The expression of the Nkx2.2 gene is involved in the organization of the alar-basal boundary in the forebrain of vertebrates. Its expression in different diencephalic and telencephalic regions, helped to define distinct progenitor domains in mouse and chick. Here we investigated the pattern of Nkx2.2 protein distribution throughout the development of the forebrain of the anuran amphibian, Xenopus laevis. We used immunohistochemical and in situ hybridization techniques for its detection in combination with other essential territorial markers in the forebrain. No expression was observed in the telencephalon. In the alar hypothalamus, Nkx2.2 positive cells were scattered in the suprachiasmatic territory, but also in the supraopto-paraventricular area, as defined by the expression of the transcription factor Orthopedia (Otp) and the lack of xDll4. In the basal hypothalamus Nkx2.2 expressing cells were localized in the tuberal region, with the exception of the arcuate nucleus, rich in Otp expressing cells. In the diencephalon it was expressed in all three prosomeres (P1-P3) and not in the zona limitans intrathalamica. The presence of Nkx2.2 expressing cells in P3 was restricted to the alar portion, as well as in prosomere P2, whereas in P1 the Nkx2.2 expressing cells were located in the basal plate and identified the alar/basal boundary. These results showed that Nkx2.2 and Sonic hedgehog are expressed in parallel adjacent stripes along the anterior-posterior axis. The results of this study showed a conserved distribution pattern of Nkx2.2 among vertebrates, crucial to recognize subdivisions that are otherwise indistinct, and supported the relevance of this transcription factor in the organization of the forebrain, particularly in the delineation of the alar/basal boundary of the forebrain.

Major common features have been reported for the organization of the basal telencephalon in amniotes, and most characteristics were thought to be acquired in the transition from anamniotes to amniotes. However, gene expression, neurochemical, and hodological data obtained for the basal ganglia and septal and amygdaloid complexes in amphibians (anamniotic tetrapods) have strengthened the idea of a conserved organization in tetrapods. A poorly characterized region in the forebrain of amniotes has been the bed nucleus of the stria terminalis (BST), but numerous recent investigations have characterized it as a member of the extended amygdala. Our study analyzes the main features of the BST in anuran amphibians to establish putative homologies with amniotes. Gene expression patterns during development identified the anuran BST as a subpallial, nonstriatal territory. The BST shows Nkx2.1 and Lhx7 expression and contains an Islet1-positive cell subpopulation derived from the lateral ganglionic eminence. Immunohistochemistry for diverse peptides and neurotransmitters revealed that the distinct chemoarchitecture of the BST is strongly conserved among tetrapods. In vitro tracing techniques with dextran amines revealed important connections between the BST and the central and medial amygdala, septal territories, medial pallium, preoptic area, lateral hypothalamus, thalamus, and prethalamus. The BST receives dopaminergic projections from the ventral tegmental area and is connected with the laterodorsal tegmental nucleus and the rostral raphe in the brainstem. All these data suggest that the anuran BST shares many features with its counterpart in amniotes and belongs to a basal continuum, likely controlling similar reflexes, reponses, and behaviors in tetrapods.

Expression patterns of Nkx2.1 and Islet-1 (Isl1), which encode transcription factors that are key in the regionalization of the forebrain, were analyzed by combined immunohistochemical methods in young adult specimens of two lungfishes (Neoceratodus forsteri and Protopterus dolloi) and a urodele amphibian (Pleurodeles waltl). We aimed to get insights into the possible organization of the forebrain in the common ancestor of all tetrapods because of the pivotal phylogenetic significance of these two groups, being lungfishes the closest living relatives of tetrapods, and representing urodeles a model of simple brain organization with most shared features with amniotes. These transcription factors display regionally restricted expression domains in adult (juvenile) brains that are best interpreted according to the current prosomeric model. The regional patterns observed serve to identify regions and compare between the three species studied, and with previous data reported mainly for amniotes. We corroborate that Nkx2.1 and Isl1 expressions have very similar topologies in the forebrain. Common features in all sarcopterygians (lungfishes and tetrapods) have been observed, such as the Isl1 expression in most striatal neurons, whereas Nkx2.1 is restricted to migrated interneurons that reach the ventral pallium (VP). In the pallidal derivatives, the combination of both markers allows the identification of the boundaries between the ventral septum, the bed nucleus of the stria terminalis (BST) and the preoptic commissural region. In addition, the high Isl1 expression in the central amygdala (CeA), its boundary with the lateral amygdala (LA), and the scattered Nkx2.1 expression in the medial amygdala (MeA) are also shared features. The alar and basal hypothalamic territories, and the prethalamus and posterior tubercle (TP) in the diencephalon, have maintained a common pattern of expression. This regional distribution of Isl1 and Nkx2.1 observed in the forebrain of urodeles and lungfishes contributes further to our understanding of the first terrestrial vertebrates and their ancestors.

Cladistians are a group of basal nonteleost actinopterygian fishes that represent an interesting group for the study of primitive brain features, most likely present in the ancestral Osteichthyes. We have investigated the catecholaminergic (CA) systems in the brain of two representative cladistian species, the bichir Polypterus senegalus and the reedfish Erpetoichthys calabaricus, by means of antibodies against tyrosine hydroxylase (TH; the first enzyme in the synthesis of catecholamines) and dopamine (DA). Double immunohistofluorescence was performed for simultaneous detection of TH with nitric oxide synthase, choline acetyltransferase, calbindin, calretinin, and serotonin, aiming to accurately establish the localization of the CA neurons and to assess possible interactions between these neuroactive substances. All forebrain CA groups of cladistians are dopaminergic, whereas noradrenergic cells are located within the rhombencephalon. Distinct groups of DA immunoreactive (DA-ir) cells were observed in the olfactory bulb, subpallium, and preoptic area of the telencephalon. Hypothalamic groups were detected in the suprachiasmatic nucleus, retrotuberal and retromamillary areas and, in particular, the paraventricular organ showed immunoreactivity to dopamine but not to TH. Diencephalic DA-ir groups were detected in the prethalamus, posterior tubercle, and pretectum. A small DA-ir cell population was observed in the midbrain tegmentum only in Polypterus. CA cell groups were also located in the locus coeruleus, solitary tract nucleus, and area postrema within the rhombencephalon, the spinal cord, and the retina. The comparison of these results with other vertebrates, using a neuromeric analysis, shows highly conserved traits in all vertebrates studied but also evidences particular characteristics of actinopterygian fishes.

The onset and developmental dynamics of Pax3, Pax6, and Pax7 expressions were analyzed by immunohistochemical techniques in the central nervous system (CNS) of embryos, larvae, and recently metamorphosed juveniles of the urodele amphibian Pleurodeles waltl. During the embryonic period, the Pax proteins start being detectable in neuroepithelial domains. Subsequently, they become restricted to subsets of cells in distinct brain regions, maintaining different degrees of expression in late larvae and juvenile brains. Specifically, Pax6 is broadly expressed all along the urodele CNS (olfactory bulbs, pallium, basal ganglia, diencephalon, mesencephalic tegmentum, rhombencephalon, and spinal cord) and the developing olfactory organ and retina. Pax3 and Pax7 are excluded from the rostral forebrain and were usually observed in overlapping regions during embryonic development, whereas Pax3 expression is highly downregulated as development proceeds. Thus, Pax3 is restricted to the roof plate of prosomere 2, pretectum, optic tectum, rhombencephalon, and spinal cord. Comparatively, Pax7 was more conspicuous in all these regions. Pax7 cells were also found in the paraphysis, intermediate lobe of the hypophysis, and basal plate of prosomere 3. Our data show that the expression patterns of the three Pax genes studied are overall evolutionarily conserved, and therefore could unequivocally be used to identify subdivisions in the urodele brain similar to other vertebrates, which are not clearly discernable with classical techniques. In addition, the spatiotemporal sequences of expression provide indirect evidence of putative migratory routes across neuromeric limits and the alar-basal boundary.

The present study represents a detailed spatiotemporal analysis of the localization of calbindin-D28k (CB) and calretinin (CR) immunoreactive structures in the brain of Xenopus laevis throughout development, conducted with the aim to correlate the onset of the immunoreactivity with the development of compartmentalization of distinct subdivisions recently identified in the brain of adult amphibians and primarily highlighted when analyzed within a segmental paradigm. CR and CB are expressed early in the brain and showed a progressively increasing expression throughout development, although transient expression in some neuronal subpopulations was also noted. Common and distinct characteristics in Xenopus, as compared with reported features during development in the brain of mammals, were observed. The development of specific regions in the forebrain such as the olfactory bulbs, the components of the basal ganglia and the amygdaloid complex, the alar and basal hypothalamic regions, and the distinct diencephalic neuromeres could be analyzed on the basis of the distinct expression of CB and CR in subregions. Similarly, the compartments of the mesencephalon and the main rhombencephalic regions, including the cerebellum, were differently highlighted by their specific content in CB and CR throughout development. Our results show the usefulness of the analysis of the distribution of these proteins as a tool in neuroanatomy to interpret developmental aspects of many brain regions.