Recombinant Danio rerio Basic leucine zipper and W2 domain-containing protein 1-B (bzw1b)

What is the predicted function of BZW1B in zebrafish based on homology studies?

Based on information about the BZW family proteins, BZW1B likely functions as a eukaryotic translation factor. Similar to the related BZW2, it may be involved in the regulation of protein synthesis and various cellular processes that require strict regulation . The protein is likely localized in the cytoplasm and may be expressed in multiple tissues throughout the body, with potential higher expression in specific tissues such as the heart, placenta, skeletal muscle, and certain brain regions, as observed with BZW2 .

How is recombinant BZW1B typically produced for research applications?

Recombinant zebrafish BZW1 protein can be produced using various expression systems, with the yeast expression system being particularly advantageous. This system provides an economical and efficient eukaryotic platform for both secretion and intracellular expression . The yeast system allows for post-translational modifications such as glycosylation, acylation, and phosphorylation, ensuring native protein conformation and functionality .

For purification, the recombinant protein is typically tagged with a His-tag, enabling affinity chromatography purification to achieve purity levels exceeding 90% . Alternative expression systems include E. coli, mammalian cells, or baculovirus infection, each with different advantages regarding cost, production time, and protein quality .

What are optimal approaches for manipulating BZW1B expression in zebrafish embryos?

Zebrafish embryos offer significant advantages for genetic manipulation due to their accessibility and transparency. The most effective approach for studying BZW1B function involves microinjection of early-stage embryos with either overexpression constructs or knockdown reagents . Solutions containing genetic material can be delivered into the blastomeres (embryonic cells on top of the yolk) either through direct injection or via the yolk, utilizing natural cytoplasmic movements to distribute the material .

For transient manipulation, morpholinos targeting bzw1b mRNA can be used, while CRISPR-Cas9 components can generate more stable genetic modifications. Following genetic manipulation, careful quantification of embryonic phenotypes is essential to elucidate BZW1B's genetic mechanisms in development . Temperature control during these experiments is crucial, as zebrafish embryos show temperature-dependent physiological responses that could confound experimental results .

How can plate-based respirometry assess functional impacts of BZW1B manipulation?

The XF96 Seahorse extracellular flux analyzer platform offers a powerful approach to investigate the bioenergetic effects of BZW1B manipulation in zebrafish embryos . This methodology allows for the parallel measurement of up to 92 individual embryos, imposing less shearing stress compared to traditional chamber-based respirometry .

For optimal results, embryos should be dechorionated to enhance respiration measurements and improve responses to mitochondrial inhibitors and uncouplers . Sedation with tricaine is recommended to reduce variability in oxygen traces caused by spontaneous activity . Standard mitochondrial inhibitor concentrations for 48-50 hpf embryos include:

This methodology enables measurement of various respiratory parameters including basal respiration, ATP turnover, proton leak, and maximal respiratory capacity, potentially revealing BZW1B's effects on energy metabolism .

What proteomic approaches can reveal BZW1B function in zebrafish?

Proteomic analysis provides a comprehensive approach to study the effects of BZW1B manipulation in zebrafish embryos . Comparative proteomics between wild-type and BZW1B-modified zebrafish can reveal downstream effects on protein expression patterns and identify functional networks. Mass spectrometry-based approaches can identify proteins that interact with BZW1B, helping to elucidate its role in cellular processes.

For temporal studies, analyzing proteome changes at different developmental stages in response to BZW1B manipulation can reveal stage-specific functions. Integration with transcriptomic data provides a more comprehensive understanding of BZW1B's role in cellular regulation and development .

How might BZW1B influence zebrafish brain function and cognitive abilities?

Recent research has revealed that zebrafish possess more sophisticated cognitive abilities than previously thought, including the ability to create mental maps of their environment . This opens intriguing possibilities for studying BZW1B's potential role in neural function and cognition.

Zebrafish exhibit remarkably quick reaction times (approximately 10 milliseconds) to stimuli, suggesting pre-computation of environmental maps . This rapid response system provides an excellent model to investigate whether BZW1B affects neural processing and behavioral responses. Behavioral assays can be designed to test if BZW1B manipulation alters escape responses, environmental mapping abilities, or learning and memory functions.

The zebrafish optical tectum may contain 3D spatial representations similar to those in the mammalian superior colliculus, offering a translational model to study BZW1B's potential role in visual processing and spatial cognition . Integration with transgenic zebrafish lines expressing fluorescent reporters in specific neural populations can help elucidate BZW1B's role in neural development and function.

What considerations are important when designing temperature-dependent studies of BZW1B function?

Temperature significantly impacts zebrafish embryo physiology and potentially BZW1B function. Research has shown that zebrafish embryos exhibit distinct respiratory responses to temperature variations, with stable respiration rates across a certain thermal window . When designing temperature-dependent studies of BZW1B, researchers should consider:

• Pre-exposure effects: Pre-exposing embryos to warmer temperatures can extend their thermal window of stable embryonic respiration rates toward higher temperatures .

• Metabolic mechanisms: At lower temperatures, slow respiration appears to be caused by low ATP turnover rather than limited mitochondrial oxidative power, suggesting temperature-dependent changes in cellular metabolism .

• Mitochondrial efficiency: At higher temperatures, increasing proton leak impacts the consumption of proton motive force and limits mitochondrial efficiency, potentially affecting BZW1B function in energy regulation .

• Life cycle bottlenecks: Temperature sensitivity varies across developmental stages, making it important to identify the most vulnerable stages for studying BZW1B's role in thermal adaptation .

How can advances in zebrafish cloning technology facilitate BZW1B research?

The development of reproducible nuclear transfer cloning protocols in zebrafish offers significant advantages for BZW1B research . This technology enables the preservation of valuable zebrafish lines with specific BZW1B modifications or mutations. The Zebrafish International Resource Center (ZIRC), which currently houses and distributes thousands of genetically modified zebrafish lines, could potentially utilize cloning technology to more efficiently maintain and distribute BZW1B-modified lines .

Advanced cloning techniques can help create genetically identical backgrounds for studying BZW1B function, reducing experimental variability. The combination of cloning with existing cryopreservation methods could allow for long-term storage and revival of valuable BZW1B-modified zebrafish lines, facilitating longitudinal studies and resource sharing within the research community .

What controls are essential when studying BZW1B function in zebrafish?

When designing experiments to study BZW1B in zebrafish, several critical controls must be incorporated:

• Biological controls:

  • Wild-type uninjected embryos from standardized lines (e.g., AB strain)

  • Embryos injected with control proteins or control morpholinos/CRISPR guides

  • Rescue experiments where BZW1B function is restored after knockdown

• Technical controls:

  • Verification of protein expression or knockdown efficacy through Western blotting or qPCR

  • Assessment of off-target effects through multiple targeting approaches

  • Consistent developmental staging of embryos

  • Temperature control, particularly important given the temperature sensitivity of zebrafish embryos

• Physiological controls:

  • When performing respirometry measurements, control for potential confounding factors such as spontaneous movement by using tricaine sedation

  • For dechorionated embryos, ensure consistent handling to prevent physical damage

How should researchers interpret variable phenotypes resulting from BZW1B manipulation?

Variation in phenotypes following BZW1B manipulation may stem from multiple factors:

• Dosage effects: Different levels of BZW1B expression or knockdown efficiency may result in varying phenotypic outcomes.

• Developmental timing: The effects of BZW1B manipulation likely vary depending on the developmental stage, requiring careful temporal analysis.

• Temperature effects: Given the temperature sensitivity of zebrafish embryos, phenotypic outcomes may be influenced by experimental temperature conditions .

• Genetic background: The zebrafish strain used (e.g., AB strain) may influence phenotypic manifestations of BZW1B manipulation .

• Compensatory mechanisms: Zebrafish may activate molecular pathways that compensate for BZW1B dysfunction, potentially masking or altering expected phenotypes.

Careful documentation of all experimental conditions and systematic phenotypic assessment are essential for meaningful interpretation of results.

What potential roles might BZW1B play in zebrafish disease models?

Based on information about the related BZW2 protein, BZW1B may have significant roles in disease processes that could be modeled in zebrafish:

• Cancer models: The related BZW2 shows upregulation in various cancers, correlating with increased severity and mortality . BZW1B might have similar roles in zebrafish cancer models, potentially affecting cell proliferation or tumor progression.

• Viral infection responses: Given that BZW2 has been found to interact with SARS-CoV-2 , BZW1B might play roles in host-pathogen interactions that could be studied in zebrafish infection models.

• Developmental disorders: As a potential translation factor, BZW1B likely influences protein synthesis during development, making it a candidate gene for developmental disorders.

• Bioenergetic dysfunction: The established methods for assessing mitochondrial function in zebrafish embryos provide tools to investigate BZW1B's potential role in metabolic disorders .

How might integrative multi-omics approaches advance BZW1B research?

Future research on BZW1B would benefit from integrative approaches combining:

• Genomics: CRISPR-Cas9 gene editing to create precise modifications in the bzw1b gene locus.

• Transcriptomics: RNA-seq analysis to identify genes and pathways affected by BZW1B manipulation.

• Proteomics: Mass spectrometry-based approaches to identify BZW1B-interacting proteins and downstream effects on the proteome .

• Metabolomics: Analysis of metabolic changes resulting from BZW1B manipulation, particularly in conjunction with respirometry data .

• Behavioral phenomics: Systematic analysis of behavioral changes in response to BZW1B manipulation, leveraging the sophisticated cognitive abilities of zebrafish .