The eggshell is required for meiotic fidelity, polar-body extrusion and polarization of the C. elegans embryo

Background Fertilization restores the diploid state and begins the process by which the single-cell oocyte is converted into a polarized, multicellular organism. In the nematode, Caenorhabditis elegans, two of the earliest events following fertilization are secretion of the chitinous eggshell and completion of meiosis, and in this report we demonstrate that the eggshell is essential for multiple developmental events at the one-cell stage. Results We show that the GLD (Germline differentiation abnormal)-1-regulated hexosamine pathway enzyme, glucosamine-6-phosphate N-acetyltransferase (GNA)-2, is required for synthesis of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), the substrate for eggshell chitin synthesis by chitin synthase-1 (CHS-1). Furthermore, while chs-1(RNAi) or combined RNAi with the chitin-binding proteins, CEJ-1 and B0280.5, does not interfere with normal meiotic timing, lagging chromosomes are observed at meiosis, and polar-body extrusion fails. We also demonstrate that chitin, and either CEJ-1 or B0280.5, are essential for the osmotic/permeability barrier and for movement of the sperm pronucleus/centrosome complex to the cortex, which is associated with the initiation of polarization. Conclusion Our results indicate that the eggshell is required in single-cell C. elegans development, playing an essential role in multiple actin-dependent early events. Furthermore, the earliest meiotic roles precede osmotic barrier formation, indicating that the role of the eggshell is not limited to generation of the osmotic barrier.


Background
During oocyte development, inhibition of mitogen-activated protein kinase (MAPK) signaling causes arrest at meiotic prophase I. Subsequent maturation, which results in breakdown of the germinal vesicle (nuclear envelope) and assembly of the meiotic spindle, requires relief of MAPK inhibition. In Caenorhabditis elegans, this relief is provided by major sperm protein (MSP), budded off from sperm stored in the spermatheca [1,2]. MSP displaces ephrin bound to oocyte VAB-1 receptors, resulting in MAPK activation and oocyte maturation. MSP also binds non-VAB receptors on oocyte and gonadal sheath cell membranes, inducing ovulation of the mature oocyte into the spermatheca, the site of fertilization. Fertilization activates the anaphase-promoting complex/cyclosome (APC/ C), triggering progression past anaphase I [3]. Concomitantly, fertilization signals the rapid assembly of a chiti-nous eggshell that surrounds the developing embryo until hatching.
The nematode eggshell can have up to five layers, although in most species, including C. elegans, it is a trilamellate structure, comprised of an outer vitelline layer, a middle chitin-containing layer and an inner lipid-rich layer [4][5][6][7]. Detailed ultrastructural studies of the C. elegans eggshell are lacking. However, electron micrographic studies of Ascaris lumbricoides [8] revealed that shortly after sperm penetration, the outer plasma membrane-like layer separates from the egg cytoplasm, resulting in a dense outer vitelline layer. Underlying the vitelline layer is a structureless zone that subsequently becomes filled with chitin and protein, resulting in the formation of the mechanically resistant middle layer of the shell. Specific proteins in this middle layer have not been identified, but proteins with chitin-binding domains are likely candidates. In C. elegans, these include T10E10.4, F23F12.8, M03E7.4, R02F2.4, K04H4.2A, C39D10.7, W03F11.1, W02A2.3, H02I12.1, B0280.5 and CEJ-1, proteins predicted to have Peritrophin-A domains, a conserved chitinbinding domain found in peritrophic matrix proteins of insects and in animal chitinases [9,10]. Two of these proteins, CEJ-1 and B0280. 5, have recently been shown to bind chitin and to be modified by chondroitin addition [11]. Moreover, chondroitin deficiency in squashed vulva (Sqv) mutants results in embryos in which the space between the eggshell and the embryo is missing, and in which cytokinesis at the one-cell stage is defective [12]. These results suggest that CEJ-1 and B0280.5 are likely to play an important role as components of the eggshell.
Coincident with the deposition of chitin and protein into the middle layer of the shell, the inner proteolipid layer of the eggshell (the proposed permeability barrier) is formed by extrusion of embryonic cytoplasmic refringent granules, and the first polar body is extruded into this layer. By the time of pseudocleavage in the one-cell embryo, the trilamellate eggshell is separated from the embryo plasma membrane by a clear zone, which may be the precursor of the perivitelline fluid (PVF) that surrounds later stage embryos. In 3-day-old Ascaris suum embryos, the PVF has been shown to contain a number of proteins, including the fatty acid-binding protein, As-p18, which has been suggested to play a role in maintaining the barrier function of the inner eggshell layer [13].
Chitin ([(GlcNAcβ1-4GlcNAc) n ]) is polymerized from the sugar nucleotide donor, uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), synthesized by the hexosamine pathway ( Figure 1) [14]. Eggshell chitin production places a sudden and high demand on this pathway, with as much as 50% of embryonic glycogen proposed to be required for UDP-GlcNAc synthesis in Ascaris megalo-cephala [15]. The C. elegans eggshell can be removed at the two-cell stage, without interrrupting development, at least until gastrulation [16]. This has led to the suggestion that the function of the chitinous eggshell is restricted to mechanical support of the developing embryo from gastrulation onwards. However, an earlier embryonic lethal phenotype results from RNA interference (RNAi), with enzymes catalyzing any one of the five hexosamine pathway steps leading to chitin synthesis ( Figure 1) [17][18][19][20]. Furthermore, transcripts for two key enzymes in this pathway, glucosamine-6-phosphate N-acetyltransferase (GNA)-2 and glutamine synthetase (GLN)-5, are tightly regulated by the germline translational repressor, GLD-1 ( Figure 1) [21,22]. Taken together, these results suggest that the chitinous eggshell is developmentally essential before the two-cell stage.
Many of the developmental events that follow fertilization require asymmetric or focal actomyosin contraction, which may depend on eggshell assembly. For example, polar-body cytokinesis is highly asymmetric and depends on non-muscle myosin (NMY)-2 enriched at the contractile ring [23,24]. Polar-body extrusion also requires the actin/myosin cross-linking protein, ANI-1, the myosin light chain homologue, MLC-4, and the microfilament organizing proteins, profilin (PFN)-1 and CYK (Cytokinesis defect)-1 [23][24][25]. Following polar-body extrusion, the embryonic cortex undergoes a period of membrane ruffling that is dependent on focal enrichment of the actomyosin cytoskeleton at the base of the ruffles [26]. Ruffling requires ANI-1, NMY-2, PFN-1 and CYK-1, and is coincident with cytoplasmic flow and the movement of the sperm pronucleus/centrosome complex (SPCC) towards the cortex [27]. SPCC-cortical association marks the posterior of the embryo [28], and is associated with the initiation of anterior-posterior (A-P) polarization, which culminates in an asymmetric first cell division, generating two daughter cells (AB and P 1 ) with distinct developmental potentials [29,30]. Coincident with SPCC-cortical association is the local clearing of NMY-2 foci and local relaxation of actomyosin contraction. The resultant noncontractile cortex expands anteriorly to the approximate halfway point, terminating in the pseudocleavage furrow, which demarcates the smooth PAR (Partitioning abnormal)-2/PAR-1-binding posterior cortex from the contractile PAR-3/PAR-6/PKC-3-binding anterior cortex [26,30]. This highly asymmetric contraction and P-A cortical flow is associated with an opposing A-P cytoplasmic flow, which transports cytoplasmic determinants, including P granules, to the posterior [30,31].
Progression past meiosis metaphase I requires activation of the APC/C. Complete loss of function of genes encoding APC subunits results in Mat (metaphase to anaphase transition-defective) embryos with either no polarization or reversed polarization [28]. Partial loss of function of APC can support the completion of meiosis, but results in embryos with combined polarity and osmotic-sensitivity defects (Pod phenotype of mat 1-3, emb-27, emb-30) [46,47]. The Pod class is diverse, including not only APC subunit genes, but also genes encoding a coronin-like actin-binding protein (POD-1), two genes encoding enzymes required for fatty-acid biosynthesis [acetylCoA carboxylase (POD-2) and NADPH-dependent cytochrome P450 reductase (EMB-8)] and a gene encoding the Rho family GTPase, CDC-42 [5,32,[48][49][50]. In this study, we identify gna-2(qa705) as a novel Pod mutant, and demonstrate that eggshell chitin and two functionally redundant chitin-binding proteins, CEJ-1 and B0280.5, are essential for error-free segregation at meiosis, for extrusion of the polar bodies and for SPCC association with the cortex. These results form the basis of a model of C. elegans embryonic development that includes an essential role for the eggshell in actomyosin-dependent events at the onecell stage.

Meiosis and polar-body extrusion are defective in gna-2(qa705)
Live in utero recordings of gna-2(qa705) embryos expressing tubulin::GFP showed meiotic spindles that wax and wane with normal timing ( Figure 3A,C). Meiosis I+II was almost identical for wild type (29 min) and gna-2(qa705) (31 min). Furthermore, the time during which a meiosis II spindle could be visualized in wild type (8 min) and gna-2(qa705) (12 min) ( Figure 3A,B; bottom rows) is consistent with the timing for wild type reported by Liu et al [53], (13 min), but very different from the timing for cul-2(RNAi) (meiosis II spindle still present at 36 min), which has a perdurant meiosis II spindle.
As an independent measure of meiotic timing, we examined embryos expressing histone H2B::GFP ( Figure 3D,F). Results for wild-type meiotic timing (13 min for meiosis I, 25 min for meiosis II) were similar to those previously reported for wild type (15-17 min for meiosis I, 30-31 min for meiosis II) [52,53]. Furthermore, gna-2(qa705) had meiosis I and meiosis II timings similar to wild type. This is very different from results reported for cul-2(RNAi) or zyg-11(RNAi), in which meiosis I timing was normal, but meiosis II time was extended (60-75 min) [52,53]. Combined with the tubulin::GFP results, the histone H2B::GFP results support the conclusion that gna-2(qa705) does not have a perdurant meiosis II spindle. Using the histone H2B::GFP, we detected stray chromatin (distinct from the two masses segregated at anaphase) in 8% of gna-2(qa705) embryos at anaphase I, and 15% of gna-2(qa705) embryos at anaphase II, suggesting chromosome nondisjunction ( Figure 3E, red arrow, Table 1). Additionally, a polar body was not extruded following anaphase I, and all maternal DNA entered meiosis II, resulting in 12 sister chromatid pairs at prophase II (100% of embryos), rather than the normal number of 6 ( Figure  3D, 17 min time point, Table 1). A second polar body formed after anaphase II, but was not extruded (100% of embryos; Table 1). Whereas polar-body extrusion was defective, maternal and paternal pronuclei decondensed and met with normal timing ( Figure 3D, yellow asterisk, Figure 3F). The unextruded second polar body ( Figure 3D, red asterisk) often migrated to meet the pronuclei, although its migration usually lagged behind that of the maternal pronucleus.

gna-2(qa705) is a novel Pod gene
Live in utero recording also showed that the SPCC failed to associate closely with the cortex in gna-2(qa705) (69% of embryos) (Table 1, Figure 4). In wild-type embryos, SPCC-cortical association correlates with the initiation of polarization, which results in the anterior spread of a PAR-1,2-binding smooth posterior cortex that culminates in the pseudocleavage furrow. In addition to showing failed SPCC-cortical association, a pseudocleavage furrow was never observed in gna-2(qa705) (100% of embryos; Table  1, Figure 4). Furthermore, cortical polarity was defective in gna-2(qa705) embryos, as indicated by a failure to segregate PAR-3 to the anterior cortex ( Figure 5A), and cortical PAR-2 was reduced and mislocalized following GNA-2 depletion by gna-2 RNAi ( Figure 5A, Table 2; 150 mM KCl treatment). Cytoplasmic polarity was also defective, as posterior localization of P granules was not observed and PIE-1 was undetectable ( Figure 5A). Differential interference contrast (DIC) imaging (Leica Leitz DMRB) revealed that gna-2(qa705) embryos had some form of eggshell ( Figure 5B, arrows); however, the shell was deficient in chitin and other lectin-reactive glycoconjugates ( Figure 2F,G) and did not maintain a rigid oval shape in the uterus. gna-2(qa705) embryos were also osmotically sensitive, swelling in H2O and shrinking in 300 mM KCl, and were permeable to the lipophilic dye, FM 4-64, consistent with an osmotic/permeability barrier defect ( Figure 5B,C). Together with the polarity defects, this identifies gna-2(qa705) as a novel Pod mutant. It is possible that polarization defects in Pod mutants might be secondary to osmotic defects, as has been shown for the cytokinesis defect of cyk-3 mutants [54]. As such, the chitinous eggshell may be essential to provide an osmotically appropriate environment for A-P polarization. To determine if GNA-2-deficient embryos could polarize if provided with synthetic osmotic support, worms expressing PAR-2::GFP were subjected to gna-2 RNAi, and embryos were dissected and incubated in minimal embryonic growth medium (EGM) [55]. EGM provided little rescue of polarization ( Table 2), suggesting that polarization defects in GNA-2-deficient embryos may be dissociable from osmotic sensitivity. However, it is also possible gna-2(qa705) has normal meiotic timing, but fails to extrude polar bodies and has 12 sister chromatids at meiosis II that EGM did not provide adequate and/or timely osmotic support.

RNAi with chitin synthase-1 phenocopies gna-2(qa705)
The C. elegans genome encodes two chitin synthase (chs) homologues. chs-2 is expressed in the pharynx and head neurons of larvae and adults, and chs-1 is expressed in the hermaphrodite germline, in eggs and in dauer larvae [56,57]. Consistent with its expression during early development, chs-1 RNAi blocked eggshell chitin production and resulted in a highly penetrant embryonic lethality that resembled gna-2(qa705) ( Figure 6A,B). Embryos were fragile, osmotically sensitive and FM 4-64-permeable ( Figure 6C,D). Meiotic timing was normal ( Figure 3F), but embryos failed to extrude the polar bodies and had 12 sister chromatid pairs at meiosis II (100% of embryos; Table 1). Furthermore, stray chromatin detected at anaphase II (10% of embryos) suggested meiotic chromosome segregation defects (Table 1). Additionally, the SPCC failed to associate closely with the cortex (30% of embryos) and a pseudocleavage furrow was not detected (100% of embryos) (Table 1, Figure 4). PAR-3 and P granules were mislocalized and PIE-1 was undetectable ( Figure  6E), showing that polarization was defective. Moreover, PAR-2::GFP was undetectable or mislocalized, and incubation in minimal EGM did not rescue localization (Figure 6E, Table 2). The nearly identical phenotypes of gna-2(qa705) and chs-1(RNAi) support the conclusion that chitin deficiency is responsible for the gna-2(qa705) Mel phenotype. The Pod phenotype of hypomorphic alleles of genes encoding APC subunits might suggest that APC activity is required for chitin synthesis. However, while mat-2(ax76) embryos at the non-permissive temperature (25°C) were arrested at meiotic metaphase I, they still produced a chitinous eggshell, showing that chitin synthesis does not depend on APC activity ( Figure 6F). This result also indicates that the Pod phenotype of hypomorphic APC alleles does not result from chitin deficiency.
WGA-G and MBK-2 colocalize in the germline, and WGA-G was intact in mbk-2(pk1427) ( Figure 8B), a putative null allele, suggesting that WGA-G could be required upstream of MBK-2 localization. In that case, disruption of embryonic WGA-G by chs-1 RNAi might also prevent MBK-2 localization. Hermaphrodites expressing MBK-2::GFP were subjected to chs-1 RNAi, and germline and embryos were costained with a GFP antibody and WGA-TRITC. Cortical MBK-2 was undetectable in oocytes and in embryos following chs-1 RNAi ( Figure 8C). In contrast, neither MBK-2 nor WGA-G was mislocalized following cej-1+B0280.5(coRNAi) ( Figure 8D). Chitin was not detected in oocytes in the germline, using the highly specific chitin-binding probe, CBD-F ( Figure 8E), but chs-1 RNAi disrupted oocyte localization of MBK-2. The simplest explanation for these findings is that chs-1 RNA or CHS-1, rather than chitin itself, is required for MBK-2 localization. In this regard, chitin synthases [58] and the homologous vertebrate hyaluronan synthases [59], are unusual in their localization to the plasma membrane, rather than the endoplamic reticulum/Golgi apparatus. Saccharomyces cerevesiae chitin synthase-III is found in membranous organelles called chitosomes, and chitin synthase activation probably requires both enzyme phosphorylation and transport of chitosomes to the plasma membrane [58,60,61]. Therefore, one possibility is that MBK-2 may be localized to the oocyte cortex by virtue of its association with a CHS-1-containing compartment. Alternatively, chitin may be present in oocytes in the germline at a level that was too low to detect, or in a form that was masked by binding to a germline-expressed chitin-binding protein.

Discussion
The eggshell is required for actin-dependent events at meiosis Treatment of C. elegans embryos with latrunculin A, a drug that inhibits actin polymerization by sequestering Gactin, prevents polar-body extrusion and results in a phenotype of 12 sister chromatids at meiotic metaphase II [62]. Embryos deficient in PFN-1, an actin-binding protein, have a similar phenotype [62]. Here we demonstrate that in spite of normal meiotic timing, gna-2(qa705), chs-1(RNAi) or cej-1+B0280.5(coRNAi) embryos have a latrunculin A-like meiotic phenotype, with failed polarbody extrusion, and 12 sister chromatid pairs at meiosis II. Therefore, a critical role of the eggshell in meiosis appears to be support of actin-dependent polar-body extrusion.
C. elegans males arise at a frequency of about 0.5%, by non-disjunction of the X chromosome, and a phenotype showing a high incidence of males (Him) is a common finding in mutants with meiotic chromosome-segregation defects [63]. gna-2(qa705) rescued by extrachromosomal array was Him, and stray chromatin was detected at meiosis I and meiosis II, in some gna-2(qa705) embryos. Furthermore, chs-1(RNAi) or cej-1+B0280.5(coRNAi) embryos also had stray chromatin at meiosis. These findings indicate that the eggshell is required for faithful meiotic chromosome segregation. Meiotic spindles localized to the embryonic cortex in gna-2(qa705), chs-1(RNAi) or cej-1+B0280.5(coRNAi) embryos and meiosis occurred with normal timing, suggesting that gross meiotic spindle defects did not underlie the chromosome-segregation defects. However, it is possible that more subtle defects in spindle microtubule structure could explain the lagging chromosome phenotype. In starfish, and perhaps also in Xenopus and mouse [64][65][66], actin is essential for chromosome capture by the spindle during chromosome segregation. Therefore, the chromosome segregation defects identified in this study may also reflect a requirement for the eggshell in supporting actin-dependent chromosome capture.  -2(qa705), chs-1(RNAi) and  cej-1+B0280.5(coRNAi). Columns of images are a developmental series of individual wild-type, gna-2(qa705), chs-1(RNAi) or cej-1+B0280.5(coRNAi) embryos, from meiosis II metaphase to pronuclear meeting. In wild type, the sperm pronucleus (sp) touches the cortex (indicated by a red arrow) and a pseudocleavage furrow can be seen (indicated by white arrows) before and during pronuclear migration. In gna-2(qa705), chs-1(RNAi) or cej-1+B0280.5(coRNAi) a sperm pronucleus (sp) decondenses but fails to associate closely with the cortex (indicated by a red arrow), and a pseudocleavage furrow was not seen. Maternal (ma) DNA (11-12 sister chromatid pairs can be seen at meiosis II metaphase in gna-2(qa705) and chs-1(RNAi)). Images are of live embryos expressing histone H2B::GFP, recorded in utero.
FM 4-64 dye permeability is a reliable indicator of osmotic sensitivity in Pod mutants, and in wild-type embryos treated with a laser pulse to perforate the eggshell [5]. In wild-type embryos, the first polar body stained with FM 4-64, demonstrating that it is external to the barrier. Therefore, meiosis I and extrusion of the first polar body precede development of the osmotic/permeability barrier. Accordingly, the defects in meiosis I segregation and first polar-body extrusion in gna-2(qa705), chs-1(RNAi) and cej-1+B0280.5(coRNAi) are not likely to be secondary to osmotic defects. CEJ-1 and B0280.5 bind chitin [11]; furthermore, they are substituted with chondroitin chains and are predicted to be mucins, which are highly hydrophilic and hydrating glycoproteins [11]. Chondroitin proteoglycans are required in the C. elegans hermaphrodite to prevent collapse of the developing vulva [12]. Furthermore, they are essential for polar-body extrusion and for the separation of the embryonic plasma membrane from the eggshell [12]. Therefore, analogous to the role of chondroitin-proteoglycans in the developing vulva, CEJ-1/B0280.5 may support actomyosin-mediated cytokinesis of polar bodies by generating a hydrated matrix.

The eggshell is required for polarization, an actomyosindependent process
SPCC-cortical association failed in gna-2(qa705), chs-1(RNAi) and cej-1+B0280.5(coRNAi), and pseudocleavage furrow formation was not detected. Furthermore, in fixed embryos, cortical PAR-3 was not asymmetric, cortical PAR-2 was reduced and mislocalized, cytoplasmic P granules were not confined to the posterior, and cytoplasmic PIE-1 was undetectable. A perdurant meiotic spindle has been proposed as a possible explanation for reversal of GNA-2 deficiency results in a polarity-osmotic defective (Pod) phenotype polarization in cul-2(RNAi) [53]. However, in this study, we determined that meiotic spindle duration and meiotic timing were normal; therefore, the polarization defects associated with deficiency of eggshell chitin or CEJ-1/ B0280.5 cannot be a consequence of an extended meiosis II and/or a perdurant meiosis II spindle.
The importance of actomyosin contraction in polarization is underscored by the previous observation that loss of function of actin-associated proteins, such as POD-1, NMY-2, MLC-4 or PFN-1, prevents both cytoplasmic flow and polarization [5,36,67,68]. Our results demonstrate that actomyosin-dependent polarization also requires eggshell chitin and CEJ-1/B0280.5. While most gna-2(qa705), chs-1(RNAi) and cej-1+B0280.5(coRNAi) embryos showed failed SPCC-cortical association, in a few embryos the SPCC appeared to associate closely with the cortex, but pseudocleavage was not seen. These results suggest that in addition to being required for SPCC movement to or association with the cortex, the eggshell may also be required subsequently for the anterior-directed cortical-domain movement that follows SPCC-cortical association.

Pod mutants and the osmotic barrier
PAR mutants are polarity-defective, but osmotically insensitive. Therefore, polarization is not required for development of the osmotic barrier. In contrast, polarity is usually defective in osmotically sensitive mutants, and the Pod phenotype results from deficiency of a diverse group of proteins, including POD-1 (an actin-binding protein), APC subunits (MAT-1-3, EMB-27,30), enzymes required for fatty-acid synthesis (POD-2, an acetylCoA carboxylase, EMB-8, an NADPH-cytochrome P450 reductase, F32H2.5, a fatty acid synthase) [5,46,49], a ubiquitin Cterminal hydrolase required for actin-dependent processes (CYK-3) [54] and proteins required for eggshell synthesis (GNA-2, CHS-1, CEJ-1/B0280.5) (this study). These results indicate that polarization may require an osmotically appropriate environment, and that Pod-class polarity defects could be secondary to osmotic sensitivity. The results of our study, and those of Rappleye et al [49] for emb-8(hc69), emb-8(RNAi), pod-2(RNAi) or fatty-acid synthase (F32H2.5)(RNAi), showing that polarization was not rescued by incubation in isotonic medium, suggest that in some Pod mutants polarity defects may not simply be a consequence of osmotic defects. Intriguingly, Rappleye et al [49] also determined that pod-2(ye60) supplemented with long-chain fatty acids (C16-C22) were normally polarized when dissected in isotonic medium. Unfortunately, however, in that study it was not reported whether polarity was defective in embryos dissected in non-isotonic medium, probably reflecting the fact that osmotically sensitive embryos often burst (or crenate) without osmotic support. Therefore, while the results showed that a combination of lipid supplementation and osmotic support rescued polarization in pod-2(ye60), it is not clear whether the rescue was provided by the lipids or by the isotonic medium. Additionally, the osmotically sensitive mutants emb-8(t1462) and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (F08F8.2)(RNAi) were normally polarized when dissected in isotonic medium but no data were presented for embryos dissected in non-isotonic medium. As such, the question of whether or not polarization defects of Pod  mutants are secondary to osmotic sensitivity remains unresolved.
As has been suggested for the ascaroside layer in Ascaris species [6], a role of the osmotic barrier in the C. elegans embryo may be to prevent desiccation after the egg is laid. The molecule(s) responsible for this barrier has not been identified. It may include lipid molecules of the inner eggshell layer playing a role analogous to that of ascarosides. However, EM images of pod-1(ye11) showed that all three eggshell layers appeared intact, but embryos were osmotically sensitive and dye-permeable [5]. Therefore, it seems likely that the C. elegans permeability barrier resides inside the inner eggshell layer, as suggested by our results showing that the first polar body (putatively in the inner eggshell layer) stains with FM 4-64, while the second does not. A deficiency of HMG-CoA reductase causes osmotic sensitivity, suggesting that the C. elegans osmotic barrier requires cholesterol [49]. Furthermore, as long-chain fatty acids [palmitate, stearate, oleate, linoleate, arachidonate, docosohenaenoate or phosphatidylinositol (linoleic + palmitoleic)] were insufficient to rescue the osmotic sensitivity of pod-2(ye60) [49], the barrier may require shorter-chain fatty acids or other malonyl CoA-derived chs-1(RNAi) results in a gna-2(qa705)-like Pod phenotype molecules. In this study, we determined that chs-1(RNAi) or cej-1+B0280.5(coRNAi) embryos have defects in the osmotic/permeability barrier. These results suggest that the chitinous middle layer of the eggshell is required for synthesis/extrusion of the inner eggshell layer, which could be the osmotic barrier itself, or is required for subsequent synthesis/extrusion of molecule(s) comprising the osmotic barrier.

The eggshell may provide a "skeleton" to support actomyosin contraction in the one-cell embryo
The eggshell is required for multiple early developmental events, including meiosis, polar-body extrusion, osmotic barrier function and polarization. However, while the eggshell is physically associated with the embryonic plasma membrane at meiosis I, by polarization, additional layers separate the chitinous eggshell from the embryonic plasma membrane. This indicates that the embryonic plasma membrane is in contact with different extracellular molecules at different stages in the development of the one-cell embryo, which may suggest that the eggshell serves distinct functions at different stages. For example, during meiosis I, when it is closely associated with the plasma membrane, the eggshell might act as a physical scaffold for cortical molecules overlying the meiotic spindle. Subsequently, during polar-body extrusion, the eggshell might provide a fluid space into which the polar bodies can be extruded. Additionally, from second polarbody extrusion onwards, the eggshell could be required primarily to provide an osmotic barrier. Moreover, as long-chain fatty-acid supplementation suggests that membrane properties are crucial for polarization [49], the eggshell might be required during polarization for integrity of the plasma membrane. While these or other distinct, separate roles could underlie the requirement for the eggshell in multiple early developmental processes, a common feature of these events may explain, at least in part, the requirement for the eggshell. Specifically, as meiosis, polar-body extrusion and polarization all appear to be actomyosin-dependent, it seems likely that the eggshell supports actomyosin-dependent contractile events.
At both the cellular and the whole-animal level, some form of skeletal support usually facilitates contractile events. For example, in motile mammalian cells, dynamic adhesion to the extracellular matrix supports the pulling forces of the actomyosin network that move the cell forward [70]. At the whole-animal level, skeletal-muscle contraction is facilitated by a skeleton, which can be internal or external, and rigid or hydrostatic. For example, in vertebrates and "hard-shelled" arthropods, contraction requires muscle attachment to the bony skeleton or to a rigid exoskeleton, respectively. In animals that lack a rigid skeleton, a hydrostatic skeleton can provide a zone of incompressible fluid to transmit the force of muscle contraction (reviewed by Chapman [71]). By way of example, coelomic fluid in annelids is propelled forward by a wave of contraction of circular and longitudinal muscles, which results in a net movement of the worm in the direction of coelomic fluid flow [71,72]. Furthermore, while crustaceans normally depend on a rigid exoskeleton for body muscle contraction, a hydrostatic skeleton is used during the post-molt, soft-shell stage [73]. Hydrostatic skeletons can also be utilized within the context of a rigid 'con-tainer'. For example, fluid in the gut of the nematode Aphelenchoides acts as a hydrostatic skeleton that facilitates movement either of gut contents or of the whole animal, depending on the coordination of body-wall muscle contraction. Specifically, when localized regions of dorsal and ventral body muscles contract in phase, gut contents are moved; conversely, a wave of alternating dorsal and ventral contraction results in sinusoidal locomotion of the worm.
CEJ-1 and B0280.5 bind chitin, and it is possible that they also bind, directly or indirectly, to plasma-membrane molecules that link to the actomyosin cytoskeleton. In this way, the eggshell and/or molecules in the PVF could act as a dynamic adhesive matrix to anchor focal actomyosin contraction during meiosis, polar-body extrusion and polarization, analogous to the role played by the extracellular matrix in supporting focal adhesion in migrating cells or extending axonal growth cones. Conversely, or additionally, as CEJ-1 and B0280.5 are chondroitin-modified and have multiple mucin domains, their role may reflect an ability to generate a fluid-filled space.
In this case, transmission of actomyosin contractile forces in the one-cell C. elegans embryo may depend on the hydrated eggshell/PVF acting as a hydrostatic skeleton. This would be analogous to the situation in jumping spiders, where it has been proposed that a hydrostatic skeleton within a rigid exoskeleton allows application of a large amount of force to a small deformation, resulting in a forceful and quick movement [71]. In the case of polarbody extrusion, localized asymmetric force application may facilitate cytokinesis. Similarly, during the actomyosin-dependent ruffling stage that is coincident with movement of the large SPCC to the cortex, fluid translocated into the ingressing ruffles (and away from the nonruffling adjacent membrane) might be required to generate sufficient force to propel the SPCC to the cortex. Figure 9 presents a summary of how early developmental events may be dependent on GNA-2, CHS-1 and the eggshell: (A) following relief of GLD-1-mediated gna-2 translational repression in oocytes in the proximal germline, GNA-2 is required for synthesis of UDP-GlcNAc for (B) eggshell chitin synthesis by CHS-1. (C) Chitin binds the chondroitin-modified chitin-binding proteins, CEJ-1 and/or B0280.5, made available following relief of GLD-1 translational repression. CEJ-1 and/or B0280.5 bind water, resulting in the development of a hydrated zone, which is required for faithful chromosome segregation at meiosis (I and II). (D) The hydrated zone is also required for actomyosin-dependent extrusion of the first polar body and, perhaps, for secretion of the inner layer of the eggshell. (E) After first polar-body extrusion, the eggshell is needed for exocytosis/secretion of the molecule(s) of the osmotic/permeability barrier. There is no direct evidence that secretion of the barrier is actomyosin-dependent. However, deficiency of the actin-binding protein, POD-1, results in a Pod phenotype combined with exocytosis/endocytosis defects [5], and our study shows that the timing of barrier extrusion is temporally coincident with that reported for redistribution of cortical actin into foci [69]. These findings are consistent with the possibility that osmotic/permeability barrier extrusion is actomyosin-dependent. The eggshell is also required for subsequent actomyosin-dependent extrusion of the second polar body. F) Following pronuclear decondensation, the eggshell is necessary for the SPCC to associate closely with the cortex, which is associated with the initiation of polarization. G) The eggshell may also be required during the ensuing phase of actomyosin-dependent development of asymmetric cortical PAR domains

Methods
Standard methods were followed for molecular biology and nematode culture [63,74]. Unless otherwise stated, wild type refers to N2 worms fed OP50 bacteria.

Additional strains
The following additional strains were also constructed: XA781
To determine hatchling number, single L1/L2 larvae (P o ) were transferred to individual plates and allowed to lay eggs. Total hatchling number/hermaphrodite was determined by aspirating F1 larvae prior to adulthood. For experiments quantifying % Him, the P o was transferred to a new plate each day until no new hatchlings were observed, and the sex of the hatchlings on each progeny plate was determined at the L4 or adult stage. Results were analysed by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. Hatchling number was determined, rather than brood size or % of brood that hatched, because brood-size measurements would have been inaccurate for the following reasons: (i) embryos with a defective eggshell are fragile and sometimes break as they are laid; these embryos would have be excluded from the brood count; (ii) embryos with a defective eggshell are often laid as part of a large cohesive mass, which precludes accurate quantification of the number of embryos in the mass; and (iii) gna-2(qa705) and chs-1(RNAi) embryos are pale brown and non-refractile, making them easily confused with (unfertilized) oocytes.
To test biochemical rescue of the gna-2(qa705) Mel phenotype, young adult Unc-55 hermaphrodites were picked from gld-1(q485)/gna-2(qa705) unc55 (e1170)I plates. UDP-GlcNAc was dissolved in water (10, 250, 100 or 250 mM) and injected into the rachis of a single gonad arm as described previously [75]. The number of hatchlings from each injected hermaphrodite was quantified by transferring individual hatchlings to single plates. Hermaphrodites throwing any wild-type or Gld-1 progeny were excluded from further analysis.

HPLC
For HPLC analysis 1000 adult hermaphrodite wild type or gna-2(qa705) were collected in M9 buffer, washed three times in M9, incubated for 30 min at room temperature (RT), washed three times in H 2 O, resuspended in 50 μl H 2 O, frozen in dry ice/ethanol and stored at -80°C for no longer than 1 week. Samples were thawed rapidly, sonicated for 20 seconds, centrifuged, and the supernatant filtered and injected onto an anion exchange column (Zorbax SAX; Agilent Technologies, Chromatographic Specialities Inc., Brockville, ON). Detection was by ultraviolet absorption at 254 nm. UDP-GlcNAc, UDP GalNAc, UDP-Glc and UDP-Gal were quantified and UDP-GlcNAc and UDP-GalNAc were expressed as a percentage of UDP-Glc + UDP-Gal
To examine meiotic spindles, meiotic DNA segregation, polar-body extrusion, SPCC-cortical association, pseudocleavage furrow formation and pronuclear migration, wild-type or gna-2(qa705) hermaphrodites expressing tubulin::GFP or histone H2B::GFP were transferred to agarose pads, and embryos were examined in utero, using a Leica DMLSFA confocal microscope. The effect on meiotic DNA segregation, polar-body extrusion, SPCC-cortical association, pseudocleavage furrow formation and pronuclear migration of chs-1(RNAi) or cej-1+B0280. 5(coRNAi) was also determined in worms expressing histone H2B::GFP. For meiosis duration (determined with tubulin::GFP), timing was defined as follows: • Meiosis I: time from entry into the spermatheca until tubulin::GFP waned to a small spot (the tubulin::GFP sig-nal does not wane to undetectable before the meiosis II spindle waxes). Time from entry into the spermatheca was used instead of nuclear envelope breakdown (NEBD) because it is easily and objectively assessed without DIC optics, and because it excludes any oocytes that undergo NEBD but fail to be ovulated (and fertilized) in a timely manner.
• Meiosis II: time from the end of meiosis I (defined above) until the meiosis II spindle waned to a very pale structure.
• Meiosis I + II: time from entry into the spermatheca until the meiosis II spindle waned to a very pale structure.
As an additional way to determine meiotic timing, we also examined chromosome segregation using histone H2B::GFP. Timing was defined as follows: • Meiosis I: time from entry into the spermatheca until chromatin masses were separated (anaphase I).
The eggshell in development of the one-cell C. elegans embryo • Meiosis II: time from entry into the spermatheca until chromatin masses were separated a second time (anaphase II).
• Pronuclear meeting: time from entry into the spermatheca until maternal and paternal pronuclei met.
Failure of the SPCC to associate closely with the cortex was scored as any embryo in which the sperm pronucleus (visualized with histone H2B::GFP) failed to come within half of the sperm pronuclear diameter of the cortex. Results were analysed by t test (tubulin::GFP experiment) or one-way ANOVA followed by Tukey's multiple comparison test (histone H2B::GFP experiment). Lipophilic dye permeability was tested by DIC/UV microscopy of living embryos dissected in 150 mM KCl containing 30 μM

Authors' contributions
WLJ isolated and characterized gna-2(qa705), and performed microscopy and RNAi experiments. AK performed microinjections and conducted the suppressor screen. Plasmids and C. elegans strains were generated by WLJ and AK, and HPLC was conducted by JWD. WLJ, AK and JWD conceived of the study, and the manuscript was drafted by WLJ, with AK and JWD revising it critically. All authors read and approved the final manuscript.