A multi-layered and dynamic apical extracellular matrix shapes the vulva lumen in Caenorhabditis elegans

Jennifer D. Cohen, Alessandro P. Sparacio, Alexandra C. Belfi, Rachel Forman-Rubinsky, David H. Hall, Hannah M. Maul-Newby, Alison R. Frand, Meera V. Sundaram

Posted on: 6 May 2020 , updated on: 14 May 2020

Preprint posted on 15 April 2020

Article now published in eLife at http://dx.doi.org/10.7554/eLife.57874

Vulval lumen formation during morphogenesis depends on the effort of apical ECM factors that dynamically and temporally assemble in a cell-dependent fashion

Selected by Matus Lab

Categories: cell biology, developmental biology

Contributing writers: Maryam A. Azmi, Michael A. Q. Martinez, Taylor N. Medwig-Kinney, Nicholas J. Palmisano, and David Q. Matus

Background

Multicellular tubulogenesis is largely mediated by intraluminal forces, such as hydrostatic pressure, and/or apical extracellular matrix (aECM) factors that are secreted into the intraluminal space. The aECM is believed to act either like a reservoir for water molecules to promote the expansion of the lumen, or to assemble into specialized structures called fibrils that shape the lumen. Caenhorhabditis elegans is one of the few model organisms in which tubulogenesis can be studied in vivo with unprecedented resolution. In C. elegans, the intestine (Leung et al. 1999), excretory canal (Sundaram and Buechner 2016), and vulva (Estes et al. 2009) have all served as models for studying mechanisms of lumen formation. Cohen et al. vastly augment our understanding of vulval tubulogenesis by exploring a previously uncharted territory—the vulval luminal matrix—via confocal and transmission electron microscopy. This preprint reveals the spatiotemporal dynamics of the aECM within this luminal matrix and along the vulval tube, as well as the molecular factors that make up the aECM, including chondroitin proteoglycans and sheath matrix proteins.

Key Findings

Sheath matrix proteins assemble on apical surfaces in a vulval cell type-dependent manner

To understand the differences between the matrices produced between primary (1°; vulE and vulF) and secondary (2°; vulA, vulB1, vulB2, vulC, and vulD) vulval cell types, the authors analyzed changes in the localization of sheath matrix proteins in lin-12/Notch gain-of-function (lin-12(d)) and loss-of-function (lin-12(0)) mutants. lin-12(d) mutants have only 2° cell types, while lin-12(0) have only 1° cell types (Greenwald et al. 1983; Sternberg and Horvitz 1989). Both lin-12(0) and lin-12(d) mutants have misshapen vulval lumens, suggesting that each vulval cell type recruits essential chondroitin proteoglycans and sheath matrix proteins to their surfaces. The authors found that lin-12 mutants had changes in the localization of zona pellucida (ZP) domain proteins. For example, they found that LET-653(ZP), a shortened form of LET-653 containing only the ZP domain, bound to all apical surfaces in lin-12(0) mutants, but to none in lin-12(d) mutants. Conversely, they found that NOAH-1 was strongly absent from the apical membranes in lin-12(0) mutants but was strongly bound to the dorsal-most apical surfaces in lin-12(d) mutants. It was also found that both lin-12(0) and lin-12(d) lack core fibrils. Therefore, 1° and 2° cell types recruit different sheath matrix proteins to their apical surfaces, which attaches to the core fibrillar network needed for completion of vulval tubulogenesis.

The sheath matrix protein FBN-1 functions during vulval eversion

Cohen et al. introduced the importance of sheath matrix proteins in the later stages of vulval morphogenesis. The initial assessment of fibrillin-related protein, FBN-1, localization showed its loss from the lumen over 1° vulval cells during vulva eversion. Further observation of fbn-1 mutants demonstrated normal vulval morphogenesis until the mid-L4 stage. However, at later L4/adult molt stages, the mutants had improper vulva eversion where vulA and vulB1/B2 cells formed abnormal bulges. These findings indicate that FBN-1 functions predominantly in vulval eversion rather than inflation, thus confirming the diverse roles of chondroitin proteoglycans in vulval shaping.

Chondroitin proteoglycans and sheath matrix proteins exhibit both lumen expanding and constricting functions during vulval tubulogenesis

Through examination of double mutants for mig-22, which encodes a chondroitin sulfate synthase, and the sheath matrix factor, let-653, the authors show that the relationship between these proteins changes over time. Specifically, they find that initially, antagonism between the two proteins exists, with MIG-22 promoting lumen inflation and LET-653 restraining it. However, later in vulval development, they function cooperatively to restrict expansion of the dorsal-most region of the vulval lumen and to promote vulval eversion and cuticle formation. Taken together, these results demonstrate that the roles of aECM factors can be highly context- and time-dependent, allowing for regulation of the balance between expansion and constriction forces to properly shape the vulva.

Working model

Cohen et al. elegantly show that vulval tubulogenesis relies on dynamic cellular events that cooperate together in a spatial and temporal fashion. Vulval tubulogenesis initially depends on chondroitin proteoglycans, such as FBN-1, that form a granular matrix, which promotes lumen expansion (A). In a cell-type specific manner, sheath matrix proteins, such as LET-653, are then temporally assembled and disassembled on the apical surfaces of each mature vulval cell (B). Next, a core fibrillar structure within the center of the vulval lumen attaches to the sheath matrices, via ventrolateral fibrillar structures, generating the forces needed to shape the vulva (C). Lastly, the lumen narrows into a slit that is lined by cuticle (D).

Why we chose the preprint

This preprint is an excellent example of the strengths of C. elegans as a developmental model organism. Its invariant cell lineage, highly stereotyped progression of vulval morphogenesis, and visual amenability allowed for this beautiful study of aECMs during vulval lumen formation. The authors use a combination of endogenous translational reporters and confocal microscopy to visualize the dynamic expression and localization of aECM proteins, specifically comparing the expression patterns between two subtypes of vulval cells: 1˚ and 2˚ fated. Furthermore, using alternative tissue fixation methods paired with transmission electron microscopy (TEM), the authors reveal ultrastructural morphology of preserved aECM structure with unprecedented detail.

How this work moves the field forward

This study expands upon previous work showing the relationship between the pushing forces of chondroitin and constricting forces of actin-myosin by 2˚ vulval cells to expand the vulval lumen. The study introduces new strategies for quantifying vulval lumen dimensions, which can be further adapted to measure other lumen types, and uses high pressure freezing and freeze substitution methods to observe and analyze the ultrastructural features of the vulval luminal matrix. Importantly, this study visualizes the dynamic localization of aECM factors in a cell-type dependent fashion and shows how the expanding and constricting properties of these factors combine together to shape and mold the vulval into its mature form.

Overall, the work done in this preprint establishes the vulval lumen as a powerful model for understanding lumen formation and for studying the aECM using modern cell biological and genetic approaches paired with live-cell imaging. This work is also exciting in that it sets the stage for future studies that can further bridge the link between the gene regulatory networks (GRN) that control cell specification, such as those that involve lin-11 and lin-29 (Ririe et al. 2008), and the tissue remodeling events that lead to morphogenesis, which may include the expression of specific aECM modules—in some ways reminiscent of a “hox-code” for aECM within a defined lineage. It will be fascinating to see if lumen aECM is patterned using similar mechanisms in other tissues and taxa.

Questions for the authors

Do the vulB1 cells secrete multivesicular bodies into the interstitial space, and if so, what is so special about the vulB1 cells that they have this function?
Have the authors considered using transcriptional reporters to identify cell(s) of origin for potentially secreted molecules?
We are all super excited to see our favorite cell, the anchor cell, potentially generating extracellular vesicles (EVs) and we were wondering how early during uterine-vulval morphogenesis do EVs become detectable by TEM and what might they contain? Matrix metalloproteinases and other proteases to facilitate chemical degradation of the basement membrane and/or signaling molecules for interactions with adjacent uterine and vulval cells?

References

Cohen, J. D., Sparacio, A. P., Belfi, A. C., Forman-Rubinsky, R., Hall, D. H., Maul-Newby, H. M., Frand, A. R., and Sundaram, M. V. (2020). A multi-layered and dynamic apical extracellular matrix shapes the vulva lumen in Caenorhabditis elegans. bioRxiv. doi:10.1101/2020.04.14.041483.

Estes, K. A. and Hanna-Rose, W. (2009). The anchor cell initiates dorsal lumen formation during C. elegans vulval tubulogenesis. Dev Biol., 328(2), 297-304. doi:10.1016/j.ydbio.2009.01.034

Greenwald, I. S., Sternberg, P. W., and Horvitz, H. R. (1983). The lin-12 locus specifies cell fates in Caenorhabditis elegans. Cell, 34(2), 435-444. doi:10.1016/0092-8674(83)90377-X

Leung, B., Hermann, G. J. and Priess, J. R. (1999). Organogenesis of the Caenorhabditis elegans intestine. Dev Biol., 216(1), 114-134. doi:10.1006/dbio.1999.9471

Ririe, T. O., Fernandes, J. S., and Sternberg, P. W. (2008). The Caenorhabditis elegans vulva: a post-embryonic gene regulatory network controlling organogenesis. Proc. Natl. Acad. Sci. USA, 105(51), 20095-20099. doi:10.1073/pnas.0806377105

Sternberg, P. W., and Horvitz, H. R. (1989). The combined action of two intercellular signaling pathways specifies three cell fates during vulval induction in C. elegans. Cell, 58(4), 679-693. doi:10.1016/0092-8674(89)90103-7

Sundaram, M. V. and Buechner, M. (2016). The Caenorhabditis elegans Excretory System: A Model for Tubulogenesis, Cell Fate Specification, and Plasticity. Genetics, 203(1), 35- 63. doi:10.1534/genetics.116.189357

doi: https://doi.org/10.1242/prelights.20179

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Author's response

Meera Sundaram shared

David:

During your summary of uterine-vulval morphogenesis at the beginning of the results section, there’s a set of three papers about the cellular mechanisms that mediate basement membrane gap expansion that might be nice to include to fill in the gap between the vulval cue and toroid formation, as it is written now. Also, just as an aside, the primary VPCs don’t need to divide to release the cue, they just need to be specified as primary (if you arrest them they still make the cue). This work came from the Sherwood lab during and shortly after my postdoc in in the lab. In Ihara et al. 2011, they showed that the D cell holds onto the basement membrane in an integrin-dependent fashion, limiting the size of the expanding gap. In Matus et al. 2014, we showed that the D cell exits one round of division before all the other VPCs in an evolutionarily conserved fashion—it never divides in all rhabditid nematodes surveyed to date (>50 species!). In that paper, we also suggest that it is the terminal division of the E and F cells that helps open up the gap, if you prevent that terminal division, then they hold onto the BM. Finally, we showed that integrin is likely functioning through laminin to facilitate D cell-BM adhesion. After I left the Sherwood lab, they published a beautiful paper, McClatchey et al. 2016, where they tease out the uterine cell mechanism for controlling the BM gap, via Notch-dependent dystroglycan regulation.

Response: Yes, these are all great papers! We were trying to be succinct in the background but we will try to get a little more detail in there. I did notice that some integrin manipulations can give a phenotype not unlike the mig-22;let-653 double mutant appearance. There are certainly going to be both basement membrane-derived forces and apical matrix forces acting in concert to shape that dorsal lumen.

Michael:

What kind of ECM shapes the uterine lumen beginning at L4.4? (Figure 2) Response: Almost nothing is known about this. But the lumen blows up pretty fast and by TEM we can see a matrix in there that is probably responsible.
What do you believe explains the variable expression of ZP proteins FBN-1, LET-653, and NOAH-1 during the early stages of vulva morphogenesis? (Figure 3D) Response: I think it’s just a matter of expression being low as matrix incorporation is just beginning—it is hovering around the limits of our detection.
Why is the expression pattern different between LET-653 and LET-653(ZP)? (Figure 3D) What isoforms did they examine? Response: Our Gill et al. 2016 paper showed that the LET-653 PAN domains are responsible for binding the luminal core fibrils, and that PAN-mediated binding seems to compete with ZP-mediated apical membrane association. All 3 splice isoforms of LET-653 include both PAN and ZP domains—they differ in the size of the mucin-like linker between the ZPn and ZPc subdomains.
We are all super excited to see our favorite cell, the anchor cell, potentially generating extracellular vesicles (EVs) and we were wondering how early during uterine-vulval morphogenesis do EVs become detectable by TEM and what might they contain? Matrix metalloproteinases and other proteases to facilitate chemical degradation of the basement membrane and/or signaling molecules for interactions with adjacent uterine and vulval cells? (Figure 4F) Response: I agree the EVs are super interesting! We have not done TEM on younger specimens to know when they first appear, and we don’t yet have a way to see them by light microscopy. We can’t know for sure that they come from the AC, but they are only in that layer of matrix found right next to it.
Are these EVs or multivesicular bodies, as they are still contained within the plug? (Figure 5F). Response: I’d like to get more images of the plug at this stage, but based on what we have seen so far, there seems to be diverse types of things in there (more diverse than what we saw in the younger Figure 4 specimen).
There is no hymen in lin-12(0) and lin-12(d). What does this mean for the AC? (Figure 6). Response: The lin-12(d) mutants we used did not have an AC. But I’m not sure what is going on with the AC/hymen in lin-12(0) mutants. There are many things we would like to look at more carefully in these mutants, including what really happens during eversion to lead to the “blip” phenotype (Greenwald labspeak for the protruding vulva).

Taylor:

Have you considered using transcriptional reporters to identify cell(s) of origin for potentially secreted molecules? (Figure 6) Response: In Gill et al. 2016, we analyzed a let-653 transcriptional reporter and it was expressed in all L4 vulva cell types. In Forman-Rubinsky et al. 2017 we analyzed an lpr-3 transcriptional reporter and it seemed restricted to secondary cells. We haven’t looked carefully at the dynamics of those, or looked at the other genes yet, but definitely it would be nice to tie the signaling and transcription factors to matrix gene expression.

Nicholas:

Do the vulB1 cells secrete multivesicular bodies into the interstitial space, and if so, what do you think is so special about the vulB1 cells that they have this function? Response: The vulB1 cells are not the only ones that have MVBs—those MVBs are all over the place, even in the hypodermis. They seem to be especially abundant as worms approach the molt. Michel Labouesse’s lab has suggested that they are involved in apical secretion of hedgehog-like proteins, but they could also be involved in matrix degradation/recycling.

Maryam:

How was the plug determined to have an “AC-like” matrix? (Figure 5B and 5F) Response: The L4.4/5 “AC matrix” and the L4.8/9 “plug” are both electron-dense, fine-grained matrices located adjacent to the AC hymen and containing EVs. We don’t know the origin or contents of these matrices, so can’t really know if they are molecularly the same. Perhaps we should word this more cautiously.
Were there differences in Box 2 width between the WT, mig-22 mutants and mig-22;let-653 double mutants? (Figure 11B) Response: Yes, Box2 width is significantly smaller in mig-22 mutants compared to WT, and let-653;mig-22 have an intermediate Box2 width. All the measurement data are in Supplemental Figure 3, but maybe we should add these data to the main figure.

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A multi-layered and dynamic apical extracellular matrix shapes the vulva lumen in Caenorhabditis elegans

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