Nerea Moreno

LIM-homeodomain genes as developmental and adult genetic markers of Xenopus forebrain functional subdivisions

We have investigated the expression patterns of five LIM‐homeodomain (LIM‐hd) genes, x‐Lhx1, x‐Lhx2, x‐Lhx5, x‐Lhx7, and x‐Lhx9 in the forebrain of the frog Xenopus laevis during larval development and in the adult. The results were analyzed in terms of neuromeric organization of the amphibian brain and of combinatorial LIM‐hd code and showed that LIM‐hd developmental transcription factors are particularly powerful to highlight the coherence of several groups or nuclei, to delineate subdivisions, and/or to clarify structures that are still a matter of debate. Among other findings, we bring substantial evidence for the following: (1) a dual origin of olfactory bulb neurons, based on x‐Lhx5 expression; (2) the existence of a ventral pallium in frog, based on x‐Lhx9 expression; (3) a multiple (pallial and subpallial) origin for the nuclei of the amygdaloid complex, based on distinct combinations of the five studied genes; (4) a clear homology between the Xenopus medial pallium and the mammalian hippocampus, based on x‐Lhx2 pattern; and (5) a confirmed prosomeric organization of the diencephalon, based on alternating x‐Lhx1/5 and x‐Lhx2/9 expressions. In addition, the important expression levels for LIM‐hd factors found throughout development and in the adult brain suggest a role for these genes in development and maintenance of neuronal specification and phenotype, as for example in the case of x‐Lhx7 and cholinergic neurons. Moreover, following LIM‐hd patterns throughout development points out to some of the migrations and morphogenetic movements, which give rise to the adult structures. Finally, the detailed description of the LIM‐hd code in the developing and adult Xenopus forebrain provides interesting cues for the possible mechanisms of evolution of the vertebrate forebrain. J. Comp. Neurol. 472:52–72, 2004.

Evolution of the amygdaloid complex in vertebrates, with special reference to the anamnio-amniotic transition

Numerous studies over the last few years have demonstrated that the amygdaloid complex in amniotes shares basic developmental, hodological and neurochemical features. Furthermore, homologous territories of all the main amygdaloid subdivisions have been recognized among amniotes, primarily highlighted by the common expression patterns for numerous developmental genes. Thus, derivatives from the lateral pallium, ventral pallium and subpallium constitute the fundamental parts of the amygdaloid complex. With the development of new technical approaches, study of the precise neuroanatomy of the telencephalon of the anuran amphibians (anamniotes) has been possible. Current embryological, hodological and immunohistochemical evidence strongly suggests that most of the structures present in amniotes are recognizable in these anamniotes. These investigations have yielded enough results to support the notion that the organization of the anuran amygdaloid complex includes subdivisions with their origin in ventral pallial and subpallial territories; a strong relationship with the vomeronasal and olfactory systems; abundant intra‐amygdaloid connections; a main output centre involved in the autonomic system; recognizable amygdaloid fibre systems; and distinct chemoarchitecture. Therefore, the new ideas regarding the amygdaloid evolution based on the recent findings in anamniotes, and especially in anurans, strongly support the notion that basic amygdaloid structures were present at least in the brain of ancestral tetrapods organized following a basic plan shared by tetrapods.

Hodological characterization of the medial amygdala in anuran amphibians

Early studies in anuran amphibians defined the amygdala as a single unit that only later could be subdivided into medial and lateral parts with the achievement of sensitive immunohistochemical and tracing techniques. However, the terminology used was often misleading when comparing with “homologous” amygdaloid nuclei in amniotes. Recently, the basal telencephalon of anurans has been demonstrated to be more complex than previously thought, and distinct amygdaloid nuclei were proposed on the basis of immunohistochemistry. Moreover, developmental data are increasing that support this notion. In the present study, we analyzed the patterns of afferent and efferent connections of the medial amygdala (MeA; formerly amygdala pars lateralis), considered as the main target of the vomeronasal information from the accessory olfactory bulb, as in other vertebrates. By means of axonal transport of dextran amines, the afferent and efferent connections of the MeA were traced in Rana perezi and Xenopus laevis under in vivo and in vitro conditions. Largely similar results were found in both species. The results showed abundant intratelencephalic and extratelencephalic connections that were readily comparable to those of other tetrapods. Most of these connections were reciprocal and, in particular, the strong relation of the MeA with the hypothalamus, via the stria terminalis, was demonstrated. Immunohistochemical techniques showed staining patterns that revealed abundant peptidergic afferents to the MeA, as well as minor inputs containing other neurotransmitters such as catecholamines. Double‐labeling experiments demonstrated that the peptidergic fibers that reach the MeA originate in the ventral hypothalamus, whereas the catecholaminergic innervation of the MeA arises in the caudal extent of the posterior tubercle. Taken together, the results about connectivity in our study support the comparison of the MeA in anurans with its counterparts (and similarly named) amygdaloid nuclei in amniotes. Most of the hodological features of the medial amygdala seem to be shared by those tetrapods with well‐developed vomeronasal systems. J. Comp. Neurol. 466:389–408, 2003.

Localization and connectivity of the lateral amygdala in anuran amphibians.

On the basis of chemoarchitecture and gene expression patterns in the amphibian amygdaloid complex, new subdivisions have been proposed and compared with their counterparts in amniotes. Thus, a portion of the ventral pallium of anurans has been tentatively named “lateral amygdala” (LA) and compared with the basolateral complex of mammals. To strengthen the putative homology, we have analyzed the pattern of afferent and efferent connections of the LA in the anurans Rana perezi and Xenopus laevis. Tract‐tracing techniques with dextran amines were used under in vivo and in vitro conditions. The results showed important connections with the main olfactory bulb, via the lateral olfactory tract. In addition, abundant intratelencephalic connections, via the rostral branch of the stria terminalis, were revealed, involving mainly the basal ganglia, septal nuclei, bed nucleus of the stria terminalis, and especially other amygdaloid nuclei. Nontelencephalic connections were found from the dorsal thalamus and parabrachial area and, in particular, from the hypothalamus through the caudal branch of the stria terminalis. All these results strongly suggest that the LA in anurans is a multimodal area in the ventral pallium that shares many hodological features with the amygdaloid ventropallial derivatives of the basolateral complex of amniotes. J. Comp. Neurol. 479:130–148, 2004.

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.

Development and evolution of the subpallium

Among vertebrates, the ventral part of the telencephalon called the subpallium presents common basic developmental, hodological, neurochemical and functional features. It is genetically specified by expression of Dlx genes; its progenitor zones contribute a huge variety of neuronal cell types throughout the telencephalon; it is the origin and substrate of multiple and complex migration and navigation pathways during embryogenesis; and its derivatives, i.e. the basal ganglia and the amygdaloid complex, are highly conserved through evolution. Comparative developmental studies point to a largely common basic plan to generate the subpallium in vertebrates, including comparable progenitor domains and similar migratory cellular movements. In the course of telencephalic evolution however, slight variations have occurred, and the subpallium has probably represented a source for significant novelties and diversification in vertebrate forebrain anatomy and physiology.

Regionalization of the telencephalon in urodele amphibians and its bearing on the identification of the amygdaloid complex.

The brain of urodele amphibians has formed the basis for numerous comparative neuroanatomical studies because its simplified arrangement of neurons and fibers was considered to represent the basic pattern common to all tetrapods. However, on the basis of classical histological techniques many common features shared by the brain of amniotes could not be identified in the anamniotic amphibians. Recently, the combined analysis of the chemoarchitecture and hodology has demonstrated that the brain, and particularly the telencephalon, of anuran amphibians shares all major basic features with amniotes. In the present study, we have conducted a series of immunohistochemical detections for telencephalic regional markers (nitric oxide synthase (NOS), γ-amino butyric acid (GABA), Islet-1 (Isl1), and Nkx2.1) that were useful tools for unraveling telencephalic organization in other vertebrates. In addition, the combination of tract-tracing techniques with dextran amines to demonstrate olfactory secondary centers, hypothalamic projections, and brainstem connections has served to propose subdivisions within the amygdaloid complex. The results of the present analysis of the urodele telencephalon using a multiple approach have demonstrated, among other features, the presence of a ventral pallial region, striatopallidal subdivision in the basal ganglia, and three main components of the amygdaloid complex. Therefore, in spite of its apparently simple organization, within the telencephalon of urodeles it is possible to identify most of the features observed in amniotes and anurans that are only revealed with the use of combined modern techniques in neuroanatomy.

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.

Evidences for tangential migrations in Xenopus telencephalon: developmental patterns and cell tracking experiments.

Extensive tangential cell migrations have been described in the developing mammalian, avian, and reptilian forebrain, and they are viewed as a powerful developmental mechanism to increase neuronal complexity in a given brain structure. Here, we report for the first time anatomical and cell tracking evidence for the presence of important migratory processes in the developing forebrain of the anamniote Xenopus laevis. Combining developmental gene expression patterns (Pax6, Nkx2.1, Isl1, Lhx5, Lhx9, and Dll3), neurotransmitter identity (GABA, NOS, ChAT), and connectivity information, several types of putative migratory cell populations and migration routes originating in the ventral pallium and the subpallium are proposed. By means of in vivo cell tracking experiments, pallio‐subpallial and subpallio‐pallial migrating neurons are visualized. Among them, populations of Nkx2.1+ striatal interneurons and pallial GABAergic interneurons, which also express the migratory marker doublecortin, are identified. Finally, we find that these tangentially migrating pallial interneurons travel through an “isl1‐free channel” that may guide their course through the subpallium. Our findings strongly suggest that the developing Xenopus telencephalon shares many similarities with amniotes in terms of neuronal specification and migrations. However, some differences are discussed, particularly with regard to the evolution of the pallium.

Spatio-temporal expression of Pax6 in Xenopus forebrain

x-Pax6 gene expression has been previously demonstrated in the Xenopus forebrain at early developmental stages and, in particular, it was analyzed in relation to putative migratory cells in the primordial septum and olfactory bulbs. Here we investigated the pattern of x-Pax6 expression during embryonic and larval stages and in the adult. To gain more insight into the exact localization of x-Pax6 labeled cells, we used combinations of x-Pax6 with other telencephalic markers of the Xenopus forebrain such as Islet-1, Nkx2.1, and tyrosine hydroxylase. The results, analyzed within the frame of the current prosomeric model of the vertebrate forebrain, demonstrated robust expression in the pallium and the prethalamic diencephalic segment, from early embryonic stages. In addition, starting at the end of the embryonic period, x-Pax6 was detected in the subpallial mantle (but not in the proliferating cells of the ventricular/subventricular zones). The pattern of distribution in the pallium narrows through larval development and in the adult, whereas in the subpallium the expression was gradually followed and identified into the dorsal part of the septum, corroborating in Xenopus a mixed pallial/subpallial origin of the neurons in this region. The findings show that the strongly conserved features of x-Pax6 expression through forebrain development shared by all amniote vertebrates are also present in the anamniote amphibians as a common characteristic of the forebrain organization of tetrapods.