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DEVELOPMENTAL DYNAMICS 208:244–254 (1997)
Expression of the Mouse Fibronectin Gene and
Fibronectin-lacZ Transgenes During Somitogenesis
ROBERT A. PERKINSON AND PAMELA A. NORTON*
Department of Medicine, Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pennsylvania 19107
ABSTRACT
Fibronectins (FNs) are essential for the proper development of embryonic
mesenchymal tissues. A lacZ reporter gene has
been fused to 4.9 kbp of DNA from the rat FN gene
58 flanking region, and this construct has been
microinjected into fertilized mouse embryos to
investigate the cis elements needed for the temporal and spatial regulation of FN in vivo. Histochemical staining of embryos for b-galactosidase
activity demonstrated that four independent lines
shared a specific pattern of lacZ expression, reflecting the activity of the fibronectin sequences
contained within the transgene. Specifically,
somites stained positively for lacZ, but expression was spatially and temporally non-uniform,
with higher levels in more caudal somites after a
total of ca. 13 somite pairs had formed. This
rostral-caudal gradient of lacZ expression in
somites of embryos beyond this stage resembled
the distribution of endogenous FN mRNA, as
detected by whole mount in situ hybridization.
The transgene was not expressed in the developing heart where endogenous FN mRNA was detected. Unexpectedly, highly localized staining
was observed within the neural tube beginning at
ca. E10–10.5, and two of the lines exhibited additional areas of staining due to the individual
integration sites. Thus, the 4.9 kbp FN fragment
appears to recapitulate closely the complex pattern of FN expression observed during somitogenesis. A smaller fragment of 0.9 kbp also directed
lacZ expression in caudal somites at E9.5, suggesting that these sequences are sufficient to establish the spatio-temporal pattern. Dev. Dyn. 208:
244–254, 1997. r 1997 Wiley-Liss, Inc.
Key words: fibronectin; somite; transgenic mice;
beta-galactosidase; in situ hybridization
INTRODUCTION
Fibronectins (FNs) are a family of large adhesive
glycoproteins that possess the ability to interact with
cells via integrin cell surface receptors as well as with
other extracellular matrix components such as collagen
and heparin (Hynes, 1990; Mosher, 1989; Paolella et al.,
1993). FNs are present in vertebrate embryos at many
sites of cell migration, and they have been postulated to
be important for these processes (Adams and Watt,
r 1997 WILEY-LISS, INC.
1993; DeSimone, 1994; Dufour et al., 1988; Hynes,
1994). Direct evidence for functional roles of FNs has
been provided by perturbation experiments and genetic
manipulation. Anti-FN antibodies or peptides that interfere with FN-integrin interactions can disrupt gastrulation of amphibian and avian embryos, and impede
migration of mouse primary mesodermal cells (Boucaut
et al., 1984a,b; Harrisson et al., 1993; Johnson et al.,
1992; Klinowska et al., 1994). In contrast, mouse
embryos homozygous for a disruption of the FN gene
appear to gastrulate normally, but fail to form somites
and exhibit other mesenchymal defects as well as
defects in heart development (George et al., 1993). The
failure to form somites is interesting in light of previous
work implicating FNs in this process. FN accumulates
in the region of the segmental plate that represents the
site of active somitogenesis, adjacent to the caudalmost somite, as well as within and surrounding the
somites themselves (Duband et al., 1987; Lash et al.,
1984; Ostrovsky et al., 1983; Sternberg and Kimber,
1986). Addition of FN to presomitic mesoderm has been
observed to enhance somite formation in culture (Lash
et al., 1984). FN has also been implicated in heart
formation and in cardiac cushion cell migration (Linask
and Lash, 1988), and thus cardiac defects in the homozygous null mice were not surprising. In addition,
evidence suggests that FNs promote the migration of
neural crest cells through and around the somites via
interactions with integrin cell surface receptors (Boucaut et al., 1984b; Duband et al., 1991; Duband et al.,
1986; Dufour et al., 1988; Krotoski et al., 1986; Rovasio
et al., 1983). In the adult, FN is a constituent of many
basement membranes and FN levels are increased
rapidly and locally during wound healing and in response to other fibrotic stimuli, suggesting an important role in these processes. However, the early death of
the FN-null mice precludes assessment of the need for
FN in later organogenesis or in the adult.
The foregoing observations regarding the distribution of FNs suggest that the single copy Fn1 gene is
regulated in a complex manner. The DNA elements
needed for cell type-specific regulation of FN have
begun to be identified from transfection studies and in
Contract Grant sponsor: National Institutes of Health; Contract
Grant number: GM-46402.
*Correspondence to: Pamela A. Norton, Department of Medicine,
Thomas Jefferson University, 1020 Locust Street, Philadelphia, PA
19107.
Received 19 April 1996; Accepted 11 October 1996
FIBRONECTIN AND SOMITOGENESIS
vitro studies using extracts of cells and tissues (Dean,
1989; Dean et al., 1989; Miao et al., 1993; Muro et al.,
1992; Nakajima et al., 1992; Perkinson et al., 1996;
Singh and Kanungo, 1991; Sporn and Schwarzbauer,
1995). However, the use of transgenic mice has proven
necessary to identify the full complement of sequences
needed for correct tissue-specific regulation of many
genes (Kitsis and Leinwand, 1992). For instance, detailed analysis of the globin gene cluster has revealed
that proper erythroid-specific, copy number dependent
expression requires the presence of locus control regions that are 30–40 kbp from the coding regions
(Dillon and Grosveld, 1993). This indicates that a
complete description of the regulation of FN expression
during embryogenesis and pathologic processes requires in vivo study.
The E. coli lacZ gene has been used extensively as a
reporter gene to investigate the sequences required to
confer regulated expression during embryonic development (Bonnerot and Nicolas, 1993). The lacZ gene
product, b-galactosidase, is a cell autonomous marker
that is readily detected and that is functionally neutral
in many cell types (Beddington et al., 1989; Goring et
al., 1987). For these reasons, we chose to use a lacZ
reporter gene to investigate the cis elements needed for
the temporal and spatial regulation of the FN gene in
transgenic mice. Previously, we established that a
fragment of 4.9 kbp from the 58 end of the rat FN gene
directs the expression of the lacZ reporter in rodent
fibroblasts [pFNZ4.9 (Perkinson et al., 1996)]; we and
others have also demonstrated that shorter FN fragments are also functional in such cells (Miao et al.,
1993; Nakajima et al., 1992; Sporn and Schwarzbauer,
1995). In the present report, we describe the phenotype
of mice that contain FN-lacZ transgenes pFNZ4.9 and
pFNZ0.9 during the interval within which somitogenesis occurs. Histochemical staining for the lacZ gene
product, b-galactosidase, reveals that the FN fragment
directs the expression of lacZ in developing somites in a
fashion that closely parallels endogenous FN mRNA, as
detected by in situ hybridization. However, the transgene fails to recapitulate the complete repertoire of
expression of the endogenous gene, as detailed below.
RESULTS
Whole Mount In Situ Hybridization
Determination of FN mRNA Distribution
We wished to employ lacZ as a reporter gene to
identify sequences from the fibronectin gene that confer
cell and tissue specific gene expression. However, interpretation of patterns of transgene expression requires
comparison with expression of the endogenous Fn1
gene. The distribution of FN in the extracellular matrix
may not coincide precisely with the location of cells that
express the gene, due to differences in deposition or
turnover. Thus, we evaluated FN mRNA levels by
whole mount in situ hybridization. Embryos were isolated and fixed at E8.0–10.5, and hybridized to digoxigenin-labelled RNA probes either complementary (anti-
245
sense) or identical (sense control) to mouse FN mRNA.
At ca. E8.0, the presomitic mesoderm hybridized intensely with the antisense probe, with lower levels of
staining in somites and in the heart (Fig. 1a). All
somites were labelled, and the somewhat more strongly
stained caudal somites are indicated; the more rostral
2–3 somites are partially obscured by amnion remnants. At E9.5, specific labelling was detected in the
caudal somites and presomitic mesoderm, with weak
staining in the heart (Fig. 1b). At E10.5, significant
background was present in these large specimens, but
strong staining was observed with the antisense probe
in the pre-somitic mesoderm and the caudal-most
somites (Fig. 1c). These regions were not labelled with
the sense control (Fig. 1d). Thus, it appears that FN
mRNA levels are high prior to overt somite formation
then decline as somites mature.
Structure of the FN-lacZ Reporter Gene and
Identification of Transgenic Mice
We initially chose to test a fairly large segment of 58
flanking DNA from the FN gene for its ability to be
expressed in vivo. A ca. 4.9 kbp fragment of the rat FN
gene was isolated from a lambdaphage clone (Patel et
al., 1987). This fragment includes ca. 4.8 kbp of 58
flanking sequence, the transcription start site and 136
nucleotides of 58 untranslated sequence. The sequence
of the 58 flanking region is incomplete, but the proximal
half has been determined in three overlapping segments (Nakajima et al., 1992; Patel et al., 1987; Sporn
and Schwarzbauer, 1995). In general, the three reports
are in agreement in regions of overlap. However, a short
region of sequence divergence occurs between 21908
and 21897, with a PstI site present in only one of the
two sequences, as shown in Figure 2b. The 4.9 kbp
fragment that we isolated also lacks the PstI site at
21908 relative to the start of transcription. To determine which sequence variant corresponds to the endogenous FN gene, we performed PCR analysis. The
forward primer corresponds to sequences that lie near a
PstI site at ca. 24.8 kbp relative to the transcription
start site (unpublished data); the reverse primer lies
within the most 58 region of sequence concordance (see
Fig. 2a). Amplification of rat genomic DNA resulted in a
2.5 kbp fragment that was identical in size to that
derived from the plasmid template (Fig. 2c, compare
lanes 2 and 3). In addition, the products amplified from
both genomic DNA and plasmid were cleaved by BstXI
to yield fragments of 2.0 and 0.5 kbp (data not shown;
refer to map on Fig. 2a). Thus, the 4.9 kbp fragment
retains the proper sequence organization of the rat FN
gene, indicating that the sequence of Sporn and Schwarzbauer, extending from 22547 to 21080 and lacking
the PstI site, is the correct one. Because the 4.9 kbp PstI
fragment was derived from the same lambdaphage as
the subclones that these workers sequenced, it very
likely contains all the sequence features that they
describe.
246
PERKINSON AND NORTON
Fig. 1. Distribution of endogenous FN mRNA. FN mRNA distribution
was examined by whole mount in situ hybridization using digoxigenin
labelled RNA probes transcribed from mouse FN cDNA; a–c, antisense
probe, d, sense probe. a: Lateral view of E8.5 embryo. Note the strong
staining in the posterior region (P) with lower levels of staining in somites
(arrowheads) and heart (H). b: Lateral view of E9.5 embryo. Hybridization
is strongest in presomitic mesoderm (P) but is also apparent in caudal
somites. c: Lateral view of E10.5 embryo; the head has been removed.
While background is higher at this stage, staining is evident in the
presomitic mesoderm of the tail (P) and in caudal somites. d: Dorsal view
of E10.5 embryo; the head has been removed. Note lack of staining of
presomitic mesoderm (P) and adjacent somites.
Plasmid pFNZ4.9 contains a lacZ reporter gene
flanked on the 58 end by the 4.9 kbp fragment and on
the 38 end by 0.8 kbp sequence from the 38 end of the rat
FN gene [Fig. 2a (Perkinson et al., 1996)]. Thus, FN
sequences provide signals for the start of transcription
and for polyadenylation, but the entire FN coding
region has been replaced by lacZ. This plasmid was
digested with NotI to remove vector sequences and the
gel-purified insert DNA was injected into fertilized
one-cell mouse embryos, which were then implanted
into foster mothers. Screening of 41 pups by PCR
analysis of tail DNA identified four mice that had
acquired FNZ4.9 DNA (Table 1). All four founders were
bred to C3H mice, and F1 progeny were assayed as
pups (DNA) or as embryos (lacZ expression). This
initial analysis demonstrated that the transgene was
transmitted in all four lines and that it was expressed
in the mice. In three lines, the frequency of positives
was significantly less than 50%, suggesting the founders
were mosaics. The similar frequency of positives detected by the two methods indicates that the lacZ assay
reliably identified transgenic individuals. The estimated copy number of the transgene is also indicated.
In subsequent generations, all four lines transmitted
the transgene at the expected Medelian frequency (data
Fig. 2. Structure of the FN-lacZ transgene. a: Diagram of FNZ4.9 DNA
microinjected into embryos. The start of transcription is indicated by the
arrow. Open bar, FN 58 flanking sequences; stippled box, FN 58 untranslated sequences; line, lacZ; shaded box, FN 38 sequences, with the site of
polyadenylation indicated. The entire transgene is approximately 8.5 kbp
in length. b: Comparison of the rat FN sequence reported by Sporn and
Schwarzbauer (top) and Nakajima et al. (bottom) at positions ca. 21,900
kbp relative to the transcription start site. The former sequence continues
upstream, whereas the latter truncates at the PstI site (underlined). The
sequences are identical beginning at the italicized residues and continuing 38. c: PCR of FN from rat genomic DNA and pFNZ4.9. Lane 1, no
template DNA; lane 2, rat genomic DNA; lane 3, plasmid pFNZ4.9 DNA. In
lanes 1 and 2, 20 µl of each PCR reaction using the eLongase enzyme
mixture was loaded; in lane 3, 5 µl of a reaction using Taq DNA
polymerase was loaded. At left, positions of molecular size markers are
indicated (kbp).
247
FIBRONECTIN AND SOMITOGENESIS
TABLE 1. Summary of FNZ4.9 Transgene Transmission to F1 Generation Animals
Est. copy number
DNA detectiona
lacZ detectionb
Transgene positive/Total
Male 4
2
6/22
13/31
Male 23
4–5
4/29
2/17
Male 24
6–7
6/35
1/8
Female 30
2–3
15/34
NT
19/53
6/46
7/43
15/34
aDNA detection,
transgene detected by PCR of genomic DNA from tails.
detection, transgene detected by staining embryos with X-gal.
NT, not tested.
blacZ
not shown). Analyses of lacZ expression in the F1 mice
and subsequent generations are detailed below.
Features of lacZ Reporter Gene Expression
Common to All Lines
Starting at E8.0, embryos were isolated, fixed and
stained with X-gal to detect b-galactosidase expression.
Several individuals from lines 24 and 30 are shown in
Figure 3; these two lines were indistinguishable, and
the staining features described were also observed in
embryos derived from lines 23 and 4. By E8.0, FN-null
mutant embryos have a deficiency of mesoderm and fail
to initiate somitogenesis (George et al., 1993), indicating a requirement for FN at or prior to this stage. At
E8.0, we observed lacZ expression in the head-fold
mesenchyme and in the first somite pairs (Fig. 3a),
consistent with the reported normal distribution of FN
at this stage (George et al., 1993). A similar situation
was observed at ca. E8.5–9.0 (Fig. 3b), with staining in
all somite pairs (ca. 8–12) but with diminished staining
in the head region. In slightly more advanced embryos
(ca. E9.5, 13 or more somite pairs), lacZ expression was
most evident in newly formed somites, but was diminished in the older, rostral somites (Fig. 3c). Thus,
formation of $ca. 13 somite pairs and the nearcompletion of turning coincided with a transition in the
pattern of FNZ4.9 transgene expression. It was noted
that X-gal staining was present within the rostral
portion of the presomitic mesoderm, where somitogenesis is occurring (Fig. 3c and d). Dissection of the caudal
somites revealed that the transgene appeared to be
expressed in cells dispersed throughout the somite
(date not shown). Note that no X-gal staining was
observed in the developing heart (Fig. 3b and c).
By day 10.5, newly forming somites continued to be
positive for transgene expression (Fig. 3e). In addition,
a blue stripe was evident in the lateral view, beginning
in the cranial region and extending to the region of the
hind limb bud. Somewhat more dorsal views demonstrated two internal bilateral stripes, most readily
visible through the roof of the fourth ventricle at the
back of the head (Fig. 3f ). Transverse sections of
stained embryos revealed that these bilateral tracts
were within the neural tube (see below). Non-transgenic littermates were completely negative for X-gal
staining. The pattern of staining in the neural tube and
in the most posterior somites persisted through days
E11.5–12.5 (data not shown). Thus, the FN sequences
present in the FNZ4.9 transgene direct lacZ expression
in the somites, but not in the heart; the expression in
the central nervous system is discussed below.
Line Specific Features of Reporter
Gene Expression
The staining pattern described above is shared by all
four lines. Lines 30 and 24 exhibited only this pattern of
staining, but lines 4 and 23 exhibited additional regions
of distinct, line specific staining. Characterization of
these lines has been less extensive, and was restricted
largely to stages E9.5–10.5. In addition to the common
staining pattern, embryos of line 23 exhibited variable
X-gal staining near the crown of the head, in the facial
region and in the fore and hind limbs (not shown).
E10.5 embryos of line 4 exhibited intense staining in
the developing forelimb which was strongest in the
anterior and dorsal regions of the limb (Fig. 4). No
staining of the forelimbs at this stage has been observed by in situ hybridization with the antisense FN
probe (data not shown). The hind limb bud was stained
much less intensely, even when a similar size and
developmental stage was reached (data not shown).
Additional staining was observed in the area where the
cerebellum will form, and in more rostral somites;
neither is typical of endogenous FN mRNA (Fig. 1c and
data not shown). The staining patterns are summarized
in Table 2. We attribute the staining patterns unique to
lines 4 or 23 to the different integration sites of
the transgenes in these lines, as they are not consistent with the pattern of expression of the endogenous
gene.
Ectopic Expression of the FN-lacZ Transgene
in the Neural Tube
An E10.5 embryo of line 4 similar to those shown in
Figure 4 that had been stained with X-gal was postfixed
and sectioned. Transverse sections across the region
just rostral to the forelimb bud were reacted with
anti-FN antibody and horseradish peroxidase-conjugated secondary antibody. FN staining was distributed
widely, including within the heart and surrounding the
neural tube. However, there was no obvious colocalization of the brown peroxidase reaction product
with the blue X-gal staining within the ventro-lateral
248
PERKINSON AND NORTON
TABLE 2. Summary of X-gal Staining Patterns of FNZ4.9
Transgenic Mice
Line
4
23
24
30
Heart
2
2
2
2
Somites
1
1
1
1
Neural
tube
1
1
1
1
Other
forelimb, head
limbs, head
2
2
within the neural tube (Fig. 1a and not shown). Thus,
the combined data indicate that FNZ4.9 transgene
accurately reflects expression of the endogenous gene in
the somites, but not in other tissues.
A 0.9 kb Fragment Is Sufficient to Confer
Somite-Specific Expression
Fig. 3. Expression of the FNZ4.9 transgene in E8.0–10.5 embryos of
lines 24 and 30. Embryos from the two lines were stained with X-gal at
various developmental stages; representative individuals are shown. a:
E8.0 embryo with 2–3 somites. Note staining in head folds (arrows). b:
Lateral view of an E9.0 embryo with ca. 11 somites. The embryo is
incompletely turned, bringing both columns of somites into view. Note
similar staining of somites at all levels, and lack of staining in the heart
(labelled with adjacent H). c: Lateral view of an E9.25 embryo with ca. 15
somites. Staining in rostral somites has diminished, and there is lack of
staining in the heart (H). d: Higher magnification view of caudal end of an
E9.25 embryo. Note that staining is present in the presomitic mesoderm,
caudal to the last visible somite, the boundaries of which are defined by
the arrows. e: Lateral view of an E10.5 embryo. Staining is observed in
newly formed caudal somites, and as well as within the neural tube (see
text). f: A more dorsal view of an E10.5 embryo, revealing the bilateral
neural tube staining.
neural tube (Fig. 5). Thus, FN does not appear to
accumulate at a specific site within the neural tube. In
addition, high levels of FN mRNA were not detected
Two transgenics were obtained with pFNZ0.9, which
contains only 880 bp of FN promoter sequence upstream of the transcription start site. Lines were established from each founder (designated lines 255 and
272), and F1 and F2 progeny analyzed for lacZ expression at E9–11. Some individuals from both lines exhibited X-gal staining in caudal somites and presomitic
mesoderm; Figure 6a shows an individual from line 255
with staining similar to that seen for FNZ4.9 mice at a
comparable stage (compare to Fig. 3c). A total of 12
E9.0–9.5 individuals were positive for somitic and
presomitic mesoderm staining out of 15 total positive
for X-gal staining. In contrast, the somite staining
pattern was less well preserved in later embryos; only
8/19 total E10-12 X-gal positives exhibited any staining
in caudal somites, and staining tended to be weak
relative to that seen with FNZ4.9 embryos. However, a
different pattern was seen in some individuals beginning at E10, with staining of lateral structures that
may represent the dorsal root ganglia. The intensity of
staining is highly variable, from almost none to very
strong (Fig. 6b and c); such variation was observed in
both lines and extreme examples were found within
single litters. Staining of head structure was also
observed, but no staining was detected in the heart, and
the ectopic staining within the neural tube was diminished relative to the FNZ4.9 mice. These staining
features were never observed in studies of the FNZ4.9
mice. Single individuals were found with both somitic
and lateral staining patterns, suggesting that they are
not mutually exclusive. Note that the in situ hybridization data did not reveal any evidence for lateral expression of FN (compare Fig. 1b and c with Fig. 6b and c).
Thus, the 0.9 kbp fragment was sufficient to establish
transgene expression in caudal somites and the presomitic mesoderm in embryos of E9–9.5, but that this
was not sufficient to reliably direct expression to nascent somites at later times.
DISCUSSION
Four independent lines of mice were obtained that
contain the FNZ4.9 transgene. Studies of embryos
FIBRONECTIN AND SOMITOGENESIS
249
Fig. 4. Expression of the FNZ4.9 transgene in E10.5 embryos from
line 4. Embryos were collected at E10.5 and stained with X-gal. Lateral
and dorsal views of line 4 embryos. Note the staining in caudal somites
and in the neural tube. Strongly stained forelimbs are marked by arrows.
revealed that the FN sequences present in the transgene are sufficient to establish and maintain an appropriate rostral-caudal gradient of expression in the
somitic mesoderm. However, the transgene is not expressed in the developing heart although endogenous
FN mRNA is present [Fig. 1 and (ffrench-Constant and
Hynes, 1988)]. A truncated version of this transgene
also directed reporter gene expression to the somites,
but the instability of the phenotype over time suggests
that some regulatory elements have been removed.
it is interesting that FN expression is high during the
compaction process.
The lacZ-positive cells appeared to be distributed
throughout the somite. This distribution is not in
precise agreement with reports of the FN protein
distribution in the chick and mouse, where FN has been
reported to surround the somites, with less material
within the somite (Duband et al., 1987; Duband et al.,
1986; Krotoski et al., 1986; Ostrovsky et al., 1983;
Stepp et al., 1994; Sternberg and Kimber, 1986). However, b-galactosidase is a cell-autonomous marker; the
cells that synthesize the protein retain it. In contrast,
FN is an extracellular protein, and its distribution may
not reflect its pattern of synthesis, due to differential
deposition or turnover in the extracellular matrix.
Alternatively, there may be reduced immunoreactivity
of the FN in certain regions, possibly due to masking by
other molecules.
Most of the studies regarding the localization of FN
protein (see above) and mRNA (ffrench-Constant and
Hynes, 1989; ffrench-Constant and Hynes, 1988) in
somites have been done in the chick. Although the
mouse and chick are likely to be very similar, our study
has revealed that FN gene expression undergoes a
distinct transition during somitogenesis. Beyond the
ca. 12 somite stage, the caudal-most somites express
much higher levels of FN mRNA and b-galactosidase
compared to the more rostral structures. We might thus
expect that FN distribution in the matrix will vary with
developmental stage and axial level. In agreement with
this idea, another group has described a shift in the
localization of FN in older, more rostral somites in the
mouse (Sternberg and Kimber, 1986). It is also of
interest that differences have been observed in the
expression of cadherin-11 and N-cadherin in newly
formed vs. older somites (Kimura et al., 1995). Thus, it
FNs Gene Expression During Somitogenesis
An early defect manifested by FN-null mice is the
absence of somites, along with a general deficiency of
mesoderm (George et al., 1993). Interestingly, we observed that FN mRNA levels were elevated in the
presomitic mesoderm, consistent with a role for FN in
somitogenesis. Somites are formed from the paraxial
mesoderm, beginning between E8.0 and E8.5, with a
new pair forming at approximately hourly intervals
(Tam, 1981). It has been proposed that FN is involved
during early somitogenesis, at the compaction stage
that precedes epithelialization (Lash et al., 1984).
Expression of the lacZ reporter and FN mRNA was
evident in the pre-somitic mesoderm (Figs. 2 and 4), as
is FN protein (Duband et al., 1987; Lash et al., 1984;
Ostrovsky et al., 1983). The domain of FN mRNA
expression extends more caudally than that of lacZ,
which may reflect mRNA accumulation prior to the
synthesis of significant levels of protein. Alternatively,
expression of the transgene may be slightly delayed
with respect to the endogenous gene; such a phenomenon has been reported for the MyoD gene (Asakura et
al., 1995). We have shown previously that the activity of
the FN promoter is increased with increased cell density in mouse fibroblasts (Perkinson et al., 1996). Thus,
250
PERKINSON AND NORTON
show that the FN gene is induced in newly formed
somites, and that lacZ expression appears somewhat
elevated in the anterior portion of each somite (Fig. 3d).
These findings are consistent with a role for FNs in
crest cell migration, although FN protein has been
reported to be evenly distributed throughout the somite
(Krotoski et al., 1986). However, our data are consistent
with formation of a transient gradient.
FN Gene Expression in Other Regions
Fig. 5. Expression of the endogenous FN gene. Immunohistochemical localization of FN. An E10.5 embryo from line 4 was stained with X-gal,
postfixed, then embedded in paraffin and sectioned. This transverse
section was incubated with polyclonal anti-FN antibody, and detected via
horseradish peroxidase conjugated secondary antibody. Note reactivity
surrounding the heart (H, bottom) and the neural tube (N, top), and the
lack of co-localization within the neural tube between FN (brown) and
b-galactosidase (blue).
may be important to evaluate several developmental
stages of both chick and mouse embryos to detect any
transitions that occur in the expression of various
adhesion molecules.
Neural crest cells migrate from the dorsal surface of
the neural tube in an axial wave that follows somitogenesis. Immunolocalization studies suggested that FN
plays a role in neural crest cell migration through and
around the somites. Later studies demonstrated that
FN promotes neural crest cell migration, and that
specific antibodies and peptides disrupt FN-cell interactions (Boucaut et al., 1984a,b; Duband et al., 1991;
Duband et al., 1986; Dufour et al., 1988; Krotoski et al.,
1986; Rovasio et al., 1983). If FN is involved in crest cell
migration, it must be present prior to crest cell migration, and should be concentrated in the anterior portion
of the somite (Bronner-Fraser et al., 1991). Our data
The FNZ4.9 transgene contains ca. 4.9 kbp of DNA 58
to the transcription start site, 0.1 kbp of 58 untranslated sequence and less than 1.0 kb of 38 sequences;
these are sufficient to confer appropriate FN gene
expression in newly forming somites. Regulatory elements that have been identified as important for FN
promoter function include an enhancer element that
lies nearly 2.0 kbp from the start site (Sporn and
Schwarzbauer, 1995), as well as more proximal elements that are important for promoter activity in
cultured cells (Bowlus et al., 1991; Dean et al., 1989;
Miao et al., 1993; Muro et al., 1992; Nakajima et al.,
1992). All these elements are present in the FNZ4.9
transgene; the absence of lacZ expression in the early
embryonic heart indicates that the construct probably
lacks one or more as yet unidentified tissue-specific
sequence element. The posited additional regulatory
element(s) could lie further upstream of the transcription start site than the nearly 5.0 kbp included in
FNZ4.9. Alternatively, it could lie within the transcribed region. A DNAseI hypersensitive site within the
first intron is present in at least one adult tissue where
FN is expressed (liver) and absent from one where the
gene is largely silent (cerebrum) (Singh and Kanungo,
1991). Analyses of the FNZ0.9 transgene indicates that
,1.0 kbp is sufficient to direct reporter gene expression
in the somites, but that maintenance of gene expression
is more variable. It is possible that the variable phenotype of FNZ0.9 embryos at later stages is a consequence
of removing the fibroblast enhancer (Sporn and Schwarzbauer, 1995). Additional constructs will be needed to
test the role of these individual elements.
Staining in the neural tube was unexpected; typically, FN protein has not been detected within the
neural tube, and we did not detect significant levels of
mRNA. However, FN and its receptor were detected in
one study (Duband et al., 1986), and an in situ hybridization study in the chick noted labelling in precisely
this area when an embryo of a similar developmental
stage was examined (ffrench-Constant and Hynes,
1989). Thus, it is possible that the endogenous FN gene
normally is expressed in a restricted fashion in the
neural tube, but that expression of the FN-lacZ transgene is enhanced, possibly due to the absence of a
silencer element. One trivial explanation, that heterologous sequences were fused during cloning to proximal
FN promoter elements, is discounted by the PCR data
shown in Figure 1c. The identity of the labelled cells
has not been established, but based on their ventral
FIBRONECTIN AND SOMITOGENESIS
251
Fig. 6. Expression of the FNZ0.9 transgene in E9.5–10.5 embryos.
Embryos from lines 255 and 272 were stained with X-gal at various
developmental stages; representative individuals are shown. a: Lateral
view of an E9.5 embryo with ca. 20 somites. Staining in presomitic
mesoderm (P) and caudal somites is evident and there is lack of staining
in the heart (H). b: Frontal-lateral view of a ca. E10.0 embryo. Staining is
observed internally and in the cranial region, and very weak staining is
seen laterally in the vicinity of the more rostral somites. c: Lateral view of
an E10.5 embryo. Lateral staining is very strong in this individual.
location, they might represent a motor neuron subset.
This raises the possibility that the expression of the
FNZ4.9 transgene is under the influence of one or more
members of the LIM homeobox genes, which appear to
define subgroups of motor neurons in a combinatorial
fashion (Tsuchida et al., 1994). Further studies are
necessary to establish the identity of the transgenepositive cells as well as the sequences responsible for
this highly restricted pattern of expression.
defined as E0.5.) Embryos from timed matings of
C3H/HeJ mice (Taconic Farms) were dissected in phophate buffered saline (PBS), 2.0 mM EGTA. Extraembryonic membranes were removed, and older embryos were decapitated. Embryos were fixed in fresh 4%
paraformaldehyde, 2.0 mM EGTA in PTW (PBS, 0.1%
Tween 20) at 4°C for 5–18 hr, and stored in methanol at
220°C for up to 1 month.
Rehydrated embryos were treated with 10 µg/ml
Proteinase K (Gibco/BRL) in PTW for 10–20 min,
depending on the age of the embryo, then post-fixed for
20 min in 4% paraformaldehyde, 0.1% glutaraldehyde
in PTW. Embryos were washed with PTW and equilibrated in hybridization mix (50% formamide, 1.3 3 SSC,
5 mM EDTA, 50 µg/ml yeast RNA, 0.2% Tween-20, 0.5%
CHAPS, 100 µg/ml heparin), then prehybridized in 1.0
ml of hybridization mix for 2 hr at 65°C. Embryos were
incubated overnight at 65°C in 1.0 ml of prewarmed
hybridization mix containing 0.1–0.25 µg/ml digoxygenin-labelled RNA probe. Following hybridization,
embryos were washed twice at 65°C for 30 min with 1.5
ml hybridization mix, then equilibrated with MABT
(100 mM sodium maleate, pH 7.5, 150 mM NaCl, 0.1%
Tween). Embryos were gently rocked at room temperature for 1 hr in 1.5 ml MABT, 2% Boehringer Blocking
Reagent (BBR), then for 1.5 hr with 1.5 ml MABT, 2%
BBR, 20% heat-treated sheep serum. Antibody binding
was overnight at 4°C in 1.0 ml MABT, 2% BBR, 20%
serum and 1/2,000th dilution of alkaline phosphataseanti-DIG antibody (Boehringer). Embryos were washed
three times with MABT, twice with NTMT (0.1 M NaCl,
0.1 M Tris-Cl, pH 9.5, 0.05 M MgCl2, 0.1% Tween-20),
and transferred to 1.5 ml NTMT/NBT/BCIP (NTMT,
0.225 mg p-nitro blue tetrazolium chloride, 0.115 mg
5-bromo-4-chloro-3-indolyl phosphate). To stop color
EXPERIMENTAL PROCEDURES
Digoxygenin-Labelled RNA Probe Preparation
A 480 bp EcoRI-HindIII mouse fibronectin cDNA
fragment was subcloned into pBS2 (Stratagene); this
fragment includes 27 bp of the alternative EIIIB exon
(Górski et al., in press). Labelled RNA probes were
synthesized by in vitro transcription of the EcoRI or
HindIII linearized DNA with T3 or T7 RNA polymerase,
respectively. A 20 µl in vitro transcription reaction
contained 1 µg DNA, 2 µl of NTP label mix [10 mM ATP,
10 mM CTP, 10 mM GTP, 6.5 mM UTP, 3.5 mM
digoxygenin-labelled UTP (Boehringer Mannheim)], 1
µl of RNase Block (40 units, Stratagene) and 1 µl of
RNA polymerase (50 units, Stratagene). Reactions were
incubated at 37°C for 2 hr then stopped with 2 µl of 0.2
M EDTA, pH 8.0. The RNA was precipitated with 2.5 µl
of 4M LiCl and 75 µl of 100% ethanol at 220°C
overnight. The RNA was collected by centrifugation,
washed once with 70% ethanol, dried, and stored in 100
µl H2O and 1 µl of RNase inhibitor.
Whole Mount In Situ Hybridization With
Non-radioactive Detection
Whole mount in-situ hybridizations were performed
on E9.5–E10.5 mouse embryos essentially as described
(Henrique et al., 1995). (The date of the vaginal plug is
252
PERKINSON AND NORTON
development, embryos were washed with PTW, refixed,
dehydrated and stored in 100% methanol.
Construction of the Transgene and Verification
of Its Structure
The plasmids pFNZ4.9 and pFNZ0.9 contain a lacZ
reporter flanked by sequences from the rat FN gene,
and the construction of these plasmids and their characterization in cultured cells has been reported elsewhere
(Perkinson et al., 1996). Briefly, a 4.9 kilobase pair
(kbp) PstI fragment was excised from lrFN-9 (Patel et
al., 1987) and inserted upstream of lacZ; this fragment
includes the transcription initiation site and 136 nucleotides of 58 untranslated sequence. A SacII-HindIII
fragment containing most of the 38 untranslated sequence and 204 nucleotides beyond the site of polyA
addition was excised from lrFN-1 (Patel et al., 1987)
and inserted downstream of lacZ. Thus, lacZ transcripts from pFNZ4.9 should contain 136 nucleotides of
58 and 513 nucleotides of 38 untranslated sequence
derived from rat FN (the complete regions are 207 and
691 nucleotides, respectively). The lacZ reporter was
designed so as to be translated efficiently in eukaryotic
cells, and the b-galactosidase fusion protein that results is typically nuclear or peri-nuclear in localization
(McInnis et al., 1995). Similarly, pFNZ0.9 contained a
StuI-PstI fragment of 880 bp inserted upstream of the
lacZ reporter.
To confirm the structure of the 58 flanking region of
the rat FN gene, primers were designed based on the
sequence of the furthest upstream portion of the 4.9 kbp
fragment for the forward primer (unpublished data)
and on published sequence for the reverse primer
(Nakajima et al., 1992; Sporn and Schwarzbauer, 1995).
Forward (58-GATTCTGTGCATGGGAGC-38) and reverse (58-CCACAAATATGGCAGATC-38) primers were
used in PCR reactions with Taq DNA polymerase
(Stratagene) or eLongase Polymerase mixture (Gibco/
BRL), following manufacturer’s recommended conditions. Templates were either 0.2 µg rat liver genomic
DNA (gift of D. DeSimone) or 25 pg of plasmid DNA;
amplification was for 35 cycles (94°C, 30 sec; 52°C, 30
sec; and 68 or 72°C, 3 min). Aliquots were analyzed by
agarose gel electrophoresis and visualized by photographing the ethidium bromide stained gel. The photograph was scanned and the TIFF image was reversed
with NIH Image.
Production and Breeding of Trangenic Mice
Prior to microinjection, the FN-lacZ fragment was
separated from vector sequences by digestion with NotI
(pFNZ4.9) or NotI plus BglII (pFNZ0.9) and gel purification. Microinjection of DNA into one-cell mouse embryos and embryo transfer was performed at the Jefferson Cancer Institute Transgenic Mouse Facility.
Embryos were derived by mating C57BL/6J 3 C3H/
HeJ F1 hybrids. In subsequent generations, transgenic
individuals were bred to C3H males and females.
Embryos for lacZ analyses were derived from timed
matings or from mice sacrificed when obviously pregnant. In the latter case, all the members of the litter
were examined for length, number of somites (in the
case of younger embryos) or appearance of limbs (older
embryos), which were used as criteria to establish the
approximate gestational age of the group (Kaufman,
1992).
Detection of lacZ Transgene
Pups were weaned at 3 to 4 weeks of age and tail
biopsies were performed essentially as described (Hogan
et al., 1994) and resuspended in 100 µl TE (10 mM Tris,
pH 8.0, 0.1 mM EDTA). PCR was carried out in a
volume of 100 µl using 1.0 µl genomic DNA, 200 µM
each dNTP, 10 µl 10 3 reaction buffer and 2.5 U Taq
DNA Polymerase (Perkin-Elmer/Cetus or Stratagene)
and 50 pmol of each lacZ-specific primer (forward,
58-CGTAATAGCGAAGAGGCCCG-38; reverse, 58-GCCCGTTGCACCACAGATGA-38). Amplification was for
30 cycles (1 min, 94°C; 1 min, 62°C; and 1 min, 72°C).
Twenty microliter aliquots were analyzed by agarose
gel electrophoresis and visualized by staining with
ethidium bromide. A 363 bp product was obtained with
individuals whose genome contained a copy of the
transgene. Transgene copy number was determined by
dot blot hybridization analysis of DNA isolated from F1
progeny mice, using a random primer labelled fragment
of lacZ as a probe; standard protocols were followed
(Sambrook et al., 1989). Blots were exposed to a Molecular Dynamics PhophorImager screen for quantitation.
Histochemical Assay for b-Galactosidase
Non-transgenic C3H females were mated to transgenic males; the date of plug is considered E0.5 (embryonic day 0.5). Females were sacrificed at E8–12 and
embryos were dissected out in PBS. Treatment of
embryos was similar to that described (Bonnerot and
Nicolas, 1993; Hogan et al., 1994). Embryos were fixed
in 10 ml of cold freshly prepared 4% paraformaldehyde
in PBS containing 0.02% NP40, 0.01% sodium deoxycholate, 2 mM MgCl2, and 5 mM EGTA (PBS rinse mix),
according to age (from 5 min for E8 to 20 min for E12.5).
After fixation, embryos were washed three times with
10 ml of PBS rinse mix at room temperature. Staining
was in PBS containing 50 mM K3Fe(CN) 6, 50 mM
K4Fe(CN) 6, 0.02% NP40, 0.01% sodium deoxycholate, 2
mM MgCl2, 1 mM EGTA and 1.0 mg/ml X-gal (5-bromo4-chloro-3-indolyl-b-D-galactoside; stock, 40 mg/ml in
dimethylformamide) at 37°C from 2 to 48 hr. Following
color development, embryos were post-fixed in 4%
paraformaldehyde in PBS. In some cases, embryos were
dehydrated in ethanol, and cleared in methyl salicylate.
Immunohistochemistry
Embryos that had been postfixed following X-gal
staining were dehydrated, embedded in paraffin and
7.0 µm serial sections were cut and dried. Prior to
immunodetection, sections were deparaffinized, and
rehydrated into PBS. After blocking with 5% dry milk
FIBRONECTIN AND SOMITOGENESIS
powder in Tris-buffered saline, sections were incubated
first with a polyclonal antibody directed against mouse
FN (Telios). Following three washes in PBS, horseradish peroxidase-conjugated goat anti-rabbit IgG (BioRad) was added. After three washes, substrate (0.025%
diaminobenzidine, 0.03% H2O2 ) was applied, and development was monitored visually.
ACKNOWLEDGMENTS
We thank A. Gehris for help with generating sections,
K. Cheah for providing detailed protocols for lacZ
detection, and R. McInnis for technical assistance during the early stages of this project. We are grateful to D.
Henrique, D. Ish-Horowitz, S. Sporn and J. Schwarzbauer for communicating data prior to publication and
to R. Hynes for the lambdaphage clones. Many thanks
go to our colleagues V. Bennett, A. Gehris, and G.
Grunwald for helpful comments on manuscript.
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