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Background: Lung branching morphogenesis is characterized by epithelial-mesenchymal interactions and mediated by complex signaling that ultimately defines the airway conducting system. Retinoic Acid (RA) signaling is crucial for lung organogenesis and modulates proximal-distal patterning and branching morphogenesis. However, how signaling networks and energy metabolism work together to shape embryonic lung development remains unexplored. Here, we investigate how RA signaling influences the metabolic profile of lung branching. Methods: We performed ex vivo lung explant culture using embryonic chicken lungs (Gallus gallus) treated with DMSO, 1 uM RA (stimulation), or 10 uM of BMS493 (inhibition). Explant culture media was collected, and the consumption/production of extracellular metabolites was evaluated by 1H-NMR spectroscopy. Lung explants were analyzed to determine mitochondrial respiration and biogenesis. Proliferation was assessed using an EdU‐based assay. Western blot, qPCR, and in situ hybridization were performed to assess the expression of metabolic/signaling components. Results: Upon stimulation, RA signaling increases lung branching, maintaining proper morphology. Besides, there is an increase in the amount of glucose directed into pyruvate and succinate rather than into alanine or lactate production. On the other hand, RA signaling inhibition decreases lung branching, induces a cystic-like phenotype, and promotes mitochondrial function. Proliferation status and lactate dehydrogenase expression are both regulated by RA signaling. Furthermore, RA signaling controls fatty acid metabolism through an AMPK-dependent mechanism. Conclusions: This report shows that RA signaling modulates the energy metabolism of lung branching morphogenesis and highlights the importance of metabolic regulation in this phase, contributing to understanding the etiology of congenital lung disorders.

Cell fate and growth require one-carbon units for the biosynthesis of nucleotides, methylation reactions, and redox homeostasis, provided by one-carbon metabolism. Consistently, defects in one-carbon metabolism lead to severe developmental defects, such as neural tube defects. However, the role of this pathway during brain development and in neural stem cell regulation is poorly understood. To better understand the role of one carbon metabolism we focused on the enzyme Serine hydroxymethyl transferase (Shmt), a key player of the one-carbon cycle, during Drosophila brain development. We show that although loss of Shmt does not cause obvious defects in the central brain, it leads to severe phenotypes in the optic lobe. The shmt mutants have smaller optic lobe neuroepithelia partly justified by increased apoptosis. Additionally, shmt mutant neuroepithelia have morphological defects, failing to form a lamina furrow likely explaining the observed absence of lamina neurons. These findings show that one-carbon metabolism is critical for the normal development of neuroepithelia, and consequently for the generation of neural progenitor cells and neurons. These results propose a mechanistic role for one-carbon during brain development.

Funding Information: We would like to thank Tiago Baptista and Hugo Silva for experimental help. We thank Florence Janody and Rita Teodoro for critical reading of the manuscript. We thank Juergen Knoblich for the Miranda antibody. We thank the cytometry and fly facilities at NOVA Medical School for technical support and CONGENTO: consortium for genetically tractable organisms (LISBOA-01-0145-FEDER-022170); Bloomington Drosophila Stock Center (NIH P40OD018537) and Vienna Drosophila Resource Center (VDRC) [52 ], for the stocks used in this study; the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. Funding Information: Open access funding provided by FCT|FCCN (b-on). This work was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (H2020-ERC-2017-STG-GA 759853-StemCellHabitat); by Wellcome Trust and Howard Hughes Medical Institute (HHMI-208581/Z/17/Z-Metabolic Reg SC fate); EMBO Installation grant (H2020-EMBO-3311/2017/G2017) and by Fundação para a Ciência e Tecnologia (IF/01265/2014/CP1252/CT0004, EXPL/BIA-BID/1394/2021 and 2020.05639.BD to A.R.R). This work was supported by iNOVA4Health—UIDB/04462/2020 and UIDP/04462/2020, and by the Associated Laboratory LS4FUTURE (LA/P/0087/2020), two programs financially supported by Fundação para a Ciência e Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior. Publisher Copyright: © 2023, The Author(s). Tumor cells have an increased demand for nutrients to sustain their growth, but how these increased metabolic needs are ensured or how this influences tumor formation and progression remains unclear. To unravel tumor metabolic dependencies, particularly from extracellular metabolites, we have analyzed the role of plasma membrane metabolic transporters in Drosophila brain tumors. Using a well-established neural stem cell-derived tumor model, caused by brat knockdown, we have found that 13 plasma membrane metabolic transporters, including amino acid, carbohydrate and monocarboxylate transporters, are upregulated in tumors and are required for tumor growth. We identified CD98hc and several of the light chains with which it can form heterodimeric amino acid transporters, as crucial players in brat RNAi (bratIR) tumor progression. Knockdown of these components of CD98 heterodimers caused a dramatic reduction in tumor growth. Our data also reveal that the oncogene dMyc is required and sufficient for the upregulation of CD98 transporter subunits in these tumors. Furthermore, tumor-upregulated dmyc and CD98 transporters orchestrate the overactivation of the growth-promoting signaling pathway TOR, forming a core growth regulatory network to support brat IR tumor progression. Our findings highlight the important link between oncogenes, metabolism, and signaling pathways in the regulation of tumor growth and allow for a better understanding of the mechanisms necessary for tumor progression. publishersversion published