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A 4D single-cell protein atlas of transcription factors delineates spatiotemporal patterning during embryogenesis

An Author Correction to this article was published on 08 December 2021

This article has been updated

Abstract

Complex biological processes such as embryogenesis require precise coordination of cell differentiation programs across both space and time. Using protein-fusion fluorescent reporters and four-dimensional live imaging, we present a protein expression atlas of transcription factors (TFs) mapped onto developmental cell lineages during Caenorhabditis elegans embryogenesis, at single-cell resolution. This atlas reveals a spatiotemporal combinatorial code of TF expression, and a cascade of lineage-specific, tissue-specific and time-specific TFs that specify developmental states. The atlas uncovers regulators of embryogenesis, including an unexpected role of a skin specifier in neurogenesis and the critical function of an uncharacterized TF in convergent muscle differentiation. At the systems level, the atlas provides an opportunity to model cell state–fate relationships, revealing a lineage-dependent state diversity within functionally related cells and a winding trajectory of developmental state progression. Collectively, this single-cell protein atlas represents a valuable resource for elucidating metazoan embryogenesis at the molecular and systems levels.

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Fig. 1: A developmental protein expression atlas describing hundreds of TFs in nearly all lineage-mapped embryonic cells.
Fig. 2: Spatiotemporal patterning of embryogenesis by lineage-, tissue- and time-specific TFs.
Fig. 3: Spatiotemporal TF cascades of cell lineage differentiation.
Fig. 4: Expression-guided discovery of new regulators of spatiotemporal fate patterning.
Fig. 5: Lineage-dependent diversity of cell states within functionally related cells.
Fig. 6: Systematic undirectional state transition during cell differentiation.

Data availability

All data generated or analyzed in this study are included in the article (Extended Data files and Supplementary Tables). The 4D live imaging data generated and analyzed that support the findings of this study are deposited in BioStudies at https://www.ebi.ac.uk/biostudies/studies/S-BIAD56/. The unprocessed and processed quantitative expression of all TFs in all traced cells at each time point are deposited in Zenodo at https://doi.org/10.5281/zenodo.4737593 (ref. 163). All processed data generated in this study and their visualization are also available at http://dulab.genetics.ac.cn/TF-atlas/. Source data are provided with this paper.

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Acknowledgements

We thank M. -Q. Dong, National Institute of Biological Sciences, for providing CRISPR–Cas9 reagents, and Caenorhabditis Genetics Center for providing some strains. This work was supported by grants from the ‘Strategic Priority Research Program’ of the Chinese Academy of Sciences to Z.D. (XDB19000000), the National Natural Science Foundation of China to Z.D. (31771598, 32061143010 and 31722035) and to X.M. (31900578), and the State Key Laboratory of Molecular Developmental Biology, China to Z.D.

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Authors and Affiliations

Authors

Contributions

Z.D., X.M. and Z.Z. conceived the project and designed the study; X.M., L.X., Z.D., W.X., Y.K., Y.Z., G.W. and Y.W. conducted the experiments and generated the data. Z.Z. and Z.D. performed the data analysis. Z.D. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Zhuo Du.

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Competing interests

The authors declare no competing interests.

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Peer review information Nature Methods thanks David Matus, John I. Murray, Marta Shahbazi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Madhura Mukhopadhyay was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Generation of protein-fusion fluorescent reporter of TFs.

a, Selection of 290 high-confidence TFs as target genes with human homologs or worm-specific TFs with potential interest in cell differentiation. Right: TF classes based on typical domains. Classes with less than three TFs are not included. b, Maximum projections of 3D images showing cellular protein expression of representative TFs. 4D imaging of each strain was repeated in ≥2 embryos, showing similar expression patterns. Scale bar, 10 µm. c, Fosmid-based protein-fusion reporters. Two hundred two protein-fusion reporters of 185 TFs have been generated by the ModENCODE projects in which GFP (green) was fused to a TF in a fosmid by recombineering, and the fosmid was then integrated into the C. elegans genome14,15. d, Tagging endogenous TFs with mNeonGreen/GFP by CRISPR/Cas9-mediated gene knock-in. A homologous recombination (HR) template sequence containing the coding sequence of mNeonGreen/GFP (green) flanked by 800-1,000 bp sequence homologous to the endogenous TF locus was used to guide HR following cleavage by Cas9 at the N- or C-terminals of the TF gene. (e) Flowchart showing the sources of 291 protein-fusion reporters for 266 TFs.

Extended Data Fig. 2 High quality of cellular protein expression of TFs.

a, Distribution of the Pearson correlation coefficient (R) for experimental replicates of TF expression assays. b, Representative examples showing the consistency of cellular TF expression between replicates. c, Comparison of cellular TF expression between different fosmid-based protein-fusion reporter strains that tag the same TF. d, Comparison of cellular TF expression between fosmid-based (top) and knockin-based (bottom) reporter strains that tag the same TF. e, Comparison of the measured TF expression to that previously reported for 30 TFs selected from the literature in cell tracks (a series of temporally ordered mother-daughter cells) leading to terminal cells. f, Pie charts comparing the distribution of the status of lineage identity (ID) assignment of all embryonic cells (n = 1341) between scRNA-seq and protein expression data. g, Comparison of the precision and sensitivity of mRNA and protein expression results at different expression cut-offs. The true positive (TP), false-positive (FP), and false-negative (FN) rates are calculated to determine the sensitivity and precision as TP/(TP + FN) and TP/(TP + FP), respectively. h, Correlation between mRNA and protein expression in identical cells. ρ and P-value were calculated by a two-sided Spearman’s rank correlation, n = 253,159.

Source data

Extended Data Fig. 3 Spatiotemporal patterning of cell states by lineage-, tissue-, and time-specific TFs.

a, Tree visualization of binarized expression (green) of representative lineage-specific TFs in lineaged cells. b, Representative example showing single-cell expression of three anterior-posterior asymmetric TFs. Purple and blue bars shown on the bottom of the cell lineage trees indicate the lineages produced by anteriorly and posteriorly localized progenitor cells following a division, respectively. c, Enrichment of the overlap between tissue-specific TFs identified in this study and by29. Statistics: One-tailed hypergeometric test. d, Protein expression of representative tissue-specific TFs in lineaged cells. Barcodes on the bottom highlight cells of specific tissue types. e, Tree visualization of the cellular expression patterns of TFs before (top) and after (bottom) switching progenitor cell fate by RNAi against specific fate specifiers. Arrows indicate fate transformations. Similar results were observed in ≥2 embryos. f, Relative enrichment of tissue-specific genes in the targets of tissue-specific TFs. In each tissue, relative enrichment was measured as the ratio of the frequency of genes specifically expressed in this tissue to that of genes specifically expressed in other tissues. Tissue-specific genes identified by29 were used. g, Tree visualization of the number of transiently-expressed TFs in each lineaged cell. The instances in which a TF is expressed before degradation were used to calculate the number. h, Left: single-cell expression of two transiently-expressed TFs. Right: changes in TF expression levels before and after degradation in indicated cells. i, Heatmap showing the contribution of transient expression to the net difference in TF expression between mother and daughter cells (Mother-daughter cell pair number n = 1,200). j, Percent of distinguishable mother-daughter states under various criteria before and after considering transient TF expression. Statistics: Two-tailed Wilcoxon signed-rank test. k, Distribution of the fraction of specifically expressed TFs in all TFs that distinguish each cell from individual other cells. l, Each curve shows the fractions of cells (averaged from 1000 times of simulations) whose TF expression is distinct from all other cells under different definitions (number of TFs showing differential binary expression) after removing a certain number of specifically-expressed TFs.

Source data

Extended Data Fig. 4 Construction of spatiotemporal TF cascades.

a, Tree visualization of the cell lineage leading to corresponding progenitors (colored circles) of defined tissue types. b, Spatiotemporal modules of cell lineage differentiation. Each column represents the development of a tissue progenitor cell lineage (spatial module). Each row represents the classification of the regulation of each progenitor cell lineage into three temporal modules (color gradient). c, Schematic of calculating the similarity in single-cell expression of a given gene. d, Single-cell expression similarity between 30 pairs of TFs known to function in the same pathway24,34,127,132,151,164,165. e, Comparison of expression similarity between genes in the same pathway (n = 30) and between randomly selected gene pairs (n = 30). The boxplot shows the median with IQR and whiskers extending to 1.5× the IQR; outliers are plotted as points. Statistics: Two-tailed Wilcoxon signed-rank test. f, Tree visualization of cellular expression of PHA-4 and CEH-27 in each lineaged cell. The barcode at the bottom indicates the pharyngeal cells. g, Changes in cellular CEH-27 protein expression in pha-4(zu225) embryos. Above showing cellular expression of CEH-27 protein in lineaged cells of wild-type embryos with vertical lines on the bottom indicating cells in which loss or reduction of CEH-27 expression was detected in pha-4(zu225) embryos. Numbers indicate the penetrance of expression changes.

Extended Data Fig. 5 ELT-1 regulates LIN-32 expression in multiple cell lineages.

a, Single-cell protein expression of ELT-1 and LIN-32. b, Top: Neuronal-specific TFs targeted by ELT-1. Bottom: ELT-1 binding pattern near the lin-32 gene. c, Comparison of single-cell mNeonGreen::LIN-32 presence in all analyzed elt-1(ok1002)/+ and elt-1(ok1002) embryos in lineaged cells. d, Representative micrographs showing loss and gain of mNeonGreen::LIN-32 expression and in cells from various lineages. Similar results were observed in ≥3 embryos. Scale bar, 2 μm. The center Z planes of corresponding cells were identified using mCherry signals and cropped from embryos. Figure organization is as in Fig. 4b. e, Representative micrographs showing loss and gain of cell death in elt-1(ok1002) embryos. Similar results were observed in ≥4 embryos. Scale bar, 2 μm. Programmed cell death was determined by a characteristic sequence of morphological changes in the nucleus of the corresponding cell. The center Z planes of corresponding cells were identified using mCherry signals and cropped from embryos. Figure organization is as in Fig. 4d.

Extended Data Fig. 6 ELT-1 regulates SOX-2 expression in multiple cell lineages.

a, Single-cell protein expression of ELT-1 and SOX-2::mNeonGreen. The color-coded barcode on the bottom highlights the neuronal and skin cells. b, Left: loss and gain of SOX-2:: mNeonGreen expression in corresponding cell lineages of elt-1(ok1002) embryos. Similar results were observed in ≥2 embryos. Numbers at the bottom indicate the penetrance of each phenotype. c, Comparison of single-cell SOX-2::mNeonGreen presence in all analyzed elt-1(ok1002)/+ and elt-1(ok1002) embryos in corresponding cell lineages.

Extended Data Fig. 7 M03D4.4 converges muscle differentiation in the body wall and pharynx.

a, Pan-muscle specificity of M03D4.4. Left: cell types of M03D4.4-expressing cells (apoptotic cells were not considered). Right: coverage of M03D4.4-expressing cells in different muscle cell types. b, Single-cell protein expression of M03D4.4::GFP in cell lineages before and after switching fates of specific progenitor cells. The homeotic transformation of ABala, ABalp, ABara, and ABarp cell fates to C (ABa-to-4C) was induced by mex-3(RNAi). The homeotic transformation of ABa cell fate to EMS (ABa-to-EMS) was induced by mex-5(RNAi). Similar results were observed in ≥2 embryos. c, Comparison of single-cell protein expression of M03D4.4 and other known regulatory TFs (PAL-1, HND-1, UNC-120, HLH-1) in the C and D lineages. d, Comparison of single-cell protein expression of PHA-4, TBX-2, and M03D4.4 in ABalpa, ABara, and MS lineages that produce pharyngeal muscle cells. For c, d, similar results were observed in ≥2 embryos for each reporter strain. e, Inferred regulatory relationship between M03D4.4 and other regulators. f, Comparison of single-cell M03D4.4 protein expression in corresponding cell lineages between all analyzed wild-type and pal-1(RNAi) embryos. g, Comparison of single-cell M03D4.4 protein expression in corresponding cell lineages between all analyzed wild-type and pha-4(zu225) embryos. h, Enrichment of proteins preferentially expressed in pharyngeal and body muscle cells40 in the ChIP-seq targets of M03D4.4. Statistics: One-tailed hypergeometric test. i, Enrichment of genes involved in body muscle development and function41 in the ChIP-seq targets of M03D4.4. Statistics: One-tailed hypergeometric test. j, Enrichment of RNAi phenotypes in M03D4.4 target genes. Statistics: One-tailed hypergeometric test, Benjamini-Hochberg corrected P-value. k, Generation of a M03D4.4 loss of function allele. A 1390-bp deletion that removes exons 4-6 and most of exon 7 was induced by CRISPR/Cas9-mediated gene editing.

Source data

Extended Data Fig. 8 Lineage-dependent cell state diversity within each tissue.

a, UMAP plot of neuronal (left, n = 41 for sensory neuron; 54 for inter neuron; 52 for motor neuron) and pharyngeal (right, n = 15 for pharyngeal neuron; 37 for pharyngeal muscle) subtypes based on TF expression. Parameters: min_dist = 0.8, n_neighbors = 5. b, Distribution of tissue types (colors) across terminal cells. c, Comparison of intra-tissue state divergences between all cells and between cells of each tissue progenitor cell lineage (cell pair numbers from left to right: Neu, n = 17,391; 300; 55; 21; 21; 55; 210; 3; 21; 66; 55; 36; 21; 45; 66; 36; 15. Pha, n = 2,415; 36; 55; 6; 91; 6; 6; 3; 10; 10; 10. Ski, n = 4,005; 6; 6; 28; 6; 6; 6; 6; 6; 6; 6; 6; 6; 6. Mus, n = 2,346; 120; 120; 120; 6; 6). The boxplots show the median with IQR and whiskers extending to 1.5 × the IQR; outliers are plotted as points. Statistics: Two-tailed Mann–Whitney U test. d, Changes in intra-tissue state divergence in terminal cells as a function of cell lineage distance in corresponding tissues. State divergence at certain cell lineage distance was not included because of a small number of eligible cells (n < 3). Cell pair numbers from left to right: Pha n = 24; 48; 73; 133; 220; 220; 156; 416; 1,125. Ski n = 32; 61; 82; 124; 297; 13; 254; 773; 126. Mus n = 31; 52; 94; 204; 22; 355; 128; 720. Int n = 4; 8; 18; 36. The boxplots show the median with IQR and whiskers extending to 1.5× the IQR; outliers are plotted as points. e, Changes in intra-sub-tissue state divergence in terminal neuronal and pharyngeal cells as a function of cell lineage distance. Neuronal and pharyngeal cells were divided into subtypes according to known functional subdivisions. Cell pair numbers from left to right: Motor neuron n = 10; 19; 36; 57; 42; 166; 69; 420; 507. Sensory neuron n = 4; 7; 15; 14; 32; 14; 99; 147; 336. Inter neuron n = 7; 18; 31; 75; 116; 119; 268; 589. Pharyngeal muscle n = 5; 13; 22; 27; 62; 45; 80; 70; 342. Pharyngeal neuron n = 5; 11; 15; 16; 50. The boxplots show the median with IQR and whiskers extending to 1.5× the IQR; outliers are plotted as points. f, Tissue and lineage compositions of 20 de novo identified state clusters. Individual tissue progenitor cell lineages were used as the unit to represent the lineage compositions of cells in each state cluster with hyphens linking left-right symmetric lineages. g, Lineage distribution of cells from representative clusters. Connected lines indicate left-right symmetric lineages.

Extended Data Fig. 9 Lineage-restricted expression of tissue-specific TFs.

a, Comparison of tissue coverage of each lineage-specific TFs in cells of all five broad tissue types. b, Expression of tissue-specific TFs (rows) in each clonal cell lineage of corresponding tissues (columns). c, Heatmap showing pair-wise state divergence (range 0-1) between all multipotent progenitor cells (n = 140) calculated using lineage-specific TFs. Cells are ordered first by lineage and then by generation. Cells before the 26-cell stage were not included due to a small number of expression TFs.

Extended Data Fig. 10 State transitions during embryogenesis.

a,b, Comparison of state divergence of cells at each cell generation to the initial and terminal state for cells that differentiate into different tissue types (a) and for cells that are from different cell lineages (b). Comparison numbers from left to right: n = 1.303; 490; 550; 403; 48 for tissue; n = 553; 543; 471; 533; 295; 48; 267; 84 for lineage. c, Heatmap showing the difference between SDm-t and SDd-t calculated by using single-cell transcriptomes following cell divisions (cell track number n = 382, ordered by lineage). d, Comparison of SDm-d at different developmental stages. Mother-daughter cell pair numbers from left to right: n = 20; 50; 102; 176; 334; 343. The boxplot shows the median with IQR and whiskers extending to 1.5× the IQR; outliers are plotted as points. Each dot is the result of a mother-daughter cell comparison. e, Correlation between SDm-d and the difference between SDm-t and SDd-t (each dot represents a comparison, n = 1,938). R and P-value were calculated by a two-sided Pearson’s correlation. Results for the last round of cell division were not included because, by definition, SDm-d equals SDm-t − SDd-t in these cells.

Supplementary information

Supplementary Information

Supplementary Notes 1–6, Supplementary Figs. 1–5 and references.

Reporting Summary

Supplementary Table 1

Selection of TFs. The table lists the names, sources, conservation and related information of all 609 high-confidence TFs encoded in the C. elegans genome (compiled from the wTF 2.0 database and WormBase followed by manual curation) and the selection criteria of 290 TFs as the target of this study.

Supplementary Table 2

Reporter verification and all strains used in this study. a, The sources and verification of fluorescent reporters. b, The strain names and associated genotypes of all dual-fluorescent reporters of TFs. c, The other strains and associated reagents used in this study.

Supplementary Table 3

Cellular expression pattern of all TFs and quality controls. a, Expression levels of 291 protein-fusion TF reporters (for 266 TFs) in 1,204 lineaged cells. TF reporters marked in yellow indicate those that exhibit phenotypic abnormalities of cell proliferation or cell position (sheets 3 and 4). b, The quantitative expression of all TFs in multiple embryos (≥2) for the same reporter strain up to the 350-cell stage. c, Cells in each TF reporter strain that exhibits reproducible defects in cell cycle length. d, Cells in each TF reporter strain that exhibits reproducible cell position defects. e, Previous RNA-seq expression of the TFs that were identified as exhibiting sporadic expression in this study. f, The results of comparing the expression patterns of TFs reported in this study to those reported in the literature. We only considered TFs for which expression patterns in specific lineages, tissue types, cells and developmental stages were previously described. g, The correlation coefficient of single-cell protein expression of TFs between this and previous studies. h, The results of cell-by-cell expression comparisons for 30 TFs with well-documented cellular expression patterns.

Supplementary Table 4

TFs exhibiting various expression specificity. a, All identified lineage-specific TFs, enriched lineages and enrichment scores. b, All identified A–P asymmetric TFs and related bias scores. c, All identified tissue-specific TFs, enriched tissue types and enrichment scores. d, The enriched RNAi phenotypes for the ChIP–seq target genes of each tissue-specific TF. Yellow highlights those phenotypes that are related to the function of corresponding tissue. e, All TFs with transient expression. ‘1’ denotes a TF that is expressed in a mother cell but absent in at least one of its daughters.

Supplementary Table 5

The spatiotemporal cascade of TFs. a, The assignment of TFs into spatiotemporal modules. b, All relationships between TFs inferred based on expression similarity and previous ChIP–seq experiments. c, The inferred TF cascade for each tissue progenitor cell lineage.

Supplementary Table 6

Embryonic cell differentiation phenotypes in TF mutants. a, Changes in cellular expression of CEH-27 in pha-4(zu225) embryos. The table lists cellular expression levels of CEH-27 in all analyzed wild-type and pha-4(zu225) embryos. b, Changes in cellular expression of LIN-32 and SOX-2 in elt-1(ok1002) embryos. The table lists cellular expression levels of LIN-32 and SOX-2 in all analyzed elt-1(ok1002)/+ and elt-1(ok1002) embryos. c, Changes in cellular expression of M03D4.4 in pal-1(RNAi) and pha-4(zu225) embryos. The Table lists changes in cellular expression levels of M03D4.4 in all analyzed embryos upon pal-1 and pha-4 perturbations.

Supplementary Table 7

State clusters of terminal cells. The table lists the lineage and functional name, tissue type and clustering results of all terminally differentiated cells.

Supplementary Table 8

State transitions during cell lineage differentiation. a, State divergences of progenitor cells at all stages to the initial and terminal states. b, State divergences between mother and daughter cells following each cell division.

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Ma, X., Zhao, Z., Xiao, L. et al. A 4D single-cell protein atlas of transcription factors delineates spatiotemporal patterning during embryogenesis. Nat Methods 18, 893–902 (2021). https://doi.org/10.1038/s41592-021-01216-1

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