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First published online 15 February 2006
doi: 10.1242/dev.02256

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

Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA.

^* Author for correspondence (e-mail: vandoren{at}jhu.edu)

Accepted 16 December 2005

	SUMMARY

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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. Previously, we have shown that 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. Here, we present an in vivo structure-function analysis 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. We further show that expression of DE-cadherin in the gonad is regulated post-transcriptionally and that 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.

Key words: fear of intimacy, shotgun, DE-cadherin, Zinc transport, ZIP proteins, Post-transcriptional gene regulation, Drosophila

	INTRODUCTION

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The formation of complex organs from individual tissues is an essential developmental process that requires coordination of many different types of cellular events. Extensive cell-cell contact and communication ensures that these events are well orchestrated. The Drosophila gonad is an ideal model for studying organogenesis. Formation of this organ requires that germ cells and somatic gonadal precursors (SGPs), which are initially specified far apart from each other, undergo a complex morphogenetic program to form a properly patterned embryonic gonad. This organ then further differentiates into either an ovary or testis.

Germ cells are initially formed at the posterior pole of Drosophila embryos during the syncytial blastoderm stage. They are carried inside the embryo during gastrulation, where they migrate through the midgut epithelium into the mesoderm to contact SGPs (reviewed by Starz-Gaiano and Lehmann, 2001). SGPs are specified in bilateral clusters within three parasegments of the embryo (PS10-12) (Brookman et al., 1992) and are initially identified by their expression of Eyes Absent (EYA) and Zinc-finger homeodomain protein 1 (ZFH-1) (Boyle et al., 1997; Broihier et al., 1998). The posterior of male gonads contains an additional group of male-specific SGPs (msSGPs) specified in a separate location (PS13) (DeFalco et al., 2003). Once germ cells contact SGPs, these two distinct cell types sort away from neighboring mesodermal cells, forming a contiguous tissue along PS10-12. Germ cells and SGPs then undergo two concurrent, but separable, morphogenetic processes (germ cell ensheathment and gonad compaction) to form a properly patterned organ (Jenkins et al., 2003). As SGPs contact germ cells, they undergo dramatic cell shape changes to extend thin cellular protrusions, which ensheath individual germ cells (Jenkins et al., 2003). At the same time, the associated germ cells and SGPs compact together in PS10 to form a round organ with a distinct boundary (Boyle and DiNardo, 1995; Brookman et al., 1992; Jenkins et al., 2003).

Although many genes involved in germ cell migration and SGP specification have been characterized, only three downstream genes have been identified that specifically coordinate the morphogenetic movements of gonad formation. traffic jam encodes a Maf transcription factor required for germ cells and SGPs to intermingle properly in both embryonic and adult gonads, and may regulate the adhesive properties of SGPs (Li et al., 2003). The homophilic cell-adhesion molecule Drosophila E-cadherin [DE-cadherin, encoded by the shotgun (shg) locus (Tepass et al., 1996; Uemura et al., 1996)] is also important for regulating cell adhesion in the gonad. DE-cadherin is upregulated in the SGPs during gonad formation and is required for both gonad compaction and germ cell ensheathment (Jenkins et al., 2003). fear of intimacy (foi) is also required for gonad formation (Moore et al., 1998; Van Doren et al., 2003) and foi mutants exhibit a similar phenotype to shg mutants. foi and shg are both required for a specific aspect of tracheal development (tracheal branch fusion), suggesting that these genes may participate in a common pathway to coordinate tissue morphogenesis (Van Doren et al., 2003). Consistent with this, DE-cadherin protein levels are greatly reduced in foi mutant gonads (Jenkins et al., 2003), suggesting that foi may act upstream of DE-cadherin to regulate gonad formation. How DE-cadherin is regulated during gonad morphogenesis, and how this is influenced by FOI, has not been previously explored.

FOI (dZIP6/dSLC39A6) is a member of the Zrt, IRT-like protein (ZIP) family of ion transporters (Mathews et al., 2005) that control zinc influx into the cytoplasm from outside the cell or from intracellular stores (Gaither and Eide, 2001). Conserved from fungi to humans, the ZIP family is defined by its conserved transmembrane (TM) structure and several regions of homology (Eng et al., 1998) (Fig. 1B). These proteins often have a histidine-rich cytoplasmic loop, and some have a histidine-rich N-terminal extracellular domain (Fig. 1A). The most conserved region within the LIV-1 subfamily, to which FOI belongs, is known as the `signature sequence' or HELP domain and is predicted to contain two amphipathic -helices with several highly conserved histidine residues (Fig. 1B). Previously, we have shown that FOI is a zinc-specific transporter, and have identified key conserved regions that are crucial for zinc transport activity (Mathews et al., 2005).

Although it is clear how zinc transporters can act at the cellular level to regulate zinc homeostasis, it is more challenging to understand the complex phenotypes caused by changes in zinc transporter function. Little is known about the in vivo roles of the ZIP family in animals; however, recent work indicates that these proteins are essential for embryonic development and adult homeostasis. Two ZIP family members, FOI and zebrafish LIV1 (zSLC39A6), are required for specific morphogenetic events during embryonic development (Pielage et al., 2004; Van Doren et al., 2003; Yamashita et al., 2004). In addition, mutations in human ZIP4 (SLC39A4) are responsible for the genetic disorder acrodermatitis enteropathica (AE), the symptoms of which include epidermal lesions, gastrointestinal defects and infant mortality (Kury et al., 2003; Wang et al., 2002). AE can be treated with increased dietary zinc, and some human ZIP4 mutations associated with AE affect zinc transport activity (Wang et al., 2004), indicating that defects in zinc regulation are crucial to this disease. However, it is still unclear whether zinc can be a regulator of developmental events and if ZIP proteins act primarily through their zinc transport activity or have other essential functions relevant for their in vivo roles.

Here, we use an in vivo rescue assay to analyze FOI function during gonad formation. In particular, we investigate whether the function of FOI as a zinc transporter is crucial for its role in development by studying mutant forms of FOI that are defective for zinc transport. In addition, we investigate how FOI acts to regulate DE-cadherin protein expression in the gonad, and uncover a surprising role for post-transcriptional regulation in generating the proper expression pattern of DE-cadherin.

	MATERIALS AND METHODS

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Fly stocks
The following fly stocks were used: wild type, fafl (ru st faf lacZ e ca) (Moore et al., 1998) and w¹¹¹⁸; foi alleles; foi^20.71 and foi^16.33 (Moore et al., 1998); UAS-mCD8::GFP and tubulin-GAL4 (Lee and Luo, 1999); shg^R69, (Godt and Tepass, 1998), tub-DE-cad and shg^R69, tub-DE-cad flies [a gift from P. Rorth (Pacquelet et al., 2003)]; twist-GAL4 (mesoderm) (Baylies and Bate, 1996); breathless-GAL4 (trachea) (Shiga et al., 1996); and shg^k03401 flies (obtained from the Bloomington Stock Center).

Plasmids
GAL4-dependent, C-terminal hemagglutinin-epitope (HA) tagged FOI (UAS-foi) is as previously described (Van Doren et al., 2003). Site-directed mutations were introduced into the UAS-foi construct using QuikChange (Stratagene): D308A, H554A, D551A/D558A, T557P, H583A/H587A, E584A/E588A/D591A and Y646A (this work) (Mathews et al., 2005). N was generated using a PCR-based strategy to delete amino acids D23-D254 (Q22GRAK255). CATSUP TM6-8 was generated using a PCR-based strategy to replace TM6-8 in FOI with the CATSUP TM6-8 domain. Cloning details available upon request.

Immunolabeling
Antibody staining conducted as previously described (Jenkins et al., 2003; Moore et al., 1998), except for -DCAD2 immunolabeling where embryos were devitellinized by sonication (Patel, 1994) in PBS containing 0.1% Triton X-100 with a 3 second constant pulse using a Branson Sonifier 250 (setting 1). Antibodies used (dilutions, source): rabbit -VAS (1:5,000, R. Lehmann), chick -VAS (1:5000, K. Howard), rabbit -ß-GAL (1:10,000, Capel), mouse -ß-GAL (1:10,000, Promega), rabbit -GFP (1:2,000, Torrey Pines Biolabs), rabbit -ZFH-1 (1:5,000, R. Lehmann), mouse -EYA 10H6 (1:25, N. Bonini, Developmental Studies Hybridoma Bank, DSHB), mouse -NRT BP106 (1:10, C. Goodman, DSHB), rat -DE-cadherin (DCAD2, 1:20, T. Uemura, DSHB), sheep -Digoxigenin (1:2000, Roche Diagnostics) and mouse -HA (0.4 µg/ml, Roche Diagnostics). Secondary antibodies (Molecular Probes or Jackson Laboratory) were used at 1:500. Homozygous mutant embryos were identified using TM3, Ubx-lacZ (ß-GAL). Embryos staged according to Campos-Ortega and Hartenstein (Campos-Ortega and Hartenstein, 1985). shg mutant embryos were aged for 20 hours at 18°C after egg laying to yield collections of embryos stage 15 and older. Embryos were mounted in 70% glycerol containing 2.5% DABCO (Sigma) and visualized using a Zeiss LSM 510 meta confocal microscope.

In vivo rescue assay
Mutant UAS-foi transgenic lines were generated (Rubin and Spradling, 1982) and lines with expression levels similar to wild-type UAS-foi were selected by crossing to breathless-GAL4 and immunolabeling histochemically with -HA. To express UAS-foi in a foi mutant, foi^20.71, UAS-foi/TM3, Ubx-lacZ flies were crossed to foi^20.71, twist-GAL4/TM3, Ubx-lacZ. Controls include foi mutants with the UAS-foi transgene or GAL4 driver alone. Rescue of gonad compaction was examined in at least two independent transgenic lines, analyzing at least 80 gonads for each line. Gonads were considered rescued when similar to wild type. A single representative line was chosen for further analysis of gonad compaction (-ZFH-1, -VAS), germ cell ensheathment (-NRT, -VAS) and DE-cadherin expression (-DCAD2, -VAS), and at least 30 gonads were analyzed. Rescue was scored as the presence of a greater amount of germ cell ensheathment or gonad-specific DE-cadherin expression than normally observed in foi mutants (i.e. not necessarily rescued to wild-type levels).

FOI subcellular localization and membrane topology
UAS-foi constructs were expressed in Schneider S2 cells by co-transfection with actin-GAL4 as described (Van Doren et al., 2003). Protein localization and C-terminal membrane topology were assessed using immunolabeling of permeabilized and non-permeabilized cells, followed by staining with DAPI to label DNA, as previously described (Mathews et al., 2005; Van Doren et al., 2003). Cells were visualized using a Zeiss Axiovert 5100 2TV microscope equipped for deconvolution microscopy (Deltavision, Applied Precision).

Whole-mount fluorescence in situ hybridization
The shg antisense probe was synthesized by digesting pBS-DEcad (gift from V. Hartenstein) with HindIII and transcribing with T3 RNA polymerase (Promega) using digoxigenin-labeled UTP (Boehringer-Mannheim). Embryos were fixed and devitellinized, as for immunostaining. Whole-mount in situ hybridization was performed as previously described (Lehmann and Tautz, 1994), except that instead of Proteinase K treatment, embryos were permeabilized in PBS containing 0.1% Tween-20 (PBTw) with two 3-second continuous pulses using a Branson Sonifier 250 and washed in PBTw. The in situ was developed with the HNPP Fluorescent Detection Set (Roche Diagnostics) according to the manufacturer's instructions. Following this a standard antibody staining was performed (as described above), except the NGS-blocking steps were removed. Embryos were mounted in 70% glycerol containing 2.5% DABCO (Sigma) and visualized using a Zeiss LSM 510 meta confocal microscope. At least 40 gonads were analyzed for each stage and genotype.

	RESULTS

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Zinc transport activity is essential for gonad coalescence and DE-cadherin expression
To determine how FOI affects gonad morphogenesis and DE-cadherin expression, we conducted a structure-function analysis of FOI using an in vivo transgenic rescue assay. Previously, we have shown that expression of wild-type or epitope-tagged versions of FOI can rescue the gonad coalescence defects observed in foi mutants (Mathews et al., 2005; Van Doren et al., 2003). Here, we use this assay to analyze the ability of mutant forms of FOI to rescue gonad compaction, germ cell ensheathment and DE-cadherin expression. Importantly, we have previously tested a number of these mutant forms of FOI for their ability to function in zinc transport (Mathews et al., 2005). This allows us to determine whether it is the zinc transport activity of FOI, or some other function, that is important for regulating gonad morphogenesis and DE-cadherin expression.

Mutations in FOI
We generated a series of mutations in FOI (Fig. 1A,B) within the context of a C-terminal hemagglutinin (HA)-tagged version of the protein that retains wild-type activity in vivo (Van Doren et al., 2003). To determine the importance of the histidine-rich N-terminal domain, we deleted this region (N). To assess whether the primary sequence of the TM domains, or just their transmembrane structure, is essential for FOI function, we mutated specific conserved amino acids in TM domains 2 (D309A) and 7 (Y646A), and also replaced TM domains 6-8 of FOI with TM domains 6-8 from the related Drosophila ZIP protein, CATSUP (Stathakis et al., 1999) (CAT TM6-8). Finally, we altered the highly conserved `HELP' domain to determine the importance of conserved histidine residues (H554A and H583A/H587A), and to investigate whether disrupting the predicted amphipathic (D551A/D558A and E584A/E588A/D591A) or alpha-helical (T557P) nature of this domain would affect FOI function.

View larger version (54K): [in this window] [in a new window]   Fig. 1. FOI is a member of the ZIP family of zinc transporters. (A) Diagram of the FOI protein with conserved domains highlighted. Mutations introduced into HA-FOI are specified. (B) Sequence alignment of highly conserved domains (as indicated) of representative ZIP proteins from different subgroups. Adapted, with permission, from Mathews et al. (Mathews et al., 2005). Alternative nomenclature for these same proteins is included in the lower alignment. Residues tested for function in gonad morphogenesis are indicated (brackets link mutations introduced together). (C) Deconvolution images of Drosophila S2 cells transfected with the indicated UAS-foi constructs and immunolabeled with -HA (red) before permeabilization. DAPI (blue) labels the nuclei. Scale bar: 10 µm.

Rescue of gonad phenotypes
We next assessed the ability of mutant forms of FOI to rescue gonad defects observed in homozygous foi-mutant embryos. First, we examined gonad compaction, the process by which the germ cells and SGPs condense to form a round organ (Jenkins et al., 2003). Compaction was quantified using an immunohistochemical stain of Vasa (VAS), a germ cell-specific antigen (Fig. 2A), as performed previously (Van Doren et al., 2003). In addition, the association between germ cells and SGPs was characterized in embryos immunolabeled with antibodies recognizing VAS and ZFH1 (Fig. 2B). Proteins with mutations in the N-terminal, TM (except the Y646A mutation in TM7) and HELP domains of FOI were all defective in their ability to rescue the gonad compaction defects observed in foi mutants (Fig. 2A). In most cases, SGPs and germ cells remained loosely associated along several embryonic parasegments (e.g. Fig. 2E,F), similar to foi mutant controls (Fig. 2C), although some mutant proteins (e.g. E584A/E588A/D591A) exhibited rescued gonad compaction in a small percentage of embryos (Fig. 2A). These data indicate that the N-terminal, TM and HELP domains are essential for FOI function during gonad compaction. Only the Y646A mutant rescued the foi mutant gonad compaction defect with high penetrance (Fig. 2A). In embryos expressing Y646A, germ cells and SGPs properly associated with one another in a round gonad (Fig. 2G), similar to embryos expressing wild-type HA-FOI (Fig. 2D).

We next assessed germ cell ensheathment, the process in which SGP extensions surround individual germ cells in the gonad (Jenkins et al., 2003). Embryos were immunolabeled with anti-Neurotactin (anti-NRT) to reveal the SGP cell surface, including the extensions around germ cells (Jenkins et al., 2003). Unlike expression of HA-FOI, which fully rescues the germ cell ensheathment defects observed in foi mutant gonads, none of the mutant FOI proteins comparably rescued this phenotype (Fig. 3A). A fraction of embryos expressing either the Y646A or E582A/E588A/D591A proteins exhibited rescue of germ cell ensheathment (Fig. 3F,G compared with B,D), but most embryos expressing these or other mutant forms of FOI exhibited no rescue and were indistinguishable from foi mutant controls (Fig. 3E, compare with 3C). Together, these data indicate that the N-terminal, TM and HELP domains are all required for germ cell ensheathment.

Finally, we examined the ability of mutant versions of FOI to rescue DE-cadherin protein expression in the gonad. DE-cadherin is expressed at high levels in the gonad of wild-type embryos (Fig. 4B) but is greatly reduced in foi mutants (Fig. 4C) (Jenkins et al., 2003). Three of the mutant FOI proteins (T557P, E584A/E588A/D591A and Y646A) rescued at least some DE-cadherin expression in the gonad (Fig. 4A,F,G). However, although DE-cadherin localization appeared normal in embryos expressing the Y646A protein, it appeared weaker and more diffuse in embryos expressing the T557P or E584A/E588A/D591A proteins. It is possible that the DE-cadherin protein observed in these embryos is mislocalized or inactive, consistent with the poor degree of rescue observed with the T557P and E584A/E588A/D591A proteins in our other assays. The remaining mutant forms of FOI were unable to rescue any DE-cadherin expression (Fig. 4A,E), indicating that the N-terminal, HELP and TM domains are all required for proper DE-cadherin expression during gonad coalescence.

View larger version (90K): [in this window] [in a new window]   Fig. 2. Rescue of gonad compaction. Expression of indicated FOI proteins in foi-mutant embryos. (A) Graph indicates percentage of fully compacted gonads, as judged by -VAS staining (average of two independent lines +s.d.; asterisk indicates construct for which three independent insertion lines were tested). (B-G) Confocal images of representative stage 15-16 gonads immunolabeled with -VAS (germ cells, red) and -ZFH-1 (SGPs, green). (B) Wild type, (C) foi mutant, (D-G) foi mutants with the indicated HA-FOI proteins expressed. Only expression of HA-FOI (D) or Y646A (G) rescues gonad compaction. Insets in C and E represent the most severe phenotype observed for these two genotypes; the full panels represent a less severe, but more representative, phenotype. Scale bars: 10 µm.

View larger version (96K): [in this window] [in a new window]   Fig. 3. Rescue of germ cell ensheathment. Expression of indicated FOI proteins in foi-mutant embryos. (A) Graph indicates percentage of gonads with ensheathed germ cells (as judged with -NRT). Only expression of wild-type HA-FOI fully rescues germ cell ensheathment. (B-G) Confocal images of representative stage 15-16 gonads immunolabeled with -NRT (SGP cell surface). Insets show location of germ cells (-VAS, blue) relative to SGPs (-NRT, red). (B) Wild type, (C) foi mutant, (D-G) foi mutants with the indicated HA-FOI proteins expressed. (F,G) Examples of gonads in which germ cell ensheathment is rescued in these genotypes (i.e. more ensheathment than observed in foi mutants). Arrows indicate examples of germ cell ensheathment. Perimeter of gonad is outlined. Scale bars: 10 µm.

View larger version (111K): [in this window] [in a new window]   Fig. 4. Rescue of DE-cadherin expression. Expression of indicated FOI proteins in foi-mutant embryos. (A) Graph indicates the percentage of gonads with higher levels of DE-cadherin expression than foi-mutant controls. (B-G) Confocal images of representative stage 15-16 gonads, immunolabeled with -DCAD2 (DE-cadherin). Insets show positions of germ cells (-VAS, blue) relative to DE-cadherin (-DCAD2, red). (B) Wild type, (C) foi mutant, (D-G) foi mutants with the indicated HA-FOI proteins expressed. Wild type (B), HA-FOI (D), T557P (F) and Y646A (G) gonads express DE-cadherin, while foi20.71 (C) and H554A (E) gonads do not. DE-cadherin expression in gonads expressing T557P (F) is more diffuse than in wild type (B). DE-cadherin in neighboring tracheal branches is labeled (tr). Perimeter of gonad is outlined. Scale bars: 10 µm.

foi affects shg (DE-cadherin) RNA levels
As our data suggest that the zinc transport activity of FOI is required for proper DE-cadherin protein levels in the gonad, we wanted to determine how FOI and zinc might regulate DE-cadherin expression. Previously, it has been shown that zebrafish LIV-1, which is highly similar to FOI, can regulate the activity of the transcription factor SNAIL, a known regulator of DE-cadherin expression (Yamashita et al., 2004). To determine if FOI affects DE-cadherin transcript levels in the gonad, we analyzed shg mRNA expression using fluorescent in situ hybridization. In wild-type embryos, shg RNA was first detected in the gonad just prior to gonad coalescence (stage 13; Fig. 6A,A'), and was also detected at later stages after gonad coalescence (Fig. 6C,C'), similar to what is observed with DE-cadherin protein (Jenkins et al., 2003). shg RNA was primarily observed in the cells immediately surrounding the germ cells (SGPs), but was not observed in the germ cells themselves. shg RNA was also observed in other tissues that express high levels of DE-cadherin protein, including the salivary gland, dorsal epidermis and gut (Tepass et al., 1996). In foi mutants, gonad-specific shg RNA was not observed at either early (stage 13; Fig. 6B,B') or later stages (through stage 16; Fig. 6D,D'), but was still observed in other tissues where DE-cadherin is normally expressed (W.R.M. and M.V.D., unpublished). Thus, within the gonad, FOI appears to regulate DE-cadherin at least in part by regulating shg RNA levels.

The decreased levels of shg RNA observed in foi mutants might reflect decreased levels of shg transcription, instability of the shg mRNA, or both. To investigate shg transcription, we analyzed the expression of a shg enhancer-trap, for which accumulation of ß-galactosidase (ß-GAL) is an indicator of shg promoter activity. Wild-type gonads exhibited slightly increased expression of this enhancer-trap in SGPs, compared with surrounding tissues, just prior to gonad coalescence (stage 13) (A.B.M. and M.V.D., unpublished) and this was maintained throughout the rest of embryogenesis (Fig. 6E). No such increase was observed in foi mutant gonads (Fig. 6F), indicating that foi may have some effect on shg transcription. However, the upregulation of the shg enhancer trap in the gonad and the change in foi mutants appear much less dramatic than we observed for the endogenous shg RNA and DE-cadherin protein. This suggests that DE-cadherin might also be regulated at the post-transcriptional level.

View larger version (22K): [in this window] [in a new window]   Fig. 5. The zinc transport activity of FOI is required for proper gonad morphogenesis in vivo. Graph represents a comparison of the amount of FOI function exhibited by each mutant version of FOI, relative to the activity of wild-type HA-FOI. Data include zinc transport activity, as assayed by zinc uptake in cultured cells [blue, data from Mathews et al. (Mathews et al., 2005)], and in vivo rescue activity, as judged by gonad compaction (red), germ cell ensheathment (gray) and DE-cadherin expression (green) (data from Figs 2, 3, 4 but normalized to HA-FOI controls). The zinc transport ability of each FOI protein directly correlates with its ability to rescue the foi gonad phenotype in vivo.

Next, we examined DE-cadherin protein in embryos where tub-DE-cad was the only source of zygotic DE-cadherin expression [tub-DE-cad expressed in embryos homozygous for a protein null mutant of shg (shg^R69)] (Godt and Tepass, 1998; Pacquelet et al., 2003). Surprisingly, in shg^R69; tub-DE-cad embryos, we observed that DE-cadherin protein was present at much higher levels in the gonad than in the surrounding tissue, even though it was being expressed from the ubiquitous tubulin promoter. DE-cadherin was expressed in a majority of gonads (92%, n=41, Fig. 7D), and the coalescence defects observed in shg mutants were largely rescued in these embryos. Gonad-specific DE-cadherin expression is due to the tub-DE-cad transgene and not residual maternally contributed DE-cadherin, as zygotic shg^R69 mutant embryos alone exhibited little or none of the typical DE-cadherin staining in the gonad (Fig. 7C). The residual DE-cadherin `ring' staining observed in the shg^R69 mutant gonad is also present in embryos in which the shg gene is deleted and is not associated with the normal partners of DE-cadherin such as Armadillo/ß-catenin (Jenkins et al., 2003). Furthermore, gonad-specific DE-cadherin expression is unlikely to be due to gonad-specific transcription from the tubulin promoter, as examination of a related alpha-tubulin84B promoter construct (tubulin-GAL4 crossed to UAS-mCD8::GFP) revealed that the activity of the tubulin promoter is actually lower in the gonad than in surrounding tissues (Fig. 7E). Thus, we conclude that the gonad-specific pattern of DE-cadherin expression is generated by post-transcriptional regulation.

View larger version (105K): [in this window] [in a new window]   Fig. 6. shg mRNA is reduced in foi mutants. (A-D) Confocal images of embryos labeled by fluorescent in situ hybridization to reveal shg RNA (red) and immunolabeled with -VAS (germ cells, green). (A'-D') shg RNA channel alone. shg RNA is observed in SGPs of wild-type embryos both before (A, stage 13) and after (C, stage 15) gonad coalescence, but is not detected in foi-mutant gonads at either stage (B, stage 13; D, stage 15). (E,F) Confocal images of stage 15 wild-type (E) and foi-mutant (F) gonads heterozygous for the shgk03401 enhancer-trap immunolabeled with -ß-GAL (enhancer trap, green) and -VAS (germ cells, blue). SGPs were identified using -EYA (not shown). (E',F') Identical images showing only -ß-GAL. SGPs in wild-type gonads (E') exhibit slightly increased shgk03401 expression compared with surrounding tissues, while SGPs in foi-mutants (F') exhibit weaker expression than surrounding tissues. The perimeter of gonad is outlined. Scale bar: 10 µm.

Interestingly, the tub-DE-cad transgene was still able to rescue the gonad phenotype in some foi-mutant embryos. Although foi mutant gonads rarely appear wild type (4%, n=42), when assessed for both SGP and germ cell morphology, 22% (n=86) of foi^20.71, tub-DE-cad embryos exhibited wild-type gonads (Fig. 7H; data not shown). The remaining embryos had gonads similar to foi mutants (Fig. 7G), and some also contained germ cells that retained a protrusive morphology. In general, the foi^20.71, tub-DE-cad embryos that exhibited increased DE-cadherin expression in the gonad appeared to be those with rescued gonad coalescence (compare Fig. 7F-H). Thus, the relatively low-level rescue of the foi mutant phenotype by tub-DE-cad may reflect the reduced expression of DE-cadherin protein in foi mutants or, alternatively, could reflect DE-cadherin-independent effects of foi. Regardless, expression of DE-cadherin was sufficient to rescue the foi mutant phenotype in some cases, indicating that one of the principal ways that foi affects gonad formation is through regulating DE-cadherin.

Finally, we wanted to investigate whether the post-transcriptional effects of foi on DE-cadherin expression occur at the level of the DE-cadherin protein or RNA. To do this, we investigated the pattern of shg RNA accumulation from the tub-DE-cad transgene. When tub-DE-cad was expressed in wild-type embryos, we observed a high level of shg RNA accumulation in the gonad by in situ hybridization (W.R.M. and M.V.D., unpublished). tub-DE-cad also restored shg RNA accumulation to a shg^R69 mutant (Fig. 7J, compared with I, 81% of shg^R69; tub-DE-cad embryos exhibited high levels of shg RNA in the gonad, n=38). By contrast, when tub-DE-cad was expressed in foi-mutant embryos, shg RNA accumulation in the gonad was greatly reduced (Fig. 7K), and was detectable in only 24% of the gonads (Fig. 7L, n=42). This is comparable with the fraction of gonads that exhibited high levels of DE-cadherin protein expression in the foi^20.71, tub-DE-cad background. Thus, expression of DE-cadherin using the tub-DE-cad transgene is unable to fully restore shg RNA accumulation in foi-mutant gonads. As the activity of a related alpha-tubulin84B promoter construct (tubulin-GAL4) is unaffected in foi mutants (W.R.M. and M.V.D., unpublished), foi is likely to affect shg RNA stability.

View larger version (155K): [in this window] [in a new window]   Fig. 7. foi regulates shg at the post-transcriptional level. (A-D,F-H) Confocal sections of stage 15-16 embryos immunostained with -ZFH-1 (SGPs, red) and -DCAD2 (DE-cadherin, green). The right-hand panels show DE-cadherin channel alone. Gonads and germ cells are indicated with broken lines and asterisks, respectively. Both wild-type (A) and wild-type, tub-DE-cad (B) embryos exhibit high levels of DE-cadherin in the gonad. (C) shgR69 mutant gonad lacking most DE-cadherin expression, but with some -DCAD2 immunoreactivity in large ring-like structures that fail to co-localize with armadillo (Jenkins et al., 2003). (D) shgR69; tub-DE-cad gonad with high levels of DE-cadherin concentrated around germ cells where they are ensheathed by SGPs. (E) Confocal section of a stage 15 embryo, in which tubulin-GAL4 drives expression of UAS-mCD8::GFP, immunolabeled with -GFP (green) and -EYA (SGPs, red). The SGPs express less GFP than the surrounding mesodermal cells. (F,H) foi20.71, tub-DE-cad. DE-cadherin is most often seen as weak punctae between germ cells (F) or is completely absent in gonads. In some embryos (H), more DE-cadherin protein is detected. (G) Little or no DE-cadherin expression is observed in the SGPs of foi20.71 mutant gonads. msSGPs (posterior/right in this gonad) still express DE-cadherin in foi mutants, as has previously been reported (Jenkins et al., 2003). (I-L) Confocal sections of stage 15-16 embryos, labeled by fluorescent in situ hybridization to reveal shg RNA (red) and immunolabeled with -VAS (green, germ cells). Right-hand images represent shg RNA alone. Very little shg RNA immunoreactivity is observed in shgR69 mutant gonads (I), but shg RNA is restored in shgR69; tub-DE-cad (J). tub-DE-cad fails to restore shg RNA expression to most foi20.71 mutant gonads (K), but a minority have shg RNA restored (L). Scale bar: 10 µm.

	DISCUSSION

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Zinc transport activity of FOI is essential for gonad morphogenesis
Previously, it was unclear whether ZIP family members regulate developmental processes by acting as zinc transporters or through some other unidentified function. Our 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 (Fig. 5). 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, we would expect to have identified conserved residues that affect these two activities independently. 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. We conclude 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.

Our in vivo analysis is also informative for revealing domains that are essential for FOI function. We find that, even though the N-terminal extracellular domain of FOI shows little sequence conservation with other family members (Mathews et al., 2005), and some ZIP family members lack an extended N-terminal domain, this domain is nevertheless important for FOI function. We also find that, 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, we analyzed several characteristics of the highly conserved HELP domain in FOI (which may or may not have TM structure) (Mathews et al., 2005). The predicted amphipathic -helical nature of this domain appears to be crucial, as 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. As 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.

Regulation of DE-cadherin by FOI
shg and foi are both required for proper gonad and tracheal morphogenesis (Uemura et al., 1996; Van Doren et al., 2003), and foi regulates DE-cadherin expression in the gonad (Jenkins et al., 2003). 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 (A.B.M., J. Weyers and M.V.D., unpublished). 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, we found that expression of DE-cadherin was sufficient to partially rescue foi mutant gonads.

As both DE-cadherin protein and shg RNA levels are reduced in foi mutant gonads, we investigated whether foi affects DE-cadherin transcription. Our 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 et al., 2004). Examples of how zinc transporters and zinc can modulate the function of transcription factors include regulating zinc-dependent DNA-binding domains (e.g. zinc fingers) (Dalton et al., 1997), regulating non-DNA binding zinc-finger domains (Bird et al., 2003) and affecting transcription factor subcellular localization (Smirnova et al., 2000; Yamashita et al., 2004).

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 (Gumbiner, 2000). Here, we present clear evidence that DE-cadherin is regulated at the post-transcriptional level in the embryonic gonad. Expression of DE-cadherin from the general tubulin promoter (tub-DE-cad) (Pacquelet et al., 2003) was sufficient to restore gonad-specific DE-cadherin protein accumulation in shg mutants (Fig. 7). Recent work suggests that DE-cadherin localization within the ovary is also regulated partly through a post-transcriptional mechanism (Becam et al., 2005). Thus, post-transcriptional regulation may be sufficient to generate tissue-specific patterns of DE-cadherin expression in many contexts. tub-DE-cad was 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, as foi affects the gonad-specific accumulation of shg RNA from tub-DE-cad (Fig. 7), 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.

Role of ZIP proteins in development and disease
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 (Fig. 8). 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 et al., 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 et al., 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 (Nieto, 2002). As human LIV-1 is strongly expressed in breast cancer cell lines (Manning et al., 1988), and has been implicated in breast cancer metastasis (Manning et al., 1994), 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 (Tanaka-Matakatsu et al., 1996). As FOI is also required for this process (Van Doren et al., 2003), it 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 (Boyle and DiNardo, 1995), we have not observed changes in DE-cadherin expression during gonad coalescence in esg mutants (S. Le Bras and M.V.D., unpublished), 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, as discussed above, we have 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 (Ohkubo and Ozawa, 2004).

View larger version (15K): [in this window] [in a new window]   Fig. 8. Models of zinc regulation of DE-cadherin. Human and zebrafish LIV1 proteins may function as zinc transporters to activate SNAIL, a repressor of E-cadherin transcription. Drosophila FOI may activate Escargot (an activator of DE-cadherin) in the trachea, and is required for both DE-cadherin transcription and RNA stability in the gonad.

	ACKNOWLEDGMENTS

 
We thank A. Pacquelet and P. Rorth for the generous gift of the tubulin-DE-cadherin flies, and our many other colleagues who contributed essential reagents as indicated in the Materials and methods. We thank the Bloomington Stock Center (Indiana University) for fly stocks and the Developmental Studies Hybridoma Bank (University of Iowa) for antibodies. We thank Michael McCaffery and the JHU integrated imaging center for providing essential microscopy resources. We thank Kyle Cunningham, Jonathan Shaw and members of the Van Doren laboratory for critical reading of the manuscript. This work was supported by the National Institutes of Health (GM63023) and the Pew Charitable Trust.

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