A Window into Mammalian Basement Membrane Development: Insights from the mTurq2-Col4a1 Mouse Model
Posted on: 30 October 2023 , updated on: 3 November 2023
Preprint posted on 27 September 2023
Categories: cell biology, developmental biology, genetics
Background
Basement membranes are thin, specialized extracellular matrixes surrounding most tissues and underlying the basal side of epithelia (1). These protein networks provide cells a substrate to attach as well as important biophysical and biochemical information (2). During morphogenesis, basement membranes are remodeled to allow cells and tissues to grow and change shape (3). These basement membrane dynamics are poorly understood in mammals. This is mainly due to the lack of models in which the core basement membrane components can be endogenously labeled.
The mouse skin is a suitable tissue for studying cellular and tissue behaviors. Skin explants allow easy ex vivo culturing and the use of high-resolution microscopy. The periodically invaginating hair follicles can be used to analyze basement membrane dynamics in the context of the budding of a tissue. The epidermis consists of five different layers: stratum basale, stratum spinosum, stratum granulosum, stratum lucidum, and stratum corneum. The basement membrane underlies the basal epithelium layer and faces the dermis. It contains the major basement membrane component, Collagen IV. Collagen IV is expressed exclusively in the basement membrane and is encoded by six different genes in mammals (Collagen4A1-6).
The preprint discussed here offers an in-depth exploration of basement membrane development using a novel transgenic mouse model.
Key Findings:
A novel mTurq2-Col4a1 transgenic mouse line
In contrast to invertebrate genetic model organisms, there are only a few genetic mouse lines that enable visualization of basement membrane components. This is a gap that needs to be filled in order to understand the role of the basement membrane in mammalian development. The authors introduced the mTurq2-Col4a1 mouse model which allows real-time visualization of basement membrane development. Specifically, the authors used CRISPR/Cas9 to generate a fluorescent fusion protein of Col4a1, the ubiquitously expressed collagen IV subunit. Considering the brightness and chemical properties of the mTurquoise2 fluorophore that was used, this model offers a novel way to study the dynamic changes that occur during basement membrane formation.
Validation of mTurq2-Col4a1 functionality
mTurq2-Col4a1 transgenic mice are not homozygously viable. Nevertheless, the authors provide a series of experimental results they obtained confirming the proper localization and functionality of this protein: the mTurq2-Col4a1 protein is incorporated into the basement membrane in the correct spatial distribution, colocalizes with Collagen IV and Perlecan antibody staining, and heterozygous mice show the same basement membrane thickness as control animals in transmission electron microscopy (TEM) images. Taken together, the mTurq2-Col4a1 mouse model is a suitable tool for studying basement membrane dynamics.
Figure 1: (A) Schematic illustration with mTurquoise2-tagged Collagen4a1 and Collagen 4 heterotrimer. (B) Collagen4a1 locus with insertion site of mTurquoise2. (C-E’’) Fluorescence signal of mTurq2-Col4a1 colocalizes with anti-Collagen IV and anti-Perlecan antibody staining in the adult skin.
Basement membrane dynamics
To study the dynamics of the basement membrane during development at the highest possible resolution, the authors developed a planar-sagittal multiview imaging approach. Skin explants were folded over an agarose gel to image in XY and XZ planes at the same time. This allowed the authors to perform long-term live imaging of basement membrane dynamics during the morphogenesis of hair follicles.
Unequal Collagen IV density
Previous studies have shown perforated and thinned basement membranes during budding and branching morphogenesis (4,5). Surprisingly, in this study the mTurq2-Col4a1 intensity was higher at the actively growing base of developing hair follicles than in the neighboring region.
The basement membrane is highly pliable
When epidermal cells undergo cell division, they do not detach from the basement membrane. Moreover, the basement membrane becomes deformed by the dividing rounding cells, indicating a high pliability of the basement membrane during hair follicle development.
Figure 2: (B) Schematic illustration of planar-sagittal multiview imaging. (C) Long-term live imaging of developing hair follicles (cells in red, Col4a1 in cyan). (D, E) Protein density of Col4a1 is higher in the invaginating region of developing hair follicles than in the interfollicular epidermis. (F, G) Dividing cells stay attached and deform the basement membrane.
High stability of Collagen IV
Finally, the authors could show high stability of Collagen IV using a Fluorescence Recovery After Photobleaching (FRAP) approach.
Importance
This preprint is a significant contribution to the field of basement membrane biology. The novel mouse model and imaging techniques used in this study provide a new perspective on basement membrane development. A functional collagen IV fusion protein will likely advance the study of basement membranes in mice to the same extent that it did in Drosophila for invertebrates. This preprint yields surprising new results. The key findings that Collagen IV protein density is increased during hair follicle invagination, that the basement membrane is highly pliable and that cells maintain adhesion even during cell division are important new insights for the community.
Question for the authors
- mTurq2-Col4a1 mice are unviable when homozygous, suggesting that it is a hypomorphic allele. Do you expect defects in specific tissues that are more likely to require the mechanical properties of Collagen IV?
- As you pointed out in your manuscript, studies in Drosophila and C. elegans show rapid turnover with a half-life of about 7 hours (6,7). Your FRAP experiment includes data until 560 seconds after photobleaching, although you also show long-term live-imaging data in Figure 5 with a time span of 350 minutes. Would it be possible to analyze the recovery of Collagen IV over a longer period of time?
References
- Jayadev R, Sherwood DR. Basement membranes. Curr Biol. 2017 Mar 20;27(6):R207-R211. https://doi: 10.1016/j.cub.2017.02.006
- Yurchenco PD. Basement membranes: cell scaffoldings and signaling platforms. Cold Spring Harb Perspect Biol. 2011 Feb 1;3(2):a004911. https://doi: 10.1101/cshperspect.a004911
- Töpfer U. Basement membrane dynamics and mechanics in tissue morphogenesis. Biol Open. 2023 Aug 15;12(8):bio059980. https://doi: 10.1242/bio.059980
- Spurlin JW, Siedlik MJ, Nerger BA, Pang MF, Jayaraman S, Zhang R, Nelson CM. Mesenchymal proteases and tissue fluidity remodel the extracellular matrix during airway epithelial branching in the embryonic avian lung. Development. 2019 Aug 19;146(16):dev175257. https://doi: 10.1242/dev.175257
- Harunaga JS, Doyle AD, Yamada KM. Local and global dynamics of the basement membrane during branching morphogenesis require protease activity and actomyosin contractility. Dev Biol. 2014 Oct 15;394(2):197-205. https://doi: 10.1016/j.ydbio.2014.08.014
- Keeley DP, Hastie E, Jayadev R, Kelley LC, Chi Q, Payne SG, Jeger JL, Hoffman BD, Sherwood DR. Comprehensive Endogenous Tagging of Basement Membrane Components Reveals Dynamic Movement within the Matrix Scaffolding. Dev Cell. 2020 Jul 6;54(1):60-74.e7. https://doi: 10.1016/j.devcel.2020.05.022
- Matsubayashi Y, Sánchez-Sánchez BJ, Marcotti S, Serna-Morales E, Dragu A, Díaz-de-la-Loza MD, Vizcay-Barrena G, Fleck RA, Stramer BM. Rapid Homeostatic Turnover of Embryonic ECM during Tissue Morphogenesis. Dev Cell. 2020 Jul 6;54(1):33-42.e9. https://doi: 10.1016/j.devcel.2020.06.005
doi: https://doi.org/10.1242/prelights.35875
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