Lung branching morphogenesis is accompanied by temporal metabolic changes towards a glycolytic preference

Hugo Fernandes-Silva,Rute S Moura,Jorge Correia-Pinto,Marco G Alves,Pedro F Oliveira,Henrique Araújo-Silva,Ana M Silva

doi:10.1186/s13578-021-00654-w

Hugo Fernandes-Silva, Rute S Moura + Show 5 more

Open Access

https://doi.org/10.1186/s13578-021-00654-w

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Abstract

BackgroundLung branching morphogenesis is characterized by epithelial-mesenchymal interactions that ultimately define the airway conducting system. Throughout this process, energy and structural macromolecules are necessary to sustain the high proliferative rates. The extensive knowledge of the molecular mechanisms underlying pulmonary development contrasts with the lack of data regarding the embryonic lung metabolic requirements. Here, we studied the metabolic profile associated with the early stages of chicken pulmonary branching.MethodsIn this study, we used an ex vivo lung explant culture system and analyzed the consumption/production of extracellular metabolic intermediates associated with glucose catabolism (alanine, lactate, and acetate) by 1H-NMR spectroscopy in the culture medium. Then, we characterized the transcript levels of metabolite membrane transporters (glut1, glut3, glut8, mct1, mct3, mct4, and mct8) and glycolytic enzymes (hk1, hk2, pfk1, ldha, ldhb, pdha, and pdhb) by qPCR. ldha and ldhb mRNA spatial localization was determined by in situ hybridization. Proliferation was analyzed by directly assessing DNA synthesis using an EdU-based assay. Additionally, we performed western blot to analyze LDHA and LDHT protein levels. Finally, we used a Clark-Type Electrode to assess the lung explant's respiratory capacity.ResultsGlucose consumption decreases, whereas alanine, lactate, and acetate production progressively increase as branching morphogenesis proceeds. mRNA analysis revealed variations in the expression levels of key enzymes and transporters from the glycolytic pathway. ldha and ldhb displayed a compartment-specific expression pattern that resembles proximal–distal markers. In addition, high proliferation levels were detected at active branching sites. LDH protein expression levels suggest that LDHB may account for the progressive rise in lactate. Concurrently, there is a stable oxygen consumption rate throughout branching morphogenesis.ConclusionsThis report describes the temporal metabolic changes that accompany the early stages of chicken lung branching morphogenesis. Overall, the embryonic chicken lung seems to shift to a glycolytic lactate-based metabolism as pulmonary branching occurs. Moreover, this metabolic rewiring might play a crucial role during lung development.

Highlights

Lung branching morphogenesis is characterized by epithelial-mesenchymal interactions that ulti‐ mately define the airway conducting system
Lung branching morphogenesis is accompanied by temporal metabolite changes To describe the metabolic alterations that occur during the early stages of chicken pulmonary branching, in vitro lung explant culture was performed using stages b1, b2, and b3 (1, 2, or 3 secondary buds formed per bronchus, respectively) that correspond to the first three branching stages (Fig. 1a); new branches are clearly seen with the epithelial marker l-cam (Fig. 1a)
The culture system was performed for 48 h and refreshed at 24 Hours (D1) (24 h); medium was collected at 0 Hours (D0) (0 h; to be used as reference/control), D1, and 48 Hours (D2) (48 h), and analyzed by 1H Nuclear Magnetic Resonance (1H-NMR) spectroscopy

Summary

Introduction

Lung branching morphogenesis is characterized by epithelial-mesenchymal interactions that ulti‐ mately define the airway conducting system Throughout this process, energy and structural macromolecules are necessary to sustain the high proliferative rates. Pulmonary branching morphogenesis is an intricate process governed by epithelial-mesenchymal interactions and is dependent on complex signaling events. This process occurs throughout the early stages of embryonic. Gallus gallus, the primordial lung appears around day 3 of embryogenesis as a protuberance from the primitive foregut [2] During this process, the mesobronchus grows distally, and the new secondary bronchi sprout laterally into the surrounding mesenchymal compartment [1, 3]. FGF (Fibroblast Growth Factor), WNT (Wingless-related Integration Site), SHH (Sonic Hedgehog), and Retinoic Acid signaling pathways were described as playing critical roles in chicken pulmonary branching morphogenesis [7,8,9,10,11]

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