Jennifer Shepard

Positional cloning of zebrafish ferroportin1 identifies a conservedvertebrate iron exporter

Defects in iron absorption and utilization lead to iron deficiency and overload disorders. Adult mammals absorb iron through the duodenum, whereas embryos obtain iron through placental transport. Iron uptake from the intestinal lumen through the apical surface of polarized duodenal enterocytes is mediated by the divalent metal transporter, DMT1 (refs 1,2,3). A second transporter has been postulated to export iron across the basolateral surface to the circulation. Here we have used positional cloning to identify the gene responsible for the hypochromic anaemia of the zebrafish mutant weissherbst. The gene, ferroportin1, encodes a multiple-transmembrane domain protein, expressed in the yolk sac, that is a candidate for the elusive iron exporter. Zebrafish ferroportin1 is required for the transport of iron from maternally derived yolk stores to the circulation and functions as an iron exporter when expressed in Xenopus oocytes. Human Ferroportin1 is found at the basal surface of placental syncytiotrophoblasts, suggesting that it also transports iron from mother to embryo. Mammalian Ferroportin1 is expressed at the basolateral surface of duodenal enterocytes and could export cellular iron into the circulation. We propose that Ferroportin1 function may be perturbed in mammalian disorders of iron deficiency or overload.

Zebrafish as a cancer model system

The zebrafish, with its combination of forward genetics and vertebrate biology, has great potential as a cancer model system.

Implantation in Vivo and Retrieval of Artificial Structures Consisting of Rabbit and Human Urothelium and Human Bladder Muscle

The harvest of human bladder muscle and urothelial cells with subsequent growth may be useful for tissue replacement in genitourinary reconstruction. We previously reported the development of a system for the harvest, delivery and growth of rabbit urothelium in vivo using biodegradable polymers. We have now expanded and adapted this system for the harvest and in vivo implantation of human bladder urothelial and muscle cells. Synthetic polymer fibers of polyglycolic acid can serve as a scaffold and a delivery vehicle for the implantation of rabbit uroepithelial cells into athymic host animals. The polymers, which slowly degrade in vivo, allow the urothelial cells to survive at the implant site. We demonstrate that polyglycolic acid polymers support the proliferation of rabbit urothelial cells in situ and can serve as a maleable substrate for the creation of new urological structures that replace the degrading polymer fibers. We also show that human urothelial cells and bladder muscle cells, when implanted on polyglycolic acid fibers, from new urological structures in vivo composed of both cell types. The human cell-polymer xenografts can be recovered from host animals at extended times after implantation. These data suggest that feasibility of using polyglycolic acid polymers as substrates for the creation of human urothelial and muscle grafts for genitourinary reconstruction.

Developmental derivation of embryonic and adult macrophages.

The macrophage cell lineage continually arises from hematopoietic stem cells during embryonic, fetal, and adult life. Previous theories proposed that macrophages are the recent progeny of bone marrow–derived monocytes and that they function primarily in phagocytosis. More recently, however, observations have shown that the ontogeny of macrophages in early mouse and human embryos is different from that occurring during adult development, and that the embryonic macrophages do not follow the monocyte pathway. Fetal macrophages are thought to differentiate from yolk sac–derived primitive macrophages before the development of adult monocytes. Further support for a separate lineage of fetal macrophages has come from studies of several species, including chicken, zebrafish, Xenopus, Drosophila, and C. elegans. The presence of fetal macrophages in PU.1-null mice indicates their independence from monocyte precursors and their existence as an alternative macrophage lineage.

The zon laboratory guide to positional cloning in zebrafish.

Zebrafish genome sequencing project has improved efficiency of positional cloning in zebrafish and traditional chromosome walking by isolating large insert genomic libraries has become a past. However, the genetic principles underlying the positional cloning still form the foundation for current chromosome walking using the genome sequence assemblies and related genomic sequence and clone information. This guide intends to summarize our accumulated experience in positional cloning using the current genomic resources and tools, and provide a practical guide to positional and/or candidate cloning of mutants of interest.

Analysis of the Cell Cycle in Zebrafish Embryos

Publisher Summary This chapter discusses the analysis of the cell cycle in Zebrafish Embroys. The advantages of the zebrafish system that make it a powerful organism to study vertebrate development include external fertilization of oocytes, transparent embryos, and rapid embryonic development. These features also provide the opportunity to study early cell divisions and tissue-specific cellular proliferation. This chapter provides protocols for assays that characterize various phases of cell division in zebrafish embryos. It discusses the design of screens for mutations aVecting embryonic cell proliferation and a method to detect the novel compounds affecting the zebrafish cell cycle. Assays discussed in this chapter include: DNA content analysis by flow cytometry, whole-mount embryonic antibody staining, mitotic spindle analysis, 5-bromo-2-deoxyuridine (BrdU) incorporation, cell death analysis, and in situ hybridization with cell cycle regulatory genes. Finally, this chapter concludes with the genetic tractability of the zebrafish system that makes it an excellent organism, in which to pursue forward genetic screens for mutations or chemical screens for novel compounds that alter cell division using one or more of these cell cycle assays.