A novel group of secretory cells regulates development of the immature intestinal stem cell niche through repression of the main signaling pathways driving proliferation.

The intestinal epithelium has constant turnover throughout the life of the organ, with apoptosis of cells at the tips of folds or villi releasing cells into the lumen. Due to constant turnover, epithelial cells need to be constantly replaced. Epithelial cells are supplied by stem cell niches that form at the base of the interfold space (zebrafish) and crypts (birds and mammals). Within the adult stem cell niche of mammals, secretory cells such as Paneth and goblet cells play a role in modulation of proliferation and stem cell activity, producing asymmetric divisions. Progeny of asymmetric divisions move up the fold or villi, giving rise to all of the epithelial cell types. Although much is known about function and organization of the adult intestinal stem cell niche, less is understood about regulation within the immature stem cell compartment. Following smooth muscle formation, the intestinal epithelium folds and proliferation becomes restricted to the interfold base. Symmetric divisions continue in the developing interfold niche until stem cell progeny begin asymmetric divisions, producing progeny that migrate up the developing folds. Proliferative progeny from the developing stem cell niche begin migrating out of the niche during the third week post-embryogenesis (zebrafish) or during the postnatal period (mammals). Regulation and organization of epithelial proliferation in the immature stem cell niche may be regulated by signals comparable to the adult niche. Here we identify a novel subset of secretory cells associated with the developing stem cell niche that receive Notch signaling (referred to as NRSCs). Inhibition of the embryonic NRSCs between 74 hpf to 120 hpf increases epithelial proliferation as well as EGF and IGF signaling. Inhibition of post-embryonic NRSCs (6 hpf to 12 dpf) also increases epithelial proliferation and expression level of Wnt target genes. We conclude that NRSCs play a role in modulation of epithelial proliferation through repression of signaling pathways that drive proliferation during both embryogenesis and the post embryonic period.

Loss of ascl1a prevents secretory cell differentiation within the zebrafish intestinal epithelium resulting in a loss of distal intestinal motility.

The vertebrate intestinal epithelium is renewed continuously from stem cells at the base of the crypt in mammals or base of the fold in fish over the life of the organism. As stem cells divide, newly formed epithelial cells make an initial choice between a secretory or enterocyte fate. This choice has previously been demonstrated to involve Notch signaling as well as Atonal and Her transcription factors in both embryogenesis and adults. Here, we demonstrate that in contrast to the atoh1 in mammals, ascl1a is responsible for formation of secretory cells in zebrafish. ascl1a-/- embryos lack all intestinal epithelial secretory cells and instead differentiate into enterocytes. ascl1a-/- embryos also fail to induce intestinal epithelial expression of deltaD suggesting that ascl1a plays a role in initiation of Notch signaling. Inhibition of Notch signaling increases the number of ascl1a and deltaD expressing intestinal epithelial cells as well as the number of developing secretory cells during two specific time periods: between 30 and 34hpf and again between 64 and 74hpf. Loss of enteroendocrine products results in loss of anterograde motility in ascl1a-/- embryos. 5HT produced by enterochromaffin cells is critical in motility and secretion within the intestine. We find that addition of exogenous 5HT to ascl1a-/- embryos at near physiological levels (measured by differential pulse voltammetry) induce anterograde motility at similar levels to wild type velocity, distance, and frequency. Removal or doubling the concentration of 5HT in WT embryos does not significantly affect anterograde motility, suggesting that the loss of additional enteroendocrine products in ascl1a-/- embryos also contributes to intestinal motility. Thus, zebrafish intestinal epithelial cells appear to have a common secretory progenitor from which all subtypes form. Loss of enteroendocrine cells reveals the critical need for enteroendocrine products in maintenance of normal intestinal motility.

Toxicity and developmental defects of different sizes and shape nickel nanoparticles in zebrafish.

Metallic nanoparticles such as nickel are used in catalytic sensing, and electronic applications, but health and environmental affects have not been fully investigated. While some metal nanoparticles result in toxicity, it is also important to determine whether nanoparticles of the same metal but of different size and shape changes toxicity. Three different size nickel nanoparticle (Ni NPs) of 30, 60, and 100 nm and larger particle clusters of aggregated 60 nm entities with a dendritic structure were synthesized and exposed to zebrafish embryos assessing mortality and developmental defects. Ni NPs exposure was compared to soluble nickel salts. All three 30, 60, and 100 nm Ni NPs are equal to or less toxic than soluble nickel while dendritic clusters were more toxic. With each Ni NP exposure, thinning of the intestinal epithelium first occurs around the LD10 continuing into the LD50. LD50 exposure also results in skeletal muscle fiber separation. Exposure to soluble nickel does not cause intestinal defects while skeletal muscle separation occurs at concentrations well over LD50. These results suggest that configuration of nanoparticles may affect toxicity more than size and defects from Ni NPs exposure occur by different biological mechanisms than soluble nickel.

Mutation of smooth muscle myosin causes epithelial invasion and cystic expansion of the zebrafish intestine.

Zebrafish meltdown (mlt) mutants develop cystic expansion of the posterior intestine as a result of stromal invasion of nontransformed epithelial cells. Positional cloning identified zebrafish smooth muscle myosin heavy chain (myh11) as the responsible gene. The mlt mutation constitutively activates the Myh11 ATPase, which disrupts smooth muscle cells surrounding the posterior intestine. Adjacent epithelial cells ectopically express metalloproteinases, integrins, and other genes implicated in human cancer cell invasion. Knockdown and pharmacological inhibition of these genes restores intestinal structure in mlt mutants despite persistent smooth muscle defects. These data identify an essential role for smooth muscle signaling in the maintenance of epithelial architecture and support gene expression analyses and other studies that identify a role for stromal genes in cancer cell invasion. Furthermore, they suggest that high-throughput screens to identify regulators of cancer cell invasion may be feasible in zebrafish.

Intestinal growth and differentiation in zebrafish.

Intestinal development in amniotes is driven by interactions between progenitor cells derived from the three primary germ layers. Genetic analyses and gene targeting experiments in zebrafish offer a novel approach to dissect such interactions at a molecular level. Here we show that intestinal anatomy and architecture in zebrafish closely resembles the anatomy and architecture of the mammalian small intestine. The zebrafish intestine is regionalized and the various segments can be identified by epithelial markers whose expression is already segregated at the onset of intestinal differentiation. Differentiation of cells derived from the three primary germ layers begins more or less contemporaneously, and is preceded by a stage in which there is rapid cell proliferation and maturation of epithelial cell polarization. Analysis of zebrafish mutants with altered epithelial survival reveals that seemingly related single gene defects have different effects on epithelial differentiation and smooth muscle and enteric nervous system development.

Unique and conserved aspects of gut development in zebrafish.

Although the development of the digestive system of humans and vertebrate model organisms has been well characterized, relatively little is known about how the zebrafish digestive system forms. We define developmental milestones during organogenesis of the zebrafish digestive tract, liver, and pancreas and identify important differences in the way the digestive endoderm of zebrafish and amniotes is organized. Such differences account for the finding that the zebrafish digestive system is assembled from individual organ anlagen, whereas the digestive anlagen of amniotes arise from a primitive gut tube. Despite differences of organ morphogenesis, conserved molecular programs regulate pharynx, esophagus, liver, and pancreas development in teleosts and mammals. Specifically, we show that zebrafish faust/gata-5 is a functional ortholog of gata-4, a gene that is essential for the formation of the mammalian and avian foregut. Further, extraembryonic gata activity is required for this function in zebrafish as has been shown in other vertebrates. We also show that a loss-of-function mutation that perturbs sonic hedgehog causes defects in the development of the esophagus that parallel those associated with targeted disruption of this gene in mammals. Perturbation of sonic hedgehog also affects zebrafish liver and pancreas development, and these effects occur in a reciprocal fashion, as has been described during mammalian liver and ventral pancreas development. Together, these data define aspects of digestive system development necessary for the characterization of zebrafish mutants. Given the similarities of teleost and mammalian digestive physiology and anatomy, these findings have implications for developmental and evolutionary studies as well as research of human diseases, such as diabetes, liver cirrhosis, and cancer.