The telencephalon of the frog Xenopus based on calretinin immunostaining and gene expression patterns

To further understand the organization and evolution of the telencephalon, we analyzed in the frog Xenopus laevis the expression of the genes Distal-less-4 (Xdll-4, comparable to the mouse gene Dlx2) and GAD-67 (XGAD-67, expressed in GABAergic cells), and compared this with calretinin immunostaining and the cytoarchitecture of the telencephalon. Our results show that like in other vertebrates, the telencephalon of the frog Xenopus is divided into two major territories: a basal, subpallial region showing a high density of cells expressing Xdll-4 and XGAD-67, and a dorsal, pallial region showing only few, dispersed cells expressing these genes. The subpallial territory of the frog Xenopus includes the septum, the amphibian basal ganglia, some basal forebrain cholinergic cell groups and some amygdala nuclei. In the pallium of the frog Xenopus, medial, dorsal, lateral, and ventral parts could be distinguished, similar to those described in amniotes. In summary, the amphibian telencephalon shows a basic morphogenetic organization similar to that of amniotes, which suggests that this organization is common to the telencephalon of all tetrapods.     

Expression of the genes Emx1, Tbr1, and Eomes (Tbr2) in the telencephalon of Xenopus laevis confirms the existence of a ventral pallial division in all tetrapods

To investigate the pallial organization and the exact location and extension of the ventral pallium in amphibians, we cloned a fragment of the homeobox XenopusTbr1 (xTbr1) gene and analyzed its expression compared with that of the genes xEomes (Tbr2) and xEmx1 in the telencephalon of the frog Xenopus laevis during embryonic and larval development. The expression of xEmx1 was also analyzed in the adult frog. We compared the expression patterns of these pallial marker genes with that of the subpallial gene xDistal-less-4 (xDll4). Our results indicate that the whole pallium of Xenopus expresses the T-box genes xTbr1 and xEomes (in proliferating cells and/or mantle) during embryonic and larval development, and the expression of these genes is topographically complementary to that of xDll4 in the subpallium. In addition to their massive expression in the pallium, both xTbr1 and xEomes are expressed in a few dispersed cells in the subpallium, which may represent immigrant cells of pallial origin, because these genes are not found in the subpallial proliferating cells. On the other hand, during development xEmx1 is expressed in a large part of the pallium (proliferating and postmitotic cells) except for an area adjacent to the pallio-subpallial boundary, where xEmx1 is observed only in some mantle cells. This pallial area poor in xEmx1 expression and poor in expression of the subpallial gene xDll4, but expressing the pallial marker genes xTbr1 and xEomes, appears to represent the amphibian ventral pallium, comparable to that described in other vertebrates (Puelles et al. [2000] J. Comp. Neurol. 424:409-438). In the adult frog, the ventral pallium appears to include the rostral part of the lateral amygdalar nucleus as well as a large part of the medial amygdalar nucleus (as defined by Marín et al. [1998] J. Comp. Neurol. 392:285-312). In contrast, the caudal part of the previously termed lateral amygdalar nucleus shows strong xEmx1 expression and may be a lateral pallial derivative. The possible homology of these amphibian amygdalar nuclei is discussed. Finally, expression of xTbr1, xEomes, and xEmx1 is observed in the mitral cell layer of the olfactory bulb from early developmental stages, further supporting that this structure is a pallial derivative.     

Phylotypic expression of the bHLH genes Neurogenin2, Neurod,and Mash1 in the mouse embryonic forebrain

In the anamniote model animals, zebrafish and Xenopus laevis, highly comparable early forebrain expression patterns of proneural basic helix‐loop‐helix (bHLH) genes relevant for neurogenesis (atonal homologs, i.e., neurogenins/NeuroD and achaete‐scute homologs, i.e., Ascl/ash) were previously revealed during a particular period of development (zebrafish: 3 days; frog: stage 48). Neurogenins/NeuroD on the one hand and Ascl1/ash1 on the other hand exhibit essentially mutually exclusive spatial patterns, probably reflecting different positional information received within the neural tube, and appear to underlie glutamatergic versus GABAergic neuronal differentiation, respectively. Significant data suggest that similar complementary localizations of these proneural genes and corresponding differentiation pathways also exist in the mouse, the prominent mammalian model. The present article reports on detailed mouse brain bHLH gene expression patterns to fill existing gaps in the identification of expression domains, especially outside the telencephalon. Clearly, there are strong similarities in the complementarity of territories expressing Ascl1/Mash 1 versus neurogenins/NeuroD in the entire mouse forebrain, except for the pretectal alar plate and basal plate of prosomeres 1–3. The analysis substantiates localization of neurogenins/NeuroD in the pallium, eminentia thalami, and dorsal thalamus, and expression of Ascl1/Mash 1 in the striatal and septal subpallium, preoptic region, ventral thalamus, and hypothalamus, which is highly similar to the situation described in Xenopus and zebrafish. Thus, all three vertebrate model species display a “phylotypic stage or period” corresponding to a temporally and spatially defined control of neurogenesis during forebrain development, ultimately resulting in the differentiation of distinct populations of glutamatergic versus GABAergic neurons. J. Comp. Neurol. 518:851–871, 2010. © 2009 Wiley‐Liss, Inc.     

Understanding the basic circuitry of the cerebral hemispheres: the case of lizards and its implications in the evolution of the telencephalon

The organization of the cerebral hemispheres of mammals is characterized by corticostriatal glutamatergic projections and striatopallidal GABAergic ones, plus the descending projections of the pallium and subpallium to extratelencephalic targets. The present review of the available neuroanatomical data on the forebrain of lizards suggests that the telencephalon of reptiles also follows this basic pattern of connectivity. In addition, we show that this basic circuitry includes a pallido-cortical projection, therefore forming a cortico-striato-pallido-cortical circuit. The analysis of this circuitry for the medial, dorsal, lateral, and ventral pallial divisions in reptiles and mammals leads to the following conclusions: (1) The medial and dorsal cortices of lizards together appear to be equivalent to the medial pallium of mammals. (2) The projection from the lacertilian dorsal cortex to the striatum proper resembles the subiculo-striatal projection of mammals, rather than the isocortical projection to the caudatus-putamen. (3) Most of the dorsal striatum of reptiles is engaged in the corticostriatal circuit corresponding to the ventral pallium (the anterior dorsal ventricular ridge), and therefore, it is not equivalent to the mammalian caudatus-putamen, which is involved in the circuit of the dorsal pallium. (4) The main and accessory olfactory bulbs also follow this pattern of connections.     

Tangentially migrating GABAergic cells of subpallial origin invade massively the pallium in developing sharks

We studied the development of the GABAergic system in the telencephalon of the dogfish Scyliorhinus canicula using GABA and glutamate decarboxylase (GAD) immunocytochemistry. The earliest GABA-expressing cells appeared in the basal telencephalon (subpallium) of stage 24 embryos. Shortly after, the subpallium showed abundant GABA-expressing neuroblasts near the meningeal surface or migrating radially in the neuroepithelium. The limit between the GABA-expressing region and the remainder of the telencephalon (pallium) was sharp and coincides with the pallial/subpallial boundary. At stage 28, GABA-expressing cells with the morphology of tangentially migrating cells (showing a thick growth cone-like leading process) migrate from a dome-shaped protrusion of the lateral subpallium and extended laterally and rostrodorsally into the pallium following either a superficial route or coursing periventricularly. At later stages, abundant GABA-expressing cells were seen in various pallial regions and strings of GABA-expressing cells, possibly migrating, were also noted. The colonization of the dogfish pallium by GABA-expressing cells, originating from the subpallium, is strongly reminiscent of the palliopetal tangential migrations of GABA-expressing cells demonstrated in the telencephalon of mammals and follows similar routes. These results strongly suggest that tangential migrations of GABA-expressing cells appeared very early in vertebrate forebrain evolution.     

Pallial and subpallial derivatives in the embryonic chick and mouse telencephalon, traced by the expression of the genes Dlx-2, Emx-1, Nkx-2.1, Pax-6, and Tbr-1

Pallial and subpallial morphological subdivisions of the developing chicken telencephalon were examined by means of gene markers, compared with their expression pattern in the mouse. Nested expression domains of the genes Dlx-2 and Nkx-2.1, plus Pax-6-expressing migrated cells, are characteristic for the mouse subpallium. The genes Pax-6, Tbr-1, and Emx-1 are expressed in the pallium. The pallio-subpallial boundary lies at the interface between the Tbr-1 and Dlx-2 expression domains. Differences in the expression topography of Tbr-1 and Emx-1 suggest the existence of a novel "ventral pallium" subdivision, which is an Emx-1-negative pallial territory intercalated between the striatum and the lateral pallium. Its derivatives in the mouse belong to the claustroamygdaloid complex. Chicken genes homologous to these mouse genes are expressed in topologically comparable patterns during development. The avian subpallium, called "paleostriatum," shows nested Dlx-2 and Nkx-2.1 domains and migrated Pax-6-positive neurons; the avian pallium expresses Pax-6, Tbr-1, and Emx-1 and also contains a distinct Emx-1-negative ventral pallium, formed by the massive domain confusingly called "neostriatum." These expression patterns extend into the septum and the archistriatum, as they do into the mouse septum and amygdala, suggesting that the concepts of pallium and subpallium can be extended to these areas. The similarity of such molecular profiles in the mouse and chicken pallium and subpallium points to common sets of causal determinants. These may underlie similar histogenetic specification processes and field homologies, including some comparable connectivity patterns.     

The teleostean forebrain: a comparative and developmental view based on early proliferation, Pax6 activity and catecholaminergic organization

An improved comparative interpretation of the teleostean forebrain suggests that the dorsal tier (Vd,Vc) and ventral tier (Vv,Vl) nuclei of the ventral telencephalic area (subpallium) represent the striatum and septum, respectively. Among other arguments, a dopaminergic innervation originating in the diencephalic posterior tubercle reaches Vd and dense efferents of Vv project to the midline hypothalamus in the adult zebrafish subpallium. The adult area dorsalis telencephali represents the teleostean pallium. Regulatory genes typically expressed in the early amniote subpallium (e.g., Dlx-1) are also restricted to the presumptive zebrafish ventral telencephalic area. Further, early Pax6 protein distribution in the zebrafish telencephalon corresponds to the migrating stream noted at the pallial-subpallial boundary in amniotes, but a ventricular, radial glia-based expression in the pallium is absent. The peripherally migrated, adult diencephalic preglomerular complex of the basal plate posterior tubercle (early: M2) provides sensory inputs to the pallium. Early Pax6 protein distribution indicates that at least part of M2 may directly originate from alar plate ventral thalamic Pax6-expressing cells. Dopaminergic cells of the basal plate posterior zebrafish forebrain (P1-P3) are restricted to the ventral thalamic prosomere (P3), including those forming the adult ascending dopaminergic system. Moreover, the latter likely depend developmentally on the dorsally adjacent alar plate Pax6-expressing cells.     

An immunohistochemical study of the telencephalon of the African lungfish, Protopterus annectens

The telencephalon of the African lungfish, Protopterus annectens, was studied by immunohistochemical techniques in order to identify the major subdivisions of the telencephalon and determine the possible homologues of these subdivisions, if any, in other vertebrates. The distributions of four different neuropeptides (substance P, leucine-enkephalin, avian pancreatic polypeptide, and LANT6), a neurotransmitter (serotonin), and a neurotransmitter-related enzyme that is involved in catecholamine synthesis (tyrosine hydroxylase) were examined. The resultant labeling patterns indicated that the telencephalon of lungfish consists of three major subdivisions--a rostrally and dorsally situated olfactory bulb, a dorsally situated pallial region located caudal to the olfactory bulbs, and a ventrally situated subpallial regions. The dorsal and lateral pallial regions, which both receive secondary olfactory input, are somewhat distinct from one another cytoarchitectonically, but their immunohistochemical labeling characteristics did not differ. Thus, the lateral pallium and the dorsal pallium together appear to constitute an olfactory pallium in lungfishes. The medial pallium was found to consist of three immunohistochemically distinct subdivisions--a dorsal cell group, an intermediate cell group, and a ventral cell group. These medial pallial fields extend throughout the entire rostrocaudal extent of the medial wall of the telencephalon. Although one or more of these medial pallial cell groups may be homologous to specific portions of the medial pallium in land vertebrates, no specific similarities were observed to support any proposed one-to-one correspondences. The possibility that one or more of the medial pallial cell groups of lungfishes correspond to cell groups located in the dorsal pallium of land vertebrates could not be excluded. The subpallium is divided into lateral, medial, and caudal subdivisions. The lateral subdivision appears to be homologous to the basal ganglia of land vertebrates since it contains neuropeptide/neurotransmitter-specific neuronal populations that are characteristic of the striatal and pallidal portions of the basal ganglia of amniotes. The medial subdivision of the subpallium shows the topographic and immunohistochemical characteristics of the septal region and the nucleus accumbens region of the amniote telencephalon. The caudal subpallium does not show any distinctive immunohistochemical labeling characteristics and its possible homologue in land vertebrates is unclear.(ABSTRACT TRUNCATED AT 400 WORDS)     

Thalamo-telencephalic pathways in the fire-bellied toad Bombina orientalis

It was suggested that among extant vertebrates, anuran amphibians display a brain organization closest to the ancestral tetrapod condition, and recent research suggests that anuran brains share important similarities with the brains of amniotes. The thalamus is the major source of sensory input to the telencephalon in both amphibians and amniote vertebrates, and this sensory input is critical for higher brain functions. The present study investigated the thalamo-telencephalic pathways in the fire-bellied toad Bombina orientalis, a basal anuran, by using a combination of retrograde tract tracing and intracellular injections with the tracer biocytin. Intracellular labeling revealed that the majority of neurons in the anterior and central thalamic nuclei project to multiple brain targets involved in behavioral modulation either through axon collaterals or en passant varicosities. Single anterior thalamic neurons target multiple regions in the forebrain and midbrain. Of note, these neurons display abundant projections to the medial amygdala and a variety of pallial areas, predominantly the anterior medial pallium. In Bombina, telencephalic projections of central thalamic neurons are restricted to the dorsal striato-pallidum. The bed nucleus of the pallial commissure/thalamic eminence similarly targets multiple brain regions including the ventral medial pallium, but this is accomplished through a higher variety of distinct neuron types. We propose that the amphibian diencephalon exerts widespread influence in brain regions involved in behavioral modulation and that a single dorsal thalamic neuron is in a position to integrate different sensory channels and distribute the resulting information to multiple brain regions.     

Expression of Dbx1, Neurogenin 2, Semaphorin 5A, Cadherin 8, and Emx1 distinguish ventral and lateral pallial histogenetic divisions in the developing mouse claustroamygdaloid complex

We studied the lateral and ventral pallial divisions of the claustroamygdaloid complex by means of analysis of expression patterns of the developmental regulatory genes Tbr1, Dbx1, Neurogenin 2, Emx1, Cadherin 8, and Semaphorin 5A in mouse developing telencephalon, from embryonic day 12.5 until birth. Our results indicate that these genes help to distinguish distinct lateral and ventral pallial histogenetic divisions in the embryonic telencephalon. Tbr1 is broadly expressed in both lateral and ventral pallial histogenetic divisions (the lateroventral migratory stream plus the mantle) during early and intermediate embryonic development; its signal becomes weak in parts of the mantle during late embryonic development. Dbx1 is strongly and specifically expressed in progenitor cells (ventricular zone) of the ventral pallium during early embryonic development, but there is no signal of this gene in the rest of the pallium nor the subpallium. Neurogenin 2 and Semaphorin 5A are both expressed in a ventral subdivision of the lateroventral migratory stream (called by us the ventral migratory stream). Further, specific nuclei of the claustral complex and pallial amygdala show strong expression of Neurogenin 2 and/or Semaphorin 5A, including the ventromedial claustrum and endopiriform nuclei, the lateral and basomedial amygdalar nuclei, the anterior and posteromedial cortical amygdalar areas, plus the amygdalo-hippocampal area. We interpret these nuclei or areas of the claustroamygdaloid complex as possible derivatives of the ventral pallium. In contrast, during embryonic development the dorsolateral claustrum, the basolateral amygdalar nucleus, and the posterolateral cortical amygdalar area do not express or show weak expression of Neurogenin 2 or Semaphorin 5A, but express selectively and strongly Cadherin 8 plus Emx1, and may be derivatives of the lateral pallium. The lateral pallial and ventral pallial divisions of the claustroamygdaloid complex appear to have some different sets of connections, although this requires further investigation.     

Organization of the pallium in the fire-bellied toad Bombina orientalis. I: Morphology and axonal projection pattern of neurons revealed by intracellular biocytin labeling

The cytoarchitecture and axonal projection pattern of pallial areas was studied in the fire-bellied toad Bombina orientalis by intracellular injection of biocytin into a total of 326 neurons forming 204 clusters. Five pallial regions were identified, differing in morphology and projection pattern of neurons. The rostral pallium receiving the bulk of dorsal thalamic afferents has reciprocal connections with all other pallial areas and projects to the septum, nucleus accumbens, and anterior dorsal striatum. The medial pallium projects bilaterally to the medial pallium, septum, nucleus accumbens, mediocentral amygdala, and hypothalamus and ipsilaterally to the rostral, dorsal, and lateral pallium. The ventral part of the medial pallium is distinguished by efferents to the eminentia thalami and the absence of contralateral projections. The dorsal pallium has only ipsilateral projections running to the rostral, medial, and lateral pallium; septum; nucleus accumbens; and eminentia thalami. The lateral pallium has ipsilateral projections to the olfactory bulbs and to the rostral, medial, dorsal, and ventral pallium. The ventral pallium including the striatopallial transition area (SPTA) has ipsilateral projections to the olfactory bulbs, rostral and lateral pallium, dorsal striatopallidum, vomeronasal amygdala, and hypothalamus. The medial pallium can be tentatively homologized with the mammalian hippocampal formation, the dorsal pallium with allocortical areas, the lateral pallium rostrally with the piriform and caudally with the entorhinal cortex, the ventral pallium with the accessory olfactory amygdala. The rostral pallium, with its projections to the dorsal and ventral striatopallidum, resembles the mammalian frontal cortex.     

The distribution of GABA-containing perikarya,fibers, and terminals in the forebrain and midbrain of pigeons,with particular reference to the basal ganglia and its projection targets

Immunohistochemical techniques were used to study the distributions of glutamic acid decarboxylase (GAD) and γ-aminobutyric acid (GABA) in pigeon forebrain and midbrain to determine the organization of GABAergic systems in these brain areas in birds. In the basal ganglia, numerous medium-sized neurons throughout the striatum were labeled for GABA, while pallidal neurons, as well as a small population of large, aspiny striatal neurons, labeled for GAD and GABA. GAD+ and GABA+ fibers and terminals were abundant throughout the basal ganglia, and GABAergic fibers were found in all extratelencephalic targets of the basal ganglia. Most of these targets also contained numerous GABAergic neurons. In pallial regions, approximately 10-12% of the neurons were GABAergic. The outer rind of the pallium was more intensely labeled for GABAergic fibers than the core. The olfactory tubercle region, the ventral pallidum, and the hypothalamus were extremely densely labeled for GABAergic fibers, while GABAergic neurons were unevenly distributed in the hypothalamus. GABAergic neurons and fibers were abundant in the dorsalmost part of thalamus and the dorsal geniculate region, while GABAergic neurons and fibers were sparse (or lightly labeled) in the thalamic nuclei rotundus, triangularis, and ovoidalis. Further, GABAergic neurons were abundant in the superficial tectal layers, the magnocellular isthmic nucleus, the inferior colliculus, the intercollicular region, the central gray, and the reticular formation. GABAergic fibers were particularly abundant in the superficial tectal layers, the parvocellular isthmic nucleus, the inferior colliculus, the intercol-licular region, the central gray, and the interpeduncular nucleus. These results suggest that GABA plays a role as a neurotransmitter in nearly all fore- and midbrain regions of birds, and in many instances the observed distributions of GABAergic neurons and fibers closely resemble the patterns seen in mammals, as well as in other vertebrates. © 1994 Wiley-Liss, Inc.     

Comparative functional analysis provides evidence for a crucial role for the homeobox gene Nkx2.1/Titf-1 in forebrain evolution

Knockout of the Nkx2.1 (Titf-1) homeobox gene in the mouse leads to severe malformation and size reduction of the basal telencephalon/preoptic area and basal hypothalamus, indicating an important role of this gene in forebrain patterning. Here we show that abrogation of the orthologous gene in the frog Xenopus laevis by way of morpholino knockdown also affects the relative size of major regions in both the telencephalon (subpallium versus pallium) and diencephalon (hypothalamus versus thalamus). Remarkably, while a similar effect on the telencephalon was noted previously in Nkx2.1-knockout mice, the effect on the diencephalon seems to be specific for Xenopus. This difference may be explained by the partially dissimilar expression of the orthologous genes in the forebrain of Xenopus and mouse. In both species Nkx2.1 is expressed in the basal telencephalon/preoptic area and basal hypothalamus, but in Xenopus this gene is additionally expressed in the alar hypothalamus. Phylogenetic comparison of Nkx2.1 expression in the forebrain suggests that the expression in the basal telencephalon-preoptic region and alar hypothalamus appeared in the transition from jawless to jawed vertebrates, but the alar hypothalamic expression was later dramatically reduced during evolution to birds and mammals. Our study suggests that changes in the regulation of Nkx2.1 expression have played an important role on the evolution of forebrain development, and emphasizes the potential of the combined analysis of expression and function of master control genes in different vertebrates for unraveling the origin of brain complexity and diversity.     

Immunohistochemical organization of the forebrain in the white sturgeon, Acipenser transmontanus

The distribution of substance P (SP), leucine-enkephalin (LENK), serotonin (5HT), dopamine (DA), and tyrosine hydroxylase (TH) was examined in the forebrain of the white sturgeon in order to evaluate several anatomical hypotheses based on cytoarchitectonics, and to gain a better understanding of the evolution of the forebrain in ray-finned fishes. The subpallium of the telencephalon has the highest concentration of the neuropeptides SP and LENK, allowing the pallial-subpallial border to be easily distinguished. The distribution of dopamine is similar to that of serotonin in the subpallium, fibers positive for these transmitters are particularly dense in the dorsal and ventral divisions of the subpallium. In addition, a small population of DA- and 5HT-positive cell bodies--which appear to be unique to sturgeons--was identified at the level of the anterior commissure. The internal granular layer of the olfactory bulbs had large numbers of TH-positive cell bodies and fibers, as did the rostral subpallium. The occurrence of cell bodies positive for LENK in the dorsal nucleus of the rostral subpallium supports the hypothesis that this nucleus is homologous to the striatum in other vertebrates. This is further reinforced by the apparent origin of an ascending dopaminergic pathway from cells in the posterior tubercle that are likely homologous to the ventral tegmental area/substantia nigra in land vertebrates. Finally, the differential distribution of SP and TH in the pallium supports the hypothesis that the pallium, or area dorsalis, can be divided medially into a rostral division (Dm), a caudal division (Dp) that is the main pallial target of secondary olfactory projections, and a narrow lateral division (Dd+Dl) immediately adjacent to the attachment of the tela choroidea along the entire rostrocaudal length of the telencephalic hemisphere.