We have investigated developmental expression of the gene Egr-1, which encodes a protein containing three zinc fingers. Egr-1 like c-fos is a serum inducible, early response gene, which is co-induced with c-fos in a variety of quite different situations. A single 3.7-kb RNA was detected throughout fetal mouse development, which increased in absolute levels in total fetal RNA from 9.5 to 12.5 days post coitum (p.c.). In situ hybridization to 14.5- and 17.5-day p.c. fetal tissues demonstrated Egr-1 accumulation at several specific sites. These included mesenchymal components of the developing tooth germs and salivary and nasal glands; an ectodermally derived component of the whisker pad and developing muscle, cartilage, and bone. Expression of Egr-1 in cartilage and bone showed a strikingly similar expression to previously published reports of c-fos in these tissues. High levels of Egr-1 RNA was observed at the perichondrial interface of opposing cartilaginous elements and in interstitial cells that lie in between. Bone expression was observed in membranous bone of the head, alveolar bone around the tooth germs, and at periosteal and endochondral ossification sites in the limb bones. Our data support the idea that Egr-1 and c-fos may be coregulated in vivo and together may regulate normal development of the skeleton.

The interaction between groups of cells at specific times within localized regions is necessary for the orderly development of most vertebrate structures. This is true not only for the generation of primary tissue layers, such as the mesoderm and neural ectoderm, but also for the later formation of body organs. At present, little is known of the molecules that mediate the intercellular signalling or the intracellular pathways that respond to these signals. However, increasing evidence suggests that growth factors will play a central role in these events. Several growth factors have been implicated in one of the earliest inductive events in amphibian development (for a review, see Smith, 1989). Furthermore, tissue specific expression of a number of growth factor related molecules has been reported in the developing mouse (Jakobovits, 1986; Adamson, 1987; Wilkinson et al. 1988, 1989a).

Recently, a number of genes have been reported that show rapid transcriptional activation in response to a variety of growth factors (Cochran et al. 1983; Lau and Nathans, 1985, 1987; Milbrandt, 1987; Sukhatme et al. 1987; Almendral et al. 1988). Several of these genes, which include the proto-oncogenes, c-myc, c-fos, and c-jun, share properties of transcription factors. Thus, it seems likely that some of these genes operate in vivo to regulate gene expression in response to external cues.

We have focused our attention on a growth factor inducible gene, Egr-1 (early growth response-1, Sukhatme et al. 1987; Sukhatme et al. 1988). This gene has also been referred to as NGFI-A (Milbrandt, 1987), Krox-24 (Lemaire et al. 1988) and zif/268 (Christy et al. 1988). Egr-1 was originally identified as a rapid early response gene to a variety of polypeptides including NGF (Milbrandt, 1987; Sukhatme et al. 1988), FGF and PDGF (Christy et al. 1988), and general serum proteins (Sukhatme et al. 1988; Lemaire et al. 1988). Egr-1 encodes a 533 amino acid, 57K protein containing three putative DNA-binding zinc finger sequences (Sukhatme et al. 1987; Milbrandt, 1987; Christy eta/. 1988; Lemaire et al. 1988). Following mitogenic stimulation, Egr-1 shows a rapid, cycloheximide-independent, transcriptional activation with kinetics similar to those originally described for serum induction of c-fos (Greenberg and Ziff, 1984). However, within 30 min of stimulation of quiescent cells, Egr-1 is induced to approximately ten times the level of c-fos (Sukhatme et al. 1987). Collectively, these data point to a broad role for Egr-1 in signal transduction in diverse biological processes.

In adult mouse tissues, high levels of Egr-1 RNA are restricted to heart, brain, and lung, and lower levels to kidney and spleen (Milbrandt, 1987; Sukhatme et al. 1988; Christy et al. 1988; Lemaire et al. 1988). Several cell lines, which include PC12, P19, and F9, regulate Egr-1 expression in response to differentiation in culture (Milbrandt, 1987; Sukhatme el al. 1988). However, the actual developmental profile of Egr-1 expression is unknown. We have used in situ hybridization to localize Egr-1 RNA in the developing fetus. Egr-1 accumulates at several specific sites, some of which overlap with known sites of c-fos expression. Our results point to a coregulation of Egr-1 and c-fos expression during fetal development.

Egr-1 was cut with MspI and RsaI to generate a 700 bp 3′ nonfinger-encoding region (1553 to 2254, Sukhatme et al. 1988). The 5′ overhang at the MspI cut end was filled in using the Klenow fragment of DNA polymerase (Maniatis et al. 1982) and the blunt-ended fragment ligated into the Smal site of pUC13. The resultant plasmid was cut wit h Hindlll and Bglil (1959) and EcoRI and Sg/Ill to generate 406 bp and 295 bp Egr-1 fragments, which were subsequently subcloned into the plasmid pKS (Stratagene) to generate the plasmids p4.6 and p3.6 respectively. T7 transcription of WindlH cut p4.6 and T3 transcription of SacI cut p4.6 transcribe antisense and sense Egr-1 probes respectively. T7 transcription of EcoRI cut p3.6 and T3 transcription of Xba\ cut p3.6 transcribe sense and antisense Egr-1 probes respectively.

RNA blot analysis

RNA was prepared from fetal and adult samples using the lithium chloride urea procedure (Auffray and Rougeon, 1980). Five pg of total fetal or adult RNAs were fractionated by gel electrophoresis on 1.2% formaldehyde agarose gels (Maniatis et al. 1982), transferred to GeneScreen (DuPont) and u.v. crosslinked (Church and Gilbert, 1984). RNA blots were hybridized with a high specific activity, 32P-labelled, p3.6 Egr-1 DNA probe (Feinberg and Vogelstein, 1984) at 2×106ctsmin-1ml-1 in 50% formamide, 5 × Denhardt’s solution, 1.0M-NaCl, 0.05M-Tris-HCl pH7.5, 1% SDS, and 10% dextran sulfate overnight at 45 °C. Filters were washed three times, 20min each, in 2 × SSC, 1% SDS at room temperature, then twice in 0.2xSSC, 1 % SDS at 65°C for 1 h. Hybridization was visualized by autoradiography using Kodak AR-5 X-ray film.

In situ hybridization using single-stranded, 35S-labelled RNA probes was performed as described in detail elsewhere (Wilkinson et al. 1987).

Egr-1 transcription in fetal mouse embryos

Both Egr-1 probes, 3.6 and 4.6, hybridize exclusively to Egr-1 RNA in serum induced and adult RNAs (Sukhatme, unpublished observations). To examine whether Egr-1 was transcribed during fetal development, fetal RNAs were hybridized with probe 3.6. A single 3.7kb transcript, which showed an identical mobility to the transcript present in adult brain and heart, was detected at all stages of fetal development examined (Fig. 1). Absolute levels of Egr-1 steadily increased from 9.5 to 12.5 days post-coitum (p.c.) but showed no further increase from 12.5 to 17.5 days (p.c.). Equal loading of RNAs was verified by ethidium bromide staining and hybridization with a cytoplasmic actin probe (data not shown).

Fig. 1.

Northern blot analysis of Egr-1 expression in fetal RNA. Five micrograms of RNA from 9.5- to 17.5-day fetuses were analyzed for expression of Egr-1. A single 3.7kb RNA transcript, identical in size to the Egr-1 transcript (arrow), is present in adult brain (B) and heart (H). L, lung.

Fig. 1.

Northern blot analysis of Egr-1 expression in fetal RNA. Five micrograms of RNA from 9.5- to 17.5-day fetuses were analyzed for expression of Egr-1. A single 3.7kb RNA transcript, identical in size to the Egr-1 transcript (arrow), is present in adult brain (B) and heart (H). L, lung.

Egr-1 expression is spatially localized

To determine the sites of Egr-1 RNA accumulation, in situ hybridization was performed to sections of 10.5-, 14.5-, 17.5-day p.c. fetuses. No specific hybridization was seen with either 3.6 or 4.6 sense RNA probes. For brevity these results are omitted. In contrast, specific sites of Egr-1 RNA accumulation were detected with both antisense probes. Moreover, hybridization was indistinguishable in adjacent sections hybridized with 3.6 and 4.6 antisense probes. Thus, it is clear that the hybridization is specific to antisense probes that encode different unrelated regions of the Egr-1 transcript. As identical results were obtained with 3.6 and 4.6 probes, individual results with either probe will not be distinguished in this paper.

At 10.5 days p.c., no obvious contiguous sites of Egr-1 expression were observed (data not shown). However, by 14.5 days, Egr-1 RNAs accumulate in several specific locations.

Egr-1 expression in developing cartilage, bone and teeth

In situ hybridization of Egr-1 probes to 14.5- and 17.5day p.c. fetuses revealed high levels of Egr-1 transcripts in association with cartilaginous and bony tissues. Egr-1 RNA accumulated in epiphyseal cartilage at the articular surfaces of the developing limbs and in the interstitial cells that lie in between cartilaginous elements (Fig. 2A,B). At 17.5 days p.c., this was particularly evident in the distal regions of the forelimb (Fig. 2C-F). Egr-1 expression was also observed at and between the articular surfaces of the developing head skeleton at 14.5 days and about the midline of the developing jaw (Fig. 2G,H). In the cartilage of the nasal septum, Egr-1 was only expressed at the proximal end of the median cartilage (Fig. 2I,J). In the ribs at 14.5 days, Egr-1 showed a punctate perichondrial distribution rather than a uniform labelling of all perichondrial cells (Fig. 2K,L).

Fig. 2.

Cartilage-associated expression of Egr-1. (A,B) Egr-1 expression at the articular surfaces of the 14.5day p.c. shoulder joint. Egr-1 RNA accumulates in the perichondrial cartilage of the scapula (s) and humerus (h), as well as in the interstitial cells that lie between these elements. (C,D) Egr-1 expression in the distal forelimb at 17.5 days p.c. Expression is restricted to the regions at which cartilaginous elements abut (arrowheads). (E,F) High-power view of the carpals boxed in C. Egr-1 expression is seen in interstitial cells between the carpals and in the perichondrium. (G,H) Egr-1 expression at the surface of opposing cartilaginous elements (arrowheads) in the developing jaw at 14.5 days p.c. Expression elsewhere is also seen in cells either side of the midline of the jaw (arrows). The neural tube at this time does not express Egr- 1. (I,J) Expression of Egr-1 at the proximal end of the medial nasal cartilage (c) at 14.5 days p.c. No expression is seen in other areas of cartilage or in the olfactory epithelium (arrowheads). (K,L) Egr-1 expression in perichondrial cells of the ribs (arrows) at 14.5 days p.c. A,C,E,G,I,K - bright field illumination; B,D,F,H,J,L - dark field illumination.

Fig. 2.

Cartilage-associated expression of Egr-1. (A,B) Egr-1 expression at the articular surfaces of the 14.5day p.c. shoulder joint. Egr-1 RNA accumulates in the perichondrial cartilage of the scapula (s) and humerus (h), as well as in the interstitial cells that lie between these elements. (C,D) Egr-1 expression in the distal forelimb at 17.5 days p.c. Expression is restricted to the regions at which cartilaginous elements abut (arrowheads). (E,F) High-power view of the carpals boxed in C. Egr-1 expression is seen in interstitial cells between the carpals and in the perichondrium. (G,H) Egr-1 expression at the surface of opposing cartilaginous elements (arrowheads) in the developing jaw at 14.5 days p.c. Expression elsewhere is also seen in cells either side of the midline of the jaw (arrows). The neural tube at this time does not express Egr- 1. (I,J) Expression of Egr-1 at the proximal end of the medial nasal cartilage (c) at 14.5 days p.c. No expression is seen in other areas of cartilage or in the olfactory epithelium (arrowheads). (K,L) Egr-1 expression in perichondrial cells of the ribs (arrows) at 14.5 days p.c. A,C,E,G,I,K - bright field illumination; B,D,F,H,J,L - dark field illumination.

Particularly high levels of Egr-1 RNA were found in several areas undergoing bone formation including the periosteal regions of the developing long bones (Fig. 3A,B) and in membranous and alveolar boneforming regions in the head (Fig. 3C,D).

Fig. 3.

Egr-1 expression in bone- and tooth-forming regions. (A,B) Longitudinal section through humerus at 14.5 days p.c. Egr-1 expression is restricted to periosteal regions (arrowhead). (C,D) Egr-1 expression in alveolar bone (arrowheads) forming around incisor tooth germ (t) at 17.5 days p.c. (E,F) Longitudinal sections through first molar of upper jaw (u) and incisor of lower jaw (1) at 14.5 days p.c. Egr-1 expression is seen in the mesenchymal cells (m) associated with the developing tooth germs. The ectodermal component does not express Egr-1 (arrowheads). A,C,E - bright field illumination; B,D,F - dark field illumination; t, tongue.

Fig. 3.

Egr-1 expression in bone- and tooth-forming regions. (A,B) Longitudinal section through humerus at 14.5 days p.c. Egr-1 expression is restricted to periosteal regions (arrowhead). (C,D) Egr-1 expression in alveolar bone (arrowheads) forming around incisor tooth germ (t) at 17.5 days p.c. (E,F) Longitudinal sections through first molar of upper jaw (u) and incisor of lower jaw (1) at 14.5 days p.c. Egr-1 expression is seen in the mesenchymal cells (m) associated with the developing tooth germs. The ectodermal component does not express Egr-1 (arrowheads). A,C,E - bright field illumination; B,D,F - dark field illumination; t, tongue.

At 14.5 days p.c., Egr-1 expression was detected in the mesenchymal component of the developing tooth (Fig. 3E,F). Teeth develop following a complex series of interactions between the ectodermal cells of the jaw epithelium and the underlying neural crest-derived mesenchyme cells (Lumsden, 1987). The ectodermal component forms the enamel and the mesenchymal component, the dentine and pulp. At 14.5 days p.c., the mesenchyme of the incisors and first molar express high levels of Egr-1 (Fig. 3E,F). However, by 17.5 days, Egr-1 expression decreased, and the expression, now limited to the developing pulp, was patchy (data not shown).

Egr-1 expression in striated muscle and tendons

Egr-1 expression was seen in all areas in which striated muscle develops. However, in contrast to sections hybridized with a muscle-specific actin probe (data not shown), Egr-1 was not uniformly expressed throughout the developing muscle, but rather showed a patchy distribution. This is clear in sections through the muscles projecting to the developing limb and bones (Fig. 4A,B) and in the tongue, which is composed of a branching array of striated muscle fibers (Fig. 4C,D). Egr-1 expression was seen in both the central muscle and in the connective tissue sheath and developing tendons (Fig. 4A – D).

Fig. 4.

Egr-1 expression in striated muscles and tendons. Longitudinal sections through forelimb (A.B) and cross section of tongue (C,D) of 14.5-days-p.c. fetus. Egr-1 expression occurs in striated muscles of both limbs (m) and the tongue (arrows). Expression is non-uniform over the muscle blocks and is particularly high where tendons are developing (arrowheads). In addition to expression in the muscles and tendons, expression is also seen in interstitial cells at the articular surface of the scapula (s) and humerus (h), as documented in Fig. 2. A,C – bright field illumination; B,D – dark field illumination.

Fig. 4.

Egr-1 expression in striated muscles and tendons. Longitudinal sections through forelimb (A.B) and cross section of tongue (C,D) of 14.5-days-p.c. fetus. Egr-1 expression occurs in striated muscles of both limbs (m) and the tongue (arrows). Expression is non-uniform over the muscle blocks and is particularly high where tendons are developing (arrowheads). In addition to expression in the muscles and tendons, expression is also seen in interstitial cells at the articular surface of the scapula (s) and humerus (h), as documented in Fig. 2. A,C – bright field illumination; B,D – dark field illumination.

Egr-1 expression in the developing whiskers

The first hair follicles to develop in the mouse are the whisker pads, which provide the vibrissae. As with other hairs, these develop from an epithelial-mesenchymal interaction between the basal layer of the skin and underlying mesenchymal cells (Davidson and Hardy, 1952). Egr-1 RNA was detected in ectodermally derived cells of the inner root sheath in developing whisker follicles from 14.5 days p.c. (Fig. 5A – D). No expression was seen in outer root sheath cells, which are also ectodermally derived, in mesenchymal cells, which form the dermal papilla, or in the cortex of the hair (Fig. 5C-D). In the hair follicles of the skin, Egr-1 expression was not detected up to 17.5 days p.c.

Fig. 5.

Expression of Egr-1 in the whisker pads. (A,B) Longitudinal section through 14.5-day p.c. whisker pad. Expression is restricted to ectodermally-derived cells of the inner root sheath lying above the mesodermally-derived dermal papilla (p) and surrounded by the ectodermally-derived outer root sheath (arrowheads). (C,D) Transverse section through 17,5-day p.c. whisker pads. Expression of Egr-1 is seen in the inner root sheath (large arrowhead) but not in the central region of the hair shaft (small arrowhead). A,C – bright field illumination; B,D – dark field illumination.

Fig. 5.

Expression of Egr-1 in the whisker pads. (A,B) Longitudinal section through 14.5-day p.c. whisker pad. Expression is restricted to ectodermally-derived cells of the inner root sheath lying above the mesodermally-derived dermal papilla (p) and surrounded by the ectodermally-derived outer root sheath (arrowheads). (C,D) Transverse section through 17,5-day p.c. whisker pads. Expression of Egr-1 is seen in the inner root sheath (large arrowhead) but not in the central region of the hair shaft (small arrowhead). A,C – bright field illumination; B,D – dark field illumination.

Egr-1 expression in developing salivary and nasal glands

In addition to Egr-1 expression in the above-mentioned areas, Egr-1 RNA was also detected in developing glandular structures. In the nose, several sites of Egr-1 expression were observed in association with developing nasal glands (Fig. 6A,B). Expression was also seen in association with salivary glands at 14.5 days p.c. (Fig. 6C,D). In both cases, Egr-1 was not expressed in the epithelial component of the gland, but rather in the underlying mesenchymal component.

Fig. 6.

Expression of Egr-1 in developing nasal and salivary glands. Transverse sections through the head region of a 14.5-days-p.c. fetus. (A,B) Expression of Egr-1 in the developing olfactory region is concentrated in mesenchymal tissue surrounding the epithelium of developing nasal glands (arrowheads). No expression is seen in these sections in non-gland-associated mesenchyme (m), in the olfactory epithelium (arrows), or in nasal cartilage (c). (C,D) Egr-1 expression in the developing salivary glands is localized in condensed mesenchyme surrounding the epithelium of the developing gland (small arrowhead). Expression is not seen in the developing salivary duct (large arrowhead). A,C - bright field illumination; B,D - dark field illumination.

Fig. 6.

Expression of Egr-1 in developing nasal and salivary glands. Transverse sections through the head region of a 14.5-days-p.c. fetus. (A,B) Expression of Egr-1 in the developing olfactory region is concentrated in mesenchymal tissue surrounding the epithelium of developing nasal glands (arrowheads). No expression is seen in these sections in non-gland-associated mesenchyme (m), in the olfactory epithelium (arrows), or in nasal cartilage (c). (C,D) Egr-1 expression in the developing salivary glands is localized in condensed mesenchyme surrounding the epithelium of the developing gland (small arrowhead). Expression is not seen in the developing salivary duct (large arrowhead). A,C - bright field illumination; B,D - dark field illumination.

It is clear from the results presented here that the zinc finger-protein encoding gene, Egr-1, is transcribed in several different cell types during mammalian development. These include epithelial and mesenchymal cells, which are ectodermally or mesodermally derived. Egr-1 expression at these sites does not correlate with a simple common ancestry shared by Egr-1 -expressing cells. Rather, expression is presumably a requirement for the development of several unrelated tissues. Interestingly, Egr-1 is expressed in different sites at which epithelial mesenchymal interactions are important. These include the developing tooth germ, the whisker pad, and the salivary and nasal glands. Given the broad spectrum of growth factor-like molecules that activate Egr-1 expression in culture (Sukhatme et al. 1987, 1988), it seems possible that Egr-1 expression in vivo may be in response to growth factors that mediate epithelial-mesenchymal interactions. However, at present, little is known of the factors responsible for these cell interactions, although tissue-specific expression of a number of growth factor-like molecules has been described (Jako-bovits, 1986; Adamson, 1987; Wilkinson et al. 1988, 1989a).

Recent studies on fetal stages indicate that receptors to nerve growth factor (NGF) are not exclusively localized within neural tissue (Yan and Johnson, 1988). Receptors were found in muscle, mesenchyme surrounding salivary glands, and in the developing tooth bud and in hair follicles. Whether receptors are functional and actually respond in vivo to NGF has yet to be established, but it is interesting to note that Egr-1 is rapidly induced by NGF, during NGF-induced differentiation of PC12 cells (Milbrandt, 1987; Sukhatme et al. 1988). Thus, there may be some causal relationship between the non-neuronal distribution of NGF receptor and Egr-1 expression.

In addition to expression of Egr-1 at sites of epithelial mesenchymal interaction, high levels of Egr-1 expression were observed in cartilage and bone. Egr-1 expression in the 14.5- and 17.5-day skeleton is highly reminiscent of expression of the proto-oncogene c-fos in these tissues. In the appendicular skeleton at 17.5 days p.c., c-fos RNA-like Egr-1 is localized in the epiphyseal perichondrium (Dony and Gruss, 1987; Sandberg et al. 1988) as well as in the interstitial cells between opposing elements of the long bones and developing hands and feet, c-fos and Egr-1 are also coexpressed in the bone-forming regions of the head (Caubet and Bernaudin, 1988) as well as in periosteal and endochondral ossification sites in the long bones (Sandberg et al. 1988). Moreover, c-fos expression also occurs in mesenchymally-derived cells of the tooth germ, albeit at a somewhat later stage than shown to express Egr-1 in the present study. Thus, in situ hybridization strongly suggests that c-fos and Egr-1 share overlapping patterns of expression in vivo.

A number of previous studies have indicated that cfos and Egr-1 are coregulated. In a variety of different cell types, including fibroblasts, B and T lymphocytes, and epithelial cells (Sukhatme et al. 1987), Egr-1 and cfos share similar kinetics of induction following mitogenic stimulation. Furthermore, when PC12 cells (Milbrandt, 1987; Sukhatme et al. 1988) and bone marrow cells (Ovelettes, Sukhatme and Bonventre, unpublished observations) are stimulated to differentiate with NGF and GM-CSF, respectively, c-fos and Egr-1 are induced with similar kinetics. Coexpression is also observed following renal ischemia and in the early phase of compensatory renal hypertrophy (Sukhatme, unpublished observations). Finally, in the nervous system, c-fos and Egr-1 are induced following membrane depolarization (Sukhatme et al. 1988) and seizure (Morgan et al. 1987; Sukhatme el al. 1988). In contrast, a second serum responsive gene, Egr-2/Krox-20 (Chav-rier et al. 1988,1989; Joseph et al. 1988), which shares an almost identical zinc finger domain with Egr-1 (Lemaire et al. 1988), is not induced in NGF-treated PC12 cells (Joseph et al. 1988) and shows no overlapping expression with Egr-1 and c-fos in vivo (Wilkinson et al. 1989b).

The similar kinetics of Egr-1 and c-fos induction in these systems, which in the case of serum stimulation does not require new protein synthesis (Sukhatme et al. 1987), suggests that Egr-1 and c-fos share common transcriptional regulation. Comparison of the 5′ regulatory sequences indicates several similarities (Tsai-Morris et al. 1988; Christy et al. 1988). Egr-1 has six potential serum response elements (SRE, Treisman, 1986), which in c-fos are the sites at which the serum response factor binds to regulate c-fos transcription (Treisman, 1987). However, despite these similarities, induction through the serum response elements is unlikely to explain the coexpression of Egr-1 and c-fos in vivo since such elements are also present in the upstream regions of Egr-2/Knox-20 (Christy et al. 1989) as well as in α -actin genes, which show a markedly different developmental profile. More subtle similarities in the promoter elements of Egrl and c-fos must exist to explain their coregulation in such diverse circumstances.

In the absence of a detailed knowledge of the in vivo expression of potential transcriptional regulatory molecules, it is impossible to address their normal func-tions. In this report, we have implicated Egr-1 and c-fos together in regulating growth and differentiation of cartilage and bone, c-jun, which in association with cfos forms parts of the API transcription complex (Franza et al. 1988; Rauscher et al. 1988a,b;Chiu et al. 1988; Sassone-Corsi et al. 1988), shows a very similar expression in the skeleton (Wilkinson et al. 1989c). Thus, a detailed picture is emerging of a number of different regulatory molecules which may function in vivo at the same sites. The observation that deregulated expression of c-fos leads to abnormal bone development (Ruther et al. 1987) suggests that misexpression of Egr-1 may invoke a similar phenotype. However, it is also clear from our study that Egr-1 and c-fos do not share completely overlapping patterns of expression, as is also the case for c-fos and c-jun (Wilkinson et al. 1989c). Thus, it is likely that these and other transcription factors will interact in different combinations to bring about appropriate regulation of gene expression at diverse sites during development.

Adamson
,
E. D.
(
1987
).
Oncogenes in development
.
Development
99
,
449
471
.
Almendral
,
J. M.
,
Sommer
,
D.
,
Macdonald-Bravo
,
H.
,
Burckhardt
,
J.
,
Perera
,
J.
and
Bravo
,
R.
(
1988
).
Complexity of the early genetic response to growth factors in mouse fibroblasts
.
Mol. Cell. Biol.
8
,
2140
2148
.
Auffray
,
C.
and
Rougeon
,
F.
(
1980
).
Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA
.
Eur. J. Biochem.
107
,
303
314
.
Caubet
,
J. F.
and
Bernaudin
,
J. F.
(
1988
).
Expression of the c-fos proto-oncogene in bone, cartilage, and tooth-forming tissues during mouse development
.
Biology of the Cell
64
,
101
104
.
Chavrier
,
P.
,
Zerial
,
M.
,
Lemaire
,
P.
,
Almendral
,
J.
,
Bravo
,
R.
and
Charnay
,
P.
(
1988
).
A gene encoding a protein with zinc fingers is activated during Go/Gl transition in cultured cells
.
EMBO J. 7,29-35
.
Chavrier
,
P.
,
Janssen-Timmen
,
V.
,
Mattei
,
M.-G.
,
Zerial
,
M.
,
Bravo
,
R.
and
Charnay
,
P.
(
1989
).
Structure, chromosome location, and expression of the mouse zinc finger gene, Krox-20: Multiple gene products and coregulation with the proto-oncogene c-fos
.
Mol. Cell. Biol.
9
,
787
797
.
Chiu
,
R.
,
Boyle
,
W. J.
,
Meek
,
J.
,
Smeal
,
T.
,
Hunter
,
T.
and
Karin
,
M.
(
1988
).
The c-fos protein interacts with c-jun/AP-I to stimulate transcription of AP-1 responsive genes
.
Cell
54
,
541
552
.
Church
,
G. M.
and
Gilbert
,
W. C.
(
1984
).
Genomic sequencing
.
Proc. natn. Acad. Sci. U.S.A.
81
,
1991
1995
.
Christy
,
B. A.
,
Lau
,
L. F.
and
Nathans
,
D.
(
1988
).
A gene activated in mouse 3T3 cells by serum growth factors encodes a protein with “zinc finger” sequences
.
Proc. natn. Acad. Sci. U.S.A.
85
,
7857
7861
.
Cochran
,
B. H.
,
Reffel
,
A. C.
and
Stiles
,
C. D.
(
1983
).
Molecular cloning of gene sequences regulated by platelet-derived growth factor
.
Cell
33
,
939
947
.
Davidson
,
P.
and
Hardy
,
M. H.
(
1952
).
The development of mouse vibrissae in vivo and in vitro
.
J. Anat. Physiol.
86
,
342
356
.
Dony
,
C.
and
Gruss
,
P.
(
1987
).
Proto-oncogene c-fos expression in growth regions of fetal bone and mesodermal web tissue
.
Nature, Lond.
328
,
711
714
.
Feinberg
,
A.
and
Vogelstein
,
B.
(
1984
).
A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity
.
Analyt. Biochem.
137
,
266
267
.
Franza
,
B. R.
, Jr.
,
Rauscher
,
F. J.
III
,
Joseph
,
S. F.
and
Curran
,
T.
(
1988
).
The fos complex and fos-related antigens recognize sequence elements that contain AP-1 sites
.
Science
239
,
1150
1153
.
Greenberg
,
M. E.
and
Ziff
,
E. B.
(
1984
).
Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene
.
Nature, Lond.
311
,
433
438
.
Jakobovits
,
A.
(
1986
).
Oncogenes and Growth Control
(ed.
P.
Kahn
and
T.
Graf
), pp.
9
-
17
, Springer-Verlag, Berlin.
Joseph
,
L. J.
,
Lebeau
,
M. M.
,
Jamieson
,
G. A.
,
Acharya
,
S.
,
Shows
,
T. B.
,
Rowley
,
J. D.
and
Sukhatme
,
V. P.
(
1988
).
Molecular cloning, sequencing, and mapping of EGR 2, a human early growth response gene encoding a protein with “zinc-binding finger” structure
.
Proc. natn. Acad. Sci. U.S.A.
85
,
7164
7168
.
Lemaire
,
P.
,
Révélant
,
O.
,
Bravo
,
R.
and
Charnay
,
P.
(
1988
).
Two mouse genes encoding potential transcription factors with identical DNA-binding domains are activated by growth factors in cultured cells
.
Proc. natn. Acad. Sci. U.S.A.
85
,
4691
4695
.
Lau
,
L. F.
and
Nathans
,
D.
(
1985
).
Identification of a set of genes expressed during the Go/Gl transition of cultured mouse cells
.
EMBO J.
4
,
3145
3151
.
Lau
,
L. F.
and
Nathans
,
D.
(
1987
).
Expression of a set of growth-related immediate early genes in BALB/C 3T3 cells: Coordinate regulation with c-fos or c-myc
.
Proc. natn. Acad. Sci. U.S.A.
84
,
1184
1186
.
Maniatis
,
T.
,
Fritsch
,
E. F.
and
Sambrook
,
J.
(
1982
).
Molecular cloning: A laboratory manual,
Cold Spring Harbor Laboratory
,
Cold Spring Harbor, New York
.
Milbrandt
,
J.
(
1987
).
A nerve growth factor-induced gene encodes a possible transcriptional regulatory factor
.
Science
238
,
797
799
.
Morgan
,
J. J.
,
Cohen
,
D. R.
,
Hempstead
,
J. L.
and
Curran
,
T.
(
1987
).
Mapping patterns of c-fos expression in the central nervous system after seizure
.
Science
237
,
192
197
.
Rauscher
,
F. J. Ill
,
Sambucetti
,
L. C.
,
Curran
,
T.
,
Distel
,
R. J.
and
Spiegelman
,
B. M.
(
1988
).
A common DNA-binding site for fos protein complexes and transcription factor AP-1
.
Cell
52
,
471
480
.
Rauscher
,
F. J. Ill
,
Cohen
,
D. R.
,
Curran
,
T.
,
Bos
,
T. J.
,
Vogt
,
P. K.
,
Bohmann
,
D.
,
Tuan
,
R.
and FRANZA,
B. R.
Jr
. (
1988
).
Fos-associated protein p39 is the product of the jun protooncogene
.
Science
240
,
1010
1016
.
Ruther
,
V.
,
Garber
,
C.
,
Komitowski
,
D.
,
Müler
,
R.
and
Wagner
,
E. F.
(
1987
).
Deregulated c-fos expression interferes with normal bone development in transgenic mice
.
Nature, Lond.
325
,
412
416
.
Sandberg
,
M.
,
Vuorio
,
T.
,
Hirvonen
,
H.
,
Alitalo
,
K.
and
Vuorio
,
E.
(
1988
).
Enhanced expression of TGF-/J and c-fos mRNAs in the growth plates of developing human long bases
.
Development
102
,
461
470
.
Sassone-Corsi
,
P.
,
Lamph
,
W. W.
,
Kamps
,
M.
and
Verma
,
I. M.
(
1988
).
Fos-associated cellular p39 is related to nuclear transcription factor AP-1
.
Cell
54
,
553
560
.
Smith
,
J. C.
(
1989
).
Mesoderm induction and mesoderm-inducing factors in early amphibian development
.
Development
105
,
665
678
.
Sukhatme
,
V. P.
,
Kartha
,
S.
,
Toback
,
F. G.
,
Taub
,
R.
,
Hoover
,
R. G.
and
Tsai-Morris
,
C-H.
(
1987
).
A novel early growth response gene rapidly induced by fibroblast, epithelial and lymphocyte mitogens
.
Oncogene Res.
1
,
343
355
.
Sukhatme
,
V. P.
,
Cao
,
X.
,
Chang
,
L. L.
,
Tsai-Morris
,
C-H.
,
Stamenkovich
,
D.
,
Ferreira
,
P. C. P.
,
Cohen
,
D. R.
,
Edwards
,
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 differentiation and after cellular depolarization
.
Cell
53
,
37
43
.
Treisman
,
R. H.
(
1986
).
Identification of a protein-binding site that mediates transcriptional response of the c-fos gene to serum factors
.
Cell
46
,
567
574
.
Treisman
,
R. H.
(
1987
).
Identification and purification of a polypeptide that binds to the c-fos serum response element
.
EMBO J.
6
,
2711
2717
.
Tsai-Morris
,
C-H.
,
Cao
,
X.
and
Sukhatme
,
V. P.
(
1988
).
5’ flanking sequence and genomic structure of Egr-1, a mitogen inducible zinc finger-encoding gene
.
Nucl. Acids Res.
16
,
8835
8846
.
Wilkinson
,
D. G.
,
Bailes
,
J. A.
,
Champion
,
J. E.
and
Mcmahon
,
A. P.
(
1987
).
A molecular analysis of mouse development from 8 to 10 days post-coitum detects changes only in embryonic globin expression
.
Development
99
,
493
500
.
Wilkinson
,
D. G.
,
Peters
,
G.
,
Dickson
,
C.
and
Mcmahon
,
A. P.
(
1988
).
Expression of the FGF-related proto-oncogene int-2 during gastrulation and neurulation in the mouse
.
EMBO J.
7
,
691
695
.
Wilkinson
,
D. G.
,
Bhatt
,
S.
and
Mcmahon
,
A. P.
(
1989a
).
Expression of the FGF-related proto-oncogene int-2 suggests multiple roles in fetal development
.
Development
105
,
131
136
.
Wilkinson
,
D. G.
,
Bhatt
,
S.
,
Chavrier
,
P.
,
Bravo
,
R.
and
Charnay
,
P.
(
1989b
).
Segment-specific expression of a zinc finger in the developing nervous system of the mouse
.
Nature, Lond.
337
,
461
464
.
Wilkinson
,
D. G.
,
Bhatt
,
S.
,
Ryseck
,
R-P.
and
Bravo
,
R.
(
1989c
).
Tissue specific expression of c-jun and jun-b during organogenesis in the mouse
.
Development
106
,
465
471
.
Yan
,
Q.
and
Johnson
,
E. M.
(
1988
).
An immunohistochemical study of the nerve growth factor receptor in developing rats
.
J. Neurosci.
8
,
3481
3498
.