Summary Non-coding mutations at the far end of a large gene desert surrounding the SOX9 gene result in a human craniofacial disorder called Pierre Robin sequence (PRS). Leveraging a human stem cell differentiation model, we identify two clusters of enhancers within the PRS-associated region that regulate SOX9 expression during a restricted window of facial progenitor development at distances up to 1.45 Mb. Enhancers within the 1.45 Mb cluster exhibit highly synergistic activity that is dependent on the Coordinator motif. Using mouse models, we demonstrate that PRS phenotypic specificity arises from the convergence of two mechanisms: confinement of Sox9 dosage perturbation to developing facial structures through context-specific enhancer activity and heightened sensitivity of the lower jaw to Sox9 expression reduction. Overall, we characterize the longest-range human enhancers involved in congenital malformations, directly demonstrate that PRS is an enhanceropathy, and illustrate how small changes in gene expression can lead to morphological variation.

SUMMARY Anatomical proportions are robustly maintained in individuals that vary enormously in size, both within a species and between members of related taxa. However, the mechanisms underlying scaling are still poorly understood. We have examined this phenomenon in the context of the patterning of the ventral neural tube in response to a gradient of the morphogen Sonic hedgehog (SHH) in the chick and zebra finch, two species that differ in size during the time of neural tube patterning. We find that scaling is achieved, at least in part, by altering the sensitivity of the target cells to SHH and appears to be achieved by modulating the ratio of the repressive and activating transcriptional regulators, GLI2 and GLI3. This mechanism contrasts with previous experimental and theoretical analyses of morphogenic scaling that have focused on compensatory changes in the morphogen gradient itself.

The mechanisms used by embryos to pattern tissues across their axes has fascinated developmental biologists since the founding of embryology. Here, using single-cell technology, we interrogate complex patterning defects and define a Hedgehog (Hh)–fibroblast growth factor (FGF) signaling axis required for anterior mesoderm lineage development during gastrulation. Single-cell transcriptome analysis of Hh-deficient mesoderm revealed selective deficits in anterior mesoderm populations, culminating in defects to anterior embryonic structures, including the pharyngeal arches, heart, and anterior somites. Transcriptional profiling of Hh-deficient mesoderm during gastrulation revealed disruptions to both transcriptional patterning of the mesoderm and FGF signaling for mesoderm migration. Mesoderm-specificFgf4/Fgf8double-mutants recapitulated anterior mesoderm defects and Hh-dependent GLI transcription factors modulated enhancers at FGF gene loci. Cellular migration defects during gastrulation induced by Hh pathway antagonism were mitigated by the addition of FGF4 protein. These findings implicate a multicomponent signaling hierarchy activated by Hh ligands from the embryonic node and executed by FGF signals in nascent mesoderm to control anterior mesoderm patterning.

The application of single cell technologies to early development holds promise for resolving complex developmental phenotypes. Here we define a novel role for Hedgehog (Hh) signaling for the formation of anterior mesoderm lineages during gastrulation. Single-cell transcriptome analysis of Hh-deficient mesoderm revealed selective deficits in anterior mesoderm populations that later translate to physical defects to anterior embryonic structures including the first pharyngeal arch, heart, and anterior somites. We found that Hh-dependent anterior mesoderm defects were cell non-autonomous to Hh-signal reception.Transcriptional profiling of Hh-deficient mesoderm during gastrulation revealed disruptions to both transcriptional patterning of the mesoderm and a key FGF signaling pathway for mesoderm migration. FGF4 protein application was able to restore cellular migration during gastrulation that was decreased by Hh pathway antagonism. These findings implicate that primitive streak-mediated regulation of anterior mesoderm patterning is controlled by a multicomponent signaling hierarchy activated by Hh signaling and executed by FGF signal transduction.

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