Branching morphogenesis of the embryonic lung requires interactions between the epithelium and the mesenchyme. Previously, we reported that Sonic hedgehog (Shh) transcripts are present in the epithelium of the developing mouse lung, with highest levels in the terminal buds. Here, we report that transcripts of mouse patched (Ptc), the homologue of a Drosophila gene encoding a putative transmembrane protein required for hedgehog signaling, are expressed at high levels in the mesenchyme adjacent to the end buds. To investigate the function of SHH in lung development, Shh was overexpressed throughout the distal epithelium, using the surfactant protein-C (SP-C)-enhancer/promoter. Beginning around 16.5 dpc, when Shh and Ptc RNA levels are normally both declining, this treatment caused an increase in the ratio of interstitial mesenchyme to epithelial tubules in transgenic compared to normal lungs. Transgenic newborn mice die soon after birth. Histological analysis of the lungs at the light and electron microscope level shows an abundance of mesenchyme and the absence of typical alveoli. In vivo BrdU labeling indicates that Shh overexpression results in increased mesenchymal and epithelial cell proliferation at 16.5 and 17.5 dpc. However, analysis of CC-10 and SP-C expression reveals no significant inhibition in the differentiation of proximal and distal epithelial cells. The expression of genes potentially regulated by SHH was also examined. No difference could be observed between transgenic and control lungs in either the level or distribution of Bmp4, Wnt2 and Fgf7 RNA. By contrast, Ptc is clearly upregulated in the transgenic lung. These results thus establish a role for SHH in lung morphogenesis, and suggest that SHH normally regulates lung mesenchymal cell proliferation in vivo.
REFERENCES
REFERENCES
Alcedo
J.
, Ayzenzon
M.
, Von Ohlen
T.
, Noll
M.
, Hooper
J. E.
(1996
) The Drosophila smoothened gene encodes a seven-pass membrane protein, a putative receptor for the hedgehog signal.
Cell
86
, 221
–232
Alescio
T.
, Cassini
A.
(1962
) Induction in vitro of tracheal buds by pulmonary mesenchyme grafted on tracheal epithelium.
J. Exp. Zool
150
, 83
–94
Basler
K.
, Struhl
G.
(1994
) Compartment boundaries and the control of Drosophila limb pattern by hedgehog protein.
Nature
368
, 208
–214
Bellusci
S.
, Henderson
R.
, Winnier
G.
, Oikawa
T.
, Hogan
B. L. M.
(1996
) Evidence from normal expression and targeted misexpression that Bone Morphogenetic Protein-4 (Bmp-4) plays a role in mouse embryonic lung morphogenesis.
Development
122
, 1693
–1702
Bitgood
M. J.
, McMahon
A. P.
(1995
) Hedgehog and Bmp genes are co-expressed at many diverse sites of cell-cell interaction in the mouse embryo.
Dev. Biol
172
, 126
–138
Capdevila
J.
, Pariente
F.
, Sampedro
J.
, Alonso
J. L.
, Guerrero
I.
(1994
) Subcellular localization of the segment polarity protein patched suggests an interaction with the wingless reception complex in Drosophila embryos.
Development
120
, 987
–998
Chirgwin
J. M.
, Pryzbyla
A. E.
, MacDonald
R. J.
, Rutter
W. J.
(1979
) Isolation of biologically active ribonucleic acid from sources enriched with ribonuclease.
Biochemistry
18
, 5294
–5299
Diaz-Benjumea
F. J.
, Cohen
B.
, Cohen
S. M.
(1994
) Cell interaction between compartments establishes the proximal-distal axis of Drosophila legs.
Nature
372
, 175
–179
Fan
C.-M.
, Porter
J. A.
, Chiang
C.
, Chang
D. T.
, Beachy
P. A.
, Tessier-Lavigne
M.
(1995
) Long range sclerotome induction by Sonic hedgehog: direct role of the amino-terminal cleavage product and modulation by the cyclic AMP signalling pathway.
Cell
81
, 457
–465
Forbes
A. J.
, Lin
H.
, Ingham
P.
, Spradling
A. C.
(1996
) Hedgehog is required for the proliferation and specification of ovarian somatic cells prior to egg chamber formation in Drosophila.
Development
121
, 1125
–1135
Goldin
G. V.
, Wessels
N. K.
(1979
) Mammalian lung development: the possible role of cell proliferation in the formation of supernumerary tracheal buds and in branching morphogenesis.
J. Exp. Zool
208
, 337
–346
Goodrich
L. V.
, Johnson
R. L.
, Milenkovic
L.
, McMahon
J. A.
, Scott
M. P.
(1996
) Conservation of the hedgehog/patched signaling pathway from flies to mice: induction of a mouse patched gene by hedgehog.
Genes Dev
10
, 301
–312
Green
M. C.
(1968
) Mechanism of the pleiotropic effects of the short ear mutant gene in the mouse.
J. Exp. Zool
167
, 129
–150
Herberlein
U.
, Wolff
T.
, Rubin
G. M.
(1993
) The TGFhomolog Dpp and the segment polarity gene hedgehog are required for propagation of a morphogenetic wave in Drosophila retina.
Cell
75
, 913
–926
Hooper
J. E.
(1994
) Distinct pathways for autocrine and paracrine Wingless signalling in Drosophila embryos.
Nature
372
, 461
–464
Hooper
J. E.
, Scott
M. P.
(1989
) The Drosophila patched gene encodes aputative membrane protein required for segmental patterning.
Cell
59
, 751
–765
Hui
C. C.
, Slusarski
D.
, Platt
K. A.
, Holmgren
R.
, Joyner
A. L.
(1994
) Expression of three mouse homologs of the Drosophila segment polarity gene cubitus interruptus, Gli, Gli-2, and Gli-3, in ectoderm-and mesoderm-derived tissues suggests multiple roles during postimplantation development.
Dev. Biol
162
, 402
–13
Ingham
P. W.
(1993
) Localized hedgehog activity controls spatial limits of wingless transcription in the Drosophila embryo.
Nature
366
, 560
–562
Ingham
P. W.
, Taylor
A. M.
, Nakano
Y.
(1991
) Role of the Drosophila patched gene in positional signalling.
Nature
353
, 184
–187
Johnson
R. L.
, Rothman
A. L.
, Xie
J.
, Goodrich
L. V.
, bare
J. W.
, Bonifas
J. M.
, Quinn
A. G.
, Myers
R. M.
, Cox
D. R.
, Epstein
E. H.
, Scott
M. P.
(1996
) Human homolog of patched, a candidate gene for the basal cell nevus syndrome.
Science
272
, 1668
–1671
Kaartinen
V.
, Voncken
J. W.
, Shuler
C.
, Warburton
D.
, Bu
D.
, Heisterkamp
N.
, Groffen
J.
(1995
) Abnormal lung development and cleft palate in mice lacking TGF-3 indicates defects of epithelial-mesenchymal interaction.
Nature Genetics
11
, 415
–421
Kalina
M.
, Mason
R. J.
, Shannon
J. M.
(1992
) Surfactant protein C is expressed in alveolar type II cells but not in Clara cells of rat lung.
Am. J. Respir. Cell Mol. Biol
6
, 594
–600
King
J. A.
, Marker
P. C.
, Seung
K. J.
, Kingsley
D. M.
(1994
) BMP5 and the molecular, skeletal, and soft-tissue alterations in short ear mice.
Dev. Biol
166
, 112
–122
Korfhagen
T.
, Glasser
S.
, Wert
S.
, Bruno
M.
, Daugherty
C.
, McNeish
J.
, Stock
J.
, Potter
S.
, Whitsett
J.
(1990
) Cis-acting sequences from a human surfactant protein gene confer pulmonary-specific gene expression in transgenic mice.
Proc. Natl. Acad. Sci. USA
87
, 6122
–6126
Kulkarni
A. B.
, Huh
C.
, Becker
D.
, Geiser
A.
, Lyght
M.
, Flanders
K. C.
, Roberts
A. B.
, Sporn
M. B.
, Ward
J. M.
, Karlsson
S.
(1993
) Transforming growth factor-beta1 null mutation in mice causes excessive inflammatory response and early death.
Proc. Natl. Acad. Sci. USA
90
, 770
–774
Laufer
E.
, Nelson
C. E.
, Johnson
R. L.
, Morgan
B. A.
, Tabin
C.
(1994
) Sonic hedgehog and Fgf-4 act through a signaling cascade and feedback loop to integrate growth and patterning of the developing limb bud.
Cell
79
, 993
–1003
Lepage
T.
, Cohen
S. M.
, Diaz-Benjumea
F. J.
, Parkhurst
S. M.
(1995
) Signal transduction by cAMP-dependent protein kinase A in Drosophila limb patterning.
Nature
373
, 711
–715
Marigo
V.
, Scott
M. P.
, Johnson
R. L.
, Goodrich
L. V.
, Tabin
C. J.
(1996
) Conservation in hedgehog signaling: induction of a chicken patched homolog by Sonic hedgehog in the developing limb.
Development
122
, 1225
–1233
Nakano
Y.
, Guerrero
I.
, Hidalgo
A.
, Taylor
A.
, Whittle
J. R. S.
, Ingham
P. W.
(1989
) A protein with several possible membrane-spanning domains encoded by the Drosophila segment polarity gene patched.
Nature
341
, 508
–513
Peters
K.
, Werner
S.
, Liao
X.
, Wert
S.
, Whitsett
J.
, Williams
L.
(1994
) Targeted expression of a dominant negative FGF receptor blocks branching morphogenesis and epithelial differentiation of the mouse lung.
EMBO J
13
, 3296
–3301
Roberts
D. J.
, Johnson
R. L.
, Burke
A. C.
, Nelson
C. E.
, Morgan
B. A.
, Tabin
C.
(1995
) Sonic hedgehog is an endodermal signal inducing Bmp-4 and Hox genes during induction and regionalization of the chick hindgut.
Development
121
, 3163
–3174
Sakai
Y.
, Nelson
K. G.
, Snedeter
S.
, Bossert
N. L.
, Walker
M. P.
, McMahon
J.
, DiAugustine
R. P.
(1994
) Expression of epidermal growth factor in suprabasal cells of stratified squamous epithelia: implications for a role in differentiation.
Cell Growth and Diff
5
, 527
–535
Sasaki
H.
, Hogan
B. L. M.
(1993
) Differential expression of multiple fork head related genes during gastrulation and axial pattern formation in the mouse embryo.
Development
118
, 47
–59
Serra
R.
, Pelton
R. W.
, Moses
H. L.
(1994
) TGF1 inhibits branching morphogenesis and N-myc expression in lung bud organ cultures.
Development
120
, 2153
–2161
Shannon
J. M.
(1994
) Induction of alveolar type II cell differentiation in fetal tracheal epithelium by grafted distal lung mesenchyme.
Dev. Biol
166
, 600
–614
Shull
M. M.
, Ormsby
I.
, Kier
A. B.
, Pawlowski
S.
, Diebold
R. J.
, Yin
M.
, Allen
R.
, Sidman
C.
, Proetzel
G.
, Calvin
D.
, Annunziata
N.
, Doetschman
T.
(1992
) Targeted disruption of the mouse transforming growth factor-beta1 gene results in multifocal inflammatory disease.
Nature
359
, 693
–699
Simonet
W. S.
, Derose
M. L.
, Bucay
N.
, Nguyen
H. Q.
, Wert
S. E.
, Zhou
L.
, Ulich
T. R.
, Thomason
A.
, Danilenko
D. M.
, Whitsett
J. A.
(1995
) Pulmonary malformation in transgenic mice expressing human keratinocyte growth factor in the lung.
Proc. Natl. Acad. Sci. USA
92
, 12461
–12465
Singh
G.
, Katyal
S. L.
, Brown
W. E.
, Kennedy
A. L.
(1993
) Mouse clara cell 10-kDa (CC10) protein: cDNA nucleotide sequence and molecular basis of variation in progesterone binding of CC10 from different species.
Exp. Lung Research
19
, 67
–75
Snouwaert
J. N.
, Brigman
K. K.
, Latour
A. M.
, Malouf
N. M.
, Boucher
R. C.
, Smithies
O.
, Koller
B. H.
(1992
) An animal model for cystic fibrosis made by gene targeting.
Science
257
, 1083
–1088
Tabata
T.
, Kornberg
(1994
) Hedgehog is a signaling protein with a key role in patterning Drosophila imaginal discs.
Cell
76
, 89
–102
Ten Have-Opbroek
A. A. W.
(1991
) Lung development in the mouse embryo.
Exp. Lung Research
17
, 111
–130
Urase
K.
, Mukasa
T.
, Igarashi
H.
, Ishii
Y.
, Yasagi
S.
, Momoi
M. Y.
, Momoi
T.
(1996
) Spatial expression of Sonic hedgehog in the lung epithelium during branching morphogenesis.
Biochem. Biophys. Res. Comm
225
, 161
–166
Vanio
S.
, Karavanova
I.
, Jowett
A.
, Thesleff
I.
(1993
) Identification of BMP-4 as a signal mediating secondary induction between epithelial and mesenchymal tissues during early tooth development.
Cell
75
, 45
–58
Wert
S.
, Glasser
S. W.
, Korfhagen
T. R.
, Whitsett
J. A.
(1993
) Transcriptional elements from the human SP-C gene direct expression in the primordial respiratory epithelium of transgenic mice.
Dev. Biol
156
, 426
–443
Wessels
N. K.
(1970
) Mammalian lung development: interactions in formation and morphogenesis of tracheal buds.
J. Exp. Zool
175
, 455
–466
Winnier
G.
, Blessing
M.
, Labosky
P. A.
, Hogan
B.
(1995
) Bone Morphogenetic protein-4 (BMP-4) is required for mesoderm formation and patterning in the mouse.
Genes Dev
9
, 2105
–2116
Zhou
L.
, Dey
C. R.
, Wert
S. E.
, Whitsett
J. A.
(1996
) Arrested lung morphogenesis in transgenic mice bearing an SP-C- TGF-1 chimeric gene.
Dev. Biol
175
, 227
–238
This content is only available via PDF.
© 1997 by Company of Biologists
1997
