c-jun and junB are cellular genes related to the viral oncogene v-jun and encode members of the AP-1 transcription factor gene family. These genes have been implicated in the control of the G0/G1 transition in fibroblasts. Here, we have investigated the potential roles of c-jun and junB during fetal growth and organogenesis in the mouse by in situ hybridization analysis of their expression patterns, c-jun expression is detected throughout organogenesis, and transcripts are detected in many tissues, although in restricted cell populations within developing cartilage, gut and the central nervous system (CNS). In cartilage, c-jun expression is associated with rapidly proliferating perichondrial cells, but occurs in postmitotic motor neurones in the CNS. junB expression is initiated between 14 · 5 and 17 · 5 days of development, and is restricted to differentiating epidermal cells and endodermal gut epithelium. These data suggest that c-jun and junB have distinct, tissue-specific roles in cell proliferation and differentiation during fetal development.

There is increasing evidence that proto-oncogenes implicated in the control of cell proliferation may also have other crucial roles in differentiation and development (Adamson, 1987; Nusse, 1988). This view is based on the finding that the normal developmental expression patterns of certain proto-oncogenes do not correlate with high rates of cell proliferation, but rather are expressed in a cell-type-restricted or region-specific manner. In addition, in vitro studies support the notion that some proto-oncogenes may have roles in cell differentiation (reviewed by Adamson, 1987).

A number of proto-oncogenes, including c-jun and c-fos, encode nuclear proteins that may be involved in transcriptional regulation (Bohmann et al. 1987; Angel et al. 1987; Chiu et al. 1988; Rauscher et al. 1988; Sassone-Corsi et al. 1988). c-jun and a related gene, junB (Ryder et al. 1988), have substantial sequence homology with the v-jun oncogene and encode members of the AP-1 transcription factor family (Bohmann et al. 1987; Angel et al. 1987). Although AP-1 binding sites have been found in the promoter regions of a number of genes (Jones et al. 1988), little is known of the sequence specificity or physiological role of individual members of the AP-1 family. Both c-jun and junB are strongly and rapidly upregulated (as is c-fos;Greenberg & Ziff, 1984; Kruijer et al. 1984; Muller et al. 1984; Bravo et al. 1987) on the addition of growth factors to quiescent fibroblasts (Ryder et al. 1988; Ryseck et al. 1988; Quantin & Breathnach, 1988), and thus these genes may be involved in specific gene transcription during the Go to Gi transition in the cell cycle. Furthermore, the finding that AP-1 and c-fos proteins can form a complex suggests that they may act cooperatively in cells where they are coexpressed. It seems likely that c-jun has tissue-specific roles since RNA blot analysis detected transcripts only in certain tissues (lung, brain, thymus, intestine and testes) of the adult mouse (Ryseck et al. 1988).

To investigate whether c-jun and junB might have roles in mouse development, we have analysed the pattern of their expression by in situ hybridization during fetal growth and organogenesis. We report a restricted expression pattern of these genes which is suggestive of multiple and distinct tissue-specific roles.

RNA blots

Poly (A)+ RNA was electrophoresed on agarose gels in the presence of formaldehyde and blotted onto Genescreen (Dupont-NEN) as described (Wilkinson et al. 1987a). Single-stranded RNA probes were prepared and used for hybridization in 50% formamide, 1m-sodium chloride, 10 × Denhardt’s, 10% dextran sulphate, 100 μg μl-1 yeast RNA at 55 °C, followed by washing at a final stringency of 50% formamide, 2 × SSC at 65°C; the high stringency wash is under the same conditions as used for in situ hybridization. The sequences used for probes corresponded to TV-terminal regions of c-jun (residues 1 –451; Ryseck et al. 1988) and junB (residues 335 –903), which have less than 68% sequence similarity and do not include the conserved putative DNA-binding region (Ryseck et al. 1988). Previous studies have established the specificity of the c-jun (Ryseck et al. 1988) and junB (Ryder et al. 1988) probes on RNA blots.

In situ hybridization

In situ hybridization of mouse embryo sections with 35S-labelled RNA probes and high stringency washing was performed as described (Wilkinson et al. 1987ab). Control hybridizations with sense-strand probes gave no signals above background except, on occasions, over liver.

c-jun and junB transcripts in mouse fetuses

RNA blot analysis with junB probe detected a ∼2 ·0kb transcript in 17 ·5 day mouse fetuses which comigrates with that present in serum-stimulated fibroblasts (Fig. 1, lanes 1,4). As anticipated, this RN A was not detected in quiescent fibroblasts (Ryder et al. 1988), but, suprisingly, was also not detected in RNA from 14-5 day fetuses. Thus, junB RNA accumulation is strongly upregulated between 14 ·5 and 17 ·5 days of development. In contrast, c-jun transcripts are detected both in 14 ·5 day and 17 ·5 day fetuses, although the relative levels are upregulated during this period (Fig. 1, lanes 7,8). Two c-jun transcripts of ∼2 ·7 and ∼ 3 ·2kb are detected in fetal RNA and serum-stimulated fibroblasts, but not in quiescent fibroblasts (Fig. 1, lanes 5 –8). These multiple transcripts have been shown to be due to alternative polyadenylation sites in fibroblasts (Ryseck et al. 1988). These data suggest that similar c-jun and junB transcripts exist in fetuses and serum-stimulated fibroblasts, and that they are subject to regulation during fetal growth and organogenesis.

Fig. 1.

Expression of c-jun and junB RNA in mouse fetuses. RNA blots were hybridized with junB probe (lanes 1 –4), and then (without removal of junB signal) with c-jun probe (lanes 5 –8) to examine transcripts in mouse fetuses. RNA samples from serum-stimulated NIH3T3 fibroblasts (1,5), quiescent fibroblasts (2,6), 14-5 day mouse embryos (3,7) and 17 ·5 day embryos (4,8) were analysed. junB probe detects a ∼ 2 ·0 transcript, indicated as ‘b’. c-jun probe detects two transcripts of ∼ 2 ·7kb and 3 ·2 kb, indicated as ‘a’; previous work has shown these two transcripts to be due to alternative polyadenylation sites (Ryseck et al. 1988). Note that the autoradiographic exposure shown in lanes 1 –4 is greater than that of lanes 5 –8 in order to detect junB transcripts, which are present in fetal RNA at lower relative levels than c-jun transcripts. Hybridization of the blots with actin coding region probe revealed similar levels of actin RNA in each of the fetal RNA samples (data not shown).

Fig. 1.

Expression of c-jun and junB RNA in mouse fetuses. RNA blots were hybridized with junB probe (lanes 1 –4), and then (without removal of junB signal) with c-jun probe (lanes 5 –8) to examine transcripts in mouse fetuses. RNA samples from serum-stimulated NIH3T3 fibroblasts (1,5), quiescent fibroblasts (2,6), 14-5 day mouse embryos (3,7) and 17 ·5 day embryos (4,8) were analysed. junB probe detects a ∼ 2 ·0 transcript, indicated as ‘b’. c-jun probe detects two transcripts of ∼ 2 ·7kb and 3 ·2 kb, indicated as ‘a’; previous work has shown these two transcripts to be due to alternative polyadenylation sites (Ryseck et al. 1988). Note that the autoradiographic exposure shown in lanes 1 –4 is greater than that of lanes 5 –8 in order to detect junB transcripts, which are present in fetal RNA at lower relative levels than c-jun transcripts. Hybridization of the blots with actin coding region probe revealed similar levels of actin RNA in each of the fetal RNA samples (data not shown).

To examine the localization of c-jun and junB transcripts during fetal development, we have used in situ hybridization, c-jun expression was found to be relatively widespread, although found only in specific cell populations within certain tissues, whereas junB transcripts were highly restricted. These sites of expression are described below.

c-jun expression in developing cartilage

c-jun transcripts were detected in perichondrial cells throughout the 14 ·5 day fetus, for example in developing limb (Fig. 2A –D) and ribs (Fig. 3A,B). These proliferating cells are the precursors to terminally differentiating cartilage which does not express c-jun (Fig. 2A –D, Fig. 3A,B). Expression also occurred in growth zones of cartilage at 17 ·5 days of development, although at this stage similar levels of c-jun transcripts were found in surrounding mesenchymal tissue, including mesodermal web cells (Fig. 2E,F).

Fig. 2.

c-jun expression in the developing limb. Tissue sections of 14 ·5 day (A –D) and 17 ·5 day (E,F) mouse fetuses were used for in situ hybridization with c-jun probe. Bright-field (A,C,E) and dark-field (B,D,F) photographs of forelimb are shown; C,D are higher-power photographs of the limb shown in A,B. pc, perichondrial cells; c, cartilage; me, mesenchyme.

Fig. 2.

c-jun expression in the developing limb. Tissue sections of 14 ·5 day (A –D) and 17 ·5 day (E,F) mouse fetuses were used for in situ hybridization with c-jun probe. Bright-field (A,C,E) and dark-field (B,D,F) photographs of forelimb are shown; C,D are higher-power photographs of the limb shown in A,B. pc, perichondrial cells; c, cartilage; me, mesenchyme.

Fig. 3.

c-jun expression in developing muscle. Tissue sections of 14 ·5 day fetuses were used for in situ hybridization with c-jun probe (A,B,D,E) or cardiac actin probe (Minty et al. 1982) to reveal developing muscle (C,F). Bright-held (A,D) and dark-field (B,C,E,F) photographs of ribs (A –C) and diaphragm (D –F) are shown, m, muscle; pc, perichondrial cells; c, cartilage; d, diaphragm; 11, liver; lu, lung. Note that the intense localized signals over liver are due to refraction of light by erythrocytes, not silver grains.

Fig. 3.

c-jun expression in developing muscle. Tissue sections of 14 ·5 day fetuses were used for in situ hybridization with c-jun probe (A,B,D,E) or cardiac actin probe (Minty et al. 1982) to reveal developing muscle (C,F). Bright-held (A,D) and dark-field (B,C,E,F) photographs of ribs (A –C) and diaphragm (D –F) are shown, m, muscle; pc, perichondrial cells; c, cartilage; d, diaphragm; 11, liver; lu, lung. Note that the intense localized signals over liver are due to refraction of light by erythrocytes, not silver grains.

c-jun expression in developing muscle

Examination of 14 ·5 and 17 ·5 day embryos hybridized with c-jun probe suggested that expression occurred in skeletal muscle. This was confirmed by revealing the presence of skeletal muscle by hybridization of adjacent sections with cardiac actin probe; c-jun transcripts were detected in skeletal muscle throughout the embryo, for example the intercostal muscles of the ribs (Fig. 3A –C) and the diaphragm (Fig. 3D-F). In contrast, c-jun expression was not detected in smooth muscle in the developing gut (see below).

c-jun expression in the nervous system

Accumulation of c-jun transcripts was detected throughout the cranial and spinal ganglia (not shown), and the olfactory epithelium (Fig. 4A,B), components of the peripheral nervous system. However, c-jun expression was highly localized in the central nervous system. Transcripts were detected in proliferating neuroepithelial cells (the ventricular layer) in the telencephalon (Fig. 4C,D), but not in the adjacent zone of postmitotic cells. Lower relative levels of c-jun transcripts were detected in the ventricular layer of other regions of the central nervous system (Fig. 4E –H, and data not shown). A number of sites of c-jun expression were observed in the midbrain, hindbrain and spinal cord (Fig. 4E –H, and data not shown). The location of these sites suggests that they correspond to motor neurones, for example the oculomotor nucleus in the midbrain (Fig. 4E,F) and the somatic and visceral motor columns in the spinal cord (Fig. 4G,H).

Fig. 4.

c-jun expression in the developing peripheral and central nervous system. Tissue sections of 14 · 5 day mouse fetuses were used for in situ hybridization with c-jun probe. Bright-field (A,C,E,G) and dark-field (B,D,F,H) photographs of olfactory epithelium (A,B), telencephalon (C,D), midbrain (E,F) and spinal cord (G,H) are shown. A-D are longitudinal sections and E-H are transverse. oe, olfactory epithelium; v, ventricular layer of cerebral cortex; om, oculomotor neurones in midbrain; m, visceral and somatic motor neurones in the ventral spinal cord.

Fig. 4.

c-jun expression in the developing peripheral and central nervous system. Tissue sections of 14 · 5 day mouse fetuses were used for in situ hybridization with c-jun probe. Bright-field (A,C,E,G) and dark-field (B,D,F,H) photographs of olfactory epithelium (A,B), telencephalon (C,D), midbrain (E,F) and spinal cord (G,H) are shown. A-D are longitudinal sections and E-H are transverse. oe, olfactory epithelium; v, ventricular layer of cerebral cortex; om, oculomotor neurones in midbrain; m, visceral and somatic motor neurones in the ventral spinal cord.

Other sites of c-jun expression

c-jun transcripts were detected in a number of visceral organs of 14 ·5 and 17 ·5 day embryos, including lung (Fig. 3D,E), gut (see below), kidney, and adrenal gland (not shown). Notably, c-jun expression was not detected in liver (Fig. 3D,E).

junB and c-jun expression in developing gut and skin junB transcripts were not detected by in situ hybridization to 14 ·5 day fetuses (data not shown), and were found only in the developing gut and skin at 17 ·5 days. In the gut and oral cavity, junB transcripts were restricted to the endoderm, for example, in the squamous epithelium of the forestomach (Fig. 5A,B). In contrast, c-jun expression was not found in the endoderm, or in the adjacent layer of smooth muscle, but rather was detected in the surrounding mesoder-mally derived mesenchymal tissue (Fig. 5C). In the skin, junB expression was found in the outermost layer, the stratum corneum, but not in basal layers of proliferating epidermal cells (Fig. 5D,E). The stratum corneum consists of terminally differentiating cells and first arises at 16 days of development (Hanson, 1947), consistent with the absence of epidermal expression of junB at 14 ·5 days, c-jun expression occurred in a distinct, punctate pattern in epidermis (Fig. 5F).

Fig. 5.

junB and c-jun expression in gut and skin. Tissue sections of 17 · 5 day fetuses were hybridized with junB (A,B,D,E) or c-jun (C,F) probe. Bright-field (A,D) and dark-field (B,C,E,F) photographs of forestomach (A – C) and epidermis (D – F) are shown, en, endoderm; mm, muscularis mucosae; me, stomach mesenchyme; sc, stratum corneum; b, basal layers of epidermis.

Fig. 5.

junB and c-jun expression in gut and skin. Tissue sections of 17 · 5 day fetuses were hybridized with junB (A,B,D,E) or c-jun (C,F) probe. Bright-field (A,D) and dark-field (B,C,E,F) photographs of forestomach (A – C) and epidermis (D – F) are shown, en, endoderm; mm, muscularis mucosae; me, stomach mesenchyme; sc, stratum corneum; b, basal layers of epidermis.

Understanding of the normal function of c-jun and junB requires knowledge of their expression patterns. In view of the presence of AP-l-binding sites upstream of a number of functionally unrelated genes, it seems likely that collectively AP-l-like proteins may influence gene expression in many tissues and be involved in multiple biological processes. However, it is possible that individual members of the AP-1 transcription factor family may have distinct, tissue-specific roles, and the developmental expression patterns reported here are consistent with this scenario, c-jun has a relatively widespread expression, although transcripts are restricted to specific cell populations in certain tissues. In contrast, junB has a distinct and highly restricted expression pattern, suggestive of this gene having a more specialized role and regulating different target genes from c-jun.

The association of c-jun and junB expression with the resumption of fibroblast cell growth (Ryder et al. 1988; Ryseck et al. 1988; Quantin & Breathnach, 1988) suggests that these genes could be involved in cell proliferation as part of a network of growth-factor-responsive genes. Indeed, such a role is consistent with the detection of c-jun expression in certain proliferating cell populations, notably in perichondrial cells and the ventricular layer of the telencephalon. Perichondrial cells are also sites of c-fos expression (Dony & Gruss, 1987), so, in view of the physical association of AP-1 and c-fos protein (Chiu et al. 1988; Rauscher et al. 1988; Sassone-Corsi et al. 1988), it is possible that these gene products could act cooperatively in tissue-specific cell proliferation, c-fos expression also occurs in mesodermal web cells (Dony & Gruss, 1987), and this tissue also expresses c-jun. However, c-fos transcripts have not been reported in many of the other sites of c-jun expression, so it seems likely that the latter may also act independently of c-fos. Furthermore, our data suggest that coexpression of junB and c-fos does not occur during development.

The expression of c-jun in certain postmitotic cells, for example motor neurones, suggests a role of this gene in cell differentiation rather than in proliferation in at least this tissue. This restriction suggests that c-jun may be involved in the transcriptional regulation of genes expressed in motor, but not sensory, neurones. Similarly, although rapid proliferation of epidermal cells occurs in basal layers of the skin, junB expression occurs in the superficial stratum corneum which consists of terminally differentiating cells. It is intriguing that the other site of junB expression is the gut endoderm, since this epithelium is contiguous with, and shares a number of structural and functional properties with, the epidermis (Parakkal, 1967). In particular, it may be significant that these epithelial tissues are sites of keratin gene expression (reviewed by Fuchs, 1988), and it is pertinent to ascertain whether junB might be directly involved in transcriptional control of these genes.

Overall, our observations suggest that c-jun expression may be associated with tissue-specific cell proliferation, as also proposed for the c-fos proto-oncogene (Dony & Gruss, 1987). On the other hand, it seems likely that c-jun and junB also have roles in cell differentiation: c-jun in motor neurones and junB in skin and gut epithelia. An increasing number of other genes initially discovered in the context of cell growth and/or tumorigenesis, including c-src (Sorge et al. 1984) and int-2 (Wilkinson et al. 1989), have developmental expression patterns suggestive of roles in cell differentiation or phenotype. The rapid upregulation of c-jun and junB RNA levels by growth factors in fibroblasts suggests that they mediate specific gene transcription in response to extracellular signals. We envisage that these members of the AP-1 family form part of a cascade of regulatory interactions during development that lead to appropriate gene transcription in specific cell populations, and that this may involve combinatorial action with other transcription factors. Thus, understanding of the significance of the patterns of c-jun and junB regulation reported here will require analysis of the biological effects of perturbing their expression in appropriate cell types and identification of their presumed target genes.

We thank Patrick Charnay for the gift of fibroblast RNA samples, and David Sassoon for suggesting the use of cardiac actin probe. Rolf-Peter Ryseck was supported by an EMBL postdoctoral fellowship.

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