Ruth Morona

Subdivisions of the turtle Pseudemys scripta hypothalamus based on the expression of regulatory genes and neuronal markers

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. J. Comp. Neurol., 2012;520:453–478.

Calbindin-D28k and calretinin expression in the forebrain of anuran and urodele amphibians: Further support for newly identified subdivisions

A general pattern of organization of the forebrain shared by amphibians, mainly anurans, and amniotes has been proposed considering the relative topography of the territories, their connectivity, and their neurochemistry. These criteria were needed because the amphibians possess limited cell migration from the ventricle that precludes a parcellation into circumscribed nuclei. In the present study we have tested the identity of most newly described forebrain territories in anurans and urodeles with regard to their content in calbindin‐D28k (CB) and calretinin (CR). By means of immunohistochemistry, these proteins were demonstrated to be particularly abundant and specifically distributed in the amphibian forebrain and were extremely useful markers for delineating nuclear boundaries otherwise indistinguishable. In the telencephalon, labeled cells in the pallium allowed the identification of particular regions with marked differences between anurans and urodeles, whereas the subpallium showed more conservative patterns of distribution. In particular, the components of the amygdaloid complex and the basal ganglia were distinctly labeled. The distribution in the nonevaginated secondary prosencephalon and diencephalon showed abundant common features between anurans and urodeles, highlighted using the prosomeric model for the comparison. In the pretectum, thalamus, and prethalamus of urodeles, the CB and CR staining was particularly suitable for the identification of diverse structures within the simple periventricular gray layer. However, the analysis across species also revealed a considerable degree of heterogeneity, even within comparatively well‐defined neuronal populations. Therefore, the content of a particular calcium binding protein in a neuronal group is not a fully reliable criterion for considering homologies. J. Comp. Neurol. 511:187–220, 2008.

Lungfishes, Like Tetrapods, Possess a Vomeronasal System

The vomeronasal system (VNS) is an accessory olfactory system that in tetrapod vertebrates is composed of specific receptor neurons in the nasal organ and a set of centers in the forebrain that receive and relay the information consecutively towards the hypothalamus. Thus, only in tetrapods the VNS comprises a discrete vomeronasal (Jacobsons) organ, which contains receptor cells that are morphologically distinct from those of the olfactory epithelium and use different transduction mechanisms. The axons of the vomeronasal receptors in tetrapods project to the accessory olfactory bulb (AOB) in the rostral telencephalon. Secondary vomeronasal connections exist through the medial amygdala to the hypothalamus. Currently, the lungfishes are considered the closest living relatives of tetrapods. Here we show that the African lungfish, Protopterus dolloi, has epithelial crypts at the base of the lamellae of the olfactory epithelium that express markers of the vomeronasal receptors in tetrapods. The projections of these crypts allow us to identify an AOB on the lateral margin of the main olfactory bulb. The projections of this AOB reach a region that is topologically, hodologically, and immunohistochemically identical to the medial amygdala and could represent its homolog. Neurons of this putative medial amygdala were demonstrated to project to the lateral hypothalamus, as they do in tetrapods. All these features that lungfishes share with tetrapods indicate that lungfishes have the complete set of brain centers and connections involved in processing vomeronasal information and that these features were already present in the last common ancestor of lungfishes and tetrapods.

Immunohistochemical localization of calbindin‐D28k and calretinin in the brainstem of anuran and urodele amphibians

Calbindin‐D28k (CB) and calretinin (CR) are calcium binding proteins present in distinct sets of neurons; they act as buffers regulating the concentration of intracellular calcium. CB and CR immunohistochemistry was studied in the brainstem of anuran and urodele amphibians in combination with other markers (choline acetyltransferase, tyrosine hydroxylase, and nitric oxide synthase), which served to clarify the localization and signature of many cell groups. CR labeled the retinorecipient layers of the optic tectum, and CB and CR labeled distinct tectal cell populations. The two proteins were largely complementary in the torus semicircularis and marked auditory and lateral line sensory regions, depending on the species. CB and CR in the mesencephalic and isthmic tegmentum specified the boundaries of basal and medial longitudinal bands. In the cerebellum, CB labeled Purkinje cells in all species, whereas CR was mainly found in fibers and labeled Purkinje cells only in Rana. In the parabrachial region, CB and CR allowed the distinction of the laterodorsal tegmental nucleus, isthmic nucleus, locus coeruleus, and rostral octavolateral nuclei. The distribution of CB‐ and CR‐immunoreactive cells in the reticular formation and central gray was consistent with the current models of brainstem segmentation in amphibians. CR was found in the auditory fibers and nuclei in Rana and in mechanosensory lateral line fibers in Xenopus and urodeles, whereas CB mainly labeled vestibular fibers and nuclei in all species. These results highlight the anatomical complexity of the amphibian brainstem and help in an understanding of its regional organization that is not cytoarchitectonically evident. J. Comp. Neurol. 515:503–537, 2009.

Characterization of the bed nucleus of the stria terminalis in the forebrain of anuran amphibians

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. J. Comp. Neurol. 520:330–363, 2012.

Characterization of the hypothalamus of Xenopus laevis during development. I. The alar regions

The patterns of expression of a set of conserved developmental regulatory transcription factors and neuronal markers were analyzed in the alar hypothalamus of Xenopus laevis throughout development. Combined immunohistochemical and in situ hybridization techniques were used for the identification of subdivisions and their boundaries. The alar hypothalamus was located rostral to the diencephalon in the secondary prosencephalon and represents the rostral continuation of the alar territories of the diencephalon and brainstem, according to the prosomeric model. It is composed of the supraoptoparaventricular (dorsal) and the suprachiasmatic (ventral) regions, and limits dorsally with the preoptic region, caudally with the prethalamic eminence and the prethalamus, and ventrally with the basal hypothalamus. The supraoptoparaventricular area is defined by the orthopedia (Otp) expression and is subdivided into rostral and caudal portions, on the basis of the Nkx2.2 expression only in the rostral portion. This region is the source of many neuroendocrine cells, primarily located in the rostral subdivision. The suprachiasmatic region is characterized by Dll4/Isl1 expression, and was also subdivided into rostral and caudal portions, based on the expression of Nkx2.1/Nkx2.2 and Lhx1/7 exclusively in the rostral portion. Both alar regions are mainly connected with subpallial areas strongly implicated in the limbic system and show robust intrahypothalamic connections. Caudally, both regions project to brainstem centers and spinal cord. All these data support that in terms of topology, molecular specification, and connectivity the subdivisions of the anuran alar hypothalamus possess many features shared with their counterparts in amniotes, likely controlling similar reflexes, responses, and behaviors. J. Comp. Neurol. 521:725–759, 2013.

Embryonic genoarchitecture of the pretectum in Xenopus laevis: a conserved pattern in tetrapods.

Networked gene activities control the evolutionarily conserved histogenetic organization of the central nervous system of vertebrates. Genoarchitectonic studies contribute to the analysis of each morphogenetic field by identifying distinct progenitor domains and corresponding derivatives whose pattern of gene expression shows a unique combinatory code. Previous studies in the pretectal region (caudal diencephalon) have defined three anteroposterior genoarchitectonic domains that are conserved in birds and mammals. Here, we have studied the embryonic pretectal genoarchitecture in the amphibian Xenopus laevis, in order to determine whether it is possible to define a comparable anteroposterior tripartition of the amphibian pretectal area. The expression patterns of 14 genes mapped from early embryonic stages to metamorphic climax allowed us to define the boundaries of the pretectum, the expected precommissural, juxtacommissural, and commissural anteroposterior domains, and some dorsoventral subdivisions. Taken together, our data provide evidence for a conserved pattern of pretectal domains and subdomains, shared by amniotes and amphibian anamniotes (tetrapods), understandable as part of a general Bauplan in vertebrates. J. Comp. Neurol. 519:1024–1050, 2011.

Immunohistochemical localization of calbindin-D28k and calretinin in the spinal cord of Xenopus laevis

Immunohistochemical techniques were used to investigate the distribution and morphology of neurons containing the calcium‐binding proteins calbindin‐D28k (CB) and calretinin (CR) in the spinal cord of Xenopus laevis and determine the extent to which this organization is comparable to that of mammals. Most CB‐ and CR‐containing neurons were located in the superficial dorsal gray field, but with distinct topography. The lateral, ventrolateral, and ventromedial fields also possessed abundant neurons labeled for either CB or CR. Double immunohistofluorescence demonstrated that a subpopulation of dorsal root ganglion cells and neurons in the dorsal and ventrolateral fields contained CB and CR. By means of a similar technique, a cell population in the dorsal field was doubly labeled only for CB and nitric oxide synthase (NOS), whereas in the ventrolateral field colocalization of NOS with CB and CR was found. Choline acetyltransferase immunohistochemistry revealed that a subpopulation of ventral horn neurons, including motoneurons, colocalized CB and CR. The involvement of CB‐ and CR‐containing neurons in ascending spinal projections was demonstrated combining the retrograde transport of dextran amines and immunohistochemistry. Cells colocalizing the tracer and CB or CR were quite numerous, primarily in the dorsal and ventrolateral fields. Similar experiments demonstrated supraspinal projections from CB‐ and CR‐containing cells in the brainstem and diencephalon. The distribution, projections, and colocalization with neurotransmitters of the neuronal systems containing CB and CR in Xenopus suggest that CB and CR are important neuromodulator substances with functions conserved in the spinal cord from amphibians through mammals. J. Comp. Neurol. 494:763–783, 2006.

Expression patterns of Pax6 and Pax7 in the adult brain of a urodele amphibian, Pleurodeles waltl.

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. J. Comp. Neurol. 521:2088–2124, 2013.

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.