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Molecular Endocrinology, doi:10.1210/me.2007-0052
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Molecular Endocrinology 21 (6): 1430-1442
Copyright © 2007 by The Endocrine Society

An Adenosine Triphosphatase of the Sucrose Nonfermenting 2 Family, Androgen Receptor-Interacting Protein 4, Is Essential for Mouse Embryonic Development and Cell Proliferation

Fu-Ping Zhang, Andrii Domanskyi, Jorma J. Palvimo, Hannu Sariola, Juha Partanen and Olli A. Jänne

Biomedicum Helsinki, Institute of Biomedicine, Physiology (F.-P.Z., A.D., J.J.P., O.A.J.) and Developmental Biology (H.S.), University of Helsinki, and Developmental Biology Program (J.P.), Institute of Biotechnology, University of Helsinki, FI-00014 Helsinki, Finland; Department of Medical Biochemistry (J.J.P.), University of Kuopio, FI-70211 Kuopio, Finland; and Department of Clinical Chemistry (O.A.J.), Helsinki University Central Hospital, FI-00290 Helsinki, Finland

Address all correspondence and requests for reprints to: Olli A. Jänne, M.D., Ph.D., Biomedicum Helsinki, Institute of Biomedicine, University of Helsinki, Haartmaninkatu 8, FI-00014, Helsinki, Finland. E-mail: olli.janne{at}helsinki.fi.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
An adenosine triphosphatase of the sucrose nonfermenting 2 protein family, androgen receptor-interacting protein 4 (ARIP4), modulates androgen receptor activity. To elucidate receptor-dependent and -independent functions of ARIP4, we have analyzed Arip4 gene-targeted mice. Heterozygous Arip4 mutants were normal. Arip4 is expressed mainly in the neural tube and limb buds during early embryonic development. Arip4–/– embryos were abnormal already at embryonic d 9.5 (E9.5) and died by E11.5. At E9.5 and E10.5, almost all major tissues of Arip4-null embryos were proportionally smaller than those of wild-type embryos, and the neural tube was shrunk in some Arip4–/– embryos. Dramatically reduced cell proliferation and increased apoptosis were observed in E9.5 and E10.5 Arip4-null embryos. Mouse embryonic fibroblasts (MEFs) isolated from Arip4–/– embryos ceased to grow after two to three passages and exhibited increased apoptosis and decreased DNA synthesis compared with wild-type MEFs. Comparison of gene expression profiles of Arip4–/– and wild-type MEFs at E9.5 revealed that putative ARIP4 target genes are involved in cell growth and proliferation, apoptosis, cell death, DNA replication and repair, and development. Collectively, ARIP4 plays an essential role in mouse embryonic development and cell proliferation, and it appears to coordinate multiple essential biological processes, possibly through a complex chromatin remodeling system.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
WE HAVE PREVIOUSLY ISOLATED and characterized an androgen receptor-interacting protein, termed ARIP4, a member of the sucrose nonfermenting 2 (SNF2) protein family. ARIP4 sequence similarity to known proteins is restricted to the centrally located SNF2 adenosine triphosphatase (ATPase) domain (1). ARIP4 interacts with the androgen receptor (AR) in vitro and in cultured yeast and mammalian cells and activates AR-dependent transcription from minimal promoters. Our previous studies have indicated that the amino-terminal region of ARIP4 mediates its interaction with AR and that this interaction does not depend on DNA binding. ARIP4 is an active ATPase, and double-stranded DNA and single-stranded DNA enhance its catalytic activity. ATPase-deficient ARIP4 mutants behave as trans-dominant-negative regulators of AR function (1). The kinetic parameters of ARIP4-catalyzed ATP hydrolysis are similar to those of BRG-1 and SNF2h, two members of the SNF2-like protein family, whereas the catalytic activity of ARIP4 is about 10 times lower that that of the other two ATPases (2). Unlike some other members of the SNF2 family, ARIP4 does not form large protein complexes in vivo or remodel mononucleosomes in vitro. Furthermore, ARIP4 is covalently modified by sumoylation, and mutation of six potential SUMO attachment sites abolished the ability of ARIP4 to bind DNA, hydrolyze ATP, and activate AR function (2).

The Arip4 gene is ubiquitously expressed in adult mouse, and Arip4 mRNA is abundant in liver, kidney, and testis (1). Recent study found that dual-specificity tyrosine (Y)-phosphorylation regulated kinase 1A (Dyrk1A), a mammalian homolog of the Drosophila minibrain gene involved in neuronal development and in adult brain physiology, interacts with ARIP4 in yeast and mammalian cells. Dyrk1A and ARIP4 are coexpressed in the mouse brain and colocalized in hippocampal and cerebellar neurons. Inhibition of endogenous Dyrk1A and ARIP4 expression by short interfering RNA-mediated knockdown showed that both proteins are able to activate synergistically AR- and glucocorticoid receptor (GR)-dependent transcription (3). Transgenic mouse with overexpression of the Dyrk1a gene showed a deficit in visuospatial learning and memory (4, 5, 6). Dyrk1a-null mice presented a general growth delay and died during midgestation. Dyrk1a+/– mice showed decreased neonatal viability and a significant reduction of body size from birth to adulthood, as well as malformation of several brain regions (7).

The precise physiological function of ARIP4 has remained elusive. In this study, we have investigated the biological role of ARIP4 through targeted inactivation of the Arip4 gene in mice. Remarkably, loss of Arip4 expression causes embryonic lethality, and the embryos die by embryonic d 11.5 (E11.5). Arip4-null embryos are smaller than wild-type and heterozygous embryos, and the growth potential of Arip4–/– mouse embryonic fibroblasts (MEFs) is greatly reduced. Our findings imply that ARIP4 plays an important role during the embryonic development and cell growth and proliferation during development.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Generation and Characterization of Arip4 Gene Trap Allele
The mouse Arip4 gene was inactivated by gene trapping. Homologous recombination in embryonic stem (ES) cells was first identified by 5'-rapid amplification of cDNA ends as previously described (8), and confirmed by PCR using ARIP4Se1-ARIP4Anti1 and ARIP4Se1-En1 primer pairs (Fig. 1AGo). We generated 11 male and five female Arip4 chimeric mice by the morula aggregation method. The male chimeras were bred with ICR females, and germline transmission was obtained from 10 of them. Arip4+/– mice appeared to be healthy and were fertile.


Figure 1
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  		Fig. 1. Gene Trap Disruption of the Mouse Arip4 Gene

A, Schematic representation of the putative exon and intron structures of the genomic locus, the integration site of the PT1ßgeo gene trap reporter relative to Arip4, and location of the PCR primers used for genotyping to detect wild-type and trapped alleles, respectively. B, Northern blot analysis of ARIP4 mRNA expression in wild-type ES cells and ES cells trapped with PT1ßgeo. C, qRT-PCR detection of Arip4 mRNA in E10.5 wild-type and Arip4–/– embryos. Expression levels were normalized to Williams-Beuren syndrome chromosome region 1 homolog (Wbscr1) and calculated relative to wild-type embryos (=1). Each bar corresponds to mean ± SEM for two independent experiments. D, PCR analysis of genomic DNA extracted from the yolk sacs of embryos at E10.5. PCR products were amplified by primer pairs ARIP4Se1-ARIP4Anti1 [corresponding to wild-type (WT) allele; 316 bp] and ARIP4Se1-En1 [corresponding to mutant (MT) allele; 367 bp], which are indicated in A.

 
Arip4 Expression during Embryogenesis
To examine the expression level of Arip4 in ES cells carrying the Arip4 gene trap allele, Northern blot analysis was carried out by using a cRNA probe specific to Arip4 mRNA. The result showed that the amount of Arip4 mRNA in ES cells with one trapped Arip4 allele was reduced to about one half compared with wild-type ES cells. No truncated Arip4 mRNA species were detected in ES cells with the trapped Arip4 gene even after an extended exposure time (Fig. 1BGo). We also collected E10.5 embryos homozygous for the Arip4 mutation and isolated total RNA. Quantitative RT-PCR (qRT-PCR) detected ARIP4 mRNA in wild-type embryos, but not in Arip4–/– embryos (Fig. 1CGo). Collectively, our data indicate that expression of the Arip4 transcript from the gene trap allele is below detection limit, and thus the gene trap insertion is likely to represent a null mutation of Arip4. Figure 1DGo depicts the PCR strategy used to genotype Arip4 targeted embryos or mice with two specific primer pairs.

A basic local alignment search tool search of the National Center for Biotechnology Information est_mouse database (http://www.ncbi.nim.gov:80/BLAST) found ARIP4 expressed sequence tags isolated from embryos as early as at two-cell stage. The trapped vector was designed in such a fashion that lacZ expression is driven by the endogenous Arip4 promoter. This strategy resulted in the expression of ß-galactosidase (ß-gal) in lieu of ARIP4. To determine the spatiotemporal expression pattern of Arip4 in early-stage embryos, whole-mount ß-gal staining was performed in Arip4 heterozygous and homozygous embryos (Fig. 2Go). At E8.5, staining was found in the neural tube. At later stages of development (E9.5–E12.5), expression was mainly detected in the neural tube and limbs. From E14.5 onward, lacZ expression was virtually ubiquitous. To further confirm the Arip4 expression pattern, whole-mount immunohistochemical staining with anti-ARIP4 antibody was carried out on E10.5 embryos, and the results were consistent with those by ß-gal staining (supplemental Fig. S1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org).


Figure 2
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  		Fig. 2. Expression of Arip4 during the Embryonic Development

Whole-mount ß-gal staining with blue color of E8.5 (A), E9.5 (B), E10.5 (C), and E12.5 (D) embryos. Arip4 is mainly expressed in neural tubes and limb buds during the embryonic development. E and F, Representative transverse section of ß-gal-stained E10.5 embryo counterstained with eosin. F is higher magnification of E. For each gestation time point, at least five embryos were analyzed, and the representative results are shown. HF, Headfold; NT, neural tube; Y, yolk sac; LB, limb bud; N, neuroepithelium; H, heart; PC, pericardial cavity; Not, notochord. Bar, 200 µm.

 
Targeted Deletion of Arip4 Causes Early Embryonic Death
Genotype analysis of multiple litters of Arip4+/– intercrosses at 3–4 wk after birth indicated that no Arip4–/– mice were born (Table 1Go), raising the possibility that ablation of the Arip4 gene results in early lethality. To determine the age at which Arip4-null embryos die, we performed a timed mating analysis. Up to E9.5, Arip4+/+, Arip4+/–, and Arip4–/– embryos were present at the expected Mendelian ratios (Table 2Go), indicating that ARIP4 is not required for early embryonic development and implantation. However, the proportion of Arip4–/– embryos started to decline around E10.5, and none could be found beyond E12.5, although there was evidence of embryo resorption.


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  		Table 1. Genotypes of Offspring from Arip4 Heterozygote Mating

 

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  		Table 2. Genotypes of Embryos from Arip4 Heterozygote Mating

 
At E9.5, some mutant embryos were smaller than wild-type or heterozygous littermates. At E10.5, Arip4–/– embryos appeared abnormal, and their body sizes were significantly reduced compared with wild-type and heterozygous littermates (Fig. 3AGo). Histological analyses were performed to characterize further the mutant embryos. Paraffin-embedded fixed sections derived from E9.5 and E10.5 embryos were examined after staining with hematoxylin and eosin. Histological analyses of E9.5 and E10.5 embryos did not reveal any obvious developmental defects, but almost all of the major tissues in Arip4-null embryos were proportionally smaller than those in wild-type embryos (Fig. 3Go, B–E), and in some Arip4–/– embryos the neural tube was shrunk. By contrast, heterozygous embryos of the same litters presented no significant reduction in body size or morphological alterations relative to controls. Chorioallantoic placentas of Arip4–/– embryos appeared to have normal histology (supplemental Fig. S2, published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org).


Figure 3
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  		Fig. 3. Growth Retardation of Arip4–/– Embryos

A, Gross morphology of Arip4+/+, Arip4+/–, and Arip4–/– littermates at E10.5. B–E, Histological analysis of Arip4+/+ (B and D) and Arip4–/– (C and E) embryos at E10.5. At least five embryos of each genotype were analyzed, and the representative transverse sections stained with hematoxylin and eosin are shown. FB, Forebrain; HB, hindbrain; N, neuroepithelium; BA, branchial arch; H, heart; PC, pericardial cavity; OpV, optic vesicle; OtV, otic vesicle; Not, notochord. Bar, 200 µm.

 
Deficiency of Arip4 Increases Apoptosis and Reduces Cell Proliferation
Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) staining was used to detect apoptotic cells in E10.5 embryos. The proportion of apoptotic cells was significantly increased in Arip4-null embryos compared with wild-type embryos, especially in the neural tube (Fig. 4Go). We also analyzed the proliferative status of cells in embryos by immunohistochemical phospho-histone H3 staining. The number of phospho-histone H3-positive cells in Arip4–/– embryos was much lower than in wild-type embryos (Fig. 5Go). Similar results were obtained when E9.5 embryos were examined. Likewise, immunohistochemical staining for another cell proliferation marker, Ki-67, demonstrated a greatly reduced number of proliferating cells in the neuroepithelium of Arip4–/– embryos (supplemental Fig. S3, published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org).


Figure 4
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  		Fig. 4. TUNEL Staining of E10.5 Arip4+/+ (A–D) and Arip4–/– (E–H) Embryos

The number of apoptotic cells is significantly increased in Arip4–/– embryos, especially in neural tubes compared with Arip4+/+ embryos. At least five embryos of each genotype were analyzed, and the representative immunostained sections are shown. B, D, F, and H are higher magnifications of A, C, E, and G. FB, Forebrain; N, neuroepithelium; BA, branchial arch; H, heart; LB, limb bud; OpV, optic vesicle. Bar, 200 µm.

 

Figure 5
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  		Fig. 5. Phospho-Histone H3 Immunohistochemical Staining of E10.5 Arip4+/+ (A–D) and Arip4–/– (E–H) Embryos

The number of positive immunostained cells is lower in Arip4–/– embryos compared with Arip4+/+ embryos, indicating a decreased rate of cell proliferation. At least five embryos of each genotype were analyzed, and the representative immunostained sections are shown. B, D, F, and H are higher magnifications of A, C, E, and G. FB, Forebrain; N, neuroepithelium; BA, branchial arch; H, heart; OpV, optic vesicle. Bar, 200 µm.

 
To study further the effect of Arip4 mutation on cell proliferation, we isolated MEFs from wild-type and Arip4–/– E9.5 embryos. Arip4–/– MEFs ceased to grow beyond passage 2–3; by contrast, wild-type MEFs continued growing up to passage 5–6 and were not studied after that. For the cell proliferation assay, wild-type and Arip4–/– fibroblasts (both at passage 2) were plated at the same cell densities. Arip4–/– MEFs exhibited significantly decreased cell proliferation in comparison with wild-type MEFs (Fig. 6AGo). Flow cytometric analysis revealed a significantly higher proportion of apoptotic cells in Arip4–/– MEFs population compared with wild-type MEFs (Fig. 6BGo).


Figure 6
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  		Fig. 6. Increased Apoptosis and Reduced Proliferation in Arip4–/– Cells

MEFs were derived from Arip4+/+ and Arip4–/– embryos at E9.5, cultured, and analyzed in cell proliferation assay (A) and by flow cytometry (B) as described in Materials and Methods. Representative FACS histograms demonstrate increased proportion of apoptotic (sub-G1) cells in Arip4–/– population. Cell proliferation was calculated relative to wild-type MEFs (=100%). Each bar corresponds to mean ± SEM for at least two independent experiments. Statistical significance determined by using Student’s unpaired t test is indicated; **, P < 0.01.

 
Together, these data suggest that disruption of the Arip4 gene results in increased apoptosis and reduced cell proliferation during embryogenesis.

Potential ARIP4 Target Genes in Embryonic Fibroblasts
To gain mechanistic insights into developmental and cellular functions of ARIP4, we used a genome-wide approach to identify ARIP4 target genes in MEFs. Microarray analysis was performed on Arip4–/– and Arip4+/+ MEFs. Of a total of over 20,000 features analyzed on the Agilent Mouse Development Oligo Microarray, the expression of 1,263 annotated genes was increased (809 genes) or decreased (454 genes) by a factor of more than 2 in Arip4-null compared with wild-type Arip4 MEFs. In addition to key regulators of cell growth and proliferation, various genes involved in DNA replication and repair, apoptosis and cell death, neurogenesis, transcription factors, and development were affected (supplemental Table S2, published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org), suggesting that ARIP4 is involved in a variety of biological processes.

To confirm the microarray data, the expression levels of several selected genes in Arip4–/– and Arip4+/+ MEFs were analyzed by qRT-PCR (Table 3Go). Among the analyzed targets were the genes related to apoptosis and cell death, Tnfrsf19, Pea15, Tia1, Bad, Casp9, Ier3, Zac1; regulators of cell growth and proliferation, Fgfr1, Figf, Igf1, Pdgfra; mammalian development and transcription factors, Pax6, Msx2, Hoxa10. Ctbp1 and Dyrk1a were specifically selected due to the interaction of the corresponding proteins with ARIP4. The changes in expression of these genes in Arip4–/– MEFs obtained from qRT-PCR analysis were consistent with the microarray results (Table 3Go).


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  		Table 3. Quantitative RT-PCR Verification of Microarray Results for Selected Genes

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
ARIP4 belongs to the SNF2 family of proteins that are involved in chromatin remodeling, DNA repair, and homologous recombination (1). ARIP4 interacts with AR and modulates its activity in vitro and in vivo (2). The results of the present work show that ARIP4 is essential for embryonic development, and it appears to be involved in the processes of apoptosis and cell proliferation. Embryonic lethality in Arip4-null mice is likely to be independent of the ability of ARIP4 to modulate AR function, because Ar gene expression has been reported to commence at E12.5 of mouse development (9). Thus, AR-independent functions, at least those during embryonic development, appear to be physiologically more important than the functions of ARIP4 as an AR coregulator.

On the basis of the sequence similarity in the ATPase domain, the SNF2 protein family can be divided into two major groups. The first group includes true ATP-dependent chromatin remodelers involved in transcriptional regulation, whereas the proteins from the second group, despite exhibiting some chromatin remodeling activity, are generally involved in DNA repair and homologous recombination (10, 11, 12). ARIP4 belongs to this latter group and has the closest sequence similarity to ATRX and Rad54. Interestingly, mutation of the human ATRX gene results in ATR-X syndrome with mental retardation (13, 14). Conditional Atrx knockout mouse using the cre-loxP recombination system controlled by Forkhead box G1 and Nestin promoters, which direct Cre recombinase expression to either forebrain or central nervous system neuroprogenitors, respectively, showed that ATRX is critical for neuronal survival in the developing cortex and hippocampus (15). Another example of a human syndrome associated with mental retardation caused by mutations in a chromatin remodeling protein and leading to transcriptional dysregulation is the Rubinstein-Taybi syndrome, which results from mutations in the CREB-binding protein gene, encoding a transcriptional coregulator also involved in steroid hormone signaling pathways (16, 17). By contrast, Rad54 knockout mice are viable and healthy without gross developmental or phenotypic abnormalities. Nevertheless, mouse Rad54–/– cells are sensitive to ionizing radiation, mitomycin C, and methyl methanesulfonate treatment, and homologous recombination in Rad54–/– cells is reduced compared with wild-type cells (18).

The physiological role of ARIP4 has remained elusive. Using gene-trap mutagenesis in mouse ES cells, we generated Arip4-deficient mice. Heterozygous Arip4+/– mice are viable and seem to be normal. We cannot exclude the possibility that Arip4+/– mice exhibit a mild phenotype. Recently, we have demonstrated that Sertoli cells of Arip4+/– mice have reduced levels of androgen-regulated Rhox5 mRNA (19). No Arip4–/– mice were born from heterozygous breeding, suggesting that Arip4–/– homozygous mice were unable to survive during embryonic development. Further analysis revealed that Arip4–/– mice died during embryogenesis, between E10.5 and E12.5. From E9.5 onward, homozygous embryos exhibited growth retardation with reduction of body size. Morphological analysis revealed that Arip4–/– embryos were proportionally smaller than wild-type littermates, indicating that ARIP4 plays an essential role during embryonic development. Many reported mutated embryos that die during this period present growth retardation defects, and, for most of these embryos, the cause of death is unknown. In general, the major developmental abnormalities leading to death around the period of organogenesis include defective yolk sac circulation and failure to establish chorioallantoic placenta, both resulting in poor embryonic blood circulation (20, 21). With regard to Arip4-null embryos, yolk sac and chorioallantoic placenta appear to be normal, indicating that the early lethality is unlikely to be due to the failure of embryonic blood circulation.

During the embryonic development, Arip4 is mainly expressed in the neural tube and limb buds, as revealed by LacZ expression driven by Arip4 regulatory sequences and immunohistochemistry with anti-ARIP4 antibody, suggesting that ARIP4 plays an important role during early stages of mouse nervous system development. Consistent with the expression pattern of Arip4, significant decrease in the number of proliferating neuroepithelial cells and increase in apoptotic cells in neural tubes was detected in Arip4-null embryos. Thus, loss of the Arip4 gene could lead to a nervous system defect related to a reduction in the number of neural precursors and perhaps deregulation of neural differentiation. However, ARIP4 function is not limited to neuroepithelium, as revealed by analyses of both Arip4 mutant embryos and MEFs isolated from Arip4-null mice.

Recent studies demonstrated that the Dyrk1a gene product, involved in neuronal development and in adult brain physiology, interacts with ARIP4 in yeast and mammalian cells (3). ARIP4 together with Dyrk1A appears to be capable of influencing both AR- and GR-mediated transactivation in vivo in the mammalian brain, and ARIP4 and Dyrk1A are coexpressed and colocalized in hippocampal neurons in adult mouse brain (3). Transgenic mice with Dyrk1a gene overexpression showed a deficit in visuospatial learning and memory (4, 5, 6). Whole-mount in situ hybridization experiments have shown the presence of Dyrk1a transcripts in the neural tubes at E9.5 during embryonic development. Similar to Arip4 mutated mice, the mice bearing Dyrk1a mutations exhibit a general growth delay and die during early organogenesis around E10.5. Dyrk1a–/– embryos present body size reduction to one third to one half of that in wild-type littermates at E9.5. In contrast to Arip4-null embryos, the morphology of brain vesicles, heart, and liver primordium in Dyrk1a–/– embryos shows a delay in structural development. Immunostaining with the anti-ß-tubulin antibody, which specifically stains differentiating neuronal cells, was significantly reduced in the neural tube of E9.5 Dyrk1a–/– embryos, indicating either a defect in neuroblast proliferation or a delay in the maturation of the nervous system (7). Given the fact that AR and GR are also present in this region (22, 23), ARIP4 together with Dyrk1A, AR, and GR may play important roles during mouse neural development.

Deletion of Arip4 increased apoptosis and decreased cell proliferation in mutant embryos, which is further supported by experiments performed on MEFs. Flow cytometric analysis indicated a significantly higher proportion of apoptotic cells in Arip4–/– MEFs than in wild-type MEFs. Arip4–/– MEFs ceased to grow after passages 2–3 and showed increased apoptosis and attenuated cell proliferation. Microarray analysis of total mRNA expression profiles in Arip4–/– and wild-type embryonic fibroblasts revealed that expression of several apoptosis- and cell proliferation-related genes is deregulated in Arip4–/– MEFs. Deletion of ARIP4 also affects expression of several genes involved in DNA replication and repair, neurogenesis, and development. Proapoptotic Bad and Casp9 were found among the up-regulated genes. Bad belongs to BH3-only family of proteins involved in control of apoptosis (24), whereas caspase-9 is an initiator caspase usually activated in stress apoptotic pathway. Activation of caspase-9 leads to robust proteolysis and cell death (25). In addition, a cell death-related gene Tnfrsf19 (also called Troy or Taj), the overexpression of which induces apoptosis (26) and inhibits neuronal differentiation (27), was highly up-regulated in Arip4–/– MEFs. By contrast, several growth factor genes including Igf1 and Pdgfra, were down-regulated in Arip4–/– MEFs. Targeted deletion of Igf1 in mice leads to growth retardation and developmental abnormalities (28), whereas deletion of Pdgfra results in increased apoptosis of neural crest cells, severe developmental abnormalities, growth retardation, and embryonic death at midgestation (29). Thus, the microarray data suggest that Arip4 deletion results in activation of several apoptosis-related genes and inhibition of expression of some growth factors. However, deregulation of some genes may be secondary due to general proliferation defects in Arip4–/– fibroblasts. Additional studies are required to clarify the precise mechanisms leading to induction of apoptosis and inhibition of proliferation in Arip4–/– MEFs.

Some SNF2 family proteins are involved in the regulation of cell proliferation and apoptosis (30, 31). For example, embryos lacking functional Brg1 or Snf2h genes die during the periimplantation stage around E3.5 and E6.5 due to defects in cell proliferation (32, 33). Furthermore, blastocyst culture experiments have shown that loss of either of the two genes leads to growth arrest and cell death in both the trophectoderm and inner cell mass (33). By contrast, Brm-null mice develop normally, probably due to the compensation provided by Brg1. Nevertheless, fibroblasts derived from Brm-null mice exhibit proliferation defects (34). Similar to Brg1 and Snf2h expression, Arip4 mRNA is detectable already in ES cells (Fig. 1BGo). In contrast to Brg1- and Snf2h-null embryos that die during the periimplantation stages (32, 33), Arip4–/– embryos survive until midgestation. The above differences imply that SNF2 family proteins play distinct roles during the mammalian embryonic development. The studies of Brg1- and Brm-null mice suggest that these SNF2 family proteins are important for negative regulation of cell cycle progression. Brg1 heterozygous mice are susceptible to spontaneous tumor development, and Brm-null mice are heavier than controls and exhibit an increased mitotic index in liver tissue (32, 34). We have monitored heterozygous Arip4 mutants and wild-type littermates of both sexes for up to 18 months, and we have not observed tumor formation in any of these mice.

In conclusion, consequences of Arip4 gene inactivation in mice indicate that ARIP4 has an essential role in early organogenesis, most likely regulating cell growth and proliferation. Arip4-null embryos display growth retardation, with the majority of organs and tissues being smaller than those of wild-type littermates. Impaired cell proliferation in Arip4–/– embryos is reflected by reduced phospho-histone H3 staining of embryonal tissues and impaired proliferation capacity of MEFs. Conditional Arip4 gene targeted mice are required to address ARIP4 functions in specific cell types or tissues during development.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Creation of Arip4 Gene-Targeted Mice
ES cells with the trapped Arip4 gene were obtained from Gesellschaft für Strahlung und Umweltforschung Center for Environment and Health, Institute of Mammalian Genetics (Neuherberg, Germany). Briefly, mouse embryonic stem cells were electroporated with the promoterless splice-acceptor type vector, PT1ßgeo, and selected in G418 as previously described (8). Resistant clones were isolated, and the site of integration was determined by 5'-rapid amplification of cDNA ends as previously described (8). ES cells with the trapped Arip4 gene (A016E03) were used to generate chimeric mice by the standard morula aggregation method. Male chimeras were bred on the ICR genetic background to test for germline transmission. Mice and embryos were genotyped by PCR by using tail or yolk sac genomic DNA with primers for the wide-type allele (ARIP4Se1, 5'-TCAGGCTTGGCATTCTTCCA-3'; ARIP4Anti1, 5'-AAAAATTGGGGGGGAAACACCAC-3') and for the targeted allele (ARIP4Se1, 5'-TCAGGCTTGGCATTCTTCCA-3'; En1, 5'-CGCCATACAGTCCTCTTCA-CATC-3') (Fig. 1Go).

All mice were handled in accordance with the institutional policy for animal care at the University of Helsinki. All animal protocols were approved by the University of Helsinki Review Board for Animal Experiments.

Histological Analyses
Arip4+/– mice were mated, and the morning when the vaginal plug was found was defined as E0.5. Embryos were dissected in PBS, and a minimum of three embryos of each genotype (Arip4+/+, Arip4+/–, and Arip4–/–) from at least three litters were collected at E9.5 and E10.5. Embryos were fixed in 4% paraformaldehyde (PFA) at 4 C overnight, dehydrated, and embedded in paraffin. Paraffin blocks were sectioned at 5-µm thickness, and stained with hematoxylin and eosin (BDH, Poole, UK).

Culture of MEFs
E9.5 embryos were minced into small pieces and cultured in individual wells on 48-well dishes in DMEM containing 15% fetal calf serum, glutamine, penicillin, and streptomycin (each 25 U/ml) for 4 d. Cells were trypsinized and replated, and nonadherent cells were removed 1 h later.

RNA Isolation and Blotting
Total RNA was isolated using NucleoSpin RNA II kit (Macherey-Nagel, Diiren, Germany) or Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Ten micrograms of total RNA from ES cells were resolved on 1.2% formaldehyde denaturing agarose gel and transferred onto nylon membrane (Hybond-XL; Amersham Biosciences, Uppsala, Sweden) as described previously (35). The blot was hybridized with a 32P-labeled antisense cRNA probe (1.7 x 106 cpm/ml) for 2 h at 68 C in ULTRAhyb buffer (Ambion, Austin, TX). After high stringency washes with 2x standard saline citrate (SSC) (1x SSC is 0.15 M NaCl, 0.015 M sodium citrate) and 0.1% sodium dodecyl sulfate (twice for 5 min at 68 C), and with 0.1x SSC and 0.1% sodium dodecyl sulfate, the membranes were exposed to Fuji x-ray films at –70 C for 24–72 h. 32P-Labeled cRNA was synthesized using a Riboprobe Synthesis II kit (Promega, Madison, WI), [{alpha}-32P]UTP (Amersham Biosciences), and ARIP4 cDNA (corresponding to nucleotides 189-1025 of GenBank sequence NM_030730) subcloned in the pGEM4Z vector as template.

Microarray Analysis
MEFs were isolated from E9.5 wide-type and Arip4–/– embryos, and total RNA was extracted from MEFs at passage 3 by NucleoSpin RNA II kit. Briefly, 20 µg of total RNA was converted to double-strand cDNA, which was then used to generate cRNA labeled with Cy3-dUTP (wild-type Arip4) and Cy5-dUTP (Arip4-null) using the Fluorescent Direct Label kit (Agilent Technologies, Palo Alto, CA) according to the manufacturer’s instructions. Linearly amplified Cy3- or Cy5-labeled cRNA was purified, fragmented, and subjected to hybridization with Mouse Development Oligo Microarray slides (Agilent Technologies) representing over 20,000 features. The fluorescence intensities on the slides were analyzed using a laser confocal scanner and Feature Extraction Software (Agilent Technologies). Low-quality measurements (i.e. the signal was <2.6 times the background, >50% of pixels of the spot were saturated, or either the spot or the background was not uniform) were excluded from the analysis. After background subtraction, the ratio was calculated by dividing the average intensity of each spot in Arip4-null RNA hybridization by the average intensity of the corresponding spot in wild-type Arip4 RNA hybridization. Transcripts that had a mean of log-transformed ratios below –0.3 (–2-fold change) or above 0.3 (2-fold change), and differed significantly from zero (P < 0.05 as assessed with t test) were regarded as differentially expressed.

qRT-PCR
The cDNA was synthesized using random hexamer primers with SuperScript III first-strand synthesis kit (Invitrogen). For genomic DNA contamination control, RT reactions without reverse transcriptase were included. Quantitative PCR was performed using a LightCycler rapid thermal cycler system (Roche Diagnostics, Lewes, UK) in a 20-µl volume with 3.4 mM MgCl2, 1 µM forward and reverse primers (supplemental Table S1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org), and the LightCycler-DNA Master SYBR Green I mix (Roche Diagnostics) according to the manufacturer’s instructions. PCR included an initial 10-sec denaturation step (95 C) followed by 41–45 cycles of 10-sec denaturation (95 C), 5-sec annealing (58–65 C), 10-sec extension (72 C), and 5-sec SYBR Green I signal measuring (80–86 C) steps. To detect possible nonspecific amplification, a DNA melting step was included after completion of the PCR cycles. The resulting curves were quantified using LightCycler analysis software (Roche Diagnostics) according to the manufacturer’s instructions.

Embryo Whole-Mount ß-Gal Staining and Immunohistochemistry
Embryos were dissected in PBS, fixed in 4% PFA containing 2 mM MgCl2 and 1.25 mM EGTA at 4 C for 1 h, washed in staining buffer [0.1 M Na-phosphate (pH 7.3), 2 mM MgCl2, 0.01% Na-deoxycholate, and 0.02% Triton X-100] and stained in buffer containing 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, and 1 mg/ml X-gal (Sigma-Aldrich, St. Louis, MO) at room temperature for 24–36 h. Subsequently, embryos were postfixed in 4% PFA at 4 C overnight and stored in 70% ethanol.

For immunostaining, mouse embryos stained with X-gal were washed in PBS and incubated 2 h in PBSMT buffer (PBS, 2% skimmed milk, and 0.5% Triton X-100), and further incubated overnight with anti-ARIP4 K7991a1 antibody (19) diluted 1:250 in PBSMT buffer. After several washes in PBSMT, embryos were incubated overnight with fluorescein-conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted 1:250 in PBSMT. Subsequently, the embryos were washed in PBS and photographed under fluorescence microscope.

Immunohistochemical Staining
Five-micrometer sections were mounted onto SuperFrost Plus slides (Menzel, Braunschweig, Germany), dewaxed, and rehydrated. To block endogenous peroxidase activity, slides were incubated in 3% hydrogen peroxide in methanol. Slides were boiled in 10 mM sodium citrate (pH 6.0) for antigen retrieval, washed in Tris-buffered saline (TBS), and blocked in TBS containing 1% BSA (Sigma-Aldrich) and 3% normal goat serum (Vector Laboratories, Burlingame, CA). Slides were incubated with rabbit polyclonal anti-phospho-histone H3 antibody (Upstate, Lake Placid, NY; 1:250 dilution) or with anti-Ki-67 antibody (NeoMarkers, Fremont, CA; 1:2000 dilution) overnight at 4 C. After washing three times with TBS, the second antibody, biotinylated anti-rabbit IgG diluted 1:200, was applied on sections and incubated for 1 h at room temperature. Visualization of the reaction was carried out using the Vectastain Elite ABC and peroxidase diaminobenzidine substrate kits (Vector Laboratories) according to the manufacturer’s instructions. Slides were dehydrated in ascending ethanol series and mounted in Permount mounting medium (Fisher Chemicals, Fair Lawn, NJ).

TUNEL Staining
TUNEL staining was performed by using DeadEnd Colorimetric TUNEL system kit (Promega) according to the manufacturer’s instructions. Briefly, 5-µm embryo sections were deparaffinized and rehydrated. The tissue was permeabilized with 20 µg/ml proteinase K for 15 min and incubated with terminal deoxynucleotidyl transferase enzyme and biotinylated nucleotides at 37 C for 60 min. Incorporated biotinylated nucleotides were bound to horseradish peroxidase-labeled streptavidin and visualized with peroxidase diaminobenzidine system. Sections were counterstained with hematoxylin, dehydrated in ascending ethanol series, and mounted in Permount mounting medium (Fisher Chemicals).

Flow Cytometry and Cell Proliferation Assay
Flow cytometric analysis of MEFs was performed as described (36). Briefly, cells were fixed with ice-cold 70% ethanol overnight, washed with PBS, and resuspended in PBS containing 30 µg/ml propidium iodide (Sigma-Aldrich) and 30 µg/ml RNase A (Roche Diagnostics). After a 60-min incubation at 37 C, the cells were analyzed with a FACSArray flow cytometer (BD Biosciences, Franklin Lakes, NJ). For the cell proliferation assay wild-type and Arip4–/– MEFs at passage 2 were plated onto 96-well plates at the same cell densities (5 x 103 or 1 x 104 cells per well in each case), incubated 24 h at 37 C and 5% CO2, and assayed using a CellTiter96AQ Non-Radioactive Cell Proliferation Assay kit (Promega) according to the manufacturer’s instructions.


   ACKNOWLEDGMENTS
 
We thank Mikael Björklund for the help with FACS assay, and Leena Pietilä, Katja Kiviniemi, Maiju Aatalo, Eija Koivunen, Anne Reijula, Kirsi Salonen, and the Transgenic Mouse Facility at Biomedicum Helsinki for technical assistance.


   FOOTNOTES
 
This work was supported by Academy of Finland, Finnish Foundation for Cancer Research, Sigrid Jusélius Foundation, Helsinki University Central Hospital, Biocentrum Helsinki, and European Union (Contract LSHM-CT-2005-018652).

The authors have nothing to disclose.

First Published Online March 20, 2007

Abbreviations: AR, Androgen receptor; ARIP4, androgen receptor-interacting protein 4; ATPase, adenosine triphosphatase; Dyrk1A, dual-specificity tyrosine (Y)-phosphorylation regulated kinase 1A; E11.5, embryonic d 11.5; ES cell, embryonic stem cell; ß-gal, ß-galactosidase; GR, glucocorticoid receptor; MEF, mouse embryonic fibroblast; PFA, paraformaldehyde; qRT-PCR, quantitative RT-PCR; SNF2, sucrose nonfermenting 2; SSC, standard saline citrate; TBS, Tris-buffered saline; TUNEL, terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling.

Received for publication January 26, 2007. Accepted for publication March 15, 2007.


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