Analysis of Gene Expression and Evolution in
Amphioxus
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Navin R. Mahadevan |
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Recent interest has arisen in the study of the invertebrate cephalochordate, amphioxus, because of its status as the closest living relative to true vertebrates. Many of its genes have been sequenced and several orthologous genes have been discovered in humans and mice. Specifically, the T-box gene family has excited interest since its discovery during the early 1990's because of its highly conserved sequence that codes for a DNA-binding motif known as T-box. Expression of these genes is typically seen in distinct patterns during embryogenesis. Comparison of these patterns to those caused by orthologous genes in mice and humans can offer valuable insight into the evolution of developmental genes and help to reconstruct the evolutionary history of vertebrate development.
This study used two different methods to study vertebrate developmental evolution. First, whole mount in situ hybridization experiments were conducted on amphioxus embryos at different stages of development to test for the expression of two different T-box genes, Tbx 2/3 and Tbx 1/10. For this, digoxygenin-labeled sense and antisense RNA probes were synthesized from a clone of the gene and introduced into the embryo in solution. The sense probes, having the same sequence as the message, naturally would not bind to any mrna present in the embryo. Thus the embryos exposed to this probe acted as controls, ensuring that only specific RNA binding would be scored in the experimental embryos. The antisense RNA, having code complementary to the message, would bind to any expressed gene (RNA) in the developing embryo. Antibodies were added that bound to digoxygenin-marked double stranded RNA molecules, and then a stain was added to highlight these areas of expression for view under the microscope. Expression patterns in amphioxus were then compared to those found in mice and humans to identify any morphological differences in expression between the species, thus tracing the gene's evolutionary history.
It was found that expression of Tbx 2/3 starts very early in the embryo, as a ring around the circumference in the early blastula. These cells continue on to form the lip of the blastopore during gastrulation. These blastopore cells are fated to form mesendoderm and indeed, during the neurula stage, expression was seen in the posterior mesendoderm as well as the ventral gut. In the larva, expression continues in the tail, but at an ever-weakening signal as the mesendoderm differentiates into either mesoderm or endoderm. Expression also continues in the ventral gut as well as in neural cells, highlighting a structure that is similar to the human pineal gland. Tbx 1/10 is not seen in the early stages of embryogenesis; however, expression begins in late gastrula stage and is seen in the ventral part of the stomites during neurula stage. It is hypothesized that these cells are the precursors to scleratome in vertebrates. The gene is also expressed in earlier larva demarcating a region that can be considered a primitive kidney. Expression of Tbx 1/10 disappears in later larvae.
In a separate experiment, phylogenetic trees of six amphioxus genes were constructed to trace the evolution of orthologous genes found in tetrapods today. Sequences of amphioxus proteins, as well as similar proteins from other species ranging from zebrafish to humans, were recovered from NCBI's online database. These sequences were then aligned so that conserved sequence could be compared against each other. The aligned sequences were fed into a tree-building program, and a phylogenetic tree was constructed showing the evolutionary relationships between amphioxus genes and their tetrapod orthologs.
In 1998, due to mounting evidence based on phylogenetic study of amphioxus genes, it was hypothesized that the entire genome duplicated itself sometime in the early evolution of vertebrates. Evidence gathered during this study seems to support this idea. It was found that out of the seven genes studied, five had two human and mouse orthologs. However, this does not fully confirm the genome duplication hypothesis, as 1 to 1 and 1 to 3 relationships were also discovered.
In the future, sections of embryos can be taken so as to determine with greater accuracy the exact region of gene expression. Furthermore more whole mount in situ hybridization studies can be conducted and more trees can be built from different amphioxus genes. This further research can help prove (or disprove) the theory of whole genome duplication and can help answer the question of why vertebrates exist as they do today.
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