Abstract
SummaryA fundamental question in developmental biology is how the early embryo establishes the spatial coordinate system that is later important for the organization of the embryonic body plan. Although we know a lot about the signaling and gene-regulatory networks required for this process, much less is understood about how these can operate to pattern tissues in the context of the extensive cell movements that drive gastrulation. In zebrafish, germ layer specification depends on the inheritance of maternal mRNAs [1, 2, 3], cortical rotation to generate a dorsal pole of β-catenin activity [4, 5, 6, 7, 8], and the release of Nodal signals from the yolk syncytial layer (YSL) [9, 10, 11, 12]. To determine whether germ layer specification is robust to altered cell-to-cell positioning, we separated embryonic cells from the yolk and allowed them to develop as spherical aggregates. These aggregates break symmetry autonomously to form elongated structures with an anterior-posterior pattern. Both forced reaggregation and endogenous cell mixing reveals how robust early axis specification is to spatial disruption of maternal pre-patterning. During these movements, a pole of Nodal signaling emerges that is required for explant elongation via the planar cell polarity (PCP) pathway. Blocking of PCP-dependent elongation disrupts the shaping of opposing poles of BMP and Wnt/TCF activity and the anterior-posterior patterning of neural tissue. These results lead us to suggest that embryo elongation plays a causal role in timing the exposure of cells to changes in BMP and Wnt signal activity during zebrafish gastrulation.Video
Highlights
Explants from Tbx16:GFP reporter embryos [18] revealed mesoderm specification within the elongating end of the aggregate (Figures 1B–1F; Video S1), accompanied by polarized expression of tbxta (Figures 1G–1K). These results showed how symmetry breaking and mesoderm patterning can occur within explants of embryonic cells separated from the yolk
Our results demonstrate that the three primary germ layers can form and pattern in the absence of continued signals from the yolk and the yolk syncytial layer (YSL)
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
Summary
METHOD DETAILSIn vitro culture of zebrafish explants Zebrafish embryos were incubated at 28C in E3 media dechorionated either manually or using pronase (1mg/mL). When embryos reached the 256 cell stage, the embryonic cells were explanted from the yolk using an eyelash tool. These tools are made by Current Biology 30, 2984–2994.e1–e3, August 3, 2020 e1. A pescoid is defined as a total embryonic mass explant including all the blastomeres. When animal or vegetal pieces of differing sizes were taken, a small animal explant was taken from the animal pole of the embryo, with the remaining blastomeres being taken as labeled a large vegetal piece. When large regions from the animal pole were taken, the remaining blastomere explant was defined as a small vegetal piece. Comparison of sizes by diameter at 5hpc were made using the Fiji line drawing tool and compared to similar explants at 1hpc in [3] and 2 somite stage [15]
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