Biology subjects

Burgess, S. M.

Publications and source records attributed to Burgess, S. M..

At least 19 recordsLinked to original sources

bioRxiv2025

Complete de novo assembly and re-annotation of the zebrafish genome

The zebrafish (Danio rerio) is widely used in vertebrate research, but its reference genome assembly has contained extensively unresolved regions across both euchromatic and heterochromatic compartments. The previous reference genome assembly, GRCz11, consisted of 19,725 contigs assembled into 1,917 scaffolds. Recent advances in both long-read sequencing technologies and genome assembly algorithms have made "complete" genome assemblies possible for the first time. We used homozygous fish from two lab strains, "Tubingen" and "AB," for de novo genome assemblies. The new assemblies incorporated 7% more genomic sequence than GRCz11 and an additional 130 million bases of previously unassembled sequence. RefSeq annotation incorporating newly generated Iso-Seq cDNA sequences have added notable increases in mRNAs (68%), lncRNAs (47%), and misc_RNAs (1099%). Two assemblies have been elevated to reference genome status (GRCz12tu and GRCz12ab). We generated an additional 40 draft haplotypes to create a zebrafish pangenome resource and demonstrate its utility for variant analysis.

genomics↗

bioRxiv2025

Tnfrsf10 Signaling is Required to Maintain the Stem Cell Niche in the Zebrafish Lateral Line

Regenerative capacity varies significantly among species and across different organs. In mammals, the inner ear exhibits a limited ability to regenerate hair cells, whereas the lateral line sensory organ of zebrafish demonstrates apparently limitless capability to spontaneously regenerate hair cells following injury. However, the specific cell populations representing the stem cells in the neuromast and the mechanism of stem cell self-renewal remain unclear. In this study, we show that a mutation in tnfrsfa led to the depletion of neuromast sensory organs and impairment of hair cell regeneration in zebrafish. The decrease of cells peripheral to the neuromasts in tnfrsfa mutants indicates that these peripheral cells, particularly cells called "mantle cells", serve as the stem cells within the neuromast. Furthermore, our findings indicate that the ligands Tnfsf10 and Tnfsf10l3 (TRAIL) act through Tnfrsfa activating the NF-{kappa}B signaling pathway in the mantle cells in a non-cell-autonomous manner. NF-{kappa}B signaling is crucial for maintaining the stem cell identity of the mantle cells through activation or maintenance of sox2 expression. These findings demonstrate a critical role for TNF superfamily signaling in stem cell maintenance. HighlightsO_LIZebrafish tnfrsfa maintains the stem cell niche in neuromasts and is necessary for hair cell regeneration of the lateral line C_LIO_LITnfrsfa is essential for mantle cell proliferation during lateral line development and hair cell regeneration C_LIO_LITnfsf10 and Tnfsf10l3 signal through Tnfrsfa, activating NF-{kappa}B signaling and Sox2 expression in the mantle cells in a non-cell-autonomous manner C_LIO_LITnfrsfa regulates the development of neuromast independently of the Notch signaling pathway C_LI

developmental biology↗

bioRxiv2025

Zebrafish lack a strong meiotic checkpoint response to defects in chromosome synapsis

The synaptonemal complex (SC) is a meiosis-specific structure that aligns homologous chromosomes and facilitates the repair of meiotic DNA double-strand breaks (DSBs). Defects in SC assembly or unrepaired DSBs trigger a prophase I checkpoint to prevent the formation of aneuploid gametes. The strength of these checkpoints varies among species and between sexes. Whether zebrafish (Danio rerio) have prophase I surveillance mechanisms that monitor chromosome synapsis and/or meiotic DSB repair has not been explored. To investigate how defects in SC formation affect gametogenesis in zebrafish, mutations in genes encoding two structural components of the SC, syce2 and sycp1 were examined. While syce2 and sycp1 fish exhibit defects in both synapsis and DSB repair, the two mutants show different reproductive outcomes. syce2 mutant females and males produce a significant percentage of normal progeny. In contrast, sycp1 mutant females produce fewer normal offspring, while sycp1 mutant males are infertile, with spermatocytes arrested at metaphase I. Notably, offspring from syce2 and sycp1 mutant mothers show extensive somatic mosaic aneuploidy, indicating that defects in the meiotic machinery can lead to genome instability during embryogenesis. Our findings suggest that a checkpoint monitoring the progression of synapsis may be weak or absent in the zebrafish, resembling the situation in yeast, plants, and Drosophila, rather than in mice and the nematode Caenorhabditis. elegans where robust mechanisms exists to silence unpaired chromosomes leading to meiocyte apoptosis. Author SummaryMeiosis is a specialized cell division that reduces chromosome numbers for the production of haploid gametes and is necessary for sexual reproduction. During meiosis prophase I, precise coordination is required for key events such as the formation and repair of DNA double-strand breaks and the synapsis of homologous chromosomes, which is mediated by the synaptonemal complex. Specialized checkpoint pathways monitor the proper execution of these events. Errors in either process can induce a checkpoint response leading to cell death or the production of aneuploid gametes. Here, we demonstrate that the checkpoint monitoring synapsis is weak--or potentially absent--in zebrafish, suggesting that a robust synapsis checkpoint is not conserved across all vertebrates. We discuss this finding in the context of the teleost-specific whole genome duplication and the widespread heterogametic switching (e.g., XY to ZW systems) within this diverse clade, which includes over 30,000 species.

cell biology↗

bioRxiv2025

Transposon insertion causes ctnnb2 transcript instability that results in the maternal effect zebrafish ichabod (ich) mutation

The maternal-effect mutation ichabod (ich) results in ventralized zebrafish embryos due to impaired induction of the dorsal canonical Wnt-signaling pathway. While previous studies linked the phenotype to reduced ctnnb2 transcript levels, the causative mutation remained unidentified. Using long-read sequencing, we discovered that the ich phenotype stems from the insertion of a non-autonomous CMC-Enhancer/Suppressor-mutator (CMC-EnSpm) transposon in the 3UTR of the gene. Through reporter assays, we demonstrate that while wild type ctnnb2 mRNAs exhibit remarkably high stability throughout the early stages of development, the insertion of the transposon dramatically reduces transcript stability. Genome-wide mapping of the CMC-EnSpm transposons across multiple zebrafish strains also indicated ongoing transposition activity in the zebrafish genome. Our findings not only resolve the molecular basis of the ich mutation but also highlight the continuing mutagenic potential of endogenous transposons and reveal unexpected aspects of maternal transcript regulation during early zebrafish development.

developmental biology↗

bioRxiv2024

Neuronal activation in the axolotl brain promotes spinal cord regeneration

The axolotl retains a remarkable capacity for regenerative repair and is one of the few vertebrate species capable of regenerating its brain and spinal cord after injury. To date, studies investigating axolotl spinal cord regeneration have placed particular emphasis on understanding how cells immediately adjacent to the injury site respond to damage to promote regenerative repair. How neurons outside of this immediate injury site respond to an injury remains unknown. Here, we identify a population of dpErk+/etv1+ glutamatergic neurons in the axolotl telencephalon that are activated in response to tail amputation. Importantly, the activation of these neurons facilitates successful tail regeneration by promoting axon extension into the newly regenerated tissue. We demonstrate that these dpErk+ neurons extend axons into the hypothalamus, which has a well-established role in hormone production. Furthermore, these neurons upregulate the neuropeptide neurotensin in response to injury, which in turn stimulates the production of multiple hormones in the hypothalamus to promote regenerative repair. Together, these findings identify a unique population of neurons in the axolotl brain whose activation is necessary for successful spinal cord regeneration, and sheds light on how neurons outside of the immediate injury site respond to a spinal cord injury.

developmental biology↗

bioRxiv2023

The reference genome of the paradise fish (Macropodus opercularis)

Over the decades, a small number of model species, each representative of a larger taxa, have dominated the field of biological research. Amongst fishes, zebrafish (Danio rerio) has gained popularity over most other species and while their value as a model is well documented, their usefulness is limited in certain fields of research such as behavior. By embracing other, less conventional experimental organisms, opportunities arise to gain broader insights into evolution and development, as well as studying behavioral aspects not available in current popular model systems. The anabantoid paradise fish (Macropodus opercularis), an "air-breather" species from Southeast Asia, has a highly complex behavioral repertoire and has been the subject of many ethological investigations, but lacks genomic resources. Here we report the reference genome assembly of Macropodus opercularis using long-read sequences at 150-fold coverage. The final assembly consisted of {approx}483 Mb on 152 contigs. Within the assembled genome we identified and annotated 20,157 protein coding genes and assigned {approx}90% of them to orthogroups. Completeness analysis showed that 98.5% of the Actinopterygii core gene set (ODB10) was present as a complete ortholog in our reference genome with a further 1.2 % being present in a fragmented form. Additionally, we cloned multiple genes important during early development and using newly developed in situ hybridization protocols, we showed that they have conserved expression patterns.

genomics↗

bioRxiv2022

Stat3 has a different role in axon growth during development than it does in axon regeneration after injury

Signal transducer and activator of transcription 3 (STAT3) is essential for neural development and regeneration as a key transcription factor and mitochondrial activator. In this study, using zebrafish posterior lateral line (PLL) axons, we demonstrate that Stat3 plays distinct roles in PLL axon embryonic growth and regeneration. In zebrafish stat3 mutants the PLL axons were truncated during embryonic growth. Expressing stat3 in neurons of the PLL ganglion rescued their axon growth suggesting stat3 promoted PLL axon growth in a cell-autonomous manner. Using Jak/Stat signaling inhibitors or modified Stat3, we found Jak/Stat signaling was dispensable for PLL axon growth. We showed that Stat3 was co-localized with mitochondria in PLL axon and lower ATPase activity in stat3 mutant embryos than wild-type embryos. The PLL axon growth was affected by mitochondrial modulators indicating that Stat3 regulated PLL axon growth through mitochondrial Stat3 function. Mutation of stat3 or treating with Jak/Stat signaling inhibitors retarded PLL axon regeneration and Schwann cell migration. Taken together, Stat3 is required for embryonic PLL axon growth by regulating the ATP synthesis efficiency of mitochondria, whereas Stat3 stimulates PLL axon regeneration through regulating Schwann cell migration via Jak/Stat signaling. Our findings show a new mechanism of Stat3 in axon growth and regeneration. Author SummaryNeurons are one of the most specialized cell type in our body. A slender axon used for transmitting information between neuron and target cell extends from the cell body. Axon extension during both development and regeneration is regulated by intrinsic and extrinsic factors. In this study, we used the zebrafish posterior lateral line (PLL) axons which extends a long distance to study the regulation of axon extension. Signal transducer and activator of transcription 3 (STAT3) is a key transcription factor in Jak/Stat signaling and an activator for mitochondria functions. Our results demonstrate that Stat3 plays distinct roles in PLL axons development and regeneration. During embryonic development, Jak/Stat signaling is dispensable for PLL axons extension, whereas the mitochondrial Stat3 is required for embryonic axon growth through promoting ATP synthesis. During PLL axons regeneration, Jak/Stat is essential for Schwann cells migration which mediates axon regeneration.

developmental biology↗

bioRxiv2022

A regulatory network of Sox and Six transcription factors initiate a cell fate transformation during hearing regeneration in adult zebrafish

Using adult zebrafish inner ears as a model for sensorineural regeneration, we performed a targeted ablation of the mechanosensory receptors in the utricle and saccule and characterized the single-cell epigenome and transcriptome at consecutive time-points following hair cell ablation. Using deep learning on the regeneration-induced open chromatin sequences, we were able to identify unique, cell-specific transcription factor (TF) motif patterns enriched in the raw data. We correlated enhancer activity with gene expression to identify gene regulatory networks. A clear pattern of overlapping Sox- and Six-family transcription factor gene expression and binding motifs was detected, suggesting a combinatorial program of TFs driving regeneration and cell identity. Pseudo-time analysis of single-cell transcriptomic data demonstrated that the support cells within the sensory epithelium changed cell identity to a more pluripotent "progenitor" cell population that could either differentiate into hair cells or return to a support cell identity. We showed that sox2 becomes enriched in the progenitor cells and is reduced again when the cells differentiate in either direction. Analysis of the scATAC-seq data identified a 2.6 kb DNA sequence element upstream of the sox2 promoter that dynamically changed in accessibility during hair cell regeneration. When deleted, the upstream regulator of sox2 showed a dominant phenotype that resulted in a hair cell regeneration-specific deficit in both the lateral line and adult inner ear. HIGHLIGHTSIntegrated scRNA-seq and scATAC-seq of the adult zebrafish inner ear during hair cell regeneration. Support cells transition to an intermediate cell type that can differentiate to hair cells. Coordinated expression of Sox and Six transcription factors are key to hearing regeneration. Sox transcription factors trigger the regeneration response in the support cells while Sox and Six factors cooperate during hair cell differentiation. Deletion of an upstream enhancer that controls the timing of sox2 expression causes regeneration-specific defects in hearing regeneration.

genomics↗

bioRxiv2021

The Nup2 meiotic-autonomous region relieves autoinhibition of Nup60 to promote progression of meiosis and sporulation in Saccharomyces cerevisiae

During meiosis, chromosomes undergo dramatic changes in structural organization, nuclear positioning, and motion. Although the nuclear pore complex has been shown to affect genome organization and function in vegetative cells, its role in meiotic chromosome dynamics has remained largely unexplored. Recent work in the budding yeast Saccharomyces cerevisiae demonstrated that the mobile nucleoporin Nup2 is required for normal progression through meiosis I prophase and sporulation in strains where telomere-led chromosome movement has been compromised. The meiotic autonomous region (MAR), a short fragment of Nup2 responsible for its role in meiosis, was shown to localize to the nuclear envelope via Nup60 and to bind to meiotic chromosomes. To understand the relative contribution these two activities have on MAR function, we first carried out a screen for MAR mutants defective in sporulation and found that all the mutations disrupt interaction with both Nup60 and meiotic chromosomes. Moreover, nup60 mutants phenocopy nup2 mutants, exhibiting similar nuclear division kinetics, sporulation efficiencies, and genetic interactions with mutations that affect the telomere bouquet. Although full-length Nup60 requires Nup2 for function, removal of Nup60s C-terminus allows Nup60 to bind meiotic chromosomes and promote sporulation without Nup2. In contrast, binding of the MAR to meiotic chromosomes is completely dependent on Nup60. Our findings uncover an inhibitory function for the Nup60 C-terminus and suggest that Nup60 mediates recruitment of meiotic chromosomes to the nuclear envelope, while Nup2 plays a secondary role counteracting Nup60s autoinhibition.

genetics↗

bioRxiv2021

Development of motor neurons and motor activity in zebrafish requires F-actin nucleation by Fmn2b

BackgroundCytoskeletal remodelling plays a pivotal role in the establishment of neuronal connectivity during development and in plasticity in adults. Mutations in the cytoskeleton regulatory protein Formin-2 (Fmn2) are associated with neurodevelopmental disorders like intellectual disability, though its function in neuronal morphogenesis has not been characterised in vivo. ResultsHere we develop a loss-of-function model for fmn2b, the zebrafish orthologue of Fmn2, using CRISPR/Cas9-mediated gene editing. fmn2b mutants display motor deficits starting from the earliest motor responses in the embryo. We find that fmn2b is expressed in spinal motor neurons and its loss reduces motor neuron innervation of the axial muscles without affecting myotome integrity. The translocation of caudal primary (CaP) motor neuron outgrowth is compromised in fmn2b mutants, while rostral primary (RoP) motor neurons have missing soma or stall at the horizontal myoseptum. Strikingly, axon collateral branching of the motor neurons is severely compromised and results in reduced synaptic coverage of the myotome. Rescue experiments identify the requirement for Fmn2-mediated actin nucleation for motor neuron outgrowth and arborisation. ConclusionsThe zebrafish loss-of-function model of Fmn2 reveals the specific requirement of F-actin polymerisation by Fmn2 in neuromuscular development. It also underscores the role of Fmn2 in motor neuropathies, especially as a proportion of individuals harbouring mutations in Fmn2 present with hypotonia.

neuroscience↗

bioRxiv2021

The miR-430 locus with extreme promoter density is a transcription body organizer, which facilitates long range regulation in zygotic genome activation

In anamniote embryos the major wave of zygotic genome activation (ZGA) starts during the mid-blastula transition. This major wave of ZGA is facilitated by several mechanisms, including dilution of repressive maternal factors and accumulation of activating transcription factors during the fast cell division cycles preceding the mid-blastula transition. However, a set of genes escape global genome repression and are activated substantially earlier, during what is called, the minor wave of genome activation. While the mechanisms underlying the major wave of genome activation have been studied extensively, the minor wave of genome activation is little understood. In zebrafish the earliest expressed RNA polymerase II (Pol II) transcribed genes are activated in a pair of large transcription bodies depleted of chromatin, abundant in elongating Pol II and nascent RNAs (Hadzhiev et al., 2019; Hilbert et al., 2021). This transcription body includes the miR-430 gene cluster required for maternal mRNA clearance. Here we explored the genomic, chromatin organisation and cis-regulatory mechanisms of the minor wave of genome activation occurring in the transcription body. By long read genome sequencing we identified a remarkable cluster of miR-430 genes with over 300 promoters and spanning 0.6 Mb, which represent the highest promoter density of the genome. We demonstrate that the miR-430 gene cluster is required for the formation of the transcription body and acts as a transcription organiser for minor wave activation of a set of zinc finger genes scattered on the same chromosome arm, which share promoter features with the miR-430 cluster. These promoter features are shared among minor wave genes overall and include the TATA-box and sharp transcription start site profile. Single copy miR-430 promoter transgene reporter experiments indicate the importance of promoter-autonomous mechanisms regulating escape from global repression of the early embryo. These results together suggest that formation of the transcription body in the early embryo is the result of high promoter density coupled to a minor wave-specific core promoter code for transcribing key minor wave ZGA genes, which are required for the overhaul of the transcriptome during early embryonic development.

developmental biology↗

bioRxiv2021

Vestibular and auditory hair cell regeneration following targeted ablation of hair cells with diphtheria toxin in zebrafish

Millions of Americans experience hearing or balance disorders due to loss of hair cells in the inner ear. The hair cells are mechanosensory receptors used in the auditory and vestibular organs of all vertebrates as well as the lateral line systems of aquatic vertebrates. In zebrafish and other non-mammalian vertebrates, hair cells turn over during homeostasis and regenerate completely after being destroyed or damaged by acoustic or chemical exposure. However in mammals, destroying or damaging hair cells results in permanent impairments to hearing or balance. We sought an improved method for studying hair cell damage and regeneration in adult aquatic vertebrates by generating a transgenic zebrafish with the capacity for targeted and inducible hair cell ablation in vivo. This model expresses the human diphtheria toxin receptor (hDTR) gene under the control of the myo6b promoter, resulting in hDTR expressed only in hair cells. Cell ablation is achieved by an intraperitoneal injection of diphtheria toxin (DT) in adult zebrafish or DT dissolved in the water for larvae. In the lateral line of 5 dpf zebrafish, ablation of hair cells by DT treatment occurred within 2 days in a dose-dependent manner. Similarly, in adult utricles and saccules, a single intraperitoneal injection of 0.05 ng DT caused complete loss of hair cells in the utricle and saccule by 5 days post-injection. Full hair cell regeneration was observed for the lateral line and the inner ear tissues. This study introduces a new method for efficient conditional hair cell ablation in adult zebrafish inner ear sensory epithelia (utricles and saccules) and demonstrates that zebrafish hair cells will regenerate in vivo after this treatment.

developmental biology↗

bioRxiv2021

Homologous locus pairing is a transient, diffusion-mediated process in meiotic prophase

The pairing of homologous chromosomes (homologs) in meiosis is essential for distributing the correct numbers of chromosomes into haploid gametes. In budding yeast, pairing depends on the formation of 150-200 Spo11-mediated double-strand breaks (DSBs) that are distributed among 16 homolog pairs, but it is not known if all, or only a subset of these DSBs, contribute to the close juxtaposition of homologs. Having established a system to measure the position of fluorescently tagged chromosomal loci in 3D space over time, we analyzed locus trajectories to determine how frequently, and how long, loci spend colocalized or apart. Continuous imaging revealed highly heterogeneous cell-to-cell behavior of foci, with the majority of cells exhibiting a "mixed" phenotype where foci move into and out of proximity, even at late stages of prophase, suggesting that the axial structures of the synaptonemal complex may be more dynamic than anticipated. The observed plateaus of the mean-squared change in distance (MSCD) between foci informed the development of a biophysical model of two diffusing polymers that captures the loss of centromere linkages as cells enter meiosis, nuclear confinement, and the formation of Spo11-dependent linkages. The predicted number of linkages per chromosome in our theoretical model closely approximates the small number (~2-4) of estimated synapsis-initiation sites, suggesting that excess DSBs have negligible effects on the overall juxtaposition of homologs. These insights into the dynamic in-terchromosomal behavior displayed during homolog pairing demonstrate the power of combining time-resolved in vivo analysis with modeling at the granular level. Significance StatementEssential for sexual reproduction, meiosis is a specialized cell division required for the production of haploid gametes. Critical to this process is the pairing, recombination, and segregation of homologous chromosomes (homologs). While pairing and recombination are linked, it is not known how many linkages are sufficient to hold homologs in proximity. Here, we reveal that random diffusion and the placement of a small number of linkages are sufficient to establish the apparent "pairing" of homologs. We also show that colocalization between any two loci is more dynamic than anticipated. Our study is the first to provide observations of live interchromosomal dynamics during meiosis and illustrates the power of combining single-cell measurements with theoretical polymer modeling.

genetics↗

bioRxiv2020

Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

Vascular permeability triggered by inflammation or ischemia promotes edema, exacerbates disease progression, and impairs tissue recovery. Vascular endothelial growth factor (VEGF) is a potent inducer of vascular permeability. VEGF plays an integral role in regulating vascular barrier function physiologically and in pathologies, such as cancer, ischemic stroke, cardiovascular disease, retinal conditions, and COVID-19-associated pulmonary edema and sepsis, which often leads to acute lung injury, including acute respiratory distress syndrome. However, after initially stimulating permeability, VEGF subsequently mediates angiogenesis to repair damaged tissue. Consequently, understanding temporal molecular regulation of VEGF-induced vascular permeability will facilitate developing therapeutics that achieve the delicate balance of inhibiting vascular permeability while preserving tissue repair. Here, we demonstrate that VEGF signals through signal transducer and activator of transcription 3 (STAT3) to promote vascular permeability. Specifically, we show that genetic STAT3 ablation reduces vascular permeability in STAT3-deficient endothelium of mice and VEGF-inducible zebrafish crossed with CRISPR/Cas9 generated genomic STAT3 knockout zebrafish. Importantly, STAT3 deficiency does not impair vascular development and function in vivo. We identify intercellular adhesion molecule 1 (ICAM-1) as a STAT3-dependent transcriptional regulator and show VEGF-dependent STAT3 activation is regulated by JAK2. Pyrimethamine, an FDA-approved anti-microbial agent that inhibits STAT3-dependent transcription, substantially reduces VEGF-induced vascular permeability in zebrafish, mouse, and human endothelium. Indeed, pharmacologically targeting STAT3 increases vascular barrier integrity using two additional compounds, atovaquone and C188-9. Collectively, our findings suggest that the VEGF, VEGFR-2, JAK2, and STAT3 signaling cascade regulates vascular barrier integrity, and inhibition of STAT3-dependent activity reduces VEGF-induced vascular permeability in vertebrate models. Key PointsO_LIGenetic STAT3 deficiency in VEGF-inducible zebrafish and mice reveals that VEGF signals through STAT3 to promote vascular permeability C_LIO_LIPyrimethamine, a clinically available agent that inhibits STAT3 activity, reduces VEGF-induced vascular permeability in preclinical models C_LI

molecular biology↗

bioRxiv2020

High-throughput phenotypic characterization of zebrafish CRISPR mutants of DNA repair genes

A systematic knowledge of the roles of DNA repair genes at the level of the organism has been limited due to the lack of appropriate experimental techniques. Here, we generated zebrafish loss-of-function mutants for 32 DNA repair and replication genes through multiplexed CRISPR/Cas9-mediated mutagenesis. High-throughput phenotypic characterization of our mutant collection revealed that three genes (atad5a, ddb1, pcna) are essential for proper embryonic development and hematopoiesis; seven genes (apex1, atrip, ino80, mre11a, shfm1, telo2, wrn) are required for growth and development during juvenile stage and six genes (blm, brca2, fanci, rad51, rad54l, rtel1) play critical roles in sex development. Furthermore, mutation in six genes (atad5a, brca2, polk, rad51, shfm1, xrcc1) displayed hypersensitivity to DNA damage agents. Further characterization of atad5a-/- mutants demonstrate that Atad5a is required for normal brain development and hematopoiesis. Our zebrafish mutant collection provides a unique resource for understanding of the roles of DNA repair genes at the organismal level.

developmental biology↗

bioRxiv2020

Rad21l1 cohesin subunit is dispensable for spermatogenesis but not oogenesis in zebrafish

Meiosis produces haploid gametes that will give rise to the next diploid generation. Chromosome segregation errors occurring at one or both meiotic divisions result in aneuploidy, which can lead to miscarriages or birth defects in humans. During meiosis I, ring-shaped cohesin complexes play important roles to aid in the proper segregation of homologous chromosomes. While REC8 is a specialized meiosis-specific cohesin that functions to hold sister chromatids together, the role of its vertebrate-specific paralog, RAD21L, is poorly understood. Here we tested if Rad21l1, the zebrafish homolog of human and mouse RAD21L, is required for meiotic chromosome dynamics during oogenesis and spermatogenesis. We found that Rad21l1 is an abundant component of meiotic chromosomes where it localizes to both the chromosome axes and the transverse filament of the synaptonemal complex (SC). Knocking out rad21l1 causes nearly the entire mutant population to develop as fertile males, suggesting the mutation triggers a sex reversal from female to male due to a failure in oocyte production. The rad21l1-/- mutant males display normal fertility at sexual maturity. Sex reversal was partially suppressed in the absence of tp53, suggesting that the rad21l1-/- mutation causes defects leading to a Tp53 dependent response, specifically in females. The rad21l1-/-;tp53-/- double mutant females produced elevated rates of decomposing eggs and deformed offspring compared to tp53-/- controls. This response, however, is not linked to a defect in repairing Spo11-induced double-strand breaks since deletion of Spo11 does not suppress the sex reversal phenotype. Overall, our data highlight an exceptional sexually dimorphic phenotype caused by knocking out a meiotic-specific cohesin subunit. We propose that Rad21l1 is required for maintaining the integrity of meiotic chromatin architecture during oogenesis. Author SummaryA prominent symptom of age-linked reproductive decline in women is the increased rate of miscarriage and birth defects due to aneuploidy. Aneuploidy can arise when chromosomes fail to segregate properly during meiosis, the process of creating haploid gametes from a diploid germ cell. Oocyte progression normally arrests prior to anaphase I, after homologous chromosomes have formed crossovers, but before ovulation, which triggers the first round of segregation. This prolonged arrest makes oocytes especially vulnerable to degradation of meiotic chromosome structure and homolog connections over time. Cohesin complexes play a major role in maintaining the meiotic chromosome architecture. Here we assess the role of the vertebrate-specific Rad21l1 cohesin subunit in zebrafish. We find that while males appear mostly unaffected by loss of Rad21l1, oocyte production is massively compromised, leading to sex reversion to males. Sex reversion can be partially prevented in the absence of Tp53, demonstrating that loss or Rad21l1 leads to a Tp53-dependent response in oocytes. Strikingly, double mutant rad21l1 tp53 females produce large numbers of poor quality eggs and malformed offspring. This demonstrates a cohesin-linked vulnerability in female meiosis not present in males and sheds light on a potential mechanism associated with the decline in female reproductive health.

genetics↗

bioRxiv2020

Cnr2 is important for Ribbon Synapse Maturation and Function in Hair Cells and Photoreceptors

The role of the cannabinoid receptor 2 (CNR2) is still poorly described in sensory epithelia. We found strong cnr2 expression in hair cells (HCs) of the inner ear and the lateral line (LL), a superficial sensory structure in fish. Next, we demonstrated that sensory synapses in HCs were severely perturbed in zebrafish larvae lacking cnr2. Appearance and distribution of presynaptic ribbons and calcium channels (Cav1.3) were profoundly altered in mutant animals. Clustering of membrane-associated guanylate kinase (MAGUK) in post-synaptic densities (PSDs) was also heavily affected, suggesting a role for cnr2 for maintaining the structure of the sensory synapse. Furthermore, vesicular trafficking in HCs was strongly perturbed suggesting a retrograde action of the endocannabinoid system (ECs) mediated via cnr2 that was modulating HC mechanotransduction. We found similar perturbations in retinal ribbon synapses. Finally, we showed that larval swimming behaviors after sound and light stimulations were significantly different in mutant animals. Thus, we propose that cnr2 is critical for the processing of sensory information in the developing larva.

neuroscience↗

bioRxiv2020

A metabolic shift to glycolysis promotes zebrafish tail regeneration through TGF-β dependent dedifferentiation of notochord cells to form the blastema

Mammals are generally poor at tissue regeneration, in contrast, fish maintain a high capacity for regenerating complex tissues after injury. Using larval zebrafish, we show that tail amputation triggers an metabolic shift to glycolysis in cells surrounding the notochord as they reposition to the amputation site. Blocking glycolysis prevents the fin from regenerating after amputation due to the failure to form a normal, pluripotent blastema. We performed a time series of scRNA-sequencing on regenerating tails under normal conditions or in the absence of glycolysis. Strikingly, we detected a transient cell population in the single cell analysis that represents notochord sheath cells undergoing a TGF-{beta} dependent dedifferentiation and epithelium-to-mesenchyme transition to become pluripotent blastema cells. We further demonstrated that the metabolic switch to glycolysis is required for TGF-{beta} signaling and blocking either glycolysis or TGF-{beta} receptors results in aberrant blastema formation through the suppression of essential EMT mediators such as snai1.

developmental biology↗