DISSERTATION RESEARCH: Mutant models reveal latent developmental potential with roles in evolutionary change

本项目将研究导致鱼鳍骨骼形态复杂的遗传因素。骨模式的形成源于骨数量和骨连接的增加，这类研究对于理解进化和发育过程非常重要。在进化过程中，端对端排列的骨骼数量的增加有助于鳍变成四肢，并有助于灭绝的脊椎动物在陆地上定居。在发育过程中，正常的功能和运动取决于形成适当的骨数量和连接，而发育缺陷可能导致严重的损害。目前，导致鳍骨骼细化形成肢体的机制尚不清楚。为了确定相关的遗传因素，研究人员将研究模式物种斑马鱼的菌株，这些物种的基因变化会导致新的鳍骨的形成。他们还将调查两种自然发生的变异，gar和bowfin，它们有不同数量的骨头从肩膀延伸出来。这些互补的方法将揭示调节鳍和四肢骨骼生长和形成的机制，并展示简单的基因变化如何导致鳍形成四肢。了解骨模式的形成和发育机制对人类健康和发育异常具有重要意义，并可能为陆地生命的进化提供线索。此外，该项目将为美国原住民本科生提供研究机会。虽然四足脊椎动物增加了肢体近端-远端（P-D）轴上的骨骼数量，但斑马鱼等硬骨鱼减少了胸鳍骨架；沿P-D轴只发现了一根长骨。研究人员对斑马鱼进行了前向突变筛选，并分离出一种新的突变体，这种突变体沿着鳍的P-D轴形成多余的骨头，其过程与四足动物四肢的发育过程类似。拟议的研究将把突变分析的结果扩展到自然种群，以确定突变是否通过激活祖先遗传机制而产生多余的骨骼。RNA测序转录组分析将用于比较野生型和突变型斑马鱼胸鳍发育中的基因表达水平，以及两种在P-D轴发育中表现出自然变异的物种。该分析将揭示与P-D轴细化相关的差异基因表达模式。差异表达基因将在斑马鱼中进行测试，以确定它们的修饰表达是否对鳍形态有影响。这项研究将阐明在发育过程中如何产生加性表型变化，并确定在进化和疾病中形成额外鳍/肢骨的候选途径。

This project will investigate the genetic factors that cause elaborated bone pattern in fish fins. Bone pattern elaboration results from increases in bone numbers and connections, and such research is important for understanding processes of both evolution and development. In evolution, increase in the number of bones arranged end-to-end helped transform fins into limbs and aided the colonization of land by extinct vertebrates. In development, normal function and movement depends upon formation of the proper bone numbers and connections, and defective development can lead to severe impairment. Currently, the mechanisms that led to elaboration of the fin skeleton to form limbs are unknown. To identify the genetic factors involved, the researchers will study strains of the model species, zebrafish, that have genetic changes that result in the formation of new fin bones. They will also investigate naturally occurring variation in two species, gar and bowfin, that have different numbers of bones extending from the shoulder. These complementary approaches will reveal the mechanisms regulating growth and formation of the skeleton of fins and limbs, and show how simple genetic changes can lead to the formation of limbs from fins. Understanding the mechanisms of bone pattern elaboration and development has implications for human health and developmental abnormalities, and may provide clues for the evolution of life on land. Additionally, the project will provide research opportunities for Native American undergraduates. While tetrapod vertebrates have increased the number of bones along the proximo-distal (P-D) axis of the limb, teleost fishes such as the zebrafish have reduced the pectoral fin skeleton; only a single long bone is found along the P-D axis. The researchers performed a forward mutagenesis screen in zebrafish and isolated a novel mutant that forms supernumerary bones along the P-D axis of the fin through a process similar to that seen in the development of tetrapod limbs. The proposed research will extend results from mutant analysis to natural populations to determine if the mutation is producing supernumerary bones by activating ancestral genetic mechanisms. Transcriptome profiling by RNA sequencing will be used to compare gene-expression levels in the developing pectoral fins of wild-type and mutant zebrafish, as well as two species that exhibit natural variation in P-D axis elaboration. This analysis will reveal patterns of differential gene expression that correlate with P-D axis elaboration. Differentially expressed genes will be tested in the zebrafish to determine if their modified expression has an effect on fin morphology. This study will elucidate how additive phenotypic changes arise in development and identify candidate pathways by which additional fin/limb bones are formed in both evolution and disease.