Evolution of Cavefish Eye Degeneration

项目摘要智力价值：洞穴动物视力丧失的进化和发育机制尚不清楚。这一建议继续了Jeffery博士对硬骨鱼眼睛退化的研究，这种硬骨鱼有眼的表面栖息形式(表层鱼，SF)和许多无眼的洞穴栖息形式(穴居鱼，CF)。Cf形成眼球原基，但由于晶状体细胞凋亡，它们停止生长并退化。他之前的资助结果表明：(1)Hedgehog(HH)信号在CF前线被增强，并通过诱导晶状体凋亡来控制眼睛的退化；(2)分子伴侣Hsp90在CF晶状体中被特异性激活并参与晶状体凋亡；(3)HH在胚胎中线的表达逐渐限于味蕾发育；以及(4)受HH信号正调控的基因(如在视囊泡中的pax2.1a和在神经板中的nkx2.1a/b)的表达在CF中被扩大。可能补偿视觉的建设性特征，包括味蕾、嗅神经束和球茎的增加，以及腹侧端脑和下丘脑，也在CF中进化。这一延续方案探索了一种假设，即由于HH中线信号的增强，眼睛已经退化，尽管对下游靶基因的多重营养积极和消极影响导致了建设性性状和退化性状之间的发育权衡。具体目标是：(1)确定HH信号是否通过影响Dlx5(和/或Pitx3)和Pax6转录因子之间的拮抗和/或通过调节Hsp90表达和协调地促进嗅觉发育来控制晶状体细胞凋亡；(2和3)确定HH信号增强是否与(2)CF中味蕾数量增加和(3)CF中腹侧端脑和下丘脑增大有关；以及(4)通过基因分析确定味觉、嗅觉和下丘脑增强是否与眼睛退化作为发育权衡而耦合。继续工作中使用的大多数方法和试剂，包括一个由100多个Astianax cDNA组成的小组，都是在之前的拨款中获得的，杰弗里博士的初步结果表明，他的假设是可信的，并准备好进行更深入的研究。这项研究的结果有望为CF眼退化的机制提供新的见解。更广泛的影响：对洞穴动物的研究促进了发现和理解，因为它们为进化发育生物学的研究和教育提供了独特的视角。两个假说，中性突变和自然选择，已经被提出来解释退化的变化，如洞穴动物的眼睛丧失，但直到最近，很少或没有实验支持这两个假说。这些实验首次表明，失明是自然选择通过多效性HH信号的积极和消极影响对适应性性状产生的间接影响。多效性效应可能会对发展的演变产生普遍的影响。除了HH途径外，其他关键的发育信号通路都有负向和正向调控靶标，并有可能通过发育权衡来产生进化变化。通过多效性产生的进化变化在很大程度上被低估了，因为它是一种引导发育中微进化变化的机制，这些研究旨在更多地阐明这一过程。这项研究的另一个广泛影响是研究和教育的持续整合，这是通过让本科生参与原创研究，通过培训博士后和博士后研究人员，以及通过实验室扩展到高中科学项目来实现的。所有这些活动都包括不同性别和代表性不足的少数群体。洞穴鱼实验室和群体将继续是向高中生教授和理解进化生物学的有用和高度赞赏的当地资源，也将继续作为他们短期监督科学项目的资源。洞穴鱼眼睛的退化是形态变化的一个很好的例子，这种变化在实验上是容易处理的，发生在最近，并受到进化论的解释。最后，洞穴鱼群的影响超出了这个实验室，它为其他研究人员提供了支持，并在国际上推动了类似群体的产生。

Project SummaryIntellectual Merit: The evolutionary and developmental mechanisms responsible for the loss of vision in cave animals are not understood. This proposal continues Dr. Jeffery's research on eye degeneration in the teleost Astyanax mexicanus, which has an eyed surface-dwelling form (surface fish, SF) and many eyeless cave-dwelling forms (cavefish, CF). CF form eye primordia but they arrest in growth and degenerate due to lens apoptosis. The results of his previous grant showed that: (1) Hedgehog (Hh) signaling is enhanced along the CF anterior midline and controls eye degeneration by inducing lens apoptosis, (2) the molecular chaperone Hsp90 is specifically activated in the CF lens and required for lens apoptosis, (3) Hh expression along the embryonic midline is gradually restricted to developing taste buds, and (4) expression of genes positively regulated by Hh signals (e. g. pax2.1a in the optic vesicles and nkx2.1a/b in the neural plate) are expanded in CF. Constructive traits that may compensate for vision, including increases in taste buds, olfactory nerve tracts and bulbs, and the ventral telencephalon and hypothalamus, have also evolved in CF. This continuation proposal explores the hypothesis that eyes have degenerated due to enhanced Hh midline signaling though pleiotrophic positive and negative effects on downstream target genes resulting in developmental tradeoffs between constructive and regressive traits. The specific objectives are: (1) to determine whether Hh signaling controls lens apoptosis by affecting an antagonism between the Dlx5 (and/or Pitx3) and Pax6 transcription factors and/or by regulating Hsp90 expression and coordinately increasing olfactory development, (2 and 3) to determine whether enhanced Hh signaling is responsible for (2) the increased number of taste buds in CF and (3) for enlargement of the ventral telencephalon and hypothalamus in CF, and (4) to determine by genetic analysis whether gustatory, olfactory, and hypothalamic enhancement are coupled to eye degeneration as developmental tradeoffs. Most of the methodologies and reagents to be used in the continued work, including a panel of more than 100 Astyanax cDNAs, have been acquired in the previous grants, and Dr. Jeffery has preliminary results showing that his hypothesis is plausible and ready for more intensive investigation. The results of this study are expected to provide new insights into the mechanisms responsible for CF eye degeneration.Broader Impacts: Studies of cave animals advance discovery and understanding because they provide unique perspectives to research and education in evolutionary developmental biology. Two hypotheses, neutral mutation and natural selection, have been advanced to explain regressive changes such as eye loss in cave animals, but until recently there was little or no experimental support for either of them. These experiments on Astyanax CF are the first to suggest that eye loss is an indirect effect of natural selection on adaptive traits via the positive and negative effects of pleiotropic Hh signals. Pleiotropic effects may have general implications in the evolution of development. In addition to the Hh pathway, other critical developmental signaling pathways have negative and positive regulated targets and the potential to generate evolutionary changes by developmental tradeoffs. The generation of evolutionary changes via pleiotropy is largely underappreciated as a mechanism for directing microevolutionary changes in development, and these studies are designed to shed more light on this process. Another broad impact of this research is the continued integration of research and education, which is accomplished by involving undergraduate students in original research, by training pre-doctoral and post-doctoral investigators, and by the laboratory's outreach to high school science programs. All of these activities include different genders and underrepresented minorities. The cavefish laboratory and colony will continue to be a useful and highly appreciated local resource for teaching and understanding evolutionary biology to high school students, and will also continue to serve as a resource for their short supervised science projects. The degeneration of the cavefish eye is an excellent example of a morphological change that is experimentally tractable, has occurred very recently, and is subject to interpretation through evolutionary theory. Finally, the cavefish colony has an impact beyond this laboratory, serving to support other researchers and springboard the generation of similar colonies internationally.