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Genetic deletion of genes in the cerebellar rhombic lip lineage can stimulate compensation through adaptive reprogramming of ventricular zone-derived progenitors

Alexandre Wojcinski, Morgane Morabito, Andrew K. Lawton, Daniel N. Stephen, Alexandra L. Joyner
doi: https://doi.org/10.1101/383778
Alexandre Wojcinski
developmental Biology Program, Sloan Kettering Institute, New York, NY
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Morgane Morabito
developmental Biology Program, Sloan Kettering Institute, New York, NY
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Andrew K. Lawton
developmental Biology Program, Sloan Kettering Institute, New York, NY
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Daniel N. Stephen
developmental Biology Program, Sloan Kettering Institute, New York, NY
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Alexandra L. Joyner
developmental Biology Program, Sloan Kettering Institute, New York, NYBiochemistry, Cell and Molecular Biology Program, Weill Cornell Graduate School of Medical Sciences, New York, NY, 10065, USA
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  • For correspondence: joynera@mskcc.org
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Abstract

Background The cerebellum is a foliated posterior brain structure involved in coordination of motor movements and cognition. The cerebellum undergoes rapid growth postnataly due to Sonic Hedgehog (SHH) signaling-dependent proliferation of ATOH1+ granule cell precursors (GCPs) in the external granule cell layer (EGL), a key step for generating cerebellar foliation and the correct number of granule cells. Due to its late development, the cerebellum is particularly vulnerable to injury from preterm birth and stress around birth. We recently uncovered an intrinsic capacity of the developing cerebellum to replenish ablated GCPs via adaptive reprogramming of Nestin-expressing progenitors (NEPs). However, whether this compensation mechanism occurs in mouse mutants affecting the developing cerebellum and could lead to mis-interpretation of phenotypes was not known.

Methods We used two different approaches to remove the main SHH signaling activator GLI2 in GCPs: 1) our mosaic mutant analysis with spatial and temporal control of recombination (MASTR) technique to delete Gli2 in a small subset of GCPs; 2) An Atohl-Cre transgene to delete Gli2 in most of the EGL. Genetic Inducible Fate Mapping (GIFM) and live imaging were used to analyze the behavior of NEPs after Gli2 deletion.

Results Mosaic analysis demonstrated that SHH-GLI2 signaling is critical for generating the correct pool of granule cells by maintaining GCPs in an undifferentiated proliferative state and promoting their survival. Despite this, inactivation of GLI2 in a large proportion of GCPs in the embryo did not lead to the expected dramatic reduction in the size of the adult cerebellum. GIFM uncovered that NEPs do indeed replenish GCPs in Gli2 conditional mutants, and then expand and partially restore the production of granule cells. Furthermore, the SHH signaling-dependent NEP compensation requires Gli2, demonstrating that the activator side of the pathway is involved.

Conclusion We demonstrate that a mouse conditional mutation that results in loss of SHH signaling in GCPs is not sufficient to induce long term severe cerebellum hypoplasia. The ability of the neonatal cerebellum to regenerate after loss of cells via a response by NEPs must therefore be considered when interpreting the phenotypes of conditional mutants affecting GCPs.

Abbreviations

CB
Cerebellum
SHH
Sonic Hedgehog
EGL
External Granule Layer
GCP
Granule Cell Precursor
NEP
Nestin-expressing progenitor
MASTR
mosaic mutant analysis with spatial and temporal control of recombination
GIFM
Genetic Inducible Fate Mapping
VZ
ventricular zone
PC
Purkinje cell
IGL
internal granule cell layer
HH
Hedgehog
Ihh:
Indian hedgehog
Dhh:
Desert hedgehog
PTCH1
Patched1
SMO
Smoothened
Ci
cubitus interruptus
A
Activator
R
Repressor
CKO
conditional knockout
P
Postnatal day
Tm
Tamoxifen
IHC
immunohistochemistry
GFP
Green Fluorescent Protein
het
Heterozygous
TUNEL
Terminal deoxynucleotidyl transferase dUTP nick end labeling
PCL
Purkinje cell layer
ISH
in situ hybridization
WT
Wild type
TDTom
Tandem Dimeric derivative of DsRed
ML
Molecular layer
  • Abbreviations

    CB
    Cerebellum
    SHH
    Sonic Hedgehog
    EGL
    External Granule Layer
    GCP
    Granule Cell Precursor
    NEP
    Nestin-expressing progenitor
    MASTR
    mosaic mutant analysis with spatial and temporal control of recombination
    GIFM
    Genetic Inducible Fate Mapping
    VZ
    ventricular zone
    PC
    Purkinje cell
    IGL
    internal granule cell layer
    HH
    Hedgehog
    Ihh:
    Indian hedgehog
    Dhh:
    Desert hedgehog
    PTCH1
    Patched1
    SMO
    Smoothened
    Ci
    cubitus interruptus
    A
    Activator
    R
    Repressor
    CKO
    conditional knockout
    P
    Postnatal day
    Tm
    Tamoxifen
    IHC
    immunohistochemistry
    GFP
    Green Fluorescent Protein
    het
    Heterozygous
    TUNEL
    Terminal deoxynucleotidyl transferase dUTP nick end labeling
    PCL
    Purkinje cell layer
    ISH
    in situ hybridization
    WT
    Wild type
    TDTom
    Tandem Dimeric derivative of DsRed
    ML
    Molecular layer
  • Copyright 
    The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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    Posted August 02, 2018.
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    Genetic deletion of genes in the cerebellar rhombic lip lineage can stimulate compensation through adaptive reprogramming of ventricular zone-derived progenitors
    Alexandre Wojcinski, Morgane Morabito, Andrew K. Lawton, Daniel N. Stephen, Alexandra L. Joyner
    bioRxiv 383778; doi: https://doi.org/10.1101/383778
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    Genetic deletion of genes in the cerebellar rhombic lip lineage can stimulate compensation through adaptive reprogramming of ventricular zone-derived progenitors
    Alexandre Wojcinski, Morgane Morabito, Andrew K. Lawton, Daniel N. Stephen, Alexandra L. Joyner
    bioRxiv 383778; doi: https://doi.org/10.1101/383778

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