Note: Descriptions are shown in the official language in which they were submitted.
B~P ~ile No. 3153-066/LNK 2 0 8 5 2 91
Title: Novel Receptor Tyrosine Rinase
FIELD OF THE INVENTION
The invention relates to a novel tyrosine kinase receptor
protein and isoforms thereof, DNA segments encoding the
novel protein and isoforms thereof, and uses of the
protein and DNA segments.
~,
BA~KGROUND O~ THE INV~NTION
Transmembrane receptor tyrosine kinases (RTKs) comprise a
large and evolutionarily conserved family of structurally
related proteins capable of transducing extracellular
signals to the cytoplasm. The latent oncogenic potential
of these molecules and the molecular mechanisms by which
they function in signalling pathways have been the subject
of extensive study.
In addition, genetic and biochemical analyses of a variety
of developmental mutants have led to recognition of the
pivotal roles played by RTK-mediated signalling pathways
in the regulation of cell determination, migration, and
proliferation. Notable examples in Drosophila include the
role of sevenless and its ligand, bride of sevenless, in
R7 photoreceptor determination (Kramer, H., Cagan, R.L. &
Zipursky, S.L. (1991), Nature, 35~, 207-212), and of
DER/flb in early morphogenetic events during gastrulation
(Schejter, E.D. & Shilo, B.-Z. ~1989), Cell, 56,
1093-1104). Similarly, in the mouse, loss of function
mutations at the W/c-kit (Geissler, E.N., Rayn, M.A. &
Housman, D.E. (1988), Cell, 55, 185-192; Chabot, B.,
Stephenson, D.A., Chapman, V.M., Besmer, P. & Bernstein,
A. (1988), Nature, 335, 88-89) and Sl (Russell, E.S.
(1979), Adv.Genet., 20, 357-459) loci have revealed the
importance of the Kit receptor and its ligand in
melanogenesis, hematopoiesis, and gametogenesis (Dubreuil,
P., Rottapel, R., Reith, A.D., Forrester, L. &
Bernstein, A. (1990), Ann. N.Y. Acad. Sci., 599, 58-65;
.
~' '.'. '
208~291
Williams, D.E., Eisenman, J., Baird, A., Rauch, C.,
Ness, K.V., March, C.J., Park, L.S., Martin, U.,
Mochizuki, D.Y., Boswell, H.S., Burgess, G.S., Cosman, D.
& Lyman, S.D. (1990), Cell, 63, 167-174; Copeland, N.G.,
Gilbert, D.J., Cho, B.C., Donovan, P.J., Jenkins, N.A.,
Cosman, D. Anderson, D., Lyman, S.D. & Williams, D.E.
(1990), Cell, 63, 175-183 and Flanagan, J.G. & Leder, P.
(1990), Cell, 63, 185-194) while a deletion in the gene
encoding PDGFR-~ has been correlated with the Patch
mutation, which also causes a defect in melanogenesis (
Stephenson, D.A., Mercola, N., Anderson, E., Wang, C.,
Stiles, C.D., Bowen-Pope, D.F. & Chapman, V.M. (1991),
Proc.Natl.Acad.Sci., 88, 6-10). These o~servations,
together with others (reviewed in Pawson, T. & Bernstein,
A. (1991), ~rends Gen., 6, 350-356), have established the
importance of receptor-ligand interactions in the
regulation of development.
Angiogenesis in both the embryo and adult requires the
differentiation, proliferation, and migration of
endothelial cells. Tissue transplantation studies with
quail/chick chimeras have established that the
developmental cues for both endothelial cell
differentiation and proper patterning of vessels are
extracellular and not pre-programmed within the cell
(Noden, D.N. (1988) Development, 103, 121-140) Several
peptide hormones, such as bFGF, VEGF and PD-EGF, have been
shown to have both mitogenic and chemotactic effects on
cultured endothelial cells (see Tomasi, V., Manica, F. &
Spisni, E. (1990), sioFactors, 2, 213-217; Klagsbrun, M.
& D'Amore, P. (1991), Annu.Rev.Physiol., 53, 217-239, for
reviews). However, many of these factors also show
similar effects on other cell types, implying that
receptors for these factors are also expressed by such
cells.
Studies have demonstrated that both tyrosine kinase
.~ , ~ . ~ . . - - .
:. .- . : , .. ~, , . .: - - . .. ~;
. . : :: -^ .
.
. .
208~291
activity and phosphotyrosine-containing proteins are
increased in embryonic chicken heart relative to the adult
(Maher, P.A. (1991). J.Cell Biol., 112, 955-963), and that
inhibitors of kinase activity impede inductive processes
during in vitro differentiation of cardiac explants
derived from chicken embryos (Runyan, R.B., Potts, J.D.,
Sharma, R.V., Loeber, C.P., Chiang, J.J. & Bhalla, R.C.
(199O), Cell Reg., 1, 301-313).
SUMMARY OF TH~ INVENTION
The present inventors have identified and characterized a
receptor tyrosine kinase that plays a critical role in
murine cardiogenesis. The heart forms early in mouse
embryogenesis and its development is known to be
accompanied by the differentiation from mesoderm of
myocytes and endothelial cells that subsequently form the
myocardium and endocardium, respectively (Manasek, F.J.
(1976), in The Cell Surface in Animal Embryogenesis and
Development, p.545-598, Elsevier/North-Holland Biomedical
Press; Kaufman, N.H. & Navaratnam, V. (1981), J.Anat.,
133, 235-246). There have not hitherto been any reports
of directed screens for tyrosine kinases expressed during
murine cardiogenesis.
In particular, the present inventors using reverse
transcription coupled to the polymerase chain reaction
(RT-PCR) isolated from murine embryonic heart a cDNA,
designated tek, whose deduced amino acid sequence
corresponds to a novel RTK. The tek locus of mouse was
mapped to chromosome 4. The present inventors have also
shown by in situ hybridization that tek is expressed in
the endocardium as well as the endothelial lining of the
vasculature. tek was also found to be expressed in both
mature endothelial cells and their progenitors, suggesting
that the signalling pathways regulated by tek may be
important to both the determination and proli~eration of
cells of the endothelial lineage.
. ,
.:
,
.
2085291
The present invention therefore provides a purified and
isolated DNA segment having a sequence which codes for a
receptor tyrosine kinase protein which is expressed only
in cells of endothelial lineage, or an oligonucleotide
fragment of the DNA segment which is unique to the
receptor tyrosine kinase protein of the invention. In a
preferred embodiment of the invention, the purified and
isolated DNA segment has the sequence as shown in
Figure 1.
The invention also contemplates a double stranded
nucleotide sequence comprising a DNA segment of the
invention or an oligonucleotide fragment thereof hydrogen
bonded to a complementary nucleotide base sequence.
The invention further contemplates a recombinant molecule
comprising a DNA segment of the invention or an
oligonucleotide fragment thereof and an expression control
sequence operatively linked to the DNA segment or
oligonucleotide fragment. A transformant host cell
including a recombinant molecule of the invention is also
provided.
Still further, this invention provides plasmids which
comprise the DNA segment of the invention.
, ~
The invention further provides a method of preparing a
novel receptor tyrosine kinase protein or isoforms thereof
utilizing the purified and isolated DNA segments of the
invention. The method comprises culturing a transformant
host cell including a recombinant molecule comprising a
DNA segment of the invention and an expression control
sequence operatively linked to the DNA segment, in a
suitable medium until the protein is formed and thereafter
isolating the protein.
The invention further broadly contemplates a substantially
.. ., . ~ ,
: .... ~ ,.,
208~291
pure receptor tyrosine kinase protein or a part thereof, --^}
which is expressed only in cells of endothelial lineage.
The invention also permits the construction of nucleotide
probes which are unique to the novel receptor tyrosine
kinase protein of the invention or a part of the protein.
Thus, the invention also relates to a probe comprising a
nucleotide sequence coding for a protein, which displays
the properties of the novel receptor tyrosine kinase of
the invention or a peptide unique to the protein. The
probe may be labelled, for example, with a radioactive
substance and it may be used to select from a mixture of
nucleotide sequences a nucleotide se~uence coding for a
protein which displays the properties of the novel
receptor tyrosine kinase protein of the invention.
lS The invention provides a method for identifying ligands
which are capable of binding to the novel receptor
tyrosine kinase protein of the invention, isoforms
thereof, or part of the protein, comprising reacting the
novel receptor kinase protein of the invention, isoforms
thereof, or part of the protein, with at least one ligand
which potentially is capable of binding to the protein,
isoform or part of the protein, under conditions which
permit the formation of ligand-receptor protein complexes,
and assaying for ligand-receptor protein complexes, for
free ligand or for non-complexed proteins. In a preferred
embodiment of the method, ligands are identified which are
- capable of binding to and activating the novel receptor
tyrosine kinase protein of the invention, isoforms
thereof, or part of the protein. The ligands which bind to
and activate the novel receptor tyrosine kinase receptor
of the invention are identified by assaying for protein
tyrosine kinase activity i.e by assaying for
phosphotyrosine.
In addition, the invention provides a method of using the
.
- ~ ,. .. ..
.. . ~ .
208~291
novel proteins of the invention for assaying a medium for
the presence of a substance that affects a tek effector
system. In accordance with one embodiment, a method is
provided which comprises providing a known concentration
of a receptor tyrosine kinase protein of the invention,
incubating the protein with a ligand which is capable of
binding to the protein and a suspected agonist or
antagonist substance under conditions which permit the
formation of ligand-receptor protein complexes, and
assaying for ligand-receptor protein complexes, for free
ligand or for non-complexed proteins.
The methods of the invention make it possible to screen a
large number of potential ligands for their ability to
bind to the novel receptor proteins of the present
invention. The methods of the invention will also be
useful for identifying potential stimulators or inhibitors
of angiogenesis.
DESCRIPTION OF THE DRANINGS
The invention will be better understood with reference to
the drawings in which:
Figure 1 shows the nucleotide and deduced amino acid
se~uence of the novel receptor tyrosine kinase protein of
the invention;
Figure 2 shows the nucleotide and deduced amino acid
sequence of a 1601 bp DNA segment of the invention;
Figure 3 shows a comparison of a portion of the deduced
amino acid sequence of the novel receptor tyrosine kinase
protein of the invention with that of other tyrosine
kinases;
Figure 4 shows a Northern blot hybridization analysis of
expression of the DNA segment of the invention in 12.~ day
! ' .', ' ~ , , .
2085291
-- 7 --
murine embryonic heart;
Figure 5 shows the in situ hybridization analysis of
expression of the DNA segment of the invention in the 12.5
day embryo;
Figure 6 shows the expression of the DNA segment of the
invention precedes that of von Willebrand factor in 8.S
day embryos;
Figure 7 shows expression of the DNA segment of the
invention in whole mount embryostA., B., and C.);
expression in Day 8.0 embryos (D.); mRNA distribution in
a Day 9.5 embryo (E.); and En2 expression in a Day 8
embryo (F.);
Figure 8 shows the expression of the DNA segment of the
invention precedes that of von Willebrand factor in the
developing leptomeninges and in particular the absence of
immunohistochemical staining of von Willebrand factor in
Day 12.5 leptomeninges (A); in situ detection of tek
expression in Day 12.5 leptomeninges(B); staining of von
Willebrand factor in Day 14.5 leptomeninges (C);
Figure 9 shows the expression of tek in adult vasculature
and in particular bright field illumination of a section
through the upper heart region of a 3 week-old mouse
hybridized with an [35S]-labelled tek probe (A); bright
field illumination showing tek expression in endothelial
cells lining the artery and vein respectively (B) and (C);
and
Figure 10 shows the hierarchy of the endothelial cell
lineage.
DETAILED D~SCRIPTION OF T~ INV~NTION
The present inventors have isolated a novel protein
: : , , . :
' : , .. , :
2085291
-- 8 --
tyrosine kinase designated tek, expressed during murine
cardio~enesis. By analyzing the segregation of an AccI
restriction site polymorphism in AKR/J:DBA recombinant
inbred mice, the present inventors mapped the tek locus to
chromosome 4, between the brown and pmv-23 loci. This
region is syntenic with human chromosomal regions lp22-23,
9q31-33, and 9p22-13. In mice and humans, these regions
do not contain any previously described loci known to be
involved with the biology of the endothelial cell lineage
(Lyon, M.F. & Searle, A.G. Genetic Variants and Strains of
the Laboratory Mouse, New York:Oxford University Press,
1989, 2nd, Ed.; O'Brien, 1990).
The novel gene products of the invention were identified
as mouse receptor tyrosine kinase protein based on the
structural homology of the protein to the known mouse and
human tyrosine kinases. The deduced amino acid sequence of
tek predicts that it encodes a putative receptor tyrosine
kinase that contains a 21 amino acid kinase insert and
which is most closely related in its catalytic domain to
FGFR1 (mouse fibroblast growth factor) and the product of
the ret proto-oncogene.
Northern blot hybridization analysis of RNA from 12.5 day
embryonic heart using the 1.6 kb cDNA as probe suggested
2S that the tek locus gives rise to at least 4 different
transcripts of approximately 4.5, 2.7, 2.2, and 0.8 kb.
Differential splicing of primary transcripts is known to
occur for several genes encoding RTKs, including met
(Rodrigues, G.A., Naujokas, M.A. & Park, M. (1991),
Mol.Cell.Biol., 11, 2962-2970), trkB (Middlemas, D.S.,
Lindberg, R.A. & Hunter, T. (1991), Nol.Cell.Biol., 11,
143-153), ret (Tahira, T., Ishizaka, Y., Itoh, F.,
Sugimura, T. & Nagao, N. (1990), Oncogene, 5, 97-102), and
flg (Reid et al., 1990, Proc.Natl.Acad.Sci.,87,1596-1600;
Bernard, O., Li, M. & Reid, H.H. (1991), Proc.Natl.Acad
- Sci.USA, 88, 7625-7629; Eisemann, A., Ahn, J.A., Graziani,
- :.
- - - .
- ,, : , . : , . ~. . :: ~ ~ ,.
. ~ .
208~291
G., Tronick, S.R. & Ron, D. (1991), Oncogene, 6,
1195-1202; Fujita, H., Ohta, N., Kawasaki, T. & Itoh, N.
(1991), Biochem. Biophis. Res. Comm., 174, 946-951; Meng,
B. & Reid, H.H. (1991), Proc.Natl.Acad.Sci., 7625-7629),
favoring the possibility that at least some of the
smaller transcripts hybridizing with the tek cDNA are
differentially spliced. The 4.5 kb tek transcript is of
the appropriate size to encode a molecule with an
extensive extracellular domain. In contrast, the smallest
transcript, at 0.8 kb, is sufficient to encode only a
significantly truncated version of the protein. Since
this transcript was detected with a probe comprised
entirely of sequences from the catalytic domain and 3
untranslated region, it is possible that the 0.8 kb
message codes for an isoform completely lacking an
extracellular domain. Truncated molecules of this type
have recently been shown to be encoded by the trkB gene in
rats (Niddlemas et al., 1991, Mol.Cell.Biol., 11, 143-153)
and by pdgfb in murine ES cells (Vu, T.H., Nartin, G.R.,
Lee, P., Mark, D., Wang, A. & Williams, L.T. (1989),
Mol.Cell.Biol., 9, 4563-4567). These small isoforms may
act as catalytically deregulated molecules during periods
of rapid growth (Middlemas et al., 1991). The detection
of multiple tek transcripts may indicate potential
differential expression of different tek isoforms during
embryogenesis.
In the adult and all stages of embryonic development
examined, tek expression was restricted to cells of the
endothelial lineage. Specifically, in situ hybridization
analysis of adult tissues, as well as sectioned and whole
mount embryos, showed that tek is specifically expressed
in the endocardium, the leptomeninges and the endothelial
lining of the vasculature from the earliest stages of
their development. Moreover, examination of the
morphology of tek-expressing cells, and staging of tek
expression relative to that of the endothelial cell marker
,:
:`
2085291
-- 10 --
von Willebrand factor, revealed that tek is expressed
prior to von Willebrand factor and appears to mark the
embryonic progenitors of mature endothelial cells. Thus,
tek encodes a novel putative receptor tyrosine kinase that
may be critically involved in the determination and/or
maintenance of cells of the endothelial lineage.
Overall, the pattern of expression observed in sectioned
and whole mount mouse embryos was similar to that
described previously for quail embryos stained with a
monoclonal antibody specific for cells of the endothelial
lineage (Pardanaud, L., Altmann, C., Kitos, P.,
Dieterlen-Lievre, F. & Buck, C.A. (1987). Development,
100, 339-349; Coffin, J.D. & Poole, T.J. (1988).
Development, 102, 735-748). Thus, it is likely that
orchestration of vascularization in the two vertebrate
species is very similar. Studies on cell lineage
relations carried out primarily in the chick (Noden, D.M.
(1989), Am.Rev.Respir.Dis., 140, 1097-1103, and Noden,
D.M. (1990), Ann. N.Y. Acad. Sci., 1, 236-249; O'Brien,
S.J. Genetic Maps, Locus Maps of Complex Genomes. Cold
Spring Harbor Laboratory Press, 1990) have established
that endothelial cells are derived from angioblasts, which
migrate from mesoderm and populate the embryo with
precursor cells that eventually contribute to the
formation of the intraembryonic blood vessels.
Figure 10 shows the hierarchy of the endothelial cell
lineage. Horizontal bars denote the relationship between
cellular determination and onset of expression of tek and
von Willebrand factor within the lineage (adapted from
(Wagner, R.C. (1980). Adv.Microcirc., 9, 45-75). In the
yolk sac, angioblasts are thought to originate from
hemangioblasts, ill-defined cells of mesenchymal origin
that are also believed to give rise to primitive blood
cells in the developing blood islets. In the embryo, on
the other hand, angioblasts are thought to arise directly
208~291
from cells of the mesenchymal anlage (Wagner, 1980).
The present inventors' work suggested that tek is
expressed in the presumptive precursors of endothelial
cells, the angioblasts. First, tek expression was
detected in both von Willebrand factor-positive cells as
well as cells that appear to be progenitors of endothelial
cells. Second, tek expression was observed in cells of
non-endothelial morphology that in the avian system have
been identified previously as angioblasts. It may also be
significant that in the 8.5 day embryo, tek expression was
identified in cells extending beneath the ventral surface
of somites (Figure 6J). Analysis of serial sections
revealed that some of these tek-expressing cells were
actually contiguous with the somites. These cells may
correspond to those described by Beddington, R.S.P. &
Nartin, P. (1989), Mol.Cell.Med., 6, 263-274 who showed
in mouse tissue transplantation studies that lacZ-
expressing somite tissue, while devoid of endothelial
cells prior to transplantation, possess cells capable of
migrating and contributing to the host vasculature. Taken
together, the present inventors' work suggests that tek
expression may constitute the earliest mammalian
endothelial cell lineage marker described to date.
The restricted expression of tek, imposes constraints on
the cellular range of activity of the putative Tek ligand,
and suggests that the tek locus probably plays unique and
important roles in the determination, migration, or
proliferation of cells of the endothelial lineage.
As hereinbefore mentioned, the present inventors have
identified and sequenced a cDNA sequence encoding a novel
receptor tyrosine kinase protein designated tek. The DNA
sequence and deduced amino acid sequence are shown in
Figure l. The DNA sequence and deduced amino acid
sequence of a 1601 bp segment are shown in Figure 2.
2085291
- 12 -
DNA segments of the present invention encoding the novel
receptor tyrosine kinase protein of the present invention
or related or analogous sequences may be isolated and
sequenced, for example, by synthesizing cDNAs from
embryonic heart RNA by RT-PCR using degenerate
oligonucleotide primers which amplify tyrosine kinase
sequences such as the two degenerate tyrosine kinase
oligonucleotide primers described by Wilks, A.F. ((1989)
Proc.Natl.Acad.Sci., 86, 1603-1607) and analyzing the
sequences of the clones obtained following amplification.
DNA segments of the present invention encoding the novel
receptor tyrosine kinase protein of the present invention
may also be constructed by chemical synthesis and
enzymatic ligation reactions using procedures known in the
art.
It will be appreciated that the invention includes
nucleotide or amino acid sequences which have substantial
sequence homology with the nucleotide and amino acid
sequences shown in Figures 1 and 2. The term ~sequences
having substantial sequence homology'l means those
nucleotide and amino acid sequences which have slight or
inconsequential sequence variations from the sequences
disclosed in Figures 1 and 2 i.e. the homologous sequences
function in substantially the same manner to produce
substantially the same polypeptides as the actual
sequences. The variations may be attributable to local
mutations or structural modifications.
It will also be appreciated that a double stranded
nucleotide sequence comprising a DNA segment of the
invention or an oligonucleotide fragment thereof, hydrogen
bonded to a complementary nucleotide base sequence, an RNA
made by transcription of this doubled stranded nucleotide
sequence, and an antisense strand of a DNA segment of the
invention or an oligonucleotide fragment of the DNA
segment, are contemplated within the scope of the
,
,: - , :
2085291
- 13 -
invention.
A number of unique restriction se~uences for restriction
enzymes are incorporated in the DNA segments identified in
Figures 1, and these provide access to nucleotide
sequences which code for polypeptides unique to the
receptor tyrosine kinase protein of the invention.
DNA sequences unique to the receptor tyrosine kinase
protein of the invention or isoforms thereof, can also be
constructed by chemical synthesis and enzymatic ligation
reactions carried out by procedures known in the art.
The DNA segment of the present invention having a sequence
which codes for the receptor tyrosine kinase protein of
the invention, or an oligonucleotide fragment of the DNA
segment may be incorporated in a known manner into a
recombinant molecule which ensures good expression of the
protein or part thereof. In general, a recombinant
molecule of the invention contains the DNA segment or an
oligonucleotide fragment thereof of the invention and an
expression control sequence operatively linked to the DNA
segment or oligonucleotide fragment. The DNA segment of
the invention or an oligonucleotide fragment thereof, may
be incorporated into a plasmid vector, for example, pECE.
The receptor tyrosine kinase protein or isoforms or parts
thereof, may be obtained by expression in a suitable host
cell using techniques known in the art. Suitable host
cells include prokaryotic or eukaryotic organisms or cell
lines, for example, yeast, E. coli and mouse NIH 3B cells
may be used as host cells. The protein or parts thereof
may be prepared by chemical synthesis using techniques
well known in the chemistry of proteins such as solid
phase synthesis (Merrifield, 1964, J. Am. Chem. Assoc.
85:2149-2154) or synthesis in homogenous solution
(Houbenweyl, 1987, Nethods of Organic Chemistry, ed. E.
Wansch, Vol. 15 I and II, Thieme, Stuttgart).
. . ~
' ' ' .
2085291
- 14 -
The DNA segments of the invention or oligonucleotide
fragments of the DNA segments, allow those skilled in the
art to construct nucleotide probes for use in the
detection of nucleotide sequences in biological materials.
A nucleotide probe may be labelled with a radioactive
label which provides for an adequate signal and has
sufficient half-life such as 32p, 3H, 14C or the like.
Other labels which may be used include antigens that are
recognized by a specific labelled antibody, fluorescent
compounds, enzymes, antibodies specific for a labelled
antigen, and chemiluminescers. An appropriate label may
be selected having regard to the rate of hybridization and
binding of the probe to the nucleotide to be detected and
the amount of nucleotide available for hybridization.
The nucleotide probes may be used to detect genes,
preferably in human cells, that encode proteins related to
or analogous to the receptor tyrosine kinase protein of
the invention.
The receptor tyrosine kinase protein of the invention or
parts thereof, may be used to prepare monoclonal or
polyclonal antibodies. Conventional methods can be used
to prepare the antibodies. As to the details relating to
the preparation of monoclonal antibodies reference can be
made to Goding, J.W., Nonoclonal Antibodies: Principles
and Practice, 2nd Ed., Academic Press, London, 1986. The
polyclonal or monoclonal antibodies may be used to detect
the receptor tyrosine kinase protein of the invention in
various biological materials, for example they may be used
in an Elisa, radioimmunoassay or histochemical tests.
Thus, the antibodies may be used to quantify the amount of
a receptor tyrosine kinase protsin of the invention in a
sample in order to determine its role in particular
cellular events or pathological states.
The finding of a novel receptor tyrosine kinase which is
: :: : ~ -- ................ : . :. .:......... :
:: : :- : .. : .:.... :: . .
208~291
- 15 -
only expressed in cells of the endothelial lineage permits
the identification of substances i.e. ligands, which may
affect angiogenesis and/or maintenance of cells of the
endothelial lineage. Therefore, in accordance with a
method of the invention ligands, and natural and synthetic
derivatives of such ligands, which are capable of binding
to the receptor tyrosine kinase protein of the invention,
isoforms thereof, or part of the protein may be
identified. The method involves reacting the novel
receptor kinase protein of the invention, isoforms
thereof, or part of the protein with at least one ligand
which potentially is capable of binding to the protein,
isoform or part of the protein, under conditions which
permit the formation of ligand-receptor protein complexes,
and assaying for ligand-receptor protein complexes, for
free ligand or for non-complexed proteins.
The ligand-receptor protein complexes, free ligand or non-
complexed proteins receptor-ligand complex, may be
isolated by conventional isolation techniques, for
example, salting out, chromatography, electrophoresis, gel
filtration, fractionation, absorption, polyacrylamide gel
electrophoresis, agglutination, or combinations thereof.
To facilitate the assay of the components, antibody
against the receptor protein or the ligand, or a labelled
receptor protein, or a labelled ligand may be utilized.
The receptor protein or ligand may be labelled with
various enzymes, fluorescent materials, luminescent
materials and radioactive materials. Examples of suitable
enzymes include horseradish peroxidase, biotin, alkaline
phosphatase, ~-galactosidase, or acetylcholinesterase;
examples of suitable fluorescent materials include
umbelliferone, fluorescein, fluorescein isothiocyanate,
rhodamine, dichlorotriazinylamine fluorescein, dansyl
chloride or phycoerythrin; an example of a luminescent
material includes luminol; and examples of suitable
- ~ ,
- ~ , , ; ,
:~ . :
2085291
_ 16 -
radioactive material include radioactive phosphorous 32p,
iodine I125, I131 or tritium.
Radioactive labelled materials may be prepared by
radiolabeling with 12~I by the chloramine-T method
(Greenwood et al, Biochem. J. 89:114, 1963), the
lactoperoxidase method (Marchalonis et al, Biochem. J.
124:921, 1971), the Bolton-Hunter method (Bolton and
Hunter, Biochem. J. 133:529, 1973 and Bolton Review 18,
Amersham International Limited, Buckinghamshire, England,
1977), the iodogen method (Fraker and Speck, Biochem.
Biophys. Res. Commun. 80:849, 1978), the Iodo-beads method
(Markwell Anal. Biochem. 125:427, 1982) or with tritium by
reductive methylation (Tack et al., J. Biol. Chem.
255:8842, 1980).
Known coupling methods (for example Wilson and Nakane, in
~Immunofluorescence and Related Staining Techniques",
W. Knapp et al, eds, p. 215, Elsevier/North-Holland,
Amsterdam & New York, 1978; P. Tijssen and E. Kurstak,
Anal. Biochem. 136:451, 1984) may be used to prepare
enzyme labelled materials. Fluorescent labelled materials
may be prepared by reacting the material with
umbelliferone, fluorescein, fluorescein isothiocyanate,
dichlorotriazinylamine fluorescein, dansyl chloride,
derivatives of rhodamine such as tetramethyl rhodamine
isothiocyanate, or phycoerythrin.
The receptor protein or ligand used in the method of the
invention may be insolubilized. For example, the receptor
protein or ligand may be bound to a suitable carrier.
Examples of suitable carriers are agarose, cellulose,
dextran, Sephadex, Sepharose, carboxymethyl cellulose
polystyrene, filter paper, ion-exchange resin, plastic
film, plastic tube, glass beads, polyamine-methyl vinyl-
ether-maleic acid copolymer, amino acid copolymer,
ethylene-maleic acid copolymer, nylon, silk, etc. The
, -, - - . . . ,, :-.
.
:
- . :: - , .:,: -
208529~
_ 17 -
carrier may be in the shape of, for example, a tube, test
plate, beads, disc, sphere etc.
The insolubilized receptor protein or ligand may be
prepared by reacting the material with a suitable
insoluble carrier using known chemical or physical
methods, for example, cyanogen bromide coupling.
Conditions which permit the formation of ligand-receptor
protein complexes may be selected having regard to factors
such as the nature and amounts of the ligand and the
receptor protein.
In a preferred embodiment of the method, ligands are
identified which are capable of binding to and activating
the novel receptor tyrosine kinase protein of the
invention. In this method the ligands which bind to and
activate the novel receptor tyrosine kinase receptor of
the invention are identified by assaying for protein
tyrosine kinase activity i.e by assaying for
phosphorylation of the tyrosine residues of the receptor.
Protein tyrosine kinase activity may be assayed using
known techniques such as those using antiphosphotyrosine
antibodies and labelled phosphorous. For example,
immunoblots of the complexes may be analyzed by
autoradiography (32P-labelled samples) or may be blocked
and probed with antiphosphotyrosine antibodies as
described in Koch, C.A. et al (1989) Mol. Cell. Biol. 9,
4131-4140.
As hereinbefore mentioned, the invention provides a method
of using the novel proteins of the invention for assaying
a medium for the presence of a substance that affects a
tek effector system. In particular the method may be use
to detect a suspected agonist or antagonist of a tek
effector system. The agonist or antagonist may be an
.
:, - :., : . . ~ . , ~ -. -
- . ~ ., . : ,
, . . . - , : : : -.. .
2085291
- 18 -
endoqenous physiological substance or it may be a natural
or synthetic drug.
The term " tek effector system~ used herein refers to the
interactions of a ligand, and the receptor tyrosine kinase
protein of the invention, and includes the binding of a
ligand to the receptor protein or any modifications to the
receptor associated therewith, to form a ligand/receptor
complex and activating tyrosine kinase activity thereby
affecting signalling pathways, particularly those involved
in the regulation of angiogenesis.
In accordance with one embodiment, a method is provided
which comprises providing a known concentration of a
receptor tyrosine kinase protein of the invention,
isoforms thereof, or part of the protein, incubating the
protein, isoforms thereof, or part of the protein, with a
ligand which is capable of binding to the protein,
isoforms thereof, or part of the protein, and a suspected
agonist or antagonist substance under conditions which
permit the formation of ligand-receptor protein complexes,
and assaying for ligand-receptor protein complexes, for
free ligand or for non-complexed proteins.
The ligand-receptor complex, free ligand or non-complexed
proteins may be assayed as described above. Suitable
ligands used in the assay method may be identified using
the methods described above. The ligand may be a natural
ligand or a synthetic derivative having similar biological
activity.
The invention also makes it possible to screen for
antagonists that inhibit the effects of an agonist of a
tek effector system, but do not have any biological
activity in the tek effector system. Thus, the invention
may be used to assay for a substance that competes for the
same ligand-binding site on the novel receptor tyrosine
' . ~ . .
: .. : : . .. - ,:. .: , :
~52~1
-- 19 --
kinase protein of the invention.
It will be understood that the substances that can be
assayed using the methods of the invention may act on one
or more of the binding site on the receptor tyrosine
kinase or the ligand, including agonist binding sites,
competitive antagonist binding sites, non-competitive
antagonist binding sites or allosteric sites.
The methods of the invention make it possible to screen a
large number of potential ligands for their ability to
iO bind to the novel receptor tyrosine kinase protein of the
present invention. The methods of the invention are
therefore useful for identifiying potential stimulators or
inhibitors of angiogenesis.
The following non-limiting examples are illustrative of
the present invention:
EXAMPLES
The following materials and methods were utilized in the
investigations outlined in the examples:
DNAs
ARR/J, DBA, and AKR/J x DBA recombinant inbred mouse DNAs
were obtained from Jackson Labs (Bar Harbor, Maine),
digested with AccI, blotted to Zeta-Probe nylon membrane
(Bio-Rad), and probed with the 1.6 kb tek cDNA labelled by
random priming (Feinberg, A.P. & Vogelstein, B. (19B3)
Analyt.Biochem., 132, 6-13). Hybridization was performed
overnight at 65 in 200 mM sodium phosphate pH7.0, 7%
sodium dodecyl sulfate (SDS), 1% bovine serum albumin
(BSA), and 1 mN EDTA. Filters were washed twice at 55 in
2 x SSC (1 x SSC= O.lS M NaCl, 0.015 M sodium citrate
pH7.0) and 0.1% SDS and twice in 0.2 x SSC and 0.1~ SDS,
and exposed overnight to Kodak XAR-5 film.
. -~ . ~ .
- . .: .: ,-, .; . .:. . .- : :
;
. -
,
2085231
- 20 -
Nice
Embryos and adult mouse tissues were obtained from random
bred CD-l stocks (Charles River, Quebec). Embryos were
staged as Day 0.5 on the morning of a vaginal plug.
RNA purification and analysis
Total RNA was extracted from pools of 30 to 40 Day 9.5 and
12.5 murine embryonic hearts with RNAzol (CINNA/BlOTECX
Lab. Int.), with some added modifications. Briefly,
tissues were washed with ice cold phosphate buffered
saline (PBS) and homogenized in 2.5 ml of RNAzol.
Chloroform (250 ~1) was added and the tubes were mixed
vigorously and then chilled on ice for 15 min. The
suspension was centrifuged for 15 min at 4~ after which the
aqueous phase was collected and re-extracted twice more
with phenol/chloroform/isoamyl alcohol (25:24:1;
vol:vol:vol). The RNA was precipitated with an equal
volume of isopropanol, collected by centrifugation, and
the pellet resuspended in diethylpyrocarbonate (DEPC)-
treated 0.4 N sodium acetate, pH 5.2. The RNA were then
reprecipitated with two Yolumes of 95~ ethanol, washed
with 70% and 95% ethanol, dried, and resuspended in DEPC
treated 0.3 N sodium acetate, pH5.2. The RNA
concentration was determined and the RNA stored at -70
until use.
Poly A - containing RNA was purified from a pool of 100 to
150 Day 12.5 murine embryonic hearts with a Quic~Prep mRNA
isolation kit (Pharmacia) as outlined by the supplier.
For Northern blot hybridization, 5 ~g of poly A
containin~ RNA from 12.5 day embryonic heart was
electrophoresed through a formaldehyde-agarose gel and
blotted to a Zeta-Probe nylon membrane (Bio-Rad) according
to established protocols (Sambrook et al., 1989, Molecular
Cloning. Cold Spring Harbor Laboratory Press). The
membrane was hybridized with 2 [ 32p ~ -labelled antisense
riboprobe synthesized from the 1. 6 kb tek cDNA in run off
- . : . ~ -: . . ::
~, . ' :`.
208~291
- 21 -
reactions with SP6 RNA polymerase (Promega).
Reverse Transcription Coupled to the Polymerase Chain
Reaction (RT-PCR)
First strand cDNA was synthesized in a total reaction
volu~e of 20 ~1 containing 20 ~g of total RNA, 200 units
of Mo-MLV-reverse transcriptase (BRL), either 1 ~g of
oligo-d(T~18 (Day 12.5 RNA) (Boerhinger Mannheim) or 2 ~g
of random hexamer primers (Day 9.5 RNA) (Boerhinger
Nannheim), 1 x PCR buffer (Cetus), 2.5 mN MgCl2, 1 mM of
dNTPs (Pharmacia), 40 units of RNAsin (Promega), and 12.5
mM dithiothreitol. The RNA was heated to 65C for 10 min
and cooled quickly on ice prior to addition to the
reaction components. The reaction was allowed to proceed
for 1 h at 37 and then terminated by heating for 5 min at
95. For PCR, the reaction mixture was adjusted to a final
volume of 100 ~1 containing 1 x PCR buffer, 1.5 mM NgCl2,
800 ~N dNTPs, and 1 ~g of each of the two degenerate
tyrosine kinase oligonucleotide primers described by
Wilks, A.F. (1989) Proc.Natl.Acad.Sci., 86, 1603-1607.
Amplification was performed with a Ericomp thermocycler
using the following parameters: denaturation for 2 min at
94, annealing for 2 min at 42, and extension for 4 min at
63. After 40 cycles, the reaction products were collected
by ethanol precipitation and electrophoresed through at 2%
low-melt agarose (Sea Plaque) gel. In most cases a band
of approximately 200 bp was visible within a background
smear of ethidium bromide staining. This band was excised
and recovered by three cycles of freeze-thaw in 100 ~1 of
water. 10 ~1 of this solution was then subjected to a
second round of PCR under the same conditions described
above.
Cloning and sequencing of RT-PCR products.
After the second round of amplification, 10 ~1 of the
reaction mixture were analyzed on a gel for successful
:: .
. . . - . . .
.
: .
~: -- ,. ~
2~85291
- 22 -
amplification. The remaining 90 ~1 were then ethanol
precipitated, digested with EcoRI and BamHI, gel purified,
and ligated to pGEM7Zf+ (promega) digested with the same
enzymes. The ligation mixture was then transformed into
MV1190 competent cells, individual amF~ colonies picked,
plasmid DNA prepared, and the cDNA inserts analyzed by
single track dideoxynucleotide sequencing (Sanger, F.,
Nicklen, S. & Coulson, A.R. (1977). Proc. Natl.Acad. Sci.,
74, 5463-5467). A single representative clone of each
multiple isolate was sequenced in its entirety. Of the 58
clones analyzed, roughly 10% showed no sequence identity
to tyrosine kinases and were disregarded.
Isolation of additional tek cDNA sequences.
Approximately 106 plaques from an amplified, random primed
13.5 day murine embryonic AgtlO cDNA library were
hybridized with the 210 bp tek PCR product labelled with
[32P]-dCTP by PCR. Hybridization was carried out overnight
at 55 in 50% formamide, 10~ dextran sulfate (Pharmacia),
0.5 % BLOTTO, 4 x SSPE (1 x SSPE= 0.18 N NaCl, 10 mM
NaH2PO4, 1 mM EDTA, pH7.4), 100 ~g/~l sheared salmon sperm
DNA, and 2 x 106 cpm/ml of probe. Filters were washed at
55~ twice in 2 x SSC containing 0.1% SDS and twice in 0.2
x SSC containing 0.1% SDS, dried, and exposed overnight to
Kodak XAR-5 film. One clone was isolated from this screen
and was found to contain a 1.6 kb cDNA. The sequence of
the 1.6 kb cDNA was determined by the method of Sanger et
al. (1977) from a set of anchored deletions generated with
a standardized kit (Erase - A - Base, Promega).
In situ hybridization
Embryos isolated on Day 12.5 were dissected away from all
extraembryonic tissues whereas embryos at earlier time
points were recovered in utero. Embryos and adult tissues
were fixed overnight in 4% paraformaldehyde, dehydrated
with alcohols and xylenes, and embedded in paraffin.
Tissues were sectioned at 6 ~m thickness and mounted on 3-
,, : - .: . ~
`' - . - . '
-: , - ::: :,:~ .
....
- 23 - 208~291
aminopropyltriethoxysilane treated slides (Sigma). After
removal of paraffin the samples were treated with
predigested pronase (Boerhinger Mannheim), acetylated with
triethanolamine, dehydrated, and hybridized according to
the protocol described by Frohman, N.B., Boyle, M. &
Martin, G.R. ~1990), Development, 110, 589-607.
Dark and bright field photomicroscopy was performed with
a Leitz Vario Orthomat 2 photomicroscopic system.
Ad;acent sections probed with a tek sense probe produced
no detectable signal above background.
Whole-mount in situ hybridizations were performed using a
modification of existing procedures (Tautz, D. & Pfeifle,
C. (1989). Chromosoma, 98,81-85;Hemmati-Brivanlou, A.,
Franck, D., Bolce, M.E., Brown, B.D., Sive, H.L. &
Harland, R.M. (1990). Development, 110, 325-330; Conlon
and Rossant, in prep.). The hybridization of single-
stranded RNA probes labelled with digoxigenin was detected
with antidigoxigenin antibodies coupled to alkaline
phosphatase. The En2 cDNA was prepared as set forth in
Joyner A.L. & Martin, G.R. (1987). Genes and Dev., 1, 29-
38 and expression of En2 is described in Davis, C.A.,
Holmyard, D.P., Millen, K.J. & Joyner, A.L. (1991)
Development, 111:, 287-298.
Immunohistochemistry
Sections were stained immunohistochemically for von
Willebrand factor with a commercially available kit
(Biomeda). After color development, slides were
counterstained with Harris hematoxylin.
EXANPLE I
Isolation and characterization of tek
To identify and characterize tyrosine kinases expressed
during murine cardiogenesis, cDNAs were synthesized from
9.5 and 12.5 day embryonic heart RNA by RT-PCR using
. : : :
208529~
- 24 -
degenerate oligonucleotide primers previously demonstrated
to amplify tyrosine kinase sequences preferentially
(Wilks, A.F. 1989, Proc.Natl.Acad.Sci., 86, 1603-1607).
Considerable cellular differentiation and morphogenesis
have occurred within the cardiac region of the embryo by
Day 9.5. At this stage the heart has developed from the
primordial mesoderm cells of the cardiac plate into a
primitive bent tube structure, consisting of two
endothelial tubes enclosed within the developing
myocardium. Between Day 9.5 and 12.5 the heart undergoes
additional complex morphological changes in association
with the formation of the four chambers and septa
characteristic of the adult heart. Sequence analysis of
58 clones obtained following amplification revealed that
whereas roughly 10~ did not contain sequence similarities
to protein kinases the remainder corresponded to 5
distinct cDNAs (Table 1 - Identity and number of tyrosine
kinase cDNA clones recovered from Day 9.5 and 12.5 murine
embryonic heart by RT-PCR). Four of these cDNAs
represented previously characterized tyrosine kinases
including, bmk, c-src, c-abl, and the platelet derived
growth factor receptor ~-subunit (pdgfrb). The isolation
of bmk, c-src, and c-abl is consistent with the broad
tissue distribution of these kinases (Wang, J.Y.J. &
Baltimore, D. (1983). Nol.Cell.Biol., 3, 773-779;
Ben-Neriah et al., (1986J. Cell, 44, 577-586; Holtzman,
D., Cook, W. & Dunn, A. (1987). Proc.Natl.Acad.Sci., 84,
8325-8329; Renshaw, M.W., Capozza, M.A. & Wang, J.Y.J.
(1988). Mol.Cell.Biol., 8, 4547-4551). The recovery from
embryonic heart of pdgfrb at a relatively high frequency
may indicate that pdgfrb plays an important role in
cardiogenesis, as has been suggested by recent studies
demonstrating that the addition of PDGF-BB to explants of
axolotol cardiac field mesoderm stimulates the production
of beating bodies (Muslin, A.J. & Williams, L.T. (1991~.
Development, 112, 1095-1101) the fifth cDNA, which was
also isolated at high frequency, was novel and for reasons
- ' ~ , ` . ' '' ~. , , ~ . . ,
208529~
- 25 -
that will become clear below was designated tek. ThQ 210
bp RT-PCR-derived tek clone was subsequently used to
isolate additional te~ cDNA sequences.
Figure 1 shows the nucleotide and deduced amino acid
sequence of tek. Figure 2 shows the nucleotide sequence of
a 1.6 kb tek cDNA isolated from a 13.5 day mouse embryo
cDNA library. Translation of this sequence reveals a
single large open reading frame that terminates with TAG
at nucleotide 907, followed by 696 nucleotides of 3
untranslated sequence. Several features of the deduced
amino acid sequence suggest that the 1.6 kb tek cDNA
encodes the cytoplasmic portion of a transmembrane RTK,
consisting of the catalytic domain followed by a short
carboxy-terminal tail of 33 amino acid residues.
Figure 3 shows a comparison of the deduced amino acid
sequence of tek with that of other tyrosine kinases;
Identical sequences are denoted by periods. Dashes were
added to allow for optimal alignment. The kinase insert
and conserved regions of the catalytic domain are
indicated beneath the aligned sequences (Hanks, S.K.,
Quinn, A.N. & Hunter, T. (1988), Science, 241, 52).
Comparative sequences shown are for human Ret (Takahashi,
M. & Cooper, G.M. (1987). Mol.Cell.Biol., 7, 1378-1385),
and Jtkl4 (Partanen, J., Nakela, T.P., Alitalo, R.,
Lehvaslaiho, H. & Alitalo, R. (1990) Proc.Natl.Acad.Sci.,
87, 8913-8917) and murine Flg (Reid, H.H., Wilks, A.F. &
Bernard, O. (1990) Proc.Natl.Acad.Sci., 87, 1596-1600).
As shown in Figure 3, the putative kinase domain contains
several sequence motifs conserved among tyrosine ~inases,
including the tripeptide motif DFG, which is found in
almost all known kinases, and the consensus ATP-binding
site motifs GXGXXG followed by AXK 16 amino acid residues
downstream (Hanks et al ., 1988). Transmembrane RTK's
possess a methionine residue within the motif NMAIESL of
208~291
- 26 -
conserved region VIII of the catalytic domain (Hanks et
al., 1988) as does tek, and the catalytic domain is
interrupted by a putative 21 amino acid kinase insert, a
structural motif not found in cytoplasmic tyrosine kinases
(Hanks et al., 1988).
Comparison with other tyrosine kinases (Figure 3) reveals
that the deduced tek amino acid sequence shows 42~
sequence identity to the mouse fibroblast growth factor
receptor Flg (Reid et al., 1990; Safran, A., Avivi, A.,
Orr-Urtereger, A., Neufeld, G., Lonai, P., Givol, D. &
Yarden, Y. (1990). Oncogene, 5, 635-643, Sambrook, J.,
Fritsch, E.F. & Maniatis, T. (1989). Molecular Cloning.
Cold Spring Harbor Laboratory Press) and 45~ to the
transmembrane RTK encoded by the human c-ret proto-
oncogene (Takahashi & Cooper, G.M. (1987). Mol.Cell.Biol.,7, 1378-1385). In addition, striking sequence identity is
observed to a 65 amino acid residue sequence encoded by
Jtkl4, a putative tyrosine kinase cDNA isolated from
differentiating human K562 cells by RT-PCR (Partanen, J.,
Nakela, T.P., Alitalo, R., Lehvaslaiho, H. & Alitalo, K.
(1990) Proc.Natl.Acad.Sci., 87, 8913-8917). Taken
together, the results suggest that tek encodes a novel
RTK.
EXAMPLE II
Chromosomal mapping of the tek locus
Mapping of the tek locus was accomplished by monitoring
the strain distribution pattern of an AccI restriction
site polymorphism in recombinant inbred (RI) mouse strains
derived from matings between AKR/J (A) and DBA/2J (D)
mice. The tek cDNA detects bands of 6.5, 6.1, 1.3 and
6.5, 3.1, 1.3 kb in DNA from the A and D strains,
respectively. Southern blot hybridization analysis of DNA
from 24 RI mice with the 1.6 kb cDNA probe, and comparison
of the segregation pattern with the Jackson Laboratory
data base, revealed 95.8% cosegregation between tek and
- . ,
,, :
~ . . . -., . . : .
~ .
2085291
- 27 -
both brown and pmv-23, two loci that have previously been
localized to mouse chromosome 4 (Lyon & Searle, 1989).
Table 2 shows the cosegregation of the tek, brown, and
pmv-23 loci in A x D strains. In Table 2 for each RI
strain, the symbol shown indicates the presence of an
allele characteristic of the progenitor from which the
strain was derived (A, AKR/J; D, DBA/2J). These data place
tek between the ~rown and pmv-23 loci within 3.8+1.9
centimorgans of each interval.
EXAMPLE III
Multiple tek-related transcripts are expressed in
embryonic heart
tek expression in embryonic heart was examined by Northern
blot hybridization using an antisense probe darived from
the 1.6 kb tek cDNA. Figure 4 shows a Northern blot
hybridization analysis of tek expression in 12.5 day
murine embryonic heart; Arrows on the left denote the
position of migration of 28 S and 18 S ribosomal RNAs
obtained from adjacent lane loaded with total RNA. Figure
4 shows that the tek probe detects 4 transcripts of 4.5,
2.7, 2.2, and 0.8 kb in size in cardiac RNA from 12.5 day
mouse embryos. These hybridizing species vary
considerably in signal intensity, suggesting that they may
differ in relative abundance, with expression of the 2.7
and 2.2 kb transcripts occurring at significantly higher
levels than the 4.5 and 0.8 kb RNAs. While the exact
relationship among these transcripts is unclear, it is
possible that they arise by differential splicing, since
the 1.6 kb tek cDNA detects a single genomic locus in
mouse DNA by Southern blot hybridization at the same
stringency.
EXAMPLE IV
In situ localization of tek expression during mouse
embryogenesis
To determine which cell types express tek during
`
2085291
- 28 -
development, RNA in situ hybridization analyses were
performed on mouse embryos with an antisense riboprobe
synthesized from the 1.6 kb tek cDNA.
Figure 5 shows the in situ hybridization analysis of tek
expression in the 12.5 day embryo; A. Dark field
illumination of a para-sagittal section. Bar: 600~m. B.
and C. Bright and dark field illumination respectively, of
the heart region taken from a mid-sagittal section. Bar:
300 ~m. IV and VI, fourth and sixth aortic arches; A,
atrium; BA, basilar artery; CV, caudal vein; E,
endocardium; L, liver; M, leptomeninges; Ma, mandible; Ny,
myocardium; PC, pericardial cavity; RA, renal artery; SS,
sino-auricular septum; SV, sinus venosus; V, ventricle.
Figure 5A shows that in 12.5 day mouse embryos, expression
of tek is readily detected in the heart, the leptomeninges
lining the brain and spinal cord, and the inner lining of
major blood vessels, including the caudal vein and basilar
and renal arteries. In addition, thin bands of
hybridization are observed in the intersomite regions,
corresponding to tek expression in the intersegmental
vessels. Close examination of the region of the
developing heart (Figure 5B and 5C) reveals that tek is
expressed in the endocardium, as well as in cells lining
the lumina of the atria, the IV and VI aortic arches, the
sinus venosus, ` and the sino-auricular septum. In
addition, tek expression is observed in numerous small
blood vessels perforating the liver and mandible. These
observations, together with the overall pattern of
hybridiæation seen in the 12.5 day embryo, demonstrate
that tek is expressed in the endothelial cells of the
tunica interna, the innermost lining of the blood vessels;
hence the designation tunica interna endothelial cell
kinase, tek.
Nore detailed information on tek expression was obtained
, . . . ~.
- . :. :-
.,
2085291
- 29 -
through analysis of sections from earlier de~elopmental
stages. Hybridization to 6.5 and 7 day embryos revealed
that while tek is expressed strongly in the inner lining
of the small blood vessels and capillaries of the maternal
decidua, no expression is observed in either the embryo
itself or the ectoplacental cone. The absence of tek
expression at these stages is consistent with the fact
that at 6.5 to 7 days the embryo contains only a small
amount of mesoderm from which endothelial cells are known
to be derived.
Figure 6 shows the expression of tek precedes that of von
Willebrand factor in 8.5 day embryos. Adjacent transverse
sections through an 8.5 day embryo fixed in utero were
either hybridized in situ with an [35S]-labelled tek probe
or stained immunohistochemically for von Willebrand
factor. A. Bright field illumination of tek expression,
Bar: 300 ~m. B. Dark field illumination of section in A.
C. High magnification of a blood island, slightly out of
the field shown in A, depicting silver grains over flat,
elongated cells of endothelial-like morphology, Bar: 50
~m. D. Adjacent section to A at higher magnification
showing absence of expression of von Willebrand factor in
the embryo, Bar: 100 ~m. E. Adjacent section to A at
higher magnification showing expression of von Willebrand
factor in the endothelial lining of the blood vessels of
the maternal decidua. Bar: 200 ~m. F. High magnification
of cephalic region in A showing silver grains over a
large, round cell of angioblast-like morphology ~arrow).
Bar: 50 ~m. G. Bright field illumination of a sagittal
section of an 8.5 day embryo hybridized in situ with an
[35S]-labelled tek probe. Bar: 300 ~m. H. Dark field
illumination of G. I. Higher magnification of heart
region in A showing silver grains over cells with
endothelial- and angioblast-like morphology in the
developing endocardium. Bar: 100 ~m. J. Higher
magnification of somite region in A showing tek-expressing
~: :
.- ~: ~ ' '':
`` 2085291
- 30 -
cells extending beneath, and possibly from, the ventral
surface of the somites. Bar: 100 ~m. A, amnion; Ag,
presumptive angioblast; BI, blood island; D, maternal
decidua; DA, dorsal aorta; E, endocardium; Ec,
ectoplacental cone; En, endothelial cell; G, foregut; HV,
head vein; NF, neural fold; S, somite; Y, yolk sac.
RNA in situ analysis of 8.0 day embryos revealed that tek
expression first becomes detectable in the developing yolk
sac and a few small clusters of cells in the cephalic
mesenchyme. This expression becomes more pronounced by
Day 8.5, at which time significant hybridization can be
observed in the mesodermal component of the amnion (outer
cell layer) and yolk sac (inner cell layer), as well as in
- the developing endocardium and the inner lining of the
head veins and dorsal aortae (Figure 6A and 6B). In
addition, sagittal sections reveal numerous focal areas of
hybridization throughout the cephalic mesenchyme in
regions thought to contain developing vasculature, as well
as a small number of tek-expressing cells extending
beneath the ventral surface of the somites (Figure 6H and
6J).
Whole mount in situ hybridization analysis confirmed and
extended the above observations, as well as provided a
three dimensional perspective on tek expression during
embryogenesis. Figure 7 shows tek expression in whole
mount embryos; A., B., C. and D. tek expression in Day
8.0 embryos. E. tek mRNA distribution in a Day 9.5
embryo. F. En2 expression in a Day 8 embryo. I, II, III,
first, second and third aortic arches; DA. dorsal aorta;
E, endocardium; G, foregut pocket; H, heart; IS,
intersegmental vessel; My, myocardium; ; NF, neural fold;
OT; otic vesicle; V, vitelline vein; Y, yolk sac. Bars:
250 ~m.
Consistent with the observations with sectioned material,
, . : : , ., . ."
20852~1
- 31 -
localized tek expression was not observed on embryonic
Day 7. The first detectable expression was seen about the
time of first somite formation when signal was observed in
the yolk sac, head mesenchyme, and heart. In Day 8.5
embryos, tek was found to be expressed in these same
areas, and in the paired dorsal aortae, the vitelline
veins, and in the forming intersegmental vessels
(Figure 7). By this time, tek expression was clearly
confined to blood vessels within the embryo. On Day 9,
tek expression was seen in addition, in the aortic arches
and expression was very striking in the endocardium
(Figure 7E). Control hybridizations with an En-2 probe
demonstrated the specificity of tek RNA detection (Figure
7F).
BXAMPLE V
Expression of tek in endothelial cell progenitors
The observation that tek is expressed between Day 8.0 and
8.5 in focal regions thought to represent developing blood
vessels raised the possibility that tek might be expressed
in endothelial cell progenitors. Indeed, close inspection
of hybridized sections from 8 to 8.5 day embryos revealed
that while the expression of tek in the maternal decidua
is restricted to cells of an endothelial cell morphology,
tek expressing cells in the embryo are of two
morphologically distinct cell types. In the developing
blood islands of the yolk sac, where tek expression is
first detected, silver grains are localized predominantly
to elongated cells with characteristic endothelial cell
morphology (Figure 6C). In contrast, within the cephalic
mesenchyme, silver grains are frequently observed over
large, round cells that, on the basis of similar
morphology to cells described during avian embryogenesis
(Pardanaud et al., 1987; Coffin & Poole, 1988; Noden,
1989; Noden, 1991), correspond to angioblasts, the
presumptive progenitor of endothelial cells (Figure 6F).
: ,- : - :: ,. -
- 32 - 208~291
Both cell types are observed in the developing endocardium
(Figure 6I) which, at later stages, is known to contain
only fully mature endothelial cells.
To characterize more precisely the staging of tek
expression within the endothelial lineage, sections
adjacent to those used for in situ hybridization were
stained immunohistochemically for von Willebrand factor,
a well characterized marker of mature endothelial cells
(Jaffe, E.A., Hoyer, L.W. & Nachman, R.L. (1973).
J.Clin.Invest., 52, 2757-2764; Hormia, M., Lehto, V.-P. &
Virtanen, I. (1984), Eur.J.Cell.Biol., 33, 217-228).
Figure 6B and H shows that whereas tek is expressed in
both the maternal decidua and the embryo at Day 8.5,
expression of von Willebrand factor is observed only in
the tek-expressing, vascular endothelial cells of the
maternal decidua (Figure 6D and 6E). Hence tek expression
precedes that of von Willebrand factor during
embryogenesis. The same scenario is observed at later
developmental stages during vascularization of individual
organs.
Figure 8 shows the expression of tek precedes that of von
Willebrand factor in the developing leptomeninges; A.
Absence of immunohistochemical staining of von Willebrand
factor in Day 12.5 leptomeninges. Arrow denotes a large
blood vessel faintly positive for von Willebrand factor.
B. In situ detection of tek expression in Day 12.5
leptomeninges. C. Staining of von Willebrand factor in Day
14.5 leptomeninges. Day 14.5 leptomeninges were positive
for tek expression (not shown). M, leptomeninges. Bars:
200 ~m.
Figure 8 shows that in the 12.5 day embryo, the developing
leptomeninges hybridizes strongly with tek but fails to
stain positive for von Willebrand factor. By Day 14.5,
however, expression of von Willebrand factor can be
:- . , : .. .- . .
2~85291
readily detected in the leptomeninges. Assuming that
there is not a significant lag between transcription and
translation of von Willebrand factor, these observations,
together with those on the morphology of tek-expressing
cells, suggest that tek is expressed in both mature
endothelial cells and their progenitors.
EXAMPLE VI
tek is expressed in adult vasculature
While the above results establish that tek is expressed
during vascularization of the embryo, it was also of
interest to determine whether expression of tek is
maintained in endothelial cells of the adult. In situ
hybridization analysis of a section through the heart
region of a 3 week-old mouse revealed that tek is
expressed in the endocardium as well as in the
endothelial lining of major blood vessels, both arteries
and veins, connecting with the adult heart (Figure 9).
Figure 9 shows the expression of tek in adult vasculature.
A. Bright field illumination of a section through the
upper heart region of a 3 week-old mouse hybridized with
an [35S]-labelled tek probe. Bar: 20 ~m. B. and C.
Bright field illumination showing tek expression in
endothelial cells lining the artery and vein respectively.
Bar: 1 ~m. Immunohistochemical staining of adjacent
sections revealed that structures positive for tek
expression also stained positive for von Willebrand
factor. A, artery; Bl, extravasated blood; T, trachea; V,
vein.).
The intensity of the hybridization signal observed for
these structures is considerably lower than that observed
for the endocardium and blood vessels of 12.5 day embryos
hybridized and processed in parallel. This could indicate
that mature endothelial cells, which are thought to be
:. - . -:
.
.
. . ., : ~
: - ;
208~2~1
- 34 -
resting, have a different quantitative or qualitative
requirement for expression of tek.
'. '"' ~ ' - ' :
2085291
-- 35 --
Table~; Protein qroslne kinase cDNAs isolated by RT PCR
Emblyonic Age
(Days) . cDNA
tek pd~rb cobl c~ brr~lc
9.5 2f~ 7 2
12.5 5 10
- .
.. , , , , . . . . :,
--;. , - . - .
. . ~ . ~, ~ - -
,, : , .. . ..
2085291
-- 36 --
TABLI~ 2. Cosegreg~tion of the tck, bn7w~, and pmv-23 loei in A x D strains.
.
A x D strain
Locu~ 1 2 3 6 7 8 9 l0lll2l3l4lsl6l8202læ23242s262728
-
tek D D A D D A A A D A D A D D D D A D D A D D D D
brown D D A D D A A A D A A A D D D D A D A A D D D D
pmv-23 D D A D D A D A D A D D D A D D A D D A D D D A
