Recombinant Danio rerio Pescadillo (pes), partial

Basic Research Questions

• What is Pescadillo (pes) in Danio rerio and what are its primary functions?

Pescadillo (pes) is a gene in zebrafish (Danio rerio) that encodes a protein essential for nucleolar assembly, ribosome biogenesis, and cell proliferation. In vertebrates, this protein localizes to distinct substructures of the interphase nucleus, particularly nucleoli, which are the sites of ribosome biogenesis . During mitosis, pescadillo closely associates with the periphery of metaphase chromosomes and, by late anaphase, becomes associated with nucleolus-derived foci and prenucleolar bodies .

Mutation studies have revealed that disruption of the pescadillo gene blocks expansion of multiple tissues in developing zebrafish embryos, indicating its critical role in controlling cell proliferation . Additionally, pescadillo function is required for both proper oligodendrocyte progenitor formation (by regulating cell cycle progression) and normal levels of myelin gene expression .

Mouse embryos lacking pescadillo arrest at morula stages of development, with nucleoli failing to differentiate and ribosome accumulation being inhibited . This developmental arrest demonstrates the protein's essential role in early vertebrate development through its impact on ribosome biogenesis and nucleologenesis.

• How do recombinant Danio rerio pescadillo proteins from different expression systems compare?

Recombinant Danio rerio pescadillo can be produced in various expression systems, each with distinct characteristics affecting protein structure, functionality, and application suitability. The table below summarizes key differences:

When selecting an expression system for pescadillo research, consider:

• The specific requirements of your experimental design

• The importance of post-translational modifications for your application

• The potential impact of host cell contaminants

• Whether your study focuses on structure or function

For functional rescue experiments in zebrafish, mammalian cell-derived protein may provide the most physiologically relevant option, while E. coli-derived protein might be sufficient for antibody generation or structural studies .

• What are the optimal storage and handling protocols for recombinant Danio rerio pescadillo?

Proper storage and handling of recombinant pescadillo is critical for maintaining protein integrity and experimental reproducibility. Follow these evidence-based protocols:

Storage conditions:

• Store at -20°C for standard storage

• For extended storage periods, conserve at -20°C or -80°C

• For working aliquots, store at 4°C for up to one week

• Avoid repeated freezing and thawing, which is not recommended

Reconstitution methodology:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (optimally 50%) for long-term storage

• Prepare small aliquots for single use to avoid repeated freeze-thaw cycles

Shelf life considerations:

• Liquid form: approximately 6 months at -20°C/-80°C

• Lyophilized form: approximately 12 months at -20°C/-80°C

• Shelf life is influenced by storage state, buffer ingredients, storage temperature, and the stability of the protein itself

Implementing these practices will help maintain protein stability and functionality throughout your experimental timeline.

• What experimental approaches can be used to study pescadillo's role in oligodendrocyte formation?

Research has established that mutation of pescadillo disrupts oligodendrocyte formation in zebrafish through several mechanisms. To investigate this role comprehensively, implement these methodological approaches:

Genetic manipulation strategies:

• Utilize established pescadillo mutant lines (e.g., vu166) for loss-of-function studies

• Implement CRISPR/Cas9 for generating specific mutations in pescadillo domains

• Use morpholino knockdown for temporal control of pescadillo expression

• Design rescue experiments with recombinant pescadillo to confirm specificity

Visualization and quantification methods:

• Employ transgenic reporter lines (e.g., Tg(olig2:EGFP)) to visualize oligodendrocyte progenitor cells

• Perform in situ hybridization to assess myelin gene expression (e.g., mbp, plp1b)

• Use immunohistochemistry to visualize myelin sheaths and oligodendrocyte morphology

• Implement time-lapse imaging of transgenic lines to track oligodendrocyte development

Cell cycle and proliferation analysis:

• BrdU or EdU incorporation assays to quantify cell proliferation

• Cell cycle phase analysis through flow cytometry of dissociated cells

• Quantification of oligodendrocyte progenitor numbers at different developmental stages

• Assessment of cell cycle regulator expression in pescadillo-deficient contexts

Functional assays:

• Electrophysiology to measure conduction velocity in pescadillo mutants

• Behavioral assays to assess functional consequences of myelin defects

• Electron microscopy to evaluate myelin ultrastructure

• Molecular analysis of myelin protein expression levels

When analyzing data from these experiments, distinguish between pescadillo's effects on oligodendrocyte progenitor formation versus myelin gene expression, as both processes are affected by pescadillo disruption .

• How do zebrafish housing conditions impact pescadillo-focused research outcomes?

Zebrafish housing conditions significantly influence experimental outcomes in pescadillo research by affecting baseline physiology, stress levels, and developmental parameters. Standardize these critical factors:

Housing density impacts:

• Maintain optimal stocking density of 5 fish/L to minimize stress-related variables

• Higher densities can increase stress and potentially alter gene expression patterns

• Stress responses may confound cell proliferation studies involving pescadillo

Nutrition considerations:

• Provide nutritionally rich feeds including rotifers, which have been shown to mitigate anxiety-like behaviors

• Dietary composition influences development and can affect ribosome biogenesis

• In feeding studies, spirulina supplementation (4%) showed optimal growth performance for Danio rerio, which could impact developmental studies

Breeding and embryo handling:

• Zebrafish naturally breed at dawn; set up breeding at the beginning of their light period

• For pescadillo developmental studies, collect embryos promptly and maintain at 28.5°C

• Consider that embryos hatch naturally from chorions after 48-72 hours post-fertilization, but dechorionation can assist with mounting and imaging

Strain considerations:

• Different strains (TU, TL, AB) may have varying baseline pescadillo expression or function

• Strains show different susceptibility to experimental manipulations

• Document and standardize strain usage across experiments for reproducibility

Implementing these standardized housing protocols will minimize variability and enhance the reliability of pescadillo research outcomes.

Advanced Research Questions

• What molecular mechanisms underlie pescadillo's role in nucleolar assembly and ribosome biogenesis?

Pescadillo plays critical roles in nucleolar assembly and ribosome biogenesis through several sophisticated molecular mechanisms that can be investigated using advanced techniques:

Nucleolar assembly pathway:

• During mitosis, pescadillo associates closely with the periphery of metaphase chromosomes

• By late anaphase, pescadillo relocates to nucleolus-derived foci and prenucleolar bodies

• This dynamic localization pattern suggests pescadillo functions as a scaffold protein during nucleolar reformation after mitosis

• Mouse embryos lacking pescadillo show failure of nucleolar differentiation, indicating its essential role in nucleologenesis

Ribosome biogenesis involvement:

• Pescadillo appears critical for pre-rRNA processing, particularly in the maturation of large ribosomal subunit RNA

• It likely participates in the assembly of the 60S ribosomal subunit

• Disruption of pescadillo function leads to inhibited accumulation of ribosomes in mouse embryos

• The protein may coordinate multiple steps in ribosome synthesis, from rRNA transcription to subunit assembly

Experimental approaches to investigate these mechanisms:

• Ribosome profiling: Compare ribosome assembly in wild-type versus pescadillo-mutant zebrafish using polysome gradient analysis

• Nucleolar proteomics: Identify pescadillo-interacting proteins through affinity purification followed by mass spectrometry

• RNA-protein interaction analysis: Implement CLIP-seq to map pescadillo binding sites on pre-rRNAs

• High-resolution imaging: Utilize super-resolution microscopy to visualize pescadillo's subnucleolar localization during different cell cycle phases

For comprehensive analysis, combine these approaches with genetic manipulation strategies to determine which domains of pescadillo are essential for its various functions in nucleolar assembly versus ribosome biogenesis.

• How does pescadillo function in cell cycle regulation, and what methodologies best reveal these mechanisms?

Pescadillo's role in cell cycle regulation is multifaceted and critical for proper development. Research shows that pescadillo protein levels increase in rodent hepatocytes as they enter the cell cycle, suggesting a regulatory function in proliferation .

Cell cycle regulatory mechanisms:

• Pescadillo likely functions at the G1/S phase transition

• Disruption of pescadillo function results in cell cycle arrest, as observed in mouse embryos that arrest at morula stages when lacking pescadillo

• In zebrafish, mutation of pescadillo blocks expansion of various tissues in developing embryos, consistent with a cell proliferation defect

• Specifically in oligodendrocyte development, pescadillo regulates cell cycle progression affecting progenitor formation

Recommended methodological approaches:

• Cell synchronization studies: Compare cell cycle progression in pescadillo-deficient versus wild-type cells after synchronization

• Protein interaction mapping: Identify pescadillo interactions with cell cycle regulators using proximity labeling approaches

• ChIP-seq analysis: Determine if pescadillo associates with chromatin at specific cell cycle-regulated genes

• Phosphoproteomics: Investigate cell cycle-dependent phosphorylation of pescadillo

Experimental design considerations:

• Include multiple timepoints across the cell cycle

• Implement live cell imaging with cell cycle phase markers

• Compare tissue-specific effects, as certain tissues may be more sensitive to pescadillo disruption

• Correlate cell cycle defects with nucleolar functions to distinguish primary from secondary effects

When investigating oligodendrocyte-specific effects, combine BrdU labeling with oligodendrocyte lineage markers to specifically track progenitor proliferation in pescadillo mutant contexts .

• What are the best practices for rescue experiments using recombinant pescadillo in pescadillo-mutant zebrafish?

Rescue experiments provide definitive evidence for gene specificity and function. When designing rescue experiments with recombinant pescadillo in zebrafish models, implement these methodological best practices:

Delivery strategies comparison:

Experimental design considerations:

• Include appropriate controls:

  • Uninjected pescadillo mutants

  • Wild-type siblings with same treatment

  • Injection of unrelated protein/mRNA as specificity control

  • Dose-response experiment to determine optimal concentration

• Multiple phenotype assessment:

  • Quantify oligodendrocyte progenitor numbers using transgenic reporters

  • Measure myelin gene expression via qPCR or in situ hybridization

  • Assess nucleolar morphology through immunofluorescence

  • Evaluate ribosome biogenesis markers

  • Measure cell proliferation in affected tissues

• Rescue specificity analysis:

  • Test domain-specific mutants to map functional regions

  • Compare rescue efficacy of pescadillo from different expression systems

  • Implement tissue-specific rescue to determine cell autonomy

Data interpretation framework:

• Complete rescue strongly suggests direct replacement of missing function

• Partial rescue may indicate indirect effects, dosage issues, or multiple functions

• Differential rescue of distinct phenotypes (e.g., cell number vs. gene expression) suggests separable functions

• Timing-dependent rescue provides insights into developmental windows of pescadillo requirement

This systematic approach will yield rigorous evidence for pescadillo function while minimizing confounding variables.

• How can advanced imaging techniques be optimized for studying pescadillo dynamics in zebrafish?

Zebrafish embryos offer exceptional optical properties for advanced imaging studies. To optimize imaging of pescadillo dynamics, implement these evidence-based protocols:

Sample preparation optimization:

• Mounting methods: Use low-melt agarose (0.8-1.2%) to immobilize embryos while maintaining viability

• Anesthetization: Apply tricaine (0.016% working solution) to prevent movement; avoid excessive amounts which can kill embryos

• Dechorionation: Remove chorion at appropriate stages (naturally occurs at 48-72 hpf) for better optical access

• Embryo orientation: Position embryos appropriately for specific tissues of interest (lateral for spinal cord oligodendrocytes)

Imaging modality selection:

• Confocal microscopy for high-resolution 3D imaging of pescadillo localization

• Light sheet microscopy for long-term, low-phototoxicity imaging of developmental processes

• Super-resolution microscopy for sub-diffraction visualization of nucleolar substructures

• Two-photon microscopy for deep tissue imaging with reduced photodamage

Vessel and mounting considerations:

• Round bottom plates help center embryos for easier automated imaging

• Glass-bottom dishes provide superior optical properties for high-resolution imaging

• Consider specific mounting needs for specialized microscopy techniques

Technical optimization protocols:

• Minimize exposure to light during sample preparation to prevent yolk sac darkening

• For time-lapse imaging, reduce laser power to minimum effective levels

• Implement focus drift compensation for long-term imaging

• For nucleolar studies, combine pescadillo visualization with nuclear and nucleolar markers

Advanced reporter system design:

• Generate transgenic lines with fluorescently tagged pescadillo under endogenous promoter

• Implement photoconvertible fluorescent protein fusions to track protein movement

• Use FRET-based reporters to monitor pescadillo interactions in vivo

These optimized imaging protocols will enable high-quality visualization of pescadillo dynamics in developing zebrafish, particularly in contexts relevant to oligodendrocyte formation and ribosome biogenesis.

• What experimental approaches are most effective for analyzing pescadillo's role in ribosome biogenesis?

Given pescadillo's established role in ribosome biogenesis, comprehensive analysis requires specialized techniques focused on nucleolar function and ribosome assembly:

Nucleolar morphology assessment:

• Immunofluorescence microscopy: Visualize nucleolar markers (fibrillarin, nucleolin) in wild-type versus pescadillo-mutant embryos

• Electron microscopy: Examine ultrastructural changes in nucleolar components

• Live imaging: Track nucleolar dynamics using fluorescently tagged nucleolar proteins

Pre-rRNA processing analysis:

• Northern blotting to detect accumulation of pre-rRNA intermediates

• Quantitative RT-PCR to measure levels of specific pre-rRNA species

• RNA-seq to comprehensively profile rRNA processing defects

• FISH (fluorescent in situ hybridization) to visualize pre-rRNA localization

Ribosome biogenesis quantification:

• Polysome gradient analysis: Fractionate cellular lysates to assess ribosomal subunit assembly and polysome formation

• Ribosome profiling: Sequence ribosome-protected fragments to examine translation efficiency

• Mass spectrometry: Quantify ribosomal proteins and assembly factors in pescadillo-deficient contexts

Protein synthesis assessment:

• Puromycin incorporation assays to measure global protein synthesis rates

• SUnSET (Surface Sensing of Translation) methodology to visualize translation in situ

• Metabolic labeling with amino acid analogs followed by click chemistry detection

Experimental design for zebrafish studies:

• Compare pescadillo mutants (vu166) with wild-type siblings at matched developmental stages

• Analyze heterozygous versus homozygous embryos to assess dosage effects

• Implement tissue-specific analysis focusing on regions with high pescadillo expression

• Include rescue experiments with recombinant pescadillo to confirm specificity

When interpreting results, consider that ribosome biogenesis defects may have secondary effects on cell cycle progression and differentiation, particularly in rapidly developing systems like zebrafish embryos.

• How can protein-protein interaction studies with recombinant pescadillo reveal its functional networks?

Understanding pescadillo's protein interaction network is essential for elucidating its multifunctional roles. Implement these methodological approaches using recombinant pescadillo:

Affinity-based interaction screening:

• Co-immunoprecipitation (Co-IP): Use tagged recombinant pescadillo to identify interacting proteins from zebrafish embryo lysates

• Pull-down assays: Immobilize recombinant pescadillo on appropriate matrices to capture binding partners

• Tandem affinity purification: Implement sequential purification steps using dual-tagged pescadillo for increased specificity

Proximity-based approaches:

• BioID: Fuse pescadillo to a biotin ligase to identify proteins in close proximity in vivo

• APEX: Couple pescadillo with engineered peroxidase for proximity labeling

• Cross-linking mass spectrometry: Identify interaction interfaces with residue-level resolution

Biophysical interaction characterization:

• Surface plasmon resonance (SPR): Measure binding kinetics between immobilized pescadillo and potential partners

• Isothermal titration calorimetry (ITC): Determine binding thermodynamics and stoichiometry

• Microscale thermophoresis: Detect interactions based on changes in thermophoretic mobility

Interaction validation strategies:

• Confirm key interactions using multiple independent methods

• Map interaction domains using truncated or mutated pescadillo variants

• Assess co-localization of pescadillo and partners in zebrafish cells

• Determine functional consequences of disrupting specific interactions

Expression system considerations:

• For structural studies: E. coli-derived protein may be sufficient

• For post-translationally modified interactions: Mammalian cell-derived protein provides more authentic modifications

• For nucleolar interaction studies: Compare different expression sources to identify modification-dependent interactions

These complementary approaches will generate a comprehensive interaction network for pescadillo, revealing mechanisms through which it coordinates nucleolar assembly, ribosome biogenesis, and cell cycle regulation in zebrafish development.

• What comparative approaches between zebrafish pescadillo and human PES1 yield translational insights?

Comparative analysis between zebrafish pescadillo and human PES1 provides valuable translational insights. Implement these methodological approaches to leverage evolutionary conservation for biomedical applications:

Sequence and structural conservation analysis:

• Zebrafish pescadillo shares significant sequence homology with human PES1

• Conserved domains likely indicate functional conservation

• Divergent regions may reflect species-specific adaptations

• Analysis of post-translational modification sites can reveal conserved regulatory mechanisms

Functional conservation assessment:

• Cross-species rescue experiments: Test if human PES1 can rescue zebrafish pescadillo mutants

• Domain swapping: Create chimeric proteins to map functionally equivalent regions

• Interaction partner comparison: Identify conserved versus species-specific binding partners

• Subcellular localization studies: Compare nucleolar targeting and dynamics

Disease modeling applications:

• Zebrafish pescadillo mutants can model aspects of ribosome biogenesis disorders (ribosomopathies)

• Human PES1 mutations identified in disease contexts can be introduced into zebrafish pescadillo

• Pharmacological screens in zebrafish can identify compounds that modulate pescadillo function with potential therapeutic applications

Experimental design considerations:

• Use appropriate expression systems for cross-species protein production

• Implement equivalent methodologies when comparing across species

• Account for developmental timing differences between zebrafish and human systems

• Consider tissue-specific functions that may vary between species

Translational research workflow:

• Identify human disease mutations affecting PES1

• Generate equivalent mutations in zebrafish pescadillo

• Characterize phenotypic outcomes in zebrafish models

• Test rescue with wild-type human PES1

• Screen for compounds that ameliorate mutant phenotypes

This comparative approach maximizes the translational value of zebrafish pescadillo research while acknowledging the limitations of cross-species extrapolation.