Pleiotropic and isoform-specific functions for Pitx2 in superior colliculus and hypothalamic neuronal development.

Transcriptional regulation of gene expression during development is critical for proper neuronal differentiation and migration. Alternative splicing and differential isoform expression have been demonstrated for most mammalian genes, but their specific contributions to gene function are not well understood. In mice, the transcription factor gene Pitx2 is expressed as three different isoforms (PITX2A, PITX2B, and PITX2C) which have unique amino termini and common DNA binding homeodomains and carboxyl termini. The specific roles of these isoforms in neuronal development are not known. Here we report the onset of Pitx2ab and Pitx2c isoform-specific expression by E9.5 in the developing mouse brain. Using isoform-specific Pitx2 deletion mouse strains, we show that collicular neuron migration requires PITX2AB and that collicular GABAergic differentiation and targeting of hypothalamic projections require unique Pitx2 isoform dosage. These results provide insights into Pitx2 dosage and isoform-specific requirements underlying midbrain and hypothalamic development.

The ascending tectofugal visual system in amniotes: new insights.

Ascending tectal axons carrying visual information constitute a fiber pathway linking the mesencephalon with the dorsal thalamus and then with a number of telencephalic centers. The sauropsidian nucleus rotundus and its mammalian homologue(s) occupy a central position in this pathway. The aim of this study was analyzing the rotundic connections in reptiles and birds in relation with comparable connections in mammals, by using biotinylated dextran amines and the lipophilic carbocyanine dye DiI as tracing molecules. In general, rotundic connections in reptiles and birds are quite similar, especially with regards to pretectal and tectal afferences; as a novel finding, we describe varicose fibers arising from nucleus rotundus that reached the developing chick striatum. In addition, this study described the dorsal claustrum as a novel telencephalic target for the suprageniculate nucleus in mammals. Overall, telencephalic projections from the posterior/intralaminar complex of the mammalian thalamus can be compared with the telencephalic projections of the reptilian nucleus rotundus. With the exception of the isocortical connections, the mouse suprageniculate nucleus shares a number of afferent and efferent connections with the sauropsidian nucleus rotundus. Especially significant were the suprageniculate fibers reaching the striatum and then following to reach pallial derivatives such as the lateral amygdala (ventral pallium) and the dorsal claustrum (lateral pallium). These connections can be compared with the rotundic fibers reaching the ventromedial part of the anterior dorsal ventricular ridge in reptiles/entopallium in birds (ventral pallium) and the dorsolateral part of the anterior dorsal ventricular ridge in reptiles (lateral pallium), and probably the mesopallium in birds.

Light experience induces differential asymmetry pattern of GABA- and parvalbumin-positive cells in the pigeon's visual midbrain.

The formation of functional and morphological asymmetries within the pigeon's tectofugal system depends on left-right differences in visual input during embryonic development. This asymmetric stimulation presumably affects activity-dependent differentiation processes within the optic tectum. Behavioral studies reveal that prehatch light stimulation asymmetry influences both left- and right-hemispheric processes in a differential way. Thus, we have to assume divergent effects on both hemispheres. This study represents an attempt to test the hypothesis that embryonic light asymmetry induces different, cell-type-specific effects in the left and the right optic midbrain. Since it is likely that inhibitory interneurons play a critical role in the establishment of asymmetries, we examined in both sides of the brain the soma sizes of GABA- and parvalbumin- (PV) immunoreactive (ir) cells of the tectum and the magnocellular isthmic nucleus in controls and in dark-incubated animals. No cell size asymmetries of magnocellular isthmic neurons were found in either dark-incubated or control birds. Dark-incubation also prevented the establishment of lateralized differences in GABAergic and PV-positive tectal cells. However, in control birds GABAergic cells displayed larger somata in the left tectum, whereas PV-ir neurons were enlarged within the right tectum. This complementary asymmetry pattern suggests that PV- and GABA-ir tectal cells represent different cellular populations which react differently to visual input. Thus, our data show that visual lateralization does not result from a mere growth promoting effect that enhances differentiation within the behaviorally dominant left side, but is constituted by different cell type-specific circuits which are divergently adjusted in the left and in the right tectum.

Targeting axons to specific fiber tracts in vivo by altering cadherin expression.

In brain development, neurons have to be connected with specific postsynaptic neurons to establish functional neuronal circuits. Cadherins are cell adhesion molecules, which mark developing neuronal circuits. Each member of this class of molecules is expressed only on a restricted set of fiber fascicles that connect gray matter structures to form functional neural circuits. In view of their expression patterns, cadherins have been postulated to play a functional role in the proper establishment of fiber connections. We chose the chicken optic tectum to analyze the instructive potential of cadherins in axonal pathfinding. Three tectofugal pathways, the tectothalamic, tectobulbar, and tectoisthmic tracts, exit the dorsal mesencephalon via the brachium of the superior colliculus, a large fiber structure, which can be divided in specific subtracts that are characterized by the selective expression of N-cadherin, cadherin-7, cadherin-6B, or R-cadherin. By using in vivo electroporation, we overexpressed each of the cadherins in tectal projection neurons between embryonic days 6 and 11. Cotransfection with green fluorescent protein expression plasmid allowed us to assess the pathway choice, which the transgenic axons had made. Quantification based on confocal laser scanning microscopic images revealed that transgenic axons selectively fasciculated with tectofugal tracts specified by the matching type of cadherin. This is the first direct evidence that cadherins mediate differential axonal pathfinding in vivo, possibly by a preferentially homotypic adhesive mechanism.

Role of cdk5 and tau phosphorylation in heterotrimeric G protein-mediated retinal growth cone collapse.

During axonal growth, repulsive guidance cues cause growth cone collapse and retraction. In the chick embryo, membranes from the posterior part of the optic tectum containing ephrins are original collapsing factors for axons growing from the temporal retina. We investigated signal transduction pathways in retinal axons underlying this membrane-evoked collapse. Perturbation experiments using pertussis toxin (PTX) showed that membrane-induced collapse is mediated via G(o/i) proteins, as is the case for semaphorin/collapsin-1-induced collapse. Studies with Indo-1 revealed that growth cone collapse by direct activation of G(o/i) proteins with mastoparan did not cause elevation of the intracellular Ca(2+) level, and thus this signal transduction pathway is Ca(2+) independent. Application of the protein phosphatase inhibitor okadaic acid alone induced growth cone collapse in retinal culture, suggesting signals involving protein dephosphorylation. In addition, pretreatment of retinal axons with olomoucine, a specific inhibitor of cdk5 (tau kinase II), prevented mastoparan-evoked collapse. Olomoucine also blocks caudal tectal membrane-mediated collapse. These results suggest that rearrangement of the cytoskeleton is mediated by tau phosphorylation. Immunostaining visualized complementary distributions of tau phospho- and dephosphoisoforms within the growth cone, which also supports the involvement of tau. Taking these findings together, we conclude that cdk5 and tau phosphorylation probably lie downstream of growth cone collapse signaling mediated by PTX-sensitive G proteins.

Cyclic nucleotide-gated cation channel expression in embryonic chick brain.

Cyclic nucleotide-gated cation channels mediate sensory transduction in vertebrate photoreceptors and olfactory epithelium. These channels are also present in some non-sensory cells, but little is known of their physiological roles outside sensory systems. Using in situ hybridization we found that cyclic nucleotide channel mRNA is expressed specifically in the embryonic chicken forebrain, thalamus, optic tectum, basal midbrain and hindbrain, as well as in the branchial arches, limb buds and skin. Cyclic nucleotide gated channels may thus contribute to development or to cellular differentiation in the brain and in other tissues.

Graded and lamina-specific distributions of ligands of EphB receptor tyrosine kinases in the developing retinotectal system.

Molecular gradients have been postulated to control the topographic mapping of retinal axons in their central targets. Based initially on their expression patterns, and more recently on functional studies, members of the EphA subfamily of receptor tyrosine kinases and their ephrin-A ligands have been implicated in the guidance of retinal axons along the anterior-posterior axis of the chick optic tectum. The report that a receptor of the EphB subfamily, EphB2/Cek5/Nuk/Sek3, is expressed in a high ventral to low dorsal gradient in the developing chick retina and is present on ganglion cell axons suggests that it may be involved in the mapping of retinal axons along the corresponding dorsal-ventral axis of the tectum. To address this issue, we have determined the expression and distribution of ephrin-B1/LERK-2/Cek5-L and ephrin-B2/LERK-5/Htk-L/ELF-2, ligands for EphB2, in the developing chick retinotectal system using riboprobes, immunocytochemistry, and receptor affinity probes. Both ephrin-B1 and ephrin-B2 transcripts are expressed in a high dorsal to low ventral gradient in the developing retina, complementary to the distribution of EphB2. Ephrin-B1 and ephrin-B2 proteins are predominantly found in the developing plexiform layers, suggesting a role in the development of intraretinal connections. Neither protein is detected on ganglion cell axons. In tectum, ephrin-B1 transcripts are expressed in a high dorsal to low ventral gradient in the neuroepithelium and the protein is present along the processes of radial glia and is concentrated at their endfeet in the stratum opticum, at the time retinal axons are growing through it. This distribution of ephrin-B1 suggests that it influences retinal axon mapping along the dorsal-ventral tectal axis and may also be involved in intratectal development. In contrast, ephrin-B2 transcripts and protein are localized to the deeper retinorecipient laminae in the tectum at the time retinal axons begin to arborize in them, suggesting that this ligand may influence the laminar patterning of retinal axon terminations.

The embryological development of primary visual centres in the turtle Emys orbicularis.

The development of the primary visual centres was studied in a series of embryos of the turtle, Emys orbicularis, incubated at 25 degrees C. The differentiation of both visual and nonvisual diencephalic and mesencephalic structures takes place entirely within the 2nd quarter of the period of incubation; this finding appears to be consistent with previous descriptions of the embryology of 2 other chelonian species, Lepidochelys and Chelydra. Two successive waves of migration, each dividing into internal and external sheaves, are involved in the formation of the structures of the diencephalon and mesencephalon. The primary visual centres, which comprise 2 hypothalamic, 5 thalamic and 5 pretectal zones of retinal projections, together with the 2 superficial layers of the tectum and a single tegmental projection zone, all have their origin in the external sheaf of the 1st wave of migration. The finding that the adult nucleus geniculatus lateralis dorsalis, pars ventralis arises from one of the migrations of the dorsal thalamus is discussed in the context of the debate over the possible homologues of the mammalian geniculostriate visual pathway.

Laminar specificity of extrinsic cortical connections studied in coculture preparations.

The formation of specific neural connections in the cerebral cortex was studied using organotypic coculture preparations composed of subcortical and cortical regions. Morphological and electrophysiological analysis indicated that several cortical efferent and afferent connections, such as the corticothalamic, thalamocortical, corticocortical, and corticotectal connections, were established in the cocultures with essentially the same laminar specificity as that found in the adult cerebral cortex, but without specificity of sensory modality. This suggests the existence of a cell-cell recognition system between cortical or subcortical neurons and their final targets. This interaction produces lamina-specific connections, but is probably insufficient for the formation of the modality-specific connections.

Retinal and tectal connections of embryonic nucleus superficialis magnocellularis and its mature derivatives in the chick.

In a companion paper (Puelles et al, this issue), the cytoarchitectonic development of the thalamic primordium called nucleus superficialis magnocellularis (SM) and its adult configuration in the chick were studied, correcting the misinterpretations that have impeded proper study of this neuronal group. Given its superficial position in the diencephalon, in contact with the optic tract and neighbouring retinorecipient grisea (SS, GV), as well as with the tecto-recipient n. rotundus, SM was suspected to have connections with centers of the visual pathway. In this paper we report the existence of a non-topographic retinal projection over the superficial adult derivate of SM (n. interstitialis tractus opticus, ITO) and a non-topographic, diffuse projection of the whole SM-derived population (area perirotundica, ApR, and ITO) onto the optic tectum. The latter was demonstrated throughout the late embryonic period in which SM loses its embryonic unitary character and becomes dispersed into its ill-defined, definitive adult portions (ITO, ApR). Golgi-like HRP- or DiI-labeling of SM cells showed a protracted immature appearance of their dendrites, expressed coincidently with a capacity to translocate superficially into the optic tract.

Subplate neurons pioneer the first axon pathway from the cerebral cortex.

During the development of the nervous system, growing axons must traverse considerable distances to find their targets. In insects, this problem is solved in part by pioneer neurons, which lay down the first axonal pathways when distances are at a minimum. Here the existence of a similar kind of neuron in the developing mammalian telencephalon is described. These are the subplate cells, the first postmitotic neurons of the cerebral cortex. Axons from subplate neurons traverse the internal capsule and invade the thalamus early in fetal life, even before the neurons of cortical layers 5 and 6, which will form the adult subcortical projections, are generated. During postnatal life, after the adult pattern of axonal projections is firmly established, most subplate neurons disappear. These observations raise the possibility that the early axonal scaffold formed by subplate cells may prove essential for the establishment of permanent subcortical projections.

[Development of the trout visual system (Salmo irideus) (author's transl)]. / Le développement du systéme visuel primaire de Salmo irideus.

The organization of the primary visual system of the larval trout (Salmo irideus) was studied at different stages of development: 5 days before hatching, 1 and 2 months post-hatching, by Fink and Heimer's method and radioautography. After ablation of the right retina or injection of 14C Proline (0,05 microCi in 0,5 microliter) in the right eye, animals were kept at 13 degrees C for 6 hours to 7 days and fixed according to the technic used. We also stained transverse sections of larval stages (14 days before hatching to hatching stage) with Bodian's method. The results show that the retino-tectal fibers are precocious. They are present five days before hatching. The retino-thalamic projections appear at hatching. The geniculate nucleus has a mesencephalic origin. The radioautograms show this structure at hatching. The optic tectum differentiates progressively latero-medially and from the anterior level to the posterior level. Among the 3 correspondent optic tectal layers of the adult (superficial, intermediate and deep), the second is present at hatching while the two others appear progressively between one and two months. A comparison of the fish brain with that of other vertebrates is difficult. We try to discuss some problems of homology (geniculate nucleus, pretectum, accessory optic centre).

Development of the diencephalon in the rat. IV. Quantitative study of the time of origin of neurons and the internuclear chronological gradients in the thalamus.

Groups of pregnant rats were injected with two successive daily doses of 3H-thymidine from gestational days 13 and 14 (E13 + 14) until the day before birth (E21 + 22). With this progressively delayed comprehensive labelling procedure we determined the time of origin of neurons in the nuclei of the epithalamus, thalamus, and ventral thalamus. The zona incerta, subthalamic nucleus, reticular nucleus, posterior nucleus, and ventral lateral geniculate nucleus are composed of the earliest arising neurons (E13, or before, to E15). The neurons of the lateral habenular nucleus are produced between days E13--16. The neurons of the medial geniculate and lateral geniculate nuclei, the ventrobasal and ventrolateral complexes, and the nucleus lateralis, pars posterior, arise rapidly on days E14--15; the medial geniculate nucleus with a peak on day E14, the others with a peak on day E15. Neurons of a group of nuclei, with ill-defined boundaries medial to the sensory relax nuclei, arise apparently on days E15--16, with a peak on day E15; these may represent the intralaminar nuclei. The next group is generated on days E15--16 but with peak formation time on day E16; this includes the anteroventral, anterodorsal, anteromedial and mediodorsal nuclei. The rhomboid, reuniens and paratenial nuclei, and the paraventricular nucleus, pars anterior, arise next (E16--17). The medial habenular nucleus forms last and over a protracted period (E15--19). With their lengthy generation time the lateral and medial habenular nuclei resemble more the nuclei of the hypothalamus than the nuclei of the dorsal thalamus.

Prenatal development of central optic pathways in albino rats.

The development of the central optic projections in albino rat fetuses has been studied using light and electron microscopic degeneration techniques and the horseradish peroxidase method for demonstrating axonal projections of neurons. The first optic axons to reach the region of the optic chiasm arrive at day 15. By day 16, a substantial optic chiasm is seen and the optic tract can be traced into the epithalamus, having first passed through the ventral lateral geniculate nucleus and a thin lamina of cells which is thought to correspond to part of the future dorsal geniculate nucleus. A growth rate of 80-100 mum per hour is estimated for the fastest growing axons. By day 16-1/3 the first axons have entered the anterior border of the superior colliculus and in the next day have grown across the entire rostrocaudal extent with the exception of the medial and lateral edges. The optic axons are recognized at day 17 as bundles lying just below the surface, but in older animals they come to lie deeper, as the whole layer of optic innervation broadens. The first synapses to be formed in the superior colliculus (some of them of optic origin) appear on day 17. Subsequently, there is a gradual increase in the number of contacts, the great majority being formed by optic axons. Compared with previous studies on Xenopus and chick, one of the most striking features of the development of the central visual connections in the rat is the relatively long time before the first optic axons reach the brain and the speed with which they innervate the central structures once they have arrived.