a functional study of mir-124 in the developing neural tube

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

those transfected with miR-124mt (F).

Figure S5. Ectopic miR-124 causes basal lamina defects. (A) Overexpression of miR-

124 sometimes resulted in the disintegration of basal laminae throughout the transfected

side (arrows). (B,C) Transfection of miR-124mt did not affect basal lamina integrity or

the expression of integrin β1 (ITGB1). (D,E) Quantifications of ITGB1 fluorescence

intensity. Corresponding areas on the transfected (R) side and control (L) side were

measured, shown by white-bordered boxes. Total pixels in the R-box were divided by

those in the L-box (R/L). The fluorescence intensity of DAPI serves as a control.

Figure S6. miR-124-binding sites in LAMC1 and ITGB1 3'-UTRs. (A) Panel (a)

illustrates possible base-pairings between miR-124 and chick (gg) LAMC1 3'-UTR.

Sequences in blue shade are miR-124, those in red shade are fragments of LAMC1 3'-

UTR. There are 3 potential miR-124-binding sites in LAMC1 3'-UTR, with the last 2 sites

being closely linked. Panel (b) shows alignments of the three miR-124-binding sites in

human (hs), mouse (mm), and chick (gg) LAMC1 3'-UTRs. Nucleotides that can base-

pair with miR-124 are shaded in red. Underlined nucleotides are seed matches. Colons (:)

indicate nucleotides conserved in all three species. (B) Possible base-pairings between

Cao et al. 4

miR-124 and chick (gg) ITGB1 3'-UTR (a) and alignments of the two potential miR-124-

binding sites found in human, mouse, and chick ITGB1 3'-UTRs (b).

Table S1. Quantifications of basal lamina defects. In every embryo, the distance

between each examined sections was ≥60 µm to avoid counting the same defect multiple

times. “Defects frequency” is obtained by dividing the number of defective sections with

that of the sections examined in each embryo.

Table S2. Summary of LAMC1 and ITGB1 RNAi experiments. Out of 4 LAMC1-

RNAi constructs (Lam1-4) and 3 ITGB1-RNAi constructs (Itg1,2, and 4) tested, only

Lam4 and Itg4 (bold letters) caused basal lamina defects. “RFPRNAi” is the empty

vector into which target sequences were inserted. “Start” refers to the location of the first

nucleotide of the target sequence within the coding sequence (CDS) or 3'-UTR of

LAMC1 or ITGB1 mRNA.

Additional materials and methods

Immunocytochemistry

The following antibodies were used: rabbit anti-Sox2 (Chemicon, 1:500), rat anti-BrdU

(Accurate, 1:200), mouse anti-Tuj1 (Covance, 1:1,000), mouse anti-NeuN (Chemicon,

1:200), mouse anti-p27/Kip1 (Pharmingen, 1:50), rabbit anti-Caspase-3 (Cell Signaling,

1:400), and rabbit anti-GFP (Molecular Probes, 1:1,000). The following mouse antibodies

Cao et al. 5

were obtained from Developmental Studies Hybridoma Bank in the supernatant form:

integrin β1 (V2E9, 1:10), laminin 1 (3H11, 1:100), BEN (1:200), Neurofilament (3A10,

1:100), MNR2 (81.5C10, 1:50), Lim1/2 (4F2, 1:50), Lim3 (67.4E12, 1:50), Isl1/2

(39.4D5, 1:50). For BrdU staining, sections were first treated with 2N HCl at 37ºC for 30

min, neutralized with 0.1M boric acid, pH8.5, then processed as regular

immunocytochemistry. The rat anti-BrdU antibody was incubated with the sections for 72

hrs at 4°C. All other primary antibodies were incubated overnight.

miRNA overexpression constructs

The following primers were used to amplify mir-124a-2 from mouse genomic DNA:

forward 5'-TCCAACACCGGAGACCTACAACTCTAGGAGTAGGGACTC, reverse

5'-CTTAAAAAAT GAGACCGAATCAATGCGAGGGGTCCTTGT. The following

primers were used to amplify mir-20 from human (293T cell) genomic DNA: forward 5'-

TCCAACACCGGAGACCTGATGGTGGCCTGCTATTTCC, reverse 5'-

CTTAAAAAAT GAGACCATGGATTTGCACAGCAGAATATC. PCR products were

digested with Bsa I (bold letters are BsaI sites) and cloned into pENTR/U6 (Invitrogen).

To generate miR-124mt, two steps of QuikChange site directed mutagenesis (Stratagene)

were performed. The oligo pairs for the first mutagenesis were 5'-

CCGTGTTCACAGCGGACCAGTATTTAATGTCATACAATTAAGGCAC and the

antisense strand, those for the second step were

CGGACCAGTATTTAATGTCATACAAT ATT GGCACGCGGTGAATGCC and the

antisense strand. Bold letters are the mutations introduced.

Cao et al. 6

miRNA sensors

pSensor was constructed using pd4EGFP-N1 (Clontech) as the parental plasmid. The

detailed map and sequence for pSensor will be provided upon request. mRFP construct

was a gift from Roger Tsien. To construct pSensor-124, oligo pairs of miR-124

complementary sequence (sense 5'-

GGCCGCTGGCATTCACCGCGTGCCTTAAGGCGCGCCTGGCATTCACCGCGTG

CCTTAAGGCCGG, antisense 5'-

CCTTAAGGCACGCGGTGAATGCCAGGCGCGCCTTAAGGCACGCGGTGAATGC

CAGC, bold letters are NotI and FseI sticky ends) were annealed and cloned after

d4EGFP, those of the scrambled sequence (sense 5'-

GGCCGCCGTAGACGTCTTGCCTACGTACGGCGCGCCCGTAGACGTCTTGCCT

ACGTACGGCCGG, antisense 5'-

CCGTACGTAGGCAAGACGTCTACGGGCGCGCCGTACGTAGGCAAGACGTCTA

CGGC) after mRFP.

ITGB1 and Ngn2 overexpression constructs

Chick cDNAs were prepared from total RNAs of HH24-25 embryos with SuperScript II

reverse transcriptase (Invitrogen). ITGB1 cDNA (2.4 kb) was then amplified as two

fragments. The primers for the 5' half (1 kb) were: forward 5'-

GAAAGATATC ATGGCCGAGACTAATTTAACATTGCTCACG and reverse 5'-

GTTCTCACTAAGCTT CTGTACCAGATGAGC. The primers for the 3' half (1.4 kb)

were: forward 5'- CTCATCTGGTACAGAAGCTT AGTGAGAAC and reverse 5'-

GAAATCTAGA TTATTTTCCCTCATATTTAGGATTGACCAC (bold letters are

Cao et al. 7

restriction sites). The 5' and 3' PCR products were digested with EcoRV/HindIII and

HindIII/XbaI, respectively, and subcloned into the EcoRV/XbaI sites of pMIW3.1, a

vector modified from pMIW (Muramatsu et al. 1997). Expression of the cloned ITGB1 is

driven by the chick β-actin promoter. The Ngn2 expression plasmid has been described

(Lee et al. 2005).

Luciferase-UTR constructs

Chick LAMC1 3'-UTR (2.4 kb) was amplified as two fragments from chick genomic

DNA. The primers for the 5' half (1.3 kb) were: forward 5'-

GAAATCTAGA GGGTGTGCTGGGGGTGGAGGGTATTCCC and reverse 5'-

GAAATA ATTAAT AGCACCAGTGTGTGTCAG. The primers for the 3' half (1.1 kb)

were: forward 5'-CTGACACACACTGGTGCTATTAAT TATTTC and reverse 5'-

CTAAAGTCGACAATGCTTGAAAGAATCCCAAGGTATAAGG (bold letters are

restriction sites). The 5' and 3' PCR products were digested with XbaI/AseI and

AseI/SalI, respectively, and subcloned into the XbaI/SalI sites of pTK-Luc. Chick ITGB1

3'-UTR (1.2 kb) was amplified with the following two primers: forward 5'-

GAAATCTAGA ACACTGCTTGTGTAACTTCACGACAC and reverse 5'-

TAAAT GTCGACTACAAGATAAAGAGACGGCATGCAAG, and subcloned into

XbaI/SalI sites of pTK-Luc.

miRNA Northern blot

The Northern probe for miR-124 was 5'-TGGCATTCACCGCGTGCCTTAA. The probe

for miR-124mt was 5'-TGGCATTCACCGCGTGCCAATA. Total RNA was extracted

Cao et al. 8

with TRIZOL (Invitrogen) according to the manufacturer’s manual. 30 µg of total RNA

was loaded onto each lane of 15% acrylamide denaturing gels. A detailed protocol can be

found on Bartel lab’s website

(http://web.wi.mit.edu/bartel/pub/protocols/miRNA_Nrthrns_Protocol.pdf).

miRNA in situ hybridization

The probe for miR-124 was 5'-TGGCATTCACCGCGTGCCTTA, where bold letters

are “locked” nucleic acids (LNA). The control probe was 5'-

AATTCCGTGCGCCACTTACGG. No significant match of the control probe was found

by chick genomic BLAST. Both probes were purchased from Proligo. Hybridization was

performed on 20 µm cryosections at 60ºC. A detailed protocol is available at Exiqon’s

website (http://www.exiqon.com/uploads/Frozen_sections_in_situ_hybridization(2).pdf).

No signal was seen with the control probe (data not shown).

LAMC1 and ITGB1 RNAi

The RNAi constructs were generated using a recently developed chick RNAi system

pRFPRNAiC (Das et al. 2006). The 22-nucleotide target sequences for LAMC1 and

ITGB1 were chosen using www.genscript.com/ssl-bin/app/rnai. Four LAMC1 and three

ITGB1 target sequences were tested (Table S2). Each RNAi plasmid was diluted to 1.0

µg/µl and transfected into the chick neural tube by in ovo electroporation.

In situ hybridization of LAMC1 and ITGB1

Cao et al. 9

For LAMC1 in situ hybridization, an XbaI/MscI fragment of chick LAMC1 3'-UTR,

corresponding to the first 565 bp of chick LAMC1 3'-UTR, was subcloned from pTK-

Luc-LAMC1-UTR into the XbaI/EcoRV sites of pBluscript SK. For the antisense probe,

the template plasmid was digested with XbaI and transcribed with T7 polymerase using

the Riboprobe Combination System-T3/T7 (Promega). For the sense (control) probe, the

template was digested with HindIII and transcribed with T3 polymerase. For ITGB1 in

situ hybridization, an XbaI/EcoRI fragment of chick ITGB1 3'-UTR, corresponding to the

first 637 bp of chick ITGB1 3'-UTR, was subcloned from pTK-Luc-ITGB1-UTR into the

XbaI/EcoRI sites of pBluscript SK. For the antisense probe, the template was digested

with XbaI and transcribed with T7 polymerase. For the sense probe, the template was

digested with EcoRI and transcribed with T3 polymerase. Hybridization was carried out

on 20 µm frozen sections at 60ºC according to a method described previously (Ma et al.

1996).

Luciferase assays

293T cells were seeded in 24-well plates to ~80% confluency 6 hrs before transfection.

For each well, the following plasmids were mixed and transfected with Fugene 6 (Roche)

according to manufacturer’s manual: 100 ng of Luciferase-UTR construct, 20 ng of pRL-

TK (Promega), and 5-50 ng of miRNA expression plasmid or the vector pENTR. Each

transfection was triplicated. 36-40 hrs after transfection, cells were lysed with 100 µl/well

of passive lysis buffer (Promega). 10 µl of cell lysate from each well was used to measure

luciferase activities with the Dual-luciferase reporter assay system (Promega). Each

experiment was repeated 2-3 times.

Cao et al. 10

Image acquisition and analysis

Fluorescent images were acquired with Nikon Eclipse TE300 microscope and BioRad

Radiance 2100 laser scanning system. Bright field images were acquired with Nikon

Eclipse E800 microscope and RT Slider Spot camera (Diagnostic Instruments).

Fluorescence intensity was measured with MetaMorph. ImagePro was used to quantify

BrdU+ cells.

References:

Das, R.M., Van Hateren, N.J., Howell, G.R., Farrell, E.R., Bangs, F.K., Porteous, V.C.,

Manning, E.M., McGrew, M.J., Ohyama, K., and Sacco, M.A. 2006. A robust

system for RNA interference in the chicken using a modified microRNA operon.

Dev. Biol. 294: 554-563.

Lee, S.K., Lee, B., Ruiz, E.C., and Pfaff, S.L. 2005. Olig2 and Ngn2 function in

opposition to modulate gene expression in motor neuron progenitor cells. Genes

Dev. 19: 282-294.

Ma, Q., Kintner, C., and Anderson, D.J. 1996. Identification of neurogenin, a vertebrate

neuronal determination gene. Cell 87: 43-52.

Muramatsu, T., Mizutani, Y., Ohmori, Y., and Okumura, J.-i. 1997. Comparison of three

nonviral transfection methods for foreign gene expression in early chicken

embryos in ovo. Biochem. Biophys. Res. Commun. 230: 376-380.

2’-O-methyl-20as, 20 hpe

D

LNA-124scr, 45 hpe

A


C


B

GENESDEV/2006/073635

Figure S1

Cao et al.

Ngn2, 20 hpe

Tuj1

GF

P D

AP

I

Ngn2, 45 hpe

E F

miR

-124,

45 h

pe

p27/Kip1 GFP DAPI

C

Tuj1 GFP DAPI

B

NeuN GFP DAPI

D

Sox2 GFP

A

miR

-124, 45 h

pe


Figure S2

Cao et al.

TU

NE

L G

FP

DA

PI

miR-124, 20 hpe

A

miR-124mt +pENTR +

0miR-124 +

1

2

3

6

Tra

nsfe

cte

d

vs.

contr

ol sid

e

B

4

5

++

+

20 hpe 45 hpe

C

miR-124, 20 hpe

E

miR-124, 45 hpe

Caspase

GF

P n=5 (2-5 sections/embryo)

Total sections examined: 20

Electroporated side:

Caspase /GFP : 64

Caspase /GFP : 10

Control side: Caspase : 10

+ +

+ -

+

miR-124, 20 hpeD

Caspase

GF

P D

AP

I


Figure S3

Cao et al.

aPKC GFP DAPI β-catenin GFP DAPI

FD E

miR

-124, 45 h

pe

Lim1/2 GFP DAPI Lim3 GFP DAPI Isl1/2 GFP DAPI

miR

-124, 45 h

pe

miR

-124m

t, 4

5 h

pe

A B C

β-catenin GFP DAPI


Figure S4

Cao et al.

miR-124mt, 45 hpe

B

miR-124mt, 20 hpe

ITG

B1 G

FP

C

miR-124, 45 hpe

Lam

inin

GF

P D

AP

I A

D

ITG

B1 G

FP

miR-124, 20 hpe

L R

R/L

ITGB1: 77%

DAPI: 98%

miR-124mt, 20 hpe

E

RL

R/L

ITGB1: 95%

DAPI: 97%


Figure S5

Cao et al.

uggcuau g gugccuuaag a

accggug c cacggaauug g

aau

53

3'miR-124

gg UTR

u

2221

3'

cuggc ca u cgugccuu a

g aaccc

accg gu g gcacggaa ug c

aau u

uucacuuu ugccuuaa

aaguggcg acggaauuc

c g u

3'gcca

A (a) LAMC1 3'-UTR and miR-124

50 aggccacagagugccuugahs UTR 2640 uggaccuuuuauaugugccuucacuuuagcuguuugccuua-

46 aggccucg--gugccuugamm UTR 2399 uggcccuuuucaaugugccuucacuuuagcuguuugccuuaa

53 uggcuauggagugccuuaagg UTR 2221 uggccccacaguacgugccuucacuucagcuuuuugccuuaa

(b) LAMC1 3'-UTR alignment

::: ::::::: :

::: ::::::: :

::: :: : :::::::::::: :::: :::::::::

::: :: : :::::::::::: :::: :::::::::

B

(b) ITGB1 3'-UTR alignment

226hs UTR 1081

232mm UTR 1036aggucacagugugccuuuu aaucuuguuuuaagugccuuuu

262gg UTR 1061aggucacauggugccuuuu auccuuguuuuaagugccuuuu

:::::::: :::::::::

:::::::: :::::::::

: ::::::::::::::::::

: ::::::::::::::::::


Figure S6

Cao et al.

aggucacauugugccuuuu

ggucac gugccuua g

ccagug cacggaag gg a

u

262

3'

u

ca

uu

uu

a

(a) ITGB1 3'-UTR and miR-124

gg UTR

miR-124

uu gugccuuaa

gg cacggaaaccguaag

1061

3'

u

cgu

uu

uu

auccuugu

auucuuguuuuaagugccuuua

a functional study of mir-124 in the developing neural tube

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