The body plan of the mammalian embryo is shaped through the process of gastrulation, an early developmental event that transforms an isotropic group of cells into an ensemble of tissues ordered with reference to three orthogonal axes 1 . While model organisms have provided much insight into this process, we know very little about gastrulation in humans due to the difficulty of obtaining embryos at such early stages of development, as well as to the ethical and technical restrictions that limit the feasibility of observing gastrulation ex vivo 2 . Here we show that human embryonic stem cells can be used to generate gastruloids: three dimensional multicellular aggregates that differentiate to derivatives of the three germ layers organised spatiotemporally, without additional extra-embryonic tissues. Human gastruloids undergo elongation along an anteroposterior axis and, using spatial transcriptomics, we show that they exhibit patterned gene expression. This includes a somitogenesis signature that suggests that 72 hour human gastruloids exhibit features of Carnegie Stage 9 embryos 3 . Our study represents a new, experimentally tractable model system to reveal and probe human-specific regulatory processes occurring during axial organisation in early development.The body plan of mammalian embryos emerges through interactions of sequential cell fate decisions and morphogenetic events, which have hitherto been difficult to observe in humans.Human Embryonic Stem Cells (hESCs) 4 have opened up opportunities for studying early fate decisions, and have hinted at the existence of regulatory mechanisms specific to humans 5,6 . But, in contrast to the embryo, where proportionate populations interact with one another to generate tissues and organs, differentiation in adherent culture is heterogeneous and favours a limited number of cell types 7 . Seeding hESCs on micropatterned surfaces yields coordinated patterns of gene expression, but without the axial organization characteristic of embryos 8 . However, when mouse ESCs are aggregated in suspension under defined conditions, they generate 'gastruloids': a three-dimensional, in vitro model of mammalian development, which exhibits an embryo-like spatiotemporal organization of gene expression 9,10 . We hypothesised that similar human gastruloids could be derived from hESCs. Generation of human gastruloidsWhen hESCs in 2D culture were treated with Chiron, a Wnt agonist, for one day before seeding defined numbers in low-adherence plates in the presence of Chiron, they formed compact, spherical aggregates within a few hours (Fig. 1a, Extended Data Fig. 1a-c). These aggregates progressively broke symmetry and formed elongated structures, with maximal elongation at 72-96h (Fig. 1a-d). On average, ~66% of aggregates from each experiment displayed an elongated morphology at 72h. Although some of the structures remained TOP award (NWO-CW 714.016.001), and the Foundation for Fundamental Research on Matter, financially supported by NWO (FOM-14NOISE01) to S.C.v.d.B., A.A. and A.v.O.. This w...

CRISPR/Cas9 efficiently induces targeted mutations via non-homologous-end-joining but for genome editing, precise, homology-directed repair (HDR) of endogenous DNA stretches is a prerequisite. To favor HDR, many approaches interfere with the repair machinery or manipulate Cas9 itself. Using Medaka we show that the modification of 5’ ends of long dsDNA donors strongly enhances HDR, favors efficient single-copy integration by retaining a monomeric donor conformation thus facilitating successful gene replacement or tagging.

Summary A 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 Abstract

Since publication, an error has been identified in Figure S1C. In the initial production of this figure, we had mistakenly duplicated the column under the heading ''L15 + 3% FBS'' to the left of ''L15 + 10% FBS.'' We have removed the second L15 + 3% FBS column to resolve this image duplication. We also duplicated images at 5 hpc L15 +3% FBS and L15 + 10% FBS. We have checked the metadata and origin of this image and confirmed it originates from the L15 + 10% FBS condition. As this error occurred in figure production and not during data analysis, this error has not impacted the conclusions of this experiment. This error has now been corrected online. The authors apologize for this error and any confusion it may have caused.