A detailed 3D MRI brain atlas of the African lungfish Protopterus annectens.

The study of the brain by magnetic resonance imaging (MRI) in evolutionary analyses is still in its incipient stage, however, it is particularly useful as it allows us to analyze detailed anatomical images and compare brains of rare or otherwise inaccessible species, evolutionarily contextualizing possible differences, while at the same time being non-invasive. A good example is the lungfishes, sarcopterygians that are the closest living relatives of tetrapods and thus have an interesting phylogenetic position in the evolutionary conquest of the terrestrial environment. In the present study, we have developed a three-dimensional representation of the brain of the lungfish Protopterus annectens together with a rostrocaudal anatomical atlas. This methodological approach provides a clear delineation of the major brain subdivisions of this model and allows to measure both brain and ventricular volumes. Our results confirm that lungfish show neuroanatomical patterns reminiscent of those of extant basal sarcopterygians, with an evaginated telencephalon, and distinctive characters like a small optic tectum. These and additional characters uncover lungfish as a remarkable model to understand the origins of tetrapod diversity, indicating that their brain may contain significant clues to the characters of the brain of ancestral tetrapods.

Distribution of the transcription factor islet-1 in the central nervous system of nonteleost actinopterygian fish: Relationship with cholinergic and catecholaminergic systems.

Islet-1 (Isl1) is one of the most conserved transcription factors in the evolution of vertebrates, due to its continuing involvement in such important functions as the differentiation of motoneurons, among other essential roles in cell fate in the forebrain. Although its functions are thought to be similar in all vertebrates, the knowledge about the conservation of its expression pattern in the central nervous system goes as far as teleosts, leaving the basal groups of actinopterygian fishes overlooked, despite their important phylogenetic position. In order to assess the extent of its conservation among vertebrates, we studied its expression pattern in the central nervous system of selected nonteleost actinopterygian fishes. By means of immunohistochemical techniques, we analyzed the Isl1 expression in the brain, spinal cord, and sensory ganglia of the cranial nerves of young adult specimens of the cladistian species Polypterus senegalus and Erpetoichthys calabaricus, the chondrostean Acipenser ruthenus, and the holostean Lepisosteus oculatus. We also detected the presence of the transcription factor Orthopedia and the enzymes tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) to better locate all the immunoreactive structures in the different brain areas and to reveal the possible coexpression with Isl1. Numerous conserved features in the expression pattern of Isl1 were observed in these groups of fishes, such as populations of cells in the subpallial nuclei, preoptic area, subparaventricular and tuberal hypothalamic regions, prethalamus, epiphysis, cranial motor nuclei and sensory ganglia of the cranial nerves, and the ventral horn of the spinal cord. Double labeling of TH and Isl1 was observed in cells of the preoptic area, the subparaventricular and tuberal hypothalamic regions, and the prethalamus, while virtually all motoneurons in the hindbrain and the spinal cord coexpressed ChAT and Isl1. Altogether, these results show the high degree of conservation of the expression pattern of the transcription factor Isl1, not only among fish, but in the subsequent evolution of vertebrates.

Expression of SATB1 and SATB2 in the brain of bony fishes: what fish reveal about evolution.

Satb1 and Satb2 belong to a family of homeodomain proteins with highly conserved functional and regulatory mechanisms and posttranslational modifications in evolution. However, although their distribution in the mouse brain has been analyzed, few data exist in other non-mammalian vertebrates. In the present study, we have analyzed in detail the sequence of SATB1 and SATB2 proteins and the immunolocalization of both, in combination with additional neuronal markers of highly conserved populations, in the brain of adult specimens of different bony fish models at key evolutionary points of vertebrate diversification, in particular including representative species of sarcopterygian and actinopterygian fishes. We observed a striking absence of both proteins in the pallial region of actinopterygians, only detected in lungfish, the only sarcopterygian fish. In the subpallium, including the amygdaloid complex, or comparable structures, we identified that the detected expressions of SATB1 and SATB2 have similar topologies in the studied models. In the caudal telencephalon, all models showed significant expression of SATB1 and SATB2 in the preoptic area, including the acroterminal domain of this region, where the cells were also dopaminergic. In the alar hypothalamus, all models showed SATB2 but not SATB1 in the subparaventricular area, whereas in the basal hypothalamus the cladistian species and the lungfish presented a SATB1 immunoreactive population in the tuberal hypothalamus, also labeled with SATB2 in the latter and colocalizing with the gen Orthopedia. In the diencephalon, all models, except the teleost fish, showed SATB1 in the prethalamus, thalamus and pretectum, whereas only lungfish showed also SATB2 in prethalamus and thalamus. At the midbrain level of actinopterygian fish, the optic tectum, the torus semicircularis and the tegmentum harbored populations of SATB1 cells, whereas lungfish housed SATB2 only in the torus and tegmentum. Similarly, the SATB1 expression in the rhombencephalic central gray and reticular formation was a common feature. The presence of SATB1 in the solitary tract nucleus is a peculiar feature only observed in non-teleost actinopterygian fishes. At these levels, none of the detected populations were catecholaminergic or serotonergic. In conclusion, the protein sequence analysis revealed a high degree of conservation of both proteins, especially in the functional domains, whereas the neuroanatomical pattern of SATB1 and SATB2 revealed significant differences between sarcopterygians and actinopterygians, and these divergences may be related to the different functional involvement of both in the acquisition of various neural phenotypes.

Analysis of Islet-1, Nkx2.1, Pax6, and Orthopedia in the forebrain of the sturgeon Acipenser ruthenus identifies conserved prosomeric characteristics.

The distribution patterns of a set of conserved brain developmental regulatory transcription factors were analyzed in the forebrain of the basal actinopterygian fish Acipenser ruthenus, consistent with the prosomeric model. In the telencephalon, the pallium was characterized by ventricular expression of Pax6. In the subpallium, the combined expression of Nkx2.1/Islet-1 (Isl1) allowed to propose ventral and dorsal areas, as the septo-pallidal (Nkx2.1/Isl1+) and striatal derivatives (Isl1+), respectively, and a dorsal portion of the striatal derivatives, ventricularly rich in Pax6 and devoid of Isl1 expression. Dispersed Orthopedia (Otp) cells were found in the supracommissural and posterior nuclei of the ventral telencephalon, related to the medial portion of the amygdaloid complex. The preoptic area was identified by the Nkx2.1/Isl1 expression. In the alar hypothalamus, an Otp-expressing territory, lacking Nkx2.1/Isl1, was identified as the paraventricular domain. The adjacent subparaventricular domain (Spa) was subdivided in a rostral territory expressing Nkx2.1 and an Isl1+ caudal one. In the basal hypothalamus, the tuberal region was defined by the Nkx2.1/Isl1 expression and a rostral Otp-expressing domain was identified. Moreover, the Otp/Nkx2.1 combination showed an additional zone lacking Isl1, tentatively identified as the mamillary area. In the diencephalon, both Pax6 and Isl1 defined the prethalamic domain, and within the basal prosomere 3, scattered Pax6- and Isl1-expressing cells were observed in the posterior tubercle. Finally, a small group of Pax6 cells was observed in the pretectal area. These results improve the understanding of the forebrain evolution and demonstrate that its basic bauplan is present very early in the vertebrate lineage.

Analysis of pallial/cortical interneurons in key vertebrate models of Testudines, Anurans and Polypteriform fishes.

The organization of the pallial derivatives across vertebrates follows a comparable elementary arrangement, although not all of them possess a layered cortical structure as sophisticated as the cerebral cortex of mammals. However, its expansion along evolution has only been possible by the development and coevolution of the cellular networks formed by excitatory neurons and inhibitory interneurons. Thus, the comparative analysis of interneuron types in vertebrate models of key evolutionary significance will provide important information, due to the extraordinary anatomical sophistication of their interneuron systems with simpler behavioral implications. Particularly in mammals, the main consensus for classifying interneuron types is based on non-overlapping markers, which do not form a single population, but consist of several distinct classes of inhibitory cells showing co-expression of other markers. In our study, we analyzed immunohistochemically the expression of the main markers like somatostatin (SOM), parvalbumin (PV), calretinin (CR), calbindin (CB), neuropeptide Y (NPY) and/or nitric oxide synthase (NOS) at the pallial regions of three different models of Osteichthyes. First, we selected two tetrapods, one amniote from the genus Pseudemys belonging to the order Testudine, at the base of the amniote diversification and with a three-layered simple cortex, and the Anuran Xenopus laevis, an anamniote tetrapod with a non-layered evaginated pallium, and finally the order Polypteriform, a small fish group at the base of the actinopterygian diversification with an everted telencephalon. SOM was the most conserved interneuron type in terms of its distribution and co-expression with other markers such as CR, in contrast to PV, which showed a different pattern between the models analyzed. In addition, the SOM expression supports a homological relationship between the medial pallial derivatives in all the models. CR and CB expressions in the tetrapods were observed, particularly, CR expressing cells were detected in the medial and the dorsal pallial derivatives, in contrast to CB, which appeared only in discrete scattered populations. However, the pallium of Polypteriforms fishes was almost devoid of CR cells, in contrast to the important number of CB cells observed in all the pallial regions. The NPY immunoreactivity was detected in all the pallial domains of all the models, as well as cells coexpressing CR. Finally, the pallial nitrergic expression was also conserved, which allows to postulate the homological relationships between the ventropallial and the amygdaloid derivatives. In summary, even in basal pallial models the neurochemically characterized interneurons indicate that their first appearance took place before the common ancestor of amniotes. Thus, our results suggest a shared pattern of interneuron types in the pallium of all Osteichthyes.

Amphibian thalamic nuclear organization during larval development and in the adult frog Xenopus laevis: Genoarchitecture and hodological analysis.

The early patterning of the thalamus during embryonic development defines rostral and caudal progenitor domains, which are conserved from fishes to mammals. However, the subsequent developmental mechanisms that lead to the adult thalamic configuration have only been investigated for mammals and other amniotes. In this study, we have analyzed in the anuran amphibian Xenopus laevis (an anamniote vertebrate), through larval and postmetamorphic development, the progressive regional expression of specific markers for the rostral (GABA, GAD67, Lhx1, and Nkx2.2) and caudal (Gbx2, VGlut2, Lhx2, Lhx9, and Sox2) domains. In addition, the regional distributions at different developmental stages of other markers such as calcium binding proteins and neuropeptides, helped the identification of thalamic nuclei. It was observed that the two embryonic domains were progressively specified and compartmentalized during premetamorphosis, and cell subpopulations characterized by particular gene expression combinations were located in periventricular, intermediate and superficial strata. During prometamorphosis, three dorsoventral tiers formed from the caudal domain and most pronuclei were defined, which were modified into the definitive nuclear configuration through the metamorphic climax. Mixed cell populations originated from the rostral and caudal domains constitute most of the final nuclei and allowed us to propose additional subdivisions in the adult thalamus, whose main afferent and efferent connections were assessed by tracing techniques under in vitro conditions. This study corroborates shared features of early gene expression patterns in the thalamus between Xenopus and mouse, however, the dynamic changes in gene expression observed at later stages in the amphibian support mechanisms different from those of mammals.

Pax6 expression highlights regional organization in the adult brain of lungfishes, the closest living relatives of land vertebrates.

The Pax6 gene encodes a regulatory transcription factor that is key in brain development. The molecular structure of Pax6, the roles it plays and its patterns of expression in the brain have been highly conserved during vertebrate evolution. As neurodevelopment proceeds, the Pax6 expression changes from the mitotic germinal zone in the ventricular zone to become distributed in cell groups in the adult brain. Studies in various vertebrates, from fish to mammals, found that the Pax6 expression is maintained in adults in most regions that express it during development. Specifically, in amphibians, Pax6 is widely expressed in the adult brain and its distribution pattern serves to highlight regional organization of the brain. In the present study, we analyzed the detailed distribution of Pax6 cells in the adult central nervous system of lungfishes, the closest living relatives of all tetrapods. Immunohistochemistry performed using double labeling techniques with several neuronal markers of known distribution patterns served to evaluate the actual location of Pax6 cells. Our results show that the Pax6 expression is maintained in the adult brain of lungfishes, in distinct regions of the telencephalon (pallium and subpallium), diencephalon, mesencephalon, hindbrain, spinal cord, and retina. The pattern of Pax6 expression is largely shared with amphibians and helps to understand the primitive condition that would have characterized the common ancestors to all sarcopterygians (lobe-finned fishes and tetrapods), in which Pax6 would be needed to maintain specific entities of subpopulations of neurons.

Comparative Analysis of the Organization of the Catecholaminergic Systems in the Brain of Holostean Fishes (Actinopterygii/Neopterygii).

Living holosteans, comprising 8 species of bowfins and gars, form a small monophyletic group of actinopterygian fishes, which are currently considered as the sister group to the enormously numerous teleosts and have largely been neglected in neuroanatomical studies. We have studied the catecholaminergic (CAergic) systems by means of antibodies against tyrosine hydroxylase (TH) and dopamine (DA) in the brain of representative species of the 3 genera included in the 2 orders of holostean fishes: Amia calva (Amiiformes) and Lepisosteus platyrhincus, Lepisosteus oculatus, and Atractosteus spatula (Lepisosteiformes). Different groups of TH/DA-immunoreactive (ir) cells were observed in the olfactory bulb, subpallium, and preoptic area of the telencephalon. Hypothalamic groups were labeled in the suprachiasmatic nucleus, tuberal (only in A. calva), retrotuberal, and retromamillary areas; specifically, the paraventricular organ showed only DA immunoreactivity. In the diencephalon, TH/DA-ir groups were detected in the prethalamus, posterior tubercle, and pretectum. In the caudal hindbrain, the solitary tract nucleus and area postrema presented TH/DA-ir cell groups, and also the spinal cord and the retina. Only in A. calva, particular CAergic cell groups were observed in the habenula, the mesencephalic tegmentum, and in the locus coeruleus. Following a neuromeric analysis, the comparison of these results with those obtained in other classes of fishes and tetrapods shows many common traits of CAergic systems shared by most vertebrates and in addition highlights unique features of actinopterygian fishes.

Pattern of nitrergic cells and fibers organization in the central nervous system of the Australian lungfish, Neoceratodus forsteri (Sarcopterygii: Dipnoi).

The Australian lungfish Neoceratodus forsteri is the only extant species of the order Ceratodontiformes, which retained most of the primitive features of ancient lobe finned-fishes. Lungfishes are the closest living relatives of land vertebrates and their study is important for deducing the neural traits that were conserved, modified, or lost with the transition from fishes to land vertebrates. We have investigated the nitrergic system with neural nitric oxide synthase (NOS) immunohistochemistry and NADPH-diaphorase (NADPH-d) histochemistry, which yielded almost identical results except for the primary olfactory projections and the terminal and preoptic nerve fibers labeled only for NADPH-d. Combined immunohistochemistry was used for simultaneous detection of NOS with catecholaminergic, cholinergic, and serotonergic structures, aiming to establish accurately the localization of the nitrergic elements and to assess possible interactions between these neurotransmitter systems. The results demonstrated abundant nitrergic cells in the basal ganglia, amygdaloid complex, preoptic area, basal hypothalamus, mesencephalic tectum and tegmentum, laterodorsal tegmental nucleus, reticular formation, spinal cord, and retina. In addition, low numbers of nitrergic cells were observed in the olfactory bulb, all pallial divisions, lateral septum, suprachiasmatic nucleus, prethalamic and thalamic areas, posterior tubercle, pretectum, torus semicircularis, cerebellar nucleus, interpeduncular nucleus, the medial octavolateral nucleus, nucleus of the solitary tract, and the dorsal column nucleus. Colocalization of NOS and tyrosine hydroxylase was observed in numerous cells of the ventral tegmental area/substantia nigra complex. Comparison with other vertebrates, using a neuromeric analysis, reveals that the nitrergic system of Neoceratodus shares many neuroanatomical features with tetrapods and particularly with amphibians.

Organization of the catecholaminergic systems in two basal actinopterygian fishes, Polypterus senegalus and Erpetoichthys calabaricus (Actinopterygii: Cladistia).

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.

Comparative Analysis of Nkx2.1 and Islet-1 Expression in Urodele Amphibians and Lungfishes Highlights the Pattern of Forebrain Organization in Early 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.

Regional chemoarchitecture of the brain of lungfishes based on calbindin D-28K and calretinin immunohistochemistry.

Lungfishes are the closest living relatives of land vertebrates, and their neuroanatomical organization is particularly relevant for deducing the neural traits that have been conserved, modified, or lost with the transition from fishes to land vertebrates. The immunohistochemical localization of calbindin (CB) and calretinin (CR) provides a powerful method for discerning segregated neuronal populations, fiber tracts, and neuropils and is here applied to the brains of Neoceratodus and Protopterus, representing the two extant orders of lungfishes. The results showed abundant cells containing these proteins in pallial and subpallial telencephalic regions, with particular distinct distribution in the basal ganglia, amygdaloid complex, and septum. Similarly, the distribution of CB and CR containing cells supports the division of the hypothalamus of lungfishes into neuromeric regions, as in tetrapods. The dense concentrations of CB and CR positive cells and fibers highlight the extent of the thalamus. As in other vertebrates, the optic tectum is characterized by numerous CB positive cells and fibers and smaller numbers of CR cells. The so-called cerebellar nucleus contains abundant CB and CR cells with long ascending axons, which raises the possibility that it could be homologized to the secondary gustatory nucleus of other vertebrates. The corpus of the cerebellum is devoid of CB and CR and cells positive for both proteins are found in the cerebellar auricles and the octavolateralis nuclei. Comparison with other vertebrates reveals that lungfishes share most of their features of calcium binding protein distribution with amphibians, particularly with salamanders.

Immunohistochemical Localization of DARPP-32 in the Brain of Two Lungfishes: Further Assessment of Its Relationship with the Dopaminergic System.

The distribution of DARPP-32 (a phosphoprotein related to the dopamine D1 receptor) has been widely used as a means to clarify the brain regions with dopaminoceptive cells, primarily in representative species of tetrapods. The relationship between dopaminergic and dopaminoceptive elements is frequently analyzed using the catecholamine marker tyrosine hydroxylase (TH). In the present study, by means of combined immunohistochemistry, we have analyzed these relationships in lungfishes, the only group of sarcopterygian fishes represented by 6 extant species that are the phylogenetically closest living relatives of tetrapods. We used the Australian lungfish Neoceratodus forsteri and the African lungfish Protopterus dolloi. The DARPP-32 antibody yields a distinct and consistent pattern of neuronal staining in brain areas that, in general, coincide with areas that are densely innervated by TH-immunoreactive fibers. The striatum, thalamus, optic tectum, and torus semicircularis contain intensely DARPP-32-immunoreactive cell bodies and fibers. Cells are also located in the olfactory bulbs, amygdaloid complex, lateral septum, pallidum, preoptic area, suprachiasmatic nucleus, tuberal hypothalamic region, rostral rhombencephalic reticular formation, superior raphe nucleus, octavolateral area, solitary tract nucleus, and spinal cord. Remarkably, DARPP-32-immunoreactive fibers originating in the striatum reach the region of the dopaminergic cells in the mesencephalic tegmentum and represent a well-established striatonigral pathway in lungfishes. Double immunolabeling reveals that DARPP-32 is present in neurons that most likely receive TH input, but it is absent from the catecholaminergic neurons themselves, with the only exception of a few cells in the suprachiasmatic nucleus of Neoceratodus and the solitary tract nucleus of Protopterus. In addition, some species differences exist in the localization of DARPP-32 cells in the pallium, lateral amygdala, thalamus, prethalamus, and octavolateral area. In general, the present study demonstrates that the distribution pattern of DARPP-32, and its relationship with TH, is largely comparable to those reported for tetrapods, highlighting a shared situation among all sarcopterygians.

Gene expression analysis of developing cell groups in the pretectal region of Xenopus laevis.

Our previous analysis of progenitor domains in the pretectum of Xenopus revealed three molecularly distinct anteroposterior subdivisions, identified as precommissural (PcP), juxtacommissural (JcP), and commissural (CoP) histogenetic domains (Morona et al. [2011] J Comp Neurol 519:1024-1050). Here we analyzed at later developmental stages the nuclei derived from these areas, attending to their gene expression patterns and histogenesis. Transcription-factor gene markers were used to selectively map derivatives of each domain: Pax7 and Pax6 (CoP); Foxp1 and Six3 (JcP); and Xiro1, VGlut2, Ebf1, and Ebf3 (PcP). Additional genoarchitectural information was provided by the expression of Gbx2, NPY, Lhx1, and Lhx9. This allowed both unambiguous characterization of the anuran pretectal nuclei with regard to their origin in the three early anteroposterior progenitor domains, and their comparison with counterparts in the chick and mouse pretectum. Our observations demonstrated a molecular conservation, during practically all the stages analyzed, for most of the main markers used to define genoarchitecturally the main derivatives of each pretectal domain. We found molecular evidence to propose homologous derivatives from the CoP (olivary pretectal, parvocellular, and magnocellular posterior commissure and lateral terminal nuclei), JcP (spiriformis lateral and lateral terminal nuclei), and PcP (anterior pretectal nucleus) to those described in avian studies. These results represent significant progress in the comprehension of the diencephalic region of Xenopus and show that the organization of the pretectum possesses many features shared with birds. J. Comp. Neurol. 525:715-752, 2017. © 2016 Wiley Periodicals, Inc.

Organization of the nitrergic neuronal system in the primitive bony fishes Polypterus senegalus and Erpetoichthys calabaricus (Actinopterygii: Cladistia).

Cladistians are a group of basal actinopterygian fishes that constitute a good model for studying primitive brain features, most likely present in the ancestral bony fishes. The analysis of the nitrergic neurons (with the enzyme nitric oxide synthase; NOS) has helped in understanding important aspects of brain organization in all vertebrates studied. We investigated the nitrergic system of two cladistian species by means of specific antibodies against NOS and NADPH-diaphorase (NADPH-d) histochemistry, which, with the exception of the primary olfactory and terminal nerve fibers, labeled only for NADPH-d, yielded identical results. Double immunohistochemistry was conducted for simultaneous detection of NOS with tyrosine hydroxylase, choline acetyltransferase, calbindin, calretinin, and serotonin, to establish accurately the localization of the nitrergic neurons and fibers and to assess possible interactions between these neuroactive substances. The pattern of distribution in both species showed only subtle differences in the density of labeled cells. Distinct groups of NOS-immunoreactive cells were observed in pallial and subpallial areas, paraventricular region, tuberal and retromammillary hypothalamic areas, posterior tubercle, prethalamic and thalamic areas, optic tectum, torus semicircularis, mesencephalic tegmentum, interpeduncular nucleus, superior and middle reticular nuclei, magnocellular vestibular nucleus, solitary tract nucleus, nucleus medianus magnocellularis, the spinal cord and amacrine cells in the retina. Large neurons in cranial nerve sensory ganglia were also labeled. The comparison of these results with those from other vertebrates, using a neuromeric analysis, reveals a conserved pattern of organization of the nitrergic system from this primitive fish group to amniotes, including mammals.

Prepatterning and patterning of the thalamus along embryonic development of Xenopus laevis.

Previous developmental studies of the thalamus (alar part of the diencephalic prosomere p2) have defined the molecular basis for the acquisition of the thalamic competence (preparttening), the subsequent formation of the secondary organizer in the zona limitans intrathalamica, and the early specification of two anteroposterior domains (rostral and caudal progenitor domains) in response to inducing activities and that are shared in birds and mammals. In the present study we have analyzed the embryonic development of the thalamus in the anuran Xenopus laevis to determine conserved or specific features in the amphibian diencephalon. From early embryonic stages to the beginning of the larval period, the expression patterns of 22 markers were analyzed by means of combined In situ hybridization (ISH) and immunohistochemical techniques. The early genoarchitecture observed in the diencephalon allowed us to discern the boundaries of the thalamus with the prethalamus, pretectum, and epithalamus. Common molecular features were observed in the thalamic prepatterning among vertebrates in which Wnt3a, Fez, Pax6 and Xiro1 expression were of particular importance in Xenopus. The formation of the zona limitans intrathalamica was observed, as in other vertebrates, by the progressive expression of Shh. The largely conserved expressions of Nkx2.2 in the rostral thalamic domain vs. Gbx2 and Ngn2 (among others) in the caudal domain strongly suggest the role of Shh as morphogen in the amphibian thalamus. All these data showed that the molecular characteristics observed during preparttening and patterning in the thalamus of the anuran Xenopus (anamniote) share many features with those described during thalamic development in amniotes (common patterns in tetrapods) but also with zebrafish, strengthening the idea of a basic organization of this diencephalic region across vertebrates.

Expression and function of the LIM-homeodomain transcription factor Islet-1 in the developing and mature vertebrate retina.

The LIM-homeodomain transcription factor Islet-1 (Isl1) has been widely used as a marker of different subtypes of neurons in the developing and mature retina of vertebrates. During retinal neurogenesis, early Isl1 expression is detected in the nuclei of neuroblasts that give rise to ganglion, amacrine, bipolar, and horizontal cells. In the mature retina, Isl1 expression is restricted to the nuclei of ganglion cells, cholinergic amacrine cells, ON-bipolar cells, and subpopulations of horizontal cells. Recent studies have explored the functional mechanisms of Isl1 during specification and differentiation of these retinal cell types. Thus, conditional inactivation of Isl1 in the developing mouse retina disrupts retinal function, and also results in optic nerve hypoplasia, marked reductions in mature ganglion, amacrine, and bipolar cells, and a substantial increase in horizontal cells. Furthermore, conditional knockout shows delayed ganglion cell axon growth, ganglion cell axon guidance error, and ganglion cell nerve fiber defasciculation. These data together suggest a possible role for Isl1 in the early differentiation and maintenance of different vertebrate retinal cell types. This review examines whether the expression pattern of Isl1 during vertebrate retinal development is conserved across vertebrate species, and discusses current understanding of the developmental functions of Isl1 in retinogenesis.

Conserved localization of Pax6 and Pax7 transcripts in the brain of representatives of sarcopterygian vertebrates during development supports homologous brain regionalization.

Many of the genes involved in brain patterning during development are highly conserved in vertebrates and similarities in their expression patterns help to recognize homologous cell types or brain regions. Among these genes, Pax6 and Pax7 are expressed in regionally restricted patterns in the brain and are essential for its development. In the present immunohistochemical study we analyzed the distribution of Pax6 and Pax7 cells in the brain of six representative species of tetrapods and lungfishes, the closest living relatives of tetrapods, at several developmental stages. The distribution patterns of these transcription factors were largely comparable across species. In all species only Pax6 was expressed in the telencephalon, including the olfactory bulbs, septum, striatum, and amygdaloid complex. In the diencephalon, Pax6 and Pax7 were distinct in the alar and basal parts, mainly in prosomeres 1 and 3. Pax7 specifically labeled cells in the optic tectum (superior colliculus) and Pax6, but not Pax7, cells were found in the tegmentum. Pax6 was found in most granule cells of the cerebellum and Pax7 labeling was detected in cells of the ventricular zone of the rostral alar plate and in migrated cells in the basal plate, including the griseum centrale and the interpeduncular nucleus. Caudally, Pax6 cells formed a column, whereas the ventricular zone of the alar plate expressed Pax7. Since the observed Pax6 and Pax7 expression patterns are largely conserved they can be used to identify subdivisions in the brain across vertebrates that are not clearly discernible with classical techniques.

Identification of striatal and pallidal regions in the subpallium of anamniotes.

The telencephalic basal ganglia (BG) of amniotes consist of two subdivisions, striatum and pallidum, which share many features, including development, cell types, neurotransmitter organization and hodology. In particular, these two subdivisions during development are defined on the basis of discrete gene expression patterns (genoarchitecture or genoarchitectonics). The characterization of the BG in the subpallium of representatives of the different classes of anamniote vertebrates was first approached in studies dealing with their localization, hodology and main neurochemical characteristics. Thus, it was proposed that an impressive degree of conservation exists across species. New insights can be gained by the comparative analysis of the expression of conserved transcription factors that distinctly define the striatal and pallidal components of the BG in all vertebrates. In addition, the expression of other genes that characterize neighboring regions of the BG is also useful to define the boundaries of each subdivision. Following this approach, we have analyzed the BG in the brain of juvenile representatives of amphibians, lungfishes, holosteans, Polypteriformes and Chondrichthyes. In addition, we briefly review previous data in teleosts and agnathans. The markers used include islet 1 and Dlx as striatal markers, whereas Nkx2.1 is essential for the identification of the pallidum. In turn, Pax6 and in particular Tbr1 are expressed in the pallium. These markers have been systematically analyzed in combination with neuronal markers of specific subpallial territories and cell populations, such as tyrosine hydroxylase, γ-aminobutyric acid, nitric oxide synthase, substance P and enkephalin. The results highlight that many genes share common distribution patterns and are arranged in conserved combinations whose identification has served to define homologies between components of the BG in distant species.

Immunohistochemical analysis of Pax6 and Pax7 expression in the CNS of adult Xenopus laevis.

Pax6 and Pax7 are transcription factors essential for the development of the CNS. In addition, increasing data, mainly obtained in amniotes, support that they are expressed in subsets of neurons in the adult, likely playing a role in maintaining neuron type identity. In the present study we analyzed the detailed distribution of Pax6 and Pax7 cells in the adult CNS of Xenopus laevis. Immunohistochemistry with antibodies that are required for high-resolution analysis of Pax-expressing cells was conducted. A wide distribution of Pax6 and Pax7 cells throughout the CNS was detected, with distinct patterns that showed only slight overlapping. Only Pax6 was expressed in the telencephalon, including the olfactory bulbs, septum, striatum and amygdaloid complex. In the diencephalon, Pax6 and Pax7 were distinct in the alar and basal parts, respectively, of prosomere 3. Large numbers of Pax6 and Pax7 cells were distributed in the pretectal region (alar plate of prosomere 1) but only Pax6 cells extended into basal plate. Pax7 specifically labeled cells in the optic tectum, including the ventricular zone, and Pax6 cells were the only cells found in the tegmentum. Pax6 was found in most granule cells of the cerebellum and Pax7 expression was found only in the ventricular zone. In the rostral rhombomere 1, Pax7 labeling was detected in cells of the ventricular zone of the alar plate, but numerous migrated cells were located in the basal plate, including the griseum centrale and the interpeduncular nucleus. Pax6 cells also formed a column of scattered neurons in the reticular formation and were found in the octavolateral area. The rhombencephalic ventricular zone of the alar plate expressed Pax7. Dorsal Pax7 cells and ventral Pax6 cells were found along the spinal cord separated from the ventricle, which did not show immunoreactivity. Our results show that the expression of Pax6 and Pax7 is widely maintained in the adult brain of Xenopus, like in urodele amphibians and in contrast to the situation described in amniotes. Therefore, in amphibians these transcription factors seem to be needed to maintain specific entities of subpopulations of neurons in the adult CNS.