Single-cell spatial mapping reveals reproducible cell type organization and spatially-dependent gene expression in gastruloids

Reviewer #1 (Public review):

Summary:

The authors performed seqFISH in 26 gastruloids and performed a variety of computational analyses on these novel spatial data sets. Whilst the data is valuable and the computational concepts useful (exposure index, L-metric, ... ), the article falls short on novelty and is written using a very clunky language, often with contradictory conclusions.

Major issues:

(1) The authors did well in explaining and detailing the provenance of data and the individual experiments performed. However, their 26 gastruloid data still constitute a very limited sampling from their total organoids: one experiment pooled 4 plates at an 80-94% success rate; 6 different aggregation experiments were done, making a total of 1843 gastruloids, sampled 26 (~1-2%). A simple IF stain of 2-3 markers in a bigger sample could have given a more accurate picture of specific domains of interest and their proximity. Regardless, more information should be given about the existing samples: variation across experimental batches, differences between 300-cell vs 100-cell gastruloids that were used.

(2) Language in the manuscript should be revised. Overall the manuscript is very long, descriptive and written "impressions and beliefs" are often not adequately justified and indeed can be contradictory, e.g. in Section 1: the title states "cell types' locations ...are consistent", a few sentences down we find "there was substantial variation" and "within range of what would be considered a 'morphologically normal' gastruloid". "quite consistent", "compelling patterning", "we don't believe"... these types of expressions are best avoided and replaced with data or used and bolstered with quantitative numbers such as percentages when a given cutoff is used. Another example: "location of each cell type relative to gastruloid morphology was quite consistent the posterior region ... mainly consisted in NMPs." Given T expression in the posterior, this result phrased as such appears quite inflated, in fact, looking at cell types in Figures S1, 2a/b/c, this reviewer would state they are all but consistent and indeed it takes sophisticated analyses to find a pattern (of sorts) beyond the coarse domains expected!

(3) Figure 6 is one of the most valuable parts of the work, as the authors use the battery of analyses developed to investigate the variable and not-so-robust endothelial clusters in gastruloids. However, this investigation is still very preliminary, and it should be further linked with known biology. It is still unclear what the unique organization of this cell type is (circularity isn't convincing) and whether any signalling cues of adjacent cells could explain it. Is there any evidence that more mature endodermal cell types are generated (like the suggested "liver") to give rise to endothelial cells? It would certainly be interesting to perform IF for this cell type together with mesodermal and endodermal markers to validate seqFISH predictions on a bigger sample.

(4) Figures 1c and 6b need statistical significance assessments.

(5) The article should include an analysis of Hox colinearity expression in these gastruloids as a validation of the system.

Reviewer #2 (Public review):

Summary:

This manuscript presents an ambitious and technically challenging spatial-transcriptomic atlas of 26 gastruloids using seqFISH. The authors introduce quantitative metrics (mixing score, exposure index, L-metric / scL-metric, spatial L-metric, triplets) to characterize spatial organization at multiple scales. The dataset is valuable, and several analyses are original, particularly the rank-based L-metric family for mutual exclusivity.

Strengths:

The authors generate one of the most detailed spatial transcriptomic datasets of gastruloids to date. They propose creative computational metrics (L-metric/scL-metric) to quantify mutual exclusivity of gene expression without predefined thresholds, and they explore organizational principles from single-cell topology to cluster-level structure. Many observations align well with known gastruloid biology, such as posterior robustness and anterior variability. The writing is generally clear, and the figures are rich.

Weaknesses:

Several central claims rely on metrics whose computation and justification are insufficiently explained, making it difficult to assess how robust or interpretable the results are. Many choices in the analysis appear arbitrary or are insufficiently motivated (normalization schemes, choice of parameters such as the number of neighbors, the distance cutoffs, hierarchical clustering setup, and so on). The interpretations of spatial consistency, gene-program inference, and endothelial heterogeneity are plausible but might be stronger than the evidence currently supports.

The manuscript would benefit from stronger benchmarking, quantification of uncertainty, and explicit controls for known artifacts in spatial transcriptomics (e.g., spillover, 2D slicing, cell type assignment entropy). The biological insights are promising, but since several depend on methodological assumptions that have not yet been demonstrated to be stable, they would benefit from clearer methodological explanation.

The work is rich and could become a reference dataset. Then, clarifying and validating the quantitative methods will considerably strengthen the impact and reliability of the conclusions.

Reviewer #3 (Public review):

Summary:

Triandafillou and colleagues report a single-cell resolved spatial atlas of gene expression of 26 gastruloids. While previous work had analyzed either single-cell gene expression or spatially coarse-grained patterns of gene expression (van den Brink et al, 2020), the authors here use multiplexed sequential RNA FISH (seqFISH) to create the first gastruloid atlas, which is simultaneously spatially and cellularly resolved. This atlas adds to a growing list of resources cataloging gastruloid development (see also Suppinger et al 2023).

To analyze this dataset, the authors also describe a novel analytical framework. Their analysis centers around the 'L-metric', which measures the degree to which pairs of genes are either coexpressed or mutually exclusive. While this metric is similar to calculating correlations in gene expressions, it has important differences (including that it can, in principle, be asymmetric; although the authors symmetrize much of their analysis). In addition to the gene-centric L-metric analysis, the authors also analyze cells in their dataset according to the cell type entropy (an information-theoretical measure of confidence in cell type assignment) and the 'exposure index' (a measure of the similarity of nearest cellular neighbors).

Using this framework, the authors focus their analysis on two major features of development. The first is the differentiation of the bipotent neuromesodermal progenitor (NMP) cells in the posterior of the gastruloid into either presomitic mesoderm (PSM) or spinal cord SC lineages. They use L-metric analysis to compare overlap in marker genes used to separate NMP, PSM, and SC fates. They highlight that L-metric analysis can recover spatial patterns of gene expression (without explicit spatial information) and discern subtle features of marker genes beyond simple binning of cell types (e.g., that Epha5 expression in anterior NMPs may predict future SC differentiation).

The second is the formation of endothelial (spatial) clusters within the gastruloid. The authors highlight two subtypes of endothelial clusters: (1) smaller clusters within the somitic anterior region, and (2) larger clusters associated with endoderm. While the authors discern some subtle differences in gene expression between these two clusters, their different spatial patterns suggest a potential physiological difference that would not be captured in traditional droplet microfluidic-based scRNAseq pipelines.

Overall, this manuscript is a sophisticated and technically sound study that will provide a valuable beachhead for future studies of developmental patterning in gastruloids and organoids.

Strengths:

The major strengths of this study are the overall technical sophistication of the data set and analysis, as well as its potential generalizability to other developmental systems (both in vitro and in vivo). The data are extensively analyzed and reasonably interpreted, and this atlas makes good use of the variability in gastruloid development to extract the statistical structure of developmental processes. The L-metric offers a parameter-free tool to analyze transcriptomic datasets that could overcome the pitfalls of other approaches.

Weaknesses:

The major limitations of this study are the depth and novelty of the developmental processes studied. The authors provide very convincing proof-of-concept that their data set can recover known features of gastruloid development, including NMP differentiation and endothelial development. However, further analysis and/or investigation would be required to discover new principles of gastruloid development and patterning.