Drosha regulates neurogenesis by controlling Neurogenin2 ... · differentiation of progenitors in...

Drosha regulates neurogenesis by controlling Neurogenin2 expression

independent of microRNAs

Philip Knuckles1, Miriam A. Vogt1,2,, Sebastian Lugert1,2, Marta Milo3, Mark M. W. Chong4,6,

Guillaume M. Hautbergue7, Stuart A. Wilson7, Dan R. Littman4,5, and Verdon Taylor1,2

1 Department of Molecular Embryology, Max Planck Institute of Immunology, Stubeweg 51, 79108

Freiburg, Germany. 2 Embryology and Stem Cell Biology, Department of Biomedicine, University of Basel,

Mattenstrasse 28, CH–4058 Basel, Switzerland 3 NIHR Cardiovascular Biomedical Research Unit, Sheffield Teaching Hospitals NHS Trust,

University of Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom 4 Molecular Pathogenesis Program, Kimmel Center for Biology and Medicine, Skirball Institute of

Biomolecular Medicine, New York University School of Medicine, New York, New York 10016, USA 5 Howard Hughes Medical Institute, New York University School of Medicine, New York, New York

10016, USA 6 The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia 7 Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank,

Sheffield S10 2TN, United Kingdom.

Nature Neuroscience: doi:10.1038/nn.3139

Supplementary Figure 1: Drosha is expressed by cells through the developing dorsal

telencephalon and knockdown results in loss of VZ progenitor status. Lineage tracing of

Hes5::Cre expressing cells by electroporation

a. In situ mRNA hybridization showing Drosha mRNA expression in the dorsal telencephalon of

E14.5 mouse embryo. Drosha RNA levels seem to show a dynamic expression pattern during the

differentiation of progenitors in the VZ to SVZ/IZ and differentiating immature neurons in the marginal

zone (MZ). Sense control for the in situ mRNA hybridization confirming specificity.


b. Scheme of Hes5::Cre construct expressing Cre–recombinase from the Hes5 regulatory

elements1. A pCAGGs Cre–reporter allele mouse line was used for lineage tracing of Hes5::Cre

expressing cells.

c. Scheme of experimental procedure, in utero electoroporation of Hes5::Cre vector and

pCAGGs::mCherry as transfection reporter into VZ progenitors into E13.5 pCAGGs::floxed–STOP–

eGFP mouse embryos. Embryos were returned to the mother and sacrificed 2 days later at E15.5.

d. Transfection of control pCAGGs::floxed–STOP–eGFP embryos with Hes5::Cre and

pCAGGs::mCherry constructs. mCherry+ cells are present within the VZ and SVZ/IZ 48 hours post–

transfection. GFP expression reflects recombination of the pCAGGs::floxed–STOP–eGFP allele. As

expected of Notch signaling progenitors, most are still within the VZ and few have commenced

differentiation and migrated to the SVZ/IZ. The mCherry+GFP- cells in the SVZ/IZ represent Hes5–

neural progenitors that commenced differentiation and exited the VZ.

Scale bars = 100 µm.



Supplementary Figure 2: Drosha KD induces neural progenitors differentiation

a. Quantification of the efficiency of the Drosha shRNA knockdown in N2A cells by qRT–PCR

relative to shRNAs directed against GFP as a control. Functional control shRNAs were expressed in

all of the experiments to monitor for Dicer and RISC quenching effects that could result from high–

level shRNA expression. Verification of shRNA efficiency of Drosha KD in N2A cells by Western blot

analysis using GAPDH as a loading control.

b. shRNA Drosha knockdown (KD) results in neural progenitors down regulating Pax6 and exiting

the VZ. Transfected cells labeled by mCherry expression.

c. Quantification of Pax6 expression by Drosha KD cells relative to control

d. shRNA–mediated Drosha KD results in precocious exit of progenitors from the VZ and loss of

the progenitor marker Sox2.

e. Quantification of Sox2 expressing Drosha KD cells relative to control shRNA (GFP) expressing

cells.

f. Transdominant negative (TN) Drosha phenocopies Drosha KD inducing neural progenitors exit

from the VZ.

g. Quantification of Pax6 expression by TN Drosha cells relative to control.

Ventricular zone – VZ, subventricular zone – SVZ, intermediate zone – IZ, marginal zone – MZ.

tTest * P<0.05, ** P<0.001. Error bars represent s.e.m. Scale bars = 20 µm.


Supplementary Figure 3: Drosha KD disrupts cellular morphology which can lead to VZ

dysplasia

a. VZ neural progenitors normally display a polarized and radial morphology, express progenitor

markers including GLAST and have apically localized γ–Tubulin expression. Drosha KD cells show a

rounded morphology synonymous with a loss of polarized morphology (*). GLAST expression is

disturbed in the KD cells and γ–Tubulin is no longer predominantly at the apical end feet of the radial

progenitors but looks to be distributed into the ventricular zone.

b. In regions where many cells were transfected, the cytoarchitecture of control shRNA expressing

cells was not disturbed. Filamentous actin revealed by phaloidin staining is prominent at apical surface

of the VZ. In regions where many Drosha KD cells are present, the cytoarchitecture of the cells is

disturbed, progenitors no longer form a clear apical boundary to the VZ and the cortical actin

cytoskeleton is distributed throughout the VZ.

Ventricular zone – VZ, subventricular zone – SVZ, intermediate zone – IZ. Scale bars = 20 µm.


Supplementary Figure 4: Drosha KD neural progenitors down–regulate Notch signaling

a. Scheme of the Hes5::GFP transgene expressing eGFP from the Hes5 regulatory elements1. 1.6

kb of the mouse Hes5 gene and 1.4 kb of downstream sequence including exons I, II and III and

endogenous introns. The transgene uses the endogenous Hes5 5–prime and 3–prime untranslated

regions including the Hes5 polyadenylation signal. Transfection of Hes5::GFP mouse embryo and

tracing of cells with pCAGGs::mCherry vector (red).


b. Hes5::GFP expression is restricted to the VZ progenitors. VZ neural progenitors transfected with

control shRNA against renilla luciferase retain Notch signaling. The majority of the Drosha KD cells

leave the VZ. Boxed areas are shown in high magnification in c.

c. VZ neural progenitors transfected with control shRNAs retain Hes5::GFP expression. Many

Drosha KD cells show low levels of Hes5::GFP expression compared to untransfected VZ neural

progenitors indicative of differentiation.

Scale bars in a and b = 100 µm in c = 20 µm.


Supplementary Figure 5: Drosha cKO results in increased cell death over time

a. Drosha KD does not induce Caspase 3 activation of neural progenitors 24 hours or 48 hours (not

shown) post–transfection.

b. Four days after Drosha cKO cells migrated to the cortical plate (CP) and show a normal

neuronal morphology (i and ii). Some cells in the SVZ/IZ show aberrant morphology and cell debris is

evident (iii and iv) indicative of cell death (arrow).

c. Four days after Dicer cKO few cells are found in the cortical plate (CP) and massive cell death

results in remnant cell debris in the SVZ/IZ (v–vii; arrows). Most of the remaining mCherry cells in the

Dicer cKO lack DAPI staining (arrows).

d. Four days post–transfection most control cells have entered the cortical plate (CP) and show a

normal neuronal morphology (viii). Cells in the SVZ/IZ are reminiscent of migratory neurobasts en

route to the CP (ix).

e. Four days post–transfection (E17.5), Drosha KD cells migrate to the cortical plate (CP) and

TUNEL staining of transfected cells increases similar to the effect of inactivation Dicer and miRNA

function. Hence, loss of Drosha also reflects Dicer and miRNA requirements in immature neurons in

the cortical plate.

Scale bars = 20 µm.


Supplementary Figure 6: Drosha KD does not induce increased expression of the basal

progenitor marker Tbr2 even though Drosha KD cells exit the VZ reduces neurospherogenic

NSC self–renewal

a. Drosha KD cells exit the VZ and enter the SVZ/IZ. Most Drosha cells migrate to the IZ outside

the Tbr2 expression domain (circled with dotted line).

b. Quantification of Tbr2 expression by Drosha KD cells relative to control.

c. Quantification of Drosha KD cells in the IZ relative to control.

d. Quantification of Tbr2 expression by Drosha KD cells relative to control 48 hours post–

transfection.


e. Scheme of procedure to isolate electroporated (red) embryonic day (E) 14.5 (shRNA KD) or

E15.5 (Drosha cKO analysis) neural progenitors for qRT–PCR and TaqMan miRNA analysis. Wild

type embryos were electroporated with Dorsha shRNA or control (GFP) shRNA expression vectors

and analyzed at E14.5. Droshaflox/flox and Droshaflox/wt embryos were electroporated with Hes5::Cre

vector and analyzed at E15.5.

f. Scheme of the experimental procedure. neural progenitors isolated from Droshaflox/flox, Dicerflox/flox,

Droshaflox/wt and Dicerflox/wt mice were expanded as neurospheres. Dissociated neural progenitors were

infected with Adeno–Cre viruses to induce recombination of the Drosha and Dicer alleles.

g. Conditional knockout (cKO) through Adeno–Cre infection of neural progenitors results in a loss

of Drosha protein in neurosphere 5 days post–infection (1°) and after passaging (2°) compared to

infected Droshaflox/wt NSCs.

Error bars represent s.e.m. Scale bar = 20 µm.


Supplementary Figure 7: Drosha KD and DGCR8 KD do not cause a global reduction in

mature miRNA levels after 28 hours but miRNA levels reduce in NSCs by 5 days after Drosha

cKO

In order to address whether Drosha KD and DGCR8 KD had a global effect on mature miRNA, we

performed qRT–PCR for specific miRNAs known to be expressed during neural development and

represented by miR15a and miR30d.

a. Quantification of relative miR15a and miR30d levels in Drosha KD and DGCR8 KD cells isolated

from electroporated cells by FACS and compared to control shRNA expressing neural progenitors.

Neither Drosha KD nor DGCR8 KD cells showed significant changes in miR15a or miR30d levels 24

hours post–KD even at the point where phenotypic changes are clear. Control cell miRNA levels were

set to 1.

b. Numerical depiction of the qRT–PCR analysis shown in a.

c. Delta CT plot of relative miRNA expression profiles of control (Droshaflox/wt) (y–axis) and Drosha

cKO (x–axis) neural progenitor cells. Correlation coefficient R2 = 0.2091 indicates major changes in

miRNA levels 5 days after Drosha cKO. Control NSCs express 135 miRNAs whereas Drosha cKO

NSCs express 44 and the expression of these is reduce compared to controls (supplementary Tables

2 and 3).

Error bars represent s.e.m.


Supplementary Figure 8: shRNA Ngn2 KD can rescue Drosha KD neural progenitors

a. Co–expression of shRNAs targeting Ngn2 and Drosha rescues neural progenitors which then

show a normal distribution in the VZ and SVZ/IZ compared to control cells expressing a scrambled

shRNA.

b. Quantification of cell distribution following simultaneous Drosha KD and Ngn2 KD showing the

rescue of VZ neural progenitors relative to control (scrambled shRNA).

Error bars represent s.e.m. Scale bars = 20 µm.


Supplementary Figure 9: Drosha interacts with Ngn2 mRNA

a. Scheme of the cross–linked immunoprecipitation (CLIP) procedure leading to analysis of bound

RNAs by qRT–PCR.

b. Gel analysis of amplicons for Ngn2 and DGCR8 after CLIP with Drosha (WT) and TN Drosha

(TN). Flag–tagged GFP was CLIPed as a control. The agarose gels are representative experiments

performed in duplicate (2 lanes per CLIP). CLIP RT+ shows amplification of precipitated Ngn2 and

DGCR8 RNAs. CLIP RT– and Input RT+ are controls for RNA amplification and absence of genomic

DNA contamination and equal amounts of input RNA into the CLIP experiments in each sample. The

specificity of the amplicons was verified by sequencing.


Figure 10: Drosha and the MP have dual roles for regulating the neural progenitor proteome

a. Drosha and DGCR8 are central components of the microprocessor catalyzing the processing of

pri–miRNA to pre–miRNA during miRNA biogenesis in the nucleus. The pre–miRNA is exported to the

cytoplasm and further processed by Dicer and loaded onto the RNA–induced silencing complex

(RISC) before targeting to mRNAs (upper). We present evidence that the MP can bind hairpins in

mRNAs directly, here shown for Ngn2 mRNA, and cleave the RNA into fragments that are degraded

and thus regulate the proteome through RNA destabilization in a Dicer– and miRNA–independent

fashion (lower).

b. Scheme of Hes and Ngn2 expression dynamics in neural stem/progenitor cells. Hes genes are

under the transcriptional control of canonical Notch signaling and Hes proteins repress their own gene

transcription resulting in an oscillatory expression2,3. Hes proteins repress Ngn2 transcription4,5. Ngn2

mRNA is destabilized by Drosha and the MP to prevent mRNA accumulation during neural

stem/progenitor cell maintenance. Loss of Notch signaling results in loss of Hes expression and relief

of Ngn2 transcriptional repression. Combined with a reduction in MP–mediated destabilization of its

mRNA and the associated increase in protein, Ngn2 promotes neurogenic differentiation of

stem/progenitor cells. The signals that positively regulate Ngn2 transcription are not well understood.


Assay Type Drosha cKO Drosha ctrl Delta Ct cKO Delta Ct Ctrl Delta Delta

CT FC CKO vs

Ctrl mmu-miR-9-4373285 Target 18.4614 20.3614 -7.5579 -6.5355 -1.0224 1.0224 mmu-miR-19b-4373098 Target 18.5662 19.6961 -7.4531 -7.2008 -0.2523 0.2523 mmu-miR-17-4395419 Selected Control 19.0576 19.8194 -6.9617 -7.0775 0.1158 -0.1158 mmu-miR-223-4395406 Target 19.625 19.4865 -6.3943 -7.4104 1.0161 -1.0161 mmu-miR-106a-4395589 Target 20.3632 21.0729 -5.6561 -5.824 0.1679 -0.1679 mmu-miR-16-4373121 Target 20.4092 21.1878 -5.6101 -5.7091 0.099 -0.099 mmu-miR-20a-4373286 Target 20.4631 21.3925 -5.5562 -5.5044 -0.0518 0.0518 mmu-miR-125b-5p-4373148 Target 20.9473 22.9957 -5.072 -3.9012 -1.1708 1.1708 mmu-miR-484-4381032 Target 21.2445 21.8291 -4.7748 -5.0678 0.293 -0.293 mmu-miR-93-4373302 Target 21.4789 22.8872 -4.5404 -4.0097 -0.5307 0.5307 mmu-miR-26a-4395166 Target 21.6381 23.4289 -4.3812 -3.468 -0.9132 0.9132 mmu-miR-19a-4373099 Target 21.6459 22.9473 -4.3734 -3.9496 -0.4238 0.4238 mmu-miR-30c-4373060 Target 21.7945 23.1467 -4.2248 -3.7502 -0.4746 0.4746 mmu-miR-191-4395410 Target 22.2844 23.6069 -3.7349 -3.29 -0.4449 0.4449 mmu-miR-126-3p-4395339 Target 22.6353 25.0856 -3.384 -1.8113 -1.5727 1.5727 mmu-miR-24-4373072 Target 22.6784 23.6911 -3.3409 -3.2058 -0.1351 0.1351 mmu-miR-30b-4373290 Target 22.7049 24.1375 -3.3144 -2.7594 -0.555 0.555 mmu-miR-135b-4395372 Target 22.7323 24.253 -3.287 -2.6439 -0.6431 0.6431 mmu-miR-92a-4373013 Selected Control 22.8331 23.7309 -3.1862 -3.166 -0.0202 0.0202 mmu-miR-125a-5p-4395309 Target 23.2743 23.9707 -2.745 -2.9262 0.1812 -0.1812 mmu-miR-342-3p-4395371 Target 23.3159 24.4412 -2.7034 -2.4557 -0.2477 0.2477 mmu-let-7g-4395393 Target 23.5034 25.4701 -2.5159 -1.4268 -1.0891 1.0891 mmu-let-7i-4395332 Target 23.6637 25.7888 -2.3556 -1.1081 -1.2475 1.2475 mmu-miR-708-4395452 Target 23.6824 24.6198 -2.3369 -2.2771 -0.0598 0.0598 mmu-miR-106b-4373155 Target 23.7977 25.65 -2.2216 -1.2469 -0.9747 0.9747 mmu-miR-301b-4395730 Target 23.9164 25.1334 -2.1029 -1.7635 -0.3394 0.3394 mmu-let-7e-4395517 Target 23.9744 24.3863 -2.0449 -2.5106 0.4657 -0.4657 mmu-let-7c-4373167 Target 24.0272 24.3138 -1.9921 -2.5831 0.591 -0.591 mmu-miR-301a-4373064 Target 24.1738 25.2642 -1.8455 -1.6327 -0.2128 0.2128 mmu-miR-138-4395395 Target 24.1812 25.6477 -1.8381 -1.2492 -0.5889 0.5889 mmu-miR-331-3p-4373046 Target 24.2343 25.3728 -1.785 -1.5241 -0.2609 0.2609 mmu-miR-140-4373374 Target 24.323 26.3221 -1.6963 -0.5748 -1.1215 1.1215


mmu-miR-30e-4395334 Target 24.3483 25.8348 -1.671 -1.0621 -0.6089 0.6089 mmu-miR-186-4395396 Target 24.3554 24.9145 -1.6639 -1.9824 0.3185 -0.3185 mmu-miR-26b-4395167 Target 24.4184 25.4644 -1.6009 -1.4325 -0.1684 0.1684 mmu-miR-384-5p-4395732 Target 24.7516 26.7314 -1.2677 -0.1655 -1.1022 1.1022 mmu-miR-204-4373094 Target 24.839 27.6971 -1.1803 0.8002 -1.9805 1.9805 mmu-miR-218-4373081 Target 24.9413 26.3087 -1.078 -0.5882 -0.4898 0.4898 mmu-miR-135a-4373140 Target 24.9824 25.9437 -1.0369 -0.9532 -0.0837 0.0837 mmu-miR-130a-4373145 Target 25.0149 27.5795 -1.0044 0.6826 -1.687 1.687 mmu-miR-30a-4373061 Target 25.1335 26.7692 -0.8858 -0.1277 -0.7581 0.7581 mmu-miR-532-5p-4380928 Target 25.15 26.6247 -0.8693 -0.2722 -0.5971 0.5971 mmu-miR-15b-4373122 Target 25.187 26.4919 -0.8323 -0.405 -0.4273 0.4273 mmu-miR-340-5p-4395369 Target 25.216 27.0567 -0.8033 0.1598 -0.9631 0.9631 mmu-miR-222-4395387 Target 25.2397 26.157 -0.7796 -0.7399 -0.0397 0.0397 mmu-miR-146a-4373132 Target 25.3335 26.5118 -0.6858 -0.3851 -0.3007 0.3007 mmu-miR-195-4373105 Target 25.342 26.5889 -0.6773 -0.308 -0.3693 0.3693 mmu-miR-20b-4373263 Target 25.4534 26.9017 -0.5659 0.0048 -0.5707 0.5707 mmu-let-7d-4395394 Target 25.6376 26.9765 -0.3817 0.0796 -0.4613 0.4613 mmu-miR-181a-4373117 Target 25.6399 27.3997 -0.3794 0.5028 -0.8822 0.8822 mmu-miR-126-5p-4373269 Target 25.6646 27.4168 -0.3547 0.5199 -0.8746 0.8746 mmu-miR-25-4373071 Target 25.674 26.6693 -0.3453 -0.2276 -0.1177 0.1177 mmu-miR-99b-4373007 Target 25.7552 27.5757 -0.2641 0.6788 -0.9429 0.9429 mmu-miR-127-4373147 Target 25.9156 28.0093 -0.1037 1.1124 -1.2161 1.2161 mmu-miR-434-3p-4395734 Target 25.9788 27.9008 -0.0405 1.0039 -1.0444 1.0444 mmu-let-7b-4373168 Target 25.9919 26.3961 -0.0274 -0.5008 0.4734 -0.4734 mmu-miR-132-4373143 Target 25.9928 27.0557 -0.0265 0.1588 -0.1853 0.1853 mmu-miR-103-4373158 Target 25.998 28.4867 -0.0213 1.5898 -1.6111 1.6111 mmu-miR-409-3p-4395443 Target 26.0099 27.3146 -0.0094 0.4177 -0.4271 0.4271 mmu-miR-449a-4373207 Target 26.0193 26.8969 0 0 0 0 mmu-miR-29a-4395223 Target 26.1016 28.4055 0.0823 1.5086 -1.4263 1.4263 mmu-miR-335-5p-4373045 Target 26.1863 27.9615 0.167 1.0646 -0.8976 0.8976 mmu-miR-744-4395435 Target 26.1894 27.0738 0.1701 0.1769 -0.0068 0.0068 mmu-miR-320-4395388 Target 26.4814 27.431 0.4621 0.5341 -0.072 0.072 mmu-miR-130b-4373144 Target 26.4885 28.4773 0.4692 1.5804 -1.1112 1.1112 mmu-miR-672-4395438 Target 26.5274 27.3443 0.5081 0.4474 0.0607 -0.0607 mmu-miR-411-4381013 Target 26.5712 29.8937 0.5519 2.9968 -2.4449 2.4449 mmu-miR-203-4373095 Target 26.5802 27.7021 0.5609 0.8052 -0.2443 0.2443


mmu-miR-124-4373295 Target 26.6027 28.8466 0.5834 1.9497 -1.3663 1.3663 mmu-miR-18a-4395533 Target 26.6219 28.0325 0.6026 1.1356 -0.533 0.533 mmu-miR-30d-4373059 Target 26.6552 28.0963 0.6359 1.1994 -0.5635 0.5635 mmu-miR-652-4395463 Target 26.6566 28.5434 0.6373 1.6465 -1.0092 1.0092 mmu-miR-328-4373049 Target 26.7557 26.7963 0.7364 -0.1006 0.837 -0.837 mmu-miR-532-3p-4395466 Target 26.7781 28.0451 0.7588 1.1482 -0.3894 0.3894 mmu-miR-139-5p-4395400 Target 26.7842 27.5444 0.7649 0.6475 0.1174 -0.1174 mmu-miR-872-4395375 Target 26.7858 28.6288 0.7665 1.7319 -0.9654 0.9654 mmu-miR-137-4373301 Target 26.8633 28.8026 0.844 1.9057 -1.0617 1.0617 mmu-miR-376c-4395580 Target 26.8883 29.0362 0.869 2.1393 -1.2703 1.2703 mmu-miR-335-3p-4395296 Target 26.8999 27.9734 0.8806 1.0765 -0.1959 0.1959 mmu-miR-21-4373090 Target 26.9898 28.9043 0.9705 2.0074 -1.0369 1.0369 mmu-miR-15a-4373123 Target 26.9909 28.497 0.9716 1.6001 -0.6285 0.6285 mmu-miR-145-4395389 Target 27.0952 29.1965 1.0759 2.2996 -1.2237 1.2237 mmu-miR-539-4378103 Target 27.1979 29.2676 1.1786 2.3707 -1.1921 1.1921 mmu-miR-323-3p-4395338 Target 27.241 28.3661 1.2217 1.4692 -0.2475 0.2475 mmu-miR-340-3p-4395370 Target 27.2726 28.7055 1.2533 1.8086 -0.5553 0.5553 mmu-miR-322-4378107 Target 27.2993 28.9119 1.28 2.015 -0.735 0.735 mmu-let-7a-4373169 Target 27.3535 27.6536 1.3342 0.7567 0.5775 -0.5775 mmu-miR-224-4395683 Target 27.5324 30.0064 1.5131 3.1095 -1.5964 1.5964 mmu-miR-296-5p-4373066 Target 27.6169 28.2216 1.5976 1.3247 0.2729 -0.2729 mmu-miR-28-4373067 Target 27.6791 29.3222 1.6598 2.4253 -0.7655 0.7655 mmu-miR-344-4373340 Target 27.8391 29.9988 1.8198 3.1019 -1.2821 1.2821 mmu-miR-431-4395173 Target 27.8404 30.7376 1.8211 3.8407 -2.0196 2.0196 mmu-miR-210-4373089 Target 27.843 28.6841 1.8237 1.7872 0.0365 -0.0365 mmu-miR-101a-4395364 Target 27.8875 29.5052 1.8682 2.6083 -0.7401 0.7401 mmu-miR-298-4395728 Target 27.9517 29.4436 1.9324 2.5467 -0.6143 0.6143 mmu-miR-27a-4373287 Target 27.9667 31.8951 1.9474 4.9982 -3.0508 3.0508 mmu-miR-128a-4395327 Target 28.077 28.6723 2.0577 1.7754 0.2823 -0.2823 mmu-miR-150-4373127 Target 28.0971 29.3432 2.0778 2.4463 -0.3685 0.3685 mmu-miR-199a-3p-4395415 Target 28.1666 32.2452 2.1473 5.3483 -3.201 3.201 mmu-miR-382-4373019 Target 28.1826 30.6738 2.1633 3.7769 -1.6136 1.6136 mmu-miR-379-4373349 Target 28.2172 31.041 2.1979 4.1441 -1.9462 1.9462 mmu-miR-487b-4378102 Target 28.4069 30.3838 2.3876 3.4869 -1.0993 1.0993 mmu-miR-676-4386776 Target 28.4207 29.5456 2.4014 2.6487 -0.2473 0.2473 mmu-miR-142-3p-4373136 Target 28.4762 29.9978 2.4569 3.1009 -0.644 0.644


mmu-miR-486-4378096 Target 28.4798 30.7982 2.4605 3.9013 -1.4408 1.4408 mmu-miR-495-4381078 Target 28.493 29.6158 2.4737 2.7189 -0.2452 0.2452 mmu-miR-27b-4373068 Target 28.5084 30.4675 2.4891 3.5706 -1.0815 1.0815 mmu-miR-574-3p-4395460 Target 28.5107 29.3871 2.4914 2.4902 0.0012 -0.0012 mmu-miR-410-4378093 Target 28.5612 30.2332 2.5419 3.3363 -0.7944 0.7944 mmu-miR-221-4373077 Target 28.5833 30.6564 2.564 3.7595 -1.1955 1.1955 mmu-miR-29c-4395171 Target 28.5855 30.588 2.5662 3.6911 -1.1249 1.1249 mmu-miR-494-4395476 Target 28.6086 30.1887 2.5893 3.2918 -0.7025 0.7025 mmu-miR-376a-4373347 Target 28.6487 31.3621 2.6294 4.4652 -1.8358 1.8358 mmu-miR-467a-4395717 Target 28.7034 31.0265 2.6841 4.1296 -1.4455 1.4455 mmu-miR-370-4395386 Target 28.7774 30.2349 2.7581 3.338 -0.5799 0.5799 rno-miR-345-3p-4395762 Target 28.8428 29.3727 2.8235 2.4758 0.3477 -0.3477 mmu-miR-425-4380926 Target 28.8441 31.1741 2.8248 4.2772 -1.4524 1.4524 mmu-miR-192-4373108 Target 28.8673 29.9953 2.848 3.0984 -0.2504 0.2504 mmu-miR-324-5p-4373052 Target 28.9663 30.3036 2.947 3.4067 -0.4597 0.4597 mmu-miR-129-3p-4373297 Target 28.9726 31.5239 2.9533 4.627 -1.6737 1.6737 mmu-miR-151-3p-4373304 Target 28.9832 30.2675 2.9639 3.3706 -0.4067 0.4067 mmu-miR-324-3p-4395639 Target 29.0006 29.8299 2.9813 2.933 0.0483 -0.0483 mmu-let-7f-4373164 Target 29.084 29.0871 3.0647 2.1902 0.8745 -0.8745 mmu-miR-497-4381046 Target 29.086 31.0431 3.0667 4.1462 -1.0795 1.0795 mmu-miR-181c-4373115 Target 29.1038 31.6846 3.0845 4.7877 -1.7032 1.7032 mmu-miR-193b-4395597 Target 29.1386 30.321 3.1193 3.4241 -0.3048 0.3048 mmu-miR-345-5p-4395658 Target 29.1869 31.0061 3.1676 4.1092 -0.9416 0.9416 mmu-miR-152-4395170 Target 29.2108 32.2119 3.1915 5.315 -2.1235 2.1235 mmu-miR-98-4373009 Target 29.2159 29.6229 3.1966 2.726 0.4706 -0.4706 mmu-miR-134-4373299 Target 29.2635 29.7618 3.2442 2.8649 0.3793 -0.3793 mmu-miR-543-4395487 Target 29.4153 32.0205 3.396 5.1236 -1.7276 1.7276 mmu-miR-143-4395360 Target 29.5191 31.8481 3.4998 4.9512 -1.4514 1.4514 mmu-miR-34b-3p-4395748 Target 29.5227 30.9113 3.5034 4.0144 -0.511 0.511 mmu-miR-185-4395382 Target 29.5456 31.4036 3.5263 4.5067 -0.9804 0.9804 mmu-miR-23b-4373073 Target 29.5586 31.7324 3.5393 4.8355 -1.2962 1.2962 mmu-miR-107-4373154 Target 29.5711 29.0028 3.5518 2.1059 1.4459 -1.4459 mmu-miR-148a-4373130 Target 29.575 31.333 3.5557 4.4361 -0.8804 0.8804 mmu-miR-350-4395660 Target 29.5801 30.6211 3.5608 3.7242 -0.1634 0.1634 mmu-miR-380-5p-4395731 Target 29.5871 31.4611 3.5678 4.5642 -0.9964 0.9964 mmu-miR-136-4395641 Target 29.5961 31.0157 3.5768 4.1188 -0.542 0.542


mmu-miR-188-5p-4395431 Target 29.5967 29.7687 3.5774 2.8718 0.7056 -0.7056 mmu-miR-155-4395701 Target 29.6207 29.8531 3.6014 2.9562 0.6452 -0.6452 mmu-miR-194-4373106 Target 29.6383 31.2141 3.619 4.3172 -0.6982 0.6982 mmu-miR-667-4386769 Target 29.7044 30.5369 3.6851 3.64 0.0451 -0.0451 mmu-miR-342-5p-4395657 Target 29.7118 29.8739 3.6925 2.977 0.7155 -0.7155 mmu-miR-361-4373035 Target 29.9323 31.1634 3.913 4.2665 -0.3535 0.3535 mmu-miR-503-4395586 Target 29.9698 32.0239 3.9505 5.127 -1.1765 1.1765 mmu-miR-674-4395193 Target 29.9925 31.7673 3.9732 4.8704 -0.8972 0.8972 mmu-miR-434-5p-4395711 Target 30.0131 32.9387 3.9938 6.0418 -2.048 2.048 mmu-miR-148b-4373129 Target 30.0598 32.6876 4.0405 5.7907 -1.7502 1.7502 mmu-miR-7a-4378130 Target 30.0691 31.7365 4.0498 4.8396 -0.7898 0.7898 mmu-miR-187-4373307 Target 30.1152 31.7949 4.0959 4.898 -0.8021 0.8021 mmu-miR-337-5p-4395645 Target 30.1762 31.8111 4.1569 4.9142 -0.7573 0.7573 rno-miR-207-4381096 Target 30.1901 32.0325 4.1708 5.1356 -0.9648 0.9648 mmu-miR-491-4381053 Target 30.2382 32.4693 4.2189 5.5724 -1.3535 1.3535 mmu-miR-433-4373205 Target 30.345 30.0527 4.3257 3.1558 1.1699 -1.1699 mmu-miR-214-4395417 Target 30.3802 32.3149 4.3609 5.418 -1.0571 1.0571 mmu-miR-671-3p-4395433 Target 30.3861 30.8003 4.3668 3.9034 0.4634 -0.4634 mmu-miR-501-3p-4381069 Target 30.4618 32.3193 4.4425 5.4224 -0.9799 0.9799 mmu-miR-31-4373331 Target 30.4845 30.6513 4.4652 3.7544 0.7108 -0.7108 mmu-miR-369-5p-4373195 Target 30.5653 32.8656 4.546 5.9687 -1.4227 1.4227 mmu-miR-448-4373206 Target 30.5924 33.4481 4.5731 6.5512 -1.9781 1.9781 mmu-miR-687-4386750 Target 30.5932 32.2899 4.5739 5.393 -0.8191 0.8191 mmu-miR-488-4381074 Target 30.5976 31.4746 4.5783 4.5777 0.0006 -0.0006 mmu-miR-544-4395680 Target 30.6041 31.7677 4.5848 4.8708 -0.286 0.286 rno-miR-351-4395764 Target 30.6548 32.2113 4.6355 5.3144 -0.6789 0.6789 mmu-miR-200b-4395362 Target 30.6633 35 4.644 8.1031 -3.4591 3.4591 mmu-miR-365-4373194 Target 30.6742 31.9836 4.6549 5.0867 -0.4318 0.4318 mmu-miR-296-3p-4395212 Target 30.8209 31.4598 4.8016 4.5629 0.2387 -0.2387 mmu-miR-182-4395729 Target 30.8769 32.649 4.8576 5.7521 -0.8945 0.8945 mmu-miR-337-3p-4395662 Target 30.8862 32.1415 4.8669 5.2446 -0.3777 0.3777 mmu-miR-429-4373355 Target 30.8909 35 4.8716 8.1031 -3.2315 3.2315 mmu-miR-34a-4395168 Target 30.8968 31.2128 4.8775 4.3159 0.5616 -0.5616 mmu-miR-339-3p-4395663 Target 30.9639 31.4789 4.9446 4.582 0.3626 -0.3626 mmu-miR-351-4373345 Target 30.9791 32.8098 4.9598 5.9129 -0.9531 0.9531 mmu-miR-376b-4395582 Target 31.0688 35 5.0495 8.1031 -3.0536 3.0536


mmu-miR-362-3p-4395746 Target 31.1434 33.1137 5.1241 6.2168 -1.0927 1.0927 mmu-miR-685-4386748 Target 31.1654 35 5.1461 8.1031 -2.957 2.957 mmu-miR-339-5p-4395368 Target 31.2391 33.6169 5.2198 6.72 -1.5002 1.5002 mmu-miR-325-4395640 Target 31.2808 35 5.2615 8.1031 -2.8416 2.8416 mmu-miR-680-4381079 Target 31.3067 31.5497 5.2874 4.6528 0.6346 -0.6346 mmu-miR-450a-5p-4395414 Target 31.5311 35 5.5118 8.1031 -2.5913 2.5913 mmu-miR-500-4395736 Target 31.5417 33.7489 5.5224 6.852 -1.3296 1.3296 mmu-miR-451-4373360 Target 31.5558 32.8827 5.5365 5.9858 -0.4493 0.4493 mmu-miR-384-3p-4395733 Target 31.6841 35 5.6648 8.1031 -2.4383 2.4383 mmu-miR-331-5p-4395344 Target 32.003 31.6835 5.9837 4.7866 1.1971 -1.1971

Supplementary Table 1. Microarray analysis and comparative quantification of miRNAs in Drosha cKO and control neural progenitors 48 hours post-

inactivation

The expression levels of the miRNA assays were quantified using the Relative Quantification (RQ) method. The data was normalized by geometric mean normalization,

using as normalizers miRNAs that were detected as stable across both assays. Stability analysis was conducted using normFinder6. miR-17 and miR-92a were used as

normalizers. Relative quantities for each miR assays (delta Ct) were calculated after normalization. Detected miRs (Ct<32) were then compared across samples.



CT FC cKO vs

Ctrl mmu-miR-484-4381032 Target 22.9391 22.6874 -1.7193 -0.1924 -1.5269 1.5269 mmu-miR-17-4395419 Selected Control 26.9573 21.4116 2.2989 -1.4681 3.767 -3.767 mmu-miR-19b-4373098 Target 27.0034 21.0038 2.3449 -1.8759 4.2208 -4.2208 mmu-miR-16-4373121 Target 27.0495 21.4188 2.3911 -1.4609 3.852 -3.852 mmu-miR-320-4395388 Target 27.1013 26.7921 2.4429 3.9123 -1.4694 1.4694 mmu-miR-9-4373285 Target 27.1492 20.6654 2.4908 -2.2143 4.7051 -4.7051 mmu-miR-685-4386748 Target 27.5107 28.1845 2.8523 5.3048 -2.4525 2.4525 mmu-miR-680-4381079 Target 27.5714 28.5307 2.913 5.651 -2.738 2.738 mmu-miR-106a-4395589 Target 27.9413 22.7994 3.2828 -0.0803 3.3631 -3.3631 mmu-miR-30c-4373060 Target 29.1251 22.7436 4.4666 -0.1361 4.6027 -4.6027 mmu-miR-20a-4373286 Target 29.2219 23.3422 4.5634 0.4625 4.1009 -4.1009 mmu-miR-191-4395410 Target 29.3884 23.2596 4.73 0.3798 4.3502 -4.3502 mmu-miR-30b-4373290 Target 29.4358 23.5771 4.7773 0.6974 4.0799 -4.0799 mmu-miR-344-4373340 Target 29.4929 29.1872 4.8344 6.3075 -1.4731 1.4731 rno-miR-207-4381096 Target 29.5381 29.3796 4.8796 6.4998 -1.6202 1.6202 mmu-miR-687-4386750 Target 29.6647 29.0679 5.0063 6.1882 -1.1819 1.1819 mmu-miR-24-4373072 Target 29.6784 24.0926 5.02 1.2128 3.8072 -3.8072 mmu-miR-93-4373302 Target 29.7668 23.4621 5.1083 0.5823 4.526 -4.526 mmu-miR-19a-4373099 Target 29.9412 23.6943 5.2828 0.8146 4.4682 -4.4682 mmu-miR-92a-4373013 Selected Control 30.0612 25.1792 5.4028 2.2994 3.1034 -3.1034 mmu-miR-574-3p-4395460 Target 30.5666 28.2686 5.9081 5.3889 0.5192 -0.5192 mmu-miR-146a-4373132 Target 30.6392 25.122 5.9807 2.2423 3.7384 -3.7384 mmu-miR-125a-5p-4395309 Target 30.6778 24.675 6.0194 1.7952 4.2242 -4.2242 mmu-miR-384-5p-4395732 Target 30.7178 25.6471 6.0594 2.7673 3.2921 -3.2921 mmu-miR-328-4373049 Target 30.7344 27.9262 6.0759 5.0465 1.0294 -1.0294 mmu-miR-26a-4395166 Target 30.9208 24.2632 6.2623 1.3834 4.8789 -4.8789 mmu-miR-222-4395387 Target 30.9218 26.935 6.2634 4.0553 2.2081 -2.2081 mmu-miR-132-4373143 Target 30.9907 24.9023 6.3322 2.0225 4.3097 -4.3097 mmu-miR-125b-5p-4373148 Target 31.1626 23.8771 6.5041 0.9974 5.5067 -5.5067 mmu-miR-494-4395476 Target 31.1757 29.0942 6.5172 6.2144 0.3028 -0.3028 mmu-miR-34a-4395168 Target 31.2038 26.8436 6.5453 3.9639 2.5814 -2.5814 mmu-miR-331-3p-4373046 Target 31.2391 26.6738 6.5806 3.7941 2.7865 -2.7865 mmu-let-7g-4395393 Target 31.2414 25.3768 6.583 2.4971 4.0859 -4.0859


mmu-miR-195-4373105 Target 31.394 26.3943 6.7356 3.5146 3.221 -3.221 mmu-miR-29a-4395223 Target 31.4399 25.4365 6.7815 2.5568 4.2247 -4.2247 mmu-miR-342-3p-4395371 Target 31.4475 26.6631 6.789 3.7834 3.0056 -3.0056 mmu-miR-667-4386769 Target 31.5949 30.6659 6.9364 7.7862 -0.8498 0.8498 mmu-miR-26b-4395167 Target 31.6567 26.4236 6.9983 3.5439 3.4544 -3.4544 mmu-miR-21-4373090 Target 31.7067 26.7024 7.0482 3.8227 3.2255 -3.2255 mmu-miR-15b-4373122 Target 31.845 25.7071 7.1866 2.8274 4.3592 -4.3592 mmu-miR-186-4395396 Target 31.8605 25.4825 7.202 2.6028 4.5992 -4.5992 mmu-miR-130a-4373145 Target 31.9531 26.6492 7.2946 3.7694 3.5252 -3.5252 mmu-miR-30a-4373061 Target 32.0946 27.1567 7.4361 4.2769 3.1592 -3.1592

Supplementary Table 2. Microarray analysis and comparative quantification of miRNAs in Drosha cKO and control NSCs 5 days post inactivation


miR-17 and miR-92a were used as normalizers. Stability analysis was conducted using normFinder6. Relative quantities for each miRNA assays (delta Ct) were calculated

after normalization. Detected miRNAs (Ct<32) were then compared across samples relative to miRNAs expressed in the Drosha cKO.



CT FC cKO vs

Ctrl mmu-miR-9-4373285 Target 27.1492 20.6654 2.4908 -2.2143 4.7051 -4.7051 mmu-miR-19b-4373098 Target 27.0034 21.0038 2.3449 -1.8759 4.2208 -4.2208 mmu-miR-17-4395419 Selected Control 26.9573 21.4116 2.2989 -1.4681 3.767 -3.767 mmu-miR-16-4373121 Target 27.0495 21.4188 2.3911 -1.4609 3.852 -3.852 mmu-miR-484-4381032 Target 22.9391 22.6874 -1.7193 -0.1924 -1.5269 1.5269 mmu-miR-30c-4373060 Target 29.1251 22.7436 4.4666 -0.1361 4.6027 -4.6027 mmu-miR-106a-4395589 Target 27.9413 22.7994 3.2828 -0.0803 3.3631 -3.3631 mmu-miR-191-4395410 Target 29.3884 23.2596 4.73 0.3798 4.3502 -4.3502 mmu-miR-20a-4373286 Target 29.2219 23.3422 4.5634 0.4625 4.1009 -4.1009 mmu-miR-93-4373302 Target 29.7668 23.4621 5.1083 0.5823 4.526 -4.526 mmu-miR-30b-4373290 Target 29.4358 23.5771 4.7773 0.6974 4.0799 -4.0799 mmu-miR-19a-4373099 Target 29.9412 23.6943 5.2828 0.8146 4.4682 -4.4682 mmu-miR-125b-5p-4373148 Target 31.1626 23.8771 6.5041 0.9974 5.5067 -5.5067 mmu-miR-24-4373072 Target 29.6784 24.0926 5.02 1.2128 3.8072 -3.8072 mmu-miR-26a-4395166 Target 30.9208 24.2632 6.2623 1.3834 4.8789 -4.8789 mmu-miR-125a-5p-4395309 Target 30.6778 24.675 6.0194 1.7952 4.2242 -4.2242 mmu-miR-132-4373143 Target 30.9907 24.9023 6.3322 2.0225 4.3097 -4.3097 mmu-miR-146a-4373132 Target 30.6392 25.122 5.9807 2.2423 3.7384 -3.7384 mmu-miR-92a-4373013 Selected Control 30.0612 25.1792 5.4028 2.2994 3.1034 -3.1034 mmu-let-7g-4395393 Target 31.2414 25.3768 6.583 2.4971 4.0859 -4.0859 mmu-miR-29a-4395223 Target 31.4399 25.4365 6.7815 2.5568 4.2247 -4.2247 mmu-miR-186-4395396 Target 31.8605 25.4825 7.202 2.6028 4.5992 -4.5992 mmu-miR-384-5p-4395732 Target 30.7178 25.6471 6.0594 2.7673 3.2921 -3.2921 mmu-miR-15b-4373122 Target 31.845 25.7071 7.1866 2.8274 4.3592 -4.3592 mmu-let-7i-4395332 Target 34.207 25.7257 9.5485 2.846 6.7025 -6.7025 mmu-miR-532-5p-4380928 Target 35.1368 25.8988 10.4783 3.0191 7.4592 -7.4592 mmu-miR-30e-4395334 Target 32.1232 25.916 7.4648 3.0362 4.4286 -4.4286 mmu-let-7e-4395517 Target 32.2617 25.9237 7.6032 3.044 4.5592 -4.5592 mmu-miR-335-3p-4395296 Target 32.7321 26.2209 8.0737 3.3412 4.7325 -4.7325 mmu-miR-106b-4373155 Target 32.7258 26.299 8.0673 3.4192 4.6481 -4.6481 mmu-miR-140-4373374 Target 33.2786 26.3335 8.6201 3.4538 5.1663 -5.1663 mmu-miR-195-4373105 Target 31.394 26.3943 6.7356 3.5146 3.221 -3.221 mmu-miR-26b-4395167 Target 31.6567 26.4236 6.9983 3.5439 3.4544 -3.4544


mmu-miR-210-4373089 Target 40 26.5677 15.3416 3.6879 11.6537 -11.6537 mmu-miR-130a-4373145 Target 31.9531 26.6492 7.2946 3.7694 3.5252 -3.5252 mmu-miR-342-3p-4395371 Target 31.4475 26.6631 6.789 3.7834 3.0056 -3.0056 mmu-miR-331-3p-4373046 Target 31.2391 26.6738 6.5806 3.7941 2.7865 -2.7865 mmu-miR-21-4373090 Target 31.7067 26.7024 7.0482 3.8227 3.2255 -3.2255 mmu-miR-34b-3p-4395748 Target 32.3168 26.7308 7.6583 3.8511 3.8072 -3.8072 mmu-miR-335-5p-4373045 Target 32.8831 26.7647 8.2247 3.885 4.3397 -4.3397 mmu-miR-320-4395388 Target 27.1013 26.7921 2.4429 3.9123 -1.4694 1.4694 mmu-miR-34a-4395168 Target 31.2038 26.8436 6.5453 3.9639 2.5814 -2.5814 mmu-miR-193b-4395597 Target 33.9793 26.8789 9.3208 3.9992 5.3216 -5.3216 mmu-let-7c-4373167 Target 33.7858 26.9293 9.1273 4.0496 5.0777 -5.0777 mmu-miR-222-4395387 Target 30.9218 26.935 6.2634 4.0553 2.2081 -2.2081 mmu-miR-155-4395701 Target 34.1056 27.0139 9.4471 4.1342 5.3129 -5.3129 mmu-let-7d-4395394 Target 32.7975 27.1112 8.1391 4.2315 3.9076 -3.9076 mmu-miR-30a-4373061 Target 32.0946 27.1567 7.4361 4.2769 3.1592 -3.1592 mmu-miR-672-4395438 Target 32.9461 27.3244 8.2877 4.4446 3.8431 -3.8431 mmu-miR-29c-4395171 Target 33.7144 27.3529 9.056 4.4731 4.5829 -4.5829 mmu-miR-744-4395435 Target 40 27.6176 15.3416 4.7379 10.6037 -10.6037 mmu-miR-138-4395395 Target 40 27.7229 15.3416 4.8432 10.4984 -10.4984 mmu-miR-872-4395375 Target 33.8184 27.7409 9.1599 4.8612 4.2987 -4.2987 mmu-miR-301b-4395730 Target 32.6106 27.7602 7.9521 4.8805 3.0716 -3.0716 mmu-miR-301a-4373064 Target 33.3698 27.9099 8.7114 5.0301 3.6813 -3.6813 mmu-miR-328-4373049 Target 30.7344 27.9262 6.0759 5.0465 1.0294 -1.0294 mmu-miR-99b-4373007 Target 35.8841 27.9684 11.2257 5.0887 6.137 -6.137 mmu-miR-30d-4373059 Target 33.8374 27.9764 9.1789 5.0967 4.0822 -4.0822 mmu-miR-340-5p-4395369 Target 34.7529 28.0808 10.0945 5.201 4.8935 -4.8935 mmu-miR-652-4395463 Target 33.9874 28.109 9.3289 5.2292 4.0997 -4.0997 mmu-miR-685-4386748 Target 27.5107 28.1845 2.8523 5.3048 -2.4525 2.4525 mmu-miR-322-4378107 Target 35.8141 28.1942 11.1556 5.3144 5.8412 -5.8412 mmu-miR-574-3p-4395460 Target 30.5666 28.2686 5.9081 5.3889 0.5192 -0.5192 mmu-miR-130b-4373144 Target 33.6623 28.3642 9.0039 5.4845 3.5194 -3.5194 mmu-miR-139-5p-4395400 Target 32.284 28.4874 7.6255 5.6077 2.0178 -2.0178 mmu-miR-680-4381079 Target 27.5714 28.5307 2.913 5.651 -2.738 2.738 mmu-miR-181a-4373117 Target 33.374 28.7194 8.7156 5.8396 2.876 -2.876 mmu-miR-18a-4395533 Target 40 28.7482 15.3416 5.8685 9.4731 -9.4731 mmu-miR-532-3p-4395466 Target 40 28.7905 15.3416 5.9107 9.4309 -9.4309


mmu-miR-25-4373071 Target 40 28.8019 15.3416 5.9222 9.4194 -9.4194 mmu-let-7b-4373168 Target 32.7155 28.8322 8.0571 5.9525 2.1046 -2.1046 mmu-miR-15a-4373123 Target 34.1502 28.8385 9.4918 5.9587 3.5331 -3.5331 mmu-miR-497-4381046 Target 40 28.8533 15.3416 5.9735 9.3681 -9.3681 mmu-miR-103-4373158 Target 36.2282 28.8643 11.5698 5.9846 5.5852 -5.5852 mmu-miR-28-4373067 Target 40 28.962 15.3416 6.0823 9.2593 -9.2593 mmu-miR-34c-4373036 Target 36.0352 29.0023 11.3767 6.1225 5.2542 -5.2542 mmu-let-7a-4373169 Target 40 29.0202 15.3416 6.1405 9.2011 -9.2011 mmu-miR-687-4386750 Target 29.6647 29.0679 5.0063 6.1882 -1.1819 1.1819 mmu-miR-494-4395476 Target 31.1757 29.0942 6.5172 6.2144 0.3028 -0.3028 mmu-miR-344-4373340 Target 29.4929 29.1872 4.8344 6.3075 -1.4731 1.4731 rno-miR-207-4381096 Target 29.5381 29.3796 4.8796 6.4998 -1.6202 1.6202 mmu-miR-129-3p-4373297 Target 40 29.5001 15.3416 6.6204 8.7212 -8.7212 mmu-miR-126-3p-4395339 Target 34.1156 29.5432 9.4571 6.6634 2.7937 -2.7937 mmu-miR-27a-4373287 Target 40 29.5736 15.3416 6.6939 8.6477 -8.6477 mmu-miR-151-3p-4373304 Target 40 29.6037 15.3416 6.724 8.6176 -8.6176 mmu-miR-296-5p-4373066 Target 34.4901 29.6447 9.8317 6.765 3.0667 -3.0667 mmu-miR-221-4373077 Target 40 29.6464 15.3416 6.7667 8.5749 -8.5749 mmu-miR-467a-4395717 Target 40 29.6605 15.3416 6.7808 8.5608 -8.5608 mmu-miR-676-4386776 Target 40 29.6733 15.3416 6.7935 8.5481 -8.5481 mmu-miR-188-5p-4395431 Target 32.561 29.7036 7.9026 6.8239 1.0787 -1.0787 mmu-miR-192-4373108 Target 33.3435 29.7559 8.6851 6.8762 1.8089 -1.8089 mmu-miR-20b-4373263 Target 35.5989 29.9041 10.9405 7.0244 3.9161 -3.9161 mmu-miR-425-4380926 Target 40 30.0416 15.3416 7.1619 8.1797 -8.1797 mmu-let-7f-4373164 Target 40 30.146 15.3416 7.2663 8.0753 -8.0753 mmu-miR-135b-4395372 Target 40 30.177 15.3416 7.2972 8.0444 -8.0444 mmu-miR-101a-4395364 Target 40 30.2111 15.3416 7.3314 8.0102 -8.0102 rno-miR-351-4395764 Target 34.9192 30.2117 10.2607 7.332 2.9287 -2.9287 mmu-miR-98-4373009 Target 34.8111 30.4669 10.1526 7.5871 2.5655 -2.5655 mmu-miR-671-3p-4395433 Target 40 30.4852 15.3416 7.6055 7.7361 -7.7361 mmu-miR-298-4395728 Target 40 30.5153 15.3416 7.6356 7.706 -7.706 mmu-miR-365-4373194 Target 40 30.5593 15.3416 7.6795 7.6621 -7.6621 mmu-miR-135a-4373140 Target 35.8226 30.5752 11.1642 7.6955 3.4687 -3.4687 mmu-miR-128a-4395327 Target 40 30.6093 15.3416 7.7296 7.612 -7.612 mmu-miR-350-4395660 Target 40 30.6267 15.3416 7.747 7.5946 -7.5946 mmu-miR-667-4386769 Target 31.5949 30.6659 6.9364 7.7862 -0.8498 0.8498


mmu-miR-339-3p-4395663 Target 40 30.6749 15.3416 7.7952 7.5464 -7.5464 mmu-miR-674-4395193 Target 40 30.7366 15.3416 7.8569 7.4847 -7.4847 mmu-miR-503-4395586 Target 40 30.7912 15.3416 7.9114 7.4302 -7.4302 mmu-miR-361-4373035 Target 40 30.795 15.3416 7.9153 7.4263 -7.4263 mmu-miR-203-4373095 Target 40 30.8183 15.3416 7.9385 7.4031 -7.4031 mmu-miR-383-4381093 Target 35.9351 30.8578 11.2766 7.9781 3.2985 -3.2985 mmu-miR-491-4381053 Target 40 31.0011 15.3416 8.1214 7.2202 -7.2202 mmu-miR-27b-4373068 Target 40 31.0481 15.3416 8.1684 7.1732 -7.1732 mmu-miR-194-4373106 Target 33.228 31.0482 8.5696 8.1685 0.4011 -0.4011 rno-miR-345-3p-4395762 Target 40 31.062 15.3416 8.1823 7.1593 -7.1593 mmu-miR-708-4395452 Target 36.2945 31.0795 11.6361 8.1997 3.4364 -3.4364 mmu-miR-340-3p-4395370 Target 35.5094 31.0944 10.851 8.2147 2.6363 -2.6363 mmu-miR-324-3p-4395639 Target 40 31.1373 15.3416 8.2576 7.084 -7.084 mmu-miR-324-5p-4373052 Target 40 31.353 15.3416 8.4733 6.8683 -6.8683 mmu-miR-187-4373307 Target 40 31.3575 15.3416 8.4778 6.8638 -6.8638 mmu-miR-185-4395382 Target 40 31.3837 15.3416 8.504 6.8376 -6.8376 mmu-miR-345-5p-4395658 Target 40 31.473 15.3416 8.5933 6.7483 -6.7483 mmu-miR-218-4373081 Target 40 31.5141 15.3416 8.6344 6.7072 -6.7072 mmu-miR-204-4373094 Target 40 31.6076 15.3416 8.7279 6.6137 -6.6137 mmu-miR-124-4373295 Target 40 31.6369 15.3416 8.7571 6.5845 -6.5845 mmu-miR-467b-4381084 Target 40 31.6566 15.3416 8.7769 6.5647 -6.5647 mmu-miR-488-4381074 Target 40 31.7294 15.3416 8.8496 6.492 -6.492 mmu-miR-199a-3p-4395415 Target 40 31.7429 15.3416 8.8631 6.4785 -6.4785 mmu-miR-148b-4373129 Target 40 31.8528 15.3416 8.9731 6.3685 -6.3685 mmu-miR-152-4395170 Target 40 31.8844 15.3416 9.0046 6.337 -6.337 mmu-miR-351-4373345 Target 40 31.9276 15.3416 9.0478 6.2938 -6.2938 mmu-miR-325-4395640 Target 40 31.9506 15.3416 9.0708 6.2708 -6.2708 mmu-miR-197-4373102 Target 32.7982 31.9899 8.1397 9.1102 -0.9705 0.9705 mmu-miR-148a-4373130 Target 40 32.0496 15.3416 9.1698 6.1718 -6.1718


Supplementary Table 3. Microarray analysis and comparative quantification of miRNAs in Drosha cKO and control NSCs 5 days post inactivation


miR-17 and miR-92a were used as normalizers. Stability analysis was conducted using6. Relative quantities for each miRNA assays (delta Ct) were calculated after

normalization. Detected miRNAs (Ct<32) were then compared across samples relative to miRNAs expressed in the control.

References 1 Basak, O. & Taylor, V. Identification of self-replicating multipotent progenitors in the embryonic nervous system by high Notch activity and Hes5 expression. Eur J

Neurosci 25, 1006-1022, (2007). 2 Kageyama, R., Niwa, Y., Shimojo, H., Kobayashi, T. & Ohtsuka, T. Ultradian oscillations in Notch signaling regulate dynamic biological events. Curr Top Dev Biol

92, 311-331, (2010). 3 Kageyama, R., Ohtsuka, T., Shimojo, H. & Imayoshi, I. Dynamic regulation of Notch signaling in neural progenitor cells. Curr Opin Cell Biol. 21, 733-740., (2009). 4 Masamizu, Y. et al. Real-time imaging of the somite segmentation clock: revelation of unstable oscillators in the individual presomitic mesoderm cells. Proc Natl

Acad Sci U S A 103, 1313-1318, (2006). 5 Kageyama, R., Ohtsuka, T. & Kobayashi, T. The Hes gene family: repressors and oscillators that orchestrate embryogenesis. Development 134, 1243-1251,

(2007). 6 Andersen, C. L., Jensen, J. L. & Orntoft, T. F. Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach

to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res. 64, 5245-5250., (2004).


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