Abstract
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.
Highlights
Morphogens are signaling molecules that convey positional information and dictate cell fates during development
In addition to paracrine signaling over several cell diameters, other models which do not rely on extracellular transport, including a relay model in which signaling induces the transcription of the gene encoding the morphogen in neighboring cells have been suggested to explain Nodal signaling in the early vertebrate embryo[5,18,19,20,21]
We found that cNodal heterozygotes and homozygotes give rise to normal fate patterns indistinguishable from wild type (WT) patterns (Fig. 1d, Supplementary Fig. 4a, b), while gastruloids made with Nodal knockout human embryonic stem cells (hESCs) lack the mesodermal layer[28] (Supplementary Fig. 3a), consistent with the phenotype of Nodal−/− mice[7]
Summary
Morphogens are signaling molecules that convey positional information and dictate cell fates during development. We visualize and quantitatively measure the levels of endogenous Nodal and Lefty1/2 mRNA and protein in human embryonic stem cells (hESCs) and in a model of human germ layer patterning, micropatterned hESC-based (2D) gastruloids[23,24]. In this model, exogenous BMP signaling triggers a cascade of signaling through the BMP, Wnt, and Nodal pathways causing germ layer differentiation. We show that propagation of the Nodal signal requires that the Nodal gene be intact in receiving cells, suggesting a transcriptional relay model As this relay causes Nodal activity to spread inwards in the gastruloid, Lefty is transiently expressed at the signaling front. This suggests that the function of Lefty during gastrulation is to control the timing and spread of the Nodal signaling wave to properly induce mesendoderm
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