B-Cell Translocation Gene 2 (Btg2) Regulates Vertebral Patterning by Modulating Bone Morphogenetic Protein/Smad Signaling

Btg2 is a primary p53 transcriptional target gene which mayfunction as a coactivator-corepressor and/or an adaptor moleculethat modulates the activities of its interacting proteins. Wehave generated Btg2-null mice to elucidate the in vivo functionof Btg2. Btg2-null mice are viable and fertile but exhibit posteriorhomeotic transformations of the axial vertebrae in a dose-dependentmanner. Consistent with its role in vertebral patterning, Btg2is expressed in the presomitic mesoderm, tail bud, and somitesduring somitogenesis. We further provide biochemical evidencethat Btg2 interacts with bone morphogenetic protein (BMP)-activatedSmads and enhances the transcriptional activity of BMP signaling.In view of the genetic evidence that reduced BMP signaling causesposteriorization of the vertebral pattern, we propose that theobserved vertebral phenotype in Btg2-null mice is due to attenuatedBMP signaling.

INTRODUCTION

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Introduction

B-cell translocation gene 2 (Btg2, also known as Tis21, Pc3,or APRO1) belongs to the antiproliferative (APRO) family, whichis classified by the conserved APRO homology boxes A and B separatedby 20 to 25 nonconserved amino acids (19, 36). A large bodyof studies has implicated the role of Btg2 in cell growth, differentiation,survival, and senescence. Overexpression of exogenous Btg2 caninduce cell cycle arrest by either Rb-dependent (12) or -independentmechanisms (17). Expression studies have suggested that theantiproliferative property of Btg2 may play a critical rolein the procession of precursor neuronal and osteoblast cellsfrom proliferation to differentiation status during development(13, 30). In addition, Btg2 expression was necessary to provideprotection against apoptosis in terminally differentiated neuronalcells (6, 8). Other reports have demonstrated that DNA-damagingagents induced Btg2 in a p53-dependent manner (7, 31). Consistentwith that finding, it was also shown that the Btg2 promotercontains functional p53-responsive elements (31) and that Btg2is a primary p53 transcriptional target gene (14).

A growing number of reports have suggested that Btg2 functionsas a coactivator-corepressor and/or an adaptor molecule thatmodulates the activities of its interacting proteins. Both Btg2and its closest family member, Btg1, have been demonstratedto bind and to positively modulate the activity of the proteinarginine methyltransferase 1, Hoxb9 (a homeobox protein), andCaf1 (carbon carbonylate repressor 4 [CCR4]-associated factor1) (18, 28, 29, 32). It was suggested that the interactionsof Btg proteins with a general transcription multisubunit complexCCR4 through Caf1 might be involved in the transcriptional regulationof the genes involved in the control of the cell cycle (22).Tob1, another ARPO member, was shown to interact with bone morphogeneticprotein (BMP)-activated Smads and to inhibit BMP/Smad signalingin osteoblasts (42). Targeted disruption of Tob1 resulted inincreased osteoblasts and bone mass as well as a predispositionto tumorigenesis (41, 42).

Immunohistological studies have shown that Btg2 is highly expressedin kidney proximal tubules, lung alveolar bronchial epithelia,and prostate acinar cells (21) and that diminished Btg2 expressionhas been correlated with cancerous states (9, 11, 35). Whilenumerous data have implicated the role of antiproliferative,p53 target Btg2 as a tumor suppressor and a regulator of differentiationof multiple cell lineages, there has been an absence of studiesconfirming its in vivo function. Canzoniere et al. recentlyshowed that Btg2 overexpression in neuronal tissues of transgenicmice led to a marked increase in the number of postmitotic neuronsand to the impairment of cerebellar development (2). This resultsupports the previously suggested role of Btg2 in controllingthe transition of neuronal precursor cells from the proliferativeto the differentiative stage in their development and maturation(2, 13).

In this paper, we demonstrate that Btg2 is expressed in thepresomitic mesoderm, tail bud, and somites during somitogenesisstages and is necessary for the normal patterning of axial vertebrae.We show that BMP signaling has an antagonistic effect on Gdf11/Acvr2bsignaling in vertebral patterning. We further show that Btg2can interact with BMP-activated Smads and enhance BMP/Smad signaling.We propose the impaired BMP/Smad signaling as a potential mechanismunderlying the posterior vertebral transformation in Btg2-deficientmice.

MATERIALS AND METHODS

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Materials and Methods

A Btg2 knockout strain was generated at the University of Florida.Generation of Bmpr2 knockout mice was previously reported (1).Mice were maintained under standard specific-pathogen-free conditions,and all animal procedures performed were reviewed and approvedby the University of Florida Institutional Animal Care and UseCommittee.

Targeting vector construction and ES cell targeting. A bacterial artificial chromosome (BAC) clone containing theentire Btg2 gene was isolated by screening the mouse 129Sv BAClibrary (Incyte Genomics, St. Louis, Mo.) with PCR primers generatedfrom exons I and II of the Btg2 gene. Upon cloning, restrictionenzyme mapping, and sequencing, we identified exons I and IIof the BAC subclones (Fig. 1a). The targeting vector was generatedby sequential subclonings of the 5' and 3' homology arms intopPNT vector (38). A 3.0-kb HindIII-XbaI fragment containingexon I and a 4.7-kb EcoRI-EcoRI fragment were sequentially insertedinto the EcoRI and XhoI sites, respectively, of the pPNT vector.The targeting vector resulted in the replacement of the entireexon II, including the region containing APRO boxes A and B,with the neomycin resistance cassette. Linearized Btg2 targetingvector was then electroporated into 4 x 10⁷ J1 embryonic stem(ES) cells, and G418- and fialuridine-resistant colonies wereselected. Approximately 400 G418- and fialuridine-resistantcolonies were randomly picked for homologous recombination screeningby genomic Southern blot analysis. Upon initial screening witha 3' probe followed by confirmation with a 5' probe and Southernblot analysis (Fig. 1b), one ES clone was identified to containthe correctly targeted Btg2 locus. ES cells from the positiveclone were injected into blastocysts on the C57BL/6 (B6) strain.Chimeric male mice were mated with females on the B6 background.

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FIG. 1. Gene targeting of the Btg2 locus. (a) Diagram showing the Btg2 WT genetic locus, the corresponding targeting construct, and the resulting targeted mutant allele. Two exons are indicated by shaded boxes. Both 5' and 3' probe regions for genomic Southern screening are indicated, as are the expected sizes of fragments from WT and mutant Btg2 loci. Restriction enzyme sites for BamHI (sites labeled B), EcoRI (E), HindIII (H), PstI (P), and XbaI (X) are indicated. Restriction enzyme sites in brackets indicate the nullification of the recognition sequence. Neomycin resistance (neo^r) and thymidine kinase (tk) cassettes are in the opposite direction of the Btg2 gene. KO, knockout. (b) Genomic Southern analysis of Btg2^+/^– crossing. Genomic DNA from WT and Btg2^+/^– and Btg2^–^/^– mice was digested with EcoRI and BamHI and subsequently hybridized with 5' and 3' probes. (c) Northern blot analysis of Btg2^+/^– crossing. Note the absence of the Btg2 transcript in the kidney of the Btg^–^/^– mutant.

Skeleton preparation and whole-mount in situ hybridization. Newborn pups either from Btg2^+/^– females mated with Btg2^+/^–or Btg2^–^/^– males or from Acvr2b^+/^– Bmpr2^+/^–females mated with Acvr2b^–^/^– males were subjectedto skeleton preparations as previously described (24). For studyingthe expression patterns of Btg2 in developing mouse embryos,wild-type (WT) embryos, ranging from embryonic day 9.0 (E9.0)to E14.5, were collected. Antisense Btg2 RNA probe was generatedby using a digoxigenin-UTP labeling kit (Roche Diagnostics,Indianapolis, Ind.), and whole-mount in situ hybridization wasperformed as described previously (40).

Transfection and reporter assays. C2C12 cells were transiently transfected with SBE₄-, ARE-, orBRE-luc and the internal control pCMV-ß-gal with orwithout the Btg2 expression vector (1 µg/well) in six-wellplates by using Lipofectin (Invitrogen, Rockville, Md.) accordingto the manufacturer's instructions. Twelve hours after transfection,cells were treated with medium containing 50 ng of BMP-2/mlfor 18 h. Luciferase activity was quantified by using an enhancedluciferase assay kit (BD Biosciences, Franklin Lakes, N.J.).Values were normalized with ß-galactosidase activity.All assays were performed in triplicate, and results are givenas the means (± standard errors) of three independenttransfections.

Immunoblotting and immunoprecipitation. 293T cells were used for the detection of protein-protein interactionsin vivo. 293T cells were transiently transfected with the indicatedplasmids. Forty-eight hours after transfection, cells were lysedin a buffer containing 25 mM HEPES (pH 7.5), 150 mM NaCl, 1%Triton X-100, 10% glycerol, 5 mM EDTA, and a protease inhibitorcocktail (Roche Diagnostics). Extracts were separated by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferredto polyvinylidene difluoride membranes; they were then probedwith polyclonal or monoclonal antisera, followed by horseradishperoxidase-conjugated anti-rabbit or anti-mouse IgG, respectively,and visualized by chemiluminescence (Pierce Biotechnology, Rockford,Ill.) according to the manufacturer's instructions. The GSTpull-down assay was performed by incubating glutathione-Sepharose4B beads (Amersham Biosciences, Piscataway, N.J.) with eachextract for 1 h. After the beads were washed four times withthe buffer used for cell solubilization, immunoblots assayswere performed.

RESULTS AND DISCUSSION

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Results and Discussion

Generation of Btg2-deficient mice. In order to investigate the in vivo function of Btg2 in mice,we have performed gene targeting experiments. Btg2 transcriptencodes 158 amino acids (aa) in two exons separated by a smallintron (Fig. 1a) (10). The first exon contains sequence encodingthe translation start site and 48 aa, while the second exoncontains sequence for the remaining 110 aa. To generate theBtg2-null mutation, we designed a targeting vector in whichthe entire second exon, containing APRO homology boxes A andB, was replaced with a neomycin resistance cassette (Fig. 1a).Homologous recombination was confirmed by genomic Southern blotanalysis using both 5' and 3' probes (Fig. 1b), and the absenceof Btg2 transcript in homozygous Btg2 mutants (Btg2^–^/^–)was confirmed by Northern blot analysis (Fig. 1c).

Btg2 is not essential for the viability of mice. Btg2 appeared dispensable for the viability of embryonic developmentand postnatal life, as we observed the Mendelian ratio of viableoffspring at the weaning age from Btg2^+/^– crosses. Bothmale and female Btg2^–^/^– mice were normal in appearanceand fertile. Furthermore, female Btg2^–^/^– mice nurturedtheir offspring normally, indicating that Btg2 is unessentialfor mammary gland development. Flow cytometry analysis of lymphoiddevelopment in thymus and spleen with antibodies specific forCD4, CD8, CD45R, and CD19 showed no obvious differences betweenBtg2^–^/^– and wild-type mice (data not shown). Inaddition, genotoxic agent-induced growth arrest was not significantlydifferent in murine embryonic fibroblasts isolated from Btg2^–^/^–and WT embryos (data not shown).

To investigate whether Btg2 played an essential role in cerebellardevelopment, we examined the cerebella of Btg2^–^/^–mice at postnatal day 4 (PN4) and at two months of age. Themorphologies, sizes, and cell densities of cerebella of Btg2^–^/^–were grossly indistinguishable from those of their littermatecontrols (data available on request). In PN4 cerebella, a comparablenumber and thickness of granule cell progenitors in the externalgranular layer were observed for Btg2^–^/^– mice andfor their littermate controls (unpublished data). The lengthsand sizes of the cerebellar lobules, as well as the cellularmorphologies and densities of the cerebella of adult Btg2^–^/^–mice were also normal (unpublished data). A detailed molecularand immunological analysis of cerebellar development may furtheraddress whether Btg2 is essential in regulating genes, suchas cyclin D1 or Math1, involved in the proliferation and differentiationof granule cell progenitors during cerebellar development. Histologicalexaminations of various organs and tissues of 9-week-old Btg2^–^/^–mice (n = 3) found no obvious abnormalities, with the exceptionof the vertebral columns as detailed below.

Btg2^–^/^– mice exhibit posterior homeotic transformation of axial vertebrae. During the expansion of the Btg2 mutant line, one Btg2^+/^–mouse displayed paralysis of the hind limbs at weaning and thenecropsy result showed an obliquely positioned lumbar vertebra.This finding, together with the previous report of the interactionof BTG2 with Hoxb9 (29), which is involved in the specificationof axial vertebrae, led us to investigate the developmentaldefects of axial skeletons for Btg2^–^/^– mice. Theaxial vertebrae are categorized as cervical, thoracic, lumbar,sacral, or caudal depending on their position along the anteroposterioraxis and on their morphological characteristics. For example,the thoracic vertebrae are characterized by the attachment ofthe ribs, whereas the sacral vertebrae are fused to each otherto form a structure called the sacrum. The numbers of vertebraein each category are mostly invariable within a species. Micehave 7 cervical (C), 13 thoracic (T), and 6 lumbar (L) vertebrae,represented by the C7-T13-L6 vertebral pattern (Fig. 2a). Wefound that the majority of Btg2^–^/^– mice exhibitedabnormal vertebral patterns.

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FIG. 2. Posterior transformation of axial vertebrae in Btg2^–^/^– mice. (a to e) Axial skeletons and vertebrosternal ribs (insets) of wild-type (a) and Btg2^–^/^– (b to e) newborn pups. In wild-type mice, the thoracic, lumbar, and sacral vertebrae begin at V8, V21, and V27, respectively (a). In Btg2^–^/^– mice, the first rib (r1) is attached to V7 instead of V8 (b and c) and is joined to the second rib ventrally at the rostral end of the first sternebra (b, inset). The asterisks indicate the ribs attached in V20, which are either absent (b) or in the form of minute rudiments (d and e). The sacral vertebrae begin at V26 instead of V27 (b).

Specifically, Btg2^–^/^– mice displayed a series ofposterior vertebral transformations (Table 1). As shown in Fig.2b and c, the first set of ribs of Btg2^–^/^– micewere attached to the seventh vertebra (V7), indicating the transformationof C7 to T1. The T13-to-L1 transformation was also apparent,as the rib rudiments in the 20th vertebra (V20) of Btg2^–^/^–mice were minute or completely absent (Fig. 2b to e). In addition,V26, which forms L6 in wild-type mice, was transformed to S1in Btg2^–^/^– mice (Fig. 2b). Interestingly, a significantnumber of the Btg2^+/^– mice also exhibited similar, butless intense, vertebral abnormalities compared to Btg2^–^/^–mice, indicating a haploinsufficiency effect (Table 1).

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TABLE 1. Posterior vertebral transformation in Btg2^–/– mice

Dynamic Btg2 expression in somitogenesis stage embryos. Each vertebra is derived from two adjacent somites (5), whichform bilaterally on either side of the neural tube. The firstsomite appears shortly after gastrulation. Thereafter, new somitesare added at a relatively constant rate from the anterior endof the presomitic mesoderm (PSM), while new mesoderm is beingadded concomitantly at the posterior end of PSM from the primitivestreak and then from the tail bud (27). It has been shown thatthe positional information of a vertebra is determined in thesomitomeres of the PSM even before they become mature somites(23, 26).

Btg2 expression was detected in the PSM region of E9 embryos(Fig. 3a). PSM and tail bud-specific expression of Btg2 continueduntil E12.5 (Fig. 3b to e) and faded away when somitogenesiswas completed (Fig. 3f and g). Btg2 transcripts were also detectedin the somites of E10.5 to E12.5 embryos (Fig. 3c' to e'). Btg2transcripts on the dorsal side of the neural tube emerged atE9 and faded away starting at E13.5 (Fig. 3a' to g'). Btg2 expressionin the PSM-tail bud region and somites of embryos during activesomitogenesis is consistent with its role in vertebral patterning.

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FIG. 3. Whole-mount in situ hybridization showing a dynamic Btg2 expression pattern in PSM-tail bud regions, somites, and the neural tubes. Lateral (a to b) and dorsal (a' to g') views of E9 to E14.5 embryos hybridized with Btg2 antisense probe are shown. Btg2 transcripts were specifically detected in the PSM region at E9 (a) and in the tail buds (indicated by asterisks) at subsequent stages (b to e). Tail bud-specific expression disappeared from E13.5 embryos (f and g). Btg2 transcripts were detected also on the dorsal side of the neural tubes from E9 to E13.5. This neural tube expression peaked at E11.5 and faded away by E13.5. Expressions in the somite (S) were also visible in E10.5 to E12.5 embryos. The inset in panel d' shows Btg2 expression in developing rib-like streaks in the trunk at E11.5.

It has been shown that Btg2 interacts with and increases thetranscriptional activity of Hoxb9 (29). If the loss of interactionbetween Btg2 and Hoxb9 were the cause of vertebral defects inBtg2^–^/^– mice, we should have observed an anteriorvertebral transformation consistent with most Hox gene-deficientmutants (4, 39). In this regard, the posterior vertebral transformationin Btg2^–^/^– is unlikely due to the attenuated transcriptionalactivities of Hox genes.

BMP type II receptor (Bmpr2) haploinsufficiency attenuates vertebral defects in Acvr2b^–^/^– mice. It was previously demonstrated that activin type IIB receptor(Acvr2b)-null (Acvr2b^–^/^–) mice exhibited an anteriorvertebral transformation resulting in the C7-T16-L6 vertebralpattern, with nine vertebrosternal (VS) ribs (24) (Fig. 4a andc). We have also shown that additional heterozygous deletionof its subfamily receptor Acvr2 gene in the Acvr2b^–^/^–background intensified the Acvr2b^–^/^– phenotype ofvertebral transformation, exhibiting the C7-T17-L7 pattern,with 10 VS ribs, and that Gdf11 is the likely ligand for Acvr2and Acvr2b (20, 25). In the course of studying the genetic interactionsbetween Acvr2b and BMP type II receptor (Bmpr2), we discoveredthat Acvr2b^–^/^– Bmpr2^+/^– mice showed alleviatedvertebral defects compared to Acvr2b^–^/^– mice. Asshown in Fig. 4b and Table 2, the transformation of V23 to T16was incomplete and V29 was transformed to S1 instead of L6 inthe majority of Acvr2b^–^/^– Bmpr2^+/^– mice. Furthermore,eight ribs, instead of nine, were attached to the sternum (Fig.4c and d; Table 2). These data indicate that there are antagonisticroles for BMP and Gdf11 signaling in vertebral development andthat attenuated BMP signals cause posteriorization of the vertebralpatterns.

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FIG. 4. Representative axial skeletons of Acvr2b^–^/^– Bmpr2^+/^– newborn pups. (a and b) Axial skeletons of Acvr2b^–/– (a) and Acvr2b^–/– Bmpr2^+/– (b) mice. Transformation of V23 to T16 in Acvr2b^–/– mice (a) was incomplete in Acvr2b^–/– Bmpr2^+/– mice (b). Asterisks and inset in panel b show small rib rudiments, indicating the incomplete transformation of V23 to the thoracic rib. V29, which mostly forms L6 in Acvr2b^–/– (a), formed S1 in Acvr2b^–/– Bmpr2^+/– (b). (c and d) Sternum and vertebrosternal ribs of Acvr2b^–/– (c) and Acvr2b^–/– Bmpr2^+/– (d) mice, showing eight VS ribs in Acvr2b^–/– Bmpr2^+/– mice, instead of nine.

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TABLE 2. Vertebral transformation in Acvr2b^–/– and Acvr2b^–/– Bmpr2^+/– mice

Btg2 enhances BMP/Smad-mediated transcriptional activation and interacts with Smad1 and Smad8. Another APRO family member, Tob1, has been shown to antagonizeBMP/Smad signaling. Tob1 deficiency leads to enhanced BMP/Smad-dependentosteoblast function and to an increased incidence of spontaneoustumor formation (41, 42). We have investigated whether Btg2also has a similar modulating effect on BMP or transforminggrowth factor ß (TGF-ß) signaling as Tob1.Based on the posterior transformation phenotype, we anticipatedthat Btg2 would either enhance BMP signaling, which is mediatedby Smad1, Smad5, and/or Smad8, or inhibit Gdf11 signaling, whichis mediated by Smad2 and/or Smad3 (25). To test this hypothesis,we analyzed the effect of Btg2 on the transcriptional activitiesof TGF-ß/activin/Gdf11- or BMP-responsive promoters.Using the artificial SBE₄-Luc reporter, which contains fourtandem repeats of Smad-binding elements and measures the Smad3-and Smad4-specific response (43), we showed that overexpressionof Btg2 has no effect on TGF-ß-induced transcriptionalactivity (Fig. 5a). To address the effect of Btg2 on a Smad2-dependentpromoter, we examined induction of the activin response element(ARE) from the Xenopus Mix.2 gene (16). Cotransfection of Btg2with the ARE-Luc reporter and FAST1 on C2C12 cells showed thatBtg2 has no effect on the TGF-ß-induced activity ofARE-Luc (Fig. 5b). To address whether Btg2 has an effect onBMP-induced transcriptional activity, we tested BMP-responsiveelement (BRE), which is found in the mouse Id1 gene and respondsto BMP signals but not to TGF-ß or to activin (15).When the BMP-responsive element BRE-Luc was used, Btg2 enhancedBMP-2-induced transcriptional activity in a dose-dependent manner(Fig. 5c and d).

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FIG. 5. BTG2 activates BMP-dependent transcription and associates with Smad1 or Smad8 in vivo. (a and b) Effect of Btg2 on TGF-ß1-induced transcription of SBE₄-Luc (a) and ARE-Luc reporters (b). C2C12 cells were transiently transfected with a control vector (pCMV-ß-gal) or Btg2 (1 µg/well) along with either SBE₄-Luc (a) or ARE reporter plus FAST1 (b). (c and d) Effect of Btg2 on BMP-induced transcription of BRE-Luc reporter. (c) C2C12 cells were transiently transfected with the control vector or Btg2 (1 µg/well) along with BRE-Luc. The cells were treated with 50 ng of BMP-2/ml (black bars) or left untreated (white bars). (d) C2C12 cells were transiently transfected with the control vector or increasing amounts of Btg2 along with BRE-Luc. Luciferase activity was normalized to ß-galactosidase activity and plotted as the mean and standard deviation for triplicates from a representative experiment. (e) GST-BTG2 was transfected into 293T cells with the Flag-tagged Smad1 or Smad5 or Myc-tagged Smad8 construct. Cell extracts were subjected to GST pull-down assay using glutathione-Sepharose 4B beads, followed by immunoblotting with anti-Flag or anti-Myc antibody. Expression of GST, GST-BTG2, and Smads was monitored as indicated.

To examine the possibility that Btg2 interacts directly withSmad proteins in vivo, 293T cells were transfected with Flag-taggedSmad1 or Smad5 or Myc-tagged Smad8 along with GST-tagged Btg2.The transfected cells were then subjected to pull-down withglutathione-Sepharose 4B beads, followed by immunoblotting withFlag or Myc antibodies. As shown in Fig. 5e, Btg2 showed stronginteractions with Smad1 or Smad8 but not with Smad5. The interactionbetween Btg2 and Smad1 or Smad8 was not increased in the presenceof BMP-2 (data not shown).

These results suggest that the posterior vertebral transformationphenotype observed with Btg2^–^/^– mice might be dueto the attenuation of BMP signaling. To test whether additionalheterozygous mutation of the Bmpr2 gene in Btg2^–^/^–mice increases the severity of the posterior transformationphenotype, we compared the vertebral pattern of Btg2^–^/^–Bmpr2^+/^– (n = 11) with Btg2^–^/^– (n = 7) littermates.However, no difference in the severities of the phenotypes betweenthe two groups was found.

In this report, we have shown that Btg2 is expressed in thePSM-tail bud region and in developing somites with dynamic expressionpatterns and that Btg2 is involved in vertebral patterning viaactivating BMP signaling. Our results support the hypothesisthat Btg2 may function as a coactivator-repressor and/or adaptormolecule in regulating the transcription status of various targetgenes. The posterior homeotic transformation phenotype of Btg2^–^/^–mice is reminiscent of those found in several knockout micedeficient in Polycomb group (PcG) genes, which are requiredin order to maintain repression of Hox genes (33). A remarkablysimilar posterior transformation phenotype was also found inmice deficient in E2f6, a transcriptional repressor, which wasfound to associate with a number of PcG proteins (34, 37). Interactionsbetween TGF-ß-activated Smad proteins with E2f familyproteins have been demonstrated (3). We speculate that Btg2may play an adaptor function in the interaction of BMP-activatedSmads and E2f6 in the PcG complex. Our results also suggestthe intriguing possibility that two APRO family members, Btg2and Tob1, may have a balancing role in BMP signaling: Btg2 activatesBMP signaling, whereas Tob1 inhibits it (42).

ACKNOWLEDGMENTS

We thank Jennifer Embury and Naime Akdede for the necropsy analysisof Btg2^–^/^– mice.

This study was supported in part by NIH grant HL64024 to S.P.O.and by Korea Heath 21 R&D project grant 02-PJ10-PG8 EC01-0028to I.K.L.

FOOTNOTES

* Corresponding author. Mailing address: Department of Physiology and Functional Genomics, University of Florida, 1600 SW Archer Rd., Room D533d, Gainesville, FL 32610. Phone: (352) 392-8197. Fax: (352) 846-0270. E-mail: ohp{at}phys.med.ufl.edu.

REFERENCES

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