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Molecular and Cellular Biology, August 2000, p. 6127-6137, Vol. 20, No. 16
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Activin beta C and beta E Genes Are Not Essential for Mouse Liver Growth, Differentiation, and Regeneration

Anthony L. Lau,1,2 T. Rajendra Kumar,1,3 Katsuhiko Nishimori,1,dagger Jeffrey Bonadio,4,Dagger and Martin M. Matzuk1,2,3,5,*

Departments of Pathology,1 Molecular and Cellular Biology,3 and Molecular and Human Genetics5 and Program in Developmental Biology,2 Baylor College of Medicine, Houston, Texas 77030, and Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 481094

Received 13 March 2000/Returned for modification 19 April 2000/Accepted 16 May 2000


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The liver is an essential organ that produces several serum proteins, stores vital nutrients, and detoxifies many carcinogenic and xenobiotic compounds. Various growth factors positively regulate liver growth, but only a few negative regulators are known. Among the latter are the transforming growth factor beta  (TGF-beta ) superfamily members TGF-beta 1 and activin A. To study the function of novel activin family members, we have cloned and generated mice deficient in the activin beta C and beta E genes. Expression analyses demonstrated that these novel genes are liver specific in adult mice. Here, we show by RNase protection that activin beta C transcripts are present in the liver beginning at embryonic day 11.5 (E11.5) whereas activin beta E expression is detected starting from E17.5. Gene targeting in embryonic stem cells was used to generate mice with null mutations in either the individual activin beta C and beta E genes or both genes. In contrast to the structurally related activin beta A and beta B subunits, which are necessary for embryonic development and pituitary follicle-stimulating hormone homeostasis, mice deficient in activin beta C and beta E were viable, survived to adulthood, and demonstrated no reproductive abnormalities. Although activin beta C and beta E mRNAs are abundantly expressed in the liver of wild-type mice, the single and double mutants did not show any defects in liver development and function. Furthermore, in the homozygous mutant mice, liver regeneration after >70% partial hepatectomy was comparable to that in wild-type mice. Our results suggest that activin beta C and beta E are not essential for either embryonic development or liver function.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Growth factors and hormones play an extremely important role in regulating biological processes from patterning of the early embryo to regulating the function of tissues and organs. The largest family of growth factors is the transforming growth factor beta  (TGF-beta ) superfamily of secreted dimeric proteins (12). Members of this family include activins, TGF-beta s, bone morphogenetic proteins (BMPs), and growth differentiation factors and demonstrate diverse functions including roles in left-right asymmetry, skeletal development, reproduction, and oncogenesis (29, 52). Both activins (beta -beta dimers) and inhibins (alpha -beta dimers) have historically been shown to be regulators of follicle-stimulating hormone (FSH) secretion from the pituitary gland (54). These earlier results were later confirmed by in vivo analysis of activin beta B-, activin receptor type IIA (ActRIIA)-, and alpha -inhibin-deficient mice (32-34). In studies involving Xenopus laevis oocytes (36), activins were tested for their ability to induce mesoderm formation. However, this observation is not true for mice (35), suggesting that the results obtained with X. laevis oocyte injection experiments may be due to nonphysiological effects of the activin ligands.

The adult liver detoxifies the blood through the actions of various enzymes, synthesizes normal serum proteins such as the acute-phase proteins and albumin, and produces bile, which is critical for normal fat absorption (9). Activin A and TGF-beta 1 have been shown to affect liver growth and function. These two proteins can inhibit mitogen-induced DNA synthesis in hepatocytes (5, 41, 51, 57), induce hepatocellular apoptosis in vitro (7, 21, 42, 48), and stimulate glycogenolysis from cultured hepatocytes (39). In vivo, pharmacological levels (e.g., intravenous infusion of recombinant activin A [21, 48]) or pathophysiologically high levels of activins (8, 30, 32) cause a reduction in liver mass by inducing hepatocellular necrosis around the central vein.

Liver regeneration occurs following liver injury that results in loss of liver mass. The liver regenerates by a process of hypertrophy and a near-synchronous proliferation of the remaining cells through several cycles of replication. Several cytokines are thought to play early roles in the regeneration process: interleukin-1 (IL-1), IL-6, and tumor necrosis factor alpha (reviewed in reference 38). IL-6 has been shown to play a critical role in the progression of liver regeneration but not for initiation (10). While the interleukins and tumor necrosis factor alpha are thought to be important during the early stages of liver regeneration, TGF-beta 1 to -3 and activins are thought to be negative regulators during this process (27, 38), since mRNAs encoding these ligands are up-regulated during this process (3, 57). However, this latter hypothesis has not yet been tested in vivo.

Recently, our groups (15, 28) and others (19, 43, 47) have cloned three new members of the TGF-beta superfamily which demonstrate highest amino acid identity in the mature peptide region to the activin (beta A and beta B) subfamily. Two of these new members, designated activin beta C (actbeta C) and activin beta E (actbeta E), have been cloned in mammals (15, 19, 28, 47), while the third, activin beta D, has been found only in X. laevis (43). Weak induction of a secondary axis has been observed when activin beta D mRNA is injected into the ventral blastomeres of Xenopus embryos (43), but activin beta C and beta E have not yet been functionally tested by any in vitro or in vivo bioassays. Expression of actbeta C and actbeta E is primarily liver specific in the adult (14, 28, 47), unlike activin beta A and activin beta B, which are widely expressed in multiple tissues in rodents (16, 37) and humans (53).

Based on the highly restricted tissue expression pattern, we hypothesized that activins beta C and beta E may play critical roles in liver physiology. To compare the in vivo functions of these novel liver-restricted mammalian activin beta C and beta E genes to those of the known activin beta A and beta B genes, we generated null mutations in actbeta C, actbeta E, or both genes in mice. Our studies show that activin beta C and beta E are not essential for liver development, liver function, or reproduction.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Construction of targeting vectors and generation of mutant mice. The replacement targeting vector for actbeta C contained 5.7 kb of sequence for the 5' homology arm, a PGKhprt selectable marker cassette which replaced a 1.7-kb BamHI-BamHI region of the locus including exon 2 of the activin beta C gene, 4.5 kb of sequence for the 3' homology arm, and an MC1tk expression cassette for negative selection (Fig. 1B). Twenty-five micrograms of the KpnI-linearized targeting vector was electroporated into 107 AB2.1 embryonic stem (ES) cells (a gift of Allan Bradley, Baylor College of Medicine). ES cells were then selected in medium containing hypoxanthine-aminopterin-thymidine and 1-(2'-deoxy-2'-fluoro-beta -D-arabinofuranosyl)-5'-iodouracil (FIAU). Culturing of ES cells and collection and injection of blastocysts have been previously described (33). For genomic Southern blot analysis, HindIII-digested DNA was transferred to a GeneScreen Plus nylon membrane (NEN Life Science Products, Boston, Mass.) and probed with an external ~300-bp NcoI-XbaI fragment (5' probe). An external ~380-bp StuI-BamHI fragment (3' probe) was also used to distinguish the wild-type and activin beta C null (actbeta Cm1) alleles (Fig. 1B).

The actbeta E targeting vector was composed of a 4.85-kb EcoRI-BamHI fragment as the 5' homology arm, a loxP-PGKneo-loxP selectable marker cassette (a gift from Richard Behringer, University of Texas-M. D. Anderson Cancer Center) that replaced a 1.7-kb BamHI-BamHI region of the locus including the entire coding region of activin beta E, a 1.8-kb BamHI-SpeI fragment as the 3' homology arm, and an MC1tk selectable marker cassette. This vector was linearized with KpnI, and 25 µg of the linearized plasmid was electroporated into 107 beta C45-F1 cells, an AB2.1 ES cell line heterozygous for the actbeta Cm1 allele, which was generated as described above. The mutant ES cells were selected in G418 and FIAU. HindIII, which distinguishes the various targeted and wild-type alleles, was used as the diagnostic restriction enzyme. The probes were a 5' external ~250-bp BamHI-EcoRI fragment and an internal ~500-bp BamHI-XhoI fragment (Fig. 1D).

Chimeras were generated by blastocyst injections of the mutant ES cells, per standard methods (33). Chimeric males were mated to C57BL/6 females for the generation of F1 hybrid mice or to 129S5/SvEvBrd female mice to generate F1 inbred mice.

RNA isolation. Timed matings of wild-type C57BL/6 mice were established, and embryonic livers were collected at various time points for RNA expression analysis. RNA collected at embryonic day 11.5 (E11.5) was liver enriched by using only trunk tissue (i.e., rostral and caudal tissues were excluded). Adult tissues from wild-type 129/SvEv-C57BL/6 hybrid mice were also collected. When necessary, tissues from several mice were pooled. Tissues were immediately homogenized in RNA STAT-60 (Leedo Medical Laboratories, Houston, Tex.) using a Tissue Tearor electronic homogenizer (Biospec Products, Bartlesville, Okla.). RNA was extracted per Leedo Medical Laboratories' instructions and stored under 70% ethanol at -80°C until required. Some samples were stored in RNA Later (Ambion, Austin, Tex.) until required for processing in accordance with the manufacturer's instructions.

RNase protection assay. Probe plasmids were obtained to analyze the RNA expression levels of actbeta C, actbeta E, beta -actin (Ambion), and junB (Ambion) and beta 2-microglobulin levels using RNase protection assays. beta 2-Microglobulin was used as a quantitative control since its expression is constant during liver regeneration (17). The plasmids containing probe DNA fragments generated in the laboratory were sequenced bidirectionally for accuracy. The mouse activin beta C probe was composed of a BamHI-EcoRI fragment from the 3' untranslated region (+1184 to +1434 of the cDNA; GenBank accession no. U40773) cloned from a 129/SvEv genomic library as previously described (28). The mouse activin beta E probe fragment was derived from exon 2 (+750 to +972 of the cDNA; GenBank accession no. U96386) and was generated using PCR primers actbeta E-F2 (5'-GAGACCACTATGTAGACTTCC) and actbeta E-R4 (5'-AGAGAGAGGCCTTCGTGCAGT). Two different mouse beta 2-microglobulin probes were used. For embryonic studies, the beta 2-microglobulin probe beta 2-MG3 (+1 to +317 of the cDNA) was derived from pHuActbeta 2m (a gift from Elizabeth Bikoff, Harvard University [2]). For all other experiments, a second beta 2-microglobulin probe (beta 2-MG5, +114 to +306 of the cDNA; GenBank accession no. X01838) was isolated by reverse transcription-PCR using forward primer beta 2-MGF1/H3 (5'-ACTGCAAGCTTAACACAG-3'), which inserts a HindIII site at the SnaBI site, and the reverse primer beta 2-MGR1 (5'-TAACTCTGCAGGCGTATG-3').

Linearized probe plasmids were transcribed in vitro to produce antisense RNA using the Ribomax in vitro transcription kit (Promega, Madison, Wis.). Since some messages are more abundant than others, cold UTP was added to all reactions to limit the protected band intensities to approximately the same range. The amount of cold UTP added was as follows: actbeta C, actbeta E, and junB, 0.1 nmol of cold UTP; beta -actin and beta 2-microglobulin, 2.5 nmol of cold UTP. The [alpha -32P]UTP-radiolabeled RNA probes were purified by gel electrophoresis and eluted at 37°C overnight in elution buffer as described by the manufacturer (RPAII kit; Ambion).

The RPAII RNase protection assay kit (RPAII kit) was used as described by the manufacturer with minor modifications. In brief, 2.5 µl of total RNA (2 µg/µl) and 3 µl of probe mix were added to the hybridization buffer. The mixture was denatured at 95°C for 3 min. The reaction mixtures were then incubated overnight at 45°C followed by digestion with a 1:100 dilution of the RNase cocktail, containing RNase A and RNase T1, at 37°C for 1 h. The protected RNA-RNA hybrids were precipitated and separated on a nondenaturing 5% acrylamide gel except for the junB probe, which was separated on an 8% acrylamide gel. Gels were vacuum dried at 80°C, exposed, and visualized by autoradiography.

Following autoradiography, dried gels were also exposed to a Storage Phosphor Screen (Molecular Dynamics, Sunnyvale, Calif.). Exposed screens were then read with a Storm 860 scanner (Molecular Dynamics), and resulting images were analyzed by ImageQuaNT version 4.2a software (Molecular Dynamics). Data, following integration, were analyzed by Student's t test using Microsoft Excel98 (Microsoft Corp., Redmond, Wash.) for statistical significance. A P value of <0.05 was considered significant for all statistical analyses. For RNase protection assays of RNA collected after partial hepatectomy, a minimum of three mice were used at each time point, and each sample was analyzed in duplicate.

Serum analysis. Adult 129/SvEv-C57BL/6 hybrid mice at 42 to 45 days were anesthetized using Metofane (Schering-Plough Animal Health, Union, N.J.), and blood was obtained by closed cardiac puncture. Serum was separated in Microtainer tubes (Becton Dickinson, Franklin Lakes, N.J.) and stored at -20°C. Serum samples were analyzed for alanine aminotransferase, aspartate aminotransferase, albumin, random glucose, and total protein by the Comparative Pathology Laboratory (Baylor College of Medicine) on a Roche Cobas MIRA analysis machine. Serum FSH levels were measured by a rat FSH radioimmunoassay with a sensitivity of 10 ng/ml using a National Institute of Diabetes and Digestive and Kidney Diseases kit (National Hormone and Pituitary Distribution Program, National Institute of Diabetes and Digestive and Kidney Disease, National Institutes of Health) as previously described (26). Sera from five to six male mice of each genotype were analyzed for FSH levels.

Morphological and histological analysis. Mice were anesthetized and sacrificed by cervical dislocation, and tissues were harvested immediately. The wet weights of relevant tissues from mice 42 to 45 days of age were recorded. For testis weights, 11 to 37 mice were analyzed per genotype. All tissues except for testis were fixed overnight at 4°C in 10% buffered formalin (pH 7.2). The testis samples were fixed overnight in Bouin's reagent and washed extensively in 70% ethanol. In some cases, prostate glands were also collected from >1-year-old mice and placed into formalin. The fixed tissue was then embedded in paraffin and sectioned at 5 µm for staining in hematoxylin and eosin for the liver. The ovaries and testes were stained with hematoxylin-periodic acid-Schiff reagent. Embedding and staining were performed per standard procedure by the Baylor College of Medicine Pathology Core Services Laboratory.

Partial hepatectomy. The partial hepatectomy procedure was performed as described previously with minor modifications (18). Adult 129 × Sv/Ev-C57BL/6 hybrid female mice, 9 to 12 weeks of age and weighing 18 to 22 g, were included in the experiment. IsoFlo (isoflurane; Abbott Laboratories, North Chicago, Ill.) was used as the anesthetic. The partial hepatectomy procedure removes the left medial, right medial, and left lateral lobes. The right half of the medial lobe was removed with one ligature. Both the left half of the medial lobe and the left lateral lobe were removed with a second ligature. During the procedure, care was taken to prevent damage to the gall bladder and the surrounding ducts. Only mice that had >70% of the liver removed were used in our analyses.


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Expression of two novel activins in the embryonic liver. The cloning and tissue expression patterns of the actbeta C and actbeta E genes in adult mice have been previously reported by us and others (14, 28, 47). Since both genes appear to be primarily liver specific, we analyzed the temporal expression profiles of actbeta C and actbeta E in the embryonic liver using RNase protection (Fig. 1A). Expression of actbeta C was readily detectable by E11.5 and reached a maximum in the adult at 9 weeks. In contrast, actbeta E was first detectable at E17.5 and appeared to be maximally expressed at birth (Fig. 1A).


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  		FIG. 1.   Expression of activin beta C and activin beta E in the livers of embryos and adult mice and gene targeting constructs for activins beta C and beta E. (A) Expression of actbeta C and actbeta E in mouse embryonic and adult livers was detected using RNase protection assays. beta 2-Microglobulin was used as the internal control. Five micrograms of total RNA from C57BL/6 mice was hybridized to probes specific for activin beta C, activin beta E, and beta 2-microglobulin. Shown is a representative autoradiograph repeated in four independent experiments. (B) The targeting strategy used to delete exon 2 of the mouse activin beta C gene. The construct contains an MC1tk expression cassette for negative selection and a PGKhprt expression cassette for positive selection. Homologous recombination will delete all of the coding sequence in exon 2. (C) Southern blot analysis of tail DNA from F2 mice at weaning. The 3' probe identifies the wild-type 13.8-kb band and the 8.3-kb mutant band. (D) The targeting construct to delete all of the coding exons of the actbeta E gene. Homologous recombination within the homology arms will replace the entire coding region of activin beta E with a floxed (vertical-box-flanked) PGKneo-positive selectable marker cassette. The MC1tk cassette is used for negative selection. Due to the close physical proximity of activin beta C and beta E loci on the same chromosome, ES cells carrying the actbeta Cm1 mutation (cell line beta C45-F1, which was used previously for germ line transmission of actbeta Cm1) were electroporated. (E) Southern blot analysis showing weaned F2 mice homozygous for the actbeta Em1 mutation (19.2-kb band) or the double mutant allele (actbeta Cm1-actbeta Em1; 13.8-kb band). The wild-type band migrates at 5.3 kb. H3, HindIII; B, BamHI; Xba, XbaI; RI, EcoRI; WT, wild type.

Targeting of the actbeta C and actbeta E genes. To determine the function of activin beta C and activin beta E, we generated ES cell lines containing single mutations in each of the two genes and a linked (double) mutation in both genes. A serial targeting strategy was employed since the actbeta C and actbeta E genes are separated by 5.5 kb on mouse chromosome 10 (14, 47). Initial targeting deleted the entire coding region of actbeta C exon 2, which encodes the mature (active) peptide domain (actbeta Cm1), to produce a putative null allele (Fig. 1B and C). For the second targeting event, in which a putative null mutation in activin beta E (actbeta Em1) was generated, we used ES cells carrying the actbeta Cm1 allele instead of wild-type ES cells (Fig. 1D and E). Targeting of the actbeta Cm1 heterozygous cell line with the activin beta E targeting vector would generate two sets of chromosomes, a cis set and a trans set, with respect to the actbeta Cm1 and actbeta Em1 alleles. If targeting of the actbeta E locus occurs on the same chromosome as the previously targeted actbeta C locus, then a cis set of chromosomes will be generated (actbeta Cm1, actbeta Em1/+, +), eventually allowing us to generate double-homozygous mutant mice. Conversely, if targeting of the actbeta E locus occurred on the wild-type chromosome (trans; actbeta Cm1, +/+, actbeta Em1), then the two mutations segregated and permitted us to generate homozygous mice carrying only the actbeta Em1 allele. The actbeta Em1 allele is also predicted to be a null allele since both coding exons have been replaced (Fig. 1D). Utilizing the above strategy, these constructs were electroporated into ES cells, and subsequently, chimeric mice were generated. Germ line transmission of two cell lines for each mutation (actbeta Cm1, actbeta Em1, and the double mutant actbeta Cm1-actbeta Em1) was achieved (Fig. 1C and D).

General phenotypes. Heterozygous mice were intercrossed, and all lines were bred separately. The offspring from the heterozygous crosses were genotyped at 3 weeks of age, and all lines genotyped showed the expected Mendelian ratio of 1:2:1 (Table 1). Therefore, activin beta C and/or activin beta E is not required during embryogenesis. At 42 days, homozygous mutant mice from all three mutations were grossly indistinguishable from their control littermates, indicating that growth and development were unaffected by the individual or combined null mutations.

                              
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  		TABLE 1.   Genotype distribution from crosses of various mutant mice

Since actbeta C and actbeta E are closely linked on the same chromosome, the integration of a selectable marker cassette into one locus could have effects on the transcription of the other locus. We examined the expression of both genes in the actbeta Cm1/actbeta Cm1, actbeta Em1/actbeta Em1, and double-homozygous mutant mice. The expression of actbeta C was not changed in actbeta Em1/actbeta Em1 mice (Fig. 2A and C). However, there appeared to be a slight elevation of actbeta E message in the actbeta Cm1/actbeta Cm1 mice, although the difference was not statistically significant (P > 0.22) when data were quantitated because of the high variability of expression of actbeta E in wild-type mice (Fig. 2B and D). Homozygous mutant mice lacked any detectable expression from the corresponding gene, further demonstrating that our gene targeting strategy produced actbeta C and actbeta E null alleles.


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  		FIG. 2.   Analysis of activin beta C and beta E expression in actbeta Cm1/actbeta Cm1, actbeta Em1/actbeta Em1, and actbeta Cm1-actbeta Em1 homozygous mice. RNase protection was used to examine the expression of actbeta C (A) and actbeta E (B) RNA expression in actbeta Cm1/actbeta Cm1, actbeta Em1/actbeta Em1, and actbeta Cm1-actbeta Em1 homozygous mutant mice. beta 2-Microglobulin was used as the internal control. Representative lanes from a single experiment are shown. Phosphorimaging was used to quantitate the expression of actbeta C (C) and actbeta E (D). All values were normalized to the average ratio of activin subunit expression to beta 2-microglobulin expression in wild-type mice. The bars show means ± standard errors of the means. Values were not statistically significant (P > 0.05) compared to the wild type. n = 5 for all genotypes. WT, wild type; actbeta C, activin beta C; actbeta E, activin beta E; actbeta C-beta E, activin beta C-beta E; beta 2-µg, beta 2-microglobulin.

Despite the restricted expression pattern of both actbeta C and actbeta E genes, liver lobular morphology and histology appeared normal at the light microscopy level in the homozygous mutant mice (Fig. 3). We analyzed if there were changes in liver growth in the null mice by comparing the ratio of wet liver mass to body mass. Even though related members such as TGF-beta 1 and activin A have been implicated in growth regulation of hepatocytes (5, 7, 21, 41, 42, 48, 51, 57), mice deficient in either actbeta C or actbeta E or both show no statistically significant differences in liver mass/body mass ratio compared to their wild-type littermates (Fig. 4).


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  		FIG. 3.   Histological analysis of livers from 42-day-old wild-type and null mice. Liver tissue from C57BL/6-129/SvEv hybrid mice was formalin fixed and stained with hematoxylin and eosin. The liver histology of the homozygous mutant mice compared to that of wild-type mice showed the expected liver lobular organization of the hepatic parenchyma. All sections were photographed at a magnification of ×100. (A) Wild-type mouse (WT); (B) actbeta Cm1/actbeta Cm1 mouse (actbeta C-/-); (C) actbeta Em1/actbeta Em1 mouse (actbeta E-/-); (D) actbeta Cm1-actbeta Em1 (actbeta C-beta E-/-) homozygous mutant mouse. CV, central vein; PT, portal triad.


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  		FIG. 4.   Liver mass/body mass ratios of male and female knockout mice compared to those of wild-type littermates at 42 to 45 days of age. Each point represents the mean ± standard error of the mean. There was no ratio from the homozygous mutant groups that statistically differed significantly from the wild-type ratio (P > 0.05). Mice analyzed in this study are as follows: activin beta C, wild-type males, n = 7; actbeta Cm1/actbeta Cm1 males, n = 16; wild-type females, n = 6; actbeta Cm1/actbeta Cm1 females, n = 5; activin beta E, wild-type males, n = 9, actbeta Em1/actbeta Em1 males, n = 13; wild-type females, n = 7; actbeta Em1/actbeta Em1 females, n = 15; actbeta Cm1-actbeta Em1, wild-type males, n = 10; actbeta Cm1-actbeta Em1 -/- males, n = 17; wild-type females, n = 6; actbeta Cm1-actbeta Em1 -/- females, n = 12.

Liver function analysis. Although there appeared to be no morphological or histological differences between the wild-type and the homozygous mutant mice, a defect in liver function could still exist. Activin A has been shown to affect glycogenolysis in cultured hepatocytes (39). To test whether the liver-restricted activin beta C and activin beta E genes may function similarly in the liver, various components in the serum obtained from 42-day-old male and female mice were analyzed (Table 2). Markers for hepatocellular damage, such as alanine aminotransferase and aspartate aminotransferase, were not significantly different between wild-type mice and null mice. Female actbeta Cm1/actbeta Cm1 mice demonstrated a significant decrease in serum albumin levels (P < 0.04). In contrast, both actbeta Cm1/actbeta Cm1 males and actbeta Cm1-actbeta Em1 double homozygous females have normal albumin levels. There were no differences in random glucose levels among the groups (Table 2). These results suggest that adult mice at 42 days lacking activin beta C and beta E have normal liver function.

                              
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  		TABLE 2.   Serum analysis of wild-type and homozygous mutant mice to study liver functiona

To check which phenotypes, if any, would eventually develop later at adult stages in the mutant mice, mice were observed for more than 6 months. Weekly weight data were considered as a gross indicator of overall health. There were no significant differences between wild-type mice and any of the homozygous mutant mice for up to 6 months (data not shown). Homozygous mutant mice survived for more than 1 year and were indistinguishable from wild-type cage mates (data not shown). This suggests that activin beta C and activin beta E do not play an essential role in regulating systemic processes related to liver metabolism.

Regulation of FSH secretion. Since activin beta A and beta B are known regulators of the hypothalamic-pituitary-gonadal axis, we analyzed the serum FSH and reproductive tracts from homozygous mutant mice. Serum FSH levels in male actbeta Cm1/actbeta Cm1 (30.1 ± 2.9 ng/ml), actbeta Em1/actbeta Em1 (35.5 ± 5.1 ng/ml), and actbeta Cm1/actbeta Em1 homozygous (31.2 ± 2.1 ng/ml) mice were not statistically different (P > 0.05 by Student's t test) from those of male wild-type mice (27.7 ± 2.7 ng/ml). Morphology and histology of the gonads from the null mice were normal (data not shown). Testis weights at 6 weeks of age were not significantly different (P > 0.05) between wild-type (82.3 ± 1.4 mg) and actbeta Cm1/actbeta Cm1 (86.7 ± 2.5 mg), actbeta Em1/actbeta Em1 (79.7 ± 2.0 mg), and actbeta Cm1-actbeta Em1 homozygous (79.2 ± 2.4 mg) mice. Furthermore, the male and female homozygous mutant mice bred normally, showed no reproductive defects, and produced normal litter sizes (Table 1). Thus, in contrast to activin beta A and beta B, activin beta C and activin beta E are not essential regulators of FSH secretion or reproductive function.

Partial hepatectomy in activin beta C and beta E knockout mice. Since there was no gross liver phenotype associated with the loss of actbeta C and/or actbeta E under normal physiological conditions, we performed 70% partial hepatectomy on these homozygous mutant mice to create a physiological stress paradigm and induce liver regeneration. To estimate the mass of the liver prior to the partial hepatectomy procedure, the average liver mass/body mass ratio in hybrid female mice for each mutation was calculated (wild-type; 0.040 ± 0.004, n = 31; actbeta Cm1/actbeta Cm1, 0.041 ± 0.003, n = 31; actbeta Em1/actbeta Em1, 0.043 ± 0.004, n = 22; and actbeta Cm1-actbeta Em1 -/-; 0.043 ± 0.001, n = 21). We analyzed the expression of actbeta C, actbeta E, beta -actin, and junB at various time points (0, 3, 6, 12, 24, 48, and 72 h) after partial hepatectomy.

In wild-type (Fig. 5A and C) and activin beta E knockout (Fig. 5B and C) mice, actbeta C expression tended to be reduced 3 to 6 h following partial hepatectomy (Fig. 5C). By 12 h, activin beta C expression appeared to be induced and appeared to peak at 24 h after partial hepatectomy. By 72 h, the actbeta C RNA was near basal levels (Fig. 5C). On the other hand, actbeta C levels in actbeta Em1/actbeta Em1 mice did not show a trend of increased induction (Fig. 5C). Though there was a trend toward lowered induction kinetics of actbeta C expression in actbeta Em1/actbeta Em1 mice, the values were not statistically significant (e.g., wild type at 0 h versus wild type at 24 h, P > 0.18).


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  		FIG. 5.   Expression of actbeta C before and after partial hepatectomy. (A and B) Activin beta C expression levels in the whole or remnant livers were determined before partial hepatectomy (0 h) and at several time points (3, 6, 12, 24, 48, and 72 h) after partial hepatectomy in wild-type (WT) (A) and actbeta Em1/actbeta Em1 (actbeta E-/-)(B) mice. Representative autoradiographs are shown. (C) Autoradiographs were quantitated and visualized as fold increase over the 0-h point ± standard error of the mean for each genotype. There was no significant difference between time zero and all other time points (P > 0.05). The following numbers of mice were analyzed per time point: three to five (wild type) and three to four (actbeta Em1/actbeta Em1). beta 2-µg, beta 2-microglobulin.

Next, we examined the expression of actbeta E during liver regeneration following partial hepatectomy (Fig. 6). Like activin beta C expression, actbeta E expression tended to be reduced 3 h after partial hepatectomy in both wild-type (Fig. 6A and C) and actbeta Cm1/actbeta Cm1 mice (Fig. 6B and C). Activin beta E was rapidly induced and appeared to peak around 6 h following partial hepatectomy in wild-type mice. Expression of actbeta E was still high at 12 h and dropped to near-basal levels by 48 h in wild-type mice. However, in actbeta Cm1/actbeta Cm1 mice, actbeta E expression appeared to peak between 6 and 24 h after the surgery (Fig. 6C).


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  		FIG. 6.   Expression of actbeta E before and after partial hepatectomy. (A and B) Remnant or whole livers from wild-type (WT) (A) and actbeta Cm1/actbeta Cm1 (actbeta C-/-) (B) mice were collected at 0, 3, 6, 12, 24, 48, and 72 h after partial hepatectomy. Representative autoradiographs are shown. Expression of actbeta E in the livers was assayed by RNase protection with beta 2-microglobin (beta 2-µg) as the internal control. (C) The protected bands were quantitated and visualized as fold increase over the 0-h point ± standard error of the mean for each genotype. An asterisk denotes time points where expression of actbeta E in activin beta C knockout or wild-type mice was statistically significantly different from the time zero value for the same genotype (P < 0.05) by Student's t test. The numbers of mice analyzed at each time point were three to four for wild type and actbeta Cm1/actbeta Cm1.

In addition, we examined the expression of junB (Fig. 7), an immediate-early gene that has been previously characterized in the regenerating liver of wild-type C57BL/6 mice (10). Basal expression of junB in the whole liver was barely detectable in actbeta Em1/actbeta Em1 and actbeta Cm1-actbeta Em1 mutant mice compared to wild-type or actbeta Cm1/actbeta Cm1 mice (Fig. 7C and D). However, during liver regeneration, the kinetics of junB expression were similar between wild-type (Fig. 7A and E) and homozygous mutant (Fig. 7B, C, D, and E) mice. Interestingly, a second low-level junB expression peak was seen at the 48-h point in actbeta Em1/actbeta Em1 and actbeta Cm1-actbeta Em1 homozygous mutant mice (Fig. 7C and D).


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  		FIG. 7.   Expression of junB before and after partial hepatectomy. The expression of junB and the internal control beta 2-microglobulin (beta 2-µg) in the liver was analyzed by RNase protection. (A to D) Representative autoradiographs from wild-type (WT) (A), actbeta Cm1/actbeta Cm1 (actbeta C-/-) (B), actbeta Em1/actbeta Em1 (actbeta E-/-) (C), and actbeta Cm1-actbeta Em1 knockout (actbeta C-beta E-/-) mice (D) are shown. (E) Quantitative results were obtained by normalizing values to the ratio of junB to beta 2-microglobulin at time zero for each genotype (mean ± standard error of the mean). Asterisks denote values that were statistically significantly different from the value for time zero of the same genotype (P < 0.05). The number of mice was three to four for all genotypes at all time points.

We also characterized the expression of beta -actin, a growth-related gene (17), during liver regeneration in wild-type mice. In general, beta -actin expression in the null mice was very similar compared to its expression in wild-type mice from 0 to 72 h after partial hepatectomy (data not shown).


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

In this study, we have used gene targeting to determine the essential in vivo functions of the novel activin subunits, activin beta C and activin beta E. Mice deficient in these two genes, either singly or in combination, appear grossly normal. Despite the dynamic embryonic expression patterns of these two subunit mRNAs, mice with either single or double mutations in these genes were recovered in the expected Mendelian ratios, showing that these genes are dispensable for normal embryonic and postnatal development. Since there was no embryonic or perinatal lethality and no sign of mandibular defects, these data strongly suggest that lack of activin beta C and/or activin beta E signaling is not the cause of the perinatal defects seen in ActRIIA knockout mice (34). Deleting actbeta C and/or actbeta E also does not produce phenotypes similar to those of activin receptor type IIB-deficient mice, the majority of which die perinatally from axial and left-right asymmetry defects (44). The receptors that transduce the activin C and/or activin E signals are not known. Our mutant mice can be a good resource, combined with recombinant activin C, E, or CE, to identify their cognate receptors, assuming that they will be up-regulated in the absence of ligands.

Loveland et al. (31) have shown that actbeta C is expressed in human and rat gonads, and others have shown that human activin beta C is expressed in prostate, kidney, heart, and ovary among other tissues listed in the UniGene database (http://www.nbci.nlm.nih.gov/UniGene/). However, our studies using Northern blot analysis (28) and RNase protection have not detected any actbeta C message in mouse ovary or testis. Histological analysis of other tissues, including the heart, testis, ovary, and prostate, did not show any gross differences. Additional phenotypes were not seen in homozygous mutant mice over 1 year of age, suggesting that activin beta C and beta E are not critical regulators of any systemic processes.

We tested whether activin beta C and activin beta E play essential roles in the regulation of FSH secretion from the pituitary gland since activin beta A and beta B were originally isolated for this activity. We did not detect any differences in the serum FSH levels among the various mutants. This is in contrast to the results we have observed with other knockout models of activins, inhibins, and the activin signaling pathway (33, 34, 55). As predicted by the current model, mice lacking alpha -inhibin and activin beta B have higher serum FSH levels (33, 55), while ActRIIA knockout mutant mice show a decrease in serum FSH levels (34). Therefore, unlike activins A and B, activin beta C and activin beta E are not essential (positive) regulators of pituitary FSH.

Normally, liver size is proportional to body habitus. Numerous studies have previously shown in different organisms (including humans), that, upon transplantation, the liver will modulate its size to "fit" the body (27). Another good example of this modulation is the restorative hyperplasia that occurs during liver regeneration (18, 27, 38). Many positive growth factors for hepatocytes such as hepatocyte growth factor/scatter factor, epidermal growth factor, insulin-like growth factor 1, and norepinephrine are known, but very few negative regulators have been discovered (27, 38). TGF-beta 1 has been shown to negatively regulate hepatocyte proliferation (5, 41, 50, 51), although TGF-beta 1 is inhibitory only 72 h after partial hepatectomy (46). Activin A has also been implicated as a negative regulator since injection of recombinant activin A intraportally into the regenerating liver delayed liver regeneration. In contrast, follistatin, an activin beta -subunit binding protein, injected into the portal vein accelerated liver regeneration (24). Both activin beta A and TGF-beta 1 genes are induced during liver regeneration, but with different kinetics. Expression of activin beta A is low in the quiescent liver but is rapidly up-regulated by 24 h after partial hepatectomy (57). TGF-beta 1 expression is also low in the quiescent liver, but during liver regeneration induced by partial hepatectomy, expression increases from 3 h to peak at 48 to 72 h (3). These initial studies suggested a negative regulatory role for the TGF-beta superfamily ligands during liver regeneration. We have also observed an increase in actbeta C and actbeta E message levels in alpha -inhibin knockout mice (data not shown), further suggesting that these genes play roles during liver regeneration. We did not observe an increased rate of liver regeneration in the activin beta C and beta E mutant mice, but the induction patterns of activin beta C and beta E appeared to be affected in the corresponding null mice (Fig. 5 and 6). This suggests that the induction of each of these genes is mutually dependent on that of the other. However, this dependency occurs only during liver regeneration, since basal levels of activin beta C and beta E are normal in the corresponding mutant mice (Fig. 2). Despite the change in activin beta C and beta E expression profiles during liver regeneration, the mRNA expression profiles of junB and beta -actin suggest that liver regeneration initiates and progresses normally in the homozygous mutant mice.

Since the liver is the major site for the production of acute-phase proteins, activin beta C and activin beta E may play a role during the acute-phase response. TGF-beta superfamily ligands have also been shown to affect inflammation and the acute-phase response. One of the phenotypes seen in the TGF-beta 1 knockout mouse is a hyperstimulated inflammatory response (49). Activin A has also been shown to affect inflammatory response through inhibition of IL-6 action (45, 58) and was isolated biochemically based on this function (4). Activin A may play a role in wound repair since activin beta A and beta B mRNA is induced at the site of wounding (20) and skin wound healing is enhanced if activin beta A is expressed from the human keratin K14 promoter in the skin (40). Further experiments are needed to understand what role(s) actbeta C and actbeta E may play during acute-phase response and wound repair.

The expression of many members of the TGF-beta superfamily and their signaling components in the liver suggests highly redundant roles. Expression of TGF-beta 1, TGF-beta 2, TGF-beta 3 (22), BMP-6 (23), BMP-9 (6), growth differentiation factor 10 (11), and activin beta A (16, 37) among others has all been detected in the liver. BMP-9 has also been shown to be a liver-restricted gene (6). Previous studies have suggested a degree of promiscuity between different receptor and ligand combinations (1, 13, 56). This suggests possible large overlaps between the functions of multiple members of the TGF-beta superfamily and their downstream signaling components, especially in such an important organ as the liver.

In conclusion, we have generated and characterized mutant mice lacking actbeta C and actbeta E genes. Targeted overexpression of activin beta C and beta E in the livers of transgenic mice and the production of functional recombinant activin C, E, and CE proteins will be invaluable in the future to study gain-of-function effects of these two genes in vivo and in vitro. Kron et al. have recently reported the production of recombinant human activin beta C protein using an insect expression system (25). Purified recombinant active protein can be tested in several functional assays such as hepatocellular apoptosis. Production of anti-activin beta C and anti-activin beta E antibodies is under way, and the antibodies will be useful for immunohistochemistry to localize the protein. We are also interested in analyzing the circulating levels of activins C and E and identifying the tissues to which these novel activins specifically bind.


    ACKNOWLEDGMENTS

We thank P. Wang and Q. Guo for technical assistance; R. R. Behringer, A. Bradley, G. Eichele, M. J. Finegold, and S. Varani for help and advice in these experiments; and C. Brown for help, advice, and critical review of the manuscript.

This work is supported in part by National Institutes of Health grant HD32067 and a sponsored research agreement from the Genetics Institute. A.L.L. is a student in the Program of Developmental Biology, supported in part by National Science Foundation grant BIR-9413237. K.N. was funded in part by the Yoshida Science Foundation.


    FOOTNOTES

* Corresponding author. Mailing address: Baylor College of Medicine, Department of Pathology, One Baylor Plaza, Houston, TX 77030. Phone: (713) 798-6451. Fax: (713) 798-5833. E-mail: mmatzuk{at}bcm.tmc.edu.

dagger Present address: Laboratory of Molecular Biology, Tohoku University, Graduate School of Agricultural Science, Aoba-ku, Sendai 981-8555, Japan.

Dagger Present address: Selective Genetics, Inc., San Diego, CA 92121.


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Abstract
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Results
Discussion
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