Paracrine signals maintain developmental states and create cell fate patterns and influence differentiation outcomes in human embryonic stem cells (hESCs) Systematic investigation of morphogen signaling is hampered by the difficulty of disentangling endogenous signaling from experimentally applied ligands. Here, we grow hESCs in micropatterned colonies of 1-8 cells ('µColonies') to quantitatively investigate paracrine signaling and the response to external stimuli. We examine BMP4-mediated differentiation in µColonies and standard culture conditions and find that in µColonies, above a threshold concentration, BMP4 gives rise to only a single cell fate, contrary to its role as a morphogen in other developmental systems. Under standard culture conditions BMP4 acts as a morphogen but this requires secondary signals and particular cell densities. We find that a 'community effect' enforces a common fate within µColonies, both in the state of pluripotency and when cells are differentiated, and that this effect allows a more precise response to external signals. Using live cell imaging to correlate signaling histories with cell fates, we demonstrate that interactions between neighbors result in sustained, homogenous signaling necessary for differentiation.

Abstract:Paracrine signals maintain developmental states and create cell-fate patterns in vivo, and influence differentiation outcomes in human embryonic stem cells (hESCs) in vitro. Systematic investigation of morphogen signaling is hampered by the difficulty of disentangling endogenous signaling from experimentally applied ligands. Here, we grow hESCs in micropatterned colonies of 1-8 cells ("Colonies") to quantitatively investigate paracrine signaling and the response to external stimuli. We examine BMP4-mediated differentiation in Colonies and standard culture conditions and find that in Colonies, above a threshold concentration, BMP4 gives rise to only a single cell fate, contrary to its role as a morphogen in other developmental systems. Under standard culture conditions, BMP4 acts as morphogen, but this effect requires secondary signals and particular cell densities. We further find that a "community effect" enforces a common fate within Colonies both in the state of pluripotency and when cells are differentiated, and that this effect allows more precise response to external signals. Using live cell imaging to correlate signaling histories with cell fates, we demonstrate that interactions between neighbors result in sustained, homogenous signaling necessary for differentiation.Keywords: human embryonic stem cells, micropatterning, BMP4 pathway, differentiation mechanisms Summary Statement (15-30 words): We quantitatively examined signaling and differentiation in hESC colonies of varying size treated with BMP4. We show that secondary signals result in morphogen and community effects that determine cell fates.

Morphogens are signaling molecules that convey positional information and dictate cell fates during development. Although ectopic expression in model organisms suggests that morphogen gradients form through diffusion, little is known about how morphogen gradients are created and interpreted during mammalian embryogenesis due to the combined difficulties of measuring endogenous morphogen levels and observing development in utero. Here we take advantage of a human gastruloid model to visualize endogenous Nodal protein in living cells, during specification of germ layers. We show that Nodal is extremely short range so that Nodal protein is limited to the immediate neighborhood of source cells. Nodal activity spreads through a relay mechanism in which Nodal production induces neighboring cells to transcribe Nodal. We further show that the Nodal inhibitor Lefty, while biochemically capable of long-range diffusion, also acts locally to control the timing of Nodal spread and therefore of mesoderm differentiation during patterning. Our study establishes a paradigm for tissue patterning by an activator-inhibitor pair.

Morphogens are signaling molecules that convey positional information and dictate cell fates during development. Little is known about how morphogen gradients are created and interpreted during mammalian embryogenesis. Here we take advantage of a human gastruloid model to visualize endogenous Nodal protein in living cells. We show that Nodal is extremely short range so that Nodal protein is limited to the immediate neighborhood of source cells. Nodal activity spreads through a relay mechanism in which Nodal production induces neighboring cells to transcribe Nodal. We further show that the Nodal inhibitor Lefty, while biochemically capable of long-range diffusion, also acts locally to control the timing of Nodal spread and therefore of mesoderm differentiation during patterning. Our study establishes a novel paradigm for tissue patterning by an activator-inhibitor pair.

Living cells rely on numerous protein-protein, RNA-protein and DNA-protein interactions for processes such as gene expression, biomolecular assembly, protein and RNA degradation. Single-molecule microscopy and spectroscopy are ideal tools for real-time observation and quantification of nucleic acids-protein and protein-protein interactions. One of the major drawbacks of conventional single-molecule imaging methods is low throughput. Methods such as sequencing by synthesis utilizing nanofabrication and single-molecule spectroscopy have brought high throughput into the realm of single-molecule biology. The Pacific Biosceinces RS2 sequencer utilizes sequencing by synthesis within nanophotonic zero mode waveguides. A number of years ago this instrument was unlocked by Pacific Biosciences for custom use by researchers allowing them to monitor biological interactions at the single-molecule level with high throughput. In this capability letter we demonstrate the use of the RS2 sequencer for real time observation of DNA-to-RNA transcription and RNA-protein interactions. We use a relatively complex model – transcription of structured ribosomal RNA from E. coli and interactions of ribosomal RNA with ribosomal proteins. We also show evidence of observation of transcriptional pausing without the application of an external force (as is required for single-molecule pausing studies using optical traps). Overall, in the unlocked, custom mode, the RS2 sequencer can be used to address a wide variety of biological assembly and interaction questions at the single-molecule level with high throughput. This instrument is available for use at the Center for Integrated Nanotechnologies Gateway located at Los Alamos National Laboratory.