Hedgehog Signaling in Intestinal Development and Homeostasis.

Annu Rev Physiol. 2020 Oct 09;:

Authors: Walton KD, Gumucio DL

Abstract
The hedgehog (Hh) signaling pathway plays several diverse regulatory and patterning roles during organogenesis of the intestine and in the regulation of adult intestinal homeostasis. In the embryo, fetus, and adult, intestinal Hh signaling is paracrine: Hh ligands are expressed in the endodermally derived epithelium, while signal transduction is confined to the mesenchymal compartment, where at least a dozen distinct cell types are capable of responding to Hh signals. Epithelial Hh ligands not only regulate a variety of mesenchymal cell behaviors, but they also direct these mesenchymal cells to secrete additional soluble factors (e.g., Wnts, Bmps, inflammatory mediators) that feed back to regulate the epithelial cells themselves. Evolutionary conservation of the core Hh signaling pathway, as well as conservation of epithelial/mesenchymal cross talk in the intestine, has meant that work in many diverse model systems has contributed to our current understanding of the role of this pathway in intestinal organogenesis, which is reviewed here. Expected final online publication date for the Annual Review of Physiology, Volume 83 is February 10, 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

PMID: 33035430 [PubMed - as supplied by publisher]

RYK-mediated filopodial pathfinding facilitates midgut elongation.

Development. 2020 Sep 29;:

Authors: Wang S, Roy JP, Tomlinson AJ, Wang EB, Tsai YH, Cameron L, Underwood J, Spence JR, Walton K, Stacker SA, Gumucio DL, Lechler T

Abstract
Between embryonic day 10.5 to 14.5, active proliferation drives rapid elongation of the murine midgut epithelial tube. Within this pseudostratified epithelium, nuclei synthesize DNA near the basal surface and move apically to divide. After mitosis, the majority of daughter cells extend a long, basally oriented filopodial protrusion, building a de novo path along which their nuclei can return to the basal side. WNT5A, secreted by surrounding mesenchymal cells, acts as a guidance cue to orchestrate this epithelial pathfinding behavior, but how this signal is received by epithelial cells is unknown. Here, we investigated two known WNT5A receptors, ROR2 and RYK. We found that epithelial ROR2 is dispensable for midgut elongation. However, loss of Ryk phenocopies the Wnt5a -/- phenotype, perturbing post-mitotic pathfinding and leading to apoptosis. These studies reveal that the ligand-receptor pair, WNT5A-RYK, acts as a navigation system to instruct filopodial pathfinding, a process critical for continuous cell cycling to fuel rapid midgut elongation.

PMID: 32994164 [PubMed - as supplied by publisher]

Controlled modelling of human epiblast and amnion development using stem cells.

Nature. 2019 09;573(7774):421-425

Authors: Zheng Y, Xue X, Shao Y, Wang S, Esfahani SN, Li Z, Muncie JM, Lakins JN, Weaver VM, Gumucio DL, Fu J

Abstract
Early human embryonic development involves extensive lineage diversification, cell-fate specification and tissue patterning1. Despite its basic and clinical importance, early human embryonic development remains relatively unexplained owing to interspecies divergence2,3 and limited accessibility to human embryo samples. Here we report that human pluripotent stem cells (hPSCs) in a microfluidic device recapitulate, in a highly controllable and scalable fashion, landmarks of the development of the epiblast and amniotic ectoderm parts of the conceptus, including lumenogenesis of the epiblast and the resultant pro-amniotic cavity, formation of a bipolar embryonic sac, and specification of primordial germ cells and primitive streak cells. We further show that amniotic ectoderm-like cells function as a signalling centre to trigger the onset of gastrulation-like events in hPSCs. Given its controllability and scalability, the microfluidic model provides a powerful experimental system to advance knowledge of human embryology and reproduction. This model could assist in the rational design of differentiation protocols of hPSCs for disease modelling and cell therapy, and in high-throughput drug and toxicity screens to prevent pregnancy failure and birth defects.

PMID: 31511693 [PubMed - indexed for MEDLINE]

Microengineered human amniotic ectoderm tissue array for high-content developmental phenotyping.

Biomaterials. 2019 09;216:119244

Authors: Nasr Esfahani S, Shao Y, Resto Irizarry AM, Li Z, Xue X, Gumucio DL, Fu J

Abstract
During early post-implantation human embryogenesis, the epiblast (EPI) within the blastocyst polarizes to generate a cyst with a central lumen. Cells at the uterine pole of the EPI cyst then undergo differentiation to form the amniotic ectoderm (AM), a tissue essential for further embryonic development. While the causes of early pregnancy failure are complex, improper lumenogenesis or amniogenesis of the EPI represent possible contributing factors. Here we report a novel AM microtissue array platform that allows quantitative phenotyping of lumenogenesis and amniogenesis of the EPI and demonstrate its potential application for embryonic toxicity profiling. Specifically, a human pluripotent stem cell (hPSC)-based amniogenic differentiation protocol was developed using a two-step micropatterning technique to generate a regular AM microtissue array with defined tissue sizes. A computer-assisted analysis pipeline was developed to automatically process imaging data and quantify morphological and biological features of AM microtissues. Analysis of the effects of cell density, cyst size and culture conditions revealed a clear connection between cyst size and amniogenesis of hPSC. Using this platform, we demonstrated that pharmacological inhibition of ROCK signaling, an essential mechanotransductive pathway, suppressed lumenogenesis but did not perturb amniogenic differentiation of hPSC, suggesting uncoupled regulatory mechanisms for AM morphogenesis vs. cytodifferentiation. The AM microtissue array was further applied to screen a panel of clinically relevant drugs, which successfully detected their differential teratogenecity. This work provides a technological platform for toxicological screening of clinically relevant drugs for their effects on lumenogenesis and amniogenesis during early human peri-implantation development, processes that have been previously inaccessible to study.

PMID: 31207406 [PubMed - indexed for MEDLINE]

Signals and forces shaping organogenesis of the small intestine.

Curr Top Dev Biol. 2019;132:31-65

Authors: Wang S, Walton KD, Gumucio DL

Abstract
The adult gastrointestinal tract (GI) is a series of connected organs (esophagus, stomach, small intestine, colon) that develop via progressive regional specification of a continuous tubular embryonic organ anlage. This chapter focuses on organogenesis of the small intestine. The intestine arises by folding of a flat sheet of endodermal cells into a tube of highly proliferative pseudostratified cells. Dramatic elongation of this tube is driven by rapid epithelial proliferation. Then, epithelial-mesenchymal crosstalk and physical forces drive a stepwise cascade that results in convolution of the tubular surface into finger-like projections called villi. Concomitant with villus formation, a sharp epithelial transcriptional boundary is defined between stomach and intestine. Finally, flask-like depressions called crypts are established to house the intestinal stem cells needed throughout life for epithelial renewal. New insights into these events are being provided by in vitro organoid systems, which hold promise for future regenerative engineering of the small intestine.

PMID: 30797512 [PubMed - indexed for MEDLINE]

Opening the black box: Stem cell-based modeling of human post-implantation development.

J Cell Biol. 2019 02 04;218(2):410-421

Authors: Taniguchi K, Heemskerk I, Gumucio DL

Abstract
Proper development of the human embryo following its implantation into the uterine wall is critical for the successful continuation of pregnancy. However, the complex cellular and molecular changes that occur during this post-implantation period of human development are not amenable to study in vivo. Recently, several new embryo-like human pluripotent stem cell (hPSC)-based platforms have emerged, which are beginning to illuminate the current black box state of early human post-implantation biology. In this review, we will discuss how these experimental models are carving a way for understanding novel molecular and cellular mechanisms during early human development.

PMID: 30552099 [PubMed - indexed for MEDLINE]

Radial WNT5A-Guided Post-mitotic Filopodial Pathfinding Is Critical for Midgut Tube Elongation.

Dev Cell. 2018 07 16;46(2):173-188.e3

Authors: Wang S, Cebrian C, Schnell S, Gumucio DL

Abstract
The early midgut undergoes intensive elongation, but the underlying cellular and molecular mechanisms are unknown. The early midgut epithelium is pseudostratified, and its nuclei travel between apical and basal surfaces in concert with cell cycle. Using 3D confocal imaging and 2D live imaging, we profiled behaviors of individual dividing cells. As nuclei migrate apically for mitosis, cells maintain a basal process (BP), which splits but is inherited by only one daughter. After mitosis, some daughters directly use the inherited BP as a "conduit" to transport the nucleus basally, while >50% of daughters generate a new basal filopodium and use it as a path to return the nucleus. Post-mitotic filopodial "pathfinding" is guided by mesenchymal WNT5A. Without WNT5A, some cells fail to tether basally and undergo apoptosis, leading to a shortened midgut. Thus, these studies reveal previously unrecognized strategies for efficient post-mitotic nuclear trafficking, which is critical for early midgut elongation.

PMID: 30016620 [PubMed - indexed for MEDLINE]