Development of midline cell types and commissural axon tracts requires Fgfr1 in the cerebrum

The adult cerebral hemispheres are connected to each other by specialized midline cell types and by three axonal tracts: the corpus callosum, the hippocampal commissure, and the anterior commissure. Many steps are required for these tracts to form, including early patterning and later axon pathfinding steps. Here, the requirement for FGF signaling in forming midline cell types and commissural axon tracts of the cerebral hemispheres is examined. Fgfr1, but not Fgfr3, is found to be essential for establishing all three commissural tracts. In an Fgfr1 mutant, commissural neurons are present and initially project their axons, but these fail to cross the midline that separates the hemispheres. Moreover, midline patterning defects are observed in the mutant. These defects include the loss of the septum and three specialized glial cell types, the indusium griseum glia, midline zipper glia, and glial wedge. Our findings demonstrate that FGF signaling is required for generating telencephalic midline structures, in particular septal and glial cell types and all three cerebral commissures. In addition, analysis of the Fgfr1 heterozygous mutant, in which midline patterning is normal but commissural defects still occur, suggests that at least two distinct FGF-dependent mechanisms underlie the formation of the cerebral commissures.     

Telencephalic and diencephalic origin of radial glial processes in the developing preoptic area/anterior hypothalamus

Neuronal birth-dating sudies using [³H]thymidine have indicated that neurons in the preoptic area/anterior hypothalamus (POA/AH) are derived primarily from progenitors in proliferative zones surrounding the third ventricle. Radial glial processes are potential guides for neuronal migration, and their presence and orientation during development may provide further information about the origin of cells in the POA/AH. In addition to determining the orientation of radial glial fibers, we examined the relationship of neurons with identified birth dates to radial glial processes in the developing POA/AH of ferrets. Neuronal birth dates were determined by injecting ferret fetuses with bromodeoxyuridine (BrdU) at several different gestational ages; brains were taken from ferret kits at subsequent prenatal ages. Sections were processed for immunocytochemistry to reveal vimentin or glial fibrillary acidic protein in radial glia, or BrdU-labeled cell nuclei. Numerous radial glial processes extended from the lateral ventricles through ventral portions of the septal region to the pial surface of the POA/AH. These fibers both encapsulated and coursed ventrally through and around the anterior commissure of ferret, rat, and mouse fetuses. These ventrally directed fibers were less evident at older ages. In double-labeled sections from ferrets, BrdU-labeled cells in the dorsal POA/AH were often aligned in the same dorsal-ventral orientation as adjacent radial glial fibers. We suggest that a subset of neurons, originating in telencephalic proliferative zones, migrates ventrally along radial glial guides into the dorsal POA/AH. © 1995 John Wiley & Sons, Inc.     

FAK deficiency in cells contributing to the basal lamina results in cortical abnormalities resembling congenital muscular dystrophies

Targeted deletion of focal adhesion kinase (fak) in the developing dorsal forebrain resulted in local disruptions of the cortical basement membrane located between the neuroepithelium and pia-meninges. At disruption sites, clusters of neurons invaded the marginal zone. Retraction of radial glial endfeet, midline fusion of brain hemispheres, and gliosis also occurred, similar to type II cobblestone lissencephaly as seen in congenital muscular dystrophy. Interestingly, targeted deletion of fak in neurons alone did not result in cortical ectopias, indicating that fak deletion from glia is required for neuronal mislocalization. Unexpectedly, fak deletion specifically from meningeal fibroblasts elicited similar cortical ectopias in vivo and altered laminin organization in vitro. These observations provide compelling evidence that FAK plays a key signaling role in cortical basement membrane assembly and/or remodeling. In addition, FAK is required within neurons during development because neuron-specific fak deletion alters dendritic morphology in the absence of lamination defects.     

Transient Neuronal Populations Are Required to Guide Callosal Axons: A Role for Semaphorin 3C

The corpus callosum (CC) is the main pathway responsible for interhemispheric communication. CC agenesis is associated with numerous human pathologies, suggesting that a range of developmental defects can result in abnormalities in this structure. Midline glial cells are known to play a role in CC development, but we here show that two transient populations of midline neurons also make major contributions to the formation of this commissure. We report that these two neuronal populations enter the CC midline prior to the arrival of callosal pioneer axons. Using a combination of mutant analysis and in vitro assays, we demonstrate that CC neurons are necessary for normal callosal axon navigation. They exert an attractive influence on callosal axons, in part via Semaphorin 3C and its receptor Neuropilin-1. By revealing a novel and essential role for these neuronal populations in the pathfinding of a major cerebral commissure, our study brings new perspectives to pathophysiological mechanisms altering CC formation.     

Anatomy and physiology of neurons composing the commissural ring nerve of the cricket, Acheta domesticus

The commissural ring nerve (RN) of the cricket Acheta domesticus links together the two cercal motor nerves of the terminal abdominal ganglion. It contains the axons of almost 100 neurons including two bilateral clusters of eight to 13 ventrolateral neurons and approximately 75 neurons with midline somata within the terminal abdominal ganglion. The ventrolateral neurons have an ipsilateral dendritic arborization within the dorsal neuropil of the ganglion and their axons use the RN as a commissure in order to enter the contralateral nerves of the tenth ganglionic neuromere. In contrast, most midline neurons have bifurcating axons projecting bilaterally into the neuropil of the ganglion as well as into the RN where they often branch extensively before entering the contralateral tenth nerves. Most RN neurons have small, non-spiking somata with spike initiation zones distant from the soma. Many midline neurons also produce double-peaked spikes in their somata, indicative of multiple spike initiation zones. Spontaneous neuronal activity recorded extracellularly from the RN reveals several units, some with variable firing patterns, but none responding to sensory stimuli. The RN is primarily composed of small (50 nm diameter) axon profiles with a few large (0.5-1 microm diameter) profiles. Occasionally, profiles of nerve terminals containing primarily small clear vesicles and a few large dense vesicles are observed. These vesicles can sometimes be clustered about an active zone. We conclude that the primary function of the RN is to serve as a peripheral nerve commissure and that its role as a neurohemal organ is negligible. J. Exp. Zool. 286:350-366, 2000.Copyright 2000 Wiley-Liss, Inc.     

Gliogenesis in the embryonic brain of the grasshopper Schistocerca gregaria with particular focus on the protocerebrum prior to mid-embryogenesis

I investigate the pattern of gliogenesis in the brain of the grasshopper Schistocerca gregaria prior to mid-embryogenesis, with particular focus on the protocerebrum. Using the glia-specific marker Repo and the neuron-specific marker HRP, I identify three types of glia with respect to their respective positions in the brain: surface glia form the outmost cell layer ensheathing the brain; cortex glia are intermingled with neuronal somata forming the brain cortex; and neuropil glia are associated with brain neuropils. The ontogeny of each glial type has also been studied. At 24 % of embryogenesis, a few glia are observed in each hemisphere of the proto-, deuto- and tritocerebrum. In each protocerebral hemisphere, such glia form a cluster that expands rapidly during later development. Closer examination reveals proliferative glia in such clusters at ages spanning from 24 to 36 % of embryogenesis, indicating that glial proliferation may account for the expansion of the clusters. Data derived from 33–39 % of embryogenesis suggest that, in the protocerebrum, each type of glia is likely to be generated by its respective progenitor-forming clusters. Moreover, the glial cluster located at the anterior end of the brain can give rise to both surface glia and cortex glia that populate the protocerebrum via subsequent migration. Proliferation is observed for all three glial types, indicating a possible source for the glia.     

Dissection of the faint little ball (flb) phenotype: determination of the development of the Drosophila central nervous system by early interactions in the ectoderm.

The complex embryonic phenotype of mutations in the faint little ball (flb) locus, encoding the Drosophila EGF receptor homolog (DER), was dissected by temperature shifts of a temperature-sensitive allele. We show that the phenotype can be resolved into at least five components, which are temporally and spatially distinct. Most notably, the central nervous system (CNS) phenotype is determined at two separate phases. A severe collapse results from early defects in the DER-expressing ectodermal cells from which neuroblasts and midline glial cells deaminate. We thus suggest that DER activity is crucial for interactions that occur in the ectoderm at an early stage, and determine the fate of neuronal and glial cell lineages. This finding explains how a severe CNS phenotype is generated in flb embryos, in spite of the absence of expression of the protein in neuronal cells. In a second phase, during germ band retraction, the flb function is required specifically in the three pairs of midline glial cells (MG). In the absence of a functional DER protein, these cells die or fail to differentiate correctly, resulting in a fused commissure phenotype.     

Ontogeny of identified cells from the median domain in the embryonic brain of the grasshopper Schistocerca gregaria

In this paper, we propose an ontogeny for previously identified cells from the median domain in the midline of the embryonic brain of the grasshopper Schistocerca gregaria. The so-called lateral cells (LCs) are characteristically located laterally within the median domain at its border with the protocerebral hemispheres. The LC occurs singly and can be identified in the early embryo on the basis of their expression of the cell surface lipocalin Lazarillo. Using immunocytochemical, dye injection, electron microscopical and histological methods, we show that these LC are neurons and derive as postmitotic cells directly from the epithelium of the median domain. Further, they and the other identified cells of the median domain such as the protocerebral commissure pioneers (PCP), co-express the Mes-3 antigen, consistent with a derivation from the mesectodermal germ layer of the embryo. Subsequent to axogenesis, electron microscopy reveals that these Mes-3-expressing LC fasciculate with the co-expressing PCPs within the developing protocerebral commissure. We present a model for the origin of all these cells based on histological data and bromodeoxyuridine incorporation. The model suggests a delamination of cells from the mesectoderm followed by a migration to their ultimate sites within the median domain.     

Glial interactions with neurons during Drosophila embryogenesis

A monoclonal antibody (Mab5B12) demonstrating specificity for glial cells within the central and peripheral nervous systems of Drosophila has been used in combination with neural-specific antibodies to study the early organization of the Drosophila embryo. The embryonic central nervous system of Drosophila contains cells within the ventral midline that are recognized by monoclonal antibody 5B12. These cells are not recognized by either a polyclonal antiserum to horse radish peroxidase, which recognizes several antigens on the surface of Drosophila neurons, or Mab22C10, which recognizes an antigen specific to the peripheral nervous system. Mab5B12-positive cells lie dorsal both to the developing anterior and posterior commissures in each thoracic and abdominal segment and to the supraoesophageal commissure. They ensheath these commissures in later stage embryos. Other Mab5B12-positive cells lie dorsolateral to the CNS and send processes laterally to the lateral sensilla during axonogenesis in the PNS. These cells surround the axons of the intersegmental and segmental nerves. Other cells that line the advancing ectoderm during dorsal closure and surround the anal pads also express the Mab5B12 antigen. Neuronal cell cultures derived from Drosophila gastrulae contain cells expressing the Mab5B12 antigen. These cells can be found separate or in close association with neuronal clusters and their axons.     

Building the central complex of the grasshopper Schistocerca gregaria: axons pioneering the w, x, y, z tracts project onto the primary commissural fascicle of the brain

The central complex is a major neuropilar structure in the insect brain whose distinctive, modular, neuroarchitecture in the grasshopper is exemplified by a bilateral set of four fibre bundles called the w, x, y and z tracts. These columns represent the stereotypic projection of axons from the pars intercerebralis into commissures of the central complex. Each column is established separately during early embryogenesis in a clonal manner by the progeny of a subset of four identified protocerebral neuroblasts. We report here that dye injected into identified pioneers of the primary brain commissure between 31 and 37% of embryogenesis couples to cells in the pars intercerebralis which we identify as progeny of the W, X, Y, or Z neuroblasts. These progeny are the oldest within each lineage, and also putatively the first to project an axon into the protocerebral commissure. The axons of pioneers from each tract do not fasciculate with one other prior to entry into the commissure, thereby prefiguring the modular w, x, y, z columns of the adult central complex. Within the commissure, pioneer axons from columnar tracts fasciculate with the growth cones of identified pioneers of the existing primary fascicle and do not pioneer a separate fascicle. The results suggest that neurons pioneering a columnar neuroarchitecture within the embryonic central complex utilize the existing primary commissural scaffold to navigate the brain midline.     

Embryonic development of the sensory innervation of the clypeo-labral complex: further support for serially homologous appendages in the locust

The clypeo-labrum, or upper lip, of insects is intimately involved in feeding behavior and is accordingly endowed with a rich sensory apparatus. In the present study we map the temporal appearance of all major clusters of sensory cells on this structure in the locust during the first half of embryogenesis. The identities of these sensory cell clusters were defined according to the origin of the branching point of their axons from the labral sensory nerve as seen at mid-embryogenesis. The first sensory cells to differentiate from the labral epithelium do so at stereotypic sites beginning at around 32% of embryogenesis. Bilaterally symmetrical clusters of differentiated neurons rapidly appear and pioneering of the labral sensory nerve on each side is performed by a specific cell from each cluster. This cell directs its axon anteriorly towards a bilaterally symmetrical pair of cells, the frontal commissure pioneers, on either side of the developing frontal ganglion. The final trajectory of the sensory nerve within the labrum closely matches the pattern of Repo-expressing glial cells. The majority of the sensory cell clusters differentiate during embryogenesis, but the number of sensory cells in some clusters are modified significantly during postembryonic development. Comparing the innervation pattern of the clypeo-labrum with that of other mouthparts and the leg at mid-embryogenesis, we find a striking similarity in organization which we interpret as support for the homologous appendage hypothesis.     

Suppression by antisense mRNA demonstrates a requirement for the glial fibrillary acidic protein in the formation of stable astrocytic processes in response to neurons

The glial fibrillary acidic protein (GFAP) is a glial-specific intermediate filament protein, which is expressed in astrocytes in the central nervous system, as well as in astrocytoma cell lines. To investigate the function of GFAP, we have studied the human astrocytoma cell line, U251, which constitutively expresses GFAP and vimentin in the same 10-nm filaments. These cells respond to neurons in vitro in the same way as primary astrocytes: they withdraw from the cell cycle, support neuronal cell survival and neurite outgrowth, and they extend complex, GFAP-positive processes. To determine the role of GFAP in these responses, we have specifically suppressed its expression by stably transfecting the U251 cells with an antisense GFAP construct. Two stable antisense cell lines from separate transfections were isolated and were shown to be GFAP negative by Northern and Western blot analyses, and by immunofluorescence studies. The antisense cell lines were inhibited in their ability to extend significant glial processes in response to neurons. In culture with primary neurons, the average increase in process length of the U251 cells was nearly 400%, as compared to only 14% for the antisense transfectants. The other neuron induced responses of astrocytes, i.e., proliferative arrest and neuronal support, were not affected in these cell lines. These data support the conclusion that the glial-specific intermediate filament protein, GFAP, is required for the formation of stable astrocytic processes in response to neurons.     

A Comparison of the Polypeptide Pattern Between a Brain Primary Culture and Fractions of Bulk-Isolated Glial Cells

Abstract: This paper reports on the electrophoretic protein/polypeptide pattern of a rat brain primary culture. For comparison, the polypeptide pattern of neuronal and glial enriched fractions from adult rat brain and cerebral hemispheres from newborn and adult rat have been analysed. Water-soluble and SDS-extractable polypeptide fractions appeared and/or increased in amount in the cultures until confluency. The polypeptide pattern of the cultures most resembled that of the glial cell fractions, showing some of this fraction's specificity. Removal of fetal calf serum and addition of 0.1 mM dibutyryl cyclic adenosine monophosphate (dB-cAMP) produced few changes in the electrophoretic pattern. The study thus provides evidence in favour of the astroglial nature of the brain primary culture. It also shows that the cells undergo some maturation in the culture.     

Development of the anterior commissure in the opossum: Midline extracellular space and glia coincide with early axon decussation

While the anterior commissure has been shown to be an important route of information transfer in the forebrain, relatively little is known about its anatomical development. Glial substrates and extracellular spaces have been associated with the maturation of other large-fiber tracts, such as the corpus callosum and retinofugal pathway. The present study examined early stages in the maturation of the commissure in the gray short-tailed opossum, Monodelphis domestica. Monodelphis offspring are born after a short 14-day gestation, and, unlike in rats and mice, the anterior commissure develops entirely during the postnatal period. A number of techniques were employed: the carbocyanine dye Dil was used to label early axons in the region, semithin plastic sections were used to examine the extracellular environment of the developing commissure, and immunocytochemistry for glial fibrillary acidic protein (GFAP) was used to characterize glial components. Results suggest that the first commissural fibers that cross the midline pass through a region of large extracellular spaces and may use GFAP-immunoreactive cells and processes as guides during their midline decussation. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 403–414, 1997.     

Frizzled-3a and slit2 genetically interact to modulate midline axon crossing in the telencephalon

The anterior commissure forms the first axon connections between the two sides of the embryonic telencephalon. We investigated the role of the transmembrane receptor Frizzled-3a in the development of this commissure using zebrafish as an experimental model. Knock down of Frizzled-3a resulted in complete loss of the anterior commissure. This defect was accompanied by a loss of the glial bridge, expansion of the slit2 expression domain and perturbation of the midline telencephalic-diencephalic boundary. Blocking Slit2 activity following knock down of Frizzled-3a effectively rescued the anterior commissure defect which suggested that Frizzled-3a was indirectly controlling the growth of axons across the rostral midline. We have shown here that Frizzled-3a is essential for normal development of the commissural plate and that loss-of-function causes Slit2-dependent defects in axon midline crossing in the embryonic vertebrate forebrain. These data supports a model whereby Wnt signaling through Frizzled-3a attenuates expression of Slit2 in the rostral midline of the forebrain. The absence of Slit2 facilitates the formation of a midline bridge of glial cells which is used as a substrate for commissural axons. In the absence of this platform of glia, commissural axons fail to cross the rostral midline of the forebrain.     

Transient cellular structures in developing corpus callosum of the human brain

The corpus callosum connects two cerebral hemispheres as the most voluminous fiber system in the human brain. The developing callosal fibers originate from immature pyramidal neurons, grow through complex pathways and cross the midline using different substrates in transient fetal structures. We analyzed cellular structures in the human corpus callosum on postmortem brains from the age of 18 weeks post conception to adult, using glial fibrillary acidic protein, neuron-specific nuclear protein, and chondroitin sulphate immunocytochemistry. We found the presence of transient cellular structures, callosal septa, which divide major fiber bundles and ventrally merge with subcallosal zone forming grooves for callosal axons. The callosal septa are composed of glial fibrillary acidic protein reactive meshwork, neurones and the chondroitin sulphate immunoreactive extracellular matrix. The developmental window of prominence of the callosal septa is between 18-34 weeks post conception which corresponds to the period of most intensive growth of callosal axons in human. During the early postnatal period the callosal septa become thinner and shorter, lose their neuronal and chondroitin sulphate content. In conclusion, transient expression of neuronal, glial and extracellular, growing substrate in the callosal septa, as septa itself, indicates their role in guidance during intensive growth of callosal fibers in the human brain. These findings shed some light on the complex morphogenetic events during the growth of the corpus callosum and represent normative parameters necessary for studies of structural plasticity after perinatal lesions.     

GUANYL CYCLASE IN CHICK EMBRYO BRAIN CELL CULTURES: EVIDENCE OF NEURONAL LOCALIZATION

Abstract— Guanyl cyclase activity was studied in dissociated chick embryo brain cell cultures presenting different ratios of neuronal to glial elements. The cultures containing neurons in substantial numbers always had higher guanyl cyclase activities than those consisting mainly of glial cells. No guanyl cyclase activity could be found in cultures made up of pure glial or meningeal cells. These results provide further evidence for our conclusion based on subcellular fractionation studies (G oridis & M organ , 1973), that brain guanyl cyclase might be overwhelmingly concentrated in neurons. Guanyl cyclase activity of chick embryo cerebral hemispheres increased sixfold between day 12 and day 16 after fertilization; an increase, though of much smaller magnitude, was also seen in cultured cells of the same age.