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A transient apical extracellular matrix relays cytoskeletal patterns to shape permanent acellular ridges on the surface of adult C. elegans [1]

['Sophie S. Katz', 'Department Of Biological Chemistry', 'David Geffen School Of Medicine', 'University Of California', 'Los Angeles', 'California', 'United States Of America', 'Trevor J. Barker', 'Department Of Genetics', 'University Of Pennsylvania Perelman School Of Medicine']

Date: 2022-10

Epithelial cells secrete apical extracellular matrices to form protruding structures such as denticles, ridges, scales, or teeth. The mechanisms that shape these structures remain poorly understood. Here, we show how the actin cytoskeleton and a provisional matrix work together to sculpt acellular longitudinal alae ridges in the cuticle of adult C. elegans. Transient assembly of longitudinal actomyosin filaments in the underlying lateral epidermis accompanies deposition of the provisional matrix at the earliest stages of alae formation. Actin is required to pattern the provisional matrix into longitudinal bands that are initially offset from the pattern of longitudinal actin filaments. These bands appear ultrastructurally as alternating regions of adhesion and separation within laminated provisional matrix layers. The provisional matrix is required to establish these demarcated zones of adhesion and separation, which ultimately give rise to alae ridges and their intervening valleys, respectively. Provisional matrix proteins shape the alae ridges and valleys but are not present within the final structure. We propose a morphogenetic mechanism wherein cortical actin patterns are relayed to the laminated provisional matrix to set up distinct zones of matrix layer separation and accretion that shape a permanent and acellular matrix structure.

Animal surfaces are often decorated with intricately shaped structures such as denticles, ridges, or scales that are composed of extracellular matrix materials. We don’t understand how those extracellular materials get sculpted into appropriate shapes, but most models propose that cellular protrusions initiate the process. Here we investigated the formation of nematode adult alae, which are three racing stripe-like cuticle ridges that run along the left and right sides of the body. We found that alae development requires the actin cytoskeleton and a set of temporary matrix components, both of which organize into longitudinal stripes that presage the final structure. Using electron microscopy, we saw no evidence for cell membrane protrusion or folding that would explain a ridged matrix pattern. Instead, we observed that the first sign of alae formation is the appearance of four small separations between different layers of the temporary matrix. We propose that cytoskeletal patterns are relayed to the to the temporary matrix to trigger delamination and subsequent zonal differences in matrix accumulation that establish the alternating valley and ridge pattern of the alae.

Funding: This work was supported by the National Science Foundation (IOS1258218 to ARF, https://www.nsf.gov/ ), the National Institutes of Health (R01GM125959 and R35GM136315 to MVS, OD010943 to DHH, and training awards GM007185 to SK and T32 AR007465 to JDC, https://www.nih.gov/ ), and by a Dissertation Year Fellowship from the UCLA Graduate Division (to SK, https://grad.ucla.edu/ ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2022 Katz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Here, we examined how the actin cytoskeleton and the provisional matrix work together to sculpt the alae of adult C. elegans. Transient actomyosin-dependent narrowing of the seam surface accompanies deposition of the provisional matrix at early stages of alae formation, but it does not appear to buckle the apical membrane. Instead, longitudinal actin filament bundles (AFBs) at the seam cortex align with ultrastructural delaminations and future valleys that flank alae ridges. Actin is required to pattern the provisional matrix into longitudinal bands, and the provisional matrix is required to establish demarcated zones of matrix layer adhesion and separation which ultimately give rise to alae ridges and their intervening valleys, respectively. We propose a morphogenetic mechanism wherein cortical actin patterns are relayed to the laminated provisional matrix to set up distinct zones of matrix layer separation and accretion, thereby shaping a permanent and acellular matrix structure.

Recent work has shown that a ZP-rich provisional matrix precedes formation of each C. elegans cuticle [ 25 ]. In the embryo, the first cuticle is synthesized beneath a provisional matrix termed the embryonic sheath [ 23 , 26 , 27 ]. During the molt cycle, epidermal cells secrete a new provisional apical matrix beneath each pre-molt cuticle before synthesis of the post-molt cuticle ( Fig 1A ) [ 20 , 28 ]. Components of the provisional matrix are removed before or along with the pre-molt cuticle. This transient provisional matrix may help maintain tissue and body shape during the molting process. The provisional matrix also influences the organization of the subsequent cuticle and shapes the alae of the L1, dauer and adult stages, potentially by acting as a scaffold for further matrix deposition [ 18 – 20 , 28 ].

Actomyosin-dependent forces have been indirectly implicated in alae formation at the L1 and dauer stages, but the specific steps involved have not been characterized in detail. L1 cuticle formation occurs in the context of actomyosin-driven seam cell apical constriction, which elongates the embryo and narrows the entire body shape [ 23 , 24 ]. Similarly, dauer larvae are radially-constricted compared to the preceding larval stage [ 16 , 19 , 22 ]. Cuticulin mutants that lack alae at one or both of these stages also have wider seam cells and a dumpy body shape [ 13 ]. Based on these observations, Sapio et al (2005) proposed that cross-linking of stage-specific ZP proteins shrinks the innermost cuticle layer to narrow the lateral epidermis and buckle the outer cuticle layers above it, forming the dauer alae. However, this "cuticle buckling" model does not readily explain how alae form in the adult, where overall volume increases while the general body shape remains similar to that of the preceding L4 stage ( Fig 1A ).

(A) L4-to-Adult development. Top schematics show anatomy of the seam and hyp7 syncytia. Bottom schematics show apical matrices. The provisional matrix (purple) is secreted beneath the L4 cuticle (gray) prior to, or during, synthesis of the new adult cuticle (black). (B) Lateral view of the adult cuticle. Micrographs show adult alae as visualized by DiI staining and DIC. Magenta lines indicate dark bands corresponding to valleys in both the DiI and DIC images. Scale bar: 5 μm. (C) Transverse view of adult. Cartoon (top) shows how alae are positioned relative to the seam and hyp7 syncytia. Transmission electron micrograph (bottom) shows the three alae ridges (yellow arrowheads). The underlying seam syncytium is false-colored in blue. Scale bar: 500 nm. (D) When viewed by DIC imaging, alae on the developing adult cuticle gradually became visible underneath the L4 cuticle. Brackets indicate regions above seam syncytia where longitudinal ridges could be detected. N2 animals were grown at 20°C and staged based on vulva tube morphology [ 31 ]. Alae were detected in 0/24 L4.4, 3/9 L4.5, 11/13 L4.6, 24/26 L4.7, 15/15 L4.8, and 16/16 adults imaged. When detected at L4.5 or L4.6, the alae were very subtle, as shown. Scale bars: 5 μm.

The C. elegans body cuticle is a multi-layered aECM composed mainly of collagens [ 14 ]. C. elegans sheds and replaces its cuticle by molting to progress between each of its four larval stages and to enter adulthood [ 15 – 17 ]. The cuticle of each stage is unique in structure: Longitudinal acellular ridges or "alae" form above the lateral (seam) epidermis in L1s, dauer larvae, and adults, but not in the intervening L2, L3, or L4 stages ( Fig 1 ) [ 1 ]. Therefore, alae patterns must be generated de novo, rather than propagated from one life stage to the next across the molts. The number, size, and shape of alae ridges also varies among L1s, dauer larvae, and adults [ 1 ]. The basis of these stage-specific morphologies is unclear, except for the differential requirements for specific aECM proteins, including Zona Pellucida (ZP)-domain cuticulin proteins and various provisional matrix components [ 13 , 18 – 22 ].

In some cases, the shape of an aECM structure is molded at least in part by the shape of the underlying epithelium at the time of matrix deposition. For example, denticles and taenidial ridges on insect cuticles originate as actin-based cellular protrusions that subsequently become coated with aECM [ 2 , 11 , 12 ]. The cellular protrusions eventually withdraw, leaving the rigid aECM structures in place. Differences in matrix composition affect not only denticle or taenidia shape but also the apical domain architecture within the underlying cells, suggesting mechanical connections among the aECM, apical junctions, and the actin cytoskeleton. However, these mechanical links between aECM and the cytoskeleton have yet to be fully elucidated. Furthermore, some complex aECM structures such as nematode alae are not obviously associated with cellular protrusions [ 1 , 13 ], raising the question of how such acellular structures are shaped.

Apical extracellular matrices (aECMs) line all epithelial surfaces in contact with the environment. These aECMs vary in composition, but typically contain a mix of proteins, carbohydrates and lipids that are organized into recognizable layers. Some aECMs are soft and gel-like, but others form more rigid structures with characteristic shapes, such as the hook-like denticles or cuticle ridges of insects and nematodes [ 1 , 2 ] or the scales on butterfly wings [ 3 ]. Examples of aECM in humans include mucin- and proteoglycan-rich linings within many tube lumens [ 4 – 6 ]; the tectorial membrane, a flexible aECM sheet that relays sound waves within the inner ear [ 7 ]; hair, an amalgamation of keratinized cells and extracellular macromolecules [ 8 ]; and tooth enamel, a composite of calcium phosphate minerals [ 9 ]. Mutations that affect component matrix proteins cause various disease phenotypes [ 4 , 6 , 7 , 10 ]. Despite the widespread functional and medical significance of the aECM, the cellular and molecular mechanisms that sculpt apical matrices are not well understood.

Results

Morphogenesis of the adult-stage alae begins midway through the L4 stage The C. elegans epidermis consists of cells and multinucleate syncytia that together synthesize most of the external cuticle [17,29]. The lateral seam and adjacent hyp7 syncytium are the two largest tissues and are connected by apical-lateral junctions analogous to those found in vertebrates and insects [30] (Fig 1A). The seam cells undergo stem cell-like asymmetric divisions early in each larval stage: anterior daughters fuse with hyp7, while posterior daughters remain in place and reconnect. During L4, seam cells exit the cell-cycle, fuse into bilateral syncytia, and ultimately synthesize alae—three key events that mark the L4-to-adult transition [17]. Adult alae consist of three longitudinal ridges that decorate the cuticle overlying the seam [1] (Fig 1). Alae ridges stain prominently with the lipophilic fluorescent dye DiI [32] (Fig 1B). Alae also can be seen with Differential Interference Contrast (DIC) microscopy as alternating dark and light stripes, which we confirmed correspond to the three ridges and four flanking valleys, respectively (Fig 1B). As viewed by transmission electron microscopy (TEM), alae are acellular structures and protrude approximately 0.5 microns above the rest of the cuticle surface (Fig 1C). We were able to visualize the timing of adult alae formation using DIC microscopy of L4 animals staged based on developing vulva tube morphology (Fig 1D). Longitudinal stripes on the lateral surface first became visible at stages L4.5-L4.6. The stripes were initially subtle but gradually became more prominent at later stages. Thus, morphogenesis of the alae began well before the L4-to-adult molt and appeared to be a gradual rather than abrupt process.

Actomyosin filaments in both the seam and hyp7 shape the adult-stage alae To test the role of the actin cytoskeleton in forming alae, we used bacterial-mediated RNA-interference (RNAi) to silence actin genes in developing larvae and later examined the lateral surfaces of young adults by DIC microscopy and staining with DiI. Five C. elegans genes encode actin monomers. During the L4 stage, epidermal cells and syncytia express act-2 most highly, with some evidence for expression of act-1, -3 and -4 at other stages [33,34]. To simultaneously knock down act-1, -2, -3 and -4, we selected an act-2-derived dsRNA trigger complementary to all four transcripts (Materials and Methods). Further, we customized and applied an established experimental paradigm to selectively knock down actin in either the seam or hyp7. This system involved tissue-specific expression of RDE-1, a worm homolog of Argonaute, in rde-1(ne219) mutants otherwise insensitive to siRNAs [35]. This approach bypassed much of the embryonic and larval lethality associated with systemic actin(RNAi) over the full course of development. Waiting until the L2 stage to deliver actin dsRNAs (an approach we term "attenuated RNAi") also bypassed much of this lethality, allowing larvae to develop into small adults. Attenuated actin(RNAi) or preferential knockdown of actin in either the seam or hyp7 resulted in patches of disorganized adult-stage alae (Fig 2A and 2C and 2D). The most severely disorganized regions were widened and lacked continuous ridges entirely, instead showing a haphazard arrangement of short fragments. Other regions retained the outer dorsal and ventral ridges but showed breaks and misorientations within the central alae ridge, resulting in a “braid-like” appearance. Larger gaps in the alae (> 5 microns long) sometimes flanked the disorganized regions, and in these cases the underlying epidermis may have been disrupted due to earlier defects in seam cell reconnection, as previously described [36]. Because cell division and fusion were not of interest in this study, our approaches were designed to minimize such defects and our analysis prioritized extended regions of alae disorganization over any large gaps in the alae (Fig 2D, Materials and Methods). The abovementioned alae deformities were not observed in rde-1 null mutants fed actin dsRNAs or in wild-type animals fed short dsRNAs transcribed from the vector. The fact that knockdowns in either the seam or hyp7 caused similar deformities suggests that actin networks within both syncytia work cooperatively to shape the adult-stage alae. PPT PowerPoint slide

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TIFF original image Download: Fig 2. Knockdown of actin, NM II, or AJ components results in alae disorganization. (A) Tissue-specific and whole animal actin knockdowns (strains JK537, ARF281, ARF330, ARF408, N2, 25°C). Pseam is the promoter of egl-18 (previously known as elt-5) [37]. Phyp7 is the promoter of dpy-7 [38]. (B) NM II mutants and knockdowns (strains WM179, WM180, 25°C). For both A and B, representative DIC images show structures on the lateral surface of young adults of indicated genotypes. White brackets demarcate the region of interest. Yellow and magenta lines label presumptive ridges and adjacent valleys in normal alae. Arrows point to tortuous sub-structures; arrowheads, minor deformities. Asterisk labels large gap. Green dashed lines label severely disorganized regions with no remaining longitudinal pattern. (C) DiI staining of the cuticle in actin and NM II knockdowns, with symbols as above. (D) Prevalence of deformed alae. Disorganized refers to alae that are fragmented and/or contain mis-oriented or tortuous ridges (arrows, green dashed lines). Gaps are regions >5 microns wide that lack alae entirely (asterisks). Minor deformities include smaller breaks and divots (arrowheads). Values are weighted averages from two independent trials; N: sample size. *** P<0.001 for all pairwise comparisons of prevalence of seemingly normal alae in knockdowns and mock-treated specimens; Fisher’s exact test with Bonferroni’s correction for multiple comparisons. (E-F) AJ mutants or knockdowns (strains N2 and PE97, 25°C), and prevalence of deformed alae, as above. ***P < 0.001. Scale bars: 5 μm. https://doi.org/10.1371/journal.pgen.1010348.g002 Non-muscle myosin (NM II) often partners with actin to generate morphogenetic forces [39,40]. The nmy-1 and nmy-2 genes of C. elegans both encode heavy chains of NM II that are 47% identical in primary sequence to human NMHC-IIB and expressed in the epidermis [33,41]. We used nmy-1(RNAi) and conditional alleles of nmy-2 to partially inactivate NM II. Fragmented and disorganized alae were observed on most nmy-1(RNAi); nmy-2(ts) double mutants cultivated at restrictive temperature (Fig 2B–2D). In contrast, only minor deformities in the alae were observed in nmy-1(RNAi) or nmy-2(ts) single mutants, although nmy-2(ts) defects were greatly enhanced by expression of an F-actin biosensor and junction marker (see below) (Fig 2D). The combinatorial effect of nmy-1(RNAi) and nmy-2(ts) suggests that these paralogs make redundant contributions to a morphogenetic mechanism involving actomyosin-dependent forces.

Apical Junction (AJ) components that interact with actin networks shape the adult alae Actomyosin filaments often attach to cell membranes at cell-cell junctions [39,40]. To evaluate the role of AJs in patterning the adult alae, we similarly used RNAi and a hypomorphic allele to knock down key AJ components while larvae developed and then examined the lateral surface of young adults. HMP-1/α-catenin is the actin-binding component of cadherin-catenin complexes (CCCs) that mechanically link the various epidermal cells of C. elegans [30,42,43]. The Zonula Occludens (ZO) homolog ZOO-1 cooperatively recruits actin bundles to AJs [44]. Defective alae were observed on the surface of more than one third of surviving hmp-1(fe4) hypomorphic mutants, and these defects were further enhanced by simultaneous knockdown of zoo-1 (Fig 2E and 2F). Thus, genetic manipulations known to impede the transmission of mechanical forces through AJs interfered with morphogenesis of adult-stage alae.

Longitudinal AFBs in the seam presage the pattern of adult alae The seam-specific UTRNCH::GFP marker revealed that striking changes in actin appearance accompanied seam cell division, fusion, narrowing, and widening (Figs 3B and 3D and S1). As seam cells reconnected following their last round of cell division, cortical actin networks remodeled to orient longitudinally. At the onset of narrowing (~L4.3), four longitudinal AFBs assembled at the cortex of the seam syncytia; two of these AFBs co-localized with AJM-1::mCHERRY along the dorsal and ventral junctions with hyp7, while the other two AFBs were located more medially. At the narrowest seam stage (L4.3-L4.4), often only three longitudinal AFBs were detected, suggesting the medial AFBs had moved closer together and potentially joined. As the seam widened again (L4.5-L4.7), four longitudinal AFBs were again observed. Medial AFBs began to disassemble at the L4.7-L4.8 stages (Fig 3B), after the time that alae first become visible by DIC (Fig 1D). Breaks in the outer junctional AFBs also appeared at this time, and spikes of F-actin that apparently crossed the dorsal and ventral junctions became more prominent. This progressive transition from continuous longitudinal to discontinuous transverse F-actin structures along the margins might reflect a concurrent transition in the net direction of force propagation between the seam and hyp7. In summary, the transient narrowing of seam syncytia midway through L4 coincided with dynamic reorganization of the bulk of cortical F-actin into four longitudinal AFBs. This pattern is not what we would expect for a typical apical constriction process, where AFBs are usually either isotropic or aligned in the direction of tissue narrowing [49]. However, these distinctive arrangements are reminiscent of the adult alae pattern of three longitudinal ridges and four flanking valleys, which first manifest while these AFB patterns are still present.

Provisional matrices are patterned into longitudinal bands during seam widening To examine the timing and patterns of provisional matrix deposition, we visualized the lipocalin LPR-3 and the ZP-domain proteins LET-653, FBN-1, and NOAH-1 using functional translational fusions expressed from the endogenous loci or from extrachromosomal transgenes [27,53] (Materials and Methods) (Fig 7A). Provisional matrix proteins were visible over the seam syncytium during the L4.3-L4.4 stages, suggesting they are secreted prior to and/or during the period of seam narrowing (Fig 7B). The provisional matrix proteins initially appeared diffuse and unpatterned, but over the next few hours, each protein resolved into a characteristic pattern of longitudinal bands that correspond to developing alae ridges or their associated valleys and borders (Fig 7B–7D). LPR-3 and LET-653 bands appeared in mid-L4, before alae became clearly detectable by DIC. LPR-3 specifically marked three developing ridges, while LET-653 marked four valleys (Fig 7B). NOAH-1 bands resolved slightly later and marked both three ridges and four valleys in different z-planes (Fig 7B–7D). The distance between the NOAH-1 apical ridge signal and the subapical "valley" signal was 0.9 microns, which is greater than the height of the mature alae; from this we infer that subapical NOAH-1 may sit at the plasma membrane below the new adult cuticle. FBN-1 was the last factor to become patterned, very close to the L4-Adult molt, and marked only valleys (Fig 7B–7D). Each protein also showed a different timeline of disappearance, but all disappeared from the alae region in adults (Fig 7E). Therefore, these provisional matrix proteins are present during the period when alae are first being patterned and formed, but they are not permanent components of these ridge structures. PPT PowerPoint slide

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TIFF original image Download: Fig 7. Provisional matrix patterns presage the ridges and valleys of adult-stage alae. (A) Diagrams of provisional matrix components. (B) Representative confocal slices show the dynamic distributions of indicated fusions in animals from mid- to late-L4. LET-653::SfGFP (strain UP3746, 20°C). SfGFP::LPR-3 (strains UP3666 or UP3693, 20°C). NOAH-1::SfGFP (strain ARF503 25°C). FBN-1::mCHERRY (strain ARF379, 25°C). For NOAH-1::SfGFP, both apical and sub-apical confocal slices from the same animal are shown; 1/8 and 3/8 late L4 animals showed only the "ridge" or only the "valley" pattern, respectively, and 4/8 showed both patterns simultaneously. (C) Airyscan-processed images of late L4s, showing that apical NOAH-1 aligns with alae ridges and sub-apical FBN-1 aligns with valleys, as seen by DIC. (D) Schematic shows interpretation of apical patterns as alae ridges and sub-apical patterns as valleys. Correspondingly, on all micrographs, yellow lines indicate developing alae ridges and white lines indicate flanking valleys. (E) Provisional matrix factors disappear in adults. Confocal slices from same strains shown in A, at 20°C 24 hours after mid-L4 stage. All images are representative of at least n = 5 per marker per stage. Scale bars: 5 μm. https://doi.org/10.1371/journal.pgen.1010348.g007

Actin is required to pattern the provisional matrix into longitudinal bands To understand the relationships between the seam longitudinal AFBs and the overlying longitudinal bands of provisional matrix, we combined our seam actin sensors with different matrix fusions (Fig 8A–8C). In narrowed seams, when only one medial AFB could be resolved, SfGFP::LPR-3 was cleared from a thin band directly overlying that medial AFB (Fig 8A). A similar largely offset relationship was observed between the AFBs and the 3 apical bands of LPR-3 or NOAH-1 in later L4 animals (Fig 8A–8C). Together, these data suggest that AFBs underlie nascent valleys surrounding each developing ridge. PPT PowerPoint slide

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TIFF original image Download: Fig 8. Actin is required to pattern the provisional matrix. (A-C) Spatial relationships between seam AFBs and provisional matrix bands. Maximum intensity projections of animals expressing the designated actin sensor and matrix fusion. Brackets indicate seam region. Arrows point to medial AFBs. Images are representative of at least n = 6 per strain per stage. (A,B) LPR-3 apical bands are largely offset from AFBs (strains UP4127 and UP4170, 20°C). For A, only specimens where the UTRNCH::dsRed sensor detected medial AFBs in addition to junctional AFBs could be assessed. (C) NOAH-1::SfGFP apical bands are largely offset from AFBs (strain UP4114, 20°C). D) actin RNAi disrupts LPR-3 provisional matrix patterns. Standard methods for bacterially-induced actin RNAi were used, and surviving animals were imaged at the L4.5-L4.7 stage (strain UP3666, 20°C). Middle panel shows example of a patchy and faint pattern. Right panels show examples of disorganized patterns. (E) Quantitation of SfGFP::LPR-3 patterns after actin depletion. (F) actin RNAi disrupts NOAH-1 provisional matrix patterns. The attenuated actin RNAi protocol was used, and animals were imaged at the late L4 stage (Strain ARF503, 25°C). 5/8 specimens showed alae abnormalities that matched the aberrant NOAH-1 pattern, while 3/8 had normal alae and normal NOAH-1 bands. Scale bars: 5 μm. https://doi.org/10.1371/journal.pgen.1010348.g008 To test if AFBs might pattern the provisional matrix, we determined the effect of actin RNAi on provisional matrix patterns. Following actin knockdown, SfGFP::LPR-3 often appeared greatly disorganized or localized to misoriented or braid-like structures, rather than longitudinal bands (Fig 8D and 8E). Furthermore, the remaining SfGFP::LPR-3 bands frequently contained many small breaks and regions that were faint and ill-defined (Fig 8D and 8E). Similarly, in older larvae in which disorganized alae ridges were becoming detectable by DIC, NOAH-1::SfGFP marked the misoriented ridges (Fig 8F). Finally, in L3 larvae where cortical actin networks are more isotropic, both LPR-3 and NOAH-1 were present but remained diffuse and unpatterned over the seam (S1 Fig). We conclude that the L4-specific cortical actin networks are required to pattern the provisional matrix into continuous longitudinal bands to initiate alae formation, and that loss or mis-patterning of the provisional matrix can explain mis-patterning of the permanent alae ridges.

Ultrastructure of developing alae reveals alternating sites of separation and adhesion among apical matrix layers To characterize changes in the ultrastructure of the lateral epidermis and overlying apical matrices that happen while the alae take shape, we turned to transmission electron microscopy (TEM), using high pressure fixation to best preserve the fragile matrix [54,55]. We collected transverse sections through the mid-body of 10 distinct mid-L4 specimens, inspected the corresponding micrographs, and ordered the specimens by inference based on matrix appearance and comparisons to our DIC (Fig 1D) and confocal (Figs 3 and 4 and 7) image timelines. The micrographs shown in Fig 9 represent distinct steps in alae morphogenesis. PPT PowerPoint slide

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TIFF original image Download: Fig 9. Ultrastructure of developing alae reveals differential matrix separation vs. adhesion. (A-F) TEM micrographs of mid- to late-L4 wild-type (N2) specimens arranged by inferred age (total N = 10). Transverse cuts through the mid-body are shown. See Fig 1C for cartoon rendering of perspective. Seam cell is false-colored in blue. White arrowheads indicate adherens junctions between the seam and hyp7 syncytia. Scale bars: 200 nm. (A) ~L4.3-L4.4. The seam cell is highly constricted, with its narrowest point ~500nm in width. Black arrowheads indicate electron dense provisional matrix material on apical surfaces of both seam and hyp7 syncytia. (B) ~L4.4-L4.5. Magenta arrowheads indicate four regions of provisional matrix separation. Asterisk marks a vesicle in transit across seam membrane. (C) ~L4.4-L4.5. Yellow arrowheads indicate three regions of provisional matrix adhesion at nascent alae tips. Extracellular vesicles (asterisks) are present in the matrix over hyp7. C’) Regions of matrix separation are enlarged compared to panel C, which is another body region from the same specimen. Many extracellular vesicles (asterisks) and a larger membrane-bound structure (arrow) are present within the future adult cuticle. (D) ~L4.6-L4.7. Discernable alae ridges have formed and contain electron dense material at their tips. Matrix fibrils connect these ridges to the L4 cuticle, while additional L4-cuticle-attached matrix protrudes down into the intervening gaps. (E) ~L4.8. Maturing alae have grown in length and width, and valleys have narrowed. The central ridge still maintains a discernable connection to the L4 cuticle. https://doi.org/10.1371/journal.pgen.1010348.g009 In two inferred L4.3-L4.4 stage specimens (Fig 9A), the subapical region of the seam cell (at the adherens junctions) appears very narrow and pinched (0.5–1 micron wide), with hyp7 pushing in on both sides, consistent with apical constriction. Dark electron-dense extracellular material sits beneath the L4 cuticle, lining the apical plasma membrane of both the seam cell and hyp7; this material likely corresponds to the provisional matrix, which is deposited at this stage (Figs 7 and 8). The seam cell apical membrane and L4 cuticle remain flat in these specimens. However, in a third specimen that also appears to be ~L4.4, the seam cell apical surfaces are still flat, yet the L4 cuticle has seemingly buckled into three deep folds (S2 Fig). This latter animal has a large break in the L4 cuticle at the vulva lumen, which may have released mechanical constraints on the tissue and matrix. Alternatively, cuticle buckling could be a normal but transient response to initial seam constriction. In three specimens inferred to be just slightly older, about L4.4 to L4.5 stage (Fig 9B–9C’), the seam apical surface is narrow (0.9–1.1 microns) and the apical membrane and L4 cuticle are flat, but four discrete regions of separation appear between matrix layers over the seam. These separations define three intervening regions of remaining matrix adhesion, which we infer correspond to the future alae ridges. When comparing two images from different body regions in one of these specimens (Fig 9C–9C’), the separations are larger, and points of adhesion narrower, as ridges become more apparent. Dark electron-dense material lines the top and bottom surfaces of the separations, suggesting that both separations and adhesions occur between layers composed of the freshly deposited provisional matrix. Another feature of these three specimens is the presence of many membrane-bound vesicles or organelles near the seam and hyp7 apical membranes, including in the apparent extracellular space where new adult cuticle is forming (Fig 9B–9C’). The extracellular vesicles (EVs) range in size from ~15 nm (the typical size of exosomes [56]) to >600 nm (resembling migrasomes [57]). These EVs may contain materials for building or modifying the alae and cuticle. In the four oldest specimens, inferred to range from L4.6 to L4.8, the seam cell is wider (1.5–3 microns) and in three of the specimens it has sunk internally, below the level of hyp7 (Fig 9D and 9E). The seam cell contains many lamellar structures resembling lysosomes or lysosome-related organelles (LROs) (Fig 10C). The nascent adult cuticle underneath the L4 cuticle shows progressively larger alae ridges, with deeper and narrower valleys separating the ridges. Small points of connection remain between these adult alae ridges and the thinning L4 cuticle above. Much additional matrix material has accreted at the base of the L4 cuticle and protrudes downward in a pattern that is complementary to that of the adult alae. This material is presumably "valley-localized" provisional matrix (Fig 7C and 7D) that will be removed along with the old L4 cuticle at the molt. From these TEM data, we draw several key conclusions. First, many changes in matrix appearance occur immediately following the apex of seam narrowing. These changes are accompanied by the presence of vesicle populations, including EVs, that suggest active secretion by both the seam and hyp7 syncytia. Second, the seam apical membrane appears smooth throughout all stages of alae formation, with no evidence of upward protrusions or folds. However, the seam does sink internally as alae form, suggesting some downward force. Third, formation of alae involves differential separation vs. adhesion of matrix layers within the provisional matrix zone, with four regions of separation and three intervening regions of adhesion echoing the spacing of longitudinal AFBs within the widening seam and the banding patterns observed for provisional matrix proteins.

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