[Forebrain regionalization mechanisms]. / Mecanismos de la formación de regiones en el cerebro anterior.

Where do the regions of the forebrain come from? The paradigm that has come to light in the last ten years is that of a neural plate subdivided by soluble factors which (through the formation of gradients or of a molecular framework) transmits positional information to the neural stem cells. Some of those soluble factors are Fgf8, Sonic hedgehog and proteins of the BMP family. The neural stem cells interpret positional information in terms of the expression of combinations of certain transcription factors. Such combinations would finally be responsible for the formation of specific neural lineages (cortical, thalamic, etc.) from specific neuroepithelial regions. In addition, this primary positional information can make certain regions of the neural primordium (secondary organizers) secrete new soluble factors. These would in turn give rise to new positional information, spatially more restricted, so that the whole process would repeat itself to confer detail to a certain area. This wealth of new knowledge is already helping us to understand the cause of some brain malformations, and maybe we will soon be able to apply it to the early diagnosis and the prevention of such conditions.

Mecanismos de la formación de regionesen el cerebro anterior / Forebrain regionalization mechanisms

¿De dónde vienen las regiones del cerebro anterior? El paradigma que va saliendo a la luz en los últimos diez años es el de una placa neural subdivida por factores solubles que (a través de gradientes o de un entramado molecular) transmiten información posicional a las células madre neurales. Algunos de esos factores solubles son Fgf8, Sonic hedgehog y proteínas de la familia BMP.Las células madre neurales interpretan la información posicional en términos de expresión de combinaciones de factores de la transcripción. Tales combinaciones serían finalmente responsables de la formación de linajes neuronales específicos (corticales, talámicos, etc.) a partir de regiones neuroepiteliales específicas. Además, la información posicional `primaria' puede hacer que determinadas regiones del primordio neural (organizadores secundarios) segreguen nuevos factores solubles. Éstos darían a su vez lugar a nueva información posicional, de influencia más restringida, y así se repetiría el proceso localmente, añadiendo `detalle' a una cierta región. El torrente de nuevos conocimientos adquiridos mediante genética molecular nos está ayudando a comprender la causa de algunas malformaciones congénitas cerebrales, y tal vez pronto se pueda aplicar también a su diagnóstico precoz y a su prevención (AU)

Expression of Foxb1 reveals two strategies for the formation of nuclei in the developing ventral diencephalon.

Fork head b1 (Foxb1; also called Fkh5, HFH-e5.1, Mf3) is a winged helix transcription factor gene whose widespread early expression in the developing neural tube is soon restricted to the ventral and caudal diencephalon. During diencephalic neurogenesis, Foxb1 is expressed in one patch of neuroepithelium comprising a large mammillary portion and a smaller tuberal portion. The labeled cells coming from this patch contribute to nuclear formation by means of two different strategies: (1) caudally, the young neurons aggregate and settle immediately, giving rise to the nuclei of the mammillary body; (2) rostrally, the young neurons separate from the neuroepithelium forming a trail of cells which spans the mantle layer mediolaterally and which will give rise to two separate cell groups (the dorsal premammillary and part of the lateral hypothalamic area). Our results show the elaborate, regionalized histogenetic mechanisms necessary for the differentiation of the caudal diencephalon; moreover, they suggest that specifically labeled populations, arising from specifically labeled neuroepithelial patches and giving place to specific brain nuclei could be a common mechanism to build complex, nonlaminar regions of the forebrain.

Winged helix transcription factor Foxb1 is essential for access of mammillothalamic axons to the thalamus.

Our aim was to study the mechanisms of brain histogenesis. As a model, we have used the role of winged helix transcription factor gene Foxb1 in the emergence of a very specific morphological trait of the diencephalon, the mammillary axonal complex. Foxb1 is expressed in a large hypothalamic neuronal group (the mammillary body), which gives origin to a major axonal bundle with branches to thalamus, tectum and tegmentum. We have generated mice carrying a targeted mutation of Foxb1 plus the tau-lacZ reporter. In these mutants, a subpopulation of dorsal thalamic ventricular cells "thalamic palisade" show abnormal persistence of Foxb1 transcriptional activity; the thalamic branch of the mammillary axonal complex is not able to grow past these cells and enter the thalamus. The other two branches of the mammillary axonal complex (to tectum and tegmentum) are unaffected by the mutation. Most of the neurons that originate the mammillothalamic axons suffer apoptosis after navigational failure. Analysis of chimeric brains with wild-type and Foxb1 mutant cells suggests that correct expression of Foxb1 in the thalamic palisade is sufficient to rescue the normal phenotype. Our results indicate that Foxb1 is essential for diencephalic histogenesis and that it exerts its effects by controlling access to the target by one particular axonal branch.

Gli3 is required for Emx gene expression during dorsal telencephalon development.

Dentate gyrus and hippocampus as centers for spatial learning, memory and emotional behaviour have been the focus of much interest in recent years. The molecular information on its development, however, has been relatively poor. To date, only Emx genes were known to be required for dorsal telencephalon development. Here, we report on forebrain development in the extra toes (Xt(J)) mouse mutant which carries a null mutation of the Gli3 gene. This defect leads to a failure to establish the dorsal di-telencephalic junction and finally results in a severe size reduction of the neocortex. In addition, Xt(J)/Xt(J) mice show absence of the hippocampus (Ammon's horn plus dentate gyrus) and the choroid plexus in the lateral ventricle. The medial wall of the telencephalon, which gives rise to these structures, fails to invaginate during embryonic development. On a molecular level, disruption of dorsal telencephalon development in Xt(J)/Xt(J) embryos correlates with a loss of Emx1 and Emx2 expression. Furthermore, the expression of Fgf8 and Bmp4 in the dorsal midline of the telencephalon is altered. However, expression of Shh, which is negatively regulated by Gli3 in the spinal cord, is not affected in the Xt(J)/Xt(J) forebrain. This study therefore implicates Gli3 as a key regulator for the development of the dorsal telencephalon and implies Gli3 to be upstream of Emx genes in a genetic cascade controlling dorsal telencephalic development.

Pax2/5 and Pax6 subdivide the early neural tube into three domains.

The nested expression patterns of the paired-box containing transcription factors Pax2/5 and Pax6 demarcate the midbrain and forebrain primordium at the neural plate stage. We demonstrate that, in Pax2/5 deficient mice, the mesencephalon/metencephalon primordium is completely missing, resulting in a fusion of the forebrain to the hindbrain. Morphologically, in the alar plate the deletion is characterized by the substitution of the tectum (dorsal midbrain) and cerebellum (dorsal metencephalon) by the caudal diencephalon and in the basal plate by the replacement of the midbrain tegmentum by the ventral metencephalon (pons). Molecularly, the loss of the tectum is demonstrated by an expanded expression of Pax6, (the molecular determinant of posterior commissure), and a rostral shift of the territory of expression of Gbx2 and Otp (markers for the pons), towards the caudal diencephalon. Our results suggest that an intact territory of expression of Pax2/5 in the neural plate, nested between the rostral and caudal territories of expression of Pax6, is necessary for defining the midbrain vesicle.

Fjx1, the murine homologue of the Drosophila four-jointed gene, codes for a putative secreted protein expressed in restricted domains of the developing and adult brain.

The Drosophila gene four jointed (fj) codes for a secreted or cell surface protein important for growth and differentiation of legs and wings and for proper development of the eyes. Here we report the cloning of the mouse four-jointed gene (fjx1) and its pattern of expression in the brain during embryogenesis and in the adult. In the neural plate, fjx1 is expressed in the presumptive forebrain and midbrain, and in rhombomere 4, however a small rostral/medial area of the forebrain primordium is devoid of expression. Expression of fjx1 in the neural tube can be divided into three phases. (1) In the embryonic brain fjx1 is expressed in two patches of neuroepithelium: in the midbrain tectum and the telencephalic vesicles. (2) In fetal and early postnatal brain fjx1 is expressed mainly by the primordia of layered telencephalic structures: cortex (ventricular layer and cortical plate), olfactory bulb (subependymal layer and in the mitral cell layer). In addition expression is observed in the superior colliculus. (3) In the adult, fjx1 is expressed by neurones evenly distributed in the telencephalon (isocortex, striatum, hippocampus, olfactory bulb, piriform cortex), in the Purkinje cell layer of the cerebellum, and numerous medullary nuclei. In the embryo, strong expression can further be seen in the apical ectodermal ridge of fore- and hindlimbs and in the ectoderm of the branchial arches.

The fork head transcription factor Fkh5/Mf3 is a developmental marker gene for superior colliculus layers and derivatives of the hindbrain somatic afferent zone.

Fork head-5 (Fkh5; also known as Mf3 and TWH) is a transcription factor of the winged helix family. As part of an extended project to understand the function of this protein in the developing mouse brain, in the present work we have used Fkh5/Mf3 expression as a marker to study the development of the midbrain and hindbrain. In the midbrain, Fkh5/Mf3 is expressed in the superior colliculus, in the ventricular layer of the inferior colliculus and in the isthmus. In the superior colliculus, Fkh5/Mf3 is expressed by cells of layers 4a and 4c since early in development. In the hindbrain, Fkh5/Mf3 is a longitudinal marker (as opposed to a transverse or rhombomeric one), since it labels nuclei belonging to the somatic afferent zone (ventral cochlear nucleus, cuneate and external cuneate nuclei, principal and spinal nuclei of the trigeminal). In addition, Fkh5/Mf3 is expressed by the developing endopiriform nucleus and by the olivary pretectal nucleus. The results suggest that Fkh5/Mf3 has an early role in the lamination of the tectum and in the longitudinal differentiation of the hindbrain.

GABA-immunoreactive cells of the cortical primordium contribute to distinctly fated neuronal populations.

The roles of GABA during development, as either a putative neurotransmitter or a nonsynaptic trophic factor, are being discussed intensely in recent literature. We offer an anatomical framework to better understand these possible roles in the developing cerebral cortex. During the early development of the cerebral cortex, GABA-containing cells constitute an outstanding cell population in the primordial plexiform layer, but they later distribute into at least four compartments. These include (1) Cajal-Retzius cells in layer I and (2) the subplate cells. Certain of these GABA-containing cell groups may disappear either by ceasing their expression of GABA, dilution in a growing brain volume, or cell death, possibilities that are reviewed here. The chemical tags that characterize Cajal-Retzius cells, both in the forming isocortex and Ammon's horn, are discussed. Another cell population that also belongs to the primordial plexiform layer is formed by (3) the tangentially migrating cells of the deep intermediate layer. These migrate away from the isocortical primordium to invade, and contribute cells to, the forming stratum oriens of the Ammon's horn. Since these cells cross cortical area boundaries, their tangential migration is relevant to the issue of cortical area specification during development. Finally, GABA-immunoreactive cells in the developing cortical plate are considered to be (4) the future GABAergic interneurons. A hypothetical mechanism is presented here to explain their acquisition of laminar positions, which is known to take place simultaneously, and with an identical "inside-out gradient," to the pyramidal cells generated contemporarily.

Apaf1 (CED-4 homolog) regulates programmed cell death in mammalian development.

The cytosolic protein APAF1, human homolog of C. elegans CED-4, participates in the CASPASE 9 (CASP9)-dependent activation of CASP3 in the general apoptotic pathway. We have generated by gene trap a null allele of the murine Apaf1. Homozygous mutants die at embryonic day 16.5. Their phenotype includes severe craniofacial malformations, brain overgrowth, persistence of the interdigital webs, and dramatic alterations of the lens and retina. Homozygous embryonic fibroblasts exhibit reduced response to various apoptotic stimuli. In situ immunodetection shows that the absence of Apaf1 protein prevents the activation of Casp3 in vivo. In agreement with the reported function of CED-4 in C. elegans, this phenotype can be correlated with a defect of apoptosis. Our findings suggest that Apaf1 is essential for Casp3 activation in embryonic brain and is a key regulator of developmental programmed cell death in mammals.

Pax-2 in the chiasm.

Pax genes are expressed in specific patterns in the nervous system during development and in the adult. Recent findings suggest a link between the expression of Pax-2 and axonal guidance. Mice with a targeted deletion of Pax-2 are an excellent tool for studying axonal pathfinding at the molecular level, especially with respect to the optic chiasm. The date reviewed here suggest that Pax-2 regulates the expression of surface molecules involved in contact attraction and that the mutual regulation of the expression of Pax-2 and the Sonic hedgehog gene is of importance in the formation of the chiasm region.

Expression of Meis2, a Knotted-related murine homeobox gene, indicates a role in the differentiation of the forebrain and the somitic mesoderm.

Knotted (Kn) genes are expressed within restricted domains of the plant meristems and play a key role in the control of plant morphogenesis. We have isolated the Kn-related gene Meis2 in mouse, which labels the lateral somitic compartment and its derivatives during early mouse embryogenesis and later becomes a marker for the dorso-ectodermal region overlying cells of the paraxial mesoderm. Meis2 is also highly expressed in specific areas of the developing central nervous system from embryonic day 9 (e9) onward. In later developmental stages, a strong expression is detectable in differentiating nuclei and regions of the forebrain, midbrain, hindbrain, and spinal cord. This temporal and spatial expression pattern suggests that Meis2 may play an important role in the cascade of induction leading to somitic differentiation as well as in brain regionalization and histogenesis.

Survival of inner ear sensory neurons in trk mutant mice.

Analysis of trkB-/-; trkC-/- double mutant mice revealed that peripheral and central inner ear sensory neurons are affected in these mice. However, a substantial amount of cochlear and vestibular neurons survive, possibly due to maintenance or upregulation of TrkA expression. To clarify the function of the TrkA receptor during development of the cochlear and vestibular ganglion we analysed trkA-/- mice and the expression of this receptor in inner ear sensory neurons of trkB-/-; trkC-/- animals. TrkA homozygous mutant mice showed normal numbers of neurons and no TrkA expression was detected in neurons of trkB-/-; trkC-/- double mutant mice. We conclude that TrkA is not essential for inner ear development and that in the absence of any of the known catalytic Trk receptors peripheral inner ear sensory neurons are prone to undergo cell death or must use a different signaling mechanism to survive.

Conserved biological function between Pax-2 and Pax-5 in midbrain and cerebellum development: evidence from targeted mutations.

The development of two major subdivisions of the vertebrate nervous system, the midbrain and the cerebellum, is controlled by signals emanating from a constriction in the neural primordium called the midbrain/hindbrain organizer (Joyner, A. L. (1996) Trends Genet. 12, 15-201). The closely related transcription factors Pax-2 and Pax-5 exhibit an overlapping expression pattern very early in the developing midbrain/hindbrain junction. Experiments carried out in fish (Krauss, S., Maden, M., Holder, N. & Wilson, S. W. (1992) Nature (London) 360, 87-89) with neutralizing antibodies against Pax-b, the orthologue of Pax-2 in mouse, placed this gene family in an regulatory cascade necessary for the development of the midbrain and the cerebellum. The targeted mutation of Pax-5 has been reported to have only slight effects in the development of structures derived from the isthmic constriction, whereas the Pax-2 null mutant mice show a background-dependent phenotype with varying penetrance. To test a possible redundant function between Pax-2 and Pax-5 we analyzed the brain phenotypes of mice expressing different dosages of both genes. Our results demonstrate a conserved biological function of both proteins in midbrain/hindbrain regionalization. Additionally, we show that one allele of Pax-2, but not Pax-5, is necessary and sufficient for midbrain and cerebellum development in C57BL/6 mice.

Neurogranin in the development of the rat telencephalon.

We have used a novel antibody to map the distribution of the protein kinase C substrate protein RC3/neurogranin during the development of the rat telencephalon. Neurogranin appearance in the rat brain is biphasic: it shows an early stage of anatomically restricted, low-intensity expression, and a juvenile stage of anatomically widespread, high-intensity expression. Most of the structures that express neurogranin during development conserve it in the adult stage. Neurogranin expression starts on embryonic day 18 in two different sites-the amygdalar primordium and in the piriform cortex-and is confined to these structures until the first postnatal day (P1). On P1, neurogranin expression increases dramatically in intensity, and appears in the olfactory cortex, isocortex, subiculum and hippocampus. In the striatum, expression starts on P1 and extends to the caudoputamen and parts of the globus pallidus and septum. Particularly complex patterns of labelling can be seen in the amygdala and cerebral cortex. Cortical layers showing early expression are the presumptive layers 4 and 5 in the somatosensory cortex, and layers 2 and 5 in the anterior cingulate and agranular insular cortices. Immunoreactivity is found mostly in cell bodies during the early and juvenile stages, but by the end of the first postnatal week it starts being more apparent in the neuropil. This phenomenon probably reflects the intracellular translocation of neurogranin to distal parts of the dendrites and dendritic spines. This process culminates by the end of the second postnatal week, when the adult pattern is reached. According to the timing and anatomy of its distribution, expression of neurogranin seems to be independently regulated in each telencephalic region by specific signalling mechanisms. It is proposed, on this basis, that neurogranin could be implicated in neuronal differentiation and synaptogenesis during telencephalic development.

Model of forebrain regionalization based on spatiotemporal patterns of POU-III homeobox gene expression, birthdates, and morphological features.

In situ hybridization was used to map spatiotemporal expression patterns of the four known intronless POU-III transcription factor genes Brn-1, Brn-2, Brn-4, and Tst-1 in the developing rat forebrain vesicle, beginning on embryonic day 10. The results indicate that the proliferation layers (ventricular and subventricular) and mantle layer of the forebrain neural tube each display a strikingly unique pattern of regionalized POU-III expression. Within a particular region, or layer within a region, none to all four of the mRNAs may be detected, and during development a particular mRNA in a particular region displays one of five expression patterns, or a combination of these patterns, which may be described as conserved, lost, transient, acquired, or redeployed expression. In the developing brain as a whole, Brn-1 and Brn-2 early on display somewhat different spatial expression patterns that converge to essential identity in the adult, whereas Brn-4 expression is initially broad and becomes much more restricted in the adult, and Tst-1 expression expands greatly through development. Usually, though not always, expression patterns tend to correlate with major histological features in the forebrain (often internal or external sulci associated with proliferation zones), and little evidence for waves of expression moving through the whole forebrain over time was obtained. Thus, clear differences in hybridization intensity often are observed between the cerebral cortex, basal telencephalic nuclei, hypothalamus, ventral thalamus, dorsal thalamus, and pretectal region. In contrast, transverse bands of hybridization extending from the roof to the floor of the forebrain, corresponding to proposed neuromeres, were not observed with these probes. The results suggest that POU-III transcription factors help define specific regions in the early neuroepithelium as well as different cellular phenotypes in the ventricular, subventricular, and mantle layers of specific regions later in development. Thus, the functions of these regulatory proteins may be different in proliferating neuroepithelial cells, young neurons, and mature neurons and appear to be region-specific.

Localization of neuropeptide Y and its C-terminal flanking peptide in human renal tissue.

We produced and characterized three anti-C-flanking peptides of neuropeptide Y (CPON) monoclonal antibodies. The Ka for these antibodies ranged from 0.4 to 0.8 x 10(8) l/mol with an IC50 for CPON(1-30) at about 20 nM as determined by ELISA. All these antibodies are IgG1 and recognize the 16-30 part of CPON. These antibodies and a specific anti-NPY monoclonal antibody were used to study the localization of CPON and NPY in the human kidney. The avidin-biotin technique was employed. NPY and CPON immunoreactivities were present in large amount in the renal tubules of the human kidney but not in the glomeruli. No labeling was found within the renal arterioles and veins, but some immunoreactivity was evidenced in the perivascular area. Because no specific receptor for CPON has been described to date, the presence of this peptide in the tubules may be due to a tubular reabsorption or perhaps to a local synthesis of pro-NPY.

Distribution of calbindin and parvalbumin in the developing somatosensory cortex and its primordium in the rat: an immunocytochemical study.

Immunocytochemical techniques were used to analyze the distribution of the calcium-binding proteins calbindin and parvalbumin during the pre- and postnatal development of the rat somatosensory cortex. Calbindin occurs in most early differentiated neurons that form the primordial plexiform layer at embryonic day 14. This expression in transient; during the perinatal period, calbindin becomes immunologically undetectable within the structures derived from the primordial plexiform layer, i.e., the prospective layers I and VIb. Immunoreactive neurons are also absent from adult layers I and VIb. Calbindin is also detected in a second population of neurons which, from embryonic day 18 onwards, distributes diffusely within the cortical plate. Some neurons of this population show morphological traits of immaturity, while others show complete dendritic arborization. The definitive pattern of distribution of calbindin-immunoreactive neurons is achieved by postnatal day 22. Infragranular layers contain intensely-immunoreactive cells whose numerical density decreases during postnatal development, whereas in supragranular layers similar neurons are interspersed among numerous faintly-stained neurons. Parvalbumin is detected for the first time at postnatal day 6, within a small group of neurons located in cortical layer V, and extends afterwards through the whole thickness of the cerebral cortex. At this same postnatal stage, groups of immunoreactive puncta are also found in layer IV of the somatosensory cortex; these puncta increase in density progressively and, at embryonic day 13, immunoreactive cells appear also grouped at this level. At this postnatal age, parvalbumin immunostaining delineates the somatosensory map in cortical layer IV. From this stage to adulthood, the number of immunoreactive neurons increases in the whole thickness of the somatosensory cortex. Barrels in layer IV become less distinct as immunoreactive cells and processes invade the septa. Layer IV in the adult somatosensory cortex appears more densely populated by parvalbumin immunoreactive neurons and puncta than in the surrounding areas.