Collagen polarization provides a structural memory for the elongation of epithelial anlage

Hiroko Katsuno-Kambe, Jessica L. Teo, Robert J. Ju, James E. Hudson, Samantha J. Stehbens, Alpha S. Yap

Preprint posted on 28 February 2021 https://www.biorxiv.org/content/10.1101/2021.02.28.433274v1.full#disqus_thread

Article now published in eLife at http://dx.doi.org/10.7554/eLife.67915

A walk down memory lane - collagen I polarization provides a structural memory for tissue elongation by stimulating cell proliferation

Selected by Ilaria Di Meglio

Categories: biophysics, cell biology, developmental biology

Background

During development, simple epithelia undergo morphogenesis to generate complex 3D organs through deformations like folding, elongation and/or branching. Branching morphogenesis generates organs required for the exchange of gases, solutes and nutrients, such as the airways of the lung, kidney tubes, and salivary glands. Initially, epithelia simply grow until developmental signals instruct branching or bifurcations of the tissue to generate tubular organs. While the decision to branch may be regulated by developmental signals, tissue elongation can take hours or even days, timescales which are much longer than those of the underlying signaling pathways. But in addition to signaling pathways, morphogenesis is also driven by physical cues which may bridge the gap between these two timescales. The immediate microenvironment of these tissues, specifically the extracellular matrix (ECM) that surrounds them, has received growing interest in the field with respect to its role as a driver of morphogenesis. In fact, the ECM – composed primarily of proteins, water and polysaccharides – is not just a physical scaffold for tissues; it is a complex chemical and physical signaling platform required for normal tissue morphogenesis [1]. In this study, Katsuno-Kambe and colleagues investigate the specific role of the ECM component type I collagen, the dominant form of collagen during epithelial tubulogenesis, in driving mammary epithelial anlage elongation.

Key findings

Given the complexity and inaccessibility of in vivo model systems to study this link, the authors turn to 3D organotypic cultures of human MCF10A mammary epithelial cells as an in vitro model for mammary anlage elongation. They first seeded epithelial cells in Matrigel, a basement membrane-like hydrogel that promotes proliferation into multicellular aggregates but not symmetry breaking. Aggregates were then re-embedded in type I collagen, the major component in the stromal environment during mammary tubulogenesis, which promoted proliferation into elongated solid cords of non-polarized cells. Thus, collagen I matrix contributes to symmetry breaking of aggregates into cord-like structures.

Prior to investigating the specific role of the ECM, the authors assessed which cellular processes – migration, polarization or proliferation – contributed to epithelial elongation. While tracking of cell nuclei ruled out migration, proliferation was evidently required for anlage elongation. Indeed, staining for Ki-67, a marker for proliferating cells, revealed a significant difference in proliferation, with 41% Ki-67 positive cells in elongating and only 19% in non-elongating parts of the epithelium. In line with this, inhibition of proliferation with two cell cycle inhibitors, Mitomycin C and aphidicolin, inhibited elongation.

Next, aggregates were embedded in fluorescently labelled collagen to investigate the contribution of the ECM. Measurement of the coherency of fibrils (co-orientation in the same direction) showed that coherency increased with symmetry breaking and was markedly greater around elongating protrusions of aggregates compared to aggregates that remain spherical. Treatment with both Mitomycin C or aphidicolin reduced this coherency, as did inhibition of cell contractility, emphasizing the need for proliferation during elongation and for forces exerted by aggregates to reorganize their local ECM. In addition to coherency, the authors observed increased anisotropy of collagen fibrils around elongating regions. Thus, collagen anisotropy increases around elongating aggregates and polarizes in the direction of elongation.

Could collagen polarization provide the directional cue required for further aggregate elongation? To test this possibility, the authors exogenously stretched the collagen gel to polarize it independently of cell-generated forces. In these experiments, aggregate elongation was observed as a delayed response to external stretch, with preferential elongation along the axis of stretch applied. Importantly, when collagen polarization was reversed, the impact of the stretch on aggregates was disrupted, suggesting that the external stretch applied requires collagen polarization to promote elongation. To rule out the possibility that elongation resulted from cell-intrinsic mechanisms in response to stretch, aggregates were extracted from gels immediately after stretch and re-embedded in unstretched gels with randomly oriented collagen fibers. In this scenario, cells retained no memory of the stretch and aggregates elongated within the same timescale as in control (unstretched) conditions. Thus, collagen polarization, and not cell-intrinsic mechanisms activated by stretching, promotes aggregate elongation and orients the axis of symmetry-breaking. Importantly, measurement of Ki-67 staining in control unstretched gels, aggregates in stretched gels that were re-embedded, or aggregates in stretched gels that were allowed to float, confirmed that collagen polarization, rather than just transfer into collagen, stimulated cell proliferation which was then required for anlage elongation.

Finally, the authors assessed which signaling pathways mediated the proliferative response. Inhibition of β1-integrin, a collagen I receptor, and downstream ERK signaling during gel-stretching experiments blocked proliferation and reduced the proportion of aggregates that elongated, respectively. Thus, β1-integrin signaling and ERK1/2 signaling mediate the proliferative signal in response to collagen polarization, thereby promoting symmetry-breaking of the epithelial aggregates.

**Figure 1.** Model for collagen polarization as a structural memory for epithelial anlage elongation. The model illustrates how initially, (i) epithelia anlage exert isotropic force patterns on collagen fibrils, after which (ii) anisotropies in force associated with symmetry-breaking of the aggregate exert strain on the collagen fibrils which (iii) promote elongation of the anlage via increased proliferation. Adapted from Figure 7 (Katsuno-Kambe *et al*).

Why I liked this preprint

Overall, I really enjoyed reading this preprint because it presents a relatively simple set of experiments that unravel a rather complex link between the mechanics of the ECM and cell proliferation in driving mammary tissue morphogenesis. Another aspect that immediately captured my attention was the use of two scaffolds to grow and study anlage elongation, a Matrigel substrate followed by a type I collagen gel. While Matrigel has been the gold standard scaffold used to grow and study 3D culture models, it is a commercially available hydrogel that presents a number of disadvantages, one of these being that its composition is often ill-defined. Thus, the physical and chemical cues imposed by the ECM throughout morphogenesis may be masked by the inhomogeneity within and between Matrigel scaffolds. Instead, Katsuno-Kambe and colleagues transfer their culture into a collagen I gel in this study, which not only better recapitulates native ECM, it can be used to reliably unravel the specific contribution of the ECM for elongation of epithelial anlage.

Tags: collagen, elongation, epithelia, tubulogenesis

Posted on: 19 March 2021 , updated on: 26 March 2021

doi: https://doi.org/10.1242/prelights.27716

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Author's response

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1. To recapitulate symmetry breaking, the authors first embed cells within Matrigel and then re-embed them in a collagen I gel. What happens if cells are not embedded in Matrigel but instead immediately in collagen I matrix on day 1? In the physiological (in vivo) conditions, does the composition and/or amount of the ECM therefore change during mammary gland development?

We found that if isolated MCF10A cells are immediately embedded in collagen I gels, then they form elongated cord-like structures as they proliferate. In fact, it was the contrast between this phenotype and that seen when cells were grown in Matrigel de novo (where they eventually form soccer ball-like cysts) that prompted our experiments. We wondered if multicellular aggregates retained some morphogenetic plasticity, especially if isotropic aggregates could be persuaded to break symmetry and elongate.

Physiologically, mammary gland development has been studied extensively during puberty. There, the duct epithelium is covered by a thick basement membrane which contains mainly type IV collagen and laminin. In contrast, the basement membrane around the terminal end bud (TEB) is very thin. The TEB leads the process of ductal elongation into the mammary fat pad, which contains type 1 collagen. So, based on our findings, we’d be inclined to postulate that contact with collagen 1 in the fat pad facilitates cell proliferation for the elongation process.

2. In addition to assessing the specific role of collagen I on anlage elongation, have you assessed the importance of matrix compliance on this process? Would you expect a difference between stiff ECM (less compliant) versus softer, more compliant, ECM?

The question of compliance is important, but not one that we have explored experimentally. It has been reported that bundling and co-alignment of collagen fibers may increase their stiffness. So, we do speculate that increased stiffness might be a parameter of collagen polarization that is being “read” by the cells to stimulate proliferation. In the present experiments we used gels with a collagen concentration of 1 mg/ml. Aggregates could also elongate in gels of up to 3 mg/ml, which would be predicted to be stiffer. But changing collagen concentration can also affect pore size of the gel, making it a maneuver which is difficult to interpret.

3. The authors inhibit proliferation, a simple yet elegant way to show that it is required for anlage elongation, but they do not investigate the opposite scenario by inducing increased proliferation (or over-activating proteins like ERK or YAP) within their model system. Could this represent a way to assess and recapitulate certain disease conditions?

Driving cell proliferation. What really struck us was that the increase in cell proliferation was regional, being confined to the sections of the aggregate that underwent elongation. On reflection, this makes teleological sense, as increased proliferation throughout the initially-rounded aggregate would cause it to expand in all directions, rather than elongate. To test the opposite scenario that you propose, it would be necessary to increase proliferation regionally within the aggregates. Whether this is sufficient to induce elongation would be an interesting question (though one that might be better the subject of a study in itself). It is worth emphasizing that we also saw a tendency for cells divide along the axis of elongation. And, as a small point of clarification, we’d note that cells do move in the elongating region; but we did not see an increase in migration that would suggest that it was principally responsible for elongation. So, while our current data lead us to argue that regionally-increased proliferation is what mediates between polarized collagen and aggregate elongation, these other factors may collaborate.

4. Is the 4h stretching time and % of stretch from original length of the gel physiologically relevant? Do you expect a different effect on the collagen fibers and anlage elongation if the rate of stretching and speed are controlled?

The duration and degree of gel stretch was chosen empirically. Stretching the gel for up to 24 hours yielded the same degree of collagen polarization as 4 hours of stretching. Our current data suggest that the degree of stretch is not directly responsible for causing the aggregates to elongate; rather stretch is mediated via collagen polarization. We would be very interested in feedback from the community, especially if phenomena akin to collagen polarization have been seen as epithelial anlage elongate in vivo.

Thanks again!

Hiroko and Alpha

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Collagen polarization provides a structural memory for the elongation of epithelial anlage

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