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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.

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.

We investigated whether gamma-amino butyric acidergic (GABAergic) cell populations correlate positionally with specific Dlx-expressing histogenetic territories in an anamniote tetrapod, the frog Xenopus laevis. To that end, we cloned a fragment of Xenopus GAD67 gene (xGAD67, expressed in GABAergic neurons) and compared its expression with that of Distal-less-4 gene (xDll-4, ortholog of mouse Dlx2) in the forebrain at late larval and adult stages. In Xenopus, GABAergic neurons were densely concentrated in xDll-4-positive territories, such as the telencephalic subpallium, part of the hypothalamus, and ventral thalamus, where nearly all neurons expressed both genes. In contrast, the pallium of Xenopus generally contained dispersed neurons expressing xGAD67 or xDll-4, which may represent local circuit neurons. As in amniotes, these pallial interneurons may have been produced in the subpallium and migrated tangentially into the pallium during development. In Xenopus, the ventral division of the classic lateral pallium contained extremely few GABAergic cells and showed only low signal of the pallial gene Emx1, suggesting that it may represent the amphibian ventral pallium, homologous to that of amniotes. At caudal forebrain levels, a number of GABAergic neurons was observed in several areas (dorsal thalamus, pretectum), but no correlation to xDll-4 was observed there. The location of GABAergic neurons in the forebrain and their relation to the developmental regulatory genes Dll and Dlx were very similar in Xenopus and in amniotes. The close correlation in the expression of both genes in rostral forebrain regions supported the notion that Dll/Dlx are among the genes involved in the acquisition of the GABAergic phenotype.

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 [19]. 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.

In this study, we review data on the existence of comparable divisions and subdivisions in the telencephalon of different groups of tetrapods based on expression of some developmental regulatory genes, having a particular focus in the comparison of the anuran amphibian Xenopus and the mouse. The available data on Xenopus, mouse, chick and turtle indicate that apparently all tetrapod groups possess the same molecularly distinct divisions and subdivisions in the telencephalon. This basic organization was likely present in the telencephalon of stem tetrapods. Each division/subdivision is characterized by expression of a unique combination of developmental regulatory genes, and appears to represent a self-regulated and topologically constant histogenetic brain compartment that gives rise to specific groups of cells. This interpretation has an important consequence for searching homologies, since a basic condition for cell groups in different vertebrates to be considered homologous is that they originate in the same compartment. However, evolution may allow individual cell groups derived from comparable (field homologous) subdivisions to be either similar or dissimilar across the vertebrate groups, giving rise to several possible scenarios of evolution, which include both the evolutionary conservation of similar (homologous) cells or the production of novel cell groups. Finally, available data in the lamprey, a jawless fish, suggest that not all telencephalic subdivisions were present at the origin of vertebrates, raising important questions about their evolution.