Previous Article | Next Article ![]()
Molecular and Cellular Biology, August 2006, p. 6149-6156, Vol. 26, No. 16
0270-7306/06/$08.00+0 doi:10.1128/MCB.00298-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Department of Physiological Chemistry, Graduate School of Comprehensive Human Sciences and Institute of Basic Medical Sciences, University of Tsukuba, 1-1-1 Ten-nohdai, Tsukuba 305-8575, Japan,1 Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongoh, Bunkyo-ku, Tokyo 113-0033, Japan,2 Department of Veterinary Anatomy, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1 Yayoi Bunkyo-ku, Tokyo 113-8657, Japan,3 Stem Cell Project,4 Infectious Diseases Project,5 Biomembrane Signalling Project,6 Core Technology & Research Center, Laboratory of Mouse Model for Human Heritable Diseases, The Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan,7 Department of Pathology and Immunology, Akita University School of Medicine, Akita 010-8543, Japan,8 Center for Developmental Genetics and Department of Pharmacology, Stony Brook University, New York, New York 11794-51409
Received 17 February 2006/ Returned for modification 28 March 2006/ Accepted 3 June 2006
|
|
|---|
|
|
|---|
To address this issue, we have generated and analyzed ARF6 knockout mice. The results obtained in this study revealed that ARF6/ embryos exhibit abnormal liver development characterized by reduced size and aberrant structure, due to defective hepatic cord formation, and we identify a specific signaling pathway that appears to be responsible for the abnormal development.
|
|
|---|
![]() View larger version (21K): [in a new window] |
FIG. 1. Targeted
disruption of the ARF6 gene. (A) Schematic
representation of ARF6 targeting. The genomic structure of the
ARF6 gene is shown at the top. Noncoding regions of exons are
represented by filled boxes, and the open reading frame is represented
by the hatched box. RV, EcoRV; A, ApaI. (B to D) Genotypes were
verified by Southern blotting (B), PCR (C), and Western blotting of
total lysates prepared from embryonic fibroblasts of each genotype
(D).
|
Antibodies, chemicals, and probes. Anti-albumin antibody was purchased from Bethyl Laboratories, anti-Liv2 from MBL, anti-c-Met from Santa Cruz Biotechnology, Texas Red-conjugated streptavidin from Perkin Elmer Life Sciences, hepatocyte growth factor (HGF) from Sigma-Aldrich, anti-Ter119 and anti-CD45 from BD Pharmingen, anti-active caspase 3 from Cell Signaling, and rhodamine-phalloidin from Molecular Probes. A polyclonal anti-ARF6 antiserum was a generous gift of J. G. Donaldson.
In situ hybridization and histochemistry. To synthesize a probe to detect ARF6 mRNA expression, an ARF6 cDNA, a generous gift of K. Nakayama, was digested with HincII, subcloned into pBluescript II SK(+), and then digested with BglII. Using the digested plasmid, a digoxigenin-labeled antisense probe corresponding to nucleotides 507 to 992 of the ARF6 gene was generated using a DIG RNA-labeling kit (Roche). Whole-mount in situ hybridization was carried out as described previously (12), and the stained embryos were sectioned horizontally to analyze ARF6 mRNA expression in the liver.
For immunohistochemical analysis, embryos were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), embedded in optimal cutting temperature compound (Sakura Finetechnical), sectioned, and stained with antibodies. Bromodeoxyuridine (BrdU)-positive cells were detected using a BrdU in situ detection kit (BD Pharmingen) according to the manufacturer's protocol. Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining was performed using the DeadEnd fluorometric TUNEL system (Promega) according to the manufacturer's protocol. For double staining of hepatocytes and erythroid cells with their specific antibodies (the anti-albumin and Ter119 antibodies, respectively) and TUNEL, the anti-albumin and anti-Ter119 antibodies were detected using biotin-conjugated secondary antibodies, and visualization was performed using Texas Red-conjugated streptavidin. To enhance the anti-albumin and anti-Liv2 antibody signals, a VECTASTAIN Elite ABC kit (Vector Laboratories) was used.
Culture of fetal hepatocytes. Livers dissected from embryonic day 13.5 (E13.5) wild-type and ARF6/ embryos were minced and liver cells dissociated by being sequentially incubated in liver perfusion medium and liver digest medium (Invitrogen). Dissociated cells were seeded on collagen-coated dishes or cover glasses and incubated for 2 h in the basic medium, which consists of Eagle minimum essential medium, 1 mM sodium pyruvate, nonessential amino acid solution, 500 U/ml penicillin, 50 µg/ml streptomycin, and 0.1 µM dexamethasone, supplemented with insulin-transferrin-selenium -X and 10% fetal calf serum (FCS), during which time the fetal hepatocytes attached. The cells were then washed with the basic medium to remove hematopoietic cells and dead cells and further incubated overnight in the basic medium supplemented with 10% FCS. The cells were again washed with the basic medium to completely remove the remaining hematopoietic cells. The purity of fetal hepatocytes thus obtained was more than 90%, as assessed by immunostaining using the anti-albumin antibody.
Assay of hepatic cord-like structure formation of fetal hepatocytes. The HGF-induced cord-like structure formation assay was performed as previously reported (13) with minor modifications. Briefly, fetal hepatocytes cultured on collagen-coated dishes were detached with liver perfusion medium and liver digest medium and replated on 1.2 mg/ml collagen gel. After 2 h, the attached cells were washed, overlaid with collagen gel, and incubated in the basic medium supplemented with 10% FCS for 24 h. The cells were then incubated without or with 25 ng/ml of HGF in the basic medium supplemented with 10% FCS for 24 h. After fixation with 4% paraformaldehyde in PBS, the cells were observed using light microscopy. To assess the morphologies of the colonies and to count the number of cells in each colony, fixed cells were stained for actin and nuclei using rhodamine-phalloidin and DAPI (4',6'-diamidino-2-phenylindole), respectively. Colonies composed of fewer than 10 cells were assessed. Colonies that formed cord-like structures in response to HGF stimulation were defined as those elongated more than 70 µm on the long axis, with protrusions at the tips of the colonies. Under nonstimulated conditions, fewer than 25% of the colonies exhibited extension defined as such.
Pulldown assay of activated ARF6. To analyze ARF6 activation, ARF6-GTP pulldown assays were carried out as previously reported (20). Briefly, 5 x 105 fetal hepatocytes seeded on collagen-coated dishes in the basic medium were cultured overnight. After being washed, fetal hepatocytes were further incubated for 4 h and then stimulated with or without 25 ng/ml HGF for 2 min. Cells were harvested and lysed using a lysis buffer composed of 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 2 mM MgCl2, 0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate, 1% Triton X-100, 10% glycerol, 1 µg/ml aprotinin, and 1 µg/ml leupeptin. Lysate supernatants were mixed with glutathione-Sepharose beads preconjugated with glutathione S-transferase-GGA31-226 and incubated for 30 min with gentle rotation. The beads were washed three times with the washing buffer composed of 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 2 mM MgCl2, 1% NP-40, 10% glycerol, 1 µg/ml aprotinin, and 1 µg/ml leupeptin, and the ARF6-GTP bound to the glutathione S-transferase-GGA31-226 beads eluted using sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer. The eluted, active form of ARF6 was detected by Western blotting with anti-ARF6 antiserum.
Hematopoietic cell culture. Hematopoietic cells prepared from E13.5 embryonic livers were cultured in StemPro34 supplemented with mouse stem cell factor (100 ng/ml), interleukin-3 (10 ng/ml), and interleukin-6 (10 ng/ml). After 3 days, cells were harvested and stained with annexin V-fluorescein isothiocyanate (FITC) (BD Pharmingen) according to the manufacturer's protocol. To evaluate the extent of apoptosis, the ratios of annexin V-positive cells to propidium iodide (PI)-negative cells were analyzed by flow cytometry.
Proliferation assay of fetal hepatocytes. Proliferation of cultured fetal hepatocytes was analyzed by [3H]thymidine incorporation. Fetal hepatocytes were seeded on collagen-coated dishes and cultured overnight in the presence or absence of 25 ng/ml HGF in the medium consisting of minimum essential medium, 1 mM sodium pyruvate, nonessential amino acid solution, 500 U/ml penicillin, and 50 µg/ml streptomycin. Cells were extensively washed and further cultured for 6 h in the presence of 5 µCi/ml [3H]thymidine. Cells were then fixed with 10% trichloroacetic acid for 30 min on ice. After being washed with PBS, samples were treated with 1 N NaOH for 20 min at 37°C followed by 1 N HCl treatment for neutralization. Samples thus prepared were harvested, and the [3H]thymidine incorporated was measured by liquid scintillation counting.
|
|
|---|
|
View this table: [in a new window] |
TABLE 1. Genotypes
of progeny
|
![]() View larger version (40K): [in a new window] |
FIG. 2. Defective
liver formation in ARF6 knockout embryos. (A to C) Appearance
of E13.5 embryos. Note the reduction in the size of the
ARF6/
embryonic liver (C, black arrowhead). The white arrowhead in panel C
indicates edema at the head. Scale bars, 1 mm. (D to H) Livers
dissected from E13.5 embryos. Images shown are views of embryonic
livers from the top (D and E), bottom (F), and side (G and H). Note the
aberrant structures of the lobes in the
ARF6/
livers as well as the reductions in size (E, F, and H). White
arrowheads in panels F, G, and H indicate the outlines of liver lobes.
Scale bars, 1 mm. (I) Body weights of E13.5
embryos. Data shown are the means ± SDs. +/+,
n = 12; +/, n = 21;
/, n = 9. (J) The ratios
of liver weight to body weight for E13.5 embryos. Data shown
are the means ± SDs. +/+, n =
12; +/, n = 21;
/, n = 9. An asterisk denotes
statistical significance (P
< 0.005).
(K) Whole-mount in situ hybridization analysis of E10.5
ARF6+/+ (+/+, left
image) and
ARF6/
(/, right image) embryos using an antisense
ARF6 probe. The arrowhead indicates the liver, which stained
intensely with the probe. Scale bar, 1 mm. (L and M)
Higher-magnification images of the whole-mount-in situ-hybridized
embryos shown in panel K. Scale bars, 100 µm. (N and O)
Sections of whole-mount-in situ-hybridized E10.5
ARF6+/+ (N) and
ARF6/ (O)
embryos using an antisense ARF6 probe. Scale bars, 100
µm. L, liver; Fl, forelimb bud; G,
gut.
|
![]() View larger version (50K): [in a new window] |
FIG. 3. Hypocellularity
and induction of apoptotic cell death in the
ARF6/
embryonic liver. (A and B) Hematoxylin and eosin staining of liver
sections prepared from E13.5 wild-type (Wt) and
ARF6/
embryos. Note the hypocellularity in the
ARF6/
embryonic liver. (C to F) BrdU-incorporated cells in E13.5 wild-type (C
and E) and
ARF6/ (D
and F) embryonic livers were detected with anti-BrdU antibody (brown)
and counterstained with hematoxylin (blue). The boxed areas in panels C
and D are shown at higher magnification in panels E and F,
respectively. (G to L) Liver sections prepared from E13.5 wild-type and
ARF6/
embryos were stained with PI (to visualize nuclei [G and H]) and TUNEL
(as an indicator of apoptosis [I and J]). The boxed areas in panels G
and H were merged to present both channels (K and L). PI staining is
visualized as red and TUNEL-positive cells as green. (M)
Activation of caspase 3 in E13.5 embryonic livers and hearts was
analyzed by Western blotting of total lysates with the anti-active
caspase 3 antibody. Scale bars, 100 µm (A to D and G to J) and
50 µm (E, F, K and
L).
|
![]() View larger version (59K): [in a new window] |
FIG. 4. Analysis
of apoptotic cell death in
ARF6/
embryonic liver. (A and B) E13.5
ARF6/
embryonic liver sections were double stained with TUNEL (green) and
antialbumin (to identify hepatocytes [red]) (A) or
anti-Ter119 antibodies (to identify erythroid cells [red]) (B).
TUNEL-positive and -negative cells are indicated by arrowheads and
arrows, respectively. (C and D) Liver sections prepared from E10.0
wild-type (Wt) (C) and
ARF6/ (D)
embryos were double stained with TUNEL (green) and PI (red). Arrowheads
represent TUNEL-positive liver cells, and dotted lines show the
outlines of the livers. (E) In vitro-cultured fetal
hepatocytes prepared from E13.5 embryonic livers were stained with
TUNEL. TUNEL-positive cells were counted to calculate the percentages
of apoptotic cells (n = 3). Data shown are the means
± SDs. (F) Hematopoietic cells prepared from E13.5
embryonic livers were cultured for 3 days. To evaluate the extent of
apoptosis, the harvested cells were stained with annexin V-FITC and the
percentages of annexin V-positive cells in PI-negative cells assessed
by flow cytometry (n = 3). Data shown are the means
± SDs. G, gut; L, liver; scale bars, 10 µm
(A and B) and 50 µm (C and
D).
|
![]() View larger version (85K): [in a new window] |
FIG. 5. Defective
hepatic cord formation in
ARF6/
embryonic liver. (A to H) Liver sections prepared from wild-type (Wt)
(left panels) and
ARF6/
(right panels) embryos at E9.0 (A and B), E10.0 (C and D), E11.5 (E and
F), and E13.5 (G and H) were stained with anti-Liv2 (A to D) and
antialbumin (E to H) antibodies. LD, liver diverticulum; G, gut; scale
bars, 50
µm.
|
![]() View larger version (22K): [in a new window] |
FIG. 6. Involvement
of ARF6 in HGF-induced hepatic cord-like structure formation.
(A) Activation of ARF6 by HGF stimulation of cultured fetal
hepatocytes (n = 3). (B) Suppression of
HGF-induced cord-like structure formation in
ARF6/ fetal
hepatocytes. Scale bar, 100 µm. (C) Quantification of
the HGF-induced cord-like structure formation of fetal hepatocyte
colonies. Data shown are the means ± SEMs (n =
4). The asterisk denotes statistical significance (P <
0.005). (D) Effect of ARF6 deficiency on HGF-induced
proliferation of fetal hepatocytes, as assessed by
[3H]thymidine incorporation. Data shown are the means
± SDs (n =
3).
|
|
|
|---|
Although the molecular mechanism by which ARF6 regulates the cascade of signaling required for hepatic cord formation remains to be clarified, it is plausible that ARF6 functions by regulating membrane morphology and/or cell migration through controlling actin cytoskeletal reorganization, which is a well-known role for it in model systems (1, 6, 9, 16, 18). This idea is derived from the observation that elongation and branching of the E10.0 hepatic epithelial sheets were abnormal in the ARF6/ embryonic liver (Fig. 5D). Alternatively, membrane trafficking regulated by ARF6 might also be critical for hepatic cord formation, as suggested by reports that ARF6-regulated internalization of E-cadherin enhances the motility of epithelialized cells (14) and that the level of E-cadherin decreases in migrating fetal hepatocytes during liver development (24).
Disruption of HGF-dependent cord-like structure formation in ARF6/ fetal hepatocytes in the in vitro assay system was not complete (Fig. 6B and C), suggesting that HGF utilizes another signaling system, as well as the ARF6-mediated signaling pathway, to couple HGF signaling to cord-like structure formation. If this is true and HGF is the critical physiological factor for hepatic cord formation, then disruption of hepatic cord formation in ARF6/ embryos should be less severe than that in HGF knockout embryos. However, the severity of the liver developmental defect in ARF6/ embryos was almost the same as that reported for HGF knockout embryos (21). These observations lead us to speculate that, in addition to HGF, other hepatic cord formation-promoting factors most likely exist. Supporting this assumption, it has previously been reported that epidermal growth factor and transforming growth factor ß also induce hepatic cord-like structure formation in vitro (13, 19). In addition, it has been reported that transforming growth factor ß type III receptor-deficient mouse embryos display defects in the ultrastructure of the liver (25). Finally, previous reports demonstrating that ARF6 is absolutely required for these types of growth factor-induced cell functions in other settings (4, 9) also support the hypothesis described above.
In the present study, we demonstrated that the smaller sizes and hypocellularity of the ARF6/ embryonic livers were attributable to the progression of liver cell apoptosis (Fig. 3G to M); proliferation of fetal hepatocytes in vitro and in vivo was not impaired by ARF6 deficiency (Fig. 3C to F and 6D), inconsistent with prior reports that ARF6 is essential for cytokinesis (22, 23). This apparent discrepancy suggests the existence of compensatory or redundant mechanisms that promote cytokinesis not only in hepatocytes but in most if not all other cell types. Moreover, we would emphasize that the induction of apoptosis in the ARF6/ embryonic liver, which is not cell lineage specific (Fig. 4A and B), does not seem to be the primary consequence of ARF6 deficiency. This conclusion is supported by the observation that abnormal liver architecture (Fig. 5C and D), but not apoptosis (Fig. 4C and D), was observed at earlier stages (E10.0) of embryonic liver development, and cultured ARF6/ fetal hepatocytes or hematopoietic cells prepared from E13.5 embryos did not exhibit increased apoptosis (Fig. 4E and F). Instead, we would propose that the induction of apoptosis of fetal hepatocytes and of hematopoietic cells that would normally come to coreside in the cord niche might be attributable secondarily to an aberrant fetal liver microenvironment that is unsupportive for liver cell survival, due to incomplete hepatic cord formation. Such an aberrant microenvironment could cause the activation of caspase 3, resulting in the induction of apoptosis, although the details of this death response remain to be elucidated.
ARF6/ embryos were frequently found to be anemic, which might be responsible for the mid- to late-gestational lethality (data not shown). Considering that we observed progressive and substantial apoptosis of hematopoietic cells in the embryonic liver (Fig. 4), it is quite reasonable to speculate that anemia caused in ARF6/ embryos is attributable to progressive apoptosis of hematopoietic cells that is triggered by the defect of liver formation, as was suggested for HGF knockout mice (21). We cannot rule out, however, an additional effect on hematopoiesis as well. Nonetheless, the lethality observed for ARF6/ embryos during mid- to late gestation appears attributable to the observed defects in liver formation. This idea is consistent with the observation that there was some variation in the stage of lethality of ARF6/ embryos (Table 1), since variation was also observed in the severity of the liver formation defect (data not shown).
In conclusion, this report provides
evidence for the first time that ARF6 physiologically functions in
liver development by regulating hepatic cord formation. In addition to
this function, ARF6 may also be involved in other physiological and
pathological events, such as development and functions of postnatal
tissues and metastasis of tumor cells that, like fetal hepatocytes
during liver development, require actin cytoskeletal reorganization.
ARF6 could also be involved in many other settings, e.g., pathogenesis
of the Vibrio cholerae bacterium-induced diarrhea through the
promotion of ADP-ribosylation of Gs
(10) by cholera toxin in
intestinal epithelial cells. To clarify these functions, it will be
necessary to generate conditional knockout mice, since
ARF6-null mice invariably exhibit embryonic
lethality.
We thank H. Nishina and K. Miyazawa for valuable comments and advice. We are also greatly appreciative to J. Penninger, J. G. Donaldson, and K. Nakayama for their generous gifts of E14K ES cells and a mouse 129/Ola genomic library, the anti-ARF6 antiserum, and ARF6 cDNA, respectively.
|
|
|---|
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»