|Center for Regenerative Therapies TU Dresden
|Interdisciplinary Center for Neuroscience, Frankfurt
All talks will take place in Campus Biotech, H8-01 Auditorium.
|8:30 - 9:00
|Welcome Coffee / Posters installation
|9:00 - 9:10
|Julien Bertrand & Alessandro De Simone
|KEYNOTE 1 (Chair: Julien Bertrand)
|9:10 - 9:50
Control of organ growth: what can we learn from the posterior lateral line primordium.
The mechanisms controling organ growth are essential during embryo development, as well as during homeostasis to prevent tumor formation. While the Hippo signaling pathway has been shown to play a central role in both contexts, it is not yet clear how the Hippo effectors Yap1 and Taz are precisely regulated. We use the posterior lateral line primordium (pLLP), a migrating group of epithelial cells forming sensory organs, to understand how tissue growth is controlled during organ formation. The pLLP is highly dynamic and easily accessible for imaging, making it ideal to track biological processes underlying tissue morphogenesis.
|SESSION 1: early development (Chair: Olga Afonso)
|9:50 - 10:10
|Shayan Shami Pour
Yolk granule fusion and microtubule aster formation regulate cortical granule exocytosis in zebrafish oocytes.
Dynamic reorganization of the cytoplasm is key to many core cellular processes, such as cell division and cell polarization. Cytoskeletal rearrangements are thought to constitute the main drivers of cytoplasmic flows and reorganization. In contrast, remarkably little is known about how dynamic changes in size and shape of cell organelles affect cytoplasmic organization. Here, we show that within the maturing zebrafish oocyte, the surface localization of cortical granules and Rab11, a master regulator of exocytosis, is achieved by the combined activities of yolk granule fusion to the centre and microtubule aster formation and flows to the oocyte cortex, which collectively enable cortical granule exocytosis and the subsequent chorion elevation thus preparing the oocyte for fertilization.
|10:10 - 10:30
Role of Palmitoylating Enzymes in Cilia Formation and Function in the Zebrafish Model.
zDHHCs form a family of 23 enzymes performing palmitoylation, a reversible lipid modification, that regulates protein trafficking and turnover in the cell. We hypothesized that zDHHC enzymes could be involved in maintaining the unique protein composition of the cilia membrane. We studied the expression of zDHHC family and observed that among all zDHHCs, zDHHC4 was the most abundant in most hair cell types. We confirmed this observation experimentally and showed that downregulation of zDHHC4 leads to a “cilia” phenotype in zebrafish larvae, misexpression of important cilia markers (spaw, atoh1a) and reduction of cristae hair cells number. To better understand zDHHC4’s role in cilia’s development and function, the project is currently focusing on identifying the molecular targets of zDHHC4.
|10:30 - 11:10
|Coffee Break (Posters/Exhibitors)
|SESSION 2: Vascular and Neural biology (Chair:Julien Bertrand)
|11:10 - 11:30
Junctional dynamics and cell-cell interactions underlying vascular tube formation require signaling by PI3-Kinase α.
Vertebrates depend on a functional vasculature to transport oxygen and nutrients to all tissues of the body and to remove waste products from them. Most vascular tubes display multicellular architecture, i.e. there is always at least two endothelial cells outlining the vessel lumen. During development, endothelial cells in early vessels rearrange and intercalate, thereby transforming the vessel architecture from uni- to multicellular. It was recently reported that abrogation of PI3-Kinase α signaling impairs multicellular tube formation in both mouse retina and zebrafish embryos. Here, we show specific local activity of PI3-Kinase α at endothelial cell-cell interfaces and how genetic loss of PI3-Kinase α prohibits cell intercalation movements by destabilizing de novo junctional contacts.
|11:30 - 11:50
Biallelic variants in BRF2 are associated with craniofacial anomalies and cognitive impairment.
Biallelic variants in BRF2 are associated with craniofacial anomalies and cognitive impairment BRF2 encodes a TFIIB-like transcription factor, which recruits the RNA-polymerase III to transcribe specific noncoding RNAs. We identified biallelic variants in BRF2 in 7 individuals from 3 families presenting a variable phenotype ranging from craniofacial anomalies to developmental delay depending on the combination of variants. In silico 3D protein modelling and cellular assays show functional impairment of these variants. Zebrafish knocked down for the orthologous brf2 presented with behavioural and morphological anomalies which could be complemented by the human BRF2 mRNA. Our data support the pathogenicity of the identified BRF2 variants, linking another RNA-polymerase III subunits to brain anomalies.
|11:50 - 12:10
|Joaquin Navajas Acedo
Spatiotemporal emergence of somato-sensory neuron diversity.
How neuron diversity emerges during development at the cellular and molecular levels is poorly understood. In zebrafish, the primary somatosensory system of Rohon-Beard (RB) neurons disappears and is replaced by the DRG, but our results contradict this 100-year-old paradigm. Our work, combining imaging and single-cell transcriptomics across development, shows RBs possess complex neuronal diversity at three levels: transcriptome, axial distribution and interindividual distribution. In toto cell tracking shows RBs possess simple cell lineages, making possible to link neuron diversity to cell behaviors in development. Current work aims to use spatial transcriptomics and manipulations to comprehensively describe the molecular cascade necessary for RB development and diversification
|12:10 - 12:30
A key role in controlling brain excitability falls to excitatory amino acid transporter 2 (EAAT2) that removes glutamate from the synaptic cleft after a synaptic event.
Knockout of eaat2a in zebrafish leads to spontaneous epileptic seizures, but paradoxically mutant larvae display reduced neuronal activity between seizures. In order to investigate this hypoactivity, we performed a transcriptome analysis of larval brains. Preliminary results point towards upregulation of anticonvulsant neuropeptides, particularly galanin.
|12:30 - 12:45
|Swiss Zebrafish Society meeting
|12:45 - 14:00
|Lunch Break (Posters/Exhibitors)
|KEYNOTE 2 (Chair: Alessandro De Simone)
|14:00 - 14:40
Functional restoration of the regenerating adult zebrafish retina.
Zebrafish can regenerate many organs and tissues, including the central nervous system (CNS). Within the CNS-derived neural retina, light lesions cause a loss of photoreceptors and subsequent activation of Müller glia, acting as retinal stem cells. Müller glia-derived progenitors differentiate and eventually restore retinal anatomy within four weeks. However, little is known about how light lesions impair vision functionally, and how well visual function is restored during regeneration in adult animals. We applied novel quantitative behavioral assays to assess restoration of visual function during homeostasis and regeneration in adult zebrafish. In a vision-dependent social preference test, vision is massively impaired early after lesion, but returns to pre-lesion levels within 7 days after lesion. In a quantitative optokinetic response assay with different degrees of difficulty, similar to vision tests in humans, we find that vision for easy conditions with high contrast and low level of detail, as well as color vision, is restored around 7-10 days post lesion. Under more demanding low contrast and high detail conditions, vision is regained only later from 14 days post lesion onwards. In parallel work, we studied electrophysiological recovery of stimulus-dependent gCaMP-conductances in regenerating cones. We conclude that vision, based on contrast sensitivity, spatial resolution and color perception, is fully regenerated in adult zebrafish retina in a gradual manner.
|SESSION 3: Regeneration and Stem Cells (Chair: Alessandro De Simone)
|14:40 - 15:00
The regeneration-responsive element careg monitors activation of Müller glia after MNU-induced damage of photoreceptors in the zebrafish retina.
Zebrafish can regenerate their damaged photoreceptors through the activation of Müller glia. Using the MNU-treatment injury model, we identified that the transgenic reporter careg, a marker of regenerating fin and heart, also participates in retina restoration in zebrafish. scRNAseq analysis of regenerating retinas revealed distinct molecular signatures of careg:EGFP expressing and non-expressing Müller glia, suggesting their heterogenous responsiveness to the regenerative program. Dynamics of ribosomal protein S6 phosphorylation showed that the TOR signaling became progressively switched from Müller glia to progenitors. In conclusion, the careg reporter detects activated Müller glia, and provides a common marker of regeneration-competent cells in diverse zebrafish organs.
|15:00 - 15:20
Investigating Injury Types and Intercellular Communication in Zebrafish Heart Regeneration.
Zebrafish hearts can regenerate regardless of the age or injury method used, but the effect of different injury methods and intercellular interaction is not clear. We used bioinformatics to compare transcriptomics data from injuries - genetic ablation, resection, and cryoinjury. For intercellular communication, we developed a ligand-receptor network using differential genes from three studies and identified important nodes using network theory and natural language processing. We show a core regeneration pathway and the crucial role of fibroblasts. We are confirming our results using knockout lines and present them as web apps for the community.
|15:20 - 15:40
p16.1/.2’s roles in hematopoiteic progenitors’ commitment towards lymphoid fate?
Blood is regenerated by a cell population known as hematopoietic stem cells (HSCs). We identified si:ch211-214p16.1 and si:ch211-214p16.2 – shortened as p16.1 and p16.2 – as two undescribed genes specifically expressed by emerging HSCs in the dorsal aorta of the zebrafish embryo. Knock-down of both genes using splice morpholinos leads to a general decrease of lymphopoiesis: this translates by a loss of lymphoid precursors in the CHT at 48hpf, a delay of thymus seeding at 60hpf, as well as a decreases of ikaros and rag1 signal in the thymus in situ hybridization at 4 and 5 days post fertilization, respectively. Transcriptomics data suggest that p16.1 and p16.2 could be interactors of rac1l, an ortholog of rac1, a Rho-GTPase shown to be involved in T-cell differentiation in mice.
|15:40 - 16:00
The role of basal keratinocytes keratins in zebrafish epidermis differentiation.
Keratin expression ‘switches’ are a hallmark of skin differentiation. To further investigate an active role of keratins in the differentiation process, we are studying the developing zebrafish epidermis, which consists of the outer periderm cell layer and the more “stem-like” basal keratinocyte cell layer. Analysis of the basal cell layer reveals that a subset of basal keratinocyte keratins shows a temporal and spatial restricted pattern in contrast to other components of the embryonic skin, such as E-Cadherin and the transcription factor tp63. To further investigate the role of those keratins in the differentiation process, we are using CRISPR-Cas9 to knock-out basal keratinocytes keratins
|16:00 - 16:30
|Coffee Break (Posters/Exhibitors)
|SESSION 4: New labs in Switzerlands (Chair: Stephan Neuhauss)
|16:30 - 16:50
|Alessandro De Simone
How does a regenerating scale reach its final size?
Regeneration poses key questions regarding how signals are organized in time and space to recover a body part of the right size. We tackle these questions with a quantitative approach using the zebrafish scale as model system. Scales are dermal bone disks, whose bone-forming cells regenerate through phases of differentiation, proliferation and then hypertrophy. Dynamic Fgf/Erk signaling regulate these processes of proliferation and hypertrophy. As cells proliferate, Erk is uniformly high. At the transition to hypertrophy, Erk switches off. Thereafter, cell growth is driven by a series of expanding Erk activity waves. How does the Erk switch-off relate to the transition from proliferation to hypertrophy? How is wave generation tuned so that scale cells reach their appropriate size?
|16:50 - 17:10
The spark of life. Initiating transcription in embryos.
The localization of transcriptional machinery in specialized transcription bodies is a hallmark of gene expression in eukaryotic cells. In spite of the attention these bodies have received in recent years, it is not so clear how they form, and if and how they affect gene expression. It is generally difficult to address these questions because bodies are often small, short-lived, and highly abundant. Furthermore, without knowing the sequence that nucleates their formation, it is impossible to perturb them specifically. We have taken advantage of two prominent transcription bodies that mark the onset of transcription during zebrafish embryogenesis to address some of these questions. Here, I will talk about our latest results regarding the formation and function of transcription bodies.
|Concluding remarks – Poster awards
|Farewell Happy Hour
Sorry, we are sold out.
Hematopoietic stem cells (HSCs) arise as a rare population from the hemogenic endothelium and subsequently colonise the zebrafish caudal hematopoietic tissue (CHT), the foetal liver in mammals, where they expand. We previously showed that the transcription factor tfec is strongly involved in HSCs expansion in the CHT. ATAC-seq analysis identified, in tfec intron 2, a potential enhancer that could be bound by a number of transcription factors. Among these feature the MAF family genes which are highly conserved throughout species. We focus on mafa (c-Maf in mammals) which is expressed in the vascular niche. We show that mafa regulates tfec expression. We aim to further elucidate the role of mafa in regulating HSCs, by deciphering the genetic network controlled by mafa in endothelial cells.
PTEN-induced putative kinase 1 (PINK1) is a well-characterized regulator of mitochondrial quality control through mitophagy and its mutations are associated with recessive Parkinson’s disease. However, little is known about its functions in normal and malignant hematopoiesis in vertebrates. Here we aim to unravel the roles of PINK1 in definitive hematopoiesis and its underlying mechanisms using zebrafish (Danio rerio). In this study, we utilized CRISPR/Cas9 system to generate pink1 knockout zebrafish model and PINK1-deficient leukemia cell line. We found that pink1 deficiency activated autophagy in hematopoietic cells and promoted definitive hematopoiesis in zebrafish embryos, which can be alleviated by canonical autophagy inhibition. Further, the proteomic and metabolic analysis revealed.
Fibrinogen plays a critical role in hemostasis. It has two copies of A_, B_ and _ polypeptides. A_E is an A_ isoform with an extra C-terminal domain (_EC). _EC is conserved in vertebrates but its function is unclear. In zebrafish embryos most fibrinogen molecules contain A_E, but in the AB background a mutation in the fibrinogen A_ chain gene (fga) selectively prevents A_E production and results in slower blood clotting. This is corrected by expression of A_E, but not A_. To characterize the function of _EC we are studying structure-activity relationships using expression of mutated A_E in fga-/- zebrafish larvae and measuring clotting times and cellular interactions. We are also developing tools to purify recombinant zebrafish fibrinogen to measure fibrinogen clotting parameters in vitro.
Chemical tRNA modifications are critical for accurate and efficient translation, and their absence can lead to codon-specific translation. Wobble uridine (U34) is a key example of such modifications, but their role and synthesis in vertebrate development remain unclear.
Using zebrafish, we investigate the expression pattern of modifying enzyme genes by in situ hybridization and RNA-seq. We find that certain pathways exhibit tissue-specific expression and that the enzymes work coordinated during embryogenesis. Also, the mutant larvae of U34 modifying enzymes by CRISPR-Cas9 showed morphological differences in brain size and lower survival rates than the wild type. This suggests that the fish rely on U34 modifications especially during early development, with less influence in adults.
Fin regeneration has been extensively studied in zebrafish, but little is known about regulators of this process in distant fish taxa, such as the Poeciliidae family. Here, we use platyfish to investigate several aspects of fin regeneration. Firstly, blocking the BMP type-I receptor suppressed phospho-Smad1/5 immunoreactivity, and impaired fin regeneration after blastema formation, with no bone or actinotrichia restoration. In addition, the wound epidermis displayed extensive thickening associated with expanded Tp63, suggesting abnormal tissue differentiation. Our data adds to increasing evidence for the integrative role of BMP signaling in epidermal and skeletal tissue formation during fin regeneration and expands knowledge of common regenerative mechanisms in diverse clades of teleosts.
Drug repurposing has become an important strategy overall and particularly in response to the COVID-19 pandemic. However, often the effects of well-studied clinically approved drugs are poorly documented in the context of embryonic development. This may have important consequences for the prescription during pregnancy. We developed a zebrafish screening platform assesses side effects on cardiovascular development of FDA approved drugs used in clinical practice to treat COVID-19 and their immune modulatory effect upon spike protein treatment. We also assessed if zebrafish can be infected with Coronavirus from the Sars-Cov-2 family.
Glucose is vital for metabolism, ATP production, and biosynthesis. Photoreceptors use glucose and lactate to meet their energy needs. The energy metabolism and collaborative metabolic environments are crucial for proper retinal function. However, the precise dynamics, particularly in vivo, are poorly understood. We studied glucose uptake and lactate production in the retina. In Glut and Ldh mutant retinas, preventing glucose entry or lactate production, we found reduced glycolysis, morphological alterations, and vision loss. Mitochondrial metabolism couldn't compensate, emphasizing the essential role of glucose and lactate. This study sets the stage for using zebrafish to elucidate metabolic patho-mechanisms underlying photoreceptor degeneration and retinal diseases
The visual pigment 11-cis retinal (RAL) is fundamental for photoreceptor light absorption and the recycling of 11-cis RAL is vital for the vertebrate neuroretina. The Retinal G-coupled protein Receptor (RGR) is a non-visual opsin that is known to be involved in the recycling of 11-cis RAL, however the precise function is still debated. Herein, the goal is to address the role of RGR in the recycling of 11-cis RAL, using the zebrafish larval retina. Starting with the identification of rgra expression pattern, the protein localization of RGRa and the generation of respective knock-out line, we ultimately aim to characterize the physiology and morphology of rgr mutants and suggest a model for the recycling of 11-cis RAL including RGR.
Endothelial cell polarity is essential for initiation of vascular lumen formation. However, it remains largely unclear how the junctional and apical proteins are properly segregated. Here, we studied the cellular activities and protein dynamics with a photoconvertible version of VE-cadherin (Cdh5-mClav) in the vasculature. our study reveals that formation of the vascular lumen requires a balance of actomyosin contractility between apical and junctional domains that regulating the movement junctional complexes between different compartments.
Neurotoxicity occurs when exposure to chemicals alters the normal function of the nervous system. It is often assessed by measuring behavioral alterations, as they reflect the combined action of neuronal, neuroendocrine and neuromuscular signals. However, it remains unclear whether these alterations are transient or reversible, and to what extent they reflect specific changes in the nervous system. We address these questions, by studying zebrafish larvae behavior upon exposure to neurotoxic substances, followed by depuration in clean water to assess recovery potential. We analyze molecular, cellular and structural changes in the nervous system in order to investigate mechanistic links.
Fibroblasts are a heterogeneous cell-population with key roles in tissue development, homeostasis and repair. Challenging the view of fibroblasts as “solitary cells”, we propose that dynamic cell-cell interactions play a central role in their coordinated differentiation and ability to adapt to environmental changes. Using in toto imaging, we have defined measures for fibroblast organization and connectivity using point-cloud and network representations, respectively. These allow characterization of global fibroblast distribution and connectivity across different developmental stages using features defined for individual cells. Integrating transcriptional data on fibroblast sub-type markers will allow us to address the role of cell heterogeneity in network organization and vice versa.
Many signals important for morphogenesis have been discovered, but it is unclear how they are organized to coordinate regeneration. We address this question in zebrafish scales, using live imaging. After scale loss, a new bone is deposited by a monolayer of osteoblasts, which forms through differentiation, followed by proliferation, and finally hypertrophy. Erk signaling is a key regulator of cell proliferation. At the transition from proliferation to hypertrophy, Erk switches from a high to a low activity state. In this project, we ask how Erk instructs proliferation and eventually stops it. We test the working hypothesis that Erk is inactivated by compressive forces arising as cells get crowded.
Microglia are specialized phagocytes of the brain that can engulf and remove apoptotic neurons. These dying cells release a number of critical instructive signals, such as ATP, ADP, and UDP. Previous studies have shown that ATP induces chemotaxis via the microglial GPCR P2RY12, while UDP promotes neuronal phagocytosis via GPCR P2RY6 in culture. Despite this, however, little is known about the spatiotemporal regulation of these receptors and their role in controlling specific microglial activities. Using quantitative imaging and perturbations in zebrafish larvae, we aim to understand how P2RY6 and P2RY12 signalling contribute to the recognition and engulfment of apoptotic neurons and whether activating these pathways can trigger specific responses in these potent brain phagocytes.
Ciliopathies like Joubert Syndrome (JS) arise from dysfunctional primary cilia. Neurological defects seen in many ciliopathies, e.g., the cerebellar malformation in JS, highlight the crucial yet poorly understood role for primary cilia in CNS development. Using the zebrafish JS models cc2d2aw38 and talpid3i264, we show reduced Arl13b-positive primary cilia in various regions of the CNS, including the cerebellum. Despite this, we see no CNS morphological anomalies and normal Purkinje and eurydendroid cell layers. Nevertheless, gene set enrichment analysis of whole larval RNA-seq data indicates that zebrafish JS mutants show differentially expressed gene sets enriched for synaptic function or voltage-gated ion channel genes, and thus may exhibit underlying defects in neuronal function.
To investigate occurrence of the FLASH effect using various quality of beam, wt zebrafish (AB) embryos were irradiated with electron, proton and photon beams at various dose and dose rates. Zebrafish embryos (n>20; 4-4.40hpf) were irradiated at 8-12Gy with eRT6/Oriatron, 5.5MeV electrons at 0.1 (conventional), 1400 and >106Gy/s (FLASH, 1 pulse); transmission proton irradiations at 0.1, 1 (conventional) and 1400Gy/s (FLASH), 235MeV with Gantry 1 (continuous cyclotron beam) and Xrad 225CX/225KeV photons-Cu Filter 3mm-13mA-2.27Gy/min (conventional). Dosimetry was performed as described in (Jorge, 2019; Christensen, 2021 and Nesteruk, 2021). Survival and development were monitored as well as cell death and proliferation. The development of embryos was not impaired by 8Gy delivered in 1 pulse.
Cardiovascular diseases (CVD) account for 17.9M deaths/year. Platelets are prominent actors in CVD, but our understanding of factors regulating their behaviour is incomplete. Several microRNAs (miRNAs) are abundant in platelets and associated with their reactivity, including miR-223. To study the role of miR-223 in thrombocytes, the cellular equivalent of platelets in zebrafish, we developed a transgenic zebrafish model with altered thrombocyte miR-223 levels and monitored thrombus formation by laser-induced blood vessel injury. Increased thrombocyte miR-223 shortens the time to first thrombocyte binding at injury sites, and increases the number of cells exiting a thrombus after initial binding. This demonstrates a role for miR-223 in thrombocyte attachment kinetics and thrombus stability.
Cancer is without a doubt one of the major epidemiologies of the current era and one of the main causes of death worldwide, leading to 10 million fatalities in 2020. Among the most common types of cancer are those that develop in the lung, breast, prostate, and colon/rectal, and due to their high frequency, these are also some of the most studies types of cancer.
In regeneration, a lost or damaged organ is rebuilt to its original size and shape. To study how signals control the cell behaviors necessary for regeneration, we use the zebrafish scale has a model system. In the latest phase of scale regeneration, scales grow by hypertrophy, i.e., cell grow without proliferation. Hypertrophy is instructed by a series of concentric waves of activity of the Extracellular Signal- Regulated kinase (Erk). I am investigating what stops wave generation once scales reach their correct final size. To this end, I am characterizing how wave properties change as scales approach the end of their regeneration. I am aiming at perturbing this process by generating tools to selectively perturb the pattern of Erk activation and generate ectopic waves.
Somitogenesis is a dynamic developmental process unique to vertebrate species, whereby the anterior-posterior embryonic axis becomes sequentially segmented into somites. The formation of these somites entails fast/sharp molecular transitions that are orchestrated by a molecular oscillator. By applying single cell genomics in zebrafish, we are reconstructing the spatiotemporal map of cell states and transitions that lead to the formation of somites. The integration of RNA and ATAC data will enable us to gather a comprehensive picture of these fast transitions, understand which genes and transcription factors are involved and what architecture of molecular events leads to the correct formation of somites.
Our studies in the zebrafish retina show that knock out of two postsynaptic members of the Excitatory Amino Acid Transporters (EAATs) group, eaat5b and eaat7 causes impaired responses to stimuli in the short- and long-wavelength ranges, implying a wavelength-specific tuning of these two proteins. Imaging the whole-retina distribution of these transporters, we discovered that they are differentially expressed in the outer plexiform layer and particularly in a region essential for prey capture. To assess if these two proteins are related to known wavelength-specific behaviors, we created behavioral assays targeting short- and long-wavelengths. Taken together, our work puts Eaat5b and Eaat7 as potentially important factors for adjusting light integration dynamics through the inner retina.
Gliomas are the most common malignant tumors of the nervous system. Patient tumors are highly heterogeneous; thus, patient prognosis remains poor, which highlights the need new personalized therapies. Here, we propose an assay to test combination of drugs and new potential molecules in patient-derived microtumors in zebrafish. We cultivated glioma tumoral cells from patients, labeled and injected them into zebrafish to study their progression. Given the complexity and unique characteristics of each patient tumor, their cells behaved and responded to drugs differently. The microtumors were followed with light-sheet fluorescent microscopy and its volumes quantified. This assay allows for co-clinical dynamic screening of response to different treatments for each patient.
Gene expression is crucial. It has been shown that transcription occurs in transcription bodies, which are difficult to study due to their small size and the impossibility of specifically disrupting them, which is why their impact on transcriptional output is still unclear. During the activation of transcription in fish embryos, however, two prominent transcription bodies emerge, which can be specifically disrupted. Using live imaging and nascent RNA-seq, we found that transcription bodies regulate transcription by accumulating pause-release machinery creating a favorable environment for transcription locally, while depriving genes elsewhere from the same machinery.
Difficulties to conserve lipid droplets (LD) in larvae has limited their use in lipid metabolism research. 750 larvae were entered at 5 dpf in two high fat diet interventions (HFD) or a control diet. Whole larvae neutral lipid content was measured using Oil Red O. Using a novel embedding and cryosectioning protocol, we characterized muscle and liver LD. Survival and growth were not impacted by HFD. Significant increases in neutral lipids were observed at 15, 18 and 21 dpf. At 21 dpf, HFD increased intramuscular LD size but not number. The increase of LD number in liver was associated with the emergence of fibrosis. In conclusion, our novel protocol confirmed the preservation of organ morphology and allowed subcellular LD volume quantification.
Previous analyses on blood vessel formation and anastomosis in zebrafish have shown that junctional remodeling is central to many aspects of morphogenetic endothelial cell-cell interactions. Cell rearrangement is mediated by a particular structure, called junction-based lamellipodia (JBL), which are thought to provide a tractile force for junction elongation. Using pharmacological inhibition and novel fluorescent reporters we are investigating regulatory and physical mechanisms underlying the respective steps of JBL function. In particular we identified the actomyosin contractility as an important basis for junctional ring elongation
m6A is the most abundant internal modification of eukaryotic mRNA. It is introduced by a complex containing the catalytic subunit Mettl3 and two additional members: Mettl14 and Wtap.m6A has been linked to multiple developmental processes. Understanding the molecular mechanism of how m6A modulates these processes remains a fundamental challenge. To identify these mechanisms, we deleted Mettl3 in zebrafish. Mettl3-/- fish die within 28 days post fertilization. Using single-cell RNAseq, we found that multiple eye-specific cells are underrepressented in the mutants and was confirmed by histological analysis. Excitingly, we found that mutant cells adapt to m6A absence through autoregulation of Wtap splicing. Our work provides a framework for understanding how m6A functions during vertebrate development.
The stress response is an organism’s reaction to a stressor, triggering a cascade of physiological changes aiming to restore the organism’s homeostasis. In teleosts, the HPI axis, as well as the HSC axis plays an important role in those responses, ultimately leading to behavior alterations. There are numerous behavioral responses of fish to a negative stressful stimulus, that are commonly measured, unfortunately, little is known about reactions to a positive stimulus, e.g., a feed reward in fish. In this study, the effects of a positive (feed reward) and a negative stressor (netting above water surface for 1 min) on the behavior of male and female zebrafish were examined.