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Organization of the mouse dorsal thalamus based on topology, calretinin immnunostaining, and gene expression
Brain Research Bulletin, Vol. 57, Nos. 3/4, pp. 439 – 442, 2002 Copyright © 2002 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/02/$–see front matter
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Organization of the mouse dorsal thalamus based on topology, calretinin immnunostaining, and gene expression Gertrudis Gonza´lez, Luis Puelles and Loreta Medina* Department of Morphological Sciences, Faculty of Medicine, University of Murcia, Murcia, Spain ABSTRACT: To better understand the organization and evolution of the dorsal thalamus, we have made a first approach to analyze the possible histogenetic compartments of the mammalian dorsal thalamus using mouse embryos. For that, we have analyzed the expression of the proneural gene Math4a and the protein calretinin. Our results suggest the existence of rostrodorsal, caudoventral, and ventral compartments in the embryonic dorsal thalamus of the mouse, which partly parallel the dorsoventral histogenetic tiers postulated in the dorsal thalamus of sauropsids [8,27]. The rostrodorsal compartment of the mouse dorsal thalamus is characterized by expression of Math4a, and it appears to include sensory and motor thalamic nuclei projecting to the dorsal pallium (isocortex). This compartment appears equivalent to the lemnothalamus proposed by Butler [6] in tetrapods based on hodological grounds. The caudoventral and ventral compartments of the mouse dorsal thalamus lack expression of Math4a in the mantle, but they are characterized by several populations of calretinin-immunorective neurons that show projections to the claustroamygdaloid region in the ventrolateral pallium. More studies will be needed to analyze if the compartments proposed in this study represent true histogenetic units, and to find homologous developmental fields in all vertebrates. © 2002 Elsevier Science Inc.
location, input from the retina, and output to a primary visual area located in the dorsal pallium in all amniotes studied [3,19]. However, other proposed homologies remain a matter of debate. This is the case of the tecto-recipient nucleus rotundus of the dorsal thalamus of birds and reptiles (sauropsids), which projects to a ventrolateral pallial territory within the dorsal ventricular ridge or DVR [1,2,11,13,14,23]. This nucleus of sauropsids has been proposed to be homologous to the mammalian tecto-recipient pulvinar/lateral posterior thalamic complex that projects to the dorsal pallium [14,15]. Some authors, however, argue against this homology because of the different topological position and the different input and outputs of these sauropsidian and mammalian thalamic centers [8,12,25]. One of the reasons for this controversy is the complexity that the dorsal thalamus has separately reached in mammals and birds, which makes it difficult to understand the true topological relations of their multiple cell groups, and to compare them to the simpler thalamic scheme present in modern reptiles or in amphibians. Recently, it has been suggested that tetrapods may share a number of primary hodological or histogenetic divisions in the dorsal thalamus. The proposed hodological divisions of the tetrapod dorsal thalamus are the collothalamus and lemnothalamus [6]. On the other hand, other authors have suggested that the dorsal thalamus of birds and reptiles shows four different histogenetic divisions: an anteroventral area plus dorsal, intermediate, and ventral tiers [8,27]. It is unknown whether these histogenetic units are present in mammals. As a first approach to analyze this, we have studied the possible compartmental organization of the mammalian developing dorsal thalamus using mouse embryos as a model, by way of topological criteria, calretinin immunostaining, and some gene expression data.
KEY WORDS: Histogenetic compartments, Lemnothalamus, Collothalamus, Math4a, Claustroamygdaloid region, Ventrolateral pallium.
INTRODUCTION In all amniotes (mammals, birds, and reptiles), the dorsal thalamus contains specific centers that project to the telencephalic pallium, providing it with information needed to carry out a large variety of functions, from perception and motor control to learning and memory. This explains why the increase in size and complexity of pallial territories during evolution has always been accompanied by an increase of the corresponding dorsal thalamic centers. To understand the evolution of the pallium, neurobiologists have tried to compare and homologize these thalamic cell groups among amniotes during the last century [3,4,6,7,19,32]. This task has been complicated by the presence of different numbers of dorsal thalamic nuclei in different species. These studies, nevertheless, have shown the existence of some comparable thalamic centers and circuits to the pallium in mammals, birds, and some reptiles, such as the dorsal lateral geniculate nucleus, which shows a similar
MATERIAL AND METHODS Mouse embryos of 14.5 days post-coitum were used for the present study. They were fixed in 4% paraformaldehyde, and the brains were processed for in situ hybridization following a procedure previously described [28]. The brains were sectioned on a vibratome at 150 m thick in either the frontal or sagittal planes. Some series of sections were hybridized using digoxigenin-labeled riboprobes to detect the expression of the proneural gene Math4a [10,31] or the homeotic gene Dlx-5 [5,9]. Parallel sections and some of the hybridized sections were immunostained for calreti-
* Address for correspondence: Dr. Loreta Medina, Departamento de Ciencias Morfolo´gicas, Facultad de Medicina, Universidad de Murcia, 30100 Murcia, Spain. Fax: 34-968 363955; E-mail: [email protected]
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FIG. 1. (A), (B) Sagittal section throughout the thalamus of an E14.5 mouse embryo hybridized for either Dlx-5 (A) or Math4a (B) (dark blue), and immunostained for calretinin (brown). (C), (D) Frontal sections throughout the thalamus of the mouse embryo at an intermediate (C) or caudal (D) level, hybridized for Math4a (dark blue) and immunostained for calretinin (brown). Abbreviations: A, anterior nuclear complex; BG, basal ganglia; Cx, cerebral cortex; cv, caudoventral compartment; DLG, dorsal lateral geniculate nucleus; DT, dorsal thalamus; ET, epithalamus; fr, fasciculus retroflexus; LP, lateral posterior nucleus; MG, medial geniculate nucleus; MI, midline and intralaminar nuclei; PT, pretectum; rd, rostrodorsal compartment; S, septum; SC, superior colliculus; sm, stria medullaris; SPF, subparafascicular nucleus; v, ventral compartment; VB, ventrobasal nuclear complex; VLG, ventral lateral geniculate nucleus; VT, ventral thalamus. Scale ⫽ 0.2 mm.
nin, using a rabbit anti-calretinin antiserum (1:2000; Swant, Bellinzona, Switzerland). RESULTS AND DISCUSSION As seen in sagittal sections, the thalamus of the mouse embryo can be perfectly delineated by the presence of two fiber tracts (see Fig. 1): (a) the stria medullaris, whose fibers are immunolabeled for calretinin and run rostrodorsally over the thalamus ending in the habenular region; and (b) the fasciculus retroflexus, mostly negative for calretinin, which runs dorsoventrally from the habenular region into the tegmentum just rostral to the interprosomeric p1–p2 boundary, separating the thalamus from the pretectum. The expression of the homeotic gene Dlx5 as well as calretinin immunostaining allows a clear distinction between the ventral and dorsal thalami (Fig. 1). The ventral thalamus is characterized by expression of Dlx5, and it lacks calretinin immunostaining (Figs. 1A, B). On the contrary, the dorsal thalamus lacks expression of Dlx5 but
contains several cell populations immunostained for calretinin (Fig. 1). The cell populations of the dorsal thalamus immunostained for calretinin give rise to immunoreactive projections that traverse the ventral thalamus in their way to the telencephalon (Figs. 1B, C). The distinction between the ventral and dorsal thalamus based on Dlx genes or calretinin immunostaining was previously observed by other authors [5,17,22]. In addition, our results on Math4a reveal a differential expression of this gene within the dorsal thalamus (Figs. 1B, C). Based on topology, calretinin immunostaining, and the expression of the proneural gene Math4a, the dorsal thalamus of the mouse embryo is apparently divided into the following three major domains, which we tentatively call compartments (Fig. 1): 1. A rostrodorsal compartment, characterized by the expression of Math4a (with a decreasing mediolateral gradient), which is medially (periventricularly) poor in calretinin but laterally contains cells and neuropil moderately stained for calretinin. This
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compartment appears to include the anterior nuclear complex and some specific sensory and motor centers, such as the dorsal lateral geniculate nucleus and the ventrobasal complex (DLG and VB in Fig. 1C). These cell groups are known to project to specific dorsal pallial (isocortical) territories [6,7,21]. Part of the projections from the dorsal geniculate nucleus to the dorsal pallium are calretinin-immunoreactive (Fig. 1C). 2. A caudoventral compartment, characterized by lack of Math4a expression (Fig. 1). This compartment shows a medial (periventricular) part rich in calretinin-immunostained cells and neuropil, and a lateral part with dispersed calretinin-immunostained cells (Fig. 1). A dorsal extension of this compartment appears to reach the habenular region and bounds on the origin of the fasciculus retroflexus. This compartment apparently includes the midline and intralaminar nuclei, which show calretininimmunostained projections to the pallium, and at least part of the posterior nuclear complex (Fig. 1B, C). These nuclei are known to receive tectal (superior collicular) input, and some of them show specific projections to the claustroamygdaloid region [21,30], which is included in the ventrolateral pallium of mammals [26]. 3. A smaller ventral compartment, adjacent to the alar-basal boundary, characterized by the presence of cells and neuropil densely immunostained for calretinin (v in Fig. 1). This compartment appears to include the subparafascicular nucleus and other nuclei of the posterior intralaminar group, as well as the medial geniculate nucleus. These nuclei receive inferior collicular input and project to the amygdala and the temporal part of the dorsal pallium [21].
to understand the components and evolution of this dorsal thalamic domain. Regarding the proposed caudoventral and ventral compartments of the mouse dorsal thalamus, more studies are needed to know if they represent true morphogenetic domains and are equivalent to any of the sauropsidian dorsoventral tiers. As noted above, the proposed caudoventral compartment apparently contains some nuclei that establish a link between the superior colliculus and the ventrolateral pallium. This is the case of the posterior nuclei, part of which receive input from intermediate/deep collicular layers [34], and it has been shown to project to the claustrum [30], in the ventrolateral pallium [26]. Based on these connections, the posterior nuclei, or part of them, have been proposed to be comparable to the nucleus rotundus of sauropsids [12,25]. For this to be true, both the posterior nuclei of mammals and the nucleus rotundus of sauropsids must be formed in the same morphogenetic domain of the dorsal thalamus. Unfortunately, this is unknown for the moment. In reptiles and birds, the dorsal thalamus has been divided into primary dorsoventral tiers based on topology and the distribution of calcium-binding proteins [8], or cadherins [27]. In the mouse postnatal thalamus, several cadherin subtypes show restricted expression patterns [33], but it is at present unknown what is the exact relation between these cadherins and the compartments defined by Math4a and calretinin in the mouse, and the tiers proposed in birds and reptiles. More studies will be needed for establishing homologous primary histogenetic divisions of the dorsal thalamus in all vertebrates, and for that purpose, expression patterns of transcription factors or other early regulatory genes will be helpful.
This tentative divisional scheme of the mouse thalamus partly parallels the postulated dorsal, intermediate, and ventral compartments of sauropsids, but it needs to be confirmed by further studies that show that the proposed divisions really represent true histogenetic domains. To analyze this, expression patterns of transcription factors or regulatory genes may be helpful. The fact that the rostrodorsal compartment expresses the proneural gene Math4a (also known as Neurogenin 2) is of interest. Neurogenins are mouse homologues of the Drosophila atonal gene, and they are involved in neurogenesis and in neuroblast determination [10,31]. This suggests that Math4a may play a role in the early specification and development of the cell groups in the rostrodorsal domain. Another domain expressing the homeotic gene Nkx-2.2 has been described in the developing dorsal thalamus in mouse, which is adjacent to both the alar-basal boundary and to the zona limitans intrathalamica (the boundary between the dorsal and ventral thalamus) [16,20,29]. Nkx-2.2 has been suggested to play a role in the specification of the ventral phenotypes within the dorsal thalamus [20]. Comparative data from our laboratory on Nkx-2.2 in the avian dorsal thalamus indicate that this gene is expressed in derivatives of the anteroventral histogenetic area [18,24]. This suggests that the Nkx-2.2 transcription factor may characterize the intergeniculate leaflet of mammals and may be expressed outside of the rostrodorsal, caudoventral, and ventral compartments recognized with Math4a and calretinin. The present results indicate that the nuclei in the Math4aexpressing domain appear to be sensory and motor cell groups that project extensively to the dorsal pallium [21], and this may be a general feature for the cell groups formed within this domain. Thus, this domain appears comparable to the concept of lemnothalamus proposed by Butler based on hodological grounds [6]. If this is so, and if Math4a is shown to play an important role in the specification of the rostrodorsal domain, studies on the expression of the homologue of this gene in other vertebrates would help
ACKNOWLEDGEMENT
Supported by the Seneca Foundation (PB/25/FS/99) and the Spanish Ministry of Science and Technology (MCYT, DGI BFI2000-1359-C0202).
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Organization of the mouse dorsal thalamus based on topology, calretinin immnunostaining, and gene expression Gertrudis Gonza´lez, Luis Puelles and Loreta Medina* Department of Morphological Sciences, Faculty of Medicine, University of Murcia, Murcia, Spain ABSTRACT: To better understand the organization and evolution of the dorsal thalamus, we have made a first approach to analyze the possible histogenetic compartments of the mammalian dorsal thalamus using mouse embryos. For that, we have analyzed the expression of the proneural gene Math4a and the protein calretinin. Our results suggest the existence of rostrodorsal, caudoventral, and ventral compartments in the embryonic dorsal thalamus of the mouse, which partly parallel the dorsoventral histogenetic tiers postulated in the dorsal thalamus of sauropsids [8,27]. The rostrodorsal compartment of the mouse dorsal thalamus is characterized by expression of Math4a, and it appears to include sensory and motor thalamic nuclei projecting to the dorsal pallium (isocortex). This compartment appears equivalent to the lemnothalamus proposed by Butler [6] in tetrapods based on hodological grounds. The caudoventral and ventral compartments of the mouse dorsal thalamus lack expression of Math4a in the mantle, but they are characterized by several populations of calretinin-immunorective neurons that show projections to the claustroamygdaloid region in the ventrolateral pallium. More studies will be needed to analyze if the compartments proposed in this study represent true histogenetic units, and to find homologous developmental fields in all vertebrates. © 2002 Elsevier Science Inc.
location, input from the retina, and output to a primary visual area located in the dorsal pallium in all amniotes studied [3,19]. However, other proposed homologies remain a matter of debate. This is the case of the tecto-recipient nucleus rotundus of the dorsal thalamus of birds and reptiles (sauropsids), which projects to a ventrolateral pallial territory within the dorsal ventricular ridge or DVR [1,2,11,13,14,23]. This nucleus of sauropsids has been proposed to be homologous to the mammalian tecto-recipient pulvinar/lateral posterior thalamic complex that projects to the dorsal pallium [14,15]. Some authors, however, argue against this homology because of the different topological position and the different input and outputs of these sauropsidian and mammalian thalamic centers [8,12,25]. One of the reasons for this controversy is the complexity that the dorsal thalamus has separately reached in mammals and birds, which makes it difficult to understand the true topological relations of their multiple cell groups, and to compare them to the simpler thalamic scheme present in modern reptiles or in amphibians. Recently, it has been suggested that tetrapods may share a number of primary hodological or histogenetic divisions in the dorsal thalamus. The proposed hodological divisions of the tetrapod dorsal thalamus are the collothalamus and lemnothalamus [6]. On the other hand, other authors have suggested that the dorsal thalamus of birds and reptiles shows four different histogenetic divisions: an anteroventral area plus dorsal, intermediate, and ventral tiers [8,27]. It is unknown whether these histogenetic units are present in mammals. As a first approach to analyze this, we have studied the possible compartmental organization of the mammalian developing dorsal thalamus using mouse embryos as a model, by way of topological criteria, calretinin immunostaining, and some gene expression data.
KEY WORDS: Histogenetic compartments, Lemnothalamus, Collothalamus, Math4a, Claustroamygdaloid region, Ventrolateral pallium.
INTRODUCTION In all amniotes (mammals, birds, and reptiles), the dorsal thalamus contains specific centers that project to the telencephalic pallium, providing it with information needed to carry out a large variety of functions, from perception and motor control to learning and memory. This explains why the increase in size and complexity of pallial territories during evolution has always been accompanied by an increase of the corresponding dorsal thalamic centers. To understand the evolution of the pallium, neurobiologists have tried to compare and homologize these thalamic cell groups among amniotes during the last century [3,4,6,7,19,32]. This task has been complicated by the presence of different numbers of dorsal thalamic nuclei in different species. These studies, nevertheless, have shown the existence of some comparable thalamic centers and circuits to the pallium in mammals, birds, and some reptiles, such as the dorsal lateral geniculate nucleus, which shows a similar
MATERIAL AND METHODS Mouse embryos of 14.5 days post-coitum were used for the present study. They were fixed in 4% paraformaldehyde, and the brains were processed for in situ hybridization following a procedure previously described [28]. The brains were sectioned on a vibratome at 150 m thick in either the frontal or sagittal planes. Some series of sections were hybridized using digoxigenin-labeled riboprobes to detect the expression of the proneural gene Math4a [10,31] or the homeotic gene Dlx-5 [5,9]. Parallel sections and some of the hybridized sections were immunostained for calreti-
* Address for correspondence: Dr. Loreta Medina, Departamento de Ciencias Morfolo´gicas, Facultad de Medicina, Universidad de Murcia, 30100 Murcia, Spain. Fax: 34-968 363955; E-mail: [email protected]
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FIG. 1. (A), (B) Sagittal section throughout the thalamus of an E14.5 mouse embryo hybridized for either Dlx-5 (A) or Math4a (B) (dark blue), and immunostained for calretinin (brown). (C), (D) Frontal sections throughout the thalamus of the mouse embryo at an intermediate (C) or caudal (D) level, hybridized for Math4a (dark blue) and immunostained for calretinin (brown). Abbreviations: A, anterior nuclear complex; BG, basal ganglia; Cx, cerebral cortex; cv, caudoventral compartment; DLG, dorsal lateral geniculate nucleus; DT, dorsal thalamus; ET, epithalamus; fr, fasciculus retroflexus; LP, lateral posterior nucleus; MG, medial geniculate nucleus; MI, midline and intralaminar nuclei; PT, pretectum; rd, rostrodorsal compartment; S, septum; SC, superior colliculus; sm, stria medullaris; SPF, subparafascicular nucleus; v, ventral compartment; VB, ventrobasal nuclear complex; VLG, ventral lateral geniculate nucleus; VT, ventral thalamus. Scale ⫽ 0.2 mm.
nin, using a rabbit anti-calretinin antiserum (1:2000; Swant, Bellinzona, Switzerland). RESULTS AND DISCUSSION As seen in sagittal sections, the thalamus of the mouse embryo can be perfectly delineated by the presence of two fiber tracts (see Fig. 1): (a) the stria medullaris, whose fibers are immunolabeled for calretinin and run rostrodorsally over the thalamus ending in the habenular region; and (b) the fasciculus retroflexus, mostly negative for calretinin, which runs dorsoventrally from the habenular region into the tegmentum just rostral to the interprosomeric p1–p2 boundary, separating the thalamus from the pretectum. The expression of the homeotic gene Dlx5 as well as calretinin immunostaining allows a clear distinction between the ventral and dorsal thalami (Fig. 1). The ventral thalamus is characterized by expression of Dlx5, and it lacks calretinin immunostaining (Figs. 1A, B). On the contrary, the dorsal thalamus lacks expression of Dlx5 but
contains several cell populations immunostained for calretinin (Fig. 1). The cell populations of the dorsal thalamus immunostained for calretinin give rise to immunoreactive projections that traverse the ventral thalamus in their way to the telencephalon (Figs. 1B, C). The distinction between the ventral and dorsal thalamus based on Dlx genes or calretinin immunostaining was previously observed by other authors [5,17,22]. In addition, our results on Math4a reveal a differential expression of this gene within the dorsal thalamus (Figs. 1B, C). Based on topology, calretinin immunostaining, and the expression of the proneural gene Math4a, the dorsal thalamus of the mouse embryo is apparently divided into the following three major domains, which we tentatively call compartments (Fig. 1): 1. A rostrodorsal compartment, characterized by the expression of Math4a (with a decreasing mediolateral gradient), which is medially (periventricularly) poor in calretinin but laterally contains cells and neuropil moderately stained for calretinin. This
ORGANIZATION OF THE MOUSE DORSAL THALAMUS
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compartment appears to include the anterior nuclear complex and some specific sensory and motor centers, such as the dorsal lateral geniculate nucleus and the ventrobasal complex (DLG and VB in Fig. 1C). These cell groups are known to project to specific dorsal pallial (isocortical) territories [6,7,21]. Part of the projections from the dorsal geniculate nucleus to the dorsal pallium are calretinin-immunoreactive (Fig. 1C). 2. A caudoventral compartment, characterized by lack of Math4a expression (Fig. 1). This compartment shows a medial (periventricular) part rich in calretinin-immunostained cells and neuropil, and a lateral part with dispersed calretinin-immunostained cells (Fig. 1). A dorsal extension of this compartment appears to reach the habenular region and bounds on the origin of the fasciculus retroflexus. This compartment apparently includes the midline and intralaminar nuclei, which show calretininimmunostained projections to the pallium, and at least part of the posterior nuclear complex (Fig. 1B, C). These nuclei are known to receive tectal (superior collicular) input, and some of them show specific projections to the claustroamygdaloid region [21,30], which is included in the ventrolateral pallium of mammals [26]. 3. A smaller ventral compartment, adjacent to the alar-basal boundary, characterized by the presence of cells and neuropil densely immunostained for calretinin (v in Fig. 1). This compartment appears to include the subparafascicular nucleus and other nuclei of the posterior intralaminar group, as well as the medial geniculate nucleus. These nuclei receive inferior collicular input and project to the amygdala and the temporal part of the dorsal pallium [21].
to understand the components and evolution of this dorsal thalamic domain. Regarding the proposed caudoventral and ventral compartments of the mouse dorsal thalamus, more studies are needed to know if they represent true morphogenetic domains and are equivalent to any of the sauropsidian dorsoventral tiers. As noted above, the proposed caudoventral compartment apparently contains some nuclei that establish a link between the superior colliculus and the ventrolateral pallium. This is the case of the posterior nuclei, part of which receive input from intermediate/deep collicular layers [34], and it has been shown to project to the claustrum [30], in the ventrolateral pallium [26]. Based on these connections, the posterior nuclei, or part of them, have been proposed to be comparable to the nucleus rotundus of sauropsids [12,25]. For this to be true, both the posterior nuclei of mammals and the nucleus rotundus of sauropsids must be formed in the same morphogenetic domain of the dorsal thalamus. Unfortunately, this is unknown for the moment. In reptiles and birds, the dorsal thalamus has been divided into primary dorsoventral tiers based on topology and the distribution of calcium-binding proteins [8], or cadherins [27]. In the mouse postnatal thalamus, several cadherin subtypes show restricted expression patterns [33], but it is at present unknown what is the exact relation between these cadherins and the compartments defined by Math4a and calretinin in the mouse, and the tiers proposed in birds and reptiles. More studies will be needed for establishing homologous primary histogenetic divisions of the dorsal thalamus in all vertebrates, and for that purpose, expression patterns of transcription factors or other early regulatory genes will be helpful.
This tentative divisional scheme of the mouse thalamus partly parallels the postulated dorsal, intermediate, and ventral compartments of sauropsids, but it needs to be confirmed by further studies that show that the proposed divisions really represent true histogenetic domains. To analyze this, expression patterns of transcription factors or regulatory genes may be helpful. The fact that the rostrodorsal compartment expresses the proneural gene Math4a (also known as Neurogenin 2) is of interest. Neurogenins are mouse homologues of the Drosophila atonal gene, and they are involved in neurogenesis and in neuroblast determination [10,31]. This suggests that Math4a may play a role in the early specification and development of the cell groups in the rostrodorsal domain. Another domain expressing the homeotic gene Nkx-2.2 has been described in the developing dorsal thalamus in mouse, which is adjacent to both the alar-basal boundary and to the zona limitans intrathalamica (the boundary between the dorsal and ventral thalamus) [16,20,29]. Nkx-2.2 has been suggested to play a role in the specification of the ventral phenotypes within the dorsal thalamus [20]. Comparative data from our laboratory on Nkx-2.2 in the avian dorsal thalamus indicate that this gene is expressed in derivatives of the anteroventral histogenetic area [18,24]. This suggests that the Nkx-2.2 transcription factor may characterize the intergeniculate leaflet of mammals and may be expressed outside of the rostrodorsal, caudoventral, and ventral compartments recognized with Math4a and calretinin. The present results indicate that the nuclei in the Math4aexpressing domain appear to be sensory and motor cell groups that project extensively to the dorsal pallium [21], and this may be a general feature for the cell groups formed within this domain. Thus, this domain appears comparable to the concept of lemnothalamus proposed by Butler based on hodological grounds [6]. If this is so, and if Math4a is shown to play an important role in the specification of the rostrodorsal domain, studies on the expression of the homologue of this gene in other vertebrates would help
ACKNOWLEDGEMENT
Supported by the Seneca Foundation (PB/25/FS/99) and the Spanish Ministry of Science and Technology (MCYT, DGI BFI2000-1359-C0202).
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