A Developmental Approach to Forebrain Organization in Elasmobranchs: New Perspectives on the Regionalization of the Telencephalon
Isabel Rodríguez-Moldes
Department of Cell Biology and Ecology, University of Santiago de Compostela, Santiago de Compostela , Spain
thyans, as this structure develops by evagination (as in most other vertebrates), whereas in most osteichthyans (bony fishes), it develops by eversion, a markedly different process. Among chondrichthyans, the Lesser Spotted Dogfish Scyli-orhinus canicula (Elasmobranchii) appears to offer the most potential as a model species for study. Developmental stud-ies of Scyliorhinus have revealed a segmentary pattern in the developing forebrain, similar to that described in other ver-tebrates, as well as the occurrence of tangential cell migra-tion within the telencephalon, especially in relation to the pallial-subpallial boundary. These observations indicate that major morphogenetic processes thought to be a hallmark of
It is essential to consider chondrichthyans (cartilaginous fishes) in analyzing ancestral brain organization because this radiation represents the out-group to all other living gna-thostomes (jawed vertebrates). It is particularly crucial to un-derstand the evolution of the telencephalon in chondrich-
Published online: September 2, 2009
Isabel Rodríguez-Moldes Departamento de Biología Celular y Ecología, Edificio CIBUS Universidad de Santiago de Compostela, ES–15782 Santiago de Compostela (Spain) Tel. +34 981 563 100, ext. 16950, Fax +34 981 528 006 E-Mail [email protected]
Forebrain studies in cartilaginous fishes are highly important in any analysis of vertebrate brain evolution. Cartilaginous fishes (Chondrichthyans) represent an an-cient gnathostome radiation, which includes sharks, skates and rays (Elasmobranchii) and chimaeras (Holo-cephala). Because of their key position as an out-group to all other living gnathostomes, they are essential in assess-ing the ancestral condition of brain organization. They are particularly crucial to studies of telencephalic evolu-tion, as the development of the telencephalon in chon-drichthyans is very similar to that in sarcopterygians (which include the lobe-finned fishes and tetrapods), but very different from that in most other fishes (i.e, the ac-tinopterygians or ray-finned fishes) that comprise the sis-ter group of sarcopterygians. In chondrichthyans, the tel-encephalon develops by a morphogenetic process known as evagination, which implies protrusion towards the outside and expansion of the lateral telencephalic walls, but in actinopterygians the lateral walls of the telenceph-alon turn out along their dorsal margins and then extend laterally by a process called eversion. It has been proposed that telencephalic eversion evolved as a consequence of intracranial space constraints which caused the telen-cephalon to be squeezed between the nasal epithelia [Striedter and Northcutt, 2006].
The study of the elasmobranch central nervous system has been almost sinfully neglected in history; however, in an important work, Smeets et al. [1983] described in de-tail the organization of the mature forebrain (and other brain regions) in representatives from four groups of chondrichthyans: batoids (a ray), holocephala (a chimae-ra), galeomorph sharks (the Lesser Spotted Dogfish, Scy-liorhinus canicula ) and squalomorh sharks (the Spiny Dogfish, Squalus acanthias ). The section on the telen-cephalon has also been revised [Smeets, 1990]. The work-shop where this paper was originally presented was held in honor of Professor Rudolf Nieuwenhuys [see Nieuwen-huys, 2009], and it would be compounding the sin not to acknowledge his tremendous contributions. One of Prof. Nieuwenhuys’ greatest works, the extraordinary three-
volume set on the central nervous system of vertebrates, also includes a chapter on the central nervous system in cartilaginous fishes [Smeets, 1998] that is essential to an understanding of central nervous system organization in chondrichthyans. Finally, R. Glenn Northcutt has also contributed significantly to studies of the forebrain in chondrichthyan fishes [Northcutt, 1978, 1989; Hofmann and Northcutt, 2008], particularly in showing the impor-tance of sampling brain variation among different spe-cies and recognizing adaptive patterns.
Experimental anatomical studies demonstrating the existence of non-olfactory connections in the telencepha-lon of elasmobranchs [Ebbesson and Schroeder, 1971; Schroeder and Ebbesson, 1974; Graeber et al., 1978; Lui-ten, 1981a, b; Smeets and Northcutt, 1987], and thoserevealing that only a restricted area of the telencephalon receives secondary olfactory fibers [Hofmann and Northcutt, 2008; see also Smeets, 1990, 1998 for reviews] have been especially important, as they refuted the classic conception that elasmobranchs have a simple, exclusively olfactory telencephalon. With the improvement of im-munohistochemical techniques, and the development of antibodies against diverse neuroactive substances, the complexity of the telencephalon in elasmobranchs has been increasingly revealed. The studies of Meredith and Smeets [1987] which focused on the forebrain and mid-brain in Raja, using immunomarkers for dopamine, and Northcutt and colleagues [1988] concerning the telen-cephalon in Squalus, using markers for monoamines and neuropeptides, have been particularly important, as they indicated homologies with important mammalian cen-ters, such as the basal ganglia. These and other studies have greatly contributed to our knowledge of telence-phalic organization in elasmobranchs.
The Neuromeric Model of the Forebrain in Fishes
Most literature on elasmobranch brains reflects the classic subdivision of the anterior brain (derived from the prosencephalon or forebrain) into two vesicles: the telen-cephalon and the diencephalon. The diencephalon is fur-ther subdivided from dorsal to ventral into four parts: epithalamus, dorsal thalamus, ventral thalamus, and hy-pothalamus. The telencephalon is subdivided into three parts: the telencephalic hemispheres, with a dorsal part, or pallium, and a ventral part, or subpallium; the olfac-tory bulbs; and the telencephalon impar, i.e., that part of the telencephalon with an undivided ventricle in the midline. In addition, there is the preoptic area, which is
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generally assigned to the diencephalon, although its tel-encephalic origin has been recognized.
In 1993, Puelles and Rubenstein reconsidered a seg-mentary subdivision of the brain according to transverse and longitudinal domains, and they proposed a segmen-tal model for the diencephalon (caudal prosencephalon) and the secondary prosencephalon, both subdivisions of the early or primary prosencephalon. According to this model, the diencephalon is subdivided into three proso-meres (p1–p3), which contain the pretectum (p1), the thalamus (dorsal thalamus, p2) and the prethalamus (ventral thalamus, p3); the secondary prosencephalon comprises the hypothalamus, the preoptic area, and the telencephalon. The model, first proposed in mammals, has also been applied to other amniotes and has been very useful in the search for homologies. In fishes, this model has been used in studies reinterpreting the organization of the forebrain in lampreys [Pombal and Puelles, 1999; Pombal et al., 2009], zebrafishes [Wullimann and Pu-elles, 1999; Mueller and Wullimann, 2009] and lungfish-es [González and Northcutt, 2009] on the basis of the distribution of some neural markers. These species rep-resent jawless vertebrates (lamprey) and bony fishes (ze-brafish and lungfish), respectively; they do not represent all groups of fishes. In cartilaginous fishes, studies of forebrain segmentation are very scarce, although neuro-meric segmentation of a shark brain was described as ear-ly as 1906 by Von Kupffer, who examined 10-mm em-bryos of Squalus acanthias and identified three proso-meric segments: one telencephalic segment and two diencephalic segments (parencephalon and synencepha-lon). More recently, segmentary organization of a shark forebrain was suggested [Ferreiro-Galve et al., 2008], based on stage 31 embryos of Scyliorhinus canicula . These studies are still too limited to provide an accurate de-scription of the segmentary organization of the forebrain in elasmobranchs.
The Lesser Spotted Dogfish, Scyliorhinus canicula , as
a Model Species
Much of our information on mature brain organiza-tion in elasmobranchs comes from morphological stud-ies of the Spiny Dogfish Squalus acanthias [Northcutt et al., 1988; Smeets, 1998]. In addition, there are classic de-velopmental studies of the brain in Squalus [Von Kupffer, 1906; Holmgren, 1922]. Members of this genus have great disadvantages for modern developmental studies, how-ever, because they are viviparous, intolerant of captivity
in aquaria, and have a long embryonic period (2 years) and a low reproduction rate (1–7 embryos per female).
In the last few years, most if not all studies of develop-ment in elasmobranches have been carried out on a small shark, the Lesser Spotted Dogfish, Scyliorhinus canicula [for a review, see Coolen et al., 2009]. There are several reasons why this species is a good candidate for a ‘model organism.’ Abundant embryonic material can be ob-tained throughout the year, and the eggs can be easily maintained in laboratory conditions until hatching. This, together with the transparency of the egg shells, makes it possible to obtain embryonic series. In fact, series of the normal development have been available since the work of Ballard et al. [1993].
Among chondrichthyans, studies of brain organiza-tion in galeomorph sharks (such as S. canicula ) are espe-cially critical because these sharks have elaborated brains [Northcutt, 1977], similar to those of teleosts and amni-otes, and are thus more likely to reveal homologies be-tween chondrichthyans and other vertebrates. In elabo-rated shark brains [Northcutt, 1977], the neuronal cell bodies migrate away from the periventricular matrix during development and form more or less distinct cell groups, whereas in laminated shark brains (i.e., those of squalomorph sharks, such as Squalus ), most neurons re-main periventricular, i.e., close to the place where they proliferated [Northcutt, 1977]. Northcutt’s designations of laminar and elaborated shark brains were reviewed and designated Type I and Type II by Butler and Hodos [2005].
The application of modern as well as classical tech-niques has allowed researchers to determine how the ma-ture forebrain of S. canicula (hereafter referred to as the Lesser Spotted Dogfish) is organized. Experimental stud-ies have provided significant information on telencephal-ic connections in chondrichthyans [Smeets, 1982, 1983], and the Golgi method has shown a variety of cell types in the telencephalon [Manso and Anadón, 1993]. In addi-tion, immunohistochemical techniques have provided a detailed picture of forebrain organization in the Lesser Spotted Dogfish, revealing neuronal systems that contain modulatory neuroactive substances, such as calbindin D-28k [Rodriguez-Moldes et al., 1990], FMRFamide [Val-larino et al., 1991; Chiba et al., 1991], neuropeptide Y [Chiba and Honma, 1992], substance P [Rodríguez-Mol-des et al., 1993], Met- and Leu-enkephalin [Vallarino et al., 1994], Thyrotropin-releasing hormone [Teijido et al., 2002] and choline acetyltransferase [Anadón et al., 2000].
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Finally, as an element most important for a model or-ganism, the entire genome of the Lesser Spotted Dogfish may shortly be sequenced and available, thanks to the ef-forts of the group led by Sylvie Mazan at the Institut of Transgenosis of Orléans (France). In fact, the only mod-ern and molecular information we have on the early de-velopment of the central nervous system in chondrich-thyans comes from the studies of this group, whose mem-bers have analyzed the expression of developmental genes such as Pax6 [Derobert et al., 2002a], Emx [Derobert et al., 2002b], and Otx [Sauka-Spengler et al., 2001; Plouhinec et al., 2005] in embryos of the Lesser Spotted Dogfish from gastrulation to early organogenesis. Further sup-port for the use of this species as a model organism has been provided by Coolen et al. [2009].
Periods of Forebrain Development in the Lesser
Spotted Dogfish
Three periods can be distinguished in the develop-ment of the forebrain in this species. During the first pe-riod (embryos from stages 22 to 27), the walls are thin and enclose a wide ventricle ( fig. 1 A). No layering is ob-served in these neuroepithelial walls, which consist of a proliferating neuroepithelium with bipolar cells radially arranged, some migrating radially ( fig. 1 B). The second period (embryos from stages 28 to 31) is characterized by increasing thickness of the walls due to the beginning of the three-layered organization characteristic of the walls in developing vertebrate brains. Three zones can be rec-ognized during this period: the proliferating ventricular zone (VZ); the intermediate zone (IZ), where most of the somata of the bipolar cells are located; and the marginal zone (MZ) formed by abundant tangentially oriented processes ( fig. 1 C, D). Most bipolar cells are migrating radially, but in some regions (ventral telencephalon, see below) tangentially migrating cells are also present. Dur-ing stage 31, important morphological changes take place in the anterior part of the head, caused by the formation and expansion of the rostrum ( fig. 1 E). When stage 32 begins, the first half of embryonic development has tak-en place, and the forebrain has reached the point where its organization is relatively mature ( fig. 2 A). At this point, the third period begins, and it extends through the second half of the embryonic period, which includes stages 32 to 34, and postembryonic stages (juveniles and adulthood) ( fig. 2 A, B). At stage 32, the neuronal cell bodies have already migrated away from the periventric-ular matrix, and the main forebrain populations ob-
served in juveniles and adults are already recognizable. The third period, therefore, can be considered a period of maturation.
Regionalization of the Diencephalon (Caudal
Prosencephalon) in the Lesser Spotted Dogfish
The use of immunomarkers of the GABAergic system as antibodies against GABA and the synthesis enzyme, the glutamic acid decarboxylase (GAD), has shed light on forebrain regionalization in this dogfish, as they seem to be good markers for some boundaries and domains [Car-rera et al., 2006, 2008a; Ferreiro-Galve et al., 2008]. More-over, the expression of the Pax6 gene, which is considered one of the genes responsible for forebrain regionalization in vertebrates, reveals a pattern consistent with that ob-served in other fishes [Wullimann and Rink, 2001] and highlights the presence of longitudinal and transverse boundaries, such as the alar-basal, diencephalic-mesen-cephalic, and pallial-subpallial boundaries, as well as some prosomeric domains.
The segmental organization of the forebrain is evident in stage 31 of these dogfish embryos, based on the distri-bution of Pax6 and some markers of early and late neu-ronal differentiation, such as GAD, calretinin, tyrosine hydroxylase (TH) and serotonin (5-HT) [Ferreiro-Galve et al., 2008]. Considering the anatomical landmarks that are evident in both developing and mature dogfish fore-brains, and the pattern of distribution of the aforemen-tioned markers, the identification of forebrain subdivi-sions can be extended to embryonic and postembryonic stages in the developmental periods described above (figs. 1A, C, E; 2A, B). The nomenclature used throughout the paper follows that of Puelles and Rubenstein [2003].
The alar-basal boundary along the caudal diencepha-lon corresponds to a longitudinal strip of GABAergic and Pax6-expressing cells, which continues along the mesen-cephalon ( fig. 3 A–C). As in other vertebrates, the sonic hedgehog protein is located in the basal plate and the floor plate of the diencephalon in this dogfish and allows recognition of the alar-basal boundary. Some transverse boundaries are also recognizable. The sharp limit be-tween the caudal extension of the prosencephalic Pax6 domain and the negative mesencephalic alar plate, the optic tectum, marks the diencephalic-mesencephalic boundary ( fig. 3 C). The fasciculus retroflexus is a con-spicuous transverse landmark that courses along the boundary between prosomeres p1 and p2 ( fig. 2 B), and the zona limitans intrathalamica is recognized as the
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wedge of GABAergic cells that extends between proso-meres p2 and p3 ( fig. 3 A, B) [see also Carrera et al., 2006; Ferreiro-Galve et al., 2008]. This transverse boundary can be identified also by the presence of sonic hedgehog protein.
The extension of prosomeres p1-p3 in embryonic and postembryonic stages ( fig. 1 A, C, E; 2 A, B) is mainly based on the distribution of Pax6 cells, which are absent from p2 (or, possibly, restricted to the ventricle and thus
less obvious), but which form a conspicuous domain in the pretectum (alar plate of p1) and in the prethalamus (alar plate of p3), and also extend through the p3 basal plate ( fig. 3 C). Pax6 cells are also observed dorsal to the preoptic region, in an area that corresponds to the emi-nentia thalami. This is in concordance with observations in other vertebrates [Puelles et al., 2000; Wullimann and Mueller, 2004]. Based on the continuity between the Pax6 cell groups of the eminentia thalami and those of the
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Fig. 1. Schematic drawings of sagittal sec-tions through the brains of Lesser Spotted Dogfish embryos at stages 25 ( A ), 29 ( C ) and 31 ( E ) and representation of the cellu-lar organization in the prosencephalic walls at the first ( B ) and second ( D ) embry-onic periods. In the sagittal drawings, the alar-basal boundary (dotted line) and the diencephalic prosomeres (transverse seg-ment) are noted. The suggested location of the pallial-subpallial boundary (dash-dot-ted line) and the boundary between the mesencephalon and the rhombencephalon (dashed line) are also indicated. For abbre-viations, see list. Scale bars: 200 � m.
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prethalamus, both groups are included in the alar part of p3 in this dogfish ( fig. 1 A, C, E; 2 A, B; 3 C). This agrees with the prosomeric model in its modern iteration [Pu-elles and Rubenstein, 2003], which considers the eminen-tia thalami as part of p3, not the dorsal part of p4.
In addition to Pax6 cells, p1 also contains GABAergic cells in the alar plate (pretectum), whereas its basal plate includes the large calretinin-expressing cells of the nu-cleus of the medial longitudinal fascicle, which lie dorsal to the catecholaminergic (TH-immunoreactive) cells of the rostral part of the ventral tegmental area. In contrast to the absence of Pax6, the alar plate of p2 (thalamus) contains a group of calretinin-expressing cells from stage 31 onwards. In the basal regions of p3, the group of Pax6 cells overlaps with the abundant TH-ir popula-tion of the posterior tubercle and the dorsal hypothala-mus.
No clear limit between the diencephalon (caudal pros-encephalon) and the secondary prosencephalon (hypo-
thalamus and telencephalon) has been noted in this dog-fish, but the rostral extension of the Pax6 cell group of the eminentia thalami can be used as the virtual boundary ( fig. 1 A, C, D; 2 A, B).
Regionalization of the Secondary Prosencephalon
in the Lesser Spotted Dogfish
Following the prosomeric model, the hypothalamus (also considered the rostral part of the diencephalon) is included in the secondary prosencephalon. The hypothal-amus in adult Lesser Spotted Dogfish, as in other verte-brates, is characterized by the abundance of catechol-aminergic, serotoninergic, and peptidergic groups, which are mainly formed by cerebrospinal fluid-contacting neu-rons. The few existing studies of the ontogeny of these hypothalamic systems in elasmobranchs reveal the exis-tence of large temporal differences in their development.
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Fig. 2. Schematic drawings of sagittal sec-tions through the brains of Lesser Spotted Dogfish embryos at stage 32 ( A ) and as ju-veniles ( B ) showing the alar-basal bound-ary (dotted line) and the diencephalic pro-someres (transverse segment). The sug-gested location of the pallial-subpallial boundary (dash-dotted line) and the boundary between the mesencephalon and the rhombencephalon (dashed line) are also indicated. For abbreviations, see list. Scale bars: 1 mm ( A ), 2.5 mm ( B ).
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The catecholaminergic cells of the hypothalamus of the Lesser Spotted Dogfish differentiates early (stage 26), compared to structures of the mesencephalon and the rhombencephalon (stages 30–31) [Carrera et al., 2005]. The opposite happens with the hypothalamic serotonin-ergic cells, which differentiate late (stage 31) in relation to the rhombencephalic serotoninergic cells (stage 26) [Car-rera et al., 2008b]. From stage 31, the hypothalamus in this species contains abundant serotoninergic and cate-
cholamine-synthesizing (TH-immunoreactive) cells. TheTH-ir groups lie dorsal to the periventricular serotonin-ergic cell groups, occupying a location that probably cor-responds to the dorsal limit of the hypothalamus. The hypothalamic peptidergic system, which contains neuro-peptide Y, also develops late [Chiba et al., 2002], appar-ently during the second developmental period. Cells con-taining calretinin are also abundant in the embryonic and mature hypothalamus.
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Fig. 3. Photomicrographs of sagittal ( A–C ) and transverse ( D ) sections through the forebrain of Lesser Spotted Dogfish embryos at stages 26 ( A , B ), 29 ( C ) and 31 ( D ) showing the distribution of GAD ( A , B ) and Pax6 ( D ) im-munoreactive cells. Small arrows in A–C mark the strip of cells related to the alar-basal boundary. Arrowheads in C indicate the diencephalic-mesencephalic juncture. Large arrows in D mark the pallial-subpallial boundary. The level of the transverse section in D is indicated in figure 1E. For abbreviations, see list. Scale bars: 200 � m. * = Lateral/ventral pallial region with very scarce Pax6 cells.
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The preoptic area is generally considered a subdivision of the secondary prosencephalon, although according to the modern iteration of the prosomeric model, it forms part of the basal telencephalon (subpallium). In the Less-er Spotted Dogfish, this appears to be possible, taking into account the distribution of GABAergic cells. During the first and second periods of forebrain development, GABA, which is considered a subpallial marker in early develop-mental stages, is synthesized in most cells that extend through the basal part of the forebrain, from a level just anterior to the optic stalk (the presumptive preoptic area) to a very rostral telencephalic level, where the sharp de-crease in GABA might represent the pallial-subpallial boundary. Because the density of GABAergic somata de-creases during the third period of forebrain development, differences between preoptic and subpallial territories cannot be discerned with this marker, but when an em-bryo enters the prehatching phase (stages 33 and 34) sero-toninergic cells differentiate in the walls of the preoptic recess to form a small population that is also observed in adults [Carrera et al., 2008b]. The preoptic area of this dogfish has been well studied in relation to the neurose-cretory system [Vallarino et al., 1990; Meur ling et al., 1996] because it is in this region that one finds the neurons of the magnocellular preoptic nucleus, which forms part of the classical neurosecretory system, together with sev-eral populations of cells that contain diverse hypophysio-tropic neuropeptides [for a review, see Smeets, 1998].
During the first and second developmental periods, a dorsal part, or pallium, and a ventral part, or subpallium, can be distinguished in the telencephalon of the Lesser Spotted Dogfish, based on differences in the distribution of GABAergic and Pax6-expressing cells. During the first period, the subpallium contains abundant GABA-ex-pressing cells, whereas these cells are absent from the pal-lium [Carrera et al., 2008a]. In contrast, Pax6 cells are abundant in the pallium but absent in the subpallium [Ferreiro-Galve et al., 2008]. The situation is basically similar during the second period, with some interest-ing exceptions, especially at the pallium-subpalliuminterface. Here, some GABAergic prolongations extend throughout the marginal subpallial walls and continue rostromedially along the external walls of the ventral pal-lium [Carrera et al., 2008a]. As GABAergic cells are ab-sent from the dorsal telencephalon, these prolongations must belong to the GABAergic subpallial cells. Later in the second period, not only prolongations but also bipo-lar GABAergic cells with leading processes ending in growth cones are also seen at the pallium-subpallium boundary, extending radially and tangentially. Still later,
strings of GABAergic cells are seen in the pallium ex-tending periventricularly or superficially. Together these observations suggest the existence of GABAergic cells of subpallial origin that are migrating tangentially toward lateral and dorsal pallial regions [Carrera et al., 2008a]. This reveals the very early appearance of tangential mi-grations in vertebrate forebrain evolution. During the second period, Pax6-expressing cells are also abundant in the pallium and in a dense band that is mediolaterally oriented and follows the boundary between the pallium and the subpallium ( fig. 3 D). From this band, scattered Pax6-expressing cells appear to emerge towards subpal-lial regions. Thus, in the late second period, the boundary between the pallium and the subpallium is a region where subpallial GABAergic cells begin to migrate tangentially to the pallium and, probably, Pax6-expressing pallial cells begin to migrate tangentially to the subpallium.
The source of these tangentially migrating GABAergic cells in the subpallium is still unclear, but there is evidence that during the second period they could emerge from bi-lateral protrusions of the telencephalic walls, protrusions that form a conspicuous bilateral eminence. The ventral half of this bilateral eminence contains a distribution of GABAergic cells that is similar to that in the subpallium, whereas the distribution of Pax6 cells in the dorsal half is similar to that in the pallium. Therefore, the dorsal part appears to be of pallial origin, whereas the ventral part appears subpallial, with the pallial-subpallial boundary in between. GABAergic neuroblasts migrate tangentially to the pallium mainly along the subventricular walls of the eminences [Carrera et al., 2008a].
Although more studies are necessary, the presence of tangentially migrating cells in the lateral eminence sug-gests that this structure in sharks could be related to the ganglionic eminences in mammals, where the striatal and pallidal neurons arise. As early as 1922, Holmgren de-scribed three swellings in the lateral walls of the forebrain ventricle in Squalus (two rostral and one caudal), which he believed represented the striatum. He called them ‘stri-atal swellings’, and he first observed them in embryos of 8 cm in body length, when ‘the definitive arrangement of the forebrain is attained in principle.’ He also indicated that the swellings are strongly developed in adult Squalus and contain a ventricular nucleus formerly called the epi-striatum. In embryos of the Lesser Spotted Dogfish as well, the lateral eminence appears to be related to the stri-atum, as it represents the primordium of the region that in adults contains the nucleus identified by Smeets et al. [1983] as the striatum. It is also worth noting that in mam-mals, pallial cells come mostly from the medial, ‘pallidal’
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ganglionic eminence. This and other issues must be re-solved by further developmental studies.
Close to the band of Pax6 cells that might correspond to the pallial-subpallial boundary, the incipient olfactory bulbs emerge as protrusions of the lateroventral telence-phalic walls. These primordial bulbs, in which Pax6 cells are nearly absent, appear to arise from a lateral/ventral pallial region which, in contrast with the rest of the pal-lium, is practically devoid of Pax6-expresing cells (aster-isk in fig. 3 D). Although Pax6 cells are lacking in the pri-mordial olfactory bulb, they are abundant in the develop-ing and mature olfactory bulb of late embryos and postembryonic stages (juveniles and adults).
In the pallium of the Lesser Spotted Dogfish, three distinct areas can be clearly discerned: the dorsal palli-um, the medial pallium, and the lateral pallium. The Pax6 negative region, which is surrounded by the primor-dial olfactory bulb, the pallium, and the subpallium, could represent the primordial lateral pallium, although it is also possible that it represents the origin of a fourth pallial subdivision: the ventral pallium.
Concluding Remarks
These observations reveal an ancient origin for major morphogenetic processes – such as the tangential migra-tion of telencephalic cells – that were long thought to be a hallmark of mammalian brains. They also reveal the importance of studies on elasmobranchs to determine the ancestral condition of brain organization in gnathos-tomes. In addition, it is clear that the Lesser Spotted Dog-fish can serve as an important model species from which to gain insights into the origin of the complex organiza-tion of gnathostome brains.
Acknowledgements
I thank E. Candal, I. Quintana, S. Pose and, especially, I. Car-rera and S. Ferreiro-Galve for their help with various aspects of this work. This work was sponsored by grants from the Spanish Ministry of Science and Technology-FEDER (BFU2007-61154) and Xunta de Galicia (PGIDIT07PXIB200102PR).
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