Mapping cell migrations and fates in a gastruloid model to the human primitive streak

Abstract

Although fate maps of early embryos exist for nearly all model organisms, a fate map of the gastrulating human embryo remains elusive. Here, we use human gastruloids to piece together a rudimentary fate map for the human primitive streak (PS). This is possible because differing levels of BMP, WNT and NODAL lead to self-organization of gastruloids into homogenous subpopulations of endoderm and mesoderm, and comparative analysis of these gastruloids, together with the fate map of the mouse embryo, allows the organization of these subpopulations along an anterior-posterior axis. We also developed a novel cell tracking technique that detected robust fate-dependent cell migrations in our gastruloids comparable with those found in the mouse embryo. Taken together, our fate map and recording of cell migrations provides a first coarse view of what the human PS may resemble in vivo.

Keywords: Fate map; Gastruloid; Human; Migration; Primitive streak; Stem cell.

Fig. 1.

Mapping gastruloid fates to the human PS. (A) Reproduction of the mouse PS fate map (based on Tam and Behringer, 1997) with inferred signaling gradients of BMP, WNT3A and Nodal (Tam and Loebel, 2007; Arnold and Robertson, 2009). (B) Dorsal representation of the human PS fate map from the Carnegie Collection (O'Rahilly and Müller, 1987). (C) Mapping of gastruloids stimulated with BMP4, WNT3A, WNT3A+SB or WNT3A+activin to the Carnegie Collection stage 7 human PS. Gastruloids were fixed after 48 h and stained for the indicated markers. As each staining is radially symmetric, only a section from r=0 to r=R (500 μm) is shown. (D) Same as C, but comparing the expression profiles of the indicated markers in the differentiation conditions with their expression profiles in pluripotent, undifferentiated micropatterns. (E) Summary of marker expression. The OTX2/DE-Mid box with lighter yellow indicates that expression of OTX2 is less than that observed in other cells in other conditions. APS, anterior PS; DE - Ant., anterior definitive endoderm; DE - Mid, mid-streak definitive endoderm; DE - Post., posterior definitive endoderm; ExM, extra-embryonic mesoderm; LM, lateral mesoderm; Org., organizer; PM, paraxial mesoderm; PPS, posterior PS; PrEct, presumptive ectoderm.

Fig. 2.

Directed cell migration in the PS region. (A) Cloning strategy for the RUES2-KiKGR-RFP657-H2B line. (B) Using a digital micromirror, annular regions of 1000 μm diameter micropatterned RUES2-KiKGR-RFP657-H2B colonies were selectively exposed to 405 nm light for 3 s and permanently switched from green to red fluorescence. (C) Row 1 shows unconverted KikGR fluorescence (green) and converted KikGR fluorescence (red) at 0 h in each of the three annular regions. All other rows show only converted KikGR fluorescence (red) and the far-red histone nuclear marker (gray) at 52 h. In all conditions, significant movement of cells in the A₃ region is observed. (D) Quantification of C. To measure the degree of spread of the A₃ region migration in each condition we also computed the root mean square of each A₃ distribution (σ₃, overlaid in blue). Note that this number is not related to the variability or reproducibility of the cell movements, which instead can be inferred from the difference in heights of neighboring bins, as each bin represents an independent sampling of cells. For the A₃ region of each stimulation condition these differences are especially small compared with their respective bin heights, thus showing that the movements are robust. Dotted lines indicate the initial centers of each photoconverted region.

Fig. 3.

3D gastruloid morphology. (A) Radial cross-sections of RUES2-KiKGR-RFP657-H2B micropatterns photo-converted in region A₃ and stimulated with WNT3A, WNT3A+activin, WNT3A+SB or BMP4 for 52 h. In all conditions the photo-converted cells (red) can be seen to be migrating under the inner epiblast or ectoderm-like region (gray). (B) Hand-drawn depiction of 3D structure of gastruloids inferred from A. (C) Radial cross-sections of RUES2 gastruloids stimulated with WNT3A, WNT3A+activin, WNT3A+SB or BMP4, fixed and stained at 52 h for the indicated markers. As can be seen by comparison with A and B, the migratory cells are differentiated to mesoderm or endoderm and express PS markers. One can also see that in all conditions except BMP4 a basement layer of collagen IV separates the bottom migrating cells from the undifferentiated epiblast-like cells on top.

Fig. 4.

Mapping gastruloid cell migrations and fates to the human PS. (A) Diagram summarizing the fates and 3D structure of each type of gastruloid at 52 h and mapping to the human embryo (indicated by positions 1-4). As indicated by the arrowheads, we believe the edge of the epiblast/PrEct region in each gastruloid corresponds to the medial part of the in vivo PS, and that our migrations (indicated by arrows) therefore occur medially to laterally. APS, anterior PS; DE - Ant., anterior definitive endoderm; DE - Mid, mid-streak definitive endoderm; DE - Pos., posterior definitive endoderm; Epi., epiblast; ExM, extra-embryonic mesoderm; LM, lateral mesoderm; nuc., nucleus; Org., organizer; PM, paraxial mesoderm; PPS, posterior PS; PrEct, presumptive ectoderm.