Live Imaging and Genetic Dissection of Basement Membrane Development and Repair

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.

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.

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.