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Gα13 Signals via p115RhoGEF Cascades Regulating JNK1 and Primitive Endoderm Formation

2004; Elsevier BV; Volume: 279; Issue: 52 Linguagem: Inglês

10.1074/jbc.m407581200

1083-351X

Yi-Nan Lee, Craig C. Malbon, Hsien‐yu Wang,

Melanoma and MAPK Pathways

The heterotrimeric G-protein G13 mediates the formation of primitive endoderm from mouse P19 embryonal carcinoma cells in response to retinoic acid, signaling to the level of activation of c-Jun N-terminal kinase. The signal linkage map from MEKK1/MEKK4 to MEK1/MKK4 to JNK is obligate in this Gα13-mediated pathway, whereas that between Gα13 and MEKKs is not known. The overall pathway to primitive endoderm formation was shown to be inhibited by treatment with Clostridium botulinum C3 exotoxin, a specific inactivator of RhoA family members. Constitutively active Gα13 was found to activate RhoA as well as Cdc42 and Rac1 in these cells. Although constitutively active Cdc42, Rac1, and RhoA all can activate JNK1, only the RhoA mutant was able to promote formation of primitive endoderm, mimicking expression of the constitutively activated Gα13. Expression of the constitutively active mutant form of p115RhoGEF (guanine nucleotide exchange factor) was found to activate RhoA and JNK1 activities. Expression of the dominant negative p115RhoGEF was able to inhibit activation of both RhoA and JNK1 in response to either retinoic acid or the expression of a constitutively activated mutant of Gα13. Expression of the dominant negative mutants of RhoA as well as those of either Cdc42 or Rac1, but not Ras, attenuated Gα13-stimulated as well as retinoic acid-stimulated activation of all three of these small molecular weight GTPases, suggesting complex interrelationships among the three GTPases in this pathway. The formation of primitive endoderm in response to retinoic acid also could be blocked by expression of dominant negative mutants of RhoA, Cdc42, or Rac1. Thus, the signal propagated from Gα13 to JNK requires activation of p115RhoGEF cascades, including p115RhoGEF itself, RhoA, Cdc42, and Rac1. In a concerted effort, RhoA in tandem with Cdc42 and Rac1 activates the MEKK1/4, MEK1/MKK4, and JNK cascade, thereby stimulating formation of primitive endoderm. The heterotrimeric G-protein G13 mediates the formation of primitive endoderm from mouse P19 embryonal carcinoma cells in response to retinoic acid, signaling to the level of activation of c-Jun N-terminal kinase. The signal linkage map from MEKK1/MEKK4 to MEK1/MKK4 to JNK is obligate in this Gα13-mediated pathway, whereas that between Gα13 and MEKKs is not known. The overall pathway to primitive endoderm formation was shown to be inhibited by treatment with Clostridium botulinum C3 exotoxin, a specific inactivator of RhoA family members. Constitutively active Gα13 was found to activate RhoA as well as Cdc42 and Rac1 in these cells. Although constitutively active Cdc42, Rac1, and RhoA all can activate JNK1, only the RhoA mutant was able to promote formation of primitive endoderm, mimicking expression of the constitutively activated Gα13. Expression of the constitutively active mutant form of p115RhoGEF (guanine nucleotide exchange factor) was found to activate RhoA and JNK1 activities. Expression of the dominant negative p115RhoGEF was able to inhibit activation of both RhoA and JNK1 in response to either retinoic acid or the expression of a constitutively activated mutant of Gα13. Expression of the dominant negative mutants of RhoA as well as those of either Cdc42 or Rac1, but not Ras, attenuated Gα13-stimulated as well as retinoic acid-stimulated activation of all three of these small molecular weight GTPases, suggesting complex interrelationships among the three GTPases in this pathway. The formation of primitive endoderm in response to retinoic acid also could be blocked by expression of dominant negative mutants of RhoA, Cdc42, or Rac1. Thus, the signal propagated from Gα13 to JNK requires activation of p115RhoGEF cascades, including p115RhoGEF itself, RhoA, Cdc42, and Rac1. In a concerted effort, RhoA in tandem with Cdc42 and Rac1 activates the MEKK1/4, MEK1/MKK4, and JNK cascade, thereby stimulating formation of primitive endoderm. Elucidating the signal linkage map for the pathways that promote differentiation and development is a major goal of cell biology. Heterotrimeric G-proteins (G-proteins) occupy a central role in cell signaling, linking the most populous class of cell surface receptors to a smaller but diverse set of effectors that includes adenylylcyclases, ion channels, phospholipase C, Tec kinases, and members of the mitogen-activated protein kinase (MAPK) 1The abbreviations used are: MAPK, mitogen-activated protein kinase; PE, primitive endoderm; RA, retinoic acid; HA, hemagglutinin; GST, glutathione S-transferase; tPA, tissue plasminogen activator; CA, constitutively active; DN, dominant negative; PAK, p21-activated kinase; JNK, c-Jun N-terminal protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; MEKK, MEK kinase; GEF, guanine nucleotide exchange factor. cascades (1Morris A.J. Malbon C.C. Physiol. Rev. 1999; 79: 1373-1430Crossref PubMed Scopus (396) Google Scholar). In the area of differentiation and development, G-proteins play essential roles (2Malbon C.C. Biochem. 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Nishida M. Tanabe S. Takagahara S. Kozasa T. Mori Y. Nagao T. Kurose H. Mol. Pharmacol. 2003; 63: 478-488Crossref PubMed Scopus (73) Google Scholar), thrombin (18Marinissen M.J. Servitja J.M. Offermanns S. Simon M.I. Gutkind J.S. J. Biol. Chem. 2003; 278: 46814-46825Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), lysophosphatidic acid (11Sayas C.L. Avila J. J. Neurosci. 2002; 22: 6863-6875Crossref PubMed Google Scholar), polycystin-1 (30Parnell S.C. Magenheimer B.S. Maser R.L. Zien C.A. Frischauf A.M. Calvet J.P. J. Biol. Chem. 2002; 277: 19566-19572Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar), catecholamines (α1-adrenergic) (31Mao J. Yuan H. Xie W. Simon M.I. Wu D. J. Biol. Chem. 1998; 273: 27118-27123Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar), serotonergic ligands (32McGrew L. Chang M.S. Sanders-Bush E. Mol. Pharmacol. 2002; 62: 1339-1343Crossref PubMed Scopus (60) Google Scholar), thromboxanes (31Mao J. Yuan H. Xie W. Simon M.I. Wu D. J. Biol. Chem. 1998; 273: 27118-27123Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar), angiotensin-II (33Rattan S. Puri R.N. Fan Y.P. Exp. Biol. Med. 2003; 228: 972-981Crossref PubMed Scopus (37) Google Scholar), CCK-A (34Le Page S.L. Bi Y. Williams J.A. Am. J. Physiol. Cell Physiol. 2003; 285: C1197-C1206Crossref PubMed Scopus (33) Google Scholar), and the morphogen retinoic acid (27Jho E.H. Davis R.J. Malbon C.C. J. Biol. Chem. 1997; 272: 24468-24474Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 28Jho E.H. Malbon C.C. J. Biol. Chem. 1997; 272: 24461-24467Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The P19 embryonal carcinoma cells were established from a mouse blastoderm and are multipotent cells capable of forming all three germ layers (35McBurney M.W. Jones-Villeneuve E.M. Edwards M.K. Anderson P.J. Nature. 1982; 299: 165-167Crossref PubMed Scopus (575) Google Scholar). The P19 cells respond to various agents that can promote differentiation to primitive endoderm (PE), mesoderm, and ectoderm as well as to neuron-like cells (36Jones-Villeneuve E.M. McBurney M.W. Rogers K.A Kalnins V.I. J. Cell Biol. 1982; 94: 253-262Crossref PubMed Scopus (679) Google Scholar) and beating cardiac myocytes (37Bain G. Ray W.J. Yao M. Gottlieb D.I. BioEssays. 1994; 16: 343-348Crossref PubMed Scopus (235) Google Scholar, 38McBurney M.W. Int. J. Dev. Biol. 1993; 37: 135-140PubMed Google Scholar). Low concentrations of the well known morphogen, retinoic acid, promote PE formation via a Gα13-dependent pathway, highlighting a role of Gα13 also observed in early mouse development (27Jho E.H. Davis R.J. Malbon C.C. J. Biol. Chem. 1997; 272: 24468-24474Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 28Jho E.H. Malbon C.C. J. Biol. Chem. 1997; 272: 24461-24467Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Mice deficient in Gα13 expression display vascular system defects and intrauterine death (39Offermanns S. Mancino V. Revel J.P. Simon M.I. Science. 1997; 275: 533-536Crossref PubMed Scopus (293) Google Scholar). Specific features of the signal linkage map from Gα13 to PE formation have been elucidated, including obligate roles for activation of c-Jun, JNK1, MEK1 and MKK4, MEKK1, and MEKK4 (27Jho E.H. Davis R.J. Malbon C.C. J. Biol. Chem. 1997; 272: 24468-24474Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 28Jho E.H. Malbon C.C. J. Biol. Chem. 1997; 272: 24461-24467Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 29Kanungo J. Potapova I. Malbon C.C. Wang H. J. Biol. Chem. 2000; PubMed Google Scholar). In the current work, we have elucidated the effector of Gα13 as the p115RhoGEF that activates the small molecular weight GTPase RhoA. Interrupting signaling at the level of p115RhoGEF, RhoA, Cdc42, or Rac1 leads to a loss of RA signaling to JNK and failure of the embryonal P19 cells to form primitive endoderm. Cell Culture and Differentiation—The P19 embryonal carcinoma cells were purchased from the American Type Culture Collection (Rockville, MD). Both the stable transfectants and the wild-type clones were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Hyclone, Logan, UT) in a humidified atmosphere of 6% CO2. P19 cells cultured as monolayers on tissue culture plates in Dulbecco's modified Eagle's medium with 10% serum were induced to primitive endoderm by the addition of 10 nm all-trans retinoic acid (Sigma) for 2-4 days. Plasmids and Transfections—The pCMV5 plasmid harboring the Q226L mutant form of Gα13 was obtained from Dr. Alfred Gilman (Pharmacology, University of Texas Southwestern Medical School, Dallas, TX). For empty vector controls the pCDNA3 plasmid was employed. Expressed proteins were epitope-tagged either with the hemagglutinin antigen (HA-tagged) or with c-Myc (Myc-tagged). The HA-tagged versions of constitutively active p115-RhoGEF (pCMV5-HA-p115-ΔNΔ C-RhoGEF, residues 249-802) and of the dominant negative p115-RhoGEF (pCMV5-ΔDH-RhoGEF with excision of residues 466-547) were gifts from Dr. Gideon Bollag, ONYX Pharmaceuticals, Richmond, CA. The HA-tagged versions of the constitutively active Cdc42 (pCDNA3-HA-Cdc42Hs(Q61L)) and dominant negative Cdc42 (pCDNA3-HA-Cdc42Hs(T17N)) as well as the constitutively active Rac1 (pCDNA3-HA-Rac1(Q61L)) and dominant negative Rac1(pCDNA3-HA-Rac1(T17N)) plasmids were a gift from Dr. Richard A. Cerione (Department of Molecular Medicine, Cornell University, Ithaca, NY). The c-Myc-tagged version of the dominant negative RhoA (pCDNA3-Myc-RhoA(T19N)) plasmid was a gift from Dr. Dafna Bar-Sagi (Department of Molecular Genetics and Microbiology, SUNY-Stony Brook, Stony Brook, NY) and subsequently was engineered with three HA tags to replace the c-Myc tag. The constitutively active version of RhoA (pCDNA3-myc-RhoA(Q63L)) plasmid was a gift from Dr. Alan Hall (CRC Oncogene and Signal Transduction Group, Medical Research Council Laboratory for Molecular Cell Biology, University College London, UK). The assays of activated GTPases made use of pulldown assays using GST fusion proteins that contain domains that bind only the activated forms of each. The plasmids employed were the pGEX-GST-PAK1CRIB plasmid (harboring the Cdc42/Rac1 binding domain of PAK1, residues 70-149) and the pGEX-GST-RBD plasmid (harboring the RhoA binding domain of Rokα, residues 809-1062), both gifts from Dr. Lu-Hai Wang (Department of Microbiology, Mount Sinai School of Medicine, New York, NY). The P19 cells were transfected with one or more plasmids using Lipofectamine. Stably transfected P19 clones were selected in the presence of the neomycin analog, G418 (400 μg/ml). Immunoblotting—Samples (10-50 μg of protein/lane) of total cell lysates were subjected to electrophoresis in SDS on 10% polyacrylamide gels. The resolved proteins were transferred electrophoretically to nitrocellulose blots. The blots were stained with primary antibodies and the immune complexes made visible by use of a second antibody coupled with horseradish peroxidase and developed using the enhanced chemiluminescence method. The antibodies were purchased from the following sources: anti-JNK1, anti-RhoA, and anti-Cdc42 antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); anti-RAC1 antibody from Upstate Biotechnology (Lake Placid, NY); anti-HA antibodies from Roche Applied Science); and anti-cytokeratin endo A (PE marker) antibody (TROMA-1) from the Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IO). Immunoprecipitation of JNK and Activity Assays—The immunoprecipitation reactions and solid-state assay of JNK were performed as detailed earlier (27Jho E.H. Davis R.J. Malbon C.C. J. Biol. Chem. 1997; 272: 24468-24474Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) using the rGST-c-Jun amino-terminal fusion protein as substrate for JNK activity assay. The expression vector for rGST-c-Jun was generously provided by Dr. Roger Davis (Howard Hughes Medical Institute, University of Massachusetts Medical Center, Worcester, MA). Assay of Activated RhoA, Rac1, and Cdc42—The assay was performed essentially as described earlier (40van Triest M. Bos J.L. Methods Mol. Biol. 2004; 250: 97-102PubMed Google Scholar). The synthesis of the fusion polypeptides GST-RBD and GST-PAK1CRIB encoded in their respective plasmids was induced in Escherichia coli with 0.4 mm isopropyl-β-galactopyranoside at 30 °C for 4 h and affinity-purified using 50% slurry of glutathione-Sepharose 4B (Amersham Biosciences) according to the batch purification method suggested by the manufacturer. Indirect Immunofluorescence Staining of TROMA—The staining of the endoderm-specific marker antigen cytokeratin endo A by the monoclonal antibody TROMA-1 was performed as described (41Liu T. Lee Y.N. Malbon C.C. Wang H.Y. J. Biol. Chem. 2002; 277: 30887-30891Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The P19 cells were cultured, stained, and subjected to analysis by epifluorescence microscopy as described previously. As the differentiated cells often grow from monolayers to whorls of cells with multiple layers, the indirect immunofluorescence and phase contrast images may not appear to be "in focus." This artifact is unavoidable under these conditions of differentiation. Data Analysis—For all of the experiments reported, the data are compiled from at least three independent, replicate experiments performed on separate cultures on separate occasions with highly reproducible results. The indirect immunofluorescence and phase contrast images are of representative fields of interest. C. botulinum C3 Exotoxin Blocks RA-induced Formation of Primitive Endoderm—Treating P19 embryonal carcinoma (P19) cells with C. botulinum C3 exotoxin (C3) for 30 h or expression of C3 exotoxin in these cells blocked the ability of RA to promote transition of these embryonal cells to primitive endoderm, as established by assay of the expression of cytokeratin endo A (endo A), a marker protein specific for PE (Fig. 1A). Endo A expression, measured using the monoclonal antibody TROMA-1, increased more than 10-fold in response to RA, but this response was blocked by treatment with C3 exotoxin. Indirect immunofluorescent staining of endo A in P19 cells treated with RA in the absence (+RA) and the presence (+RA, +C3) of C3 exotoxin provides another demonstration that RhoA activity is essential for PE formation in response to RA (Fig. 1B). Analysis of the activity of another hallmark protein of PE formation, tissue plasminogen activator (tPA), also demonstrated the ability of C3 to attenuate the formation of PE in response to RA (Fig. 1C). The activation of JNK by RA treatment (27Jho E.H. Davis R.J. Malbon C.C. J. Biol. Chem. 1997; 272: 24468-24474Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 28Jho E.H. Malbon C.C. J. Biol. Chem. 1997; 272: 24461-24467Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 29Kanungo J. Potapova I. Malbon C.C. Wang H. J. Biol. Chem. 2000; PubMed Google Scholar), an obligate step in RA-induced PE formation, was inhibited also by treating the cells with C3 exotoxin (Fig. 1D). Taken together these studies demonstrate RhoA to be an obligate element in the signaling from RA to activation of JNK and to formation of PE. JNK1 Activation and PE Formation Are Stimulated by Expression of Constitutively Active RhoA—The formation of PE in response to RA can be mimicked in P19 cells by the transient expression of the constitutively active Q226L mutant of Gα13 (QLGα13) (27Jho E.H. Davis R.J. Malbon C.C. J. Biol. Chem. 1997; 272: 24468-24474Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 28Jho E.H. Malbon C.C. J. Biol. Chem. 1997; 272: 24461-24467Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The ability of the C3 exotoxin to block PE formation focused our attention on RhoA, the best known target of C3 exotoxin action. We examined the activation status of RhoA and two other GTPases, Cdc42 and Rac1, in P19 cells stimulated to form PE either by treatment with RA or by the transient expression of QLGα13 (Fig. 2). The activation status of RhoA, Cdc42, and Rac1 was probed using assays that specifically pull down only the activated forms of each GTPase. Treatment with RA resulted in robust activation of all three GTPases (Fig. 2A). Transient expression of activated QLGα13 mutant stimulated activation of all three GTPases, mimicking the action of RA (Fig. 2B). Treating cells with RA provokes an activation of JNK1 activity (Fig. 2C), as previously shown (27Jho E.H. Davis R.J. Malbon C.C. J. Biol. Chem. 1997; 272: 24468-24474Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). We explored whether the activation of any one of the GTPases alone, like the treatment with RA (Fig. 2C), was sufficient to activate c-Jun N-terminal kinase. The expression of the constitutively active (CA) mutants of each of the GTPases, RhoA (Q63LRhoA), Cdc42 (Q61LCdc42), and Rac1 (Q61LRac1), provoked a strong activation of JNK1 activity, nearly equivalent to that stimulated in response to RA (Fig. 2C). Expression of the CA mutant of Ras (Q61LRas), in contrast, provoked no stimulation of JNK1 activity. The levels of expression of each of the GTPases in cells expressing QLGα13 or treated with RA were essentially unchanged (data not shown). The most interesting extension of this strategy was analysis of the ability of the expressed CA GTPases to stimulate the overall pathway and, like RA, promote the formation of primitive endoderm (Fig. 2D). Whereas expression of the CA-Cdc42, CA-Rac1, and CA-RhoA each led to robust activation of JNK1 activity (Fig. 2C), only the expression of CA-RhoA was able to promote the formation of primitive endoderm (Fig. 2D). Expression of CA-Ras also failed to stimulate the formation of PE. The formation of PE must require signals from RhoA in addition to JNK activation and those mediated by Cdc42 and/or Rac1. Furthermore, expression of DN-RhoA, DN-Cdc42, or DN-Rac1 blocks JNK activation in response to treatment of the cells with the morphogen RA (Fig. 2E). These observations suggest that activation of just a single GTPase, RhoA, is sufficient to mimic either the action of RA or the transient expression of the QLGα13 on the formation of PE. Expression of p115RhoGEF Mutants Regulates JNK1 Activation and PE Formation—The exciting possibility was explored that the guanine nucleotide exchange factor for RhoA p115RhoGEF, known to be an effector of Gα13, functions in the signaling from RA to PE formation. Constitutively active p115RhoGEF, lacking the N- and C-terminal regulatory domains (p115RhoGEF, 249-802), was expressed in cells, and formation of primitive endoderm (Fig. 3A) and the activity of JNK1 were assayed (Fig. 3B). Expression of CA-p115RhoGEF provokes the formation of primitive endoderm (Fig. 3A) and activation of JNK1 (Fig. 3B), just as did expression of QLGα13 (27Jho E.H. Davis R.J. Malbon C.C. J. Biol. Chem. 1997; 272: 24468-24474Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 28Jho E.H. Malbon C.C. J. Biol. Chem. 1997; 272: 24461-24467Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Because guanine nucleotide exchange factors often regulate the activity of multiple GTPases, it was important to test whether the expression of the dominant negative mutant of RhoA (T19N mutant, DN-RhoA) attenuates the ability of the CA-p115RhoGEF to stimulate JNK1 activity (Fig. 3C). Expression of DN-RhoA sharply blocked the activation of JNK1 in response to CA-p115RhoGEF. Expression of either DN-Cdc42 (T17N mutant) or DN-Rac1 (T17N mutant) suppresses the ability of CA-p115RhoGEF to activate JNK1 (Fig. 3, D and E), whereas expression of DN-Ras has no effect on the activation of JNK1 in response to CA-p115RhoGEF (Fig. 3F). RA treatment stimulates JNK1 activity, essential for the P19 embyronal cells to commit to a primitive endoderm phenotype. We explored whether the transient expression of DN-p115RhoGEF, lacking the DH domain residues (Δ466-547), would influence the ability of RA to activate JNK1. Expression of DN-p115RhoGEF, but not the expression of DN-PDZRho-GEF, blocks activation of JNK1 in response to RA (Fig. 3G). The expression of DN-p115RhoGEF similarly blocks the activation of JNK1 in response to expression of the constitutively active Gα13, whereas expression of DN-PDZRhoGEF has no effect on the activation of JNK1 in response to expression of the constitutively active Gα13 (Fig. 3H). DN Mutants of RhoA, Cdc42, and Rac1 Block the Ability of Q226L Gα13 to Activate All Three GTPases—The relationships among these small molecular weight GTPases in the signaling cascade from Gα13 to JNK activation and to PE formation in response to RA were investigated. Cells expressing QLGα13 were transiently transfected with dominant negative mutant forms of RhoA, Cdc42, and Rac1 and the activities of the GTPases determined using the pulldown assays specific for each GTPase (Fig. 4). Expression of QLGα13 results in activation of all three GTPases, as shown earlier (Fig. 2B). Transient expression of the dominant negative mutant form of RhoA (DN-RhoA) blocked the activation of endogenous RhoA as well as the activation of Cdc42 and Rac1 in response to expression of QLGα13 (Fig. 4A). The ability of DN-RhoA to impact the activation of RhoA, Cdc42, and Rac1 suggests that Cdc42 and Rac1 could be downstream of RhoA in this pathway, because RhoA is the effector for p115RhoGEF. Expression of DN-Cdc42 blocks the ability of QLGα13 to active Cdc42, as expected. Surprisingly, the expression of DN-Cdc42 was found to also block the activation of RhoA and of Rac1 in response to QLGα13 (Fig. 4B). DN-Rac1 expression blocked the ability of QLGα13 to activate Rac1 but surprisingly was found to block also the activation of Cdc42 and RhoA in response to QLGα13 (Fig. 4C). Taking into consideration the fact that p115RhoGEF activates only RhoA, these data suggest that RhoA can signal to the other GTPases, whose activation appears to be obligate for signaling from QLGα13. Expression of DN Mutants of GTPases Blocks RA-stimulated Activation—We explored whether expression of DN mutants of GTPases altered GTPase activation in response to stimulation by the morphogen RA (Fig. 5).