Department of Molecular Genetics, College of
Medicine, University of Illinois at Chicago, Chicago, Illinois
60607-7170
winged helix/forkhead box deoxyribonucleic acid binding domain; hepatocyte nuclear factor-3/forkhead homolog; homeodomain box; Nkx2.1; GATA; Gli; Pod1
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INTRODUCTION |
MOUSE LUNG BUD FORMATION INITIATES on
day 9.5 postcoitum (pc) from the laryngotracheal groove
and involves mesenchymal-epithelial cell interactions, which include
paracrine growth factor stimulation that induces cellular
proliferation, migration, and differentiation (74). Lung
branching morphogenesis involves migration of foregut endoderm-derived
epithelial cells into the surrounding splanchnic mesoderm, resulting in
formation of the respiratory bronchioles and the terminal alveolar
sacs, which integrate with the endothelial capillary bed
(74). During mouse lung development, the pseudoglandular stage (days 9.5-16.6 pc) is characterized by
formation of the bronchial and respiratory bronchiole tree, which is
lined with undifferentiated epithelial cells juxtaposed to the
splanchnic mesoderm (130). By day 12 pc of
mouse lung development, branching of the bronchial buds gives rise to
the left lung lobe and the four lobes of the right lung. There is
extensive branching of the distal epithelium and mesenchyme during the
canalicular stage (days 16.6-17.4 pc),
resulting in formation of terminal sacs lined with epithelial cells
integrating with the mesoderm-derived vasculature. The terminal sac
stage [day 17.5 to postnatal day (PD)
5] of lung development is characterized by a coordinated
increase in terminal sac formation and vasculogenesis in conjunction
with the differentiation of alveolar epithelial type I and II cells.
The alveolar stage (PD5 to PD30) of postnatal lung development features
maturation of the terminal respiratory sacs into alveolar ducts and
sacs. At the end of this developmental process, the mature respiratory system is lined with epithelial cells possessing distinct pulmonary functions, which vary in their distribution from the proximal to distal
airway (130). Squamous epithelial cells line the larynx, and the upper airways are populated by a mixture of ciliated columnar and mucus-secreting goblet cells with foci of pulmonary neuroendocrine cells, whereas Clara cells predominate in the lower airways. The distal
alveolar sacs are populated with surfactant protein (SP) A- and
SP-C-secreting type II epithelial cells and type I epithelial cells
that form tight junctions with the pulmonary endothelial cells,
facilitating gas exchange with the circulatory system.
Branching morphogenesis of the lung involves mesenchymal-epithelial
signaling that induces cellular proliferation, migration, and
subsequent transcriptional activation of lung-specific genes. The lung
mesenchyme possesses growth factor receptors, which respond to protein
ligands secreted by the adjacent endoderm or epithelial cells.
Sonic hedgehog (Shh)-deficient [(
/)] mice
exhibit fusion of the tracheoesophageal tube, loss of asymmetry of the
lung (appears as one lobe), and diminished expansion of the alveolar
region (69). Activation of the Gli transcription factors
through the Shh signaling transduction pathway plays an important role
in lung morphogenesis (38, 86, 93, 110, 136). Transgenic mouse studies in which the SP-C gene-regulatory
region was used to increase distal epithelial cell expression of either
Shh (6) or keratinocyte growth factor (119)
resulted in overproliferation of lung mesenchyme, leading to defects in
branching morphogenesis. Appropriate expression of bone morphogenetic
protein-4 (BMP-4) (8, 132), hepatocyte growth factor (HGF)
(87), and fibroblast growth factor (FGF)-10
(7) is critical in regulating pulmonary epithelial cell
proliferation, migration, and branching morphogenesis of the lung.
Fgf10(/) mice die immediately after birth due to disruption of pulmonary branching morphogenesis, and this phenotype is
coincident with severe reductions in the expression of Shh and BMP-4
(lung endoderm) and mesenchymally derived Wnt2 (114). In
combination with in vitro lung culture studies (114, 131), analysis of Fgf10(/) mice revealed that FGF-10 is
involved in the induction of Shh, BMP-4, and Wnt2 signaling molecules,
all of which are essential for lung development. Moreover, transgenic mouse studies (95, 125) have provided further evidence
that the FGF signaling pathway is critical for airway branching and pulmonary epithelial differentiation. These studies identified several
growth factors and signaling molecules that play important roles in
lung morphogenesis during mouse embryonic development.
Vascular endothelial growth factor (VEGF) is expressed in the endoderm
or ectoderm and acts in a paracrine fashion on adjacent mesoderm tissue
to induce proliferation, cell migration, and angioblast differentiation
toward endothelial cell lineage (17, 105). This
developmental process is important for the formation of new blood
vessels de novo (vasculogenesis) or from preexisting vessels (angiogenesis). Targeted disruption of the VEGF gene
produces mutant embryos that display impaired blood island formation
and delayed endothelial cell differentiation, leading to abnormal blood
vessel development (20, 35). VEGF is the ligand for fms-like receptor tyrosine kinase [Flt-1; VEGF receptor-1 (VEGFR-1)] and receptor tyrosine kinase Flk-1 (VEGFR-2), which are expressed in
the primitive endothelium derived from mesoderm (105).
Ablation of the Flk1 gene inhibits vasculogenesis and
formation of angioblast cells in the blood islands (115),
whereas disruption of the Flt1 gene allows the formation of
angioblasts but inhibits their assembly into functional blood vessels
(36). Other receptor tyrosine kinases involved in blood
vessel formation include the Tie1 and Tie2
(TEK) genes, which are expressed in the lateral and
extraembryonic mesoderm of the developing embryo (34, 58).
Mice containing targeted Tie1 gene disruption have defects
in endothelial cell function and blood vessel formation, leading to
pulmonary edema and hemorrhage (99, 112). Targeted
ablation of the Tie2 (TEK) gene leads to defects
in endothelial cell proliferation and migration, which cause inhibition
of angiogenesis (112). Ligand stimulation of the
endothelial receptors Tie-1 and Tie-2 therefore plays an important role
in vascular remodeling (140). In the lung, overexpression of VEGF in the respiratory epithelium stimulated vasculogenesis in
transgenic mouse lungs, but its elevated expression resulted in
aberrant vessel formation and increased expression of Flk-1 and Tie-1
(146). Identification of lung mesenchymal transcription factors mediating expression of these receptor tyrosine kinases will
therefore provide insights regarding pulmonary vasculogenesis and
angiogenesis during embryonic development or after acute lung injury.
The major function of these signaling pathways is to potentiate the
activity or expression of mesenchyme- or endoderm-specific transcription factors in the developing lung. These, in turn, bind
cooperatively to distinct promoter regions and activate target gene
expression. The dynamic changes in gene expression during lung
development are critical to mediate lung morphogenesis, which involves
extensive cellular proliferation, migration, and establishment of
appropriate positioning of respiratory epithelial cells with the
mesenchyme-derived endothelial cells. The molecular events involved in
the process of lung morphogenesis have been reviewed recently by
Warburton et al. (130). We focus our review on the role of
transcription factors in lung morphogenesis and the maintenance of
differentiated gene expression. We include forkhead box (Fox) A2
[FoxA2; hepatocyte nuclear factor (HNF)-3
], FoxF1 [HNF-3/forkhead homolog (HFH)-8; forkhead-related activator (FREAC)-1], and FoxJ1 (HFH-4) transcription factors; Nkx2.1 homeodomain or thyroid
transcription factor (TTF)-1; homeodomain box (Hox) A5; the zinc finger
Gli transcription factors (related to Drosophila cubitus
interruptus); the basic helix-loop-helix (bHLH) Pod1; and GATA
transcription factors. We summarize the phenotypes of transgenic and
knockout mouse models, which define important functions of these
transcription factors in mouse lung development.
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IDENTIFICATION OF HNF-3 IN REGULATING LUNG EPITHELIAL CELL-SPECIFIC
TRANSCRIPTION |
Functional analysis of the regulatory regions of
hepatocyte-specific genes served as an important model system and
determined that hepatocyte-specific gene transcription is dependent on
the recognition of multiple DNA binding sites by distinct families of
HNFs as well as by widely distributed transcription factors (21). These studies also revealed that detectable promoter
activity required synergistic interactions among multiple HNF proteins and that this requirement plays an important role in maintaining cell-specific gene expression (22, 27-32, 37, 45, 82, 91, 107, 108). The HNF-3
, -3, and -3
proteins were
originally identified as mediating transcription of hepatocyte-specific
genes (28, 64, 65) and sharing homology in the winged
helix/forkhead DNA binding domain (25, 73). The HNF-3
and HNF-3 proteins share 93% amino acid homology in the winged
helix DNA binding domain, bind to the same DNA consensus sequences
(Fig. 1), and are potent transcriptional
activators (64, 65, 89). The HNF-3 proteins
possess a conserved NH2-terminal transcriptional activation
domain that is critical for mediating protein interactions with other
HNF transcription factors (41). An essential HNF-3 transcriptional activation domain resides within the COOH-terminal 100-amino acid residues (Fig. 1), which contain the functionally important conserved region II and III sequences (90, 100). Interestingly, a recent study (129) demonstrated that the
region II sequences in the HNF-3 COOH-terminal domain can associate
with the human homolog of the Drosophila Groucho
transcriptional repressor proteins, which suggests the possibility that
HNF-3 proteins may also function to inhibit transcription in cell types
expressing Groucho proteins. Furthermore, functional analysis of the
HNF-3 protein demonstrated that the nuclear localization function
resides within the winged helix DNA binding domain (100),
which is a general feature displayed by other forkhead transcription
factors (9, 43).
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Fig. 1.
Functional protein domains
(A) and DNA binding consensus sequences (B and
C) of the lung forkhead box (Fox) transcription factors.
A: schematic diagram of the forkhead/winged helix DNA
binding domain [percent amino acid homology; region I (magenta)] of
the 5 lung Fox transcription factors that contain the nuclear
localization signal (NLS) (100). Indicated on the
hepatocyte nuclear factor (HNF)-3 (also known as FoxA1) and HNF-3
(also known as FoxA2) proteins are their conserved
NH2-terminal [region IV/V (purple)] and COOH-terminal
[conserved region II (red) and region III (orange)] transcriptional
activation domains (TADs) (90, 100). Also shown are the
TADs (various colors) for HNF-3/forkhead homolog (HFH)-8 [also known
as forkhead-related activator (FREAC-1) and FoxF1], HFH-4 (also known
as FoxJ1), and HFH-11B (also known as FoxM1B and Trident) proteins
(43, 44, 68, 71, 97, 143). Nos. at bottom,
amino acid residues defining the functional protein domain.
B: amino acid sequence alignment of HNF-3 compared with
HNF-3, HFH-4, HFH-8, and HFH-11B winged helix/forkhead DNA binding
domains, which are presented in decreasing order of homology
(percentages on right) (26). Orange dots,
conserved amino acid residues; orange letters, conservative
changes; green letters, nonconservative changes. The positions of the
helices (blue), wing structures (brown), the -sheets (S1-S3;
orange), and turn (T') are indicated on the sequence. Also indicated
is the 20-amino acid region involved in dictating the DNA binding
specificity region. Structure-function studies with chimeric winged
helix proteins demonstrated that swapping this 20-amino acid region
between 2 winged helix proteins is capable of altering DNA binding
specificity (89). C: DNA binding consensus
sequences for the lung Fox transcription factors. The consensus DNA
binding sequence for each of the Fox proteins was determined by
PCR-mediated DNA binding site selection with recombinant glutathione
S-transferase winged helix protein with a partially
degenerate oligonucleotide as previously described (68, 89, 97,
98). V is not T; W is A or T; R is G or A; Y is T or C; N is any
nucleotide; D is not C; H is not G.
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Functional analysis of the regulatory region of lung-specific
genes also demonstrated that normal promoter activity required synergistic interaction of multiple cell-specific transcription factors
in conjunction with inducible and widely expressed transcription factors (10, 14, 15, 113, 122). These promoter studies (10, 11, 14, 42, 49, 113) demonstrated an important role for HNF-3 and HNF-3 proteins in regulating transcription of
SP and Clara cell secretory protein (CCSP) genes
required for bronchiolar and type II epithelial cell function
(Table 1). A transfection study
(108) demonstrated that Hnf3 gene
expression is stimulated by interferon (IFN)- through promoter
recognition by the IFN regulatory factor-1 protein (108).
Subsequent CCSP promoter studies indicated that IFN- induction of
CCSP gene transcription involves promoter activation
by the HNF-3 and signal transducer and activator of
transcription (STAT) proteins (70). Furthermore, HNF-3 regulates promoter expression of the Nkx homeodomain
transcription factor TTF-1 (47), which, in turn, regulates
transcription of the SP genes (14, 19, 39,
139).
Hnf3 and Hnf3 genes display
overlapping expression patterns during lung morphogenesis (Table 1),
but Hnf3 is not expressed in the developing lung
(83, 149). Because of the functional redundancies of the
HNF-3 and HNF-3 proteins in pulmonary epithelial cells, the
function of these transcription factors during lung morphogenesis was
not elucidated from analysis of mice containing targeted disruptions of
the Hnf3 genes. The Hnf3(/)
mice display hypoglycemia due to reduced pancreatic expression of
glucagon, but they exhibit normal lung development (50,
117). Likewise, the in vivo role of HNF-3 in lung
morphogenesis remains unknown because homozygous null
Hnf3 mouse embryos die in utero 9.5 days pc before
lung morphogenesis (1, 134).
Hnf3(/) embryos exhibit defects in the
formation of the node, notochord, foregut endoderm, visceral endoderm,
and neurotube (Table 2). With the availability of mice containing the LoxP-targeted HNF-3 locus (123), the use of Cre recombinase technology for
generating pulmonary epithelium-specific targeted disruption of the
Hnf3 gene will allow examination of the role of
HNF-3 in lung morphogenesis with either wild-type or
Hnf3(+/) mouse backgrounds.
During lung development, HNF-3 protein is expressed at higher levels
in epithelial cells lining the proximal airways and at lower levels in
the distal type 2 epithelial cells (149). Transgenic
SP-C-Hnf3 mice were generated that express high
levels of HNF-3 in the distal airway epithelial cells, which
disrupted the normal decreasing gradient of HNF-3 in these cells
during lung development (148). In the most severe
phenotype, the embryonic lungs consisted of primitive tubules, which
were lined with undifferentiated columnar epithelial cells that
intensely stained positive for the HNF-3 protein (Table 2).
Increased expression of HNF-3 in the distal respiratory epithelium
caused a striking inhibition in branching morphogenesis and
vasculogenesis of the lung, which is coincident with diminished
expression of E-cadherin and VEGF in these cells
(148). These transgenic mouse studies indicated that
maintaining precise levels of HNF-3 is of critical importance in
normal branching morphogenesis of the lung.
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EXPRESSION OF FORKHEAD BOX TRANSCRIPTION FACTORS IN THE LUNG |
Rodent HNF-3 (28, 64, 65) and Drosophila
homeotic forkhead proteins (133) were the first identified
members of an extensive family of transcription factors that shares
homology in the winged helix DNA binding domain (25). The
HNF-3/winged helix/forkhead proteins are a growing family of
transcription factors that play important roles in cellular
proliferation and differentiation (55) and have recently
been renamed as the forkhead box (Fox) family (51). With
PCR amplification of rodent organ cDNA with primers made to conserved
amino acid sequences in the winged helix DNA binding domain, a number
of new Fox family members were isolated from a variety of different
mouse tissues (26, 52, 98). Several Fox genes
that are expressed in the mouse lung were isolated (Table 1),
including HNF3/forkhead homolog-8 (Hfh8; also known as Freac1 or Foxf1), which is expressed in
the mesenchyme of the developing and adult mouse lung (71,
97); forkhead 6 (Fkh6; also known as
Foxl1), the expression of which is observed in embryonic lung mesenchyme (48); Hfh4 (Foxj1),
which is expressed in the ciliated epithelial cells of the developing
and adult lung (12, 18, 24, 126), and Hfh11
(Foxm1, Trident, Win), the expression of which is restricted to proliferating cells of the embryonic lung
(143) and is also reactivated after lung injury
(54).
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MESENCHYMAL EXPRESSION OF HFH-8 (FOXF1) IN DEVELOPING LUNG |
To identify the cellular expression pattern of the HFH8
(Freac1 or Foxf1) gene during mouse embryonic
development, in situ hybridization of mouse embryo paraffin sections
was performed with 33P-labeled antisense HFH-8 RNA probe
(97). After hybridization, stringent washes and
autoradiography, dark-field microscopy was used to visualize
HFH-8-expressing cells in the tissues (Fig. 2, B,
D, F, H, and J). These
studies demonstrate that HFH-8 expression initiates during
mouse gastrulation on day 7 pc in the extraembryonic mesoderm, the allantois, and the lateral mesoderm that arises from the
primitive streak region (See Table 1). Abundant HFH-8 expression
continues in the lateral mesoderm-derived somatopleuric and
splanchnopleuric mesoderm (Fig. 2, A and B),
which contribute to endothelial cell formation in the embryo proper
(92). At the onset of organogenesis at 9.5 days pc, HFH-8
expression is restricted to the splanchnic mesoderm contacting the
embryonic gut and presumptive lung bud (Fig. 2, C and
D), suggesting that it may participate in the
mesenchymal-epithelial induction of lung and gut morphogenesis
(71, 97). HFH-8 expression continues in lateral
mesoderm-derived tissue throughout mouse development and includes the
mesenchymal cells of the oral cavity, esophagus, trachea, lung, gut,
dorsal aorta, and intersomitic arteries (Fig. 2,
E-H) but not of the head (71,
97). In day 18.5 pc embryos, HFH-8 is restricted to
the distal mesenchyme of the lung and the muscle layer of the bronchus,
but its signals are absent in the epithelial cells and mesenchyme of
large vessels (Fig. 2, I and J).
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Fig. 2.
HFH-8 expression patterns in developing mouse embryo
as shown by in situ hybridization with 33P-labeled
antisense HFH-8 RNA probe of sectioned paraffin-embedded 8 (A and B)-, 9.5 (C and D)-,
13.5 (E and F)-, 16 (G and
H)-, and 18.5 (I and J)-day postcoitum
mouse embryos. After hybridization, stringent washes, and
autoradiography, dark-field microscopy (B, D,
F, H, and J) was used to visualize
HFH-8-expressing cells in the tissues. A and B:
in transverse sections of 8-day postcoitum mouse embryos, HFH-8 was
expressed in derivatives of the lateral and extraembryonic mesoderm.
C and D: in transverse sections of 9.5-day
postcoitum mouse embryos, HFH-8 was expressed in the splanchnic
mesoderm (SM) juxtaposed to the lung bud [LB; derived from foregut
(Fg) endoderm (en)]. HFH-8 expression was observed in splanchnopleure
(Sp) and somatopleure (So) mesoderm near the dorsal aorta (DA) and in
yolk sac mesoderm (YSM) and amnion (am). E and F:
in sagittal sections of 13.5-day postcoitum mouse embryo, HFH-8
expression was restricted to mesenchyme derived from the lateral
mesoderm. HFH-8 was expressed in the mesenchyme surrounding the
incisors [upper incisors (UI)] and in the mesenchyme of the tongue
(T), oropharynx (Op), epiglottis (Ep), esophagus (Es), trachea (Tr),
DA, intersomitic arteries (IA), lung (Lu), stomach (S), and intestine
(In). No hybridization is found in the heart (He), liver (L), or
vasculature (V) of the brain. G and H: in
sagittal sections of 16-day postcoitum mouse embryo, HFH-8 continued in
the mesenchyme of the lung, stomach, and intestine. I and
J: in 18.5-day embryonic mouse lung, HFH-8 expression was
found in the mesenchyme of the alveoli and bronchus (Br). No HFH-8
hybridization signals were observed in the epithelial cells of the
bronchioles (b) or in the mesenchyme of the arterioles (a). K, kidney;
NT, neurotube; PC, peritoneal cavity; CC, costal cartilage.
(A-H are reprinted from Mech Dev
69. Peterson RS, Lim L, He H, Overdier D, and Costa RH. The winged
helix transcriptional activator HFH-8 is expressed in the mesoderm of
the primitive streak stage of mouse embryos and its cellular
derivatives, p. 53-69. Copyright 1997, with permission from
Elsevier Science.)
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In more recent studies, we generated a targeted disruption of the mouse
Hfh8 gene in which the winged helix DNA binding domain was
replaced by an in-frame insertion of a nuclear-localizing -galactosidase gene (Kalinichenko VV, Lim L, Whitsett JA,
Clark J, and Costa RH, unpublished observations). Expression of the -galactosidase gene was under the control of the HFH-8
DNA-regulatory sequences, and thus staining for -galactosidase
enzyme activity allowed identification of HFH-8-expressing cells. They
were found to be colocalized with platelet endothelial cell adhesion
molecule-1-positive alveolar endothelial cells and with -smooth
muscle actin-positive peribronchiolar smooth muscle cells, but
pulmonary blood vessels lacked detectable HFH-8 staining
(54). These expression studies with adult
Hfh8(+/) lungs demonstrate that HFH-8 expression is restricted to the alveolar endothelial cells and the smooth muscle surrounding the bronchioles. However, our immunohistochemical staining
data cannot rule out the possibility that HFH-8 is also expressed in
alveolar myofibroblasts. Consistent with the early expression pattern
in the extraembryonic and lateral mesoderm-derived tissues,
Hfh8(/) mice die in utero (Kalinichenko VV, Lim L, Whitsett JA, Clark J, and Costa RH, unpublished observations). Moreover, Hfh8(+/) mice exhibit defects in lung
morphogenesis and function, suggesting that wild-type levels of HFH-8
are necessary for normal lung development.
We determined the DNA binding consensus sequence of HFH-8 using
recombinant HFH-8 protein and PCR-mediated DNA site selection (97). Identification of this DNA binding consensus
sequence revealed that HFH-8 binding sites are present in the promoter regions of genes critical for lung morphogenesis, mesenchyme
proliferation, mesenchymal-epithelial signaling, and angiogenesis or
vasculogenesis (see Table 3 for potential
Hfh8 target genes). The HFH-8 protein therefore potentially
regulates mesenchymal expression of the platelet-derived growth factor
receptors (118, 120), which are required for alveolar
structure formation (16), and receptor tyrosine kinase
Tie-1, VEGFR-1 (Flt-1), and vascular endothelial (VE)-cadherin,
which are required for endothelial cell function and assembly of
endothelial cells into functional blood vessels (36, 58, 99, 112,
127); HFH-8 binding sites were also found in the promoter
regions of mesenchyme-signaling Hgf and Bmp4
genes, the expression of which is critical for lung morphogenesis (8, 87) and extracellular matrix protease genes (Table 3, urokinase-type plasminogen activator, matrix metalloproteinase-1, and
collagenase), which are involved in cell migration or cellular repair
from injury. Interestingly, HFH-8 may regulate expression of the lung
mesenchymal transcription factors homeodomain Hoxa5 and
Hoxb5 genes, and they share overlapping embryonic expression with HFH-8 in the mesenchyme of the distal tips of the developing lung
(5, 13). Moreover, Hoxa5(/) mice show
respiratory tract defects attributed to decreases in pulmonary
epithelial cell expression of TTF-1, HNF-3 and N-myc
(5), suggesting that HFH-8 may also regulate
mesenchyme-mediated lung epithelial cell development through
regulation of the Hoxa5 gene.
Functional analysis of the HFH-8 protein with cotransfection assays
with the Hfh8-dependent reporter gene identified a cell type-specific activation domain (Fig. 1A) that resides in
the COOH-terminal region of the protein (71, 97).
Interestingly, this HFH-8 transcriptional activation domain also
exhibits homology with FoxF2 (FREAC-2; Lun), a Fox transcription factor
that is expressed in pulmonary epithelial cell lines and also shares
amino acid identity with the HFH-8 winged helix DNA binding motif
(44, 76). A second activation domain that included
conserved regions II and III was shown to activate transcription in
undifferentiated cell lines but did not function in differentiated lung
cell lines (44, 71).
Cytokines stimulate the cell type-specific expression of the P-selectin
gene, the expression of which mediates cell adhesion of leukocytes to
the endothelium and their subsequent extravasation to the underlying
injured tissue (135). Cotransfection studies suggest that
HFH-8, which is constitutively expressed in alveolar endothelial cells
and peribronchiolar smooth muscle cells, may participate in cell
type-specific activation of P-selectin in response to cytokines
(97). Moreover, HFH-8 potentially regulates expression of cytokine and chemokine genes that are involved in recruiting inflammatory cells, including interleukin-8,
interleukin-1, monocyte chemoattractant protein-1, and eotaxin (see
Table 3).
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THE FOX TRANSCRIPTION FACTOR HFH-4 (FOXJ1) IS REQUIRED FOR
DIFFERENTIATION OF CILIATED EPITHELIAL CELLS |
An in situ hybridization study (40) demonstrated that
HFH-4 (FoxJ1) initiated expression in the proximal bronchiolar
epithelial cells of the mouse embryonic lung 15.5 days pc, before the
appearance of ciliated epithelial cells. Expression of HFH-4 mRNA was
also observed in the ependymal cells and choroid epithelia on day
11 pc, which is several days before detectable HFH-4 levels in the mouse embryonic lung (68). Later in lung development,
immunohistochemical staining demonstrates that the HFH-4 protein
colocalizes with -tubulin IV-positive ciliated epithelial cells of
the proximal airways (bronchioles, bronchi, and trachea; see Table 1)
(12, 126). HFH-4 protein levels were also observed in
ciliated epithelial cells of the esophagus, nose paranasal sinuses,
ovaries, testis, and developing kidneys and in the ependymal cells
lining the spinal chord and ventricles of the brain (12, 94,
126). In the adult mouse, HFH-4 expression continues in the
ciliated epithelial cells of the lung and respiratory system, in the
choroid plexus, and in stage-specific spermatocytes of the testis.
Consistent with the role of HFH-4 in mediated formation of ciliated
epithelial cells during mouse embryogenesis, Hfh4(/) mice lacked staining for left-right dynein protein and 9+2 microtubules (motile type of cilia) in proximal epithelial cells of the lung and in
the ventricles of the brain (18, 24). The
Hfh4(/) mice displayed perinatal lethality because they
were deficient in ciliated epithelial cells lining the pulmonary
bronchioles and ventricles, leading to defects in lung function and
hydrocephalus (Table 2). HFH-4 is also transiently expressed in
the monociliated cells of the node, which plays an important role
in orchestrating gastrulation. Although cilia were present on the
Hfh4-deficient node cells, HFH-4 expression is required for
left-right asymmetry of the internal organs and Hfh4(/)
mice exhibit randomized situs inversus (internal organs display either
left or right asymmetry). These genetic studies underscore the
importance of HFH-4 in mediating differentiation of the ciliated
epithelial cell lineage and in left-right asymmetry decisions during
embryonic development.
A transgenic mouse study (124) in which HFH-4 protein was
ectopically expressed in the distal respiratory epithelial cells resulted in defects of lung branching morphogenesis. Moreover, the
distal airways were lined with atypical cuboidal or columnar epithelial
cells. In support of the role of HFH-4 in mediating differentiation of
ciliated epithelial cells, the atypical columnar cells that expressed
high levels of the Hfh4 transgene stained positive
for -tubulin IV, a marker for ciliated epithelial cells (Table 2).
Although these transgenic pulmonary epithelial cells still expressed
the transcription factors TTF-1 and HNF-3, they no longer expressed
nonciliated epithelial marker genes including SP-C,
SP-B, and CCSP. Ectopic expression of HFH-4 in
the developing mouse lung therefore promoted differentiation toward the
ciliated epithelial cellular lineage and inhibited expression of
nonciliated epithelial marker genes.
HFH-4 protein is a potent transcriptional activator, with two
activation domains at the NH2 terminus and COOH terminus of the protein (Fig. 1), and deletion of the conserved region II sequences
caused a 25% reduction in transcriptional activity (68). HFH-4 binds to a consensus DNA binding sequence that is distinct from
other Fox transcription factors (Fig. 1) in that it prefers guanine
residues instead of purines in the core sequence and is less tolerant
of nucleotide changes at the 3'-end of the binding site
(68). It is interesting to note that although different Fox transcription factors bind to a similar core consensus sequence, slight nucleotide changes either 3' or 5' to this core sequence can
alter binding specificity between different forkhead family members
(89). Although the Fox proteins show strong amino acid identity in the recognition helix (helix 3), each transcription factor
is capable of interacting with distinct DNA binding sites (Fig. 1).
Structure-function studies with chimeric winged helix recombinant
proteins demonstrated that a 20-amino acid region adjacent to the
recognition helix can alter DNA binding specificity (see Fig.
1B), and these amino acid sequences are the most divergent within the winged helix DNA binding domain (89). Moreover,
nuclear magnetic resonance structure analysis of the HFH-2 winged helix domain demonstrates that this DNA specificity region is able to fold
into a fourth helical structure and functions to reposition the
recognition helix into the DNA major groove (73).
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EXPRESSION OF FKH6 (FOXL1) TRANSCRIPTION FACTOR IN THE LUNG |
During mouse embryogenesis, Fkh6 expression overlaps with that of
HFH-8 in the mesenchyme of the developing lung, stomach, gut, tongue,
and teeth (48). The expression pattern of Fkh6 differs
from HFH-8 in that it is also expressed in the mesenchyme of the
kidney, temporal bones, nasal cavity, and otic and optic capsules. In
adult mice, Fkh6 expression is extinguished in the lung, but its
expression continues in the kidney, stomach, and small and large
intestines. Despite the Fkh6 expression pattern in developing lung
mesenchyme, the Fkh6(/) mice exhibit no defects in lung
morphogenesis or function (53). Fkh6(/)
mice die postnatally at 1 mo because of defects in the intestinal
villus or crypt structures resulting from diminished mesenchymal
expression of transforming growth factor-/activin family members
(BMP-2 and BMP-4).
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EXPRESSION OF HFH-11B (FOXM1B) IS INDUCED AFTER ADULT LUNG INJURY |
The human Fox transcription factor HFH-11B (also known as Trident
and FoxM1b) is a proliferation-specific transcription factor that
shares 39% amino acid homology with the HNF-3 winged helix DNA binding
domain (59, 141, 143). HFH-11B contains a potent COOH-terminal transcriptional activation domain that
possesses several phosphorylation sites for M phase-specific
kinases as well as proline-glutamic acid-serine-threonine (PEST)
sequences (Fig. 1A) that mediate rapid protein degradation
(59, 141, 143). HFH-11B is expressed in proliferating
embryonic cells (including the lung), but its levels diminish
postnatally during terminal differentiation (59, 143).
HFH-11 expression continues in proliferating cells of adult tissue,
primarily in the thymus, testis, small intestine, and colon (59,
143). Although HFH-11 expression is markedly induced during
cellular proliferation, its promoter region displays only a marginal
fourfold stimulation in response to serum, suggesting that increased
HFH-11 mRNA stability also plays a role in its increased levels during
proliferation (60, 143). Furthermore, HFH-11B function is
regulated by nuclear translocation because transgenic HFH-11B protein
remains cytoplasmic in quiescent liver and proliferative signals induce
HFH-11B nuclear localization (142).
HFH-11 expression is essential for normal embryonic development as
evidenced by the perinatal lethal phenotype exhibited by Hfh11/Trident(/) mice (61). Consistent with
a role in mediating cell cycle progression,
Hfh11/Trident-deficient embryos display an abnormal
polyploid phenotype in embryonic hepatocytes and cardiomyocytes (day 13 pc), suggesting that HFH-11 expression is required
to link DNA replication with mitosis (61). Reactivation of
hepatic HFH-11B levels during liver regeneration occurs at the
G1/S transition of the cell cycle, and its levels remain
elevated throughout the period of proliferation (143). A
liver regeneration study with transgenic mice (142) that
prematurely expressed hepatic levels of HFH-11B revealed that the mice
displayed an 8-h acceleration of hepatocyte entry into the S phase,
resulting from earlier expression of cell cycle-regulatory genes.
Expression of HFH-11B is also reactivated by proliferative signals in
the adult rat lung after intratracheal administration of keratinocyte
growth factor (143). A more recent mouse lung injury study
(54) has demonstrated that butylated hydroxytoluene (BHT)
lung injury also reactivates expression of HFH-11B in epithelial and
mesenchymal cells during the period of lung replication and repair
(54). In a manner similar to that described for liver regeneration, this study determined that BHT-mediated lung injury stimulates expression of the HFH-11B transcription factor, suggesting that HFH-11B participates in cellular proliferation during lung injury
repair. In the same BHT lung injury model, epithelial expression of
HNF-3 remained unchanged, whereas a 65% reduction in HFH-8 mRNA
levels was observed during the period of mesenchymal cell proliferation
repair (54).
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THE HOMEOBOX TRANSCRIPTION FACTOR NKX 2.1 (TTF-1) IS REQUIRED FOR
LUNG MORPHOGENESIS |
The Nkx homeodomain transcription factor TTF-1 (also known as
Nkx2.1 and thyroid-specific enhancer-binding protein) is expressed in
the endoderm-derived epithelial cells of the presumptive and developing
lungs and thyroid glands as well as in the embryonic diencephalon
(39, 67). Immunohistochemical staining of developing mouse
lung reveals TTF-1 staining in the proximal and distal airway epithelia
and, at later stages of lung development, in the distal alveolar
epithelial cells (149). Functional analysis of promoter and enhancer regions of the SP, T1, and
CCSP genes has implicated TTF-1 as critical for their
transcriptional activation (14, 19, 39, 103, 137, 139,
147). Interestingly, cotransfection assays have demonstrated
that TTF-1 promoter activity is stimulated by both the HNF-3
(47) and GATA-6 (116) transcription factors, which may play an important role in stimulating Ttf1 gene
expression during lung morphogenesis.
Consistent with the role of TTF-1 in lung development,
Ttf1(/) mice display severe impairment in branching
morphogenesis of the lung and in development of the thymus and
pituitary glands (56). They also exhibit pronounced
defects in ventral forebrain formation. The Ttf1-deficient
lungs develop only into the main stem bronchi and lack development of
the distal alveolar region, suggesting that their formation is arrested
in the early pseudoglandular stage of lung development (56,
75). These Ttf1-deficient pulmonary epithelial cells
fail to express nonciliated marker genes, including differentiated
SP-B, SP-C, and CCSP (Table 2), and
display reduced BMP-4 levels, which may contribute to the defect in
lung branching morphogenesis (75). More recent examination
of Ttf1(/) mice demonstrated that they lacked formation
of septa between the trachea and the esophagus, leading to a common
tracheoesophageal tube connecting the pharynx with the stomach
(75). Furthermore, the developmentally arrested
Ttf1-deficient lungs are connected to the atypical
tracheoesophageal tube through the bronchi, which resembles the human
pathological phenotype termed the tracheoesophageal fistula
(75). This inhibition in tracheoesophageal septum
formation is similar to that observed with the cubitus interruptus
Gli2(/) mouse in a heterozygous Gli3
background (86) and the Shh(/) mice
(69). These studies indicate the importance of TTF-1 in the development of the lungs and the respiratory system.
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HOX TRANSCRIPTION FACTORS IN LUNG MORPHOGENESIS |
A number of distinct Hox transcription factors are expressed in
the presumptive lung during mouse embryogenesis, and their expression
levels decrease as the mouse embryo reaches gestation (5, 13,
128). The Hox genes share homology in the
helix-turn-helix motif but vary among family members in their ability
to bind to DNA as either a monomer or dimer (23, 72). The
Hoxb3 and Hoxb4 genes are expressed in the
mesenchyme of the trachea, bronchi, and distal lung, whereas
Hoxa5, Hoxb2, and Hoxb5 are restricted to the
distal lung mesenchyme, suggesting a role in branching morphogenesis (Table 1). Consistent with an important role
in mesenchymal-epithelial interactions, the Hoxa5(/)
mice display improper tracheal formation and impaired lung branching
morphogenesis, leading to tracheal occlusions, diminished surfactant
expression, and thickening of alveolar walls (5). Loss of
mesenchymal expression of the Hoxa5 gene caused a disruption
in mesenchymal-epithelial signaling, leading to decreases in TTF-1,
HNF-3, and N-myc expression in the pulmonary epithelial cells (Table
2). Future mouse gene targeting studies will allow determination of the
role of the Hox genes in branching morphogenesis and
mesenchymal-epithelial signaling during lung development.
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ROLE OF THE ZINC FINGER GATA TRANSCRIPTION FACTORS IN LUNG
MORPHOGENESIS |
The GATA transcription factors were first identified as regulating
hematopoietic genes and share homology in their DNA binding domains
that contain two zinc finger motifs (reviewed in Refs. 78,
88). In situ hybridization studies (3, 66)
demonstrated that GATA-4 is expressed in the heart, gut endoderm,
intestinal epithelium, liver, testis, and ovaries. GATA-4 expression is
induced by retinoic acid differentiation of F9 cells into visceral or parietal endoderm and in embryoid body-induced differentiation of
embryonic stem cells into visceral endoderm (3).
Consistent with this embryonic expression pattern,
Gata4(/) embryos die shortly after gastrulation and
exhibit defects in heart morphogenesis and in foregut endoderm and
visceral endoderm formation (63, 79). This phenotype
suggests that GATA-4 expression is required for foregut endoderm
specification and may play an early role in the development of
foregut endoderm-derived organs (145). Future
studies involving tetraploid rescue of the visceral endoderm defect
will allow examination of the role of GATA-4 in gut endoderm morphogenesis and possibly in lung morphogenesis.
The GATA-5 and GATA-6 transcription factors display nonoverlapping
expression patterns in the developing lung; GATA-6 expression is
restricted to the bronchiolar epithelial cells of the lung (84), whereas GATA-5 is expressed in the smooth muscle
cells of the large airways (85). The GATA transcription
factors display similar expression patterns in developing heart,
allantois, and gut epithelial cells, but at later stages of
heart development, GATA-5 levels diminish. Their expression patterns
differ in that GATA-6 expression is found in the primitive streak
mesoderm and in Reichert's membrane (Table 1). Targeted disruption of
the Gata5 gene leads to vaginal and uterine defects in
females, but the mice display no defects in lung morphogenesis
(81). Gata6(/) embryos die during
gastrulation from defects in extraembryonic tissue, and, therefore, its
role in lung development remains unknown. Gata6 may likely
play a role in lung morphogenesis given the fact that it regulates
expression of TTF-1 (116), which is essential for lung
formation. Future experiments with Cre/LoxP technology will allow cell
type-specific ablation of the Gata6 gene and determination of its role in lung morphogenesis and function.
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CUBITUS INTERRUPTUS GLI TRANSCRIPTION FACTORS IN LUNG MORPHOGENESIS |
The zinc finger Gli transcription factors are homologs of the
Drosophila segment polarity gene cubitus
interruptus and mediate transcriptional induction in response to
Shh signaling (38, 46).
Gli3XtJ/Gli3XtJ mice, a
naturally occurring mouse mutation in the Gli3 gene, exhibit
defects in the right medial, right caudal, and accessory lobes of the
lungs (38). Targeted disruption of the Gli2
gene results in a perinatal lethal phenotype with diminished lung
proliferation and branching, leading to fusion of the four right
lung lobes into one lobe (77, 86). This lung phenotype is
coincident with diminished expression of the Shh receptor Patch and the
isoform Gli1 (Table 2). Gli2(/) mice also
display severe skeletal and neuronal defects, including hypoplastic
trachea and esophagus (77). By contrast, no defects
were observed in the transcriptionally inactive
Gli1(zfd/zfd) mouse mutation, which
deleted the exons encoding the zinc finger DNA binding
domain (zfd) (93). Interestingly, Gli1(zfd/zfd),Gli2(zfd/+)
mice, but not
Gli1(zfd/zfd),Gli3(zfd/+) mice, die soon after birth and have multiple defects, including development of smaller lungs, suggesting that the Gli1 and
Gli2 genes have redundant functions (Table 2).
Gli1(zfd/zfd), Gli2(zfd/zfd) double-mutant mice have more severe lung defects that are similar to
those found with the Shh(/) mice in which the lung
develops but displays inhibition of branching morphogenesis
(69).
A more severe lung defect is observed with the Gli2 gene
deficiency analyzed in a Gli3 heterozygous background
(86). Gli2(/),Gli3(+/) embryonic mouse lungs are more hypoplastic, and the right and left
lobes fail to separate (86). These mice have defective tracheoesophageal septum formation and possess a single
tracheoesophageal tube, which connects the pharynx with the stomach,
resembling the phenotype observed with either the Shh(/)
(69) or Ttf1 (Nkx 2.1)(/) mice
(75). The most severe phenotype is exhibited by the
Gli3(/),Gli2(/) mice, which display a
complete absence of lung, trachea, and esophagus and smaller stomach,
liver, and pancreas (86). Interestingly, the phenotype of
Gli3(/),Gli2(/) mice is more severe than
that in Shh(/) mice (69), suggesting that
these Gli transcription factors are not only regulated by Shh but may
also be controlled by other signal transduction pathways.
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THE BASIC HELIX-LOOP-HELIX POD1 TRANSCRIPTION FACTOR IN LUNG
MORPHOGENESIS |
Pod1 is a bHLH transcription factor in which the helix-loop-helix
domain mediates protein association, allowing formation of either
homodimeric and heterodimeric proteins, which then interact with DNA
though the basic amino acid region (80). Pod1 is
abundantly expressed in the mesenchyme of developing organs of the
mouse embryo, including the lung, kidney, gut, and heart and in
glomerular visceral epithelial cells (podocytes) (101,
102). Pod1(/) mice exhibit a perinatal lethal
phenotype, displaying hypoplastic lungs that lack development of the
alveolar region and kidneys that are deficient in mature glomeruli
(101). Although Pod1 is exclusively expressed in the
mesenchyme and podocytes, major defects are observed in the adjacent
epithelia and include abnormalities in epithelial cell differentiation
and branching morphogenesis (Table 2). Pod1 therefore appears to be
essential for regulating genes involved in mesenchymal-epithelial
interactions, which are critical for the morphogenesis of the lung and kidney.
The work in our laboratory was supported National Heart, Lung, and
Blood Institute Grant HL-62446; National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-54687; and National Institute of
General Medical Sciences Grant GM-43241.
Address for reprint requests and other correspondence: R. H. Costa, Dept. of Molecular Genetics (M/C 669), Univ. of Illinois at
Chicago, College of Medicine, 900 S. Ashland Ave, Rm. 2220 MBRB,
Chicago, IL 60607-7170 (E-mail: Robcosta{at}uic.edu).
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ABSTRACT
INTRODUCTION
