B RA I N R ES E A RC H R EV IE W S 5 3 (2 0 0 7) 1 7–2 6

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s r e v

Review

Patterning the developing diencephalon

Youngshin Lim, Jeffrey A. Golden ⁎

Department of Pathology and Laboratory Medicine, Abramson Research Center, Rm. 516h, Children's Hospital of Philadelphia,
34th and Civic Center Boulevard, Philadelphia, PA 19104, United States

A R T I C LE I N FO AB S T R A C T

Article history: The diencephalon is the embryonic precursor to the caudal forebrain. The major
Accepted 9 June 2006 diencephalic derivative is the thalamus, which functions as a relay station between the
Available online 31 July 2006 cortex and lower nervous system structures. Although the diencephalon has been
recognized as a vital brain region, our understanding of its development remains
Keywords: superficial. In this review, we discuss recent progresses in understanding one essential
Forebrain aspect of diencephalic development, diencephalic patterning. Signaling centers identified in
Diencephalon the zona limitans intrathalamica and along the dorsal and ventral midlines have emerged
Patterning as essential organizers in diencephalic patterning. The cumulative data reveal that the
Anterior–posterior diencephalon shares some developmental principles with more caudal brain regions,
Dorsal–ventral whereas other mechanisms are unique to this region.
© 2006 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2. AP patterning of the diencephalon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.1. Is the diencephalon a segmental structure? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2.2. The ZLI as a signaling center for diencephalic AP patterning . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.3. The anterior and posterior boundaries of the diencephalon: telencephalon–diencephalon boundary
and diencephalon–mesencephalon boundary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
3. DV development of the diencephalon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
3.1. Common themes for DV patterning: the floor plate and roof plate . . . . . . . . . . . . . . . . . . . . . . . . 23
4. Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

1. Introduction known as the prosencephalon (forebrain), mesencephalon

(midbrain), and rhombencephalon (hindbrain). The prosence-
In vertebrates, the anterior neural epithelium undergoes mor- phalon becomes further divided into the telencephalon and
phological subdivisions to generate vesicle-like structures diencephalon. The telencephalon gives rise to the cerebral

⁎ Corresponding author. Fax: +1 215 590 3709.

E-mail address: goldenj@mail.med.upenn.edu (J.A. Golden).

0165-0173/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.brainresrev.2006.06.004
18 BR A I N R ES E A RC H R EV IE W S 5 3 (2 0 0 7) 1 7–2 6

cortex, basal ganglia, and hippocampus, whereas the dience-

phalon develops into the thalamus, epithalamus, and pre-
tectum in the mature brain (the status of the hypothalamus as
a part of the diencephalon structure remains controversial).
Thus, the telencephalon and diencephalon are the embryonic
anlagen of higher cognition and integration centers in the
brain. Although these two regions of the forebrain are
functionally linked, their structural organization is distinct.
For example, in mammals, the thalamus (the major dience-
phalic derivative) consists of clusters of neurons organized
into nuclei, which are morphological and functional units,
Fig. 1 – Schematic diagrams showing the relationship
whereas the cerebral cortex (telencephalic derivative) is a
between the thalamic sensory relay nuclei and the
laminar sheet of neurons where each layer serves specific
corresponding cortical areas of the mouse brain. (A) Coronal
function. Therefore, one can presume that distinct develop-
section of the caudal forebrain illustrating sensory relay
mental strategies must be utilized by these two brain regions.
nuclei in the thalamus. Colored nuclei indicate major sensory
Although many studies have focused on molecular regulation
nuclei and the color code matches with the corresponding
of telencephalic development (for reviews, see Campbell, 2003;
cortical areas in B where each nucleus sends projection.
Grove and Fukuchi-Shimogori, 2003; Rallu et al., 2002; Ruben-
Ventrobasal nucleus, which sends projections to the
stein et al., 1998; Schuurmans and Guillemot, 2002), relatively
somatosensory cortex, is not shown here because of the
few studies have addressed development of the diencephalon.
section level. (B) The sensory and motor areas of the mouse
The key feature of diencephalic development is the
forebrain. Dashed line indicates plane of section in panel A.
formation of nuclei along the anterior–posterior (AP) and
Color code matches with the corresponding thalamic nuclei
dorsal–ventral (DV) axes. In the diencephalon, neural progeni-
in panel A. A1, primary auditory cortex; dLGN, dorsal lateral
tor cells undergo proliferation in the ventricular zone of the
geniculate nucleus; vLGN, ventral lateral geniculate nucleus;
third ventricle. Once they exit the cell cycle, post mitotic cells
M1, primary motor cortex; MGN, medial geniculate nucleus;
migrate to the mantle zone and they aggregate into various
S1, primary somatosensory cortex; SN, substantia nigra; V1,
clusters called nuclei. Differentiated neurons in each nucleus
primary visual cortex (anatomy and nomenclature based on
have distinct morphologies, employ specific neural transmit-
the mouse brain atlas by Hof et al., 2000).
ters, and generate neural connection to different regions of the
brain (Jones et al., 1997). In order to build accurate neural
circuits, it is essential to form each nucleus in the correct
location and at the appropriate time in development. Based on Lin et al., 1999; Nakagawa et al., 1999; Nothias et al., 1998;
birthdating studies in mammals, it has been hypothesized Pallas, 2001). Therefore, considering its intimate relationship
that the progenitor cells in the ventricular zone (the germinal with the cortex as well as its function as an independent brain
epithelium) are organized as mosaic patches, each giving rise structure, understanding diencephalic development provides
to neurons in specific nuclei of the diencephalon (Altman and many insights into brain development.
Bayer, 1988). As a result, it was assumed that the initial In this review, we attempt to integrate the current data
requirement for the accurate allocation of each nucleus is the addressing the molecular mechanisms underlying diencepha-
precise establishment of AP and DV identities in the neural lic development to the existing studies on AP and DV
progenitor cells. Studies from the spinal cord suggest that the patterning of the diencephalon. For AP patterning, several
progenitor cells acquire their regional identities through studies debating whether or not the diencephalon is a
transcription factor expression codes, which are regulated by segmented structure will be discussed along with the forma-
secreted signaling molecules such as Shh (Jessell, 2000). tion and function of a diencephalic signaling center, the ZLI
Although it has not been studied as thoroughly, a similar (zona limitans intrathalamica). Also, molecular patterning at
mechanism has been suggested in the diencephalon (Hashi- the anterior and posterior boundaries of the diencephalon will
moto-Torii et al., 2003). be covered. For DV patterning, we will focus on the two
The diencephalic derivatives, and the thalamic nuclei in signaling centers, the floor plate and roof plate, and the
particular, have intricate connections with functional areas in signaling molecules that they produce.
the cortex (Jones et al., 1997). For instance, sensory relay nuclei
in the thalamus – including ventrobasal, lateral geniculate,
and medial geniculate nuclei – send projections specifically to 2. AP patterning of the diencephalon
somatosensory, visual, and auditory cortices, respectively (Fig.
1) (Jones et al., 1997; Lopez-Bendito and Molnar, 2003). One of the fundamental questions in neural development is
Furthermore, the thalamocortical projections play essential how the neuroepithelia along the AP neuraxis is parceled into
roles in shaping cortical area development (Kaas et al., 1999; the prosencephalon, mesencephalon, rhombencephalon, and
Pallas, 2001). Although clearly important, the timing of this spinal cord. This question has become inseparable from a
influence is controversial; thalamocortical projections do not second question, how is neural fate first specified during early
appear to influence the initial cortical area specification but development (Bainter et al., 2001; Stern, 2001, 2002; Wilson and
are necessary for proper differentiation of cortical areas Edlund, 2001)? One hypothesis with considerable support is
(Cohen-Tannoudji et al., 1994; Kaas et al., 1999; Miyashita- that the neural induction is tightly coupled with axial
 B RA I N R ES E A RC H R EV IE W S 5 3 (2 0 0 7) 1 7–2 6 19

patterning such that neural induction signal endows the cells
with rostral “forebrain-like” character, as well as with neural
identity (Stern, 2001, 2002). According to this hypothesis, cells
with caudal character are generated by reprogramming (or
transforming) of rostral cells to a more caudal identity (Foley et
al., 2000; Muhr et al., 1997, 1999; Nieuwkoop et al., 1952).
Once the initial global AP pattern is set up, the neural tube
undergoes further local regionalization. Local AP patterning is
best understood in the hindbrain, which is segmentally
patterned (Cooke and Moens, 2002; Lumsden, 2004; Lumsden
and Krumlauf, 1996; Trainor and Krumlauf, 2000). Each seg-
ment (rhombomere) along the AP axis of the hindbrain has a
unique molecular code that defines its functional organization
(Lumsden, 2004). Compared to the hindbrain, AP patterning in
the forebrain is less clear, partially due to its complexity, which
is especially true for the telencephalon. The diencephalon,
however, is structurally more similar to the mid- and hind-
brain. Given its structural similarity to the hindbrain, it is
worth considering whether the diencephalon develops accord-
ing to a similar plan utilized by the hindbrain.
2.1. Is the diencephalon a segmental structure?
The first question that must be answered for comparing the
developmental plan of the diencephalon to that of the
hindbrain is whether it is also segmented during development.
In the well-studied body plan of insects, segmentation is
clearly recognized as transverse subdivision along the AP axis.
In vertebrates, the somites and branchial arches are obvious
examples of segmented structures (Dubrulle and Pourquie,
2004; Graham and Lumsden, 1993; Pourquie, 2001). The
vertebrate hindbrain also shares this organizational plan
(Lumsden, 2004). The advantage of segmentation is that the
whole structure can be made in modules, simplifying the or-
chestration of development through separation of cell popula-
tions into distinct functional units. This “separation of cell
populations” (compartmentalization) has been well studied in
the fly imaginal disc, where the existence of developmental
compartments was first revealed (Garcia-Bellido et al., 1973).
Genetic mosaic analyses demonstrated, across the middle of
the wing disc, the existence of a smooth, linear boundary
restricting cell behavior that did not correlate with any
morphologically visible landmarks (Garcia-Bellido et al.,
1973). Clones of cells respected this boundary, remaining res-
tricted to either the anterior or posterior compartment.
In vertebrates, the anterior embryonic neuroepithelium
contains a series of segment-like transient bulges, known as
neuromeres. In higher vertebrates, neuromeres are particu-
larly noticeable in the developing hindbrain (rhombencepha-
lon) where they are called rhombomeres. Although recognized
by embryologists for many years, their significance had been
puzzling (Lumsden and Keynes, 1989; Niel, 1918). Evidences
supporting these segment-like bulges (rhombomeres) being
lineage-restricted compartments first came from lineage-
tracing studies using dye injections into the chick embryo
(Fraser et al., 1990). Much like the Drosophila imaginal disc,
most clones of cells within each rhombomere respected the
border between rhombomeres (Fraser et al., 1990; Garcia-
Bellido et al., 1973). These data led to the notion that the
rhombomeres are lineage-restricted compartments and thus
the hindbrain is a true segmental structure.
Finding that the morphologically defined rhombomeres
represented lineage-restricted compartments prompted simi-
lar studies in the forebrain. Figdor and Stern (1993) proposed
that the diencephalon is composed of four distinct AP seg-
ments, based on the analyses of morphology, differential
distribution of neuronal antigens, scaffolds of axon fascicles,
and lineage-tracing experiment in the chick embryo. D1,
immediately posterior to the telencephalon (T), is the most
anterior segment followed by D2, D3, and D4; D4 is the most
posterior segment abutting the mesencephalon (M) (Fig. 2A)
(Figdor and Stern, 1993). D1 refers to the ventral thalamus and

Fig. 2 – Schematic diagrams comparing three proposed models of the embryonic chick diencephalon subdivision. (A) D1–D4
indicate four segments in the developing diencephalon (from Figdor and Stern, 1993). D1 includes the ventral thalamus
(prethalamus in updated terminology) and hypothalamus; D2 contains the dorsal thalamus (thalamus in updated term),
habenula, stria medullaris; D3 refers to the anterior part of the pretectum; D4 is the posterior part of the pretectum. (B) P1–P3
indicate three prosomeres (segments) in the developing diencephalon (prosomeric model by Puelles and Rubenstein, 2003). P1
contains pretectum; P2 refers to the thalamus (formerly dorsal thalamus) and epithalamus; P3 includes the prethalamus
(formerly ventral thalamus). Although the hypothalamus is not included in the diagram, it is considered as the anterior
diencephalon in this model. (C) VT, DT, AS, and PS represent four subdivisions, but not segments, in the embryonic
diencephalon (model by Larsen et al., 2001). VT and DT are called PT and T, respectively, in updated terminology. In this model,
the hypothalamus is not included in the diencephalon. Note that these diagrams are modified from each original diagram for
the purpose of comparison. Some boundaries were estimated to compensate for differences in stages studied from the original
reports. AS, anterior synencephalon; DT, dorsal thalamus; Mes, mesencephalon; PS, posterior synencephalon; PT,
prethalamus; T, thalamus; Tel, telencephalon; VT, ventral thalamus; ZLI, zona limitans intrathalamica.
20 BR A I N R ES E A RC H R EV IE W S 5 3 (2 0 0 7) 1 7–2 6
hypothalamus; D2 includes dorsal thalamus, habenula, and
stria medullaris; D3 is the anterior part of the pretectum; the
posterior part of the pretectum containing posterior commis-
sure and its associated tract make up D4. Targeted injections of
carbocyanine dyes, labeling small groups of cells within
neuromeres, as well as intracellular injection showed that
adjacent groups of cells do not mix across boundaries (Figdor
and Stern, 1993). Therefore, the authors concluded that the
diencephalon is composed of lineage-restricted segments
similar to the hindbrain.
Another feature of the segmentation is that each unit or
segment is defined by the expression of a specific set of genes,
such as Hox genes in the case of the hindbrain (Lumsden,
2004). In a series of studies comparing gene expression with
anatomic domains, Puelles and Rubenstein proposed the
prosomeric model of forebrain development (Puelles and
Rubenstein, 1993; Rubenstein et al., 1994). In this model, they
suggested that the developing prosencephalon is composed of
segmental units called prosomeres. According to their initial
model, the forebrain contained six prosomeres arranged along
the AP axis (Rubenstein et al., 1994). The posterior three (P1–
P3) subdivided the diencephalon, whereas the anterior three
(p4–p6) subdivided the secondary prosencephalon. The sec-
ondary prosencephalon included the hypothalamus, which is
considered the most anterior part of the diencephalon,
ventrally, and telencephalon dorsally. This model provided
an important framework for numerous studies on forebrain
pattern formation, establishing an evolutionarily conserved
topographical map with a corresponding gene expression re-
lationship in the vertebrate neural tube. However, some of
their data conflicted with those of several other groups,
creating controversy as to the number and boundaries of pro-
someres (Figdor and Stern, 1993; Larsen et al., 2001). The
difficulty defining prosomeric boundaries arose mainly in the
secondary prosencephalon and the adjoining part of the
diencephalon due largely to complicated morphogenesis
during telencephalic development. For example, according to
their definition, each prosomeric boundary ought to be
transverse and “complete”—implying that it must span the
entire neural tube from the roof plate to floor plate. However,
several boundaries, especially those postulated in the
hypothalamus and telencephalon, were ambiguous. These
concerns resulted in a revision of the prosomeric model that
no longer includes the secondary prosencephalon as a pro-
someric structure (Puelles and Rubenstein, 2003). In the
diencephalon, P1–P3 are still considered as prosomeric
structures with slight modification in P2 and P3: P3 includes
the prethalamus (formerly called ventral thalamus) and emi-
nentia thalamus; P2 refers to the thalamus (formerly dorsal
thalamus) and epithalamus; and P1 is the most posterior part
of the diencephalon containing the pretectum (Fig. 2B) (Puelles
and Rubenstein, 2003). Finally, they suggest that each
prosomere is not necessarily a lineage-restricted compart-
ment and that lineage restriction between segment is not
necessarily required for the definition of the segmentation
(Puelles and Rubenstein, 2003). Taken together, this model
supports the division of the diencephalon, except the hypo-
thalamus, into three segmental prosomeres.
Although anatomic and gene expression data supported
the concept of diencephalic segmentation, retroviral-based
lineage analyses refuted the strict concept of a compartment
(Arnold-Aldea and Cepko, 1996; Golden and Cepko, 1996).
These studies demonstrated that clones, labeled with replica-
tion-incompetent retrovirus infection, dispersed throughout
the diencephalon, without respecting prosomeric or neuro-
meric boundaries (Arnold-Aldea and Cepko, 1996; Golden and
Cepko, 1996). They directly conflicted with previous work using
carbocyanine dyes and intracellular injections of lineage
tracers (Figdor and Stern, 1993). At least two explanations
exist to reconcile the discrepancies between these studies.
First, the initial linage restriction between neuromeres is pre-
sent but after a yet to be defined developmental stage the
boundary disappears. This explanation accounts for lineage
restriction early in development as observed in the previous
study (Figdor and Stern, 1993), whereas when examined later
in development, there is breeching of these segmental
boundaries (Golden and Cepko, 1996). Another explanation is
that a relatively small subset of clones crosses neuromeric
boundaries early in development and such a subset of clones
may have been missed using the techniques employed in the
earlier study. The latter explanation is similar to that observed
in the hindbrain where a small percentage of clones violate
rhombomeric boundaries (Birgbauer and Fraser, 1994). In
either case, during dispersion, some clones in the diencepha-
lon do not respect segmental boundaries. Of note, in the
rhombencephalon, where cells respect rhombomere borders
early development, cell migration beyond these borders, espe-
cially after differentiation, is well recognized (Lumsden, 2004).
The diencephalic segmentation model was recently chal-
lenged by another study by Larsen et al. (2001). As opposed to
reiterative segments, they proposed that the diencephalon is
composed of five unique domains: from the anterior to
posterior, the ventral and dorsal thalamus (which are sub-
divided domains of the parencephalon and are called pre-
thalamus and thalamus in updated terms), the ZLI, and the
anterior and posterior synencephalon (Fig. 2C). Their data fail
to support morphological or molecular evidence of reiteration
through these five domains, and that the boundary cells of
these domains do not share any common gene expression
(Larsen et al., 2001). Furthermore, their fluorescent dye-tracing
studies find only the ZLI to be a true compartment. It should be
noted that the ZLI is considered as a compartment in this
study, not a border between two domains as it is generally
considered in other studies (Echevarria et al., 2003). They
concluded that the lack of overtly reiterated features, the lack
of uniformity in the expression of boundary markers, and the
lack of compartmentalization based on lineage analyses
indicate that the diencephalon should not be regarded as a
segmented structure.
2.2. The ZLI as a signaling center for diencephalic AP
patterning
In the compartmental structures of insects, such as the fly
wing disc, compartment boundary cells serve not only as a
barrier to cell intermixing, but also as a signaling center that
influences the fate of surrounding tissues and thus, an
“organizer” (Irvine and Rauskolb, 2001). Organizers have also
been identified at various border regions in the vertebrate
neural tube (Echevarria et al., 2003; Martinez, 2001; Wurst and
 B RA I N R ES E A RC H R EV IE W S 5 3 (2 0 0 7) 1 7–2 6 21

Bally-Cuif, 2001). For example, the isthmic organizer (IsO)
forms at the boundary between the mid- and hindbrain and
exerts a key influence on regional AP patterning (Martinez,
2001; Wurst and Bally-Cuif, 2001).
In the diencephalon, the ZLI forms the boundary between
the prethalamus and thalamus at the neural tube stage in
addition to its putative compartmental organization at earlier
stages as described above (Fig. 3). Based on the expression of
numerous signaling molecules, including Shh, Wnts, and Fgfs,
the ZLI has been hypothesized as a signaling center (Echevarria
et al., 2003). Evidences supporting this hypothesis have come
from several recent studies (Kiecker and Lumsden, 2004; Vieira
et al., 2005). Using tissue grafting, Vieira et al. (2005) showed
that the ZLI can induce thalamic fate. They demonstrated that
the ectopic ZLI placed in the mesencephalon can induce the
thalamic marker, Gbx2, while repressing Mab21, a mesence-
phalic marker. The ectopically induced ZLI was noted to
express Shh, similar to the endogenous one, and the organizer
activity of the ZLI was recapitulated using transplanted Shh-
expressing cells. A second study using in ovo electroporation
provided further support for the role of the ZLI-based Shh in
regulating diencephalic regional identity (Kiecker and Lums-
den, 2004). They demonstrated that Shh is both necessary and
sufficient for Dlx2 and Gbx2 expressions, both markers for the
ZLI flanking regions, prethalamus and thalamus, respectively.
Furthermore, distinct competences for Shh response were
found between the prethalamus (anterior to ZLI) and thalamus
(posterior to ZLI). The transcription factor Irx3, which is
expressed posterior to the ZLI, was sufficient to provide the
prethalamus with thalamic competence in response to Shh.
These two studies indicate that the ZLI indeed functions as an
organizer of diencephalic AP patterning and that Shh signal
mediates this organizer activity of the ZLI (Kiecker and
Lumsden, 2004; Vieira et al., 2005).
Wnt signaling from the ZLI also appears to be important for
AP patterning. Wnt3 and Wnt3a are expressed in the dorsal
region of the thalamus with their expression domains
abutting the ZLI (Braun et al., 2003; Roelink and Nusse, 1991;
Salinas and Nusse, 1992), whereas Wnt8b is expressed in the
ZLI itself (Garcia-Lopez et al., 2004). Canonical Wnt signaling is
necessary and sufficient to induce thalamic specific gene
expression, and the absence of Wnt signaling, achieved
through pharmacological inhibitors and through the Wnt
antagonist (Dkk), results in prethalamic differentiation (Braun
et al., 2003). This is consistent with the results in Wnt co-
receptor (LRP6) mutant mice that have defects in thalamic
neural precursor domains whereas the prethalamic markers
remain largely intact (Zhou et al., 2004). Together, these data
support the involvement of Wnt signaling in posteriorization
of the diencephalon. However, the endogenous Wnt ligand(s)
working in the ZLI remains to be identified. In addition to Shh
and Wnt, Fgf signaling also has been implicated in ZLI
function, although its specific role has not been further cla-
rified (Walshe and Mason, 2003).
Despite the importance of the ZLI, the mechanism of its
formation remains poorly understood. The ZLI initiates at the
ventral midline where the prechordal neural plate (labeled by
Six3) and epichordal neural plate (labeled by Irx3) meet; from
there it extends dorsally (Echevarria et al., 2003). The
juxtaposition of these two tissues has been postulated as an
essential component of ZLI specification (Braun et al., 2003;
Echevarria et al., 2003; Hashimoto-Torii et al., 2003; Kobayashi
et al., 2002; Larsen et al., 2001). Recently, the in vivo interaction
between these domains (prechordal/epichordal) was demon-
strated to be responsible for the specification of the ZLI (Vieira
et al., 2005). It was shown that the interaction between these
two domains induced a permissive property to express Shh,
an important signaling molecule for ZLI function. However, it
remains to be elucidated if and how the interaction between
Six3 and Irx3 plays a role in specification of the ZLI.
At the early neural tube stage, it has been reported that
the putative ZLI forms from a lunatic fringe (L-fng) negative,
Wnt8b-expressing wedge-shaped area in the prosencephalon
(Fig. 3A) (Kiecker and Lumsden, 2005; Larsen et al., 2001;
Zeltser et al., 2001). This wedge-shaped compartment is
flanked by L-fng expression domains both anteriorly and
posteriorly. Although this L-fng-free compartment has been
suggested to progressively narrow down to a band of cells
that later defines the ZLI, the fate map study using quail-
chick chimera did not observe any evident “narrow down”

Fig. 3 – Signaling centers for AP and DV patterning in the developing diencephalon. (A) The ZLI is initially specified in early
development (approximately stages 12–13 in the chick) by broad Wnt8+ and L-fng− domain. This domain narrows to form a
definitive ZLI later (see the same colored area labeled as ZLI in panel B). Juxtaposition of the Six3+ and Irx3+ domains establish
the AP boundaries of the ZLI. (B) The roof plate and floor plate are known as signaling centers for DV patterning. Yellow-labeled
area corresponds to the alar plate, whereas orange represents the basal plate. The hypothalamus is not included in the
diencephalon in this diagram due to its controversial origin (see text). DT, dorsal thalamus; Hy, hypothalamus; Mes,
mesencephalon; PrT, pretectum; PT, prethalamus; T, thalamus; Tel, telencephalon; VT, ventral thalamus; ZLI, zona limitans
intrathalamica.
22 BR A I N R ES E A RC H R EV IE W S 5 3 (2 0 0 7) 1 7–2 6
processes (Garcia-Lopez et al., 2004). Thus, it remains to be
elucidated how the whole compartment collapses to a band
of cells. The role of L-fng in setting up the lateral ZLI
boundary has been recently reported in chick embryo (Zeltser
et al., 2001). Ectopic expression of the L-fng in the ZLI directed
cells to sort out of the ZLI and repressed ZLI formation
(Zeltser et al., 2001). These results suggest that the limits of
the ZLI along the AP axis are restricted by these adjacent
domains of L-fng expression. Similar mechanism exists in
Drosophila where fng also regulates compartment segregation
and stabilizes the position of a signaling center at the DV
compartment boundary of the wing disc (Irvine and Raus-
kolb, 2001; Rauskolb et al., 1999).
In addition to elucidate the mechanism controlling the ZLI
formation along the AP axis, it is also important to consider
its formation in respect to the DV axis of the neural tube.
Given its expression within the ZLI, Shh expression has been
recognized as an indicator of the definitive ZLI. In the early
neural tube stage, Shh is limited to the basal plate of the
diencephalon and a small domain of expression extends
dorsally into the basal part of the presumptive ZLI epithe-
lium. This expression domain expands further dorsally as the
development proceeds. Recently, Shh from the basal plate
was found to be required for the formation and dorsal
progression of the ZLI (Zeltser, 2005). It has also been
observed that the formation of the ZLI can be opposed by
yet to be identified signals from the dorsal midline of the
diencephalon, which may account for the ZLI failing to reach
the dorsal midline (Zeltser, 2005).
In summary, the ZLI acts as a compound signaling center
that regulates AP development of the diencephalon through
interactions of various pathways including Shh, Fgf, and Wnt,
but the exact role of each signaling pathway and a possible
interaction between these pathways remains to be clarified.
The formation of the ZLI along the AP and DV axes can be
regulated by multiple factors including the juxtaposition of
pre- and epichordal plate, L-fng expression, Shh signal from
the basal plate, and unidentified signal(s) from the dorsal
midline.
2.3. The anterior and posterior boundaries of the
diencephalon: telencephalon–diencephalon boundary
and diencephalon–mesencephalon boundary
The prosencephalic vesicle, as discussed above, develops into
the telencephalon and diencephalon, but no morphological
delineation between these two regions exists until approxi-
mately stages 12–13 in the chick (Garcia-Lopez et al., 2004).
Even in later stages, the morphological boundary does not span
across the entire neural tube; although apparent dorsally, it is
not clear ventrally (reviewed in Trujillo et al., 2005). The
obscure telencephalon–diencephalon boundary (TDB) in ven-
tral at least partially reflects the anatomical complexity of this
region. Molecular markers, such as Lhx genes and SFRP
(secreted frizzled related protein, a putative inhibitor of Wnt
signaling), can provide complementary boundary information
to the morphological landmarks (Bachy et al., 2001; Kim et al.,
2001a,b), but discrepancies exist among interpretations of
molecular marker data (Alvarez-Bolado et al., 1995; Larsen et
al., 2001). Thus, both morphological landmarks and gene
expression data so far do not clearly define a complete TDB.
Furthermore, the controversy over hypothalamic designation
as part of the diencephalon further complicates the subject.
The hypothalamus was included in the diencephalon (D1
domain) defined by Figdor and Stern (1993), but excluded by
Larsen et al. (2001). Puelles and Rubenstein's (2003) model
separates the hypothalamus from the p3 domain and leaves it
as a part of the secondary prosencephalon, which is not
considered as a prosomeric structure but still considered as
part of the anterior diencephalon. Lineage studies also indicate
that there is no lineage restriction between the telencephalon
and diencephalon (Golden and Cepko, 1996; Larsen et al., 2001).
The posterior boundary of the diencephalon is more
obvious in comparison to the TDB, both morphologically and
molecularly. As early as stage 10 in the chick, the prosence-
phalic vesicle, of which the posterior limit coincides with the
diencephalic boundary based on fate map studies (Garcia-
Lopez et al., 2004), can be distinguished from the mesence-
phalic vesicle. Molecular makers delineating the DMB include
Pax6, Pax2, and Engrailed (En); the posterior limit of early Pax6
expression and the anterior limit of Pax2 and En2 have been
shown to coincide with the DMB (Araki and Nakamura, 1999;
Mastick et al., 1997; Puschel et al., 1992; Warren and Price,
1997). Not only do they mark this boundary, but they also play
roles in DMB formation. Pax6 mutants (small eye mutants) fail
to form a normal DMB (Mastick et al., 1997), likely due to a
failure of En1 and Pax2 repression in the diencephalon in the
absence of Pax6 (Matsunaga et al., 2000). Engrailed is also
involved in DMB formation by repressing diencephalic fate
(Araki and Nakamura, 1999). Misexpression of En in the
diencephalon caused a rostral shift of the DMB, Pax6 repres-
sion, and transformation of the dorsal diencephalon into the
mesencephalon. On the contrary, VP16-Engrailed construct
(dominant positive form of Engrailed) expression caused a
caudal shift of the DMB and ectopic Pax6 expression in the
mesencephalon (Araki and Nakamura, 1999). In addition to the
formation of the DMB, Engrailed (En2/En3) also plays a role in
maintaining it along with Fgf8 (Scholpp et al., 2003). Consistent
with these morphological and molecular data, lineage studies
also provide evidences that the DMB is a clear lineage
restriction boundary (Larsen et al., 2001).
3. DV development of the diencephalon
Understanding DV patterning in the diencephalon is more
difficult than in more caudal regions as a result of the neural
tube curvature in this region. The cephalic neural tube under-
goes several flexures, which results in complicated topological
relationship between anatomic structures relative to the AP
and DV axes of the neural tube, thus generating confusing
positional terminology. For example, ventral and dorsal
thalami (conventional nomenclature) do not actually indicate
DV domains but rather refer to AP domains of the diencepha-
lon (Fig. 3B). To avoid misguidance from this confusing
nomenclature, an updated terminology by Puelles and Ruben-
stein (2003) replaces the ventral thalamus with the prethala-
mus and the dorsal thalamus with the thalamus. Thus, the
true DV axis of the diencephalon must be considered with
reference to the true AP axis of the neural tube. The DV field
 B RA I N R ES E A RC H R EV IE W S 5 3 (2 0 0 7) 1 7–2 6
can be divided into four areas similar to more caudal neural
tube derivatives. These divisions are, from ventral to dorsal,
the floor plate, basal plate, alar plate, and roof plate (Fig. 3B).
The floor plate consists of ventral midline cells that function as
an embryonic organizing center producing secreted signaling
molecules. Although the floor plate shares common features
along the entire neural axis, the floor plate of the forebrain is
somewhat distinct from that of the spinal cord, with respect to
morphology, molecular constituents, cell types, cellular origin/
ontogeny, inductive pathway, and timing of induction (Placzek
and Briscoe, 2005). The basal and alar plates are located in
between the floor plate and roof plate, and most of the nuclei in
the mature diencephalon are derived from these two regions.
The roof plate resides in the dorsal midline and its progenitors
are induced in lateral regions of the closing neural tube. Similar
to the floor plate, the roof plate is a signaling center which
affects patterning of the adjacent regions. Unlike the roof plate
in the spinal cord, some cells in the forebrain roof plate
differentiate to form structures such as the choroid plexus and
pineal gland.
3.1. Common themes for DV patterning: the floor plate
and roof plate
Numerous studies in the spinal cord have established that the
floor plate and roof plate produce morphogens such as Shh
and Bmps, respectively, and that these morphogens influence
DV patterning (Wilson and Maden, 2005). For example, in the
ventral spinal cord, a gradient of Shh regulates homeodomain
transcription factors expressions in progenitor cells (Jessell,
2000). Distinct combinations of transcription factor expres-
sion, set up by Shh signaling, confer a unique DV identity to
groups of ventral neural progenitor cells, generating five
different classes of neurons (V0–V3: interneurons; MN: motor
neurons) (Briscoe and Ericson, 2001; Jessell, 2000; Shirasaki
and Pfaff, 2002). In the dorsal spinal cord, there are six
different neural progenitor cell groups that are classified
according to the expression of bHLH proteins (Math, Mash, and
Ngn) and LIM homeodomain proteins (Lbx and Lmx). TGF
superfamily members provide positional information in some
of these cell groups by setting borders of expression of
homeodomain genes in an analogous fashion to the role of
Shh in the ventral spinal cord (Helms and Johnson, 2003;
Timmer et al., 2002; Wine-Lee et al., 2004). In the diencepha-
lon, there have been similar efforts to identify region specific
transcription factors along the DV axis (Hashimoto-Torii et al.,
2003; Lim and Golden, 2002; Nakagawa and O'Leary, 2001).
Several studies have correlated transcription factor expres-
sion between early embryonic neural domains and later
mature nuclei, suggesting that positional allocation of each
nucleus along the DV axis occurs during early neurogenesis
analogous to the early assignment of DV identity in the spinal
cord (Hashimoto-Torii et al., 2003; Lim and Golden, 2002;
Nakagawa and O'Leary, 2001).
Emerging studies suggest that genes involved in DV
patterning are conserved between the spinal cord and the
diencephalon (Bach et al., 2003; Hashimoto-Torii et al., 2003;
Lim et al., 2005). Evidence supporting this contention comes
from a recent study of Sox14 and Gbx2, which are expressed in
discrete domains along the DV axis of the diencephalon
23
(Hashimoto-Torii et al., 2003). These transcription factors are
regulated by different concentrations of Shh, and distinct Gli
molecules mediate their differential response to Shh (Hashi-
moto-Torii et al., 2003). This study suggests that DV regiona-
lization in the diencephalon exploits Shh in a similar fashion to
the ventral spinal cord. What makes the same signaling
molecule act uniquely at different sites along the AP axis?
Some insight into this problem has emerged by considering
cooperative signaling of several molecules (Dale et al., 1997; Ye
et al., 1998). For instance, Bmp7 cooperates with Shh for ventral
forebrain development, whereas Fgfs do so in the midbrain
(Dale et al., 1997; Ye et al., 1998). In this respect, it is interesting
to note that the floor plate cells, the source of Shh, express
different sets of signaling molecules depending on their
position along the AP axis (Placzek and Briscoe, 2005). Floor
plate cells spanning from the prospective anterior diencepha-
lon to the hindbrain show unique expression of signaling
molecules such as Fgf, Bmp, and Egf-cfc (epidermal growth
factor-cripto-FRL-cryiptic) families, in addition to Shh and
netrin, whereas the floor plate cells in the spinal cord only
express Shh and netrin (Chapman et al., 2002; Colas and
Schoenwolf, 2000; Dale et al., 1999; Ding et al., 1998; Furuta et
al., 1997; Karabagli et al., 2002; Mahmood et al., 1995; Shen et al.,
1997). The exact role of these molecules in diencephalic pat-
terning remains to be defined.
Similar to the floor plate, several studies have indicated
that the roof plate is a signaling center for the dorsal
diencephalon. Disruption of the dorsal midline of the posterior
diencephalon affects the development of the dorsolateral
aspect of p1 including the normal expression of the dorsolat-
eral markers, Pax6, pax7, and Lim1 (Bach et al., 2003). These
defects in dorsolateral gene expression interfere with the
development of structures derived from this region including
the posterior commissure and the subcommissural organ.
Additional support for signaling along the dorsal midline
comes from the analysis of ectopic En1 expression (Louvi and
Wassef, 2000). Transgenic mice expressing En1 in the dorsal
midline from the diencephalon through the spinal cord, under
the control of the Wnt1 promoter, exhibit misspecification of
the roof plate cells. This defect in the roof plate results in
commissural axonal pathfinding errors and leads to the
defects in structures derived from the dorsal neuroepithelium
such as subcommissural organ and pineal gland. They
corroborated these results in Wnt1 mutant mice (Wnt1sw/sw)
and electroporation studies in chick. These data, together with
the study by Bach et al. (2003), demonstrate that the dorsal
diencephalic midline is necessary to specify neural tissue
adjacent to the roof plate.
The roof plate of the diencephalon expresses candidate
inductive signaling molecules such as Bmps and Wnts. Despite
the fact that BMP signaling is required for DV patterning at
other levels of the neural tube and that Bmps are expressed in
the dorsal diencephalon, their role in diencephalic patterning
remains uncertain. Unlike in the spinal cord, Bmp ligands and
Bmp receptors are expressed both in the ventral and dorsal
midline of the diencephalon, suggesting a complex role in DV
patterning (Lim and Golden, unpublished data). Ectopic activa-
tion of Bmp signaling through constitutively active Bmpr1s can
alter transcription factor expression, which perturbs later
diencephalic nuclear development (Lim et al., 2005). However,
24 BR A I N R ES E A RC H R EV IE W S 5 3 (2 0 0 7) 1 7–2 6
loss-of-function experiment using dominant-negative Bmpr1s
found no defects in diencephalic nuclear organization (Lim et
al., 2005). These loss-of-function data are consistent with the
results in the telencephalon, both in the conditional Bmpr1a;
Bmpr1b compound mutant mice and chick explant culture
studies (Gunhaga et al., 2003; Hebert et al., 2002). Together,
these data suggest that, unlike in the spinal cord, Bmp signaling
may not be required for global DV patterning in the forebrain.
However, we cannot exclude the possibility that the negative
data presented in these studies may reflect limitations in each
experimental system or potential redundancies in Bmp ligands
and receptors (Chen and Massague, 1999; Ebendal et al., 1998;
Hebert et al., 2003).
4. Conclusions and perspectives
The embryonic diencephalon employs a well-organized strat-
egy during development that resembles that of more caudal
neural tube derivatives: subdividing its homogenous field into
discrete domains along AP and DV axes and providing cells
with positional identity through local signaling centers. Along
the AP axis, several subregions can be identified with distinct
transcription factor expression and morphological boundaries.
However, these subregions do not conform to traditional seg-
ments due to a lack of shared, reiterated features between
domains. Furthermore, the lack of lineage restriction, with a
possible exception of the ZLI, further supports that the dien-
cephalon may not be a conventional segmental structure, al-
though some authors do not consider lineage restriction as
strict criteria for segmentation (Puelles and Rubenstein, 2003).
With respect to signaling centers along the AP axis of the
diencephalon, the ZLI emerges as an important organizer in
this region. An open question remains as to how the Lunatic
fringe negative domain (putative ZLI anlagen) shrinks down to
a narrow band. It is also necessary to identify the dorsal mid-
line signal opposing the ZLI progression from the basal plate
dorsally.
Along the DV axis, the roof plate and floor plate serve as key
signaling centers. Whereas Shh is required for ventral pattern-
ing, the role of Bmp signal remains uncertain for dorsal spe-
cification. This is partly due to its expression both in the dorsal
and ventral midlines, and the potential redundancy both in the
ligands and receptors. Future studies are required to clarify the
exact role of Bmps in diencephalic DV patterning. The
identification of more transcription factors will help to classify
neural progenitor cells with distinct DV identity. Eventually, it
needs to be elucidated the link between specific gene expres-
sion domain and generation of subtype-specific neurons, such
as the link shown between the Dlx expression domain in the
basal forebrain and the origins of interneurons in the telen-
cephalon (reviewed in Panganiban and Rubenstein, 2002) or
the link of the Lmx1a/Msx1-positive domain and dopaminergic
neuron development in the mesencephalon (Andersson et al.,
2006). Still many challenges remain for the investigators in this
field, especially the challenges to explore the mechanism be-
hind the development of intricate nuclear organization from a
simple neural epithelium, and beyond this, how connectivity is
established and how the diencephalon participates in specify-
ing cortical regions.
Acknowledgments
We would like to thank the Golden lab members for the
comments on the manuscript. We appreciate Sue Marone's
help for the illustrations.
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