Functional depletion of the alternative splicing factors Muscleblind-like (MBNL 1 and 2) is at the basis of the neuromuscular disease myotonic dystrophy type 1 (DM1). We previously showed the efficacy of miRNA downregulation in Drosophila DM1 model. Here, we screen for miRNAs that regulate MBNL1 and MBNL2 in HeLa cells. We thus identify miR-23b and miR-218, and confirm that they downregulate MBNL proteins in this cell line. Antagonists of miR-23b and miR-218 miRNAs enhance MBNL protein levels and rescue pathogenic missplicing events in DM1 myoblasts. Systemic delivery of these "antagomiRs" similarly boost MBNL expression and improve DM1-like phenotypes, including splicing alterations, histopathology, and myotonia in the HSALR DM1 model mice. These mammalian data provide evidence for therapeutic blocking of the miRNAs that control Muscleblind-like protein expression in myotonic dystrophy.

The orexins (hypocretins) are peptides found primarily in neurons of the hypothalamus of all vertebrates. Many differences were reported about the precise location of orexin containing cells and their projections throughout the brain in different species. However, there are few direct cross-species comparisons. Previous studies in anuran amphibians have also reported notable species differences. We examined and directly compared the distribution of orexinergic neurons and fibers within the brains of representatives of the three amphibian orders, anurans, urodeles and gymnophionans. Simultaneous detection of orexins and tyrosine hydroxylase was used to assess the precise location of the orexins in the brain and to evaluate the possible influence of the orexin system on the catecholaminergic cell groups. Although some differences were noted, a common pattern for the distribution of orexins in amphibians was observed. In all species, most immunoreactive neurons were observed in the suprachiasmatic nucleus, whereas the cells in the preoptic area and the tuberal region were more variable. Orexin immunoreactive fibers in the brain of all species included abundant fibers throughout the preoptic area and hypothalamus, whereas moderate amounts of fibers were present in the pallium, striatum, septum, thalamus, optic tectum, torus semicircularis, rhombencephalon and spinal cord. The use of double immunohistochemistry in amphibians revealed orexinergic innervation in dopaminergic and noradrenergic cell groups, such as the midbrain tegmentum, locus coeruleus and nucleus of the solitary tract, as was previously reported in mammals.

Previous studies in amphibians yielded contradictory results about the distribution of calbindin-D28k (CB) and calretinin (CR) in retinal neurons, most likely due to the different antibodies used. The present comparative study aimed to characterize the distribution of CB and CR in relation to retinal neurons in six species of anuran and urodele amphibians by using the same immunohistochemical protocol with specific poly- and monoclonal antibodies. CB was specifically found in cones, in subpopulations of bipolar and amacrine cells and in sparse neurons in the ganglion cell layer. All photoreceptors were negative for CR, whereas subpopulations of horizontal, bipolar and amacrine cells as well as cells in the ganglion cell layer contained CR. CB/CR colocalization occurred in some amacrine cells and in cells of the ganglion cell layer, with variable proportions among species. Most of the ganglion cells identified by retrograde labeling from the optic nerve contained CB and/or CR. Cholinergic cells, visualized by choline acetyltransferase (ChAT) immunoreactivity, constituted a subpopulation of the CR-positive amacrine cells in anurans and a high percentage (40-90%) of cholinergic cells were CR immunoreactive in urodeles. CB/ChAT colocalization was between 10 and 30% in anurans and lower in urodeles (7-10%). Finally, CB colocalized in 6-8% of the tyrosine hydroxylase (TH) amacrine cells only in anurans, whereas CR and TH colocalized in 5% of TH cells in the urodele retina. Our data suggest a specific pattern for CB and CR distribution in the retinal neurons of amphibians comparable to amniotes in some cell types showing, however, peculiar features not observed previously in other 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.

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.

Neuropeptide FF-like immunoreactive (NPFFir) cells and fibers were analyzed through development of Xenopus laevis. The first NPFFir cells appeared in the embryonic hypothalamus, which projected to the intermediate lobe of the hypophysis, the brainstem and spinal cord. Slightly later, scattered NPFFir cells were present in the olfactory bulbs and ventral telencephalon. In the caudal medulla, NPFFir cells were observed in the nucleus of the solitary tract only at embryonic and early larval stages. Abundant NPFFir cells and fibers were demonstrated in the spinal cord. The sequence of appearance observed in Xenopus shares many developmental features with mammals although notable differences were observed in the telencephalon and hypothalamus. In general, NPFF immunoreactivity developed earlier in amphibians than in mammals.

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.

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 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 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.

Expression patterns of Pax6, Pax7, and, to a lesser extent, Pax3 genes were analyzed by a combination of immunohistochemical techniques in the central nervous system of adult specimens of the urodele amphibian Pleurodeles waltl. Only Pax6 was found in the telencephalon, specifically the olfactory bulbs, striatum, septum, and lateral and central parts of the amygdala. In the diencephalon, Pax6 and Pax7 were distinct in the alar and basal parts, respectively, of prosomere 3. The distribution of Pax6, Pax7, and Pax3 cells correlated with the three pretectal domains. Pax7 specifically labeled cells in the dorsal mesencephalon, mainly in the optic tectum, and Pax6 cells were the only cells found in the tegmentum. Large populations of Pax7 cells occupied the rostral rhombencephalon, along with lower numbers of Pax6 and Pax3 cells. Pax6 was found in most granule cells of the cerebellum. 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. Our results show that the expression of Pax6 and Pax7 is widely maintained in the brains of adult urodeles, in contrast to the situation in other tetrapods. This discrepancy could be due to the generally pedomorphic features of urodele brains. Although the precise role of these transcription factors in adult brains remains to be determined, our findings support the idea that they may also function in adult urodeles.

The complexity of the pallium during evolution has increased dramatically in many different respects. The highest level of complexity is found in mammals, where most of the pallium (cortex) shows a layered organization and neurons are generated during development following an inside-out order, a sequence not observed in other amniotes (birds and reptiles). Species-differences may be related to major neurogenetic events, from the neural progenitors that divide and produce all pallial cells. In mammals, two main types of precursors have been described, primary precursor cells in the ventricular zone (vz; also called radial glial cells or apical progenitors) and secondary precursor cells (called basal or intermediate progenitors) separated from the ventricle surface. Previous studies suggested that pallial neurogenetic cells, and especially the intermediate progenitors, evolved independently in mammalian and sauropsid lineages. In the present study, we examined pallial neurogenesis in the amphibian Xenopus laevis, a representative species of the only group of tetrapods that are anamniotes. The pattern of pallial proliferation during embryonic and larval development was studied, together with a multiple immunohistochemical analysis of putative progenitor cells. We found that there are two phases of progenitor divisions in the developing pallium that, following the radial unit concept from the ventricle to the mantle, finally result in an outside-in order of mature neurons, what seems to be the primitive condition of vertebrates. Gene expressions of key transcription factors that characterize radial glial cells in the vz were demonstrated in Xenopus. In addition, although mitotic cells were corroborated outside the vz, the expression pattern of markers for intermediate progenitors differed from mammals.

The organization of the somatostatin-like-immunoreactive (SOM-ir) structures in the brain of anuran and urodele amphibians has been well documented, and significant differences were noted between the two amphibian orders. However, comparable data are not available for the third order of amphibians, the gymnophionans (caecilians). In the present study, we analyzed the anatomical distribution of SOM-ir cells and fibers in the brain of the gymnophionan Dermophis mexicanus. In addition, because of its known relationship with catecholamines in other vertebrates, double immunostaining for SOM and tyrosine hydroxylase was used to investigate this situation in the gymnophionan. Abundant SOM-ir cell bodies and fibers were widely distributed throughout the brain. In the telencephalon, pallial and subpallial cells were labeled, being most numerous in the medial pallium and amygdaloid region. Most of the SOM-ir neurons were found in the preoptic area and hypothalamus and showed a clear projection to the median eminence. Less conspicuously, SOM-ir structures were found in the thalamus, tectum, tegmentum, and reticular formation. Both SOM-ir cells and fibers were demonstrated in the spinal cord. The double-immunohistofluorescence technique revealed that catecholaminergic neurons and SOM-ir cells are largely intermingled in many brain regions but form totally separated populations. Many differences were found between the distribution of SOM-ir structures in Dermophis and that in anurans or urodeles. Some features were shared only with anurans, such as the abundant pallial SOM-ir cells, whereas others were common only to urodeles, such as the organization of the hypothalamohypophysial SOM-ir system. In addition, some characteristics were found only in Dermophis, such as the localization of the SOM-ir spinal cells and the lack of colocalization of catecholamines and SOM throughout the brain. Therefore, any conclusions concerning the SOM system in amphibians are incomplete without considering evidence for gymnophionans.

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.

Myotonic dystrophy type 1 (DM1) is a rare neuromuscular disease caused by expansion of unstable CTG repeats in a non-coding region of the DMPK gene. CUG expansions in mutant DMPK transcripts sequester MBNL1 proteins in ribonuclear foci. Depletion of this protein is a primary contributor to disease symptoms such as muscle weakness and atrophy and myotonia, yet upregulation of endogenous MBNL1 levels may compensate for this sequestration. Having previously demonstrated that antisense oligonucleotides against miR-218 boost MBNL1 expression and rescue phenotypes in disease models, here we provide preclinical characterization of an antagomiR-218 molecule using the HSALR mouse model and patient-derived myotubes. In HSALR, antagomiR-218 reached 40-60 pM 2 weeks after injection, rescued molecular and functional phenotypes in a dose- and time-dependent manner, and showed a good toxicity profile after a single subcutaneous administration. In muscle tissue, antagomiR rescued the normal subcellular distribution of Mbnl1 and did not alter the proportion of myonuclei containing CUG foci. In patient-derived cells, antagomiR-218 improved defective fusion and differentiation and rescued up to 34% of the gene expression alterations found in the transcriptome of patient cells. Importantly, miR-218 was found to be upregulated in DM1 muscle biopsies, pinpointing this microRNA (miRNA) as a relevant therapeutic target.

Skeletal muscle symptoms strongly contribute to mortality of myotonic dystrophy type 1 (DM1) patients. DM1 is a neuromuscular genetic disease caused by CTG repeat expansions that, upon transcription, sequester the Muscleblind-like family of proteins and dysregulate alternative splicing of hundreds of genes. However, mis-splicing does not satisfactorily explain muscle atrophy and wasting, and several other contributing factors have been suggested, including hyperactivated autophagy leading to excessive catabolism. MicroRNA (miR)-7 has been demonstrated to be necessary and sufficient to repress the autophagy pathway in cell models of the disease, but the origin of its low levels in DM1 was unknown. We have found that the RNA-binding protein Musashi-2 (MSI2) is upregulated in patient-derived myoblasts and biopsy samples. Because it has been previously reported that MSI2 controls miR-7 biogenesis, we tested the hypothesis that excessive MSI2 was repressing miR-7 maturation. Using gene-silencing strategies (small interfering RNAs [siRNAs] and gapmers) and the small molecule MSI2-inhibitor Ro 08-2750, we demonstrate that reducing MSI2 levels or activity boosts miR-7 expression, represses excessive autophagy, and downregulates atrophy-related genes of the UPS system. We also detect a significant upregulation of MBNL1 upon MSI2 silencing. Taken together, we propose MSI2 as a new therapeutic target to treat muscle dysfunction in DM1.

The symptoms of Myotonic Dystrophy Type 1 (DM1) are multi-systemic and life-threatening. The neuromuscular disorder is rooted in a non-coding CTG microsatellite expansion in the DM1 protein kinase (DMPK) gene that, upon transcription, physically sequesters the Muscleblind-like (MBNL) family of splicing regulator proteins. The high-affinity binding occurring between the proteins and the repetitions disallow MBNL proteins from performing their post-transcriptional splicing regulation leading to downstream molecular effects directly related to disease symptoms such as myotonia and muscle weakness. In this study, we build on previously demonstrated evidence showing that the silencing of miRNA-23b and miRNA-218 can increase MBNL1 protein in DM1 cells and mice. Here, we use blockmiR antisense technology in DM1 muscle cells, 3D mouse-derived muscle tissue, and in vivo mice to block the binding sites of these microRNAs in order to increase MBNL translation into protein without binding to microRNAs. The blockmiRs show therapeutic effects with the rescue of mis-splicing, MBNL subcellular localization, and highly specific transcriptomic expression. The blockmiRs are well tolerated in 3D mouse skeletal tissue inducing no immune response. In vivo, a candidate blockmiR also increases Mbnl1/2 protein and rescues grip strength, splicing, and histological phenotypes.

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.

The patterns of distribution of a set of conserved brain developmental regulatory transcription factors and neuronal markers were analyzed in the subpallium of the juvenile turtle, Pseudemys scripta. Immunohistochemical techniques were used with a combination of primary antibodies for the identification of the main boundaries and subdivisions in the basal telencephalon. In the basal ganglia, the combinatorial expression on Pax6, Nkx2.1, and GABA was a powerful tool for the identification of the nucleus accumbens, the dorsal portion of the striatum, and the pallidal regions. It was also possible to suggest migratory streams of neurons from the pallidum into the striatal regions. On the basis of GABA, Pax6, Tbr1, tyrosine hydroxylase, Darpp32, and Nkx2.1 combinatorial expression patterns, the boundaries of the septal subdivisions and their embryological origin were assessed. In particular, the bed nucleus of the stria terminalis was identified. Within the amygdaloid complex, the striatal central amygdala was characterized by Pax6 expression, whereas Orthopedia gene expression highlighted, at least, a subdivision of the medial amygdala. A newly identified preoptic commissural area and the boundaries of the preoptic area were assessed, mainly by the localization of Nkx2.1 expression. Finally, additional data were obtained by combining immunohistochemistry and tracing techniques on the interneuronal nature of the cholinerginergic, nitrergic, and Nkx2.1-positive striatal cells. Taken together, all the results of the present study allowed recognizing main features in the organization of the subpallium in reptiles that, in most cases, are shared with other amniotes and amphibians.

The patterns of distribution of a set of conserved brain developmental regulatory transcription factors and neuronal markers were analyzed in the hypothalamus of the juvenile turtle, Pseudemys scripta. Combined immunohistochemical techniques were used for the identification of the main boundaries and subdivisions in the optic, paraventricular, tuberal, and mammillary hypothalamic regions. The combination of Tbr1 and Pax6 with Nkx2.1 allowed identification of the boundary between the telencephalic preoptic area, rich in Nkx2.1 expression, and the prethalamic eminence, rich in Tbr1 expression. In addition, at this level Nkx2.2 expression defined the boundary between the telencephalon and the hypothalamus. The dorsalmost hypothalamic domain was the supraoptoparaventricular region that was defined by the expression of Otp/Pax6 and the lack of Nkx2.1/Isl1. It is subdivided into rostral, rich in Otp and Nkx2.2, and caudal, only Otp-positive, portions. Ventrally, the suprachiasmatic area was identified by its catecholaminergic groups and the lack of Otp, and could be further divided into a rostral portion, rich in Nkx2.1 and Nkx2.2, and a caudal portion, rich in Isl1 and devoid of Nkx2.1 expression. The expressions of Nkx2.1 and Isl1 defined the tuberal hypothalamus, whereas only the rostral portion expressed Otp. Its caudal boundary was evident by the lack of Isl1 in the adjacent mammillary area, which expressed Nkx2.1 and Otp. All these results provide an important set of data on the interpretation of the hypothalamic organization in a reptile, and hence make a useful contribution to the understanding of hypothalamic evolution.