The Intestinal Epithelium - Fluid Fate and Rigid Structure From Crypt Bottom to Villus Tip

Abstract

The single-layered, simple epithelium of the gastro-intestinal tract controls nutrient uptake, coordinates our metabolism and shields us from pathogens. Despite its seemingly simple architecture, the intestinal lining consists of highly distinct cell populations that are continuously renewed by the same stem cell population. The need to maintain balanced diversity of cell types in an unceasingly regenerating tissue demands intricate mechanisms of spatial or temporal cell fate control. Recent advances in single-cell sequencing, spatio-temporal profiling and organoid technology have shed new light on the intricate micro-structure of the intestinal epithelium and on the mechanisms that maintain it. This led to the discovery of unexpected plasticity, zonation along the crypt-villus axis and new mechanism of self-organization. However, not only the epithelium, but also the underlying mesenchyme is distinctly structured. Several new studies have explored the intestinal stroma with single cell resolution and unveiled important interactions with the epithelium that are crucial for intestinal function and regeneration. In this review, we will discuss these recent findings and highlight the technologies that lead to their discovery. We will examine strengths and limitations of each approach and consider the wider impact of these results on our understanding of the intestine in health and disease.

Keywords: cancer; differentiation; intestine; organoid; plasticity; regeneration; single cell; stem cell.

FIGURE 1

Structure of the intestine. (A) The intestine is organized in crypt-villus units. At the bottom of the crypt, in the stem cell zone crypt-base columnar cells (CBCs) act as stem cells of the tissue and are intercalated between Paneth cells. Paneth cells are the primary niche of CBCs and provide them with Notch ligands, EGF, and WNTs to support their continuous proliferation. At the same time Paneth cells also produce anti-microbial products to protect CBCs. In the Transit Amplifying zone (TA zone) the highly proliferative absorptive and slow dividing secretory progenitors differentiate to their respective lineage. The ratio between absorptive and secretory progenitors is controlled via lateral inhibition. Epithelial cells moving from the crypt bottom toward the villus encounter several opposing signaling gradients, among them WNT and BMP. WNT signals, which are necessary for the stemness of CBCs, are higher at the crypt bottom and gradually decrease toward the villus, while increasing BMP levels induce differentiation and gradual fate changes as cells rise up toward the villus tips. These signaling gradients are shaped by mesenchymal populations, such as fibroblasts or telocytes. Distinct populations with differing secretory profiles constitute the mesenchymal stem cell niche adjacent to crypts or induce continuous fate changes along the villus. Gray solid arrows indicate cells with Notch activity. (B) Cell fate determination in the intestinal epithelium. Once CBCs leave the stem cell zone, they start to differentiate either toward the absorptive or the secretory fate depending on Notch signals. Secretory progenitor cells can give rise to Paneth cells, goblet cells, Tuft cells and enteroendocrine cells, while absorptive progenitors can give rise to microfold cells and enterocytes. However, fate changes are not unidirectional and can be reverted upon appropriate environmental stimuli, such as tissue damage. Likewise, certain intestinal epithelial populations (e.g., enterocytes, EE cells, and goblet cells) dynamically acquire and lose different functions and thus cell identities in the course of their lives due to the instructive capacity of changing environments that they traverse as they move along crypt and villus. Black solid arrows indicate cell fate decisions during the differentiation process and gray dotted arrows indicate documented plasticity events by distinct cell populations.

FIGURE 2

(A) Topology of epithelial and mesenchymal cell populations across the crypt-villus axis. Distinct populations of epithelial and mesenchymal cells can be encountered at specific positions along the crypt villus axis. CBCs located at the crypt bottom, proliferate and can give rise to all epithelial cell types of the intestine. Secretory populations exist at various positions across the crypt-villus axis, including Paneth cells (crypt bottom) that protect and nurture CBCs, Tuft (villus) and goblet (crypt + villus) cells that coordinate inflammatory responses, as well as hormone-producing enteroendocrine cells (crypt + villus). Absorptive progenitors give rise to enterocytes and M cells. Enterocytes located at different parts of the villus are linked to distinct functions such as amino-acid (aa) and carbohydrate transport and lipid uptake. M cells are mainly located above Peyer’s patches and their main role is to transport antigens to the antigen-presenting cells underneath them for further processing. Stromal cells provide structural support to the tissue and provide epithelial cells with signaling molecules, regulating important processes such as proliferation and differentiation. Several fibroblast populations located at the crypt bottom in close proximity to the stem cell zone have been linked to production of WNTs and RSPO, which are essential for stem cell maintenance. Telocytes have varying secretory profiles depending on their position along the crypt-villus axis. A subset of telocytes found under the crypt produce canonical WNT ligands and RSPO3. However, telocytes locally concentrated at the villus base and tips and are linked to production of BMP ligands that promote differentiation of epithelial cells. (B) Effects of stromal cell-derived signals on intestinal epithelial cells. Stromal cells produce various signaling molecules affecting the behavior of intestinal epithelial cells. Telocytes and fibroblasts located near the stem cell zone secrete WNT ligands and RSPO to maintain stemness of CBCs, while WNT antagonists and BMP inhibitors, produced by myocytes and crypt-associated telocytes establish the limits that distinguish the stem cell zone from the rest of the crypt. Upon damage, fibroblast-derived PGE₂ drives the regeneration of stem cells via the YAP signaling axis. BMPs produced mainly by telocytes found in the villus induce differentiation and zonation of enterocytes, enteroendocrine cells and potentially other cell types as they migrate from the villus base toward the tip. Likewise, inflammatory signals derived from immune cells drive stem cell expansion and proliferation, instruct cell fate decisions and introduce strong differentiation biases toward secretory cell lineages so that tissue’s homeostasis is re-established after damage of the intestinal epithelium.

FIGURE 3

Comparison of the main methods to study spatio-temporal relationships.

FIGURE 4

Comparison of different organoid systems to assess fate determination and plasticity in homeostasis and disease.

FIGURE 5

Plasticity of the intestinal epithelium upon different challenges. (A) Calorie restriction. Long-term fasting induces morphological changes in the intestine associated with reduced villus-length. It also affects the stem cell zone, by inducing an increase in the populations of CBCs and Paneth cells and decrease in TA cells. (B) Nutrient overabundance. High-fat diet affects the stem cell compartment, as it induces an increase in the number of CBCs and decrease in Paneth cells. This was linked to the acquisition of Notch independence by CBCs as they produce their own Notch ligands to stimulate Notch signaling. (C) Damage-induced plasticity. Severe damage of the epithelium can lead to profound inflammation that in turn activates group 3 innate lymphoid cells (ILC3), which produce IL-25 to support CBC proliferation. Alternatively, ILC3s can also promote tissue regeneration by CBCs via an IL-25 independent mechanism, which involves the activation of YAP signaling in epithelial cells. This effect is most likely mediated by a stromal population that reacts to ILC3 activation with release of IL-11. If CBCs have been damaged or eliminated in the course of the insult, differentiated epithelial cells can fall back into the niche and de-differentiate to restart tissue replenishment. (D) Infection-related plasticity. Upon infection, Tuft and goblet cells are activated to produce anti-microbial products. Also, Tuft cells secrete IL-25 that activates ILC2s which in turn secrete Il-13. IL-13 acts on epithelial cells and strongly favors differentiation to Tuft and goblet cells, which results in Tuft and goblet cell hyperplasia.

FIGURE 6

Comparison of the main technologies available to perform lineage tracing.