LRRN1 Antibody

What is LRRN1 and why is it important in developmental research?

LRRN1, also called NLRR1 (Neuronal leucine-rich repeat protein 1), is a 95 kDa type I transmembrane glycoprotein with eleven leucine-rich repeats (LRRs) in its extracellular domain, alongside Ig-like and fibronectin type III domains . It plays critical roles in multiple developmental processes that make it valuable for research:

In cochlear development, LRRN1 regulates medial boundary formation in the organ of Corti, specifically influencing the precise patterning of inner hair cells (IHCs) along the neural boundary . This regulation occurs through interactions with the Notch signaling pathway, where LRRN1 appears to enhance Notch signaling across the medial boundary . During embryonic development, LRRN1 is expressed in motor neurons and somatic myoblasts, with particularly important functions in embryonic mouse somites around E9.5 .

In stem cell biology, LRRN1 is abundantly expressed on human embryonic stem cell (hESC) surfaces before differentiation into embryoid bodies . Research demonstrates it maintains pluripotency by regulating the protein stability of key pluripotency factors (OCT4, NANOG, and SOX2) through prevention of their nuclear export and subsequent degradation .

These diverse developmental roles make LRRN1 an important target for research in regenerative medicine, developmental biology, and hearing disorders.

What are the available types of LRRN1 antibodies and their applications?

Several types of LRRN1 antibodies are available for research, each with distinct characteristics and optimal applications:

For cochlear development studies, the sheep anti-human/mouse LRRN1 (R&D Systems AF4990) has been successfully used at 0.1 μg/ml for immunostaining in cochlear whole-mounts . This antibody has demonstrated high specificity in identifying the single row of cells that forms the medial border of the developing organ of Corti.

For stem cell research, antibodies detecting the extracellular domain (aa 26-631) of LRRN1 are particularly useful for surface marker studies, as approximately 90.7% of hESCs express LRRN1 on their surface before differentiation .

For functional studies, the monoclonal antibody TB0777#281-6 has shown significant inhibitory effects on cell growth in neuroblastoma models and xenograft tumors, suggesting therapeutic potential beyond basic research applications .

How should researchers validate LRRN1 antibodies for experimental use?

Proper validation of LRRN1 antibodies is critical for ensuring experimental reliability and reproducibility. A comprehensive validation strategy should include:

Positive and Negative Controls

Use embryonic mouse tissues (particularly E9.5 somites) as positive controls, as they show strong LRRN1 expression . For negative controls, Lrrn1 knockout tissues are ideal . The literature describes Lrrn1−/− mutant mice with both 2-bp and 4-bp deletion alleles that can serve as excellent negative controls .

Cross-Reactivity Assessment

Test for cross-reactivity with related proteins, particularly other leucine-rich repeat family members. This is especially important for polyclonal antibodies that might recognize conserved domains .

Multiple Detection Methods

Validate antibodies across different applications (IHC, WB, flow cytometry) when possible. For example, if an antibody works well for Western blot, confirm its specificity in immunohistochemistry before using it for spatial expression studies .

Functional Validation

For monoclonal antibodies like TB0777#281-6 that show functional effects (cell growth inhibition), validate these effects across multiple cell lines and confirm they're specific to LRRN1 targeting .

Epitope Mapping

Where possible, understand which domain of LRRN1 your antibody recognizes. Antibodies targeting different regions (LRR domains vs. Ig or fibronectin domains) may provide different results in functional studies . The polyclonal antibody HPA011071, for instance, targets a specific PrEST antigen sequence that should be considered when interpreting results .

Proper validation should also include titration experiments to determine optimal concentrations for each application, as recommended dilutions vary significantly (from 0.04 μg/ml for Western blot to 10 μg/ml for immunofluorescence) .

What factors affect LRRN1 antibody performance in immunohistochemistry?

Multiple factors can significantly impact the performance of LRRN1 antibodies in immunohistochemistry, requiring careful optimization:

Fixation Methods

For embryonic tissues where LRRN1 is highly expressed, use 4% paraformaldehyde fixation, but limit fixation time to preserve antigenicity . Overfixation can mask epitopes, especially in the extracellular domain of LRRN1.

Antigen Retrieval

Heat-induced epitope retrieval (HIER) at pH 6 is recommended for many LRRN1 antibodies, particularly for formalin-fixed tissues . This step is crucial for exposing epitopes that may be masked during fixation.

Blocking Conditions

Use 5-10% normal serum from the species of your secondary antibody. For polyclonal LRRN1 antibodies, more stringent blocking may be required to reduce background. For stem cell surface staining, avoid detergents that could disrupt membrane epitopes .

Antibody Concentration and Incubation

LRRN1 antibody concentrations vary widely by application. For the sheep anti-human/mouse LRRN1 antibody, 0.1 μg/ml has been effective for cochlear tissue staining , while higher concentrations (10 μg/mL) have been used for embryonic mouse somites .

Tissue-Specific Considerations

For cochlear tissues, careful microdissection and handling are essential to preserve the delicate structure of the organ of Corti . For embryonic tissues, cryosections often yield better results than paraffin sections for LRRN1 detection .

Detection Systems

For fluorescent detection, use directly conjugated secondary antibodies with minimal cross-reactivity. The literature shows successful visualization using NorthernLights™ 557-conjugated Anti-Sheep IgG Secondary Antibody for LRRN1 detection in embryonic tissues .

The quality of LRRN1 immunostaining can be enhanced by overnight incubation at 4°C rather than shorter incubations at room temperature, particularly for developmental tissues where precise spatial localization is critical .

How can I quantify LRRN1 expression in developmental studies?

Quantification of LRRN1 expression in developmental studies requires specific approaches depending on the research question and tissue context:

For Cochlear Development Studies

When analyzing LRRN1's role in boundary formation in the organ of Corti, researchers have employed region-specific quantification methods . Count extra inner hair cells (IHCs) and inner phalangeal cells (IPhCs) in defined cochlear regions (apical, middle, and basal) and normalize to cochlear length (e.g., per 1 mm) . This approach allows comparison between mutant and wild-type phenotypes while accounting for positional differences along the cochlear spiral.

For Lineage Tracing Experiments

When using Lrrn1-CreERT2; Rosa26 tdTomato mice for lineage tracing, quantify the proportion of different cell types labeled relative to the total labeled cells . This method is particularly useful when cell labeling is sparse, as is often the case with temporally controlled Cre induction.

Statistical Analysis

For LRRN1 expression analysis, researchers have successfully applied:

• Independent, two-tailed, unequal variance Student's t-tests for comparing cell counts between genotypes

• Holm–Šidák correction for multiple comparisons to maintain statistical validity

• Two-way ANOVA when comparing effects across both developmental stages and cell types, followed by Tukey's multiple comparison test

Imaging and Quantification Tools

For accurate quantification, use confocal microscopy with z-stack imaging to capture the full depth of the tissue, particularly important for three-dimensional structures like the cochlea . Employ automated cell counting software when possible, but validate with manual counts in subsets of samples.

When comparing LRRN1 expression across different developmental timepoints, it's essential to process and image all samples under identical conditions to allow for valid comparisons of expression intensity .

What special considerations apply when using LRRN1 antibodies in stem cell research?

Stem cell research presents unique challenges and opportunities for LRRN1 antibody applications:

Surface vs. Intracellular Detection

LRRN1 is abundantly expressed on hESC surfaces before differentiation, with approximately 90.7% of hESCs positive for LRRN1 compared to only 11.8% of embryoid body outgrowth cells . For surface detection, use gentle dissociation methods (non-enzymatic when possible) and avoid detergents that could disrupt membrane integrity.

Pluripotency State Correlation

When studying LRRN1's role in pluripotency maintenance, correlate LRRN1 expression with established pluripotency markers (OCT4, NANOG, SOX2) . Because LRRN1 affects protein stability of these factors without altering their mRNA levels, assess both transcription (qPCR) and protein expression (Western blot, immunostaining).

Differentiation Dynamics

Track LRRN1 expression during differentiation using flow cytometry with careful gating strategies. The sharp decrease in LRRN1 during differentiation makes it a potential marker for monitoring pluripotency status .

Functional Studies

When conducting LRRN1 knockdown experiments in stem cells, assess:

• Self-renewal capacity through colony formation assays

• Differentiation bias through germ layer marker expression

• Protein half-lives of pluripotency factors using cycloheximide chase experiments

• AKT phosphorylation status, as LRRN1 appears to function through this pathway

Technical Considerations

For flow cytometry applications, include appropriate controls (FMO, viability dyes) and consider using a two-color approach to distinguish surface from total LRRN1. For immunoprecipitation studies, use lysis conditions that effectively solubilize this transmembrane protein while preserving critical interactions.

Research has demonstrated that LRRN1 silencing causes decreased self-renewal capacity and skewed differentiation toward endoderm/mesoderm lineages, providing important phenotypic benchmarks for knockdown studies .

How does LRRN1 interact with the Notch signaling pathway in development?

Understanding LRRN1's interaction with the Notch pathway is critical for developmental studies, particularly in cochlear research:

Mechanistic Relationship

LRRN1 appears to enhance Notch signaling across the medial boundary of the developing organ of Corti . This enhancement is crucial for proper formation of the single row of inner hair cells and suppression of the conversion of adjacent nonsensory cells into hair cells and supporting cells.

Genetic Interaction Evidence

Research has demonstrated that deletion of Lrrn1 leads to boundary formation disruptions similar to those caused by reduced Notch activity . These disruptions manifest as ectopic inner hair cells and supporting cells. More compelling evidence comes from Lrrn1−/−; Notch1+/− mutant mice, which show interaction effects between these pathways .

Pharmacological Validation

Gamma-secretase inhibitors like DAPT, which block Notch signaling, produce phenotypes similar to Lrrn1 deletion, further supporting their functional relationship . This pharmacological approach provides complementary evidence to genetic studies.

Co-localization Studies

For studying LRRN1-Notch co-localization, researchers should:

• Use sequential immunostaining with carefully selected antibody combinations

• Employ sheep anti-human/mouse LRRN1 (R&D Systems AF4990) at 0.1 μg/ml

• Select Notch antibodies depending on which aspect of signaling is being investigated (cleaved vs. full-length Notch1)

• Use confocal microscopy with adequate resolution to visualize boundary structures

Quantification Approaches

When analyzing LRRN1-Notch interactions quantitatively, researchers have:

• Counted ectopic inner hair cells per cochlear region

• Measured the width of the prosensory domain

• Applied statistical tests (Student's t-tests with appropriate corrections) to compare genotypes

This interaction between LRRN1 and Notch signaling has significant implications for understanding boundary formation in development and potential applications in regenerative approaches for hearing disorders .

What are the best fixation and permeabilization protocols for LRRN1 detection?

Optimal fixation and permeabilization protocols for LRRN1 detection vary by application and tissue type:

For Immunohistochemistry/Immunofluorescence

Cochlear whole-mounts and cryosections have been successfully processed using:

• Fixation with 4% paraformaldehyde

• Overnight incubation with primary antibodies at 4°C

• For sheep anti-human/mouse LRRN1 (R&D Systems AF4990), a concentration of 0.1 μg/ml has proven effective

For Flow Cytometry

When detecting LRRN1 on stem cell surfaces:

• For surface detection: Avoid fixation when possible; if needed, use mild fixation (1% PFA for 10 minutes)

• For intracellular detection: Fix with 2-4% PFA followed by permeabilization with 0.1% saponin or 0.1% Triton X-100

• Include appropriate blocking steps (2-5% serum from the species of the secondary antibody)

For Western Blot

Recommended protein extraction conditions:

• Lysis buffers containing 1% NP-40 or Triton X-100 effectively solubilize this transmembrane protein

• Include protease inhibitor cocktails to prevent degradation

• For hard-to-lyse tissues, consider RIPA buffer with brief sonication

Tissue-Specific Considerations

For embryonic tissues:

• Use shorter fixation times (10-15 minutes) to preserve antigenicity

• For immersion-fixed frozen sections of embryonic mouse somites (E9.5), researchers have successfully used 10 μg/mL anti-LRRN1 antibody with overnight incubation at 4°C

For stem cells:

• When studying surface LRRN1, avoid permeabilization entirely

• For assessment of both surface and intracellular pools, consider a differential staining approach

Optimizing these protocols is essential as over-fixation can mask LRRN1 epitopes, particularly in the extracellular domain, while insufficient fixation may compromise tissue morphology and produce artifactual staining .

How can I distinguish between different LRRN1 isoforms in experimental systems?

Distinguishing between LRRN1 isoforms or modified forms requires specific technical approaches:

Potential LRRN1 Variants

The literature indicates at least one potential alternate start site at Met286 that could produce a truncated protein . Additionally, as a glycoprotein, LRRN1 may exhibit different glycosylation states that appear as distinct bands in Western blot analysis.

Western Blot Approaches

To distinguish between isoforms:

• Use gradient gels (4-15% or 4-20%) for better resolution of closely migrating bands

• Run reduced and non-reduced samples side-by-side to identify forms maintained by disulfide bonds

• Consider enzymatic deglycosylation (PNGase F treatment) to distinguish glycosylation variants from true isoforms

• Use different antibodies targeting distinct epitopes to confirm band identity

PCR-Based Methods

For mRNA isoform detection:

• Design primers flanking potential alternative splice sites

• Use isoform-specific primers when sequence information is available

• Consider qRT-PCR to quantify relative abundance of different isoforms

Mass Spectrometry

For definitive isoform identification:

• Immunoprecipitate LRRN1 from tissues of interest

• Perform tryptic digestion and LC-MS/MS analysis

• Map peptides to theoretical sequences of potential isoforms

Controls and Validation

To confirm isoform identity:

• Use recombinant LRRN1 protein standards with known sequence

• Include Lrrn1 knockout tissues as negative controls

• Consider tissues from different developmental stages as some isoforms may be stage-specific

When analyzing Western blot results showing multiple bands, consider that LRRN1's typical molecular weight is around 95 kDa , but post-translational modifications (particularly glycosylation) can significantly alter its apparent molecular weight.

What are the critical parameters for successful Western blot detection of LRRN1?

Successful Western blot detection of LRRN1 requires attention to several critical parameters:

Sample Preparation

LRRN1 is a transmembrane protein, requiring effective solubilization:

• Use lysis buffers containing 1% NP-40 or Triton X-100

• Include both protease and phosphatase inhibitors

• For membrane-enriched fractions, consider using stronger detergents (0.5-1% SDS)

• Avoid excessive heating during sample preparation (65°C is often sufficient)

Gel Electrophoresis

For optimal resolution:

• Use 8-10% polyacrylamide gels that provide good separation around LRRN1's molecular weight (95 kDa)

• Consider gradient gels (4-15%) if studying potential isoforms or modified forms

• Include positive controls (tissues known to express LRRN1, such as embryonic neural tissues)

• Load appropriate protein amounts (typically 20-50 μg of total protein)

Transfer Conditions

For efficient transfer of this larger protein:

• Extended transfer times (1-2 hours) or overnight transfer at lower voltage

• Methanol concentration in transfer buffer may need optimization (10-20%)

• Consider using nitrocellulose membranes with 0.45 μm pore size rather than 0.2 μm

Antibody Selection and Dilution

The polyclonal anti-LRRN1 antibody HPA011071 has been validated for Western blot at working concentrations of 0.04-0.4 μg/ml . Primary antibody incubation is typically performed overnight at 4°C for optimal results.

Blocking and Detection

For reduced background:

• 5% non-fat dry milk in TBST is generally effective for blocking

• BSA (3-5%) may provide better results for some antibodies

• For enhanced sensitivity, consider using HRP-conjugated secondary antibodies with enhanced chemiluminescent detection

Troubleshooting Common Issues

If detecting multiple bands or no signal:

• Verify sample preparation (membrane proteins often require specialized extraction)

• Assess antibody specificity using knockout controls when possible

• Consider protein degradation as a source of multiple bands

• For glycoproteins like LRRN1, enzymatic deglycosylation can help identify the core protein

Working concentration optimization is crucial, as too high concentrations can lead to non-specific binding while too low concentrations may yield insufficient signal .

How can I conduct LRRN1 knockdown experiments effectively?

Effective LRRN1 knockdown experiments require careful design and appropriate controls:

Knockdown Methods

Several approaches have been documented in the literature:

• Short hairpin RNA (shLRRN1) has been successfully used in hESCs to study pluripotency effects

• Genetic models including Lrrn1−/− mice with 2-bp or 4-bp deletions have been generated for developmental studies

• Combinatorial models (Lrrn1−/−; Notch1+/−) have been used to study pathway interactions

Experimental Design

For shRNA knockdown:

• Design multiple shRNA sequences targeting different regions of LRRN1

• Include non-targeting shRNA controls with similar GC content

• Validate knockdown efficiency at both mRNA (qRT-PCR) and protein (Western blot) levels

• Consider inducible knockdown systems for temporal control

For genetic models:

• Careful genotyping protocols have been described for Lrrn1 mutants

• Heterozygous littermates serve as appropriate controls

• For combinatorial studies with Notch pathway, Lrrn1+/+; Notch1+/− animals provide critical comparison groups

Phenotypic Analysis

Different experimental systems require specific analysis approaches:

For stem cell studies:

• Assess self-renewal capacity and differentiation potential

• Measure protein levels and half-lives of pluripotency factors (OCT4, NANOG, SOX2)

• Evaluate AKT phosphorylation status

• Examine nuclear vs. cytoplasmic localization of pluripotency factors

For cochlear development:

• Quantify ectopic inner hair cells and supporting cells

• Analyze across different cochlear regions (apex, middle, base)

• Apply appropriate statistical tests (independent, two-tailed, unequal variance Student's t-tests with Holm–Šidák correction)

Validation and Rescue

• Confirm phenotypes with multiple knockdown constructs

• Include rescue experiments with shRNA-resistant LRRN1 constructs

• For developmental studies, compare pharmacological manipulation (e.g., Notch inhibition) with genetic approaches

Research has demonstrated that LRRN1 silencing leads to decreased self-renewal capacity and skewed differentiation toward endoderm/mesoderm lineages, providing expected phenotypes for validation .

How should I interpret LRRN1 expression data across different developmental stages?

Interpreting LRRN1 expression across developmental stages requires consideration of its dynamic expression patterns and multiple functions:

Developmental Timing Considerations

LRRN1 shows stage-specific expression patterns:

• In mouse embryonic somites, strong expression is observed at E9.5

• In cochlear development, LRRN1 is expressed before organ of Corti formation in the row of cells that will form its medial border

• In embryonic stem cells, LRRN1 expression is high (90.7% positive cells) but decreases dramatically during differentiation (11.8% in embryoid body outgrowth)

Tissue-Specific Expression Patterns

When analyzing expression data:

• Compare expression within the same tissue across timepoints rather than between different tissues

• Consider the spatial context, as LRRN1 often marks specific boundaries or cell populations

• Use lineage tracing approaches like Lrrn1-CreERT2; Rosa26 tdTomato to track LRRN1-expressing cells and their progeny

Quantification Methods

For accurate developmental comparisons:

• In cochlear studies, normalize cell counts to cochlear length (e.g., per 1 mm) to account for growth

• For lineage tracing, calculate the proportion of each cell type relative to total labeled cells

• Apply appropriate statistical tests when comparing across stages (e.g., two-way ANOVA comparing both stage and cell type)

Developmental Function Interpretation

Different expression patterns reveal distinct functions:

• In cochlear development, LRRN1 regulates boundary formation through Notch pathway interaction

• In stem cells, LRRN1 maintains pluripotency by preventing nuclear export and degradation of key factors

• Expression in embryonic mouse somites suggests roles in early segmentation and patterning

When studying developmental dynamics, the timing of LRRN1 induction or suppression relative to other developmental markers can provide insights into its role in determining cell fate decisions and tissue patterning .

What controls are essential when studying LRRN1 antibody specificity?

Rigorous control experiments are crucial for validating LRRN1 antibody specificity:

Genetic Controls

The gold standard for antibody validation:

• Lrrn1 knockout tissues as negative controls

• Lrrn1 heterozygous tissues to assess gene dosage effects on staining intensity

• Tissue-specific conditional knockouts to verify regional specificity

• The literature describes Lrrn1−/− mutants with 2-bp and 4-bp deletions that can serve as excellent negative controls

Peptide Competition

To verify epitope specificity:

• Pre-incubate antibody with excess recombinant LRRN1 or immunizing peptide

• Compare staining with and without competition

• Specific signal should be significantly reduced or eliminated

Biological Controls

Leverage known expression patterns:

• Positive tissue controls: Embryonic mouse somites (E9.5), developing cochlea, human embryonic stem cells

• Negative tissue controls: Adult tissues with minimal LRRN1 expression

• Developmental series showing expected up/downregulation during key transitions

Technical Controls

Essential for methodology validation:

• Secondary antibody-only controls to assess non-specific binding

• Isotype controls for monoclonal antibodies

• Multiple antibodies targeting different LRRN1 epitopes should show consistent patterns

• Orthogonal methods (RNA in situ hybridization) to confirm expression patterns

Procedural Variations

To distinguish real signal from artifacts:

• Test multiple fixation and antigen retrieval protocols

• Compare different antibody concentrations (titration series)

• Evaluate different detection systems (direct vs. indirect, enzymatic vs. fluorescent)

For Western blot applications, additional controls include:

• Recombinant LRRN1 protein as a positive control

• Multiple antibodies targeting different epitopes

• Molecular weight verification (approximately 95 kDa)

These comprehensive controls ensure that staining patterns attributed to LRRN1 truly represent the protein of interest rather than technical artifacts or cross-reactivity .

How can I optimize LRRN1 antibody staining in difficult tissues?

Optimization of LRRN1 staining in challenging tissues requires tissue-specific adaptations:

For Cochlear Tissues

The delicate architecture of the organ of Corti presents special challenges:

• Careful microdissection and handling to preserve structure

• Gentle fixation (4% PFA for 1-2 hours) to maintain antigenicity while preserving morphology

• Extended primary antibody incubation (overnight at 4°C) using sheep anti-human/mouse LRRN1 at 0.1 μg/ml

• Careful washing to remove background without damaging the sensory epithelium

For Embryonic Tissues

Early developmental stages often require specialized approaches:

• For mouse somites (E9.5), immersion fixation of frozen sections has proven effective

• 10 μg/mL anti-LRRN1 antibody with overnight incubation at 4°C

• NorthernLights™ 557-conjugated secondary antibodies provide good signal-to-noise ratio

• DAPI counterstaining helps orient the tissue

For Stem Cells

Surface marker detection requires modified protocols:

• Avoid harsh detergents when studying surface LRRN1

• Consider live cell staining for surface epitopes

• Use gentle fixation (1-2% PFA for 10-15 minutes) if fixation is necessary

• Include careful blocking steps to reduce background

Antigen Retrieval Optimization

For fixed tissues with masked epitopes:

• Heat-induced epitope retrieval (HIER) at pH 6 is recommended for many LRRN1 antibodies

• Test multiple retrieval buffers (citrate pH 6, EDTA pH 8, Tris-EDTA pH 9)

• Optimize retrieval duration (10-30 minutes)

• Allow slow cooling to room temperature after retrieval

Signal Amplification Methods

For tissues with low LRRN1 expression:

• Tyramide signal amplification can enhance detection sensitivity

• Polymer-based detection systems often provide better signal than traditional ABC methods

• Consider multilabel immunofluorescence with spectral unmixing for tissues with high autofluorescence

These optimization strategies have enabled successful LRRN1 detection even in challenging contexts like the precise boundary regions of the developing cochlea and early embryonic structures .

What are the best methods to co-localize LRRN1 with other developmental markers?

Effective co-localization of LRRN1 with other developmental markers requires careful experimental design:

Antibody Selection and Validation

For reliable co-localization:

• Choose primary antibodies raised in different host species to avoid cross-reactivity

• For LRRN1, the sheep anti-human/mouse antibody (R&D Systems AF4990) pairs well with rabbit or mouse antibodies against other markers

• Validate each antibody individually before attempting co-localization

• Test for potential cross-reactivity between all components of the staining system

Sequential Staining Protocol

For markers with conflicting requirements:

• Complete staining for the most sensitive marker first

• Fix briefly after first round of detection to stabilize the signal

• Proceed with subsequent marker detection

• Include single-stained controls processed in parallel

Imaging Considerations

For optimal co-localization analysis:

• Use confocal microscopy with appropriate controls for spectral bleed-through

• Acquire z-stacks to capture the full three-dimensional relationship between markers

• Apply consistent settings across all samples to allow valid comparisons

• Consider super-resolution techniques for closely adjacent markers

Specific Co-Localization Combinations

Quantitative Co-Localization Analysis

For rigorous spatial relationship assessment:

• Calculate standard co-localization coefficients (Pearson's, Manders' overlap)

• For boundary studies, analyze the precise spatial relationship between markers

• In the developing cochlea, analyze the relationship between LRRN1 and the single row of inner hair cells at the medial boundary

In cochlear development studies, co-localization of LRRN1 with Notch pathway components has provided crucial insights into its role in boundary formation through Notch signaling enhancement .