Lineage and Spatial Pre-patterns Are Lost due to Extensive Cell Divisions and Cell Mixing

(A–D’) Disassociation (A) and reaggregation (B) of explanted cells results in tbxta expression (n = 8/8; expressed/total imaged; C and C’) and, infrequently, elongation of the aggregate (n = 2 observed; D and D’). Cells in full embryonic explants undergo rapid cell divisions as seen in images acquired on SPIM.

(E–F’) Number of cells in pescoids (E) counted based on image segmentation (black curve), as seen in images acquired on SPIM (F and F’). Dashed curves are estimates of the number of cells, starting from the number of spots segmented at t = 0, if all cells divided synchronously every 20 min (blue line) or only a random sub-population (<50%) of cells divided every 20 min (orange line).

(F and F’) Cells in pescoid divide randomly with no preference for direction of division, leading to mixing of cells.

(G–I’) Injecting embryos with fluorescent high-molecular-weight dextran at the 64-cell stage, (H–I) labeling marginal cells prior to making pescoids demonstrates that these labeled cells spread across the entire pescoid within (H’–I’) 5 h of explanting. These cells also show a high level of intermixing of labeled and unlabeled cells. n = 10, all demonstrating cell mixing. (G–I) Images shown as maximum projections. (I’) Shown as central 2-μm slice.

(J–L’) This is also shown in pescoids injected at the one-cell stage with Kikume mRNA and then a small population of cells labeled by photo conversion at 1 hpc. Explants were reimaged at 3 hpc to assay for label mixing (n = 6 replicates, all demonstrating cell mixing). (K–K’) Images are shown as maximum projections at 1 and 3 hpc. (L–L’) spot detection of labelled and unlabelled cells in 3D rendering of (K–K’) demonstating cell mixing at 1 and 3 hpc.

(N–Q’) HCR staining of animal cap explants and pescoids at 5 hpc reveals a similar expression of eomesodermin (pescoids 4/4; animal caps 6/6), mxtx2 (pescoids 3/5; animal caps 4/5), tbxta (pescoids 5/6; animal caps 3/4), and goosecoid (pescoids 3/6; animal caps 2/4). In (N)–(Q), n = gene expression observed/total imaged.

(R) This finding is further supported by (R and R’) clear expression of a Tbx16::GFP reporter in both animal caps (4/6; expression/imaged from heterozygous in-cross) and full pescoid explant at 7 hpc (2/4; expression/imaged from heterozygous in cross).

Further replicates of labeling experiments (G–L) demonstrating cell mixing are shown in Figure S2. A comparison of animal cap size explants is available in Figure S3. The SPIM data can be viewed in Video S3. Scale bar represents 50 μm.

Nodal Signaling Is Upstream of PCP-Driven Convergence and Extension, which Drives Elongation

(A–C) The first signaling event that polarizes to a single point within the pescoid is that of Nodal signaling, demonstrated through (A–C) polarization of phospho-Smad 2/3 activity. This is shown in composite color images and as pSmad2/3 signal inverted images (A’–C’; 2 hpc n = 4/8; 3 hpc n = 5/8; 5 hpc n = 4/6; total with polarized signal/total imaged).

(D and E) We also observe polarized (D) ndr1 and (E) ndr2 expression in the elongation at 5hpc (ndr1 n = 4/6; ndr2 n = 4/6; expression observed/total imaged; total with polarized signal/total imaged).

(F–I) Treatment with the Nodal inhibitor SB-505124 between (G) 1 and 3 hpc inhibits elongation of the explants and (H) to a lesser extent when applied between 5 and 7 hpc when compared to controls at 7 hpc (F and I).

(J) The PCP components wnt11f2 and fzd2 are observed expressed in the elongating end of the pescoid with the spatial organization of these domains reflected in (J’) surface renderings of the HCR signal.

(K and K’) Inhibition of Nodal signaling between 1 and 3 hpc results in loss of wnt11f2 and frz2 expression.

(L–O) Inhibition of convergence and extension movements using (M) blebbistatin or (N) injection at the one-cell stage of a dominant-negative dishevelled construct blocks elongation when compared to controls (L and O), further supporting that elongation is caused by convergence and extension movements.

Elongation is demonstrated not be to be caused by polarized cell division in Figure S2. Explant sizes and the ability to form mesoderm are shown in Figure S3. Polarization of Nodal signaling with the absence of polarized β-catenin or FGF (ppERK) is further shown in Figure S4. Further time-lapse data from signal reporters are available in Video S4. Scale bar represents 50 μm.

PCP-Dependent Elongation Is Required for Regulating Exposure to BMP and Wnt Activity

(A–F) Expression of bmp7 (A–C) and TCF::GFP (D–F) as a time course at 2 hpc, 5 hpc, and 7 hpc reveals both signaling domains are spread across the explant evenly at early time points and are restricted to either end of the explant by 7 hpc. TCF::GFP assayed by HCR against GFP mRNA for immediate reporter activity readout is shown (2 hpc bmp7 2/3, tcf.:gfp 4/6; 5 hpc bmp7 5/5, tcf.:gfp 10/10; 7 hpc bmp7 4/4, tcf.:gfp 4/5; expression observed/total imaged).

(G–M, O–R, and T–W) The time course reveals the isolation of the BMP and Wnt domains to either end of the explant allows expression of hindbrain marker (G–I) krox20 in a characteristic two-stripe pattern. (G) Initially no expression is observed (n = 0/6; expression/total imaged), followed by (H) expression of a single stripe (n = 7/8; expression/total imaged) at 7 hpc and then (I) two stripes by 10 hpc (n = 5/8 expression/total imaged). Inhibition of convergence and extension using dominant-negative dishevelled injected at the one-cell stage (O–R) and treatment with 2.5 μM blebbistatin (T–W) reveals that the Wnt/TCF and BMP domains do not separate as observed in the (J–M) control.

(N, S, and X) Description of these profile quantitatively through normalization of the long axis of the explant and normalization of signal intensity between 0 and 1 (n = minimum 7 per condition; line represents mean). The lack of low BMP moderate Wnt/TCF domain can be observed in the central region of the explants compared to control. (N) displays a control profile, (S) displays the profile of a DEP+ explant, and (X) displays a profile of an explant treated with Blebbistatin.

(N’, S’, and X’) The lack of low BMP moderate Wnt/TCF domains results in significantly altered patterns of krox20 with no double-stripe patterns observed other than in controls. Scale bar represents 50 μm. Time-lapse data from signal reporters are available in Video S4.

Axial Patterning Can Occur in the Absence of Yolk

(A–K) Explants of early embryonic blastomeres taken at the (A) 256-cell stage demonstrate elongation and mesendodermal induction visualized through expression of a (B–F) Tbx16::GFP reporter (n = 4/8; heterozygous in-cross) and (G–K) tbxta mRNA (n = 1 hpc 4/4; 2 hpc 5/6; 3 hpc 5/6; 5 hpc 6/8; 7 hpc 6/6).

(L and M) At the opposite pole to tbxta expression at 7 hpc, (L) bmp4 expression is observed at the opposing end of the aggregate to tbxta expression with (M) sox32 expression found in the center of some aggregates, but those without sox32 expression are able to elongate (n = 3/8; sox32 positive explants/assayed elongating explants).

(N) In the high bmp4 domain at 7 hpc, gata4 expression is observed (n = 8/9), with protocadherin8 (n = 8/9) expression more posteriorly and noto furthest posterior from the bmp4 expression domain (n = 7/9).

(O) Germ cell markers (O and O’) nanos and vasa are also observed coexpressed in cells within the core of the aggregates in the sox32-positive domain (n = 5/5) at 7 hpc.

Elongation is further quantified and shown to occur in a range of media in Figure S1. Time-lapse data of bright-field and Tbx16::GFP explants can be found in Video S1. Slice views, 3D views and surface renderings of in situ HCR data are available in Video S2. n = expression observed/total imaged. Scale bars represent 50 μm (A–K’) and 70 μm (L–O’).