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The role of metabolism in the regulation of neural stem cell fate and proliferation in Drosophila melanogaster
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RESUMO: As células estaminais dependem de uma regulação metabólica rígida para manter o equilíbrio entre a autorrenovação e a diferenciação, um processo essencial para o desenvolvimento adequado dos tecidos e para a manter a homeostase. O metabolismo de um carbono é uma via central que liga a disponibilidade de nutrientes a processos celulares vitais, incluindo a biossíntese de nucleótidos, o equilíbrio redox e a regulação epigenética. Apesar da sua relevância estar amplamente documentada em sistemas mamíferos, o papel específico do metabolismo de um carbono nas células estaminais neurais e no desenvolvimento do cérebro permanece pouco caracterizado. Esta lacuna é ainda mais evidente em modelos invertebrados, particularmente no contexto do desenvolvimento neural em Drosophila melanogaster (referenciado como Drosophila). Para colmatar esta falha, utilizámos Drosophila como organismo modelo, para investigar os mecanismos através dos quais o metabolismo de um carbono regula o destino das células estaminais neurais e o desenvolvimento do tecido neural.
Começámos por avaliar o impacto da desregulação do metabolismo de um carbono na diferenciação e proliferação das NSCs da Drosophila, denominadas neuroblastos (NBs), no cérebro central (CB). Através do uso de RNA de interferência específica para as enzimas do ciclo de um carbono, silenciámos oito enzimas-chave deste ciclo e analisamos os efeitos na proliferação, crescimento e diferenciação dos NBs. Identificámos as enzimas Serina hidroximetiltransferase (Shmt), a timidilato sintetase (Ts) e a Adenosilhomocisteinase (Ahcy) como essenciais para a diferenciação adequada dos NBs, uma vez que a sua supressão prolongou o tempo de vida dos NBs e comprometeu a progressão normal das suas linhagens, especialmente nos NBs do tipo II. Estes resultados evidenciam o papel fundamental do metabolismo de um carbono na promoção da diferenciação atempada e na manutenção das linhagens celulares, talvez através da síntese de nucleótidos e à preservação da estabilidade epigenética.
Adicionalmente, investigámos se o metabolismo de um carbono também desempenha algum papel relevante no desenvolvimento neural precoce, focando-nos no lóbulo óptico (LO), onde as células neuroepiteliais (NE) transitam para NBs durante a fase larval. Demonstrámos que a Shmt é essencial para a proliferação, sobrevivência e organização das células NE. A falta de Shmt nos lóbulos ópticos revelou um aumento do apoptose, redução da expansão neuroepitelial e falha na formação da lamina furrow, culminando na perda de neurónios da lâmina. Estes resultados revelam um papel mais amplo do metabolismo de um carbono na manutenção da integridade epitelial, no suporte à função das células estaminais e na garantia da formação neuronal.
Para explorar de forma mais aprofundada a dinâmica metabólica subjacente à regulação das células estaminais, desenvolvemos um biossensor geneticamente codificado para detectar os níveis intracelulares de S-adenosilmetionina (SAM), o principal doador de grupos metil no ciclo de um carbono. Usando um sistema sintético derivado de levedura desenhamos versões do biossensor fluorescente (SFB) e luminescente (SLB). Estes biossensores foram testados em células S2 de Drosophila e em células P19 mamíferas. Embora o sensor baseado em luciferase necessite de otimizações adicionais, o SFB permitiu a deteção eficaz de SAM intracelular, estabelecendo as bases para futuros perfis metabólicos em tecidos vivos.
Em suma, esta tese demonstra que o metabolismo de um carbono um é um regulador central das transições das células estaminais neurais e da morfogénese dos tecidos em Drosophila melanogaster. Ao combinar manipulação genética e análise do desenvolvimento, mostramos que as vias metabólicas estão profundamente interligadas com o tempo de desenvolvimento, a especificação da linhagem e a competência das células estaminais. Este trabalho não só permite o entendimento do controlo metabólico da neurogénese, como também fornece novas ferramentas e abordagens para investigar como o metabolismo celular se relaciona com o desenvolvimento e a doença.
ABSTRACT: Stem cells rely on tightly regulated metabolic programs to balance self-renewal and differentiation, an equilibrium essential for tissue development and homeostasis. One-carbon metabolism is a central pathway that links nutrient availability to vital cellular processes, including nucleotide biosynthesis, redox balance, and epigenetic regulation. While its importance has been established and extensively studied in mammalian systems, the specific role of one-carbon metabolism in neural stem cells (NSCs) and brain development remains poorly defined. This knowledge gap is even more pronounced in invertebrate models, particularly in the context of neural development in Drosophila melanogaster (termed Drosophila). To address this, we used Drosophila as a model to investigate how one-carbon metabolism regulates NSC fate and neural tissue development. We started by investigating how disrupting one-carbon metabolism affects Drosophila NSC (called neuroblast (NB)) differentiation and proliferation in the central brain (CB). Utilizing lineage-specific RNA interference, we knocked down eight key enzymes within the one-carbon cycle and evaluated their impact on NB proliferation, growth, and differentiation. Among these, we identified Serine hydroxymethyltransferase (Shmt), thymidylate synthase (Ts), Adenosylhomocysteinase (Ahcy) as essential for proper NB differentiation, as their downregulation caused prolonged NB lifespan and disrupted normal lineage progression, with particularly impacts on type II NBs. These findings highlight the crucial role of one-carbon metabolism in promoting timely differentiation and preserving lineage commitment, likely through supporting nucleotide synthesis and maintaining epigenetic stability. Furthermore, we investigated whether one-carbon metabolism also plays a role in early neural development, focusing on the optic lobe (OL), where neuroepithelial (NE) cells transition into NBs during larval stages. Here, we demonstrate that shmt is essential for NE cell proliferation, survival, and tissue organization. Shmt-deficient optic lobes displayed increased apoptosis, reduced neuroepithelial expansion, and a failure to form the lamina furrow, ultimately leading to the loss of lamina neurons. These results reveal a broader role for one-carbon metabolism in maintaining epithelial integrity, supporting stem cell function, and ensuring neuron formation. To further explore the metabolic dynamics underlying stem cell regulation, we developed a genetically encoded biosensor to detect intracellular levels of S-adenosylmethionine (SAM), the primary methyl donor in the one-carbon cycle. Using a synthetic yeast-derived MetJ-based system, we engineered both fluorescent (SFB) and luminescent (SLB) biosensor versions, validated in Drosophila S2 cells and mammalian P19 cells. Although the luciferase-based sensor required further refinement, the SFB successfully enabled intracellular SAM detection, laying the groundwork for future metabolic profiling in live tissues. Altogether, this thesis demonstrates that one-carbon metabolism is a critical regulator of neural stem cell transitions and tissue morphogenesis in Drosophila melanogaster. By combining genetic manipulation, developmental analysis, we show that metabolic pathways are tightly intertwined with developmental timing, lineage specification, and stem cell competence. This work not only advances our understanding of the metabolic control of neurogenesis
ABSTRACT: Stem cells rely on tightly regulated metabolic programs to balance self-renewal and differentiation, an equilibrium essential for tissue development and homeostasis. One-carbon metabolism is a central pathway that links nutrient availability to vital cellular processes, including nucleotide biosynthesis, redox balance, and epigenetic regulation. While its importance has been established and extensively studied in mammalian systems, the specific role of one-carbon metabolism in neural stem cells (NSCs) and brain development remains poorly defined. This knowledge gap is even more pronounced in invertebrate models, particularly in the context of neural development in Drosophila melanogaster (termed Drosophila). To address this, we used Drosophila as a model to investigate how one-carbon metabolism regulates NSC fate and neural tissue development. We started by investigating how disrupting one-carbon metabolism affects Drosophila NSC (called neuroblast (NB)) differentiation and proliferation in the central brain (CB). Utilizing lineage-specific RNA interference, we knocked down eight key enzymes within the one-carbon cycle and evaluated their impact on NB proliferation, growth, and differentiation. Among these, we identified Serine hydroxymethyltransferase (Shmt), thymidylate synthase (Ts), Adenosylhomocysteinase (Ahcy) as essential for proper NB differentiation, as their downregulation caused prolonged NB lifespan and disrupted normal lineage progression, with particularly impacts on type II NBs. These findings highlight the crucial role of one-carbon metabolism in promoting timely differentiation and preserving lineage commitment, likely through supporting nucleotide synthesis and maintaining epigenetic stability. Furthermore, we investigated whether one-carbon metabolism also plays a role in early neural development, focusing on the optic lobe (OL), where neuroepithelial (NE) cells transition into NBs during larval stages. Here, we demonstrate that shmt is essential for NE cell proliferation, survival, and tissue organization. Shmt-deficient optic lobes displayed increased apoptosis, reduced neuroepithelial expansion, and a failure to form the lamina furrow, ultimately leading to the loss of lamina neurons. These results reveal a broader role for one-carbon metabolism in maintaining epithelial integrity, supporting stem cell function, and ensuring neuron formation. To further explore the metabolic dynamics underlying stem cell regulation, we developed a genetically encoded biosensor to detect intracellular levels of S-adenosylmethionine (SAM), the primary methyl donor in the one-carbon cycle. Using a synthetic yeast-derived MetJ-based system, we engineered both fluorescent (SFB) and luminescent (SLB) biosensor versions, validated in Drosophila S2 cells and mammalian P19 cells. Although the luciferase-based sensor required further refinement, the SFB successfully enabled intracellular SAM detection, laying the groundwork for future metabolic profiling in live tissues. Altogether, this thesis demonstrates that one-carbon metabolism is a critical regulator of neural stem cell transitions and tissue morphogenesis in Drosophila melanogaster. By combining genetic manipulation, developmental analysis, we show that metabolic pathways are tightly intertwined with developmental timing, lineage specification, and stem cell competence. This work not only advances our understanding of the metabolic control of neurogenesis
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One-carbon metabolism Neural Stem Cells Drosophila melanogaster Neuroblast Neurogenesis Central Brain Optic Lobe Neuroepithelial cells Shmt Ts Ahcy Stem Cell differentiation SAM Biosensor