Development

In ascidian embryos, gene expression from the zygotic genome begins between the 8- and 16-cell stages. While most zygotic genes are expressed in specific cell lineages at these stages, transcription factors that provide spatial cues for establishing specific expression patterns are insufficient to activate target genes at normal physiological levels. Gata.a is a transcription factor that provides spatial cues for targets expressed specifically in the animal hemisphere. Intriguingly, it is also required for physiological-level expression of many zygotic genes expressed in the vegetal hemisphere. In the present study, we found that Tbx21 and Klf6/7 augment the latter function of Gata.a. To determine the global extent of genes under control of these factors, we identified genes zygotically activated in early embryos using RNA-sequencing of BrU-labelled zygotic mRNAs. Our results revealed that approximately 80% of all zygotically activated genes were under control of these three factors. That is, together, Gata.a, Tbx21, and Klf6/7 are necessary to regulate target gene expression at physiological levels. This requirement for a specific set of broadly distributed factors resembles those of pioneer transcription factors that trigger zygotic genome activation (ZGA) in other animals, including flies and vertebrates. Regulatory factors involved in ZGA vary among animals, and our results indicate that ascidians use a distinct set of transcription factors for ZGA.

During preimplantation development in mice, cells of the inner cell mass undergo a cell fate decision to become either Epiblast (Epi) or Primitive Endoderm (PrE) cells. Cell fate patterns during this stage range from an alternating pattern at the beginning to the separation of Epi and PrE at the end. Several mechanisms guiding this decision and pattern formation have been proposed, including intra- and intercellular signalling, cell division and cell sorting. The current understanding is that signalling generates the cell fates and subsequent sorting introduces the spatial cell fate separation. We used agent-based modelling to investigate whether cell differentiation and cell sorting can act concurrently and how their relative contributions to pattern formation may change over time. Comparing our model to experimental data for mouse blastocysts and ICM organoids, we find two mechanistic regimes that can produce the experimentally observed spatial separation: (i) simultaneous long-range intercellular signalling and cell sorting, and (ii) a gradual transition from short-range signalling to cell sorting, in which the timing is mediated via reducing cell fate plasticity. While the second agrees better with existing experimental evidence for late blastocysts, the first might still be relevant for early and mid blastocysts. Together, our results refine the sequential view of Epi/PrE patterning by showing that fate specification and cell sorting can be dynamically coupled, with their relative contributions changing over the course of blastocyst development.

Vascular Endothelial Zinc Finger 1 (VEZF1) is essential for embryonic development, but its role in pluripotency exit remains unclear. Previous work showed that Vezf1-deficient ESCs exhibit impaired differentiation, reduced Dnmt3b expression, and genome-wide hypomethylation. Here, we show that Vezf1-/- ESCs fail to efficiently repress the pluripotency transcriptional program during differentiation, a defect that persists after ectopic Dnmt3b expression. Genome-wide analysis revealed VEZF1 occupancy at regulatory regions of genes involved in several developmental signaling pathways, including MAPK, WNT, and Hippo, as well as at some pluripotency-associated genes. Many VEZF1-bound MAPK genes showed reduced expression in undifferentiated Vezf1-/-ESCs, suggesting that VEZF1 activity contributes to transcriptional competence required for efficient pluripotency exit. VEZF1 loss also led to widespread acquisition of new CTCF sites associated with developmental signaling, a subset of which overlapped VEZF1-bound regulatory regions. CTCF depletion had only limited effects on the expression of the VEZF1-bound MAPK genes examined, indicating that increased CTCF occupancy alone is insufficient to explain their reduced expression in Vezf1-/- ESCs. Together, our findings identify a DNMT3B-independent function of VEZF1 in facilitating the exit from pluripotency and establishing transcriptional competence for differentiation, while revealing a potential role for VEZF1 in regulating CTCF occupancy during developmental state transitions.

Reelin is a secreted extracellular matrix protein that regulates neuronal migration and layer formation in the developing brain, yet its role in retinal development remains incompletely defined. Here, we investigated Reelin function in retinal lamination using wild-type and Reeler (Reln-/-) mice, combining stage-resolved RNA in situ hybridization, immunohistochemistry, and single-nucleus RNA sequencing. We show that Reln is dynamically expressed in ganglion cell layer and inner nuclear layer neurons during retinal development and persists in discrete adult neuronal populations. Loss of Reelin leads to widespread defects in retinal organization affecting both neurons and Muller glia. In Reln-/- retinas, Muller glia exhibit reduced Glul positive extensions, indicating impaired glial scaffold maturation. Early-born neuronal populations are also disrupted, with altered spatial organization markers associated with retinal ganglion cell differentiation within the ganglion cell layer at postnatal day 9. Horizontal cells are significantly reduced with dorsal-predominant vulnerability, while cone photoreceptors are generated in normal numbers but show incomplete positioning within the outer nuclear layer. Together, these findings identify Reelin as a key regulator of retinal lamination that coordinates neuronal positioning with Muller glia morphogenesis, extending its canonical role in brain development to the vertebrate retina.

During mouse embryogenesis, three sequential waves of cell polarization occur in the 8-cell-morula trophectoderm (TE), E3.5-E4.5 primitive endoderm (PrE), and E4.5-E5.5 epiblast (Epi), driving cell fate determination and morphogenic events that lay the foundation for embryonic development. However, it is not clear whether a general regulator that functions in all three waves of polarization exist. Here, we demonstrate that transcriptional factor THAP11 is such a general regulator. Using a novel mouse model combining genetic knockout and an inducible degron system, we demonstrate that stage-specific acute THAP11 depletion causes the failure of the three waves cell polarization, resulting in defective blastocyst formation, PrE maturation, and Epi lumenogenesis, respectively. Mechanistically, THAP11 does not directly regulate pluripotency genes but drive polarization through both stage-specific and conserved chromatin-bindings that commonly activate polarity programs, including Golgi vesicle transport. THAP11 activates polarity genes by promoting chromatin accessibility at their promoters. THAP11, together with stage-specific regulators, sequentially paces the timing of cell polarization. Collectively, our study establishes THAP11 as a general regulator of embryonic morphogenesis.

MyoD plays a central role in determining the skeletal muscle lineage in vertebrate embryos. The core enhancer (CE) and distal regulatory region (DRR) are the only known MyoD enhancers, and together, they recapitulate all major aspects of MyoD expression in the embryo. However, knocking out each enhancer individually has only modest effects on MyoD expression. Here, we show that embryos lacking both enhancers maintain muscle-specific MyoD expression, indicating the existence of unknown MyoD regulatory elements. Precision run-on sequencing together with available ChIP-seq and DNase I hypersensitivity datasets identified three new candidate enhancer regions within 96 kb of MyoD 5 flanking sequences. Transgenic analysis revealed that DNA elements at -36 and -60 kb are active in all muscle-forming regions, each recapitulating aspects of endogenous MyoD expression. Enhancer activities in muscle regulatory factor-deficient mice suggest that they are components of the auto- and cross-regulatory circuitry that maintains MyoD expression. Analysis of Hi-C data showed that the entire -96 kb region constitutes a loop domain, within which multiple interactions between elements and with the MyoD gene were detected. A larger loop domain delimited by CTCF sites was also identified from -96 kb to +220 kb relative to the MyoD transcriptional start site. These data indicate that the newly identified enhancers are key components of a cis regulatory network that controls the activation and maintenance of MyoD expression in the embryo. One sentence summaryCis regulation of MyoD transcription during development

Fat and Dachsous cadherins are large transmembrane proteins that regulate tissue growth and planar cell polarity across species. In mammals, Fat4 is known to bind Dchs1, but whether it also binds Dchs2 is unclear. Here we show that Dchs2 binds Fat4 in a trans-heterophilic manner at cell-cell contacts, mirroring the Fat4-Dchs1 interaction. In the developing mouse retina, Fat4 and Dchs1 expression peaks embryonically and declines after birth, whereas Dchs2 expression begins postnatally and persists into adulthood, with all three co-expressed from birth through the second postnatal week. While Fat4 and Dchs1 mutants show no defects in retinal lamination or cell-type composition, loss of Dchs2 increases number of cones, reduces Muller glia number, and results in disorganization of photoreceptor outer segments. Electron microscopy shows that Dchs2 mutant photoreceptors fail to form the regular nuclear columns seen in controls, with disrupted stacking and breaks in the outer limiting membrane. These findings identify Dchs2 as a novel Fat4 ligand and reveal a distinct, non-redundant requirement for Dchs2 in photoreceptor organization and outer limiting membrane integrity in the mature retina.

Gastrulation is thought to be driven primarily by forces generated within individual cells. These cell-intrinsic forces collectively induce tissue-scale flows and transform the monolayered embryo into a multilayered structure. However, as the embryo constitutes a mechanically closed system, these flows must be balanced by regions of resistance or anchoring to enable asymmetric morphogenesis. In the Drosophila embryo, integrin-mediated attachment of the blastoderm to the vitelline envelope has been shown to stabilize germ band extension at the organismal scale. Disrupting such an attachment leads to a characteristic twisting phenotype. Yet, how this attachment shapes concurrent global morphogenetic events remains unclear. We discovered that the integrin -subunit scab, which mediates the attachment, is expressed in three different regions of the cellular blastoderm near prominent invagination events. Through a combination of light-sheet imaging, genetic and mechanical perturbations, we demonstrate that integrin-enhanced friction is essential for unidirectional tissue flows in those regions, with effects including cephalic furrow positioning and epithelial stability. Guided by a minimal physical model, we further show that multiple attachment sites enhance the robustness and reproducibility of global tissue movements. Together, our results indicate that Drosophila gastrulation emerges from a balance between cell-intrinsic force generation and spatially distributed adhesion to the surrounding envelope, which together shape tissue flows at the embryo scale.

Adult schistosomes can survive for decades in the hostile niche of the host vasculature, yet the mechanisms that support tissue repair and resilience remain poorly understood. Here, we show that both mechanical and chemical injury trigger robust proliferative responses in adult parasites and display distinct spatial patterns. We identify a bHLH transcription factor that responds specifically to mechanical injury and is required for injury-induced stem cell proliferation and parenchymal cell renewal. Downstream, we find a WD40 repeat-containing protein that acts within a subset of stem cells to mediate this regenerative program. Together these findings detail an injury-responsive regenerative program in schistosomes that supports both their longevity and resilience in vivo.

Developmental transcription factors often have overlapping functions, making it difficult to define the distinct roles of individual factors during lineage specification. We investigated the partially redundant transcription factors TBX-35 and CEH-51 in the Caenorhabditis elegans embryonic MS mesodermal lineage using 4D lineage tracing, reporter imaging, genetics, and single-cell RNA sequencing. In tbx-35 mutants, MS descendants showed progressively slower cell cycles and a division pattern that increasingly resembled the cousin C lineage. Fate-regulator expression also shifted toward C-like features, including ectopic pal-1 and expanded HLH-1 expression, although mutant cells did not simply adopt normal C-lineage positions. Loss of tbx-35 also impaired a later MS-dependent Notch induction in the AB lineage while leaving an earlier induction intact. CEH-51 showed a different pattern of activity whereby its protein became enriched in anterior MS daughters, and ceh-51 mutants produced later, more restricted lineage defects that were strongest in descendants of cells with higher CEH-51 levels. Single-cell profiling identified overlapping but nonidentical sets of genes dependent on the two factors. TBX-35-dependent changes were strongest at earlier stages, whereas CEH-51-dependent genes became more prominent later and were enriched in anterior MS sublineages. Finally, temperature-shift experiments determined that the severity and onset of tbx-35 mutant phenotypes depend on the maternal temperature environment and cannot be explained by differences in residual CEH-51 expression. These findings reveal that TBX-35 and CEH-51 contribute differently across the MS lineage and that reliable mesoderm development is supported by overlapping zygotic and maternal regulatory inputs.

The olfactory epithelium of adult mammals contains two populations of stem cells that support its remarkable ability to regenerate neuronal and non-neuronal cell types throughout life. How olfactory epithelial cell types are established during development, however, is not well understood. Here, we use genetic lineage tracing and single-cell RNA sequencing of the perinatal mouse olfactory epithelium to construct a developmental trajectory consisting of multiple lineages. We identify transitional states and lineage relationships between individual cells and establish Ascl1+ cells as the primary multipotent progenitors in the perinatal olfactory epithelium. Further, Ascl1+ cells become progressively restricted in their cell fate capacity over developmental time and appear to be transcriptionally primed toward specific lineages. We also predict signaling pathways that may contribute to lineage plasticity and niche permissiveness. Together, these results contribute to our understanding of how cell-intrinsic and-extrinsic signals contribute to the establishment of a stem cell niche.

A key requirement to address fundamental questions in developmental biology is the ability to visualise and manipulate endogenous signalling proteins in vivo. Here we generate such an enabling experimental tool for the highly conserved Hippo pathway and its transcriptional effector Yap1, which converts mechanical and chemical cues into transcriptional output during embryogenesis and regeneration. We generate and characterise a Yap1- mGreenLantern (Yap1-mGL) endogenous CRISPR/Cas9-mediated knock-in line that enables 4D analysis of Yap1 dynamics in medaka. We resolve Yap1- mGL expression and nuclear/cytoplasmic localisation in real time, revealing the onset of reporter fluorescence before gastrulation, tissue and cell-type specific Yap1-mGL localisation during somitogenesis and injury-induced Yap1 upregulation during larval spinal cord regeneration. Using the nanobody-based degron system, we degrade the Yap1 protein in vivo, recapitulating the genetic loss-of-function mutant phenotype. Combining endogenous tags with nanobody-based degrons offers a powerful approach to visualise and acutely perturb endogenous protein function in vivo.

Lamins are the major structural components of the nuclear lamina with a variety of roles in development and organogenesis. However, the function of lamins during trophoblast development, the first lineage to differentiate during mouse embryogenesis, remains unexplored. By utilizing an in vitro trophoblast stem cell differentiation model in a lamin null setting, we uncover that lamins maintain expression of genes related to trophoblast differentiation, while repressing genes involved in maintaining trophoblast stem cell stemness and off-lineage development. By deleting different combinations of lamins in mice, we show that both lamin triple-knockout and lamin-A and -B1 (lamin-A/B1) double-knockout result in placental defects, including reduced placenta size and disrupted placental organogenesis at embryonic day (E)9.5. At this stage, lamin-A/B1 are expressed in trophoblast giant cells of the placenta, and lamin-A/B1 loss leads to their impaired maturation in vivo. Lamin-A/B1 double knockout trophoblast giant cells exhibit reduced nuclear size along with a reduction of DNA damage signaling foci, suggesting a role for lamins in supporting trophoblast giant cell polyploidization. Similar to the transcriptional dysregulation observed during differentiation of lamin triple knockout trophoblast stem cells in vitro, lamin-A/B1 knockout in vivo results in downregulation of genes related to trophoblast giant cell function and upregulation of off-lineage genes. Our results suggest lamins are required for placental organogenesis by maintaining polyploidization and lineage-associated transcriptional programs in trophoblast giant cells.

The initiation of meiosis in germ cells is largely regulated by extrinsic cues from the gonadal environment, but the logic of these signals remains poorly understood in non-mammalian vertebrates. Retinoic acid has long been considered a principal meiosis-inducing signal, yet recent genetic and reconstitution studies indicate that retinoic acid alone is insufficient. In mice, bone morphogenetic protein 2 cooperates with retinoic acid to establish the oogenic program through the bone morphogenetic protein-responsive transcriptional regulator Zglp1, but whether this regulatory logic is conserved beyond mammals is unknown. Here, using cultured chicken (Gallus gallus) primordial germ cells, we show that bone morphogenetic protein 2 cooperates with retinoic acid to promote a meiotic-entry transcriptional program. Retinoic acid alone induced a limited retinoic acid-responsive state, whereas combined treatment reduced the primordial germ cell program and activated early meiotic genes. The resulting transcriptome matched the premeiotic-to-meiotic-entry transition of the embryonic ovary in a single-cell atlas of chicken germ cells. We also generated a genome-edited primordial germ cell line carrying an SYCP3 promoter-green fluorescent protein reporter as a platform for dissecting meiotic-entry signals in culture. Comparative genomic analysis revealed Gallus-specific pseudogenization of ZGLP1, which is intact in closely related galliform species. Retinoic acid and bone morphogenetic protein may therefore act through a different downstream regulator in chicken. Article summaryEggs and sperm are produced by meiosis, a specialized cell division that starts during embryonic development. Retinoic acid, a signal derived from vitamin A, was long thought to be enough to start meiosis, but on its own it is not. In mice, a second signal, bone morphogenetic protein 2 (BMP2), works alongside retinoic acid to push germ cells toward the egg-producing program. We asked whether birds use the same combination. Giving both signals to chicken primordial germ cells in culture switched on genes for egg development and for the first steps of meiosis; retinoic acid alone did not. Birds and mammals last shared an ancestor more than 300 million years ago, so the pairing of these two signals appears to be an old feature of vertebrate germ cells. The cells stopped short of completing meiosis, which means other signals are still missing. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=169 SRC="FIGDIR/small/740788v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1b47deaorg.highwire.dtl.DTLVardef@b6a61dorg.highwire.dtl.DTLVardef@153f4c1org.highwire.dtl.DTLVardef@e9c641_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cerebellar output neurons transmit the results of cerebellar computation to other brain regions. In most vertebrates, cerebellar output neurons form discrete nuclei, whereas in teleosts they are represented by eurydendroid cells, which differ markedly in morphology and anatomical organization. How these divergent output systems are developmentally related remains unclear. Here, we show that zebrafish eurydendroid cells arise from atoh1a-expressing progenitors, including a population expressing olig2, revealing a developmental origin shared with cerebellar output neurons in other vertebrates. atoh1a is required for eurydendroid cell differentiation and regulates lhx9 and lhx2b, which promote robo1 and robo3 expression and proper axon guidance. Our findings also suggest a role for Reelin signaling in eurydendroid cell positioning. Together, these findings reveal conserved developmental features shared by cerebellar output neurons across vertebrates, as well as mechanisms underlying the distinct anatomical organization of teleost eurydendroid cells.

How progenitors can faithfully self-assemble to build new organs in adulthood is a central unsolved problem in regeneration biology. The planarian eye is a model for uncovering principles of de novo organogenesis during regeneration. In this process, pluripotent adult stem cells specify into ovo+ migratory progenitors which target to the eye field due to Wnt and FGFRL signals produced from bodywall muscle, then differentiate into photoreceptor neurons (PRNs) and pigmented optic cup (OC) cells, followed by organization of these two cell types in an opposing pattern that completes eye organ maturation. We used scRNA-seq to characterize eye progenitor transcriptomes during regeneration, followed by FISH validation, and RNAi screening to identify regulators of eye regeneration. This uncovered a function for the discoidin domain receptor ddr-1 in preventing the premature terminal differentiation of migratory eye progenitors. DDRs encode collagen receptors, and bodywall muscle is the major source of collagen in planarians, suggesting eye progenitors likely interface with muscle ECM for their proper deployment in regeneration. We further identified a novel eye morphogenesis phenotype from knockdown of tmem132-1, causing failed heterotypic adhesion between eye cell types. TMEM132 factors are conserved cell-surface receptors that mediate adhesion and regulate migration, and human TMEM132 mutations are associated with several neurobehavioral diseases. Therefore, planarian tmem132-1 likely directly regulates the capture and assembly of migratory progenitors into mature eyes during regeneration. Together, these results define new steps in the eye regeneration pathway and suggest ancient roles for DDR and TMEM132 in progenitor migration and coordinated organ assembly.

Regenerating tissues like the axolotl (Ambystoma mexicanum) limb draw from many cell types, but isolating specific cell populations remains a bottleneck. Standard approaches rely on enzymatic dissociation and fluorescence-activated cell sorting (FACS), which are slow, costly, and require specialized instrumentation. We adapted INTACT (Isolation of Nuclei Tagged in specific Cell Types) to the axolotl, generating a transgenic line (INTACT-olotl) expressing Sun1-sfGFP in limb connective tissue, the population that carries limb positional information, dedifferentiates following injury, and forms the majority of the regenerative blastema. Combining an optimized nuclei isolation protocol with anti-GFP nanobody capture enriched labeled nuclei from ~34.6% of input to 80-89% purity. Relative to dissociation and FACS, this recovers roughly 30-fold more target nuclei in less than half the time and without a cytometer. Bead-bound nuclei are directly applied to combinatorial-barcoding snRNA-seq, eliminating fluorescence-activated nuclei sorting (FANS). Profiling INTACT+ connective tissue during TGF{beta} inhibition revealed a loss of extracellular matrix remodeling with upregulated pro-fibrotic and inflammatory gene programs, resembling an unresolved wound environment. These changes reverse and regeneration resumes upon drug washout, indicating that functional TGF{beta} signaling is required for the transition from wound resolution to blastema formation.

The organisation of myelinated axons into specialised domains is essential for saltatory conduction and depends on the polarised distribution of neuronal and glial cell-adhesion molecules, including neurofascins. However, how these domains assemble and refine in vivo remains unclear, as longitudinal imaging of endogenous proteins within intact nervous systems remains challenging. Here, we generated knock-in zebrafish in which endogenous neuronal and glial neurofascins are fused to fluorescent proteins, which enabled live-imaging without perturbing node assembly or introducing overexpression artefacts. Using these reporters, we visualised neuronal and glial neurofascin dynamics, and the formation of nodes of Ranvier and paranodes in vivo. We find that nascent nodes progressively compact in developing peripheral nerves, and that this process depends on glial neurofascin. Our novel toolkit for imaging endogenous neurofascins reveals a glia-dependent mechanism that shapes the developmental refinement of nodal architecture after their initial assembly. Our findings imply that glia-driven modulation of paranodes can be a mechanism for nervous systems to regulate circuit function.

Pericyte specification and expansion must be coordinated with vascular growth to establish a functional cerebrovascular network, yet how endothelial signals control these distinct developmental transitions remains incompletely understood. Here, using genetic manipulation, single-cell transcriptomic analysis, and longitudinal in vivo imaging in zebrafish, we identify ligand-specific endothelial Notch signaling as a mechanism coordinating brain pericyte development. Endothelial Jag2b is required for efficient pericyte specification from pericerebral progenitor pools and subsequently promotes Notch-dependent pericyte proliferation and population expansion. By contrast, endothelial Dll4 restrains pericyte proliferation and functionally opposes Jag2b-dependent expansion, demonstrating that distinct endothelial Notch ligands generate different developmental outputs. Endothelial Notch activation increases jag2b expression, and genetic interaction analyses establish Jag2b as an important mediator of Notch-dependent pericyte expansion. Moreover, genetic disruption of piezo1 reduces brain pericyte abundance, whereas endothelial jag2b expression rescues this phenotype, placing Jag2b downstream of Piezo1-dependent hemodynamic signaling. Together, these findings reveal a ligand-specific endothelial Notch program that integrates hemodynamic cues with endothelial-pericyte communication to coordinate the specification and expansion of brain pericytes during cerebrovascular development.

Hematopoietic stem cells (HSCs) arise from the hemogenic endothelium in the dorsal aorta through the endothelial-to-hematopoietic transition, a tightly regulated developmental process requiring coordinated signaling from the developing hematopoietic niche. Wnt and Notch signaling are essential for HSC specification, with Wnt functioning upstream of Notch; however, the molecular mechanisms by which these pathways are coordinated during HSC development remain poorly understood. Here, we identify Disheveled 2 (Dvl2), a key intracellular Wnt scaffold protein, as an essential regulator of HSC identity specification in zebrafish. Loss of dvl2 impaired HSC specification and reduced Notch signaling in the dorsal aorta, despite normal canonical Wnt activity in the developing endothelium, suggesting that Dvl2 regulates HSC specification independently of endothelial beta-catenin signaling. Hemogenic endothelial-specific expression of the Notch intracellular domain rescued HSC defects in dvl2 morphants, demonstrating that Dvl2 functions upstream of Notch activation in the hemogenic endothelium during HSC specification. In addition, loss of dvl2 reduced primitive macrophages and neutrophil populations. Restoration of primitive myeloid cells through gata1a knockdown rescued both endothelial Notch signaling and HSC defects, demonstrating that Dvl2 is also required for the supportive immune cells that induce HSC identity. Together, these findings reveal that Dvl2 coordinates hemogenic endothelial and immune niche cues to control HSC identity specification.