Interleukin-7 (IL-7) and IL-7 splice variants affect differentiation of human neural progenitor cells.

Alternative splicing of pre-mRNA increases proteomic diversity, a crucial mechanism in defining tissue identity. We demonstrate differentially spliced interleukin (IL)-7 in distinct anatomic areas in the adult, in developing human brains and in normal human neuronal progenitor (NHNP) cells. IL-7c (c, the canonical form spanning all six exons) or its variants IL-7 delta 5, delta 4 or delta 4/5 were cloned and expressed as recombinant proteins. IL-7 and splice variants were able to shift the differentiation of NHNP cells as compared with the diluent control (P<0.01) defined by anti-beta (III)-tubulin and glial fibrillary acidic protein expression, with different degrees (IL-7c>delta 4/5>IL-7 delta 5); IL-7 delta 4 exhibited a significantly weaker potency. Differentiation was confirmed by transcriptome analysis of IL-7c-stimulated neural NHNP cells, resulting in 58 differentially expressed genes; some of these are involved in neural differentiation, for example, the developmentally regulated transcription factor krüppel-like factor 12, musashi 2, a translational regulator of cell fate or the sonic hedgehog receptor patch 1. This suggests that IL-7 influences neural development at a molecular level by participating in human brain architecture through glia cell formation: a paradigm that alternative splicing in cytokines, for example, for IL-7, has a physiological role in human organ development and progenitor cell differentiation.

Mass spectrometrical analysis of human serine racemase in foetal brain.

We analysed human serine racemase for the first time from human foetal brain by mass spectrometrical methods, MALDI MS and MS/MS. The detection of human serine racemase from a transient area of human foetal brain, the perireticular nucleus, that is suggested to be mainly involved in guidance of corticofugal and thalamocortical fibers, may be a clue for the important role of this enzyme in neuronal migration and brain development via regulation of NMDA receptor activity.

Calcium-binding proteins in the human developing brain.

Recent studies have demonstrated that antibodies against the calcium-binding proteins (CaBPs) parvalbumin (PV), calbindin (CB), and calretinin (CR) are appropriate tools for demonstrating transient features and developmental changes of human fetal brain organization as well as for detecting specific alterations in pathologically altered specimens. CB and CR are abundantly expressed in various nerve cell types of the subplate in the second half of gestation. The subplate being an outstandingly wide zone subjacent to the cortical plate, it is a "waiting compartment" for various cortical afferents that reside here prior to entering the cortical plate. The cortical plate (future layers II-VI of the cerebral cortex) contains only CR-ir neurons until the 6th gestational month. In the 7th and 8th month, cortical CB- and PV-ir interneurons are observed in deeper portions of the cortical plate. Cajal-Retzius cells of layer I are CR-immunolabeled from the 4th month onwards. Fetal hydrocephalus causes severe alterations of CB- and PV-ir neurons in the subplate and the cortical plate: shrinkage of ir neurons, loss of process labeling and in most severe cases, entire loss of immunolabeling. Such alterations, which cannot be detected in Nissl-stained sections, indicate distinct impairment of neuronal function. The ganglionic eminence being a prominent part of the telencephalic proliferative zone persists nearly throughout the entire fetal period. Between 16 and 24 weeks of gestation, CR-ir cells are found in the center and, in a higher number, in the periphery, i.e., the mantle zone, of the ganglionic eminence. The mantle zone also exhibits CB-ir cells. These observations support experimental data showing that CR-ir precursor cells leave the ganglionic eminence to migrate towards the cerebral cortex. The CR- and CB-ir neurons of the mantle zone most probably represent an intermediate target for outgrowing axons. This notion is supported by the observation that SNAP (synaptosomal associated protein) 25-ir fibers coming from the intermediate zone terminate upon CR-ir cells in the mantle zone. Within the amygdaloid complex, immature, migrating CR- or CB-ir neurons are observed in the 5th and 6th gestational month. In the 8th and 9th month, anti-CR and anti-CB mark different subsets of interneurons as well as a small proportion of pyramidal projection neurons. The different subsets of interneurons are likely to be functionally different with regard to their connectivities. Considering studies in the literature, it is obvious that CR is transiently expressed in pyramidal cells. Moreover, diffuse (neuropil) CB and CR immunolabeling, which is found in different intensities in the various amygdaloid subdivisions, displays distinct redistribution during development, an observation indicating reorganization of afferent inputs. The sequential arrival of various afferent fiber systems in the two compartments of the striatum (patch and matrix compartment) is reflected by changing patterns of diffuse CB immunolabeling: During the second half of gestation, the patches are labeled and postnatally a changeover to matrix labeling is seen. The thalamic reticular complex reveals prominent transient features seen in PV and CR immunopreparations. Four subdivisions become obvious: the main portion, the perireticular nucleus, the medial subnucleus, and the pregeniculate nucleus. The PV- and CR-ir perireticular nucleus, not visible in the mature brain, is a distinct fetal entity located within the internal capsule. The main portion of the reticular complex is much more prominent in the fetus than in the adult and displays transitory CR expression. The most probable developmental role of the reticular complex is to provide guiding cues for outgrowing axons from or into the dorsal thalamus. The basal nucleus of Meynert and the hypothalamic tuberomamillary nucleus both provide extrathalamic projections to the cerebral cortex. The sequential differentiation of the two nuclei can be demonstrated using anti-CB and anti-PV. The basal nucleus strongly expresses CB and appears to be mature distinctly earlier than the PV-ir tuberomamillary nucleus. Antisera against CaBPs clearly demonstrate that the magnocellular part of the red nucleus located in the mesencephalic tegmentum is outstanding in the fetal and perinatal brain and inconspicuous in the adult. In particular, CB is the most abundant CaBP in this portion of the red nucleus. The dominance of the magnocellular part over the parvocellular part may be a substrate for a specific transitory pattern of motor behavior. On the whole, CaBPs mark the transient architectonic organization of the brain, which is involved in the establishment of transitory neuronal circuitries. The latter are essential for the formation of mature projections. Detailed data on the normal organization of the transient structures are required for the evaluation of alterations occurring in the fetal and perinatal brain. The transient structures are sites of predilection for alteration caused by hypoxia-ischemia, hemorrhage, or hydrocephalus.

Expression of a kinase anchoring protein 79 and synaptophysin in the developing human red nucleus.

Our previous study showed that in the human fetal and neonatal brain, the magnocellular and parvocellular parts of the red nucleus can be well delineated by calcium-binding proteins. To study the development of rubral afferents, the expression of A kinase anchoring protein 79 (AKAP79) and synaptophysin (SYN) was examined in the human fetal red nucleus. It was found that during prenatal development both AKAP79 and SYN expression increased gradually although a major alteration in the distribution of the proteins within the two compartments of the red nucleus was not observed. In AKAP79 immunopreparations, the magnocellular part became well demarcated from 23 weeks of gestation onwards and both parts showed punctate immunolabelling with moderate to high packing densities of immunoreactive cells. SYN immunoreactivity with a punctate appearance was, however, mainly located in the parvocellular part. It was evenly distributed throughout the compartment at 14-22 weeks of gestation, and then from 23 weeks to the time of birth, there was a pericellular arrangement of SYN. Our observations are mainly in line with connectivity data regarding the red nucleus.

Expression of calbindin and calretinin in the human ganglionic eminence.

The conspicuous ganglionic eminence representing a part of the telencephalic proliferative zone contains neuroblasts of the striatum. Recently it has been found to contribute significantly to the class of interneurons in the cerebral cortex. Subpopulations of cortical interneurons contain calretinin and calbindin. The expression of calretinin and calbindin in the ganglionic eminence and adjacent areas has been investigated immunocytochemically during fetal development using the brains of 10 infants ranging in age from 16 and 26 weeks gestation. Between 16 and 20 weeks gestation, numerous calretinin-immunreactive nerve cells are found in the ganglionic eminence, particularly in the mantle and the intermediate zones. The number of calretinin-immunoreactive cells decreases gradually from 21 weeks gestation onwards. Larger calbindin-immunoreactive cells are seen in the ganglionic eminence, and their number increases from 20 weeks gestation in the mantle zone. These results may indicate that calretinin-immunoreactive precursor cells, found in the ganglionic eminence, tangentially migrate toward the cortex. Moreover, the mantle zone displaying a specific calretinin and calbindin immunolabeling may represent an intermediate target for outgrowing axons. The findings are discussed with regard to central nervous system complications in preterm infants involving the ganglionic eminence.

Development-related expression of AKAP79 in the striatal compartments of the human brain.

The expression of AKAP79 which tethers regulatory proteins within postsynaptic densities has been studied in the two striatal compartments, i.e. patches and matrix, at different stages of the developing human brain by means of immunohistochemistry. The two striatal compartments exhibit various intensities of diffuse immunolabelling and a different number of immunoreactive nerve cells. From the 14th to 20th gestational week a nearly homogeneous distribution of immunoreactive structures in the two compartments of the striatum is seen. Thereafter, a decrease in immunoreactive structures within the matrix is observed (22nd-25th week, intermediate stage). From the 27th week onwards the patch compartment contains distinctly more immunoreactive puncta and nerve cells. Thus, the patches stand out clearly in the immunopreparations. This distribution pattern does not change during proceeding development. AKAP79-immunoreactive nerve cells closely resemble those constituting the class of medium-sized inhibitory projection neurons that receive the dopaminergic input of the striatum. Literature data suggest that AKAP79 may be functionally attributed to dopaminergic inputs. Accordingly, the patterns of AKAP79 expression can at least in part be correlated with the sequential occurrence of dopaminergic innervation. The mature matrix containing a dopaminergic innervation being as dense as in the patches displays distinctly less AKAP79-immunoreactive neurons and puncta than the patches. This discrepancy might indicate that a subpopulation of matrix neurons may, despite dopaminergic input, not express AKAP79.

Effect of fetal hydrocephalus on the distribution patterns of calcium-binding proteins in the human occipital cortex.

Neuronal pathology in the cerebral cortex (CC) of fetal hydrocephalus brains is quite subtle when applying routine Nissl staining. This study is aimed at investigating alterations of interneurons which can be immunolabelled by antibodies against the calcium-binding proteins calretinin (CR), calbindin (CB) and parvalbumin (PV). The subplate (SP) subjacent to the cortex anlage is included as this transient zone plays a pivotal role in the establishment of cortical connections. Nine occipital lobes from cases of fetal hydrocephalus and 9 controls were categorized according to age: 21-25 weeks of gestation (group 1) and 32-36 weeks (group 2). No differences in the distribution, number and morphology of CR-immunoreactive (ir) neurons are seen when comparing hydrocephalic with control brains of group 1. In severely altered hydrocephalic brains of group 2, the distribution and number of CR-, PV- and CB-ir nerve cells are not altered; however, they appear shrunken and processes are less immunolabelled. In extremely altered tissue PV-, CB-ir neurons cannot be detected, whereas the number of CR-ir somata is not reduced. The data indicate that subpopulations of interneurons of the CC may be differentially damaged. The alterations observed in the SP may implicate a possible impairment of transient neuronal circuitries that are essential for the development of cortical connections. On the whole, these neuronal alterations may account for residual deficits observed after shunting.

Expression of leukemia inhibitory factor in the ganglionic eminence of the human fetal brain after bleedings.

Bleedings in the ganglionic eminence (GE) being a prominent domain of the telencephalic proliferative zone is a frequent complication of preterm infants. Such bleedings may induce cellular responses in the vicinity of the lesion. Using immunohistochemistry, this study demonstrates for the first time that leukemia inhibitory factor (LIF) belonging to the family of neuropoetic cytokines is expressed in GE cells up to 200 microm from the lesion (six brains, gestational age 22-26 weeks); whereas control brains do not exhibit any LIF-immunoreactive cells in the GE. The expression of LIF in cases with bleedings may interfere with normal developmental processes taking place within the ganglionic eminence.

Differential expression of calcium-binding proteins in the red nucleus of the developing and adult human brain.

The adult human red nucleus consists of two parts: (1) the parvocellular part, which is clearly separated from (2) the magnocellular part. The latter and its rubrospinal projection is known to be rudimentary in the adult human brain. Information concerning the fetal or neonatal features of the red nucleus is sparse. This study is aimed at providing a detailed account of the distribution of three calcium-binding proteins: calretinin (CR), calbindin (CB), and parvalbumin (PV), which are known to be expressed in distinct neuronal populations. Special attention has been paid to transient phenomena. CB was the most abundant protein in the magnocellular part in fetal and perinatal brains; immunoreactive (ir) neurons appeared numerous and densely packed. In the adult only few and widely spaced ir nerve cells were present. CR-expression largely corresponds to that of CB, except that fewer neurons were immunolabelled. In double-labellings the majority of neurons expressed both CB and CR; a moderate number of nerve cells solely expressing CR was present in the magnocellular part. PV-ir fibers and a moderate number of small cells were observed in the fetal, perinatal as well as the adult parvocellular part. A few PV-ir neurons were seen in the magnocellular part of the fetal and perinatal brains. Our results indicated that: (1) the magnocellular and parvocellular parts of the red nucleus were well-demarcated portions from fetal life onwards, thus a dominance of the parvocellular part over the magnocellular occurred during development; (2) the magnocellular part was more prominent in the fetal period than in adulthood; (3) neurons in the red nucleus were heterogeneous with respect to the immunoreactivities towards the three calcium-binding proteins examined; (4) the transient prominence of the magnocellular part might be a substrate for a specific transitory pattern of motor behaviour.

Transient expression of synaptogyrin in the ganglionic eminence of the human fetal brain.

The ganglionic eminence (GE) representing a conspicuous bulb-like elevation of the telencephalic proliferative zone has recently been shown to be involved in the establishment of cortical connections. This study demonstrates the presence of synaptogyrin-immunoreactivity in a large number of cell bodies of the human GE between 12 and 20 weeks of gestation. From the 20th week onwards synaptogyrin expression sharply declines. No immunoreactive structures are detectable in the 23rd week or later. As the GE persists nearly throughout the entire fetal period these results show that its neurochemical features change distinctly in the course of development. The synaptogyrin-immunoreactive GE-cells may form an early corticopedal connection which provides a scaffold for outgrowing cortical axons.

Transient structures of the human fetal brain: subplate, thalamic reticular complex, ganglionic eminence.

Morphological features of the subplate, the thalamic reticular complex and the ganglionic eminence, which represent three major transient structures of the human fetal forebrain, are summarized with special reference to their functional roles. The subplate harboring various neuronal types is an outstandingly wide zone subjacent to the cortical plate in the human fetal brain. Within the subplate various cortical afferents establish synaptic contacts for a prolonged period before entering the cortical plate. Therefore, the subplate is regarded as a "waiting compartment" which is required for the formation of mature cortical connections. Next to the thalamic reticular nucleus, within the fibers of internal capsule, the perireticular nucleus is located which has been established as a distinct entity during development. Its various neuronal types express a number of different neuroactive substances. Perinatally, the perireticular nucleus is drastically reduced in size. It is involved in the guidance of corticofugal and thalamocortical fibers. The ganglionic eminence is a conspicuous proliferative area that persists throughout nearly the entire fetal period. In the human fetal brain it extends medially upon the dorsal thalamic nuclei which receive precursor cells from the ganglionic eminence. Postmitotic cells in the marginal zone of the ganglionic eminence serve as an intermediate target for growing axons. On the whole, all three structures establish transient neural circuitries that may be essential for the formation of adult projections. The characteristics of the three transient structures are particularly relevant for developmental neuropathology as these structures may be damaged in disorders that preferentially occur in preterm infants.

Distribution of SNAP-25 in transient neuronal circuitries of the developing human forebrain.

The distribution of SNAP-25 is demonstrated within prominent transient structures in the developing human forebrain. During early fetal development SNAP-25 is mainly expressed in axons of the intermediate zone and the internal capsule. The fibers appear directed towards the mantle zone of the ganglionic eminence and the perireticular nucleus located within the internal capsule. Cells of these two areas are shown to interact with SNAP-25 immunoreactive structures with the aid of double-labellings. The SNAP-25 immunoreactive fibers may represent corticofugal axons which contact the perireticular nucleus and ganglionic eminence which are regarded as intermediate targets providing a scaffold for growing axons. Anti-SNAP-25, thus, is an appropriate marker of intermediate targets which are involved in brain injuries of preterm infants.

Changing distribution patterns of synaptophysin-immunoreactive structures in the human dorsal striatum of the fetal brain.

Within the striatum two compartments, matrix and patches, can be distinguished by differences in the expression of neuroactive substances, afferent and efferent connections and time of neurogenesis. The present study was done to demonstrate the pattern of synaptophysin (SYN) expression which is indicative of synaptogenesis in the human fetal striatum (15th-32nd weeks of gestation) with special reference to developmental changes. From the 15th to the 22nd gestational weeks an intense diffuse SYN immunolabelling of striatal patches is observed. In the matrix SYN-immunoreactive fiber bundles are seen until the 20th week. Thereafter, the matrix is nearly devoid of SYN-immunoreactive structures. From the 28th week of gestation the matrix contains diffuse SYN immunoreactivity which gradually becomes as intense as that of the patches. The latter, thus, can no longer be delineated in the 30th week. The results show that fibrous SYN immunolabelling most probably indicating intra-axonal transport of synaptic vesicles can only be observed during the first half of gestation. Moreover, it becomes obvious that the patch compartment can selectively be visualized by anti-SYN until the 28th week. This pattern may correspond to the early dopaminergic innervation from the substantia nigra which is known to reach the developing patches. From the 28th week a transition from patchy to diffuse immunolabelling is seen. The increase in matrix labelling may be due to the occurrence of new neuronal contacts. The changeover from patchy to homogeneous SYN immunolabelling takes place distinctly earlier than changes in the distribution of other neuroactive substances described before.

Expression of MAP1a and MAP1b in the ganglionic eminence and the internal capsule of the human fetal brain.

The expression of microtubule-associated proteins 1a and 1b (MAP1a and 1b) were investigated in two transient structures, the ganglionic eminence (GE) being a prominent part of the telencephalic proliferative zone and the perireticular nucleus (PR) within the internal capsule (IC). Anti-MAP1a immunolabels PR neurons from 18 weeks of gestation (wg) onwards, whereas anti-MAP1b immunolabels long IC fibers between 18 and 22 wg. MAP1b is further present in thalamic fibers that seem to terminate at the medial margin of the GE, in a moderate number of cells of the GE and its medial extension, the gangliothalamic body (GTB). From 26 to 33 wg MAP1b is expressed in short fiber bundles of the IC, a few MAP1b-positive cells are seen in the GE. MAP1a has so far been described to appear in differentiated neurons and to be related to late developmental events. However, the transient PR being involved in axonal guidance as an intermediate target shows a precocious MAP1a-expression. The MAP1b-finding that thalamocortical fibers accumulate at the GE-margin indicates that this region represents an intermediate target for these fibers. The short MAP1b fiber bundles found in the IC are in accordance with cell culture experiments showing that MAP1b is concentrated in distal parts of outgrowing axons.

Interleukin-6 receptor is highly expressed in the ganglionic eminence of the human fetal brain.

The expression of interleukin-6 receptor (IL-6R) has been investigated immunohistochemically in seven fetal brains with special reference to the ganglionic eminence (GE), a part of the telencephalic proliferative zone. Between the 22nd and 28th gestational week the GE stands out conspicuously due to its very high amount of intensely IL-6R-immunostained cells. Adjacent brain areas exhibit only very weak immunoreactivity. Between the 32nd and 36th week a moderate IL-6R immunolabelling is seen in the remnants of the GE. IL-6 may activate the immature IL-6R-positive cells to secrete a protease which is likely to be involved in GE involution and, perhaps, in the development of hemorrhage frequently occurring in the GE of premature infants.

Distribution patterns of vimentin-immunoreactive structures in the human prosencephalon during the second half of gestation.

Neuronal migration is guided by long radially oriented glial fibres. During late stages of development radial glial cells are transformed into astrocytes. A predominant intermediate filament protein within radial glial cells and immature astrocytes is vimentin. In this study fetal brain sections were used to demonstrate the transient features of vimentin-positive radial glia. In the lower half of the cerebral wall of the 6th gestational month bundles, curvature, and crossing of vimentin-positive fibres are regularly seen. Moreover, fibres terminating on vessels are observed. In the upper half fibres are radially oriented; when ascending towards the pial surface the number and diameter of fibres appears conspicuously decreased. Radially aligned fibres display numerous varicosities. In the 8th month the bulk of vimentin-positive fibres is encountered next to the ganglionic eminence and below isocortical cerebral fissures. The dentate gyrus is conspicuous due to its high amount of immunolabelled fibres. Furthermore, densely packed fibres are visible within the internal and external capsule and in the vicinity of the anterior commissure. Radial glial somata are found in the proliferative areas as well as in the adjacent white matter. In the latter location bipolar, monopolar and stellate vimentin-positive cells are present. The results demonstrate an area-specific distribution pattern of vimentin-positive structures which can be correlated with migrational events. Areas maturing late in development for instance, reveal dense immunolabelling in the 8th month. The orientation and position of radial fibres point to an additional developmental role of these fibres, i.e. their involvement in the guidance of growing axons. Moreover, the arrangement and morphology of vimentin-positive fibres, such as retraction of fibres or occurrence of varicosities, are indicative of degenerative events. Accordingly, a transformation of radial glial somata, their displacement towards the white matter and finally the growth of stellate processes can clearly be demonstrated.