S100A4 Mouse

What is the molecular structure of mouse S100A4 and how does it compare to other species?

Mouse S100A4 is a 12 kDa protein consisting of 101 amino acids with two calcium-binding EF-hand domains located at amino acid positions 12-47 and 50-85. The protein shares high sequence homology with other mammalian species, specifically 96% with rat, 93% with human, and 90% with bovine S100A4 . This high conservation across species indicates the protein's fundamental biological importance.

When conducting cross-species studies, researchers should note that despite this high homology, species-specific differences may still affect protein-protein interactions, particularly in the variable regions outside the calcium-binding domains.

What is the typical expression pattern of S100A4 in normal mouse tissues?

Contrary to earlier assumptions that S100A4 expression is restricted to fibroblasts, the protein is expressed in multiple cell types. S100A4 is found in monocytes, macrophages, T lymphocytes, and various epithelial cells . It is also robustly expressed in differentiating fiber cells of the ocular lens .

For experimental design, it's important to use multiple antibody validation techniques when studying S100A4 expression patterns. Cross-reactivity with other S100 family members can be an issue, so researchers should employ both monoclonal and polyclonal antibodies alongside mRNA expression analysis to confirm protein expression in specific cell types .

How do researchers generate and confirm S100A4 knockout mouse models?

S100A4 knockout (S100A4−/−) mice have been established through standard gene targeting approaches. Confirmation of knockout should involve both genotyping and protein expression analysis. Researchers should verify absence of S100A4 using:

• PCR-based genotyping

• Western blot analysis of tissues known to express S100A4

• Immunohistochemistry of multiple tissues

• RT-qPCR to confirm absence of S100A4 mRNA

When interpreting phenotypes, researchers should be aware that S100A4-deficient mice typically develop normally but may exhibit late-onset phenotypes such as cortical cataracts . Additionally, these mice display compromised immune responses to mucosal antigens, indicating the importance of S100A4 in immune function .

How does S100A4 expression in mouse models correlate with metastatic potential?

S100A4 expression levels directly correlate with metastatic potential in mouse tumor models. In transgenic mouse studies, animals overexpressing S100A4 showed no increase in primary tumor formation but displayed markedly increased frequency of lung metastasis when crossed with mice harboring a tumorigenic background .

For metastasis studies, researchers should implement:

• Quantitative assessment of S100A4 expression levels using RT-qPCR and western blotting

• Immunohistochemical analysis comparing primary tumors with metastatic lesions

• In vivo imaging techniques to track metastatic spread in real-time

• Comparison of S100A4 expression in matched primary and metastatic samples

The metastasis-promoting effect of S100A4 has been shown to be independent of primary tumor development, suggesting separate molecular mechanisms for tumorigenesis versus metastatic spread .

What are effective approaches to target S100A4 in mouse cancer models?

Researchers investigating S100A4 as a therapeutic target in mouse cancer models can employ several strategies:

• Genetic approaches:

  • Conditional knockout systems (Cre-loxP) to study tissue-specific effects

  • siRNA or shRNA knockdown for transient suppression

  • CRISPR/Cas9 genome editing for permanent modification

• Pharmacological approaches:

  • Small molecule inhibitors that disrupt calcium binding

  • Peptide inhibitors targeting S100A4-protein interactions

  • Antibodies against extracellular S100A4

• Evaluation methods:

  • Primary tumor growth measurements

  • Quantification of metastatic burden

  • Survival analysis comparing intervention groups

  • Molecular pathway analysis to confirm mechanism of action

When designing these studies, researchers should consider both intracellular and extracellular functions of S100A4, as the protein operates through multiple mechanisms depending on its cellular location .

How does S100A4 deficiency affect lens development and what are the molecular mechanisms involved?

S100A4 knockout mice develop late-onset cortical cataracts, indicating an essential role in lens homeostasis. Transcriptome profiling of S100A4−/− lenses reveals molecular mechanisms characterized by:

• Aberrant upregulation of photoreceptor and Müller glia-specific genes

• Increased expression of the olfactory sensory neuron-specific gene S100A5

• Altered histone methylation patterns, specifically changes in trimethylated H3K27 and H3K4

Research methodologies to investigate these mechanisms should include:

• RNA-seq analysis of lens tissue at multiple developmental stages

• ChIP-seq to map histone modification patterns

• Immunohistochemical verification of aberrantly expressed proteins

• Lens clarity measurements with age progression

Pathway analysis of differentially expressed genes in S100A4−/− lenses has identified Crx and Nrl transcription factors as significant upstream regulators, suggesting that S100A4 suppresses retinal gene expression during lens differentiation through epigenetic mechanisms .

What techniques are recommended for studying S100A4's role in regulating gene expression in the eye?

When investigating S100A4's role in regulating gene expression in ocular tissues, researchers should employ:

• Epigenetic analysis techniques:

  • ChIP-seq for histone modifications (particularly H3K27me3 and H3K4me3)

  • ATAC-seq to assess chromatin accessibility

  • DNA methylation analysis of CpG islands in promoters of affected genes

• Transcription factor analysis:

  • ChIP-PCR for specific transcription factors (Crx, Nrl) identified in pathway analyses

  • Electrophoretic mobility shift assays to confirm protein-DNA interactions

  • Reporter gene assays to verify functional effects on transcription

• Tissue-specific analyses:

  • Laser capture microdissection to isolate specific cell populations

  • Single-cell RNA-seq to detect cell-type-specific effects

  • In situ hybridization to localize mRNA expression patterns

The discovery that many upregulated genes in S100A4−/− lenses possess promoters with high-density CpG islands bearing specific trimethylation marks suggests that S100A4 may regulate gene expression through interaction with epigenetic machinery .

What evidence supports S100A4's potential as a mucosal adjuvant in mouse models?

Recent research has identified S100A4 as a potent mucosal adjuvant with several advantageous properties:

• Comparable or superior efficacy to cholera toxin (CT), a standard mucosal adjuvant

• No adverse reactions observed after administration

• Ability to elicit antigen-specific antibody responses that persist for over 6 months

• Induction of pulmonary cytotoxic T cell responses

• No stimulation of antibodies against itself (self-tolerance as an adjuvant)

Experimental evidence shows that intranasal immunization with recombinant S100A4 and model antigens (ovalbumin) or clinically relevant antigens (SARS-CoV-2 spike protein) resulted in:

• Robust antigen-specific IgG (total, IgG1, IgG2c) responses in serum

• Strong mucosal IgA responses in lung exudate, broncho-alveolar lavage fluid, vaginal lavage, and feces

• Sustained antibody responses for more than 6.5 months

What mechanisms underlie S100A4's adjuvant activity and how can researchers assess them?

S100A4's adjuvant activity appears to operate through several mechanisms that can be assessed using specific research techniques:

• Prolonged antigen residence:

  • Real-time fluorescence optical imaging with fluorescently labeled antigens

  • Quantification of antigen retention in nasal tissue over time

• Enhanced antigen presentation:

  • Flow cytometry to track migration of antigen-presenting cells

  • Assessment of dendritic cell activation markers

  • T cell activation assays using S100A4-pulsed dendritic cells

• Germinal center induction:

  • Microscopic examination of lymphoid tissues

  • Novel label-free MALDI-TOF mass spectrometry for measuring germinal center activity

  • Flow cytometric analysis of germinal center B cells

• Safety assessment:

  • Histological examination of olfactory bulb for inflammation

  • Behavioral testing to assess olfactory function

  • Long-term monitoring for adverse effects

Notably, S100A4-deficient mice show severely compromised antigen-specific immune responses, which can be restored by engrafting wild-type dendritic cells, highlighting the importance of S100A4 expression in antigen-presenting cells for effective adaptive immune responses .

How can researchers effectively track S100A4-mediated cellular responses in real-time?

For real-time tracking of S100A4-mediated cellular responses, researchers should consider these advanced methodologies:

• Live-cell imaging approaches:

  • Fluorescent protein tagging of S100A4 (GFP, mCherry) for subcellular localization

  • FRET-based sensors to detect S100A4 protein-protein interactions

  • Calcium imaging to correlate S100A4 activity with calcium fluctuations

• In vivo tracking systems:

  • Intravital microscopy to observe S100A4-expressing cells in living tissues

  • Bioluminescence imaging of S100A4 promoter activity using luciferase reporters

  • PET or SPECT imaging with radiolabeled antibodies against S100A4

• Molecular dynamics:

  • Photoactivatable or photoconvertible S100A4 fusions to track protein movement

  • FLIP/FRAP techniques to measure protein mobility and turnover

  • Optogenetic control of S100A4 expression for temporal studies

The combination of these approaches allows for comprehensive understanding of both spatial and temporal aspects of S100A4 function in complex biological systems.

What are the most sensitive methods for detecting low levels of S100A4 expression in mouse tissues?

For detecting low-abundance S100A4 expression, researchers should employ these highly sensitive techniques:

• Nucleic acid detection:

  • Digital droplet PCR for absolute quantification of S100A4 mRNA

  • RNAscope in situ hybridization for single-molecule detection in tissue sections

  • Single-cell RNA-seq to identify rare S100A4-expressing cell populations

• Protein detection:

  • Highly-sensitive ELISA with signal amplification

  • Proximity ligation assay for in situ protein detection

  • Mass spectrometry with targeted multiple reaction monitoring

  • Immunoprecipitation followed by western blot for enrichment of low-abundance protein

• Functional assays:

  • Reporter cell lines with amplified readouts (e.g., luciferase) driven by the S100A4 promoter

  • CRISPR activation systems to enhance detection of endogenous S100A4 expression

  • Calcium-binding assays to detect functional S100A4 in cell/tissue lysates

When using these techniques, appropriate controls are essential, including S100A4 knockout tissues as negative controls and known high-expressing tissues as positive controls.