The apical ECM of the excretory duct and pore is contiguous with that of the epidermis

The apical ECM and cytoskeleton differ significantly between different tube types in the excretory system. The excretory canal cell resembles the C. elegans gut in that it is not lined by cuticle, but has a specialized apical cytoskeleton containing the FERM domain protein ERM-1 (Gobel et al., 2004; Van Furden et al., 2004) (Fig. 1B,C). It also requires a set of specialized ‘exc’ gene products for its lumenal maintenance (Buechner et al., 1999; Buechner, 2002). By contrast, the duct and pore do not appear to express ERM-1 or most exc genes, but the mature duct and pore lumens are lined by a collagenous cuticle that is contiguous with that of the epidermis (Nelson and Riddle, 1984) (Fig. 1A-E). However, the bulk of cuticle secretion does not occur until late in embryogenesis (Costa et al., 1997; Johnstone and Barry, 1996), after the duct and pore have taken their mature shapes.

To examine the pre-cuticular duct and pore ECM, we analyzed existing transmission electron micrographs of 1.5-fold embryos (Fig. 1F-J). At this stage, the embryonic epidermis is lined by a thin apical ECM termed the ‘embryonic sheath’ (Priess and Hirsh, 1986), which later becomes an outer layer of the L1 cuticle (Costa et al., 1997). Outside the sheath, four additional ECM layers were visible that together constitute the eggshell and are secreted by the embryo soon after fertilization (Benenati et al., 2009; Rappleye et al., 1999). The innermost of these layers was a sac-like structure that encased the entire embryo; it was closely apposed to the sheath in most regions, but separated from the sheath at points where the embryo bends inward, including at the excretory pore opening (Fig. 1I). Within the nascent excretory pore and duct lumen, a very thin lining of gray material was visible that may correspond to a sheath-like ECM. The remainder of the duct and pore lumenal space, as well as the lumen of the canal cell, was filled with fibrous electron-dense material (Fig. 1I,J). This fibrous ECM material disappeared from the duct and pore by 6 hours later, at which time the cuticle lining of the duct and pore had been secreted (Fig. 1D,E). In summary, TEM analysis revealed the presence of two apical ECM layers within the duct and pore before cuticle secretion; both of these layers are morphologically distinct from the innermost layers of the epidermal ECM with which they are in contact.

let-4 and egg-6 encode transmembrane proteins with extracellular leucine-rich repeats

To identify genes important for excretory duct and pore development or maintenance, we searched for mutants with defects in these tubes. let-4(mn105) mutants previously were reported to have a rod-like lethal phenotype indicative of excretory system defects (Buechner et al., 1999; Meneely and Herman, 1979), making let-4 a candidate of interest. We isolated egg-6(cs67) in an ethyl methane sulfonate (EMS) mutagenesis screen for rod-like lethal mutants (see Materials and methods); a second allele, egg-6(ok1506), was obtained from the C. elegans gene knockout consortium (Moerman and Barstead, 2008).

let-4(mn105) is a recessive, loss-of-function mutation and caused highly, but not completely, penetrant lethality (Fig. 2A). The majority of mutants died as early L1 larvae with excretory defects. A smaller percentage of mutants died as embryos. Approximately 2% of mutants were ‘escapers’ that survived to adulthood and were fertile, but exhibited defects in locomotion and egg-laying. The progeny of these escaper homozygotes had the same rate of lethality as progeny from heterozygous mothers, indicating that there was no maternal effect on lethality (Fig. 2A).

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Fig. 2.

let-4 and egg-6 encode related transmembrane proteins with extracellular leucine-rich repeats. (A) Genetic analyses and transgenic rescue experiments. For transgene rescue experiments, progeny were collected from let-4(mn105) homozygotes carrying csEx173 (C44H4 cosmid +sur-5p::GFP) and a separately marked transgene of interest, or from egg-6(ok1506)/hT2[qIs48] heterozygotes carrying a marked transgene of interest. Rescue was determined by scoring each hatched worm for L1 lethality. For let-4; lin-48p::LET-4(+) rescue experiment, progeny were obtained from let-4 heterozygous mothers carrying lin-48p::LET-4(+). (B) let-4 and egg-6 genomic regions and rescue fragments. Note that paralog sym-1 is immediately adjacent to let-4. (C) Predicted protein structures and mutant lesions. The LET-4 and EGG-6 LRR domains share 49% similarity. GenBank accession numbers are JQ292907 (let-4) and JQ292908 (egg-6).

egg-6(cs67) and egg-6(ok1506) are also recessive loss-of-function mutations and both caused fully penetrant L1 lethality owing to excretory defects (Fig. 2A). Animals rescued for this zygotic lethality by an egg-6(+) transgene (see below) gave 100% dead embryos in the next generation, revealing a maternal egg-6 requirement. Embryos lacking maternal egg-6 arrested at the ∼40 cell stage and had fragile eggshells (data not shown).

We positionally cloned let-4 and found that it corresponded to the gene sym-5/C44H4.2, which encodes a predicted type I transmembrane protein with 14 extracellular leucine-rich repeat (LRR) domains and a short cytoplasmic tail (Fig. 2B,C). let-4(mn105) mutants had a C to T nucleotide change in the fourth exon of C44H4.2, introducing a stop codon into the 11th LRR. A 5.3 kb genomic fragment encompassing C44H4.2 and no other genes rescued mn105 lethality. RNAi against C44H4.2 also recapitulated some aspects of the let-4 phenotype (see below). Although C44H4.2 has been previously called sym-5 (synthetic lethal with mec-8) based on genetic interactions with the mec-8 splicing factor observed in RNAi experiments (Davies et al., 1999), the let-4 gene name pre-dates those studies. Therefore, we refer to C44H4.2 as LET-4.

We positionally cloned cs67 and found that it corresponded to the gene egg-6/K07A12.2, which encodes an LRR transmembrane protein related to LET-4 (Fig. 2B,C). egg-6 was independently identified and named based on its eggshell-defective RNAi phenotype (A. Singson, personal communication) (Carvalho et al., 2011; Sonnichsen et al., 2005). cs67 mutants had a C to T nucleotide change in the eighth exon of egg-6/K07A12.2, introducing a stop codon into the extracellular domain. cs67 failed to complement egg-6(ok1506), which deletes 1678 bp of the coding region, completely eliminating the LRR domain. A 10.5 kb genomic fragment encompassing K07A12.2 and no other genes rescued cs67 and ok1506 zygotic lethality.

LET-4 and EGG-6 belong to the large family of extracellular LRR (eLRR) proteins, which includes many proteins involved in cell adhesion, ECM interactions and signaling. LET-4 and EGG-6 specifically belong to the ‘eLRR only’ or ‘eLRRon’ subgroup (Dolan et al., 2007), because they contain no other recognizable domains. Mice have 52 eLRRon proteins, including LRRTM1-3, which are involved in synaptic junction formation or stabilization (Brose, 2009; de Wit et al., 2009; Ko et al., 2009; Linhoff et al., 2009; Siddiqui et al., 2010) and the small leucine-rich proteoglycans (SLRPs), which modulate collagen matrix assembly (Kalamajski and Oldberg, 2010). In addition to LET-4 and EGG-6, C. elegans has 15 other members of the eLRRon family, including SYM-1 (Davies et al., 1999). The LRR domain of LET-4 is more similar to that of SYM-1 (53%) than to that of EGG-6 (49%), but all three proteins cluster together within the C. elegans eLRRon family (Dolan et al., 2007).

LET-4::GFP and EGG-6::GFP localize to the apical (luminal) side of the duct, pore and other external epithelia

To visualize the localization of LET-4 and EGG-6, we generated fusion proteins by inserting GFP at the LET-4 or EGG-6 C-terminus within our genomic rescue fragments. Both the LET-4::GFP and EGG-6::GFP fusion proteins rescued lethality of the corresponding mutants, indicating that all required regulatory elements were included in the transgenes and that the tagged proteins were functional (Fig. 2A,B).

LET-4::GFP and EGG-6::GFP were expressed in a subset of epithelial cells, including epidermal, vulval and rectal cells and the excretory duct and pore (Fig. 3; supplementary material Fig. S1). EGG-6::GFP was also observed in some neurons (supplementary material Fig. S1). Expression began around the ventral enclosure stage of embryogenesis and continued through larval development, but then decreased in adulthood. Expression was absent from internal epithelia such as the gut and pharyngeal tubes (Fig. 3C-F). LET-4::GFP was transiently expressed in the excretory canal cell at the 1.5-fold stage (Fig. 3C), but no longer visible in this cell by hatch. Notably, with the exception of the canal cell, the epithelia that expressed LET-4 and EGG-6 were those that would eventually become cuticle-lined.

In almost all epithelia where they were expressed, LET-4::GFP and EGG-6::GFP appeared strongly apically enriched (Fig. 3; supplementary material Fig. S1). In the excretory duct and pore, LET-4::GFP and EGG-6::GFP lined the luminal membrane (Fig. 3A,B; supplementary material Fig. S1). In the epidermis, both fusions were distributed across the apical surfaces of most dorsal and ventral epidermal cells but were observed more weakly or variably in the lateral (seam) epidermis (Fig. 3G-H). Neither fusion was strongly enriched at apical junctions based on co-visualization with DLG-1/Discs Large::mcherry (Fig. 3B,D,F-H). Both fusions partially overlapped with but did not strongly colocalize with transepidermal intermediate filaments at hemidesmosomes (supplementary material Fig. S1 and data not shown). Both fusions were present in many large puncta, potentially representing a vesicular compartment trafficking to or from the membrane. In summary, LET-4 and EGG-6 topology and apical localization suggest a configuration in which the LRR domains extend into the apical ECM, but localization is not limited to known sites of epidermal-apical ECM attachments.

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Fig. 3.

LET-4::GFP and EGG-6::GFP localize to the apical domains of the excretory duct, pore and epidermal cells. (A-B″) In L1 larvae, LET-4::GFP and EGG-6::GFP localize apically within the excretory duct and pore. Symbols as in Fig. 1. (A) LET-4::GFP. (A′) lin-48p::mCherry labels the duct cell cytoplasm. (A″) Overlay. (B) EGG-6::GFP. (B′) DLG-1::mCherry marks apical junctions of the duct and pore cells. (B″) Overlay. (C-H″′) In 1.5-fold (C,E,G,H) and threefold (D,F) stage embryos, LET-4::GFP (C,D,G) and EGG-6::GFP (E,F,H) localize apically within the epidermis. Apical junctions are marked with DLG-1::mCherry. (C-F) Mid-plane. Note LET-4::GFP expression on the outer surface of the canal cell (asterisk in C). (G-H) Surface view. Scale bars: 5 μm.

Interestingly, the one exception to the apical localization of LET-4::GFP was the excretory canal cell. At the 1.5-fold stage, when LET-4::GFP was transiently expressed in the canal cell, LET-4::GFP localized uniformly around the plasma membrane and not to the developing internal lumen (Fig. 3C,C′). Although the significance of this expression is unclear, we speculate that the unique localization pattern reflects molecular differences in the apical domain of the canal cell versus the apical domains of other LET-4::GFP-expressing cells.

let-4 and egg-6 are required to maintain junction and lumen integrity in the excretory duct and pore

The majority of let-4(mn105) mutants and all egg-6(cs67) or egg-6(ok1506) mutants arrested as L1 larvae with excretory defects (Fig. 2A). The overall morphology and junctional pattern of the excretory system appeared initially normal in mutant threefold embryos, but became detectably abnormal shortly before hatching (Fig. 4; supplementary material Fig. S2). The first detectable abnormality was a swelling of the canal cell lumen in the region proximal to the canal-duct junction (Fig. 4C-E). Subsequently, the duct and pore cells separated from each other and the pore autocellular junction disappeared (Fig. 4F-H). Remnants of junction material sometimes remained at the separation points, suggesting junction breakage. The duct-canal junction and duct cell body remained intact, and the duct lumen often swelled considerably. Canal lumen swelling was a secondary consequence of defects in the duct and pore, as the excretory phenotype was rescued by lpr-1p-driven LET-4(+) or EGG-6(+) transgenes expressed specifically in the duct, pore and epidermal cells but not in the canal cell (Fig. 2A). Neither let-4 nor egg-6 was rescued by dpy-7p-driven transgenes expressed in the pore and epidermal cells or by lin-48p-driven transgenes expressed in the duct. Thus, let-4 and egg-6 are required in the excretory duct and pore, but not in the canal cell, and are required for both lumen and junction maintenance.

To confirm these interpretations for let-4 mutants, and to visualize the narrow lumen of the canal cell, duct cell and pore cell directly, we performed TEM of serial thin sections. We analyzed three wild-type and nine let-4 mutant embryos at the late threefold stage, surrounding the window when defects first become visible by light microscopy. In 5/9 let-4 threefold embryos, all three tube cells were still connected and the lumen was continuous, with no apparent distortions. Intercellular apical junctions appeared normal (Fig. 4J), as did the cuticular lining of the duct and pore (Fig. 4K). Because 97% of let-4 embryos eventually display excretory defects, we infer that these embryos would have displayed defects shortly thereafter, had they been allowed to mature. The absence of any detectable junction or luminal defect in these embryos indicates that initial steps of junction formation, lumen growth and cuticle secretion are fairly normal in let-4 mutants.

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Fig. 4.

let-4 and egg-6 are each required to maintain junction integrity between the excretory duct and pore. (A-H) Late threefold embryos (C,D) or early L1 larvae (A,F,G) expressing apical junction marker AJM-1::GFP. L1 larvae also express duct and pore cell marker dct-5p::mCherry. The arrowhead indicates the canal-duct junction; the arrow indicates the pore autojunction. The dotted line indicates the pharynx, which is also labeled by AJM-1::GFP. (A) Wild-type L1 larval excretory system. (B) Schematic interpretation of A. (C,D) Junction morphology initially appears normal in let-4(mn105) (C) and egg-6(ok1506) (D) mutants, but the canal cell lumen is beginning to swell (asterisk). (E) Schematic interpretation of C,D. (F,G) In older let-4 (F) and egg-6 (G) mutants, the duct and pore cells have separated (carets mark ends of cells) but contain junction remnants, and the pore autocellular junction has disappeared. Swelling of the canal cell lumen is more pronounced (asterisk). (F′,G′) Red channels from F and G, respectively. (H) Schematic interpretation of F,G. (I-M″) TEM images of the threefold excretory system. Duct cell is yellow; pore cell, blue; lu indicates lumen; cut, cuticle. (I,J) At threefold, 5/9 let-4 embryos still appeared normal at the connection between the duct and the pore cell (compare wild-type (I) to let-4 (J). The arrows indicate dark junctional material. (K) The duct cell lumen diameter also appeared normal (compare with wild-type, Fig. 1D). (L) In some let-4 embryos that lacked duct-pore connectivity, the duct lumen behind the break was swollen and the apical membrane had separated from the cuticle lining. (M-M″) Adjacent serial sections documenting duct lumen termination. A bolus of cuticle-like material is present near the termination point (M). Scale bars: 5 μm in A-H; 1 μm in I-M.

In 4/9 let-4 threefold embryos, the duct and pore appeared to have separated already, as we had also observed by confocal microscopy. In one of these embryos, the existing duct lumen and the canal lumen appeared normal. In another, the duct lumen appeared normal, but the canal lumen was greatly enlarged. In the remaining two embryos, the duct lumen diameter was enlarged proximal to the duct cell body, and the apical membrane in this region had separated from the cuticle lining (Fig. 4L). Because the cuticle ring had a normal diameter in these cases, we infer that lumen distortion occurred subsequent to cuticle secretion. In two embryos, we were able to trace the duct lumen to its premature termination within the duct process (Fig. 4M). We were unable to recognize the excretory pore cell in three of these embryos, suggesting that the pore lacked its characteristic autocellular junction and lumen. Our interpretation is that duct and pore separation leads to pore collapse and lumen retraction, and that duct and canal cell lumen swelling behind the break is a secondary consequence of excretory fluid backup. Thus the primary defect in let-4 mutants appears to be a failure to maintain the duct-pore intercellular junction.

Paralogs let-4 and sym-1 function redundantly to maintain epidermal junction integrity during embryonic elongation

A small proportion of let-4 mutant or let-4 RNAi embryos ruptured during elongation and failed to hatch (Fig. 2A, Fig. 5A). This phenotype reflected a semi-redundant role of let-4 and its closest paralog, sym-1. Like LET-4, SYM-1 also is expressed in epidermal cells, but unlike LET-4, SYM-1 lacks a transmembrane domain and is secreted into the apical ECM (Davies et al., 1999). Whereas essentially all sym-1 embryos developed normally, ∼100% of sym-1; let-4(RNAi) embryos ruptured (Fig. 5A). The rare embryos that did not rupture swelled abnormally as they approached hatching, suggesting a defect in osmotic integrity (Fig. 5B). Similar osmotic defects were seen in mec-8; let-4(RNAi) embryos (Fig. 5A), the basis for the alternative let-4 name ‘sym-5’ (Davies et al., 1999).

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Fig. 5.

let-4 and sym-1 are redundantly required to maintain junction integrity in the epidermis. (A) Quantification of embryonic lethality after let-4 or egg-6 RNAi. ^*Embryonic lethal phenotype varied. Most mec-8; let-4(RNAi) embryos swelled. egg-6(RNAi) embryos arrested at ∼40 cell stage. (B) Rare sym-1; let-4(RNAi) embryos that did not rupture swelled as they approached hatching. (C-O′) AJM-1::GFP (C-I) and HMR-1::GFP (J-O) apical junction markers appeared normal in sym-1 control embryos (C,D,J,K) and sym-1; let-4(RNAi) embryos (E,F,L,M) at the 1.5-fold stage (C,E,J,L) and at the threefold stage before rupture (D,F,K,M). (G,H,N) Most sym-1; let-4(RNAi) embryos began to extrude epidermal cells (asterisk) and rupture after elongating to the threefold stage. (I,O) Terminal arrest phenotype. Scale bars: 5 μm.

egg-6 zygotic mutants did not rupture, egg-6 RNAi did not show genetic interactions with sym-1 (Fig. 5A), and egg-6 apparently could not compensate for loss of let-4 and sym-1 despite being expressed in the epidermis (Fig. 3E,F,H). lpr-1p::LET-4(+) also failed to rescue egg-6 excretory defects (Fig. 2A), indicating that let-4 cannot compensate for loss of egg-6. Thus, whereas LET-4 and SYM-1 have some redundant requirements, LET-4 and EGG-6 have unique requirements.

Epidermal rupture can be caused by excessive actin-myosin contractile activity during embryonic elongation (Wissmann et al., 1999; Diogon et al., 2007; Gally et al., 2009) or by defects in structural components of the epidermal junctions (Costa et al., 1998; Totong et al., 2007; Lockwood et al., 2008). In sym-1; let-4(RNAi) embryos, AJM-1::GFP and HMR-1/cadherin::GFP localized normally before rupture (Fig. 5E,F,L,M). Junctions did not appear distorted or break during the early steps of elongation as in known mutants with increased contractile activity (Diogon et al., 2007). Instead, in temporal analyses, most embryos elongated to the threefold stage before rupturing (Fig. 5F,M). Rupture occurred focally and was preceded by local junction distortions and small apical bulges as individual seam cells left the plane of the epithelium (Fig. 5G,H,N). After rupture, remaining non-ruptured regions of the epidermis still had normal junction morphology. Thus, as for the excretory system, we found no evidence for defects in junction establishment. Rather, LET-4 and SYM-1 are required to prevent epidermal junction breaks during the latter part of embryogenesis. Notably, this is the time frame when cuticle secretion begins and epidermal-ECM interactions must be remodeled (Costa et al., 1997).

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Fig. 6.

The cytoplasmic and transmembrane domains of LET-4 are dispensable for function. (A-C″) L4 larvae expressing lpr-1p::LET-4 deletion constructs tagged with GFP. lin-48p::mCherry labels the entire cytoplasm of the duct cell. (A) LET-4::GFP localizes along the lumen of the duct cell. (B) signalseqGFP::LET-4(ΔLRR) is dispersed throughout the duct cell cytoplasm. (C) Similarly to A, LET-4(ΔC-term)::GFP localizes along the lumen of the duct cell. (D) let-4(mn105) rescue data. At least two independent transgenic lines were scored per construct, and non-transgenic siblings (gray) were scored as negative controls. low, injected at 2.5 ng/μl; high, injected at 5 ng/μl. LET-4(ΔLRR) contains LET-4 amino acids 398-773; LET-4(ΔC-term) 1-713; LET-4(ΔTM) 1-688. Scale bar: 5 μm.

The LET-4 transmembrane and cytoplasmic domains are dispensable for function

Some eLRRon proteins, including SYM-1, lack a transmembrane domain, and are secreted into the apical ECM (Davies et al., 1999). Furthermore, although EGG-6 has a predicted transmembrane domain, some proportion of EGG-6::GFP was still secreted, as it accumulated between the embryo and eggshell (supplementary material Fig. S1). To ask if LET-4 must be tethered to the membrane and to identify domains important for its function, we deleted the transmembrane (TM), cytoplasmic (Cterm) or extracellular LRR domains in the context of an lpr-1p::LET-4 transgene construct tagged with GFP to visualize localization within the excretory duct (Fig. 6). LET-4(ΔLRR) failed to rescue let-4 lethality; furthermore, the fusion protein was not apically enriched (Fig. 6B,D). By contrast, LET-4(ΔCterm) efficiently rescued let-4 lethality and appeared properly localized (Fig. 6C,D). LET-4(ΔTM) appeared toxic to embryos and we were able to obtain only a few transgenic lines with very low, undetectable levels of expression. LET-4(ΔTM) transgenes were apparently expressed, however, since they partially rescued let-4 larval lethality (Fig. 6D). We conclude that the LRR domains are required for proper LET-4 function and localization, whereas the cytoplasmic domain is dispensable, and that tethering of LET-4 to the membrane is not absolutely required for function.

let-4, egg-6 and sym-1 are required for proper apical ECM organization

The above studies suggested that LET-4, EGG-6 and SYM-1 all might function extracellularly as part of the apical ECM. Although the excretory duct and pore lumen ECM appeared morphologically indistinguishable from wild type in let-4 mutants (Fig. 4K, Fig. 7A), several abnormalities in the epidermal apical ECM were observed in eLRRon mutants. First, in 5/5 let-4 embryos at the 1.5-fold stage, TEM analysis revealed a large gap between the inner eggshell layer and the epidermal embryonic sheath layer (Fig. 7A), suggesting disorganization of one or both of these layers. Second, in most let-4 and egg-6 mutant threefold embryos, many globular structures accumulated between the embryo and the eggshell (Fig. 7B-D). These globules contained cytoplasm, as they were marked by GFP in transgenic embryos expressing cytoplasmic GFP reporters (Fig. 7B). In let-4 TEMs, these cytoplasts appeared membrane-bound and were positioned between the nascent cuticle and the inner eggshell layer (Fig. 7D), indicating that cell fragments had been shed before cuticle secretion; this is consistent with a defect in a protective function of the embryonic sheath. Third, most let-4, egg-6 and sym-1 mutant L1 larvae showed abnormal permeability to dye (Fig. 7E-J), indicating a defect in larval cuticle organization. A requirement for eLRRon proteins in apical ECM organization is further supported by the eggshell defects observed after depletion of maternal egg-6 (Carvalho et al., 2011; Sonnichsen et al., 2005) (A. Singson, personal communication). In summary, eLRRon single mutants show penetrant and early, but relatively mild, defects in apical ECM organization. We propose that eLRRon proteins function primarily to organize the apical ECM, and that defects in apical ECM lead secondarily to defects in epithelial junction maintenance.

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Fig. 7.

let-4, egg-6 and sym-1 are required for proper apical ECM organization. (A) (i) TEM image of 1.5-fold let-4 mutant, adjacent to the pore opening. Canal cell is colored red; duct cell, yellow; pore cell, blue. The distance between the inner eggshell layer (orange arrows) and the embryonic sheath layer (green arrows) was increased compared with wild type (Fig 1I). (ii) At 1.5-fold, let-4 lumen morphology and lumen ECM (line) were indistinguishable from wild type (compare with Fig. 1I,J). (B-C) DIC image of extra-embryonic cytoplasts in let-4(mn105) (B,B′) and egg-6(ok1506) threefold embryos (C). Cytoplasts are labeled with vha-1p::GFP in let-4 (B′). (D) TEM image of extra-embryonic cytoplasts in a let-4 threefold embryo. (E-I′) Hoescht dye permeability assay. DIC (E-I) and fluorescence (E′-I′) images of L1 larvae treated with Hoechst dye, 0.7 seconds exposure time. (J) Quantification of dye permeability. Scale bars: 5 μm.