Cao et al. 1 A Functional Study of miR-124 in the Developing Neural Tube Xinwei Cao 1 , Samuel L. Pfaff 2,4 , and Fred H. Gage 1,3 Supplemental materials Figure legends Figure S1. Inhibiting miR-124 in the chick neural tube. (A) Transfection of LNA- 124scr had no effect on miR-124 ISH signals at 45 hpe. (B-D) Transfection of 2'-O- methyl miR-124 antisense oligonucleotides (2'-O-methyl-124as) at 200 μM caused a decrease in miR-124 ISH signals at 20 hpe (B, arrows). A much weaker effect was observed at 45 hpe (C). 2'-O-methyl miR-20 antisense oligonucleotides (2'-O-methyl- 20as) had no effect on miR-124 ISH signals (D). Figure S2. Overexpressing miR-124 does not promote overt neuronal differentiation. (A-D) Ectopic miR-124 did not have a significantly effect on the expression of the neural progenitor marker Sox2 or neuronal markers Tuj1, p27/Kip1, and NeuN at 45 hpe, although transfected spinal cords were often disorganized with some cells aberrantly located inside the ventricle (arrowheads). (E-F) Overexpression of Ngn2 caused a dramatic upregulation of Tuj1 at both 20 and 45 hpe.
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A Functional Study of miR-124 in the Developing Neural Tube
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Cao et al. 1
A Functional Study of miR-124 in the Developing Neural Tube
Xinwei Cao1, Samuel L. Pfaff2,4, and Fred H. Gage1,3
Supplemental materials
Figure legends
Figure S1. Inhibiting miR-124 in the chick neural tube. (A) Transfection of LNA-
124scr had no effect on miR-124 ISH signals at 45 hpe. (B-D) Transfection of 2'-O-
methyl miR-124 antisense oligonucleotides (2'-O-methyl-124as) at 200 µM caused a
decrease in miR-124 ISH signals at 20 hpe (B, arrows). A much weaker effect was
observed at 45 hpe (C). 2'-O-methyl miR-20 antisense oligonucleotides (2'-O-methyl-
20as) had no effect on miR-124 ISH signals (D).
Figure S2. Overexpressing miR-124 does not promote overt neuronal
differentiation. (A-D) Ectopic miR-124 did not have a significantly effect on the
expression of the neural progenitor marker Sox2 or neuronal markers Tuj1, p27/Kip1,
and NeuN at 45 hpe, although transfected spinal cords were often disorganized with some
cells aberrantly located inside the ventricle (arrowheads). (E-F) Overexpression of Ngn2
caused a dramatic upregulation of Tuj1 at both 20 and 45 hpe.
Cao et al. 2
Figure S3. Overexpression of miR-124 leads to increased cell death. (A) TUNEL
assay showed that ectopic miR-124 led to increased apoptosis. (B) Quantifications of
TUNEL signals at 20 and 45 hpe. For the “20 hpe” time point, 5 miR-124-, 3 miR-
124mt-, and 3 pENTR-transfected embryos were analyzed. For each embryo, TUNEL
signals on the transfected side (2-5 sections from each miR-124 embryo and 5 sections
from each miR-124mt and pENTR embryo) were added together and divided by the sum
of the control side. For the “45 hpe” time point, 5 miR-124-, 5 miR-124mt-, and 4
pENTR-transfected embryos (3 sections from each embryo) were analyzed. At 45 hpe,
endogenous apoptosis is taking place on the ventral neural tube of some sections. For
such sections, only the TUNEL signals on the dorsal neural tube were counted. At 20
hpe, an increase in apoptosis was observed with both ectopic miR-124 and miR-124mt,
but the former had a stronger apoptotic effect. At 45 hpe, only ectopic miR-124, but not
miR-124mt, caused a significant increase in cell death. (C-E) Overexpression of miR-124
led to increased immunoreactivities of cleaved Caspase-3 at 20 and 45 hpe. In panel E,
enlargements of the boxed area are shown on the right side of the dashed line with arrows
illustrating colocalizations of cleaved Caspase-3 and GFP. Panel D is a quantification of
Caspase-3+ cells in miR-124-transfected spinal cords at 20 hpe. On the electroporated
side, the majority of Caspase+ cells (64 out of 74) were also GFP+, while the number of
Caspase+/GFP- cells was same as that of Caspase+ cells on the control side, suggesting
that the apoptotic effect of ectopic miR-124 is cell-autonomous.
Figure S4. Ectopic miR-124 causes abnormal cell migration into the ventricle and
disruptions of luminal adherens junctions. (A-C) Immunostainings for Lim1/2, Lim3,
Cao et al. 3
and Isl1/2 showed that some post-mitotic neurons had aberrantly settled inside the
ventricle of miR-124-transfected spinal cords (arrowheads). (D-F) Immunostainings for
aPKC and β-catenin, components of adherens junctions, showed disruptions in luminal
adherens junctions in miR-124-transfected spinal cords (D and E, arrowheads) but not in