Patterning by morphogen gradients relies on the capacity to generate reproducible distribution profiles. Morphogen spread depends on kinetic parameters, including diffusion and degradation rates, which vary between embryos, raising the question of how variability is controlled. We examined this in the context of Toll-dependent dorsoventral (DV) patterning of the Drosophila embryo. We find that low embryo-to-embryo variability in DV patterning relies on wntD, a Toll-target gene expressed initially at the posterior pole. WntD protein is secreted and disperses in the extracellular milieu, associates with its receptor Frizzled4, and inhibits the Toll pathway by blocking the Toll extracellular domain. Mathematical modeling predicts that WntD accumulates until the Toll gradient narrows to its desired spread, and we support this feedback experimentally. This circuit exemplifies a broadly applicable induction-contraction mechanism, which reduces patterning variability through a restricted morphogen-dependent expression of a secreted diffusible inhibitor.

Establishment of morphogen gradients in the early Drosophila embryo is challenged by a diffusible extracellular milieu, and by rapid nuclear divisions that occur at the same time. To understand how a sharp gradient is formed within this dynamic environment, we followed the generation of graded nuclear Dorsal protein, the hallmark of pattern formation along the dorso-ventral axis, in live embryos. The dynamics indicate that a sharp extracellular gradient is formed through diffusion-based shuttling of the Spaetzle (Spz) morphogen that progresses through several nuclear divisions. Perturbed shuttling in wntD mutant embryos results in a flat activation peak and aberrant gastrulation. Re-entry of Dorsal into the nuclei at the final division cycle plays an instructive role, as the residence time of Dorsal in each nucleus is translated to the amount of zygotic transcript that will be produced, thereby guiding graded accumulation of specific zygotic transcripts that drive patterned gastrulation. We conclude that diffusion-based ligand shuttling, coupled with dynamic readout, establishes a refined pattern within the diffusible environment of early embryos.

Buffering variability in morphogen distribution is essential for reproducible patterning. A theoretically proposed class of mechanisms, termed “distal pinning,” achieves robustness by combining local sensing of morphogen levels with global modulation of gradient spread. Here, we demonstrate a critical role for morphogen sensing by a gene enhancer, which ultimately determines the final global distribution of the morphogen and enables reproducible patterning. Specifically, we show that, while the pattern of Toll activation in the early Drosophila embryo is robust to gene dosage of its locally produced regulator, WntD, it is sensitive to a single-nucleotide change in the wntD enhancer. Thus, enhancer properties of locally produced WntD directly impinge on the global morphogen profile.

13Establishment of morphogen gradients in the early Drosophila embryo is challenged by a 14 diffusible extracellular milieu, and rapid nuclear divisions that occur at the same time. To 15 understand how a sharp gradient is formed within this dynamic environment, we followed the 16 generation of graded nuclear Dorsal (Dl) protein, the hallmark of pattern formation along the 17 dorso-ventral axis, in live embryos. We show that a sharp gradient is formed through 18 extracellular, diffusion-based morphogen shuttling that progresses through several nuclear 19 divisions. Perturbed shuttling in wntD mutant embryos results in a flat activation peak and 20 aberrant gastrulation. Re-entry of Dl into the nuclei at each cycle refines the signaling output, 21 by guiding graded accumulation of the T48 transcript that drives patterned gastrulation. We 22 conclude that diffusion-based ligand shuttling, coupled with dynamic readout, establishes a 23 refined pattern within the diffusible environment of early embryos. 24 25 the BMP gradient in the Xenopus embryo, where it acquired additional features that allow 54 scaling of the gradient with embryo size (Ben-Zvi et al., 2014;Ben-Zvi et al., 2008). 55Compelling evidence for shuttling was provided by comparing mutant phenotypes with the 56 predictions made by computational models (Ben-Zvi et al., 2008;Eldar et al., 2002; Haskel-57 Ittah et al., 2012). It was also demonstrated that ligand produced ectopically in one part of the 58 embryo can be translocated to and endocytosed in the normal activation domain (Reversade 59 and De Robertis, 2005;Wang and Ferguson, 2005). Experimentally, these data were obtained 60 through the analysis of fixed embryos. Yet, the essence of the shuttling mechanism resides in 61 its dynamics. What is the time-frame during which the gradient is established? How fast is 62 gradient formation relative to its readout? Is the gradient stably formed, or is it subject to 63 subsequent cycles of refinements? Insight into these questions requires monitoring the dynamic 64 distribution of the morphogen within single embryos. 65 Furthermore, the shuttling mechanism makes a number of counter-intuitive predictions 66 regarding the dynamics of pattern formation. In particular, it predicts that signaling at the edge 67 of the source will initially increase, as ligand begins to accumulate, but will subsequently be 68 reduced, since ligand is continuously being shuttled to the center of the field. This non-69 monotonic behavior is a defining property of the shuttling mechanism that concentrates ligand, 70 but is absent from other diffusion-based mechanisms establishing a graded pattern. In a certain 71 parameter range, shuttling also predicts transient formation of a double-peak pattern within the 72 gradient, again a prediction that is absent from naïve gradient-forming mechanisms. 73 Uncovering such features is again possible only by monitoring the dynamics of gradient 74 formation in live embryos. 75The ability to observe the dynamics of morphogen gradient formatio...