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


Development 133, 1143-1153 (2006)
Published by The Company of Biologists 2006


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

Wendy R. Mathews, Daniel Ong, Allison B. Milutinovich and Mark Van Doren*

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


Figure 1
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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., 2005Go). 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 {alpha}-HA (red) before permeabilization. DAPI (blue) labels the nuclei. Scale bar: 10 µm.

 

Figure 2
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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 {alpha}-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 {alpha}-VAS (germ cells, red) and {alpha}-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.

 

Figure 3
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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 {alpha}-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 {alpha}-NRT (SGP cell surface). Insets show location of germ cells ({alpha}-VAS, blue) relative to SGPs ({alpha}-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.

 

Figure 4
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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 {alpha}-DCAD2 (DE-cadherin). Insets show positions of germ cells ({alpha}-VAS, blue) relative to DE-cadherin ({alpha}-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.

 

Figure 5
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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., 2005Go)], 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.

 

Figure 6
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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 {alpha}-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 {alpha}-ß-GAL (enhancer trap, green) and {alpha}-VAS (germ cells, blue). SGPs were identified using {alpha}-EYA (not shown). (E',F') Identical images showing only {alpha}-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.

 

Figure 7
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Fig. 7. foi regulates shg at the post-transcriptional level. (A-D,F-H) Confocal sections of stage 15-16 embryos immunostained with {alpha}-ZFH-1 (SGPs, red) and {alpha}-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 {alpha}-DCAD2 immunoreactivity in large ring-like structures that fail to co-localize with armadillo (Jenkins et al., 2003Go). (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 {alpha}-GFP (green) and {alpha}-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., 2003Go). (I-L) Confocal sections of stage 15-16 embryos, labeled by fluorescent in situ hybridization to reveal shg RNA (red) and immunolabeled with {alpha}-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.

 

Figure 8
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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.

 





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