] after measurement of

the particle volume fraction assuming fish blood composition is

similar to that of humans.

Lidocaine Treatment

Heart rates of experimental embryos were decreased by dosage-

dependent exposure to lidocaine added to the bathing solution.

Lidocaine was drawn from the stock solution (1% stock, Abbott

Laboratories) and diluted into wells containing artificial pond

water and approximately five embryos. Embryos were exposed to

lidocaine for 24 h beginning at 31 hpf, the developmental stage

marked by the transition from unidirectional to bidirectional flow.

Assays of valve morphology and function were carried out at

96 hpf, a stage in which all wild-type fish hearts have at least one

well-developed valve leaflet. Surviving embryos (

80%) were

washed three times, placed in artificial pond water, and incubated

at 28.5

u

C until being imaged (4 dpf). Normalized heart rates were

calculated by dividing the heart rates of individuals (

= 30)

exposed to lidocaine by the heart rates of individuals under control

conditions. Heart rates were measured after 1 h of continuous

exposure to lidocaine (Figure 2A).

Temperature

Zebrafish heart rates are regulated by ambient temperature.

Unless otherwise noted, embryos were incubated at 28.5

u

C (VWR

Scientific incubator, model 2030). To increase heart rates, a higher

temperature (32 or 34

u

C) incubator (Thermolyne, model 37900)

was used. Edema and abnormal cardiogenesis were observed when

embryos were raised at 16

u

C, 20

u

C, and 35

u

Morpholine Oligonucleotides

Two MOs targeted against the putative translational site of

klf2a

were obtained from Gene Tools LLC (5

-gtaaaatcgttccactcaaagc-

cat-3

-MO1; 5

-agctgagatgcatggacctgtccag-3

-MO2). MOs were

dissolved in 5 mM Hepes (pH 7.6) and were injected into one-cell

stage embryos (total amount of 7 or 15 ng per embryo). We found

that the two MOs induced the same range of malformations (valve

malformation: 40%,

= 15 for MO1; 52%,

= 36 for MO2;

edema: 33%,

= 84 for MO1; 36%,

= 86 for MO2). The

specificity of each MO was assessed using a standard eGFP fusion

approach where the eGFP sequence (pEGFP-N1, Clontech) was

fused by amplifying eGFP via PCR using primers containing the

target sequence of each MO and a sp6 sequence in order to

translate the PCR product (mMESSAGE mMACHINE sp6,

Ambion) (Figure S10). Control embryos were injected with a

similar amount of a standard mismatch MO provided by Gene

Tools LLC (5

-agGtgaCatgcatCgacctCtcgag-3

). The specificity of

this MO was addressed using the eGFP fusion approach (Figure

S11), and its effect on valve development was analyzed using

Tg(flk1:EGFP)

embryos (Figure S11). Specificity of the MOs was

further assessed by analyzing the ability of

klf2a

mRNA

overexpression to rescue the MO-induced edema. A total of

100 pg of mRNA was coinjected with 15 ng of each MO, and

edema was scored at 32 hpf (Figure S10). MOs to

gata1

gata2

and

gata1/2

were used as in [34],

cx36.7

as in [15], and

myh6

as in [13].

In Situ Hybridization

ISHs were performed as described in [53] using the following

probes:

cmlc2

bmp4

(both provided by L. Trinh, California

Institute of Technology),

notch1b

(provided by M. Lardelli,

University of Adelaide),

nppa

(provided by T. Zhong, Vanderbilt

Medical School), and

klf2a

probe (obtained by PCR amplification

of the plasmid IRBOp991B0734D provided by RPDZ, Berlin).

Valve Development Assay

A random sample of experimentally manipulated embryos was

imaged at 96 hpf and scored based on the presence of valve

leaflets. A focal plane with the atrium, ventricle, and AV canal in

view was chosen to illustrate the phenotype. In cases where leaflets

were difficult to identify (

2%), the presence or absence of

transvalvular retrograde flow was used to determine abnormal or

normal valvulogenesis, respectively.

Cell Shape Assay

A random sample of experimentally manipulated

Tg(flk1:EGFP)

embryos was imaged at 72 hpf, and a section plan of 10

m

m was

made using the substack maker plugin with Image J. Cell shapes

were reconstructed in three dimensions using the contour surface

key in Imaris (Bitplane). A minimum of two embryos and ten cells

in each condition were reconstructed. We then calculated the ratio

of the length of the two longest sides and used a

-test for two

proportions to perform the statistical analysis.

Real-Time RT-PCR

At 56 hpf, embryonic hearts were dissected in egg water after

MO injection or lidocaine treatment. Two to three batches of ten

hearts for each condition were pooled, and RNA was extracted

using Trizol. RT was performed using the same amount of

extracted mRNA and further tested by RT-PCR using the 96-well

plate ABI 7000 QPCR machine in a SYBR Green (Bio-Rad)

assay. The fold changes were calculated by the DCt method using

a reference gene (zebrafish TBP) and plotted as a percentage of

expression normalized to control. ANOVA tests were performed

using Instat (Graphpad Software, Inc).

Supporting Information

Figure S1

Oscillatory flow is observed in the AV canal

before valves become functional.

(A–D) Confocal scans of

hearts (ventral view, anterior to the top) at four developmental

stages showing the morphology of the developing heart between

36 and 120 hpf. The endocardial tissue in the AV canal at 48 hpf

in shown by the arrow in (C). Valve leaflets appear at 84 hpf and

are mature by 120 hpf. The black box underlines the location of

blood flow analysis for each stage (A–D). Scale bar indicates

m

m. (E–H) Transvalvular flow direction over time shows that

mature valve leaflets are necessary to prevent retrograde flow in

the heart. Anterograde flow from the atrium to ventricle is shown

in black, retrograde flow from the ventricle to the atrium in red,

and no flow between the chambers is shown in white.

Found at: doi:10.1371/journal.pbio.1000246.s001 (2.39 MB TIF)

Figure S2

klf2a

expression is localized to the endothe-

lial cells of the AV canal.

(A) Brightfield image of

klf2a

mRNA

distribution at 48 hpf using NBT-BCIP revelation. (B) Maximal

intensity projection of 15 sections obtained by confocal microscopy

(633-nm excitation wavelength) reveals the specific expression

domain of

klf2a

to the innermost cell layer of the heart. (C) Profile

plot of the pixel intensity measured along the bottom white line in

(B) showing increased signal in the AV canal (white arrows). (D

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November 2009 | Volume 7 | Issue 11 | e1000246

and E) Drawings locating the endothelial (e) and myocardial (m)

layer on the picture. (F, I, and J) Maximal intensity projection of

ten sections obtained by confocal microscopy (633-nm excitation

wavelength) reveals that the specific expression domain of

klf2a

increases and becomes brighter to the innermost cell layer of the

heart at during the valve elongation stage (60 hpf). (G) By

comparison, expression of

cmlc2

labels the myocardium and not

the endothelium. (H) Same imaging procedure using an embryo

not labeled with NBT-BCIP showing no staining.

Found at: doi:10.1371/journal.pbio.1000246.s002 (6.54 MB TIF)

Figure S3

Decreased blood cells number do not affects

heart chamber patterning as well as head and trunk

vasculogenesis.

(A, B, F, G, K, and L)

nppa

and

bmp4

expression

is unaffected in

gata1

(F and G) and

gata2

(K and L) morphants

compared to controls (A and B) showing that heart chambers and

AV canal patterning is normal when blood cell numbers decrease.

(C–E, H–J, and M–O) GFP expression in

(

flk1:EGFP)

delimitates the cardiovascular system as it is limited to every

endothelial cells in the embryo (C, H, and M). Details of the head

(D, I, and N) and trunk (E, J, and O) vasculature in controls (C–E),

gata1

(H–J), and

gata2

(M–O) show that no obvious malformation

of the cardiovascular system is visible when blood cell number

decreases. Arrows in (D, I, and N) point to the fourth branchial

arch; arrows in (E, J, and O) point to secondary sprouts of the

trunk cardiovascular wiring. Panels (C, H, and M) are each a

composite of two original images.

Found at: doi:10.1371/journal.pbio.1000246.s003 (7.34 MB TIF)

Figure S4

Quantitative analysis of the blood flow

observed in the AV canal at 48 hpf.

(A) Shear stress

estimated in the AV canal at 48 hpf. (B) Recapitulative table of

the different flow features observed in the AV canal after the

different treatments done in this paper. The energy expenditure of

blood (E) required by blood cells going through the AV canal was

calculated during the retrograde and anterograde flow portions of

the heart cycle. It directly depends on the magnitude of the wall

shear stress (WSS) and provides an estimate of the amount of WSS

received by a single cell by taking into account the period of

stimulation and the wall shear force intensity generated at each

heart beat (see Materials and Methods). (C) Normalized flow

velocity observed in

gata

morphants. (D) Outline summarizing the

experimental outcome of decreasing oscillatory flow by decreasing

blood viscosity (

gata1

and

gata2

MO). The color code for gene

expression is the same as in Figure 2.

Found at: doi:10.1371/journal.pbio.1000246.s004 (2.77 MB TIF)

Figure S5

Decreased retrograde flow via changes in

contractility affects valve morphogenesis.

(A–H) Flow

pattern at 48 hpf and associated confocal sections of the valve-

forming region at 96 hpf in (A) control, (B)

cx36.7

(see also Video

S6), (C)

myh6

(Video S6), (D)

ttna

(Video S6) knock downs, and (E)

in the

silent heart

(

sih

) mutants.

myh6

ttna

, and

sih

inactivation leads

to a dramatic decrease in the RFF and valve defects, whereas

cx36.7

knock down has an almost normal RFF and valves

compared to the control. (F–H)

klf2a

expression in (F) control, (G)

cx36.7

, and (H)

myh6

morphants. Absence of

klf2a

expression was

observed in

myh6

morphants (41%,

= 36) (H), but normal

expression levels were observed in

cx36.7

morphants (75%,

= 50)

(G). These two populations were significantly different (

a

= 0.1). (I)

Energy expenditure comparison between control,

gata1

, and

myh6

morphants during the retrograde, anterograde, or both flow

direction phases. The apparition of valve dysgenesis coincides with

a low energy expenditure during phases of retrograde flow rather

than a reduction of the overall energy expenditure during phases

of anterograde and retrograde flow. (J) Proportionally decreased

RFF through treatment with

cx36.7

myh6

,or

ttna

MOs leads to an

increase in valve defects. The maximal effect is observed in no flow

(

sih

) or no RFF (

ttna

) conditions.

Found at: doi:10.1371/journal.pbio.1000246.s005 (2.26 MB TIF)

Figure S6

Strong phenotype triggered by lidocaine

treatment.

(A) Control conditions (B) After treatment with

lidocaine, 17% (

= 36) embryos do not have endothelial tissue

thickening. (C and D)

bmp4

expression in (C) lidocaine-treated and

(D) untreated embryos. In treated embryos, the heart tube is very

immature, a situation very similar to that observed in the no-flow

conditions reported in [14]. Such embryos were not used for flow

analyses or qPCR, nor were they tested for valve morphogenesis at

later stages. White arrow points to the AV canal.

Found at: doi:10.1371/journal.pbio.1000246.s006 (5.54 MB TIF)

Figure S7

notch1b

expression after lidocaine treatment.

notch1b

is expressed at the AV boundary in control embryos (A and

C) and after 5 h of lidocaine treatment (100%,

= 47; (B)) but

disappears after 15 h of lidocaine treatment (61%,

= 36; (D)).

Anterior is to the top.

Found at: doi:10.1371/journal.pbio.1000246.s007 (1.44 MB TIF)

Figure S8

Expression of

notch1b

bmp4

, and

cmlc2

control ([A, C, and E], respectively) and

klf2a

MO-

treated ([B, D, and F], respectively) embryos.

A strong

phenotype after

klf2a

MO treatment is visible in a minority

fraction of embryos treated with

klf2a

MO, which display

immature heart growth (13%,

= 20). In these strongly affected

embryos, the heart tube morphology is similar to that observed in

conditions were blood flow is suppressed (see [7]); they were not

used for flow analysis, qPCR, or for scoring valve morphogenesis

at later stages.

Found at: doi:10.1371/journal.pbio.1000246.s008 (1.60 MB TIF)

Figure S9

Expression of three marker genes at 36 hpf in

the heart of normal and

klf2a

morphants.

(A and B)

cmlc2

expression is essentially normal in the

klf2a

morphants, showing

that chamber specification occurs independently of

klf2a

. (C and

bmp4

mRNA distribution at 36 hpf showing that expression is

normal in the MO-treated embryo in the AV node region at that

stage. (E and F)

notch1b

expression decreases in the AV boundary

of the

klf2a

morphants at 36 hpf (

= 24, 63%; compare expression

at tip of arrows). Arrows point to the AV boundary in all panels.

Found at: doi:10.1371/journal.pbio.1000246.s009 (2.53 MB TIF)

Figure S10

Validation of the MO strategy.

(A–L) MOs

against

klf2a

block the translation of eGFP fusion proteins carrying

their target sequences. (M–X) Control MO (a mismatch of MO2)

do inhibit the translation of eGFP fusion proteins carrying its

target sequence (M–R), whereas MOs directed against

klf2a

cannot

block the translation of the target sequence of the mismatch MO

(S–X), validating the specificity of each MO.

Found at: doi:10.1371/journal.pbio.1000246.s010 (5.16 MB TIF)

Figure S11

(A–E) Injection of

klf2a

mismatch morpho-

lino does not affect valve invagination and cell shape.

(F)

Overexpression of

klf2a

mRNA rescues

klf2a

MO-mediated

phenotype. (A–B’) Comparison of the valve phenotype between

the different treatment affecting valvulogenesis using

(

flk1:egfp

)

at 72 hpf. GFP is expressed in the endothelial cell layer and

highlights the developing valves. (A, B, and B’) control embryo, (C,

D, and D’)

klf2a

mismatch morphant. (B’ and D’) Schematic

representation of the panels (B and D) outlining the endothelial

cells within valve-forming region (yellow) and the heart lumen

(white). Mismatch MO injection leads to a normal ingression of

the endothelial cells and cuboidal cell rearrangement showing that

Reversing Blood Flows during Valvulogenesis

PLoS Biology | www.plosbiology.org

November 2009 | Volume 7 | Issue 11 | e1000246

leaflet invagination occurs properly and that there is no

nonspecific effects due to MO injection. (F) Percentage of rescue

obtained after overexpression of

klf2a

mRNA concomitantly with

klf2a

MO (

= 115 for MO1,

= 49 for MO2) compared with

klf2a

MO injected embryos (

= 84 for MO1 and

= 88 for MO2). A,

atrium; V, ventricle.

Found at: doi:10.1371/journal.pbio.1000246.s011 (1.70 MB TIF)

Video S1

Transvalvular flow changes dramatically

during cardiac morphogenesis.

Heartbeats in BODIPY-

ceramide-stained embryos from four developmental stages (36, 72,

84 and 120 hpf) are shown. At each stage, the age, period length,

and transvalvular flow direction are shown. Valve leaflets begin to

develop by 84 hpf and are mature by 120 hpf. Transvalvular

retrograde flow exists until mature valve leaflets develop.

Found at: doi:10.1371/journal.pbio.1000246.s012 (5.58 MB

MOV)

Video S2

Three dimensional reconstruction of

klf2a

expression in the AV canal endothelium.

Found at: doi:10.1371/journal.pbio.1000246.s013 (2.37 MB

MOV)

Video S3

Transvalvular flow changes in the AV canal

and atrium at 36 hpf, 48 hpf, and 56 hpf in wild-type

embryos (which also serve as controls [CTL]).

Found at: doi:10.1371/journal.pbio.1000246.s014 (8.86 MB AVI)

Video S4

Transvalvular flow changes in the A–V canal

gata1

morphants,

gata2

morphants and

gata1/2

morphants at 48 hpf.

The RFF is increased in

gata1

and

gata1/2

compared to controls (see Video S3), whereas the RFF in

gata2

morphants is decreased compared to controls,

gata1

, and

gata1/2

morphants.

Found at: doi:10.1371/journal.pbio.1000246.s015 (8.34 MB

MOV)

Video S5

Hematocrit is severely reduced in

gata2

morphants at 48 hpf.

Blood cells traveling in an eye capillary

in control (top panel in the video) and

gata2

morphant (bottom

panel of the video).

Found at: doi:10.1371/journal.pbio.1000246.s016 (0.60 MB

MOV)

Video S6

Transvalvular flow changes in the AV canal in

cx36.7,

ttna

, and

myh6

morphants at 48 hpf.

Found at: doi:10.1371/journal.pbio.1000246.s017 (9.25 MB

MOV)

Video S7

Transvalvular flow changes in the AV canal of

control embryos at 3 Hz, 2.4 Hz, 1.5 Hz, and 1.2 Hz.

Wild-type hearts at 48 hpf display normal RFF when incubated at

normal temperatures but display reduced RFF when incubated at

elevated temperatures (which artificially increases the heart rate)

and when incubated at low temperatures (which artificially

decreases the heart rate).

Found at: doi:10.1371/journal.pbio.1000246.s018 (9.70 MB

MOV)

Video S8

Reduced oscillatory flow leads to heart valve

dysgenesis.

Wild-type hearts at 96 hpf have functional valve

leaflets that prevent retrograde flow across the AV valve. Fish

exposed to reduced oscillatory flow through lidocaine exposure

experience valve dysgenesis. Retrograde flow results from the

absence of valve leaflets. Fish exposed to lidocaine and incubated

at elevated temperatures, restoring the natural heart rate, do not

experience valve dysgenesis.

Found at: doi:10.1371/journal.pbio.1000246.s019 (8.40 MB

MOV)

Video S9

Three-dimensional cell shape in the AV canal

of transgenic

(

flk1:EGFP)

embryos at 72 hpf in a

control embryo, in

gata1

gata2

, and

klf2a

morphants,

and in a lidocaine-treated embryo.

GFP is expressed in the

endothelial cell layer (in white) and the yellow shape highlights the

cell shape of one cell in the developing valves.

Found at: doi:10.1371/journal.pbio.1000246.s020 (3.56 MB

MOV)

Acknowledgments

We are grateful to L. Trinh for sharing reagents and for providing probes,

M. Lardelli, T. Zhong, and H. Clevers for ISH probes, the members of the

Beckman Biological Imaging Center for discussions, the Bronner-Fraser

laboratory for sharing tools and reagents, and Shigehisa Hirose for

providing an aliquot of

cx36.7

morpholino.

Author Contributions

The author(s) have made the following declarations about their

contributions: Conceived and designed the experiments: JV ASF ML

MG SEF. Performed the experiments: JV ASF DW DP. Analyzed the data:

JV ASF ML DW MG SEF. Contributed reagents/materials/analysis tools:

DW. Wrote the paper: JV ASF ML MG SEF. Movie crafting: JV ASF ML.

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