Cell lineage-specific transcriptome analysis for interpreting cell fate specification of proembryos

Abstract

In Arabidopsis, a zygote undergoes asymmetrical cell division that establishes the first two distinct cell types of early proembryos, apical and basal cells. However, the genome-wide transcriptional activities that guide divergence of apical and basal cell development remain unknown. Here, we present a comprehensive transcriptome analysis of apical and basal cell lineages, uncovering distinct molecular pathways during cell lineage specification. Selective deletion of inherited transcripts and specific de novo transcription contribute to the establishment of cell lineage-specific pathways for cell fate specification. Embryo-related pathways have been specifically activated in apical cell lineage since 1-cell embryo stage, but quick transcriptome remodeling toward suspensor-specific pathways are found in basal cell lineage. Furthermore, long noncoding RNAs and alternative splicing isoforms may be involved in cell lineage specification. This work also provides a valuable lineage-specific transcriptome resource to elucidate the molecular pathways for divergence of apical and basal cell lineages at genome-wide scale.

Fig. 1. Overall analyses of the transcriptomes of zygote, apical, and basal cell lineages of early proembryos.

a Isolated living apical and basal cell lineages of early proembryos. Bar = 10 µm. b, c Hierarchical clustering (b) and principal component analysis (c) of the transcriptomes of zygote, apical, and basal cell lineages of early proembryos. d Pearson’s correlation between the transcriptomes of zygote, apical, and basal cell lineages of early proembryos. CW cell wall, Zy24 zygote at 24 h after pollination, AC apical cell, BC basal cell, 32E embryo proper of 32-cell proembryo, 32S suspensor of 32-cell proembryo. Same abbreviations were also used in the following figures.

Fig. 2. Global temporal dynamics of gene expression during apical and basal cell lineage development.

a, b Scatterplot showing temporal dynamics of gene expression in the process of apical (a) and basal (b) cell lineage specification. Pink dots indicate upregulated genes, and blue dots indicate downregulated genes. c Graph showing the number of differentially expressed genes between AC and Zy24 or between BC and Zy24. d Graph showing the number of differentially expressed genes between 32E and AC or between 32S and BC. e, f Comparisons of upregulated genes between AC/Zy24 and 32E/AC (e) or between BC/Zy24 and 32S/BC (f). g, h GO analysis of upregulated genes in apical (g) and basal cell linage (h) at 1-cell and 32-cell embryo stages, respectively.

Fig. 3. Lineage specific transcriptome analysis reveals progressive apical and basal cell lineage differentiation during early embryogenesis.

a Volcano plots display differentially expressed genes between apical and basal cell lineages at 1-cell and 32-cell embryo stages. b, c Comparisons of upregulated genes (b) or downregulated genes (c) between AC/BC and 32E/32S. d, e Expression profiles of the apical cell lineage- (d) and basal cell lineage-maintained (e) genes in egg cells, zygotes, apical, and basal cell lineages of early proembryos. Pink lines (d) and blue lines (e) indicate the mean expression level of all genes in different groups. f, g GO enrichment analysis of apical cell lineage- (f) and basal cell lineage-maintained (g) genes, respectively. EC egg cell, Zy14 zygote at 14 h after pollination.

Fig. 4. Expression pattern of selected apical cell lineage-maintained genes validates the transcriptomic analysis.

a, b Spatio-temporal expression of ACL1 (a) and ACL2 (b) in early embryogenesis. Zy zygote, 1C 1-cell embryo, 4C 4-cell embryo, 8C 8-cell embryo, 32C 32-cell embryo, AC apical cell, BC basal cell, EP embryo proper, Sus suspensor. Bar = 10 μm.

Fig. 5. Expression pattern of selected basal cell lineage-maintained genes validates the transcriptomic analysis.

a, b Spatio-temporal expression of BCL1 (a) and BCL2 (b) in early embryogenesis. Zy zygote, 1C 1-cell embryo, 4C 4-cell embryo, 8C 8-cell embryo, 32C 32-cell embryo, AC apical cell, BC basal cell, EP embryo proper, Sus suspensor. Bar = 10 μm.

Fig. 6. The expression of lncRNAs and alternative splicing isoforms during the process of apical and basal cell lineage specification.

a Venn diagram of the lncRNAs in five different cell types. b The length of known and novel lncRNAs identified in early embryos. Whiskers in box plots indicate the minimum and maximum length of lncRNAs. c Proportions of different types of lncRNAs identified in early embryos. d Hierarchical clustering of zygote, apical, and basal cell lineages of early proembryos based on the expression of lncRNAs. e MA plots depict differentially expressed lncRNAs between apical and basal cell, or between 32E and 32S. f Overlap analysis of upregulated or downregulated lncRNAs between AC/BC and 32E/32S. g Heatmap showing the expression of apical cell lineage- and basal cell lineage-maintained lncRNAs in early embryogenesis. h, i The number of alternative splicing events (h) and genes (i) in different cells. j The number of genes with different transcripts in each cell type. k Overlap analysis of alternative splicing transcripts identified in apical and basal cell lineages.

Fig. 7. The regulatory networks of diverse transcription factors in apical and basal cell lineages of early proembryos.

a Venn diagram showing expressed TF genes in AC, BC, 32E, and 32S (FPKM ≥ 1). b Scatterplot showing differentially expressed TF genes between AC and BC, and between 32E and 32S. c Overlap analysis of upregulated or downregulated TF genes between AC/BC and 32E/32S. d, e Identification of TF genes that are coexpressed with apical cell lineage- (d) and basal cell lineage-maintained (e) genes in early embryogenesis. Orange lines (d) and purple lines (e) indicate the TF genes in each group. f, g Enrichment of TF DNA binding motifs within the promoter regions of upregulated and downregulated genes between AC/BC (f) and 32E/32S (g). h, i Predicated regulatory networks between enriched TFs and their potential targeted genes in apical (h) and basal cell (i) lineages.