The emergence of circadian timekeeping in the intestine.

The circadian clock is a molecular timekeeper, present from cyanobacteria to mammals, that coordinates internal physiology with the external environment. The clock has a 24-h period however development proceeds with its own timing, raising the question of how these interact. Using the intestine of Drosophila melanogaster as a model for organ development, we track how and when the circadian clock emerges in specific cell types. We find that the circadian clock begins abruptly in the adult intestine and gradually synchronizes to the environment after intestinal development is complete. This delayed start occurs because individual cells at earlier stages lack the complete circadian clock gene network. As the intestine develops, the circadian clock is first consolidated in intestinal stem cells with changes in Ecdysone and Hnf4 signalling influencing the transcriptional activity of Clk/cyc to drive the expression of tim, Pdp1, and vri. In the mature intestine, stem cell lineage commitment transiently disrupts clock activity in differentiating progeny, mirroring early developmental clock-less transitions. Our data show that clock function and differentiation are incompatible and provide a paradigm for studying circadian clocks in development and stem cell lineages.

Single-cell resolution of the adult zebrafish intestine under conventional conditions and in response to an acute Vibrio cholerae infection.

Vibrio cholerae is an aquatic bacterium that causes severe and potentially deadly diarrheal disease. Despite the impact on global health, our understanding of host mucosal responses to Vibrio remains limited, highlighting a knowledge gap critical for the development of effective prevention and treatment strategies. Using a natural infection model, we combine physiological and single-cell transcriptomic studies to characterize conventionally reared adult zebrafish guts and guts challenged with Vibrio. We demonstrate that Vibrio causes a mild mucosal immune response characterized by T cell activation and enhanced antigen capture; Vibrio suppresses host interferon signaling; and ectopic activation of interferon alters the course of infection. We show that the adult zebrafish gut shares similarities with mammalian counterparts, including the presence of Best4+ cells, tuft cells, and a population of basal cycling cells. These findings provide important insights into host-pathogen interactions and emphasize the utility of zebrafish as a natural model of Vibrio infection.

Mechanisms of epithelial growth and development in the zebrafish intestine.

The intestinal epithelium is a complex tissue monolayer composed of regionally and functionally specialized intestinal epithelial cells. Given epithelial exposure to harsh and varied luminal conditions, epithelial cells continuously regenerate to sustain the barrier against environmental factors, including microbial invaders. Multipotent intestinal stem cells are essential to epithelial regenerative capacity, generating a programed mixture of absorptive and secretory cell types. Mechanisms of epithelial growth and differentiation in response to endogenous or external stressors remain under investigation. In this review, we highlight the zebrafish, Danio rerio, as a potent model of intestinal epithelial development and function. We describe epithelial composition and key regulators of epithelial renewal to promote the zebrafish as an investigative tool to study epithelial development and growth. We also highlight areas for discovery, particularly in the context of stress-dependent regulation of epithelial function.

Immune regulation of intestinal-stem-cell function in Drosophila.

Intestinal progenitor cells integrate signals from their niche, and the gut lumen, to divide and differentiate at a rate that maintains an epithelial barrier to microbial invasion of the host interior. Despite the importance of evolutionarily conserved innate immune defenses to maintain stable host-microbe relationships, we know little about contributions of stem-cell immunity to gut homeostasis. We used Drosophila to determine the consequences of intestinal-stem-cell immune activity for epithelial homeostasis. We showed that loss of stem-cell immunity greatly impacted growth and renewal in the adult gut. In particular, we found that inhibition of stem-cell immunity impeded progenitor-cell growth and differentiation, leading to a gradual loss of stem-cell numbers with age and an impaired differentiation of mature enteroendocrine cells. Our results highlight the importance of immune signaling in stem cells for epithelial function in the adult gut.

A cell atlas of microbe-responsive processes in the zebrafish intestine.

Gut microbial products direct growth, differentiation, and development in animal hosts. However, we lack system-wide understanding of cell-specific responses to the microbiome. We profiled cell transcriptomes from the intestine, and associated tissue, of zebrafish larvae raised in the presence or absence of a microbiome. We uncovered extensive cellular heterogeneity in the conventional zebrafish intestinal epithelium, including previously undescribed cell types with known mammalian homologs. By comparing conventional to germ-free profiles, we mapped microbial impacts on transcriptional activity in each cell population. We revealed intricate degrees of cellular specificity in host responses to the microbiome that included regulatory effects on patterning and on metabolic and immune activity. For example, we showed that the absence of microbes hindered pro-angiogenic signals in the developing vasculature, causing impaired intestinal vascularization. Our work provides a high-resolution atlas of intestinal cellular composition in the developing fish gut and details the effects of the microbiome on each cell type.

Myt1 Kinase Couples Mitotic Cell Cycle Exit with Differentiation in Drosophila.

The Drosophila midgut is an excellent system for characterizing cell cycle regulation in the context of tissue homeostasis. Two major progenitor cell types populate the midgut: mitotic intestinal stem cells and their post-mitotic daughters, enteroblasts. Although regulatory networks that control stem cell proliferation are well characterized, how enteroblast mitotic-cell-cycle exit is coordinated with endocycle entry and enterocyte specification remains poorly defined. Myt1 is a conserved Cdk1 inhibitory kinase that regulates mitotic timing during animal development. Here, we use myt1-null mutants and cell-specific RNA interference to investigate Myt1 function in stem cells and enteroblast progenitors. Myt1 depletion alters cell cycle kinetics and promotes ectopic stem cell and enteroblast mitoses at the expense of enteroblast-enterocyte differentiation. These aberrant enteroblast mitoses rely upon cyclin A, implicating Myt1 inhibition of cyclin A/Cdk1 as a mechanism for the coupling mitotic exit with differentiation in enteroblasts.