CDX4 Antibody

What is CDX4 and what applications are CDX4 antibodies validated for?

CDX4 is a transcription factor belonging to the caudal-related homeobox gene family that functions as a regulator of Hox gene expression and plays critical roles in hematopoiesis, particularly in primitive stem and progenitor cells . CDX4 is preferentially expressed in primitive stem and progenitor cells, with expression downregulated in more differentiated cell types .

CDX4 antibodies have been validated for multiple research applications:

• Western Blotting (WB): Multiple antibodies demonstrate specific detection of CDX4 protein with observed molecular weights typically around 37-42 kDa, despite the calculated molecular weight of 30 kDa

• Immunohistochemistry (IHC): Both on tissue sections and whole-mount specimens

• Chromatin Immunoprecipitation (ChIP): Used for studying CDX4 interactions with promoter regions

• Electrophoretic Mobility Shift Assays (EMSA): For analyzing DNA-protein interactions involving CDX4

• Enzyme-Linked Immunosorbent Assay (ELISA): Several antibodies are validated for this application

• Immunofluorescence (IF): For cellular localization studies

What controls should be incorporated when working with CDX4 antibodies?

Implementing appropriate controls is essential for valid interpretation of CDX4 antibody experiments:

Positive Controls:

• Cell lines: SKOV-3 cells, Sp2/0 cells, and C6 cells show detectable CDX4 expression

• Tissues: Mouse thymus tissue for western blot applications

• Developmental models: Paraformaldehyde-fixed zebrafish embryos (1 day post-fertilization) for whole-mount immunohistochemistry

Negative Controls:

• ChIP experiments: Chromatin precipitated with an irrelevant antibody serves as a specificity control

• EMSA: Unlabeled oligonucleotide competitors with mutations in the CDX4-binding consensus sequence

• Cell types: Most terminally differentiated cells lack CDX4 expression and can serve as biological negative controls

Additional Validation Controls:

• Input chromatin as a positive control for ChIP experiments

• Oligonucleotide competitors with irrelevant sequences from other promoters for binding specificity

• Base pair-swapping mutations or scrambled control sequences when using shRNA approaches

How does antibody selection impact CDX4 detection in different species?

Antibody selection significantly impacts successful CDX4 detection across species:

Species Reactivity Patterns:

• Human: Most commercial antibodies show validated reactivity

• Mouse: Several antibodies demonstrate cross-reactivity

• Rat: Some antibodies are validated for rat samples

• Zebrafish: Specific antibodies are available for developmental studies in zebrafish embryos

• Other species: Some antibodies show predicted reactivity with cow, dog, horse, pig, and rabbit samples based on sequence homology

Epitope Considerations:

• C-terminal antibodies: Recognize the C-terminal region of CDX4 (e.g., ABIN2777577)

• Internal region antibodies: Target various internal domains (AA 71-170, AA 181-230, etc.)

• N-terminal antibodies: Target the N-terminal region

Cross-reactivity with other CDX family members (CDX1, CDX2) should be evaluated when selecting antibodies, particularly for studies in systems where multiple CDX proteins may be expressed.

What are the optimal methods for detecting CDX4 expression in hematopoietic cells?

Several complementary methods have been optimized for detecting CDX4 expression in hematopoietic cells:

Real-time Quantitative RT-PCR (qRT-PCR):

• Use CDX4-specific primers designed to avoid cross-reactivity with other CDX family members

• Studies have successfully detected CDX4 expression in 23% of AML patients using this method

• Appropriate reference genes must be carefully selected for hematopoietic samples

Flow Cytometry:

• Combined with markers such as KDR and CD1d to identify CDX4-expressing hemogenic mesoderm

• Co-staining with erythroid markers like CD71 and Ter119 can identify specific erythroid populations

Western Blotting:

• Expected molecular weight: 30 kDa (calculated), but typically observed at 37-42 kDa

• Recommended antibody dilutions: 1:500-1:2000 for most applications

• Sample lysis conditions must adequately preserve nuclear proteins

Single-cell RNA Sequencing:

• Can identify rare CDX4-expressing populations in heterogeneous cell mixtures

• Has successfully identified CDX4+ populations during human pluripotent stem cell differentiation

How does CDX4 contribute to normal and malignant hematopoiesis?

CDX4 plays distinct roles in both normal hematopoietic development and malignant transformation:

Normal Hematopoiesis:

• CDX4 is expressed in primitive stem and progenitor cells

• It regulates Hox gene expression, particularly genes involved in erythroid development

• CDX4 expression in hemogenic mesoderm marks cells with multi-lineage potential

• Experimental studies show constitutive expression increases erythroid cell formation in vitro (643-fold increase)

Malignant Hematopoiesis:

• CDX4 expression has been detected in 23% (10/44) of AML patients

• Expression patterns vary by cytogenetic subtype:

  • 3/4 patients with trisomy 8

  • 3/7 patients with t(15;17)(q22;q11~21)

  • 3/16 patients with normal cytogenetics

• Aberrant expression induces acute erythroid leukemia (AEL) in mouse models

• CDX4-induced AEL is characterized by:

  • Downregulation of genes associated with erythroid differentiation

  • Suppression of terminal erythroid differentiation

  • Upregulation of stemness-related genes

The dual role in normal development and leukemogenesis makes CDX4 an important target for understanding both normal hematopoiesis and hematological malignancies.

What is the relationship between CDX4 and Hox gene expression in the hematopoietic system?

The CDX4-Hox regulatory axis represents a critical regulatory network in hematopoiesis:

Regulatory Mechanism:

• CDX4 functions as an upstream regulator of Hox gene expression

• Constitutive CDX4 expression leads to upregulation of multiple Hox genes compared to controls

• HoxA10 activates CDX4 transcription, and CDX4 activates HOXA10 transcription, creating a regulatory feedback loop

Differential Hox Regulation by CDX Family Members:

• Both CDX2 and CDX4 upregulate Hox genes, but with different patterns

• Hoxb3 and Hoxb4 are significantly higher expressed in CDX4-induced AEL compared to CDX2-induced AML

• Hoxb4 has been previously shown to induce erythroid colony formation in humans

Molecular Interaction Studies:

• CDX4 does not interact with its own promoter in vivo

• CDX4 binds to specific cis-elements in the HOXA10 promoter (-124 to -140 bp)

• This binding can be detected through chromatin immunoprecipitation and EMSA techniques

CD1d-derived CD34+ cells expressing CDX4 robustly express HOXA7/9 , indicating specific Hox gene targets depending on cellular context.

What molecular mechanisms underlie CDX4-induced acute erythroid leukemia?

Research has elucidated several mechanisms through which CDX4 contributes to acute erythroid leukemia (AEL) development:

Transcriptional Dysregulation:

• CDX4 suppresses expression of genes associated with erythroid differentiation

• Gene expression analyses show upregulation of genes involved in stemness and leukemogenesis

• Downregulation of target genes of Gata1 and Gata2, which are responsible for erythroid differentiation

Differentiation Blockade:

• CDX4 increases the percentage of immature GFP+CD71+Ter119- erythroid cells with a parallel decrease of more mature GFP+CD71+Ter119+ cells

• This indicates a partial block in erythroid differentiation at a specific developmental stage

Hox Gene Modulation:

• CDX4 induces differential Hox gene expression compared to CDX2

• Hoxb3 and Hoxb4, which are associated with erythroid development, are significantly higher expressed in CDX4-induced AEL

Co-occurring Genetic Alterations:

• Whole-exome sequencing identified recurrent mutations enriched for transcription factors involved in erythroid lineage specification

• Mutations in TP53 target genes, similar to those reported in patients with AEL

This interplay between CDX4 overexpression, Hox gene dysregulation, and co-occurring mutations collectively contributes to the development of AEL.

What experimental designs are most effective for studying the functional role of CDX4 in hematopoiesis?

Based on published research, several experimental approaches have proven effective for studying CDX4's role in hematopoiesis:

In Vitro Models:

• Retroviral overexpression in mouse hematopoietic stem and progenitor cells (HSPCs)

  • Liquid expansion cultures to assess proliferation rates

  • Colony-forming cell (CFC) assays with serial replating to assess self-renewal

  • Spleen colony-forming unit (CFU-S) assays to evaluate progenitor activity

In Vivo Models:

• Transplantation of CDX4-transduced bone marrow HSPCs into mice

  • Long-term follow-up (>300 days) to assess leukemia development

  • Secondary transplantation to confirm leukemia-propagating cell activity

  • Multi-organ analysis for leukemic infiltration

Molecular Interaction Studies:

• Chromatin immunoprecipitation (ChIP) to identify direct CDX4 binding targets

• Electrophoretic mobility shift assays (EMSA) to study DNA-protein interactions

• Reporter assays with promoter constructs to analyze transcriptional regulation

Gene Expression Manipulation:

• Constitutive expression through retroviral transduction

• shRNA-mediated knockdown to assess loss-of-function effects

• Comparative studies between CDX4 and related factors like CDX2

Human Cell Models:

• Analysis of CDX4 expression in patient samples

• Human pluripotent stem cell differentiation to study CDX4 in early hematopoietic development

• Single-cell RNA sequencing to identify rare CDX4+ populations

These complementary approaches provide comprehensive insights into CDX4 function from molecular interactions to disease development.

How can contradictory findings about CDX4 expression in different cell types be reconciled?

Reconciling contradictory findings about CDX4 expression requires careful consideration of several factors:

Methodological Variables:

Biological Context Considerations:

• Developmental stage: CDX4 expression is highest in primitive cells and decreases with differentiation

• Disease state: Expression patterns in normal vs. leukemic cells differ significantly

• Species differences: Expression patterns may vary between human, mouse, and zebrafish models

Experimental Approach Strategies:

• Use multiple complementary detection methods to validate expression findings

• Perform detailed time-course analyses to capture dynamic expression changes

• Include appropriate positive and negative controls for each methodology

• Consider the impact of culture conditions on gene expression patterns

• Employ single-cell approaches to identify rare CDX4-expressing populations

For example, apparent contradictions in CDX4 expression in adult bone marrow can be reconciled by recognizing that CDX4+ cells represent a rare subpopulation that may be diluted in bulk analyses but detectable through single-cell or enrichment approaches .

What is the current understanding of the CDX4-HOXA10 regulatory circuit in normal and malignant hematopoiesis?

The CDX4-HOXA10 regulatory circuit represents a sophisticated feedback mechanism in hematopoiesis:

Bidirectional Regulation Mechanism:

• HoxA10 activates CDX4 transcription through binding to a specific cis element (-139 to -150 bp) in the CDX4 promoter

• CDX4, in turn, activates HOXA10 transcription by binding to a Cdx-binding site (-124 to -140 bp) in the HOXA10 promoter

• This creates a positive feedback loop that can amplify expression of both factors

Molecular Evidence:

• Chromatin immunoprecipitation confirms in vivo binding of these factors to their respective targets

• EMSA studies demonstrate specific protein-DNA interactions with these regulatory elements

• Reporter assays with wild-type and mutant binding sites confirm functional significance of the interaction

• CDX4 does not regulate its own promoter directly, as shown in chromatin immunoprecipitation experiments

Functional Implications:

• This regulatory circuit may maintain expression of both factors in specific cellular contexts

• Disruption of this circuit could contribute to dysregulated hematopoiesis

• The positive feedback loop explains how aberrant expression of either factor could lead to sustained dysregulation

Implications in Malignancy:

• Aberrant activation of this circuit contributes to leukemogenesis

• The sustained expression of both factors promotes a differentiation block

• This circuit operates within a broader network of homeobox gene regulation

Understanding this regulatory loop provides potential therapeutic targets for interventions in CDX4-expressing malignancies.

How should CDX4 antibodies be optimized for immunohistochemistry applications?

Optimizing CDX4 antibodies for immunohistochemistry requires attention to several parameters:

Antibody Selection:

• Choose antibodies specifically validated for IHC applications

• Consider polyclonal antibodies for enhanced sensitivity (e.g., GTX128752 for zebrafish studies)

• Verify species reactivity matches your experimental model

Sample Preparation:

• For whole-mount zebrafish embryos: Paraformaldehyde fixation works effectively

• Antigen retrieval: Tris-HCl buffer, pH 9.0, 20 min at 70°C has been successfully employed

• For tissue sections: Standard formalin fixation and paraffin embedding protocols

Protocol Optimization:

• Antibody dilution: Follow manufacturer recommendations (e.g., 1:100 for whole-mount zebrafish)

• Incubation conditions: Typically overnight at 4°C for whole-mount specimens

• Detection system: Secondary antibodies appropriate for the host species

• Background reduction: Use appropriate blocking solutions (typically 5-10% serum from secondary antibody species)

Controls:

• Positive control: Use tissues known to express CDX4 (e.g., zebrafish embryos, AML samples)

• Negative control: Omit primary antibody or use tissues known to lack CDX4 expression

• Peptide competition: Pre-incubate antibody with immunizing peptide to verify specificity

Visualization:

• For fluorescent detection: Use appropriate filters and counterstains

• For chromogenic detection: Optimize substrate development time

What are the critical factors in designing experiments to study CDX4-HOXA10 interactions?

Designing robust experiments to study CDX4-HOXA10 interactions requires attention to these critical factors:

Chromatin Immunoprecipitation (ChIP) Design:

• Antibody selection: Use ChIP-validated antibodies with demonstrated specificity

• Controls: Include input chromatin as positive control and irrelevant antibody precipitation as negative control

• Primer design: Design primers spanning the CDX4 binding site in the HOXA10 promoter (-124 to -140 bp) and the HOXA10 binding site in the CDX4 promoter (-139 to -150 bp)

• Quantification: Use qPCR with standard curves for accurate quantification

EMSA Experimental Design:

• Probe design: Use radiolabeled, double-stranded oligonucleotides representing binding sites

• Competition assays: Include unlabeled wild-type and mutant competitors to demonstrate specificity

• Supershift assays: Use antibodies against CDX4 or HOXA10 to confirm complex identity

• Controls: Include irrelevant oligonucleotide competitors and irrelevant antibodies

Reporter Assay Considerations:

• Construct design: Include wild-type and mutant binding sites in reporter constructs

• Controls: Use empty vector controls and constructs with non-binding mutant sequences

• Gain/loss of function: Test effects of overexpression and knockdown of CDX4 and HOXA10

• Normalization: Include internal control reporters to normalize for transfection efficiency

Co-expression Studies:

• Cell selection: Use cells with appropriate context (e.g., hematopoietic progenitors)

• Manipulation approaches: Consider both overexpression and knockdown strategies

• Analysis methods: Evaluate effects on target gene expression and cellular phenotypes

• Controls: Include appropriate vector controls and scrambled shRNA controls

What are the implications of CDX4 expression in acute myeloid leukemia patient samples?

The detection of CDX4 expression in acute myeloid leukemia (AML) patient samples has several implications:

Diagnostic and Prognostic Considerations:

• CDX4 expression has been detected in 23% (10/44) of AML patients

• Expression patterns vary by cytogenetic subtype:

  • 3/16 patients with normal cytogenetics

  • 3/4 patients with trisomy 8

  • 3/7 patients with t(15;17)(q22;q11~21)

  • 1/2 patients with t(9;11)(p22;q23)

• No CDX4 expression was detected in patients with inv(16) or complex karyotypes

• This distribution suggests potential as a biomarker for specific AML subtypes

Disease Mechanism Insights:

• CDX4 expression is strongly associated with acute erythroid leukemia (AEL)

• Gene expression analyses show CDX4 upregulates genes involved in stemness and leukemogenesis

• CDX4 induces a proteomic profile that overlaps with primitive human erythroid progenitors

• Whole-exome sequencing identified mutations in transcription factors involved in erythroid lineage specification

Therapeutic Implications:

• The CDX4-HoxA10 regulatory circuit represents a potential therapeutic target

• Disrupting this positive feedback loop might restore normal differentiation

• Targeting downstream effectors of CDX4 could provide alternative approaches

• Understanding CDX4's role in erythroid differentiation block may guide development of differentiation therapies

How does the expression of CD1d relate to CDX4 function in hematopoietic development?

Recent research has revealed an important relationship between CD1d and CDX4 in hematopoietic development:

Co-expression Pattern:

• Single-cell RNA sequencing of human pluripotent stem cell differentiation identified a CDX1/2/4+ CD1d+ mesodermal population

• CD1d, a non-canonical MHC receptor typically found on antigen-presenting cells, shows enrichment in CDX4-high clusters

• CD1d expression correlates with high CDX1/2/4 expression during early development

Functional Significance:

• KDR+CD1d+ mesoderm efficiently gives rise to hemogenic endothelium with erythroid, myeloid, and lymphoid potential

• CD1d serves as a surface marker for isolating CDX4-expressing hemogenic mesoderm populations

• CD1d-derived CD34+ cells robustly express HOXA7/9, indicating a specific developmental program

Research Applications:

• CD1d can be used as a surface marker to isolate CDX4-expressing populations for functional studies

• Combined analysis of CD1d and CDX4 may provide insights into the earliest stages of definitive hematopoiesis

• The CD1d/CDX4 relationship offers new approaches to studying hematopoietic specification from pluripotent stem cells

Potential Mechanisms:

• CD1d may function in creating a microenvironment conducive to CDX4 expression

• CDX4 might directly or indirectly regulate CD1d expression

• Both factors may be co-regulated by upstream developmental signals

This emerging relationship between CD1d and CDX4 opens new avenues for understanding and manipulating early hematopoietic development.