Recombinant Danio rerio Ubiquitin carboxyl-terminal hydrolase 16 (usp16), partial

What is Ubiquitin Carboxyl-Terminal Hydrolase 16 (USP16) and what are its primary functions in zebrafish?

USP16 in zebrafish belongs to the ubiquitin-specific peptidase family that catalyzes the removal of ubiquitin from protein substrates. This deubiquitinase specifically targets histone H2A at lysine 120 (H2AK119Ub), a modification associated with transcriptional repression. By removing this ubiquitin mark, USP16 functions as a transcriptional coactivator . The deubiquitination of histone H2A is essential for proper chromosome segregation during mitosis and serves as a prerequisite for subsequent histone H3 phosphorylation at serine 10 (H3S10ph) . Zebrafish USP16 shares substantial homology with human USP16, making it a valuable model for studying deubiquitination pathways relevant to human biology and disease .

How does the structure of zebrafish USP16 compare to the human ortholog?

Zebrafish USP16 maintains the core structural features found in human USP16, including:

• A zinc-finger ubiquitin-binding domain (ZnF-UBP) that recognizes the C-terminal region of ubiquitin

• A catalytic domain (CD) with the characteristic hand-like USP fold consisting of palm, thumb, and fingers subdomains

• A catalytic triad for enzymatic activity

While both proteins share high sequence homology in the catalytic regions, some regulatory phosphorylation sites differ between species. For instance, the Ser552 phosphorylation site important for nuclear import of human USP16 during mitosis is not conserved in zebrafish . This suggests that zebrafish may employ alternative mechanisms for regulating USP16 localization and activity during cell division, an important consideration when extrapolating experimental findings between species.

What expression systems are commonly used to produce recombinant zebrafish USP16?

Recombinant zebrafish USP16 can be produced in multiple expression systems, each offering distinct advantages for different experimental applications:

These recombinant forms are typically produced with affinity tags (such as His-tags) to facilitate purification, as demonstrated with other UCH proteins . Selection of the appropriate expression system depends on the specific experimental requirements, particularly regarding post-translational modifications and enzymatic activity.

How can I optimize purification protocols for recombinant zebrafish USP16 to maintain enzymatic activity?

Purification of enzymatically active zebrafish USP16 requires careful consideration of buffer conditions and purification strategies:

• Affinity chromatography: His-tagged USP16 can be efficiently purified using Ni-NTA chromatography, with typical yields reaching 18 mg of pure active enzyme per 100 ml culture broth when using secretory systems . Add imidazole (10-20 mM) in the binding buffer to minimize non-specific binding.

• Buffer optimization: Maintain pH 6.0-7.5 during purification, as this range has been shown to be optimal for UCH stability and activity in similar systems. Include protease inhibitors to prevent autodegradation .

• Reducing agents: Include DTT or β-mercaptoethanol (1-5 mM) in all buffers to maintain the catalytic cysteine in reduced state, which is critical for enzymatic activity.

• Temperature considerations: Perform all purification steps at 4°C to minimize protein denaturation and maintain activity.

• Activity verification: Confirm enzymatic activity using ubiquitin-AMC or ubiquitin fusion protein substrates. For example, a ubiquitin-magainin fusion protein system can be used to assess cleavage specificity, with subsequent mass spectrometry analysis to verify precise cleavage at the carboxyl terminus of ubiquitin .

Purified USP16 can be stabilized by adding glycerol (10-20%) for long-term storage at -80°C, with minimal loss of activity over several months.

What are the optimal experimental conditions for assessing zebrafish USP16 deubiquitinating activity in vitro?

Effective assessment of zebrafish USP16 deubiquitinating activity requires carefully controlled experimental conditions:

• Buffer composition: Use 50 mM HEPES pH 7.5, 100 mM NaCl, 1 mM DTT, 1 mM EDTA, and 5% glycerol as a standard reaction buffer.

• Substrate selection: For nucleosomal substrates (preferred by USP16), use purified zebrafish or human nucleosomes containing ubiquitinated H2A. Alternatively, synthetic ubiquitin chains or ubiquitin-AMC can be used for preliminary activity screening .

• Reaction monitoring: Monitor H2A deubiquitination using:

  • Western blotting with anti-ubiquitin or anti-H2AK119Ub antibodies

  • HPLC analysis of reaction products

  • Mass spectrometry to confirm precise cleavage sites

• Controls: Include:

  • Heat-inactivated USP16 (negative control)

  • A general deubiquitinase such as USP2 (positive control)

  • Specific USP16 inhibitors when available (specificity control)

• Kinetic analysis: Determine Km and kcat values using varying substrate concentrations (0.1-10 μM) and enzyme concentrations (10-100 nM).

The specific activity of purified recombinant zebrafish USP16 should be comparable to that of human USP16, with efficient cleavage of ubiquitin fusion proteins observed at enzyme:substrate ratios of approximately 1:50 to 1:100 under optimal conditions .

How can I design CRISPR/Cas9 experiments to study USP16 function in zebrafish development?

Designing effective CRISPR/Cas9 experiments for zebrafish USP16 requires careful target selection and validation:

• Target site selection:

  • Target exons encoding the catalytic domain (particularly exons containing the catalytic triad residues)

  • Use CHOPCHOP or similar tools to identify guide RNAs with high on-target and low off-target scores

  • Design at least 2-3 different sgRNAs targeting different regions to control for off-target effects

• Experimental design:

  • Inject Cas9 mRNA/protein with sgRNAs into single-cell zebrafish embryos

  • Include control groups: uninjected, Cas9-only, and non-targeting sgRNA

  • Screen F0 embryos for phenotypes and gene editing efficiency by T7E1 assay or direct sequencing

  • Raise potential founders to establish stable mutant lines

• Phenotype analysis:

  • Examine nephron development using pronephros markers like pax2a and podocyte-specific genes (wt1a, wt1b, podxl)

  • Assess histone H2A ubiquitination status by immunohistochemistry

  • Analyze cell cycle progression in tissues with high proliferation rates

  • Evaluate gene expression changes in targets known to be regulated by H2A deubiquitination

• Verification of mutant lines:

  • Confirm mutations by sequencing

  • Validate loss of USP16 protein by Western blot

  • Rescue experiments by mRNA injection of wild-type USP16 to confirm specificity of phenotypes

When analyzing USP16 mutant phenotypes, it is essential to distinguish between direct effects of USP16 loss and secondary effects due to cardiac or vascular defects, as these can indirectly affect kidney development in zebrafish .

What strategies can be employed to overcome insolubility issues when expressing recombinant zebrafish USP16?

Insolubility of recombinant zebrafish USP16 can be addressed through several strategies:

• Expression system optimization:

  • Switch to secretory expression in Pichia pastoris, which has proven successful for similar UCH enzymes, with yields of up to 210 mg/l under optimized conditions

  • Test expression in multiple strains: P. pastoris Mut^s strain tends to produce higher yields of recombinant UCH proteins than Mut⁺ strain

  • Adjust induction conditions: for P. pastoris, maintain pH 6.0 in BMMY/methanol medium during induction phase

• Protein engineering approaches:

  • Express individual domains separately (ZnF-UBP domain, catalytic domain)

  • Remove predicted disordered regions that may contribute to aggregation

  • Create fusion constructs with solubility-enhancing tags (MBP, SUMO, or Thioredoxin)

• Refolding strategies:

  • For inclusion bodies, develop a denaturation/refolding protocol using step-wise dialysis

  • Include L-arginine (0.5-1 M) or low concentrations of urea (1-2 M) to enhance refolding efficiency

  • Add zinc during refolding to ensure proper formation of the ZnF-UBP domain

• Buffer optimization for purification:

  • Test detergents (0.05-0.1% Triton X-100) to increase solubility

  • Include stabilizing agents like glycerol (10-20%)

  • Optimize salt concentration (typically 100-500 mM NaCl)

Western blot analysis using anti-USP16 antibodies can be used to track protein expression and solubility across different conditions, enabling systematic optimization of expression parameters .

How can I distinguish between the functions of USP16 and other deubiquitinases in zebrafish models?

Distinguishing the specific functions of USP16 from other deubiquitinases in zebrafish requires multiple complementary approaches:

• Selective inhibition strategies:

  • Design substrate-trapping mutants by mutating the catalytic cysteine residue of USP16

  • Use CRISPR/Cas9 to create precise point mutations that disrupt catalytic activity without affecting protein expression

  • Employ morpholino knockdown with careful validation of specificity through rescue experiments

• Substrate specificity analysis:

  • Compare H2A deubiquitination patterns between USP16 and related enzymes (USP21, USP22)

  • Perform proteomic analysis to identify unique USP16 substrates versus those targeted by multiple DUBs

  • Use artificial ubiquitin substrates with different chain linkages to characterize enzymatic preferences

• Genetic interaction studies:

  • Create double knockdown/knockout models combining USP16 with other DUBs

  • Assess phenotypic enhancement or suppression to map functional relationships

  • Use epistasis analysis to determine pathway relationships

• Temporal and spatial expression analysis:

  • Compare expression patterns of USP16 with other DUBs during zebrafish development

  • Use conditional knockout strategies to disrupt USP16 function in specific tissues or developmental stages

  • Employ tissue-specific rescue to determine where USP16 function is required

When analyzing pronephros development in zebrafish, carefully distinguish between direct effects of USP16 manipulation and indirect effects due to cardiac dysfunction, as fluid flow defects can independently affect kidney development . Note that unlike other deubiquitinases, USP16 does not deubiquitinate histone H2B , providing one clear functional distinction.

What controls should be included when studying zebrafish USP16 in pronephros development?

Robust experimental design for studying USP16's role in zebrafish pronephros development requires comprehensive controls:

• Genetic manipulation controls:

  • Use at least two independent methods for USP16 manipulation (e.g., morpholino knockdown and CRISPR/Cas9)

  • Include mismatch/scrambled morpholinos or non-targeting sgRNAs as negative controls

  • Perform rescue experiments with wild-type USP16 mRNA to confirm specificity

  • Include catalytically inactive USP16 mutants in rescue experiments to determine if enzymatic activity is required

• Cardiovascular function controls:

  • Monitor cardiac contractility and vascular flow, as defects in these systems can indirectly affect pronephros development

  • Include tnnt2a knockdown controls, which affect cardiac contractility without directly altering USP16 function

  • Assess fluid flow through the pronephros using fluorescent dextran injections

• Developmental markers:

  • Track multiple pronephros markers to distinguish between tubulogenesis defects and cell fate specification issues

  • Monitor expression of pax2a (tubule marker) and podocyte markers (wt1a, wt1b, podxl)

  • Include polarity markers to assess epithelial organization (prkcι, prkcζ) as these affect tubule formation

• Cell behavior controls:

  • Analyze mesenchymal to epithelial transition (MET) markers to determine if USP16 affects this critical process

  • Assess cell proliferation and apoptosis rates to distinguish between defects in tissue growth versus differentiation

  • Monitor cell migration patterns, particularly for podocytes, which require normal fluid flow for proper positioning

When interpreting results, it is crucial to distinguish between primary effects of USP16 manipulation and secondary effects due to altered cardiac function or fluid flow. This can be achieved by comparing USP16 morphant/mutant phenotypes with those caused by disruption of intraflagellar transport (e.g., ift88 knockdown), which eliminates pronephros fluid output through a different mechanism .

How can comparative analysis between zebrafish and human USP16 inform therapeutic development?

Comparative analysis of zebrafish and human USP16 provides valuable insights for therapeutic development:

• Conserved mechanisms and targets:

  • Both zebrafish and human USP16 specifically deubiquitinate H2AK119Ub, suggesting evolutionary conservation of this key regulatory function

  • The catalytic domains show high structural similarity, including the unusual Cys-His-Ser catalytic triad (instead of the typical Cys-His-Asn/Asp arrangement found in most USPs)

  • This conservation supports the use of zebrafish as a model for screening compounds targeting the catalytic mechanism

• Species-specific regulatory differences:

  • Regulatory phosphorylation sites, including Ser552 which controls nuclear import of human USP16 during mitosis, are not conserved in zebrafish

  • These differences suggest that while catalytic inhibitors may work across species, compounds targeting regulatory mechanisms may require species-specific design

• Validation pathway for therapeutic candidates:

  • Initial high-throughput screening of compound libraries against recombinant human and zebrafish USP16

  • Secondary validation in zebrafish embryos to assess in vivo efficacy and toxicity

  • Tertiary validation in human cell lines to confirm cross-species activity

  • Final evaluation in disease models (e.g., zebrafish models of disorders associated with epigenetic dysregulation)

• Disease relevance:

  • The role of USP16 in regulating H2A deubiquitination connects it to gene expression control and cell cycle progression

  • These functions make USP16 a potential target for conditions involving abnormal cell proliferation or epigenetic dysregulation

  • Zebrafish models allow rapid assessment of USP16 inhibitors on embryonic development, providing early indications of potential developmental toxicity

Careful analysis of chemical inhibitor effects on both zebrafish and human USP16 can identify compounds with therapeutic potential while highlighting species-specific considerations for their development and application.

What insights do zebrafish USP16 studies provide about the evolution of deubiquitinating enzymes across vertebrates?

Zebrafish USP16 studies reveal important evolutionary insights about deubiquitinating enzymes:

• Structural conservation:

  • The core catalytic domain architecture of USP16 is highly conserved across vertebrates, from fish to mammals, suggesting strong evolutionary pressure to maintain its fundamental enzymatic function

  • Both zebrafish and human USP16 contain the unusual Cys-His-Ser catalytic triad, distinguishing them from most USPs that utilize a Cys-His-Asn/Asp arrangement

  • This conservation indicates the early emergence and functional importance of this variant catalytic mechanism in vertebrate evolution

• Regulatory divergence:

  • While catalytic regions show high conservation, regulatory elements display greater divergence

  • Key phosphorylation sites, such as Ser552 in human USP16, are not conserved in zebrafish, suggesting species-specific regulation of nuclear import and activity during mitosis

  • This divergence suggests that common ancestors had the core enzymatic function, but regulatory mechanisms evolved separately in different vertebrate lineages

• Substrate recognition patterns:

  • Both zebrafish and human USP16 preferentially recognize nucleosomal substrates and specifically target H2AK119Ub

  • This conservation of substrate specificity across ~450 million years of evolutionary divergence highlights the fundamental importance of this epigenetic regulatory mechanism

  • Neither appears to deubiquitinate histone H2B, maintaining this functional specificity across vertebrate evolution

• Functional specialization:

  • Comparative studies between zebrafish USP16 and other DUBs provide insights into how functional specialization occurred during evolution

  • The maintenance of distinct roles for different DUBs (e.g., USP16, USP21, USP22) across vertebrates suggests early functional divergence followed by conservation of specialized roles

These evolutionary insights not only enhance our understanding of USP16 biology but also provide context for interpreting experimental results across species and identifying conserved targets for potential therapeutic intervention.

How can I integrate zebrafish USP16 studies with multi-omics approaches to understand its broader biological functions?

Integrating zebrafish USP16 studies with multi-omics approaches enables comprehensive characterization of its biological functions:

• Genomics integration:

  • Perform ChIP-seq for H2AK119Ub in wild-type and USP16-depleted zebrafish embryos to identify genomic regions directly regulated by USP16 activity

  • Combine with ATAC-seq to correlate changes in H2A ubiquitination with chromatin accessibility

  • Use CUT&RUN or CUT&Tag for higher resolution mapping of H2AK119Ub patterns in specific tissues or developmental stages

• Transcriptomics applications:

  • Compare RNA-seq profiles from wild-type, USP16 morphant/mutant, and rescued embryos to identify genes and pathways dependent on USP16 activity

  • Perform single-cell RNA-seq to identify cell type-specific effects, particularly in the developing pronephros

  • Use temporal transcriptomics to map dynamic changes in gene expression during development in response to USP16 manipulation

• Proteomics strategies:

  • Employ immunoprecipitation coupled with mass spectrometry (IP-MS) to identify USP16 interaction partners in zebrafish

  • Perform ubiquitin remnant profiling to identify all substrates affected by USP16 depletion

  • Use SILAC or TMT labeling to quantify global proteome changes in response to USP16 manipulation

  • Compare interactomes between zebrafish and human USP16 to identify conserved and divergent interaction networks

• Integrative analysis framework:

  • Correlate H2AK119Ub patterns with gene expression changes to identify direct regulatory targets

  • Map protein-protein interactions onto affected pathways to build functional networks

  • Use pathway enrichment analysis to identify biological processes most dependent on USP16 activity

  • Develop computational models that integrate multi-omics data to predict USP16 function across different tissues and developmental stages

This integrated approach can reveal how USP16's molecular function in histone deubiquitination connects to broader physiological roles in development, particularly in the context of pronephros formation and function in zebrafish, while identifying conserved mechanisms relevant to human biology .

What emerging technologies could enhance our understanding of zebrafish USP16 function?

Several cutting-edge technologies can significantly advance zebrafish USP16 research:

• Advanced genome editing approaches:

  • Prime editing for precise introduction of point mutations to study specific functional domains without complete protein loss

  • Inducible CRISPR systems (e.g., photoactivatable or chemical-inducible Cas9) for temporal control of USP16 disruption

  • Base editing technologies to introduce specific codon changes that alter catalytic activity or regulatory sites

• Live imaging innovations:

  • CRISPR-based endogenous tagging of USP16 with fluorescent proteins to monitor expression and localization in real-time

  • Implementation of FRET-based sensors to track USP16 activity in living embryos

  • Light-sheet microscopy for long-term, non-invasive imaging of USP16-expressing cells during zebrafish development

  • Optogenetic tools to control USP16 activity or localization with spatial and temporal precision

• Single-cell and spatial transcriptomics:

  • Single-cell RNA-seq combined with lineage tracing to map USP16-dependent developmental trajectories

  • Spatial transcriptomics methods (e.g., Slide-seq, MERFISH) to map gene expression changes in their anatomical context

  • Integration with chromatin accessibility mapping at single-cell resolution to correlate USP16 activity with epigenetic regulation

• Structural biology advances:

  • Cryo-EM analysis of USP16 in complex with nucleosomal substrates to understand the molecular basis of specificity

  • AlphaFold2 and RoseTTAFold predictions validated with hydrogen-deuterium exchange mass spectrometry to map functional domains

  • Time-resolved structural studies to capture conformational changes during the catalytic cycle

These technologies would address current limitations in understanding USP16 dynamics and provide unprecedented insights into how this deubiquitinase functions in zebrafish development, particularly in the context of pronephros formation and function .

How might USP16 function in zebrafish relate to regenerative medicine applications?

The study of USP16 in zebrafish offers unique insights for regenerative medicine applications:

• Regenerative capacity connection:

  • Zebrafish possess remarkable regenerative abilities, including the capacity to regenerate fins, heart tissue, and neural structures

  • USP16's role in regulating histone H2A ubiquitination impacts gene expression programs that could be critical for regenerative responses

  • Comparing USP16 activity in regenerative contexts between zebrafish and mammals could identify regulatory differences that contribute to differential regenerative capacity

• Stem cell regulation mechanisms:

  • USP16 is known to regulate the self-renewal of hematopoietic stem cells in mammals

  • In zebrafish, studying how USP16 affects stem cell populations during development and regeneration could reveal conserved mechanisms for stem cell maintenance and differentiation

  • Manipulation of USP16 activity could potentially enhance stemness or direct differentiation in therapeutic contexts

• Epigenetic reprogramming insights:

  • The deubiquitination of H2AK119Ub by USP16 counteracts Polycomb-mediated gene silencing, a process central to cellular reprogramming

  • Understanding how this process is regulated in regeneration-competent zebrafish tissues could inform strategies to enhance regenerative potential in human tissues

  • Comparative studies of USP16-regulated genes during zebrafish regeneration could identify key regulatory networks to target in regenerative medicine

• Therapeutic strategy development:

  • Small molecule modulators of USP16 activity identified in zebrafish screens could be developed as tools to enhance regenerative responses

  • Cell-based therapies might benefit from transient USP16 modulation to promote specific differentiation programs

  • Tissue engineering approaches could incorporate insights from USP16-regulated processes in zebrafish development to improve outcomes

The regenerative capability of zebrafish makes them an exceptional model for studying how epigenetic regulators like USP16 contribute to tissue repair and regeneration, with direct implications for developing new approaches to regenerative medicine in humans .

What are the most promising approaches for developing selective inhibitors of zebrafish USP16 for research applications?

Development of selective zebrafish USP16 inhibitors requires systematic approaches:

• Structure-based design strategies:

  • Utilize AlphaFold2 predictions and experimental structures of USP domains to identify unique features of the zebrafish USP16 catalytic site

  • Focus on the unusual Cys-His-Ser catalytic triad, which distinguishes USP16 from most other USP family members

  • Design compounds that exploit structural differences between USP16 and related deubiquitinases

  • Incorporate covalent warheads that target the catalytic cysteine with high specificity

• High-throughput screening approaches:

  • Develop fluorescence-based activity assays using ubiquitin-AMC or FRET-based substrates for primary screening

  • Implement cell-based secondary screens in zebrafish cell lines expressing fluorescently-tagged H2A to monitor deubiquitination

  • Establish counter-screens against related USPs to ensure selectivity

  • Validate hits using recombinant protein biochemistry and zebrafish embryo phenotypic assays

• Fragment-based drug discovery:

  • Screen fragment libraries against purified zebrafish USP16 using NMR, thermal shift assays, or X-ray crystallography

  • Identify binding fragments that target allosteric sites unique to USP16

  • Optimize fragments through medicinal chemistry to improve potency and selectivity

  • Combine fragments targeting different sites to create highly selective inhibitors

• Substrate-mimetic inhibitors:

  • Design peptidomimetics based on the unique interaction between USP16 and its preferred substrate (H2AK119Ub)

  • Incorporate non-hydrolyzable isopeptide bond mimetics to create transition state analogs

  • Focus on the nucleosomal context preferred by USP16 to enhance selectivity over other DUBs

Promising inhibitor candidates should be validated in zebrafish embryos to confirm their ability to recapitulate genetic loss-of-function phenotypes, particularly in pronephros development . The most effective compounds will likely need to balance potency, selectivity, and cell permeability while demonstrating minimal off-target effects in physiological systems.

How should researchers approach replication and validation when studying zebrafish USP16?

Robust replication and validation of zebrafish USP16 studies requires comprehensive methodological considerations:

• Multiple manipulation approaches:

  • Employ at least two independent methods for USP16 manipulation (e.g., morpholino knockdown, CRISPR/Cas9 knockout, dominant-negative overexpression)

  • Validate each approach with appropriate controls (mismatch morpholinos, off-target analysis for CRISPR)

  • Perform rescue experiments with wild-type USP16 to confirm phenotype specificity

  • Include catalytically inactive USP16 mutants in rescue experiments to distinguish between enzymatic and scaffolding functions

• Comprehensive phenotypic analysis:

  • Examine multiple tissues and developmental processes, not just primary tissues of interest

  • Assess both morphological and molecular phenotypes using standardized protocols

  • Document complete developmental trajectories rather than single time points

  • Employ quantitative metrics where possible to enable statistical validation

• Cross-species validation:

  • Compare findings with mammalian cell culture models to identify conserved mechanisms

  • Validate key results in other fish models where appropriate

  • Consider evolutionary context when interpreting species-specific differences

• Technical validation benchmarks:

  • Verify antibody specificity through knockout controls and competitive binding assays

  • Confirm recombinant protein activity through multiple independent assays

  • Validate RNA-seq or other omics findings with independent techniques (qRT-PCR, Western blotting)

  • Implement blinded analysis protocols for subjective phenotypic assessments

By implementing these rigorous validation approaches, researchers can ensure that findings related to zebrafish USP16 function are robust, reproducible, and relevant to broader understanding of deubiquitination processes in vertebrate development and disease .

What are the key considerations when translating findings from zebrafish USP16 studies to human disease contexts?

Translating zebrafish USP16 findings to human disease contexts requires careful consideration of several factors:

• Evolutionary conservation assessment:

  • Evaluate sequence and structural homology between zebrafish and human USP16 (particularly in catalytic domains)

  • Compare regulatory mechanisms, noting that key phosphorylation sites like Ser552 in human USP16 are not conserved in zebrafish

  • Assess conservation of interaction partners and signaling pathways

  • Validate key findings in both systems when possible

• Physiological context differences:

  • Consider differences in organ complexity and function (e.g., pronephros versus metanephric kidney)

  • Account for differences in developmental timing and lifespan

  • Recognize that regenerative capacity differs dramatically between zebrafish and humans

  • Evaluate how these differences might impact the relevance of specific USP16 functions

• Disease modeling limitations:

  • Acknowledge that zebrafish models may not fully recapitulate human disease complexity

  • Consider that compensatory mechanisms may differ between species

  • Recognize that pharmacokinetics and drug responses vary across species

  • Implement parallel studies in human cell lines or tissues when possible

• Translational pathway design:

  • Establish clear translational benchmarks (e.g., biomarkers that work across species)

  • Develop staged validation approaches moving from zebrafish to mammalian models

  • Consider humanized zebrafish models for specific applications

  • Integrate findings with human genetic and clinical data when available

Through careful attention to these considerations, researchers can maximize the translational value of zebrafish USP16 studies while recognizing their limitations, ultimately advancing our understanding of USP16-related pathways in human health and disease .