Cell lineage-guided mass spectrometry reveals increased energy metabolism and reactive oxygen species in the vertebrate organizer.

Molecular understanding of the vertebrate Organizer, a tissue center critical for inductive signaling during gastrulation, has so far been mostly limited to transcripts and a few proteins, the latter due to limitations in detection and sensitivity. The Spemann-Mangold Organizer (SMO) in the South African Clawed Frog (X. laevis), a popular model of development, has long been known to be the origin of signals that pattern the mesoderm and central nervous system. Molecular screens of the SMO have identified several genes responsible for the ability of the SMO to establish the body axis. Nonetheless, a comprehensive study of proteins and metabolites produced specifically in the SMO and their functional roles has been lacking. Here, we pioneer a deep discovery proteomic and targeted metabolomic screen of the SMO in comparison to the remainder of the embryo using high-resolution mass spectrometry (HRMS). Quantification of ~4,600 proteins and a panel of targeted metabolites documented differential expression for 460 proteins and multiple intermediates of energy metabolism in the SMO. Upregulation of oxidative phosphorylation and redox regulatory proteins gave rise to elevated oxidative stress and an accumulation of reactive oxygen species in the SMO. Imaging experiments corroborated these findings, discovering enrichment of hydrogen peroxide in the SMO. Chemical perturbation of the redox gradient perturbed mesoderm involution during early gastrulation. HRMS expands the bioanalytical toolbox of cell and developmental biology, providing previously unavailable information on molecular classes to challenge and refine our classical understanding of the Organizer and its function during early patterning of the embryo.

The 15-min (Sub)Cellular Proteome.

Single-cell mass spectrometry (MS) opens a proteomic window onto the inner workings of cells. Here, we report the discovery characterization of the subcellular proteome of single, identified embryonic cells in record speed and molecular coverage. We integrated subcellular capillary microsampling, fast capillary electrophoresis (CE), high-efficiency nano-flow electrospray ionization, and orbitrap tandem MS. In proof-of-principle tests, we found shorter separation times to hinder proteome detection using DDA, but not DIA. Within a 15-min effective separation window, CE data-independent acquisition (DIA) was able to identify 1,161 proteins from single HeLa-cell-equivalent (∼200 pg) proteome digests vs. 401 proteins by the reference data-dependent acquisition (DDA) on the same platform. The approach measured 1,242 proteins from subcellular niches in an identified cell in the live Xenopus laevis (frog) embryo, including many canonical components of organelles. CE-MS with DIA enables fast, sensitive, and deep profiling of the (sub)cellular proteome, expanding the bioanalytical toolbox of cell biology. Authorship Contributions: P.N. and B.S. designed the study. L.R.P. collected the X. laevis cell aspirates. B.S. prepared and measured the samples. B.S. and P.N. analyzed the data and interpreted the results. P.N. and B.S. wrote the manuscript. All the authors commented on the manuscript.

Dilute to Enrich for Deeper Proteomics: A Yolk-Depleted Carrier for Limited Populations of Embryonic (Frog) Cells.

Abundant proteins challenge deep mass spectrometry (MS) analysis of the proteome. Yolk, the source of food in many developing vertebrate embryos, complicates chemical separation and interferes with detection. We report here a strategy that enhances bottom-up proteomics in yolk-laden specimens by diluting the interferences using a yolk-depleted carrier (YODEC) proteome via isobaric multiplexing quantification. This method was tested on embryos of the South African Clawed Frog (Xenopus laevis), where a >90% yolk proteome content challenges deep proteomics. As a proof of concept, we isolated neural and epidermal fated cell clones from the embryo by dissection or fluorescence-activated cell sorting. Compared with the standard multiplexing carrier approach, YODEC more than doubled the detectable X. laevis proteome, identifying 5,218 proteins from D11 cell clones dissected from the embryo. Ca. ∼80% of the proteins were quantified without dropouts in any of the analytical channels. YODEC with high-pH fractionation quantified 3,133 proteins from ∼8,000 V11 cells that were sorted from ca. 2 embryos (1.5 µg total, or 150 ng yolk-free proteome), marking a 15-fold improvement in proteome coverage vs the standard proteomics approach. About 60% of these proteins were only quantifiable by YODEC, including molecular adaptors, transporters, translation, and transcription factors. While this study was tailored to limited populations of Xenopus cells, we anticipate the approach of "dilute to enrich" using a depleted carrier proteome to be adaptable to other biological models in which abundant proteins challenge deep MS proteomics.

Biological mass spectrometry enables spatiotemporal 'omics: From tissues to cells to organelles.

Biological processes unfold across broad spatial and temporal dimensions, and measurement of the underlying molecular world is essential to their understanding. Interdisciplinary efforts advanced mass spectrometry (MS) into a tour de force for assessing virtually all levels of the molecular architecture, some in exquisite detection sensitivity and scalability in space-time. In this review, we offer vignettes of milestones in technology innovations that ushered sample collection and processing, chemical separation, ionization, and 'omics analyses to progressively finer resolutions in the realms of tissue biopsies and limited cell populations, single cells, and subcellular organelles. Also highlighted are methodologies that empowered the acquisition and analysis of multidimensional MS data sets to reveal proteomes, peptidomes, and metabolomes in ever-deepening coverage in these limited and dynamic specimens. In pursuit of richer knowledge of biological processes, we discuss efforts pioneering the integration of orthogonal approaches from molecular and functional studies, both within and beyond MS. With established and emerging community-wide efforts ensuring scientific rigor and reproducibility, spatiotemporal MS emerged as an exciting and powerful resource to study biological systems in space-time.

Microanalytical Mass Spectrometry with Super-Resolution Microscopy Reveals a Proteome Transition During Development of the Brain's Circadian Pacemaker.

During brain development, neuronal proteomes are regulated in part by changes in spontaneous and sensory-driven activity in immature neural circuits. A longstanding model for studying activity-dependent circuit refinement is the developing mouse visual system where the formation of axonal projections from the eyes to the brain is influenced by spontaneous retinal activity prior to the onset of vision and by visual experience after eye-opening. The precise proteomic changes in retinorecipient targets that occur during this developmental transition are unknown. Here, we developed a microanalytical proteomics pipeline using capillary electrophoresis (CE) electrospray ionization (ESI) mass spectrometry (MS) in the discovery setting to quantify developmental changes in the chief circadian pacemaker, the suprachiasmatic nucleus (SCN), before and after the onset of photoreceptor-dependent visual function. Nesting CE-ESI with trapped ion mobility spectrometry time-of-flight (TOF) mass spectrometry (TimsTOF PRO) doubled the number of identified and quantified proteins compared to the TOF-only control on the same analytical platform. From 10 ng of peptide input, corresponding to <∼0.5% of the total local tissue proteome, technical triplicate analyses identified 1894 proteins and quantified 1066 proteins, including many with important canonical functions in axon guidance, synapse function, glial cell maturation, and extracellular matrix refinement. Label-free quantification revealed differential regulation for 166 proteins over development, with enrichment of axon guidance-associated proteins prior to eye-opening and synapse-associated protein enrichment after eye-opening. Super-resolution imaging of select proteins using STochastic Optical Reconstruction Microscopy (STORM) corroborated the MS results and showed that increased presynaptic protein abundance pre/post eye-opening in the SCN reflects a developmental increase in synapse number, but not presynaptic size or extrasynaptic protein expression. This work marks the first development and systematic application of TimsTOF PRO for CE-ESI-based microproteomics and the first integration of microanalytical CE-ESI TimsTOF PRO with volumetric super-resolution STORM imaging to expand the repertoire of technologies supporting analytical neuroscience.

Mapping protein-exopolysaccharide binding interaction in Staphylococcus epidermidis biofilms by live cell proximity labeling.

Bacterial biofilms consist of cells encased in an extracellular polymeric substance (EPS) composed of exopolysaccharides, extracellular DNA, and proteins that are critical for cell-cell adhesion and protect the cells from environmental stress, antibiotic treatments, and the host immune response. Degrading EPS components or blocking their production have emerged as promising strategies for prevention or dispersal of bacterial biofilms, but we still have little information about the specific biomolecular interactions that occur between cells and EPS components and how those interactions contribute to biofilm production. Staphylococcus epidermidis is a leading cause of nosocomial infections as a result of producing biofilms that use the exopolysaccharide poly-(1→6)-ß-N-acetylglucosamine (PNAG) as a major structural component. In this study, we have developed a live cell proximity labeling approach combined with quantitative mass spectrometry-based proteomics to map the PNAG interactome of live S. epidermidis biofilms. Through these measurements we discovered elastin-binding protein (EbpS) as a major PNAG-interacting protein. Using live cell binding measurements, we found that the lysin motif (LysM) domain of EbpS specifically binds to PNAG present in S. epidermidis biofilms. Our work provides a novel method for the rapid identification of exopolysaccharide-binding proteins in live biofilms that will help to extend our understanding of the biomolecular interactions that are required for bacterial biofilm formation.

Time-resolved quantitative proteomic analysis of the developing Xenopus otic vesicle reveals putative congenital hearing loss candidates.

Over 200 genes are known to underlie human congenital hearing loss (CHL). Although transcriptomic approaches have identified candidate regulators of otic development, little is known about the abundance of their protein products. We used a multiplexed quantitative mass spectrometry-based proteomic approach to determine protein abundances over key stages of Xenopus otic morphogenesis to reveal a dynamic expression of cytoskeletal, integrin signaling, and extracellular matrix proteins. We correlated these dynamically expressed proteins to previously published lists of putative downstream targets of human syndromic hearing loss genes: SIX1 (BOR syndrome), CHD7 (CHARGE syndrome), and SOX10 (Waardenburg syndrome). We identified transforming growth factor beta-induced (Tgfbi), an extracellular integrin-interacting protein, as a putative target of Six1 that is required for normal otic vesicle formation. Our findings demonstrate the application of this Xenopus dataset to understanding the dynamic regulation of proteins during otic development and to discovery of additional candidates for human CHL.

Deep Proteome of the Developing Chick Midbrain.

The epithelial-to-mesenchymal transition (EMT) and migration of cranial neural crest cells within the midbrain are critical processes that permit proper craniofacial patterning in the early embryo. Disruptions in these processes not only impair development but also lead to various diseases, underscoring the need for their detailed understanding at the molecular level. The chick embryo has served historically as an excellent model for human embryonic development, including cranial neural crest cell EMT and migration. While these developmental events have been characterized transcriptionally, studies at the protein level have not been undertaken to date. Here, we applied mass spectrometry (MS)-based proteomics to establish a deep proteomics profile of the chick midbrain region during early embryonic development. Our proteomics method combines optimal lysis conditions, offline fractionation, separation on a nanopatterned stationary phase (µPAC) using nanoflow liquid chromatography, and detection using quadrupole-ion trap-Orbitrap tribrid high-resolution tandem MS. Identification of >5900 proteins and >450 phosphoproteins in this study marks the deepest coverage of the chick midbrain proteome to date. These proteins have known roles in pathways related to neural crest cell EMT and migration such as signaling, proteolysis/extracellular matrix remodeling, and transcriptional regulation. This study offers valuable insight into important developmental processes occurring in the midbrain region and demonstrates the utility of proteomics for characterization of tissue microenvironments during chick embryogenesis.

Cell Lineage-Guided Microanalytical Mass Spectrometry Reveals Increased Energy Metabolism and Reactive Oxygen Species in the Vertebrate Organizer.

Molecular understanding of the vertebrate Organizer, a tissue center critical for inductive signaling during gastrulation, has so far been limited to transcripts and some proteins due to limitations in detection and sensitivity. The Spemann-Mangold Organizer (SMO) in the South African Clawed Frog ( X. laevis ), a popular model of development, has long been discovered to induce the patterning of the central nervous system. Molecular screens on the tissue have identified several genes, such as goosecoid, chordin, and noggin, with independent ability to establish a body axis. A comprehensive study of proteins and metabolites produced in the SMO and their functional roles has been lacking. Here, we pioneer a deep discovery proteomic and targeted metabolomic screen of the SMO in comparison to the rest of the embryo using liquid chromatography high-resolution mass spectrometry (HRMS). Quantification of ∼4,600 proteins and a panel of metabolites documented differential expression for ∼450 proteins and multiple intermediates of energy metabolism in the SMO. Upregulation of oxidative phosphorylation (OXPHOS) and redox regulatory proteins gave rise to elevated oxidative stress and an accumulation of reactive oxygen species in the Organizer. Imaging experiments corroborated these findings, discovering enrichment of hydrogen peroxide in the SMO tissue. Chemical perturbation of the redox gradient affected mesoderm involution during early tissue movements of gastrulation. HRMS expands the bioanalytical toolbox of cell and developmental biology, providing previously unavailable information on molecular classes to challenge and refine our classical understanding of the Organizer and its function during early patterning of the embryo.

Case Report: Brainstem angiocentric glioma presenting in a toddler child-diagnostic and therapeutic challenges.

Introduction: Angiocentric gliomas (AG) in brainstem location are exceedingly rare and might cause differential diagnostic problems and uncertainty regarding the best therapeutic approach. Hereby, we describe the clinicopathological findings in a brainstem AG presenting in a toddler child and review the literature. Case report: A 2-year-old boy presented with 5 weeks history of gait disturbances, frequent falls, left-sided torticollis and swallowing problems. MRI head showed a T2-hyperintense, partly exophytic mass lesion centred in the pontomedullary region, raising the possibility of diffuse midline glioma. The exophytic component was partially resected by suboccipital craniotomy, leaving intact the infiltrative component. Ventriculoperitoneal shunt was implanted due to postoperative hydrocephalus. Histological examination revealed a moderately cellular tumour consisted of bland glial cells infiltrating the brain parenchyma and radially arranged around the blood vessels. By immunohistochemistry, the tumour strongly expressed S100 and GFAP in addition to intense nestin positivity, while OLIG2 was negative in the perivascular tumour cells. DNA methylation array profiled the tumour as "methylation class diffuse astrocytoma, MYB or MYBL1-altered subtype B (infratentorial)" and an in-frame MYB::QKI fusion was identified by RNA sequencing, confirming the diagnosis of angiocentric glioma. The patient has been initially treated with angiogenesis inhibitor and mTOR inhibitor, and now he is receiving palliative vinblastine. He is clinically stable on 9 months follow-up. Conclusion: Brainstem AG may cause a diagnostic problem, and the surgical and oncological management is challenging due to unresectability and lack of response to conventional chemo-radiation. In the future, genetically-tailored therapies might improve the prognosis.

Initial recommendations for performing, benchmarking and reporting single-cell proteomics experiments.

Analyzing proteins from single cells by tandem mass spectrometry (MS) has recently become technically feasible. While such analysis has the potential to accurately quantify thousands of proteins across thousands of single cells, the accuracy and reproducibility of the results may be undermined by numerous factors affecting experimental design, sample preparation, data acquisition and data analysis. We expect that broadly accepted community guidelines and standardized metrics will enhance rigor, data quality and alignment between laboratories. Here we propose best practices, quality controls and data-reporting recommendations to assist in the broad adoption of reliable quantitative workflows for single-cell proteomics. Resources and discussion forums are available at https://single-cell.net/guidelines .

Microanalysis of Brain Angiotensin Peptides Using Ultrasensitive Capillary Electrophoresis Trapped Ion Mobility Mass Spectrometry.

While the role of the renin-angiotensin system (RAS) in peripheral circulation is well characterized, we still lack an in-depth understanding of its role within the brain. This knowledge gap is sustained by lacking technologies for trace-level angiotensin detection throughout tissues, such as the brain. To provide a bridging solution, we enhanced capillary electrophoresis (CE) nanoflow electrospray ionization (ESI) with large-volume sample stacking and employed trapped ion mobility time-of-flight (timsTOF) tandem HRMS detection. A dynamic pH junction helped stack approximately 10 times more of the sample than optimal using the field-amplified reference. In conjunction, the efficiency of ion generation was maximized by a cone-jet nanospray on a low sheath-flow tapered-tip nano-electrospray emitter. The platform provided additional peptide-dependent information, the collision cross section, to filter chemical noise and improve sequence identification and detection limits. The lower limit of detection reached sub-picomolar or ∼30 zmol (∼18,000 copies) level. All nine targeted angiotensin peptides in mouse tissue samples were detectable and quantifiable from the paraventricular nucleus (PVN) of the hypothalamus even after removal of circulatory blood components (perfusion). We anticipate CE-ESI with timsTOF HRMS to be broadly applicable for the ultrasensitive detection of brain peptidomes in pursuit of a better understanding of the brain.

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing (Frog) Embryo.

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design efficient remedies for diseases. Technologies enabling accurate identification and quantification of diverse types and large numbers of proteins would provide still missing information on molecular mechanisms orchestrating tissue and organism development in space and time. Here, we present a mass spectrometry-based protocol that enables the measurement of thousands of proteins in identified cell lineages in Xenopus laevis (frog) embryos. The approach builds on reproducible cell-fate maps and established methods to identify, fluorescently label, track, and sample cells and their progeny (clones) from this model of vertebrate development. After collecting cellular contents using microsampling or isolating cells by dissection or fluorescence-activated cell sorting, proteins are extracted and processed for bottom-up proteomic analysis. Liquid chromatography and capillary electrophoresis are used to provide scalable separation for protein detection and quantification with high-resolution mass spectrometry (HRMS). Representative examples are provided for the proteomic characterization of neural-tissue fated cells. Cell-lineage-guided HRMS proteomics is adaptable to different tissues and organisms. It is sufficiently sensitive, specific, and quantitative to peer into the spatio-temporal dynamics of the proteome during vertebrate development.

Capillary Electrophoresis Mass Spectrometry for Scalable Single-Cell Proteomics.

Understanding the biochemistry of the cell requires measurement of all the molecules it produces. Single-cell proteomics recently became possible through advances in microanalytical sample preparation, separation by nano-flow liquid chromatography (nanoLC) and capillary electrophoresis (CE), and detection using electrospray ionization (ESI) high-resolution mass spectrometry (HRMS). Here, we demonstrate capillary microsampling CE-ESI-HRMS to be scalable to proteomics across broad cellular dimensions. This study established proof-of-principle using giant, ∼250-µm-diameter cells from embryos of the frog Xenopus laevis and small, ∼35-µm-diameter neurons in culture from the mouse hippocampus. From ∼18 ng, or ∼0.2% of the total cellular proteome, subcellular analysis of the ventral-animal midline (V11) and equatorial (V12) cells identified 1,133 different proteins in a 16-cell embryo. CE-HRMS achieved ∼20-times higher sensitivity and doubled the speed of instrumental measurements compared to nanoLC, the closest neighboring single-cell technology of choice. Microanalysis was scalable to 722 proteins groups from ∼5 ng of cellular protein digest from identified left dorsal-animal midline cell (D11), supporting sensitivity for smaller cells. Capillary microsampling enabled the isolation and transfer of individual neurons from the culture, identifying 37 proteins between three different cells. A total of 224 proteins were detected from 500 pg of neuronal protein digest, which estimates to a single neuron. Serial dilution returned 157 proteins from sample amounts estimating to about half a cell (250 pg protein) and 70 proteins from ca. a quarter of a neuron (125 pg protein), suggesting sufficient sensitivity for subcellular proteomics. CE-ESI-HRMS complements nanoLC proteomics with scalability, sensitivity, and speed across broad cellular dimensions.

Patch-Clamp Proteomics of Single Neurons in Tissue Using Electrophysiology and Subcellular Capillary Electrophoresis Mass Spectrometry.

Understanding of the relationship between cellular function and molecular composition holds a key to next-generation therapeutics but requires measurement of all types of molecules in cells. Developments in sequencing enabled semiroutine measurement of single-cell genomes and transcriptomes, but analytical tools are scarce for detecting diverse proteins in tissue-embedded cells. To bridge this gap for neuroscience research, we report the integration of patch-clamp electrophysiology with subcellular shot-gun proteomics by high-resolution mass spectrometry (HRMS). Recording of electrical activity permitted identification of dopaminergic neurons in the substantia nigra pars compacta. Ca. 20-50% of the neuronal soma content, containing an estimated 100 pg of total protein, was aspirated into the patch pipette filled with ammonium bicarbonate. About 1 pg of somal protein, or ∼0.25% of the total cellular proteome, was analyzed on a custom-built capillary electrophoresis (CE) electrospray ionization platform using orbitrap HRMS for detection. A series of experiments were conducted to systematically enhance detection sensitivity through refinements in sample processing and detection, allowing us to quantify ∼275 different proteins from somal aspirate-equivalent protein digests from cultured neurons. From single neurons, patch-clamp proteomics of the soma quantified 91, 80, and 95 different proteins from three different dopaminergic neurons or 157 proteins in total. Quantification revealed detectable proteomic differences between the somal protein samples. Analysis of canonical knowledge predicted rich interaction networks between the observed proteins. The integration of patch-clamp electrophysiology with subcellular CE-HRMS proteomics expands the analytical toolbox of neuroscience.

Single-Cell Mass Spectrometry of Metabolites and Proteins for Systems and Functional Biology.

Molecular composition is intricately intertwined with cellular function, and elucidation of this relationship is essential for understanding life processes and developing next-generational therapeutics. Technological innovations in capillary electrophoresis (CE) and liquid chromatography (LC) mass spectrometry (MS) provide previously unavailable insights into cellular biochemistry by allowing for the unbiased detection and quantification of molecules with high specificity. This chapter presents our validated protocols integrating ultrasensitive MS with classical tools of cell, developmental, and neurobiology to assess the biological function of important biomolecules. We use CE and LC MS to measure hundreds of metabolites and thousands of proteins in single cells or limited populations of tissues in chordate embryos and mammalian neurons, revealing molecular heterogeneity between identified cells. By pairing microinjection and optical microscopy, we demonstrate cell lineage tracing and testing the roles the dysregulated molecules play in the formation and maintenance of cell heterogeneity and tissue specification in frog embryos (Xenopus laevis). Electrophysiology extends our workflows to characterizing neuronal activity in sections of mammalian brain tissues. The information obtained from these studies mutually strengthen chemistry and biology and highlight the importance of interdisciplinary research to advance basic knowledge and translational applications forward.

Data-Dependent Acquisition Ladder for Capillary Electrophoresis Mass Spectrometry-Based Ultrasensitive (Neuro)Proteomics.

Measurement of broad types of proteins from a small number of cells to single cells would help to better understand the nervous system but requires significant leaps in sensitivity in high-resolution mass spectrometry (HRMS). Microanalytical capillary electrophoresis electrospray ionization (CE-ESI) offers a path to ultrasensitive proteomics by integrating scalability with sensitivity. Here, we systematically evaluate performance limitations in this technology to develop a data acquisition strategy with deeper coverage of the neuroproteome from trace amounts of starting materials than traditional dynamic exclusion. During standard data-dependent acquisition (DDA), compact migration challenged the duty cycle of second-stage transitions and redundant targeting of abundant peptide signals lowered their identification success rate. DDA was programmed to progressively exclude a static set of high-intensity peptide signals throughout replicate measurements, essentially forming rungs of a "DDA ladder." The method was tested for ∼500 pg portions of a protein digest from cultured hippocampal (primary) neurons (mouse), which estimated the total amount of protein from a single neuron. The analysis of ∼5 ng of protein digest over all replicates, approximating ∼10 neurons, identified 428 nonredundant proteins (415 quantified), an ∼35% increase over traditional DDA. The identified proteins were enriched in neuronal marker genes and molecular pathways of neurobiological importance. The DDA ladder enhances CE-HRMS sensitivity to single-neuron equivalent amounts of proteins, thus expanding the analytical toolbox of neuroscience.

Mass spectrometry based proteomics for developmental neurobiology in the amphibian Xenopus laevis.

The South African clawed frog (Xenopus laevis), a prominent vertebrate model in cell and developmental biology, has been instrumental in studying molecular mechanisms of neural development and disease. Recently, high-resolution mass spectrometry (HRMS), a bioanalytical technology, has expanded the molecular toolbox of protein detection and characterization (proteomics). This chapter overviews the characteristics, advantages, and challenges of this biological model and technology. Discussions are offered on their combined use to aid studies on cell differentiation and development of neural tissues. Finally, the emerging integration of proteomics and other 'omic technologies is reflected on to generate new knowledge, drive and test new hypotheses, and ultimately, advance the understanding of neural development during states of health and disease.

Altering metabolite distribution at Xenopus cleavage stages affects left-right gene expression asymmetries.

The left-right (L-R) axis of most bilateral animals is established during gastrulation when a transient ciliated structure creates a directional flow of signaling molecules that establish asymmetric gene expression in the lateral plate mesoderm. However, in some animals, an earlier differential distribution of molecules and cell division patterns initiate or at least influence L-R patterning. Using single-cell high-resolution mass spectrometry, we previously reported a limited number of small molecule (metabolite) concentration differences between left and right dorsal-animal blastomeres of the eight-cell Xenopus embryo. Herein, we examined whether altering the distribution of some of these molecules influenced early events in L-R patterning. Using lineage tracing, we found that injecting right-enriched metabolites into the left cell caused its descendant cells to disperse in patterns that varied from those in control gastrulae; this did not occur when left-enriched metabolites were injected into the right cell. At later stages, injecting left-enriched metabolites into the right cell perturbed the expression of genes known to: (a) be required for the formation of the gastrocoel roof plate (foxj1); (b) lead to the asymmetric expression of Nodal (dand5/coco); or (c) result from asymmetrical nodal expression (pitx2). Despite these perturbations in gene expression, we did not observe heterotaxy in heart or gut looping at tadpole stages. These studies indicate that altering metabolite distribution at cleavage stages at the concentrations tested in this study impacts the earliest steps of L-R gene expression that then can be compensated for during organogenesis.