Cellular transitions during cranial suture establishment in zebrafish.

Cranial sutures separate neighboring skull bones and are sites of bone growth. A key question is how osteogenic activity is controlled to promote bone growth while preventing aberrant bone fusions during skull expansion. Using single-cell transcriptomics, lineage tracing, and mutant analysis in zebrafish, we uncover key developmental transitions regulating bone formation at sutures during skull expansion. In particular, we identify a subpopulation of mesenchyme cells in the mid-suture region that upregulate a suite of genes including BMP antagonists (e.g. grem1a) and pro-angiogenic factors. Lineage tracing with grem1a:nlsEOS reveals that this mid-suture subpopulation is largely non-osteogenic. Moreover, combinatorial mutation of BMP antagonists enriched in this mid-suture subpopulation results in increased BMP signaling in the suture, misregulated bone formation, and abnormal suture morphology. These data reveal establishment of a non-osteogenic mesenchyme population in the mid-suture region that restricts bone formation through local BMP antagonism, thus ensuring proper suture morphology.

Modeling the roles of cohesotaxis, cell-intercalation, and tissue geometry in collective cell migration of Xenopus mesendoderm.

Collectively migrating Xenopus mesendoderm cells are arranged into leader and follower rows with distinct adhesive properties and protrusive behaviors. In vivo, leading row mesendoderm cells extend polarized protrusions and migrate along a fibronectin matrix assembled by blastocoel roof cells. Traction stresses generated at the leading row result in the pulling forward of attached follower row cells. Mesendoderm explants removed from embryos provide an experimentally tractable system for characterizing collective cell movements and behaviors, yet the cellular mechanisms responsible for this mode of migration remain elusive. We introduce a novel agent-based computational model of migrating mesendoderm in the Cellular-Potts computational framework to investigate the respective contributions of multiple parameters specific to the behaviors of leader and follower row cells. Sensitivity analyses identify cohesotaxis, tissue geometry, and cell intercalation as key parameters affecting the migration velocity of collectively migrating cells. The model predicts that cohesotaxis and tissue geometry in combination promote cooperative migration of leader cells resulting in increased migration velocity of the collective. Radial intercalation of cells towards the substrate is an additional mechanism contributing to an increase in migratory speed of the tissue. Model outcomes are validated experimentally using mesendoderm tissue explants.

Redox heterogeneity in mouse embryonic stem cells individualizes cell fate decisions.

Pluripotent embryonic stem cells (ESCs) can develop into any cell type in the body. Yet, the regulatory mechanisms that govern cell fate decisions during embryogenesis remain largely unknown. We now demonstrate that mouse ESCs (mESCs) display large natural variations in mitochondrial reactive oxygen species (mitoROS) levels that individualize their nuclear redox state, H3K4me3 landscape, and cell fate. While mESCs with high mitoROS levels (mitoROSHIGH) differentiate toward mesendoderm and form the primitive streak during gastrulation, mESCs, which generate less ROS, choose the alternative neuroectodermal fate. Temporal studies demonstrated that mesendodermal (ME) specification of mitoROSHIGH mESCs is mediated by a Nrf2-controlled switch in the nuclear redox state, triggered by the accumulation of redox-sensitive H3K4me3 marks, and executed by a hitherto unknown ROS-dependent activation process of the Wnt signaling pathway. In summary, our study explains how ESC heterogeneity is generated and used by individual cells to decide between distinct cellular fates.

Cohesin composition and dosage independently affect early development in zebrafish.

Cohesin, a chromatin-associated protein complex with four core subunits (Smc1a, Smc3, Rad21 and either Stag1 or 2), has a central role in cell proliferation and gene expression in metazoans. Human developmental disorders termed 'cohesinopathies' are characterized by germline variants of cohesin or its regulators that do not entirely eliminate cohesin function. However, it is not clear whether mutations in individual cohesin subunits have independent developmental consequences. Here, we show that zebrafish rad21 or stag2b mutants independently influence embryonic tailbud development. Both mutants have altered mesoderm induction, but only homozygous or heterozygous rad21 mutation affects cell cycle gene expression. stag2b mutants have narrower notochords and reduced Wnt signaling in neuromesodermal progenitors as revealed by single-cell RNA sequencing. Stimulation of Wnt signaling rescues transcription and morphology in stag2b, but not rad21, mutants. Our results suggest that mutations altering the quantity versus composition of cohesin have independent developmental consequences, with implications for the understanding and management of cohesinopathies.

A fibronectin gradient remodels mixed-phase mesoderm.

Physical processes ultimately shape tissue during development. Two emerging proposals are that cells migrate toward stiffer tissue (durotaxis) and that the extent of cell rearrangements reflects tissue phase, but it is unclear whether and how these concepts are related. Here, we identify fibronectin-dependent tissue stiffness as a control variable that underlies and unifies these phenomena in vivo. In murine limb bud mesoderm, cells are either caged, move directionally, or intercalate as a function of their location along a stiffness gradient. A modified Landau phase equation that incorporates tissue stiffness accurately predicts cell diffusivity upon loss or gain of fibronectin. Fibronectin is regulated by WNT5A-YAP feedback that controls cell movements, tissue shape, and skeletal pattern. The results identify a key determinant of phase transition and show how fibronectin-dependent directional cell movement emerges in a mixed-phase environment in vivo.

Ectopic Activation of Fgf8 in Dental Mesenchyme Causes Incisor Agenesis and Molar Microdontia.

Putatively, tooth agenesis was attributed to the initiation failure of tooth germs, though little is known about the histological and molecular alterations. To address if constitutively active FGF signaling is associated with tooth agenesis, we activated Fgf8 in dental mesenchyme with Osr-cre knock-in allele in mice (Osr2-creKI; Rosa26R-Fgf8) and found incisor agenesis and molar microdontia. The cell survival assay showed tremendous apoptosis in both the Osr2-creKI; Rosa26R-Fgf8 incisor epithelium and mesenchyme, which initiated incisor regression from cap stage. In situ hybridization displayed vanished Shh transcription, and immunostaining exhibited reduced Runx2 expression and enlarged mesenchymal Lef1 domain in Osr2-creKI; Rosa26R-Fgf8 incisors, both of which were suggested to enhance apoptosis. In contrast, Osr2-creKI; Rosa26R-Fgf8 molar germs displayed mildly suppressed Shh transcription, and the increased expression of Ectodin, Runx2 and Lef1. Although mildly smaller than WT controls prenatally, the Osr2-creKI; Rosa26R-Fgf8 molar germs produced a miniature tooth with impaired mineralization after a 6-week sub-renal culture. Intriguingly, the implanted Osr2-creKI; Rosa26R-Fgf8 molar germs exhibited delayed odontoblast differentiation and accelerated ameloblast maturation. Collectively, the ectopically activated Fgf8 in dental mesenchyme caused incisor agenesis by triggering incisor regression and postnatal molar microdontia. Our findings reported tooth agenesis resulting from the regression from the early bell stage and implicated a correlation between tooth agenesis and microdontia.

A human pluripotent stem cell-based somitogenesis model using microfluidics.

Emerging human pluripotent stem cell (hPSC)-based embryo models are useful for studying human embryogenesis. Particularly, there are hPSC-based somitogenesis models using free-floating culture that recapitulate somite formation. Somitogenesis in vivo involves intricately orchestrated biochemical and biomechanical events. However, none of the current somitogenesis models controls biochemical gradients or biomechanical signals in the culture, limiting their applicability to untangle complex biochemical-biomechanical interactions that drive somitogenesis. Herein, we develop a human somitogenesis model by confining hPSC-derived presomitic mesoderm (PSM) tissues in microfabricated trenches. Exogenous microfluidic morphogen gradients imposed on the PSM tissues cause axial patterning and trigger spontaneous rostral-to-caudal somite formation. A mechanical theory is developed to explain the size dependency between somites and the PSM. The microfluidic somitogenesis model is further exploited to reveal regulatory roles of cellular and tissue biomechanics in somite formation. This study presents a useful microengineered, hPSC-based model for understanding the biochemical and biomechanical events that guide somite formation.

Diverse Fgfr1 signaling pathways and endocytic trafficking regulate mesoderm development.

The fibroblast growth factor (FGF) pathway is a conserved signaling pathway required for embryonic development. Activated FGF receptor 1 (FGFR1) drives multiple intracellular signaling cascade pathways, including ERK/MAPK and PI3K/AKT, collectively termed canonical signaling. However, unlike Fgfr1-null embryos, embryos containing hypomorphic mutations in Fgfr1 lacking the ability to activate canonical downstream signals are still able to develop to birth but exhibit severe defects in all mesodermal-derived tissues. The introduction of an additional signaling mutation further reduces the activity of Fgfr1, leading to earlier lethality, reduced somitogenesis, and more severe changes in transcriptional outputs. Genes involved in migration, ECM interaction, and phosphoinositol signaling were significantly downregulated, proteomic analysis identified changes in interactions with endocytic pathway components, and cells expressing mutant receptors show changes in endocytic trafficking. Together, we identified processes regulating early mesoderm development by mechanisms involving both canonical and noncanonical Fgfr1 pathways, including direct interaction with cell adhesion components and endocytic regulation.

A Spatio-Temporal-Dependent Requirement of Sonic Hedgehog in the Early Development of Sclerotome-Derived Vertebrae and Ribs.

Derived from axial structures, Sonic Hedgehog (Shh) is secreted into the paraxial mesoderm, where it plays crucial roles in sclerotome induction and myotome differentiation. Through conditional loss-of-function in quail embryos, we investigate the timing and impact of Shh activity during early formation of sclerotome-derived vertebrae and ribs, and of lateral mesoderm-derived sternum. To this end, Hedgehog interacting protein (Hhip) was electroporated at various times between days 2 and 5. While the vertebral body and rib primordium showed consistent size reduction, rib expansion into the somatopleura remained unaffected, and the sternal bud developed normally. Additionally, we compared these effects with those of locally inhibiting BMP activity. Transfection of Noggin in the lateral mesoderm hindered sternal bud formation. Unlike Hhip, BMP inhibition via Noggin or Smad6 induced myogenic differentiation of the lateral dermomyotome lip, while impeding the growth of the myotome/rib complex into the somatic mesoderm, thus affirming the role of the lateral dermomyotome epithelium in rib guidance. Overall, these findings underscore the continuous requirement for opposing gradients of Shh and BMP activity in the morphogenesis of proximal and distal flank skeletal structures, respectively. Future research should address the implications of these early interactions to the later morphogenesis and function of the musculo-skeletal system and of possible associated malformations.

Identification of endothelial and mesenchymal FOXF1 enhancers involved in alveolar capillary dysplasia.

Mutations in the FOXF1 gene, a key transcriptional regulator of pulmonary vascular development, cause Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins, a lethal lung disease affecting newborns and infants. Identification of new FOXF1 upstream regulatory elements is critical to explain why frequent non-coding FOXF1 deletions are linked to the disease. Herein, we use multiome single-nuclei RNA and ATAC sequencing of mouse and human patient lungs to identify four conserved endothelial and mesenchymal FOXF1 enhancers. We demonstrate that endothelial FOXF1 enhancers are autoactivated, whereas mesenchymal FOXF1 enhancers are regulated by EBF1 and GLI1. The cell-specificity of FOXF1 enhancers is validated by disrupting these enhancers in mouse embryonic stem cells using CRISPR/Cpf1 genome editing followed by lineage-tracing of mutant embryonic stem cells in mouse embryos using blastocyst complementation. This study resolves an important clinical question why frequent non-coding FOXF1 deletions that interfere with endothelial and mesenchymal enhancers can lead to the disease.

Differential cellular stiffness across tissues that contribute to Xenopus neural tube closure.

During the formation of the neural tube, the primordium of the vertebrate central nervous system, the actomyosin activity of cells in different regions drives neural plate bending. However, how the stiffness of the neural plate and surrounding tissues is regulated and mechanically influences neural plate bending has not been elucidated. Here, we used atomic force microscopy to reveal the relationship between the stiffness of the neural plate and the mesoderm during Xenopus neural tube formation. Measurements with intact embryos revealed that the stiffness of the neural plate was consistently higher compared with the non-neural ectoderm and that it increased in an actomyosin activity-dependent manner during neural plate bending. Interestingly, measurements of isolated tissue explants also revealed that the relationship between the stiffness of the apical and basal sides of the neural plate was reversed during bending and that the stiffness of the mesoderm was lower than that of the basal side of the neural plate. The experimental elevation of mesoderm stiffness delayed neural plate bending, suggesting that low mesoderm stiffness mechanically supports neural tube closure. This study provides an example of mechanical interactions between tissues during large-scale morphogenetic movements.

On mechanical phase and form.

Epithelial folding is a fundamental biological process that requires epithelial interactions with the underlying mesenchyme. In this issue of Cell, Huycke et al. investigate intestinal villus formation. They discover that water-droplet-like behavior of mesenchymal cells drives their coalescence into uniformly patterned aggregates, which generate forces on the epithelium to initiate folding.

Mapping mouse axial progenitor dynamics in vitro.

In this issue of Developmental Cell, Bolondi et al. systematically assesses neuro-mesodermal progenitor (NMP) dynamics by combining a mouse stem-cell-based embryo model with molecular recording of single cells, shedding light on the dynamics of neural tube and paraxial mesoderm formation during mammalian development.

Differential proliferation regulates multi-tissue morphogenesis during embryonic axial extension: integrating viscous modeling and experimental approaches.

A major challenge in biology is to understand how mechanical interactions and cellular behavior affect the shapes of tissues and embryo morphology. The extension of the neural tube and paraxial mesoderm, which form the spinal cord and musculoskeletal system, respectively, results in the elongated shape of the vertebrate embryonic body. Despite our understanding of how each of these tissues elongates independently of the others, the morphogenetic consequences of their simultaneous growth and mechanical interactions are still unclear. Our study investigates how differential growth, tissue biophysical properties and mechanical interactions affect embryonic morphogenesis during axial extension using a 2D multi-tissue continuum-based mathematical model. Our model captures the dynamics observed in vivo by time-lapse imaging of bird embryos, and reveals the underestimated influence of differential tissue proliferation rates. We confirmed this prediction in quail embryos by showing that decreasing the rate of cell proliferation in the paraxial mesoderm affects long-term tissue dynamics, and shaping of both the paraxial mesoderm and the neighboring neural tube. Overall, our work provides a new theoretical platform upon which to consider the long-term consequences of tissue differential growth and mechanical interactions on morphogenesis.

Salivary gland developmental mechanics.

The salivary gland undergoes branching morphogenesis to elaborate into a tree-like structure with numerous saliva-secreting acinar units, all joined by a hierarchical ductal system. The expansive epithelial surface generated by branching morphogenesis serves as the structural basis for the efficient production and delivery of saliva. Here, we elucidate the process of salivary gland morphogenesis, emphasizing the role of mechanics. Structurally, the developing salivary gland is characterized by a stratified epithelium tightly encased by the basement membrane, which is in turn surrounded by a mesenchyme consisting of a dense network of interstitial matrix and mesenchymal cells. Diverse cell types and extracellular matrices bestow this developing organ with organized, yet spatially varied mechanical properties. For instance, the surface epithelial sheet of the bud is highly fluidic due to its high cell motility and weak cell-cell adhesion, rendering it highly pliable. In contrast, the inner core of the bud is more rigid, characterized by reduced cell motility and strong cell-cell adhesion, which likely provide structural support for the tissue. The interactions between the surface epithelial sheet and the inner core give rise to budding morphogenesis. Furthermore, the basement membrane and the mesenchyme offer mechanical constraints that could play a pivotal role in determining the higher-order architecture of a fully mature salivary gland.

Defective mesenchymal Bmpr1a-mediated BMP signaling causes congenital pulmonary cysts.

Abnormal lung development can cause congenital pulmonary cysts, the mechanisms of which remain largely unknown. Although the cystic lesions are believed to result directly from disrupted airway epithelial cell growth, the extent to which developmental defects in lung mesenchymal cells contribute to abnormal airway epithelial cell growth and subsequent cystic lesions has not been thoroughly examined. In the present study using genetic mouse models, we dissected the roles of bone morphogenetic protein (BMP) receptor 1a (Bmpr1a)-mediated BMP signaling in lung mesenchyme during prenatal lung development and discovered that abrogation of mesenchymal Bmpr1a disrupted normal lung branching morphogenesis, leading to the formation of prenatal pulmonary cystic lesions. Severe deficiency of airway smooth muscle cells and subepithelial elastin fibers were found in the cystic airways of the mesenchymal Bmpr1a knockout lungs. In addition, ectopic mesenchymal expression of BMP ligands and airway epithelial perturbation of the Sox2-Sox9 proximal-distal axis were detected in the mesenchymal Bmpr1a knockout lungs. However, deletion of Smad1/5, two major BMP signaling downstream effectors, from the lung mesenchyme did not phenocopy the cystic abnormalities observed in the mesenchymal Bmpr1a knockout lungs, suggesting that a Smad-independent mechanism contributes to prenatal pulmonary cystic lesions. These findings reveal for the first time the role of mesenchymal BMP signaling in lung development and a potential pathogenic mechanism underlying congenital pulmonary cysts.

Congenital disorders are medical conditions that are present from birth. Although many congenital disorders are rare, they can have a severe impact on the quality of life of those affected. For example, congenital pulmonary airway malformation (CPAM) is a rare congenital disorder that occurs in around 1 out of every 25,000 pregnancies. In CPAM, abnormal, fluid-filled sac-like pockets of tissue, known as cysts, form within the lungs of unborn babies. After birth, these cysts become air-filled and do not behave like normal lung tissue and stop a baby's lungs from working properly. In severe cases, babies with CPAM need surgery immediately after birth. We still do not understand exactly what the underlying causes of CPAM might be. CPAM is not considered to be hereditary ­ that is, it does not appear to be passed down in families ­ nor is it obviously linked to any environmental factors. CPAM is also very difficult to study, because researchers cannot access tissue samples during the critical early stages of the disease. To overcome these difficulties, Luo et al. wanted to find a way to study CPAM in the laboratory. First, they developed a non-human animal 'model' that naturally forms CPAM-like lung cysts, using genetically modified mice where the gene for the signaling molecule Bmpr1a had been deleted in lung cells. Normally, Bmpr1a is part of a set of the molecular instructions, collectively termed BMP signaling, which guide healthy lung development early in life. However, mouse embryos lacking Bmpr1a developed abnormal lung cysts that were similar to those found in CPAM patients, suggesting that problems with BMP signalling might also trigger CPAM in humans. Luo et al. also identified several other genes in the Bmpr1a-deficient mouse lungs that had abnormal patterns of activity. All these genes were known to be controlled by BMP signaling, and to play a role in the development and organisation of lung tissue. This suggests that when these genes are not controlled properly, they could drive formation of CPAM cysts when BMP signaling is compromised. This work is a significant advance in the tools available to study CPAM. Luo et al.'s results also shed new light on the molecular mechanisms underpinning this rare disorder. In the future, Luo et al. hope this knowledge will help us develop better treatments for CPAM, or even help to prevent it altogether.

A single-cell atlas of pig gastrulation as a resource for comparative embryology.

Cell-fate decisions during mammalian gastrulation are poorly understood outside of rodent embryos. The embryonic disc of pig embryos mirrors humans, making them a useful proxy for studying gastrulation. Here we present a single-cell transcriptomic atlas of pig gastrulation, revealing cell-fate emergence dynamics, as well as conserved and divergent gene programs governing early porcine, primate, and murine development. We highlight heterochronicity in extraembryonic cell-types, despite the broad conservation of cell-type-specific transcriptional programs. We apply these findings in combination with functional investigations, to outline conserved spatial, molecular, and temporal events during definitive endoderm specification. We find early FOXA2 + /TBXT- embryonic disc cells directly form definitive endoderm, contrasting later-emerging FOXA2/TBXT+ node/notochord progenitors. Unlike mesoderm, none of these progenitors undergo epithelial-to-mesenchymal transition. Endoderm/Node fate hinges on balanced WNT and hypoblast-derived NODAL, which is extinguished upon endodermal differentiation. These findings emphasise the interplay between temporal and topological signalling in fate determination during gastrulation.

Inflow Tract Development.

The development of the inflow tract is undoubtedly one of the most complex remodeling events in the formation of the four-chambered heart. It involves the creation of two separate atrial chambers, the formation of an atrial/atrioventricular (AV) septal complex, the incorporation of the caval veins and coronary sinus into the right atrium, and the remodeling events that result in pulmonary venous return draining into the left atrium. In these processes, the atrioventricular mesenchymal complex, consisting of the major atrioventricular (AV) cushions, the mesenchymal cap on the primary atrial septum (pAS), and the dorsal mesenchymal protrusion (DMP), plays a crucial role.

Epicardium and Coronary Vessels.

Congenital anomalies and acquired diseases of the coronary blood vessels are of great clinical relevance. The early diagnosis of these conditions remains, however, challenging. In order to improve our knowledge of these ailments, progress has to be achieved in the research of the molecular and cellular mechanisms that control development of the coronary vascular bed. The aim of this chapter is to provide a succint account of the key elements of coronary blood vessel development, especially in the context of the role played by the epicardium and epicardial cellular derivatives. We will discuss the importance of the epicardium in coronary blood vessel morphogenesis, from the contribution of the epicardially derived mesenchyme to these blood vessels to its role as an instructive signaling center, attempting to relate these concepts to the origin of coronary disease.

TBX3 is essential for establishment of the posterior boundary of anterior genes and upregulation of posterior genes together with HAND2 during the onset of limb bud development.

During limb bud formation, axis polarities are established as evidenced by the spatially restricted expression of key regulator genes. In particular, the mutually antagonistic interaction between the GLI3 repressor and HAND2 results in distinct and non-overlapping anterior-distal Gli3 and posterior Hand2 expression domains. This is a hallmark of the establishment of antero-posterior limb axis polarity, together with spatially restricted expression of homeodomain and other transcriptional regulators. Here, we show that TBX3 is required for establishment of the posterior expression boundary of anterior genes in mouse limb buds. ChIP-seq and differential gene expression analysis of wild-type and mutant limb buds identifies TBX3-specific and shared TBX3-HAND2 target genes. High sensitivity fluorescent whole-mount in situ hybridisation shows that the posterior expression boundaries of anterior genes are positioned by TBX3-mediated repression, which excludes anterior genes such as Gli3, Alx4, Hand1 and Irx3/5 from the posterior limb bud mesenchyme. This exclusion delineates the posterior mesenchymal territory competent to establish the Shh-expressing limb bud organiser. In turn, HAND2 is required for Shh activation and cooperates with TBX3 to upregulate shared posterior identity target genes in early limb buds.