Vertebrate chordates have innovations not shared by non-vertebrate chordates, including neural crest, neurogenic placodes, skeletal features, and an amplified genome. The project's long-term objective is to learn how vertebrates evolved from non-vertebrate chordates. This work tests the hypothesis that vertebrates evolved the ability of neural plate border genes to turn on neural crest specifiers. The project tests predictions of the hypothesis in Oikopleura dioica, a larvacean with a fully sequenced genome, using novel methods to alter hundreds of embryos at once. Larvaceans are ubiquitous marine organisms that are considered basal chordates that contribute substantially to the biomass and carbon cycling of oceans. Oikopleura orthologs of neural crest-related genes will be cloned and in situ hybridization will be used to learn their expression patterns. Neural crest-related gene regulatory networks in Oikopleura will be dissected by performing gain- and loss-of-function experiments with Oikopleura orthologs of key crest-related genes, and results by in situ hybridization and genomic microarray will be evaluated. Public understanding will be enhanced of the roles of larvaceans in the environment and in the investigation of vertebrate origins. Achieving these aims will define developmental mechanisms that vertebrates evolved to develop a full blown neural crest and illuminate mechanisms by which evolutionary novelties arise. Because larvaceans, especially Oikopleura dioica, remove large quantities of the greenhouse gas carbon dioxide from the atmosphere for long-term storage in ocean depths, a better understanding of larvacean biology will help illuminate understanding of global warming. The project will educate school children about the role of larvaceans in ecology and the science of evolutionary biology and will mentor representatives of underrepresented groups, particularly minority undergraduates.

Project Report

The Problem: Vertebrates possess features not shared by other Chordates, including neural crest, placodes, an enlarged brain, cartilage and mineralized skeletons, and an amplified genome. Our goal is to learn how evolved genetic changes produced derived developmental mechanisms leading to novel vertebrate features. We study the non-vertebrate chordate Oikopleura dioica, a tiny but abundant (thousands of individuals per cubic meter) organism found in all seas (Fig. 1). Climate Change: Oikopleura eats photosynthetic algae that take carbon dioxide from the atmosphere and use it to secrete a mucus house that they discard 12 times a day. Old houses fall to the ocean floor, thereby removing carbon to long-term storage consisting of 10% of the ocean’s primary productivity, thus playing an enormous role in buffering carbon dioxide levels that contribute to global warming. Chrodate Body Plan: Chordates require developmental signaling by retinoic acid (RA), a metabolite of Vitamin A, but Oikopleura lacks genes for RA synthesis, degradation, and reception. How can this be? Our experiments revealed that RA treatment does not alter anterior-posterior signaling in Oikopleura as it does in vertebrates, showing -- surprisingly – development of a chordate body plan without RA. Thyroid Evolution: The thyroid regulates energy expenditure and growth. The Oikopleura endostyle (Fig. 1) shares features with the thyroid but little is known about its anterior-posterior patterning. Results showed that Oikopleura Otx, Pax2/5/8, and Hox1 genes were expressed in the organ’s anterior, middle, and posterior as in vertebrates. Expression analysis suggested that some ancestral gene functions were maintained by one vertebrate gene duplicate (say Otx1 or Pax2) and others were retained by other vertebrate copies (say Otx2 or Pax5). We found that Otx, Pax2/5/8, and Hox1 related genes have similar patterns in Oikopleura and in vertebrates for both endostyle and nervous system, showing that this gene set was in the developmental gene toolkit for axial patterning more than 600 million years ago. Gene Duplication and Novel Functions: Duplicated genes generally either disappear as one copy vanishes or are retained after evolving beneficial functions (neofunctionalization) or, as we suggested, after partitioning original functions (subfunctionalization). Pax2/5/8 genes duplicated after Oikopleura and vertebrate lineages diverged (Fig. 2) but ancestral functions were uncertain. Our expression studies showed for endostyle, pharynx and hindgut that ancestral functions partitioned differently in Oikopleura and its relatives, the ascidians. Novel heart expression likely evolved by neofunctionalization. Expression in the endostyle, at sites of epithelial remodeling, and in sensory tissues mimics the separate Pax2, Pax5 and Pax8 genes in vertebrates and may indicate that these functions arose in the chordate common ancestor. This work provides insights into the evolutionary development of heart, thyroid, pharynx, mouth and placodes. Further, it supports the controversial conclusion that a gill arch of Oikopleura corresponds to the vertebrate ear placode and that the ancestral Pax2/5/8 gene functioned to engineer epithelial fusions and perforations, including gill slits. Evolution of Retinoic Acid Signaling: Enzymes that synthesize RA provide the first step in regulating RA signaling. Adh3 is the only enzyme of its RA-synthesizing family found in non-vertebrates, but its role in RA biosynthesis is uncertain. If Adh3 plays a role in RA synthesis, then why wasn’t it lost like other RA machinery genes? Results, unexpectedly, showed that Oikopleura preserved a functional Adh3 gene that was expressed like other non-vertebrate chordates. We conclude that Adh3 was not preserved for its conserved role in RA synthesis and that a role in RA synthesis in vertebrates would be due to neofunctionalization. Oikopleura Genome: Oikopleura, despite its chordate body plan and sister relationship to vertebrates, has the smallest animal genome. What genomic changes occurred as the Oikopleura genome dwindled? We sequenced Oikopleura genomic DNA from Oregon to compare to Norway. Results showed that many genome features shared by all animals, including transposon diversity, developmental gene repertoire, gene order on chromosomes, and the organization of expressed non-coding parts of genes (introns), are shattered in Oikopleura. Because many DNA regulatory elements are conserved in sequence without encoding protein (conserved non-coding elements, CNEs), we studied CNEs in Oikopleura. No CNEs were shared by Oikopleura and vertebrates but many were shared between Oregon and Norway (Fig. 3), and thus provide candidates to test for regulatory function. CNEs were systematically located in introns that are larger than average, likely because their presence prohibited intron shortening. How can Oikopleura preserve chordate developmental regulation without sharing regulatory sequences? A New EvoDevo Paradox: The genomic and developmental aspects of our work pose a critical paradox for understanding evolutionary developmental mechanisms: What causes genome stability across nearly all animals despite enormous developmental plasticity (compare a sponge to a human) versus the enormous genome plasticity in Oikopleura despite its developmental genetic stability? This is work for the future.

Agency
National Science Foundation (NSF)
Institute
Division of Integrative Organismal Systems (IOS)
Type
Standard Grant (Standard)
Application #
0719577
Program Officer
Steven L. Klein
Project Start
Project End
Budget Start
2007-08-15
Budget End
2011-07-31
Support Year
Fiscal Year
2007
Total Cost
$605,999
Indirect Cost
Name
University of Oregon Eugene
Department
Type
DUNS #
City
Eugene
State
OR
Country
United States
Zip Code
97403