Lee Gyunghee G, Peterson Aidan J, Kim Myung-Jun, Shimell MaryJane, O'Connor Michael B, Park Jae H
Department of Biochemistry, Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee, United States of America.
Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota, United States of America.
PLoS One. 2025 May 30;20(5):e0318406. doi: 10.1371/journal.pone.0318406. eCollection 2025.
Evolutionarily conserved transforming growth factor β (TGF-β) signaling is used in both vertebrates and invertebrates to regulate a variety of developmental and cellular processes. The baboon (babo) gene encoding a Drosophila type-I TGF-β receptor produces three isoforms via alternative splicing: BaboA, BaboB, and BaboC. In this study, we generated three fly lines, each carrying an isoform-specific GFP tag, and another line with a GFP conjugated at the C-terminus common to all isoforms. Using these lines, we assessed (1) whether the tagged proteins function properly in rescue assays and (2) how the isoform expression is regulated in various tissues including the central nervous system (CNS). A Gal4 knock-in line in the babo locus was also characterized for reporter expression, mutant phenotypes, and isoform-specific knockdown phenotypes. We found that the C-terminal tag does not interrupt the subcellular targeting and functions of the tagged isoforms, but the internal isoform tags do so in a cell- and isoform-specific fashion. Nevertheless, our results demonstrated that these tags faithfully reflect endogenous expression of individual isoforms. Certain cell types express single or multiple isoforms at different levels, suggesting that alternative splicing could determine the isoform types and their levels depending on cell (or tissue) type. The larval CNS displays distinct patterns of two isoforms, BaboA and BaboC. BaboC is mostly expressed in neural cells originating during embryogenesis, while BaboA is broadly expressed in neural cells produced from both embryonic and postembryonic stages. Assays of both isoform-specific mutants and cell-specific knockdown of individual isoforms revealed broad roles played by BaboA in postembryonic neurogenesis and differentiation of precursor neurons, remodeling processes of persisting larval neurons, and metamorphic CNS reorganization, which are essential for establishing of the adult CNS. Taken together, this study demonstrates that the GFP-tagged lines permit visualization of endogenous expression of individual isoforms, which further provides clues about cell- and stage-specific functions played by each isoform.
进化上保守的转化生长因子β（TGF-β）信号通路在脊椎动物和无脊椎动物中都用于调节多种发育和细胞过程。编码果蝇I型TGF-β受体的狒狒（babo）基因通过可变剪接产生三种异构体：BaboA、BaboB和BaboC。在本研究中，我们构建了三个果蝇品系，每个品系携带一种异构体特异性的绿色荧光蛋白（GFP）标签，以及另一个在所有异构体共同的C末端缀合有GFP的品系。利用这些品系，我们评估了：（1）标记蛋白在拯救实验中是否能正常发挥功能；（2）异构体表达在包括中枢神经系统（CNS）在内的各种组织中是如何受到调控的。还对babo基因座中的一个Gal4敲入品系进行了报告基因表达、突变体表型和异构体特异性敲低表型的表征。我们发现C末端标签不会干扰标记异构体的亚细胞定位和功能，但内部异构体标签会以细胞和异构体特异性的方式干扰。尽管如此，我们的结果表明这些标签忠实地反映了各个异构体的内源性表达。某些细胞类型以不同水平表达单一或多种异构体，这表明可变剪接可以根据细胞（或组织）类型决定异构体的类型及其水平。幼虫中枢神经系统显示出两种异构体BaboA和BaboC的不同模式。BaboC主要在胚胎发生期间起源的神经细胞中表达，而BaboA广泛表达于胚胎期和胚胎后期产生的神经细胞中。对异构体特异性突变体和单个异构体的细胞特异性敲低的分析揭示了BaboA在胚胎后期神经发生、前体神经元分化、持续存在的幼虫神经元重塑过程以及变态中枢神经系统重组中发挥的广泛作用，这些对于建立成体中枢神经系统至关重要。综上所述，本研究表明绿色荧光蛋白标记的品系能够可视化各个异构体的内源性表达，这进一步为每种异构体所发挥的细胞和阶段特异性功能提供了线索。
