Axis Specification in Zebrafish Is Robust to Cell Mixing and Reveals a Regulation of Pattern Formation by Morphogenesis: Current Biology | Current Biology

The ability of pescoids to undergo symmetry breaking and elongation in the absence of a pre-pattern led us to question how cells behave in early pescoids. We used light sheet imaging (selective plane illumination microscopy [SPIM]) of pescoids immediately after harvesting. Cells undergo several rounds of rapid division and increases by about 20-fold over a period of 2.5 h (Figures 2E and 2F; Video S3). In order to understand the contribution of cell division patterns to cell rearrangement in the explants, we estimated the increase in the number of cells in two scenarios: (1) if all cells were to divide synchronously every 20 min (Figure 2E, blue graph) or (2) only a random sub-population (Figure 2E, orange graph). Compared to these estimates, it is clear that there is a degree of asynchrony in the rate of divisions across the early pescoids. Analysis of the direction of division further showed that there is no spatial pattern of divisions (Figure 2F) and, as a result of which, prior lineage-based pre-patterns would become homogenized owing to these rapid, asynchronous divisions within the pescoids before elongation. To determine whether cell rearrangements in the pescoids might contribute to the erasure of any pre-existing spatial pre-pattern, we performed labeling of marginal cells using fluorescent high-molecular-weight dextran, injected at the 64-cell stage, prior to explanting (Figures 2G–2I’). After 5 h post-culture, these labeled cells were observed distributed throughout the pescoid, with high levels of intermixing between labeled and unlabeled cells (Figures 2I’ and S2B). To demonstrate this further, small photo-labels of cells at one edge of the pescoid were made at 1 hpc and their distribution observed 3 h later (Figures 2J–2L’). In all cases, a complete mixing of labeled and unlabeled cells was observed (Figures 2K’, 2L, and S2A). These results reveal that pescoids undergo extensive cell mixing at early stages that effectively remove any pre-pattern that could be produced from the inheritance of maternal mRNAs at the vegetal pole. As a consequence, we expected that cells containing maternal mRNAs that initially reside in a vegetal domain would move to more animal regions as a consequence of this cell mixing. Indeed, hybridization chain reaction (HCR) staining of animal caps and 5-hpc pescoids revealed the presence of the maternally deposited mRNAs eomesodermin (Figures 2N and 2N’) and mxtx2 (Figures 2O and 2O’), as well as expression of the early mesodermal marker tbxta (Figures 2P and 2P’) and marker of the embryonic shield, goosecoid (Figures 2Q and 2Q’) in both whole-embryo explants (pescoids) and animal caps. In addition, time-lapse movies of animal caps showed a clear expression of the Tbx16:GFP reporter (Figures 2R and 2R’). Together, these results suggested to us that the maternally inherited mesodermal specification markers are present in both vegetal and animal blastomeres by the 256-cell stage.

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  Video S3. Ubiquitous H2B-GFP-Labeled Explant Imaged on a SPIM with a Time Resolution of 2.5 min, Related to Figure 2

The inheritance of the maternal mRNAs mxtx2 and eomesodermin to the animal pole was surprising and suggests that both animal and vegetal regions of the early blastoderm might have similar potentials to undergo elongation. When taking both animal and vegetal (Figure S3A) regions at a range of sizes, we found no bias toward vegetal explants in their ability to elongate, although tissue size was a key factor (Figure S3B). Indeed, complete pescoids elongated to a much greater extent than either poles alone, suggesting that a complete set of cells was important for pescoid elongation. To test whether this size dependency might explain the absence of elongation in animal caps that are smaller than pescoid explants, we dissociated and reaggregated 2 animal caps together. In some cases, this led to the formation of a protrusion; however, this was again dependent on aggregate size. In no cases were protrusions observed in single reaggregated animal caps (Figures S3D and S3E). This size dependency was also confirmed when cutting un-dissociated pescoids into smaller portions (Figure S3F). Individual quarters were able to express tbxta, but not elongate (Figure S3G). Taken together, these results suggest that aggregate size and not the gene expression state of cells within the aggregate is the major determining factor for early embryonic cells to elongate.

Having established that pescoids can specify mesendoderm even when cells are rearranging their initial spatial positions, we next sought to determine whether associated signaling pathways can also form polarized expression in this context. We fixed pescoids at intermediate stages between initial culture and protrusion formation at 5 hpc and stained for phospho-Smad 2/3 (Figures 3A–3C, S4A, and S4B), β-catenin (Figures S4C and S4D), and diphosphorylated ERK-1 and ERK-2 (Figure S4E). At 5 hpc, we observe an association of Smad2/3 signaling with the elongated pole is (Figure 3A), together with the expression of the Nodal ligands ndr1 and ndr2 (Figures 3D and 3E). We also observe a clear association of the Nodal activity with elongation from the imaging of pescoids taken from embryos transgenic for an activin response element driving GFP expression [

] (Video S4). Furthermore, blocking Nodal receptor activity between 1 and 3 hpc with SB505124 inhibits pescoid elongation (Figures 3G and 3I), compared to controls (Figures 3F and 3I). The requirement for Nodal activity is increased at early stages as later treatments between 5 and 7 hpc have a lesser effect on elongation (Figure 3H). This importance of Nodal signaling in driving explant elongation is in line with recent work showing that Nodal signaling is sufficient to drive the elongation of animal caps [

22

• Williams M.L.K.
• Solnica-Krezel L.

Nodal and planar cell polarity signaling cooperate to regulate zebrafish convergence and extension gastrulation movements.

] and an inheritance of Nodal activity from the germ ring in blastoderm explants similar to those presented here [

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• Schauer A.
• Pinheiro D.
• Hauschild R.
• Heisenberg C.-P.

Zebrafish embryonic explants undergo genetically encoded self-assembly.

].