The current understanding of mammalian kidney development is largely based on mouse models. Recent landmark studies revealed pervasive differences in renal embryogenesis between mouse and human. The scarcity of detailed gene expression data in humans therefore hampers a thorough understanding of human kidney development and the possible developmental origin of kidney diseases. In this paper, we present a single-cell transcriptomics study of the human fetal kidney. We identified 22 cell types and a host of marker genes. Comparison of samples from different developmental ages revealed continuous gene expression changes in podocytes. To demonstrate the usefulness of our data set, we explored the heterogeneity of the nephrogenic niche, localized podocyte precursors, and confirmed disease-associated marker genes. With close to 18,000 renal cells from five different developmental ages, this study provides a rich resource for the elucidation of human kidney development, easily accessible through an interactive web application.

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.

Gastruloids have emerged as highly usefulin vitromodels of mammalian gastrulation. One of the most striking features of 3D gastruloids is their elongation, which mimics the extension of the embryonic anterior-posterior axis. Although axis extension is crucial for development, the underlying mechanism has not been fully elucidated in mammalian species. Gastruloids provide an opportunity to study this morphogenic processin vitro. Here, we measure and quantify the shapes of elongating gastruloids and show, by Cellular Potts model simulations, that a combination of convergent extension and differential adhesion can explain the observed shapes. We reveal that differential adhesion alone is insufficient and also directly observe hallmarks of convergent extension by time-lapse imaging of gastruloids. Finally, we show that gastruloid elongation can be abrogated by inhibition of the Rho kinase pathway, which is involved in convergent extensionin vivo. All in all, our study demonstrates, how gastruloids can be used to elucidate morphogenic processes in embryonic development.

Over the past decade, exciting progress in stem cell research has led to the development of organoids as a new system to model human development and disease in vitro. Organoids are three dimensional (3D) cell cultures, derived from primary tissue or stem cells, which recapitulate in vivo biology more closely than conventional cell culture systems. Live fluorescence imaging is an invaluable tool to observe organoid development or their response to perturbations. However, imaging organoids over time remains challenging, due to their large size, demanding culture conditions and light sensitivity. Light-sheet microscopy is ideal to overcome these challenges, due to the restriction of light exposure to the focal plane. Most light-sheet microscope designs, however, require the specimen to be embedded in agarose cylinders or small capillaries, making them unsuitable for organoid systems. Here, we present the design of a light-sheet microscope that overcomes this limitation. We adopted an inverted microscope setup [1], which we optimized for the use with organoids. In the inverted design of Strnad et al. [1], both the illumination and detection objective face upward and are immersed in water. This configuration allows various 3D cell culture systems to be imaged and cultured in a liquid medium. In their design, however, the illumination of the specimen is limited to one side, which might not be sufficient for organoids that can reach a size of millimeters. In our design we included the option to extend the setup with a second illumination objective opposite to the first. As a result, the orientation of the objectives changed compared to the design of Strnad et al., while keeping the sample holder equally accessible (see Figure 1). Furthermore, we also adapted the design of Strnad et al. to enable imaging in the incubator for a long time. To prevent the electronic