Biomedical subjects

Luis Puelles

Publications and source records attributed to Luis Puelles.

35 records · Page 2Linked to original sources

PubMed2003

Expression of the genes GAD67 and Distal-less-4 in the forebrain of Xenopus laevis confirms a common pattern in tetrapods.

We investigated whether gamma-amino butyric acidergic (GABAergic) cell populations correlate positionally with specific Dlx-expressing histogenetic territories in an anamniote tetrapod, the frog Xenopus laevis. To that end, we cloned a fragment of Xenopus GAD67 gene (xGAD67, expressed in GABAergic neurons) and compared its expression with that of Distal-less-4 gene (xDll-4, ortholog of mouse Dlx2) in the forebrain at late larval and adult stages. In Xenopus, GABAergic neurons were densely concentrated in xDll-4-positive territories, such as the telencephalic subpallium, part of the hypothalamus, and ventral thalamus, where nearly all neurons expressed both genes. In contrast, the pallium of Xenopus generally contained dispersed neurons expressing xGAD67 or xDll-4, which may represent local circuit neurons. As in amniotes, these pallial interneurons may have been produced in the subpallium and migrated tangentially into the pallium during development. In Xenopus, the ventral division of the classic lateral pallium contained extremely few GABAergic cells and showed only low signal of the pallial gene Emx1, suggesting that it may represent the amphibian ventral pallium, homologous to that of amniotes. At caudal forebrain levels, a number of GABAergic neurons was observed in several areas (dorsal thalamus, pretectum), but no correlation to xDll-4 was observed there. The location of GABAergic neurons in the forebrain and their relation to the developmental regulatory genes Dll and Dlx were very similar in Xenopus and in amniotes. The close correlation in the expression of both genes in rostral forebrain regions supported the notion that Dll/Dlx are among the genes involved in the acquisition of the GABAergic phenotype.

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PubMed2003

Neuronal differentiation patterns in the optic tectum of the lizard Gallotia galloti.

This study examines in detail the sequences of morphological differentiation and deduces mode of migration into specific layers of all types of neurons present in the optic tectum of the lizard Gallotia galloti. It complements previous similar work on tectal histogenesis in the chick. It was found that the neuronal population diversity in the lizard tectum can be reduced by developmental analysis to three neuroblast classes, called Types I, II and III. These classes correspond closely to those present in the developing avian tectum. Neurons belonging to each developmental class were characterized by their initial polarity, mode of translocation into the mantle layer and pattern of sprouting of primary axonal and dendritic processes. Each class produced along time a subset of the cell types distinguished in the mature tectum. Some aspects of sauropsidian tectal histogenesis are also common of other vertebrates, suggesting that fundamental mechanisms of tectal neuronal differentiation are conserved in tetrapods. Analysis of evolutive differences of tectal structure points to changes affecting the layering and perhaps the population size of specific cell types. Whereas tectal cell-type homology can be easily fundamented on embryological evidence and seems to be consistent with hodological and, to some extent, functional homology, the periventricular, central and superficial strata of the tectum are heterogeneous in cellular composition in different species and therefore represent analogous, rather than homologous entities.

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PubMed2003

The relationship between hodological and cytoarchitectonic organization in the vestibular complex of the 11-day chicken embryo.

To understand the relationship between structure and function in specific brain regions, it is necessary to ascertain which anatomical features are physiologically relevant. Physiological studies of brain function traditionally have been set in the context of anatomical features based on cytoarchitectonics and myeloarchitectonics, but the relationship between structure and function in this context can be complex. Alternative schemes of anatomical organization, such as that based on hodology (the mapping of projections) may provide greater insight. Here, we make a direct comparison of the hodological and the cytoarchitectonic organization of the vestibular complex in the mid-term chicken embryo, using retrograde tracing and three-dimensional reconstruction. In one set of experiments, vestibulospinal and vestibulo-ocular neuron groups were selectively labeled with biotin dextran-amines and aligned with the cytoarchitectonically defined vestibular nuclei in alternating sections that were then combined into intercalated three-dimensional models. This allowed a semiquantitative analysis of the apportionment of individual hodological groups among cytoarchitectonic nuclei. In another set of experiments, vestibulospinal and vestibulo-ocular neuron groups were labeled differentially with fluorescent dextran-amines, three-dimensionally reconstructed, and subjected to a quantitative analysis of spatial overlap. Our results provide the first three-dimensional representation and quantitative analysis of the hodological compartmentalization of the vestibular complex (the "hodological mosaic"). They also show directly how each hodologically defined neuron group relates to the conventional vestibular nuclei, underscoring the fact that the units of the hodological mosaic do not bear a one-to-one correspondence to the cytoarchitectonic nuclear divisions. Some hodologically defined groups are localized to restricted portions of a nucleus, whereas others overlap multiple nuclei. Thus, hodology and cytoarchitectonic features appear to be separately regulated in the vestibular complex of the chicken embryo, possibly through different sets of positional specification mechanisms. The three-dimensional representations we present here provide a foundation for integrating anatomical, physiological, developmental, and evolutionary studies of the vestibular system.

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PubMed2003

Six3 repression of Wnt signaling in the anterior neuroectoderm is essential for vertebrate forebrain development.

In vertebrate embryos, formation of anterior neural structures requires suppression of Wnt signals emanating from the paraxial mesoderm and midbrain territory. In Six3(-/-) mice, the prosencephalon was severely truncated, and the expression of Wnt1 was rostrally expanded, a finding that indicates that the mutant head was posteriorized. Ectopic expression of Six3 in chick and fish embryos, together with the use of in vivo and in vitro DNA-binding assays, allowed us to determine that Six3 is a direct negative regulator of Wnt1 expression. These results, together with those of phenotypic rescue of headless/tcf3 zebrafish mutants by mouse Six3, demonstrate that regionalization of the vertebrate forebrain involves repression of Wnt1 expression by Six3 within the anterior neuroectoderm. Furthermore, these results support the hypothesis that a Wnt signal gradient specifies posterior fates in the anterior neural plate.

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PubMed2003

Forebrain gene expression domains and the evolving prosomeric model.

The prosomeric model attributes morphological meaning to gene expression patterns and other data in the forebrain. It divides this territory into the same transverse segments (prosomeres) and longitudinal zones in all vertebrates. The axis and longitudinal zones of this model are widely accepted but controversy subsists about the number of prosomeres and their nature as segments. We describe difficulties encountered in establishing continuity between prosomeric limits postulated in the hypothalamus and intra-telencephalic limits. Such difficulties throw doubt on the intersegmental nature of these limits. We sketch a simplified model, in which the secondary prosencephalon (telencephalon plus hypothalamus) is a complex protosegment not subdivided into prosomeres, which exhibits patterning singularities. By contrast, we continue to postulate that prosomeres p1-p3 (i.e. the pretectum, thalamus and prethalamus) are the caudal forebrain.

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PubMed2002

Cortical excitatory neurons and glia, but not GABAergic neurons, are produced in the Emx1-expressing lineage.

By homologous recombination of an internal ribosome entry site and Cre recombinase coding region into the 3'-untranslated region of the mouse Emx1 gene, we have generated a strain of mice, Emx1(IRES)cre, that expresses the Cre recombinase in a spatial and temporal pattern like that observed for Emx1. When mated to reporter strains, these mice are a sensitive means to fate-map the Emx1-expressing cells of the developing forebrain. Our results demonstrate that radial glia, Cajal-Retzius cells, glutamatergic neurons, astrocytes, and oligodendrocytes of most pallial structures originate from an Emx1-expressing lineage. On the other hand, most of the pallial GABAergic neurons arise outside the Emx1-expressing lineage. Structures that are located near the basal ganglia (e.g., the amygdala and endopiriform nuclei) are not uniformly derived from Emx1-expressing cells.

3' Untranslated Regions↗

PubMed2002

Mesencephalic and diencephalic afferent connections to the thalamic nucleus rotundus in the lizard, Psammodromus algirus.

The present work is an analysis of the afferent projections to the thalamic nucleus rotundus in a lizard, both at the light- and electron-microscopic level, using biotinylated dextran amine (BDA) as a neuroanatomical tracer. This study has confirmed previously reported afferent projections to nucleus rotundus in reptiles and has also identified a number of new cellular aggregates projecting to this dorsal thalamic nucleus. After BDA injections into nucleus rotundus, retrogradely labelled neurons were observed consistently within the following neuronal groups in the midbrain and the diencephalon: (i) the stratum griseum centrale of the optic tectum; (ii) the nucleus subpretectalis in the pretectum; (iii) the nucleus ansa lenticularis posterior, the posterior nucleus of the ventral supraoptic commissure, and the posteroventral nucleus, in the dorsal thalamus and (iv) the lateral suprachiasmatic nucleus and part of the reticular complex in the ventral thalamus. Tectal axons entering nucleus rotundus were fine and varicose and formed exclusively asymmetric synaptic contacts, mainly on small dendritic profiles. Rotundal neurons had symmetric synapses made by large boutons probably of nontectal origin. After comparing our results with those in other reptiles, birds and mammals, we propose that the sauropsidian nucleus rotundus forms part of a visual tectofugal pathway that conveys mesencephalic visual information to the striatum and dorsal ventricular ridge, and is similar to the mammalian colliculo-posterior/intralaminar-striatoamygdaloid pathway, the function of which may be to participate in visually guided behaviour.

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PubMed2002

Expression from a Dlx gene enhancer marks adult mouse cortical GABAergic neurons.

In this paper we analyse the expression pattern of a zebrafish dlx4/6 enhancer/reporter construct in embryonic transgenic mice. We show that the pattern of LacZ/beta-galactosidase in cells that tangentially migrate from the ganglionic eminences to the cerebral cortex is identical to that of various subpallial markers, namely Dlx and GAD genes, that are known to label this population. Because beta-galactosidase activity persists long after expression of the Dlx genes and the transgene becomes undetectable, we were able to analyse the beta-galactosidase-positive cell population of the mature cortex through X-gal staining and immunohistochemistry. We show that this population is largely identical with the adult cortical and hippocampal interneuron population, providing further evidence for their subpallial origin.

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PubMed2002

Fate map of the chicken neural plate at stage 4.

A detailed fate map was obtained for the early chick neural plate (stages 3d/4). Numerous overlapping plug grafts were performed upon New-cultured chick embryos, using fixable carboxyfluorescein diacetate succinimidyl ester to label donor chick tissue. The specimens were harvested 24 hours after grafting and reached in most cases stages 9-11 (early neural tube). The label was detected immunocytochemically in wholemounts, and cross-sections were later obtained. The positions of the graft-derived cells were classified first into sets of purely neural, purely non-neural and mixed grafts. Comparisons between these sets established the neural plate boundary at stages 3d/4. Further analysis categorized graft contributions to anteroposterior and dorsoventral subdivisions of the early neural tube, including data on the floor plate and the eye field. The rostral boundary of the neural plate was contained within the earliest expression domain of the Ganf gene, and the overall shape of the neural plate was contrasted and discussed with regard to the expression patterns of the genes Plato, Sox2, Otx2 and Dlx5 (and others reported in the literature) at stages 3d/4.

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PubMed2002

Patterning of the basal telencephalon and hypothalamus is essential for guidance of cortical projections.

We have investigated the mechanisms that control the guidance of corticofugal projections as they extend along different subdivisions of the forebrain. To this aim, we analyzed the development of cortical projections in mice that lack Nkx2-1, a homeobox gene whose expression is restricted to two domains within the forebrain: the basal telencephalon and the hypothalamus. Molecular respecification of the basal telencephalon and hypothalamus in Nkx2-1-deficient mice causes a severe defect in the guidance of layer 5 cortical projections and ascending fibers of the cerebral peduncle. These axon tracts take an abnormal path when coursing through both the basal telencephalon and hypothalamus. By contrast, loss of Nkx2-1 function does not impair guidance of corticothalamic or thalamocortical axons. In vitro experiments demonstrate that the basal telencephalon and the hypothalamus contain an activity that repels the growth of cortical axons, suggesting that loss of this activity is the cause of the defects observed in Nkx2-1 mutants. Furthermore, analysis of the expression of candidate molecules in the basal telencephalon and hypothalamus of Nkx2-1 mutants suggests that Slit2 contributes to this activity.

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PubMedDate not supplied

Patch/matrix patterns of gray matter differentiation in the telencephalon of chicken and mouse.

The mammalian striatum, a subpallial area, consists of two compartments (patches/striosomes and matrix) that differ in their neuronal birth dates, connectivity, neurochemistry, and molecular make-up. For example, members of the cadherin family of adhesion molecules (cadherin-8 and OL-protocadherin) are differentially expressed by the striosomes and the striatal matrix. A patch/matrix type of organization also has recently been found in the ventral hyperstriatum and the neostriatum of the chicken pallium, where cell clusters of similar birthdates ("isochronic" clusters) are surrounded by a matrix of cells that are born at a different time. Immunostaining with antibodies against cadherins reveals a similar arrangement of cell clusters. In the avian neostriatum, cadherin-7-positive cell clusters ("islands") are surrounded by a matrix of cells that express R-cadherin. The islands coincide, at least in part, with the isochronic cell clusters, as shown by pulse-labeling with bromodeoxyuridine. Likewise, isochronic clusters of the hyperstriatum ventrale relate to patchy heterogeneities in the cadherin-7 immunoreactivity pattern. Cadherins are known to mediate the aggregation and sorting of cells during development in many organs. Their differential expression by isochronic cell populations in the mammal subpallium and avian pallium suggests a common morphogenetic mechanism that regulates the formation of the patch/matrix patterns in these regions.

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PubMedDate not supplied

Field homology as a way to reconcile genetic and developmental variability with adult homology.

The theoretical and developmental fundament of field homology is here examined, particularly as applied by the authors to comparative neurobiology. Preliminary considerations explore conceptual differences between sameness (homology) and similarity. The source of sameness (the biological evolutionary relationship properly sought in homology analysis) is thought to lie in morphostatic evolutionary and morphogenetic processes, which constrain organismal variation at the level of its fundamental structural organization (Bauplan). This occurs via regulation of the branching mode of the morphogenetic sequence or epigenetic landscape. Of fundamental importance in this context is the role of developmental (morphogenetic) fields. The latter concept is analyzed in its general properties and is postulated to underpin the stability of the developing Bauplan down to the ultimate conserved details. Developmental fields subdivide during ontogenesis into ever smaller fields in a complex hierarchy, defining at each stage the developmental entities which are subjected to regulatory, morphostatic effects via the genome and indirect phenotypic selection. These fields thus represent the natural characters for considerations of embryonic homology, and underlie adult homology, rather than arbitrarily selected embryonic parts. Field subdivision proceeds into the constitution of individually specified cell populations. Field regulatory properties, however, do not extend to all differentiation phenomena observed in embryos. This means there is a limit to the applicability of field homology analysis, leaving space for biological variation and convergence outside of proper homology relationships. Genetic and developmental variability are compensated by the regulatory functions of the developmental fields insofar as they relate to correct Bauplan construction. These ideas suggest the convenience of a more systematic use of field homology methods, which start with appropriately identified developmental fields to expand knowledge on adult homology (sameness) and eventually also on accessory structural and functional similarities or differences.

Aging↗

PubMedDate not supplied

The telencephalon of the frog Xenopus based on calretinin immunostaining and gene expression patterns.

To further understand the organization and evolution of the telencephalon, we analyzed in the frog Xenopus laevis the expression of the genes Distal-less-4 (Xdll-4, comparable to the mouse gene Dlx2) and GAD-67 (XGAD-67, expressed in GABAergic cells), and compared this with calretinin immunostaining and the cytoarchitecture of the telencephalon. Our results show that like in other vertebrates, the telencephalon of the frog Xenopus is divided into two major territories: a basal, subpallial region showing a high density of cells expressing Xdll-4 and XGAD-67, and a dorsal, pallial region showing only few, dispersed cells expressing these genes. The subpallial territory of the frog Xenopus includes the septum, the amphibian basal ganglia, some basal forebrain cholinergic cell groups and some amygdala nuclei. In the pallium of the frog Xenopus, medial, dorsal, lateral, and ventral parts could be distinguished, similar to those described in amniotes. In summary, the amphibian telencephalon shows a basic morphogenetic organization similar to that of amniotes, which suggests that this organization is common to the telencephalon of all tetrapods.

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PubMedDate not supplied

A neural plate fate map at stage HH4 in the chick: methodology and preliminary data.

This paper centers on the design of a perfected methodology for establishing a fate map of the chick neural plate at stages 3d/4, projected upon the closing neural tube (stages 9-11). The principal aim was to saturate the area of interest with overlapping small isochronic and homotopic grafts (100-300 cells), in order to later derive firmer conclusions from the detailed comparisons thus made possible. We used an ocular grid centered on Hensen's node for the localization of the grafts. Chick embryos in New culture were used as donors and hosts, to evade potential differences in intercalation or proliferation behavior between quail and chick cells. Donor tissue was labelled with the non-diffusing fluorescein derivative carboxyfluorescein diacetate succinimidyl ester, later visualized by fluorescence microscopy at various timepoints during survival and by a sensitive whole-mount immunocytochemical protocol after fixation. We present only preliminary data of the ongoing mapping, illustrating well-delimited patches of graft-derived cells which can be identified across the neural/non-neural epiblast continuum, or across the dorsoventral dimension of the neural tube wall.

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PubMedDate not supplied

Organization of the mouse dorsal thalamus based on topology, calretinin immnunostaining, and gene expression.

To better understand the organization and evolution of the dorsal thalamus, we have made a first approach to analyze the possible histogenetic compartments of the mammalian dorsal thalamus using mouse embryos. For that, we have analyzed the expression of the proneural gene Math4a and the protein calretinin. Our results suggest the existence of rostrodorsal, caudoventral, and ventral compartments in the embryonic dorsal thalamus of the mouse, which partly parallel the dorsoventral histogenetic tiers postulated in the dorsal thalamus of sauropsids. The rostrodorsal compartment of the mouse dorsal thalamus is characterized by expression of Math4a, and it appears to include sensory and motor thalamic nuclei projecting to the dorsal pallium (isocortex). This compartment appears equivalent to the lemnothalamus proposed by Butler in tetrapods based on hodological grounds. The caudoventral and ventral compartments of the mouse dorsal thalamus lack expression of Math4a in the mantle, but they are characterized by several populations of calretinin-immunorective neurons that show projections to the claustroamygdaloid region in the ventrolateral pallium. More studies will be needed to analyze if the compartments proposed in this study represent true histogenetic units, and to find homologous developmental fields in all vertebrates.

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PubMedDate not supplied

Gbx2 expression in the late embryonic chick dorsal thalamus.

The expression pattern of the transcription factor gene Gbx2 in the forebrain of chicken embryos (embryonic day 14) was mapped using digoxigenin-labeled riboprobes and compared with the expression of the transcription factors Pax6 and Nkx2.2. The topographic analysis of Gbx2 expression on coronal and sagittal sections discriminated the positions and boundaries of diverse neuronal nuclei belonging to the dorsal thalamus from neighboring territories (the epithalamus, ventral thalamus, pretectum, and the underlying basal plate). The differential expression of Gbx2 within the dorsal thalamus clearly corresponds with the existence of four primary subdivisions identified in a previous study from this laboratory [13]: the anteroventral region and dorsal, intermediate, and ventral tiers. The subhabenular region turned out not to express Gbx2; this possibly implies it needs to be distinguished as a fifth separate dorsal thalamus subdivision.

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PubMedDate not supplied

The avian griseum tectale: cytoarchitecture, NOS expression and neurogenesis.

The griseum tectale (GT) is a retinorecipient layered formation located in the rostral alar midbrain, just behind the constriction that separates it from the diencephalon (pretectum). Tritiated-thymidine autoradiographic data on neuronal birthdates show that the GT cell population starts to be generated at HH21 and most neurons are born by stage HH25, accumulating within a primordial periventricular layer, which shows strong nitric oxide synthase immunoreactivity at later stages of development. There is a barely noticeable rostrocaudal neurogenetic and differentiation gradient across the GT, which seems to continue into that of the neighboring optic tectum. The GT layering develops gradually by radial migration of its postmitotic neurons between stages HH26 and HH35. The structure of the mature GT can be divided into periventricular, central, and superficial layers, similarly to the adjacent optic tectum, but it shows different layering aspects, particularly in the retinorecipient superficial layer.

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