During development, a single cell divides and differentiates to become a complete multicellular organism. Understanding normal development is essential to understanding both developmental defects and cancers, in which developmental programs have been reactivated resulting in uncontrolled growth and metastasis. Amphioxus (Branchiostoma floridae) is proving an excellent model for development as it is closely related to vertebrates but much simpler both structurally and genomically. The amphioxus genome is fully sequenced and there are extensive libraries of clones of genes expressed in development. Thus, identifying developmental genes and gene networks is proving to be easier in amphioxus than in vertebrate model organisms. Despite these advantages, amphioxus is not widely studied because embryos have only been available during a limited breeding season. The present study builds upon recent breakthroughs in amphioxus husbandry to establish protocols for the continuous culture of amphioxus in the laboratory as well as methods for genetically manipulating amphioxus to determine gene functions. Successful laboratory culture of amphioxus will allow genetic lines of amphioxus to be established, as has been done for such vertebrate models as the mouse and zebrafish, but at lower cost. Thus, the project will open up new avenues for rapidly and economically solving basic developmental processes. All methods and resources developed will be made publically available. The results will be published in peer-reviewed journals and presented at scientific meetings. The work will also provide unique educational opportunities for a graduate student, two undergraduates, and 1-2 high school students per year. Finally, the project will strengthen international collaborations between research laboratories in the U.S. and Taiwan, establish a new web resource for the developmental biology community, and be incorporated into educational outreach activities through the Birch Aquarium in San Diego and the University of Colorado Museum of Natural History. Information about the project will be accessible via www.colorado.edu/eeb/facultysites/medeiros/Home.html.

Project Report

The importance of amphioxus: Amphioxus, an invertebrate chordate closely related to vertebrates, is a key model organism for understanding the fundamental genetic basis of vertebrate development, including humans. A nearly 7-fold increase in the number of papers/year on amphioxus between 1987 and 2013 is due largely to our discovery in 1988 that amphioxus can be induced to spawn in the laboratory during summer, though only on days when they would normally spawn in nature. Amphioxus is a key model for vertebrate development because it is very like a vertebrate, but much simpler both structurally and genomically. It resembles a small fish without fins, jaws or eyes. Its development is controlled by genetic programs very much like those that pattern vertebrate embryos, but whereas all vertebrates underwent two or more rounds of whole genome duplications leading to 3 or 4 copies of most developmental genes, the amphioxus genome is unduplicated, greatly facilitating understanding the function of individual genes. The goal of this research: The goal of the present research was to put the Florida amphioxus Branchiostoma floridae into continuous breeding culture in the laboratory and make it possible to obtain gametes and embryos any day of the year. Background: Amphioxus burrows in clean sand in bays, feeding on microscopic algae. Normally, the animals (sexes are separate) breed in summer when the water warms up. On days when they breed, they swim out of the sand after sunset and broadcast sperm and eggs. Embryos and larvae develop in the water column. In about 3 weeks, they burrow in the sand. Experimental design: This grant supported one technician and involved undergraduates in the research. For most of the undergraduates, it was their first lab experience. Supplies are relatively inexpensive: cultures of phytoplankton from the Bigelow laboratory in Maine, phytoplankton nutrients, jars for algal culture, air pumps and air stones, and plastic bins for $3-$6 each, petri dishes for raising larvae and fluorescent lights. Animals were maintained at room temperature on a light dark cycle of 14 hr light and 10 hrs dark per day. They were kept behind black curtains so that the dark period began at noon. When animals spawned, they would do so about ½ hour after the lights were turned off. A moonlight cycle was also provided with an inexpensive moonlight computer from Fishbowl Innovations, Seattle WA. Feeding experiments: Each experiment had 10 animals in 1.5 l of seawater. Of eight types of phytoplankton (brown: Isochrysis, Tisochrysis, Pavlova, Thalassiosira, green: Dunaliella, Tetraselmis, Platymonas and red: Rhodomonas), the brown algae promoted maximum gonad growth and the most frequent spawnings. (As the animals are transparent, gonads can be measured on living animals). Pumping brown algae into the cultures round the clock promoted gonad growth more than feeding twice/day. Induction of Spawning. To induce spawning, we put animals in a room with the temperature adjusted to 17oC (62oF) for a few days to a month or more. 25 animals were kept in 8l of seawater. Half the water was changed daily. The water was aerated, and 200 ml of mixed brown algal cultures in late log phase was added twice/day. When animals were returned to room temperature 25oC (77oF), 5-80% bred 24-36 hours later-- independent of the time of year. Raising embryos: We raised embryos in petri dishes until they were about 5 mm long, and then moved them to larger containers as they grew. They reached breeding size (1 inch) in about six months. This compares favorably with 3-4 months in nature. Larvae are fed twice a day on brown phytoplankton. We currently have the 3rd generation in the laboratory. Scientific significance: Breeding amphioxus in the laboratory year-round is a major breakthrough--the final step needed to make amphioxus an effective model organism for developmental studies including understanding the basics of gene networks, birth defects and cancer, and addressing the broader question of how vertebrates arose from their invertebrate ancestors. Broader aspects: Benefits to society: Amphioxus a very promising organism for understanding the genetic programs that pattern human embryos, and THE organism for addressing the question of how humans arose historically—where did a complex human brain come from? Why are simple animals like amphioxus unable to reason? It is ideal to compare with vertebrates to understand the basic framework on which the vertebrates were built. Training undergraduates including underrepresented minorities: This research has involved 18 undergraduates--13 at present. These 13 include three Mexican-americans, one Philippino, and one south-east Asian (Vietnamese ancestry). Eleven are women. Summary: This research has been highly successful. We now have Branchiostoma floridae in continuous breeding culture in the laboratory and have generated three consecutive generations of animals. This has paved the way for generating mutant strains.

Agency
National Science Foundation (NSF)
Institute
Division of Integrative Organismal Systems (IOS)
Application #
1160880
Program Officer
Steven Klein
Project Start
Project End
Budget Start
2012-04-01
Budget End
2014-03-31
Support Year
Fiscal Year
2011
Total Cost
$143,329
Indirect Cost
Name
University of California-San Diego Scripps Inst of Oceanography
Department
Type
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92093