In many species, leaves are initiated at the flanks of shoot meristems. Usually, subsequent growth mainly occurs in the plane of the leaf blade, which leads to the formation of a bifacial leaf with dorso-ventral identities. In a classical set of surgical experiments in potato meristems, Sussex provided evidence that dorsoventrality depends on a signal emanating from the meristem centre. Although these results could be reproduced in tomato, this concept has been debated. We revisited these experiments in Arabidopsis where a range of markers are available to target the precise site of ablation. Using specific markers for organ founder cells and dorsoventral identity, we were unable to perturb the polarity of leaves and sepals long before organ outgrowth. While results in Solanaceae suggested that dorsoventral patterning was unstable during early development, we find that in Arabidopsis the local information contained within and around the primordium is able to withstand major invasive perturbations, long before polarity is fully established.

ABSTRACT Heterochromatin-related epigenetic mechanisms, such as DNA methylation, facilitate pairing of homologous chromosomes during the meiotic prophase of mammalian spermatogenesis. In pro-spermatogonia, de novo DNA methylation plays a key role in completing meiotic prophase and initiating meiotic division. However, the role of maintenance DNA methylation in the regulation of meiosis, especially in the adult, is not well understood. Here, we reveal that NP95 (also known as UHRF1) and DNMT1 – two essential proteins for maintenance DNA methylation – are co-expressed in spermatogonia and are necessary for meiosis in male germ cells. We find that Np95 - or Dnmt1 -deficient spermatocytes exhibit spermatogenic defects characterized by synaptic failure during meiotic prophase. In addition, assembly of pericentric heterochromatin clusters in early meiotic prophase, a phenomenon that is required for subsequent pairing of homologous chromosomes, is disrupted in both mutants . Based on these observations, we propose that DNA methylation, established in pre-meiotic spermatogonia, regulates synapsis of homologous chromosomes and, in turn, quality control of male germ cells. Maintenance DNA methylation, therefore, plays a role in ensuring faithful transmission of both genetic and epigenetic information to offspring.

ABSTRACT FGF signaling is involved in mesoderm induction in members of deuterostomes (e.g. tunicates, hemichordates), but not in flies and nematodes, in which it has a role in mesoderm patterning and migration. However, we need comparable studies in other protostome taxa in order to decipher whether this mesoderm-inducing function of FGF extends beyond the lineage of deuterostomes. Here, we investigated the role of FGF signaling in mesoderm development in three species of lophophorates, a clade within the protostome group Spiralia. Our gene expression analyses show that the mesodermal molecular patterning is conserved between brachiopods and phoronids, but the spatial and temporal recruitment of transcription factors differs significantly. Moreover, the use of the inhibitor SU5402 demonstrates that FGF signaling is involved in different steps of mesoderm development, as well as in morphogenetic movements of gastrulation and axial elongation. Our findings suggest that the mesoderm-inducing role of FGF extends beyond the group of deuterostomes.

ABSTRACT Movement of epithelial cells in a tissue occurs through neighbor exchange and drives tissue shape changes. It requires intercellular junction remodeling, a process typically powered by the contractile actomyosin cytoskeleton. This has been investigated mainly in homogeneous epithelia, where intercalation takes minutes. However, in some tissues, intercalation involves different cell types and can take hours. Whether slow and fast intercalation share the same mechanisms remains to be examined. To address this issue, we used the fly eye, where the cone cells exchange neighbors over ∼10 h to shape the lens. We uncovered three pathways regulating this slow mode of cell intercalation. First, we found a limited requirement for MyosinII. In this case, mathematical modeling predicts an adhesion-dominant intercalation mechanism. Genetic experiments support this prediction, revealing a role for adhesion through the Nephrin proteins Roughest and Hibris. Second, we found that cone cell intercalation is regulated by the Notch pathway. Third, we show that endocytosis is required for membrane removal and Notch activation. Taken together, our work indicates that adhesion, endocytosis and Notch can direct slow cell intercalation during tissue morphogenesis.

ABSTRACT The focal adhesion protein Kindlin2 is essential for integrin activation, a process that is fundamental to cell-extracellular matrix adhesion. Kindlin 2 ( Fermt2 ) is widely expressed in mouse embryos, and its absence causes lethality at the peri-implantation stage due to the failure to trigger integrin activation. The function of kindlin2 during embryogenesis has not yet been fully elucidated as a result of this early embryonic lethality. Here, we showed that kindlin2 is essential for neural crest (NC) formation in Xenopus embryos. Loss-of-function assays performed with kindlin2- specific morpholino antisense oligos (MOs) or with CRISPR/Cas9 techniques in Xenopus embryos severely inhibit the specification of the NC. Moreover, integrin-binding-deficient mutants of Kindlin2 rescued the phenotype caused by loss of kindlin2 , suggesting that the function of kindlin2 during NC specification is independent of integrins. Mechanistically, we found that Kindlin2 regulates the fibroblast growth factor (FGF) pathway, and promotes the stability of FGF receptor 1. Our study reveals a novel function of Kindlin2 in regulating the FGF signaling pathway and provides mechanistic insights into the function of Kindlin2 during NC specification.

ABSTRACT Genetic screens are designed to target individual genes for the practical reason of establishing a clear association between a mutant phenotype and a single genetic locus. This allows for a developmental or physiological role to be assigned to the wild-type gene. We previously observed that the concurrent loss of Pax6 and Polycomb epigenetic repressors in Drosophila leads the eye to transform into a wing. This fate change is not seen when either factor is disrupted separately. An implication of this finding is that standard screens may miss the roles that combinations of genes play in development. Here, we show that this phenomenon is not limited to Pax6 and Polycomb but rather applies more generally. We demonstrate that in the Drosophila eye-antennal disc, the simultaneous downregulation of Pax6 with either the NURF nucleosome remodeling complex or the Pointed transcription factor transforms the head epidermis into an antenna. This is a previously unidentified fate change that is also not observed with the loss of individual genes. We propose that the use of multi-gene knockdowns is an essential tool for unraveling the complexity of development.

During development, gene expression regulates cell mechanics and shape to sculpt tissues. Epithelial folding proceeds through distinct cell shape changes that occur simultaneously in different regions of a tissue. Here, using quantitative imaging in Drosophila melanogaster , we investigate how patterned cell shape changes promote tissue bending during early embryogenesis. We find that the transcription factors Twist and Snail combinatorially regulate a multicellular pattern of lateral F-actin density that differs from the previously described myosin-2 gradient. This F-actin pattern correlates with whether cells apically constrict, stretch, or maintain their shape. We show that the myosin-2 gradient and F-actin depletion do not depend on force transmission, suggesting that transcriptional activity is required to create these patterns. The myosin-2 gradient width results from a gradient in RhoA activation that is refined through the balance between RhoGEF2 and the RhoGAP C-GAP. Our experimental results and simulations of a 3D elastic shell model show that tuning gradient width regulates tissue curvature.

Absence of a specialized wound epidermis is hypothesized to block limb regeneration in higher vertebrates. However, the factors preventing its formation in regeneration-incompetent animals are poorly understood. To characterize the endogenous molecular and cellular regulators of specialized wound epidermis formation in Xenopus laevis tadpoles, and the loss of their regeneration-competency during development, we used single-cell transcriptomics and ex vivo regenerating limb cultures. Transcriptomic analysis revealed that the specialized wound epidermis is not a novel cell state, but a re-deployment of the apical-ectodermal-ridge (AER) program underlying limb development. Enrichment of secreted inhibitory factors, including Noggin , a morphogen expressed in developing cartilage/bone progenitor cells, are identified as key inhibitors of AER cell formation in regeneration-incompetent tadpoles. These factors can be overridden by Fgf10 , which operates upstream of Noggin and blocks chondrogenesis. These results indicate that manipulation of the extracellular environment and/or chondrogenesis may provide a strategy to restore regeneration potential in higher vertebrates.

ABSTRACT During heart development, epicardial cells residing within the outer layer undergo epithelial-mesenchymal transition (EMT) and migrate into the underlying myocardium to support organ growth and morphogenesis. Disruption of epicardial EMT results in embryonic lethality, yet its regulation is poorly understood. Here, we report epicardial EMT within the mesothelial layer of the mouse embryonic heart at ultra-high resolution using scanning electron microscopy combined with immunofluorescence analyses. We identified morphologically active EMT regions that associated with key components of the extracellular matrix, including the basement membrane-associated proteoglycan agrin. Deletion of agrin resulted in impaired EMT and compromised development of the epicardium, accompanied by downregulation of Wilms’ tumor 1. Agrin enhanced EMT in human embryonic stem cell-derived epicardial-like cells by decreasing β-catenin and promoting pFAK localization at focal adhesions, and promoted the aggregation of dystroglycan within the Golgi apparatus in murine epicardial cells. Loss of agrin resulted in dispersal of dystroglycan in vivo , disrupting basement membrane integrity and impairing EMT. Our results provide new insights into the role of the extracellular matrix in heart development and implicate agrin as a crucial regulator of epicardial EMT.

ABSTRACT Optimal embryonic development plays a major role in the health of an individual beyond the developmental stage. Nutritional perturbation during development is associated with cardiovascular and metabolic disease later in life. With both nutritional uptake and overall growth being risk factors for eventual health, it is necessary to understand not only the behavior of the processes during development but also their interactions. In this study, we used differential equations, image analyses, curve fittings, parameter estimation and laboratory experiments to quantify the rate of yolk absorption and its effect on early development of a vertebrate model ( Danio rerio ). Findings from this study establish a nonlinear functional relationship between nutrient absorption and early fish growth. We found that the rate of change in fish length and yolk utilization is logistic, that is the yolk decays rapidly for a period of time before leveling out. An interesting finding from this study is that yolk utilization reaches its maximum at 84 h post-fertilization. We validated our mathematical models against experimental observations, making them powerful tools for replication and future simulations.

ABSTRACT Angiogenesis in the developing mammalian retina requires patterning cues from astrocytes. Developmental disorders of retinal vasculature, such as retinopathy of prematurity (ROP), involve arrest or mispatterning of angiogenesis. Whether these vascular pathologies involve astrocyte dysfunction remains untested. Here, we demonstrate that the major risk factor for ROP – transient neonatal exposure to excess oxygen – disrupts formation of the angiogenic astrocyte template. Exposing newborn mice to elevated oxygen (75%) suppressed astrocyte proliferation, whereas return to room air (21% oxygen) at postnatal day 4 triggered extensive proliferation, massively increasing astrocyte numbers and disturbing their spatial patterning prior to the arrival of developing vasculature. Proliferation required astrocytic HIF2α and was also stimulated by direct hypoxia (10% oxygen), suggesting that astrocyte oxygen sensing regulates the number of astrocytes produced during development. Along with astrocyte defects, return to room air also caused vascular defects reminiscent of ROP. Strikingly, these vascular phenotypes were more severe in animals that had larger numbers of excess astrocytes. Together, our findings suggest that fluctuations in environmental oxygen dysregulate molecular pathways controlling astrocyte proliferation, thereby generating excess astrocytes that interfere with retinal angiogenesis.

ABSTRACT In the face, symmetry is established when bilateral streams of neural crest cells leave the neural tube at the same time, follow identical migration routes and then give rise to the facial prominences. However, developmental instability exists, particularly surrounding the steps of lip fusion. The causes of instability are unknown but inability to cope with developmental fluctuations are a likely cause of congenital malformations, such as non-syndromic orofacial clefts. Here, we tracked cell movements over time in the frontonasal mass, which forms the facial midline and participates in lip fusion, using live-cell imaging of chick embryos. Our mathematical examination of cell velocity vectors uncovered temporal fluctuations in several parameters, including order/disorder, symmetry/asymmetry and divergence/convergence. We found that treatment with a Rho GTPase inhibitor completely disrupted the temporal fluctuations in all measures and blocked morphogenesis. Thus, we discovered that genetic control of symmetry extends to mesenchymal cell movements and that these movements are of the type that could be perturbed in asymmetrical malformations, such as non-syndromic cleft lip. This article has an associated ‘The people behind the papers’ interview .

Cardiac Purkinje cells (PCs) comprise the most distal portion of the ventricular conduction system (VCS) and are essential for synchronous activation of the ventricular myocardium. Contactin-2 (CNTN2), a member of the immunoglobulin superfamily cell adhesion molecules (IgSF-CAMs), was previously identified as a marker of the VCS. Through differential transcriptional profiling we discovered two additional highly enriched IgSF-CAMs in the VCS, NCAM-1 and ALCAM. Immunofluorescence staining showed dynamic expression patterns for each IgSF-CAM during embryonic and early post-natal stages, but ultimately all three proteins became highly enriched in mature PCs. Mice deficient in NCAM-1, but not CNTN2 or ALCAM, exhibited defects in Purkinje cell gene expression and VCS patterning, as well as cardiac conduction disease. Moreover, using ST8sia2 and ST8sia4 knockout mice, we show that inhibition of post-translational modification of NCAM-1 by polysialic acid (PSA) disrupts trafficking of sarcolemmal intercalated disc proteins to Purkinje cell junctional membranes and abnormal expansion of the extracellular space between apposing Purkinje cells. Taken together, our data provide novel insights into the complex developmental biology of the ventricular conduction system.

ABSTRACT During lung development, epithelial branches expand preferentially in a longitudinal direction. This bias in outgrowth has been linked to a bias in cell shape and in the cell division plane. How this bias arises is unknown. Here, we show that biased epithelial outgrowth occurs independent of the surrounding mesenchyme, of preferential turnover of the extracellular matrix at the bud tips and of FGF signalling. There is also no evidence for actin-rich filopodia at the bud tips. Rather, we find epithelial tubes to be collapsed during early lung and kidney development, and we observe fluid flow in the narrow tubes. By simulating the measured fluid flow inside segmented narrow epithelial tubes, we show that the shear stress levels on the apical surface are sufficient to explain the reported bias in cell shape and outgrowth. We use a cell-based vertex model to confirm that apical shear forces, unlike constricting forces, can give rise to both the observed bias in cell shapes and tube elongation. We conclude that shear stress may be a more general driver of biased tube elongation beyond its established role in angiogenesis. This article has an associated ‘The people behind the papers’ interview.

Zic-r.a, a maternal transcription factor, specifies posterior fate in ascidian embryos. However, its direct target, Tbx6-r.b , does not contain typical Zic-r.a binding sites in its regulatory region. Using an in vitro selection assay, we found that Zic-r.a binds to sites dissimilar from the canonical motif, by which it activates Tbx6-r.b in a sub-lineage of muscle cells. These sites with non-canonical motifs have weak affinity for Zic-r.a; therefore, it activates Tbx6-r.b only in cells expressing Zic-r.a abundantly. Meanwhile, we found that Zic-r.a expressed zygotically in late embryos activates neural genes through canonical sites. Because different zinc-finger domains of Zic-r.a are important for driving reporters with canonical and non-canonical sites, it is likely that the non-canonical motif is not a divergent version of the canonical motif. In other words, our data indicate that the non-canonical motif represents a motif distinct from the canonical motif. Thus, Zic-r.a recognizes two distinct motifs to activate two sets of genes at two timepoints in development.

ABSTRACT The vertebrate retina is generated by retinal progenitor cells (RPCs), which produce >100 cell types. Although some RPCs produce many cell types, other RPCs produce restricted types of daughter cells, such as a cone photoreceptor and a horizontal cell (HC). We used genome-wide assays of chromatin structure to compare the profiles of a restricted cone/HC RPC and those of other RPCs in chicks. These data nominated regions of regulatory activity, which were tested in tissue, leading to the identification of many cis -regulatory modules (CRMs) active in cone/HC RPCs and developing cones. Two transcription factors, Otx2 and Oc1, were found to bind to many of these CRMs, including those near genes important for cone development and function, and their binding sites were required for activity. We also found that Otx2 has a predicted autoregulatory CRM. These results suggest that Otx2, Oc1 and possibly other Onecut proteins have a broad role in coordinating cone development and function. The many newly discovered CRMs for cones are potentially useful reagents for gene therapy of cone diseases.

ABSTRACT Ret signaling promotes branching morphogenesis during kidney development, but the underlying cellular mechanisms remain unclear. While Ret -expressing progenitor cells proliferate at the ureteric bud tips, some of these cells exit the tips to generate the elongating collecting ducts, and in the process turn off Ret . Genetic ablation of Ret in tip cells promotes their exit, suggesting that Ret is required for cell rearrangements that maintain the tip compartments. Here, we examine the behaviors of ureteric bud cells that are genetically forced to maintain Ret expression. These cells move to the nascent tips, and remain there during many cycles of branching; this tip-seeking behavior may require positional signals from the mesenchyme, as it occurs in whole kidneys but not in epithelial ureteric bud organoids. In organoids, cells forced to express Ret display a striking self-organizing behavior, attracting each other to form dense clusters within the epithelium, which then evaginate to form new buds. The ability of forced Ret expression to promote these events suggests that similar Ret -dependent cell behaviors play an important role in normal branching morphogenesis.

ABSTRACT A key step in the activation of canonical Wnt signaling is the interaction between β-catenin and Tcf/Lefs that forms the transcription activation complex and facilitates the expression of target genes. Eukaryotic initiation factor 4A3 (EIF4A3) is an ATP-dependent DEAD box-family RNA helicase and acts as a core subunit of the exon junction complex (EJC) to control a series of RNA post-transcriptional processes. In this study, we uncover that EIF4A3 functions as a Wnt inhibitor by interfering with the formation of β-catenin/Tcf transcription activation complex. As Wnt stimulation increases, accumulated β-catenin displaces EIF4A3 from a transcriptional complex with Tcf/Lef, allowing the active complex to facilitate the expression of target genes. In zebrafish embryos, eif4a3 depletion inhibited the development of the dorsal organizer and pattern formation of the anterior neuroectoderm by increasing Wnt/β-catenin signaling. Conversely, overexpression of eif4a3 decreased Wnt/β-catenin signaling and inhibited the formation of the dorsal organizer before gastrulation. Our results reveal previously unreported roles of EIF4A3 in the inhibition of Wnt signaling and the regulation of embryonic development in zebrafish.

ABSTRACT In mammalian ovaries, immature oocytes are reserved in primordial follicles until their activation for potential ovulation. Precise control of primordial follicle activation (PFA) is essential for reproduction, but how this is achieved is unclear. Here, we show that canonical wingless-type MMTV integration site family (WNT) signaling is pivotal for pre-granulosa cell (pre-GC) activation during PFA. We identified several WNT ligands expressed in pre-GCs that act in an autocrine manner. Inhibition of WNT secretion from pre-GCs/GCs by conditional knockout (cKO) of the wntless ( Wls ) gene led to female infertility. In Wls cKO mice, GC layer thickness was greatly reduced in growing follicles, which resulted in impaired oocyte growth with both an abnormal, sustained nuclear localization of forkhead box O3 (FOXO3) and reduced phosphorylation of ribosomal protein S6 (RPS6). Constitutive stabilization of β-catenin (CTNNB1) in pre-GCs/GCs induced morphological changes of pre-GCs from a squamous into a cuboidal form, though it did not influence oocyte activation. Our results reveal that canonical WNT signaling plays a permissive role in the transition of pre-GCs to GCs, which is an essential step to support oocyte growth.

ABSTRACT Signaling centers, or organizers, regulate many aspects of embryonic morphogenesis. In the mammalian molar tooth, reiterative signaling in specialized centers called enamel knots (EKs) determines tooth patterning. Preceding the primary EK, transient epithelial thickening appears, the significance of which remains debated. Using tissue confocal fluorescence imaging with laser ablation experiments, we show that this transient thickening is an earlier signaling center, the molar initiation knot (IK), that is required for the progression of tooth development. IK cell dynamics demonstrate the hallmarks of a signaling center: cell cycle exit, condensation and eventual silencing through apoptosis. IK initiation and maturation are defined by the juxtaposition of cells with high Wnt activity to Shh -expressing non-proliferating cells, the combination of which drives the growth of the tooth bud, leading to the formation of the primary EK as an independent cell cluster. Overall, the whole development of the tooth, from initiation to patterning, is driven by the iterative use of signaling centers.