Genes & Development

Med12 and Med13 are components of the kinase module of the mediator complex. Mutations of Med12 and Med13 have been associated with neurodevelopmental disorders and various cancers. However, their functions in neural development are not well understood. Here we show that in the developing Drosophila brain, Med12 and Med13 are required to prevent tumorigenic dedifferentiation of intermediate neural progenitors (INPs) and maintain neural stem cell (NSC) self-renewal. We further demonstrate that Med12 and Med13 prevent INP dedifferentiation by coordinating with a subset of core mediator complex subunits to mediate the activation of genes required for INP fate commitment. In contrast, during the maintenance of NSC self-renewal, Med12 and Med13 antagonize the function of a different subset of core mediator complex subunits. Together, our findings reveal that Med12 and Med13 perform two distinct functions in neural progenitors by coordinating with one subset of core mediator complex subunits while antagonizing another. HighlightsO_LILoss of Med12 and Med13 causes dedifferentiation of intermediate neural progenitors C_LIO_LIMed12 and Med13 mediate the activation of target genes of PntP1 C_LIO_LILoss of Med12 and Med13 leads to premature loss of neural stem cells C_LIO_LIMed12 and Med13 act with one subset of core mediator subunits but oppose another C_LI eTOC blurbZhu and his colleagues show that Med12 and Med13 promote cell fate commitment of intermediate neural progenitor cells and self-renewal of neural stem cells. Med12 and Med13 perform these two distinct functions by coordinating with one subset of core mediator complex subunits while opposing another to regulate the expression of different target genes.

BRD4 is a member of the bromodomain and extraterminal domain containing protein family, primarily known for regulating transcriptional elongation and enhancer activity. Heterozygous loss-of-function mutations in BRD4 cause craniofacial and neurodevelopmental impairments. However, it remains unclear why mutations in this general transcriptional activator lead to specific neurodevelopmental defects. Using an in vitro human embryonic brain development model, we demonstrate that BRD4 functions as a repressor of Polycomb-regulated developmental and neuronal genes. Acute degradation or loss-of-function mutations in BRD4 in human embryonic stem cells and neuronal lineage deregulate immediate-early genes important for learning and memory formation. We show that BRD4 interacts with components of the noncanonical Polycomb repressive complex PRC1.6 and co-occupies PRC1.6-bound and bivalently marked promoters (H3K27me3 and H3K4me3). We further demonstrate that H3K14ac and H3K23ac recruit BRD4 to bivalent chromatin via its second bromodomain (BD2). These interaction and co-occupancy data suggest BRD4 could contribute to recruitment or maintenance of PRC1.6 and EED at specific sets of genes. As a result, BRD4 represses key developmental and neuronal transcription factors, as well as genes required for learning and memory formation. Single-cell RNA-seq and single-cell CUT&Tag analyses in unguided neuronal organoids confirm that BRD4 loss-of-function mutations lead to increased expression of Polycomb-regulated developmental transcription factor families, including ZIC, HOX, PAX, SOX, and POU. Additionally, single-cell chromatin accessibility data reveal that BRD4 mutations increase accessibility at transcription factor motifs normally repressed by BRD4. Neuronal organoids with BRD4 mutations lead to altered neuronal cell fate, particularly increased differentiation towards diencephalic and retinal pigment epithelium, including the appearance of eye-like pigmentation. Together, these findings uncover a critical role for BRD4 in preventing the premature activation of developmental transcription factors, providing new mechanistic insights into the pathogenesis of congenital neurodevelopmental disorders. HighlightsO_LIAcute degradation of BRD4 leads to upregulation of bivalently marked developmental and neuronal genes. C_LIO_LIBRD4 interacts with components of the PRC1.6 complex and EED. C_LIO_LIBRD4 represses PRC1.6-repressed and bivalent genes C_LIO_LIH3K14ac and H3K23ac roles in recruiting BRD4 to bivalent chromatin C_LIO_LIBRD4-BD2 mutations lead to premature upregulation of neuronal genes, altering neuronal cell fate C_LI

DNA packaging into heterochromatin is a fundamental mechanism of transcriptional silencing. However, how heterochromatin regulates neurogenesis in the developing cerebral cortex remains poorly understood. A defining feature of heterochromatin is trimethylation of histone H3 lysine 9 (H3K9me3), catalyzed by the H3K9 methyltransferases SETDB1, SUV39H1, and SUV39H2. Here, we generate a cortex-specific triple knockout mouse model lacking Setdb1, Suv39h1, and Suv39h2 to interrogate the collective functions of H3K9 methyltransferases and H3K9me3 during corticogenesis. Loss of H3K9 methyltransferases disrupts cell-cycle dynamics and cortical neurogenesis, resulting in microcephaly. We show that H3K9me3 is associated with the silencing of distinct gene families, lineage-inappropriate genes, and transposable elements, and that its loss is accompanied by local chromatin opening and enhanced transcription factor occupancy. Our findings suggest that H3K9me3 regulates neurogenesis in part by silencing the growth-inhibitory gene Cdkn1c in intermediate progenitors. These results underscore the critical role of heterochromatin in the temporal control of neurogenesis.

Mouse meander tail (mea) mutations produce kinked tails and selective malformation of the cerebellum anterior compartment. The anterior cerebellum defects are cell autonomous with respect to granule cell precursors, but the molecular basis has not been known. Myb-like, SWIRM, and MPN domain containing protein 1 (MYSM1) is a chromatin-associated deubiquitinase that promotes gene expression by removing monoubiquitin from histone H2A, among other targets. Loss of MYSM1 function in mice or humans results in bone marrow failure with defective maturation of B cell lineages. Here we show that extant mea alleles have mutations in Mysm1 and cause both neurological and hematological phenotypes, as do new non-complementing endonuclease-mediated mutations. Multimodal single-nucleus assays show Mysm1 effects on gene expression in several lineages and on the proportion of granule cell precursors by E14.5. Intriguingly, Mysm1 orthologs have been independently lost in several animal and fungal lineages, including yeast, flies, and nematodes. These results unite previously disconnected literature and demonstrate a requirement for MYSM1 activity in compartment-specific development of the cerebellum and suggest potential for compensatory pathways. Significance StatementPerturbations to core regulatory machinery often produce pleiotropic effects and even intensively studied systems can have significant phenotypic effects that were not assessed in models developed for a different purpose. Here we show that classical meander tail mice, characterized by ankylosing spondylitis in tail vertebrae and a cerebellum malformation that defined the anterior-posterior compartment boundary, have mutations in Mysm1, encoding a histone 2A deubiquitinase. We show pigmentation defects and hematopoietic abnormalities that model human disease. While Mysm1 mutations change gene expression patterns in many cerebellar cell types, they selectively decrease the proportion of granule cell lineages. Recurrent loss of Mysm1 orthologs across fungal and animal phylogenies suggests the potential for bypass mechanisms.

AbstractBRCA1-linked cancer genomes contain abundant genome-wide [~]10 kb Group 1 tandem duplications (TDs) that are drivers of tumorigenesis. Group 1 TD formation is recapitulated at a chromosomal Tus/Ter site-specific replication fork barrier in DNA end resection-defective mouse embryonic stem (mES) cells lacking Brca1 exon 11. To explore relationships between DNA end resection and Group 1 TD formation, we analyzed Brca1 coiled coil (CC) domain mutants--separation-of-function alleles that are impaired for homologous recombination but competent for DNA end resection. Notably, Brca1 CC mutants retain the ability to suppress Group 1 TDs in the Tus/Ter system and in a mouse model of Brca1-linked tumorigenesis. These data show that Brca1 CC domain mutant cancers follow a path of tumorigenesis distinct from that of other pathogenic Brca1 alleles. FANCM is a TD co-suppressor, the loss of which is synthetic lethal/sick in combination with Brca1 exon 11 mutation. In contrast, Fancm deletion is well-tolerated by Brca1 CC mutant mES cells. Thus, Group 1 TD formation and Fancm synthetic lethality are linked phenotypes related to defective BRCA1-mediated DNA end resection.

In synovial sarcoma, the BAF subunit SS18 is fused to SSX, a transcriptional repressor, generating the oncogenic SS18::SSX fusion protein. Incorporation of SS18::SSX into BAF complexes leads to their aberrant retargeting to Polycomb-repressed genes via SSX, while simultaneously altering their composition and activity. The presence of BAF at Polycomb target sites is widely assumed to be essential for gene activation. Here, we directly tested the requirement for BAF activity in synovial sarcoma cell survival and SS18::SSX-driven transcription. Using targeted degradation of BAF ATPase subunits and deletion of core components, we show that BAF loss has modest effects on sarcoma cell viability and does not impede SS18::SSX target gene expression. Consistently, deletion of the BAF ATPase subunit Smarca4 does not impair SS18::SSX-driven tumor formation in vivo. Using domain-specific SS18::SSX mutants, we further demonstrate that the fusion can activate oncogenic transcription independently of BAF interaction, and that this activity depends on the C-terminal QPGY-rich domain of SS18. Mechanistically, SS18::SSX promotes transcription by engaging the histone acetyltransferase EP300, independently of BAF. Accordingly, pharmacologic degradation of EP300/CREBBP suppresses SS18::SSX-driven transcriptional programs and impairs synovial sarcoma cell survival. Together, these findings challenge the view that BAF activity is required for SS18::SSX-mediated transcriptional activation and demonstrate that aberrant Polycomb target gene expression is sustained through recruitment of transcriptional coactivators in the absence of BAF. Our work reveals new therapeutic vulnerabilities in synovial sarcoma and suggests broader relevance for targeting coactivator-dependent transcription in fusion-driven cancers. HighlightsO_LIBAF degradation does not alter SS18::SSX-activated transcriptional programs C_LIO_LIDirect SS18::SSX transcriptional activation is independent of BAF interaction C_LIO_LIThe SS18 C-terminus engages the co-activator EP300 to promote gene expression C_LIO_LISmall molecule degraders of EP300/CREBBP abolish SS18::SSX-mediated transcription C_LI

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.

Boys experience an overall increased incidence of several childhood cancers, including medulloblastoma, a clinically heterogeneous cerebellar tumor. In subtypes of Group 3 and Group 4 medulloblastoma, males are three times more prevalent than females. As medulloblastoma is suspected to initiate during fetal development, we hypothesized that this sex bias reflects a combination of prenatal, sex-specific developmental processes and somatic alterations. To test these hypotheses, we compiled a large multi-omics dataset from children with medulloblastoma, which revealed sex-specific alterations, including frequent loss of the inactive X chromosome in females with Group 4. Generation of a sex-matched single-cell transcriptome atlas of the developing murine cerebellum enabled investigation of putative developmental factors underlying sex bias. Progenitors giving rise to Group 3/4 subgroups were more abundant, more proliferative, and harbored more open chromatin for recruitment of LMX1A and OTX2, master transcription factors defining Group 3/4 identity. Advanced genetically engineered mouse models and human cerebellar organoids were leveraged to determine whether sexual dimorphism arises from intrinsic or extrinsic factors. These models showed that the XY genotype contributed to the phenotype, but the predominant effect was driven by presence of the male gonadal hormone testosterone. Our findings provide a sex-specific genetic and neurodevelopmental explanation for male bias in an aggressive pediatric brain tumor. Outcomes from this study may inform novel treatment strategies delivered according to sex and are likely to be broadly applicable to other sex-biased malignancies arising in early life. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/714163v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@3a06faorg.highwire.dtl.DTLVardef@1a01bb7org.highwire.dtl.DTLVardef@7bc9c2org.highwire.dtl.DTLVardef@fb206d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Transcription factors (TFs) bind to enhancers and recruit H3K4me1 methyltransferase KMT2D, chromatin remodeler BAF, and H3K27 acetyltransferase p300 to activate transcription. However, the role of chromatin modifiers in regulating de novo binding of TFs on enhancers remains unclear. Using a robust nuclear translocation system, we show that the muscle lineage-determining TF MyoD binds to chromatin pervasively within one hour, with half of induced MyoD binding sites co-occupied by KMT2D, BAF, and p300. On the majority of these MyoD+ enhancers, acute depletion of KMT2D or short-term inhibition of BAF or p300 enzymatic activity markedly reduces de novo binding of MyoD as well as that of KMT2D, BAF, and p300. On enhancers with intact MyoD binding despite these perturbations, we observe a cooperative recruitment among chromatin modifiers. Similar interdependent relationships are observed between the signal-dependent TF Glucocorticoid Receptor and KMT2D, BAF, and p300. Together, our findings show that chromatin modifiers are not only downstream effectors but also required for de novo binding of TFs on enhancers, refining a model of enhancer establishment as a process governed by functional cooperation rather than a strict hierarchy. Bullet pointsO_LIAcute KMT2D depletion disrupts de novo binding of MyoD, BAF, and p300 on enhancers. C_LIO_LIBAF and p300 enzymatic activities are required for de novo binding of MyoD, BAF, KMT2D, and p300 on enhancers. C_LIO_LICooperative binding of KMT2D, BAF, and p300 on MyoD+ enhancers. C_LIO_LIGR displays interdependencies with chromatin modifiers KMT2D, BAF, and p300 on enhancers. C_LI

MicroRNAs (miRNAs) are key post-transcriptional regulators of cell state transitions, yet their function in early human brain development is largely unknown. Here, we present a longitudinal analysis of miRNA function in developing human forebrain organoids. We show that mRNAs and miRNAs expression mirrors known developmental gene programs and that miRNA biogenesis peaks at neural commitment. To test the function of miRNAs in regulating commitment, we impaired their biogenesis at defined stages. miRNA disruption during pre-neuronal commitment caused severe patterning defects, whereas post-commitment perturbation had minimal impact on forebrain identity. We show that miRNA loss during pre-commitment increased WNT and BMP signaling, thus shifting cell fates towards non-forebrain identity such as midbrain/hindbrain. These effects could be partially rescued by expressing five miRNAs. Our findings uncover a critical time window where miRNAs regulate morphogen signaling in early human neurodevelopment, establishing them as essential temporal determinants of cell fate and brain regional identity.

The ability to clear transcription complexes from DNA is especially important after DNA damage that produces replication stress. Using the bacterium E. coli, we show here that mutations in RNA polymerase that reduce termination, inhibitors of Rho-dependent termination, and inversion of a highly transcribed ribosomal RNA operon both enhance sensitivity to the quinolone ciprofloxacin (CPX); and identify two transcription factors, SspA and RapA, that impact these effects in opposite ways.We demonstrate that the rapA promoter is induced by CPX, independent of the LexA/RecA SOS response but is dependent on DnaA. Previous work has shown that RapA is expressed highest in rapidly growing cells whereas SspA levels respond to starvation.The factors have opposing effects on tolerance to chronic exposure to CPX, with RapA promoting cell growth and SspA inhibiting it. Functional SspA is also required for the CPX toxicity of the rRNA operon inversion; in sspA{Delta} mutants it has no negative consequence. In otherwise wild-type cells, loss of RapA has little effect except in strains lacking RNase HI, the enzyme that removes RNA/DNA hybrids from DNA. However, in cells lacking SspA, RapA strongly promotes survival, suggesting that SspA must block positive effects of RapA on tolerance. The RapA requirement for CPX tolerance is not relieved by RNase HI overexpression and therefore RapA must be not be merely preventing R-loop formation. RapA also in some way promotes the use of RNA loops to initiate DNA replication in the absence of DnaA. We propose that SspA stabilizes stalled or post-termination RNAP/DNA complexes and that the presence of SspA prevents RapA release of these complexes.

SPO11 forms hundreds of double-strand breaks (DSBs) to initiate meiotic recombination that is normally error-free. However, SPO11 activity can be mutagenic when one chromatid incurs closely spaced DSBs (double cuts), especially when DSBs are dysregulated by loss of the ATM kinase. De novo indels and structural variants can arise via end joining at double cuts within a single hotspot (microdeletions) or at adjacent hotspots separated by at least 30 kb, as we now show, sometimes accompanied by ectopic insertions of double-cut fragments. Here, we investigate how meiotic DSB end processing influences end joining. In MRE11-deficient mouse spermatocytes, which do not resect their DSBs, deletions at double cuts occur readily, with end-joining breakpoint profiles closely matching SPO11 DSB profiles. Microdeletions suggest that two DSBs can be as close as [~]21 bp. The tyrosyl DNA phosphodiesterase TDP2 contributes to both deletion formation and ectopic insertion of double-cut fragments, presumably by removing SPO11 from DNA ends prior to joining. Finally, observations suggest a cooperative role for MRE11 and ATM in locally regulating DSB distributions. Our findings provide insight into the mechanism of de novo mutation origin, emphasizing the role of meiotic DSBs in shaping genome evolution.

Temporary disruptions to epigenetic mechanisms can misroute development and permanently alter cell fate. In particular, it was recently shown that transient loss of Polycomb silencing in flies irreversibly reprograms cells toward cancer (Parreno et al. 2024). Whether somatic dysfunction in parents can create multi-generational heritable susceptibility to tumorigenesis is unknown. In eutelic organisms like Caenorhabditis elegans, adult somatic cells no longer divide, precluding somatic cancer, yet tumors can still form in the continuously dividing germline. Here, we show that disruption of coelomocytes, somatic scavenger cells, just in C. elegans mothers, provokes transgenerationally heritable germline tumorigenesis that persists for multiple generations in genetically wild-type descendants. We found that when the coelomocytes phagocytic activity dysfunctions, it impairs clear out of RNA from body fluids, and thus disrupts systemic RNA homeostasis, allowing excess somatic RNAs to access the germline, and leading to widespread transcriptional and small RNA dysregulation and transgenerational loss of germline identity. Converging lines of evidence point towards small RNAs being the heritable agents carrying the pathological information. Together, these findings highlight mechanisms which maintain systemic RNA homeostasis as an important protective barrier against heritable tumorigenesis.

DNA-RNA hybrids (R-loops) form transiently on the genome and regulate cellular homeostasis. They also influence genome editing outcomes, highlighting their therapeutic potential in vivo. This protocol enables high-resolution mapping of DNA-RNA hybrids directly from frozen mouse tissues. Following tissue homogenisation and lysis, genomic DNA is extracted, digested and DNA-RNA hybrids are isolated using the hybrid-specific S9.6 monoclonal antibody. The purified hybrids are then processed for whole-genome sequencing to generate R-loop profiles. For complete details on the use and execution of this protocol, please refer to Puzzo, Crossley et al1. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=188 SRC="FIGDIR/small/716701v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1c0e72borg.highwire.dtl.DTLVardef@188cd77org.highwire.dtl.DTLVardef@695c1corg.highwire.dtl.DTLVardef@11e6eff_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOgraphical abstractC_FLOATNO C_FIG

Brain tumor (Brat) is a Drosophila TRIM-NHL protein required for embryogenesis and neural stem cell differentiation. Although structural and biochemical studies established that the Brat NHL domain specifically binds RNA, the in vivo requirement for this activity has not been directly tested. Here, we used structure-guided mutagenesis and genome engineering to determine whether RNA recognition is essential for Brat function during development. The direct interaction between Brats NHL domain and RNA containing Brat Binding Sites (BBS) can be abolished by alanine substitution of three separate residues on the NHL surface. We introduced these point mutations into the endogenous brat locus by CRISPR-mediated Scarless Gene Editing to generate three independent RNA-binding defective mutant (RBDmt) alleles. Complementation tests demonstrated that each allele behaves as a strong loss-of-function mutation: homozygotes and hemizygotes are inviable, and RBDmt alleles fail to complement classical brat null and hypomorphic alleles. Lethal phase analysis revealed death predominantly during late larval and pupal stages, consistent with known brat alleles. Consistent with the namesake brat phenotype, RBDmt larval brains exhibited widespread expression of neuroblast markers and a marked reduction of neuronal differentiation. In embryos, these alleles failed to complement female sterile brat alleles and recapitulated characteristic abdominal segmentation defects. Finally, RT-qPCR showed increased expression of endogenous Brat target mRNAs in mutant larvae, consistent with loss of Brat-mediated repression. Together, these results demonstrate that direct RNA binding is the essential molecular activity of Brat and that post-transcriptional regulation of Brat target mRNAs underlies its critical roles across development.

N6-methyladenosine (m6A) is the most abundant internal modification in mRNA and has been shown to regulate gene expression through the binding of specific reader proteins, such as YTHDF2, which promotes mRNA decay. Previous studies indicate that YTHDF2 has relatively weak intrinsic RNA-binding affinity, suggesting that additional factors may facilitate its association with target transcripts. Here, we show that the C2H2-Zinc Finger protein, ZNF121, binds mRNA in cells and physically interacts with YTHDF2 in the cytoplasm. We demonstrate that approximately 80% of ZNF121-bound mRNAs are also YTHDF2 targets, and that their binding sites highly correlate with each other. Loss of ZNF121 impairs YTHDF2 binding to shared targets and increases their stability, independent of the presence of m6A modifications. Moreover, co-regulated transcripts are enriched for cell-cycle-related pathways, and ZNF121 depletion leads to elevated expression of the oncogene MDM2, implicating ZNF121 in growth control and the DNA damage response. Our findings identify ZNF121 as a cofactor that enhances YTHDF2-mediated mRNA regulation and reveal a previously unknown layer of control in mRNA decay.

Proper polyadenylation site (PAS) selection is critical for RNA isoform determination. Core spliceosomal components, including U1 snRNP, regulate PAS choice, but whether they work with other splicing factors in this role remains unclear. Here, we establish that the splicing factor SRSF1 regulates PAS selection independently of and through interactions with U1 snRNP. Independent of U1 snRNP, SRSF1 binds RNA near proximal PASs within 3 UTRs to promote their usage, and, in line with this observation, breast cancer tumors with altered SRSF1 levels display shifted 3'-end selection. In conjunction with U1 snRNP, SRSF1 acts on PASs through U1 snRNP-mediated SRSF1-Pol II interactions. Consistent with co-transcriptional regulation, SRSF1 reduces the Pol II elongation index and limits transcription readthrough. Together, our results reveal that SRSF1 shapes RNA isoform determination beyond its canonical role in splicing, through a combination of direct RNA binding and U1 snRNP-dependent coordination with Pol II.

The cerebellar rhombic lip neurogenic niche is critical for glutamatergic neurogenesis, and dysregulated differentiation of rhombic lip lineage cells is hypothesized to cause medulloblastoma (MB), a malignant pediatric cancer lacking targeted therapies. Humans have an expanded rhombic lip subventricular zone compartment not seen in mice or macaques, and the common Group 4 subtype of MB is hypothesized to arise by dysregulation of the EOMES+ unipolar brush cells (UBC) generated by this compartment. However, it is unknown whether humans have unique UBC populations not seen in the mouse and, if so, whether Group 4 MB tumour cells resemble these UBC populations. We integrated 336,598 human and mouse single-cell transcriptomes of the developing cerebellum (9-21 post-conception weeks in humans and E10-P14 in mice) and identified two subpopulations of UBC that are enriched in the human samples, relative to mice. One of these populations, which we term UBC 1, predominates at 11 post-conception weeks, shows upregulation of the Eyes Shut Homolog (EYS) transcript, is predicted to be driven by OTX2, SOX4, and SOX11, and is enriched for axonogenesis and neurodifferentiation pathways. We analyzed 27,735 single-cell transcriptomes from eight MB tumours and recapitulated the observation that Group 4 MB cells best resemble UBC. We then found that two-thirds of Group 3 and Group 4 MB UBC-like cells best resemble human-enriched UBC 1 cells. Gene regulatory network analysis revealed that top regulators of Group 4 MB tumour cells include EOMES, SOX4, and SOX11. Our work provides initial evidence for human-enriched UBC states, and suggests that SOX4 and SOX11 may drive neurogenesis in UBC and gene expression in a subset of Group 4 MB tumour cells. Our findings shed light on genetic determinants of human cerebellar expansion and suggest that human models may be needed to recapitulate the oncogenesis of Group 3 and 4 MB.

AbstractCytoplasmic RNA granules, including stress granules, P bodies, neuronal RNA granules, and germ granules, are essential for RNA storage and regulation across a wide range of organisms. However, dissecting the contributions of individual factors to granule function is challenging because of the interdependence of components in vivo. This is especially true for DEAD-box helicases, common regulators of mRNA granules, whose specific contributions remain unclear. In this study, we developed a synthetic approach to de novo generate germ granules, enabling us to identify the minimal machinery needed for RNA localization and translational activation. Using a self-assembling PopTag-based scaffold derived from Caulobacter fused to the RNA-binding domain (RBD) of the germplasm organizer Oskar, we found that the recruitment of endogenous germ granule mRNAs (nanos and pgc) depended on the DDX4 protein Vasa. By employing orthogonal RNA tethering approaches, we demonstrate that Vasa is both necessary and sufficient for localized mRNA translation. Consistent with these findings, acute depletion of Vasa from endogenous germ granules specifically reduced Nanos translation without affecting mRNA localization, confirming Vasa as a core factor linking RNA recruitment to localized translational activation. These in vivo reconstitution experiments reveal a two-component module in which a scaffold RBD and the Vasa helicase, but not other DEAD-box helicases, enable RNP condensates to accumulate specific RNAs and promote their translation. Overall, our study uncovers previously unrecognized functions of an RNA helicase within ribonucleoprotein condensates and demonstrates the power of synthetic biology to analyze complex biomolecular condensates in living organisms.

Tissue-resident macrophages are increasingly recognized for their roles in promoting organogenesis, yet how macrophages are involved in fetal ovarian development remains unclear. In particular, little is known about ovarian macrophage ontogeny and how it relates to germ cell entry into meiosis and establishment of the oocyte reserve. Here we combine temporally-controlled lineage tracing of yolk-sac erythro-myeloid progenitors, fetal HSC-derived progenitors, and postnatal monocytes to map multi-wave seeding and remodeling of ovarian macrophages across fetal and early postnatal life. We identify three major resident subsets defined by MHCII and CSF1R that display distinct expansion kinetics and persistence, and we show that CCR2-dependent monocyte recruitment is required for efficient maturation of postnatal macrophage populations. Functionally, transient or sustained depletion of CSF1R fetal macrophages perturbs ovarian vascular growth and triggers precocious meiotic initiation without overt loss of germ cells, leading to persistent, premature meiotic progression. Extending macrophage depletion into late gestation disrupts perinatal physiological germ cell attrition despite rapid postnatal macrophage repopulation. Together, our findings establish ovarian macrophages as stage-specific regulators that couple immune ontogeny to ovarian morphogenesis and germ cell quality control during establishment of the oocyte reserve. One Sentence SummaryOvarian macrophages are required for the proper timing of germ cell meiotic entry and progression, vascular growth, and for the physiological clearance of germ cells during establishment of the oocyte reserve in perinatal stages.