Midkine Mouse

What is Midkine and why is it important in mouse developmental studies?

Midkine is a small secreted heparin-binding protein (13-18 kD) that belongs to the neurite growth-promoting factor family, along with pleiotrophin (PTN). It was named "midkine" because it was originally identified as a cytokine highly expressed during mid-gestation in many organs of the mouse, particularly the kidneys, heart, and brain . Midkine promotes growth through effects on cell proliferation, migration, and differentiation, making it a critical factor in developmental biology . Mouse models are particularly valuable for studying Midkine because they demonstrate high expression levels (approximately 10-fold greater than in humans across cell types) and have well-characterized expression patterns throughout development .

How does the structure of mouse Midkine compare to human Midkine?

Mouse and human Midkine share significant structural similarities. The mRNA and protein structures are similar, with an amino acid sequence predicted to have 83% homology between species . Both consist of 121 amino acids and are highly endowed with positively charged basic amino acids (arginine, lysine, and histidine) . The protein structure is composed of N-terminal and C-terminal halves linked by five disulfide bonds, with the C-terminal portion containing a strong conformation-dependent heparin binding site that is crucial for neurite extension and plasminogen activator activities, but not for promoting cell survival .

What are the primary receptors for Midkine in mouse models?

Midkine interacts with several receptors in mouse models, which complicates understanding its specific roles. Key receptors include:

• Syndecans (particularly Syndecan-1 and Syndecan-3)

• Protein tyrosine phosphatase ζ (PTPζ)

• Low-density lipoprotein receptor-related protein (LRP)

• Anaplastic lymphoma kinase (ALK)

• Notch2

Syndecan-3 appears particularly important in neural tissues, with expression data showing it is more highly expressed than Syndecan-1 in both mouse and human brain tissues . Researchers should note that Midkine's interaction with multiple receptors (each having numerous potential ligands) limits the ability to delineate specific activities through simple receptor blockade or knockout studies .

What is the temporal and spatial expression pattern of Midkine during mouse development?

Midkine expression during mouse development follows a distinct pattern:

• Detected as early as embryonic day 5 (E5) in the ectoderm, allantois, and chorion of placental tissues

• By E8.5, expression is found throughout the whole mouse embryo and extra-embryonic membranes

• Strong mRNA expression throughout the developing cortical plate at E14.5 and E15.5

• Expression in jaw, hindlimb bud, skin, placental capillary endothelial cells, brain, and spinal cord at E14.5

• By E17 in rats (comparable developmental stage in mice), Midkine immunoreactivity emerges radially from the ventricular zone into the telencephalon, with most intense expression in the intermediate zone and subventricular zones

• Expression decreases after birth, unlike Pleiotrophin which increases from birth and persists into adulthood

For reference, gene expression profiling data across different brain regions and developmental stages is available in Figures 2 and 5 of the cited research .

How does Midkine expression differ across cell types in the mouse brain?

Cell-type specific analysis using RNA-sequencing has revealed considerable variation in Midkine expression across neural cell types. Data from mouse brain tissues shows that Midkine expression is approximately 10-fold greater across all cell types compared to human counterparts . In the embryonic mouse brain at E14.5-E15.5, Midkine mRNA is strongly expressed throughout the developing cortical plate . During later developmental stages, single-cell RNA-sequencing has revealed that Midkine and Pleiotrophin are upregulated by Müller glia during later stages of development in retinal tissues . Following injury, Midkine is dramatically upregulated in mature Müller glia in chick retinas but interestingly downregulated in mouse retinas .

What are the phenotypic consequences of Midkine knockout in mice?

Midkine knockout (KO) mice exhibit several distinctive phenotypic characteristics:

• Delayed hippocampal development, shown by a transient abnormal increase in calretinin in the granule cell layer of the dentate gyrus

• Increased anxiety and impaired working memory as assessed via elevated plus maze test and y-maze test, respectively

• Reduced striatal dopamine content, suggesting increased vulnerability for behavioral disorders such as schizophrenia and autism

• Auditory deficits, which are more severe in Midkine/Pleiotrophin double knockout mice

Most notably, Midkine/Pleiotrophin double knockout (DKO) mice:

• Are born at one-third the expected frequency based on Mendelian segregation, suggesting embryonic lethality prior to E14.5

• Present with severe postnatal growth retardation (50% reduction at 4 weeks of age) that is not corrected with high-calorie feeding

• Show 40-50% reduction in spontaneous locomotor activity at 4 weeks of age compared to wild-type mice

What techniques are recommended for studying Midkine expression in mouse models?

Several complementary techniques are recommended for comprehensive analysis of Midkine expression:

• RNA-sequencing and transcriptomics:

  • Single-cell RNA-sequencing offers high-resolution cell-type specific expression data

  • Resources like http://www.brainrnaseq.org/ provide valuable reference datasets

  • For embryonic expression patterns, resources like Genepaint (https://gp3.mpg.de/) and GENSAT (http://www.gensat.org/) provide validated expression data

• Immunohistochemistry/Immunofluorescence:

  • Effective for spatial localization within tissues

  • Should include controls for antibody specificity

  • Can be combined with cell-type specific markers for colocalization studies

• In situ hybridization:

  • Particularly valuable for embryonic tissues

  • Allows precise localization of mRNA expression patterns

  • Can be performed at high resolution using RNAscope technology

• Western blotting:

  • For quantitative analysis of protein levels

  • Should be normalized to appropriate housekeeping proteins

  • Can detect different Midkine isoforms

How should Midkine knockout or knockdown experiments be designed and validated?

When designing Midkine knockout or knockdown experiments:

• Generation of knockout models:

  • Consider both conventional and conditional knockout approaches

  • Currently, there are no conditional KO mice for Midkine, which limits the ability to study nuanced functions

  • Double knockout with Pleiotrophin should be considered due to functional redundancy

• Validation recommendations:

  • Confirm knockout at both mRNA (RT-qPCR) and protein (Western blot, immunohistochemistry) levels

  • Validate across multiple tissues due to tissue-specific expression

  • Check for compensatory upregulation of related proteins (particularly Pleiotrophin)

  • Assess developmental timepoints carefully, as effects may be stage-specific

• Experimental considerations:

  • Include appropriate littermate controls

  • Consider the impact of genetic background on phenotype

  • Due to potential embryonic lethality in double knockouts, carefully time experiments and consider using heterozygous breeding schemes

What methodological approaches are effective for studying Midkine's neuroprotective functions?

To investigate Midkine's neuroprotective functions:

• In vitro approaches:

  • Primary neuronal cultures subjected to oxygen-glucose deprivation

  • Excitotoxicity models using glutamate exposure

  • Assessment of apoptotic markers (cleaved caspase-3, TUNEL staining)

  • Measurement of key signaling pathways (e.g., pS6, cFos expression)

• In vivo approaches:

  • Traumatic brain injury models - assess apoptosis via cleaved caspase-3 expression

  • Retinal damage models - quantify dying neurons and reactive microglia

  • Exogenous Midkine administration with appropriate delivery methods

  • Inhibition of Midkine signaling using Na₃VO₄ to block neuroprotective effects

• Analysis methods:

  • Flow cytometry to segregate and analyze specific cell populations

  • Assessment of microglial/macrophage polarization using markers for M1 (CD16/32+) and M2 (arginase1+) phenotypes

  • mRNA analysis of inflammatory markers (TNFα, CD11b)

  • Quantification of cell death and survival

How does Midkine influence neuroinflammation in mouse models of brain injury?

Midkine plays a complex role in neuroinflammation after brain injury:

• Microglial/macrophage response:

  • In traumatic brain injury (TBI) models, Midkine knockout mice show reduced microglial/macrophage Iba1-immunoreactivity at 3 days post-injury compared to wild-type mice

  • Midkine appears to modulate the polarization of microglia, with knockout mice showing fewer pro-inflammatory M1 (CD16/32+) cells in perilesional sites and reduced mRNA levels of M1 markers (TNFα, CD11b)

  • Flow cytometry analysis reveals that Midkine knockout mice have increased levels of anti-inflammatory M2 arginase1+ microglia and M2 CD163+ macrophages

• Chemoattractant properties:

  • Midkine serves as a chemoattractant for leukocytes, potentially amplifying peripheral immune cell recruitment to injury sites

  • This may explain why Midkine knockout mice had reduced apoptosis and improved neurological outcomes following TBI

  • The effect appears to be age-dependent, with different impacts on blood-brain barrier permeability, chemokine function, and leukocyte recruitment after CNS injury

• Developmental considerations:

  • Age-dependent differences in blood-brain barrier permeability significantly impact Midkine's effects

  • In contrast to adult models, developmental models may show different inflammatory responses to Midkine modulation

What is known about Midkine's role in retinal development and injury response?

Recent research on Midkine in retinal tissues has revealed:

• Expression patterns:

  • Single-cell RNA-sequencing shows that Midkine and Pleiotrophin are upregulated by Müller glia during later stages of development in chick retina

  • After retinal damage or FGF2 and insulin treatment, Midkine is dramatically upregulated in mature Müller glia in chick retinas

  • Interestingly, Midkine is downregulated by Müller glia in damaged mouse retinas, showing species-specific responses

• Functional effects:

  • In both chick and mouse retinas, exogenous Midkine induces expression of cFos and pS6 in Müller glia

  • In chick retina, Midkine significantly decreases:

    • Numbers of dying neurons

    • Reactive microglia

    • Proliferating Müller glia-derived progenitor cells (MGPCs)

  • In mice, Midkine:

    • Promotes a small but significant increase in proliferating MGPCs in damaged retinas

    • Potently decreases the number of dying cells

• Signaling pathway interactions:

  • Inhibition of Midkine signaling with Na₃VO₄ blocks neuroprotective effects and increases cell death

  • Inhibitors of PP2A and Pak1, which are associated with Midkine signaling through integrin β1, suppressed the formation of MGPCs in damaged chick retinas

How can Midkine be utilized as a therapeutic agent in mouse models of brain injury?

Midkine shows promise as a therapeutic agent for brain injury, though methodological considerations are important:

• Delivery methods:

  • Direct intracerebroventricular or intranasal administration

  • Modified Midkine peptides with enhanced stability and brain penetration

  • Nanoparticle encapsulation for targeted delivery

  • Recombinant Midkine can be produced in various expression systems and tagged with fluorophores for tracking

• Therapeutic applications:

  • Amelioration of cell death in hypoxic-ischemic injury models

  • Modulation of glial reactivity after injury

  • Enhancement of proliferation and migration of neural precursor cells

  • Promotion of hypoxia-induced angiogenesis

• Research gaps and future directions:

  • Most studies have been conducted in vitro with limited in vivo validation

  • Need for studies in models that incorporate the maternal-placental-fetal unit, such as the precocial spiny mouse or fetal and newborn sheep

  • Investigation of potential Midkine deficiency in preterm birth scenarios, given high levels in amniotic fluid

  • Development of improved drug delivery platforms to enhance therapeutic efficacy and bioavailability

How do Midkine expression patterns differ between mouse and human brain development?

Important species differences exist in Midkine expression patterns:

• Quantitative differences:

  • Across cell types, Midkine expression is approximately 10-fold greater in mouse compared to humans

  • This substantial difference should be considered when translating findings from mouse models to human applications

• Receptor expression differences:

  • RNA sequencing suggests that Syndecan-1 expression is approximately 100-fold less in humans than in mice

  • Syndecan-3 expression is about 50-fold less in humans compared to mice

  • These differences may indicate fundamental physiological variations that prevent simple extrapolation of findings between species

• Research implications:

  • Results from rodent experiments must be interpreted cautiously due to these expression differences

  • Protein abundance or stability differences in humans may potentially counteract higher expression of mouse mRNA

  • Validation in human cell or tissue models is strongly recommended before clinical translation

What methodological challenges exist in studying Midkine function across species?

Several key challenges complicate cross-species studies of Midkine:

• Receptor redundancy and complexity:

  • Midkine interacts with several receptors, each with numerous potential ligands

  • This limits the ability to delineate activity through receptor blockade or knockout studies

  • Structural and functional similarities between Midkine and Pleiotrophin create redundancy in signaling systems

• Technical limitations:

  • Lack of conditional knockout models for Midkine limits nuanced investigation of functions

  • Complex feedback loops between Midkine and Pleiotrophin complicate interpretation of single-protein manipulations

  • Antibody specificity issues may arise when detecting Midkine across different species

• Developmental timing differences:

  • Cross-species comparisons must account for differences in developmental timing and maturation

  • Expression patterns that appear similar may occur at different relative developmental stages

  • These differences necessitate careful experimental design when comparing developmental processes across species

What should researchers consider when designing translational studies using mouse Midkine models?

For translational studies involving Midkine in mouse models:

• Model selection considerations:

  • Consider models that incorporate the maternal-placental-fetal unit for developmental studies

  • The precocial spiny mouse or fetal and newborn sheep may provide better translational value for certain questions

  • Use appropriate developmental timepoints that correspond to human developmental stages of interest

• Validation requirements:

  • Validate key findings in human cell cultures or organoids when possible

  • Consider the species differences in receptor expression when interpreting results

  • Account for the 10-fold difference in Midkine expression levels between mice and humans

• Therapeutic development suggestions:

  • Focus on functional outcomes rather than mechanistic details that may differ across species

  • Consider the potential impact of species-specific inflammatory responses

  • Develop improved drug delivery platforms to enhance therapeutic efficacy and bioavailability in target tissues

  • Evaluate dose-response relationships carefully, given the substantial expression differences between species