Generation and regeneration of Motor Neurons Flashcards
where are MNs located in the CNS
HB
ventral horn of SC
what is local processing
activation of the flexor muscle inhibits the extensor muscle (antagonistic muscle)
where is Shh produced
floorplate and notochord
MN production
neural progenitor domain pMN in the ventral neural tube
Class I/II TF
class I TFs (Pax6/Dbx2) repressed by Shh
Class II TFs (Nkx2.2/Nkx6.1) activated by Shh
Evidence to show graded Shh signalling
Ericson et al., 1997
Titration of Shh forms different neuronal subtypes:
V1 - low conc
V2 - intermediate conc
MN - high conc of Shh
use reverse anti-Shh antibody - Shh conc is 0 (no V1/V2/MN)
motor column
PGC (FoxP1 low)
LMC (FoxP1 high)
MMC (Lhx3)
HMC
sub column
LMCl (projects to dorsal limb)
LMCm (projects to ventral limb)
motor pool
rf-MP (Lhx1)
vasti-MP (Lsl1)
role of Hox genes
conserved
determine positional identity of cell types along the rostral caudal axis
males have 4 hox clusters
females have 1 hox cluster
Hox gene cluster
secondary organising centres along the R-C axis
morphogens which induce Hox genes: Gdf11, RA, FGF
FGF manipulation in chick/mammals using electroporation
FGF8 causes gain of Hoxc9/loss of hoxc6
LMCm/LMCl projections
LMC m project to ventral limb contains ephrin A TF: Isl1 Receptor: EphB
LMC l project to dorsal limb contains ephrin B TF: Lhx Receptor: EphA
ectopically expressing LMCl/m determinants
GFP (control) 50/50 projection to d/v limb
GFP+lim1 (LMC l) - most projections to dorsal limb
GFP+Isl1 - mostly ventral projections
role of Pea3
marks 2 motor pools in LMC m
dendritic morphology of Pea3/non-Pea3 pool
Pea3 pool - cresent shaped, no sensory axons, no medial projections
non-pea3 pool: project medially
pluripotent embryonic stem cells
derived from inner cell mass
forms all germ layers
in-vitro formation of MN from embryonic stem cells
express morphogens: RA/Shh (provide positional information)
ectoderm to MN formation
primative ectoderm - rostral neural - via RA - caudal neural - via Shh - MN
neural induction/caudalisation/ventralisation
what do ES derived MNs form
MMC
HMC
not PGC (controls involuntary movement)
role of doxycycline
chemical on/off switch
induces TFs: Ngn2/Isl1/Lhx3
produces transcriptional profile of NIL motor neurons generated by extrinsic factors (RA/shh)
NIP vs NIL
NIP - exist dorsally, cranial MN
NIL - exist ventrally spinal MN
NIL MN
express all markers of somatic MNs derived with extrinsic factors but lack expression of progenitor specific genes (Sox1/olig1/olig2)
transition from early embryonic identity to post mitotic MN (skip neural progenitor stage)
generation of induced MNs
mouse embryonic fibroblasts (MEFs) - expose to late progenitors (sox1/Pax6/Nkx6.1/Olig2) - MN+glia
application of induced MNs
ALS patient fibroblast - direct conversion to MN (respond to chemicals) form NMJ
amyotrophic lateral sclerosis
selective degeneration of MNs/neurons of the motor cortex - progress muscle weakness and spasticity (most of CNS unaffected)
risk of development: 1in 500
death of patient: 3-5 yrs after onset of symptoms (lose innervation of respiratory muscles)
no effective treatment
10-15% genetic cause
most commonly: defect of superoxidase dimutase 1 (SOD1) point mutation
sporadic ALS is usually polygenic
mutant SOD1
extract SOD1 from astrocytes - coexpress with hESC MN - Hb9/GFP+ cells
astro SOD1 mutant - degeneration of MNs
use of co-cultured astrocytes and MNs
ALS drug screening
limitation of induced MN
optogenetic activation of MN (blue light on ChR) replace input from spinal circuits
do not reflect normal MN pathology
in-vitro neuromuscular circuits
Osaki et al., 2018
develop in-vitro NM device using human iPSC MN (form NMJs with myofibers) - stabilised by 2 flexible pillars
in ALS, MN fail to induce myofiber contractions when stimulated with light
