Recombinant Danio rerio Disks large-associated protein 1 (dlgap1), partial

What is the basic structure and function of DLGAP1 in zebrafish?

DLGAP1 (Disks large-associated protein 1) in zebrafish functions as a scaffold protein within the postsynaptic density and is a member of the Scribble cell polarity complex. The protein contains multiple domains that facilitate protein-protein interactions, including several PDZ-binding motifs that enable its association with membrane proteins and cytoskeletal components. In zebrafish, DLGAP1 demonstrates both centrosomal and cytoplasmic localization, with its distribution pattern depending on the cell cycle stage and activation of specific signaling pathways. The centrosomal localization is particularly dynamic and can be modulated by various kinases including Jak2, SRC, MAPK, and CDK1, which promote DLGAP1 dissociation from centrosomes . This protein plays crucial roles in cellular polarity maintenance, signal transduction, and megakaryocytic differentiation processes in hematopoietic cells, suggesting conservation of function across vertebrate species. The full-length DLGAP1 cDNA sequence in zebrafish spans approximately 3197 bp, encoding a protein that shares significant homology with its mammalian counterparts.

How do AB and TL zebrafish strains differ in DLGAP1 expression patterns?

The AB and Tupfel long-fin (TL) zebrafish strains, commonly used in research, exhibit significant differences in gene expression patterns, including those related to DLGAP1 function. Studies have demonstrated that these strains differ in baseline hypothalamus-pituitary-interrenal (HPI)-axis activity, with AB showing higher activity than TL strains . This difference in HPI-axis activity influences numerous downstream genes and biological processes, potentially affecting DLGAP1 expression and function. AB strains consistently show higher expression of genes related to neurodevelopment and immune system function compared to TL strains . These differences are detectable as early as 5 days post-fertilization, indicating that strain-specific expression patterns are established during early development. The mutation in connexin 41.8 that gives TL fish their spotted rather than striped pattern may also influence brain, eye, and heart function, potentially impacting DLGAP1 expression in these tissues. Researchers must account for these strain differences when designing experiments and interpreting results, as baseline biological differences may confound experimental outcomes related to DLGAP1 function.

What are the recommended methods for cloning and expressing recombinant zebrafish DLGAP1?

The cloning and expression of recombinant zebrafish DLGAP1 can be accomplished through several validated methodological approaches. For full-length DLGAP1 cloning, researchers have successfully used PCR amplification of the complete 3197 bp cDNA sequence using high-fidelity polymerases such as Phusion Polymerase, with primers containing appropriate restriction sites (e.g., EcoRI and KpnI) for subsequent ligation into expression vectors . The full-length template cDNA can be sourced from commercially available clone repositories, such as the MGC clone ID: 9020442 (IMAGE:9020442) in pCR4-TOPO vector . For expression studies, fusion constructs such as pEGFP-DLGAP1 have been effectively employed by in-frame ligation of the DLGAP1 cDNA into vectors like pEGFP-C1, allowing for visualization of the protein's cellular localization . Truncated versions of DLGAP1 can also be generated by designing primers that target specific exons, such as using a forward primer complementary to exon 5 containing the first methionine codon in that exon for the generation of pEGFP-TrDLGAP1 . Expression can be verified through standard techniques including Western blotting, immunofluorescence, and flow cytometry, with the latter being particularly useful for quantifying transfection or transduction efficiency when using fluorescent protein tags.

What tissue-specific expression patterns of DLGAP1 have been observed in zebrafish?

The tissue-specific expression patterns of DLGAP1 in zebrafish reveal important insights into its biological functions across different organ systems. RNA sequencing studies of adult wild-type (TLAB) zebrafish germline tissues have provided valuable data on DLGAP1 expression in reproductive organs, including ovaries, oocytes, and testes . The gene expression profiling conducted through high-throughput sequencing has allowed researchers to create comprehensive expression maps of DLGAP1 across these germline tissues. In addition to germline expression, DLGAP1 has been observed in hematopoietic tissues, where it plays a role in megakaryocytic growth and differentiation . The protein's centrosomal and cytoplasmic localization in hematopoietic cells suggests tissue-specific functions related to cell division and differentiation processes. DLGAP1 expression has also been detected in neural tissues, consistent with its known role in postsynaptic density organization in other vertebrate species. When designing tissue-specific studies of DLGAP1, researchers should consider the baseline variations in gene expression between common zebrafish strains like AB and TL, as these differences may influence the interpretation of tissue-specific expression patterns.

How does DLGAP1 interact with cell signaling pathways in zebrafish development?

DLGAP1 functions as a critical node in multiple cell signaling networks during zebrafish development, integrating signals from several pathways to regulate cellular processes. Research indicates that DLGAP1 interacts with key signaling kinases, including Jak2, SRC, and MAPK, which promote its dissociation from centrosomes and alter its subcellular distribution . This dynamic localization pattern suggests that DLGAP1 serves as a downstream effector for these signaling pathways, potentially translating extracellular signals into changes in cell polarity, division, or differentiation. The protein's interaction with the cell cycle regulator CDK1 further implicates DLGAP1 in coordinating cell division processes with developmental signaling inputs . In hematopoietic cells, DLGAP1 has been shown to negatively affect the growth rate of MPL-dependent cells while supporting megakaryocytic polyploidization, demonstrating its role in regulating cell proliferation versus differentiation decisions . These findings parallel observations in other model systems where DLGAP family proteins modulate synaptic plasticity and neuronal development through interactions with multiple signaling pathways. The complex interplay between DLGAP1 and various signaling pathways suggests that it may function as a scaffold that brings together multiple signaling proteins to coordinate developmental processes in zebrafish.

What experimental approaches can be used to study DLGAP1 protein-protein interactions in zebrafish?

Several sophisticated experimental approaches can be employed to investigate DLGAP1 protein-protein interactions in zebrafish systems. Immunoprecipitation coupled with mass spectrometry represents a powerful technique for identifying novel DLGAP1 binding partners in zebrafish tissues or cell lines. This approach can be enhanced by using epitope-tagged versions of DLGAP1, such as the EGFP-DLGAP1 fusion protein, which facilitates efficient pull-down of protein complexes . The RNA immunoprecipitation (RIP) assay, which has been successfully applied to study interactions between DLGAP1-AS1 and miR-515-5p in other contexts, can be adapted to investigate potential RNA binding properties of zebrafish DLGAP1 . Proximity-based protein labeling methods, such as BioID or APEX2, can be particularly valuable for capturing transient or weak interactions that might be lost during conventional immunoprecipitation procedures. For visualizing protein interactions in living cells or tissues, fluorescence resonance energy transfer (FRET) or bimolecular fluorescence complementation (BiFC) can be employed using zebrafish-specific constructs. The dual-luciferase reporter assay, which has been used to confirm binding relationships between DLGAP1-AS1 and miR-515-5p, provides another approach for validating predicted interactions . These methodologies can be complemented by computational predictions using tools like StarBase v2.0, which has been successfully applied to identify potential binding partners for DLGAP1-related transcripts .

How do mutations or knockdown of DLGAP1 affect zebrafish development and physiology?

Mutations or knockdown of DLGAP1 in zebrafish can produce diverse phenotypic effects across multiple physiological systems due to the protein's involvement in fundamental cellular processes. Experimental approaches using shRNA-mediated knockdown have revealed that DLGAP1 downregulation impacts cellular growth dynamics in hematopoietic contexts, suggesting potential developmental consequences in tissues with high proliferative activity . Given DLGAP1's centrosomal localization and its role in megakaryocytic polyploidization, perturbations in its expression likely affect cell division and differentiation processes during embryonic and larval development. The protein's interaction with multiple signaling pathways, including those mediated by Jak2, SRC, and MAPK kinases, indicates that DLGAP1 dysfunction may disrupt signal transduction networks critical for normal development . When designing DLGAP1 knockdown or mutation studies, researchers should consider the baseline strain differences between AB and TL zebrafish, as these strains exhibit intrinsic variations in gene expression profiles related to neurodevelopment, stress responses (HPI-axis), and immune function that could confound experimental results . The strain-specific differences in behavioral responses to stimuli, such as habituation to acoustic/vibrational stimuli and light-dark induced changes in motor behavior, should also be factored into the interpretation of phenotypic outcomes following DLGAP1 manipulation .

What are the key considerations for designing CRISPR/Cas9-mediated gene editing of DLGAP1 in zebrafish?

Designing effective CRISPR/Cas9-mediated gene editing strategies for zebrafish DLGAP1 requires careful consideration of several technical factors to ensure specificity and efficiency. Target site selection is critical and should focus on functionally important domains of DLGAP1, such as PDZ-binding motifs or regions essential for centrosomal localization, while avoiding regions with sequence similarity to other genes to minimize off-target effects. Researchers should conduct thorough bioinformatic analysis using zebrafish-specific genome browsers and CRISPR design tools to identify guide RNAs with high on-target efficiency and low off-target potential. When designing guide RNAs, considerations should include the GC content (ideally 40-60%), absence of homopolymer stretches, and targeting of early exons to maximize the likelihood of generating functional knockouts. Since DLGAP1 demonstrates strain-specific expression differences between AB and TL zebrafish lines, researchers should carefully select and consistently use a single strain throughout their experiments to avoid confounding variables . For delivery methods, microinjection of Cas9 protein with synthetic guide RNAs into one-cell stage embryos typically yields higher editing efficiency compared to plasmid-based approaches. Validation strategies should include a combination of genomic PCR, T7 endonuclease I assay, high-resolution melting analysis, and ultimately sequencing to confirm the presence and nature of induced mutations.

How can researchers effectively analyze DLGAP1 expression across different developmental stages?

Effective analysis of DLGAP1 expression across zebrafish developmental stages requires a multi-faceted approach combining various molecular techniques. Quantitative real-time PCR (qRT-PCR) represents a reliable method for measuring DLGAP1 mRNA levels at different timepoints, as demonstrated in studies comparing gene expression in zebrafish tissues . When designing qRT-PCR assays, researchers should carefully select reference genes that show stable expression across developmental stages, such as EF1α or β-actin. RNA sequencing provides a more comprehensive view of expression patterns and has been successfully applied to analyze zebrafish germline tissues, enabling researchers to place DLGAP1 expression within the broader transcriptomic landscape . Whole-mount in situ hybridization offers spatial information about DLGAP1 expression in intact embryos, complementing the quantitative data from qPCR and RNA-seq. For protein-level analysis, Western blotting with specific antibodies against zebrafish DLGAP1 or epitope-tagged recombinant versions can track protein abundance, while immunofluorescence microscopy reveals subcellular localization patterns, which are particularly relevant given DLGAP1's dynamic centrosomal association . Transgenic reporter lines expressing fluorescent proteins under the control of the DLGAP1 promoter can facilitate real-time visualization of expression dynamics in living embryos. Researchers should account for strain-specific variations when comparing expression data, as AB and TL zebrafish demonstrate intrinsic differences in gene expression profiles .

What protein purification strategies are most effective for recombinant zebrafish DLGAP1?

Purification of recombinant zebrafish DLGAP1 presents several technical challenges that can be addressed through strategic protein expression and purification approaches. Affinity-based purification systems represent the most straightforward initial approach, with His-tagged or GST-tagged DLGAP1 constructs enabling single-step enrichment on nickel or glutathione resins, respectively. For more complex applications requiring higher purity, researchers can implement multi-step purification protocols combining affinity chromatography with size exclusion chromatography to separate DLGAP1 from contaminating proteins based on molecular size. Expression systems should be carefully selected based on experimental requirements; bacterial expression in E. coli provides high yields but may lack post-translational modifications, while baculovirus-infected insect cells or mammalian expression systems better preserve native protein folding and modifications. Given DLGAP1's role as a scaffold protein with multiple interaction domains, solubility can be a significant challenge during purification . This issue can be addressed by expressing functional domains separately rather than the full-length protein, using solubility-enhancing fusion partners like MBP or SUMO, or optimizing buffer conditions with stabilizing additives such as low concentrations of non-ionic detergents. For structural studies, limited proteolysis followed by mass spectrometry can identify stable domains amenable to crystallization. When expressing recombinant DLGAP1 constructs based on zebrafish cDNA, researchers should verify their sequence against the reference genome, particularly if using different zebrafish strains as source material, due to potential strain-specific variations .

What approaches can be used to study the cellular localization of DLGAP1 in zebrafish cells?

Multiple complementary approaches can be employed to investigate the cellular localization of DLGAP1 in zebrafish cells, providing insights into its functional dynamics. Fluorescent protein fusion constructs, such as the pEGFP-DLGAP1 vector, offer a powerful tool for visualizing DLGAP1 localization in living cells through confocal or wide-field fluorescence microscopy . These constructs can be transfected into zebrafish cell lines or microinjected into embryos for in vivo imaging. Immunofluorescence microscopy using antibodies specific to zebrafish DLGAP1 represents another valuable approach, allowing detection of the endogenous protein without potential artifacts from overexpression or fusion tags. Co-localization studies with markers for specific cellular compartments, such as γ-tubulin for centrosomes or PSD-95 for postsynaptic densities, can precisely define DLGAP1's subcellular distribution patterns . For temporal dynamics, time-lapse imaging of fluorescently tagged DLGAP1 can reveal how its localization changes during cell cycle progression or in response to signaling pathway activation. Super-resolution microscopy techniques, including structured illumination microscopy (SIM) or stochastic optical reconstruction microscopy (STORM), provide enhanced spatial resolution for detailed analysis of DLGAP1's organization within subcellular structures. Biochemical fractionation of zebrafish cells followed by Western blotting can complement imaging approaches by quantitatively assessing DLGAP1's distribution across cellular compartments. When designing localization studies, researchers should consider that DLGAP1's centrosomal association is dynamically regulated by various kinases, including Jak2, SRC, MAPK, and CDK1, necessitating careful control of cellular signaling states .

How should researchers interpret contradictory findings about DLGAP1 function between different zebrafish strains?

Contradictory findings regarding DLGAP1 function between different zebrafish strains require careful interpretation through a systematic analytical framework. Researchers should first acknowledge that AB and TL strains exhibit fundamental differences in baseline gene expression, particularly in pathways related to the hypothalamus-pituitary-interrenal (HPI) axis, neurodevelopment, and immune function, which might directly or indirectly affect DLGAP1 activity . These intrinsic strain differences can manifest as early as 5 days post-fertilization, potentially confounding experimental results if strain selection is not consistent across studies . When evaluating contradictory results, researchers should meticulously document and compare the specific strains used, their genetic background verification, and maintenance conditions, as laboratory-specific selective pressures can drive genetic drift over generations. Cross-validation experiments using multiple strains with clearly defined genetic backgrounds can help distinguish strain-specific effects from general DLGAP1 functions conserved across zebrafish populations. The mutation in connexin 41.8 in TL zebrafish, which affects their pigmentation pattern, may have pleiotropic effects on other physiological systems, potentially influencing DLGAP1-dependent processes . Meta-analysis approaches combining data from multiple studies while accounting for strain as a variable can help identify consistent DLGAP1 functions despite strain-specific variations. When publishing results, researchers should explicitly report the strain used and consider including comparative data from multiple strains when feasible to enhance reproducibility and translational relevance.

How can researchers troubleshoot issues with recombinant DLGAP1 expression and purification?

Troubleshooting recombinant DLGAP1 expression and purification requires systematic identification and resolution of common technical challenges. Low expression levels may result from codon usage bias between zebrafish and the expression host; this can be addressed by using codon-optimized synthetic genes or expression hosts with rare tRNA supplementation. Protein insolubility often occurs with scaffold proteins like DLGAP1 that contain multiple interaction domains; researchers can mitigate this by lowering expression temperature (16-20°C), using solubility-enhancing fusion tags (SUMO, MBP, Trx), or employing specialized E. coli strains designed for challenging proteins (e.g., Arctic Express, Rosetta-gami). Proteolytic degradation during expression or purification can be addressed by including protease inhibitor cocktails, using protease-deficient expression hosts, or optimizing purification protocols to minimize processing time. For proteins showing poor binding to affinity resins, researchers should verify tag accessibility through Western blotting or consider alternative tag positions (N-terminal versus C-terminal) or different affinity systems altogether. When expression yields remain problematic, exploring alternative expression systems such as insect cells or cell-free systems may prove beneficial for obtaining functional DLGAP1 protein. If specific domains prove particularly challenging, researchers can adopt a divide-and-conquer approach by expressing individual domains separately, as demonstrated in studies using truncated DLGAP1 constructs . For downstream applications requiring removal of affinity tags, optimization of protease cleavage conditions and subsequent separation of the cleaved tag from the target protein may require iterative refinement of buffer conditions and chromatography parameters.

What are the best practices for validating antibodies against zebrafish DLGAP1 for immunological studies?

Thorough validation of antibodies against zebrafish DLGAP1 is essential for ensuring reliable immunological studies and should follow a multi-step verification process. Initial validation should begin with Western blot analysis using both recombinant zebrafish DLGAP1 (e.g., from the pEGFP-DLGAP1 construct) and endogenous protein from zebrafish tissue lysates, confirming detection of bands at the expected molecular weight (approximately 100-120 kDa for full-length protein) . Knockdown or knockout controls using morpholinos, RNAi, or CRISPR/Cas9-edited zebrafish provide critical negative controls; antibodies should show reduced or absent signal in these samples compared to wild-type. Immunoprecipitation followed by mass spectrometry verification can confirm that the antibody is specifically capturing DLGAP1 rather than cross-reacting with related proteins. For immunohistochemistry or immunofluorescence applications, researchers should verify that the subcellular localization pattern matches known distribution characteristics of DLGAP1, such as its documented centrosomal and cytoplasmic localization in certain cell types . Cross-reactivity testing against related DLGAP family proteins (DLGAP2-5) is essential to ensure specificity within this protein family. When commercially available antibodies lack sufficient zebrafish specificity, researchers may need to develop custom antibodies using zebrafish-specific peptide sequences that differ from other species and DLGAP family members. Batch-to-batch consistency testing is recommended for long-term studies, as antibody performance can vary between production lots. Multi-antibody verification using different antibodies targeting distinct epitopes of DLGAP1 provides additional confidence when consistent results are obtained.

What emerging technologies are likely to advance DLGAP1 research in zebrafish models?

Several cutting-edge technologies are poised to significantly advance DLGAP1 research in zebrafish models in the coming years. CRISPR-based technologies beyond gene knockout, such as CRISPRa (activation) and CRISPRi (interference), will enable precise temporal and spatial control of DLGAP1 expression, allowing researchers to study its function in specific tissues or developmental stages without permanent genetic modifications. Optogenetic and chemogenetic tools adapted for zebrafish systems will permit rapid and reversible manipulation of DLGAP1 activity or localization, facilitating the study of its dynamic functions in real-time within living organisms. Single-cell RNA sequencing technologies applied to zebrafish embryos and tissues will reveal cell type-specific expression patterns of DLGAP1 and its potential antisense transcript DLGAP1-AS1, providing insights into their roles in cellular differentiation and tissue development . Advanced protein imaging techniques, including lattice light-sheet microscopy and expansion microscopy, will offer unprecedented spatiotemporal resolution for visualizing DLGAP1 dynamics within cellular structures. Proteomics approaches such as BioID, APEX proximity labeling, or thermal proximity coaggregation (TPCA) will enable comprehensive mapping of DLGAP1 protein interaction networks in different cellular contexts. CRISPR-based lineage tracing methods will allow researchers to track the developmental trajectories of cells with altered DLGAP1 expression or function. Organ-on-chip technologies incorporating zebrafish cells could provide controlled microenvironments for studying DLGAP1 function in specific tissues under defined conditions, bridging the gap between in vitro and in vivo studies.

How might comparative studies between zebrafish and human DLGAP1 inform therapeutic applications?

Comparative studies between zebrafish and human DLGAP1 hold significant potential for informing therapeutic applications across multiple disease contexts. Structural comparisons of the protein domains between species can identify conserved functional regions that represent promising targets for drug development, particularly for conditions involving synaptic dysfunction where DLGAP1 plays critical roles. The zebrafish model offers a powerful system for high-throughput screening of small molecules that modulate DLGAP1 function, with rapid developmental timelines and optical transparency facilitating efficient phenotypic assessment of compound effects. Humanized zebrafish models, where the endogenous DLGAP1 gene is replaced with the human ortholog, can provide insights into human-specific functions and serve as platforms for testing therapeutics targeted at human DLGAP1. The role of DLGAP1 in megakaryocytic growth and differentiation suggests potential applications in hematological disorders, with zebrafish models offering accessible systems for investigating these processes . Comparative studies of DLGAP1 antisense transcripts between zebrafish and humans could reveal conserved regulatory mechanisms with therapeutic relevance, particularly given the established role of DLGAP1-AS1 in cancer progression through the miR-515-5p/MARK4 signaling pathway . The zebrafish's regenerative capabilities provide unique opportunities for studying DLGAP1's potential roles in tissue repair and regeneration, with implications for regenerative medicine approaches. Understanding species-specific differences in DLGAP1 regulation and function can help predict potential off-target effects or limitations of therapeutics developed based on zebrafish models when translated to human applications.

What are the most promising applications of DLGAP1 research in developmental biology and disease modeling?

DLGAP1 research in zebrafish presents numerous promising applications across developmental biology and disease modeling domains. In neurodevelopmental research, DLGAP1's role in synaptic organization makes it relevant for modeling conditions such as autism spectrum disorders, schizophrenia, and intellectual disabilities, where postsynaptic density proteins are frequently implicated. The protein's involvement in cell polarity regulation through the Scribble complex suggests applications in studying epithelial development and diseases involving disrupted tissue architecture, with zebrafish embryos providing accessible models for visualizing these processes in real time. DLGAP1's demonstrated functions in megakaryocytic growth and differentiation indicate potential applications in hematological disease modeling, with zebrafish blood cell development offering a well-characterized system for such investigations . The relationship between DLGAP1 and its antisense transcript DLGAP1-AS1, which has been implicated in cancer progression, points to applications in cancer biology research, with zebrafish xenograft models providing platforms for studying these interactions in vivo . DLGAP1's centrosomal localization and its regulation by cell cycle-related kinases suggest relevance for studying disorders of cell division and chromosomal instability . The protein's interactions with multiple signaling pathways, including those mediated by Jak2, SRC, and MAPK kinases, indicate applications in research on diseases involving dysregulated signal transduction . Zebrafish models with modified DLGAP1 expression could serve as valuable tools for drug discovery across these disease contexts, leveraging the organism's amenability to high-throughput screening approaches.

What are the key considerations for designing rigorous DLGAP1 research in zebrafish?

Designing rigorous DLGAP1 research in zebrafish requires careful attention to several methodological factors that can significantly impact experimental outcomes and interpretability. Strain selection and consistency represent fundamental considerations, as documented differences between AB and TL strains in baseline gene expression, HPI-axis activity, and behavioral responses can confound results if not properly controlled . Researchers should clearly report the specific strain used and maintain consistent strain usage throughout a study series. Genetic background verification through SNP analysis or whole-genome sequencing is advisable for long-maintained laboratory lines to account for potential genetic drift. Appropriate controls for gene manipulation experiments should include validation of knockdown or overexpression efficiency at both mRNA and protein levels, while considering potential compensatory mechanisms that might mask phenotypes. When studying DLGAP1 subcellular localization, researchers should account for its dynamic regulation by various kinases and cell cycle stages, implementing appropriate synchronization protocols or time-course analyses to capture these dynamics . Experimental design should incorporate both sexes and multiple developmental stages when relevant, as DLGAP1 functions may vary across development and between male and female zebrafish. Statistical approaches should be predetermined with appropriate power analyses and include plans for handling outliers and missing data. Transparent reporting of all methods, including husbandry conditions, water parameters, light cycles, and feeding regimens, is essential for reproducibility, as these factors can influence zebrafish physiology and potentially affect DLGAP1-related phenotypes.