CDHR5 Mouse

Basic Research Questions

• What is CDHR5 and what is its fundamental role in mouse intestinal epithelium?

CDHR5 is a transmembrane cell adhesion protein localized to the microvillar brush border of cholangiocytes, colonocytes, enterocytes, and kidney epithelial cells. In mouse intestinal epithelium, it forms bridges between microvilli, contributing to proper brush border assembly . CDHR5 knockout studies have demonstrated its importance in maintaining intestinal tissue homeostasis, with CDHR5-deficient mice exhibiting specific intestinal phenotypes including shortened microvilli, mislocalization of brush border proteins, mucosal barrier defects, and secretory hyperplasia . Methodologically, immunohistochemistry and RT-qPCR can be used to detect and quantify CDHR5 expression in mouse tissues.

• How does CDHR5 expression differ between mouse and human tissues?

While CDHR5 is expressed in similar epithelial cell types across species, important structural differences exist. Mouse CDHR5 contains a mucin-like repeat (MLR) domain with a 3X tandem threonine-serine-proline (TSP) repeat sequence with relatively low repeat identity . In contrast, human CDHR5 has a 4X tandem repeat sequence of 31 amino acids rich in threonine, serine, and proline residues . These structural variations may influence binding properties and signaling outcomes across species. Researchers can use sequence alignment tools and structural prediction software to analyze these differences when designing cross-species studies.

• What phenotypes are observed in CDHR5-deficient mice?

CDHR5-deficient mice (CDHR5 Δ/Δ) are viable without obvious intestinal pathology but exhibit several distinct intestinal phenotypes :

• Shortening of microvilli

• Mislocalization of brush border proteins

• Mucosal barrier defects

• Secretory cell hyperplasia

When challenged with AOM/DSS (azoxymethane/dextran sodium sulfate) carcinogenesis protocol, these mice show increased tumor burden compared to wild-type counterparts . Researchers can generate CDHR5-deficient models using CRISPR/Cas9 gene editing or homologous recombination in embryonic stem cells, followed by verification through immunohistochemistry and RT-qPCR.

• How is CDHR5 involved in brush border assembly in mouse intestinal epithelium?

CDHR5 contributes to brush border assembly through its participation in the intermicrovillar adhesion complex (IMAC) . The cytoplasmic tail of CDHR5 is critical for its correct apical targeting and functional properties . CDHR5 associates with EBP50 (ezrin-radixin-moesin binding phosphoprotein 50), which helps stabilize the apical membrane and proper localization of brush border proteins . Loss of these interactions disrupts brush border assembly, resulting in reduced apical IMAC levels . Experimentally, this can be observed using immunofluorescence microscopy to track protein localization and electron microscopy to visualize ultrastructural changes in the brush border.

Intermediate Research Questions

• What is the relationship between CDHR5 and microvilli dynamics in mouse models?

CDHR5 plays a crucial role in stabilizing microvilli at epithelial cell boundaries. Research has shown that microvilli at cell margins exhibit constrained movement compared to medial microvilli, with trajectories sampling less than 2 μm² area over time versus up to 6 μm² for medial microvilli . Mean squared displacement analysis confirms that marginal microvilli are approximately 10-fold more constrained in their movement . CDHR5 contributes to this stability through transjunctional adhesion complexes that link microvilli from neighboring cells . To study these dynamics, researchers can employ live cell imaging with fluorescently tagged CDHR5 and track microvillar movement using particle tracking algorithms.

• How does CDHR5 expression affect tumor development in mouse models of colorectal cancer?

CDHR5 functions as a tumor suppressor in colorectal cancer. CDHR5-deficient mice subjected to the AOM/DSS chemical carcinogenesis protocol develop tumors with characteristics of invasive carcinomas :

• Enhanced cancer stemness

• Nuclear accumulation of activated Stat3 and β-Catenin

• Epithelial-to-mesenchymal transition (EMT)

• Upregulation of the Stat3/β-Catenin target Survivin

• Reduction in p21 expression (which is repressed by β-Catenin)

These findings demonstrate that CDHR5 inhibits colorectal tumor progression. Researchers can evaluate these markers using immunohistochemistry, Western blotting, and RT-qPCR to assess CDHR5's impact on oncogenic signaling pathways.

• What signaling pathways interact with CDHR5 in mouse intestinal tissue?

CDHR5 functions as a node connecting multiple signaling pathways:

CDHR5-deficient mice show increased numbers of secretory cells in the intestine and in tumors, characteristic of reduced Notch signaling . In silico analyses have confirmed a positive correlation between CDHR5 and major Notch targets at the RNA level in human cancers . These molecular changes can be investigated using chromatin immunoprecipitation (ChIP) to identify direct interactions with transcription factors and RNA-seq to analyze transcriptional networks.

• How do CDHR5 knockout mice respond to chemically-induced colorectal cancer?

When CDHR5-deficient mice are treated with the AOM/DSS carcinogenesis protocol, they develop significantly more tumors compared to wild-type controls . These tumors display characteristics of invasive carcinomas with enhanced cancer stemness markers and EMT features . The tumors also show nuclear accumulation of activated Stat3 and β-Catenin, along with increased expression of their target gene Survivin and decreased expression of p21 . This model provides strong evidence for CDHR5's tumor suppressive function in colorectal cancer. Researchers can quantify tumor burden using colonoscopy, histopathological analysis, and molecular profiling of tumor tissues.

Advanced Research Questions

• What are the molecular mechanisms of CDHR5-mediated tumor suppression in mouse models?

CDHR5 exerts its tumor suppressive function through multiple interconnected mechanisms:

• Regulation of Notch signaling: CDHR5 promotes Notch pathway activation, which controls cell fate determination. CDHR5 deficiency leads to reduced NICD (Notch intracellular domain) and Hes1 protein levels .

• Wnt pathway modulation: CDHR5 appears to limit β-Catenin nuclear localization and activity. In CDHR5-deficient mice, increased nuclear β-Catenin drives tumor progression .

• STAT3 pathway inhibition: CDHR5 normally suppresses STAT3 activation. CDHR5-deficient tumors show enhanced STAT3 signaling .

• Maintenance of epithelial integrity: CDHR5 inhibits EMT, a critical process in cancer progression. Loss of CDHR5 promotes EMT features in tumors .

These mechanisms can be investigated using phospho-proteomics to map signaling networks, CRISPR screens to identify genetic interactions, and chromatin accessibility assays to determine effects on gene regulation.

• How can species-specific differences in CDHR5 structure be addressed in translational research?

Significant structural variations exist in CDHR5 across species that must be considered when translating findings:

Crystallographic studies of human and mouse CDHR5 ectodomains have revealed different binding interfaces, and bead aggregation assays have shown that human and mouse intermicrovillar cadherins engage in different homophilic and heterophilic interactions . To address these differences, researchers should consider:

• Using humanized mouse models expressing human CDHR5

• Validating findings across multiple species

• Employing comparative structural biology approaches

• Conducting functional assays to confirm conservation of key interactions

• What experimental approaches can effectively study CDHR5 protein interactions in mouse tissues?

Several complementary methods can be employed to study CDHR5 interactions:

• Crystallography: X-ray crystallography has been successfully used to determine structures of mouse CDHR5 ectodomains at 2.1 Å resolution . This approach reveals molecular details of binding interfaces.

• Bead aggregation assays: These have proven valuable for identifying CDHR5 domains involved in both heterophilic and homophilic adhesion .

• Proximity labeling: BioID or APEX2 fusion proteins can identify proteins in close proximity to CDHR5 in living cells.

• Live cell imaging: Fluorescently tagged CDHR5 can be tracked to observe dynamics of microvillar interactions and protein localization .

• Co-immunoprecipitation combined with mass spectrometry: This approach can identify novel binding partners and validate known interactions.

• FRET/BRET assays: These techniques can monitor protein-protein interactions in real-time within living cells.

• How does CDHR5 expression correlate with prognosis in mouse cancer models versus human cancers?

CDHR5 expression has significant prognostic implications across multiple cancer types:

In hepatocellular carcinoma (HCC):

• CDHR5 is downregulated in HCC tissues compared to adjacent liver tissues

• CDHR5 expression correlates with tumor numbers, size, and TNM stage

• CDHR5 functions as an independent risk factor for patient survival

• In vitro experiments show CDHR5 suppresses proliferation of HCC cells

In renal cell carcinoma (RCC):

In colorectal cancer (CRC):

• CDHR5 is downregulated in human altered crypt foci, adenomas, carcinomas, and CRC liver metastasis

• CDHR5-deficient mice show increased tumor burden and more aggressive tumor features