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Developmental Biology

Thalamic development induced by Shh in the chick embryo

Claudia Vieiraa, Ana-Lila Gardab, Kenji Shimamurac, Salvador Martineza,*

aNeuroscience Institute, Miguel Hernandez University N-332, Km 87, E-03550 Alicante, SpainbCentro IVI-Murcia, Navegante Macias 5, E-30007 Murcia Spain

cDivision of Morphogenesis, Department of Embryogenesis, Institute of Molecular Embryology and Genetics, Kumamoto University,

Honjo 2-2-1, Kumamoto 860-0811, Japan

Received for publication 18 February 2005, revised 23 May 2005, accepted 24 May 2005

Available online 18 July 2005

Abstract

Patterning of the early neural tube is achieved in part by the inductive signals, which arise from neuroepithelial signaling centers. The

zona limitans intrathalamica (ZLI) is a neuroepithelial domain in the alar plate of the diencephalon which separates the prethalamus from the

thalamus. The ZLI has recently been considered to be a possible secondary organizer, effecting its inductions via sonic hedgehog (Shh), a

signaling molecule which drives morphogenetic information for the thalamus. Using experimental embryological techniques involving the

generation of chimeric embryos, we show that the formation of the ZLI in the diencephalic alar plate is due to an interaction between the

prechordal and epichordal plate neuroepithelia. We also provide evidence that Shh expression in the ZLI underlies the morphogenetic activity

of this putative diencephalic organizer. Ectopic Shh led to the auto-induction of its own gene expression in host cells, as well as to the

expression of other genes involved in diencephalic regionalization and histogenesis. Analysis of long-term surviving embryos after Shh

ectopic expression demonstrated that Shh was able to induce thalamic structures and local overgrowth. Overall, these results indicate that Shh

expressed in the ZLI plays an important role in diencephalic growth and in the development of the thalamus.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Chick; Neural tube; Diencephalon; Zona limitans intrathalamica; Sonic hedgehog; Thalamus; Secondary organizer

Introduction

The diencephalon is a cerebral region which develops

from the anterior part of the neural tube, the prosencepha-

lon. This area becomes subdivided into the telencephalon

and the hypothalamus at the anterior pole, and the

diencephalon at the posterior zone. The diencephalon is

limited by two boundaries, the most anterior one that lies

between the pedunculomammillary region and the eminentia

thalami and the posterior one that lies between the pretectum

and the mesencephalon (Martinez and Puelles, 2000; Puelles

and Rubenstein, 2003; Garcia-Lopez et al., 2004). The

diencephalon develops into two major neuroepithelial

domains known as the prethalamus and the thalamus

(Puelles and Rubenstein, 2003).

0012-1606/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.ydbio.2005.05.031

* Corresponding author. Fax: +34 965 919 555.

E-mail address: smartinez@umh.es (S. Martinez).

The zona limitans intrathalamica (ZLI) is a central

boundary in the diencephalon which appears as a transverse

ventricular ridge between the prethalamus and the thalamus at

the neural tube stages (Figs. 1A–F; Martinez and Puelles,

2000; Echevarria et al., 2003; Puelles and Rubenstein, 2003;

Garcia-Lopez et al., 2004). The ZLI corresponds to an inter-

segmental boundary in the neuroepithelium, in which neuro-

epithelial cells show clonal restriction and a low proliferation

rate (Fig. 1F; Puelles et al., 1987; Figdor and Stern, 1993;

Martinez and Puelles, 2000; Martinez et al., 1992; Larsen et

al., 2001; Echevarria et al., 2003). The complex pattern of

regulatory molecules expressed by ZLI cells in the neural

tube during development is indicative of its importance as a

possible secondary morphogenetic organizer in diencephalic

histogenesis (Fig. 1G; Echevarria et al., 2003).

The dynamic expression of Shh in the ZLI (Figs. 1B, C)

may be a consequence of the interaction between the

prechordal and epichordal plate, which first specifies the

284 (2005) 351 – 363

Fig. 1. Descriptive gene expression analysis. (A–C) ZLI specification. (A) Lateral view of a chick embryo at HH17 stained with acetylcholinesterase (AChE).

(B, C) Lateral views of chick embryos at HH17 (B) and HH19 (C) illustrating Shh gene expression. (D, E) Lateral view of chick embryos at stage HH23

showing the expression of some diencephalic genes. (D) Gbx2 in blue and Shh in red and (E) Dlx2 in blue and Shh in red. Gbx2 is a marker for the thalamus,

while Dlx2 is a marker for the prethalamus. (F) Lateral view of a chick embryo at HH23 stained with Fast blue. Fast blue injections show the low proliferation

rate of the ZLI and the basal plate cells. (G) Schematic representation of a chick embryo at stage HH23 showing some genes expressed in the forebrain and

midbrain. (H) Schematic representation showing the diencephalic nuclei. The different colors specify distinct nuclei involved in the same functional system:

orange, visual primary nuclei; green, sensorial nuclei; blue, prethalamic nuclei with a thalamo-telencephalic relay function; gray, basal plate nuclei.

Abbreviations: Ala, ansa lenticularis nucleus; BRN, basal optic root nucleus; D, diencephalon; DL, dorsolateral thalamic complex; epichor, epichordal; ET,

epithalamus; ETh, eminentia thalami; GV, geniculatus ventrolateralis nucleus; Hy, hypothalamus; IR, interstitial rostral nucleus; ITO, nucleus interstitialis of

the optic tract; LA, lateralis anterior nucleus; M, mesencephalon; os, optic stalk; ov, optic vesicle; pRA, perirotundic area; prechor, prechordal; PT, pretectum;

PTh, prethalamus; R, rotundus nucleus; Ret, reticular thalamic nucleus; RMT, retromammillar tegmentum; sR, subrotundus nucleus; T, telencephalon; TH,

thalamus; VLT, ventrolateralis nucleus; VTA, ventral tegmental area; ZLI, zona limitans intrathalamica.

C. Vieira et al. / Developmental Biology 284 (2005) 351–363352

cellular territory of the ZLI and then induces the molecular

events necessary for Shh activation in these cells (Garcia-

Lopez et al., 2003). Shh can act as a signaling molecule of

the putative ZLI organizer, regulating the expression of

developmental genes (Fig. 1G) which will specify compart-

mentalization and cell fate in the diencephalon (Fig. 1H;

Echevarria et al., 2003; Hashimoto-Torii et al., 2003;

Kiecker and Lumsden, 2004).

It has been suggested that the positioning and the

specification of the ZLI is a consequence of the interaction

between the prechordal and the epichordal epithelium

(Larsen et al., 2001; Braun et al., 2003; Echevarria et al.,

2003; Kiecker and Lumsden, 2004), but experimental

evidence in favor of this hypothesis is currently lacking.

The expression of Six3 in the prechordal epithelium and

Irx3 in the epichordal epithelium previous to Shh activation

in the diencephalic alar plate may constitute the essential

molecular events, which underlie this interaction (Kobaya-

shi et al., 2002; Echevarria et al., 2003).

Several transcription factors are expressed in the dien-

cephalon showing nested patterns to the ZLI cells (Fig. 1G).

Gbx2 and Sox14 are expressed in the thalamic mantle layer

C. Vieira et al. / Developmental Biology 284 (2005) 351–363 353

(Bulfone et al., 1993; Martinez-de-la-Torre et al., 2002;

Hashimoto-Torii et al., 2003), while Dlx2 is expressed in the

prethalamic mantle layer (Bulfone et al., 1993, Larsen et al.,

2001; Gonzalez et al., 2002). Nkx2.2 is expressed in the

neural epithelium flanking the expression of Shh in the ZLI

(Price et al., 1992, Shimamura et al., 1995).

Shh expression is a marker for the ZLI. Its expression

starts in the ventral edge of the ZLI, and then extends to all

the surface of the ZLI along the diencephalic alar plate

(Figs. 1B, C). In addition to the regionalization properties of

the ZLI, it has been postulated that Shh signaling plays a

role in neuroepithelial proliferation and thalamic growth; the

reduced size of the diencephalon in Shh mutants corrobo-

rates this hypothesis (Chiang et al., 1996). Thus, Shh

expression confers to the ZLI morphogenetic properties

which, as in the spinal cord, regulate the expression of

distinct sets of transcription factors necessary for the

sequential specification of neural progenitors in local

domains of the basal plate (Ericson et al., 1997; Briscoe et

al., 2000; for review see Ingham and McMahon, 2001;

Marti and Bovolenta, 2002). Evidence for the morphoge-

netic activity of Shh in the mesencephalic basal plate has

been reported by Agarwala’s group (Agarwala et al., 2001).

Indeed, Shh has been shown to act as a morphogen in the

spinal cord by inducing various genes in a concentration-

dependent manner (Roelink et al., 1995; Ericson et al.,

1997). Shh has also been proposed to act as a mitogen in the

expansion of cerebellar granule precursors (Dahmane and

Ruiz-i-Altaba, 1999; Wallace, 1999; Wechsler-Reya and

Scott, 1999; Oliver et al., 2005).

In the present paper, we demonstrate that the ZLI

develops in the neuroepithelium where the prechordal and

epichordal neural plates contact. We also show that Shh

expression is activated in ZLI cells and controls the

activation of several developmental genes, including itself,

involved in diencephalic regionalization and thalamic cell

fate. Ectopic expression of physiological levels of Shh

transforms mesencephalic and epithalamic regions into

organized thalamic structures. Finally, we provide evidence

that Shh signaling increases the growth of the thalamus.

Materials and methods

Microsurgery

Fertilized chick (Gallus gallus) and quail (Coturnix

coturnix japonica) eggs were incubated at 37-C in a forced

air incubator and staged according to Hamburger and

Hamilton (1951).

Heterotopic and isochronic grafts were performed using

quail or BrdU-labeled chick embryos as the source of the

prechordal neural epithelium to be implanted in the

epichordal epithelium of the host chick. Embryos were

operated at stage HH9 for chick and the equivalent stage for

quail embryos (7 somites). After microsurgery, the eggs

were sealed and kept in the incubator until HH23 (4 days of

incubation) or HH36 (10 days of incubation).

Cell aggregates of QT6-control quail fibroblasts and QT6

SHH-expressing cells (kindly provided by D. Duprez) were

implanted into the right side of the neural tube, after careful

opening of the neuroepithelium where cell aggregates were

inserted, at different positions in HH10–11 chicken

embryos. Cell aggregate preparation was performed in

accordance with Duprez et al. (Duprez et al., 1998).

Embryos were then left to develop until HH23 or HH36

and then fixed overnight in 4% paraformaldehyde in

phosphate-buffered saline solution (PBS 0.1 M, pH 7.4).

In situ hybridization

After fixation, embryos were rinsed in PBT (PBS with

0.1% Tween 20), dehydrated through an ascending meth-

anol series and stored in 100% methanol at �20-C before

being processed for in situ hybridization (ISH) as whole-

mount (HH23) or vibratome sections (HH36), as described

by Nieto et al. (Nieto et al., 1996). HH36 embryos were

embedded in 4% agarose in PBS and sectioned transversally

at 100 Am using a vibratome (Leica).

Digoxigenin and fluorescein-labeled RNA probes were

prepared from plasmids kindly provided by: J.L.R. Ruben-

stein (Dlx2, Nkx2.2, Shh, Pax6, Six3), A. Simeone (Gbx2),

G.G. Gonsalez (Mab21), G.R. Martin (Fgf8), A. McMahon

(Wnt8b), and M. Bronner-Fraser (Wnt3a). RNA-labeled

probes were detected by alkaline-phosphatase-coupled anti-

digoxigenin and anti-fluorescein antibodies (Roche Diag-

nostics, Mannheim, Germany) and NBT/BCIP was used as

a chromogenic substrate to detect the digoxigenin-labeled

probes (Boehringer, Mannheim, Germany), while INT/BCIP

was used for the detection of the fluorescein-labeled probes

(Roche Diagnostics, Mannheim, Germany). After ISH,

embryos were washed in PBT, photographed under a

dissecting microscope (Leica), and stored at 4-C in PBT

with 0.1% sodium azide. Vibratome sections were mounted

on glass slides, counterstained with neutral red and

dehydrated to absolute alcohol, cleared in xylene, and

mounted in Eukitt (O. Kindler GmbH and CO, Freiburg).

Histological analysis

Quail-chick chimeras were immunostained with a mono-

clonal anti-quail antibody (QCPN, Developmental Hybrid-

oma Bank, Iowa City, IA) like in Cobos et al. (2001).

Whole-mount embryos at HH19 were stained with

acetylcholinesterase (Martinez-de-la-Torre et al., 1990), and

embryos at HH23 were processed for Fast blue staining

dilution (Martinez et al., 1992).

BrdU staining was performed according to McConnell

and Kaznowski (1991).

Cryostat sections of short-term survival embryos with

SHH-expressing cells were counterstained with bisbenzi-

mide (Hoechst 33258, Sigma). Some chicken embryos at

C. Vieira et al. / Developmental Biology 284 (2005) 351–363354

HH36 were fixed overnight in Clarke’s fixative, dehydrated,

embedded in paraffin, and sectioned sagittally at 12 Am.

These sections were stained with cresyl violet. Subse-

quently, all sections were dehydrated to absolute alcohol,

cleared in xylene and mounted in Eukitt (O. Kindler GmbH

and CO, Freiburg), for further analysis and photography

under a dissecting microscope (Leica).

Results

Specification of the ZLI in the prosencephalic

neuroepithelium

The mechanisms which underlie the positioning of the

ZLI in the prosencephalic neuroepithelium are currently

unknown. It has been hypothesized that the ZLI develops in

the contacting zone between the prechordal and epichordal

neuroepithelium (Fig. 1; Larsen et al., 2001; Braun et al.,

2003; Echevarria et al., 2003; Hashimoto-Torii et al., 2003).

To explore this hypothesis, we grafted quail or BrdU-labeled

chick prechordal prosencephalic epithelium, exactly the

anlage of the dorsal pallium, into epichordal domains,

posterior diencephalon and mesencephalon of isochronic

chick embryos (Fig. 2). The grafts were excised with special

care with the help of our previously developed prosen-

cephalic fate maps, so as not to include epithelium which

expresses Fgf8 or Shh (Cobos et al., 2001; Garcia-Lopez et

al., 2004). As in our fate map experiments, we inserted a

grid with concentric circles into one ocular of the micro-

scope used to perform the grafts, so as to ensure the

exclusion of neuroepithelium expressing Fgf8 or Shh, as

well as to normalize the size and exact location of the graft

between different experiments. Of the 233 prechordal

transplants into the posterior diencephalon (n = 190) or

mesencephalon (n = 43), 152 cases were found to be

adequately integrated (120 in the diencephalon and 32 in the

mesencephalon). These cases were processed to study the

induction or repression of genes in the interface graft/host,

and in the grafted and host epithelial domains.

First, we analyzed the induction of Shh expression in the

contacting region 2 days after grafting. We observed that of

the 90 experimental cases, Shh expression was activated in

52 (Figs. 2A–K). Ectopically activated Shh was localized in

the graft/host interface, around the grafted epithelium,

contacting in most cases with the normal expression domain

of Shh in the basal plate (Figs. 2A–J). Anti-quail or anti-

BrdU immunostaining to detect donor cells showed that Shh

was induced in both donor and host interface cells (Fig. 2D).

The epichordal epithelium competent to express Shh was

mapped from the thalamus to the anterior rhombencephalic

region (isthmus-rhombomer1). On no occasion was Shh

expression induced in caudal graft/host interfaces localized

between r2 and r5 (Fig. 2K; n = 10). Some grafts of

epichordal into prechordal epithelium (n = 20) showed very

low integration and important structural anomalies, such as

dorsal midline opening, strong malformation, and reduction

of telencephalic derivatives. These cases were considered

inappropriate for analysis and interpretation.

It has been demonstrated that the expression of Fgf8 in

the isthmic organizer (IsO) is controlled by the interaction

of Otx2 and Gbx2 (Hidalgo-Sanchez et al., 1999; Joyner et

al., 2000; Garda et al., 2001; Martinez-Barbera et al.,

2001). Our next objective was to explore if the two kinds

of interactions, prechordal/epichordal and Otx2/Gbx2, can

act in parallel to regulate differential gene expression in

the contacting neuroepithelium. To this end, we grafted

prechordal-Otx2+ epithelium (Crossley et al., 2001) into

the posterior pole of the mesencephalic vesicle (epichor-

dal), including the isthmic constriction, a domain where

Gbx2 is strongly expressed (Garda et al., 2001). In seven

of ten cases, the graft perfectly integrated into the caudal

mesencephalon and the isthmic region. Neuroepithelial

expression of Shh and Fgf8 was detected by whole-mount

ISH in the chimeric neural tubes 48 h after grafting (Figs.

2I, J). We observed a clear induction of both genes in the

cells at the caudal graft/host interface, where the two

regulative effects have been established: prechordal–

epichordal interaction activated Shh in an anterior band

of cells and Otx2/Gbx2 interaction activated Fgf8 in a

posterior band of cells (Figs. 2I, J). When the interface

between donor and host epithelia developed as a normal or

ectopic choroidal structure, Shh induction in this interface

was not observed (Figs. 2C, G, and I).

Ectopic Shh induces molecular reorganization in the donor

and host epithelial grafts

Chimeric embryos presenting ectopic induction of Shh

were analyzed to explore additional modifications in the

molecular patterns of the grafted and host epithelium. In ‘‘ex

vivo’’ experiments and in electroporation assays, Shh has

been shown to modulate genetic expression at both sides of

the ZLI (Braun et al., 2003; Hashimoto-Torii et al., 2003;

Kiecker and Lumsden, 2004). However, the possible

inductive activity of Shh as a signaling molecule of the

ZLI has not yet been explored either at physiological levels

or in experiments in vivo. Thus, the induced expression of

Shh at the graft/host interface is a useful experimental model

to analyze effects of ectopic Shh in the diencephalic and

mesencephalic neuroepithelium.

In two cases, the graft included caudal prechordal

neuroepithelium, thus carrying some Fgf8, which caused

the growth of the grafted epithelium. These grafts developed

as a vesicle localized in the thalamus. This vesicle showed

an integration zone encircled by the expression of Shh. The

rostral component of this circle corresponded to the ZLI,

while ventrally the interface contacted with the basal plate

expression of Shh, and caudally a new Shh positive stripe

invaded the alar plate from the basal plate (Figs. 2F, G). The

normal antero-posterior pattern of expression of Pax6, i.e.,

positive in the thalamus and pretectum and negative in the

Fig. 2. Grafting of prechordal prosencephalic epithelium into epichordal prosencephalon (E–G) or mesencephalon (A–D, H–O). All embryos were operated at

stage HH9 and fixed at stage HH23. The prechordal grafts are delineated by white or black dots. (A, E, H, L) Schematic representations of the grafting

procedure. (B, C) Lateral views of a control embryo (B) and an experimental embryo (C) showing Shh in blue and Pax6 in red. The prechordal grafted

epithelium in C (PG) induced the midbrain alar plate (M and M*) to express Shh (ZLI*). (D) Transverse section of the embryo represented in panel C where

BrdU incorporation delineates the prechordal graft and Shh expression indicates the ectopic ZLI. (F) Dorsal view of an experimental embryo showing

expression of Shh in blue and Pax6 in red. The grafted vesicle mimics the di-mesencephalic pattern of Pax6 expression. (G) Lateral view of the embryo

represented in (F). (I) Open ventricular view of an experimental embryo with Shh in red and Fgf8 in blue. (J) Magnification of the neuroepithelium where

ectopic induction of both Shh and Fgf8 was observed in the graft/host contacting area. (K) Dorsal view of the rhombencephalon in an experimental embryo

illustrating expression of Shh in blue and Pax6 in red. The prechordal grafts in all of these experimental embryos induced an ectopic ZLI which expressed Shh.

(M–O) Lateral views of embryos illustrating expression of Mab21 in blue. (M) A control embryo; (N, O) experimental embryos in which the prechordal graft

(encircled by dots) inhibited the expression of Mab21 in the mesencephalon (arrows). Is*, ectopic isthmus; Is, isthmus; M*, ectopic mesencephalon; pal,

pallium; PG, prechordal graft; PT*, ectopic pretectum; r1, rhombomere 1; r2, rhombomere 2; r3, rhombomere 3; r4, rhombomere 4; Rh, rhombencephalon;

subpal, subpallium; TH*, ectopic thalamus; ZLI*, ectopic zona limitans intrathalamica; ZLIc, contralateral zona limitans intrathalamica.

C. Vieira et al. / Developmental Biology 284 (2005) 351–363 355

mesencephalon, was reproduced in the graft-derived vesicle,

but in a transverse axis, i.e., in an orthogonal distribution in

relation to the host pattern (Fig. 2F). These two grafts under

the influence of the surrounding ZLI were induced to

develop into thalamic (Pax6 positive) and mesencephalic

(Pax6 negative) regions. In most of the chimeric embryos

(n = 50), the graft developed as a small vesicle integrated

into the pretectum and midbrain. The chick Mab21 encodes

a homolog of the C. elegans mab-21 gene that is involved in

cell fate determination (Mariani et al., 1998, 1999) and is

expressed in pretectal and mesencephalic neuroepithelium

(personal observation). Thus, we analyzed the expression of

Mab21 in the host after prechordal grafts. The experiments

showed a repression zone of Mab21 around the graft in all

the cases (n = 17; Figs. 2L–O).

Gbx2 is known to be expressed in thalamic mantle layer

cells. Numerous data have demonstrated a positive regu-

lation of Shh upon Gbx2 (Hashimoto-Torii et al., 2003;

C. Vieira et al. / Developmental Biology 284 (2005) 351–363356

Kiecker and Lumsden, 2004). Accordingly, we found that

Gbx2 expression was activated in the host diencephalon

underlying the prechordal graft (n = 6; Figs. 3A, E).

Sections of these embryos showed that Gbx2 was exclu-

sively induced in host tissue (Fig. 3EV–EVVV).Molecular changes generated by ectopic Shh have only

been analyzed at short survival times and induction or

repression of gene expression was interpreted as a sign of

change in diencephalic regionalization, with consequences

in thalamic histogenesis (Hashimoto-Torii et al., 2003;

Kiecker and Lumsden, 2004). With a view to verifying if

these suggested structural variations do indeed occur in the

induced neuroepithelium, we analyzed some grafted

embryos at long survival periods (HH36). Our specific

interest was to explore if the ectopic induction of Shh-

related genes could generate structural changes in specific

brain regions. In five chimeric embryos with prechordal

grafts into the midbrain, we detected loss of cortical layering

characteristic of the tectal cortex in the areas around the

graft (Figs. 3C, D, G, H compare with control sections B,

F). This mesencephalic tissue, under the influence of ectopic

ZLI, does not develop tectal cortex, but develops a

cytoarchitectonically immature thalamus, with abundant

post-mitotic cells in the subventricular area and a homoge-

neous cell distribution in the mantle layer. The grafted

territory always showed a telencephalic pallial structure

(Figs. 3G, H compare with control section I).

Fig. 3. Induction of the thalamic marker Gbx2 by prechordal grafts in the mesence

HH36 (B–D, F– I). The prechordal grafts are delineated by black or white dots. (

prechordal graft in the mesencephalon induced the ectopic expression of Gbx2 in th

violet staining to show the normal anatomy of the mesencephalic alar plate. Panel

G, H) Sagittal sections of experimental embryos at HH36 with a prechordal graft in

and panel G is a high-power magnification of the square represented in panel

magnification of the square represented in panel D. QCPN staining shows the p

mesencephalic alar plate. Arrows in panels C and D delimit the mesencephalic-ind

at HH35 with cresyl violet staining to show the normal anatomy of the dorsal thala

diencephalon (I). (E) Magnification of the embryo represented in panel A illustrat

by dots. (EV–EVVV) Serial sections of the embryo represented in panel E. In panels

induced expression of Gbx2. G, graft; IC, inferior colliculus; ML, mantle lay

superficiale; Slu, nucleus semilunaris; SV, subventricular zone; TeO, optic tectum

Physiological levels of ectopic Shh regulate gene expression

in the neuroepithelium

Our previous and present experiments strongly suggest

that Shh is the candidate for the morphogenetic activity of

the ZLI. To study this activity, we implanted quail

fibroblasts that express Shh (Duprez et al., 1998) in different

places of the chick neural tube. First, we examined the

possible autoregulative effect of Shh on its own expression.

When we implanted SHH-expressing cells in the dience-

phalic epithelium, ectopic expression of Shh was detected in

donor and host cells (n = 22; Figs. 4A, B, D, H, I, and Q).

Control experiments consisted of implanting QT6-control

cells and using the contralateral side as an internal control.

Subsequently, we explored the effect of Shh on genes

expressed in the diencephalic neuroepithelium. In the

diencephalon, Pax6 is not expressed in the ZLI and basal

plate, precisely where Shh is expressed. Similarly, Pax6

expression was not observed when we implanted SHH-

expressing cells in the pretectum (n = 8/10; Figs. 4C–E),

suggesting that secreted Shh in the ZLI inhibits Pax6 in the

anterior thalamus.

We then examined the effect of Shh ectopic expression on

Nkx2.2. This homeodomain transcription factor is expressed

in the diencephalon separating the basal from the alar plate

and flanking the expression of Shh in the ZLI. When we

implanted SHH-expressing cells in the diencephalon poste-

phalon. Embryos were operated at HH9 and fixed at HH23 (A, E–EVVV) or atA) Lateral view of an experimental embryo showing Gbx2 expression. The

e pretectum. (B, F) Sagittal sections of control embryos at HH36 with cresyl

F is a high-power magnification of the square represented in panel B. (C, D,

the mesencephalic alar plate. Panels C and G are stained with cresyl violet,

C. Panels D and H are stained with QCPN and panel H is a high-power

rechordal graft while cresyl violet staining shows the distinct layers of the

uced area showed in panels G and H. (I) Sagittal section of a control embryo

mus. Tectal induced areas (G, H) show similar structural development to the

ing Gbx2 expression and BrdU incorporation; the grafted tissue is encircled

A and E, arrows point to the grafted tissues and arrowheads indicate the

er; SGC, stratum griseum centrale; SGFS, stratum griseum et fibrosum

; VE, ventricular epithelium.

Fig. 4. Transplantation of SHH-expressing cells results in induction/repression of genes (Nkx2.2, Six3, Pax6, Mab21), which mark various transverse

subdivisions of the diencephalon/mesencephalon. Embryos were operated at HH9 and fixed at HH26 (A, B) or HH23 (C–T). (A) Lateral view of an

experimental embryo showing SHH-expressing cells implanted into the dorsal diencephalon (arrow). (B) A high-power magnification of the area where SHH-

expressing cells were detected. (C, D) Lateral view of the same experimental embryo showing in panel C Pax6 expression in blue and in panel D the expression

of Pax6 in blue and Shh in red. The arrowhead in panel D indicates the ZLI area devoid of Pax6, which is partially occupied by normal Shh expression. (E)

Dorsal view of an experimental embryo illustrating Pax6 expression. The inhibition of Pax6 by the SHH-expressing cells implanted in the pretectum (arrow) is

clearly apparent. (F–H) Lateral views of the same experimental embryo in which panel F is the contralateral side, panel G is the experimental side of the

embryo with Nkx2.2 expression in blue and panel H also shows Shh expression in red. The SHH-expressing cells in the pretectum induced ectopic expression

of Nkx2.2 (arrows), while normal expression of both genes is indicated in the basal plate by an arrowhead. (HV–HVVVV) Transverse sections of the embryo

represented in panel H showing the expression of Shh induced in the tissue and the ectopic expression of Nkx2.2. Panels HV and HVV are high-power photos inwhich donor cells (arrows) expressing Shh induce Shh and Nkx2.2 in host cells (arrowheads). Bisbenzimide staining in panel HVV allows an identification of

donor cells. Panel HVVVV is a high-power magnification of the section illustrating ectopic expression of both genes represented by the arrow in panel HVVV, whilenormal expression is represented by an arrowhead. (I, J) Lateral view of experimental embryos in which ectopic induction of Nkx2.2 is observable caudally (I)

and rostrally (J) to the ZLI (arrows). Lateral (K) and dorsal (L) views of an experimental embryo in which ectopic expression of Nkx2.2 crossed the dorsal

midline. (M, N) Lateral view of an embryo where panel N is the experimental side and panel M the contralateral side, showing Six3 expression (blue) and Shh

expression (red). The SHH-expressing cells induced the ectopic expression of Six3 (arrow). (O) Transverse section of the embryo represented in panel N in

which the arrow indicates the ectopic expression of Six3 and the arrowheads indicate the quail cells. Lateral (P–S) and dorsal (T) views of experimental

embryos showing Mab21 in blue and Shh in red. Panels Q and S represent the experimental side and panels P and R the control side. In the experimental side,

the inhibition ofMab21 (arrows) can be clearly seen. ET, epithalamus; M, mesencephalon; Pi, pineal gland; PT, pretectum; PTh, prethalamus; T, telencephalon;

TH, thalamus; ZLI, zona limitans intrathalamica.

C. Vieira et al. / Developmental Biology 284 (2005) 351–363 357

rior to the ZLI, we observed a strong induction of Nkx2.2

(n = 6/6; Figs. 4F–I). Cryostat sections of these embryos

showed that Shh and Nkx2.2 expression was induced in the

epithelium close to the SHH-expressing cells (arrow in Fig.

4H–HVVVV), dorsal to their normal expression domain (arrow-

head in Fig. 4H–HVVV). The staining of these same sections

with bisbenzimide allowed us to detect the quail SHH-

expressing cells, which further confirmed their local relation

to the ectopic Shh effects (Fig. 4HV, HVV). When the cells

where implanted in the roof plate, Nkx2.2 expression

extended across the dorsal midline through the induced

domain (Figs. 4K, L). Since Nkx2.2 is expressed at both

sides of the ZLI, we explored the possibility of inducing

ectopic Nkx2.2 expression rostral to the ZLI (n = 4/4). We

detected Nkx2.2 induction in the telencephalon (Fig. 4J).

Six3 is a transcription factor expressed in the tele-

ncephalon and the thalamus, caudally bordering the

expression of Shh in the ZLI (Fig. 4M). When we

ectopically expressed Shh in the thalamus, we observed an

induction of Six3 in the caudal thalamic epithelium (n = 3/4;

C. Vieira et al. / Developmental Biology 284 (2005) 351–363358

Fig. 4N). Sections from these embryos, counterstained with

bisbenzimide, showed the quail nucleus of the SHH-

expressing cells (arrowheads in Fig. 4O) in relation to

Six3 ectopic expression (arrow in Fig. 4O). Finally, Mab21

is expressed in the mesencephalon and in the pretectum

(Figs. 4P, R). When we ectopically expressed Shh in the

pretectum, we were able to inhibit the expression of Mab21

(n = 9/11; Figs. 4Q, S, T).

Cellular specification in the thalamus is differentially

regulated by Shh on both sides of the ZLI

Dlx2 and Gbx2 are homeobox genes which are differ-

entially expressed in the diencephalon on both sides of the

ZLI. Dlx2 is expressed in the prethalamus and Gbx2 in the

thalamus. Dlx2 expression is initiated near the basal plate at

early stages of development (Fig. 5A), like the case for

Gbx2 (Hashimoto-Torii et al., 2003). When the expression

of Shh extends dorsally in the alar plate, the expression of

Dlx2 and Gbx2 accompanies Shh into the alar diencephalon

(Figs. 5B, C; Hashimoto-Torii et al., 2003). This suggests a

possible direct control of the expression of these two genes

by Shh. When we ectopically expressed Shh anterior to the

ZLI, we were capable of inducing Dlx2 in cells around

grafted cells (n = 8/9; Fig. 5F, compare with control in D),

but never posterior to the ZLI, despite the presence there of

inserted cells (n = 10/13; Fig. 5E, compare with control in

D). Sections of these embryos showed the normal expres-

Fig. 5. Shh and Dlx2 expression during early development of the prethalamus an

(A–F) Lateral views of embryos showing Dlx2 in blue and Shh in red. (A–C) E

showing that the expression of Dlx2 in the alar plate accompanies the expression o

in panels E and F. (E) An experimental embryo in which SHH-expressing cells can

In this case, the cells induced the ectopic expression of Dlx2 (arrow) and the norma

A transverse section of the embryo shown in panel F. The arrowhead indicates the

ectopic expression of Dlx2 induced by Shh. (G–I) Embryos illustrating expressio

sion of Dlx2 (arrowhead in Fig. 5FV) and the Dlx2 induced

by ectopic Shh in grafted and host cells (arrow in Fig. 5FV).Conversely, when we ectopically expressed Shh posterior to

the ZLI, we were able to induce Gbx2 expression in host

cells (n = 7/8; Figs. 6B–F, compare with control in A) in

agreement to our results in chimeric embryos. Since the

source of Shh is homogeneous in these experiments, it is

likely that the asymmetric effect is due to distinct tissue

competence anterior and posterior to the ZLI. Finally, we

also studied the effect of Shh in relation to Fgf8 (n = 4/6;

Fig. 5G), Wnt3a (n = 2/3; Fig. 5H), and Wnt8b (n = 3/4;

Fig. 5I). The expression of these genes was unaltered with

respect to their normal expression patterns.

Shh induces the development of an ectopic thalamus

Since physiological levels of Shh were found to modify

gene expression in the diencephalon and mesencephalon,

we next examined the histogenic consequences of these

molecular modifications.

To study the long-term effect of Shh on brain develop-

ment, embryos were kept alive after cell implantation until

E10 (n = 22). We detected ectopic overgrowth of the

caudal telencephalic or diencephalic tissues, creating a

supernumerary vesicle which in most cases expressed

Gbx2 (n = 18; Figs. 6G–K). Morphological analysis of

sections from these cases showed that ectopic thalamic

nuclei, such as the dorsolateral thalamic complex and the

d induction of Dlx2 expression by transplantation of SHH-expressing cells.

mbryos at different developmental stages (A) HH17, (B) HH23, (C) HH25

f Shh in the ZLI. (D) Control embryo for the experimental embryos showed

be detected in the pretectum (arrow), but there is no induction of Dlx2. (F)

l expression of Dlx2 in the preoptic area (arrowhead) could be observed. (FV)normal expression of Dlx2 in the preoptic area, while the arrow indicates the

n of Shh in red and Fgf8 (G), Wnt3a (H), Wnt8b (I) in blue.

Fig. 6. Ectopic SHH-expressing cells induce Gbx2 expression in posterior diencephalic host tissue. (A,B) Lateral views of an embryo in which panel A is the

control side and panel B the experimental side showing expression of Gbx2. The SHH-expressing cells induced ectopic expression of Gbx2 (arrow). (C)

Transverse section of the embryo represented in panel B stained with bisbenzimide showing the ectopic expression of Gbx2 near the quail cells that express Shh

(arrows delineate the quail cells). (D–F) Lateral views of experimental embryos illustrating expression of Gbx2 in blue and Shh in red (D) or Gbx2 alone (E,

F). In all cases, the ectopic induction of Gbx2 (arrows) is readily apparent. (G–K) Experimental embryos at HH36. In the lateral views, an ectopic structure is

detected in the dorsal diencephalon (asterisk); in transverse sections showing Gbx2 expression and neutral red staining, these ectopic structures are seen to

belong to the thalamus. Lateral (L) and dorsal (M) views of experimental embryos showing expression of Mab21 in blue and Shh in red. In both cases, the

inhibition of Mab21 expression (arrows) can be seen. (N) Transverse section of an HH36 embryo in which SHH-expressing cells were implanted into the

mesencephalon, altering in this way the mesencephalic alar plate. The arrows delimit the mesencephalic-induced area in which tectal layering is not detectable.

Cb, cerebellum; DLT*, ectopic dorsal lateral thalamus; DLT, dorsal lateral thalamus; R*, ectopic rotundus nucleus.

C. Vieira et al. / Developmental Biology 284 (2005) 351–363 359

rotundus nucleus, appeared as supernumerary structures

substituting the epithalamic region (Figs. 6G–K). SHH-

expressing cell implantation in the mesencephalon also

produced local overgrowth, but Gbx2 was never observed

in this tissue (n = 4; Fig. 6N). In this overgrowth, tectal

layering was completely lost (Fig. 6N), suggesting that the

area in which Mab21 was downregulated at earlier

developmental stages (Figs. 6L, M), changed its histogenic

fate probably into caudal diencephalon (pretectum).

Discussion

Specification of the ZLI

The ZLI is a transverse boundary which separates

thalamic and prethalamic compartments of the prosence-

phalon. It has been described in the neural tube of different

vertebrate species (revised in Puelles et al., 1987; Puelles,

2001). It is detectable very early in development by

functional and histological approaches in mouse and chick

embryos (Puelles et al., 1987; Shimamura and Rubenstein,

1997), long before the expression of specific genes, such as

Shh and Sim1 (Shimamura et al., 1995; Fan et al., 1996). In

conjunction with the progression of Shh expression in the

ZLI, additional properties typical of a secondary organizer

appear at this level, with the Shh protein acting as the

signaling molecule (Kiecker and Lumsden, 2004). The ZLI

then controls the normal development of the diencephalon

by planar induction, as occurs in spinal cord regionalization

by Shh secreted in the floor plate (Briscoe et al., 2000).

The formation of this limit in the alar plate of the

diencephalon has been proposed to be a consequence of an

interaction between the prechordal neural plate (Six3

positive) and the epichordal neural plate (Irx3 positive)

(Kobayashi et al., 2002; Braun et al., 2003; Kiecker and

Lumsden, 2004). However, the in vivo interaction between

these genes (Six3/Irx3) or domains (prechordal/epichordal)

has not yet been demonstrated to be responsible for the

specification of the ZLI in its natural place. In the present

study we show, through in ovo grafting experiments, that an

ectopic prechordal/epichordal interaction can specify an

ectopic ZLI in the neuroepithelium. Then, Six3 and Irx3

positive domains represent different molecular neuroepithe-

C. Vieira et al. / Developmental Biology 284 (2005) 351–363360

lial areas that drive the necessary information to activate an

ectopic ZLI development. Moreover, we observed the

induction of Shh surrounding the graft and restricted to

the bordering graft cells. Although the graft was integrated

dorsally, far from the basal/alar plate limit, the expression of

Shh extended from the basal plate towards the area around

the graft. This demonstrates that the interaction between

these two domains induces a permissive property in the two

tissues to express Shh. Moreover, the expansion of the

expression domain of Shh to include the graft/host region

suggests that a possible repressive effect on Shh expression

in this domain had been reduced or blocked. Recently, it has

been proposed that the expression of Otx2 is fundamental to

restrict Shh expression to the basal plate of the midbrain

(Puelles et al., 2003). Therefore, we can speculate that, in

our experiments, Otx2 expression can be reduced between

the induced and the normal expression of Shh, suggesting

also a negative influence of Shh over Otx2 expression. The

induction of Shh in the alar plate, a primary Otx2 positive

epithelium, by ectopic SHH-expressing cells (Fig. 7A) and

the low expression of Otx2 in the Shh expressing domains

support this interpretation.

The ZLI as the thalamic organizer: molecular interactions

The dynamic expression of some genes which are

responsible for diencephalic regionalization and cell fate

could be dependent on Shh expression in the ZLI.

Shimamura’s group has demonstrated using ex vivo experi-

ments how Shh expression in the ZLI regulates Sox14 and

Gbx2 expression in the thalamic mantle layer (Hashimoto-

Torii et al., 2003). Recently, Kiecker and Lumsden (2004)

have partially reproduced these results in ovo, showing also

modifications of Pax6 expression by Shh ectopic over-

expression at both sides of the ZLI. However, it is difficult

to interpret their results since the corresponding control and

experimental embryos showed paradoxical gene expression

patterns in the basal plate that can be a source of artifactual

effects. Moreover, anatomical references were not evident in

the narrow fields shown in their figures. Our experimental

designs permit us to localize the area of ectopic Shh

expression, without affecting large neuroepithelial domains

and the basal plate. On the contrary, their experimental

approach, gene electroporation, usually generates gene over-

Fig. 7. Model of gene expression responses to signals from the zona

limitans intrathalamica (ZLI) and isthmic organizer (IsO). Single rectan-

gular boxes represent neuroepithelial cells and different colors label

heterogenic molecular domains in the neuroepithelium. (A) Schematic

interpretation of the observed effects generated by SHH-expressing cells.

(B) Schematic representation of the possible planar molecular interactions

at the level of neuroepithelial cells in the ZLI and IsO organizers.

Extracellular ‘‘diffusible’’ effects of signaling molecules (Shh and Fgf8) in

the experimental model (SHH-expressing cells) and secondary organizers

(ZLI and IsO) have been represented by interactive arrows (activation or

repression) in the lower part of each scheme. Intracellular effects have been

represented inside the schematized cells. Neuroepithelial position and

molecular background is represented by horizontal bars.

C. Vieira et al. / Developmental Biology 284 (2005) 351–363 361

expression in the electroporated epithelium that can also be

a source of additional effects. Our results are generated by

induction of Shh expression in both grafted cells and in the

host neuroepithelium, therefore not related to Shh over-

expression.

We set out to explore the effect of physiological levels of

Shh on the diencephalic alar epithelium. Since results from

grafting experiments suggested that thalamic patterning was

under the control of the ZLI, we implanted SHH-expressing

cells to explore molecular changes in this area. We observed

that Shh induced its own expression in the contacting host

epithelium, suggesting an autoregulative mechanism of

induction and maintenance of its own expression in ZLI

cells. However, the mechanisms which restrict Shh expres-

sion to the ZLI are still unknown. A good candidate in this

regard is Otx2, which has previously been considered as a

negative regulator of Shh expression in the alar plate. In

addition to this auto-inductive effect, Shh (from exogenous

SHH-expressing cells and endogenous Shh+-host cells)

induced Nkx2.2 expression in flanking neuroepithelial cells.

Other neuroepithelial genes whose expression was regulated

by Shh included: Six3, which was induced in the thalamus

and Pax6 and Mab21 which were downregulated in the

thalamus and the pretectum-mesencephalon, respectively. In

the mantle layer of the thalamus, Gbx2 expression was

activated, whereas Dlx2 was ectopically induced in the

prethalamic mantle layer. These data strongly suggest that

Shh is the signaling molecule, which mediates the organizer

activity of the ZLI. The graded repression of Pax6

expression in the thalamus, close to the ZLI, and the

recapitulation of specific gene expression patterns in ectopic

sites suggest that the morphogenetic properties of the ZLI

are indeed generated by Shh. The fact that Dlx2 was only

activated anterior to the ZLI and Gbx2 posterior to the ZLI

is indicative of a differential potential of anterior and

posterior neuroepithelia (Fig. 7A).

ZLI control of cell fate and growth in the thalamus

An ectopic source of physiological levels of Shh induced

reproducible anatomical alterations in the neuroepithelium.

This is the first time that structural effects in the brain due to

ectopic expression of Shh have been explored. Results from

previous studies are restricted to the first few days after

experimentation and the effects on brain regionalization and

structure have been exclusively speculations, based on

altered gene expression patterns. In the present study, we

report two long-term structural effects: (1) overgrowth of the

area where SHH-expressing cells were implanted, and (2)

changes in brain cyto- and myelo-architecture. It is known

that Shh can act as a mitogen in the expansion of granule

cell precursors in the cerebellum (Dahmane and Ruiz-i-

Altaba, 1999; Wallace, 1999; Wechsler-Reya and Scott,

1999), in addition to regulating dorsal brain growth by

controlling precursor proliferation (Dahmane et al., 2001).

These effects could in part be achieved via the regulation of

cyclin D1 (Ishibashi and McMahon, 2002). In addition,

these authors reported that the lack of Fgf15 expression in

the thalamus, regulated by Shh, is possibly involved in the

diencephalic alterations seen in the Shh knockout. However,

we did not observe any modifications in the expression of

Fgf8 or Fgf19 (Wright et al., 2004), as well as other

candidate genes, such as Wnt3a or Wnt8b, in response to

SHH-expressing cell implantation (Figs. 5G–I; data not

shown).

The epithalamic area was chosen as the region of the

diencephalon in which SHH-expressing cells were

implanted for some long-term survival experiments. We

have demonstrated that supernumerary dorsal thalamic

nuclei developed in place of the habenular region (a typical

structure of the epithalamus). These structures expressed

Gbx2, a marker of thalamic cells (Martinez-de-la-Torre et

al., 2002). The cytoarchitecture of the mesencephalic cortex

flanking the induced area was found in other experiments to

be substantially altered in that normal cell layering did not

develop, suggesting a change in the cytogenetic program

towards a more nuclear-like, rather than cortical structure.

A number of genes, such as Pax6 (Warren and Price,

1997), Emx2, and Otx2 (Suda et al., 2001), have been

shown to influence diencephalic regionalization. However,

members of other molecular families including Dlx, Wnts,

Bmps, and Fgfs may also participate in this process.

Multiple and varied upstream and downstream interactions

between Shh and these molecules may underlie the

generation of the different nuclear areas which make up

the complex anatomy of the diencephalon. The induction of

ectopic structures which recapitulate normal topologic

relations supports the importance of topology in gene

expression domains and its relation to a morphogenetic

activity of Shh as a signaling molecule.

In conclusion, we present evidence that neuroepithelial

interaction between the prechordal/epichordal epithelia,

which constitute two differently specified territories, is the

cellular process which underlies the specification of the ZLI,

in much the same way as Otx2/Gbx2 expressing territories

participate in the development of the IsO. The organizer

properties are driven by signaling molecules secreted by

organizer cells, i.e., Shh in the ZLI and Fgf8 in the IsO,

which control the development of the neighbouring regions

by means of morphogenetic properties (Fig. 7B). The results

of the present study indicate that the ZLI is the organizer of

the diencephalon, regulating the gene expression patterns

which determine prethalamic and thalamic regionalization.

These events are necessary to generate nuclear and cellular

diversity, which is the basis of the complex functional

properties of this brain region.

Acknowledgments

We thank R. Garcia-Lopez for cresyl violet staining; M.

Rodenas, F. Almagro, and M. Bonete for technical

C. Vieira et al. / Developmental Biology 284 (2005) 351–363362

assistance. Work supported by the grants: UE QLRT-1999-

31625; QLRT-2000-02310; FCT/MCES; DIGESIC-MEC

PM98-0056; BFI2002-02979; GV CTDIA/2002/91. The

authors would like to express their thanks to the agency

ACTS (Academic Consulting and Translating Services;

http://www.euskalnet.net/acts) for having corrected the

English of this paper.

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