Genes & Development

Faithful genome inheritance during meiosis relies on crossover repair of double-strand DNA breaks (DSBs) to connect homologous chromosomes and direct their proper segregation. The formation of crossover-specific recombination intermediates and accumulation of pro-crossover factors occurs at an extremely limited subset of DSB sites, necessitating that the subset of recombination sites designated to become crossovers reliably mature into crossovers. Here we identify C. elegans disordered protein COSA-2 as crucial for meiotic crossover maturation. COSA-2 abruptly concentrates at crossover intermediates in late pachytene nuclei, where it colocalizes and associates with other pro-crossover factors. COSA-2 is dispensable for early loading of crossover factors and for crossover designation, but is required for maintenance of pro-crossover factors at crossover-designated sites and for focal enrichment of factors initially distributed throughout the synaptonemal complex. We define a COSA-2 execution point during late pachytene wherein crossover intermediates transition from a vulnerable state (in which they require COSA-2 to avoid being dismantled) to a state where COSA-2 and local crossover-factor enrichment are no longer required to connect homologs. We propose that COSA-2 scaffolds privileged DNA repair compartments that promote crossover-factor accumulation and protect crossover intermediates until completion of repair, thereby ensuring that crossover-designated sites reliably mature into crossovers.

Co-transcriptional splicing is a conserved feature of eukaryotic gene expression. However, establishing the functional nature of this process has been difficult. Here using high throughput CRISPR/Cas9 screens we surprisingly find that SF3B3, the third largest subunit of the U2 snRNP complex, is a major regulator of RNA Pol II pause release and processivity. Remarkably, the absence of SF3B3 dramatically perturbs transcription but U2 snRNP assembly and RNA splicing remains unaffected. Mechanistically, SF3B3 coordinates the chromatin occupancy of transcriptional kinases (CDK9/12/13) alongside the PAF1c and Integrator complexes to regulate Pol II. Structure / function analyses of SF3B3 revealed that a metazoan specific 18aa sequence within its disordered tail phenocopies its loss and mediates the physical association and stability of SF3B5. We show that loss of SF3B5 mirrors SF3B3 deficiency suggesting this submodule, although resident within the U2 snRNP complex, evolved to primarily coordinate RNA Pol II in a splicing-independent manner.

Heterochromatin Protein 1 (HP1) is a fundamental component of constitutive heterochromatin, forming subnuclear condensates whose regulation and function remain poorly understood. Here, we present an image-based CRISPR screen targeting nuclear factors that identifies splicing as a pivotal pathway regulating HP1 condensates. We discovered that unspliced intronic RNA modulates HP1 condensates by interacting co-transcriptionally with HP1. By modulating the intron content, RNA processing restricts HP1-RNA interactions at chromatin, thus enabling heterochromatin organization. Disruption of HP1 condensates due to enhanced interactions with unspliced RNA leads to loss of heterochromatin and the activation of stress response protective genes. We propose that RNA is a central component of heterochromatin that modulates HP1 condensates, and that RNA processing enzymes act as a surveillance mechanism for condensates by dynamically regulating the network of multi-valent interactions between RNA and chromatin factors. This model underscores the crosstalk between chromatin organization, transcription, and RNA processing, potentially governing broader nuclear functions.

Animal chemosensory systems discriminate between complex mixtures of chemical compounds by leveraging large repertoires of chemoreceptor proteins. To organize information from many receptors, these systems often employ a one-receptor-type per cell organization (Fulton et al. 2024). This structure requires a gene regulatory mechanism that can coordinate the generation of many distinct types of sensory neurons, each expressing a different receptor (Yusuf and Monahan 2024; Danoff et al. 2025; Pourmorady and Lomvardas 2022; Bashkirova and Lomvardas 2019; Monahan and Lomvardas 2015; Brann and Datta 2020). Here we show that testis-expressed gene 15 (Tex15) is a critical component in an epigenetic mechanism that safeguards diverse olfactory receptor (OR) choice in the mouse olfactory system. Tex15 is required to inhibit an initial wave of olfactory receptor gene transcription that occurs as olfactory sensory neuron progenitors differentiate. In Tex15 knockout (Tex15-/-) mice, this repression fails: the early-transcribed OR genes get expressed at abnormally high levels, get chosen at high rates, and come to dominate the neuronal population. This results in a profound reduction in the diversity of OR gene choice and disrupted spatial patterning of the olfactory epithelium, while maintaining the expression of one receptor per cell. These changes in OR gene expression are accompanied by reduced deposition of H3K9me3 heterochromatin on OR genes and the preferential formation of interchromosomal enhancer hubs around the dominant ORs. We propose that Tex15 directs a heterochromatin-based transcriptional repression mechanism that counter-balances early OR transcription, thereby preventing the heterogeneous onset of OR transcription from skewing OR choice.

Adult zebrafish rapidly recover glucose homeostasis after {beta}-cell loss, but the cellular basis and regulatory mechanisms that enable this response remain unclear. Here we combine single-cell transcriptomics, single-cell chromatin accessibility profiling, paired multiome analysis and functional perturbation to define early pancreatic recovery after {beta}-cell ablation. We show that, during the first month after injury, insulin production is restored predominantly by sst1.1+ {delta}1-cells rather than by rapid reconstitution of canonical {beta}-cells. Following ablation, {delta}1-cells adopt a bihormonal hybrid state and induce metabolic, secretory and {beta}-cell-associated gene programs. Systematic comparison of chromatin accessibility across endocrine cell types reveals that these {delta}1-cells are uniquely close to {beta}-cells and exhibit open chromatin at {beta}-cell enhancers in the steady state. Moreover, hybrid-cell formation occurs without major chromatin remodeling, with {beta}-cell associated loci being already accessible in {delta}1-cells before {beta}-cell injury. A comparable permissive state is present in medaka but not in human {delta}-cells, suggesting that restricted insulin accessibility may represent a barrier to endocrine plasticity in the human pancreas. In zebrafish, {delta}1-cells also show evidence of metabolic remodeling after {beta}-cell loss, including rapid accumulation of neutral lipids. Finally, gene regulatory network analysis and perturbation identify meis1a/b as required regulators of {delta}1 hybrid-cell formation after {beta}-cell loss. Together, our results define pre-existing chromatin accessibility, metabolic remodeling and instructive transcriptional regulation as key features of early functional recovery after {beta}-cell loss in the adult zebrafish pancreas.

ABSTRACTGATA6 and GATA4 play key roles in pancreatic development and are essential to maintain the classical transcriptional program in pancreatic ductal adenocarcinoma (PDAC). Using genetic mouse models we show that, in contrast to GATA6, GATA4 is dispensable for the maintenance of acinar homeostasis in the adult pancreas. Deletion of Gata4 in mice expressing mutant Kras in the embryonic pancreas (KG4C) leads to PDAC development in the absence of tissue remodeling, pancreatic intraepithelial neoplasia (PanIN), or other canonical precursor lesions present in Gata4-proficient (KC) mice. Similar observations were made when Gata4 was selectively inactivated in adult, Kras-mutant, acinar cells. We identify Pale Acinar Lesions (PALes) as a previously unrecognized pancreatic lesion, distinct from acino-ductal metaplasia (ADM) and PanINs, present in KC and KG4C mice but not in wild type mice. PALes display weak expression of acinar and ductal markers and lack mucins; they have lower proliferation rates than PanINs. RNA-seq and ChIP-seq reveal that GATA4 and GATA6 partially share genomic binding sites and transcriptomic effects, but they exert opposing influences on mutant Kras-induced, haematopoietic cell-dependent, transcriptional inflammatory programs. Adenoviral-mediated pancreatic expression of IL17 restored the formation of ductal lesions in KG4C mice but failed to rescue PanIN development. Our data indicate that GATA4 functions through the coordinated action of multiple inflammatory factors that are required for ADM/PanIN formation but are dispensable for PDAC development. Collectively, these findings challenge current paradigms of PDAC initiation and progression.

In animals, plants, and some fungi, Polycomb Repressive Complex 2 (PRC2) catalyzes trimethylation of histone H3 lysine 27 (H3K27me3) to establish transcriptionally repressed chromatin. Here, we identify the histone acetyltransferase RTT109 as a key regulator of PRC2-repressed domains in the model fungus Neurospora crassa. Although RTT109 interacts with the VPS75 homolog Nucleosome Assembly Factor 2 (NAF-2), we show that proper structure and function of PRC2-methylated chromatin require RTT109 catalytic activity but are independent of NAF-2 and H3K56 acetylation. We further demonstrate that H3K27me3 can be stably propagated over multiple rounds of mitosis in the absence of sequence-specific PRC2 targeting, and that RTT109 is essential for maintenance of the repressed state. These findings uncover a replication-linked mechanism for epigenetic memory and establish RTT109 as a key regulator of Polycomb-mediated chromatin inheritance.

Granule cell precursors (GCPs) drive the major postnatal expansion of the cerebellum and are the cells of origin of sonic hedgehog medulloblastoma (SHH MB). Although GCPs are often treated as a uniform population, increasing evidence suggests they are heterogeneous, and whether specific subpopulations show distinct tumorigenic competence remains unclear. Here, we identified a rare Nestin-expressing GCP subpopulation in the normal early postnatal cerebellum and showed that it is spatially restricted, molecularly distinct and highly competent to form tumors. These cells are enriched in the posterior external granule layer and co-express Atoh1. Using different SHH MB mouse models, we showed that when this rare subpopulation of GCPs is targeted they can give rise to SHH MB with an efficiency comparable to targeting a larger number of Atoh1-expressing GCPs and that tumors derived from Nestin-expressing GCPs arise in the posterior-lateral cerebellum. Single cell RNA sequencing revealed that Nestin-expressing GCP have a transcriptome indicating reduced neuronal differentiation and enrichment for stem cell genes compared to bulk GCPs and more closely align with SHH MB cells. Together, our findings reveal functionally important heterogeneity within the GCP lineage and suggest that SHH MB arises preferentially from a small subpopulation of GCPs that express Nestin.

25S nonfunctional RNA decay (NRD) eliminates 60S ribosomal subunits carrying inactivating mutations in the RNA. However, how cells identify defective subunits has not been described. We recently showed that the zinc-finger protein Reh1 is the last assembly factor to be released from a nascent 60S subunit. We now show that in yeast Reh1 is required for the degradation of 25S NRD substrates. 25S rRNAs carrying mutations in the catalytic center, A2820G or U2954A (A2451 and U2585, respectively in E coli numbering), are unstable in wildtype cells but are fully stabilized when REH1 is deleted. However, not all 25S rRNA mutations are recognized by Reh1. Ribosomes with a truncated L1 stalk engage in translation but cannot support viability. These ribosomes display a half-life indistinguishable from wild-type rRNA, suggesting that yeast does not have a robust surveillance system for such mutant ribosomes. Deletion of REH1 also has no impact on the levels of defective 18S rRNA. These results indicate that Reh1 and 25S NRD are specific for mutations in or near the catalytic center of the ribosome.

Neuronal maturation is associated with extensive changes in gene expression and chromatin organization. However, the molecular mechanisms that control the epigenetic landscape in terminally differentiated neurons remain poorly understood. Here, we show that maturing cerebellar granule cells undergo a striking and specific increase in the levels of the repressive histone modification H3K27me3 across different genomic regions, including individual genes, broad intergenic regions, and gene clusters. The accumulation of H3K27me3 coincides with a developmental switch from EZH2 to EZH1 and colocalizes with H3K36me2 and DNA non-CpG methylation. Using mice with a conditional deletion in the catalytic domain of EZH1, we demonstrate that the maintenance of H3K27me3 in mature neurons depends on EZH1. Unexpectedly, an almost complete loss of H3K27me3 in postmitotic GCs induces minimal changes in gene expression and chromatin accessibility at 7 months of age. Using single-nucleus RNA sequencing (snRNAseq) from the mouse neocortex, we show that, similarly to GCs, the loss of EZH1-mediated H3K27me3 also has a minimal impact on cortical neuron gene expression. The amino acid composition of EZH1 suggests reduced sensitivity to H3K36 methylation, providing a potential basis for its activity in chromatin contexts that are not permissive for EZH2. Together, our results show that a postmitotic switch from EZH2 to EZH1 establishes novel chromatin domains in neurons with a minimal role in transcriptional maintenance.

While many studies of developmental control have focused on gene activation, less is known about the extent to which regulatory programs are actively repressed in progenitor cells. We previously showed that trimethylation of histone H3 at lysine 9 (H3K9me3) is a repressive mark that is remodeled on protein-coding genes when endodermal progenitors transition to liver and pancreatic {beta} cell fates. Yet whether H3K9me3 is dynamic at promoters and enhancers has not been determined. Here we find that promoters of liver-specific genes are strongly enriched for H3K9me3 in undifferentiated progenitors, whereas such enrichment is not observed at promoters of more broadly expressed liver genes. We further show that enhancers specific to differentiated tissues--including liver, islet, and cerebral cortex--are strongly enriched for H3K9me3 in their corresponding tissue stem and progenitor cells. In hepatoblasts, H3K9me3 contributes to maintaining the undifferentiated state by restricting FOXA2 and HNF4 from binding to most enhancers, while there remain thousands of H3K9me3-marked enhancers where the factors are not restricted from binding. Our findings illustrate how H3K9me3-mediated heterochromatinization can restrict transcription factor engagement in progenitor cells to prevent inappropriate activation during early development. H3K9me3 at enhancers that allow transcription factor binding may reflect developmental competence.

SMC complexes influence virtually all DNA-dependent processes by organizing the genome via the process of loop extrusion. Although transcription has been implicated in regulating SMC complex function, the underlying mechanisms remain unclear. Further, the directionality of loop extrusion observed in biochemical experiments has been difficult to reconcile with the chromatin condensation observed in cells. Here, we use a quiescent yeast model to uncover the relationship between condensin loop extrusion and transcription. Condensin gradually relocates to transcribed gene promoters during quiescence entry, allowing us to dissect condensin targeting mechanisms temporally. Through targeted degradation experiments, we discover that topological stress generated by transcription leads to single-stranded DNA accumulation at promoters, and that these RPA-bound regions are loading sites and extrusion barriers for condensin. We further use a condensin mutant to determine that condensin extrudes loops asymmetrically in cells. We propose that antagonism by RPA universally regulates SMC complex function.

Organismal tolerance of ionizing radiation is a complex trait whose genetic basis has been studied extensively, in large part due to its significance to human health and technological advancement. Conventional mutant screens in model organisms have revealed the paramount role of DNA damage response (DDR) and repair pathways in determining tolerance to ionizing radiation. However, uncovering natural genetic variation in radiotolerance is also of critical importance, as individual differences are associated with the differential susceptibility to cancer as well as differential response to radiation treatment. Genetic variation that underlies phenotype differences in natural populations often occurs in distinct genes and pathways as compared to the genes of major effect revealed by mutant screens, owing to the impact of natural selection on the former. We therefore sought to isolate natural variation in radiotolerance of Drosophila melanogaster by performing extreme QTL mapping. We generated a large genetically diverse multiparental population and exposed 3rd instar larvae to a semi-lethal dose or ionizing radiation. By sequencing surviving adults and comparing their haplotypes to unexposed controls from the same population, we identified a single major effect QTL spanning the 3rd chromosome centromere. The QTL contains 34 genes, none of which are previously implicated in radiotolerance. We interrogated the impact of these genes on radiotolerance through forward genetic analysis and RNA-seq. Our findings implicate diverse processes in radiotolerance including cell-cycle regulation and innate immune function.

Centromeres are essential for chromosome segregation, yet in many genomes they are composed entirely of rapidly evolving repetitive DNA, embedded in other repetitive DNA that forms pericentromeric heterochromatin. Due to the difficulties of manipulating these repeat-rich regions, how the relative size of pericentromeric repeat regions influences chromosome segregation remains an open question. Here, we take advantage of the tractable Schizosaccharomyces pombe system by combining population-level analysis, complete long-read assemblies, and engineered near-isogenic strains to test how pericentromeric repeat copy number affects chromosome biology in its native context. We find that pericentromeric dh/dg arrays on chromosome 3 vary almost tenfold in size among natural S. pombe isolates, ranging from 35 to 265 kb. We converted this natural diversity into an experimental system of nearly isogenic strains that primarily differ in pericentromere size (35 to >350 kb). We found that pericentromere size does not alter baseline growth under standard conditions. However, larger pericentromeres alter transcriptional output and sensitize cells to spindle stress. We show that this spindle-stress phenotype depends on heterochromatin: loss of the H3K9 methyltransferase Clr4 abolishes size-dependent differences, whereas artificial targeting of the Chromosomal Passenger Complex to heterochromatin partially rescues the defect. Thus, we find that larger pericentromeres act as sinks for limiting regulatory factors, weakening their effective concentration at centromeres and compromising faithful chromosome segregation under stress. These results establish that naturally occurring copy-number variation within repetitive pericentromeric DNA is not merely noise, but a functional source of variation in chromosome segregation and gene regulation. Our work provides an experimentally tractable framework for understanding how repeat expansion in centromere-proximal heterochromatin influences chromosome behavior across eukaryotes.

Germinal centers (GCs) support physiological B-cell mutagenesis and are considered lymphoma-permissive; nevertheless, lymphoma development is uncommon. Human in situ follicular neoplasia (ISFN) captures this paradox: premalignant B-cells can persist within GCs for prolonged periods without progressing to overt lymphoma. We found that human ISFN, but not normal GCs, are infiltrated by CD8 T-cells, suggesting that premalignant GC B-cells are locally immune-surveilled. Using mouse models that separate early premalignant fitness from lymphoma-associated evolution, we show that fitness-enhanced premalignant GC B-cells expand transiently but are selectively eliminated by infiltrating cytotoxic CD8 T-cells, while normal GC B-cells are spared. By contrast, evolved premalignant GC B-cells retain their fitness but disable productive CD8 T-cell cytotoxic differentiation, allowing persistence and lymphoma-like transcriptional and genomic evolution. These findings establish GCs as active immune-surveillance sites and show that progression from premalignancy to lymphoma requires both enhanced GC fitness and escape from local immune control. Key findingsGCs undergo active immune-surveillance to detect premalignant B-cells. Premalignant GC B-cells trigger cytotoxic CD8 T-cell responses. Lymphoma-associated evolution enables immune-escape within GCs. Fitness and immune-escape drive evolution from premalignancy to lymphoma. BlurbGerminal centers are considered lymphoma-permissive; however, progression from premalignancy is uncommon. Using models of human in situ follicular neoplasia, Zhang et al. demonstrate that infiltrating CD8 T-cells actively eliminate premalignant GC B-cells. Co-occurrence of lymphoma-like alterations blocks this cytotoxic T-cell response, driving immune escape and lymphoma evolution.

SUMMARYMeiotic recombination initiates with DNA double-strand breaks (DSBs) repaired as either crossovers (COs) or non-crossovers. Across eukaryotes, MSH4/MSH5 (MutS{gamma}) licenses DSB repair intermediates, directing repair into the class I CO pathway via recruitment of MLH1/MLH3 (MutL{gamma}). In mammals, excess MutS{gamma} sites relative to final MutL{gamma} foci suggest additional MutS{gamma} functions, including directing repair through the minor class II CO pathway. We investigated the role of a mammalian-specific 38-amino acid C-terminal domain of MSH5 using mice lacking this domain (Msh5{Delta}C/{Delta}C). Spermatocytes and oocytes load MSH4 normally to achieve CO licensing in zygonema, but these numbers decline precipitously in pachynema, leading to dramatically reduced MutL{gamma} foci and associated pro-CO factors HEI10 and CNTD1. Despite this, licensing factors RNF212B and MutS{gamma}-associated kinase CDK4 remain persistently upregulated in pachynema. Strikingly, the switch from licensing-associated CDK4 to CO-site-associated CDK2 fails to occur in Msh5{Delta}C/{Delta}C mice, even at residual class I CO events. The result is rapid germ cell death prior to prophase I completion in both sexes. Thus, the loss of the MSH5 C-terminus functionally uncouples the regulatory proteins that define the stepwise patterning of class I COs. Our findings reveal novel early roles for the C-terminus of mammalian MSH5 in converting licensed DSB repair intermediates to designated class I COs.

The three-dimensional (3D) genome architecture is highly elastic, adapting to nuclear processes such as transcription and the DNA damage response (Dekker & Mirny 2016; Carre-Simon & Fabre 2021). Nucleotide excision repair (NER) acts within this chromatin context to detect and repair mutagenic lesions induced by ultraviolet (UV) irradiation (Sancar 2016). UV irradiation has been shown to induce restructuring of the 3D genome across multiple scales, including chromatin compartments, domains, and loops. However, the extent to which NER activity contributes to this remodelling is unresolved, as the only prior study tracking such UV-induced changes was limited to repair-proficient cells (Kaya & Adebali 2025). Here, by combining genome-wide chromatin profiling of repair-deficient human cells with loop extrusion simulations, we show that lesion-stalled RNA polymerase II (RNAPII) and repair-associated barriers constrain loop extrusion. These events counter the loop-lengthening effects of UV-induced transcriptional shutdown, leading to shorter chromatin loops and reinforced chromatin domains that facilitate efficient lesion recognition and repair. The contribution by NER machinery underscores 3D genome reorganisation as an active mechanism both initiated and harnessed by DNA repair, rather than a passive consequence of DNA damage. The contribution by RNAPII extends its role beyond activating transcription-coupled repair to promoting a genome-wide repair-permissive state. Together, these findings advance our understanding of how nuclear processes coordinate on a shared chromatin substrate to preserve genome integrity.

In Schizosaccharomyces pombe, the conserved CHD remodeler Mit1 function within the SHREC remodeler-deacetylase complex (a homolog of the metazoan Mi-2/NuRD complex), which is essential for H3K9 methylation-dependent heterochromatin establishment. However, the mechanism by which remodeler activity promotes silencing is unknown. Current models posit a hierarchical relationship between histone modifications and remodeler activity, with Mit1 acting exclusively downstream of H3K9 methylation. Here, we challenge this model by showing that tethering Mit1 at an ectopic site within euchromatin is sufficient to initiate heterochromatin assembly and generate extended domains of de novo H3K9 methylation. This process requires the Mit1 catalytic activity but does not involve direct physical interaction with Clr4, suggesting Mit1-mediated nucleosome remodeling creates a chromatin context that enhances Clr4 function. Using a genome-wide deletion screen, we determined that Mit1-initiated silencing requires all core heterochromatin factors and is critically dependent on Clr4 dosage. Furthermore, Mit1 activity facilitates heterochromatin spreading at subtelomeric regions and promotes H3K9 methylation at novel genomic sites implicated in cellular adaptation. Together, our findings support a model in which remodeler-writer pairs, analogous to reader-writer pairs, constitute conserved regulatory modules through which nucleosome organization directs the establishment of heritable epigenetic states.

During organogenesis, epithelial tissues undergo extensive three-dimensional (3D) remodeling while simultaneously generating specialized cell types. Whether these transient architectural states actively instruct lineage allocation remains unclear. Here we identify a morphogenetic stage in which resolution of epithelial stratification is required for lineage allocation and establishment of endocrine cell mass. We show that loss of the Hippo pathway regulator Merlin disrupts lumen morphogenesis and prevents formation of the transient 3D epithelial architecture that characterizes normal pancreas development. Failure to establish this architectural state alters lineage allocation, impairing acinar differentiation, markedly reducing adult endocrine cell mass, and disrupting glucose homeostasis. Mosaic analyses reveal that these lineage defects arise non-cell autonomously, demonstrating that epithelial architecture itself instructs cell fate decisions. Mechanistically, Merlin coordinates PI3K-regulated polarized membrane trafficking required for apical membrane biogenesis and lumen formation. Together, these findings identify Merlin-dependent membrane trafficking as a mechanism coupling epithelial morphogenesis to lineage allocation and demonstrate that transient developmental architectures can determine the cellular composition of mature organs.

Convergence and extension (C&E) cell movements promote anteroposterior axis extension and narrow both neuroectodermal and mesodermal tissues during gastrulation. Mediolateral cell intercalation is largely responsible for this morphogenetic process in vertebrates, but evidence suggests that additional cell-extrinsic forces generated by surrounding tissues contribute to the shaping of many structures. In zebrafish, for example, mechanical forces generated by the anteriorly migrating prechordal plate cooperate with tissue-autonomous cell intercalations to promote extension of the notochord. Here we propose a novel model for notochord morphogenesis by which mechanical epiboly forces within the enveloping layer are transmitted to the posterior end of the notochord via a cluster of dorsal forerunner cells (DFCs) that physically links the two. We found that scattered or absent DFCs caused by loss of crb2a or sox32, respectively, reduces notochord C&E and exacerbates axis extension defects in planar cell polarity signaling-deficient embryos. Using an automated image segmentation and cell shape analysis pipeline, we show that cells within the posterior notochord fail to properly elongate when DFCs are scattered or absent. Finally, we demonstrate that loss of crb2a and sox32 fails to disrupt C&E of zebrafish embryonic explants in which no epiboly occurs and all extension is driven by cell-intrinsic behaviors. Together, these findings support a model in which DFCs facilitate mechanical coupling of the enveloping layer to the posterior notochord during epiboly to ensure its robust morphogenesis during gastrulation.