A spatiotemporal transcriptomic atlas of the mouse placenta reveals glycogen cell-mediated metabolic support essential for fetal viability

Reviewer #1 (Public review):

In this manuscript, the authors combine single-nucleus RNA sequencing with spatial transcriptomics to generate a spatiotemporal atlas of mouse placental development and explore the role of glycogen trophoblast cells in fetal viability. The study integrates several computational approaches, including trajectory analysis, regulatory network inference, and spatial mapping, together with histology and glycogen measurements. Based on these analyses, the authors propose that glycogen trophoblast cells provide metabolic support that is important for maintaining placental function and fetal survival.

One of the main strengths of the study is the quality and scope of the dataset. The integration of snRNA-seq with Stereo-seq spatial transcriptomics provides a detailed view of placental organization across regions and developmental stages. This type of combined spatial and transcriptional analysis is still relatively rare in placental biology and represents an important contribution to the field. The atlas itself will likely be a valuable resource for future studies.

Another strength is the effort to connect transcriptional findings with tissue-level validation. The glycogen staining and biochemical measurements support the interpretation that glycogen trophoblast cells contribute to placental metabolic function. The spatial analyses identifying macrophage accumulation in the labyrinth region of mutant placentas are also interesting and illustrate how spatial approaches can reveal microenvironmental changes that are difficult to detect otherwise.

The main limitation of the study is that the conclusion that glycogen cells are essential mediators of metabolic support for fetal viability remains partly indirect. The transcriptomic and spatial data strongly suggest a role for these cells, but it is still difficult to determine whether glycogen cell dysfunction is the primary cause of fetal lethality or a consequence of broader placental abnormalities. Clarifying this point would strengthen the central message of the paper.

Similarly, the macrophage accumulation observed in the labyrinth appears consistent with a response to tissue stress or injury, but its relationship to glycogen cell function is not fully explained. A clearer discussion of whether this represents a primary mechanism or a secondary effect would improve the interpretation.

Overall, this is a strong dataset and a useful spatial atlas of placental development. The study provides convincing descriptive insight into glycogen trophoblast biology, and with some clarification of the mechanistic conclusions, the manuscript will be even stronger.

Reviewer #2 (Public review):

This manuscript constructs a spatiotemporal transcriptomic atlas (STAMP) of the mouse placenta from E9.5-E18.5 by integrating Stereo-seq and snRNA-seq, and identifies two glycogen trophoblast cell (GC) subtypes (GC-1 and GC-2), a spatial transition from the junctional zone (JZ) to the decidua, and metabolic defects in Ano6-null placentas including GC persistence, glycogen accumulation, reduced glycogenolysis metabolites, and partial rescue by maternal glucose supplementation. The breadth of the dataset and the integration of atlas construction with PAS/TEM/LC-MS analyses are impressive, and the study has the potential to provide a valuable resource for the placental biology community.

However, in its current form, the central claim that "GC-mediated metabolic support is essential/indispensable for fetal viability" is not sufficiently disentangled from the complex phenotype of a global Ano6 knockout model. In addition, the stage-level biological replication in the atlas and the claim of "single-cell resolution" require more careful presentation. Therefore, while the study is interesting and potentially impactful, substantial revisions are required, particularly to recalibrate the strength of the conclusions and causal interpretations.

Major comments

(1) The most significant concern is that the manuscript overinterprets the phenotype observed in a global Ano6 knockout as direct evidence that GC glycogen metabolism is essential for fetal viability. The authors themselves report multiple severe placental abnormalities in the knockout, including reduced placental size and weight, structural defects in the labyrinth, impaired vascularization, and accumulation of abnormal regions. Previous studies cited in the manuscript also indicate that Ano6 deficiency leads to defects in syncytiotrophoblast formation, impaired maternofetal exchange, and perinatal lethality.

In this context, the current data support an association between GC metabolic defects and fetal lethality, but do not establish that GC glycogen metabolism is the primary causal driver. The conclusion should therefore be moderated (e.g., "contributes to" rather than "is essential for"), unless additional placenta-specific or GC-specific functional validation is provided.

(2) Maternal glucose supplementation is an interesting functional experiment, but in its current form, it provides supportive rather than definitive mechanistic evidence. While survival improves (from ~3% to ~10%), the rescue remains partial. Moreover, the readouts are largely limited to metabolite restoration (glucose, G1P, G6P) in the placenta and fetal liver.

To support a stronger causal claim, the authors should assess whether glucose supplementation also rescues: placental morphology (especially labyrinth structure), GC number and PAS staining, ultrastructural glycogen features (TEM), fetal growth and developmental outcomes.

(3) The atlas is constructed from nine placentas across developmental stages, suggesting limited biological replication per stage. It remains unclear how robust the observed temporal trends are to litter effects, sex differences, or sectioning variability.

Furthermore, the "single-cell resolution" is not directly measured but inferred via image segmentation and reference-based mapping (e.g., TACCO). This should be more explicitly stated, as it represents computational inference rather than direct single-cell measurement.

The authors should:
- clearly report biological replicates per stage (including litter and sex),
- demonstrate reproducibility of key patterns across independent samples,
- refine the wording to reflect segmentation- and reference-based single-cell inference.

(4) The proposed developmental trajectory (JZ progenitor → GC precursor → GC-1 → GC-2) and the claim of GC migration from JZ to decidua are based on spatial distribution and computational trajectory analyses (Monocle, CytoTRACE).

While this is a compelling model, it remains inferential. The language throughout the manuscript should be softened (e.g., "consistent with spatial transition" rather than "migration"). Ideally, additional experimental validation, such as stage-resolved RNAscope/immunostaining quantification or lineage tracing, would strengthen this claim.

(5) The manuscript concludes that ANO6 deficiency leads to impaired glycogen utilization, based primarily on the observation that differentiation markers and glycogenolytic enzyme transcripts are unchanged.

However, this demonstrates what is not altered rather than what is mechanistically responsible for the defect. A more direct mechanistic link is needed, such as changes in enzyme activity, altered intracellular localization, effects on ion homeostasis or membrane biology.

(6) The statistical framework requires clarification. Several analyses use n = 4-8 placentas or "independent experiments," but it is unclear whether these represent independent litters or multiple samples from the same dam.

Given the risk of pseudoreplication in placental studies, the authors should define whether n refers to placentas or litters, report the number of dams per genotype, and ensure appropriate statistical treatment (e.g., litter-based analysis or mixed-effects models).

Author response:

eLife Assessment

This valuable study reports a spatiotemporal atlas of mouse placental development and explores the role of glycogen trophoblast cells in fetal viability. Solid data are presented to support the main conclusion. This work will be of great interest to developmental DNA reproductive biologists.

We thank the editors for this positive and balanced assessment of our study. We are encouraged that the spatiotemporal mouse placental atlas and the functional analysis of glycogen trophoblast cells were considered valuable, and that the data were viewed as providing solid support for the main conclusions.

In the revised manuscript, we will further clarify the scope of these conclusions, particularly regarding the contribution of GC-associated glycogen metabolism to fetal viability in the global Ano6 knockout model. We will also refine the wording where needed to ensure that the mechanistic interpretation accurately reflects the strength of the available evidence.

Public Reviews:

Reviewer #1 (Public review):

In this manuscript, the authors combine single-nucleus RNA sequencing with spatial transcriptomics to generate a spatiotemporal atlas of mouse placental development and explore the role of glycogen trophoblast cells in fetal viability. The study integrates several computational approaches, including trajectory analysis, regulatory network inference, and spatial mapping, together with histology and glycogen measurements. Based on these analyses, the authors propose that glycogen trophoblast cells provide metabolic support that is important for maintaining placental function and fetal survival.

One of the main strengths of the study is the quality and scope of the dataset. The integration of snRNA-seq with Stereo-seq spatial transcriptomics provides a detailed view of placental organization across regions and developmental stages. This type of combined spatial and transcriptional analysis is still relatively rare in placental biology and represents an important contribution to the field. The atlas itself will likely be a valuable resource for future studies.

Another strength is the effort to connect transcriptional findings with tissue-level validation. The glycogen staining and biochemical measurements support the interpretation that glycogen trophoblast cells contribute to placental metabolic function. The spatial analyses identifying macrophage accumulation in the labyrinth region of mutant placentas are also interesting and illustrate how spatial approaches can reveal microenvironmental changes that are difficult to detect otherwise.

The main limitation of the study is that the conclusion that glycogen cells are essential mediators of metabolic support for fetal viability remains partly indirect. The transcriptomic and spatial data strongly suggest a role for these cells, but it is still difficult to determine whether glycogen cell dysfunction is the primary cause of fetal lethality or a consequence of broader placental abnormalities. Clarifying this point would strengthen the central message of the paper.

Similarly, the macrophage accumulation observed in the labyrinth appears consistent with a response to tissue stress or injury, but its relationship to glycogen cell function is not fully explained. A clearer discussion of whether this represents a primary mechanism or a secondary effect would improve the interpretation.

Overall, this is a strong dataset and a useful spatial atlas of placental development. The study provides convincing descriptive insight into glycogen trophoblast biology, and with some clarification of the mechanistic conclusions, the manuscript will be even stronger.

We thank the reviewer for this constructive assessment of our manuscript. We are pleased that the reviewer recognized the quality and scope of the dataset, particularly the integration of snRNA sequencing with Stereo-seq spatial transcriptomics to generate a spatiotemporal atlas of mouse placental development. We also appreciate the reviewer’s view that this atlas represents a valuable resource for the placental biology and developmental biology communities. We also appreciate the reviewer’s important point that the causal relationship between glycogen trophoblast cell dysfunction, placental metabolic impairment, and fetal viability should be presented with appropriate caution. In the revised manuscript, we will clarify that our data support a strong association between impaired glycogen trophoblast cell function, altered placental glycogen metabolism, and fetal lethality in the global Ano6 knockout model, but do not by themselves establish glycogen trophoblast dysfunction as the sole or primary cause of fetal loss. We will revise the relevant sections to avoid overstatement and to distinguish more clearly between direct experimental evidence, correlative spatial-transcriptomic observations, and mechanistic interpretation. Similarly, we agree that the macrophage accumulation observed in the labyrinth region is most appropriately interpreted as a spatially localized immune or tissue-stress response in the mutant placenta. In the revised manuscript, we will expand the discussion to clarify that, while this observation may reflect downstream consequences of placental dysfunction and altered tissue homeostasis, the current data do not establish macrophage accumulation as a primary mechanism linking glycogen trophoblast defects to fetal lethality. We will therefore frame this finding as an important microenvironmental alteration revealed by the spatial atlas, rather than as definitive evidence of a direct causal pathway.

Reviewer #2 (Public review):

This manuscript constructs a spatiotemporal transcriptomic atlas (STAMP) of the mouse placenta from E9.5-E18.5 by integrating Stereo-seq and snRNA-seq, and identifies two glycogen trophoblast cell (GC) subtypes (GC-1 and GC-2), a spatial transition from the junctional zone (JZ) to the decidua, and metabolic defects in Ano6-null placentas including GC persistence, glycogen accumulation, reduced glycogenolysis metabolites, and partial rescue by maternal glucose supplementation. The breadth of the dataset and the integration of atlas construction with PAS/TEM/LC-MS analyses are impressive, and the study has the potential to provide a valuable resource for the placental biology community.

However, in its current form, the central claim that "GC-mediated metabolic support is essential/indispensable for fetal viability" is not sufficiently disentangled from the complex phenotype of a global Ano6 knockout model. In addition, the stage-level biological replication in the atlas and the claim of "single-cell resolution" require more careful presentation. Therefore, while the study is interesting and potentially impactful, substantial revisions are required, particularly to recalibrate the strength of the conclusions and causal interpretations.

Major comments

(1) The most significant concern is that the manuscript overinterprets the phenotype observed in a global Ano6 knockout as direct evidence that GC glycogen metabolism is essential for fetal viability. The authors themselves report multiple severe placental abnormalities in the knockout, including reduced placental size and weight, structural defects in the labyrinth, impaired vascularization, and accumulation of abnormal regions. Previous studies cited in the manuscript also indicate that Ano6 deficiency leads to defects in syncytiotrophoblast formation, impaired maternofetal exchange, and perinatal lethality.

In this context, the current data support an association between GC metabolic defects and fetal lethality, but do not establish that GC glycogen metabolism is the primary causal driver. The conclusion should therefore be moderated (e.g., "contributes to" rather than "is essential for"), unless additional placenta-specific or GC-specific functional validation is provided.

(2) Maternal glucose supplementation is an interesting functional experiment, but in its current form, it provides supportive rather than definitive mechanistic evidence. While survival improves (from ~3% to ~10%), the rescue remains partial. Moreover, the readouts are largely limited to metabolite restoration (glucose, G1P, G6P) in the placenta and fetal liver.

To support a stronger causal claim, the authors should assess whether glucose supplementation also rescues: placental morphology (especially labyrinth structure), GC number and PAS staining, ultrastructural glycogen features (TEM), fetal growth and developmental outcomes.

(3) The atlas is constructed from nine placentas across developmental stages, suggesting limited biological replication per stage. It remains unclear how robust the observed temporal trends are to litter effects, sex differences, or sectioning variability.

Furthermore, the "single-cell resolution" is not directly measured but inferred via image segmentation and reference-based mapping (e.g., TACCO). This should be more explicitly stated, as it represents computational inference rather than direct single-cell measurement.

The authors should:

- clearly report biological replicates per stage (including litter and sex),

- demonstrate reproducibility of key patterns across independent samples,

- refine the wording to reflect segmentation- and reference-based single-cell inference.

(4) The proposed developmental trajectory (JZ progenitor → GC precursor → GC-1 → GC-2) and the claim of GC migration from JZ to decidua are based on spatial distribution and computational trajectory analyses (Monocle, CytoTRACE).

While this is a compelling model, it remains inferential. The language throughout the manuscript should be softened (e.g., "consistent with spatial transition" rather than "migration"). Ideally, additional experimental validation, such as stage-resolved RNAscope/immunostaining quantification or lineage tracing, would strengthen this claim.

(5) The manuscript concludes that ANO6 deficiency leads to impaired glycogen utilization, based primarily on the observation that differentiation markers and glycogenolytic enzyme transcripts are unchanged.

However, this demonstrates what is not altered rather than what is mechanistically responsible for the defect. A more direct mechanistic link is needed, such as changes in enzyme activity, altered intracellular localization, effects on ion homeostasis or membrane biology.

(6) The statistical framework requires clarification. Several analyses use n = 4-8 placentas or "independent experiments," but it is unclear whether these represent independent litters or multiple samples from the same dam.

Given the risk of pseudoreplication in placental studies, the authors should define whether n refers to placentas or litters, report the number of dams per genotype, and ensure appropriate statistical treatment (e.g., litter-based analysis or mixed-effects models).

We thank the Reviewer for the careful evaluation of our manuscript and for recognizing the breadth of the STAMP dataset and the value of integrating spatial transcriptomics, snRNA-seq, PAS, TEM and LC-MS analyses.

We agree that the current manuscript overstates some mechanistic conclusions. In the revision, we will moderate the central claim and more clearly acknowledge that the global Ano6 knockout model has complex placental defects.

Comment 1: Causality in the global Ano6 knockout model

We agree that our current data do not prove that GC glycogen metabolism is the primary cause of fetal lethality in the global Ano6 knockout model. In the revised manuscript, we will avoid presenting GC dysfunction as the sole causal mechanism. We will replace stronger terms such as “essential” or “indispensable” with more measured wording such as “contributes to” or “supports.” We will frame impaired GC-associated glycogen metabolism as one important component of Ano6-null placental dysfunction.

Comment 2: Maternal glucose supplementation

We agree that maternal glucose supplementation provides supportive, but not definitive, mechanistic evidence. In the revision, we will describe the partial survival rescue more cautiously and will not use it as proof of GC-specific causality. Where possible, we will also assess whether glucose supplementation affects additional phenotypes, including fetal growth, placental morphology, GC abundance and PAS/glycogen readouts.

Comment 3: Biological replication and single-cell resolution

We agree that the replication structure and the wording of “single-cell resolution” need clarification. We will report the number of placentas, litters and available sex information for each stage. We will also revise the wording to make clear that the spatial single-cell annotation is based on image segmentation and snRNA-seq reference mapping, rather than direct single-cell measurement by Stereo-seq alone.

Comment 4: GC trajectory and spatial transition

We agree that the proposed GC trajectory and JZ-to-decidua transition remain inferential. We will soften the language throughout the manuscript, using terms such as “spatial transition,” “redistribution,” or “consistent with migration” rather than stating that migration has been directly proven.

Comment 5: Mechanism of impaired glycogen utilization

We agree that unchanged GC markers and glycogenolytic enzyme transcripts do not reveal the direct mechanism. In the revision, we will state more clearly that these data argue against gross GC differentiation defects or transcriptional loss of glycogenolytic enzymes, but that the direct mechanism may involve enzyme activity, localization, ion homeostasis or ANO6-dependent membrane biology.

Comment 6: Statistical framework

We agree that the statistical framework needs clearer reporting. We will define what each n represents, including placenta, section, litter, dam or independent experiment, and will revise the analysis or description where needed to minimize concerns about pseudoreplication.

Overall, we appreciate these comments and will use them to make the revised manuscript more precise, transparent and appropriately cautious.