A spatially resolved single cell atlas of human gastrulation

Richard C.V. Tyser, Elmir Mahammadov, Shota Nakanoh, Ludovic Vallier, Antonio Scialdone, Shankar Srinivas

Preprint posted on 21 July 2020 https://www.biorxiv.org/content/10.1101/2020.07.21.213512v1

Article now published in Nature at http://dx.doi.org/10.1038/s41586-021-04158-y

How to build a human embryo? All you need to know and more in this single cell atlas of human gastrulation.

Selected by Martin Estermann

Categories: cell biology, developmental biology, evolutionary biology, genetics, genomics, molecular biology

Background:

After fertilization, the morula cells differentiate to form the blastula, formed by the outer cells (trophoblast) and the inner cell mass. Posteriorly the inner cells differentiate into the epiblast and the hypoblast to form a bilaminar disk. During gastrulation, the two layers (epi and hypoblast) are reorganized into three germ layers: the endoderm (internal layer), ectoderm (external layer) and the mesoderm (middle layer). These three layers will give rise to all the organs and structures of the body.

Several events can be identified during gastrulation, the first being the formation of the primitive streak, from the anterior to the posterior region of the embryo. This determines the midline of the body, separating left and right and establishing the caudal and cranial regions. The epiblast cells converge at the midline and ingress at the primitive streak, positioning themselves in between the original epiblast cells (ectoderm) and the hypoblast (endoderm), forming the mesoderm.

Our understanding of human gastrulation is based mostly on studies in animal models like mouse, rabbit or chicken, or on limited collections of fixed whole samples or histological sections. Human gastrulation samples are relatively rare because this process starts around 14 days post fertilization, a period when women might not know they are already pregnant. To overcome the lack of human samples, scientists developed cultured in vitro human models, such as human embryonic stem cells or gastruloids that resulted in a greater understanding of this developmental process. Despite many advantages, these models are not completely perfect at recapitulating the human gastrulation process.

In this research, the authors were able to obtain a karyotypically normal human male embryo at the gastrulation stage (Carnegie stage 7) through the Human Developmental Biology resource (http://www.hdbr.org). This embryo was intact, and contained both the embryonic disk with amniotic cavity, the connecting stalk and the yolk sac. In order to obtain the most information possible from such a precious sample, the authors performed single-cell RNA sequencing to characterize the transcriptome of each of the different cells present in the embryo. This allowed them to not only identify the different cells present in the gastrula, but also to study the genetic regulation of this process. This resource will act as a reference database to expand our current understanding of human early embryogenesis (accessible at http://www.human-gastrula.net) and will improve the accuracy of the different in vitro methodologies to match the in vivo development.

Key findings:

1) Gastrulation at single cell resolution

In order to obtain the most information with this unique sample, the authors performed single-cell RNA sequencing to characterize the transcriptome of each of the different embryonic cells present in the sample. One of the caveats of this technique is that when generating a single cell suspension, the spatial information is lost. To overcome this obstacle, the authors dissected the embryo in three different sections that were processed independently: the yolk sac, the rostral and the caudal region of the embryonic disk (Fig. 1A). This helped roughly know the embryonic origin of the different sequenced cells.

In total, 1195 cells were sequenced, 665 from the caudal embryonic disk, 340 from the rostral embryonic disk and 190 from the yolk sac (Fig. 1B). The different cells were represented in a two-dimensional space by transcriptomic similarity: the more similar two cells’ transcriptomes are, the closer they will be in the graph. In addition, cell clusters/populations can be identified using the differentially expressed genes among cells (Fig. 1C). In this embryo, the three embryonic layers – endoderm, ectoderm and mesoderm – were detected, consistent with the gastrulation process, and a total number of 11 different main cell populations were identified.

Fig. 1. Human gastrulation at single cell resolution. (A) Dissections performed in the human gastrula to isolate the yolk sac and rostral and caudal regions of the embryonic disk. Bidimensional distribution of all the different cells color-coded by sample of origin (B) or by the different cell population/clusters (C). (Preprint Fig. 1d, 2a and 2c).

2) Cellular differentiation at single cell resolution

In addition to cellular composition, the developmental differentiation of the different cell types can be inferred using RNA velocity and trajectory analysis. RNA velocity of all the sequenced cells revealed a distinctive separation of the 3 different germ layers (Fig. 2A). Mesoderm (top) and endoderm (bottom) separated into two distinctive branches, whereas the ectoderm formed a small separated cluster in a third dimension. This large separation of mesoderm and endoderm over the second dimension (DC2) is consistent with their different developmental origins.

During gastrulation, the epiblast cells that ingress at the primitive streak will form the mesoderm. In contrast, the epiblast cells that do not ingress, will form the ectoderm. To study this developmental process, RNA velocity was performed using the epiblast, primitive streak, ectoderm and nascent mesoderm clusters (Fig. 2B). As expected, the epiblast bifurcated into two trajectories, one towards the mesoderm (right), via the primitive streak and the other towards the ectoderm (left) (Fig. 2C). This analysis not only allowed to organize the developmental origins of the cell types but also to identify changes in the cells’ transcriptomes associated with these pathways. This provided novel information about gastrulation and cell specification in humans and identified differences compared to other mammalian models like mouse.

Fig. 2. Cellular differentiation at single cell resolution. (A) RNA velocity vectors overlaid on diffusion map of cells from all 11 clusters identified in the human gastrula. (B) Diffusion map and RNA velocity vectors of 4 clusters: epiblast, primitive streak, nascent mesoderm and ectoderm. (C) Diffusion map showing the two differential epiblast trajectories towards Ectoderm (dpt~0) or Mesoderm (dpt~1). (Preprint Fig. 2d and 4a).

3) Human hematopoiesis occurs earlier than expected

In the initial 11 cell populations/clusters, one of the identified populations were the hemogenic endothelial progenitors (Fig. 3A dashed line). Deeper analysis into this cluster showed that it had a mixed expression profile of endothelial, myeloid and erythrocyte markers, suggesting that it can contain more than one different cell population. This population was then reclustered, identifying four different cell types: myeloid progenitors, endothelial cells, blood progenitors (expressing megakaryocyte and erythroid cell markers) and erythroid-myeloid progenitors (EMPs) (Fig 3B and C). EMPs constitute the second wave of macrophage progenitors. In mouse, these cells differentiate later (embryonic day 8.5) than the equivalent gastrulation stage to the human embryo (embryonic day 7-7.5). This suggests that human hematopoiesis is advanced when comparing it to mouse development. It is interesting that, although human gastrulation takes longer to occur than in mouse, hematopoietic cellular differentiation progresses over similar timescales in human and mouse. This suggest that different developmental processes (gastrulation and hematopoietic differentiation) are not in synchrony with each other.

Fig. 3. Identification of early blood progenitor in human gastrula. (A) Bi-dimensional distribution (UMAP plot) of all the different sequenced cells, containing the hemogenic endothelial progenitors and erythrocytes (dashed line) (B) Re-clustering of the hemogenic endothelial progenitors and erythrocytes clusters revealed 4 different subclusters in the hemogenic endothelial progenitors’ population, represented by different colours. (C) heatmap showing the expression of different cell specific markers. (Preprint Fig. 2a and 5e).

Why I chose this paper:

Human and mouse gastrulas are very different in morphology, which generates a problem when extrapolating data from animal models to humans. Being aware of the limitations of the human embryonic/gastrulation samples, I think that the authors made an outstanding choice to perform single cell RNA sequencing. This allowed to obtain the most detailed database of a human gastrula till date at single cell resolution. In addition, all the data generated from this research is highly accessible to the rest of the scientific community and the public in general (http://www.human-gastrula.net), making it a great resource to continue interrogating the data and improve our knowledge of human embryogenesis. I believe this research will have a direct impact on current in vitro methodologies used to study gastrulation, increasing their accuracy to recapitulate the in vivo process.

Future directions / questions for the authors:

One of the hardest decisions is to choose the single cell sequencing technology to use. Smart-seq2 allows higher sequencing depths but less cells are sequenced when comparing with Chromium, resulting in a lower detection of rare cell types. Was there any expected/reported cell type in human gastrulas that was missing from your analysis?
You demonstrated that some developmental processes, for example gastrulation and hematopoietic differentiation, are not in synchrony with each other, indicating that embryonic stages are not completely equivalent among species. Should we re-think development from a time (organ/tissue/cell specific) perspective?

Tags: development, embryo, gastrulation, human, scrna-seq, single cell, transcriptomics

Posted on: 10 September 2020

doi: https://doi.org/10.1242/prelights.24536

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Author's response

Shankar Srinivas and Antonio Scialdone shared

Q: One of the hardest decisions is to choose the single cell sequencing technology to use. Smart-seq2 allows higher sequencing depths but less cells are sequenced when comparing with Chromium, resulting in a lower detection of rare cell types. Was there any expected/reported cell type in human gastrulas that was missing from your analysis?

A: Given the higher sensitivity of Smart-Seq2 over 10X and the relatively limited number of cells in our sample at this stage, using Smart-seq2 was an easy choice. There are a few cell types we didn’t categorically identify in our dataset, but that might be simply because they are not transcriptionally distinct enough at this stage rather than rare/non-existent. For example, our ‘Ectodem’ cluster almost certainly contains cells from both extraembryonic ectoderm (amniotic ectoderm) as well as a few cells from the early embryonic ectoderm.

Q: You demonstrated that some developmental processes, for example gastrulation and hematopoietic differentiation, are not in synchrony with each other, indicating that embryonic stages are not completely equivalent among species. Should we re-think development from a time (organ/tissue/cell specific) perspective?

A: Even within a single organism, from individual to individual, there is variability between the development of different organs. For example, somite number is often used to stage embryos when studying a different organ (say the heart), but there is not a precise link between these two systems. Therefore, for a particular stage of heart development, the embryo might have a range of somite numbers. This has led to the development of organ specific staging criteria. When comparing across different species, this type of variability is compounded by species specific differences in development. This type of variability is noted in the literature and in stage definitions for model systems (eg. the Lawson and Wilson staging of mouse development in the revised edition of Kaufmann’s Atlas) and we just need to remember to not be overly rigid in thinking about stage definitions.

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A spatially resolved single cell atlas of human gastrulation

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