Open annotations (there are currently
Altmetric provides a collated score for online attention across various platforms and media.
Mac-/Lactosylceramide regulates intestinal homeostasis and secretory cell fate commitment by facilitating Notch signaling
https://doi.org/10.7554/eLife.106184.3
evidence that glucosylceramide synthase (GlcT), a rate-limiting enzyme for glycosphingolipid (GSL) production, plays a role in the differentiation of intestinal cells. Mutations in GlcT compromise Notch signaling in the
intestinal stem cell lineage, resulting in the formation of enteroendocrine tumors. Further data suggest that a homolog of glucosylceramide synthase also influences Notch signaling in the mammalian intestine. While the outstanding strengths of the initial genetic and downstream pathway analyses are noted, there are minor weaknesses in the data regarding the potential role of this pathway in Delta trafficking. Nevertheless, this study opens the way for future mechanistic studies addressing how specific lipids modulate Notch signaling activity.
https://doi.org/10.7554/eLife.106184.3.sa0
: Findings that have theoretical or practical implications beyond a single subfield
: Appropriate and validated methodology in line with current state-of-the-art
During the peer-review process the editor and reviewers write an eLife Assessment that summarises the significance of the findings reported in the article (on a scale ranging from landmark to useful) and the strength of the evidence (on a scale ranging from exceptional to inadequate).
Learn more about eLife Assessments
Cell-to-cell communication via Delta-Notch signaling is widely used in various tissues and organs to regulate development and patterning; however, the mechanisms regulating Notch signaling for precise cell fate decisions remain poorly understood. Similar to mammals, the intestinal stem cells in the adult
midgut generate both absorptive and secretory cell progeny, guided by differential levels of Notch activation. Here we performed a forward genetic screen in
and identified glucosylceramide synthase (GlcT), a rate-limiting enzyme for glycosphingolipid (GSL) production, whose mutation causes the development of secretory cell tumors. Genetic analysis of the GSL synthesis pathway, combined with metabolite rescue experiments, revealed that the tumor formation is linked to a deficiency in Mactosylceramide/Lactosylceramide. This deficiency impaired the endocytic recycling of the Delta, subsequently reducing Notch signaling activation. Conditional knockout of
, in mouse small intestine caused an excessive differentiation of goblet cells, phenotypes similar to these caused by Notch inhibition. Our study suggests an evolutionarily conserved role for a specific GSL metabolite in modulating Notch signaling during stem cell fate decisions and provides a molecular connection between ceramide metabolism and Notch signaling in regulating tissue homeostasis and tumor formation.
Notch signaling is an evolutionarily conserved pathway in metazoans that mediates local cell–cell interactions through the membrane-tethered ligand Delta (Dl) and the membrane receptor Notch. This pathway transduces signals from the cell surface to the nucleus, regulating the transcription of target genes (
). Dl-Notch signaling controls various cell fates and developmental processes, with mutations in the pathway implicated in numerous human diseases, including congenital defects and cancer (
Artavanis-Tsakonas et al., 1999
). Given that Notch can transduce, amplify, and consolidate molecular differences to influence cell fate decisions, it is crucial to understand how the strength of the Notch signaling pathway is precisely regulated to ensure proper cell fate determination and tissue development.
Notch signaling is known as a major regulator of cell fate decisions in both mammalian and
intestinal stem cell (ISC) lineages, as its activity determines the binary fate of intestinal progenitor cells. A high level of Notch activity promotes the specification of absorptive enterocytes, while low or absent Notch activity favors the specification of secretory cell types (
ISC lineage in the adult midgut is relatively simple, comprising only one type of secretory cell: enteroendocrine cells (EEs). This simplicity makes it an attractive experimental system for studying Notch signaling in cell fate decisions during epithelial renewal (
). The fly ISCs, which specifically express Dl, periodically generate two types of committed daughter cells: enteroblasts, which experience high levels of Notch activation and are primed for the specification of absorptive enterocytes, and enteroendocrine progenitor cells (EEPs), which receive low levels of Notch activation. Typically, EEPs undergo one round of mitosis before terminal differentiation, resulting in the formation of EE pairs (
). The loss of Notch signaling results in a complete blockage of enteroblast differentiation, leading to the accumulation of ISCs, EEPs, and EEs in the intestinal epithelium, producing an ‘EE tumor’ phenotype (
). It is suggested that the expression level of Dl in individual ISCs varies, leading to differential levels of Notch activation in the immediate daughter cells (
). This variation in Dl expression may result from dynamic bidirectional Dl-Notch signaling between ISCs and EEPs, which in parallel or in conjunction with an intrinsic feedback mechanism in ISCs, guides a periodic activation of an EE fate inducer Scute, thereby periodic generation of EEPs from ISCs (
). The transient activation of Scute induces irreversible Prospero expression in EEPs through a Phyl-Sina-Ttk69 regulatory cascade, initiating terminal differentiation into EEs (
). During the period of Scute inactivation, ISCs only generate enterocyte-committed enteroblasts by default via Dl-Notch mediated lateral inhibition (
). In newly enclosed flies, increased lipid intake from food can alter the membrane trafficking of Dl, subsequently increasing the frequency of EE specification from ISCs (
). Thus, both intrinsic and extrinsic mechanisms are involved in regulating Dl-Notch signaling to orchestrate stem cell fate decisions.
To better understand fate regulation in the
ISC lineage, here we conducted a forward mosaic genetic screen in the adult
midgut, focusing on the right arm of the second chromosome to identify genes whose mutations could disrupt ISC fate decisions. From this screen, we identified several complementation groups that result in EE tumor phenotypes. While most of the identified gene loci correspond to known components of the Notch signaling pathway, we discovered one gene locus not previously associated with Notch signaling. Further analyses revealed a specific GSL metabolite that regulates Notch signaling and cell fate decisions in the intestinal epithelium, and this function appears to be conserved from
We performed EMS mutagenesis on chromosome 2R (carrying FRTG13/42B) and used the Mosaic Analysis with a Repressible Cell Marker (MARCM) system to induce mutant clones in the adult midgut (
). We screened approximately 10,000 lethal lines and identified a total of 12 mutations across six complementation groups that exhibited a ‘small cell tumor’ phenotype, characterized by a significant accumulation of diploid cells within the clones (
). Co-staining with the ISC marker Dl and the EE cell marker Pros revealed that most of these tumors contained an excessive number of ISCs and EEs, the EE tumor phenotype commonly observed in Notch mutant clones (
). Further genetic mapping showed that three complementation groups were associated with genes known to function in the Notch pathway:
). One complementation group, comprising two alleles (
gene (see details in ‘Materials and methods’), while the remaining two complementation groups, each consisting of one allele, remain undetermined (
encodes glucosylceramide synthase, an enzyme that catalyzes the formation of