DHH (C23II) Mouse

What is Desert Hedgehog (DHH) and how does it differ from other hedgehog proteins?

Desert Hedgehog (DHH) belongs to the highly conserved Hedgehog family of proteins involved in multiple developmental processes. In mammals, there are three hedgehog family members: Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh), each with distinct tissue expression patterns and developmental roles . DHH shares approximately 74% amino acid sequence identity with mouse Indian and Sonic hedgehog in the N-terminal peptide region . The most significant differences lie in their expression patterns and tissue-specific functions, with DHH being predominantly expressed in Sertoli cells of the testes, Schwann cells of peripheral nerves, and ovarian granulosa cells .

Unlike Shh, which has broader developmental roles across numerous tissues, DHH has more specialized functions in gonadal development and peripheral nerve formation. Methodologically, researchers should consider these tissue-specific expression patterns when designing experiments targeting DHH signaling.

What is the significance of the C23II notation in recombinant mouse DHH?

The C23II notation in recombinant mouse DHH indicates a specific modification where the cysteine at position 23 has been substituted with isoleucine. This recombinant form encompasses the N-terminal fragment spanning from Cys23 to Gly198, with the modification Cys23Ile-Ile and an additional N-terminal methionine . This modification is significant because:

• It affects the protein's ability to undergo autocatalytic processing

• It may alter the binding characteristics to receptors like Patched

• It provides a more stable recombinant protein for experimental applications

Researchers should be aware that this modified version may exhibit slightly different activity compared to native DHH, although it maintains the core functional domains necessary for signaling activities.

How is DHH processed post-translationally and what impact does this have on its function?

DHH, like other hedgehog proteins, is synthesized as a 45 kDa precursor that undergoes autocatalytic cleavage to generate a 19 kDa N-terminal fragment . This N-terminal fragment remains membrane-associated due to post-translational modifications including cholesterol addition to its C-terminus and palmitate attachment to its N-terminus .

The processing sequence is:

• Translation of full-length precursor protein

• Autocatalytic cleavage mediated by the C-terminal domain

• Addition of cholesterol to the newly formed C-terminus of the N-terminal fragment

• Palmitoylation of the N-terminus

• Membrane association of the processed signaling molecule

These lipid modifications are crucial for proper DHH signaling as they:

• Restrict diffusion of the protein

• Enable formation of multimeric complexes

• Facilitate long-range signaling through extracellular vesicles

For experimental purposes, researchers must consider that recombinant versions may lack these modifications, potentially affecting activity and diffusion properties.

What is the canonical signaling pathway of DHH and how does it differ from other hedgehog proteins?

DHH signals through the canonical hedgehog pathway by binding to the Patched (Ptch) receptor, releasing Smoothened (SMO) from inhibition, and ultimately activating Gli transcription factors .

The canonical pathway proceeds as follows:

• DHH binds to Patched receptor on target cells

• This binding relieves Patched-mediated inhibition of Smoothened

• Activated Smoothened triggers intracellular signaling cascades

• Gli family transcription factors translocate to the nucleus

• Target gene transcription is initiated, including pathway components like Gli1 and Ptch

While the basic mechanism is shared with Shh and Ihh, DHH has some distinct features:

• DHH binds both Patched and Patched 2 receptors

• DHH also interacts with Hedgehog interacting protein (Hip)

• DHH has lower potency in activating pathway components compared to Shh

• DHH exhibits more restricted expression patterns and tissue-specific functions

Methodologically, researchers can assess pathway activation through quantitative PCR analysis of Gli1 and Ptch1 expression, as these are reliable readouts of hedgehog pathway activity.

What roles does DHH play in testicular development and spermatogenesis?

DHH plays critical roles in testicular development and spermatogenesis through several mechanisms :

• DHH is produced by Sertoli cells and acts on surrounding cells in the developing testis

• It induces steroidogenic factor 1 expression, which is instrumental in promoting Leydig cell differentiation

• DHH signaling promotes the deposition of basal lamina surrounding seminiferous tubules

• It contributes to proper organization of the testicular compartments

In humans, mutations in DHH are associated with pure gonadal dysgenesis, highlighting its essential role in normal gonadal development . Experimental approaches to study DHH function in testicular development include:

• Conditional knockout models with Sertoli cell-specific deletion of DHH

• Ex vivo testicular explant cultures treated with recombinant DHH protein

• Imaging of basal lamina formation in response to DHH signaling

These approaches allow researchers to dissect the temporal and cell-type specific roles of DHH in testicular development.

How does DHH function in peripheral nerve development and maintenance?

DHH is expressed by Schwann cells in the peripheral nervous system and is upregulated following nerve injury . Its functions in peripheral nerves include:

• Promoting the formation of the connective tissue sheath surrounding peripheral nerves

• Inducing the expression of Patched and Hip in nerve fibroblasts

• Contributing to nerve regeneration after injury

• Maintaining the integrity of the peripheral nerve architecture

Experimental approaches to study DHH in peripheral nerves include:

• Sciatic nerve crush injury models to examine DHH upregulation

• Immunohistochemical analysis of DHH expression in peripheral nerves

• Assessment of nerve conduction velocity in DHH knockout models

• Analysis of connective tissue formation around nerves in response to DHH signaling

The presence of DHH can be detected in neuronal cell bodies and processes of mouse brain using immunohistochemical techniques with specific antibodies like the Goat Anti-Mouse Desert Hedgehog/Dhh N-Terminus Antibody .

What are the optimal methods for detecting DHH expression in tissue samples?

Detection of DHH expression in tissue samples can be accomplished through several complementary techniques, each with specific considerations:

Immunohistochemistry (IHC):

• Fixed frozen sections yield excellent results for DHH detection

• Recommended protocol: Use Anti-Mouse Desert Hedgehog/Dhh N-Terminus Antibody at 5 μg/mL concentration, incubated overnight at 4°C

• Visualization systems like HRP-DAB provide clear staining of neuronal cell bodies and processes

• Counterstaining with hematoxylin helps visualize tissue architecture

Western Blotting:

• Effective for detecting recombinant DHH proteins

• Cross-reactivity with other hedgehog family members should be assessed

• Antibodies directed against the N-terminus of DHH provide specific detection

RT-qPCR:

• Allows quantitative assessment of DHH mRNA expression

• Requires careful primer design to avoid amplification of homologous regions in other hedgehog genes

• Reference genes should be validated for the specific tissue being analyzed

For researchers working with mouse models, neuronal tissues, testicular samples, and peripheral nerves are particularly relevant for DHH expression analysis.

How should recombinant mouse DHH (C23II) N-Terminus be reconstituted and stored for optimal activity?

Proper reconstitution and storage of recombinant mouse DHH (C23II) N-Terminus is critical for maintaining its biological activity:

Reconstitution:

• Lyophilized protein should be reconstituted at 500 μg/mL in sterile PBS

• For carrier-free preparations, avoid buffers containing BSA

• Addition of DTT may help maintain protein stability by preventing oxidation of cysteine residues

Storage:

• Use a manual defrost freezer and avoid repeated freeze-thaw cycles

• Upon receipt, store immediately at recommended temperatures

• Aliquot reconstituted protein to minimize freeze-thaw cycles

• Working solutions can typically be stored at 4°C for up to one week

Activity Considerations:

• Biological activity should be tested after reconstitution

• The half-life of activity may vary depending on experimental conditions

• The effective concentration for biological activity is typically <20 μg/mL

Researchers should validate protein activity in their specific experimental systems, as the effective concentration may vary across different cell types and assays.

What cell-based assays are most appropriate for evaluating DHH functional activity?

Several cell-based assays can be utilized to evaluate the functional activity of DHH:

Osteoblast Differentiation Assay:

• C3H10T1/2 cells are commonly used as they respond to hedgehog signaling by differentiating into osteoblasts

• Alkaline phosphatase activity serves as a reliable readout of osteoblast differentiation

• Both colorimetric and fluorometric methods can quantify alkaline phosphatase activity

• Dose-response curves with recombinant DHH can determine IC50 values

Pathway Activation Assays:

• Quantitative PCR analysis of Gli1 and Patched-1/2 mRNA expression

• Reporter assays using Gli-responsive elements driving luciferase expression

• These assays typically show activity at IC50 concentrations below 10 μM for effective DHH preparations

Cell Proliferation Assays:

• Certain cell types like granulosa cells respond to DHH with increased proliferation

• BrdU incorporation or MTT assays can measure proliferative responses

• Controls with pathway inhibitors (e.g., cyclopamine) can confirm specificity

For all assays, appropriate positive controls (such as recombinant Shh) and negative controls should be included to validate assay performance.

How can DHH signaling be targeted in experimental models of peripheral nerve injury?

Targeting DHH signaling in peripheral nerve injury models presents several experimental approaches:

Gain-of-function approaches:

• Local application of recombinant DHH protein to injury sites

• Viral vector-mediated overexpression of DHH in Schwann cells

• Conditional transgenic models with inducible DHH expression

• Ex vivo nerve explants treated with recombinant DHH

Loss-of-function approaches:

• Conditional knockout models using Schwann cell-specific Cre drivers

• siRNA or shRNA-mediated knockdown of DHH in Schwann cells

• Application of DHH-neutralizing antibodies to injured nerves

• Small molecule inhibitors of hedgehog pathway components

Readouts of nerve regeneration:

• Immunohistochemical analysis of myelin formation

• Electrophysiological assessment of nerve conduction velocity

• Behavioral testing of sensory and motor function

• Analysis of connective tissue sheath formation around regenerating axons

DHH upregulation following nerve injury suggests a potential therapeutic role in promoting nerve regeneration . Researchers should carefully time their interventions, as the temporal dynamics of DHH expression after injury are critical for optimal outcomes.

What are the implications of DHH mutations in human reproductive disorders and how can mouse models elucidate these mechanisms?

Human mutations in DHH are associated with pure gonadal dysgenesis and reproductive disorders . Mouse models can elucidate the mechanisms underlying these conditions through several approaches:

Humanized mouse models:

• Introduction of human DHH mutations into the mouse genome

• Analysis of gonadal development and fertility

• Comparison of phenotypes between mouse and human conditions

• Assessment of compensatory mechanisms in different genetic backgrounds

Mechanistic analyses:

• Examination of steroidogenic factor 1 induction in Leydig cells

• Analysis of basal lamina formation around seminiferous tubules

• Assessment of spermatogonial stem cell maintenance

• Evaluation of Sertoli cell-germ cell interactions

Translational approaches:

• Testing potential therapeutic interventions targeting downstream pathways

• Analysis of timing-dependent effects of DHH signaling restoration

• Evaluation of cell transplantation approaches for restoring fertility

• Biomarker development for early detection of DHH-related disorders

These mouse models can provide insights into the critical windows of DHH signaling during gonadal development and identify potential intervention points for human reproductive disorders.

How does DHH interact with other signaling pathways in development and disease?

DHH interacts with multiple signaling pathways, creating complex regulatory networks:

Interactions with other hedgehog family members:

• Potential redundancy or compensation between DHH, Shh, and Ihh

• Differential receptor binding affinities and downstream signaling

• Tissue-specific co-expression patterns and functional overlap

Cross-talk with other developmental pathways:

• Wnt signaling pathway interaction affects cell fate decisions

• Notch signaling coordinates with DHH during Schwann cell development

• BMP signaling modulates DHH effects on osteoblast differentiation

• Steroid hormone signaling interacts with DHH in reproductive tissues

Pathological pathway interactions:

• In ovarian cancer, DHH upregulation correlates with proliferative index and poor prognosis

• Inflammatory signaling pathways may alter DHH expression after nerve injury

• Metabolic pathways can influence cholesterol modification of DHH protein

Experimental approaches to study these interactions include:

• Multi-omics analyses of tissue samples with altered DHH signaling

• Combinatorial treatment with pathway-specific agonists/antagonists

• Conditional knockout models targeting multiple pathway components

• Proteomics approaches to identify DHH-interacting proteins

Understanding these pathway interactions is crucial for developing targeted therapies for DHH-related disorders.

What are common challenges in differentiating between DHH and other hedgehog protein activities in experimental systems?

Differentiating between DHH and other hedgehog protein activities presents several challenges:

Sequence and structural similarities:

• Mouse DHH shares 74% amino acid sequence identity with mouse Ihh and Shh in the N-terminal domain

• Similar receptor binding mechanisms make functional discrimination difficult

• Common downstream signaling pathways activate similar target genes

Cross-reactivity issues:

Technical solutions:

• Use knockout or knockdown models to eliminate specific hedgehog proteins

• Employ isoform-specific blocking antibodies for selective inhibition

• Design PCR primers targeting unique regions to distinguish mRNA expression

• Utilize mass spectrometry for definitive protein identification

Experimental design considerations:

• Study tissues with predominant expression of a single hedgehog protein

• Use recombinant proteins with confirmed specificity

• Include appropriate controls for each hedgehog family member

• Validate findings across multiple detection methods

How can researchers address variability in DHH activity across different experimental systems?

Variability in DHH activity across experimental systems can be addressed through:

Standardization approaches:

• Establish consistent reconstitution protocols for recombinant proteins

• Validate each lot of recombinant DHH for activity before use

• Develop standard curves with known active compounds

• Use internal controls across experiments

Cell system considerations:

• Different cell types may express varying levels of pathway components

• Receptor expression levels (Patched, Patched 2, Hip) affect sensitivity

• Primary cells versus cell lines may show different response thresholds

• Species differences must be considered in cross-species experiments

Environmental factors:

• Media composition affects DHH solubility and receptor binding

• Serum components may contain inhibitors or potentiators

• Cell density influences paracrine signaling efficiency

• Temperature and pH affect protein stability and receptor binding

Analytical approaches:

• Quantitative dose-response curves establish EC50/IC50 values

• Time-course experiments identify optimal response windows

• Multiple readouts of pathway activation confirm activity

• Statistical methods account for inherent biological variability

By addressing these factors systematically, researchers can minimize variability and increase reproducibility of DHH-related experiments.

What are the optimal controls for validating DHH antibody specificity in immunohistochemistry and Western blotting?

Validating DHH antibody specificity requires rigorous controls:

Positive controls:

• Recombinant mouse DHH protein (Catalog # 733-DH) for Western blotting

• Tissues with known DHH expression (testes, peripheral nerves, ovaries)

• Cells transfected with DHH expression constructs

• Tissues from wild-type animals compared to conditional knockouts

Negative controls:

• Tissues from DHH knockout animals (primary negative control)

• Irrelevant tissues known not to express DHH

• Antibody pre-absorption with recombinant DHH protein

• Isotype control antibodies at matching concentrations

Cross-reactivity assessment:

• Test against recombinant Shh and Ihh proteins

• Examine tissues with known expression of other hedgehog proteins

• Simultaneous detection with multiple hedgehog-specific antibodies

• Western blot molecular weight comparison (slight differences exist)

Technical validation:

• Multiple antibodies targeting different DHH epitopes

• Correlation of protein detection with mRNA expression

• Testing across different fixation and processing methods

• Titration experiments to determine optimal antibody concentration

For immunohistochemistry specifically, perfusion-fixed frozen sections of mouse brain and spinal cord (dorsal roots) serve as excellent positive control tissues .

What emerging technologies could advance our understanding of DHH signaling dynamics?

Several emerging technologies hold promise for advancing our understanding of DHH signaling dynamics:

Single-cell technologies:

• Single-cell RNA sequencing to identify DHH-responsive cell populations

• Single-cell proteomics to detect cell-specific pathway activation

• Mass cytometry to quantify pathway component expression at single-cell resolution

• Spatial transcriptomics to map DHH expression and signaling in tissue context

Advanced imaging approaches:

• Live-cell imaging with fluorescent DHH reporters

• Super-resolution microscopy to visualize DHH localization

• FRET/FLIM techniques to detect protein-protein interactions in real-time

• Light-sheet microscopy for whole-organ imaging of DHH signaling

CRISPR-based technologies:

• CRISPR activation/inhibition systems for temporal control of DHH expression

• Base editing for precise introduction of DHH mutations

• CRISPR screens to identify novel DHH pathway components

• In vivo CRISPR delivery for tissue-specific pathway modulation

Computational approaches:

• Machine learning algorithms to predict DHH binding partners

• Systems biology modeling of pathway dynamics

• Structural biology predictions of DHH-receptor interactions

• Multi-omics data integration for comprehensive pathway analysis

These technologies will enable more nuanced understanding of context-dependent DHH signaling and potentially reveal new therapeutic targets.

How might targeting DHH signaling be leveraged for regenerative medicine applications?

DHH signaling presents several promising avenues for regenerative medicine applications:

Peripheral nerve regeneration:

• DHH expression is upregulated following nerve injury

• Recombinant DHH could enhance Schwann cell-mediated nerve repair

• Biomaterial scaffolds delivering DHH might guide regenerating axons

• Combination therapies targeting DHH and other regenerative pathways

Gonadal tissue engineering:

• DHH's role in testicular development suggests applications in fertility restoration

• In vitro spermatogenesis protocols could benefit from DHH supplementation

• Engineered testicular organoids with DHH-expressing Sertoli cells

• Treatment of conditions associated with DHH mutations

Cancer therapeutics:

• DHH upregulation in ovarian cancer correlates with poor prognosis

• DHH pathway inhibitors might supplement existing cancer therapies

• Targeting DHH-expressing cancer stem cells could reduce recurrence

• Biomarkers based on DHH pathway activation may guide treatment selection

Translational considerations:

• Recombinant protein stability and delivery methods

• Temporal aspects of DHH signaling during regeneration

• Potential off-target effects on other tissues expressing pathway components

• Combination with cell-based therapies for synergistic effects

Early research suggests that the therapeutic window and dosing regimens will be critical factors in successful DHH-based regenerative therapies.

What are the current gaps in our understanding of DHH function compared to other hedgehog family members?

Despite significant advances, several knowledge gaps remain in our understanding of DHH compared to other hedgehog family members:

Structural and functional specificity:

• Molecular basis for differential receptor binding between DHH and other hedgehogs

• Structural determinants of DHH's more restricted tissue activity

• Differences in post-translational processing efficiency

• Species-specific variations in DHH function

Evolutionary aspects:

• Evolutionary history of functional divergence among hedgehog proteins

• Conservation of DHH-specific functions across species

• Adaptive significance of maintaining three separate hedgehog genes

• Comparative analysis of DHH function in non-mammalian vertebrates

Pathological roles:

• Comprehensive understanding of DHH in disease states beyond gonadal dysgenesis

• DHH contributions to peripheral neuropathies

• Potential involvement in metabolic disorders

• DHH in aging-related tissue degeneration

Regulatory mechanisms:

• Epigenetic regulation of DHH expression

• Non-coding RNAs targeting DHH signaling

• Post-translational modifications beyond lipidation

• Receptor trafficking and turnover in DHH-responsive cells

Addressing these knowledge gaps will require integrated approaches combining structural biology, developmental biology, and pathological investigations across multiple experimental systems.

DHH Protein Characteristics and Sequence Homology

Data compiled from search results

Experimental Detection Methods for DHH

Data compiled from search result

DHH Biological Functions in Development and Disease