BIOLOGICAL OVERVIEW

Gonad formation requires specific interactions between germ cells and specialized somatic cells, along with the elaborate morphogenetic movements of these cells to create an ovary or testis. A large-scale screen for mutations affecting gonad formation in Drosophila (Moore, 1998) identified mutations that affect the coalescence of the embryonic gonad. Germ cell migration occurs normally in these mutants, and the germ cells are able to correctly associate with the gonadal mesoderm. However, the germ cells fail to form the tight cluster typically found in a properly coalesced gonad, and instead remain only loosely aligned. Three such mutant lines were identified that exhibit similar phenotypes in the gonad and trachea, and all three form a single complementation group. Because the germ cells fail to become intimately associated with one another in the gonad in these mutants, the gene represented by this complementation group was termed fear of intimacy (foi) (Van Doren, 2003).

foi is of particular interest because it affects gonad formation without affecting gonad cell identity, and is therefore specifically required for the morphogenesis of this organ. foi is also required for tracheal branch fusion during tracheal development. E-cadherin/shotgun is similarly required for both gonad coalescence and tracheal branch fusion, suggesting that E-cadherin and Foi cooperate to mediate these processes. foi encodes a member of a novel family of transmembrane proteins that includes the closely related human protein LIV1. The finding that Foi is a cell-surface protein required in the mesoderm for gonad morphogenesis sheds light on the function of this new family of proteins and on the molecular mechanisms of organogenesis (Van Doren, 2003).

Germ cells follow a complex developmental program in order to form the gametes and give rise to the next generation of a species. In animals, much of germ cell development takes place in the gonads, where specialized somatic cells create the unique environment necessary for germ cell differentiation. Thus, proper gonad formation is crucial for germ cell development and reproductive health. Gonad formation is also an excellent system for studying basic questions of morphogenesis: how different cell types recognize one another and undergo the cellular movements required to form properly patterned tissues and organs. There are at least two different types of cellular movements that are required for gonad formation. The first is individual cell migration -- the primordial germ cells migrate from their site of origin to make contact with the cells of the somatic gonad. The second is coordinated tissue morphogenesis -- the germ cells and somatic gonadal cells together coalesce to form the embryonic gonad. Little is known about how such cellular movements combine to produce the gonad, or, indeed, any organ (Van Doren, 2003).

In Drosophila, germ cells initially form as the pole cells at the posterior end of the embryo. The movements of gastrulation bring these cells into the interior of the embryo where they are contained in the posterior endoderm. From this location, the germ cells actively migrate out of the endoderm and into the mesoderm, and make contact with specific mesodermal derivatives that will give rise to the somatic gonad or gonadal mesoderm. The gonadal mesoderm forms from three clusters of mesodermal cells on each side of the embryo. These cells are specified in the eve domain of the dorsolateral mesoderm, and form only in parasegments (PS) 10-12 because of the action of the homeotic gene abdA. Approximately 10 cells form in each cluster, and are recognizable by their expression of the nuclear proteins Eyes absent (Eya) and Zfh1. The three clusters of gonadal mesoderm join to form a single band of cells across PS10-12 at the same time the germ cells complete their migration and specifically associate with these cells (Van Doren, 2003 and references therein).

In the next step of gonad formation, the germ cells and gonadal mesoderm cells undergo a dramatic rearrangement to coalesce in PS10 and form a spherically shaped embryonic gonad. Although this process has not previously been studied in detail, early work suggests that some gonadal mesoderm cells form a sheath around the germ cells, while other mesodermal cells remain intermingled with them. It has also been shown that the gonadal mesoderm does not require the germ cells for gonad formation, and a properly patterned gonad can form in embryos that completely lack germ cells. Thus, the gonadal mesoderm cells can independently undergo the morphogenetic movements of gonad coalescence, suggesting that they play an active role in this process, while the germ cells may be more passive. Although the gonadal mesoderm is specified from PS10-12, the gonad forms in PS10. Thus, it appears that gonadal mesoderm cells move with the germ cells from more posterior segments to PS10 to form the embryonic gonad (Van Doren, 2003).

Although a considerable amount is known about how gonadal mesoderm cell identity is established, little is known about how this identity is translated into the cell-cell interactions and cellular movements required for gonad morphogenesis. The phenotypic and molecular characterization of fear of intimacy shows that it is required for gonad coalescence but not for gonad cell identity. Thus, the Foi protein may play a specific role in gonad morphogenesis. Foi is a transmembrane protein localized to the cell surface and is a member of a new family of proteins that have been well-conserved evolutionarily. Additional studies point to a common molecular mechanism at work in both gonad and tracheal morphogenesis, and both E-cadherin and Foi appear to cooperate to mediate this common mechanism (Van Doren, 2003).

The gonadal mesoderm in foi mutants was examined and this tissue was found to be defective in its ability to undergo the morphogenetic movements of gonad coalescence. In the strongest mutant phenotype, the cells of the gonadal mesoderm do not coalesce with the germ cells, and instead can be seen extending into the other tissues of the embryo. foi mutants are clearly defective in gonadal mesoderm morphogenesis independent of the germ cells (Van Doren, 2003).

A crucial question is whether foi affects gonadal mesoderm coalescence by altering the identity of these cells or by affecting their ability to carry out the appropriate morphogenetic program. foi is shown not to affect the identity of the gonadal mesoderm and, instead, affects gonad coalescence by interfering with the process of morphogenesis downstream of cellular identity (Van Doren, 2003).

foi mutants exhibit a highly-specific gonad phenotype. Not only are molecular markers for the germ cells and somatic cells of the gonad still expressed, but these cells undergo the initial morphogenic movements required for gonad formation, including the proper association of the germ cells and gonadal mesoderm. What is defective is the ability of these cells together to undergo the transition from a loosely associated tissue to the tightly compacted and patterned embryonic gonad. There are several morphogenetic processes that could contribute to such a transition in tissue architecture. Foi does not appear to be affecting cell death or cell division: no dramatic changes are observed in cell number between wild-type and foi-mutant gonads. Instead, it is likely that Foi is affecting changes in cell-cell contact or cell shape that may be required for gonad coalescence. Coalescence of the gonad does not require the presence of the germ cells, indicating that the gonadal mesoderm may be 'driving' this process. It has been found that foi is required in the mesoderm. Thus, the current hypothesis is that Foi is essential for changes in cell-cell contact or cell shape within the gonadal mesoderm that mediate the transition of this tissue from an uncoalesced to a coalesced gonad (Van Doren, 2003).

Sequence database analysis indicates that the FICL family of transmembrane proteins is ancient in origin, yet has expanded in animals to include multiple family members and independent subgroups that are likely to have diverged functions. Although members of the FICL family are well represented in the databases, little is known about the function of any family member. Loss-of-function mutations in bacterial (M. xanthus) and yeast (S. cerevisae) family members are viable with no growth defects on rich medium, but have apparently not been further analyzed. In Arabidopsis, mutations in IAR1 confer resistance to high levels of conjugated auxins (Lasswell, 2000), and IAR1 is therefore likely to be important for the uptake or metabolism of these hormone derivatives. In Drosophila, mutations in Catsup lead to elevated catecholamine levels due to increased activity of the rate-limiting enzyme in this pathway, tyrosine hydroxylase (TH) (Stathakis, 1999). Thus, the Catsup protein may act as a negative regulator of TH activity. Virtually nothing is known about how this family of proteins functions at the molecular level to control such apparently different cellular processes (Van Doren, 2003 and references therein).

The data indicate that Foi is a cell-surface protein and is required in the mesoderm for gonad coalescence. This suggests several models for how Foi might be acting at the molecular level. First of all, Foi might act in cell adhesion, either directly via its extracellular domains or by regulating the activity of a cell adhesion molecule such as E-cadherin. The lack of clear sequence homology within the putative extracellular N-terminal domain in the FICL family suggests that either this domain is not acting in protein-protein interaction, or that different FICL family members have very different binding partners. Foi might also be involved in contacting and regulating the cytoskeleton, which is likely to mediate the changes in cellular morphology observed during gonad coalescence. Such a role might include affecting cytoskeletal changes in response to signals or providing contact between the cytoskeleton and the cell-surface or cell-cell junctions. Finally, Foi might act in sending or receiving a signal that is required for the onset of gonad coalescence. In this capacity, Foi might act non-autonomously in the surrounding non-gonadal mesoderm to produce a signal to the gonad, or autonomously within the gonadal mesoderm to respond to this signal and initiate gonad morphogenesis (Van Doren, 2003).

Recently, epitope-tagged versions of two other FICL family members, ermelin and KE4, have been reported to localize to the endoplasmic reticulum when expressed in tissue culture (Suzuki, 2002). The data in both tissue culture and in embryos with functional HA-Foi transgenes indicate that Foi is localized to the cell surface. Thus, different FICL family members may have distinct subcellular localizations (Van Doren, 2003).

Since FICL family members are predicted to have multiple transmembrane domains, an interesting possibility is that these proteins act as channels, either alone or as homo- or hetero-multimers. For example, gonad morphogenesis might be initiated or coordinated by an intercellular signal that involves membrane transport by Foi or cell adhesion might be regulated by transport of a required ion or small molecule effector. In support of the channel model, the TM domains of Foi show sequence homology with other FICL family members. This homology appears to be more extensive than would be necessary to simply retain TM character, and suggests that the primary sequence of these domains is critical for some aspect of Foi function, such as the formation of a transmembrane channel. Sequence comparisons have revealed some homology between the ZIP family of metal transporters and members of the FICL family (Eng, 1998). However, there are many regions of homology that discriminate between the ZIP and FICL families, and there are several 'true' ZIP family members in both the human and Drosophila genome databases. Thus, the ZIP and FICL families may be evolutionarily related in a more distant manner, but this does not necessarily indicate that the FICL proteins will also be metal transporters. Whether FICL family members act as channels at all, and what their substrates might be, are interesting questions for future analysis (Van Doren, 2003).

foi and E-cadherin share similar mutant phenotypes in gonad coalescence and tracheal branch fusion. This suggests that there is a common molecular mechanism at work in both gonad and tracheal morphogenesis, and that E-cadherin and Foi may be cooperating to mediate this common mechanism. In the gonad, E-cadherin-based cell adhesion might act to promote proper cell-cell contacts required for coalescence and gonad organization. An important aspect of the mechanism of action of Foi may be to somehow modulate E-cadherin based cell adhesion. In support of this, E-cadherin expression has been found to increase in the gonadal mesoderm at the time that coalescence begins, and E-cadherin expression in the gonad has been found to be drastically reduced in foi mutants (Van Doren, 2003).

The relationship between Foi and E-cadherin is particularly interesting since the closest homolog of Foi in humans, LIV1, was identified as an estrogen-responsive gene in breast cancer cells (Manning, 1988). LIV1 expression has been correlated with mammary tumor metastasis (Manning, 1994). E-cadherin is also known to play an important role in regulating metastatic potential in a variety of human cancers, with downregulation being correlated with increased metastasis and upregulation being found at the site of secondary tumor formation. The analysis of Foi in Drosophila suggests that LIV1 and E-cadherin may be working together during breast cancer progression (Van Doren, 2003).

Gonad formation and gametogenesis are essential for the fundamental process of sexual reproduction, and are therefore likely to be evolutionarily conserved. There are many parallels between gonad formation in mammals and in Drosophila, and these parallels may well extend to the molecular level. Formation of the mouse gonad, for example, involves very similar stages of germ cell migration, association between germ cells and gonadal mesoderm, and gonad coalescence as seen in Drosophila. Furthermore, it has recently been shown that E-cadherin has a role in mouse gonad formation, and appears to function in the germ cells for their proper coalescence into the developing gonad. A role for E-cadherin in Drosophila gonad coalescence has been demonstrated, although the evidence points to roles for E-cadherin in both the germ cells and the gonadal mesoderm. It is intriguing to speculate that a foi homolog may also function with E-cadherin in mouse gonad formation. Thus, as has been true for other developmental processes, understanding the mechanisms of gonad formation in Drosophila may provide a molecular picture of how this process works in other species (Van Doren, 2003).

GENE STRUCTURE

cDNA clone length - 3622

Bases in 5' UTR - 637

Exons - 6

Bases in 3' UTR - 879

PROTEIN STRUCTURE

Amino Acids - 706

Structural Domains

Recombination mapping indicated that foi is located on the left arm of chromosome 3 (3-25.2) and complementation tests identified two transposon insertion lines that fail to complement the phenotype of independent foi alleles. Experiments were performed to excise one of these transposon insertions, which demonstrate that the transposon is responsible for the foi phenotype in this line. Molecular analysis revealed that both transposons had inserted into the 5' untranslated region of the same transcription unit, producing a 4 kb RNA as judged by Northern blot. Several cDNAs corresponding to this transcript were identified and the longest of these, which was 3.85 kb, was completely sequenced. Sequence analysis of genomic DNA from three independent ethylmethane sulfonate-induced foi alleles reveals that all have nonsense mutations in the large open reading frame present in this cDNA. These data, combined with the ability of this cDNA to rescue the foi mutant phenotype in transgenic animals, led to the conclusion that the foi transcription unit had been identifed (Van Doren, 2003).

The Foi protein is predicted to be 706 amino acids in length and to have an N-terminal signal sequence and at least six transmembrane domains (TM1-3 and TM4-6). The highly conserved 'HELP domain' is weakly predicted by some algorithms to contain an additional 1-2 TM spans, and so the mature Foi protein is likely to have 6-8 TM spans in total. Foi also contains a histidine rich N-terminal domain and a short C-terminal tail (Van Doren, 2003).

Homology searches with Foi reveal that it is part of a larger family of proteins that are conserved from yeast to humans. Although only one Foi-related sequence currently appears in the genome databases of the fungi S. cerevisae and S. pombe and the plant A. thaliana, multiple family members are found in the genomes of animals such as Drosophila (four members), C. elegans (eight members), and humans (six members). In animals, this family can be divided into two subgroups, one more closely related to Foi and a second that is more related to another Drosophila protein Catsup (Stathakis, 1999). For example, Drosophila Foi is more closely related to human LIV1 (Manning, 1988) than it is to Drosophila Catsup. Likewise, Drosophila Catsup is more closely related to human KE4 (Ando, 1996) than it is to Drosophila Foi. Thus, this family seems to have split into two subgroups prior to the divergence of protostome and deuterostome metazoans. Since the founding members of this family include Foi, IAR1 (Arabidopsis) (Lasswell, 2000), Catsup and LIV1, this family of proteins will be referred to as the FICL family (Van Doren, 2003).

Members of the FICL family share several regions of sequence homology as well as an overall similar domain structure and predicted membrane topology. They each have a long N terminus that is histidine rich and contains several putative glycosylation sites, but does not show sequence homology. The TM domains, however, share considerable sequence homology. This homology appears to be more extensive than would be necessary to maintain their transmembrane character, and there are a number of invariant residues in these domains. Thus, these sequences may play a more specific role in FICL protein function in addition to their structural role in forming TM spans (Van Doren, 2003).

The most highly conserved region of the FICL family is the 'HELP' domain (named after a conserved amino acid cluster usually found in this domain). This domain is the region that shows the highest sequence identity throughout the family, and is also part of a larger domain family found in prokaryotes (ProDom analysis). This domain is 75% identical (90% similar) in Drosophila Foi and human LIV1, and 33% identical (47% similar) in Foi and the prokaryotic M. xanthus GufA protein (McGowan, 1993). Although the FICL family has clearly been well conserved across a broad evolutionary spectrum, little is known about how these proteins function at the molecular level (Van Doren, 2003).

date revised: 30 June 2003

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.