Development

Robust tissue growth control requires long-range communication between the rate of progenitor addition and tissue expansion. However, the regulatory mechanisms that couple these two processes are unknown. In zebrafish, notochord morphogenesis is a principal driver of axis extension through the combined actions of posterior progenitor addition and anterior vacuolation. To elucidate how progenitor dynamics and vacuole-driven cell expansion interact to shape notochord development, we first generated a mathematical model that links progenitor addition rate to the progressive expansion of cells from anterior-to-posterior to simulate vacuolation rate. Comparing this with empirical measurements, we find that progenitor incorporation together with vacuolation, produces a linear gradient in nearest neighbour distance. We next explored the role of YAP/TAZ in regulating the rate of progenitor addition in mutants for YAP/TAZ inhibitor vgll4b. We find that vgll4b expression and YAP activity are enriched in posterior midline progenitors. Loss of vgll4b elevates YAP signaling, enhances progenitor addition, restricts vacuole expansion, and--after a transient buffering phase--compromises A-P axis elongation. These results support a long-range feedback mechanism linking progenitor recruitment to vacuolation, enabling the notochord to balance cellular input with volumetric expansion, thereby maintaining tissue proportions. Graphical abstract. Proposed working model O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/706348v2_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@6c3c6dorg.highwire.dtl.DTLVardef@1f34eb0org.highwire.dtl.DTLVardef@b33a21org.highwire.dtl.DTLVardef@ad582b_HPS_FORMAT_FIGEXP M_FIG C_FIG A. Schematic representation of the authors proposed model illustrating how increased YAP activation in notochord progenitors of vgll4b mutants leads to enhanced progenitor addition to the notochord. This increased incorporation subsequently compromises the ability of notochord cells to undergo proper vacuolation, resulting in reduced axial elongation compared with control embryos.

The meninges are a multilayered connective tissue that supports and protects the brain and skull, yet their developmental origins and signaling functions remain poorly understood. Here, we establish zebrafish as a tractable model for defining meningeal development and function across larval and adult stages. Using a restricted foxc1b:Gal4 reporter, we resolve the spatiotemporal emergence of meningeal fibroblasts and demonstrate a conserved, dosage-sensitive requirement for Foxc1 activity during meningeal formation. A targeted pharmacological screen identifies Wnt signaling as essential for timely establishment of the primary meninx. To define adult meningeal diversity, we integrate single-cell multiome profiling with spatial validation and identify multiple transcriptionally distinct populations organized into layered compartments, including pial, arachnoid, dural, and periosteal dura fibroblasts. Finally, inducible larval ablation of meningeal cells reveals limited regenerative capacity following widespread loss and leads to persistent defects in calvarial osteogenesis and brain architecture, including reduced osteoblast differentiation at bone fronts and disrupted tissue organization at sites lacking meningeal recovery. Together, these findings define key features of zebrafish meninges and provide a framework for dissecting meningeal development, regeneration, and meninges-dependent signaling in vivo. Summary StatementThis study uses zebrafish to show how the tissues surrounding the brain develop early and guide proper formation of both the brain and skull.

Pericytes are mural cells that provide support to the endothelium of small blood vessels. Pericyte soma are regularly spaced along vessels, and their processes overlap only slightly. Given that vessel patterning is imprecise, we explore the interplay between vessel growth and pericyte recruitment that leads to even pericyte spacing. After recruitment to the zebrafish brain central arteries (CtAs), pericytes undergo rapid expansion, followed by morphological differentiation. Blocking angiogenesis by reducing Gpr124 (Wnt) or Vegf signaling reduces the length of the vessel network and the number of pericytes, preserving spacing, suggesting proportional recruitment of pericytes to cover the network and the territorial nature of pericytes. However, these initial brain pericytes have low proliferation rates. We demonstrate that additional pericytes are recruited firstly through migration of col5a1- and later col1a2-expressing fibroblasts into the brain. These second-wave pericytes retain some fibroblast properties and show elevated col1a2 levels in a model of pericyte loss (notch3 mutants). Our data provide new insights into the developmental timing, expansion, and novel origins of late-arriving brain pericytes during embryogenesis. SUMMARY STATEMENTThis article demonstrates that brain pericytes originate from multiple sources, including fibroblast-derived populations, and how pericyte numbers are adjusted in proportion to vessel development.

Visualizing protein expression dynamics with high temporal resolution is essential for understanding how cells acquire specific fates and functions during development, where key decisions can occur within minutes. Conventional direct fluorescent tagging often fails to capture these rapid changes in protein expression due to the relatively slow fluorophore maturation time. Indirect epitope-based labeling strategies offer a promising alternative, yet only a limited number of these systems have been developed and used in the context of multicellular organisms. Here, we evaluate and combine four epitope-based indirect labeling systems for live-imaging of proteins in C. elegans: the SunTag, Frankenbody, MoonTag and AlfaTag systems. Each system uses a fluorescently labeled high-affinity single-chain antibody or nanobody to recognize short peptide epitopes fused to a protein of interest, enabling immediate visualization of newly synthesized proteins. We demonstrate that all four systems specifically label epitope-tagged endogenous proteins and show no detectable cross-reactivity when used in dual-color combinations, enabling simultaneous visualization of distinct proteins within the same embryo. In addition, we show that the SunTag system offers three major advantages over direct labeling: earlier detection of proteins, enhanced sensitivity through signal amplification (as illustrated by CAM-1) and less impact on the function (as demonstrated for ERM-1). Together, this expanded toolkit of epitope-based labeling systems offers many new opportunities for visualizing rapid protein dynamics and for dissecting how their dynamics drive cell fate decisions during development. SUMMARYThe development of epitope-labeling systems has improved live-imaging quality of proteins. Unfortunately, limited systems exist for multicellular organisms to study protein expression in the context of development. Here, we expand the epitope-labeling toolbox for C. elegans by combining SunTag or Frankenbody with MoonTag or AlfaTag. Our data indicates that these systems simultaneously visualize different endogenous proteins without cross-reactivity. Moreover, the SunTag system shows advantages over direct labeling: earlier detection, enhanced sensitivity through signal amplification and less impact on protein function. This expanded epitope-labeling toolbox in C. elegans provides opportunities for accurate visualization of different proteins that drive cell fate decisions. O_FIG O_LINKSMALLFIG WIDTH=155 HEIGHT=200 SRC="FIGDIR/small/703904v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@449fe0org.highwire.dtl.DTLVardef@15c68cforg.highwire.dtl.DTLVardef@1e51ff8org.highwire.dtl.DTLVardef@196114d_HPS_FORMAT_FIGEXP M_FIG C_FIG

1The stereotyped internalization of two endodermal precursors during early Caenorhabditis elegans gastrulation enables quantitative dissection of cell ingression mechanics. Experimental work has shown that apical constriction drives Ea and Ep ingression, and several molecular features involved have been identified. Yet, no integrative mechanical analysis has assessed how these elements collectively produce the observed behavior. To address this, we combined biomechanical simulations with a comprehensive dataset of 3D-segmented cell meshes, some with cortical protein distributions, to analyze the mechanics of ingression in its in-vivo context. Our analysis shows the process starts shortly after birth of the ingressing cells. A cortical flow drives the formation of an E-cadherin-rich structure at the apical Ea-Ep interface, which contributes to localizing the buildup of apical tension. Simulations show that medioapical actomyosin contraction can reproduce the observed ingression movements and suggest force transmission to neighboring cells via a friction-based molecular clutch at the apical ring of contact. A series of concurrent cell divisions facilitates ingression, and their stereotyped planar orientation also contributes. Furthermore, we observe an embryo-wide movement of cells during gastrulation. This movement resembles a flow, suggesting that local force generation leads to global rearrangements via internal pressure changes. Finally, at the end of ingression, detailed microscopy shows that neighboring cells actively close the gastrulation cleft by forming a rosette-like configuration and extending actin-rich protrusions. In conclusion, our integrated mechanical description of gastrulation shows that successful ingression is driven by apical constriction and supported by localized friction-based force transmission, coordinated stereotyped cell divisions, and the resulting global tissue flow.

Investigating single-cell dynamics and morphology in tissues and embryos requires highly accurate quantitative analysis of microscopy images. Despite significant advances in the field of bioimage analysis, even the most sophisticated segmentation and tracking algorithms inevitably produce errors (e.g. : over segmentation, missing objects, miss-connected objects). Although error rate may be small, their propagation throughout a time-lapse sequence has catastrophic effects on the accuracy of tracking and extraction of single cell parameters. Extracting single cell temporal information in the context of tissue/embryo requires thus expert curation to identify and correct segmentation errors. In the movies commonly used in developmental biology and stem cell research, both the number of imaged cells and the duration of recording are large, making this manual correction task extremely time-consuming. This has now become a major bottleneck in the fields of development, stem cell biology and bioimage analysis. We present here EpiCure (Epithelial Curation), a versatile tool designed to streamline and accelerate manual curation of segmentation and tracking in 2D movies of large epithelial tissues. EpiCure uses temporal information and morphometric parameters to automatically identify segmentation and tracking errors and provides user-friendly tools to correct them. It focuses on ergonomics and offers several visualization options to help navigating in movies of tissue covering a large number of cells, speeding up the detection of errors and their curation. EpiCure is highly interoperable and supports input from a wide range of segmentation tools. It also includes multiple export filters, enabling seamless integration with downstream analysis pipelines. In this paper, using movies from several animal models, we highlight the importance of curating cell segmentation and tracking for accurate downstream analysis, and demonstrate how EpiCure helps the curation process for extracting accurate single cell dynamics and cellular events detection, making it faster and amenable on large dataset.

Cajal-Retzius cells (CR cells) are the earliest born neurons in the cerebral cortex, and have been implicated in regulating neuronal migration and development of circuitry. A major source of CR cells is the cortical hem, a signaling center at the dorsal telencephalic midline. The hem functions as the hippocampal organizer via canonical WNT signaling and hem progenitors are therefore exposed to high levels of WNT ligands. We tested whether constitutive stabilization of {beta}-Catenin (gain of function, GOF) in the mouse cortical hem progenitors supports CR cell production. We find that although neurons are produced from the hem, they do not acquire molecular features of CR cell identity. The trajectory of differentiation examined using single-cell transcriptomics reveals that immature CR cells normally display a Tbr2+ stage, which is absent upon {beta}-Catenin GOF. These data indicate that CR progenitors in the hem are sensitive to levels of stabilized {beta}-Catenin and that a Tbr2+ stage may be important for the acquisition of CR cell identity.

Adult hydrozoan cnidarians undergo extensive tissue turnover, generating neural cell types including nematocytes (stinging cells) and gland cells from interstitial stem cells (i-cells) expressing stemness proteins such as Piwi and Nanos. The contribution of i-cells during embryogenesis, however, has been unclear. Here we address the origin of neural cells during development of the Clytia hemisphaerica planula larva. Marker gene in situ hybridisation revealed that Piwi/Nanos1-expressing cells within the early gastrula presumptive endoderm generate a substantial pool of nematoblasts, a few of which migrate and differentiate in the planula ectoderm. Some neurogenic and neuronal markers, however, showed a markedly distinct expression profile, developing within a basal layer of the aboral/lateral ectoderm during gastrulation. Embryo bisection and lineage tracing experiments confirmed that sensory neurons and secretory cell types derive from gastrula ectoderm, while nematocytes and at least some ganglionic neurons derive from i-cells. Knockdown and inhibitor treatments revealed steps in neuron and nematocyte development regulated by Wnt-{beta}-catenin. We conclude that two distinct neurogenesis pathways operate during Clytia embryogenesis, one involving aboral ectoderm delamination, and one generating mainly nematocytes from i-cell-like precursors. Summary statementDuring embryogenesis in the hydrozoan Clytia neural cell types derive both from Piwi/Nanos expressing "i-cells" and from ectodermal delamination during gastrulation.

Tubular organs present a common solution to fluid transport in multicellular organisms. They often arise by an initial bulging of flat epithelial progenitor cells, which then undergo branching morphogenesis. Here, we present 3 cooperative programs fully defining the Drosophila airway progenitor field and their roles in early morphogenesis linking the radial pattern of the 2-dimensional (2D) field to the proximo-distally patterning of the 3D tubes. We previously showed that extrinsic Hedgehog (Hh) and intrinsic POU-Homeobox TF Ventral-veinless (Vvl)/Drifter/U-turn dominantly drive the transcriptional program toward the distal airway cell identity at the expense of a proximal program specified by the GATA TF grain (grn). Both programs require the basic-HLH-POU TF trachealess (trh) (Matsuda et. al, 2015). Whereas trh is not essential for primordia invagination, we show that in hh vvl double mutants, the oval-shaped primordia frequently remain at the 2D plane, retaining trh expression in a grn dependent manner. Therefore, hh and vvl are the principal regulators of progenitor invagination independent of trh. Each of the 3 regulators, Trh, Vvl and Grn fulfills only complementary or compensatory functions in transcription and morphogenesis but their combinations functionally define the airway progenitor field. We further provide a comprehensive description for allocating the airway progenitors on the body coordinates, involving dorsal Decapentaplegic/BMP signaling along the dorso-ventral axis and subsequent radial EGFR signaling along the proximo-distal axis. The presence of 3 complementary, regulatory programs in early gene expression and morphogenesis of the simple Drosophila airways may reflect the vital needs for respiration, and their influence on the evolution of various strategies in tubular organ development.

Congenital heart defects frequently arise from alterations in the elongation of the cardiac outflow tract (OFT). Proper elongation of the OFT depends on the coordinated deployment of progenitor cells from the second heart field (SHF) and on dynamic interactions with the extracellular matrix (ECM). Among ECM components, fibronectin (Fn1) and tenascin-C (TnC) have emerged as key regulators of cardiac morphogenesis. Studies in mouse embryos have shown that mesodermal Fn1 is required to maintain proper TnC localization within SHF cells. To study heart development, mammalian models are challenging to use because of their in utero development. This limitation highlights the need for alternative models with external development, where direct observation is possible; however, in these systems, the cellular organization of the SHF and the dynamics of its ECM environment remain poorly characterized Here, we investigated the cellular and extracellular architecture of SHF cells localized to the dorsal pericardial wall (DPW) during heart development in Xenopus laevis. We show that SHF cells undergo a stage-dependent transition from a predominantly monolayered organization at NF35 to a multilayered structure at NF42. This transition is accompanied by dynamic remodeling of the ECM, characterized by increased expression of Fn1, TnC, and Collagen I (ColI) and by redistribution of ECM components within the DPW. Functional experiments revealed that depletion of Fn1 disrupts cardiac morphogenesis, leading to shortening of the OFT and reduced ventricular size. Moreover, loss of Fn1 decreases TnC and ColI levels and alters the spatial organization of TnC within the DPW, indicating that Fn1 is required for proper ECM assembly within the SHF cells. These findings identify Fn1 as a key regulator of ECM assembly within the DPW and highlight how ECM remodeling contributes to the organization of SHF progenitor cells during OFT elongation. Altogether, we demonstrated that Xenopus laevis is a powerful model for studying ECM-driven mechanisms of cardiac morphogenesis.

The blood-brain barrier (BBB) protects the brain from circulating metabolites and plays central roles in neurological diseases. Endothelial cells (ECs) of the BBB are enwrapped by mural cells including pericytes and vascular smooth muscle cells (vSMCs) that regulate angiogenesis, vessel stability and barrier function. To explore mural cell control of the BBB, we investigated neurovascular phenotypes in zebrafish pdgfrb mutants that lack brain pericytes and vSMCs. As expected, mutants showed an altered cerebrovascular network with mispatterned capillaries. Unexpectedly, mutants displayed no BBB leakage at larval stages of development. This demonstrates that pericytes and vSMCs do not control BBB function in developing zebrafish. Instead, we observed juvenile and adult BBB disruption occurring at "hotspot" focal hemorrhages at large vessel aneurysms. ECs at leakage hotspots showed induction of caveolae on abluminal surfaces and structural defects including basement membrane thickening and disruption. Our work suggests that capillary pericytes regulate cerebrovascular patterning in development and vSMCs of major arteries protect from hemorrhage and BBB breakdown in older zebrafish. The fact that young zebrafish have a functional BBB in the absence of mural cells calls for renewed interrogation of mural cell control of the BBB throughout vertebrate evolution.

Animal development is a complex process that requires the coordination of a plethora of pathways in space and time. In several species, the availability of tissue explants has provided a simplified context that facilitates mechanistic investigations, particularly into dynamic events. Here, we demonstrate that extruded C. elegans gonads are a viable tissue explant system for this model organism. Using live-cell imaging, we show that C. elegans gonad explants retain many tissue properties that have been documented in vivo, including mitosis, meiosis, apoptosis and gametogenesis. We further show that C. elegans explants are acutely responsive to treatment by the microtubule depolymerizing drug nocodazole. Our work thus reveals C. elegans gonad explants as a new system in which live-cell imaging and acute drug treatment can be combined to decipher the mechanisms governing germline development.

Long-term live imaging of growing samples with light-sheet fluorescence microscopy provides unique insights into development, but morphogenesis often displaces features of interest outside the microscopes field of view (FOV), calling for automated methods to track these features and update the microscopes FOV in real time. Existing solutions, which typically rely on local or global intensity distributions, struggle to follow specific features robustly throughout morphogenesis, leading to truncated or incomplete datasets. Here, we present a light-sheet live tracking tool (LiLiTTool) that maintains user-defined regions of interest (ROI) within the FOV throughout extended imaging sessions. LiLiTTool uses Cotracker3, a state-of-the art deep learning-based motion predictor, augmented by sensor fusion with a trained object-detector. This enables robust compensation for drift, rotation, and deformation during morphogenesis, while meeting the timing constraints of live acquisition. We validated LiLiTool by integrating with the Viventis LS1 microscope, achieving sub-second processing and stable tracking of growing zebrafish embryos over many hours. LiLiTTool supports multi-ROI tracking in 3D, enabling simultaneous monitoring of multiple features within the same embryo and in multiple embryos during a single acquisition. LiLiTTool is modular and openly available on GitHub and as a napari plugin for post-acquisition tracking. By enabling precise, adaptive, and scalable real-time imaging, LiLiTTool advances smart microscopy approaches and provides the developmental biology community with a practical tool for capturing reliable spatio-temporal information in growing embryos or other morphogenetic systems.

Epithelial morphogenesis and homeostasis rely on dynamic remodeling of cell-cell junctions. Tricellular junctions (TCJs) at cell vertices are key sites that control epithelial permeability and plasticity, yet how TCJs are remodeled remains unclear. In the follicular epithelium (FE) in Drosophila ovaries, TCJs open transiently in a process called patency to allow passage of yolk proteins for uptake by the oocyte. We investigated how a gradient of TGF-{beta} signaling activity suppresses patency in a graded manner across the FE. We show that TGF-{beta} signaling blocks patency in a cell-autonomous manner by strengthening E-Cadherin (E-Cad)-based adhesion through inducing E-Cad transcription and preventing its removal from cell vertices. In parallel, TGF-{beta} signaling activates myosin II through Rho-Rok signaling. However, myosin II activity is dispensable for TGF-{beta}-mediated suppression of patency. We show that TGF-{beta} signaling controls TCJ remodeling in follicle cells primarily by reinforcing E-Cad-based adhesion, in part through upregulating p120-catenin. Our findings disentangle the roles of adhesion and actomyosin contractility in maintaining TCJ integrity and reveal how a tissue-scale morphogen gradient is translated into graded epithelial permeability.

Early neural development involves a combination of planar signals from the vertebrate organiser and vertical signals from its derived structures, the prechordal plate and notochord. However, the relative contribution of each structure to neural development is not clear. Here, we isolate anterior tissues from the primitive streak at successively later stages of development, to identify the extent of patterning that can occur prior to, during, and after the formation of the organiser and its later derivatives. Our results show that acquisition of neural identity occurs gradually and that exposure to planar signals from the developing node is necessary for neural plate specification. We also show that planar node-derived signals are required for AP patterning in isolated anterior tissues and give evidence that early neural tissue is of anterior character which subsequently becomes caudalised by signals (in part) from the developing node. However, anterior neural identity is lost without long-term contact with vertical signals from the axial mesendoderm. These results reveal a previously unappreciated level of autonomy in anterior neural development in the absence of node derived tissues. Summary statementCulture of isolated anterior tissues from the chick embryo reveal the roles of planar and vertical organiser signals for neural specification and anteroposterior patterning and maintenance.

Asymmetric cell division in the epidermal stem cells of Caenorhabditis elegans, known as seam cells, relies on the Wnt/{beta}-catenin asymmetry pathway to generate daughter cells with distinct fates. However, whether components of this pathway components are transcriptionally regulated during these divisions remains unclear. Here, we employ single molecule fluorescence in situ hybridisation to quantify mRNA distributions of key Wnt pathway components during L2 symmetric and asymmetric seam cell divisions. We find that transcripts encoding the negative regulators pry-1/Axin and apr-1/APC are enriched in posterior daughter cells, while those encoding the positive regulators sys-1/{beta}-catenin, wrm-1/{beta}-catenin, and lit-1/NLK, along with the transcription factor pop-1/TCF, are enriched in anterior daughter cells. Strikingly, molecular asymmetries are already evident following the L2 symmetric division, with anterior and posterior daughters exhibiting distinct levels of Wnt component expression and Wnt pathway activation. These mRNA distributions are surprising considering the established protein localisations that underpin the Wnt asymmetry model and suggest extensive post-divisional transcriptional regulation. We further demonstrate that pop-1 asymmetric expression depends on Wnt signalling activity, supporting a model in which transcriptional feedback reinforces cell fate decisions. Investigation of protein distributions using knock-in reporters for PRY-1 and CAM-1 showed that protein accumulation patterns at L2 are consistent with transcript levels. Our findings uncover pervasive transcriptional feedback within the Wnt pathway that likely contributes to robust fate specification and reveal molecular heterogeneity with potential functional consequences for lineage behaviour.

In vertebrate peripheral nerves, damaged axons can regrow after injury, but the outcomes of regeneration are variable and often incomplete. Schwann cells in injured nerves are important for repair, but their actions at different positions and stages of nerve repair are not well understood. We have investigated the roles of Schwann cells in a larval zebrafish nerve injury model, in which nerves are visible in living animals during development, the initial injury response, and regrowth of the transected axons. After mechanical injury, distal Schwann cells adopt a repair phenotype characterized by changes in marker expression, elongation, and ability to guide axons across the injury site. In contrast, proximal Schwann cells are not sufficient to guide axons across the injury site, and they associate with axons that regrow along aberrant paths. In erbb2 mutants lacking Schwann cells, developmental axon growth is normal, but after transection, axonal regrowth is greatly slowed and often misdirected. By examining animals with nerves partially populated by Schwann cells, we find that axons can regrow through regions devoid of Schwann cells, provided that at least one distal Schwann cell is at the injury site. Timelapse imaging reveals that distal Schwann cells extend processes toward the injury site, which contact and guide axons regrowing from the proximal nerve stump. In irf8 mutants lacking macrophages, debris from transected axons is cleared on schedule, and axonal regrowth is normal. Our studies demonstrate that Schwann cells immediately distal to the injury site have a unique and essential role in axonal regrowth. Main PointsO_LIAfter nerve transection in larval zebrafish, proximal and distal Schwann cells have distinct functions at injury site C_LIO_LIA single distal repair Schwann cell is sufficient for axonal regrowth C_LIO_LIAxonal regrowth is normal in mutants without macrophages C_LI

Neural crest (NC) cells are dynamic embryonic stem cells that undergo an epithelial-to-mesenchymal transition (EMT) and alter their cell states from tightly adherent to migratory and invasive during early development. While EMT transcriptional programs are well characterized, how cytoskeletal architecture is developmentally patterned across EMT states remains poorly understood. Here, we present a spatial and temporal atlas of - and {beta}-tubulin isotype gene expression during NC EMT in the chick embryo. Single cell RNA-sequencing reveals diversity in tubulin isotype gene expression from ubiquitous (TUBA1A, TUBA1B) to cell type specific (TUBAL3, TUBB4B). In addition, we identified novel enrichment of several tubulin isotypes in NC and NC-associated clusters (TUBB3, TUBA3E, TUBG1). Using fluorescent in situ hybridization chain reaction (HCR), we focus on NC EMT and migration states to validate and spatially resolve these expression patterns. Additional characterization in differentiated cells reveals tubulin gene expression in specific neuronal and myogenic populations. We further identify expression of the microtubule motor genes KIF11 and DYNC1LI1 within neural tube and NC populations, suggesting coordinated regulation of microtubule composition and cargo transport capacity. Together, these data establish that vertebrate NC EMT is accompanied by systematic reprogramming of tubulin gene expression and provide a developmental resource for investigating cytoskeletal control of cell state transitions. SUMMARY STATEMENTThis study defines when and where distinct tubulin genes are expressed during neural crest epithelial-to-mesenchymal transition in the chicken embryo providing a resource for understanding cytoskeletal organization across embryonic cell state changes.

Binding of Fibroblast growth factor (FGF) to a heparan sulfate proteoglycan (HSPG) is required for paracrine FGF signaling. To improve our understanding of FGF:HSPG association, we developed a method to monitor export of the Drosophila FGF ortholog Branchless (Bnl) in vivo. We detected Bnl on the surface of approximately 10% of Bnl-producing cells, but Bnl on the surface of cells depleted of HS was much reduced. HS depletion also non-autonomously decreased the activity of cytonemes that extend from cells that receive Bnl. These results are consistent with the idea that Bnl export to the cell surface is regulated, that intracellular binding of an HSPG to Bnl in producing cells is essential for export, and that cells that take up Bnl actively participate in its release from producing cells. SummaryLevels of FGF exported to the surface of FGF-expressing cells are dependent on intracellular heparan sulfate proteoglycans.

To form new sprouts during angiogenesis, endothelial cells coordinate migration through biophysical and biomechanical signaling with each other and the micro-environment. In zebrafish embryos, a key example of this process is intersegmental vessel (ISV) formation, where endothelial tip cells sprout dorsally from the dorsal aorta, elongate between somites, and connect to form the dorsal longitudinal anastomotic vessel (DLAV). While various factors coordinate ISV pathfinding, such as vascular endothelial growth factor (VEGF), semaphorin signaling, and integrin-mediated adhesion, the role of extracellular matrix (ECM) mechanics and distribution remains incompletely understood. Here, we combined in vivo timelapse imaging with mathematical modeling to study how ECM components influence endothelial migration. Experimentally, morpholino single knockdowns of laminin or fibronectin slowed ISV elongation but most vessels eventually reached the DLAV. To analyze potential effects of ECM mechanics on sprout progression, we developed a hybrid mathematical model of ISV elongation. The model combines a mass-spring based representation of a network of ECM fibers with an experimentally validated Cellular Potts Model of endothelial cell behavior that described cell elongation and migration, integrin-based mechanosensitive adhesion formation, and chemical signaling through VEGFs and semaphorins. For baseline parameters, this model predicts that the ECM helps to confine the extending sprout to the intersegmental space. After reducing the stiffness of the ECM network, ISVs sprouted more slowly than in baseline conditions, consistent with our experimental observation. After further reduction of ECM stiffness, the model predicted increased fusion of ISVs, a behavior reminiscent of the behavior of endothelial cells in many in vitro models and in our previous in silico models. Guided by these data and predictions, we hypothesized that ECM in the intersomitic space guides endothelial selforganization during ISV pathfinding. In agreement with this hypothesis and model predictions, combined knockdown of laminin and fibronectin produced network-like endothelial arrangements and errant pathfinding of ISVs. Importantly, re-expression of fibronectin using chimeric fibronectin mRNA substantially restored ISV organization in the triple knockdown backgrounds, supporting that the severe disorganization reflects specific loss of fibronectin-dependent function rather than nonspecific injection effects. Altogether, our results suggest a model of guided self-organization for ISV formation, in which the ECM and chemical guidance cues including semaphorins confine self-organized endothelial network formation to the intersegmental space.