Recombinant Xenopus laevis ORM1-like protein 1 (ormdl1)

How does ormdl1 expression change during Xenopus development?

While specific ormdl1 expression patterns have not been extensively characterized, developmental expression analysis in Xenopus can be approached through methods similar to those used for other proteins. Based on research approaches documented for other Xenopus proteins, expression analysis typically involves:

• Temporal expression profiling through developmental stages (from oocyte to tadpole)

• Spatial expression mapping using in situ hybridization

• Tissue-specific expression quantification using RT-PCR or RNA-seq

Many Xenopus proteins show maternal expression patterns, with transcripts stored in oocytes before fertilization, similar to what has been observed with DP-1 and DP-2 proteins that "encode maternally stored transcripts that are expressed during early development" . A comprehensive developmental expression profile would be valuable for understanding ormdl1's potential roles during embryogenesis.

What is known about ormdl1's molecular interactions in Xenopus cellular pathways?

While direct information about ormdl1 interactions in Xenopus is limited, research approaches used to study other Xenopus proteins provide a framework for investigation. Molecular interaction studies could include:

• Co-immunoprecipitation assays to identify binding partners

• Yeast two-hybrid screening to detect direct protein-protein interactions

• Proximity labeling approaches (BioID, APEX) to map the local interactome

Studies of transcription factors in Xenopus have demonstrated complex interaction networks that integrate developmental signaling, such as the DP proteins that "interact combinatorially with E2F proteins to generate an array of DNA binding complexes that integrate cell-cycle progression with the transcription apparatus" . Similar comprehensive interaction mapping for ormdl1 would help place it within cellular regulatory networks.

What are the most effective approaches for functional characterization of ormdl1 in Xenopus laevis?

Functional characterization of ormdl1 would benefit from multiple complementary approaches:

• Loss-of-function studies: CRISPR/Cas9-mediated knockout or antisense morpholino oligonucleotides can be employed to examine the developmental consequences of ormdl1 depletion. This approach has been successfully used in Xenopus for other proteins, as seen in the study where "whole-brain electroporation was performed on late stage 46 tadpoles with 2 μg/μl pSOX2-bd::turboGFP and 0.4mM antisense morpholino oligonucleotide tagged with lissamine fluorophores" .

• Gain-of-function studies: Microinjection of synthetic mRNA encoding ormdl1 into Xenopus embryos can reveal phenotypes associated with overexpression.

• Structure-function analysis: Expression of mutated versions of ormdl1 can identify critical domains for function.

• Tissue-specific manipulation: Targeted expression or knockout in specific tissues can reveal context-dependent functions.

A comprehensive functional characterization would involve phenotypic analysis at the cellular, tissue, and organismal levels following these interventions.

How can transcriptomic and proteomic approaches be applied to study ormdl1 function?

Multi-omics approaches provide powerful tools for understanding ormdl1 function in broader cellular contexts:

• RNA-seq analysis following ormdl1 manipulation: This approach can identify downstream transcriptional effects, similar to methods used in neural progenitor cell studies where "transcriptomic profiling of neural progenitor cells and newly generated neurons with RNA-seq [was used] to identify differentially expressed transcripts" .

• Proteomics of ormdl1-associated complexes: Immunoprecipitation followed by mass spectrometry can identify the protein interaction network.

• Phosphoproteomics: Analysis of phosphorylation changes following ormdl1 manipulation can reveal impacts on signaling pathways.

• Single-cell transcriptomics: This approach can reveal cell-type specific responses to ormdl1 perturbation.

Analysis should include rigorous bioinformatic pipelines to identify significantly altered pathways and biological processes, with validation of key findings through independent experimental approaches.

What genetic approaches can be used to generate ormdl1 mutant Xenopus models?

Several genetic approaches have proven effective for generating mutant Xenopus models:

• CRISPR/Cas9 genome editing: This has become the method of choice for generating specific mutations in Xenopus. The approach typically involves "CRISPR-Cas9 genetic modification... using a single-guide RNA (sgRNA) that targeted exon 1" of the gene of interest, as demonstrated in studies of prominin-1 .

• Verification of mutations: "Blood samples or heterozygous F1 embryos [can be] used for genomic DNA extraction to confirm and characterize CRISPR-mediated indels by Sanger sequencing" .

• Germline transmission: To establish stable mutant lines, "Matings between animals that had confirmed indels by blood draw [can be] performed to obtain F1 progeny" .

• Tissue-specific mutagenesis: For studies focused on specific tissues, targeted delivery of CRISPR components through electroporation can be effective.

The establishment of both F0 mosaic animals and stable F1 lines provides complementary advantages for studying acute and long-term consequences of ormdl1 disruption.

What are the optimal conditions for expressing and purifying recombinant Xenopus laevis ormdl1?

The expression and purification of recombinant Xenopus laevis ormdl1 requires careful optimization:

• Expression systems:

  • Bacterial expression (E. coli): Suitable for non-glycosylated protein domains

  • Insect cell expression (Sf9, Hi5): Better for mammalian proteins requiring folding assistance

  • Mammalian expression (HEK293, CHO): Optimal for proteins requiring complex post-translational modifications

• Purification strategy:

  • Affinity tags: His-tag, GST, or FLAG tag for initial capture

  • Ion exchange chromatography: For charge-based separation

  • Size exclusion chromatography: For final polishing and buffer exchange

• Quality control:

  • SDS-PAGE with Coomassie staining to assess purity

  • Western blotting to confirm identity

  • Mass spectrometry for accurate molecular weight determination

  • Circular dichroism to verify proper folding

Commercial sources like CUSABIO TECHNOLOGY LLC provide recombinant Xenopus laevis ormdl1 , but custom expression and purification protocols may be necessary for specific research applications requiring modifications or specialized tags.

What experimental designs are most effective for studying ormdl1 developmental functions?

Effective experimental designs for studying ormdl1 developmental functions include:

• Temporal manipulation studies:

  • Early embryonic knockdown through morpholino injection at 1-2 cell stage

  • Inducible systems (e.g., heat shock promoters) for stage-specific manipulation

  • Time-lapse imaging to track developmental consequences

• Spatial manipulation approaches:

  • Targeted injections to affect specific blastomeres

  • Tissue-specific promoters for localized expression

  • Electroporation for region-specific delivery, such as "whole-brain electroporation... with 2 μg/μl pSOX2-bd::turboGFP"

• Functional readouts:

  • Morphological phenotyping

  • Molecular marker analysis

  • Behavioral assays for later stage functional assessments

  • Live imaging of cellular behaviors

• Experimental controls:

  • Rescue experiments to confirm specificity

  • Non-targeting morpholinos or guide RNAs as negative controls

  • Multiple target sequences to confirm consistent phenotypes

A combination of these approaches is ideal for comprehensive functional characterization across developmental stages.

What imaging techniques are most informative for ormdl1 localization studies?

Multiple imaging approaches provide complementary information about ormdl1 localization:

• Fixed tissue imaging:

  • Immunofluorescence with antibodies against ormdl1 or epitope tags

  • Fluorescence in situ hybridization for mRNA localization

  • Super-resolution microscopy (STORM, STED) for nanoscale localization

• Live imaging approaches:

  • Fluorescent protein fusions (GFP, mCherry) for dynamic studies

  • Photoactivatable or photoconvertible tags for pulse-chase experiments

  • FRAP (Fluorescence Recovery After Photobleaching) for mobility assessment

• Subcellular co-localization:

  • Counter-staining with organelle markers

  • Multi-channel confocal microscopy

  • Proximity ligation assays for interaction studies

• Whole animal imaging:

  • Tissue clearing techniques for deep imaging

  • Light sheet microscopy for rapid volumetric imaging

  • In vivo time-lapse imaging as demonstrated for neural progenitor cells where "For in vivo live-cell time-lapse imaging, whole-brain electroporation was performed on late stage 46 tadpoles"

The appropriate combination of techniques depends on the specific research questions regarding ormdl1 localization, dynamics, and interactions.

How should researchers interpret ormdl1 expression patterns in the context of Xenopus development?

Interpretation of ormdl1 expression patterns should consider multiple contextual factors:

What approaches help differentiate between direct and indirect effects in ormdl1 functional studies?

Distinguishing direct from indirect effects requires multiple complementary approaches:

• Temporal analysis:

  • Early vs. late effects after ormdl1 manipulation

  • Time-course experiments to establish sequence of events

  • Inducible systems for temporal control

• Molecular approaches:

  • Direct binding assays (ChIP, EMSA) for DNA interactions

  • Protein-protein interaction studies (co-IP, BiFC)

  • In vitro reconstitution to test sufficiency

• Genetic interaction studies:

  • Epistasis analysis with related pathway components

  • Double knockdown/mutant analysis

  • Rescue experiments with specific pathway components

• Systems-level analysis:

  • Network analysis of transcriptomic/proteomic data

  • Pathway enrichment analysis

  • Mathematical modeling of potential regulatory networks

These approaches collectively provide a framework for establishing causal relationships and distinguishing primary from secondary effects of ormdl1 manipulation.

How can researchers address potential redundancy with other ORM family members in functional studies?

Addressing functional redundancy requires systematic approaches:

• Comprehensive expression analysis:

  • Comparative expression profiling of all ORM family members

  • Co-expression analysis to identify potential compensatory mechanisms

  • Single-cell analysis to detect cell-type specific co-expression

• Multiple gene targeting:

  • Simultaneous knockdown/knockout of multiple family members

  • Graduated depletion to identify threshold effects

  • Combinatorial targeting to identify synergistic relationships

• Domain-specific analysis:

  • Identification of unique vs. shared functional domains

  • Domain-swap experiments to test functional equivalence

  • Structure-function analysis of family-specific features

• Evolutionary analysis:

  • Comparative studies across species with different numbers of paralogs

  • Analysis of selection pressures on different family members

  • Identification of species-specific adaptations in function

These approaches collectively provide a framework for understanding the unique and redundant functions of ormdl1 within the broader ORM protein family context.

What are the most promising research directions for ormdl1 in Xenopus laevis models?

Future research on ormdl1 in Xenopus laevis should focus on:

• Comprehensive characterization of expression patterns throughout development

• Generation of specific antibodies and reporter lines for dynamic studies

• CRISPR-mediated genome editing to create stable mutant lines

• Integration with multi-omics approaches to place ormdl1 in broader cellular networks

• Comparative studies with mammalian models to identify conserved functions

Particular attention should be given to potential roles in ER homeostasis, calcium signaling, and sphingolipid metabolism, which are conserved functions of ORM family proteins across species. The unique advantages of the Xenopus system, including "excellent experimental system to investigate how sensory experience modulates neural development because tadpoles receive and [process sensory information]" , position it well for integrative studies of ormdl1 function in development and physiology.

How can findings from Xenopus laevis ormdl1 studies be translated to other model systems and human health?

Translation of findings from Xenopus to other systems involves:

• Comparative genomics:

  • Identification of conserved domains and regulatory elements

  • Analysis of evolutionary conservation of interaction networks

  • Mapping of functional motifs across species

• Cross-species validation:

  • Testing of key findings in mammalian cell culture

  • Generation of equivalent mutations in mouse models

  • Correlation with human genetic studies

• Disease relevance assessment:

  • Analysis of ormdl1 variants in human disease databases

  • Functional testing of disease-associated variants in Xenopus

  • Development of phenotypic assays relevant to human pathologies

• Therapeutic development pipelines:

  • Xenopus-based screens for pathway modulators

  • Validation of potential therapeutic targets

  • Development of biomarkers for pathway activity