Historic obstacles and emerging opportunities in the field of developmental metabolism - lessons from Heidelberg.

The field of developmental metabolism is experiencing a technological revolution that is opening entirely new fields of inquiry. Advances in metabolomics, small-molecule sensors, single-cell RNA sequencing and computational modeling present new opportunities for exploring cell-specific and tissue-specific metabolic networks, interorgan metabolic communication, and gene-by-metabolite interactions in time and space. Together, these advances not only present a means by which developmental biologists can tackle questions that have challenged the field for centuries, but also present young scientists with opportunities to define new areas of inquiry. These emerging frontiers of developmental metabolism were at the center of a highly interactive 2023 EMBO workshop 'Developmental metabolism: flows of energy, matter, and information'. Here, we summarize key discussions from this forum, emphasizing modern developmental biology's challenges and opportunities.

Revisiting the role of metabolism during development.

An emerging view emphasizes that metabolism is highly regulated in both time and space. In addition, it is increasingly being recognized that metabolic pathways are tightly connected to specific biological processes such as cell signaling, proliferation and differentiation. As we obtain a better view of this spatiotemporal regulation of metabolism, and of the molecular mechanisms that connect metabolism and signaling, we can now move from largely correlative to more functional studies. It is, therefore, a particularly promising time to revisit how metabolism can affect multiple aspects of animal development. In this Review, we discuss how metabolism is mechanistically linked to cellular and developmental programs through both its bioenergetic and metabolic signaling functions. We highlight how metabolism is regulated across various spatial and temporal scales, and discuss how this regulation can influence cellular processes such as cell signaling, gene expression, and epigenetic and post-translational modifications during embryonic development.

Rewiring of embryonic glucose metabolism via suppression of PFK-1 and aldolase during mouse chorioallantoic branching.

Adapting the energy metabolism state to changing bioenergetic demands is essential for mammalian development accompanying massive cell proliferation and cell differentiation. However, it remains unclear how developing embryos meet the changing bioenergetic demands during the chorioallantoic branching (CB) stage, when the maternal-fetal exchange of gases and nutrients is promoted. In this study, using metabolome analysis with mass-labeled glucose, we found that developing embryos redirected glucose carbon flow into the pentose phosphate pathway via suppression of the key glycolytic enzymes PFK-1 and aldolase during CB. Concomitantly, embryos exhibited an increase in lactate pool size and in the fractional contribution of glycolysis to lactate biosynthesis. Imaging mass spectrometry visualized lactate-rich tissues, such as the dorsal or posterior neural tube, somites and head mesenchyme. Furthermore, we found that the heterochronic gene Lin28a could act as a regulator of the metabolic changes observed during CB. Perturbation of glucose metabolism rewiring by suppressing Lin28a downregulation resulted in perinatal lethality. Thus, our work demonstrates that developing embryos rewire glucose metabolism following CB for normal development.

Temporal regulation of Lin28a during mammalian neurulation contributes to neonatal body size control.

BACKGROUND: The timing of developmental events is tightly regulated along a time axis for normal development. Although the RNA-binding protein Lin28a plays a crucial role in the regulation of developmental timing in Caenorhabditis elegans, how the timing of Lin28a expression affects the rate and/or duration of developmental events during mammalian development remains to be addressed. RESULTS: In this study, we discovered that the timing and the duration of Lin28a expression affect embryonic growth. During the neurulation stage of mouse development, endogenous Lin28a levels start to drop. When Lin28a expression was maintained transiently using the inducible tetracycline-regulated gene expression (Tet-ON) system [doxycycline (Dox)-inducible Lin28a transgenic (iLin28a Tg) mice] with Dox administration at E8.5 and E9.5, it resulted in neonatal lethality, increased body weight (organomegaly), and an increased number of caudal vertebrae at birth. On the other hand, Lin28a induction only at E8.5 caused neonatal lethality and organomegaly, but did not affect the caudal vertebra number. Of note, although Dox treatment before or after neurulation still caused neonatal lethality, it neither caused organomegaly nor the increased caudal vertebra number in iLin28a Tg neonates. CONCLUSIONS: Temporal regulation of Lin28a expression during neurulation affects developmental events such as cessation of axial elongation and embryonic growth in mice.

Mammalian embryos show metabolic plasticity toward the surrounding environment during neural tube closure.

Developing embryos rewire energy metabolism for developmental processes. However, little is known about how metabolic rewiring is coupled with development in a spatiotemporal manner. Here, we show that mammalian embryos display plasticity of glucose metabolism in response to the extracellular environment at the neural tube closure (NTC) stage, when the intrauterine environment changes upon placentation. To study how embryos modulate their metabolic state upon environmental change, we analyzed the steady-state level of ATP upon exposure to extrauterine environments using both an enzymatic assay and a genetically encoded ATP sensor. Upon environmental changes, NTC-stage embryos exhibited increased ATP content, whereas embryos before and after NTC did not. The increased ATP in the NTC-stage embryos seemed to depend on glycolysis. Intriguingly, an increase in mitochondrial membrane potential (ΔΨm) was also observed in the neural ectoderm (NE) and the neural plate border of the non-neural ectoderm (NNE) region. This implies that glycolysis can be coupled with the TCA cycle in the NE and the neural plate border depending on environmental context. Disrupting ΔΨm inhibited folding of the cranial neural plate. Thus, we propose that embryos tune metabolic plasticity to enable coupling of glucose metabolism with the extracellular environment at the NTC stage.

Glycolytic flux-signaling controls mouse embryo mesoderm development.

How cellular metabolic state impacts cellular programs is a fundamental, unresolved question. Here, we investigated how glycolytic flux impacts embryonic development, using presomitic mesoderm (PSM) patterning as the experimental model. First, we identified fructose 1,6-bisphosphate (FBP) as an in vivo sentinel metabolite that mirrors glycolytic flux within PSM cells of post-implantation mouse embryos. We found that medium-supplementation with FBP, but not with other glycolytic metabolites, such as fructose 6-phosphate and 3-phosphoglycerate, impaired mesoderm segmentation. To genetically manipulate glycolytic flux and FBP levels, we generated a mouse model enabling the conditional overexpression of dominant active, cytoplasmic PFKFB3 (cytoPFKFB3). Overexpression of cytoPFKFB3 indeed led to increased glycolytic flux/FBP levels and caused an impairment of mesoderm segmentation, paralleled by the downregulation of Wnt-signaling, reminiscent of the effects seen upon FBP-supplementation. To probe for mechanisms underlying glycolytic flux-signaling, we performed subcellular proteome analysis and revealed that cytoPFKFB3 overexpression altered subcellular localization of certain proteins, including glycolytic enzymes, in PSM cells. Specifically, we revealed that FBP supplementation caused depletion of Pfkl and Aldoa from the nuclear-soluble fraction. Combined, we propose that FBP functions as a flux-signaling metabolite connecting glycolysis and PSM patterning, potentially through modulating subcellular protein localization.

Neural tube closure and embryonic metabolism.

Neural tube closure (NTC) is an embryonic process during formation of the mammalian central nervous system. Disruption of the dynamic, sequential events of NTC can cause neural tube defects (NTD) leading to spina bifida and anencephaly in the newborn. NTC is affected by inherent factors such as genetic mutation or if the mother is exposed to certain environmental factors such as intake of harmful chemicals, maternal infection, irradiation, malnutrition, and inadequate or excessive intake of specific nutrients. Although effects of these stress factors on NTC have been intensively studied, the metabolic state of a normally developing embryo remains unclear. State-of-the art mass spectrometry techniques have enabled detailed study of embryonic metabolite profiles and their distribution within tissues. This approach has demonstrated that glucose metabolism is altered during NTC stages involving chorioallantoic branching. An understanding of embryonic metabolic rewiring would help reveal the etiology of NTD caused by environmental factors.

HIF-1α-PDK1 axis-induced active glycolysis plays an essential role in macrophage migratory capacity.

In severely hypoxic condition, HIF-1α-mediated induction of Pdk1 was found to regulate glucose oxidation by preventing the entry of pyruvate into the tricarboxylic cycle. Monocyte-derived macrophages, however, encounter a gradual decrease in oxygen availability during its migration process in inflammatory areas. Here we show that HIF-1α-PDK1-mediated metabolic changes occur in mild hypoxia, where mitochondrial cytochrome c oxidase activity is unimpaired, suggesting a mode of glycolytic reprogramming. In primary macrophages, PKM2, a glycolytic enzyme responsible for glycolytic ATP synthesis localizes in filopodia and lammelipodia, where ATP is rapidly consumed during actin remodelling processes. Remarkably, inhibition of glycolytic reprogramming with dichloroacetate significantly impairs macrophage migration in vitro and in vivo. Furthermore, inhibition of the macrophage HIF-1α-PDK1 axis suppresses systemic inflammation, suggesting a potential therapeutic approach for regulating inflammatory processes. Our findings thus demonstrate that adaptive responses in glucose metabolism contribute to macrophage migratory activity.