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. 2024 Sep 9;59(17):2347-2363.e9.
doi: 10.1016/j.devcel.2024.05.014. Epub 2024 Jun 5.

An integrated approach identifies the molecular underpinnings of murine anterior visceral endoderm migration

Shifaan Thowfeequ  1 , Jonathan Fiorentino  2 , Di Hu  1 , Maria Solovey  3 , Sharon Ruane  1 , Maria Whitehead  4 , Felix Zhou  5 , Jonathan Godwin  1 , Yentel Mateo-Otero  6 , Bart Vanhaesebroeck  4 , Antonio Scialdone  7 , Shankar Srinivas  8
Affiliations

An integrated approach identifies the molecular underpinnings of murine anterior visceral endoderm migration

Shifaan Thowfeequ et al. Dev Cell. .

Abstract

The anterior visceral endoderm (AVE) differs from the surrounding visceral endoderm (VE) in its migratory behavior and ability to restrict primitive streak formation to the opposite side of the mouse embryo. To characterize the molecular bases for the unique properties of the AVE, we combined single-cell RNA sequencing of the VE prior to and during AVE migration with phosphoproteomics, high-resolution live-imaging, and short-term lineage labeling and intervention. This identified the transient nature of the AVE with attenuation of "anteriorizing" gene expression as cells migrate and the emergence of heterogeneities in transcriptional states relative to the AVE's position. Using cell communication analysis, we identified the requirement of semaphorin signaling for normal AVE migration. Lattice light-sheet microscopy showed that Sema6D mutants have abnormalities in basal projections and migration speed. These findings point to a tight coupling between transcriptional state and position of the AVE and identify molecular controllers of AVE migration.

Keywords: anterior visceral endoderm; cell migration; embryonic patterning; mouse embryogenesis; phosphoproteomics; semaphorin signaling; single-cell transcriptomics.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Identification and validation of anterior and posterior VE markers (A) Schematic summarizing the isolation and the transcriptional characterization of VE cells. (B) Uniform manifold approximation and projection (UMAP) plots of cells from 5.5-dpc (n = 40) and 6.25-dpc (n = 11) embryos, clustered into five groups at each stage: early- or late-anterior visceral endoderm (AVE), rest of the VE surrounding the epiblast or ExE (emVE and exVE, respectively), the epiblast (Epi), the extra-embryonic ectoderm (ExE). (C) Heatmap of normalized log expression levels of known marker genes for all identified cell types. (D) First two diffusion components (DC1 and DC2) of early- or late-AVE and emVE cells, colored according to cluster (left) and diffusion pseudotime (dpt) coordinate (right). Mean standardized expression of the genes belonging to high-in-AVE and low-in-AVE groups. (E) Spatial expression of selected high-in-AVE markers, using HCR (Efna5) or immunofluorescence (KRT19 and DBN1). Hhex-GFP marks the AVE. (F) Spatial expression of selected low-in-AVE markers (Efnb1 and Nrg1). Cer1 expression marks the AVE. In (E) and (F), blue lines indicate the position of the AVE, and the orange asterisk marks the posterior. Scale bars represent 20 μm, and embryos are orientated anterior to the left. See also Tables S1, S2, S3, S4, and S13.
Figure 2
Figure 2
Proteomic and phosphoproteomic landscape of the post-implantation/pre-gastrulation embryo (A) Scatterplot of log2 fold change between the embryonic and abembryonic halves, in proteomics vs. phosphoproteomics data. Each marker represents a protein, and those corresponding to genes from the high-in-AVE group are highlighted in red. Pearson’s correlation coefficient = 0.36; p = 1.3 × 10−3. (B) Dot plots of gene expression (scRNA-seq) corresponding to (phospho)proteins that are differentially expressed between the embryonic and abembryonic halves only in one dataset (sets outlined in A). (C) Surface renderings, showing expression of KRT8 (n = 7) and phospho(Ser23)-KRT8 (n = 5) visualized by immunofluorescence in 6.5-dpc embryos. Hhex-GFP marks the AVE. Scale bars represent 20 μm. (D) Kinase-substrate bipartite networks predicted for substrate proteins upregulated in the embryonic (left) or abembryonic (right) half in the phosphoproteomics dataset. Dot plots of gene expression (scRNA-seq) are shown for the corresponding kinases and substrates. See also Tables S5, S6, S7, S8, S14, S15, S16, and S17.
Figure 3
Figure 3
Symmetry breaking along the anterior-posterior axis in direct comparison to AVE migration (A) Expression patterns of Cer1 and Wnt3 in the AVE and emVE clusters, as a function of diffusion pseudotime. (B) Volume renderings showing changes to the Wnt3 expression domain (visualized by HCR) relative to Cer1-expressing AVE cells, in pre-migration (n = 6), mid-migration (n = 16), and post-migration (n = 27) embryos. (C) Quantification of the VE Cer1 and Wnt3 HCR signals in the anterior and posterior regions of pre- (n = 6), mid- (n = 8), and post-migration (n = 9) embryos. Expression levels are presented as an anterior:posterior (A:P) ratio, with 1 indicating balanced expression of the two markers. Data are represented as mean ± SEM. (D) Expression patterns of Nodal in AVE and emVE clusters, as a function of diffusion pseudotime. (E) Optical sections through embryos showing the distinct expression of Nodal in the epiblast and the VE relative to that of Cer1 at 5.5 (n = 6) and 6.25 (n = 4) dpc. Magnifications of the boxed anterior and posterior regions are shown underneath to highlight distinct Nodal expression in VE and epiblast. (F) Volume renderings showing changes in Nodal and T expression relative to the position of Cer1-expressing AVE cells in pre-migration (n = 3), mid-migration (n = 6), and post-migration (n = 3) embryos. In (A) and (D), each black dot represents a cell; the red line shows the fit obtained from a generalized additive model (GAM). In (B), (E), and (F), scale bars represent 20 μm; and embryos are orientated anterior to the left. See also Videos S1, S2, S3, and S4.
Figure 4
Figure 4
Origin and fate of the AVE (A) PAGA graph computed from RNA velocities and projected on the first two diffusion components of a diffusion map of the AVE and emVE clusters, combining the data from both stages. (B) Direct comparison of short-term lineage-labeled AVE cells (expressing the Hhex-GFP reporter) and the contemporaneous expression of the endogenous Hhex transcript (visualized by HCR) in 5.5-dpc (n = 7) and 6.25-dpc (n = 5) embryos. Embryos are rotated by ∼20° about their PD axis to show the full anterior surface along with lateral sides. (C) Volume renderings directly comparing changes to the expression domains of the AVE markers Cer1, Hhex, and Lefty1 in embryos before (n = 13), during (n = 18), and after (n = 8) AVE migration. Embryos are orientated anterior to the left. (D) Schematic summarizing changes to Cer1, Hhex, and Lefty1 expression domains throughout the VE because of AVE migration. Scale bars represent 20 μm. See also Videos S5 and S6.
Figure 5
Figure 5
Spatial mapping of the transcriptional heterogeneity seen within the AVE and exVE clusters (A) Computational strategy used for VE sub-clustering of 10× scRNA-seq data (Nowotschin et al.17), using gene expression features extracted from our Smart-seq2 data. The AVE cluster was selected for further sub-clustering analysis. (B) UMAP plots of cells (data from Nowotschin et al.17) belonging to the AVE at 5.5 dpc (top) and 6.5 dpc (bottom), colored according to sub-clusters (Figure S4 and STAR Methods for AVE identification in these data). (C) Violin plots showing Cer1, Lefty1, and Dkk1 normalized log expression in cells grouped according to the clusters in (B), for both stages. (D) Heatmap showing normalized log expression of the top genes upregulated in the late-AVE-medial cluster and the AVE-lateral cluster (10 each), obtained through a differential expression analysis between the two clusters at 6.5 dpc. (E) Volume renderings showing the expression domains of Cer1, Lefty1, and Dkk1, used to distinguish between the sub-clusters of the AVE at 5.5 (n = 8) and 6.5 (n = 3) dpc. (F) Heatmap showing normalized log expression of the top 10 genes upregulated in the exVE-proximal cluster and the exVE-distal cluster at 6.5 dpc. Nrg1 (which ranked 13th in the exVE-proximal cluster by adjusted p value) was manually added to the list. (G) UMAP plots showing sub-clusters within the exVE at 6.5 dpc (left) and of cells belonging to the exVE cluster at 5.5 (center) and 6.5 dpc (right), colored according to normalized log expression of Nrg1 and Efnb1. (H) Volume renderings showing changes to the expression domains of Efnb1 and Nrg1 between 5.5 (n = 6) and 6.5 (n = 4) dpc in comparison with Cer1-expressing AVE cells. Orange and red lines represent, for Efnb1 and Nrg1, respectively, the proximal-to-distal extent of their expression domains. Scale bars represent 20 μm, and embryos are orientated anterior to the left. See also Tables S9, S10, S11, and S12 and Videos S2 and S6.
Figure 6
Figure 6
Semaphorin signaling is required for correct AVE migration (A) Schematic of computational and experimental strategy used to discover signaling pathways important for AVE migration. (B) Plot showing ligand-receptor pairs (LRPs) containing at least one high-in-AVE component (blue), at 5.5 and/or 6.25 dpc. Arrows indicate the direction of signaling. (C) Violin plots showing normalized log expression levels of Sema6d and Plxna1 in AVE, emVE, and Epi clusters, at 5.5 dpc (top), and the intercellular communication pattern associated with SEMA6D:PLXNA1 (bottom). (D) Rigid body and scaling registration used to remove drift and growth from confocal time-lapse movies for phenotyping of AVE migration with MOSES analysis. (E) MOSES motion saliency maps computed from reverse and forward tracking (STAR Methods) to identify spatial location of motion sources (left) and sinks (right) from a wild-type and Sema6d homozygous mutant (Sema6d-KO) embryo. The position of the median source/sink relative to the origin and boundary of motion is marked. (F) Snapshots of the mesh connecting initially neighboring superpixels when MOSES is applied to track AVE migration (forward and reverse) in a wild-type and Sema6d-KO embryo. (G) Plots of motion signatures (mesh strain vs. time) extracted from tracking AVE migration in wild-type and Sema6d-KO embryos (n = 7 each), which summarize the extent of mesh deformation based on forward and reverse tracking. (H) Plot of the first two principal components (PC1 vs. PC2) after applying principal-component analysis to the values of the mesh strains extracted from the concatenated forward and reverse time-lapse tracking of AVE migration in wild-type (WT) and Sema6d-KO (MUT) embryos. In parentheses is the percentage of explained variance. (H′) and (H″) represent the loadings of PC1 and PC2, respectively, for the mesh strains obtained from the forward (top) and the reverse (bottom) tracking. The monotonic increase of PC1 suggests that it represents overall AVE movement. The non-monotonic behavior of PC2, with a stationary point at ∼120 min, indicates that it represents the rate at which AVE cells slow down. (I) Maximum intensity projections of high-resolution lattice light-sheet time-lapse frames from cultured wild-type and Sema6d-KO embryos, showing the emergence and retraction of basal projections by migrating Hhex-GFP-positive AVE cells (green). Arrowheads point to basal projections at their measured maximum lengths. (J) Plot showing difference (p = 0.03; nested ANOVA) in the maximum length of basal projections from wild-type (n = 9 embryos, n = 76 projections) and Sema6d-KO mutant embryos (n = 9, n = 66 projections). Data are represented as mean ± SEM. See also Videos S7 and S8.
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