InteractiveFly: GeneBrief

fear of intimacy: Biological Overview | Functional Analysis | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

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).

REGULATION

Zinc is essential for many cellular processes, and its concentration in the cell must be tightly controlled. The Zrt/IRT-like protein (ZIP) family of zinc transporters have recently been identified as the main regulators of zinc influx into the cytoplasm; however, little is known about their in vivo roles. fear of intimacy (foi) encodes a putative member of the ZIP family that is essential for development in Drosophila. This study demonstrates that FOI can act as an ion transporter in both yeast and mammalian cell assays and is specific for zinc. Insight into the mechanism of action of the ZIP family was provided through membrane topology and structure-function analyses of FOI. This work demonstrates that Drosophila FOI is closely related to mammalian ZIP proteins at the functional level and that Drosophila represents an ideal system for understanding the in vivo roles of this family. In addition, this work indicates that the control of zinc by ZIP transporters may play a critical role in regulating developmental processes (Mathews, 2005).

The membrane topology for a ZIP family member was assessed within the context of a full-length protein that retains biological function in vivo. The N terminus of FOI is extracellular, which is consistent with the presence of a predicted signal peptide and findings that the N terminus is glycosylated. The middle region of FOI is intracellular, which agrees with computer predictions that there are 3 TM domains between the N terminus and this region and with observations that a potential glycosylation site in this region is not used. Finally, the finding that the C terminus of FOI is extracellular indicates that there are an odd number of TM domains between the middle region and the C terminus. If the 3 strongly predicted TM domains (TM6-8) are correct, this indicates that the signature sequence domain cannot contain a single TM domain as predicted by many computer algorithms (Mathews, 2005).

It remains an open question as to whether the signature sequence domain of FOI contains 0 or 2 TM domains. Most computer predictions favor 0 TM domains for FOI, but the homology with the ZIP I/II family, in which TM4 and TM5 are more strongly predicted, favors the presence of 2 TM domains. In an attempt to address this, a single glycosylation consensus site was inserted between the two predicted amphipathic helices (Regions A and B) of the signature sequence domain of the N-terminal deleted FOI, which is not otherwise glycosylated. The engineered site can be glycosylated only if the signature sequence domain contains 2 TM domains, making the loop between the helices extracellular. However, no evidence for glycosylation of this site was found in the in vitro translation/glycosylation assay. Also a proline was inserted in place of the threonine in Region A (T556P) of the signature sequence domain within the full-length HA-FOI protein. If Region A normally forms a TM domain, then this proline might disrupt its helical nature and alter the membrane topology or subcellular localization of FOI. However, the FOI T556P protein localizes normally to the cell surface of cultured S2 cells and has an extracellular C terminus. Although these negative results are insufficient for concluding that the signature sequence domain does not contain TM character, they do raise the possibility that the two predicted amphipathic helices of this region might remain cytoplasmic or that they do not fully traverse the membrane (Mathews, 2005).

ZIP proteins are defined by their family-specific homology in conserved domains and common protein structure. Because the conserved domains and overall protein structure are likely to be functionally important, the importance of some of these features in FOI was tested and it was found that they are crucial for zinc transport. The signature sequence domain, the most highly conserved domain in the ZIP family, contains two histidine residues that have been conserved throughout the entire family. Mutating a single one of these residues (H554A) has a drastic effect on zinc transport by FOI in both yeast and mammalian zinc transport assays. Mutation of the homologous residue in Arabidopsis IRT1 (H197A), a ZIP I/II family member with broad cation specificity, blocked the function of this protein in yeast assays designed to test transport of different cations. Together these data suggest that this histidine is crucial for ion transport by all ZIP family members and thus is likely involved in coordinating ions through a membrane pore. Mutation of the second highly conserved histidine also blocked the function of IRT1 (H224A), indicating that both of these histidines are essential for ion transport (Mathews, 2005).

Other structural features and conserved residues are specific for individual subfamilies within the larger ZIP family. It was of particular interest to determine whether these features are also involved in zinc transport or reflect other subfamily-specific functions. One feature unique to LIV-1 proteins is their large N terminus that is glycosylated and rich in histidines. The work indicates that this domain is critical for zinc transport by FOI. Additionally, several disease-causing mutations in human acrodermatitis enteropathica patients have been mapped to the N terminus of hZIP4, and one of these mutations, P200L, causes reduced zinc influx by mouse ZIP4. This histidine-rich domain may function as a zinc-binding domain to induce protein conformational changes required for ion transport or to increase local zinc concentrations. The importance of the N terminus in LIV-1 proteins indicates a functional divergence between ZIP subgroups, perhaps affecting the mechanism of ion specificity, transport, or regulation (Mathews, 2005).

In the LIV-1 subfamily, the signature sequence domain has many more conserved charged residues than are present in the ZIP I/II subfamily and is predicted to form two amphipathic alpha helices. In Region B, LIV-1 subfamily members have three conserved acidic residues, whereas ZIP I/II subfamily members have one or none. Mutation of these three acidic residues (E584A/E588A/D591A) reduces zinc transport levels by FOI, suggesting that the amphipathic nature of this region is essential for zinc transport. Interestingly, the mutation of a single acidic residue in this region of IRT1 (E228A) also affected function in yeast transport assays. Together the data suggest that both the amphipathic nature of the signature sequence domain and its specific histidine residues are crucial for ion transport function. Differences between the ZIP subfamilies also are found in TM1-3 and TM6-8. In TM2, the LIV-1 subfamily has a conserved aspartic acid, whereas the ZIP I/II subfamily usually has threonine in this position. Mutation of this residue (D308A) has a strong effect on the ability of FOI to act as a zinc transporter. Thus, the conserved primary sequence within the TM domains of the ZIP family is clearly important for transport function in addition to providing the necessary TM structure (Mathews, 2005).

A number of features of FOI that represent characteristic differences between the LIV-1 and ZIP I/II subfamilies were examined. In each case, alteration of these features affects the ability of FOI to function as a zinc transporter. It is concluded that these differences do not reflect other subfamily-specific functions for these proteins that are independent of zinc transport and that the different subfamilies are likely to share similar functions. These differences may instead reflect subtle changes in ion specificity or in the mechanism of zinc transport between the subfamilies (Mathews, 2005).

Because ZIP family members play an important role in both development and human disease, it is crucial to understand the in vivo functions of these proteins. The work demonstrates that Drosophila FOI is a zinc transporter with similar ion specificity to other LIV-1 subfamily members. Essential features and sequence identities are conserved between FOI and mammalian ZIP family members, and these features are critical for ion transport. Thus, FOI functions in a similar or identical manner to other family members and represents an excellent opportunity to study the biological roles of this family in a genetically tractable system. In addition, FOI strongly prefers zinc over other cations tested, indicating that zinc plays an important role in regulating developmental processes (Mathews, 2005).

Two ZIP family members known developmental roles: Drosophila FOI and Zebrafish LIV-1. These proteins may act, at least in part, by affecting the expression or function of the calcium-dependent cell adhesion molecule E-cadherin. Like FOI, E-cadherin is required in Drosophila for both gonad and trachea morphogenesis, and expression of E-cadherin is strongly reduced in foi mutants. Thus, FOI may affect morphogenesis primarily by affecting E-cadherin. Similarly, the role of zLIV-1 in Zebrafish gastrulation may be because of effects on E-cadherin expression. zLIV-1 regulates the activity of the zinc-finger transcription factor Snail, which is a known regulator of E-cadherin. This indicates that ZIP family members can regulate E-cadherin at the transcriptional level by modulating intracellular zinc concentration. It is possible that zinc can also regulate E-cadherin in other ways, for example by directly binding to the E-cadherin protein to affect its function, or by regulating the activity of metalloproteinases that are known to cleave the extracellular domain of E-cadherin. One possibility that this work excludes is that FOI affects the calcium-dependent function of E-cadherin by acting as a calcium transporter to regulate extracellular levels of calcium. Even a 50-fold molar excess of calcium failed to compete with zinc for transport by FOI. Thus, FOI likely affects E-cadherin function primarily by affecting the intracellular and/or extracellular concentration of zinc (Mathews, 2005).

Interestingly, the roles of ZIP family members in disease may also be due in part to effects on cell-cell adhesion and cadherin function. hLIV-1 is strongly expressed in breast cancer cell lines, and hLIV-1 expression has been correlated with the potential of breast cancer tumors to metastasize. E-cadherin is a critical regulator of metastasis in breast and other cancers. Furthermore, E-cadherin and other cadherin family members are critical for integrity and function of the epidermis. Thus, the dermatological lesions that are prevalent in acrodermatitis enteropathica patients with mutations in hZIP4 may represent effects on cadherin function. Although this is highly speculative, the regulation of cadherin function by ZIP proteins represents the type of biological role for this family that can be identified and studied in model organisms like Drosophila (Mathews, 2005).

Zinc transport activity of Fear of Intimacy is essential for proper gonad morphogenesis and DE-cadherin expression

Embryonic gonad formation involves intimate contact between germ cells and specialized somatic cells along with the complex morphogenetic movements necessary to create proper gonad architecture. Gonad formation in Drosophila requires the homophilic cell-adhesion molecule Drosophila E-cadherin (DE-cadherin), and also Fear of Intimacy (FOI), which is required for stable accumulation of DE-cadherin protein in the gonad. In vivo structure-function analysis is presented of FOI that strongly indicates that zinc transport activity of FOI is essential for gonad development. Mutant forms of FOI that are defective for zinc transport also fail to rescue morphogenesis and DE-cadherin expression in the gonad. Expression of DE-cadherin in the gonad is regulated post-transcriptionally and foi affects this post-transcriptional control. Expression of DE-cadherin from a ubiquitous (tubulin) promoter still results in gonad-specific accumulation of DE-cadherin, which is strongly reduced in foi mutants. This work indicates that zinc is a crucial regulator of developmental processes and can affect DE-cadherin expression on multiple levels (Mathews, 2006).

It has been unclear whether ZIP family members regulate developmental processes by acting as zinc transporters or through some other unidentified function. These data now indicate that FOI regulates gonad formation through its zinc transporter activity. The ability of the mutant forms of FOI to rescue gonad morphogenesis and DE-cadherin expression corresponds directly with their ability to function as zinc transporters. Mutations that strongly affect the zinc transport activity of FOI (e.g., H554A) also strongly reduce the ability of FOI to rescue gonad morphogenesis and DE-cadherin expression. Mutations that only partially affect the zinc transport activity of FOI (e.g., Y646A) retain some ability to rescue gonad morphogenesis and DE-cadherin expression. If FOI affects gonad formation through a function separate from zinc transport, identification of conserved residues that affect these two activities independently would have been expected. This was not the case. Indeed, even single amino acid changes in very different regions of FOI (e.g., D308A and H554A) affect both zinc transport and gonad morphogenesis. It is concluded that the zinc transporter function of FOI is essential for gonad morphogenesis and regulation of DE-cadherin. This reveals a crucial role for zinc regulation in development and suggests that other ZIP family members with developmental roles (e.g., zebrafish LIV1) may also act via zinc transport (Mathews, 2006).

In vivo analysis is also informative for revealing domains that are essential for FOI function. Even though the N-terminal extracellular domain of FOI shows little sequence conservation with other family members, and some ZIP family members lack an extended N-terminal domain, this domain is nevertheless important for FOI function. In addition to their TM character, the specific sequence of the TM domains is crucial for FOI function. Mutations that are not predicted to affect the TM structure of FOI, such as mutating a single acidic residue in TM2 (D308A) or replacing TM6-8 of FOI with similar TM domains from the related protein CATSUP (CAT TM6-8), still disrupt the in vivo rescue activity of FOI. Finally, several characteristics were analyzed of the highly conserved HELP domain in FOI (which may or may not have TM structure). The predicted amphipathic alpha-helical nature of this domain appears to be crucial, since altering the pattern of acidic residues (D551A/D558A and E584A/E588A/D591A) or inserting a helix contorting proline residue (T557P) disrupts FOI function. In addition, conserved histidines in this domain are essential (H554A and H583A/H587A), and mutating even a single histidine has a dramatic effect in vivo. Since FOI is a zinc transporter, it is likely that the specific sequences of the TM domains form the proper membrane pore for zinc, while histidines in the N-terminal and HELP domains act to coordinate zinc before and during transport (Mathews, 2006).

shg and foi are both required for proper gonad and tracheal morphogenesis, and foi regulates DE-cadherin expression in the gonad. DE-cadherin protein levels are not reduced in foi mutants simply because the gonad has failed to coalesce; other mutations blocking gonad coalescence do not affect DE-cadherin. Thus, it is likely that foi affects DE-cadherin more directly and this is an important aspect of how foi functions in gonad and tracheal development. In support of this, it was found that expression of DE-cadherin was sufficient to partially rescue foi mutant gonads (Mathews, 2006).

As both DE-cadherin protein and shg RNA levels are reduced in foi mutant gonads, whether foi affects DE-cadherin transcription was investigated. Analysis of a shg enhancer-trap suggests that some aspects of DE-cadherin regulation by foi may be at the transcriptional level. Recently, it has been shown that a related ZIP protein, zebrafish LIV1, can regulate the activity of the Zn-finger transcription factor SNAIL, which may also influence E-cadherin expression (Yamashita, 2004; Mathews, 2006).

However, although a majority of studies focus on transcriptional regulation of E-cadherin, it is likely that this essential cell-adhesion molecule is often regulated at many levels, including through post-transcriptional and post-translational mechanisms. This study presents clear evidence that DE-cadherin is regulated at the post-transcriptional level in the embryonic gonad. Expression of DE-cadherin from a general tubulin promoter (tub-DE-cad) is sufficient to restore gonad-specific DE-cadherin protein accumulation in shg mutants. Recent work suggests that DE-cadherin localization within the ovary is also regulated partly through a post-transcriptional mechanism. Thus, post-transcriptional regulation may be sufficient to generate tissue-specific patterns of DE-cadherin expression in many contexts. tub-DE-cad is much less able to restore DE-cadherin protein to the gonad in foi mutants. This indicates that FOI is required for positive, post-transcriptional regulation of DE-cadherin. One component of this regulation is likely to act on shg RNA stability, since foi affects the gonad-specific accumulation of shg RNA from tub-DE-cad, but does not affect the activity of the tubulin promoter. Thus, the steady-state pattern of shg RNA accumulation does not merely reflect shg promoter activity but may have a significant post-transcriptional component. In principle, zinc could regulate the activity of RNA-binding proteins that affect RNA stability in the same way it regulates DNA-binding transcription factors. In addition, DE-cadherin may be further regulated at the protein level in the gonad, such as through regulation of translation or protein stability (Mathews, 2006).

Recent in vivo work on several ZIP proteins suggests that these zinc transporters play essential roles in development and disease that may broadly involve regulation of cell-cell adhesion. In zebrafish, regulation of SNAIL by LIV1 is essential for the anterior migration of zebrafish organizer cells and may regulate E-cadherin expression in this tissue (Yamashita, 2004). According to this model, LIV1 activates SNAIL activity, which leads to downregulation of E-cadherin and the decreased cell adhesion necessary for cell migration (Yamashita, 2004). Interestingly, SNAIL is also thought to be an important regulator of E-cadherin during the progression and metastasis of certain cancers, such as breast cancer. As a tumor gains metastatic potential, SNAIL expression is upregulated and E-cadherin is downregulated. Since human LIV-1 is strongly expressed in breast cancer cell lines, and has been implicated in breast cancer metastasis, it may function to activate the activity of SNAIL as a transcriptional repressor of E-cadherin, again allowing for cell migration and metastasis. A similar, but opposite, relationship may exist in the Drosophila tracheal system, where the SNAIL family member Escargot (ESG) is a positive regulator of E-cadherin during the fusion of neighboring tracheal branches. Since FOI is also required for this process, FOI may act by promoting ESG activity. In this case, FOI and ESG would activate DE-cadherin expression, which is necessary for cell-cell attachment during tracheal branch fusion. In the gonad, FOI is also positively required for DE-cadherin expression. Although ESG is present in the gonad, no changes have been observed in DE-cadherin expression during gonad coalescence in esg mutants, indicating that some other target for regulation by FOI and zinc must exist in this tissue. An important theme in the action of ZIP proteins may be to influence the activity of zinc-regulated transcription factors, with cell-cell adhesion molecules being important targets of such regulation. However, it was found that additional, post-transcriptional mechanisms are crucial in the gonad for regulation of DE-cadherin protein expression by FOI. Thus, it will be very important to analyze the contribution of post-transcriptional regulation of E-cadherin to other developmental and disease processes. Indeed, there is even evidence that the same crucial factor, SNAIL, can influence post-transcriptional regulation (Mathews, 2006).

An important issue relevant to the role of zinc and zinc transporters in development and disease is whether they play an instructive or permissive role. Is zinc merely required at a minimum threshold level in various tissues or does regulation of intracellular zinc concentration play a signaling role at specific times and places? Existing evidence suggests that zinc may play an instructive role. Both Drosophila foi and zebrafish LIV1 have highly tissue-specific patterns of expression and affect the development of selected tissues, while others remain unaffected. Mammalian ZIP and Cation Diffusion Facilitator family members also have tissue-specific expression patterns. Thus, zinc transporters have the necessary spatial and temporal resolution to play an instructive role. In addition, zinc transporters have clear roles as modulators of intracellular signals. They have the capacity to modulate signaling pathways, for example the ras pathway, and can influence transcription factor activity and gene expression. Because the zinc transport activity of FOI is crucial for its developmental role, it is likely to act by modulating zinc concentration. Thus, zinc has the potential to be an important and dynamically regulated signaling molecule during development and adult homeostasis (Mathews, 2006).

DEVELOPMENTAL BIOLOGY

Embryonic

To begin to address how Foi might act in gonad coalescence, the expression pattern of the foi transcript was examined. foi mRNA that is likely to be maternal in origin is found throughout the early embryo with a higher concentration present at the posterior pole. This posteriorly localized RNA is taken up by the pole cells (future germ cells) as they form, while the remaining maternal transcript is degraded. Although the localization of foi transcript to the germ cells is intriguing, no function for this maternal RNA has yet been found. Offspring that lack maternal foi activity (produced from homozygous germline clones in mosaic females) show no developmental defects and grow up to be fertile adults. In addition, the gonad and tracheal phenotypes associated with removing foi activity both maternally and zygotically are not more severe than the zygotic phenotypes alone (Van Doren, 2003).

foi shows zygotic expression in a number of tissues, including general expression during gastrulation that is stronger in the invaginating mesoderm. Slightly later, the general expression is reduced, while high-level expression appears in the anterior and posterior endoderm primordia. During the time of gonad coalescence (stage 14), broad expression is observed in the embryo, including the mesoderm, whereas foi is less highly-expressed in the epidermis (Van Doren, 2003).

Since the Foi protein is predicted to contain multiple transmembrane spans, it should be localized either to the cell surface, or to a membrane bound cellular compartment. Although no antisera that recognize the endogenous Foi protein has yet been raised, the subcellular localization of Foi was examined using epitope-tagged versions of the protein. Three versions of Foi were generated where the hemagglutinin (HA) epitope tag was placed either in the N-terminal domain, the domain between TM1-3 and TM4-6, or the C-terminal domain. Constructs expressing these proteins were then transfected into Drosophila tissue culture cells (Schneider S2) and the subcellular localization of Foi was determined by immunofluorescence using anti-HA antibodies. Foi is localized to the cell surface, and very little staining is observed intracellularly. Foi co-localizes with a control plasma membrane protein (CD8-GFP), confirming its cell surface localization. It is unlikely that the cell surface localization is due to overwhelming a system for localizing Foi to a subcellular compartment, because little Foi protein is detected intracellularly and even weakly expressing cells show Foi on the cell surface. Identical results were obtained for all three HA-tagged versions of Foi, making it also unlikely that the epitope tag is interfering with the normal subcellular localization of Foi. Finally, both the N-terminal and C-terminal epitope tagged versions of Foi are able to rescue the foi-mutant phenotype in a transgenic rescue assay, indicating that these proteins retain wild-type activity (Van Doren, 2003).

To observe the subcellular localization of Foi in the embryo, transgenic lines were generated expressing HA-Foi from a Gal4-responsive promoter (UAS), and this was used to express HA-Foi in specific tissues in the embryo. HA-Foi expression was first examined in the germ cells, since the large size and spherical shape of these cells allows for a more accurate assessment of subcellular localization. HA-Foi is clearly localized to the surface of these cells. To examine HA-Foi in the somatic cells of the gonad, it was expressed throughout the embryonic mesoderm. Although the small size of these cells makes subcellular localization difficult to assess, a restricted staining pattern was observed that is consistent with HA-Foi being at the cell surface. HA immunoreactivity is found along the borders between the gonadal mesoderm and the germ cells, and around each germ cell. The gonadal mesoderm cells were found to extend cellular projections that ensheath each individual germ cell. HA-Foi appears to localize to these cellular projections. HA-Foi shows significant co-localization with the membrane associated protein Discs large (Dlg), further indicating that Foi is present at the cell surface in the gonadal mesoderm. Finally, HA-Foi expression was examined in the trachea. HA-Foi is preferentially localized to the cell periphery, and can also be observed on cellular extensions between the fusion tip cells during tracheal branch fusion. Thus, an epitope-tagged version of Foi that is competent to rescue Foi activity exhibits cell surface localization in both tissue culture cells and the embryo. Although Foi is likely to also be present in the secretory pathway on its way to the plasma membrane, the localization of Foi suggests that it normally functions as a cell-surface protein (Van Doren, 2003).

The foi RNA expression pattern does not specifically indicate in which tissues foi might be acting. To further address this issue, and to verify the identification of the foi transcription unit, attempts were made to rescue the foi-mutant phenotype using tissue-specific Foi expression. These experiments were done largely with non-HA-tagged versions of Foi, but similar rescue is observed with N- and C-terminally HA-tagged versions of Foi. Expression of UAS-Foi in the mesoderm of a foi mutant is sufficient to rescue the gonad coalescence defect. Expression of UAS-Foi in the germ cells is unable to rescue this phenotype. Thus, foi is required within the mesoderm for gonad coalescence. Expression of UAS-Foi within tracheal cells is able to rescue the tracheal fusion defect of foi mutants; however, mesodermal expression is also able to rescue tracheal defects. Whether rescue of the tracheal phenotype by the mesoderm Gal4 represents a non-autonomous role for foi, or is due to low-level expression of this driver in the trachea, can be addressed in the future with more traditional genetic mosaic analysis (Van Doren, 2003).

EFFECTS OF MUTATION

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; all three exhibit similar phenotypes in the gonad and trachea, and all 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 (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 what is interpreted to be 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. In embryos exhibiting a weaker phenotype, the gonadal mesoderm appears to partially coalesce but this process is incomplete, resulting in misshapen gonads. To determine if gonadal mesoderm coalescence was being blocked because the germ cells are in some way defective, the morphogenesis of the gonadal mesoderm was examined in embryos lacking germ cells. oskar (osk) is required for germ cell formation and specific alleles of osk cause complete germ cell loss without affecting other aspects of embryonic development. In such mutants, the gonadal mesoderm coalesces normally, even though these embryos lack germ cells. However, in osk foi double mutants, the gonadal mesoderm fails to coalesce, as is the case in foi mutants where germ cells are present. Thus, foi mutants are clearly defective in gonadal mesoderm morphogenesis independent of the germ cells (though this experiment does not exclude the possibility of an additional role for foi in the germ cells themselves) (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. To address this, the expression of a number of molecular markers for the gonadal mesoderm were examined, including the 412 retrotransposon RNA, Wnt2, Hmgcr, Zfh1 and Eya. In all cases, the expression of these markers in the gonadal mesoderm is unchanged in foi mutants, indicating that foi does not affect the identity of these cells. Furthermore, the cells of the gonadal mesoderm exhibit their normal behavior prior to coalescence. In foi mutants, these cells still form in three independent clusters that then join to form a single band of cells, similar to wild type. They also act as the target cells for germ cell migration, specifically associating with germ cells at embryonic stages 12-13 and remaining associated at later stages. This is in contrast to mutations in genes such as eya that affect gonadal mesoderm identity; the germ cells do not remain associated with the gonadal mesoderm in these mutants. It is concluded that foi does not affect the identity of the gonadal mesoderm and, instead, affects gonad coalescence by interfering with the process of morphogenesis downstream of cellular identity. Molecular markers for germ cell identity were examined [vasa (vas) and Iswi], and the identity of the germ cells were found to be similarly unaffected in foi mutants (Van Doren, 2003).

foi mutants die at the end of embryogenesis, but no general defects were observed in the development of a variety of different tissues analyzed, including the nervous system, midgut, musculature and embryonic cuticle pattern. One other tissue, however, was found that exhibits defects in foi mutants: the developing tracheal system. The tracheal network develops from individual groups of cells that form tracheal branches within different segments of the embryo. Some of these branches must fuse with branches from neighboring segments to make a continuous network of tubules throughout the embryo. This process is controlled by the terminal cell in the fusion branches, termed the fusion tip cell. During tracheal branch fusion, fusion tip cells from neighboring branches specifically adhere to one another and form a lumen between them (Van Doren, 2003).

In foi mutants, tracheal cells appear to differentiate and form tracheal branches normally, but lateral trunk (LT) tracheal branch fusion is disrupted. By labeling the tracheal cells with a cell surface marker (CD8-GFP), it was observed that the fusion tip cells from some neighboring branches fail to meet properly and form a lumen between them. This phenotype is highly penetrant. Thin cellular extensions are sometimes observed between fusion tip cells of failed fusions, suggesting that these cells retain the ability to recognize one another. However, the main cell bodies are clearly displaced from one another and have not become closely associated as is observed in wild type. Rather than fusing and forming a lumen with the appropriate partners, the defective branches appear to remain independent, but are still capable of extending ganglionic branches (GBs) ventrally, which is the appropriate behavior for the properly fused LT branch. Thus, no evidence is seen that the tracheal branches are defective in cell migration or branch extension in foi mutants, and the defect appears more specific to the process of branch fusion. To address further whether the fusion tip cells retained their proper identity, the expression of the fusion tip cell marker escargot (esg) was analyzed in foi mutants using an enhancer trap in this gene. This marker is still expressed in foi mutants, even in fusion tip cells from branches that fail to fuse. Thus, as is observed for the gonad, foi mutants show defects in tracheal morphogenesis but not in cell identity (Van Doren, 2003).

The homotypic cell adhesion molecule E-cadherin has also been shown to be essential for the process of tracheal branch fusion. The similarity of the foi and E-cadherin (shotgun) mutant phenotypes in the trachea prompted an examination of the role of E-cadherin in gonad morphogenesis. Interestingly, it was found that shg mutant embryos do indeed exhibit defects in gonad coalescence. In mutant embryos, the gonadal mesoderm begins to coalesce with the germ cells, but the gonads are misshapen and coalescence often does not proceed to completion. This phenotype closely resembles the 'weak' phenotype observed in foi mutants. One possible explanation for shg exhibiting a weaker gonad phenotype than foi is the substantial maternal contribution of shg. Since shg is required for oogenesis, it was not possible to analyze embryos where this maternal contribution was removed (Van Doren, 2003).

In most animal species, germ cells require intimate contact with specialized somatic cells in the gonad for their proper development. The establishment of germ cell-soma interaction during embryonic gonad formation has been analyzed in Drosophila; somatic cells undergo dramatic changes in cell shape and individually ensheath germ cells as the gonad coalesces. Germ cell ensheathment is independent of other aspects of gonad formation, indicating that separate morphogenic processes are at work during gonadogenesis. The cell-cell adhesion molecule Drosophila E-cadherin is essential both for germ cell ensheathment and gonad compaction, and is upregulated in the somatic gonad at the time of gonad formation. Differential cell adhesion contributes to cell sorting and the formation of proper gonad architecture. In addition, Fear of Intimacy, a novel transmembrane protein, is also required for both germ cell ensheathment and gonad compaction. E-cadherin expression in the gonad is dramatically decreased in fear of intimacy mutants, indicating that Fear of Intimacy may be a regulator of E-cadherin expression or function (Jenkins, 2003).

Gonad coalescence in Drosophila is the rearrangement of germ cells and somatic gonadal precursors (SGPs) from a broad association stretching across three parasegments (10, 11 and 12) of the embryo to a tight cluster of cells located in PS10. In this process, germ cells become enclosed in the environment that will nurture them as they adopt stem cell fates and begin gametogenesis. Germ cell-soma contact in the embryonic gonad is already extensive, with each germ cell becoming surrounded by somatic cell membrane. Furthermore, E-cadherin plays a key role in this and other aspects of gonad formation. Detailed analysis of gonad coalescence has shown that it can be subdivided into two processes: gonad compaction and germ cell ensheathment. In gonad compaction, SGPs and germ cells physically condense together to create a rounded organ. Germ cell ensheathment is characterized by the dramatic shape changes of SGPs that produce thin cellular extensions that surround the germ cells. Germ cells lack cellular extensions during gonad compaction, and need not be present for compaction to occur. This suggests that SGPs provide the 'driving force' behind the movements of compaction and germ cells play a more passive role (Jenkins, 2003).

Several pieces of data indicate that gonad compaction and germ cell ensheathment are distinct, separable events. Germ cell ensheathment is already apparent at stage 13, prior to the onset of compaction. In addition, compaction proceeds normally in agametic embryos, despite a lack of germ cell ensheathment. Furthermore, in mutants that affect gonad coalescence (shg, foi), examples of gonads with no ensheathment but a high degree of compaction have been observed, and also gonads with good ensheathment but little compaction. Thus, gonad compaction and germ cell ensheathment are independent processes that together contribute to the proper architecture of the coalesced embryonic gonad. Both of these processes require the adhesion molecule E-cadherin (Jenkins, 2003).

Embryos with mutations in the fear of intimacy gene share several gonad defects with shg mutant embryos, including defects in gonad compaction and germ cell ensheathment. Both genes are also required for tracheal branch fusion, suggesting that Drosophila E-cadherin and FOI may work together to promote all of these processes. Consistent with this, E-cadherin protein levels are severely reduced within the gonads of foi mutants. E-cadherin expression is reduced in SGPs, which display defective behaviors in foi mutants. Thus, gonad defects in foi mutants correlate strongly with the cells in which E-cadherin expression is most affected, suggesting that this may be the cause of the foi mutant phenotype (Jenkins, 2003).

There are several possible models for how FOI, a cell surface, multipass transmembrane protein, might be affecting the levels of E-cadherin protein. FOI could act as a receptor or channel that signals the beginning of coalescence. Upregulation of E-cadherin in the SGPs could require such a signal. Or, FOI might act to localize E-cadherin complexes to sites of germ cell-soma and soma-soma contact within the gonad. As such, FOI could act during the export of E-cadherin to the cell surface, or to localize E-cadherin to specific sites of cell-cell contact. Alternatively, FOI might affect E-cadherin levels by affecting its function as a cell adhesion molecule. It has been suggested that the stability of E-cadherin is tightly linked to its function in adhesion complexes, with reduced E-cadherin function leading to a faster turnover of the protein. FOI might modulate E-cadherin function by acting as a co-factor itself on the cell surface, or by acting as a transporter to alter the concentration of a small molecule modulator of E-cadherin adhesion, such as Ca²⁺ (Jenkins, 2003).

Fear-of-intimacy controls glial cell migration

Development of complex organs depends on intensive cell-cell interactions, which help coordinate movements of many cell types. In a genetic screen aimed to identify genes controlling midline glia migration in the Drosophila nervous system, mutations in the gene kästchen were identified. During embryogenesis kästchen is also required for the normal migration of longitudinal and peripheral glial cells. During larval development, kästchen non-cell autonomously affects the migration of the subretinal glia into the eye disc. During embryonic development, kästchen not only affects glial cell migration but also controls the migration of muscle cells toward their attachment sites. In all cases, kästchen apparently functions in terminating or restricting cell migration. The molecular nature of the gene was identifed by performing transgenic rescue experiments and by sequence analysis of mutant alleles. Kästchen corresponds to fear-of-intimacy (foi) that was identified in a screen for genes affecting germ cell migration, suggesting that Foi-Kästchen is more generally involved in regulating cell migration. It encodes a member of an eight-transmembrane domain protein family of putative Zinc transporters or proteases. The topology of the Foi protein was determined by using antisera against luminal and intracellular domains of the protein and evidence is provided that it does not act as a Zinc transporter. Genetic evidence suggests that one of the functions of foi may be associated with hedgehog signaling (Pielage, 2004).

During the development of the nervous system, cell migration can be observed for several different classes of glial cells. Within the embryonic CNS, midline glial cells have to migrate along cell processes of the VUM neurons to generate the regular pattern of anterior and posterior commissures found in every neuromere. The presence of the neuronal substrate is crucially important to initiate and guide glial migration. Similarly, glial cells covering the longitudinal connectives that emerge from laterally located glioblasts have to migrate toward the CNS midline where they interact with neuronal growth cones that will eventually pioneer the longitudinal tracts. The Slit-Roundabout ligand-receptor system that controls axonal pathfinding across the CNS midline also regulates some aspects of glial migration (Pielage, 2004).

In the larval PNS, the axons of the developing photoreceptors project through the optic stalk to the brain. Glial cells are born in the optic lobes and migrate through the optic stalk toward photoreceptor cells. Recent analysis has shown that these glial cells are guided by a signal released from developing photoreceptor cells in the eye disc. Part of the signaling mechanism may be conveyed by the signaling molecule Hedgehog and the casein kinase Igamma Gilgamesh that prevents precocious glial cell migration (Hummel, 2002). Glial cell migration also plays an important role within the developing optic lobes. However, the molecules guiding this migration have not yet been identified (Pielage, 2004).

Over the last years, a genetic approach has been conducted to search for mutations affecting the migration of midline glial cells. Several genes were found to be required to regulate specification and cell number of the midline glia. Subsequent phenotypic analyses showed that only few of the genes are required for the migration process per se. The most striking example is given by the kästchen gene, which affects the migration of several cell types: glial cells, primordial germ cells, somatic muscle, and tracheal cells. A detailed phenotypic analysis of the kästchen mutant phenotype and evidence is provided that Kästchen acts non-cell autonomously to terminate glial cell migration. Sequence analysis and rescue data demonstrate that kästchen is allelic to fear-of-intimacy (Pielage, 2004).

To determine the temporal and spatial expression pattern of foi, whole mount in situ hybridization experiments were performed using digoxigenin-labeled RNA probes. Expression can first be detected in the unfertilized egg and is maternally supplied. During blastoderm stages, higher levels of expression are detected in the PGCs. At germband extension, expression levels appear to increase in all epithelial folds and in the primordium of the posterior midgut as well as in the forming stomodeum. In the extended germband stage, mesodermal cells start to spread over the inner surface of the embryo. Concomitantly, foi expression becomes elevated and is detectable in the mesoderm until stage 13/14. In addition, expression is found in the developing nervous system. At the end of embryonic development, elevated expression is detectable in the lateral chordotonal organs in the abdominal segments. The foi-associated P-element insertion l(3)j8E8 can be used as an enhancer detector, since it carries a lacZ reporter gene. Expression of this enhancer trap can first be detected in the extended germband stage in mesodermal progenitor cells. Later in development, the foi enhancer is active in the ectoderm where an upregulation occurs in the posterior part of each segment. The domains of elevated foi expression correspond in part to the sites of engrailed expression (Pielage, 2004).

The fused commissure phenotype that led to the identification of foi mutants is indicative for defects in the migration of midline glial cells. For example, in pointed mutant embryos, the midline glial cells fail to differentiate and are generally found anterior to the fused commissures. In no case they intermingle between anterior and posterior commissures, which thus appear fused (Pielage, 2004).

In foi mutant embryos, midline glial cells are specified in their normal number and are able to migrate but finally fail to intercalate in between segmental commissures. Thus, foi does not affect early phases of glial migration but rather regulates later aspects because mutant glial cells often follow their normal path; compared to pointed mutants, they end up posterior in the neuromere. The formation of the VUM axons that are the neuronal substrate along which midline glial cells migrate is not affected in foi mutant embryos, suggesting that the interaction between glial and neuronal cells is impaired (Pielage, 2004).

In addition to the midline phenotype, a reduction in the size of longitudinal connectives and abnormal fasciculation was noted. These phenotypes are often associated with defects in longitudinal glial cells. In wild-type embryos, the longitudinal glial cells are born in the lateral cortex and migrate toward the forming connectives, in part guided by the Slit-Roundabout signaling system. Once they contact the connectives, they migrate in anterior and posterior directions to evenly cover the longitudinal axon tracts. In mutant foi embryos, glial cells are initially specified in normal number. However, during stages 15-16, migration defects can be noted. In extreme cases, no or only few glial cells are found in some hemineuromeres, whereas in the directly neighboring hemineuromeres increased numbers of glial cells are found. Similar to the midline glia phenotype, the longitudinal glial cells do not lose their general ability to migrate but they are not able to find and associate with their appropriate targets (Pielage, 2004).

To address whether foi affects glial migration in a more general manner, the peripheral glial cell phenotype of foi mutants was determined. The PNS of the Drosophila embryo has a simple structured organization. Forty-four sensory neurons are organized in four clusters and project their axons into the ventral nerve cord and about 30 motoneurons send their axons from the CNS toward specific muscle targets. In the PNS, two main nerve branches can be distinguished, the segmental and the intersegmental nerves. These nerves must be isolated from the surrounding hemolymph to allow electrical conductance. Most of the peripheral glial cells that perform this function are born within the ventral nerve cord from identified glioblasts. During late stage 13, these glial cells migrate from the ventral nerve cord to very stereotyped positions along the peripheral nerves. In foi mutants, the neuronal components of the PNS form relatively normally. The peripheral glial cells are born in normal number but migrate to ectopic positions such as the dorsal branch of the trachea system, where they are never found in wild type. In some cases, migration is not initiated and glial cells accumulate at the CNS-PNS transition zone (Pielage, 2004).

Glial cell migration is not only relevant for embryonic development but also plays an important function during later developmental stages. During late larval stages, subretinal glial cells invade the eye disc from the optic stalk. Migration of these glial cells is regulated by still unknown cues and terminates just posterior to the morphogenetic furrow. To address the question whether foi affects this glial migration process, mutant larval eye discs were generated using the eyFlp Minute technique. This technique adds a growth disadvantage to non-mutant cells and leads to eye discs that essentially consist of only mutant tissue. The migration of these glial cells was analyzed in foi mutant eye discs by simultaneously staining for the photoreceptor axons and the glial cells. All foi alleles analyzed led to a similar phenotype. The progression of the morphogenetic furrow was disturbed and did not advance as far anterior as in wild-type eye discs. Concurrently, a stream of glial cells migrates across the morphogenetic furrow apparently not respecting the signals that normally terminate glial cell migration. When small mutant foi clones were induced, no abnormal phenotypes were noted. Both the morphogenetic furrow progression and the glial cell migration proceeded normally. This suggests that foi acts non-cell autonomously in eye discs to control morphogenetic furrow progression and glial cell migration (Pielage, 2004).

Because foi mutations affect germ cell migration and glial cell migration, foi appears to be more generally required for the control of cell migration. To further test this hypothesis, the development of the somatic musculature was examined. Following a series of fusion events, the developing muscle fibers migrate toward their attachment sites, the apodemes. This process, which is reminiscent of axonal growth cone migration, is impaired in foi mutant embryos. In wild-type embryos, the lateral transverse muscles (muscles 21-24) migrate to stereotyped positioned tendon cells in the lateral body wall. In mutant foi embryos, these muscles appear to ignore their wild-type attachment sites and continue to grow ventrally to reach ectopic positions. The distortion of motoraxon projection is likely due to the mesodermal phenotype (Pielage, 2004).

The induction of large foi mutant eye clones results in a prominent adult compound eye phenotype. Anterior eye structures are affected and those remaining have a rough appearance. In addition, the formation of the dorsal head case is severely affected. The formation of the bristles is impaired and the ocelli are often missing. The eye phenotype is reminiscent of some aspects of a hedgehog mutant phenotype. Interestingly, hedgehog has been shown to regulate glial migration in the developing eye disc. Phenotypic analyses have suggested that foi and hedgehog might be interacting during eye development. Homozygous hedgehog^bar3 (hh¹) flies are viable and show an eye phenotype reminiscent of mutant foi eyes. The anterior portion of the eye is missing due to a premature stop of morphogenetic furrow progression. To analyze a possible genetic interaction between hedgehog and foi, the effects of a 50% reduction of foi was examined in a hedgehog mutant background. hh¹/hh¹ mutant eyes develop only 9-10 rows of photoreceptor cells. When one copy of foi was removed in this genetic background, the phenotype was enhanced so that only 6-7 rows of photoreceptor cells form, suggesting that the two genes may interact during eye development (Pielage, 2004).

When foi and hedgehog indeed interact, one may expect further phenotypic similarities. To address this question in more detail, germ line clones were generated using the FRT ovo^D technique and the development of the embryonic nervous system was analyzed as well as the pattern of the larval cuticle. It was possible to induce a sufficient number of germline clones only when using the hypomorphic O1-41 mutation; for the strong mutation B1-89, only very few embryos were obtained, indicating some requirements of foi during oogenesis. In both cases, however, paternal rescue was noted. Maternal and zygotic mutant foi embryos displayed a wide range of mutant phenotypes. In about 50% of the cases, a severely disrupted neural development resembling a mutant hedgehog phenotype was noted. In one quarter of the embryos, there was a very strong foi phenotype. In the remaining embryos, no development was observed. To compare foi and hedgehog phenotypes, cuticle preparations were also analyzed. Wild-type larvae show denticle belts in every segment and have a characteristic head skeleton. Mutant hedgehog larvae lack a well-differentiated head skeleton and are characterized by a lawn of denticles. Maternal and zygotic mutant foi embryos display a range of phenotypes. In the most extreme situation, the animals lack the head skeleton and have expanded ventral denticle belts. The other embryos showed a reduced degree of severity, which can be judged by defects in the head skeleton. Thus, during embryogenesis, foi and hedgehog mutants share at least some phenotypic traits supporting the notion that both genes may in part act in a common pathway (Pielage, 2004).

By sequence analogy, Foi belongs to a family of Zinc transporters whose members all share Zinc ion binding capabilities (Gaither, 2001; Taylor, 2003). Within the N-terminus, a conserved cysteine spacing motif can be noted suggesting a conserved tertiary structure (Taylor, 2003). The deduced protein sequence was further analyzed using different algorithms. Following an N-terminal signal peptide are eight possible transmembrane domains. The C-terminal transmembrane domains show similarities to Zn²⁺ transporters (ZIP domain), transmembrane domains 4 and 5 are most conserved and are characterized by a HELP motif, that is similar to the active center of some metalloproteases (MMP). To confirm the subcellular localization and topology of Foi, two polyclonal antisera were generated against different domains of the protein. None of the antisera could detect the endogenous protein in whole mount preparations of embryos or imaginal discs. However, it was possible to detect Foi protein in Drosophila embryos following overexpression using the GAL4 system. The majority of the protein appeared to be localized at the cell membrane, and some vesicular structures were also noted. Similarly, when Foi was overexpressed in S2 cells, the majority of the protein could be detected on the cell membrane. The protein is not detected in untransfected cells and has an apparent molecular weight of about 70 kDa. Because Foi is a multipass transmembrane domain protein, the orientation of membrane integration was determined with the different antisera using unfixed, nonpermeabilized S2 cells. A staining signal was detected only when the N-terminal antisera was used. To test whether Foi might function as a Zn²⁺ transporter, Newport green stainings were performed to measure Zn²⁺ uptake in dependence of the Foi concentration. Irrespective of the level of the Foi protein, no alterations were noted in the level of Newport green staining, indicating that Foi might be not required for Zn²⁺ homeostasis. However, due to a lack of appropriate tools, it was not possible to perform a positive control (Pielage, 2004).

How could foi affect migration? foi mutations were initially isolated due to their fused commissure phenotype, which is indicative for defective midline glial cell migration. Among the collection of mutants identified in this large scale mutagenesis, foi appeared unique, since it did not completely block midline glia migration as do, for example, mutations in pointed. Rather foi is required to control and stop cell migration. This phenotypic trait is also found in other migratory cells. Termination of migration may be a cell autonomous property but is very likely to be regulated by extracellular signals. The genetic data are consistent with a possible interaction of foi with components of the hedgehog signaling cascade. Loss of foi function leads to phenotypes resembling aspects of the hedgehog mutant phenotype. This is particularly evident during head development, where both ommatidial development as well as head capsule formation is affect by foi and hedgehog. Furthermore, gene dose experiments support the interaction of the two genes (Pielage, 2004).

Hedgehog is an evolutionary well-conserved signaling molecule that controls a wide range of cellular interactions. Within the eye disc, Hedgehog not only controls the differentiation of neuronal cells units but also directly regulates the migration of glial cells that invade the eye disc epithelium from the optic stalk. In addition, Hedgehog is required for germ cell migration (Pielage, 2004).

During development of the vertebrate nervous system, Hedgehog controls neuronal migration as well as growth cone navigation. In the peripheral nervous system, ectopic Hedgehog blocks migration of trigeminal precursors whereas loss of hedgehog function leads to a migration of the trigeminal precursor cells to ectopic targets. In vitro experiments show that this might be in part a consequence of changes in cell adhesion. Interestingly, Hedgehog affects migration of neural crest cells independent of the canonical Patched-Smoothened signaling pathway. To assay whether Hedgehog affects glial migration through the canonical pathway, a dominant negative version of the Patched receptor was expressed in developing glial cells but no glial migration phenotype was obtained. Similarly, the loss of components of the canonical Hedgehog pathway in glial cells does not lead to a migration phenotype. Thus, as observed for neural crest cells, Hedgehog may not be acting through Patched to control glial migration in Drosophila (Pielage, 2004).

How could Foi interact with Hedgehog? Foi is predicted to encode a multiple pass transmembrane protein containing eight transmembrane domains. It belongs to the so-called ZIP family (Zrt, Irt-like proteins), a large group of at least 86 members that bind or transport zinc. The ZIP family can be further subdivided into four subgroups. The largest group has recently been termed LZT subfamily (LIV-1 subfamily of ZIP transporters) and currently comprises 36 members (Taylor, 2003). However, all LZT members are also characterized by a so-called HELP motif that defines the active site of matrix metalloproteases (MMP). Because this catalytic zinc-binding motif is predicted to be in the transmembrane domain V, Foi may be a new transmembrane protease (Pielage, 2004).

Intramembrane proteolysis has recently been recognized as an important control mechanism found throughout evolution. Bacteria use it to generate extracellular pheromones or to liberate transcription factors within the cell. Similarly, eukaryotic cells utilize intramembrane proteolysis in diverse cellular processes such as lipid metabolism, response to unfolded proteins, and cell differentiation (Pielage, 2004).

The experiments demonstrate that it is unlikely that Foi is regulating Zn²⁺ homeostasis. The genetic studies identified the Hedgehog signaling cascade as a possible candidate of Foi function. The analysis of foi mutant eye clones demonstrates that Foi affects eye development in a non-cell autonomous manner. This suggests that Foi is required for the generation of the signaling molecule and not for the perception of the signal. The Hedgehog protein is generated as a 45-kDa precursor that undergoes autocatalytic processing to generate an active 22-kDa N-terminal protein. It has been reported that Zinc ions can influence autocatalytic properties of inteins, which exhibit similar autocatalytic processing as Hedgehog. However, no changes were detected in the zinc ion concentration following Foi overexpression. Alternatively, Foi may regulate the activity of a yet unidentified signaling pathway, since not all mutant phenotypes can easily be explained by a lack of hedgehog signaling. Future experiments will allow a determination of whether Foi activates parts of the Hedgehog signal transduction cascade or other signaling systems by functioning as an intramembrane protease (Pielage, 2004).

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Search PubMed for articles about Drosophila fear of intimacy

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date revised: 25 October 2023

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