Perspective: From mutants to mechanisms? Assessing the candidate gene paradigm in evolutionary biology.

The generation of mutants in model organisms by geneticists and developmental biologists over the last century has occasionally produced phenotypes that are startlingly reminiscent of those seen in other species. Such extreme mutations have generally been dismissed by evolutionary geneticists since the "modern synthesis" as irrelevant to adaptation and speciation. But only in recent years has information on the molecular bases of mutant phenotypes become widely available, and thus work on testing the relevance of such extreme mutations to the generation of phylogenetic diversity has just begun. Here we evaluate whether evolutionary mimics are, in fact, useful for pinpointing the genetic differences that distinguish morphological variants generated during evolution. Examples come from both plants and animals, and range from intraspecific to interordinal taxonomic ranges. The use of mutationally defined candidate genes to predict evolutionary mechanisms has so far been most fruitful in explaining intraspecific variants, where it has been effective in both plants and animals. In several cases these efforts were facilitated or supported by parallel results from quantitative trait loci studies, in which natural alleles controlling continuous variation in developmental model organisms were mapped to mutationally defined genes. However, despite these successes the approach's utility seems to rapidly decay as a function of phylogenetic distance. This suggests that the divergence of developmental genetic systems is great even in closely related organisms and may become intractable at larger distances. We discuss this result in the context of what it teaches us about development, the future prospects of the candidate gene approach, and the historical debate over process in micro- and macroevolution.

Developmental system drift and flexibility in evolutionary trajectories.

The comparative analysis of homologous characters is a staple of evolutionary developmental biology and often involves extrapolating from experimental data in model organisms to infer developmental events in non-model organisms. In order to determine the general importance of data obtained in model organisms, it is critical to know how often and to what degree similar phenotypes expressed in different taxa are formed by divergent developmental processes. Both comparative studies of distantly related species and genetic analysis of closely related species indicate that many characters known to be homologous between taxa have diverged in their morphogenetic or gene regulatory underpinnings. This process, which we call "developmental system drift" (DSD), is apparently ubiquitous and has significant implications for the flexibility of developmental evolution of both conserved and evolving characters. Current data on the population genetics and molecular mechanisms of DSD illustrate how the details of developmental processes are constantly changing within evolutionary lineages, indicating that developmental systems may possess a great deal of plasticity in their responses to natural selection.

Regulatory elements required for development of caenorhabditis elegans hermaphrodites are conserved in the tra-2 homologue of C. remanei, a male/female sister species.

The Caenorhabditis elegans hermaphrodite is essentially a female that produces sperm. In C. elegans, tra-2 promotes female fates and must be repressed to achieve hermaphrodite spermatogenesis. In an effort to learn how mating systems evolve, we have cloned tra-2 from C. remanei, the closest gonochoristic relative of C. elegans. We found its structure to be similar to that of Ce-tra-2 but its sequence to be divergent. RNA interference demonstrates that Cr-tra-2 promotes female fates. Two sites of tra-2 regulation are required for the onset of hermaphrodite spermatogenesis in C. elegans. One, the MX region of TRA-2, is as well conserved in C. remanei as it is in C. briggsae (another male/hermaphrodite species), suggesting that this control is not unique to hermaphrodites. Another, the DRE/TGE element of the tra-2 3' UTR, was not detected by sequence analysis. However, gel-shift assays demonstrate that a factor in C. remanei can bind specifically to the Cr-tra-2 3' UTR, suggesting that this translational control is also conserved. We propose that both controls are general and do not constitute a novel "switch" that enables sexual mosaicism in hermaphrodites. However, subtle quantitative or qualitative differences in their employment may underlie differences in mating system seen in Caenorhabditis.

Apextrin, a novel extracellular protein associated with larval ectoderm evolution in Heliocidaris erythrogramma.

During the evolution of direct development in the sea urchin Heliocidaris erythrogramma major modifications occurred, which allowed the precocious formation of adult-specific structures and led to a novel larval body that surrounds these structures. The HeET-1 gene was isolated in a differential screen for transcripts enriched in the early embryos of H. erythrogramma relative to those of its indirect-developing congener, H. tuberculata. HeET-1 was unique among the three genes found in that no homologous transcript was detected in H. tuberculata total embryonic RNA blots. To verify this apparently extreme differential expression of the HeET-1 genes in Heliocidaris, we isolated the HeET-1 homologue from H. tuberculata genomic DNA and used it to probe blots of poly(A)+ RNA prepared from H. tuberculata embryos. It is expressed in H. tuberculata embryos at levels undetectable by this technique. The predicted amino acid sequence of HeET-1 suggested that it encodes a novel secreted protein. To assess the function of HeET-1, we raised polyclonal antisera to the HeET-1-encoded protein. We find that it is present in eggs in a type of secretory vesicle and that this maternal pool is gradually secreted after fertilization. As cells acquire apical-basal polarity in the blastula the protein becomes localized to the apical extracellular matrix, leading us to name the protein apextrin. The apical extracellular localization of apextrin is maintained in the columnar cells of the larval ectoderm until their internalization at metamorphosis. Ingressing mesenchyme cells rapidly endocytose apextrin upon leaving the vegetal plate. Comparison with fibropellin III, an apical lamina component, suggests that apextrin is an extracellular protein that is in tighter association with the plasma membrane than is the hyalin layer or apical lamina. We propose that apextrin is involved in apical cell adhesion and that its high level of expression may represent an adaptive cooption necessary for strengthening the large H. erythrogramma embryo.

Application of intravascular brachytherapy: utilization of beta-irradiation source delivery systems within a community-based cardiac center.

PURPOSE: There are numerous clinical studies ongoing to assess the outcome, physics, and radiobiology of intravascular brachytherapy and its effect on the reduction of the rate of restenosis after balloon angioplasty procedures. The present study reports on the experience of two different delivery systems as utilized in the community hospital setting. METHODS AND MATERIALS: Patients were enrolled into one of four ongoing trials at our institution: the Novoste Beta-Cath trial, the Novoste Stents and Radiation Therapy trial (START), the Novoste START 40/20 trial, and the Guidant Intimal Hyperplasia Inhibition with Beta In-stent Trial (INHIBIT). The Novoste studies utilized the Novoste Beta-Cath System with 90Sr/Y, and the Guidant INHIBIT trial used 32P. Enrollments into the various trials were determined by inclusion and exclusion criteria specified by each protocol. Randomization was conducted per criteria as determined by the participating study protocol. RESULTS: Forty-two patients were enrolled in total. Thirty-four were included in the Novoste trials and eight in the Guidant study, according to availability of the trial. Assessment of practicality of treatment was dependent primarily on treatment duration and extension of time of catheterization procedures by the addition of intravascular radiation. Average dwell time within the Novoste trials was 3 min 40 s, and 7 min 46 s for patients in the Guidant study. No acute complications were observed in any of the trials. CONCLUSIONS: Intravascular brachytherapy can be performed in the community hospital setting without compromising the efficiency of balloon angioplasty procedures. Pending long-term outcome data and FDA approval for specific delivery systems, endovascular brachytherapy in community hospital cardiac catheterization laboratories can be realized in an efficient and timely manner.

Isolation and characterization of three mRNAs enriched in embryos of the direct-developing sea urchin Heliocidaris erythrogramma: evolution of larval ectoderm.

The Australian sea urchin Heliocidaris erythro-gramma utilizes a derived direct developmental mode that evolved 8-12 million years ago. From a differential screen we have isolated a small set of cDNAs corresponding to genes more greatly expressed in embryos of H. erythrogramma than in those of its indirect-developing nearest relative, H. tuberculata. The method was biased towards abundant transcripts and did not allow detection of modifications of usage of highly conserved gene family members. Three differentially expressed abundant transcripts were found that potentially encode secreted proteins. Two of these, the arylsulfatase HeARS and the putative lectin HeEL-1, were identifiable as homologues of known proteins. Another gene, HeET-1, may be exclusively expressed in the H. erythrogramma embryo. In situ hybridization experiments demonstrate that all three transcripts are localized to the ectoderm. Two of them, HeET-1 and HeEL-1, are transcribed in an identical domain comprising the larval ectoderm. This region of gene expression has acquired a novel columnar cytology during the evolution of the H. erythrogramma embryo. The third sequence, HeARS, encodes an arylsulfatase homologue. Its expression is uniform in the gastrula, but as the rudiment develops it accumulates to the greatest extent in the invaginating vestibular ectoderm. Through comparisons with indirect-developing species, we show that this concentration of arylsulfatase mRNA in the rudiment is a novel feature of H. erythrogramma development. These data suggest that H. erythrogramma has a unique arrangement of ectodermal gene expression territories. We propose that these reflect larval adaptations that have occurred in the lineage leading to H. erythrogramma, and enabled the evolution of direct development.