The Drosophila fussel gene is required for bitter gustatory neuron differentiation acting within an Rpd3 dependent chromatin modifying complex
The Drosophila fussel gene is required for bitter gustatory neuron differentiation acting within an Rpd3 dependent chromatin modifying complex
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DOI：
10.1371/journal.pgen.1007940
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发表时间：
2019-02-01
期刊：
影响因子：
4.5
通讯作者：
Schneuwly, Stephan
中科院分区：
文献类型：
--
作者：
Rass, Mathias;Oestreich, Svenja;Schneuwly, Stephan
Ski/ sno1蛋白家族成员被归类为原癌基因，在脊椎动物和无脊椎动物中作为TGF-ss/ bmp通路的负调控因子。该蛋白家族的一个新成员是fussel (fuss)，它是人类功能性Smad抑制元件（fussel-15和fussel-18）的果蝇同源物。我们和其他人已经证明Fuss与SMAD4相互作用，并且过表达导致Dpp信号的强烈抑制。然而，为了能够表征果蝇的内源性Fuss功能，我们已经通过CRISPR/Cas9系统生成了许多最先进的工具，包括抗Fuss抗体、特异性Fuss - gal4系和Fuss突变果蝇系。Fuss是一种核有丝分裂后蛋白，主要在中间神经元中表达，Fuss突变体完全存活，没有任何明显的发育表型。为了确定fuss突变可能影响的靶基因或靶细胞，我们在成年果蝇中进行了靶向DamID实验，揭示了fuss在苦味神经元中的功能。我们充分表征了fuss在成年长鼻中的表达，并通过使用食物选择测定，我们能够证明fuss突变体在检测苦味化合物方面表现出缺陷。这与味觉受体基因表达（Gr33a, Gr66a, Gr93a）的减少有关，这为行为表型提供了分子联系。此外，Fuss与Rpd3相互作用，味觉神经元中Rpd3的下调导致了Fuss表达的缺失。令人惊讶的是，在幼虫中枢神经系统中，没有发现Fuss与磷酸化的Mad共定位，排除了Fuss直接参与Dpp/BMP信号传导的可能性。在这里，我们提供了味觉苦味神经元中Fuss功能的第一个令人兴奋的联系。虽然味觉受体已经被很好地表征，但对于味觉神经元的分化和成熟知之甚少。因此，这项工作揭示了Fuss是在染色质修饰复合体中作用的苦味神经元适当分化的关键因素。Ski/ sno1蛋白已被发现是将鸡成纤维细胞转化为癌细胞的原癌基因。它们被发现在胚胎和成人组织中普遍表达，并干扰TGF-ss/BMP信号传导。最近，一组蛋白质被发现属于同一蛋白质家族，即功能性Smad抑制元件（Fussel）。它们具有高度受限的，主要是神经元的表达模式，这表明与Ski/ snow相比，它们具有不同的功能重要性。我们使用果蝇作为模型生物来表征高度特异性的神经元表达模式，并在果蝇基因中创建了敲除突变。令人惊讶的是，fuss突变体是完全可行的，但它们在苦味感知方面表现出缺陷，事实上，我们可以证明fuss在苦味感知神经元中特异性表达，在那里它影响了它们的终端分化，使这些细胞对苦味化合物不敏感。为了了解Fuss蛋白家族的分子功能，我们开始了蛋白质相互作用的研究，发现Fuss蛋白是染色质修饰复合体的一部分，这对成年神经系统中神经元的正常分化似乎很重要，因此，将果蝇作为研究Fuss蛋白家族分子功能不可或缺的模型。
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.Author summary Ski/Sno proteins have been discovered as proto-oncogenes transforming chicken fibroblasts into cancer cells. They have been found to be ubiquitously expressed in embryonic and adult tissues and to interfere with TGF-ss/BMP signaling. More recently, a group of proteins has been discovered which belongs to the same protein family, the functional Smad suppressing elements (Fussel). They have a highly restricted, mainly neuronal expression pattern suggesting different functional importance compared to Ski/Sno. We have used Drosophila as a model organism to characterize the highly specific neuronal expression pattern and created knock-out mutations within the Drosophila fuss gene. Surprisingly, fuss mutants are fully viable, but they show defects in bitter taste perception, and indeed, we could prove that Fuss is expressed specifically in bitter sensing neurons, where it affects their terminal differentiation making these cells insensitive for bitter compounds. To understand the molecular process involved in Fuss function we started protein interaction studies and could show, that Fuss forms part of a chromatin modifying complex, which seems to be important for the proper differentiation of neurons in the adult nervous system, therefore, assigning Drosophila as an indispensable model to study the molecular function of the Fuss protein family.