- Email: [email protected]
Gbx2 expression in the late embryonic chick dorsal thalamus
Brain Research Bulletin, Vol. 57, Nos. 3/4, pp. 435– 438, 2002 Copyright © 2002 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/02/$–see front matter
PII S0361-9230(01)00721-3
Gbx2 expression in the late embryonic chick dorsal thalamus Margaret Martı´nez-de-la-Torre,1* Ana-Lila Garda,2 Eduardo Puelles1 and Luis Puelles1 1
Department of Morphological Sciences, University of Murcia, Murcia, Spain; and 2Instituto de Neurociencias, Faculty de Medicina, Campus de San Juan (UMH), Alicante, Spain
ABSTRACT: The expression pattern of the transcription factor gene Gbx2 in the forebrain of chicken embryos (embryonic day 14) was mapped using digoxigenin-labeled riboprobes and compared with the expression of the transcription factors Pax6 and Nkx2.2. The topographic analysis of Gbx2 expression on coronal and sagittal sections discriminated the positions and boundaries of diverse neuronal nuclei belonging to the dorsal thalamus from neighboring territories (the epithalamus, ventral thalamus, pretectum, and the underlying basal plate). The differential expression of Gbx2 within the dorsal thalamus clearly corresponds with the existence of four primary subdivisions identified in a previous study from this laboratory [13]: the anteroventral region and dorsal, intermediate, and ventral tiers. The subhabenular region turned out not to express Gbx2; this possibly implies it needs to be distinguished as a fifth separate dorsal thalamus subdivision. © 2002 Elsevier Science Inc.
formed, as recently described [2,8,13]. The DT of sauropsids includes, in addition, an anteroventral histogenetic domain that builds a perirotundic neuronal population comparable to the mammalian intergeniculate leaflet [6,11] and other minor DT formations (see below). MATERIALS AND METHODS Chicken eggs were incubated in a forced air incubator until E14 (or stage 40 [4]. The embryos were fixed in 4% paraformaldehydephosphate-buffered saline (PBS) at 4°C for 48 h and the brains were dissected free of meninges. Some of these late-embryonic brains were embedded in 4% agarose in PBS, and the blocks were sectioned in a vibratome at 100-m thickness, whereas other brains where cryoprotected and cut into frozen sections of 60-m thickness. In situ hybridization (ISH) was performed using digoxigenin-labeled anti-sense riboprobes for chicken Gbx2 (from A. Simeone), Pax6 or Nkx2.2 (from J.L.R. Rubenstein). The ISH proceeded as described either by [5] or [15].
KEY WORDS: Patterning, Forebrain development, Histogenesis, Diencephalon, Prosomeres.
INTRODUCTION The homeobox transcription factor Gbx2 is expressed very early during neural development, and it is essential for the establishment of the isthmic organizer that directs the regionalization of anterior hindbrain and posterior midbrain [7]. The early expression pattern of Gbx2 in chicken embryos is first associated with the posterior neural plate (stage HH4, [4]) and, shortly afterwards, with the anlagen of cerebellar and isthmic regions, at the mid-hindbrain transition zone [3,10,14]. At stages HH20-23 (E3– 4), additional Gbx2 expression appears selectively in the dorsal thalamus (DT) and in the basal telencephalon [10]. The function of Gbx2 is essential for the normal histogenesis of the DT, as has been shown by the Gbx2 knockout in the mouse [9]. This makes this gene a useful marker for a comparative analysis of the DT, because it would be expected to be needed as well in the formation of the DT (as a field) in other vertebrates. In this paper we have analyzed the expression of Gbx2 in the chicken dorsal thalamus at embryonic day 14 (E14; when structural development is finished). Our interest was to define the boundaries of the DT with respect to the epithalamus, ventral thalamus, pretectum, and underlying basal plate (DT is an alar domain [12]). Moreover, we wanted to see if the major primary histogenetic subdivisions of the DT, namely the dorsal, intermediate and ventral tiers, express the Gbx2 signal differentially. These subfields of the DT are the site where specific nuclei are
RESULTS AND DISCUSSION A schematic outline of the area of interest with the expected subdivisions is shown in Fig. 1A. A cross-section through the DT reveals the restriction of Nkx2.2 expression to migrated derivatives of the anteroventral region, forming a perirotundic complex (av; Fig. 1B; [6,11]). Figure 1C shows the topographic relationships of DT with the epithalamus or habenula, ventral thalamus and pretectum in such cross-sections (DT, VT, PT, Hab; Fig. 1C). Our results on Gbx2 are shown in coronal and sagittal section series (Figs. 2, 3). Neither the epithalamic habenular nuclei (Hab), nor the subhabenular nuclei (Sh) express Gbx2 (Figs. 2A,B,3C,E,F). This correlates basically with the description in the mouse [1], but adds the negative data on the subhabenular complex, widely thought to belong to the DT. If this assignation holds, the Gbx2-negative subhabenular region might be considered a separate primary subdivision of the DT. Alternatively, if Gbx2 expression is taken as the criterium for defining DT, the subhabenular region would need to be assigned to the epithalamus. Moreover, the dorsal Gbx2-negative area caudally extends laterally in a thin subpial population of very small neurones (Fig. 2B), recently identified as the superficial microcellular nucleus [13]. The superficial microcellular nucleus selectively expresses a strong Pax6 signal. This feature is shared with the habenular region (SMi, Hab
* Address for correspondence: Margaret Martı´nez-de-la-Torre, Department of Morphological Sciences, Faculty of Medicine, University of Murcia, Murcia, 30100, Spain. Fax: 34-968-363955; E-mail: [email protected]
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MARTI´NEZ-DE-LA-TORRE ET AL. in Fig. 1C and data not show), perhaps indicating a common histogenetic origin. Nuclei formed within all three DT dorsoventral tiers express Gbx2 with different levels of signal, possibly revealing differential regulation of its expression (d, i, v in Figs. 2A–G,3A–F). In general, the intermediate tier derivatives (i.e., the nucleus rotundus, the nucleus triangularis, and the nucleus dorsolateralis posterior) tend to show low expression, the dorsal tier nuclei show variable signal (stronger in the anterior dorsal nuclei) and an intense expression characterized the ventral tier derivatives (i.e., the nucleus ovoidalis). Among the postulated derivatives of the anteroventral area, nucleus subrotundus also strongly expresses Gbx2, while the perirotundic area (PRA) and the nucleus interstitialis tr.opticus (ITO) do not (these elements strongly express Nkx2.2, Fig. 1B). This difference raises the question whether the subrotundic nucleus may belong to the ventral tier instead to the anteroventral domain. The boundaries of DT with the ventral thalamus and pretectum are well respected by the Gbx2-positive domain (Figs. 2,3), though a few Gbx2-positive neurons seem to disperse along the exiting thalamotelencephalic fibers into the adjacent ventral thalamus (Figs. 2C,D). The boundary of the DT with the basal plate tegmentum placed underneath it lies along an obliquely inclined plane best appreciated in pure transverse sections (not shown). A topologically ventralmost Gbx2-positive nucleus, probably belonging to the ventral tier (according to its strong signal), appears in caudal cross-sections (Z in Figs. 2G–I,3C,D). It was not distinguished in previous studies on the avian DT and may be homologous to the reptilian nucleus Z, as suggested by its interstitial topography relative to the toro-ovoidal tract (tovt; Fig. 3C), and its position close to interstitial nuclei of the tectothalamic tract -the posterior nucleus of the ventral supraoptic commissure and the posterointermediate nucleus (nALp, see [1]). The latter two nuclei do not express Gbx2 at all, and may be understood as derivatives of the anteroventral complex, because they express Nkx2.2, as other anteroventral derivatives do (not shown). ACKNOWLEDGEMENTS
The present study was supported by a DGICYT grant PB98-0397. A-L.G. was supported by DGI 1FD 1997-2090.
REFERENCES
FIG. 1. Delimitation of the avian dorsal thalamus and its subdivisions. (A) Diagram of the avian diencephalon in a lateral view (rostral oriented to the left), according to the prosomeric model. It shows the topography of the dorsal thalamus (DT) as a component of the alar plate, wedged between the ventral thalamus (VT) and the pretectum (PT), and covered by the epithalamus, represented by the habenular and subhabenular regions (Hab, Sh). Inside the DT, four histogenetic fields are postulated, represented by the anteroventral area or region, adjacent to the zona limitans intrathalamica (p2/p3 interprosomeric boundary), and dorsal, intermediate and ventral DT tiers. Note that conventional coronal sections through the DT will cut first the dorsal tier elements and last the ventral tier elements, always in the presence of anteroventral and epithalamic derivatives. This schema can best be compared with sagittal sections (see Fig. 3). (B) Coronal section reacted with Nkx2.2 n situ hybridization (ISH), illustrating the peritotundic arrangement of the migrated derivatives of the anteroventral area. The external and internal boundaries of the DT are indicated with continuous and dash lines, respectively. (C) Lower magnification of coronal section through the DT. This section shows a faint ISH reaction for Nkx2.2 in
1. Bulfone, A. P.; Puelles, L.; Porteus, M. H.; Frohman, M. A.; Martin, G. R.; Rubenstein, J. R. L. Spatially restricted expression of Dlx-1, Dlx-2 (Tes-1), Gbx-2 and Wnt-3 in the embryonic day 12.5 mouse forebrain defines potential transverse and longitudinal segmental boundaries. J. Neurosci. 13:3155–3172; 1993. 2. Davila, J. G.; Guirado, S.; Puelles, L. Expression of calcium-binding proteins in the diencephalon of the lizard Psammodromus algirus. J. Comp. Neurol. 427:67–92; 2000. 3. Garda, A. L.; Echevarria, D.; Martinez, S. Neuroepithelial co-expression of Gbx2 and Otx2 precedes Fgf8 expression in the isthmic organizer. Mech. Dev. 101:111–118; 2001. 4. Hamburguer, H.; Hamilton, H. L. A series of normal stages in the development of the chick embryo. J. Morphol. 88:49 –92; 1951. 5. Henrique, D.; Adam, J.; Myat, A.; Chitnis, A.; Lewis, J.; Ish-Horowicz, D. Expression of a Delta homologue in prospective neurons in the chick. Nature 375:787–790; 1995.
the perirotundic anteroventral derivatives (ITO/pRA) and a strong ISH reaction for Pax6 in the ventrolateral habenula (Hab) and the superficial microcellular nucleus (SMi). Pax6 also appears expressed in the ventral thalamus (VT).
GBX2 GENE IN CHICK DORSAL THALAMUS
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FIG. 2. Series of coronal sections at embryonic day 14, showing rostrodorsal to caudoventral sections (A–I) across the chicken DT, reacted by ISH for Gbx2. While all the DT expresses the marker, there are distinct differences in the level of signal detected in the different nuclei and tiers. The internal boundaries are marked with dashed white lines. Note a strong signal in the nucleus ovoidalis and neighboring ventral tier grisea in (D)–(G), a rather low signal in nucleus rotundus (but higher in its anteroventral subregion) and other intermediate tier formations in (C)–(G), and strong to moderate signal in dorsal tier components: the anterior dorsal nuclei placed close to the zona limitans boundary with the VT show strong signal—(A,B)—whereas the posterior dorsal nuclei close to the pretectum show a lower signal—(C,D,E). (D–F) The nucleus subrotundus is strongly positive for Gbx2, but the periotundic formation is wholly negative (compare Fig. 1B). The habenular, subhabenular, and superficial microcellular nuclei are negative (A–D), as are likewise the posteroventral, posterointermediate, and posterior supraoptic commissure nuclei [PV, nALp, and PCSV, respectively, in (G–I)]. In contrast, the newly identified avian nucleus Z expresses strongly Gbx2 (G–I). See List of Abbreviations.
6. Martı´nez, S.; Alvarado-Mallart, R. M.; Martı´nez-de-la-Torre, M.; Puelles, L. Retinal and tectal connections of embryonic nucleus superficialis magnocellularis and its mature derivatives in the chick. Anat. Embryol. 183:235–243; 1991. 7. Martinez, S. The isthmic organizer and brain regionalization. Int. J. Dev. Biol. 45:367–371; 2001. 8. Min-Suk, Y. P.; Puelles, L.; Redies, C. Formation of cadherin-expressing brain nuclei in diencephalic alar plate divisions. J. Comp. Neurol. 421:461– 480; 2000. 9. Miyashita-Lin, E. H.; Wassarman, K. M.; Martinez, S.; Rubenstein, J. L. R. Early neocortical regionalization in the absence of thalamic innervation. Science 285:906 –909; 1999. 10. Niss, K. L. A. Expression of the homeobox gene GBX2 during chicken development. Mech. Dev. 76:151–155; 1998. 11. Puelles, L.; Guille´ n, M.; Martı´nez-de-la-Torre, M. Observations on the fate of nucleus superficialis magnocellularis of Rendahl in the avian diencephalon, bearing on the organization and nomenclature of neighbouring retinorecipient nuclei. Anat. Embryol. 183:221–233; 1991.
12. Puelles, L.; Rubenstein, J. L. R. Expression patterns of homeobox and other putative regulatory genes in the embryonic mouse forebrain suggest a neuromeric organization. Trends Neurosci. 16:472– 479; 1993. 13. Redies, C.; Ast, M.; Nakagawa, S.; Takeichi, M.; Martinez-de-laTorre, M.; Puelles, L. Morphologic fate of diencephalic prosomeres and their subdivisions revealed by mapping cadherin expression. J. Comp. Neurol. 421:481–514; 2000. 14. Shamim, H. I. M. Expression of Gbx-2 during early development of the chick embryo. Mech. Dev. 76:157–159; 1998. 15. Shimamura, K.; Rubenstein, J. L. R. Inductive interactions direct early regionalization of the mouse forebrain. Development 124:2709 –2718; 1994.
LIST OF ABBREVIATIONS av, anteroventral area d, dorsal tier DT, dorsal thalamus
MARTI´NEZ-DE-LA-TORRE ET AL.
438
FIG. 3. Series of sagittal sections at embryonic day 14, showing lateral to medial sections (A–G) across the chicken DT, reacted by in situ hybridization for Gbx2 (weak neutral red counterstain). The expression domain on the whole respects the interprosomeric boundaries with the ventral thalamus (VT) and pretectum (PT) (compare Fig. 1A). The DT tiers are identified (d, i, v); note the most intense expression of the marker in the ventral tier (v), the lowest signal in the intermediate tier (i) and a variable signal from rostral to caudal in the dorsal tier (d). The nucleus subrotundus is strongly positive [SR in (B–F)]. Note also the negative habenular and subhabenular regions [Hab/Sh in (C–F)] and the positive nucleus Z at the topologically ventralmost part of the DT [Z in (C,D)]. See List of Abbreviations.
Hab, i, ITO, nALp, Ov, PCSV, pRA, PT, PV,
habenula intermediate tier nucleus interstitialis tr.opticus nucleus ansa lenticularis posterior (or nucleus posterointermedius) nucleus ovoidalis posterior nucleus of the ventral supraoptic commissure perirotundic area pretectum posteroventral nucleus
R, SP, Sh, SMi, SR, Tov, v, VT, Z, zl,
nucleus rotundus nucleus subpretectalis subhabenular nuclei superficial microcellular nucleus subrotundic nucleus toro-ovoidal tract ventral tier ventral thalamus nucleus Z zona limitans intrathalamica
PII S0361-9230(01)00721-3
Gbx2 expression in the late embryonic chick dorsal thalamus Margaret Martı´nez-de-la-Torre,1* Ana-Lila Garda,2 Eduardo Puelles1 and Luis Puelles1 1
Department of Morphological Sciences, University of Murcia, Murcia, Spain; and 2Instituto de Neurociencias, Faculty de Medicina, Campus de San Juan (UMH), Alicante, Spain
ABSTRACT: The expression pattern of the transcription factor gene Gbx2 in the forebrain of chicken embryos (embryonic day 14) was mapped using digoxigenin-labeled riboprobes and compared with the expression of the transcription factors Pax6 and Nkx2.2. The topographic analysis of Gbx2 expression on coronal and sagittal sections discriminated the positions and boundaries of diverse neuronal nuclei belonging to the dorsal thalamus from neighboring territories (the epithalamus, ventral thalamus, pretectum, and the underlying basal plate). The differential expression of Gbx2 within the dorsal thalamus clearly corresponds with the existence of four primary subdivisions identified in a previous study from this laboratory [13]: the anteroventral region and dorsal, intermediate, and ventral tiers. The subhabenular region turned out not to express Gbx2; this possibly implies it needs to be distinguished as a fifth separate dorsal thalamus subdivision. © 2002 Elsevier Science Inc.
formed, as recently described [2,8,13]. The DT of sauropsids includes, in addition, an anteroventral histogenetic domain that builds a perirotundic neuronal population comparable to the mammalian intergeniculate leaflet [6,11] and other minor DT formations (see below). MATERIALS AND METHODS Chicken eggs were incubated in a forced air incubator until E14 (or stage 40 [4]. The embryos were fixed in 4% paraformaldehydephosphate-buffered saline (PBS) at 4°C for 48 h and the brains were dissected free of meninges. Some of these late-embryonic brains were embedded in 4% agarose in PBS, and the blocks were sectioned in a vibratome at 100-m thickness, whereas other brains where cryoprotected and cut into frozen sections of 60-m thickness. In situ hybridization (ISH) was performed using digoxigenin-labeled anti-sense riboprobes for chicken Gbx2 (from A. Simeone), Pax6 or Nkx2.2 (from J.L.R. Rubenstein). The ISH proceeded as described either by [5] or [15].
KEY WORDS: Patterning, Forebrain development, Histogenesis, Diencephalon, Prosomeres.
INTRODUCTION The homeobox transcription factor Gbx2 is expressed very early during neural development, and it is essential for the establishment of the isthmic organizer that directs the regionalization of anterior hindbrain and posterior midbrain [7]. The early expression pattern of Gbx2 in chicken embryos is first associated with the posterior neural plate (stage HH4, [4]) and, shortly afterwards, with the anlagen of cerebellar and isthmic regions, at the mid-hindbrain transition zone [3,10,14]. At stages HH20-23 (E3– 4), additional Gbx2 expression appears selectively in the dorsal thalamus (DT) and in the basal telencephalon [10]. The function of Gbx2 is essential for the normal histogenesis of the DT, as has been shown by the Gbx2 knockout in the mouse [9]. This makes this gene a useful marker for a comparative analysis of the DT, because it would be expected to be needed as well in the formation of the DT (as a field) in other vertebrates. In this paper we have analyzed the expression of Gbx2 in the chicken dorsal thalamus at embryonic day 14 (E14; when structural development is finished). Our interest was to define the boundaries of the DT with respect to the epithalamus, ventral thalamus, pretectum, and underlying basal plate (DT is an alar domain [12]). Moreover, we wanted to see if the major primary histogenetic subdivisions of the DT, namely the dorsal, intermediate and ventral tiers, express the Gbx2 signal differentially. These subfields of the DT are the site where specific nuclei are
RESULTS AND DISCUSSION A schematic outline of the area of interest with the expected subdivisions is shown in Fig. 1A. A cross-section through the DT reveals the restriction of Nkx2.2 expression to migrated derivatives of the anteroventral region, forming a perirotundic complex (av; Fig. 1B; [6,11]). Figure 1C shows the topographic relationships of DT with the epithalamus or habenula, ventral thalamus and pretectum in such cross-sections (DT, VT, PT, Hab; Fig. 1C). Our results on Gbx2 are shown in coronal and sagittal section series (Figs. 2, 3). Neither the epithalamic habenular nuclei (Hab), nor the subhabenular nuclei (Sh) express Gbx2 (Figs. 2A,B,3C,E,F). This correlates basically with the description in the mouse [1], but adds the negative data on the subhabenular complex, widely thought to belong to the DT. If this assignation holds, the Gbx2-negative subhabenular region might be considered a separate primary subdivision of the DT. Alternatively, if Gbx2 expression is taken as the criterium for defining DT, the subhabenular region would need to be assigned to the epithalamus. Moreover, the dorsal Gbx2-negative area caudally extends laterally in a thin subpial population of very small neurones (Fig. 2B), recently identified as the superficial microcellular nucleus [13]. The superficial microcellular nucleus selectively expresses a strong Pax6 signal. This feature is shared with the habenular region (SMi, Hab
* Address for correspondence: Margaret Martı´nez-de-la-Torre, Department of Morphological Sciences, Faculty of Medicine, University of Murcia, Murcia, 30100, Spain. Fax: 34-968-363955; E-mail: [email protected]
435
436
MARTI´NEZ-DE-LA-TORRE ET AL. in Fig. 1C and data not show), perhaps indicating a common histogenetic origin. Nuclei formed within all three DT dorsoventral tiers express Gbx2 with different levels of signal, possibly revealing differential regulation of its expression (d, i, v in Figs. 2A–G,3A–F). In general, the intermediate tier derivatives (i.e., the nucleus rotundus, the nucleus triangularis, and the nucleus dorsolateralis posterior) tend to show low expression, the dorsal tier nuclei show variable signal (stronger in the anterior dorsal nuclei) and an intense expression characterized the ventral tier derivatives (i.e., the nucleus ovoidalis). Among the postulated derivatives of the anteroventral area, nucleus subrotundus also strongly expresses Gbx2, while the perirotundic area (PRA) and the nucleus interstitialis tr.opticus (ITO) do not (these elements strongly express Nkx2.2, Fig. 1B). This difference raises the question whether the subrotundic nucleus may belong to the ventral tier instead to the anteroventral domain. The boundaries of DT with the ventral thalamus and pretectum are well respected by the Gbx2-positive domain (Figs. 2,3), though a few Gbx2-positive neurons seem to disperse along the exiting thalamotelencephalic fibers into the adjacent ventral thalamus (Figs. 2C,D). The boundary of the DT with the basal plate tegmentum placed underneath it lies along an obliquely inclined plane best appreciated in pure transverse sections (not shown). A topologically ventralmost Gbx2-positive nucleus, probably belonging to the ventral tier (according to its strong signal), appears in caudal cross-sections (Z in Figs. 2G–I,3C,D). It was not distinguished in previous studies on the avian DT and may be homologous to the reptilian nucleus Z, as suggested by its interstitial topography relative to the toro-ovoidal tract (tovt; Fig. 3C), and its position close to interstitial nuclei of the tectothalamic tract -the posterior nucleus of the ventral supraoptic commissure and the posterointermediate nucleus (nALp, see [1]). The latter two nuclei do not express Gbx2 at all, and may be understood as derivatives of the anteroventral complex, because they express Nkx2.2, as other anteroventral derivatives do (not shown). ACKNOWLEDGEMENTS
The present study was supported by a DGICYT grant PB98-0397. A-L.G. was supported by DGI 1FD 1997-2090.
REFERENCES
FIG. 1. Delimitation of the avian dorsal thalamus and its subdivisions. (A) Diagram of the avian diencephalon in a lateral view (rostral oriented to the left), according to the prosomeric model. It shows the topography of the dorsal thalamus (DT) as a component of the alar plate, wedged between the ventral thalamus (VT) and the pretectum (PT), and covered by the epithalamus, represented by the habenular and subhabenular regions (Hab, Sh). Inside the DT, four histogenetic fields are postulated, represented by the anteroventral area or region, adjacent to the zona limitans intrathalamica (p2/p3 interprosomeric boundary), and dorsal, intermediate and ventral DT tiers. Note that conventional coronal sections through the DT will cut first the dorsal tier elements and last the ventral tier elements, always in the presence of anteroventral and epithalamic derivatives. This schema can best be compared with sagittal sections (see Fig. 3). (B) Coronal section reacted with Nkx2.2 n situ hybridization (ISH), illustrating the peritotundic arrangement of the migrated derivatives of the anteroventral area. The external and internal boundaries of the DT are indicated with continuous and dash lines, respectively. (C) Lower magnification of coronal section through the DT. This section shows a faint ISH reaction for Nkx2.2 in
1. Bulfone, A. P.; Puelles, L.; Porteus, M. H.; Frohman, M. A.; Martin, G. R.; Rubenstein, J. R. L. Spatially restricted expression of Dlx-1, Dlx-2 (Tes-1), Gbx-2 and Wnt-3 in the embryonic day 12.5 mouse forebrain defines potential transverse and longitudinal segmental boundaries. J. Neurosci. 13:3155–3172; 1993. 2. Davila, J. G.; Guirado, S.; Puelles, L. Expression of calcium-binding proteins in the diencephalon of the lizard Psammodromus algirus. J. Comp. Neurol. 427:67–92; 2000. 3. Garda, A. L.; Echevarria, D.; Martinez, S. Neuroepithelial co-expression of Gbx2 and Otx2 precedes Fgf8 expression in the isthmic organizer. Mech. Dev. 101:111–118; 2001. 4. Hamburguer, H.; Hamilton, H. L. A series of normal stages in the development of the chick embryo. J. Morphol. 88:49 –92; 1951. 5. Henrique, D.; Adam, J.; Myat, A.; Chitnis, A.; Lewis, J.; Ish-Horowicz, D. Expression of a Delta homologue in prospective neurons in the chick. Nature 375:787–790; 1995.
the perirotundic anteroventral derivatives (ITO/pRA) and a strong ISH reaction for Pax6 in the ventrolateral habenula (Hab) and the superficial microcellular nucleus (SMi). Pax6 also appears expressed in the ventral thalamus (VT).
GBX2 GENE IN CHICK DORSAL THALAMUS
437
FIG. 2. Series of coronal sections at embryonic day 14, showing rostrodorsal to caudoventral sections (A–I) across the chicken DT, reacted by ISH for Gbx2. While all the DT expresses the marker, there are distinct differences in the level of signal detected in the different nuclei and tiers. The internal boundaries are marked with dashed white lines. Note a strong signal in the nucleus ovoidalis and neighboring ventral tier grisea in (D)–(G), a rather low signal in nucleus rotundus (but higher in its anteroventral subregion) and other intermediate tier formations in (C)–(G), and strong to moderate signal in dorsal tier components: the anterior dorsal nuclei placed close to the zona limitans boundary with the VT show strong signal—(A,B)—whereas the posterior dorsal nuclei close to the pretectum show a lower signal—(C,D,E). (D–F) The nucleus subrotundus is strongly positive for Gbx2, but the periotundic formation is wholly negative (compare Fig. 1B). The habenular, subhabenular, and superficial microcellular nuclei are negative (A–D), as are likewise the posteroventral, posterointermediate, and posterior supraoptic commissure nuclei [PV, nALp, and PCSV, respectively, in (G–I)]. In contrast, the newly identified avian nucleus Z expresses strongly Gbx2 (G–I). See List of Abbreviations.
6. Martı´nez, S.; Alvarado-Mallart, R. M.; Martı´nez-de-la-Torre, M.; Puelles, L. Retinal and tectal connections of embryonic nucleus superficialis magnocellularis and its mature derivatives in the chick. Anat. Embryol. 183:235–243; 1991. 7. Martinez, S. The isthmic organizer and brain regionalization. Int. J. Dev. Biol. 45:367–371; 2001. 8. Min-Suk, Y. P.; Puelles, L.; Redies, C. Formation of cadherin-expressing brain nuclei in diencephalic alar plate divisions. J. Comp. Neurol. 421:461– 480; 2000. 9. Miyashita-Lin, E. H.; Wassarman, K. M.; Martinez, S.; Rubenstein, J. L. R. Early neocortical regionalization in the absence of thalamic innervation. Science 285:906 –909; 1999. 10. Niss, K. L. A. Expression of the homeobox gene GBX2 during chicken development. Mech. Dev. 76:151–155; 1998. 11. Puelles, L.; Guille´ n, M.; Martı´nez-de-la-Torre, M. Observations on the fate of nucleus superficialis magnocellularis of Rendahl in the avian diencephalon, bearing on the organization and nomenclature of neighbouring retinorecipient nuclei. Anat. Embryol. 183:221–233; 1991.
12. Puelles, L.; Rubenstein, J. L. R. Expression patterns of homeobox and other putative regulatory genes in the embryonic mouse forebrain suggest a neuromeric organization. Trends Neurosci. 16:472– 479; 1993. 13. Redies, C.; Ast, M.; Nakagawa, S.; Takeichi, M.; Martinez-de-laTorre, M.; Puelles, L. Morphologic fate of diencephalic prosomeres and their subdivisions revealed by mapping cadherin expression. J. Comp. Neurol. 421:481–514; 2000. 14. Shamim, H. I. M. Expression of Gbx-2 during early development of the chick embryo. Mech. Dev. 76:157–159; 1998. 15. Shimamura, K.; Rubenstein, J. L. R. Inductive interactions direct early regionalization of the mouse forebrain. Development 124:2709 –2718; 1994.
LIST OF ABBREVIATIONS av, anteroventral area d, dorsal tier DT, dorsal thalamus
MARTI´NEZ-DE-LA-TORRE ET AL.
438
FIG. 3. Series of sagittal sections at embryonic day 14, showing lateral to medial sections (A–G) across the chicken DT, reacted by in situ hybridization for Gbx2 (weak neutral red counterstain). The expression domain on the whole respects the interprosomeric boundaries with the ventral thalamus (VT) and pretectum (PT) (compare Fig. 1A). The DT tiers are identified (d, i, v); note the most intense expression of the marker in the ventral tier (v), the lowest signal in the intermediate tier (i) and a variable signal from rostral to caudal in the dorsal tier (d). The nucleus subrotundus is strongly positive [SR in (B–F)]. Note also the negative habenular and subhabenular regions [Hab/Sh in (C–F)] and the positive nucleus Z at the topologically ventralmost part of the DT [Z in (C,D)]. See List of Abbreviations.
Hab, i, ITO, nALp, Ov, PCSV, pRA, PT, PV,
habenula intermediate tier nucleus interstitialis tr.opticus nucleus ansa lenticularis posterior (or nucleus posterointermedius) nucleus ovoidalis posterior nucleus of the ventral supraoptic commissure perirotundic area pretectum posteroventral nucleus
R, SP, Sh, SMi, SR, Tov, v, VT, Z, zl,
nucleus rotundus nucleus subpretectalis subhabenular nuclei superficial microcellular nucleus subrotundic nucleus toro-ovoidal tract ventral tier ventral thalamus nucleus Z zona limitans intrathalamica