Gridlock, a localized heritable vascular patterning defect in the zebrafish.

We are using the zebrafish, Danio rerio, to identify genes that generate and pattern the vertebrate vasculature. We have isolated a recessive mutation, gridlockm145 (grlm145) in which blood flow to the tail is impeded by a localized vascular defect. Using a novel microangiographic method, we show that the blockade is in the anterior trunk, where the paired lateral dorsal aortae normally merge to form the single midline aorta. Arterial-venous shunts and collateral vessels develop in most mutant embryos, bypassing the lesion and reconstituting caudal blood flow. The grl defect resembles coarctation of the aorta, a human congenital cardiovascular malformation of unknown aetiology, in the location of the lesion and its consequences and in the mutants' dependence on collateral vessels for survival.

A genetic screen for vascular mutants in zebrafish reveals dynamic roles for Vegf/Plcg1 signaling during artery development.

In this work we describe a forward genetic approach to identify mutations that affect blood vessel development in the zebrafish. By applying a haploid screening strategy in a transgenic background that allows direct visualization of blood vessels, it was possible to identify several classes of mutant vascular phenotypes. Subsequent characterization of mutant lines revealed that defects in Vascular endothelial growth factor (Vegf) signaling specifically affected artery development. Comparison of phenotypes associated with different mutations within a functional zebrafish Vegf receptor-2 ortholog (referred to as kdr-like, kdrl) revealed surprisingly varied effects on vascular development. In parallel, we identified an allelic series of mutations in phospholipase c gamma 1 (plcg1). Together with in vivo structure-function analysis, our results suggest a requirement for Plcg1 catalytic activity downstream of receptor tyrosine kinases. We further find that embryos lacking both maternal and zygotic plcg1 display more severe defects in artery differentiation but are otherwise similar to zygotic mutants. Finally, we demonstrate through mosaic analysis that plcg1 functions autonomously in endothelial cells. Together our genetic analyses suggest that Vegf/Plcg1 signaling acts at multiple time points and in different signaling contexts to mediate distinct aspects of artery development.

Zebrafish dracula encodes ferrochelatase and its mutation provides a model for erythropoietic protoporphyria.

Exposure to light precipitates the symptoms of several genetic disorders that affect both skin and internal organs. It is presumed that damage to non-cutaneous organs is initiated indirectly by light, but this is difficult to study in mammals. Zebrafish have an essentially transparent periderm for the first days of development. In a previous large-scale genetic screen we isolated a mutation, dracula (drc), which manifested as a light-dependent lysis of red blood cells [1]. We report here that protoporphyrin IX accumulates in the mutant embryos, suggesting a deficiency in the activity of ferrochelatase, the terminal enzyme in the pathway for heme biosynthesis. We find that homozygous drc(m248) mutant embryos have a G-->T transversion at a splice donor site in the ferrochelatase gene, creating a premature stop codon. The mutant phenotype, which shows light-dependent hemolysis and liver disease, is similar to that seen in humans with erythropoietic protoporphyria, a disorder of ferrochelatase.

Imaging blood vessels and lymphatic vessels in the zebrafish.

Blood vessels supply tissues and organs with oxygen, nutrients, cellular, and humoral factors, while lymphatic vessels regulate tissue fluid homeostasis, immune trafficking, and dietary fat absorption. Understanding the mechanisms of vascular morphogenesis has become a subject of intense clinical interest because of the close association of both types of vessels with pathogenesis of a broad spectrum of human diseases. The zebrafish provides a powerful animal model to study vascular morphogenesis because of their small, accessible, and transparent embryos. These unique features of zebrafish embryos permit sophisticated high-resolution live imaging of even deeply localized vessels during embryonic development and even in adult tissues. In this chapter, we summarize various methods for blood and lymphatic vessel imaging in zebrafish, including nonvital resin injection-based or dye injection-based vessel visualization, and alkaline phosphatase staining. We also provide protocols for vital imaging of vessels using microangiography or transgenic fluorescent reporter zebrafish lines.

Characterization of two frizzled8 homologues expressed in the embryonic shield and prechordal plate of zebrafish embryos.

We have isolated and characterized two complete cDNA clones, Zfz8a and Zfz8b, which encode zebrafish Frizzled (Fz) homologues. The predicted protein sequences, spanning 579 and 576 amino acid residues for ZFz8a and ZFz8b, respectively, were highly homologous (78%) to each other and contained an extracellular cysteine-rich domain and seven transmembrane domains that are well conserved in Fz receptor protein members. In comparison with other Fz family members, ZFz8a and ZFz8b showed the highest homology with mouse Fz8 (MFz8), sharing 84 and 76% amino acid identity, respectively. The presence of Zfz8a and Zfz8b transcripts was detected by in situ hybridization in zebrafish embryos from the 512 cell stage, and their appearance in the future dorsal region could be observed before embryos reached the 30% epiboly stage. At shield stage, Zfz8a transcripts were expressed in both epiblast and shield whereas expression of Zfz8b was only detected in the embryonic shield. During gastrula stages, both Zfz8a and Zfz8b transcripts were found in anterior dorsal regions of the involuting mesendoderm (future prechordal plate). By the 2- to 3-somite stage, expression of both Zfz8a and Zfz8b was restricted to the prechordal plate and prospective anterior neurectoderm, although expression of the Zfz8a gene was no longer present in the most anterior portion of the prechordal plate, the polster. In one-eyed pinhead mutant embryos, which lack prechordal plate, both Zfz8a and Zfz8b transcripts were reduced, confirming the prechordal plate specificity of Zfz8a and Zfz8b gene expression. These results provide an additional evidence supporting the role of Wnt signaling in organizer-mediated axial patterning.

Studying vascular development in the zebrafish.

The zebrafish, a genetically accessible vertebrate with an externally developing, optically clear embryo, is ideally suited for in vivo functional dissection of the embryonic development of the circulatory system. Here, we review the advantages of the zebrafish as a model system for studying vascular development, and describe genetic and experimental tools, methods and resources that have been developed to exploit these advantages. We also discuss briefly how some of these tools and methods can be brought to bear on problems of relevance to human health.

Fulfilling the social contract between medical schools and the public.

To gain a better understanding of the effects of medical schools related to transformations in medical practice, science, and public expectations, the Association of American Medical Colleges (AAMC) established the Advisory Panel on the Mission and Organization of Medical Schools (APMOMS) in 1994. Recognizing the privileges academic medicine enjoys as well as the power of and the strain on its special relationship with the American public, APMOMS formed the Working Group on Fulfilling the Social Contract. That group focused on the question: What are the roles and responsibilities involved in the social contract between medical schools and various interested communities and constituencies? This article reports the working group's findings. The group describes the historical and philosophical reasons supporting the concept of a social contract and asserts that medical schools have individual and collective social contracts with various subsets of the public, referred to as "stakeholders." Obligations derive implicitly from the generous public funding and other benefits medical school receive. Schools' primary obligation is to improve the nation's health. This obligation is carried out most directly by educating the next generation of physicians and biomedical scientists in a manner that instills appropriate professional attitudes, values, and skills. Group members identified 27 core stakeholders (e.g., government, patients, local residents, etc.) and outlined the expectations those stakeholders have of medical schools and the expectations medical schools have of those stakeholders. The group conducted a survey to test how leaders at medical schools responded to the notion of a social contract, to gather data on school leaders' perceptions of what groups they considered their schools' most important stakeholders, and to determine how likely it was that the schools' and the stakeholders expectations of each other were being met. Responses from 69 deans suggested that the survey provoked thinking about the broad issue of the social contract and stakeholders. Leaders on the same campuses disagreed about what groups were the most important stakeholders. Similarly, the responses revealed a lack of national consensus about the most important stakeholders, although certain groups were consistently included in the responses. The group concludes that medical school leaders should examine their assumptions and perspectives about their institutions' stakeholders and consider the interests of the stakeholders in activities such as strategic planning, policymaking, and program development.

How medical schools can maintain quality while adapting to resource constraints.

To gain a better understanding of the effects on medical schools of ongoing transformations in medical practice, science, and public expectations, the Association of American Medical Colleges (AAMC) formed the Advisory Panel on the Mission and Organization of Medical Schools (APMOMS) in 1994. Six working groups were appointed to address different issues of importance. This article is a report of the findings and recommendations of the Working Group on Adapting to Resource Constraints. That group was charged to consider how leaders in academic medicine can respond to the challenges of external forces and the anticipated diminishing of resources, and to focus on medical schools and how they can maintain quality while reengineering to effect needed changes. The group members developed their thinking within four categories: size of the academic enterprise; organizational models and their relationships to the clinical enterprise; faculty tenure and compensation; and partnerships with capital-intensive entities. Three recommendations for action, to which the APMOMS unanimously agreed, were made to the AAMC, which has already acted upon them in ways described in the article. The group also developed a series of "ideas for consideration," which represent a range of the members' perspectives. The working group did not seek (and probably could not have obtained) unanimous agreement on many of the issues that these ideas focus upon. The ideas are presented as a series of resolutions designed to stimulate discussion and foster better-informed planning.