Download Lbx1 marks a subset of interneurons in chick hindbrain and spinal cord

Mechanisms of Development 101 (2001) 181±185
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Gene expression pattern
Lbx1 marks a subset of interneurons in chick hindbrain and spinal cord
Frank R. Schubert a,1,*, Susanne Dietrich b,1, Roy C. Mootoosamy b,
Susan C. Chapman a, Andrew Lumsden a
a
MRC Centre for Developmental Neurobiology, King's College London, 4th Floor New Hunt's House, Guy's Campus, London SE1 9RT, UK
b
Department of Craniofacial Development, King's College London, Floor 27 Guy's Tower, London SE1 9RT, UK
Received 21 September 2000; received in revised form 6 November 2000; accepted 7 November 2000
Abstract
The putative transcription factor Lbx1 is expressed in the mantle zone of the hindbrain and spinal cord caudal to rhombomere 1, in a
speci®c domain of the alar plate. The Lbx1 domain overlaps with the expression domains for Tlx3 and partially with the domains for Pax2/
Lim1. The ventral border of the Lbx1 domain coincides with the ventral border of the dorsalmost Serrate1 stripe in the ventricular zone. The
latter borders the intermediate stripe of both Delta and Lunatic fringe expression. The Lbx1 domain contains differentiated interneurons that
project into the lateral longitudinal fasciculus. q 2001 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Lbx1; LH2B; Isl1; Lim1; Tlx3; Pax2; Serrate-1; Delta-1; Lunatic fringe; Vertebrates; Chick; CNS; Hindbrain; Spinal cord; Neurons; Interneurons;
Differentiation; Speci®cation
1. Results
The vertebrate CNS is thought to be rostrocaudally
patterned by a combinatorial code of transcription factors,
expressed in overlapping domains (Puelles and Rubenstein,
1993; Kessel, 1994; Lumsden and Krumlauf, 1996). Similarly, overlapping expression domains of transcription
factors have been suggested to set up the dorsoventral architecture of the CNS, thereby specifying subtypes of motorneurons (Varela-Echavarria et al., 1996) and interneurons
(Burrill et al., 1997; Liem et al., 1997; Briscoe et al., 2000).
We have isolated the chick homologue of the Drosophila
ladybird gene, Lbx1 (Dietrich et al., 1998). Lbx1 has been
studied so far primarily with respect to its expression in
migratory hypaxial muscle precursors in chick (Dietrich et
al., 1998) and mouse (Jagla et al., 1995; Schafer and Braun,
1999; Dietrich et al., 1999; Gross et al., 2000; Brohmann et
al., 2000). Here, we have used comparative in situ hybridization, antibody staining and retrograde labelling to characterize the Lbx1 expressing cells in the chick hindbrain and
spinal cord.
Lbx1 expression commences at stage HH16 (Hamburger
and Hamilton, 1951) in a few scattered cells in the dorsal
hindbrain (not shown). As more cells began to express the
* Corresponding author.
E-mail address: [email protected] (F.R. Schubert).
1
These authors contributed equally to the work.
gene at HH17 and HH18, the staining becomes a continuous, bilateral stripe in the alar plate of hindbrain and spinal
cord (Figs. 1A and 2A, blue staining). At later stages, the
domain broadens (Fig. 1B,C, blue staining), but is maintained at least until HH26 with some upregulation in rhombomeres 3±6 (not shown). At all stages analyzed, the Lbx1
domain resides in the mantle zone of hindbrain and spinal
cord. To precisely localize the Lbx1 domain, we doublelabelled the embryos with Serrate-1. This gene, besides an
expression domain bordering the ¯oor plate, labels the
ventricular zone of hindbrain and spinal cord in two invariant bilateral stripes (Fig. 1A,B,D-N, red staining; Myat et
al., 1996). The ventral boundary of the Lbx1 stripe corresponds with the dorsalmost Serrate stripe (Fig. 1A,B,DI,O), dorsally adjacent to the intermediate stripe for both
Delta-1 (Fig. 1C, red staining) and Lunatic fringe (Fig.
1O, red staining; summarised in Fig. 2H). Unlike Serrate1 (Fig. 1B), Lbx1 is not expressed rostral to rhombomere 2
(Fig. 1 A-D). In rhombomere 2 itself, the Lbx1 domain is
split into two bilateral stripes (Fig. 1B,C,E), while further
caudally only one broad stripe is visible (Fig. 1F±I,O).
To establish the position of Lbx1 positive cells with
respect to some of the known markers for the dorsoventral
subdivision of the CNS and for neuronal subtypes, we
compared the expression pattern of Lbx1 with those of
LH2B, Isl1, Lim1, Tlx genes and Pax2. In the spinal cord,
LH2B, Isl1 and Lim1 de®ne three columns of dorsal interneurons named D1-D3, respectively (Liem et al., 1997).
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F.R. Schubert et al. / Mechanisms of Development 101 (2001) 181±185
Fig. 1. Comparative expression analysis of Lbx1. (A±C) Flat mounted hindbrains at HH18 (A) and HH24 (B,C), labelled with Lbx1 in blue and Serrate-1 (A,B)
or Delta-1 (C) in red. Anterior is to the top, ventral is in the midline, dorsal is to the sides. Note the continuous expression domain of Lbx1 posterior to
rhombomere 1. The ventral border of the Lbx1 stripe and the dorsalmost Serrate-1 stripe coincide (striped arrows). The Serrate stripes are con®ned to areas
negative for Delta-1 (C, arrowheads). (D±H) Cross sections through the hindbrain of a HH17 embryo stained for Lbx1 (blue) and Serrate-1 expression (red), the
rhombomeres are indicated (r1±r7). Only right halves of the sections are shown; dorsal is to the top. In rhombomere 1, only Serrate-1 is expressed (red arrow).
In the other rhombomeres, Lbx1 signals are found in the mantle zone, reaching as far ventral as the dorsalmost Serrate-1 stripe in the ventricular zone (striped
arrows). (I-N) Cross sections through the lumbar spinal cord of HH24 embryos, labelled in blue with Lbx1 (I), a Tlx1 homeobox probe in the spinal cord
detecting Tlx3 (J), Lim1 (K), Pax2 (L), Isl1 (M), LH2B (N) and in red with Serrate-1 (I±N). Dorsal is to the top. Similar to the hindbrain, the ventral extreme of
the Lbx1 domain corresponds to the ventral border of the dorsalmost Serrate-1 domain (J, striped arrow). The ventral Tlx3 domain resides dorsal to the Serrate-1
stripe (J, separate arrows). The ventral Lim1 domain (K) and the medial Pax2 domain (L) reach the level of the dorsal Serrate-1 stripe (striped arrows), not
overlapping with the expression domains of Isl1 (M) and LH2B (N). (O) Cross-section through the lumbar spinal cord of a HH22 embryo labelled with Lbx1 in
blue and Lunatic fringe in red, dorsal is to the top. Note that the Lbx1 stripe borders the Lunatic-positive region in the ventricular zone (arrows). The scale bar in (A)
corresponds to 500 mm in (A±C). The scale bar in (D) corresponds to 50 mm in (D±H). The scale bar in (I) corresponds to 50 mm in (I±O).The red arrows in (D±N)
indicate the level of the dorsalmost Serrate-1 stripe; the red arrow in (O) marks the border of the intermediate Lunatic stripe. Expression of the marker in blue at the
same dorsoventral level is indicated by striped arrows. Abbreviations: fp, ¯oor plate; r1±r7 rhombomeres 1±7; spc, spinal cord.
F.R. Schubert et al. / Mechanisms of Development 101 (2001) 181±185
183
Fig. 2. Relation of Lbx1 expression and position of interneurons. (A) Flat mounted hindbrain at HH17, same orientation as Fig. 1 (A±C). Note the Lbx1 staining
bordering the zone rich in dorsoventrally orientated axons and overlaying the developing lateral longitudinal fasciculus (arrowheads). (B±E) Cross-sections
through rhombomeres r2 (B), r4 (C), r6 (D) at HH17 and the lumbar spinal cord (spc) at HH20 (E), orientation as in Fig. 1 (D±O). Lbx1 staining resides at the
same dorsoventral level as the exit points for cranial and spinal nerves. Note that the neuro®lament expressing cells within the Lbx1 domain project ventrally
rather than towards the exit points (arrows). (F±G) Retrograde labelling of Lbx1-positive neurons. Longitudinally projecting neurons were back-labelled with
¯uorescent Dextran (red) as indicated in (F). (G) Magni®cation of the area framed in (F). The Lbx1 domain (black) yields neurons (arrowheads) whose axons
project into the lateral longitudinal fasciculus on the ispilateral side. Note that the labelled cell bodies locate rostral to the labelled site indicating that their
axons project caudally. (H) Schematic representation of markers for dorsal interneurons. Interneuron-markers for the mantle zone: dark blue, LH2B; yellow,
Lim1; green, Isl1; purple, Pax2; orange Tlx3; turquoise, Lbx1. Markers for the ventricular zone: red, Serrate-1; grey-hatched, Dll1 together with Lunatic fringe.
Note that the neural expression of Lbx1, LH2B, Lim1, and Pax2 is restricted to the CNS, while Isl1 and Tlx3 are also expressed in the dorsal root ganglia (DRG).
The scale bar in (A) corresponds to 100 mm. The scale bar in (B) corresponds to 50 mm in (B±E). The scale bar in (G) corresponds to 100 mm. Abbreviations: V,
trigeminal nerve; VII, facial nerve; VI, abducens nerve; IX, glossopharyngeal nerve; DRG, dorsal root ganglion, llf, lateral longitidinal fasciculus; others as in
Fig. 1.
Tlx3 marks D2 interneurons and uncharacterized interneurons located more ventrally (Logan et al., 1998), and Pax2
labels D3 interneurons and two interneuron populations
located at the sulcus limitans and further ventrally within
the basal plate (Burrill et al., 1997). In the spinal cord at
HH19 (not shown) and HH24 (Fig. 1I±O), we found the
ventral domain labelled by the Tlx1 homeobox probe (in
the spinal cord recognising Tlx3) embedded within the
expression domain of Lbx1 (compare Fig. 1I,J). In addition,
the Lbx1 domain partially overlaps in its dorsal aspect with
the medial Lim1 stripe (Fig. 1K), which apparently is
congruent with the dorsalmost Pax2 stripe (Fig. 1L).
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F.R. Schubert et al. / Mechanisms of Development 101 (2001) 181±185
Ventrally, the Lbx1 domain exceeds the Tlx3 domain (Fig.
1J) and partially overlaps with the ventral Lim1 stripe (Fig.
1K, arrow) and the medial Pax2 stripe (Fig. 1L, arrow). At
all stages, Lbx1 is expressed ventral to the dorsal interneurons de®ned by LH2B (Fig. 1N), the dorsal Lim1 stripe (Fig.
1K), Isl1 (Fig. 1M) and the dorsal Tlx3 stripe (Fig. 1J). Thus,
Lbx1 labels a domain that yields the ventral, Tlx3-positive
interneurons as well as the bordering Lim1/Pax2- positive
interneurons (summarised in Fig. 2H).
To investigate whether the cells expressing Lbx1 are
indeed differentiated neurons, we simultaneously labelled
HH17-24 ¯at mounted hindbrains and whole embryos for
Lbx1 mRNA (Fig. 2A±E, blue staining) and intermediate
neuro®lament protein (Fig. 2A±E, brown staining). Within
the Lbx1 domain, cells were found double-labelled with the
anti-neuro®lament antibody. (Fig. 2B±E and data not
shown). At HH17, Lbx1 expression marks the dorsal
extreme of a zone rich in dorsoventrally orientated axons
(Fig. 2A). Furthermore, Lbx1 signals overlie the developing
lateral longitudinal fasciculus (Fig. 2A, arrowhead). Finally,
the Lbx1 staining is closely associated with the entry/exit
point of the HH17 cranial nerves in rhombomeres 2, 4, 6 and
7 (Fig. 2B±D and not shown), and the HH20 dorsal root
entry zones of the spinal cord (Fig. 2E). Neuro®lamentpositive cells within the Lbx1 domain appear to project
axons ventrally (Fig. 2B±D, arrows).
To investigate whether Lbx1 expressing neurons also
send axons into the underlying lateral longitudinal fasciculus, we labelled longitudinally projecting axons unilaterally
with rhodamine-labelled ®xable dextran (Fig. 2F), and
marked Lbx1 expressing cells by in situ hybridization.
Retrogradely labelled cell bodies were found within the
Lbx1 domain, in rhombomere 4 rostral to the labelled site
(Fig. 2G, arrowheads). Thus, the Lbx1 expression domain
contains ipsilaterally descending interneurons like the
lateral vestibulospinal tract group (Clarke and Lumsden,
1993; Auclair et al., 1999). The spatiotemporal expression
of Lbx1 is summarized in Fig. 2H.
2. Methods
The probe for chick Lbx1 has been described elsewhere
(Dietrich et al., 1998). The probe for Tlx1/3 was a similar
PCR fragment derived from the Tlx1 homeobox (Logan et
al., 1998). Delta-1 (Henrique et al., 1995) and Serrate-1
(Myat et al., 1996) were donated by J. Lewis, Lunatic fringe
(Laufer et al., 1997) was a gift from C. Tabin. The LH2B
(Nohno et al., 1997) probe we received from J.C. IzpisuaBelmonte, Isl1 and Lim1 (Tsuchida et al., 1994) were a gift
from T. Jessell, and Pax2 (Herbrand et al., 1998) was
donated by E. Bober. Neuro®lament protein was detected
with the RMO270 antibody (Zymed). In situ hybridization
was performed as described (Dietrich et al., 1998), and was
followed by standard immunohistochemistry to detect
neuro®lament protein, where applicable. Longitudinally
projecting neurons were labelled by cutting the hindbrain
unilaterally transversally, and applying rhodamine-labelled
®xable dextran (Molecular Probes).
Acknowledgements
We thank Eva Bober, Juan-Carlos Izpisua-Belmonte,
Tom Jessell, Julian Lewis, and Cliff Tabin for probes. We
are particularly grateful to Monica Ensini for helpful
suggestions on the dorsoventral organisation of LIM gene
expression patterns. Work was funded by the Wellcome
Trust and the MRC.
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