Mutations in the gene encoding the Survival Motor Neuron (SMN) protein are responsible for autosomal recessive proximal spinal muscular atrophy (SMA). SMN orthologues have been identified in the nematode worm C. elegans and the yeast Schizosaccharomyces pombe but, to date, no human paralogues have been described. An SMN-related protein (SMNrp) gene is described that encodes a novel protein of 239 amino acids, which has recently been identified as a constituent of the spliceosome complex and designated SPF30. Significant similarity to the SMN protein is apparent only within a central region of SMNrp that represents a tudor domain. The SMNrp/SPF30 gene has been mapped to chromosome 10q23. It is differentially expressed, with abundant levels in skeletal muscle. An exclusively nuclear localization for SMNrp in cultured cells and muscle sections was revealed using GFP fusion constructs and thereafter confirmed with a polyclonal antibody raised against SMNrp. Overexpression of SMNrp as a fusion protein in HeLa cells in culture induced dose-dependent apoptosis with positive TUNEL staining. In addition to a possible role for this protein as a pro-apoptotic factor, SMN and its related protein share significant similarities in sequence and cellular function (Talbot, 1998).
Spinal muscular atrophy (SMA) is a neurodegenerative disease of spinal motor neurons caused by reduced levels of functional survival of motor neurons (SMN) protein. SMN is part of a macromolecular complex that contains the SMN-interacting protein 1 (SIP1) and spliceosomal Sm proteins. Although it is clear that SIP1 as a component of this complex is essential for spliceosomal uridine-rich small ribonucleoprotein (U snRNP) assembly, the role of SMN and its functional interactions with SIP1 and Sm proteins are poorly understood. This study shows that the central region of SMN comprising a tudor domain facilitates direct binding to Sm proteins. Strikingly, the SMA-causing missense mutation E134K within the tudor domain severely reduced the ability of SMN to interact with Sm proteins. Moreover, antibodies directed against the tudor domain prevent Sm protein binding to SMN and abolish assembly of U snRNPs in vivo. Thus, these data show that SMN is an essential U snRNP assembly factor and establish a direct correlation between defects in the biogenesis of U snRNPs and SMA (Bühler, 1999).
Arginine residues in RG-rich proteins are frequently dimethylated posttranslationally by protein arginine methyltransferases (PRMTs). The most common methylation pattern is asymmetrical dimethylation, a modification important for protein shuttling and signal transduction. Symmetrically dimethylated arginines (sDMA) have until now been confined to the myelin basic protein MBP and the Sm proteins D1 and D3. The human Sm protein B/B' and one of the Sm-like proteins, LSm4, contain sDMA in vivo as shown by mass spectrometry and protein sequencing. The symmetrical dimethylation of B/B', LSm4, D1, and D3 decisively influences their binding to the Tudor domain of the 'survival of motor neurons' protein (SMN): inhibition of dimethylation by S-adenosylhomocysteine (SAH) abolishes the binding of D1, D3, B/B', and LSm4 to this domain. A synthetic peptide containing nine sDMA-glycine dipeptides, but not asymmetrically modified or nonmodified peptides, specifically inhibits the interaction of D1, D3, B/B', LSm4, and UsnRNPs with SMN-Tudor. Recombinant D1 and a synthetic peptide can be methylated in vitro by both HeLa cytosolic S100 extract and nuclear extract; however, only the cytosolic extract produced symmetrical dimethylarginines. Thus, the Sm-modifying PRMT is cytoplasmic, and symmetrical dimethylation of B/B', D1, and D3 is a prerequisite for the SMN-dependent cytoplasmic core-UsnRNP assembly. This demonstration of sDMAs in LSm4 suggests additional functions of sDMAs in tri-UsnRNP biogenesis and mRNA decay. These findings also have interesting implications for the understanding of the aetiology of spinal muscular atrophy (Brahms, 2001).
The SMN protein, which is linked to spinal muscular atrophy (SMA), plays an important role in the assembly of the spliceosomal small nuclear ribonucleoprotein complexes. This function requires binding of SMN to the arginine-glycine (RG) rich C-terminal tails of the Sm proteins, which contain symmetrically dimethylated arginine residues (sDMA) in vivo. Using NMR titrations, it is shown that the SMN Tudor domain recognizes these sDMAs in the methylated RG repeats. Upon complex formation a cluster of conserved aromatic residues in the SMN Tudor domain interacts with the sDMA methyl groups. Two high resolution structures of the uncomplexed SMN Tudor domain are presented, a 1.8Å crystal structure and an NMR structure that has been refined against a large number of backbone and side-chain residual dipolar couplings. The backbone conformation of both structures is very similar, however, differences are observed for the cluster of conserved aromatic side-chains in the sDMA binding pocket. In order to validate these variations a novel application of residual dipolar couplings was introduced for aromatic rings. Structural information can be derived from aromatic ring residual dipolar couplings, even in the presence of internal motions such as ring flipping. These residual dipolar couplings and ring current shifts independently confirm that the SMN Tudor domain adopts two different conformations in the sDMA binding pocket. The observed structural variations may play a role for the recognition of sDMAs (Sprangers, 2003).
Although most patients with spinal muscular atrophy (SMA) are homozygous for deletion of the SMN1 gene, some patients bear one SMN1 copy with a subtle mutation. Detection of such an intragenic mutation may be helpful not only in confirming diagnosis but also in elucidating functional domains of the SMN protein. In this study, a novel mutation was identiifed in SMN1 of two Japanese patients with type I SMA. DHPLC and sequencing analysis revealed that they harbored a point mutation in SMN1 exon 3, 275G --> C, leading to tryptophan-to-serine substitution at amino acid 92 (W92S) at the N-terminal of SMN Tudor domain. In-vitro protein binding assays showed that the mutation severely reduced interaction of the domain with SmB protein and fibrillarin, suggesting that it impairs the critical function of SMN. In conclusion, a novel mutation, W92S, in the Tudor domain was found to affect the interaction of SMN with the target proteins (Kotani, 2007).
The Tudor domain is an approximately 60-amino acid structure motif in search of a function. This study shows that the Tudor domains of the spinal muscular atrophy gene product SMN, the splicing factor 30 kDa (SPF30), and the Tudor domain-containing 3 (TDRD3) proteins interacted with arginine-glycine-rich motifs in a methylarginine-dependent manner. The Tudor domains also associated with methylarginine-containing cellular proteins, providing evidence that methylated arginines represent physiological ligands for this protein module. In addition, it is reported that spliceosomal small nuclear ribonucleoprotein particles core Sm proteins accumulates in the cytoplasm when arginine methylation is inhibited with adenosine dialdehyde or in the presence of an excessive amount of unmethylated arginine-glycine-rich peptides. These data provide in vivo evidence in support of a role for arginine methylation in the proper assembly and localization of spliceosomal Sm proteins (Côté, 2005).
The coactivator-associated arginine methyltransferase CARM1 is recruited by many different transcription factors as a positive regulator. To understand the mechanism by which CARM1 functions, attempts were made to isolate its substrates. A small-pool screening approach was developed for this purpose and CA150, SAP49, SmB, and U1C were identified as splicing factors that are specifically methylated by CARM1. CA150, a molecule that links transcription to splicing, interacts with the Tudor domain of the spinal muscular atrophy protein SMN in a CARM1-dependent fashion. Experiments with an exogenous splicing reporter and the endogenous CD44 gene revealed that CARM1 promotes exon skipping in an enzyme-dependent manner. The identification of splicing factors that are methylated by CARM1, and protein-protein interactions that are regulated by CARM1, strongly implicates this enzyme in the regulation of alternative splicing and points toward its involvement in spinal muscular atrophy pathogenesis (Cheng, 2007).
Blimp1 (see Drosophila Blimp-1), a transcriptional repressor, has a crucial role in the specification of primordial germ cells (PGCs) in mice at embryonic day 7.5 (E7.5). This SET-PR domain protein can form complexes with various chromatin modifiers in a context-dependent manner. Blimp1 has a novel interaction with Prmt5, an arginine-specific histone methyltransferase that mediates symmetrical dimethylation of arginine 3 on histone H2A and/or H4 tails (H2A/H4R3me2s). Prmt5 has been shown to associate with Tudor, a component of germ plasm in Drosophila melanogaster. Blimp1-Prmt5 colocalization results in high levels of H2A/H4 R3 methylation in PGCs at E8.5. However, at E11.5, Blimp1-Prmt5 translocates from the nucleus to the cytoplasm, resulting in the loss of H2A/H4 R3 methylation at the time of extensive epigenetic reprogramming of germ cells. Subsequently, Dhx38, a putative target of the Blimp1-Prmt5 complex, is upregulated. Interestingly, expression of Dhx38 is also seen in pluripotent embryonic germ cells that are derived from PGCs when Blimp1 expression is lost. This study demonstrates that Blimp1 is involved in a novel transcriptional regulatory complex in the mouse germ-cell lineage (Ancelin, 2006).
Biological responses to histone methylation critically depend on the faithful readout and transduction of the methyl-lysine signal by 'effector' proteins, yet an understanding of methyl-lysine recognition has so far been limited to the study of histone binding by chromodomain and WD40-repeat proteins. The double tudor domain of JMJD2A, a Jmjc domain-containing histone demethylase, binds methylated histone H3-K4 and H4-K20. The double tudor domain has an interdigitated structure, and the unusual fold is required for its ability to bind methylated histone tails. The cocrystal structure of the JMJD2A double tudor domain with a trimethylated H3-K4 peptide reveals that the trimethyl-K4 is bound in a cage of three aromatic residues, two of which are from the tudor-2 motif, whereas the binding specificity is determined by side-chain interactions involving amino acids from the tudor-1 motif. This study provides mechanistic insights into recognition of methylated histone tails by tudor domains and reveals the structural intricacy of methyl-lysine recognition by two closely spaced effector domains (Huang, 2006).
The post-translational modification of histones regulates many cellular processes, including transcription, replication and DNA repair. A large number of combinations of post-translational modifications are possible. This cipher is referred to as the histone code. Many of the enzymes that lay down this code have been identified. However, so far, few code-reading proteins have been identified. This study describes a protein-array approach for identifying methyl-specific interacting proteins. Not only chromo domains but also tudor and MBT domains bind to methylated peptides from the amino-terminal tails of histones H3 and H4. Binding specificity observed on the protein-domain microarray was corroborated using peptide pull-downs, surface plasma resonance and far Western blotting. Thus, these studies expose tudor and MBT domains as new classes of methyl-lysine-binding protein modules, and also demonstrates that protein-domain microarrays are powerful tools for the identification of new domain types that recognize histone modifications (Kim, 2006).
53BP1 is a key transducer of the DNA damage checkpoint signal, which is required for phosphorylation of a subset of ATM substrates and p53 accumulation. After cell irradiation, the 53BP1 N-terminal region is phosphorylated. Its two C-terminal BRCT motifs interact with p53. Its central region is required and sufficient for 53BP1 foci formation at DNA strand breaks and for 53BP1 binding to the kinetochore. It contains an RG-rich segment and interacts with DNA in vitro. The major globular domain of the 53BP1 central region adopts a new structural motif composed of two tightly packed Tudor domains and a C-terminal alpha helix. A unique surface essentially located on the first Tudor domain is involved in the binding to 53BP1 RG-rich sequence and to DNA, suggesting that the Tudor tandem can act as an adaptor mediating intramolecular as well as intermolecular protein-protein interactions and protein-nucleic acid associations (Charier, 2004).
The mechanisms by which eukaryotic cells sense DNA double-strand breaks (DSBs) in order to initiate checkpoint responses are poorly understood. 53BP1 is a conserved checkpoint protein with properties of a DNA DSB sensor. The structure of the domain of 53BP1 that recruits it to sites of DSBs has been solved. This domain consists of two tandem tudor folds with a deep pocket at their interface formed by residues conserved in the budding yeast Rad9 and fission yeast Rhp9/Crb2 orthologues. In vitro, the 53BP1 tandem tudor domain bound histone H3 methylated on Lys 79 using residues that form the walls of the pocket; these residues were also required for recruitment of 53BP1 to DSBs. Suppression of DOT1L, the enzyme that methylates Lys 79 of histone H3, also inhibited recruitment of 53BP1 to DSBs. Because methylation of histone H3 Lys 79 was unaltered in response to DNA damage, it is proposed that 53BP1 senses DSBs indirectly through changes in higher-order chromatin structure that expose the 53BP1 binding site (Huyen, 2004).
The human p100 protein is a vital transcription regulator that increases gene transcription by forming a physical bridge between promoter-specific activators and the basal transcription machinery. The tudor and SN (TSN) domain of p100 interacts with U small nuclear ribonucleoprotein (snRNP) complexes, suggesting a role for p100 in the processing of precursor messenger RNA. The crystal structure of the p100 TSN domain was determined to delineate the molecular basis of p100's proposed functions. The interdigitated structure resembles a hook, with a hinge controlling the movement and orientation of the hook. These studies suggest that a conserved aromatic cage hooks methyl groups of snRNPs and anchors p100 to the spliceosome. These structural insights partly explain the distinct roles of p100 in transcription and splicing (Shaw, 2007).
Transcription and pre-mRNA splicing are the key nuclear processes in eukaryotic gene expression, and identification of factors common to both processes has suggested that they are functionally coordinated. p100 protein has been shown to function as a transcriptional co-activator for several transcription factors. p100 consists of staphylococcal nuclease (SN)-like and Tudor-SN (TSN) domains of which the SN-like domains have been shown to function in transcription, but the function of TSN domain has remained elusive. This study identified interaction between p100 and small nuclear ribonucleoproteins (snRNP) that function in pre-mRNA splicing. The TSN domain of p100 specifically interacts with components of the U5 snRNP, but also with the other spliceosomal snRNPs. In vitro splicing assays revealed that the purified p100, and specifically the TSN domain of p100, accelerates the kinetics of the spliceosome assembly, particularly the formation of complex A, and the transition from complex A to B. Consistently, the p100 protein, as well as the separated TSN domain, enhanced the kinetics of the first step of splicing in an in vitro splicing assay in dose-dependent manner. Thus these results suggest that p100 protein is a novel dual function regulator of gene expression that participates via distinct domains in both transcription and splicing (Yang, 2007).
Characteristic ribonucleoprotein-rich granules, called nuages, are present in the cytoplasm of germ-line cells in many species. In mice, nuages are prominent in postnatal meiotic spermatocytes and postmeiotic round spermatids, and are often called chromatoid bodies at the stages. Mouse tudor repeat-1 (Mtr-1) encodes a MYND domain and four copies of the tudor domain. Multiple tudor domains are a characteristic of the Tudor protein, a component of Drosophila nuages. Mtr-1 is expressed in germ-line cells and is most abundant in fetal prospermatogonia and postnatal primary spermatocytes. The MTR-1 protein is present in the cytoplasm of prospermatogonia, spermatocytes, and round spermatids, and predominantly localizes to chromatoid bodies. This study shows that an assembled form of small nuclear ribonucleoproteins (snRNPs), which usually function as spliceosomal complexes in the nucleus, accumulate in chromatoid bodies, and form a complex with MTR-1. When expressed in cultured cells, MTR-1 forms discernible granules that co-localize with snRNPs in the cell plasm during cell division. The deletion of multiple tudor domains in MTR-1 abolishes the formation of such granules. These results suggest that MTR-1, which would provide novel insights into evolutionary comparison of nuages, functions in assembling snRNPs into cytoplasmic granules in germ cells (Chuma, 2003).
Embryonic patterning and germ-cell specification in mice are regulative and depend on zygotic gene activities. However, there are mouse homologues of Drosophila maternal effect genes, including vasa and tudor, that function in posterior and germ-cell determination. A targeted mutation in Tudor domain containing 1/mouse tudor repeat 1 (Tdrd1/Mtr-1), a tudor-related gene in mice, leads to male sterility because of postnatal spermatogenic defects. TDRD1/MTR-1 predominantly localizes to nuage/germinal granules, an evolutionarily conserved structure in the germ line, and its intracellular localization is downstream of mouse vasa homologue/DEAD box polypeptide 4 (Mvh/Ddx4), similar to Drosophila vasa-tudor. Tdrd1/Mtr-1 mutants lack, and Mvh/Ddx4 mutants show, strong reduction of intermitochondrial cement, a form of nuage in both male and female germ cells, whereas chromatoid bodies, another specialized form of nuage in spermatogenic cells, are observed in Tdrd1/Mtr-1 mutants. Hence, intermitochondrial cement is not a direct prerequisite for oocyte development and fertility in mice, indicating differing requirements for nuage and/or its components between male and female germ cells. The result also proposes that chromatoid bodies likely have an origin independent of or additional to intermitochondrial cement. The analogy between Mvh-Tdrd1 in mouse spermatogenic cells and vasa-tudor in Drosophila oocytes suggests that this molecular pathway retains an essential role(s) that functions in divergent species and in different stages/sexes of the germ line (Chuma, 2006; full text of article).
The germ-line cells of many animals possess a characteristic cytoplasmic structure termed nuage or germinal granules. In mice, nuage that is prominent in postnatal male germ cells is also called intermitochondrial cement or chromatoid bodies. TDRD1/MTR-1, which contains Tudor domain repeats, is a specific component of the mouse nuage, analogously to Drosophila Tudor, a constituent of polar granules/nuage in oocytes and embryos. TDRD6 and TDRD7/TRAP, which also contain multiple Tudor domains, specifically localize to nuage and form a ribonucleoprotein complex together with TDRD1/MTR-1. The characteristic co-localization of TDRD1, 6 and 7 was disrupted in a mutant of mouse vasa homologue/DEAD box polypeptide 4 (Mvh/Ddx4), which encodes another evolutionarily conserved component of nuage. In vivo over-expression experiments of the TDRD proteins and truncated forms during male germ cell differentiation showed that a single Tudor domain is a structural unit that localizes or accumulates to nuage, but the expression of the truncated, putative dominant negative forms is detrimental to meiotic spermatocytes. These results indicate that the Tudor-related proteins, which contain multiple repeats of the Tudor domain, constitute an evolutionarily conserved class of nuage components in the germ-line, and their localization or accumulation to nuage is likely conferred by a Tudor domain structure and downstream of Mvh, while the characteristic repeated architecture of the domain is functionally essential for the differentiation of germ cells (Hosokawa, 2007).
mRNA localization is a powerful mechanism for targeting factors to different regions of the cell and is used in Drosophila to pattern the early embryo. The parasitoid wasp Nasonia (Hymenoptera) undergoes long germ development similar to that of Drosophila, yet is evolutionarily very distant from flies (> 200 MY) and lacks bicoid. During oogenesis of Nasonia, mRNA localization is used extensively to replace the function of the bicoid gene for the initiation of patterning along the antero-posterior axis. Nasonia localizes both caudal and nanos to the posterior pole, whereas giant mRNA is localized to the anterior pole of the oocyte; orthodenticle1 (otd1) is localized to both the anterior and posterior poles. The abundance of differentially localized mRNAs during Nasonia oogenesis provided a unique opportunity to study the different mechanisms involved in mRNA localization. Through pharmacological disruption of the microtubule network, it was found that both anterior otd1 and giant, as well as posterior caudal mRNA localization was microtubule-dependent. Conversely, posterior otd1 and nanos mRNA localized correctly to the posterior upon microtubule disruption. However, actin is important in anchoring these two posteriorly localized mRNAs to the oosome, the structure containing the pole plasm. Moreover, knocking down the functions of the genes tudor and Bicaudal-D mimics disruption of microtubules, suggesting that tudor’s function in Nasonia is different from flies, where it is involved in formation of the pole plasm (Olesnicky, 2007).
Both the Drosophila and Nasonia ovariole are meroistic, meaning that the nurse cells and oocyte are both of germ cell descent and originate from the same primordium, but differentiate during subsequent cell divisions. As each ovarian follicle develops and is positioned more distally along the ovariole, the nurse cells remain attached to one another and to the oocyte through ring canals, which arise from incomplete cleavage during cell division. The 16 sister cells that make up each germline cyst result from four of these incomplete divisions. An egg chamber forms comprising of 15 nurse cells and the oocyte, surrounded by the somatic follicle cells, which form an epithelial layer around the oocyte. Nurse cells produce metabolites and other factors that transit through the ring canals to accumulate in the oocyte (Olesnicky, 2007).
The Drosophila oocyte is specified early during oogenesis as a result of the asymmetric segregation of the fusome, an organelle that connects the 16 cells. Once the oocyte has been specified, the polarity of the oocyte microtubule network becomes extremely dynamic and undergoes a major reorganization resulting from communication between the oocyte and follicle cells. This reorganization is essential to localize maternal mRNAs that will generate the axes of the embryo. At first, microtubule minus ends extend from the nurse cells into the oocyte toward a microtubule organizing center (MTOC) localized at the posterior pole of the oocyte, near its nucleus. Later, however, the posterior MTOC disassembles while multiple MTOCs form toward the anterior of the growing oocyte. At this stage, the microtubules are therefore pointing from the plus end at the posterior of the oocyte to the minus end at the anterior. mRNAs and the oocyte nucleus utilize the polarity of the microtubules to localize to the anterior or posterior pole (Olesnicky, 2007).
Nasonia oogenesis presents striking similarities to that of Drosophila. It is divided into five morphologically distinct stages. In stage 1, the nurse cells and oocyte are indistinguishable until they begin to segregate, with the oocyte lying towards the posterior of the follicle. By stage 2, the nurse cells and a smaller oocyte are clearly distinguishable, as a constriction forms between the oocyte and its supporting nurse cells. At this stage, the oocyte nucleus is positioned in the center of the cell. The oocyte continues to grow throughout stage 3 until it becomes larger than its accompanying nurse cells. Concomitantly, the oocyte nucleus migrates to the dorsal anterior cortex of the developing oocyte, as in Drosophila. Later, during stage 4, the nurse cells begin to degenerate as they empty all their contents into the oocyte. In the final stage (5), a vitelline membrane is constructed around the embryo (Olesnicky, 2007).
This study shows that the localization of four maternal mRNAs is achieved using at least 2 distinct mechanisms. It is shown that, during Nasonia oogenesis, microtubules play a major role in oocyte polarity and in the control of anterior localization of otd1 and gt mRNA and the posterior localization of cad mRNA. In contrast, the actin cytoskeleton is important for anchoring the oosome and is therefore essential for the localization of nanos and otd1 mRNA to the posterior pole of the oocyte (Olesnicky, 2007).
It is proposed that Nasonia utilizes two basic mechanisms for the localization of mRNA, a microtubule-dependent mechanism and an actin-dependent, microtubule-independent one. Anterior localization of gt and otd1 mRNA, as well as posterior localization of cad mRNA, all rely on a similar microtubule-dependent mechanism while posterior localization of otd1 and nos mRNAs relies on actin. In wild-type follicles, cad and gt mRNAs are initially localized, while later in oogenesis this localization is relaxed to achieve a more graded mRNA distribution. otd1 anterior mRNA, although not graded, is also localized loosely in wild-type follicles. nos mRNA localization and posteriorly localized otd1 mRNA, however, are tightly localized to the posterior in a microtubule-independent manner. Interestingly, in freshly laid embryos both posterior otd1 mRNA and nos mRNA are localized to the oosome. Maintaining localization of these two posteriorly localized mRNAs relies on the actin cytoskeleton. Additionally, actin might be required to anchor the oosome to the posterior pole of the oocyte, as well as to trap mRNA to the oosome. It is therefore likely that both mRNAs are localized to structures within the germ plasm, resulting in a tight localization that is maintained throughout oogenesis and early embryogenesis and does not rely extensively on microtubules (Olesnicky, 2007).
Piwi proteins are essential for germline development, stem cell self-renewal, epigenetic regulation, and transposon silencing. They bind to a complex class of small noncoding RNAs called Piwi-interacting RNAs (piRNAs). Mammalian Piwi proteins such as Mili are localized in the cytoplasm of spermatogenic cells, where they are associated with a germline-specific organelle called the nuage or its derivative, the chromatoid body, as well as with polysomes. To investigate the molecular mechanisms mediated by Mili, Mili-interacting proteins were sought. This study reports that Mili specifically interacts with Tudor domain-containing protein 1 (Tdrd1), a germline protein that contains multiple domains. This RNA-independent interaction is mediated through the N-terminal domain of Mili and the N-terminal region of Tdrd1 containing the myeloid Nervy DEAF-1 (MYND) domain and the first two Tudor domains. In addition, Mili positively regulates the expression of the Tdrd1 mRNA. Furthermore, Mili and Tdrd1 mutants share similar spermatogenic defects. However, Tdrd1, unlike Mili, is not required for piRNA biogenesis. These results suggest that Mili interacts with Tdrd1 in the nuage and chromatoid body. This interaction does not contribute to piRNA biogenesis but represents a regulatory mechanism that is critical for spermatogenesis (Wang, 2009).
Home page: The Interactive Fly © 2006 Thomas Brody, Ph.D.
date revised: 1 August 2007
The Interactive Fly resides on the
Society for Developmental Biology's Web server.