Development

3.0Stromal cells can direct epithelial differentiation during organ development; however, these pathways remain poorly defined. FGF signaling is essential for submandibular salivary gland development, and FGF2 can regulate proacinar cell differentiation in organoids through autocrine signaling in stromal cells. We performed scRNA Seq and identified stromal cell subsets expressing Fgf2 and Fgf10 that also express Pdgfr. When combined with epithelial cells in organoids, MACS-sorted PDGFR+ cells sufficiently promoted proacinar differentiation. Gene expression analysis revealed FGF2 activates the gene Bmp7 in the stroma. BMP7 could replace stromalsignaling and stimulate epithelial acinar differentiation but not branching. However, in the absence of FGF2, pathway analysis revealed that the stromal cells differentiated into myofibroblasts. Myofibroblast differentiation was induced when we treated organoids with TGF{beta}1, which also prevented proacinar differentiation. Conversely, FGF2 reversed TGF{beta}s effects. Dissecting pathways driving acinar differentiation will facilitate development of regenerative therapies. 2.0 Summary StatementEmbryonic salivary glands contain multiple stromal cell populations. FGF2 maintains the stromal Pdgfr+ population in-vitro. The PDGFR+ stromal cells drive early epithelial secretory cell differentiation using BMP7.

Organ morphogenesis relies on a tightly regulated interplay between cell proliferation, migration, and differentiation. Emerging evidence suggests that mechanical forces act alongside molecular signals to orchestrate tissue patterning, yet how these diverse inputs are integrated remains poorly understood. The zebrafish posterior lateral line offers a powerful in vivo model for studying how cellular behaviors and mechanosensitive signaling are spatiotemporally coordinated during organogenesis. Here, we identify the transcription factor Sox2 as a key regulator of lateral line morphogenesis, influencing the positioning, size, and number of neuromasts, the sensory organs of the lateral line. Loss of Sox2 leads to increased lateral line primordium cell proliferation, disrupted rosette assembly, and smaller neuromasts positioned more posteriorly along the body axis, while Sox2 overexpression produces opposite phenotypes. Sox2 functions in part by repressing Yap/Taz signaling in the primordium. Reduced Yap/Taz activity results in more anterior neuromast deposition and premature termination of primordium migration. Furthermore, we show that as the primordium expands through cell-proliferation, increased cell-junction tension activates Yap/Taz, thereby influencing lateral line development. Reducing overproliferation in sox2-/- embryos diminishes the elevated Yap/Taz activity, supporting a model in which Sox2 limits proliferation to suppress Yap/Taz signaling and ensure proper primordium morphogenesis. These findings uncover a biomechanical feedback loop in which Sox2 regulates morphogenesis by modulating tissue tension and mechanosensitive signaling.

The central nervous system (CNS) contains myriads of different types of cells produced from multipotent neural progenitors. Neural progenitors acquire distinct cell identities depending on their spatial position, but they are also influenced by temporal cues to give rise to different cell populations over time. For instance, the progenitors of the cerebral neocortex generate different populations of excitatory projection neurons following a well-known sequence. The Notch signaling pathway plays crucial roles this process but the molecular mechanisms by which Notch impacts progenitor fate decisions have not been fully resolved. Here, we show that Notch signaling is essential for neocortical and hippocampal morphogenesis, and for the development of the corpus callosum and choroid plexus. Our data also indicate that, in the neocortex, Notch controls projection neuron fate determination through the regulation of two microRNA (miRNA) clusters that include let-7, miR-99a/100, and miR-125b. Our findings collectively suggest that balanced Notch signaling is crucial for telencephalic development and that the interplay between Notch and miRNAs is critical to control neocortical progenitor behaviors and neuron cell fate decisions.

Leydig cells (LCs) are the major androgen-producing cells in the testes. They arise from steroidogenic progenitors, whose origins, maintenance and differentiation dynamics remain largely unknown. Here, we identified Wnt5a as a specific marker of steroidogenic progenitors, whose expression begins at around E11.5-E12.5 in interstitial cells of the fetal mouse testis. In vivo lineage tracing indicates that Wnt5a-expressing progenitors are initially present in large numbers in the fetal testis and then progressively decrease as development progresses. We provide evidence that Wnt5a-expressing cells are bona fide progenitors of peritubular myoid cells as well as fetal and adult LCs, contributing to most of the LCs present in the fetal and adult testis. Additionally, we show in the adult testis that Wnt5a expression is restricted to a subset of LCs exhibiting a slow but noticeable clonal expansion, revealing hitherto unappreciated proliferation of fully differentiated LCs as a contribution to the adult LC pool.

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.

The cerebral cortex is a multi-layered structure generated through the migration of neural precursors from their birthplace in the ventricular zone to their destination within the cortical plate. Neuronal migration defects are responsible for many human pathologies collectively called neuronal migration disorders, which include subcortical band heterotopia and cobblestone brain (COB) malformation. One example of a protein involved in a neuronal migration disorder is the echinoderm microtubule-associated protein-like 1 (EML1) protein, one of six members of the mammalian EML family. Absence of EML1 protein results in subcortical band heterotopia in mice and humans. Here, we report that absence of the paralogous protein EML3 leads to delayed embryonic development and small size, and a COB-like phenotype with neuronal ectopias in the dorsal telencephalon. We found that EML3 is expressed in the neuroepithelium and meningeal mesenchyme when those tissues participate in pial basement membrane (PBM) formation. Transmission electron microscopy demonstrated that the extracellular matrix of the PBM is structurally abnormal in Eml3 null mice when the first radially migrating neurons arrive. The reduced structural integrity of the PBM leads to focal over-migration of neurons into the subarachnoid space. These findings strengthen the link between the EML protein family and cortical neuronal migration defects by identifying Eml3 as the first EML family member whose absence leads to over-migration of neuroblasts. Moreover, we report the first COB-like phenotype with PBM structural defects when a single microtubule-associated protein is deleted.

Vascular endothelial cells in vivo are exquisitely regulated by their local environment, which is disrupted or absent when using methods such as FACS sorting of cells isolated from animals or in vitro cell culture. Here, we profile the gene expression patterns of undisturbed endothelial cells in living animals using a novel "AngioTag" zebrafish transgenic line that permits isolation of actively translating mRNAs from endothelial cells in their native environment. This transgenic line uses the endothelial cell-specific kdrl promoter to drive expression of an epitope tagged Rpl10a 60S ribosomal subunit protein, allowing for Translating Ribosome Affinity Purification (TRAP) of actively translating endothelial cell mRNAs. By performing TRAP-RNAseq on AngioTag animals, we demonstrate strong enrichment of endothelial specific genes and uncover novel endothelial genes and unique endothelial gene expression signatures for different adult organs. Finally, we generated a versatile "UAS:RiboTag" transgenic line to allow a wider array of different zebrafish cell and tissue types to be examined using TRAP-RNAseq methods. These new tools offer an unparalleled resource to study the molecular identity of cells in their normal in vivo context.

How tissue shape and therefore function is encoded by the genome remains in many cases unresolved. The tubes of the salivary glands in the Drosophila embryo start from simple epithelial placodes, specified through the homeotic factors Scr/Hth/Exd. Previous work indicated that early morphogenetic changes are prepatterned by transcriptional changes, but an exhaustive transcriptional blueprint driving physical changes was lacking. We performed single-cell-RNAseq-analysis of FACS-isolated early placodal cells, making up less than 0.4% of cells within the embryo. Differential expression analysis in comparison to epidermal cells analysed in parallel generated a repertoire of genes highly upregulated within placodal cells prior to morphogenetic changes. Furthermore, clustering and pseudo-time analysis of single-cell-sequencing data identified dynamic expression changes along the morphogenetic timeline. Our dataset provides a comprehensive resource for future studies of a simple but highly conserved morphogenetic process of tube morphogenesis. Unexpectedly, we identified a subset of genes that, although initially expressed in the very early placode, then became selectively excluded from the placode but not the surrounding epidermis, including hth, grainyhead and tollo/toll-8. We show that maintaining tollo expression severely compromised the tube morphogenesis. tollo is likely switched off to not interfere with key Tolls/LRRs that are expressed and function in the placode.

During vertebrate neurogenesis, a progressive transition from symmetric proliferative to asymmetric neurogenic divisions is critical to balance growth and differentiation. Using single-cell RNA-seq data from chick embryonic neural tube, we identify the cell cycle regulator Cdkn1c as a key regulator of this transition. While Cdkn1 is classically associated with neuronal cell cycle exit, we show that its expression initiates at low levels in neurogenic progenitors. Functionally targeting the onset of this expression impacts the course of neurogenesis: Cdkn1c knockdown impairs neuron production by favoring proliferative symmetric divisions. Conversely, inducing a low-level CDKN1c misexpression in self-expanding progenitors forces them to prematurely undergo neurogenic divisions. CDKN1c exerts this effect primarily by inhibiting the cyclin D-CDK complex and lengthening G1 phase duration. We propose that Cdkn1c acts as a dual driver of the neurogenic transition whose low level of expression first controls the progressive entry of progenitors into neurogenic modes of division before a higher expression mediates cell cycle exit in daughter cells. This highlights that the precise control of neurogenesis regulators expression levels sequentially imparts distinct functions, and is essential for proper neural development.

Molecular embryology studies have established that the patterning of the gastrula-stage mouse embryo is dependent on a regulatory network where the WNT, BMP and NODAL signalling pathways cooperate. Still, important aspects of their respective contributions to this process remain unclear. Here, studying their impact on the spatial organization and the developmental trajectories of micro-patterned Epiblast Like Cells (EpiLC) colonies, we show that when BMP is present, it dominates NODAL and WNT and imposes a posterior character to the colonies differentiation. However, the use of two Nodal mutant cell lines allowed us to show that prior to BMP action, NODAL is required to establish the mesendodermal lineage. The fact that mutant phenotypes were more severe in vitro than in vivo suggests that embryonic phenotypes are partially rescued by ligands of extra-embryonic or maternal origin. Our work demonstrates the complementarity of micro-patterned EpiLC colonies to embryological approaches.

A Wnt activity gradient is essential for the formation of the anterior-posterior (AP) axis in all vertebrates. The relationship between the dynamics of Wnt signaling and specification of AP coordinates is difficult to study in mammalian embryos due to the inaccessibility of developing embryos and the difficulty of live imaging. Here, we developed an in vitro model of human neuroectoderm patterning, where the AP axis self-organizes along the radius of a micropatterned human pluripotent stem cell colony. We used this system to study the quantitative relationship between Wnt signaling in space and time and the resulting AP patterns. We found that rather than a smoothly varying gradient along the axis, signaling is elevated in midbrain compared to either surrounding region. The timing, rather than the amplitude or duration, of the Wnt response played the most important role in setting axial coordinates. These results establish a simple system for studying the patterning of the human nervous system and elucidate how cells interpret Wnt dynamics to determine their position along the AP axis.

Developing tissues are sequentially patterned by extracellular signals that are turned on and off at specific times. In the zebrafish hindbrain, fibroblast growth factor (Fgf) signalling has different roles at different developmental stages: in the early hindbrain, transient Fgf3 and Fgf8 signalling from rhombomere 4 is required for correct segmentation, whereas later, neuronal Fgf20 expression confines neurogenesis to specific spatial domains within each rhombomere. How the switch between these two signalling regimes is coordinated is not known. We present evidence that the promyelocytic leukaemia zinc finger (Plzf) transcription factor is required for this transition to happen in an orderly fashion. Plzf expression is high in the early anterior hindbrain, then gradually upregulated posteriorly and confined to neural progenitors. In mutants lacking functional Plzf, fgf3 expression fails to be downregulated and persists until a late stage, resulting in excess and more widespread Fgf signalling during neurogenesis. Accordingly, the spatial pattern of neurogenesis is disrupted in plzf mutants. Our results reveal how the distinct stage-specific roles of Fgf signalling are coordinated in the zebrafish hindbrain.

During the development of the cerebral cortex, neurons are generated directly from radial glial cells or indirectly via basal progenitors. The balance between these division modes determines the number and types of neurons formed in the cortex thereby affecting cortical functioning. Here, we investigate the role of primary cilia in this process. We show that a mutation in the ciliary gene Inpp5e leads to a transient increase in direct neurogenesis and subsequently to an overproduction of layer V neurons in newborn mice. Loss of Inpp5e also affects ciliary structure coinciding with increased Akt and mTOR signalling and reduced Gli3 repressor levels. Genetically re-storing Gli3 repressor rescues the decreased indirect neurogenesis in Inpp5e mutants. Overall, our analyses reveal how primary cilia determine neuronal subtype composition of the cortex by controlling direct vs indirect neurogenesis. These findings have implications for understanding cortical malformations in ciliopathies with INPP5E mutations.

Neural tube morphogenesis provides a dynamic setting in which to study epithelial cell behaviours. Members of the claudin family of tight junction proteins regulate apical epithelial cell behaviors at all steps of neural tube development. We discovered that CLDN3, expressed in the non-neural ectoderm but not the neural ectoderm, is required to mediate neural fold fusion in chick embryos, particularly in the spinal region of the embryo. Depleting CLDN3 affects apical protein localization and apical domain morphology. Here, we used live imaging to re-examine the process of neural fold fusion in the cranial and spinal regions of the embryo and assessed biomechanical parameters of the non-neural ectoderm that are dependent on CLDN3. Our live imaging confirmed previous reports that unlike neural fold fusion in the cranial region and posterior neuropore, the spinal region does not depend on progressive fusion driven by "zippering" cell behaviors but instead fuses in a multi-step process where contact occurs simultaneously at multiple points along the anterior-posterior axis. We and others refer to the process of spinal neural fold fusion as "buttoning" to highlight the differences in cell behaviors from those observed during zippering (van Straaten et al., 1993). CLDN3-depletion decreased the rate of progression of neural fold buttoning within the spinal region. Using cell segmentation analyses we confirmed that CLDN3 depletion decreased the apical cell area of cells at the edges of the neural folds but not of lateral cells in the non-neural ectoderm. CLDN3 depletion increased pMLC staining within the apical domain of the cell, coinciding with a decrease in cell area, suggesting increased epithelial tension. Laser ablation studies revealed that the non-neural ectoderm of CLDN3-depleted embryos exhibits higher tension during neural fold fusion. We showed that treatment with the myosin II inhibitor blebbistatin is sufficient to partially rescue the neural fold fusion defects in CLDN3-depleted embryos. This work provides further evidence for the importance of non-neural ectodermal tissue tension in neural fold fusion and suggests that loss of CLDN3 may alter tissue tension through cytoskeletal regulation pathways within the apical domain. This work supports that CLDN3 contributes to neural fold fusion and epithelial tissue tension during neural fold fusion via modifications to the apical cytoskeleton. SummaryWe found that the tight junction protein Claudin-3 (CLDN3) plays a role in regulating tissue tension, by directing actomyosin contraction and apical cell shape/size changes essential for chick neural fold fusion.

Pluripotent cells reorganise themselves into an epithelium before they initiate differentiation, but it is not clear how these two events are mechanistically linked. Here we use quantitative imaging approaches to measure cellular rearrangements that accompany exit from naive pluripotency. We show that competence to epithelialise, like competence to differentiate, is a regulated process. The pro-differentiation transcription factor Tcf15 prospectively identifies cells that are competent to epithelialise. We identify early upregulation of the laminin receptor integrin alpha3 prior to differentiation and show that Tcf15 helps to regulate this change. Finally, we show that Tcf15 identifies and is required for efficient differentiation of a primed subpopulation of pluripotent cells. We conclude that competence to epithelialise is actively regulated and linked to differentiation-competence through the transcription factor Tcf15.

Kidney development is known to be driven by interactions between stromal, nephron and ureteric epithelium progenitors in the nephrogenic niche. In contrast, the epithelial nephrons generated in this environment have largely been considered a product of niche rather than an active participant in the signalling interactions that maintain it. However, knockout of Wnt4, a gene required for nephron formation and stromal development, results in hypoplastic kidneys. We hypothesised that the forming nephron may play a role in maintaining the nephrogenic niche. In support of this hypothesis, conditional deletion of Wnt4 from the nephron lineage resulted in nephron progenitor dispersal and death, reduced branching morphogenesis and nephron progenitor cell number. Bulk and single cell transcriptional profiling of Wnt4 mutant kidneys revealed a downregulation of BMP signalling effectors Id1, and Id3 in nephron progenitor cells, implicating Wnt4 target BMP4 as a paracrine signal mediating feedback from the committing nephron. Recombinant BMP4 restored nephron progenitor compaction in cultured Wnt4 mutant kidneys and blocked differentiation in wildtype controls mirroring the role of BMP7-MAPK signalling in progenitor self-renewal. Our data supports a revised model of the nephrogenic niche in which forming nephrons promote progenitor maintenance and branching morphogenesis, in part via paracrine BMP4 signalling under the control of Wnt4. This requirement for nephron-derived signals for maintenance of the nephrogenic niche provides new mechanistic insight into kidney morphogenesis and human renal hypodysplasia phenotypes associated with deleterious WNT4 mutations.

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.

The human heart is infamous for not healing after infarction in adults, prompting biomedical interest in species that can regenerate damaged hearts. In such animals as zebrafish and neonatal mice, cardiac repair relies on remaining heart tissue supporting cardiomyocyte proliferation. Natural de novo cardiogenesis in post-embryonic stages thus remains elusive. Here we show that the tunicate Ciona, an ascidian among the closest living relatives to the vertebrates, can survive complete chemogenetic ablation of the heart and loss of cardiac function, and recover both cardiac tissue and contractility. As in vertebrates, Ciona heart regeneration relies on Bone Morphogenetic Protein (BMP) signaling-dependent proliferation of cardiomyocytes, providing insights into the evolutionary origins of regenerative cardiogenesis in chordates. Remarkably, prospective lineage tracing by photoconversion of the fluorescent protein Kaede suggested that new cardiomyocytes can emerge from endodermal lineages in post-metamorphic animals, providing an unprecedented case of regenerative de novo cardiogenesis. Finally, while embryos cannot compensate for early losses of the cardiogenic lineage, forming heartless juveniles, developing animals gain their regenerative ability during metamorphosis, uncovering a fundamental transition between deterministic embryogenesis and regulative post-embryonic development.

Nodal and Wnt signalling play an important role in the emergence of the mammalian body plan, primarily by orchestrating gastrulation. While the literature suggests they cooperate to build the primitive streak, their individual contributions remain poorly understood. Using gastruloids, we found that Wnt/{beta}-catenin drives a genetic program characteristic of the late primitive streak, promoting the development of posterior body structures in a time and dose-dependent manner. Conversely, Nodal activates a distinct transcriptional module resembling the early streak. By engineering gastruloids with varying levels of Nodal signalling, we demonstrate that a decreasing temporal gradient of Nodal activity is critical for establishing the anterior body, with higher Nodal levels producing more anterior structures in a concentration-dependent manner. Our findings suggest that Nodal and Wnt act antagonistically, initiating distinct developmental modules within the primitive streak. This antagonism is likely the core mechanism driving the early body plan in mammals.

The widespread regenerative capacity of plants is mediated by the ability of specialized cells to reprogram their fate, but the sequential cellular states of regenerating plant cells remain an open question. Here, we characterize the trajectory of cellular reprogramming using single-cell RNA/ATAC-seq, imaging, and mutant analysis. The earliest events were dependent on repressive chromatin modification, where Multiome and genetic analysis showed that Class I histone deacetylases (HDACs) HDA9 and HDA19 were needed to shut down old identities and to prevent a runaway stress response. Cell division mediates a second step needed for the acquisition of many new identity markers, where division rates were tuned by DOF transcription factor OBP1 accelerating and SMR5, 7, and 10 decelerating division rates hours later. The results show how plants actively mediate the loss of remnant identities within hours of injury and then tune cell division rates to rapidly reprogram cells to new identities.