TYRO3 Mouse

What is TYRO3 and where is it expressed in mouse tissues?

TYRO3 is a receptor tyrosine kinase from the TAM (TYRO3, AXL, MERTK) family with significant neuroprotective functions. In mice, TYRO3 is highly expressed in neuronal populations, particularly in the arcuate nucleus of the hypothalamus, ventromedial nucleus, and at lower levels in the paraventricular nucleus. It shows strong expression in the hippocampus (CA1, CA3 regions and distinct cells within the dentate gyrus), serotonergic neurons in the raphe nuclei, and cerebellar cells (granule and Purkinje cells) . TYRO3 is also specifically expressed in Schwann cells but not in dorsal root ganglion (DRG) neurons . Additionally, TYRO3 appears to play a role in testis development as indicated by research on embryonic mouse gonads .

What are the primary phenotypes of TYRO3 knockout mice?

TYRO3 knockout mice exhibit reduced myelin thickness in the sciatic nerve, as evidenced by increased g-ratio (the ratio between axon diameter and outer diameter of myelinated fiber) . At 2 weeks of age, TYRO3-knockout mice show an average g-ratio of 0.69±0.048 compared to 0.66±0.053 in controls, with similar differences persisting in 2-month-old mice (0.65±0.058 in knockouts vs. 0.61±0.056 in controls) . These mice also show decreased expression levels of myelin markers like myelin protein zero (MPZ/P0) in sciatic nerve extracts and impaired formation of myelin basic protein (MBP)-positive myelin segments in dorsal root ganglion cultures . Unlike anorexia (anx/anx) mouse models, no abnormalities in size or appetite have been reported specifically for TYRO3-/- mice, suggesting possible compensatory mechanisms in appetite regulatory circuitry .

How should researchers design experiments to study TYRO3's role in myelination?

When investigating TYRO3's role in myelination, researchers should implement both in vitro and in vivo approaches due to potential discrepancies between these systems. In vitro studies using dorsal root ganglion (DRG) cultures from TYRO3-knockout mice show more pronounced impairments in myelin formation compared to the less severe phenotypes observed in vivo in sciatic nerves . This difference may result from varying growth factor availability or extracellular matrix composition between the two environments.

A comprehensive experimental design should include:

• Electron microscopy analysis of myelin thickness and g-ratio measurements

• Immunoblotting for myelin markers (MBP, MPZ/P0)

• Immunofluorescence staining of cultured DRGs

• Analysis of myelination-associated transcription factors

• Assessment of TYRO3 downstream signaling partners (particularly Fyn)

Researchers should examine multiple time points (as phenotypes were detected from 2 weeks to 2 months of age) and consider potential compensatory mechanisms from other TAM receptors .

What molecular mechanisms underlie TYRO3's effect on neuronal survival in mouse models?

TYRO3 exerts its neuroprotective effects through interaction with the nonreceptor tyrosine kinase Fyn, which was identified as a binding partner of the TYRO3 intracellular domain through affinity chromatography . In TYRO3-knockout mice, Fyn activity is downregulated, suggesting that TYRO3 regulates myelination through Fyn activation . This represents a newly recognized receptor-linked signaling mechanism controlling Schwann cell myelination.

The neuroprotective role of TYRO3 is particularly evident in appetite regulatory circuitry. In anx/anx mice (a mouse model of anorexia), wild-type TYRO3 transgenes delay hypothalamic Npy+ neuron degeneration, suggesting TYRO3 helps maintain these neurons . It's important to note that the absence of similar phenotypes in TYRO3-/- mice may be due to compensation mechanisms, as research on AgRP/Npy+ neuron ablation shows that gradual loss allows for circuit adaptation, whereas acute loss disrupts feeding behavior .

How do TYRO3 knockout phenotypes interact with other gene mutations in mice?

The interaction between TYRO3 and other genetic mutations reveals important functional redundancies and synergies:

• TYRO3 and other TAM receptors: Triple knockouts of TYRO3/AXL/MERTK exhibit more severe phenotypes, including cellular degeneration in the neocortex, hippocampus, cerebellum, and retina, illustrating redundant reinforcing neuroprotective roles .

• TYRO3 and MERTK: Loss of TYRO3 accelerates retinal degeneration in MERTK-/- mice, suggesting a compensatory protective role .

• TYRO3 and AXL: Mice lacking both AXL and TYRO3 show compromised migration and survival of gonadotrophin release hormone (GnRH)-expressing neurons into the hypothalamus .

• TYRO3 and anx mutation: The R7W-TYRO3 variant (C19T mutation in the signal sequence) acts as a strain-specific modifier of anx phenotypes. Wild-type TYRO3 transgenes doubled weight and lifespans of anx/anx mice relative to non-transgenic anx/anx littermates, while the R7W-TYRO3 variant could not provide this rescue .

This suggests researchers should carefully consider genetic background and potential modifier genes when interpreting TYRO3 knockout phenotypes.

What is the specific role of TYRO3 in mouse testis development?

TYRO3, along with other TAM family members (AXL and MERTK), appears to have redundant roles in regulating germ cell development during early testis development . Inhibition of the TAM family in embryonic day 11.5 (E11.5) ex vivo cultured male mouse gonads results in reduced germ cell numbers due to decreased proliferation and increased apoptosis .

How does TYRO3 contribute to the anorexia (anx/anx) mouse model phenotypes?

TYRO3 plays a significant role in the anorexia (anx/anx) mouse model, serving as an important modifier of the anx phenotype. Transgenic expression of wild-type TYRO3 in anx/anx mice doubled their weight and lifespans compared to non-transgenic anx/anx littermates . At postnatal day 21 (P21), anx/anx mice transgenic for TYRO3-GFP or human TYRO3 showed significant body weight increases (average 82.3±3.6%) compared to non-transgenic anx/anx littermates .

Wild-type TYRO3 expression delayed hypothalamic Npy+ neuron degeneration in anx/anx mice, suggesting a neuroprotective role in appetite regulatory circuitry . Interestingly, the R7W-TYRO3 variant (containing a C19T mutation in the signal sequence) could not rescue these phenotypes, indicating that proper TYRO3 function is critical for this neuroprotective effect .

The study also noted abnormal RNA localization of the mutated TYRO3 in anx/anx brains at P19, with C19T variant TYRO3 RNA appearing concentrated in cell soma and often in aggregates, suggesting that proper RNA localization is important for TYRO3 function .

What roles does TYRO3 play in mouse nervous system myelination?

TYRO3 plays a crucial role in promoting myelination in the mouse peripheral nervous system through its interaction with Fyn tyrosine kinase. TYRO3 knockout mice exhibit reduced myelin thickness in the sciatic nerve, quantified by an increased g-ratio . The phenotype is evident from 2 weeks to at least 2 months of age, indicating a persistent requirement for TYRO3 in maintaining proper myelination .

TYRO3 is specifically expressed in Schwann cells (the peripheral myelin-forming glial cells) but not in DRG neurons . Cultures from TYRO3 knockout mice show impaired formation of myelin segments positive for myelin basic protein (MBP), and decreased expression of myelin protein zero (MPZ/P0) is observed in TYRO3-knockout sciatic nerve extracts .

Mechanistically, TYRO3 acts through its binding partner Fyn, a nonreceptor tyrosine kinase. Fyn activity is down-regulated in TYRO3-knockout mice, and ablating Fyn in mice similarly results in reduced myelin thickness . This TYRO3-Fyn signaling axis represents a receptor-linked mechanism controlling Schwann cell myelination.

What are the most effective methods to study TYRO3 expression patterns in mouse tissues?

Based on the research, the most effective methods to study TYRO3 expression patterns include:

For comprehensive analysis, researchers should combine these techniques to assess both RNA and protein expression at different developmental stages. Special attention should be paid to subcellular localization, as abnormal RNA localization was observed in mutant TYRO3 (C19T variant) .

What experimental systems best model TYRO3 function in myelination studies?

For studying TYRO3's role in myelination, researchers should consider:

• Ex vivo DRG cultures: Dorsal root ganglion cultures from TYRO3 knockout mice showed impaired formation of myelin segments, making this a sensitive system for detecting TYRO3-dependent myelination . These cultures can be immunostained for myelin markers like MBP.

• In vivo sciatic nerve analysis: Electron microscopy analysis of sciatic nerves allows for precise measurement of myelin thickness through g-ratio quantification (ratio between axon diameter and outer diameter of myelinated fiber) . This is essential for confirming in vivo relevance of findings.

• Primary Schwann cell cultures: Given that TYRO3 is specifically expressed in Schwann cells, isolated primary cultures allow for cell-autonomous studies of TYRO3 function .

• Transgenic rescue experiments: Using transgenic expression of wild-type or mutant TYRO3 in knockout backgrounds can help determine structure-function relationships .

Researchers should consider both in vitro and in vivo approaches, as phenotypes may differ between these systems. The time point of analysis is also critical, as myelin defects in TYRO3 knockout mice were observed from 2 weeks to 2 months of age .

How can researchers effectively manipulate TYRO3 signaling in mouse experimental models?

Researchers can employ several approaches to manipulate TYRO3 signaling in mouse models:

• Knockout models: Complete genetic deletion of TYRO3 provides insights into its necessity for normal function .

• Conditional knockout models: Tissue-specific or temporally controlled deletion can help distinguish primary from secondary effects and overcome potential developmental compensation.

• Transgenic expression: Overexpression of wild-type or mutant forms can be used for rescue experiments or to study gain-of-function effects, as demonstrated with TYRO3-GFP and human TYRO3 transgenes in anx/anx mice .

• Pharmacological inhibition: TAM family inhibitors like BMS-777607 or LDC1267 can be used in ex vivo gonad cultures to study acute inhibition effects .

• Downstream signaling manipulation: Since Fyn was identified as a binding partner and downstream effector of TYRO3, manipulating Fyn can provide insights into TYRO3 signaling mechanisms .

• Point mutations: Specific mutations like R7W-TYRO3 (C19T) can be used to study structure-function relationships .

Researchers should consider possible redundancy with other TAM receptors and may need to generate compound knockouts to observe certain phenotypes .

What are the current contradictions in TYRO3 mouse model data that require resolution?

Several contradictions in TYRO3 research require further investigation:

• Phenotypic differences between single and compound knockouts: While triple TAM receptor knockouts show severe neurodegeneration, single TYRO3 knockout mice show limited phenotypes, suggesting complex redundancy that is incompletely understood .

• In vitro versus in vivo phenotype discrepancies: TYRO3 knockout mice show more pronounced myelination defects in DRG cultures compared to sciatic nerves, suggesting environmental factors may modulate TYRO3 function .

• Absence of metabolic phenotypes in TYRO3 knockouts: Despite TYRO3's apparent role in maintaining Npy+ neurons in the appetite regulatory circuitry (demonstrated through rescue experiments in anx/anx mice), TYRO3 knockout mice don't show reported abnormalities in size or appetite . This suggests either compensatory mechanisms or subtleties in phenotype that previous studies missed.

• Temporal dynamics of TYRO3 function in testis development: While TYRO3 knockout affects gene expression at E12.5 (reduced germ cell genes, increased Sertoli cell genes), these expression levels normalize by E14.5, leaving questions about the longer-term consequences of these transient changes .

Resolving these contradictions will require more sophisticated genetic approaches, careful temporal analyses, and consideration of potential compensation mechanisms.

What advanced technical approaches are emerging for TYRO3 functional studies in mice?

Emerging technical approaches for TYRO3 functional studies include:

• Single-cell RNA sequencing: This can provide higher resolution of cell type-specific TYRO3 expression and responses to TYRO3 manipulation, particularly important given the cell-specific expression patterns observed .

• Temporally controlled conditional knockouts: Using systems like tamoxifen-inducible Cre/loxP to delete TYRO3 at specific developmental stages can help distinguish developmental versus maintenance roles and overcome compensatory mechanisms.

• CRISPR/Cas9 genome editing: Creating precise point mutations (like the R7W-TYRO3 variant) to study structure-function relationships without the variability of transgenic approaches.

• Phosphoproteomics: Given TYRO3's role as a tyrosine kinase receptor, comprehensive analysis of phosphorylation changes in knockout or overexpression models can identify novel signaling pathways.

• Ex vivo organ culture systems: As demonstrated with embryonic gonad cultures , these systems allow for controlled manipulation of TYRO3 signaling while maintaining tissue architecture.

• Intravital imaging: Real-time visualization of myelination processes in TYRO3 mutant versus wild-type mice could provide dynamic insights into how TYRO3 affects Schwann cell behavior.

• Receptor-specific ligand manipulation: Developing tools to selectively activate or block TYRO3 without affecting other TAM receptors would help distinguish TYRO3-specific functions.

How should researchers approach the redundancy between TAM family receptors in mouse studies?

When addressing TAM receptor redundancy, researchers should consider:

• Compound knockout strategy: Generate double (TYRO3/AXL, TYRO3/MERTK) or triple knockouts to uncover phenotypes masked by redundancy . Previous studies show that while single knockouts have limited phenotypes, double and triple knockouts reveal more severe defects.

• Tissue-specific analysis: Focus on tissues where TYRO3 shows unique expression patterns distinct from AXL and MERTK, such as specific neuronal populations in the arcuate nucleus, dentate gyrus, and serotonergic neurons .

• Molecular specificity examination: Identify TYRO3-specific binding partners (like Fyn) that may not interact with other TAM receptors to uncover unique signaling pathways .

• Acute versus chronic manipulation: Use pharmacological inhibitors (like BMS-777607 or LDC1267) for acute inhibition studies , which may reveal functions before compensation occurs, compared with genetic knockout models where developmental compensation may mask phenotypes.

• Quantitative expression analysis: Determine the relative expression levels of each TAM receptor in tissues of interest, as the predominant receptor may play a more significant role.

• Rescue experiments: Test whether overexpression of other TAM family members can rescue TYRO3 knockout phenotypes to determine functional equivalence.

• Ligand specificity studies: Examine whether TYRO3 responds to specific ligands not shared by other TAM receptors, which could reveal context-dependent functions.