developmental effects of ectopic expression of the glucocorticoid

MOLECULAR AND CELLULAR BIOLOGY,0270-7306/99/$04.0010

Oct. 1999, p. 7106–7122 Vol. 19, No. 10

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Developmental Effects of Ectopic Expression of the GlucocorticoidReceptor DNA Binding Domain Are Alleviated by an Amino Acid

Substitution That Interferes with Homeodomain BindingJUN MING WANG,1 GRATIEN G. PREFONTAINE,2 MADELEINE E. LEMIEUX,1 LOUISE POPE,1

MARIE-ANDREE AKIMENKO,3* AND ROBERT J. G. HACHE1,3,4*

Department of Medicine,1 Graduate Program in Biochemistry,2 Department of Cellular and Molecular Medicine,3 andDepartment of Biochemistry, Microbiology, and Immunology,4 The Loeb Health Research Institute at the Ottawa

Hospital, University of Ottawa, Ottawa, Ontario, Canada K1Y 4E9

Received 10 May 1999/Returned for modification 21 June 1999/Accepted 24 June 1999

Steroid hormone receptors are distinguished from other members of the nuclear hormone receptor familythrough their association with heat shock proteins and immunophilins in the absence of ligands. Heat shockprotein association represses steroid receptor DNA binding and protein-protein interactions with othertranscription factors and facilitates hormone binding. In this study, we investigated the hormone-dependentinteraction between the DNA binding domain (DBD) of the glucocorticoid receptor (GR) and the POU domainsof octamer transcription factors 1 and 2 (Oct-1 and Oct-2, respectively). Our results indicate that the GR DBDbinds directly, not only to the homeodomains of Oct-1 and Oct-2 but also to the homeodomains of several otherhomeodomain proteins. As these results suggest that the determinants for binding to the GR DBD areconserved within the homeodomain, we examined whether the ectopic expression of GR DBD peptides affectedearly embryonic development. The expression of GR DBD peptides in one-cell-stage zebra fish embryos severelyaffected their development, beginning with a delay in the epibolic movement during the blastula stage andfollowed by defects in convergence-extension movements during gastrulation, as revealed by the abnormalpatterns of expression of several dorsal gene markers. In contrast, embryos injected with mRNA encoding a GRpeptide with a point mutation that disrupted homeodomain binding or with mRNA encoding the DBD of theclosely related mineralocorticoid receptor, which does not bind octamer factors, developed normally. Moreover,coinjection of mRNA encoding the homeodomain of Oct-2 completely rescued embryos from the effects of theGR DBD. These results highlight the potential of DNA-independent effects of GR in a whole-animal model andsuggest that at least some of these effects may result from direct interactions with homeodomain proteins.

Nuclear hormone receptors comprise a broadly distributedclass of transcriptional regulators implicated in diverse physi-ological processes (4, 54). Many nuclear receptors play majorroles in development; these include the control of patterningand tissue differentiation. Nuclear receptors are marked by ahighly conserved DNA binding domain (DBD) comprised oftwo Cys4 zinc fingers (48, 54). They also contain a less wellconserved C-terminal region that confers ligand responsive-ness to the transcriptional regulatory functions of many recep-tors.

Most nuclear receptors are constitutive transcriptional reg-ulators whose transcriptional regulatory potential is altered byassociation with ligands. Steroid hormone receptors, however,are distinguished by their association into transcriptionally in-active heat shock protein (hsp)- and immunophilin-containingcomplexes in the absence of steroidal ligands (4, 63). Associ-ation into hsp complexes prevents steroid receptor DNA bind-ing by physically masking the DBD. hsp association also altersthe conformation of the steroid receptor ligand binding do-main in a manner that facilitates ligand binding (62, 63). In-

deed, hsp association appears to be required for ligand bindingby the glucocorticoid receptor (GR).

In addition to direct regulation of transcription throughDNA response elements, it is becoming increasingly apparentthat many important functions of nuclear receptors are medi-ated through protein-protein interactions with other transcrip-tion factors in the absence of DNA binding (30, 37). Thephysiological importance of these activities has recently beenhighlighted by a report that mice with a mutation in the GRthat compromised DNA binding are viable (69).

Interestingly, several protein-protein interactions betweennuclear receptors and other sequence-specific transcriptionfactors are mediated through receptor DBDs (13, 20, 55, 76).The best characterized of these interactions occurs with AP-1(29, 38, 53). Several nuclear hormone receptors, including GR(29), androgen receptor (AR) (75), estrogen receptor a (ERa)(100), thyroid hormone receptor (108), and retinoic acid re-ceptors and retinoid X receptors (81), form complexes withAP-1 that block the interaction of AP-1 with its normal re-sponse elements. At least for GR, the receptor–AP-1 complexis redirected to composite response elements specific for theGR–AP-1 complex. In addition, GR can bind to and repressthe DNA binding of NF-kB (76). For steroid hormone recep-tors, these interactions are dependent upon the dissociation ofhsp complexes and thus are sensitive to both steroids andsteroid antagonists (29).

Recently, we determined that three steroid hormone recep-tors, GR, AR, and progesterone receptor (PR), associatedthrough their DBDs with the POU DBDs of octamer transcrip-

* Corresponding author. Mailing address for Marie-Andree Aki-menko: The Loeb Health Research Institute, 725 Parkdale Ave., Ot-tawa, Ontario, Canada K1Y 4E9. Phone: (613) 761-5142. Fax: (613)761-5036. E-mail: [email protected]. Mailing address for Robert J. G.Hache: The Loeb Health Research Institute, 725 Parkdale Ave., Ot-tawa, Ontario, Canada K1Y 4E9. Phone: (613) 761-5142. Fax: (613)761-5036. E-mail: [email protected].

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tion factors 1 and 2 (Oct-1 and Oct-2, respectively) (64, 65). Incontrast, ERa, mineralocorticoid receptor (MR), and severalother nuclear receptors failed to interact with Oct-1 and Oct-2in transfected cells. These results indicated that Oct-1 andOct-2 binding was restricted within the nuclear hormone re-ceptor superfamily. For GR, AR, and PR, octamer factor bind-ing was strictly dependent upon the dissociation of free recep-tors from hsp-immunophilin complexes that followed ligandbinding. Functionally, binding to GR, AR, and PR affected theDNA targeting of the octamer factors in the cell by preferen-tially increasing their binding to octamer motifs in the tran-scriptional regulatory regions of steroid hormone-responsivereporter genes (11, 64, 65, 97). There also has been a report forGR that this interaction may simultaneously decrease the oc-cupancy of octamer motifs in transcriptional regulatory regionslacking steroid hormone response elements (46). For rat GR,C500Y and L501P substitutions in the second zinc finger of theDBD abrogated octamer factor binding and their recruitmentto DNA (65).

Like GR, POU transcription factors comprise a large familyof transcriptional regulators with a highly conserved DBD (73).The POU domain is bipartite, consisting of POU-specific andPOU homeodomain motifs that bind cooperatively to the 59and 39 halves of DNA response elements (43). Homeoboxgenes encode master developmental control proteins involvedin virtually all aspects of pattern formation and tissue differ-entiation in the developing embryo (70, 72, 73, 101).

Several homeobox genes involved in the first steps of theestablishment of the vertebrate body axis during gastrulationhave been characterized. Homeobox genes such as goosecoid(8, 9, 32, 92), floating head or Xnot (95, 98), GSX (51), Xlim-1(94), Otx2 (61, 89), Xtwin (49), and Siamois (22, 39, 49, 52) areexpressed in the organizer and show dorsalizing propertieswhen ectopically expressed in the embryo. The homeoboxgenes Xvent-1 and Vox (also named Xvent-2) have been shownto be involved in ventral fate specification (27, 59, 78). Most ofthese genes are activated between the midblastula transitionand the onset of gastrulation. However, some of them, such asgoosecoid (92), Otx2 (61, 89), and Xtwin (49), as well as anumber of POU factors (58, 60, 79, 80) are expressed as ma-ternal factors prior to being zygotically expressed. Despite theimportant functions of these genes at the onset of gastrulation,little is known about the role of maternal homeodomain pro-teins during cleavage of the vertebrate embryo and the role ofthe maternal and zygotic homeodomain proteins prior to gas-trulation.

Glucocorticoids are teratogenic (1). The most frequent cor-relate of embryonic exposure is cleft palate (26). In mammals,the expression of GR has been detected beginning at a stageequivalent to day 9.5 of mouse embryogenesis (18, 42). Fur-ther, the down regulation of GR mRNA at the final differen-tiation stages of tissues in which it is expressed is suggestive ofa morphogenetic role (42). Indeed, insertional inactivation ofthe GR gene results in mice that die soon after birth due to adefect in the final stages of lung development (17). Studieswith Xenopus also indicate a role for GR at the earliest stagesof embryogenesis. GR mRNA is abundant in Xenopus oocytesbut is rapidly degraded during the early cleavage stages ofembryogenesis. GR transcripts are reexpressed prior to thecompletion of gastrulation and become localized to the dorsalectoderm (25).

In this study, we report the Oct-1 homeodomain and theOct-2 homeodomain (Oct-2HD) to be necessary and sufficientfor the direct binding of Oct-1 and Oct-2 to the GR DBD.L501P-sensitive GR binding to the homeodomain was not lim-ited to Oct-1 and Oct-2 but also was observed for several other

homeodomain proteins, including zebra fish (Danio rerio) dlx2and hoxd4. Intriguingly, the expression of GR DBD but notMR DBD peptides in one- or two-cell-stage zebra fish embryosspecifically affected embryonic development at or before thetime of blastoderm migration in a manner that correlated withthe L501P-sensitive binding of the GR DBD to maternal andembryonic factors. Moreover, these developmental defectswere rescued by the coexpression of a peptide containing Oct-2HD. These results establish the GR DBD as a molecular probefor important developmental events occurring near the timewhen embryonic transcription is initiated and predict an im-portant role for homeodomain proteins that bind to the GRDBD during these events.

MATERIALS AND METHODS

Plasmids. pGEM7Z-Oct-1 was produced by insertion of the HindIII/BamHIOct fragment from pCGN-Oct-1 (96) into the corresponding sites of pGEM7Z(Promega). pGEX2T-GR (amino acids [aa] 407 to 568) and pGEX2TGRC500Y(aa 407 to 556) were constructed by insertion of the BamHI/EcoRI fragmentfrom pSP64X568 (71) and the BamHI/SmaI fragment from pT7C500Y (77),respectively, in frame into the appropriate restriction sites of pGEX2T (Phar-macia). pTL2HAOct-2 was produced by first inserting the Oct-2 XbaI (blunted)/BamHI fragment from pCGN-Oct-2 (96) into pACT-2 (Clontech) to acquire ahemagglutinin epitope and then recloning with BglII/XhoI into the correspond-ing sites of pTL2. pTL2OCT2DHD was created by removing the EagI/PstI DNAfragment corresponding to the homeodomain (aa 296 to 359) and religating it inframe. pTL2-OCT2DPOU and pTL2-OCT2DSP were created through a similardeletion and religation strategy to remove aa 152 to 349 and aa 152 to 286,respectively. pGEX2TGR-PKA vectors (wild type [WT], C500Y, L501P, C460Y,and C460Y-L501P; aa 407 to 550) were constructed by insertion of an oligonu-cleotide encoding a protein kinase A (PKA) recognition motif, LARRASYP,into the StyI/EcoRI sites of plasmid pGEX2T-GR. pGEX2T-OCT2HD (aa 294to 377) was constructed by insertion of a 14-bp linker into the BamHI/EagI sitesof pGEX2T-OCT2POU (aa 195 to 377) (65). Expression plasmids for dlx2(pGEX3X-dlx2 and pTL2-dlx2), hoxd4 (pTL2-hoxd4), and msxB (pGEX3X-msxB; aa 135 to 196) were as described previously (107), as were the pGEX2T-PrdHD, pGEX2T-FtzHD, and pGEX2T-OtdHD constructs (104). pGEX2T-HoxC4 (aa 148 to 227) was subcloned from a human Jurkat T-cell cDNA library.pCGNOCT-1, pCGNOCT-2, and GR plasmids pT7X556 (aa 407 to 556),pT7C460Y, pT7R489R, pT7L501P, and pT7C500Y have been described previ-ously (71, 96). The GR double-mutant plasmid pT7C460Y/L501P was con-structed by replacing the XhoI/SphI DNA fragment from pT7L501P with thecorresponding fragment from pT7C460Y. pET-11a-MR-DBD and pET-11a-OCT2HD were constructed by insertion of the fragments containing the MRDBD (aa 567 to 700) and Oct-2HD (aa 294 to 377) coding sequences created byPCR from pGALO-MR-DBD (64) and pGEX2T-OCT2HD, respectively, inframe into pET-11a (Novagen; the NheI/BclI and NheI/BamHI sites, respective-ly). The plasmids used for making the in situ probes have been described pre-viously as pBluescript SK for ntl (82), shh (44), flh (95), MyoD (102), gsc (92), andbmp4 (57) and pBluescript KS for axial (93).

GST pull-down and immunoprecipitation protein binding assays. GlutathioneS-transferase (GST) fusion proteins were expressed, purified, and tested in aGST pull-down assay as previously described (65). The 35S-Met-labeled proteinswere produced by in vitro translation (Promega TNT coupled in vitro transcrip-tion-translation kit) with either T7 or SP6 RNA polymerase. To produce C-terminal Oct-1 protein truncations, plasmid DNAs were restricted prior to invitro translation. To assay for binding, labeled proteins were incubated with 0.5mg of GST fusion protein attached to glutathione-Sepharose in binding buffer(20 mM HEPES [pH 7.9], 60 mM KCl, 12% glycerol, 1.5 mM EDTA, 1 mMdithiothreitol, 0.15 mM phenylmethylsulfonyl fluoride [PMSF], 0.1% NonidetP-40 [NP-40]) for 2 h at 4°C. Following extensive washing with binding buffer, thesamples were eluted by boiling for 5 minutes in sodium dodecyl sulfate (SDS)sample buffer prior to SDS-polyacrylamide gel electrophoresis (PAGE) analysis.The gels were dried and visualized by fluorography.

For coimmunoprecipitation assays, in vitro-translated Oct-2FL, Oct-2DSP,Oct-2DHD, Oct-2DPOU, dlx2, and hoxd4 were incubated with c-myc-tagged GRimmunoprecipitated with antibody 9E10 from nuclear extracts prepared fromdexamethasone-treated, stably expressing SF-7 fibroblasts or the parental cellline as previously described (65). Following extensive washing with bindingbuffer, the bound proteins were resolved by SDS-PAGE and visualized by fluo-rography. Myc-GR loading in each experiment was confirmed by Western blotanalysis with antibody 9E10 and visualized by enhanced chemiluminescence(Amersham). Phosphorimager quantification (Bio-Rad model GS-525) of theSDS-polyacrylamide gels and the immunoblots was done with background sub-traction.

For the direct binding assay, GST-GR WT and mutants tagged with a PKAphosphorylation site were expressed in bacteria. The GST fusion proteins werefirst immobilized on glutathione-Sepharose in TEGz50 buffer (50 mM Tris [pH

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7.5], 50 mM NaCl, 10% glycerol, 0.5 mM EDTA, 50 mM ZnCl2, 0.5 mM PMSF)plus 1% Triton X-100 and then labeled with [g-32P]ATP by a kinase reaction withthe catalytic subunit of PKA (Sigma) in HMK buffer (20 mM Tris [pH 7.5], 100mM NaCl, 12 mM MgCl2, 1 mM dithiothreitol) for 30 min at 30°C. The reactionwas terminated by the addition of 1 ml of stop buffer (10 mM NaPO4, 10 mMNa4O7P2, 10 mM EDTA, 2 mg of bovine serum albumin [BSA] per ml). Thelabeled proteins were eluted in TEGz50 buffer (23) plus 1% Triton X-100 bythrombin (Sigma) cleavage. Approximately 5 ng of the 32P-labeled peptides wasincubated with immobilized GST fusion proteins in binding buffer in the pres-ence of 2 mg of BSA per ml and 1 mM PMSF. Following extensive washing, thebound proteins were eluted in SDS sample buffer, resolved by SDS-PAGE, andvisualized by autoradiography. The input GST-homeodomain proteins and BSAwere resolved by SDS-PAGE and visualized by Coomassie blue staining.

Tissue cultures and transient transfections. CHO-K1 cells (American TypeCulture Collection [ATCC]) were maintained in a-minimal essential mediumsupplemented with 10% fetal bovine serum (Life Technologies). SF-7 and clonalcell lines (65) were maintained in Dulbecco’s modified Eagle’s medium supple-mented with 10% fetal bovine serum.

For the mammalian two-hybrid assay, bait plasmids pCGN-Oct-2, pTL2-hoxd4, pTL2-dlx2, and pSG5CREB were cotransfected with pGAL4-GR-DBD,pGAL4-GRL501P, or pGALO and pG5E1BCAT into CHO cells (ATCC) withLipofectamine (10 ml per 60-mm dish; 50 ng of the chloramphenicol acetyltrans-ferase [CAT] reporter, 50 ng of the GAL4 expression plasmid, and 25 ng of thebait plasmid). Cells were harvested 48 h posttransfection. The CAT activities ofcytoplasmic extracts were determined by standard protocols and are expressed asfold induction of GAL4-GR DBD or GAL4-GRL501P over GALO. pRSV-bGAL(50 ng) was cotransfected to provide an internal control for transfection effi-ciency. All transfections and CAT assays were performed in duplicate in at leastthree independent assays. Error bars represent the standard errors of the mean.

Fish and embryos. Zebra fish eggs were obtained by natural spawning from acolony of fish derived from stock from a local fish supplier. Embryos were raisedat a constant 28°C temperature in system water or in embryo medium by stan-dard methods (103). Developmental stages are given in hours postfertilization(p.f.).

In vitro transcription and microinjection. mRNAs of the GR DBD wereproduced in vitro by linearizing the pT7 plasmids with EcoRV, pET-11a-OCT2HD with BamHI, and pET-11a-MR-DBD with HindIII and transcribingwith T7 polymerase by use of an mRNA capping kit (Stratagene, La Jolla, Calif.).Following ethanol precipitation, the RNAs were resuspended in filtered distilledwater. The purified mRNAs were quantified on a Bio-Rad Gel Doc systemfollowing agarose gel electrophoresis. Injections of 0.2 to 0.4 nl of synthetic,capped RNA at concentrations of 100 to 600 mg/ml were given to one- ortwo-cell-stage zebra fish embryos by use of backfilled capillaries (Flaming/Brownmicropipette puller; Sutter Instrument Co., Novato, Calif.) and a pressure-pulsedmicroinjector (PV830 Pneumatic Picopump; WPI, Sarasota, Fla.). Injected em-bryos were raised in embryo medium and monitored at 2-h intervals and at othertimes as specificallly indicated. Death and developmental abnormalities wererecorded to 24 h p.f. Embryos were photographed with a Leica stereomicro-scope. Embryos stored for in situ analysis were fixed in phosphate-buffered saline(pH 7.4) containing 4% paraformaldehyde overnight at 4°C, the chorion wasmanually removed, and the samples were stored in methanol at 220°C.

In situ hybridizations. In situ hybridization with whole-mount zebra fish em-bryos was performed as described previously (2, 3) by use of nonhydrolyzedantisense RNA probes. Enzymes used for linearization and for transcription forprobe synthesis were as follows: ntl (82), XhoI and T7 RNA polymerase (T7); shh(44), BglII and T7; flh (95), EcoRI and T7; axial (93), DraI and T3; MyoD (102),EcoRI and T7; gsc (92), BamHI and T7; and bmp4 (57), EcoRI and T7.

Far-Western analysis of proteins binding to the GR DBD in embryonic ex-tracts. Embryo lysates were prepared from zebra fish embryos collected betweencell cycles 8 and 10 (early blastula stages) and from embryos at 50% epiboly(early gastrula stage). The dissected embryos were placed in cell lysis buffer (25mM HEPES [pH 7.9], 100 mM KCl, 20% glycerol, 0.2 mM PMSF, 2 mM EDTA,2 mM DTT, 0.01% NP-40) (1 ml per embryo) at 4°C, and the cells were dispersedby vortexing. An equal volume of 23 gel loading buffer (100 mM Tris-HCl, [pH6.8], 200 mM DTT, 4% SDS, 0.2% bromophenol blue, 20% glycerol) was added.Lysate (25 mg) from the early blastula or the early gastrula stage was loaded andresolved by SDS–12% PAGE. Following electrophoresis, the proteins weretransferred to polyvinylidene difluoride membranes (Millipore). Denaturationand renaturation of filter-bound proteins were accomplished by immersing themembranes in 6 M guanidine HCl, gently agitating the mixture at room temper-ature for 10 min, and renaturing the samples by 1:1 serial dilutions with binding

buffer (5, 7). The membrane was washed in binding buffer for 10 min, and thefilter was blocked with 3% BSA (wt/vol) for a minimum 1 h at 20°C. The filterwas then incubated with 32P-labeled GRC460Y (GR carrying a C460Y substitu-tion) or GRC460Y/L501P (GR carrying C460Y and L501P substitutions) (in bind-ing buffer containing 0.3% BSA; the final specific activity was about 106 cpm perml) overnight at 4°C. After five 15-min washes with binding buffer, the filter wasexposed to X-AR film (Kodak).

RESULTS

The octamer factor homeodomain is sufficient for C500Y-and L501P-sensitive GR DBD binding in vitro. To begin todelimit the requirements within full-length Oct-1 and Oct-2 forbinding to steroid receptors, we examined the binding of C-terminally truncated forms of Oct-1 to the GR DBD (aa 407 to568) by using a GST pull-down assay (Fig. 1A). In vitro-trans-lated full-length Oct-1 bound specifically to the WT GR DBDfused to GST but did not interact with GST-GRC500Y (Fig. 1A,lanes 1 and 2). Deletion of the Oct-1 C terminus up to thehomeodomain had only a slight effect on the interaction withGR (Fig. 1A, lane 3). Further truncation into the homeodo-main, however, abrogated binding (Fig. 1A, lanes 4 and 5). Inan additional experiment, it was confirmed that binding of theGR DBD to the Oct-1 homeodomain is distinct from thebinding previously reported for herpes simplex virus protein 16(VP16), as substitutions in the Oct-1 homeodomain that havebeen shown previously to disrupt VP16 binding (47) had noeffect on GR binding in this assay (66).

Similarly, immunoprecipitation assays performed with invitro-translated Oct-2 constructs containing internal deletionsand full-length GR with an N-terminal c-myc tag in crudenuclear extracts prepared from murine SF-7 cells indicatedthat deletion of Oct-2HD was sufficient to eliminate GR bind-ing (Fig. 1B). While full-length Oct-2 bound strongly to immu-noprecipitated GR (Fig. 1B, lane 1), deletion of the entirePOU domain or the POU homeodomain alone from full-length Oct-2 eliminated binding (Fig. 1B, lanes 3 and 4). In-terestingly, deletion of the POU-specific domain alone de-creased binding to the GR DBD somewhat (Fig. 1B, lane 2).This result may suggest a role for the POU-specific domain inGR binding. However, as the result was not supported insubsequent experiments, it would seem more likely that itreflects a decrease in the accessibility of the homeodomain toGR when the adjacent POU-specific domain was deleted fromOct-2.

To resolve whether the octamer factor homeodomain wassufficient for GR binding and to determine whether bindingwas direct, we performed a binding assay with purified com-ponents that had been expressed in bacteria. WT and L501P-substituted GR DBDs (aa 407 to 550) containing PKA recog-nition sequences were expressed as GST fusion proteins,labeled with 32P by use of PKA, and then separated from theGST moiety by cleavage with thrombin. The purified GR DBDpeptides were tested for binding to the complete Oct-2 POUdomain (aa 194 to 377) or to Oct-2HD (aa 294 to 377) ex-pressed as GST fusion proteins and bound to glutathione-Sepharose (Fig. 2). The WT GR DBD bound strongly to boththe complete POU domain and the POU homeodomain alone

FIG. 1. The association of Oct-1 and Oct-2 with GR is lost upon deletion of the Oct homeodomains. (A) Binding of in vitro-translated, 35S-Met-labeled Oct-1peptides to the WT GR DBD (aa 407 to 568; lanes 2 to 5) or the GR DBD with a C500Y substitution (lane 1), expressed as GST fusion proteins. Lanes 6 to 9 showthe signal obtained with 10% of the labeled proteins added to the binding assay. (B) Binding of full-length, 35S-labeled, in vitro-translated Oct-2 (lane 1), Oct-2 deletionconstructs lacking the POU-specific domain (DSP) (lane 2), the POU homeodomain (DHD) (lane 3), and the complete POU domain (DPOU) (lane 4), and fireflyluciferase (Luc; lane 5) to full-length GR with a c-myc epitope tag. Samples were immunoprecipitated with c-myc antibody 9E10 from whole-cell extracts prepared fromdexamethasone-treated, stably transfected murine SF-7 fibroblasts. Lanes 6 to 10 show the signal obtained with 10% of the labeled proteins added to the binding assay.In both panels A and B, bound peptides were resolved by SDS-PAGE (12% gels) and visualized by fluorography.



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(Fig. 2, lanes 2 and 3), but no binding was detected to GSTalone (Fig. 2, lane 4). Further, the L501P substitution in theGR DBD prevented binding (Fig. 2, lanes 6 to 8). Thus, Oct-2HD was sufficient for direct binding to the GR DBD in vitro.

As the homeodomain is remarkably well conserved, we nextbecame interested in determining whether GR might also binddirectly to the homeodomains of other proteins. To first test

this possibility in vitro, we expressed the homeodomains ofseveral proteins as GST fusion proteins (Fig. 3A) and testedthem for binding to 32P-labeled GR DBD peptides (Fig. 3Band C). Remarkably, all of the homeodomains tested bound tothe WT GR DBD peptides (Fig. 3B, lanes 2 to 8), but noneinteracted significantly with the GR DBD peptide containingthe L501P substitution (Fig. 3C, lanes 2 to 8). By comparisonwith the input GST-homeodomain proteins, binding was stron-gest to the HoxC4 homeodomain (Fig. 3B, lane 4) and tofull-length dlx2 (Fig. 3B, lane 3) and was weakest to the Prdhomeodomain.

L501P-sensitive binding of GR to homeodomain proteins invivo. To determine whether GR DBD binding to full-lengthhomeodomain proteins could be detected in the cell, we se-lected two zebra fish homeodomain proteins, hoxd4 and dlx2,which are unrelated outside the homeodomain and only dis-tantly related to Oct-1 and Oct-2 within the homeodomainfamily, for further study. hoxd4 is a close zebra fish relative ofhuman HoxC4, whose homeodomain bound to the GR DBD(see above). First, we compared the interaction of the GRDBD with hoxd4 and dlx2 in a one-hybrid assay performedwith CHO cells (Fig. 4). In these experiments, the activity ofthe CAT reporter gene used was dependent upon a specificassociation of the GAL4-GR DBD fusion proteins and thefull-length homeodomain proteins. Transactivation was medi-ated by the natural transcriptional activation domains withinOct-2, hoxd4, and dlx2. Expression of the GAL4 DBD alone orfused to the GR DBD peptides had no significant effect ontranscription of the CAT reporter gene (66). However, coex-pression of full-length Oct-2 with the WT GAL4-GR DBDfusion protein potentiated CAT activity six- to eightfold abovethe level obtained with the GAL4 DBD alone. This inductionin activity was completely sensitive to the L501P mutation inthe GR DBD, as coexpression of Oct-2 with the GAL4-

FIG. 2. The GR DBD binds directly to Oct-2HD. GST-tagged WT GR DBDand GRL501P DBD (aa 407 to 550) containing PKA consensus phosphorylationsites were expressed in and purified to homogeneity from Escherichia coli andlabeled with 32P by use of PKA. Signals obtained with 10% of the 32P-labeled GRand GRL501P DBDs added to the binding assay are shown in lanes 1 and 5. The32P-labeled GR peptides were tested for binding to the POU domain and POUhomeodomain of Oct-2 or to GST alone (lanes 2 to 4 and 6 to 8, respectively).MW, molecular weight (in thousands).

FIG. 3. The GR DBD binds directly to the homeodomains of several pro-teins. (A) Coomassie blue-stained SDS-polyacrylamide gel of 0.5 mg of BSA(lane 1) and GST fusion proteins containing human Oct-2HD (lane 2), full-lengthzebra fish dlx2 (lane 3), and the homeodomains of human HoxC4 (lane 4),Drosophila paired (Prd, lane 5), Drosophila orthodenticle (Otd, lane 6), zebra fishmsxB (lane 7), and Drosophila fushi tarazu (Ftz, lane 8). MW, molecular weight(in thousands). (B and C) Autoradiographs of SDS-polyacrylamide gels showingthe binding of 32P-labeled GR DBD (B) and GRL501P DBD (C) peptides to theGST fusion proteins shown in panel A. Lanes 1 show the signal from 10% of the32P-labeled peptides added to each incubation, while lane 9 shows binding to aglutathione-Sepharose extract from mock-transformed bacterial cells.

FIG. 4. One-hybrid analysis of the L501P-sensitive interaction of the GRDBD with full-length Oct-2, hoxd4, and dlx2 in mammalian cells. The GR DBD,the DBD with an L501P substitution fused to the GAL4 DBD, or the GAL4DBD alone was coexpressed with Oct-2, hoxd4, dlx2, or CREB in CHO cells. At48 h after transfection, transcription from a reporter gene with an E1B minimalpromoter and five GAL4 binding sites was assessed by a CAT assay. The data areexpressed as the fold induction of CAT activity in the presence of GAL-GRDBDL501P (hatched bars) and GAL-GR DBD (solid bars) versus in the presenceof GAL4 DBD (GALO). The error bars represent the standard error of themean of three to five independent experiments performed in duplicate (P ,0.02).

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GRL501P fusion protein had no effect on reporter gene expres-sion.

Coexpression of full-length hoxd4 and dlx2 with theGAL4-GR DBD fusion proteins resulted in similar GRL501P-sensitive activation of transcription from the E1B promoter. Incontrast, coexpression of the bZip transcription factor CREBhad no significant effect on reporter gene activity under theseconditions. These results indicated that specific binding to theGR DBD in the cell occurred for at least two homeodomainproteins outside the octamer transcription factor subfamily.

To confirm further the potential for interactions betweenfull-length GR and full-length homeodomain proteins, we ex-amined the ability of in vitro-translated dlx2 and hoxd4 to bindto full-length, ligand-activated GRs extracted from the nucleusof murine fibroblasts (Fig. 5). Previously, we had demonstratedthat full-length GR and both Oct-1 and Oct-2 associated in thisassay in a manner that was completely sensitive to the GRL501Pmutation (64). Similarly, both dlx2 and hoxd4 were observed tobind WT GR from extracts prepared from dexamethasone-treated cells (Fig. 5A, lanes 2 and 6). In contrast, no bindingwas observed with extracts prepared from cells expressingGRL501P (Fig. 5A, lanes 3 and 7) or with extracts preparedfrom control parental cells lacking a stably expressed GR (Fig.5A, lanes 4 and 8).

Together, these results indicated, through three indepen-

dent assays, that a peptide encompassing the GR DBD couldbind directly to the homeodomains of distantly related home-odomain proteins. Further, in all these assays, binding wassensitive to an L501P substitution in the DNA-contacting a-he-lix of the second zinc finger.

L501P-sensitive, DNA-independent effects of the GR DBDon early embryogenesis in zebra fish. The homeodomain me-diates DNA binding and protein-protein interactions that de-termine a large component of homeodomain protein functionin the cell (73). Recent gene replacement experiments havesuggested that DNA-independent functions of GR are impor-tant components of the steroid response (69). The GR DBDcontains several interfaces for protein-protein interactions thatparadoxically may mediate at least some of these DNA-inde-pendent functions of GR (37). The diverse nature of GRDBD-homeodomain binding observed in our molecular assayssuggested that ectopic expression of the GR-DBD in wholeanimals might affect the normal activities of at least some ofthe proteins that can be contacted by the GR DBD. Further,parallel experiments with the L501P substitution would beexpected to reveal effects specific to the surface involved inhomeodomain binding.

To test our hypothesis, we examined the consequences fordevelopment of introducing mRNAs encoding GR DBD pep-tides into one- or two-cell-stage zebra fish embryos. Zebra fishare particularly well suited for studies examining the develop-mental consequences of the ectopic expression of peptidesduring embryogenesis (19, 41). Eggs are fertilized externally,and proteins can be reliably expressed by microinjection ofmRNA immediately following fertilization. Analysis of devel-opmental abnormalities that may be induced is facilitated bythe optical clarity and rapid development of the embryos (40).Further, preliminary microinjection experiments with mRNAencoding green fluorescent protein (GFP) established that the

FIG. 5. The binding of full-length GR in nuclear extracts to full-length ze-brafish dlx2 and hoxd4 is sensitive to GRL501P. (A) Binding of full-length, 35S-labeled, in vitro-translated dlx2 (lanes 2 and 3) and hoxd4 (lanes 6 and 7) to GRand GRL501P immunoprecipitated with c-myc antibody 9E10 from whole-cellextracts prepared from dexamethasone-treated, stably transfected murine SF-7fibroblasts. Lanes 4 and 8 show binding to extracts prepared from untransfectedSF-7 cells, while lanes 1 and 5 show 10% of the in vitro-translated homeodomainproteins added to the binding assay. (B) Western blot of the GRs immunopre-cipitated from stably transfected SF-7 cells expressing WT GR and GRL501P andfrom untransfected SF-7 cells (lanes 9 to 11). Below each lane in panels A andB are the counts obtained following phosphorimager analysis.

FIG. 6. Microinjection of mRNA encoding a GR peptide with the homeodo-main binding surface into one- or two-cell-stage zebra fish embryos interfereswith embryogenesis in an L501P-sensitive manner. The outcome at 24 h isdisplayed for one- or two-cell-stage embryos injected with 0.2 to 0.4 nl of RNAs(600 mg/ml) encoding GR mutant peptides with a GR DBD backbone (aa 407 to556), an antisense transcript of the WT GR, or the MR DBD (aa 567 to 700) orwith saline alone. The peptides encoded by the RNAs injected are listed at theleft, followed by the total number of embryos injected (n) and the total numberof independent injection series performed with each sample. To the right, thepercentage of embryos failing to survive for 24 h following injection is indicatedby dark gray bars, while the percentage of embryos with malformations visibleunder the stereomicroscope is indicated by light gray bars. The error bars indi-cate the standard deviations for independent trials (for GRC460Y and GRK489Rcompared to the controls, the P value was ,0.001).



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proteins encoded from the microinjected mRNAs in our ex-periments were expressed uniformly through 24 h of develop-ment in almost all cells of the embryos (99).

All of the GR constructs expressed in the embryos in our

experiments contained the primary GR nuclear localizationsequence to ensure that the peptides expressed would be con-centrated in the embryonic nuclei. Further, to exclude effectson development that might result from the binding of GRDBD to DNA, the GR DBD peptides expressed also containedamino acid substitutions C460Y, K489R, and/or L501P, whichcompromise the DNA binding of GR (77). Although C460Yand K489R interfere with GR DNA binding, they were notobserved to affect binding to the POU domain of Oct-1 (65).

In the first experiments, the status of the microinjected em-bryos was examined 24 h after injection, at the end of thesegmentation period. The results of these experiments arepresented in Fig. 6 and 7 and in Table 1.

Microinjection of mRNAs encoding aa 407 to 556 of GRwith a C460Y or K489R substitution severely affected embry-

FIG. 7. Examples of the four major classes of visible defects in zebra fish embryos 24 h following injection with GRC460Y. From microinjected embryos, the chorionwas manually removed, and the samples were photographed under a stereomicroscope at a magnification of 363. Anterior is to the left; dorsal is to the top. (A) Anormally developed control embryo injected with saline. (B to D) Examples of embryos lacking a distinct axis. (E) Embryo with a curved tail. (F) Embryo lacking botha head and a tail. (G) Embryo missing a head and with a truncated tail. The arrowheads indicate the axial mesoderm (B to D), and the arrow indicates the somites(C). Scale bar: A, 130 mm; B to G, 106 mm.

TABLE 1. Dose response of the microinjection of GR DBD-encoding mRNA into zebra fish embryos

pg of GRC460Yinjected/embryo

%a of embryos at 24 h showing:

Failure to survive Visible malformations

180 53 6 4.2 24 6 3.430 30.3 6 4.6 43 6 6.8

a Mean 6 standard deviation from at least four independent injections.



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onic survival (Fig. 6). Fewer than 50% of the embryos injectedwith either mRNA survived to 24 h. In contrast, only approx-imately 10% of embryos microinjected with saline or an anti-sense GR DBD-encoding mRNA failed to develop to 24 h.This value is well within the percentage of growth arrest that isexpected to result from the mechanical manipulation andpuncture of the embryos in this procedure (6, 24). Moreover,the mean time to arrest for the embryos injected with the testmRNAs was 8 to 10 h, during late gastrulation, while the meantime to arrest for the control embryos was approximately 12 h(99). Remarkably, the development of embryos microinjectedwith mRNA encoding GR DBD with an L501P mutation or acombination of C460Y and L501P mutations was indistinguish-able from that of the control embryos (Fig. 6). Moreover, theeffects on development were highly specific to expression of theGR DBD. In contrast to the severe effects of the ectopic ex-pression of the GR DBD peptides, microinjection of mRNAencoding comparable DBD peptide from MR, which differsfrom GR by only 4 aa within the core of the DBD but whichdoes not interact with the octamer factor homeodomains(65), resulted in completely normal embryonic developmentthat was indistinguishable from that of all of the controls(Fig. 6).

In addition, over 50% of the embryos that were microin-jected with the GRC460Y- and GRK489R-encoding mRNAs andthat survived to 24 h were afflicted with axial abnormalities thatwere readily visible under the stereomicroscope. In contrast,malformations of embryos microinjected with L501P-substi-tuted and C460Y- and L501P-substituted GR DBDs and withthe MR DBD occurred at the same low frequency as thatobserved for embryos microinjected with antisense mRNA orsaline.

Several examples of the developmental abnormalities ob-served at 24 h p.f. for embryos injected with the GRC460Yconstruct are shown in Fig. 7. The predominant phenotypeobserved (46% 6 7%) was dramatic underdevelopment, withembryos lacking a unitary body axis as well as differentiatedanteroposterior structures (Fig. 7B to D). These embryos gen-erally exhibited distinct notochord-like regions that were sep-arated by lobes of yolk. Many embryos (25% 6 5%) containeda nearly normal axis in the trunk region but lacked head andtail structures (Fig. 7F and G), while the rest exhibited defectsprimarily in the development of the tail (29% 6 8%) (Fig. 7E).

The severity of the phenotypes observed correlated directlywith the quantity of GR DBD mRNA microinjected into theembryos (Table 1). A decrease in the concentration of theGRC460Y peptide injected from 600 to 100 mg/ml led to a 40%decrease in the number of embryos whose development wasarrested prior to 24 h. In addition, at 100 mg/ml there was aconcomitant increase in the percentage of severely malformedembryos that survived to 24 h.

Together, these results provided a strong indication that theregion of the GR DBD including the homeodomain bindinginterface specifically interfered with some early events impor-tant for the axial development of the embryos.

Expression of the GRC460Y peptide perturbs the formationof the axial mesoderm. The phenotype of the malformed em-bryos uniformly suggested that the defects induced by the GRDBD peptides most likely resulted from defects in the forma-tion of the axial mesoderm, in particular, notochord formation.To examine in greater detail the nature of the effect of the GRpeptides on the early development of the embryo, we analyzedthe expression of several markers for the early development ofthe axial mesoderm and notochord by in situ hybridization withmicroinjected whole-mount embryos.

The zebra fish no tail (ntl) gene is a member of the T-box

gene family (82–84) and is likely the orthologue of the mam-malian Brachyury gene, a developmental control gene whichencodes a transcription factor directly implicated in the for-mation of the primitive streak (68, 105). In zebra fish, ntl isrequired for notochord and tail formation (28, 82).

In wild-type embryos, ntl expression is first detected at thelate blastula stage (4 h p.f.) in a few dispersed cells at the dorsalside of the blastula (84). Later, ntl is expressed in cells of thepresumptive mesoderm of the germ ring (or marginal zone)and at the early gastrula stage (5.5 h p.f.). It is also activated incells of the embryonic shield at the dorsal midline (Fig. 8A).The expression of ntl is specifically maintained in the axialmesoderm as cells migrate away from the blastoderm margin(Fig. 8F). By the end of gastrulation, ntl expression is confinedto the developing notochord cells in the axial mesoderm and toboth axial and nonaxial cells in the developing tail bud (Fig.8K). After 24 h of development, ntl expression is localized tothe notochord cells of the tail (Fig. 8P).

The pattern of expression of ntl in embryos injected withRNAs encoding GR DBDs with the C460Y and L501P muta-tions was similar to that observed in control embryos at alldevelopmental stages (Fig. 8E, J, and O). In contrast, micro-injection of RNA encoding GR DBD with the C460Y muta-tion severely affected ntl expression from the earliest stagesanalyzed. For the majority of the embryos, blastoderm migra-tion over the yolk cell had not yet reached 50% epiboly, asexpected at 5.5 h p.f. (Fig. 8B to D). In a large proportion ofthe GRC460Y-injected embryos, ntl transcripts were found in afew cells at the dorsal margin of the blastoderm, a patternsimilar to that observed in normal embryos at 4 h p.f. (84),suggesting a developmental delay in these embryos (Fig. 8B).Strikingly, in the rest of the embryos, ntl expression was eitherinterrupted in the marginal zone (Fig. 8C) or was seen as asmall indentation (Fig. 8D) at the dorsal side of the embryos.These observations were suggestive of defects in the formationof the embryonic shield.

At 8.5 h p.f., while ntl expression in GRC460Y-injected em-bryos appeared essentially normal in cells of the germ ring,expression in the notochord precursors was affected to variousdegrees (Fig. 8G to I). In the less affected embryos, the domainof ntl-expressing cells in the axial mesoderm was wider and lessanteriorly extended than in the control embryos, a findingwhich may have reflected defects in the convergence and ex-tension movements of the cells during gastrulation (compareFig. 8F and G). In more affected embryos, in addition to adelay in development, it seemed that the shield area was de-void of ntl-expressing cells (Fig. 8H and I). Nevertheless, in-volution movement probably occurred, since rudiments ofstripes of cells extending anteriorly were visible starting fromthe extremities of the germ ring surrounding the normal posi-tion of the shield (Fig. 8H and I). These kind of patterns wouldhave presumably led to embryos (if viable) with partially du-plicated axes.

At later stages (12 h), ntl expression revealed that mostsurviving embryos had a short and kinked notochord axis (Fig.8L and M), suggesting that extension movements toward theanterior part of the embryos may have been affected. Someembryos at the tail bud stage had two distinct short duplicatedaxes as a possible consequence of convergence defects duringthe gastrula stage (Fig. 8N). Finally, among the embryos sur-viving to 24 h p.f., we observed the presence of a short sec-ondary notochord axis expressing ntl and developing from thetail region of the embryos, a finding which may have been theresult of a late duplication of the notochord axis during theelongation period of the embryos (Fig. 8Q).

To confirm and expand on the observations obtained with



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ntl, we examined the expression of other markers of the axialmesoderm in the microinjected embryos (Fig. 9). These mark-ers included sonic hedgehog (shh), which encodes a signalingprotein involved in the differentiation and patterning of vari-ous embryonic tissues, including the notochord and the floorplate cells of the neural tube (44); the homeobox gene floatinghead (flh), the zebra fish ortholog of the Xenopus gene Xnot,which is involved in notochord development (95); and axial,the ortholog of the mouse HNF-3b gene, which encodes atranscription factor of the winged-helix family and which hasbeen shown to be essential for the development of the axialmesoderm (93) (Fig. 9A, E, and I). Injection of zebra fishembryos with RNAs encoding GR DBDs carrying the C460Yand L501P mutations did not significantly alter the expressionof these markers (Fig. 9D, H, and L). In contrast, the expres-sion of the GRC460Y peptide severely affected the pattern ofexpression of shh, flh, and axial in the developing axial meso-derm in ways that were strikingly similar to those observed for

ntl (Fig. 9B, C, F, G, J, and K). Together, these results stronglysupport our conclusion that the GR DBD peptides interferedwith early embryonic developmental processes required for thenormal formation of the axial mesoderm.

To examine whether the GR DBD peptides also affecteddevelopment beyond the axial mesoderm, we analyzed theexpression of MyoD, a gene encoding a basic helix-loop-helixprotein expressed in presomitic mesoderm during gastrulationand then in somites (Fig. 9M to P) (21, 102). We observed thatthe expression of MyoD in 10-h zebra fish embryos injectedwith RNA encoding the GRC460Y/L501P peptide did not signif-icantly differ from that in control embryos (Fig. 9M and P). Atthis stage, MyoD transcripts are normally found in two elon-gating rows of cells, the adaxial cells, adjacent to the axialmesoderm. These cells differentiate into the slow muscle fibersof the zebra fish myotome (21). In contrast, the pattern ofexpression of MyoD in GRC460Y-injected embryos showed var-ious degrees of disruption, ranging from more widely sepa-

FIG. 8. The expression of the GRC460Y peptide in zebra fish embryos disrupts the pattern of no tail expression during embryogenesis. In situ hybridization ofwhole-mount zebra fish embryos with a no tail antisense RNA probe at the indicated times after fertilization is shown. Embryos were injected at the one- or two-cellstage with saline (mock) (A, F, K, and P) or mRNAs encoding the GR DBD (aa 407 to 556) with C460Y (B to D, G to I, L to N, and Q) or C460Y and L501P (E,J, and O) mutations. Three examples of the hybridization patterns observed upon expression of the GRC460Y peptide at each time point are shown. Dorsal views areshown in panels A and C to O, while an animal pole view is shown in panel B and a lateral view of the tail is shown in panels P and Q. The arrow in panel Q indicatesthe secondary notochord axis. a, axial mesoderm; gr, germ ring; es, embryonic shield; n, notochord. Scale bar: A to O, 100 mm; P and Q, 70 mm.



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rated stripes of adaxial cells (Fig. 9N) to a complete disruptionof MyoD expression (Fig. 9O), reflecting the defects observedin the patterns of the axial markers.

As our analysis of axial markers indicated defects in theformation of the embryonic shield, we extended our analysisfurther to examine the expression of goosecoid (gsc), a ho-meobox gene that has been proposed to participate in theestablishment and maintenance of the organizer and shield(16, 84, 92). Furthermore, gsc is expressed very early duringzebra fish embryogenesis; it is first maternally and ubiquitouslyexpressed in the embryo, and then zygotic transcripts are de-tected at 4 h p.f., shortly after the midblastula transition (84,92). Therefore, it is a valuable marker for analyzing the devel-opmental effects of GR peptides prior to gastrulation.

At the late blastula stage, gsc is expressed as a patch of cellsat the margin of the blastoderm (84, 92). At the onset ofgastrulation, the domain of gsc expression is limited to the

shield region (Fig. 10A). As was observed with the other axialmarkers, only embryos injected with RNA encoding theGRC460Y/L501P peptide reproduced the normal pattern of ex-pression of gsc at that stage (Fig. 10C).

Even taking into account the developmental delay of theseembryos, the pattern of expression of gsc differed from thenormal expression of gsc observed in noninjected embryos atthe late blastula stage. Indeed, at 5.5 h p.f., a time at whichembryos injected with the GRC460Y-encoding RNA had anappearance normally associated with 4.7 h p.f. (30% epiboly),gsc was expressed only in a very small subset of cells in thedorsal area of the embryo but was not localized at the embry-onic margin (Fig. 10B), as expected for WT embryos at 30%epiboly. This observation lends further credence to the sugges-tion that the misexpression of the GRC460Y peptide affectedevents occurring very early during development.

As gastrulation proceeds, gsc transcripts are restricted to the

FIG. 9. Whole-mount in situ hybridization showing the expression of developmental markers for axial mesoderm (a) (A to L) and paraxial mesoderm (p) (M toP) formation in 12-h embryos. Embryos were injected at the one- or two-cell stage with saline (mock) (A, E, I, and M) or mRNAs encoding the GR DBD (aa 407 to556) with C460Y (B, C, F, G, J, K, N, and O) or C460Y and L501P (D, H, L, and P) mutations. Dorsal views of the patterns of expression of sonic hedgehog (shh, Ato D), floating head (flh, E to H), axial (I to L), and MyoD (M to P) in 12-h embryos are shown. In GRC460Y/L501P-injected embryos (D, H, L, and P), the patterns ofexpression of shh, flh, and axial in the developing notochord and of MyoD in the paraxial mesoderm do not differ significantly from those observed in control embryos(A, E, I, and M). In contrast, in GRC460Y-injected embryos, the shh, flh, axial, and MyoD patterns of expression are highly perturbed (B, C, F, G, J, K, N, and O). tb,tail bud. Scale bar: A to P, 140 mm.



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anterior edge of the progressing shield and start to be ex-pressed in a rostral crescent of cells of the prechordal plate andsome cells of the anterior part of the dorsal midline (Fig. 10D).While embryos injected with GRC460Y/L501P-encoding RNAhad a gsc expression pattern similar to that of control embryos,GRC460Y-injected embryos always had a wider gsc domain ofexpression in the dorsal midline and rarely showed the char-

acteristic crescent-shaped expression in the prechordal platecells (Fig. 10E and F).

To determine whether, in addition to the observed dorsaldefects, the expression of the GRC460Y peptide also generatedventral defects in the embryos, we analyzed the expression ofthe bone morphogenetic gene, bmp4 (41, 57), which is involvedin the ventral patterning of zebra fish embryos (Fig. 11). At the

FIG. 10. Whole-mount in situ hybridization showing the expression of goosecoid in microinjected embryos. Embryos were injected at the one- or two-cell stage withsaline (mock) (A and D) or mRNAs encoding the GR DBD (aa 407 to 556) with C460Y (B and E) or C460Y and L501P (C and F) mutations. Patterns of expressionof goosecoid at 5.5 h (A to C) and 12 h (D to F) are shown. Dorsal views are shown in panels A, B, and D to F, while an animal pole view is shown in panel C. Injectionof GR DBDC460Y/L501P failed to modify gsc expression at all developmental stages (compare panels A, D, C, and F). (A and C) At the onset of gastrulation, gsc isexpressed in the embryonic shield on the dorsal part of the embryo (indicated by an arrowhead in panel A). (B) Blastoderm migration in most GRC460Y-injectedembryos is delayed compared to that in control embryos and GRC460Y/L501P-injected embryos, and gsc expression is greatly reduced. (D and F) gsc expression is confinedto the prechordal plate (indicated by an arrow in panel D) and cells of the anterior part of the dorsal midline (indicated by an arrowhead in panel D). (E) InGRC460Y-injected embryos, gsc is expressed in a cluster of cells without any distinct pattern; in particular, no rostral crescent corresponding to the prechordal mesodermis visible. Scale bar: A to F, 106 mm.

FIG. 11. Whole-mount in situ hybridization showing the expression of bmp4 in microinjected embryos at the shield stage. Embryos were injected at the one- ortwo-cell stage with saline (mock) (A) or mRNAs encoding the GR DBD (aa 407 to 556) with C460Y (B) or C460Y and L501P (C) mutations. Animal pole views ofembryos are oriented with their ventral area to the left and their dorsal area to the right. Note that the bmp4 pattern of expression is affected only in the dorsal regionof GRC460Y-injected embryos. The arrow indicates the position of the embryonic shield. Scale bar: A to C, 105 mm.



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beginning of gastrulation (shield stage, 6 h p.f.), bmp4 isstrongly expressed on the ventral region of embryos, especiallyin the marginal zone (Fig. 11A). In addition, bmp4 transcriptsare also found on the lateral areas and in the inner cells of theembryonic shield (41, 57). We observed, at the shield stage, alateral expansion of the domain of bmp4 expression on thedorsal region of GRC460Y-injected embryos (Fig. 11B). In con-trast, bmp4 expression on the ventral part of these embryosappeared unchanged. Embryos injected with saline or withGRC460Y/L501P-encoding RNA showed the normal bmp4 pat-tern of expression (Fig. 11C). Similarly, the expression ofbmp2, another member of the bone morphogenetic gene fam-ily, and eve-1, a homeobox gene related to the Drosophilaeven-skipped gene, whose patterns of expression on the ventro-lateral areas of embryos at the early gastrula stage overlapextensively with that of bmp4 (33, 57), remained unchanged inGRC460Y-injected embryos (99). These results suggest that thedevelopmental effects of GR DBD peptide expression may berestricted to the dorsal region of zebra fish embryos.

Developmental defects induced by ectopic expression of theGR DBD are rescued by coexpression of Oct-2HD. To begin toassess the specificity of the developmental defects induced bythe GR DBD peptides, we tested whether the phenotypesinduced by the GRC460Y peptide could be influenced by thecoexpression of a homeodomain peptide. For this experiment,we injected the mRNA encoding GRC460Y alone (180 pg) ortogether with a twofold excess of the mRNA encoding Oct-2HD (360 pg). Oct-2HD was selected for these experiments as itbinds poorly to DNA in the absence of the POU-specific do-main but binds avidly to the GR DBD. The results were strik-ing (Fig. 12). The coexpression of Oct-2HD almost completelyrescued the embryos from the effects of GRC460Y. Fully 65% ofthe coinjected embryos developed without visible defects to

24 h p.f., compared to the nearly 75% of embryos injected withthe GRC460Y-encoding mRNA alone that failed to develop ordisplayed the severely malformed phenotypes illustrated inFig. 7.

The effect of the Oct-2HD-encoding mRNA appeared tooccur in direct opposition to the effect of the GRC460Y peptiderather than by nonspecifically influencing GRC460Y produc-tion. Coinjection of the same amounts of Oct-2HD-encodingmRNA and GFP-encoding mRNA had no effect on the level ofexpression of GFP compared to injection of GFP-encodingmRNA alone, and coinjection of GFP-encoding mRNA andGRC460Y-encoding RNA had no effect on the phenotypes ob-tained (99). Further, as the concentration of the Oct-2HD-encoding mRNA was decreased within the same total mRNAconcentration, the effects of GRC460Y reappeared proportion-ally (99). These results do not in any way prove that the de-velopmental effects of the GR peptides were mediated throughbinding to embryonic homeodomain proteins. However, theydo strongly suggest that the defects resulting from the embry-onic expression of the GR DBD arise from GR DBD-medi-ated protein-protein interactions that overlap with the bindingof the GR to a homeodomain.

Last, our molecular analysis suggested that specific bindingto a protein or proteins expressed in the early embryo inter-fered with their normal function at a time coincident with theonset of embryonic transcription. To obtain direct evidence insupport of this hypothesis, cell lysates prepared from early-blastula- and early-gastrula-stage zebra fish embryos were ex-amined for specific binding to WT and GRL501P DBD peptidesby a far-Western approach (Fig. 13). Extracts prepared fromearly-blastula-stage embryos collected between zygotic cell cy-cles 8 and 11 (approximately between 2.25 and 2.75 h p.f.) werecomposed entirely of proteins derived from maternal mRNAs

FIG. 12. Microinjection of mRNA encoding Oct-2HD into one- or two-cell-stage zebra fish embryos rescues the developmental defects induced by GRC460Y. Theoutcome at 24 h is displayed for one- or two-cell-stage embryos injected with 0.2 to 0.4 nl of RNA encoding GRC460Y (600 mg/ml), saline alone, or RNAs encodingGRC460Y and Oct-2HD (1,200 mg/ml). The peptides encoded by the RNAs injected are listed at the left, followed by the total number of embryos injected (n) and thetotal number of independent injection series performed with each sample. To the right, the percentage of embryos failing to survive for 24 h following injection isindicated by dark gray bars (D), the percentage of embryos with malformations visible under the stereomicroscope is indicated by light gray bars (M), and the percentageof normal embryos is indicated by white bars (N). The error bars indicate the standard deviations for independent trials (for GRC460Y and GRC460Y plus Oct-2compared to the controls, the P value was ,0.001).



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(35). In contrast, in extracts prepared from early-gastrula-stageembryos (collected at 50% epiboly), there was also a strongrepresentation of proteins derived from mRNA transcribedfrom the embryo genome (35, 36, 40).

Following electrophoresis in duplicate on a single gel, trans-fer to a nylon membrane, and renaturation, duplicate sampleswere incubated with equal numbers of counts of 32P-labeledGRC460Y and GRC460Y/L501P peptides labeled to the same spe-cific activity. Exposure of the membrane revealed the presenceof several nuclear factors that were preferentially recognizedby the GRC460Y peptide in several independent experiments.In the extract from the early blastula, two bands at 31 and 28kDa bound the GRC460Y peptide (Fig. 13B, lane 1). Whilebinding to the 31-kDa band was only modestly affected by theL501P mutation, the signal at 28 kDa decreased markedly (Fig.13B, lane 3). Thus, the GR DBD bound specifically to at leastone maternally expressed factor in a manner that was sensitiveto the L501P substitution.

In the extract from the gastrula, the intensity of the signalsobtained with the GRC460Y peptide at 31 and 28 kDa increasedseveralfold (Fig. 13B, lane 2). However, although equalamounts of proteins from blastula and gastrula lysates wereexamined, we cannot rule out the possibility that the increasein signal intensity at 31 and 28 kDa reflected an increase in thenumber of cells expressing these proteins at the gastrula stagerather than an increase in protein synthesis per cell. In additionto the 28- and 31-kDa signals, a third L501P-sensitive signal, at45 kDa, was also detected. Interestingly, in this extract, the 31-kDa signal was highly sensitive to the L501P substitution in theGR DBD peptide (Fig. 13B, lane 4), suggesting that the signaloriginated from a factor different from that in the extract fromthe early blastula. Thus, it appears that the L501P-sensitive de-velopmental defects induced by the expression of the GR DBDmay arise from specific interactions with proteins encoded byeither or both maternal or zygotically transcribed RNAs.

DISCUSSION

Many studies have demonstrated the ability of nuclear hor-mone receptor DBDs to enter into productive protein-protein

interactions with other transcription factors. Elsewhere, wehave demonstrated that three steroid hormone receptors, GR,AR, and PR, can interact productively with the Oct-1 andOct-2 proteins through their DBDs to recruit them to DNAbut that MR is unable to interact with Oct-1 and Oct-2 underthe same conditions (64). In the present work, we determinedthat, when ectopically expressed following mRNA microinjec-tion into one- or two-cell-stage zebra fish embryos, GR but notMR DBD peptides severely perturbed the development of theaxial mesoderm. These effects correlated directly with theL501P-sensitive direct binding of GR to the homeodomains ofseveral homeodomain proteins in vitro and in transfected cellsand were rescued by the coexpression of Oct-2HD in embryos.While the full extent of the influence of GR-homeodomainbinding on homeodomain protein activity in vivo remains to beproven, the sensitivity in our experiments of the developmentaldefects to the same L501P substitution that abrogated GR-homeodomain binding in tissue culture cells is highly sugges-tive of a causal linkage to the developmental effects.

DBD-mediated nuclear receptor-homeodomain protein bind-ing influences homeodomain protein function. The home-odomain contains a highly conserved DNA binding domain.Homeodomain proteins generally bind to DNA sequencescontaining a highly redundant core motif. However, alone,most homeodomain proteins bind DNA only with a relativelylow affinity. In many instances, therefore, homeodomain pro-tein targeting to specific DNA response elements in the cellhas been shown to be dependent upon protein-protein inter-actions with other transcription factors that promote the local-ization of the homeodomain proteins to specific transcriptionalregulatory regions. For example, Hox homeodomain proteinsfrom Hox gene complexes gain DNA binding specificity andaffinity through cooperative binding with the divergent home-odomain protein Pbx1 (87), and abdB-like Hox proteins stabi-lize DNA binding via the homeodomain protein Meis1 (88). Ithas also been demonstrated that Pbx1 and Meis1 dimerize anddisplay distinctive DNA binding specificities (14). In anotherexample, the human homeodomain protein Phox1 has beenshown to impart serum-responsive transcriptional activity to

FIG. 13. Far-Western analysis of the binding of GRC460Y and GRC460Y/L501P peptides to proteins extracted from early zebra fish embryos. (A) Protein lysates wereprepared from early-blastula-stage and early-gastrula-stage dissected zebra fish embryos from which the chorion had been removed. A Coomassie blue-stainedSDS–12% polyacrylamide gel analysis of 25 mg of each extract is shown. (B) Following transfer to nylon membranes, the extracts were denatured and renatured andthe membranes were hybridized with either GRC460Y (lanes 1 and 2) or GRC460Y/L501P (lanes 3 and 4) peptides labeled with 32P to the same specific activity at a PKAphosphorylation site added to the C termini of the peptides. Incubation, washing, and exposure to autoradiographic film of the membranes were performed identicallyfor each probe.



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the c-fos serum response element by interacting with the serumresponse factor (90). Another homeodomain protein, the car-diogenic Nkx-2.5 factor, is recruited by serum response factorto activate cardiac alpha-actin gene transcription in murinefibroblasts (15). Nkx-2.5 also cooperates with GATA-4, a zincfinger transcription factor, to activate the transcription of thecardiac alpha-actin and atrial natriuretic factor genes (50, 85);in both cases, the protein interaction region has been mappedto the Nkx-2.5 homeodomain (15, 50, 85).

Protein-protein interactions with factors other than GRhave also been shown to influence the DNA targeting of POUdomain proteins beyond the increase in DNA binding speci-ficity provided by the POU-specific domain. For example, theformation of a ternary complex among Oct-1, host cell factor,and herpes simplex virus protein 16 redirects Oct-1 from oc-tamer motifs to TAATGARAT sequences (45). Additionally,the transcription factor Ets-1, a nuclear phosphoprotein in-volved in cell proliferation, functionally and physically interactswith GHF-1/Pit-1 to direct transcription from the prolactinpromoter (10).

While it is noteworthy that all of the homeodomain proteinstested in vitro in our experiments bound directly to the GRDBD in an L501P-sensitive manner and that the phenotypederived from the ectopic expression of the GR DBD was res-cued by the coexpression of Oct-2HD, the extent of GR-home-odomain binding in vivo remains to be established. In partic-ular, the degree to which the GR DBD binds productively tohomeodomain proteins in the cell under physiological condi-tions will need to be evaluated for each potential interaction.While the high degree of conservation within the homeodo-main may permit broad-based binding to the GR DBD in vitroand in transient overexpression assays, it would seem moreprobable that productive interactions in vivo would be tightlyrestricted within glucocorticoid-responsive tissues to the subsetof homeodomain proteins with a physiologically relevant affin-ity for GR. The first example of such a physiologically relevanteffect appears to be the interaction among GR, Oct-1, andOct-2, which seems to be crucial for the responsiveness ofmouse mammary tumor virus to steroid hormones (11, 64, 65).

AR and PR also bind to the POU domains of Oct-1 andOct-2 and promote their binding to the mouse mammary tu-mor virus promoter in transfected cells (64). Therefore, it mayseem tempting to speculate more broadly that nuclear recep-tors generally bind to and affect homeodomain protein func-tion. However, other experiments reinforce the expectationthat nuclear receptor-homeodomain protein binding in situwill be found to occur at the individual, rather than at thefamily, level. For example, although GR, AR, and PR appearto interact productively with Oct-1 and Oct-2, several othernuclear receptors, including ERa, MR, retinoid X receptor aretinoic acid receptor a, and FTZ-F1a, were unable to interactwith Oct-1 and Oct-2 in two hybrid experiments with trans-fected cells (64).

Nevertheless, we suggest that the apparently general natureof the potential for direct GR DBD-homeodomain bindinghighlighted in our experiments may reflect a broadly conservedability of nuclear hormone receptors to enter, on an individualbasis, into productive interactions with specific homeodomainproteins. Further, a conserved basic mechanism of protein-protein binding may underlie specific interactions between in-dividual proteins from each family. In this regard, we note arecent report that the Drosophila FTZ-F1 nuclear receptorbinds through its DBD to the homeodomain of fushi tarazu(FTZ) in a way that promotes the cooperative binding of FTZto transcriptional regulatory regions also containing DNAbinding sites for FTZ-F1 (106). Similarly, the POU domains of

Brn-3a and Brn-3b interact with the DBD of ERa to differen-tially modulate transcription from estrogen response elements(12).

Ectopic expression of the GR DBD disrupts critical eventsthat control the earliest stages of embryogenesis. The expres-sion of GR DBD peptides compromised for DNA binding byC460Y or K489R point mutations after microinjection ofmRNA into one- or two-cell-stage zebra fish embryos affecteddevelopment prior to the completion of the blastula stage in amanner that predominantly led to embryonic death duringgastrulation. Remarkably, these effects were completely sensi-tive to the L501P substitution that abrogated GR-homeodo-main binding. Further, the coexpression of Oct-2HD counter-acted the effects of the GR DBD to allow normal embryonicdevelopment.

Our analysis of the affected embryos suggests that the phe-notypes and the defects in gene expression observed between5.5 and 8.5 h p.f. are most likely to have arisen from an L501P-sensitive, GR DBD-mediated defect in the normal cell move-ments that occur through the blastula and gastrula stages andthat are at the origin of the formation of the embryonic axis. Atthe blastula stage, during epibolic movement, cells of the blas-toderm migrate toward the vegetal pole of the embryo. Thesemovements occurred normally in embryos microinjected withcontrol RNA and RNAs encoding GR DBDs with L501P andwith C460Y and L501P substitutions. In contrast, the spread-ing of the blastoderm toward the vegetal pole was clearlydelayed following the microinjection of a GR DBD-encodingRNA lacking the L501P substitution.

This observation suggested that the defects were initiated ator before the early phase of epiboly. The blastula stage is acrucial developmental stage during which, concomitant withthe acquisition of cell motility, zygotic transcription is activatedduring the midblastula transition period that initiates at cellcycle 10 of zebra fish embryogenesis (35). However, the mo-lecular events underlying the onset of transcription in thezygote are not presently understood. Our results raise theinteresting possibility that the GRC460Y peptide affected thefunction of key maternal proteins required for the initiation ofzygotic transcription and/or interacted with and affected thedevelopmental function of one or more of the earliest zygoticproteins. Further, they also may reflect the specific interfer-ence of the GR DBD with the transcriptional regulatory ac-tivity of homeodomain proteins at the onset of zygotic tran-scription.

However, it is also possible that the defects observed duringepiboly were mediated at a level other than transcription. Theymay reflect the consequences of GR DBD peptide effects onproteins acting at an even earlier developmental stage, such asduring the early phase of the blastula stage or during thecleavage period (0 to 2.25 h). Indeed, far-Western analysis ofpotential GR DBD binding factors indicated the potential forboth maternally and zygotically expressed proteins to be rec-ognized by the GR DBD in an L501P-sensitive manner. Fur-ther experiments, including analyses of cell death and cellproliferation at these early times of development, are beingpursued to more clearly localize the onset of the effects of theGR DBD peptides.

Interestingly, the GRC460Y-injected embryos share someearly phenotypic characteristics with some zebra fish epibolymutants isolated in zebra fish mutagenesis screens (34, 56)which illustrated that both maternal and embryonic contribu-tions are essential for controlling early embryonic cell move-ments. Indeed, Kane et al. described the characterization offour recessive epiboly mutations which, when homozygous,result in a slowdown and arrest of epiboly around midepiboly;



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these mutations are lethal during or shortly after gastrulation(34). The similarity of the phenotypes of some of these mutantswith the defects observed in the GRC460Y-injected embryossuggests that the ectopic expression of GR DBD peptides mayhave interfered with proteins involved in the same pathways asthose affected in these mutants. Thus, GR DBD peptides mayoffer a means to identify key components of the machineryresponsible for early morphogenetic movements.

The consequences of the early L501P-sensitive events in-duced by the GR DBD peptides became more prominent asembryogenesis progressed. In zebra fish, gastrulation is char-acterized by several morphogenetic movements, including in-volution of the cells at the embryonic margin and convergenceand extension movements that reshape the blastula embryo.Our present analysis does not allow us to comment on poten-tial defects in the involution movement, which marks the onsetof gastrulation. To obtain this information, a detailed analysisof cell movement at the margin of the embryo by use of No-marski optics and time lapse will be necessary.

However, the localization of the expression of axial meso-dermal markers surrounding but not within the normal field ofthe embryonic shield is suggestive of L501P-sensitive inhibitionof cell migration or convergence to the organizer field in af-fected embryos. Moreover, the blunting of the anterior exten-sion and the widening of the axial mesoderm in GRC460Y-expressing embryos, which in extreme cases led to axialduplication, are also suggestive of defects in convergenceand/or extension movements. Alternatively, it is also possiblethat the widening of the domains of expression of the axialmarkers observed in the GRC460Y-injected embryos originatedfrom an increased number of dorsal cells. However, the induc-tion of axial duplication in many embryos is more likely to beconsistent with the hypothesis of incomplete convergence oflaterally and ventrally located cells toward the dorsal midline,as an increase in cell number would be expected to enlarge thewidth of only a single axis.

The combination of defects in convergence and extensionmovements during gastrulation could account for the pheno-type of the surviving 24-h embryos lacking anterior and poste-rior structures. Interestingly, this phenotype resembles thatwhich has been observed for a number of gastrulation mutantsthat remain to be molecularly characterized, including mutantsshowing defects in convergence and extension movements(56, 91).

Finally, we note with interest that the defects that we haveobserved in the development of zebra fish embryos upon ex-pression of the GR DBD with a C460Y or K489R substitutionresemble the effects observed upon the overexpression of full-length nuclear receptors in Xenopus embryos (25, 26, 67, 86).In preliminary studies, the overexpression of full-length GRin Xenopus embryos resulted in developmental defects startingduring the early blastula stage (25, 26). Interestingly, whilethese defects were also lethal prior to the completion of thegastrula stage, they were entirely dependent upon steroidtreatment.

The defects observed in our experiments also overlappedextensively with the defects observed upon the overexpressionof retinoid X receptor and thyroid hormone receptor in Xeno-pus embryos (67). In these experiments, the amounts of recep-tors required to generate the severe phenotypes decreasedmarkedly when the embryos were treated with triiodothyro-nine (67). Moreover, the occurrence of phenotypic defects inthese experiments and other, related transgenic experiments inwhich the role of the DBD was investigated was dependent onthe presence of an intact receptor DBD (31, 67, 74, 86). Ourresults suggest that, rather than resulting strictly from the

DNA-dependent properties of the nuclear receptors used inthese studies, these phenotypes also may be determined by theDNA-independent actions of the receptor DBDs.

Our far-Western and one-hybrid analysis results suggest thatthe identity of the key factors controlling early morphogeneticevents that are targeted by GR may be expected to be revealedby expression library screening approaches that detect specificprotein binding. Experiments to further localize the onset ofthe developmental defects and to identify the L501P-sensitivemolecular targets of the GR DBD are ongoing.

ACKNOWLEDGMENTS

We thank the many people who provided the plasmids used in thiswork including, in particular, Q. Long, T. Zerucha, and M. Ekker butalso K. Yamamoto, W. Herr, C. Schild-Poulter, D. Grunwald, M. Hal-pern, V. Korzh, U. Strahle, M. Tada, D. Wilson, and E. Weinberg. Weare grateful to the members of the Hache laboratory and to Y. Le-febvre and M. Ekker for critical comments on the manuscript.

This work was supported by an operating grant from the MedicalResearch Council of Canada. J.M.W. has been funded by an L. Simi-novitch postdoctoral fellowship from The Loeb Health Research In-stitute at the Ottawa Hospital and a fellowship from the NaturalSciences and Engineering Research Council of Canada. G.G.P. is therecipient of an MRC studentship. M.E.L. holds a junior postdoctoralfellowship from the National Cancer Institute of Canada. R.J.G.H. isa Medical Research Council of Canada scientist.

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