要約のみTohoku University小金澤雅之課

Md Tanveer Ahsan

English

Tohoku University博士（生命科学）博士学位論文 (Thesis(doctor))要約のみthesi

Md, Tanveer Ahsan

Tohoku University Repository (TOUR) / 東北大学機関リポジトリ

Calmodulin-binding transcription factor shapesthe male courtship song in Drosophila著者 Md Tanveer Ahsa学位授与機関 Tohoku University学位授与番号 11301URL http://hdl.handle.net/10097/00126436      博士論文（要約）   Calmodulin-binding transcription factor shapes the male courtship song in Drosophila （雄の求愛歌の特性決定に関与する croaker 遺伝子の同定とその機能の解析）         平成 30年度 東北大学大学院生命科学研究科 生命機能科学専攻 Md. Tanveer Ahsan    1 Introduction Many animals utilize acoustic signals or songs to attract mates. During courtship, Drosophila melanogaster males vibrate a wing to produce characteristic sounds called pulse and sine song, respectively. However, genetic and neural mechanisms regulating courtship songs have not been fully elucidated. Males of the Drosophila melanogaster mutant croaker (cro) generate a polycyclic pulse song dissimilar to a monocyclic song typical of wild-type males during courtship. However, cro has not been molecularly mapped to any gene in the genome. In this study, I molecularly and functionally characterized the cro locus to understand how the nervous system shapes the male courtship song.   Results In this study, I demonstrated that cro is a mutation in the gene encoding the Calmodulin-binding transcription activator (Camta). A previous study mapped cro to the chromosomal location 45DE on the 2nd chromosome. Based on this knowledge, I performed genetic complementation tests with chromosomal deficiencies located around this cytological region. Some of the tested deficiencies produced the cro like phenotype when placed in trans to cro. The deficiency mapping data placed the cro locus between the cytological positions 45D8 and 45E3. In this cytological interval, three genes have been annotated, i.e., bruchpilot (brp), Wnt2 and Camta. To narrow down the gene responsible for the cro phenotype, I carried out a rescue experiment by introducing an intact cro locus into the mutant chromosome with a BAC clone (BAC-CH321-22B08) that contains the entire Camta locus, but neither brp nor Wnt2 locus. The BAC clone rescued the cro mutant phenotype, suggesting that cro is allelic to Camta. Since cro is a P-element insertion mutant, I next determined the P-element insertion point by two independent methods: plasmid rescue and inverse PCR. Both methods suggested that the cro mutant chromosome has a P-element insertion in  2 the fourth intron of the Camta gene. To further validate this result, I collected several available fly lines having a transposon insertion in the Camta locus to conduct genetic complementation tests with cro. Indeed, some of the chromosomes carrying a transposon insertion (e.g., MiMICMI04570) failed to rescue the cro phenotype, indicating that the cro phenotype is rescued from deficits of Camta. This notion is supported by the qPCR experiment that showed that the Camta mRNA amount is significantly reduced in the cro mutant. I performed a knockdown experiment with UAS-Camta RNAi using several brain cell specific GAL4 drivers. Two pan-neural GAL4 (elav-GAL4 and nSyb-GAL4) induced polycyclic songs typical of cro mutants whereas Glia specific repo-GAL4 did not when used to drive UAS-Camta RNAi, indicating the role of Camta in neurons. To further narrow down the neural subset responsible for the cro phenotype, I performed cro knockdown in fru-GAL4 or dsx-GAL4 positive cells, because the core portion of the male courtship circuit is composed of fru and/or dsx expressing neurons. fru-GAL4-driven Camta RNAi expression induced polycyclic songs, indicating that fru[+] neurons are involved in inducing the cro phenotype. Cells positive for dsx-GAL4 had no effect on pulse song upon knocking down of Camta. It has been postulated that courtship decision-making is made in the brain, whereas the motor pattern generator for courtship song resides in the ventral nerve cord (VNC). To determine where in the central nervous system (CNS) cro is required for normal song generation, I performed head specific knockdown of Camta in fru[+] cells  with OtdFLP, which releases GAL4 from GAL80-mediated repression by flipping the STOP sequence out from tub>GAL80> only in the head. Knockdown of Camta in fru[+] neurons in the head induced polycyclic pulse song, indicating their role in pulse song generation. Taking into account the observation that the head-restricted cro knockdown in fru-GAL4-expressing neurons induced polycyclic pulse song, I tested whether cro knockdown in a few fru-expressing brain neurons known to have a pivotal  3 role in courtship song generation can induce polycyclic pulse song. I first examined pIP10 neurons, which represent laterally paired descending interneurons with the command fiber-like function to initiate male courtship upon artificial activation. These interneurons have been reported to trigger pulse song generation when forcibly activated. Notably, cro knockdown specifically targeted to pIP10 neurons by the VT40556-GAL4 did not induce polycyclic pulse song during male courtship toward a female. Next, I examined the effect of cro knockdown in mcALa neurons, which have been implicated in the ordering of courtship elementary actions. Interestingly, the mcALa-specific intersectional combination p52a-GAL4 and fruFLP led to the generation of polycyclic pulse song, suggesting a new role for mcALa, i.e., shaping pulse song. In contrast, P1-targetted cro knockdown by the intersection of NP2631-GAL4 and fruFLP did not induce polycyclic pulse song during male courtship. To decipher whether cro function is required for shaping pulse song during courtship actions or in the development of the courtship song circuit, I also conducted a stage-restricted cro knockdown experiment. Knockdown of Camta in developmental stages resulted in cro phenotype indicating its role in shaping the song circuit in developmental stages.  Conclusion I demonstrated that the cro chromosome carries a P-element insertion in the fourth intron of the Camta locus, which decreases the amount of Camta mRNA in the cro mutant. Camta knockdown in a defined set of cells suggested that cro acts in fru[+] mcALa neurons in the brain to shape monocyclic pulse song. This finding points to the role of brain in pulse song shaping, which has been ascribed to the motor pattern generator for courtship song locating in the mesothoracic segment of the ventral nerve cord (e.g., the TN1 cluster). It would be thus interesting to examine the neural circuit mechanism whereby mcALa controls the action of the song pattern generator in the VNC to generate  4 monocyclic songs. Since cro has been implicated in the generation of the species difference in courtship songs between D.sechellia and D.simulans, it would also be interesting to examine an evolutionary divergence in the cro locus as a substrate of different courtship songs among species. This possibility can be tested by the experiment, where the entire Camta locus of one species replaces the counterpart in the other species to see whether this cro gene exchange is accompanied by the exchange of song characteristics.   

Calmodulin-binding transcription factor shapes the male courtship song in Drosophila

博士学位論文の要旨及び審査結果の要旨 (Summary of Thesis(DR))othe

モハンマド, タンビール アーサン

Calmodulin-binding transcription factor shapesthe male courtship song in Drosophila著者 Md Tanveer Ahsa号 17学位授与機関 Tohoku University学位授与番号 生博第385号URL http://hdl.handle.net/10097/00126471  氏名（本籍地） 学 位 の 種 類 学 位 記 番 号 学位授与年月日 学位授与の要件 研 究 科 ， 専 攻  論 文 題 目    博士論文審査委員  モハンマド タンビール アーサン Md Tanveer Ahan  博士（生命科学） 生博第３８５号 令和元年８月８日 学位規則第４条第１項該当 東北大学大学院生命科学研究科 （博士課程）生命機能科学専攻 Calmodulin-binding transcription factor shapes  the male courtship song in Drosophila （雄の求愛歌の特性決定に関与する croaker遺伝子の同定とその機能の解析）  （主査）  准教授 小金澤 雅之 教授  東谷 篤志 教授  安部 健太郎 准教授 山方 恒宏                     論文内容の要旨 Introduction Many animals utilize acoustic signals or songs to attract mates. During courtship, Drosophila melanogaster males vibrate a wing to produce characteristic sounds called pulse and sine song, respectively. However, genetic and neural mechanisms regulating courtship songs have not been fully elucidated. Males of the Drosophila melanogaster mutant croaker (cro) generate a polycyclic pulse song dissimilar to a monocyclic song typical of wild-type males during courtship. However, cro has not been molecularly mapped to any gene in the genome. In this study, I molecularly and functionally characterized the cro locus to understand how the nervous system shapes the male courtship song.   Results In this study, I demonstrated that cro is a mutation in the gene encoding the Calmodulin-binding transcription activator (Camta). A previous study mapped cro to the chromosomal location 45DE on the 2nd chromosome. Based on this knowledge, I performed genetic complementation tests with chromosomal deficiencies located around this cytological region. Some of the tested deficiencies produced the cro like phenotype when placed in trans to cro. The deficiency mapping data placed the cro locus between the cytological positions 45D8 and 45E3. In this cytological interval, three genes have been annotated, i.e., bruchpilot (brp), Wnt2 and Camta. To narrow down the gene responsible for the cro phenotype, I carried out a rescue experiment by introducing an intact cro locus into the mutant chromosome with a BAC clone (BAC-CH321-22B08) that contains the entire Camta locus, but neither brp nor Wnt2 locus. The BAC clone rescued the cro mutant phenotype, suggesting that cro is allelic to Camta. Since cro is a P-element insertion mutant, I next determined the P-element insertion point by two independent methods: plasmid rescue and inverse PCR. Both methods suggested that the cro mutant chromosome has a P-element insertion in the fourth intron of the Camta gene. To further validate this result, I collected several available fly lines having a transposon insertion in the Camta locus to conduct genetic complementation tests with cro. Indeed, some of the chromosomes carrying a transposon insertion (e.g., MiMICMI04570) failed to rescue the cro phenotype, indicating that the cro phenotype is rescued from deficits of Camta. This notion is supported by the qPCR experiment that showed that the Camta mRNA amount is significantly reduced in the cro mutant. I performed a knockdown experiment with UAS-Camta RNAi using several brain cell specific GAL4 drivers. Two pan-neural GAL4 (elav-GAL4 and nSyb-GAL4) induced polycyclic songs typical of cro mutants whereas Glia specific repo-GAL4 did not when used to drive UAS-Camta RNAi, indicating the role of Camta in neurons. To further narrow down the neural subset responsible for the cro phenotype, I performed cro knockdown in fru-GAL4 or dsx-GAL4 positive cells, because the core portion of the male courtship circuit is composed of fru and/or dsx expressing neurons. fru-GAL4-driven Camta RNAi expression induced polycyclic songs, indicating that fru[+] neurons are involved in inducing the cro phenotype. Cells positive for dsx-GAL4 had no effect on pulse song upon knocking down of Camta. It has been postulated that courtship decision-making is made in the brain, whereas the motor pattern generator for courtship song resides in the ventral nerve cord (VNC). To determine where in the central nervous system (CNS) cro is required for normal song generation, I performed head specific knockdown of Camta in fru[+] cells  with OtdFLP, which releases GAL4 from GAL80-mediated repression by flipping the STOP sequence out from tub>GAL80> only in the head. Knockdown of Camta in fru[+] neurons in the head induced polycyclic pulse song, indicating their role in pulse song generation. Taking into account the observation that the head-restricted cro knockdown in fru-GAL4-expressing neurons induced polycyclic pulse song, I tested whether cro knockdown in a few fru-expressing brain neurons known to have a pivotal role in courtship song generation can induce polycyclic pulse song. I first examined pIP10 neurons, which represent laterally paired descending interneurons with the command fiber-like function to initiate male courtship upon artificial activation. These interneurons have been reported to trigger pulse song generation when forcibly activated. Notably, cro knockdown specifically targeted to pIP10 neurons by the VT40556-GAL4 did not induce polycyclic pulse song during male courtship toward a female. Next, I examined the effect of cro knockdown in mcALa neurons, which have been implicated in the ordering of courtship elementary actions. Interestingly, the mcALa-specific intersectional combination p52a-GAL4 and fruFLP led to the generation of polycyclic pulse song, suggesting a new role for mcALa, i.e., shaping pulse song. In contrast, P1-targetted cro knockdown by the intersection of NP2631-GAL4 and fruFLP did not induce polycyclic pulse song during male courtship. To decipher whether cro function is required for shaping pulse song during courtship actions or in the development of the courtship song circuit, I also conducted a stage-restricted cro knockdown experiment. Knockdown of Camta in developmental stages resulted in cro phenotype indicating its role in shaping the song circuit in developmental stages.  Conclusion I demonstrated that the cro chromosome carries a P-element insertion in the fourth intron of the Camta locus, which decreases the amount of Camta mRNA in the cro mutant. Camta knockdown in a defined set of cells suggested that cro acts in fru[+] mcALa neurons in the brain to shape monocyclic pulse song. This finding points to the role of brain in pulse song shaping, which has been ascribed to the motor pattern generator for courtship song locating in the mesothoracic segment of the ventral nerve cord (e.g., the TN1 cluster). It would be thus interesting to examine the neural circuit mechanism whereby mcALa controls the action of the song pattern generator in the VNC to generate monocyclic songs. Since cro has been implicated in the generation of the species difference in courtship songs between D.sechellia and D.simulans, it would also be interesting to examine an evolutionary divergence in the cro locus as a substrate of different courtship songs among species. This possibility can be tested by the experiment, where the entire Camta locus of one species replaces the counterpart in the other species to see whether this cro gene exchange is accompanied by the exchange of song characteristics.              論文審査結果の要旨  ショウジョウバエ属では行動の種差が、種間の生殖隔離に寄与していることが少なくない。特に、雄の発する求愛歌の特徴を雌が聞き分け、それにより交尾をするか否かの意思決定が左右されることが知られている。しかし、その重要性にも拘らず、求愛歌を生み出す遺伝子や神経回路の種差については、その多くが未解明である。本研究では、キイロショウジョウバエを用いて求愛歌の生成に寄与する遺伝子を同定するとともに、その機能の場を細胞レベルで特定することを目指した。  キイロショウジョウバエの求愛歌はパルス歌とサイン歌の 2タイプからなる。このうち、パルス歌に異常の生ずる突然変異体として、croaker (cro)が知られている。野生型の雄ではパルスのピークは一個（monocyclic）であるが、cro変異体雄のパルスには二つのピークを持つ（polycyclic）ものが高い頻度で含まれる。プラスミドレスキュー法と inverse PCRにより cro原因遺伝子をクローニングし、Calmodulin-binding transcription factor (Camta)をコードする遺伝子を特定した。実際、正常型 Camtaを含む BAC cloneを cro変異体に導入すると、パルス歌の異常が救済され、Camta座を欠く欠失染色体は cro変異を相補しなかった。CamtaRNAiを組織特異的に発現させて cro変異体と同様の表現型を誘導する実験から、ニューロンでの Camtaノックダウンが有効であることを見出した。加えて、雄の求愛行動回路形成においてマスターレギュレータとして働く fruitless (fru)遺伝子に着目し、その発現ニューロンに限定した Camtaノックダウンを行うと、cro様表現型が現れた。そこで fru発現細胞の一部のみを標的としたノックダウンする実験を行い、mcALと呼ばれる30個のニューロンでの Camtaのノックダウンが、cro様のパルス歌の異常を生じさせることが明らかとなった。 この様に、求愛歌産生に寄与する遺伝子と神経細胞の特定に大きな成果を挙げており、この事実は Tanveer Ahsanが自立して研究活動を行うに必要な高度の研究能力と学識を有することを示している。したがって，Tanveer Ahsan提出の論文は，博士（生命科学）の博士論文として合格と認める。  

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雄の求愛歌の特性決定に関与するcroaker遺伝子の同定とその機能の解析

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