Award details

Emergence of functional polarity in a tubular epithelium: a mechanistic study

Reference	BB/N001281/1
Principal Investigator / Supervisor	Dr Barry Denholm
Co-Investigators / Co-Supervisors
Institution	University of Edinburgh
Department	Centre for Discovery Brain Sciences
Funding type	Research
Value (£)	352,499
Status	Completed
Type	Research Grant
Start date	04/07/2016
End date	03/01/2020
Duration	42 months

Abstract

The epithelial tubes of our bodies, such as those of the lung, mammary gland and kidney (to give just a few examples) have a functional polarity written into their proximo-distal (P-D) axis, i.e. they have distinct segments of different cell-types carrying out specialised functions. The sequence of these segments is important and often underscores the emergent properties of the system (e.g. in the kidney, ability to produce and concentrate urine). Unlike the other main body axes, where rudimentary axes are inherited from the unfertilised egg, the P-D axis is established de novo within the developing organism. The specification of the P-D axis is an intriguing developmental problem, which is not well understood in most contexts. Using the simple epithelial tubule of the Drosophila renal system (the Malpighian tubule - MpT) we will determine the mechanism(s) that establish the P-D axis during development, and its relationship to how cell differentiation is patterned to bring about functional polarity in the organ. We conceptualise three alternative P-D axis-specifying mechanisms and investigate whether, either individually or in combination, they define the MpT P-D axis. These mechanisms are: (a) Radial patterning within the 2-D MpT primordium; (b) Asymmetric morphogenetic signalling; (c) Cellular birth order. In addition, we have identified several transcription factors (TFs) that are regionally expressed along the P-D axis (which appear to correlate to specific physiological segments). We hypothesise these TFs are the targets of the upstream developmental mechanisms, which in turn act to pattern the channels, transporters and receptors that ultimately execute the physiological and homeostatic activities of the tubule. Using genetic methods and physiological assays we will identify the developmental mechanisms and gene networks that map out the emergence of functional polarity along the P-D axis of MpTs.

Summary

Many of the tissues in our bodies are built up around complex arrays of cellular tubes, which permit the entry, exit, and transport of life-sustaining molecules such as oxygen, glucose, and water. But these tubes do more than simple convey materials to and fro - they also carry out vital roles in modifying the material passing through them. Functional differences along epithelial tubes are a prevalent feature in the organs in our bodies; the origin of these differences is underpinned by regional cell differentiation. The microscopic unit that makes up our kidney - the nephron - is a good example. One end is specialised to filter the blood, producing the primary urine. The tubular system that receives this urine has distinct segments with cells differing in their transport properties and permeabilities to water, salts and other compounds. It is these sequences of activity along the proximal-to-distal (P-D) axis of the nephron - a property we define as 'functional polarity' - that allows the kidney to produce and concentrate urine. The mechanisms that orchestrate the emergence of functional polarity are not well understood for most organs. This proposal focuses on how functional polarity is established along the P-D axis of the simple cellular tubule in the fruit fly (Drosophila) renal system. The insect renal system or Malpighian tubule (MpT) is the major organ for excretion, removal of wastes and toxins, and it controls the body's salt and water budget. MpTs are simple cellular tubes that contain a handful of different cell types, and a small number of cells overall (~120 in Drosophila). Yet this simplicity belies several distinct and vitally important activities. These include (but are not limited to) secretion of urine, reabsorption from the urine (to retrieve valuable materials, and to regulate fluid and salt) and control of the body's calcium levels. These different activities are carried out in spatially restricted segments of the tubule. Their order of execution underscores organ function (e.g. it is clearly important to produce urine before modification can take place). The simplicity of the MpT, its experimental tractability, and an ability to measure function, makes it an ideal subject to tackle the fundamental question outlined here. We will investigate three alternative mechanisms that might specify the P-D axis: (a) Overlapping chemical signals act to establish a 'dartboard-like pattern' of wedges and concentric rings of differential gene expression superimposed onto a flat sheet of cells. When the tubule telescopes-out from the sheet as a 3-D structure, the bullseye, mid ring- and outer ring-cells develop alternative identities based on these concentric rings of gene activity. (b) Asymmetric chemical signal(s) are issued from the base (P) and tip (D) of the developing tubule. Cells experience differing concentrations of the signal(s) depending on their position along the P-D axis, and use this to guide their differentiation. (c) Cell proliferation in the tubule is tightly controlled and occurs sequentially in distinct phases. This birth order acts to controls differentiation: with early-, mid- and late-born cells developing into alternative cell-types. We will also investigate an identified set of 'control genes', known as transcription factors (TFs), which have segment specific activities. We suggest these TFs are activated by the developmental mechanism(s) outlined above, and these in turn, activate genes encoding the channels and transport proteins that ultimately execute the activities of secretion, reabsorption and calcium handling. Using a variety of genetic methods to manipulate and mark different sets of cells, we will identify the networks and connections between genes that map out the emergence of functional polarity along the P-D axis of MpTs. We anticipate our work will lead to a better understanding of the development and function of the tubular organs of our own bodies.

Impact Summary

Although this project is a fundamental study and it is difficult to determine where impact might fall, we suggest three potential areas: (1) As the insect MpT is the fastest fluid-secreting epithelium known (Maddrell, 1991 Bioessays) it represents an ideal model to study the development of a transporting epithelium and to analyse transport functions. Our previous work on insect excretory tissues (e.g. Weavers et al., 2009 Nature; Denholm et al., 2013 Development) has revealed universal aspects of organ structure and function, which have contributed to our understanding of some of our own organs such as the kidney. We believe the work outlined here will reveal further insight into organ development and physiology that will be of wide relevance. For example, DACH (a vertebrate orthologue of dachshund - one of our chosen P-D axis patterning genes) is regionally expressed in the developing renal tubules of the mouse and human nephron, suggesting a role in P-D patterning, although its precise role has not been investigated (Ayres et al., 2001 Genomics; Kozmick et al., 1999 Dev. Genes Evol; Li et al., 2003 Nature). These data suggest similar mechanisms might govern P-D patterning generally across tubular epithelia. For these reasons, the genes identified as having important roles in the MpT could reveal genes with critical activities in our own organs, and therefore might have future impact in biomedicine by contributing to our understanding of tubulopathies. (2) Insects are the most successful and biodiverse group of animals on earth. They have (i) economic importance, as food or silk producers, as pests, biofoulers and invasive species, and (ii) biomedical importance, as disease and parasite vectors. The practical economic value of understanding insect physiology is very clear from the history of work on insect hormones, leading to hormone analogue insecticides, and more recent work, for example on insect olfaction leading to an understanding of insect repellent action. We cannot predict a direct impact of our work on applied insect biology, but we believe that this proposal addresses an area of insect biology that has considerable potential for practical application. The MpTs are major organs for excretion and osmoregulation and have been shown to be both a target tissue for insecticide action (Maddrell, 1971 Nature), and the primary tissue for insecticide detoxification in insects (Yang et al., 2007 Physiological Genomics). It follows that our understanding of MpT physiology is central to developing methods and strategies for control and manipulation of these species. Thus this work might provide an entry point for control/manipulation, e.g. the molecular mechanisms underpinning fluid-secretion or toxin transport may suggest 'druggable' targets to perturb or enhance MpT function. (3) Although members of the general public are unlikely to be immediate users of the research we plan to undertake, we recognise the importance of letting them know about the kind of research we do and why we do it (after all, it is the general public who ultimately pays for Research Council-funded research). Whilst the value of using simple models (like fruit flies) to understand complex biology is clear to many scientists, it is not immediately obvious to all. Through a series of outreach and public engagement activities we aim to convey important biological concepts and to break down preconceptions about using simple models, whilst at the same time striving to inform, entertain, and inspire.

Committee	Research Committee C (Genes, development and STEM approaches to biology)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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