DEVELOPMENTAL DYNAMICS 207235-252 (1996)
Expression Patterns of I&, Id2, and Id3 Are Highly
Related But Distinct From That of Id4 During
YALE JEN, KATIA MANOVA, AND ROBERT BENEZRA
Cell Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York 10021
helix-loop-helix (dnHLH) proteins inhibit the ac-
tivities of bHLH transcription factors in diverse
cell lineages (Benezra et al. 119901 Cell 61:49-59;
Christy et a1 119911 Proc. Natl. Acad. Sci. U.S.A.
88:1815-1819; Sun et a1 119911 Mol. Cell Biol. 11:
5603-5611; Riechmann et al.  Nucleic Acids
Res. 22749-755). Currently, there are four mem-
bers in the dnHLH family, Idl, Id2, Id3, and Id4. In
this report, we have performed a detailed compar-
ative in situ hybridization analysis to examine
their expression pattern during post-gastrula-
tional mouse development. Idl, 2, and 3 are ex-
pressed in multiple tissues, whereas Id4 expres-
sion can only be detected in neuronal tissues and
in the ventral portion of the epithelium of the de-
veloping stomach. The regions where Id13 genes
are expressed, such as gut, lung, kidney, tooth,
whisker, and several glandular structures, are un-
dergoing active morphogenetic activities. The ex-
pression patterns of Idl, 2, and 3 overlap in many
organs, except in the tissues derived from primi-
tive gut. In the latter, Id1 and Id3 signals are de-
tected in the mesenchyme surrounding the epi-
thelium, whereas Id2 is expressed within the
epithelium. The difference in the patterns of ex-
pressions of Id13 and Id4 suggest that the domi-
nant negative transcriptional activity of these two
subclasses of the Id family may have different
physiological consequences. o 1996 Witey-Liss, Inc.
The murine dominant negative
Key words: Mouse Embryogenesis, Helix-Loop-
Helix, Id, In Situ Hybridization, Dif-
Eukaryotic transcription factors can be divided into
groups defined by shared sequences and presumed
structural similarities. The group which contains the
basic helix-loop-helix (bHLH) domain constitutes an
evolutionarily conserved family that has been impli-
cated in cell type determination (for review see Wein-
traub et al., 1991). The bHLH proteins form either
homo- or hetero-dimers through the HLH dimerization
domain and bind to their target DNA sequences to reg-
One of the mechanisms that regulates the available
0 1996 WILEY-LISS, INC.
pool of bHLH dimers in cells is the sequestration of the
bHLH proteins by the dominant negative HLH
(dnHLH) proteins (for review see Kadesch, 1992). The
dnHLH proteins possess an HLH motif but do not con-
tain the adjacent stretch of basic amino acids which is
required for DNA binding and therefore form non-func-
tional heterodimers with bHLH proteins. The Id pro-
teins from both mammals, and Xenopus (Wilson and
Mohun, 1995; Zhang et al., 1995) and the Drosophila
extramachrochaetae ( e m ) gene (Ellis, 1994) are the
members of the dnHLH class identified thus far and all
are capable of inactivating bHLH proteins by direct
physical interaction. Currently, there are four mem-
bers in the mammalian Id family: Idl, Id2 (Sun et al.,
19911, Id3 (Christy et al., 19911, and Id4 (Riechmann et
al., 1994). The Xenopus homologs of Id2 and Id3 have
also been identified (Wilson and Mohun, 1995; Zhang
et al., 1995). The mammalian Id genes appear to an-
tagonize the activity of bHLH proteins involved in
many developmental processes, such as myogenesis
(Jen et al., 1992; Kurobayashi et al., 1994), myelopoie-
sis (Kreider et al., 1992), lymphopoiesis (Wilson et al.,
1991), bone morphogenesis (Ogata et al., 1993), kidney
glomerular mesangial cell development (Simonsen, et.
al., 1993), trophoblast development (Janatpour et al.,
1994), as well as in cell cycle progression (Hara et al.,
The embryonic expression pattern of Id1 has been
determined by in situ hybridization analysis (Duncan
et al., 1992, 1994; Evans and O'Brien, 1993; Wang et
al., 1992). Id1 expression appears in many tissues and
its expression roughly correlates with the less differ-
entiated state of the cells. Id2 and Id3 expression pat-
terns have been documented during embryonic neuro-
genesis only (Neuman et al., 1993; Ellmeier et al.,
1992; Nagata and Todokoro, 1994). The Id4 embryonic
expression pattern has not been reported.
In this study, we have examined the expression pat-
tern of all four Id genes by in situ hybridization of
adjacent sections in order to determine precise regions
of overlap or differences in the pattern of expression of
this gene family. Our results suggest that Idl, 2, and 3
Received January 5, 1996; accepted May 24, 1996.
Address reprint requestslcorrespondence to Robert Benezra, Cell
Biology Program, Memorial Sloan Kettering Cancer Center, 1275
York Avenue, New York, NY 10021.
JEN ET AL.
are expressed in multiple tissues at the sites which
undergo active morphogenic activities, whereas Id4 ex-
pression is mainly detected in neuronal tissues as well
as in more differentiated regions of several other tissue
types. In addition to providing a useful catalogue of the
patterns of Idl-4 gene expression, these results also
suggest that the consequences of dominant negative
regulation of transcription factor activity may be dif-
ferent for different Id family members.
Probes for Idl-4 were generated outside of the con-
served HLH domain to ensure specificity (see Materials
and Methods). Northern analysis using each of these
probes confirmed that they were specific for the appro-
priate RNA species (data not shown). Furthermore, in
situ analysis indicated that some tissues only hybridize
to one of the four probes consistent with the idea that
the specificity of the probes was maintained under in
situ hybridization conditions. Sense RNA probes were
used in the in situ hybridization analysis as negative
controls. None of the sense probes showed any signals
above background. However, the Id4 probe gave a
lower signalhoise ratio compared to the other three
probes. Note that a summary of the expression pattern
of all four Id genes is presented in Table 1.
During gastrulation, all of the Id genes, except Id4,
are expressed in a specific manner (Jen et al., unpub-
lished data): Id1 and 3 are mainly detected in the inner
cell mass derived tissues, whereas Id2 expression ap-
pears in the trophoectodermal derivatives. The overall
postgastrulational expression pattern of Id genes de-
scribed in this study is as follows: Id1 (Wang et al.,
1992; Duncan et al., 1992; Evans and O'Brien, 1993;
this work), Id2 and Id3 are expressed in multiple tis-
sues, and the expression patterns among them exhibit
extensive overlap (Fig. 1.1-4). For example, the signal
of these three Id genes can be detected in the mandib-
ular arch, somites, and in the mesenchyme surround-
ing the dorsal side of the developing stomach. The Id4
signal can be detected mainly in neuronal tissues and
in the ventral side of the stomach epithelium during
early stages (Fig. 1.4). Later in development, several
other tissues also acquire Id4 expression.
Expression in Developing Cartilage and Bone
Embryonic skeletal development is composed of
three distinct lineages (for a review see Erlebacher et
al., 1995): the craniofacial skeleton is derived from the
neural crest cells; the somitic derived sclerotome gen-
erates both the axial skeleton and the lateral plate
mesoderm. The latter will later form the appendicular
skeleton. Idl, 2, and 3 are expressed during skeletoge-
nesis in all three lineages, and their expression pat-
terns are very similar (Fig. 2A-0). By 8.5 dpc, all three
Id mRNAs can be detected in the ridges of the unclosed
head folds and the surrounding cranial mesenchyme,
due presumably to the migrating neural crest cells
(data not shown). By 9.5 dpc, the expression of the
three Id genes is detected in the branchial arches (data
not shown), and the expression persists (Fig. 1.1-3) as
more craniofacial structures are being developed. The
craniofacial skeletal structures become more distinct
at 14.5 dpc and the messages of the three Id genes can
be found in many sites undergoing chondrogenesis or
cartilage formation, such as the otic capsule, nasal sep-
tum, and many cranal bones (Fig. 2K,L,M, and N). No
significant level of Id4 can be detected in the craniofa-
cia1 skeletal structures (Fig. 2K,O).
During axial skeleton development, Id1 (Wang et al.,
1992; Evans and OBrien, 1993; this work), Id2, and Id3
signals are first observed in the 10.5 dpc somites. The
somitic expression of these Id genes is mainly located in
the sclerotome, and very weak signals appear in the
dermatome. The intervening myotome is devoid of any
Id signals (data not shown) (for Idl, see Wang et al.,
1992; Evans and OBrien, 1993; this work). In the 11.5
dpc sclerotome, the expression of all three Id genes ap-
pears in the rostral half which contains less cells (Fig.
2A-D). Later, the rostral sclerotome will give rise to the
center of the vertebrae, which also expresses the same
Id genes (data not shown). The more densely packed
caudal sclerotome, which later forms the intervertebral
disk, is negative for Id messages. From 12.5 dpc, the
perichondria surrounding the developing ribs also ex-
press Idl, 2, and 3. At later stages, both the ribs and the
vertebrae acquire some weak Id4 expression (data not
shown). No significant Id4 message can be detected in
the sclerotome (Fig. 2A,E) and its derivatives.
Id expression in the appendicular skeletal develop
ment is similar to that of the axial skeletal lineages:
Idl, 2, and 3 are first expressed in the limb buds be-
tween 9.5 to 10.5 dpc (data not shown). By 12.5 dpc, the
expression of the three Id genes can be detected in both
(1) the interdigital mesenchyme, which later under-
goes apoptosis (Hammar and Mottet, 1971) in order to
remove the intervening web between the digits, and (2)
in the blastemal condensations where the chondrifica-
tion has just started (Rugh, 1990) (Fig. 2P-S). Later,
all four Id genes are expressed in the hypertrophic car-
tilage and the newly forming bony diaphysis of many
long bones (Fig. 2FJ) and digits in 16.5 dpc embryos.
However, the level of Id4 expression is just slightly
above background (Fig. 2F,J).
Craniofacial and trunk dermis are derived from neu-
ral crest and the dermatome portion of the somite, re-
spectively. Low levels of Id1 (Evans and OBrien, 1993;
this work), Id2, and Id3 are expressed in the developing
dermis (data not shown).
Expression in the Epithelial-Mesenchymal
Interacting Craniofacial Structures
The developing whisker follicle and tooth are both
regulated by the sequential and reciprocal interactions
between the cranial neural crest-derived mesenchyme
and the facial or oral epithelial placodes (Lumsden,
1988; Hardy, 1992; Panaretto, 1993). The early mor-
phological events of tooth development are the invagi-
EXPRESSION OF Id FAMILY IN MOUSE
TABLE 1. Expression of Each Id Gene i n the 1 1 . 5 - t o 16.5-Day Mouse Embryo"
Id1 Id2 Id3
Septum and valves
Epith. of otic vesicle
sur. mes. +
sur. mes. +
Epith. and sm. mus. +
selected neurons +
stir. mes. +
selected neurons +
Mature neurons -
Mature neurons +
Mature neurons -
Nasal processes 11.5D
Mes. adjacent to
supporting cells 5
supporting cells t
Postmitotic neurons +
supporting cells t
Dental papilla mes.
dermal sheath, and
outer root sheath
Inner root sheath
aEpith. = epithelium; Mes = mesenchyme; Sm.mus. = smooth muscle; Sur. mes. = surrounding mesenchyme. Relative level
of mRNA expression is indicated by: - = no expression; 5 = trace expression; + = mRNA is expressed. ND = not
JEN ET AL.
exhibit an expression pattern which partially overlaps
that of the Id genes. Examples are E2A (Roberts et al.,
19931, M-twist (Wolf et al., 1991), Purdzis (Burgess et
al., 19951, Sclerazis (Cserjesi et al., 19951, and HES-1
(Sasai et al., 1992). M-twist is noteworthy since like
Idl, M-twist can inhibit muscle differentiation in tissue
culture (Hebrok et al., 1995). M-twist expression can be
detected in the sclerotorne, mesenchymal cells in the
head, branchial arches, and limb bud, and in cardiac
cushions. The expression pattern of M-twist in the
somite, limb bud, and craniofacial structures is mutu-
ally exclusive with respect to the expression pattern of
myogenic factor, myfs (Hebrok et al., 1995; Ott et al.,
1991; Wolf et al., 1991) analogous to the relationship
between Id1 and myE in the same structures (Wang et
al., 1992; Evans and O'Brien, 1993). Based on these
results, it will be of interest to further investigate the
possible relationship between Id and M-twist. Interest-
ingly, the expression of Myf5, as well as Idl, Id2, and
Id3 in branchial arches, occurs early and precedes myo-
genesis. This observation suggests that Idl, which is
also expressed in the brachial arches, can prevent the
myogenic regulatory factors (MRFs) from activating
downstream differentiation genes as well as antago-
nize the expression of MRFs in the somites (see Jen et
al., 1992; Wang et al., 1992).
In neuronal tissues, Id4 expression appears in differ-
entiated neurons, unlike Id1 and Id3 which are found
in mitotically active neuroblasts (Jen et al., unpub-
lished data). In addition, Id4 expression in other tissues
is more limited than that of Idl-3, and is present in
more differentiated regions. If we assume that all of the
Id genes can inhibit other bHLH proteins from binding
DNA, these results would suggest that Id4 may target
a distinct set of proteins leading to different physiolog-
ical consequences. The close similarity in the expres-
sion pattern of Id3 and Idl, on the other hand, suggests
a redundancy between these two genes. The existence
of such a redundancy could be due to the selective ad-
vantage of cumulative function and/or of higher fidel-
ity (Thomas, 1993). The actual functional relationship
among the different members of Id gene family will
need to be resolved by loss-of-function genetic manip-
MATERIALS AND METHODS
Generation of Each Id Specific Clones
All Id gene specific clones were generated by PCR
amplifying the region of each Id gene, which was de-
void of the HLH domain, followed by cloning each Id
specific fragment into pBluescriptKS (Stratagene, La
Jolla, CA). The templates used for generating Idl, 2,
and 3 fragments are clone pMH18AR (Benezra et al.,
1990), Id2 genomic clone (Sun et al., 1991), and
HLH462 phagmid (Christy et al., 19911, respectively.
The total genomic DNA from the tail of the C57BL/
CBA mice was used as template to generate Id4 specific
fragments. The oligonucleotide sequences, which were
used for generating Id4 specific fragments, are derived
from the available Id4 cDNA sequences in Genbank.
The Id1 specific clone, pBS/KS-Idlsp, contains an in-
sert of 350 bp which corresponds to the nucleotides 390
to 742 of the Id1 cDNA. The Id2 specific clone, pBS/
KS-Idasp, contains an insert of 195 bp which corre-
sponds to the nucleotides 1 to 195 of the Id2 cDNA. The
Id3 specific clone, pBS/KS-Id3sp, contains an insert of
270 bp which corresponds to the nucleotides 365 to 635
of the Id3 cDNA. The Id4 specific clone, pBS/KS-Id4sp,
contains an insert of 260 bp which corresponds to the
nucleotides 665 to 945 of the Id4 cDNA.
(-y-35S)UTP labeled riboprobes were used for all in
situ hybridization analysis. Antisense Id1 specific ribo-
probes were generated by linearizing pBS/KSXdlsp
with XbaI and transcribing by T3 RNA polymerase.
The control sense probes can be generated by lineariz-
ing with XhoI and transcribing by T7 RNA poly-
merase. The Id2 specific antisense riboprobes were gen-
erated by linearizing pBS/KSId2sp with EcoRI and
transcribing by T7 RNA polymerase. The control sense
probes were generated by linearizing with B a d 1 and
transcribing by T3 RNA polymerase. The Id2 full
length cDNA antisense riboprobes which generated
from linearizing Id2 cDNA clones, pId2k (Sun et al.,
19911, with XhoI, and transcribing with SP6 RNA poly-
merase, were also used in in situ hybridization analy-
sis. Both Id2 specific and full length probes gave an
identical specific signal in the in situ hybridization
analysis. However, compared to the Id2 specific probes,
the full length Id2 riboprobes gave much stronger sig-
nals and have been used for most of the in situ hybrid-
ization analysis. The Id3 specific probes were gener-
ated by linearizing pBS/KS-Id3sp with Sac1 and
transcribing by T3 RNA polymerase. The control sense
probes were generated by linearizing with EcoRI and
transcribing by T7 RNA polymerase. The Id4 specific
probes were generated by linearizing pBS/KS-Id4sp
with XbaI and transcribing by T3 RNA polymerase.
The control sense probes can be generated by lineariz-
ing with EcoRI and transcribing by T7 RNA poly-
Staging of Mouse Embryos
Embryos obtained from the mating between C57BL/
CBA F1 mice were used for all the in situ hybridization
analysis. The day when the vaginal plugs was detected
was counted as 0.5 days postcoitum (dpc).
In Situ Hybridization
Preparations of embryonic tissue sections, slide hy-
bridization, and the stringency of washes were the
same as described before (Wang et al., 1992). Briefly,
embryos from 4.5 to 8.5 dpc were retained inside the
surrounding decidua, and the embryos of older ages
were removed from the decidua and fixed in freshly
prepared cold 4% paraformaldehyde in phosphate-
buffer saline overnight. For larger embryos, the fixa-
EXPRESSION OF Id FAMILY IN MOUSE
tion were carried out under vacuum to ensure that the
tissues were properly fixed. Tissues were then slowly
dehydrated and embedded into paraffin blocks. Serial
sections (5-7 krn) were collected on glass microscope
"subbed" slides. Slides with attached sections were de-
paraffinized, rehydrated, Proteinase K treated, acety-
lated, and dehydrated. The hybridizations were carried
out at 52°C for > 16 hr in 50% deionized formamide, 0.3
M NaC1,20 mM Tris-HC1 (pH7.4),5 mM EDTA, 10 mM
NaH,PO, (pH8.01, 10% dextran sulfate, 1 X Den-
hardt's, 500 kg/ml yeast RNA, 0.1 M DTT with 50,000
dpm/ml 35S-labeled RNA probe. After hybridization,
the slides were washed in the following order: (1) 5 x
SSC, 10 mM D!L" at 50°C for 30 min; (2) 50% forma-
mide, 2 x SSC, 0.1 M DlT at 65°C for 20 min; (3) twice
with washing solution (0.1 M Tris-HC1 tpH7.51, 0.4 M
NaC1, 50 mM EDTA) for 10 min at 37°C; (4) washing
solution with 20 kg/ml of RNaseA at 37°C for 1 hr; (5)
washing solution for 5 min at 37°C; (6) repeat the 50%
formamide wash once; (7) 2 x SSC at 37°C for 15 min;
and finally (8) 0.1 x SSC at 37°C for 15 min. Then, the
slides were dehydrated rapidly and processed for stan-
dard autoradiography using Kodak emulsion NTB-2
and exposed for 5 to 28 days at 4°C. After developing in
Kodak D-19 developer and fixer, the sections were
stained with hematoxylin and eosin and mounted with
Cytoseal mounting media 60. Analysis were carried
out using both light and darkfields, or Nomarski optics
on a Zeiss Axiophot microscope (Zeiss, Thornwood,
Y. J. is grateful to Dr. David Sassoon for help with
the in situ analysis. Y. J. and K. M. also thank Dr.
David Lyden for his encouragement. R. B. would like to
dedicate this manuscript to Hal. This work was sup-
ported by grants from the National Science Foundation
(IBN-9118977) and the National Cancer Institute (P30-
NOTE ADDED IN PROOF:
During the preparation of this manuscript, two arti-
cles describing the expression patterns of Id3 and Id4
during mouse embryogenesis were published. The re-
sults are consistent with those presented here. These
articles are (1) Ellmeier and Weith (1995) Dev. Dyn.
203:163-173; and (2) Riechman and Sablitzky (1995)
Cell Growth Differ. 62337-843.
Bard, J. B., McConnell, J. E., and Davies, J. A. (1994) Toward a
genetic basis for kidney development. Mech. Dev. 483-11.
Benezra, R., Davis, R. L., Lockshon, D., Turner, D. L., and Weintraub,
H. (1990) The protein Id: A negative regulator of helix-loop-helix
DNA binding proteins. Cell 61:49-59.
Bockman, D. E., and Kirby, M. L. (1984) Dependence of thymus de-
velopment on derivatives of the neural crest. Science 223:498-500.
Burgess, R., Cserjesi, P., Ligon, K. L., and Olson, E. N. (1995) Paraxis:
A basic helix-loop-helix protein expressed in paraxial mesoderm
and developing somites. Dev. Biol. 168:296-306.
Christy, B. A,, Sanders, L. K., Lau, L. F., Copeland, N. G., Jenkins, N.
A., and Nathans, D. (1991) An Id-related helix-loop-helix protein
encoded by a growth factor-inducible gene. Roc. Natl. Acad. Sci.
Coffin, J. D., Harrison, J., Schwartz, S., and Heimark, R. (1991) An-
gioblast differentiation and morphogenesis of the vascular endothe-
lium in the mouse embryo. Dev. Biol. 148:51-62.
Cserjesi, P., Brown, D., Ligon, K. L., Lyons, G. E., Copeland, N. G.,
Gilbert, D. J., Jenkins, N. A., and Olson, E. N. (1995) Sclemxis: A
basic helix-loop-helix protein that prefigures skeletal formation
during mouse embryogenesis. Development 121:1099-1110.
Davidson, P., and Hardy, M. H. (1952) The development of mouse
vibrissae in vivo and in vitro. J. Anat. 86:342-356.
Duncan, M., DiCicco-Bloom, M., Xiang, X., Benezra, B., and Chada,
K. (1992) The gene for the helix-loop-helix protein, Id, is specifically
expressed in neural precursors. Dev. Biol. 154:l-10.
Duncan, M. K., Shimamura, T., and Chada, K. (1994) Expression of
the helix-loop-helix protein, Id, during branching morphogenesis in
the kidney. Kidney Int. 46~324-332.
Echelard, Y., Epstein, D. J., stJacques, B., Shen, L., Mohler, J., Mc-
Mahon, J. A., and McMahon, A. P. (1993) Sonic Hedgehog, a mem-
ber of a family of putative signaling molecules, is implicated in the
regulation of CNS polarity. Cell 75:1417-1430.
Ekblom, P. (1989) Developmentally regulated conversion of mesen-
chyme to epithelium. FASEB. J. 3:2141-2150.
Ellis, H. M. (1994) Embryonic expression and function of the Drosoph-
ilu helix-loop-helix gene, eztramacrochaetae. Mech. Dev. 47:65-72.
Ellmeier, W., Aguzzi, A., Kleiner, E., Kurzbauer, R., and Weith, A.
(1992) Mutually exclusive expression of a helix-loop-helix gene and
N-myc in human neuroblastomas and in normal development.
EMBO J. 11:2563-2571.
Erlebacher, A., Filvaroff, E. H., Gitelman, S. E., and Deryneck, R.
(1995) Toward a molecular understanding of skeletal development.
Evans, S. M., and OBrien, T. X. (1993) Expression of the helix-loop-
helix factor Id during mouse embryonic development. Dev. Biol.
Fukagawa, M., Suzuki, N., Hogan, B. L. M., and Jones, C. M. (1994)
Embryonic expression of mouse Bone Morphogenetic Protein-1
(BMP-l), which is related to the Drosophila Dorsoventral gene tol-
loid and encodes a putative astacin metalloendopeptidase. Dev.
Hammar, S. P., and Mottet, N. K. (1971) Tetrazolium salt and elec-
tron-microscopic studies of cellular degeneration and necrosis in the
interdigital areas of the developing chick limb. J. Cell Sci. 8:229-
Hara, E., Yamaguchi, T., Nojima, H., Ide, T., Campisi, J., Okayama,
H., and Oda, K. (1994) Id-related genes encoding HLH proteins are
required for G, progression and are repressed in senescent human
fibroblasts. J. Biol. Chem. 269:2139-2145.
Hardy, M. H. (1992) The secret life of the hair follicle. Trends Genet.
Hebrok, M., Wertz, K., and Fuchtbauer, E.-M. (1995) M-twist is an
inhibitor of muscle differentiation. Dev. Biol. 165:537-544.
Janatpour, M. J., McMaster, M. T., Israel, M. A., and Fisher, S. J.
(1994) Expression of the transcriptional negative regulator Id2 is
modulated during human cytotrophoblast differentiation. Mol. Biol.
Cell 5(Suppl.) 453a.
Jen, Y., Weintraub, H., and Benezra, R. (1992) Overexpression of Id
protein inhibits the muscle differentiation program: In vivo associ-
ation of Id with E2A proteins. Genes Dev. 61466-1479.
Jones, C. M., Lyons, K. M., and Hogan, B. L. M. (1991) Involvement of
Bone Morphogenetic Protein4 (BMP4) and Vgr-1 in morphogene-
sis and neurogenesis in the mouse. Development 111:531-542.
Kadesch, T. (1992) Helix-loop-helix proteins in the regulation of im-
munoglobulin gene transcription. Immunol. Today 13:31-36.
Katagiri, T., Yamaguchi, A., Komaki, M., Ah, E., Takahashi, N.,
Ikeda, T., Rosen, V., Wozney, J. M., Fujisawa-Sehara, A., and Suda,
T. (1994) Bone Morphogenetic Protein-2 converts the differentation
pathway of C2C12 myoblasts into the osteoblast lineage. J. Cell
JEN ET AL.
Kaufman, M. H. (1992) “The Atlas of Mouse Development.” New
York: Academic Press.
Kirby, M. L., Gale, T. F., and Stewart, D. E. (1983) Neural crest cells
contribute to aorticopulmonary separation. Science 220:1059-1061.
Kreider, B., Benezra, R., Roverea, G., and Kadesch, T. (1992) Inhibi-
tion of myeloid differentiation by the helix-loop-helix protein Id.
Kurobayashi, M., Dutta, S., and Kedes, L. (1994) Serum inducible
factors binding to an activating transcription factors motif regulate
transcription of the Id2A promoter during myogenic differentiation.
J. Biol. Chem. 269:31162-31170,
Lumsden, A. G. S. (1988) Spatial organization of the epithelium and
the role of neural crest cells in the initiation of the mammalian
tooth germ. Developpment 103 (Suppl.):155-169.
Lyons, K. M., Pelton, R. W., and Hogan, B. L. M. (1989) Pattern of
expression of murine Vgr-1 and BMP-2a RNA suggest that trans-
forming growth factor-p-like genes coordinately regulate aspects of
embryonic development. Genes Dev. 3:1657-1668.
Lyons, K. M., Pelton, R. W., and Hogan, B. L. M. (1990) Organogen-
esis and pattern formation in the mouse: RNA distribution patterns
suggest a role for Bone Morphogenetic Protein-2A (BMP-2A). De-
Lyons, K. M., Hogan, B. L. M. and Robertson, E. J. (1995) Colocaliza-
tion of BMP7 and BMP2 RNAs suggests that these factors cooper-
atively mediate tissue interactions during murine development.
Mech. Dev. 50:71-83.
McMahon, A. P., Champion, J. E., McMahon, J. A., and Sukhatme, V.
P. (1990) Developmental expression of the putative transcription
factor Egr-1 suggests that Egr-1 and c-fos are coregulated in some
tissues. Development 108:281-287.
Nagata, Y., and Todokoro, K. (1994) Activation of helix-loop-helix
proteins Idl, Id2 and Id3 during neural differentiation. Biochem.
Biophys. Res. Commun. 1991355-1362.
Neuman, T., Keen, A., Zuber, M. X., Kristjansson, G. I., Gruss, P., and
Nornes, H. 0. (1993) Neuronal expression of regulatory helix-loop-
helix factor Id2 gene in mouse. Dev. Biol. 160:186-195.
Noden, D. M. (1989) Embryonic origins and assembly of blood vessels.
Am. Rev. Respir. Dis. 140:1097-1103.
Noden, D. M. (1991) Origins and patterning of avian outflow tract
endocardium. Development 111:867-876.
and Mizuno, T. (1981) Mesenchymal control over elon-
gating and branching morphogenesis in salivary gland develop-
ment. J Embryol. Exp. Morphol. 66209-221.
Ogata, T., Wozney, J. M., Benezra, R., and Noda, M. (1993) Bone
morphogenetic protein 2 enhances expression of a gene Id (inhibitor
of differentiation), encoding a helix-loop-helix molecule in osteo-
blast-like cells. Proc. Natl. Acad. Sci. U.S.A. 90:9219-9222.
Ott, M.-O., Bober, E., Lyons, G., Arnold, H., and Buckingham, M.
(1991) Early expression of the myogenic regulatory gene, myf-5, in
precursor cells of skeletal muscle in the mouse embryo. Develop-
Panaretto, B. A. (1993) Gene expression of potential morphogens dur-
ing hair follicle and tooth formation: A Review. Reprod. Fertil. Dev.
Riechmann, V., Van Cruchten, I., and Sablizky, F. (1994) The expres-
sion patten of Id4, a novel dominant negative helix-loop-helix pro-
tein, is distinct from Idl, Id2 and Id3. Nucleic Acids Res. 22749-
Roberts, V. J., Steenbergen, R., and Murre, C. (1993) Localization of
E2A mRNA expression in developing and adult rat tissues. Roc.
Natl. Acad. Sci. U.S.A. 90:7583-7587.
Roman, J., Little, C. W., and McDonald J. A. (1991) Potential role of
RGD-binding integrins in mammalian lung branching morphogen-
esis. Development 112:551-558.
Rugh, R. (1990) “The Mouse: Its Reproduction and Development.”
Oxford Univ. Press.
Sasai, Y., Kageyama, R., Tagawa, Y., Shigemoto, R., and Nakanishi,
S. (1992) Two mammalian helix-loop-helix factors structurally re-
lated to Drosophila hairy and Enhancer of split. Genes Dev. 6:2320-
Simon-Assmann, P., and Kedinger, M. (1993) Heterotypic cellular CQ-
operation in gut morphogenesis and differentiation. Semin. Cell
Simonsen, M. S., Rooney, A., and Herman, W. H. (1993) Expression
and differential regulation of Idl, a dominant negative regulator of
basic helix-loop-helix transcription factors, in glomerular mesan-
gial cells. Nucleic Acids Res. 215767-5774.
Stuart, E. T., Kioussi, C., and Gruss, P. (1994) Mammalian PAX
genes. Ann. Rev. Genet. 28:117-140.
Sukhatme, V. P., Cao, X., Chang, L. L., Tsai-Morris, C. H., Stamen-
kovich, D., Ferreira, P. C. P., Cohen, D. R., Edward, s. A., Shows, T.
B., Curran, T., LeBeau, M. M., and Adamson, E. D. (1988) A zinc
finger-encoding gene coregulated with c-fos during growth and dif-
ferentiation and after cellular depolarization. Cell 53:37-43.
Sun, X.-H., Copeland, N. G., Jenkins, N. A,, and Baltimore, D. (1991)
Id proteins Id1 and Id2 selectively inhibit DNA binding by one class
of helix-loop-helix proteins. Mol. Cell Biol. 11:5603-5611.
Thomas, J. H. (1993) Thinking about genetic redundancy. Trends
Tournay, O., and Benezra, R. (1996) Transcription of the dominant
negative helix-loop-helix protein Id1 is regulated by a protein com-
plex containing the early growth response gene EGR-1. Mol. Cell
Wall, N. A., Blessing, M., Wright, C. V. E., and Hogan, B. L. M. (1993)
Biosynthesis and in vivo localization of the Decapentaplegic-Vg-
related protein, DVR-6 (Bone Morphogenetic Protein-6). J. Cell
Wang, Y., Benezra, R., and Sassoon, D. (1992) Id expression during
mouse development: A role in morphogenesis. Dev. Dyn. 194:222-
Weintraub, H., David, R., Tapscott, S., Thayer, M., Krause, M., Ben-
ezra, R., Blackwell, T. K., Turner, D., Rupp, R., Hollenberg, S.,
Zhuang, Y., and Lassar, A. (1991) The MyoD gene family, nodal
point during specification of the cell linage. Science 251:761-766.
Wilson, R. B., Kiledjian, M., Shen, C. P., Benezra, R., Zwollo, P.,
Dymecki, S. M., Desiderio, S. V., and Kadesch, T. (1991) Repression
of immunoglobulin enhancers by the helix-loop-helix protein Id
Implication by B lymphoid development. Mol. Cell Biol. 11:6185-
Wilson, R., and Mohun, T. (1995) XIdx, a dominant negative regulator
of bHLH function in early Xenopw embryos. Mech. Dev. 49:211-
Wolf, C., Thisse, C., Stoetzel, C., Thisse, B., Gerlinger, P., and Perrin-
Schmitt, F. (1991) The M-twist gene of Mus is expressed in subsets
of mesodermal cells and is closely related to the Xenopus X-twi and
Drosophila twist genes. Dev. Biol. 143:363-373.
Zhang, H., Reynaud, S., Kloc, M., Etkin, L. D., and Spohr, G. (1995) Id
gene activity during Xenopus embryogenesis. Mech. Dev. 50119-