Single-Cell Transcriptional Profiling of the Adult Corticospinal Tract Reveals Forelimb and Hindlimb Molecular Specialization

Golan, Noa; Sd, Kauer; Db, Ehrlich; Ravindra, Neal G.; Dijk, David van; Wb, Cafferty

doi:10.1101/2021.06.02.446653

Cited by 6 publications

(8 citation statements)

References 57 publications

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“…Our screen was completed in crym-GFP transgenic mice treated with retrograde tracers injected into the contralateral cervical enlargement after PyX. While the crym-GFP transgenic line is superior in labeling CSNs versus tracers, it may not represent complete CSN coverage (Arlotta et al, 2005;Fink et al, 2015;Golan et al, 2021) and could preferentially label CSNs innervating the cervical cord. Thus, the bulk sequencing from our screen would have undersampled plastic lumbar CSNs.…”

Section: Corticospinal Neuron Subtype-specific Therapies?mentioning

confidence: 99%

Inositol Polyphosphate-5-Phosphatase K (Inpp5k) Enhances Sprouting of Corticospinal Tract Axons after CNS Trauma

Kauer

Fink

et al. 2022

J. Neurosci.

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Failure of CNS neurons to mount a significant growth response after trauma contributes to chronic functional deficits after spinal cord injury. Activator and repressor screening of embryonic cortical neurons and retinal ganglion cells in vitro and transcriptional profiling of developing CNS neurons harvested in vivo have identified several candidates that stimulate robust axon growth in vitro and in vivo. Building on these studies, we sought to identify novel axon growth activators induced in the complex adult CNS environment in vivo. We transcriptionally profiled intact sprouting adult corticospinal neurons (CSNs) after contralateral pyramidotomy (PyX) in nogo receptor-1 knock-out mice and found that intact CSNs were enriched in genes in the 3-phosphoinositide degradation pathway, including six 5-phosphatases. We explored whether inositol polyphosphate-5-phosphatase K (Inpp5k) could enhance corticospinal tract (CST) axon growth in preclinical models of acute and chronic CNS trauma. Overexpression of Inpp5k in intact adult CSNs in male and female mice enhanced the sprouting of intact CST terminals after PyX and cortical stroke and sprouting of CST axons after acute and chronic severe thoracic spinal contusion. We show that Inpp5k stimulates axon growth in part by elevating the density of active cofilin in labile growth cones, thus stimulating actin polymerization and enhancing microtubule protrusion into distal filopodia. We identify Inpp5k as a novel CST growth activator capable of driving compensatory axon growth in multiple complex CNS injury environments and underscores the veracity of using in vivo transcriptional screening to identify the next generation of cell-autonomous factors capable of repairing the damaged CNS.

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Section: Corticospinal Neuron Subtype-specific Therapies?mentioning

confidence: 99%

Inositol Polyphosphate-5-Phosphatase K (Inpp5k) Enhances Sprouting of Corticospinal Tract Axons after CNS Trauma

Kauer

Fink

et al. 2022

J. Neurosci.

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“…Most of these studies, however, were performed in early postnatal or adult mice, using virus or CTB injections. [41][42][43][44] While these approaches do offer superior labeling efficiency compared to our method and-by extension-higher expected cell numbers, they do so at the expense of temporal resolution. Moreover, they only allow for the probing of developmental time points at which the wiring architecture of neuromuscular circuits has already been well established.…”

Section: Discussionmentioning

confidence: 91%

“…To do so, techniques combining single‐cell sequencing with retrograde labeling have already been developed in recent years, in order to decipher the transcriptional logic of neuronal wiring. Most of these studies, however, were performed in early postnatal or adult mice, using virus or CTB injections 41‐44 . While these approaches do offer superior labeling efficiency compared to our method and—by extension—higher expected cell numbers, they do so at the expense of temporal resolution.…”

Section: Discussionmentioning

confidence: 99%

A method to investigate muscle target‐specific transcriptional signatures of single motor neurons

Berki

Sacher

Fages

et al. 2022

Developmental Dynamics

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Background: Motor neurons in the vertebrate spinal cord have long served as a paradigm to study the transcriptional logic of cell type specification and differentiation. At limb levels, pool-specific transcriptional signatures first restrict innervation to only one particular muscle in the periphery, and get refined, once muscle connection has been established. Accordingly, to study the transcriptional dynamics and specificity of the system, a method for establishing muscle target-specific motor neuron transcriptomes would be required. Results: To investigate target-specific transcriptional signatures of single motor neurons, here we combine ex-ovo retrograde axonal labeling in midgestation chicken embryos with manual isolation of individual fluorescent cells and Smart-seq2 single-cell RNA-sequencing. We validate our method by injecting the dorsal extensor metacarpi radialis and ventral flexor digiti quarti wing muscles and harvesting a total of 50 fluorescently labeled cells, in which we detect up to 12,000 transcribed genes. Additionally, we present visual cues and cDNA metrics predictive of sequencing success. Conclusions:Our method provides a unique approach to study muscle targetspecific motor neuron transcriptomes at a single-cell resolution. We anticipate that our method will provide key insights into the transcriptional logic underlying motor neuron pool specialization and proper neuromuscular circuit assembly and refinement.

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“…Single cell RNA sequencing technologies are rapidly expanding awareness of cellular diversity in the nervous system. Single-cell and -nuclei data exist for various regions in the adult and developing mouse nervous system including retina (Tran et al, 2019), dorsal root ganglion (Renthal et al, 2020), spinal cord (Russ et al, 2021), cortex (la Manno et al, 2021; Yao et al, 2021), and CST (Golan et al, 2021). Here we aimed to broaden understanding of supraspinal cell types spanning forebrain, midbrain, and hindbrain, and to test whether transcriptionally unique profiles associate with established anatomical classifications.…”

Section: Discussionmentioning

confidence: 99%

“…In the murine nervous system single cell datasets have identified ever finer distinctions between subtypes of neurons withing numerous regions including spinal cord, retina, sensory ganglia, and cortex (Renthal et al, 2020; Russ et al, 2021; Tran et al, 2019; Yao et al, 2021). Apart from recent datasets in corticospinal tract (CST) neurons, however, little is known the transcriptional profiles that characterize neurons that project axons to different spinal targets, and the degree to which anatomical distinctions between the locations of supraspinal cell bodies are associated with distinct patterns of gene expression (Golan et al, 2021; Sahni et al, 2021b).…”

Section: Introductionmentioning

confidence: 99%

Single nuclei analyses reveal transcriptional profiles and marker genes for diverse supraspinal populations

Beine

Wang

Tsoulfas

et al. 2022

Preprint

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The mammalian brain contains numerous neurons distributed across forebrain, midbrain, and hindbrain that project axons to the lower spinal cord and work in concert to control movement and achieve homeostasis. Extensive work has mapped the anatomical location of supraspinal cell types and continues to establish specific physiological functions. The patterns of gene expression that typify and distinguish these disparate populations, however, are mostly unknown. Here we combined retrograde labeling of supraspinal cell nuclei with fluorescence activated nuclei sorting and single nuclei RNA sequencing analyses to transcriptionally profile neurons that project axons from the mouse brain to lumbar spinal cord. We identified fourteen transcriptionally distinct cell types and used a combination of established and newly identified marker genes to assign an anatomical location to each. To validate the putative marker genes, we visualized selected transcripts and confirmed selective expression within lumbar-projecting neurons in discrete supraspinal regions. Finally, we illustrate the potential utility of these data by examining the expression of transcription factors that distinguish different supraspinal cell types and by surveying the expression of receptors for growth and guidance cues that may be present in the spinal cord. Collectively these data establish transcriptional differences between anatomically defined supraspinal populations, identify a new set of marker genes of use in future experiments, and provide insight into potential differences in cellular and physiological activity across the supraspinal connectome.

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Single-Cell Transcriptional Profiling of the Adult Corticospinal Tract Reveals Forelimb and Hindlimb Molecular Specialization

Cited by 6 publications

References 57 publications

Inositol Polyphosphate-5-Phosphatase K (Inpp5k) Enhances Sprouting of Corticospinal Tract Axons after CNS Trauma

Inositol Polyphosphate-5-Phosphatase K (Inpp5k) Enhances Sprouting of Corticospinal Tract Axons after CNS Trauma

A method to investigate muscle target‐specific transcriptional signatures of single motor neurons

Single nuclei analyses reveal transcriptional profiles and marker genes for diverse supraspinal populations

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