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

ABSTRACT

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.

Summary: Participants of the 2023 EMBO workshop on Developmental Metabolism outline the zeitgeist of the field by summarizing key technological advances and trends in the scientific mindset.

Introduction

Metabolism consolidates a plethora of chemical processes that must occur within an organism to grow, maintain life and reproduce. Through such processes, organisms transform energy and matter to fuel their energetic needs and build complex structures from pools of small metabolites. During development, metabolites not only provide energy and building blocks for macromolecules, but they also influence cell signaling events and gene expression networks across space and time. Studies of developmental metabolism are, therefore, inherently interdisciplinary and can encompass fields that not only include biology, chemistry and nonequilibrium physics, but also span math, ecology and global public health.

When Joseph Needham wrote his history of chemical embryology (i.e. developmental metabolism) in the 1930s (Needham, 1931), studies of metabolism were an important and popular focus of developmental biologists (for examples, see Beadle and Ephrussi, 1936; Warburg, 1908; Fink, 1925). Advances in respirometry, calorimetry and chromatography allowed scientists to explore how pools of individual chemicals were reordered to form complex lifeforms. However, as the advent of modern genetics and molecular biology highlighted the importance of cell- and tissue-specific signaling in growth and development, studies of developmental metabolism became increasingly difficult. For example, when developmental biologists were examining the products of gene expression using techniques such as in situ hybridization and immunofluorescence (Tautz and Pfeifle, 1989; Ortiz Hidalgo, 2022), nearly all metabolites within central carbon metabolism remained undetectable at the single-cell level. Similarly, while genetic techniques such as mosaic analysis and conditional knockouts (Germani et al., 2018; Yochem and Herman, 2003; García-Otín and Guillou, 2006) enabled precise investigations of how gene products can act in cell nonautonomous mechanisms, intercellular and interorgan metabolic communication studies remained rudimentary. These historic obstacles, however, are now succumbing to new technological and computational advances. As highlighted in the EMBO workshop ‘Developmental metabolism: flows of energy, matter, and information’, entirely new fields of inquiry are opening within this interdisciplinary field. Here, we summarize emerging tools and scientific opportunities that were the focus of this highly interactive workshop.

How do set-points of metabolic states regulate development?

Metabolism exhibits tissue- and life stage-specific patterns. To achieve such metabolic set-points during development, metabolism is regulated dynamically in time and space by intrinsic developmental programs and extrinsic environmental cues (Miyazawa and Aulehla, 2018). Spatiotemporal regulation of metabolism is essential to meet the bioenergetic demand of embryos for ATP and biomass production (Tennessen and Thummel, 2011; Agathocleous et al., 2012; Song et al., 2017; Djabrayan et al., 2019; Rodríguez-Nuevo et al., 2022; Mandal et al., 2005); however, the bioenergetic costs and constraints of each cellular process remain largely unknown (Yang et al., 2021b). These unanswered questions represent an exciting and relatively unexplored area of developmental biology. Moving forward, our field must ask how developing organisms allocate their energy/nutrient budget to each developmental process, and how such metabolic constraints control development. This fundamental question has been revisited by combining the theory of nonequilibrium physics with technical advances that allow for precise metabolite measurements (Rodenfels et al., 2019, 2020; Yang et al., 2021a; Cadart et al., 2023), giving insights into, for example, the energetic cost of cell division and developmental progression. Simultaneously, it is increasingly clear that metabolism tightly regulates signal transduction pathways and gene expression networks that drive growth and development (Miyazawa and Aulehla, 2018).

Many of the talks at EMBO focused on key advances in understanding how metabolic flux and metabolite levels modulate developmental regulatory mechanisms. An increasing number of studies have demonstrated that the metabolic state of cells modulates the epigenome through protein post-translational modifications (PTMs) because the PTM substrates and co-factors are products of metabolic pathways and can be rate-limiting factors for these modifications (Reid et al., 2017; Finley, 2023; Samo et al., 2021). For example, these mechanisms play an important role in mammalian pre-implantation stage embryos where spatiotemporal regulation of the citric acid cycle and glucose metabolism contribute to zygotic genome activation and cell fate specification, respectively (Nagaraj et al., 2017; Chi et al., 2020). Similarly, glucose metabolism has also recently been found to influence mesoderm specification by regulating protein glycosylation (Cao et al., 2023 preprint). These are but a few examples demonstrating the direct link between metabolites and the molecular basis of development. With the constant discovery of new metabolic PTMs, such as histone lactylation by lactate (Figlia et al., 2020), we anticipate that this will be a rich area of discovery.

In addition to PTMs, metabolic control of the intracellular biochemical environment can have dramatic effects on signal transduction (Oginuma et al., 2020; Diaz-Cuadros et al., 2023; Spannl et al., 2020; MacColl Garfinkel et al., 2023). For example, glycolysis is linked to the activation of Wnt signaling via the regulation of intracellular pH, which impacts the acetylation state of β-catenin (Oginuma et al., 2020). Excitingly, a recent study has demonstrated that mitochondrial oxidative metabolism is involved in establishing embryonic patterning via the stabilization of hypoxia inducible factor 1a (Hif1a) protein (MacColl Garfinkel et al., 2023). Such findings are striking, because the intracellular biochemical environment is expected to have pleiotropic effects on biochemical reactions operating within cells. As developmental biologists continue studying these phenomena, we highlight the need for future studies to determine how metabolic processes are used in developing systems without compromising other processes.

Finally, both metabolites and metabolic enzymes can function as signaling molecules to directly influence signal transduction and gene expression (Miyazawa et al., 2022; Liu et al., 2023; Enzo et al., 2015; Hudry et al., 2019; Takata et al., 2023; Miyazawa et al., 2024 preprint; Stapornwongkul et al., 2023 preprint; Wahl et al., 2013; Erb and Kliebenstein, 2020; Samo et al., 2021; Kosmacz et al., 2020). Although metabolic signaling is well known to regulate cellular anabolic and catabolic reactions [e.g. AMP-activated protein kinase (AMPK) and mTOR signaling pathways (González et al., 2020)], recent findings have expanded to the regulation of developmental signaling pathways. For example, the glycolytic intermediate fructose 1,6-bisphosphate, which serves as a sentinel for glycolytic flux across species, has recently been discovered to functionally link glycolytic flux and Wnt signaling to control the timing of mouse embryo mesoderm segmentation (Miyazawa et al., 2024 preprint, 2022). These findings, many of which were discussed at the EMBO workshop, demonstrate the essential role of metabolism in directly regulating development, and highlight an ongoing need to refine our ability to measure and visualize changes in metabolic flux and metabolite abundance within developing systems.

Technological advances

Studies of developmental metabolism primarily focus on the metabolic mechanisms that generate complex structures from relatively simple molecules. Thus, observing and investigating the thermodynamics and biochemistry of metabolism during development offers the possibility to decipher complex structure biogenesis, one step at a time, as complexity increases. Therein lies the biggest current technological challenge – the picture is never complete. Small perturbations within the developing organism have indirect pervasive influences and we are not able to assess all the consequences on a cell, organism or on a temporal scale. Recent technological advances provide a more refined picture of energy flow and metabolic flux, both at the intracellular and organismal levels, holding the potential to tackle these issues of complexity. In this regard, the latest developments in single-cell and spatial-omics technologies, including spatial metabolomics, as well as the advent of lifetime imaging technology and fluorescence resonance energy transfer (FRET)-based biosensors, now provide the ability to observe small molecule metabolites in space and time (Subramanian et al., 2020; Bressan et al., 2023; Alves et al., 2015; Yang et al., 2021a,b; Datta et al., 2020; Fang et al., 2023; Alexandrov, 2023). When these technologies are complemented with flux analyses using stable isotope tracers (Jang et al., 2018), developmental biologists can explore, for the first time, the dynamic metabolic state of individual cells and tissues in vivo (Bulusu et al., 2017; Miyazawa et al., 2017; Solmonson et al., 2022). The power of this combined approach was evident throughout the EMBO workshop, illustrated by recent metabolic studies of mammalian somitogenesis; by combining FRET imaging studies and flux analysis, several studies have demonstrated that, within the presomitic mesoderm, glycolytic activity is graded along the anteroposterior axis and functionally linked to regulation of Wnt signaling – an elegant example of how exploring the spatial and temporal changes in metabolite abundance can open new fields of inquiry (Bulusu et al., 2017; Miyazawa et al., 2022; Oginuma et al., 2020, 2017).

Although the ability to visualize and quantify metabolites in space and time represents essential advances in determining how metabolic networks arrange themselves during development, our ability to exploit these technologies has been limited by how little is known about the energetic costs of embryogenesis, juvenile growth, maturation and reproduction; such information is essential for understanding how metabolism fuels these processes and how energy is used to build complex structures. As noted at the EMBO workshop, developmental biologists are addressing this key question by using classical approaches to study the energetics of developing systems. Through a combination of isothermal calorimetry (to measure heat dissipation), respirometry and phosphorescent probe-based extracellular flux analysis (Rodenfels et al., 2019; Arunachalam et al., 2023; Mookerjee et al., 2017; Cadart et al., 2023), a quantitative understanding of energy metabolism in developing systems is emerging. Such information may lead to the discovery of the conserved metabolic features that drive development in the embryo and beyond.

In addition to experimental advances, discussions among the attendees often focused on the need to align these advances to ensure quality standards of methods and analyses, thus increasing reliability across approaches and species. The conclusion from these discussions was that, first, the community needs quality control instruments for collecting and handling scientific data; second, our field should embrace the implementation of the FAIR (findability, accessibility, interoperability and reusability) principle (Wilkinson et al., 2016); and third, we need to consolidate quantitative data with a mechanistic understanding of developmental processes. Together, these goals in experimental quality control and data accessibility are essential for generating reproducible findings but will also lay a foundation for theoretical models, which represent powerful tools for recognizing patterns, and predicting coherences and outcomes over large metabolic networks (Berg et al., 2023).

Theoretical challenges in developmental metabolism

Developmental metabolism is an inherently complex endeavor that involves understanding how the changes in metabolic networks are coordinated with gene regulatory networks, cell fate decisions, interorgan communication and various other dynamic processes. As discussed during the EMBO workshop, theoretical modelling can provide a powerful tool in untangling complexity of a biological system and identifying relevant parameters within large-scale metabolic networks (i.e. the key parameters that might represent only a small subset of the entire system). Although an ever-increasing amount of experimental data fosters extensive computational models, fundamental theoretical challenges must be addressed to successfully use these data to elucidate the principles of metabolic networks, energetic demands of cellular processes, spatial organization of metabolism and their broader effects on developmental biology.

A broad understanding of chemical reaction networks is crucial in elucidating metabolic reprogramming, the influence of metabolism in cell fate determination, and the dynamic regulation and homeostasis of metabolic fluxes. Metabolic control analysis and similar structural characterizations have demonstrated the power of theory in identifying robust properties of biochemical networks (Fell, 1997; Reder, 1988; Okada and Mochizuki, 2016). However, open questions remain on how to establish global frameworks for characterizing flux and output sensitivity in real-world metabolic networks, while also extending the scope of control analysis in multistable systems that switch between metabolic states – an essential consideration for studies of metabolic reprogramming. Although coarse-graining metabolic interactions could simplify the complexity, maintaining physical consistency is essential. Towards this goal, recent studies integrating thermodynamic principles with chemical reaction network theory have provided frameworks for thermodynamically consistent coarse-graining of metabolic networks (Rao and Esposito, 2016; Avanzini et al., 2023; Dal Cengio et al., 2023). Thus, a future challenge for theoretical studies of developmental metabolism is to achieve a thermodynamic framework of metabolic control and reprogramming by blending these earlier ideas from different disciplines.

Another major focus of theoretical research in developmental metabolism is physical bioenergetics, which aims to determine the energetic demand and efficiency of cellular processes and whether/how metabolic requirements become a constraining factor for growth (Scott et al., 2010; Ilker and Hinczewski, 2019; Lynch and Trickovic, 2020). Several recent studies have illustrated bioenergetic estimations for gene expression (Lynch and Marinov, 2015), microRNA regulation (Ilker and Hinczewski, 2024) and morphogenetic programs (Song and Hyeon, 2021). Over the next decade, it would be important to combine nonequilibrium physics with biochemistry and cell biology to eventually build a ‘thermodynome’, a comprehensive account of energetic costs of biological function and how they vary between species, akin to omics. Close interdisciplinary collaborations in theory and experiments will allow the disentanglement of the contributions from different biological processes to energy expenditure in organisms.

Although the importance of theoretical modeling might seem abstract to many developmental biologists, the problems that can be tackled with such approaches are directly relevant to the study of developmental metabolism. For example, classic studies by Max Kleiber revealed that, across a wide range of species, the total rate of energy expenditure of an organism (i.e. metabolic rate) scales with approximately ¾ power of body mass (Kleiber, 1932). Recent work in planarians has also validated this as an intraspecific behavior (Thommen et al., 2019). Kleiber's law remains puzzling because it posits neither linear scaling with body mass nor a surface law. However, there are notable theoretical hypotheses based on transport constraints in organisms, suggesting an interplay between body plan and metabolism (West et al., 1997; Banavar et al., 1999). However, these theories have not been conclusively validated and we still lack a clear picture of how such universal scaling behavior arises in animal energetics. Moving forward, novel multiscale theoretical models, combined with the new technologies for studying metabolism hold the potential for discovering the mechanisms that underlie Kleiber's law and other developmental processes.

With these questions in mind, discussions at EMBO focused on how theory can bridge phenomena across different scales, with the goal of understanding the interplay between metabolic fluxes and biological structures in cells and tissues. These discussions highlighted notable theoretical advances in bacterial and yeast systems, which use experimental observations combined with computational models such as flux balance analysis (Orth et al., 2010), whole-cell simulations of bacteria (Thornburg et al., 2022) and coarse-grained models that capture essential metabolism features (Basan et al., 2015). Moreover, it can be interesting to explore mathematical and conceptual parallels in developmental and evolutionary processes on how metabolism is interconnected with multicellular organization. Together, interdisciplinary approaches will be key to elucidating how metabolism and information processing organize together in organisms.

A new era in model organism research – balancing classic and emerging systems

Both technological and theoretical studies of developmental metabolism are conducted using a very small subset of the global biome. As described above and highlighted at the EMBO workshop, unicellular organisms such as bacteria and yeast represent powerful models for understanding how cell growth and proliferation are coordinated with changes in metabolic networks (Papagiannakis et al., 2017; Takhaveev et al., 2023). Similarly, model organisms that have long functioned as the ‘workhorses’ of genetic research (e.g. Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Mus musculus and Arabidopsis thaliana) are not only essential for exploring the interplay between genes and metabolism but also serve as powerful genetic frameworks for developing new tools and techniques. This relatively small number of experimental systems represent the bulk of modern studies in developmental metabolism (Doke and Dhawale, 2015; Zhao et al., 2022; Madden et al., 2020). However, advances in metabolomics and genomics, as well as in measuring energetics and metabolite abundance, have significantly expanded the suite of organisms that can be used to study developmental metabolism effectively. Thus, the organism best suited to study the problem at hand can be used – whether that be classic developmental systems, such as sea urchins or Xenopus laevis, emerging model systems, such as planaria, or potentially even wild populations.

As an extension of these discussions, the advantages of in vitro systems must not be ignored by the developmental metabolism community, as the ability to observe development in a dish represents an experimental system that is relatively fast, inexpensive, easy to manipulate and – depending on the model – subject to fewer ethical concerns (Beydag-Tasöz et al., 2023; Zhao et al., 2022) . In addition, in vitro systems offer unique advantages for studying metabolism, such as the use of defined media, compatibility with isotope tracing and the ability to use imaging tools (such as fluorescent sensors); these all open new possibilities. Similarly, stem cell-based models of embryo development offer invaluable insights beyond the possibilities of classical models (Tyser et al., 2021; Sozen et al., 2022). They can help to reveal the interplay between metabolism and the major signaling pathways, such as those that drive the development of ectoderm, mesoderm and endoderm (Stapornwongkul et al., 2023 preprint; Cao et al., 2023 preprint; Dingare et al., 2024; Luque et al., 2023 preprint). Finally, the advent of artificial cells holds the potential to revolutionize the way metabolism is studied at the level of a single cell (Guan et al., 2018), because this system provides a unique means of manipulating metabolic parameters using a bottom-up approach that is impossible in other systems.

Even though new technologies allow developmental biologists to study a seemingly limitless number of systems, we anticipate that well-defined model organisms will remain the gold standard. The unparalleled genetic resources, well-annotated knowledge bases (Alliance of Genome Resources Consortium, 2024) and intense community support enable studies that are not feasible in other systems, irrespective of the advances noted above. However, as increasing ethical concerns and legal restrictions hover over the use of vertebrate animals in applied and also basic research (Doke and Dhawale, 2015), well-established invertebrate systems and in vitro models are becoming the focus of new approach methods (PrecisionTox Consortium, 2023). Our field should embrace these new research directions because they hold the potential to maintain the prominence of valuable genetic systems while addressing novel scientific but also societal challenges.

In conclusion, we left EMBO with the impression that deciphering the complexity of developmental metabolism requires classic genetic models, new systems ideally suited to ask specific questions, and a theoretical framework capable of interpreting very large datasets and exploiting this information to identify new and exciting questions.

Exploring developmental metabolism through the lens of human disease

The interdependence between metabolism and development has been evident for decades within the context of human disease. Many rare diseases stem from metabolic disruption, thus studying the link between disease phenotypes and developmental metabolism can complement and inform ongoing model organism research (reviewed by Nissenkorn et al., 2001; Erez and DeBerardinis, 2015; Illsinger and Das, 2010). Although the developmental phenotypes associated with aberrant metabolic flux are variable, some phenotypic commonalities have emerged, including neurocognitive disorders and neuromotor delay, neural tube defects, and congenital heart defects (BoAli et al., 2018; Hedermann et al., 2021; Wu et al., 2023; Keuls et al., 2023). Consistent with these findings, recent studies have demonstrated that metabolic signaling events are essential in the early embryonic patterning and function of the brain and heart (MacColl Garfinkel et al., 2023; Licznerski et al., 2020).

The effects of metabolic patterning and disease on the heart and brain may seem unsurprising considering the high energetic demands of both tissues. However, the importance of metabolism in embryonic development is underscored by this targeted effect, as the heart and brain are crucial for systemic interorgan communication, both in terms of system monitoring by the nervous system, as well as information flow (e.g. molecular signaling via the bloodstream) (Ivanovitch et al., 2017; Schussler et al., 2021). The effect of abnormal metabolism on these organs highlights the interaction between metabolic signaling and the development of these essential tissues (Casimir et al., 2024).

The relationship between metabolism and embryogenesis is also apparent from the interplay between metabolic disease and human development. For example, diabetic pregnancies are associated with increased risk of fetal complications and congenital malformations, with the greatest impacts observed in the cardiovascular and central nervous systems (Ornoy et al., 2015; Bhandari et al., 2024; Nold and Georgieff, 2004). Notably, neural tube defects, hydrocephalus and heart defects, such as transposition of large vessels and ventricular septum defects, are prevalent among infants of diabetic mothers (Ornoy et al., 2015; Nold and Georgieff, 2004), as is perinatal mortality (Melamed and Hod, 2009). Although advances in early diagnosis and management have reduced perinatal mortality, challenges persist regarding prematurity and pre-term delivery, and the offspring of diabetic mothers face increased risks of metabolic syndrome, diabetes and insulin resistance (Boney et al., 2005; Ornoy et al., 2015).

Our field is poised to address this global health crisis as research into the disease etiology of gestational diabetes and diabetes mellitus types I and II can provide better metabolic screening and prevention measures, allowing for earlier intervention and advice before, during and after pregnancy. At the cellular level, identifying biomarkers could provide new tools for early detection of abnormal metabolic conditions during pregnancy – a crucial step to prevent worsening maternal and fetal effects (Saravanan, 2020). Finally, emerging evidence demonstrates that maternal and paternal genetics, behavior and diet influence the development and health of subsequent generations via poorly understood epigenetic mechanisms (Santilli and Boskovic, 2023; Lempradl, 2020). We must recognize that our ability to improve the lives of parents, pregnant people and their offspring depends on studies in the clinic and at the bench. Better interdisciplinary communication will improve the chances of bettering the lives of our community members.

Addressing global challenges

Biomedical research foundations support many developmental biologists, so our research programs tend to emphasize human disease models. Although such foci are important, the global challenges facing humanity require a better understanding of how growth and metabolism are coordinately regulated in a rapidly changing environment. Global climate change will inevitably disrupt biomes by altering the timing, rate and resiliency of developmental systems (Gilbert, 2021). Moreover, the stresses imposed by temperature extremes, changes in precipitation and modified nutrient distribution are exacerbated by widespread environmental pollution (Fuller et al., 2022). Developmental biologists must expand our traditional foci to address problems that threaten human and environmental health, and studies of developmental metabolism should be at the forefront of these efforts.

The EMBO workshop included discussions highlighting the potential for studying development in the context of global issues. In particular, plant developmental metabolism must be given increased prominence. As referenced throughout this Spotlight, key questions in developmental metabolism, such as the concept of metabolic set-points, the role of metabolites as signaling molecules and the influence of metabolic flux on chromatin modification, are being addressed in plants (Erb and Kliebenstein, 2020; Kosmacz et al., 2020; Samo et al., 2021; Wahl et al., 2013). As climate change disrupts agriculture and ecosystems (Lippmann et al., 2019), such studies of plant development are of utmost global importance. Similarly, our community needs to address biodiversity issues, with global insect decline as a prime example: increasing global temperatures and widespread pesticide use are severely altering the developmental trajectories and reproductive capacity of insects (Gandara et al., 2024 preprint; Pawar et al., 2024). Future studies should explore how environmental stress reduces the metabolic plasticity of developing systems and use this information to push for regulatory changes in environmental pollution and habitat conservation – we, as a community, must begin to assert our expertise for societal change (Gilbert, 2021).

Finally, we encourage our community to expand our view of the science that fits within the field of developmental metabolism. For example, oceanic phytoplankton blooms represent some of the most important biological communities on the planet, and their metabolism influences biogeochemical cycles of elements (Kuhlisch et al., 2024). The flow of metabolites between the microorganisms in these communities is reminiscent of the movement of metabolites among different cells in a developing plant or animal. Thus, metabolite transfer between species of proliferating phytoplankton could serve as a powerful model for understanding how small-molecule transfer supports growth and development in multicellular systems.

In conclusion, the EMBO workshop ‘Developmental metabolism: flows of energy, matter, and information’ emphasized how far the field of developmental metabolism has advanced, sparked lively debates and highlighted current challenges.