NRP1 Mouse

What is the expression pattern of NRP1 in mouse tissues?

NRP1 is abundantly expressed on the surface of various cell types in mice. In vascular tissue, it's predominantly expressed in endothelial cells where it forms heterocomplexes with VEGFR2. NRP1 is also expressed in smooth muscle cells, particularly in gastrointestinal smooth muscle. Additionally, NRP1 expression has been documented in immune cells and neuronal tissues.

Expression can be detected through:

• Immunofluorescent staining of tissues (effective in ear skin, back skin, and other organs)

• Western blotting of tissue lysates (lung lysates provide reliable detection)

• qPCR for mRNA expression analysis

Protein detection typically shows NRP1 at approximately 120-140 kDa molecular weight, though an 80 kDa protein of unknown origin may also be detected by some antibodies .

Confirmation of NRP1 deletion should involve multiple methods:

• Protein level verification:

  • Western blot analysis of tissue lysates (lung tissue provides reliable results)

  • Quantification shows significant reduction in NRP1 protein levels in knockout mice

• Tissue-specific verification:

  • Immunofluorescent staining of target tissues

  • Complete loss of NRP1 can be visualized in tissues such as ear and back skin

• Functional verification:

  • Assessing phenotypic changes characteristic of NRP1 deletion

  • For endothelial knockout, examining vascular permeability responses

  • For smooth muscle knockout, evaluating contractility parameters

For inducible models, verification should be performed after the induction period to confirm temporal deletion efficiency .

How does NRP1 deletion affect VEGFA-induced vascular permeability in different tissues?

The effect of NRP1 deletion on VEGFA-induced vascular permeability shows remarkable tissue specificity, presenting a complex research area:

• Tissue-dependent responses:

  • Ear dermis: Endothelial-specific NRP1 deletion (NRP1 iECKO) increases VEGFA-induced vascular leakage

  • Trachea and back skin: NRP1 deletion reduces VEGFA-induced permeability by approximately 75%

  • Kidney, skeletal muscle, and heart: NRP1 appears to play a passive role in VEGFA-induced leakage

• Quantitative analysis methods:

  • Measure lag period (time from stimulation to leakage onset)

  • Assess rate of dextran extravasation

  • Analyze disruption of endothelial cell-cell junctions

• Paradoxical findings between approaches:

  • Global pharmacological NRP1 inhibition using blocking antibodies reduces vascular leakage in ear dermis

  • This contrasts with increased leakage observed in endothelial-specific genetic deletion

  • Global genetic deletion (NRP1 iKO) results match antibody blocking rather than EC-specific deletion

These findings demonstrate that NRP1 can function as a positive regulator, negative regulator, or passive component in vascular permeability depending on tissue context and experimental approach .

What are the mechanisms behind NRP1-mediated gastrointestinal smooth muscle contractility?

NRP1 plays a crucial role in maintaining gastrointestinal smooth muscle contractility, particularly in aging mice:

• Key phenotypic changes in NRP1 SMKO mice:

  • Significant reduction in intestinal length by 6 months of age

  • Development of severe constipation by 18 months

  • Intestinal enlargement consistent with chronic intestinal pseudo-obstruction

  • Thinning of intestinal smooth muscle layers

• Molecular mechanisms:

  • Reduced expression of contractile proteins in smooth muscle cells

  • Specifically decreased expression of smooth muscle myosin heavy chain (smMHC) isoform SMB

  • Significant increase in small-conductance calcium-activated potassium channel 3 (SK3/KCa2.3)

  • SK3/KCa2.3 is known to negatively regulate smooth muscle contraction

• Experimental approaches for assessment:

  • Contractility assays of isolated intestinal segments

  • Protein expression analysis of contractile apparatus components

  • Histological assessment of muscle layer thickness

  • Functional transit studies to measure GI motility

The age-dependent nature of this phenotype suggests NRP1 is essential for maintaining, rather than establishing, the contractile phenotype of visceral smooth muscle cells .

How do experimental approaches to global versus tissue-specific NRP1 deletion yield different results?

The discrepancies between global and tissue-specific NRP1 deletion outcomes present a methodological challenge:

• Contrasting phenotypes:

  • In ear skin, endothelial-specific deletion increases VEGFA-induced permeability

  • Global deletion (both genetic and antibody-mediated) decreases permeability

  • In trachea and back skin, both approaches reduce permeability

• Methodological considerations:

  • Developmental compensation: Global knockouts may activate compensatory mechanisms absent in tissue-specific models

  • Cell-cell interactions: NRP1 on non-endothelial cells may influence endothelial function through juxtacrine signaling

  • Temporal aspects: Acute (antibody) versus chronic (genetic) loss of function yields different results

• Experimental design recommendations:

  • Combine multiple approaches (genetic deletion, antibody blocking)

  • Include both global and tissue-specific models

  • Employ acute inducible systems to minimize developmental compensation

  • Analyze multiple tissue beds to account for organotypic differences

This research challenge highlights the importance of using complementary approaches and careful interpretation of seemingly contradictory results .

What role does NRP1 play in immune regulation and autoimmune disease models?

NRP1 serves important functions in immune regulation, particularly in the context of autoimmune diseases:

• NRP1 in experimental autoimmune encephalomyelitis (EAE):

  • Tissue-specific deletion of NRP1 in CD4+ T cells results in increased EAE severity

  • NRP1-deficient mice exhibit preferential TH-17 lineage commitment

  • Regulatory T cell (Treg) functionality is decreased in NRP1-deficient models

• Mechanistic insights:

  • NRP1-expressing CD4+ T cells suppress effector T-cell proliferation

  • NRP1-mediated suppression can be inhibited by TGF-β blockade

  • NRP1-mediated suppression is independent of IL-10 signaling

• Experimental approaches:

  • Generation of retroviral GFP vector containing mouse Nrp1 cDNA

  • Isolation of CD4+ T cells from naïve myelin basic protein (MBP)-specific T cells

  • Assessment of T cell differentiation patterns and cytokine production

  • In vivo and in vitro suppression assays

These findings suggest NRP1 is essential for maintaining peripheral tolerance, and its absence can lead to unchecked autoimmune responses .

How can contradictory findings about NRP1 function in different studies be reconciled?

Contradictory findings regarding NRP1 function, particularly in vascular biology, can be approached methodologically:

• Sources of experimental variability:

  • Model differences: Global versus conditional knockouts yield different results

  • Tissue specificity: NRP1 functions differently in ear skin versus trachea or back skin

  • Temporal factors: Acute versus chronic loss of function produces different phenotypes

  • Cell-type interactions: Non-autonomous effects when multiple cell types express NRP1

• Reconciliation strategies:

  • Side-by-side comparisons: Test multiple models under identical conditions

  • Rescue experiments: Re-express NRP1 in knockout backgrounds to confirm specificity

  • Domain-specific mutations: Target specific functional domains rather than whole protein

  • Single-cell approaches: Analyze cell-type specific responses within heterogeneous tissues

• Data interpretation framework:

  • Consider NRP1 as a context-dependent modulator rather than a simple positive/negative regulator

  • Analyze both kinetic parameters (response onset, duration) and magnitude of effects

  • Map tissue-specific receptor and ligand expression patterns

For example, when studying vascular permeability, researchers should employ both antibody blockade and genetic deletion approaches in the same tissue beds under identical stimulation conditions .

What are the optimal experimental methods for studying NRP1 function in vivo?

Several experimental approaches are particularly valuable for studying NRP1 function in vivo:

• Vascular permeability assessment:

  • Intravital microscopy using fluorescent dextran tracers

  • Quantitative analysis of both temporal aspects (lag period) and magnitude (rate of extravasation)

  • Miles assay for macroscopic assessment of vascular leakage

• Smooth muscle function evaluation:

  • Intestinal transit assays using fluorescent tracers

  • Ex vivo contractility measurements of isolated muscle segments

  • Histological assessment of muscle layer thickness and organization

• Immune function analysis:

  • T cell proliferation and suppression assays

  • Cytokine production profiling

  • Adoptive transfer experiments with NRP1-deficient versus wild-type T cells

• Molecular interaction studies:

  • Co-immunoprecipitation to detect NRP1-VEGFR2 complexes

  • Proximity ligation assays for detecting protein-protein interactions in situ

  • FRET-based approaches for monitoring receptor interactions in live cells

The optimal approach depends on the specific research question, but combining multiple methodologies provides the most robust results .

How does the cytoplasmic domain of NRP1 contribute to its signaling functions?

The cytoplasmic domain of NRP1 plays specific roles in receptor trafficking and signaling:

• Structure and interactions:

  • Contains a C-terminal SEA motif

  • Functions as a PDZ binding domain

  • Mediates binding to synectin (GIPC1)

• Functional consequences:

  • Regulates endocytic trafficking of both NRP1 and VEGFR2

  • Removal delays VEGFR2 endocytosis following VEGFA binding

  • Leads to enhanced surface retention of VEGFR2

  • Reduces phosphorylation of tyrosine (Y)1175 in VEGFR2

• Experimental approaches:

  • Generation of cytoplasmic domain deletion mutants

  • Analysis of receptor internalization kinetics

  • Assessment of downstream signaling pathway activation

  • Comparison with full NRP1 knockouts to distinguish domain-specific functions

Understanding the cytoplasmic domain's function is particularly important because it reveals how NRP1 acts as a modulator of VEGFR2 activation and downstream signaling pathways rather than simply as a ligand-binding co-receptor .

What is the current understanding of NRP1's role in olfactory system development?

The role of NRP1 in olfactory system development, particularly regarding glomerular positioning, remains controversial:

• The NRP1 gradient model:

  • Original hypothesis proposed NRP1 forms an anterior-low to posterior-high gradient in the olfactory bulb

  • OR-derived cAMP signals were thought to determine NRP1 expression levels

  • This gradient was proposed to determine anterior-posterior patterning of glomeruli

• Contradictory findings:

  • Recent studies using conditional NRP1 knockout mice observed various configurations for M71 glomeruli

  • Glomerular positions do not undergo the simple anterior shift previously reported

  • These findings contradict the original model of NRP1-dependent anterior-posterior patterning

• Current research approaches:

  • Use of gene-targeted mouse strains with reporters for specific odorant receptors

  • Analysis of glomerular positioning in conditional NRP1 knockout backgrounds

  • 3D reconstruction of glomerular maps in the olfactory bulb

The current evidence suggests that while NRP1 may influence olfactory system development, its role in glomerular positioning is more complex than initially proposed .

How might NRP1 be targeted in therapeutic approaches for vascular disorders?

Understanding NRP1's complex role in vascular biology suggests potential therapeutic applications:

• Targeting considerations:

  • Tissue-specific effects require precise targeting approaches

  • Global inhibition may produce different effects than cell-type specific approaches

  • Temporal aspects of intervention may determine efficacy

• Potential therapeutic applications:

  • Vascular permeability modulation: NRP1 blocking antibodies reduce VEGFA-induced vascular leakage in specific tissues

  • Tumor angiogenesis: Several reports link increased NRP1 expression in tumors to poor prognosis

  • Inflammatory conditions: NRP1's role in immune regulation suggests potential for targeting inflammatory disorders

• Experimental therapeutic approaches:

  • Antibody-based blocking strategies

  • Small molecule inhibitors targeting specific NRP1 domains

  • Cell-type specific delivery systems

Understanding the relationship between NRP1 and VEGFA-VEGFR2 signaling has potential therapeutic benefit, particularly in conditions involving pathological vascular permeability or angiogenesis .

What are common technical challenges in studying NRP1 function in mice?

Researchers face several technical challenges when investigating NRP1:

• Genetic model limitations:

  • Global knockout is embryonic lethal, necessitating conditional approaches

  • Potential off-target effects of Cre expression in conditional models

  • Incomplete recombination in conditional systems

• Tissue-specific variability:

  • Divergent phenotypes between tissue beds complicate interpretation

  • Requires analysis of multiple tissues to establish comprehensive understanding

  • Need for standardized methodologies across different vascular beds

• Protein detection challenges:

  • Some antibodies may detect an 80 kDa protein of unknown origin

  • Confirmation of specific signal requires knockout controls

  • Recommended use of multiple detection methods (Western blot, immunofluorescence)

• Functional assay standardization:

  • Vascular permeability assays require precise quantification of both temporal and magnitude parameters

  • Ex vivo contractility measurements need standardized tension and stimulation protocols

  • Immune function assays benefit from defined cell populations and culture conditions

Addressing these challenges requires rigorous experimental design, appropriate controls, and combining multiple complementary approaches .

How should researchers interpret contradictory data between global and tissue-specific NRP1 knockout models?

When faced with contradictory results between global and tissue-specific knockouts:

• Analytical framework:

  • Consider non-cell-autonomous effects of NRP1 in global knockouts

  • Evaluate potential developmental compensation mechanisms

  • Assess timeline differences between acute and chronic loss of function

• Experimental validation approaches:

  • Complement genetic models with pharmacological interventions

  • Use inducible systems to minimize developmental effects

  • Perform tissue-specific rescue experiments

• Interpretation guidelines:

  • View contradictions as revealing of complex biology rather than experimental failures

  • Map tissue-specific expression of NRP1 interaction partners

  • Consider microenvironmental factors that might influence NRP1 function

The contrasting phenotypes observed between global and endothelial-specific NRP1 deletion in ear skin vascular permeability exemplify this challenge, suggesting important juxtacrine signaling between endothelial and non-endothelial cells .