The <i>Drosophila fussel</i> gene is required for bitter gustatory neuron differentiation acting within an Rpd3 dependent chromatin modifying complex

Members of the Ski/Sno protein family are classified as proto-oncogenes and act as negative regulators of the TGF-ß/BMP-pathways in vertebrates and invertebrates. A newly identified member of this protein family is fussel (fuss), the Drosophila homologue of the human functional Smad suppressing elements (fussel-15 and fussel-18). We and others have shown that Fuss interacts with SMAD4 and that overexpression leads to a strong inhibition of Dpp signaling. However, to be able to characterize the endogenous Fuss function in Drosophila melanogaster, we have generated a number of state of the art tools including anti-Fuss antibodies, specific fuss-Gal4 lines and fuss mutant fly lines via the CRISPR/Cas9 system. Fuss is a predominantly nuclear, postmitotic protein, mainly expressed in interneurons and fuss mutants are fully viable without any obvious developmental phenotype. To identify potential target genes or cells affected in fuss mutants, we conducted targeted DamID experiments in adult flies, which revealed the function of fuss in bitter gustatory neurons. We fully characterized fuss expression in the adult proboscis and by using food choice assays we were able to show that fuss mutants display defects in detecting bitter compounds. This correlated with a reduction of gustatory receptor gene expression (Gr33a, Gr66a, Gr93a) providing a molecular link to the behavioral phenotype. In addition, Fuss interacts with Rpd3, and downregulation of rpd3 in gustatory neurons phenocopies the loss of Fuss expression. Surprisingly, there is no colocalization of Fuss with phosphorylated Mad in the larval central nervous system, excluding a direct involvement of Fuss in Dpp/BMP signaling. Here we provide a first and exciting link of Fuss function in gustatory bitter neurons. Although gustatory receptors have been well characterized, little is known regarding the differentiation and maturation of gustatory neurons. This work therefore reveals Fuss as a pivotal element for the proper differentiation of bitter gustatory neurons acting within a chromatin modifying complex.Ski/Sno蛋白家族（Ski/Sno protein family）成员被归类为原癌基因，在脊椎动物与无脊椎动物中均作为转化生长因子β/骨形态发生蛋白（TGF-β/BMP）信号通路的负调控因子发挥功能。该家族新鉴定的成员为fussel（简称fuss），即人类功能性Smad抑制元件fussel-15与fussel-18的果蝇同源物。本团队及其他研究团队已证实，Fuss可与SMAD4相互作用，且其过表达会强烈抑制Dpp信号通路。然而，为阐明黑腹果蝇（Drosophila melanogaster）内源性Fuss的生物学功能，我们借助CRISPR/Cas9系统构建了一系列前沿研究工具，包括抗Fuss抗体、特异性fuss-Gal4果蝇品系以及fuss突变体果蝇品系。Fuss主要定位于细胞核，属于有丝分裂后蛋白，主要在中间神经元中表达；fuss突变体果蝇可正常存活且无明显发育表型。为鉴定fuss突变体中受影响的潜在靶基因或细胞类型，我们在成虫果蝇中开展了靶向DamID实验，结果揭示了fuss在苦味味觉神经元中的功能。我们完整表征了成虫果蝇喙部的fuss表达模式，并通过食物选择实验证实，fuss突变体在苦味物质检测方面存在缺陷。该表型与味觉受体基因（Gr33a、Gr66a、Gr93a）的表达水平下调相关，为这一行为表型提供了分子层面的关联依据。此外，Fuss可与Rpd3相互作用，在味觉神经元中下调rpd3的表达可重现Fuss表达缺失所导致的表型。令人意外的是，在幼虫中枢神经系统中并未观察到Fuss与磷酸化Mad的共定位，这排除了Fuss直接参与Dpp/BMP信号通路的可能性。本研究首次阐明了Fuss在苦味味觉神经元中的功能关联。尽管味觉受体的功能已得到充分研究，但目前对于味觉神经元的分化与成熟机制仍知之甚少。因此，本工作揭示Fuss作为染色质修饰复合物（chromatin modifying complex）中的关键调控元件，在苦味味觉神经元的正常分化过程中发挥核心作用。