Endogenous FGFs drive ERK-dependent cell fate patterning in 2D human gastruloids.

The role of FGF is the least understood of the morphogens driving mammalian gastrulation. Here we investigated the function of FGF in a stem cell model for human gastrulation known as a 2D gastruloid. We found a ring of FGF-dependent ERK activity that closely follows the emergence of primitive streak (PS)-like cells but expands further inward. We showed that this ERK activity pattern is required for PS-like differentiation and that loss of PS-like cells upon FGF receptor inhibition can be rescued by directly activating ERK. We further demonstrated that the ERK-ring depends on localized activation of basally localized FGF receptors (FGFR) by endogenous FGF gradients. We confirm and extend previous studies in analyzing expression of FGF pathway components, showing the main receptor to be FGFR1 and the key ligands FGF2/4/17, similar to the human and monkey embryo but different from the mouse. In situ hybridization and scRNA-seq revealed that FGF4 and FGF17 expression colocalize with the PS marker TBXT but only FGF17 is maintained in nascent mesoderm and endoderm. FGF4 and FGF17 reduction both reduced ERK activity and differentiation to PS-like cells and their derivatives, indicating overlapping function. Thus, we have identified a previously unknown role for FGF-dependent ERK signaling in 2D gastruloids and possibly the human embryo, driven by a mechanism where FGF4 and FGF17 signal through basally localized FGFR1 to induce PS-like cells.

Time-integrated BMP signaling determines fate in a stem cell model for early human development.

How paracrine signals are interpreted to yield multiple cell fate decisions in a dynamic context during human development in vivo and in vitro remains poorly understood. Here we report an automated tracking method to follow signaling histories linked to cell fate in large numbers of human pluripotent stem cells (hPSCs). Using an unbiased statistical approach, we discover that measured BMP signaling history correlates strongly with fate in individual cells. We find that BMP response in hPSCs varies more strongly in the duration of signaling than the level. However, both the level and duration of signaling activity control cell fate choices only by changing the time integral. Therefore, signaling duration and level are interchangeable in this context. In a stem cell model for patterning of the human embryo, we show that signaling histories predict the fate pattern and that the integral model correctly predicts changes in cell fate domains when signaling is perturbed. Our data suggest that mechanistically, BMP signaling is integrated by SOX2.

The many dimensions of germline competence.

Primordial germ cell (PGC) specification is the first step in the development of the germline. Recent work has elucidated human-mouse differences in PGC differentiation and identified cell states with enhanced competency for PGC-like cell (PGCLC) differentiation in vitro in both species. However, it remains a subject of debate how different PGC competent states in vitro relate to each other, to embryonic development, and to the origin of PGCs in vivo. Here we review recent literature on human PGCLC differentiation in the context of mouse and non-human primate models. In contrast to what was previously thought, recent work suggests human pluripotent stem cells (hPSCs) are highly germline competent. We argue that paradoxical observations regarding the origin and signaling requirements of hPGCLCs may be due to local cell interactions. These confound assays of competence by generating endogenous signaling gradients and spatially modulating the ability to receive exogenous inductive signals. Furthermore, combinatorial signaling suggests that there is no unique germline competent state: rather than a one-dimensional spectrum of developmental progression, competence should be considered in a higher dimensional landscape of cell states.

Spatial Single Cell Analysis of Proteins in 2D Human Gastruloids Using Iterative Immunofluorescence.

During development, cell signaling instructs tissue patterning, the process by which initially identical cells give rise to spatially organized structures consisting of different cell types. How multiple signals combinatorially instruct fate in space and time remains poorly understood. Simultaneous measurement of signaling activity through multiple signaling pathways and of the cell fates they control is critical to addressing this problem. Here we describe an iterative immunofluorescence protocol and computational pipeline to interrogate pattern formation in a 2D model of human gastrulation with far greater multiplexing than is possible with standard immunofluorescence techniques. This protocol and computational pipeline together enable imaging followed by spatial and co-localization analysis of over 27 proteins in the same gastruloids. We demonstrate this by clustering single cell protein expression, using techniques familiar from scRNA-seq, and linking this to spatial position to calculate spatial distributions and cell signaling activity of different cell types. These methods are not limited to patterning in 2D gastruloids and can be easily extended to other contexts. In addition to the iterative immunofluorescence protocol and analysis pipeline, Support Protocols for 2D gastruloid differentiation and producing micropatterned multi-well slides are included. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Iterative immunofluorescence Basic Protocol 2: Computational analysis pipeline Support Protocol 1: Generating micropatterned multi-well slides Support Protocol 2: Differentiation of 2D gastruloids.

The time integral of BMP signaling determines fate in a stem cell model for early human development.

How paracrine signals are interpreted to yield multiple cell fate decisions in a dynamic context during human development in vivo and in vitro remains poorly understood. Here we report an automated tracking method to follow signaling histories linked to cell fate in large numbers of human pluripotent stem cells (hPSCs). Using an unbiased statistical approach, we discovered that measured BMP signaling history correlates strongly with fate in individual cells. We found that BMP response in hPSCs varies more strongly in the duration of signaling than the level. However, we discovered that both the level and duration of signaling activity control cell fate choices only by changing the time integral of signaling and that duration and level are therefore interchangeable in this context. In a stem cell model for patterning of the human embryo, we showed that signaling histories predict the fate pattern and that the integral model correctly predicts changes in cell fate domains when signaling is perturbed. Using an RNA-seq screen we then found that mechanistically, BMP signaling is integrated by SOX2.

Efficient differentiation of human primordial germ cells through geometric control reveals a key role for Nodal signaling.

Human primordial germ cells (hPGCs) form around the time of implantation and are the precursors of eggs and sperm. Many aspects of hPGC specification remain poorly understood because of the inaccessibility of the early postimplantation human embryo for study. Here, we show that micropatterned human pluripotent stem cells (hPSCs) treated with BMP4 give rise to hPGC-like cells (hPGCLC) and use these as a quantitatively reproducible and simple in vitro model to interrogate this important developmental event. We characterize micropatterned hPSCs up to 96 hr and show that hPGCLC populations are stable and continue to mature. By perturbing signaling during hPGCLC differentiation, we identify a previously unappreciated role for Nodal signaling and find that the relative timing and duration of BMP and Nodal signaling are critical parameters controlling the number of hPGCLCs. We formulate a mathematical model for a network of cross-repressive fates driven by Nodal and BMP signaling, which predicts the measured fate patterns after signaling perturbations. Finally, we show that hPSC colony size dictates the efficiency of hPGCLC specification, which led us to dramatically improve the efficiency of hPGCLC differentiation.

In humans and other animals, eggs and sperm are unique cells that pass on genetic material to the next generation. They originate from a small group of cells called primordial germ cells that form early in life in the developing embryo. Several different signal molecules including ones known as BMP4, Wnt, and Nodal, instruct certain cells in the embryo to become primordial germ cells. The process by which primordial germ cells are made in humans is very different to how primordial germ cells are made in mice and other so-called model animals that are commonly used in research. This has made it more challenging to uncover the details of the process in humans. Fortunately, new methods have recently been created that mimic aspects of how human embryos develop using human stem cells in a laboratory dish, providing an opportunity to gain a deeper understanding of how human germ cells form. Jo et al. used a technique called micropatterning to control the shape and size of groups of human stem cells growing in a laboratory dish. Treating these cells with a signal known as BMP4 gave rise to cells that resembled primordial germ cells. The team then used this system as a model to study how primordial germ cells form in humans. The experiments found that reducing Wnt signals in stem cells stopped primordial germ cells from forming in response to BMP4, confirming that Wnt signals made by the cells in response to BMP4 are essential. However, this block was overcome by providing the stem cells with another signal called Nodal. This suggests that the role of Wnt signaling in primordial germ cell formation is in part indirect by switching on Nodal in stem cells. Defects in eggs and sperm may lead to infertility, therefore, the findings of Jo et al. have the potential to help researchers develop new fertility treatments that use eggs or sperm grown in a laboratory from the patients' own stem cells. Such research would benefit from first developing a better understanding of how to make primordial germ cells.