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

Abstract

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.

Fig 1. Timeline of adult alae formation.

(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.

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.

Fig 3. Formation of longitudinal AFBs and transient narrowing of seam syncytia precede the appearance of alae.

(A) Diagram depicting seam (magenta) and hyp7 syncytia in an L4 larva. Green, apical junctions between syncytia. Red box indicates region of interest (ROI) imaged below. The seam-specific promoter egl-18p was used to drive UTRNCH::GFP expression. (B) Representative confocal projections show UTRNCH::GFP (magenta) and AJM-1::mCHERRY (green) signals captured at the indicated stages (strain ARF404, 25°C). Magenta lines label AFBs. Arrows point to AJs between seam (s) cell cousins about to fuse. Chevrons point to spikes of F-actin crossing AJs. Images are representative of at least n = 6 animals per stage, except n = 4 young adults. (C) Quantification of apical width of seam syncytia at indicated stages (strain SU93, 20°C). Values are the distance between AJM-1::GFP marked AJs, representing the mean of 6 measurements per worm. Bars: mean and s.d. *P ≤ 0.05, **P ≤ 0.01; Mann-Whitney test. (D) Enlarged ROIs delineated by brackets in (B). Scale bars: 5 μm.

Fig 4. Longitudinal AFBs assemble in hyp7 as the seam narrows, whereas circumferential AFBs form parallel arrays as the seam widens.

(A) Diagram as in Fig 3A except hyp7 is magenta while seam is white. The hyp7-specific promoter dpy-7p was used to drive UTRNCH::dsRED expression. (B) Inverted confocal fluorescence micrographs show UTRNCH::dsRED detected at the indicated stages (strain ARF385, 25°C). L4.2: Arrow indicates F-actin at a presumptive seam-seam junction before fusion. Horizontal magenta lines label longitudinal AFBs along the lateral margins. Vertical magenta lines label circumferential AFBs (CFBs). Images are representative of n = 9 or more animals per stage. Scale bar, 10 μm. (C) Longitudinal AFBs in hyp7 overlap with AJs. (D) Quantitation of cortical CFB alignment in lateral (thick) region of hyp7. Values are averages of 6 ROIs per worm. ****P ≤ 0.0001, *P ≤ 0.01, Anova with Tukey’s correction for multiple comparisons.

Fig 5. Longitudinal AFBs and contours of seam syncytia depend on NM II.

(A) Endogenous NMY-2::GFP aligns with junctional AFBs at the seam-hyp7 margins and with the medial AFB in narrowed (L4.3-L4.4) seam syncytia. AFBs were marked by Pegl-18::UTRNCH::dsRED (strain ARF500, 20°C). (B) Endogenous NMY-1::GFP also faintly marks seam-hyp7 margins (strain ML2540, 20°C). Both A and B show single confocal slices representative of at least 6 animals per stage. (C) Representative confocal maximum projections show F-actin and AJs in seam syncytia of mid-L4 animals that expressed Pegl-18::UTRNCH::GFP and AJM-1::mCHERRY (strain ARF404, 25°C). Magenta lines label longitudinal AFBs. Arrows point to extensions of seam over hyp7. Asterisks label aggregated F-actin. Arrowheads point to fragmented medial AFBs. Scale bar: 5 μm. (D) Quantitation of seam width—values are the area surrounded by AJs normalized to imaged interval. Bars signify mean and sd; ****P ≤ 0.0001, ***P ≤ 0.001, ** P ≤ 0.01, ordinary Anova with Tukey’s test for multiple comparisons. Images and measurements from two independent trials; total sample sizes as indicated.(E) Quantitation of UTRNCH::GFP patterns. *P <0.01, ***P <0.0001, Fisher’s Exact test.

Fig 6. Provisional matrix components are required for patterning the alae.

(A) DIC images of alae in RNAi knockdowns or mutants of the indicated genotypes (Strains N2 and ARF251, 25°C; strains UP3184 and UP3452, 20°C). Arrows indicate disorganized regions. Bracket indicates a large gap where no alae are present. (B) Quantification of alae defects, as in Fig 2D. ****P<0.0001, Fisher’s exact test. ^Data reproduced from [20]. ^#Numbers here are an under-estimate of the true penetrance of alae defects because not all mosaics would have lost lpr-3 in the seam lineage (see Materials and Methods). (C) At L4.4 stage, seam width (visualized with AJM-1::GFP) is similar between WT (strain SU93) and let-653(cs178) mutants (strain UP3184), both at 20°C. (D) Measurements were performed as in Fig 3B, using either AJM-1::GFP or the actin sensor UTRNCH::GFP to assess seam margins. WT datapoints for AJM-1::GFP are re-used from Fig 3B. let-653 genetically interacted with the actin sensor to widen the seam. ns = not significant. *P<0.05, Mann-Whitney test. (E) At L4.4 stage, longitudinal AFBs (visualized with Pseam::UTRNCH::GFP) were present in both WT (strain ARF505) and let-653(cs178) mutants (strain UP4195), both at 20°C.

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.

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.

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.

Fig 10. The provisional matrix component LET-653 is required for patterned adhesion versus separation of matrix layers.

(A-C) TEM micrographs of let-653(cs178) (A,B, strain UP3342) or wild-type (C, N2) L4 specimens. Transverse cuts through the mid-body are shown. Seam cell is false-colored in blue. Scale bars: 200 nm. Boxed regions are shown at higher magnification in A’-C’. (A, A’) There is no clear distinction of adhesive vs. separated matrix regions in this mid-L4 let-653 specimen, despite the appearance of nascent alae ridges. (B, B’) Mis-shapen alae ridges have completely separated from the L4 cuticle of this late L4 let-653 specimen. Large vesicle structures (yellow arrows) also fill the seam cell, suggesting abnormal protein trafficking, We previously reported an accumulation of unusually large vesicles in vulF vulva cells of this same specimen [53]. (C, C’) Many lysosomes or related lamellar organelles (white arrows) appear within the seam cell in wild-type late L4 specimens (N = 4).

Fig 11. A cytoskeletal relay model for development of the adult alae.

Seam is blue, hyp7 peach. Cortical actin networks depicted in red, NM II in yellow, and AJs between seam and hyp7 in black. (A) Graphical representation of cortical actin networks across the larval-to-adult transition. (1) Epidermis prior to longitudinal AFB assembly; (2) Seam narrowing. Longitudinal AFBs (with associated NM II) and poorly aligned hyp7 CFBs are present. The most medial seam AFBs are closely spaced and often unresolvable; (3) Seam widening. Longitudinal AFBs and well-aligned hyp7 CFBs are present. NM II is concentrated at the outer (junctional) AFBs; (4) AFB disassembly and enlargement of transverse actin spikes. (B) Graphical representation of transverse cross-sections through the seam, hyp7, and overlying matrices during early (top), mid (middle), and late (bottom) stages of alae formation. (Early) Seam narrowing and deposition of provisional matrix layers (purples). (Mid) Matrix layer separation over AFBs and beginnings of matrix patterning. Both the seam and hyp7 syncytia secrete EVs. (Late) As alae take shape, distinct provisional matrix components (purples) and cuticle components (grey) accumulate within different subdomains. LPR-3 and collagens concentrate at ridges, LET-653 (and later FBN-1) at valleys, and NOAH-1 at both ridges and valleys.