Recapitulating mammalian embryonic development in vitro is a major challenge in 1 biology. It has been shown that gastruloids 1-5 and ETX embryos 6 can display hallmarks 2 of gastrulation in vitro. However, these models fail to progress beyond spatially 3 segregated, yet amorphous cellular assemblies. Systems such as organoids 7 do show tissue 4 stratification and organogenesis, but require adult stem cells or exogeneous induction of 5 specific cell fates, and hence do not reflect the emergent organization of embryonic 6 development. Notably, gastruloids are derived exclusively from embryonic stem cells 7 (ESCs), whereas, in vivo, crucial patterning cues are provided by extraembryonic cells 8 . 8Here, we show that assemblies of mouse ESCs (mESCs) and extraembryonic endoderm 9 (XEN) cells can develop beyond gastrulation and produce a central hallmark of 10 organogenesis: stratified neural epithelia resembling a neural tube, which can be further 11 differentiated to cerebral cortex-like tissue. By single-cell RNA-seq, we show that our 12 2 model has a larger cell type diversity than existing models, and that mESCs and XEN 13 cells impact each other's differentiation. XEN cells promote neural tube formation 14 through local inhibition of primitive streak formation. In turn, the presence of mESCs 15 drives XEN cells to resemble visceral endoderm, which envelops the embryo in vivo. This 16 study provides a model system to investigate neurulation and extraembryonic endoderm 17 development, and may serve as a starting point to generate embryo models that advance 18 further toward the formation of the vasculature, nervous system, and digestive tube. 19 20We first implemented the original mouse gastruloid protocol 1 in which mESCs are aggregated 21 in N2B27 media and exposed to a pulse of WNT signaling for 24 h. After 96 h, this protocol 22 resulted in elongated gastruloids. As reported before 1-3 , gastruloids contained localized 23 primitive streak-and neural progenitor-like compartments, marked by Brachyury (T) and 24 SOX2, respectively (Fig. 1b, inset). We then adapted the gastruloid protocol by co-aggregating 25 XEN cells with mESCs, keeping all other conditions the same (Fig. 1a). After 96 h, the 26 resulting aggregates again showed T-positive and SOX2 positive compartments (Fig. 1b). 27However, in striking contrast with standard gastruloids, SOX2-positive cells were now 28 organized in stratified epithelia surrounding one or multiple lumina. The frequency of these 29 tubular structures depended on the fraction of XEN cells (Fig. 1c, Extended Data Fig. 1a). At 30 a XEN:mESC ratio of 1:3 we observed the concurrence of SOX2-positive tubes and T-positive 31 cells in the majority of aggregates. Since the canonical pluripotency marker OCT4 was not 32 expressed (Extended Data Fig. 1b), we hypothesized that the observed structures resemble 33 neural tubes. The presence of N-cadherin and absence of E-cadherin in the tubes (Fig. 1d) is 34 consistent with the known switch from E-to N-cadherin during neural differentiation in ...

Stem-cell derived in vitro systems, such as organoids or embryoids, hold great potential for modeling in vivo development. Full control over their initial composition, scalability, and easily measurable dynamics make those systems useful for studying specific developmental processes in isolation. Here we report the formation of gastruloids consisting of mouse embryonic stem cells (mESCs) and extraembryonic endoderm (XEN) cells. These XEN-enhanced gastruloids (XEGs) exhibit the formation of neural epithelia, which are absent in gastruloids derived from mESCs only. By single-cell RNA-seq, imaging, and differentiation experiments, we demonstrate the neural characteristics of the epithelial tissue. We further show that the mESCs induce the differentiation of the XEN cells to a visceral endoderm-like state. Finally, we demonstrate that local inhibition of WNT signaling and production of a basement membrane by the XEN cells underlie the formation of the neuroepithelial tissue. In summary, we establish XEGs to explore heterotypic cellular interactions and their developmental consequences in vitro.

The G4C2 hexanucleotide repeat expansion in the c9orf72 locus is one among a plethora of mutations associated with amyotrophic lateral sclerosis. It accounts for the majority of disease cases. The exact processes underlying the pathology of this mutation remain elusive, yet recent evidence suggests a mechanism that disrupts axonal trafficking. Here, we used a neuronal cell line with and without the G4C2 repeats, and implemented time-resolved local mean squared displacement analysis to characterize the motion of lysosomes inside neurites. Neurites were either aligned along chemically patterned lines, or oriented randomly on the substrate. We confirmed that in the presence of the G4C2 repeats, lysosome motion was affected. Lysosomes had a smaller reach exhibited lower velocity, especially inside aligned neurites. At the same time they became more active with increasing length of the G4C2 repeats when the neurites were randomly oriented. The duration of diffusive and super-diffusive lysosome transport remained unaffected for both neurite geometries and for all lengths of the repeats, but the displacement and velocity was decreased on varying the repeat number and neurite geometry. Lastly, the ratio of anterograde/retrograde/neutral trajectories was affected disparately for the two neurite geometries. Our observations support the hypothesis that impaired axonal trafficking emerges in the presence of the G4C2 hexanucleotide repeat expansion.