Recombinant Mouse Cadherin-7 (Cdh7), partial

What is Cadherin-7 and how is it classified among cadherins?

Cadherin-7 (Cdh7) belongs to the type II classic cadherin subfamily of calcium-dependent cell adhesion molecules. Vertebrate classic cadherins are divided into type I and type II subtypes, which are expressed in brain subdivisions (including prosomeres, rhombomeres, and progenitor domains) and in specific neuronal circuits in region-specific patterns . Cadherin-7 is closely clustered on a chromosome with cadherin-19 (cad19) and cadherin-20 (cad20) . Unlike type I cadherins such as N-cadherin, cadherin-7 demonstrates distinct adhesive properties and developmental expression patterns.

What are the key structural domains of Cadherin-7?

Cadherin-7, like other classic cadherins, contains:

• Multiple extracellular cadherin repeat (CR) domains

• A transmembrane domain

• A cytoplasmic domain that interacts with catenins

The full-length cadherin-7 protein functions as a transmembrane protein, while alternative splicing can generate a soluble isoform that lacks the transmembrane domain. In chicken embryos, this soluble isoform contains a 49-bp insertion that causes a frameshift and introduces a premature stop codon before the transmembrane domain . This results in a protein consisting only of the extracellular domains of full-length cadherin-7 .

How does Cadherin-7 expression change during embryonic development?

Cadherin-7 demonstrates a dynamic expression pattern during embryonic development. In chicken embryos:

• Expression begins at early developmental stages, with variant transcripts detected in the rostral half of the trunk by stage 17

• The soluble isoform reaches levels of approximately one-tenth to one-fifth of the full-length cadherin-7 in the rostral half

• After this stage, the expression level of the variant form in the trunk at wing level remains relatively constant until E9, maintaining a consistent ratio to the full-length cadherin-7

• In E5 chicken embryos, the soluble variant form is expressed transiently and strongly in dermomyotomes, then at the dorsal and ventral lips and in the myotome

• Faint signals of the variant form are detected in proximal dorsal and ventral roots and in dorsal root ganglia

This expression pattern suggests developmental regulation of cadherin-7 isoforms during embryogenesis, particularly in tissues undergoing active morphogenesis.

What is the relationship between Cadherin-7 and neural crest cell development?

Neural crest cell (NCC) development involves multiple morphogenetic processes including epithelial-to-mesenchymal transition (EMT), cell migration, cell aggregation, and cell differentiation . These processes are associated with a strictly regulated pattern of cadherin synthesis:

• Premigratory NCCs produce N-cadherin and cadherin-6B

• Both are downregulated when cells separate from the neural tube and begin producing cadherin-7

• When NCCs reaggregate and differentiate at specific sites, they resume N-cadherin expression

This sequential expression pattern suggests that cadherin-7 plays a specific role in the migratory phase of neural crest development, while N-cadherin is associated with more stable cellular aggregations.

How does Cadherin-7 differ functionally from N-cadherin?

Despite both being cadherins, N-cadherin (type I) and cadherin-7 (type II) demonstrate significant functional differences:

These differences suggest that cadherin-7 mediates more dynamic adhesions that are compatible with cell migration, while N-cadherin establishes more stable intercellular connections .

What is the role of the soluble Cadherin-7 isoform?

The soluble cadherin-7 isoform generated by alternative splicing plays an inhibitory role in cell adhesion. Key findings include:

• Cell aggregation of L-cad7 cells (expressing full-length cadherin-7) was significantly slower in the presence of conditioned medium containing the soluble variant

• This inhibition was calcium-dependent, consistent with cadherin function

• Co-immunoprecipitation experiments demonstrated that the variant protein directly interacts with full-length cadherin-7

• In fractionation studies of E5 chicken embryos, the majority of the variant form was found in the soluble fraction, with a small amount in the membranous fraction

This represents the first demonstration of a soluble form of type I and II cadherins generated by alternative splicing rather than proteolysis, suggesting a novel regulatory mechanism for cadherin-mediated adhesion .

What are effective approaches for studying Cadherin-7 function in vitro?

Several methodologies have proven effective for investigating cadherin-7 function:

• Cell aggregation assays: Used to quantify cadherin-7-mediated adhesion, measured by calculating the index Nt/No (where No is the initial cell number before aggregation and Nt is the total particle number at incubation time t)

• Transfection studies: Generate stable cell lines expressing full-length cadherin-7 (e.g., L-cad7) or transient expression of variant forms (e.g., COS variant or 293 variant cells)

• Immunofluorescence staining: Visualize cadherin-7 expression and localization using specific antibodies such as CCD7-1 (for both forms) or anti-variant antibodies (specific to the variant form)

• Immunoprecipitation: Confirm protein-protein interactions, such as between soluble and full-length cadherin-7 isoforms

• Cell fractionation: Separate soluble and membranous fractions to determine the subcellular localization of cadherin-7 isoforms

• Cell migration assays: Compare migratory behaviors of cells expressing different cadherins on substrates like fibronectin

How can researchers effectively detect and distinguish between different Cadherin-7 isoforms?

To effectively differentiate between full-length and soluble cadherin-7 isoforms:

• PCR-based detection:

  • Design primers that span the alternative splicing region (49-bp insertion in chicken)

  • Use competitive PCR with appropriate competitors for quantitative analysis

• Immunological detection:

  • Use antibodies that recognize both isoforms (e.g., CCD7-1)

  • Generate isoform-specific antibodies (e.g., anti-variant antibodies directed against the unique C-terminal peptide of the variant form)

• Western blotting:

  • Full-length cadherin-7 appears at approximately 105 kDa

  • The soluble variant appears at approximately 73 kDa

  • Degradation products may be detected at approximately 80 kDa

• Subcellular fractionation:

  • The full-length form predominantly localizes to the membranous fraction

  • The soluble variant predominates in the soluble fraction, with small amounts in the membranous fraction (likely due to interaction with the full-length form)

How does Cadherin-7 influence cell migration during development?

Cadherin-7 plays a critical role in regulating cell migration during development, particularly in neural crest cells:

• Dynamic adhesion properties: Cadherin-7 mediates transient cell-cell contacts that allow cells to maintain cohesion while still migrating effectively

• Matrix-dependent regulation: The adhesive phenotype of cadherin-7-expressing cells is regulated by the extracellular matrix environment, which also controls migratory behavior

• Differential signaling: Cadherin-7, unlike N-cadherin, permits fibronectin-dependent signaling pathways to remain active, supporting cell migration

• In vivo migration: When grafted into embryos, cadherin-7-expressing cells disperse efficiently into embryonic structures, whereas N-cadherin-expressing cells remain at or near the graft site

These properties suggest that cadherin-7 expression provides a balance between cell-cell adhesion and migration capability, which is essential for developmental processes involving collective cell migration.

What is the relationship between Cadherin-7 and the extracellular matrix during cell migration?

The interaction between cadherin-7-mediated adhesion and the extracellular matrix, particularly fibronectin (FN), is complex:

• Dual adhesion systems: Cadherin-7-expressing cells can simultaneously engage in cell-cell adhesion and cell-matrix adhesion

• Conditional stability: Cadherin-7-mediated contacts between cells are transient when cells migrate on fibronectin but become stabilized if FN-cell interactions are perturbed

• Signaling crosstalk: The extent of FN-dependent focal adhesion kinase (FAK) phosphorylation is affected by prior engagement in cadherin-mediated adhesion, though less so for cadherin-7 than for N-cadherin

• Migration regulation: The transient contacts observed specifically in cadherin-7-expressing cells appear to be important in the control of cell motility on matrix components

This interplay between cadherin-7 and extracellular matrix components provides a mechanism for fine-tuning cell migration during developmental processes.

How might Cadherin-7 research contribute to understanding developmental disorders?

Understanding cadherin-7 function has potential implications for developmental disorders:

• Neural crest-related disorders: Given cadherin-7's role in neural crest migration, its dysfunction could contribute to neurocristopathies (disorders of neural crest development)

• Neuronal circuit formation: As cadherin-7 is expressed in specific neuronal circuits, alterations in its function might affect circuit assembly and function

• Cell migration defects: Abnormalities in cadherin-7-mediated cell migration could potentially contribute to a range of developmental anomalies

• Adhesion-migration balance: Disorders involving improper balance between cell adhesion and migration might involve cadherin-7 dysfunction

Research into cadherin-7's precise roles in these processes could provide insights into the etiology of related developmental disorders and potentially suggest therapeutic approaches.

What are the emerging techniques for investigating Cadherin-7 function in complex tissues?

Several advanced techniques are emerging for studying cadherin-7 in complex developmental contexts:

• CRISPR/Cas9 genome editing: For generating precise modifications to cadherin-7 genes in model organisms

• Organoid systems: For studying cadherin-7 function in three-dimensional tissue-like structures that better mimic in vivo development

• Live imaging techniques: For visualizing cadherin-7-expressing cells in real-time during morphogenetic processes

• Single-cell transcriptomics: For analyzing cadherin-7 expression at the single-cell level during development

• Optogenetic approaches: For spatiotemporally controlled manipulation of cadherin-7 function in developing tissues

These techniques promise to provide deeper insights into the complex roles of cadherin-7 in tissue morphogenesis and development.