Ex Vivo Oculomotor Slice Culture from Embryonic GFP-Expressing Mice for Time-Lapse Imaging of Oculomotor Nerve Outgrowth

Whitman, Mary C.; Bell, Jéssica; Nguyen, Elaine; Engle, Elizabeth C.

doi:10.3791/59911

Cited by 5 publications

(1 citation statement)

References 29 publications

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“…This could include comparative histology of brain and CN development of cleared embryos, and comparative growth properties of affected and unaffected neurons in dissociated culture, explant culture, and live 10.3389/fnins. 2023.1226181 Frontiers in Neuroscience frontiersin.org imaging in oculomotor slice culture (Whitman et al, 2019). Cryo-EM could be pursued to determine wildtype and mutant protofilament number and heterodimer angles.…”

Section: Future Directionsmentioning

confidence: 99%

TUBB3 and KIF21A in neurodevelopment and disease

2023

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Neuronal migration and axon growth and guidance require precise control of microtubule dynamics and microtubule-based cargo transport. TUBB3 encodes the neuronal-specific β-tubulin isotype III, TUBB3, a component of neuronal microtubules expressed throughout the life of central and peripheral neurons. Human pathogenic TUBB3 missense variants result in altered TUBB3 function and cause errors either in the growth and guidance of cranial and, to a lesser extent, central axons, or in cortical neuronal migration and organization, and rarely in both. Moreover, human pathogenic missense variants in KIF21A, which encodes an anterograde kinesin motor protein that interacts directly with microtubules, alter KIF21A function and cause errors in cranial axon growth and guidance that can phenocopy TUBB3 variants. Here, we review reported TUBB3 and KIF21A variants, resulting phenotypes, and corresponding functional studies of both wildtype and mutant proteins. We summarize the evidence that, in vitro and in mouse models, loss-of-function and missense variants can alter microtubule dynamics and microtubule-kinesin interactions. Lastly, we highlight additional studies that might contribute to our understanding of the relationship between specific tubulin isotypes and specific kinesin motor proteins in health and disease.

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Section: Future Directionsmentioning

confidence: 99%

TUBB3 and KIF21A in neurodevelopment and disease

2023

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A cell type-aware framework for nominating non-coding variants in Mendelian regulatory disorders

Lee,

Ayers,

Kosicki

et al. 2024

Nat Commun

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Unsolved Mendelian cases often lack obvious pathogenic coding variants, suggesting potential non-coding etiologies. Here, we present a single cell multi-omic framework integrating embryonic mouse chromatin accessibility, histone modification, and gene expression assays to discover cranial motor neuron (cMN) cis-regulatory elements and subsequently nominate candidate non-coding variants in the congenital cranial dysinnervation disorders (CCDDs), a set of Mendelian disorders altering cMN development. We generate single cell epigenomic profiles for ~86,000 cMNs and related cell types, identifying ~250,000 accessible regulatory elements with cognate gene predictions for ~145,000 putative enhancers. We evaluate enhancer activity for 59 elements using an in vivo transgenic assay and validate 44 (75%), demonstrating that single cell accessibility can be a strong predictor of enhancer activity. Applying our cMN atlas to 899 whole genome sequences from 270 genetically unsolved CCDD pedigrees, we achieve significant reduction in our variant search space and nominate candidate variants predicted to regulate known CCDD disease genes MAFB, PHOX2A, CHN1, and EBF3 – as well as candidates in recurrently mutated enhancers through peak- and gene-centric allelic aggregation. This work delivers non-coding variant discoveries of relevance to CCDDs and a generalizable framework for nominating non-coding variants of potentially high functional impact in other Mendelian disorders.

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Decreased ACKR3 (CXCR7) function causes oculomotor synkinesis in mice and humans

Whitman

Miyake

Nguyen

et al. 2019

Human Molecular Genetics

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Oculomotor synkinesis is the involuntary movement of the eyes or eyelids with a voluntary attempt at a different movement. The chemokine receptor CXCR4 and its ligand CXCL12 regulate oculomotor nerve development; mice with loss of either molecule have oculomotor synkinesis. In a consanguineous family with congenital ptosis and elevation of the ptotic eyelid with ipsilateral abduction, we identified a co-segregating homozygous missense variant (c.772G>A) in ACKR3, which encodes an atypical chemokine receptor that binds CXCL12 and functions as a scavenger receptor, regulating levels of CXCL12 available for CXCR4 signaling. The mutant protein (p.V258M) is expressed and traffics to the cell surface but has a lower binding affinity for CXCL12. Mice with loss of Ackr3 have variable phenotypes that include misrouting of the oculomotor and abducens nerves. All embryos show oculomotor nerve misrouting, ranging from complete misprojection in the midbrain, to aberrant peripheral branching, to a thin nerve, which aberrantly innervates the lateral rectus (as seen in Duane syndrome). The abducens nerve phenotype ranges from complete absence, to aberrant projections within the orbit, to a normal trajectory. Loss of ACKR3 in the midbrain leads to downregulation of CXCR4 protein, consistent with reports that excess CXCL12 causes ligand-induced degradation of CXCR4. Correspondingly, excess CXCL12 applied to ex vivo oculomotor slices causes axon misrouting, similar to inhibition of CXCR4. Thus, ACKR3, through its regulation of CXCL12 levels, is an important regulator of axon guidance in the oculomotor system; complete loss causes oculomotor synkinesis in mice, while reduced function causes oculomotor synkinesis in humans.

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Ex Vivo Oculomotor Slice Culture from Embryonic GFP-Expressing Mice for Time-Lapse Imaging of Oculomotor Nerve Outgrowth

Cited by 5 publications

References 29 publications

TUBB3 and KIF21A in neurodevelopment and disease

TUBB3 and KIF21A in neurodevelopment and disease

A cell type-aware framework for nominating non-coding variants in Mendelian regulatory disorders

Decreased ACKR3 (CXCR7) function causes oculomotor synkinesis in mice and humans

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