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Tenascin-C Lines the Migratory Pathways of Avian
Primordial Germ Cells and Hematopoietic Progenitor Cells
Department of Neurobiology and Anatomy, Bowman Gray School of Medicine, Medical Center Blud, Winston-Salem, North
Carolina 27157 (K.K.A.), and Department of Cell Biology and Human Anatomy, University of California, Davis, California
95616 (R.P.T.)
Tenascin-C is a large hexameric
extracellular matrix glycoprotein associated with
epithelial-mesenchymal interactions, connective
tissue development, and the formation of the central nervous system. Tenascin-C also lines the
pathways followed by migrating avian neural
crest cells, although its role in neural crest morphogenesis remains unclear. In vitro, tenascin-C
interferes with cell-fibronectin interactions, and
promotes the motility of many cell types including
the neural crest. To determine if tenascin-C is a
consistent component of matrices through which
invasive embryonic cells migrate, we have investigated if tenascin-Cis associated with 2 additional
populations of motile, embryonic cells: primordial
germ cells and hematopoietic progenitor cells. We
have found that HNK-1, a monoclonal antibody
used as a marker of neural crest, also stains avian
primordial germ cells. Double-label immunohistochemistry reveals that tenascin-C is found in the
mesenchyme adjacent to the ventral half of the
dorsal aorta where the primordial germ cells penetrate the vessel wall, and both tenascin-C and fibronectin are present in the extracellular matrix
through which the primordial germ cells migrate
to reach the genital ridges. Unlike fibronectin,
which is found throughout the splanchnic mesoderm, tenascin-C is concentrated in the proximal
part of the splanchnicregion where the primordial
germ cells are concentrated. In embryos where the
gonadal anlagen are surgically removed before the
primordial germ cells leave the bloodstream, ectopic primordial germ cells were found exclusively
in head and trunk mesenchyme containing tenascin-C. Like primordial germ cells, a subset of hematopoietic progenitor cells migrate through the
mesenchyme ventral to the dorsal aorta where
they form hematopoietic clusters. Others bud directly into the lumen of the aorta. Anti-tenascin-C
stains the mesenchyme surrounding the migrating
cells as well as the basal surfaces of the cells that
appear to be budding into the lumen. In situ hybridization with a tenascin-C-specificcDNA probe
shows that the major sources of the tenascin-C
mRNA in this region are the hematopoietic progenitor cells themselves as well as the cells in the
wall of the ventral aorta. mRNAs encoding 3 major
splice variants of tenascin-C were identified by reverse transcriptase polymerase chain reaction
(PCR) in the embryonic aorta and adjacent mesenchyme dissected from both the region of primordial germ cell and hematopoietic precursor cell
migration. These experiments indicate that tenascin-Cis a component of the migratory environment
for many motile cells in the early embryo, where it
has the potential to mediate cell-fibronectin interactions. o 1996 Wiley-Lisa, Inc.
Key words: Tenascin, Development, Extracellular matrix, Cytotactin, Cell motility,
Primordial germ cell, Hematopoietic
progenitor cell
Extracellular matrix (ECM) molecules play an essential role during cell migration (Thiery et al., 1985;
Adams and Watt, 1993). These molecules are known to
affect cell proliferation and differentiation, as well as
promote cell motility and define migratory pathways.
For example, immunohistochemistry has revealed that
the glycoprotein tenascin-C is found along the migratory pathways of avian neural crest cells, where it is
concentrated in the rostra1 half of each somite at a time
when the neural crest invades this structure (Tan et
al., 1987; Mackie et al., 1988b). In vitro studies have
shown that neural crest cells can migrate on substrata
coated with tenascin-C, and that tenascin-C added to
the culture medium will cause neural crest cells that
have attached to adhesive ECM to round up (Halfter et
al., 1989). These observations and others (ChiquetEhrismann et al., 1988; Lotz et al., 1989) have led to
the notion that tenascin-C promotes motility by helping cells detach from their substratum and move on.
Studies on a variety of pathologies involving invasive
cell behavior also support this hypothesis. For example, tenascin-C is upregulated at the margins of healing wounds (Mackie et al., 1988a) and is found in the
Received November 30, 1995;accepted February 16,1996.
Address reprint requests/correspondence to R.P. Tucker, Dept of
Cell Biology and Human Anatomy, School of Medicine, University of
California, Davis, CA 95616-8643.
stroma surrounding many types of tumors (Mackie et
al., 1987; Borsi et al., 1992).
Here, we have attempted to understand the function
of tenascin-C during cell migration by studying 2 populations of motile cells in the embryonic day (E) 3 chick
that share many properties with the neural crest: the
primordial germ cells (PGCs), and hematopoietic progenitor cells (HPCs). PGCs originate extraembryonically, and were first identified in the germinal crescent
of the E l chick where they enter the forming bloodstream (Swift, 1914). They circulate passively until E3
when they adhere to a region of the ventral wall of the
dorsal aorta. They eventually pass through the wall of
the vessel and migrate through the splanchnic mesoderm en route to the genital ridges.
In the E3 chick, the ventrolateral walls of the dorsal
aorta are thickened by basophilic cells (DieterlenLihvre and Martin, 1981). As determined by immunohistochemistry, these cells are HPCs (Cormier et al.,
1986). In E4 chick embryos, dense paraaortic hematopoietic foci are scattered in the mesenchyme from the
mesonephros to the lung rudiment (Dieterlen-Lievre
and Martin, 1981). ChicWquail chimeras (Martin et al.,
1980) and aortic cultures (Cormier et al., 1986) show
that the aortic HPCs are the source of the hematopoietic clusters. The cells from the paraaortic foci subsequently migrate to colonize the thymus of the E6 chick.
The thymus emits a chemoattractant to guide the foci
cells (Ben Slimane et al., 1983), but the early pathway
from the aorta is as yet uncharacterized.
Here, we have used immunohistochemistry, in situ
hybridization, and reverse-transcriptase polymerase
chain reaction (RT-PCR), to determine if tenascin-C is
part of the ECM associated with PGCs and HPCs in the
chick embryo. We found that tenascin-C is concentrated in the ECM through which these populations of
embryonic migratory cells pass, implicating this glycoprotein in a general role associated with promoting cell
The Antibody HNK-1 is a Marker of Chick PGCs
The established marker for avian PGCs is the PAS
reaction (Clawson and Domm, 1962; Fujimoto et al.,
1976). The PAS reaction, however, does not allow for
double-labeling studies with tenascin-C antibodies. We
have found that the monoclonal antibody HNK-1 can
be used as a marker for chick PGCs. Whole blood
smears from stage 13-14 chick embryos contain cells
with the typical size and granulated appearance of
PGCs that are positive for HNK-1 (Fig. lA,B). Cells
that display the “teardrop” shape of a white blood cell
are not positive for the HNK-1 antigen (Fig. lC,D). In
addition, PGCs were isolated on a Ficoll gradient; half
of the isolated cells were stained with PAS and the
other half with HNK-1. Large, granular cells positive
for PAS (Fig. 1E) are also positive for HNK-1 (Fig.
lF,G). The HNK-1 staining is punctate, and appears to
be on the cell surface.
Chick PGCs are also positive for HNK-1 in situ.
When sections containing PGCs were found by PAS
staining, adjacent sections were stained with HNK-1.
Similarly shaped cells in the same regions are positive
for both PAS and HNK-1 (Fig. lH,J). The HNK-1stained cells exhibit the cell-surface, punctate pattern
observed in vitro. The staining is not as intense as the
staining of nearby neural crest cells, but is consistent
and well above background. Thus, HNK-1 can be used
to identify not only the neural crest in the avian embryo, but also PGCs.
Tenascin Lines the Pathways of Normal and
Ectopic Chick PGCs
Cross-sections of E3 chicks were stained with PAS to
locate PGCs. Adjacent sections were double-labeled
with a combinations of either anti-tenascin-C and
HNK-1, or tenascin-C and fibronectin antibodies.
HNK-Utenascin-C double-labeling shows PGCs in the
mesenchyme ventral to the dorsal aorta and in the
splanchnic mesoderm (Fig. 2A,D). Tenascin-C is abundant in the ECM around the ventral wall of the aorta
and continues ventrally into the splanchnic mesoderm
where it is concentrated in or near the endoderm basement membrane (Fig. 2B). Higher magnification demonstrates that tenascin-C is in the splanchnic mesoderm corresponding to areas where PGCs are found
(Fig. 2D,E). Anti-fibronectin staining shows that fibronectin is also present, but is not confined to the PGC
pathway (Fig. 2C). Controls with secondary antibody
alone show no staining (Fig. 2F). Thus, tenascin-C is
concentrated in the ECM in regions where PGCs are
found, in contrast to the widespread distribution of fibronectin.
If the caudal third of stage 12-14 embryos is removed in ovo, and the embryo is allowed to develop for
1 more day, PGCs leave the bloodstream elsewhere
(Nakamura et al., 1991). To determine if tenascin-C is
an element common to both normal and ectopic PGC
pathways, the distribution of tenascin-C around the
“lost” cells was examined.
Transverse sections through the hemi-embryos
stained with PAS show that the PGCs leave the bloodstream and move into the ectopic areas as described by
Nakamura et al. (1991). If adjacent sections are stained
with tenascin-C antibodies, it is apparent that the
PGCs are always located in tenascin-C-rich matrices.
For example, PGCs were found in patches of tenascinC-rich matrix in the perioptic mesenchyme (Fig. 3A,B).
Near the developing hindbrain, an ectopic PGC was
found in ECM where tenascin-C is concentrated (Fig.
3D,E). Outside the head region, the PGCs were found
in the anterior half of the somites (Fig. 3G) where the
neural crest are also found (Fig. 31). As previously described (Tan et al., 1987; Mackie et al., 1988b), tenascin-C is restricted to the anterior half of the somites at
this time. In all of these regions, fibronectin has a widespread distribution (Fig. 3C,F); PGCs were not found in
mesenchyme stained by anti-fibronectin and not anti-
Fig. 1. HNK-1 is a marker of chick PGCs. A-D: Whole blood smear
from a stage 13 chick embryo. A: A PGC recognized by morphological
criteria viewed with phase optics. B: The same cell, but not surrounding
cells in the smear, is stained by the monoclonal antibody HNK-1. C: A
white blood cell in the same smear. D: This cell is not positive for HNK-1.
E-G: PGCs isolated on a Ficoll density gradient are stained by PAS. E:
The isolated cells are PAS-positive. F: Another cell from the same frac-
tion viewed with phase contrast optics shows the characteristic size and
granular appearance of a PGC. G: The cell shown in F is also positive for
HNK-1. H: PGCs (arrows) that have just left the bloodstream are darkly
stained by PAS in a cross-section through the notochord (n) and paired
aorta (a) of an E3 chick embryo. I: In an adjacent section, cells in the
same region with the same shape as the PAS-positive cells are stained
by HNK-1 (arrows).
tenascin-C. Note that the tenascin-C immunoreactivity
seen in each of the regions where ectopic PGCs were
found was identical to that reported previously (Tan et
al., 1987; Mackie et al., 1988b; Tucker, 1991).
gion. Consistent with our immunohistochemistry, a hybridization signal was seen in the endothelium around
the entire perimeter of the aorta, but was more prevalent in the ventral half (Fig. 4E).In sections containing
ventrolateral hematopoietic foci, the hybridization signal was strongest over the foci themselves, indicating
that the HPCs or immediately adjacent cells are a major source of tenascin-C (Fig. 4F). Control sections incubated with plasmid DNA show background hybridization levels (Fig. 4G).
Tenascin-C is Found Near Migrating HPCs
Anti-HNK-1 transiently marks HPCs found in the
ventrolateral clusters in the wall of the dorsal aorta
(Thiery et al., 1985). Most of the cells in the intraaortic
foci bud into the lumen of the aorta upon maturation
(Fig. 4A,C), but a subset emigrate from the aorta into
the surrounding mesenchyme (Fig. 4C). Immunohistochemistry shows that tenascin-C is concentrated in the
mesenchyme through which the HPCs migrate (Fig.
4B,D). Tenascin-C also cradles the HPCs that are detaching from the wall of the aorta (Fig. 4D).
In situ hybridization with a tenascin-C-specific
cDNA (cTn8) was carried out to determine the cellular
origins of tenascin-C in the aortic hematopoietic re-
Several Tenascin-C Splice Variants are Present
in the ECM of the Two Migratory Pathways
Tenascin-C has several splice variants that have different properties in vitro (for reviews see Tucker et al.,
1994b; Leprini et al., 1994). To determine which splice
variants may be expressed near migrating PGCs and
HPCs, RT-PCR was done on poly(A) RNA isolated from
the migratory mesenchyme of the PGCs and HPCs. In
Fig. 2. Tenascin-C lines the PGC migratory pathway. A: A low-magnification overview of a frozen cross section through the trunk of an E3
chick embryo stained with HNK-1. HNK-1 stains the neural crest cells on
either side of the neural tube (nt) and the notochord (no). PGCs are in the
splanchnic mesoderm just ventral to the paired aorta (a). The region in
the box is shown at higher magnification in 0. 8: The same section
shown in A was also stained with an antiserum to tenascin-C. Tenascin-C
is part of the ECM surrounding the neural crest, as well as in the mesoderm surrounding the paired aorta (a). There is no tenascin-C immunoreactivity in the extraembryonic membranes (arrowhead) or in the
splanchnic mesoderm ventral to the level occupied by PGCs (arrow). The
latter regions are shown at higher magnification in E. C: An adjacent
section stained with anti-fibronectin. Fibronectin is widely distributed
throughout mesenchymal ECM, including extraembryonic membranes
(arrowhead) and the splanchnic mesoderm ventral to the PGCs (arrow).
D:The PGC migratory pathway stained with HNK-1. PGCs are present in
the splanchnic mesoderm (arrow). E: The same section shown in D
stained with anti-tenascin-C. Tenascin-C is present in or near the basement membranes lining the splanchnic mesoderm (arrowheads). The
PGC shown in D is near or in contact with a patch of tenascin-C-rich ECM
(arrow). F: The same region of an adjacent section stained with secondary antibody alone to control for background fluorescence and nonspecific staining.
both regions, PCR products corresponding to 3 splice
variants of tenascin-C were found (Fig. 5).
cent and Thiery, 1984; Tucker et al., 1984; Rickman et
al., 1985; Bronner-Fraser, 1986; Loring and Erickson,
1987; Erickson e t al., 1989) and HPCs (Thiery et al.,
1985);here we show that avian PGCs are stained with
HNK-1, both in vitro and in situ. Double-label immunohistochemistry with HNK-1 and tenascin-C antibod-
The monoclonal antibody HNK-1 is a n established
marker of neural crest cells (Vincent et al., 1983; Vin-
Fig. 3. Ectopic PGCs are found in tenascin-C-rich ECM. A-C: Sections through the perioptic mesenchyme of an E3 chick embryo following
surgical removal of the gonadal anlagen. A: PAS staining was used to
identify ectopic PGCs (arrowheads) in the mesenchyme (m) near the
pigment epithelium (pe), neural retina (nr) and diencephalon (di). B: An
adjacent section stained with an antibody to tenascin-C shows that the
ectopic PGCs (approximated by asterisks) are found in tenascin-C-rich
ECM. C: The same section shown in B was stained with anti-fibronectin.
Fibronectin is also part of the ECM surrounding the ectopic PGCs. D-F:
Cross-sections through the developing hindbrain (hb), cardinal vein (c)
and nearby notochord (n) of an E3 embryo following removal of the
gonadal anlagen. D: PAS reveals an ectopic PGC (arrowhead) in the
44 1
mesenchyme (m) near the cardinal vein. E: An adjacent section (slightly
compressed) stained with tenascin-C antibody shows tenascin-C in the
ECM surrounding the ectopic PGC (approximated by the asterisk) adjacent to the cardinal vein. F: The same section stained with anti-fibronectin. G-I: Frontal frozen sections through the trunk of an E3 embryo following ablation of the gonadal anlagen. G: PAS staining reveals an
ectopic PGC (arrowhead) in the anterior sclerotome (a), but not the posterior sclerotome (p) or dermamyotome (dm) of a somite. H: An adjacent
section stained with tenascin-C antibodies shows tenascin-C in the anterior sclerotome. I: The same section stained with HNK-1 shows the
neural crest cells in the anterior sclerotome.
Fig. 4. A subset of HPCs make tenascin-C and migrate into tenascinC-rich mesenchyme surrounding the aorta. A-D: Double-label immunohistochemistry with HNK-1 and tenascin-C antiserum on a frozen crosssection through an E3 chick embryo. A: HPCs (arrowhead) lining the
ventral wall of the aorta (a) are stained by HNK-1. Neural crest cells on
either side of the aorta are also brightly stained, as is the notochord (n).
These HPCs are shown at a higher magnification in C. 6: The same
section stained with a tenascin-C antibody shows tenascin-C in the ECM
surrounding the aorta, especially in the mesenchyme just ventral to the
aorta. C: A higher magnification of the HPCs stained by HNK-1. Many of
the cells are lining the aorta, where they appear to be budding into the
lumen of the vessel (arrowhead). Other HPCs appear to be migrating into
the adjacent mesenchyme (arrows). 0: The same region shown in C
stained with anti-tenascin-C. Tenascin-C lines the basal surface of the
HPCs (arrowhead) and is present in the ECM through which the HPCs
migrate. E-G: Darkfield images of nearby sections following in situ hybridization and autoradiography. E: The tenascin-C cDNA probe cTn8
labels the notochord (n) and the endothelium of the aorta (a). F: In a
region where 2 ventrolateral clusters of HPCs are found, the tenascin-C
cDNA probe cTn8 labels the clusters intensely (arrows). G: A third section incubated with a control DNA (pUC) shows low background levels.
ies were done to characterize the ECM through which
the PGCs migrate. Both monoclonal and polyclonal tenascin-C antibodies reveal tenascin-C in the ECM surrounding migrating PGCs. This staining is particularly strong around the ventral side of the aorta, but
also stretches into the splanchnic mesoderm ventral to
the aorta where the PGCs are migrating to the genital
ridges. We were unable to use in situ hybridization to
determine the cellular origins of this tenascin-C because of unusually high background levels in this part
4 540
4 264
Fig. 5. Transcripts encoding 3 splice variants of tenascin-C were
identified in poly(A) RNA isolated from the HPC and PGC migratory pathways by RT-PCR. Primers spanning the tenascin-C variable domain were
used to amplify products with the anticipated sizes of 264 b.p., 540 b.p.
and 1,060 b.p., correspondingto no variable repeats, one variable repeat,
and 3 variable repeats, respectively. RT-PCR products amplified from
poly(A) RNA isolated from the HPC migratory pathway (lane 1) and the
PGC migratory pathway (lane 2) show that the splice variant profile is
similar in both regions. The 1,060 b.p. band is present in lane 2 but
appears very faint in this micrograph. Hash marks on the left indicate size
standards. From top to bottom: 2,072 b.p., 600 b.p., 300 b.p., 100 b.p.
of the embryo (results not shown). However, RT-PCR
demonstrates that the mRNAs encoding at least three
tenascin-C splice variants are present in the PGCs
andlor the tissues lining their migratory pathway.
Tenascin-C in the PGC migratory pathway could be
doing several things, including marking the region of
the aorta where the PGCs leave the bloodstream, thus
triggering their active phase of migration. Alternatively, tenascin-C could be interacting with other ECM
molecules such as fibronectin to facilitate PGC migration. The first role seems unlikely. In vitro studies have
suggested that the factor responsible for guiding the
PGCs is a chemoattractant emitted by the genital
ridges (Kuwana et al., 1986; Godin et al., 1990) and
that TGF-P1 can mimic this effect (Godin and Wylie,
1991). Paraaortic tenascin-C immunoreactivity is not
specific to the area of PGC attachment and migration,
but instead is present around the dorsal aorta at all
axial levels examined. Thus, it is unlikely that tenascin-C is the signal for PGCs to leave the bloodstream
and begin their active phase of migration. It is possible
that specific variants of tenascin-C could guide the
PGCs; however, RT-PCR shows that each of the major
splice variants is expressed a t axial levels where the
PGCs leave the aorta as well as at more rostra1 levels
(i.e., where the HPCs are forming).
It is possible that tenascin-C contributes to a matrix
that supports PGC migration, perhaps by interfering
with cell-fibronectin interactions (Chiquet-Ehrismann
et al., 1988; Lotz et al., 1989). Fibronectin has been
found in the PGC pathway in chick (Urven et al., 1989),
Xenopus (Heasman et al., 1981) and mouse (ffrenchConstant et al., 1991). Here, we show that anti-fibronectin staining was not confined to the PGC pathway,
but was widespread throughout the chick embryo. In
contrast, individual HNK-1-positive PGCs were frequently seen close or in contact with strands of antitenascin-C-stained ECM. Little or no anti-tenascin-C
staining was seen ventral to the pathways followed by
the PGCs. In addition, ectopic PGCs are encountered
exclusively in regions stained by both tenascin-C and
fibronectin antibodies. The location of the ectopic cells
supports the hypothesis that a combination of tenascin-C and fibronectin forms an ideal substratum for cell
migration. However, one must consider that most of
the tenascin-C in the head region is probably expressed
by the cranial neural crest (Tucker and McKay, 1991).
It is possible that the PGCs are responding to the same
migratory cues as the neural crest, and that the colocalization of tenascin-C and ectopic PGCs is a coincidence of this comigration. This hypothesis could be
tested in a future study by a double ablation of the
neural crest and the genital ridges. Finally, it has been
shown that premigratory PGCs respond to fibronectin
differently than postmigratory PGCs (ffrench-Constant, 1991). It is possible that PGCs express tenascin-C upon activation, thus changing their affinity for
The HPCs also migrate from the ventral wall of the
aorta into the surrounding mesenchyme. Anti-tenascin-C staining is robust throughout the ventral mesenchyme a t this axial level. We have also shown that like
the neural crest (Tucker and McKay, 1991), HPCs are
potentially the source of much of the surrounding tenascin-C, thus making it unlikely that tenascin-C actually directs the pathways followed by the HPCs. Most
HPCs bud into the lumen of the aorta; one role oftenascin-C made by HPCs may be to help them detach
from the fibronectin-rich substratum and/or each other
as they mature, thus regulating the release of cells into
the bloodstream.
Previous studies have shown that tenascin-C surrounds migrating neural crest cells (Tan et al., 1987;
Mackie et al., 1988b; Epperlein et al., 1988; BronnerFraser, 1988). This tenascin-C is probably made by the
neural crest cells themselves, as tenascin-C can be detected in culture medium conditioned by the neural
crest, and in situ hybridization shows that migrating
neural crest cells are positive for tenascin-C transcripts
(Tucker and McKay, 1991). Here, we show tenascin-C
in the ECM surrounding normal and ectopic PGCs, as
well as migrating HPCs. In the case of the PGCs, tenascin-C is present in a subset of the ECM stained with
antibodies to fibronectin. These observations demonstrate that tenascin-C is a component common to the
ECM surrounding diverse populations of migratory
embryonic cells, thus supporting a role for tenascin-C
in promoting cell motility in a general way, perhaps by
interfering with cell-fibronectin interactions.
Animals, Sectioning and Immunostaining
Fertile chicken eggs (Hubbard Farms, Statesville,
NC) were kept in a humid incubator at 37°C for 55-72
hours. For immunohistochemistry, in situ hybridization and PAS staining whole embryos were fixed in
ice-cold 4% paraformaldehydetphosphate-bufferedsaline (PBS) solution for 5 hours and then cryoprotected
in 20% sucrose in PBS overnight. After embedding in
Tissue Tek (Miles, Elkhart, IN) using a slurry of dry ice
and 2-methylbutane, 10-15 pm sections were cut on a
Bright cryostat and put on poly-L-lysine (Sigma, St.
Louis, Mobcoated slides. The sections were air-dried
for 20 minutes and then stored a t -70°C until needed.
All antibodies used in this study have been previously characterized. The monoclonal antibody M1 recognizes a constant region of tenascin-C (Chiquet and
Fambrough, 1984; Hybridoma Bank, Rockville, MD).
Undiluted hybridoma supernatants were used. The
rabbit anti-chicken tenascin-C polyclonal antiserum
(Chiquet-Ehrismann et al., 1986) used in double-labeling studies was a generous gift from Dr. Eleanor J.
Mackie (RVC, London). The mouse monoclonal antibody HNK-1 recognizes a carbohydrate epitope and can
be used to identify neural crest cells (e.g., Vincent and
Thiery, 1984). The hybridoma was acquired from the
American Type Culture Collection (Rockville, MD).
Undiluted hybridoma supernatants were used. The
anti-human plasma fibronectin polyclonal antibody
was purchased from Accurate Chemical and Scientific
(Westbury, NY) and diluted 1500, while TRITC and
FITC-conjugated secondary antibodies were obtained
from Hyclone Laboratories. Sections to be stained were
rehydrated in PBS for 5 minutes and then blocked in
0.5% bovine serum albumin (BSA) in PBS for 15 minutes before secondary antibody, diluted 1 5 0 in PBS,
was added. After 5 hours the slides were rinsed for 15
minutes in PBS and mounted in 50% glycerol in PBS
containing 1 pg ml-' Hoechst nuclear dye (Boehringer
Mannheim, Idianapolis, IN). If double-labeling studies
were done, appropriate quantities of both primary and
then secondary antibodies were applied t o the sections
a t the same time. PAS staining was performed as described by Mowry (1963). Immunostaining of isolated
PGCs was done in a manner similar to the immunohistochemistry described above, except the cells were
stained in a microcentrifuge tube. HNK-1 was added to
the tube for 1 hour; secondary antibodies were allowed
to incubate for 20 minutes. The cells were rinsed in
PBS by gentle centrifugation.
pellet was then added to 200 pl of a 6.3% FicolUmedium
199 solution and layered on 900 pl of 16% Ficoll/medium 199 in a glass culture tube. To isolate the PGCs,
the gradient was centrifuged at 800 x g for 30 minutes
in a swinging bucket rotor. The PGCs collected from
the interface were diluted 1.5-fold with medium 199
and centrifuged for 3 minutes at 400 x g. The PGC
pellet was rinsed 3 times with medium 199 by centrifugation.
In Situ Hybridization
The chick tenascin-C-specific cDNA probe cTn8 (a
generous gift, from Dr. Ruth Chiquet-Ehrismann,
Basel) was previously described by Pearson et al.
(1988). In situ hybridization was carried out as described by Tucker et al. (1994a). In brief, sections were
cut as described above and prehybridized for 1 hour at
room temperature in prehybridization buffer (Sigma)
diluted 1:1with autoclaved deionized water and 20 mM
2-mercaptoethanol (Sigma). Sections were then dehydrated by dipping into 100% ethanol and air-dried.
cTn8 was labeled with [35SldCTP(Amersham, Arlington Heights, IL) using a Prime-a-Gene kit (Promega,
Madison, WI). Unincorporated nucleotides were removed using a G-50 spin column (Worthington Biochemical, Freehold, NJ) 5 x lo6 c.p.m. of boiled probe
was suspended in 20 pl hybridization buffer (Sigma)
containing 50%deionized formamide (Fisher Scientific,
Pittsburgh, PA), 20 mM Tris pH 8.0, 0.1% sarkosyl
(Fisher), and 1%dextran sulfate (Sigma), and applied
to the section. The sections were allowed to incubate at
42°C for 18 hours. After removing the coverslips, the
sections were washed with 1 x SSC and 20 mM 2-mercaptoethanol for 1 hour a t room temperature and 1
hour a t 42°C. The slides were allowed t o dry and then
were processed by dipping in NTB2 autoradiography
emulsion (Kodak, Rochester, NY). After developing,
the slides were mounted in 50% glycerol in PBS and 1
pg ml-' Hoechst nuclear dye.
To dissect the HPC and PGC migratory mesenchyme, E3 chicks were taken from the egg, rinsed in
PBS and placed in an agar-coated 10-cm petri dish. The
aorta can be easily followed throughout the embryo a t
this stage. To isolate the HPC migratory region, the
mesenchyme around the aorta rostra1 to the vitelline
vein was dissected free using tungsten needles. The
mesenchyme through which the PGCs move was removed in a similar manner, but only from axial levels
caudal t o the vitelline veins. The dissected tissue was
transferred immediately to an autoclaved microcentriPGC Isolation
fuge tube and placed in liquid nitrogen. Poly(A) RNA
PGCs were isolated from stage 13-14 (Hamburger was isolated from the dissected tissue using a Micro
and Hamilton, 1951) chick blood using methods modi- Fast Trak kit (Invitrogen, San Diego, CA).
To induce ectopic PGC migration, the gonadal anlafied from Chang et al. (1992). Blood from the vitelline
veins was collected using an ultrafine glass pipette and gen were removed prior to PGC colonization. Eggs were
suspended in 1ml of medium 199 (Sigma). The suspen- set to obtain stage 12-13 embryos, and candled to desion was centrifuged at 200 x g for 3 minutes. The termine the position of the blastodisc within the egg.
ral crest cell migration in avian embryos using monoclonal antiWindows were made over this site using an electric
body HNK-1. Dev. Biol. 115:44-53.
sander, keeping the shell membrane intact. After
M. (1988) Distribution and function of tenascin durswabbing the perimeter of the window and the mem- Bronner-Fraser,
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(1992) Simple method for isolation of primordial germ cells from
and the surrounding membranes, the embryo was
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makers’ forceps. After moistening the embryo with a Chiquet-Ehrismann, R., Kalla, P., Pearson, C.A., Beck, K., and Chidrop of sterile saline, the egg was sealed with cellotape
quet, M. (1988) Tenascin interferes with fibronectin action. Cell
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ing and staining as described above. In all, 8 embryos
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survived the surgery to be analyzed by PAS and imDieterlen-Lievre, F., and Martin, C. (1981) Diffuse intraembryonic
munohistochemical staining.
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Reverse Transcriptase PCR
Epperlein, H.H., Halfter, W., and Tucker, R.P. (1988) The distribution
of fibronectin and tenascin along migratory pathways of the neural
To determine which tenascin-C splice variants are
crest in the trunk of amphibian embryos. Development 103:743present in the PGC and HPC migratory pathway, RT756.
PCR from the isolated poly(A) RNA from the dissected Erickson, C.A., Loring, J.F., and Lester, S.M. (1989) Migratory pathways of HNK-1-immunoreactive neural crest cells in the rat emmesenchyme was performed with primers described in
bryo. Dev. Biol. 134:112-118.
Tucker et al. (1994b). These primers, TNSVA and ffrench-Constant,
C., Hollingsworth, A., Heasman, J., and Wylie, C.
TNSVB, correspond to the beginning of the fifth and
(1991) Response to fibronectin of mouse primordial germ cells besixth fibronectin-type I11 repeats of chick tenascin-C;
fore, during and after migration. Development 113:1365-1373.
i.e., they span the variable domain (Tucker et al., Fujimoto, T., Ukeshima, A., and Kiyofuji, R. (1976) The origin, migration and morphology of the primordial germ cells in the chick
1994a). A 264 b.p. product corresponds to a tenascin-C
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transcript without the variable domain. Products of Godin, I., and Wylie, C.C. (1991) TGF-PI inhibits proliferation and
540 b.p. and 1,060 b.p. correspond to transcripts encodhas chemotropic effect on mouse primordial germ cells in culture.
Development 113:1451-1457.
ing tenascin-C with 1 and 3 variable repeats, respectively. All nucleotides, enzymes and buffers were sup- Godin, I., Wylie, C., and Heasman, J. (1990) Genital ridges exert
long-range effects on mouse primordial germ cell numbers and diplied in a reverse transcriptase PCR kit (Perkin Elmer,
rection of migration in culture. Development 108:357-363.
Nonvalk, CT). The reactions were carried out as rec- Halfter, W., Chiquet-Ehrismann, R., and Tucker, R.P. (1989) The efommended by the manufacturer.
fect of tenascin and embryonic basal lamina on the behavior and
This work was done in partial fulfillment of a doctoral degree a t the Department of Neurobiology and
Anatomy at the Bowman Gray School of Medicine,
with funds provided by the National Science Foundation (BNS-9021124) and the UCD School of Medicine.
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