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Forkhead Transcription Factor HFH-4 Expression Is

Temporally Related to Ciliogenesis


Erica N. Blatt, Xiu Hua Yan, Mary K. Wuerffel, Daniel L. Hamilos, and Steven L. Brody
Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri

Members of the forkhead/winged-helix family of transcription factors are expressed in tissue-specific pat-
terns and play critical roles in development and cell differentiation. The expression of forkhead family
member hepatocyte nuclear factor-3/forkhead homologue 4 (HFH-4) has been localized by RNA-blot
analysis and in situ hybridization to the proximal airway of the lung (trachea, bronchi, and bronchioles)
with onset at mouse embryonic day (E) 14.5 and is present in the choroid plexus, ependymal cells, oviduct,
and testis. We hypothesized that the restricted expression of HFH-4 messenger RNA suggests a function
common to these tissues and therefore a cell-specific role for HFH-4. Accordingly, an anti–HFH-4 anti-
body was generated and used for cell-specific localization of protein expression to begin to identify the
functions of HFH-4. We found HFH-4 expression in proximal airway ciliated epithelial cells, but not Clara
cells or alveolar epithelial cells. HFH-4 was also expressed in ciliated epithelial cells of the nose and para-
nasal sinuses, choroid plexus, ependyma, and oviduct. In developing mouse lung, HFH-4 expression was
initially detected in airway epithelial cells at E15.5, before the appearance of cilia, and at later stages was
localized to epithelial cells with cilia. In the testis, HFH-4 expression in spermatids was coincident with
stage-specific generation of flagella. The temporal relationship of HFH-4 expression to the development of
cilia and flagella, and the restricted expression in ciliated epithelial cells, suggest that this transcription fac-
tor has a role in regulation and maintenance of the ciliated cell phenotype in epithelial cells. Blatt, E. N.,
X. H. Yan, M. K. Wuerffel, D. L. Hamilos, and S. L. Brody. 1999. Forkhead transcription factor
HFH-4 expression is temporally related to ciliogenesis. Am. J. Respir. Cell Mol. Biol. 21:168–176.

The lung contains diverse types of epithelial cells in the factors, including the homeodomain thyroid transcription
proximal (trachea, bronchi, and bronchioles) and distal factor (TTF)-1 and the forkhead/winged-helix protein hepa-
(alveoli) airways. This diversity allows the airway epithe- tocyte nuclear factor (HNF)-3b, also have important func-
lial cells to carry out a wide variety of functions required tions in airway epithelial cell differentiation (6–8). Both
for host defense and gas exchange (1, 2). In the developing TTF-1 and HNF-3b are expressed in early developing lung
lung, airway epithelial cell differentiation occurs after the epithelial cells of proximal and distal airways, and altered
lung bud begins to branch (1, 3). Molecular mechanisms expression of these factors results in a failure of normal
that direct differentiation of airway epithelial cells during airway epithelial cell differentiation (9, 10). These obser-
development are not well understood. Secreted growth vations suggest that transcription factors play central roles
factors such as fibroblast growth factors, transforming in establishing the general pattern of epithelial cell differ-
growth factor-b, and epithelial growth factor appear to be entiation in the lung.
important in branching and epithelial cell differentiation, Members of the forkhead/winged-helix family of tran-
particularly in the alveolar epithelium (4, 5). Transcription scription factors are expressed in a tissue-specific manner
and have critical functions in cell differentiation and regu-
(Received in original form February 9, 1999 and in revised form March 5,
lation of specialized cellular function (11). For example,
1999 ) forkhead factors BF-1 and BF-2 specify differentiation of
Address correspondence to: Steven L. Brody, Washington University distinct regions of the brain, MFH-1 is critical in skeletogen-
School of Medicine, Box 8052, 660 S. Euclid Ave., St. Louis, MO 63110. esis, and whn (mutated in the nude mouse) is responsible
E-mail: sbrody@pulmonary.WUSTL.edu for hair growth and thymus development (12–14). In the
Abbreviations: Clara cell secretory protein, CCSP; complementary DNA, lung, forkhead factors HNF-3a and b have been shown to
cDNA; embryonic day, E; fluorescein isothiocyanate, FITC; glutathione regulate the expression of airway epithelial cell–specific
S-transferase, GST; HNF-3/forkhead homologue 4, HFH-4; hepatocyte genes for surfactant protein B and the Clara cell secretory
nuclear factor, HNF; immunoglobulin, Ig; mesenger RNA, mRNA; olfac-
tory marker protein, OMP; phosphate-buffered saline, PBS; room tem- protein (CCSP) (7, 8). However, HNF-3a and b are widely
perature, RT. expressed throughout endoderm-derived cells in most or-
Am. J. Respir. Cell Mol. Biol. Vol. 21, pp. 168–176, 1999 gans, indicating that these proteins alone do not direct
Internet address: www.atsjournals.org lung-specific epithelial cell differentiation (15). Instead,
Blatt, Yan, Wuerffel, et al.: HFH-4 Expression in Ciliated Cells 169

forkhead factors that are more restricted in expression will tration was determined using the Bio-Rad protein assay
likely be important for differentiation of specific popula- solution (Bio-Rad, Hercules, CA).
tions of airway epithelial cells.
We and others have previously described the cloning Antibody Specificity Testing
and expression of HNF-3/forkhead homologue 4 (HFH-4), A rat HFH-4 cDNA containing the complete open reading
a third forkhead/winged-helix transcription factor that is ex- frame (1,266 base pairs) was constructed by polymerase
pressed in airway epithelial cells. HFH-4 has been cloned chain reaction (PCR) amplification using two overlapping
from mouse, rat, and human; and, in contrast to HNF-3a rat HFH-4 cDNA clones (17) as templates, Vent DNA
and b, it has a restricted pattern of expression (16–21). polymerase (New England Biolabs, Beverly, MA), and a
Northern analysis and in situ hybridization demonstrate 59 oligomer containing an XbaI site (59-TCTAGAGCA-
that HFH-4 messenger RNA (mRNA) is restricted to the GACATGGCGGAGAGC) and 39 oligomer containing a
epithelium of lung, oviduct, choroid plexus and ependyma BamHIsite (59-GGATCCCTGACTTGAGCACTGTCC).
of the brain, and the seminiferous tubules of the testis (17, The PCR product was subcloned into pCR3 (Invitrogen,
19, 21). Previously, we used in situ hybridization to demon- Carlsbad, CA). The cDNA sequences containing the open
strate that expression of HFH-4 mRNA is developmen- reading frame of the rat HNF-3a and HNF-3b (kindly pro-
tally regulated in proximal airway epithelial cells with on- vided by J. Darnell, Jr., Rockefeller University, New York,
set in the mouse lung at embryonic day (E) 14.5 (17). A NY) were removed from parent vectors and subcloned into
similar pattern of HFH-4 gene expression occurs in devel- pBSKS II (Strategene, La Jolla, CA) at XhoI and HindIII
oping human lung (21). Although HFH-4 recognizes a restriction sites (27). The cDNA sequences for HFH-4,
DNA consensus sequence in these tissues, the in vivo gene HNF-3a, and HNF-3b were in vitro translated in the pres-
targets of HFH-4 are not established (19). The restricted ence of 35[S]-labeled methionine using rabbit reticulocyte
pattern of HFH-4 expression suggests that HFH-4 has a lysates (TNT; Promega, Madison, WI) and the appropriate
specific role in regulation of differentiation of epithelial RNA polymerase (T3 or T7). The appropriate sizes of in
cells in the airway and other tissues. A role in epithelial vitro–translated proteins were confirmed by polyacryla-
cell differentiation is also suggested by recent reports by mide gel electrophoresis (PAGE) and autoradiography.
others and us that targeted deletion of HFH-4 results in an Translated proteins were separated on 7.5% sodium dode-
absence of cilia in the airway (22, 23). cyl sulfate polyacrylamide gels, transferred to nitrocellulose
The cellular heterogeneity of tissues expressing HFH-4 membranes (Hybond-ECL; Amersham, Arlington Heights,
mRNA makes it difficult to determine the specific type of IL), blocked (1 h, room temperature [RT]) in Tris-buff-
cell expressing HFH-4. In turn, this makes identification of ered saline with Tween-20 (Tris, 20 mM; NaCl, 137 mM;
a function for HFH-4 difficult. For example, the proximal Tween-20, 0.1%; pH 7.6) containing nonfat milk (5%) and
airway of the lung contains ciliated, basal, intermediate, incubated (48C, overnight) with anti–HFH-4 antibody (1:500)
mucus, and Clara cells (1, 24), all or some of which might in blocking solution. Primary antibody binding was de-
express HFH-4. Therefore, we developed a polyclonal tected using a chemiluminescence detection system (ECL;
HFH-4 antibody and used this antibody in combination Amersham).
with other epithelial cell markers to immunolocalize
HFH-4 expression. These studies revealed that HFH-4 Immunohistochemistry
protein expression is restricted to ciliated cells and tempo- Developmental stage–specific mouse tissues were ob-
rally related to ciliogenesis. Characterization of expression tained from timed pregnancies determined using the pres-
in developing organs suggests that HFH-4 plays a role in ence of a vaginal plug at E0.5. Human samples were ob-
regulation of genes important for epithelial cell differenti- tained from the Pathology Department at Barnes-Jewish
ation and ciliogenesis. Hospital with permission of the Human Studies Commit-
tee at Washington University (both St. Louis, MO).
Materials and Methods Mouse tissues for immunohistochemical evaluation were
fixed in 10% buffered formalin, embedded in paraffin, and
Antibody Generation sectioned (6 mM thick). Paraffin-embedded sections were
Rat HFH-4 complementary DNA (cDNA) coding for deparaffinized in a D-limonene-based clearing solution
amino acids 1–117 was subcloned downstream and in- (Stephens Scientific, Riverdale, NJ) for 15 min and then
frame with glutathione S-transferase (GST) at the XhoI rehydrated in graded ethanol. Antigen retrieval was then
site of pGEX4T1 (Pharmacia, Uppsala, Sweden) to gener- performed by placing slides containing the sections in a
ate the GST–HFH-4 (1–117) plasmid. The plasmid was plastic container with antigen unmasking solution (Vector
used to express GST–HFH-4 (1–117) in Escherichia coli Laboratories, Burlingame, CA). After boiling for 3 min in
(strain B12), and the fusion protein was purified by affinity a microwave oven, samples were subjected to continued
chromatography using glutathione sepharose beads (Phar- low heat to maintain boiling for two consecutive 5-min pe-
macia) as described previously (25). The eluted protein riods, followed by cooling (1 h) to RT. For peroxidase-
was injected into rabbits with 1 ml of Freund’s complete based antibody detection, endogenous peroxidase was in-
adjuvant. Rabbit serum was harvested and the immuno- activated by incubation in hydrogen peroxide (3%) in
globulin (Ig) G fraction was isolated by precipitation with phosphate-buffered saline (PBS) (5 min, RT). Samples
caprilic acid (26). Polyclonal anti–HFH-4 antibody was af- were washed twice in PBS and incubated (30 min, RT) in a
finity-purified by binding to purified GST–HFH-4 (1–117) blocking solution containing 2% fish gel (Sigma, St. Louis,
protein linked to glutathione sepharose. Antibody concen- MO) in PBS. The samples were then incubated (48C, over-
170 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 21 1999

polyclonal anti–HFH-4 antibody, immunoblot analysis was


performed using lysates from in vitro–translated HFH-4,
HNF-3a, and HNF-3b cDNAs (Figure 1A). In vitro trans-
lation of HFH-4 labeled with 35[S]methionine resulted in a
58-kD product. Immunoblots of in vitro–translated HFH-4
(in the absence of methionine labeling) and control fork-
head proteins incubated with the anti–HFH-4 antibody
demonstrated a specific band at approximately 58 kD in
the HFH-4 sample and no cross-reactivity with control
forkhead proteins (Figure 1B). No bands were detected
when control IgG antibody was substituted for anti–HFH-4
antibody (not shown). These data indicate specific binding
Figure 1. Immunoblot detection of in vitro HFH-4 expression. of the anti–HFH-4 antibody to HFH-4 protein and not to
(A) Lysates containing in vitro–translated cDNA sequences of HNF-3 homologue proteins expressed in lung epithelial
forkhead proteins HNF-3a, HNF-3b, or HFH-4 labeled with cells.
35
[S]methionine and analyzed by PAGE and autoradiography.
(B) Lysates from A of in vitro–translated proteins (in the absence HFH-4 Expression in Adult Lung
of 35[S]methionine) evaluated by immunoblot analysis using poly- To determine whether HFH-4 is expressed in a specific
clonal anti–HFH-4 antibody demonstrate identification of HFH-4 population of epithelial cells in the lung, sections of adult
without cross-reactivity. Protein standards are indicated.
mouse, rat, and human lungs were subjected to immuno-
histochemical analysis with anti–HFH-4 antibody. HFH-4
was expressed in a subpopulation of proximal airway epi-
thelial cells in the trachea, bronchi, and bronchioles, but
night) with control (IgG) antibody or primary antibody in
not in alveolar epithelial cells (Figures 2A and 2D). A sim-
blocking solution. Primary antibodies (and dilutions used)
ilar pattern of staining was seen in adult rat and human
included: rabbit antirat HFH-4 (1:500 dilution); rabbit an-
lungs (not shown). HFH-4 expression was more abundant
tirat HNF-3b (28) (1:500; kindly provided by R. Costa, Uni-
in the nuclei than in the cytoplasm of epithelial cells. The
versity of Illinois, Chicago, IL); rabbit antimouse CCSP (29)
pattern of HFH-4 expression was distinct from expression
(1:500; kindly provided by F. DeMayo, Baylor University,
of HNF-3b that was present in all proximal airway epithe-
Houston, TX); goat antimouse olfactory marker protein
lial cells and in distal airway type II pneumocytes (Figure
(OMP) (30) (1:4,000; kindly provided by F. Margolis, Uni-
2B) as previously shown (6). No staining was seen in sam-
versity of Maryland, Baltimore, MD); and b-tubulin IV
ples incubated with control IgG antibodies (Figure 2C).
(1:250; BioGenex, San Ramon, CA). For light microscopy,
Examination of immunoperoxidase localization of HFH-4
sections were washed and incubated (30 min, RT) with a
by light microscopy revealed that HFH-4 was not detected
biotinylated secondary antibody that was detected with
in endothelium, interstium, or alveolar epithelium (Figure
the avidin-biotin complex using peroxidase or alkaline
2D). Cell types expressing HFH-4 appeared to be ciliated
phosphatase reagents (ABC Elite; Vector Laboratories).
cells and not Clara cells (Figures 2A and 2D), as identified
Sections were counterstained with hematoxylin.
by cell morphology (24). To identify cilia on respiratory
For immunofluorescent localization, slides containing par-
cells, the expression of b-tubulin IV in cilia was used as pre-
affin-embedded samples were prepared as described above
viously described (31, 32). Comparison of b-tubulin IV and
but goat antimouse fluorescein isothiocyanate (FITC)–
HFH-4 expression by immunofluorescence indicated that
labeled (1:200 dilution) or donkey antirabbit indocarbocy-
HFH-4 expression is restricted to ciliated cells (Figure 2E).
anine (Cy3)–labeled (1:800 dilution) secondary antibodies
We also performed immunostaining of mouse lung sections
(both from Jackson ImmunoResearch Laboratories, West
with anti-CCSP and HFH-4 antibodies. Dual localization
Grove, PA) were used. After washing in PBS, slides were
revealed that expression of HFH-4 and CCSP in the mouse
mounted with anti-fade media (Vectashield; Vector Labo-
airway are in two mutually exclusive cell populations (Fig-
ratories) and stored at 2208C. In each experiment, parallel
ure 2F). Thus, HFH-4 is expressed in a specific population
samples were incubated with a control antibody (IgG of
of proximal airway epithelial cells that are ciliated.
appropriate species) and enzymatic or fluorescence detec-
To determine whether HFH-4 may be related to differ-
tion consistently revealed no immunostaining. Photographs
entiation status associated with malignant transformation
of images were electronically digitized by scanning and
of cells derived from the airway epithelium, HFH-4 ex-
composed in Photoshop (Adobe Systems, San Jose, CA).
pression was also evaluated in primary human lung carci-
nomas. Specimens from surgical resections of primary lung
Results
carcinomas, including squamous cell carcinoma (n 5 1),
Anti–HFH-4 Antibody Generation adenocarcinoma (n 5 5), and bronchoalveolar carcinoma
Polyclonal anti–HFH-4 antibody was generated in rabbit (n 5 1), were stained with anti–HFH-4 antibodies. Al-
by immunization with a GST–HFH-4 fusion protein con- though HFH-4 expression was detected in bronchial epi-
taining the N-terminal region of rat HFH-4. This HFH-4 thelial cells in normal areas of lung, HFH-4 expression was
amino acid sequence has high homology (93%) to the hu- not detected in any of the malignant cell types (not shown).
man HFH-4 sequence and does not include the DNA These observations suggest that HFH-4 is expressed spe-
binding domain (17, 21). To evaluate the specificity of the cifically in well-differentiated, normal airway epithelial cells.
Blatt, Yan, Wuerffel, et al.: HFH-4 Expression in Ciliated Cells 171

Figure 2. HFH-4 expression in


adult mouse lung. Immunolocal-
ization of HFH-4 and localization
of HFH-4 with airway epithelial
cell–specific markers. Immunoper-
oxidase staining (brown) demon-
strates (A) HFH-4 expression in a
subpopulation of proximal airway
epithelial cells. (B) HNF-3b ex-
pression in all proximal airway
cells and in type II pneumocytes
(arrows). (C) Control IgG. (D)
Low-magnification view of HFH-4
expression in bronchi (b), and not
in alveolar epithelium (a) or en-
dothelium (v). (E) Immunofluo-
rescent localization of HFH-4 (Cy3)
and b-tubulin IV (FITC; cilia-spe-
cific marker) expression in ciliated
cells. (F) HFH-4 expression (im-
munoperoxidase) and CCSP ex-
pression (alkaline phosphatase; blue) detected in unique populations (F). Sections counterstained with hematoxylin (A–D). Scale bar in
F is 19 mM in A, B, C, and F; 48 mM in D; and 16 mM in E.

HFH-4 Expression in Upper Airway of immotile cilia (33). The neuroepithelial olfactory cells
Nose and Paranasal Sinuses were identified by the expression of OMP in the superior
Epithelial cells that line the upper respiratory tract (nose regions of the nasal cavity, the vomernasal organ, and the
and paranasal sinuses) include ciliated, squamous, and neuronal cells within the vomer bone of the nasal septum
secretory cells that are histologically similar to epithelial (Figure 3A) (30, 34). The sensory, nonmotile cilia of the
cells of the bronchi (33) but have not been previously eval- olfactory epithelium, which have a structure different from
uated for HFH-4 expression by in situ hybridization. We the motile cilia of the respiratory epithelium, were not de-
found that HFH-4 protein is expressed in the ciliated cells tected by b-tubulin IV but were identified by light micros-
of the nose and paranasal sinuses in a predominant nu- copy (35, 36). Simultaneous anti–HFH-4 and anti-OMP
clear pattern, similar to HFH-4 expression in ciliated epi- immunostaining demonstrated that olfactory epithelial
thelial cells of the lung (Figures 3A–3C). cells do not express HFH-4 (Figures 3A and 3C). Hence,
The nose also contains neuroepithelial cells with bipo- HFH-4 expression is associated with respiratory cells with
lar neurons that terminate in an olfactory knob linked to motile-type cilia that contain nine outer doublet microtu-

Figure 3. HFH-4 expression in nose


and paranasal sinuses. Immunoper-
oxidase or immunofluorescent de-
tection of HFH-4 and other epithe-
lial cell markers. (A) The distribution
of HFH-4 expression (immunoper-
oxidase, brown) and OMP (alkaline
phosphatase, blue) in mouse nose
and paranasal sinuses. (Boxed ar-
eas indicate locations of higher
magnification in B and serial sec-
tion in C). (B) Immunoperoxidase
detection of HFH-4 expression in
ciliated epithelial cells of the nose.
(C) Immunofluorescent detection
of HFH-4 (Cy3) and b-tubulin IV
(FITC) expression in ciliated epi-
thelial cells of nose but not in olfac-
tory epithelial cells that contain sen-
sory cilia (not detected by b-tubulin
IV) and express OMP ( arrow-
heads). (D) HFH-4 expression in ciliated epithelial cells of a human nasal polyp. (E) HFH-4 expression in nonciliated nasal epithelial
cells of an E15.5 mouse. Sections were counterstained with hematoxylin (A, B, D, and E). Scale bar in E is 140 mM in A, 6 mM in B, 14
mM in C, 19 mM in D, and 70 mM in E.
172 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 21 1999

Figure 4. HFH-4 expression in mouse


oviduct and brain. (A) HFH-4 ex-
pression (immunoperoxidase, brown)
in epithelial cells of oviduct. (B) De-
tail of oviduct from A demonstrat-
ing HFH-4 expression in ciliated but
not nonciliated cells. (C) Immuno-
fluorescent localization of HFH-4
(Cy3) and b-tubulin IV (FITC) ex-
pression in oviduct epithelial cells
(asterisks indicate lumen). (D) Im-
munoperoxidase detection of HFH-
4 expression in brain choroid plexus
at E15.5. (E) HFH-4 expression in
adult brain ependymal cells of the
lateral ventricle. (F) Immunofluo-
rescent localization of HFH-4 and
b-tubulin IV expression in ependy-
mal cells. Sections were counter-
stained with hematoxylin (A, B, D,
and E). Scale bar in F is 19 mM in A;
6 mM in B; 18 mM in C, D, and F;
and 13 mM in E.

bules with dynein arms and an inner microtubule pair (9 1 HFH-4 expression in nasal epithelial cells of the mouse
2), but not with sensory cilia that contain only the nine was detected at E15.5 (Figure 3E) but cilia were not de-
outer doublets (without dynein arms or the inner pair; 9 1 tected on these cells when examined by light microscopy.
0) (2, 36–38). This indicates a further restriction of HFH-4 However, at birth, the epithelial cells of the nose and para-
expression. nasal sinuses were ciliated as determined by expression of
The epithelium of human nasal polyps obtained from b-tubulin IV (not shown). These observations indicate
patients with advanced chronic sinusitis (Figure 3D) is his- that HFH-4 can be present in nonciliated epithelial cells,
tologically similar to normal upper airway respiratory epi- where expression may precede the appearance of cilia on
thelium (39). Immunostaining of nasal polyps with anti– these cells.
HFH-4 antibody demonstrated that the ciliated epithelial
cells of the nasal polyp also express HFH-4 (Figure 3D). HFH-4 Expression in Extrarespiratory Tissues
Thus, although nasal polyps represent abnormally prolif- Other tissues shown to express HFH-4 mRNA include
erating tissue, the pattern of HFH-4 expression is similar oviduct, choroid plexus, and brain ependymal cells (17,
to that in normally differentiated upper airway. 19). To identify cells in these tissues expressing HFH-4

Figure 5. HFH-4 expression in de-


veloping mouse lung. Immunoper-
oxidase or immunofluorescent de-
tection of HFH-4 and other epithelial
cell markers. (A) E14.5 lung evalu-
ated by immunoperoxidase (brown)
for HFH-4 shows absent expression.
(B) E14.5 serial section at E14.5 im-
munostained for HNF-3b expres-
sion. (C) HFH-4 expression at E15.5.
(D) E15.5 lung (serial section, higher
magnification) demonstrating low
levels of b-tubulin IV (FITC) ex-
pression in some HFH-4 (Cy3)–ex-
pressing cells. (E) HFH-4 expres-
sion in newborn (NB) lung. (F) NB
lung (serial section, higher magnifi-
cation) demonstrating more abun-
dant b-tubulin IV expression on
HFH-4 expressing cells. Sections
were counterstained with hematox-
ylin (A–C and E). Scale bar in F is
48 mM in A–C and E, and 24 mM in
D and F.
Blatt, Yan, Wuerffel, et al.: HFH-4 Expression in Ciliated Cells 173

protein, immunostaining with the anti–HFH-4 antibody lin IV (Figure 5F). Thus, stage-specific evaluation of
was also performed. The oviduct, the most proximal re- HFH-4 and b-tubulin IV expression in the developing lung
gion of the fallopian tube, is lined with epithelial cells con- suggests that HFH-4 expression is temporally associated
taining motile cilia (37). HFH-4 is highly expressed in with ciliogenesis.
proximal oviduct within almost every epithelial cell lining
the lumen (Figure 4A). In distal oviduct there are fewer HFH-4 Expression during Spermiogenesis
ciliated cells, and in these regions HFH-4 expression is Spermatids develop flagella containing microtubules ar-
found in fewer cells (not shown) (37). As in the respiratory ranged in a 9 1 2 pattern, identical to that of motile cilia of
tract, HFH-4 is expressed specifically in ciliated cells and the airway (37). Sperm maturation occurs in a wavelike
in a predominantly nuclear pattern (Figure 4B). Secretory, fashion within the seminiferous tubules of the testis where
nonciliated cells of the oviduct do not express HFH-4 (Fig- spermatocytes mature to become spermatids that develop
ure 4B). Dual localization of HFH-4 and b-tubulin IV con- flagella (spermiogenesis) and are released as spermatozoa.
firmed the relationship between HFH-4 expression and The cycles of sperm maturation were identified in cross
the ciliated cell phenotype (Figure 4C). A similar pattern sections of tubules using the 14-stage system of Leblond
of HFH-4 expression is present in human oviduct (not and Clermont based on the morphologic appearance of
shown). germ cells (41). HFH-4 was previously shown by in situ hy-
The motile cilia on the cells of the choroid plexus and bridization to be expressed in spermatids during stages I to
ependymal cells that line the ventricles of the brain are VI within the seminiferous tubules of the testis (17).
thought to assist in local mixing of cerebral spinal fluid Using anti–HFH-4 antibodies, a stage-dependent pat-
(40). In the developing choroid plexus, HFH-4 expression tern of nuclear and cytoplasmic expression of HFH-4 was
is present at E15.5 (Figure 4D). The ependymal cells lining detected during cycles of sperm maturation. Before the
the ventricles of the brain also express HFH-4 in a pre- maturation of spermatids and formation of flagella, HFH-4
dominantly nuclear pattern (Figure 4E). Dual staining of was weakly expressed in the cytoplasm of spermatocytes
brain tissues to detect HFH-4 and b-tubulin IV confirmed (Figure 6A). After meiosis, spermatids expressed abun-
that HFH-4 expressing cells are ciliated (Figure 4F). Thus, dant HFH-4 in the nucleus during stages III through VI
HFH-4 expression is present in cells that have motile cilia (Figures 6B and 6C). Early spermatids with nuclear HFH-4
in both respiratory and nonrespiratory tissues. also expressed cytoplasmic b-tubulin IV as the flagella was
HFH-4 expression was also evaluated by immunohis- initially formed (Figure 6F). (At this stage, b-tubulin IV
tochemistry in adult mouse tissues that do not contain mo- was also expressed in the manchette in the innermost layer
tile cilia. HFH-4 expression is not detected in sections from of more mature spermatids, present simultaneously with
heart, liver, spleen, kidney, skeletal muscle, skin, stomach, the new spermatids [Figure 6F] and at stages XI though
small intestine, large intestine, and bladder (data not shown). XIII [Figure 6H] as previously described [42].) After stage
This further confirms the relationship of HFH-4 to organs VII, HFH-4 expression in spermatids was extinguished
containing cells with motile cilia. while development of the flagella continued (Figure D).
b-tubulin IV expression in flagella of mature spermatozoa
HFH-4 Expression in Developing Lung was present in the central lumen at stage VII, immediately
On the basis of the observation that HFH-4 expression before spermatozoa release (Figure 6G). At these stages,
was restricted to ciliated epithelial cells, we next examined HFH-4 expression in the nucleus of spermatids was no
the temporal relationship between HFH-4 expression and longer present, but was expressed in the cytoplasm of sper-
the appearance of cilia during airway epithelial cell differ- matocytes (Figures 6D, 6E, 6G, and 6H). These observa-
entiation in developing lung. Previously, in situ hybridiza- tions demonstrate that HFH-4 expression in the nuclei of
tion marked the onset of HFH-4 mRNA in the airway epi- early spermatids precedes the complete formation of fla-
thelium at the late pseudoglandular stage (E14.5) in the gella in mature spermatids. This suggests that nuclear ex-
mouse lung (17). With anti–HFH-4 antibody, HFH-4 ex- pression of HFH-4 is associated with early production of
pression was not detected at E14.5 (Figure 5A). In con- flagella but that there is no persistent expression of HFH-4
trast, in a serial section of E14.5 lung, abundant HNF-3b in maturing spermatids during later flagella development.
was expressed in the airway epithelial cells (Figure 5B) as
previously demonstrated (6). At E14.5, cilia were not
present, as indicated by absent expression of b-tubulin IV Discussion
in airway epithelial cells (not shown). One day later, at This study demonstrates that forkhead/winged-helix tran-
E15.5, HFH-4 was expressed in approximately half of the scription factor HFH-4 expression is localized to ciliated
proximal airway epithelial cells (Figure 5C). At E15.5 epithelial cells and is temporally related to ciliogenesis. A
b-tubulin IV expression was first detected, restricted to a role for HFH-4 in ciliogenesis is demonstrated by the tem-
small number of airway epithelial cells that express HFH-4 poral relationship of HFH-4 to the appearance of cilia in
(Figure 5D). Tracking HFH-4 and b-tubulin IV expression developing lung and flagella during spermiogenesis. Cili-
in the developing airway epithelial cells from E17.5 to ated epithelial cells are thought to arise in the large airway
E18.5 revealed that the number of HFH-4–expressing cells from undifferentiated columnar cells and basal cells and in
that also express b-tubulin IV gradually increased. At bronchioles from undifferentiated cells and Clara cells, but
birth, HFH-4 was present throughout the proximal airway molecular markers to identify progenitor cells have not
in a pattern similar to adult lung (Figure 5E) and now al- been identified (43, 44). Ciliogenesis during lung develop-
most every cell that expresses HFH-4 also expresses b-tubu- ment has previously been identified by b-tubulin IV ex-
174 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 21 1999

for HFH-4 in ciliogenesis. This role is substantiated by the


recent observations by others and us that targeted deletion
of HFH-4 in the mouse results in an absence of cilia in the
airway (22, 23). Thus, HFH-4 is the first reported tran-
scription factor expressed specifically in ciliated cells and
related to ciliogenesis. A role for HFH-4 in a fundamental
process such as ciliogenesis is consistent with the central
biologic functions demonstrated for other winged-helix/
forkhead proteins (11, 13, 14, 46).
The characterization of HFH-4 protein expression ex-
tends the previous observations of HFH-4 mRNA in in
situ hybridization (17, 19). First, antibody localization of
HFH-4 demonstrates that expression is specifically within
the epithelial cells of the proximal airway, upper respira-
tory tract, choroid plexus, ependymal cells, oviduct, and
testis. Second, among epithelial cells expressing HFH-4
mRNA, HFH-4 protein expression is localized specifically
to ciliated cells. HFH-4 also has a nuclear pattern of ex-
pression in these cells that could not be determined by in
situ hybridization. Third, localization of HFH-4 expression
with other epithelial cell–specific markers demonstrates
the temporal relationship of HFH-4 expression to cell dif-
ferentiation toward the ciliated cell phenotype in the air-
way. And fourth, HFH-4 protein localization uncovers a
complex pattern of cytoplasmic and nuclear HFH-4 ex-
pression in the cells of the seminiferous tubules of the tes-
tis, strongly suggesting a regulated role in spermiogenesis
coincident with the development of flagella—a structural
equivalent of the cilia in sperm.
The pattern of HFH-4 protein expression in sperm mat-
Figure 6. Expression of HFH-4 in adult mouse testis. Localiza- uration was more complex than that demonstrated by in
tion of HFH-4 expression by immunoperoxidase (brown; A–E)
situ hybridization. HFH-4 mRNA expression in the testis
or immunofluorescence (Cy3; F–H) and b-tubulin IV (FITC; F–
H) to detect development of flagella in spermatids within semi-
was detected in the earliest stages of spermatid maturation
niferous tubules. (A) HFH-4 is weakly expressed in the cytoplasm (stages I through VI), when HFH-4 protein was also de-
of secondary spermatocytes, but not expressed in spermatids (in- tected in the nucleus. However, HFH-4 protein was also
nermost cell layer, stage I) during early maturation. (B) HFH-4 present in the cytoplasm of spermatocytes (stages VII
expression in the nucleus of spermatids at stages III through IV. through XIV) before differentiation into spermatids. The
(C) Less-abundant HFH-4 expression in nucleus of spermatids at presence of HFH-4 protein in the cytoplasm of spermato-
stages V to VI. (D) Absent HFH-4 expression in spermatids (in- cytes is not consistent with genetic analysis showing absent
ner layer of cells), now present in the cytoplasm of spermatocytes HFH-4 mRNA in the testis of mutant mice that have de-
at stages VII to VIII. (E) Absent HFH-4 expression in sperma- fects in meiosis (17, 18). This apparent inconsistency may
tids and weaker cytoplasmic HFH-4 expression in spermatocytes
be accounted for by protein sharing made possible by in-
at stages XI through XIII. (F) Immunofluorescent localization of
HFH-4 and b-tubulin IV (FITC) expression demonstrates HFH-
tercellular bridges between germ cells and demonstrated
4 expression in the nucleus of early spermatids associated with for other DNA-binding proteins (47). The regulation of
b-tubulin IV (FITC) expression in the cytoplasm (arrrowheads). nuclear localization of HFH-4 may be through a putative
(G) b-tubulin IV expression in flagella (tails massed at the center nuclear localization signal (KKRRLPPVH) that is con-
of the tubule) of mature spermatozoa just before release and ab- served within the DNA-binding domain of other forkhead
sent HFH-4 expression in spermatozoa and spermatids at stages proteins (48). The transient nuclear expression of HFH-4
VII to VIII. (H) Absent HFH-4 expression in maturing sperma- in spermiogenesis is also in contrast to the persistent nu-
tids with prominent b-tubulin IV expression in the manchette clear expression of HFH-4 in ciliated epithelial cells. Per-
(man). HFH-4 is expressed in the cytoplasm of spermatocytes at sistent nuclear expression may be important for ongoing
stages XI through XIII. Sections counterstained with hematoxy-
cell responses or for maintenance of ciliogenesis in cells
lin (A–E). The stage of spermiogenesis in the individual tubules
is indicated. Scale bar in E is 46 mm in A, B, C, D, and E; and 29
undergoing continuous low-level loss of cilia—a process
mm in F, G, and H. not necessary in sperm. Shared and unique functions of
HFH-4 in spermiogenesis and ciliogenesis remain to be
elucidated.
The in vivo molecular targets for HFH-4 activation are
pression at E19 in mice, and by electron microscopy at E20 unknown. Although DNA binding sequences for HFH-4
in rats and at gestational wk 12 to 18 in humans (32, 38, have been identified, these are seemingly not related to
45). Thus, cilia appearance consistently follows HFH-4 ex- proteins involved in ciliogenesis (19). HFH-4 expression is
pression in mice and humans (21), consistent with a role most directly related to cells with motile cilia rather than
Blatt, Yan, Wuerffel, et al.: HFH-4 Expression in Ciliated Cells 175

sensory cilia (36). Sensory or nonmotile cilia lack dynein plantation expression patterns indicate a role for the mouse forkhead/
HNF-3 a, b and g genes in determination of the definitive endoderm,
arms as well as the central microtubule pair (central appa- chordamesoderm and neuroectoderm. Development 119:567–578.
ratus) that interacts with the dynein arms, suggesting that 16. Clevidence, D. E., D. G. Overdier, W. Tao, Q. Xiabing, L. Pani, E. Lai, and
the genes coding for central apparatus proteins may be R. H. Costa. 1993. Identification of nine tissue-specific transcription fac-
tors of the hepatocyte nuclear factor 3/forkhead DNA-binding-domain
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proteins comprising the central apparatus have been iso- 17. Hackett, B. P., S. L. Brody, M. Liang, I. D. Zeitz, L. A. Bruns, and J. D. Git-
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Acknowledgments: The authors thank their colleagues at Washington Univer- 22. Chen, J., H. J. Knowles, J. L. Hebert, and B. P. Hackett. 1998. Mutation of
sity: R. Mecham (assistance with antibody production and purification), M. the mouse hepatocyte nuclear factor/forkhead homologue 4 gene results in
Botney (for providing human lung tissues), and D. C. Look (for helpful discus- an absence of cilia and random left-right asymmetry. J. Clin. Invest. 102:
sions); J. Darnell, Jr. (Rockefeller University) for HNF-3a and HNF-3b cDNA 1077–1082.
plasmids; and for providing antibodies, R. Costa (University of Illinois, Chi- 23. Brody, S. L., E. N. Blatt, X. H. Yan, M. K. Wuerfell, S. D. Shapiro, and D. L.
cago), F. DeMayo (Baylor University), and F. Margolis (University of Mary- Hamilos. 1999. Relationship of transcription factor HFH-4 to ciliogenesis
land). This work was supported, in part, by a grant to one author (E.N.B) from in airway epithelial cells. Am. J. Respir. Crit. Care Med. (Abstr.) (In press)
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