Biology subjects

Lee, A. S.

Publications and source records attributed to Lee, A. S..

At least 19 recordsLinked to original sources

bioRxiv2026

RNA m6A mediates de novo DNA methylation and heritable epigenetic silencing in human cells

DNA methylation is the most abundant epigenetic modification on the human genome and changes dynamically during embryogenesis, aging, and disease. How DNA methylation is established de novo remains incompletely understood. Using orthogonal CRISPR platforms to initiate de novo DNA methylation coupled with genome-wide CRISPR screens and proximity-dependent proteomics, we discover the interplay between RNA m6A modifications and de novo DNA methylation to program heritable epigenetic memory in human cells. The RNA m6A writer complex is recruited to chromatin with DNA methyltransferases and histone repressors during the transient establishment phase of heritable epigenetic silencing. Inhibition of RNA m6A during early pluripotent transition leads to defects in de novo DNA methylation, resulting in upregulation of transposable elements that pose a threat to genome integrity during early development. Our work reveals the vital coordination between the epitranscriptome and de novo DNA methylation to establish proper heritable epigenetic silencing in human cells.

genetics↗

bioRxiv2026

CTL1 (SLC44A1) regulates myelin lipid composition and architecture in Schwann cells

Myelin formation requires extensive membrane lipid synthesis, yet how myelinating glia acquire the choline needed for this process remains incompletely understood. Choline transporter-like protein 1 (CTL1; SLC44A1) is highly expressed in Schwann cells, and CTL1 deficiency in oligodendrocytes impairs CNS myelination. Here, we examined the role of CTL1 in peripheral nerve myelination using Schwann cell-specific Ctl1 knockout mice. Unexpectedly, loss of Ctl1 did not alter Schwann cell differentiation, the number of myelinated axons, or myelin thickness, but increased myelin abnormalities, including in-foldings and out-foldings. Lipidomic analysis revealed selective alterations in myelin lipid composition, particularly among long-chain lipid species, together with triglyceride accumulation in whole nerves. CTL1 deficiency also increased mTORC1-associated S6 and mTORC2-associated AKT S473 phosphorylation. Transcriptomic analysis revealed downregulation of gene programs associated with fatty acid {beta}-oxidation, triglyceride catabolism, and oxidative phosphorylation. Following peripheral nerve injury, Ctl1-deficient Schwann cells generated a normal repair response and efficiently remyelinated regenerated axons despite altered mTOR signaling. Together, these findings demonstrate that CTL1 is not required for overall peripheral myelin production but contributes to maintaining normal myelin lipid composition and architecture, revealing an unexpected ability of Schwann cells to sustain myelination despite disruption of CTL1-dependent choline metabolism

neuroscience↗

bioRxiv2026

Transcriptional regulation by Tbx2 paralogs creates diversity within photoreceptor subtypes

Photoreceptors are retinal neurons whose subtype-specific opsin expression and mosaic organization enable parallel encoding of spectral and spatial visual information. Yet, the transcriptional mechanisms establishing and maintaining subtype identity remain poorly understood. We investigated the role of the T-box transcription factors tbx2a and tbx2b in zebrafish photoreceptor development and found evidence for paralog subfunctionalization across photoreceptor lineages. Tbx2b is required for UV-cone generation and prevents fate switching toward rods, while Tbx2a is constrained to UV-cone generation without involving rods. These factors also maintain cone identity by repressing inappropriate M-cone fate. Despite major changes in photoreceptor composition, tbx2 mutants retain luminance- and motion-driven vision, whereas prey capture is abolished only in tbx2b mutants and not tbx2a mutants. Our findings identify the tbx2 paralogs as key regulators of cone-subtype generation and maintenance and reveal how developmental programs diversify photoreceptor fate to appropriately support visually-guided behaviors important for survival.

developmental biology↗

bioRxiv2026

Evolution of a core ribosomal innovation in octopus

Much of biology focuses on how genetic changes mediate new functions, but less attention is given to adaptations in other steps of the central dogma. Octopuses exhibit complex nervous systems and sophisticated behaviors that rival vertebrates, but via an entirely divergent evolutionary history. Here, we serendipitously discovered that octopus ribosomes contain a structural break in the core ribosomal RNA that is unique among all animals. This break site enhances translation fidelity to reduce miscoding and subsequent protein aggregation, even when engineered into evolutionarily distant bacterial ribosomes. Furthermore, high fidelity translation by octopus ribosomes supports proteomic stability during extensive RNA editing observed in cephalopods, suggesting synergy between distinct non-canonical modes of gene regulation. This adaptation emerged in recently derived octopuses with expanded nervous systems, thereby revealing a mechanism that could broadly support the evolution of novel organismal traits.

biochemistry↗

bioRxiv2026

Enteroviral epitope mimicry enables NK cell-mediated targeting of ASPH in hepatocellular carcinoma

Cancer development is shaped by host-microbe interactions, including viral infections. While several viruses are established oncogenic drivers, their potential protective roles in cancer remain unclear. Here we identify a dominant antibody response to CE1, a consensus epitope of enterovirus and rhinovirus, that is associated with reduced hepatocellular carcinoma (HCC) incidence and mortality. Anti-CE1 antibodies selectively recognize HCC cells and mediate anti-tumor activity through NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). Mechanistically, anti-CE1 antibodies cross-react with aspartate {beta}-hydroxylase (ASPH), with CE1-ASPH sequence homology underpinning tumor recognition and cytotoxicity. Clinically, ASPH is aberrantly upregulated in HCC and correlates with inferred NK cell-associated ADCC activity and improved survival in CE1-seropositive patients. Collectively, these findings reveal a mechanism by which antiviral humoral immunity confers cancer protection through molecular mimicry and highlight anti-CE1 immunity as a potential therapeutic strategy in HCC.

cancer biology↗

bioRxiv2025

Preoptic Neurons that Regulate Hibernation Entry

Evolution of seasonal hibernation has enabled mammals to survive harsh conditions by entering a state of prolonged hypometabolism and hypothermia with body temperatures as low as 0-4{degrees}C1-6. Despite decades of physiological studies, the genetic tools to study hibernation have remained limited and the mechanisms that induce hibernation entry are still unknown. Focusing on the brain, we map state-dependent neuronal activity across the hibernation cycle in Syrian hamsters and identify the hypothalamic anterior preoptic area (aPOA) as a key regulator of hibernation entry. Single-nucleus RNA and chromatin profiling provided a map of neuronal populations present in the hamster POA and enabled the discovery and design of an enhancer AAV that selectively targets hibernation-associated aPOA subpopulations. Using this genetic approach, we show that Samd3-positive aPOA glutamatergic neurons are necessary for entry into hibernation and that their activation is sufficient to induce a prolonged hypothermic state with associated nesting behavior. Together, we identify the first neuronal population that controls entry into hibernation, opening new avenues for investigating and manipulating the metabolic and physiological mechanisms underlying this extreme state of "suspended animation" and its potential applications in aging and disease.

neuroscience↗

bioRxiv2025

Large-scale discovery of neural enhancers for cis-regulation therapies

CRISPR-based gene activation (CRISPRa) has emerged as a promising therapeutic approach for neurodevelopmental disorders (NDD) caused by haploinsufficiency. However, scaling this cis-regulatory therapy (CRT) paradigm requires pinpointing which candidate cis-regulatory elements (cCREs) are active in human neurons, and which can be targeted with CRISPRa to yield specific and therapeutic levels of target gene upregulation. Here, we combine Massively Parallel Reporter Assays (MPRAs) and a multiplex single cell CRISPRa screen to discover functional human neural enhancers whose CRISPRa targeting yields specific upregulation of NDD risk genes. First, we tested 5,425 candidate neuronal enhancers with MPRA, identifying 2,422 that are active in human neurons. Selected cCREs also displayed specific, autonomous in vivo activity in the developing mouse central nervous system. Next, we applied multiplex single-cell CRISPRa screening with 15,643 gRNAs to test all MPRA-prioritized cCREs and 761 promoters of NDD genes in their endogenous genomic contexts. We identified hundreds of promoter- and enhancer-targeting CRISPRa gRNAs that upregulated 200 of the 337 NDD genes in human neurons, including 91 novel enhancer-gene pairs. Finally, we confirmed that several of the CRISPRa gRNAs identified here demonstrated selective and therapeutically relevant upregulation of SCN2A, CHD8, CTNND2 and TCF4 when delivered virally to patient cell lines, human cerebral organoids, and a humanized mouse model of hTcf4. Our results provide a comprehensive resource of active, target-linked human neural enhancers for NDD genes and corresponding gRNA reagents for CRT development. More broadly, this work advances understanding of neural gene regulation and establishes a generalizable strategy for discovering CRT gRNA candidates across cell types and haploinsufficient disorders.

genomics↗

bioRxiv2025

Deletion of Cacna1c (CaV1.2) in D1-expressing cells elicits divergent sex-specific effects on aversive and spatial memories

Dopamine signaling is critical for cognitive and emotional regulation and is implicated in multiple neuropsychiatric disorders. One downstream effector of dopamine is the L-type calcium channel CaV1.2, encoded by the risk gene CACNA1C. Genome-wide association studies have consistently linked CACNA1C single nucleotide polymorphisms to schizophrenia, bipolar disorder, and related conditions. We previously showed that homozygous deletion of Cacna1c in dopamine receptor 1 (D1)-expressing cells enhances remote (30 days post-training) contextual fear memory in male mice. Here, we extend these findings by examining sex- and gene dosage-specific behavioral consequences of Cacna1c loss in D1 cells. We find that D1-Cacna1c deletion produces a sex- and gene dosage-dependent effect on fear memory. In males, homozygous loss of D1-Cacna1c heightens remote contextual fear at 30-days post-training, replicating prior findings, whereas partial loss had no effect. Cue-associated fear memory remained unaffected across genotypes. In contrast, females exhibited heightened contextual fear with both heterozygous and homozygous D1-Cacna1c loss at 24-hrs, 7-days, and 30-days post-training, indicating increased sensitivity to contextual aversive learning. Cue-associated fear memory was higher at 24-hrs but normalized at later time points in females. In the Water Y-maze, males with heterozygous or homozygous D1-Cacna1c loss showed impaired spatial memory at 7-days post-training, whereas females were unaffected. D1-Cacna1c deletion reduced locomotor activity selectively in females during the initial 5-mins of a 60-min session, with no genotype effects in males. Social interaction and anxiety-like behavior were unchanged across groups. Together, these findings highlight the interplay between dopamine receptor signaling and calcium channel function in shaping sex-dependent aspects of memory.

neuroscience↗

bioRxiv2025

Giant DNA viruses encode a hallmark translation initiation complex of eukaryotic life

In contrast to living organisms, viruses were long thought to lack protein synthesis machinery and instead depend on host factors to translate viral transcripts. Here, we discover that giant DNA viruses encode a distinct and functional IF4F translation initiation complex to drive protein synthesis, thereby blurring the line between cellular and acellular biology. During infection, eukaryotic IF4F on host ribosomes is replaced by an essential viral IF4F that regulates viral translation, virion formation, and replication plasticity during altered host states. Structural dissection of viral IF4F reveals that the mRNA cap-binding subunit mediates exclusive interactions with viral mRNAs, constituting a molecular switch from translating host to viral proteins. Thus, our study establishes that viruses express a eukaryotic translation initiation complex for protein synthesis, illuminating a series of evolutionary innovations to a core process of life.

microbiology↗

bioRxiv2025

Replication stress alters CENP-A nucleosome stability during S phase

1.The maintenance of centromere identity is essential for the proper segregation of chromosomes during cell division. Centromere identity is epigenetically specified by centromeric histone H3 variant CENP-A, and its retention during DNA replication is facilitated by HJURP chaperone. Replication stress disrupts replication fork progression and can negatively influence the interactions between histone chaperone network necessary for retention and deposition of parental and new histones, respectively. In this study we investigate the role of replication stress response on centromere inheritance. We define changes in centromere-associated proteins that govern stability of centromeric and canonical nucleosomes through proximity labeling coupled with affinity purification mass spectrometry. We identified that under replication stress, CENP-A-containing chromatin strongly enriches for SWI/SNF chromatin remodeling proteins ATRX. We show that depletion of ATRX and its associated histone H3.3 chaperone DAXX results in the loss CENP-A retention in S-phase and loss persists into the subsequent cell cycle. Altogether our findings provide insight into how replication stress negatively influences centromeric chromatin instability and delineates a function of DAXX-ATRX complex in maintaining centromere inheritance during DNA replication.

biochemistry↗

bioRxiv2025

Direct Readout of Multivalent Chromatin Reader-Nucleosome Interactions by Nucleosome Mass Spectrometry

Histone post-translational modifications (PTMs) often serve as distinct recognition sites for the recruitment of chromatin-associated proteins (CAPs) for epigenome regulation. While CAP-PTM interactions have been extensively studied using histone peptides, this cannot consider the regulatory potential of multi-site binding on intact nucleosomes. To overcome this limitation, we applied Nucleosome Mass Spectrometry (Nuc-MS), a native Top-Down MS approach that enables controlled disassembly of intact CAP:nucleosome (CAP:nuc) complexes to provide a direct readout of the contained histone proteoforms. As proof of principle, we show the BPTF PHD-BD native tandem reader requires coincident H3K4me3K9acK14acK18ac for effective nucleosome engagement. We extend our approach to explore how the BRD4 (native BD1-BD2), DNMT3A-MPP8 (chimeric PWWP-CD), and PtSHL (native BAH-BD) tandem readers interact with endogenous nucleosomes. Each reveals distinct enrichment profiles: BRD4 favoring di- and tri-acetylated histone H4 proteoforms, whereas DNMT3A-MPP8 and PtSHL preferentially interact with hypermethylated H3 proteoforms. Of note the latter enriches combinatorial {H3K4me3K27me3} on the same histone tail in HeLa chromatin, and thus expands the potential biology of this widely studied bivalent signature. By directly characterizing CAP:nuc complex composition with combinatorial PTM information in a single readout, Nuc-MS serves as a new approach to discover the modifications driving binding, and therefore primary candidates to explore for structural biology and genomic studies. O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/651740v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@8815e9org.highwire.dtl.DTLVardef@5685b5org.highwire.dtl.DTLVardef@643881org.highwire.dtl.DTLVardef@1b9bc20_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFor TOC only.C_FLOATNO Synopsis. Nuc-MS provides the ability to control the disassembly of chromatin-associated protein-nucleosome complexes (CAP-nuc) and delineate the histone proteoforms driving tandem reader domain binding. C_FIG

biochemistry↗

bioRxiv2025

Nucleosome context regulates chromatin reader preference

Chromatin is more than a simple genome packaging system, and instead locally distinguished by histone post-translational modifications (PTMs) that can directly change nucleosome structure and / or be "read" by chromatin-associated proteins to mediate downstream events. An accurate understanding of histone PTM binding preference is vital to explain normal function and pathogenesis, and has revealed multiple therapeutic opportunities. Such studies most often use histone peptides, even though these cannot represent the full regulatory potential of nucleosome context. Here we apply a range of complementary and easily adoptable biochemical and genomic approaches to interrogate fully defined peptide and nucleosome targets with a diversity of mono or multivalent chromatin readers. In the resulting data, nucleosome context consistently refined reader binding, and multivalent engagement was more often regulatory than simply additive. This included abrogating the binding of the Polycomb group L3MBTL1 MBT to histone tails with lower methyl states (me1 or me2 at H3K4, H3K9, H3K27, H3K36 or H4K20); and confirmation that the CBX7 chromodomain and AT-hook-like motif (CD-ATL) tandem act as a functional unit to confer specificity for H3K27me3. Further, in vitro nucleosome preferences were confirmed by in vivo reader-CUT&RUN genomic mapping. Such data confirms that more representative chromatin substrates provide greater insight to biological mechanism and its disorder in human disease.

biochemistry↗

bioRxiv2025

Sex-biased Transcriptome in in vitro Produced Bovine Early Embryos

BackgroundMorphologic sex differences between males and females typically emerge after the primordial germ cell migration and gonad formation, although sex is determined at fertilization based on chromosome composition. A key debated sexual difference is the embryonic developmental rate, with in vitro produced male embryos often developing faster. However, the molecular mechanisms driving early embryonic sex differences remain unclear. ResultsTo investigate the transcriptional sex difference during early development, in vitro produced bovine blastocysts were collected and sexed by PCR. A significant male-biased development was observed in expanded blastocysts. Ultra-low input RNA-seq analysis identified 837 DEGs, with 231 upregulated and 606 downregulated in males. Functional enrichment analysis revealed male-biased DEGs were associated with metabolic regulation, whereas female-biased DEGs were related to female gonad development, sex differentiation, inflammatory pathways, and TGF-beta signaling. Comparing X chromosome and autosome expression ratio, we found that female-biased DEGs contributed to the higher X-linked gene dosage, a phenomenon not observed in male embryos. Moreover, we identified the sex-biased transcription factors and RNA-bind proteins, including pluripotent factors such as SOX21 and PRDM14, and splicing factors FMR1 and HNRNPH2. Additionally, we revealed 1,555 significantly sex-biased differential alternative splicing (AS), predominantly skipped exons, mapped to 906 genes, with 59 overlapping with DEGs enriched in metabolic and autophagy pathways. By incorporating novel isoforms from long reads sequencing, we identified 1,151 sex-biased differentially expressed isoforms (DEIs) associated with 1,017 genes. Functional analysis showed that female-biased DEIs were involved in the negative regulation of transcriptional activity, while male-biased DEIs were related to energy metabolism. Furthermore, we identified sex-biased differential exon usage in DENND1B, DIS3L2, DOCK11, IL1RAPL2, and ZRSR2Y, indicating their sex-specific regulation in early embryo development. ConclusionThis study provided a comprehensive analysis of transcriptome differences between male and female bovine blastocysts, integrating sex-biased gene expression, alternative splicing, and isoform dynamics. Our findings indicate that enriched metabolism processes in male embryos may contribute to the faster developmental pace, providing insights into sex-specific regulatory mechanisms during early embryogenesis. Plain English summaryMale and female early embryos develop at different speeds, with male embryos often developing faster than female embryos. However, the reasons behind these early differences remain unclear. In this study, we examined gene activity in bovine embryos to uncover the biological factors regulating these early sex differences. We collected in vitro-produced bovine blastocysts, examined their sex, and confirmed that male embryos develop faster. By analyzing global gene activity, including alternative splicing, which allows one gene to code for multiple RNA isoforms and proteins, we found distinct gene expression profiles between male and female embryos. Male embryos showed higher activity in genes related to metabolism and cellular functions, while female embryos had increased activity in genes associated with female-specific gonad development and gene expression regulation. We also examined differences in how genes on the X chromosome were expressed. Female embryos had higher X-linked gene expression, which may contribute to sex-specific developmental regulation. Additionally, we identified sex-specific transcription factors and RNA-binding proteins that regulate early embryo development, some of which are known to control pluripotency and gene splicing. Overall, our study provides new insights into how gene activity shapes early sex differences, suggesting that enhanced metabolism in male embryos may be a key driver of their faster developmental rate. HighlightsO_LIMale embryos develop faster due to increased gene expression in metabolism pathways C_LIO_LIFemale embryos exhibit higher X-linked gene expression, suggesting X-dosage compensation plays a role in early development C_LIO_LISex-biased alternative splicing events contribute to embryonic metabolism, autophagy, and transcriptional regulation in embryos C_LIO_LISex-biased isoform diversity contributes to distinct developmental regulation in male and female embryos C_LIO_LIKey pluripotency factors (SOX21, PRDM14) and splicing regulators (FMR1, HNRNPH2) drive sex-specific gene expression C_LI

developmental biology↗

bioRxiv2024

Polymorphic tandem repeats shape single-cell gene expression across the immune landscape

Tandem repeats (TRs) - highly polymorphic, repetitive sequences across the human genome - are important regulators of gene expression but remain underexplored due to challenges in accurate genotyping and analysis1. Here, we generate new whole genome and single-cell RNA sequencing from >5.4 million blood-derived cells across 1,925 individuals in two cohorts [Cuomo et al., accompanying manuscript], and perform meta-analysis to characterize the impact of variation in >1.7 million TR loci on immune cell type-specific gene expression. We identify >69,000 single-cell expression TR loci (sc-eTRs), 30.7% of which are specific to one of 28 immune cell types, and reveal dynamic regulatory effects using cell-state inference. Matched single-cell ATAC sequencing profiles from >3.4 million nuclei in 922 individuals [Xue et al., accompanying manuscript]. uncover chromatin accessibility QTLs for nearly one-third of expression-associated TRs, supporting coordinated effects on cis-regulatory architecture. Fine-mapping implicates 1,490 TRs as candidate causal drivers of gene expression in 6.1% of tested genes, and colocalization analyses highlight >200 genes in which TRs likely mediate genetic associations with immune and hematological traits. Together, these results provide a genome-wide, multiomic view of TR-mediated regulation in the human immune system, establishing TRs as key contributors to cell type-specific regulatory variation and complex trait architecture.

genomics↗

bioRxiv2024

Cerebellar output neurons impair non-motor behaviors by altering development of extracerebellar connectivity

The capacity of the brain to compensate for insults during development depends on the type of cell loss, whereas the consequences of genetic mutations in the same neurons are difficult to predict. We reveal powerful compensation from outside the cerebellum when the excitatory cerebellar output neurons are ablated embryonically and demonstrate that the minimum requirement for these neurons is for motor coordination and not learning and social behaviors. In contrast, loss of the homeobox transcription factors Engrailed1/2 (EN1/2) in the cerebellar excitatory lineage leads to additional deficits in adult learning and spatial working memory, despite half of the excitatory output neurons being intact. Diffusion MRI indicates increased thalamo-cortico-striatal connectivity in En1/2 mutants, showing that the remaining excitatory neurons lacking En1/2 exert adverse effects on extracerebellar circuits regulating motor learning and select non-motor behaviors. Thus, an absence of cerebellar output neurons is less disruptive than having cerebellar genetic mutations.

neuroscience↗

bioRxiv2024

Temperature and photoperiod differentially impact maternal phenotypes in diapause egg-laying Aedes albopictus mosquitoes

BackgroundAedes albopictus (Skuse 1894) mosquitoes can transmit deadly arboviruses and are globally invasive due to their ability to survive in both tropical and temperate climates. Although adults cannot survive harsh winters, females are capable of anticipating seasonal change and producing overwintering diapause (DP) eggs that remain in a state of arrested development over the winter and hatch when favorable conditions return in the spring. Previous work has shown that shortening photoperiod (day length) alone is sufficient for DP induction. While decreasing temperatures can facilitate DP entry, temperature signals alone are not sufficient to induce DP. Methodology/Principal FindingsTo identify maternal phenotypes predictive of DP egg production, we characterized aspects of maternal physiology and behavior to identify those that correlate with DP egg production and changes in photoperiod, versus changes in temperature. Neither changes in temperature nor photoperiod impacted protein preference, blood meal consumption, or total number of eggs produced per female. Egg retention and oviposition timing were influenced by temperature, independent of DP egg production. However, females housed under short photoperiod conditions showed increased starvation resistance, despite showing similar levels of locomotor activity and internal stores of triacylglycerols, glucose, glycogen, and trehalose compared to females housed in long photoperiods. Conclusions/SignificanceThese results suggest that temperature and photoperiod differentially affect maternal phenotypes and identify starvation resistance as a maternal phenotype that is influenced by photoperiod and can be used to predict DP egg status. AUTHOR SUMMARYAedes albopictus mosquitoes can survive in temperate climates because females are able to detect changes in temperature and day length in the autumn to produce "diapause" eggs that are provisioned with extra nutrients to survive harsh winters by remaining in a state of arrest and hatching in the spring. Although temperature and day length normally change in concert with each other throughout the year, studies have shown that day length is the most important cue used by female mosquitoes to initiate diapause egg production. In this study, the authors examined aspects of feeding and reproduction to determine which of these correlated with day length. Many aspects of reproduction were influenced by temperature; however, housing female mosquitoes under autumn-like short day conditions led to increased starvation resistance. Surprisingly these females survive longer even though they do not appear to expend less energy compared to females housed in spring-like long day conditions at the same temperature. This suggests that the mosquito mothers undergo changes in their metabolism that can be used predict whether she will produce eggs that can survive the winter. This new knowledge may lead to new targets to disrupt seasonal reproduction in mosquitoes and limit the parts of the world where they can survive.

physiology↗

bioRxiv2023

Engrailed transcription factors direct excitatory cerebellar neuron diversity and survival

The excitatory neurons of the three cerebellar nuclei (eCN) form the primary output for the cerebellar circuit. The medial eCN (eCNm) were recently divided into molecularly defined subdomains in the adult, however how they are established during development is not known. We define molecular subdomains of the eCNm using scRNA-seq and spatial expression analysis and show they evolve during embryogenesis to resemble the adult. Furthermore, the eCNm is transcriptionally divergent from the rest of the eCN by E14.5. We previously showed that loss of the homeobox genes En1 and En2 leads to death of a subset of embryonic eCNm. We demonstrate that mutation of En1/2 in embryonic eCNm results in cell death of specific posterior eCNm molecular subdomains and loss of TBR2 (EOMES) expression in an anterior subdomain, as well as reduced synaptic gene expression. We further reveal a similar function for EN1/2 in mediating TBR2 expression, neuron differentiation and survival in the two other cerebellar excitatory neuron types. Thus, our work defines embryonic eCNm molecular diversity and reveals conserved roles for EN1/2 in the cerebellar excitatory neuron lineage.

developmental biology↗

bioRxiv2023

Autism candidate gene rbm-26 (RBM26/27) regulates MALSU-1 to protect against mitochondrial dysfunction during axon development.

Mitochondrial dysfunction is thought to be a key component of neurodevelopmental disorders such as autism, intellectual disability, and ADHD. However, little is known about the molecular mechanisms that protect against mitochondrial dysfunction during neurodevelopment. Here, we address this question through the investigation of rbm-26, the C. elegans ortholog of the RBM27 autism candidate gene, which encodes an RNA-binding protein whose role in neurons is unknown. We report that RBM-26 (RBM26/27) protects against axonal defects by negatively regulating expression of the MALS-1 (MALSU1) mitoribosomal assembly factor. Autism-associated missense variants in RBM-26 cause a sharp decrease in RBM-26 protein expression along with defects in in axon overlap and axon degeneration that occurs during larval development. Using a biochemical screen, we identified the mRNA for the MALS-1 mitoribosomal assembly factor as a binding partner for RBM-26. Loss of RBM-26 function causes a dramatic overexpression of mals-1 mRNA and MALS-1 protein. Moreover, genetic analysis indicates that this overexpression of MALS-1 is responsible for the mitochondrial and axon degeneration defects in rbm-26 mutants. These observations reveal a mechanism that regulates expression of a mitoribosomal assembly factor to protect against axon degeneration during neurodevelopment.

neuroscience↗