C/EBP is a positive-acting transcription factor important for hepatocyte-specific expression present not only in hepatocytes but also a limited number of other cell types in adult mice. By Northern blot analysis and in situ hybridization experiments with mouse embryos and adult tissues, we first detected C/EBP mRNA in hepatocytes at the 13th gestational day, when no other cell types give detectable signals and thus by this test C/EBP is, at least in the embryo, a ‘liver-specific’ factor. Only trace amounts of C/EBP were seen in the yolk sac and no mRNA was detectable in choroid plexus in either embryos or adult animals. Both these cell types produce some proteins (e.g. albumin, transthyretin, a-l antitrypsin and others) that are also made in the liver where C/EBP is important for their production; thus either fewer factors or different factors govern yolk sac and choroid plexus production of these proteins. C/EBP mRNA was not detected in fetal brain but was present in several regions of the adult mouse brain again emphasizing that this factor does not appear to have a very early embryologic role. In the adult brain, it was most concentrated in CAI to CA4 regions of the hippocampus, in cerebellar Purkinje cells, and in layer II and III of the cortex.

In adult animals, transcription of many genes is sharply limited to particular cell types and transcriptional activation requires the action of positive-acting transcription factors (Johnson and Mcknight, 1989). These findings suggest two possibilities for cell-specific regulation that are not mutually exclusive: (1) positive transcription factors may be present in all cells but modified post-translationally in a cell-specific fashion; or (2) transcription factors may be themselves transcribed in a cell-specific manner. The latter, cell-specific (or cell-limited) transcription of transcription factors, portends a cascade of regulation starting with the fertilized egg and developing sequentially to establish the transcriptional program of a specialized adult cell type. From studies of genes expressed only or mainly in hepatocytes, a series of DNA-binding proteins that are enriched in hepatocytes compared to other cell types have been identified (Courtois et al. 1987; Babiss et al. 1987; Costa et al. 1988a; Costa et al. 1988b; Lichtsteiner et al. 1987;Monaci et al. 1988; Frain et al. 1989). For one of them, C/EBP (CCAAT/enhancer binding protein; Johnson et al. 1987; Landschulz et al. 1988), we have shown that transcriptional control underlies its limited distribution in adult mouse tissues (Xanthopoulos et al. 1989).

An important first step in understanding the physiological functions of a given protein in an animal is to acquire knowledge about its cell or tissue distribution. However, analysis of whole organs for the presence of a factor obviously fails to provide information concerning which cell type or what fraction of the cells contains the factor. We have therefore used in situ hybridization to study the cellular distribution of C/EBP mRNA in various adult organs as well as embryos at various stages. C/EBP was detected in the embryonic liver by the 13th gestational day at which time it is not present at significant concentrations in any other cell type. In addition, the liver remains the most abundant site of C/EBP expression throughout adult life. In situ hybridization experiments also revealed that C/EBP is found in several regions of the adult brain, the hippocampus being the most prominent site of expression. Finally, we discuss the possible meaning of these findings for ideas of combinatorial action of transcription factors in cellspecific regulation.

Animais

C3H/C57BJ hybrid mice purchased from Jackson Laboratory were used for all the studies in this paper. The animals were kept in a specific pathogen-free facility in the laboratory animal research center at Rockefeller University.

Preparation of RNA

Organs were removed from animals and used immediately for RNA preparation by the guanidinium isothiocyanate method (Chirgwin et al. 1979). For adult tissue samples, female mice over 10 weeks old and averaging 25 grams in body weight were used. Ten animals were used for brain dissection under a dissecting microscope. For prenatal samples, mice were set up for mating and the presence of the vaginal plug in the morning were dated as day 1. For each time point, ten to forty embryos were dissected and pooled together to obtain sufficient RNA.

Northern blot analysis

As described previously (Kuo and Darnell, 1989), 20pg of total RNA was denatured with glyoxal, electrophoresed in 1% agarose gel, electrotransferred (Smith et al. 1984) onto nitrocellulose and probed with 32P-labelled DNA probes at 55°C overnight. After washing with O.lxSSC, 0.1% SDS for 3–5 min at 65 °C, filters were exposed to Kodak XAR film at − 80°C with intensifying screen.

In situ hybridization

The procedure was basically the same as described previously (Kuo et al. 1988). Prefixation in 4% paraformaldehyde was followed by cryoprotection (30% sucrose) and cryostat sectioning. Serial sections 5–10 /on thick were collected onto poly-L-lysine-coated slides. After pretreatment and acetylation, 15 μ l of the hybridization solution containing 105cts min−1 of 35S-labelled riboprobe was placed on the slide, covered with cover slip, sealed with rubber cement and stored in a humidified chamber for hybridization. Hybridization was at 55°C for 4–16h. Washing with 0.2 × SSC at 60°C was followed by treatment with 5 μ gml−1 of RNAse A in 2xSSC at 37°C. The slices were dehydrated, coated with Kodak NTB-2 emulsion (diluted 1:1 with H2O) and exposed from 1 day to 3 weeks. (Adult TTR signals in liver and choroid plexus were seen within 1 day; adult liver signals for C/EBP required 5 to 7 days and fetal liver signals for C/EBP required 3 weeks). After development, the slides were counterstained with either hematoxylin-eosin or Giemsa stain and covered with permount. Bright- and dark-field microscopic pictures were taken using a Nikon Diaphot and a regular 35 mm camera.

DNA manipulation and probe synthesis

DNA manipulation and purification were according to either Maniatis et al. 1982 or Ausubel et al. 1987. The DNA probe for mouse C/EBP was à 1.1 KB Pstl-Sstl fragment of mouse C/EBP cDNA clone (Xanthopoulos et al. 1989). For Northern analysis, the gel-purified fragment was labelled by random priming (Feinberg and Vogelstein, 1983) with 32P-dATP. The 35S-labelled riboprobes were generated with SP6 or T7 RNA polymerase using linearized template.

Developmental occurrence of C/EBP mRNA

C/EBP binds to a number of sites required for maximal hepatoma-specific expression of the transthyretin (TTR), α-l antitrypsin and albumin genes (Costa et al. 1988b; Costa et al. 1988a; Lichtsteiner et al. 1987). In addition, C/EBP protein produced in vivo will stimulate transcription of a test gene on a second plasmid in cotransfection experiments (Friedman et al. 1989; Christy et al. 1989; Herrera et al. 1989).

We have previously shown that the three genes mentioned above are transcribed actively in 15 and 17 day fetuses (Powell et al. 1984) and mouse albumin gene expression in the liver has been detected by as early as the 13th gestational day of mouse development (Tilghman and Beyayew, 1982; Panduro et al. 1987; Camper et al. 1989). At the same time, the yolk sac endoderm also expresses these genes (Meehan et al. 1984; Fig. 3). We therefore tested the course of C/EBP expression during embryogenesis. RNA was isolated from liver at various stages during development and analyzed by Northern blots (Fig. 1) C/EBP mRNA was detected by this technique in 17 day fetal liver. From this time on until adulthood, the steady-state level of C/EBP mRNA remained relatively the same with no more than a twofold fluctuation. As a control albumin and TTR mRNAs were tested and they exhibited fairly constant levels of expression in these samples. Other mRNAs produced in adult liver e.g. contrapsin, a protease inhibitor, showed a large surge of expression at the time of birth, possibly in response to the increased level of glucocorticoid in the blood at this time (Panduro et al. 1987).

Fig. 1

Developmental regulation of C/EBP mRNA accumulation in mouse liver Northern blot analysis of 20 pg of mRNA from various embryonic stages was carried out first with a 32P-labeled C/EBP probe. After autoradiography the filter was then stripped successively for hybridization with each probe. Two exposures (3h and 30 h) were used for the TTR probe to show that the 13 day fetal sample (13F) was intact. 13F, 13 days fetal liver; 17F, 17 days fetal liver; TF, term fetal liver; 2D, 5D and 2W, 2 days, 5 days and 2 weeks neonatal liver; AD, adult liver samples.

Fig. 1

Developmental regulation of C/EBP mRNA accumulation in mouse liver Northern blot analysis of 20 pg of mRNA from various embryonic stages was carried out first with a 32P-labeled C/EBP probe. After autoradiography the filter was then stripped successively for hybridization with each probe. Two exposures (3h and 30 h) were used for the TTR probe to show that the 13 day fetal sample (13F) was intact. 13F, 13 days fetal liver; 17F, 17 days fetal liver; TF, term fetal liver; 2D, 5D and 2W, 2 days, 5 days and 2 weeks neonatal liver; AD, adult liver samples.

In situ hybridization studies of C/EBP in developing mouse embryo

Detection of C/EBP mRNA by Northern blot analysis from very early embryos was difficult partly because of the scarcity of RNA and the difficulty of obtaining pure tissue samples. For these reasons and in order to localize the cells containing C/EBP mRNA, we carried out in situ hybridization to further examine C/EBP expression during mouse development.

Mouse embryos from the 8th gestational day to term were examined using 35S-labelled riboprobes. Both sense and antisense probes were made for C/EBP, albumin and TTR to correlate their expression. Even though the albumin mRNA was detected in day 12 embryonic liver (data not shown), the earliest time we were able to detect C/EBP convincingly was day 13 in the embryonic liver (Fig. 2 A and B). No signal observed elsewhere in the 13 day embryo. The positive signals for C/EBP (or for albumin and TTR, not shown) in the 13 day liver were patchy presumably because, at this early stage, 50% of the cells in the liver are not hepatocytes but hematopoietic cells (Fig. 2, A and B). We examined especially carefully for expression in fetal gut, choroid plexus and yolk sac because some of the proteins made in hepatocytes are made in those cells. By day 13, the albumin mRNA was detectable not only in fetal liver but also in fetal gut and in yolk sac endoderm (Meehan et al. 1984; Fig. 3, A and B) while the TTR mRNA gave a strong signal in choroid plexus and a definite but weaker signal than albumin in yolk sac endoderm and was not present in the gut (Fig. 3C to F). We did not detect significant levels of C/EBP mRNA in any of these embryonic structures by in situ hybridization (Fig. 3, G and H). This does not rule out the possibility of the presence of a very low level of C/EBP in these developing tissues but does indicate that the ratio of C/EBP mRNA to albumin or TTR mRNA in fetal liver is much higher than it is in yolk sac or choroid plexus. At most a very low level of C/EBP mRNA was detected in the yolk sac RNA after a very long exposure of a Northern blot (Fig. 4), again supporting the conclusion that the yolk sac contained a much lower level of C/EBP mRNA than fetal liver.

Fig. 2

In situ hybridization of C/EBP antisense probe to mouse embryo sections. The mouse embryos were collected from timed pregnant mothers by Cesarean section. They were immersion-fixed in 4% paraformaldehyde and cryoprotected in 30% sucrose before cryosectioning. In situ hybridization was done as described in the Materials and methods. A and B, 13 days fetus; C and D, 15 days fetus; E and F, adult liver. A, C and E, bright-field; B, D and F, dark-field microscopic pictures. Liv, liver; He, heart; S, stomach; Lu, lung; V, vertebrae; CV, central vein; PV, portal vein. Magnifications: A through D, 100 ×; E and F, 200 ×. (Probes in the sense orientation gave no specific signals in this and other experiments.)

Fig. 2

In situ hybridization of C/EBP antisense probe to mouse embryo sections. The mouse embryos were collected from timed pregnant mothers by Cesarean section. They were immersion-fixed in 4% paraformaldehyde and cryoprotected in 30% sucrose before cryosectioning. In situ hybridization was done as described in the Materials and methods. A and B, 13 days fetus; C and D, 15 days fetus; E and F, adult liver. A, C and E, bright-field; B, D and F, dark-field microscopic pictures. Liv, liver; He, heart; S, stomach; Lu, lung; V, vertebrae; CV, central vein; PV, portal vein. Magnifications: A through D, 100 ×; E and F, 200 ×. (Probes in the sense orientation gave no specific signals in this and other experiments.)

Fig. 3

In situ hybridization of albumin and transthyretin antisense riboprobe in the 13 day mouse embryo The preparation of the mouse embryo is the same as described in Fig. 2. A and B, albumin probe; C to F, TTR probe; G and H, C/EBP probe. Y, yolk sac; Ch, choroid plexus; Pl, placenta; Em, embryo. A, C and E, bright-field; B, D and F, dark-field. Magnifications: A, B, E, and F, lOOx; C, D, G, and H, 40 × ×.

Fig. 3

In situ hybridization of albumin and transthyretin antisense riboprobe in the 13 day mouse embryo The preparation of the mouse embryo is the same as described in Fig. 2. A and B, albumin probe; C to F, TTR probe; G and H, C/EBP probe. Y, yolk sac; Ch, choroid plexus; Pl, placenta; Em, embryo. A, C and E, bright-field; B, D and F, dark-field. Magnifications: A, B, E, and F, lOOx; C, D, G, and H, 40 × ×.

Fig. 4

C/EBP mRNA in the yolk sac. Total cytoplasmic RNA was isolated from yolk sac membrane dissected out from six pregnant mice at 17 days gestation (See Materials and methods). Northern blot analysis was as described in the legend of Fig. 1.

Fig. 4

C/EBP mRNA in the yolk sac. Total cytoplasmic RNA was isolated from yolk sac membrane dissected out from six pregnant mice at 17 days gestation (See Materials and methods). Northern blot analysis was as described in the legend of Fig. 1.

The in situ hybridization signal for C/EBP mRNA in the developing liver was stronger by day 15 (Fig. 2, C and D). The pattern remained relatively the same from this time on into adulthood (Fig. 2, E and F) and was uniformly distributed within the liver acinus in late fetal, neonatal and adult animals. In the region of the portal triad where blood vessels and connective tissue are more plentiful, nonparenchymal cells lacked any C/EBP signal (Fig. 2, E and F).

C/EBP expression in the mouse brain

In a previous report, we described Northern blot analysis for C/EBP that showed at most a low signal in total brain tissue. But during the examination for hybridization to choroid plexus cells (discussed below), we did detect the C/EBP mRNA by in situ hybridization of brain slices (Xanthopoulos et al. 1989). We have now identified the location of the major C/EBP signals in the brain. As a first step, we dissected the central nervous system of ten adult female mice into four regions: cerebellum, forebrain, hippocampus and a central segment containing the midbrain, thalamus and corpus callosum. RNA was isolated from these regions and analyzed by Northern blots. C/EBP mRNA was detectable at low levels in all four regions (Fig. 5), but by densitométrie scanning we estimated the level to be 10% or less that seen in the liver (glutamine synthetase, GS and transthyretin (TTR) probes were used to test the intactness of RNA. The TTR signals are due to choroid plexus cells in the ventricles and thus are absent completely from the forebrain. Glial cells express GS and signals in each brain section are apparent.)

Fig. 5

C/EBP mRNA in different regions of the brain. RNA was isolated from various regions of the brain dissected out from ten adult female mice as described in the Materials and methods. Northern blot analysis is as described in the legend of Fig. 1.

Fig. 5

C/EBP mRNA in different regions of the brain. RNA was isolated from various regions of the brain dissected out from ten adult female mice as described in the Materials and methods. Northern blot analysis is as described in the legend of Fig. 1.

We then examined by in situ hybridization specific cells for C/EBP expression. In the hippocampus where the level is the highest in the CNS, neurons in CAI, CA2, CA3, CA4 regions and in the dentate gyrus were all positive for C/EBP (Fig. 6A and B). In the cerebellum (Fig. 6C and D), Purkinje cells contained the highest level of C/EBP, but neurons in the molecular layer and granular layer were also weakly positive while some astrocytes in the molecular layer were clearly negative (Fig. 6E, arrowhead). In basal ganglia, midbrain, caudate nucleus, paraventricular zone, and many of the deep nuclei all contained neurons that were weakly positive (data not shown). In forebrain, a few positive neurons were seen distributed throughout all layers but were somewhat more concentrated in layer II and III (Fig. 6, F and G). Although choroid plexus cells that surround the lateral ventricles express very large amounts of TTR mRNA (Dickson et al. 1985; Fig. 3C and D shows this in fetal choroid plexus cells), and this gene has at least three binding sites for C/EBP in its regulatory regions, only background or at most a very low level of C/EBP mRNA was seen in the choroid plexus cells of either fetus (Fig. 3G,H) or adult (Fig. 6H and I). For example the choroid plexus cells in adults have less C/EBP than cells in the dentate gyrus (DG in Fig. 6H).

Fig. 6

In situ hybridization of C/EBP antisense probe in mouse brain. The preparation of the mouse brain for in situ hybridization is the same as described in Fig. 2. A and B, horizontal sections of hippocampus; C, D, and E, cerebellar folium; F and G, frontal cortex, layer 1 to VI are from top to bottom; H and I, regions of the lateral ventricle. Pu, Purkinje cells; DG, dentate gyrus; Ch, choroid plexus; Mol, molecular layer; Gr, granular layer; W, white matter. A, C, E, F and H, bright field; B, D, G and I, dark field. Magnifications: A to B and F through I, lOOx; C and D, 40×; E,200×.

Fig. 6

In situ hybridization of C/EBP antisense probe in mouse brain. The preparation of the mouse brain for in situ hybridization is the same as described in Fig. 2. A and B, horizontal sections of hippocampus; C, D, and E, cerebellar folium; F and G, frontal cortex, layer 1 to VI are from top to bottom; H and I, regions of the lateral ventricle. Pu, Purkinje cells; DG, dentate gyrus; Ch, choroid plexus; Mol, molecular layer; Gr, granular layer; W, white matter. A, C, E, F and H, bright field; B, D, G and I, dark field. Magnifications: A to B and F through I, lOOx; C and D, 40×; E,200×.

While the C/EBP mRNA was clearly present in the adult CNS, we failed to detect C/EBP mRNA in prenatal nervous tissues. The earliest time we observed convincing in situ signals in the brain was at 10 days postnatally. As in the adult, the signal was strongest in the hippocampal regions (data not shown). In the mouse, it is well-known that considerable growth and development of the hippocampus occurs postnatally.

Studies with the C/EBP transcription factor have helped to settle an important fundamental question about gene regulation in mammals: are transcription factors that participate in cell-specific gene activation in one cell type (e.g. hepatocytes) limited in their cell distribution perhaps even to a single cell type? By Northern blot analysis, C/EBP was found in a limited number of tissues including the liver (Xanthopolous et al. 1989; Birkenmeier et al. 1989) and by nuclear run-on analysis we found strong transcriptional signals for C/EBP only in liver and fat cells but not elsewhere. Thus C/EBP is not cell-specific but cell-limited in adults and this limitation is based on transcriptional regulation. The present study further shows that C/EBP is present in the 13 day fetal liver when organogenesis is well under way but not earlier. Moreover, there was no signal in any other tissue at this time. Thus C/EBP at 13 days of fetal life is a ‘liver-specific’ factor. These observations support the notion that C/EBP is probably important in specifying the hepatocyte-specific phenotype. Presumably it is transcriptionally activated during organogenesis and not during earlier embyrogenesis. Other mammalian transcription factors known to be cell-limited may also be transcriptionally controlled during cell specialization and have cell-limited distribution. Pit-1, an anterior pituitary-gland-specific factor (Bodner et al. 1988; Ingraham et al. 1988), Oct-2, a lymphoid-specific factor (Staudt et al. 1986; Staudt et al. 1988; Scheidereit et al. 1988) and HNF1, a liverspecific factor (Courtois et al. 1987; Frain et al. 1989) are candidates for such regulation.

This study also emphasizes that, at this stage in our knowledge of the regulation and distribution of transcription factors, we still have a great deal to learn, particularly about the logic underlying the distribution of a specific trancription factor. For example, in the adult animal, C/EBP is present in fat cells and intestinal cells as well as in liver. Because of this distribution, McKnight and colleagues (Birkenmeier et al. 1989; Christy et al. 1989) were led to suggest the following possibility: these three tissues have an important role in assimilation and metabolism of energy-yielding nutrients. They proposed therefore that C/EBP has as a principle function the regulation of genes participating in energy transduction or storage. While some of the genes affected by C/EBP might fall into this category, it is plain that many others affected by this regulator do not-e.g. transthyretin, albumin and crl-antitrypsin. In addition, we now describe the localized appearance of C/EBP in several regions of the brain. It seems at this point that attempts to impute biochemical interrelations to group of genes served by particular regulatory factors may well be fruitless.

This report serves to provide evidence on another widely discussed point that has not yet been illustrated by experimental findings. To the degree that the number of transcriptional factors is limited, there must be combinations of factors to produce variable patterns of gene expression from cell to cell. For instance C/EBP is present in fat cells as well as liver, but many of the set of genes that is regulated (in part) by C/EBP in the liver (e.g. albumin, TTR) are silent in fat cells presumably because the proper combination of factors is not there. On the other hand, TTR is a prominent product of the liver and 30-fold higher levels are formed in the choroid plexus (Dickson et al. 1985); if all the factors, including TTR, that are necessary in liver were also present in the choroid plexus then why are not many other hepatocyte expressed genes also active in the choroid plexus? Our results help to explain this conundrum. The present experiments show that the choroid plexus lacks detectable levels of C/EBP. In transgenic animals, the known C/EBP sites (and other sites for liver-enriched factors) are sufficient for hepatocyte expression of TTR but not for choroid plexus expression (Yan et al. 1990). Indeed sections of the TTR upstream region other than those with known binding sites for proteins in hepatocytes are required for choroid plexus expression and presumably choroid plexus-enriched proteins drive TTR expression even better than C/EBP and other hepatocyte enriched factors. Thus one combination of factors in the liver produces albumin a-1 antitrypsin and transthyretin expression and another factor or factors allows high levels of transthyretin expression but not most other hepatocyte-expressed genes (Yan et al. 1990).

For the yolk sac endodermal cells a different situation exists. These cells have been regarded as serving as an ‘early fetal liver’ because they produce so many of the secreted proteins that hepatocytes do (Meehan et al. 1984). However, it is clear that C/EBP is not present or present at only a low level in yolk sac endodermal cells. Either some of the other liver-specific factors nucleus HNF1,3,4; (Costa et al. 1989) or some additional yolk sac specific factors must account for this transcription of the ‘liver-specific’ mRNAs in the yolk sac endodermal cells. At any rate it seems assured that variation in the distribution of factors producing ‘workable’ combinations is required for a controlled program of cellspecific gene expression.

Finally we note without being able to provide any illuminating comments that some hippocampal neurons contain abundant C/EBP. These cells have been the object of recent attention because they also contain products of certain proto-oncogenes that play a role in transcriptional controls (c-fos; Sagas et al. 1988; Morgan et al. 1987). So far as we are aware there is no evidence that these cells are any more active in mRNA synthesis than other neurons although this might be the simplest basis for the presence of mRNAs for a variety of transcription factors. The total pattern of distribution of C/EBP mRNA in the brain does not correlate with the distribution of any known neurotransmitters or receptors. As probes for additional transcription factors (for example Pit-1, Bm-1, Bm-2 and Brn-3; He et al. 1989) become available will we be confronted with the presence of a larger and larger number of factors in certain limited regions of the brain such as hippocampus or will we find a constantly variable pattern of distribution among a variety of nerve cells? More importantly will any of these descriptive studies reveal useful clues about nerve cell specificity?

With the possibilities for such studies mounting almost daily, the facts will soon begin to accumulate. However, the interpretations and the ultimate understanding of the logic behind the participation of certain factors to regulate certain genes in certain cells may not be so soon in coming.

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