Andrew Freddo | Dr Katherine D. Walton | Professor Deborah L. Gumucio – An Absorbing Tale of the Intestine Unfolds
Understanding the mechanisms behind the development of the small intestine will help aid discovery of new therapies targeting intestinal disorders. Andrew Freddo and his colleagues at the University of Michigan Medical School are working to understand these life-threatening disorders that currently have limited treatment options.
The Small Intestine’s Large Surface Area
The digestive system is composed of a set of organs that perform complex functions to ensure that the body absorbs all the essential nutrients from food while eliminating undigested components and waste products. Once food passes through the mouth, oesophagus and stomach, it enters the small intestine. This structure is a narrow, long tube of about 2 cm in diameter that is so named because it is half the diameter of the large intestine that follows it.
Seemingly contrary to its name, the small intestine actually represents the longest part of the digestive tract, extending to a length of over 20 feet in humans, a length that provides the necessary surface area for effective nutrient absorption. In addition to its length, the small intestine has two other mechanisms for maximising its absorptive surface.
First, the inside wall of the small intestine is convoluted – it is lined with finger-like projections called villi. There are around 3–7 million villi in the human small intestine, each measuring roughly 0.5–1.6 mm in length, that increase the surface area available for absorption of nutrients by 6-8–fold.
Second, the absorptive surface of the epithelium that makes up these villi has microscopic membrane protrusions called microvilli that form a brush like structure, or brush border, leading to an additional 10-fold increase in the intestinal surface area. Together, the length of the intestine, the multiple folds and the presence of villi and microvilli leads to an estimated total absorptive surface area of over 30 square meters (over 320 square feet) in the human adult, an area that ensures that essential nutrients are effectively absorbed by the body.
Significant loss of this enormous surface area can lead to malabsorption and intestinal failure, either through stunted growth in intestinal length during the development of the small intestine during embryogenesis, as in in congenital short bowel syndrome, or severe digestive disorders in adults, such as celiac disease, where many villi are lost. Understanding the mechanisms behind the development of small intestinal length, emergence of villi, and generation of microvilli will provide information needed to develop new therapies targeting these life-threatening disorders, which currently have limited treatment options.
‘Our lab is searching for a cellular and molecular understanding of how the small intestine develops. In particular, the process of villus formation, which occurs in foetal life, is essential to generate the enormous amount of surface area that is required for proper digestion of food and absorption of nutrients.’
The generation of the intestine in vertebrates is a stepwise process that begins early in embryonic development, with the formation of a single layer of epithelial cells that is moulded into a tube with a flat surface. This tube elongates rapidly and, at a specific stage in foetal life, the surface of the tube begins to convolute to form the villi. Concurrently, a thick lawn of microvilli is generated on the inner (or lumenal) surface of each epithelial cell.
Though the length and girth of villi can be modified after birth, providing some compensation in intestinal shortening, the number of villi present in an individual is thought to be largely established at the time of birth. Thus, to learn how villi are generated, it is necessary to study their emergence during foetal life.
Redefining the Structure of the Intestinal Epithelium
Professor Deborah Gumucio works in the departments of Cell and Developmental Biology and Internal Medicine at the University of Michigan. Her research focuses on understanding the cellular and molecular basis for intestinal development.
With graduate student Andrew Freddo and Dr Katherine Walton, the team is studying the key mechanisms that drive villus formation in mice. This complex process requires constant communication between the epithelial cells that line the intestinal lumen and a loose layer of underlying cells, collectively called the mesenchyme. The intestinal mesenchyme includes multiple cell types, including blood vessels, fibroblasts or connective tissue, nerves and muscle cells.
Freddo describes how, ‘our lab is searching for a cellular and molecular understanding of how the small intestine develops. In particular, the process of villus formation, which occurs in foetal life, is essential to generate the enormous amount of surface area that is required for proper digestion of food and absorption of nutrients.’
Epithelia throughout the body can be classified into multiple different types based on the shapes of the cells and the number of cell layers that comprise the epithelium. The different shapes include: squamous or flattened, cuboidal or square and columnar or tall, rectangular shaped cells.
Layers are described as simple, containing one layer, stratified, containing several layers, or pseudostratified. A pseudostratified layer gives the appearance of several layers because the cell nuclei, the cellular structure that houses genetic material, are staggered within the epithelial sheet but it is actually a single layer of cells. This is in contrast to other simple epithelia, in which all nuclei are neatly aligned.
It was previously thought that epithelial cells that line the small intestine early in foetal life, before the villi are formed, were multi-layered or stratified. With improved imaging techniques to track the shape of cells during development, Professor Gumucio’s laboratory redefined the classification of the intestinal epithelial cells as a pseudostratified structure, meaning that all cells have both an apical and basal surface.
The team also found that within the epithelial sheet, the cell nuclei move back and forth between the top, the lumenal or apical surface, and the bottom, the basal surface, near the basement membrane, of the epithelial cells.
This movement occurs as the cells divide and the staggered nature of the nuclei gives the false impression of multiple cell layers. Indeed, in pseudostratified cells, the nuclei at the bottom surface synthesise DNA, then travel to the top surface to undergo mitosis, where the cells divide into daughter cells, and then return to the basement surface to restart the cycle. This constant movement of nuclei is called interkinetic nuclear migration (INM), and the process is closely coupled to cell division and growth of the intestine.
Defining Villus Borders Utilising Mechanical Forces
Freddo is currently working in Professor Gumucio’s lab to investigate cellular dynamics during the emergence of villi in foetal life. During villus development, the pseudostratified epithelium described above becomes converted into a columnar epithelium and the flat lumenal surface of the pseudostratified epithelial tube becomes convoluted as the villi emerge.
Freddo has studied the role of cell to cell signalling events between the epithelial cells and the underlying mesenchymal layer during this process and has proposed that intraepithelial compression forces drive the formation of regularly patterned boundaries that separate the emerging villus domains.
Several important signalling pathways transmit signals to embryonic cells to direct them to differentiate properly. One of these, the ‘hedgehog’ pathway, is essential for the cellular communication between the epithelial and mesenchymal layers that drives the formation of villi.
Another member of Professor Gumucio’s team, Dr Katherine Walton, Assistant Research Professor in Cell and Developmental Biology at the University of Michigan, has published work describing how hedgehog signalling molecules, secreted by epithelial cells, lead to the formation of clusters of underlying mesenchymal cells.
These clusters form a patterned array, which spreads down the entire intestine in a proximal to distal wave over approximately 36 hours, a staggering rate that translates to generation of one cluster every 5 minutes. The clusters then signal back to the overlying epithelium, causing epithelial cells above them to change shape from pseudostratified, tall and thin, to columnar, shorter and fatter, a process that causes the epithelium to buckle, generating an emerging villus.
Freddo and his colleagues showed that the cluster-driven change in the shape of the epithelial cells that lie over each mesenchymal cluster generates compressive forces on the surrounding cells located between the clusters. Importantly, cells within these pressurised regions at this time are still pseudostratified and undergoing INM, with many actively dividing.
Normally, when a cell is dividing (marked in red in the above figure), it becomes spherical to sort its chromosomes and divide into two cells. In a pseudostratified epithelium, this normally happens adjacent to the lumenal surface. However, in regions that lie between the expanding epithelium above clusters, circumferential pressure within the epithelium causes dividing cells to rapidly dip beneath the apical surface (indicated with the arrows in the above figure). Since the apical portion of these dividing cells is still connected to the surrounding apical surface, this creates an invagination or fold in the apical membrane. These invaginations represent the boundaries between emerging villi.
‘Our goal is to apply what we learn to develop methods to make new surface area for patients born without a large portion of their small intestine or who have lost it to surgery.’
Based on these observations, the team collaborated with other investigators with expertise in mathematical modelling, graduate student Suzanne Shoffner and Drs Santiago Schnell, Shiva Rudraraju and Krishna Karikapati, to develop a computational model that faithfully recapitulates these events.
The model revealed that three key events are required for the formation of the invaginations, including: a) pressure from the expansion of the clusters; b) changes in the stiffness of the epithelial apical surface due to cell rounding; and c) the vertical displacement of the rounded mitotic cell. Together, the work demonstrates how signalling molecules, such as hedgehog, can cause epithelial cell shape changes that then produce mechanical forces which generate villus boundaries.
More information can be gained from the study of systems in which villus development is perturbed. Ezrin, an important protein found on the cell’s outer surface, is known to be involved in proper villi formation, because Ezrin-deficient mice exhibit villi that are fused together, as if their boundaries are not properly formed.
Freddo’s preliminary data indicate that the lack of Ezrin also leads to perturbation of epithelial cell shape as well as formation of an abnormally stratified epithelium instead of the pseudostratified epithelium normally observed during development. Additional studies of Ezrin-deficient intestines have the potential to reveal more information about the complex process of villus formation.
Towards Bioengineered Intestines
Freddo hopes to one day identify target molecules or strategies that can help engineer intestines for patients. He says that, ‘our goal is to apply what we learn to develop methods to make new surface area for patients born without a large portion of their small intestine or who have lost it to surgery.’
Bioengineered intestines could also be used as efficient in vitro models for studying basic intestinal cell biology. Several groups are working toward this goal using three main approaches. A slurry of adult whole intestines, including all cell types, form the basis of Tissue Engineered Small Intestines (TESI).
Adult intestinal stem cells also have been used to develop an epithelial layer independent of an underlying mesenchyme. Finally, embryonic stem cells, which are still capable of forming a large variety of cell types, can be induced to form both the endoderm and mesoderm layers that make up the intestine.
However, despite these multiple approaches, developing these bioengineered intestines is still in the early stages and the models are imperfect – no villus formation has been observed in vitro. However, when these cultured tissues are transplanted into animals, villus development does occur. It is thought that molecules and cell types found in the host likely provide the necessary signals, and forces, that drive the generation of villi in these constructs. However, further work is needed to identify the source of these cues.
Freddo has a positive outlook on the future work, ‘our next steps are to understand the mechanism by which physical forces and signal-induced cell shape changes can cause dramatic surface area expansion, and to utilise molecular and physical methods to try to manipulate this process. With this understanding, it may eventually be possible to engineer sheets of cells to fold into proper villus structures, so as to be useful as a therapeutic option for patients lacking a significant portion of their small intestine.’
Meet the researchers
Andrew M. Freddo
University of Michigan
Ann Arbor, MI
Andrew Freddo is currently an MD/PhD candidate at the University of Michigan with expected graduation in May 2018, after which he will be completing residency training in combined Internal Medicine-Pediatrics at the University of Colorado. He has won several awards for his PhD work, including the Ruth L. Kirchstein National Research Service Award for Individual Predoctoral MD/PhD fellows. Andrew Freddo’s research focuses on understanding the role of cell division and intraepithelial forces in driving villi formation in the small intestine.
Dr Katherine D. Walton
Department of Cell and Developmental Biology
University of Michigan
Ann Arbor, MI
Dr Katherine Walton obtained her PhD in Cell and Molecular Biology at Duke University, North Carolina, after which she joined the department of Cell and Developmental Biology at the University of Michigan as a postdoctoral researcher. She currently serves as a research assistant professor in the same department. The goal of Dr Walton’s research is to understand the cellular and molecular mechanisms that drive intestinal organogenesis in order to create engineered intestines for people with short bowel syndrome.
T: (+1) 734 647 0171
Professor Deborah L. Gumucio, PhD
James Douglas Engel Professor of Cell and Developmental Biology
Department of Cell and Developmental Biology
University of Michigan
Ann Arbor, MI
Professor Deborah Gumucio completed her PhD in 1986 and then pursued postdoctoral studies at the University of Michigan. She joined the Department of Anatomy and Cell Biology at Michigan in 1991 and was promoted to Associate Professor in 1996 and to full Professor in 2002. Professor Gumucio founded the Center for Organogenesis in 1995 and established the Bioartography Project in 2005 as a fundraiser and public outreach program. Her major research focus is investigation of intestinal organogenesis, with emphasis on morphogenic and signalling events.
T: (+1) 734 647 0172
National Institutes of Health (NIH) – NIDDK F30 DK100125. Predoctoral Fellowship for Andrew Freddo (PI: Gumucio), A novel Ezrin-dependent cell division drives villus formation.
NIH – NIDDK R01 DK089933 (PI- Gumucio), Morphogenesis of the Fetal Intestinal Epithelium.
NIH – R01 DK089933 (PI: Gumucio), Morphogenesis of the fetal intestinal epithelium – National Institutes of Diabetes, Digestive and Kidney Diseases for Dr Katherine Walton.
AM Freddo, SK Shoffner, Y Shao, K Taniguchi, AS Grosse, MN Guysinger, S Wang, S Rudraraju, B Margolis, K Garikipati, S Schnell and DL Gumucio, Coordination of signaling and tissue mechanics during morphogenesis of murine intestinal villi: a role for mitotic cell rounding, Integrative Biology, 2016,8, 918–928.