lin-39 Antibody
Description
Introduction to LIN-39 Antibody
LIN-39 antibody is a specialized immunological tool developed to detect and study the LIN-39 protein, a Caenorhabditis elegans homolog of the Hox gene Deformed (Dfd) in Drosophila and mammalian HoxD4. This antibody has been instrumental in elucidating LIN-39's roles in vulval development, neuronal specification, and cell fate regulation .
Vulval Precursor Cell (VPC) Development
LIN-39 antibody has been critical in mapping LIN-39 expression during vulval morphogenesis:
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Prevention of Cell Fusion: LIN-39 represses the fusogen EFF-1 in VPCs (P(3–8).p) during larval stages, preventing fusion with hypodermal cells. Loss of LIN-39 leads to ectopic cell fusion, as shown via immunofluorescence .
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Regulation of Cell Proliferation: LIN-39 collaborates with Wnt signaling to promote vulval cell division. Antibody staining revealed LIN-39 expression in unfused VPCs, correlating with their mitotic activity .
Table 1: LIN-39 Antibody Applications in Vulval Studies
Cholinergic Motor Neurons (MNs)
LIN-39 antibody studies in the ventral nerve cord (VNC) demonstrated:
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Synergy with UNC-3 and MAB-5: LIN-39 cooperates with these transcription factors to activate cholinergic terminal identity genes (e.g., unc-129, acr-2). Depletion of LIN-39 reduces expression of these genes .
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Post-Developmental Role: Auxin-inducible LIN-39 degradation in adults led to loss of cholinergic markers, confirming its continuous requirement .
GABAergic Neurons
In VD/VC neurons, LIN-39 antibody staining revealed:
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Collaboration with UNC-30/PITX to activate GABAergic identity genes (e.g., ser-2, flp-11) .
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Direct binding to promoters of VD/VC-specific genes via ChIP-Seq .
Technical Validation and Experimental Use
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Specificity: LIN-39 antibody detects endogenous protein in nuclei of mid-body neurons and vulval muscles, validated in lin-39::GFP reporters .
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ChIP-Seq Utility: Used to identify LIN-39 binding sites in promoters of target genes (e.g., eff-1, vab-23), revealing conserved Hox-binding motifs .
Table 2: LIN-39 Antibody Validation Data
| Assay Type | Target Tissue | Outcome | Citation |
|---|---|---|---|
| Immunostaining | Vulval Muscles | Nuclear LIN-39 expression in adults | |
| ChIP-qPCR | VPCs and Neurons | Direct binding to eff-1 and vab-23 promoters |
Evolutionary Insights and Comparative Studies
LIN-39 antibody studies in Pristionchus pacificus highlighted functional divergence:
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In C. elegans, LIN-39 prevents VPC fusion, while in P. pacificus, it inhibits apoptosis in homologous cells .
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Regulatory sequence differences in lin-39 promoters underlie species-specific roles .
Research Limitations and Future Directions
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Antibody Cross-Reactivity: No data exists on cross-reactivity with other Hox proteins (e.g., MAB-5).
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Dynamic Expression: Temporal resolution of LIN-39 expression during larval transitions remains underexplored.
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
LIN-39 is a transcription factor that binds to the consensus 5'-TGATNNAT(G/T)(G/A)-3' PBC/Hox motif of target genes, thereby regulating their expression. It binds to the PBC/Hox motif within the lineage enhancer region of sem-2, promoting cell fate specification in the postembryonic mesoderm (M lineage). LIN-39 also regulates mig-13 expression, which controls the asymmetric distribution of the actin cytoskeleton-binding protein COR-1 in Q neuroblasts. This, in turn, governs the polarity of Q neuroblast migration and subsequent mid-body region-specific development. Furthermore, LIN-39, in conjunction with the orphan nuclear receptor NHR-25, regulates vulval precursor cell (VPC) differentiation.
LIN-39's roles in development are multifaceted, as evidenced by the following research findings:
- LIN-39 is a permissive factor maintaining vulval precursor cell cycle machinery expression, ensuring their proliferative competence. It also initiates lateral NOTCH signaling in adjacent VPCs, overcoming LIN-31 Forkhead-mediated cell cycle inhibition. (PMID: 27475488)
- MAB-5 plays a non-autonomous role in regulating QR and QL descendant migrations, a function partially masked by redundancy with LIN-39. EGL-5/Abdominal-B, another Hox gene, also appears to non-autonomously regulate Q descendant migrations. Elevated MAB-5 levels in body wall muscle (BWM) can influence the spon-1 promoter near Q cells. (PMID: 27225683)
- LIN-26 (hypodermal fate regulator) and ELT-6 (GATA factor) positively regulate lin-39 expression in embryonic VPC precursors. (PMID: 24885717)
- LIN-39 establishes neuroblast anterior polarity by activating mig-13, while MAB-5 inhibits this polarity by suppressing lin-39 and mig-13 expression. (PMID: 23784779)
- LIN-39 and EFL-3 cell-type specifically regulate egl-1 (BH3-only cell death gene) transcription and cell fate determination. (PMID: 21596899)
- lin-39 transcriptional upregulation is dependent on Ras signaling. (PMID: 16412617)
- LIN-39 and CEH-20 (downstream of RTK/Ras and Wnt pathways) integrate signals to the LIN-12/Notch cascade, regulating vulval cell fates. (PMID: 17084835)
- The GLI protein family influences somatic cell fates by regulating the expression of Hox genes, including lin-39. (PMID: 19361495)
Q&A
Basic Research Questions
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What experimental systems are best suited for studying LIN-39 antibody specificity in C. elegans?
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Use transgenic lines with reporter constructs (e.g., lin-39::GFP fusions) to visualize endogenous LIN-39 expression patterns. For example, a 10-kb regulatory region in construct pKG1 replicates native expression in the ventral midbody and vulval muscles, while deletions (e.g., pKG2) reveal ectopic expression patterns .
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Validate antibody specificity via immunofluorescence co-localization with GFP-tagged LIN-39 in vulval precursor cells (VPCs) .
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How do I validate LIN-39 antibody performance across developmental stages?
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Compare antibody staining with transcriptional reporters in embryos, larvae, and adults. Weak expression in adult vulval muscles (observed in pKG2 deletions) suggests regulatory elements downstream of the transcription start site influence stage-specific expression .
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Use loss-of-function mutants (e.g., lin-39(n1760)) as negative controls to confirm antibody signal absence .
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Advanced Research Questions
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How do conflicting data on LIN-39 regulation by Wnt vs. Ras signaling arise, and how can they be resolved?
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Experimental design: Employ dual-pathway inhibition (e.g., bar-1/β-catenin mutants for Wnt, mpk-1 RNAi for Ras/MAPK). Transcriptional upregulation of lin-39::GFP in P6.p is Ras-dependent but Wnt-independent .
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Data contradiction: Ectopic expression in pKG2 constructs (no promoter) suggests baseline regulatory activity downstream of Wnt. Use quantitative imaging to distinguish pathway-specific effects .
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What methodologies identify LIN-39 interaction partners in vulval development?
Methodological Challenges and Solutions
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How to address low signal-to-noise ratios in LIN-39 antibody staining?
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What computational tools predict LIN-39 binding sites in non-coding regulatory regions?
