Recombinant Danio rerio Serine/threonine-protein phosphatase 2A regulatory subunit B' subunit gamma (ppp2r3c)

What are the predicted biological functions of ppp2r3c in zebrafish?

Zebrafish ppp2r3c is predicted to be involved in several critical biological processes:

• Cytoskeleton organization

• Lymphocyte homeostasis

• Positive regulation of B cell differentiation

• Regulation of dephosphorylation events

The protein is predicted to enable metal ion binding activity and to be located in both the cytoplasm and nucleus, with specific activity in the centrosome . This suggests a multifunctional role in cellular signaling and structure maintenance. The human ortholog of this gene is implicated in spermatogenic failure 36, indicating potential conservation of reproductive functions across species .

How does ppp2r3c relate to other phosphatase regulatory subunits in zebrafish?

Ppp2r3c belongs to the PP2A (Protein Phosphatase 2A) family, which represents one of the major serine/threonine phosphatases in eukaryotic cells. In zebrafish, as in other vertebrates, PP2A functions as a heterotrimeric complex consisting of:

• A catalytic C subunit

• A structural A subunit

• A variable regulatory B subunit

Ppp2r3c belongs to the B'' subfamily of regulatory subunits, which influence substrate specificity and subcellular localization of the PP2A holoenzyme. Unlike other phosphatases such as Pten that function as both lipid and protein phosphatases with distinct roles in angiogenesis and development , ppp2r3c is primarily involved in protein dephosphorylation regulation and has predicted roles in cytoskeletal organization and immune cell development .

What experimental approaches are optimal for studying ppp2r3c function in zebrafish embryonic development?

For investigating ppp2r3c function in zebrafish embryonic development, researchers should consider implementing a multi-faceted approach:

• Gene knockdown/knockout strategies:

  • Morpholino antisense oligonucleotides for transient knockdown

  • CRISPR/Cas9 genome editing for generating stable mutant lines

  • Conditional knockout systems for temporal control of gene expression

• Rescue experiments:

  • Microinjection of synthetic mRNA encoding wild-type ppp2r3c at the one-cell stage (300 pg recommended based on similar phosphatase studies)

  • Use of phosphatase activity mutants to determine which specific activities (if any) rescue developmental phenotypes

• Live imaging:

  • Utilize the transparent nature of zebrafish embryos for in vivo imaging of developmental processes

  • Consider using the casper strain (nacre and roy orbison double mutant) for enhanced transparency throughout development

  • Generate transgenic reporter lines using the Tol2 Transposase system for visualizing specific cell types

• Protein-protein interaction studies:

  • Co-immunoprecipitation to identify binding partners

  • Proximity ligation assays to verify interactions in situ

  • Yeast two-hybrid screening to identify novel interactors

Since ppp2r3c is predicted to be involved in cytoskeleton organization and immune cell development , particular attention should be paid to these processes during embryogenesis.

How can phosphatase activity of recombinant ppp2r3c be measured and distinguished from other phosphatases?

Measuring and distinguishing the specific phosphatase activity of recombinant ppp2r3c requires careful experimental design:

• In vitro phosphatase assays:

  • Use synthetic phosphopeptide substrates containing phosphorylated serine/threonine residues

  • Measure dephosphorylation through colorimetric detection of released phosphate (malachite green assay)

  • Include appropriate controls with specific phosphatase inhibitors:

    • Okadaic acid (inhibits PP2A at low concentrations)

    • Calyculin A (inhibits both PP1 and PP2A)

    • Fostriecin (selective PP2A/PP4 inhibitor)

• Holoenzyme reconstitution:

  • Express and purify recombinant ppp2r3c with His-tag

  • Combine with purified PP2A catalytic (C) and scaffold (A) subunits

  • Compare activity of the reconstituted holoenzyme with that of the catalytic subunit alone

• Substrate specificity profiling:

  • Employ phosphoproteomic approaches to identify specific substrates

  • Use peptide arrays containing diverse phosphorylated sequences

  • Compare dephosphorylation patterns with those of other phosphatases

A critical consideration is ensuring that the recombinant protein maintains proper folding and post-translational modifications. The yeast-expressed recombinant ppp2r3c with His-tag may be suitable for initial studies, but mammalian expression systems might better preserve native protein characteristics for functional studies.

What are the most effective transgenic approaches for studying ppp2r3c in the context of hematopoiesis?

Given the predicted role of ppp2r3c in lymphocyte homeostasis and B cell differentiation , specialized transgenic approaches can be employed to study its function in hematopoiesis:

• Lineage-specific fluorescent reporter lines:

  • Generate double transgenic lines expressing:

    • Hematopoietic stem cell markers (e.g., cd41:GFP)

    • B-cell lineage markers (e.g., rag1:mCherry)

  • Use the Tol2 transposase system for efficient transgenesis

  • Combine with ppp2r3c knockout/knockdown to visualize effects on specific populations

• Inducible expression systems:

  • Employ heat shock promoters (hsp70) to control temporal expression

  • Use Gal4/UAS or Cre/loxP systems for cell type-specific expression

  • Create dominant-negative constructs to selectively inhibit ppp2r3c function

• Transplantation assays:

  • Label donor cells with fluorescent markers

  • Transplant cells from ppp2r3c-modified embryos into casper recipients for enhanced visibility

  • Perform competitive transplantation assays with mixed wild-type and ppp2r3c-deficient cells

• CRISPR screening approaches:

  • Implement targeted screens for ppp2r3c-interacting genes in hematopoietic lineages

  • Use pooled CRISPR libraries targeting phosphatase-related pathways

  • Analyze effects on B cell differentiation through flow cytometry and single-cell sequencing

For optimal results, researchers should take advantage of zebrafish embryonic transparency and rapid development when designing experiments . Cell sorting techniques combined with transcriptomic analysis can provide additional insights into the molecular mechanisms by which ppp2r3c influences hematopoietic lineage specification.

What is the optimal expression system for producing functional recombinant Danio rerio ppp2r3c?

The choice of expression system for recombinant ppp2r3c production depends on experimental requirements:

For most zebrafish ppp2r3c research applications, the yeast expression system represents a practical compromise, providing sufficient yield with appropriate eukaryotic modifications . For studies requiring absolutely native conformation and activity, co-expression with other PP2A subunits in mammalian cells may be necessary to ensure proper complex formation and activity.

How can researchers effectively use microinjection to study ppp2r3c function in zebrafish embryos?

Microinjection is a powerful technique for studying ppp2r3c function in zebrafish embryos:

• mRNA injection protocol:

  • Synthesize capped mRNA encoding wild-type or mutant ppp2r3c using in vitro transcription

  • Optimize concentration (300 pg has been effective for similar phosphatase studies)

  • Inject at the one-cell stage for ubiquitous distribution

  • Include fluorescent marker (e.g., mCherry-tagged construct) to verify expression

• Morpholino design considerations:

  • Target translation start site or splice junctions

  • Validate specificity through rescue experiments with morpholino-resistant mRNA

  • Use 1-3 ng doses to minimize off-target effects

• CRISPR/Cas9 delivery:

  • Co-inject Cas9 mRNA (150 pg) with target gRNAs (50-100 pg)

  • Design gRNAs targeting critical functional domains

  • Screen founders by targeted sequencing or heteroduplex mobility assays

• Experimental controls:

  • Include non-injected controls from same clutch

  • Use mismatch or standard control morpholinos

  • For rescue experiments, include phosphatase-dead mutants

  • Perform dose-response studies to establish optimal concentrations

• Phenotypic evaluation:

  • Monitor development at standard time points (24, 48, 72 hpf)

  • Focus on expected phenotypes based on predicted functions:

    • Cytoskeletal organization defects

    • Lymphoid development abnormalities

    • Centrosomal abnormalities

For combining with transgenic approaches, researchers working with ppp2r3c should consider using Tg(kdrl:eGFP) lines to visualize vascular development , particularly if investigating potential roles in angiogenesis by analogy with other phosphatases like Pten .

What approaches can be used for tissue-specific manipulation of ppp2r3c expression in zebrafish?

Tissue-specific manipulation of ppp2r3c expression requires specialized genetic tools:

• Gal4/UAS system implementation:

  • Generate driver lines expressing Gal4 under tissue-specific promoters:

    • rag1 promoter for B cell lineage

    • lck promoter for T cell lineage

    • kdrl promoter for vascular endothelium

  • Create UAS:ppp2r3c-mCherry responder lines for visualization

  • Cross driver and responder lines to achieve tissue-specific expression

• Cre/loxP conditional approaches:

  • Generate floxed ppp2r3c alleles using CRISPR/Cas9 genome editing

  • Create tissue-specific Cre driver lines

  • Implement inducible systems (CreERT2) for temporal control

  • Cross with reporter lines for lineage tracing

• Cell transplantation techniques:

  • Generate mosaic embryos by transplanting cells at blastula stage

  • Label donor cells with fluorescent markers for tracking

  • Use casper recipient embryos for enhanced visibility throughout development

  • Compare cell-autonomous versus non-cell-autonomous effects

• Localized CRISPR delivery:

  • Inject Cas9 protein with sgRNAs into specific tissues using glass micropipettes

  • Electroporate Cas9 ribonucleoprotein complexes into target tissues

  • Use tissue-specific promoters to drive Cas9 expression

For analyzing the effects of tissue-specific manipulation, researchers should take advantage of zebrafish transparency by implementing advanced imaging techniques such as light-sheet microscopy for long-term in vivo tracking of labeled cells . This approach is particularly valuable for studying dynamic processes like cytoskeletal reorganization and immune cell migration, which are relevant to ppp2r3c function .

How can researchers differentiate between direct and indirect effects of ppp2r3c manipulation?

Distinguishing direct from indirect effects of ppp2r3c manipulation requires systematic experimental design:

• Temporal analysis approaches:

  • Perform time-course experiments following ppp2r3c manipulation

  • Use inducible systems (heat shock, chemical induction) to control timing

  • Monitor immediate early gene responses (within 1-2 hours)

  • Compare early versus late phenotypes to establish cause-effect relationships

• Phosphoproteomic profiling:

  • Compare phosphorylation patterns at multiple time points after ppp2r3c manipulation

  • Identify primary dephosphorylation targets (immediate changes)

  • Map secondary signaling cascade effects (delayed changes)

  • Create temporal phosphorylation networks to distinguish direct from indirect targets

• Rescue experiment strategies:

  • Perform targeted rescue with specific pathway components

  • Create phosphomimetic mutations in suspected direct targets

  • Use small molecule inhibitors of downstream pathways

  • Compare partial versus complete phenotypic rescue

• In vitro validation:

  • Perform direct dephosphorylation assays with purified components

  • Use recombinant ppp2r3c protein with His-tag with potential substrates

  • Implement proximity-dependent labeling techniques (BioID, APEX) to identify proteins in close proximity to ppp2r3c in vivo

When interpreting results, researchers should consider that as a regulatory subunit of PP2A, ppp2r3c likely functions by modulating the catalytic activity or substrate specificity of the PP2A holoenzyme rather than possessing intrinsic phosphatase activity. This distinguishes it from dual-function phosphatases like Pten that have both lipid and protein phosphatase activities with distinct developmental roles .

What controls and validations are critical when studying ppp2r3c in zebrafish models?

Implementing appropriate controls and validations is essential for robust ppp2r3c research:

• Genetic manipulation controls:

  • Include multiple independent morpholinos or CRISPR guide RNAs

  • Validate knockdown/knockout efficiency at both mRNA and protein levels

  • Perform rescue experiments with wild-type ppp2r3c mRNA

  • Generate and characterize multiple independent mutant lines

• Phenotypic validation approaches:

  • Compare morpholino phenotypes with stable mutant phenotypes

  • Use dose-response relationships to establish specificity

  • Document phenotypes at standardized developmental stages

  • Implement quantitative phenotypic measurements rather than subjective assessments

• Biochemical validation requirements:

  • Confirm protein expression and localization using specific antibodies

  • Verify PP2A complex formation through co-immunoprecipitation

  • Validate phosphatase activity using in vitro and cell-based assays

  • Test substrate specificity using phosphopeptide arrays

• Cross-species validation strategies:

  • Compare zebrafish findings with mammalian cell culture models

  • Test functional conservation with human PPP2R3C

  • Verify orthologous gene function through cross-species rescue experiments

  • Correlate findings with available human genetic data (especially regarding spermatogenic failure 36)

When interpreting results from zebrafish models, researchers should consider the advantages of this model system, including transparent embryos for in vivo imaging and rapid development , while acknowledging potential limitations in directly translating findings to mammalian systems.

How can researchers address data inconsistencies in ppp2r3c functional studies?

When encountering inconsistent results in ppp2r3c studies, researchers should implement a systematic troubleshooting approach:

• Technical variability assessment:

  • Standardize embryo staging and data collection timepoints

  • Control for genetic background differences between zebrafish lines

  • Implement blinded scoring of phenotypes

  • Use statistical power calculations to ensure adequate sample sizes

• Genetic compensation mechanisms:

  • Investigate potential upregulation of paralogs or related genes

  • Compare acute (morpholino) versus chronic (mutant) loss of function

  • Perform transcriptomic analysis to identify compensatory pathways

  • Consider generating double or triple knockouts of related genes

• Experimental condition variables:

  • Standardize temperature conditions (zebrafish development is highly temperature-dependent)

  • Control for maternal contribution in early embryonic studies

  • Consider circadian rhythms and developmental timing

  • Document and control water quality parameters

• Data reconciliation approaches:

  • Create detailed phenotypic catalogs with penetrance information

  • Implement quantitative rather than binary phenotype assessments

  • Use multiple complementary techniques to validate key findings

  • Develop computational models to reconcile apparently contradictory data

When publishing results, researchers should transparently report all experimental conditions and observed variability. For studies involving recombinant ppp2r3c protein, documentation of protein purity (>90% for commercial preparations) , expression system, and any tags or modifications is essential for reproducibility.

What emerging technologies could advance ppp2r3c research in zebrafish models?

Several cutting-edge technologies offer promising avenues for advancing ppp2r3c research:

• Single-cell multi-omics approaches:

  • Apply single-cell RNA sequencing to identify cell type-specific responses to ppp2r3c manipulation

  • Combine with single-cell ATAC-seq to map regulatory changes

  • Implement spatial transcriptomics to preserve tissue context information

  • Develop cell type-specific phosphoproteomics to identify direct substrates

• Advanced genome editing technologies:

  • Apply base editing for precise point mutations without double-strand breaks

  • Implement prime editing for targeted insertions and replacements

  • Develop conditional knockin strategies for temporal control

  • Create allelic series to study structure-function relationships

• Advanced imaging modalities:

  • Implement lattice light-sheet microscopy for long-term, non-phototoxic imaging

  • Apply super-resolution techniques to visualize subcellular localization

  • Develop FRET-based sensors for PP2A activity in live embryos

  • Use optogenetic tools to manipulate ppp2r3c function with spatial precision

• In silico approaches:

  • Develop computational models of PP2A regulatory networks

  • Apply machine learning to predict phenotypic outcomes

  • Create integrative multi-scale models connecting molecular events to developmental phenotypes

  • Implement systems biology approaches to understand pathway crosstalk

The transparency of zebrafish embryos makes them particularly suitable for advanced imaging techniques , while their genetic tractability facilitates implementation of sophisticated genome editing approaches. Combining these technologies with the casper strain for enhanced transparency throughout development could provide unprecedented insights into ppp2r3c function in vivo.

How might understanding ppp2r3c function contribute to human disease research?

Research on zebrafish ppp2r3c has several potential implications for human disease:

• Reproductive disorders:

  • The human ortholog of ppp2r3c is implicated in spermatogenic failure 36

  • Zebrafish models could elucidate mechanisms underlying this condition

  • Potential applications in male infertility diagnostics and therapeutics

  • Insights into conserved phosphatase functions in gametogenesis

• Hematopoietic disorders:

  • Given ppp2r3c's predicted role in lymphocyte homeostasis and B cell differentiation

  • Potential relevance to lymphoproliferative disorders and immunodeficiencies

  • Models for studying B cell development and function

  • Therapeutic targeting potential in B cell malignancies

• Developmental disorders:

  • PP2A dysregulation is associated with numerous developmental abnormalities

  • Zebrafish models could reveal specific roles of the B'' regulatory subunit

  • Insights into cytoskeletal organization during development

  • Potential relevance to centrosome-related developmental disorders

• Therapeutic development platforms:

  • Zebrafish embryos as screening platforms for modulators of PP2A activity

  • High-throughput phenotypic screens for pathway-specific compounds

  • Development of B subunit-specific PP2A modulators

  • Validation of therapeutic candidates in a vertebrate model

Translational studies should consider both the conservation of PP2A complex components between zebrafish and humans and potential species-specific differences in regulatory mechanisms. The zebrafish model offers unique advantages for in vivo visualization of developmental and disease processes , complementing mammalian models and cell culture systems.