HES7 Antibody
Description
Definition and Overview of HES7 Antibody
The HES7 antibody is a specialized immunological tool targeting the HES7 protein, a member of the hairy and enhancer of split (HES) family of basic helix-loop-helix (bHLH) transcription factors. HES7 functions as a transcriptional repressor critical for somitogenesis, the process of embryonic segmentation that forms the vertebral column and associated tissues . Antibodies against HES7 enable researchers to study its expression, localization, and regulatory roles in developmental biology and disease models.
Protein Features
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Gene ID: 84667 (Human), with orthologs in mouse (85% sequence identity) .
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Domains: Contains bHLH motifs for DNA binding and dimerization, and WRPW motifs for transcriptional repression .
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Post-Translational Modifications: Degraded via the ubiquitin-proteasome system, with a half-life of ~23 minutes in fibroblasts and <20 minutes in presomitic mesoderm (PSM) .
Antibody Immunogen
Common immunogen sequences include PPPPHSQDGAPKAPLPPPPAFWRPWP, corresponding to residues in the human HES7 protein .
Applications of HES7 Antibodies
HES7 antibodies are validated for:
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Immunohistochemistry (IHC): Detects HES7 protein waves in the PSM during somitogenesis .
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Chromatin Immunoprecipitation (ChIP): Confirms HES7 binding to its own promoter and Lfng regulatory regions .
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Immunofluorescence (IF): Visualizes cyclic expression patterns in mouse embryos .
Role in Somitogenesis
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Cyclic Expression: HES7 protein oscillates in the PSM with a 2-hour periodicity, driving the segmentation clock .
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Negative Feedback: HES7 represses its own transcription and Lfng expression, essential for synchronized somite formation .
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3'UTR Dependency: Knock-in mice lacking Hes7 3'UTR show disrupted mRNA stability, reduced protein levels, and somite defects .
Mechanistic Insights
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Ubiquitination: HES7 degradation is mediated by the ubiquitin-proteasome system, confirmed via cycloheximide chase and ubiquitination assays .
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Transcriptional Control: Wnt/β-catenin and Tbx6 pathways regulate Hes7 promoter activity, with Tbx6 binding critical for PSM-specific expression .
Validation and Quality Control
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Specificity: Validated in Hes7-null mice, showing no cross-reactivity .
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Functional Assays: Antibodies used in ChIP confirmed in vivo binding to Hes7 and Lfng promoters .
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Consistency: Multiple vendors report >85% antigen sequence identity across human, mouse, and rat orthologs .
Importance in Developmental Biology
HES7 antibodies have been pivotal in elucidating:
Product Specs
**Constituents:** 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Mutations in HES7 have been specifically linked to defects in vertebral, heart, and neural tube development. This observation offers a valuable diagnostic tool for patients with Spondylocostal Dysostosis (SCD) and provides guidance for molecular genetic testing. PMID: 23897666
- Research suggests that the genes MESP2, HES7, and DUSP6 may not be involved in the etiopathogenesis of sporadic and non-syndromic Congenital Scoliosis in the Chinese Han population. PMID: 22744456
- Two novel missense mutations in HES7 have been identified in a family exhibiting Spondylocostal Dysostosis. PMID: 20087400
- The R25W missense mutation within HES7 has been established as a causative factor for Spondylocostal Dysostosis. PMID: 18775957
Q&A
What are the primary applications of the HES7 antibody in developmental biology?
The HES7 antibody is primarily used to study the dynamic expression patterns of the HES7 protein during somitogenesis. Whole-mount immunostaining techniques have demonstrated its specificity in detecting HES7 protein within the presomitic mesoderm (PSM) but not in other regions, such as formed somites . This specificity allows researchers to correlate protein localization with mRNA expression domains, providing insights into transcriptional and post-transcriptional regulation mechanisms. Additionally, the antibody facilitates studies on protein stability and degradation pathways, which are crucial for understanding the oscillatory nature of HES7 expression .
How is the specificity of the HES7 antibody validated?
The specificity of the HES7 antibody is validated through multiple approaches. Immunostaining experiments have shown that the antibody reacts specifically with HES7 protein in wild-type embryos but not in HES7-null mutants . Furthermore, Western blot analysis using hemagglutinin (HA)-tagged recombinant HES7 protein confirms its ability to detect polyubiquitinated forms of the protein . Such validations ensure that the antibody accurately represents native protein levels and modifications.
What experimental techniques are commonly used with the HES7 antibody?
The most common techniques include whole-mount immunostaining, Western blotting, chromatin immunoprecipitation (ChIP), and multiplex hybridization chain reaction (HCR) imaging . These methods allow researchers to visualize protein localization, assess stability and degradation pathways, analyze promoter interactions, and quantify gene expression levels within complex tissues. For example, ChIP analysis has demonstrated that HES7 protein interacts with its own promoter region as well as with other cyclic gene promoters like Lfng .
How does post-translational modification affect HES7 protein stability?
Post-translational modifications significantly influence HES7 protein stability. Studies have shown that its half-life is approximately 23 minutes under normal conditions but can be extended when proteasome inhibitors are applied . The ubiquitin-proteasome system plays a central role in degrading HES7 protein efficiently within the PSM, ensuring its oscillatory expression aligns with somitogenesis cycles .
What are the challenges in detecting oscillatory expression patterns of HES7?
Detecting oscillatory expression patterns requires precise timing and sensitivity due to the rapid turnover of both mRNA and protein. Techniques such as time-lapse imaging combined with intron-specific probes have been employed to capture real-time transcriptional activity . Additionally, multiplex imaging methods like hybridization chain reaction (HCR) provide high-resolution spatial data on mRNA distribution within embryonic tissues .
How does intronic delay contribute to oscillatory expression of HES7?
Intronic delay is a critical factor in maintaining oscillatory gene expression. The presence of introns slows down transcriptional processing, thereby introducing a temporal lag between transcription initiation and mRNA maturation . This delay ensures synchronization between transcriptional cycles and protein degradation rates, which is essential for periodic repression by bHLH factors like Hes7 .
What role does Fgf4 play in regulating HES7 levels during somitogenesis?
Fgf4 has been identified as a key regulator of HES7 levels within the PSM. Experimental data show that Fgf4 mutants exhibit reduced intensity and altered distribution of HES7 mRNA spots compared to controls . This reduction disrupts normal segmentation patterns, leading to vertebral defects. Advanced imaging techniques such as multiplex HCR have been instrumental in quantifying these changes at subcellular resolution .
How can chromatin immunoprecipitation (ChIP) be used to study promoter interactions involving HES7?
ChIP analysis using anti-HES7 antibodies enables researchers to identify direct interactions between the HES7 protein and its promoter region in vivo . This technique has revealed that stabilized forms of Hes7 interact with cyclic gene promoters like Lfng, influencing transcriptional activity during segmentation cycles . Such findings highlight the dual role of Hes7 as both a transcriptional repressor and an activator under specific conditions.
What experimental models are available for studying mutations affecting HES7 function?
Various genetic models have been developed to study mutations impacting HES7 function. For instance, hypomorphic Wnt3a mutants demonstrate downregulation of Hes7 expression due to impaired Notch signaling pathways . Additionally, null alleles for Hes7 provide a baseline for validating antibody specificity and assessing gene function during embryonic development .
How do contradictory data regarding Notch signaling influence interpretations of Hes7 regulation?
Contradictory findings regarding Notch signaling necessitate careful experimental design and data analysis. While some studies suggest that Rbpj binding sites are essential for Hes7 promoter activation, others indicate posterior Hes7 expression occurs independently of Notch signaling . Researchers must consider factors such as promoter construct designs, cellular context, and compensatory pathways when interpreting these results.
Experimental Design Considerations
When designing experiments involving the HES7 antibody, researchers should prioritize techniques that provide temporal resolution and spatial accuracy. Time-lapse imaging combined with intron-specific probes offers valuable insights into transcriptional dynamics . Similarly, multiplex imaging methods like hybridization chain reaction (HCR) enable simultaneous visualization of multiple gene expression domains within intact tissues .
Data Analysis Strategies
Quantitative analysis tools such as Imaris modeling software are essential for interpreting complex datasets generated from imaging experiments . These tools allow researchers to create volumetric models based on fluorescence intensity thresholds, facilitating precise quantification of gene expression levels across different developmental stages.
Addressing Data Contradictions
To resolve contradictions in data related to Hes7 regulation, researchers should employ complementary approaches such as luciferase reporter assays and ChIP analysis . Combining these methods provides a more comprehensive understanding of promoter activity and protein-DNA interactions under varying experimental conditions.
