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Cell Growth & Differentiation Vol. 11, 163-171, March 2000
© 2000 American Association for Cancer Research

Expression of the A-raf Proto-Oncogene in the Normal Adult and Embryonic Mouse1

Jennifer C. A. Luckett, Martin B. Hüser, Nikolaos Giagtzoglou2, Jane E. Brown and Catrin A. Pritchard3

Department of Biochemistry [J. C. A. L., M. B. H., N. G., C. A. P.] and Division of Biomedical Services [J. E. B.], University of Leicester, Leicester LE1 7RH, United Kingdom


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
We have determined the expression pattern of the A-raf proto-oncogene in the embryonic and adult mouse. Western blot analysis of protein lysates from tissues of adult mice show that p69A-raf is ubiquitously expressed, but that levels of expression vary among different tissues. To determine the cell-specific expression pattern of A-raf, we generated transgenic mice expressing the ß-galactosidase reporter gene from the A-raf promoter. We show that A-raf expression is highly specific within a given tissue, and we identify cell types expressing this gene in the adult testis, epididymis, vas deferens, seminal vesicle, ovary, oviduct, bladder, kidney, intestine, heart, spleen, thymus, and cerebellum. In the embryo, ubiquitous expression of the reporter gene is observed, but the highest levels of expression are specifically detected in the embryonic heart at stages 9.5–11.5 days post-coitum.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Raf proteins are serine/threonine protein kinases that are activated transiently in response to the binding of extracellular ligands to specific classes of cell surface receptors (1) . Receptor classes that regulate Raf activity include receptor tyrosine kinases, seven transmembrane receptors that couple to G-proteins, and cytokine receptors that regulate cytosolic tyrosine kinases (2 , 3) . Raf proteins are involved in intracellular signal transduction pathways that transmit signals from activated receptors. One of the best characterized signal transduction cascades involving Raf is the Ras/Raf/MEK4 /ERK pathway, which is evolutionarily conserved in metazoan organisms (4) . This pathway consists of small GTP binding proteins of the Ras family that interact with and stimulate Raf activity (5, 6, 7, 8, 9, 10) . Activated Raf proteins then phosphorylate and activate the MEKs, dual specificity kinases that phosphorylate and activate a group of kinases known as ERKs or p42/p44 mitogen-activated protein kinases (11, 12, 13) . Activated ERKs regulate a number of proteins that have a profound effect on cell physiology including members of the Ets-family of transcription factors (14 , 15) . The ensuing regulation of genes such as c-fos (14 , 15) , HB-EGF (16) , and cyclin D1 (17 , 18) plays a central role in the control of proliferation, differentiation, apoptosis, and the control of cell shape and motility, as well as in the aberrant cell behavior displayed by tumor cells (3 , 18, 19, 20, 21) .

In mammals, the Raf family comprises three members, Raf-1, A-Raf, and B-Raf, that share a high degree of sequence similarity (2) . All Raf proteins share three conserved regions embedded in variable sequences. However, they have a number of amino acid differences within the conserved regions that generate functional differences. All three Rafs are linked to activated cell surface receptors through their ability to associate with Ras.GTP. This has the effect of translocating them to the plasma membrane, where they are activated (22, 23, 24, 25) . However, Raf-1 also requires tyrosine and serine kinase signals for full activation, whereas B-Raf does not (25 , 26) . The mechanism of regulation of A-Raf appears to be similar to Raf-1 in that it requires Ras.GTP binding and tyrosine and serine phosphorylation for full activation (25) . Yet a number of differences between A-Raf and Raf-1 have been observed. In primary rat ventricular myocytes, powerful hypertrophic agonists such as phorbol esters and ET-1 strongly activate both Raf-1 and A-Raf, whereas weaker hypertrophic agonists, such as acidic fibroblast growth factor and insulin-like growth factor-1, fail to activate A-Raf but activate Raf-1 (27 , 28) . Furthermore, Raf-1 activation induced by ET-1 in these cells is inhibited by activation of cAMP-dependent protein kinase A, whereas A-Raf activation by ET-1 is less affected (27) . In primary bone marrow cells, both A-Raf and Raf-1 are activated by interleukin 3. However, in this situation, Raf-1 activation was found to be insensitive to phosphatidylinositol 3-kinase inhibition, whereas A-Raf activation was blocked by inhibition of phosphatidylinositol 3-kinase (29) .

Raf proteins also differ in their ability to activate downstream effectors. The three Raf isoforms phosphorylate and activate downstream kinases (MEKs and ERKs) with quite different potencies: B-Raf is a strong MEK activator, A-Raf a weak MEK activator, and Raf-1 is intermediate between the two (25 , 30) . B-Raf appears to be the major activator of MEK in neural cells, despite both B-Raf and Raf-1 being expressed and activated in these cells (31, 32, 33) . Apart from MEK, yeast two-hybrid studies have identified a number of proteins that may be alternative substrates for specific Raf isoforms. A-Raf, but not B-Raf or Raf-1, has been shown to bind to the regulatory ß subunit of Casein Kinase 2 (CK2ß; Refs. 34 and 35 ) as well as to M2-PK (35) .5 A-Raf kinase activity has been shown to be enhanced upon coexpression with CK2ß in baculovirus cells (34 , 35) , and coexpression of A-Raf with M2-PK increases tetramerization and activity of M2-PK (35) .5

That the Raf kinases have unique properties is supported by the generation of mice deficient in each of these proteins, which display grossly different phenotypes. Mice with a knockout mutation of the A-raf gene, which is located on the X chromosome, die 1–6 weeks after birth because of a progressive failure to thrive (36) . They feed normally but are hyperactive and also demonstrate movement abnormalities. The pathophysiological basis of these phenotypes has not yet been determined. By contrast, mice deficient for B-Raf die in embryogenesis at 12.5 days p.c. of vascular rupture caused by abnormal patterns of endothelial cell apoptosis (37) . Mice with a hypomorphic allele of raf-1 die perinatally of lung and skin abnormalities (38) .

Any study of Raf function must take into consideration the expression patterns of the raf genes in the particular cell type under study. Previous expression studies have largely been restricted to Northern analysis (39) . In these studies, it was shown that raf-1 is ubiquitously expressed and that B-raf expression is restricted to germ cells and neuronal tissue. Subsequent reverse transcription-PCR studies have detected B-raf transcripts in all tissues (40) . Northern analysis showed that A-raf mRNA is produced in all cell types, but levels of expression vary enormously (39) . Highest levels of A-raf mRNA are present in tissues of the urogenital tract, particularly the epididymis, but low or barely detectable levels of mRNA are produced in the cerebral cortex, cerebellum, spinal cord, stomach, lung, and skin. In situ hybridization analysis of A-raf mRNA has so far been performed only on the testis and epididymis, and these studies show a diffuse distribution of signal throughout all cells in these tissues (41 , 42) . The A-raf promoter contains three potential GREs (43) , and in cell transfection assays the A-raf promoter was found to be dexamethasone inducible (44) . These observations have led to the suggestion that the A-raf gene is steroid hormone inducible and is important for urogenital function (43 , 44) . However, the A-Raf-deficient mice are fully fertile, and kidney function is normal (36) . This observation has prompted a more detailed investigation of the cell type-specific expression of A-raf in the embryonic and adult mouse. Here, we have used Western blot analysis to show that the A-Raf protein exhibits a similar pattern of expression to A-raf mRNA. In addition, we have generated transgenic mice expressing the ß-gal reporter gene from the A-raf promoter. These mice have been used to provide the first description of the cell-specific expression pattern of A-raf in the embryo and adult tissue.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Western Blot Analysis of Protein Lysates from Adult Mouse Tissues.
Protein lysates were prepared from a variety of tissues obtained from male and female mice, 6 weeks of age, derived from the outbred MF-1 strain as described in "Materials and Methods." Twenty µg of each extract were analyzed by probing Western blots with an anti-p69A-raf antibody (Fig. 1)Citation . This antibody is highly specific for p69A-Raf because it does not detect any of the other Raf proteins or a band at Mr 69,000 in tissues derived from A-Raf-deficient mice (36) . The A-Raf protein was detected in all of the tissues examined, but levels of expression varied enormously. Highest levels of expression were observed in components of the male reproductive tract, particularly the epididymis and seminal vesicle. Medium levels of expression were observed in a number of other urogenital tissues, i.e., testis, vas deferens, ovary, bladder, uterus, and kidney, as well as in a number of nonurogenital tissues such as heart, liver, and intestine. Lower levels of expression were present in pancreas, lung, cerebellum, thymus, and spleen, and barely detectable levels of expression were observed in the cerebral cortex and skeletal muscle. By scanning the autoradiograms, we estimate that the protein is present at ~10-fold lower levels in skeletal muscle and 20-fold lower levels in cerebral cortex than in testis (Fig. 1B)Citation . Highest levels of expression were observed in the epididymis and seminal vesicle, with A-Raf being expressed at 2-fold higher levels in epididymis and 3-fold higher levels in seminal vesicle than the testis (Fig. 1B)Citation .



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Fig. 1. Western blot analysis of p69A-raf in adult mouse tissues. Western blot analysis was performed as described in "Materials and Methods" on 20 µg of total protein lysate prepared from each tissue. The results of this analysis are shown in A. Equal protein loadings were confirmed by analyzing the Ponceau S-stained blots. By scanning the autoradiographs, it was possible to construct a bar graph indicating the amount of p69A-raf in each tissue (B).

 
Generation of Transgenic Mice Expressing the ß-gal Reporter Gene from the A-raf Promoter.
Available antibodies for A-Raf do not give A-Raf-specific expression patterns in immunohistochemical studies of mouse tissues.6 Therefore, to investigate the cell-specific expression of A-raf in adult and embryonic tissue, transgenic mice were generated expressing the ß-gal reporter gene from the A-raf promoter. We identified a 5.1-kb SacI fragment, which contains the mouse A-raf minimal promoter region, ~2 kb of DNA upstream of the minimal promoter, and the first four exons of the coding region (see "Materials and Methods" and Ref. 43 ). The restriction map and exon structure of this fragment is illustrated in Fig. 2Citation . We cloned this 5.1-kb SacI fragment next to the ß-geo reporter gene (45) . The A-raf:ß-geo DNA fragment was electroporated into ES cells, and transfected colonies were selected with G418. Most colonies expressed ß-gal activity and, from these, five were chosen that had single integration events based on Southern analysis (data not shown). These were microinjected into day 3.5 embryos, and several chimeric animals were derived. Germ-line transmission was obtained from one ES cell clone, and a transgenic line was established by breeding to the MF-1 mouse strain. The expression pattern of the reporter gene was analyzed in tissues from the transgenic line as well as chimeras from the other four ES clones.



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Fig. 2. Restriction map of promoter region of the mouse A-raf gene. The restriction map of the 5.1-kb SacI fragment used for construction of the A-raf:ß-geo transgene is indicated. The location of the promoter region, transcription initiation site, and first four coding exons were located on this map by using the data of Lee et al. (43). The A-raf:ß-geo transgene was constructed by cloning this SacI fragment next to the ß-geo gene from the plasmid pß-geobpA (45), creating an in-frame fusion between exon 4 of A-raf and the ß-gal gene. S, SacI; E, EcoRI; H, HindIII; P, PstI. The black bars beneath the restriction map represent exons, and the hatched bar represents the minimal promoter region mapped by Lee et al. (43).

 
Expression of the A-raf:ß-geo Transgene in Adult Mouse Tissues.
Expression of the transgene was analyzed either in whole adult tissues or in sections of adult tissues derived from the transgenic line and chimeras from four different ES clones. Table 1Citation summarizes these data. Background staining as a result of endogenous ß-gal activity was also analyzed in wild-type MF-1 mice. The data show that the expression pattern of the transgene is highly consistent between the transgenic lines and chimeras, and little background staining in wild-type tissues was observed. Highest levels of expression were observed in tissues of the male and female reproductive tracts, although lower levels of staining were observed in a number of nonurogenital tissues (Table 1)Citation .


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Table 1 Expression of transgene in adult tissues from transgenic line and chimeras

Tissues were collected from male and female transgenic, chimeric, or wild-type animals and were either stained with X-gal as whole tissues or sectioned and then stained with X-gal.

 
After 4 h in the X-gal stain, expression of the transgene was observed in the seminal vesicle and epididymis, and after 16 h, expression was detected in the testis and vas deferens of the transgenic line and chimeras (Table 1Citation and Fig. 3, A–DCitation ). Although staining of wild-type epididymis and vas deferens was occasionally observed, this was only detected after longer incubation times with the stain (Table 1)Citation . Cross-section analysis showed that expression in the testis was specific for the seminiferous tubules as well as for the interstitial cells of Leydig (Fig. 3C)Citation . Staining of the fibroblast cells of the basement membrane was not observed. Within the seminiferous tubules, staining of the primary and secondary spermatocytes was observed, but it was not possible to distinguish whether the Sertoli cells also expressed the transgene because of the diffuse nature of the stain. Expression in the epididymis, vas deferens, and seminal vesicle was specific to the pseudostratified columnar epithelial layers, but no staining of any other cell type in these tissues was observed (Fig. 3D)Citation . In the female reproductive tract, expression was observed in the ovary and oviduct after 16 h and in the uterus after 30 h in the X-gal stain (Table 1)Citation . Upon staining of the ovary and oviduct, highly regionalized expression was observed (Fig. 3, E–H)Citation . Halos of staining in the maturing follicles in the ovary were observed, and very little blue coloration was detected in any other part of this tissue (Fig. 3, E and F)Citation . Cross-section analysis showed that this expression was restricted to the granulosa cells of the stratified epithelium of the maturing follicles of the ovary (Fig. 3G)Citation . ß-gal staining was observed throughout the oviduct, although intensive staining was observed of the infundibulum, the funnel shaped opening at the end of the oviduct which is drawn out into fimbrae (Fig. 3, E, F, and HCitation ). Cross-section analysis showed that expression in the infundibulum was restricted to the ciliated columnar epithelial cell layer, and there was no expression in the smooth muscle or connective tissue (Fig. 3H)Citation .



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Fig. 3. Expression of the A-raf:ß-geo transgene in male and female reproductive tissues. Whole tissues (A, B, E, and F) and 15-µm sections (C, D, G, and H) were fixed, washed, and stained with X-gal for up to 16 h. Bar, 25 µm. A, male testis and epididymis derived from transgenic animal. B, male testis and epididymis derived from chimera (ES clone 33). C, longitudinal section of testis derived from chimera. L, Leydig cells; ST, seminiferous tubule. This section was counterstained with eosin. Expression of the transgene is clearly observed in the developing sperm of the seminiferous tubules and the interstitial cells of Leydig. No staining of the epithelial layer of the basement membrane was observed. D, longitudinal section of epididymis from a transgenic animal. Expression of the transgene is observed at a high level in the columnar epithelial cells of the convoluted tubules but in no other cell type. E, ovary and oviduct derived from transgenic line. O, ovary; Ov, oviduct; I, infundibulum. Staining of the maturing follicles in the ovary is observed. Intense staining is also observed in the infundibulum, adjacent to the ovary. F, ovary and oviduct derived from chimera (ES clone 20). G, cross-section of maturing follicle of ovary derived from transgenic animal. A high level of expression is observed in the granulosa cells of the maturing follicle. No staining of the rest of the ovary is observed. H, cross-section of infundibulum derived from transgenic animal. Staining of the ciliated epithelial cells is observed, but there is no staining of the smooth muscle or connective tissue layers.

 
ß-gal staining of the kidney and bladder of the transgenic line and chimeras was observed after 16 h in the stain (Table 1Citation and Fig. 4, A–DCitation ). Cross-section analysis showed that the transgene is expressed in the kidney cortex and renal pelvis but not in the medulla (Fig. 4C)Citation . In the kidney cortex, highest levels of expression were observed in the cuboidal epithelial cells of the proximal and distal convoluted tubules as well as in the glomerulus, but staining was not detected in the epithelium of the collecting tubules or loop of Henle (data not shown). In the renal pelvis, staining of the papillary ducts was observed (data not shown), and in the bladder, expression was specifically observed of the transitional epithelium but there was no expression in the smooth muscle (Fig. 4D)Citation .



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Fig. 4. Expression of A-raf in adult mouse tissues. Whole tissues (A and B) and sections (C–I) were fixed, washed, and stained with X-gal for up to 30 h. All sections were 15 µm thick, except for the kidney section in C, which was 35 µm thick. The cerebellum section in J was 10 µm thick and was subjected to in situ hybridization with an A-raf riboprobe. A, whole kidney from transgenic line. B, whole kidney from chimera (ES clone 33). C, longitudinal section of kidney from transgenic animal. ß-gal staining of the kidney cortex and renal pelvis is observed, but there was no staining of the medulla. D, longitudinal section of collapsed bladder from transgenic animal. Staining of the transitional epithelium is observed, but there is no staining of the smooth muscle. Bar, 40 µm. E, longitudinal section of heart from transgenic animal. Staining is observed in cardiac muscle throughout the myocardium of all four chambers. Bar, 25 µm. F, longitudinal section of colon from a transgenic animal. Expression of the transgene is observed in the inner and outer smooth muscle layers, but there is no staining of the submucosa or mucosa. Bar, 25 µm. G, longitudinal section of thymus from a transgenic animal. Staining of the thymocytes is observed. Bar, 40 µm. H, longitudinal section of spleen from transgenic animal. Staining of the red pulp, but not the white pulp, is observed. Bar, 40 µm. I, section of cerebellum from transgenic animal. Expression of the transgene was observed in Purkinje cells, but no staining of cells in the molecular or granular layers was detected. The section was counterstained with eosin. Bar, 100 µm. J, in situ hybridization of cerebellum section from MF-1 mouse with A-raf riboprobe. As with the transgene, expression of the endogenous A-raf gene is specifically observed in the Purkinje cells. Bar, 100 µm.

 
In the remaining adult tissues tested, low levels of expression of the transgene were observed, and in some cases, staining was only observed after 30 h in the stain (Table 1)Citation . Again, consistent patterns of expression were observed between the transgenic line and chimeras, with little background staining in wild-type animals (Table 1)Citation . In the heart, ubiquitous staining was observed throughout the musculature, reflecting expression of A-raf mRNA in the myocardium of all four chambers (Fig. 4E)Citation . Expression of the transgene was also observed throughout the muscularis externa of the small and large intestine. Staining was observed in the inner circular and outer longitudinal muscle layers but not in the myenteric plexus, the submucosa, or mucosa (Fig. 4F)Citation . In the thymus, staining of thymocytes located in the cortex of the thymus was observed (Fig. 4G)Citation , and in the spleen, expression was specifically observed of the red pulp, which contains the splenic cords (Fig. 4H)Citation . In the cerebellum, staining of the cell body of the Purkinje cells was clearly observed in the transgenic line, and there was no staining of the molecular or granular layers (Fig. 4I)Citation . However, no staining of the Purkinje cells was observed in the cerebellum of any of the chimeras (Table 1)Citation . To confirm whether the staining in the transgenic line reflects the true expression pattern of A-raf, in situ hybridization was performed on the cerebellum of wild-type animals using a digoxigenin antisense riboprobe specific for A-raf. The data obtained clearly complement the ß-gal staining data because expression of A-raf in the Purkinje cells was specifically observed (Fig. 4J)Citation . No ß-gal staining was observed in the lung or liver of the transgenic line or chimeras, and very little staining of the cerebral cortex was observed (Table 1)Citation .

Expression of the A-raf:ß-geo Transgene in Mouse Embryos.
To obtain transgenic embryos, females homozygous for the transgene were mated to wild-type MF-1 males or to males homozygous for the transgene. Matings were timed such that embryos were obtained at various stages of gestation from 7.5–11.5 days p.c. Embryos were dissected from yolk sacs, fixed, and stained in situ with X-gal (see "Materials and Methods"). At 7.5 days p.c., staining was observed throughout the epiblast and at lower levels in the extraembryonic tissues and ectoplacental cone (Fig. 5A)Citation . At 10.5 days p.c., after 30 h in the X-gal stain, ß-gal expression was observed throughout the embryo (Fig. 5B)Citation , indicating that, as in adult tissue, the transgene is ubiquitously expressed. This same staining pattern was also obtained with embryos at 9.5 days p.c. and 11.5 days p.c. The abdominal region consistently appeared to be most intensively stained in these embryos. To observe this in more detail, we incubated embryos in the X-gal stain for a shorter period. After 16 h, the embryonic heart was the only structure visibly stained in the 11.5 days p.c. embryo (Fig. 5C)Citation . Sagittal sections of a 11.5-day p.c. embryo were prepared and stained with X-gal for 16 h (Fig. 5D)Citation . After this time, specific staining of the walls of each chamber of the heart was observed as well as the sinus venosus, which ultimately becomes part of the right atrium and coronary sinus. No staining of any other embryonic tissue was observed.



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Fig. 5. Expression of the A-raf:ß-geo transgene in embryonic development. Embryos were prepared and stained as described in "Materials and Methods." A, 7.5-day p.c. embryo stained for 24 h. Expression is observed primarily in the epiblast and at a lower level in extraembryonic tissues. B, 10.5-day p.c. embryo stained for 30 h. ß-gal staining is observed throughout the embryo, with highest levels of staining in the abdominal region. C, 11.5-day p.c. embryo stained for 16 h. H, heart. After developing the stain for a shorter length of time, ß-gal staining is specifically observed in the embryonic heart. Very little staining is observed in other embryonic tissues. D, sagittal section of heart region of 11.5-day p.c. embryo. SV, sinus venosus. After developing the stain for 16 h, expression of the transgene was observed in the walls of the four chambers of the heart as well as the sinus venosus. No staining of any other embryonic tissue is observed. Bar, 100 µm.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Previous studies of the expression pattern of the A-raf gene by Northern blot analysis has indicated that mRNA expression of this gene is highest in tissues comprising the urogenital tract (39) . Our Western blot analysis is entirely consistent with this view, suggesting that there is little, if any, regulation of this gene at the posttranscriptional level (Fig. 1)Citation . We detected highest levels of p69A-raf in components of the male reproductive tract such as the epididymis and seminal vesicle. High levels of p69A-raf were also detected in testis, ovary, uterus, bladder, and kidney. Apart from urogenital tissues, a number of nonurogenital tissues express significant levels of this protein, including liver, heart, intestine, spleen, thymus, and cerebellum. As with the Northern data, both cerebral cortex and skeletal muscle express little if any of the protein.

The high urogenital expression of A-raf has led to the view that this gene is steroid hormone inducible (39 , 43 , 44) . Analysis of the A-raf promoter supports this observation because sequencing of the minimal promoter region between -59 and +93 has revealed a region low in G+C content without CAAT and TATA consensus sequences but with two GRE and one thyroid hormone response element. A third GRE and two more thyroid hormone response elements are located further upstream (43) . Lee et al. (44) have further shown that a vector containing 93 bp of exon 1 and 1.4 kb of DNA upstream of the A-raf gene subcloned upstream of a promoterless reporter construct can be transactivated by dexamethasone in HeLa cells cotransfected with a vector expressing a wild-type glucocorticoid receptor. The glucocorticoid receptor also forms multiple complexes with the A-raf promoter, and the GREs appear to be important for these interactions (44) . The expression pattern of the A-raf reporter transgene described in this report mimics the Northern and Western blot data (Figs. 3Citation and 4)Citation . This suggests that regulatory elements contained within the 5.1-kb SacI genomic fragment used in the A-raf transgene are sufficient to define the tissue specificity of A-raf mRNA and protein and that there are probably no other regulatory elements within the body of the A-raf gene or 3' to it.

Analysis of tissues from the transgenic line and chimeras from four different ES clones shows that the expression pattern of the A-raf:ß-geo construct is highly consistent between them all, indicating that there is no positional-effect variation because of the site of insertion of the transgene in the different ES cell lines. Absence of detectable expression of the transgene in the cerebellum of all the chimeras may be explained by a low level contribution of ES cells to this tissue in chimeric animals. One inconsistency with our data is that the A-Raf protein is expressed in liver and lung (Fig. 1)Citation , but no expression of the transgene in the liver or lung of the transgenic line or chimeras was detected (Table 1)Citation . The reason for this is not clear, but it may suggest that the regulatory elements required for expression in these two tissues are not present in the transgene. Our expression data also suggest that there is a switch in the pattern of expression of A-raf during development from a high level of expression in the embryonic heart to a lower level of expression in the adult heart and high levels of expression in the adult urogenital system. Further expression analysis is required to fully understand the mechanism behind and reasons for this developmental switch.

Despite the steroid hormone-inducible nature of the A-raf gene in adult tissues, its function in steroid hormone-responsive tissue is not clear. Studies of A-Raf-deficient mice have so far revealed no major defect in male or female reproduction or in the urinary system (36) , although minor defects may still exist. This is most likely to have arisen because the major function of A-Raf in the urogenital tract in these mice is compensated by other Raf kinases or MEK kinases expressed in these tissues. Both Raf-1 and B-Raf are ubiquitously expressed (39 , 40) , and therefore, it is possible that one or the other can overcome the absence of A-Raf. Instead, the A-Raf-deficient mice demonstrate progressive wasting, despite normal feeding, suggesting that they either have a metabolic defect or that they do not absorb sufficient nutrients (36) . They also have motor coordination defects, suggesting neuronal dysfunction (36) . These observations have prompted this more detailed analysis of the expression pattern of A-raf.

We have discovered that, despite its expression in all tissues, A-raf expression is highly cell type specific. It is expressed in a disparate range of different cell types derived from all embryonic lineages. However, it is expressed at highest levels in the epithelia of the male and female reproductive tracts, which are of mesodermal origin. A-raf appears to be expressed at particularly high levels in simple or pseudostratified columnar epithelial cells as found in the fimbrae of the infundibulum, the maturing ovarian follicles, the epididymis, vas deferens, and seminal vesicles, cells that are often ciliated. The ciliated cells in the fimbrae of the infundibulum are vital for capturing and conducting oocytes toward the uterus, and in the epididymis, the ciliated cells are involved in maturation and transport of spermatazoa. The high level of expression of A-raf in these cells suggests that it may be important for maintenance of ciliary action.

The expression pattern of A-raf in nonurogenital tissue as revealed in this report could be more relevant to the A-Raf-deficient phenotype. The expression of A-raf in the Purkinje cells of the cerebellum is intriguing in light of the movement difficulties of the A-Raf-deficient mice. Similarly, the expression of A-Raf in the smooth muscle layer of the intestine may be related to the wasting phenotype of these mice. Expression of A-Raf in the musculature of the embryonic and adult heart is particularly interesting given previous observations on the involvement of A-Raf in cardiac myocyte hypertrophy. The Raf/MEK/ERK cascade is vital for mediating the transcriptional changes associated with hypertrophy, and A-Raf may play a unique role in this process. Both Raf-1 and A-Raf are activated in response to powerful hypertrophic agonists (27 , 28) . However, A-Raf appears to be an important mediator of the ERK cascade when cAMP levels are elevated because Raf-1 activation is reduced considerably in the presence of cAMP, whereas A-Raf activation is less affected (27) . No histological abnormalities have been observed in the cerebellum, intestine, or heart of the A-Raf-deficient mice despite intensive investigations (36) . However, physiological studies of these organs in the A-Raf-deficient mice is now warranted on the basis of the results presented here.

Perhaps the only common characteristic between the cell types expressing A-Raf is that they appear to be those requiring a high level of metabolic activity. Ciliated epithelial cells require a high level of metabolism to maintain ciliary action. In the kidney, A-raf is expressed at high levels in the epithelial cells of the proximal and distal convoluted tubules of the kidney cortex, which are involved in the active transport of salts (Fig. 4C)Citation . A-raf is not expressed in the ascending and descending limbs or in the loop of Henle in the medulla, which are involved in the passive diffusion of water, urea, and salt (Fig. 4C)Citation . In addition, in the 9.5–11.5-day p.c. embryo, highest levels of expression by far are observed in the embryonic heart. The significance of this novel finding is not clear, but various lines of evidence indicate that it may be related to the metabolism of the embryo at various stages of development. Studies of embryos explanted and cultured in vitro have shown that glycolysis is the main energy source in the 8.5–11.5-day p.c. embryos, whereas in the later embryo, there is decreased glycolysis and increased activity of the Krebs cycle (46) . Cox and Gunberg (47) have shown that glycolysis is required as the main energy source to maintain contractions of the early rat embryo heart, but at later stages of embryogenesis, when the blood circulation is more established, the embryonic heart shifts in dependence to the higher energy yields of the Krebs cycle and oxidative phosphorylation (47) .

The high level of expression of A-raf observed in these metabolically active cells may be a reflection of the involvement of A-Raf in the regulation of glycolysis through its association with M2-PK. A-Raf, but not Raf-1 or B-Raf, regulates the activity of this glycolytic enzyme by converting it from its dimeric form to the more active tetrameric form (35) .5 Conceivably, the wasting phenotype of the A-Raf-deficient mice may be related to abnormal regulation of M2-PK. This isoform of PK is expressed at high levels in tumor cells, and transformed cells exhibit increased rates of M2-PK activity (35 , 48) . In addition, A-Raf and M2-PK cooperate in cell transformation after coexpression in NIH3T3 cells (35) .5 These data suggest that A-Raf may play a critical role in the regulation of metabolism through M2-PK, which may be important for ensuring energy production in tumor cells under hypoxic conditions. Further studies of the expression pattern of A-Raf in relation to M2-PK are now critical for examining the role of the interaction between these two proteins in the development of tumors as well as for further unraveling the pathophysiological basis of the A-Raf-deficient phenotype.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Western Blot Analysis.
Freshly isolated mouse tissues were homogenized in Gold lysis buffer, and Triton X-100 soluble lysates were obtained by two centrifugation steps of the homogenates at 13,000 rpm for 15 min at 4°C (13 , 30) . Protein concentrations were measured with the bicinchoninic acid protein assay kit (Pierce), and equal amounts of cell lysates were electrophoresed through 6% polyacrylamide gels. After transfer to 0.45 µm nitrocellulose filters (Schleicher and Schuell), Western blots were stained with Ponceau S stain (0.5% w/v made in 5% w/v trichloroacetic acid), and protein loadings were compared. The blots were then incubated with a 1:1000 dilution of anti-p69A-raf polyclonal antiserum (Upstate Biotechnology, Inc.) for at least 1 h at room temperature. Although this antibody detects a number of other proteins, it has been shown previously to detect an A-Raf-specific band at Mr 69,000 (36) . The filters were washed in Tris-buffered saline (TBS) containing 0.05% (v/v) Tween 20 and incubated with 1:5000 dilutions of an anti-rabbit secondary antibody coupled to horseradish peroxidase (Sigma) for 1 h at room temperature. After washing with TBS/Tween, the enhanced chemiluminescence detection system (Pierce) was used to visualize antigen-antibody complexes. Autoradiographs were scanned to assess the relative abundance of p69A-raf in each tissue.

Generation of Transgenic Mice.
The A-raf promoter and transcription initiation site have been identified previously by sequencing and by performing assays for promoter activity in transient transfection assays (43) . These studies located minimal promoter activity between nucleotides -59 and +93. We located the promoter region to the 5.1-kb SacI fragment (Fig. 2)Citation by hybridization of an oligonucleotide within the minimal promoter region to mouse A-raf lambda phage. The SacI fragment was then ligated in-frame to the ß-geo gene isolated from the plasmid pß-geobpA (45) , which consists of a 3.1-kb region encoding the lacZ gene fused to the 1.0-kb neoR gene and a 0.2-kb polyadenylation signal. In the final construct, the ß-geo reporter gene was expressed from the A-raf promoter as a fusion with the first 84 amino acids of the A-Raf protein. ES cells were grown, and transgenic mice were generated by the methods described by Torres and Kühn (49) . The A-raf:ß-geo construct was linearized with NotI and SnaBI, and the plasmid insert was purified from its backbone vector by agarose gel electrophoresis. One µg of insert DNA was electroporated into 1 x 107 E14.1 ES cells, and transfected colonies were selected with G418. The ES cells were cultured on a feeder layer of mitotically inactive embryonic fibroblasts. After 7 days growth under drug selection, 50 colonies were picked, trypsinized, and transferred to feeder layers on 48-well plates. A portion was propagated for subsequent analysis of genomic DNA and for ß-gal expression assays using the kit provided by Stratagene. Colonies 20, 33, 42, 49, and 62 were microinjected into blastocysts derived from the F1 offspring of C57BL/6 x CBA strain intercrosses. Several chimeras were obtained from all colonies. However, only chimeras from clone 62 transmitted the transgene through the germ line, and from these a transgenic line was established by breeding to the MF-1 mouse strain.

X-gal Staining of Adult Tissues and Embryos.
Tissues were collected from adult transgenic and wild-type animals, washed in PBS containing 0.01% (v/v) FCS, and fixed in 4% (w/v) paraformaldehyde/0.2% (v/v) gluteraldehyde in phosphate buffer [0.1 M sodium phosphate (pH 7.3), 2 mM magnesium chloride, and 5 mM ethylene glycol tetraacetic acid] at room temperature for 20 min. They were then washed twice in wash buffer [phosphate buffer containing 0.01% (w/v) sodium deoxycholate and 0.02% (w/v) Nonidet P40]. Embryos were collected from transgenic females at various stages after gestation and treated in the same way as the adult tissues, except that fixation was allowed to proceed for 10–15 min. For sectioning, tissues and embryos were embedded in Tissue Tek-OCT compound (Sakura) in cryogenic moulds and solidified on a dry ice-ethanol bath. Cryostat sections at 15–35-µm thickness were thaw mounted onto microscope slides pretreated with silane. Sections were fixed in 4% (w/v) paraformaldehyde in phosphate buffer at room temperature for 10 min and then washed twice in wash buffer. Whole tissues and sections were stained in reaction buffer made fresh each time, consisting of wash buffer with 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, and 1 mg/ml X-gal for 2–24 h at 37°C. For the embryos and whole tissues, reactions were stopped by washing twice in wash buffer, and they were then stored in PBS. Sections were washed twice in wash buffer and mounted in glycerol-gelatin (Sigma). Prior to mounting, some of the sections were counterstained with eosin. Stained tissues and sections were photographed under a Nikon SMZ-U dissection microscope or a Nikon TMS-F compound microscope.

In Situ Hybridization.
A fragment of mouse A-Raf cDNA spanning amino acid residues 1–84 was subcloned into pBluescript SKII+, and digoxigenin-labeled antisense and sense riboprobes were prepared by T7 and T3 in vitro transcription reactions using standard procedures. For in situ hybridization, the method described by Braissant et al. (50) was followed. Ten-µm tissue sections were thaw mounted onto silane-coated microscope slides and fixed in 4% (w/v) paraformaldehyde made in DEPC.PBS for 10 min and then rinsed twice in DEPC.PBS. Sections were then treated twice with active 0.1% (v/v) DEPC.H2O for 15 min, equilibrated with 5x SSC for 15 min, and prehybridized for 2 h at 58°C in hybridization mix consisting of 50% formamide, 5x SSC, and 40 µg/ml salmon sperm DNA. They were then hybridized overnight with 20 µl of hybridization mix containing 400 ng/ml probe in a hybridization chamber containing 5x SSC and 50% formamide secured under a piece of Nescofilm. The next day, slides were washed for 30 min in 2x SSC at room temperature, twice for 1 h in 0.1x SSC at 65°C, then equilibrated for 5 min in buffer 1 [100 mM Tris (pH 7.5), 150 mM NaCl]. Sections were incubated with the anti-digoxigenin antibody (Boehringer-Mannheim) diluted 1:10,000 in buffer 1 containing 0.5% (w/v) blocking reagent (Boehringer-Mannheim). Excess antibody was removed by washing three times in buffer 1, and the sections were equilibrated in buffer 2 [100 mM Tris-HCl (pH 9.5), 100 mM NaCl, and 50 mM MgCl2] for 10 min. Color was developed overnight at 37°C in buffer 2 containing 75 mg/ml 4-nitro blue tetrazolium chloride and 50 mg/ml 5-bromo-4-chloro-3-inolyl-phosphate. The reactions were stopped in 10 mM Tris-HCl, EDTA (pH 8.0), and the slides were mounted in glycerol-gelatin aqueous mountant. Stained sections were photographed under a Nikon TMS-F compound microscope.


    Acknowledgments
 
We thank Felix Beck, Joe Tucci, David Critchley, and Sue Monkley for support, advice, and comments on the manuscript. We are also grateful to Sue Figgitt for preparing ES cells for injection, Steve Sheardown for providing the ß-geo plasmid, and Susan Giblett for help with the tissue sections. We are indebted to members of the Division of Biomedical Services at Leicester for their support.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the award of a Royal Society fellowship (to C. A. P.) and a BBSRC studentship (to J. C. A. L.). This study was completed in partial fulfillment for a PhD (J. C. A. L.). Back

2 Present address: Foundation of Research and Technology, Institute of Molecular Biology and Biotechnology, P. O. Box 1527, Vassilika Vouton, GR 71110 Heraclion, Crete, Greece. Back

3 To whom requests for reprints should be addressed, at Department of Biochemistry, University of Leicester, University Road, Leicester LE1 7RH, United Kingdom. Phone: 44-0-116-252-3452; Fax: 44-0-116-252-5097; E-mail: cap8{at}le.ac.uk Back

4 The abbreviations used are: MEK, mitogen/ERK kinase; ERK, extracellular signal-regulated kinase; ß-gal, ß-galactosidase; p.c., post-coitum; M2-PK, pyruvate kinase isoform M2; GRE, glucocorticoid response element; ES, embryonic stem; DEPC, diethyl pyrocarbonate; X-gal, 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside; ET-1, endothelin-1; cAMP, cyclic AMP. Back

5 U. Rapp, University of Wuerzburg, Germany, personal communication. Back

6 C. A. Pritchard, unpublished results. Back

Received for publication 6/30/99. Revision received 2/11/00. Accepted for publication 2/14/00.


    References
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 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
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