Vertebrate ancient opsin Antibody

What is Vertebrate Ancient (VA) opsin and what is its evolutionary significance?

Vertebrate Ancient (VA) opsin belongs to the G-protein coupled receptor family within the opsin subfamily. First isolated from Atlantic salmon and subsequently found in numerous non-mammalian vertebrates, VA opsin represents one of the most ancient photoreceptive molecules in the vertebrate lineage . Recent comprehensive genomic surveys have identified 291 VA opsin genes from 246 non-mammalian species, showing high conservation among non-mammals but complete absence in mammalian genomes .

VA opsin functions as a green-sensitive photopigment with maximum absorption at approximately 500 nm when bound to 11-cis-retinal . Evolutionarily, VA opsin emerged before many other visual opsins, and its conservation suggests critical functions in non-visual photoreception throughout vertebrate evolution . The widespread distribution of VA opsin in the deep brain of diverse vertebrates indicates an ancient photoreception system that likely predated the evolution of lateral eyes .

What is the difference between VA and VAL opsin?

VA and VAL (VA-Long) opsin are alternative splicing variants encoded by the same gene. They share a common core sequence in the membrane-spanning domains (M1-Q303) but differ significantly in their C-terminal cytoplasmic tails . This boundary between the common and isoform-specific regions corresponds exactly to a conserved splice site found among vertebrate opsin genes .

The functional difference between these variants is substantial. VAL-opsin with bound 11-cis-retinal forms a functional green-sensitive photopigment (λmax ~500 nm), whereas VA-opsin exhibits no photosensitivity even in the presence of 11-cis-retinal . This difference likely results from the C-terminal region's role in proper protein folding and stability, as the proximal region of the C-terminal tail has been shown to play a critical role in the proper folding of rhodopsin .

Where is VA opsin expressed in vertebrates?

VA opsin exhibits specific expression patterns across different vertebrate tissues:

Brain Expression:

• In zebrafish, functionally active VAL-opsin is localized in a limited number of cells surrounding the diencephalic ventricle of the central thalamus, distributed over approximately 200 μm along the rostrocaudal axis .

• In Atlantic salmon, VA opsin is strongly expressed in the pineal organ and in bilateral columns of subependymal cells in the epithalamus .

• In birds, VA opsin is found in the hypothalamus, where it may regulate circadian and reproductive responses to photoperiod .

Retinal Expression:

• VAL-opsin is expressed in a subset of non-GABAergic horizontal cells in the zebrafish retina .

• The VAL-opsin-expressing horizontal cells are regularly distributed in the retinal layer with a dorsoventral gradient (stronger expression in the dorsal retina) .

Studies have confirmed VA opsin expression in fish, amphibians, reptiles, and birds, but not in mammals, which appear to have lost this opsin during evolution .

What is the function of VA opsin in vertebrates?

VA opsin primarily functions in non-visual photoreception, serving several physiological roles:

The multiple sites of expression (brain, pineal, retina) suggest that VA opsin serves as a versatile photosensory molecule that contributes to various aspects of light-dependent physiology in non-mammalian vertebrates .

How can I distinguish between VA and VAL opsin in immunohistochemistry?

To differentiate between VA and VAL opsin in immunohistochemical studies, researchers should use antibodies targeting distinct regions of these proteins:

• C-terminal Specific Antibodies: Develop antibodies (like the "VC antibody") against the C-terminal tail unique to VAL-opsin (Q303-M377) . These antibodies will detect only VAL-opsin and not VA-opsin, as demonstrated in immunoblot experiments where VC antibody bound to recombinant VAL-opsin (~50 kDa) but not to VA-opsin .

• N-terminal Common Antibodies: Use antibodies (like the "VN antibody") targeting the N-terminal region (M1-S31) common to both variants . These will detect both VA and VAL opsins.

• Comparative Analysis: Compare staining patterns between adjacent sections using both antibody types. Regions positive for both VN and VC antibodies contain VAL-opsin, while areas positive for VN but negative for VC antibodies may contain VA-opsin .

• Western Blot Verification: Confirm the distinction through Western blotting, where VAL-opsin resolves at approximately 50 kDa compared to the smaller VA-opsin .

How can I validate the specificity of a VA opsin antibody?

Rigorous validation of VA opsin antibodies requires multiple complementary approaches:

Biochemical Validation:

• Perform Western blotting on tissues known to express VA opsin (fish brain/retina) to confirm detection of proteins at the expected molecular weight (~50 kDa for VAL-opsin) .

• Test antibodies on recombinant VA and VAL opsins expressed in heterologous systems (e.g., 293S cells) to verify specific recognition .

Immunohistochemical Controls:

• Include pre-absorption controls by incubating the antibody with the immunizing peptide before tissue application; this should eliminate specific staining .

• Replace primary antibody with preimmune serum or IgG fraction at equivalent concentration as negative controls .

• Use multiple antibodies targeting different epitopes of VA opsin to confirm consistent staining patterns .

Cross-Validation:

• Compare immunohistochemical results with in situ hybridization for VA opsin mRNA.

• Verify cell-type specificity through co-localization studies with established cell markers (e.g., GAD for GABAergic neurons) .

What tissues should I include as positive and negative controls for VA opsin immunostaining?

Positive Control Tissues:

Negative Control Tissues:

• Mammalian brain tissues (mammals lack VA opsin)

• GABAergic horizontal cells in zebrafish retina (shown to be VAL-negative)

• Muscle or other non-neural tissues from the same species

• Zebrafish central brain regions outside the thalamus

Process all control tissues alongside experimental samples using identical protocols to ensure valid comparisons.

What are the optimal tissue preparation and immunohistochemistry protocols for VA opsin detection?

Based on successful studies, the following protocol elements are recommended for VA opsin detection:

Tissue Preparation:

• Fix tissues in 4% paraformaldehyde in phosphate buffer (pH 7.4) for 12-24 hours at 4°C .

• Cryoprotect in sucrose solution (15-30%) until tissues sink.

• Embed in OCT compound and prepare cryosections (10-20 μm thickness) .

Immunohistochemistry Protocol:

• Pretreat sections with blocking solution containing 1.5% normal serum and 0.3% Triton X-100 in PBS .

• Incubate with primary antibody (VC antibody at 0.6 μg/ml or VN antibody at 0.4 μg/ml) diluted in blocking solution for 3 days at 4°C .

• Rinse thoroughly with PBS.

• Incubate with appropriate secondary antibody (e.g., horse anti-mouse IgG conjugated with FITC or Texas Red) for 20 hours at 4°C .

• Wash with PBS.

• Counterstain nuclei with TO-PRO-3 (0.2 μM) if desired .

• Mount sections using a 1:1 mixture of 50% glycerol in PBS and Vectashield Mounting Medium .

This extended incubation approach has proven effective for detecting the relatively low abundance VA opsin in brain and retinal tissues.

How can I co-localize VA opsin with other markers in the same tissue section?

For successful co-localization studies with VA opsin:

• Multiple Immunofluorescence:

  • Use primary antibodies from different host species (e.g., mouse anti-VA opsin with rabbit anti-GAD) .

  • Apply species-specific secondary antibodies conjugated to spectrally distinct fluorophores (FITC, Texas Red) .

• Sequential Staining Protocol:

  • For primary antibodies from the same species, use sequential staining with complete blocking between rounds.

  • Consider direct conjugation of one primary antibody to eliminate cross-reactivity issues.

• Imaging and Analysis:

  • Use confocal microscopy with appropriate controls for spectral overlap.

  • In zebrafish retina studies, successful co-localization of VA opsin and GAD demonstrated that VA opsin-expressing horizontal cells are distinct from GABAergic horizontal cells .

• Alternative Approaches:

  • Combine immunohistochemistry for VA opsin with in situ hybridization for other markers when antibody compatibility is challenging.

  • For triple labeling, include a nuclear counterstain like TO-PRO-3 to provide cellular context .

How do I troubleshoot weak or non-specific staining with VA opsin antibodies?

For Weak Staining:

• Extend primary antibody incubation time (e.g., 3 days at 4°C as used in successful zebrafish studies) .

• Optimize detergent concentration for better penetration (0.3% Triton X-100 proved effective) .

• Consider antigen retrieval if epitopes may be masked by fixation.

• Use signal amplification systems (avidin-biotin complex or tyramide signal amplification).

• Ensure tissue sections are sufficiently thin (10-20 μm) for adequate antibody penetration .

For Non-specific Staining:

• Increase blocking reagent concentration (use 1.5-3% normal serum) .

• Perform more stringent washing (longer duration, more frequent changes).

• Pre-absorb antibody with tissues known to cause cross-reactivity.

• Test multiple antibodies targeting different epitopes of VA opsin.

• Verify secondary antibody specificity by omitting primary antibody entirely .

Common Pitfalls:

• Over-fixation can mask epitopes.

• Insufficient blocking leads to high background.

• Inappropriate detergent concentration affects membrane protein accessibility.

• Cross-reactivity with other opsins due to sequence similarities.

Are there cross-reactivity issues with other opsins when using VA opsin antibodies?

Cross-reactivity is a significant concern with VA opsin antibodies due to sequence similarities within the opsin family. The opsin superfamily includes visual opsins (rhodopsin and cone opsins) and non-visual opsins (pinopsin, parapinopsin, parietopsin, and the newly discovered QB opsin) .

To address potential cross-reactivity:

• Epitope Selection: Target unique regions like the C-terminal tail of VAL-opsin, which shows minimal sequence homology to other opsins . The VC antibody region (Q303-M377) was specifically chosen for this reason .

• Sequence Analysis: Perform sequence alignment between your target VA opsin and other opsins expressed in the same tissues to identify potential cross-reactive epitopes .

• Validation Experiments:

  • Western blot analysis should show a single band of appropriate size (~50 kDa for VAL-opsin) .

  • Pre-absorption controls with peptides from both VA opsin and related opsins can reveal cross-reactivity.

  • Parallel staining with established antibodies against other opsins (rod opsin, cone opsins) helps identify overlap .

The search results indicate that properly validated VA opsin antibodies show distinct staining patterns from other opsin antibodies in salmon brain, suggesting specificity can be achieved with careful antibody selection .

What are the evolutionary implications of VA opsin distribution across vertebrate species?

The evolutionary distribution of VA opsin provides key insights into vertebrate photoreception evolution:

Phylogenetic Distribution:

• VA opsin is highly conserved among non-mammalian vertebrates but absent in mammals, suggesting selective loss in the mammalian lineage .

• Recent comprehensive surveys identified 291 VA opsin genes from 246 non-mammalian species, demonstrating remarkable conservation .

• Paralogs resulting from whole genome duplications in teleost fishes (VA.a and VA.b) have been retained, suggesting important non-redundant functions .

Evolutionary Implications:

• Ancient Origin: VA opsin likely existed in the common ancestor of all vertebrates, as evidenced by its presence in lampreys and conservation across diverse lineages .

• Functional Specialization: The expression of VA opsin in deep brain photoreceptors across diverse species indicates an evolutionarily conserved non-visual photoreception system predating lateral eyes .

• Alternative Splicing: The VA/VAL splicing system represents the first example of alternative splicing generating opsin isoforms with distinct amino acid sequences and functional properties, revealing an evolutionary mechanism for photopigment diversification .

• Comparative Distribution: The simultaneous presence of VA opsin with other non-visual opsins in similar brain regions across species suggests evolutionary conservation of deep brain photoreception networks .

This evolutionary distribution pattern indicates that non-visual photoreception was a fundamental capability in early vertebrates that has been selectively maintained or modified across different lineages.