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INTRODUCTION |
Development of the mammalian palate is a complex process involving
the migration and differentiation of neural crest derivatives in the
cranial region of the embryo. Palatal structures arise from the first
branchial arches, paired swellings of neural crest mesenchyme. The
formation of the secondary palate first becomes evident on gestational
day (gd)1 12 in the mouse
embryo, with the appearance of the palatal shelves, bilateral growths
of tissue extending vertically from the roof of the oronasal cavity
along either side of the tongue. The palatal shelves continue to grow
in this orientation until gd14, when they become reoriented to lie
horizontally above the tongue in a process known as elevation.
Following elevation, the opposing shelves contact each other and fuse
to form a continuous structure separating the oral and nasal cavities.
Disruption of the growth and morphological differentiation of these
facial primordia through teratological insult, abnormal gene activity
or regulation, or a combination of these factors can lead to palatal
malformations, which are a relatively common birth defect in humans.
(1, 2).
In an effort to identify genes with possible roles in regulating facial
development, we have used the differential display method (3, 4) to
examine gene expression in the embryonic murine palate. This analysis
has led to the identification of a novel gene transcript, expressed
peripherally in the palate and quite strongly in the presumptive nasal
epithelium of the embryo, with a striking spatially restricted pattern
of expression. This transcript also shows strong expression in the
adult murine lung. In this study, we describe the identification,
expression pattern, and sequence of this novel gene transcript, which
we refer to as plunc (palate, lung,
and nasal epithelium clone).
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EXPERIMENTAL PROCEDURES |
Animals--
Pregnant females of mouse strain ICR (Ace Animals,
Horsham, PA) were killed by cervical dislocation on gd12-14, which
represents the period of embryonic palatal morphogenesis. Embryos were
removed from the mothers, and palatal tissue was harvested by
dissection. Total RNA was prepared by the acid guanidinium
thiocyanate-phenol/chloroform extraction method of Chomczynski and
Sacchi (5) and quantitated by measurement of optical density at 260 nm.
Reverse Transcription and Differential mRNA
Display--
Differential display was performed essentially according
to the method described by Liang and Pardee (3) and co-workers (4).
Briefly, 2 µg of total RNA from gd13 and gd14 embryonic palate
tissues was subjected to reverse transcription using an anchored
oligo(dT) primer (dT12-GC) to prime the reaction. Following reverse transcription, 2 µl of each reaction was subjected to PCR
using the same anchored oligo(dT) primer and an arbitrary 10-base
olignucleotide (5'-CTGATCCATG-3'). 10 µCi of [33P]dCTP
(NEN Life Science Products) was included with the PCR reactions to
allow for subsequent visualization of the amplification products by
autoradiography. PCR was performed using the following profile: 94 °C for 2 min (1 cycle); 94 °C for 30 s, 40 °C for 2 min, and 72 °C for 30 s (40 cycles); and 72 °C for 5 min (1 cycle). PCR products were then subjected to electrophoresis on a 6%
nondenaturing sequencing gel. The gel was dried onto Whatman 3MM paper
under vacuum and subjected to autoradiography. As a control for
possible DNA contamination, a sample of RNA from gd13 mandible prepared in the same manner as the palate RNA was subjected to PCR without prior
reverse transcription. Autoradiographs were examined for bands present
exclusively in either gd13 or gd14 palate samples. To be considered as
a candidate for further analysis, bands had to be present in both of
the duplicate PCR lanes for each sample and could not correspond to any
of the bands generated by PCR of the nonreverse transcribed mandible
RNA, because any bands in this sample must be derived from
contaminating DNA.
Elution of Amplified Fragments from Gels and
Reamplification--
Bands representing differentially expressed
transcripts were cut from the dried gels and transferred to 0.5-ml
polypropylene tubes. The gel slices were rehydrated for 10 min in 100 µl of water and boiled for 15 min. The supernatants were transferred to clean 0.5-ml tubes, following which the DNA was precipitated by the
addition of 8 µl of 3 M potassium acetate (pH 5.2), 2.5 µl of 20 mg/ml glycogen, and 300 µl of absolute ethanol. After an
overnight incubation at
20 °C, samples were centrifuged for 20 min
in a microcentrifuge at 4 °C to collect the eluted DNA. The eluted
bands were then reamplified using PCR to obtain sufficient material for
subcloning, using the same primers and amplification profile as the
initial reaction. No 33P-labeled nucleotide was included in
the reamplification reaction mixtures.
Subcloning of Reamplified cDNA Fragments--
The
reamplified fragments were subcloned into the pGEM-T vector (Promega)
and transformed into competent JM109 Escherichia coli cells.
Recombinant plasmids were initially identified by blue/white selection.
The presence of inserts in these plasmids was confirmed by removal of
the insert by digestion with NcoI and PstI.
Plasmids that contained inserts were subjected to sequencing, which was
performed by the Thomas Jefferson University DNA Synthesis and
Sequencing facility. Sequence analysis was performed using the BLAST
sequence analysis program (6).
Northern Blot Analysis--
Total RNA from embryonic palate and
brain was subjected to electrophoresis on 1% agarose/2.2 M
formaldehyde denaturing gels. Following electrophoresis the gels were
soaked briefly in water and transferred to nitrocellulose (Schleicher & Schuell) by capillary action using 10× SSC to effect transfer. RNA was
cross-linked to nitrocellulose filters using a Stratagene ultraviolet
cross-linker, and the blots were stored at
20 °C until ready for
use. For RNA analysis of adult tissues, a commercial multiple tissue
blot (CLONTECH number 7762-1) was probed according
to the manufacturer's instructions.
Library Screening--
A mouse heart cDNA library (
ZAP II
adult mouse heart cDNA library, Stratagene, La Jolla, CA) was
screened under high stringency conditions using the cDNA probe
isolated from the differential display gel. Positive plaques were
picked from plates and subjected to secondary and tertiary screenings
to ensure purity of the clones obtained from the screening. Positive
clones were sequenced by the Thomas Jefferson University DNA Synthesis
and Sequencing Facility.
In Situ Hybridization of Mouse Tissues--
Harvested mouse
embryos (gd13-15) and adult mouse tissues were fixed in 4%
paraformaldehyde/phosphate-buffered saline (pH 7.4) at 4 °C
overnight and processed under RNase-free conditions for standard
paraffin embedding. Serial coronal sections of embryonic heads were
used to survey plunc mRNAs expressed during craniofacial development. To detect expression elsewhere during mouse development, embryo bodies were sectioned in both transverse and saggital
orientations. Finally, after our Northern analysis detected high levels
of plunc message in the adult mouse lung, the entire
thoracic contents of two pregnant female mice were dissected out, fixed
in toto, and completely sectioned for in situ analysis.
All sections were processed for RNA hybridization and emulsion
autoradiography according to the detailed protocol of Wawersik and
Epstein (7). Two overlapping plunc clones (6-1 and 16-3), representing three-quarter-length and full-length cDNAs,
respectively, were used to generate sense and antisense
35S-labeled riboprobes. Each probe was synthesized at
1-2 × 106 cpm/µl and diluted to apply 3-5 × 106 cpm/slide in 50 µl of hybridization buffer. After
stringent washing, RNase treatment, dehydration, and preliminary
autoradiography, the slides were dipped in emulsion and exposed in
darkness at 4 °C for 5-7 days before developing. Developed slides
were counterstained with hematoxylin and eosin and viewed using
sequential bright field and dark field illumination on a Jenaval
microscope. All photographs were taken on Ektachrome 64T or 160T color
slide film and scanned into Adobe Photoshop 3.0TM.
For analysis of plunc expression in the adult nasopharynx,
four heads from 2-month-old mice were perfused with
paraformaldehyde/phosphate-buffered saline, decalcified in 0.5 M EDTA for 2-3 weeks at 4 °C, and sliced coronally with
a clean razor blade for preparation as whole mounts. Whole mount
in situ hybridization was performed with digoxigenin-labeled riboprobes according to Wilkinson (8).
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RESULTS |
Differential Display Analysis of Gene Expression in the
Palate--
Total RNA was prepared from palatal tissue dissected from
gd13 (prior to palatal shelf elevation) and gd14 (following shelf elevation and fusion) mouse embryos of strain ICR. A portion of a
differential display gel comparing gd13 and gd14 RNA is shown in Fig.
1. Comparison of banding patterns
revealed several differentially expressed genes. At least two bands
were present in the gd13 lanes (Fig. 1, bands 4 and
5) that were not observed in the gd14 lanes, whereas three
bands were present on gd14 (Fig. 1, bands 1,
2, and 3) and not gd13. None of these bands
appeared in control reactions not subjected to reverse transcription.
These bands therefore represent cDNA fragments of up to five genes
that show developmental regulation of expression in embryonic palatal
tissue between gd13 and gd14. These bands were eluted from the
differential display gel and subjected to a second round of PCR, using
the same primers as before. Of the five bands identified on the
original gel, two (Fig. 1, bands 1 and 2) were
reamplified by PCR. The band 1 gene was successfully subcloned into the
plasmid vector pGEM-T (Promega).

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Fig. 1.
Differential display of mouse embryonic
palate RNA. Duplicate PCR reactions were prepared from each
reverse transcription reaction and were run in adjacent gel lanes. A
gd13 mandible RNA sample prepared in exactly the same manner as the
palate RNA was subjected to PCR without reverse transcription as a
control (lanes 1 and 2). Lanes 3 and
4 represent gd13 palate RNA samples; lanes 5 and
6 represent gd14 palate RNA. Bands representing
differentially expressed transcripts are marked with
arrows.
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Confirmation of Differential Expression in the Developing
Palate--
We confirmed the differential expression of the band 1 gene in the developing palate by Northern blot analysis (Fig.
2A). A radiolabeled band 1 cDNA probe hybridized to a single mRNA that is only weakly
expressed in gd13 palate tissue but is strongly expressed in gd14
palate. This message continues to be expressed strongly in the palate
on gd15 and gd16 (data not shown). The mobility of this mRNA was
approximately that of an endogenous GAPDH transcript (9), allowing us
to estimate the size of the band 1 transcript to be approximately 1.2 kilobases.

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Fig. 2.
Northern analysis of band 1 gene expression
in embryonic and adult mouse tissues. A, the
upper panel shows equal amounts (20 µg) of total RNA
isolated from embryonic gd13 palate (lane 1) gd13 brain
(lane 2), gd14 brain (lane 3), and gd14 palate
(lane 4) probed with the band 1 cDNA probe as described
under "Experimental Procedures." The probe hybridizes to an
mRNA species approximately 1.2 kilobases in length, based on its
mobility compared with a GAPDH loading control (lower panel,
same blot reprobed with a murine GAPDH probe). B, the
upper panel shows a commercially prepared blot representing
a variety of mRNAs from adult mouse tissues (~2 µg
mRNA/lane). A 32P-labeled plunc cDNA
probe (clone 6-1) shows a single band hybridizing in the lung lane at
approximately 1.2 kilobases (kb). The lower panel
shows the same blot hybridized with a murine -actin control probe.
Note that the size of the -actin transcripts depends on the tissue
type, and some tissues express more than one form.
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To determine whether band 1 gene expression was specific to the palate
or might be expressed elsewhere in the embryonic head, we included RNA
samples prepared from gd13 and gd14 total brain tissue (Fig.
2A). The band 1 probe did not hybridize to any mRNA species expressed in gd13 or gd14 brain RNA. Other embryonic RNAs were
not examined.
Isolation of cDNA Clones--
Preliminary Northern blot
examination of band 1 expression in the adult mouse indicated high
levels of expression in the heart (data not shown). Although subsequent
Northern blots failed to confirm this observation, we obtained several
homologous, overlapping clones containing the band 1 sequence from
screening a mouse heart cDNA library. Initial screening of the
library was accomplished using the band 1 probe isolated from the
differential display gel. Two positive clones were obtained from this
screen (Fig. 3). These clones were 851 base pairs (clone 6-1) and 783 base pairs (clone 14-1) in length and
covered the entire band 1 sequence as well as several hundred bases of
additional 5' sequence. Because this still left a discrepancy between
the length of the cDNA sequence and the apparent length of the
mRNA observed on the Northern blots, we performed a second
screening of the cDNA library using clone 6-1 as a probe. This
screening yielded four more positive clones, including one (16-3)
containing an open reading frame that we believe represents the
full-length coding sequence (Fig. 3). Overall, the cDNAs identified
by library screening comprise a total overlapping sequence of 1113 base
pairs, which is in good agreement with the length of the embryonic
mRNA species observed by Northern blotting.

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Fig. 3.
Schematic representation of the
plunc cDNA showing the open reading frame region,
the putative 5'- and 3'-untranslated regions (UTR),
and the polyadenylated region, as well as representations of the
overlapping clones obtained from library screening to yield the
full-length plunc cDNA sequence. Solid
lines indicate portions of the clones for which only one strand
was sequenced; boxes show portions for which double stranded
sequence information was obtained.
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Expression in Adult Tissues--
We repeated our Northern analysis
of adult mouse tissues using a commercially prepared multiple tissue
blot (see "Experimental Procedures"). A 32P-labeled
cDNA probe from the clone 6-1 hybridized to a single mRNA
species approximately 1.2 kilobases in length expressed in the adult
lung only (Fig. 2B). No other tissues examined showed expression of this gene. Based on the embryonic and adult expression patterns, we gave the gene represented by our clones the working name
of plunc (for palate and lung
clone).
Expression Pattern during Mouse Embryogenesis--
Given the
temporal expression pattern suggested by the differential display and
the embryonic Northern blot, we performed in situ
hybridization on several gd14 embryos (Fig.
4). Antisense RNA probes from two
plunc clones (6-1 and 16-3) produced identical hybridization
patterns. In the embryonic head at this stage plunc is
exclusively expressed in parts of the presumptive nasal epithelium, including the lateralmost epithelium on the dorsal aspect of the palatal shelves. In the embryonic body, very weak expression was observed in left and right lobes of the developing thymus. No other
embryonic tissues showed any tendency to hybridize with the antisense
probes. Sections probed with either of the sense transcripts gave no
detectable signals.

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Fig. 4.
In situ hybridization of embryonic
mouse tissues with 35S-labeled plunc
riboprobes. Paired images show bright field
(hematoxylin-eosin stain, left) and dark field
(right) views of coronal sections. A,
B, and D show bright field and dark field views
of the same tissue section; C shows similar sections from
different embryos. All specimens are gd14 littermates. The order of
images (A-D) mimics an antero-posterior series of slices
through the embryonic head. A, plunc signal
appears cranially in the ventrolateral folds of the nasal conchae and
the lateral sides of the nasal columella. B,
plunc is also expressed in the nasal turbinates
(top) and on the lateral nasal walls just dorsal to the
palatal shelves. In this embryo one palatal shelf is elevated, whereas
the other is still ventrally directed. Note plunc expression
in the "corner" where the elevated palatal shelf makes a 90-degree
angle with the lateral nasal wall. C, more posteriorly,
expression is found in discrete segments along the lateral nasal walls,
the mid-lateral part of the nasal columella, and the ventrolateral
extremities of the columella. In this embryo the palatal shelves are
fully elevated and fused. D, plunc is strongly
expressed where the lateral nasal walls fuse with the ventrolateral
extremities of the nasal columella, forming pared sinuses above
(s) and a common nasal passage below. np, common
nasal passage; ns, nasal septum; *, palatal
shelf; s, sinus cavity; t, nasal turbinate.
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Although the developing nasal epithelium is continuous,
plunc expression is not. This mRNA species showed
multiple segments of strong signal in the presumptive nasal epithelium,
separated by gaps where only background levels were detected. These
gaps were complex but symmetrical, appearing in the same pattern on each side of the nasal septum. Although the exact configuration of the
signal varied depending on the level of section, we observed a
consistent expression pattern within our set of embryos
(n = 6).
Proceeding from anterior to posterior through coronal sections of the
embryonic head (gd14), plunc is first expressed on the lateral walls of the nasal columella and the ventro-lateral corners of
the nasal conchae (Fig. 4A). More posteriorly, these corner patches of expression come to lie on the dorso-lateral presumptive nasal epithelium of the palatal shelves (Fig. 4B). At the
same level, another region of plunc expression was detected
dorsally in the epithelium of the developing nasal turbinates. Finally, cells expressing plunc mRNA were observed at the
ventro-lateral extremities of the nasal columella (Fig. 4C).
Thus at least four distinct areas of plunc expression (two
on the nasal columella, one on the nasal turbinates, and one on the
lateral nasal walls) are found on each side of the nasal septum during
this developmental period. The intervening epithelial areas
consistently showed no detectable signal.
Although plunc expression is limited to the dorso-lateral
epithelium of the developing palatal shelves and is not present at the
midline junction, plunc transcripts appear at the site of
another important craniofacial fusion. Anteriorly in the face, the
ventral part of the nasal columella contacts the elevated palatal
shelves to establish the secondary palate and two paired nasal
cavities. More posteriorly, however, the columella is free of the
secondary palate and fuses instead with the lateral nasal walls,
forming a common nasal passage below and two ethmoidal sinuses above.
In sections of this posterior region, we observed plunc
transcripts in both the lateral nasal wall and the ventral corners of
the nasal columella as these areas come together (Fig. 4D).
The bands of plunc expression eventually oppose each other and merge as the adjacent epithelia fuse. After fusion,
plunc signal is absent from the sinus cavities but continues
to be expressed in two small spots on the roof of the common nasal
passage (not shown).
Expression Pattern in Adult Tissues--
Expression of
plunc in the mature mouse nasal epithelium was confirmed by
whole mount in situ hybridization with an antisense probe
from the 6-1 clone. plunc signal in the mature nasal
structures parallels that of the gd14 embryo, consisting of discrete
epithelial bands on the exposed surfaces of the nasal columella,
turbinates, and common nasal passage (Fig.
5).

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Fig. 5.
Whole mount in situ
hybridization of adult mouse tissues with digoxigenin-labeled
plunc riboprobes. A, coronal slice
through the anterior nasal cavities. plunc expression
appears as dark bands on the epithelium of the nasal septum
(ns) and nasal turbinates (nt). B, a
more posterior slice showing plunc expression restricted to
a subset of the epithelium in the ventral part of the nasal
turbinates.
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Hybridization of 35S-labeled probes to adult mouse thoracic
sections also confirmed strong plunc expression in the
murine respiratory passages and lungs. Unlike the "broken" pattern
in the embryonic nasal passages, plunc is uniformly
expressed in the entire lining of the adult trachea (Fig.
6A). This continuous
expression persists all the way down the tracheal tube and past its
division into left and right bronchial passages, each of which shows an
unbroken ring of plunc-expressing cells (Fig.
6B). The signal becomes abruptly weaker as these main
bronchial passages branch again and ramify throughout the lung lobes
(Fig. 6C). In all areas of expression plunc
transcripts were found exclusively in the outermost layer of epithelial
cells (Fig. 6D). All terminal bronchioles, respiratory bronchioles, and lung alveoli were negative for plunc
expression.

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Fig. 6.
In situ hybridization of adult
mouse tissues with 35S-labeled plunc
riboprobes. Paired images show bright field
(left) and dark field (right) views of the same
tissue section. A, a cross-section through the adult
thoracic cavity shows strong plunc expression in the entire
lining of the trachea (tr). B, a more posterior
section shows the tracheal tube divided into left and right bronchial
passages (lb and rb); plunc expression
also lines these divided airways. C, as the main bronchial
passages narrow and enter the lung, plunc expression in the
bronchial epithelium abruptly falls to background levels. D,
detail of the tracheal tube (from A) showing hybridization
signal (dense black grains) exclusively in the
pseudostratified columnar epithelium. e, esophagus;
lb, left bronchus; lu, lung; tr,
trachea; rb, right bronchus.
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As in the embryo, we also detected weak expression of plunc
in the adult thymus (not shown). Both left and right lobes of this
organ showed small clumps or "islands" of positive cells within a
larger, background mass of thymocytes. No other sectioned organs or
tissues (including esophagus, heart, major vessels, pericardium, and
lung pleura) showed any plunc hybridization signal.
Sequence Analysis of plunc cDNAs--
Sequencing of the band 1 cDNA fragment isolated from the differential display gel showed it
to be 255 base pairs in length. The sequences of the reverse
transcription and PCR primers were faithfully reproduced at the 5' and
3' ends of the fragment. A consensus polyadenylation signal sequence
beginning 22 bases upstream of the poly(A) tail was also noted.
The sequence of the complete plunc cDNA is shown in Fig.
7. This sequence contains an open reading
frame of 834 base pairs (278 codons), giving a putative protein product
of molecular mass 28,618 Da. Additionally, there is a 55-base pair
5'-untranslated sequence and a 207-base 3'-untranslated sequence.
Comparison of the complete sequence with entries in the
GenBankTM data base using the BLAST algorithm (6) showed no
significant homologies to known sequences at the nucleotide level. A
search of the dBEST data base showed 100% identity between
plunc and three murine expressed sequence tags
(GenBankTM accession numbers AA028768, cloned from a gd19
fetal mouse cDNA library and corresponding to plunc
residues 680-1076; AA763873, cloned from an adult mouse thymus
cDNA library and corresponding to plunc residues
11-481; and AA880683, cloned from an adult mouse lung cDNA library
and corresponding to plunc residues 602-1013). The sources
of these ESTs are consistent with the expression patterns we have
observed for plunc. Unfortunately, these sequence tags gave
no clues as to the identity of plunc, nor did a search of GenBankTM using these ESTs provide any additional
significant homologies to known genes. Significant homologies were also
detected between plunc and two other sequence tags
identified by screening of a human olfactory epithelial cell library
(GenBankTM accession numbers N27741 and N23239). Again,
searching of the GenBankTM data base using these sequences
as input did not reveal any further homologies with known
sequences.

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Fig. 7.
Nucleotide sequence and predicted amino acid
sequence of plunc. The repeated Pro-Leu-Pro-Leu
motif is shown in italics. Consensus sequences for protein
kinase C (SLK) and casein kinase II (SFVD and SGLD) are indicated by
solid underlines. N-Glycosylation sequences are
indicated by dotted underlines. Leucine residues identified
as part of a leucine zipper motif are shown in bold type, as
is a polyadenylation consensus sequence.
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Significant homologies at the amino acid level were observed with two
salivary gland proteins: the von Ebner minor salivary gland protein
(GenBankTM accession number U46068) and the parotid
secretory protein (PSP) precursor (Refs. 11 and 12;
GenBankTM accession number U79414). Comparisons of these
amino acid sequences are shown in Fig. 8.
The highest degree of homology was seen in the amino-terminal regions
of these proteins; of the first 15 amino acids of plunc and
murine PSP, 12 are identical and 2 show conservative amino acid
substitutions. The bovine PSP, BSP30, showed 10 amino acid identities
and 2 conservative substitutions in the same region, whereas von Ebner
minor salivary gland protein showed 6 identities and 4 conservative
substitutions. Interestingly, this region is reported to be part of the
PSP signal sequence (11), which is not part of the mature protein.
Other homologous regions of the amino acid sequence are characterized
by conservative amino acid substitutions more than amino acid
identities between the proteins.

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Fig. 8.
Amino acid sequence homologies between the
putative plunc gene product, von Ebner minor salivary
gland protein, and members of the PSP family. Homologous sequences
were identified by comparing the putative plunc protein
sequence with proteins in the GenBankTM data base using the
BLAST algorithm (6). Identical amino acid residues are indicated by
asterisks; conservative amino acid substitutions are
indicated by a plus sign. Hyphens indicate gaps introduced
into the protein sequences to allow optimal alignment. A
shows the region of greatest homology between plunc, the von
Ebner salivary gland protein, and the PSP family members. B
shows the homology of the amino-terminal sequence of the putative
plunc gene product to the signal sequences of the PSPs and
the von Ebner protein.
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Analysis of the predicted amino acid sequence using the PROSITE program
(14) found several functionally significant motifs (Fig. 7), including
consensus sequences for phosphorylation by protein kinase C (15, 16)
and casein kinase II (17), two N-glycosylation sites (18,
19), and a leucine zipper (20-22). A portion of the leucine zipper is
contained within the region homologous to the PSP signal sequence, so
the functional significance of this motif in this protein is questionable.
An interesting and apparently unique repeating sequence pattern is also
present in the amino region of this protein. This pattern consists of
six amino acids of the sequence Gly-(Leu/Pro/Gln)-(Pro/Leu)-Leu-Pro-Leu and is repeated four times, beginning with residue 23, with two amino
acids spacing between the repeated elements. A search of GenBankTM found no such amino acid motifs represented in
this data base; whether this repeating sequence is of functional
significance or is a coincidental occurrence is presently unknown.
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DISCUSSION |
plunc is a novel gene transcript with a unique spatial
and temporal pattern of expression in the embryonic and adult mouse. Expression in the mouse embryo is first noted on gd14 on or about the
time of palatal shelf elevation and fusion and is confined to discrete
regions of the nasal epithelium. These regions of expression are
associated with the dorsal palate, nasal septum, and nasal conchae. The
transcript is also expressed strongly in the tracheal and bronchial
epithelium of the adult mouse lung.
The discovery of a new gene raises the corresponding question of the
function of the gene. Because plunc is expressed only at the
margins of the palatal shelves, well removed from the sites of shelf
contact and fusion, we think it unlikely that plunc plays a
role in this aspect of facial morphogenesis. plunc
expression is, however, associated with areas of presumptive fusion of
adjacent epithelia as the nasal columella and lateral nasal walls form the sinus cavities and the common nasal passage. It is possible that
plunc may serve in some regulatory aspect of this
morphogenetic event.
Examination of the predicted amino acid sequence of the
plunc protein indicates that it is a secreted molecule; the
related PSP is a major protein component of saliva (11). Thus one
alternative hypothesis is that plunc is not a morphogenetic
factor but a component of nasal or respiratory mucus. The
pseudostratified columnar epithelia of the nasal and respiratory
passages are provided with scattered, isolated goblet cells, and
subepithelial mucous glands that produce a variety of proteinaceous
secretions (23). Although plunc is abundantly expressed in
certain regions of these epithelia, the in situ
hybridization signal does not specifically match the distribution of
either of these cell types. The fact that plunc transcripts are absent from the smaller respiratory passages and lung alveoli also
makes it unlikely that this gene product is a secretion involved in gas
exchange (i.e. not a surfactant).
The potential relationship between the PSPs and plunc
provides little insight into plunc function, because the
functions of the PSPs themselves remain unknown. PSP expression in the
murine parotid glands is coordinately regulated with salivary amylase (12); whether this indicates that the functions of these proteins are
complementary remains to be determined. The reduced expression of the
bovine PSP, BSP30, has been linked to resistance to pasture bloat
disease in cattle (10). Again, the potential significance of this
observation for embryonic development and epithelial cell function is
unknown. The strict spatial and temporal regulation of expression of
both the PSPs and plunc, however, implies that these gene
products are important in defining the functions of the expressing tissues.
The onset of plunc expression coincides with differentiation
of the presumptive nasal epithelium into mature nasal epithelial cells
(13). We also note the persistent expression of plunc in the
adult nasopharynx as well as the fact that sequence tags with high
homology to plunc have been identified in human olfactory epithelium. These observations lead us to speculate that the
association of plunc with epithelial cells of the developing
palate and nasopharynx may be related to the differentiation and
maintenance of an as yet unidentified subpopulation of epithelial cells.
In the embryonic and adult nasopharynx, plunc shows a
surprisingly discontinuous pattern of expression in a continuous
epithelial sheet. Because no obvious histological features distinguish
regions of epithelial cells that express plunc from those
that do not, the basis for this pattern of expression is unknown. These
observations suggest that the nasal epithelium, relatively uniform in
appearance, may contain functionally distinct regions defined on the
basis of differential gene expression. An examination of the molecular regulation of the plunc transcript should prove useful in
testing this hypothesis.
Unlike the pattern observed in the nasal epithelium, plunc
expression in the lower respiratory tract is continuous throughout the
trachea and upper bronchi. As in the nasopharynx, however, there is no
histological demarcation indicating the limit of plunc expression in the bronchial epithelium. The distinction between plunc-expressing and nonexpressing cells may be present only
at the molecular level, and it will be interesting to determine whether the same factors that govern plunc expression in the
tracheobronchial epithelium also operate in the nasal epithelium.
Sequence analysis of plunc also revealed an apparently novel
repeating amino acid motif, Leu-Pro-Leu-Pro-Leu. This repeated sequence
is likely to represent the amino terminus of the mature protein
following removal of the presumptive signal sequence. Because no other
known proteins were found to share this motif, its functional
significance here remains speculative. A targeted search for other new
proteins that may share this sequence element should be useful in
determining whether this sequence serves an important role in
plunc function.
Our goal in undertaking these studies was to identify candidate genes
involved in the control of craniofacial morphogenesis. Although
plunc is unlikely to play a role in this process, the expression pattern of this novel transcript indicates a previously unsuspected level of complexity in the nasal and respiratory
epithelium. Determination of the function of plunc and the
mechanisms underlying its highly restricted pattern of expression
represent the next steps in determining the regionally specified
functions of these tissues.