Phenotypes In Zebrafish Research Articles

A mutualistic model bacterium is lethal to non-symbiotic hosts via the type VI secretion system.

What makes a bacterium pathogenic? Since the early days of germ theory, researchers have categorized bacteria as pathogens or non-pathogens, those that cause harm and those that do not, but this binary view is not always accurate. Vibrio fischeri is an exclusive mutualistic symbiont found within the light organs of Hawaiian bobtail squid. This symbiotic interaction requires V. fischeri to utilize a range of behaviors and produce molecules that are often associated with pathogenicity. This juxtaposition of employing "pathogenic" behaviors for a symbiotic relationship led the field to focus on how V. fischeri establishes a beneficial association with its host. In this study, we observe that V. fischeri induces mortality in zebrafish embryos and Artemia nauplii. Non-lethal doses of V. fischeri leads to zebrafish growth delays and phenotypes indicative of disease. Our data also provide evidence that the conserved type VI secretion system on chromosome I (T6SS1) plays a role in the V. fischeri -induced mortality of zebrafish embryos and Artemia nauplii. These results support the hypothesis that the V. fischeri T6SS1 is involved in eukaryotic cell interactions. Despite its traditional view as a beneficial symbiont, we provide evidence that V. fischeri is capable of harming aquatic organisms, indicating its potential to be pathogenic toward non-symbiotic hosts.

Zebrafish in-vivo study reveals deleterious activity of human TBC1D24 genetic variants linked with autosomal dominant hearing loss

Hearing loss is a common sensory impairment with a heterogeneous genetic etiology. Genetic variants in the TBC1D24 gene have recently emerged as an important cause of the non-syndromic autosomal dominant hearing loss (ADHL). However, the molecular mechanism behind the TBC1D24-associated ADHL is unknown. Using a zebrafish model, we investigated involvement of TBC1D24 in hearing and the functional effects of the associated ADHL-causing genetic variants. We show that the morpholino-mediated knock-down of Tbc1d24 resulted in defective ear kinocilia structure and reduced locomotor activity of the embryos. The observed phenotypes were rescued by a wild-type TBC1D24 mRNA but not by a mutant mRNA carrying the ADHL-causing variant c.553G>A (p.Asp185Asn), supporting its pathogenic potential. CRISPR-Cas9-mediated knock-out of tbc1d24 led to mechanosensory deficiency of lateral line neuromasts. Overexpression of TBC1D24 mRNA resulted in developmental abnormalities associated with ciliary dysfunction and mesendodermal mispatterning. We observed that the ADHL-causing TBC1D24 variants: c.553G>A (p.Asp185Asn); c.1460A>T (p.His487Leu), c.1461C>G (p.His487Gln) or a novel variant c.905T>G (p.Leu302Arg) alleviated the effect of overexpression, indicating that these variants disrupt the TBC1D24 function. Furthermore, the zebrafish phenotypes correspond to the severity of ADHL. Specific changes in ear structures upon TBC1D24 overexpression further highlighted its tissue-specific role in ciliary function and inner ear development. Our findings provide functional evidence for the pathogenic potential of the ADHL-causing TBC1D24 variants and lead to new insights into the function of TBC1D24 in cilia morphogenesis.

Zebrafish as a model organism to study sporadic Alzheimer's disease: Behavioural, biochemical and histological validation

Alzheimer's disease (AD) is a global burden to the healthcare system with no viable treatment options till date. Rodents and primates have been extensively used as models for understanding AD pathogenesis and identifying therapeutic targets. However, the focus is now shifting towards developing alternate models. Zebrafish is emerging as a preferred model for neurodegenerative conditions because of its simple nervous system, highly conserved genome and short duration required to model disease condition. The present study is aimed to develop streptozotocin (STZ)-induced model of sporadic AD (sAD) in zebrafish. STZ was administered to adult zebrafish (4–6 mo) at different doses (1 to 50 mg/kg body weight, intracerebroventricularly). Kaplan-Meier survival analysis revealed time and dose dependent mortality in the zebrafish administered with STZ. Based on survival analysis, 1 to 10 mg/kg body weight of STZ was selected for behavioural, molecular and histological studies. STZ administered fish had anxiety-like and stress behaviour in novel tank and light/dark preference tests. STZ-induced cognitive and memory deficits assessed using novel object recognition and spatial alternation tests. Further, expression of markers of amyloidogenic pathway (appa and bace1) were increased in terms of mRNA and protein levels in a time and dose dependent manner following STZ administration. However, expression of non-amyloidogenic pathway mediator (adam10) was reduced at both mRNA and protein level. Histological assessment using hematoxylin and eosin, and Nissl stain revealed loss of neurons in STZ administered fish. The ratio of phosphor-tauser396/total-tau was increased in STZ administered fish. Based on these findings, 5 mg/kg body weight of STZ was found to be most appropriate dose to exhibit sAD phenotype. Mass spectrometric analysis confirmed the presence of amyloid beta oligomers in brains of STZ administered fish. Transmission electron microscopy also showed the presence of higher order insoluble amyloid fibrils with twists. Immunohistochemical analysis revealed amyloid beta deposits in brain of STZ administered fish. Golgi-cox staining indicated decreased number of dendrites, whereas microglia had increased density, span ratio, soma area and lacunarity. The results of the present study demonstrate presence of AD hallmarks and phenotype in zebrafish 7 days post STZ administration (5 mg/kg). The study validates the potential of STZ-induced sAD in zebrafish as a reliable model for studying pathophysiology and rapid screening of therapeutic molecules against sAD.

Epigenetic regulation by polycomb repressive complex 1 promotes cerebral cavernous malformations

Cerebral cavernous malformations (CCMs) are anomalies of the cerebral vasculature. Loss of the CCM proteins CCM1/KRIT1, CCM2, or CCM3/PDCD10 trigger a MAPK-Krüppel-like factor 2 (KLF2) signaling cascade, which induces a pathophysiological pattern of gene expression. The downstream target genes that are activated by KLF2 are mostly unknown. Here we show that Chromobox Protein Homolog 7 (CBX7), component of the Polycomb Repressive Complex 1, contributes to pathophysiological KLF2 signaling during zebrafish cardiovascular development. CBX7/cbx7a mRNA is strongly upregulated in lesions of CCM patients, and in human, mouse, and zebrafish CCM-deficient endothelial cells. The silencing or pharmacological inhibition of CBX7/Cbx7a suppresses pathological CCM phenotypes in ccm2 zebrafish, CCM2-deficient HUVECs, and in a pre-clinical murine CCM3 disease model. Whole-transcriptome datasets from zebrafish cardiovascular tissues and human endothelial cells reveal a role of CBX7/Cbx7a in the activation of KLF2 target genes including TEK, ANGPT1, WNT9, and endoMT-associated genes. Our findings uncover an intricate interplay in the regulation of Klf2-dependent biomechanical signaling by CBX7 in CCM. This work also provides insights for therapeutic strategies in the pathogenesis of CCM.

The people behind the papers - Joey Ghersi and Stefania Nicoli.

Akt is believed to be important for vascular development, but complex tissue-specific loss-of-function studies have been difficult in mice because of embryonic lethality. In a new study, Wenping Zhou, Joey Ghersi, Stefania Nicoli and colleagues report the phenotype of a complete Akt mutant zebrafish and its role in arterial specification via Notch signaling. To learn more about their work, we spoke with corresponding authors Joey Ghersi, Assistant Professor at Centre Hospitalier Universitaire (CHU) Sainte-Justine, Canada, and Stefania Nicoli, Associate Professor Tenure at Yale University, USA.

Zebrafish (Danio rerio) behavioral phenotypes are not underscored by different gut microbiomes.

Although bold and shy behavioral phenotypes in zebrafish (Danio rerio) have been selectively bred and maintained over multiple generations, it is unclear if they are underscored by different gut microbiota. Using the microbiota-gut-brain concept, we examined the relationship between gut microbiota and the behavioral phenotypes within this model animal system to assess possible gut microbe-mediated effects on host behavior. To this end, we amplified and sequenced 16S rRNA gene amplicons from the guts of bold and shy zebrafish individuals using the Illumina Miseq platform. We did not record any significant differences in within-group microbial diversity nor between-group community composition of the two behavioral phenotypes. Interestingly, though not statistically different, we determined that the gut microbial community of the bold phenotype was dominated by Burkholderiaceae, Micropepsaceae, and Propionibacteriaceae. In contrast, the shy phenotype was dominated by Beijerinckaceae, Pirelullacaeae, Rhizobiales_Incertis_Sedis, and Rubinishaeraceae. The absence of any significant difference in gut microbiome profiles between the two phenotypes would suggest that in this species, there might exist a stable core gut microbiome, regardless of behavioral phenotypes, and possibly, a limited role for the gut microbiota in modulating this selected-for host behavior. This study characterized the gut microbiomes of distinct innate behavioral phenotypes of the zebrafish (that are not considered dysbiotic states) and did not rely on antibiotic or probiotic treatments to induce changes in behavior. Such studies are crucial to our understanding of the modulating impacts of the gut microbiome on normative animal behavior.

Context-dependent hyperactivity in syngap1a and syngap1b zebrafish models of SYNGAP1-related disorder.

SYNGAP1-related disorder (SYNGAP1-RD) is a prevalent genetic form of Autism Spectrum Disorder and Intellectual Disability (ASD/ID) and is caused by de novo or inherited mutations in one copy of the SYNGAP1 gene. In addition to ASD/ID, SYNGAP1 disorder is associated with comorbid symptoms including treatment-resistant-epilepsy, sleep disturbances, and gastrointestinal distress. Mechanistic links between these diverse symptoms and SYNGAP1 variants remain obscure, therefore, our goal was to generate a zebrafish model in which this range of symptoms can be studied. We used CRISPR/Cas9 to introduce frameshift mutations in the syngap1a and syngap1b zebrafish duplicates (syngap1ab) and validated these stable models for Syngap1 loss-of-function. Because SYNGAP1 is extensively spliced, we mapped splice variants to the two zebrafish syngap1a and b genes and identified mammalian-like isoforms. We then quantified locomotory behaviors in zebrafish syngap1ab larvae under three conditions that normally evoke different arousal states in wild-type larvae: aversive, high-arousal acoustic, medium-arousal dark, and low-arousal light stimuli. We show that CRISPR/Cas9 indels in zebrafish syngap1a and syngap1b produced loss-of-function alleles at RNA and protein levels. Our analyses of zebrafish Syngap1 isoforms showed that, as in mammals, zebrafish Syngap1 N- and C-termini are extensively spliced. We identified a zebrafish syngap1 α1-like variant that maps exclusively to the syngap1b gene. Quantifying locomotor behaviors showed that syngap1ab mutant larvae are hyperactive compared to wild-type but to differing degrees depending on the stimulus. Hyperactivity was most pronounced in low arousal settings, and hyperactivity was proportional to the number of mutant syngap1 alleles. Syngap1 loss-of-function mutations produce relatively subtle phenotypes in zebrafish compared to mammals. For example, while mouse Syngap1 homozygotes die at birth, zebrafish syngap1ab-/- survive to adulthood and are fertile, thus some aspects of symptoms in people with SYNGAP1-Related Disorder are not likely to be reflected in zebrafish. Our data support mutations in zebrafish syngap1ab as causal for hyperactivity associated with elevated arousal that is especially pronounced in low-arousal environments.

Sodium valproate effects on the morphological and neurobehavioral phenotype of zebrafish

The anticonvulsant sodium valproate (SV) is frequently administered as a medicament but bears several negative effects in case of exposure during development. We analyzed extensively these early development effects of using the zebrafish model. Zebrafish embryos were exposed as eggs to two sublethal concentrations of SV, 10 and 25 mg/L. A general embryo toxicity analysis revealed extended anomalies in the cardiovascular system, and in the craniofacial and the spinal skeleton, as well as high mortality, in the embryos exposed to SV. The teratogenic potential of SV was confirmed in hacthed larvae by morphometric and cartilage profile analysis. Last, neurobehavioral impairments due to SV were highlighted in subjects’ activity, anxiety, response to stimulations, habituation learning, and daily synchronization of locomotor activity, overall mirroring typical phenotypes associated with autistic spectrum disorders. In conclusion, our results confirmed the presence of extended and multifaced impacts of exposure to SV during development.

Creatine and l-carnitine attenuate muscular laminopathy in the LMNA mutation transgenic zebrafish

Lamin A/C gene (LMNA) mutations contribute to severe striated muscle laminopathies, affecting cardiac and skeletal muscles, with limited treatment options. In this study, we delve into the investigations of five distinct LMNA mutations, including three novel variants and two pathogenic variants identified in patients with muscular laminopathy. Our approach employs zebrafish models to comprehensively study these variants. Transgenic zebrafish expressing wild-type LMNA and each mutation undergo extensive morphological profiling, swimming behavior assessments, muscle endurance evaluations, heartbeat measurement, and histopathological analysis of skeletal muscles. Additionally, these models serve as platform for focused drug screening. We explore the transcriptomic landscape through qPCR and RNAseq to unveil altered gene expression profiles in muscle tissues. Larvae of LMNA(L35P), LMNA(E358K), and LMNA(R453W) transgenic fish exhibit reduced swim speed compared to LMNA(WT) measured by DanioVision. All LMNA transgenic adult fish exhibit reduced swim speed compared to LMNA(WT) in T-maze. Moreover, all LMNA transgenic adult fish, except LMNA(E358K), display weaker muscle endurance than LMNA(WT) measured by swimming tunnel. Histochemical staining reveals decreased fiber size in all LMNA mutations transgenic fish, excluding LMNA(WT) fish. Interestingly, LMNA(A539V) and LMNA(E358K) exhibited elevated heartbeats. We recognize potential limitations with transgene overexpression and conducted association calculations to explore its effects on zebrafish phenotypes. Our results suggest lamin A/C overexpression may not directly impact mutant phenotypes, such as impaired swim speed, increased heart rates, or decreased muscle fiber diameter. Utilizing LMNA zebrafish models for drug screening, we identify l-carnitine treatment rescuing muscle endurance in LMNA(L35P) and creatine treatment reversing muscle endurance in LMNA(R453W) zebrafish models. Creatine activates AMPK and mTOR pathways, improving muscle endurance and swim speed in LMNA(R453W) fish. Transcriptomic profiling reveals upstream regulators and affected genes contributing to motor dysfunction, cardiac anomalies, and ion flux dysregulation in LMNA mutant transgenic fish. These findings faithfully mimic clinical manifestations of muscular laminopathies, including dysmorphism, early mortality, decreased fiber size, and muscle dysfunction in zebrafish. Furthermore, our drug screening results suggest l-carnitine and creatine treatments as potential rescuers of muscle endurance in LMNA(L35P) and LMNA(R453W) zebrafish models. Our study offers valuable insights into the future development of potential treatments for LMNA-related muscular laminopathy.

P53 inhibitor or antioxidants reduce the severity of ethmoid plate deformities in zebrafish Type 3 Treacher Collins syndrome model

Treacher Collins syndrome-3 (TCS-3) is a rare congenital craniofacial disorder attributed to variants in the RNA pol I subunit C (POLR1C). The pathogenesis of TCS-3 linked to polr1c involves the activation of apoptosis-dependent p53 pathways within neural crest cells (NCCs). This occurs due to disruptions in ribosome biogenesis, and the restoration of polr1c expression in early embryogenesis effectively rescues the observed craniofacial phenotype in polr1c-deficient zebrafish. Clinical variability in TCS patients suggests interactions between genes and factors like oxidative stress. Elevated production of reactive oxygen species (ROS) in epithelial cells may worsen phenotypic outcomes in TCS individuals. Our study confirmed excessive ROS production in facial regions, inducing apoptosis and altering p53 pathways. Deregulated cell-cycle and epithelial-to-mesenchymal transition (EMT) genes were also detected in the TCS-3 model. Utilizing p53 inhibitor (Pifithrin-α; PFT-α) or antioxidants (Glutathione; GSH and N-Acetyl-L-cysteine; NAC) effectively corrected migrated NCC distribution in the pharyngeal arch (PA), suppressed oxidative stress, prevented cell death, and modulated EMT inducers. Crucially, inhibiting p53 activation or applying antioxidants within a specific time window, notably within 30 h post-fertilization (hpf), successfully reversed phenotypic effects induced by polr1c MO.

Use of All-Male cyp17a1-Deficient Zebrafish (Danio rerio) for Evaluation of Environmental Estrogens.

Natural and synthetic environmental estrogens (EEs) are widespread and have received extensive attention. Our previous studies demonstrated that depletion of the cytochrome P450 17a1 gene (cyp17a1) leads to all-testis differentiation phenotype in zebrafish and common carp. In the present study, cyp17a1-deficient zebrafish with defective estrogen biosynthesis were used for the evaluation of EEs, as assessed by monitoring vitellogenin (vtg) expression. A rapid and sensitive assessment procedure was established with the 3-day administration of estradiol (E2), followed by examination of the transcriptional expression of vtgs in our cyp17a1-deficient fish. Compared with the control fish, a higher E2-mediated vtg upregulation observed in cyp17a1-deficient zebrafish exposed to 0.1 μg/L E2 is known to be estrogen receptor-dependent and likely due to impaired in vivo estrogen biosynthesis. The more responsive vtg expression in cyp17a1-deficient zebrafish was observed when exposed to 200 and 2000 μg/L bisphenol A (BPA) and perfluoro-1-octanesulfonate (PFOS). The estrogenic potentials of E2, BPA, and PFOS were compared and assessed by the feminization effect on ovarian differentiation in cyp17a1-deficient zebrafish from 18 to 50 days postfertilization, based on which a higher sensitivity of E2 in ovarian differentiation than BPA and PFOS was concluded. Collectively, through the higher sensitivity to EEs and the capacity to distinguish chemicals with different estrogenic potentials exhibited by the all-male cyp17a1-deficient zebrafish with impaired estrogen biosynthesis, we demonstrated that they can be used as an excellent in vivo model for the evaluation of EEs. Environ Toxicol Chem 2024;43:1062-1074. © 2024 SETAC.

Leucine-Rich Repeat in Polycystin-1 Suppresses Cystogenesis in a Zebrafish (Danio rerio) Model of Autosomal-Dominant Polycystic Kidney Disease.

Mutations of PKD1 coding for polycystin-1 (PC1) account for most cases of autosomal-dominant polycystic kidney disease (ADPKD). The extracellular region of PC1 contains many evolutionarily conserved domains for ligand interactions. Among these are the leucine-rich repeats (LRRs) in the far N-terminus of PC1. Using zebrafish (Danio rerio) as an in vivo model system, we explored the role of LRRs in the function of PC1. Zebrafish expresses two human PKD1 paralogs, pkd1a and pkd1b. Knockdown of both genes in zebrafish by morpholino antisense oligonucleotides produced phenotypes of dorsal-axis curvature and pronephric cyst formation. We found that overexpression of LRRs suppressed both phenotypes in pkd1-morphant zebrafish. Purified recombinant LRR domain inhibited proliferation of HEK cells in culture and interacted with the heterotrimeric basement membrane protein laminin-511 (α5β1γ1) in vitro. Mutations of amino acid residues in LRRs structurally predicted to bind laminin-511 disrupted LRR-laminin interaction in vitro and neutralized the ability of LRRs to inhibit cell proliferation and cystogenesis. Our data support the hypothesis that the extracellular region of PC1 plays a role in modulating PC1 interaction with the extracellular matrix and contributes to cystogenesis of PC1 deficiency.

Induction of Paraquat-Mediated Parkinsonian Phenotype in Zebrafish.

Paraquat (PQ) is a well-known neurotoxin closely associated with neurodegenerative Parkinson's disease (PD). Zebrafish are utilized as a model for PD research because of their well-defined neuropathology and locomotor behavior. Here, we highlight protocols for inducing PD using PQ and analyzing locomotor activity in adult zebrafish. Basic Protocol 1 details the treatment of adult male zebrafish with 60 mg/kg PQ via intraperitoneal injection to induce a PD-like phenotype, followed by the steps to perform a locomotor assay. Basic Protocol 2 provides step-by-step guidance for processing the acquired videos in ToxTrac software to understand the locomotor parameters of 0.9% saline- and 60 mg/kg PQ-injected adult zebrafish. The simplicity of the treatment strategy, low-cost video acquisition setup, and free video processing make these protocols accessible without prior experience. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Development of Parkinson's disease features in adult zebrafish Basic Protocol 2: ToxTrac analysis for locomotor assay.

Hox genes control homocercal caudal fin development and evolution.

Ancient bony fishes had heterocercal tails, like modern sharks and sturgeons, with asymmetric caudal fins and a vertebral column extending into an elongated upper lobe. Teleost fishes, in contrast, developed a homocercal tail characterized by two separate equal-sized fin lobes and the body axis not extending into the caudal fin. A similar heterocercal-to-homocercal transition occurs during teleost ontogeny, although the underlying genetic and developmental mechanisms for either transition remain unresolved. Here, we investigated the role of hox13 genes in caudal fin formation as these genes control posterior identity in animals. Analysis of expression profiles of zebrafish hox13 paralogs and phenotypes of CRISPR/Cas9-induced mutants showed that double hoxb13a and hoxc13a mutants fail to form a caudal fin. Furthermore, single mutants display heterocercal-like morphologies not seen since Mesozoic fossil teleosteomorphs. Relaxation of functional constraints after the teleost genome duplication may have allowed hox13 duplicates to neo- or subfunctionalize, ultimately contributing to the evolution of a homocercal tail in teleost fishes.

JM-20 potently prevents the onset of caffeine-induced anxiogenic phenotypes in zebrafish (Danio rerio)

Anxiety is among the most prevalent mental disorders present in the general population. Benzodiazepines are the most commonly prescribed drugs for the treatment of anxiety. Using zebrafish as a model organism, we investigated the anxiolytic activity of JM-20, a novel hybrid molecule with a 1,5-benzodiazepine ring fused to a dihydropyridine moiety. Firstly, we carried out some assays to analyze the possible toxicity mediated by JM-20. For this, zebrafish were exposed to different JM-20 concentrations (0-5 μM) for 96 h. Then, using the novel tank test, we evaluated both locomotor and anxiety-like behavior of the animals. Furthermore, brain, liver and plasma were removed to assess toxicity parameters. JM-20 exposure did not cause changes on novel tank, and also did not alter brain viability, hepatic LDH and plasma ALT levels. Afterward, we investigated whether a pre-exposure to JM-20 would prevent the anxiogenic effect evoked by caffeine. In the novel tank test, caffeine significantly decreased the time spent at the top, as well as the number of transitions to the top area. Moreover, caffeine decreased both the total and average time spent in the lit area, as well as increased the number of risk episodes evaluated by the light-dark test. Whole-body cortisol levels were also increased by caffeine exposure. Interestingly, pre-treatment with JM-20 abolished all alterations induced by caffeine. The anxiolytic effect profile of JM-20 was similar to those found for diazepam (positive control). Our findings show, for the first time, the anxiolytic effect of JM-20 in zebrafish, and its relationship with cortisol regulation.

Lens autophagy protein ATG16L1: a potential target for cataract treatment.

Rationale: Cataract is the leading cause of blindness and low vision worldwide, yet its pathological mechanism is not fully understood. Although macroautophagy/autophagy is recognized as essential for lens homeostasis and has shown potential in alleviating cataracts, its precise mechanism remains unclear. Uncovering the molecular details of autophagy in the lens could provide targeted therapeutic interventions alongside surgery. Methods: We monitored autophagic activities in the lens and identified the key autophagy protein ATG16L1 by immunofluorescence staining, Western blotting, and transmission electron microscopy. The regulatory mechanism of ATG16L1 ubiquitination was analyzed by co-immunoprecipitation and Western blotting. We used the crystal structure of E3 ligase gigaxonin and conducted the docking screening of a chemical library. The effect of the identified compound riboflavin was tested in vitro in cells and in vivo animal models. Results: We used HLE cells and connexin 50 (cx50)-deficient cataract zebrafish model and confirmed that ATG16L1 was crucial for lens autophagy. Stabilizing ATG16L1 by attenuating its ubiquitination-dependent degradation could promote autophagy activity and relieve cataract phenotype in cx50-deficient zebrafish. Mechanistically, the interaction between E3 ligase gigaxonin and ATG16L1 was weakened during this process. Leveraging these mechanisms, we identified riboflavin, an E3 ubiquitin ligase-targeting drug, which suppressed ATG16L1 ubiquitination, promoted autophagy, and ultimately alleviated the cataract phenotype in autophagy-related models. Conclusions: Our study identified an unrecognized mechanism of cataractogenesis involving ATG16L1 ubiquitination in autophagy regulation, offering new insights for treating cataracts.

A Novel Role for the Longevity-Associated Protein SLC39A11 as a Manganese Transporter.

The identification of aging- and longevity-associated genes is important for promoting healthy aging. By analyzing a large cohort of Chinese centenarians, we previously found that single-nucleotide polymorphisms (SNPs) in the SLC39A11 gene (also known as ZIP11) are associated with longevity in males. However, the function of the SLC39A11 protein remains unclear. Here, we found that SLC39A11 expression is significantly reduced in patients with Hutchinson-Gilford progeria syndrome (HGPS). In addition, we found that zebrafish with a mutation in slc39a11 that significantly reduces its expression have an accelerated aging phenotype, including a shortened average lifespan, muscle atrophy and reduced swimming, impaired muscle regeneration, gut damage, and abnormal morphology in the reproductive system. Interestingly, these signs of premature aging were more pronounced in male zebrafish than in females. RNA-sequencing analysis revealed that cellular senescence may serve as a potential mechanism for driving this slc39a11 deficiency-induced phenotype in mutant zebrafish. Moreover, immunofluorescence showed significantly increased DNA damage and reactive oxygen species signaling in slc39a11 mutant zebrafish. Using inductively coupled plasma mass spectrometry (ICP-MS), we found that manganese significantly accumulates in slc39a11 mutant zebrafish, as well as in the serum of both global Slc39a11 knockout and hepatocyte-specific Slc39a11 knockout mice, suggesting that this metal transporter regulates systemic manganese levels. Finally, using cultured human fibroblasts, we found that both knocking down SLC39A11 and exposure to high extracellular manganese increased cellular senescence. These findings provide compelling evidence that SLC39A11 serves to protect against the aging process, at least in part by regulating cellular manganese homeostasis.

Myeloid cells interact with a subset of thyrocytes to promote their migration and follicle formation through NF-κB

The pathogenesis of thyroid dysgenesis (TD) is not well understood. Here, using a combination of single-cell RNA and spatial transcriptome sequencing, we identify a subgroup of NF-κB-activated thyrocytes located at the center of thyroid tissues in postnatal mice, which maintained a partially mesenchymal phenotype. These cells actively protruded out of the thyroid primordium and generated new follicles in zebrafish embryos through continuous tracing. Suppressing NF-κB signaling affected thyrocyte migration and follicle formation, leading to a TD-like phenotype in both mice and zebrafish. Interestingly, during thyroid folliculogenesis, myeloid cells played a crucial role in promoting thyrocyte migration by maintaining close contact and secreting TNF-α. We found that cebpa mutant zebrafish, in which all myeloid cells were depleted, exhibited thyrocyte migration defects. Taken together, our results suggest that myeloid-derived TNF-α-induced NF-κB activation plays a critical role in promoting the migration of vertebrate thyrocytes for follicle generation.

Genetic evidence for differential functions of figla and nobox in zebrafish ovarian differentiation and folliculogenesis

FIGLA and NOBOX are important oocyte-specific transcription factors. Both figla-/- and nobox-/- mutants showed all-male phenotype in zebrafish due to increased dominance of the male-promoting pathway. The early diversion towards males in these mutants has precluded analysis of their roles in folliculogenesis. In this study, we attenuated the male-promoting pathway by deleting dmrt1, a key male-promoting gene, in figla-/- and nobox-/- fish, which allows a sufficient display of defects in folliculogenesis. Germ cells in figla-/-;dmrt1-/- double mutant remained in cysts without forming follicles. In contrast, follicles could form well but exhibited deficient growth in nobox-/-;dmrt1-/- double mutants. Follicles in nobox-/-;dmrt1-/- ovary could progress to previtellogenic (PV) stage but failed to enter vitellogenic growth. Such arrest at PV stage suggested a possible deficiency in estrogen signaling. This was supported by lines of evidence in nobox-/-;dmrt1-/-, including reduced expression of ovarian aromatase (cyp19a1a) and level of serum estradiol (E2), regressed genital papilla (female secondary sex characteristics), and more importantly the resumption of vitellogenic growth by E2 treatment. Expression analysis suggested Nobox might regulate cyp19a1a by controlling Gdf9 and/or Bmp15. Our discoveries indicate that Figla is essential for ovarian differentiation and follicle formation whereas Nobox is important for driving subsequent follicle development.

Fgf22 and Fgfr2b are required for neurogenesis and gliogenesis in the zebrafish forebrain

Fibroblast growth factors (Fgfs) play crucial roles in various developmental processes including brain development. We previously identified Fgf22 in zebrafish and found that fgf22 is involved in midbrain patterning during embryogenesis. Here, we investigated the role of Fgf22 in the formation of the zebrafish forebrain. We found that fgf22 was essential for determining the ventral properties of the telencephalon and diencephalon but not for cell proliferation. In addition, the knockdown of fgf22 inhibited the generation of glutamatergic neurons, γ–aminobutyric acid (GABA)ergic interneurons and astrocytes. Recently, Fgf signaling has received much attention because of its importance in the pathogenesis of multiple sclerosis, in which oligodendrocytes and myelin are destroyed. However, the effects of each Fgf on oligodendrocytes remain largely unknown. Therefore, we also investigated the role of Fgf22 in oligodendrocyte development and explored whether there is a difference between Fgf22 and other Fgfs. Knockdown of fgf22 promoted the generation of oligodendrocytes. Conversely, overexpression of fgf22 inhibited the generation of oligodendrocytes. Furthermore, the forebrain phenotypes of fgfr2b knockdown zebrafish were remarkably similar to those of fgf22 knockdown zebrafish. This establishes the Fgf22-Fgfr2b axis as a key ligand‒receptor partnership in neurogenesis and gliogenesis in the forebrain. Our results indicate that Fgf22 has a unique function in suppressing oligodendrocyte differentiation through Fgfr2b without affecting cell proliferation.