Extraembryonic endoderm cells induce neuroepithelial tissue in gastruloids

Abstract

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 ...

Dynamics of anteroposterior axis establishment in a mammalian embryo-like system

“…between mouse and gastruloid While the more differentiated cell types emerging in gastruloids have already been mapped to their respective in vivo counterparts as part of previous studies [15,46,47,48], incipient germ layer formation has thus far not been explicitly addressed from such a comparative perspective. Therefore, we conducted mutual nearest neighbor (MNN)-based integration of our gastruloid scRNA-seq dataset from 24h-72h with a previously published mouse sc-atlas [49] (Figure 5a,b).…”

Development in a Dish—In Vitro Models of Mammalian Embryonic Development

“…Gastruloids and similar structures (see above) are therefore important models of posterior neural tube development ( Figure 2D ). As discussed above, gastruloids form neural tube-like structures when embedded in Matrigel or surrounded by a layer of extraembryonic cells ( Bérenger-Currias et al, 2020 ; van den Brink et al, 2020 ; Veenvliet et al, 2020 ).…”

“…These gastruloids, remarkably similar in shape and organization to the posterior end of embryos, are referred to as trunk-like structures ( Figure 1 ). To compensate for the absence of extraembryonic tissue in gastruloids, Bérenger-Currias et al (2020) modified the gastruloid protocol by aggregating both mESCs and XEN cells. These aggregates induced neural tube-like structures that have a neural progenitor-like transcriptome and were shown to recapitulate neural tube development ( Figure 1 ).…”

“…More recent attempts to co-culture different organoids aim at addressing this by bringing multiple “organs” together ( Koike et al, 2019 ). In addition, embryo-like model systems resembling gastrulating embryos can be directed toward specific trajectories and allow organogenesis within a multi-tissue and multi-organ context ( Bérenger-Currias et al, 2020 ; Rossi et al, 2021 ). While such models have to be optimized and developed further, they will be of great benefit for the investigation of organogenesis during embryonic development.…”

Translating Embryogenesis to Generate Organoids: Novel Approaches to Personalized Medicine

“…datasets provide evidence on the emergence of neural tube and somites recapitulating early organogenesis ( van den Brink et al., 2020 ; Veenvliet et al, 2020 ). Culturing mouse gastruloids with an underlying extracellular matrix led to the formation of structures resembling somites and neural tube ( van den Brink et al., 2020 ; Veenvliet et al, 2020 ), whereas another report suggested that mixing gastruloids and extra-endodermal cells can lead to the formation of the neural tube ( Bérenger-Currias et al, 2020 ). Recently, gastruloids have been generated from self-organizing hESCs, which can also differentiate to three germ layers ( Moris et al., 2020 ).…”