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Reproduction (2005) 129 557-564
DOI: 10.1530/rep.1.00638
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RESEARCH

SH2 domain-mediated activation of an SRC family kinase is not required to initiate Ca2+ release at fertilization in mouse eggs

Lisa M Mehlmann and Laurinda A Jaffe

Department of Cell Biology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, Connecticut 06032, USA

Correspondence should be addressed to L M Mehlmann or L A Jaffe; Email: lmehlman{at}neuron.uchc.edu or ljaffe{at}neuron.uchc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
SRC family kinases (SFKs) function in initiating Ca2+ release at fertilization in several species in the vertebrate evolutionary line, but whether they play a similar role in mammalian fertilization has been uncertain. We investigated this question by first determining which SFK proteins are expressed in mouse eggs, and then measuring Ca2+ release at fertilization in the presence of dominant negative inhibitors. FYN and YES proteins were found in mouse eggs, but other SFKs were not detected; based on this, we injected mouse eggs with a mixture of FYN and YES Src homology 2 (SH2) domains. These SH2 domains were effective inhibitors of Ca2+ release at fertilization in starfish eggs, but did not inhibit Ca2+ release at fertilization in mouse eggs. Thus the mechanism by which sperm initiate Ca2+ release in mouse eggs does not depend on SH2 domain-mediated activation of an SFK. We also tested the small molecule SFK inhibitor SU6656, and found that it became compartmentalized in the egg cytoplasm, thus suggesting caution in the use of this inhibitor. Our findings indicate that although the initiation of Ca2+ release at fertilization of mammalian eggs occurs by a pathway that has many similarities to that in evolutionarily earlier animal groups, the requirement for SH2 domain-mediated activation of an SFK is not conserved.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
At fertilization, Ca2+ elevation in the egg cytosol establishes a block to polyspermy, and also causes the resumption of the cell cycle, leading to embryonic development (Ducibella et al. 2002, Runft et al. 2002, Hyslop et al. 2004, Markoulaki et al. 2004). In mammals (Miyazaki et al. 1992, 1993), as well as other species in the vertebrate evolutionary line (see Runft et al. 2002), the Ca2+ is released from the egg’s endoplasmic reticulum by inositol trisphosphate (IP3), but the mechanisms leading to IP3 production are incompletely understood. SRC family kinases (SFKs) function in this signalling pathway in echinoderms (Jaffe et al. 2001, O’Neill et al. 2004), ascidians (Runft & Jaffe 2000), fish (Wu & Kinsey 2002, Kinsey et al. 2003), and frogs (Sato et al. 1996, 2000), but whether the same is true in mammals is uncertain.

For echinoderms, the evidence for the function of SFKs in fertilization signalling includes immune complex kinase assays (Kinsey 1996, Abassi et al. 2000, O’Neill et al. 2004), fertilization-dependent association of SFKs with phospholipase C (PLC){gamma} (Giusti et al. 1999a, Kinsey & Shen 2000, Runft et al. 2004), inhibition of Ca2+ release by dominant negative SFK constructs (Giusti et al. 1999b, 2003, Abassi et al. 2000, Kinsey & Shen 2000, O’Neill et al. 2004), and stimulation of PLC{gamma}-dependent Ca2+ release by injection of SRC protein or SFK activating antibodies (Giusti et al. 2000, 2003). These findings support a model, for echinoderm species, in which sperm–egg interaction activates an SFK, which directly or indirectly activates PLC{gamma}, leading to IP3 production and Ca 2+ release; some but not all aspects of this pathway have been established for ascidians, fish, and frogs.

For mammals, tyrosine phosphorylation of several proteins has been found to increase within 1–3 h after fertilization of rat eggs; however, it is not known if this is due to activation of SFKs or other tyrosine kinases (Ben-Yosef et al. 1998). Tyrosine kinase inhibitors have been applied during mouse fertilization, and this delayed the initiation of Ca2+ release, but the inhibitors used were not specific for SFKs (Dupont et al. 1996). Also, it is not known whether the delay was due to direct effects on the initiation of Ca2+ release, or to indirect effects on the sperm prior to its binding and fusion with the egg. In a recent study, such effects on sperm physiology were avoided by injecting sperm or sperm extracts into eggs, rather than applying sperm to the outside of eggs as occurs normally (Kurokawa et al. 2004). PP2 (4-amino-5(4-chlorophenyl)-7-(t-butyl)pryazolo[3,4-d]pyrimidine), which inhibits SFKs, did not inhibit Ca2+ release in response to injection of sperm or sperm extracts into the mouse egg cytoplasm (Kurokawa et al. 2004). Although this experiment argued against a role for SFKs in initiation of Ca2+ release, critical signalling events related to sperm–egg membrane binding and fusion could have been bypassed, because the experiment tested the effect of PP2 on the response to sperm injection vs the response to the multistep process of fertilization.

In the work to be described here, we determined which SFKs are present in mouse eggs, and then microinjected the eggs with membrane impermeant inhibitors of these SFKs. By use of microinjection, we were able to isolate effects on the egg from effects on the sperm, thus allowing us to examine if inhibition of SFKs in the egg inhibited Ca2+ release during normal fertilization.

Mammalian genomes encode eight different SFKs: SRC, FYN, YES, FGR, LYN, LCK, HCK, and BLK (Bolen et al. 1991), and several different SFKs can function in activation of PLC{gamma}, leading to Ca2+ release (Quek et al. 2000, Rhee 2001, Ozdener et al. 2002). We used immunoblotting to test which of the SFK proteins are present in mouse eggs, and then used the Src homology 2 (SH2) domains of these SFKs as dominant negative inhibitors, to investigate the function of SFKs in mediating Ca2+ release at fertilization. Although many signalling proteins contain SH2 domains, sequence differences within these ~100 amino acid regions confer specificity on their binding to phosphotyrosine-containing sequences in other proteins (Songyang & Cantley 1995). Because excess SFK SH2 domains can interfere with the interaction of SFKs with their binding partners, intracellular injection of SH2 domains has been an effective and specific way of inhibiting SFK activation in fibroblasts (Roche et al. 1995) as well as eggs (Giusti et al. 1999b, Abassi et al. 2000, Kinsey & Shen 2000, Runft & Jaffe 2000, Sette et al. 2002, Kinsey et al. 2003, O’Neill et al. 2004). Single cell kinase assays of zebrafish eggs injected with FYN SH2 domains have shown that the SH2 domains prevent the increase in SFK activity at fertilization; they prevent the stimulation of SFK activity, rather than directly inhibiting the kinase activity, since addition of the SH2 domains to an in vitro kinase assay had very little effect on kinase activity (Kinsey et al. 2003).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
Preparation of gametes
Fully grown, immature mouse oocytes were collected from NSA (CF-1) mice (Harlan, Indianapolis, IN, USA) after killing the mice using CO2. Oocytes were collected in minimal essential medium (MEM) supplemented with 250 µM dbcAMP (Mehlmann & Kline 1994). Oocytes were washed into MEM without dbcAMP to induce oocyte maturation; such in vitro matured eggs exhibit a normal pattern of Ca2+ oscillations following fertilization (Mehlmann et al. 2001). Sperm were collected from the caudal epididymides and vas deferens in Whittingham’s medium (Hogan et al. 1994) containing 3% (w/v) bovine serum albumin (BSA) (Mehlmann & Kline 1994), and were allowed to capacitate for ≥90 min before use. For fertilization, the zona was removed from eggs using acid Tyrode’s solution (Mehlmann et al. 1996). Eggs in Whittingham’s medium containing no BSA were adhered to a glass coverslip coated with Cell-Tak (Collaborative Research, Bedford, MA, USA) that formed the bottom of a chamber fitting into a heated microscope stage perfused with 5% CO2 (Medical Systems Corp., Greenvale, NY, USA). Sperm were applied at a final concentration of ~2–5 x 105 sperm/ml. Eggs of starfish (Asterina miniata) were obtained and fertilized as previously described (Carroll et al. 1997).

Immunoblotting
Immunoblotting was performed as described previously (Mehlmann et al. 1998). Aliquots of mouse eggs were frozen in liquid N2 after removal of excess medium, and stored at –80 °C until use. Cell lysates used as positive controls were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Primary antibodies are listed in Table 1Go.


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  		Table 1 Primary antibodies used for immunoblotting.
 
SH2 domain experiments
Plasmids encoding the glutathione S-transferase fusion proteins GST-FYN SH2 (chicken, amino acids 148–251) and GST-YES SH2 (mouse, amino acids 142–265) were obtained from K Vuori (Cancer Center, The Burnham Institute, La Jolla, CA, USA) and D Flynn (MBR Cancer Center, West Virginia University, Morgantown, WV, USA) respectively. The amino acid sequence of the SH2 domain of chicken FYN is 99% identical to that of mouse FYN (SH2 domain region as defined in Xu et al. 1997). SH2 domain proteins were produced in bacteria, and purified, dialyzed, and concentrated as described previously (Carroll et al. 1997). Zona-intact eggs were quantitatively microinjected using pipettes backfilled with mercury (Mehlmann & Kline 1994, Jaffe & Terasaki 2004). Concentrations of injected substances were calculated based on cytoplasmic volumes of 200 pl and 3000 pl for mouse and starfish eggs respectively. Ca2+ changes were measured using calcium green 10 kDa dextran (Molecular Probes, Eugene, OR, USA), at a final concentration of 16 µM (mouse) or 10 µM (starfish) (Carroll et al. 1997, Mehlmann et al. 1998). For injection of FYN and YES SH2 domains into mouse eggs, the stock solution contained 10 mg/ml FYN SH2, 10 mg/ml YES SH2, and 330 µM calcium green dextran in phosphate-buffered saline. Injections of the SH2 domain fusion proteins into the mouse eggs were made 2–7 h before insemination. The delay between injection and insemination was a consequence of performing the injections using zona-intact eggs (the eggs were held in place by use of a suction pipette that attaches to the zona). We then removed the zonae with low pH medium, and afterwards allowed the eggs to equilibrate in medium of normal pH for at least 2 h prior to insemination (fertilization is inefficient if the zona is present). Although degradation of the SH2 domains during the 2–7 h period between injection and insemination is a possible concern, we consider this to be unlikely, since SH2 domains of PLC{gamma} are stable in the egg cytoplasm for at least 6.5 h (Mehlmann et al. 1998).

Experiments with SU6656
SU6656 (2-oxo-3-(4,5,6,7-tetrahydro-1H-indol-2-ylmethylene)-2,3-dihydro-1H-indole-5-sulfonic acid dimethylamide; Calbiochem) was prepared as a 10 mM stock in dimethyl sulfoxide, and then diluted to 10 µM in MEM. Oocytes that had been incubated with SU6656 were observed using a BioRad MRC600 confocal microscope, with a 40 x /1.2 numerical aperture water immersion objective (Carl Zeiss, Inc., Thornwood, NY, USA). Fluorescence was excited using the 488 nm line of a krypton/argon laser, and detected using a 515 nm long pass filter.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
FYN and YES proteins are present in mouse eggs, but other SFKs are not detectable
A previous study has shown that FYN protein is present in mouse eggs (Sette et al. 2002), but it has been uncertain whether other SFK proteins are present as well. To determine which of the eight SFKs are expressed in mouse eggs, immunoblotting was used to compare eggs with cell lines known to express each of these proteins. In addition to FYN, YES protein was detected in mouse eggs (Fig. 1AGo), using two different antibodies. FYN and YES proteins have also been found in rat eggs (Talmor-Cohen et al. 2004a).



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  		Figure 1 Immunoblots of SFK proteins in mouse eggs and control lysates of cell lines. All egg lanes contained 2.5 µg protein (100 eggs). See Table 1Go for antibodies used. (A) Eggs contain FYN and YES proteins; YES was detected with two different antibodies. BD; BD 610375. (B) No other SFK proteins were detected in eggs. Jurkat, PC-12, BT-20, Namalwa and RAW 264.7; positive control cell lines.

 
Other SFK proteins were not detectable in mouse eggs, under conditions where these SFKs were detectable in comparable amounts of control cell protein (Fig. 1BGo). The absence of a detectable amount of SRC protein in mouse eggs was surprising, since SRC is present in rat eggs (Talmor-Cohen et al. 2004a). However, based on three independent experiments in which we observed a strong SRC signal from 1 µg of a control cell lysate, and no SRC signal from 2.5–5 µg of mouse egg lysate, we concluded that mouse eggs contain little if any SRC protein. LCK was also of particular interest since it was previously reported to be present based on immunofluorescence and an immune complex kinase assay (Mori et al. 1991), and RT-PCR (Mori et al. 1992). However, these studies did not test for the presence of LCK protein by immunoblotting, so a possible explanation for the difference in our findings could be antibody non-specificity in the previous work. We concluded from our immunoblots that the major SFK proteins present in mouse eggs are FYN and YES.

Ca2+ release at fertilization of mouse eggs does not require SH2 domain-mediated SFK activation
To investigate if FYN and YES function in the signalling pathway leading to Ca2+ release at fertilization in mouse eggs, we injected eggs with a mixture of FYN and YES SH2 domain proteins (Fig. 2AGo). The FYN SH2 domain is an effective inhibitor of mouse egg activation in response to injection of a truncated form of a receptor tyrosine kinase (tr-KIT) (Sette et al. 2002), and in sea urchin (Abassi et al. 2000, Kinsey & Shen 2000), starfish (Giusti et al. 1999b), ascidian (Runft & Jaffe 2000), and fish (Kinsey et al. 2003) eggs, 2.5–25 µM FYN SH2 partially or completely inhibits Ca2+ release at fertilization. We confirmed the effectiveness of the FYN SH2 domain by showing that, as previously described (Giusti et al. 1999b), it inhibited Ca2+ release at fertilization in starfish eggs (Fig. 2B and CGo). We also tested the YES SH2 domain in starfish eggs; seven eggs injected with 2.5–25 µM YES SH2 all showed either no Ca2+ release or a delayed and reduced Ca2+ release when fertilized, compared with controls injected with the Ca2+ indicator only (Fig. 2DGo). SH2 domains of non-SRC family tyrosine kinases (ABL, SYK, ZAP-70) are not inhibitory (Giusti et al. 1999b, Abassi et al. 2000, Kinsey & Shen 2000, Runft & Jaffe 2000, Kinsey et al. 2003, O’Neill et al. 2004).



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  		Figure 2 FYN and YES SH2 domains inhibit Ca2+ release at fertilization in starfish but not mouse eggs. (A) GST-SH2 domain fusion proteins used for microinjection. 10% SDS-PAGE gel stained with Coomassie blue; 1 µg protein per lane. (B) Ca2+ release at fertilization in a starfish egg. (C) Inhibition of Ca2+ release at fertilization in a starfish egg by injection of FYN SH2, at a cytoplasmic concentration of 1.0 mg/ml = 25 µM. (D) Inhibition of Ca2+ release at fertilization in a starfish egg by injection of YES SH2, at a cytoplasmic concentration of 0.5 mg/ml = 12 µM. (E) Ca2+ release at fertilization in a mouse egg. (F) No inhibition of Ca2+ release at fertilization in a mouse egg by injection of a mixture of FYN and YES SH2 domains, each at a cytoplasmic concentration of 0.5 mg/ml = 12 µM. Traces in (B–F) show calcium green fluorescence as a function of time. Asterisks indicate the action potential, which marks the time of sperm–egg fusion in starfish eggs (McCulloh & Chambers 1992).

 
Injection of mouse eggs with a mixture of FYN and YES SH2 domains (12 µM each) did not inhibit Ca2+ release at fertilization (Fig. 2E and FGo). Of seven eggs tested, six showed Ca2+ release, and the time between sperm addition and the Ca2+ rise was 14 ± 5 min (means±S.D.). Four of five control eggs injected with the Ca2+ indicator only showed Ca2+ release, beginning at 14 ± 2 min after sperm addition. The amplitude of the Ca2+ release and the pattern of oscillations were also the same for FYN SH2 + YES SH2 domain-injected eggs vs controls. In another series of experiments, Ca2+ release at fertilization was unaffected by injection of 25 µM FYN SH2 domain protein (six eggs). As discussed above, our control experiments showed that both FYN and YES SH2 domains were effective SFK inhibitors in starfish eggs, and previously published work has also indicated that, at a comparable concentration, FYN SH2 domains are effective inhibitors in mouse eggs stimulated with tr-KIT (Sette et al. 2002). Thus, we concluded that in mouse fertilization, Ca2+ release in the egg does not depend on SH2 domain-mediated activation of an SRC.

Intracellular compartmentalization of the SFK inhibitor SU6656
To examine the SFK dependence of Ca2+ release at fertilization of mouse eggs using a different method, we attempted to use a small molecule inhibitor of SFK activity, SU6656 (Blake et al. 2000). We chose not to use another commonly used SFK inhibitor, PP2, because preliminary experiments showed that PP2 inhibited mouse sperm motility.

To determine if SU6656 was an effective inhibitor in an egg known to require SFK activity for Ca2+ release at fertilization, we initially applied it to starfish eggs; surprisingly, SU6656 (10 µM) had no inhibitory effect on fertilization-induced Ca2+ release (n = 4 eggs). SU6656 is fluorescent, so it was possible to check that it entered the oocyte cytoplasm by fluorescence microscopy. These observations showed that SU6656 did enter the cytoplasm but, within the cytoplasm, it was compartmentalized within vesicles near the oocyte surface (Fig. 3AGo). This sequestration may explain why it was not an effective SFK inhibitor. Likewise, in mouse oocytes, SU6656 was not distributed homogeneously within the cytoplasm, indicating compartmentalization within organelles (Fig. 3BGo). For this reason, we did not use this inhibitor to test the SFK dependence of Ca2+ release at fertilization in mouse eggs. These findings suggest caution in the use of SU6656 as an inhibitor of SFKs. Since SU6656 compartmentalizes within the cytoplasm, it may not be accessible to the inner surface of the plasma membrane, where SFKs function in transducing extracellular signals.



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  		Figure 3 SU6656 is compartmentalized in starfish and mouse oocytes. (A) A starfish oocyte that had been incubated for 80 min in 10 µM SU6656 in sea water before imaging. The confocal section shows that SU6656 is localized in vesicles near the oocyte surface. (B) A mouse oocyte that had been incubated for 120 min in 10 µM SU6656 in MEM containing 250 µM dbcAMP before imaging. The confocal section shows that SU6656 is localized in compartments throughout the cytoplasm and concentrated around the prophase nucleus (germinal vesicle). Scale bars = 20 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
At fertilization in species of the vertebrate evolutionary line, Ca2+ is released from the egg’s endoplasmic reticulum by a process requiring IP3, and in non-mammalian vertebrates, an SFK functions in this pathway (Runft et al. 2002, see Introduction). The present results show that Ca2+ release at fertilization in mouse eggs is not inhibited by excess SH2 domains of the two SFK proteins found in the eggs, FYN and YES. These findings indicate that the signalling events leading to IP3-dependent Ca2+ release are fundamentally different in eggs of mice, compared with those of echinoderms (Giusti et al. 1999b, 2003, Abassi et al. 2000, Kinsey & Shen 2000, O’Neill et al. 2004,), ascidians (Runft & Jaffe 2000), and fish (Kinsey et al. 2003), where SFK SH2 domains do inhibit Ca2+ release at fertilization. Other differences between echinoderms and mammals are that in echinoderms (Carroll et al. 1997, 1999, Shearer et al. 1999), but not mice (Mehlmann et al. 1998), PLC{gamma} SH2 domains inhibit Ca2+ release at fertilization, and the time between sperm fusion and Ca2+ release is greater in mouse eggs (~1–5 min) compared with echinoderm eggs (~4–12 s) (see Runft et al. 2002).

Although our work argues against a role for SH2 domain-dependent activation of an SFK in initiating Ca2+ release at fertilization in mouse eggs, other studies indicate that SFKs may function downstream of Ca2+ to cause the resumption of meiosis that occurs at fertilization. Injection of constitutively active FYN protein causes metaphase II-arrested mouse and rat eggs to resume meiosis (Sette et al. 2002, Talmor-Cohen et al. 2004b), and the SFK kinase inhibitors PP2 and SU6656 inhibit the resumption of meiosis in rat eggs in response to the Ca2+ ionophore ionomycin (Talmor-Cohen et al. 2004a). FYN kinase is localized to the meiotic spindle of rat eggs (Talmor et al. 1998, Talmor-Cohen et al. 2004a,b), and SU6656 causes disintegration of the meiotic spindle (Talmor-Cohen et al. 2004b); in addition, tubulin coimmunoprecipitates with FYN in ionomycin-activated rat eggs (Talmor-Cohen et al. 2004b). All of these findings support a role for FYN in the Ca2+-dependent activation of meiosis at fertilization of mammalian eggs, but leave open the question of how Ca2+ release is initiated.

Much of the recent work on egg activation at fertilization in mammalian eggs has concerned the hypothesis that a substance from the sperm cytoplasm that enters the egg cytoplasm as a consequence of sperm–egg fusion could be the activator of the Ca2+ release pathway (see Runft et al. 2002). Current candidates include tr-KIT (Sette et al. 2002) and PLC{zeta} (Cox et al. 2002, Saunders et al. 2002, Fujimoto et al. 2004, Kouchi et al. 2004, Yoda et al. 2004, Knott et al. 2005). Our findings argue against tr-KIT, since FYN SH2 domains inhibit Ca2+ release in response to tr-KIT injection (Sette et al. 2002), but not in response to fertilization.

PLC{zeta} has several properties consistent with an egg-activating function. (1) The protein is present in sperm (Cox et al. 2002, Saunders et al. 2002) and is localized to the part of the sperm head that first enters the egg at fertilization (Fujimoto et al. 2004). (2) Sperm extract fractions containing PLC{zeta} correlate with fractions that can activate eggs (Fujimoto et al. 2004). (3) PLC{zeta} exhibits high PLC activity at low free Ca2+ levels as found in unfertilized eggs (Kouchi et al. 2004). (4) Microinjection of PLC{zeta} RNA or protein causes Ca2+ oscillations in eggs, identical to those at fertilization (Cox et al. 2002, Saunders et al. 2002, Kouchi et al. 2004, Yoda et al. 2004). (5) Immunodepletion of sperm extracts with a PLC{zeta} antibody removes the ability of the extract to cause Ca2+ release (Saunders et al. 2002). (6) Fertilization of eggs with sperm from transgenic mice expressing short hairpin RNAs that target PLC{zeta} results in a decreased duration of the Ca2+ transients (Knott et al. 2005). However, it is not known whether other proteins might have been removed from the sperm extract by the PLC{zeta} antibody that was used for immunodepletion, and the short hairpin RNAs had only a slight effect on the initiation of the Ca2+ transients. In addition, some uncertainty remains as to whether a single sperm contains sufficient PLC{zeta} protein (Saunders et al. 2002, Fujimoto et al. 2004, Kouchi et al. 2004) or PLC activity (Mehlmann et al. 2001, Kouchi et al. 2004) to initiate Ca2+ release. Issues contributing to this uncertainty include the use of partially purified recombinant protein and incompletely characterized antibodies for the quantification of PLC{zeta} in sperm.

Nevertheless, PLC{zeta} is at present the best supported candidate for a sperm factor causing mammalian egg activation. Regardless of what the sperm factors that activate eggs turn out to be, our results have indicated that these molecules are likely to differ among species that do or do not require SH2 domain-mediated SFK activation in the signalling pathway.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
Antibodies and DNA constructs were generously provided to us by the colleagues listed in the Materials and Methods and Table 1Go. We thank Mark Terasaki for help with the confocal microscopy, and Linda Runft and Kathy Foltz for reading the manuscript.

Funding

The work was supported by a fellowship from the Lalor Foundation to L M and NIH grant HD14939 to L A J. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scienctific work.


   Footnotes
 
Received 19 December 2004
First decision 31 January 2005
Accepted 15 February 2005


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

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