Unraveling the genetic architecture of congenital vertebral malformation with reference to the developing spine.

Congenital vertebral malformation, affecting 0.13-0.50 per 1000 live births, has an immense locus heterogeneity and complex genetic architecture. In this study, we analyze exome/genome sequencing data from 873 probands with congenital vertebral malformation and 3794 control individuals. Clinical interpretation identifies Mendelian etiologies in 12.0% of the probands and reveals a muscle-related disease mechanism. Gene-based burden test of ultra-rare variants identifies risk genes with large effect sizes (ITPR2, TBX6, TPO, H6PD, and SEC24B). To further investigate the biological relevance of the genetic association signals, we perform single-nucleus RNAseq on human embryonic spines. The burden test signals are enriched in the notochord at early developmental stages and myoblast/myocytes at late stages, highlighting their critical roles in the developing spine. Our work provides insights into the developmental biology of the human spine and the pathogenesis of spine malformation.

Ependymal polarity defects coupled with disorganized ciliary beating drive abnormal cerebrospinal fluid flow and spine curvature in zebrafish.

Idiopathic scoliosis (IS) is the most common spinal deformity diagnosed in childhood or early adolescence, while the underlying pathogenesis of this serious condition remains largely unknown. Here, we report zebrafish ccdc57 mutants exhibiting scoliosis during late development, similar to that observed in human adolescent idiopathic scoliosis (AIS). Zebrafish ccdc57 mutants developed hydrocephalus due to cerebrospinal fluid (CSF) flow defects caused by uncoordinated cilia beating in ependymal cells. Mechanistically, Ccdc57 localizes to ciliary basal bodies and controls the planar polarity of ependymal cells through regulating the organization of microtubule networks and proper positioning of basal bodies. Interestingly, ependymal cell polarity defects were first observed in ccdc57 mutants at approximately 17 days postfertilization, the same time when scoliosis became apparent and prior to multiciliated ependymal cell maturation. We further showed that mutant spinal cord exhibited altered expression pattern of the Urotensin neuropeptides, in consistent with the curvature of the spine. Strikingly, human IS patients also displayed abnormal Urotensin signaling in paraspinal muscles. Altogether, our data suggest that ependymal polarity defects are one of the earliest sign of scoliosis in zebrafish and disclose the essential and conserved roles of Urotensin signaling during scoliosis progression.

Rassf7a promotes spinal cord regeneration and controls spindle orientation in neural progenitor cells.

Spinal cord injury (SCI) can cause long-lasting disability in mammals due to the lack of axonal regrowth together with the inability to reinitiate spinal neurogenesis at the injury site. Deciphering the mechanisms that regulate the proliferation and differentiation of neural progenitor cells is critical for understanding spinal neurogenesis after injury. Compared with mammals, zebrafish show a remarkable capability of spinal cord regeneration. Here, we show that Rassf7a, a member of the Ras-association domain family, promotes spinal cord regeneration after injury. Zebrafish larvae harboring a rassf7a mutation show spinal cord regeneration and spinal neurogenesis defects. Live imaging shows abnormal asymmetric neurogenic divisions and spindle orientation defects in mutant neural progenitor cells. In line with this, the expression of rassf7a is enriched in neural progenitor cells. Subcellular analysis shows that Rassf7a localizes to the centrosome and is essential for cell cycle progression. Our data indicate a role for Rassf7a in modulating spindle orientation and the proliferation of neural progenitor cells after spinal cord injury.

Zebrafish: an important model for understanding scoliosis.

Scoliosis is a common spinal deformity that considerably affects the physical and psychological health of patients. Studies have shown that genetic factors play an important role in scoliosis. However, its etiopathogenesis remain unclear, partially because of the genetic heterogeneity of scoliosis and the lack of appropriate model systems. Recently, the development of efficient gene editing methods and high-throughput sequencing technology has made it possible to explore the underlying pathological mechanisms of scoliosis. Owing to their susceptibility for developing scoliosis and high genetic homology with human, zebrafish are increasingly being used as a model for scoliosis in developmental biology, genetics, and clinical medicine. Here, we summarize the recent advances in scoliosis research on zebrafish and discuss the prospects of using zebrafish as a scoliosis model.

Cardiotoxicity of Zebrafish Induced by 6-Benzylaminopurine Exposure and Its Mechanism.

6-BA is a common plant growth regulator, but its safety has not been conclusive. The heart is one of the most important organs of living organisms, and the cardiogenesis process of zebrafish is similar to that of humans. Therefore, based on wild-type and transgenic zebrafish, we explored the development of zebrafish heart under 6-BA exposure and its mechanism. We found that 6-BA affected larval cardiogenesis, inducing defective expression of key genes for cardiac development (myl7, vmhc, and myh6) and AVC differentiation (bmp4, tbx2b, and notch1b), ultimately leading to weakened cardiac function (heart rate, diastolic speed, systolic speed). Acridine orange staining showed that the degree of apoptosis in zebrafish hearts was significantly increased under 6-BA, and the expression of cell-cycle-related genes was also changed. In addition, HPA axis assays revealed abnormally expressed mRNA levels of genes and significantly increased cortisol contents, which was also consistent with the observed anxiety behavior in zebrafish at 3 dpf. Transcriptional abnormalities of pro- and anti-inflammatory factors in immune signaling pathways were also detected in qPCR experiments. Collectively, we found that 6-BA induced cardiotoxicity in zebrafish, which may be related to altered HPA axis activity and the onset of inflammatory responses under 6-BA treatment.

Cilia regulate meiotic recombination in zebrafish.

Meiosis is essential for evolution and genetic diversity in almost all sexual eukaryotic organisms. The mechanisms of meiotic recombination, such as synapsis, have been extensively investigated. However, it is still unclear whether signals from the cytoplasm or even from outside of the cell can regulate the meiosis process. Cilia are microtubule-based structures that protrude from the cell surface and function as signaling hubs to sense extracellular signals. Here, we reported an unexpected and critical role of cilia during meiotic recombination. During gametogenesis of zebrafish, cilia were specifically present in the prophase stages of both primary spermatocytes and primary oocytes. By developing a germ cell-specific CRISPR/Cas9 system, we demonstrated that germ cell-specific depletion of ciliary genes resulted in compromised double-strand break repair, reduced crossover formation, and increased germ cell apoptosis. Our study reveals a previously undiscovered role for cilia during meiosis and suggests that extracellular signals may regulate meiotic recombination via this particular organelle.

Ciliogenesis requires sphingolipid-dependent membrane and axoneme interaction.

Cilium formation and regeneration requires new protein synthesis, but the underlying cytosolic translational reprogramming remains largely unknown. Using ribosome footprinting, we performed global translatome profiling during cilia regeneration in Chlamydomonas and uncovered that flagellar genes undergo an early transcriptional activation but late translational repression. This pattern guided our identification of sphingolipid metabolism enzymes, including serine palmitoyltransferase (SPT), as essential regulators for ciliogenesis. Cryo-electron tomography showed that ceramide loss abnormally increased the membrane-axoneme distance and generated bulged cilia. We found that ceramides interact with intraflagellar transport (IFT) particle proteins that IFT motors transport along axoneme microtubules (MTs), suggesting that ceramide-IFT particle-IFT motor-MT interactions connect the ciliary membrane with the axoneme to form rod-shaped cilia. SPT-deficient vertebrate cells were defective in ciliogenesis, and SPT mutations from patients with hereditary sensory neuropathy disrupted cilia, which could be restored by sphingolipid supplementation. These results reveal a conserved role of sphingolipid in cilium formation and link compromised sphingolipid production with ciliopathies.

Distribution pattern of floor plate neurons in zebrafish.

The floor plate (FP) is a critical signaling center for the development of neural tube and body axis, and is localized at the ventral midline of the neural tube. Multiple types of neurons are present in the floor plate, while the distribution pattern of these neuronal cells remains unclear. By using transgenic zebrafish lines that specifically label different neuronal cells, we investigated the distribution pattern of these neurons in the floor plate region. Our results showed that foxj1a, sox2, clusterin and gfap genes were expressed in the medial floor plate (MFP), consisting of a single row of cells. The cerebrospinal fluid-contacting neurons (CSF-cNs), also named as Kolmer-Agduhr interneurons (KA' and KA" neurons), were located on the lateral sides of MFP. The foxj1a and pkd2l1 genes were expressed in the KA" neurons, which were located to the ventral terminal gap of wedge-shaped MFP cells. The neighboring KA" neurons were separated by neurons expressing Gfap, Olig2 or Sox2. In contrast, the KA' neurons were positive for Foxj1a +/Pkd2l1+/Olig2+, and were localized to the dorsal side of KA" neurons. Similarly, the Sox2 or Olig2 expressing neurons were intermingled with KA' neurons along the anterior-posterior axis in these regions. Further pharmaceutical treatment demonstrated that interference of Notch signaling resulted in the abnormal distribution of floor plate neurons together with strong dorsal body curvature at 3 days post fertilization in the zebrafish larvae. Our data showed the gene expression patterns and relative positions of the floor plate neurons; and suggested a potential role of Notch signaling during floor plate development.

Distinct Patterns of Automatic and Controlled Incongruent Information Processing in the Human Brain.

It is a fundamental ability to discriminate incongruent information in daily activity. However, the underlying neural dynamics are still unclear. Using stereoelectroencephalography (SEEG), in this study, we investigated the fine-grained and different states of incongruent information processing in patients with refractory epilepsy who underwent intracranial electrode implantation. All patients performed a delayed match-to-sample paradigm in the sequential pairs of visual stimuli (S1 followed by S2). Participants were asked to discriminate whether the relevant feature of S2 was identical to S1 while ignoring the irrelevant feature. The spatiotemporal cortical responses evoked by different conditions were calculated and compared, respectively, in the context of brain intrinsic functional networks. In total, we obtained SEEG recordings from 241 contacts in gray matter. In the processing of irrelevant incongruent information, the activated brain areas included the superior parietal lobule, supramarginal gyrus, angular gyrus, inferior temporal gyrus, and fusiform gyrus. By comparing the relevant incongruent condition with the congruent condition, the activated brain areas included the middle frontal gyrus, superior temporal gyrus, middle temporal gyrus, posterior superior temporal sulcus, and posterior cingulate cortex. We demonstrated the dynamics of incongruent information processing with high spatiotemporal resolution and suggested that the process of automatic detection of irrelevant incongruent information requires the involvement of local regions and relatively few networks. Meanwhile, controlled discrimination of relevant incongruent information requires the participation of extensive regions and a wide range of nodes in the network. Furthermore, both the frontoparietal control network and default mode network were engaged in the incongruent information processing.

E2f4 is required for intestinal and otolith development in zebrafish.

E2f4 is a multifunctional transcription factor that is essential for many cellular processes. Although the role of E2f4 during cell cycle progression has been investigated in great detail, less is known about E2f4 during embryonic development. Here, we investigated the role of E2f4 during zebrafish development. Zebrafish e2f4 mutants displayed ectopic otolith formation due to abnormal ciliary beating in the otic vesicle. The beating defects of motile cilia were caused by abnormal expression of ciliary motility genes. The expression of two genes, lrrc23 and ccdc151, were significantly decreased in the absence of E2f4. In addition to that, e2f4 mutants also displayed growth retardation both in the body length and body weight and mostly died at around 6 months old. Although food intake was normal in the mutants, we found that the microvilli of the intestinal epithelia were significantly affected in the mutants. Finally, the intestinal epithelia of e2f4 mutants also displayed reduced cell proliferation, together with an increased level of cell apoptosis. Our data suggested a tissue-specific role of E2f4 during zebrafish development, which is distinct from the traditional views of this protein as a transcription repressor.

Fluorene-9-bisphenol exposure decreases locomotor activity and induces lipid-metabolism disorders by impairing fatty acid oxidation in zebrafish.

AIMS: Fluorene-9-bisphenol (BHPF), as a substitute for bisphenol A, is used in many industries in daily life. Many studies have clarified its effects as an endocrine disruptor on organisms, but its effect on lipid metabolism of zebrafish larvae is not clear. Patients with non-alcoholic fatty liver disease (NAFLD) are more susceptible to external pollutants. It is not clear how BHPF perturbs lipid metabolism or promotes NAFLD progression. MAIN METHODS: We explored the biological effects of BHPF on locomotor activity, inflammatory response, endoplasmic reticulum (ER) stress and lipid metabolism in zebrafish, especially in the mechanism of lipid homeostasis disorder. In addition, the role of BHPF in the progression of non-alcoholic fatty liver disease (NAFLD) was further explored. KEY FINDINGS: We found that high concentration (100 nmol/L) BHPF caused retarded growth, mild lipid accumulation and reduced the locomotive activity of zebrafish larvae, accompanied by a decrease in endogenous cortisol level. At the same time, it caused the full activation of inflammation and ER stress. Rescue experiments by 25(OH)D3 demonstrated that high concentration of BHPF caused defects in 1,25(OH)2D3 metabolic pathway through downregulation of cyp2r1, which further damaged pgc1a-mediated fatty acid oxidation and mitochondrial function, resulting in lipid accumulation. In summary, exposure to BHPF could damage lipid homeostasis and worsen the diet-induced NAFLD. SIGNIFICANCE: Our findings provide new insights into the role of BHPF in development of overweight and obesity and also improve understanding of its toxicological mechanism. Our results play a warning role in the administration of environmental pollutants.

Loss of Ift74 Leads to Slow Photoreceptor Degeneration and Ciliogenesis Defects in Zebrafish.

Cilia are microtubule-based structures projecting from the cell surface that perform diverse biological functions. Ciliary defects can cause a wide range of genetic disorders known collectively as ciliopathies. Intraflagellar transport (IFT) proteins are essential for the assembly and maintenance of cilia by transporting proteins along the axoneme. Here, we report a lack of Ift74, a core IFT-B protein, leading to ciliogenesis defects in multiple organs during early zebrafish development. Unlike rapid photoreceptor cell death in other ift-b mutants, the photoreceptors of ift74 mutants exhibited a slow degeneration process. Further experiments demonstrated that the connecting cilia of ift74 mutants were initially formed but failed to maintain, which resulted in slow opsin transport efficiency and eventually led to photoreceptor cell death. We also showed that the large amount of maternal ift74 transcripts deposited in zebrafish eggs account for the main reason of slow photoreceptor degeneration in the mutants. Together, our data suggested Ift74 is critical for ciliogenesis and that Ift proteins play variable roles in different types of cilia during early zebrafish development. To our knowledge, this is the first study to show ift-b mutant that displays slow photoreceptor degeneration in zebrafish.

Biodiversity-based development and evolution: the emerging research systems in model and non-model organisms.

Evolutionary developmental biology, or Evo-Devo for short, has become an established field that, broadly speaking, seeks to understand how changes in development drive major transitions and innovation in organismal evolution. It does so via integrating the principles and methods of many subdisciplines of biology. Although we have gained unprecedented knowledge from the studies on model organisms in the past decades, many fundamental and crucially essential processes remain a mystery. Considering the tremendous biodiversity of our planet, the current model organisms seem insufficient for us to understand the evolutionary and physiological processes of life and its adaptation to exterior environments. The currently increasing genomic data and the recently available gene-editing tools make it possible to extend our studies to non-model organisms. In this review, we review the recent work on the regulatory signaling of developmental and regeneration processes, environmental adaptation, and evolutionary mechanisms using both the existing model animals such as zebrafish and Drosophila, and the emerging nonstandard model organisms including amphioxus, ascidian, ciliates, single-celled phytoplankton, and marine nematode. In addition, the challenging questions and new directions in these systems are outlined as well.

Heme-binding protein CYB5D1 is a radial spoke component required for coordinated ciliary beating.

Coordinated beating is crucial for the function of multiple cilia. However, the molecular mechanism is poorly understood. Here, we characterize a conserved ciliary protein CYB5D1 with a heme-binding domain and a cordon-bleu ubiquitin-like domain. Mutation or knockdown of Cyb5d1 in zebrafish impaired coordinated ciliary beating in the otic vesicle and olfactory epithelium. Similarly, the two flagella of an insertional mutant of the CYB5D1 ortholog in Chlamydomonas (Crcyb5d1) showed an uncoordinated pattern due to a defect in the cis-flagellum. Biochemical analyses revealed that CrCYB5D1 is a radial spoke stalk protein that binds heme only under oxidizing conditions. Lack of CrCYB5D1 resulted in a reductive shift in flagellar redox state and slowing down of the phototactic response. Treatment of Crcyb5d1 with oxidants restored coordinated flagellar beating. Taken together, these data suggest that CrCYB5D1 may integrate environmental and intraciliary signals and regulate the redox state of cilia, which is crucial for the coordinated beating of multiple cilia.

Disrupted intraflagellar transport due to IFT74 variants causes Joubert syndrome.

PURPOSE: Ciliopathies are a group of disorders caused by defects of the cilia. Joubert syndrome (JBTS) is a recessive and pleiotropic ciliopathy that causes cerebellar vermis hypoplasia and psychomotor delay. Although the intraflagellar transport (IFT) complex serves as a key module to maintain the ciliary structure and regulate ciliary signaling, the function of IFT in JBTS remains largely unknown. We aimed to explore the impact of IFT dysfunction in JBTS. METHODS: Exome sequencing was performed to screen for pathogenic variants in IFT genes in a JBTS cohort. Animal model and patient-derived fibroblasts were used to evaluate the pathogenic effects of the variants. RESULTS: We identified IFT74 as a JBTS-associated gene in three unrelated families. All the affected individuals carried truncated variants and shared one missense variant (p.Q179E) found only in East Asians. The expression of the human p.Q179E-IFT74 variant displayed compromised rescue effects in zebrafish ift74 morphants. Attenuated ciliogenesis; altered distribution of IFT proteins and ciliary membrane proteins, including ARL13B, INPP5E, and GPR161; and disrupted hedgehog signaling were observed in patient fibroblasts with IFT74 variants. CONCLUSION: IFT74 is identified as a JBTS-related gene. Cellular and biochemical mechanisms are also provided.

Melatonin alleviates the deterioration of oocytes and hormonal disorders from mice subjected to glyphosate.

Glyphosate (Gly) is the herbicide widely used in agricultural fields and landscaping. Mammalian exposure to glyphosate could cause neurotoxicity, blood, liver, kidney, endocrine, reproductive, genetic and other toxic effects. Melatonin (MT) is a neuroendocrine hormone secreted mainly by the pineal gland. It is unknown whether MT can improve reproductive defects and hormonal disorders in mice exposed to Gly. In this study, mice were exposed to 250 and 500 mg/kg Gly by intragastric administered and 15 mg/kg MT was treated via intraperitoneal injection for 7 days. Our results shown that the weight gain of mice, the ovarian coefficient and the ATP content in the ovaries were decreased significantly. Gly-treated oocytes showed that the first polar body extrusion failed, the level of oxidative stress increased, and the mitochondrial membrane potential (MMP) decreased. Subsequently, our results showed that increased expression level of BAX protein, reduced expression of BCL-2 protein, ATG12 and LC3 protein expression increased in ovaries after Gly treatment. At the same time, Gly exposure led to abnormal expression of Hypothalamic-Pituitary-Thyroid (HPT) axis-related genes and disrupted hormone homeostasis. After the injection of 15 mg/kg MT, the oocytes showed decreased oxidative stress level, increased mitochondrial membrane potential, incremental ATP content in the ovaries and the hormone levels were approached to the control group. Thus, our results demonstrated that melatonin can improve oocyte quality and maintain hormone homeostasis in mice exposed to glyphosate.

Melatonin Reverses 10-Hydroxycamptothecin-Induced Apoptosis and Autophagy in Mouse Oocyte.

10-Hydroxycamptothecin (HCPT) is a widely used anticancer drug that induces cytotoxicity by triggering the cell apoptotic pathway. Studies have shown that HCPT has harmful effects on normal cells, but whether HCPT affects the development of mouse oocytes in vitro has not been reported. First, this study investigated the development of oocytes exposed to 60 µM HCPT in vitro. In the HCPT-treated group, the first polar body extrusion (PBE) rate of oocytes decreased, spindle morphology was abnormal, DNA double-strand break, oxidative stress level increased, and mitochondrial distribution was abnormal. The apoptosis and autophagy levels of oocytes in the HCPT-treated group were detected by qRT-PCR and western blot. Compared with the control group, the expressions of key regulators of oocyte apoptosis (bax, caspase-3) and autophagy (lc3, beclin, ATG12) pathway were increased in the HCPT-treated group. HCPT treatment induced apoptosis and autophagy in oocytes. Melatonin (MT) can protect cell structure, prevent DNA damage, and reduce the content of peroxides. So we wondered whether MT could ameliorate the harmful effects of mouse oocytes induced by HCPT. Interestingly, the addition of 1 mM MT can protect oocytes from HCPT toxicity to some extent. Compared with the HCPT group, the addition of 1 mM MT increased the PBE ratio of oocytes, decreased ROS levels, and decreased spindle abnormalities and DNA breakage ratio. In summary, these results revealed that HCPT exhibited adverse effects on mouse oocyte maturation and quality, and MT administration alleviated the negative influence of HCPT.

Morpholino-Mediated Knockdown of Ciliary Genes in Euplotes vannus, a Novel Marine Ciliated Model Organism.

Cilia are highly conserved organelles present in almost all types of eukaryotic cells, and defects in cilia structure and/or function are related to many human genetic disorders. Single-celled ciliated protists, which possess diverse types of cilia, are remarkable model organisms for studying cilia structures and functions. Euplotes vannus is a representative ciliate with many intriguing features; for example, it possesses extensively fragmented somatic genomes and a high frequency of + 1 programmed ribosomal frameshifting. However, the molecular mechanisms underlying these remarkable traits remain largely unknown, mainly due to the lack of efficient genetic manipulation tools. Here, we describe the first application of a morpholino-based strategy to knockdown gene expression in E. vannus. Through interfering with the function of two ciliary genes, ZMYND10 and C21ORF59, we showed that these two genes are essential for the ciliary motility and proliferation of E. vannus cells. Strikingly, both ZMYND10- and C21ORF59-knockdown cells developed shorter cilia in the ventral cirri, a special type of ciliary tuft, suggesting a novel role for these genes in the regulation of cilia length. Our data provide a new method to explore gene function in E. vannus, which may help us to understand the functions of evolutionarily conserved cilia-related genes as well as other biological processes in this intriguing model.