Lhx2 and Lhx9 Determine Neuronal Differentiation and
Compartition in the Caudal Forebrain by Regulating Wnt
Daniela Peukert1,2, Sabrina Weber1, Andrew Lumsden2, Steffen Scholpp1*
1Karlsruhe Institute of Technology (KIT), Institute of Toxicology and Genetics (ITG), Karlsruhe, Germany, 2MRC Centre of Developmental Neurobiology, King’s College
London, United Kingdom
Initial axial patterning of the neural tube into forebrain, midbrain, and hindbrain primordia occurs during gastrulation. After this
patterning phase, further diversification within the brain is thought to proceed largely independently in the different primordia.
Here we show that proper neuronal differentiation of the thalamus requires Lhx2 and Lhx9 function. In Lhx2/Lhx9-deficient
zebrafish embryos the differentiation process is blocked and the dorsally adjacent Wnt positive epithalamus expands into the
signaling alter the expression of the thalamus specific cell adhesion factor pcdh10b and lead subsequently to a striking anterior-
posterior disorganization ofthe caudal forebrain. We therefore suggest thatafterinitial neural tube patterning,neurogenesiswithin
a brain compartment influences the integrity of the neuronal progenitor pool and border formation of a neuromeric compartment.
Citation: Peukert D, Weber S, Lumsden A, Scholpp S (2011) Lhx2 and Lhx9 Determine Neuronal Differentiation and Compartition in the Caudal Forebrain by
Regulating Wnt Signaling. PLoS Biol 9(12): e1001218. doi:10.1371/journal.pbio.1001218
Academic Editor: William A. Harris, University of Cambridge, United Kingdom
Received May 10, 2011; Accepted November 2, 2011; Published December 13, 2011
Copyright: ? 2011 Peukert et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: D.P., S.W., and S.S. are funded by the DFG Emmy Noether grant 847/2, and A.L. is supported by the Medical Research Council (MRC). The publication
was supported by Open Access Publishing Fund of Karlsruhe Institute of Technology (KIT). The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Abbreviations: cTh, caudal thalamus; DMB, diencephalic-mesencephalic border; Eth, embryonic epithalamus; hpf, hours post fertilization; IGL, intergeniculate
leaflet; ISH, in situ hybridizations; Lhx, LIM homeobox; MDO, mid-diencephalic organizer; MZ, mantle zone; rTh, rostral thalamus; SVZ, subventricular zone; vLGN,
ventral lateral geniculate; VZ, ventricular proliferation zone; ZLI, zona limitans intrathalamica.
* E-mail: email@example.com
Segmentation is a fundamental step during vertebrate brain
development. It involves patterning of the cranial neural tube into
distinct and segregated transverse units aligned serially along the
longitudinal axis . The most important prerequisite for
segmentation are borders between the successive neuromeres to
allow individual regionalization, growth, and acquisition of distinct
functional identity. This process may be hindered in an embryonic
brain by the fact that it rapidly increases in size and complexity.
Molecular mechanisms underlying segmentation have been
studied during development of the relatively simple hindbrain
region [2,3]. Expression patterns of many regulatory genes also
suggest a neuromeric organization of the embryonic forebrain
[4,5]. Recent studies support a segmental forebrain bauplan with
three prosomeres (P1–P3) (reviewed in ). Based on morphology
and gene expression the alar plate of the diencephalon is divided
into the prethalamus (P3), thalamus (P2), and pretectum (P1). The
epithalamus including epiphysis and habenular nuclei are part of
P2. The border between prethalamus and thalamus is defined by
compartment borders with the interposed narrow region known as
the zona limitans intrathalamica (ZLI). Extracellular cell adhesion
proteins such as Tenascin within the ZLI have been suggested to
mediate lineage restriction between the ZLI and the anteriorly
adjacent prethalamus and posteriorly adjacent thalamus [6–8].
Similarly, the diencephalic-mesencephalic border (DMB), at the posterior
limit of the pretectum, has been identified as a compartment
boundary where, in addition to Tenascin, an Eph-ephrin
dependent mechanism has been suggested to maintain cell
segregation [6,9,10]. Recent fate mapping studies suggest that
the border between the thalamus and the pretectum may also be
lineage restricted . However, little is known about a possible
mechanism leading to cell lineage restriction between these
compartments. The embryonic thalamus (P2) becomes subdivided
into two molecularly distinct domains: the rostral thalamus (rTh)
marked by expression of the proneural gene Ascl1 and the caudal
thalamus (cTh), which expresses Neurog1 [12–14]. In tetrapods,
the rTh contributes to the majority of the GABAergic neurons in
the thalamus including ventral lateral geniculate (vLGN) and
intergeniculate leaflet (IGL), whereas the caudal thalamus gives
rise to predominately glutamatergic nuclei projecting to the
LIM homeobox (Lhx) genes regulate developmental processes
at multiple levels including tissue patterning, cell fate specifica-
tion, and growth . These selector genes act as highly similar
and highly conserved paralogs. They show a restricted expression
pattern in the developing caudal forebrain in frog and mouse;
Lhx1/Lhx5 mark the rTh and the pretectum, whereas expression
PLoS Biology | www.plosbiology.org1December 2011 | Volume 9 | Issue 12 | e1001218
of the Apterous group of Lhx2/Lhx9 is confined to the cTh [19–
22]. In the mouse, Lhx2 function is required for the acquisition of
neuronal identity in different regions such as the telencephalon
and nasal placode [23,24]. In the cortex, Lhx2 is required to limit
the adjacent cortical hem, which expresses BMP as well as
canonical Wnts. Both signaling pathways orchestrate hippocam-
pal development [25,26]. This suggests that Lhx2-mediated
neurogenesis is involved in maintaining the integrity of cortex. In
the diencephalon, the Lhx2/Lhx9 positive cTh is also enriched in
Wnt signaling pathway components in monkeys . Corre-
spondingly, this region is located next to sources of canonical Wnt
ligands at the mid-diencephalic organizer (MDO), the signal-generat-
ing population in the ZLI, and at the diencephalic roof plate
[8,28]. Although the arrangement of these two Wnt positive
organizers and the Lhx2/Lhx9 expression pattern in the adjacent
Wnt receiving tissue is similar to that in the cortex, our
knowledge on their function during diencephalon development
is still lacking. During early patterning, Wnt signaling was
suggested to have an influence on induction of the thalamus [29–
31], but the function of Wnts during regionalization remains
After initial anterior-posterior patterning of the neural tube
during gastrulation, it is believed that brain segments develop
largely independently. Here we show that Lhx2 and Lhx9 are
redundantly required to drive neurogenesis in the zebrafish
thalamus. Furthermore, we show that neuronal differentiation
mediated by Lhx2/Lhx9 has an impact on maintenance of the
thalamus boundaries. Lhx2/Lhx9 restrict the expression of the cell
adhesion factor Pcdh10b to the thalamus and therefore sustain the
thalamus as a true developmental compartment. Thus, Lhx2/
Lhx9 is required for proper development of the thalamus, the core
relay station in the brain, and for the integrity of the entire caudal
In zebrafish, the Apterous group of LIM genes contains three
members: lhx2a, lhx2b, and lhx9 . Lhx2a is expressed only in
the early-born olfactory relay neurons , whereas Lhx2b
resembles the expression pattern of Lhx2 as described in other
model organisms. To facilitate species comparison, Lhx2b is named
as Lhx2 throughout the article.
Fine Mapping of the Temporal and Spatial Expression of
Lhx2 and Lhx9 in the Caudal Diencephalon
To explore neuronal differentiation in the thalamus, we
examined the expression dynamics of lhx2 and lhx9 at early stages
of caudal forebrain development (Figures 1 and S1). We detect
expression of lhx9 in the diencephalon first at 30 hpf (primordial
stage 15; Figure 1a, asterisk), while at 42 hpf (high-pec stage), the
lhx9 expression domain broadens and an overlapping domain of
lhx2 expression becomes apparent (Figure 1b). At 48 hpf (long-pec
stage), lhx2 and lhx9 are co-expressed in the thalamus (Figure 1c,
asterisk). This expression is maintained at later stages (Figure S1).
A cross-section validates the overlap of Lhx2 and Lhx9 positive
At 48 hpf, lhx9 expression is in proximity to, but with a distinct
(Figure 1d,d9). In order to determine the fate of cells in this shh
and lhx9 negative domain, we cloned the zebrafish homolog of the hey-
like transcription factor (helt). Helt has been described as a specific
marker of the prospective GABA interneurons of the rostral
thalamus (rTh), pretectum, and midbrain [34,35] and is required
for the formation of these interneurons in the mouse mesenceph-
alon . The expression domain of helt abuts the rostral, ventral,
and caudal extent of the lhx9 expression domain (Figure 1e,e9).
Complementary to the helt expression, we find an overlap with
glutamatergic neurons marked by vglut2.2 at 3 dpf (Figure S1).
This suggests that lhx9 marks the caudal thalamus (cTh) and is
absent in the GABAergic rTh and pretectum in zebrafish. The
ßHLH factor neurogenin1 is strongly expressed in an intermediate
layer of the neuroepithelium of the cTh, most likely the
subventricular zone (Figure 1f,f9). Expression of neurog1 abuts the
expression of lhx9 in the cTh. The medial part of the lhx9
expression domain overlaps with the expression of the differen-
tiation marker id2a (Figure 1g,g9). The expression domain of the
thalamus-specific post-mitotic neuronal marker lef1 [16,37]
overlaps entirely with lhx9 (Figure 1h,h9). The dorsal limit of the
Lhx9 domain is adjacent to that of Wnt3a, a marker of the central
epithalamus (Figure 1i,i9). Nevertheless, the lhx9 expression
domain overlaps with the expression of the Wnt target axin2 in
the diencephalic alar plate (Figure S1), suggesting that Wnt
expression at the epithalamus/MDO might be required to activate
the Wnt signaling cascade in the thalamic territory.
Thus, we can define Lhx2/Lhx9 as a marker for post-mitotic
neurons of the thalamic mantle zone in zebrafish at 48 hpf.
in thalamic neuroepithilium
Incomplete Development of Thalamic Neurons in Lhx2
and Lhx9-Deficient Embryos
At 48 hpf key markers for neurogenesis in the zebrafish brain
are expressed in a pattern representing best comparability with
amniote brains . Therefore, we chose this stage for the
following analyses. To address the function of Lhx2 and Lhx9 in
the developing caudal thalamus, we used an antisense Morpho-
lino-based knock-down strategy (Figure S2). Neither lhx22/2
zebrafish mutant embryos (beltv24)  (n=13) nor single
morphant embryos for either lhx2 or lhx9 (n=29) are visibly
The thalamus is the interface between the body and the
brain. It connects sensory organs with higher brain areas
and modulates processes such as sleep, alertness, and
consciousness. Our knowledge about the embryonic
development of this central relay station is still fragment-
ed. Here, we show that the transcription factors Lhx2 and
Lhx9 are essential for the development of the relay
thalamus. Zebrafish embryos lacking Lhx2/Lhx9 have
stalled neurogenesis - neuronal progenitor cells accumu-
late but do not complete their differentiation into thalamic
neurons. In addition, we find that the neighboring Wnt-
expressing epithalamus expands into the space containing
mis-specified thalamus in these embryos. We identified a
which is controlled by canonical Wnt signaling. Altered
Wnt-dependent Pcdh10b function in Lhx2/Lhx9-deficient
embryos leads to intermingling of the thalamus and
adjacent brain compartments and consequently regional-
ization within the caudal forebrain is lost. Organization of
the developing CNS into molecularly distinct but transient
segments and the implications for regional differentiation
are well established for the developing hindbrain. We
conclude that this applies to caudal forebrain too: Lhx2
and Lhx9 emerge as crucial factors driving neurogenesis
and maintaining the regional integrity of the caudal
forebrain. These are two prerequisites for the formation
of this important relay station in the brain.
Function of Lhx2 and Lhx9 in the Caudal Forebrain
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distinguishable from uninjected wild type embryos (Figure S2)
similar to the situation in the Lhx2 knock-out mouse. However,
lhx2/lhx9 double morphant embryos showed significant disruption
of thalamic structure (Figure 2). This is consistent with their
overlapping expression domains in the diencephalon (Figure 1)
and suggests a functional redundancy within the Apterous group
during caudal thalamus development. Therefore, we focused on
an approach to reduce both Lhx2 and Lhx9 messages simulta-
neously by generating double morphant embryos. In addition, we
analyzed the lhx9 knock-down morphant in the zebrafish lhx2
Figure 1. Dynamic expression pattern of lhx2 and lhx9 during regionalization of the caudal forebrain. A double in situ hybridization
approach for thalamic development. Embryos were mounted laterally (a, b, c, etc.) or sectioned and the left hemisphere is shown (c9, d9, e9, etc.).
Plane of section is indicated in the previous picture with black arrowheads. Asterisks mark the position of the thalamus. Marker genes and stages are
indicated (a, b), all other embryos (c–i9) are 48 hpf. lhx2 expression is stained in red and lhx9 is stained in blue. lhx9 expression is revealed in the
thalamus at 30 hpf (a). At 42 hpf, lhx9 expression increases and lhx2 expression is detectable ventro-posteriorly within the lhx9 domain (b). At 48 hpf,
lhx2 and lhx9 overlap in the Th (c) and cross-section analysis reveals an overlap of both markers within the mantle zone of the thalamus (c9). The shh-
positive mid-diencephalic organizer (MDO) is located anterior to the lhx9 positive thalamus (d), and a cryo-section reveals a gap between both
expression domains (d9). Helt expression in the rostral thalamus (rTh) and pretectum (PTec) abuts the lhx9 expression (e, e9). neurog1 marks the
thalamic territory (f) and cross-section in (f9) shows that neurog1 marks the subventricular zone (SVZ; white bar) and does not overlap with the
expression domain of lhx9 in the mantle zone. The thalamus expression domain of lhx9 overlaps with the pattern of id2a in the medial part of the
mantle zone (g, g9, black bar). lef1 as a marker of post-mitotic thalamic neurons shows co-expression with lhx9 in the MZ (i, i9; black bar). Notably, lhx9
expression is seen also in the epiphysis (Ep). The thalamic lhx9 expression domain abuts the wnt3a expression domain in the epithalamus (ETh, g, g9).
ETh, epithalamus; HyTh, hypothalamus; Mtz; marginal tecal zone; pTu, posterior tuberculum; Tec, tectum; Tel, telencephalon.
Function of Lhx2 and Lhx9 in the Caudal Forebrain
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mutant background. To define the step in thalamic neuronal
differentiation that is dependent on Lhx2/Lhx9 function, we
analyzed the expression of the following set of thalamus-specific
markers: the neurogenic marker deltaA , the ßHLH factor
neurog1, marking early thalamic progenitors , a regulator of
neuronal differentiation id2a, and a marker for mature thalamic
neurons lef1 [16,42], the caudal thalamus-specific homeobox gene
gbx2 [43,44], and the pan-neuronal marker elav-like 3 (formerly Hu
antigen C) . These markers can be allocated to three layers in
a neuroepithelium in zebrafish: the ventricular proliferation zone
(VZ) is positive for deltaA, the intermediate or subventricular zone
(SVZ) zone is marked by neurog1, and the post-mitotic mantle zone
(MZ) by elavl3 .
At 48 hpf, we observe a lateral expansion of the deltaA positive
ventricular zone in
Figure 2a–b9). Likewise, the expression of the proneural factor
neurog1 (n=18) in the subventricular zone expands laterally
(Figure 2c–d9). Consequently, the expression of the post-mitotic
thalamic neuronal markers id2a (19/31) and lef1 (13/20) is
significantly reduced (Figure 2e–h). Interestingly, the Shh-
dependent homeobox transcription factor gbx2 (n=25) as well as
the Wnt mediator tcf7l2 show no alteration in compound
morphant embryos (Figures 2i,j9, S3). The pan-neuronal marker
elavl3 is decreased in the mantle zone (3/5; Figure 2k,l). This
suggests that DeltaA and Neurog1 positive thalamic progenitors
need Lhx2/Lhx9 function to proceed with neuronal differentiation
To validate our knock-down strategy and to restrict our analysis
temporally and spatially to the thalamus after 24 hpf, we adapted
the electroporation technique to the zebrafish system. We were
thereby able to deliver DNA unilaterally into the neural tube by
pulsed electric stimulation at 24 hpf (Figure 3a) and analyze the
thalamus at 48 hpf (Figure 3b). Electroporation of EGFP DNA
leads to neither molecular nor morphological alteration of the
forebrain/midbrain area (Figure 3c,d; n=15). Based on previous
experiments, we asked if Lhx2 function is sufficient for the
induction of post-mitotic thalamic neurons in the Lhx2/Lhx9-
double-deficient embryos. Therefore, we re-introduced Lhx2
function unilaterally in the thalamus of Lhx2/lhx9 morphant
embryos at 24 hpf corresponding to the endogenous onset of Lhx2
expression (Figure 1). At 48 hpf, the loss of id2a (7/19), lef1 (3/15),
and Elavl3:GFP (8/15) expression within the thalamus of lhx2/lhx9
morphant embryos was restored in the electroporated hemisphere
at 48 hpf (Figure 3f,h,j). It seems that the laterally expanded
epithalamus of morphant embryos can be restored in the
electroporated hemisphere (arrowheads).
Therefore, we conclude that Lhx2/Lhx9 function is crucial for
neurogenesis in the caudal thalamus. Furthermore, Lhx2 alone
can compensate for the loss of Lhx2 and Lhx9, suggesting a
redundant function between these paralogs during thalamic
neurogenesis. Finally, local electroporation is a valid tool to
validate the specificity of a knock-down approach in zebrafish.
Thalamic Neurogenesis Is Required to Limit the MDO and
In the next set of experiments we analyzed the consequence of
Lhx2/Lhx9 deficiency on adjacent tissues: the mid-diencephalic
organizer (MDO) and the embryonic epithalamus (ETh). We find
that in morphant embryos the expression domain of lmx1b.1, a
marker for the MDO and the Eth, expands ventro-posteriorly into
the thalamus at 36 hpf (31/36; Figure 4a–b9). Similarly, the
expression domains of wnt3a (89/141) and wnt1 (8/11) also expand
(Figures 4c–d9, S3). A cross-section reveals that the wnt3a
expression is induced ectopically lateral to the habenula,
presumably in the thalamic territory (Figure 4d9, arrow) although
the forming habenula remains wnt3a negative . To test
Figure 2. Differentiation of thalamic neurons is stalled in lhx2/lhx9 morphant embryos. Analysis of embryos for neuronal differentiation in
double morphant embryos at 48 hpf, lateral view (a, b, c, etc.), and cross-section of left hemispheres (a9, b9, c9 etc.) of the same embryo are shown.
The expression domain of the neuronal precursor deltaA at the ventricular zone (VZ) is vigorously expanded in double morphant embryos (a–b9).
Expression of the progenitor marker neurog1 marking the subventricular zone (SVZ, white bars) is also broadened in lhx2/lhx9 morphant embryos
compared to control embryos (c–d9). However, the thalamus-specific terminal differentiation markers, id2a and lef1, are down-regulated in the mantle
zone of the cTh (MZ, black bars) of Lhx2/Lhx9-deficient embryos (e–h9). The postmitotic marker gbx2 shows no alteration in the compound morphant
embryos (i–j9). The number of cells expressing the pan-postmitotic neuronal marker elavl3 is strongly decreased in the double morphant embryos
shown by a confocal microscope section of an transgenic Elavl3:GFP transgenic embryos (k, l). The deltaA and neurog1 positive precursor pool in the
ventricular/subventricular zone (blue and green domain) expands on the expense of the post-mitotic thalamic neurons (red domain) in the mantle
zone in lhx2/lhx9 morphant embryos (m, n). ETh, epithalamus; HyTh, hypothalamus; MDO, mid-diencephalic organizer; MZ, mantle zone; pTu,
posterior tuberculum; VZ, ventricular zone.
Function of Lhx2 and Lhx9 in the Caudal Forebrain
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whether the expanded Wnt expression affects thalamic develop-
ment, we first monitored Wnt activity in the diencephalon. Here,
we analyzed the expression pattern of the pan-canonical Wnt
target gene axin2 at 24 hpf, 48 hpf, and at 72 hpf. As expected, we
were not able to detect expansion of axin2 expression prior to onset
of Lhx2/Lhx9 expression in the thalamus (Figure S3). From 48hpf,
axin2 expression is progressively increased in the thalamus of
Lhx2/Lhx9-deficient embryos (35/53; Figures 4e–f9, S3). We
confirmed these results using a Wnt reporter zebrafish line
76TCFsiam:GFP, which expresses GFP under the control of
seven repetitive TCF-responsive elements driving a minimal
promoter. The GFP expression is detectable around known
canonical Wnt sources in the diencephalon—that is, the MDO/
ETh area (Figure 4g,h). Lhx2/Lhx9 morphant embryos show
expanded GFP expression in the thalamus (23/35; Figure 4g9,h9).
In summary, we find that the knock-down of Lhx2/Lhx9 in
zebrafish embryos results in an expansion of the epithalamic
expression domain of Wnt ligands. This leads to an enhancement
of Wnt signaling in the diencephalon, predominantly in the
Protocadherin10b Is a Thalamus-Specific Wnt Target
To address the consequences of the loss of Lhx2/Lhx9 and the
subsequent upregulation of Wnt signaling on the integrity of the
caudal diencephalon, we analyzed the expression pattern of
regionally expressed cell adhesion factors in the caudal forebrain.
We find that the expression of the cell adhesion molecule,
protcadherin10b (pcdh10b), starts in the cTh during late somitogenesis
(Figure S4). At 48 hpf, pcdh10b is predominantly expressed in the
progenitor layer, non-overlapping with the post-mitotic lhx2/lhx9
positive neurons (Figure 5a,a9). The expression domain of pcdh10b
abuts dorsally the expression domain of the epithalamus including
the wnt3a expression domain (Figure 5b,b9) and posteriorly with
the domain of the pretectal marker gsx1 (Figure 5c,c9). Thus,
pcdh10b marks specifically caudal thalamic progenitors at 48 hpf.
To investigate the functional interaction between Lhx2/Lhx9
and Pcdh10b, we electroporated lhx2 DNA unilaterally into the
caudal diencephalon. Overexpression of Lhx2 proved to be
sufficient to inhibit pcdh10b expression in the ventricular zone of
the thalamus (16/36; Figure 5c,c9). Furthermore, the thalamic
expression domain of pcdh10b in lhx2/lhx9-deficient embryos
expands into the mantle zone of the cTh (17/23, Figures 5d9,e9,
S4). This suggests a repressor function of Lhx2 on pcdh10b
expression. Interestingly, and beyond a direct repressor effect in
situ, pcdh10b also expanded posteriorly into the normally Lhx2/
Lhx9 negative pretectum (Figure 5d,e).
How do we explain this non-autonomous expansion of pcdh10b
following knock-down of Lhx2/Lhx9? We wondered whether this
could be linked to increased Wnt signaling in the diencephalon of
Lhx2/Lhx9-depleted embryos. Therefore, we altered canonical
Wnt signaling by treating embryos with small molecule effectors of
the Wnt signaling pathway such as the activator, BIO (a GSK3ß
Figure 3. Lhx2 promotes thalamic neurogenesis. At 24 hpf, DNA
(indicated in red) was injected into the brain ventricle followed by
electroporation approach (a). To validate the specificity and efficiency,
we targeted one hemisphere of the thalamus territory with EGFP DNA
at 24 hpf. We find a co-localization with the thalamus-specific marker
barhl2:mCherry at 48 hpf (b). Analysis of cross-sections reveal that
electroporation of EGFP DNA does not alter the expression of lef1 in wt
embryos (c). Furthermore, we find strong down-regulation of lef1 in
lhx2/lhx9 morphant embryos, which is not altered by EGFP DNA
electroporation (d). After electroporation of lhx2 DNA, we observe an
unaltered expression of id2a, lef1, and Elavl3-GFP expression within the
endogenous expression site in the electroporated hemispheres (e, g, i).
Electroporated side was identified by an ISH against lhx2 mRNA in red.
However, electroporation of lhx2 DNA at 24 hpf can restore the
expression of id2a, lef1, and Elavl3-GFP in Lhx2/Lhx9-deficient embryos
(f, g, j; asterisk). Notably, electroporation of Lhx2 can ectopically induce
id2a expression in the basal plate—that is, in the pTu (f). pTu, posterior
tuberculum; RP, roof plate, Tec, tectum; Tel, telencephalon.
Function of Lhx2 and Lhx9 in the Caudal Forebrain
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inhibitor) . To mimic the situation in lhx2/lhx9 morphant
embryos, and to avoid gross malformation due to altered
patterning during gastrulation, we started ectopic activation of
Wnt signaling at 16 hpf and treated the embryos up to 48 hpf. In
treated embryos we see ectopic induction of axin2 expression at 48
hpf (Figure S4), an expansion of pcdh10b expression into the
pretectum (30/36; Figure 5f,f9) similar to the outcome from Lhx2/
Lhx9 depletion. In BIO treated embryos, the expression pattern of
the principal signal of the MDO, shh, and the patterning marker
pax6a are unaltered excluding pleomorphic effects of the treatment
Following these results, we analyzed the expression of pcdh10b in
embryos carrying a mutation in the Wnt pathway inhibitor Axin1
. Although axin1 mutants lack most of the telencephalon and
the eyes (Figure S4), we find an enlarged expression domain of
pcdh10b in the cTh at 48 hpf (Figure 5g,g9). Accordingly, we
treated embryos with the Wnt signaling antagonist IWR-1 (a
tankyrase inhibitor, Figure S4)  from 16 hpf to 48 hpf.
Inhibition of Wnt signaling exhibits a decrease of pcdh10b
expression (55/58; Figure 5h,h9). To validate these results, we
used a heatshock inducible transgenic fish line to overexpress the
canonical Wnt antagonist Dickkopf1, Dkk1 (Figure S4) [50,51] at
10 hpf. Indeed, we find a similar decrease of pcdh10b expression
(Figure 5i,i9). This effect is seen before, but not after, endogenous
pcdh10b induction, suggesting that Wnt signaling is required for
induction of pcdh10b but not for its maintenance (Figure S4).
To dissect the regulatory contribution of Lhx2/Lhx9 and Wnt
signaling to pcdh10b expression, we reduced Wnt3a function in
Lhx2/Lhx9-deficient embryos (Figure 5j,j9). Interestingly, here we
do not find the posterior expansion of the pcdh10b expression
domain into the pretectum (40/84; Figure 5i). However, we still
observe the expansion of pcdh10b into the neuronal layer (40/84;
In summary, these data suggest that Wnt signaling, most likely
by Wnt3a, induces expression of pcdh10b in the caudal thalamus
and Lhx2/Lhx9 are able to limit pcdh10b expression to the
progenitor zone (Figure 5k). Furthermore, ectopic upregulation of
Wnt signaling is able to induce pcdh10b expression also in the
ventricular zone of the pretectum.
Protocadherin10b Mediates Lineage Restriction and
To study the consequences of altered Pcdh10b levels in the
developing caudal forebrain, we analyzed the maintenance of the
border zone between thalamus and pretectum in Lhx2/Lhx9
morphant embryos and Pcdh10b-deficient embryos (Figure 6 and
Figure S5). We used five different sequential approaches from the
onset of neuronal differentiation at 42 hpf to the formation of a
mature thalamus at 4 dpf.
Firstly, we analyzed thalamus-specific GFP expression in the
Gbx2:GFP transgenic zebrafish line (Figure 6a–c9) . In
embryos deficient for Lhx2/Lhx9, we observe that GFP-positive
cells in the ventricular zone of the pretectum become detached
from the Gbx2:GFP positive thalamus (8/14; Figure 6b,b9, white
arrow), suggesting the loss of lineage restriction at the thalamus/
prectectum boundary and the spread of thalamic cells into the
pretectum. Assuming this to be the case, we next asked if different
levels of pcdh10b are required to maintain lineage restriction at this
border. Therefore, we interfered with Pcdh10b function by using a
Morpholino antisense approach for Pcdh10b . In pcdh10b
morphant embryos we find Gbx2:GFP positive cells ectopically in
the pretectal progenitor layer (18/25; Figure 6c,c9, white arrows).
Figure 4. Knock-down of Lhx2/Lhx9 leads to an expansion of the Wnt positive epithalamus. A lateral view (a, b, c, etc.) and a cross-
section (a9, b9, c9, etc.) of the left hemisphere of the same embryo at 48 hpf are displayed. Thalamus is marked by asterisks. Section plane of the cross-
section is indicated by black arrowheads. In control MO injected embryos, lmx1b.1 expression domain marks the MDO and the dorsal RP (a, a9). Knock-
down of Lhx2/Lhx9 leads to an expansion of both areas into the thalamic territory (b, b9). wnt3a marks the epiphysis but not the habenula territory (c,
c9). In Lhx2/Lhx9-deficient embryos, wnt3a expression is ectopically activated in the dorsal part of the thalamus (d, d9). Subsequently the expression
of Wnt target genes such as axin2 (e, e9) as well as the Wnt reporter line 76TCF-Xla Siam:GFP ia4 (g, g9) shows an expanded expression domain in
compound morphant embryos (f, f9 and h, h9). Ep, epiphysis; Hb, habenula; pTu, posterior tuberculum.
Function of Lhx2 and Lhx9 in the Caudal Forebrain
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Secondly, we examined the separation of thalamic and pretectal
domains by the regional expression of the transcription factors lhx9
and gsx1 (Figure 6d–f9). Knock-down of Lhx2/Lhx9 (11/16) or
Pcdh10b (46/73) leads to significant intermingling of lhx9 positive
thalamic cells and gsx1 positive pretectal cells (Figure 6e–f9, white
Thirdly, considering the relay thalamus being mainly glutama-
tergic whereas the central pretectum remains mainly GABAergic,
we looked at the localization of the ßHLH factors Tal1 and
Neurog1. Tal1 marks the inhibitory neurons of the rTh and
pretectum, whereas glutamatergic progenitors express Neurog1
We find the specification of ectopic Tal1 positive neurons in the
territory of the caudal thalamus in Lhx2/Lhx9 double morphant
embryos as well as in Pcdh10b-deficient embryos (Figure 6h–i9).
Figure 5. Expression and regulation of protocadherin10b in the thalamus. Lateral views and corresponding cross-sections of the left
hemisphere of the same embryo at 48 hpf are displayed. Exceptions are a horizontal section in (c9) and dorsal view in (d). Asterisks mark the thalamic
territory. pcdh10b expression abuts the expression domain of lhx9 in the mantle zone (MZ, black bar; a, a9). The roof plate marker, wnt3a, is adjacently
expressed to the pcdh10b expression in the thalamus (b, b9). Expression of pcdh10b in the thalamus abuts posteriorly the expression domain of gsx1
and therefore respects the border to the pretecum (c) shown in a dorsal view (c9). Overexpression of lhx2 DNA via electroporation leads to a unilateral
downregulation of pcdh10b expression (dorsal view, d; d9). Control embryos show pcdh10b expression in the cTh (d, d9). In lhx2 mutant embryo
knocked-down for lhx9, pcdh10b expression expands into the pretectum (e), and the ventricular expression expands into the MZ (e9, white bar).
Treatment of embryos with the Wnt signaling agonist BIO from 16 hpf to 48 hpf leads to an expansion of pchd10b expression into the pretectum (f,
white arrow), however the expanded VZ is not detectable (f9, white bar). Although the gross morphology is altered, pcdh10b expression shows similar
broadening in axin1 mutant embryos (g, g9). Consequently, blocking of Wnt signaling by IWR-1 treatment from 16 hpf to 48 hpf leads to a severe
downregulation of pcdh10b (h, h9). Embryos with ubiquitous expression of the Wnt inhibitor Dkk1 after heat shock activation at 10 hpf leads to a
downregulation of pcdh10 expression at 48 hpf (i, i9). Knock-down of Wnt3a in the Lhx2/Lhx9-double-deficient embryos leads to a rescue of the
expansion into the pretectum (j), however the lateral expansion of the VZ is still detectable (j9). Canonical Wnt signaling—that is, Wnt3a—is required
for induction of pcdh10b expression in the thalamic ventricular zone, whereas Lhx2/Lhx9 inhibits pcdh10b expression in the mantle zone of the cTh
Function of Lhx2 and Lhx9 in the Caudal Forebrain
PLoS Biology | www.plosbiology.org7 December 2011 | Volume 9 | Issue 12 | e1001218
Fourthly, we analyzed the expression of Gad1, a marker of
inhibitory GABAergic neurons by fluorescent ISH at 3 dpf
(Figure 6j). In both Lhx2/Lhx9-deficient embryos (4/8) and
pcdh10b morphant embryos (6/10) gad1 positive cells are mis-
located within the glutamatergic caudal thalamic domain
(Figure 6k–l9; white arrows, Figure S5).
Fifthly, we studied the anatomy of the caudal forebrain by
analyzing areas of clustered cell nuclei at 4 dpf. In wild type
embryos, we observe demarcations between prethalamus and
thalamus (the ZLI), between the thalamus and the pretectum, and
between the pretectum and the midbrain (the diencephlic-mesencephlic
border; DMB) (Figure 6m). The observed anatomical compartition
Figure 6. Protocadherin10b is required to maintain integrity of thalamus. Dorsal views of the left hemisphere of embryo at 42 hpf (a–c), 48
hpf (d–i), and 3 dpf (j–l) are displayed. To visualize orientation of the figures, small sketches accompany the experiments showing the thalamus (Th) in
dark grey and the rostral thalamus (rTh)/pretectum (PTec) in light grey. At 4 dpf, the anatomy of the caudal forebrain is visualized by a confocal
microscopy analysis of ubiquitous nuclei staining by Sytox green (m–o). At 42 hpf, gbx2:GFP expression marks the thalamus as well as the position of
the diencephalic-mesencephalic border (DMB) by the position of the posterior commissure (PC). Knock-down of Lhx2/Lhx9 leads to the appearance
of gbx2:GFP positive cells posterior to endogenous expression domain (b, white arrow). In embryos knocked down for Pcdh10b, thalamic gbx2:GFP
cells appear similarly to (b) in the pretectum (c, white arrows). Analysis of lhx2/lhx9 morphant embryos and pcdh10b morphant embryos by a double
ISH approach for lhx9/gsx1 (d–f). lhx9 marks the thalamus and gsx1 the pretectum seen in a dorsal view (d). In Lhx2/Lhx9 morphant embryos, the
expression pattern of lhx9 and gsx1 intermingles (e, white arrow) similar to the phenotype observed in pcdh10b morphant embryos (f; white arrows).
Confocal sectioning of lhx2/lhx9 double morphant embryos in vivo reveals mixing between Tal1:GFP positive and the neurog1:RFP positive cells in
the cTh (g–h9, white arrows). A similar intermingling phenotype is detectable in pcdh10b morphant embryos at 48 hpf (i, i9). At 3 dpf, the rTh is
marked by gad1 by a fluorescent ISH (j, j9). After knock-down of Lhx2/Lhx9, gad1 positive cells can be found in the territory of the cTh (k, white
arrows); furthermore, in Pcdh10b-deficient embryos, gad1 positive can also be found in the cTh (l, l9). Lateral views of the caudal forebrain show three
cell nuclei loose border zones: the border between prethalamus and thalamus, the ZLI (white dashed lines), the one between the thalamus and the
pretectum (red arrows), and the one between pretectum and midbrain DMB (white dashed lines). The border zone between the thalamus and the
pretectum is not detectable in lhx2/lhx9 morphant embryos (n). Similarly, this demarcation is also missing in pcdh10b morphant embryos (o), whereas
the ZLI and the DMB are not affected. Tec, tectum; Teg, tegmentum.
Function of Lhx2 and Lhx9 in the Caudal Forebrain
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correlates with the described genetic profile of these territories
(Figure S5). In lhx2/lhx9 morphant embryos, the demarcation
between the thalamus and pretectum is not detectable, although
the ZLI and the DMB are unaltered (Figure 6n). In pcdh10b
morphant embryos, we are not able to identify the boundary
between pretectum and thalamus (Figure 6o), while the ZLI and
DMB are still visible. We hypothesize that similar adhesive
properties in the thalamus and in the pretectum lead to a loss of
separation of these brain parts. Thus, we conclude that a Pcdh10b
positive thalamus and a Pcdh10b negative pretectum are required
to establish a border between these compartments.
Development of Thalamic Relay Neurons
The molecular mechanisms that control the orderly series of
developmental steps leading to mature thalamic neurons are
poorly understood. Although numerous transcription factors are
specifically expressed in the thalamus , only a few have been
functionally characterized such as Gbx2, Neurog2, and Her6.
Gbx2 knock-out mice show disrupted differentiation of the
thalamus by the absence of thalamus-specific post-mitotic
neuronal markers Id4 and Lef1, and subsequently lack cortical
innervation by thalamic axons . Although Neurog2-knock-out
mice show a similarly severe failure in neuronal connectivity to the
cortex, the expression of Lhx2, Id2, and Gbx2 is unchanged in these
mice, suggesting that in the absence of Neurog2 thalamic neurons
are not re-specified at the molecular level . In contrast, Her6
regulates the thalamic neurotransmitter phenotype by repressing
neurog1 function and subsequently the glutamatergic lineage. By
contrast, Her6 function is a prerequisite for Ascl1a-positive
interneuron development in the GABAergic rostral thalamus .
Here, we investigate the function of conserved Lhx2 and Lhx9
expression during thalamic development. Lim-HD genes form
paralogs such as Lhx1 and Lhx5, and Lhx2 and Lhx9 . These
pairs have been implicated in various aspects of forebrain
development. Lhx1/Lhx5 influence Wnt activity by promoting
the expression of the Wnt inhibitors sFRPs. This local Lhx-
mediated Wnt inhibition is required in the extra embryonic tissue
for proper head formation  and establishment of the
prethalamus . The Apterous group, Lhx2 and Lhx9, is
required for multiple steps during neuronal development. Lhx2 is
required in mouse for maintenance of cortical identity and to
confine the cortical hem, allowing proper hippocampus formation
in the adjacent pallium [26,56]. However, Lhx2 function during
diencephalic development is still under debate. Although the
Apterous genes are already present in the nervous system of the
cephalochordate Amphioxus—that is, AmphiLhx2/9 —and
co-expression of Lhx2 and Lhx9 has been documented in the
diencephalon of vertebrates, such as zebrafish (here), Xenopus
[20,22], and mouse , their function in the thalamus has
remained unclear. Recent studies of Lhx2 mutant mice showed no
alteration during thalamic neuronal regionalization . Further-
more, the function of Lhx9 has not been described, but the
expression pattern suggests a role during forebrain development
and possibly in parcellation of the thalamus .
Here, we show that single knock-down of Lhx2 or Lhx9 has no
diencephalic phenotype with the markers analyzed (Figure S2),
comparable to the Lhx2 knock-out mouse, but that simultaneous
knock-down of both Lhx2 and Lhx9 leads to stalling of thalamic
neurogenesis at the late progenitor stage (Figure 2). Furthermore,
the activation of Lhx2 alone is sufficient to compensate for the loss
of both Lhx2 and Lhx9 (Figure 3). Our results suggest that Lhx2 is
functionally redundant to Lhx9 to ensure proper thalamic
development. In contrast to other vertebrates, zebrafish embryos
show co-expression of Lhx2 and Lhx9 in the telencephalon until
48 hpf (Figure 1), which could again suggest redundancy .
Indeed the pallium is less affected in the lhx22/2 mutant fish
compared to loss of the neocortex in Lhx22/2 mutant mice
[39,59]. Furthermore, in the Lhx9 negative nasal placode, the
knock-out of Lhx2 has been shown to lead to a similar neuronal
In the thalamus, Lhx2/Lhx9 may regulate genes that are
essential to complete neuronal development, such that neurons do
not reach the terminal neuronal stage. In Lhx2/Lhx9 morphant
embryos, we find that the expression of deltaA, neurog1, as well as
pcdh10b is increased. During neuronal development in fish,
Neurog1 has been shown to activate delta genes directly by
binding several E-box motives in the delta promoter region .
This suggests that in Lhx2/Lhx9 morphant embryos, neuronal
progenitor development is arrested at the level of deltaA/neurog1
expression. Consistently, terminal thalamic neuronal markers such
as Id2a and Lef1 are absent in Lhx2/Lhx9 morphant embryos.
Interestingly, both of these markers have been shown to be
activated by Wnt signaling [61,62]. Although local Wnt activity is
upregulated locally in the lhx2/lhx9 morphant embryos, these
target genes are not transcribed, suggesting that Lhx2/Lhx9
thalamic neuronal differentiation is coupled to a second compe-
tence phase for Wnt signaling. Also, the late and restricted onset of
Lhx2/Lhx9 expression in the thalamus and their requirement for
Id2a and Lef1 expression may explain the thalamic neuronal
specificity of the Wnt target lef1. Thus, we propose that Lhx2/
Lhx9 are essential determinants for cells to reach the late stage of
thalamic neuronal development.
In the spinal cord, Lim HD factors together with ßHLH factors
have been shown to be required for cell cycle exit . The Lim
containing factor Isl-1 and Lhx3 together with the ßHLH factors
Neurog2 and NeuroM act in a combinatorial manner to directly
trigger motor neuron differentiation. In the thalamus, we find a
similar process: Lhx2/Lhx9 inhibit the expression of progenitor
markers such as pcdh10b and activate the expression of postmitotic
differentiation markers such as id2a, lef1, and elavl3. Interestingly,
proper differentiation of thalamic neurons is required to restrict
the MDO and dorsal roof plate (Figure 7), a finding that reflects the
conversion of neocortex in Lhx2 knock-out mice. Here, the Gdf7
positive cortical hem expands at the expense of the neocortex .
This supports the hypothesis that proper neuronal differentiation is
required to maintain brain compartments and their borders.
Wnt Signaling, Pcdh10, and Cell Adhesion
In the mid-diencephalon, the central source of patterning cues is
the MDO. Here, three different signaling pathways merge: Shh,
Fgf, and Wnt . Shh signaling has been shown to induce
proneural genes such as Ascl1 in the rostral thalamus and Neurog1
in the caudal thalamus (cTh) [12,13,65] and a set of transcription
factors assigning specific properties to the developing thalamic cells
[14,21,66–68]. Furthermore, Fgf signaling influences the develop-
ment of the rTh  and parts of cTh, the motor learning area
. Interestingly, although the mid-diencephalon expresses a set
of canonical and non-canonical Wnt ligands and receptors
[27,28], the function of Wnt signaling is not clear. Wnt signaling
seems to be required for mediating thalamic identity in chick
embryonic explants  and mutation of the Wnt co-receptor
Lrp6 leads to a severe reduction of thalamic tissue in mice .
Here, we show that Wnt signaling from the MDO and the roof
plate influence compartition of the caudal diencephalon. The
canonical Wnt signaling pathway plays a pivotal role in mediating
adhesiveness and the key effector of the Wnt pathway, b-catenin,
Function of Lhx2 and Lhx9 in the Caudal Forebrain
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was initially discovered for its role in cell adhesion [71,72]: it
promotes adhesiveness by binding to the transmembrane, Ca2+-
dependent homotypic adhesion molecule cadherin, and links
cadherin to the intracellular actin cytoskeleton. Although several
classes of molecules are involved in morphogenetic events,
cadherins appear to be the major group of adhesion molecules
mediating formation of boundaries in the developing CNS .
After a phase of ubiquitous expression, cadherins display a very
distinct expression pattern in the neural tube . In the
developing diencephalon, classical cadherins, such as Chd2,
Chd6b, and Chd7, mark presumptive nuclear gray matter
structures within developmental compartments . Still, these
studies so far are not able to explain the different compartition in
the caudal forebrain.
Here, we describe the expression pattern of the non-clustered
protocadherin, pcdh10b, in the developing diencephalon and show
that it marks the ventricular zone of the thalamus at mid-
somitogenesis (Figure S5). During somitogenesis, pcdh10b modu-
lates cell adhesion and regulates movement of the paraxial
mesoderm and somite segmentation . We find that the border
of pcdh10b expression co-localizes with the border between
thalamus and pretectum during diencephalic regionalization
(Figure 5). Furthermore, we could link Pcdh10b expression to
canonical Wnt signaling. In chick, some hallmarks of lineage
restriction for the border between thalamus and pretectum have
been observed previously; for example, vimentin and chondroitin
sulfate proteglycans are strongly enriched at this border. Similar to
the anatomical observation in fish (Figure 6j–l), the chick neural
tube shows a morphological ridge where interkinetic movement is
disrupted . However, there are conflicting data from direct
analyses of cell lineages in the caudal chick forebrain regarding cell
compartment borders between thalamus and pretectum [6,76].
This may be explained by the different stages of analysis. In other
vertebrate models, Pcdh10 expression has been reported only at
later stages in development, in chicken HH28, and in mouse E15
[77,78], arguing against a comparable role in these model
organisms. However, Pcdh10 together with Pcdh8, 12, 17, 18,
and19 belongtoa structurally
non-clustered d2 protocadherins, and several members indeed
show an expression pattern during somitogenesis in mouse .
Although we have not carried out direct lineage restriction
experiments by tracing small cell clones at the border, we suggest
that the thalamic area intermingles with the pretectum when both
areas express similar levels of this adhesion molecule (Figure 7).
Our data are supported by the fact that pcdh10b knock-down or
overexpression also lead to a similar phenotype in somite
development . Similarly in Gbx2 knock-out mice, thalamus
cells start to intermingle with pretectum cells . Interestingly,
these authors observe a non-cell autonomous function for this
transcription factor and claim a restriction mechanism mediated
by an unknown cell adhesion factor. We suggest that, as for Lhx2/
Lhx9, Gbx2 is required for the acquisition of proper neuronal
identity and the lack of Gbx2 may lead to a similar sequence of
events—that is, expansion of the Wnt-positive roof plate and
alteration in pcdh10b expression. This hypothesis should be tested
in the Gbx2 knock-out mouse. Notably, as pcdh10b is also
expressed in hindbrain rhombomeres  its function should be
determined during differentiation in this well-studied segmented
part of the neural tube; should compartment formation in the
caudal forebrain and hindbrain turn out to involve similar
molecular effectors, we may reach a unifying mechanism for
compartition of the neuraxis—whether it be in the generation of
single units (thalamus, pretectum) or iterated modules (rhombo-
Thus, we suggest that Lhx2/Lhx9 is required for neurogenesis
within the thalamus and is important to maintain longitudinal axis
patterning of the CNS also at later stages. Alteration of
neurogenesis in a brain part affects the development of the
neighboring parts and thus leads to loss of the integrity over
Materials and Methods
Maintenance of Fish
Breeding zebrafish (Danio rerio) were maintained at 28uC on a
14 h light/10 h dark cycle . To prevent pigment formation,
embryos were raised in 0.2 mM 1-phenyl-2-thiourea (PTU,
Sigma) after 24 hpf. The data we present in this study were
acquired from analysis of wild-type zebrafish of KCL (KWT) and
of the ITG (AB2O2) as well as the transgenic zebrafish lines;
tal1:GFP , hs-dkk1:GFP , elavl3:GFP , GA079:RFP ,
shh:RFP, neurog1:RFP , gbx2:GFP , and the belladonna
zebrafish mutant line with a loss of lhx2  and masterblind
mutant line carrying a mutation in axin1 . In bel/lhx2 mutants,
a 22 bp deletion in the third exon causes a frame-shift and
therefore a stop codon after the second LIM domain. Embryos
were staged  and ages are listed as hours post fertilization (hpf).
Transient knock-down of gene expression was performed as
described in . We used the following Morpholino-antisense
oligomeres (MO, Gene Tools) at a concentration of 0.5 mM: lhx2
MO (59-GCT TTT CTC CTA CCG TCT CTG TTT C-39), lhx9
MO (59-AGG TGT TCT GAC CTG CTG GAG CCG T-39),
wnt3a MO , and pcdh10b MO . The injection of MO
oligomers was performed into the yolk cell close to blastomeres at
one-cell or two-cell stage. For electroporation, embryos were
Figure 7. Function of Lhx2/Lhx9 during thalamic neurogenesis
and regionalization of the caudal forebrain. The schematic
drawing shows a 3-D view of the left hemisphere of the caudal
diencephalon and body axis. In Lhx2/Lhx9-deficient embryos, the
ventricular and subventricular zone of the thalamus (blue) expands
laterally into the mantle zone (red). Furthermore, the Wnt positive
epithalamus (green) expands ventrally into the misspecified MZ.
Subsequently, upregulation of Wnt signaling in the mid-diencephalon
may lead to intermingling of thalamus and pretectum by altered
localization of Pcdh10b (yellow/blue stripes).
Function of Lhx2 and Lhx9 in the Caudal Forebrain
PLoS Biology | www.plosbiology.org 10December 2011 | Volume 9 | Issue 12 | e1001218
manually dechorionated and mounted laterally in 1.5% low
melting-point agarose at 24 hpf. We locally injected 0.5 mg/ml
GAP43-GFP DNA solution or 1 mg/ml pCS2+lhx2 DNA 
solution in the III brain ventricle. The positive charged anode was
positioned on top of the diencephalon, whereas the negative
cathode was positioned underneath the diencephalon (Figure 3).
For electroporation, we used a platinum/iridium wire with a
0.102 mm diameter (WPI Inc.). During the electroporation
procedure the embryo was kept in 16Ringer as conductive fluid.
We used the stimulator CUY21 (Nepa Gene Ltd.) with the
following stimulation parameters: 24 V voltage square wave pulse,
4 ms pulse length, 2 ms pulse interval, delivered three times.
Settings are based on the published electroporation approaches in
To manipulate Wnt signaling in vivo, we used BIO 
((29Z,39E)-6-Bromo-indirubin-39-oxime, TOCRIS Bioscience or
IWR-1 ; SIGMA) as pharmacological agonist and antagonist
of the Wnt signaling pathway. For Wnt signaling analyses,
embryos were dechorionated at 16 hpf (15–17-somite stage) and
incubated with 4 mM of BIO in 1% DMSO, 40 mM IWR-1 in
0.2% DMSO, or with 1% DMSO only.
Prior to staining, embryos were fixed in 4% paraformaldehyde/
PBS at 4uC overnight for further analysis.
Whole-mount mRNA in situ hybridizations (ISH) were
performed as described in . Antisense probes were generated
from RT-PCR products for the following probes with primer pairs
(forward/reverse): lhx2b, 59-AGT GCG TCT CAC GGA AAT
CT-39/59-GCA TCC ATG ATC GGT CTT CT-39; lhx9, 59-
CGT TGG AGA AAG TGG ACT GG-39/59-TGG TGA AGA
ATT CCG ATC AA-39; sema3d, 59-GCT GCA GAA ATC TCC
TCG TC-39/59-ATT TTG CAC AAG TGG GCA TT-39; helt,
59-CCA AAA AGC TCG CCT TTA ATC-39/59-AAC ATA
TTA AGA CGT ATT TAC AGA GCA-39; lmx1b.1, 59-GAC
AAC AGC CGG GAT AAA AA-39/59-CCA TCC GAT TGG
ACA TTA CC-39.
The expression pattern and/or antisene RNA probes have been
described for shha (formerly known as shh; ), gsx1 , pax6a ,
gbx2 , axin2 , lef1 , wnt3a , dla , id2a ,
lmx1b.1 , pcdh10b , gad1 (gad67) , and vglut2.2 .
Post-ISH, embryos were re-fixed in 4% paraformaldehyde/PBS
at 4uC overnight and transferred to 15% sucrose/PBS and kept for
8 h at 4uC. For embedding, embryos were transferred to a mould
filled with 15% sucrose/7.5% gelatine/PBS at 42uC for 10 min.
The moulds were kept overnight at 4uC, frozen in liquid nitrogen
on the following day, and stored at 280uC until required. Frozen
blocks were sectioned coronal with 16 mm thickness on the
To reveal neurons that have initiated axogenesis, we used a
monoclonal antibody against acetylated tubulin (Sigma, T-6793)
in a concentration of 1:20 as described in .
For visualizing cell nuclei, embryos were fixed in 4%
paraformaldehyde/PBS at room temperature for 2 h and
transferred in 16 PBS. Fixed brains were hemisected and
incubated in 25 mM SYTOX nucleic acid stain (Invitrogen)
overnight. After washing in 16PBS brains were mounted laterally
for confocal imaging analysis.
Prior to imaging, embryos were deyolked, dissected, and
mounted in 70% (v/v) glycerol/PBS on slides with cover slips.
Images were taken on Olympus SZX16 microscope equipped with
a DP71 digital camera by using the imaging software Cell A. For
confocal analysis, embryos were embedded for live imaging in
1.5% low-melting-point agarose (Sigma-Aldrich) dissolved in 16
Ringer’s solution containing 0.016% tricaine at 48 hpf. Confocal
image stacks were obtained using the Leica TCS SP5 X confocal
laser-scanning microscope. We collected a series of optical planes
(z-stacks) to reconstruct the imaged area. Rendering the volume in
three dimensions provided a view of the image stack at different
angles. The step size for the z-stack was usually 1–2 mm and was
chosen upon calculation of the theoretical z-resolution of the 406
objective. Images were further processed using Imaris 4.1.3
development. A double in situ hybridization approach was used
for analysis. All embryos were mounted laterally with stages
indicated, except (d9) is a dorsal view and (g9) is a cross-section of
the left hemisphere. lhx9 reveals an onset of expression in the
thalamus (Th) at 22 hpf (a, asterisk), limited anteriorly by shh, a
marker of the MDO and posteriorly by gsx1, a marker of the
pretectum (PTec). At 28 hpf, lhx2 shows an onset of expression in
the thalamus (b, asterisk). Within the thalamus, at 28 hpf helt marks
the rostral thalamus (rTh) and the pretectum (c), however the lhx9
expression domain shows no overlap with the helt domain. The
epithalamus is marked by the Wnt ligand, wnt3a, and the
expression of the Wnt reporter 76TCF-siam:GFP (d). The dorsal
view reveals lateral a stronger expression of gfp-mRNA in
comparison to the wnt3a pattern (d9). At 48 hpf, lhx2 and lhx9
show specific expression patterns in the telencephalon (Tel),
thalamus (asterisk), and ventral to the tectum (Tec), indicated by
the overlapping expression domain of pax6a, marking the alar plate
of the forebrain during development (e, f). axin2 expression in the
thalamus co-localizes with the lhx9 expression. (g, g9). vglut2.2, a
marker of glutamatergic neurons in the relay thalamus (cTh), shows an
overlapping expression domain with lhx9 (h). Both genes, lhx2 and
lhx9, mark the thalamus at 3 dpf (i). ETh, epithalamus; HyTh,
hypothalamus; MDO, mid-diencephalic-organizer; PTec, pretec-
tum; RP, roof plate; rTh, rostral thalamus; Tec, tectum ; Tel,
Expression pattern of lhx2 and lhx9 during thalamus
forebrain development. To validate the efficiency of the lhx2 and
lhx9 splice-site Morpholino-antisense oligomere approach, we
isolated cDNA from injected and non-injected embryos at 48 hpf.
A PCR approach, with primers flanking exon1 and exon2 of lhx2,
demonstrates a suppression of the splicing event of intron1 (1.5 kb)
in five individual embryos injected with lhx2 MO (emb1–5)
compared to a control embryo (con, 221 bp) (a). A similar effect is
demonstrated in injected lhx9 MO embryos 1–4 (emb1–4; b),
which display a non-splicing event of intron1 (993 bp), compared
to control embryos (con, 231 bp) (b). An antibody against
acetylated tubulin shows midline crossing axons anterior (AC,
anterior commissure) and posterior (POC, post-optic commissure)
in the telencephalon (c). In lhx2/lhx9 double morphant embryos,
both commissures do not cross the midline (arrow, d). Single in situ
hybridizations of embryos at 48 hpf are displayed by a lateral view
(e–l). Knock-down of Lhx2 and Lhx9 leads to a decrease of sema3d
expression in postoptic commissure (POC, arrow; e, f). The
morphant analysis of single knock-down, either lhx2 or lhx9, shows
that lef1 expression is unaltered in the thalamus (h, j), compared to
the control embryos (g, i, k). In the lhx2 mutant embryos, lef1
expression in the thalamus shows a weak alteration (l). HyTh,
Efficient knock-down of lhx2 and lhx9 during
Function of Lhx2 and Lhx9 in the Caudal Forebrain
PLoS Biology | www.plosbiology.org11 December 2011 | Volume 9 | Issue 12 | e1001218
hypothalamus; MDO, mid-diencephalic-organizer; pTu, posterior
tuberculum; RP, roof plate; Tec, tectum; Tel, telencephalon.
neuron differentiation. A single in situ hybridization approach was
used for analysis and all embryos were mounted laterally except in
(I9, j9) showing cross-section of left hemispheres. Stages are
indicated. In Lhx2/Lhx9-deficient embryos, lef1 expression in the
thalamus (asterisk) is unaltered at 24 hpf but down-regulated at 3
dpf (a–d). Similarly, the Wnt target gene axin2 shows no alteration
in Lhx2/Lhx9-deficient embryos at 20 hpf (e, f), however at 3 dfp
an up-regulation can be detected in the mid-diencephalon (g, h).
In control MO embryos, wnt1 is expressed at 48 hpf in the roof
plate (RP) and lhx2/lhx9 morphant embryos display an expansion
of the wnt1 expression domain into the thalamic territory (i–j9). In
contrast tcf7l2 shows no alteration in the expression pattern at the
same stage in the caudal forebrain. HyTh, hypothalamus; pTu,
posterior tuberculum; RP, roof plate; Tec, tectum; Tel, telen-
lhx2/lhx9 morphant embryos show defect in thalamic
regulation. All embryos are analyzed by a single in situ
hybridization approach and mounted laterally, with stages
indicated, except (c9) shows a cross-section and the left hemisphere
is displayed. In the thalamus (asterisk) pcdh10b reveals an onset of
expression in segmentation phase (18 hpf), which increases during
development (a, b). Knock-down of Lhx2/Lhx9 leads to an
expansion of pcdh10b expression into the pretectum (pTec, c), as
well as of the ventricular zone (VZ, white bar, c9). Black
arrowheads indicate the plane of a cross-section. To validate the
efficiency of pharmacological treatment with the Wnt signaling
agonist BIO or antagonist IWR-1, we also analyzed under the
same conditions the Wnt target gene axin2. Treatment with the
Wnt signaling agonist BIO demonstrates an up-regulation of axin2,
displayed lateral (d, e). Axin2 expression is upregulated in axin1
mutant embryo masterblind (mbl, f). The treatment of embryos with
the Wnt signaling antagonist IWR-1 leads to a loss of axin2 in the
diencephalon (g, h). We find a similar reduction of axin2 expression
in embryos expressing Dkk1 post-heat-shock at 16 h (i). Treatment
of embryos with the Wnt agonist BIO has no effect in the
expression of shh or pax6a in the forebrain (j–m). In contrast,
embryos treated with the antagonist IWR-1 after endogenous
pcdh10b induction between 24 hpf and 48 hpf show no change in
pcdh10b expression pattern. HyTh, hypothalamus; pTec, pretec-
The thalamic expression of protocadherin10b and its
tum; pTu, posterior tuberculum; RP, roof plate; Tec, tectum; Tel,
SYTOX nuclei staining. Analyses at 48 hpf, lateral view (a, b, c)
and dorsal sections of left hemispheres (d–f9) are shown. Lhx9
marks the thalamus (a) and gsx1 the pretectum (a). In lhx2/lhx9
and pcdh10b morphant embryos, the expression domains overlap.
A similar intermingling of expression domains is visible in embryos
stained for vglut2.2 and gad1 (d–f9). Embryos have been analyzed at
4 dpf by a confocal microscopy analysis of ubiquitous nuclei
staining by Sytox (g–j0). The analyzed section of the lateral view
except dorsal view (h9) is indicated by a schematic drawing (insert).
A sytox staining in green reveals structures of the forebrain and
midbrain (g). To confirm the position of the thalamus, we analyzed
the shh:RFP transgenic line, marking the MDO anterior to the
thalamus (Th, b, b9). The position of the thalamus and pretectum
(PTec) was mapped in the neurog1:RFP transgenic line (i–i0). To
distinguish between the caudal thalamus (cTh) and pretectum, we
also analyzed the tal1:GFP transgenic line. It labels GABAergic
neurons of the rostral thalamus (rTh) and pretectum and therefore
identifies the cell nuclei loose border zone between thalamus and
pretectum (j–j0). ETh, epithalamus; HyTh, hypothalamus; MDO,
mid-diencephalic-organizer; PC, posterior commissure; PG, preg-
lomerular complex; PTec, pretectum; PTh, prethalamus; pTu,
posterior tuberculum; RP, roof plate; rTh, rostral thalamus; Tec,
tectum; Tel, telencephalon; Th, thalamus.
Mapping of the diencephalon in larval stage via
We would like to thank F. Argenton (Padua, Italy), M. Brand (CRT
Dresden, Germany), F. Mu ¨ller (Birmingham, U.K.), T. Murakami
(Gunma, Japan), H. Okamoto (RIKEN, Saitama, Japan), U. Stra ¨hle
(KIT), and S. Wilson (UC London) for fish lines and plasmids. J. Gilthorpe
(UCMM, Sweden) helped us to establish the electroporation technique in
fish. We got valuable input on the manuscript from G. Davidson (KIT) and
C. Kiecker and J. Clarke (both MRC, KC London).
The author(s) have made the following declarations about their
contributions: Conceived and designed the experiments: DP AL SS.
Performed the experiments: DP SW SS. Analyzed the data: DP SS.
Contributed reagents/materials/analysis tools: DP SW SS. Wrote the
paper: AL SS.
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