tracheal occlusion increases the rate of epithelial ...vfleury/articles/trachealocclusion.pdf ·...

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M E C H A N I S M S O F D E V E L O P M E N T x x x ( 2 0 0 8 ) x x x – x x x

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MOD 2898 No. of Pages 11, Model 7

22 November 2007 Disk UsedARTICLE IN PRESS

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Tracheal occlusion increases the rate of epithelial branchingof embryonic mouse lung via the FGF10-FGFR2b-Sprouty2pathway

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Mathieu unbekandta,1, Pierre Marie Del Moralb,1, Frederic Salab, Saverio Belluscib,David Warburtonb, Vincent Fleurya,*

aGroupe Matiere Condensee et Materiaux, Universite de Rennes 1, 35042 Rennes, FrancebDevelopmental Biology Program, The Saban Research Institute of Childrens Hospital Los Angeles and the Centre for Cranofacial Molecular

Biology, Keck School of Medicine and School of Dentistry, University of Southern California, Los Angeles, CA 90027, USA
P A R T I C L E I N F O
Article history:

Received 21 March 2007

Received in revised form

16 September 2007

Accepted 31 October 2007

Keywords:

Lung development

Tracheal occlusion

Mechanotransduction

Branching morphogenesis

FGF10

Congenital diaphragmatic hernia

0925-4773/$ - see front matter � 2007 Publisdoi:10.1016/j.mod.2007.10.013

* Corresponding author. Tel.: +33 2 23 23 55E-mail address: vincent.fleury@univ-renn

1 These authors contributed equally to this

Please cite this article in press as: unbekaDev. (2008), doi:10.1016/j.mod.2007.10.0

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A B S T R A C T

Tracheal occlusion during lung development accelerates growth in response to increased

intraluminal pressure. In order to investigate the role of internal pressure on murine early

lung development, we cauterized the tip of the trachea, to occlude it, and thus to increase

internal pressure. This method allowed us to evaluate the effect of tracheal occlusion on

the first few branch generations and on gene expression. We observed that the elevation

of internal pressure induced more than a doubling in branching, associated with increased

proliferation, while branch elongation speed increased 3-fold. Analysis by RT-PCR showed

that Fgf10, Vegf, Sprouty2 and Shh mRNA expressions were affected by the change of intra-

luminal pressure after 48 h of culture, suggesting mechanotransduction via internal pres-

sure of these key developmental genes. Tracheal occlusion did not increase the number

of branches of Fgfr2�/� mice lungs nor of wild type lungs cultured with Fgfr2b antisense

RNA. Tracheal occlusion of Fgf10LacZ/� hypomorphic lungs led to the formation of fewer

branches than in wild type. We conclude that internal pressure regulates the FGF10-

FGFR2b-Sprouty2 pathway and thus the speed of the branching process. Therefore pressure

levels, fixed both by epithelial secretion and boundary conditions, can control the branch-

ing process via FGF10-FGFR2b-Sprouty2.

� 2007 Published by Elsevier Ireland Ltd.
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1. Introduction

Branching morphogenesis is a developmental process

common to many organs (lung, kidney, pancreas, mammary

and salivary glands), that is highly conserved during evolu-

tion, generating tree-like structures with a high surface to vol-

ume ratio. Human lung is formed by 23 successive branch

generations, leading to at least 5 million branches that give

hed by Elsevier Ireland L

98.es1.fr (V. Fleury).

work.

ndt, M. et al, Tracheal13

rise to a final alveolar gas diffusion surface of 70 square me-

ters in the adult. At early developmental stages (pseudoglan-

dular stage), the lung is principally composed of three

compartments: the epithelium, a unicellular layer forming

the branches that continuously secretes fluid during develop-

ment, the surrounding mesenchyme and the outer mesothe-

lium. Mouse lung development begins at embryonic day 9.5

(E9.5), where a complex network of mesenchymal-epithelial

td.

occlusion increases the rate of epithelial branching ..., Mech.

mailto:[email protected]

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molecular interactions induces this structure (Warburton

et al., 2005; Shannon and Hyatt, 2004). Grafting experiments

of pulmonary mesenchyme to tracheal epithelium, which

normally does not branch, is able to induce supernumerary

budding (Alescio and Cassini, 1962), showing that branching

of the epithelium is regulated by soluble factors expressed

by the mesenchyme. Conversely, grafting of tracheal mesen-

chyme to distal lung epithelium inhibits the branching pro-

cess (Wessells, 1970). Moreover, these epithelial-

mesenchymal interactions are necessary to pattern blood

vessels, lymphatics, cartilage, smooth muscle and other cell

types resulting from mesenchymal differentiation, thus lead-

ing to lung functional efficiency. Advances in molecular biol-

ogy have made possible the discovery of many of the key

genes involved in this mechanism.

Fibroblast Growth Factor 10 (Fgf10), originally detected at a

high level at E14 in the mesenchyme (Yamasaki et al., 1996)

plays a critical role during lung development (Bellusci et al.,

1997a; Park et al., 1998). Fgf10 null embryos exhibit lung agen-

esis where the primordial buds are forming but are unable to

branch (Sekine et al., 1999). Fibroblast Growth Factor receptor

2b (Fgfr2b) knockout mice show a similar phenotype (Peters

et al., 1994), suggesting that FGFR2b is a main receptor for

FGF10. Moreover, FGF10 coated beads have been shown to

have a chemotactic effect on embryonic mouse lung epithe-

lium (Park et al., 1998). FGF10 addition to embryonic mouse

lung epithelium in culture also accelerated the rate of branch-

ing (Bellusci et al., 1997a). FGF10 is dynamically expressed in

distal mesenchyme clusters, in front of future new branches

sites, acting on distal epithelium mainly via FGFR2b in vivo.

However, it has been shown in vitro that FGF10 is also able

to bind FGFR1b (Ornitz et al., 1995). Thus, the proliferative

and chemotactic role of FGF10 on epithelial structures is

linked to branch formation. Interestingly, Fgf10 is a well-con-

served functional ortholog of Branchless while Fgfr2b is func-

tionally orthologous to Breathless in Drosophila tracheal

morphogenesis (Sutherland et al., 1996).

Sonic Hedgehog (Shh) is expressed by the lung epithelium

and has a mitogenic effect on the mesenchyme via its receptor

Patched (Ptc) (Urase et al., 1996; Pepicelli et al., 1998; Bellusci

et al., 1997b; Van Tuyl and Post, 2000). Ptc transduction activates

both the transcriptional repressor Smo (Smoothened) and Hip

(Hedgehog interacting protein) that negatively regulate Shh

expression and activity (Chuang and Mc Mahon, 1999). Shh null

mutation results in a profound pulmonary hypoplasia and a

failure of tracheo-oesophagal septation. Shh�/� lungs exhibit

a more widespread pattern of Fgf10 expression, as opposed to

the spatially restricted pattern in normally developing lungs

(Pepicelli et al., 1998). SHH acts as an inhibitor of Fgf10 at the

transcriptional level and is necessary to spatially restrict

Fgf10 expression pattern so that branches may form as local-

ized extensions of the epithelium. At the distal tip of the epithe-

lium, where branching will occur, SHH is restricted by Hip,

thereby de-repressing Fgf10 expression.

Sprouty2 is an FGF10 inducible negative regulator of FGF

receptor signaling and is expressed by the distal epithelium,

in a complementary pattern to Fgf10 (Hacohen et al., 1998;

Mailleux et al., 2001). The dyad Fgf10/Sprouty2 is thus a major

coupling mechanism involved in the regulation of the branch-

ing process as an activator-inhibitor pair.

Please cite this article in press as: unbekandt, M. et al, TrachealDev. (2008), doi:10.1016/j.mod.2007.10.013

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One of the key genes involved in the pulmonary vasculo-

genesis process is Vascular Endothelial Growth Factor (VEGF).

Vegf is expressed by the distal epithelium and acts on mesen-

chymal cells via its receptor Flk1. VEGF triggers the prolifera-

tion of Flk1 positive cells that comprise the endothelial cell

progenitors (Yamashita et al., 2000; Del Moral et al., 2006).

Lung morphogenesis is therefore ‘‘coordinated’’ by dyads

of morphogenetic and inhibitory genes, including Fgf10/Spro-

uty2, Bmp4/Gremlin, Shh/Hip, and Wnt/Dkk1 that mediate epi-

thelial-mesenchymal interactions to finely regulate the

branching pattern.

However, lung growth is sensitive to variations of the con-

text, including boundary conditions and internal pressure

variations, which impact the genetically programmed

branching pattern (Fleury et al., 2005). During development,

embryonic lung epithelium continuously produces a fluid into

its lumen, thereby creating an internal pressure within the

epithelial tubes. This pressure has been shown to play an

important role in lung morphogenesis. Several studies

showed that the occlusion of the embryonic trachea increases

lung internal pressure by approximately twofold, leading to

an acceleration of lung growth and branching morphogene-

sis, while conversely drainage of this fluid decreases lung

growth and development in the fetal sheep lung (Alcorn

et al., 1977; Blewett et al., 1996; Moessinger et al., 1990). This

effect has also been shown in murine whole-organ culture

(Bullard et al., 1997), where an impact on developmental gene

expression such as Parathyroid Hormone (Pth) has also been

reported (Cilley et al., 2000).

Congenital Diaphragmatic Hernia (CDH) is a common

developmental disease (1 over 2200 births), consisting of a

failure of the closure of the diaphragm, leading to the entry

of the viscera in the chest, which obstruct lung growth and

eventually resulting in lung hypoplasia. Tracheal occlusion

has been evaluated to reverse lung hypoplasia in develop-

mental diseases such as CDH in the fetal sheep (DiFiore

et al., 1994; Nardo et al., 1995). Clinical studies have also been

performed on human fetuses (Harrison et al., 2003; Deprest

et al., 2004) with variable success, suggesting that tracheal

occlusion could be a way to cure CDH patients.

To test how intraluminal pressure interacts with the lung

developmental molecular pathways, we performed tracheal

occlusion of E12.5 embryonic mouse lungs, by cauterization

of the tip of the trachea, and observed the development of

branching in culture for 48h. Our work suggests that at early

stages of lung development, intraluminal pressure plays a

critical role. The occlusion of the trachea induced a doubling

in the branching rate and strongly affects lung developmental

signaling pathways like the Fgf10/Fgfr2b/Sprouty2 pathway.

We show that internal pressure regulates lung growth and

branching rate by controlling Fgf10 expression level.

2. Results

2.1. Effect of tracheal occlusion on branch formation

E12.5 embryonic mouse lungs were carefully cauterized at

the level of the tip of the trachea. The induced occlusion of

the trachea leads to an increase of the intraluminal pressure


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because the distal epithelium produces a fluid that, due to the

cauterization, cannot escape through the trachea. These

lungs were cultured for 24 and 48 h and compared to non-oc-

cluded lungs, kept in the same culture conditions, which were

used as controls (Fig. 1A). The average number of distal

branches for control lungs before culture was 24 ± 5 (n = 21)

and 25 ± 4 (n = 17) for the cauterized ones. Therefore, no sig-

nificant difference existed in the number of distal branches

between the two experimental sets prior to culture

(P = 0.442). After 24 h of culture, the average number of newly

formed branches was 20 ± 4 (n = 21) for cauterized lungs and

7 ± 2 (n = 17) for the control lungs (Fig. 1B). Thus a 3-fold in-

crease in the rate of branch formation was observed in cauter-

ized lungs after 24 h of culture. From 24 to 48 h of culture, the

average number of new formed branches was 33 ± 17 (n = 6)

for the cauterized lungs and 15 ± 4 (n = 6) for the control. Cau-

terization led to a 2-fold increase in average of the number of

new branches between 24 and 48 h of culture. Thus tracheal

occlusion led to more than a doubling of the branching rate

in comparison to control lungs. Therefore, a clearly marked

phenotype was observed in cauterized lungs at 48 h. The total

number of branches was doubled, as compared to controls

and the branch diameter was concomitantly reduced by

30%, going from 92 ± 3 microns in control lungs to 72 ± 11

microns in cauterized lungs (Fig. 1C). However, the global size

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Fig. 1 – (A) The morphological effect of increased internal press

arrow shows the cauterized trachea. A significant increase (3-fo

The average number of total new branches per day in cauterized

occurs due to the increase of intraluminal pressure by cauteriza

and cauterized lungs after 48 h corresponding to the red box in (

inter-branch length appears shorter than in control lungs.


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of the cauterized lungs remained similar to the control

ones.

2.2. Effect of tracheal occlusion on branch growth speed

To study the effect of the increase of intraluminal pressure

on growth speed, we performed embryonic lung growth mov-

ies. Fig. 2 displays the different steps occurring during a

branching event.

In order to measure the growth speed, we defined a length

that we called L corresponding to the distance between the

centre of the apex of a branch to the mesothelial capsule

(the border of the organ). Three steps in the dichotomous

branching process can be observed. During the first phase,

the branch extends and grows toward the capsule (Fig. 2A,

1–4). Thus the distance L decreases over time. This is the elon-

gation step. Then the distance L remains constant and the

branch expands, acquiring a typical ‘‘T’’ shape. This phase

corresponds to a stop step (Fig. 2A, 5–8). There is a stopping

distance called L0, at which branches cease to extend. Then

the branch divides in two and the centre of the branch re-

cedes. The distance L then increases corresponding to the

branching step (Fig. 2A, 9–12).

The variation of the distance L in time allows us to distin-

guish between the phases of the branching process. Fig. 2B

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ure on cauterized lungs as compared to control lungs. Black

ld) in the number of branches occurs in cauterized lungs. (B)

and control lungs. A 2- or 3-fold increase of branching events

tion. (C) Details of distal branches morphology of the control

A). More branches are visible in the cauterized lungs and the


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Fig. 2 – (A) The evolution in time, each image being separated by an interval of 1 h and 30 min, of the distal tip of a cauterized

lung branch. To study the branching process, we defined a length L corresponding to the distance from the centre of the apex

of a branch to the capsule (the border of the organ). This distance L is represented on image 1 of (A). Distinct phases can be

observed during lung branch growth. From 1–4, the branch grows towards the capsule and the distance L decreases,

corresponding to the elongation phase. From 5–8, the branch stops and its sides progressively dilate, corresponding to the

stop phase. Finally, from 9–12, the branch divides in two and the centre of the branch apex goes backwards relative to the

pleura and the distance L increases, corresponding to the branching phase. (B) The measurements of the distance L of a

control lung branch. The elongation phase (in green), stop phase (in blue) and branching phase (in red) are clearly distinct.

The measurements were fitted linearly and the average growth speed of each phase corresponds to the slope. (C) The average

growth speed in each phase of cauterized and control lung branches. In the elongation phase, the average growth speed is

2.7 ± 1.1 microns/h for control lungs and 9.5 ± 2 microns/h for clamped lungs. In the stop phase, the average growth speed is

0.4 ± 0.4 microns/h for control lungs and �0.1 ± 0.8 microns/h for cauterized lungs. During the branching phase, the average

growth speed is 5.1 ± 0.8 microns/h for control lungs and 5.0 ± 2.2 microns/h for cauterized lungs. The increase of internal

pressure leads to a threefold increase in the elongation phase growth speed.Q2




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displays the evolution of the distance L as a function of the

culture time for a branch of a control lung. We measured

the average branch growth speed in control and cauterized

lungs during the different steps of the branching process

(Fig. 2C). During the elongation step, the average growth

speed is 3 ± 1 microns/h for control lungs and 10 ± 2 mi-

crons/h for cauterized lungs. During the stop step, the average

growth speed is 0.4 ± 0.4 microns/h for control lungs and

�0.1 ± 0.8 microns/h for cauterized lungs. During the branch-

ing step, the average growth speed is 5 ± 1 microns/h for con-

trol lungs and 5 ± 2 microns/h for cauterized lungs. Thus

cauterization principally affects the elongation step, which

is more than three times faster in cauterized lungs as com-

pared to control lungs (see Fig. 3).

The stopping distance L0 is affected by the increase of

intraluminal pressure and shortens of more than 50% (from

62 ± 8 microns in control lungs to 20 ± 4 microns in cauter-

ized lungs). Hence tracheal occlusion leads to a 3-fold de-

crease of the stopping distance L0. Thus, tracheal


occlusion leads to branches growing out faster and closer

to the capsule.

2.3. Effect of tracheal occlusion on cell proliferation

To determine if cell proliferation was involved in the occlu-

sion-induced phenotype, Phosphorylated Histone-H3 and

phosphorylated ERK stainings of control and cauterized lung

sections were performed. Histone H3 phosphorylation is a mar-

ker of chromosome condensation during cell mitosis, while ERK

phosphorylation marks the activation by growth factors of the

receptor tyrosine kinases involved in the Mitogen-Activated

Protein Kinase (MAPK) pathway. A significant increase in lung

cell proliferation was observed in cauterized lungs as compared

to control lungs. Mesenchymal as well as epithelial cell prolifer-

ations were affected. In control lungs 7.59 ± 1.7% of epithelial

cells and 8.63 ± 1.07% of mesenchymal cells were positive for

Phosphorylated Histone-H3 immunostaining. In cauterized

lungs 13.1 ± 1.56% of epithelial cells (1.8-fold increase) and


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Fig. 3 – Phosphorylated Histone-H3 and phosphorylated ERK immunostainings of 5 lm sections of control and cauterized

lungs cultured during 48 h. A high increase in the number of proliferative cells can be observed in cauterized lungs as

compared to controls. Mitotic cells are both mesenchymal and epithelial. The increase of intraluminal pressure in the

cauterized lungs induces a raise of the percentage of phosphorylated Histone-H3 positive cells. 7.59 ± 1.7% epithelial cells

and 8.63 ± 1.07% mesenchymal cells were stained in the control lungs and 13.1 ± 1.56% epithelial cells and 12.5 ± 1.4%

mesenchymal cells were stained in the control lungs. Phosphorylated ERK staining shows a similar increase in epithelial

proliferation. 3.47 ± 1.04% of epithelial cells were stained in the cauterized lungs and 16.21 ± 3.11% of epithelial cells were

stained in the control lungs.




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12.5 ± 1.4% of mesenchymal cells (1.5-fold increase) were Phos-

phorylated Histone-H3 positive (all p < 0.05). Thus, both epithe-

lial and mesenchymal proliferation rates were increased in

cauterized lungs. Tracheal occlusion induced an increase in

proliferation of the epithelium almost two times greater than

in control lungs. Phosphorylated ERK staining showed a higher

activation of ERK MAPK pathways in epithelial cells of cauter-

ized lungs as compared to control ones. In control lungs

3.47 ± 1.04% of epithelial cells were positive for Phosphorylated

ERK staining whereas 16.21 ± 3.11% of epithelial cells (4.7-fold

increase) were stained in cauterized lungs. Therefore, tracheal

occlusion strongly affects lung cell proliferation, suggesting that

the observed effect on lung morphogenesis involves pressure-

sensitive growth factor pathways

2.4. Effect of tracheal occlusion on gene expression

To test how key growth factor pathways known to be in-

volved in lung branching morphogenesis may be modified

by the increase of lung intraluminal pressure, we analyzed

by semi quantitative RT-PCR the changes in gene expression

induced by the cauterization. Analysis was done for Fgf10,

Shh, Sprouty2 and Vegf expression (Fig. 4), corrected to b-actin

expression level. Fgf10 expression was increased by 53% after

48 h of culture in cauterized lungs compared to control lungs,

Shh expression by 45%, Vegf by 37%, but Sprouty2 expression

decreased by 27% in cauterized lungs compared to controls.

Thus tracheal occlusion of mouse embryonic lungs at early


stages of development strongly affects expression levels of

key developmental genes that are involved in the branching

process (Fgf10, Sprouty2 and Shh) as well as in mesenchymal

development and differentiation (Shh, Vegf).

2.5. Effect of tracheal occlusion on blood vessel formation

As shown by RT-PCR experiments, Vegf expression is in-

creased by the increased intraluminal pressure. Blood vessel

development was analyzed using Flk1nlacZ/+ lungs staining

using X-gal. E11.5 Flk1nlacZ/+ lungs were dissected and cauter-

ized before being placed in culture. After 24 h, cauterized

lungs exhibited a significantly more developed blood vessel

network with a higher number of capillary blood vessels

and anastomoses as compared to control lungs (Fig. 5). Thus,

the increase of intraluminal pressure led to an acceleration of

vascular development in the lung.

2.6. Analysis of Fgf10 and Sprouty2 expression by wholemount in situ hybridization

Semi quantitative RT-PCR showed that the increase of

intraluminal pressure leads to an increase of 53% in Fgf10

expression and a decrease of 27% of Sprouty2 expression.

We analyzed the effect of cauterization on the Fgf10 and Spro-

uty2 spatial expression using whole mount in situ hybridiza-

tion (Fig. 6). The increase of intraluminal pressure led to a

striking change in the spatio-temporal expression of Fgf10.


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Fig. 5 – LacZ staining of control (A) and cauterized lungs (B)

of Flk1nlacZ/+ E11.5 transgenic mice after 24 h of culture.

Blood vessel network appears more ramified and developed

in cauterized lung as compared to control. The increase of

intraluminal pressure by cauterization leads to an

acceleration of blood vessel network formation and

development.

Fig. 6 – Fgf10 and Sprouty2 whole mount in situ hybridization

of control (left panel) and cauterized (right panel) lungs after

48 h of culture (lungs were put in culture at E12.5). As

observed with RT-PCR, the Fgf10 expression is higher in

cauterized lungs than in controls while the expression of

Sprouty2 is decreased in cauterized lungs. In the control,

Fgf10 is principally expressed in the distal mesenchyme in

front of the growing branches. The cauterized lung displays

a high increase of Fgf10 expression and a change in its

localization, Fgf10 being more widespread in the internal

mesenchyme and surrounds the newly forming branches.

Fig. 4 – Tracheal occlusion affects gene expression of

cauterized lungs as compared to controls. After 48 h of

culture semi quantitative RT-PCR (A) showed an increase of

Fgf10 (53%), Shh (45%) and Vegf (37%) mRNA expression and

a decrease of Sprouty2 (27%) mRNA expression in E12.5

cauterized embryonic mouse lungs cultured during 48 h as

compared to controls. b-actin mRNA expression level was

used for quantification. (B) These variations of gene

expression. Control lung mRNA expression is fixed at 100%

and variations in cauterized lungs genetic expression are

represented correspondingly.




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The level of expression of Fgf10 was higher in cauterized

lungs as compared to controls, thus confirming the RT-PCR re-

sults. Moreover in cauterized lungs, Fgf10 expression ap-

peared more widespread in the mesenchyme around the

epithelial branches and not only restricted to the distal mes-

enchyme as in control lungs. Whole mount in situ hybridiza-

tion of Sprouty2 showed a decrease of its expression at the

epithelial tips in cauterized lungs (Fig. 6) as observed with

the RT-PCR experiments (see Fig. 7).


T2.7. Fgfr2-IIIb�/� embryonic mouse lung fails to respondto tracheal occlusion

The cauterization of the trachea of wild type mouse embry-

onic lungs at early stages led to a high increase in branch num-

ber. One key gene involved in the branching process is Fgf10.

The increase of internal pressure induced a 50% overall in-

crease in Fgf10 expression. A close relationship between pres-

sure and FGF10 therefore seems to exist. In order to have a

closer look at this pathway, we decided to perform tracheal

occlusion experiments on Fgfr2-IIIb�/� lungs. FGFR2-IIIb is a

transmembrane protein expressed by the epithelium, acting

as a receptor of the mesenchymally expressed FGF10. Null mu-

tants for Fgfr2-IIIb exhibit a similar lung phenotype to Fgf10 null

embryonic lungs. Tracheal occlusion of the Fgfr2-IIIb�/� lungs

as compared to controls led to a dilation of the primordial buds.

This dilation is evidence of the increase of internal pressure

caused by cauterization of the tracheal tip. However no new

branches formed, indicating that the increase of internal pres-

sure was unable, without the functional FGF10-FGFR2IIb path-

way, to generate new branches. Thus tracheal occlusion did not

rescue the Fgfr2-IIIb�/� phenotype. Therefore, FGF10 signaling

is required both for branching per se as well as the response

to intraluminal pressure.

2.8. Embryonic lung culture in presence of Fgfr2-IIIbantisense RNA

In order to further confirm that internal pressure affects

lung branching morphogenesis via the FGF10/FGFR2B path-


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Fig. 7 – E12.5 Fgfr2IIIb�/� transgenic mice lungs display a

very strong phenotype with no branch formation. Only the

primordial buds are formed and are unable to branch.

Cauterization leads to a dilation of these primordial buds

with no branch formation. Intraluminal pressure elevation

is not able to rescue Fgfr2-IIIb�/� phenotype. The observed

dilation of cauterized Fgfr2IIIb�/� mice lungs is consistent

with an increase of lung internal pressure. FGF10 signaling

by epithelial cells via FGFR2IIIb is needed for lung branching

morphogenesis and pressure is unable by itself to induce

branching events.

Fig. 8 – (A—C) The growth of an E12.5 cauterized lung every

24 h. Panel D displays an E12.5 cauterized lung. After 24 h of

culture, antisense Fgfr2-IIIb RNA was added to its culture

medium (D). From 24 to 48 h of culture this lung formed only

2 new branches (E) while cauterized lungs form an average

33 new branches during this period. The addition of

antisense Fgfr2-IIIb RNA leads to the inhibition of Fgfr2-IIIb

expression and of the lung branching despite the increase of

internal pressure by trachea cauterization.




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Rway, we cultured cauterized wild type E12.5 lungs in presence

or absence of antisense Fgfr2-IIIb RNA to block Fgfr2-IIIb

expression. After 24 h of culture, Fgfr2-IIIb antisense RNA

was added to the culture medium. The addition of oligodeg-

oxynucleotides led to strong inhibition of the branching pro-

cess (Fig. 8). Lungs cultured in these conditions formed 2 ± 1

new branches from 24 to 48 h of culture, whereas cauterized

lungs in control conditions formed 33 ± 17 new branches dur-

ing this period. RT-PCR showed that the addition of Fgfr2-IIIb

antisense in the culture medium lead to substantial inhibi-

tion of the expression of Fgfr2-IIIb. These results further sup-

port the necessity for FGF signaling to respond to increased

intraluminal hydraulic pressure.

2.9. Fgf10 hypomorphic lung culture

Internal pressure and the FGF10/FGFR2B pathway there-

fore appear to be linked during embryonic lung branching

morphogenesis. As an additional test of this hypothesis, we

cauterized the trachea of Fgf10LacZ/� embryonic lungs that ex-

press 70% lower levels of Fgf10 than wild type lungs and exhi-


bit hypomorphic, significantly decreased epithelial branching.

After 48 h of culture, we observed a 3.5 ± 0.2-fold increase in

the number of branches for the wild type cauterized lungs.

In contrast, cauterized hypomorph lungs exhibited a

2.4 ± 0.1-fold of increase in the number of branches (Fig. 9).

Thus, the 70% decrease of the level of Fgf10 expression in

the hypomorph lungs induces a significant decrease in the

number of branches formed in response to tracheal

cauterization.

3. Discussion

Trachea occlusion leads to more than a doubling in

branching rate and a threefold increase in elongation growth

speed of the first few generations of airways in early embry-

onic mouse lung. This robust effect confirms the crucial role

of internal pressure on lung development and growth speed.

It also affects lung development globally as shown by the fas-

ter formation of the blood vessel network, implying that a glo-

bal mesenchymal-epithelial-endothelial interaction network

is affected. Hence, a modest (2-fold) increase of internal pres-

sure leads to markedly accelerated growth and lung morpho-

genesis. Conversely, a decrease of internal pressure by

draining of fluid leads to lung hypoplasia and to a decrease

in branching rate (Alcorn et al., 1977). Internal pressure can

therefore control embryonic lung branching events, both by


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Fig. 9 – Cauterization experiments of a wild type lung (left

panels) and of a Fgf10 LacZ/� lung at E12.5 put in culture for

48 h. The Fgf10 LacZ/� lungs express 70% less Fgf10 than wild

type lungs. Cauterization of these lungs decreased the

number of formed branches. During the 48 h of culture a

3.5 ± 0.2-fold increase in the number of branches is

observed in the wild type cauterized lungs, while Fgf10 LacZ/

� lungs exhibit a 2.4 ± 0.1-fold increase in the number of

branches. The low level of Fgf10 expression decreases the

number of newly formed branches.




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Edecreasing and increasing rates of branching. This suggests a

compound effect on branching rate and lung development of

internal pressure. Thus, for normal lung development, inter-

nal pressure has to be controlled and regulated.

The main factors fixing pressure levels are epithelial fluid

secretions and boundary conditions (presence of other

branches, distance to the output, external pressure and laryn-

geal resistance). Moreover, previous studies on the effect of

tracheal occlusion (Alcorn et al., 1977; Blewett et al., 1996)

show that the net lung internal liquid hydraulic pressure

(the difference between internal pressure and atmospheric

pressure) increases by a factor of about 2- to 3-fold in lungs

with occluded tracheas as compared to control ones. This in-

crease of internal pressure corresponds closely to the average

number of new branches per day obtained in our experi-

ments. This result suggests that the branching rate and the

epithelial growth speed vary roughly linearly with the gradi-

ent of pressure, within a physiological, non-traumatic range

of pressure increase.

Key lung developmental genes such as Fgf10, Shh, Sprouty2

and Vegf are affected in cauterized lungs as compared to con-

trol ones. RT-PCR measurements were done on total lung

mRNA, whereas Fgf10 mRNA expression is only restricted to

clusters of distal mesenchyme cells and Sprouty2 is restricted

to the distal epithelium, both representing a small subpopula-

tions of all lung cells. In the same way, Shh as well as Vegf are

only expressed by certain epithelial cells. Therefore whole


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lung RT-PCR might underestimate the real variations in

mRNA production by averaging to total cells the variations

in mRNA concentrations. Fgf10 and Sprouty2 whole mount

in situ hybridizations confirmed the increase of Fgf10 expres-

sion and the decrease of Sprouty2 expression in cauterized

lungs observed by RT-PCR experiments. The increase of inter-

nal pressure by trachea cauterization also leads to a change in

the spatio-temporal expression of Fgf10, which appears more

widespread around the branches, particularly internal ones,

than in control lungs. Due to the link of Fgf10 expression with

the internal pressure, the Fgf10 in situ hybridization could be

interpreted as an indirect strain map in the mesenchyme,

convoluted first by the kinetics of molecular expressions

and by the staining dynamics. The increase in Shh expression,

which has a mitogenic effect on mesenchymal cells, and to-

gether with increased Fgf10 expression, which is involved in

epithelial growth, is coherent with the cell proliferation re-

sults. Both mesenchymal and epithelial cell proliferation are

affected by the increase of intraluminal pressure. However,

epithelial cell proliferation is increased more than mesenchy-

mal cell proliferation in cauterized lungs as compared to con-

trols. This differential cell proliferation may explain why

there appears to be a differential effect on epithelial branch-

ing versus the apparent thinning of the mesenchyme. The

global size (diameter) of the lungs does not increase in cauter-

ized lungs in culture, however they are thicker. The percent-

age of the surface area devoted to epithelial branches

increases, while epithelial lumen diameter significantly de-

creases and growth speed increases.

While Fgf10 expression is increased, the expression of its

inducible inhibitor Sprouty2 is decreased as observed by RT-

PCR and by whole-mount in situ hybridization. The net effect

is likely a very important increase of net Fgf10 signaling.

These variations in mRNA production suggest the possibility

of regulation of primordial lung developmental genes via

the intraluminal pressure. Vegf expression in lung cells has

previously been shown to be positively affected by stretch

(Muratore et al., 2000). Tracheal cauterization experiments

hence show that this effect can be observed on cultured

embryonic lungs.

Mechanical forces can lead to changes in gene expression

(Farge, 2003; Muratore et al., 2000). However, little is known

about genes able to induce Fgf10 expression and thus how

internal pressure and cell shape or membrane deformation

could regulate its expression. Fgf10+/� mouse lungs show de-

fects in mesenchymal cell differentiation as well as in blood

vessel networks, cartilage ring formation, and smooth muscle

cell differentiation (Mailleux et al., 2005). This suggests that

Vegf may possibly be induced directly by the FGF10 signaling

pathway, implying that expression of these two genes could

be linked. Such a correlation could be important to match

lung epithelial growth with its vasculature (Del Moral et al.,

2006).

Fgfr2-IIIb�/� cauterized lungs show only a dilation of the

primordial buds without generation of new branches. Internal

pressure increases as shown by luminal dilation but is unable,

without the functional FGF10-FGFR2-IIIb pathway to generate

branches. This clearly suggests that the formation of an in-

creased number of branches and the global acceleration of

development via elevation of internal pressure is transduced


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through the FGF10 pathway. Moreover, the inhibition of Fgfr2-

IIIb expression in cauterized lungs in culture, by antisense

RNA, also halts the lung branching process. Thus, the in-

crease in the number of branches observed in cauterized

lungs is likely due to the mechano-sensitivity of Fgf10 expres-

sion. An increase of internal pressure leads to a higher stress

in the distal mesenchyme and thus to an increase in Fgf10

expression and in the number of branches. This speculation

is supported by cauterization experiments of Fgf10lacZ/�

embryonic mouse lungs. These lungs formed on average

40% less branches than wild type cauterized lungs, despite

the same increase of internal pressure.

The link between FGF10-FGFRIII2b-SPRY2 and lung inter-

nal pressure gives an interesting clue in the complex process

of lung branching morphogenesis. Cells are submitted to

compressive and tensile stresses by the intraluminal pres-

sure, which up-regulate Fgf10 expression. Compressive and

tensile stresses are related to the mechanical properties of

the tissue (compression is mostly transmural, while stretch

is in-plane). An increase of internal pressure leads to an in-

creased expression of Fgf10 and thus to accelerated epithelial

growth speed, as observed in the trachea cauterization

experiments.

We suggest that Fgf10 is regulated by lung internal pres-

sure not only by affecting its production rate but also by

changing its localization and thus its dynamic pattern of

expression. Distal epithelial cells are continuously producing

a fluid. This fluid induces pressure at the level of the branch

tips. This pressure affects distal mesenchymal and epithelial

cells and thus induces Fgf10 expression. By this mean, during

epithelial growth, the pressure peak is always in front of the

growing branch and Fgf10 is induced in front of the tip in a dy-

namic pattern, leading to branch growth.

To conclude, we were able by cauterization of the tip of

the trachea to occlude it and thus to observe the role of

internal pressure in mouse lung branching development

at early embryonic stages in culture. Tracheal ligation at

E12.5 leads to a significant increase in branching and glo-

bal acceleration of lung growth and development. Varia-

tions of internal pressure affect the mRNA expression of

key lung signaling pathways such as Fgf10-Fgfr2IIIb-Sprou-

ty2, as well as Shh and Vegf. We observed an increase of

the growth speed of lung branching morphogenesis that

is clearly mediated by the FGF10-FGFR2B-Sprouty2 signaling

pathway.
C 579580581582583584585586587588589590591592593594595
UN4. Experimental procedures

4.1. Lung cultures

Wild type, Fgfr2-IIIb�/� (De Moerlooze et al., 2000) andFlk1nLacZ/+ (Shalaby et al., 1995) E12.5 mouse embryos were dis-sected in Hanks’ Balanced Salt solution (Gibco, Grand Island,NY). The morning of the finding of the vaginal plug was des-ignated E0.5. Lung primordials were isolated and placed on8.0 lm Nuclepore Track-Etch membrane (Whatman) and cul-tured in 500 mL of DMEM F12 medium (Gibco, Grand Island,NY) supplemented with 50 U/ml penicillin/streptomycin and10% fetal bovine serum. Dishes were incubated at 37 �C and5% CO2 for 24 or 48 h.


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The tracheas of 21 lungs were carefully cauterized using a sur-gical cauterizer (Allegiance Healthcare Corporation) and com-pared to 17 non-cauterized lungs used as controls. Number ofdistal branches was counted for each lung of the two experimen-tal sets, at t = 0 h, after 24 h, and again after 48 h of culture. In or-der to obtain the number of newly formed branches per day foreach lung, the number of distal branches at 24 h of culture wassubtracted from the number of branches when the lungs wereput in culture, and the number of distal branches at 48 h was sub-tracted from the number of distal branches at 24 h.

4.2. Histone-H3 and Phosphorylated ERKimmunofluorescence

Via standard protocol, slides were treated with different anti-bodies. Rabbit anti-Phospho-Histone H3 was used at the dilutionof 1:100, and rabbit anti-Phospho—p44/42 MAPK (Erk1 and Erk2)was used at the dilution of 1:60 (Cell Signaling Laboratories). Cy-3 conjugated goat anti-rabbit (Jackson Immunoresearch Laborato-ries) diluted 1:200 were used as secondary antibodies. (All sam-ples were mounted with Dapi-labeled Vectashield� andphotomicrographs were taken). 4 control and 4 cauterized lungsections were analyzed.

Epithelial and mesenchymal cell counting were based on mor-phological appearance.

4.3. Embryonic lung growth development movies

E12.5 control and cauterized embryonic mouse lungs were putin culture during 48 h under the objective of a Leica Live Imaginginverted microscope in its chamber regulated at 37 �C, 5% CO2 and100% humidity. The distance L, corresponding to the distance be-tween the centre of the apex of a branch and the capsule, and itsevolution during the time of culture was measured for 5 growingbranches per lung movie. For each branch and for each step of thegrowth (elongation, stop and branching), the data of L as a func-tion of time were fitted linearly. The slope of this fit gave thegrowth speed of a branch for each phase. These measurementswere then averaged to give the average values of the growth speedin each step.

4.4. RT-PCR

After 48 h of culture, total mRNA was isolated from individuallung cultures using Trizol reagent (Invitrogen) following the man-ufacturer’s instructions. Spectrometry was used to determineRNA concentration. Superscript first-strand synthesis systemfor RT-PCR (Invitrogen) was used to generate the cDNA. Semi-quantitative RT-PCR was performed for b-actin, Fgf10, Shh, Vegfand Sprouty2. b-actin mRNA expression level was used forquantification.

The primers used for amplification were as follows:b-actin forward primer, 5 0-GTGGGCCGCTCTAGGCACCAA-3 0; b-

actin reverse primer, 5 0-CGGTTGGCCTTAGGGTTCAGGG-3 0 (pre-dicted size 250 bp).

Fgf10 forward primer, 5 0-CACATTGTGCCTCAGCCTTTCC-3 0;Fgf10 reverse primer, 5 0-CCTGCCATTGTGCTGCCAGTTAA-3 0 (pre-dicted size 505 bp).

mSprouty2 forward primer, 5 0-TGTGAGGACTGTGGAAGTGC-3 0;mSprouty2 reverse primer,

5 0-TTTAAGGCAACCCTTGCTGG-3 0 (predicted size 300 bp).Shh forward primer, 5 0-GGAAAACACTGGAGCAGACC-3 0 ; Shh

reverse primer,5 0-CCACGGAGTTCTCTGCTTTC-3 0 (predicted size 309 bp).Vegf forward primer, 5 0-ATGTGACAAGCCAAGGCGGTG-3 0, Vegf

reverse primer,5 0-TGGCGATTTAGCAGCCAGATA-3 0, (predicted size 300 bp).


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4.5. b-galactosidase expression

E11.5 Flk1nLacZ/+ embryonic lungs were cultured for 48 h, andincubated at 37 �C in X-gal substrate solution until staining ap-peared as described in Hogan et al., 1994.

4.6. Fgf10 Whole mount in situ hybridization

Fgf10 Whole-mount in situ hybridization was done as previ-ously described (Winnier et al., 1995). A 584 bp Fgf10 cDNA (Bellu-sci et al., 1997b) and a 948 bp mouse Spry2 cDNA were used astemplate for the synthesis of digoxigenin-labeled riboprobes.

To determine relative expression levels in cauterized lungscompared with controls, the lungs were all processed in the sametube, for both Fgf10 and Sprouty2 in situ hybridizations.

4.7. Fgfr2-IIIb Antisense RNA

Digoxigenin-labeled riboprobes corresponding to the anti-sense RNA of the exon IIIB of the Fgfr2 gene were synthesizedusing a pBluescript IISK+ plasmid (Stratagene) containing themurine cDNA of this gene, as previously described (De Moerloozeet al., 2000). The probe was generated using a DIG RNA labeling kit(SP6/T7/T3) (Roche) and introduced in the culture medium of cau-terized lungs after 24 h of culture.

4.8. Fgf10 LacZ/� embryonic lungs

For simplicity, Fgf10+/�; Mlc1vnLacZ-24+/�mice are herein referredto as Fgf10LacZ/–(Kelly et al., 2001). Fgf10LacZ/– mouse embryos weregenerated by crossing Fgf10+/� mice with Mlc1vnLacZ-24+/� mice(Kelly et al., 2001). E12.5 Fgf10LacZ/� lungs were isolated and the tra-cheas cauterized prior to being put in culture for 48 h. The totalnumber of branches was counted manually when placed in cultureafter 24 and 48 h, and were compared to the number of branchescounted in wild type cauterized cultured lungs.

4.9. Statistics

The data in the present study were expressed as means ± SD,and the significance of variances between means was evaluatedby Student’s t test and/or ANOVA. p values less than 0.05 wereconsidered to be statistically significant.

Acknowledgements

We thank Dr. Zoher Gueroumi, Dr. Georges Baffet and Dr.

Anmnemiek J.M. Cornelissen for helpful discussions and

two anonymous referees for their comments. This work was

supported in part by HL75773 (D.W.), HL 44977 (D.W.), HL

44060 m(D.W.), HL 60231 (D.W.), HL 074832-01 (S.B.), American

Lung Association (S.B.), American Heart Assocmiation

(P.D.M.), by the CNRS (V.F.) and by a French ministrym mmof

research fellowship (M.U.).
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