Gastric epithelial stem cells in development, homeostasis and regeneration.

The stem/progenitor cell pool is indispensable for the development, homeostasis and regeneration of the gastric epithelium, owing to its defining ability to self-renew whilst supplying the various functional epithelial lineages needed to digest food efficiently. A detailed understanding of the intricacies and complexities surrounding the behaviours and roles of these stem cells offers insights, not only into the physiology of gastric epithelial development and maintenance, but also into the pathological consequences following aberrations in stem cell regulation. Here, we provide an insightful synthesis of the existing knowledge on gastric epithelial stem cell biology, including the in vitro and in vivo experimental techniques that have advanced such studies. We highlight the contributions of stem/progenitor cells towards patterning the developing stomach, specification of the differentiated cell lineages and maintenance of the mature epithelium during homeostasis and following injury. Finally, we discuss gaps in our understanding and identify key research areas for future work.

Unveiling the Influence of Tumor Microenvironment and Spatial Heterogeneity on Temozolomide Resistance in Glioblastoma Using an Advanced Human In Vitro Model of the Blood-Brain Barrier and Glioblastoma.

Glioblastoma (GBM) is the most common primary malignant brain cancer in adults with a dismal prognosis. Temozolomide (TMZ) is the first-in-line chemotherapeutic; however, resistance is frequent and multifactorial. While many molecular and genetic factors have been linked to TMZ resistance, the role of the solid tumor morphology and the tumor microenvironment, particularly the blood-brain barrier (BBB), is unknown. Here, the authors investigate these using a complex in vitro model for GBM and its surrounding BBB. The model recapitulates important clinical features such as a dense tumor core with tumor cells that invade along the perivascular space; and a perfusable BBB with a physiological permeability and morphology that is altered in the presence of a tumor spheroid. It is demonstrated that TMZ sensitivity decreases with increasing cancer cell spatial organization, and that the BBB can contribute to TMZ resistance. Proteomic analysis with next-generation low volume sample workflows of these cultured microtissues revealed potential clinically relevant proteins involved in tumor aggressiveness and TMZ resistance, demonstrating the utility of complex in vitro models for interrogating the tumor microenvironment and therapy validation.

Role of Wnt signaling in the maintenance and regeneration of the intestinal epithelium.

The intestinal epithelium plays a key role in digestion and protection against external pathogens. This tissue presents a high cellular turnover with the epithelium being completely renewed every 5days, driven by intestinal stem cells (ISCs) residing in the crypt bases. To sustain this dynamic renewal of the intestinal epithelium, the maintenance, proliferation, and differentiation of ISCs must be precisely controlled. One of the central pathways supporting ISC maintenance and dynamics is the Wnt pathway. In this chapter, we examine the role of Wnt signaling in intestinal epithelial homeostasis and tissue regeneration, including mechanisms regulating ISC identity and fine-tuning of Wnt pathway activation. We extensively discuss the contribution of the stem cell niche in maintaining Wnt signaling in the intestinal crypts that support ISC functions. The integration of these findings highlights the complex interplay of multiple niche signals and cellular components sustaining ISC behavior and maintenance, which together supports the immense plasticity of the intestinal epithelium.

Targeted ablation of Lgr5-expressing intestinal stem cells in diphtheria toxin receptor-based mouse and organoid models.

Intestinal cells marked by Lgr5 function as tissue-resident stem cells that sustain the homeostatic replenishment of the epithelium. By incorporating a diphtheria toxin receptor (DTR) cassette linked to the Lgr5 coding region, native Lgr5-expressing cells are susceptible to ablation upon DT administration in vivo. A similar strategy can be used for Lgr5-expressing cells within organoids established from DTR models. Together, these in vivo and in vitro approaches will facilitate dissection of the roles of Lgr5-expressing cells residing in different tissue compartments. For complete details on the use and execution of this protocol, please refer to Tan et al. (2021).

A monoastral mitotic spindle determines lineage fate and position in the mouse embryo.

During mammalian development, the first asymmetric cell divisions segregate cells into inner and outer positions of the embryo to establish the pluripotent and trophectoderm lineages. Typically, polarity components differentially regulate the mitotic spindle via astral microtubule arrays to trigger asymmetric division patterns. However, early mouse embryos lack centrosomes, the microtubule-organizing centres (MTOCs) that usually generate microtubule asters. Thus, it remains unknown whether spindle organization regulates lineage segregation. Here we find that heterogeneities in cell polarity in the early 8-cell-stage mouse embryo trigger the assembly of a highly asymmetric spindle organization. This spindle arises in an unusual modular manner, forming a single microtubule aster from an apically localized, non-centrosomal MTOC, before joining it to the rest of the spindle apparatus. When fully assembled, this 'monoastral' spindle triggers spatially asymmetric division patterns to segregate cells into inner and outer positions. Moreover, the asymmetric inheritance of spindle components causes differential cell polarization to determine pluripotent versus trophectoderm lineage fate.

Cytoskeletal control of early mammalian development.

The cytoskeleton - comprising actin filaments, microtubules and intermediate filaments - serves instructive roles in regulating cell function and behaviour during development. However, a key challenge in cell and developmental biology is to dissect how these different structures function and interact in vivo to build complex tissues, with the ultimate aim to understand these processes in a mammalian organism. The preimplantation mouse embryo has emerged as a primary model system for tackling this challenge. Not only does the mouse embryo share many morphological similarities with the human embryo during its initial stages of life, it also permits the combination of genetic manipulations with live-imaging approaches to study cytoskeletal dynamics directly within an intact embryonic system. These advantages have led to the discovery of novel cytoskeletal structures and mechanisms controlling lineage specification, cell-cell communication and the establishment of the first forms of tissue architecture during development. Here we highlight the diverse organization and functions of each of the three cytoskeletal filaments during the key events that shape the early mammalian embryo, and discuss how they work together to perform key developmental tasks, including cell fate specification and morphogenesis of the blastocyst. Collectively, these findings are unveiling a new picture of how cells in the early embryo dynamically remodel their cytoskeleton with unique spatial and temporal precision to drive developmental processes in the rapidly changing in vivo environment.

Keratins are asymmetrically inherited fate determinants in the mammalian embryo.

To implant in the uterus, the mammalian embryo first specifies two cell lineages: the pluripotent inner cell mass that forms the fetus, and the outer trophectoderm layer that forms the placenta1. In many organisms, asymmetrically inherited fate determinants drive lineage specification2, but this is not thought to be the case during early mammalian development. Here we show that intermediate filaments assembled by keratins function as asymmetrically inherited fate determinants in the mammalian embryo. Unlike F-actin or microtubules, keratins are the first major components of the cytoskeleton that display prominent cell-to-cell variability, triggered by heterogeneities in the BAF chromatin-remodelling complex. Live-embryo imaging shows that keratins become asymmetrically inherited by outer daughter cells during cell division, where they stabilize the cortex to promote apical polarization and YAP-dependent expression of CDX2, thereby specifying the first trophectoderm cells of the embryo. Together, our data reveal a mechanism by which cell-to-cell heterogeneities that appear before the segregation of the trophectoderm and the inner cell mass influence lineage fate, via differential keratin regulation, and identify an early function for intermediate filaments in development.

Expanding Actin Rings Zipper the Mouse Embryo for Blastocyst Formation.

Transformation from morula to blastocyst is a defining event of preimplantation embryo development. During this transition, the embryo must establish a paracellular permeability barrier to enable expansion of the blastocyst cavity. Here, using live imaging of mouse embryos, we reveal an actin-zippering mechanism driving this embryo sealing. Preceding blastocyst stage, a cortical F-actin ring assembles at the apical pole of the embryo's outer cells. The ring structure forms when cortical actin flows encounter a network of polar microtubules that exclude F-actin. Unlike stereotypical actin rings, the actin rings of the mouse embryo are not contractile, but instead, they expand to the cell-cell junctions. Here, they couple to the junctions by recruiting and stabilizing adherens and tight junction components. Coupling of the actin rings triggers localized myosin II accumulation, and it initiates a tension-dependent zippering mechanism along the junctions that is required to seal the embryo for blastocyst formation.