BIP, a BRAM-interacting protein involved in TGF-b signalling, regulates body length in Caenorhabditis elegans

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1 BIP, a BRAM-interacting protein involved in TGF-b signalling, regulates body length in Caenorhabditis elegans Katsura Sugawara 1, Kiyokazu Morita 1, Naoto Ueno 1 and Hiroshi Shibuya 1,2, * 1 Division of Morphogenesis, Department of Developmental Biology, National Institute for Basic Biology, Okazaki , Japan 2 Department of Molecular Cell Biology, Medical Research Institute, Tokyo Medical and Dental University, Kanda-Surugadai , Chiyoda , Japan Abstract Background: The TGF-b superfamily has diverse biological activities and is involved in the early development of animals. We previously identified a novel family member, BMP receptor associated molecule BRAM), which binds to the intracellular domain of BMP type IA receptor and is involved in the BMP signalling pathway. Results: To identify novel molecules involved in TGFb signalling pathways, we performed yeast two-hybrid screening using BRAM as bait. From a Xenopus cdna library, we cloned a cdna encoding 693 amino acids and containing the motif for an oxysterol binding protein OSBP), which we designated BRAM interacting protein BIP). We then isolated a BIP homologue from the Caenorhabditis elegans that encodes 733 amino acids and also contains the OSBP-like motif. Immunoprecipitation and Western blotting studies revealed that C. elegans BIP could interact with the C. elegans BRAM homologues BRA-1 and BRA-2. C. elegans BIP was expressed in pharyngeal muscle, hypodermis and several neuronal cells, an expression pattern overlaps with those of BRA-1 and BRA-2. Finally, we found that inhibition of BIP expression in C. elegans by double stranded RNA interference produces a Sma phenotype. Conclusions: BIP was isolated using the yeast twohybrid systems. BIP may function in the TGF-b pathway and regulate body length in C. elegans. Introduction In recent years, it has been revealed that many growth factors and signal transducers are involved in the early development of animals. In particular, the transforming growth factor-b TGF-b)superfamily has been shown to mediate diverse biological activities such as cell proliferation, embryonic patterning and cell-type specification Heldin et al. 1997; Attisano & Wrana 1998; Derynck et al. 1998; Padgett et al. 1998; Whitman 1998; Massague 2000). TGF-b signal transduction pathways are well conserved among many organisms. TGF-b or a related ligand binds to its cognate type II receptor, and this ligand-receptor then complexes with the type I receptor. The type II receptor possesses a Communicated by: Eisuke Nishida * Correspondence: Department of Molecular Cell Biology, Medical Research Institute, Tokyo Medical and Dental University, Kanda-Surugadai , Chiyoda , Japan. shibuya.mcb@mri.tmd.ac.jp serine/threonine kinase activity that phosphorylates and activates the type I receptor. The activated type I receptor then phosphorylates and activates the Smad proteins, which transduce the signal to the nucleus. Other proteins that mediate TGF-b signalling include TGF-b activated kinase TAK1) Yamaguchi et al. 1995), which is involved in the mitogen-activated protein kinase MAPK)cascade, and TAK1 binding protein 1 TAB1) Shibuya et al. 1996). In the nematode Caenorhabditis elegans, at least three TGF-b related ligands, DAF-7, DBL-1 and UNC-129, have been reported Patterson & Padgett 2000). UNC- 129 is involved in the axon pathfinding Colavita et al. 1998). DAF-7 is involved in the daf the dauer larva formation)pathway, which regulates dauer larva formation in response to scarcity of food and high pheromone or overcrowding conditions Ren et al. 1996). The type I and type II receptors for DAF-7 are DAF-1 and DAF-4, respectively. Binding of DAF-7 to DAF-4 causes activation of DAF-1, which in turn phosphorylates the intracellular components DAF-8 q Blackwell Science Limited Genes to Cells 2001) 6, 599±

2 K Sugawara et al. and DAF-14. These components then associate with DAF-3, resulting in transduction of the signal to the nucleus and induction of the biological responses required for dauer formation. DBL-1 plays a role in the sma pathway, which regulates body length in C. elegans Suzuki et al. 1999). The receptors for DBL-1 are SMA-6 type I receptor)and DAF-4 type II receptor), and the intracellular transducers for the sma pathway are SMA-2, SMA-3 and SMA-4. Once activated, SMA-6 phosphorylates SMA-2 and SMA-3, which then associate with SMA-4, and these intracellular components translocate to the nucleus. SMA-2, SMA-3 and SMA-4 also regulate male sensory tale ray patterning in a pathway involving MAB-21 Morita et al. 1999). Recently, several novel molecules that modulate the TGF-b signalling pathway have been identified. Smad6 and Smad7 have been shown to negatively regulate TGF-b signalling in vertebrates. Smad7 binds to either TGF-b or BMP receptor complexes, and prevents the association and phosphorylation of the Smads Nakao et al. 1997). Smad6 inhibits BMP signalling by interacting with the receptor or by binding to phosphorylated Smad1 and inhibiting its association with Smad4 Imamura et al. 1997). In this way, these regulators function as part of a negative feedback system in the TGF-b pathways. In addition, a novel class of molecule, called the Smad anchor for receptor activation SARA)has been identified Tsukazaki et al. 1998). SARA interacts with unphosphorylated Smad2 and recruits it to the TGF-b receptor complex. Once Smad2 has been phosphorylated by the type I receptor, it dissociates from SARA and interacts with Smad4 to form Smad2/Smad4 complexes, which in turn translocate to the nucleus. Another component, the BMP receptor associated protein BRAM), was identified as an intermediate between the BMP receptor and the TAK1 signalling pathway Kurozumi et al. 1998). BRAM was found to associate with the intracellular domain of BMP type IA receptor and also to associate with TAB1 in mammalian cells. In C. elegans, two homologues for BRAM, BRA-1 and BRA-2, have also been identified. BRA-1 is involved in the daf pathway and BRA-2 is involved in the sma pathway Morita et al. 2001), and they act as negative regulators for TGF-b signalling in C. elegans. We performed yeast two-hybrid screening to isolate BRAM-interating proteins, with the idea that such proteins may participate in the TGF-b signalling pathway and play a role in the early development of animals. We identified a protein we termed BRAMinteracting protein, or BIP, and found that it can form a complex with either BRA-1 or BRA-2. Loss-offunction BIP by double-stranded RNA interference exhibited a Sma phenotype. Our results suggest that BIP may play a role in TGF-b signalling. Results Isolation of cdna clones encoding proteins associated with BRAM To obtain molecules involved in the TGF-b superfamily signal transduction pathway, we used the yeast two-hybrid system with human BRAM as bait. From < transformants, we obtained 12 clones which encoded the identical protein. We next screened a Xenopus cdna library using the cdna obtained in the two-hybrid screen and obtained the full-length cdna. This cdna encoded a novel protein of 693 amino acids containing an oxysterol binding protein OSBP)motif EQVSHHPPVSA, Fig. 1A). We designated the molecule BRAM-interacting protein, or BIP. A further database search revealed the existence of both human and nematode C. elegans BIP homologues. Identification of C. elegans BIP Since two BRAM homologues, BRA-1 and BRA-2 have been identified in C. elegans and shown to function in TGF-b signalling, we focused our analysis on the C. elegans homologue of BIP. To determine the structure of C. elegans BIP, first we sequenced several BIP EST clones yk504f2, yk318d4 and yk154e4). Of these clones, yk504f2 contained the longest putative open reading frame. Next, we determined the 5 0 -end of BIP mrna by RT-PCR analysis. We used two primers described in Experimental procedures, i.e. the spliced-leader sequence SL-1)primer and the antisense primer see Experimental procedures)located within the putative coding region of BIP. With these primers, we obtained a PCR product and found that there was no start codon between the SL-1 sequence and the putative start codon of yk504f2. Examination of the PCR product confirmed that the C. elegans BIP encodes 733 amino acids and contains an OSBP-like motif in the middle of the protein Fig. 1B). The BIP gene was mapped to ZK on chromosome X of C. elegans. A comparison of the genomic and cdna sequences indicated that BIP consists of 11 exons Fig. 1C). Interestingly, the most C-terminal amino acid of the OSBP motif in C. elegans BIP is serine, whereas in every other known OSBP motif this amino 600 Genes to Cells 2001) 6, 599±606 q Blackwell Science Limited

3 BRAM interacting protein Figure 1 Structural analysis of BIP. A) Predicted amino acid sequence of Xenopus BIP. Oxysterol binding protein motif is indicated with an underline. B)Predicted amino acid sequences of C. elegans BIP. C. elegans BIP encodes 733 amino acids and contains an oxysterol binding protein-like motif underline). C) Genomic structure of C. elegans BIP. Eleven exons are shown in boxes separated by introns. C. elegans BIP gene was mapped to ZK on chromosome X and its first exon was mapped to cosmid F19D8, which is neighbouring to cosmid ZK1086. acid is alanine. We do not have any data showing that BIP actually functions as an oxysterol binding protein. BIP interacts with BRA-1 and BRA-2 in vitro To examine whether BIP interacts with BRA-1 and/or BRA-2, we performed immunoprecipitation studies. Flag-tagged BIP protein and HA-tagged BRA-1 or BRA-2 were co-expressed in COS-7 cells. As shown in Fig. 2, both the BRA-1-HA and BRA-2-HA fusion proteins were efficiently immunoprecipitated with anti-flag antibody, demonstrating that BRA-1 and BRA-2 each interact with BIP. To delineate the regions of BRA-1 and BRA-2 that bind to BIP, we constructed N-terminal and C-terminal truncated mutants of BRA-1 and BRA-2. The N-terminaldeleted forms of both BRA-1 and BRA-2 were able to interact with BIP, showing that the highly conserved C-terminal halves of BRA-1 and BRA-2 are responsible for their association with BIP. BIP expression pattern in C. elegans To examine the localization of BIP expression in vivo, we fused the BIP promoter region to a cdna encoding GFP protein, and transgenic animals carrying q Blackwell Science Limited Genes to Cells 2001) 6, 599±

4 K Sugawara et al. BRA-1 is expressed in neuronal cells and BRA-2 is expressed in pharyngeal muscle, hypodermis and intestine data not shown). These data suggest that the expression pattern of BIP overlaps with those of BRA-1 and BRA-2, and that these molecules have the potential to interact in vivo. Figure 2 C. elegans BIP associates with BRA-1 and BRA-2. COS-7 cells were transiently co-transfected with Flag-tagged BIP and HA-tagged BRA-1 or BRA-2. A)Interaction between BIP and BRA-1. COS-7 cells were transiently co-transfected with HA-tagged BRA-1, BRA-1 DN and BRA-1DC, and Flag-tagged BIP. B)Interaction between BIP and BRA-2. COS-7 cells were transiently co-transfected with HA-tagged BRA-2, BRA-2 DN and BRA-2DC, and Flag-tagged BIP. Cell lysates were subjected to immunoprecipitation using the anti- Flag antibody and then immunoblotting using the anti-ha antibody. Expression of BRA-1 and BRA-2 was measured by anti-ha immunoblotting of samples from cell lysates. the BIP promoter-driven GFP construct were generated. GFP was expressed strongly in pharyngeal muscle, hypodermis and some neuronal cells Fig. 3). This expression pattern was consistent from larval to adult stage animals data not shown). The expression in pharyngeal muscle was also confirmed by wholemount in situ hybridization Fig. 3E). In contrast, Inhibition of BIP function by double-stranded RNA interference causes Sma phenotype To investigate the role of BIP in C. elegans, we used double-stranded RNA interference dsrnai) Fire et al. 1998)to inhibit BIP function. Synthesized double-stranded RNA encoding BIP was injected into wild-type or DBL-1 over-expressing animals, and the F1 phenotypes were observed. Interference of the wild-type animals by dsrna reproducibly caused a Sma phenotype Fig. 4). Moreover, whereas animals over-expressing DBL-1 normally exhibit a Lon long) phenotype, this phenotype was rescued by injection of the BIP dsrna Fig. 5). Furthermore, we examined the effects of BIP dsrna on lon-1 animals. Lon-1 is one of the target genes in the sma pathway and the lossof-function of the gene shows Lon phenotype. The injection of BIP dsrna had no effect on lon-1 data not shown), suggesting that BIP was hypostatic to lon- 1. Since BIP could interact with BRA-1 as well as BRA-2, we also examined the effect of dsrnai on phenotypes relating to the daf pathway; however, no obvious phenotypes were observed data not shown). These results suggest that C. elegans BIP regulates C. elegans body length, possibly through an interaction with BRA-2. Discussion We performed a yeast two-hybrid screening with the goal of obtaining molecules involved in TGF-b superfamily signalling pathways. Previously, we had identified BRAM by yeast two-hybrid screening and found that it associates with the intracellular domains of both BMP type IA receptor and TAB1 in mammalian cells. However, the precise role of BRAM in the early development of animals was not well understood. To clarify the role of BRAM, we searched for BRAM-interacting proteins using yeast two-hybrid screening and identified a BRAM-interacting protein from a Xenopus cdna library. Xenopus BIP encodes 693 amino acids and contains an oxysterol binding protein motif E-[KQ]-x-S-H-[HR]-P-P-x-[STACF]- A). Oxysterols are oxygenated derivatives of cholesterol 602 Genes to Cells 2001) 6, 599±606 q Blackwell Science Limited

5 BRAM interacting protein Figure 3 The expression of a BIP::GFP fusion. The vector including upstream regulatory sequences and GFP gene were injected into the gonadal syncytia of hermaphrodites. A)BIP::GFP expression of an adult hermaphrodite. BIP was expressed in pharyngeal muscle and hypodermis. B)DIC micrograph of A). C) BIP::GFP was also expressed in several neuronal cells in tail region of an adult hermaphrodite arrowhead). D) DIC micrograph of C). E) in situ hybridization of BIP. A strong signal was observed in pharyngeal muscle. and have diverse biological activities such as effects on sphingolipid metabolism, platelet aggregation, apoptosis and protein prenylation Schroepfer 2000). Recently, it has been revealed that a specific group of oxysterols activates transcription through the nuclear orphan receptor LXRa, resulting in the regulation of cholesterol metabolism Janowski et al. 1996). Oxysterol binding proteins are present in many organisms, such as human, Mus musculus, C. elegans, Drosophila melanogaster, Neurospora crassa, Schizosaccharomyces pombe, Shaccharomyces cerevisiae and Arabidopsis thaliana. In animals, oxysterol binding proteins might repress the gene for HMG-CoA reductase, which is the key enzyme for cholesterol synthesis. Therefore, Xenopus BIP might regulate not only the TGF-b pathway but also cholesterol metabolism: controlling both the accumulation of extracellular matrix and the composition of cholesterol in the cell membrane. In a search of several gene databases, we also found a C. elegans homologue of BIP. To identify the structure of C. elegans BIP, we analysed the nucleotide sequence of the cdna and several EST clones. C. elegans BIP was found to encode 733 amino acids and consist of 11 exons. Interestingly, C. elegans BIP contains an oxysterol binding protein-like motif, EQVSHHPPVSS, but unlike every other known example, the most C-terminal amino acid of this region is not alanine but serine. Whether this motif functions as an oxysterol binding protein motif or affects cholesterol metabolism needs to be further investigated. In C. elegans, two BRAM homologues, BRA-1 and BRA-2 had previously been identified and shown to participate in the TGF-b signalling pathway: BRA-1 has been shown to be involved in the DAF-7 daf pathway and BRA-2 in the DBL-1 sma pathway K.M. & N.U. unpublished results). Both BRA-1 and BRA-2 show a significant amino acid identity with human BRAM, particularly in their C-terminal regions, suggesting that their relationship with BIP in C. elegans may be similar to that between BRAM and BIP in vertebrates. To confirm this, we performed q Blackwell Science Limited Genes to Cells 2001) 6, 599±

6 K Sugawara et al. Figure 4 Double-stranded RNA interference of BIP caused Sma phenotype. Synthesized double stranded RNA was injected to the gonadal syncytia of adult hermaphrodites. The body length of F1 progenies were measured. A)Body length of wildtype animals 1.18 ^ 0.09 mm, mean ^ SD). B) Body length of dsrna injected animals 1.00 ^ 0.09 mm, mean ^ SD). immunoprecipitation and Western blotting, and showed that C. elegans BIP could associate with either BRA-1 or BRA-2. To show that these proteins interact in vivo, we examined the expression pattern of BIP in C. elegans. We created transgenic worms carrying a green fluorescent protein GFP)reporter driven by the BIP promoter BIP::GFP), and observed that GFP was Figure 5 BIP dsrnai suppressed the phenotype of dbl-1 11). A)Body length of dbl-1 11)animals 1.53 ^ 0.06 mm, mean ^ SD). B) Body length of BIP dsrna injected dbl-1 11) animals 1.33 ^ 0.08 mm, mean ^ SD). expressed in pharyngeal muscle, hypodermis and some neuronal cells. This pattern resembles that of BRA-1, which negatively regulates the daf pathway, and is expressed in amphid neurones, and BRA-2, which is involved in the sma pathway, regulates body length in C. elegans, and is expressed in pharyngeal muscle, hypodermis and the intestine. In addition, SMA-6, which encodes the type I receptor of the sma pathway, is expressed in pharyngeal muscle and intestine cells Krishna et al. 1999). These results suggested that BIP may be involved in the daf and/or the sma pathways. To further investigate the role of BIP in C. elegans, we performed double-stranded RNA interference 604 Genes to Cells 2001) 6, 599±606 q Blackwell Science Limited

7 BRAM interacting protein dsrnai). Injection of BIP dsrna caused a Sma phenotype in wild-type animals, and suppressed the Lon phenotype caused by over-expression of DBL-1, suggesting that BIP participates in the sma pathway. To more fully elucidate the role of BIP in the regulation of body length, we need to undertake genetic analysis and binding studies examining the relationships between BIP and other molecules in the sma pathway, such as SMA-6, SMA-2, SMA-3 and SMA-4. In the present study, we also examined whether inhibition of BIP function caused a Daf phenotype, but no Daf phenotype was apparent. However, this does not necessarily rule out a role for BIP in the daf pathway, since some neuronally expressed genes appear to be refractory to dsrnai. To better clarify the roles of BIP in the daf pathway, we need to create mutant BIP animals. Experimental procedures Yeast two-hybrid screening and cdna cloning of Xenopus BIP Human BRAM was cloned in-frame into the LexA-DNA binding domain vector, pbtm116. The resulting plasmid pbtm116-hbram was used as bait. The yeast reporter strain L40 expressing this bait was transformed with a Xenopus cdna library inserted into the activation domain vector pact2 Clontech). Transformants were screened for the activation of HIS reporter genes. cdna inserts from positive clones were identified by restriction mapping and further characterized by DNA sequencing. To obtain full length Xenopus BIP cdna, a Xenopus cdna library Stratagene)carried in the Lambda ZAP II vector was screened under stringent conditions with a 1.5 kb cdna insert isolated in the two-hybrid screen and labelled with a random-priming kit Amersharm Pharmacia Biotech). The positive phage clones were excised according to the manufacturer's instruction and the cdnas were sequenced in the pbluescript vector. Identification of C. elegans BIP cdna structure To identity the C. elegans BIP cdna structure, we sequenced several EST clones courtesy of Y. Kohara). Furthermore, to determine the 5 0 untranslated region, we performed PCR with C. elegans cdna as a template. We used the following primers, Sl-1: 5 0 -GGTTTAATTACCCAAGTTTGAG-3 0 anti-sense: 5 0 -CGTTTCTTCTCGTCGCGATACT-3 0 PCR products were cloned and identified by DNA sequencing. Mammalian cell expression plasmid construction The whole coding region of C. elegans BIP was inserted into the mammalian expression vector pcmv5 which produces fusion proteins joined to a Flag tag at the N-terminus. For BRA-1 and its truncated version, the cdna encoding the whole coding region was subcloned into the vector pdha, which fuses two copies of the DNA sequence encoding the HA epitope in-frame to the COOH-terminus of the insert. HA-tagged cdna fragments were next cloned into the mammalian expression vector pcdna3 Invitrogen). For construction of BRA-2, HAtagged BRA-2, BRA-2 DC amino acids 1±128)and BRA- 2 DN amino acids 129±214)were amplified by PCR with primers containing the appropriate restriction sites. The correct PCR products were cloned into the expression vector pcs2. Antibodies, immunoprecipitation and Western blotting The monoclonal antibody against the haemagglutinin HA) epitope Y-11 was purchased from Santa Cruz. The anti-flag epitope was obtained from Sigma. COS-7 cells were transiently transfected with the constructs by the calcium phosphate method. The harvest and lysis of the cells, immunoprecipitation and Western blotting were performed as described Kurozumi et al. 1998). C. elegans strains All strains were cultured by standard method. The following strains were used: wild-type C. elegans variety Bristol strain N2), dbl-1 11) ctls40 Suzuki et al. 1999). Expression pattern of C. elegans BIP For the expression pattern studies, 4.2 kb fragments including upstream regulatory sequences were amplified by PCR and the identified clones were used to generate a translational fusion with the GFP gene in the vector ppd95.67 courtesy of A. Fire). The resulting vector was injected into the gonadal syncytia of C. elegans hermaphrodites. We performed whole mount in situ hybridization as described Mochii et al. 1999). Double stranded RNA interference dsrnai) and body length measurement of adult worms For the loss-of-function studies using dsrnai Fire et al. 1998), a yk504f2 cdna clone gifted by Y. Kohara)was used as a template. The plasmid was linearized by appropriate restriction enzymes and RNA was synthesized by mmessage mmachine kit Ambiion)with T3 and T7 primers. After the reaction, each reaction mixture was mixed and maintained at 65 8C for 10 min and annealed with each other at 37 8C for 30 min. Synthesized double stranded RNA was injected to the gonadal syncytia of adult hermaphrodites. F1 progenies were analysed for their phenotype. Body length measurement was performed as described Morita et al. 1999). q Blackwell Science Limited Genes to Cells 2001) 6, 599±

8 K Sugawara et al. Acknowledgements We thank M. Lamphier for a critical reading of the manuscript. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and Japan Society for the Promotion of Science. References Attisano, L. & Wrana, J.L. 1998)Mads and Smads in TGF b signalling. Curr. Opin. Cell Biol. 10, 188±194. Colavita, A., Krishna, S., Zheng, H., Padgett, R.W. & Culotti, J.G. 1998)Pioneer axon guidance by UNC-129, a C. elegans TGF-b. Science 31, 706±709. Derynck, R., Zhang, Y. & Feng, X.H. 1998)Smads: transcriptional activators of TGF-b responses. Cell 95, 737±740. Fire, A., Xu, S., Montgomery, M.K., et al. 1998)Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806±811. Heldin, C.H., Miyazono, K. & ten Dijke, P. 1997)TGF-b signalling from cell membrane to nucleus through SMAD proteins. Nature 390, 465±471. Imamura, T., Takase, M., Nishihara, A., et al. 1997)Smad6 inhibits signalling by the TGF-b superfamily. Nature 389, 622±626. Janowski, B.A., Willy, P.J., Devi, T.R., Falck, J.R. & Mangelsdorf, D.J. 1996)An oxysterol signalling pathway mediated by the nuclear receptor LXR a. Nature 383, 728±731. Krishna, S., Maduzia, L.L. & Padgett, R.W. 1999)Specificity of TGFb signaling is conferred by distinct type I receptors and their associated SMAD proteins in Caenorhabditis elegans. Development 126, 251±260. Kurozumi, K., Nishita, M., Yamaguchi, K., et al. 1998) BRAM1, a BMP receptor-associated molecule involved in BMP signalling. Genes Cells 3, 257±264. MassagueÂ, J. 2000)How cells read TGF-b signal. Nature Rev. Mol. Cell Biol. 1, 169±178. Mochii, M., Yoshida, S., Morita, K., Kohara, Y. & Ueno, N. 1999)Identification of transforming growth factor-bregulated genes in Caenorhabditis elegans by differential hybridization of arrayed cdnas. Proc. Natl. Acad. Sci. USA 96, 15020± Morita, K., Chow, K.L. & Ueno, N. 1999)Regulation of body length and male tail ray pattern formation of Caenorhabditis elegans by a member of TGF-b family. Development 126, 1337±1347. Morita, K., Shimizu, M., Shibuya, H. & Ueno, N. 2001)A DAF-1-binding protein BRA-1 is a negative regulator of DAF- 7TGF-b signaling. Proc. Natl. Acad. Sci. USA 98, 6284±6288. Nakao, A., Afrakhte, M., Moren, A., et al. 1997)Identification of Smad7, a TGFb-inducible antagonist of TGF-b signalling. Nature 389, 631±635. Padgett, R.W., Das, P. & Krishna, S. 1998)TGF-b signaling, Smads, and tumor suppressors. Bioessays 20, 382±390. Patterson, G.I. & Padgett, R.W. 2000)TGFb-related pathways. Roles in Caenorhabditis elegans development. Trends Genet. 16, 27±33. Ren, P., Lim, C.S., Johnsen, R., et al. 1996)Control of C. elegans larval development by neuronal expression of a TGF-b homolog. Science 274, 1389±1391. Schroepfer, G.J. Jr 2000)Oxysterols: modulators of cholesterol metabolism and other processes. Physiol. Rev. 80, 361±554. Shibuya, H., Yamaguchi, K., Shirakabe, K., et al. 1996)TAB1: an activator of the TAK1 MAPKKK in TGF-b signal transduction. Science 272, 1179±1182. Suzuki, Y., Yandell, M.D., Roy, P.J., et al. 1999)A BMP homolog acts as a dose-dependent regulator of body size and male tail patterning in Caenorhabditis elegans. Development 126, 241±250. Tsukazaki, T., Chiang, T.A., Davison, A.F., Attisano, L. & Wrana, J.L. 1998)SARA, a FYVE domain protein that recruits Smad2 to the TGFb receptor. Cell 95, 779±791. Whitman, M. 1998)Smads and early developmental signaling by the TGFb superfamily. Genes Dev. 12, 2445±2462. Yamaguchi, K., Shirakabe, K., Shibuya. H., et al. 1995) Identification of a member of the MAPKKK family as a potential mediator of TGF-b signal transduction. Science 270, 2008±2011. Received: 11 January 2001 Accepted: 9 April Genes to Cells 2001) 6, 599±606 q Blackwell Science Limited