Recombinant Danio rerio ORM1-like protein 3 (ormdl3)

What is the evolutionary significance of ORMDL3 in Danio rerio compared to human ORMDL3?

ORMDL3 belongs to a highly evolutionarily conserved gene family with homologs found across species ranging from yeast to vertebrates. The ORMDL gene family encodes transmembrane proteins anchored in the endoplasmic reticulum (ER) . The significant level of conservation seen in ORMDL proteins across diverse lineages strongly suggests functional importance throughout evolution. Human ORMDL genes (ORMDL1, ORMDL2, and ORMDL3) share 80-84% positional identity at the protein level, with 116 out of 153 amino acid residues conserved across all three sequences . This high degree of conservation extends to zebrafish ORMDL3, making Danio rerio an excellent model for studying the fundamental functions of this protein. The maintenance of duplicate copies of ORMDL genes in different lineages further supports their functional significance . This evolutionary conservation enables researchers to use zebrafish as a tractable model system to understand fundamental ORMDL3 functions that likely translate to humans.

How does ORMDL3 expression differ across tissues during zebrafish development?

While the search results don't provide specific data on zebrafish ORMDL3 expression patterns, we can infer from studies in other organisms that ORMDL3 likely exhibits developmental stage-specific expression patterns. In humans, ORMDL genes are expressed ubiquitously in adult and fetal tissues . Similarly, the Drosophila ORMDL homolog is expressed throughout embryonic and larval stages, particularly in ectodermally derived tissues .

In zebrafish, tracking ORMDL3 expression during development can be accomplished through various techniques:

• Whole-mount in situ hybridization using RNA probes specific to ORMDL3

• Transgenic reporter lines with fluorescent proteins driven by the ORMDL3 promoter

• Quantitative PCR analysis of different tissues at various developmental stages

Researchers studying ORMDL3 in zebrafish should consider establishing a comprehensive expression map across developmental stages, as this would provide valuable insights into potential tissue-specific functions of this protein.

What subcellular localization pattern does ORMDL3 exhibit in zebrafish cells?

Based on the conserved nature of ORMDL proteins, zebrafish ORMDL3 is expected to localize to the endoplasmic reticulum (ER) membrane. Studies in other organisms have established that the ORMDL gene family encodes transmembrane proteins anchored in the ER . This subcellular localization in zebrafish can be confirmed through:

• Fluorescent tagging of recombinant ORMDL3 combined with confocal microscopy

• Co-localization studies with known ER markers

• Subcellular fractionation followed by western blotting

Understanding the precise subcellular localization is critical for interpreting functional studies, as it provides context for potential interaction partners and signaling pathways influenced by ORMDL3.

What are the optimal expression systems for producing recombinant Danio rerio ORMDL3?

For successful production of recombinant Danio rerio ORMDL3, researchers should consider several expression systems, each with distinct advantages:

Bacterial expression systems (E. coli):

• Advantages: Rapid growth, high protein yield, cost-effective

• Limitations: May lack appropriate post-translational modifications, potential for inclusion body formation

• Optimization: Use strains designed for membrane protein expression (e.g., C41/C43); consider fusion tags to enhance solubility

Yeast expression systems:

• Advantages: Eukaryotic processing, suitable for membrane proteins

• Relevance: Particularly appropriate since yeast contains ORMDL homologs, suggesting compatible cellular machinery

• Note: Yeast knockout experiments have shown that human ORMDL homologs can rescue phenotypes in yeast ORMDL mutants, demonstrating functional conservation

Insect cell expression systems:

• Advantages: Advanced eukaryotic processing, good for complex proteins

• Considerations: Higher cost, more complex protocols

Mammalian cell expression systems:

• Advantages: Most authentic post-translational modifications

• Considerations: Highest cost, lower yields, longer timeline

The choice of expression system should be guided by the specific experimental requirements, particularly whether the recombinant protein needs to maintain native conformation and functionality.

How can zebrafish embryos be effectively used for studying ORMDL3 function through transgenic approaches?

Zebrafish embryos provide an excellent model for studying ORMDL3 function due to their transparency and rapid development. Several transgenic approaches can be employed:

Cre-lox recombination system:

• Enables conditional activation or inactivation of ORMDL3 in specific tissues

• Can be combined with cell-lineage tracing to monitor the descendants of cells expressing ORMDL3

• Light-activated Cre recombinase variants allow for precise spatiotemporal control of recombination

CRISPR-Cas9 genome editing:

• For generating knockout or knockin ORMDL3 zebrafish lines

• Can be used to introduce specific mutations corresponding to human disease variants

• Potential for tissue-specific CRISPR using restricted promoters

Transgenic reporter lines:

• Fusion of ORMDL3 promoter with fluorescent proteins to visualize expression patterns

• Fusion of ORMDL3 protein with fluorescent tags to monitor subcellular localization

Morpholino knockdown:

• For rapid assessment of ORMDL3 loss-of-function phenotypes

• Should be validated with genetic mutants to confirm specificity

These approaches can be particularly powerful when combined with the cell lineage tracing tools mentioned in the search results, which allow for tracking cells expressing ORMDL3 throughout development .

What are the key considerations for designing primers for zebrafish ORMDL3 cloning and expression analysis?

When designing primers for zebrafish ORMDL3 cloning and expression analysis, researchers should consider:

For cloning applications:

• Include appropriate restriction sites compatible with your expression vector

• Add Kozak sequence (GCCACC) before the start codon for efficient translation

• Consider codon optimization for your expression system

• Account for the addition of purification tags (His, GST, etc.) if needed

• Ensure proper reading frame is maintained

For expression analysis (qPCR):

• Design primers spanning exon-exon junctions to avoid genomic DNA amplification

• Aim for amplicon size of 80-150 bp for optimal qPCR efficiency

• Match primer Tm values (ideally between 58-62°C)

• Test primer specificity against other ORMDL family members (ORMDL1, ORMDL2)

• Include appropriate reference genes for normalization (e.g., ef1α, β-actin)

General considerations:

• Check for secondary structures and primer-dimers

• Ensure GC content is between 40-60%

• Avoid runs of identical nucleotides, especially guanines

• Verify primer specificity using BLAST against the zebrafish genome

Careful primer design is essential for successful amplification, cloning, and accurate expression analysis of zebrafish ORMDL3.

How can comparative studies between human and zebrafish ORMDL3 inform our understanding of asthma pathogenesis?

Comparative studies between human and zebrafish ORMDL3 can provide valuable insights into asthma pathogenesis through multiple approaches:

Functional conservation analysis:

• Human ORMDL3 polymorphisms (particularly rs7216389 and rs4378650) have been strongly associated with childhood asthma risk

• Testing equivalent mutations in zebrafish ORMDL3 can help determine functional conservation

• Cross-species rescue experiments to assess functional equivalence (similar to the yeast studies where human ORMDL proteins rescued yeast knockouts)

Inflammatory pathway investigation:

• Zebrafish models expressing variant forms of ORMDL3 can be used to study inflammatory responses

• The transparency of zebrafish embryos allows real-time visualization of immune cell recruitment and inflammatory processes

Transcriptomic and proteomic comparisons:

• Comparing expression profiles in zebrafish models with altered ORMDL3 expression to human asthmatic bronchial samples

• Identification of conserved downstream pathways affected by ORMDL3 dysregulation

Developmental impact assessment:

• Since asthma often develops in childhood, zebrafish provide an excellent model to study how ORMDL3 variants affect early lung development

• Cell lineage tracing in zebrafish can help identify how ORMDL3 expression patterns during development might predispose to later respiratory issues

This comparative approach leverages the genetic and physiological similarities between species while taking advantage of the experimental tractability of the zebrafish model.

What are the most effective approaches for studying ORMDL3 protein interactions in zebrafish models?

Studying ORMDL3 protein interactions in zebrafish requires sophisticated methodologies that maintain the protein's native environment as an ER membrane protein:

Proximity-based labeling techniques:

• BioID or TurboID fused to ORMDL3 to identify proximal proteins in living zebrafish cells

• APEX2 peroxidase-based proximity labeling for temporal control of labeling reactions

Co-immunoprecipitation optimized for membrane proteins:

• Crosslinking approaches to stabilize transient interactions

• Detergent optimization critical for maintaining ORMDL3 in its native conformation

• Tandem affinity purification for increased specificity

FRET/BRET-based interaction studies:

• For investigating specific hypothesized interactions in living cells

• Particularly useful for monitoring dynamic changes in protein interactions under different conditions

Split-protein complementation assays:

• Using split-GFP or split-luciferase fusions to detect protein-protein interactions in vivo

Interactome analysis specific to ER membrane:

• Subcellular fractionation to enrich for ER membrane before interaction studies

• Correlation with known ER stress response pathways

These approaches should be combined with functional validation through genetic manipulation of identified interaction partners to establish biological relevance of the interactions.

How does ORMDL3 function in ER stress responses in zebrafish, and how can this be experimentally assessed?

Based on ORMDL3's localization to the ER membrane and studies in other organisms, it likely plays a role in ER stress responses in zebrafish. This can be experimentally assessed through:

ER stress induction experiments:

• Treating zebrafish embryos with known ER stress inducers (tunicamycin, thapsigargin, DTT)

• Comparing responses in wild-type vs. ORMDL3 mutant or overexpressing zebrafish

• This approach is supported by yeast studies showing that ORMDL knockouts exhibit increased sensitivity to tunicamycin and dithiothreitol, known ER stress inducers

UPR pathway analysis:

• Monitoring key UPR (unfolded protein response) markers in response to ORMDL3 manipulation:

  • XBP1 splicing

  • ATF6 translocation

  • PERK phosphorylation

  • BiP/GRP78 upregulation

Calcium homeostasis assessment:

• Real-time calcium imaging in zebrafish embryos with altered ORMDL3 expression

• ER calcium store measurements using specific indicators

Lipid composition analysis:

• Lipidomics to assess changes in ER membrane composition

• Focus on sphingolipids and ceramides, which have been linked to ORMDL function

Transgenic reporter systems:

• Creating zebrafish lines with UPR element-driven fluorescent reporters

• Visualizing ER stress responses in real-time during development

These approaches could reveal how ORMDL3 contributes to ER homeostasis and stress responses, potentially informing its role in asthma pathogenesis.

What are common challenges in expressing recombinant Danio rerio ORMDL3 and how can they be overcome?

Expressing recombinant Danio rerio ORMDL3 presents several challenges typical of membrane proteins:

Challenge: Poor expression yields
Solutions:

• Test multiple expression systems (bacterial, yeast, insect, mammalian)

• Optimize codon usage for the expression host

• Consider fusion partners that enhance expression (MBP, SUMO, Trx)

• Adjust induction conditions (temperature, inducer concentration, time)

Challenge: Protein misfolding and aggregation
Solutions:

• Lower expression temperature (16-20°C for E. coli)

• Co-express with molecular chaperones

• Use specialized strains designed for membrane proteins

• Include mild detergents during lysis and purification

Challenge: Toxicity to expression host
Solutions:

• Use tightly controlled inducible promoters

• Consider auto-induction systems to gradually express protein

• Test lower-copy-number vectors

• The fact that human ORMDL proteins can rescue yeast ORMDL knockouts suggests yeast might be a compatible expression host

Challenge: Difficult purification due to membrane localization
Solutions:

• Optimize detergent screening (start with mild non-ionic detergents)

• Consider extraction using amphipols or nanodiscs to maintain native conformation

• Use on-column detergent exchange during purification

• Test different solubilization conditions and times

Challenge: Verifying proper folding and functionality
Solutions:

• Develop functional assays based on known activities

• Use circular dichroism to assess secondary structure

• Thermal shift assays to evaluate stability

• Binding assays with known interaction partners

These approaches can be modified based on specific experimental outcomes and requirements.

How can researchers distinguish between the effects of ORMDL3 and other ORMDL family members in zebrafish studies?

Distinguishing between ORMDL family members in zebrafish requires careful experimental design:

Sequence-specific genetic manipulation:

• CRISPR-Cas9 targeting of unique regions of ORMDL3

• Verification of specificity by sequencing and assessing expression of all ORMDL family members

• Design compensatory rescue constructs with synonymous mutations to resist CRISPR targeting

Isoform-specific knockdown:

• Morpholinos targeting unique splice junctions or UTRs of ORMDL3

• siRNA/shRNA with verified specificity

• Rescue experiments with ORMDL3-specific constructs to confirm phenotype specificity

Expression analysis tools:

• Isoform-specific qPCR primers designed to unique regions

• Specific antibodies confirmed for selectivity (if available)

• RNA-seq analysis with isoform-level quantification

Functional compensation assessment:

• Sequential knockout/knockdown of individual ORMDL family members

• Combinatorial approaches to assess redundancy

• Overexpression of specific isoforms in various knockout backgrounds

Tissue-specific approaches:

• Leverage any differences in expression patterns between ORMDL family members

• Use tissue-specific promoters to manipulate ORMDL3 in relevant tissues

This issue of distinguishing between family members is particularly relevant given the high sequence similarity between ORMDL proteins (80-84% identity between human ORMDL proteins) and likely similar conservation in zebrafish.

What statistical approaches are most appropriate for analyzing phenotypic data from zebrafish ORMDL3 studies?

Analyzing phenotypic data from zebrafish ORMDL3 studies requires robust statistical approaches tailored to the specific experimental design:

For developmental phenotypes:

• Kaplan-Meier survival analysis for mortality data

• Chi-square tests for categorical phenotypes (e.g., presence/absence of specific defects)

• Mixed-effects models for longitudinal measurements accounting for clutch effects

For gene expression studies:

• ANOVA with post-hoc tests for comparing multiple conditions

• Consider false discovery rate correction for RNA-seq or other high-throughput data

• Principal component analysis for visualizing global expression patterns

• Gene set enrichment analysis to identify affected pathways

For behavioral analyses:

• Repeated measures ANOVA for behavioral time-course data

• Non-parametric tests if normality assumptions are violated

• Hidden Markov models for complex behavioral sequences

Sample size considerations:

• Power analysis based on preliminary data

• Account for clutch-to-clutch variability

• Consider nested designs to account for non-independence of embryos from the same parents

Controlling for confounding factors:

• Randomization of treatment groups

• Blinding for phenotype scoring

• Inclusion of appropriate controls (wild-type siblings, non-targeting CRISPR, etc.)

• Technical replicates vs. biological replicates distinction

How might zebrafish ORMDL3 models contribute to development of novel therapeutics for asthma?

Zebrafish ORMDL3 models offer unique advantages for therapeutic development for asthma:

High-throughput drug screening:

• Utilize transgenic zebrafish with fluorescent reporters linked to ORMDL3-dependent pathways

• Screen chemical libraries for compounds that normalize ORMDL3-induced phenotypes

• Assess effects on inflammatory responses in real-time

• The transparency of zebrafish embryos enables direct visualization of drug effects on relevant tissues

Genetic modifier screens:

• Identify genes that suppress or enhance ORMDL3-associated phenotypes

• These modifiers represent potential therapeutic targets

• CRISPR-based screens in zebrafish carrying ORMDL3 variants associated with asthma

Mechanism-based therapeutic development:

• Detailed understanding of ORMDL3's role in ER stress may reveal novel intervention points

• Test compounds that modulate ER stress pathways altered by ORMDL3 variants

• Leverage the observed association between ORMDL3 SNPs and asthma risk to develop targeted approaches

Personalized medicine applications:

• Generate zebrafish models with specific human ORMDL3 variants

• Test therapeutic responses in variant-specific models

• The strong association of SNPs like rs7216389 with asthma (odds ratio 1.44, meta-analysis p<0.00001) suggests variant-specific approaches may be valuable

Alternative delivery methods testing:

• Assess inhaled vs. systemic delivery of potential therapeutics

• Evaluate tissue-specific drug targeting approaches

Zebrafish models provide a bridge between in vitro studies and mammalian models, allowing for rapid, cost-effective therapeutic development.

What are promising approaches for investigating the interplay between ORMDL3 and environmental factors in zebrafish models?

Investigating ORMDL3-environment interactions in zebrafish can provide valuable insights into asthma pathogenesis:

Exposure models:

• Develop standardized protocols for exposing zebrafish to relevant environmental factors:

  • Allergens (house dust mite extract, pollens)

  • Air pollutants (particulate matter, ozone)

  • Respiratory viruses

  • Microbiome alterations

Transgenic reporter systems:

• Create zebrafish lines with ORMDL3 promoter-driven reporters to monitor environmental effects on expression

• Develop reporters for inflammatory pathways downstream of ORMDL3 to visualize responses

Gene-environment interaction assessment:

• Compare responses to environmental exposures between wild-type and ORMDL3 variant zebrafish

• Factorial experimental designs to identify synergistic effects

• Time-course studies to determine critical developmental windows for interactions

Epigenetic mechanisms:

• Investigate how environmental exposures might alter ORMDL3 regulation through:

  • DNA methylation analysis

  • Histone modification profiling

  • Chromatin accessibility assessment

Systems biology approaches:

• Multi-omics integration (transcriptomics, proteomics, metabolomics) following environmental exposures

• Network analysis to identify key nodes in ORMDL3-dependent response pathways

This research direction is particularly relevant given that asthma is understood to result from interactions between genetic susceptibility factors (like ORMDL3 variants) and environmental exposures.

How can cell-lineage tracing techniques be optimized for studying ORMDL3 expression dynamics during zebrafish development?

Cell-lineage tracing techniques offer powerful tools for understanding ORMDL3 expression dynamics:

Photoactivatable systems optimization:

• Utilize Cre-lox systems activated by light for precise spatiotemporal control

• Optimize light exposure parameters for minimal phototoxicity while achieving complete recombination

• The search results mention that "direct activation of Cre recombinase with light would allow for facile, rapid, and high spatiotemporal control of DNA recombination for study in the developing zebrafish embryo"

Multicolor lineage tracing:

• Implement Brainbow/Confetti systems under ORMDL3 promoter control

• Optimize spectral separation for clearly distinguishing multiple lineages

• Develop image analysis pipelines for tracking large numbers of labeled cells

Temporal control enhancements:

• Utilize split Cre recombinase systems that can be activated by light illumination

• Optimize the dimerization parameters of photo-responsive proteins to achieve rapid and complete recombination

• Address challenges of dimer dissociation that can occur after illumination

Integration with single-cell approaches:

• Combine lineage tracing with single-cell RNA-seq to determine transcriptional states of ORMDL3-expressing lineages

• Develop protocols for isolating fluorescently labeled cells for molecular analysis

Four-dimensional imaging optimization:

• Establish imaging parameters that minimize phototoxicity during long-term tracking

• Develop computational approaches for cell tracking in complex tissues

• Optimize mounting and immobilization methods for extended live imaging

These optimized techniques would provide unprecedented insights into how ORMDL3 expression patterns during development might contribute to asthma susceptibility and other conditions.

How do functional differences between zebrafish and human ORMDL3 impact experimental design and data interpretation?

Understanding the functional differences between zebrafish and human ORMDL3 is critical for translational research:

Sequence and structural comparison:

• While ORMDL proteins are highly conserved across species, even small differences may affect function

• Human ORMDL family members share 80-84% identity , suggesting similar conservation levels with zebrafish ORMDL3

• Key functional domains and motifs should be compared to ensure relevance of zebrafish models

Expression pattern differences:

• Human ORMDL3 is ubiquitously expressed , but zebrafish may have tissue-specific expression patterns

• Developmental timing of expression may differ between species

• These differences must be accounted for when designing experiments and interpreting results

Interaction partner conservation:

• Verify whether key ORMDL3 interaction partners identified in humans are conserved in zebrafish

• Different interaction networks may lead to distinct functional outcomes

• Yeast studies showing functional rescue by human ORMDL proteins suggest fundamental functions are conserved across species

Physiological differences:

• Zebrafish respiratory system differs anatomically from human lungs

• Inflammatory responses and immune cell types have both similarities and differences

• Careful consideration of which aspects of asthma pathophysiology can be modeled in zebrafish

Experimental design considerations:

• Include human ORMDL3 expression in zebrafish models alongside zebrafish ORMDL3

• Conduct parallel experiments in mammalian and zebrafish systems when possible

• Consider using humanized zebrafish models for specific applications

These considerations ensure appropriate translation of findings from zebrafish to human health applications.