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Expression of Multiple CD44 Isoforms in the Apical
Ectodermal Ridge of the Embryonic Mouse Limb
Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, Massachusetts 02111
Previous immunohistochemical
studies have shown that CD44 is highly enriched
within the apical ectodermal ridge of the developing limb (Wheatley et al. [19931 Development 119
295-3061, but the particular isoforms of CD44 were
not identified. We show here that CD44s (standard
or “hemopoietic” isoform) and several CD44 variants, especially V3-Vl0, VPV10, and V6V10, are
concentrated in the apical ectodermal ridge in the
early mouse limb. Since CD44s is a major cell surface receptor for hyaluronan, we compared its localization with that of hyaluronan. In the early
limb bud, hyaluronan is distributed throughout
the mesoderm but is absent from all regions of the
ectoderm. Hyaluronan is especially enriched in
the basement membrane separating ectoderm and
mesoderm, except beneath the apical ectodermal
ridge where it is absent. Since CD44s is a known
endocytic receptor for hyaluronan, its presence in
ridge ectoderm could lead to degradation of hyaluronan destined for the neighboring region of
basement membrane, thus facilitating interaction
of the ridge with underlying mesoderm. The CD44
(V3-VlO) isoform found in the ridge is expressed
elsewhere as a proteoglycan with heparan sulfate
chains that bind fibroblast growth factors. Since
fibroblast growth factors are present in the ridge
and are essential for limb morphogenesis, CD44
(V3-V10) is likely to act as a cofactor or modulator
in the growth-promotingaction or maintenance of
the ridge. o ISSS Wiley-Liss, Inc.
Key words: Mouse limb development, CD44 isoforms, Hyaluronan
with cytoskeletal proteins (Lacy and Underhill, 1987;
Bourguignon et al., 1993; Tsukita et al., 1994) and contains serine residues that can be phosphorylated
(Neame et al., 1992). Exons 6-15 of CD44 (usually
termed V1-V10) are alternatively spliced into the
membrane-proximal region to give numerous additional isoforms (Screaton et al., 1993; Sherman et al.,
1994). CD44 is also extensively glycosylated, with both
oligosaccharide and glycosaminoglycan side chains,
thus generating further diversity among the various
CD44 isoforms.
Although it is clear that hyaluronan is a major
ligand for CD44s (Aruffo et al., 1990; Culty et al.,
19901, CD44 variants do not usually bind hyaluronan
as avidly as CD44s (Jackson et al., 1995). However,
interactions within the plasma membrane can influence binding to hyaluronan in a positive or negative
fashion (He et al., 1992; Lesley et al., 1992, 1993). As
yet, other ligands for most CD44 variants have not
been clearly established, except that proteoglycan
forms of CD44 bind extracellular matrix proteins (Jalkanen and Jalkanen, 1992; Faassen et al., 1992) and
growth factors (Tanaka et al., 1993; Bennett et al.,
1995) via their glycosaminoglycan side chains. Of special interest is the recent finding that CD44 variants
incorporating the V3 exon are preferentially conjugated with heparan sulfate side chains that bind basic
fibroblast growth factor (FGF-2) and other heparinbinding growth factors (Jackson et al., 1995;Bennett et
al., 1995).
CD44 isoforms containing variant exons are commonly found in normal adult and fetal epithelia (Terpe
et al., 1994; Mackey et al., 19941, in several types of
leukocytes (Arch et al., 1992; Mackay et al., 19941, and
in metastatic cancer cells (Gunthert et al., 1991; Rudy
et al., 1993). Immunohistochemistry has been used to
document the presence of CD44 during specific stages
of development of a variety of tissues and organs (Underhill, 1993; Wheatley et al., 1993; Pavasant et al.,
1994). One of the major conclusions to emerge from the
latter work, together with several cellular and biochemical studies (Orkin et al., 1982; Culty et al., 1992,
Hua et al., 1993; Underhill et al., 19931, is that CD44s
mediates internalization of hyaluronan en route to in-
CD44 is a widely distributed integral cell surface
glycoprotein expressed in many adult and embryonic
tissues (Kennel et al., 1993; Wheatley et al., 1993;
Lesley et al., 1993). There is a single gene for CD44
which, in the mouse, contains 20 exons. Exons 1-5,
16-18, and 20 give rise to CD44s (standard or “hemopoietic” form of CD44), the predominant isoform
present in most tissues. This isoform contains an
extracellular domain with an N-terminal hyaluronanbinding site and a membrane-proximal region, a transReceived January 25, 1996;accepted May 22, 1996.
membrane domain, and a C-terminal cytoplasmic doAddress reprint requestslcorrespondence to Bryan P. Toole, Departmain (Stamenkovic et al., 1989; Goldstein et al., 1989; ment
of Anatomy and Cellular Biology, Tufts University School of
Zhou et al., 1989). The cytoplasmic domain interacts Medicine, Boston, MA 02111.
product obtained using RNA from the rest of the limb
tissues was -110 bp, corresponding to CD44s (Fig. lC,
lane 3).
The PCR products detected above were cloned into
the pCRII vector, and then plasmids were prepared and
digested with EcoR1. The inserts were subjected to agarose electrophoresis to confirm their size and purity
(Fig. 1D) and fully sequenced. The sequences were then
compared with published mouse genomic sequences for
the various CD44 isoforms (Screaton et al., 1993). The
sequences of the 110-bp products obtained from RNA
derived from AER-enriched tissue or from the remaining limb tissues corresponded in both cases to the expected regions of exons 5 and 16 of CD44, thus confirming the presence of CD44s in these tissues. The two
larger products of -750 bp and -1,000 bp, detected
only in the AER-enriched tissue, were found to contain
the same partial exon 5 and 16 sequences as above plus
the entire V6-V10 and V4-V10 groups of exons, respectively. In addition we cloned and fully sequenced
several minor products from the AER-enriched tissue;
these isoforms contained exons V4N6N9-Vl0, V4l
V8-Vl0, V7-VIO, V8-VIO (E-form), V9, and V9-10
(Fig. 2).
To verify the above results and to obtain additional
information regarding the types of CD44 variants
present in the AER region of the limb, we used additional RT-PCR strategies. First, we used sense primer
2 from the experiments above (Fig. lB), together with
antisense primers corresponding to portions of exons
V3 through V10, as described in Experimental Procedures and Table 1. In this series of experiments two
major products were obtained (Fig. 3A); these products
corresponded in size to the CD44 variants containing
exons V4-V10 and V6-V10 and were verified as such
by partial sequencing, thus confirming our results
above. Second, we used primer 1 from above (Fig. 1B)
Identification of Several CD44 Isoforms in the
with sense primers from exons V1 through V10. An
Distal Region of the Early Limb Bud
additional major amplified product was obtained (Fig.
Approximately 100 E l 0 mouse limbs were dissected 3B, lane 4)) which was sequenced completely and ideninto two regions, one of which contained the AER plus tified as corresponding to the CD44 variant containing
a small amount of adherent mesoderm; the other re- exons V3-V10. In both series, several other minor
gion included the rest of the limb (see Fig. 1A). Total variants were detected and confirmed, by partial seRNA was then prepared from separate pools of these quencing, to be the same as those found by the first
limb regions, and reverse transcription-polymerase strategy. Also, small amounts of CD44(V10),and CD44
chain reaction (RT-PCR)was performed as described in (V2-V3) were found.
In summary, CD44 isoforms expressed in AER-enExperimental Procedures, using primers 1and 2 which
flank the site of insertion of variant exons in CD44 riched tissue from E l 0 mouse limbs include CD44s,
(Fig. 1B). If CD44s is the only isoform present in these CD44 (V3-V10), CD44 (V4-V10) and CD44 (V6-VlO).
tissues, the RT-PCR product would be 110 bp in size Several other minor variants are also present. The maand would include portions of exon 5 and exon 16 of jor isoform expressed in the rest of the limb is CD44s.
CD44. If isoforms other than CD44s are present, larger
products would be obtained. As shown in Figure lC, Localization of CD44 Isoform mRNAs in the
lane 2, three significant amplified products were ob- AER by In Situ Hybridization
The dissected regions of the E l 0 limbs used above for
tained using RNA prepared from the AER-enriched tissue. One of these was -110 bp and thus corresponded RT-PCR were cross-contaminated to a small extent.
to CD44s; however, the other two products were larger, Thus, for example, it is possible that the variants de-750 bp and -1,000 bp. Several minor bands were also tected in the AER-enriched tissue could have been deobtained between 200 and 700 bp. The only significant rived from a small amount of contaminacing subecto-
tracellular degradation. A second conclusion is that
CD44 is often present at sites of epithelial-mesenchyma1 interactions that are known to be important in
morphogenesis (Underhill, 1993; Wheatley et al.,
1993). However, the CD44 isoforms present in these
developing tissues have not been identified or specifically localized, although preliminary data obtained by
size estimation (Wheatley et al., 1993) and polymerase
chain reaction (PCR) (Ruiz et al., 1995) indicate that
many isoforms are likely to be present during morphogenesis of a number of tissues and organs.
The studies of Wheatley et al. (1993) demonstrated a
striking localization of CD44 to the apical ectodermal
ridge (AER) of the mouse limb bud. The AER is known
to interact with distal limb mesoderm and thereby control growth and shape during early stages of limb bud
morphogenesis (Saunders, 1948; Summerbell et al.,
1973). FGF-2, FGF-4, and FGF-8 are present in the
AER and appear to mediate at least part of the function
of the AER (Niswander et al., 1993; Fallon et al., 1994;
Crossley and Martin, 1995; Ochiya et al., 1995). Since
V3-containing isoforms of CD44 have been shown to
bear heparan sulfate side chains that bind FGF (Bennett et al., 1995), it would be of particular interest to
know whether these forms are present in the AER. In
addition, whereas there is considerable evidence that
hyaluronan plays an important role in the chick embryo limb (Toole et al., 1991), a recent study failed to
detect hyaluronan in the developing mouse limb (Fenderson et al., 1993). Since CD44s is a likely component of
the AER (Wheatley et al., 1993), the relative distribution of hyaluronan and CD44s is also of significant interest. Because of the above issues, the current study
was conducted to establish the nature of CD44 isoforms
during morphogenesis of the mouse embryo limb.
Dissection o f E l 0 limb b u d
beneath AER
Plane o f dissectlon
CD44 gene:
Fig. 1. Identification of CD44 isoforms in AER-enriched tissue by RfPCR. A: Diagram of E l 0 mouse limb with the AER shown in longitudinal
section. The AER-enriched region was dissected along the thick line, thus
separating the AER and a small amount of attached mesoderm from the
rest of the limb. Total RNA was then prepared from these two areas for
RT-PCR. B: Diagram of CD44 gene showing the exons of CD44s (sl-s9)
and of the variant isoforms (VI-VIO), as well as the locations of antisense primer 1 and sense primer 2. C: Agarose electrophoresis of RTPCR products. Lane 1: 100-bp DNA ladder. Lane 2: RT-PCR using total
RNA from the AER-enriched tissue. Lane 3: RT-PCR using total RNA
from the remaining tissues of the limb. Arrowheads indicate the major
bands: CD44 (V4-V10), CD44 (V6-VIO), and CD44s in lane 2 and
CD44s in lane 3. 0 : pCRll clones of RT-PCR products. Lane 1: 100-bp
DNA ladder. Lanes 2-1 1: pCRll clones containing inserls corresponding
to the RT-PCR products from CD44s, CD44 (V4-V10), CD44 (V6-V10),
CD44 (V4N6N9-V10), CD44 (V4N8-V10), CD44 (V7-V10), CD44
(VS-VlO), CD44 (V9), CD44 (V9-VIO), and CD44 (V3-V10), respectively.
CD44 isoform
RT-PCR products
$5 $6
$5 U4 U5 U6 U7 U8 U 9 U l O s 6
s 5 U 6 U 7 US U9 U 1 0 s 6
6 5 U 4 U 6 U9U10a6
t 5 U 4 U 8 U9UtOs6
$5 U7 U8 U9 U10 $6
s 5 U S U 9 UlOs6
$5 U 9 s 6
$5 U9 U10 $ 6
U9-UI 0
U3 u4 U5 U6 U7 U8 U 9 U 1 0 s 6
Fig. 2. CD44 isoforms identified by RT-PCR, pCRll cloning, and sequencing. lsoforms were identified as described in Figure 1, and comparison of sequences was obtained with published CD44 genomic sequences (Screaton et al., 1993). The major RT-PCR products identified
corresponded to CD44s. CD44 (V4-V10), and CD44 (V6-VlO); other
minor products identified were V4N6N9-VI0, V4NB-VI 0, V7-VI0,
V8-VIO (E form), VQ, and V9-VIO. An additional prominent product,
CD44 (V3-VIO), was identified as described in Figure 3.
dermal mesoderm rather than the AER itself.
Consequently we determined the precise location of
these variants by in situ hybridization using several
digoxigenin-labeled antisense riboprobes. Probe H3-5
(410 bp) is derived from the 5’ common region a€0 4 4
and would detect all isoforms. Probe V4-5 (230 bp)
contains exons V4 and V5 and would detect variants
Fig. 3. Confirmation of identities of major CD44 variants in AER-enriched tissue. A: Sense primer 2 and antisense primers from exon VIO,
V9, V8, V7, V6, V5. V4, or V3 were used in RT-PCR (lanes 2-9, respectively); lane 1, 100-bp DNA ladder. The sizes obtained for the two major
products are those expected for CD44 variants, V4-VIO and V6-VIO
(arrowheads in lanes 2-6); the arrowheads in lanes 7 and 8 indicate the
product expected for V4-V10; the arrowhead in lane 9 indicates that
expected for V3-V10. 8: Antisense primer 1 and sense primers from
exon V1, V2, V3, V4,V5, V6,V7, V8, V8, or V10 were used (lanes S11,
respectively): lane 1, DNA ladder. The size obtained for the major product in lane 4 (arrowhead) is that expected for CD44 variant, V3-V10. The
identities of the variants were conflrmed by sequencing.
CD44 (V3-V10) and CD44 (V4-VlO) but not CD44s,
CD44 (V6-V10), or several of the other minor variants.
Probe V4-7 (472 bp), probe V6-7 (242 bp), probe
V6-10 (406 bp), and probe V8-VIO (398 bp) would detect most variants but not CD44s. As controls, sense
probes corresponding to all of the above antisense
probes were used.
Figure 4 shows the results obtained with some of the
above probes for the El0 limb bud. All of the antisense
probes gave a strong signal €or mRNA in the AER,
although the short probes (V4-5 and V6-7) gave
slightly less intense signals than the longer probes.
TABLE 1. RT-PCR Primed
Antisense primers
Sense primers
T h e sequences were derived from Screaton et al.
(1993) (GenbanWEMBL accession number L13611).
Very low signals were obtained in the remaining ectoderm and in the mesoderm in all cases (Fig. 4A-C). NO
signal was obtained in any region of the limb with any
of the sense probes (e.g., see Fig. 4D). These results,
along with the results of RT-PCR, indicate that all of
the variant CD44 isoforms are virtually restricted to
the AER.
Although CD44s was detected in the mesoderm by
RT-PCR, the in situ signal observed with all of the
probes was diffuse and close to background level. This
continued to be the case at later developmental stages
prior to differentiation. As differentiation progressed
in the limb, CD44s was detected by RT-PCR and in situ
hybridization in putative osteoprogenitor cells within
the erosion zone of developing bone (also see Pavasant
et al., 1994) and in the perichondrium. The E isoform,
CD44 (VS-VlO), was also present in the perichondrium. No other variants were detected in these structures (data not shown).
Western Blot Analysis of CD44 in Limb Tissues
Although our in situ results showed that the concentration of CD44 in the mesoderm and non-AER ectoderm is low relative to the AER, our RT-PCR results
indicated that some CD44s is present outside the AER.
Unfortunately, it is not possible to design an in situ
probe specific for CD44s, and therefore we cannot directly demonstrate its presence in the AER by this
method. Thus, there is a slight possibility that CD44s
detected by PCR in the AER-enriched tissue may have
been amplified from a small amount of contaminating
mesoderm or non-AER ectoderm. To rule out this possibility we performed Western blots of extracts obtained from AER-enriched tissue and the remaining
limb tissues (mesodedectoderm). An -85-kDa pro-
tein corresponding to CD44s was readily detected as
the main CD44 isoform in the AER sample (Fig. 5).
When equivalent samples of AER and mesodedectoderm were used, CD44s was seen to be much more concentrated in the AER sample than in the remaining
tissues (compare lanes 1and 2 in Fig. 5).Thus, a t most,
a small proportion of CD44s in the AER sample could
have been derived from mesodermal or non-AER ectodermal contamination, and we conclude that CD44s is
a major CD44 isoform in the AER.
Distribution of Hyaluronan During Early Mouse
Limb Development
The results presented above indicate that one of the
major CD44 isoforms expressed by the AER is CD44s,
the hyaluronan receptor ( A d o et al., 1990; Culty et
al., 1990).This finding suggests that the distribution of
hyaluronan may be related in some way to the AER.
Consequently we used the bPG probe (see Experimental Procedures) to determine whether we could detect
hyaluronan in the mouse limb bud a t the time that the
AER is present and expresses CD44s.
We found staining for hyaluronan at a significant
level throughout the mesoderm, but not in the the ectoderm, of E9 (Fig. 6B) and E10.5 (Fig. 6D,G) limb
buds. At the latter stage there is a particularly strong
reaction for hyaluronan in the basement membrane
underlying the ectoderm of the limb buds. However
this staining is interrupted beneath the AER (Fig.
6D,G). Strong reactivity for hyaluronan persists in the
peripheral mesoderm of the limb bud (Figs. 611, but
staining decreases markedly in the condensed mesoderm prior to cadilage differentiation (data not
We also compared the distribution of hyaluronan
with CD44 by immunohistochemistry. As reported by
Wheatley et al. (1993), immunoreactivity with antiCD44 mAb was strong within the AER of the E10.5
hindlimb (Fig. 6C,E,F) but was greatly reduced in the
ectoderm of earlier (Fig. 6A) and later (Fig. 6H) stage
limb buds; this distribution is in agreement with that
of mRNA transcripts as obtained by in situ hybridization (Fig. 4). CD44 distribution as determined by immunohistochemistry or in situ hybridization was also
concordant at later stages of limb development. Immunoreactivity at all stages was similar with or without
hyaluronidase pretreatment of the sections.
Two outcomes of this study provide potentially new
insights into AER function during limb bud morphogenesis. The first of these is identification of several
CD44 isoforms that are highly enriched in the AER
relative to mesoderm and other regions of the ectoderm
of the early limb bud. The major CD44 isoforms detected in the AER, i.e., CD44s, V3-Vl0, V4-V10, and
V6-Vl0, are likely to exhibit different biochemical activities and fulfill different functions in the AER. Our
second striking observation is that the basement mem-
Fig. 4. Localization of CD44 variant mRNAs to the AER by in situ
hybridization. E l 0 mouse embryo limb buds were hybridized with digoxigenin-labeled riboprobes to common and variant regions of CD44.
A: H3-5 antisense probe to wmmon region of CD44. B: V4-7 antisense
probe. C: V4-5 antisense probe. In all three cases, as well as with the
V6-7, V6-10, and V8-10 antisense probes (not shown), strong positive
signals were obtained in and restrictedto the AER. 0: H3-5 sense probe;
no signal was detected in the AER or elsewhere in the limb. The same
negative result was obtained with the other five sense probes (not
shown). Bar = 40 Fm.
several systems is mediation of hyaluronan uptake en
route to degradation by lysosomal enzymes (Orkin et
al., 1982; Culty et al., 1992, Hua et al., 1993; Underhill
et al., 1993; Underhill, 1993; Pavasant et al., 1994).
Possibly then, CD44s on the surface of AER cells mediates removal of hyaluronan from the basement membrane region directly beneath it. Consistent with this
idea is the fact that lysosomal hyaluronidase is elevated in distal limb ectoderm relative to other regions
of the limb (Kulyk and Kosher, 1987) and would thus
degrade internalized hyaluronan in the AER region.
Another hyaluronan-binding protein that has been
implicated in endocytosis of hyaluronan is intercellular adhesion molecule-1 (ICAM) (McCourt et al.,
1994). We have surveyed several stages of mouse embryo development for the distribution of ICAM and
found that there is very low and diffuse expression in
Fig. 5. identification of CD44s in extracts of AER and mesoderm. the limb bud but high levels in some other areas of the
Extracts of AER-enriched tissue and the remaining limb mesoderm/ec- embryo (unpublished data). Thus, it is unlikely that
toderm were analyzed by SDS-PAGE and Western blotting, using anti- ICAM would be responsible for removal of hyaluronan
body KM201 against CD44. Lane 1:AER-enriched extract, 20 pg protein.
Lane 2: Mesoderm/ectodermextract, 20 pg protein. Lane 3: Mesoderm/ from the sub-AER basement membrane. The depletion
ectoderm extract, 50 pg protein. Lanes 4-6: Identical with lanes 1-3 in of hyaluronan in this region may facilitate AERmethe absence of primary antibody. The arrow indicates the position of soderm contact and subsequent morphogenesis in a
migration of CD44s.
similar manner to that of glycosaminoglycan turnover
in the basement membrane during epithelial branching (Bernfield et al., 1984).
Although it is clear that a major ligand for CD44s is
brane region immediately beneath the AER is depleted
of hyaluronan relative to the basement membrane un- hyaluronan, this is not the case for the variant CD44
isoforms (Jackson et al., 1995). We have found that the
derlying other regions of the ectoderm.
One of the most prominent isoforms of CD44 in the same CD44 isoforms as described in this study are also
AER is CD44s, a well-characterized hyaluronan recep- present in many other inductive epithelia undergoing
tor. A role that has been ascribed to this receptor in active morphogenesis, including epithelia of develop-
Fig. 6. Distributionof hyaluronan and CD44 proteinduring early mouse
limb development. In A,C,E,F, and H, mAb KM201 was used for detection
of CD44. In B,D,G,I, hyaluronan was stained with bPG. A: Longitudinal
section of E9 forelimb. Arrows indicate dispersed CD44-positive cells in
the mesoderm. The rest of the mesoderm and the ectoderm are virtually
negative. 6: Longitudinal section of E9 forelimb showing hyaluronan
present throughout the mesoderm but absent from the ectoderm (arrowheads). C,D: Cross sections of E10.5 hindlimb at high power, showing the
presenceof CD44 and the absenceof hyaluronan,respectively, in the AER
(arrowheads). Hyaluronan staining is strong in the basement membrane
(solid arrows) underlying the ectoderm except beneath the AER (open
arrows), where it is absent. E,F: Longitudinaland cross sections, respectively, of E10.5 hindlimb showing that CD44 is restricted to the AER
(arrowheads)and a few dispersed cells in the mesoderm. G: Cross section
of E10.5 hindlimb showing that hyaluronan is distributed throughout the
mesoderm, is highly enriched in the basement membrane (solid arrows)
except beneath the AER (open arrows), and is absent from the entire
ectoderm including the AER (arrowheads). H,I: Longitudinal sections of
E l 2 hindlimb, showing the absence of CD44 immunoreactivity from the
ectoderm and mesoderm and the presence of hyaluronan in the mesoderm but not the ectoderm. Bar in I = 80 pm and applies to A,B,F,G and
H: bar in D = 25 pm and applies to C; bar in E = 100 pm.
ing teeth, nose, ear, and hair follicles (Yu and Toole,
unpublished data). Some of these CD44 variants are
also expressed by adult epithelia (Terpe et al., 1994;
Mackay et al., 19941, and many are expressed by malignant carcinoma cells where they are thought to confer metastatic properties to these cells (Gunthert et al.,
1991; Rudy et al., 1993). The ligands and cellular roles
of most of these variants are not established, but it
seems very likely that they are important in some aspect of morphogenetic cell behavior.
One variant of particular interest is CD44 (V3-V10),
which was found to be present at readily detectable
levels in AER-enriched tissue. The V3 exon encodes the
optimal consensus motif for addition of heparan sulfate
side chains, and thus V3-containing isoforms are usually expressed as heparan sulfate proteoglycans. These
proteoglycans bind FGF avidly (Jackson et al., 1995;
Bennett et al., 1995). Since members of the FGF family
are localized within the AER and are critical for its
morphogenetic action (Niswander et al., 1993; Fallon
et al., 1994; Crossley and Martin, 1995; Ochiya et al.,
1995), heparan sulfate-conjugated CD44 (V3-VlO)
may be involved in retaining FGF preferentially at the
surface of AER cells and presenting it in an appropriate configuration to promote cellular proliferation in
the mesoderm immediately underlying the AER or
within the AER itself (Rapraeger et al., 1991; Yayon et
al., 1991; Spivak-Kroizman et al., 1994). It is interesting to note that other heparan sulfate-proteoglycans
that might participate in such events, i.e., syndecan
and perlecan, are not present in the AER (Solursh and
Jensen, 1988; Solursh et al., 1990; Gould et al., 1992).
Even though FGF-2 is produced throughout most of the
early limb bud ectoderm (Savage et al., 19931, it may be
unable to stimulate proliferation outside the AER, or
the region of mesoderm immediately subjacent to the
AER, because of the absence of CD44 (V3-VlO) from
non-AER limb ectodenn, The latter point together with
restriction of other fibroblast growth factors to the
AER (Niswander et al., 1993; Crossley and Martin,
1995)could provide part of the mechanism whereby the
AER controls directional outgrowth, as has been established in previous biological experiments (Saunders,
1948; Summerbell et al., 1973). Although it is still not
clear how FGF-2 is secreted, the function of FGF-2 in
non-AER ectoderm may be to regulate homeobox gene
expression, e.g., Msx-1 (Wang and Sassoon, 1995), or
extracellular matrix composition in the subectodermal
mesoderm (Knudson et al., 1995) so as to facilitate the
differentiation program unique to this region.
The role of other CD44 variants in the AER, especially V4-VIO and V6-VIO which are present in considerable amounts, is not clear. However, their restricted localization to the AER and to other instructive
epithelia (Yu and Toole, unpublished data) suggests
strongly that they participate in the function or maintenance of these specialized epithelia, possibly through
interactions with other morphogenetic factors, extracellular matrix macromolecules, or cell adhesion components.
TABLE 2. PCR Primers for In Situ Probesa
The sequences were derived from Screaton et al.
(1993) (GenbanmMBL accession number
cDNA Cloning and Sequencing
The products from PCR were either directly cloned
into the pCRII cloning vector (Version 2.3, Invitrogen,
San Diego, CA) or purified using Geneclean (BiolOl
Inc, La Jolla, CA) from 1%agarose gels, then cloned
into the pCRII cloning vector. The nucleotide sequences of the inserts were determined using the
dideoxy chain termination method with Sequenase (US
Biochemical Corp., Cleveland, OH) from doubleDetection of CD44 Isoforms by RT-PCR
stranded templates following the manufacturer's instructions.
Total RNA was isolated from mouse embryonic tissues using TRIzol reagent (Gibco-BRL)according to the Preparation of Tissue Sections
manufacturer's instructions.
Embryos were obtained from pregnant Swiss WebcDNA was synthesized using Superscript I1 RNase
H- reverse transcriptase (Gibco-BRL) following the ster mice (Taconic Laboratory, Germantown, NY), and
manufacturer's instructions. Briefly, 5 pg of total RNA embryonic age was determined from the time of apwas first incubated with 1 pl of 500 p.g/ml oligo pearance of a vaginal plug. Embryos of different stages
(dT),z-le (Gibco-BRL)at 70°C for 10 minutes, then with were fixed in 4% paraformaldehyde (Tousimis, RockSuperscript I1 RNase H- reverse transcriptase at 37°C ville, MD) in phosphate-buffered saline (PBS), washed
for 1hour, followed by 1p1 of RNase H (2.7 U/p1; Gibco- in PBS, dehydrated through 30%,70%, 95%, 100%ethBRL) at 37°C for 15 minutes to degrade the RNA tem- anol and xylene, and then embedded in paraffin wax
(Fisher, Columbia, MD). Sections (6-8 pm) were cut,
on slides, and used for immunohistochemisTwo 20-mer oligonucleotides, primers 1and 2, corresponding to sequences that flank the variant exons of try, hyaluronan-affinity histochemistry, or in situ hyCD44 (see Table l), were used for the first set of PCR bridization.
experiments. In the second set of PCR experiments,
sense primer 2 was used with antisense primers from In Situ Hybridization
Six different sets of primers, the sequences of which
exons v10, v9, v8, v7, v6, v5, v4, v3. In the third set of
PCR experiments, antisense primer 1 was used with are given in Table 2, were used in PCR to generate six
sense primers from exons v l , v2, v3, v4, v5, v6, v7, v8, different probes. These probes are 1)H3-5, composed
v9, v10 (see Table 1 for details). The variant exon se- of nucleotides 6-416 of CD448, which is common to all
quences were derived from Screaton et al. (1993) (Gen- isoforms of CD44; 2) V4-5, spanning exons V4 through
bankEMBL accession number L13611). PCR was per- V5; 3) V4-7, spanning exons V4 through V7; 4) V6-7,
formed using Ampli Taq DNA polymerase (Perkin spanning exons V6 through V7; 5) V6-10, spanning
Elmer, Norwalk, CT) according to the manufacturer's from within exon 6 to within exon 10; and 6) V8-10,
instructions. After 35 cycles a t 94°C for 30 seconds, spanning exons V8 through V10. The PCR products
55°C for 30 seconds, and 72°C for 1minute, followed by were cloned into pCRII vector separately, and then a
a final 5 minutes a t 72"C, 10 p,1 of the PCR reaction single clone was picked for each insert and sequenced
products were analyzed by 1% agarose gel electro- to confirm authenticity and orientation.
Plasmids containing the inserts were prepared using
phoresis. The remaining portions of each sample were
a Qiagen plasmid Midikit (Qiagen Inc, Chatsworth,
used for cDNA cloning.
2 12
CA). These plasmids were linearized using BamHI or
XhoI (Gibco-BRL)and transcribed with T7 or SP6 RNA
polymerase (Promega, Madison, WI) in the presence of
digoxigenin-conjugated UTP (Boehringer-Mannheim,
Indianapolis, IN) to make sense or anti-sense digoxigenin riboprobes. After transcription a t 37°C for 2 hours,
RQ1 DNase (Promega) was used to degrade the DNA
template. Digoxigenin-riboprobes were precipitated in
the presence of LiCl and ethanol. The quantity of labeled RNA was checked by dot-blotting with alkaline
phosphatase-conjugated polyclonal antibody to digoxigenin (Boehringer-Mannheim) according to the manufacturer’s instructions. Before hybridization, the
probes were denatured by heating a t 65°C for 10 minutes.
In situ hybridization was performed by adapting procedures from Rider et al. (19921, Wilkinson (1992j, and
the manufacturer’s protocols (Boehringer-Mannheim).
Briefly, paraffin sections were deparaffinized and rehydrated, treated with proteinase K and refixed with
formaldehyde, acetylated with acetic anhydride, and
dehydrated before hybridization. Hybridization was
performed overnight at 60°C. Nonspecifically bound
probe was digested with RNase, and sections were
washed in 50% formamide/lx SSC a t 60°C. Positive
signals were detected using anti-digoxigenin antibody
conjugated with alkaline phosphatase and chromogenic substrates NBT and BCIP according to the manufacturer’s instructions (Boehringer-Mannheim). The
sections were mounted in gel mount (Biomedia Corp,
Foster City, CA).
The hyaluronan-binding region of cartilage proteoglycan (aggrecan) binds strongly and specifically to hyaluronan, and thus can be used as a probe for detection
of hyaluronan in cells and tissue sections (Knudson
and Toole, 1985; Green et al., 1988).Thus, we used the
biotinylated hyaluronan-binding region of cartilage
proteoglycan (bPG, kindly provided by Dr. Charles B.
Underhill) for this purpose. After inactivation of endogenous peroxidase as for immunohistochemistry ,
sections were incubated with 2 pg/ml bPG in PBS containing 10% calf serum (Gibco-BRL, Gaithersburg,
MD) for 30 minutes a t room temperature. To determine
background staining, 2 pg/ml bPG was first mixed with
100 pg/ml hyaluronan before use; virtually no staining
was obtained when this control was used. After extensive washing with PBS, bound bPG was detected using
Vector A and B reagents (Vector Laboratories) and diaminobenzidine tetrahydrochloride dihydrate (Aldrich) according to the manufacturers’ instructions.
Tissue Extraction and Western Blotting
Approximately 200 limbs from E l 0 mouse embryos
were dissected into two parts: AER-enriched tissue and
the remaining limb tissues (mesoderdectoderm) (see
Fig. 1A). The two tissue groups were homogenized in 5
volumes of 5 mM HEPES, 2 mM MgCI,, pH 7.4, containing 2 mM PMSF, 2 pg/ml leupeptin, and 0.5 U/ml
aprotinin in Dounce homogenizers. Nuclei and cellular
debris were removed by centrifugation at 6,300g for 5
minutes. The supernatant was centrifuged at 14,OOOg
for 10 minutes, and the membrane pellets were lysed in
50 mM Tris-HC1 buffer, pH 7.4, containing 150 mM
NaCl, 5 mM EDTA, 1%Triton X-100, 0.1% sodium
dodecyl sulfate (SDS),2 mM phenylmethylsulfonyl fluoride (PMSF), 2 pg/ml leupeptin, and 0.5 U/ml aprotinin.
The membrane extracts were subjected to 10% SDSpolyacrylamide gel electrophoresis under nonreducing
conditions, then transferred to Hybond-ECL nitrocellulose membranes (Amersham, Arlington Heights, IL).
After transfer, the membranes were blocked in 1%
BSA, 1%nonfat milk, and 5 pg/ml goat IgG in 0.2%
Tween 20,0.05 M Tris-HCL, 0.15 M NaC1, pH 8.0, then
incubated with monoclonal antibody, KM201, against
CD44 (ATCC, Rockville, MD) a t 4°C overnight. After
extensive washing with 0.2% Tween 20, 0.05 M TrisHCl, 0.15 M NaC1, pH 8.0, the membranes were incubated with goat anti-rat secondary antibody conjugated
with horseradish peroxidase for 30 minutes a t room
temperature, followed by ECL detection reagents (Amersham) according to the manufacturer’s instructions.
CD44 Immunohistochemistry
For localization of CD44, we used monoclonal antibody (mAb) KM201 prepared from hybridoma cultures
(ATCC, Rockville, MD) or mAb 5D2-27 obtained form
Developmental Studies Hybridoma Bank (NICHD,
Baltimore, MD). Partially purified KM201, obtained by
50% (NH,),SO, precipitation of KM201 hybridoma culture medium was used in the experiments reported
here unless mentioned otherwise. After the sections
were rehydrated through a series of ethanol concentrations and water, they were incubated in 3% H,O, in
methanol for 1hour at room temperature to inactivate
endogenous peroxidase, and with 1%bovine serum albumin (BSA), l%nonfat milk, 10 pg/ml goat-IgG at
room temperature for 1 hour to block non-specific
staining. The sections were then incubated with 5-10
Fg/ml KM201 a t 4°C overnight. Before treatment of 3%
some sections were treated with testicular hyaluronidase (50 units/ml in 0.15 M NaC1, 0.05 M Na
acetate, pH 5.0) a t 37°C for 3 hours, but little difference
in staining was observed. Biotinylated goat anti-rat
secondary antibody followed by Vector staining reACKNOWLEDGMENTS
agents A and B (Vector Laboratories, Burlingame, CA)
We thank Drs. Tet-kin Yeo and Stuart Tobet for critand diaminobenzidine tetrahydrochloride dihydrate
(Aldrich Chemical Company, Milwaukee, WI) were ical reading of the manuscript. This research was supused according to the manufacturers’ instructions to ported by grants DE 05838 and HD 23681 from the
detect KM201 reactivity.
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