conserved mir-8/mir-200 defines a glial niche that controls … · 2017. 2. 24. · developmental...

Developmental Cell

Article

Conserved miR-8/miR-200 Definesa Glial Niche that Controls NeuroepithelialExpansion and Neuroblast TransitionJavier Morante,1,* Diana M. Vallejo,1 Claude Desplan,2 and Maria Dominguez11Instituto de Neurociencias, Consejo Superior de Investigaciones Cientıficas y Universidad Miguel Hernandez, Av Santiago Ramon

y Cajal s/n, 03550 San Juan de Alicante, Spain2Center for Developmental Genetics, Department of Biology, New York University, 100 Washington Square East, New York, NY 10003, USA*Correspondence: [email protected]

http://dx.doi.org/10.1016/j.devcel.2013.09.018

SUMMARY

Neuroepithelial cell proliferation must be carefullybalanced with the transition to neuroblast (neuralstem cell) to control neurogenesis. Here, we showthat loss of the Drosophila microRNA mir-8 (thehomolog of vertebrate miR-200 family) results inboth excess proliferation and ectopic neuroblasttransition. Unexpectedly, mir-8 is expressed in asubpopulation of optic-lobe-associated cortex gliathat extend processes that ensheath the neuroepi-thelium, suggesting that glia cells communicatewith the neuroepithelium. We provide evidence thatmiR-8-positive glia express Spitz, a transforminggrowth factor a (TGF-a)-like ligand that triggersepidermal growth factor receptor (EGFR) activationto promote neuroepithelial proliferation and neuro-blast formation. Further, our experiments suggestthat miR-8 ensures both a correct glial architectureand the spatiotemporal control of Spitz protein syn-thesis via direct binding to Spitz 30 UTR. Together,these results establish glial-derived cues as key reg-ulatory elements in the control of neuroepithelial cellproliferation and the neuroblast transition.

INTRODUCTION

The correct regulation of adult brain size and function is a funda-

mental process that requires the careful regulation of neural stem

cell numbers during early neurogenesis. Inmature tissues, neural

stem cell number is precisely controlled by signal(s) from the

surrounding neural stem cell niche. However, evidence for the

occurrence and need of a niche microenvironment during early

neurogenesis has remained elusive (Knoblich, 2008). The optic

lobe of Drosophila is a well characterized model to study early

neurogenesis (Brand and Livesey, 2011) and the neural networks

underlying vision circuits (Morante and Desplan, 2008). Each

optic lobe derives from neural stem cells generated in a stereo-

typed pattern from two columnar neuroepithelia called the inner

(IPC) and outer (OPC) proliferation centers (White and Kankel,

1978) (Figures S1A–S1C available online). The OPC generates

174 Developmental Cell 27, 174–187, October 28, 2013 ª2013 Elsev

lamina and medulla neurons (Morante et al., 2011). The emer-

gence of neuroblasts in the part of the OPC neuroepithelium

that generates the medulla is dependent on the expression of

Lethal of scute [l(1)sc], a proneural basic helix-loop-helix protein

that specifies neuroblast fate (Yasugi et al., 2008). Expression of

l(1)sc progresses like a wave that resembles the morphogenetic

furrow in the fly eye imaginal disc (Treisman and Heberlein,

1998). As in the retina, the progression of neural development

associated with the expression of L(1)sc expression is positively

regulated by epidermal growth factor receptor (EGFR) signaling

and negatively regulated by Notch signaling (Egger et al., 2010;

Ngo et al., 2010; Reddy et al., 2010; Wang et al., 2011b; Yasugi

et al., 2010). Ahead of the wave that produces neuroblasts,

uncommitted neuroepithelial cells continue to divide symmetri-

cally, expanding the pool of prospective neuroblasts.

Increased signaling from the JAK/STAT and EGFR pathways

(Ngo et al., 2010; Wang et al., 2011a; Yasugi et al., 2008), or

loss of Hippo pathway activity (Kawamori et al., 2011; Reddy

et al., 2010), or of lethal (3) malignant brain tumor (Richter

et al., 2011) all cause oversized neuroepithelium and tumor

growth associated with a delay or blockade of the emergence

of neuroblasts. In contrast, loss of the Notch pathway has

the opposite consequence, advancing neuroblast progression

and causing a premature termination of neuroepithelial growth

(Egger et al., 2010; Ngo et al., 2010; Reddy et al., 2010; Wang

et al., 2011b; Yasugi et al., 2010). These observations indicate

that the EGFR pathway affects both the rate of neuroepithelial

proliferation and the rate of transition to neuroblast, reminiscent

to the reiterated use of the EGFR/Ras pathway during retinal

development (Domınguez et al., 1998; Freeman, 1996). Thus,

signaling through the EGFR/Ras pathway provides a good

candidate for coordinating proliferation with neuroblast

emergence. However, it remains obscure how the strength

and spatiotemporal activation of the EGF receptor pathway is

regulated to ensure proper brain size and pattern.

Glial cells are essential for the maintenance of nervous sys-

tem homeostasis, and the loss of glial function induces adult

neural degeneration (Kretzschmar et al., 1997). Thus far, two

distinct layers of surface glia ensheathing the Drosophila larval

brain have been described: the perineurial sheath of astro-

cyte-like cells, which divide throughout larval development;

and a layer of subperineurial glia that form septate junctions

and act as the main blood-brain barrier (Awasaki et al., 2008;

Bainton et al., 2005; Pereanu et al., 2005; Schwabe et al.,

ier Inc.

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Developmental Cell

A MicroRNA-Dependent Niche for Early Neurogenesis

2005; Stork et al., 2008; reviewed in DeSalvo et al., 2011;

Edwards and Meinertzhagen, 2010; Hartenstein, 2011; Stork

et al., 2012). Here, we describe a glial cell layer ensheathing

the optic lobe neuroepithelium, ‘‘optic-lobe-associated cortex

glia,’’ which is distinct from the two previously characterized

surface-associated glia cell populations, the perineurial and

subperineurial glial cells. We show that these glia are the source

of the EGFR ligand Spitz whose production is controlled by a

microRNA (miRNA).

Here, we show that glia ensheathing the developing optic

lobe neuroepithelium are marked by miRNA mir-8, which con-

trols their ability to produce the EGFR ligand Spitz. miR-8 is

the sole fly homolog of the human mir-200 gene family of tumor

suppressors (Brabletz et al., 2011; Vallejo et al., 2011) that

control epithelial-to-mesenchymal reprogramming (Park et al.,

2008; Wellner et al., 2009) and the acquisition of stemness (Shi-

mono et al., 2009). Previous work on this miRNA in flies (Hyun

et al., 2009; Karres et al., 2007) has demonstrated effects on

the nervous system, motor activity, leg development, and over-

all body size. Our analyses unveil a dual role for miR-8 in optic-

lobe-associated cortex glia: miR-8 is required to increase the

size of the glia, thereby facilitating sprouting toward the neuro-

epithelium; it also negatively regulates Spitz levels by directly

binding to its 30 UTR, thereby preventing premature and excess

signaling in the underlying neuroepithelium. Consistently, loss

and gain of spitz in the miR-8+ cortex glia mimicked the neuro-

epithelial defects observed following mir-8 gain and loss,

respectively, whereas expression of spitz lacking its 30 UTR

rescued defects caused by mir-8 overexpression. Taken

together, these findings define a prominent role for an optic-

lobe-associated cortex glia in controlling EGFR signaling and

thus the balance between neuroepithelial proliferation and

neuroblast emergence.

RESULTS

The Conserved MicroRNA miR-8 Is Expressed in GliaLoss ofmir-8 is associated with increased apoptosis in the larval

Drosophila brain (Karres et al., 2007). To further investigate the

developmental role of miR-8 in neurogenesis, we analyzed cells

expressing mir-8 at larval stages using a mir-8-Gal4 enhancer

trap line (Karres et al., 2007) and a nuclear RFP reporter (UAS-

H2B::RFP, Figures 1A–1D).

We detected mir-8 expression in the central brain and optic

lobe using both mir-8-Gal4 and in situ hybridization (Figures 1A

and S1D–S1H). mir-8 was detected in cortex glia in the central

brain and in glia surrounding the optic lobe neuroepithelium

starting before late third (L3) stage (Figures 1A–1D). mir-8 was

not detected in neuroblasts or in neuroepithelial cells (Figures

1A–1D, S1E, and S1F).

We used a membrane-tagged GFP reporter (UAS-

mCD8::GFP, Figures 1E–1H) to more precisely define the struc-

ture of the miR-8+ glia in the optic lobe. Serial optical sectioning

revealed that miR-8 positive optic lobe glia lie on the surface

(Figure 1E) and in the boundary between the optic lobe and the

central brain and project long extensions (arrowheads, Figures

1F–1H). This notion is further confirmed by single-cell labeling

using mir-8-Gal4 driving a FLP-out construct (Figures 1I–1L).

Moreover, these GFP-marked clones highlighted that surface-

Developm

associated miR-8-glia cells with large and elaborate arboriza-

tions ensheathing the developing optic lobe neuroepithelium

(Figure 1J) and central brain miR-8 cortex glia that ensheath neu-

roblast and neuronal lineages (arrowheads, Figure 1L) likely orig-

inate from distinct progenitor cells as we never recovered clones

containing both types ofmiR-8 glia.We determined that a normal

brain contains on average 137.33 ± 6.53 (mean ± SEM) miR-8+

glia within the optic lobe (Figure 1A). Of those, 25.16 ± 1.99

(mean ± SEM) glia cells lie on the surface of the optic lobe and

show the elaborated morphology illustrated in Figure 1J.

miR-8+ Surface-Associated Glia Lie Underneath theSubperineurial Glial Cell LayerPrevious studies classified the surface-associated glia surround-

ing the developing Drosophila optic lobes into two populations

(Awasaki et al., 2008; Stork et al., 2008; reviewed in DeSalvo

et al., 2011; Edwards and Meinertzhagen, 2010; Hartenstein,

2011; Stork et al., 2012): the perineurial glial cells, which are

located in the outermost superficial layer and the blood-brain-

barrier (also known as subperineurial glia), beneath the perineu-

rial layer. We found that miR-8+ glia constitute a glial layer that

lies underneath the subperineurial glial cell layer as revealed

by labeling them with the miR-8 driver (mir-8>mCD8::GFP)

and the subperineurial markers Moody (Bainton et al., 2005;

Schwabe et al., 2005) (Figures 2B and 2C) and Nrx-IV-GFP

(DeSalvo et al., 2011) (Figures S2A and S2B).

We also compared the nuclei and processes of miR-8 and

subperineurial glia labeled with an UAS-DsRed reporter under

the control of moody-Gal4 (arrows in Figures 2D–2F, S2C, and

S2D) in L3 larval optic lobes. Figures 2G and 2H show the

distinctive sprouting and locations of miR-8+ and subperineurial

glia (arrowheads and arrows point to mir-8 and moody glia,

respectively) in adult optic lobes. Therefore, miR-8 expression

defines a layer of glia underneath the subperineurial glial cell

layer in both the developing and adult optic lobe. Hereafter, we

refer to these glia as ‘‘optic-lobe-associated cortex glia’’

(Figure 2I).

Glial miR-8 Controls Neuroepithelial ExpansionThe developing optic lobe progenitor region is formed by

a growing neuroepithelial zone and a ‘‘neurogenic’’ zone where

neuroblasts are generated in a wave crossing the neuroepithe-

lium. In L3 wild-type brains, the neuroepithelium can be labeled

by staining for epithelial junctional markers Patj, DE-Cadherin

(DE-Cad), or Discs large (Dlg) (Figures 3A, S3A, and S3D) (Egger

et al., 2007). Lethal (1) of scute [L(1)sc] marks the transition zone

from neuroepithelial cells to neuroblasts (Figure S3D) (Yasugi

et al., 2008), which are labeled by the expression of Miranda

(Mira) (Figures 3A and S3A) (Egger et al., 2007; Morante et al.,

2011). The expression of Mira expands across the neuroepithe-

lium as neuroblasts are generated (Yasugi et al., 2008).

In flies lacking mir-8 (mir-8D2) (Karres et al., 2007), the neu-

roepithelium of the larva is 143% overgrown (11,565.11 ±

510.65 mm2, [mean ± SEM], n = 14 optic lobes scored) as

compared with wild-type (WT) L3 neuroepithelium (7,907.54 ±

732.1 mm2, n = 13). In the subsequent stage, ectopic and

precocious neuroblasts as visualized by the ectopic unevenly

spaced Mira- and L(1)sc-positive cells scattered throughout

the enlarged neuroepithelium (Figures 3B, 3E, and S3E). This

ental Cell 27, 174–187, October 28, 2013 ª2013 Elsevier Inc. 175

Figure 1. The Conserved MicroRNA miR-8 Is Expressed in Glia

(A–D) miR-8 labels a subset of Repo (red) glial cells. (A) Whole-mount view of a L3 brain and (B–D) details of L1, L2, and L3 brains from flies carrying mir-8-Gal4

driving a nuclear UAS-H2B::RFP (green). The neuroepithelial cells (NE) are labeled by DE-Cadherin (DE-Cad, blue).

(E–H) Magnification and serial optical sections from anterior (F) to posterior (H) showing miR-8+ cortex glia structure visualized by mCD8::GFP (green). NE is

labeled for DE-Cad (blue).

(I) Scheme to generate GFP-labeled mir-8-Gal4-positive glial clones in hsp70-FLP/+; mir-8-Gal4/UAS (FRT) CD2, y+ (FRT) mCD8::GFP.

(J–L) Examples shown are the distinct classes of FLP-out clones recovered. (J) Single cell clones (glial nuclei visualized by Repo label) of surface-associated glia

overlying the NE. (K–L) Single or few cell-clones of optic-lobe-associated cortex glia (K) and central brain cortex glia that ensheath neuronal cell bodies (K) and

neuroblast (arrowheads, L). CB, central brain; IPC, inner proliferation center; OL, optic lobe; OPC, outer proliferation center. Scale bars represent 50 mm (A, F–H)

and 20 mm (B–E, J–L).

See also Figure S1.

Developmental Cell


suggests that miR-8 is required both to limit the growth and neu-

roblast formation within the neuroepithelium.

Next, we investigated the effects of glial-specific overexpres-

sion ofmir-8 (UAS-mir-8) (Vallejo et al., 2011) using the pan-glial

driver repo-Gal4 (Sepp et al., 2001). mir-8 overexpression pro-

duced defects that were opposite to those seen in mir-8-defi-

cient animals, with a 41% reduction in the size of the underlying

neuroepithelium (4,713.32 ± 509.09 mm2, n = 12) and overall brain

size (Figures 3C, 3E, and S3C). This smaller neuroepithelial size

was coupled with accelerated progression of the proneural wave

marked by L(1)sc (Figure S3F).

To test the endogenous requirement of mir-8 in optic-lobe-

associated cortex glia, we attempted to generate genetic


mosaicsofmir-8—gliabut only recovered rareclonesof singleglial

cell, possibly reflecting the notion that these glia cells grow by en-

doreplication without dividing during larval stages (Stork et al.,

2012 and see below). We then used amiR-8microRNA ‘‘sponge’’

(UAS-mir-8-Sponge(SP)::GFP) (Loyaet al., 2009) expressed in the

endogenous mir-8 domain using mir-8-Gal4. miR-8 glia-specific

depletion of mir-8 caused defects similar to those observed in

wholly nullmir-8mutant animals (Figure 3D). For example, expres-

sion of two copies of theUAS-miR-8-(SP)::GFP bymir-8-Gal4 led

to an expansion of the neuroepithelium and ectopic neuroblasts

(Mira-positive cells) scattered throughout the enlarged neuroepi-

thelium. Thus, glia-specific gain and loss of mir-8 impact on the

growth and the emergence of neuroblast in the neuroepithelium.

ier Inc.

Figure 2. Surface-Associated miR-8 Glia

Layer Lies Underneath the Subperineurial

Glia

(A–C) Images showmiR-8+ cells (mir-8>H2B::RFP,

A ormCD8::GFP, B-C) constitute a distinct class of

surface-associated glia from that of the sub-

perineurial glia as is visualized by Moody-b anti-

body (green, B andC). Arrowhead points tomiR-8+

glia and arrow to subperineurial (miR-8� Moody+)

glia.

(D–F) Subperineurial glial membranes are labeled

by moody-Gal4 driving UAS-DsRed (red, arrow)

and glia nuclei by Repo. Single (E) and double

channel confocal image (Repo and moo-

dy>DsRed: F) are shown.

(G) Expression pattern of mir-8>DsRed in an adult

medulla cortex (MeC). Arrowheads point cortex

glia.

(H) Expression pattern of moody>DsRed in an

adult medulla cortex. Arrows point to sub-

perineurial glia. Neuropil and glia in (G) and (H) are

visualized by DN-Cad (blue) and Repo (green).

Insets show whole-mount views of adult optic

lobes.

(I) Schematic overview of the three layers of sur-

face-associated in a L3 brain lobe: miR-8+ (this

study), subperineurial (SPG) and perineurial (PG)

glial layers as single cells and highlighted in

different colors. NE, neuroepithelial cells. Scale

bar represents 20 mm.

See also Figure S2.

Developmental Cell


The Size and Sprouting of Optic-Lobe-AssociatedCortex Glia Is Controlled by miR-8These observations revealed communication between glia and

the underlying neuroepithelium. They suggest that miR-8+ glia

define an anatomical niche that provides structure and/or signals

for neuroepithelial progenitors.

miR-8+ optic-lobe-associated cortex glia exhibit characteristic

large nuclear and somatic sizes (44.42 ± 2.37 mm2, mean ± SEM:

n = 91 glial cells in 10 brains) as compared with subperineurial

glia (as visualized by Moody marker) (19.29 ± 1.74 mm2, 48 cells

in six brains) and perineurial glia (12.89 ± 0.59 mm2, 100 cells in

six brains) (quantification in Figure 4A; see Experimental Proce-

dures). Overexpression ofmir-8 in all glia by repo-Gal4 increased

further the glial nuclear size and processes. See quantification in

Figure 4A and compare count in Figure 4B versus 4C in late third

3D-brain reconstructions. Thus, glial cells immediately overlying

the optic lobe neuroepithelium (Moody negative and hence pre-

sumably the endogenous miR-8 glial cell population, arrow-

heads in Figures 4E and 4G) increased their size on average

400% (184.816 ± 15.48 mm2: n = 70 cells in 10 brains) and the

Moody-positive glia on top of this layer increased their size by

�500% (109.096 ± 10.979 mm2: n = 35 cells in ten brains, arrows

in Figures 4E and 4G). See also whole-mount (Figures 4D and 4F)

Developmental Cell 27, 174–187,

and high-magnification (Figures 4E and

4G) views. Central brain cortex glia also

increased their size (Figures S4A and

S4B) in response to mir-8 overexpres-

sion. However, satellite glia, epithelial

glia, marginal glia, and medulla neuropil

glia (Hartenstein, 2011; hereafter referred to as inner glia) did

not change cell size. Thus, control inner glial size area was

11.653 ± 0.86 mm2 (n = 47 cells in six brains scored) and

13.643 ± 1.16 mm2 in repo>mir-8 brains (n = 65 cells in six brains

scored) (Figures 4D and 4F and quantification in Figure 4A).

FLP-out clones overexpressing mir-8 (Figures S4C and S4D)

recovered contained a single cell, suggesting that mir-8 overex-

pressing glial cells do not proliferate (Stork et al., 2012; Vallejo

et al., 2011). These single-cell FLP-out mir-8 expressing clones

increased glial cell size cell-autonomously. Additionally, the

number of non-miR-8 surface-associated glial cells was reduced

from 78.91 ± 2.86 perineurial (i.e., Moody-negative) glia and

17.75 ± 1.48 subperineurial (i.e., Moody+) glia in WT (n = 21 optic

lobes scored) to 9.27 ± 0.91 glia in repo>mir-8 optic lobes (n =

18), most of them being Moody+ glia (Figures 4B and 4C). The

reduction of glial cell number may result in part from migrating

defects throughout the optic stalk (see Figures S4F and S4G)

and/or inhibition of cell proliferation. The role of miR-8 in cell

growth is evolutionarily conserved because overexpression of

human mir-200c by repo-Gal4 similarly increased cell-autono-

mously surface glial size (Figure S4E).

Cell size is often proportional to the amount of nuclear DNA

and many differentiating cells increase in size by undergoing

October 28, 2013 ª2013 Elsevier Inc. 177

Figure 3. Glial miR-8 Expression Controls

Neuroepithelial Expansion

(A–C) Whole-mount views of L3 brains in WT (A),

mir-8D2 (B) and animals overexpressing mir-8 by

repo-Gal4 (C). The NE are visualized by DE-Cad

(blue) and Patj (green) and the location of neuro-

blasts by Mira (red). (B) Larger neuroepithelium in

mir-8D2 brains (arrowheads) and precocious neu-

roblast transitions (arrows). (C) Image illustrates

the smaller neuroepithelial size (as measured in E)

in animals overexpressing mir-8 by repo-Gal4.

(D) Glia-specific depletion of mir-8 by expressing

two copies of UAS-mir-8-SP::GFP with mir-8-

Gal4.

(E) Histogram shows NE area of the indicated ge-

notypes measured using ImageJ (n = 13 [repo >],

n = 14 [mir-8D2] and n = 12 [repo>mir-8] brain lobes

scored). Shown are mean ± SEM and p values

were calculated by two-tailed unpaired t test.

Scale bar represents 50 mm.

See also Figure S3.

Developmental Cell


endoreplication without cell division (Park and Asano, 2008;

Zielke et al., 2011). Indeed, key markers of endoreplication,

including the APCFzr/Cdh1–lacZ enhancer trap line (Zielke et al.,

2008), were specifically only activated in central brain and op-

tic-lobe-associated cortex glial cells positive for mir-8 and spitz

in L3 larvae (Figures 4H and S4H–S4J; see also Figure S5),

consistent with the lack of mitotic figures in these cells (Figures

4I and S4K–S4M). Indeed, in L3 brains, we could only seemitosis

in neuroepithelial cells and neuroblasts (Figure S4N).

Similar to the increased cell size observed by overexpressing

mir-8 (Figures 4F and 4G), we observed increase glial cell size

when the fly homolog of the essential endoreplication factor

cdt1 (called dup in Drosophila) (Whittaker et al., 2000) was over-

expressed by repo-Gal4 (Figure S4O), albeit the effect was less

strong than when mir-8 was overexpressed. Conversely, RNA

interference (RNAi) silencing of dup/Cdt1 or of components of

the prereplication complex (Table S1) (Park and Asano, 2008)

in glial cells reduced cell number and size of the surface glia

(14 ± 1.89 mm2 in repo>dup-IR brains; n = 30 cells in 10 brains,

Figure 4A). In these repo>dup-IR brains, the only remaining sur-

face glia (10 ± 0.81 cells, n = 10) displayed short processes as

visualized by mCD8::GFP (arrowheads in Figure 4J and 4K and

compare with Figures 4D–4G) and were positive for the subper-

ineurial marker Moody. These alterations in size and sprouting

were fully reversed by expressing mir-8 in a repo>dup-IR back-

ground (Figures 4L and 4M). Thus, miR-8 seems to influence glial

size via a mechanism that might involve the depletion of a nega-

tive regulator of endoreplication, whose identity is unknown.

The underlying neuroepithelia in brains with smaller glia in re-

po>dup-IR and repo>orc1-IR were highly disorganized as visu-

alized by Patj staining (Figures 4N and 4O) with intermingled,

ectopic neuroepithelial-neuroblast transitions, as evident by

L(1)sc and Mira labeling (Figures S4P–S4S). This nonautono-

178 Developmental Cell 27, 174–187, October 28, 2013 ª2013 Elsevier Inc.

mous effect is specific for miR-8 glial

cells because selective expression of

mir-8 transgene in the endoreplicating

subperineurial glia cells (Unhavaithaya

and Orr-Weaver, 2012) usingmoody-Gal4 resulted in overgrown

glial cells (Figures S4T–S4Y), but the growth of the underlying

neuroepithelium and the timing of neuroblast transition were

not unaffected. Thus, we concluded that the proper structure

and signaling of miR-8 glia is necessary for proper neuroepithe-

lium development. We speculate that the nonautonomous influ-

ence of miR-8 glia likely involve direct regulation of a secreted

factor(s) by the microRNA.

Optic-Lobe-Associated Cortex Glia Is a Source of theEGFR Ligand SpitzThe brain defects observed in mir-8-deficient flies resembled

those seen in argos (aos) mutants (Figures 5C and 5D), where

we observed an enlarged neuroepithelium and then in later stage

a disruption of the orderly specification of neuroblasts, with

ectopic formation of neuroblasts visualized with Mira (compare

Figure 5D with Figure S3E). The aos gene encodes a negative

regulator of EGF receptor signaling that sequesters the EGFR

ligand TGF-a-like called Spitz (Freeman et al., 1992; Klein

et al., 2004). Spitz/TGF-a-like ligand is produced as an inactive

precursor that is converted to the active form when it is pro-

cessed by the intramembrane protease Rhomboid (Rho) (Sturte-

vant et al., 1993).

In the L3 brain, the aos-lacZ (Figure 5E) and rho-lacZ

(Figure 5F) enhancer traps were specifically expressed in the

optic-lobe-associated cortex glia. Rho is generally produced

by the same cells that express the ligand Spitz and its negative

regulator Aos. Consistently, spitz expression was detected in

miR-8+ cortex glial cells as visualized using two independent

enhancer traps (Figures 5G and S5A–S5C and compare with Fig-

ures 1A and 1D) spi-lacZ and spi-Gal4 (Jiang and Edgar, 2009).

No expression of the other EGFR ligands vein/Neuregulin and

keren was observed and downregulation of their expression by

Figure 4. Structure of the Surface Glia and Their Effects on the Neuroepithelium

(A) Histogram represents the means ± SEM of the glial nuclear area of wild-type perineurial (red bar), Moody+ subperineurial (green), miR-8+ surface cortex glia

(dark blue), and inner glia (gray); the glial nuclear area of non-miR-8 surface (green), miR-8 surface cortex glia (dark blue) and inner (gray) glia upon mir-8

overexpression, and the glial nuclear area of Moody-subperineurial glia (green) and inner (gray) glia upon dup/cdt1 depletion by RNAi (dup-IR) by repo-Gal4.

(B and C) Illustrative examples of 3D reconstructions of whole-mount WT (B) and repo>mir-8 (C) L3 brains.

(D–G) Changed size in glia cells of control WT (D-E) and repo>mir-8>CD8::GFP (F-G) L3 brain lobes. Glia nuclei were visualized with Repo (D, F, red) and

neuroepithelia stained with DE-Cad (D, F) and Dlg (E, blue). Arrowheads in (E) and (G) point to miR-8+ glia (green). Subperineurial (SPG) glia are visualized by

Moody (red, arrow in E and G).

(H) APCFzr/Cdh1-lacZ (green) labels a subset of glial cells that by position agrees with miR-8+ glia.

(I) miR-8+ glia (mir-8>H2B::RFP, green) are always negative for pH3 mitotic marker (red).

(J and K) Depletion of dup by RNAi (dup-IR) in repo cells. Arrowheads in (J) point to disrupted surface glia, which are all positive for Moody (red; arrow in K).

(L–O) The glial undergrowth defect of repo>dup-IR is rescued bymir-8 overexpression (repo>dup-IR>mir-8). Glial nuclei were visualized by Repo (red; L, M), the

NE by Patj (green, N, O) and DE-Cad (blue, L–O) and neuroblasts by Mira (red, N and O). CB, central brain; IPC, inner proliferation center; OPC, outer proliferation

center; VNC, ventral nerve cord. Scale bars represent 100 mm (B and C), 50 mm (D, F, H–J, L), and 20 mm (E, G, K, M–O).

See also Figure S4 and Table S1.

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Developmental Cell 27, 174–187, October 28, 2013 ª2013 Elsevier Inc. 179

Figure 5. Optic-Lobe-Associated Cortex

Glia Is a Source of the EGFR Ligand Spitz

(A–D) Whole brain (A and C) and a magnification of

an area (B and D) from a L3 WT (A and B) and

homozygous aosD7 animals (C and D). The NE is

visualized by DE-Cad (blue) and neuroblasts by

Mira (red).

(E–G) Enhancer-traps showing the transcription of

aos-lacZ (E), rho-lacZ (F) (green staining) occurs in

glia (Repo+, red). Coexpression of spi-lacZ (green)

and mir-8>H2B::RFP (G) in optic lobe-associated

cortex glia. Scale bars represent 50 mm (A and C)

and 20 mm (B, D, E–G).

See also Figure S5.

Developmental Cell


RNA interference had no effect on the neuroepithelial phenotype

(data not shown).

We therefore investigated the defects in the neuroepithelium

following specific interference with spi and aos genes in the

miR-8+ glia. We first used the biologically active processed

form of Spitz tagged with GFP (UAS-sSpi::GFP) (Miura et al.,

2006) to activate the EGF receptor, while allowing us to track

localization of the Spi protein. This construct was expressed us-

ing mir-8-Gal4 (Figure 6). The observed effect was comparable

with that seen using repo-Gal4, which drives expression in all

glia (Figures S6A and S6B).

180 Developmental Cell 27, 174–187, October 28, 2013 ª2013 Elsevier Inc.

The presence of overexpressed

sSpi::GFP in miR-8 surface cortex glial

cells was evident in puncta overlying the

cells of the neuroepithelium and resulted

in a dramatic expansion of the neuroepi-

thelium coupled with precocious and

ectopic neuroblasts (Figures 6A and 6B),

which resembled the defects seen inmir-

8deficient flies.Due to the lethality caused

by early expression of sSpi::GFP, we used

a temperature-sensitive Gal80 construct

(tub-Gal80ts: McGuire et al., 2004) and

grew larvae at the permissive temperature

of 18�C until early L3 stage. The tempera-

ture was then increased to 31�C to inacti-

vate Gal80 and release Gal4 repression

for 24 hr in order to characterize the ef-

fects of sSpi::GFP overexpression on the

underlying neuroepithelium.

Endogenous depletion of spitz by RNA

interference (UAS-spi-IR) (Dietzl et al.,

2007) using mir-8-Gal4 (mir-8>UAS-

spi-IR) resulted in a dramatic reduction

in neuroepithelial size (Figure 6C).

These defects were less severe when

we used repo-Gal4 (Figure S6C). More-

over, expression of the spi-IR transgene

by moody-Gal4 had no effect on the

morphogenesis of the underlying neuroe-

pithelium (Figures S6G–S6J). Therefore,

the miR-8+ optic-lobe-associated cortex

glia represents an endogenous source

of Spitz that is required to control neuroe-

pithelial proliferation and neuroblast formation. In agreement

with the secreted nature of Spi protein, small clones of glial cells

null for spi (spi1) recovered using theMARCM technique (Lee and

Luo, 1999) revealed noneffect in the underlying larval neuroepi-

thelium and in the final optic lobe size, likely due to nonautono-

mous rescue by the adjacent spi+ glial cells (Figures S6K–S6M).

Spitz Is a Direct Target of miR-8Gain and loss of the EGF receptor ligand Spitz in miR-8 positive

glial cells, through loss of aos or Spitz depletion by RNAi

increased or reduced the expansion of the neuroepithelium,

Figure 6. Surface Cortex Glia-Derived Spitz

Promotes Neuroepithelial Expansion and

Neuroblast Generation

(A and B) L3 brains shown are of larvae raised at

18�C (A), or shifted to 31�C during the early to late

L3 larval stage (B) to drive expression of sSpi::GFP

by mir-8-Gal4.

(C) Inhibition of endogenous spi in miR-8 glia by

UAS-spi-IR. NE, neuroepithelial cells; Nbs, neu-

roblasts. Scale bar represents 50 mm.

See also Figure S6.

Developmental Cell


respectively, and disrupted the orderly emergence of neuroblast

transition. Because these effects were also seen in loss and gain

of mir-8 animals, we investigated whether Spitz may be directly

downregulated by miR-8. The 30 UTR of spitz mRNA contains

only one potential binding site for miR-8 that is highly conserved

across Drosophila species (Figure 7A). We validated (Figures

7B–7D) this binding site using tubulin-luciferase reporters that

contained either the full-length WT 30 UTR of spitz or a mutated

version that abolished microRNA binding. The luciferase activity

of these constructs was measured in Drosophila Schneider cells

(S2) that were cotransfected with a tub-mir-8 plasmid to express

the microRNA (Figure 7B). Luciferase activity from the reporter

containing the WT spitz-30UTR was reduced by 77% when mir-

8 was expressed. This effect was reversed when the conserved

miR-8 binding site wasmutated (Figure 7B). We also generated a

tub-eGFP sensor containing the WT or the mutated 30 UTR of

Spitz and overexpressed the mir-8 transgenes in vivo in cells

of the posterior wing compartment using engrailed (en)-Gal4

(en>UAS-mir-8>DsRed). We observed a specific decrease in

the expression of a tub-eGFP-sensor containing the WT 30

UTR of spi, whereas we found no change in the expression of

a mutated version of the sensor containing the same three point

mutations in the miR-8 binding motif that prevented downregu-

lation in S2 cells (Figures 7C and 7D).

Endogenous levels of spimRNAs were elevated 1.5-fold in the

larval brain of flies lacking mir-8 (Figure 7E), as determined by

quantitative RT-PCR (qRT-PCR) analysis, consistently with spi

being a biologically relevant target of the microRNA. Expression

of spitz transgene without its 30 UTR by repo-Gal4, and therefore

incapable of being downregulated by miR-8, fully reverted the

defects caused by mir-8 overexpression (Figures 7F and 7G).

Moreover, the growth defect in brain overexpressing mir-8 was

enhanced by further reducing the expression levels of its target

Spitz (repo>mir-8>spi-IR, Figures 7H and 7I). In addition, some

of these brains (n = 13 out of 53 brains) exhibited areas of cell

death, which lacked the typical features of apoptosis and auto-

phagy, and hence seemed to be caused by necrotic cell death.

The occurrence of these cell deaths suggest that an acute depri-

vation of spi (gain of mir-8 and downregulation by spi-IR) may

cause brain trauma or some forms of neurodegeneration. Further

comprehension of this process could have important implica-

tions in Spi-regulated processes.

Intrinsic Activation of EGFR Mimics mir-8 DefectsThese results suggest that there is communication between

miR-8 glia and the neuroepithelial progenitors likely through

Developm

the EGF pathway that sustains the growth of the neuroepithelium

and the location and extent of neuroblast emergence. Directly

altering EGFR activity in the neuroepithelium mimicked the

defects observed in mir-8 and glial-specific spi mutants (Fig-

ure 6). We used a Gal4 line that drives expression exclusively

in neuroepithelial cells (c855a-Gal4) (Egger et al., 2007) of a

ligand-independent constitutively active form of the EGFR

(c855a>UAS-EGFRAct) (Domınguez et al., 1998), or of an acti-

vated form of Ras (c855a>UAS-RasV12). As in mir-8 flies (Fig-

ure 3B), or upon glial-specific increase in Spitz expression

(through loss of aos or Spitz overexpression; Figures 5C, 5D,

and 6B), the neuroepithelium expressing the activated form of

EGFR (Figure 8B) or RasV12 (data not shown) was increased in

size and ectopic neuroblasts were found throughout the neuro-

epithelium (Figures S7A–S7C). These phenotypes are reminis-

cent to those of Spitz overexpression in glial cells and of the brain

mutant for aos or mir-8.

We confirmed that both the overgrowth and ectopic neuroepi-

thelial-neuroblast transitions were cell-autonomous responses

to the activation of the EGFR signaling cascade. We used

MARCM to generate and mark clones of cells that express

constitutively active RasV12. MARCM clones of RasV12 cells pro-

duced cell-autonomous outgrowths and ectopic neuroblasts

(Figure 8D), supporting the view that intrinsic activation of the

EGFR receptor pathway is directly involved in autonomously

stimulating growth and generating neuroblasts. Inactivation of

the EGFR pathway by the expression of dominant negative

forms of the receptor inhibited neuroepithelial growth and fewer

neuroblasts formed (Figure 8E), demonstrating that the endoge-

nous EGFRpathway is required for neuroepithelial expansion. As

shown in Figure S7G, inactivation of EGFR by the expression of

dominant negative led to a 52% smaller size of the optic lobe

(1,714.695 ± 156.499 mm2 in c855a>EGFRDN: 5 hr after puparium

formation, APF5, n = 8 brains scored) as compared withWT con-

trol (3,523.1 ± 305.18 mm2: APF5, n = 7 brains scored). These

phenotypes of overactivation of EGFR/Ras pathway resembled

disc overgrowth and formation of ectopic morphogenetic furrow

in eye imaginal discs (Domınguez et al., 1998).

The effects of inappropriate activation of the EGFR pathway

within the neuroepithelium differed from previous studies that

suggested a role of EGFR only in the step of neuroblast transition

(Yasugi et al., 2010). It has been proposed that Notch signaling

negatively controls neuroblast transition by antagonizing EGFR

signaling and hence the effects of EGFR and Notch gain and

loss of function are opposed (Egger et al., 2010; Ngo et al.,

2010; Reddy et al., 2010; Yasugi et al., 2010). However, we


BA

control mir-8 Δ2 mir-8>sSpi;tub-Gal80 ts

UTR C CACGC A U3 GACA GCC AGUAUUA CUGU UGG UCAUAAU

miR-8 3 AGAAA ACUG

5

305

spitz 3´UTR (394bp)

AAAAAA

0

0,2

0,4

0,6

0,8

1

1,2

tub-emptytub-mir-8

E

C D

en-Gal4>DsRed>mir-8

L3

tub-eGFP::spi-3’UTR wt tub-eGFP::spi-3’UTR mut

0

0,5

1

1,5

2

Rel

ativ

e lu

cife

rase

act

ivity

spi-UTR wt spi-UTR mut

F G IH

repo-Gal4>sSpi::GFP>mir-8; tub-Gal80 repo-Gal4>spi-IR>mir-8controlts

Mira DE-Cad Repo Patj DE-Cad DAPI

Rel

ativ

e sp

itz m

RN

A

Figure 7. Spitz Is a Direct Target of miR-8

(A) Computer prediction of miR-8-binding site in the spi 30 UTR (seed sequence in red).

(B) Luciferase assay in S2 cells cotransfected with tub-mir-8 or the tub-empty vector, together with a firefly luciferase vector containing the spi 30 UTR(tub-luc::spi-30UTRwt) or a luciferase vector with mutations (tub-luc::spi-30UTRmut) in the seed sequence (underlined bases in A).

(C and D) Wing discs showing overexpression of UAS-mir-8 and UAS-DsRed by en-Gal4 (red) and of GFP in a tub-eGFP::spi-30UTRwt (C) and mutated

tub-eGFP::spi-30UTRmut (D) sensors.

(E) Histogram shows the relative mRNA levels of spi determined by qRT-PCR in three independent experiments (mean + SEM) in the brains of controls, mir-8D2

mutants and flies expressing mir-8>sSpi::GFP and tub-Gal80ts.

(F and G) Rescue of the effects ofmir-8 overexpression by a Spi transgene lacking its 30 UTR. Flies reared at 18�C (F) or shifted to 31�C during the early to late L3

larval stage (G).

(H and I) NE and neuroblast transitions in controls (H) and in larvae expressing repo>mir-8>spi-IR (I). Glial cells (Repo+, green) and NE cells [(DE-Cad+, blue) or

(Patj+, yellow)]. DAPI counterstaining highlights all nuclei. Scale bar represents 20 mm (F–I).

Developmental Cell


observed that the phenotypes were not identical. As shown in

Figure 8F, in Notch loss-of-function mutations, the wave of L(1)

sc expression that marks the neurepithelial-neuroblast transition

zone moved faster whereas activation of the EGFR pathway


(c855a>UAS-EGFRAct or c855a>UAS-RasV12) gave rise to

ectopic neuroepithelial-neuroblast transition zones across the

length of the neuroepithelium (Figure 8B) and enhanced neuroe-

pithelial growth, a phenotype not observed in Notch mutants

ier Inc.

Figure 8. Intrinsic Alteration of EGFR Signaling Mimics the Defects

Induced by Altered miR-8 Expression

(A and B) Control (A) and c855a-Gal4>EGFRAct (B) neuroepithelia stained with

Dlg (blue)andwith theneuroblast transitionmarkersL(1)sc (red)andMira (green).

Developmental Cell


Developm

(Figures S7A–S7D). This inappropriate activation of the EGFR

pathway ultimately exerted profound effects on optic lobe

patterning and size (Figures S7E–S7H).

Collectively, these findings indicate that the expression of the

conserved microRNA miR-8 in a population of optic-lobe-asso-

ciated cortex glia is required for the development of the underly-

ing neuroepithelium. Moreover, our results demonstrate that

expression of Spitz in the miR-8 glia is necessary and sufficient

to sustain neuroepithelial expansion as well as the generation

of neuroblasts. We demonstrate that Spitz is a direct, biologically

relevant target of miR-8, molecularly linking proliferation and the

progression of neuroblast transition. Based on these findings,

we propose that optic-lobe-associated miR-8-Spitz cortex glia

represent an anatomical niche that provides the signals and sup-

port required for neuroepithelial cell expansion and neuroblast

formation and that the production of signals from this niche

must be tightly regulated. It is unclear whether Spi exerts these

influences directly, by activating EGFR in the neuroepithelial

cells, or via secondary signals (Figure 8G).

DISCUSSION

Our analysis reveals that the production by an optic-lobe-asso-

ciated cortex glia of the EGFR ligand Spitz is critical for coordina-

tion of neuroepithelial proliferation and the spatiotemporal

emergence of neuroblasts. External signaling from the surround-

ingmicroenvironment is a commonmechanism for the regulation

of stem cell number and behavior in mature tissues, but the

need of a niche microenvironment during early neurogenesis

was unknown.

Signaling through Surface-Associated miR-8-Spitz-Positive Cortex GliaFlies deficient for the microRNAmir-8 exhibit brain degeneration

and behavioral defects. Strikingly, the microRNA is expressed in

cortex glial cells lying underneath the blood-brain-barrier (sub-

perineurial) glial layer (Figures 1 and 2). These glia are of large

size and produce long protrusions that ensheath the developing

optic lobe neuroepithelium and can be distinguished by their

(C and D) c855a-Gal4 MARCM GFP (green) control clones (C) and those

overexpressing UAS-RasV12 (D). Neuroblasts are visualized by Mira (red) and

NE by DE-Cad (blue).

(E) Intrinsic inactivation of EGFR signaling via EGFRDN.

(F) Brains in which Notch receptor is inactivated via RNAi by c855a-Gal4 and

stained with Dlg (blue), L(1)sc (red), and Mira (green).

(G) Model of miR-8-Spitz regulatory interactions and glial-derived Spitz

in coordinating neuroepithelial expansion and neuroblast transition. miR-8

increases, in an autocrine manner, cortex glial cellular size that facilitates the

formation of long cellular processes that ensheath the underlying neuro-

epithelium. miR-8 also negatively regulates, in a paracrine manner, neuro-

epithelial growth, and neuroblast transition via direct inhibition of Spitz protein

translation. Blocking endoreplication and/or loss of miR-8 in cortex glia

releases miR-8 inhibition of Spitz levels, thereby resulting in inappropriate or

enhanced EGFR signaling. Feedback signaling via EGFR may also stimulate

the expression of rho and aos in miR-8+ positive cortex glia, thereby contrib-

uting to the fine-tuning of Spitz protein activation and secretion. Glial-to-

neuroepithelial cells signaling via secondary signal (X) is not ruled out. Scale

bar represents 20 mm.

See also Figure S7.


Developmental Cell


selective expression of the EGFR ligand spitz as well as its mod-

ulators aos and rho.

Genetic manipulation ofmir-8, spi, or aos, in this glial cell pop-

ulation unveiled cell nonautonomous influences of these glia on

the development of the underlying neuroepithelium. Similar glial

signaling to neuroblasts in the larval Drosophila brain has been

demonstrated for a class of neuroblasts that remain quiescent

until nutrient-responsive satellite and cortical glia reactivate their

proliferation (Chell and Brand, 2010; Sousa-Nunes et al., 2011).

Our study identifies another population of glia cells that sustain

growth of neuroepithelial cells and the neuroepithelial-neuro-

blast transition via a miR-8-Spitz axis.

Glial-mediated regulation of the neuroepithelium is reminis-

cent to the roles of mammalian astrocytes that are known

to potently stimulate neurogenesis in cell culture (Lim and

Alvarez-Buylla, 1999; Song et al., 2002) and a component of

endogenous neural stem cell niche in adult mammalian neuro-

genesis (Nern and Momma, 2006). Additionally, EGFR is also

implicated in glial cell proliferation in Drosophila (Read et al.,

2009; Reddy and Irvine, 2013) and human glioma often exhibits

elevated EGFR signaling.

Surface-Associated miR-8-Spitz-Positive GlialArchitecture and SignalingThe notion that miR-8-Spitz-positive cortex glia constitute an

anatomically and functionally distinct population of surface-

associated glia cells is strongly supported by the finding that

RNAi knockdown of spi in subperineurial glia using moody-

Gal4 has no effect on neuroepithelial development. Spitz protein

is converted to its active form by the Rhomboid protease, which

is also expressed by miR-8 glia. The extracellular factor Aos

limits Spitz spreading and signaling level that may influence

the effects of Spitz-EGFR in the responding neuroepithelium

(e.g., sustaining proliferation and preventing premature or

ectopic neuroblast formation). The posttranscriptional silencing

of spi mRNA by the miR-8 binding to a sequence in its 30 UTRprovides another layer of regulation to fine-tune the timing, local-

ization and/or amount of Spitz protein translation. Moreover,

the distinctive architecture of miR-8-Spitz-positive cortex glia

appears to be regulated by endoreplication regulators dup/

Cdt1 and themicroRNAmiR-8. Importantly, expression of a spitz

transgene lacking its 30 UTR (and hence unable of regulating by

miR-8) fully rescued the undergrowth defect caused by mir-8

overexpression in the glia cells. Therefore, we suggest that cor-

tex glia employ a coordinated strategy that is mediated bymiR-8

to ensure that: (1) the glia establish a correct architecture to pro-

vide a continuous layer of cortex glia cells that extend long pro-

cesses to the neuroepithelium; and (2) correct local (or temporal)

control of Spitz protein synthesis. Given the expression of rho

and aos is directly induced by EGFR signaling in other context,

a feed-back signaling via EGFR may also occur in miR-8-Spitz

positive glial cells, thereby contributing to the fine-tuning of Spitz

protein activation and secretion.

Do Optic-Lobe-Associated miR-8-Spitz Cortex GliaRepresent an Anatomical Niche?A niche typically refers to a confined anatomical location where

adult stem cells reside and provides the signals required to

sustain stem cell function and number. Niches are usually


composed of supporting cells that make physical contact with

the stem cells and act locally (Morrison and Spradling, 2008).

The optic-lobe-associated miR-8-Spitz-positive cortex glia

appear to represent a niche that contributes signals for the

growth andmorphogenesis of the neuroepithelium and that con-

stitutes a functionally distinct population of that of the blood-

brain-barrier glial cells.

Extrinsic signaling in the coordination of neuroepithelial prolif-

eration in the developing mammalian forebrain of Foxc1 mutant

mice has also been suggested. In these mice, the meninges are

reduced or absent, resulting in an expansion of the neuroepithe-

lium due to the predominance of symmetric divisions (Sie-

genthaler et al., 2009). The meninges are a source of the retinoic

acid required for the transition of neuroepithelial cells into radial

glia and neurons (Siegenthaler et al., 2009). Furthermore, menin-

geal cells secrete and organize the pial basement membrane, a

thin sheet of extracellular matrix that covers the brain and that is

enriched in a variety of growth factors. Rupture of the basement

membrane in the developing brain causes type II lissencephaly,

generating ectopic precursor clusters and cortical heterotopias

due to impaired attachment of the radial glia to the basement

membrane, resulting in a general laminar disorganization

(Haubst et al., 2006). These defects are reminiscent to those of

disrupted surface glia cells described here.

In summary our findings suggest that neuroepithelial prolifera-

tion and the onset of neuroblasts in the developing optic lobe

neuroepithelium are largely influenced by extrinsic cues via a

miR-8-dependent mechanism in the overlying glia. The reprog-

ramming of neuroepithelial cells into neural stem cells (neuro-

blasts) is associated with dramatic morphological and molecular

changes, including the loss of epithelial determinants DE-

Cadherin, Crumbs and PatJ (Egger et al., 2007; Ngo et al.,

2010; Wang et al., 2011a), and enhanced expression of the

Snail-family zinc-finger transcriptional repressor Worniu (Ashraf

et al., 2004). These changes are strikingly reminiscent of the

events that drive the epithelial-to-mesenchymal transition

(EMT), which confers a stem-like character inmammalian epithe-

lial cells and in cancer cells (Mani et al., 2008) andwhich are regu-

lated by the miR-200 family. Indeed, downregulation of human

mir-200 genes in epithelial normal and cancer cells promotes

EMTand the acquisition of ‘‘stemness’’ (Park et al., 2008;Wellner

et al., 2009) effects that are presumed to be cell-autonomous.

Our findings demonstrate that miR-8 can exert its effects non-

cell-autonomously, opening the possibility that microRNA of

the miR-200 family may play similar roles in stem cell fate niches

and/or microenvironmental regulation of metastasis.

EXPERIMENTAL PROCEDURES

Drosophila Husbandry

The lines used in this study are described in FlyBase: c855a-Gal4, en-Gal4,

mir-8NP5427-Gal4, moodyXA12-Gal4, repo-Gal4, spiNP0261-Gal4, aprk568-lacZ,

aosD7, aosw11-lacZ, APCFzr/Cdh1-lacZ, mir-8D2, rhoX81-lacZ, Nrx-IV-GFP

(Buszczak et al., 2007), spis3547-lacZ, tub-Gal80ts, UAS-mCD8::GFP, UAS-

DsRed, UAS-H2B::RFP, UAS-lacZ, UAS-aos, UAS-Dcr2, UAS-dup (gift from

B. Calvi), UAS-EGFRAct, UAS-EGFRDN1-7, UAS-keren-IR (VDRC KK104299),

UAS-mir-200c, UAS-mir-8, UAS-mir-8-SP::GFP, UAS-Notch-IR (VDRC

KK100002), UAS-spi-IR (VDRC GD3922), UAS-RasV12, and UAS-sSpi::GFP.

RNAi lines in Table S1 are described in the Supplemental Experimental

Procedures.

ier Inc.

Developmental Cell


FLP-Out Clonal Analysis with mir8-Gal4

Larvae of the genotype yw hsp70-FLP122; UAS>CD2,y+ stop>mCD8::GFP/

mir-8-Gal4 were heat shocked for 20 min at 37�C at 72 hr after egg-laying

(AEL) to fuse the UAS sequences to mCD8::GFP coding sequences by

removing the CD2, y+ stop cassette flanked by the FRT (denoted as > )

sequences, specifically in a subset of miR-8+ cells. FLP-out clones marked

by GFP were analyzed at 24 hr postinduction (96 hr AEL).

MARCM Clones

MARCM clones were generated in glial cells using repo-FLP by crossing

female virgins tub-Gal80 FRT40A; repo-Flp1C, repo-Gal4 UAS-mCD8::GFP/

TM6 (gift of C. Klambt) to males FRT40A spi1/CyO and brains were dissected

at L3 stage (6 clones in optic lobe glia in n = 69 larval brains analyzed and 13

clones in the optic lobe in n = 85 adult brains).

MARCM RasV12-expressing clones were induced by 20 min heat-shock at

37�C at 72 hr AEL and brains were dissected 24 hr postinduction (96 hr AEL)

in animals of the genotype: yw hsp70-FLP122; UAS-mCD8::GFP; FRT42D/

FRT42D tubP-Gal80; c855a-Gal4/UAS-RasV12. Control MARCM clones were

of the genotype: yw hsp70-FLP122 UAS-mCD8::GFP; FRT42D/FRT42D

tubP-Gal80; c855a-Gal4/TM2.

Antibodies and In Situ Hybridization

Brains were dissected out in cold PBS and fixed in 4%PFA for 20min (Morante

and Desplan, 2011). The following primary antibodies were used: mouse anti-

Dlg (1/100, DSHB), mouse anti-Elav (1/50, DSHB), mouse anti-Mira (1/50)

(Ohshiro et al., 2000), mouse anti-Repo (1/50, DSHB), rabbit anti-b-Gal (1/

20,000, Cappel), rabbit anti-GFP (1/1,000, Molecular Probes), rabbit anti-

Mira (1/2,000) (Ikeshima-Kataoka et al., 1997), rabbit anti-Patj (1/2,000) (Bhat

et al., 1999), rabbit anti-pH3 (1/2,000, Upstate), rat anti-DE-Cad (1/50,

DSHB), rat anti-DN-Cad (1/25, DSHB), rat anti-L(1)sc (1/800, gift from A. Car-

mena), and rat anti-Moody-b (1/200, gift from U. Gaul). Secondary antibodies

were purchased from Invitrogen and Jackson ImmunoResearch. DAPI (Invitro-

gen) counterstaining was done using a solution (0.3 mg/ml). Brains were

mounted in Vectashield (Vector Labs), maintaining their 3D configuration

and images were obtained using a Leica TCS SP2 Confocal microscope.

Locked nucleic acid (LNA)-probe in situ hybridization was performed as in

Vallejo et al. (2011) using amiR-8 probe (Exiqon) end-labeled with a DIG oligo-

nucleotide 30-end labeling kit (Roche) and hybridized at 42�C.

Glial and Neuroepithelial Area Measurements

Nuclear size of surface glia and neuroepithelial areas were measured as

described in the Supplemental Experimental Procedures.

miRNA-mRNA 30 UTR Alignment and Transgenic Constructs

The binding site for miR-8 in the spi 30 UTR was identified using the BiBiServ

server (Rehmsmeier et al., 2004). TheUAS-mir-8 construct was described pre-

viously (Vallejo et al., 2011). The tub-eGFP::spi-UTR and tub-eGFP::spi-UTR-

mut constructs were generated by cloning the full-length 30 UTR of the

Drosophila spi gene, or a mutated sequence, into the 30 end of the tub-eGFP

reporter vector (a gift from S.M. Cohen).

Luciferase Reporter Assays

For Drosophila S2 cell luciferase assays, cells were cotransfected in 24-

well plates with the tub-mir-8 plasmid (250 ng) (Karres et al., 2007), the

luciferase::spi-UTR or luciferase::spi-UTR-mut constructs (25 ng), and

the Renilla luciferase plasmid (25 ng) for normalization. Target sites in the

mutant construct were generated using the QuickChange Site-Directed

Mutagenesis kit (Stratagene). The relative luciferase activity was measured

60 hr posttransfection using the Dual-Glo Luciferase Assay system

(Promega).

qRT-PCR

Total RNAwas extracted from L3 brains using the RNAeasy-Mini Kit (QIAGEN),

and cDNA was synthesized using an oligo-dT primer and the SuperScript RT-

III Kit (Invitrogen). Quantitative real-time PCR was performed using the ABI

SYBR green system using gene-specific primers (Supplemental Experimental

Procedures) designed with Universal Probe Library Assay Design Center

(ROCHE; see https://qpcr1.probefinder.com). The dGAPDH and dUba2

Developm

primers were used to normalize the mRNA levels. The assays were performed

in an ABI7500 apparatus and analyzed using the Applied Biosystem software.

All qPCRs were performed in triplicate and the relative expression was calcu-

lated using the comparative CT method.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures,

seven figures, and one table and can be found with this article online at

http://dx.doi.org/10.1016/j.devcel.2013.09.018.

ACKNOWLEDGMENTS

We are very grateful to M. Asano, N. Baker, H. Bellen, E. Bier, R. Cagan, B.

Calvi, A. Carmena, S. Cohen, B. Edgar, M. Freeman, U. Gaul, C. Klambt, F.

Matsuzaki, R. Mann, R. Lehmann, L. Luo, P. Simpson, B. Shilo, J. Treisman,

D. van Vactor, M. Vidal, the Bloomington Stock Center, the NIG-Fly Stock Cen-

ter, the Drosophila Genetic Resource Center at Kyoto Institute of Technology,

the VDRC, and the Developmental Studies Hybridoma Bank for reagents. We

thank the TRiP at Harvard Medical School (NIH/NIGMS R01-GM084947) for

providing transgenic RNAi fly stocks used in this study. We also thank Andrea

Brand, Boris Egger, Marion Silies, Tetsuya Tabata, and members of the Des-

plan and Dominguez laboratories for in-depth discussions and suggestions

and Giovanna Exposito for helping with Imaris software. This work was sup-

ported by a Ramon y Cajal grant (RyC-2010-07155) and a Ministerio de Econ-

omia y Competitividad grant (SAF2012-31467) to J.M., a National Institutes of

Health grant (R01 EY017916) to C.D., and Spanish National grants (BFU2009-

09074 and SAF2012-35181), a Generalitat Valenciana grant (PROMETEO II/

2013/001), and Fundacion Botin to M.D.

Received: February 4, 2013

Revised: July 22, 2013

Accepted: September 19, 2013

Published: October 17, 2013

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