UGDH Mouse

What is the structure and genomic location of the mouse Ugdh gene?

The mouse Ugdh gene is composed of 10 exons spanning approximately 15 kilobases of genomic DNA . Through interspecific backcross analyses, the gene has been localized to mouse chromosome 5 at approximately 39 centimorgans, which corresponds to human chromosome 4p13-15 for the UGDH gene . The mouse Ugdh cDNA encodes a protein of 493 amino acids, which is 24-25 residues longer at its carboxyl terminus than the previously reported bovine Udpgdh sequence .

Southern blot analysis strongly indicates that Udpgdh is encoded by a single gene in the mouse genome . The gene's structure is highly conserved, reflecting the evolutionary importance of this enzyme in various developmental and metabolic processes.

What is the enzymatic function of UGDH and what pathways does it participate in?

UGDH (EC 1.1.1.22) catalyzes the oxidation of UDP-glucose to UDP-glucuronate, a reaction that involves NAD+ as a cofactor . The enzymatic reaction can be represented as:

UDP-glucose + 2 NAD+ + H2O → UDP-glucuronate + 2 NADH + 2 H+

This conversion is critical for several metabolic pathways:

UDP-glucuronate serves as an essential precursor for glycosaminoglycan synthesis, which are key components of the extracellular matrix . Research suggests that glycosaminoglycan biosynthesis may be partly regulated by the availability of activated UDP-glucuronate, as determined by relative Udpgdh expression levels .

What is the expression pattern of Ugdh in mouse embryonic and adult tissues?

Northern analyses have indicated widespread expression of the Ugdh gene in both embryonic and adult mouse tissues . The expression pattern shows specific temporal and spatial regulation during development, particularly in tissues undergoing morphogenesis.

During mouse heart development, Ugdh shows highly localized expression at sites of endocardial cushion formation preceding valve morphogenesis :

• At embryonic day 11.5 (E11.5), strong expression is observed in the endocardial cushions within the atrioventricular (AV) canal, with both endothelial cells covering the cushions and mesenchymal cells within the cushions staining positive

• At E12.5, Ugdh mRNA is present in endocardial cushions of the AV canal, dorsal mesenchymal protrusion, and outflow tract cushions

• By E14.5, specific expression is observed in the valve leaflets located in the AV canal

This specific expression pattern correlates with UGDH's essential role in embryonic development, particularly in heart valve formation and gastrulation processes .

How can I generate Ugdh knockout models in mice or mouse cell lines?

Several approaches have been successfully employed to generate Ugdh knockout models:

CRISPR-Cas9 for Cell Line Knockouts:

For generating Ugdh knockout in mouse cell lines, CRISPR-Cas9 technology has proven highly effective :

• Design guide RNAs targeting exonic regions of the Ugdh gene

• Transiently transfect cells with ribonucleoprotein (RNP) complexes of Cas9 protein and Ugdh-targeting sgRNA

• Isolate and screen clonal populations for frameshift mutations

• Validate knockout through sequencing, Western blotting, and functional assays

• Establish control populations with wild-type Ugdh from the same transfection experiment

ENU-Induced Mutagenesis for Mouse Models:

The "lazy mesoderm" (lzme) mutation that disrupts mouse Ugdh was generated through ENU (N-ethyl-N-nitrosourea) mutagenesis :

• Treat male mice with ENU to induce random mutations

• Breed treated males to identify phenotypes of interest

• Map and sequence mutations to confirm disruption of the Ugdh gene

• Maintain heterozygous carriers for experimental breeding

Conditional Knockout Approaches:

For tissue-specific or inducible Ugdh knockout:

• Generate mice with loxP sites flanking critical exons of Ugdh

• Cross with tissue-specific or inducible Cre-expressing mouse lines

• Validate knockout efficiency through tissue-specific analysis

Each approach allows researchers to study different aspects of Ugdh function, from cellular processes to whole-organism development.

What are the best methods to detect and measure UGDH activity in mouse tissues?

Several complementary methods can be employed to detect and measure UGDH activity:

Enzymatic Activity Assays:

Direct measurement of UGDH enzymatic activity involves:

• Homogenize tissues under conditions that preserve enzyme activity

• Incubate with UDP-glucose substrate and NAD+ cofactor

• Monitor NADH production spectrophotometrically at 340 nm

• Calculate specific activity using purified recombinant UGDH as a standard

Commercially available mouse Ugdh recombinant proteins can serve as positive controls for activity assays .

Metabolite Measurement via LC-MS:

For indirect assessment of UGDH activity:

• Extract metabolites from tissues or cells using appropriate solvent systems

• Separate and identify UDP-glucuronate using liquid chromatography-mass spectrometry

• Quantify UDP-glucuronate levels relative to internal standards

• Compare levels between wild-type and Ugdh-manipulated samples

Downstream Product Analysis:

Since UGDH is essential for glycosaminoglycan synthesis:

• Stain tissues for hyaluronan using biotinylated hyaluronan-binding protein

• Quantify glycosaminoglycans using dimethylmethylene blue assays

• Analyze specific glycosaminoglycan structures via mass spectrometry

In zebrafish models, knockdown of ugdh resulted in reduced hyaluronan accumulation in multiple tissues, including the heart, providing a visual readout of UGDH activity .

How can I validate UGDH expression and localization in mouse developmental studies?

A multi-technique approach is recommended for comprehensive validation:

Immunohistochemistry/Immunofluorescence:

Using specific antibodies against UGDH :

• Prepare tissue sections from developmental time points of interest

• Perform antigen retrieval if necessary

• Incubate with primary anti-UGDH antibodies

• Detect with fluorescent or enzyme-conjugated secondary antibodies

• Analyze cellular and subcellular localization

In Situ Hybridization:

For mRNA localization:

• Design antisense RNA probes complementary to Ugdh mRNA

• Apply to tissue sections from different developmental stages

• Visualize expression patterns using chromogenic or fluorescent detection

This technique has successfully revealed Ugdh expression in mouse heart tissues at E11.5, E12.5, and E14.5 .

Quantitative RT-PCR:

For quantitative assessment of Ugdh mRNA levels:

• Extract RNA from tissues or sorted cell populations

• Perform reverse transcription to generate cDNA

• Quantify Ugdh expression relative to housekeeping genes

• Compare expression across developmental stages

Western Blotting:

For protein expression levels:

• Extract proteins from tissues or cells

• Separate by SDS-PAGE and transfer to membranes

• Probe with anti-UGDH antibodies at recommended dilutions (e.g., 1:2000)

• Quantify relative expression levels

Combining these approaches provides comprehensive validation of both expression levels and spatial localization during development.

How does UGDH contribute to mouse embryonic gastrulation?

UGDH plays a critical role in mouse gastrulation, with evidence from the ENU-induced "lazy mesoderm" (lzme) mutation:

Essential Role in Cell Migration:

Embryos with disrupted Ugdh function arrest during gastrulation with severe defects in mesoderm and endoderm migration . This migration is essential for proper formation of the three primary germ layers and subsequent organogenesis.

Molecular Mechanism - FGF Signaling Dependency:

The primary mechanism through which UGDH affects gastrulation involves Fibroblast Growth Factor (FGF) signaling:

• Analysis of molecular markers indicates that FGF signaling is specifically blocked in lzme mutant embryos

• The phenotype closely resembles that of mutants in the FGF pathway

• Importantly, other essential gastrulation pathways (Nodal and Wnt3 signaling) appear to function normally in lzme embryos

Proteoglycan-Dependent Mechanism:

UGDH's enzymatic activity produces UDP-glucuronate, which is required for glycosaminoglycan synthesis. The research demonstrates that proteoglycans (containing glycosaminoglycan chains) are specifically required during mouse gastrulation to promote FGF signaling .

This establishes a direct mechanistic link between a metabolic enzyme (UGDH), extracellular matrix components (proteoglycans), and a major signaling pathway (FGF) in controlling a fundamental developmental process.

What is the specific role of UGDH in mouse heart valve development?

UGDH exhibits precise temporal and spatial expression patterns during heart valve development:

Stage-Specific Expression Pattern:

• At E11.5, strong Ugdh expression is observed in endocardial cushions within the AV canal

• At E12.5, expression continues in AV cushions, dorsal mesenchymal protrusion, and outflow tract cushions

• By E14.5, expression becomes concentrated in valve leaflets of the AV canal

This expression pattern precisely correlates with the timing of valve morphogenesis.

Functional Significance:

The expression of Ugdh precedes and coincides with critical stages of valve formation:

• Initial endocardial cushion formation

• Endocardial-to-mesenchymal transition (EndMT)

• Valve leaflet morphogenesis

Studies in zebrafish demonstrate that when ugdh is knocked down, there is reduced hyaluronan accumulation in the heart , suggesting conserved functions across vertebrates.

Molecular Mechanism:

UGDH produces UDP-glucuronate, essential for hyaluronan synthesis, which is a key component of the cardiac jelly and endocardial cushions. The spatiotemporal expression of Ugdh suggests it provides critical substrates for:

• Hyaluronan synthesis during cushion expansion

• Extracellular matrix composition necessary for cell migration

• Supporting signaling pathways required for EndMT

This demonstrates how a metabolic enzyme plays a specific morphogenetic role through production of substrates for extracellular matrix components essential for tissue development.

How does UGDH affect FGF signaling during mouse development?

UGDH has a specific and profound impact on FGF signaling during mouse development:

Direct Evidence from Ugdh Mutants:

Studies of the lzme mutation, which disrupts the Ugdh gene, provide compelling evidence for UGDH's role in FGF signaling:

• Mutant embryos arrest during gastrulation with defects resembling those seen in FGF pathway mutants

• Molecular marker analysis confirms that FGF signaling is specifically blocked in lzme mutant embryos

• Remarkably, signaling by other critical gastrulation factors (Nodal and Wnt3) remains normal

Mechanism: Proteoglycan-Dependent FGF Signaling:

The research establishes that proteoglycans, which require UDP-glucuronate produced by UGDH, are specifically required for FGF signaling during mouse gastrulation . This provides genetic evidence for a model where:

• UGDH produces UDP-glucuronate

• UDP-glucuronate is used for glycosaminoglycan synthesis

• These glycosaminoglycans form parts of proteoglycans

• Proteoglycans specifically facilitate FGF ligand-receptor interactions

• FGF signaling then directs proper cell migration during gastrulation

Pathway Specificity:

The fact that other signaling pathways remain intact in Ugdh-deficient embryos highlights a selective dependency of FGF signaling on properly formed proteoglycans. This suggests that FGF receptors or ligands have specific structural requirements for interaction with sulfated proteoglycans to achieve efficient signal transduction.

This UGDH-FGF relationship establishes a direct link between metabolic pathways and morphogenetic signaling, explaining why defects in glycosaminoglycan synthesis can lead to specific developmental abnormalities.

How does UGDH knockout affect cancer cell migration and metastasis in mouse models?

UGDH knockout has significant effects on cancer cell behavior and metastatic capacity:

Effects on Cell Migration In Vitro:

Knockout of Ugdh in highly-metastatic 6DT1 mouse mammary cancer cells revealed:

• No significant effect on in vitro proliferation rates

• Significantly impaired migration in wound healing assays, with Ugdh-KO cells taking longer to close migration gaps

• Severely reduced chemotactic migration in Boyden chamber (Transwell) assays

Impact on Metastasis In Vivo:

When orthotopically injected into syngeneic mice:

• Ugdh-KO tumors grew significantly slower than WT tumors

• Ugdh-KO injected mice developed significantly fewer lung metastases compared to WT injected mice

Metabolite Specificity:

To confirm that the observed effects were due to UDP-glucuronate depletion specifically:

• Researchers generated Uxs1-KO cells (Uxs1 converts UDP-glucuronate to UDP-xylose)

• Uxs1-KO resulted in reduced UDP-xylose and accumulation of UDP-glucuronate

• Importantly, Uxs1-KO did not affect tumor growth or lung metastasis

This confirms that UDP-glucuronate depletion, not downstream effects on UDP-xylose, is responsible for the reduced metastatic capacity of Ugdh-KO cells.

These findings establish that UDP-glucuronate biosynthesis through UGDH is critical for cancer cell migration and metastasis in mouse models of breast cancer.

What are the metabolic consequences of UGDH inhibition in mouse cancer models?

UGDH inhibition leads to several important metabolic changes in cancer cells:

Primary Metabolic Effects:

Knockout of Ugdh in mouse cancer cells results in:

• Blockage of UDP-glucuronate production, the direct product of UGDH enzymatic activity

• Potential accumulation of the substrate UDP-glucose

• Disruption of glycosaminoglycan synthesis pathways

Downstream Glycosaminoglycan Effects:

The absence of UDP-glucuronate affects multiple glycosaminoglycan types:

• Reduced hyaluronan synthesis

• Decreased production of chondroitin sulfate and heparan sulfate proteoglycans

• Altered extracellular matrix composition

Effects on Cell Phenotype:

Interestingly, Ugdh-KO in mouse breast cancer cells leads to:

This contrasts with findings in human lung cancer cells, where UGDH knockout impaired EMT, suggesting context-dependent effects of UGDH on cancer cell phenotypes.

Metabolite Specificity:

Experiments with Uxs1 knockout cells provide important insights:

• Uxs1-KO leads to reduced UDP-xylose and accumulation of UDP-glucuronate

• Despite UDP-xylose depletion, Uxs1-KO does not affect tumor growth or metastasis

• This confirms the specific importance of UDP-glucuronate, rather than downstream metabolites

These metabolic consequences highlight UDP-glucuronate as a critical metabolite for cancer cell function, particularly in processes related to metastasis.

How does UGDH expression correlate with disease progression in mouse models of cancer?

UGDH expression shows significant correlations with cancer progression in mouse models:

Expression in Primary Tumors:

In mouse models of breast cancer:

• The highly-metastatic 6DT1 mouse mammary cancer cell line expresses Ugdh

• These cells generate triple-negative orthotopic tumors with a claudin-low gene expression signature characteristic of aggressive breast cancer

• Ugdh knockout leads to slower tumor growth, suggesting UGDH expression promotes primary tumor growth

Correlation with Metastatic Potential:

The most striking correlation is between UGDH expression and metastatic capacity:

• Ugdh-KO in breast cancer cells significantly decreases metastatic capacity in syngeneic mice

• Ugdh-KO tumors produce significantly fewer lung metastases compared to WT tumors

• This suggests UGDH expression is particularly important for the metastatic phase of cancer progression

Clinical Relevance:

Findings from mouse models align with human cancer data:

• UGDH expression is associated with worse breast cancer patient survival, particularly in poor-prognosis subtypes

• High expression of genes in the UDP-glucose pathway correlates with decreased patient survival in publicly-available datasets

• This suggests translational relevance of mouse model findings to human disease

Mechanistic Basis for Correlation:

The mechanism by which UGDH promotes disease progression appears related to:

• Enhanced cell migration, which is impaired in Ugdh-KO cells

• Production of UDP-glucuronate for glycosaminoglycan synthesis

• Creation of a favorable tumor microenvironment supporting invasion and metastasis

• Specific effects on metastatic processes rather than primary cellular proliferation

These correlations highlight UGDH as both a potential biomarker for metastatic risk and a therapeutic target for preventing cancer metastasis.