InteractiveFly: GeneBrief

Otefin: | References


Gene name - Otefin

Synonyms -

Cytological map position - 55C2-55C2

Function - signaling

Keywords - nuclear lamin, nuclear envelope reassembly, Dpp pathway, germ line stem cells

Symbol - Ote

FlyBase ID: FBgn0266420

Genetic map position - 2R:14,163,108..14,164,698 [-]

Classification - LEM domain protein

Cellular location - nuclear



NCBI links: Precomputed BLAST | EntrezGene

Recent literature
Barton, L. J., Lovander, K. E., Pinto, B. S. and Geyer, P. K. (2016). Drosophila male and female germline stem cell niches require the nuclear lamina protein Otefin. Dev Biol. [Epub ahead of print] PubMed ID: 27174470
Summary:
The nuclear lamina is an extensive protein network that underlies the inner nuclear envelope. This network includes the LAP2-emerin-MAN1-domain (LEM-D) protein family, proteins that share an association with the chromatin binding protein Barrier-to-autointegration factor (BAF). Loss of individual LEM-D proteins causes progressive, tissue-restricted diseases, known as laminopathies. Mechanisms associated with laminopathies are not yet understood. This study describes one of the Drosophila nuclear lamina LEM-D proteins, Otefin (Ote), a homologue of emerin. Previous studies have shown that Ote is autonomously required for the survival of female germline stem cells (GSCs). This study demonstrates that Ote is also required for survival of somatic cells in the ovarian niche, with loss of Ote causing a decrease in cap cell number and altered signal transduction. Germ cell-restricted expression of Ote rescues these defects, revealing a non-autonomous function for Ote in niche maintenance and emphasizing that GSCs contribute to the maintenance of their own niches. Further, the requirement of Ote in the male fertility was investigated. ote mutant males become prematurely sterile as they age. Parallel to observations in females, this sterility is associated with GSC loss and changes in somatic cells of the niche, phenotypes that are largely rescued by germ cell-restricted Ote expression. Taken together, these studies demonstrate that Ote is required autonomously for survival of two stem cell populations, as well as non-autonomously for maintenance of two somatic niches. Finally, the data add to growing evidence that LEM-D proteins have critical roles in stem cell survival and tissue homeostasis.

BIOLOGICAL OVERVIEW

Nuclear envelope proteins play important roles in chromatin organization, gene regulation, and signal transduction; however, the physiological role of these proteins remains elusive. This study found that otefin (ote), which encodes a nuclear lamin, is essential for germline stem cell (GSC) maintenance. Ote, as an intrinsic factor, is both necessary and sufficient to regulate GSC fate. Furthermore, ote is required for the Dpp/BMP signaling pathway to silence bam transcription. By structure-function analysis, it was demonstrated that the nuclear membrane localization of Ote is essential for its role in GSC maintenance. Ote physically interacts with Medea/Smad4 at the bam silencer element to regulate GSC fate. Thus, this study demonstrates that specific nuclear membrane components mediate signal-dependent transcriptional effects to control stem cell behavior (Jiang, 2008).

In adult tissues, stem cells are characterized by their unique capacity to produce daughter stem cells for self-renewal as well as differentiated daughter cells for maintaining homeostasis. Understanding how the self-renewal and differentiation processes of stem cells are controlled will not only reveal the fundamental biological mechanisms that govern the formation and maintenance of tissues, but may also influence future stem cell-based therapies for regenerative medicine (Jiang, 2008).

The Drosophila ovarian germline stem cells (GSCs) within the germarium region provide an attractive system to study the regulatory mechanisms that determine stem cell fate. A typical Drosophila ovary is composed of 16-20 ovarioles, each consisting of an anterior functional unit called a germarium and a linear string of differentiated egg chambers posterior to the germarium. In the tip of the germarium, GSCs normally divide asymmetrically to ensure that one daughter remains attached to the stromal somatic cap cells (or niche cells) for self-renewal. The remaining daughter cell is displaced from the niche and becomes a cystoblast (CB), which initiates differentiation and sustains oogenesis. During this process, one gene, bag-of-marbles (bam), has been shown to act autonomously in the germline to play an instructive role in CB differentiation. In contrast, gene products, such as Piwi and Dpp, a homolog of BMP2/4 in mammals, are produced from niche cells; however, they function as maintenance factors for GSC self-renewal. It has been shown that Dpp signaling from stromal cells activates Smad signaling in GSCs, directly silences bam transcription, and blocks the formation of Bam:Bgcn complexes that would otherwise antagonize translational repression. However, the issue of how Dpp/Smad signaling is transduced in the nucleus and acts especially at the bam silencer element to repress bam transcription remains poorly understood (Jiang, 2008).

The nuclear envelope separates the nucleoplasm from cytoplasm and is composed of outer and inner membranes that are separated by the perinuclear space and joined at nuclear pore complexes. The nuclear lamina is a network of lamin polymers and lamin-associated proteins that are embedded in the inner membrane (Gruenbaum, 2005). Increasing evidence indicates that these nuclear membrane proteins play important roles in chromatin organization, gene regulation, and signal transduction at the cellular level. However, the physiological roles of these proteins remain elusive. Otefin (Ote) is one member of the 'LEM' family, which represents an important group of nuclear membrane-associated proteins that share a conserved LEM domain (Wagner, 2007). Previous studies have shown that Ote physically interacted with lamin B and YA proteins and localized at the nuclear envelope (Ashery-Padan, 1997a; Ashery-Padan, 1997b; Goldberg, 1998). Although inhibition of lamin activity by anti-lamin antibody prevented nuclear assembly in vitro, RNAi experiments demonstrated that knockdown of Ote exhibited no effect on Drosophila Kc167 cells, which suggests that Ote might not be a limiting component for the maintenance of the nuclear architecture (Wagner, 2004). Thus, the function and physiological role of Ote remain elusive (Jiang, 2008).

This study shows that otefin (ote), which encodes a nuclear lamin, is essential for GSC maintenance. Ote, as an intrinsic factor, is both necessary and sufficient for GSC maintenance by silencing bam transcription via interaction with Dpp signaling. Furthermore, nuclear membrane localization of Ote is critical for its function in the maintenance of GSC. Finally, biochemical evidence is presented to support that Ote physically interacts with Medea, a Drosophila Smad4, at the bam silencer element to regulate GSC fate. Thus, these data indicate that an integral membrane protein, the nuclear lamin Ote, functions at target gene loci to mediate BMP signal-dependent repression (Jiang, 2008).

This study has identified and characterized Otefin (Ote) as a protein the plays an important role in the regulation of GSC fate via BMP/Dpp signaling. The data support the notion that specific nuclear membrane components mediate signal-dependent transcriptional effects to control stem cell behavior (Jiang, 2008).

Observation of the abnormality and loss of germ cells in ote mutant ovaries prompted an exploration of whether ote is involved in the regulation of GSC fate. Using germline clonal analysis and rescue tests, it was demonstrated that ote plays an intrinsic role in GSC self-renewal. In addition, it was also observed that ectopic expression of ote increased the number of GSC-like cells, most likely through repression of GSC/CB differentiation. Thus, the results suggest that, like Dpp signaling, Ote is also both necessary and sufficient to regulate GSC fate. A previous study has demonstrated that knockdown of Ote by RNAi interference exhibited no effect on Drosophila Kc167 cells, suggesting that Ote might not be a limiting factor for the maintenance of the nuclear architecture in cultured cells (Wagner, 2004). Consistently, clonal data showed that ote mutant GSCs could develop into normal cysts and egg chambers rather than undergo apoptosis, suggesting that Ote plays a specific role in maintaining GSC self-renewal but not germ cell viability. As supportive evidence, it was also shown that loss of function of ote did not affect the nuclear architecture and the normal expression of other nuclear lamin components in ovaries. In addition, it was found, except in germ cells, neither overexpression nor loss of function of ote exhibited obvious defects in other developmental processes. Together, these data suggest that Ote may play a role in the maintenance of GSC and germ cell development rather than performing a general cell biological function (Jiang, 2008).

Previous studies have revealed two major signaling mechanisms, dpp-dependent bam transcriptional silencing and bam-independent translational repression, that function cooperatively in the repression of GSC differentiation. In GSCs, Pum/Nos-mediated and microRNA-mediated translational control have been proposed to repress translation of the mRNA pool that promotes GSC/CB differentiation; in contrast, Dpp signaling from the niche cells is responsible for silencing bam transcription in GSCs by activating Smad complexes that physically bind the bam silencer element. Thus, the question becomes how Ote integrates into this signal network. Several lines of genetic evidence strongly suggest that Ote acts through the Dpp signaling pathway rather than through a parallel (Dpp-independent) pathway. (1) The removal of Ote activity not only results in the loss of GSCs, but also replicates the mad or med mutant phenotypes. (2) ote suppressed the TKVca-overexpression phenotype, suggesting that the function of Dpp signaling required Ote activity in order to repress germ cell differentiation. (3) Genetic analysis showed that the ote and dpp pathway are functionally dependent on each other. Thus, these results strongly suggest that Ote serves as a positive component in the Dpp signaling pathway rather than acting through a parallel (Dpp-independent) pathway to regulate GSC fate (Jiang, 2008).

The loss of ote results in a female sterile phenotype but does not affect Dpp signaling in other developmental stages, implying that Ote regulates Dpp signaling only in the ovary. It is possible that ote plays a specific role in regulation of the Dpp pathway in ovary, but is dispensable for dpp pathway regulation in other tissues. A similar example is brinker (brk), which also functions in a tissue-specific manner. It has been shown that brk acts as a negative regulator of the dpp pathway in wing growth control; however, it is dispensable for the dpp pathway in the regulation of GSC fate. Another possibility is that ote could have a redundant function with other nuclear membrane protein(s) in the regulation of the dpp pathway in other tissues (Jiang, 2008).

Structure-function analysis revealed that nuclear membrane localization is essential for Ote function in the regulation GSC fate, the co-IP and FRET assays showed a direct interaction between Ote and Med at the nuclear membrane, and the ChIP assay verified that Ote associated with the bam silencer element in a Med-dependent manner, indicating that Ote/Med interaction might be important for recruiting the bam locus to the nuclear envelope. Combined with the data that Ote is necessary and sufficient for bam silencing in vivo, the results further suggest that Ote/Med-mediated relocalization of the bam locus to the nuclear periphery might be important for bam silencing in the regulation of GSC fate. It has been proposed that subnuclear environments at the nuclear periphery promote gene silencing and activation. Silenced regions of the genome, such as centromeres and telomeres, are statically tethered to the nuclear envelope (Ahmed, 2007). Thus, Ote/Med interaction recruiting the bam locus to the nuclear periphery that results in bam silencing may provide an interesting example to support the role of the nuclear periphery in target-gene silencing at the transcriptional level to maintain the identity of the specific type cells (Jiang, 2008).

It has been shown that Schnurri (Shn), a negatively acting Mad cofactor (Affolter, 2007), is genetically required for GSC maintenance. The biochemical evidence showed that the bam silencer element could also form a ShnCT-containing protein-DNA complex with high affinity when Dpp signaling was activated (Pyrowolakis, 2004). Thus, studies have proposed that Shn probably serves as a component in the bam silencing complexes/Smad complexes required for bam silencing, and germline stem cells are maintained by Shn recruitment to the bam silencer element. However, so far, the direct experimental evidence that loss of shn results in derepression of bam in GSCs is still lacking. Since Shn, like Ote, has tissue-specific functions mediated by its ability to confer repressive activity on Smad complexes, it will be interesting to test whether Shn acts together with Ote at the bam silencer element in GSCs (Jiang, 2008).

The LEM family represents an important group of nuclear membrane-associated proteins that share a conserved LEM domain. A number of studies have focused on the potential biochemical properties of these proteins and their relationship with nuclear assembly and cell division at the cellular level (Ashery-Padan, 1997a; Goldberg, 1998; Gruenbaum, 2005; Mattout-Drubezki, 2003). Recently, several studies revealed that certain nuclear envelope components are involved in signal transductions, such as MAN1, a nuclear membrane protein that binds Smad2 and Smad3 and antagonizes TGF-β signaling in vertebrates. These findings are in contrast with the current results indicating that Ote functions positively to regulate Dpp signaling transduction in the regulation of GSC. It has been reported that a Drosophila LEM domain protein encoded by the annotated gene CG3167, named dman1, is the putative ortholog to vertebrate MAN1 (Wagner, 2006). Similar to Ote, downregulation of dMAN1 by RNAi has no obvious effect on Kc167 cells, suggesting that the dMAN1 protein is also not a limiting component of the nuclear architecture either (Wagner, 2004; Wagner, 2006). Since ote and dman1 possess opposite roles in the regulation of TGF-β/BMP signaling, and dMan1 potentially interacts with Mad in yeast two-hybrid assays and co-IP assays in S2 cells, it would be interesting to determine whether Ote and dMan1 collaborate together to balance the self-renewal and differentiation of GSCs by controlling the proper induction of Dpp pathway activity. There is no known counterpart to Ote in mammals; however, Emerin has a domain arrangement similar to Ote, since it also contains a LEM at its N terminus and a single TM at its C terminus. It has been reported that mutations in emerin cause Emery-Dreifuss muscular dystrophy in humans; however, the molecular mechanism of these mutations and their phenotypes remain poorly understood. This study has characterized a new role in the regulation of stem cells for the nuclear lamin Otefin. It will also be interesting to determine whether nuclear lamina components in mammals, including humans, are also involved in fate determination of stem cells, as well as in mediating signal-dependent gene silencing related to human diseases (Jiang, 2008).

Interactions among Drosophila nuclear envelope proteins lamin, otefin, and YA

Lamin, Otefin, and YA are the three Drosophila nuclear envelope proteins that have been characterized in early embryos. The yeast two-hybrid system was used to explore the interactions between pairs of these proteins. The ubiquitous major lamina protein, Lamin Dm, interacts with both Otefin, a peripheral protein of the inner nuclear membrane, and YA, an essential, developmentally regulated protein of the nuclear lamina (see A model for the organization of lamin, otefin, and YA in the nuclear envelope.). In agreement with this interaction, Lamin and Otefin can be coimmunoprecipitated from the vesicle fraction of Drosophila embryos and will colocalize in nuclear envelopes of Drosophila larval salivary gland nuclei. The two-hybrid system was further used to map the domains of interaction among Lamin, Otefin, and YA. Lamin's rod domain interacts with the complete otefin protein, with otefin's hydrophilic NH2-terminal domain, and with two different fragments derived from this domain. Analogous probing of the interaction between Lamin and YA shows that the lamin rod and tail plus part of its head domain are needed for interaction with full-length YA in the two-hybrid system. YA's COOH-terminal region is necessary and sufficient for interaction with lamin. These results suggest that interactions with lamin might mediate or stabilize the localization of Otefin and YA in the nuclear lamina. They also suggest that the need for both Otefin and Lamin in mediating association of vesicles with chromatin might reflect the function of a protein complex that includes these two proteins. Since the hydrophobic COOH terminus of Otefin is required for targeting to the inner nuclear membrane, Otefin may connect with the inner nuclear membrane through its COOH terminus and with the nuclear lamina through other regions of otefin. Interaction between Otefin and Lamin may stabilize the localization of Otefin. This could be similar to the case of the lamin B receptor (LBR) in vertebrates, which has a hydrophilic NH2 terminus and a hydrophobic COOH terminus that is capable of targeting the LBR to the inner nuclear membrane. Since the NH2 terminus of the LBR alone targets a cytosolic protein to the nucleus but a type II integral protein to the inner nuclear membrane, this suggests that targeting a protein to the inner nuclear membrane requires a special domain, such as one mediating interaction with other nuclear envelope proteins (Goldberg, 1998).

Previous studies provided indirect evidence for an in vivo interaction between lamin and otefin. This evidence included the peripheral nucleoplasmic localization of both otefin and lamin, the similar levels of resistance of otefin and of lamin to extraction with Triton X-100, the finding that in early embryos both proteins remain associated with the spindle envelope during mitosis, and the finding that both lamin and otefin are required during nuclear assembly for the attachment of membrane vesicles to chromatin. In addition, in the maternal pool, otefin is associated with the same membrane vesicle fractions as lamin Dmmit. The present study has provided in vitro and in vivo evidence for such an interaction and has shown that otefin and lamin colocalize in salivary gland cell nuclei (Goldberg, 1998).

The two-hybrid experiments in yeast cells revealed that otefin and lamin can interact with one another in the absence of any other Drosophila proteins. Interaction domains delineated in the yeast two-hybrid assay suggest that otefin interacts with the lamin rod domain through otefin's hydrophilic NH2 terminus, including otefin's aa 35 to 172, which have been shown previously to stabilize otefin's localization to the nuclear envelope. Since the hydrophobic COOH terminus of otefin is required for targeting to the inner nuclear membrane, otefin may connect with the inner nuclear membrane through its COOH terminus and with the nuclear lamina through other regions of otefin. Interaction between otefin and lamin may stabilize the localization of otefin. This could be similar to the case of the lamin B receptor (LBR) in vertebrates, which has a hydrophilic NH2 terminus and a hydrophobic COOH terminus that is capable of targeting LBR to the inner nuclear membrane. Since the NH2 terminus of LBR alone targets a cytosolic protein to the nucleus but a type II integral protein to the inner nuclear membrane, this suggests that targeting a protein to the inner nuclear membrane requires a special domain, such as one mediating interaction with other nuclear envelope proteins (Goldberg, 1998).

In vitro studies have shown that the rod domain of lamin has several biological activities. The heptad repeats in the rod domain are involved in coiled-coil interactions, and sequences at both ends are involved in the head-to-tail organization of lamin filaments. The rod domain contains a chromatin binding site, and it can bind M/SAR sequences with high affinity. The current results show that the rod domain can also serve as a binding domain for otefin (Goldberg, 1998).

Distinct regions specify the targeting of Otefin to the nucleoplasmic side of the nuclear envelope

Otefin is a 45-kDa nuclear envelope protein with no apparent homology to other known proteins. It includes a large hydrophilic domain, a single carboxyl-terminal hydrophobic sequence of 17 amino acids, and a high content of serine and threonine residues. Cytological labeling located otefin on the nucleoplasmic side of the nuclear envelope. Chemical extraction of nuclei from Drosophila embryos reveals that Otefin is a peripheral protein whose association with the nuclear envelope is stronger than that of lamin. Deletion mutants of otefin were expressed in order to identify regions that direct Otefin to the nuclear envelope. These experiments revealed that the hydrophobic sequence at the carboxyl terminus is essential for correct targeting to the nuclear envelope, whereas additional regions in the hydrophilic domain of Otefin are required for its efficient targeting and stabilization in the nuclear envelope (Ashery-Padan, 1997a).

Localization and posttranslational modifications of otefin, a protein required for vesicle attachment to chromatin, during Drosophila melanogaster development

Otefin is a peripheral protein of the inner nuclear membrane in Drosophila melanogaster. During nuclear assembly in vitro, it is required for the attachment of membrane vesicles to chromatin. With the exception of sperm cells, otefin colocalizes with lamin Dm0 derivatives in situ and presumably in vivo and is present in all somatic cells examined during the different stages of Drosophila development. In the egg chamber, otefin accumulates in the cytoplasm, in the nuclear periphery, and within the nucleoplasm of the oocyte, in a pattern similar to that of lamin Dm0 derivatives. There is a relatively large nonnuclear pool of otefin present from stages 6 to 7 of egg chamber maturation through 6 to 8 h of embryonic development at 25°C. In this pool, otefin is peripherally associated with a fraction containing the membrane vesicles. This association is biochemically different from the association of otefin with the nuclear envelope. Otefin is a phosphoprotein in vivo and is a substrate for in vitro phosphorylation by cdc2 kinase and cyclic AMP-dependent protein kinase. A major site for cdc2 kinase phosphorylation in vitro was mapped to serine 36 of otefin. Together, these data suggest an essential role for otefin in the assembly of the Drosophila nuclear envelope (Ashery-Padan, 1997b).

Isolation and characterization of the Drosophila nuclear envelope otefin cDNA

The primary translation otefin gene product has a calculated mass of 45 kDa, contains many serine and threonine residues, and is mostly hydrophilic. However, in the carboxyl terminus, there is a hydrophobic region which may serve as a membrane anchoring domain. RNA blot analysis indicated that the otefin gene codes for a single poly(A+) transcript of 1.6 kilobases and that relatively large amounts of this transcript are present during developmental stages in which many nuclear divisions occur. Polyclonal antibodies raised against the cDNA translation product react with a 58-kDa mammalian nuclear envelope protein, demonstrating evolutionary conservation (Padan, 1990).

Persistence of major nuclear envelope antigens in an envelope-like structure during mitosis in Drosophila melanogaster embryos

Using monoclonal antibodies, the fate of three different nuclear envelope proteins during mitosis in Drosophila early embryos was followed by indirect immunofluorescence microscopy. Two of these proteins, lamin and otefin, a newly characterized nuclear envelope polypeptide with an apparent Mr of 53,000, are apparently present in an envelope-like structure that is present throughout mitosis. Immunoelectron microscopy of interphase nuclei indicates that otefin, like lamin, is not a component of nuclear pore complexes. In contrast with lamin and otefin, gp188, a putative pore complex component, was completely redistributed through the surrounding cytoplasm during prophase in comparable early embryo specimens and was present in an envelope only in interphase. Together with previous morphological studies, these data suggest that the entire mitotic apparatus including condensed chromosomes and spindle is enclosed by an envelope throughout mitosis during early embryogenesis in Drosophila. This 'spindle envelope' contains both lamin and otefin but probably not pore complex proteins (Harel, 1989).


REFERENCES

Search PubMed for articles about Drosophila Otefin

Affolter, M. and Basler, K. (2007) The Decapentaplegic morphogen gradient: from pattern formation to growth regulation, Nat. Rev. Genet. 8 (2007): 663-674. PubMed ID: 17703237

Ahmed, S. and Brickner, J. H. (2007). Regulation and epigenetic control of transcription at the nuclear periphery. Trends Genet. 23: 396-402. PubMed ID: 17566592

Ashery-Padan, R., Weiss, A. M., Feinstein, N. and Gruenbaum, Y. (1997a). Distinct regions specify the targeting of otefin to the nucleoplasmic side of the nuclear envelope. J. Biol. Chem. 272(4): 2493-9. PubMed ID: 8999964

Ashery-Padan, R., et al. (1997b). Localization and posttranslational modifications of otefin, a protein required for vesicle attachment to chromatin, during Drosophila melanogaster development. Mol. Cell Biol. 17(7): 4114-23. PubMed ID: 9199347

Goldberg, M., et al. (1998). Interactions among Drosophila nuclear envelope proteins lamin, otefin, and YA. Mol. Cell Biol. 18(7): 4315-23. PubMed ID: 9632815

Gruenbaum, Y., et al. (2005). The nuclear lamina comes of age. Nat. Rev. Mol. Cell Biol. 6: 21-31. PubMed ID: 15688064

Harel, A., et al. (1989). Persistence of major nuclear envelope antigens in an envelope-like structure during mitosis in Drosophila melanogaster embryos. J. Cell Sci. 94: 463-70. PubMed ID: 2517292

Jiang, X., et al. (2008). Otefin, a nuclear membrane protein, determines the fate of germline stem cells in Drosophila via interaction with Smad complexes. Dev. Cell 14(4): 494-506. PubMed ID: 18410727

Mattout-Drubezki, A. and Gruenbaum, Y. (2003). Dynamic interactions of nuclear lamina proteins with chromatin and transcriptional machinery. Cell. Mol. Life Sci. 60: 2053-2063. PubMed ID: 14618255

Padan, R., Nainudel-Epszteyn, S., Goitein, R., Fainsod, A. and Gruenbaum, Y. (1990). Isolation and characterization of the Drosophila nuclear envelope otefin cDNA. J. Biol. Chem. 265(14): 7808-13. PubMed ID: 2186029

Pyrowolakis, G., Hartmann, B., Müller, B., Basler, K. and Affolter, M. (2004). A simple molecular complex mediates widespread BMP-induced repression during Drosophila development. Dev. Cell 7(2): 229-40. PubMed ID: 15296719

Wagner, N., Schmitt, J. and Krohne G. (2004). Two novel LEM-domain proteins are splice products of the annotated Drosophila melanogaster gene CG9424 (Bocksbeutel). Eur. J. Cell Biol. 82(12): 605-16. PubMed ID: 15035436

Wagner, N., Kagermeier, B., Loserth, S. and Krohne, G. (2006). The Drosophila melanogaster LEM-domain protein MAN1. Eur. J. Cell Biol. 85(2): 91-105. PubMed ID: 16439308

Wagner, N. and Krohne, G. (2007). LEM-Domain proteins: new insights into lamin-interacting proteins. Int. Rev. Cytol. 261:1-46. PubMed ID: 17560279


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date revised: 30 January 2009

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