Live Imaging and Genetic Dissection of Basement Membrane Development and Repair
Lead Research Organisation:
King's College London
Department Name: Randall Div of Cell and Molecular Biophy
Abstract
The basement membrane, a thin layer of linked extracellular proteins (extracellular matrix), underlies nearly all epithelial cells in the human body. This specialised cellular "tarmac" is necessary for the function of its overlying cells, and an abnormal basement membrane plays a role in a number of pathologies. Despite this clinical relevance, we know little about how the basement membrane is formed. Furthermore, despite its certain damage during any type of tissue injury, we know nothing about how the basement membrane is capable of repair.
The basement membrane is composed of a number of different components, such as Collagen Type IV, which are linked by enzymatic reactions to yield a final stable structure. The production and organisation of these components is also thought to require a number of different cell-types. Due to this complexity, a complete understanding of the basement membrane requires its examination within a living organism, which until recently has been experimentally unfeasible.
In this proposal we will exploit our ability to live image basement membrane development and repair within a living organism. Fruit flies (Drosophila melanogaster) are becoming a widely utilised model system to understand the basement membrane as this animal has an assortment of extracellular matrix proteins identical to humans. Furthermore, preliminary data from our laboratory has revealed that basement membrane formation during embryogenesis can be imaged live during animal development. In this proposal we will exploit our ability to live image basement membrane development, along with our capacity in flies to knockout virtually any gene of interest, to fully dissect the mechanisms of basement membrane formation and repair.
In the first Objective we will characterise the timecourse of basement membrane formation by time-lapse microscopy. We will subsequently use strategies to specifically remove hypothesised basement membrane components and factors required for its formation within the various cells in the embryo thought to be involved in basement membrane development. This analysis will allow us to highlight the molecular mechanisms behind basement membrane formation and the relative requirement of different cells in its production.
In the subsequent Objective we will examine the basement membrane repair response. Preliminary data from the laboratory has revealed that the epithelium and underlying basement membrane can be specifically damaged in the fly by laser ablation; this leads to a healing response whereby over the course of a few hours the basement membrane hole is sealed. We will characterise this response and determine the cellular and molecular mechanisms involved in basement membrane repair. Our analysis suggests that basement membrane healing is an active cellular process that requires recruitment of fly macrophages (Drosophila inflammatory cells), and we will directly test the function of macrophages in repairing the damage. Furthermore, preliminary data reveals that the fly macrophages directly respond to damage to the basement membrane (rather than damage to the overlying epithelial cells), which we will directly test. This data suggests that a damaged basement membrane may be playing a significant role in inflammatory responses, which will have wide reaching clinical implications.
The basement membrane is composed of a number of different components, such as Collagen Type IV, which are linked by enzymatic reactions to yield a final stable structure. The production and organisation of these components is also thought to require a number of different cell-types. Due to this complexity, a complete understanding of the basement membrane requires its examination within a living organism, which until recently has been experimentally unfeasible.
In this proposal we will exploit our ability to live image basement membrane development and repair within a living organism. Fruit flies (Drosophila melanogaster) are becoming a widely utilised model system to understand the basement membrane as this animal has an assortment of extracellular matrix proteins identical to humans. Furthermore, preliminary data from our laboratory has revealed that basement membrane formation during embryogenesis can be imaged live during animal development. In this proposal we will exploit our ability to live image basement membrane development, along with our capacity in flies to knockout virtually any gene of interest, to fully dissect the mechanisms of basement membrane formation and repair.
In the first Objective we will characterise the timecourse of basement membrane formation by time-lapse microscopy. We will subsequently use strategies to specifically remove hypothesised basement membrane components and factors required for its formation within the various cells in the embryo thought to be involved in basement membrane development. This analysis will allow us to highlight the molecular mechanisms behind basement membrane formation and the relative requirement of different cells in its production.
In the subsequent Objective we will examine the basement membrane repair response. Preliminary data from the laboratory has revealed that the epithelium and underlying basement membrane can be specifically damaged in the fly by laser ablation; this leads to a healing response whereby over the course of a few hours the basement membrane hole is sealed. We will characterise this response and determine the cellular and molecular mechanisms involved in basement membrane repair. Our analysis suggests that basement membrane healing is an active cellular process that requires recruitment of fly macrophages (Drosophila inflammatory cells), and we will directly test the function of macrophages in repairing the damage. Furthermore, preliminary data reveals that the fly macrophages directly respond to damage to the basement membrane (rather than damage to the overlying epithelial cells), which we will directly test. This data suggests that a damaged basement membrane may be playing a significant role in inflammatory responses, which will have wide reaching clinical implications.
Technical Summary
Objective 1: Dissect the dynamics of basement membrane development and the mechanistic role of associated cell-types in its formation.
In Objective 1 we will characterise the dynamics of basement membrane (BM) deposition during Drosophila embryogenesis by time-lapse microscopy of fluorescently tagged BM components (we are currently examining Collagen IV-GFP, but will develop the capacity to examine other BM components). We will subsequently examine the role of various Drosophila cell-types (i.e. hemocytes and epithelial cells) in BM deposition by cell-type specific knockdown of components, exploiting the Gal4-UAS system and RNAi in flies. In collaboration with Dr. Franck Schnorrer, we will also develop and characterise additional fluorescently tagged BM matrix fly lines. This analysis will for the first time allow us to comprehensively dissect the mechanisms behind de novo formation of the basement membrane in vivo.
Objective 2: Examine the mechanisms of basement membrane repair following wounding and the relative role of associated cell-types in its re-formation.
In Objective 2 we will examine the mechanisms behind BM repair. We will induce damage to the Drosophila embryonic epithelium and underlying BM by laser ablation, and by timelapse imaging of fluorescently labelled Collagen IV (or other tagged BM components developed in Objective 1), characterise the dynamics of the repair response. Subsequently, we will functionally dissect the role of hemocytes and epithelial cells in healing the basement membrane by cell-type specific manipulation of these cells during repair using the Gal4-UAS system. As preliminary analysis suggests that Drosophila hemocytes (fly macrophages) are specifically responding to BM damage after laser ablation - rather than epithelial damage - we will also directly test this hypothesis by imaging hemocyte wound responses in the absence of a BM, and by inducing specific damage to the BM and subsequently imaging the hemocyte wound response.
In Objective 1 we will characterise the dynamics of basement membrane (BM) deposition during Drosophila embryogenesis by time-lapse microscopy of fluorescently tagged BM components (we are currently examining Collagen IV-GFP, but will develop the capacity to examine other BM components). We will subsequently examine the role of various Drosophila cell-types (i.e. hemocytes and epithelial cells) in BM deposition by cell-type specific knockdown of components, exploiting the Gal4-UAS system and RNAi in flies. In collaboration with Dr. Franck Schnorrer, we will also develop and characterise additional fluorescently tagged BM matrix fly lines. This analysis will for the first time allow us to comprehensively dissect the mechanisms behind de novo formation of the basement membrane in vivo.
Objective 2: Examine the mechanisms of basement membrane repair following wounding and the relative role of associated cell-types in its re-formation.
In Objective 2 we will examine the mechanisms behind BM repair. We will induce damage to the Drosophila embryonic epithelium and underlying BM by laser ablation, and by timelapse imaging of fluorescently labelled Collagen IV (or other tagged BM components developed in Objective 1), characterise the dynamics of the repair response. Subsequently, we will functionally dissect the role of hemocytes and epithelial cells in healing the basement membrane by cell-type specific manipulation of these cells during repair using the Gal4-UAS system. As preliminary analysis suggests that Drosophila hemocytes (fly macrophages) are specifically responding to BM damage after laser ablation - rather than epithelial damage - we will also directly test this hypothesis by imaging hemocyte wound responses in the absence of a BM, and by inducing specific damage to the BM and subsequently imaging the hemocyte wound response.
Planned Impact
The first beneficiaries will be academic in nature involving a wide range of UK and international scientists. The basement membrane is a critical component of all epithelial tissues in the human body and is discussed at length in every cell biology textbook. However, the scientific community knows surprisingly little about how the basement membrane forms or functions. Our work will be of relevance to scientists studying the biochemical makeup of the basement membrane as well as developmental biologists trying to understand basement membrane formation and function in developing animals. We also plan to disseminate our model system and novel reagents to these researchers to help them address their questions of interest within a physiologically relevant context. Indeed, the system that we are developing is one of the few that allows us to address questions surrounding the basement membrane in a living organism.
There is also likely to be more clinically oriented impact as basement membrane dysfunction leads to a number of pathologies. Indeed, there are a number of human diseases caused by basement membrane mutations. Once our system is fully developed and characterised, it will be possible for scientists to test the precise role of various human point mutations in basement membrane components in vivo within our system; this will be critical in helping these scientists understand the precise role of these mutations in human disease.
Additional clinical beneficiaries will be scientists studying tissue repair and regenerative medicine. The basement membrane will be damaged during any tissue insult and yet we have no idea regarding how it is repaired, nor do we understand the consequences of its damage. Furthermore, we hypothesise that a damaged basement membrane is playing a direct role in inflammatory responses and if this is indeed the case, our work will have wide reaching impact in helping to treat a range of inflammatory conditions. Identifying the precise basement membrane component that helps inflammatory cells to be recruited to sites of damage may give us additional clinical targets to modulate inflammatory reactions.
A final impact of this proposal concerns the goal of replacement, refinement, and reduction of animals in research. The scientific community primarily carries out wound healing studies in animals such as mice. The system that we have developed in fruit flies allows us to rapidly carry out wound healing experiments (in a non-'protected' animal) within an in vivo physiologically relevant setting. Despite this being an invertebrate system, the fruit fly has an identical makeup of basement membrane to humans, and our work can be directly extrapolated. However, mammalian models will eventually need to be utilised to understand basement membrane repair responses; knowledge from this proposal will directly help in reducing the number of animals required for these experiments by generating clearly testable hypotheses.
There is also likely to be more clinically oriented impact as basement membrane dysfunction leads to a number of pathologies. Indeed, there are a number of human diseases caused by basement membrane mutations. Once our system is fully developed and characterised, it will be possible for scientists to test the precise role of various human point mutations in basement membrane components in vivo within our system; this will be critical in helping these scientists understand the precise role of these mutations in human disease.
Additional clinical beneficiaries will be scientists studying tissue repair and regenerative medicine. The basement membrane will be damaged during any tissue insult and yet we have no idea regarding how it is repaired, nor do we understand the consequences of its damage. Furthermore, we hypothesise that a damaged basement membrane is playing a direct role in inflammatory responses and if this is indeed the case, our work will have wide reaching impact in helping to treat a range of inflammatory conditions. Identifying the precise basement membrane component that helps inflammatory cells to be recruited to sites of damage may give us additional clinical targets to modulate inflammatory reactions.
A final impact of this proposal concerns the goal of replacement, refinement, and reduction of animals in research. The scientific community primarily carries out wound healing studies in animals such as mice. The system that we have developed in fruit flies allows us to rapidly carry out wound healing experiments (in a non-'protected' animal) within an in vivo physiologically relevant setting. Despite this being an invertebrate system, the fruit fly has an identical makeup of basement membrane to humans, and our work can be directly extrapolated. However, mammalian models will eventually need to be utilised to understand basement membrane repair responses; knowledge from this proposal will directly help in reducing the number of animals required for these experiments by generating clearly testable hypotheses.
Organisations
People |
ORCID iD |
Brian Stramer (Principal Investigator) |
Publications
Díaz-De-La-Loza M
(2024)
The extracellular matrix in tissue morphogenesis: No longer a backseat driver
in Cells & Development
Gyoergy A
(2018)
Tools Allowing Independent Visualization and Genetic Manipulation of Drosophila melanogaster Macrophages and Surrounding Tissues.
in G3 (Bethesda, Md.)
José Alonso Solís-Lemus
(2019)
Macrosight: A Novel Framework to Analyze the Shape and Movement of Interacting Macrophages Using Matlab
in Journal of Imaging
Matsubayashi Y
(2017)
A Moving Source of Matrix Components Is Essential for De Novo Basement Membrane Formation
in Current Biology
Matsubayashi Y
(2020)
Rapid Homeostatic Turnover of Embryonic ECM during Tissue Morphogenesis.
in Developmental cell
Serna-Morales E
(2023)
Extracellular matrix assembly stress initiates Drosophila central nervous system morphogenesis.
in Developmental cell
Serna-Morales E
(2022)
Extracellular matrix assembly stress drives Drosophila central nervous system morphogenesis
Stramer B
(2017)
Mechanisms and in vivo functions of contact inhibition of locomotion.
in Nature reviews. Molecular cell biology
Stramer BM
(2015)
Cells on film - the past and future of cinemicroscopy.
in Journal of cell science
Description | We have been developing a model system to understand how organisms create and deposit extracellular matrix (ECM) components during animal development. ECM is essential extracellular scaffolds that are conserved among nearly all multicellular organisms, however we know virtually nothing about how cells regulate their production and construction during development. Using this system we have discovered that Drosophila macrophages are required to produce nearly all ECM components during development. Furthermore, their migration throughout the embryo is essential to evenly deposit components during development. More recently, we have shown that it is only a subset of BM components that require local delivery. For example, we have discovered that Collagen IV is insoluble during de novo formation of the ECM, while Laminin is freely diffusing. As a result, Collagen IV is totally reliant on macrophage migration for its even dispersal. When macrophage dispersal is effected, this leads specifically to uneven Collagen IV delivery, developmental defects, and embryonic lethality. Additionally, by exploiting the genetics of the Drosophila system we have developed novel approaches to examine the turnover of the extracellular matrix during development. Surprisingly, this work has reveled that extracellular matrix components, such as collagen, are rapidly turning over during embryogenesis with a half life of 7-10 hours. By analysing genes that control ECM turnover, we also revealed that constant degradation is essential for normal tissue development. |
Exploitation Route | The idea that macrophages, which are thought to be primarily immunological cells, may have other roles in animal physiology is novel. Indeed, a number of labs have recently discovered other functions for these cells during development and it is possible that mammalian macrophages may share similar functions to Drosophila macrophages in producing ECM components. The ECM is thought to be long lived and highly stable with a half life on the order of weeks to years. This work has revealed that the ECM is actually highly dynamic and undergoing constant homeostatic turnover during embryogenesis. This has led to other laboratories rethinking the dogma about ECM stability. |
Sectors | Healthcare Pharmaceuticals and Medical Biotechnology |
Description | We have engaged with the public with regards to how scientific imaging and movie generation contributes to scientific research. |
First Year Of Impact | 2015 |
Impact Types | Cultural Societal |
Description | Dynamic analysis of basement membrane during tissue morphogenesis |
Amount | $10,000 (USD) |
Organisation | Janelia Research Campus |
Sector | Public |
Country | United States |
Start | 03/2022 |
End | 04/2022 |
Description | Janelia Research Campus Advanced Imaging Center |
Amount | $10,000 (USD) |
Organisation | Howard Hughes Medical Institute |
Department | Janelia Research Campus |
Sector | Academic/University |
Country | United States |
Start | 03/2016 |
End | 03/2016 |
Title | Drosophila macrophage labeling tools |
Description | Novel Drosophila fly lines containing various ubiquitously labeled macrophages. Allows for the genetic manipulation of the fly while simultaneously imaging macrophage populations in the animal in vivo. |
Type Of Material | Model of mechanisms or symptoms - non-mammalian in vivo |
Year Produced | 2017 |
Provided To Others? | Yes |
Impact | There have already been publications showing the utility of these tools to the Drosophila macrophage community |
Description | History of time lapse microscopy in the biological laboratory |
Form Of Engagement Activity | A magazine, newsletter or online publication |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Public/other audiences |
Results and Impact | We discovered a number of original scientific films from well known scientists and had them digitised and archived at the Wellcome Trust. These films were some of the first time lapse movies generated in the biological laboratory and this discovery received significant interest from the general public. This led to a publication in a scientific journal of the history of timelapse cinematography in the biological laboratory, as well as an interview with Radio 4. |
Year(s) Of Engagement Activity | 2015 |
URL | http://blog.wellcomelibrary.org/2014/09/cells-on-film-making-movies-in-biology/ |