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Mechanisms of Development 101 (2001) 181±185 www.elsevier.com/locate/modo 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). 0925-4773/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(00)00537-2 182 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). 184 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). 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