Generation of left-right asymmetry is an integral partGeneration of left-right asymmetry during development is an integral part of the establishment of the vertebrate body plan (Capdevila et al. 2000;Mercola and Levin 2001;Wright 2001;Yost 2001;Hamada et al. 2002). Specification of the left-right axis requires multiple steps: (1) generation of an initial asymmetric signal in or near the embryonic node, (2) transfer of asymmetric signals from the node to the lateral plate mesoderm (LPM), (3) induction of an evolutionarily conserved cascade of gene expression in the left LPM, and (4) transformation of these left-right asymmetric signals into morphological asymmetries of the visceral organs. In mice, generation of the initial asymmetric signal requires directional fluid flow on the ventral surface of the node (Nonaka et al. 2002). This fluid flow is generated by motile monocilia on cells of the node, and the presence of nodal cilia is conserved in other vertebrates (Essner et al. 2002). However, the mechanism by which directional fluid flow at the node specifies orientation of the left-right axis is controversial (Stern and Wolpert 2002;Tabin and Vogan 2003). In addition, the mechanism for transfer of the initial asymmetric signal from the node to the LPM is unknown.The Notch signaling pathway is an evolutionarily conserved intercellular signaling mechanism. Mutations in Notch pathway components disrupt embryonic development in diverse multicellular organisms and cause in- Here we demonstrate that the Notch signaling pathway plays a primary role in the establishment of leftright asymmetry in mice by directly regulating expression of the Nodal gene. Embryos mutant for the Notch ligand Dll1 or doubly mutant for the Notch1 and Notch2 receptors exhibit multiple defects in left-right asymmetry. Notably, Dll1 −/− embryos do not express Nodal in the region around the node. Analysis of the enhancer regulating node-specific Nodal expression (termed the NDE) revealed the presence of binding sites for the RBP-J protein. Mutation of these sites destroyed the ability of the NDE to direct node-specific gene expression in transgenic mice. These results demonstrate that Dll1-mediated Notch signaling is essential for generation of leftright asymmetry, and indicate that perinodal expression of the Nodal gene is an essential component of left-right asymmetry determination in mice. Results and Discussion Laterality defects in Dll1 mutant and Notch1/Notch2 double-mutant mouse embryosDuring studies on the role of the Dll1 gene during somitogenesis (Zhang et al. 2002), we observed that some Dll1 −/− embryos (Hrabé de Angelis et al. 1997) exhibited reversed heart looping. We examined this phenotype more closely by performing scanning electron micros-

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.

Evolution of facial morphology arises from variation in the activity of developmental regulatory networks that guide the formation of specific craniofacial elements. Importantly, the acquisition of novel morphology must be integrated with a phylogenetically inherited developmental program. We have identified a unique region of the secondary palate associated with the periodic formation of rugae during the rostral outgrowth of the face. Rugae function as SHH signaling centers to pattern the elongating palatal shelves. We have found that a network of signaling genes and transcription factors is spatially organized relative to palatal rugae. Additionally, the first formed ruga is strategically positioned at the presumptive junction of the future hard and soft palate that defines anterior-posterior differences in regional growth, mesenchymal gene expression and cell fate. We propose a molecular circuit integrating FGF and BMP signaling to control proliferation and differentiation during the sequential formation of rugae and inter-rugae domains in the palatal epithelium. The loss of p63 and Sostdc1 expression and failed rugae differentiation highlight that coordinated epithelial mesenchymal signaling is lost in the Fgf10 mutant palate. Our results establish a genetic program that reiteratively organizes signaling domains to coordinate the growth of the secondary palate with the elongating midfacial complex.

Mammalian embryogenesis is characterized by rapid cellular proliferation and diversification. Within a few weeks, a single-cell zygote gives rise to millions of cells expressing a panoply of molecular programs. Although intensively studied, a comprehensive delineation of the major cellular trajectories that comprise mammalian development in vivo remains elusive. Here, we set out to integrate several single-cell RNA-sequencing (scRNA-seq) datasets that collectively span mouse gastrulation and organogenesis, supplemented with new profiling of ~150,000 nuclei from approximately embryonic day 8.5 (E8.5) embryos staged in one-somite increments. Overall, we define cell states at each of 19 successive stages spanning E3.5 to E13.5 and heuristically connect them to their pseudoancestors and pseudodescendants. Although constructed through automated procedures, the resulting directed acyclic graph (TOME (trajectories of mammalian embryogenesis)) is largely consistent with our contemporary understanding of mammalian development. We leverage TOME to systematically nominate transcription factors (TFs) as candidate regulators of each cell type’s specification, as well as ‘cell-type homologs’ across vertebrate evolution.